WO2019235854A1 - 간엽줄기세포를 유효성분으로 포함하는 염증 질환 예방 또는 치료용 약학 조성물 - Google Patents
간엽줄기세포를 유효성분으로 포함하는 염증 질환 예방 또는 치료용 약학 조성물 Download PDFInfo
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- WO2019235854A1 WO2019235854A1 PCT/KR2019/006817 KR2019006817W WO2019235854A1 WO 2019235854 A1 WO2019235854 A1 WO 2019235854A1 KR 2019006817 W KR2019006817 W KR 2019006817W WO 2019235854 A1 WO2019235854 A1 WO 2019235854A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
- A61K31/728—Hyaluronic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/48—Reproductive organs
- A61K35/51—Umbilical cord; Umbilical cord blood; Umbilical stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
- A61P11/06—Antiasthmatics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
Definitions
- composition for preventing or treating disease in salt comprising mesenchymal stem cells as an active ingredient
- the present invention relates to a pharmaceutical composition for preventing or treating inflammatory diseases comprising mesenchymal stem cells expressing 1 show 2.
- Inflammation is a localized protective reaction that usually appears against foreign substances or harmful stimuli.
- Direct causes of inflammation include infectious causes such as bacteria, viruses and parasites; Physical causes such as burns or irradiation; Chemical causes such as toxins and drugs; Immunoreactive causes such as allergies and autoimmune reactions.
- antibiotics are used for inflammation caused by bacterial infections
- anti-inflammatory enzymes are used for inflammations without pain and pus.
- nonsteroidal anti-inflammatory drugs are used, and in case of inflammation caused by abnormal immune system, immunosuppressants or steroids are used.
- immunosuppressive agents are directly involved in the immune system and therefore have a high incidence of side effects. Steroids also have side effects when used for long periods of time.
- mesenchymal stem cells 0611; 1 can differentiate into several cell lines, such as fat cells, bone cells and chondrocytes.
- mesenchymal stem cells have the advantage that can be separated from tissues such as dense bone, peripheral blood, adipose tissue, umbilical cord blood and amniotic membrane in addition to bone marrow.
- the inventors have confirmed that the / / 2/1 / a mesenchymal stem cell expressing ⁇ 2 shows the anti-inflammatory effects superior to the mesenchymal stem cells do not express the second bye.
- the present invention was completed by confirming that mesenchymal stem cells expressing VIII-Ul 2 in bronchial lung dysplasia-induced mouse models and Alzheimer's disease-induced mouse models exhibited excellent anti-inflammatory effects. Challenge solution
- One aspect of the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases comprising mesenchymal stem cells expressing 3 ⁇ 4 2 on the cell surface as an active ingredient.
- Another aspect of the present invention provides a method for preventing or treating an inflammatory disease comprising administering the pharmaceutical composition to a subject.
- Another aspect of the present invention provides the use of mesenchymal stem cells expressing 1 show 2 on the cell surface for the manufacture of a medicament for preventing or treating inflammatory diseases.
- Another aspect of the present invention provides a method for selecting mesenchymal stem cells having excellent inflammation inhibitory ability, including separating mesenchymal stem cells expressing 3 ⁇ 4 2 on the cell surface.
- Mesenchymal stem cells expressing VII 2 on the cell surface according to the present invention inhibit the secretion of inflammatory cytokines 1-(1) and increase the expression of anti-inflammatory markers 0) 163 and 3 ⁇ 4- 1,
- Pharmaceutical compositions comprising cells as an active ingredient can be usefully used to inhibit inflammation or treat inflammatory diseases.
- Figure 2 shows the development of ⁇ -Show 2 in 13 ⁇ 4 room 264.7 cells induced inflammatory response by treatment of This figure shows the calculated rate of mTNF-a secretion after treatment of mesenchymal stem cells (lot: G171, G28, G257) or mesenchymal stem cells (lot: G571, G43, G240) not expressing HLA-A2. .
