WO2014193060A1 - Therapeutic agent and treatment method for multiple sclerosis through concomitant administration of human bone marrow-derived mesenchymal stem cell and minocycline - Google Patents

Abstract

The present invention relates to the concomitant administration of a human bone marrow-derived mesenchymal stem cell (hBM-MSC) and minocycline, and alleviates the clinical severity of multiple sclerosis compared to a conventional separate use of minocycline, and solves the problem of exhibiting anti-inflammatory and neuron protective effects and good resistance for long term use but having toxicity to CNS. The concomitant administration of hBM-MSC and minocycline exhibits a remarkable reduction in clinical score compared to the separate use thereof. Further, the concomitant administration of hBM-MSC and minocycline strengthens immunomodulatory effects, suppresses pro-inflammatory cytokines (IFN-γ, TNF-α) and conversely increases anti-inflammatory cytokines (IL-4, IL-10). In addition, the number of apoptotic cells is confirmed to be significantly reduced in TUNEL dyeing if hBM-MSC and minocycline are concomitantly administered compared to the separate use thereof. Therefore, the concomitant administration of hBM-MSC and minocycline can be used for treating multiple sclerosis.

Description

Multiple sclerosis treatment and treatment method by co-administration of human bone marrow-derived mesenchymal stem cells and minocycline

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. As it progresses, damage to the nervous system accumulates and the recovery slows down and continues to deteriorate in the same form as chronic degenerative disease. This is called secondary progressive MS. Progressive progression with no apparent recurrence from the onset is called primary progressive MS.

Many experimental therapies have been developed to improve the survival rate of multiple sclerosis patients. There are several methods for the treatment of multiple sclerosis, such as glatiramer acetate or mitoxantrone, which are aimed at immunological aspects. Although there are some approved treatments for multiple sclerosis, many patients do not respond appropriately to these agents and thus a protocol for more effective treatment for multiple sclerosis is needed.

Experimental autoimmune encephalomyelitis (EAE) is a disease animal model for CNS autoimmune diseases and is immune to specific CNS antigens. In many clinical and histopathology, the EAE model is reported to be similar to human MS, and is most commonly used as an animal model for studying the mechanism and therapeutic effects of MS.

Myelitis refers to inflammation of the common spinal cord caused by an infection or the like. In the past, before imaging was developed, 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. There are many causes of myelitis, but 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.

The use of minocycline in EAE and multiple sclerosis weakened disease activity within 2 months after treatment and decreased gadolinium enhancement in magnetic resonance imaging (MRI). In addition, 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.

However, 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.

Mesenchymal stem cells (MSCs) are adult pluripotent cells that differentiate into mesenchymal lineages such as osteoblasts, chondrocytes, and adipocytes. Currently, MSCs have been studied in preclinical and clinical settings due to their self-renewal ability, diverse lines, and immunosuppressive capacity. Moreover, MSCs can migrate to the wound site.

Several treatment strategies have been studied for the treatment of multiple sclerosis in EAE mice, but more research is needed focusing on preventing inflammatory infiltration of cells in the CNS and / or preventing demyelination and apoptosis.

Therefore, 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.

In addition, 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. will be.

Also 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.

In order to achieve the object of the present invention as described above, 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.

In one embodiment of the present invention, the bone marrow-derived mesenchymal stem cells may be human bone marrow.

In one embodiment of the present invention, the bone marrow may be an autologous bone marrow obtained from an individual having multiple sclerosis or encephalomyelitis.

In one embodiment of the present invention, 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. The term 'pharmaceutically acceptable' 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. In addition, 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. In addition, 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. In addition, 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.

As used herein, 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. Refers to a drug used for the purpose of treatment, diagnosis, and prevention through a series of actions, such as proliferating, selecting, or otherwise altering the biological properties of a living autologous, allogeneic, or heterologous cell in order to restore it. . 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.

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.

Characteristic cytokines indicating the development of Th1 and Th2 are IFN-γ (interferon gamma), TNF-α (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.

Due to the pathogenic complexity and heterogeneity of multiple sclerosis marker combinations, co-administration is an attractive treatment strategy.

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. In addition, 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.

In the present invention, it was confirmed that the co-administration of hBM-MSC and minocycline exerted a beneficial effect in EAE mice, and the present inventors demonstrated that co-treatment treatment delayed the onset of symptoms, and also showed immune severity, inflammation and demyelination. Alleviate and enhance neuroprotective effects over each medication.

