WO2014193060A1 - Agent thérapeutique et méthode de traitement de la sclérose en plaques par une administration concomitante d'une cellule souche mésenchymateuse dérivée de la moelle osseuse humaine et de minocycline - Google Patents

Agent thérapeutique et méthode de traitement de la sclérose en plaques par une administration concomitante d'une cellule souche mésenchymateuse dérivée de la moelle osseuse humaine et de minocycline Download PDF

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WO2014193060A1
WO2014193060A1 PCT/KR2013/011476 KR2013011476W WO2014193060A1 WO 2014193060 A1 WO2014193060 A1 WO 2014193060A1 KR 2013011476 W KR2013011476 W KR 2013011476W WO 2014193060 A1 WO2014193060 A1 WO 2014193060A1
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minocycline
hbm
msc
treatment
multiple sclerosis
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전신수
유충헌
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Industry Academic Cooperation Foundation of Catholic University of Korea
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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
    • A61K31/65—Tetracyclines
    • 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
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • the present invention relates to a composition and method for preventing or treating multiple sclerosis, encephalomyelitis, etc. by administering human bone marrow-derived mesenchymal stem cells and minocycline in combination.
  • Multiple sclerosis is an inflammatory disease associated with autoimmunity that causes damage to myelin sheaths around the axon of the CNS, resulting in demyelination and loss of neurons.
  • Multiple sclerosis is characterized by various signs and symptoms, such as exacerbation after remission and recovery, and the most common type is relapsing-remitting MS.
  • secondary progressive MS Progressive progression with no apparent recurrence from the onset is called primary progressive MS.
  • EAE Experimental autoimmune encephalomyelitis
  • Myelitis refers to inflammation of the common spinal cord caused by an infection or the like.
  • the disease was thought to be a transverse (horizontal) invasion of one side of the spinal cord, hence the name acute transverse myelitis. Since then, with medical advances, it has been known that inflammation in the spinal cord may occur in the longitudinal direction of the spinal cord.
  • acute transverse myelitis is caused by an immune-mediated reaction, which is divided into two cases, one of which has no known cause and is associated with other systemic diseases.
  • Blood-Brain Barrier is a barrier that separates cerebrospinal fluid and blood and has high selective permeability to isolate the body's key regulatory centers from pathogens such as bacteria that can be transported into the blood and from potentially dangerous substances in the blood. Play a role.
  • Minocycline is a tetracycline-based antibiotic whose chemical formula is C 23 H 27 N 3 O 7 , 7-bis (dimethylamino) -1,4,4a, 5,5a, 6,11,12a-octahydro-3 It is, 10,12,12a-tetrahydroxy-1,11-dioxo-2-naphtacenecar-boxomide with molecular weight of 457.49. It has strong antibacterial activity, maintains blood concentration for a long time and can be used for oral or injection. In addition, it is a semisynthetic tetracycline derivative having blood brain barrier function, anti-inflammatory function and anti-apoptosis function, and is suitable for treating CNS-related diseases. Therefore, it is effective in delaying the progression of various degenerative neurological diseases.
  • minocycline in EAE and multiple sclerosis weakened disease activity within 2 months after treatment and decreased gadolinium enhancement in magnetic resonance imaging (MRI).
  • minocycline prevents demyelination of exons in EAE, exerts a neuroprotective effect, attenuates neuronal death, and regulates immunodifferentiation from Th1 to Th2 phenotype. Doing so reduces the infiltration of T cells into the spinal cord.
  • minocycline can cause side effects such as lupus (systemic lupus erythematosus) and serum sickness (serum sickness), and it is toxic to the CNS at high doses. Therefore, it is necessary to develop a co-administration method using low doses of minocycline.
  • MSCs Mesenchymal stem cells
  • an object of the present invention is bone marrow-derived mesenchymal stem cells (BM-MSC); And it provides a pharmaceutical composition for the prevention or treatment of multiple sclerosis or encephalomyelitis comprising a minocycline compound or a pharmaceutically acceptable salt of the compound as an active ingredient.
  • BM-MSC bone marrow-derived mesenchymal stem cells
  • BM-MSC in plasma in vitro; And treating a minocycline compound or a mixture of pharmaceutically acceptable salts of the compounds to provide a method of modulating cytokine expression to reduce Th1 cytokine or to increase Th2 anti-inflammatory cytokine.
