JP7126703B2 - Cerebral disorder therapeutic agent containing umbilical cord-derived cells - Google Patents

Description

TECHNICAL FIELD The present invention relates to therapeutic agents for brain disorders containing umbilical cord-derived cells.

Perinatal neonatal encephalopathy complicates 1 to 2 per 1,000 live births and causes lifelong cerebral palsy. .

Perinatal brain disorders that cause cerebral palsy include hypoxic-ischemic encephalopathy, cerebral hemorrhage, and periventricular leukomalacia. , activation of macrophages and associated hypercytokinemia, which is an inflammatory pathology. In particular, cerebral hemorrhage is likely to be complicated in premature infants, and the symptoms are the most severe and the life prognosis is poor.

Hypothermia therapy is considered effective as a treatment, but it is said to be effective only in 1 out of 8 to 9 children treated. Moreover, at present, there is no established treatment method other than hypothermia.

So far, clinical studies have been conducted in which autologous bone marrow-derived mesenchymal cells are directly administered to patients with cerebral palsy, and their effectiveness has been reported (Non-Patent Document 1). In addition, although there is a report of administration of umbilical cord-derived cells, it has not resulted in marked improvement in some motor functions (Non-Patent Document 2).

Cytotherapy 2013 Dec;15(12):1549-62 Cytotherapy 2015 17(2): 224-231

In cerebral disorders caused by cerebral hemorrhage or ischemia, it is necessary not only to cure the underlying hemorrhage or ischemia, but also to promote the protection or regeneration of nerve cells and the like damaged by the cause. Administration of umbilical cord-derived cells has been considered for the purpose of protecting or regenerating such nerve cells. It is unclear whether the In addition, in brain disorders, it is assumed that intrathecal administration by lumbar puncture is performed. Make it an issue.

As a result of intensive research to solve the above problems, the present inventors found that intravenous administration of a cell population containing cells prepared from umbilical cord tissue including amniotic membrane, blood vessels, perivascular tissue, and Walton's Jelly caused cerebral hemorrhage. The inventors have found that the behavior of brain disorders can be improved, and have completed the present invention.

Accordingly, the present invention relates to:
[1] A therapeutic agent for brain disorders, comprising umbilical cord-derived cells.
[2] The therapeutic agent of [1], which is for intravenous administration.
[3] The therapeutic agent of [1] or [2], wherein the umbilical cord-derived cells are cells prepared from umbilical cord tissue containing amnion, blood vessels, perivascular tissue, and Walton's Jelly.
[4] The umbilical cord-derived cells are
Cells characterized by (i) positive for CD105, CD73, CD90, CD44, HLA-class I, HLA-G5 and PD-L2, and (ii) negative for CD45, CD34, CD11b, CD19 and HLA-Class II The therapeutic agent according to any one of [1] to [3], which is
[5] The umbilical cord-derived cells are
(iii) The therapeutic agent according to any one of [1] to [4], which induces expression of any one gene and/or protein of IDO, PGE2, and PD-L1 under inflammatory conditions.
[6] The therapeutic agent of any one of [1] to [5], wherein the brain disorder is fetal or neonatal brain disorder.
[7] The therapeutic agent of any one of [1] to [6], wherein the brain disorder is cerebral palsy.
[8] (i) positive for CD105, CD73, CD90, CD44, HLA-class I, HLA-G5 and PD-L2, and (ii) negative for CD45, CD34, CD11b, CD19 and HLA-Class II, Umbilical cord-derived cells for the treatment of brain disorders.
[9] (iii) The umbilical cord-derived cell of [8], wherein the expression of any one gene and/or protein of IDO, PGE2, and PD-L1 is induced under inflammatory conditions.
[10] The umbilical cord-derived cells of [8] or [9], which are for intravenous administration.
[11] The umbilical cord-derived cells of [8] or [9], which are cells prepared from umbilical cord tissue containing amniotic membrane, blood vessels, perivascular tissue and Walton's Jelly.
[12] The umbilical cord-derived cell of any one of [8] to [11], wherein the brain disorder is fetal or neonatal brain disorder.
[13] The umbilical cord-derived cell of any one of [8] to [12], wherein the brain disorder is cerebral palsy.
[14] A method for treating brain disorders using umbilical cord-derived cells.
[15] The therapeutic method of [14], wherein the umbilical cord-derived cells are intravenously administered.
[16] The therapeutic method of [14] or [15], wherein the umbilical cord-derived cells are cells prepared from umbilical cord tissue containing amnion, blood vessels, perivascular tissue, and Walton's Jelly.
[17] The umbilical cord-derived cells are
(i) positive for CD105, CD73, CD90, CD44, HLA-class I, HLA-G5 and PD-L2, and (ii) negative for CD45, CD34, CD11b, CD19 and HLA-Class II [14] to [16].
[18] The therapeutic method of [17], wherein the umbilical cord-derived cells (iii) are induced to express IDO, PGE2, PD-L1 genes and/or proteins under inflammatory conditions.
[19] The treatment method of any one of [14] to [18], wherein the brain disorder is a fetal or neonatal brain disorder.
[20] The treatment method according to any one of [14] to [19], wherein the brain disorder is cerebral palsy.

According to the present invention, the treatment of brain disorders comprises a cell population comprising cells prepared from amniotic membrane, blood vessels, perivascular tissue and umbilical cord tissue, including Walton's Jelly, and/or is administered intravenously. It becomes possible to provide agents.

