JP2011211996A - Liposome having tumor specificity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide such a drug delivery system (DDS) medicine as specifically and efficiently adsorb-taken in by tumor cells.SOLUTION: There are provided a vector for transferring a drug into tumor cells, comprising: a liposome containing Sindbis virus or a part thereof as a constituent; a cancer-treating composition containing the drug-transferring vector sealing a drug exhibiting an antitumor effect; a tumor cell-labeling composition containing the drug-transferring vector sealing a labeling substance; a method for treating a cancer by administering the drug-transferring vector encapsulating the drug exhibiting the anti-tumor effect; and a tumor cell-labeling method for administering the drug-transferring vector encapsulating the labeling substance.

Description

  The present invention relates to a vector for introducing a drug into tumor cells. More specifically, the present invention relates to a vector for introducing a drug into tumor cells, which comprises a liposome containing Sindbis virus or a part thereof as a constituent component. Furthermore, the present invention relates to the above-mentioned vector encapsulating a drug. The present invention also relates to a composition for cancer treatment containing the above-mentioned vector encapsulating a drug exhibiting an antitumor effect, and a composition for labeling tumor cells containing the above-mentioned vector encapsulated with a labeling substance. Furthermore, the present invention provides a cancer treatment method comprising administering the above-mentioned vector encapsulating a drug exhibiting an antitumor effect, and a tumor cell comprising administering the above-mentioned vector encapsulating a labeling substance. The present invention relates to a labeling method.

  Development of technology related to drug administration that delivers drugs effectively and intensively to targeted affected areas, that is, organs, tissues, cells, pathogens, etc., so-called drug delivery systems (hereinafter sometimes referred to as DDS) has been actively developed. Has been done. DDS controls the pharmacokinetics in the body by devising the administration method and administration form of a drug, thereby enabling selective delivery of the drug to a target site. As a result, it is possible to achieve an optimal therapeutic effect by the drug and further reduction of side effects by the drug. Various DDS preparations have been developed, and among them, liposomes are attracting attention as useful (DDS) preparations.

  Liposomes are closed vesicles composed of lipid bilayers based on phospholipids that constitute the cell membranes of living organisms, and can encapsulate water-soluble or fat-soluble drugs. Therefore, liposomes are useful as a drug transporter in DDS because they have high biocompatibility and can be transported while protecting the encapsulated drug from degradation enzymes and the like in the body. On the other hand, liposomes are easily trapped by phagocytic cells of reticuloendothelial tissue (RES) centering on the liver and spleen, and as a result, they are liable to accumulate in the liver and spleen and to be discharged from the living body.

In recent years, functional liposomes having enhanced usefulness as liposome DDS preparations have been developed. For example, liposomes modified with a ligand or an antibody have been developed in order to enhance a target tissue or target targeting function (target arrival specificity). In addition, in order to improve the general properties of liposomes, such as leakage of inclusions, aggregation, and capture by RES, the surface of the liposomes may be abbreviated as water-soluble polymer polyethylene glycol (hereinafter abbreviated as PEG). Liposomes have been developed that are not easily captured by RES by being modified with a hydrophilic polymer, for example, and have the property of circulating and staying in the blood for a long time (Non-patent Document 1). PEG-modified liposomes are called stealth liposomes. PEG-modified liposomes encapsulating doxorubicin, is marketed under the trade name DOXIL ^{registered trademark,} which is used in the treatment of malignant tumors. In addition to encapsulating chemotherapeutic agents, PEG-modified liposomes are being applied for photochemotherapy with porphyrin derivatives and neutron capture therapy with boron compounds.

  On the other hand, in order to increase the efficiency of liposome introduction into the cytoplasm, an intracellular substance introduction vector produced by fusing a virus having membrane fusion ability and a liposome has been developed (Patent Document 1). An intracellular substance introduction vector prepared by fusing a virus and a liposome has a liposome-derived inner space inside, and has the same protein as the virus envelope outside the lipid bilayer. Therefore, such a liposome can introduce a drug encapsulated in its inner space into cells with the same high efficiency as a virus. As such vectors, for example, vectors obtained by fusing a liposome with Sendai virus (Hemagglutinating Virus of Japan) or vesicular stomatitis virus (Vesicular Stomatitis Vius) have been reported (Non-patent Document 2 and Patent Document 1). Vesicular stomatitis virus, or a vector in which a portion thereof is fused with a liposome, does not show erythrocyte hemolysis even under physiological conditions, and should exhibit good stability and an excellent rate of introduction of intracellular substances so that it can be used safely. Has been reported.

JP-A-11-187873.

Klibanov A.L. et al., “FEBS Letters”, 1990, Vol. 268, p.235-237. Nakagawa T. et al., “Drug Delivery System”, 1996, Volume 11, pages 411-417.

  Currently, DDS including DDS effective for cancer treatment has been actively developed, but a system that can be specifically adsorbed and taken up by tumor cells has not yet been developed.

