Classifications

• • 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
• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
  • C12N5/0662—Stem cells
  • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells

Definitions

• the present invention relates to a composition for treating arthropathy.
• Osteoarthritis is a degenerative disease characterized by the deterioration of joint cartilage and bone, triggered by various disorders of joint integrity.
• OA is a general term for joint degeneration due to multiple causes. Inflammation and pro-inflammatory responses of the innate immune system are involved in the progression of OA.
• OA develops in joints such as the knee, hip, hand, foot, and spine, and is most commonly caused by the knee joint.
• MSCs trophic actions such as cell proliferation promotion, chemoattraction, and anti-apoptotic effects have attracted attention as the main mechanism of action of MSCs.
• the trophic activity of MSCs may be triggered by extracellular matrix (ECM) production, cell-cell contact, or secretion of signaling molecules.
• ECM extracellular matrix
• EVs extracellular vesicles
• Exosomes are vesicles with a diameter of about 100 nm, synthesized and released from multivesicular bodies that fuse with the cell membrane by endocytosis.
• Exosomes are cargos surrounded by a lipid bilayer and contain various biomolecules such as nucleic acids (miRNA), proteins, and lipids. It has been revealed that exosomes play an important role as mediators of intercellular communication, in that secreted exosomes are taken up by recipient cells and their cargo contents are incorporated. It is known that exosomes released from cultured cells are present in the culture medium, but it has been difficult to completely separate them from other vesicles such as microvesicles at the time of collection. Therefore, extracellular vesicles mainly composed of exosomes are operationally defined as extracellular vesicles (EVs).
• EVs extracellular vesicles
• MSC-EVs MSC-derived EVs
• UC ultracentrifugation
• UF ultrafiltration
• TDF tangential flow filtration
• SEC size exclusion chromatography
• UF ultrafiltration
• FBS fetal bovine serum
• hPL human platelet lysate
• CDM chemically defined medium
• the object of the present invention is to provide a novel composition that can be used to treat osteoarthritis.
• the inventors have conducted intensive research into the above-mentioned problems, and have found that when mesenchymal stem cells are cultured using a medium consisting of specific components, (1) the amount of extracellular vesicles per mesenchymal stem cell increases, (2) the protein expression profile in mesenchymal stem cells is significantly different from the profile of mesenchymal stem cells cultured using existing mesenchymal stem cell culture media, (3) the resulting culture supernatant contains relatively few contaminant components (non-EV components) other than the useful components, the extracellular vesicles, (4) the obtained extracellular vesicles enhance the proliferation, migration, extracellular matrix expression, and anti-apoptotic activity of human articular chondrocytes (hAC) in vitro, and (5) the obtained extracellular vesicles can be administered to a collagenase-induced osteoarthritis mouse model to treat osteoarthritis, and the like. Based on these findings, the inventors have conducted further research and have completed the present invention. That is,
• a pharmaceutical composition for treating arthropathy comprising a composition containing extracellular vesicles derived from a culture supernatant of mesenchymal stem cells, the medium for the mesenchymal stem cells being cultured under the following conditions: (1) A pharmaceutical composition that is a chemically defined medium that does not contain any animal-derived components different from those of a subject to be treated; and (2) a number of microparticles having a particle size of 1 to 1000 nm before use is less than 1 x 10 particles/mL.
• the medium was further conditioned as follows: (3) The total protein amount contained in the medium before use is 100 ⁇ g/mL or less; The pharmaceutical composition according to [1], which satisfies one or more of the following conditions selected from (4) not containing a surfactant or containing a surfactant at less than its critical micelle concentration; and (5) containing TNF- ⁇ and TRAIL/APO2L. [3] The pharmaceutical composition according to [2], wherein the contents of TNF- ⁇ and TRAIL/APO2L in the medium (5) above are each 0.0001 to 2.0 mg/L before use.
• [4] The pharmaceutical composition according to any one of [1] to [3], wherein the number of microparticles per unit weight of protein in the extracellular vesicle-containing composition (unit: 10 9 particles/mg protein) is 10 to 1,000.
• [5] The pharmaceutical composition according to any one of [1] to [4], wherein the medium is GMP grade.
• [6] The pharmaceutical composition according to any one of [1] to [5], wherein the composition containing extracellular vesicles is a culture supernatant or a purified product thereof.
• [7] The pharmaceutical composition according to any one of [1] to [6], wherein the arthritis is osteoarthritis.
• Step 1 A step of culturing mesenchymal stem cells in a medium that satisfies the following conditions: (1) the medium is a chemically defined medium that does not contain any animal-derived components that are heterologous to the subject of treatment; and (2) the number of microparticles having a particle diameter of 1 to 1000 nm before use is less than 1 x 109 particles/mL.
• Step 2 A step of recovering a composition containing extracellular vesicles from the culture obtained in Step 1.
• the medium was further conditioned as follows: (3) The total protein amount contained in the medium before use is 100 ⁇ g/mL or less; The method for producing according to [9], which satisfies one or more of the following conditions: (4) containing no surfactant or containing a surfactant at less than its critical micelle concentration; and (5) containing TNF- ⁇ and TRAIL/APO2L. [11] The method according to [10], wherein the contents of TNF- ⁇ and TRAIL/APO2L in the medium (5) above are each 0.0001 to 2.0 mg/L before use.
