WO2010044390A1 - ポリアルキレンイミンを含むウイルス感染症治療薬 - Google Patents
ポリアルキレンイミンを含むウイルス感染症治療薬 Download PDFInfo
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- WO2010044390A1 WO2010044390A1 PCT/JP2009/067713 JP2009067713W WO2010044390A1 WO 2010044390 A1 WO2010044390 A1 WO 2010044390A1 JP 2009067713 W JP2009067713 W JP 2009067713W WO 2010044390 A1 WO2010044390 A1 WO 2010044390A1
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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
- A61K31/74—Synthetic polymeric materials
- A61K31/785—Polymers containing nitrogen
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
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- 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
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- 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
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- 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
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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- 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
- A61P31/18—Antivirals for RNA viruses for HIV
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- 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
- A61P31/20—Antivirals for DNA viruses
- A61P31/22—Antivirals for DNA viruses for herpes viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to a therapeutic agent for viral infection containing polyalkyleneimine.
- herpes simplex virus type 2 (HSV-2) is a pathogen that latently infects after initial infection and recurs to cause genital herpes. Its prevalence is high, and it is said that it is impossible to get out of latent infection for the rest of the life with current drug therapies. More recently, HSV-2 has been attracting attention as a factor for the spread and stimulation of human immunodeficiency virus (HIV) infection (see, for example, Non-Patent Document 1). Drug therapy is an important issue for AIDS countermeasures.
- nucleic acid analogs such as acyclovir and vidarabine, which are antiviral drugs
- these antiviral drugs enter the virus-infected cells and inhibit virus growth, so the virus side is likely to mutate to get out of the effects of the drug and acquire drug resistance, and treatment is long-term Therefore, it is necessary to pay attention to the emergence of drug-resistant viruses.
- a therapeutic agent for viral infection that has a different mechanism of action from conventional therapeutic agents.
- Patent Document 1 is known.
- Patent Document 1 affects various test measurement values by adding a virus inactivating agent mainly composed of polyethyleneimine having an average molecular weight in the range of 500 to 8,000 to a sample made of blood, body fluid, or the like. Without giving, the virus contained in the sample is inactivated to prevent virus infection.
- polyethyleneimine is known to have high toxicity to living bodies. Toxicity is not so much a problem when living cells are not used, but in the case of an evaluation system involving living cells, it becomes a big problem in developing new therapeutic agents, particularly in the field of medicine. For this reason, there has been little investigation so far regarding how it acts when a highly toxic polyethyleneimine is administered to a living body, particularly whether it can be used as a medicine.
- Patent document 2 is mentioned as a few examples which administered polyethyleneimine to the biological body.
- Patent Document 2 shows an immunostimulatory effect such as the induction of interferon ⁇ expression when a linear polyethyleneimine having a molecular weight of 87,000 introduced with a hydrophobic group such as a C18 acyl group is diluted and administered intraperitoneally to mice. It is described that However, the example of Patent Document 2 only shows that a linear polyethyleneimine having a specific molecular weight and having a specific hydrophobic group introduced therein is administered into the abdominal cavity of a mouse.
- Polyethyleneimine is a polymer that can have various structures and molecular weights, but other than those disclosed in Patent Document 2, for example, polyalkyleneimines having other molecular weights or branched or unmodified polyalkyleneimines
- Patent Document 2 Japanese Patent Laid-Open No. 6-80520 Japanese translation of PCT publication No. 2004-520328 Wald et al., "Journal of Infectious Diseases" (Wald et al., J. Infect. Dis., 185, p. 45-52, 2002)
- An object of the present invention is to provide a therapeutic agent for a viral infection that has a mechanism of action different from that of a conventional antiviral drug and that makes it difficult for a drug resistant virus to appear.
- the polyalkyleneimine having a predetermined molecular weight when administered to the living body, the polyalkyleneimine is adsorbed to the virus that has entered the living body, so that the virus is adsorbed on the cell membrane or enters the cell. And as a result, it has been found that an effect of inactivating the virus and treating or preventing a viral infection occurs.
- the present invention includes the following inventions.
- a pharmaceutical composition for treating or preventing a viral infection comprising a polyalkylenimine having a weight average molecular weight in the range of 300 to 400,000.
- the pharmaceutical composition according to (1) which is an external pharmaceutical composition containing polyalkylenimine in an amount of 20 mg / ml or less.
- a virus inactivating agent for living body administration comprising polyalkyleneimine.
- a viral cell adsorption inhibitor containing a polyalkyleneimine A viral cell adsorption inhibitor containing a polyalkyleneimine.
- a viral cell entry inhibitor comprising a polyalkyleneimine.
- a method for treating or preventing a viral infection comprising administering a polyalkylenimine having a weight average molecular weight in the range of 300 to 400,000.
- a method for inactivating a virus comprising administering a polyalkylenimine to a living body.
- a method for inhibiting cell adsorption of a virus comprising administering a polyalkyleneimine.
- a method for inhibiting viral cell entry comprising administering a polyalkylenimine.
- a drug-resistant virus is unlikely to appear, and a relatively inexpensive therapeutic agent for viral infection can be provided.
- a therapeutic agent for viral infection that is non-specific to the type of virus.
- the polyalkyleneimine used in the present invention is, for example, an alkyleneimine having 2 to 8 carbon atoms such as ethyleneimine, propyleneimine, butyleneimine, dimethylethyleneimine, pentyleneimine, hexyleneimine, heptyleneimine, octyleneimine, particularly 2 to It means a polymer obtained by polymerizing one or more of 4 alkyleneimines by a conventional method, and a polymer chemically modified by reacting them with various compounds.
- polyethyleneimine (PEI) is particularly preferred in the present invention.
- Polyethyleneimine is a polymer that can be synthesized by a method such as ring-opening polymerization using ethyleneimine as a catalyst with carbon dioxide, hydrochloric acid, hydrobromic acid, p-toluenesulfonic acid, aluminum chloride or boron trifluoride. Yes, it is widely used in a wide range of fields such as adhesion, coating, and lamination.
- Specific polyethyleneimines that can be used in the present invention include, for example, Epomin (registered trademark) (model number: SP-003, SP-006, SP-012, SP-018, SP- 200 and P-1000), polyethyleneimine commercially available from Wako Pure Chemical Industries, Ltd.
- distributed code 161-17831 (average molecular weight of about 600), distributor code 167-17811 (average molecular weight of about 1,800), and distributor code 164-17821 (average molecular weight about 10,000)
- polyethyleneimine catalog number 23966-2 (linear, MW 25,000)
- BASF Lupasol registered trademark
- Polyalkyleneimines including polyethyleneimine can be synthesized with various molecular weights by selecting a production method, and can be synthesized with linear or branched ones.
- linear polyethyleneimine has the formula:
- n is in the range of 7 to 10000.
- the branched polyethyleneimine is represented by the formula:
- the degree of branching of polyalkyleneimine including polyethyleneimine can be represented by the abundance ratio of primary amino group, secondary amino group and tertiary amino group present in the molecular skeleton.
- the ratio of each amino group is not particularly limited.
- the primary amino group, the secondary amino group, and the tertiary amino group are entirely contained. Those that occupy 25 to 45 mol%, 35 to 50 mol%, and 20 to 35 mol%, respectively, with respect to the amino group can be suitably used.
- primary amino group, secondary amino group and Tertiary amino groups account for 30-40 mol%, 30-40 mol%, 25-35 mol%, respectively, of which 35 mol%, 35 mol%, and 30 mol% respectively occupy the total amino groups It can be used suitably.
- the polyalkyleneimine can have various structures depending on the production method, but the polyalkyleneimine in the present invention may be either linear or branched.
- the polyalkyleneimine can have various molecular weights, but the weight average molecular weight of the polyalkyleneimine used in the present invention is in the range of 300 to 400,000. In particular, the weight average molecular weight is preferably 300 to 30,000, more preferably 2,000 to 30,000, especially 2,000 to 25,000.
- the weight average molecular weight of the polyalkyleneimine used in the present invention is most preferably in the range of 2,000 to 5,000.
- the weight average molecular weight here refers to GPC (gel permeation) using water-soluble sugars (such as maltotriose and maltohexaose manufactured by Wako Pure Chemical Industries, and pullulan P-82 manufactured by Showa Denko KK) as standard substances. Chromatography) method.
- the polyalkyleneimine may be unmodified or may be chemically modified by reacting with various compounds, that is, having a substituent introduced on the nitrogen atom.
- Polyalkyleneimines include, for example, aldehydes, ketones, alkyl halides, isocyanates, thioisocyanates, alkenes, alkynes, vinyl compounds (such as acrylonitrile), epoxy compounds (such as epichlorohydrin), cyanamides, guanidines, ureas, organic acids (such as fatty acids), acids It can be modified by reacting with an anhydride or acyl halide.