  • BM-MSC provides a cell therapy for the prevention or treatment of multiple sclerosis or encephalomyelitis comprising a minocycline compound or a pharmaceutically acceptable salt of the compound as an active ingredient.
  • the present invention is BM-MSC; And it provides a pharmaceutical composition for the prevention or treatment of multiple sclerosis or encephalomyelitis comprising a minocycline compound or a pharmaceutically acceptable salt of the compound as an active ingredient.
  • the bone marrow-derived mesenchymal stem cells may be human bone marrow.
  • the bone marrow may be an autologous bone marrow obtained from an individual having multiple sclerosis or encephalomyelitis.
  • the prevention or treatment of multiple sclerosis or encephalomyelitis is demyelination of neurons, neuroprotective effect, reduction of tissue damage, prevention of apoptosis or inflammatory It may be due to the inhibitory effect of inflammatory infiltration.
  • the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier refers to a cell or human being exposed to the composition, which is not toxic.
  • the carrier can be used without limitation so long as it is known in the art such as buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, bases, excipients, lubricants, preservatives and the like.
  • the pharmaceutical compositions of the present invention can be prepared according to techniques commonly used in the form of various formulations. For example, injectables can be prepared in the form of unit dose ampoules or multiple dose inclusions.
  • the pharmaceutical composition of the present invention may be packaged in a suitable container according to the desired purpose.
  • the pharmaceutical composition of the present invention may be administered in a suitable formulation and may be parenterally administered, for example, by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, into a target cell according to the desired method. Topical administration can be done.
  • the dosage of the pharmaceutical composition can vary depending on several factors, including the age, body weight, general health, sex, time of administration, route of administration, rate of release, drug combination and severity of the particular disease of the individual.
  • the pharmaceutical compositions of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers.
  • the BM-MSC may be administered at a dosage within the range of 2 ⁇ 10 5 cells / kg to 2 ⁇ 10 7 cells / kg, which is a therapeutically effective amount on an adult basis, but is not limited thereto.
  • the minocycline compound or a pharmaceutically acceptable salt of the compound may be administered at a dosage in the range of 0.001 to 100 mg / kg on an adult basis, but the dosage does not limit the scope of the present invention.
  • the present invention provides a method for preventing or treating multiple sclerosis or encephalomyelitis comprising administering the pharmaceutical composition to a subject.
  • the present invention relates to BM-MSC in plasma in vitro; And treating a minocycline compound or a mixture of pharmaceutically acceptable salts of the compounds to provide a method of modulating cytokine expression that reduces Th1 cytokine or increases Th2 cytokine.
  • the present invention is BM-MSC; And it provides a cell therapy for the prevention or treatment of multiple sclerosis or encephalomyelitis comprising a minocycline compound or a pharmaceutically acceptable salt of the compound as an active ingredient.
  • the term 'cell therapeutic agent' refers to a medicine (US FDA regulation) used for the purpose of treatment, diagnosis, and prevention of cells and tissues prepared by isolation, culture, and special chewing from humans, and functions of cells or tissues.
  • a medicine US FDA regulation
  • Cell therapy agents are largely classified into somatic cell therapy and stem cell therapy according to the degree of differentiation of cells, and the present invention relates in particular to stem cell therapy.
  • the present invention provides a method for preventing or treating multiple sclerosis or encephalomyelitis comprising administering the cell therapy agent to a subject.
  • the prophylactic or therapeutic method may be, but is not limited thereto, in combination with BM-MSC, minocycline or a pharmaceutically acceptable salt of the compound.
  • hBM-MSC Viability assay of the cells confirmed that hBM-MSC is affected by minocycline.
  • the minocycline-treated hBM-MSCs were characterized by flow cytometry using markers on the MSC surface and analyzed for their differentiation potential.
  • Minocycline does not affect the surface phenotype, differentiation and viability of hBM-MSCs, whereas high concentrations of minocycline affect the viability of the astrocytes.
  • EAE was induced using MOG35-55 in C57BL / 6 mice, and immunopathology assays were used to search for inflammatory cells, demyelination and neuroprotective effects.
