Pharmaceutical composition for suppressing chronic inflammation

Abstract

The present invention relates to a pharmaceutical composition for suppressing chronic inflammation, the composition comprising a small RNA derived from an iPS cell or an adipose-derived stem cell.

Description

Pharmaceutical composition for suppressing chronic inflammation

[0001] The present invention relates to a pharmaceutical composition for suppressing chronic inflammation.

[0002] In recent years, research in the field of cell therapy using iPS cells and stem cells has been actively conducted. Research is also being conducted using cell lysates rather than the cells themselves. Patent Document 1 discloses a therapeutic formulation for osteoarthritis or ligament or tendon injuries, containing as an active ingredient a filtrate of a disrupted adipose tissue-derived stem cell lysate. Patent Document 2 also discloses a skin protective agent containing as an active ingredient a filtrate of a disrupted adipose tissue-derived SVF fraction stem cells, wherein the filtrate contains one or more proteins selected from the group consisting of heat shock proteins, cytokines and / or paracrine factors, and cell surface proteins, and wherein the filtrate is obtained by filtering only through a filter with a pore size of 0.1 μm to 0.45 μm.

[0003] International Publication No. WO 2022 / 180661 International Publication No. WO 2022 / 018897

[0004] An object of the present invention is to provide a new pharmaceutical composition and the like that is effective in suppressing chronic inflammation.

[0005] As a result of intensive research aimed at solving the above problems, the present inventors discovered that small RNAs derived from iPS cells or adipose tissue-derived stem cells are effective in suppressing chronic inflammation, and thus completed the present invention.

[0006] That is, the present invention is as follows: [1] A pharmaceutical composition for suppressing chronic inflammation, comprising small RNA derived from iPS cells or adipose tissue-derived stem cells. [2] The pharmaceutical composition according to [1], wherein the chronic inflammation is multiple sclerosis, neuromyelitis optica, MOG antibody-associated disease, asthma, arthritis, psoriasis, rheumatoid arthritis, ankylosing spondylitis, vasculitis, hypertension, autoimmune fatty liver, alcoholic hepatitis, enteritis, autoimmune uveitis, cystitis, dermatitis, rhinitis, periodontitis, stomatitis, alopecia areata, or sarcopenia. [3] The pharmaceutical composition according to [1] or [2], which suppresses the expression of inflammatory cytokines. [4] The pharmaceutical composition according to [3], wherein the gene involved in the expression of inflammatory cytokines is C3, Il1b, Nos2, Selp, Vegfa, Il3, Lrp2, Cd4, Cd40lg, Fasl, or Il4. [5] The pharmaceutical composition according to any one of [1] to [4], wherein the pharmaceutical composition comprises a protoplast composition derived from iPS cells or adipose tissue-derived stem cells. [6] The pharmaceutical composition according to any one of [1] to [5], wherein the cells are human cells. [7] The pharmaceutical composition according to any one of [1] to [6], wherein the cells are 75% or more confluent. [8] The pharmaceutical composition according to any one of [1] to [7], wherein the cells have been passaged two or more times. [9] The pharmaceutical composition according to any one of [1] to [8], wherein the protoplast composition is a cell lysate.

[10] The pharmaceutical composition according to any one of [1] to [9], wherein the protoplast composition has been removed from nuclei and / or mitochondria.

[11] A pharmaceutical composition for suppressing chronic inflammation, comprising a cell lysate derived from iPS cells, wherein the cell lysate contains small RNA derived from iPS cells and has been removed from nuclei and / or mitochondria.

[12] A pharmaceutical composition for suppressing chronic inflammation, comprising a cell lysate derived from adipose tissue-derived stem cells, the cell lysate containing small RNA derived from adipose tissue-derived stem cells, and from which nuclei and / or mitochondria have been removed. The pharmaceutical composition according to

[11] or

[12] may be the pharmaceutical composition according to any one of [1] to

[10] above.

[0007] The present invention may be embodied as follows.

[13] A protoplast composition derived from iPS cells or adipose tissue-derived stem cells, from which the nucleus has been removed and which contains small RNA.

[14] The protoplast composition according to

[13] , wherein the iPS cells or adipose tissue-derived stem cells are human cells.

[15] The protoplast composition according to

[13] or

[14] , wherein the iPS cells or adipose tissue-derived stem cells are 75% or more confluent.

[16] The protoplast composition according to any of

[13] to

[15] , wherein the iPS cells or adipose tissue-derived stem cells have been passaged two or more times.

[17] The protoplast composition according to any of

[13] to

[16] , wherein the protoplast composition is a cell lysate.

[18] The protoplast composition according to any of

[13] to

[17] , from which mitochondria have been removed.

[0008] The present invention also includes the following embodiments. [A1] A method for suppressing chronic inflammation, comprising administering to a subject in need thereof an effective amount of small RNA derived from iPS cells or adipose tissue-derived stem cells, or a protoplast composition derived from iPS cells or adipose tissue-derived stem cells from which nuclei have been removed and which contains small RNA. [A2] A method for suppressing chronic inflammation, comprising administering to a subject in need thereof an effective amount of small RNA according to any of [1] to

[12] , the pharmaceutical composition according to any of [1] to

[12] , or the protoplast composition according to any of

[13] to

[18] . [B1] A protoplast composition derived from iPS cells or adipose tissue-derived stem cells from which nuclei have been removed and which contains small RNA, for use in suppressing chronic inflammation. [B2] The small RNA according to any one of [1] to

[12] , the pharmaceutical composition according to any one of [1] to

[12] , or the protoplast composition according to any one of

[13] to

[18] , for use in suppressing chronic inflammation. [C1] Use of a protoplast composition derived from iPS cells or adipose tissue-derived stem cells, comprising small RNA derived from iPS cells or adipose tissue-derived stem cells, or from which nuclei have been removed and small RNA, for suppressing chronic inflammation. [C2] Use of the small RNA according to any one of [1] to

[12] , the pharmaceutical composition according to any one of [1] to

[12] , or the protoplast composition according to any one of

[13] to

[18] , for suppressing chronic inflammation. [D1] Use of a protoplast composition derived from iPS cells or adipose tissue-derived stem cells, comprising small RNA derived from iPS cells or adipose tissue-derived stem cells, or from which nuclei have been removed and small RNA, in the manufacture of a pharmaceutical composition for suppressing chronic inflammation. [D2] Use of the small RNA according to any one of [1] to

[12] , the pharmaceutical composition according to any one of [1] to

[12] , or the protoplast composition according to any one of

[13] to

[18] in the manufacture of a pharmaceutical composition for suppressing chronic inflammation. [D3] Use of the small RNA according to any one of [1] to

[12] or the protoplast composition according to any one of

[13] to

[18] in the manufacture of a pharmaceutical composition for suppressing chronic inflammation.[D4] Use of the small RNA according to any of [1] to

[12] or the protoplast composition according to any of

[13] to

[18] in the manufacture of the pharmaceutical composition according to any of [1] to

[12] . [E1] Use of a protoplast composition derived from iPS cells or adipose tissue-derived stem cells from which nuclei have been removed and which contains small RNA, in the manufacture of a chronic inflammation suppressor. [E2] Use of the small RNA according to any of [1] to

[12] , the pharmaceutical composition according to any of [1] to

[12] , or the protoplast composition according to any of

[13] to

[18] in the manufacture of a chronic inflammation suppressor. [F1] A chronic inflammation suppressor comprising small RNA according to any of iPS cells or adipose tissue-derived stem cells or a protoplast composition derived from iPS cells or adipose tissue-derived stem cells from which nuclei have been removed and which contains small RNA. [F2] A chronic inflammation inhibitor comprising the small RNA according to any one of [1] to

[12] , the pharmaceutical composition according to any one of [1] to

[12] , or the protoplast composition according to any one of

[13] to

[18] .

[0009] The present invention can provide a pharmaceutical composition and the like that is effective in suppressing chronic inflammation.

[0010] The results of weight measurement are shown. In the vehicle group, weight loss peaked on Day 9, with some individuals experiencing significant weight loss from Day 12 onwards. From Day 18 onwards, weight loss was observed in all individuals. On the other hand, in the drug group, weight peaked on Day 9, but only one individual experienced significant weight loss, and no weight loss was observed. The results of EAE score measurement are shown. In the vehicle group, individuals began to develop pathology from Day 12 onwards, when weight loss was observed. On Day 17, three individuals had a score of 3 (complete paralysis of both hind limbs), indicating severe pathology. On the other hand, in the drug group, individuals began to develop pathology from Day 12 onwards, and although pathology developed in all individuals, the severity was milder than in the vehicle group. The results of EAE score measurement are shown. The graph shows, from top to bottom, the vehicle group, the adipose tissue-derived cell-administered group, the adipose tissue-derived cell + adipose tissue-derived cell lysate-administered group, and the non-induced group.