FIG. 1 shows the results of measuring the expression level of IDO mRNA in umbilical cord-derived cells by quantitative PCR (ratio to positive control GAPDH). FIG. 2 shows the results of measuring the protein concentration of PGE2 in the culture supernatant obtained by co-culturing MLR and umbilical cord-derived cells by ELISA. FIG. 3 shows stained images of brain tissue sections collected after administration of PBS to neonatal cerebral hemorrhage model mice. The lower figure shows an enlarged image of the frame of the upper figure, and shows an immunostained image stained with antibodies against MAP2 and GFAP. MAP2 is shown in green and GFAP in red. FIG. 4 shows a schematic diagram of the schedule for preparation, treatment and evaluation of neonatal cerebral hemorrhage model mice. A cerebral hemorrhage model was prepared by intracerebral administration of blood on day 5 after birth (Day 5), cells for treatment were administered on day 8 after birth (Day 8), and cells were administered on day 15 and day 22 after birth. It shows that evaluation was performed by MRI and behavioral evaluation was performed on the 23rd day after birth. FIG. 5 shows the results of pharmacokinetics using umbilical cord-derived cells transfected with the Luciferase gene. In vivo imaging of luminescence by Luciferase at 6 hours, 3 days, 4 days, 5 days, 7 days, 14 days and 21 days after cell administration is shown. Twenty-one days after dosing, no umbilical cord-derived cells are detectable. FIG. 6 shows mouse behavioral trajectories of the 10-minute open field test. The figure below shows a graph of the number of times of rearing behavior and the number of times of rearing behavior (traveled distance) in cerebral hemorrhage model mice and normal mice after administration of umbilical cord-derived cells (MSC) or negative control (PBS). FIG. 7 shows the measurement results of stride length (forelimb stride length, hindlimb stride length and distance between forelimbs and hindlimbs) or hindlimb angles in cerebral hemorrhage model mice and normal mice after administration of umbilical cord-derived cells (MSC) or negative control (PBS). The figure below shows the behavior of left leg abduction in each mouse as measured by the hindlimb angle. Figure 8 shows the measurement results of MRI in cerebral hemorrhage model mice after administration of umbilical cord-derived cells (MSCs) or negative control (PBS) at 15 days after birth (Figure 8A) and 22 days after birth (Figure 8B). show. FIG. 9 shows immunostained images of the periventricular tissues of untreated mice, negative control (PBS) administration, or cerebral hemorrhage model mice after administration of umbilical cord-derived cells (MSC), stained with antibodies against MAP2 and GFAP, and DAPI. MAP2 is shown in green and GFAP in red, DAPI (nuclei) in blue. FIG. 10 shows HE-stained images of periventricular tissues of untreated mice, cerebral hemorrhage model mice after administration of negative control (PBS) or after administration of umbilical cord-derived cells (MSCs). FIG. 11 shows MBP-stained images of the periventricular tissues of untreated mice, cerebral hemorrhage model mice after administration of negative control (PBS) or after administration of umbilical cord-derived cells (MSCs). Figure 12 shows histological quantitative evaluation of MBP-stained images of periventricular tissues of untreated mice, negative control (PBS) administration, or cerebral hemorrhage model mice after administration of umbilical cord-derived cells (MSC), and white matter volume ( %) is calculated. FIG. 13 shows TUNEL-stained images of periventricular tissues of untreated mice, cerebral hemorrhage model mice after administration of negative control (PBS) or after administration of umbilical cord-derived cells (MSCs). FIG. 14 shows histological quantitative evaluation of TUNEL-stained images of periventricular tissues of untreated mice, negative control (PBS) administration, or cerebral hemorrhage model mice after administration of umbilical cord-derived cells (MSC). The result of calculating the ratio (%) of is shown. FIG. 15 shows the results of behavioral evaluation (measurement of behavioral distance for 10 minutes) after administering umbilical cord-derived cells (UC-MSCs) obtained by culturing under each condition to the facial vein of a cerebral hemorrhage model mouse. indicates FIG. 16 shows the results of behavioral evaluation (measurement of the number of times of standing up) after administration of umbilical cord-derived cells (UC-MSCs) obtained by culturing under each condition to the facial vein of a cerebral hemorrhage model mouse. FIG. 17 shows the results of measuring the expression levels of BDNF and HGF mRNAs in umbilical cord-derived cells by quantitative PCR (ratio to positive control GAPDH). FIG. 18 shows the results of bead assay measurement of protein concentrations of BDNF and HGF in the culture supernatant obtained by co-culturing mouse neurons and umbilical cord-derived cells. FIG. 19 shows the results of examining the effect of administration of umbilical cord-derived cells to cerebral hemorrhage model mice (effect on serum NGF content). FIG. 20 shows the results of examining the effect of administration of umbilical cord-derived cells to cerebral hemorrhage model mice (effect on serum BDNF content). FIG. 21 shows the results of examining the effect of administration of umbilical cord-derived cells to cerebral hemorrhage model mice (effect on serum HGF content). FIG. 22 shows the results of evaluating the persistence of the cells in each organ after administration of umbilical cord-derived cells to adult mice. FIG. 23 shows the results of immunostaining (HLA-Class I and CD105) of brain sections of mice to which umbilical cord-derived cells were administered via the jugular vein.

In the present invention, the umbilical cord is a white tubular tissue that connects the fetus and the placenta, and means a tissue that does not contain the placenta and umbilical cord blood. The umbilical cord of the present invention is not particularly limited as long as it is an umbilical cord collected from mammals, and for example, umbilical cords of primate mammals. More preferably, it is a human umbilical cord.

