WO2021125841A1 - Composition pharmaceutique pour administration artérielle, comprenant une lignée de cellules souches adultes contenant des gènes de facteur de transcription neurogène à hélice-boucle-hélice de base (bhlh) - Google Patents

Composition pharmaceutique pour administration artérielle, comprenant une lignée de cellules souches adultes contenant des gènes de facteur de transcription neurogène à hélice-boucle-hélice de base (bhlh) Download PDF

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WO2021125841A1
WO2021125841A1 PCT/KR2020/018572 KR2020018572W WO2021125841A1 WO 2021125841 A1 WO2021125841 A1 WO 2021125841A1 KR 2020018572 W KR2020018572 W KR 2020018572W WO 2021125841 A1 WO2021125841 A1 WO 2021125841A1
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mannitol
pharmaceutical composition
helix
cells
stem cell
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조다롱
장다영
머라시니수바스
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(주)셀레브레인
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an adult stem cell line into which a basic helix-loop-helix (bHLH)-type neurogenic transcription factor gene has been introduced, specifically, basic helix-loop-helix;
  • bHLH basic helix-loop-helix
  • Prevention of neurological diseases comprising an adult stem cell line into which a basic helix-loop-helix (bHLH)-type neurogenic transcription factor gene is introduced, and intra-arterial administration after administration of mannitol; It relates to a pharmaceutical composition for treatment.
  • Mesenchymal stem cells are generally support cells (stroma) that help hematopoiesis in the bone marrow, and are divided into various mesodermal lineage cells including bone, cartilage, fat, and muscle cells while maintaining an undifferentiated state. Because it has the characteristic of differentiation, it is very useful as a material for developing artificial tissues.
  • mesenchymal stem cells have been reported to have the potential to differentiate into glial cells in the brain (Azizi et al., Proc. Natl. Acad. Sci. USA, 94, 4080-4085 (1998); Kopen et.al., Proc. Natl. Acad. Sci. USA, 96, 10711-10716 (1999))
  • the possibility of using mesenchymal stem cells to treat diseases of the central nervous system has been raised (Li et al., Neurosci. Lett., 315 , 67-70 (2001); and Chen., et al., Stroke 32, 1005-1011 (2001)).
  • Neurogenin and NeuroD are transcription factors of the basic helix-loop-helix (bHLH) family that play a key role in the formation of the nervous system.
  • bHLHs such as E12 or E47 It forms a complex with a protein having a structure and binds to a DNA sequence containing an E-box (CANNTG) or, rarely, an N-box. It has been shown that this binding is essential for activating the expression of tissue-specific genes that promote neuronal differentiation of transcription factors of the bHLH structure (Lee, Curr. Opni. Genet. Dev., 7, 13-20 (1997)). .
  • bHLH-type transcription factors such as neurogenin or neuroD were introduced into mesenchymal stem cells.
  • mesenchymal stem cells continuously expressing these transcription factors were prepared and differentiated into neurons at a high rate when transplanted into the brain of an animal model (Korean Patent Publication Nos. 10-2004-0016785 and 10). -2012-0130584).
  • a bHLH-type transcription factor such as Neurogenin or NeuroD
  • the effect of administering mesenchymal stem cells that continuously express these transcription factors by introducing a bHLH-type transcription factor such as Neurogenin or NeuroD may be affected by the blood-brain barrier; This can only be achieved when the therapeutic agent effectively penetrates the BBB) and reaches the lesion.
  • the blood-brain barrier acts to selectively permeate blood substances into the brain parenchyma, thereby changing the composition of permeable substances in the brain parenchyma.
  • BBB blood-brain barrier
  • this role of the blood-brain barrier reduces the effectiveness of treatment by limiting the delivery of the therapeutic agent into the brain parenchyma when the therapeutic agent is administered in brain diseases such as brain tumors.
  • the treatment effect of the therapeutic agent injected into the blood vessel did not appear as the vascular brain barrier was damaged in the acute phase due to the onset of the stroke and the resulting vascular brain barrier opening was restored.
  • Intracerebral administration is the most reliable administration method in cell transplantation, but it is not preferred in the clinical field due to dangerous complications such as seizures, chronic subconjunctival hematoma, cortical vein occlusion, and postoperative clinical deterioration.
  • Intravenous, IV has a problem of potentially reducing the therapeutic effect because most of the mesenchymal stem cells are filtered in the liver or lungs before they arrive at the desired location.
  • IA intra-arterial
  • Mannitol is a sugar alcohol widely used as a raw material for food, medicine, pharmaceuticals, and cosmetics.
  • the chemical formula is C 6 H 14 O 6 , and both D- and L-forms exist, but D-type mannitol is widely distributed in nature.
  • mannitol has a diuretic effect as an osmotic agent, and in addition, it is known to have various actions, such as changing the structure of red blood cells, reducing hematocrit, improving microcirculation, and destroying blood vessel walls.
  • the present inventors have studied a method of administering a pharmaceutical composition comprising an adult stem cell line into which a basic helix-loop-helix (bHLH)-type neurogenic transcription factor gene is introduced as an active ingredient.
  • bHLH basic helix-loop-helix
  • the prevention or treatment effect of neurological diseases, especially chronic stroke and Alzheimer's is maximized when mesenchymal stem cells into which the neurogenic transcription factor gene is introduced after administration of mannitol are administered (Intra-arterial, IA).
  • IA Intra-arterial
  • An object of the present invention is to provide a pharmaceutical composition for temporarily opening the blood-brain barrier to increase the access to the infarct site of a stem cell therapeutic agent and the therapeutic efficacy of a nervous system disease.