[0011] The pharmaceutical composition of the present invention contains small RNA derived from iPS cells or adipose tissue-derived stem cells and is used to suppress chronic inflammation.

[0012] In the present invention, small RNA derived from iPS cells or adipose tissue-derived stem cells is used as an ingredient effective in suppressing chronic inflammation. The small RNA derived from iPS cells or adipose tissue-derived stem cells is not particularly limited, but may be small RNA present in the protoplasm of iPS cells or adipose tissue-derived stem cells. Known examples of small RNA include microRNA (miRNA), short interfering RNA (siRNA), and piwi-interacting RNA (piRNA).

[0013] In the present invention, the method for obtaining small RNA derived from iPS cells or adipose tissue-derived stem cells is not particularly limited and may be carried out by a conventionally known method. The iPS cells or adipose tissue-derived stem cells from which small RNA is to be obtained may be homogenized, and small RNA may be obtained using a kit or the like. Furthermore, the iPS cells or adipose tissue-derived stem cells from which small RNA is to be obtained may be cultured appropriately to a state suitable for obtaining small RNA, and then used to obtain small RNA derived from iPS cells or adipose tissue-derived stem cells of the present invention. A commercially available kit may be used for obtaining small RNA.

[0014] In the present invention, the small RNA is not particularly limited, and may be at least one selected from the following miRNAs. The small RNA may include all of the following miRNAs, or may include at least two or more selected from the following miRNAs: hsa-miR-135a-5p-478581_mir hsa-miR-187-3p-477941_mir hsa-miR-197-3p-477959_mir hsa-miR-338-3p-478037_mir hsa-miR-504-5p-478144_mir hsa-miR-570-3p-479053_mir hsa-miR-589-5p-479073_mir

[0015] In the present invention, the small RNA is not particularly limited, but may not include at least one type selected from the following miRNAs. The small RNA may not include all of the following miRNAs, or may not include at least two or more types selected from the following miRNAs. hsa-miR-140-5p-477909_mir hsa-miR-142-3p-477910_mir hsa-miR-149-5p-477917_mir hsa-miR-217-478773_mir hsa-miR-409-5p-478872_mir hsa-miR-411-5p-478086_mir hsa-miR-486-5p-478128_mir hsa-miR-548b-3p-479018_mir hsa-miR-548d-3p-477833_mir hsa-miR-616-3p-478177_mir hsa-miR-624-3p-479108_mir hsa-miR-636-478185_mir hsa-miR-651-5p-479131_mir hsa-miR-654-3p-479135_mir hsa-miR-654-5p-478368_mir hsa-miR-30e-5p-479235_mir hsa-miR-885-5p-478207_mir hsa-miR-888-5p-479192_mir

[0016] In the present invention, small RNAs derived from iPS cells are not particularly limited, and may be at least one selected from the following miRNAs. The small RNAs derived from iPS cells may include all of the following miRNAs, or may include at least two or more selected from the following miRNAs. hsa-miR-95-3p-478213_mir hsa-miR-96-5p-478215_mir hsa-miR-105-5p-477865_mir hsa-miR-124-3p-477879_mir hsa-miR-126-3p-477887_mir hsa-miR-135a-5p-478581_mir hsa-miR-150-5p-477918_mir hsa-miR-183-5p-477937_mir hsa-miR-187-3p-477941_mir hsa-miR-197-3p-477959_mir hsa-miR-200b-3p-477963_mir hsa-miR-204-5p-478491_mir hsa-miR-302a-3p-478006_mir hsa-miR-302b-3p-478591_mir hsa-miR-302c-3p-478509_mir hsa-miR-338-3p-478037_mi hsa-miR-363-3p-478060_mi hsa-miR-489-3p-478130_mir hsa-miR-504-5p-478144_mir hsa-miR-512-5p-478972_mir hsa-miR-516a-5p-478978_mir hsa-miR-517a-3p_hsa-miR-517b-3p-479485_mir hsa-miR-518c-3p-478982_mir hsa-miR-519a-3p-479534_mir hsa-miR-519d-3p-478986_mir hsa-miR-519e-3p-479340_mir hsa-miR-520d-5p-478616_mir hsa-miR-523-3p-478994_mir hsa-miR-526b-5p-478997_mir hsa-miR-548c-3p-479537_mir hsa-miR-551b-3p-478159_mirhsa-miR-570-3p-479053_mir hsa-miR-579-3p-479059_mir hsa-miR-589-5p-479073_mir hsa-miR-627-5p-478427_mir hsa-miR-873-5p-478204_mir hsa-miR-876-3p-479186_mir hsa-miR-211-5p-478507_mir hsa-miR-520f-3p-479343_mir

[0017] In the present invention, small RNAs derived from iPS cells include, but are not limited to, the following miRNAs: hsa-miR-135a-5p-478581_mir hsa-miR-187-3p-477941_mir hsa-miR-197-3p-477959_mir hsa-miR-338-3p-478037_mir hsa-miR-504-5p-478144_mir hsa-miR-570-3p-479053_mir hsa-miR-589-5p-479073_mir Furthermore, they may contain at least one or more types selected from the following miRNAs:hsa-miR-95-3p-478213_mir hsa-miR-96-5p-478215_mir hsa-miR-105-5p-477865_mir hsa-miR-124-3p-477879_mir hsa-miR-126-3p-477887_mir hsa-miR-150-5p-477918_mir hsa-miR-183-5p-477937_mir hsa-miR-200b-3p-477963_mir hsa-miR-204-5p-478491_mir hsa-miR-302a-3p-478006_mir hsa-miR-302b-3p-478591_mir hsa-miR-302c-3p-478509_mir hsa-miR-363-3p-478060_mir hsa-miR-489-3p-478130_mir hsa-miR-512-5p-478972_mir hsa-miR-516a-5p-478978_mir hsa-miR-517a-3p_hsa-miR-517b-3p-479485_mir hsa-miR-518c-3p-478982_mir hsa-miR-519a-3p-479534_mir hsa-miR-519d-3p-478986_mir hsa-miR-519e-3p-479340_mir hsa-miR-520d-5p-478616_mir hsa-miR-523-3p-478994_mir hsa-miR-526b-5p-478997_mir hsa-miR-548c-3p-479537_mir hsa-miR-551b-3p-478159_mir hsa-miR-579-3p-479059_mir hsa-miR-627-5p-478427_mir hsa-miR-873-5p-478204_mir hsa-miR-876-3p-479186_mir hsa-miR-211-5p-478507_mir hsa-miR-520f-3p-479343_mir.

[0018] In the present invention, the small RNA derived from adipose tissue-derived stem cells is not particularly limited, and may be at least one selected from the following miRNAs. The small RNA derived from adipose tissue-derived stem cells may include all of the following miRNAs, or may include at least two or more selected from the following miRNAs. hsa-miR-29c-3p-479229_mir hsa-miR-135a-5p-478581_mir hsa-miR-187-3p-477941_mir hsa-miR-196b-5p-478585_mir hsa-miR-197-3p-477959_mir hsa-miR-198-478749_mir hsa-miR-338-3p-478037_mir hsa-miR-504-5p-478144_mir hsa-miR-548a-5p-479501_mir hsa-miR-570-3p-479053_mir hsa-miR-589-5p-479073_mir hsa-miR-597-5p-478339_mir

[0019] In the present invention, small RNAs derived from adipose tissue-derived stem cells include, but are not limited to, the following miRNAs: hsa-miR-135a-5p-478581_mir hsa-miR-187-3p-477941_mir hsa-miR-197-3p-477959_mir hsa-miR-338-3p-478037_mir hsa-miR-504-5p-478144_mir hsa-miR-570-3p-479053_mir hsa-miR-589-5p-479073_mir Furthermore, they may contain at least one or more types selected from the following miRNAs: hsa-miR-29c-3p-479229_mir hsa-miR-196b-5p-478585_mir hsa-miR-198-478749_mir hsa-miR-338-3p-478037_mir hsa-miR-597-5p-478339_mir

[0020] In the present invention, small RNA derived from iPS cells or adipose tissue-derived stem cells may be obtained from a protoplast composition derived from iPS cells or adipose tissue-derived stem cells. Furthermore, in the present invention, a protoplast composition derived from iPS cells or adipose tissue-derived stem cells may be used as the small RNA derived from iPS cells or adipose tissue-derived stem cells, and a protoplast composition from which nuclei and / or mitochondria have been removed may be used. In the present invention, a protoplast composition is a composition comprising intracellular components of iPS cells or adipose tissue-derived stem cells. The composition is not particularly limited as long as it contains components containing the protoplast of iPS cells or adipose tissue-derived stem cells, but may, for example, contain intracellular components obtained by disrupting the cell membrane. Intracellular components include components present within cells, such as nuclei, mitochondria, Golgi apparatus, cytoplasm, and vesicles.

