JP2022039056A - Pharmaceutical composition and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a pharmaceutical composition capable of delivering a nucleic acid drug to a target cell and a method for producing the same. SOLUTION: The pharmaceutical composition contains an exosome derived from an administration subject and an oligonucleotide contained in the exosome. In addition, the method for producing a pharmaceutical composition includes obtaining an exosome from a blood sample derived from an administration subject, and encapsulating the obtained exosome with an oligonucleotide. [Selection diagram] FIG. 8

Description

Article 30, Paragraph 2 of the Patent Act Applicable application is available. P3637 Website publication date September 5, 1st year of Reiwa

The present invention relates to a pharmaceutical composition and a method for producing the same.

Exosomes are membrane-bound extracellular vesicles formed in endosome compartments in eukaryotic cells. Exosomes can contain functional biomolecules such as proteins and nucleic acids and deliver them to receiving cells. For example, Patent Document 1 proposes a milk-derived exosome containing a biological therapeutic agent. It is also known that exosomes derived from tumor cells have the property of directing to specific organs.

Special Table 2019-535839 Gazette

An object of the present invention is to provide a pharmaceutical composition capable of delivering a nucleic acid drug to a target cell and a method for producing the same.

Specific means for solving the above problems are as follows, and the present invention includes the following aspects. The first aspect is a pharmaceutical composition containing an exosome derived from an administration subject and an oligonucleotide contained in the exosome. Oligonucleotides may include siRNA. Exosomes may contain exosomes derived from cancer cells to be administered, and may be derived from serum to be administered. The pharmaceutical composition may also be used in the treatment of metastatic cancer.

The second aspect is a method for producing a pharmaceutical composition, which comprises obtaining an exosome from a serum derived from an administration subject and encapsulating the obtained exosome with an oligonucleotide. Oligonucleotides may include siRNA. Exosomes may include exosomes derived from cancer cells to be administered.

A third aspect is a method for treating a disease, which comprises administering the pharmaceutical composition to a subject. The oligonucleotide contained in the pharmaceutical composition may contain siRNA. The exosome contained in the pharmaceutical composition may contain exosomes derived from cancer cells to be administered. Exosomes may be derived from the serum to be administered. Methods of treating the disease may be used to treat metastatic cancer.

A fourth aspect is a pharmaceutical composition comprising an exosome derived from an administration subject and a biological therapeutic agent contained in the exosome. The biotherapeutic agent may be one that is not naturally present in the exosomes derived from the subject. The biotherapeutic agent may be a peptide or protein or oligonucleotide.

According to the present invention, it is possible to provide a pharmaceutical composition capable of delivering a nucleic acid drug to a target cell and a method for producing the same.

It is a figure which shows the accumulation in the lung of an exosome derived from a cancer cell. It is a graph which shows the accumulation in the lung of the exosome derived from a cancer cell. It is a figure which shows the delivery property of siRNA of an exosome derived from a cancer cell to a cell. It is a graph which shows the delivery property of siRNA of an exosome derived from a cancer cell to a cell. It is a figure which shows the expression suppression of the target protein of the exosome preparation derived from a cancer cell. It is a graph which shows the expression suppression of the target protein of the exosome preparation derived from cancer cells. It is a figure which shows the suppression of cancer metastasis to the lung of the exosome preparation derived from a cancer cell. It is a graph which shows the cancer metastasis suppression to the lung of the exosome preparation derived from a cancer cell.

In the present specification, the term "process" is included in this term not only as an independent process but also as long as the intended purpose of the process is achieved even if it cannot be clearly distinguished from other processes. .. Further, the content of each component in the composition means the total amount of the plurality of substances present in the composition when a plurality of substances corresponding to each component are present in the composition, unless otherwise specified. Hereinafter, embodiments of the present invention will be described in detail. However, the embodiments shown below exemplify a pharmaceutical composition and a method for producing the same for embodying the technical idea of the present invention, and the present invention is the pharmaceutical composition and the method for producing the same. Not limited to.

