JPWO2011142484A1 - Viral infection drug containing polyalkyleneimine - Google Patents

Abstract

It is an object of the present invention to provide a novel therapeutic agent for viral infection. The present invention provides a pharmaceutical composition comprising a polyalkylenimine and at least one selected from neutral or negatively charged liposomes and chitosan. When polyalkyleneimine is used in combination with neutral or negatively charged liposomes or chitosan, the cytotoxicity of polyalkyleneimine can be reduced, or the antiviral activity can be increased.

Description

  The present invention relates to a therapeutic agent for viral infection. The present invention particularly relates to a pharmaceutical composition for treating or preventing viral infections comprising polyalkyleneimines.

In recent years, viral infections caused by various viruses have become a major problem. For example, herpes simplex virus type 2 (HSV-2) is a pathogen that latently infects after initial infection and causes recurrent 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 of infection and onset stimulation of human immunodeficiency virus (HIV) (see, for example, Non-Patent Document 1). Drug therapy is an important issue for AIDS countermeasures.
At present, nucleic acid analogs such as acyclovir and vidarabine, which are antiviral drugs, are known as therapeutic drugs for herpes. However, 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. Thus, there is a need for a therapeutic agent for viral infection that has a different mechanism of action from conventional therapeutic agents.
From the standpoint of preventing the emergence of drug-resistant viruses, it is desirable to have a virus infection therapeutic agent that can inactivate the virus before the host cell is infected with the virus, rather than entering the virus-infected cell as in the past. . As a method for inactivating a virus before infecting a host cell in vitro, for example, a method described in Patent Document 1 is known.
The invention described in Patent Document 1 adds 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, etc. In this method, the virus contained in the sample is inactivated without affecting the virus infection. However, 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 discloses an immunostimulatory effect, such as the expression of interferon γ is induced 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 a mouse. It is described that is seen. 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 There have been no known examples of experiments conducted on the effects of administration. Moreover, the possibility that the action of the polyalkyleneimine on the living body can be changed depending on the substance used in combination has never been studied.

Japanese Patent Laid-Open No. 6-80520 International Publication No. 02/053149 Pamphlet

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 novel therapeutic agent for viral infection. The object of the present invention is to reduce the cytotoxicity of polyalkyleneimine or to increase the antiviral activity, particularly in a therapeutic drug for viral infections containing polyalkyleneimine.
As a result of research, the present inventors have reduced the cytotoxicity of polyalkyleneimine by using at least one substance selected from neutral or negatively charged liposomes and chitosan in combination with polyalkylenimine, or It has been found that the antiviral activity of polyalkyleneimines is enhanced.
That is, the present invention includes the following inventions.
(1) To treat or prevent a viral infection comprising a polyalkylenimine having a weight average molecular weight in the range of 300 to 400,000, and at least one selected from neutral or negatively charged liposomes and chitosan Pharmaceutical composition.
(2) The pharmaceutical composition according to (1), comprising a polyalkylenimine having a weight average molecular weight in the range of 300 to 400,000, and at least one selected from negatively charged liposomes and chitosan.
(3) The pharmaceutical composition according to (1) or (2), comprising oligochitosan as chitosan.
(4) The pharmaceutical composition according to any one of (1) to (3), comprising at least one selected from cholesterol sulfate liposomes and oleic acid-modified liposomes as negatively charged liposomes.
(5) The pharmaceutical composition according to any one of (1) to (4), wherein the polyalkyleneimine is a linear or branched polyethyleneimine.
(6) The pharmaceutical composition according to any one of (1) to (5), wherein the viral infection is a herpesvirus infection.
According to the present invention, it is possible to provide a relatively inexpensive viral infection therapeutic agent with lower cytotoxicity or higher antiviral effect.
This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2010-112518, which is the basis of the priority of the present application.

FIG. 1 is a graph showing the results of a cytotoxicity test when polyethyleneimine and liposomes are used in combination.
FIG. 2 is a graph showing the results of a cytotoxicity test using liposomes alone.
FIG. 3 is a graph showing the results of a cytotoxicity test using a cholesterol sulfate liposome alone.
FIG. 4 is a graph showing the results of a cytotoxicity test using chitosan alone.
FIG. 5 is a graph showing the results of a cytotoxicity test when polyethyleneimine and chitosan are used in combination.
FIG. 6 is a photograph taken with a confocal laser microscope. It can be confirmed that the fluorescently labeled polyethyleneimine has been taken up into the cells.