  Liposomes that are considered useful as DDS preparations also have low tumor targeting. Therefore, tumor targeting is enhanced by EPR (Enhanced Permeability Retention) effect, modification of antibodies against tumor-associated antigens, and induction of tumor immunity by inclusion of tumor-associated antigens. EPR effects and induction of tumor immunity are secondary effects, and since effective tumor-related antigens are in the research stage, tumor targeting is still low, and improvements are required. In addition, the above-mentioned liposomes are observed to have a high drug introduction effect even in non-tumor cells when clinical application is examined, and may cause serious side effects. Therefore, it is necessary to improve tumor targeting. .

  An object of the present invention is to provide a DDS preparation that is specifically and efficiently adsorbed and taken up by tumor cells.

  In order to achieve the above-mentioned object, the inventors of the present invention focused on an oncolytic virus and conducted extensive research. The Reoviridae reovirus and Togaviridae alphavirus Sindbis virus, which the inventors have studied, specifically infect (adsorb) tumors and proliferate tumor-specifically. In particular, Sindbis virus infects numerous types of tumor cells, such as malignant melanoma, malignant lymphoma, squamous cell carcinoma, and adenocarcinoma. It is known that infection (adsorption) of these viruses to tumor cells is carried out via receptors on the tumor cell membrane, and several types of receptors have been identified. Therefore, we thought that the development of cancer-specific DDS would be possible by using ligands that bind to these receptors.

  The liposome fused with the Sindbis virus is adsorbed and ingested specifically and efficiently by tumor cells, and the liposome encapsulating a drug exhibiting an antitumor effect is a normal liposome encapsulating the drug As a result, the present invention has been achieved.

That is, the present invention relates to the following.
1. A vector for introducing a drug into a tumor cell, comprising a liposome containing Sindbis virus or a part thereof as a constituent component.
2. A vector for introducing a drug into tumor cells, comprising liposomes substantially containing Sindbis virus E1 protein and E2 protein.
3. A vector for introducing a drug into a tumor cell, wherein Sindbis virus is subjected to sucrose density gradient centrifugation to obtain E1 protein and E2 protein fractions, which are used as liposomes as constituents.
4. The vector for introducing a drug into tumor cells according to any one of 1 to 3 above, wherein the liposome is a liposome modified with polyethylene glycol.
5. The vector for introducing a drug into tumor cells according to 4 above, wherein the polyethylene glycol is polyethylene glycol having an average molecular weight of 1,000 to 5,000.
6. The vector for introducing a drug into the tumor cell according to any one of 1 to 5 above, wherein the drug is encapsulated.
7. The vector for introducing a drug into the tumor cell according to the above 6, wherein the drug is a drug exhibiting an antitumor effect.
8. The vector for introducing a drug into a tumor cell according to the above 7, wherein the drug exhibiting an antitumor effect is cisplatin.
9. The vector for introducing a drug into the tumor cell according to the above 6, wherein the drug is a labeling substance.
10. Use of the vector for introducing a drug into a tumor cell according to any one of 1 to 5 in the production of a composition for treating cancer.
11. A composition for treating cancer comprising the vector for introducing a drug into tumor cells as described in 7. or 8. above as an active ingredient.
12. A method for treating cancer, comprising administering the drug introduction vector according to 7. or 8. above.
13. Use of the vector for introducing a drug into tumor cells according to any one of 1 to 5 in the production of a composition for labeling tumor cells.
14. A tumor cell labeling composition comprising the drug introduction vector of 9.
15. A method for labeling tumor cells, comprising administering the vector for drug introduction described in 9. above.

  According to the present invention, a vector for introducing a drug into a tumor cell, which comprises a liposome containing Sindbis virus or a part thereof as a constituent component, and a cancer therapeutic composition and a tumor cell labeling composition containing the vector Can provide things.

  Since the drug introduction vector according to the present invention specifically adsorbs to tumor cells, the encapsulated drug can be transported specifically to tumor cells. Therefore, the side effect on the normal cells of the encapsulated drug is reduced.

  In addition, Sindbis virus infects humans through mosquitoes, but it is a low-pathogenic virus that only causes slight fever and can be administered systemically, so the formulation using that component has few side effects, and It is considered a safe and useful drug that can be administered systemically.

  Thus, the present invention provides a DDS preparation that is specifically and efficiently adsorbed and ingested by tumor cells and exhibits high tumor cell toxicity while reducing side effects, and is useful in the field of cancer treatment. is there.