• a composition containing extracellular vesicles derived from a culture supernatant of mesenchymal stem cells for use in the treatment of osteoarthritis comprising extracellular vesicles derived from a culture supernatant of mesenchymal stem cells, the medium of the mesenchymal stem cells being subjected to the following conditions: (1) A composition that is a chemically defined medium that does not contain any animal-derived components different from those of a subject to be treated; and (2) a composition that satisfies the following conditions before use: the number of microparticles having a particle size of 1 to 1000 nm is less than 1 x 109 particles/mL.
• the medium was further conditioned as follows: (3) The total protein amount contained in the medium before use is 100 ⁇ g/mL or less; The composition according to [A1], which satisfies one or more of the following conditions selected from (4) not containing a surfactant or containing a surfactant at less than its critical micelle concentration; and (5) containing TNF- ⁇ and TRAIL/APO2L. [A3] The composition according to [A2], wherein the content of TNF- ⁇ and TRAIL/APO2L in the medium (5) is 0.0001 to 2.0 mg/L, respectively, before use.
• [A4] The composition according to any one of [A1] to [A3], wherein the number of microparticles per unit weight of protein in the extracellular vesicle-containing composition (unit: 10 9 particles/mg protein) is 10 to 1,000.
• [A5] The composition according to any one of [A1] to [A4], wherein the medium is GMP grade.
• [A6] The composition according to any one of [A1] to [A5], wherein the extracellular vesicle-containing composition is a culture supernatant or a purified version thereof.
• [A7] The composition according to any one of [A1] to [A6], wherein the arthritis is osteoarthritis.
• [A8] The composition according to any one of [A1] to [A7], wherein the arthropathy is knee arthropathy.
• [B1] A method for treating arthropathy, comprising administering a composition containing extracellular vesicles derived from a culture supernatant of mesenchymal stem cells to a subject suffering from arthropathy, wherein the medium for the mesenchymal stem cells is cultured under the following conditions: (1) A chemically defined medium that does not contain any animal-derived components different from those of a subject to be treated; and (2) a number of microparticles having a particle size of 1 to 1000 nm in a state before use is less than 1 x 109 particles/mL.
• the medium was further conditioned as follows: (3) The total protein amount contained in the medium before use is 100 ⁇ g/mL or less; The method according to [B1], which satisfies one or more of the following conditions: (4) not containing a surfactant or containing a surfactant at less than its critical micelle concentration; and (5) containing TNF- ⁇ and TRAIL/APO2L. [B3] The method according to [B2], wherein the contents of TNF- ⁇ and TRAIL/APO2L in the medium (5) above are each 0.0001 to 2.0 mg/L before use.
• [B4] The method according to any one of [B1] to [B3], wherein the number of microparticles per unit weight of protein in the extracellular vesicle-containing composition (unit: 10 9 particles/mg protein) is 10 to 1,000.
• [B5] The method according to any one of [B1] to [B4], wherein the medium is GMP grade.
• [B6] The method according to any one of [B1] to [B5], wherein the composition containing extracellular vesicles is a culture supernatant or a purified product thereof.
• [B7] The method according to any one of [B1] to [B6], wherein the arthritis is osteoarthritis.
• [B8] The method according to any one of [B1] to [B7], wherein the arthropathy is knee arthropathy.
• [C1] A use of a composition containing extracellular vesicles derived from a culture supernatant of mesenchymal stem cells in the manufacture of a medicine for the treatment of arthropathy, the medium for the mesenchymal stem cells being cultured under the following conditions: (1) A chemically defined medium that does not contain any animal-derived components different from those of a subject to be treated; and (2) a use that satisfies the following conditions before use: the number of microparticles having a particle diameter of 1 to 1000 nm is less than 1 x 109 particles/mL.
• [C2] The medium was further conditioned as follows: (3) The total protein amount contained in the medium before use is 100 ⁇ g/mL or less; The use according to [C1], which satisfies one or more of the following conditions: (4) not containing a surfactant or containing a surfactant at less than its critical micelle concentration; and (5) containing TNF- ⁇ and TRAIL/APO2L. [C3] The use according to [C2], wherein the contents of TNF- ⁇ and TRAIL/APO2L in the medium (5) above are each 0.0001 to 2.0 mg/L before use.
• [C4] The use according to any one of [C1] to [C3], wherein the number of microparticles per unit weight of protein in the extracellular vesicle-containing composition (unit: 10 9 particles/mg protein) is 10 to 1,000.
• [C5] The use according to any one of [C1] to [C4], wherein the medium is GMP grade.
• [C6] The use according to any one of [C1] to [C5], wherein the composition containing extracellular vesicles is a culture supernatant or a purified product thereof.
• [C7] The use according to any one of [C1] to [C6], wherein the arthritis is osteoarthritis.
• [C8] The use according to any one of [C1] to [C7], wherein the osteoarthritis is knee osteoarthritis.
• the present invention provides a composition for treating knee osteoarthritis, which contains extracellular vesicles obtained by culturing human mesenchymal stem cells in a medium containing less than 1 x 109 microparticles/mL having a particle size of 1 to 1000 nm before use, and is used for treating knee osteoarthritis.
• the knee arthritis may be osteoarthritis
• the medium may contain TNF- ⁇ and TRAIL/APO2L.
