阅读说明：本技术 肌萎缩侧索硬化症治疗用医药组合物 (Pharmaceutical composition for treating amyotrophic lateral sclerosis ) 是由 本望修 佐佐木祐典 佐佐木优子 冈真一 中崎公仁 前泽理惠 于 2020-03-13 设计创作，主要内容包括：本发明涉及用于治疗肌萎缩侧索硬化症的医药组合物。更详细而言,本发明涉及特征在于含有间充质干细胞、被静脉内施予的肌萎缩侧索硬化症治疗用医药组合物。(The present invention relates to a pharmaceutical composition for the treatment of amyotrophic lateral sclerosis. More specifically, the present invention relates to a pharmaceutical composition for treating amyotrophic lateral sclerosis, which is characterized by containing mesenchymal stem cells and being administered intravenously.)

1. A pharmaceutical composition for treating Amyotrophic Lateral Sclerosis (ALS), which contains mesenchymal stem cells and is to be administered intravenously.

2. The pharmaceutical composition of claim 1, which is administered more than 2 times.

3. The pharmaceutical composition of claim 1 or 2, which is administered 10 per 1 administration⁶More than one mesenchymal stem cell.

4. The pharmaceutical composition of any one of claims 1-3, wherein the mesenchymal stem cell is a bone marrow-or blood-derived mesenchymal stem cell.

5. The pharmaceutical composition of any one of claims 1-4, wherein the mesenchymal stem cell is a mesenchymal stem cell derived from bone marrow or blood of the patient.

6. The pharmaceutical composition of any one of claims 1-5, wherein the mesenchymal stem cells are CD24 negative.

Technical Field

The present invention relates to a pharmaceutical composition for the treatment of amyotrophic lateral sclerosis. More specifically, the present invention relates to a pharmaceutical composition for treating amyotrophic lateral sclerosis, which contains mesenchymal stem cells and is to be administered intravenously.

Background

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease with selectively impaired motor nerves, a severe disease with an unknown etiology that lacks radical therapy. ALS is roughly classified into familial ALS and sporadic ALS, and it has been reported that about 22% of patients with familial ALS, which account for about 5% of the total, have a mutation in the gene of superoxide dismutase (SOD 1). In addition to SOD1, TDP43, FUS/TLS, C9orf72 and the like have been reported as causative genes of ALS.

Disruption of the Blood Brain Barrier (BBB) and the Blood Spinal Cord Barrier (BSCB) is known as one of the common mechanisms in ALS patients and animal models of ALS. The destruction of BBB/BSCB is observed at an early stage of ALS onset, and is attracting attention as one of causes of ALS because it causes various neurological damages (non-patent documents 1 to 4). ALS is associated with various symptoms such as neuroinflammation and motor nerve loss, and therefore, effective treatment methods for these various symptoms are expected.

Mesenchymal Stem cells (Mesenchymal Stem cells: MSCs) are known to have neuroprotective and BBB repair effects. The inventors of the present application have reported that improvement of motor function and repair of damaged parts are confirmed by administering MSC intravenously to patients with cerebral infarction (patent documents 1 to 3). In addition, it has been reported that by intravenously administering MSC to a patient with spinal cord injury, functional recovery, promotion of axon regeneration, and reduction of injured sites were confirmed (patent documents 2 and 3).

MSCs are known to secrete various neurotrophic factors, and among them, the effect of protecting motor nerve cells has been confirmed against glial cell line-derived neurotrophic factor (GDNF) (non-patent document 5). The inventors of the present application confirmed that the amount of GDNF in the MSC-administered group was significantly higher than that in the control group by measuring the amount of GDNF contained in brain tissue (infarct zone) of rats in a cerebral infarction model by ELISA (non-patent document 6). Gothelf et al disclose a method for treating ALS by culturing MSCs in a differentiation medium containing bFGF and PDGF to induce differentiation into cells secreting neurotrophic factors, and injecting the cells under the subarachnoid space or into muscle of ALS patients (patent document 4). Lanza et al disclose a pharmaceutical composition for treating an undesirable immune response containing mesenchymal stromal cells derived from induction of differentiation of hemangioblasts, and although ALS is also described in the list of target diseases, no specific pharmacological effect has been shown (patent document 5).