In using umbilical cord-derived cells as a therapeutic agent for brain disorders in the present invention, the umbilical cord may be an umbilical cord collected from a subject to be treated or an umbilical cord collected from a non-therapeutic subject. It is desirable to use umbilical cords collected from other than the subject of treatment from the viewpoint of not being subject to restrictions on preparation. As shown in the examples described later, the umbilical cord-derived cells of the present invention exhibit therapeutic effects without being excluded due to immune rejection or the like, even if the umbilical cord-derived cells are collected from sources other than the target of treatment. has been confirmed.

In the present invention, the umbilical cord can be collected by appropriately removing the placenta from postpartum tissues including the placenta and umbilical cord delivered by vaginal delivery and caesarean section. After removing the cord blood from the collected umbilical cord, aseptic or bacteriostatic treatment may be performed. Removal of cord blood is accomplished by rinsing with an anticoagulant solution such as a heparin-containing solution. Aseptic or bacteriostatic treatments include, but are not limited to, application of povidone-iodine, or media supplemented with one or more antibiotics and/or antimycotics such as penicillin, streptomycin, amphotericin B, gentamicin, and nystatin. Alternatively, it may be immersed in a buffer. Moreover, a step of selectively lysing erythrocytes may be included as necessary. Methods well known in the art can be used to selectively lyse red blood cells, such as incubation in hypertonic or hypotonic media with lysis with ammonium chloride.

The umbilical cord-derived cells of the present invention mean a cell population prepared using umbilical cord as a raw material, and the preparation method includes (1) cutting the umbilical cord, (2) culturing the umbilical cord segment, and (3) ) methods comprising the step of passaging are exemplified. In addition, other methods for preparing the cells include (A) a step of cutting the umbilical cord, a step of enzymatic treatment, or dissociating the tissue by both, (B) culturing the umbilical cord tissue, and (C) passage. A method is exemplified that includes the step of: The umbilical cord-derived cells may be a heterogeneous cell population instead of a uniform cell population. In addition, the umbilical cord-derived cells of the present invention may be defined as a cell population having the following characteristics;
(a) shows adhesion to plastic under culture conditions in a standard medium;
(b) positive for CD105, CD73, CD90, CD44, HLA-class I, HLA-G5 and PD-L2, negative for CD45, CD34, CD11b, CD19 and HLA-Class II, and (c) conditions of inflammation The expression of IDO, PGE2, PD-L1 genes and/or proteins is induced.

In the present invention, the term “positive” refers to comparison with a negative control cell that does not express the antigen or a negative control reaction using an antibody that does not react with the antigen by an analysis method such as flow cytometry that is detected using an antigen-antibody reaction. Similarly, negative means equal or less detection compared to a negative control reaction using negative control cells that do not express the antigen or an antibody that does not react with the antigen. means that

In the present invention, HLA-class I means HLA-A, B or C, and HLA-Class II means HLA-DR, DQ or DP.

In the present invention, inflammatory conditions refer to conditions of contact with inflammatory cytokines such as interferon γ.

In the step of (1) cutting the umbilical cord of the present invention, the umbilical cord obtained by the above-described method is cut by mechanical force (shredding force or shearing force) in a state containing amniotic membrane, blood vessels, perivascular tissue, and Walton's Jelly. can be done by Although not particularly limited, the umbilical cord segment obtained by cutting has a size of 1 to 10 mm ³ , 1 to 5 mm ³ , 1 to 4 mm ³ , 1 to 3 mm ³ or 1 to 2 mm ³ . In the step of (A) enzymatic treatment of the present invention, the umbilical cord obtained by the above method is enzymatically treated in a state containing amniotic membrane, blood vessels, perivascular tissue, and Walton's Jelly to dissociate the tissue. . Enzymatic treatments include, but are not limited to, enzyme treatments with collagenase, dispase, hyaluronidase, and the like.

The steps (2) and (B) of culturing umbilical cord segments of the present invention can be performed by seeding cut umbilical cord segments in a culture vessel and culturing in a culture medium suitable for umbilical cord-derived cells. In a preferred embodiment of step (2), the umbilical cord segment is not treated with a digestive enzyme.

In the present invention, the incubator is not particularly limited as long as it has a solid surface, but the term "solid surface" means any material that can bind to the umbilical cord-derived cells of the present invention. In certain embodiments, such material is a plastic material that has been treated (eg, treated to increase hydrophilicity) to promote binding of mammalian cells to its surface. The shape of the culture vessel having a solid surface is not particularly limited, but petri dishes, flasks, and the like are preferably used.