• the contents of the TNF- ⁇ and the TRAIL/APO2L in the medium before use may each be 0.0001 to 2.0 mg/L.
• the total amount of protein contained in the medium before use may be 100 ⁇ g/mL or less.
• the medium may be free of a surfactant. Additionally, the medium may be free of components derived from non-human animals, preferably from animals including humans.
• the present invention provides technology related to a novel composition for use in treating osteoarthritis.
• Figure 1 is a graph showing the ExoScreen assay to quantify EVs released into culture medium. ASCs were cultured in the indicated media for 24 or 48 hours, and the supernatants were analyzed by ExoScreen assay.
• Figures 1A and 1B show the signal intensity (SI) and data collected per cell for CD9 x CD9 EVs at 24 or 48 hours, respectively.
• Figures 1C and 1D show the SI and data collected per cell for CD63 x CD63 EVs at 24 or 48 hours, respectively. **, ##: p ⁇ 0.01 between the two groups connected by a bar or compared with the other group, respectively.
• C1-C4 correspond to CDM1-CDM4, respectively.
• Figure 2 shows the distribution of particles and proteins from EVs and BGs isolated from different media. EVs from each medium and concentrates (BGs) from each culture-free medium were extracted and compared.
• Figures 2A and 2B show representative captured images of particles detected by NTA and histograms of particle size distribution, respectively.
• Figures 2C and 2D show data on particle and protein concentrations for each sample.
• Figure 2E shows protein distribution by silver staining. Alb: albumin.
• Figure 2F shows the purity data of each cell culture-derived EV group, calculated as the number of particles per protein. ND (not detected) indicates that the number of particles is 20 or less per frame or the protein concentration is out of range, and is treated as 0 in statistical analysis.
• Figure 3 shows TEM images of EVs derived from each cell culture, exosome markers, and nanoplasmonic assay evaluation.
• Figure 3A shows a representative TEM image.
• Figure 3B shows the evaluation of classical exosome marker proteins and albumin by Western blotting.
• Figure 3C shows the quantitative analysis of CD9-CD63 EVs measured by ELISA. Values were calculated using CD9/CD63 fusion protein as a standard.
• FIG. 4 shows the therapeutic effects of EVs and BGs derived from each medium on proliferation, migration, ECM synthesis, and anti-apoptosis of hACs.
• FIG. 4B and FIG. 4C show stained microscopic images of migrated cells and their relative numbers, respectively, and show values relative to the control. Three fields were randomly selected for each group. Scale bar: 200 ⁇ m.
• FIG. 4F show flow cytometry analysis for apoptosis detection and occupancy by Annexin V(+)7-AAD(-) cells, defined as early apoptosis, respectively.
• NS not significant in comparison with the control group.
• FIG. 5 is a graph showing flow cytometry analysis of surface markers of MSCs.
• FIG. 6 is a photograph showing the appearance of CDM4 and a conventional medium before use.
• FIG. 7 shows the particle size distribution of CDM4 and conventional media before use.
• FIG. 8 shows protein distribution by silver staining of CDM4-EVs derived from three different donors.
• 9 shows the therapeutic effect of CDM4-EV on CIOA mice.
• KOA was induced by injecting collagenase into the right knee joint, and CDM4-EV (treated) or CDM4-BG (untreated) was injected into the same site on day 8.
• Mice were sacrificed on day 28, and the tibia was isolated from the injected knee. The contralateral tibia was used as a normal control.
• 9A and 9B show the macroscopic images of the tibial articular surface and the macroscopic cartilage damage score. Scale bar: 500 ⁇ m.
• 9C and 9D show the histological section of the medial tibia stained with Safranin O and the OARSI score. Scale bar: 200 ⁇ m.
• composition for treating arthropathy provides a pharmaceutical composition for treating arthropathy (hereinafter, sometimes referred to as the "pharmaceutical composition of the present invention"), which comprises a composition containing extracellular vesicles derived from a culture supernatant of mesenchymal stem cells.
• the medium for the mesenchymal stem cells is cultured under the following conditions:
• the medium is characterized by satisfying the following: (1) It is a chemically defined medium (CDM) that does not contain any animal-derived components different from those of the subject to be treated; and (2) Before use, the number of microparticles having a particle size of 1 to 1000 nm is less than 1 x 109 particles/mL.
• CDM chemically defined medium
• extracellular vesicle-containing composition contained in the pharmaceutical composition of the present invention contains extracellular vesicles (EVs) secreted by mesenchymal stem cells when the mesenchymal stem cells are cultured in a medium satisfying certain conditions (hereinafter, sometimes referred to as the "medium used in the present invention"), and may contain components other than the EVs contained in the culture supernatant to the extent that they do not have an undesirable effect on the therapeutic activity for arthropathy.
• the EVs are the substantial active ingredient in the treatment of arthropathy.
• the mesenchymal stem cells cultured in the medium used in the present invention are not particularly limited as long as they are derived from mammals (e.g., laboratory animals such as mice and rats, pet animals such as dogs and cats, livestock animals such as pigs, horses and cows, and primates such as humans, monkeys, orangutans and chimpanzees), but are preferably derived from the same species of animal as the animal to be treated.
• the subject of treatment is a human, and therefore cells of human origin are preferably used as mesenchymal stem cells.
