JP2007119396A - Pharmaceutical formulation for transpulmonary administration containing nanoparticle having sealed nucleic acid compound - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a pharmaceutical formulation that directly and efficiently delivers a nucleic acid compound such as nucleic acid, gene, etc., to the disease part of an intractable pulmonary diseases such as lung cancer, pulmonary fibrosis, etc., by using a nanoparticle, effectively and continuously develops actions and has high safety. <P>SOLUTION: The pharmaceutical formulation for transpulmonary administration comprises nanoparticles obtained by sealing a nucleic acid compound such as plasmid, siRNA, etc., acting on target molecules in intractable pulmonary diseases such as lung damage/pulmonary fibrosis, lung cancer, bronchial asthma, etc., in a biocompatible polymer. Consequently, the nucleic acid compound is efficiently introduced organ specifically and disease site specifically into the body. Actions are continuously developed by gradual release of the nucleic acid compound from the insides of the nanoparticles. Possibility to clinical applications is increased due to minimization of influence on the whole body. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a pharmaceutical preparation for transpulmonary administration comprising biocompatible nanoparticles in which a nucleic acid compound such as a plasmid or siRNA is encapsulated in a biocompatible polymer.

  Respiratory diseases are mainly composed of infectious diseases such as tuberculosis, depending on lifestyle, environment, aging, medical advances, etc., lung cancer, pulmonary fibrosis, bronchial asthma, chronic obstructive pulmonary disease (COPD), etc. Has become a refractory pulmonary disease.

  Lung cancer has the highest number of malignant tumor deaths per year and is on the rise. Therefore, new treatment methods such as chemotherapy and radiation therapy using anticancer agents are being developed one after another, but have not yet reached surgical treatment. In bronchial asthma, some effects have been obtained with inhaled steroids, but steroid-resistant refractory bronchial asthma is also present, so it is still not a sufficient treatment.

  As for acute lung injury and pulmonary fibrosis, many lung diseases such as acute respiratory distress syndrome (ARDS), hypersensitivity pneumonitis, interstitial pneumonia, etc. are accompanied by alveolitis, and prolongation of inflammation regardless of whether acute or chronic. And fibrosis associated with severe lung injury is an important factor in prognosis deterioration, and treatment targeting pulmonary epithelial cell damage is regarded as important, and various molecular targeted drugs are candidates. It has not been put into practical use.

  In recent years, the entire nucleotide sequence of the human genome has been deciphered, and it has been clarified that various diseases are caused by genetic diseases, and it is expected that the relationship between more diseases and genes will be elucidated in the future. And based on such information, so-called gene therapy, which fundamentally treats diseases caused by genetic diseases at the gene level, has attracted attention as a new therapeutic strategy for intractable diseases.

  In gene therapy, double-stranded RNA (siRNA) is introduced into cells to suppress the expression of defective genes with the same sequence (protein synthesis) (RNA interference), or independently from chromosomal DNA. A method of introducing existing circular DNA (plasmid DNA) into a patient's body is known.

  There are many studies on applying such gene therapy to intractable pulmonary diseases. For example, Patent Document 1 discloses a transforming growth factor beta (hereinafter referred to as TGF-beta) response element in order to reduce cell proliferative diseases such as pulmonary fibrosis, renal fibrosis, tumor formation and proliferation, and glaucoma. A pharmaceutical composition comprising a CTGF-responsive agent is disclosed. Patent Document 2 discloses a method for directly and safely and effectively suppressing the expression of tissue fibrosis by using an antisense oligonucleotide of a stress protein (heat shock protein) HSP47 gene.

Furthermore, Patent Document 3 discloses that human antibody-producing transgenic mice prepared using genetic engineering techniques are immunized with a soluble human TGF-beta II type receptor to bind to human TGF-beta II type receptor, and human TGF- It is stated that various human monoclonal antibodies that inhibit the transmission of beta signals into cells are effective for the prevention and treatment of diseases such as tissue fibrosis in various organs caused by the action of human TGF-beta. It is disclosed.
Here, the therapeutic effect of gene therapy depends on how efficiently and safely the gene is introduced and acted on the disease site.

  Currently used gene transfer methods include a viral vector method in which a transgene is incorporated into a viral DNA to create a recombinant virus, and the gene is transferred using the viral infection mechanism. However, although the gene introduction efficiency is high in the virus vector method, there is a problem of side effects caused by the virus, which is not sufficient in terms of safety.

  In addition, gene therapy is often difficult depending on the lesion site, for example, refractory lung diseases such as lung cancer, bronchial asthma, acute lung injury, and pulmonary fibrosis are the most peripheral bronchioles in lung tissue, Since it has a lesion head in the alveolar region, an effective gene transfer method has not been established yet, and it depends on other treatment methods.

  Against this background, a technique for introducing a gene using a so-called drug delivery system (hereinafter referred to as DDS) that prevents excessive drug administration by preventing the drug from being absorbed and decomposed until it reaches the affected area. Has been actively studied. DDS is a technology that wraps in a film or forms a matrix in a film agent to effectively and intensively deliver the drug to the target affected area without absorbing or decomposing the drug on the way and releasing the drug at the affected area. In addition to enhancing the therapeutic effect of the drug, there is an advantage that side effects can be reduced. Among them, the transpulmonary route that is delivered via the alveoli can be administered directly into the lung, and therefore has advantageous conditions as a treatment method for intractable lung disease.

  As a gene transfer method using DDS, a liposome method using a liposome as a carrier (vector) of a transgene has been devised and put into practical use. However, in the liposome method, since the liposome is a phospholipid, safety to the human body is high, but it is not sufficient in terms of gene introduction efficiency and sustaining effect.

  The biocompatible polymer used as a carrier material is preferably biocompatible and has low irritation and toxicity to the living body, is biocompatible, and is biodegradable by being decomposed and metabolized after administration. Moreover, it is preferable that it is the particle | grain which releases the drug to include continuously and gradually. As such a material, for example, as disclosed in Patent Documents 4 to 7, a polylactic acid / glycolic acid copolymer (hereinafter referred to as PLGA) is suitably used. It is known that PLGA can contain a drug and can be stored for a long time while maintaining the efficacy of the drug. Furthermore, it is considered that PLGA is hydrolyzed by the action of a degrading enzyme in the living body and can be sustainedly released in units of several hours to several tens of hours.

  Such nanoparticles are generally produced using a spherical crystallization method in which a drug solution dissolved in a good solvent is dropped into a poor solvent that is difficult to dissolve the drug under stirring to precipitate drug crystals. Is done. In the spherical crystallization method, nanoparticles can be formed by a physicochemical method, and the resulting nanoparticles are almost spherical, so there is no need to consider the problem of residual catalyst or raw material compounds from homogeneous nanoparticles. It can be formed easily.

  However, in patent documents 4-7, the particle | grains made into object are micro size, and the encapsulation method of the gene to them was not established. Since micro-sized particles cannot be incorporated into cells, the target of nucleic acids and genes is extremely low when applied to refractory lung disease lesions such as bronchioles and alveolar regions. Therefore, practical application was difficult. Furthermore, a specific application example to a drug for pulmonary administration for treating refractory pulmonary diseases as described above is not described, but merely suggests application to oral or inhalation administration.

  On the other hand, Non-Patent Document 1 discloses that pDNA, which is obtained by modifying the surface of PLGA nanoparticles with chitosan and encapsulating inside, is N- [1- (2,3-Dioleoyloxy) propyl] -N, N, N-trimethylammonium. A method of increasing the amount of cellular uptake and gene expression efficiency and increasing the encapsulation rate of pDNA by forming a complex with salts (DOTAP) is disclosed.

However, Non-Patent Document 1 does not suggest any application to a drug for transpulmonary administration for treating refractory lung disease as described above. Cationic lipids such as DOTAP generally improve the intracellular introduction and expression of genes, but are known to exhibit cytotoxicity and are safe for use as raw materials for drugs for pulmonary administration. Therefore, further improvements were necessary.
JP 2004-132974 A JP 2003-159087 A JP 2001-206899 A JP-T-2001-526634 Special Table 2002-544175 Japanese translation of PCT publication No. 2003-509035 JP 2005-500304 Gazette Takeshi Sakai, Hiromitsu Yamamoto, Hirofumi Takeuchi, Yoshiaki Kawashima “Abstracts of 20th Formulation and Particle Design Symposium” (November 11, 2004)

  In view of the above problems, the present invention efficiently delivers a nucleic acid compound such as a nucleic acid or gene directly to a disease site using nanoparticles for refractory lung diseases such as lung cancer and pulmonary fibrosis. An object of the present invention is to provide a highly safe pharmaceutical preparation for transpulmonary administration which is effectively and continuously expressed.

  In order to achieve the above object, a first configuration of the present invention is a pharmaceutical preparation for transpulmonary administration comprising a nucleic acid compound-encapsulated nanoparticle formed by encapsulating a nucleic acid compound in a biocompatible nanoparticle.

  The second configuration of the present invention is characterized in that in the pharmaceutical preparation of the above configuration, a nucleic acid compound is further attached to the surface of the biocompatible nanoparticle.