Pharmaceutical Composition A pharmaceutical composition comprises an exosome derived from an administration subject and an oligonucleotide contained in the exosome. The pharmaceutical composition is administered to an administration subject from which the exosome is derived, and is applied to the treatment of a disease possessed by the administration subject. By including the oligonucleotide in the exosome, the degradation of the oligonucleotide is suppressed, the stability in the living body is improved, and the cell permeability can be improved. In addition, since exosomes are derived from the administration target, low antigenicity can be achieved and high safety can be ensured.

The subject of administration of the pharmaceutical composition may be, for example, a mammal, and the mammal includes a human. Moreover, the administration subject may be a non-human animal. Further, the administration subject may be a mammal having a disease. Diseases to be administered include cancer, malignant melanoma, fundus melanoma, breast cancer, pancreatic cancer, colorectal cancer, metastatic cancer such as osteosarcoma and the like. The treatment of the disease may be any treatment given to the disease, and examples thereof include treatment, improvement, suppression of progression (prevention of deterioration), prevention, and alleviation of symptoms caused by the disease.

The exosomes constituting the pharmaceutical composition are derived from the subject to which the pharmaceutical composition is administered. Exosomes can be extracted from, for example, a biological fluid sample collected from an administration subject and a culture supernatant of cells collected from the administration subject. Examples of the biological fluid sample include blood samples such as whole blood, plasma, and serum, urine, tears, saliva, and cerebrospinal fluid. Examples of cells collected from the administration subject include tumor cells, immune cells (normal cells) and the like. Exosomes are preferably extracted from the blood sample to be administered, more preferably from serum. Extraction of exosomes can be performed using, for example, a commercially available exosome extraction kit according to the instruction manual of the kit.

Here, the term "exosome" as used herein refers to "microvesicles", "liposomes", "exosomes", "exosome-like particles", "exosome-like vesicles", "liposome membranes", "nanovectors", etc. "Exosomes", "Lactosomes", "Extracellular vesicles", "Argosomes", "Apoptotic bodies", "Epidizimosomes", "Promininosomes", "Prostasomes", "Dexosomes", "Texosomes", "Oncosomes" It embraces the term and its concept. The average particle size of exosomes may be, for example, 20 nm or more and 200 nm or less.

The exosomes constituting the pharmaceutical composition preferably contain exosomes derived from cancer cells possessed by the administration subject. By including exosomes derived from cancer cells, high organ directivity can be obtained. Exosomes derived from cancer cells include exosomes produced by cancer cells. It is known that the metastasis destination of cancer cells is specified to some extent depending on the type of primary lesion. For example, in the case of melanoma, the main metastasis destinations of cancer are the lungs, etc., in the case of intraocular melanoma, the liver, etc., in the case of breast cancer, the lungs, liver, brain, bones, etc., and the pancreas. If it is, it is the liver, etc., if it is colorectal cancer, it is the liver, etc., and if it is osteosarcoma, it is the lung, etc. Pharmaceutical compositions containing such highly organ-oriented exosomes are considered to be particularly effective in the treatment of metastatic cancer.

On the surface of exosomes derived from cancer cells, for example, tetraspanins such as CD9, CD63, CD81, adhesion factors such as integrin, ICAM-1, VCAM-1, phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, various receptors , Cholesterol, various antibodies, ceramide, etc. are considered to be present.

The oligonucleotide contained in the exosome is different from the oligonucleotide that the exosome derived from the administration subject can originally contain, and is not particularly limited as long as it is used for the treatment of a disease. Examples of the oligonucleotide include siRNA, shRNA, miRNA, siRNA expression vector, shRNA expression vector, miRNA expression vector, antisense nucleic acid, decoy nucleic acid, aptamer, CpG oligo and the like. The oligonucleotide preferably contains at least siRNA. The base sequence of the oligonucleotide is not particularly limited, and may be appropriately selected depending on the purpose of the pharmaceutical composition and the like. The oligonucleotide may be single-stranded or double-stranded.