  • the present invention includes an adult stem cell line into which a basic helix-loop-helix (bHLH) type neurogenic transcription factor gene is introduced as an active ingredient, and mannitol is administered.
  • bHLH basic helix-loop-helix
  • a pharmaceutical composition for preventing or treating a nervous system disease characterized in that the posterior artery is administered (intra-arterial administration).
  • the present invention includes an adult stem cell line into which a basic helix-loop-helix (bHLH) type neurogenic transcription factor gene is introduced, and is characterized in that it is administered intra-arterially after mannitol is administered.
  • bHLH basic helix-loop-helix
  • It relates to a pharmaceutical composition for the prevention or treatment of neurological diseases, wherein the administration of the pharmaceutical composition can overcome chronic physical disorders caused by apoptosis after stroke, including stroke, Parkinson's disease, Alzheimer's disease, Hunting It has been developed as a new therapeutic agent applicable to neurological diseases such as tone chorea, amyotrophic lateral sclerosis, and spinal cord injury, and can be widely used for clinical and various studies.
  • 1a is a photomicrograph of GFP-positive MSC and MSC/Ngn1 cells observed under a fluorescence microscope and a photomicrograph of superparamagnetic iron oxide (SPIO) labeling using Prussian blue staining.
  • SPIO superparamagnetic iron oxide
  • 1B is a photograph of brain cross-sections taken by MRI at 4 hours and 24 hours after SPIO-labeled cells were transplanted into the internal carotid artery of an animal model of chronic stroke.
  • Figure 1c is a graph quantifying the area occupied by cells remaining in the brain tissue of the animal model based on the signal for SPIO observed on the MRI image.
  • FIG. 2A is a graph showing that vascular endothelial cell permeation resistance (TEER) was measured in bEnd.3 cells treated with mannitol, and the formation of tight junctions was reduced by mannitol.
  • TEER vascular endothelial cell permeation resistance
  • Figure 2b is a graph showing the transmittance of a 150 kda fluorescent material (FITC-dextran) after treatment with mannitol in cultured bEnd.3 cells.
  • FITC-dextran 150 kda fluorescent material
  • 2c is a fluorescence micrograph showing the results of confirming the expression of ZO-1 and Claudin-5, which are close junction markers, before and after mannitol treatment in cultured bEnd.3 cells by immunocytochemistry.
  • Figure 2d is a diagram showing the results of confirming the expression level of the close junction marker in the cells after treating the cultured bEnd.3 cells with mannitol by Western blotting.
  • Figure 2e is a graph quantitatively showing the relative expression level of the close junction marker Z0-1 in the cells after treatment with mannitol in cultured bEnd.3 cells.
  • 2f is a graph quantitatively showing the relative expression level of Claudin-5, a close junction marker, in the cells after treatment with mannitol in cultured bEnd.3 cells.
  • 3A is a schematic diagram of an experiment comparing MSC-GFP and MSC/Ngn1-GFP cell mobility after treatment with mannitol in bEnd.3 forming a tight junction.
  • Figure 3b is a graph showing the results of comparison by quantifying the number of cells that have migrated to the bottom of the insert.
  • Figure 4 is a graph showing the results of comparing the measurement of the resistance of MSC and MSC / Ngn1 cells in an environment of serum (FBS) removal and hypoxic conditions.
  • Figure 5a is a diagram showing a photograph of brain tissue isolated when mannitol and Evans blue dye were injected into the tail vein (IV) and internal carotid artery (IA) in an animal model with a normal blood-brain barrier.
  • Figure 5b is a photograph showing the effect of the infusion rate on the blood-brain barrier permeability in a normal animal model when mannitol is injected into the internal carotid artery.
  • Figure 6a is a diagram showing a schematic diagram of injection of mannitol into the internal carotid artery in the animal model and the injection of Evans blue dye for each time period.
  • Figure 6b is a view showing a photograph of brain tissue separated after injection of mannitol into a normal animal model and Evans blue dye for each time period.
  • Figure 6c is a view showing a photograph of the brain tissue separated after injection of mannitol into the chronic stroke animal model and Evans blue dye for each time period.
  • FIG. 7A is a diagram illustrating a brain cross-section after selection of an animal model of stroke using MRI and transplantation of cells labeled with SPIO.
  • 7B is a graph illustrating quantification of cells remaining in the brain tissue of an animal model based on a signal for SPIO observed on an MRI image.
  • 8A is a photograph confirming the outflow of extravascular cells 1 hour, 24 hours, and 1 week after cell transplantation.
  • 8B is a graph showing the quantification of GFP-positive cells in the area around the ischemic lesion.
  • Figure 8c is a graph showing the number of stem cells distributed in the ischemic lesion hemisphere quantified using AluJ-qPCR.
  • 8D is a fluorescence micrograph showing hMT/NeuN, hMT/MAP2, and hMT/GFAP-positive cells in the brain tissue of an animal model of chronic stroke administered with MSCs and MSC/Ngn1 cells after mannitol pretreatment.
  • Figure 9a is a graph showing the results of the corner test (Corner test) in the stroke animal model.
  • Figure 9b is a graph showing the test results of the rotarod test (Rotarod test) in the stroke animal model.
  • Figure 9c is a graph showing the test results of the adhesion removal test (Adhesive removal test) in the stroke animal model.