- Examples of the substituent introduced onto the nitrogen atom of the polyalkyleneimine by modification include C 1-6 alkyl, C 1-6 hydroxyalkyl, C 1-6 alkenyl, acyl, amide (these groups are further hydroxy , Halo, cyano, nitro, acyl, amide, sulfonyl, sulfinyl, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, C 2-6 haloalkenyl, C 2-6 alkynyl, C 2-6 Haloalkynyl, C 1-6 alkoxy, C 1-6 haloalkoxy, phenyl, benzyl, C 3-6 cycloalkyl, C 3-6 cycloalkenyl and 3-6 membered saturated, partially unsaturated or aromatic heterocycle Which may be substituted by Examples of the reaction of polyalkyleneimine with various compounds are as follows.
- R is hydroxy, halo, cyano, nitro, acyl, amide, sulfonyl, sulfinyl, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, C 2-6 haloalkenyl, C 2- 6 alkynyl, C 2-6 haloalkynyl, C 1-6 alkoxy, C 1-6 haloalkoxy, phenyl, benzyl, C 3-6 cycloalkyl, C 3-6 cycloalkenyl and 3-6 membered saturation, partial A substituent selected from the group consisting of unsaturated or aromatic heterocycles)
- the modified polyalkyleneimine include polyalkyleneimine ethoxylates, particularly polyethyleneimine ethoxylates.
- the polyalkyleneimine ethoxylate is a polymer having a structure in which a polyethylene glycol chain is bonded to the nitrogen atom of the polyalkyleneimine.
- the polyalkyleneimine ethoxylate can be synthesized, for example, by reacting an epoxy compound with polyalkyleneimine. More specifically, for example, polyethyleneimine ethoxylate is an active hydrogen contained in an amino group in polyethyleneimine by feeding ethylene oxide into polyethyleneimine in a liquid under a nitrogen atmosphere at high temperature, high pressure and in the presence of a catalyst. It can be synthesized by adding ethylene oxide from the base point.
- a specific polyalkyleneimine ethoxylate that can be used in the present invention is polyethyleneimine ethoxylate having a weight average molecular weight of about 2,000 to 15,000, preferably about 4,000 to 12,000, particularly about 5,500 to 10,000. Those having a hydroxyl value measured by the phthalation method of 50 to 500 mgKOH / g, preferably 70 to 400 mgKOH / g are preferred. Among these, polyethyleneimine ethoxylate having a dendrimer structure centered on polyethyleneimine is particularly preferable. Examples of usable polyethyleneimine ethoxylates include those prepared by reacting ethylene oxide with Epomin SP-006 or SP-018 manufactured by Nippon Shokubai Co., Ltd.
- Polyalkylenimine is a very strong cationic polymer.
- proteins that exist on the surface of viruses and play a role in binding to host cells are known to be negatively charged. Therefore, when a polyalkyleneimine is administered to an environment where viruses are present, the polyalkyleneimines are electrostatically adsorbed on the surface of the virus, resulting in the virus adsorbing to the cell membrane of the host cell or entering the cell. To prevent it and inactivate the virus.
- polyalkylenimines act directly on viruses in vivo to inactivate viruses, and are particularly effective for viral infections that cannot be treated or prevented by immunostimulants that improve the immune function of the body, for example.
- the present invention has not only a pharmaceutical composition for treating or preventing viral infections, but also an action mechanism that, when administered to a living body, the activity of the virus is blocked by adsorbing to the virus that has entered the living body. It also relates to a virus inactivating agent for biological administration. In addition, it inhibits the ability of viruses to adsorb to cells by adsorbing to viruses before adsorbing to cells, and it inhibits the ability of viruses already adsorbed to enter cells. It also relates to viral cell entry inhibitors.
- polyalkyleneimine of the present invention has a non-specific antiviral action (an action to prevent and treat viral diseases by suppressing viral growth). That is, polyalkyleneimine belongs to the herpesviridae family such as herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), varicella-zoster virus (VZV) and cytomegalovirus (CMV).
- HSV-1 herpes simplex virus type 1
- HSV-2 herpes simplex virus type 2
- VZV varicella-zoster virus
- CMV cytomegalovirus
- Viruses belonging to the Orthomyxoviridae family such as viruses (collectively referred to as herpes viruses), influenza A viruses, influenza B viruses, influenza C viruses (collectively referred to as influenza viruses), human immunodeficiency viruses (HIV Viruses belonging to the retroviridae family, viruses belonging to the Paramyxoviridae family such as measles virus and mumps virus, viruses belonging to the Picornaviridae family such as poliovirus, rhinovirus and hepatitis A virus, hepatitis B virus, etc.
- Viruses belonging to the family Flaviviridae such as hepatitis C virus, Japanese encephalitis virus and West Nile virus, viruses belonging to the adenoviridae family such as human adenovirus, viruses belonging to the coronaviridae family such as coronavirus and SARS virus, rubella Viruses belonging to Togaviridae such as viruses, viruses belonging to Rhabdoviridae such as rabies virus and vesicular stomatitis virus, viruses belonging to Filoviridae such as Ebola virus, and viruses belonging to Papovaviridae such as human papilloma virus (HPV) It has an antiviral action against various viruses such as those described above, and is effective in the treatment of infectious diseases caused by infection with these viruses.
- the polyalkyleneimine of the present invention has an excellent antiviral action against herpes virus, influenza virus, human immunodeficiency virus, measles virus, poliovirus and rhinovirus, and develops upon infection with these viruses. It is effective in treating infectious diseases.
- the polyalkylenimines of the present invention are herpesviridae, orthomyxoviridae, retroviridae, paramyxoviridae, hepadnaviridae, flaviviridae, coronaviridae, togaviridae, and rhabdoviridae.
- Herpes virus has branched polyethyleneimine with a weight average molecular weight of about 2,310-4,600 or linear polyethyleneimine with a weight average molecular weight of about 25,000, and measles virus has a branched polyethyleneimine with a weight average molecular weight of about 2,310.
- polyethyleneimine ethoxylate having a weight average molecular weight of about 10,000 is particularly effective, and for coronavirus, branched polyethyleneimine having a weight average molecular weight of about 1,470 to 321,200 or polyethyleneimine ethoxylate having a weight average molecular weight of about 10,000 is particularly effective.
- the polyalkyleneimine of the present invention is effective for the prevention or treatment of herpesvirus infections such as cold sores, herpes stomatitis, herpes keratitis, genital herpes, neonatal herpes, chicken pox and herpes zoster caused by infection with herpes virus.
- herpesvirus infections such as cold sores, herpes stomatitis, herpes keratitis, genital herpes, neonatal herpes, chicken pox and herpes zoster caused by infection with herpes virus.
- herpesvirus infections such as cold sores, herpes stomatitis, herpes keratitis, genital herpes, neonatal herpes, chicken pox and herpes zoster caused by infection with herpes virus.
- it is particularly effective for the prevention or treatment of genital herpes caused by herpes simplex virus type 2 infection.
- the polyalkyleneimine of the present invention includes influenza, AIDS, measles, mumps, polio, cold syndrome, hepatitis, Japanese encephalitis, West Nile fever, SARS, rubella, rabies, Ebola hemorrhagic fever, warts and warts. It is also effective in preventing or treating cervical cancer.
- the polyalkyleneimine of the present invention can be administered as a pharmaceutical composition alone or mixed with various pharmacologically acceptable formulation adjuvants. Generally, it is administered in a dosage form suitable for use, such as oral administration, intravenous administration, topical administration, transdermal or transmucosal administration, depending on the purpose.
- a dosage form suitable for use such as oral administration, intravenous administration, topical administration, transdermal or transmucosal administration, depending on the purpose.
- Various dosage forms include, for example, oral preparations for oral administration, injections for parenteral administration including intravenous administration, or external preparations for external administration including transdermal or transmucosal administration (external preparation) Suppositories, eye drops, nasal drops, ear drops, buccal preparations or inhalants).
- an external preparation means all the chemical
- the topical administration means administration of the drug directly to the eyes, nose, ears, mouth, anus,
- the internal use includes pharmaceutically acceptable solutions, suspensions, emulsions, syrups and elixirs.
- a liquid agent can be obtained by dissolving, suspending or emulsifying an active substance containing polyalkyleneimine in a commonly used diluent (for example, purified water, ethanol or a mixture thereof).
- the liquid preparation may contain a wetting agent, suspending agent, emulsifying agent, sweetening agent, flavoring agent, fragrance, preservative or buffering agent.
- Examples of the dosage form of the external preparation include ointments, gels, creams, poultices, patches, liniments, sprays, inhalants, sprays, aerosols, eye drops and nasal drops. .
- the ointment is manufactured by a known or commonly used formulation.
- an active substance containing a polyalkyleneimine is prepared by mixing or melting in a base.
- the ointment base is selected from known or commonly used ones.