  According to a third configuration of the present invention, in the pharmaceutical preparation having the above configuration, the biocompatible polymer forming the biocompatible nanoparticle is polylactic acid, polyglycolic acid, lactic acid / glycolic acid copolymer, or lactic acid. -It is one of aspartic acid copolymers.

  A fourth configuration of the present invention is a therapeutic agent for lung injury / pulmonary fibrosis using a soluble type II TGF receptor plasmid having TGF-beta as a target molecule as the nucleic acid compound in the pharmaceutical preparation of the above configuration.

  A fifth configuration of the present invention is a therapeutic agent for lung injury / pulmonary fibrosis using a mutant MCP-1 plasmid having MCP-1 as a target molecule as the nucleic acid compound in the pharmaceutical preparation of the above configuration.

  The sixth configuration of the present invention is a therapeutic agent for lung injury / pulmonary fibrosis using siRNA against CCR2 or a plasmid expressing the siRNA as the nucleic acid compound in the pharmaceutical preparation of the above configuration.

  A seventh configuration of the present invention is a therapeutic agent for lung injury / pulmonary fibrosis using siRNA against MCP-1 or a plasmid expressing the siRNA as the nucleic acid compound in the pharmaceutical preparation having the above configuration.

  The eighth configuration of the present invention is a therapeutic agent for lung injury / pulmonary fibrosis using siRNA against Egr-1 or a plasmid expressing the siRNA as the nucleic acid compound in the pharmaceutical preparation of the above configuration.

  A ninth configuration of the present invention is a therapeutic agent for lung cancer using a p53 plasmid having a p53 protein as a target molecule as the nucleic acid compound in the pharmaceutical preparation of the above configuration.

  The tenth configuration of the present invention is a therapeutic agent for lung cancer using a soluble VEGF receptor plasmid having VEGF as a target molecule as the nucleic acid compound in the pharmaceutical preparation of the above configuration.

  The eleventh configuration of the present invention is a therapeutic agent for bronchial asthma using siRNA against IL-13 or a plasmid expressing the siRNA as the nucleic acid compound in the pharmaceutical preparation of the above configuration.

  A twelfth configuration of the present invention is a therapeutic agent for bronchial asthma using a dominant negative SOCS3 plasmid having SOCS3 as a target molecule as the nucleic acid compound in the pharmaceutical preparation of the above configuration.

  A thirteenth configuration of the present invention is a therapeutic agent for bronchial asthma using siRNA against SOCS3 or a plasmid expressing the siRNA as the nucleic acid compound in the pharmaceutical preparation having the above configuration.

  According to the first configuration of the present invention, the biocompatible nanoparticle penetrates into the cell through the mucous membrane or blood vessel in the lung, thereby efficiently allowing the nucleic acid compound enclosed in the nanoparticle to reach the affected area. Since the action can be sustained by gradually releasing the nucleic acid compound from the nanoparticles, a pharmaceutical preparation suitable as a DDS preparation used for the transpulmonary route is provided.

  In addition, according to the second configuration of the present invention, by carrying the nucleic acid compound on the surface of the nanoparticle, the total amount of encapsulation including the inside and the surface can be increased, and the dosage form can be downsized. To achieve a practical transpulmonary dose. In addition to the nucleic acid compound that is released from the inside of the nanoparticle in a sustained manner, the drug formulation is given both immediate effect and sustainability by acting on the nucleic acid compound that dissolves from the surface of the nanoparticle immediately after administration. Can do.

  Further, according to the third configuration of the present invention, in the pharmaceutical preparation of the first or second configuration, polylactic acid, polyglycolic acid, lactic acid / glycolic acid copolymer, or lactic acid is used as the biocompatible polymer. -By using any of the aspartic acid copolymers, it can be stored for a long period of time while maintaining the efficacy of the nucleic acid compound, and it can be stored for a long period of time while maintaining the efficacy of the nucleic acid compound. A pharmaceutical preparation capable of sustained release of a nucleic acid compound can be provided by decomposing DNA.

  According to the fourth configuration of the present invention, in the pharmaceutical preparation of any one of the first to third configurations, a soluble type II TGF receptor plasmid having TGF-beta as a target molecule in a nanoparticle as a nucleic acid compound By encapsulating, a pharmaceutical preparation for transpulmonary administration having an excellent therapeutic effect on lung damage and pulmonary fibrosis, which are difficult to treat lung diseases, is obtained.

  Moreover, according to the fifth configuration of the present invention, in the pharmaceutical preparation of any one of the first to third configurations, as a nucleic acid compound, a mutant MCP-1 having MCP-1 as a target molecule in a nanoparticle By encapsulating the plasmid, it becomes a pharmaceutical preparation for transpulmonary administration having an excellent therapeutic effect on lung damage and pulmonary fibrosis, which are refractory lung diseases.

  According to the sixth configuration of the present invention, in the pharmaceutical preparation of any one of the first to third configurations, as a nucleic acid compound, siRNA against CCR2 or a plasmid that expresses the siRNA is encapsulated in a nanoparticle. As a result, the pharmaceutical preparation for transpulmonary administration has an excellent therapeutic effect on pulmonary injury and pulmonary fibrosis, which are intractable pulmonary diseases.

  According to the seventh configuration of the present invention, in the pharmaceutical preparation of any one of the first to third configurations, as a nucleic acid compound, siRNA against MCP-1 or a plasmid expressing the siRNA is contained in the nanoparticles. By encapsulating, it becomes a pharmaceutical preparation for transpulmonary administration that has an excellent therapeutic effect on lung damage and pulmonary fibrosis, which are intractable pulmonary diseases.

  According to the eighth configuration of the present invention, in the pharmaceutical preparation of any one of the first to third configurations, as a nucleic acid compound, siRNA against Egr-1 or a plasmid that expresses the siRNA is contained in the nanoparticles. By encapsulating, it becomes a pharmaceutical preparation for transpulmonary administration that has an excellent therapeutic effect on lung damage and pulmonary fibrosis, which are intractable pulmonary diseases.

  According to the ninth configuration of the present invention, in the pharmaceutical preparation of any one of the first to third configurations, a p53 plasmid having a p53 protein as a target molecule is encapsulated in a nanoparticle as a nucleic acid compound. Thus, a pharmaceutical preparation for transpulmonary administration for treating lung cancer, which has an excellent therapeutic effect particularly on primary alveolar epithelial cancer.

  According to the tenth configuration of the present invention, in the pharmaceutical preparation of any of the first to third configurations, a soluble VEGF receptor plasmid having VEGF as a target molecule is encapsulated as a nucleic acid compound in a nanoparticle. By doing so, it becomes a pharmaceutical preparation for transpulmonary administration for treating lung cancer, which has an excellent therapeutic effect especially on metastatic lung cancer.

  According to the eleventh configuration of the present invention, in the pharmaceutical preparation of any of the first to third configurations, as a nucleic acid compound, siRNA against IL-13 or a plasmid that expresses the siRNA is contained in the nanoparticles. By encapsulating, it becomes a pharmaceutical preparation for transpulmonary administration that has an excellent therapeutic effect on bronchial asthma, which is an intractable pulmonary disease.

  According to the twelfth configuration of the present invention, in the pharmaceutical preparation of any one of the first to third configurations, a dominant negative SOCS3 plasmid having SOCS3 as a target molecule is encapsulated in the nanoparticle as a nucleic acid compound. As a result, the pharmaceutical preparation for transpulmonary administration has an excellent therapeutic effect on bronchial asthma, which is an intractable pulmonary disease.

  According to the thirteenth configuration of the present invention, in the pharmaceutical preparation of any one of the first to third configurations, siRNA against SOCS3 or a plasmid expressing the siRNA is encapsulated in the nanoparticle as a nucleic acid compound. As a result, the pharmaceutical preparation for transpulmonary administration has an excellent therapeutic effect on bronchial asthma, which is an intractable pulmonary disease.

  The nucleic acid compound-encapsulated nanoparticles used in the pharmaceutical preparation for transpulmonary administration of the present invention include a nano-sized particle (nanosphere) in which a nucleic acid compound such as plasmid DNA or siRNA is encapsulated in a biocompatible polymer. It is a thing. By allowing these nanoparticles to penetrate into the cells via the mucosa and blood vessels in the lungs by dry powder inhalation (DPI), the nucleic acid compound can reach the affected area directly in an organ-specific manner, and the nanoparticles can be used over a long period of time. Since the nucleic acid compound can be gradually released from the lung, it can be suitably used as a material for a DDS preparation used for the transpulmonary route.

  The method for producing the nanoparticles used in the present invention is not particularly limited as long as the method can process the nucleic acid compound and the biocompatible polymer into particles having an average particle size of less than 1,000 nm. However, since nucleic acids are decomposed when various external stresses are applied, conventional methods for preparing nanoparticles that require high shear force cannot be applied. Therefore, the spherical crystallization method which is a non-high shear force particle preparation method can be used suitably.