  • bHLH basic helix-loop-helix
  • a neurogenic transcription factor may be used as the bHLH-family transcription factor, and as a neurogenic transcription factor gene, for example, neurogenin 1 gene (GenBank Accession No. U63842, GenBank Accession No. U67776), neuro Genin 2 gene (GenBank Accession No. 76207, GenBank Accession No. AF303001), Neuro D1 gene (GenBank Accession No. U24679, GenBank Accession No. AB018693), MASH1 gene (GenBank Accession No. M95603, GenBank Accession No. L08424), CDNA obtained from nucleotide sequence information such as the MATH3 gene (GenBank Accession No. D85845) and the E47 gene (GenBank Accession No. M65214, GenBank Accession No. AF352579) can be cloned or synthesized and used, and it can be used to exhibit equivalent or similar activity If possible, some of these sequences may be modified, deleted, or substituted.
  • neurogenin 1 gene GenBank Accession No.
  • the term "adult stem cell” refers to a cell in an undifferentiated state that is differentiated into a cell of a specific tissue when necessary, and the adult stem cell line is bone marrow, adipose tissue, blood, umbilical cord blood, umbilical cord, liver. , skin, gastrointestinal tract, placenta, uterus, or stem cells derived from an abortion.
  • the adult stem cell line is a bone marrow-derived adult stem cell line, more preferably a bone marrow-derived mesenchymal stem cell.
  • Bone marrow-derived adult stem cells have several types of adult stem cells such as mesenchymal stem cells (MSCs) as well as hematopoietic stem cells capable of producing blood and lymphocytes. Among them, mesenchymal stem cells cannot proliferate in vitro. As cells capable of being easily differentiated into various cell types (adipocytes, chondrocytes, muscle cells, and bone cells) (Caplan, AI, J. Orthop. Res., 9:641-650, 1991; Beresford, JN, et al. al., J.
  • adult stem cell line into which the basic helix-ring-helix neurogenesis transcription factor gene is introduced refers to the basic helix-ring-helix neurogenesis transcription factor, preferably the neurogenin of SEQ ID NO: 1 1 Refers to an adult stem cell line into which the gene is introduced.
  • the present invention includes an adult stem cell line and mannitol into which a basic helix-loop-helix (bHLH) type neurogenesis transcription factor gene is introduced, and an adult into which the bHLH type neurogenic transcription factor gene is introduced.
  • Stem cell lines and mannitol provide a pharmaceutical composition for preventing or treating neurological diseases, characterized in that intra-arterial administration.
  • the basic helix-loop-helix (bHLH)-type neurogenic transcription factor gene may be cloned into a vector and introduced into adult stem cells.
  • vector refers to an expression vector capable of expressing a target protein in a suitable host cell, and refers to a genetic construct including essential regulatory elements operably linked to express a gene insert.
  • operably linked means that a nucleic acid expression control sequence and a nucleic acid sequence encoding a target protein are functionally linked to perform a general function.
  • the operative linkage with the recombinant vector can be prepared using a genetic recombination technique well known in the art, and site-specific DNA cleavage and ligation are performed using enzymes generally known in the art.
  • Vectors include plasmid vectors, cosmid vectors, viral vectors, and the like. Preferably, it is a viral vector.
  • Viral vectors are retroviruses, for example, Human immunodeficiency virus (HIV), Murineleukemia virus (MLV), Avian sarcoma/leukosis (ASLV), Spleen necrosis virus (SNV), Rous sarcoma virus (RSV), Mouse mammary tumor (MMTV). virus), MSV (Murine sarcoma virus) and lentivirus (Lentivirus), adenovirus (Adenovirus), adeno-associated virus (Adeno-associated virus), including vectors derived from herpes simplex virus (Herpes simplex virus), etc. not limited
  • the pharmaceutical composition of the present invention is to open the blood brain barrier (BBB), inject mannitol to effectively deliver the stem cells to the brain and central nervous system, and administer the stem cell line arterially.
  • BBB blood brain barrier
  • the delivery efficiency of therapeutic drugs is very low because the brain nervous system tissue is surrounded by the dura mater, pia mater, and blood-brain barrier (BBB).
  • BBB blood-brain barrier
  • mannitol in order to effectively deliver stem cells to the brain, mannitol is first used to open the blood-brain barrier and then the stem cells are administered to effectively deliver stem cells and prevent or treat neurological diseases. making it do Mannitol is a vasodilator drug that reduces pressure in the upper skull and can induce temporary and strong contraction of the endothelial cells forming the blood-brain barrier, so it is recommended for patients with brain diseases such as Alzheimer's disease. It is known as a drug used when administering a drug to a patient.
  • carotid artery administration is preferred.
  • the carotid artery is one of the arteries and is divided into three blood vessels, the common carotid artery, the external carotid artery (ICA), and the internal carotid artery (ICA).
  • the carotid artery administration is preferably an internal carotid artery (ICA) administration.
  • ICA internal carotid artery
  • the carotid artery administration is administered by ligating the pterygopalatine artery (PPA) before administration, but this process may be omitted depending on the patient.
  • PPA pterygopalatine artery
  • the adult stem cell line into which the neurogenesis transcription factor gene of the basic helix-ring-helix series is introduced is administered after mannitol is injected.
  • the mannitol may be injected at a dose of 0.5 to 3 g/kg, preferably at a dose of 1 to 3 g/kg, and more preferably at a dose of 1 to 2 g/kg.
  • the mannitol may be a bolus injection at a rate of 1 to 5 ml in 15 seconds, preferably a bolus injection at a rate of 1 to 4 ml in 15 seconds, more preferably Preferably, it may be a bolus injection at a rate of 1 to 2 ml in 15 seconds.
  • Bolus infusion is a fast infusion method in which a drug, drug or other compound is administered individually within a specific time period, usually within 1 to 30 minutes, to increase the concentration of a drug in the blood.