• the tissue from which the mesenchymal stem cells are derived is not particularly limited, and may be any tissue such as adipose tissue, bone marrow, umbilical cord, dental pulp or placenta, but preferably the mesenchymal stem cells are adipose-derived mesenchymal stem cells (ASC).
• ASC adipose-derived mesenchymal stem cells
• the conditions for culturing mesenchymal stem cells in the medium used in the present invention are not particularly limited as long as the mesenchymal stem cells can secrete EVs.
• those culture conditions may be adopted as long as EVs are secreted.
• the medium used in the present invention refers to a medium for secreting extracellular vesicles (EVs) used when causing cells to secrete EVs.
• EVs extracellular vesicles
• the basic components constituting the medium used in the present invention may be the same as those contained in a medium for proliferation of stem cells (e.g., a medium for proliferation of mesenchymal stem cells).
• a medium for proliferation of stem cells e.g., a medium for proliferation of mesenchymal stem cells.
• Examples of the basic components of the medium used in the present invention include amino acids, vitamins, inorganic salts, and other components.
• the amino acids include essential amino acids isoleucine, leucine, lysine, methionine, phenylalanine, threonine (threonine), tryptophan, valine, and histidine, and non-essential amino acids tyrosine, cysteine, aspartic acid, asparagine, serine, glutamic acid, glutamine, proline, glycine, alanine, and arginine;
• the vitamins include ascorbic acid, choline, myoinositol, niacinamide, pantothenic acid, pyridoxine, pyridoxal, thiamine, putrescine, biotin, cyanocobalamin, folic acid , and riboflavin; and the inorganic salts include FeSO4 , CuSO4, MnSO4 , Na2SiO3 , ZnSO4 , MgSO4 , CaCl2 , NaH2PO4 , Na2HPO
• a culture supernatant prepared using a medium containing an additive containing unknown animal-derived components such as serum is not suitable for use as a medicine because it contains impurity components other than the intended active ingredient.
• serum e.g., bovine serum or human serum
• serum has a risk of performance differences between lots, and further infection with prion diseases such as mad cow disease and human infectious diseases.
• mesenchymal stem cell culture media of a defined composition contain components that affect the proliferation and migration of human articular chondrocytes, and the production of medicines using media containing impurity components with such unintended activity should be avoided.
• the medium used in the present invention is (1) a defined-composition medium that does not contain at least animal-derived components heterologous to the subject of treatment.
• the medium is a defined-composition medium that does not contain any components derived from the animal that is the subject of treatment.
• the medium used in the present invention is (2) a medium in which the number of microparticles having a particle diameter of 1 to 1000 nm is less than 1 x 10 9 /mL before use.
• Conventional media (such as a medium in which serum is added to a basal medium, or a medium in which hormones and specific nutritional components are added to a basal medium (also called a serum-free medium)) contain many extracellular vesicle-like particles having a particle size equivalent to that of the secreted extracellular vesicles.
• a medium containing such extracellular vesicle-like particles is used during the secretion process, the presence or absence of secretion of extracellular vesicles and the amount of secretion cannot be confirmed by the physical indicators described below.
• the medium used in the present invention is a medium in which extracellular vesicle-like particles are excluded as much as possible, and preferably (2') the number of microparticles having a particle size of 1 to 1000 nm in the state before use is less than 5 x 10 8 particles/mL.
• the particle size range of the microparticles contained in the medium used in the present invention can be set according to the target extracellular vesicles, and when recovering exosomes described later, the number of microparticles in the range of 10 to 500 nm is preferably less than the above numerical value, more preferably 30 to 200 nm, and particularly preferably 50 to 150 nm. When the number of microparticles is within such a range, the degree of interference (extracellular vesicle-like particles being detected together with extracellular vesicles) when detecting extracellular vesicles by physical indicators is within an acceptable range.
• microparticle refers to nanoscale particles (1 nm to 1000 nm) that can be detected using an electron microscope or scattering by laser light.
• the number of microparticles in the medium or extracellular vesicle-containing composition used in the pharmaceutical composition of the present invention is defined as the number of particles with a particle diameter of 1 to 1000 nm per mL of sample when measured by the nanoparticle traffic analysis (NTA) method using the following measuring equipment and under the following measuring conditions.
• NTA nanoparticle traffic analysis
• NanoSight LM10 manufactured by Malvern
• Measurement conditions ⁇ Video recording time: 60 seconds
• Camera level 14 Detection threshold 7
• the number of particles may be measured under equivalent conditions to the above-mentioned measurement conditions using another measurement device that has the same measurement capability as the above-mentioned measurement device and uses the NTA method as its measurement principle. A person skilled in the art would be able to appropriately select such a measurement device and measurement conditions.
• Examples of fine particles in the medium or extracellular vesicle-containing composition used in the pharmaceutical composition of the present invention include extracellular vesicles, extracellular vesicle-like particles such as vesicles formed by lipids (e.g., liposomes), and micelles formed by surfactants.
• the medium used in the present invention preferably does not contain lipids or contains lipids at less than 100 nM (preferably less than 50 nM, more preferably less than 10 nM). This is because lipids can cause the formation of fine particles in the medium.
• lipids are required as components of cell membranes when cells grow, but the medium used in the present invention does not require lipids in the first place because cell growth is not assumed.
• lipids that can be contained in the cell growth medium are typically at least one selected from unsaturated fatty acids, saturated fatty acids, and sterols.