[0021] In the present invention, cell disruption is not particularly limited and can be carried out by a conventionally known method. Examples of cell disruption methods include freeze-thawing (a process of freezing and then thawing), ultrasonic waves, a French press, a mortar, a homogenizer, glass beads, etc. The cells used for cell disruption are not particularly limited, and for example, live cells may be used, or non-live dead cells may be used. For cell disruption, it is preferable to use a freeze-thawing or ultrasonic method, but the conditions for such methods are not particularly limited.

[0022] After the cells are disrupted, they may be filtered using a filter, filter paper, or the like. Sterilization treatment may be performed after or before filtration. After the cells are disrupted, the protoplast composition may be obtained by centrifugation.

[0023] In the present invention, the protoplasm composition may be one from which nuclei have been removed and which contains small RNA. The method for removing nuclei from a protoplasm composition obtained by cell disruption is not particularly limited and can be carried out by a conventionally known method. The method for removing nuclei may be centrifugation or the use of an antibody that recognizes nuclei. The removal of nuclei from a protoplasm composition can be confirmed by staining or microscopic observation.

[0024] When iPS cells are used as the raw material for the protoplast composition, removing the nucleus is preferable because it makes it possible to remove factors present in the nucleus and reduce side effects.

[0025] It is preferable that mitochondria have been further removed from the protoplasm composition of the present invention. The removal of mitochondria from the protoplasm composition obtained by cell disruption is not particularly limited and can be carried out by known methods. In the protoplasm composition obtained by cell disruption, mitochondria may be removed following or simultaneously with the removal of nuclei, or the removal of mitochondria may be carried out first and then the removal of nuclei. The removal of mitochondria from the protoplasm composition is not particularly limited and can be carried out by known methods. The removal of mitochondria may be carried out by centrifugation or using an antibody that recognizes mitochondria. The removal of nuclei and mitochondria may be carried out simultaneously by centrifugation. The removal of mitochondria from the protoplasm composition can be confirmed by staining or microscopic observation.

[0026] In the present invention, "nuclei and / or mitochondria removed" means that the protoplast composition does not necessarily contain nuclei and / or mitochondria, but does not necessarily mean that the protoplast composition does not contain nuclei and / or mitochondria at all. That is, the protoplast composition may have 50% or more of the nuclei and / or mitochondria removed relative to the amount of nuclei and / or mitochondria contained in intact cells (which may be dead) prior to nuclei and / or mitochondria removal, or may have 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more of the nuclei and / or mitochondria removed. Furthermore, the protoplast composition of the present invention may have 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more of the nuclei and / or mitochondria removed relative to the composition prior to nuclei and / or mitochondria removal during the process of obtaining the protoplast composition. In the present invention, removal of nuclei and / or mitochondria from a protoplasmic composition can also be confirmed by confirming a reduction in the amount of nuclei and / or mitochondria compared to the amount of nuclei and / or mitochondria that would be present in a simple cell lysate. In this case, it can also be understood that nuclei and / or mitochondria have been removed when the amount of nuclei and / or mitochondria is reduced compared to the amount of nuclei and / or mitochondria that would normally be present in cells or cell lysates before removal.

[0027] In the present invention, when small RNA or a protoplasmic composition is derived from iPS cells, the iPS cells are not particularly limited.

[0028] iPS cells refer to induced pluripotent stem cells, and are not particularly limited in terms of cell reprogramming, but are cells obtained by introducing a specific nuclear reprogramming factor into a somatic cell. The iPS cells used in the present invention are somatic cell-derived artificial stem cells that have properties similar to those of ES cells, for example, in terms of pluripotency and proliferation ability through self-replication. Reprogramming factors for obtaining iPS cells are not particularly limited, but include, for example, Oct3 / 4, Klf4, Klf1, Klf2, Klf5, Sox2, Sox1, Sox3, Sox15, Sox17, Sox18, c-Myc, L-Myc, N-Myc, TERT, SV40 large T antigen, HPV16 E6, HPV16 E7, Bmil, Lin28, Lin28b, Nanog, Esrrb, and Esrrg. iPS cells may be obtained by conventionally known methods, or available iPS cells may be used. iPS cells used are usually from one type of cell, but cells of two or more different origins may also be used.

[0029] In the present invention, when producing small RNA derived from iPS cells or a protoplast composition derived from iPS cells containing small RNA from which the nuclei have been removed, the method for culturing iPS cells is not particularly limited. The medium used for culturing iPS cells is not particularly limited, and conventionally known mediums may be used. Furthermore, the culture conditions for culturing iPS cells are not particularly limited, and conventionally known conditions may be used for culturing.

[0030] In the present invention, iPS cells for obtaining small RNA or protoplasmic compositions are preferably iPS cells that have been subcultured. ６ ~10 ８ It is preferable to carry out the process until 10 cells are obtained. ７It is advisable to culture iPS cells using the number of subcultures as a guideline. The subculture may be performed two or more times, three or more times, four or more times, five or more times, six or more times, seven or more times, eight or more times, nine or more times, or ten or more times. It is preferable to obtain small RNA or protoplasmic compositions using iPS cells that have been subcultured two or more times, 2 to 20 times, 2 to 12 times, 2 to 10 times, 2 to 5 times, or 2 to 4 times. The upper limit of the number of subcultures is not particularly limited, but may be 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, or 12 or less.

[0031] Furthermore, in the present invention, iPS cells from which small RNA or protoplast compositions are obtained are preferably iPS cells that have reached a confluence of 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more. The iPS cells may be iPS cells that have reached a confluence of 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, or 90% or less. The iPS cells of the present invention are preferably iPS cells that have reached a confluence of 75% or more, and preferably 75% to 95%, or 80% to 90%. In the present invention, iPS cells that have reached 75% or more, 75% to 95%, or 80% to 90% confluence may be detached and further subcultured to again obtain iPS cells that have reached 75% or more, 75% to 95%, or 80% to 90% confluence. The multiple times of culturing to reach confluence may be two, three, four, five, or six times.

[0032] In the present invention, when the small RNA or protoplasmic composition is derived from adipose tissue-derived stem cells, the adipose tissue-derived stem cells are not particularly limited.

[0033] Stem cells are immature cells that have pluripotency and the ability to proliferate through self-renewal, and adipose tissue-derived stem cells (ADSCs) are a type of mesenchymal stem cells.

[0034] Adipose tissue, which serves as the source of adipose tissue-derived stem cells, is a type of biological tissue composed of adipocytes, and the adipose tissue is not particularly limited. The adipose tissue may be abdominal or non-abdominal adipose tissue, and may be brown adipose tissue or white adipose tissue. The method for obtaining the adipose tissue is not particularly limited. Commercially available adipose tissue-derived stem cells may be used. The adipose tissue-derived stem cells used are usually cells derived from one type of adipose tissue, but cells of two or more different origins may also be used.

[0035] In the present invention, adipose tissue-derived stem cells for obtaining small RNA or protoplasmic compositions are preferably adipose tissue-derived stem cells that have been subcultured. ６ 5 to 10 ８ It is preferable to carry out the process until 10 cells are obtained. ７ It is advisable to culture the cells as a guideline. The subculture may be performed two or more times, three or more times, four or more times, five or more times, six or more times, seven or more times, eight or more times, nine or more times, or ten or more times. It is preferable to obtain small RNA or protoplasmic compositions using adipose tissue-derived stem cells that have been subcultured two or more times, 2 to 20 times, 2 to 12 times, 2 to 10 times, 2 to 5 times, or 2 to 4 times. The upper limit of the number of subcultures is not particularly limited, but may be 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, or 5 or less.

[0036] In the present invention, adipose tissue-derived stem cells for obtaining small RNA or protoplasmic compositions are preferably adipose tissue-derived stem cells that have reached a confluence of 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more. The adipose tissue-derived stem cells may be adipose tissue-derived stem cells that have reached a confluence of 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, or 90% or less. The adipose tissue-derived stem cells of the present invention are preferably adipose tissue-derived stem cells that have reached a confluence of 75% or more, and preferably 75% to 95%, or 80% to 90%. The adipose tissue-derived stem cells of the present invention may be detached from adipose tissue-derived stem cells that have reached 75% or more, 75% to 95%, or 80% to 90% confluence and further subcultured to obtain adipose tissue-derived stem cells that have reached 75% or more, 75% to 95%, or 80% to 90% confluence again. The multiple times of confluence may be two, three, four, five, or six times.