  • the adult stem cell line into which the neurogenesis transcription factor gene of the basic helix-ring-helix series is introduced may be administered 1 ⁇ 10 4 cells/kg to 1 ⁇ 10 9 cells/kg, preferably 1 ⁇ 10 5 Dogs/kg to 1 ⁇ 10 8 pcs/kg may be administered, and more preferably 5 ⁇ 10 5 pcs/kg to 5 ⁇ 10 7 pcs/kg may be administered.
  • the adult stem cell line into which the neurogenesis transcription factor gene of the basic helix-ring-helix series is introduced is differentiated into neurons and glial cells.
  • the pharmaceutical composition of the present invention may be administered intra-arterially for the prevention or treatment of neurological diseases.
  • the present inventors have studied various administration routes. Specifically, intracerebral administration (Intracerebral, IC) is the most reliable administration method in cell transplantation, but it is not preferred in the clinical field due to dangerous complications such as seizures, chronic subconjunctival hematoma, cortical vein occlusion, and postoperative clinical deterioration.
  • intravenous administration Intra problem of potentially reducing the therapeutic effect because most of the mesenchymal stem cells are filtered out of the liver or lungs before they arrive at the desired location.
  • neurode disease refers to a general term for various diseases related to nerves, particularly cranial nerves.
  • the nervous system disease is Parkinson's disease, Alzheimer's disease, Huntington's chorea, amyotriophic lateral sclerosis, epilepsy, schizophrenia, chronic stroke ( chronic stroke) or spinal cordinjuries, preferably Parkinson's disease, Alzheimer's disease, amyotriophic lateral sclerosis, or chronic stroke.
  • prevention refers to any action of suppressing or delaying the onset of all diseases related to neurological diseases using an adult stem cell line into which the neurogenin 1 gene is introduced.
  • treatment refers to any action that improves or beneficially alters a nervous system disease using the cell line.
  • chronic stroke can be effectively treated using the transformed stem cells of the present invention.
  • the present invention includes the step of intra-arterial administration of an adult stem cell line and mannitol into which a basic helix-loop-helix (bHLH)-type neurogenic transcription factor gene is introduced. It provides a method for preventing or treating neurological diseases.
  • bHLH basic helix-loop-helix
  • the bHLH according to the present invention may have the characteristics described above.
  • the subject may be a mammal, specifically a human.
  • Adult stem cells according to the present invention may have the characteristics as described above.
  • adult stem cell line into which the basic helix-ring-helix neurogenesis transcription factor gene is introduced refers to the basic helix-ring-helix neurogenesis transcription factor, preferably the neurogenin of SEQ ID NO: 1 1 Refers to an adult stem cell line into which the gene is introduced.
  • carotid artery administration is preferred.
  • the carotid artery is one of the arteries and is divided into three blood vessels, the common carotid artery, the external carotid artery (ICA), and the internal carotid artery (ICA).
  • the carotid artery administration is preferably an internal carotid artery (ICA) administration.
  • ICA internal carotid artery
  • the carotid artery administration is administered by ligating the pterygopalatine artery (PPA) before administration, but this process may be omitted depending on the patient.
  • PPA pterygopalatine artery
  • the adult stem cell line into which the neurogenesis transcription factor gene of the basic helix-ring-helix series is introduced is administered after mannitol is injected.
  • the mannitol may be injected at a dose of 0.5 to 3 g/kg, preferably at a dose of 1 to 3 g/kg, and more preferably at a dose of 1 to 2 g/kg.
  • the mannitol may be a bolus injection at a rate of 1 to 5 ml in 15 seconds, preferably a bolus injection at a rate of 1 to 4 ml in 15 seconds, more preferably Preferably, it may be a bolus injection at a rate of 1 to 2 ml in 15 seconds.
  • the adult stem cell line into which the neurogenesis transcription factor gene of the basic helix-ring-helix series is introduced may be administered 1 ⁇ 10 4 cells/kg to 1 ⁇ 10 9 cells/kg, preferably 1 ⁇ 10 5 Dogs/kg to 1 ⁇ 10 8 pcs/kg may be administered, and more preferably 5 ⁇ 10 5 pcs/kg to 5 ⁇ 10 7 pcs/kg may be administered.
  • the present inventors isolated mesenchymal stem cells from bone marrow, and introduced neurogenin 1 thereto to prepare mesenchymal stem cells into which neurogenin 1 was introduced.
  • the mesenchymal stem cells into which neurogenin 1 has been introduced have a higher transplantation rate than mesenchymal stem cells into which neurogenin 1 has not been introduced in acute stroke with active inflammatory response after stroke induction. This is presumed to be due to increased expression of inflammatory cytokines receptors in mesenchymal stem cells into which neurogenin 1 has been introduced, resulting in increased reactivity to inflammatory cytokines at brain injury sites.
  • mannitol was used as a drug for opening the vascular brain barrier by inducing the contraction of vascular endothelial cells.
  • the permeation resistance of vascular endothelial cells and the permeated capacity of fluorescent dextran were measured when mannitol was treated.
  • mannitol was treated with vascular endothelial cells for up to 6 hours. It was confirmed that the permeation resistance of the dextran was decreased, and the transmitted capacity of the fluorescent dextran was increased (see FIGS. 2a and 2b ).
  • mesenchymal stem cells into which neurogenin 1 was introduced were more resistant to hypoxic conditions than mesenchymal stem cells, which means that mesenchymal stem cells introduced with neurogenin 1 were chronic stroke lesions, that is, mesenchymal stem cells under hypoxic conditions. It was confirmed that it can be usefully used for the treatment of chronic stroke by showing a therapeutic effect for a long time due to higher viability and persistence than cells (see FIG. 4 ).