Most importantly, this combination is characterized by providing a medicinal use in that it enhances immunomodulatory function and improves functional recovery of multiple sclerosis patients.

Combined administration of 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.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Experimental Example

1. Preparation of hBM-MSCs and Astrocytes

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).

2. Evaluation of MSC Activity and Characteristics on Minocycline

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 was performed to identify cell surface markers. After trypsinizing minocycline treated hBM-MSC and untreated hBM-MSC, washed with phosphate buffered saline (PBS), and then differentiated into adipocyte or osteoblastic lineage of hBM-MSC. Induced in the non-contained state. Three weeks after the start of culture in induction medium with or without minocycline, differentiated cells were fixed with 10% formaldehyde.

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.

3. EAE Induction and Treatment

EAE was induced in 11-week-old female C57BL / 6 mice using MOG35-55 (Hooke Labs, Lawrence, Mass., USA), totaling 200 μg 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.

Paralysis, a clinical symptom of EAE, was measured every day from 5 days after immunization, and 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. Group 1 is PBS (n = 10), Group 2 is hBM-MSC (1.5 × 10 ⁶ cells in 100 μl PBS, n = 10), Group 3 is minocycline (10 mg / kg, intraperitoneal injection, n = 10 ), Group 4 was combined administration of hBM-MSC and minocycline (n = 10).

All measures started 7 days after immunization and minocycline was administered continuously intraperitoneally until the mice were killed.

4. Histopathology

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. For immunofluorescence, 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),

Polyclonal rabbit Iba1 (anti-ionized calciumbinding adapter molecule 1; Wako Pure Chemical Industries, Osaka, Japan),

Monoclonal rat NeuN (antineuronal nuclear antigen; Chemicon International Temecula, CA, USA),

Monoclonal rat anti-CD4 (BD Biosciences Pharmingen, CA, USA), polyclonal rabbit anti-mouse myelin basic protein (Millipore, Billerica, Mass., 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).

All pictures were taken with an LSM 700 confocal microscope (Carl Zeiss, Oberkochen, Germany).

5. ELISA measurement

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.

6. Measurement of apoptosis by TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling; Roche, Basel, Switzerland) assay

Apoptosis was visualized by 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.

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.

7. Quantitative and Statistical Analysis

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.

All statistical comparisons between the groups were analyzed by ANOVA (post-hoc) with one-way analysis of variance (BNO) and Bonferroni corrections, with p-value <0.05 being significant. Considered.

<Example 1>

Identification of the effects of minocycline on the viability, phenotype, and differentiation of hBM-MSCs

To test whether 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 μM, and the viability of astrocytes decreased at 810 μM.

Evidence of toxicity to astrocytes suggests toxicity according to the cell type of the CNS and suggests the use of low doses of minocycline for the following combined administration experiments in the body. In addition, the characteristics of minocycline-treated hBM-MSC was measured by FACS hBM-MSC surface phenotype, wild-type hBM-MSC and minocycline-treated hBM-MSC were similarly CD90, CD44, CD73 strong positive CD34, CD45, HLA-DR showed a strong negative. FACS analysis for surface markers showed no difference between wild type hBM-MSCs and minocycline treated hBM-MSCs (see FIG. 1B). The effect of minocycline on the hBM-MSC phenotype was analyzed by FACS, and wild type (black line) and minocycline treated hBM-MSC (red line) were labeled with antibodies to MSC phenotype surface markers as shown in FIG.

Minocycline did not affect the expression of MSC phenotypic surface markers, hBM-MSCs expressed CD90, CD44, CD73 and lacked CD34, CD45, HLA-DR.

The effect of minocycline on the differentiation capacity of hBM-MSC was assessed by incubating minocycline (10 μM) 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. As a result, 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. In addition, minocycline treated hBM-MSCs were transferred and implanted into the spinal cord after intravenous administration to EAE mice after intravenous administration. As a result, as shown in FIG. 8, 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 μM).

On day 5 after minocycline treatment (1.5 × 10 ⁶ ), 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). The second picture is a high magnification of the square in the (a) part of the first picture (Scale bar = 200 μm in (a).) The nucleus is stained blue with DAPI. Arrows indicate GFP positive cells (Scale bar = 1 mm in A)

These results confirmed that the viability and properties of hBM-MSC were not affected by minocycline.