In the present invention, a suitable culture medium for umbilical cord-derived cells is a basal medium supplemented with serum and/or containing albumin, transferrin, fatty acids, insulin, sodium selenite, cholesterol, collagen precursors, trace elements, 2 - May be made with the addition of one or more serum replacements such as mercaptoethanol, 3'-thiolglycerol. These media may optionally contain substances such as lipids, amino acids, proteins, polysaccharides, vitamins, growth factors, low-molecular-weight compounds, antibiotics, antifungal agents, antioxidants, pyruvic acid, buffers, and inorganic salts. may be added. The basal medium is, for example, Dulbecco's Modified Eagle's Medium (DMEM) (high glucose or low glucose), modified DMEM, DMEM/MCDB 201, Eagle's basal medium, Ham's F10 medium (F10), Ham' s F-12 medium (F12), Iscove's modified Dulbecco's (IMDM) medium, Fischer's medium, mesenchymal stem cell growth medium (MSCGM), DMEM/F12, RPMI 1640, CELL-GRO-FREE and mixed media thereof, etc. are mentioned. Examples of serum include, but are not limited to, human serum, fetal bovine serum (FBS), bovine serum, calf serum, goat serum, horse serum, porcine serum, sheep serum, rabbit serum, rat serum, and the like. When serum is used, it may be added at 5 v/v% to 15v/v%, preferably 10v/v%, to the basal medium. Examples of fatty acids include, but are not limited to, linoleic acid, oleic acid, linoleic acid, arachidonic acid, myristic acid, palmitoic acid, palmitic acid, and stearic acid. Examples of lipids include, but are not limited to, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and the like. Examples of amino acids include, but are not limited to, L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, L-glycine, and the like. Examples of proteins include, but are not limited to, ecotin, reduced glutathione, fibronectin and β2-microglobulin. Polysaccharides are exemplified by glycosaminoglycans, and among glycosaminoglycans, hyaluronic acid, heparan sulfate and the like are particularly exemplified, but not limited thereto. Growth factors include, for example, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), Examples include leukocyte inhibitory factor (LIF), basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-β), hepatocyte growth factor (HGF), connective tissue growth factor (CTGF) and erythropoietin. but not limited to: Antibiotics and/or antifungal agents include penicillin G, streptomycin sulfate, amphotericin B, gentamicin, nystatin and mixtures thereof. In steps (2) and (B), from the viewpoint of preventing the seeded umbilical cord segments from floating in the culture medium, it is desirable to hold the umbilical cord segments with a plate or the like during the culture period. As the plate, the plate described in JP-A-2015-70824 is exemplified.

In steps (2) and (B), culture is performed under 0-5% CO ₂ . Preferably, it is cultured under 2-25% O ₂ , more preferably 5-20% O ₂ . The culture is preferably carried out at 25-40°C, more preferably at 37°C.

In steps (2) and (B), cells migrate from the umbilical cord section, and the cells can be cultured in the incubator until they become 50%, 60%, 70%, 80% or more confluent. preferable. After culturing, the cells are washed to remove unbound cells and cell debris, detached with a detachment agent containing trypsin, EDTA solution, collagenase, dispase, hyaluronidase or mixtures thereof, and the cells and umbilical cord sections are removed. Only cells can be obtained as umbilical cord-derived cells by filtering the containing stripping solution using a cell strainer or the like. The obtained umbilical cord-derived cells can be seeded in the culture vessel described above and cultured using the culture medium described above.

Subsequently, in steps (3) and (C), the umbilical cord-derived cells can be appropriately grown to the required number by subculturing. For subculture, cells may be detached with a detachment agent containing trypsin, EDTA solution, collagenase, dispase, hyaluronidase, or a mixture thereof, seeded at an appropriate cell density in a separately prepared culture vessel, and culture continued. When seeding cells, typical cell densities are 100 cells/cm ² to 100,000 cells/cm ² , 500 cells/cm ² to 50,000 cells/cm ² , 1,000 to 10,000 cells /cm ² , 2,000 to 10,000 cells/cm ² and the like are exemplified. In certain aspects, the cell density is 2,000-10,000 cells/cm ² . It is preferable to adjust the period to reach appropriate confluency to be 3 to 7 days. During culturing, the medium may be changed as appropriate, if necessary.

The subculturing of steps (3) and (C) may be performed until senescence where cell division stops. From the viewpoint of therapeutic use, it is preferably subcultured 3 to 25 times, more preferably subcultured 4 to 12 times.

In order to confirm that the obtained cells are the umbilical cord-derived cells of the present invention, surface antigens and the like may be analyzed by conventional methods using flow cytometry and the like. Moreover, you may evaluate by measuring the amount of various proteins produced from the said cell.

The obtained umbilical cord-derived cells may be directly prepared for treatment or may be cryopreserved. Cryopreservation is carried out by suspending the cells in a cryopreservation solution capable of adequately preserving umbilical cord-derived cells and storing the cells at -80°C to -180°C. The cryopreservation solution is not particularly limited, but for example, an aqueous solution containing a cryoprotectant and glucose is exemplified. Examples of antifreeze agents include dimethylsulfoxide (hereinafter referred to as DMSO), dextran, glycerol, propylene glycol, and 1-methyl-2-pyrrolidone. Preferred antifreeze agents are DMSO and propylene glycol, with DMSO being particularly preferred. The cryoprotectant is preferably contained in the cryopreservation solution at 1.0 to 15.0 w/v%, more preferably 5.0 to 15.0 w/v%, and 5.0 to 12.0 w/v. %/v %, and particularly preferably 8.0 to 11.0 w/v %.

Glucose contained in the cryopreservation solution is preferably contained in the cryopreservation solution at 0.5 to 10.0 w/v%, more preferably 1.0 to 10.0 w/v%, 2 0 to 8.0 w/v% is more preferred, and 2.0 to 5.0 w/v% is particularly preferred.