  Spherical crystallization is a method in which spherical crystal particles can be designed and processed by directly controlling their physical properties by controlling the crystal generation and growth process in the final process of compound synthesis. One of the spherical crystallization methods is an emulsion solvent diffusion method (ESD method).

  The ESD method is a technique for producing nanospheres based on the following principle. In this method, two types of solvents are used: a good solvent that can dissolve PLGA or the like as a base polymer for encapsulating the drug, and a poor solvent that does not dissolve PLGA. As this good solvent, an organic solvent such as acetone that dissolves PLGA and is mixed with the poor solvent is used. And a polyvinyl alcohol aqueous solution etc. are normally used for a poor solvent.

  As an operation procedure, first, after dissolving PLGA in a good solvent, a drug solution is added and mixed in the good solvent so that the PLGA does not precipitate. When this mixed solution containing PLGA and a drug is dropped into a poor solvent while stirring, the good solvent (organic solvent) in the mixed solution rapidly diffuses and moves into the poor solvent. As a result, self-emulsification of the good solvent occurs in the poor solvent, and submicron sized good solvent emulsion droplets are formed. Furthermore, because the organic solvent continuously diffuses from the emulsion into the poor solvent due to the mutual diffusion of the good solvent and the poor solvent, the solubility of the PLGA and the drug in the emulsion droplets decreases, and finally the drug is removed. PLGA nanospheres of encapsulated spherical crystal particles are produced.

  In the above spherical crystallization method, nanoparticles can be formed by a physicochemical method, and the resulting nanoparticles are substantially spherical. Therefore, it is necessary to consider the problem of catalyst and raw material compound residues from homogeneous nanoparticles. And can be easily formed. Then, the organic solvent which is a good solvent is distilled off under reduced pressure (solvent distillation step) to obtain drug-containing nanoparticle powder. Then, the obtained powder is composited as it is or into flocculated particles that can be re-dispersed by lyophilization or the like as necessary (compositing step) to form composite particles, which are then filled into a container and filled with drug-containing nanoparticles. To do.

  The type of the good solvent and the poor solvent is determined according to the type of the nucleic acid compound to be encapsulated and is not particularly limited, but the biocompatible nanoparticle is a raw material for a pharmaceutical preparation that acts on the human body. Therefore, it is necessary to use a material that is highly safe for the human body and has a low environmental load.

  Examples of such a poor solvent include water or water to which a surfactant is added. For example, a polyvinyl alcohol aqueous solution to which polyvinyl alcohol is added as a surfactant is preferably used. Examples of surfactants other than polyvinyl alcohol include lecithin, hydroxymethylcellulose, hydroxypropylcellulose, and the like. In addition, when excess polyvinyl alcohol remains, you may provide the process (removal process) of removing polyvinyl alcohol by centrifugation etc. after a solvent distillation process.

  Examples of good solvents include halogenated alkanes, which are low-boiling and poorly water-soluble organic solvents, acetone, methanol, ethanol, ethyl acetate, diethyl ether, cyclohexane, benzene, toluene, and the like, for example, adverse effects on the environment and the human body. Only a small amount of acetone or a mixture of acetone and ethanol is preferably used.

  In addition, when trying to produce nanoparticles encapsulating nucleic acid compounds using conventional spherical crystallization methods, water-soluble nucleic acid compounds dispersed and mixed in a good solvent leak and dissolve in poor solvents, resulting in nanoparticles. Since only the macromolecule forming the nuclei is deposited, the nucleic acid compound is hardly encapsulated. Therefore, in order to increase the encapsulation rate of the nucleic acid compound inside the nanoparticles, it is preferable to add a cationic polymer to the poor solvent. When a cationic polymer is added in a poor solvent, the cationic polymer adsorbed on the nanoparticle surface interacts with the nucleic acid compound present on the emulsion droplet surface, thereby suppressing leakage of the nucleic acid compound into the poor solvent. Can be considered.

  In addition, when a cationic polymer is added to a poor solvent, the surface of the nanoparticles is modified with the cationic polymer and is positively charged. Therefore, by adding a nucleic acid compound during lyophilization, anionic properties can be added to the particle surface. The nucleic acid compound can be electrostatically supported. In this way, the total amount of encapsulation can be increased by supporting the nanoparticles on the surface. In addition to the nucleic acid compound that is released from the inside of the nanoparticle in a sustained manner, the drug formulation is given both immediate effect and sustainability by acting on the nucleic acid compound that dissolves from the surface of the nanoparticle immediately after administration. Can do.

  Examples of the cationic polymer include chitosan and chitosan derivatives, cationized cellulose in which a plurality of cationic groups are bonded to cellulose, polyamino compounds such as polyethyleneimine, polyvinylamine, and polyallylamine, polyamino acids such as polyornithine and polylysine, and polyvinylimidazole. , Polyvinylpyridinium chloride, alkylaminomethacrylate quaternary salt polymer (DAM), alkylaminomethacrylate quaternary salt / acrylamide copolymer (DAA) and the like, and chitosan or derivatives thereof are particularly preferably used.

  Chitosan is a natural polymer with many glucosamines, one of the sugars with amino groups, contained in shrimp, crabs, and insect shells. Emulsion stability, shape retention, biodegradability, biocompatibility It is widely used as a raw material for cosmetics, foods, clothing, pharmaceuticals, etc. By adding this chitosan into a poor solvent, a highly safe nucleic acid compound-encapsulated nanoparticle can be produced without adversely affecting the living body.

  It should be noted that chitosan derivatives such as N- [2-hydroxy-3- (trimethylammonio) propyl] chitosan having a higher cationic property by quaternizing a part of chitosan which is originally cationic ( Use of cationic chitosan is preferable because more anionic drug can be supported on the particle surface, and the repulsive force between the particles becomes stronger and the stability of the particles becomes higher.

  In order to improve the affinity and dispersion stability of the nucleic acid compound in a good solvent, a cationic lipid such as DOTAP may be added to the good solvent to form a complex with the nucleic acid compound. However, since the cationic lipid released in the cell may cause cytotoxicity, attention should be paid to the amount added.

  The nanoparticles obtained as described above can be combined into aggregated particles (nanocomposites) that can be redispersed when powdered by freeze drying or the like. At this time, it is preferable that the organic or inorganic substance is combined so as to be redispersible and dried together with the nanoparticles. For example, by applying sugar alcohol or sucrose, it is possible to effectively prevent variation in the encapsulation rate, and sugar alcohol or the like can be used as an excipient to improve the handleability of the nanoparticles. Examples of the sugar alcohol include mannitol, trehalose, sorbitol, erythritol, maltose, xylitol, etc. Among them, trehalose is particularly preferable.

  By this combination, nanoparticles are gathered before use and become agglomerated particles that are easy to handle, and when exposed to moisture during use, the particles return to the nanoparticles and become composite particles that restore characteristics such as high reactivity. In addition, it can replace with a freeze-drying method, can also combine by the fluidized-bed dry granulation method (For example, using Agromaster AGM by Hosokawa Micron Co., Ltd.), and can integrate again in the state which can be isolate | separated.

  The biocompatible polymer used in the present invention is desirably a biocompatible polymer that has low irritation and toxicity to the living body, is biocompatible, and is decomposed and metabolized after administration. Moreover, it is preferable that it is the particle | grain which discharge | releases the medicine to include continuously and gradually. In particular, PLGA can be suitably used as such a material. It is known that PLGA nanoparticles can contain a drug and can be stored for a long period of time while retaining the efficacy of the drug.

  In addition, the evaluation of DDS as a carrier is determined by the arrival rate to the cell, the ability to take up into the cell, and the expression of the effect within the cell. PLGA nanoparticles are the most stable, withstand inhalation, and have a high rate of passage through the mucosal layer and cell reach after deposition in the alveoli. The uptake rate into cells is highest in membrane-fused liposomes, but the uptake rate can be increased to the same level as in liposomes by modifying the surface of PLGA nanoparticles. Furthermore, it is considered that sustained release in units of several days to one month can be performed from the characteristics of PLGA hydrolysis and long-term half-life. These characteristics make PLGA nanoparticles an excellent carrier for intractable pulmonary diseases having lesions in bronchioles and alveoli.

  The molecular weight of PLGA is preferably in the range of 5,000 to 200,000, and more preferably in the range of 15,000 to 25,000. The composition ratio of lactic acid and glycolic acid may be 1:99 to 99: 1, but glycolic acid is preferably 0.333 with respect to lactic acid 1. PLGA having a lactic acid and glycolic acid content in the range of 25 wt% to 65 wt% is preferably used because it is amorphous and soluble in an organic solvent such as acetone.

  Moreover, it is preferable to modify the surface of PLGA with polyethylene glycol (PEG) because the affinity between the water-soluble nucleic acid compound and PLGA is improved and encapsulation becomes easy. Other biodegradable biocompatible polymers include polyglycolic acid (PGA), polylactic acid (PLA), polyaspartic acid, and the like. In addition, aspartic acid / lactic acid copolymer (PAL) and aspartic acid / lactic acid / glycolic acid copolymer (PALG), which are these copolymers, may be used, and charged groups such as amino acids or functionalizable groups may be used. You may have.