<Example 2>

Effect on Clinical Score of EAE Mice in Combination Administration

To evaluate the effects of co-administration in EAE mice and progress on minocycline disease, mice were given PBS, hBM-MSC, minocycline and minocycline in combination with hBM-MSC 7 days after immunization (n = 10 / group).

Behavioral tests were performed up to 50 days after immunization, and PBS treated mice developed clinical symptoms of EAE mice 10 days after immunization.

Compared with PBS treatment, hBM-MSC or minocycline treated mice delayed the onset of clinical symptoms (p = 0.0035, PBS vs. hBM-MSC treatment), and hBM-MSC treated mice had no progression to clinical symptoms of EAE. The average 12.0 ± 1.80 days after immunization. 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).

Co-administration significantly delayed the onset of clinical symptoms compared to single-dose treatment. Mice progressing with EAE took 14.5 ± 1.0 days on average after immunization (p <0.001, hBM-MSC vs. co-administration; p = 0.007). Minocycline vs. co-administration) (see FIG. 2A).

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. The highest immune score was PBS treatment: 3.640 ± 1.284, hBM-MSC treatment: 2.333 ± 0.6831, minocycline treatment: 2.033 ± 0.2582, P = 0.028, PBS vs hBM-MSC treatment, P = 0.014, PBS vs minocycline treatment. Thus, compared to PBS treatment, hBM-MSC treatment or minocycline treatment significantly reduced the average clinical score and the highest clinical score.

The mean immune score for the co-administered group was 0.7375 ± 0.3235, p = 0.005, hBM-MSC vs. Combination administration; p = 0.031, minocycline vs. Co-administration The maximum immune score for the co-administered group was also 1.1625 ± 0.3292 (p = 0.009, hBM-MSC vs. co-administration; p = 0.024, minocycline vs. co-administration, see Figures 2b, 2c). The co-administered group significantly reduced overall disease progression compared to the group administered individually.

EAE was immunized with MOG 35-55 amino acid peptide in female C57 BL / 6 mice, and as shown in FIGS. 2A, 2B and 2C, the mean of clinical scores for different EAE treatments measured daily (n = 10 / Group) is as follows (FIG. 2A). PBS treated mice progressed to EAE with an average maximum severity of grade 4. hBM-MSC or minocycline treatment reduced the severity of the disease with an average maximum severity of Grade 1. Co-administration is a mean maximum severity of Grade 1, which significantly alleviates the severity of the disease (Points, mean; bars, SE.). Mean clinical scores for the four groups were measured from 1 to 50 days after immunization. Compared with PBS treatment, 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. Columns, mean; bars, SE. * P <0.05, * * p <0.01, *** p <0.001, one-way ANOVA with post-hoc Bonferroni corrections (FIG. 2C). The results represent three independent experiments.

These results indicate that co-administration of hBM-MSC and minocycline in EAE mice alleviates immune severity and improves neuronal recovery.

<Example 3>

Effect of Combination on the Number of Inflammatory Cells in the Spinal Cord of EAE Mouse

Improved clinical score following co-administration may reduce inflammatory infiltration in the CNS. To determine the effect of co-administration on the influx of inflammatory cells, 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).

Stereoscopic analysis indicated that the individual treatment of hBM-MSC or minocycline reduced the number of infiltrating cells as compared to PBS treatment (p <0.001, PBS vs. hBM-MSC treatment; p <0.001, PBS vs. minocycline treatment) There is a much more pronounced reduction in the case of combination dosing (p = 0.002, hBM-MSC vs. co-administration; p = 0.020, minocycline vs. co-administration) (see Figure 3b).

Furthermore, there is clear evidence of significant reduction of CD4 ⁺ T cell infiltration in the lumbar spine of mice co-administered with hBM-MSC or minocycline (p <0.001, hBM-MSC vs. co-administration: p = 0.003, minocycline vs. co-administration) (see FIG. 3C).

These results are shown in Figures 3a, 3b and 3c, H & E (top panel) and CD4 staining (bottom panel) was performed to detect infiltration of mononuclear and T cells in the lumbar spinal cord of EAE mice. Arrows indicate infiltrated areas (Scale bar, 200 μm) (FIG. 3A). Statistical analysis indicated that hBM-MSC or minocycline individual treatment significantly reduced clinical scores as compared to PBS treatment. Co-administration significantly reduced the number of infiltrating cells by 20-30% compared to hBM-MSC or minocycline treatment (p <0.05) (FIG. 3b). Co-administration significantly reduced CD4 ⁺ cells by 30-40% compared to hBM-MSC or minocycline treatment (p <0.05) .Columns, mean; bars, SE. * p <0.05, ** p <0.01, *** p <0.001, one-way ANOVA with post-hoc Bonferroni corrections.) (FIG. 3C).