siRNA is a double-stranded RNA consisting of 21 to 23 base pairs. siRNA is involved in a phenomenon called RNA interference (RNAi), and by degrading mRNA, expression of a target gene can be suppressed in a sequence-specific manner. Although siRNA is a double-stranded RNA, the 3'portion of each RNA strand has a structure protruding by 2 bases. It is known that siRNA is produced by excising from a hairpin-shaped RNA or a long double-stranded RNA by an enzyme called dicer, and as a result, each strand has a phosphate group at the 5'end and a 3'. It has a structure having a hydroxy group at the end. The siRNA may have, for example, a function of suppressing cell proliferation, a function of suppressing infiltration, a function of suppressing migration, a function of suppressing immunity, and the like. Examples of the target gene of siRNA include Glypcian-3, Cyclin B1 and the like.

miRNAs are functional nucleic acids 20 to 25 bases long. MiRNAs are functional ncRNAs (non-coding RNAs) and are thought to play an important role in the biological phenomenon of regulating the expression of other genes. MiRNAs are thought to regulate target gene expression by binding to the 3'untranslated region (UTR) of the target mRNA and inhibiting translation. The miRNA may have, for example, a function of suppressing cell proliferation, a function of suppressing infiltration, a function of suppressing migration, a function of suppressing immunity, and the like. Examples of the target gene of miRNA include Glypcian-3, Cyclin B1 and the like.

The oligonucleotide contained in the exosome may be a vector expressing at least one of siRNA and miRNA. By using a vector, the effect of siRNA or miRNA can be more sustained.

The oligonucleotide contained in the exosome may be composed of a natural nucleotide residue or may be composed of a non-natural modified nucleotide residue. Non-natural modified nucleotide residues include, for example, modified phosphodiester bonds between nucleosides, modified 2'hydroxyl groups of ribose, those containing intramolecularly crosslinked ribose, purine bases and pyrimidine bases. At least one of which is modified is included. Examples of modification of the phosphodiester bond moiety include phosphorothioation, methylphosphonate, methylthiophosphonate, phosphorodithioate, phosphoramidate, peptide bond substitution and the like. Examples of modification of the 2'hydroxyl group of ribose are 2'-O-methylation, 2'-O-methoxyethylation, 2'-O-aminopropyl (AP), 2'-fluoromation, 2'-. Examples thereof include O-methylcarbamoylethylation and 3,3-dimethylallylation. Examples of intramolecular crosslinked ribose include nucleotides crosslinked at the 2'and 4'positions (2', 4'-BNA). 2', 4'-BNA includes, for example, locked nucleic acid (α-L-methyleneoxy (4'-CH ₂ -O-2') BNA or β-D-methyleneoxy (4'-), which is also called LNA. CH ₂ -O-2') BNA, ethyleneoxy (4'-(CH ₂ ) ₂ -O-2') BNA, also known as ENA), β-D-thio (4'-CH ₂ -S-2) ') BNA, Aminooxy (4'-CH ₂ -ON (R) -2') BNA (R is H or CH ₃ ), 2', 4'-Oxyamino (4'also called BNANC -CH ₂ -N (R) -O-2') BNA (R is H or CH ₃ ), 2', 4'-BNACOC, 3'amino-2', 4'-BNA, 5'-methyl BNA , Also called cEt-BNA (4'-CH (CH ₃ ) -O-2') BNA, also called cMOE-BNA (4'-CH (CH ₂ OCH ₃ ) -O-2') BNA , AmNA, amide type BNA (4'-C (O) -N (R) -2') BNA (R is H or CH ₃ ) and the like. Examples of modification of the base moiety include halogenation; methylation, ethylation, n-propylation, isopropylization, cyclopropylation, n-butylation, isobutylation, s-butylation, t-butylation, cyclobutylation. Alkylation such as; hydroxylation; amination; deaminolation; demethylation and the like.