• main lipids that constitute the cell membrane of mesenchymal stem cells include oleic acid, arachidonic acid, linoleic acid, stearic acid, and palmitic acid. Therefore, the medium used in the present invention preferably has a total concentration of oleic acid, arachidonic acid, linoleic acid, stearic acid, and palmitic acid of less than 100 nM, more preferably less than 50 nM, particularly preferably less than 10 nM, and most preferably less than 5 nM.
• the medium used in the present invention preferably does not contain a surfactant or contains a surfactant at less than the critical micelle concentration.
• surfactants are added to solubilize insoluble components such as the lipids and some nutrients in the medium or to promote cell growth.
• micelles formed by this solubilization can be detected together with extracellular vesicles as extracellular vesicle-like particles.
• the inventors have also confirmed the formation of extracellular vesicle-like particles even when the surfactant in the medium is less than the critical micelle concentration. This is presumably due to the formation of micelle-like bodies (like micelles formed to wrap around the lipids in the medium).
• the concentration of the surfactant is preferably 10 times the critical micelle concentration (1/10 of the critical micelle concentration) or less, more preferably 50 times or less, even more preferably 100 times or less, particularly preferably 500 times or less, and particularly preferably 1000 times or less.
• surfactants include Tween 80, SDS, Tergitol 7, Irgasan, and monesin.
• the critical micelle concentration of the surfactant can be measured, for example, by the following method: A surface tensiometer Sigma (manufactured by KSV Instruments) is used to perform analysis using an analysis program in the Sigma system.
• the surfactant is dropped into an aqueous medium at 0.01% increments, and the interfacial tension after stirring and standing is measured. From the obtained surface tension curve, the surfactant concentration at which the interfacial tension does not decrease even when the surfactant is dropped is calculated as the critical micelle concentration.
• the critical micelle concentration of Tween 80 in an aqueous medium was measured with a Sigma surface tensiometer and found to be 15 mg/L.
• the medium used in the present invention preferably contains a cytokine belonging to the TNF (tumor necrosis factor) family.
• the medium used in the present invention preferably contains TNF- ⁇ and TRAIL/APO2L as cytokines. These cytokines contribute to the induction of cell death, and therefore are not added to media for the growth or maintenance of cells.
• a medium suitable for the growth or maintenance of cells, which exhibits a high survival rate is used when secreting extracellular vesicles.
• the medium used in the present invention does not assume cell growth.
• the amount of extracellular vesicles secreted from the cells increases, even though cell growth and maintenance are suppressed compared to when these cytokines are not contained.
• the contents of TNF- ⁇ and TRAIL/APO2L before use are preferably 0.0001 to 2.0 mg/L, and particularly preferably 0.001 to 1.0 mg/L, respectively. When the contents of TNF- ⁇ and TRAIL/APO2L are within such ranges, damage to cells is minimized and extracellular vesicles are efficiently secreted from the cells.
• the medium used in the present invention does not need to contain growth factors (e.g., bFGF, EGF, PDGF and/or TGF- ⁇ 1) that may be contained in conventional medium. If the components of the medium are unknown, the presence or absence of the above proteins can be determined by conventionally known protein detection methods (e.g., ELISA method, Western blotting method, etc.).
• growth factors e.g., bFGF, EGF, PDGF and/or TGF- ⁇ 1
• the presence or absence of the above proteins can be determined by conventionally known protein detection methods (e.g., ELISA method, Western blotting method, etc.).
• the medium used in the present invention preferably has a total protein amount of 100 ⁇ g/mL or less before use.
• Conventional medium contains a large amount of diverse proteins for cell growth or maintenance. However, these proteins may remain in the product as impurities when purifying extracellular vesicles. Therefore, the medium used in the present invention is a medium from which proteins unnecessary for secretion of extracellular vesicles are removed as much as possible, and the total protein amount before use is preferably 50 ⁇ g/mL or less, more preferably 30 ⁇ g/mL or less, and particularly preferably 20 ⁇ g/mL or less. When the total protein amount is in such a range, the impurities during purification are within the allowable range.
• the total protein amount in the medium can be measured by a conventionally known measurement method.
• methods for measuring the total protein amount include the Qubit method, the Bradford method, the WST method, the Biuret method, the Lowry method, and the BCA method. Note that these measurement methods may be interfered with by surfactants and the like in the medium, so the medium must be appropriately diluted before measurement.
• the extracellular vesicle-containing composition contained in the pharmaceutical composition of the present invention may be the culture supernatant itself, or may be one that has been subjected to a purification process for extracellular vesicles.
• the method for purifying the culture supernatant is not particularly limited as long as it is a method that can concentrate extracellular vesicles and reduce the concentrations of other impurity components. Examples of methods for purifying the culture supernatant for extracellular vesicles include, but are not limited to, tangential flow filtration (TFF), purification by antibody affinity, size exclusion chromatography column, and density gradient ultracentrifugation.
• the purity of the extracellular vesicle-containing composition contained in the pharmaceutical composition of the present invention is defined as the purity (10 9 particles/mg protein)
• the purity of the extracellular vesicle-containing composition is usually 10 or more, preferably 100 or more, more preferably 150 or more.
• the upper limit of the purity but examples thereof include 1,000 or less, 800 or less, and 500 or less.