- FIG. 3 after treatment of mesenchymal stem cells expressing HLA-A2 control siRNA (HLA-A2 (+)-MSC + siCON) or siRNA for HLA-A2 (HLA-A2 (+)-MSC + siHLA_A2) , When co-cultured with Raw264.7 cells that induced the inflammatory response, mTNF-a secretion was measured.
- control siRNA HLA-A2 (+)- in mesenchymal stem cells expressing HLA-A2.
- HLA-A2 (+)-MSC + siHLA-A2 MSN + siCON
- HLA-A2 (+)-MSC + siHLA-A2 s iRNA for HLA-A2
- FIG. 5 is a diagram analyzing HLA-A2 expression patterns of mesenchymal stem cells (P2) subjected to two passages and mesenchymal stem cells (P6) subjected to six passages.
- FIG. 6 shows mesenchymal stem cells expressing PBS, HLA-A2 (HLA-A2 (+)-MSC), control si RNA (HLA-A2 (+)-MSC-s i CON) or HLA in a bronchiopulmonary dysplasia mouse model
- HLA-A2 (+)-MSC control si RNA
- HLA-A2 (+)-MSC-siHLA-A2 control siRNA
- HLA-A2 HLA-A2 (+)
- control si RNA HLA-A2 (+)-MSC-si CON
- HLA-A2 mesenchymal stem cells expressing PBS, HLA-A2 (+)
- control si RNA HLA-A2 (+)-MSC-si CON
- HLA-A2 mesenchymal stem cells expressing PBS, HLA-A2 (+)
- control si RNA HLA-A2 (+)-MSC-si CON
- HLA-A2 mesenchymal stem cells expressing PBS, HLA-A2 (+)
- siRNA HLA-A2 (+)-MSC-siHLA-A2
- FIG. 8 is a single or repeated administration of mesenchymal stem cells expressing physiological saline (Control) and HLA-A2 to a mouse model of Alzheimer's disease. Figures confirming the expression levels of TNF-a and IFN-Y.
- FIG. 9 is a single or repeated administration of mesenchymal stem cells expressing physiological saline (Control) and HLA-A2 to the Alzheimer's disease mouse model, and brain tissues of mice were extracted at 20 weeks and then in the tissues. Figures confirming the expression levels of TNF-a and IFN-Y.
- FIG. 10 shows physiological saline (Control) and HLA-A2 in the Alzheimer's disease mouse model.
- FIG. 11 is a single or repeated administration of mesenchymal stem cells expressing physiological saline (Control) and HLA-A2 to a mouse model of Alzheimer's disease. It is a figure confirming the expression level of the inner CD163 and Arg-1. Best mode for carrying out the invention
- One aspect of the invention provides a composition for preventing or treating inflammatory diseases comprising mesenchymal stem cells expressing HLA-A2 (human leukocyte antigen A2) on the cell surface as an active ingredient.
- HLA-A2 human leukocyte antigen A2
- the HLA-A2 is one of the serotypes belonging to the HLA-A serogroup of human leukocyte antigen (HLA), wherein the HLA is encoded by a human histocompatibility complex (MHC) gene. Glycoprotein molecule.
- HLA-A is expressed on the cell surface of all nucleated cells and platelets, and cytotoxic T cells function as an antigen recognition function to recognize and remove cells infected with viruses or tumor cells.
- the anti-inflammatory effects of human-derived mesenchymal stem cells expressing HLA-A2 have not been identified.
- the present invention is based on the results that mesenchymal stem cells expressing HLA-A2 on the cell surface have superior inflammation inhibitory ability compared to mesenchymal stem cells not expressing HLA-A2.
- mesenchymal stem cells expressing HLA-A2 on the cell surface of the present invention is 70%, 75%, 80%, 85 compared to mesenchymal stem cells not expressing HLA-A2 It is characterized by further expressing HLA-A2 more than%, 90%, or 95%.