[0037] In the present invention, the method for producing a protoplast composition is not particularly limited, but it is preferably produced by the following steps. First, iPS cells or adipose tissue-derived stem cells in a desired state are obtained by culturing, harvesting, etc. Next, preferably, the protoplast composition is produced by carrying out the following steps: (1) disrupting the iPS cells or adipose tissue-derived stem cells; and (2) centrifuging the disruption solution obtained in step (1) or the disruption solution. The obtained protoplast composition may be used as is as small RNA derived from iPS cells or adipose tissue-derived stem cells. Alternatively, the obtained protoplast composition may be subjected to centrifugation or antibody removal of nuclei and / or mitochondria, and used as a protoplast composition derived from iPS cells or adipose tissue-derived stem cells containing small RNA. Furthermore, an extraction and purification step may be carried out to obtain small RNA derived from iPS cells or adipose tissue-derived stem cells.

[0038] In the present invention, the origin of the iPS cells or adipose tissue-derived stem cells is not particularly limited, and they may be cells derived from mammals (including pet animals, livestock, and laboratory animals), but are preferably human iPS cells or adipose tissue-derived stem cells. Mammals include, but are not limited to, humans, monkeys, pigs, cows, horses, goats, sheep, dogs, cats, mice, rats, guinea pigs, and hamsters.

[0039] In the present invention, the pharmaceutical composition is used to suppress chronic inflammation. Here, suppression of chronic inflammation may mean treating, alleviating, etc., diseases or symptoms known as chronic inflammation, may mean exhibiting an inflammation-suppressing effect, or may mean preventing diseases or symptoms known as chronic inflammation. In the present invention, the pharmaceutical composition may be a chronic inflammation suppressor, may be a pharmaceutical composition for use in the treatment or prevention of chronic inflammation, or may be a pharmaceutical composition for use in the treatment or prevention of diseases or symptoms related to chronic inflammation. In the present invention, the pharmaceutical composition may be a pharmaceutical composition for use in alleviating diseases or symptoms related to chronic inflammation.

[0040] In the present invention, the inhibitory effect on chronic inflammation has been confirmed using an experimental autoimmune encephalomyelitis (EAE) model, as described in the Examples. In particular, in the Examples using an experimental autoimmune encephalomyelitis (EAE) model, it was confirmed that experimental autoimmune encephalomyelitis (EAE) did not develop, thereby confirming the preventive effect of the present invention on chronic inflammation. Examples of chronic inflammation in the present invention include, but are not limited to, multiple sclerosis, neuromyelitis optica (NMOSD), and MOG antibody-associated disease (MOGAD). Examples of chronic inflammation include, but are not limited to, asthma, arthritis, psoriasis, rheumatoid arthritis, ankylosing spondylitis, vasculitis, hypertension, autoimmune fatty liver, alcoholic hepatitis, enteritis, autoimmune uveitis, cystitis, dermatitis, rhinitis, periodontitis, stomatitis, alopecia areata, and sarcopenia.

[0041] In the present invention, small RNA and protoplasmic compositions have been confirmed to be effective in an experimental autoimmune encephalomyelitis (EAE) model, and since this animal model is known to be induced by IL-17-producing (TH17) or IFN-γ-producing (TH1) myelin-specific CD4+ cells, the chronic inflammation may be a disease associated with IL-17 and / or IFN-γ. For information on IL-17-related diseases, see, but are not limited to, the following review: de Morales, JMGR et al. (2020). Critical role of interleukin (IL)-17 in inflammatory and immune disorders: An updated review of the evidence focusing in controversies. Autoimmunity reviews, 19(1), 102429. Furthermore, for information on chronic inflammation in the present invention, see the following: Asthma Molet, S. et al. (2001). IL-17 is increased in asthmatic airways and induces human bronchial fibroblasts to produce cytokines. Journal of Allergy and Clinical Immunology, 108(3), 430-438. Osteoarthritis Attur, MG et al. (1997). Interleukin-17 upregulation of nitric oxide production in human osteoarthritis cartilage. Arthritis & Rheumatism: Official Journal of the American College of Rheumatology, 40(6), 1050-1053. Psoriasis Silfvast-Kaiser, A. et al. (2019). Anti-IL17 therapies for psoriasis. Expert Opinion on Biological Therapy, 19(1), 45-54.Rheumatoid arthritis Van Den Berg et al. (2009). IL-17 as a future therapeutic target for rheumatoid arthritis. Nature Reviews Rheumatology, 5(10), 549-553. Ankylosing spondylitis Yin, Y. et al. (2020). Efficacy and safety of IL-17 inhibitors for the treatment of ankylosing spondylitis: a systematic review and meta-analysis. Arthritis research & therapy, 22(1), 1-11. Vascular disorders (vasculitis) Csiszar, A. et al. (2003). Aging-induced proinflammatory shift in cytokine expression profile in rat coronary arteries. The FASEB journal, 17(9), 1183-1185. Hypertension Madhur, MS et al. (2010). Interleukin 17 promotes angiotensin II-induced hypertension and vascular dysfunction. Hypertension, 55(2), 500-507. Autoimmune fatty liver Terziroli Beretta-Piccoli, B. et al. (2022). Autoimmune hepatitis. Cellular & molecular immunology, 19(2), 158-176. Alcoholic hepatitis Lemmers, A. et al. (2009). The interleukin-17 pathway is involved in human alcoholic liver disease. Hepatology, 49(2), 646-657. Inflammatory / BR>-diseases (ulcerative colitis, Crohn's disease, infectious colitis, ischemic colitis) Fujino, S. et al. (2003).Increased expression of interleukin 17 in inflammatory bowel disease. Gut, 52(1), 65. Autoimmune uveitis Zhong, Z. et al. (2021). Activation of the interleukin-23 / interleukin-17 signaling pathway in autoinflammatory and autoimmune uveitis. Progress in retinal and eye research, 80, 100866. Dermatitis (atopic dermatitis, allergic rhinitis, allergic asthma: a systematic review) Hofmann, MA et al. (2021). Role of IL-17 in atopy-A systematic review. Clinical and Translational Allergy, 11(6), e12047. Periodontal disease Feng, Y. et al. (2022). Role of interleukin-17A in the pathomechanisms of periodontitis and related systemic chronic inflammatory diseases. Frontiers in Immunology, 13, 862415. Alopecia areata Ramot, Y. et al. (2018). IL-17 inhibition: is it the long-awaited savior for alopecia areata? Archives of dermatological research, 310, 383-390. Sarcopenia Xiong, L. et al. (2023). Association between Elderly Sarcopenia and Inflammatory Cytokine Interleukin-17: A Cross-Sectional Study. BioMed Research International, 2023. In the present invention, reference is also made to previously known literature regarding cystitis, dermatitis, rhinitis, stomatitis (gingivostomatitis), and the like.

[0042] In the present invention, the suppression of chronic inflammation may be achieved by suppressing the expression of inflammatory cytokines. Factors (genes) involved in the suppression of inflammatory cytokine expression are not particularly limited, but include, for example, the following: 18S, Gapdh, Hprt1, Gusb, Agtr2, Bax, Bcl2, Bcl2l1, C3, Ccl19, Ccl2, Ccl3, Ccl5, Ccr2, Ccr4, Ccr7, Cd19, Cd28, Cd34, Cd38, Cd3e, Cd4, Cd40, Cd40lg, Cd68, Cd80, Cd86, Cd8a, Csf1, Csf2, Csf3, Ctla4, Cxcl10, Cxcl11, Cxcr3, Cyp1a2, C yp7a1, Edn1, Fas, Fasl, Fn1, Gzmb, H2-Ea, H2-Eb1, Hmox1, Icos, Ifng, Ikbkb, Il10, Il12a, Il12b, Il13, Il15, Il17a, Il18, Il1a, Il1b, Il2, Il2ra, Il3, Il 4, Il5, Il6, Il7, Il9, Lrp2, Lta, Nfkb1, Nfkb2, Nos2, Prf1, Ptgs2, Ptprc, Sel e, Selp, Ski, Smad3, Smad7, Socs1, Socs2, Stat1, Stat3, Stat4, Stat6, Tbx21, Tgfb1, Tnf, Tnfrsf18, Vcam1, Vegfa, Ace, Icam1, Lif, Ly96, Nfatc3 and Nfatc4. Among these, the gene involved in the expression of inflammatory cytokines is preferably C3, Il1b, Nos2, Selp, Vegfa, Il3, Lrp2, Cd4, Cd40lg, Fas1 or Il4, and may be Cd4, Cd40lg, Fas1 or Il4.