Documents of the prior art

Patent document

Patent document 1: WO2009/034708

Patent document 2: WO2017/188457

Patent document 3: WO2018/034023

Patent document 4: WO2014/024183 (Japanese patent publication No. 2018-138044)

Patent document 5: WO2013/082543 (Japanese patent laid-open No. 2018-27954)

Non-patent document

Non-patent document 1: winkler et al 2014, Proc. Natl. Acad. Sci., Vol.111(11): ppE1035-E1042

Non-patent document 2: winkler et al, 2013, Acta neuropathology, Vol.125(1): pp111-120

Non-patent document 3: nicaise et al, 2009, Brain res, vol.1301: pp152-162

Non-patent document 4: sasaki et al 2015, Neuropathology Vol.35(6): pp518-528

Non-patent document 5: kosuge et al, 2009, Neurosci.letters, Vol.454(2): p165-169

Non-patent document 6: horita et al, 2006, J.Neurosci, Res.Vo.84(7): pp1495-1504

Disclosure of Invention

Problems to be solved by the invention

The present invention addresses the problem of providing a novel means for treating various symptoms of Amyotrophic Lateral Sclerosis (ALS).

Means for solving the problems

The present inventors have variously evaluated the natural course of symptoms from the viewpoint of motor function and histology using ALS model rats, and confirmed that reduction of symptom development can be achieved by intravenous administration of MSC in both moderate and severe stages. Further, the present inventors have studied a more efficient administration method and completed the present invention.

That is, the present invention relates to the following (1) to (6).

(1) A pharmaceutical composition for treating Amyotrophic Lateral Sclerosis (ALS), which contains mesenchymal stem cells and is to be administered intravenously.

(2) The pharmaceutical composition of (1), which is administered more than 2 times.

(3) The pharmaceutical composition of (1) or (2) which is administered 10 per 1 administration⁶More than one mesenchymal stem cell.

(4) The pharmaceutical composition according to any one of (1) to (3), wherein the mesenchymal stem cell is a mesenchymal stem cell derived from bone marrow or blood.

(5) The pharmaceutical composition according to any one of (1) to (4), wherein the mesenchymal stem cell is a mesenchymal stem cell derived from bone marrow or blood of the patient.

(6) The pharmaceutical composition of any one of (1) to (5), wherein the mesenchymal stem cell is negative for CD 24.

Effects of the invention

The pharmaceutical composition of the present invention is expected to prevent nerve damage by increasing the expression of neurotrophic factors while inhibiting the destruction of BBB/BSCB. The pharmaceutical composition of the present invention can inhibit the decrease in motor function and prolong the survival time regardless of the degree of progression of symptoms, and thus can be an effective treatment method for ALS.

Brief description of the drawings

FIG. 1: FIG. 1 shows ALS model rats (SOD1) by behavioral evaluation^G93AIntroduction of mutant genes into rats). The vertical axis is the BBB score and the horizontal axis is the number of days.

FIG. 2: figure 2 shows a tissue image (KB staining) of ALS model rats. Purple: nerve cells and glial cells (small cells); blue color: myelin sheath.

FIG. 3: figure 3 shows behavioral assessment of ALS model rats (moderate stage). (A) BBB score (solid line: MSC administration group, dotted line: vehicle (vehicle) administration group), (B) amount of change in BBB score (black: MSC administration group, white: vehicle administration group).

FIG. 4: figure 4 shows the evaluation of survival time of ALS model rats (moderate stage). (A) Kaplan-Meier curves (solid line: MSC-administered group, dotted line: vehicle-administered group), (B) survival after 40 days (black: MSC-administered group, white: vehicle-administered group).

FIG. 5: fig. 5 shows the evaluation of the number of motor nerve cells of the spinal cord of ALS model rats (moderate stage). Tissue images of anterior horn of spinal cord (lumbar cord) on day 14 after administration ((a) vehicle administration group, (B) MSC administration group, arrow being motor nerve cell), and (C) map of number of motor nerve cell (black: MSC administration group, white: vehicle administration group).

FIG. 6: FIG. 6 shows the evaluation of BBB/BSCB disruption in ALS model rats (moderate stage). Staining images of the cerebral cortex ((a) vehicle-administered group, (B) MSC-administered group), arrows are motor nerve cells); (C) the area of the leakage area of Evans blue (black: MSC administration group, white: vehicle administration group, leakage area of each part of the cerebral cortex, brain stem (medulla oblongata), cervical medulla, and lumbar medulla shown on the left).

FIG. 7: FIG. 7 shows Nrtn expression levels (fold change) obtained by quantitative RT-PCR (black: MSC-administered group, white: vehicle-administered group).

