WO2020184729A1 - 筋萎縮性側索硬化症の治療用医薬組成物 - Google Patents
筋萎縮性側索硬化症の治療用医薬組成物 Download PDFInfo
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- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
Definitions
- the present invention relates to a pharmaceutical composition for treating amyotrophic lateral sclerosis. More specifically, it relates to a pharmaceutical composition comprising mesenchymal stem cells and administered intravenously for the treatment of amyotrophic lateral sclerosis.
- ALS Amyotrophic Lateral Sclerosis
- SOD1 superoxide dismutase
- BBB blood-brain barrier
- BSCB blood-brain barrier
- MSC Mesenchymal stem cells
- Non-Patent Document 5 glial cell line-derived neurotrophic factor (GDNF) has been confirmed to have an effect of protecting motor neurons.
- GDNF glial cell line-derived neurotrophic factor
- the inventors measured the amount of GDNF contained in the brain tissue (infarct region) of the cerebral infarction model rat by ELISA, and confirmed that the amount of GDNF was significantly higher in the MSC-administered group than in the control group. (Non-Patent Document 6). Gothelf et al.
- Treated ALS by culturing MSC in a differentiation medium containing bFGF or PDGF to induce differentiation into cells that secrete neurotrophic factors, and injecting this into submucosal or intramuscular injections of ALS patients.
- a differentiation medium containing bFGF or PDGF to induce differentiation into cells that secrete neurotrophic factors, and injecting this into submucosal or intramuscular injections of ALS patients.
- Patent Document 4 Lanza et al. Disclose pharmaceutical compositions for treating unnecessary immune responses, including mesenchymal stromal cells induced to differentiate from hemangioblasts, and ALS is also included in the list of target diseases. However, no specific pharmacological effect has been shown (Patent Document 5).
- An object of the present invention is to provide a new means for treating various symptoms of amyotrophic lateral sclerosis (ALS).
- ALS amyotrophic lateral sclerosis
- ALS model rats Using ALS model rats, the inventors evaluated the natural course of symptoms from a multifaceted viewpoint of motor function and histology, and progressed symptoms by intravenous administration of MSCs in both moderate and severe stages. It was confirmed that the reduction was possible. Furthermore, a more effective administration method was investigated, and the present invention was completed.
- the present invention relates to the following (1) to (6).
- the pharmaceutical composition according to (1) which is administered twice or more. (3) in a single dose, 10 6 or more mesenchymal stem cells are administered, (1) or a pharmaceutical composition according to (2).
- the pharmaceutical composition of the present invention can be expected to prevent neuropathy by suppressing the breakdown of BBB / BSCB and increasing the expression of neurotrophic factors. Since the pharmaceutical composition of the present invention can suppress the decline in motor function and prolong the survival time regardless of the degree of symptom progression, it can be an effective therapeutic method for ALS.
- FIG. 1 shows the natural course of ALS model rats (SOD1 G93A mutant gene-introduced rats) by behavioral evaluation.
- the vertical axis is the BBB score and the horizontal axis is the number of days.
- FIG. 2 shows a histological image (KB staining) of an ALS model rat. Purple: nerve cells and glial cells (small cells), blue: myelin sheath.
- FIG. 3 shows the behavioral evaluation of ALS model rats (Modelate-stage).
- A BBB score (solid line: MSC administration group, broken line: vehicle administration group),
- B) change amount of BBB score black: MSC administration group, white: vehicle administration group).
- FIG. 4 shows an evaluation of the survival time of ALS model rats (Moderate-stage).
- FIG. 5 shows an evaluation of the number of motor neurons in the spinal cord of ALS model rats (Moderate-stage). Tissue image of the anterior horn of the spinal cord (lumbar spinal cord) 14 days after administration ((A) vehicle administration group, (B) MSC administration group, arrow indicates motor neuron), and (C) motor neuron number graph (black) : MSC administration group, white: vehicle administration group).
- FIG. 6 shows an evaluation of BBB / BSCB failure in ALS model rats (Modelate-stage).
- FIG. 7 shows the Nrtn expression level (Fold change) by quantitative RT-PCR (black: MSC administration group, white: vehicle administration group).
- FIG. 8 shows the behavioral evaluation of ALS model rats (Severe-stage) (solid line: MSC administration group, broken line: vehicle administration group).
- FIG. 9 shows an evaluation of the survival time of ALS model rats (Severe-stage) (top: MSC administration group, bottom: vehicle administration group).
- FIG. 10 shows behavioral evaluation by multiple doses of MSCs in ALS model rats (Moderate-stage).
- A BBB score (solid line: multiple MSC administration group, broken line: single MSC administration group, dotted line: vehicle administration group),
- B change in BBB score (white: multiple MSC administration group, gray: single administration) MSC administration group, black: vehicle administration group).