  • Th1 and Th2 Characteristic cytokines indicating the development of Th1 and Th2 are IFN- ⁇ (interferon gamma), TNF- ⁇ (tumor necrosis factor alpha), IL-4 (interleukin-4) and IL-10 (interleukin-10) by ELISA. TUNEL staining was used to account for cell apoptosis in the spinal cord of EAE mice.
  • hBM-MSCs have potential as therapeutic agents in neurodegenerative diseases.
  • hBM-MSC is easily obtained from the human spinal cord, and because T-cells do not recognize cell surface antigens well, the immune system can be avoided and used for transplantation.
  • hBM-MSCs show immunoregulatory functions both in vitro and in the body by inhibiting the activation and proliferation of T lymphocytes, inducing Th2 polar immune responses, and inducing endogenous treatment.
  • transplantation of hBM-MSCs can inhibit inflammation, reduce demyelination, protect nerves and exons in EAE, and provide a viable method by targeting and targeting damaged tissues.
  • this combination is characterized by providing a medicinal use in that it enhances immunomodulatory function and improves functional recovery of multiple sclerosis patients.
  • hBM-MSC and minocycline promotes migration from Th1 cytokines to Th2 cytokines, reduces the influx of inflammatory cells, inhibits demyelination, reduces cell death, enhances neuroprotective function, and enhances EAE Effective treatment is achieved by preventing disease progression in mice. In addition, the side effects of minocycline alone treatment can be minimized.
  • 1A, 1B and 1C show the effect of minocycline on the viability of hBM-MSCs.
  • 2A, 2B and 2C show that the combination treatment of hBM-MSC and minocycline alleviates the severity of clinical EAE in mice.
  • 3A, 3B and 3C show that hBM-MSC and minocycline reduce inflammation in the EAE mouse spinal cord.
  • 4A, 4B and 4C show that co-administration of hBM-MSC with minocycline reduces demyelination in the spinal cord of EAE mice.
  • 5A-5D show that co-administration reduces neuroglial activity in the spinal cord of EAE mice and protects neurons.
  • 6A and 6B show promoting balance shift from Th1 cytokine to Th2 cytokine in EAE mice by co-administration.
  • 7A and 7B show that co-administration of hBM-MSC with minocycline in EAE mouse spinal cord reduces apoptosis.
  • Figure 8 shows the identification of minocycline treated hBM-MSC in the spinal cord of EAE mice.
  • 9A and 9B show that co-administration reduces Th1 frequency and increases Th2 frequency in EAE mice.
  • Figure 10 shows the characteristics of apoptosis in the lumbar spinal cord of EAE mice.
  • hBM-MSC was purchased from Lonza (Walkersville, Ind., USA) and the cells were thawed and initiation of the culture was performed according to the manufacturer's instructions.
  • the cells were aliquoted into culture dishes and hBM-MSC basal medium and growth supplements were fed to the dispensed MSC cells (37 ° C., 5% CO 2 ).
  • Astrocytes were distributed from the American Type Culture Collection (ATCC), Manassas, VA, USA.
  • the medium was cultured in DME medium (Dulbecco 'modified Eagle' medium; Invitrogen, Carlsbad, Calif., USA) containing 10% FBS (fetal bovine serum).
  • DME medium Dulbecco 'modified Eagle' medium; Invitrogen, Carlsbad, Calif., USA
  • FBS fetal bovine serum
  • Minocycline was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved 1 mM in distilled water and filtered sterilization.
  • hBM-MSCs or astrocytes were seeded in 24-well plates (8 ⁇ 10 3 ) or 96-well plates (5 ⁇ 10 3 ), respectively.
  • the amount of minocycline was increased to confirm cytotoxicity of minocycline to hBM-MSCs or astrocytes. After 24 hours of administration, cell activity was analyzed using MTT assay (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium; Sigma-Aldrich).
  • FACS Fluorescence Activated Cell Sorting
  • Adipose cells were detected by staining intracellular lipid droplets, 0.3% Oil Red O staining for 10 minutes, bone cells were detected through calcium phosphate, and 0.2% Alizarin Red S for 20 minutes. Stained with.