FIG. 8: fig. 8 shows behavioral evaluation of ALS model rats (severe stage) (solid line: MSC-administered group, dotted line: vehicle-administered group).

FIG. 9: fig. 9 shows the evaluation of survival time of ALS model rats (severe stage) (upper: MSC-administered group, lower: vehicle-administered group).

FIG. 10: figure 10 shows behavioral assessment based on multiple administrations of MSC in ALS model rats (moderate stage). (A) BBB score (solid line: MSC multiple administration group, dotted line: MSC single administration group, dotted line: vehicle administration group), (B) amount of change in BBB score (white: MSC multiple administration group, gray: MSC single administration group, black: vehicle administration group).

FIG. 11: fig. 11 shows the evaluation of long-term motor function based on MSC multiple administrations for ALS model rats (solid line: MSC multiple administration group, dotted line: MSC single administration group, dotted line: vehicle administration group).

FIG. 12: fig. 12 shows the evaluation of survival time based on MSC multiple administrations for ALS model rats (solid line: MSC multiple administration group, dotted line: MSC single administration group, dotted line: vehicle administration group).

FIG. 13: FIG. 13 shows the evaluation of MSC administration amounts of ALS model rats (moderate stage) (day 3, day 7, day 14: vehicle administration group, low dose MSC administration group (1.0X 10) all from the left of the figure⁵N-3), conventional dose MSC administration group (1.0 × 10)⁶N-4), high dose MSC administration group (1.0 × 10)⁷N-3)).

FIG. 14: figure 14 shows the evaluation of MSC administration method for ALS model rats (moderate stage) (day 3, day 7, day 14: vehicle intramedullary administration group, MSC intravenous administration group, all from the left of the figure).

Detailed Description

1. Amyotrophic lateral sclerosis

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease in which motor nerves are selectively impaired. ALS is broadly divided into familial ALS and sporadic ALS, with the majority being sporadic (95%). ALS is progressive, with symptoms not being alleviated after the disease, muscles throughout the body gradually fail to exercise, and eventually die due to respiratory failure. At present, the pathogenesis of ALS is unknown, and no radical treatment method exists.

Recently, nerve damage caused by disruption of the Blood Brain Barrier (BBB) and the Blood Spinal Cord Barrier (BSCB) has been attracting attention as one of the causes of ALS. Given that MSCs secrete various neurotrophic factors, the present inventors have identified that intravenous administration of MSCs results in increased GDNF expression at the site of disease. And it was confirmed that intravenous administration of MSC increased the expression of neural rank protein (Neurturin) (Nrtn), one of GDNF families, at the affected site (see examples below).

2. Mesenchymal stem cells

The "mesenchymal stem cell" used in the pharmaceutical composition of the present invention is a stem cell having a multi-differentiation ability and a self-replication ability, which is present in a trace amount in mesenchymal cells of a mesenchymal tissue, and is known to have a differentiation ability to differentiate into not only connective tissue cells such as osteocytes, chondrocytes, and adipocytes, but also nerve cells and cardiomyocytes.

The source of the mesenchymal stem cells may be cells induced by differentiation of ES cells or induced pluripotent stem cells (iPS cells), or may be cells obtained by strain culture, or may be cells isolated and expanded from a living body. In the case of an organism, bone marrow, peripheral blood, umbilical cord blood, fetal embryo, brain, and the like are exemplified, but mesenchymal stem cells derived from bone marrow or blood are preferable, and bone marrow mesenchymal stem cells are particularly preferable, and among them, human bone marrow mesenchymal stem cells are preferable. Mesenchymal stem cells derived from bone marrow have the following advantages: 1) significant effects can be expected, 2) the risk of side effects is low, 3) sufficient donor cell supply can be expected, 4) non-invasive treatment is possible, autografting is possible, therefore, 5) the risk of infectious diseases is low, 6) there is no fear of immune rejection, 7) there is no ethical problem, 8) it is easy to be accepted by society, 9) it is easy to be widely planted as general medical treatment; and so on. Furthermore, bone marrow transplantation therapy is already used in clinical sites, and safety has been confirmed. Furthermore, stem cells derived from bone marrow have high migration properties, and can be administered intravenously to a target damaged tissue in addition to local transplantation, and thus a therapeutic effect can be expected.

The cells may be derived from allogeneic or autologous cells, preferably mesenchymal stem cells derived from autologous cells (cells derived from the patient himself).