  Other biocompatible polymers include polyalkylenes such as polyethylene and polypropylene, polyvinyl compounds such as polyvinyl alcohol, polyvinyl ether and polyvinyl ester, polyamide, polycarbonate, polyethylene glycol, polyethylene oxide, polyethylene terephthalate, and acrylic acid. Polymers with methacrylic acid, cellulose and other polysaccharides, and peptides or proteins, or copolymers or mixtures thereof.

  Examples of the nucleic acid compound encapsulated in the biocompatible nanoparticle of the present invention include plasmid DNA, gene, siRNA, oligonucleotide, antisense oligonucleotide, ribozyme, apseter and the like.

  Oligonucleotide has a plurality of (2-99) nucleoside phosphate esters (nucleotides ATP, GTP, UTP, or dATP, dGTP, dCTP, dTTP) in which purine or pyrimidine is β-n-glycoside-linked to the sugar. An antisense oligonucleotide is a molecule in which an antisense strand is introduced into an oligonucleotide.

  Antisense represents a base sequence that is complementary to the base sequence (sense sequence) of a messenger RNA (hereinafter referred to as mRNA) that synthesizes a protein. Genetic information is transmitted from the protein to the protein. This method of blocking the transmission of gene information using artificially synthesized DNA is called an antisense method.

  That is, if the base sequence of the mRNA of the target gene is clear, an antisense oligonucleotide complementary to the mRNA can be administered to specifically inhibit only the expression of the target gene. This antisense method is used not only as a gene analysis tool to identify gene action in the biosynthetic process by examining the gene inhibition effect of antisense, but also as a gene therapy method that suppresses diseases caused by gene abnormalities. .

  Ribozymes refer to RNA molecules that cleave RNA strands in a site-specific manner, for example. In order for the target gene to be a ribozyme substrate, a NUX sequence (N: any base, U: uracil, X: a base other than guanine) needs to exist, and if both ends form a base pair, the sequence is Is optional. Therefore, by designing a ribozyme that forms base pairs with sequences before and after the NUX sequence of viral genes and proto-oncogenes, it becomes possible to specifically suppress only the expression of the target gene. Used for gene therapy. In addition, if you target the gene whose function you want to investigate, it will be a gene analysis tool.

  An aptamer is a nucleic acid that specifically binds to a target molecule and functions as an antibody. Aptamers can be easily synthesized in large quantities, and do not depend on the conservation of the amino acid sequence of the target molecule, and have the advantage of being easily improved. Inhibition of proto-oncogene function (cancer suppression) and quantitative measurement (cancer diagnosis) Alternatively, it is expected to be used as a gene drug that mimics a physiologically active protein.

  In addition, although any one of the nucleic acid compounds may be encapsulated, if a plurality of components having different efficacy and action mechanism are encapsulated, it is expected that the efficacy of each component is promoted by the synergistic effect of each component. Furthermore, other components can be encapsulated together with the nucleic acid compound.

  The nucleic acid compound-encapsulated nanoparticles used in the present invention are not particularly limited as long as they have an average particle size of less than 1,000 nm, but the average particle size is set to 500 nm or less in order to increase the efficiency of reaching the affected area. In particular, 100 nm or less is more preferable in order to achieve a high gene expression rate when nanoparticles delivered to the target site are subjected to endocytosis of the cell membrane and taken into cells.

  When nucleic acid compound-encapsulated nanoparticles produced in this way are used as raw materials for drugs for pulmonary administration, the ability of nanoparticles to be incorporated into cells improves gene expression efficiency and directs to target molecules It is possible to provide an excellent preparation for transpulmonary administration. In addition, since a high concentration nucleic acid compound can be contained, the dosage form can be miniaturized, and the preparation can satisfy a practical transpulmonary dose. Hereinafter, specific examples of the pharmaceutical preparation for transpulmonary administration of the present invention using the target molecule in each case of lung injury / pulmonary fibrosis, lung cancer and bronchial asthma and the nucleic acid compound-encapsulating nanoparticles acting on the target molecule will be described.

[Treatment of lung injury and pulmonary fibrosis targeting TGF-beta]
A bleomycin pneumonitis / pulmonary fibrosis model using mice or rats is prepared by administering bleomycin intravenously, intraperitoneally, or transbronchially. In this model, lung damage and fibrosis are formed in 2 to 3 weeks for transbronchial administration and 4 to 6 weeks for intravenous and intraperitoneal administration. Experiments on molecular target therapy for various factors related to lung injury and lung fibrosis have been performed using this model, and TGF-beta is a major target molecule among them. In addition, apoptosis of epithelial cells is essential for fibrosis by TGF-beta, and a transcription factor called Egr-1 is essential for the apoptosis.

  Conventionally, soluble TGF-beta receptor II protein and anti-TGF-beta antibody are systemically administered intravenously or intraperitoneally, or soluble TGF-beta receptor II expression plasmid (hereinafter referred to as soluble type II TGF receptor plasmid) Attempts have been made to suppress strong lung damage and lung fibrosis caused by TGF-beta by, for example, a method of introducing and expressing a gene in the thigh muscle by in vivo electroporation. In addition, the number of inflammatory cells in bronchoalveolar lavage fluid, lung tissue findings, the number of apoptotic cells as lung epithelial damage, and the measurement of hydroxyproline, collagen, and fibrinctin in lung tissue as fibrosis indicators The effectiveness is clarified. However, the multifunctionality of TGF-beta is a problem for clinical application.

  Therefore, nanoparticles encapsulating siRNA for Egr-1 or a plasmid expressing siRNA were prepared, administered to the mouse bleomycin pneumonitis model via lung, and bronchoalveolar lavage fluid (BALF) and lung tissue were investigated for efficacy. Confirmation is expected to be effective against lung injury and lung fibrosis. In addition, it is considered that lung damage and lung fibrosis can be suppressed even when a similar type experiment is performed with a soluble type II TGF receptor plasmid enclosed in nanoparticles.

[Treatment of lung injury and pulmonary fibrosis targeting MCP-1]
On the other hand, Monocyte chemoattractant protein-1 (hereinafter referred to as MCP-1) is increased in bronchoalveolar lavage fluid and lung tissue in human pulmonary fibrosis, and CC is a receptor for MCP-1 in animal models. Effects on pulmonary fibrosis of chemokine receptor 2 (hereinafter referred to as CCR2) knockout mouse, MCP-1 neutralizing antibody, or mutant MCP-1 using the above in vivo electroporation were confirmed. It is a chemokine.

  As in the case of targeting TGF-beta, siRNA against CCR2 was encapsulated in nanoparticles and administered to the mouse bleomycin pneumonitis model, and bronchoalveolar lavage fluid (BALF) and lung tissue were examined. Pneumonitis was significantly suppressed (see Examples). In addition, a nanoparticle encapsulating a plasmid expressing siRNA against CCR2 or a mutant MCP-1 plasmid acting as a dominant negative inhibitor against MCP-1 with an N-terminal deletion of MCP-1 was prepared, and the same experiment was conducted. Even if performed, the effectiveness against pneumonitis is also expected.

[Treatment of primary alveolar epithelial cancer by introducing wild-type p53 gene]
Primary alveolar epithelial cancer (BAC) is a subtype of primary lung adenocarcinoma, but differs from other lung adenocarcinomas in terms of its intrapulmonary progression and anticancer drug sensitivity. In the advanced form, it grows in the form of replacing the alveolar epithelium along the alveolar structure, and the aeration is maintained in the tumor. In addition, drugs other than gefitinib (trade name Iressa) are poorly effective against anticancer drugs. Even when gefitinib is successful, the response period is about 12 months, and some tumors acquire resistance by causing genetic mutations. As for gefitinib, as a side effect, acute lung injury is caused in about 6%, and about 40% of cases have died, so there is a strong social refusal.

In the United States, gene therapy for BAC is in progress and the Phase I trial has been completed.
In this test, an adenovirus vector (INGN201: trade name Advexin) in which the wild-type p53 gene is recombined is administered via the respiratory tract, and a certain degree of effectiveness is obtained. However, because of the strong immunogenicity of adenovirus, there is a risk of causing lung injury via an inflammatory reaction, and since repeated administration produces viral antibodies, the possibility that the effect may weaken as a result cannot be denied.

The cytocidal effect and safety of the expressed p53 protein itself has been proven in clinical trials targeting lung cancer, but as a promoter driving the p53 gene, a relatively lung adenocarcinoma such as Secretary leukopeptidase inhibitor (SLPI) By using a specific promoter, higher safety is ensured. The experimental method of the suppressive effect on lung cancer by introduction of the wild type p53 gene and the expected effect will be described below.
(In vitro experimental system)

(Lung cancer cell line)
In this experiment, three cell lines differing in sensitivity to wild-type p53 protein (p53 gene point mutant: NCI-H157 strain, p53 gene deletion strain: NCI-H1299 strain, wild-type p53 gene strain: NCI-H460 strain) Is used.