FIG. 7 is a graph showing the transition of infection score in HSV-2 animal infection experiment 1 of Example 3.

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 2 carbon atoms. 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. Among these polyalkyleneimines, polyethyleneimine (PEI) is particularly preferable 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, boron trifluoride, or the like. 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. (sales code 161-17831 (average molecular weight about 600), sales code 167-17811 (average molecular weight about 1,800), and sales Original code 164-17821 (average molecular weight of about 10,000)), polyethyleneimine (catalog number 23966-2 (linear, MW 25,000)) commercially available from Polysciences, Inc., BASF Lupasol (registered trademark) commercially available from the company Model Number: Lupasol FG, Lupasol G20, Lupasol G35, and Lupasol PR8515) and the like.
Polyalkyleneimines including polyethyleneimine can be synthesized with various molecular weights by selecting a production method, and can be synthesized with linear or branched ones. For example, linear polyethyleneimine has the formula:
(Where n is in the range of 7 to 10,000). The branched polyethyleneimine is represented by the formula:
Can be expressed as 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. When the polyalkyleneimine of the present invention has a branched structure, the ratio of each amino group is not particularly limited. For example, the primary amino group, the secondary amino group, and the tertiary amino group are entirely contained. Those which occupy 25 to 45 mol%, 35 to 50 mol%, and 20 to 35 mol%, respectively, with respect to the amino group can be preferably used. Particularly, primary amino group, secondary amino group and A tertiary amino group occupies 30 to 40 mol%, 30 to 40 mol%, 25 to 35 mol%, particularly 35 mol%, 35 mol%, and 30 mol%, respectively, with respect to 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, and particularly preferably 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 referred to here is 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 _2-6 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 And optionally substituted with 3-6 membered saturated, partially unsaturated or aromatic heterocycles. Examples of the reaction of polyalkyleneimine with various compounds are as follows.
Wherein 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 A substituent selected from the group consisting of cycloalkenyl and 3-6 membered saturated, partially unsaturated or aromatic heterocycles)
Specific examples of 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, and has a weight average molecular weight of about 2,000 to 15,000, preferably 4,000 to 12,000. It is preferably about 5,500 to 10,000 and has a hydroxyl value measured by an anhydrous phthalation method of 50 to 500 mgKOH / g, preferably 70 to 400 mgKOH / g. 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.
Polyalkyleneimines are very strong cationic polymers. In contrast, 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. In other words, 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.
Polyalkyleneimine can improve antiviral activity and reduce cytotoxicity when used in combination with at least one selected from neutral or negatively charged liposomes and chitosan. Therefore, the pharmaceutical composition for treating or preventing the viral infection of the present invention comprises polyalkyleneimine and at least one selected from neutral or negatively charged liposomes and chitosan. Two or more of neutral or negatively charged liposomes and chitosan may be used in combination with polyalkyleneimine.
Liposomes are closed lipid vesicles (vesicles) composed of phospholipid bilayers and containing water therein. 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.
Various known lipids such as phospholipids can be used as the liposome membrane component. For example, naturally occurring phosphatidylcholine (egg yolk lecithin, soybean lecithin, etc.), phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidic acid, ceramide, sphingomyelin and their hydrogenated products, and synthetically obtained dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine Dimyristoyl phosphatidylcholine, dicetyl phosphate, dihexadecyl phosphate, dioctadecyldimethylammonium salt, stearylamine, and other artificial lipids can be used alone or in combination.
In addition, as a liposome membrane component, cholesterol, cholesterol succinic acid, cholesterol sulfate, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol, etc. to stabilize the membrane physically or chemically and regulate the fluidity of the membrane Sterols derived from these animals, or plant-derived sterols (phytosterols) such as stigmasterol, sitosterol, campesterol, and brassicasterol can be used alone or in combination.
Depending on the components used at the time of manufacture, the liposomes can be positively charged or negatively charged, or neutral (ie, positively or negatively charged). In the present invention, the liposome used in combination with the polyalkyleneimine is preferably a neutral or negatively charged liposome. Examples of neutral liposomes include those produced using neutral phospholipids such as egg yolk lecithin and soybean lecithin and neutral sterols such as cholesterol. As negatively charged liposomes, for example, in addition to neutral lipids and neutral sterols, those produced using negatively charged sterols such as cholesterol sulfate (in this specification, cholesterol sulfate is used as a membrane component). And liposomes prepared by modifying a neutral liposome prepared in advance with a fatty acid such as oleic acid. Methods for modifying liposomes with other compounds are known to those skilled in the art. The particle size of the liposome used in the present invention is not particularly limited, but is more preferably 0.15 μm or less, for example. By using in combination with neutral or negatively charged liposomes, particularly negatively charged liposomes, the polyalkyleneimine can be carried into the cell, and the antiviral action of the polyalkyleneimine can be exhibited within the cell. That is, since polyalkyleneimine is cationic, it can be adsorbed on the cell surface by itself, but it is difficult to enter the cell, but neutral or negatively charged liposomes, particularly negatively charged liposomes. By using in combination with liposomes, it is thought that it becomes possible to pass through the cell membrane and enter the cell.