- FIG. 11 shows a long-term evaluation of motor function by multiple administration of MSC in ALS model rats (Modelate-stage) (solid line: multiple MSC administration group, broken line: single MSC administration group, dotted line: vehicle administration group).
- FIG. 12 shows an evaluation of survival time by multiple administration of MSC in ALS model rats (Modelate-stage) (solid line: multiple MSC administration group, broken line: single MSC administration group, dotted line: vehicle administration group).
- FIG. 14 shows an evaluation of the MSC administration method in ALS model rats (Modelate-stage) (Day 3, Day 7, Day 14: from the left of the graph, vehicle intramedullary administration group, MSC intrathecal administration group, MSC vein). Oral administration group).
- Amyotrophic Lateral Sclerosis is a progressive neurodegenerative disease in which motor nerves are selectively impaired. ALS is broadly divided into familial ALS and sporadic ALS, most of which are sporadic (95%). ALS is progressive, and when affected, symptoms do not diminish, muscles throughout the body gradually become immobile, and eventually death occurs due to respiratory failure. At present, the cause of ALS is unknown and there is no cure for it.
- the "mesenchymal stem cells” used in the pharmaceutical composition of the present invention are stem cells having pluripotency and self-renewal ability that are present in trace amounts in stromal cells of mesenchymal tissue. , It is known that it not only differentiates into connective tissue cells such as bone cells, chondrocytes, and adipocytes, but also has the ability to differentiate into nerve cells and myocardial cells.
- mesenchymal stem cells As the source of mesenchymal stem cells, cells isolated and proliferated from living organisms, whether they are cells induced to differentiate from ES cells or induced pluripotent stem cells (iPS cells), or cells that have been established. It may be.
- iPS cells induced pluripotent stem cells
- bone marrow or blood-derived mesenchymal stem cells particularly bone marrow mesenchymal stem cells, particularly human bone marrow mesenchymal stem cells are preferable.
- Bone marrow-derived mesenchymal stem cells are 1) expected to have remarkable effects, 2) low risk of side effects, 3) expected supply of sufficient donor cells, and 4) non-invasive treatment and autologous transplantation. 5) Low risk of infection, 6) No concern about immune rejection, 7) No ethical problems, 8) Socially acceptable, 9) Widely used as general medical treatment There are advantages such as easy fixation. Furthermore, bone marrow transplantation therapy is a treatment already used in clinical practice, and its safety has been confirmed. In addition, bone marrow-derived stem cells are highly migratory, and can be expected to have a therapeutic effect by reaching the target injured tissue not only by local transplantation but also by intravenous administration.
- the cells may be derived from allogeneic cells or autologous cells, but mesenchymal stem cells derived from autologous cells (derived from the patient's own cells) are preferable.
- the mesenchymal stem cells used in the present invention are preferably in an undifferentiated state. This is because undifferentiated cells have a high proliferation rate and a high survival rate after introduction into a living body. Undifferentiated can be confirmed, for example, by negative CD24, which is a differentiation marker. The inventors have also developed a method for obtaining such cells, the details of which are described in WO2009 / 00253.
- cells separated from bone marrow fluid or the like under conditions that do not substantially come into contact with an anticoagulant are separated from allogeneic serum (preferably autologous serum; human in a human pharmaceutical composition).
- allogeneic serum preferably autologous serum; human in a human pharmaceutical composition.
- Proliferate using a medium that contains serum and does not contain anticoagulants (such as heparin) or contains very low concentrations.
- "No anticoagulant or very low concentration” means that it does not contain an effective amount of anticoagulant as an anticoagulant.
- the effective amount as an anticoagulant is usually about 20 to 40 ⁇ / mL, but in the method developed by the inventor, a blood collection tube for sampling in advance is used.
- the amount in the sample collected from the living body is less than 5 U / mL, preferably less than 2 U / mL, more preferably less than 0.2 U / mL, when culturing cells.
- the amount present in the medium is less than 0.5 U / mL, preferably less than 0.2 U / mL, more preferably less than 0.02 U / mL with respect to the volume of the medium (see WO2009 / 034708).
- the density of cells in the medium affects the nature of the cells and the direction of differentiation. In the case of mesenchymal stem cells, if the cell density in the medium exceeds 8,500 cells / cm 2 , the cell properties will change, so subculture at a maximum of 8,500 cells / cm 2 or less. Is preferable, and more preferably, subculture is performed when the number of cells reaches 5,500 / cm 2 or more.
- the method developed by the inventors uses a human serum-containing medium, it is desirable to change the medium as few times as possible in consideration of the burden on the serum donor, for example, at least once a week, more preferably weekly. Change the medium once or twice.