It is a figure explaining that tumor-specific distribution of the virus was observed in tumor-bearing nude mice intravenously injected with green fluorescent protein (GFP) -expressing Sindbis virus (in the figure, GFP-SIN (+)) . High GFP fluorescence was observed in the tumor (Tumor), but the GFP fluorescence observed in the brain (Brain), heart (Heart), lung (Lung), liver (Liver), kidney (Kidney), testis (Intestine) It was similar to that of tumor-bearing nude mice intravenously injected with GFP alone. (Example 1) Compared with Sindbis virus (SIN) hybrid liposomes encapsulating cisplatin, SIN hybrid liposomes encapsulating normal saline (NS) and non-SIN hybrid liposomes encapsulating cisplatin or NS, tumor cell lines (HSC-2 , HSC-3, RERF-LC-AI) is a diagram for explaining the high cytotoxicity. On the other hand, there was no significant difference in the cytotoxic effect on normal fibroblasts (Fibroblast). In the figure, the SIN hybrid liposome encapsulating cisplatin is indicated as SIN liposome cisplatin. In addition, SIN hybrid liposome encapsulating NS, cisplatin or non-SIN hybrid liposome encapsulating NS are denoted as SIN liposome NS, liposome cisplatin, and liposome NS, respectively. (Example 1) It is a figure explaining that rapid tumor regression was observed in the mouse | mouth which inject | poured the Sindbis virus (SIN) hybrid liposome which enclosed cisplatin. On the other hand, a large tumor (1500 to 2000 mm ³ ) was formed in mice injected with SIN hybrid liposome encapsulating normal saline (NS) or SIN hybrid liposome encapsulating cisplatin or NS. The horizontal axis represents the number of days after liposome injection, and the vertical axis represents the tumor volume. In the figure, SIN hybrid liposome encapsulating cisplatin is indicated as SIN phospho cisplatin (●). In addition, SIN hybrid liposome encapsulating NS, cisplatin, or non-SIN hybrid liposome encapsulating NS are denoted as SIN lipo NS, liposome cisplatin, and liposome NS, respectively (□, ■, Δ). (Example 1)

  The present invention relates to a vector for introducing a drug into tumor cells, which comprises a liposome containing Sindbis virus or a part thereof as a constituent component. This drug introduction vector has high tumor cell specificity, and is characterized in that the drug encapsulated in the vector can be transported specifically and efficiently to tumor cells, and further, side effects of the drug on normal cells are reduced. It also has the advantage of.

  The vector for introducing a drug into a tumor cell means a carrier for transporting the drug into the tumor cell.

  Sindbis virus is an icosahedral-like single-stranded (+) RNA virus with an envelope. The infection is transmitted by mosquitoes and infects vertebrates, but in humans it is a low pathogenic virus that only causes mild cold symptoms.

  Sindbis virus, known as Oncolytic virus, infects numerous types of tumor cells, such as malignant melanoma, malignant lymphoma, squamous cell carcinoma, and adenocarcinoma, and is specific for tumor cells. Multiply. Also, the possibility of treatment of cervical cancer cells and ovarian cancer cells by Sindbis virus has been reported (Unno Y. et al., “Cancer Therapy”, 2005, Volume 11, No. 12, p.4553-4560). Oncolytic virus refers to a virus that has the ability to bind and adsorb specifically to tumor cells, enter into tumor cells, replicate within tumor cells, and kill only tumor cells. Tumor cell-specific means that it acts selectively on tumor cells compared to normal cells and has little effect on normal cells.

  It is known that envelope proteins present in the envelope are generally involved in infection of target cells with enveloped viruses. The envelope is a lipid bilayer structure possessed by some viruses, is located on the outermost side of the virus particle, and covers the capsid containing the virus genome, which is the basic structure of the virus. The envelope is obtained by budding while the virus grows inside the infected cell and goes out of the cell while covering a biological membrane such as a cell membrane or a nuclear membrane. For this reason, it is basically derived from the lipid bilayer membrane of the host cell, but in addition to this, the virus's unique protein encoded by the viral gene is expressed on the biological membrane and then combined with the biological membrane. Incorporated into the virus particle and expressed on the surface of the virus particle as an envelope protein. Envelope proteins are glycoproteins, also called spikes, that protrude from the envelope. Envelope proteins are known to have various functions, such as the ability to bind to the receptors on the target cell side during the adsorption and entry of the virus into the target cell, and the function to avoid the host defense function of the host. It plays an important role in virus infection. In many viruses with an envelope derived from the cell membrane of the target cell, the envelope protein binds to the receptor on the target cell side, and then the viral envelope and the cell membrane fuse together to form the viral genome contained in the envelope. The containing capsid is delivered into the target cell.

  Sindbis virus is known to have envelope proteins called E1 protein and E2 protein, and these E1 protein and E2 protein are considered to be involved in specific adsorption to target cells.

  Therefore, as a vector for introducing a drug into a tumor cell comprising a liposome containing a part of Sindbis virus as a constituent, preferably a drug into a tumor cell comprising a liposome substantially containing a Sindbis virus envelope protein An example of the vector for introduction into a tumor cell is a vector for introduction, more preferably a liposome substantially comprising S1 bis virus E1 protein and E2 protein. Here, “substantially containing an envelope protein” means that other proteins derived from Sindbis virus, such as a core protein, may be included as long as the envelope protein is included. In addition, “substantially contains E1 protein and E2 protein” includes other proteins derived from Sindbis virus, such as envelope proteins and core proteins other than E1 protein and E2 protein, as long as they contain E1 protein and E2 protein. It means that it may be.

  Liposomes containing Sindbis virus or a part thereof as a constituent component may be referred to herein as Sindbis virus hybrid liposomes.

  The Sindbis virus hybrid liposome can be produced by fusing Sindbis virus or a part thereof with the liposome by a method known per se. Fusion of Sindbis virus or a part thereof and a liposome means that the lipid of Sindbis virus or a part thereof and the liposome membrane constitutes the same membrane, and the structure of the protein contained in Sindbis virus or a part thereof The treatment is performed so that the function is shown in the liposome membrane as well as when it is present in the virus particle.