• the range of the purity is usually 10 to 1,000, preferably 100 to 1,000, more preferably 150 to 1,000, or usually 10 to 800, preferably 100 to 800, more preferably 150 to 800, or alternatively usually 10 to 500, preferably 100 to 500, more preferably 150 to 500.
• the medium used in the present invention may be of GMP (Good Manufacturing Practice) grade.
• a specific example of the medium used in the present invention is a medium having the composition shown in Table 1 below. (This medium is called "CDM4" in the following examples.)
• the pharmaceutical composition of the present invention may be used for the treatment or prevention of arthropathy.
• the "arthropathy" to which the pharmaceutical composition of the present invention is applied may be any joint disease (including both inflammatory and non-inflammatory diseases) occurring in synovial joints (e.g., knee joint, shoulder joint, elbow joint, hip joint, ankle joint, wrist joint, finger joint, spinal intervertebral joint, and temporomandibular joint, etc.).
• arthropathy examples include, but are not limited to, osteoarthritis, chronic rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, sports-related joint disorders (e.g., tennis elbow), congenital bone system diseases (e.g., achondroplasia, multiple epiphyseal dysplasia, spondyloepiphyseal dysplasia, metaphyseal dysplasia, Stickler syndrome, pseudoachondroplasia, multiple exostoses, hemihypertrophy, Ollier disease, Maffucci syndrome, etc.), osteochondroma, chondroma, etc.
• the arthropathy may be osteoarthritis.
• osteoarthritis examples include, but are not limited to, knee osteoarthritis, shoulder osteoarthritis, elbow osteoarthritis, hip osteoarthritis, ankle osteoarthritis, hand osteoarthritis, finger osteoarthritis, spinal osteoarthritis, and temporomandibular osteoarthritis.
• the pharmaceutical composition of the present invention is used for the treatment or prevention of osteoarthritis, and more preferably for the treatment or prevention of knee osteoarthritis.
• the subject of application of the pharmaceutical composition of the present invention is not particularly limited as long as it is an animal that can suffer from arthropathy.
• subjects of application include test animals such as mice and rats, pet animals such as dogs and cats, livestock animals such as pigs, horses, and cows, humans, and primates such as monkeys, orangutans, and chimpanzees, with humans being particularly preferred.
• the content of the extracellular vesicle-containing composition in the pharmaceutical composition of the present invention is not particularly limited, but can be appropriately selected, for example, within the range of 10 to 10 particles/ ⁇ L in terms of the number of extracellular vesicles (represented by the number of microparticles measured by the NTA method).
• the pharmaceutical composition of the present invention may contain pharma- ceutical acceptable components other than the composition derived from the culture supernatant containing extracellular vesicles secreted by mesenchymal stem cells by culturing in the medium used in the present invention, so long as the desired therapeutic or prophylactic effect is maintained.
• pharma- ceutical acceptable components other than the composition derived from the culture supernatant containing extracellular vesicles secreted by mesenchymal stem cells by culturing in the medium used in the present invention, so long as the desired therapeutic or prophylactic effect is maintained.
• Such components may include, for example, antioxidants, buffers, bacteriostatic agents, isotonicity agents, thickeners, stabilizers, preservatives, etc.
• the pharmaceutical composition of the present invention can be formulated as an aqueous or non-aqueous injectable liquid.
• the pharmaceutical composition of the present invention can be administered to a subject parenterally.
• parenteral administration include subcutaneous injection, intramuscular injection, local injection, and intraperitoneal administration, and may be appropriately determined depending on the site and symptoms of arthropathy in the subject.
• the dosage of the pharmaceutical composition of the present invention to a subject may vary depending on the subject to be administered, the administration method, the administration form, etc. Those skilled in the art can appropriately determine the appropriate dosage.
• treatment of a disease can include not only curing the disease, but also remission of the disease, improvement of the severity of the disease, and inhibition of the progression of the disease.
• prevention of a disease includes delaying the onset of a disease in addition to preventing the onset of the disease.
• prevention of a disease can also include preventing the recurrence of the disease after treatment or delaying the recurrence of the disease after treatment.
• composition in this specification can also be replaced with “medicinal agent” or “drug”.
• the present invention also provides a method for producing a pharmaceutical composition for treating arthropathy (hereinafter, may be referred to as "the production method of the present invention”), which comprises the following steps: (Step 1) A step of culturing mesenchymal stem cells in a medium that satisfies the following conditions: (1) the medium is a chemically defined medium that does not contain any animal-derived components different from those of the subject to be treated; and (2) the number of microparticles having a particle diameter of 1 to 1000 nm before use is less than 1 x 109 particles/mL. (Step 2) A step of recovering the composition containing extracellular vesicles derived from the culture supernatant obtained in step 1.
• step 1 of the manufacturing method of the present invention the mesenchymal stem cells, their culture conditions, the medium used, etc. are the same as those described for the pharmaceutical composition of the present invention.
• a composition derived from the culture supernatant containing extracellular vesicles secreted from mesenchymal stem cells cultured in the medium used in the present invention is collected.
• the extracellular vesicle-containing composition may be the culture supernatant itself, or may be a culture supernatant from which extracellular vesicles have been purified.
• the method for collecting the culture supernatant is not particularly limited, and any method known per se (e.g., filtration, centrifugation, etc.) may be used.
• step 2 further includes the purification step.
• the method for purifying extracellular vesicles can also be the same as that described for the pharmaceutical composition of the present invention.