- the mesenchymal stem cells of the present invention express any one selected from the group consisting of CD73 (cluster of differentiation 73), CD90, ⁇ 105, CD166 and combinations thereof on the cell surface, CD14, CD34, CD45 HLA- It may not express any one selected from the group consisting of DR (human leukocyte antigen DR) and combinations thereof.
- the mesenchymal stem cells of the present invention may express more than 70% of CD73, CD90, CD105 and ⁇ 166 on the cell surface, respectively, and less than 1% of ⁇ 14, ⁇ 34, CD45 and HLA-DR, respectively. have.
- the mesenchymal stem cells of the present invention express at least 75% of HLA-A2 on the surface of the cells, at least 70% of CD73, ⁇ 90, CD105 and CD166, respectively, and express CD14, CD34, CD45 and HLA-.
- DR may be expressed at 1% or less each.
- the CD73 is also called 5′-nucleotide hydrolase (5′-nucleot idase, 5′_NT) and is an enzyme encoded by the NT5E gene. CD73 functions to convert adenosine monophosphate (AMP) to adenosine.
- 5′_NT 5′-nucleotide hydrolase
- AMP adenosine monophosphate
- CD90 is a cell surface protein composed of N-glycosylated GPI (Glycophosphat idyl inosi tol) and a single V-like immunoglobulin domain.
- GPI glycophosphat idyl inosi tol
- CD105 also called ENG (Endogl in)
- ENG Endogl in
- the CD166 is a type I transmembrane glycoprotein of the immunoglobul in superfami ly located on the cell surface. CD166 is encoded by the ALCAM gene.
- the CD14 is a pattern recognition receptor (PRR) and is one of the components of the innate immune system.
- the CD14 interacts with covalent receptors TLR 4 (Tol 1-1 ike receptor 4) and MD-2 to cope with the bacterium LPS (1 ipopolysacchar ide) in the presence of LBP (1 ipopolysacchar ide-binding protein). To combine.
- VII 34 is a membrane penetrating phospho glycoprotein (phosphog 1 ycoprot e i n) encoded by the CD34 gene in humans.
- the CD45 is a protein tyrosine phosphatase (PTP) enzyme encoded by the gene ⁇ in humans.
- PTP protein tyrosine phosphatase
- the HLA-DR is a cell surface receptor belonging to Cl ass I I of HLA, and is involved in autoimmunity, disease susceptibility and disease resistance.
- the mesenchymal stem cells can be used regardless of the origin, preferably may be derived from umbilical cord blood.
- the content of mesenchymal stem cells expressing HLA-A2 in the pharmaceutical composition is 2019/235854 1 »(: 1 ⁇ 1 ⁇ 2019/006817
- the content of mesenchymal stem cells expressing Group 2 in the pharmaceutical composition may be 5.0X10 ® / 3> ⁇ 10 7 cells / 111 b.
- 50 1 of a solution of IX 10 7 cells / 111 urine content was administered to mice.
- the pharmaceutical composition is a kind of cell therapy, and may further include a pharmaceutically acceptable carrier.
- the carrier is commonly used in the manufacture of pharmaceuticals, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrroli It may include money, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like.
- composition of the present invention may further include a pharmaceutically acceptable additive selected from the group consisting of lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and combinations thereof.
- a pharmaceutically acceptable additive selected from the group consisting of lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and combinations thereof.
- the carrier may comprise from about 1% to about 99.99% by weight, preferably from about 90% to about 99.99% by weight, based on the total weight of the pharmaceutical composition of the present invention, wherein the pharmaceutically acceptable additive is about 0.1 Weight percent to about 20 weight percent.
- the pharmaceutical composition may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and excipient according to conventional methods, or may be prepared by incorporating into a multi-dose container.