The cryopreservation solution may further contain other ingredients. Other ingredients include, for example, pH adjusters, thickeners, and the like. Examples of pH adjusters include sodium bicarbonate, HEPES, and phosphate buffers. In addition, when a phosphate buffer is not added to the Basic Stock Solution (BSS), a solution added with sodium chloride having a function of imparting a buffering capacity around a pH suitable for umbilical cord-derived cells can also be used. Among these, it is particularly preferable to use a phosphate buffer. A pH adjusting agent is preferably used as appropriate to adjust the pH of the cryopreservation solution to approximately 6.5 to 9.0, preferably 7.0 to 8.5. The phosphate buffer in the present invention includes sodium chloride, monosodium phosphate (anhydrous), monopotassium phosphate (anhydrous), disodium phosphate (anhydrous), trisodium phosphate (anhydrous), potassium chloride, and potassium dihydrogen phosphate (anhydrous), etc. It is particularly preferable to use sodium chloride, monosodium phosphate (anhydrous), potassium chloride, or potassium dihydrogen phosphate (anhydrous). The pH adjuster is preferably contained in the cryopreservation solution at 0.01 to 1.0 w/v%, more preferably 0.05 to 0.5 w/v%.

The cryopreservation solution may or may not contain natural animal-derived components. Natural animal-derived components include, for example, the serum and basal medium described above. Cryopreservation solutions are preferably free of natural animal-derived components. A cryopreservation solution that does not contain natural animal-derived components does not cause lot-to-lot quality problems of natural animal-derived components, and the various cytokines, growth factors, and hormones contained in serum are naturally present. It is possible to avoid the possibility of changes in the properties of cells in the umbilical cord tissue due to components unnecessary for cell preservation, and also to avoid the influence of components of unknown origin contained in the basal medium. Cryopreservation solutions that are free of natural animal-derived components are therefore very useful, especially in clinical use.

The cryopreservation solution may further contain a thickening agent. The thickening agent is not particularly limited as long as it can constitute a cryopreservation solution that can sufficiently preserve umbilical cord tissue. Examples of thickening agents include carboxymethyl cellulose (hereinafter referred to as CMC), carboxymethyl cellulose sodium (hereinafter referred to as CMC-Na), organic acid polymers, propylene glycol alginate, and sodium alginate. As thickeners, CMC and CMC-Na are preferred, and CMC-Na is particularly preferred. Among organic acid polymers, sodium polyacrylate is preferred. The thickening agent is preferably contained in the cryopreservation solution at 0.1 to 1.0 w/v%, more preferably 0.1 to 0.5 w/v%, and 0.2 to 0.4 w /v% is more preferred.

The cryopreservation solution is preferably an aqueous solution. The osmotic pressure of the cryopreservation solution is preferably 1000 mOsm or more, more preferably 1000 to 2700 mOsm, in order to maintain performance as a preservation solution.

A preferred cryopreservation solution is an aqueous solution containing a thickening agent, a cryoprotectant, and glucose, and free of natural animal-derived components. A more preferred example is an aqueous solution containing CMC-Na, DMSO, and glucose and free of natural animal-derived components. In a more preferred example, the cryopreservation solution contains 0.1 to 1.0 w/v% of CMC-Na, 1.0 to 15.0 w/v% of DMSO, and 0.5 to 10.0 w/v of glucose. /v % and free of natural animal-derived components.

The umbilical cord-derived cells obtained by the above method can be prepared as a therapeutic agent for brain disorders by mixing with an infusion preparation. On the other hand, the cryopreserved umbilical cord-derived cells may be suspended in a cryopreservation solution and used as a therapeutic agent for brain disorders as they are after thawing, or may be prepared as a therapeutic agent for brain disorders by mixing with an infusion preparation. good. In mixing, the culture solution or cryopreservation solution in which the cells are suspended may be mixed with the infusion preparation, etc. After the cells are separated from the solvent by centrifugation, etc., only the cells are mixed with the infusion preparation. can be In order to avoid complicated procedures, it is preferable not to include a step of culturing frozen cells after thawing, or to directly mix a cryopreservation solution in which thawed cells are suspended with an infusion preparation.

The "infusion preparation" in the present invention is not particularly limited as long as it is a solution that can be used in the treatment of humans. No. 2 liquid, No. 3 liquid, No. 4 liquid and the like can be mentioned.

The therapeutic agents for brain disorders of the present invention also include kits in which umbilical cord-derived cells and infusion preparations are combined.

The cell-mixed infusion preparation may further contain a pharmaceutically acceptable carrier. In the present invention, the carrier means a suspending agent, a solubilizing agent, a stabilizing agent, a tonicity agent, a preservative, an antiadsorption agent, a surfactant, a diluent, a medium, and a pH adjusting agent for administering the therapeutic agent. Agents, analgesics, buffers, sulfur-containing reducing agents, antioxidants and the like can be added as appropriate.

Examples of suspending agents include methyl cellulose, polysorbate 80, hydroxyethyl cellulose, gum arabic, tragacanth powder, carboxymethyl cellulose sodium, polyoxyethylene sorbitan monolaurate and the like. Solution adjuvants include polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinic acid amide, polyoxyethylene sorbitan monolaurate, macrogol, castor oil fatty acid ethyl ester, and the like. Stabilizers include dextran 40, methylcellulose, gelatin, sodium sulfite, sodium metasulfate and the like. Examples of tonicity agents include D-mannitol and sorbitol. Preservatives include, for example, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, sorbic acid, phenol, cresol, chlorocresol and the like. Examples of antiadsorption agents include human serum albumin, lecithin, dextran, ethylene oxide propylene oxide copolymer, hydroxypropyl cellulose, methyl cellulose, hydrogenated castor oil, polyethylene glycol and the like. Examples of current-containing reducing agents include N-acetylcysteine, N-acetylhomocysteine, thiochitoic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, sodium thiosulfate, glutathione, Examples include those having a sulfhydryl group such as thioalkanoic acids having 1 to 7 carbon atoms. Examples of antioxidants include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbic palmitate, L-ascorbic stearate, sodium hydrogen sulfite. , sodium sulfite, triamyl gallate, propyl gallate or sodium ethylenediamine tetraacetate (EDTA), sodium pyrophosphate, chelating agents such as sodium metaphosphate. Furthermore, inorganic salts such as sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, and sodium hydrogen carbonate; organic salts such as sodium citrate, potassium citrate, and sodium acetate; may be added as appropriate. In addition, as an anticoagulant and pH adjuster, for example, ACD-A solution (composition of sodium citrate hydrate, citric acid hydrate, glucose, etc.) may be added.