  • the transplanted cells gradually disappear, and in the later stages (1 day to 1 week), the residual mesenchymal stem cells into which neurogenin 1 was introduced was higher than that of the mesenchymal stem cells (see FIGS. 7b, 8b and 8c). This is a result consistent with that neurogenin 1 increases cell viability in hypoxic conditions (see FIG. 4 ).
  • a corner test measuring asymmetry according to brain damage to determine whether neurogenin 1 introduced mesenchymal stem cells were administered after mannitol pretreatment (corner test), a rotarod test for measuring the running time on a cylindrical device, and an adhesive removal test for attaching a tape to the front paw of an animal and measuring the time it takes to remove the tape (adhesive removal test) was performed.
  • the group to which mesenchymal stem cells into which neurogenin 1 was introduced was administered had the highest degree of recovery of nerve function in all tests conducted up to 8 weeks after administration. This means that cerebral artery administration of mesenchymal stem cells into which neurogenin 1 is introduced together with pretreatment with mannitol promotes the recovery of behavioral function in chronic stroke (see FIGS. 9A and 9B to 9C ).
  • the pharmaceutical composition of the present invention can overcome chronic physical disorders caused by apoptosis after stroke, so it can be developed as a new therapeutic agent, or can be applied to neurological diseases such as stroke, Parkinson's disease, Alzheimer's disease, spinal cord injury, etc. It can be widely used for clinical and various research, such as as a basis for cell replacement therapy and gene therapy therapy.
  • DMEM medium Gibco
  • FBS 10% FBS
  • MSCs mesenchymal stem cells isolated in Preparation Example 1-1 were maintained at 37° C. in a CO 2 incubator at intervals of 2 days in a mesenchymal stem cell medium (10% FBS + 10 ng/ml bFGF (Sigma)). + 1% penicillin/streptomycin (Gibco) + 89% DMEM) while culturing. After the cells have proliferated to a density of about 80% of the area of the culture vessel, the cells attached to the culture vessel are suspended using 0.25% trypsin/0.1 mM EDTA (Gibco), and then the medium is added and diluted 1:20. It was then subcultured in fresh medium.
  • MSCs mesenchymal stem cells
  • the sequence corresponding to the coding region was bound to the pMSCV-puro vector (Clontech) using T4 DNA ligase (Roche), and then transduced into E. coli DH5a to finally pMSCV.
  • a pMSCV-puro/hNgn1 vector in which the human neurogenin 1 (hNgn1) gene was inserted into the -puro vector was constructed.
  • the prepared pMSCV-puro/hNgn1 vector was introduced into 293T cells by calcium phosphate precipitation, and then expression was confirmed by Western blotting.
  • the pMSCV-puro/hNgn1 vector was transduced into retroviral packaging cells, PA317 (ATCC CRL-9078) or PG13 (ATCC CRL-10686) cells by calcium phosphate precipitation. After 48 hours, only the culture solution was taken and filtered through a filtration membrane having a size of 0.45 ⁇ m to obtain a neurogenin 1 retrovirus solution. The retrovirus solution was aliquoted and stored at -70°C before use.
  • Preparation Example 2-1 After culturing the mesenchymal stem cells of Preparation Example 1-2 to 70% full in a 100 mm culture vessel, 4 ml of the neurogenin 1 retrovirus solution obtained in Preparation Example 2-1 was added to polybrene (polybrene, Sigma) 8 A solution to which ⁇ g / ml was added and 10 ng / ml of basic fibroblast growth factor (basic fibroblast growth factor, bFGF, Dong-A Pharmaceutical) was added and cultured for 8 hours.
  • polybrene polybrene, Sigma
  • basic fibroblast growth factor basic fibroblast growth factor
  • the retrovirus solution was removed and 10 ml of mesenchymal stem cell medium was added and cultured for 24 hours, and then the retrovirus was re-infected. After repeating this process 1-4 times, finally mesenchymal stem cells were removed with trypsin, diluted 1:20 with a medium, and subcultured. During subculture, puromycin (puromycin, Sigma) was added to the medium at a concentration of 2 ⁇ g/ml, and selected for 2 weeks so that only retrovirus-infected cells survive.
  • puromycin puromycin, Sigma
  • mesenchymal stem cells resistant to puromycin were used as cells expressing neurogenin 1.
  • the prepared mesenchymal stem cells into which the neurogenin 1 gene was introduced (hereinafter, MSC/Ngn1) were added to DMEM (Dulbecco's modified Eagle's medium) medium with 10% fetal bovine serum (HyClone, Logan, UT, USA). and 100 unit/ml penicillin and 100 ⁇ g/ml streptomycin (Gibco, Grand Island, NY, USA) were transplanted for 5 to 7 days in a growth medium.
  • MSCs and MSC/Ngn1 were transformed with a GFP-expressing lentivirus to track the transplantation rate and differentiation into neurons in vivo.
  • MSC of Preparation Example 1-2 and MSC/Ngn1 of Preparation Example 2-2 were transformed into GFP-expressing lentivirus at 8 ⁇ g/ml of polybrene for 7 hours to obtain GFP.
  • MSC-GFP and MSC/Ngn-GFP expressing 10,000 MSC-GFP and MSC/Ngn1-GFP were cultured in 12-well plates for one day. The total number of cells was counted using a dissecting fluorescence microscope (Olympus), and the number of cells expressing GFP was measured. Both MSC-GFP and MSC/Ngn1-GFP cells showed more than 95% GFP expression rate.
  • MSC and MSC/Ngn1 were labeled with superparamagnetic iron oxide (SPIO) nanoparticles.