- the formulation may be in the form of solutions, suspensions, syrups or emulsions in oil or aqueous media, or may be in the form of extracts, powders, granules or capsules, and may further comprise dispersants or stabilizers.
- the inflammatory disease refers to a disease accompanied by inflammation.
- the inflammatory diseases include rheumatoid arthritis, atopy, asthma, allergic rhinitis, Alzheimer's disease, Diabetic nephropathy, Crohn's disease, inflammatory bowel disease, post-transplant rejection, bronchopulmonary dysplasia ⁇ ⁇ ) or chronic obstructive pulmonary disease ⁇ 1)), but is not limited thereto.
- Another aspect of the invention provides a method for preventing or treating an inflammatory disease comprising administering to a subject a pharmaceutical composition for treating an inflammatory disease comprising a mesenchymal stem cell expressing HLA-A2 on a cell surface.
- Mesenchymal stem cells expressing HLA-A2 are the same as described above in the pharmaceutical composition.
- the subject may be a mammal, specifically a human.
- the route of administration and dosage of the pharmaceutical composition may be administered to the subject in various ways and amounts depending on the condition of the patient and the presence of side effects, and the optimal method and dosage may be selected by a person skilled in the art to an appropriate range.
- the pharmaceutical composition may be administered in combination with other drugs or physiologically active substances known for the inflammatory diseases to be treated, or may be formulated in combination with other drugs.
- examples include subcutaneous, eye, intraperitoneal, intramuscular, oral, rectal, orbital, intracranial, intracranial, intravertebral, intraventricular, intramedullary, There is intranasal and intravenous administration.
- the administration may be administered one or more times, once to three times, specifically three times. In case of repeated administration, it may be administered at 1 to 56 days, 7 to 49 days, 14 to 42 days or 21 to 35 days intervals. Preferably, administration may be at 28 day intervals. If the dosage is high, it may be administered several times a day.
- the dose of HLA-A2 expressing mesenchymal stem cells is from lx 10 6 cells / object to
- the dose of HLA-A2 expressing mesenchymal stem cells is 5X 10 4 cells / kg to lx 10 7 cells / kg, 1 X 10 5 cells / kg to 7X 10 6 cells / kg, 2X 10 5 cells / kg to 5 ⁇ ⁇ 10 5 cells / kg.
- Another aspect of the invention provides the use of mesenchymal stem cells expressing HLA-A2 on the cell surface for the manufacture of a medicament for the prevention or treatment of inflammatory diseases.
- Another aspect of the present invention provides a method for selecting mesenchymal stem cells having excellent inflammation inhibitory ability, including separating mesenchymal stem cells expressing HLA-A2 on a cell surface.
- the method of separating the mesenchymal stem cells expressing HLA-A2 on the cell surface can be isolated using, for example, a flow cytometer.
- anti-X14 antibody, anti-CD34 antibody, anti-CD45 antibody, anti-HLA-DR antibody, anti-CD73 antibody, anti-CD90 antibody, anti-CD105 antibody, anti-CD166 antibody And treating the mesenchymal stem cells with the mouse anti-HLA-A2 antibody to analyze the cell surface antigens, and then assigning electric charge only to the mesenchymal stem cells expressing HLA-A2, CD73, CD90, CD105, and CD166 and Mesenchymal stem cells expressing HLA-A2 were isolated on the cell surface by passing through the cells.
- mesenchymal stem cells After cord blood-derived mesenchymal stem cells were first isolated from umbilical cord blood, 5 passages of mesenchymal stem cells (5 passages), which were passaged five times, were cryopreserved in liquid nitrogen, and several lots of mesenchymal stem cells were used. The mesenchymal stem cells, which were cryopreserved, were thawed for about 3 minutes in a constant temperature water bath at 37 ° C. Then, mesenchymal stem cells were resuspended in a -MEM medium containing 10% (v / v) FBS (fetal bovine serum) and centrifuged for 5 minutes at 1,200 rpm using a centrifuge. After the centrifugation, the supernatant was removed and the remaining mesenchymal stem cells were resuspended in PBSCphosphate buf fer sal ine.