The mesenchymal stem cells used in the present invention are preferably in an undifferentiated state. This is because the cell growth rate in an undifferentiated state and the survival rate after introduction into a living body are high. The undifferentiated state can be confirmed by, for example, being negative for CD24 as a differentiation marker. The inventors of the present application have also developed a method for obtaining such cells, and the details thereof are described in WO 2009/002503.

In the aforementioned method developed by the present inventors, cells isolated from bone marrow fluid or the like are proliferated using a medium containing the same kind of serum (preferably autologous serum; human serum in a human pharmaceutical composition) and containing no or very low concentration of an anticoagulant (heparin or the like) without substantially contacting the anticoagulant (heparin or the like). By "free of or containing an anticoagulant at a very low concentration," it is meant that an effective amount of anticoagulant is not present. Specifically, for example, if heparin or its derivative is used, the effective amount as an anticoagulant is usually about 20 to 40 μ/mL, and in the method developed by the inventors, the amount in the sample collected from the living body is reduced to less than 5U/mL, preferably less than 2U/mL, and more preferably less than 0.2U/mL by minimizing the amount previously added to the blood collection tube used for sample collection, and the amount present in the culture medium during cell culture is reduced to less than 0.5U/mL, preferably less than 0.2U/mL, and more preferably less than 0.02U/mL relative to the volume of the culture medium (see WO 2009/034708).

The density of cells in the culture medium has an effect on the properties of the cells and the directionality of differentiation. In betweenIn the case of mesenchymal stem cells, if the cell density in the medium is more than 8,500 cells/cm²The nature of the cells will vary, and is therefore preferably at most 8,500 cells/cm²Subculture is carried out in the following manner, more preferably at a rate of up to 5,500 cells/cm²Subculture was performed at the above time points.

In the method developed by the present inventors, since a human serum-containing medium is used, the number of medium exchanges is preferably reduced as much as possible in consideration of the burden on the serum donor, and for example, the medium exchanges are performed at least 1 time per week, more preferably 1 to 2 times per week.

For the culture, subculture was repeated until the total number of cells became 10⁸More than one. The number of cells required may vary depending on the intended use, and it is considered that the number of mesenchymal stem cells required for transplantation for the treatment of ischemic brain diseases such as cerebral infarction is 10⁷More than one, 10 in the invention⁶More than one. According to the method developed by the inventors of the present application, 10 days can be obtained in about 12 days⁷Individual mesenchymal stem cells.

If necessary, the expanded mesenchymal stem cells may be preserved by a method such as cryopreservation (for example, in a deep freezing refrigerator at-152 ℃) until use. In cryopreservation, a medium containing serum (preferably human serum, more preferably autologous serum), dextran, and DMSO (a medium for mammalian cells such as RPMI) is used as a cryopreservation solution. For example, cells can be suspended in a cryopreservation solution containing 20.5mL of RPMI subjected to ordinary filter sterilization and 20.5mL of autologous serum collected from a patient, 5mL of dextran, and 5mL of DMSO, and cryopreserved at-150 ℃. For example, Cryoserv available from NIPRO corporation may be used as DMSO, and LOW-MOLECULAR DEXTRAN L INJECTION (LOW MOLECULAR DEXTRAN L INJECTION) available from Otsuka may be used as DEXTRAN.

The mesenchymal stem cell prepared as described above may be a) added with a cytokine to a culture comprising the mesenchymal stem cell, confirming that the mesenchymal stem cell expresses CX3CL 1; or b) confirming the expression of EGFR and/or ITGA4 by the mesenchymal stem cells, thereby confirming the quality and function of the mesenchymal stem cells.

The "inflammatory cytokine" used herein includes TNF- α, INF γ, IL-1, IL-6, IL-8, IL-12 and IL-18, and preferably includes TNF- α, INF γ and IL-6, and more preferably a mixture of TNF- α, INF γ and IL-6.

The foregoing method may further comprise: confirming the presence of any one or more selected from the group consisting of BDNF, VEGF, and HGF in the culture (without cytokine addition). In particular, it is important to confirm the presence of BDNF and/or VEGF, and most importantly, the presence of BDNF.