  • EAE was induced in 11-week-old female C57BL / 6 mice using MOG35-55 (Hooke Labs, Lawrence, Mass., USA), totaling 200 ⁇ g MOG35-55 containing 6 mg / ml of Mycobacterium tuberculosis. It was suspended in CFA (complete Freund's adjuvant) and injected subcutaneously in 2 parts of mice. Two and 24 hours after MOG35-55 injection, mice received 100 ng of pertussis toxin intraperitoneally.
  • MOG35-55 Hooke Labs, Lawrence, Mass., USA
  • mice were converted into scores of clinical symptoms as follows. 0 for no clinical symptoms, 1 for sagging tails, 2 for partial paralysis of the hind limbs, 3 for complete paralysis of the hind limbs, 3 for complete paralysis of the hind limbs and 4 for complete paralysis of the forelimbs Or 5 if death is achieved.
  • mice were randomly divided into the following four groups.
  • mice were slaughtered at 46 days post-immunization, part of the lumbar spinal cord was isolated and frozen, H & E (hematoxylin and eosin) staining and LFB (Luxol Fast Blue) staining.
  • Immunofluorescence staining was performed to identify inflammatory cells, demyelination, and nerve loss, and glial cells were activated according to the general procedure.
  • lumbar spinal cord sections were incubated with the following antibodies at 4 ° C. overnight.
  • Monoclonal rat GFAP anti-glial fibrillary acidic protein; Millipore, Temecula, CA, USA
  • Antibody staining was visualized through anti-rabbit and anti-rat antibodies (Cy3-conjugated secondary antibodies; Jackson ImmunoResearch, West Grove, PA, USA), and the specificity of the immune response was immunized at the site where the main antibody or side antibody was missing. It was confirmed by the absence of an immunohistochemical reaction. Counterstaining of cell nuclei was performed for 10 minutes using DAPI (4-6-diamidino-2-phenyindole).
  • Plasma cytokines were determined by ELISA, and plasma was obtained from all individuals in each group 40 days after immunization. Plasma was incubated for 4 hours at room temperature in coated 96-well plates, three washes followed by addition of complex antibody at room temperature for 2 hours and incubation for 30 minutes in substrate solution. The reaction was then stopped using a stop solution.
  • the optical density of each well was measured at 450 nm using a microplate reader.
  • TUNEL assay using Cy3-conjugated streptavidin (Jackson ImmunoResearch Laboratories). Slides were immersed in distilled water at 60 ° C. for 2 hours and washed with terminal deoxynucleotidyl transferase (TdT) labeled buffer.
  • TdT terminal deoxynucleotidyl transferase
  • the TUNEL reaction mixture was dropped onto the sections with a pipette and incubated in a humidification chamber at 37 ° C. for 1 hour and terminated with a terminating buffer. Counterstaining of cell nuclei was performed by incubating for 10 minutes by adding DAPI to the sections. In addition to the features of apoptosis, the sections were overlapped with NeuN, GFAP, Ib-1, and CD4, respectively.
  • the measurements were performed by a test tube who did not know the status of each animal's action, and qualitatively analyzed 6 to 8 sites of lumbar spine obtained from each group.
  • H & E staining and LFB staining were measured using Slide Scanner for Digital Pathology (SCN400, Leica, Wetzlar, Germany). Immunofluorescence was measured by confocal microscopy. All pictures were taken with MetaMorph (Molecular Devices, Sunnyvale, CA, USA). ; version 7.5).
  • the number of infiltrating cells, the number of staining positive cells or the fluorescence intensity was expressed as the number of cells or the fluorescence intensity of each lesion site.
  • Lesion size was determined by quantitative pathology analysis of LFB comparative stained spinal cord sites. The lesion size was expressed as the area of the lesion according to the photographed objective magnification, and the data were expressed as mean ⁇ SEMs.
  • minocycline could affect the survival of hBM-MSCs and astrocytes, the two cells were each grown in media containing varying concentrations of minocycline.
  • the viability of hBM-MSC and astrocytes was analyzed by MTT 24 hours after minocycline (0-10 mM). As a result, as shown in FIG. 1A, minocycline did not affect the viability of hBM-MSC until 10 mM and reduced the viability of astrocytes at high concentration (810 mM) (Points, mean; bars, SE). .). That is, the viability of hBM-MSC was not affected until 10 ⁇ M, and the viability of astrocytes decreased at 810 ⁇ M.