(Wild type p53 gene expression plasmid)
The wild type p53 gene cDNA is prepared by preparing an expression cassette driven by CMV or hTERT (human teromelase reverse transcriptase) or SLPI (Secretory leukoprotease inhibitor) promoter, and recombining it with a mammalian expression plasmid. As a negative control, a plasmid expressing GFP (Green Fluorescent Protein) and Luciferase gene is prepared by the same method.

(Induction of apoptosis)
A complex of the above wild-type p53 expression plasmid and negative control plasmid and a nanosphere carrier (hereinafter referred to as carrier) is formed, and the complex is mixed into a cell culture medium to introduce a gene into a lung cancer cell line. Gene transfer efficiency is quantified by measuring the ratio of GFP positive cells under a fluorescence microscope. Cells in which apoptosis is induced are quantified by performing flow cytometry using an anti-Anexin-V antibody.

(Inhibition of cell proliferation)
By quantifying viable lung cancer cells after gene introduction by MTS assay, the growth inhibitory effect is evaluated by comparison with the values before introduction.

(Expected effect)
From the results of experiments using wild-type p53 gene-expressing adenovirus so far, it was confirmed that NCI-H157 strain and NCI-H1299 strain are highly sensitive to wild-type p53 gene transfer and die by a small amount of gene transfer Has been. On the other hand, the NCI-H460 strain is more resistant to the gene transfer than the previous two strains. In sensitive strains, cell apoptosis is confirmed 3 hours after gene transfer, and approximately 90% or more of the cells die after 48 hours.

The amount of wild-type p53 protein accumulated in cells varies depending on the results of complex kinetics such as gene transfer efficiency, time until gene expression after transfer, dilution of the transgene by cell division, and elimination of the transgene by intracellular metabolism. I think that. In the above experiment, the expression level reaches a peak for 48 hours after introduction when dividing cells are used, and 7 days after introduction when non-dividing cells are used. It is considered that the same result can be obtained basically by gene transfer using a carrier, and a cell-killing effect by induction of apoptosis is expected from an early stage after the transfer.
(In vivo experimental system)

(Cancer-bearing animal model)
The alveolar epithelial cancer mouse model is prepared by transplanting the above three types of lung cancer cell lines into the lungs via the airway into nude mouse lungs.

(Treatment experiment)
A complex of the wild-type p53 cDNA expression plasmid and a carrier is formed, and the complex is inhaled with an ultrasonic nebulizer or directly injected through the airway. Regarding the engraftment and proliferation of the transplanted lung cancer cells, the transplanted mice are sacrificed over time, and the histological observation of the resected lung and other organs is performed to evaluate the state of the primary lesion (lung) and other organ metastases. Moreover, in order to quantify the anti-tumor effect over time in the same mouse, an FDG-PET test is periodically performed. In addition, systemic effects are assessed by weight, arterial oxygen saturation and survival.

  In addition, a mouse having normal immunity (C57BL6) was used, and a wild-type p53 cDNA expression plasmid (using CMV, hTERT, and SLPI promoters) and a carrier complex, wild-type p53-expressing adenovirus was trans-respirated into the lung. Inject and examine histologically for injury to normal lung tissue.

(Expected effect)
The method of injecting wild-type p53 gene-expressing adenovirus directly into a tumor formed subcutaneously in nude mice showed an obvious tumor growth-inhibiting effect, but the stromal structure inside the tumor inhibited the virus infection. The effect as observed in the in vitro experiment was not obtained. However, the intrapulmonary tumor created in this animal model has a relatively small tumor volume compared to the subcutaneous tumor model, so if the inhaled or transtracheally injected plasmid carrier complex can reach the alveolar level. Sufficient antitumor effect can be expected.

  In addition, when the wild-type p53 protein is excessively expressed, it may be toxic to normal cells, so that the gene expression promoter is preferably as tumor-specific as possible. The hTERT and SLPI promoters were used as candidates. These promoters are highly expressed in many cancer types, including lung cancer, while their expression in normal cells is low, so they are also expected as general gene therapy tools.

  By encapsulating this p53 plasmid in nanoparticles and administering it to the prepared lung cancer model mouse transtracheally, it is possible to avoid problems with immunogenicity compared to the case of using a viral vector, and even to the alveolar region. Since it becomes a reachable vector, it is considered to be very effective as a DDS to BAC showing alveolar epithelial-like spread.

[Treatment of metastatic lung cancer by introduction of soluble VEGF receptor gene]
The lung is the most common metastatic organ of primary lung cancer. Chemotherapy with anti-cancer drugs is the first choice for lung cancer cases with multiple lung metastases, but generally the response rate is about the most effective anti-cancer drug combination therapy (platinum + new anti-cancer drug) in advanced stage lung cancer. The median survival time is 35% and the prognosis is poor with 12 to 15 months. The results of a phase III clinical trial using anti-VEGF antibody (Bevasizumab: trade name Avastin) were announced at the American Society of Clinical Oncology this year, and the survival period was prolonged by using Avastin in combination with carboplatin and paclitaxel. It became clear.

  Avastin has already been shown to be useful for prolonging survival in colorectal cancer, and VEGF is considered to be one of the therapeutic targets in advanced lung cancer. However, the anti-VEGF antibody needs to be repeatedly administered, and even though it is a humanized antibody, there are reports of antibody problems produced in the antibody, and although there are still a few cases, fatal gastrointestinal bleeding has been reported.

On the other hand, the soluble VEGF receptor is originally an endogenous protein and thus has a lower immunogenicity than anti-VEGF antibodies. Unlike an antibody, it is expressed by a transgene, so that the expression period and expression level may be adjusted. It has the advantage that a local high expression state can be created by the gene introduction site. The experimental method of the suppressive effect on lung cancer by introduction of the wild type p53 gene and the expected effect will be described below.
(In vitro experimental system)

(Lung cancer cell line)
In this experiment, two types of lung cancer cell lines (VEGF high producing strain: NCI-H157 strain, VEGF low producing strain: NCI-H460 strain) and normal airway cell BEAS-2B strains having different VEGF producing ability are used.

(Soluble VEGF receptor gene expression plasmid)
After isolating the extracellular region from the wild-type VEGF receptor gene cDNA, human IgG Fc is added as a tag to the C-terminus to prepare an expression cassette driven by a CMV promoter or TRE (tetracycline induction) promoter. The cassette is recombined with a mammalian expression plasmid and a long-term expression episomal vector (under study) to produce a plasmid that expresses a soluble VEGF receptor gene (hereinafter referred to as a soluble VEGF receptor plasmid). As a negative control, a plasmid expressing GFP and Luciferase genes is prepared by the same method.

(Confirmation of expression of soluble VEGF receptor)
A complex of the above soluble VEGF receptor plasmid and negative control plasmid and a carrier is formed, and the complex is mixed into a cell culture medium to introduce a gene into a normal airway cell line. Gene transfer efficiency is quantified by measuring the ratio of GFP positive cells under a fluorescence microscope. The culture solution for 72 hours after gene introduction is collected, and the expression of soluble VEGF receptor is confirmed by ELISA using an anti-human IgG Fc antibody.

(Modulation of soluble VEGF receptor expression)
In order to artificially adjust the expression level of the soluble VEGF receptor, a TRE promoter-dependent soluble VEGF receptor plasmid and a tetracycline promoter-binding protein expression plasmid are simultaneously introduced into cells to construct a so-called Tet-on system. Changes in soluble VEGF receptor concentration in the culture solution are quantified by ELISA by adding and removing tetracycline from the culture solution. In addition, since several reports have already been made about the neutralizing activity of VEGF by the soluble VEGF receptor produced from this expression cassette, the description is omitted here.

(Expected effect)
According to the results of experiments using a soluble VEGF receptor-expressing adenovirus so far, the cells into which the gene has been introduced secrete the soluble VEGF receptor into the culture medium, and the receptor exhibits the angiogenic activity of the wild-type VEGF molecule. Since inhibition has been confirmed, similar results can be expected even with gene transfer using the pharmaceutical preparation of the present invention. For artificial regulation of expression using a tetracycline-inducible promoter, add or remove tetracycline from the culture if the two expression plasmids that form a complex with the same carrier can be designed to be 1: 1. It is presumed that expression regulation is possible.

In addition, if the two types of plasmids can be introduced into cells at a certain ratio, the range of application has the potential to greatly expand. Although the ratio of a plurality of vectors can be made constant at the time of administration in the conventional method, it is not always introduced into individual cells at the same ratio as at the time of administration. Expression levels vary. However, if the compounding ratio of the two types of plasmids can be made constant when forming the complex between the nanosphere carrier and DNA, the complex will be incorporated into the cell as it is, so the introduction ratio of multiple genes at the individual cell level will be kept constant. It seems possible. This is expected to be useful in intracellular events obtained only by expression of a plurality of proteins, particularly in regulating protein expression.
(In vivo experimental system)

(Cancer-bearing animal model)
An advanced lung cancer mouse model is prepared by transplanting the above two types of lung cancer cell lines into the lungs via the airway into nude mouse lungs.