Chitosan is a kind of polysaccharide and is a polymer compound in which D-glucosamine is bonded to β-1,4. As chitosan used in the present invention, oligochitosan having a relatively small molecular weight is preferable. The degree of polymerization of the oligochitosan is, for example, preferably about 3 to 100, particularly about 5 to 50, especially about 10 to 30.
The pharmaceutical composition comprising the polyalkyleneimine of the present invention and at least one selected from neutral or negatively charged liposomes and chitosan has a nonspecific antiviral action (preventing viral diseases by inhibiting viral growth). And the action of treating. That is, the pharmaceutical composition of the present invention comprises herpesviruses such as herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), varicella-zoster virus (VZV) and cytomegalovirus (CMV). Viruses belonging to the family Orthomyxoviridae such as viruses belonging to the family (collectively referred to as herpes viruses), influenza A viruses, influenza B viruses, influenza C viruses (collectively referred to as influenza viruses), and human immunodeficiency Viruses belonging to retroviridae such as virus (HIV), viruses belonging to paramyxoviridae such as measles virus and mumps virus, viruses belonging to picornaviridae such as poliovirus, rhinovirus and hepatitis A virus, type B Viruses belonging to the family Hepadnaviridae such as flame virus, viruses belonging to Flaviviridae such as hepatitis C virus, Japanese encephalitis virus and West Nile virus, viruses belonging to the adenoviridae family such as human adenovirus, coronavirus, SARS virus Viruses belonging to the Coronaviridae family, viruses belonging to the Togaviridae family such as rubella virus, viruses belonging to the Rhabdoviridae family such as rabies virus and vesicular stomatitis virus, viruses belonging to the Filoviridae family such as Ebola virus, and human papillomaviruses It has an antiviral action against various viruses such as viruses belonging to the family of papovaviridae such as (HPV), and is effective in the treatment of infectious diseases caused by infection with these viruses. For example, the pharmaceutical composition 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. Among these, the pharmaceutical composition of the present invention includes herpesviridae, orthomyxoviridae, retroviridae, paramyxoviridae, hepadnaviridae, flaviviridae, coronaviridae, togaviridae and rhabdoviridae Have an excellent antiviral action against viruses having an envelope belonging to, such as herpes virus, measles virus, influenza virus, coronavirus and human immunodeficiency virus, and infection with these viruses It is effective for the treatment of infections caused by Also, herpes virus has a pharmaceutical composition using a branched polyethyleneimine having a weight average molecular weight of about 2,310 to 4,600 or a linear polyethyleneimine having a weight average molecular weight of about 25,000, and measles virus has a weight average molecular weight. A pharmaceutical composition using about 2,310 branched polyethyleneimine, a pharmaceutical composition using polyethyleneimine ethoxylate having a weight average molecular weight of about 10,000 for influenza virus, and a weight average molecular weight of 1 for coronavirus. , A pharmaceutical composition using a branched polyethyleneimine having a molecular weight of about 470 to 321,200 or a polyethyleneimine ethoxylate having a weight average molecular weight of about 10,000 is particularly effective.
In particular, the pharmaceutical composition 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 shingles caused by infection with herpes virus. In particular, it is particularly effective for the prevention or treatment of genital herpes caused by herpes simplex virus type 2 infection. Furthermore, the pharmaceutical composition of the present invention is effective against viruses that have acquired resistance to conventional antiviral drugs such as acyclovir. In addition to these, the pharmaceutical composition 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 pharmaceutical composition of the present invention comprises a polyalkyleneimine and at least one selected from neutral or negatively charged liposomes and chitosan as it is or mixed with various pharmacologically acceptable formulation adjuvants. Can be 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. 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). In addition, an external preparation means all the chemical | medical agents used directly to the human body except an internal medicine and an injection. The topical administration means administration of the drug directly to the eyes, nose, ears, mouth, anus, skin or mucous membrane, and includes transdermal or transmucosal administration.