  • SPIO superparamagnetic iron oxide
  • MSC-GFP and MSC/Ngn1-GFP obtained in Example 1-1 were treated with a 50 ⁇ g/ml concentration of SPIO solution together with a cell culture solution and cultured for one day, to remove excess SPIO nanoparticles. Washed 3 times.
  • an animal middle cerebral artery occlusion/reperfusion (MCAO) model was performed using the intratibial vessel occlusion method.
  • nylon suture with round coated tip was inserted into the ECA and ligated by gently pushing into the ICA. Nylon sutures were removed 120 minutes after ligation, and animals were re-anesthetized and re-perfused (MCAO). During the operation, the body temperature of the mice was kept constant at 37°C, and all surgical instruments were sterilized.
  • the prepared animal model confirmed chronic ischemic stroke on the 3rd day after MCAO through brain MRI scan and nerve severity test (Neuro Severity Score TEST, NSS test) (FIG. 7a).
  • MSCs and MSC/Ngn1 cells were administered, and migration and persistence to the chronic stroke lesion site were confirmed as follows.
  • a model having a cerebral infarction rate of 50% or more in the acute phase (3rd day) after stroke induction was selected, and at the time point (4th week) after one month, SPIO-labeled 1 ⁇ 10 6 MSCs and MSC/Ngn1 cells were suspended in 500 ⁇ l physiological saline through the internal carotid artery (ICA) and injected for at least 2 minutes.
  • ICA internal carotid artery
  • SPIO serpra paramagnetic iron oxide nanoparticles
  • stem cell DMEM medium 10% FBS (Gibco) + 10 ng/ml bFGF (Sigma Aldrich) to enable tracking and observation on MRI)
  • G + 1% penicillin/streptomycin (Gibco) + 89% DMEM (Gibco)
  • PB Prussian blue
  • the vascular endothelial cell permeation resistance which is commonly used to evaluate the functionality of the vascular brain barrier and the fluorescence of transplanted FITC-dextran (150 kda) -endothelial electrical resistance (TEER) was measured.
  • mouse cerebrovascular endothelial cell line bEnd.3 cells (CRL-2299, ATCC, Manassas VA, USA) was treated with high glucose DMEM supplemented with 10% FBS (Hyclone) and 1% penicillin/streptomycin (Gibco). The cells were cultured while the medium was added to the medium (Gibco) and the medium was replaced every 2 days.
  • bEnd.3 cells were aliquoted into transwell inserts with 8 ⁇ m pores, cultured for 5 days, and then treated with 300 mM mannitol (Sigma Aldrich) suspended in physiological saline for 6 hours.
  • Transendothelial electrical resistance was measured using a Millicell ERS-2 voltohmmeter (Merck Millipore), and 150 kda fluorescent dextran (FITC-dextran, Sigma Aldrich) was transmitted through the chamber. The capacity was confirmed.
  • FIGS. 2A and 2B a tight junction was generated on the 5th day after culturing the bEnd.3 cells, thereby increasing the vascular endothelial cell permeation resistance and the permeated dose of fluorescent dextran. Although decreased, the permeability of cerebrovascular endothelial cell tight junctions increased up to 6 hours after treatment with mannitol.
  • anti-ZO-1 (1:500, Thermo Fisher Scientific) and anti-claudin-5 (1:500, Thermo Fisher Scientific) primary antibody and then HRP (horseradish peroxidase) secondary anti-rabbit or
  • HRP horseradish peroxidase
  • the band was detected using a mouse antibody (1:10,000, Invitrogen) and Actin (1:10,000, Merck Millipore) was used as a housekeeping gene.
  • the image of the band was acquired using the ChemiDoc imaging system (Bio-Rad), and the intensity of the band was measured using the Image J program.
  • In vitro migration analysis was performed to confirm in vitro migration of MSCs and MSC/Ngn1 cells to the chronic stroke environment by passing through the vascular endothelial cell tight junction with increased permeability by mannitol treatment.
  • bEnd.3 cells 5 ⁇ 10 4 bEnd.3 cells per well were seeded into the transwell insert of FIG. 2A and cultured for 5 days.
  • hemispheres of the chronic stroke cerebrum ipsilateral hemisphere one month after stroke induction
  • PBS phosphate-buffered saline
  • the tissue was stirred at 12000 rpm for 5 min. was centrifuged, and after harvesting the supernatant, the protein in the sample was quantified using the BCA Protein Assay (ThermoFisher Scientific).
  • DMEM medium 1% penicillin
  • MSC-GFP or MSC/Ngn1-GFP cells are added to the insert in 100 ⁇ l stem cell DMEM medium [10% FBS (Gibco) + 10 ng/ml bFGF (Sigma Aldrich)) + 1% penicillin/streptomycin (Gibco) + 89% DMEM (Gibco)] and incubated at 37°C in a 5% CO 2 incubator for 24 hours.
  • cells expressing GFP that passed through the bEnd.3 cell layer and migrated to the bottom of the insert were observed with a fluorescence microscope (Olympus) and the number of cells was counted.
  • MSC and MSC/Ngn1 cells were cultured in a serum-free DMEM medium [1% penicillin/streptomycin (Gibco) + 99% DMEM (Gibco)] in a 5% CO 2 incubator at 37° C., or in a hypoxia chamber. , 2% O 2 + 5% CO 2 + 93% N 2 ) and incubated in an incubator at 37°C.
  • DMEM medium 1% penicillin/streptomycin (Gibco) + 99% DMEM (Gibco)
  • MSC cells were reduced in serum-deprived conditions (20% oxygen, serum deprivation) and hypoxic, serum-deprived conditions (2% oxygen, serum deprivation).