(Treatment experiment)
A complex of a soluble VEGF receptor plasmid and a carrier is formed, and the complex is inhaled with an ultrasonic nebulizer or injected directly via the airway. The soluble VEGF receptor concentration in blood and alveolar lavage fluid is measured over time by ELISA.

  Regarding the engraftment and proliferation of the transplanted lung cancer cells, the transplanted mice are sacrificed over time, and the histological observation of the resected lung and other organs is performed to evaluate the state of the primary lesion (lung) and other organ metastases. Moreover, in order to quantify the anti-tumor effect over time in the same mouse, an FDG-PET test is periodically performed. The inhibitory effect on tumor angiogenesis is evaluated by measuring and quantifying blood vessel density after identifying tumor blood vessels by immunostaining. In addition, systemic effects are assessed by weight, arterial oxygen saturation and survival.

  Using normal immunity mice (C57BL6), a complex of soluble VEGF receptor plasmid and carrier and soluble VEGF receptor-expressing adenovirus were transtracheally injected into the lung to histologically injure normal lung tissue. To consider.

(Expected effect)
When a soluble VEGF receptor-expressing adenovirus was administered to the thigh muscle or abdominal cavity of nude mice, the concentration of soluble receptor in the blood peaked on day 7, and it gradually decreased, but it was possible to measure the blood concentration until 4 weeks later. It was. The blood concentration in this experiment is expected to follow a similar course, but receptor production in the alveoli may occur earlier. Regarding the antitumor effect, it is already clear that the NCI-H157 strain forms a tumor in a VEGF-dependent manner, so that if the soluble VEGF receptor has reached an effective concentration, gene transfer using a carrier is sufficient for antitumor activity. Expected to be effective.

  On the other hand, side effects due to inhibition of VEGF function in normal lungs are still not fully understood, and in clinical trials using anti-VEGF antibodies, multiple deaths from hemoptysis have been reported. In the experiment used, pulmonary emphysema has been reported. In particular, in the case of gene transfer of the present invention, expression may be sustained for a long period of time, and more careful observation is necessary. In the future, it will be necessary to construct a system that can adjust the concentration of soluble receptors when adverse reactions occur. If a system using a tetracycline-inducible promoter functions in vitro, it will also be necessary to verify it in vivo. is there.

[Treatment of bronchial asthma targeting IL-13]
Interleukin-13 (hereinafter referred to as IL-13) is a cell-mediated immunity shift from Th1 to Th2 in bronchial asthma, airway hypersensitivity, goblet cell hyperplasia, airway wall fibrosis, etc. It is an important cytokine that forms the pathology in asthma. It is also known to have an important role not only in bronchial asthma but also in pulmonary fibrosis. Up to now, the importance of this is clarified by experiments using IL-13 transgenic mice, knockout mice, and neutralizing antibodies to IL-13, and application to treatment is awaited.

  The bronchial asthma model is created by inhaling ovalbumin after sensitizing the mouse with ovalbumin (ovalbumin, hereinafter abbreviated as OVA). Moreover, the inhibitory effect of bronchial asthma can be performed by investigating airway hypersensitivity, eosinophil infiltration into the airway, goblet cell hyperplasia, and airway wall thickening. A significant inhibitory effect is expected by encapsulating siRNA for IL-13 or a plasmid expressing siRNA in nanoparticles and administering it to the prepared bronchial asthma model mouse transtracheally.

[Treatment of bronchial asthma targeting SOCS3]
Th2-type cytokines such as IL-4, IL-5, and IL-13 are involved in the pathology of allergic diseases including bronchial asthma. It is considered that allergic diseases including asthma develop when naive T cells are induced to differentiate predominantly in functional CD4Th2 cells. Suppressor Of Cytokine Signaling (hereinafter referred to as SOCS) is a molecule that suppressively controls cytokine signals, and forms a family based on the similarity of its structure. It has been clarified in mouse and human systems that SOCS3 belonging to these family molecules is specifically expressed in Th2 in T cells.

  When T cells were isolated from peripheral blood and the expression of each SOCS was analyzed, SOCS3 expression was significantly higher in patients with atopic asthma and atopic dermatitis than in the healthy controls, and as the severity increased, SOCS3 Expression was increased. Furthermore, SOCS3 expression correlated with blood IgE levels, which are also markers of atopy and Th2 type responses.

  Moreover, when T cells were differentiated in vitro, SOCS expression was very characteristic in the T cell differentiation process, and SOCS3 was selectively expressed in Th2 cells. When T cell differentiation was analyzed using T cells of mice (SOCS3-Tg mice) that constantly overexpress SOCS3 specifically in T cells, Th2 differentiation was found to be higher than T cells derived from wild-type control mice. It was weakening. Furthermore, when an in vivo experiment using an allergic asthma model by OVA sensitization / exposure was performed, airway eosinophils significantly increased and airway hypersensitivity was remarkably enhanced in SOCS3-Tg mice.

  When a dominant negative (dn) mutant SOCS3 molecule (hereinafter referred to as dnSOCS3) was introduced into a T cell by retrovirus in order to examine its application to therapy, Th2 differentiation was suppressed. Therefore, suppression of Th2 differentiation is expected by encapsulating dnSOCS3 protein in nanospheres and introducing it into T cells. Furthermore, in in vivo experiments using OVA asthma model mice, by inhalation administration of dnSOCS3 encapsulated nanospheres, It is expected to suppress airway eosinophilia and airway hypersensitivity. From these facts, dnSOCS3 protein-encapsulated nanospheres, dnSOCS3 expression plasmid-encapsulated nanospheres, SOCS3-siRNA-encapsulated nanospheres, and SOCS3-siRNA expression plasmid-encapsulated nanospheres are considered to be useful as novel therapeutic methods for bronchial asthma and allergic diseases.

  By the method as described above, it is possible to control the target molecule in the alveolar epithelial cell, which was difficult to reach the nucleic acid compound sufficiently by the conventional method. In addition, since nanoparticles formed of biocompatible polymers such as PLGA are used as vectors, there is no cytotoxicity compared to the case of using viruses and liposomes, and the efficiency of gene introduction and the sustainability of the effects can be improved. .

  Therefore, for refractory pulmonary diseases such as pulmonary fibrosis and lung cancer, a revolutionary change will be brought about in the current treatment strategy in which it is necessary to use drugs that are ineffective but have inevitable side effects. The contribution is very large. Furthermore, by changing the type and route of administration of the nucleic acid compound encapsulated in the nanoparticles, it can also be used to treat other organs such as malignant tumors, arteriosclerosis, cirrhosis, and renal sclerosis. It is considered applicable.

  In addition, the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the means to be solved by the invention, and technical means disclosed in different embodiments are respectively included. Embodiments obtained by appropriate combinations are also included in the technical scope of the present invention. Hereinafter, the effects of the present invention will be described in more detail with reference to examples. In this example, as an example, the effect of suppressing lung injury / pulmonary fibrosis when siRNA-encapsulated nanoparticles against MCP-1 were used as a pharmaceutical preparation for transpulmonary administration was investigated.

[Preparation of bleomycin pneumonitis model mouse and measurement of MCP-1 kinetics]
This experiment was conducted based on the animal handling regulations of Kyushu University School of Medicine and the guidelines of the American Physiological Society. C57BL / 6 mice (male 7-8 weeks old, weight 20-25 g, KBT Oriental) were anesthetized by intraperitoneal administration of pentobarbital (Schering-Plough) 50 μL containing 1.5 U / kg bleomycin Saline solution was administered transtracheally. Mice were sacrificed on days 1, 3, 5, 7, 10, 14 after bleomycin administration, the right lung was fixed with 10% formalin solution, the left lung was snap frozen with liquid nitrogen, and used for the experiment. Stored at -80 ° C.

The frozen left lung was homomodulated with a polytron homogenizer (Kinematica) in hypotonic buffer (25 mmol HEPES, pH = 7.5, 5 mmol MgCl ₂ , 1 mmol EGTA, 1 mmol PMSF, 1 mg / mL leupeptin, and 1 mg / mL aprotinin). The mixture was centrifuged at 15,000 G for 30 minutes at 4 ° C., and the supernatant was analyzed for MCP-1 protein by ELISA.

  The protein concentration in the supernatant was measured by Bio-Rad protein assay. Each supernatant was diluted with sample buffer (133 mmol Tris-HCl, pH = 6.8, 0.1% SDS, 5% glycerol, 0.67% 2-ME, 1 μg / mL aprotinin) and boiled. Each lane of SDS-PAGE was electrophoresed with 30 μg of protein, and the protein was transferred to polyvinylidene fluoride hydrophobic membrane (Millipore). The membrane was blocked with 5% fat-free skim milk TBS-T solution, and anti-fibronectin antibody, type 1 collagen, type 3 collagen, anti-α-tubulin antibody (above, Santa Cruz Biotechnology) at 4 ° C. Reacted overnight. After washing, the mixture was reacted with a biotinylated secondary antibody for 30 minutes at room temperature. The blot was luminescent with an ECL luminescence kit (Amersham Biosciences). The blot was captured with an image scanner (GT-8700, Epson) and quantified using NIH Image 1.61 software.