Cell-mixed infusion formulations may be composed of organics such as biopolymers, inorganics such as hydroxyapatite, specifically collagen matrices, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers and chemical derivatives thereof for topical administration. may be mixed with

Examples of the administration method of the therapeutic agent for brain disorders of the present invention include intracerebral administration, intrathecal administration, intramuscular administration, subcutaneous administration, and intravenous administration. Intravenous administration is particularly preferred because it can be administered safely and stably regardless of the skill of the administering person.

The dose of the therapeutic agent for brain disorders of the present invention is the amount of cells that, when administered to a subject, can produce a therapeutic effect on the disease compared to a subject to whom the agent is not administered. A specific dosage can be appropriately determined depending on the subject's age, body weight, range of ischemia, symptoms, etc. For example, the number of umbilical cord-derived cells is 10 ⁴ to 10 ⁹ cells/kg per administration. 10 ⁴ to 10 ⁸ cells/kg body weight, 10 ⁴ to 10 ⁷ cells/kg body weight, and more preferably 10 ⁴ to 10 ⁸ cells/kg body weight.

The therapeutic agent for brain disorders of the present invention may be administered multiple times, for example, 2, 3, 4, 5 or more times. However, it may be determined as appropriate. The administration interval in the case of multiple administrations may be determined as appropriate while confirming the therapeutic effect of the subject, for example, once a day, once a week, once every two weeks, once a month or once every three months. Examples include once, once every six months, and the like.

In the present invention, encephalopathy is a comprehensive term encompassing not only acute and chronic disorders of the central nervous system but also symptoms or diseases. Acute brain injury includes, but is not limited to, cerebral hemorrhage, cerebral ischemia or cerebral infarction (including embolic and thrombotic occlusion), brain contusion, cerebral palsy, hypoxic-ischemic encephalopathy, periventricular Examples include leukomalacia and shaken baby syndrome. Cerebral hemorrhage is intracranial (epidural, subdural, subarachnoid, and intracerebral) caused by hypertension, arteriosclerosis, trauma, brain tumor, cerebral arteriovenous malformation, or cerebral arteriovenous dissection. Bleeding means. Chronic brain disorders include, but are not limited to, Alzheimer's disease, Pick's disease, Lewy body disease, progressive supranuclear palsy, multiple system atrophy, amyotrophic lateral sclerosis, degenerative ataxia, Examples include corticobasal degeneration, subacute sclerosing panencephalitis, Huntington's disease, Parkinson's disease, progressive multifocal leukoencephalopathy, and familial autonomic neuropathy.

Considering the more effective effect of the therapeutic agent provided by the present invention, indication for acute brain injury is preferred, and indication for brain injury in the fetal or neonatal period is more preferred. Examples of brain disorders in the fetal or neonatal period include cerebral hemorrhage, cerebral ischemia, cerebral contusion, cerebral palsy, hypoxic-ischemic encephalopathy, periventricular leukomalacia, and shaken baby syndrome. The treatment of brain disorders in the fetal or neonatal period also includes the treatment of secondary brain disorders caused by the primary brain disorder, such as the treatment of cerebral palsy that develops as a result of cerebral ischemia in the fetal or neonatal period. do. Treatment of secondary brain damage resulting from the first brain damage in the fetal or neonatal period is not necessarily performed at the onset of the first brain damage, e.g., one week after the onset of the first brain damage , 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year or more good. Accordingly, the present invention encompasses therapeutics administered to infants, toddlers, children or adults for brain damage secondary to primary brain damage in the fetal or neonatal period. From the viewpoint of high therapeutic effect, it is more preferable to administer the therapeutic agent of the present invention as soon as possible after brain injury. If the child is born with severe asphyxia, it is highly likely that he or she will be affected by any of the brain disorders described above. is preferably administered.

In the present invention, the term "fetus" refers to a growing child in the mother's body, from the 8th week of pregnancy until delivery, and the term "newborn" refers to a child from 0 days to less than 28 days after birth. An infant is a child aged 28 days or more and under 1 year old, an infant is a child aged 1 year or over but under 6 years old, and a child is a child aged 6 years or over but under 20 years old. and an adult means a person 20 years of age or older. Thus, the fetal or neonatal period means the period from the eighth week of gestation onwards to less than 28 days after birth.

In the present invention, the treatment of encephalopathy means not only restoring the pathology of the above-mentioned encephalopathy to normal, but also alleviating symptoms, delaying progression, delaying onset, or preventing. encompasses

The therapeutic agent for brain disorders of the present invention may be used in combination with other therapeutic agents, or may be used as a single agent.