  In its production, the Sindbis virus is inactivated. The preparation of Sindbis virus can be carried out by infecting and proliferating infectious cells known per se, and the inactivation thereof can be carried out by a method known per se, for example, ultraviolet irradiation or surfactant treatment.

  Specifically, Sindbis virus hybrid liposomes can be produced by mixing inactivated Sindbis virus and liposomes and incubating at 37 ° C. with vigorous shaking. The incubation time is generally 6 hours to 1 hour, preferably 3 to 1 hour, more preferably 2 hours. In addition, for liposomes that substantially contain Sindbis virus E1 and E2 proteins, Sindbis virus is subjected to sucrose density gradient centrifugation to obtain E1 protein and E2 protein fractions, which serve as liposomes as constituents. Can be manufactured.

  Production of liposomes can be carried out using a liposome preparation method known per se. Liposomes are classified according to their structure into multilamellar vesicles (MLV) and unilamellar vesicles. Single-layer vesicles can also have a diameter of 20-100 nm, small unilamellar vesicles (SUV), 100-1000 nm, large unilamellar vesicles (LUV), 5000-100000 nm Divided into giant unilamellar vesicles (GUVs). MLV can be prepared by vortexing. As a method for preparing SUV, an ultrasonic treatment method, an ethanol injection method, a surfactant removal method, an extrusion method and the like are known. Known methods for adjusting LUV include a reverse phase evaporation method, an ethanol injection method, a freeze-thaw method, an extrusion method, and the like. In addition, as a method for adjusting GUV, a stationary hydration method, an electrical formation method, an organic solvent dialysis method, a freeze-thawed salt dialysis method, and the like are known. MLVs are generally stable and have good retention efficiency for high-molecular substances, but are low in efficiency in introducing encapsulated drugs into cells because of their non-uniform size and multiple layers. However, MLV can be changed to SUV and further LUV by an annealing method in which incubation is performed after sonication. SUVs are uniform in size, but are difficult to retain polymeric substances and are relatively unstable. LUV has good retention efficiency for proteins, DNA, etc., but its size is not uniform. As described above, a number of methods are known for preparing liposomes. Appropriate liposomes may be selected according to the drug to be encapsulated and use, and an appropriate preparation method may be adopted for the liposomes.

  Liposomes may be multilamellar vesicles or unilamellar vesicles, but are preferably unilamellar vesicles. Although the size is not particularly limited, it is appropriate that the diameter is 10 μm to 0.1 μm, preferably 3 μm to 0.1 μm, and more preferably 0.5 μm to 0.1 μm.

  Generally, phospholipids, cholesterols, nitrogen lipids and the like are used as the constituent components of the liposome, and phospholipids are particularly preferably used.

  Examples of lipids include phospholipids, glycolipids, sterols, and saturated or unsaturated fatty acids. Specific examples of phospholipids include phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidic acid, cardiolipin, sphingomyelin, egg yolk lecithin, soy lecithin, lysolecithin and other natural phospholipids, or the like by a conventional method. Examples include hydrogenated ones. Specific examples of glycolipids include glyceroglycolipids such as sulfoxyribosyl glyceride, diglycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride, and glycosyl diglyceride, and sphingoglycolipids such as galactosyl cerebroside, lactosyl cerebroside, and gangliosi. Can be mentioned. Specific examples of sterols include sterols derived from animals such as cholesterol, cholesterol succinic acid, cholestanol, lanosterol, dihydrolanosterol, desmosterol, and dihydrocholesterol, and from plants such as stigmasterol, sitosterol, campesterol, and brassicasterol And sterols derived from microorganisms such as timosterol and ergosterol. Specific examples of the saturated or unsaturated fatty acid include saturated or unsaturated fatty acids having 12 to 20 carbon atoms such as palmitic acid, oleic acid, stearic acid, arachidonic acid, and myristic acid. Can be mentioned. These lipids can be used alone, but can also be used in combination of two or more.

  Furthermore, a membrane stabilizer can be contained as a constituent component of the liposome in order to physically or chemically stabilize the lipid membrane or to regulate the fluidity of the lipid membrane. Examples of membrane stabilizers include sterol, glycerin or fatty acid esters thereof. Examples of the sterol include the same specific examples as described above, and examples of the glycerin fatty acid ester include triolein and trioctanoin.

  In addition, an antioxidant can be included to prevent oxidation of the lipid membrane. Examples of the antioxidant include tocopherol, propyl gallate, ascorbyl palmitate, butylated hydroxytoluene and the like.

  Furthermore, a charged substance can be contained in order to impart positive charge or negative charge to the lipid membrane. Examples of the charged substance that imparts a positive charge include saturated or unsaturated aliphatic amines such as stearylamine and oleylamine, and saturated or unsaturated cationic synthetic lipids such as dioleoyltrimethylammonium propane. Examples of the charged substance that imparts a negative charge include dicetyl phosphate, cholesteryl hemisuccinate, phosphatidylserine, phosphatidylinositol, and phosphatidic acid.

  As a more specific liposome, there can be exemplified a liposome produced by mixing egg yolk phosphatidylcholine, cholesterol, and phosphatidylserine at a molar ratio of 4: 5: 1 and dehydrating and rehydrating (see Example 1).