These results confirmed that the combination of hBM-MSC and minocycline reduced inflammation in the EAE mouse spinal cord.

<Example 4>

Effect of Combination on Demyelination in the Spinal Cord of EAE Mice

To determine if tissue damage was reduced in EAE mice, 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).

Moreover, there was a significant decrease in the combination group compared to the individual group (p <0.001, PBS vs. combination; p = 0.017, hBM-MSC vs. combination; p = 0.038, minocycline vs. combination) ( 4b). In addition, immunofluorescence staining was found to be more significant MBP expression in the spinal cord of the hBM-MSC or minocycline treated group compared to the PBS treated group (p = 0.048, PBS vs. hBM-MSC treatment; p = 0.007, PBS vs Minocycline treatment).

Compared with hBM-MSC or minocycline-treated group, the label intensity of MBP positive cells in the combination group was significantly increased (p = 0.005, hBM-MSC vs. combination; p = 0.003, minocycline vs. combination) (FIG. 4c).

These results were shown in Figures 4a, 4b and 4c, the spinal cord region stained LFB (top panel) and MBP (bottom panel) was measured to detect the demyelination range in EAE mice. Arrows indicate areas where demyelination occurred (Scale bar, 200 μm.) (FIG. 4A). Statistical analysis showed that co-administration significantly reduced demyelination compared to hBM-MSC or minocycline treatment (p <0.05) (FIG. 4B). Co-administration significantly protected the number of MBP positive cells compared to hBM-MSC or minocycline treatment (Columns, mean; bars, SE. * p <0.05, ** p <0.01, *** p <0.001 , one-way ANOVA with post-hoc Bonferroni corrections (FIG. 4C). The results represent three separate experiments. These results confirmed that the combination of hBM-MSC and minocycline reduced tissue damage and alleviated clinical symptoms of EAE.

Example 5

Effects of Co-administration on Neuroinflammatory and Neurodegenerative Diseases in the EAE Mouse Spinal Cord

Neuroinflammatory and neurodegenerative were evaluated with GFAP-, Iba-1- and NeuN positive cells.

In PBS mice, intense glial activity and increased number of cells stained intensely with GFAP and Iba-1 at the lesion site were evident. The present inventors confirmed that the number of glial cells and microglia was reduced in hBM-MSC or minocycline-treated mice compared to EAE mice treated with PBS.

In the co-administration case, only a small number of GFAP- and Iba-1 positive astrocytes and microglia were observed (see FIG. 5A).

GFAP: p <0.001, PBS vs. hBMMSCs treatment; p <0.001, PBS vs. Minocycline treatment; Iba-1: p = 0.002, PBS vs. hBM-MSC treatment; p = 0.001, PBS vs. It was shown as minocycline treatment. Therefore, statistical analysis of GFAP- and Iba-1 positive cells significantly reduced the fluorescence intensity of the label and the number of activated microglial cells for activated astrocytes by hBM-MSC or minocycline treatment compared to PBS treatment. It became.

GFAP: p <0.001, hBM-MSC vs. Combination administration; p <0.001, minocycline vs. Combination administration; Iba-1: p = 0.009, hBM-MSC vs. Combination administration; p = 0.024, minocycline vs. Co-administration appeared. Therefore, there was a significant decrease in the group administered in combination compared to the case of individual treatment (see FIGS. 5B and 5C).

Next, 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.

 Statistical analysis confirmed a significant increase in the number of NeuN positive cells in the spinal cord portion of hBM-MSC or minocycline treated mice compared to PBS treatment (p = 0.016, PBS vs. hBMMSCs treatment; p = 0.004, PBS vs. minocycline treatment). ). However, there was a significant increase in the group that was co-administered compared to hBM-MSC or minocycline treatment (p <0.001, hBM-MSC vs. co-administration; p <0.001, minocycline vs. co-administration) (see Figure 5d).

These results were shown in Figures 5a to 5d, the sections were labeled with GFAP (top panel), Iba-1 (middle panel), and NeuN (bottom panel), and the immune response, respectively, astrocytes, microglia And neurons were detected. In addition, the intensity of the GFAP immune response was expressed in PBS treated EAE mice and was reduced in hBM-MSC or minocycline individually treated EAE mice (FIG. 5A). However, few astrocytes responded to the co-administered group.