- higher fatty acids or higher fatty acid esters for example, adipic acid, myristic acid, palmitic acid, stearic acid, oleic acid, adipic acid ester, myristic acid ester, palmitic acid ester, stearic acid ester or oleic acid ester
- waxes E.g., beeswax, whale wax or ceresin
- surfactants e.g., polyoxyethylene alkyl ether phosphates
- higher alcohols e.g., cetanol, stearyl alcohol or cetostearyl alcohol
- silicone oils e.g., Dimethylpolysiloxane
- hydrocarbons eg, hydrophilic petrolatum, white petrolat
- Gel is manufactured by a known or commonly used formulation. For example, it is prepared by melting an active substance containing polyalkyleneimine in a base.
- the gel base is selected from known or commonly used ones.
- lower alcohol such as ethanol or isopropyl alcohol
- gelling agent such as carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose or ethylcellulose
- neutralizing agent such as triethanolamine or diisopropanolamine
- a base selected from surfactants (eg, polyethylene glycol monostearate), gums, water, absorption promoters and anti-rash agents is used alone or in admixture of two or more.
- a preservative, an antioxidant or a flavoring agent may be included.
- Creams are manufactured by known or commonly used formulations. For example, it is produced by melting or emulsifying an active substance containing polyalkyleneimine in a base.
- the cream base is selected from known or commonly used ones. For example, higher fatty acid esters, lower alcohols, hydrocarbons, polyhydric alcohols (eg, propylene glycol or 1,3-butylene glycol), higher alcohols (eg, 2-hexyldecanol or cetanol), emulsifiers (eg, polyoxy Bases selected from ethylene alkyl ethers or fatty acid esters, etc.), water, absorption accelerators and anti-rash agents are used alone or in admixture of two or more. Furthermore, a preservative, an antioxidant or a flavoring agent may be included.
- the poultice is manufactured by a known or commonly used formulation.
- an active substance containing a polyalkyleneimine is melted in a base, and a kneaded product is spread and applied on a support.
- the poultice base is selected from known or commonly used ones.
- thickeners eg polyacrylic acid, polyvinylpyrrolidone, gum arabic, starch, gelatin or methylcellulose
- wetting agents eg urea, glycerin or propylene glycol
- fillers eg kaolin, zinc oxide, Bases selected from talc, calcium or magnesium
- water, solubilizers, tackifiers and anti-rash agents are used alone or in admixture of two or more.
- a preservative, an antioxidant or a flavoring agent may be included.
- the patch is manufactured by a known or commonly used formulation.
- an active substance containing a polyalkyleneimine is melted in a base and spread-coated on a support.
- the base for patch is selected from known or commonly used ones.
- a base selected from a polymer base, fats and oils, higher fatty acids, tackifiers and anti-rash agents is used alone or in admixture of two or more.
- a preservative, an antioxidant or a flavoring agent may be included.
- the liniment is manufactured by a known or commonly used formulation.
- an active substance containing polyalkyleneimine is added to one or more bases selected from water, alcohol (eg, ethanol or polyethylene glycol), higher fatty acid, glycerin, soap, emulsifier and suspending agent.
- bases selected from water, alcohol (eg, ethanol or polyethylene glycol), higher fatty acid, glycerin, soap, emulsifier and suspending agent.
- bases selected from water, alcohol (eg, ethanol or polyethylene glycol), higher fatty acid, glycerin, soap, emulsifier and suspending agent.
- a preservative, an antioxidant or a flavoring agent may be included.
- Sprays, inhalants, and sprays are buffers that provide isotonicity with stabilizers such as sodium bisulfite in addition to commonly used diluents, such as sodium chloride, sodium citrate or citric acid.
- stabilizers such as sodium bisulfite
- diluents such as sodium chloride, sodium citrate or citric acid.
- An isotonic agent such as sodium bisulfite
- injections are all injections and drops.
- intramuscular injection subcutaneous injection, intradermal injection, intraarterial injection, intravenous injection, intraperitoneal injection, spinal cavity injection and vein Including instillation agents.
- Injections include solid injections used by dissolving or suspending in solutions, suspensions, emulsions, and solvents for use.
- An injection is used by dissolving, suspending or emulsifying an active substance containing a polyalkyleneimine in a solvent.
- the solvent for example, distilled water for injection, physiological saline, vegetable oil, propylene glycol, polyethylene glycol, alcohols such as ethanol, and combinations thereof are used.
- this injection contains a stabilizer, a solubilizing agent (for example, glutamic acid, aspartic acid or polysorbate 80 (registered trademark)), a suspending agent, an emulsifying agent, a soothing agent, a buffering agent or a preservative. Also good.
- a sterile solid preparation for example, a lyophilized product, can be produced and used by dissolving it in sterilized or sterile distilled water for injection or other solvent before use.
- the ophthalmic solution includes ophthalmic solution, suspension type ophthalmic solution, emulsion type ophthalmic solution, in-use dissolved ophthalmic solution and eye ointment.
- These eye drops are produced according to known methods.
- an active substance containing polyalkyleneimine is used after being dissolved, suspended or emulsified in a solvent.
- a solvent for eye drops for example, sterilized purified water, physiological saline, other aqueous solvents or non-aqueous preparations for injection (for example, vegetable oil, etc.) and combinations thereof are used.
- Eye drops include isotonic agents (eg, sodium chloride, concentrated glycerin, etc.), buffering agents (eg, sodium phosphate or sodium acetate), surfactants (eg, polysorbate 80 (trade name), stearic acid)
- Polyoxyl 40 for example, polyoxyethylene hydrogenated castor oil
- stabilizer for example, sodium citrate or sodium edetate
- preservative for example, benzalkonium chloride or paraben
- an aerosol, a powder for inhalation, or a liquid for inhalation is included, and the liquid for inhalation may be used by dissolving or suspending in water or other appropriate medium at the time of use.
- inhalants are produced according to known methods.
- preservatives eg, benzalkonium chloride or parabens
- colorants eg, sodium phosphate or sodium acetate
- isotonic agents eg, chloride
- a thickener for example, cariboxyvinyl polymer, etc.
- an absorption enhancer and the like are appropriately selected as necessary.
- lubricants for example, stearic acid and its salts
- binders for example, starch or dextrin
- excipients for example, lactose or cellulose
- colorants for example, antiseptics
- An agent for example, benzalkonium chloride or paraben
- an absorption enhancer and the like are appropriately selected as necessary.
- a nebulizer for example, an atomizer or a nebulizer
- an inhalation administration device for powder medicine is usually used when administering a powder for inhalation.
- compositions for external administration include suppositories for rectal administration and pessaries for intravaginal administration, which contain an active substance containing polyalkyleneimine and are prescribed by conventional methods.
- the pharmaceutical composition containing the polyalkyleneimine of the present invention is particularly preferably for topical administration to the local area where a viral infection has developed, especially transdermal or transmucosal administration.
- Polyalkyleneimine is preferably administered externally at a concentration of 20 mg / ml or less, particularly preferably externally administered at a concentration of 0.004 mg / ml to 20 mg / ml.
- the dose is preferably about 0.001 to 50 mg per kg body weight per day based on the weight of polyalkyleneimine, for example, in the case of mammals.
- the antiviral action can be maintained while suppressing the toxicity of polyalkyleneimine.
- the toxicity of polyethyleneimine to the living body has an effect on the living body, for example, it causes weight loss and eventually weakens, and eventually causes death. Animals can cure viral infections without dying.
- the polyalkyleneimine concentration at the time of external administration is 10 mg / ml or less because toxicity can be further suppressed.
- the administration of the pharmaceutical composition containing the polyalkylenimine of the present invention may be performed either before or after the virus infection. However, when administered before or immediately after the virus infection, a good effect is exhibited. Administration to a virus-infected animal may be performed for only one day or continuously for several days. In the case of external administration, especially in the case of administration in a dosage form having a relatively high concentration of about 10 mg / ml to 20 mg / ml, especially about 20 mg / ml, even a single-day administration exhibits a sufficient effect.
- a certain number of days For example, it is preferable to administer for about 2 weeks or continuously for at least about 3 to 7 days. Administration may be once per day, or may be divided into 2-3 times or divided into 4-5 times.
- the toxicity of polyalkyleneimine can also be suppressed by modifying polyalkyleneimine.
- the toxicity to a living body can be suppressed by using polyalkyleneimine ethoxylate, particularly polyethyleneimine ethoxylate.
- the toxicity of polyalkyleneimine can also be suppressed by using polyalkyleneimine in combination with lipid particles.
- the lipid particle is a concept including particles composed of a lump of lipids, particles (micelles) whose surfaces are covered with lipids and containing lipophilic components therein, liposomes, and the like.
- the lipid particles in the present invention are preferably in the form of polymer micelles, emulsions, microspheres, nanoglobules, or liposomes.