  3 μm paraffin sections were adhered to a slide glass coated with poly-L-lysine, deparaffinized, and then the Histofine SAB-PO kit (manufactured by Nichirei) was used in a modified streptavidin-biotinylated peroxidase method described below. Immunohistochemical staining was performed. Non-specific proteins were blocked by incubation with rabbit or goat serum for 30 minutes at room temperature. The sections were reacted overnight at 4 ° C. with an anti-MCP-1 antibody (Santa Cruz Biotechnology). A control in which the specific antibody was replaced with non-immune reaction serum was used as a control. The sections were blocked with endogenous peroxidase with 0.3% active oxygen methanol solution, reacted with a biotinylated secondary antibody for 30 minutes, reacted with streptavidin-biotinylated peroxidase complex for 30 minutes, and then mounted.

  As a result of the test, the concentration of MCP-1 in lung tissue and BALF was significantly increased from 3 to 14 days after administration of bleomycin. MCP-1 was found in alveolar macrophages, type II alveolar epithelial cells, and bronchiole epithelial cells in normal mice. MCP-1 expression was strongly observed in the stroma of these cells and the inflammatory site 3 to 14 days after bleomycin administration.

[Preparation of siRNA-encapsulated PLGA nanospheres for MCP-1]
Three types of annealed siRNA oligonucleotides (manufactured by Ambion) for mouse MCP-1 and cultured mouse lung epithelial cell line LA4 ceII line and peritoneal macrophage ceIII line RAW (manufactured by ATCC) were prepared. siRNA was administered to cultured cells using an Ambion transfection kit, and the suppression of the expression of MCP-1 was confirmed by the immune cell staining and Western blotting of Example 1. Using the results, siRNA with the highest suppression efficiency was selected from the three types, and siRNA-encapsulated nanospheres were prepared. The preparation method is described below.

  An aqueous solution of 0.5% by weight polyvinyl alcohol (manufactured by Kuraray; PVA403) was prepared and used as a poor solvent. Also, 1 g of PLGA (Wako Pure Chemical; PLGA7520, lactic acid / glycolic acid = 75/25, molecular weight 20,000) was dissolved in 100 mL of acetone, and 5 mg of siRNA against MCP-1 (hereinafter referred to as MP-1 siRNA) was added thereto. 15 mL of dissolved phosphate buffer (PBS) was added and mixed to obtain a polymer solution. This solution was dropped into the above poor solvent at 4 mL / min while stirring at 40 ° C. and 400 rpm to obtain a siRNA-encapsulated PLGA nanosphere suspension. The acetone solvent was distilled off under reduced pressure for 3 hours and then lyophilized to obtain siRNA-encapsulated PLGA nanosphere powder.

  The redispersibility of the obtained powder in water was good, and the average particle size when the nanosphere powder was redispersed in purified water was 190 nm as measured by a dynamic light scattering method. Moreover, it was 0.42 weight% when the MCP-1 siRNA encapsulation rate in nanosphere powder was measured using the spectrophotometer.

Comparative Example 1

[Preparation of non-specific siRNA-encapsulated PLGA nanospheres]
As a comparative control example of MCP-1 siRNA-encapsulated PLGA nanospheres, PLGA nanospheres encapsulating non-specific siRNA (hereinafter referred to as control siRNA) were prepared. The preparation method was performed in the same manner as in Example 1.

  The redispersibility of the obtained powder in water was good, and the average particle size when the nanosphere powder was redispersed in purified water was measured by a dynamic light scattering method to be 205 nm. Moreover, it was 0.40 weight% when the control siRNA enclosure rate in nanosphere powder was measured using the spectrophotometer.

[Evaluation of nanosphere administration method using coumarin-encapsulated PLGA nanosphere]
In order to confirm the behavior of the nanosphere in the body, a coumarin-encapsulated PLGA nanosphere, which is a fluorescent label, was prepared. The preparation method is described below.

  A 2 wt% aqueous solution of polyvinyl alcohol (manufactured by Kuraray; PVA403) was prepared and used as a poor solvent. 2 g of PLGA (Wako Pure Chemical; PLGA7520, lactic acid / glycolic acid = 75/25, molecular weight 20,000) and 1 mg of coumarin (Coumarin 6; manufactured by ICN Biomedical) were dissolved in a mixture of 40 mL of acetone and 20 mL of ethanol to obtain a polymer solution. . This solution was added dropwise at 4 mL / min with stirring at 40 ° C. and 400 rpm in the above poor solvent to obtain a coumarin-encapsulated PLGA nanosphere suspension. Acetone / ethanol solvent was distilled off under reduced pressure for 2 hours, followed by centrifugation (41,000 G, −20 ° C., 20 minutes). Purified water was added to the resulting precipitate, and PLGA nanospheres were sonicated. Suspended. This operation was repeated twice to remove excess PVA. The finally obtained suspension was freeze-dried to obtain a coumarin-encapsulated PLGA nanosphere powder. The redispersibility of the obtained powder in water was good. When the particle size distribution was measured by a dynamic light scattering method, the average particle size was 240 nm.

  15.7 mg of the above-mentioned coumarin-encapsulated PLGA nanosphere was suspended in 10 mL of PBS to prepare a dosing solution. A total of 100 μL of 50 μL was inhaled from both nostrils of C57BL / 6 mice nasally for 10 days, and the uptake and expression into lung cells were confirmed with a fluorescence microscope. Before administration of the nanosphere suspension, anesthesia was performed by intraperitoneal administration of pentobarbital (manufactured by Schering-Plough). When the mice were dissected on the 14th day after administration of bleomycin, coumarin fluorescence was observed mainly in the bronchiole epithelium, and uptake was observed in the alveolar macrophages and alveolar epithelial cells. Therefore, this administration method was adopted as a method for administering MCP-1 siRNA-encapsulated PLGA nanospheres to mice.

[Inhibitory effect of pneumonitis using MCP-1 siRNA-encapsulated PLGA nanospheres]
Using the MCP-1 siRNA-encapsulated PLGA nanospheres prepared in Example 2, the inhibitory effect on bleomycin pneumonitis was investigated. The test method is described below.

  15.7 mg (siRNA: 66 μg, 5 nmol) of the MCP-1 siRNA-encapsulated PLGA nanosphere obtained in Example 2 was suspended in 10 mL of PBS to obtain an administration solution. As a comparative control, 16.5 mg (siRNA: 66 μg, 5 nmol) of the control siRNA-encapsulated PLGA nanosphere obtained in Comparative Example 1 was suspended in 10 mL of PBS to obtain a dosing solution. From day 4 to day 13 after bleomycin administration, a total of 100 μL was inhaled intranasally from both nostrils of C57BL / 6 mice every day. Before administration of the nanosphere suspension, anesthesia was performed by intraperitoneal administration of pentobarbital (manufactured by Schering-Plough). On day 14 after administration of bleomycin, mouse bronchoalveolar lavage fluid and lung tissue were collected.

(Statistical processing)
For statistical analysis, ANOVA and Sceffe's F test were used for the number of cells and protein concentration in BALF, the number of TUNEL positive cells, the amount of hydroxyproline, and the results of ELISA. For comparison of histological grade, Kruskal-Wallis and Mann-Whitney's U test were used. P <0.05 was considered significant. StatView J-4.5 (Abacus Concepts) was used as the analysis software.

(Histological examination)
After intrathoracic resection, the pulmonary circulation was flushed with physiological saline. Lung samples were fixed overnight in 10% formalin and then embedded in paraffin. Paraffin sections (thickness 3 μm) were placed on a slide glass and stained with HE. The pathological grade of inflammation and fibrosis was determined by grading the entire medial sagittal plane at 40x magnification as follows.
0; no lesion; 1; the range of inflammation and fibrosis is less than 25% of the lung; 2; 25% to 50%; 3;
In addition, the slides were evaluated for collagen deposition by Sirius red staining.

(Hydroxyproline assay)
The lung tissue snap-frozen with liquid nitrogen was freeze-dried using a freeze-dry system (manufactured by Labconco), weighed, finely crushed, and then thawed in 6N HCl at 120 ° C. for 16 hours. The amount of hydroxyproline in each sample was measured according to the protocol of Woessner.

  The alveolar wall began to thicken with neutrophil and lymphocyte infiltration 7 days after bleomycin administration. On the 14th day after administration of bleomycin, a large number of lymphocytes infiltrated into the lung stroma, and thickening of the alveolar septum, collapse of the alveolar space, and growth of fibroblasts were observed. In the MCP-1 siRNA-encapsulated nanosphere-administered group (hereinafter referred to as siRNA-administered group), bleomycin pneumonitis was significantly different in histopathological grade after 14 days compared to the control siRNA-administered group (hereinafter referred to as control group). (The number of mice of grade 3, grade 2, grade 1, and grade 0 was significant as 0, 2, 13, 4 in the siRNA administration group and 7, 12, 0, 0 in the control group, respectively. A difference (p <0.01) was observed, and when MCP-1 siRNA and control siRNA alone were administered, abnormalities were observed even after examining the mouse lung tissue after 1, 3, 7, 14, 21, and 28 days There wasn't.