The amount of oligonucleotide contained in the exosome may be appropriately selected depending on the purpose and the like. For example, it may be 0.1% by mass or more and 2.0% by mass or less with respect to the mass of the exosome, and preferably 0.2% by mass or more and 1.0% by mass or less.

The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. As the pharmaceutically acceptable carrier, any carrier commonly used in pharmaceutical preparations can be used. Examples of the carrier include a solvent, a solubilizing agent, a suspending agent, an tonicity agent, a buffering agent or a pH adjusting agent, a pain-relieving agent, and the like in a semi-solid preparation or a liquid preparation. Further, if necessary, additives such as preservatives, antioxidants, colorants, sweeteners, refreshing agents or flavoring agents, defoaming agents, and thickening agents may be contained.

Examples of the solvent in the liquid preparation include purified water, ethanol, propylene glycol, macrogol, sesame oil, corn oil, olive oil and the like. Examples of the solubilizing agent include propylene glycol, D-mannitol, benzyl benzoate, ethanol, triethanolamine, sodium carbonate, sodium citrate and the like. Examples of the suspending agent include benzalkonium chloride, carmellose, hydroxypropyl cellulose, propylene glycol, povidone, methyl cellulose, glycerin monostearate and the like. Examples of the tonicity agent include glucose, D-sorbitol, sodium chloride, D-mannitol and the like. Examples of the buffer or pH adjuster include sodium hydrogen phosphate, sodium acetate, sodium carbonate, sodium citrate and the like. Examples of the soothing agent include benzyl alcohol and the like.

Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, chlorobutanol, benzyl alcohol, sodium dehydroacetate, sorbic acid and the like. Examples of the antioxidant include sodium sulfite, ascorbic acid and the like. Examples of the colorant include edible pigments (eg, edible red No. 2 or 3, edible yellow No. 4 or 5, etc.), β-carotene and the like. Examples of the sweetener include sodium saccharin, dipotassium glycyrrhizinate, aspartame and the like. Examples of the refreshing agent or flavoring agent include l-menthol or mint water. Examples of the defoaming agent include dimethylpolysiloxane and silicon defoaming agents. Examples of the thickener include xanthan gum, tragant, methyl cellulose, dextrin and the like.

The pharmaceutical composition may contain an anticancer agent or may be used in combination with the anticancer agent, if necessary. Examples of anticancer agents include antimetabolites, molecular targeted agents, alkylating agents, plant alkaloid agents, anticancer antibiotics, platinum preparations, hormone agents, biological response regulators, immune checkpoint inhibitors and the like. Can be mentioned.

Among the anticancer agents, for example, antimetabolites include gemcitabine, cytarabine, enocitabine, tegafur, carmofur and the like. Molecular-targeted drugs include imatinib, gefitinib, sunitinib, cetuximab and the like. Examples of the alkylating agent include ifosfamide, cyclophosphamide, dacarbacin and the like. Examples of the plant alkaloid agent include docetaxel, vincristine, vindesine, and vinblastine. Examples of the anticancer antibiotic include pirarubibicin, bleomycin, mitomycin, peplomycin and the like. Examples of the platinum preparation include cisplatin, carboplatin, oxaliplatin and the like. Examples of hormonal agents include exemestane, tamoxifen, prednisolone and the like. Examples of the biological response regulator include interferon and interleukin. Immune checkpoint inhibitors include nivolumab, pembrolizumab, ipilimumab and the like.

The content of the active ingredient in the pharmaceutical composition varies depending on the dosage form, dosage and the like, and is, for example, 0.1% by mass or more and 20% by mass or less, or 0.1% by mass or more and 10% by mass or less of the whole composition. .. The administration dose of the pharmaceutical composition is appropriately selected depending on the administration subject, disease, symptom, dosage form, administration route and the like. The administration dose is, for example, when orally administered to an adult cancer patient, the active ingredient is usually about 0.1 mg or more and 500 mg or less, or about 0.5 mg or more and 100 mg or less, once or several times per day. It can be administered separately.