[0043] The pharmaceutical composition of the present invention may contain other pharmaceutically acceptable ingredients, including, but not limited to, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, physiological saline, etc.

[0044] In the present invention, chronic inflammation can be suppressed by administering small RNA derived from iPS cells or adipose tissue-derived stem cells, a protoplast composition derived from iPS cells or adipose tissue-derived stem cells, or a pharmaceutical composition of the present invention to a subject. The route of administration, dosage, and administration interval may be adjusted as appropriate. Examples of subjects include humans, monkeys, pigs, cows, horses, goats, sheep, dogs, cats, mice, rats, guinea pigs, and hamsters.

[0045] In the present invention, small RNA, protoplasmic composition, or pharmaceutical composition may be administered as prepared just before use, or as prepared in advance. When small RNA, protoplasmic composition, or pharmaceutical composition is prepared in advance, for example, it may be thawed from a frozen storage, or it may be refrigerated. When prepared just before use, the starting material may be iPS cells or adipose tissue-derived stem cells, iPS cells or adipose tissue-derived stem cells that have been cultured as specified, or a lysate of iPS cells or adipose tissue-derived stem cells.

[0046] The pharmaceutical composition of the present invention may be used as an injection. In particular, intravenous administration is preferred. When the pharmaceutical composition of the present invention is an injection, it can be easily injected into the body, which is economical in terms of surgical time and cost. Furthermore, the pharmaceutical composition of the present invention has excellent storage properties due to the use of small RNA and protoplasmic compositions, which is a novel approach. Therefore, it has the advantage of being able to be stored (especially frozen) as an injection, making it more convenient than cell therapy in which cells are directly administered.

[0047] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples in any way.

[0048] Example 1 The following materials were used for culturing iPS cells: 1. Cell culture medium Stem Flex ＴＭMedium (manufactured by Thermo Fisher Scientific) + 0.5% Penicillin / Streptomycin (manufactured by Wako) Y-27632 (manufactured by Wako) (a 10 mM solution was prepared, then aliquoted and stored frozen at -30°C. This was thawed and added to the medium to a final concentration of 10 μM (only during subculture))

[0049] 2 Culture substrate Vitronectin (manufactured by Thermo Fisher Scientific)

[0050] 3. Cell washing solution, detachment solution D-PBS(-) (manufactured by Wako) 0.5mM EDTA (UltraPure ＴＭ 0.5M EDTA, pH 8.0 (Thermo Fisher Scientific) diluted 1,000 times with D-PBS(-)

[0051] 5 Culture containers 10cm dish (manufactured by TPP) 6well-plate (manufactured by TPP) 12well-plate (manufactured by TPP)

[0052] Cell culture (human iPS cells) was performed using the following method. 1. Reactivation of frozen cells 1) Frozen iPS cell stock was thawed in a 37°C water bath. 2) The cells were suspended in culture medium (+10 mM Y-27632). 3) The cells were centrifuged at 200g for 3 minutes at room temperature. 4) The supernatant was removed, and the cells were suspended in culture medium (+10 mM Y-27632) and placed in a 6-well plate at approximately 2-3 x 10 cells. ４ cell / cm ２ 5) 5% CO ２ 6) The next day, the medium was replaced with a culture medium.

[0053] 2. Cell Recovery and Freezing 1) It was confirmed that the cells in 6) above had grown to approximately 80-90% confluence. 2) The medium was aspirated and washed once with cell washing solution. 3) 1 mL of cell detachment solution was added (per well (6-well plate)) and the plate was left to stand in an incubator at 37°C for approximately 5 minutes. 4) The state of the cells was confirmed under a microscope, and the cell detachment solution was aspirated. 5) 2 mL of cell culture medium (per well (6-well plate)) was added, the cells were detached by pipetting, thoroughly suspended, and then collected in a 15 mL centrifuge tube. 6) A portion of the cell suspension was mixed with an equal volume of trypan blue solution, and the cell number was counted using a hemocytometer. 7) The cell suspension in 5) was centrifuged at 200g for 3 minutes at room temperature. 8) The culture supernatant was removed, and the cell pellet was frozen and stored at -80°C.

[0054] 3. Extraction and Purification of Small RNA 1) Cell pellets were homogenized using TRIzol Reagent (Invitrogen). 2) Small RNA (including miRNA) was extracted and purified from the homogenized sample using PureLink miRNA Isolation Kit (Invitrogen). Total RNA was dissolved in Nuclease-Free Water (Invitrogen). Each procedure was performed according to the manual.

[0055] Below, myelin oligodendrocyte glycoprotein (MOG) was expressed in C57BL / 6J mice. ３５－５５ (MOG ３５－５５ The effect of the present invention on an experimental autoimmune encephalomyelitis (EAE) model was evaluated, which was induced by administering an emulsion of IgG1 and complete Freund's adjuvant (CFA) and pertussis toxin (PTX).

[0056] Approximately 10-week-old female C57BL / 6J mice purchased from Jackson Laboratory Japan were injected with an elicitation reagent (Hooker Kits) 11 days after the day of delivery (Day 0). ＴＭ MOG ３５－５５An autoimmune encephalomyelitis (EAE) model was created by administering EGFR / CFA emulsion and PTX (Hooke Laboratories). Food and water were provided ad libitum. 1. Emulsion administration (Day 0) 1-1. All mice were anesthetized with isoflurane. 1-2. Hooke Kits ＴＭ MOG included in ３５－５５ 100 μL each of an emulsion of PTX and CFA was administered subcutaneously at two sites. 2. PTX Administration (Day 0 and Day 1) 2-1. The dose of PTX was 195 ng / mouse. The required amount of PTX was collected and diluted to the specified volume with PBS. 2-2. 100 μL of PTX was administered intraperitoneally 1 to 6 hours after emulsion administration (Day 0). 2-3. 100 μL of PTX was administered intraperitoneally 24 hours after the first PTX administration (Day 1).

[0057] On Day 14, the vehicle group received a single intravenous administration of the vehicle administration solution, and the drug group received a single intravenous administration of the drug administration solution. The n number was 4 for each group. The vehicle administration solution was 10-fold diluted Nuclease-Free Water (not DEPC-Treated) (Invitrogen) with PBS (pH 7.4, Thermo Fisher Scientific). The drug administration solution was 2.01 x 10 ７ A small RNA extract (100 μL) derived from 114.4 μg / mL of cells was diluted 10-fold with PBS and used. Blood was collected, serum separated, and dissected for evaluation. The EAE score was calculated based on the behavior of all mice, rated on a 10-point scale. The evaluation criteria are as follows: 0: no abnormalities, 0.5: stiff tail, 1: tail weakness, 1.5: tail weakness and weakness, 2: paralysis of one limb, 2.5: paralysis of one limb and weakness of the other limb, 3: complete paralysis of both hind limbs, 3.5: recumbency, 4: moribund state, 5: death

[0058] The results are shown in Figures 1 and 2. A tendency for weight loss and suppression of the pathological condition was confirmed in the drug group, confirming the effectiveness of the drug group in the EAE animal model.