- FBS fetal bovine serum
- anti-CD14 antibody (BD pharmigen, Cat # 555397), anti-CD34 antibody pharmigen, Cat # 555822), anti-CD45 antibody (BD pharmigen, Cat # 555482), anti-HLA-DR antibody (BD pharmigen, Cat # 555811), anti-CD73 antibody (BD pharmigen, Cat # 550257), anti-CD90 antibody (Invi trogen, Cat # 12-0909-42), anti -CD105 antibody (Invi trogen, Cat # 12-1057-42), anti-CD166 antibody (BD pharmigen, Cat # 555263) and mouse anti-HLA-A2 antibody (BD pharmigen, Cat # 558570) at a temperature of 25 ° C.
- cord blood-derived mesenchymal stem cells of the G171, G028 and G257 lot express more than 70% of HLA-A2 on the cell surface.
- cord blood-derived mesenchymal stem cells of G571, G043 and G240 lot was confirmed to express less than 1% HLA-A2 on the cell surface.
- the mesenchymal stem cell group expressing HLA-A2 on the cell surface HLA-A2 (+)
- the mesenchymal stem cell group not expressing HLA-A2 on the cell surface HLA-A2 (-)
- inflammatory response was induced by treating LPS (Lipopolyssachar ide), an inflammatory stimulant, to Raw 264.7 cells, which are mouse-derived macrophages.
- LPS Lipopolyssachar ide
- the mesenchymal stem cells of each lot that had been cryopreserved were thawed for about 3 minutes in a 37 ° C thermostat. After that, the mesenchymal stem cells were resuspended in a -MEM medium containing 10% (v / v) FBS (fetal bovine serum) and centrifuge for 5 minutes at 1,200 rpm. Centrifugation. After centrifugation, the supernatant was removed and the remaining mesenchymal stem cells were resuspended in RPMI medium.
- LPS Lipopolyssachar ide
- Raw264.7 cells were co-cultured with 2 ⁇ 10 4 G171, G028, G257, G571, G043 or G240 lot mesenchymal stem cells. After 24 hours, the culture medium was collected, and analyzed using-Quantikine® mouse TNF-a Immunoassay kit (R & D Systems).
- siRNA was used to inhibit HLA-A2 expression of mesenchymal stem cells and to compare the effects of inhibiting inflammation.
- HLA-A2 (+)-MSC HLA-A2 expressing mesenchymal stem cells
- mesenchymal stem cells of G027, G074, G171, G610, G028, or G257 lot are treated with control siRNA (HLA-A2 (+)-MSC + siC0N) or HLA-A2.
- control siRNA HLA-A2 (+)-MSC + siC0N
- HLA-A2 (+)-MSC + siHLA Prepared by treatment with siRNA (HLA-A2 (+)-MSC + siHLA), and then, using RPMI medium containing LPS at a concentration of 1 ng / ⁇ i, 2X10 4 Raw264.7 cells and 2X10 in each well.
- Four prepared mesenchymal stem cells were co-cultured. After 24 hours, the culture medium was collected and analyzed for the secretion of mTNF-ci using Quantikine® mouse TNF-a Immunoassay kit (R & D Systems).
- siRNA control HLA-A2 (+)-MSC-siCON
- HLA-A2 siRNA control
- HLA-A2 siRNA control
- Bronchopulmonary dysplasia is caused by abnormal lung structure and arrest of lung development. This is one of the inflammatory diseases in which lung inflammation is caused by excessively induced pulmonary inflammation during high oxygen therapy through oxygen respirator during neonatal respiratory distress. To date, there is no fundamental treatment or treatment worldwide. Therefore, when umbilical cord blood-derived mesenchymal stem cells expressing 10 2 on the cell surface were administered to a bronchiopulmonary dysplasia mouse model, treatment effects such as immature lung tissue generation and inflammation inhibition were confirmed.