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 μm).

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).

Taken together, these results indicate that the combination of hBM-MSC and minocycline alleviates neurodegeneration in the CNS, reduces neuroinflammatory inflammation, and alleviates clinical symptoms in EAE.

<Example 6>

Effect of Th1 Cytokine and Th2 Cytokine Balance on Co-administered EAE Mice

In order to confirm that the expression of inflammatory cytokines is regulated, the inventors measured the expression of IFN-γ / TNF-α and IL-4 / IL-10 in the plasma of EAE mice by ELISA.

As a result, IFN-γ: p = 0.010, PBS vs. hBM-MSC treatment; p = 0.005, PBS vs. Minocycline treatment; TNF-α: p = 0.042, PBS vs. hBM-MSC treatment; p = 0.009, PBS vs. Minocycline treatment; IL-4: p = 0.004, PBS vs. hBM-MSC treatment; p = 0.001, PBS vs. Minocycline treatment; IL-10: p = 0.022, PBS vs. hBM-MSC treatment; p = 0.008, PBS vs. It is shown as minocycline treatment, and we found that the IL-4 / IL-10 Th2 cytokine was significantly reduced in the expression of IFN-γ / TNF-α Th1 cytokines in hBM-MSC or minocycline treated groups compared to the PBS treated group. It was confirmed that the expression of significantly increased (see Fig. 6a and 6b).

Also measured, IFN-γ: p = 0.006, hBM-MSC vs. Combination administration; p = 0.048, minocycline vs. Combination administration; TNF-α: p = 0.006, hBM-MSC vs. Combination administration; p = 0.009, minocycline vs. Combination administration; IL-4: p <0.001, hBMMSCs vs. Combination administration; p <0.001, minocycline vs. Combination administration; IL-10: p = 0.028, hBM-MSC vs. Combination administration; p = 0.043, minocycline vs. Co-administration, and the present inventors have shown a marked reduction in the expression of IFN-γ / TNF-α Th1 cytokines and IL-4 / IL- in the co-administered group compared to the respective hBM-MSC or minocycline-treated groups. It was confirmed that the expression of the 10 Th2 cytokine was significantly increased (see FIGS. 6A and 6B). The initial immune response was also at the periphery prior to the migration of immune cells into the CNS.

Plasma was isolated from 5 groups of EAE mice 40 days after immunization. Expression levels of cytokine proteins in plasma were quantified by ELISA. These results are shown in Figures 6a and 6b.

Statistical analysis showed a significant decrease in IFN-γ / TNF-α Th1 cytokines when treated with hBM-MSC or minocycline compared to PBS treatment, compared with the group treated with hBM-MSC or minocycline. Similar expression patterns were evident in Figure 6a.

Compared to the PBS treatment, 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.

We tested all therapeutic effects on the systemic immune microenvironment in an in vitro system, and the production of IFN-γ and IL-4 was evaluated by separating anti-MOG35-55 stimulated spleen from all EAE groups.

The analysis showed that IFN-γ was significantly reduced and IL-4 production was significantly increased in hBM-MSC or minocycline treated group compared to PBS treated group (IFN-γ: p = 0.006, PBS vs. hBM-MSC treatment; p <0.001, PBS vs. minocycline treatment; IL-4: p = 0.042, PBS vs. hBM-MSC treatment; p = 0.035, PBS vs. minocycline treatment).

The same pattern appears in the case of co-administration. However, it can be seen that the effect is significantly higher than the respective treatments (IFN-γ: p = 0.025, hBM-MSC vs. co-administration; p = 0.037, minocycline vs. co-administration; IL-4: p = 0.039, hBM -MSC vs. co-administration; p = 0.046, minocycline vs. co-administration) (see FIGS. 9A and 9B).

These results are shown in Figure 9, spleen cells (5 × 10 ⁵ / well) of EAE mice isolated 40 days after the immunization was stimulated by MOG35-55. The number of spleens producing MOG specific IFN-γ (FIG. 9A) / IL-4 (FIG. 9B) was determined via ELISPOT. Co-administration significantly reduced the frequency of Th1 compared to the hBM-MSC or minocycline administered individually, but increased the frequency of myelin protein specific Th2 (p <0.05). From these results, it can be seen that the co-administration of hBM-MSC and minocycline has a systemic effect on the effector of EAE.