  • Minocycline did not affect the expression of MSC phenotypic surface markers, hBM-MSCs expressed CD90, CD44, CD73 and lacked CD34, CD45, HLA-DR.
  • minocycline The effect of minocycline on the differentiation capacity of hBM-MSC was assessed by incubating minocycline (10 ⁇ M) in or without treatment for 3 weeks.
  • Minocycline did not affect the ability to differentiate into adipocyte lineage or bone formation lineage.
  • the effect of minocycline on the differentiation capacity of hBM-MSC was measured.
  • minocycline is capable of differentiating hBM-MSC into oil cell or bone cell line by staining with hBM-MSC with Oil Red O and Alizarin Red S, respectively. Did not affect.
  • These results are shown as independent experiments.
  • minocycline treated hBM-MSCs were transferred and implanted into the spinal cord after intravenous administration to EAE mice after intravenous administration.
  • minocycline-treated hBM-MSC was confirmed in the spinal cord of EAE mice, and hBM-MSC to which Ad-GFP (50 MOI) was added was treated with minocycline (10 ⁇ M).
  • hBM-MSCs were imaged with a Zeiss LSM 700 multifocal microscope and the GFP transformed hBM-MSCs on the lesion area were green and many were closely associated with blood vessels (( A) is the first picture).
  • Minocycline treated mice had an average of 12.67 ⁇ 1.0 days after immunization with clinical signs of EAE (p ⁇ 0.001, PBS vs. minocycline treatment).
  • the average clinical score of PBS treatment was 3.166 ⁇ 1.075, hBM-MSC treatment: 1.433 ⁇ 0.534, minocycline treatment: 1.223 ⁇ 0.4936, P ⁇ 0.001, PBS vs hBM-MSC treatment, P ⁇ 0.001, PBS vs minocycline treatment.
  • hBM-MSC treatment or minocycline treatment significantly reduced the average clinical score and the highest clinical score.
  • the co-administered group significantly reduced overall disease progression compared to the group administered individually.
  • hBM-MSC or minocycline individual treatment significantly reduced the average clinical score.
  • Combination administration significantly reduced the average clinical score (p ⁇ 0.05) compared to hBM-MSC or minocycline treatment (FIG. 2B).
  • the maximum clinical score of each mouse was recorded for each course of the whole experiment, and the trend of the maximum clinical score of the four groups was equal to the average clinical score (p ⁇ 0.05.
  • Improved clinical score following co-administration may reduce inflammatory infiltration in the CNS.
  • lumbar spinal cord slices in EAE mice were labeled with H & E and anti-CD4 to detect monocyte and T cell infiltration. Infiltrating cells increased in spinal white matter of PBS treated group, but these cells decreased after co-administration compared to individual treatment (see FIG. 3A).
  • lumbar spinal cord slices were labeled with LFB and anti-MBP.
  • Demyelination was identified and reduced in the lumbar spinal cord protein, and decreased significantly when combined with each individual treatment (see FIG. 4A).
  • Statistical analysis of the degree of demyelination showed a significant decrease in mice treated with hBM-MSC or minocycline alone compared to the group of PBS treated mice (p ⁇ 0.001, PBS vs. hBM-MSC treatment; p ⁇ 0.001 , PBS vs. minocycline treatment).
  • Neuroinflammatory and neurodegenerative were evaluated with GFAP-, Iba-1- and NeuN positive cells.
  • the present inventors counted the number of neurons in the gray matter of the lumbar spinal cord and measured the neuroprotective effect of each treatment. Compared with PBS treatment, the number of NeuNs in the gray matter of hBM-MSC or minocycline-treated EAE mice increased. On the other hand, co-administration resulted in a marked increase than NeuN in individually treated tissues.
  • Iba-1 identified in response to microglia, showed the same GFAP-like immune response pattern in four EAE groups, and the opposite pattern was found in all markers, NeuN (Scale bar, 200 ⁇ m).
  • Figure 5b is a statistical analysis of GFAP fluorescence, the number of Iba-1 positive cells shows that the activity of astrocytes and microglia significantly reduced in combination compared to hBM-MSC or minocycline (Fig. 5c) .