When CX3CL1 is expressed by mesenchymal stem cells by adding an inflammatory cytokine, excellent inflammatory regulation (immunoregulation) of the mesenchymal stem cells can be expected, and when EGFR and/or ITGA4 is expressed in 90% or more of the mesenchymal stem cells, excellent ability of the mesenchymal stem cells to accumulate in the damaged site can be expected. In addition, if any one of the trophic factors such as BDNF, VEGF, and HGF is present in the medium, it is expected that mesenchymal stem cells having a high neuroprotective effect are included, and the presence of BDNF and/or VEGF, particularly the presence of BDNF, can be an important indicator of MSCs having a high neuroprotective effect. Although mesenchymal stem cells secrete BDNF, VEGF and/or HGF even when unstimulated, confirmation of the secretion capacity allows evaluation of secretion from unstimulated cells and evaluation of secretion from cells stimulated by inflammatory cytokines.

In the case where the expression of CX3CL1, EGFR, ITGA4, BDNF, VEGF, and HGF is a cell surface protein such as EGFR and ITGA4, the expression at the protein level is preferably used as an index as compared with the gene level, and the measurement is preferably performed by Flow Cytometry (FCM) from the viewpoint of simplicity and sensitivity, and in the case where the expression is a secreted protein such as CX3CL1, BDNF, VEGF, and HGF, the bead measurement method is preferably used from the viewpoint of simplicity and sensitivity.

3. The pharmaceutical composition of the present invention

The pharmaceutical composition of the present invention is a pharmaceutical composition for treating ALS, which contains mesenchymal stem cells and is administered intravenously.

The higher the number of mesenchymal stem cells contained in the pharmaceutical composition of the present invention is, the more preferable, the smaller the amount of mesenchymal stem cells to be used in consideration of the administration time to a patient and the time required for culturing, the more practical. Therefore, in a preferred embodiment of the pharmaceutical composition of the present invention, the number of mesenchymal stem cells contained in 1 administration is 10⁶More than one, preferably 5 × 10⁶More than one, more preferably 10⁷More than one, more preferably 5 × 10⁷More than one, more preferably 10⁸More than one, more preferably 5 × 10⁸More than one.

The pharmaceutical composition of the present invention is an intravenous administration preparation. The intravenous administration preparation is in the form of an aqueous or nonaqueous isotonic sterile solution or suspension, and can be formulated into a suitable unit administration form by appropriately combining with, for example, a pharmacologically acceptable carrier or medium, and specifically, sterilized water, physiological saline, a culture medium (particularly, a culture medium for culturing mammalian cells such as RPMI), a physiological buffer such as PBS, a vegetable oil, an emulsifier, a suspending agent, a surfactant, a stabilizer, an excipient, a vehicle, an antiseptic, a binder, and the like.

Examples of the aqueous solution for injection include physiological saline, a medium, a physiological buffer such as PBS, an isotonic solution containing glucose and other auxiliary agents, for example, D-sorbitol, D-mannose, D-mannitol, sodium chloride, and the like, and may be used in combination with an appropriate cosolvent, for example, an alcohol (specifically, ethanol, a polyhydric alcohol, propylene glycol, polyethylene glycol), a nonionic surfactant (for example, polysorbate 80, HCO-50, and the like).

ALS to be administered as the pharmaceutical composition of the present invention is not limited to familial or sporadic conditions, and is not limited to mild symptoms or severe symptoms. In the case of mild ALS, improvement of symptoms and suppression of progression can be expected by administration of the pharmaceutical composition of the present invention, and in the case of moderate to severe ALS, suppression of progression can be expected.

The number of administration and the interval between administrations of the pharmaceutical composition of the present invention are not particularly limited. For example, 2 times or more, 3 times or more, 4 times or more, 5 times or more, as long as a therapeutic effect can be expected, administration may be repeated a plurality of times. After the first administration, the administration may be performed every time symptoms develop, or may be performed periodically. Thus, depending on the symptoms, the administration interval may be 1 month to ten years, for example, 1 month, 2 months, 3 months, 4 months, 6 months, 1 year, 2 years, 3 years to ten years. By repeated administration, the symptoms can be maintained in a favorable state, prolonging survival time.

The pharmaceutical composition of the present invention can be used in combination with known ALS therapeutic agents, for example, riluzole (riluzole), edaravone, penicillin, β lactam antibiotics, and the like.

The pharmaceutical composition of the present invention can inhibit the destruction of BBB/BSCB, promote the expression of neurotrophic factors, and inhibit nerve damage. The pharmaceutical composition of the present invention can suppress the decrease in motor function and prolong the survival time regardless of the degree of progression of symptoms.

Examples

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Reference example 1: preparation of mesenchymal Stem cells derived from rat bone marrow

The MSCs used in the following examples were prepared by the following steps according to the previous report.