It can be seen that the cytokine balance shifted significantly from Th1 cytokine to Th2 cytokine in commonly co-administered mice.

<Example 7>

Effect of Combination on Apoptosis in the Spinal Cord of EAE Mouse

To confirm that co-administration protects damaged spinal cord cells from apoptosis, apoptotic cell death was measured via TUNEL.

These results are shown in Figures 7a and 7b, apoptosis was measured by TUNEL staining, TUNEL positive cells are red, compared with DAPI is blue (Scale bar, 200 μm) (Fig. 7a) .

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).

Therefore, in the case of co-administration in EAE, 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.

These results are shown in Figure 10, 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)

Nuclei were counterstained with DAPI (blue) and arrows indicate positive cells. Scale bars in A, B, C, D = 200 μm, Scale bars in a, b, c, d = 100 μm.

These results indicate that co-administration in the spinal cord of EAE mice can reduce neuronal death.

<Example 8>

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.

Although individual treatment of hBM-MSC or low dose of minocycline has significant effects compared to PBS treatment in current EAE mice, co-administration protects EAE mice during the course of the disease, significantly alleviates the severity of the disease, Reduction, demyelination reduction, neurodegeneration and immunomodulatory strengthening function. Since the therapeutic combination of hBM-MSC and minocycline meets the criteria, their combination is reasonable in multiple sclerosis.

There are many possible mechanisms for the therapeutic effect of co-administration. The first beneficial effect of co-administration in the present invention is that it may be involved in the regulation of IFN-γ / TNF-α 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. On the other hand, Th2 cytokines have been known to be involved in alleviation and recovery.

In tissue-specific autoimmunity, cytokine balance is central to determining resistance and sensitivity. EAE susceptibility is thought to be related to the expression of the major preinflammatory Th1 cytokines, IFN-γ and TNF-α. On the other hand, Th2 cytokines such as IL-4 and IL-10 are anti-inflammatory cytokines that prevent or ameliorate disease. TNF-α / IFN-γ 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.

In the present invention, it was confirmed that the combination administration promotes the migration from Th1 to Th2 cytokines in EAE mice. Moreover, 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. The co-administered effect of hBM-MSC and minocycline goes beyond the synergistic effect of each dose.

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. In the present invention, 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.

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. In addition, it was confirmed in the present invention that 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.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Combination of bone marrow-derived mesenchymal stem cells (BM-MSC) with 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. In addition, the side effects of minocycline alone treatment can be minimized. Thus, a combination of hBM-MSC and minocycline can be used to treat multiple sclerosis.

Claims (8)

Bone marrow-derived mesenchymal stem cells (BM-MSCs); And a minocycline compound or a pharmaceutically acceptable salt of the compound as an active ingredient for the prevention or treatment of multiple sclerosis or encephalomyelitis. The method according to claim 1, The bone marrow-derived mesenchymal stem cells are pharmaceutical composition for the prevention or treatment of multiple sclerosis or encephalomyelitis, characterized in that derived from human bone marrow. The method according to claim 1, The bone marrow is an autologous bone marrow obtained from an individual having multiple sclerosis or encephalomyelitis (autologous bone marrow) characterized in that the pharmaceutical composition for the prevention or treatment of multiple sclerosis or encephalomyelitis. The method according to claim 1, The prevention or treatment of multiple sclerosis or encephalomyelitis may be performed by preventing demyelination of neurons, neuroprotective effects, reduction of tissue damage, prevention of apoptosis, or inhibition of inflammatory infiltration. Pharmaceutical composition for the prevention or treatment of multiple sclerosis or encephalomyelitis, characterized in that by. Bone marrow-derived mesenchymal stem cells (BM-MSCs) in plasma in vitro; And treating a minocycline compound or a mixture of pharmaceutically acceptable salts of the compound to reduce Th1 cytokine or increase Th2 cytokine. Bone marrow-derived mesenchymal stem cells (BM-MSCs); And a cell therapy for preventing or treating multiple sclerosis or encephalomyelitis comprising a minocycline compound or a pharmaceutically acceptable salt of the compound as an active ingredient. A method of preventing or treating multiple sclerosis or encephalomyelitis comprising administering to a subject a composition of any one of claims 1-4. A method of preventing or treating multiple sclerosis or encephalomyelitis comprising administering to a subject a cell therapy agent of claim 6.

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