- Culturing is carried out repeatedly subcultured until the total number of cells is 10 8 or more.
- the number of cells needed may vary depending on the intended use, for example, the number of mesenchymal stem cells required for transplantation for the treatment of ischemic brain diseases such as cerebral infarction 10 7 or more It is believed that 10 6 or more in the present invention. According to the method we have developed, it can be in about 12 days to obtain a 10 7 mesenchymal stem cells.
- the proliferated mesenchymal stem cells may be stored by a method such as cryopreservation (for example, in a deep freezer at ⁇ 152 ° C.) until used.
- a medium containing serum preferably human serum, more preferably autologous serum
- dextran DMSO (medium for mammalian cells such as RPMI)
- RPMI medium for mammalian cells
- cells can be cryopreserved at ⁇ 150 ° C. in a cryopreservation solution containing 20.5 mL of normal filtration sterilized RPMI and 20.5 mL of autologous serum collected from a patient, 5 mL of dextran, and 5 mL of DMSO.
- cryoserve and dextran manufactured by Nipro Co., Ltd. can use low-molecular-weight dextran L injection manufactured by Otsuka Pharmaceutical, but the DMSO is not limited thereto.
- the cytokine was added to the culture containing the mesenchymal stem cells, and it was confirmed that the mesenchymal stem cells expressed CX3CL1, or b) the interval.
- the mesenchymal stem cells express EGFR and / or ITGA4, the quality and function of the mesenchymal stem cells may be confirmed.
- Examples of the "inflammatory cytokine” used include TNF- ⁇ , INF ⁇ , IL-1, IL-6, IL-8, IL-12, IL-18, among which TNF- ⁇ , INF- ⁇ , and It preferably contains IL-6, and more preferably a mixture of TNF- ⁇ , INF ⁇ , and IL-6.
- the method may further include confirming the presence of any one or more selected from BDNF, VEGF, and HGF in the (cytokine-free) culture.
- BDNF BDNF
- VEGF vascular endothelial growth factor
- HGF vascular endothelial growth factor
- the mesenchymal stem cells express CX3CL1 by the addition of inflammatory cytokines, the mesenchymal stem cells can be expected to have an excellent inflammatory regulating action (immunity regulating action), and 90% or more of the mesenchymal stem cells have EGFR and / or.
- ITGA4 is expressed, the mesenchymal stem cells can be expected to have excellent ability to accumulate at the injured site.
- any of the nutritional factors such as BDNF, VEGF, and HGF is present in the medium, it can be expected to contain mesenchymal stem cells having a high neuroprotective effect, and among them, the presence of BDNF and / or VEGF, especially BDNF.
- the presence of may be an important indicator of MSC with high neuroprotective activity.
- Mesenchymal stem cells secrete BDNF, VEGF and / or HGF even when unstimulated, but confirmation of secretory capacity may be evaluated for secretion from unstimulated cells or from cells after inflammatory cytokine stimulation. Secretion may be assessed.
- CX3CL1, EGFR, ITGA4, BDNF, VEGF, and HGF is preferably expressed at the protein level rather than at the gene level, and in the case of cell surface proteins such as EGFR and ITGA4, it is simple and sensitive. From the point of view, it is preferable to measure using flow cytometry (FCM), and in the case of secretory proteins such as CX3CL1, BDNF, VEGF and HGF, it is preferable to use a bead assay from the viewpoint of simplicity and sensitivity.
- FCM flow cytometry
- the pharmaceutical composition of the present invention is a pharmaceutical composition for treating ALS, which contains mesenchymal stem cells and is characterized by being administered intravenously.
- the number of cells of mesenchymal stem cells contained in a single dose is 10 6 or more, preferably 5 ⁇ 10 6 or more, more preferably 10 7 or more , more preferably 5 ⁇ 10 7 or more, more preferably 10 8 or more, more preferably 5 ⁇ 10 8 or more.
- the pharmaceutical composition of the present invention is an intravenously administered preparation.
- Intravenous formulations are in the form of aqueous or non-aqueous isotonic sterile solutions or suspensions, eg, pharmacologically acceptable carriers or media, specifically sterile water, saline, media.
- physiological buffers such as PBS, vegetable oils, emulsifiers, suspensions, surfactants, stabilizers, excipients, vehicles, preservatives, binders Etc. are appropriately combined to formulate an appropriate unit administration form.
- aqueous solution for injection examples include physiological saline, medium, physiological buffer solution such as PBS, isotonic solution containing glucose and other auxiliary agents, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride and the like.
