Pharmaceutical composition for treating acute cerebral infarction

Abstract

The present invention relates to mesenchymal stem cell therapy for treating acute or subacute cerebral infarction. More specifically, the present invention relates to a pharmaceutical composition for treating acute or subacute cerebral infarction, comprising mesenchymal stem cells as an active ingredient, the pharmaceutical composition being characterized by being intravenously administered to a subject three or more times at a standard single dose.

Description

Pharmaceutical composition for treating acute cerebral infarction

[0001] Related Applications: This specification includes the contents of the specification of Japanese Patent Application No. 2024-092810 (filed June 7, 2024), from which the present application claims priority. Technical Field: The present invention relates to a mesenchymal stem cell therapy for treating acute or subacute cerebral infarction. More specifically, the present invention relates to a pharmaceutical composition for treating acute or subacute cerebral infarction, which contains mesenchymal stem cells as an active ingredient, and which is administered intravenously to a subject three or more times in a standard single dose.

[0002] Intravenous administration of mesenchymal stem cells (MSCs) has been suggested as a promising therapeutic option for cerebral infarction. However, the results of randomized clinical trials evaluating a single infusion of MSCs for acute cerebral infarction are unclear. Prasad et al. reported that in 58 patients who received a mean of 288.75 million MSCs at a median of 18.5 days after the onset of cerebral infarction, no significant differences were observed between the single MSC infusion group and the control group in terms of Barthel Index score, modified Rankin Scale, National Institutes of Health Stroke Scale (NIHSS) score, or change in infarct volume at 180 days (Non-Patent Document 1). In a more recent study, Law et al. reported that in 17 patients who received a mean of 140 million MSCs at 63.0 days after the onset of cerebral infarction, there was no improvement in neurological recovery or functional outcome at 12 months as measured by the Barthel Index score, modified Rankin Scale, or National Institutes of Health Stroke Scale (NIHSS) score, but the MSC group showed an improvement in absolute change in median infarct volume (Non-Patent Document 2). Improved approaches may be needed to achieve greater functional improvement in the treatment of stroke.

[0003] We have reported that a single intravenous administration of mesenchymal stem cells (MSCs) during the acute and chronic phases of cerebral ischemia in a rat model of cerebral infarction improves motor function (Patent Document 1, Non-Patent Documents 3-6). Mesenchymal stem cells orchestrate processes such as neuroprotection, stabilization of a disrupted blood-brain barrier, angiogenesis, remyelination of demyelinated axons, axon regeneration, and induction of neuroplasticity. Interestingly, our research has shown that in rats with chronic stroke or spinal cord injury, multiple administrations of MSCs promote greater functional improvement than a single administration of standard MSCs (Patent Document 2, Non-Patent Documents 7 and 8).

[0004] WO2009 / 034708WO2017 / 188457

[0005] Prasad et al. Intravenous autologous bone marrow mononuclear stem cell therapy for ischemic stroke: a multicentric, randomized trial. Stroke. 2014;45(12): 3618-3624.Law et al. The effects of intravenous infusion of autologous mesenchymal stromal cells in patients with subacute middle cerebral artery infarct: a phase 2 randomized controlled trial on safety, tolerability and efficacy. Cytotherapy. 2021;23(9): 833-840.Kiyose et al. Intravenous infusion of mesenchymal stem cells enhances therapeutic efficacy of reperfusion therapy in cerebral ischemia. World Neurosurg. 2021.Namioka et al. Intravenous infusion of mesenchymal stem cells for protection against brainstem infarction in a persistent basilar artery occlusion model in the adult rat. J Neurosurg. 2018: 1-9.Nagahama et al. Preservation of interhemispheric cortical connections through corpus callosum following intravenous infusion of mesenchymal stem cells in a rat model of cerebral infarction. Brain Res. 2018;1695: 37-44.Nakazaki et al. Intravenous infusion of mesenchymal stem cells inhibits intracranial hemorrhage after recombinant tissue plasminogen activator therapy for transient middle cerebral artery occlusion in rats. J Neurosurg. 2017;127(4): 917-926.Takemura et al. Repeated intravenous infusion of mesenchymal stem cells for enhanced functional recovery in a rat model of chronic cerebral ischemia. J Neurosurg. 2021: 1-10.Kurihara et al. Repeated intravenous infusion of mesenchymal stem cells enhances recovery of motor function in a rat model with chronic spinal cord injury. Brain Res. 2023;1817: 148484.

[0006] An object of the present invention is to provide a modified approach to achieve greater functional improvement in the treatment of cerebral infarction with mesenchymal stem cells.

[0007] The inventors discovered that multiple administrations of standard MSCs during the acute or subacute phase of cerebral infarction can treat cerebral infarction more effectively than a single administration of standard MSCs or a single administration of a large amount of MSCs, and completed the present invention.