The internal preparations include pharmaceutically acceptable solutions, suspensions, emulsions, syrups and elixirs. Such a solution can be obtained by dissolving, suspending or emulsifying the active substance in a commonly used diluent (for example, purified water, ethanol or a mixture thereof). Furthermore, 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 produced by a known or commonly used formulation. For example, it is prepared by kneading or melting the active substance in a base. The ointment base is selected from known or commonly used ones. For example, 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 petrolatum, purified lanolin or liquid paraffin), glycols (eg, ethylene glycol, diethylene glycol, propylene) Glycol, polyethylene glycol or macrogol), vegetable oil (eg, castor oil, olive oil, sesame oil or turpentine oil), animal oil (eg, mink oil, egg yolk oil, squalane or squalene), water, absorption enhancer, rash The base selected from the inhibitors is used alone or in combination of two or more. Further, it may contain a humectant, a preservative, a stabilizer, an antioxidant or a flavoring agent.
The gel is produced by a known or commonly used formulation. For example, the active substance is prepared by melting in a base. The gel base is selected from known or commonly used ones. For example, 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 a surfactant (for example, polyethylene glycol monostearate), gums, water, an absorption accelerator and an anti-rash agent is used alone or in admixture of two or more. Furthermore, a preservative, an antioxidant or a flavoring agent may be included.
The cream is produced by a known or commonly used formulation. For example, it is produced by melting or emulsifying an active substance 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 rash prevention 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 produced by a known or commonly used formulation. For example, it is manufactured by melting an active substance in a base, and applying it as a kneaded product on a support. The poultice base is selected from known or commonly used ones. For example, 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, magnesium, etc.), water, solubilizers, tackifiers 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 patch is produced by a known or commonly used formulation. For example, it is produced by melting an active substance in a base and spreading and coating it on a support. The base for patch is selected from known or commonly used ones. For example, 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. Furthermore, a preservative, an antioxidant or a flavoring agent may be included.
The liniment is produced by a known or commonly used formulation. For example, the active substance is dissolved, suspended, or dissolved in one or more bases selected from water, alcohol (for example, ethanol or polyethylene glycol), higher fatty acid, glycerin, soap, emulsifier and suspending agent. Produced by emulsification. Furthermore, 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. An isotonic agent such as
The injection includes all injections and also includes infusions. For example, intramuscular injection, subcutaneous injection, intradermal injection, intraarterial injection, intravenous injection, intraperitoneal injection, spinal cavity injection and vein Including instillation agents.
The injection includes a solid injection used by dissolving or suspending in a solution, suspension, emulsion, or solvent for use. An injection is used by dissolving, suspending or emulsifying an active substance in a solvent. As 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. Furthermore, 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. These are sterilized in the final step or prepared by aseptic manipulation. In addition, 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 eye drops include eye drops, suspension type eye drops, emulsion type eye drops, in-use dissolved eye drops and eye ointments. These eye drops are produced according to known methods. For example, the active substance is dissolved, suspended or emulsified in a solvent. As a solvent for eye drops, for example, sterilized purified water, physiological saline, other aqueous solvents or non-aqueous solvents for injection (for example, vegetable oil) and the like 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), etc., as appropriate It may be selected and included. These are sterilized in the final step or prepared by aseptic manipulation. In addition, an aseptic solid preparation, for example, a freeze-dried product can be produced and used by dissolving in aseptic or aseptically purified water or other solvent before use.
Inhalants include aerosols, inhalable powders or inhalable liquids, and the inhalable liquids may be used in the form of being dissolved or suspended in water or other suitable medium at the time of use. These inhalants are produced according to known methods. For example, in the case of inhalation solutions, preservatives (eg, benzalkonium chloride or parabens), colorants, buffering agents (eg, sodium phosphate or sodium acetate), isotonic agents (eg, chloride) Sodium or concentrated glycerin), a thickener (for example, carboxyvinyl polymer, etc.), an absorption enhancer, and the like, as necessary. In the case of powders for inhalation, lubricants (for example, stearic acid and its salts), binders (for example, starch or dextrin), excipients (for example, lactose or cellulose), colorants, 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) is usually used when administering an inhalation solution, and an inhalation administration device for powder medicine is usually used when administering a powder for inhalation.
Other compositions for external administration include suppositories for rectal administration and pessaries for intravaginal administration containing active substances and formulated by conventional methods.