- physiological saline medium
- physiological buffer solution such as PBS
- isotonic solution containing glucose and other auxiliary agents such as D-sorbitol, D-mannose, D-mannitol, sodium chloride and the like.
- an appropriate solubilizing agent such as alcohol, specifically ethanol, polyalcohol, propylene glycol, polyethylene glycol or a nonionic surfactant such as polysorbitol 80, HCO-50 or the like.
- the ALS to which the pharmaceutical composition of the present invention is administered may be mild or severe regardless of whether it is familial or sporadic.
- administration of the pharmaceutical composition of the present invention can be expected to improve symptoms and suppress progression, and in the case of moderate to severe ALS, suppression of progression can be expected.
- the number of administrations and the administration interval of the pharmaceutical composition of the present invention are not particularly limited. For example, it can be repeatedly administered 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. After the initial administration, it may be administered every time the symptoms progress, or it may be administered regularly. Therefore, the administration interval may be 1 month to 10 years, for example, 1 month, 2 months, 3 months, 4 months, 6 months, 1 year, 2 years, 3 years to 10 years, depending on the symptoms. .. By repeating the administration, the symptoms can be kept in good condition and the survival time can be extended.
- the pharmaceutical composition of the present invention can also be used in combination with known ALS therapeutic agents such as lithozole, edaravone, penicillin, and ⁇ -lactam antibiotics.
- known ALS therapeutic agents such as lithozole, edaravone, penicillin, and ⁇ -lactam antibiotics.
- the pharmaceutical composition of the present invention can suppress the disruption of BBB / BSCB, promote the expression of neurotrophic factors, and suppress neuropathy.
- the pharmaceutical composition of the present invention can suppress a decrease in motor function and prolong the survival time regardless of the degree of symptom progression.
- Reference Example 1 Preparation of rat bone marrow-derived mesenchymal stem cells
- the MSCs used in the following examples were prepared according to the following procedure according to the previous report. The experiment was carried out in accordance with the animal experiment management regulations of Sapporo Medical University. According to previously reported, bone marrow obtained from the femur of mature SD rats was diluted to 25 ml with Dalveco's Modified Eagle's Medium (DMEM) and heat-inactivated 10% FBS, 2 mM 1-glutamine, 100 U / ml penicillin, 0.1 mg / ml streptomycin was added and incubated in a 5% CO 2 atmosphere at 37 ° C. for 3 days (Kim S. et al., Brain Res.
- DMEM Dalveco's Modified Eagle's Medium
- Example 1 Natural course of ALS model rat 1. Behavioral Evaluation Method ALS model rats (SOD1 rats (SD Tg (SOD1G93A) L26H)) were purchased from Taconic Bioscience and behavioral evaluation was performed. SOD1 rats have a SOD1 gene mutation (SOD1G93A) and are known to reproduce the pathology of human ALS very well (https://www.taconic.com/rat-model/sodo1-rat).
- the spinal cords of rats with BBB scores of 21, 11, and 0, which are indicators of hind limb motor function evaluation were stained with Klover-Barrera ( The nerve cells and the spinal cord sheath were stained by KB staining), and changes in the number of motor neurons were observed.
- the BBB score is a method of evaluating the motor function of the hind limbs by putting a rat in an open field and moving it freely for 5 minutes (normal 21 points, the score drops due to worsening symptoms, walking difficulty at 11 points, 0 points is the worst. Cannot walk).
- ALS model rats were deeply anesthetized with an anesthetic (ketamine 100 mg / kg, xylazine 20 mg / kg) and perfused and fixed with 0.1 M phosphate buffer (4% PFA) containing PBS and 4% paraformaldehyde.
- the spinal cord was removed, cut into C1, T6, and L4 regions, respectively, and fixed overnight with 4% PFA. After fixing, paraffin embedding was performed, and a section having a thickness of 10 ⁇ m was cut out and attached to a slide glass. Then, it was stained with KB and photographed with a fluorescence microscope (KEYENCE BZ9000).
- Results Fig. 2 shows a stained image.
- motor neurons 700 ⁇ m 2 or more
- C1 cervical spinal cord
- T6 thoracic spinal cord
- L4 lumbar spinal cord
- the number of cells has decreased.
- residual nerve cells were confirmed in the cervical spinal cord and thoracic spinal cord of rats with terminal illness (BBB score 0), but nerve cells were almost completely lost in the lumbar spinal cord.
- Example 2 Effect of intravenous MSC administration in ALS model rats (Study 1: Moderate-stage) 1.
- ) was dissolved in (1.0 ⁇ 10 6 ) of MSCs, and the solution was intravenously administered from the femoral vein. 1 mL of DMEM was intravenously administered to the vehicle-administered group.