- mice within 10 hours of birth are moved to a cage of normal concentration or high concentration oxygen concentration Breeding.
- the mother mothers who gave birth were also moved to the same cage for breeding the newborn mice.
- a change in the number of newborn mice to be delivered from pregnant mother mice may occur, and considering that the average mortality rate is about 50% when the newborn mice are bred with high oxygen concentration, the final mice may be 9 in each group.
- Three mice were bred. Three of the nine animals were used for morphological analysis of lung tissue, three were alveolar solution extracts for inflammatory material identification, and the other three were used for protein analysis or protein analysis. The configuration of each group is shown in Table 2 below.
- mice On the third day of birth, newborn mice were treated with anesthetics of zoletil 50 (201 ⁇ 1 1 50) and 23.32 1 / Intraperitoneal injection.
- Mesenchymal stems treated with mesenchymal stem cells expressing PBS, HLA-A2 (HLA-A2 (+)), control siRNA (siCON) or siRNA (siHLA-A2) for HLA-A2 were then injected into the airways of newborn mice. Cells were administered. At this time, 5X10 5 mesenchymal stem cells per newborn mouse to the mesenchymal stem cell administration group to 50 PBS 0 2019/235854 1 »( ⁇ / 10 ⁇ 2019/006817 floated once).
- 5XFAD mice began to produce amyloid-beta in brain tissues at 2 months of age, and at 4 months, amyloid beta accumulates in brain tissues and at 6 months of cognitive dysfunction. (Cognitive impairment) appears. 6-month-old 5XFAD mice were mixed with zoletil, rumoon, and saline in a ratio of 4: 1: 5 and used as anesthetics. Anesthetics were intraperitoneally administered.
- a guide cannula was placed at a target position (from Bregma Anterior / Posterior: -0.22 mm, Medial / Lateral: 1.0 mm, Dorsal / Ventral: 2.5 ⁇ ), and then fixed by screwing around. Thereafter, a recovery period of 6-8 days was placed for recovery. Subsequently, the cord blood-derived mesenchymal stem cells expressing HLA-A2 were transferred to a Hamilton syringe, and then an internal cannula was placed in the guide cannula and injected into the ventricle at an infusion rate of 0.5 / min. At this time, the configuration of each group is shown in Table 3.
- an appropriate amount of anesthetic was intraperitoneally administered according to body weight, and then cardiac perfusion was performed using a peristaltic pump.
- the blood was excreted by circulating the skin, diaphragm and ribs of the mouse and circulating the PBS, which connected the pump tubing to the left ventricle, to the body.
- brain tissues were harvested and stored at -80 ° C for temperature. After lysing the brain tissue using Lysis buffer and sonicator, brain tissue lysate was diluted using a Bradford assay.
- a cytokine ELISA KIT suitable for measuring lysates such as brain tissue was used, TNF- Q ELISA KlK PeproTech, 900K54EK), IFN-y ELISA KIT (RayBio, ELM-IFNg-CL) was used to analyze the pro-inflammatory cytokine (pro-inflmmatory cytokine), CD163 ELISA KITLSBio, Cat No. LS-F9079), Arg-1 ELISA KITCLSBio, Cat No. LS-F6864] was used to analyze the anti-inflammatory cytokine (ant i-inflammatory cytokine).
- TNF-a and INF- were added to the test group in which the mesenchymal stem cells expressing HLA-A2 were expressed in a single dose and the test group repeatedly administered in the test A (12 weeks) condition and the test B (20 weeks) condition. Decreased.
- TNF-a was significantly decreased in the experimental group in which HLA-A2-expressing mesenchymal stem cells were repeatedly administered
- INF- Y was in the group in which HLA-A2-expressing mesenchymal stem cells were administered once.