[0059] Furthermore, an evaluation test of inflammatory cytokines in spleen cells in an experimental autoimmune encephalomyelitis (EAE) model was carried out. Spleen cells were prepared as follows. The reagents and instruments used are as follows. RPMI-1640 (containing L-glutamine and phenol red, manufactured by FUJIFILM Wako) 100 μm cell strainer (manufactured by Corning) 40 μm cell strainer (manufactured by Corning) ACK Lysing Buffer (manufactured by Lonza) 0.4% Trypan blue (manufactured by Thermo Fisher Scientific) Hemocytometer (OneCell Counter, manufactured by OneCell) CD4+ T Cell Isolation Kit, mouse (manufactured by Miltenyi Biotec) LS Columns (manufactured by Milltenyi Biotec) MidiMACS (Separator) (Milltenyi Biotec) Isolation buffer: 0.5% BSA (Fujifilm Wako) + 2 mM EDTA (Thermo Fisher Scientific) + D-PBS(-) (Fujifilm Wako) PluriStrainer 30 μm (PluriSelect Life Science) 1. Cell preparation Cells were prepared as follows. 1-1 Cells were obtained from the collected spleen by the following method. 1-2 A cell strainer (100 μm) was placed in a 6-well plate and the collected spleen was placed on it. 1-3 An incision was made in the spleen capsule with dissecting scissors. While dripping 3 mL of ice-cold RPMI-1640 onto the spleen to prevent it from drying out, cells were strained out of the spleen using the plunger of a 5 mL syringe. 1-4 The cells that had fallen into the well were suspended, filtered through a cell strainer (40 μm), and collected in a 50 mL centrifuge tube, then placed on ice. 1-5 The tube was centrifuged at 300 × g, 4°C, and 5 minutes, and the supernatant was aspirated. 1-6 1 mL of ACK Lysing Buffer was added, and the cells were loosened by pipetting. 1-7 The tube was left to stand at room temperature for 1 minute. 1-8 After hemolysis, 10 mL of cold RPMI-1640 was added to the spleen and suspended, and the cell suspension was transferred to a new 50 mL centrifuge tube through a 40 μm cell strainer.1-9 Centrifugation was carried out at 300 x g, 4°C, and 5 min. 1-10 After aspirating the supernatant, 2 mL of isolation buffer was added, the cells were suspended, and passed through a strainer (30 μm). 1-11 A portion of the suspension was mixed with trypan blue solution, and the cell number was counted using a hemocytometer. Of the resulting spleen cells, the maximum number of cells that could be separated using the amount of antibody cocktail provided with the kit was used for the subsequent steps. 1-12 Centrifugation was carried out again (300 x g, 4°C, 5 min). 1-13 40 μL / 1 x 10 isolation buffer was added. ７ 1-14 The antibody cocktail provided with the kit was added at 10 μL / 1 × 10 ７ 1-15 The mixture was left standing on ice for 5 minutes. 1-16 30 μL / 1×10 ７ 1-17 Anti-biotin microbeads included in the kit were added at 10 μL / 1 × 10 ７ 1-18 The suspension was left to stand on ice for 10 minutes. 1-19 If the volume of each cell suspension was less than 500 μL, isolation buffer was added to bring the volume to 500 μL. The suspension was then placed on ice. 1-20 The LS column was placed on the MidiMACS. 1-21 The LS column was washed with 3 mL of isolation buffer. 1-22 The cell suspension was added to the LS column, and the flow-through containing unlabeled cells was collected. 1-23 The LS column was washed with 3 mL of isolation buffer, and the flow-through was collected together with the above flow-through. 1-24 A portion of the suspension was mixed with trypan blue solution, and the cell count was counted using a hemocytometer. 1-25 The suspension was centrifuged at 300 × g, 4°C, and 5 minutes. The supernatant was removed, and the cell pellet was frozen and stored at -80°C. 2. Inflammatory Cytokine Gene Expression Analysis Inflammatory cytokine gene expression analysis was performed as follows. 2-1 Evaluation gene TaqMan ＴＭArray Mouse Immune Response (Thermo Fisher Scientific) 2-2 Quantitative PCR Analysis 2-2-1 1000 μL of TRIzol Reagent (Invitrogen) was added to the frozen cell pellet and homogenized. 2-2-2 Total RNA was extracted and purified (including DNase treatment) from 500 μL of the homogenized sample using RNeasy Mini Kit (QIAGEN). Total RNA was eluted using 50 μL of RNase-free water. Details of the operation were as described in the RNeasy Mini Kit (QIAGEN) manual. 2-2-3 The concentration (ng / μL) and purity ratio (A260 / A280) were confirmed using NanoDrop One (Thermo Fisher Scientific). 2-2-4 The quality of the total RNA was confirmed by agarose gel electrophoresis. 2-2-5 Using 500 ng of total RNA as a template, cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems). The synthesized cDNA was diluted with PCR Grade Water and used as the DNA template for real-time PCR. The expression level of each gene was analyzed using TaqMan Array Mouse Immune Response (Thermo Fisher Scientific). TaqMan Fast Advanced Master Mix for qPCR (Applied Biosystems) was used as the reaction reagent for real-time PCR. The StepOnePlus Real-Time PCR System (Applied Biosystems) was used.Because the StepOnePlus device used was compatible with the Fast type (0.1 mL scale), the solution in each well of TaqMan Array Mouse Immune Response (Thermo Fisher Scientific, Standard type, 0.2 mL scale) was transferred to another reaction plate (0.1 mL / well) for analysis.

[0060] As a result of real-time PCR analysis, the following 11 genes were identified as genes with the highest differences in expression levels. Expression levels of Cd4, Cd40lg, Fasl, and Il4 genes were higher than in the non-induced group, while expression levels of C3, Il1b, Nos2, Selp, and Vegfa genes were lower than in the non-induced group. Furthermore, Il3 and Lrp2 genes were below the detection limit in all individuals in the non-induced group, but were detected in the EAE group. Thus, this study identified multiple candidate genes related to inflammatory cytokines with different expression levels in CD4-positive T cells in the spleen tissue of EAE model mice.

[0061] Example 2: Cell lysates of iPS cells were prepared as follows. Genomic DNA contained in the cell lysates was quantified by real-time PCR to confirm enucleation from the cell lysates. Furthermore, mitochondrial Cytochrom c protein in the supernatant and precipitate of the cell lysates was detected by Western blot analysis to confirm the removal of mitochondria from the cell lysates.

[0062] 1. The following materials were used for extraction and purification of small RNA: ISOSPIN Liquid Sample miRNA (Nippon Gene Co., Ltd.) Nuclease-Free Water (Invitrogen) DynaMarker RNA Low II (BioDynamics Laboratory) NanoDrop One (Thermo Fisher Scientific) 2. The following materials were used for quantification of genomic DNA: Nucleic Acid Purification Kit MagExtractor -Geome- (TOYOBO Co., Ltd.) Real-time PCR was performed as follows. StepOnePlus Real-time PCR System (Applied Biosystems) Investigator Quantiplex Kit (200) (Qiagen) 3. The following materials were used to confirm mitochondrial removal. Cell lysis solution: RIPA Buffer (manufactured by Wako) 3x Sample buffer: 187.5mM Tris-HCl (pH 6.8), 6% SDS, 30% glycerol Electrophoresis gel: e-PAGEL (gradient gel, 10-20%) Iaemmli buffer: SDS Tris-glycine buffer CBB staining solution: 0.1% CBB, 30% methanol, 10% acetic acid Destaining solution: 30% methanol, 10% acetic acid Detection antibody (primary antibody): Cytochrom c Polyclonal antibody (manufactured by Proteintech) (secondary antibody): Anti-Rabbit IgG (H+L), HRP Conjugate (Promega) Chemiluminescence ImmunoStar® Zeta (Wako) X-ray film (GE Healthcare)

[0063] 1. Cell Recovery and Freezing 1) The frozen cells were euthanized as in Example 1, and the resulting cells were confirmed to have proliferated to approximately 80-90% confluence. 2) The medium was aspirated and washed once with cell washing solution. 3) 4 mL of cell detachment solution was added, and the cells were left to stand in an incubator at 37°C for 3-5 minutes. 4) The cell condition was confirmed under a microscope, and the cell detachment solution was aspirated. 5) 5 mL of cell culture medium was added, and the cells were detached by pipetting, thoroughly suspended, and then collected in a 15 mL centrifuge tube. 6) A portion of the cell suspension was mixed with an equal volume of trypan blue solution, and the cell number was counted using a hemocytometer. 7) The cell suspension from 5) was centrifuged at 200 g for 3 minutes at room temperature. 8) The culture supernatant was removed, and the cell pellet was washed with cell washing solution. 9) The cell suspension was centrifuged at 200 g for 3 minutes at 4°C. 10) Remove the supernatant, suspend the cell pellet in cell washing solution, and resuspend approximately 1 x 10 ６ The solution was adjusted to a concentration of 1000 cells / mL and stored frozen at −80° C. until use in the next step.

[0064] 2. Preparation of cell lysate 1) The frozen cell suspension from 10) above was placed in a refrigerator at 4°C and thawed. 2) After thawing, it was frozen again at -80°C. Once freezing was confirmed, it was thawed again in a refrigerator at 4°C. The freeze-thaw process was repeated in the same manner. 3) It was centrifuged at 290 x g for 5 minutes at 4°C, and the supernatant was collected in a 15 mL centrifuge tube.

[0065] 3. Enucleation and Mitochondrial Removal 1) The supernatant was centrifuged at 650 x g for 10 minutes at 4°C to remove nuclei, and the supernatant was collected in a 15 mL centrifuge tube. 2) The supernatant was centrifuged at 8,000 x g for 20 minutes at 4°C, and then collected in a 15 mL centrifuge tube and filtered through a 0.2 μm cellulose acetate membrane filter. 3) The supernatant was stored in a refrigerator at 4°C.