The invention also includes methods of treating brain disorders using umbilical cord-derived cells. Specifically, the therapeutic method of the present invention includes a therapeutic method using the above-described therapeutic agent for brain damage containing the umbilical cord-derived cells of the present invention, and the above description can be applied as a specific description. Among them, the therapeutic method of the present invention is preferably a therapeutic method in which umbilical cord-derived cells are intravenously administered. In addition, the umbilical cord-derived cells are preferably cells prepared from umbilical cord tissue containing amniotic membrane, blood vessels, perivascular tissue, and Walton's Jelly. Further, the umbilical cord-derived cells are (i) positive for CD105, CD73, CD90, CD44, HLA-class I, HLA-G5 and PD-L2, and (ii) CD45, CD34, CD11b, CD19 and HLA-Class II. is negative. When the brain disorder is brain disorder in the fetal or neonatal period, particularly cerebral palsy, the therapeutic method of the present invention exhibits remarkable effects.

Umbilical cord-derived cells Umbilical cord-derived cells were collected by the method described in Cytotherapy, 18, 229-241, 2016. Briefly, all tissue elements of the umbilical cord (including amniotic membrane, blood vessels, perivascular tissue, and Walton's Jelly) collected with the consent of the donor, with the approval of the ethics committee of the Institute of Medical Science, The University of Tokyo. ) was cut into pieces of 1 to 2 mm ³ , seeded on a culture dish, covered with cell amigo (Tsubakimoto Chain Co., Ltd.), and α-minimal essential medium supplemented with 10% fetal bovine serum (FBS) and antibiotics. obtained by a modified explant method culturing in (αMEM). The properties of the cells are plastic-adherent.

Surface antigens of umbilical cord-derived cells were positive for CD73, CD105, CD90, CD44 and HLA-class I, and negative for HLA-class II, CD34, CD45, CD19, CD80, CD86, CD40 and CD11b. In addition, HLA-G5 and PD-L2 were positive. In addition, HLA-G was weakly positive, CD49d (ITGA4) and CD184 (CXCR4) were negative to weakly positive, and CD29 (ITGB1) was positive. The presence or absence of surface antigen expression was confirmed by FACS analysis using specific antibodies against each antigen.

The umbilical cord-derived cells highly express the HGF (Hepatic Growth Factor) gene under normal conditions, and the IDO (Indoleamine 2,3-dioxygenase) gene expression under inflammatory conditions (IFN-γ 100 ng/mL). Induction was confirmed by Realtime PCR. Results for IDO are shown in FIG. In particular, HGF expression was found to be higher in umbilical cord-derived cells than in bone marrow-derived mesenchymal stem cells. In addition, it was confirmed by ELISA that secretion of PGE2 was induced by co-culturing umbilical cord-derived cells with MLR (mixed allogeneic lymphocyte reaction) (Fig. 2). In FIG. 2, "R" indicates the lymphocytes of the responder, and "S" indicates the lymphocytes of the stimulator. Lot1 to Lot4 indicate lots of umbilical cord-derived cells. PHA-L (phytohemagglutinin-L) is a non-specific inflammation-inducing reagent.

Neonatal cerebral hemorrhage model mouse An artificial cerebral hemorrhage model was prepared by injecting 20 μL of adult mouse blood into the right ventricle of a B6 Albino neonatal mouse 5 to 7 days after birth. When the cerebral hemorrhage model was imaged by MRI, enlargement of the brain ventricle was observed in the T2 image. In the hemorrhagic ventricle (affected side), more GFAP-positive fibers (red), which are intermediate filaments, were observed compared to the normal ventricle (unhealthy side) (Fig. 3).

Cell-administered umbilical cord-derived cells are obtained by collecting cells at about 4 passages, freezing them using a cryoprotectant (see Japanese Patent Laid-Open No. 2015-142559), rapidly thawing at 37°C before intravenous administration, and freezing them. The protective solution was administered as is. Three days after intracerebroventricular blood administration as a cerebral hemorrhage model, 1×10 ⁵ umbilical cord-derived cells or PBS as a negative control was injected into the facial vein of the cerebral hemorrhage model mouse. The schedule for model creation, cell administration and evaluation is shown in FIG. In addition, this experiment was performed with 3 rats in each group.

Evaluation after cell administration From the study of pharmacokinetics using umbilical cord-derived cells introduced with the Luciferase gene, it was confirmed that the administered cells reached the head 3 days after administration, as shown in FIG. rice field.

As a result of behavioral evaluation, the umbilical cord-derived cell-administered group showed an increase in the walking distance and the number of times of standing up in 10 minutes compared to the PBS-administered group (Fig. 6). Therefore, administration of umbilical cord-derived cells significantly improved motor dysfunction.

In neonatal cerebral hemorrhage model mice, left hemiplegia due to right cerebral hemorrhage causes left leg abduction, and in the PBS-administered group, the angle difference between the left and right legs tends to tilt to the left. No such tendency was observed in the group (Fig. 7). Therefore, it was confirmed that the administration of umbilical cord-derived cells contributes not only to improvement of motor function but also to alleviation of post-cerebral hemorrhage hemiplegia.

Neonatal cerebral hemorrhage (degrees III to IV) is common in premature infants and almost always progresses to cerebral palsy. The mechanism is inflammation and nerve damage due to hemorrhage, and it was suggested that umbilical cord-derived cells have the effect of improving these. However, MRI findings clearly showed no effect of administration of umbilical cord-derived cells on ventricular enlargement (Fig. 8). However, clinically, there is no correlation between imaging findings and clinical symptoms (dyskinesia), so the improvement in behavioral evaluation is considered to be due to the efficacy of umbilical cord-derived cells.