- Polymer micelles are nano-particles of several tens of nanometers that are formed by autonomous association by dispersing an amphiphilic block copolymer consisting of a hydrophilic polymer and a hydrophobic polymer in water. PEG-polyamino acid or a derivative thereof is used.
- An emulsion is a dispersion in which both solute solvents are liquid.
- a microsphere is a spherical preparation having a particle size of about several ⁇ m, and a nanosphere is a nanosphere having a nanosphere diameter size (usually 1 ⁇ m or less).
- Liposomes are closed lipid vesicles composed of phospholipid bilayers and containing water.
- the type of liposome used in the present invention is not particularly limited, and any type such as multilamellar vesicles (MLV) and single lamellar vesicles (SUV, LUV) may be used. Any known method such as a spray drying method, a reverse phase evaporation method, a surfactant removal method, or an ethanol injection method may be used. Moreover, you may adjust the particle size of the obtained liposome using an extruder, a high-pressure emulsifier, an ultrasonic wave, etc.
- phospholipids can be used as the liposome membrane component, such as egg yolk, soybean, or other naturally-derived phosphatidylcholine (lecithin), phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidic acid, oleic acid, ceramide, Sphingomyelin and hydrogenated products thereof, and synthetically obtained dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, dimyristoyl phosphatidylcholine, charged lipid dicetyl phosphate, dihexadecyl phosphate, dioctadecyldimethylammonium salt, stearylamine, and other Artificial lipids and cationic lipids can be used alone or in combination.
- phosphatidylcholine lecithin
- phosphatidylethanolamine phosphatidylinositol
- the liposome used in the present invention is particularly preferably a positively charged liposome whose surface is positively charged.
- Such positively charged liposomes can be prepared, for example, by mixing egg yolk lecithin, cholesterol and stearylamine.
- a neutral liposome composed of egg yolk lecithin and cholesterol, or a liposome obtained by coating such a neutral liposome with polyethylene glycol is particularly preferable.
- the size of the liposome used in the present invention is preferably 0.025 to 200 ⁇ m, more preferably about 0.025 to 100 ⁇ m, more preferably about 0.025 to 10 ⁇ m.
- the polyalkyleneimine can be used alone or in combination with liposomes, but may be used in combination with other active substances.
- Other active substances include known antiviral drugs such as acyclovir, valacyclovir, penciclovir, famciclovir, vidarabine, idoxuridine, sorivudine, brivudine, ganciclovir, valganciclovir hydrochloride, foscarnet sodium hydrate, oseltamivir, Zanamivir, amantadine, rimantadine, zidovudine, didanosine, zalcitabine, sanilvudine, lamivudine, abacavir, tenofovir, emtricitabine, nevirapine, efavirenz, delavirdine, indinavir, saquinavir, lopinavir, rionavir rennaviravir Iodine-
- Liposomes and other active substances may be contained in the preparation containing the polyalkyleneimine of the present invention, or administration of the preparation containing the polyalkyleneimine as a preparation different from the preparation containing the polyalkyleneimine of the present invention. It may be administered simultaneously or sequentially.
- Example 1 Inhibitory effect of external application of polyethyleneimine (SP-006 and P25K) in HSV-2 infected animals About the antiviral effect of polyethyleneimine (PEI) against herpes simplex virus type 2 (HSV-2) Infection experiments were performed.
- PEI polyethyleneimine
- HSV-2 herpes simplex virus type 2
- polyethyleneimine Nippon Shokubai Epomin (registered trademark) SP-006 (branched, weight average molecular weight by GPC method 2,310) and Polysciences polyethyleneimine (catalog number 23966-2, linear, weight) Average molecular weight 25,000: This polyethyleneimine is hereinafter referred to as “P25K”). Two types of SP-006 and P25K were adjusted to pH 7.0-7.4 with 1N hydrochloric acid.
- SP-006 is 20 mg / ml and 0.4 mg / ml (20 mg / ml is diluted 50-fold with phosphate buffered saline (PBS)), and P25K is 20 mg / ml and 0.004 mg / ml (20 mg / ml). Each ml solution was diluted 5000 times with phosphate buffered saline. As a control, phosphate buffered saline (PBS) was used.
- PBS phosphate buffered saline
- HSV-2 was inoculated locally (genital) of 5-6 week old female BALB / c mice at 20 ⁇ l per animal (containing 1 ⁇ 10 5 PFU (plaque forming unit) virus).
- Topical administration by application to the mucosa over the course of a week The results are shown in Table 1.
- HSV-2 The dose of HSV-2 was lethal for BALB / c mice, and all 13 animals died within 10 days after virus infection in the control group.
- 20 mg / ml of SP-006 and P25K were administered three times on the day of infection, the mice were obviously weakened and were not administered after the second day. Only 1 out of 3 mice treated with 20 mg / ml of SP-006 died on the first day of administration (weakness and death may be due to polyethyleneimine toxicity). In other administration groups, toxicity (change in body weight, judging from appearance) to mice was not observed.
- the amount of virus in the area 3 days after virus infection was measured by a plaque assay using a washing solution collected by washing the mouse with PBS.
- a plaque assay using a washing solution collected by washing the mouse with PBS.
- an effect of suppressing death and an effect of extending the number of days of survival were observed.
- Fig. 2 shows the change in average body weight of surviving mice, with the day of infection as 100%.
- the body weight decreased significantly on the first day due to toxicity, but recovered after the treatment was stopped and the viral infection was cured.
- the body weight gradually decreased due to viral infection, leading to the death of mice.
- polyethyleneimine is slightly toxic to mice at a concentration of about 20 mg / ml and is not suitable for continuous administration, but shows a strong therapeutic effect on genital herpes with a few administrations.
- a low concentration of 0.4 mg / ml or 0.004 mg / ml brings about a sufficient virus growth-suppressing effect to suppress onset and death without showing toxicity to mice.
- Example 2 Inhibitory effect of a complex of polyethyleneimine (SP-006) and liposome in HSV-2 infected animals
- Liposomes composed of egg yolk lecithin, cholesterol and stearylamine (5: 5: 4 in molar ratio) (total lipid 20 mg) / ml) and an aqueous solution containing SP-006 were adjusted to pH 7.0-7.4 with 1N hydrochloric acid.
- This polyethyleneimine-liposome complex contains SP-006 at a concentration of 20 mg / ml.
- This mixture was evaluated for efficacy in the genital herpes model of BALB / c mice. The experiment was performed in the same manner as in Example 1 above.
- the toxicity of polyethyleneimine to the living body can be attenuated not only by diluting and using polyethyleneimine, but also by using it together with liposomes. It was found that it was maintained.
- Example 3 HSV-2 virus inactivating action of polyethyleneimine It was examined whether the HSV-2 virus was actually inactivated by polyethyleneimine. The experiment was performed according to the following procedure.
- polyethyleneimine has no virucidal activity, it has a function of reversibly blocking virus infection by causing a loose bond (electrostatic bond) with the virus, that is, a virus inactivating action. It shows that it has.
- Comparative Example 1 As a positive control for the action of popidone iodine on HSV-2 virus , the commercially available disinfectant popidone iodine (Isodine (registered trademark) manufactured by Meiji Seika Co., Ltd., popidone iodine content 100 mg / ml) showing strong virucidal activity The same method as in Example 3 was examined.
- Isodine registered trademark
- Example 4 Cell adsorption inhibition effect of polyethyleneimine on HSV-2 virus It was examined whether the adsorption of HSV-2 virus to host cells was inhibited by polyethyleneimine. The experiment was performed according to the following procedure.
- Vero cells 24-well plate
- virus solution 200PFU / 100 ⁇ l
- polyethyleneimine solution SP-006 or P25K
- 100 ⁇ l of virus solution and polyethyleneimine solution were added to Vero cells and treated at 4 ° C for 1 hour (at 4 ° C, the virus was adsorbed on the cells, but the subsequent virus entry into the cells did not occur)
- a sample-free medium (with 0.8% methylcellulose) was overlaid.
- Table 4 shows the amount of virus adsorbed on the cells in the presence of the sample. SP-006 and P25K inhibited virus adsorption in a concentration-dependent manner.
- Example 5 Inhibitory Effect of Polyethylenimine on HSV-2 Virus Next, the inhibitory effect of polyethylenimine on the stage of HSV-2 adsorbed on host cells entering cells was examined. The experiment was performed according to the following procedure.
- Vero cells 24-well plate
- virus solution 100 PFU / 100 ⁇ l
- Comparative Example 2 Cell adsorption / invasion inhibitory effect of popidone iodine on HSV-2 virus
- the effect of popidone iodine on the adsorption of HSV-2 to host cells was examined. The experiment was performed in the same manner as in Example 4 above. As a result, the amount of virus adsorbed on the cells in the presence of the sample is shown in Table 6. Unlike polyethyleneimine, popidone iodine showed almost no concentration-dependent adsorption inhibition effect.
- HSV-2 entry inhibition effect of acyclovir was examined in the same manner as in Example 5 above.