The experiments were performed according to the animal experiment management regulations of Sapporus medical university. According to the previous report, bone marrow obtained from the femur of mature SD rat is diluted to 25ml with Dulbecco's Modified Eagle Medium (DMEM), and heat-inactivated 10% FBS, 2mM l-glutamine, 100U/ml penicillin, 0.1mg/ml streptomycin are added in 5% CO₂Incubate at 37 ℃ for 3 days under atmosphere (Kim S. et al, Brain Res.2006; 1123:27-33.Ukai R. et al, J.Neurotrauma.2007; 24: 508-. Culturing until confluent (confluent), peeling adherent cells with trypsin-EDTA, and culturing at 1 × 10⁴The density of each cell/ml was subcultured 3 times to obtain Mesenchymal Stem Cells (MSC).

Example 1: natural course of disease in ALS model rats

1. Behavioral assessment

Method

ALS model rats (SOD1 rats (SD Tg (SOD1G93A) L26H)) were purchased from Tastic Biosciences and subjected to behavioral assessment. It is known that SOD1 rats have SOD1 gene mutation (SOD1G93A), and the condition of human ALS is very well reproduced.

(https://www.taconic.com/rat-model/sod1-rat)。

Results

The onset is manifested as drooping of the tail, or abnormal gait of the hind limbs, which progresses rapidly (about 10 days) to hind limb paralysis. In the case of moderate (moderate stage: BBB score 15-11), rats typically show the earliest paralysis in a single hind limb. In the case of severe (severe stage: BBB score 10-8), although both legs are paralyzed, forelimbs can be used for ambulation. At the end stage (end stage), the rat no longer has a righting reflex (fig. 1).

2. Histological evaluation

Method

In order to histologically evaluate the natural course of disease in ALS model rats, the spinal cords of rats with 21, 11, and 0 BBB scores as hind limb motor function evaluation indices were stained for nerve cells and myelin sheath by Kluver-Barrera staining (hereinafter, KB staining), and changes in the number of motor nerve cells were observed. The BBB score is a method of evaluating the motor function of hind limbs by freely moving rats for 5 minutes in an open field (21 points for normal, 11 points for walking difficulty, and 0 point for walking disability, with the score decreasing with the worsening of symptoms).

Rats in the ALS model were deeply anesthetized with anesthetics (ketamine 100mg/kg, xylazine 20mg/kg) and fixed by perfusion with 0.1M phosphate buffer (4% PFA) containing PBS and 4% paraformaldehyde. The spinal cord was removed, dissected into C1, T6, L4 regions, and fixed in 4% PFA overnight. After fixation, paraffin embedding was performed, and a 10 μm thick section was cut out and attached to a slide glass. Then, KB staining was performed and photographs were taken with a fluorescence microscope (KEYENCE BZ 9000).

Results

The dye image is shown in fig. 2. With progression from Normal (Normal, BBB score of 21) to Moderate (model, BBB score of 11) symptoms, motor nerve cells (700 μm) in cervical (C1), thoracic (T6), and lumbar (L4) medulla²The above large cells) become small in number. In addition, although nerve cell residues were observed in the cervical and thoracic medullas of rats that became terminal symptoms (BBB score of 0), nerve cells in the lumbar medulla were almost completely eliminated.

Example 2: effect of intravenous administration of MSC in ALS model rats (study 1: intermediate stage)

1. Behavioral assessment

Method

ALS model rats with BBB score reduced to 15 were divided into 2 groups of MSC administration group (n ═ 6) and vehicle administration group (n ═ 13), in which MSC was intravenously administered from femoral vein to MSC (1.0 × 10)⁶One) dissolved in 1mL of vehicle (fresh DMEM). Vehicle administration group, 1mL of DMEM was administered intravenously. Cyclosporin a (10mg/kg) as an immunosuppressive agent was administered intraperitoneally daily for all rats from the day before MSC and vehicle administration to the end stage.

Behavioral assessment was performed using BBB scores, 2 times per week or more, and hind limb motor function was assessed by placing rats in open field for 5 minutes of free activity. The evaluator did not know to which treatment group the rat belongs.

Results

The reduction in BBB score was significantly suppressed in MSC administration group (fig. 3 (a)). In particular, in the evaluation on the administration day 3, improvement of symptoms was observed in the MSC-administered group. The amount of change in BBB score is shown. In the MSC-administered group, the BBB score varied by less than the vehicle-administered group (fig. 3 (B)).

2. Time to live

Method

Survival was evaluated 40 days after MSC or vehicle administration using a Kaplan-Meier curve for rats subjected to behavioral evaluation.