- Statistically significant decrease in (FIGS. 8 and 9). It was confirmed that the anti-inflammatory efficacy was maintained even after repeated administration through this.
- CD163 and Arg-1 (Argnase-l), which are known as anti-inflammatory cytokines in brain tissues, were analyzed by ELISA.
- CD163 and Arg-1 were increased in the HLA-A2-expressing mesenchymal stem cells and in the single-dose group.
- CD163 significantly increased HLA-A2 expressing mesenchymal stem cells in the repeated administration group
- Arg-1 increased the HLA-A2 expressing mesenchymal stem cells in a single experiment and repeated administration.
- Statistically significant increase in all experimental groups (FIGS. 10 and 11). Through this, it was confirmed that the anti-inflammatory effect was maintained over time after repeated administration.
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Priority Applications (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
SG11202011927TA SG11202011927TA (en) | 2018-06-05 | 2019-06-05 | Pharmaceutical composition comprising mesenchymal stem cells as effective ingredient for prevention or treatment of inflammatory disease |
KR1020207036318A KR20210008873A (ko) | 2018-06-05 | 2019-06-05 | 간엽줄기세포를 유효성분으로 포함하는 염증 질환 예방 또는 치료용 약학 조성물 |
CA3100471A CA3100471A1 (en) | 2018-06-05 | 2019-06-05 | Pharmaceutical composition comprising mesenchymal stem cells as effective ingredient for prevention or treatment of inflammatory disease |
CN201980038809.4A CN112261943A (zh) | 2018-06-05 | 2019-06-05 | 用于预防或治疗炎症性疾病的包含间充质干细胞作为有效成分的药物组合物 |
US16/972,118 US20210228636A1 (en) | 2018-06-05 | 2019-06-05 | Pharmaceutical composition comprising mesenchymal stem cells as effective ingredient for prevention or treatment of inflammatory disease |
AU2019283518A AU2019283518A1 (en) | 2018-06-05 | 2019-06-05 | Pharmaceutical composition comprising mesenchymal stem cells as effective ingredient for prevention or treatment of inflammatory disease |
JP2020564579A JP7480454B2 (ja) | 2018-06-05 | 2019-06-05 | 有効成分として間葉系幹細胞を含む炎症性疾患の予防又は治療のための医薬組成物 |
EP19815851.1A EP3804736A4 (en) | 2018-06-05 | 2019-06-05 | PHARMACEUTICAL COMPOSITION WITH MESENCHYMAL STEM CELLS AS AN EFFECTIVE INGREDIENT TO PREVENT OR TREAT AN INFLAMMATORY DISEASE |
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CN113616675A (zh) * | 2021-08-23 | 2021-11-09 | 上海太安堂生物医学有限公司 | 含间充质干细胞的组合物及其在治疗退行性关节炎的应用 |
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US20210228637A1 (en) | 2021-07-29 |
EP3804736A1 (en) | 2021-04-14 |
CN112261944A (zh) | 2021-01-22 |
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US20210228636A1 (en) | 2021-07-29 |
EP3804736A4 (en) | 2022-03-30 |
EP3811951A4 (en) | 2022-07-13 |
JP2021526137A (ja) | 2021-09-30 |
JP7343091B2 (ja) | 2023-09-12 |
SG11202011927TA (en) | 2020-12-30 |
CA3100471A1 (en) | 2019-12-12 |
CN112261943A (zh) | 2021-01-22 |
CA3100466A1 (en) | 2019-12-12 |
WO2019235853A1 (ko) | 2019-12-12 |
KR20210008873A (ko) | 2021-01-25 |
KR20210008101A (ko) | 2021-01-20 |
SG11202012124PA (en) | 2021-01-28 |
JP2021526135A (ja) | 2021-09-30 |
AU2019283518A1 (en) | 2021-01-14 |
AU2019283517A1 (en) | 2021-01-21 |
EP3811951A1 (en) | 2021-04-28 |
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