- Table 9 shows the amount of virus that entered the cells in the presence of acyclovir. Acyclovir did not inhibit HSV-2 entry into cells.
- Acyclovir is an agent that blocks the replication stage of HSV-2 in infected cells and is not involved in the adsorption and entry stages, which are the early stages of viral infection.
- This comparative example shows that polyethyleneimine has a different mechanism of action from acyclovir.
- Example 6 Evaluation of antiviral activity of polyethyleneimine against various viruses The antiviral activity of polyethyleneimine against herpes simplex virus types 1 and 2, measles virus, influenza A virus and human coronavirus was evaluated in vitro.
- Nippon Shokubai Epomin registered trademark
- SP-003 weight average molecular weight 1,470 by GPC method
- SP-006 weight average molecular weight 2,310 by GPC method
- SP-012 weight average molecular weight by GPC method
- SP-018 weight average molecular weight 4,600 by GPC method
- SP-200 weight average molecular weight 16,560 by GPC method
- P-1000 weight average molecular weight 321,200 by GPC method
- polyethylene prepared by the procedure described below Imine ethoxylate three types, PEIE-1, PEIE-2 and PEIE-3 was used.
- SP-003, SP-006, SP-012, SP-018, P-1000 and PEIE-3 data are in ⁇ l / ml
- SP-200, PEIE-1 and PEIE-2 data are expressed in units of ⁇ g / ml.
- test was conducted to evaluate the cytotoxicity of polyethyleneimine against each cell. The test was conducted according to the following procedure.
- Herpes simplex virus type 1 (HSV-1) According to the following procedure, the growth inhibitory effect of polyethyleneimine on herpes simplex virus type 1 was examined.
- Vero cells African green monkey kidney-derived cells
- trypsin adjusted to 1 ⁇ 10 6 cells / ml
- the virus solution was diluted appropriately with a medium so that it would be 0.2 PFU (plaque forming unit) / cell
- Polyethyleneimine was appropriately diluted in the medium (final concentrations: SP-003, SP-006, SP-012, SP-018, P-1000 and PEIE-3 are 0.0001-10 ⁇ l / ml, SP-200, PEIE -1 and PEIE-2 are 0.0001-10 ⁇ g / ml, and the same applies to [B]-[E] below)
- virus infection was performed as follows.
- Section A 25 ⁇ l of virus solution and 25 ⁇ l of polyethyleneimine were added simultaneously, and the plate was infected for 1 hour at room temperature, occasionally (about once every 5-10 minutes).
- Vero cells were cultured in a monolayer on a 35 mm dish, and the sample that had been frozen and thawed three times was diluted 10 0 -10 5 times with PBS (-) (8) Add 100 ⁇ l of diluted virus solution per dish and infect at 37 ° C. for 1 hour at room temperature (9) Methyl cellulose (MC) -added MEM medium was overlaid with 1.5 ml per dish and placed in a 37 ° C CO 2 incubator.
- the growth inhibitory effects of polyethyleneimine against these viruses are shown in Table 10 (KOS strain) and Table 11 (A4-3 strain).
- the selectivity index is 1 or more, it is considered to have antiviral activity, and if it is 10 or more, it is considered to have strong antiviral activity.
- SP-006, SP-012 and SP-018 showed a high selection index in the A section, that is, when added simultaneously with virus infection, and SP-012 was most effective .
- SP-012 was most effective for ACV resistant strains.
- Herpes simplex virus type 2 (HSV-2) The growth inhibitory effect of polyethyleneimine against herpes simplex virus type 2 was examined by the same plaque assay method as [A]. The results are shown in Table 12. SP-006, SP-012 and SP-018 showed a high selectivity index in the A section, and SP-012 was the most effective.
- MDCK cells canine kidney-derived cells
- trypsin adjusted to 1 ⁇ 10 6 cells / ml
- the virus solution was appropriately diluted with a medium to 0.2 PFU / cell
- Polyethyleneimine was diluted appropriately in the medium
- virus infection was performed as follows. Section A: 25 ⁇ l of virus solution and 25 ⁇ l of polyethyleneimine were added simultaneously, and the plate was infected for 1 hour at room temperature, occasionally (about once every 5-10 minutes).
- a diluent (medium) was added instead of polyethylene imine in section A, and the same operation as section A was performed.
- the plate was washed 3 times with PBS ( ⁇ ), and 100 ⁇ l of polyethyleneimine and 100 ⁇ l of maintenance medium were added to each well.
- MDCK cells were cultured in a monolayer on a 35 mm dish, and the supernatant of the cryopreserved culture was diluted 10 0 -10 5 times with PBS (-) (8) Add 100 ⁇ l of diluted virus solution per dish and infect at 37 ° C.
- MRC-5 cells human fetal lung-derived cells
- trypsin adjusted to 1 ⁇ 10 5 cells / ml
- the virus solution was appropriately diluted with a medium to 0.002TCID 50
- Polyethyleneimine was diluted appropriately in the medium (4) After removing the medium, virus infection was performed as follows. Section A: 25 ⁇ l of virus solution and 25 ⁇ l of polyethyleneimine were added simultaneously, and the plate was infected for 1 hour at room temperature, occasionally (about once every 5-10 minutes).
- a diluent (medium) was added instead of polyethylene imine in section A, and the same operation as section A was performed.
- Table 15 shows the results of calculating the selection index from the CC 50 value and TCID 50 value for MRC-5 cells. Almost all polyethylenimines showed a high selectivity index, with SP-012 being the most effective. PEIE-3 also showed high selectivity. In this case, the selection index was higher in the A ward than in the B ward.
- PEIE-1 171.2 g of polyethyleneimine (Epomin SP-006 manufactured by Nippon Shokubai Co., Ltd.) was charged into a 1.2 L autoclave and heated to 150 ° C. Thereafter, 167.4 g (3.8 mol) of ethylene oxide was fed to add ethylene oxide to polyethyleneimine. Subsequently, it was cooled to 80 ° C., charged with 2.5 g of a 49% KOH aqueous solution and heated to 165 ° C.
- the target PEI / PEG 22/78 (weight ratio), weight average molecular weight 5,500 by GPC method, polyethylenimine polyethoxylate (PEIE) with hydroxyl value 370 ⁇ 20mgKOH / g by anhydrous phthalation method -1) was obtained.
- PEIE polyethylenimine polyethoxylate
- PEIE-2 171.2 g of polyethyleneimine (Epomin SP-018 manufactured by Nippon Shokubai Co., Ltd.) was charged into a 1.2 L autoclave and heated to 150 ° C. Thereafter, 167.4 g (3.8 mol) of ethylene oxide was fed to add ethylene oxide to polyethyleneimine. Next, the mixture was cooled to 80 ° C., charged with 2.5 g of a 49% KOH aqueous solution, and heated to 165 ° C. While dehydrating by depressurizing to 0.05 MPa while nitrogen bubbling at a flow rate of 20 ml / min, 349.5 g (7.9 mol) of ethylene oxide was fed to add ethylene oxide to polyethyleneimine.
- the mixture was cooled to 80 ° C. and adjusted to pH 7 by adding acetic acid. 10% of pure water was added to the total amount of polyethyleneimine polyethoxylate, and the pressure was reduced to 0.005 MPa while performing nitrogen bubbling at a flow rate of 20 ml / min, and deaeration was performed for 5 hours.
- the target PEI / PEG 25/75 (weight ratio), weight average molecular weight 7,500 by GPC method, and polyethylenimine polyethoxylate (PEIE) with hydroxyl value 345 ⁇ 20mgKOH / g by anhydrous phthalation method -2) was obtained.
- PEIE-3 180.0 g of polyethyleneimine (Epomin SP-006 manufactured by Nippon Shokubai Co., Ltd.) was charged into a 1.2 L autoclave and heated to 150 ° C. Thereafter, 174.8 g (3.97 mol) of ethylene oxide was fed to add ethylene oxide to polyethyleneimine. Next, the mixture was cooled to 80 ° C., charged with 28.8 g of 49% KOH aqueous solution and heated to 165 ° C. While dehydrating by depressurizing to 0.05 MPa while nitrogen bubbling at a flow rate of 20 ml / min, 469.4 g (10.67 mol) of ethylene oxide was fed to add ethylene oxide to polyethyleneimine.
- polyethyleneimine Epomin SP-006 manufactured by Nippon Shokubai Co., Ltd.
- the temperature was lowered to 80 ° C., and the content of the autoclave was withdrawn. After the withdrawal, the amount of the remaining liquid was 173.0 g, and then 620.1 g (13.2 mol) of ethylene oxide was fed to add ethylene oxide to polyethyleneimine. Then, it cooled to 80 degreeC and added acetic acid and adjusted to pH7. 10% of pure water was added to the total amount of polyethyleneimine polyethoxylate, and the pressure was reduced to 0.005 MPa while performing nitrogen bubbling at a flow rate of 20 ml / min, and deaeration was performed for 5 hours. Light impurities are removed, and 20% of pure water is added to the total amount.