  Liposomes can be modified on the outer surface with a hydrophilic polymer. By such modification, it is possible to avoid capture of liposomes into RES and to increase the circulation residence time of liposomes in the blood, so that drug transport into tumor cells can be performed more efficiently. . Examples of the hydrophilic polymer include polyethylene glycol, polymethylethylene glycol, polyhydroxypropylene glycol, polypropylene glycol, polymethylpropylene glycol, and polyhydroxypropylene oxide. Particularly preferred is polyethylene glycol. In the formation of Sindbis virus hybrid liposomes, it is preferable to select the molecular weight of polyethylene glycol used so that the viral protein fused to the liposome membrane is not buried by polyethylene glycol. The average molecular weight of polyethylene glycol is preferably 1,000 to 5,000, more preferably 1,000 to 3,000, and still more preferably 2000. An example of such polyethylene glycol is PEG-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [Methoxy (Polyethyleneglycol) -2000]).

  Encapsulation of the drug in the Sindbis virus hybrid liposome is usually carried out in the liposome production process before fusion of the Sindbis virus or a part thereof with the liposome. That is, it is carried out by encapsulating a desired drug in a liposome and then fusing the liposome encapsulating the drug with Sindbis virus or a part thereof. Encapsulation of the drug in the liposome can be performed by a method known per se. For example, a drug can be encapsulated in a liposome by preparing a thin film from a raw material for liposome production, for example, a mixture of lipids, and suspending the thin film in a solution containing a desired drug. After mixing, ultrasonic treatment or the like may be performed as necessary. More specifically, for example, after adding a solution in which a desired drug is dissolved in a solvent to a solution in which phospholipid is dissolved in an organic solvent such as chloroform, the solvent is distilled off, and a phosphate buffer is added thereto. Additionally, after shaking, sonication and centrifugation, the supernatant can be filtered and collected.

  The drug to be encapsulated is not particularly limited, and specific examples thereof include drugs and nucleic acids that exhibit an antitumor effect. The antitumor effect means any of an action of suppressing and attenuating tumor growth, an action of suppressing and attenuating tumor invasion, and an action of killing a tumor. The drug showing antitumor effect may be any drug used for the purpose of cancer treatment, such as platinum preparations such as cisplatin and oxaliplatin, alkylating agents such as cyclophosphamide and dacarbazine, fluorouracil and tegafur, etc. Anti-metabolite antagonists, plant alkaloids such as irinotecan and paclitaxel, anticancer antibiotics such as actinomycin D and doxorubicin, hormonal agents such as anastrozole and tamoxifen, and biological response modifiers such as interferon it can. Nucleic acids include nucleic acids that encode the desired protein, nucleic acids that can suppress the expression of the desired protein, such as antisense nucleic acids, short double-stranded RNAs such as siRNA and shRNA, suicide genes, apoptosis-inducing genes, etc. Can be mentioned.

  Other preferable examples of encapsulated drugs include photosensitizers such as porphyrin derivatives used in cancer photochemotherapy. Photochemotherapy has the advantage of fewer side effects because the drug is activated only at the site irradiated with laser light after the photosensitizer is administered to the living body. Although the photosensitizer has a problem that tumor accumulation is low, by encapsulating it in the drug introduction vector of the present application, the tumor accumulation increases and a high therapeutic effect can be obtained.

  Other examples of the drug to be encapsulated include a labeling substance for labeling tumor cells. Examples of the labeling substance include compounds containing nuclides that emit γ rays such as technetium 99m. Such a compound is called an imaging agent and can be detected by accumulating it in a tumor and detecting γ-rays. By encapsulating in the drug-drug-introducing vector of the present application and using it, the imaging agent can be accumulated in a tumor-specific manner, so that the tumor can be detected with high accuracy and there are few side effects on normal tissues. . Furthermore, it is possible to detect metastatic cancer. Other examples of the labeling substance include fluorescent substances.

  The drug to be encapsulated may be one type, and two or more types can be encapsulated in combination. The amount of the drug to be encapsulated is not particularly limited, and can be appropriately changed depending on its effectiveness.

  The drug drug introduction vector according to the present invention has high tumor cell specificity, and has a feature that the drug encapsulated in the vector can be transported specifically and efficiently to tumor cells. Therefore, vector for drug introduction is esophageal cancer, lung cancer, breast cancer, stomach cancer, colorectal cancer, pancreatic cancer, liver cancer, kidney cancer, bladder cancer, solid cancer such as uterine cancer, floating cancer such as ascites cancer and pleural effusion cancer It can be applied to a wide range of cancers such as hematopoietic cancers such as leukemia and lymphoma.