- the immunosuppressant cyclosporin A (10 mg / kg) was intraperitoneally administered to all rats daily from the day before MSC and vehicle administration until the End-stage. Behavioral evaluation was performed using the BBB score, and rats were placed in an open field at least twice a week and allowed to move freely for 5 minutes to evaluate the motor function of the hind limbs. The evaluator did not know which treatment group the rat belonged to.
- administer MSC or vehicle in the same manner as above, and on the 14th day after administration, 4% Evans blue (EvB) was given to each rat to evaluate the breakdown of the blood-brain barrier / blood-brain barrier (BBB / BSCB).
- EvB Evans blue
- the patient was deeply anesthetized with an anesthetic (ketamine 100 mg / kg, xylazine 20 mg / kg) and perfused and fixed with PBS and 4% PFA.
- anesthetic ketamine 100 mg / kg, xylazine 20 mg / kg
- PBS 4% PFA.
- the brain, brain stem (medulla oblongata), cervical cord, and lumbar spinal cord were divided into four parts, each of which was embedded in an OCT compound and frozen in acetone cooled to -80 ° C.
- Nine 20 ⁇ m sections were cut out at 100 ⁇ m intervals using Cryostat and attached to a slide glass. Counterstaining was performed with DAPI, and cover slides were applied using VECTASHIELD (Vector Laboratories).
- Nrtn quantitative RT-PCR
- Quantification was performed by the qRT-PCR method, and TaqMan (registered trademark) Universal Master Mix II with Uracil-N glycosylase (UNG) and TaqMan (registered trademark) Gene Expression assay (GapNIs7), Gene Expression assay, (Thermo Fisher Scientific Inc.) was used.
- the thermal cycler protocol is as follows. After 2 minutes at 50 ° C and 10 minutes at 95 ° C, 40 cycles of 15 seconds at 95 ° C and 1 minute at 60 ° C.
- Nrtn is a member of the GDNF family, and it was confirmed that its mRNA expression was significantly increased in the MSC-administered group. From this result, it is considered that MSC transplantation exerts a neurotrophic effect on the motor neurons of the anterior horn of the spinal cord, and histologically increases the number of residual neurons, which leads to a therapeutic effect on neurological symptoms.
- Example 4 Effect of multiple administration of MSCs in ALS model rats 1.
- ALS model rats exhibited a degree of motor dysfunction in the action evaluation methods BBB score fell to 15 points, once MSC to (1.0 ⁇ 10 6 cells) every other week, in multiple intravenous Administration was performed.
- the immunosuppressant cyclosporin A (10 mg / kg) was intraperitoneally administered to all rats daily from the day before MSC and vehicle administration to the final evaluation day.
- the immunosuppressant cyclosporin A (10 mg / kg) was intraperitoneally administered to all rats daily from the day before MSC and vehicle administration to the final evaluation day.
- Example 6 Difference in effect depending on MSC administration method in ALS model rats
- was compared with the vehicle intrathecal administration group (n 3).
- 1.0 ⁇ 10 6 MSCs dissolved in 1 mL DMEM were administered from the femoral vein
- 1.0 ⁇ 10 5 MSCs were administered using a Hamilton syringe (HAMILTON). It was administered into a large tank.
- the immunosuppressant cyclosporin A (10 mg / kg) was intraperitoneally administered to all rats daily from the day before MSC and vehicle administration to the final evaluation day.
- the pharmaceutical composition of the present invention can suppress the progression of ALS and prolong the survival period by preventing the breakdown of BBB / BSCB, promoting the secretion of neurotrophic factors, and suppressing neuropathy. According to the present invention, it becomes possible to treat ALS for which there has been no effective therapeutic means so far.