The pharmaceutical composition can be administered parenterally or orally depending on its dosage form. Dosage forms that are administered parenterally include, for example, injections, infusions, eye drops, nasal agents, transpulmonary agents, and the like. Examples of the dosage form to be orally administered include liquid preparations such as syrups, emulsions and suspensions, and semi-solid preparations such as jellies.

Method for Producing Pharmaceutical Composition The method for producing a pharmaceutical composition includes a first step of obtaining an exosome from a blood sample derived from an administration subject, and a second step of encapsulating the obtained exosome with a desired oligonucleotide.

In the first step, a blood sample is collected from the administration target of the pharmaceutical composition, and exosomes are extracted from the collected blood sample. The subject of administration is, for example, a mammal, and the mammal includes a human. Moreover, the administration subject may be a non-human animal. Further, the administration subject may be a mammal having a tumor, or may be a mammal having cancer. The first step may include obtaining serum from a blood sample collected from an administration subject by a conventional method such as centrifugation. Extraction of exosomes from a blood sample can be performed using, for example, a commercially available exosome extraction kit according to the instruction manual of the kit.

In the second step, the extracted exosome is encapsulated with the desired oligonucleotide. Details of the encapsulated oligonucleotide are as described above. Examples of the method for encapsulating oligonucleotides in exosomes include a method using a gene transfer reagent and an electroporation method. A method using a gene transfer reagent is preferable. The gene transfer reagent may be appropriately selected from commercially available gene transfer reagents. Encapsulation of oligonucleotides in exosomes may be performed according to the instruction manual of a commercially available gene transfer reagent.

Treatment of Disease A treatment of a disease comprises administering to the subject an effective amount of the pharmaceutical composition. The details of the pharmaceutical composition and the administration method are as described above. The subject of treatment is, for example, a mammal, which includes a human. In addition, the target of treatment may be a non-human animal. The disease may be, for example, metastatic cancer such as malignant melanoma, fundus melanoma, breast cancer, pancreatic cancer, colorectal cancer, or osteosarcoma.

The present invention, as another aspect, is derived from an administration subject in the production of a pharmaceutical composition used for the treatment of a disease, the use of an exosome containing an oligonucleotide, and the administration subject in the treatment of a disease, and the oligonucleotide is encapsulated. The use of exosomes, which are derived from administration subjects used for the treatment of diseases, include oligonucleotide-encapsulating exosomes.

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to these Examples.

Example 1
Exosome extraction 100 to 300 μL of blood was collected from a vein in the posterior jawbone of a 6-week-old C57BL / 6 mouse (female) using an animal lancet (Goldenrod, 5.5 mm), and a blood sample derived from a normal mouse was collected. And said. Then, a mouse melanoma hypermetastatic strain (B16 / BL6) was subcutaneously transplanted into mice at 2 × 10 ⁵ cells / mouse. Fourteen days after transplantation, blood was collected in the same manner as above to obtain a blood sample derived from a cancer mouse.

After collecting the blood sample, it was centrifuged at 4 ° C. and 15000 rpm for 10 minutes, and 50 to 100 μL of serum was collected. Using the collected serum, the exosome extraction kit PureExo ^(R) (Cosmo Bio) was used to recover exosomes as follows.

Sample buffer was added to a total of 2 mL per 100 to 500 μL of serum. Take 0.3 mL each of Solution A, Solution B, and Solution C in a 1: 1: 1 ratio, mix well until a uniform layer is formed, add the entire amount to the sample, invert and mix, and then allow to stand at 4 ° C. for 30 minutes. I put it. Centrifugation was carried out at 4 ° C. and 5,000 × g for 3 minutes, the upper layer and the lower layer were pipette-removed three times, and dried at room temperature for 10 minutes, leaving a white intermediate layer from which exosomes were extracted. 1 × 4 times the volume of the recovered intermediate layer was added, resuspended by 40 vigorous pipetting, mixed with vortex, and then horizontally shaken for 3 minutes and mixed with sufficient vortex 10 times or more, and 3 sets were repeated. .. Centrifugation was carried out at 4 ° C. and 5,000 xg for 5 minutes, and the supernatant was transferred to PureExo ^(R) Colon. PureExo ^(R) Volume was centrifuged at 3,000 xg for 5 minutes, and the filtrate was designated as "exosome extract".