  In the present invention, it is possible to provide a cancer therapeutic composition containing as an active ingredient the above-mentioned drug introduction vector encapsulating a cancer therapeutic drug such as a drug or nucleic acid exhibiting an antitumor effect. The composition for cancer treatment contains a pharmaceutically acceptable carrier in addition to the drug introduction vector. Examples of the pharmaceutically acceptable carrier include pharmaceutically acceptable carriers such as water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble Examples include dextran, sodium carboxymethyl starch, pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, and lactose. . These may be used alone or in combination of two or more according to the dosage form of the drug. In addition, stabilizers, bactericides, buffers, isotonic agents, pH adjusters, and the like can be used as appropriate. Examples of the stabilizer include human serum albumin, ordinary L-amino acids, saccharides, and cellulose derivatives. The L-amino acid is not particularly limited, and may be any of glycine, cysteine, glutamic acid and the like. The saccharide is not particularly limited, for example, monosaccharides such as glucose, mannose, galactose, and fructose, sugar alcohols such as mannitol, inositol, and xylitol, disaccharides such as sucrose, maltose, and lactose, dextran, hydroxypropyl starch, chondroitin sulfate, Any of polysaccharides such as hyaluronic acid, and derivatives thereof may be used. The cellulose derivative is not particularly limited, and may be any of methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose sodium and the like. Buffering agents include boric acid, phosphoric acid, acetic acid, citric acid, ε-aminocaproic acid, glutamic acid and / or salts thereof (for example, alkali metal salts such as sodium salt, potassium salt, calcium salt, magnesium salt, etc. An alkaline earth metal salt). Examples of the isotonic agent include sodium chloride, potassium chloride, saccharides and glycerin.

  In the present invention, it is possible to provide a composition for labeling tumor cells, which contains, as an active ingredient, the above-mentioned drug introduction vector encapsulating a labeling substance for labeling tumor cells. The composition for labeling tumor cells can contain a pharmaceutically acceptable carrier in addition to the drug introduction vector.

  The composition for cancer treatment and the composition for labeling tumor cells according to the present invention can be administered parenterally by bolus injection or continuous infusion. The administration route can be administered to veins, arteries, etc., or directly to tumor tissue. Oncolytic virus is known to adsorb and enter tumor-specifically. For example, Sindbis virus is a low-pathogenic virus that only causes slight fever by infection, and systemic administration is also possible. Therefore, a preparation using the component is considered to be a safe and useful drug with few side effects and capable of systemic administration.

  The dose range of the composition according to the present invention is not particularly limited, and the effectiveness of the drug encapsulated in the drug introduction vector, the administration form, the administration route, the nature of the subject (weight, age, medical condition, and the presence or absence of other medicines) Etc.) and the judgment of the doctor in charge, etc.

  In the present invention, the drug introduction vector encapsulating a cancer treatment drug such as a drug or nucleic acid exhibiting an antitumor effect, or a pharmaceutical composition containing the drug introduction vector is administered to a subject having a cancer disease. The present invention provides a method for treating cancer.

  In the present invention, the above-mentioned drug introduction vector encapsulating a labeling substance for labeling tumor cells, and a composition containing the drug introduction vector are administered to a subject. Provide a method. Since the labeling substance accumulates specifically for tumor cells by this tumor cell labeling method, tumor cell detection can be carried out by measuring the accumulated labeling substance. Therefore, the present tumor cell labeling method can be used for a tumor cell detection method, and further can be used for diagnosis of cancer diseases and diagnosis of cancer metastasis.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated more concretely, this invention is not limited at all by the Example shown below.

  Preparation of hybrid liposomes by fusion of Sindbis virus and liposomes, adsorption specificity of the hybrid liposomes to tumor cells and ability to transport encapsulated drugs, and cytotoxicity of the hybrid liposomes encapsulating anticancer agents in vitro and in vivo The sex was examined. Hereinafter, a hybrid liposome in which a Sindbis virus and a liposome are fused may be referred to as a SIM hybrid liposome, and a liposome that is not fused with a Sindbis virus may be referred to as a non-SIM hybrid liposome.

[Materials and methods]

Cisplatin (cis-diammineplatinum (II) dichloride, Pt (NH ₃ ) ₂ Cl ₂ ), cholesterol (hereinafter sometimes abbreviated as CH), phosphatidylserine (hereinafter sometimes abbreviated as PS), and FITC-4KD (Fluoresceinisothiocyanate-4KD) was purchased from Sigma Chemical. Normal saline (hereinafter sometimes abbreviated as NS) was purchased from Otsuka Pharmaceutical Factory. Egg yolk phosphatidylcholine (hereinafter sometimes abbreviated as EPC. 99%) and polyethylene glycol derivative of phosphatidylethanolamine (PE) (PEG-DSPE2K, hereinafter abbreviated as PEP): The average molecular weight of PEG is 2000 Is obtained from Japanese fats and oils. Human lung cancer cell line RERF-LC-AI, oral cancer cell lines HSC-2 and HSC-3, Vero cells, and normal fibroblasts contain 10% fetal calf serum (FCS) and 50 units / ml penicillin and streptomycin. Cultured in Dulbecco's modified Eagle medium (DMEM). Primary cultured human keratinocytes (Normal Human Keratinocyte, sometimes abbreviated as NHK) were cultured in normal human epidermal keratinocyte medium KGM. N- (Lisaaminerhodamine B sulfonyl) -phosphatidylethanolamine (hereinafter sometimes abbreviated as N-Rh-PE), NED-PE, and Oregon Green were purchased from Invitrogen.