As a method for administering the pharmaceutical composition of the present invention, topical administration, particularly transdermal or transmucosal administration, is particularly preferred. In addition, the pharmaceutical composition of the present invention is preferably administered externally so that the polyalkyleneimine concentration is 20 mg / ml or less, and particularly preferably externally administered so that the concentration is 0.004 mg / ml to 20 mg / ml. . On the other hand, the dose is preferably about 0.001 to 50 mg per kg of body weight per day based on the weight of polyalkylenimine, for example, in the case of mammals.
Administration of the pharmaceutical composition of the present invention may be carried out either before or after viral infection, but particularly when administered before or immediately after viral infection, good effects are exhibited. Administration to a virus-infected animal may be performed for only one day or for several consecutive days. In the case of external administration, especially in the case of administration in a dosage form having a relatively high polyalkylenimine concentration of about 10 mg / ml to 20 mg / ml, especially about 20 mg / ml, even a single-day administration exhibits a sufficient effect. To do. Also, in the case of administration in a dosage form having a relatively low concentration of about 0.004 mg / ml to 10 mg / ml, particularly about 0.004 mg / ml to 1 mg / ml, especially about 0.004 mg / ml to 0.4 mg / ml. Is preferably administered continuously for a certain number of days, for example, about 2 weeks, or at least about 3-7 days. Administration may be once per day, or may be divided into 2-3 times or divided into 4-5 times.
The toxicity of the polyalkyleneimine can also be suppressed by modifying the polyalkyleneimine. For example, the toxicity to a living body can be suppressed by using polyalkyleneimine ethoxylate, particularly polyethyleneimine ethoxylate.
The pharmaceutical composition of the present invention may contain other active substances other than polyalkyleneimine, neutral or negatively charged liposomes and chitosan. 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-based disinfectants such as fuvirtide, adefovir, entecavir and ribavirin, and povidone iodine Disinfectant with viral killing effect as fine chlorhexidine and the like.
EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, this invention is not limited to these Examples.

[Example 1: Viral activity when polyethyleneimine and liposome or chitosan are used in combination]
1. Sample preparation method (1) Polyethyleneimine (PEI)
Epomin SP-012 (manufactured by Nippon Shokubai Co., Ltd., weight average molecular weight 3,610 by GPC method), which is a branched polyethyleneimine, was used. The concentration was adjusted to 100 mg / mL with Milli-Q water, and the pH was adjusted to around 7.0 using concentrated hydrochloric acid or dilute hydrochloric acid.
(2) Liposomes (Lipo: neutral liposomes)
Liposomes are modified as EPC: Ch = 5: 5 (molar ratio) using egg yolk lecithin (EPC: COATSOME NC-50, manufactured by NOF Corporation) and cholesterol (Ch: manufactured by Wako Pure Chemical Industries, Ltd., special grade). Prepared by ethanol injection method.
Each component is dissolved in an appropriate amount of ethanol (manufactured by Wako Pure Chemical Industries, Ltd.) in an eggplant-shaped flask, and the volume of the solution is about 1 mL by a rotary evaporator (EYELAN-1000, manufactured by Tokyo Science Instruments Co., Ltd.) in a 60 ° C. warm bath. Until the ethanol was distilled off. To this was added 9 mL of Milli-Q water heated to the same temperature and shaken, and then ethanol was completely distilled off again using an evaporator. The solution was adjusted to 10 mL with Milli-Q water to adjust the concentration.
(3) Oleic acid modified liposome (negatively charged liposome)
The oleic acid liposome uses egg yolk lecithin (EPC: COATSOME NC-50, manufactured by NOF Corporation), cholesterol (Ch: manufactured by Wako Pure Chemical Industries, Ltd., first grade reagent) and oleic acid, and EPC: Ch: oleic acid = 5: It was prepared by a thin film method as 5: 2 (molar ratio).
Each component was dissolved in an appropriate amount of chloroform (manufactured by Wako Pure Chemical Industries, Ltd.) in an eggplant-shaped flask, and the solution disappeared using a rotary evaporator (EYELAN-1000, manufactured by Tokyo Science Instrument Co., Ltd.) in a 60 ° C. warm bath. Chloroform was distilled off until a thin film was formed on the inner wall. To this, 9 mL of Milli-Q water heated to the same temperature was added and shaken so as to peel off the lipid on the wall surface, and then chloroform was completely distilled off again by an evaporator. The concentration of this solution was adjusted to 10 mL with Milli-Q water. In addition, if necessary, ultrasonic irradiation was performed with a bath type ultrasonic device (BRANSON 2510J-MTH, 100 W, manufactured by YAMATO) for particle size adjustment, and the particle size was adjusted. The particle size and zeta potential were measured at 25 ± 1 ° C. using an electrophoretic light scattering photometer (ELS-Z2, manufactured by Otsuka Electronics Co., Ltd.).