Results

After 40 days, more than half of the vehicle-administered groups died, while more than 80% of the MSC-administered groups survived (fig. 4).

3. Number of spinal motor nerve cells

Method

ALS model rats with BBB score reduced to 15 were divided into 2 groups of MSC-administered group (n ═ 3) and vehicle-administered group (n ═ 6), and MSC or vehicle was administered in the same manner as described above, and comparison was made on day 14 after administration with respect to motor nerve cells at anterior horn of spinal cord (lumbar cord). Cyclosporin a (10mg/kg) as an immunosuppressive agent was administered intraperitoneally daily for all rats from the day before administration of MSC and vehicle to the final evaluation day 14 days after administration. The motor nerve cells are defined as 700 μm in area²The above cells.

Results

Significantly more motor nerve cells were retained in the MSC-administered group compared to the vehicle-administered group (fig. 5).

4. Disruption of the blood brain barrier/blood brain spinal barrier (BBB/BSCB)

Method

ALS model rats with a BBB score reduced to 15 were divided into 2 groups of MSC-administered group (n ═ 4) and vehicle-administered group (n ═ 4), and MSC or vehicle was administered in the same manner as described above, and on the 14 th day after administration, 4% evans blue (EvB, Sigma, 4ml/kg) was administered to each rat from the tail vein in order to evaluate the destruction of the blood brain barrier/blood brain spinal cord barrier (BBB/BSCB). After 1 hour, deep anesthesia was applied with anesthetics (ketamine 100mg/kg, xylazine 20mg/kg) and perfusion fixation was performed with PBS and 4% PFA. After the brain and spinal cord were removed, the brain, brainstem (medulla oblongata), cervical marrow, and lumbar marrow were divided into 4 parts, each of which was embedded with an OCT compound, and frozen in acetone cooled to-80 ℃. 9 20 μm sections were cut out at 100 μm intervals using Cryostat and attached to a glass slide. Counterstaining was performed using DAPI, and VECTASHIELD(Vector Laboratories) was used to cover the coverslips. The sections were observed using a confocal microscope, and the area of evans blue that leaked out of the vessels was measured using Image J software (NIH). Ex/Em (405nm for DAPI, 488nm for FITC-lectin, 561nm for EvB; LSM780 ELYRA S.1system, Zeiss).

Results

In the cerebral cortex, brain stem (medulla oblongata), cervical marrow, and lumbar marrow, it was confirmed that the leakage of evans blue was significantly reduced in the MSC-administered group, and that the destruction of BBB/BSCB was suppressed or repaired (fig. 7).

Expression analysis of Nrtn (quantitative RT-PCR)

Method

ALS model rats with BBB score reduced to 15 were divided into 2 groups of MSC administration group (n ═ 4) and vehicle administration group (n ═ 4), MSC or vehicle was administered as described above, and 24 hours later, deep anesthesia was administered with anesthetic, and then spinal cord (lumbar spinal cord) was removed, and RNA was extracted using RNeasy Plus mini kit (QIAGEN, Venlo, The Netherlands). Quality of RNA RIN number was confirmed using Bioanalyzer RNA 6000nano kit (Agilent Technologies, Santa Clara, Calif., USA) and samples with RIN >8.0 were used only. Quantification was performed by qRT-PCR method using TaqMan (registered trademark) Universal Master Mix II with Uracil-N glycosylase (UNG) and TaqMan (registered trademark) Gene Expression assays (Gapdh, Rn 01775763-g 1; Nrtn), PRISM7500with 7500software v2.3(Thermo Fisher Scientific Inc.). Using Δ Δ Ct method, Gapdh as an internal control, the gene expression ratio of MSC-administered group to vehicle-administered group was compared (n ═ 4). The protocol for the thermal cycler is as follows.

After 2 minutes at 50 ℃ and 10 minutes at 95 ℃, 40 cycles of 95 ℃ for 15 seconds and 60 ℃ for 1 minute were carried out.

Results

In the MSC administration group, the expression level of Nrtn was significantly increased (fig. 7). Nrtn is a member of the GDNF family, whose mRNA expression was confirmed to be significantly increased in the MSC administration group. From this result, it is considered that MSC transplantation exerts a neurotrophic effect on motor nerve cells in the anterior horn of spinal cord, and histologically increases the number of remaining nerve cells, thereby exerting a therapeutic effect on neurological symptoms.