- the target PEI / PEG 5/95 (weight ratio), weight average molecular weight by GPC method 10,000, Polyethyleneimine ethoxylate (PEIE-3) having a hydroxyl value of 74.0 ⁇ 3.0 mgKOH / g was obtained by the anhydrous phthalation method.
- Example 7 Onset prevention effect by external application of polyethyleneimine (SP-006 and SP-012) in HSV-2 infected animals SP- showing high selectivity index in in vitro anti-HSV-2 activity test of Example 6 For 006 and SP-012, infection experiments using mice were performed again for the antiviral effect of these polyethyleneimines.
- SP-006 and SP-012 polyethyleneimine
- HSV-2 was inoculated locally (genital) of 5-6 week old female BALB / c mice at 20 ⁇ l per animal (containing 1 ⁇ 10 5 PFU of virus).
- SP-006, SP-012, or phosphate buffered saline at a dose of 0.2 mg or 0.02 mg per dose (20 ⁇ l) (concentration: 10 mg / ml or 1 mg / ml), 1 hour before virus infection, immediately after infection 6 hours later, 24 hours later, 48 hours later, a total of 5 times was applied to the mucosa for topical administration. No toxicity to mice was observed under this administration condition. The results are shown in Table 16.
- Example 8 Verification of no occurrence of drug-resistant virus by polyethyleneimine (SP-012) Whether herpes simplex virus type 2 (HSV-2) can be drug-resistant by administration of polyethyleneimine (SP-012) I verified.
- HSV-2 infected Vero cells were subcultured 3 times in the presence of 100 ⁇ g / ml SP-012, and further subcultured 7 times at a concentration of 200 ⁇ g / ml, and 10 surviving viruses were detected by plaque method. separated.
- a sensitivity test for SP-012 was conducted. Sensitivity was expressed as the concentration of SP-012 (IC 50 ) that inhibits virus growth by 50%.
- Table 17 As shown in Table 17, all 10 clones had an IC 50 of the same level as that of the original virus strain (wild strain), and the sensitivity to SP-012 was maintained, that is, it was judged that resistance did not occur. . From this, it is presumed that even if SP-012 is administered to a living body for a long period of time, there is a high possibility that the problem of administration interruption due to the appearance of resistant virus does not occur.
- Example 9 Comparison of the effects of polyethyleneimine (SP-012) and acyclovir on HSV-2 infected animals The same administration of herpes simplex virus type 2 (HSV-2) therapeutic drug acyclovir and polyethyleneimine (SP-012) The time course of HSV-2 proliferation in the genitals of mice when treated with dose was followed.
- HSV-2 herpes simplex virus type 2
- mice 5-6 weeks old female BALB / c mice were locally (genital) with 20 ⁇ l of HSV-2 (1x10 4 PFU / mouse to keep track of untreated control mice) ) Inoculated.
- SP-012 0.2 mg of acyclovir each time, or phosphate buffered saline, 1 hour before, 1 hour, 8 hours after infection, and twice a day from the next day (8 am, 6 pm ), Topical administration until 7 days after infection. Between 1 and 12 days after infection, the area was washed daily with phosphate buffered saline, and the amount of virus in each was measured one by one. The degree of onset and deaths were recorded for 15 days.
- Example 10 Evaluation of antiviral activity of a complex of polyethyleneimine (SP-012) and liposome against herpes simplex virus type 2 (HSV-2) A complex of polyethyleneimine (SP-012) and liposome was prepared and herpes simplex Antiviral activity against virus type 2 (HSV-2) was evaluated in vitro.
- SP-012 polyethyleneimine
- HSV-2 herpes simplex virus type 2
- Acyclovir, polyethyleneimine (SP-012), polyethyleneimine (SP-012) -neutral liposome complex, polyethyleneimine were prepared in the same manner as in Example 6 (see [B] in particular).
- SP-012-Using each sample of PEG liposome complex cytotoxicity (50% cell growth inhibitory concentration (CC 50 )), antiviral activity against herpes simplex virus type 2 (50% virus growth inhibitory concentration (IC 50 )) and the selection index (CC 50 / IC 50 ).
- the concentration range of acyclovir or polyethyleneimine was 0.02 to 2000 ⁇ g / ml during the cytotoxicity test and 0.01 to 1000 ⁇ g / ml during the antiviral activity test.
- the results are shown in Table 19.
- the complex of polyethyleneimine and neutral liposomes or PEG liposomes was found to have a higher selectivity index and a very high antiviral activity compared to the case of using polyethyleneimine alone.
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Abstract
Description