  In addition, in the siRNA administration group, collagen deposition and hydroxyproline level (MCP-1 siRNA vs control siRNA; 143 ± 6 vs 310 ± 25 mg / left lung, p <0.01), type 1 collagen, type in sirius red staining of lung tissue 3 Collagen and fibronectin expression was significantly suppressed compared to the control group.

(Lung tissue DNA damage and apoptosis)
DNA damage and apoptosis were evaluated by TUNEL method using DeadEnd colorimetric apoptosis detection system (Promega). TUNEL positive cells were counted and evaluated in 20 fields randomly selected at 200-fold magnification.

  TUNEL positive cells correlate with lung injury and fibrosis in this model. Although the cell type could not be clearly identified, in some bronchioles, alveolar epithelial cells and inflammatory cells, DNA damage and apoptosis at the inflammatory site were expressed on the 14th day of bleomycin administration. In the siRNA-administered group, a clear decrease in TUNEL positive signal (MCP-1 siRNA vs. control siRNA; 1.2 ± 0.3 vs. 6.3 ± 0.5 / field under) on the 14th day of bleomycin administration compared to the control group x200 magnification, p <0.01).

(Protein concentration in BALF, MCP-1, TNF-α, MIP-2 measurement)
The mice that had been killed were subjected to tracheostomy, and a tracheal tube was inserted and washed twice with 1 mL of sterile physiological saline. The collected solution was filtered through a monolayer of gauze, the mucus was filtered, the number of cells was counted using a hemocytometer, and the fraction of 200 cells was counted by Diff-Quick (Baxter Diagnostics) staining. The protein concentration was measured by Bio-Rad protein assay.

  MCP-1 and MIP-2 in BALF were measured with an ELISA kit (R & D systems), and TNF-α was measured with an ELISA kit (BioSouce International). For this measurement, BALF was centrifuged and the supernatant was stored at -80 ° C until use. The minimum measurement limit concentrations were 1.5 pg / mL for MIP-2 and 5 pg / mL for MCP-1 and TNF-α.

  In the siRNA administration group, the MCP-1 concentration, protein concentration, and total cell number in BALF were significantly reduced as compared with the control group. In the cell fraction, the percentage of macrophages on the 14th day after bleomycin administration was significantly increased in the siRNA administration group as compared to the control group, while the lymphocytes were significantly decreased. Neutrophils were significantly reduced on the 14th day.

  In addition, the concentrations of cytokines in BALF were significantly increased in TNF-α and MIP-2 on the 14th day after administration of bleomycin as compared with the physiological saline administration group. Furthermore, the levels of these cytokines were significantly suppressed in the siRNA administration group compared to the control group.

  From the above results, the effectiveness against pulmonary fibrosis was confirmed by intranasal inhalation administration of siRNA-encapsulated nanospheres against MCP-1 in a mouse bleomycin pneumonitis model. This method is an organ-specific and injured site-specific treatment method, and the influence on the whole body is considered to be minimal, and the possibility of clinical application is considered high.

  As described above, the pharmaceutical preparation of the present invention is an excellent preparation for transpulmonary administration with improved gene expression efficiency and high directivity for target molecules, with improved nanoparticle uptake into cells. . Therefore, although not shown here, the suppressive effect on lung cancer and bronchial asthma can be easily estimated by administration of a preparation for transpulmonary administration in which a nucleic acid compound that has been confirmed to have efficacy against lung cancer and bronchial asthma is encapsulated in nanoparticles. be able to.

  The pharmaceutical formulation for transpulmonary administration of the present invention is effective in allowing a nucleic acid compound to penetrate into a cell through a mucous membrane or blood vessel in the lung and into a cell, thereby effectively applying a nucleic acid compound to a lesion such as a bronchiole or an alveolar region. Excellent pulmonary administration that not only increases the therapeutic effect of refractory lung disease, but also reduces side effects on the human body because it can reach the concentration and gradually release the nucleic acid compound from the nanoparticles Preparation.

  In addition, by carrying the nucleic acid compound on the surface of the nanoparticle, the total amount of the nucleic acid compound including the inside and the surface of the nanoparticle can be increased. Realize quantity. In addition, by acting a nucleic acid compound that is released from the inside of the nanoparticle in a sustained manner and a nucleic acid compound that dissolves from the surface of the nanoparticle, it becomes a pharmaceutical preparation for transpulmonary administration that combines immediate effect and sustainability. .

  In addition, biomaterials that have low irritation and toxicity to the living body and are decomposed and metabolized after administration are used as the material for forming the nanoparticles, ensuring safety to the human body and the effectiveness of the nucleic acid compound. The nucleic acid compound can be stored for a long period of time while being retained, and further, the nucleic acid compound can be gradually released by the decomposition of the biocompatible polymer. In particular, it is suitable when PGA, PLA, PLGA, or PAL is used as the biocompatible polymer.

  Further, as a nucleic acid compound, a soluble type II TGF receptor plasmid having TGF-beta as a target molecule, a mutant MCP-1 plasmid having MCP-1 as a target molecule, siRNA for CCR2, MCP-1, Egr-1, or these Encapsulating the expressed plasmid provides a therapeutic agent that exhibits excellent effects on pulmonary damage and pulmonary fibrosis, which are difficult to treat lung diseases.

  In addition, if a p53 plasmid having a p53 protein as a target molecule or a soluble VEGF receptor plasmid having VEGF as a target molecule is encapsulated as a nucleic acid compound, a treatment having an excellent effect on lung cancer which is a refractory lung disease Become a medicine. The use of the p53 plasmid is particularly effective for the treatment of primary alveolar epithelial cancer, and the use of the soluble VEGF receptor plasmid is particularly effective for the treatment of metastatic lung cancer.

  In addition, siRNA against IL-13, SOCS3, or a plasmid expressing these, or a dominant negative SOCS3 plasmid targeting SOCS3 as a nucleic acid compound is excellent for bronchial asthma, which is a refractory lung disease. It becomes a therapeutic drug that shows an effect.

Claims (13)

  A pharmaceutical preparation for transpulmonary administration comprising a nucleic acid compound-encapsulated nanoparticle formed by encapsulating a nucleic acid compound in a biocompatible nanoparticle.   The pharmaceutical preparation for transpulmonary administration according to claim 1, wherein a nucleic acid compound is further attached to the surface of the biocompatible nanoparticle.   The biocompatible polymer forming the biocompatible nanoparticles is any one of polylactic acid, polyglycolic acid, lactic acid / glycolic acid copolymer, or lactic acid / aspartic acid copolymer. The pharmaceutical preparation for transpulmonary administration according to claim 1 or 2.   4. The therapeutic agent for lung injury / pulmonary fibrosis using a soluble type II TGF receptor plasmid having TGF-beta as a target molecule as the nucleic acid compound, according to any one of claims 1 to 3. Pharmaceutical formulation for transpulmonary administration.   4. The therapeutic agent for lung injury / pulmonary fibrosis using a mutant MCP-1 plasmid having MCP-1 as a target molecule as the nucleic acid compound, according to any one of claims 1 to 3. The pharmaceutical preparation for transpulmonary administration described.   The transpulmonary according to any one of claims 1 to 3, wherein the nucleic acid compound is a therapeutic agent for lung injury / pulmonary fibrosis using siRNA against CCR2 or a plasmid expressing the siRNA. Pharmaceutical formulation for administration.   4. The therapeutic agent for lung injury / pulmonary fibrosis using an siRNA against MCP-1 or a plasmid expressing the siRNA as the nucleic acid compound, according to any one of claims 1 to 3. Pharmaceutical formulation for pulmonary administration.   The pulmonary injury / pulmonary fibrosis therapeutic drug using a siRNA against Egr-1 or a plasmid expressing the siRNA as the nucleic acid compound, according to any one of claims 1 to 3. Pharmaceutical formulation for pulmonary administration.   The pharmaceutical preparation for transpulmonary administration according to any one of claims 1 to 3, wherein the nucleic acid compound is a therapeutic agent for lung cancer using a p53 plasmid having a p53 protein as a target molecule.   The pharmaceutical preparation for transpulmonary administration according to any one of claims 1 to 3, wherein the nucleic acid compound is a lung cancer therapeutic drug using a soluble VEGF receptor plasmid having VEGF as a target molecule. .   The transpulmonary administration according to any one of claims 1 to 3, wherein the nucleic acid compound is a therapeutic agent for bronchial asthma using siRNA against IL-13 or a plasmid expressing the siRNA. Pharmaceutical formulation.   The pharmaceutical preparation for transpulmonary administration according to any one of claims 1 to 3, wherein the nucleic acid compound is a therapeutic agent for bronchial asthma using a dominant negative SOCS3 plasmid having SOCS3 as a target molecule. .   The pharmaceutical preparation for transpulmonary administration according to any one of claims 1 to 3, wherein the nucleic acid compound is a therapeutic agent for bronchial asthma using siRNA against SOCS3 or a plasmid expressing the siRNA. .