  • Statistical analysis showed a significant increase in the number of NeuN positive cells in the spinal cord of EAE mice compared to hBM-MSC or minocycline (p ⁇ 0.01) .Columns, mean; bars, SE. * P ⁇ 0.05, * * p ⁇ 0.01, *** p ⁇ 0.001, one-way ANOVA with post-hoc Bonferroni corrections (FIG. 5D).
  • the inventors measured the expression of IFN- ⁇ / TNF- ⁇ and IL-4 / IL-10 in the plasma of EAE mice by ELISA.
  • the hBM-MSC or minocycline-treated group showed a marked increase in IL-4 and IL-10 protein expression levels, compared with the hBM-MSC or minocycline-treated group. Similar expression patterns were evident in (Columns, mean; bars, SE. * P ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001, one-way ANOVA with post-hoc Bonferroni corrections) (FIG. 6b) . This result represents three independent experiments.
  • apoptotic cell death was measured via TUNEL.
  • TUNEL positive cells were quantified using MetaMorph image analysis, and the number of apoptosis cells was statistically significantly reduced in the hBM-MSC or minocycline-treated group compared to the PBS-treated group. Co-administration also showed a significant reduction compared to hBM-MSC or minocycline-treated groups (p ⁇ 0.001) Columns, mean; bars, SE. ** p ⁇ 0.01, *** p ⁇ 0.001, one- way ANOVA with post-hoc Bonferroni corrections (FIG. 7b). The results also represent three independent experiments.
  • the number of apoptotic cells was decreased in the individually treated group compared to the PBS treated group (p ⁇ 0.001, PBS vs. hBM-MSC treatment; p ⁇ 0.001, PBS vs. minocycline treatment), but in combination compared to the individually treated group In one group, the number of apoptosis cells was significantly reduced (see FIGS. 7A and 7B).
  • apoptosis was reduced at the lesion site, and the inventors also confirmed that most of the apoptotic cells were double-labeled with NeuN and a neural marker. Only a few were positive for GFAP, Iba-1, or CD4.
  • the (A) picture of the first column is a double-labeled site of NeuN and TUNEL (red) shown in green in the white matter of the lumbar spine.
  • the second picture in the first column is a high magnification of the square in (A). Most dead cells are also positive for NeuN (arrow in a).
  • the second column (B) shows the double-labeled sites of GFAP (green) and TUNEL (red), which are colored green in the lumbar spinal cord.
  • the second picture in the second row is a high magnification of the rectangle in (B). Also a few dead cells are positive for GFAP (arrow in b).
  • the third column (C) shows the double-labeled sites of Iba-1 (green) and TUNEL (red) in green in the lumbar spinal cord white matter.
  • the second picture in the third column is a high magnification of the rectangle in (C).
  • the fourth column (C) shows the double-labeled regions of CD4 (green) and TUNEL (red), which are green in the lumbar spinal cord white matter.
  • the second picture in the fourth row is a high magnification of the square in (C). Also a few dead cells are positive for CD4 (arrow in d)
  • Therapeutic approaches have been attempted to improve the treatment of multiple sclerosis to identify therapies that affect new drugs or other aspects of the disease process, or use combinations of low doses of individual drugs to alleviate side effects.
  • the present inventors confirmed that high doses of minocycline reduced the activity of astrocytes, and also confirmed the effect of minocycline on hBM-MSC cells, and did not affect the activity and properties even at high doses.
  • the first beneficial effect of co-administration in the present invention is that it may be involved in the regulation of IFN- ⁇ / TNF- ⁇ and IL-4 / IL-10 expression and production in plasma and spleen cultures of EAE mice.
  • Cytokines play an important role in the development of multiple sclerosis.
  • the balance between Th1 and Th2 in the CNS is an important determinant of progression to EAE, a Th1-associated disease.
  • Th2 cytokines have been known to be involved in alleviation and recovery.
  • Th1 cytokines IFN- ⁇ and TNF- ⁇ .
  • Th2 cytokines such as IL-4 and IL-10 are anti-inflammatory cytokines that prevent or ameliorate disease.
  • TNF- ⁇ / IFN- ⁇ and IL-4 / IL-10 indicate the progression of Th1 and Th2 and are thought to play an important role in both MS and EAE disease.