変性させたポリアルキレンイミンの具体例としては、ポリアルキレンイミンエトキシレート、特にポリエチレンイミンエトキシレートが挙げられる。ポリアルキレンイミンエトキシレートとは、ポリアルキレンイミンの窒素原子上にポリエチレングリコール鎖が結合した構造を有するポリマーである。ポリアルキレンイミンエトキシレートは、例えばポリアルキレンイミンにエポキシ化合物を反応させることによって合成することができる。より具体的には、例えばポリエチレンイミンエトキシレートは、窒素雰囲気下、高温、高圧、触媒存在の条件でポリエチレンイミンにエチレンオキサイドを液中フィードすることにより、ポリエチレンイミン中のアミノ基に含まれる活性水素を基点としてエチレンオキサイドを付加させて合成することができる。本発明に用いることができる具体的なポリアルキレンイミンエトキシレートとしては、ポリエチレンイミンエトキシレートであって、重量平均分子量が2,000~15,000程度、好ましくは4,000~12,000、特に5,500~10,000程度であり、無水フタル化法で測定した水酸基価が50~500mgKOH/g、好ましくは70~400mgKOH/gであるものが好ましい。中でも、ポリエチレンイミンを中心とするデンドリマー構造を有するポリエチレンイミンエトキシレートが特に好ましい。利用可能なポリエチレンイミンエトキシレートの例としては、例えば日本触媒社製のエポミンSP-006あるいはSP-018にエチレンオキサイドを反応させて調製したものが挙げられる。
単純ヘルペスウイルス2型(HSV-2)に対するポリエチレンイミン(PEI)の抗ウイルス作用について、マウスを用いた感染実験を行った。
卵黄レシチン、コレステロール、ステアリルアミン(モル比で5:5:4)から成るリポソーム(総脂質20mg/ml)とSP-006を含む水溶液を、1N塩酸でpHを7.0-7.4に調整した。このポリエチレンイミン-リポソーム複合体には、SP-006が20mg/mlの濃度で含有されている。この混合物について、BALB/cマウスの性器ヘルペスモデルにおける有効性を評価した。実験は上記の実施例1と同様に行った。
HSV-2ウイルスが実際にポリエチレンイミンによって不活化されるかどうかを検討した。実験は以下の手順に従って行った。
(2)HSV-2(2x102PFU/100μlまたは2x104PFU/100μl)と上記(1)のポリエチレンイミン希釈液とを1:1で混合し、37℃、1時間処理した
(3)ウイルス量が2x102PFU/100μlの場合には希釈せずに、2x104PFU/100μlの場合にはPBSで100倍希釈後、プラークアッセイ法で残存ウイルス量を測定した
その結果を表2にまとめた。希釈処理を加えた場合は、希釈処理を行わなかった場合と比較すると残存ウイルス量が増加し、ウイルス不活化作用がやや弱まった。このことは、ポリエチレンイミンは殺ウイルス活性を有しないものの、ウイルスとの間に緩やかな結合(静電的結合)を起こして、可逆的にウイルスの感染を阻止する作用、すなわちウイルス不活化作用を有することを示している。
陽性対照として、強力な殺ウイルス活性を示す市販消毒薬ポピドンヨード(明治製菓社製イソジン(登録商標)、ポピドンヨード含有量100mg/ml)の殺ウイルス活性を、上記の実施例3と同様の方法で検討した。
HSV-2ウイルスがポリエチレンイミンによって宿主細胞への吸着を阻害されているかどうかを検討した。実験は以下の手順で行った。
(2)ウイルス液とポリエチレンイミン液とを100μlずつVero細胞に加え、4℃で1時間処理した(4℃では、ウイルスの細胞への吸着は起こるが、その後のウイルスの細胞内侵入は起こらない)
(3)氷冷したリン酸緩衝生理食塩水で2回洗浄後、サンプル無添加の培地(0.8%メチルセルロース加)を重層した。
次に、宿主細胞に吸着したHSV-2が細胞内に侵入する段階に対するポリエチレンイミンの阻害効果を検討した。実験は以下の手順で行った。
(3)氷冷したリン酸緩衝生理食塩水で2回洗浄後、ポリエチレンイミン(SP-006またはP25K)を添加した培地を加えて、37℃で培養した(この温度に置くことによって、4℃で吸着していたウイルスが侵入を開始する)
(4)30分後または3時間後にクエン酸バッファー(pH 3.0)で1分間処理して、その時間までに侵入していなかったウイルスを不活化した
(5)すぐに培地(0.8%メチルセルロース加)を重層した
(6)3日後にプラーク数を測定した
サンプル存在下で細胞内に侵入したウイルス量を表5に示す。SP-006およびP25Kは濃度依存的にウイルス侵入を阻害した。その効果は吸着阻害効果に比べて強力であった。また実施例2においてウイルスの不活化がみられなかった濃度であっても、ウイルス侵入阻害効果が観察された。
HSV-2の宿主細胞への吸着に及ぼすポピドンヨードの影響を検討した。実験は上記の実施例4と同様に行った。その結果、サンプル存在下で細胞に吸着したウイルス量を表6に示す。ポリエチレンイミンとは異なり、ポピドンヨードには濃度依存的な吸着阻害効果がほとんどみられなかった。
HSV-2の宿主細胞への吸着に及ぼすアシクロビルの影響を検討した。実験は上記の実施例4と同様に行った。その結果、アシクロビル存在下で細胞に吸着したウイルス量を表8に示す。ポリエチレンイミンとは異なり、アシクロビルには濃度依存的な吸着阻害効果がほとんどみられなかった。
単純ヘルペスウイルス1型および2型、麻疹ウイルス、A型インフルエンザウイルスならびにヒトコロナウイルスに対するポリエチレンイミンの抗ウイルス活性をin vitroにおいて評価した。
(2)無添加対照の細胞数を100%として、各濃度のポリエチレンイミンで処理した時の細胞数を%で表し、グラフ上で50%細胞増殖阻害濃度(CC50)を求めた
各種ウイルスに対するポリエチレンイミンの抗ウイルス活性は以下のようにして調べた。
以下の手順により、単純ヘルペスウイルス1型に対するポリエチレンイミンの増殖抑制効果を調べた。
(2)1-2日後に、単層状になっていることを確認した上で、0.2PFU(プラーク形成単位)/cellになるように、ウイルス液を適宜培地で希釈した
(3)ポリエチレンイミンを培地で適宜希釈した(終濃度:SP-003、SP-006、SP-012、SP-018、P-1000およびPEIE-3は0.0001~10μl/ml、SP-200、PEIE-1およびPEIE-2は0.0001~10μg/ml、以降の[B]~[E]においても同様)
(4)培地を除去後、ウイルス感染を以下のように行った
A区:ウイルス液25μlとポリエチレンイミン25μlを同時に加え、室温で1時間、時々(5-10分に1回程度)プレートをゆすりながら感染させた
B区:A区のポリエチレンイミンの代わりに希釈液(培地)を加えて、A区と同様の操作を行った
(5)ウイルス液を除去後、PBS(-)で3回洗浄し、ポリエチレンイミン100μlと培地100μlとを各穴に加えた
(6)24時間後に、-80℃の冷凍庫に移し、凍結融解を3回行った
(7)35mmディッシュにVero細胞を単層状に培養しておき、3回の凍結融解が終了したサンプルをPBS(-)で100-105倍に希釈した
(8)希釈したウイルス液を、1ディッシュ当り100μl加えて、37℃、1時間、室温で感染させた
(9)メチルセルロース(MC)加MEM培地を1ディッシュ当たり1.5ml重層し、37℃のCO2インキュベータに入れた
(10)1-2日後に、プラークが適当な大きさになっていることを確認して、培地を除去し、クリスタルバイオレット液で固定・染色を行い、顕微鏡下でプラーク数を計算した
(11)無添加対照区のプラーク数を100%として、ポリエチレンイミン添加区のプラークの%を計算し、片対数グラフ上で50%ウイルス増殖阻害濃度(IC50)を求めた
(12)Vero細胞に対するCC50値とIC50値から、選択指数を求めた
単純ヘルペスウイルス1型は、アシクロビル(ACV)感受性株(KOS株)と耐性株(A4-3株)とを用いた。これらのウイルスに対するポリエチレンイミンの増殖抑制効果を表10(KOS株)と表11(A4-3株)に示す。ここで選択指数は、1以上あれば抗ウイルス活性を有すると考えられ、特に10以上あれば強力な抗ウイルス活性を有すると考えられる。
[A]と同様のプラークアッセイ法により、単純ヘルペスウイルス2型に対するポリエチレンイミンの増殖抑制効果を調べた。その結果を表12に示す。SP-006、SP-012およびSP-018はA区において高い選択指数を示し、SP-012が最も効果的であった。
[A]と同様のプラークアッセイ法により、麻疹ウイルスに対するポリエチレンイミンの増殖抑制効果を調べた。その結果を表13に示す。SP-006(A区)は10以上の選択指数を示した。
以下の手順により、A型インフルエンザウイルスに対するポリエチレンイミンの増殖抑制効果を調べた。
(2)1-2日後に、単層状になっていることを確認した上で、ウイルス液を0.2PFU/cellとなるように、適宜培地で希釈した
(3)ポリエチレンイミンを適宜培地で希釈した
(4)培地を除去後、ウイルス感染を次のように行った
A区:ウイルス液25μlとポリエチレンイミン25μlを同時に加え、室温で1時間、時々(5-10分に1回程度)プレートをゆすりながら感染させた
B区:A区のポリエチレンイミンの代わりに希釈液(培地)を加えて、A区と同様の操作を行った
(5)ウイルス液を除去後、PBS(-)で3回洗浄し、ポリエチレンイミン100μlと維持培地100μlとを各穴に加えた
(6)24時間後に、-80℃の冷凍庫に移した
(7)35mmディッシュにMDCK細胞を単層状に培養しておき、凍結保存しておいた培養物の上清をPBS(-)で100-105倍に希釈した
(8)希釈したウイルス液を、1ディッシュ当り100μl加えて、37℃、1時間、室温で感染させた
(9)インフルエンザウイルスアッセイ用寒天培地を1ディッシュ当たり2ml重層し、寒天が固化してから37℃のCO2インキュベータに入れた
(10)2日後に、プラークが適当な大きさになっていることを確認して、染色後プラーク数を計算した
(11)無添加対照区のプラーク数を100%として、ポリエチレンイミン添加区のプラークの%を計算し、片対数グラフ上で50%ウイルス増殖阻害濃度(IC50)を求めた
(12)MDCK細胞に対するCC50値とIC50値から、選択指数を求めた
用いたウイルスは、A/NWS/33(H1N1)である。結果を表14に示す。選択指数10以上のポリエチレンイミンはなかったが、PEIE-3は最も効果があり、次いでSP-012、SP-006が比較的高い選択性を示した。
以下の手順により、ヒトコロナウイルスに対するポリエチレンイミンの増殖抑制効果を調べた。
(2)1-2日後に、ほぼ単層状になっていることを確認した上で、ウイルス液を0.002TCID50になるように、適宜培地で希釈した
(3)ポリエチレンイミンを適宜培地で希釈した
(4)培地を除去後、ウイルス感染を次のように行った
A区:ウイルス液25μlとポリエチレンイミン25μlを同時に加え、室温で1時間、時々(5-10分に1回程度)プレートをゆすりながら感染させた
B区:A区のポリエチレンイミンの代わりに希釈液(培地)を加えて、A区と同様の操作を行った
(5)ウイルス液を除去後、PBS(-)で3回洗浄し、ポリエチレンイミン150μlと培地150μlとを各穴に加え、36℃で処理した
(6)3日後に、細胞変性効果(CPE)(円形化および腐肉形成(rounding and sloughing))を観察して(必要に応じてその程度を記録)、-80℃の冷凍庫に移した