(4) Oligochitosan As oligochitosan, GH-020 (10-30 molecules of chitosan oligomer, manufactured by NOF Corporation) was used. It was dissolved in Milli-Q water to a concentration of 40 mg / mL, and the pH was adjusted to around 6.2.
2. Antiviral activity measurement test method Vero cells (African green monkey kidney-derived cells) were used to examine the growth inhibitory effect of each sample against herpes simplex virus type 2 (HSV-2).
Vero cells were cultured in 48-well plates. One day later, the medium was removed, and HSV-2 was infected with 0.2 PFU (plaque forming unit) / cell (one virus per 5 cells) for 1 hour at room temperature. Each sample was diluted with a medium to a predetermined concentration and then added at the same time as virus infection or immediately after virus infection. Harvested after 20 hours and viral load was quantified by plaque assay. From the results, the 50% virus growth inhibitory concentration (IC ₅₀ ) of each sample was determined.
Separately from the above, Vero cells were cultured in a 96-well plate, and a sample diluted to a predetermined concentration was added. After culturing for 72 hours, the number of viable cells was counted by trypan blue staining, and a 50% cell growth inhibitory concentration (CC ₅₀ ) was determined. A selection index was determined from the obtained CC ₅₀ value and IC ₅₀ value.
3. Test result Polyethyleneimine (SP-012) only, polyethyleneimine and liposome (Lipo) combined (including 100 mg / mL SP-012 and 20 mg / mL liposome), polyethyleneimine and oleic acid-modified liposomes combined (Including 1 mg / mL SP-012 and 2 mg / mL oleic acid-modified liposome), combined use of polyethyleneimine and oligochitosan (including 100 mg / mL SP-012 and 2 mg / mL oligochitosan), As a control, acyclovir was used as a sample to obtain a selection index. The results when samples were added simultaneously with virus infection are shown in Table 1 below. The CC ₅₀ value and the IC ₅₀ value represent the concentration of polyethyleneimine or acyclovir.
In contrast to the administration of polyethyleneimine alone, a significant increase in the selectivity index was observed particularly when liposomes, oleic acid-modified liposomes and oligochitosan were used in combination.
Next, Table 2 below shows data comparing the selectivity index when the sample was added simultaneously with the virus infection and when the sample was added immediately after the virus infection. The sample used was a polyethyleneimine (SP-012) alone or a combination of polyethyleneimine and oleic acid-modified liposomes (including 1 mg / mL SP-012 and 2 mg / mL oleic acid-modified liposomes). .
When the sample was added immediately after the virus infection rather than simultaneously with the virus infection, the selectivity index decreased by several tenths with polyethyleneimine alone, whereas the decrease in selectivity index with oleic acid-modified liposomes was 1 Stayed at around / 2. This is presumably because the use of oleic acid-modified liposomes in combination allowed the polyethyleneimine to be carried into the cells, and even the cells after virus infection were able to exert a sufficient antiviral effect.
[Example 2: Cytotoxicity when polyethyleneimine is combined with liposome or chitosan]
1. Cytotoxicity test method WST-8 assay was performed on NCI-H292 mucosal cells (derived from human lung mucinous epidermoid carcinoma).
NCI-H292 mucosal cells were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% non-immobilized FBS (Fetal bovine serum, Invitrogen) and 100 μg / mL kanamycin under conditions of 37 ° C. and 5% CO _2. did. Cells were seeded in 96-well plates 24 hours before the experiment and pre-cultured. Each sample was diluted with a medium to prepare a dilution series, which was slowly added to the cultured cells. The cells were then incubated for 24 hours at 37 ° C. The cell viability was measured by measuring the absorbance at 450 nm using Cell Counting Kit-8 (manufactured by Dojindo Laboratories). The following formula was used to calculate the cell viability.
2. Cytotoxicity when Polyethylenimine (PEI) and Cholesterol Sulfate Liposomes (Lipo (CS)) are Combined Cholesterol sulfate liposomes (negatively charged liposomes) are yolk lecithin (EPC: COATSOME NC-50, manufactured by NOF Corporation), cholesterol (Ch : Wako Pure Chemical Industries, Reagent grade 1), Cholesterol sulfate (CS: Sodium Cholesterol Sulfate, manufactured by SIGMA), EPC: Ch: CS = 5: 5: 2 (molar ratio), prepared by thin film method did.