[0066] 4. Genomic DNA Extraction 1) Frozen supernatants were thawed on ice. 2) Genomic DNA was extracted and purified from each supernatant using the Nucleic Acid Purification Kit MagExtractor -Genomic- and finally dissolved in TE solution. Details of each procedure were performed according to the manual. 5. Small RNA Extraction and Purification 1) The collected cell lysates (5 types) were thawed on ice. 2) Small RNA was extracted and purified from each cell lysate using ISOSPIN Liquid Sample miRNA. Total RNA was dissolved in nuclease-free water. Details of each procedure were performed according to the manual. 3) Yield and purity ratio were confirmed using NanoDrop One. 4) The quality of small RNA was confirmed by electrophoresis. 6. Quantification of Genomic DNA (Real-Time PCR) 1) Real-time PCR was performed using two purified genomic DNA solutions as templates and the primers included with the Investigator Quantiplex Kit. 7. Confirmation of Mitochondrial Removal (Western Blotting (Cytchrom c Detection)) 1) The precipitate (stored at -80°C) was thawed on ice. After tapping thoroughly, RIPA buffer was added, and the precipitate was dissolved by pipetting. The solution was collected in a 1.5 mL tube, placed on ice for 5 minutes, and then centrifuged at 20,000 x g for 10 minutes at 4°C. The supernatant was collected in a new 1.5 mL tube. 2) Protein concentration was measured using the DC Protein Assay. 3) One-third of the volume of 3x SDS sample buffer was added to the collected supernatant (protein extraction sample) and a portion of the supernatant, and the mixture was heated at 95°C for 5 minutes to prepare a sample for electrophoresis. 4) SDS-PAGE and Western blotting were performed using the prepared sample.

[0067] When cell lysates were stored in a 4°C refrigerator for 8 days, the amount of small RNA extracted from the cell lysate decreased by approximately 6-8% on days 2, 4, 6, and 8 compared to day 0, but no degradation of small RNA was observed over time. Furthermore, the genomic DNA content in the cell lysate decreased from 0.129 μg / mL to 0.0096 μg / mL after the enucleation and mitochondrial removal processes, suggesting that approximately 93% of the genomic DNA was removed by the enucleation and mitochondrial removal processes. CBB staining revealed no bands likely to be Cytochrome c (14 kDa) in any of the samples. Furthermore, since the cell lysates obtained in this example are believed to contain the small RNA obtained in Example 1, it is believed that the pharmacological effects in an experimental autoimmune encephalomyelitis (EAE) animal model can be confirmed by conducting evaluation experiments in an EAE animal model in the same manner as in Example 1.

[0068] The miRNAs whose expression was confirmed in iPS cells (Undetermined in hDPSC cells) were the following 39 miRNAs. hsa-miR-95-3p-478213_mir hsa-miR-96-5p-478215_mir hsa-miR-105-5p-477865_mir hsa-miR-124-3p-477879_mir hsa-miR-126-3p-477887_mir hsa-miR-135a-5p-478581_mir hsa-miR-150-5p-477918_mir hsa-miR-183-5p-477937_mir hsa-miR-187-3p-...hsa-miR-873-5p-478204_mir hsa-miR-876-3p-479186_mir hsa-miR-211-5p-478507_mir hsa-miR-520f-3p-479343_mir

[0069] Example 3 Cell lysates of adipose tissue-derived stem cells were prepared as follows: The adipose tissue-derived cells used were serial number PT-5006 (manufactured by Lonza). 1-1 Cell culture 1-1-1 Frozen cells Human adipose-derived stem cells x 1 type (1 x 10^6 cells or more / vial, transport temperature: -80°C) (manufactured by Lonza) 1-2 Extraction and purification of small RNA 1-2-1 Extraction reagents ISOSPIN Liquid Sample miRNA (manufactured by Nippon Gene Co., Ltd.) Nuclease-Free Water (manufactured by Invitrogen) DynaMarker RNA Low II (manufactured by BioDynamics Laboratory) NanoDrop One (manufactured by Thermo Fisher Scientific) 2 Test materials 2-1 Cell culture 2-1-1 Cell culture medium (For human adipose-derived stem cells) ADSC-Adipose-Derived Stem Cells Growth Medium Bullet Kit™ (manufactured by Lonza) Mesenchymal Stem Cell Growth Medium 2 (manufactured by PromoCell) 2-1-2 Cell washing solution, detachment solution, and neutralizing solution D-PBS(-) (manufactured by Wako) Trypsin / EDTA Solution (0.025% Trypsin) (manufactured by Lonza) Trypsin Neutralizing Solution (manufactured by Lonza) 2-1-3 Culture vessel 100 mm dish (Manufactured by TPP) 150 mm dish (Manufactured by TPP) 2-1-4 Cryopreservation solution Stem-cellbanker GMP grade (Manufactured by Zenoaq) 3 Test method 3-1 Cell culture (human adipose-derived stem cells) 3-1-1 Awakening of frozen cells 1) Frozen cell stock was thawed in a 37°C water bath. 2) Suspended in culture medium. 3) Centrifuged at 200g for 3 minutes at room temperature. 4) The supernatant was removed, and the cells were suspended in culture medium and placed in a 100 mm or 150 mm culture dish at a density of approximately 5 x 10 ３ cell / cm ２ 5) The cells were seeded at a cell density of 1000 x g. ２The cells were cultured at 37°C in the presence of ATP. 3-1-2 Subculture 1) It was confirmed that the cells had proliferated to approximately 80-90% confluence. 2) The medium was removed by aspirating and washed once with cell washing solution. 3) 3 mL (per 100 mm dish) of cell detachment solution was added and the dish was left to stand in an incubator at 37°C for approximately 5 minutes. 4) 3 mL (per 100 mm dish) of cell neutralizing solution was added, and the cells were thoroughly suspended by pipetting, and then collected in a 15 mL centrifuge tube. 5) The dish was centrifuged at 200 g for 3 minutes at room temperature. 6) The supernatant was removed by aspirating, and the cell pellet was suspended in culture medium. 7) A portion of the cell suspension was mixed with an equal volume of trypan blue solution, and the cell number was counted. 8) 25 mL of culture medium was added to a 150 mm culture dish, and approximately 5 x 103 cells / cm were plated. ２ 9) The cells were seeded at a cell density of 1000 kJ / well. ２ The mixture was cultured at 37°C in the presence of 9.23 x 10 ７ The cells were repeatedly subcultured until they reached 100 cells or more (because cell growth was slow after the second passage, the medium was changed to Mesenchymal Stem Cell Growth Medium 2 at the third passage. After the medium change, the growth rate increased from approximately 1.3-fold / day to approximately 2.3-fold / day). 3-1-3 Cell Recovery and Freezing 1) It was confirmed that the cells in 3-1-2 had grown to approximately 80-90% confluence. 2) The medium was removed by aspirating and the cells were washed once with cell washing solution. 3) 6 mL (per 150 mm dish) of cell detachment solution was added, and the cells were allowed to stand in an incubator at 37°C for approximately 5 minutes. 4) 6 mL (per 100 mm dish) of cell neutralizing solution was added, and the cells were thoroughly suspended by pipetting, and then collected in a 50 mL centrifuge tube. 5) Centrifugation was performed at 200 g for 3 minutes at room temperature. 6) The supernatant was removed by aspirating, and the cell pellet was suspended in culture medium. 7) A portion of the cell suspension was mixed with an equal volume of trypan blue solution, and the cell number was counted. 8) 6.03 x 10 ７ The cell suspension containing the cells was collected in a 50 mL tube. 9) The tube was centrifuged at 200 g for 3 minutes at room temperature. 10) The supernatant was removed, and the cell pellet was suspended in cell washing solution. 4 x 10 ６The resulting solution was adjusted to a concentration of 1.07 x 10 cells / mL and stored frozen at -80°C until use in step 3-2. 11) The remaining cell suspension was centrifuged at 200 g for 3 minutes at room temperature. 12) The supernatant was removed by aspirating, and a freezing solution was added to the supernatant to obtain a 1.07 x 10 cells / mL solution. ７ The cell density was adjusted to 100 cells / mL. 13) The cell suspension was dispensed in 1 mL portions into cryovials. 14) The suspension was frozen and stored at -80°C. 15) From the next day onwards, the suspension was transferred to a liquid nitrogen tank (vapor phase). 3-2 Extraction and purification of small RNA 3-2-1 Preparation of disrupted filtrate 1) The frozen cell suspension from 10) of 3-1-3 was placed in a 4°C refrigerator (or on ice) and thawed. 2) After thawing, the suspension was frozen again at -80°C. Once frozen, the suspension was thawed again in a 4°C refrigerator. The freeze-thaw process was repeated in the same manner. 3) The suspension was centrifuged at 290 x g for 5 minutes at 4°C, and the supernatant was collected in a 50 mL centrifuge tube. 3-2-2 Enucleation and Mitochondrial Removal 1) To remove nuclei, the supernatant was centrifuged at 650 x g for 10 minutes at 4°C, and the supernatant was collected in a 50 mL centrifuge tube. 2) The supernatant was centrifuged at 8,000 x g for 20 minutes at 4°C, and then collected in a 50 mL centrifuge tube. It was filtered through a 0.2 μm cellulose acetate membrane filter and stored frozen at -80°C. 3-2-3 Extraction and Purification of Small RNA 1) The frozen filtrate prepared in 3-2-2 was thawed on ice. 2) Small RNA was extracted and purified from the filtrate using ISOSPIN Liquid Sample miRNA. Total RNA was dissolved in nuclease-free water. Details of each procedure were followed in the manual. 3) The yield and purity ratio were confirmed using NanoDrop One. 4) The quality of small RNA was confirmed by electrophoresis. 3.2 x 10 ７ The cultured adipose tissue-derived cells were cultured until they became 1000 cells. A cell lysate of the cultured adipose tissue-derived cells was obtained in the same manner as in Example 2. The obtained cell lysate was added to the protoplast composition of the present invention (5.0 × 10 ６The effect of the protoplast composition of the present invention on an experimental autoimmune encephalomyelitis (EAE) model was evaluated in the same manner as in Example 1. In the treatment group, a tendency toward suppression of weight loss and pathology was confirmed, and the effect of the protoplast composition of the present invention on the EAE animal model was also confirmed.