(7)96穴プレートにほぼ単層状のMRC-5細胞を準備した
(8)3回凍結融解したウイルス液について、培地で3倍希釈系列(31-310)を作り、上記のプレートに4穴づつ、25μlを加えて感染させた(室温、1時間)
(9)培地を1穴当たり100μl加え、36℃のCO2インキュベータに入れた
(10)5日後に、CPEの有無を確認した
(11)Reed-Muench法でTCID50を求めた
(12)MRC-5細胞に対するCC50値とTCID50値から、選択指数を求めた
結果を表15に示す。ほぼすべてのポリエチレンイミンが高い選択指数を示し、SP-012が最も有効であった。PEIE-3も高い選択性を示した。この場合も、A区の方がB区よりも選択指数が高くなった。
(a)PEIE-1
ポリエチレンイミン(日本触媒社製のエポミンSP-006)172.3gを1.2Lオートクレーブに仕込み、150℃へ昇温した。その後エチレンオキサイド167.4g(3.8mol)をフィードして、ポリエチレンイミンにエチレンオキサイドを付加した。次いで、80℃まで冷却し、49%KOH水溶液を2.5g仕込み、165℃へ昇温した。20ml/分の流量で窒素バブリングしながら、0.05MPaへ減圧して脱水した後、エチレンオキサイドを443.5g(10.1mol)フィードして、ポリエチレンイミンにエチレンオキサイドを付加した。次いで80℃まで冷却し、酢酸を加えてpH7へ調整した。純水をポリエチレンイミンポリエトキシレート全量に対して10%添加し、20ml/分の流量で窒素バブリングしながら、0.005MPaへ減圧し、5時間脱気を行った。軽質不純物を除去することで、目的のPEI/PEG=22/78(重量比)、GPC法による重量平均分子量5,500、無水フタル化法による水酸基価370±20mgKOH/gのポリエチレンイミンポリエトキシレート(PEIE-1)を得た。
ポリエチレンイミン(日本触媒社製のエポミンSP-018)172.3gを1.2Lオートクレーブに仕込み、150℃へ昇温した。その後エチレンオキサイド167.4g(3.8mol)をフィードしてポリエチレンイミンにエチレンオキサイドを付加した。次いで80℃まで冷却し、49%KOH水溶液を2.5g仕込み、165℃へ昇温した。20ml/分の流量で窒素バブリングしながら、0.05MPaへ減圧して脱水した後、エチレンオキサイドを349.5g(7.9mol)フィードして、ポリエチレンイミンにエチレンオキサイドを付加した。次いで80℃まで冷却し、酢酸を加えてpH7へ調整した。純水をポリエチレンイミンポリエトキシレート全量に対して10%添加し、20ml/分の流量で窒素バブリングしながら、0.005MPaへ減圧し、5時間脱気を行った。軽質不純物を除去することで、目的のPEI/PEG=25/75(重量比)、GPC法による重量平均分子量7,500、無水フタル化法による水酸基価345±20mgKOH/gのポリエチレンイミンポリエトキシレート(PEIE-2)を得た。
ポリエチレンイミン(日本触媒社製のエポミンSP-006)180.0gを1.2Lオートクレーブに仕込み、150℃へ昇温した。その後エチレンオキサイド174.8g(3.97mol)をフィードして、ポリエチレンイミンにエチレンオキサイドを付加した。次いで80℃まで冷却し、49%KOH水溶液28.8g仕込み、165℃へ昇温した。20ml/分の流量で窒素バブリングしながら、0.05MPaへ減圧して脱水した後、エチレンオキサイドを469.4g(10.67mol)フィードして、ポリエチレンイミンにエチレンオキサイドを付加した。次いで80℃まで降温し、オートクレーブ内容液を抜出、抜出後の残液量を173.0gとした後、エチレンオキサイドを620.1g(13.2mol)フィードして、ポリエチレンイミンにエチレンオキサイドを付加した。この後、80℃まで冷却し、酢酸を加えてpH7へ調整した。純水をポリエチレンイミンポリエトキシレート全量に対して10%添加し、20ml/分の流量で窒素バブリングしながら、0.005MPaへ減圧し、5時間脱気を行った。軽質不純物を除去し、さらに純水を全量に対して20%添加し、固形分80%の水割り品として、目的のPEI/PEG=5/95(重量比)、GPC法による重量平均分子量10,000、無水フタル化法による水酸基価74.0±3.0mgKOH/gのポリエチレンイミンエトキシレート(PEIE-3)を得た。
実施例6のin vitroでの抗HSV-2活性試験において高い選択指数を示したSP-006及びSP-012を対象として、これらのポリエチレンイミンの抗ウイルス作用についてマウスを用いた感染実験を再度行った。
単純ヘルペスウイルス2型(HSV-2)がポリエチレンイミン(SP-012)の投与により薬剤耐性を有し得るか否かを検証した。
単純ヘルペスウイルス2型(HSV-2)治療薬であるアシクロビルとポリエチレンイミン(SP-012)とを同一投与量で処置した時のマウスの性器におけるHSV-2増殖の時間的過程を追跡した。
ポリエチレンイミン(SP-012)とリポソームの複合体を調製し、単純ヘルペスウイルス2型(HSV-2)に対する抗ウイルス活性をin vitroにおいて評価した。
中性のリポソーム(卵黄レシチン(EPC):コレステロール(Cho)=5:5、モル比)をポリエチレンイミン(SP-012)水溶液と混合し(最終脂質濃度20mg/ml、ポリエチレンイミン濃度100mg/ml)、1N塩酸でpHを7.0-7.4に調整した。
ポリエチレングリコール(PEG)でコートしたリポソーム(EPC:Cho:PEG=5:5:0.5、モル比)をポリエチレンイミン(SP-012)水溶液と混合し(最終脂質濃度20mg/ml、ポリエチレンイミン濃度100mg/mL)、1N塩酸でpH7.0-7.4に調整した。
実施例6(特に[B]を参照)と同様の手順により、アシクロビル、ポリエチレンイミン(SP-012)、ポリエチレンイミン(SP-012)-中性リポソーム複合体、ポリエチレンイミン(SP-012)-PEGリポソーム複合体のそれぞれのサンプルを用い、細胞毒性(50%細胞増殖阻害濃度(CC50))、単純ヘルペスウイルス2型に対する抗ウイルス活性(50%ウイルス増殖阻害濃度(IC50))および選択指数(CC50/IC50)を求めた。アシクロビルあるいはポリエチレンイミンの濃度範囲は、細胞毒性試験時は0.02~2000μg/ml、抗ウイルス活性試験時は0.01~1000μg/mlとした。その結果を表19に示す。ポリエチレンイミンと中性リポソームあるいはPEGリポソームとの複合体は、ポリエチレンイミンを単独で用いた場合と比べてより高い選択指数を示し、非常に高い抗ウイルス活性を有することがわかった。
Claims (9)
- 重量平均分子量が300~400,000の範囲であるポリアルキレンイミンを含む、ウイルス感染症を治療または予防するための医薬組成物。
- ポリアルキレンイミンを20mg/ml以下の量で含む外用医薬組成物である、請求項1に記載の医薬組成物。
- ポリアルキレンイミンが直鎖または分岐鎖のポリエチレンイミンである、請求項1または2に記載の医薬組成物。
- さらに脂質粒子を含む、請求項1~3のいずれか1項に記載の医薬組成物。
- エンベロープを有するウイルスによる感染症を治療または予防するための、請求項1~4のいずれか1項に記載の医薬組成物。
- ヘルペスウイルス感染症を治療または予防するための、請求項1~4のいずれか1項に記載の医薬組成物。
- ポリアルキレンイミンを含む、生体投与用ウイルス不活化剤。
- ポリアルキレンイミンを含む、ウイルスの細胞吸着阻害剤。
- ポリアルキレンイミンを含む、ウイルスの細胞侵入阻害剤。
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-
2009
- 2009-10-13 US US13/123,980 patent/US20110318298A1/en not_active Abandoned
- 2009-10-13 WO PCT/JP2009/067713 patent/WO2010044390A1/ja active Application Filing
- 2009-10-13 JP JP2010533892A patent/JP5465182B2/ja not_active Expired - Fee Related
- 2009-10-13 EP EP09820568A patent/EP2359835A4/en not_active Withdrawn
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Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2011142484A1 (ja) * | 2010-05-14 | 2011-11-17 | 株式会社日本触媒 | ポリアルキレンイミンを含むウイルス感染症治療薬 |
JPWO2011142484A1 (ja) * | 2010-05-14 | 2013-07-22 | 株式会社日本触媒 | ポリアルキレンイミンを含むウイルス感染症治療薬 |
JP2014505716A (ja) * | 2011-02-18 | 2014-03-06 | ヴィロノヴァ・アクチェボラーグ | B200を含んでなる、局所投与用の医薬製剤 |
WO2015020012A1 (ja) * | 2013-08-07 | 2015-02-12 | 株式会社日本触媒 | エチレンイミン重合体、およびその製造方法 |
JP2015034189A (ja) * | 2013-08-07 | 2015-02-19 | 株式会社日本触媒 | エチレンイミン重合体、およびその製造方法 |
JP2017526767A (ja) * | 2014-07-11 | 2017-09-14 | ジェンザイム・コーポレーション | 主鎖ポリアミン |
JP2020510703A (ja) * | 2017-03-17 | 2020-04-09 | ジュリ ニエミネンNIEMINEN, Jyri | 哺乳類の皮膚および粘膜の単純ヘルペス症状の治療 |
JP2017171933A (ja) * | 2017-05-12 | 2017-09-28 | 株式会社日本触媒 | エチレンイミン重合体、およびその製造方法 |
Also Published As
Publication number | Publication date |
---|---|
EP2359835A4 (en) | 2012-05-02 |
JP5465182B2 (ja) | 2014-04-09 |
JPWO2010044390A1 (ja) | 2012-03-15 |
US20110318298A1 (en) | 2011-12-29 |
EP2359835A1 (en) | 2011-08-24 |
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