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Abstract
Description
本明細書は、本願の優先権の基礎である特願2019−046572(2019年3月14日出願)の明細書に記載された内容を包含する。
技術分野
本発明は、筋萎縮性側索硬化症を治療するための医薬組成物に関する。より詳細には、間葉系幹細胞を含み、静脈内投与される、筋萎縮性側索硬化症の治療用医薬組成物に関する。
(1)筋萎縮性側索硬化症(ALS)を治療するための医薬組成物であって、間葉系幹細胞を含み、静脈内投与される医薬組成物。
(2)2回以上投与される、(1)に記載の医薬組成物。
(3)1回の投与で、106個以上の間葉系幹細胞が投与される、(1)又は(2)に記載の医薬組成物。
(4)前記間葉系幹細胞が、骨髄又は血液由来の間葉系幹細胞である、(1)~(3)のいずれかに記載の医薬組成物。
(5)前記間葉系幹細胞が、前記患者の骨髄又は血液由来の間葉系幹細胞である、(1)~(4)のいずれかに記載の医薬組成物。
(6)前記間葉系幹細胞が、CD24陰性である、(1)~(5)のいずれかに記載の医薬組成物。
筋萎縮性側索硬化症(Amyotrophic Lateral Sclerosis:ALS)は、運動神経が選択的に障害される進行性の神経変性疾患である。ALSは家族性ALSと孤発性ALSに大別されるが、大部分は孤発性(95%)である。ALSは進行性であり、罹患すると症状が軽くなることはなく、次第に全身の筋肉が動かなくなり、最終的には呼吸不全により死に至る。現在のところ、ALSの発症原因は不明であり、根本的な治療法も存在しない。
本発明の医薬組成物で使用される「間葉系幹細胞」とは、間葉系組織の間質細胞の中に微量に存在する多分化能及び自己複製能を有する幹細胞であり、骨細胞、軟骨細胞、脂肪細胞などの結合組織細胞に分化するだけでなく、神経細胞や心筋細胞への分化能を有することが知られている。
本発明の医薬組成物は、ALSを治療するための医薬組成物であって、間葉系幹細胞を含み、静脈内投与されることを特徴とする。
以下の実施例で使用するMSCは、既報にしたがい、以下の手順で調製した。
実験は、札幌医科大学の動物実験管理規定にしたがって実施した。既報に従い、成熟SDラットの大腿骨から得た骨髄をダルベッコの改変イーグル培地(DMEM)で25mlに希釈し、加熱不活化した10%FBS,2mM 1−グルタミン、100U/mlペニシリン,0.1mg/mlストレプトマイシンを添加し、5%CO2雰囲気下37℃で3日間インキュベートした(Kim S.et al.,Brain Res.2006;1123:27−33.Ukai R.et al.,J.Neurotrauma.2007;24:508−520.)。コンフルエントになるまで培養し、接着細胞をトリプシン−EDTAで剥離し、1x104個/mlの密度で3回継代培養して間葉系幹細胞(MSC)を得た。
1.行動評価
方法
ALSモデルラット(SOD1ラット(SD Tg(SOD1G93A)L26H))は、タコニックバイオサイエンスより購入し、行動評価を行った。SOD1ラットは、SOD1遺伝子変異(SOD1G93A)を有し、ヒトALSの病態を非常によく再現することが知られている
(https://www.taconic.com/rat−model/sod1−rat)。
発症はテールダウン、又は後肢異常歩行として現れ、急速に(約10日間)後肢麻痺へと進行した。中等度(Moderate−ステージ:BBBスコア15~11)では、ラットは典型的には片肢に最初の麻痺が現れた。重度(Severe−ステージ:BBBスコア10~8)では、両足が麻痺するが、前肢を使って歩くことができた。末期(End−ステージ)では、ラットは立ち直り反射ができなくなった(図1)。
方法
ALSモデルラットの自然経過を組織学的に評価するため、後肢運動機能評価の指標であるBBBスコアが21点、11点、0点のラットの脊髄を、Kluver−Barrera染色(以下KB染色)により神経細胞と髄鞘を染色し、運動神経細胞数の変化を観察した。BBBスコアはオープンフィールドにラットを入れて5分間自由に動き回らせ、後肢の運動機能を評価する方法である(正常21点、症状悪化により点数が下がり11点で歩行困難、0点がもっとも悪く歩行不可)。
図2に染色像を示す。Normal(BBBスコア21)から、Moderate(BBBスコア11)へと症状が進行すると頚髄(C1)、胸髄(T6)、腰髄(L4)のいずれにおいても運動神経細胞(700μm2以上の大きな細胞)の数が少なくなった。さらに末期症状(BBBスコア0)になったラットの頚髄、胸髄には神経細胞の残存が確認できたが、腰髄では神経細胞がほぼ完全に失われていた。
1.行動評価
方法
BBBスコアが15点に低下したALSモデルラットを、MSC投与群(n=6)とビヒクル投与群(n=13)の2群に分け、MSC投与群には1mLのビヒクル(fresh DMEM)にMSCを(1.0×106個)溶解させた溶液を、経静脈的に大腿静脈から投与した。ビヒクル投与群には1mLのDMEMを経静脈的に投与した。免疫抑制剤であるシクロスポリンA(10mg/kg)を全てのラットに対してMSC及びビヒクル投与の前日からEnd−ステージになるまで毎日腹腔内投与した。