[0070] Example 4 The following experiment was also conducted in accordance with the evaluation of the effect of the present invention on the experimental autoimmune encephalomyelitis (EAE) model in Example 1. The subjects (groups) to be administered are shown in Table 1 below. For emulsion administration (Day 0), the drug administration solution was administered to the four-times administration group on Days 2, 6, 10, and 14. For the two-times administration group, the drug administration solution was administered on Days 2 and 6, and the vehicle administration solution was administered on Days 10 and 14. The group to which the drug administration solution was administered five and two days before induction was designated the two-times administration before induction group. The group to which the drug administration solution was administered five and two days before induction, and then the drug administration solution was administered on Days 2 and 6 was designated the four-times administration before and after induction group. The two-times administration before induction group was administered with the vehicle administration solution on Days 2 and 6.

[0071] In the vehicle group, pathological symptoms began to develop on Day 12, and by Day 19, symptoms had developed in all individuals, peaking at an average score of 2.3 between Days 17 and 21. In the group administered iPS lysate four times, symptoms began to develop on Day 12, and by Day 16, symptoms had developed in all surviving individuals, with an average score of 2.7 on Days 18 and 19, a peak average score slightly higher than that of the vehicle group, but a decrease in the average score was observed between Days 20 and 21. In the group administered ADSC lysate four times, symptoms also began to develop on Day 12, and by Day 18, symptoms had developed in all surviving individuals, with an average score of 2.2 at Day 21, a peak average score lower than that of the vehicle group. In the group administered iPS lysate twice, symptoms began to appear on Day 11, but the average peak score on Day 20 was 1.7, which was lower than that of the vehicle group. One individual remained asymptomatic until the end of the study (i.e., a preventive effect was confirmed). In the group administered ADSC lysate twice, symptoms began to appear on Day 11, and the average score from Day 17 to Day 20 was 2.4, which was similar to the average peak score of the vehicle group. However, one individual remained asymptomatic until the end of the study (i.e., a preventive effect was confirmed). Furthermore, the pre-induction group administered ADSC lysate twice had an average score of 2.1, which was lower than that of the vehicle group. In the group administered iPS lysate four times before and after induction and the group administered ADSC lysate four times before and after induction, the average score was lower on Days 20 to 21 from the average peak score. The effect of the present invention was confirmed in an experimental autoimmune encephalomyelitis (EAE) model.

[0072] Example 5 In the same manner as in Example 1 to evaluate the effect of the present invention on the experimental autoimmune encephalomyelitis (EAE) model, the following experiment was also carried out by administering 1.0 × 10 adipose tissue-derived cells to the mice on Day 14. ６ When 0.932 μg / animal (6.03 × 10 cells / animal) of adipose tissue-derived cell lysates was administered, ７When two groups of 8 mice (16 mice) of adipose tissue-derived cells were administered in combination, the score further improved. The results are shown in Figure 3. Although the data are not disclosed, the average score also decreased from the peak average score on days 20 and 21 in the group administered with cell lysate derived from adipose tissue.

[0073] Example 6: Using cell lysates from adipose tissue-derived cells that had been enucleated and mitochondria-removed, small RNAs were extracted and purified in the same manner as in Example 1. Expression of the following 12 miRNAs was confirmed in adipose tissue-derived cells (undetermined in hDPSC cells). hsa-miR-29c-3p-479229_mir hsa-miR-135a-5p-478581_mir hsa-miR-187-3p-477941_mir hsa-miR-196b-5p-478585_mir hsa-miR-197-3p-477959_mir hsa-miR-198-478749_mir hsa-miR-338-3p-478037_mir hsa-miR-504-5p-478144_mir hsa-miR-548a-5p-479501_mir hsa-miR-570-3p-479053_mir hsa-miR-589-5p-479073_mir hsa-miR-597-5p-478339_mir

[0074] The following seven miRNAs were confirmed to be expressed in both iPS cells and adipose tissue-derived cells (undetermined in hDPSC cells): hsa-miR-135a-5p-478581_mir hsa-miR-187-3p-477941_mir hsa-miR-197-3p-477959_mir hsa-miR-338-3p-478037_mir hsa-miR-504-5p-478144_mir hsa-miR-570-3p-479053_mir hsa-miR-589-5p-479073_mir Furthermore, the following 18 miRNAs were not expressed in both iPS cells and adipose tissue-derived cells (expression was confirmed in hDPSC cells). hsa-miR-140-5p-477909_mir hsa-miR-142-3p-477910_mir hsa-miR-149-5p-477917_mir hsa-miR-217-478773_mir hsa-miR-409-5p-478872_mir hsa-miR-411-5p-478086_mir hsa-miR-486-5p-478128_mir hsa-miR-548b-3p-479018_mir hsa-miR-548d-3p-477833_mir hsa-miR-616-3p-478177_mir hsa-miR-624-3p-479108_mir hsa-miR-636-478185_mir hsa-miR-651-5p-479131_mir hsa-miR-654-3p-479135_mir hsa-miR-654-5p-478368_mir hsa-miR-30e-5p-479235_mir hsa-miR-885-5p-478207_mir hsa-miR-888-5p-479192_mir

Claims

1. A pharmaceutical composition comprising a cell lysate, The pharmaceutical composition for suppressing chronic inflammation, wherein the cell lysate contains small RNA derived from iPS cells or adipose tissue-derived stem cells.

2. 2. The pharmaceutical composition according to claim 1, wherein the chronic inflammation is multiple sclerosis, neuromyelitis optica, MOG antibody-associated disease, asthma, arthritis, psoriasis, rheumatoid arthritis, ankylosing spondylitis, vasculitis, hypertension, autoimmune fatty liver, alcoholic hepatitis, enteritis, autoimmune uveitis, cystitis, dermatitis, rhinitis, periodontitis, stomatitis, alopecia areata, or sarcopenia.

3. The pharmaceutical composition according to claim 1, which suppresses the expression of inflammatory cytokines.

4. The pharmaceutical composition of claim 3, wherein the gene involved in the expression of inflammatory cytokines is C3, Illb, Nos2, Selp, Vegfa, Il3, Lrp2, Cd4, Cd40lg, Fas1, or Il4.

5. The pharmaceutical composition of claim 1 , wherein the cells are human cells.

6. 10. The pharmaceutical composition of claim 1, wherein the cells are 75% or more confluent.

7. The pharmaceutical composition according to claim 1, wherein the cells have been subcultured two or more times.

8. The pharmaceutical composition of claim 1 , wherein the nuclei and / or mitochondria have been removed.

9. A pharmaceutical composition comprising a cell lysate derived from iPS cells, The pharmaceutical composition for suppressing chronic inflammation, wherein the cell lysate contains small RNA derived from iPS cells, and nuclei and / or mitochondria have been removed.

10. A pharmaceutical composition comprising a cell lysate derived from adipose tissue-derived stem cells, A pharmaceutical composition for suppressing chronic inflammation, wherein the cell lysate contains small RNA derived from adipose tissue-derived stem cells, and nuclei and / or mitochondria have been removed.