Each component was dissolved in an appropriate amount of chloroform (manufactured by Wako Pure Chemical Industries, Ltd.) in an eggplant-shaped flask, and the solution disappeared using a rotary evaporator (EYELAN-1000, manufactured by Tokyo Science Instrument Co., Ltd.) in a 60 ° C. warm bath. Chloroform was distilled off until a thin film was formed on the inner wall. To this, 9 mL of Milli-Q water heated to the same temperature was added and shaken so as to peel off the lipid on the wall surface, and then chloroform was completely distilled off again by an evaporator. The concentration of this solution was adjusted to 10 mL with Milli-Q water. In addition, if necessary, ultrasonic irradiation was performed with a bath type ultrasonic device (BRANSON 2510J-MTH, 100 W, manufactured by YAMATO) for particle size adjustment, and the particle size was adjusted. The particle size and zeta potential were measured at 25 ± 1 ° C. using an electrophoretic light scattering photometer (ELS-Z2, manufactured by Otsuka Electronics Co., Ltd.).
Cytotoxicity tests were performed according to the above procedure 1. The test of the “PEI” sample (control) was performed by preparing a dilution series in which the concentration of PEI was 5 types. The test of the “PEI + Lipo (CS) 2 mg / mL” sample is performed by preparing a dilution series in which the Lipo (CS) concentration is constant at 2 mg / mL after dilution and the PEI concentration is 5 types. It was. PEI or PEI + Lipo (CS) was preincubated at 60 ° C. for 1 hour before preparing a dilution series. The five dilution series related to the concentration of PEI were 1000 μg / mL, 750 μg / mL, 500 μg / mL, 250 μg / mL, and 100 μg / mL. The particle size of Lipo (CS) was 150 nm or less, and the zeta potential was about −50 mV.
The test results are shown in FIG. When Lipo (CS) was used in combination, cytotoxicity tended to be reduced as compared with the case where only PEI was used.
3. Cytotoxicity when Liposomes (Lipo) or Cholesterol Sulfate Liposomes (Lipo (CS)) are Administered Cytotoxicity tests were conducted using liposomes (neutral liposomes) alone. Liposomes prepared as in Example 1 1 (2) were used. The results are shown in FIG. Cytotoxicity was not observed at concentrations up to 8 mg / mL with liposomes alone.
Next, a cytotoxicity test was performed using cholesterol sulfate liposomes (negatively charged liposomes) alone. The results are shown in FIG. Liposomes showed no toxicity even at 8 mg / mL, whereas cholesterol sulfate liposomes showed strong toxicity even at 2 mg / mL. From this result, it is presumed that the cholesterol sulfate liposomes attenuated the toxicity of polyethyleneimine due to the influence of negative charges that would cause cytotoxicity alone.
4). Cytotoxicity when polyethyleneimine (PEI) and chitosan are used in combination Oligochitosan GH-020 (10-30 molecule oligomer of chitosan, manufactured by NOF Corporation) was used as chitosan. It was dissolved in Milli-Q water to a concentration of 40 mg / mL, and the pH was adjusted to around 6.2.
A cytotoxicity test was previously performed using chitosan alone. The results are shown in FIG. Chitosan alone showed high cell viability at concentrations up to 2 mg / mL. Thus, a cytotoxicity test was performed using chitosan at a concentration of 1 mg / mL or 2 mg / mL, in which cytotoxicity was not observed, in combination with PEI. The results are shown in FIG. There was no significant difference in cytotoxicity between the administration of PEI alone and the combined use of PEI and chitosan. It was revealed that the combined use of chitosan did not increase the cytotoxicity of PEI.
5. Confirmation of polyethyleneimine intracellular uptake by confocal laser microscope Each cell (NCI-H292 mucosal cell) was seeded in a 35 mm dish and pre-cultured 24 hours before the experiment. Thereafter, fluorescently labeled PEI and optionally liposomes (prepared as in Example 1, 1 (2)) were diluted with medium, slowly administered to the cells, and incubated at 37 ° C. for 3 hours. After incubation, the medium was removed and PBS (8 g NaCl, 2.9 g Na ₂ HPO ₄ · 12H ₂ O, 0.2 g KCl and 0.2 g KH ₂ PO ₄ dissolved in 1 L Milli-Q) ), And 4% formaldehyde diluted in PBS was added and incubated at room temperature for 15 minutes. Moreover, in order to prevent deterioration, the cells were covered with Aqua Poly / mount (manufactured by polysciences), and a cover glass was placed thereon. The fixed cells were observed with a confocal laser microscope (LSM5 EXCITER, manufactured by Carl Zeiss). The photographed micrograph is shown in FIG. When PEI alone was administered, coloration due to fluorescent labeling was observed throughout the cells, whereas when PEI and liposomes were used in combination, coloration was observed in areas other than the portion considered to be the cell nucleus. This suggests that PEI stays on the cell surface when PEI alone is administered, whereas PEI is taken up into cells when PEI and liposomes are used in combination. From this, it is inferred that the liposome plays a role of transporting polyethyleneimine into the cell.