• the medium components were measured before use for CDM4 used in the experiment and a conventional medium for comparison. Specifically, the particle size distribution and total protein amount were measured for CDM4 (see Table 1 above for the composition per 1000 mL of medium) and the conventional medium (serum-free medium: KBM (registered trademark) ADSC-4 (manufactured by Kohjin Bio Co., Ltd.)). As shown in Table 1, the contents of TNF- ⁇ and TRAIL/APO2L in CDM4 before use were 0.01 mg/L, respectively.
• the conventional medium contained a large amount of microparticles with a particle size of 1 to 1000 nm, whereas CDM4 contained almost no such microparticles.
• the number of microparticles with a particle size of 1 to 1000 nm in CDM4 was less than 1 x 109 particles/mL.
• the total protein amount was measured for CDM4 and conventional media using a total protein measurement kit Qubit (registered trademark) protein assay kit (manufactured by Thermo Fisher Scientific). Specifically, 0.5 to 5% of the total protein measurement reagent was added and mixed with a vortex mixer. At this time, the total protein measurement reagent was also added to the standard protein concentration solution and its serial dilution solution and mixed in the same manner. The fluorescence intensity of the samples was measured using a Qubit 4 Fluorometer (manufactured by Thermo Fisher Scientific). The total protein concentration of the sample was calculated from the fluorescence intensity of the standard protein concentration solution and its serial dilution solution. The results are shown in Table 3.
• Qubit registered trademark
• the conventional medium contained a large amount of protein, whereas the total protein amount of CDM4 was below the detection limit (12.5 ⁇ g/mL).
• ASCs Culture of human adipose-derived stem cells (ASC)
• ASCs were used as a source of EVs because they have been clinically used to treat osteoarthritis.
• Cells were obtained from the degraded tissue by centrifugation and resuspended on plastic culture dishes in an expansion medium consisting of DMEM/F-12 (Wako Pure Chemical Industries, Ltd.) supplemented with NeoSERA (Nihon Biomedical Co., Ltd.) and Human Recombinant basic fibroblast growth factor (bFGF, REPROCELL). Cells at stage 2-4 were used for all experiments. Flow cytometry analysis showed that the cells showed more than 95% positive expression of CD73, CD90, and CD105, and expression of a negative marker cocktail consisting of a mixture of CD11b/CD19/CD31/CD45/HLA-DR was ⁇ 2% (see Figure 5).
• hAC Normal human articular chondrocytes
• CDM1 KBM ADSC4, Kohjin Bio
• CDM2 MSC NutriStem XF, Sartorius
• CDM3 StemPro MSC SFM XenoFree, Gibco (a commercially available GMP-compliant serum-, animal-derived component- and phenol red-free medium).
• CDM4 A newly developed GMP-compliant medium that does not contain serum or animal-derived components, in order to avoid contamination of EVs purified using ultrafiltration with unknown substances (see Patent Nos. 7371975 and 7423016).
• the signal intensity of the PBS sample was subtracted from the experimental value as background value.
• the medium remaining in the cell culture well after the sample collection for ExoScreen assay was stained using Cell Counting Kit-8 (CCK-8, Dojindo Laboratories), and the obtained absorbance was quantified according to the protocol.
• the indicated wavelength was evaluated using Multiskan GO (Thermo Fisher Scientific).
• the (MIEV) 2018 guidelines recommend that samples isolated from non-conditioned medium without cell culture should be used as a negative control comparison to evaluate the characteristics of conditioned medium-derived EVs. Therefore, enrichments from each non-conditioned CDM were prepared as background controls (BG) using the same procedure used to isolate conditioned CDM-derived EVs. Corresponding EVs and BGs were prepared at the same enrichment ratio. Preparations were stored at 4°C for less than one month before being used in experiments.
• Nanoparticle Tracking Analysis Nanoparticle concentration and size distribution were measured using NanoSight LM10 or NS300 (Malvern). Samples were diluted to optimal concentrations (40-100 times) with contamination-free PBS and a 60-second video was recorded. The camera level was 14 and the detection threshold was 7. Protein concentration was measured using the Qubit Protein Assay Kit and Qubit Fluorometer (Invitrogen) according to the manufacturer's instructions. Not detected (ND) was defined as ⁇ 20 particles/frame by NTA or protein concentration outside the range by Qubit.
• TEM Transmission electron microscope
• ELISA Western Blotting and Enzyme-Linked Immunosorbent Assay
• the amount of EV in each EV sample was measured using a CD9/CD63 Exosome ELISA Kit for Human (Cosmo Bio) according to the manufacturer's instructions.
• the CD9/CD63 fusion protein provided with the kit was used.
• Chromogenic Nanoplasmonic Assay A colorimetric nanoplasmonic assay was used to determine the purity of the samples. EV test samples were diluted 4-fold in PBS and mixed with 6 nM gold nanoparticles (15 nm, Cytodiagnostics) in a 2:1 volume ratio. After 20 min of incubation at room temperature, the mixture was transferred to a cuvette and the UV-vis spectrum was measured from 400 to 800 nm. Test samples were replaced with PBS as a negative control and 0.2% w/v BSA as a positive control.