Example 3: effect of intravenous administration of MSC in ALS model rats (study 2: Severe phase)

Method

Presentation of BBB score reduced to 11 pointsALS model rats with severe motor impairment were divided into 2 groups, and MSC (1.0 × 10) was administered to the group (n ═ 6)⁶One) was dissolved in 1mL of vehicle (fresh DMEM) and administered intravenously from the femoral vein. In the vehicle administration group (n ═ 7), 1mL of vehicle (fresh DMEM) was intravenously administered, and motor function and survival time were evaluated. Cyclosporin a (10mg/kg) as an immunosuppressive agent was administered intraperitoneally daily for all rats from the day before MSC and vehicle administration to the end stage.

Results

For the decrease in motor function, it was significantly inhibited in MSC-administered group (n-6) compared to vehicle-administered group (n-7) (fig. 8), and survival time was also significantly prolonged (fig. 9).

Example 4: effect of multiple administration of MSC in ALS model rats

1. Behavioral assessment

Method

MSC (1.0X 10) in ALS model rats with a reduced BBB score of 15 and exhibiting moderate motor function impairment⁶One) were administered intravenously 1 time every 1 week. Motor function was evaluated for multiple administration group (n-9) based on BBB score, compared to single administration group (n-5) and vehicle administration group (n-6). Cyclosporin a (10mg/kg) as an immunosuppressive agent was administered intraperitoneally daily for all rats from the day before MSC and vehicle administration until the final evaluation day.

Results

In the multiple administration group of MSCs, improvement in motor function was seen not only in the 1 st administration, but also in the 2 nd administration (fig. 10). Both day 7 and day 14 of administration significantly inhibited motor function compared to the vehicle-administered group.

2. Long term exercise function

Method

The change in long-term motor function of the MSC multiple administration group (n ═ 7) was compared with the MSC single administration group (n ═ 6) and the vehicle administration group (n ═ 13). Cyclosporin a (10mg/kg) as an immunosuppressive agent was administered intraperitoneally daily for all rats from the day before MSC and vehicle administration to the end stage.

Results

Long-term motor function was maintained in the multiple administration group compared to the single administration group and the vehicle administration group (fig. 11).

3. Time to live

Method

Survival time of MSC multiple administration group (n ═ 7) was compared with MSC single administration group (n ═ 6) and vehicle administration group (n ═ 13).

Results

In the multiple-administration group, survival time was significantly prolonged compared to the single-administration group, the vehicle-administration group (fig. 12).

Example 5: differences in effect due to MSC administration in ALS model rats

Method

MSC administration was divided into low dose groups (1.0X 10)⁵N-3), conventional dose (1.0 × 10)⁶N-4), high dose (1.0 × 10)⁷N-3) of the 3 groups, ALS model rats with reduced BBB score to 15 were administered intravenously, and the amount of change in BBB score was compared with the vehicle-administered group (n-6).

Cyclosporin a (10mg/kg) as an immunosuppressive agent was administered intraperitoneally daily for all rats from the day before MSC and vehicle administration until the final evaluation day.

Results

In the conventional dose-administered group and the high dose-administered group, the decrease in motor function was significantly suppressed (fig. 13).

Example 6: differences in effect due to MSC administration method in ALS model rats

Method

The administration method of MSC was divided into 2 groups of intravenous administration (n ═ 3) and intramedullary administration (n ═ 3), and the change in hindlimb motor function was compared with the vehicle intramedullary administration group (n ═ 3). Intravenous administration group MSC 1.0X 10 dissolved in 1mL of DMEM was administered from the femoral vein⁶In each of the intramedullary administration groups, 1.0X 10 injection was performed using Hamilton syringe (Hamilton)⁵Individual MSC administrationTo the occipital cisterna magna. Cyclosporin a (10mg/kg) as an immunosuppressive agent was administered intraperitoneally daily for all rats from the day before MSC and vehicle administration until the final evaluation day.

Results

The decrease in motor function in the intravenously administered group was significantly suppressed in the evaluation on the 14 th day of administration. In addition, improvement of symptoms was confirmed in the intravenously administered group on the 3 rd day of administration (fig. 14).

Industrial applicability

The pharmaceutical composition of the present invention prevents the destruction of BBB/BSCB, promotes the secretion of neurotrophic factors, and inhibits nerve damage, thereby suppressing the development of ALS and prolonging the survival time. According to the present invention, ALS, which has heretofore lacked an effective therapeutic means, can be treated.

All publications, patents and patent applications cited in this specification are herein incorporated by reference as if fully set forth.