Wild-type Sindbis virus AR339 used in the present invention was provided by National Institute of Infection (Japan). The virus was propagated in primary chicken embryo fibroblasts and then passaged several times in Vero cells. This virus was used as a research stock. The GFP-expressing recombinant Sindbis virus TR339-GFP / 2A (hereinafter referred to as GFP-SIN) was derived from the cDNA clone pTR339-GFP / 2A. The cDNA clone was linearized by digestion with XhoI and a run-off transcript was produced using SP6 RNA polymerase. The RNA transcript was then introduced into C33A cells by electroporation, and the growth medium containing the virus was collected 48 hours after introduction and frozen at -80 ° C. AR339 and TR339-GFP / 2A were grown in C33A cells maintained at 37 ° C, 5% CO ₂ in DMEM containing 10% FCS, and virus titers were increased in monolayer BHK-21 cells. Measured by the plaque assay used.

Sindbis virus in vivo distribution and targeting studies were performed as described below. First, 100 μL of DMEM containing 1 × 10 ⁶ pfu of GFP-SIN was injected into the C33A tumor-bearing mouse from the jugular vein. GFP expression in the tumor was examined every 12 hours after injection. The measurement of GFP expression was carried out by using an Olympus fluorescence stereo microscope (model SZX7) equipped with a reflection illumination fluorescence unit for GFP (SZX-RFL2-GFP) in combination with a Hamamatsu ORCA-ER cooled CCD camera. At predetermined intervals, the tumor site and various organs were separated and weighed, and the accumulation of Sindbis virus in the tumor was measured. After excising the tumor and organ, they were weighed and minced with scissors and placed in a glass homogenizer containing 1 ml of 4.31% sulfosalicylic acid. GFP counts in lysates from various organs were evaluated by fluorescence measured with a plate reader (Wallac 1420 ARVOsx Multilabel Counter, manufactured by Perkin-Elmer).

  Virus purification was performed as described below. The medium containing the virus was overlaid on a 2 ml buffer containing 20% sucrose. Viral particles were precipitated from the collected medium by centrifuging at 50,000 g for 2 hours in a Ti42 rotor (Beckman). The precipitate was dissolved in 100 μl GTEN buffer (200 mM glycine, 50 mM Tris, 100 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), prepared with HCL at pH 7.5). The virus was inactivated by 45 minutes of UV irradiation. Inactivated virus cannot replicate, but the ability of the virus to adsorb and fuse remains unchanged.

  Liposomes were prepared by a dehydration rehydration method. Briefly, a lipid mixture containing EPC, cholesterol, and PS in a molar ratio of 4: 5: 1 was suspended in a solution containing chloroform and evaporated on a rotary evaporator at 50 ° C. to remove the chloroform. . N-Rh-PE or Oregon Green-DHPE was added to the mixture to label the liposome membrane. The film was hydrated with NS or 5 mM cisplatin. Thereafter, a polycarbonate filter (manufactured by Nuclepore) having a pore size of 0.4 μm was two stacked, and extrusion was performed 10 times using a pressure extruder (manufactured by Lipex Biomembranes). For the preparation of PEG-modified liposomes, the liposomes were mixed with PEG-DSPE and incubated for 30 minutes at 48 ° C. with vigorous shaking.

  Sindbis virus hybrid liposomes (SIN hybrid liposomes) were prepared by mixing PEG-modified liposomes with UV-inactivated Sindbis virus and incubating for 2 hours at 37 ° C. with vigorous shaking.

In vitro studies of cell binding and drug delivery were performed as follows. First, cancer cells were seeded and cultured in a culture dish having a cell number of 2 × 10 ⁵ and a diameter of 35 mm. After 24 hours, SIN hybrid rhodamine labeled liposomes encapsulating FITC-dextran were diluted in DMEM and added to the culture dish at a concentration of 0.3 mg total lipid per well. After 1 hour incubation at 37 ° C., the cells were washed 3 times with phosphate buffered saline (PBS, pH 7.4) to remove unbound liposomes. The bound fluorescent liposome was visualized with an Olympus fluoroview confocal laser scanning microscope (manufactured by Olympus).

Cisplatin was introduced into SIN hybrid liposomes or non-SIN hybrid liposomes in order to examine the cytotoxicity of liposomes encapsulating anticancer agents. Cancer cells were seeded in 96-well plates at a cell count of 1 × 10 ³ / well. After 24 hours, SIN hybrid liposomes containing cisplatin or NS, or non-SIN hybrid liposomes containing cisplatin or NS are diluted in DMEM to a concentration of 0.3 mg / ml total lipid, 100 μl / well is added to 8 wells, and Plates were incubated for 48 hours at 37 ° C. in a 5% CO ₂ atmosphere. Cell viability after different treatments was measured using MTT assay. Cell viability was calculated as a percentage of control.

The in vivo therapeutic effect of liposomes was examined as described below. First, RERF-LC-AI cells were collected and suspended in the medium at a concentration of 5 × 10 ⁷ / mL. Xenograft tumors were established by subcutaneous injection of 5 × 10 ⁶ tumor cells in 100 μl FCS-free culture medium into the dorsal skin of 6 week old female BALB / c nu / nu mice. Treatment began when the tumor volume reached approximately 100 mm ³ . SIN hybrid liposomes containing cisplatin or NS, or non-SIN hybrid liposomes containing cisplatin or NS were infused from the jugular vein once a week for 7 weeks at a total lipid of 0.4 mmol / kg. Tumor volume (mm ³ ) is calculated by the formula 0.873 × a × b × c, where a is the longest diameter, b is the shortest diameter, and c is the height. Mouse weights were monitored and recorded.