  • serum-deprived conditions (20% oxygen, serum deprivation)
  • hypoxic, serum-deprived conditions 2% oxygen, serum deprivation
  • MSC/Ngn1 cells are more resistant than MSC cells and have a therapeutic effect for a long time, which can be usefully used in the treatment of brain diseases such as stroke.
  • mannitol at a concentration of 25% was injected into the tail vein (IV) or intra-arterial (IA) at an amount of 5 ml/kg, and Evans blue dye at a dose of 4 mg/kg was immediately injected.
  • the heart was perfused with 0.9% physiological saline, fixed with a 10% formalin solution, and the brain was excised and photographed using a dissecting microscope (Zeiss).
  • a bolus injection of 25% mannitol at a dose of 5 ml/kg at a rate of 1.6 ml/15 seconds into the internal carotid artery of a chronic stroke animal model and normal animals 1 month after stroke induction, 5 minutes, 30 minutes , 1 hour and 2 hours later, 4 mg/kg of Evans blue dye was administered by the same route.
  • Evans blue dye administration the heart was perfused with 0.9% saline and fixed with 10% formalin solution. After that, the brain was removed and pictures were taken using a dissecting microscope (Zeiss) (FIG. 6a).
  • ischemic stroke mouse model prepared in Examples 1-3, 25%, 5 ml/kg of mannitol was injected into the internal carotid artery (ICA) of the chronic stroke animal model by 1.6 After bolus injection at a rate of ml/15 seconds, 4 mg/kg of Evans blue dye was administered by the same route after 5 minutes, 30 minutes, 1 hour, and 2 hours.
  • ICA internal carotid artery
  • the permeability of the vascular brain barrier by mannitol continued for more than 1 hour in the chronic stroke animal model.
  • the above result means that the effect of increasing the vascular-brain barrier permeability by mannitol administration is higher in the damaged brain of the ischemic model than in the normal control group.
  • Example 7 Stem cells through arterial administration and mannitol blood brain barrier Check the increase in pass
  • MSC and MSC/Ngn1 cells were administered, and the mobility and persistence to the chronic stroke lesion site were confirmed as follows.
  • MSC and MSC/Ngn1 cells were supra paramagnetic iron oxide nanoparticles (SPIO) at a dose of 50 ⁇ g/ml, Sigma Aldrich, in the same manner and conditions as in Example 1-2 before transplantation to enable tracking and observation on MRI.
  • SPIO paramagnetic iron oxide nanoparticles
  • an animal model with a cerebral infarction rate of 50% or more was selected at the initial stage (day 3) of stroke animal production, and mannitol ( 25%, Sigma Aldrich) and control (0.9% saline) were injected into the internal carotid artery (ICA) at a rate of 1.6 ml/15 sec.
  • ICA internal carotid artery
  • 1 ⁇ 10 6 MSCs and MSC/Ngn1 cells were injected into 500 cells by the same route. It was suspended in ⁇ l physiological saline and injected for 2 minutes.
  • the pterygopalatine artery (PPA) was ligated before injection of mannitol and stem cell treatment, and reperfusion was performed after injection.
  • MRI T2 star-weighted pictures were taken at 1 hour, 4 hours, 24 hours, 1 week, 4 weeks, and 8 weeks after stem cell injection, and the number of SPIO-positive pixels appearing in the whole brain tissue MRI image of the animal model was counted. quantified. Quantification was performed using the NIH Image J program.
  • TR 500 ms
  • TE 12 ms
  • average 4
  • acquisition matrix 256 ⁇ 256
  • FA 30°.
  • T2*-weighted images were obtained at 1 hour, 4 hours, 24 hours, 1 week, 4 weeks, and 8 weeks post implantation. A total of 10 slides were scanned to cover the entire rat brain. The ischemic area of each T2-weighted image was directly marked and calculated through the program (Philips DICOM Viewer R3.0 SP3).
  • the MSC/Ngn1 group injected with mannitol (+Mannitol MSC/Ngn1) showed a significant difference from the MSC group (+Mannitol MSC) and the control group (-Mannitol MSC, -Mannitol MSC/Ngn1). confirmed that.
  • the MSC/Ngn1 group injected with mannitol (+Mannitol MSC/Ngn1) showed a significant difference from the MSC group (+Mannitol MSC) and the control group (-Mannitol MSC, -Mannitol MSC/Ngn1).
  • the cortical area around the ischemic brain lesion was stained with GFP and RECA1 antibodies to confirm extravasation of the injected stem cells into the brain tissue.
  • the number of GFP-positive cells was counted and AluJ- qPCR was performed.
  • the right external carotid artery (ECA) was exposed in the model mice of Examples 1-3.
  • ECA right external carotid artery
  • MSC-GFP and MSC/Ngn-GFP prepared in Examples 1-2 were prepared at a density of 1 ⁇ 10 6 in 500 ⁇ l PBS, respectively, and the right external carotid artery (ECA) to the right internal carotid artery (ICA) was administered slowly for 2 minutes.
  • the pterygopalatine artery was temporarily closed and opened after cell administration. Blood flow in the internal carotid artery was maintained during administration.
  • saline was used instead of mannitol.
  • the tissue was incubated for 1 hour, washed twice with 1% Triton X-100/PBS, and with Alexa fluor 488- or 568-labeled anti-IgG secondary antibody (1:250, invitrogen) for 1 hour. Incubated and cross-stained for 5 minutes using Hoechst. Slide sections were mounted on a Fluoromount-G (SouthernBiotech, USA), and fluorescence images were obtained using a Zeiss LSM710 confocal microscope (Carl Zeiss, Oberkochen, Germany).