行動評価はBBBスコアを用いて行い、週に2回以上、オープンフィールドにラットを入れて5分間自由に動き回らせ後肢の運動機能を評価した。評価者はラットがどの治療群に属するか分からないようにした。
BBBスコアの低下はMSC投与群で有意に抑制された(図3(A))。特に投与3日目の評価ではMSC投与群で症状の改善が観察された。BBBスコアの変化量を示す。BBBスコアの変化量は、MSC投与群ではビヒクル投与群よりも小さかった(図3(B))。
方法
行動評価を実施したラットについて、MSCあるいはビヒクル投与後40日間の生存率をカプランマイヤー曲線を用いて評価した。
40日後にビヒクル投与群では半数以上が死亡したが、MSC投与群では80%以上が生き残った(図4)。
方法
BBBスコアが15点に低下したALSモデルラットを、MSC投与群(n=3)とビヒクル投与群(n=6)の2群に分け、上記と同様にMSCあるいはビヒクルを投与し、投与後14日目に脊髄(腰髄)前角運動神経細胞を比較した。免疫抑制剤のシクロスポリンA(10mg/kg)を全てのラットに対してMSC及びビヒクル投与の前日から投与14日後の最終評価日まで毎日腹腔内投与した。運動神経細胞の定義は面積が700μm2以上の細胞とした。
MSC投与群はビヒクル投与群に比較して有意に多くの運動神経細胞が保存されていた(図5)。
方法
BBBスコアが15点に低下したALSモデルラットを、MSC投与群(n=4)とビヒクル投与群(n=4)の2群に分け、上記と同様にMSCあるいはビヒクルを投与し、投与後14日目に、血液脳関門/血液脳脊髄関門(BBB/BSCB)の破綻を評価するため、それぞれのラットに4%エバンスブルー(EvB,Sigma,4ml/kg)を尾静脈から投与した。1時間後に麻酔薬(ケタミン100mg/kg、キシラジン20mg/kg)で深く麻酔をかけて、PBS及び4%PFAで灌流固定した。脳及び脊髄を取り出した後、脳、脳幹(延髄)、頸髄、腰髄の4部位に分けてそれぞれOCT compoundで包埋し−80℃に冷却したアセトン中で凍結させた。Cryostatを用いて20μm切片を100μm間隔で9枚切り出しスライドガラスに貼り付けた。DAPIで対比染色を行いVECTASHIELD(Vector Laboratories)を用いてカバースライドをかけた。切片は共焦点顕微鏡を用いて観察し、血管外に漏出したエバンスブルーの面積をImage Jソフトウェア(NIH)を用いて測定した。Ex/Em(405nm for DAPI,488nm for FITC−lectin,and 561nm for EvB;LSM780 ELYRA S.1 system,Zeiss)。
脳皮質、脳幹(延髄)、頚髄、腰髄いずれにおいても、MSC投与群では有意にエバンスブルーの漏出が少ないことが確認され、BBB/BSCBの破綻が抑制又は修復されていることが確認できた(図7)。
方法
BBBスコアが15点に低下したALSモデルラットを、MSC投与群(n=4)とビヒクル投与群(n=4)の2群に分け、上記と同様にMSCあるいはビヒクルを投与し、24時間後に麻酔薬で深く麻酔をかけた後、脊髄(腰髄)を取り出し、RNeasy Plus mini kit(QIAGEN,Venlo,The Netherlands)を用いてRNAを抽出した。RNAの品質はBioanalyzer RNA 6000nano kit(Agilent Technologies,Santa Clara,CA,USA)でRIN numberを確認し、RIN>8.0のサンプルのみを用いた。定量はqRT−PCR法で行い、TaqMan(登録商標)Universal Master Mix II with Uracil−N glycosylase(UNG)及びTaqMan(登録商標)Gene Expression assays(Gapdh,Rn01775763_g1;Nrtn)、PRISM7500 with 7500 software v2.3(Thermo Fisher Scientific Inc.)を使用した。ΔΔCt法を用いて、内因性コントロールはGapdhとし、ビヒクル投与群に対するMSC投与群の遺伝子発現比を比較した(n=4)。サーマルサイクラーのプロトコルは以下の通り。
50℃2分、95℃10分の後、95℃15秒、60℃1分を40サイクル。
MSC投与群で有意にNrtnの発現量が高かった(図7)。NrtnはGDNFファミリーの1つであり、そのmRNA発現が有意にMSC投与群で増加することが確認された。この結果から、MSC移植により、脊髄前角の運動神経細胞に対する神経栄養作用が発揮され、組織学的には残存神経細胞数が多くなり、それにより神経症状での治療効果に結びついたと考えられる。
方法