Introduction of siRNA As siRNA, siRNA for Glypican-3 (GPC3) was obtained from Thermo Fisher Scientific. 100 pmol of siRNA and 2 μL of gene transfer reagent Lipofectamine 2000 (Thermo Fisher Scientific) were added to an exosome extract equivalent to 250 μg of serum-derived exosome, and the mixture was allowed to stand at 37 ° C. for 30 minutes. This was used as the siRNA-introduced exosome preparation of Example 1.

Experiment 1
Evaluation of lung accumulation of exosomes derived from cancer transplant model mouse serum For exosome extracts derived from normal mice and cancer mice, a gene transfer reagent and indocyanine green (ICG) were mixed with various exosomes and allowed to stand for 12 hours. , ICG-labeled exosomes were obtained, respectively. Fourteen days after obtaining a blood sample derived from a cancer mouse, ICG-labeled exosomes were administered intravenously at 400 μg / mouse. One day after administration, the distribution of ICG-labeled exosomes in the body was measured using an in vivo photoimaging apparatus Clarvivo OPT plus (Shimadzu Corporation). The accumulation state with respect to the lung is shown in FIGS. 1 and 2. FIG. 1 (A) is an image of a lung after administration of a normal mouse-derived exosome preparation, and FIG. 1 (B) is an image of a lung after administration of a cancer mouse-derived exosome preparation. FIG. 2 shows the fluorescence intensity.

As shown in FIG. 1 (A), almost no exosomes were accumulated in the lungs after administration of the normal mouse-derived exosome preparation. On the other hand, as shown in FIG. 1 (B), exosomes were generally distributed in the lungs after administration of the cancer mouse-derived exosome preparation. As shown in FIG. 2, exosomes derived from cancer mice were significantly more agglomerated in the lung than exosomes derived from normal mice.

Experiment 2
Evaluation of intracellular uptake of exosome preparations Alexa488-siRNA (GPC3), which is a combination of the fluorescent dye Alexa Fluor ^(R) 488 and siRNA (GPC3), was obtained by requesting the synthesis of Thermo Fisher Scientific as a fluorescently labeled siRNA. .. Further, 160 pmol of Alexa488-siRNA (GPC3) and 2 μL of gene transfer reagent Lipofectamine 2000 (Thermo Fisher Scientific) were added to an exosome extract equivalent to 166 μg of serum-derived exosome, and the mixture was allowed to stand at 37 ° C. for 30 minutes. The amount was 200 μL with PBS, which was used as the siRNA-introduced exosome preparation of Experiment 2.

Using a 24-well plate, mouse melanoma cell line B16 / BL6 was seeded in 500 μL of cell culture medium supplemented with 10% FCS in DMEM at 5 × 10 ⁴ cells and cultured at 5% CO ₂ for 24 hours. The siRNA-introduced exosome preparation or PBS as a control and Alexa488-siRNA (siRNA) as a positive control were added. After continuing the culture for another 24 hours, the introduction of siRNA into the cultured cells was evaluated using a fluorescence microscope (EVOS) and flow cytometry (NovoCyte: ACEABioScience). The results are shown in FIGS. 3 and 4. FIG. 3 (A) is a fluorescence microscope image of a control, and FIG. 3 (B) is a fluorescence microscope image of cultured cells supplemented with an exosome preparation derived from cancer mice. FIG. 4 is the result of flow cytometry.

From FIGS. 3 and 4, it can be seen that siRNA is introduced into the cultured cells with high efficiency by adding the exosome preparation derived from cancer mice.