[result]

  As a result of examining the biodistribution of Sindbis virus, tumor-specific distribution (spread) of GFP-SIN was observed in tumor-bearing nude mice intravenously injected with GFP-expressing Sindbis virus (GFP-SIN) (Fig. 1). Specifically, GFP-SIN was found specifically in lysates prepared from tumor-bearing nude mouse tumors intravenously injected with GFP-SIN (FIG. 1). Almost no GFP-SIN infection was observed in other organs of the mouse.

  Next, SIN hybrid liposomes were prepared and the adsorption ability and contents transfer ability were evaluated. Specifically, tumor cells (oral cancer, lung cancer, anticancer drug resistant strains) and normal cells were incubated with rhodamine-labeled SIN hybrid liposomes encapsulating FITC-dextran or rhodamine-labeled non-SIN hybrid liposomes, and in a confocal microscope Observed. In cells incubated with rhodamine-labeled non-SIN hybrid liposomes, only a small amount of fluorescence was observed, whereas when cells were treated with rhodamine-labeled SIN hybrid liposomes, there was a large amount of rhodamine fluorescence on the cell membrane, and FITC was detected in the cytoplasm. On the other hand, the specific adsorption of liposomes to normal cell lines and the transfer of encapsulated drugs were low.

  Thus, in the tumor cell line, specific adsorption of the SIN hybrid liposome and transport of the encapsulated drug were observed, suggesting that the SIN hybrid liposome is useful as a tumor targeting drug transport carrier.

  In order to test the drug transport ability of SIN hybrid liposomes, anticancer drugs were encapsulated in liposomes, and the effects of anticancer drugs on cancer cell lines (oral cancer, lung cancer, anticancer drug resistant strains) and normal cell lines were examined. Specifically, the cytotoxicity of SIN hybrid liposomes in vitro was compared with the cytotoxicity of SIN hybrid liposomes encapsulating NS and non-SIN hybrid liposomes encapsulating cisplatin or NS. The SIN hybrid liposome encapsulating cisplatin showed significantly higher cytotoxic activity against tumor cells compared to other liposomes in a 48 hour incubation test (FIG. 2).

  Thus, in vitro studies have shown that SIN hybrid liposomes are effective for cisplatin transport to tumor cells.

This liposome encapsulating an anticancer agent was administered to tumor-bearing mice in blood to examine the in vivo tumor targeting effect. Specifically, mice bearing 100 mm ³ established tumors were randomly divided into 4 groups. Mice injected with SIN hybrid liposome encapsulating cisplatin showed rapid tumor regression. In contrast, mice injected with NS encapsulated SIN hybrid liposomes or cisplatin or NS encapsulated SIN hybrid liposomes formed large tumors (1500-2000 mm ³ ) and were finally euthanized on day 49 ( (Figure 3).

  Thus, it has been found that by using SIN hybrid liposomes, the effects of anticancer agents can be obtained in a tumor-specific manner even in vivo. This result suggests that SIN hybrid liposomes transport tumor-encapsulated drugs in vivo even in vivo.

  According to the present invention, since a drug can be specifically transported to tumor cells, a high effect with reduced side effects can be obtained in cancer treatment. Thus, the present invention is extremely useful in the field of pharmaceutical development.

Claims (15)

A vector for introducing a drug into tumor cells, comprising a liposome containing Sindbis virus or a part thereof as a constituent component. A vector for introducing a drug into tumor cells, comprising liposomes substantially containing Sindbis virus E1 protein and E2 protein. A vector for introducing a drug into a tumor cell, wherein Sindbis virus is subjected to sucrose density gradient centrifugation to obtain E1 protein and E2 protein fractions, which are used as components as liposomes. 4. The vector for introducing a drug into a tumor cell according to any one of claims 1 to 3, wherein the liposome is a liposome modified with polyethylene glycol. 5. The vector for introducing a drug into a tumor cell according to claim 4, wherein the polyethylene glycol is polyethylene glycol having an average molecular weight of 1,000 to 5,000. The vector for introducing a drug into a tumor cell according to any one of claims 1 to 5, wherein the drug is encapsulated. 7. The vector for introducing a drug into tumor cells according to claim 6, wherein the drug is a drug exhibiting an antitumor effect. 8. The vector for introducing a drug into a tumor cell according to claim 7, wherein the drug exhibiting an antitumor effect is cisplatin. 7. The vector for introducing a drug into tumor cells according to claim 6, wherein the drug is a labeling substance. Use of the vector for introducing a drug into tumor cells according to any one of claims 1 to 5 in the production of a composition for treating cancer. A composition for cancer treatment comprising the vector for introducing a drug into tumor cells according to claim 7 or 8 as an active ingredient. 9. A method for treating cancer, comprising administering the drug introduction vector according to claim 7 or 8. Use of the vector for introducing a drug into tumor cells according to any one of claims 1 to 5 in the production of a composition for labeling tumor cells. 10. A tumor cell labeling composition comprising the drug introduction vector according to claim 9. 10. A method for labeling tumor cells, comprising administering the drug introduction vector according to claim 9.