When the periventricular tissue in neonatal cerebral hemorrhage model mice was examined using a fluorescent dye antibody, the negative control (PBS administration) group showed an increase in GFAP-positive cells (glial cells; red) and MAP2-positive cells (Neuron cells). ; green), but in the group administered with umbilical cord-derived cells, many MAP2-positive cells were observed as in untreated (no bleeding) mice (Fig. 9). In addition, in the case of black-and-white images, GFAP-positive cells are cells that appear white.

In addition, in the negative control (PBS-administered) group, a decrease in the MBP-positive area indicating white matter volume was observed, but in the group administered with umbilical cord-derived cells, significant improvement was observed (Figs. 10, 11, 12). Furthermore, TUNEL-positive cells showing apoptosis were observed in the negative control (PBS-administered) group, but significantly improved in the group administered with umbilical cord-derived cells (Figs. 13 and 14). In addition, FIG. 14 digitizes the result of FIG. From the above, it was confirmed that the administration of umbilical cord-derived cells has the effect of recovering nerve cells or preventing loss of nerve cells. Note that IVH in the figure indicates intraventricular hemorrhage. The same applies to subsequent figures.

In the same manner as described above, an artificial cerebral hemorrhage model was prepared, and on day 3 after intracerebroventricular blood administration, αMEM medium (MEM administration group; IVH + UC-MSC (MEM); n = 13) or serum-free medium (Rhoto RM-administered group; IVH + UC-MSC (RM); n = 11), or PBS (PBS-administered group; IVH; n = ¹¹ ) as a negative control. Behavioral evaluation was performed by injecting into the facial vein of model mice. The results of normal mice (n=8) were shown as Control. Compared to the PBS-administered group, the umbilical cord-derived cell-administered group showed an increase in the walking distance in 10 minutes, and the RM-administering group showed a significant increase in the walking distance in 10 minutes (Fig. 15). In addition, compared to the PBS-administered group, the number of times of standing up was significantly increased in the umbilical cord-derived cell-administered group (Fig. 16). Therefore, administration of umbilical cord-derived cells significantly improved motor dysfunction.

BDNF (Brain-derived neurotrophic factor) and HGF expression levels in umbilical cord-derived cells were measured by quantitative PCR, and BDNF and HGF expression was confirmed (Fig. 17).

Mouse cerebral cortical neurons on embryonic day 16 were primary cultured and cultured for 10 hours in an 8% oxygen, glucose-free OGD (oxygen-glucose depletion) environment to create an in vitro cerebral ischemia model. Mouse neurons and umbilical cord-derived cells were co-cultured, and protein concentrations of BDNF and HGF in the supernatant were measured by bead assay. BDNF and HGF were also secreted in the supernatant of umbilical cord-derived cells alone, but the concentrations of both BDNF and HGF increased when co-cultured with mouse neurons after OGD (Fig. 18). This experiment was performed with n=1.

An artificial cerebral hemorrhage model was prepared in the same manner as described above, and the concentrations of Human-BDNF, Human-NGF (nerve growth factor) and Human-HGF were measured by bead assay using the serum of model mice. IVH(−) was performed with n=6, IVH(+)+vehicle with n=6, and IVH(+)+UC-MSCs with n=14. NGF was not observed in any group (Fig. 19), but BDNF and HGF levels were elevated in 9 out of 14 mice in the umbilical cord-derived cell administration group (Figs. 20 and 21).

24 hours and 3 weeks after administration of umbilical cord-derived cells (1×10 ⁵ ) to neonatal mice via the facial vein, the content of Human Alu element-genomic DNA was measured by quantitative PCR, and the distribution of umbilical cord-derived cells to each organ was determined. Residual evaluation was performed. Many Alu sequences were observed in the lung and brain 24 hours after administration, but no Alu sequence was detected in any organ 3 weeks after administration (Fig. 22). This experiment was performed with n=3.

In addition, immunostaining for HLA-Class I and CD105 was performed on the brains 24 hours and 3 weeks after administration of umbilical cord-derived cells (1×10 ⁵ cells) to neonatal mice, and the distribution of umbilical cord-derived cells in the brain to each organ was performed. Residual evaluation was performed. Brain sections of mice to which umbilical cord-derived cells were administered via facial vein showed double-positive cells for HLA-Class I and CD 105 in the perivascular area (Fig. 23). No double positive cells were observed. This experiment was performed with n=1.

The above results suggest that neonatal cerebral hemorrhage often develops into periventricular leukomalacia (PVL), and that administration of umbilical cord-derived cells may prevent progression to PVL. Moreover, it was thought that it could be applied to the treatment of cerebral hemorrhage caused by traffic accidents and trauma.

Claims (4)

(i) CD105, CD73, CD90, CD44, HLA-class I, HLA
- positive for G5 and PD-L2, and (ii) CD45, CD34, CD11b, CD19 and HLA-Class II
A therapeutic agent for cerebral contusion, cerebral palsy, hypoxic-ischemic encephalopathy, cerebral hemorrhage or periventricular leukomalacia in the neonatal period, comprising umbilical cord-derived cells negative for 2. The therapeutic agent according to claim 1, which is for intravenous administration. The therapeutic agent according to claim 1 or 2, wherein the umbilical cord-derived cells are cells prepared from umbilical cord tissue containing amniotic membrane, blood vessels, perivascular tissue, and Walton's Jelly. The umbilical cord-derived cells are
(iii) The therapeutic agent according to any one of claims 1 to 3, wherein the expression of any one gene and/or protein of IDO, PGE2 and PD-L1 is induced under inflammatory conditions.

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