• Cell proliferation and migration assays 3,000 hACs were seeded in a 96-well plate on day 0. After overnight culture, the medium was changed to high glucose DMEM containing 2% Exosome-depleted FBS (Gibco) and 1% Antimycotic Solution (AA, Sigma), and samples of EV, BG or vehicle control (PBS) were added at a concentration of 5 x 109 particles/ml for each of the EV group members, or equal amounts for each of the BG or control groups. Absorbance was measured using CCK-8 on days 4 and 7. The effect of hAC on migration was evaluated using CytoSelect Cell Migration Assay kit (Cell Biolab).
• hACs were cultured as micromasses ( 1x105 cells in 10 ⁇ L) on 12-well plates. After 3 hours of culture, cartilage basal medium and EV, BG samples or vehicle control (PBS) were added.
• the cartilage basal medium consisted of high glucose-containing DMEM (Nacalai), 50 ⁇ g/mL L-ascorbic acid, 40 ⁇ g/mL L-proline (Wako Pure Chemical Industries, Ltd.), 1% ITS+Premix (Corning), and 1% AA, and the addition method for each sample was the same as above. After 2 days of culture, cell masses were collected using QIAzol (Qiagen).
• CIOA Collagenase-induced osteoarthritis
• mice were randomly assigned to two groups, and the treatment group received 8 ⁇ L of EV (1 ⁇ 10 9 particles) and the control group received the same amount of BG.
• Mice were sacrificed on day 28 and the tibiae were harvested. Five randomly selected contralateral tibiae served as normal controls. The tibial articular surface was macroscopically observed and assessed using the knee cartilage damage scoring system based on the Guingamp classification (Guingamp et al., Arthritis Rheum. 1997 Sep;40(9):1670-9). Scores were assigned between 0 (normal) and 4 (worst) (Table 5).
• the medial and lateral tibial plateaus were scored separately, and the sum of the scores was the final score.
• the tibia samples were then fixed in formalin, dehydrated in successive ethanols, and decalcified in EDTA. After paraffin fixation, coronal sections of the mid-tibia were made at a thickness of 3 ⁇ m. Sections were stained with Safranin O, and the medial tibia was evaluated using the OARSI (Osteoarthritis Research Society International) semiquantitative scoring system (Glasson et al. Osteoarthritis Cartilage. 2010 Oct:18 Suppl 3:S17-23). Scores were assigned between 0 (normal) and 6 (worst) (Table 6).
• Each culture sample contained CD9-positive EVs, and when comparing samples taken at the 24-h time point, the CDM4 and CDM3 groups contained significantly more than the CDM1 and CDM2 groups (p ⁇ 0.01), with a similar pattern observed for the 48-h samples (Fig. 1A). Because each medium provided different proliferation potential to cells, when corrected for the cell number of each medium at each time point, the CDM4 group contained significantly more CD9+EVs than the other samples at all time points evaluated (p ⁇ 0.01) (Fig. 1B). The number of CD63+EVs detected was significantly higher in the CDM3 group than the other groups at both 24 and 48 hours (p ⁇ 0.01). However, when corrected for the cell number, the CDM4 group contained significantly more CD63+EVs at both time points (p ⁇ 0.01) (Fig. 1C, D). These results indicate that the CDM4 medium most efficiently promotes the release of EVs from ASCs.
• EV preparations from each conditioned medium and each uncultured medium (BG) concentrate were extracted using the TFF system. All EV samples contained some non-EV particles as determined by images captured from the NTA ( Figure 2A). Of note, BG samples from CDM1, 2, and 3 also contained particles with a peak distribution of approximately 80-140 nm in diameter, but these were not derived from cell secretions. Conversely, there were no detectable particles in BG samples in CDM4 (Fig. 2B). When comparing particle concentrations of each EV and BG, no significant differences were detected in CDM1 and 2 groups (Fig. 2C). CDM3-EV had significantly fewer particles than the CDM3-BG group (p ⁇ 0.05).
• CDM4-EV had significantly more particles than the CDM4-BG group, and no particles other than EV were detected in the latter group (p ⁇ 0.05).
• Protein concentration was assessed as an indicator of potential contamination of EV samples (Figure 2D).
• there was no detectable protein concentration for the CDM4-BG group which is also consistent with the inability to detect non-EV particles as described above.
• Gel staining was performed to confirm the protein content in each sample (Figure 2E).
• TEM revealed membranous particles with diameters of approximately 50-100 nm in all EV groups, but many rod-shaped impurities were detected, especially in CDM1, 2, and 3-EVs groups (Fig. 3A).
• CDM1, 2, and 3-EVs groups contained higher albumin amounts than the CDM4-EV group.
• CD9 and CD63 positive substances were detected by ELISA in all groups ( Figure 3C).
• the CD9 and CD63 positive EVs of CDM4-EV were about 17 to 570 times higher than those of other CDM-EVs, and a significant difference was detected (p ⁇ 0.01) ( Figure 3D).
• the number of CDM4-EVs was converted to values per particle, it was 4 to 16 times higher than the other CDMs (p ⁇ 0.05) ( Figure 3E).
• the BG group significantly promoted ECM synthesis compared to the EV group (p ⁇ 0.01).
• both BG and EV significantly inhibited apoptosis compared to the PBS control (p ⁇ 0.01).
• the present invention provides technology relating to a novel composition for use in treating arthritis (particularly osteoarthritis). Therefore, the present invention is extremely beneficial in the medical field.

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