[Example 3: HSV-2 animal infection experiment 1]
An infection experiment using mice was conducted on the antiviral effect of a combination of polyethyleneimine and liposome or chitosan against herpes simplex virus type 2 (HSV-2).
BALB / c mice (female, 6 weeks old) (n = 5) were injected subcutaneously with medroxyprogesterone (3 mg / 0.1 mL / animal) 6 and 1 day before virus inoculation. Mice were inoculated with 20 μL of HSV-2 (containing 1 × 10 ⁴ PFU of virus) per mouse (genital). The administration of the sample was continued 1 hour before infection and 12 hours after infection on the day of infection, and then twice a day (9 am, 6 pm) after the next day until 7 days after infection.
As a sample, a control consisting of PBS alone, a polyethyleneimine (SP-012) alone, a combination of polyethyleneimine and oleic acid-modified liposome (2.5 mg / mL), and polyethyleneimine and oligochitosan (GH-020, 0.2 mg / mL) was used in combination. Each sample other than the control was prepared to contain 0.2 mg / 20 μL / animal polyethyleneimine per dose.
After 1, 3, and 5 days, the area was washed with PBS, and the amount of virus therein was measured. Also, deaths and infection scores until 10 days later (0: asymptomatic, 1: local mild inflammation, 2: moderate inflammation with edema, 3: severe inflammation with exudate, 4: hind limb paralysis, 5 : Death). Table 3 shows changes in viral load and death data in each sample, and FIG. 7 shows changes in infection score.
Compared to when polyethyleneimine is administered alone, when oleic acid-modified liposomes and oligochitosan are used in combination, the amount of virus can be kept low, and HSV-2 is effective in suppressing the onset of genital herpes. It was.
[Example 4: HSV-2 infection experiment 2]
Infection experiments were carried out using mice with the aim of comparing the effect of polyethyleneimine combined with neutral liposomes on HSV-2 between pre-infection administration and post-infection administration.
BALB / c mice (female, 6 weeks old) (n = 4) were injected subcutaneously with medroxyprogesterone (3 mg / 0.1 mL / animal) 6 and 1 day before virus inoculation. Each mouse was inoculated with 20 μL of HSV-2 (containing 1 × 10 ⁴ PFU of virus) per mouse. Administration of samples on the day of infection occurred either 1 hour before infection (A) or 1 hour after infection (B). On the next day and after, the sample was administered twice a day (9 am, 6 pm), and the local area was washed with PBS on the third day of infection, and the amount of virus therein was measured. The results are shown in Table 4.
Samples include a PBS-only control, polyethyleneimine (SP-012) alone, and polyethyleneimine and neutral liposomes (prepared in Example 1, 1 (2), including 36 mg / mL liposomes) What used together was used. The dose was prepared to contain 1.0 mg / 20 μL / unit polyethyleneimine per dose.
Compared to when polyethyleneimine is administered alone, when neutral liposomes are used in combination, the first administration is administered 1 hour after infection, which is a stronger virus growth inhibitory effect than when administered 1 hour before infection. Was found to be obtained.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims (6)

  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, and at least one selected from neutral or negatively charged liposomes and chitosan .   The pharmaceutical composition according to claim 1, comprising at least one selected from polyalkylenimines having a weight average molecular weight in the range of 300 to 400,000 and negatively charged liposomes and chitosan.   The pharmaceutical composition according to claim 1 or 2, comprising oligochitosan as chitosan.   The pharmaceutical composition according to any one of claims 1 to 3, comprising at least one selected from cholesterol sulfate liposomes and oleic acid-modified liposomes as negatively charged liposomes.   The pharmaceutical composition according to any one of claims 1 to 4, wherein the polyalkyleneimine is a linear or branched polyethyleneimine.   The pharmaceutical composition according to any one of claims 1 to 5, wherein the viral infection is a herpes virus infection.