Experiment 3
Evaluation of target protein expression level by exosome preparation In Experiment 2, after adding the siRNA-introduced exosome preparation, the culture was continued for another 48 hours. Cultured cells were collected and the expression level of the target protein was evaluated by Western blotting. Western blotting was performed as follows. At the time of cell recovery, the cells were recovered as a cell lysate using a lysis buffer and electrophoresed by SDS-PAGE. Then, the protein on the gel was transferred to the PDVF membrane (transfer membrane), the antibody against the target protein was used as the primary antibody, and the protein on the transfer membrane was reacted overnight at 4 ° C. An antibody against peroxidase (enzyme) -labeled primary antibody IgG was used as a secondary antibody, and the protein on the transfer membrane was reacted again at room temperature for 1 hour. Finally, a substrate was added, and the light emission of the enzyme labeled with the secondary antibody was used to detect the protein on the transfer membrane as a band, the area of the band was calculated, and the protein was quantified. The results are shown in FIGS. 5 and 6.

From FIGS. 5 and 6, the normal mouse-derived exosome preparation and the cancer mouse-derived exosome preparation significantly suppressed the expression of the target protein with respect to the control. In addition, the cancer mouse-derived exosome preparation significantly suppressed the expression of the target protein as compared with the normal mouse-derived exosome preparation (p <0.05).

Experiment 4
Evaluation of cancer metastasis by exosome preparation Mouse melanoma cell line B16 / BL6 was transplanted into 6-week-old C57BL / 6 mice (female) at 2 × 10 ⁵ cells / mouse. On the 14th day after transplantation, 100 to 300 μL of blood was collected from a vein in the posterior part of the jawbone using an animal lancet (Goldenrod, 5.5 mm) to prepare a blood sample derived from a cancer mouse. In the same manner as above, a cancer mouse-derived exosome preparation was prepared by collecting serum from a blood sample, extracting exosomes from serum, and introducing siRNA into an exosome extract. In addition, a normal mouse-derived exosome preparation was prepared in the same manner using mice not transplanted with a melanoma cell line. As the exosome preparation derived from normal mice, a preparation in which siRNA for GPC3 was introduced (GPC3_siRNA) and a preparation in which negative control siRNA was introduced (NC_siRNA) were prepared.

For the above mice, the tumor was resected 28 days after transplantation of the mouse melanoma cell line. Then, an exosome preparation having a siRNA amount of 20 pmol / mouse was intravenously administered once every 3 days. PBS was administered as a control. Thoracotomy was performed 42 days after transplantation and the number of colonies in the lungs was counted. The results are shown in FIGS. 7 and 8. 7 (A) is a control, FIG. 7 (B) is a normal mouse-derived exosome preparation (NC_siRNA), FIG. 7 (C) is a normal mouse-derived exosome preparation (GPC3_siRNA), and FIG. 7 (D) is a cancer mouse-derived exosome preparation. It is an image of the lung to which (GPC3_siRNA) was administered.

From FIGS. 7 and 8, it can be seen that the exosome preparation introduced with GPC3_siRNA significantly suppresses lung metastasis with respect to the control.

Claims (10)

A pharmaceutical composition comprising an exosome derived from an administration subject and an oligonucleotide contained in the exosome. The pharmaceutical composition according to claim 1, wherein the oligonucleotide contains siRNA. The pharmaceutical composition according to claim 1 or 2, wherein the exosome contains an exosome derived from a cancer cell to be administered. The pharmaceutical composition according to any one of claims 1 to 3, wherein the exosome is derived from a blood sample to be administered. The pharmaceutical composition according to any one of claims 1 to 4, which is used for treating metastatic cancer. A method for producing a pharmaceutical composition, which comprises obtaining an exosome from a blood sample derived from an administration subject and encapsulating the obtained exosome with an oligonucleotide. The production method according to claim 6, wherein the oligonucleotide contains siRNA. The production method according to claim 6 or 7, wherein the exosome contains an exosome derived from a cancer cell to be administered. The production method according to any one of claims 6 to 8, wherein the pharmaceutical composition is used for the treatment of metastatic cancer. A method for treating a disease, which comprises administering the pharmaceutical composition according to any one of claims 1 to 5 to a subject.

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