  • qPCR isolated human genomic DNA from homogenized right brain hemispheres using the Qiagen kit (Cat No.69504) at 1 hour, 24 hours and 1 week after cell transplantation. Brains were stored at -80°C prior to isolation of genomic DNA. AluJ-qPCR was performed using primers having the sequences shown in Table 1 below.
  • the amplification was carried out for 40 cycles at an initial holding temperature of 95°C for 10 minutes, then 95°C for 15 seconds, 56°C for 30 seconds, and 72°C for 30 seconds, and a melting curve analysis was performed from 60°C to 95°C in 1°C units.
  • the reaction was performed in a volume of 20 ⁇ l containing 10 ⁇ l of SYBR PCR Master Mix (Thermo Fisher Scientific, A25742), 0.005 nM of each primer, and 100 ng of a DNA template diluted to a final volume with water.
  • qPCR analysis was performed using a StepOne Plus real-time PCR machine.
  • the crossing threshold (Ct) value was calculated using the StepOne software.
  • the qPCR standard curve for absolute quantification was determined using a mixture obtained by serial dilutions (10-fold serial dilutions) of genomic DNA extracted from the left brain of normal rats starting with 100 ng of human genomic DNA. qPCR was performed 4 times per sample, and the number of human cells was calculated by converting it from a standard curve using 0-100 ng human genomic DNA.
  • FIGS. 8A and 8B when mannitol injection was not performed (-Mannitol), it was confirmed that the extravasation of the injected MSCs and MSC/Ngn1 cells was not observed until 24 hours.
  • mannitol was injected (+Mannitol)
  • extravasation was observed from 24 hours after injection of MSC and MSC/Ngn1 cells, and a significant difference in the number of remaining GFP-positive cells was confirmed.
  • the MSC-administered group (+Mannitol MSC) and the control group (-Mannitol MSC, -Mannitol MSC/Ngn1) were significantly compared to those of the control group (-Mannitol MSC, -Mannitol MSC/Ngn1). It was confirmed that many cells remained.
  • MSC and MSC/Ngn1 cells were analyzed using a human-specific hMT antibody, and markers NeuN (nucleus of neurons) and MAP2 (dendritic cells of neurons) and glial cell markers that can confirm differentiation into neurons (GFAP) was analyzed by immunohistochemistry.
  • Primary antibody and subsequent secondary anti-mouse 488 or rabbit antibody 568 (1:250, Life Technologies) were used, and nuclei were detected with Hoechst (1:2,000, Thermo Fisher Scientific). The photograph was obtained using a confocal fluorescence microscope (LSM 710, Zeiss).
  • MSC/Ngn1 cells were higher than that of MSCs in the brain tissue at the first week of cell transplantation, but differentiation of these cells could not be observed.
  • Figure 8d it was confirmed that some remaining MSC cells in the brain tissue after 4 weeks were differentiated and distributed into hMT/GFAP-positive cells, whereas MSC/Ngn1 cells differentiated into hMT/NeuN and hMT/MAP2-positive neurons. could This is supported by the fact that MSC/Ngn1 has increased persistence in the body regardless of whether it is differentiated into neurons, and its resistance is increased under hypoxic and serum-free conditions.
  • mannitol and MSC/Ngn1 cells are administered, the distribution of nerve cells around the brain lesion increases, which can be usefully used in the treatment of brain diseases such as stroke.
  • MSC/Ngn1 administration through animal behavior analysis, specifically corner test, rotarod test, and adhesion removal test
  • Chronic stroke model MSC is administered instead of MSC/Ngn1
  • Neural function recovery was evaluated up to 8 weeks after administration in a chronic stroke model in which saline was injected instead of MSC/Ngn1.
  • the Corner test was used to evaluate asymmetry in rotation preference, and the animal was placed in a device consisting of two boards (50 cm ⁇ 30 cm ⁇ 30 cm) placed close at an angle of 30°. . If the animal moved into the deep corner and the whiskers hit the wall, the direction in which the animal returned was measured, and the average value was recorded in 10 trials per animal. Since normal animals rotate randomly, the unilaterality index appears close to 0, whereas in the unilateral abnormality animal model, the unilateral abnormality rotates in the damaged direction (ipsilateral). The rate of corner movement was expressed as an indicator of deficit.
  • the rotarod test is a test for measuring the running time of an experimental animal on a cylindrical device accelerated from 4 rpm to 40 rpm for 5 minutes, and it was attempted 3 times per animal and was expressed as an average value.
  • Adhesive removal test is a test for measuring the time it takes to attach a tape of 10 mm ⁇ 10 mm to the back of both forelimbs of an animal and remove the tape from both forelimbs. The cut-off time was 300 seconds.

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Abstract

La présente invention concerne une composition pharmaceutique pour la prévention ou le traitement de maladies du système nerveux, la composition pharmaceutique comprenant une lignée de cellules souches adultes contenant des gènes de facteur de transcription neurogène à hélice-boucle-hélice de base (bHLH), et est administrée par voie intra-artérielle après l'administration de mannitol. La composition pharmaceutique peut être administrée pour surmonter une incapacité physique chronique résultant de la mort cellulaire accompagnant une attaque, et peut ainsi être développée en tant que nouvel agent thérapeutique applicable à des maladies du système nerveux telles que l'accident vasculaire cérébral, la maladie de Parkinson, la maladie d'Alzheimer et le traumatisme médullaire, et peut être largement utilisée pour une application clinique et diverses études.
PCT/KR2020/018572 2019-12-20 2020-12-17 Composition pharmaceutique pour administration artérielle, comprenant une lignée de cellules souches adultes contenant des gènes de facteur de transcription neurogène à hélice-boucle-hélice de base (bhlh) WO2021125841A1 (fr)

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