  • hBM-MSCs can inhibit the activity and differentiation of T lymphocytes, induce Th2 polar immune responses, and endogenous healing (endogenous repair) Therefore, it exerts an immunomodulatory effect both in vitro and in the body.
  • endogenous healing endogenous repair
  • the second possible mechanism is to inhibit inflammation and glial activity. Inflammation is thought to be the cause of tissue damage in relapsing-remitting multiple sclerosis and EAE. Therefore, anti-inflammatory is still considered as the main therapeutic goal in early multiple sclerosis.
  • Glial activity is known to play an important role in cell destruction through the production of inflammatory cytokines and mass differentiation overwhelming surrounding cells.
  • hBM-MSC or minocycline individual treatment significantly reduced the infiltration of mononuclear and T cells and the activity of microglia and astrocytes in combination with co-administration.
  • the data presented herein are consistent with the inhibition of glial cell activity improving EAE severity, and is consistent with the fact that minocycline has a neuroprotective function by inhibiting the activity of microglia.
  • hBM-MSCs Transplantation of hBM-MSCs reduces the activity of microglia and astrocytes, providing a potential and practical means of neuroprotective effects. This is also an important factor for enhancing the therapeutic effect in EAE mice.
  • induction of neuroprotective effect of minocycline is achieved through the induction of intracellular anti-apoptotic signal transduction system, not due to anti-inflammatory activity, and this point is a combination of hBM-MSC and minocycline This is consistent with the significant reduction of apoptosis in the lesions of EAE mice.
  • BM-MSC bone marrow-derived mesenchymal stem cells
  • minocycline promotes migration from Th1 cytokines to Th2 cytokines, reduces the influx of inflammatory cells, inhibits demyelination, reduces cell death, It enhances neuroprotective function and prevents disease progression in EAE mice.
  • the side effects of minocycline alone treatment can be minimized.
  • a combination of hBM-MSC and minocycline can be used to treat multiple sclerosis.

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Abstract

La présente invention concerne l'administration concomitante d'une cellule souche mésenchymateuse dérivée de la moelle osseuse humaine (hBM-MSC) et de minocycline, et diminue la gravité clinique de la sclérose en plaques par comparaison avec une utilisation séparée classique de minocycline, et résout le problème des effets anti-inflammatoire et neuroprotecteur de même qu'a une bonne résistance pour une utilisation à long terme mais avec une toxicité pour le CNS. L'administration concomitante de la hBM-MSC et de la minocycline a pour conséquence une baisse remarquable de score clinique par comparaison avec leur utilisation séparée. En outre, l'administration concomitante de la hBM-MSC et de la minocycline renforce les effets immunomodulateurs, supprime les cytokines pro-inflammatoires (IFN-γ, TNF-α) et augmente inversement les cytokines anti-inflammatoires (IL-4, IL-10). De plus, le nombre de cellules apoptotiques est confirmé comme étant significativement diminué par coloration par la méthode TUNEL si la hBM-MSC et la minocycline sont administrées de manière concomitante par comparaison avec leur utilisation séparée. Par conséquent, l'administration concomitante de la hBM-MSC et de la minocycline peut être utilisée pour traiter la sclérose en plaques.
PCT/KR2013/011476 2013-05-28 2013-12-11 Agent thérapeutique et méthode de traitement de la sclérose en plaques par une administration concomitante d'une cellule souche mésenchymateuse dérivée de la moelle osseuse humaine et de minocycline Ceased WO2014193060A1 (fr)

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KR10-2013-0060458 2013-05-28
KR1020130060458A KR101455933B1 (ko) 2013-05-28 2013-05-28 인간골수유래 중간엽줄기세포와 미노사이클린 병용투여를 통한 다발성경화증 치료제 및 치료방법

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KR102164260B1 (ko) 2018-10-30 2020-10-12 (주)유에이치에이 메틸프레드니솔론 및 줄기세포를 유효성분으로 포함하는 다발성 경화증의 예방 또는 치료용 조성물
CN114748485B (zh) * 2022-06-06 2023-08-18 南京鼓楼医院 葫芦素b在制备治疗多发性硬化药物中的应用

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