BBB scoreが11点に低下した重度運動機能障害を呈したALSモデルラットを2群に分け、MSC投与群(n=6)には1mLのビヒクル(fresh DMEM)にMSC(1.0×106個)を溶解させ、経静脈的に大腿静脈から投与した。ビヒクル投与群(n=7)には1mLのビヒクル(fresh DMEM)を経静脈的に投与し、運動機能及び生存期間を評価した。免疫抑制剤のシクロスポリンA(10mg/kg)を全てのラットに対してMSC及びビヒクル投与の前日からEnd−stageになるまで毎日腹腔内投与した。
運動機能の低下はビヒクル投与群(n=7)に比べてMSC投与群(n=6)で有意に抑制され(図8)、生存期間も有意に延長された(図9)。
1.行動評価
方法
BBBスコアが15点に低下した中程度の運動機能障害を呈したALSモデルラットに対して、MSC(1.0×106個)を1週間おきに1回ずつ、複数回静脈内投与を行なった。複数回投与群(n=9)の運動機能をBBBスコアで評価し、単回投与群(n=5)及び、ビヒクル投与群(n=6)と比較した。免疫抑制剤のシクロスポリンA(10mg/kg)を全てのラットに対してMSC及びビヒクル投与の前日から最終評価日まで毎日腹腔内投与した。
MSCの複数回投与群では1回目だけでなく2回目の投与でも運動機能の改善が見られた(図10)。投与7日目、14日目いずれもビヒクル投与群に比べて有意に運動機能を抑制した。
方法
MSC複数回投与群(n=7)の長期的な運動機能の変化をMSC単回投与群(n=6)及びビヒクル投与群(n=13)と比較した。免疫抑制剤のシクロスポリンA(10mg/kg)を全てのラットに対してMSC及びビヒクル投与の前日からEnd−ステージになるまで毎日腹腔内投与した。
複数回投与群では単回投与群、ビヒクル投与群よりも長期間運動機能を維持することができた(図11)。
方法
MSC複数回投与群(n=7)の生存期間をMSC単回投与群(n=6)及びビヒクル投与群(n=13)と比較した。
複数回投与群では単回投与群、ビヒクル投与群よりも有意に生存期間が延長された(図12)。
方法
MSC投与量を低用量群(1.0×105個,n=3)、通常量(1.0×106個,n=4)、高用量(1.0×107個,n=3)の3群に分け、BBBスコアが15点に低下したALSモデルラットに経静脈内投与し、BBBスコアの変化量をビヒクル投与群(n=6)と比較した。
免疫抑制剤のシクロスポリンA(10mg/kg)を全てのラットに対してMSC及びビヒクル投与の前日から最終評価日まで毎日腹腔内投与した。
通常量及び高用量投与群で有意に運動機能低下が抑制された(図13)。
方法
MSCの投与方法を静脈内投与(n=3)と髄腔内投与(n=3)の2群に分け、後肢運動機能の変化をビヒクル髄腔内投与群(n=3)と比較した。静脈内投与群は1mLのDMEMに溶解したMSC 1.0×106個を大腿静脈から投与し、髄腔内投与群は1.0×105個のMSCをハミルトンシリンジ(HAMILTON)を用いて大槽内に投与した。免疫抑制剤のシクロスポリンA(10mg/kg)を全てのラットに対してMSC及びビヒクル投与の前日から最終評価日まで毎日腹腔内投与した。
投与14日目の評価では静脈内投与群で有意に運動機能低下が抑制された。また、投与3日目には静脈内投与群で症状の改善が確認された(図14)。
Claims (6)
- 筋萎縮性側索硬化症(ALS)を治療するための医薬組成物であって、間葉系幹細胞を含み、静脈内投与される医薬組成物。
- 2回以上投与される、請求項1に記載の医薬組成物。
- 1回の投与で、106個以上の間葉系幹細胞が投与される、請求項1又は2に記載の医薬組成物。
- 前記間葉系幹細胞が、骨髄又は血液由来の間葉系幹細胞である、請求項1~3のいずれか1項に記載の医薬組成物。
- 前記間葉系幹細胞が、前記患者の骨髄又は血液由来の間葉系幹細胞である、請求項1~4のいずれか1項に記載の医薬組成物。
- 前記間葉系幹細胞が、CD24陰性である、請求項1~5のいずれか1項に記載の医薬組成物。
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JP2021505167A JP7541699B2 (ja) | 2019-03-14 | 2020-03-13 | 筋萎縮性側索硬化症の治療用医薬組成物 |
US17/438,833 US20220133805A1 (en) | 2019-03-14 | 2020-03-13 | Pharmaceutical composition for treating amyotrophic lateral sclerosis |
EP20771155.7A EP3939660A4 (en) | 2019-03-14 | 2020-03-13 | PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF AMYOTROPHIC LATERAL SCLEROSIS |
SG11202110033UA SG11202110033UA (en) | 2019-03-14 | 2020-03-13 | Pharmaceutical composition for treating amyotrophic lateral sclerosis |
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