[0008] That is, the present invention relates to the following [1] to

[13] . [1] A pharmaceutical composition for treating acute or subacute cerebral infarction, comprising mesenchymal stem cells as an active ingredient, wherein a single dose is 0.3 x 10 7 ~2.0 x 10 8[1] The pharmaceutical composition according to [1], characterized in that it contains 0.3 x 10 mesenchymal stem cells and is administered intravenously to a subject three or more times. [2] The pharmaceutical composition according to [1], characterized in that it is administered three to six times. [3] The pharmaceutical composition according to [1], characterized in that it is administered three to four times. [4] A single dose of 0.3 x 10 8 ~2.0 x 10 8 pieces, preferably 0.5 x 10 8 ~2.0 x 10 8 The pharmaceutical composition according to any one of [1] to [3], comprising mesenchymal stem cells. [5] The pharmaceutical composition according to any one of [1] to [4], wherein the administration interval is 15 to 30 days, preferably 20 to 30 days. [6] The pharmaceutical composition according to any one of [1] to [5], wherein the mesenchymal stem cells are blood- or bone marrow-derived mesenchymal stem cells. [7] The pharmaceutical composition according to any one of [1] to [6], wherein the mesenchymal stem cells are human blood- or bone marrow-derived mesenchymal stem cells, more preferably human bone marrow-derived mesenchymal stem cells. [8] The pharmaceutical composition according to any one of [1] to [7], wherein the pharmaceutical composition comprises a cryopreservation solution, for example, one or more selected from DMSO, dextran, and serum (preferably allogeneic serum (human serum)). [9] The pharmaceutical composition according to any one of [1] to [8], wherein the neuroprotective effect is enhanced compared to a single administration.

[10] The pharmaceutical composition according to any one of [1] to [9], wherein neuroplasticity is promoted compared to a single administration.

[11] The pharmaceutical composition according to any one of [1] to

[10] , wherein the mesenchymal stem cells are CD24-negative.

[12] The pharmaceutical composition according to any one of [1] to

[11] , wherein the mesenchymal stem cells express CX3CL1 upon addition of an inflammatory cytokine.

[13] The pharmaceutical composition according to any one of [1] to

[12] , wherein 90% or more of the mesenchymal stem cells express EGFR and / or ITGA4.

[0009] The present invention also relates to the following [1] to

[13] . [1] A method for treating acute or subacute cerebral infarction, comprising administering mesenchymal stem cells to a subject, the method comprising administering 0.3 x 10 7 ~2.0 x 10 8[2] The method of [1], wherein the antibody is administered to a subject three to six times. [3] The method of [1], wherein the antibody is administered to a subject three to four times. [4] The method of [1], wherein the antibody is administered to a subject three to four times. [5] The method of [1], wherein the antibody is administered to a subject three to four times. [6] The method of [1], wherein the antibody is administered to a subject three to six times. [7] The method of [1], wherein the antibody is administered to a subject three to four times. [8] The method of [1], wherein the antibody is administered to a subject three to four times. [9] The method of [1], wherein the antibody is administered to a subject three to four times.

[10] The method of [1], wherein the antibody is administered to a subject three to six times.

[11] The method of

[12] , wherein the antibody is administered to a subject three to four times.

[13] The method of

[14] , wherein the antibody is administered to a subject three to 8 ~2.0 x 10 8 cells, preferably 0.5 x 10 8 ~2.0 x 10 8 [5] The method according to [1], wherein the administration interval is 15 to 30 days, preferably 20 to 30 days. [6] The method according to [1], wherein the mesenchymal stem cells are blood- or bone marrow-derived mesenchymal stem cells. [7] The method according to [1], wherein the mesenchymal stem cells are human blood- or bone marrow-derived mesenchymal stem cells, more preferably human bone marrow-derived mesenchymal stem cells. [8] The method according to [1], wherein the mesenchymal stem cells are formulated with a cryopreservation solution, for example, one or more selected from DMSO, dextran, and serum (preferably allogeneic serum (human serum)). [9] The method according to [1], wherein the neuroprotective effect is enhanced compared to a single administration.

[10] The method according to [1], wherein neuroplasticity is promoted compared to a single administration.

[11] The method according to [1], wherein the mesenchymal stem cells are CD24-negative.

[12] The method according to [1], wherein the mesenchymal stem cells express CX3CL1 upon addition of an inflammatory cytokine.

[13] The method according to [1], wherein 90% or more of the mesenchymal stem cells express EGFR and / or ITGA4.

[0010] According to the present invention, a greater functional improvement can be achieved in a method for treating acute or subacute cerebral infarction using mesenchymal stem cells.

[0011] Figure 1 illustrates the experimental protocol. MCAO was induced in 9-week-old male Sprague-Dawley rats. T2WI images obtained 3 days after MCAO were used to exclude rats with insufficient infarct area in the MCA territory. Rats were randomly assigned to four experimental groups. The vehicle group received an intravenous injection of vehicle alone (no cells; white arrow) 3 days after MCAO. The MSC-1 group received a single intravenous injection of MSCs (black arrow) on day 3 after MCAO. The MSC-3 group received multiple intravenous injections of MSCs on days 3, 10, and 17 after MCAO. The HD-MSC group received a single intravenous injection of three times the standard dose of MSCs (white arrow with horizontal line) on day 3 after MCAO. To monitor behavior, the mice were evaluated for treadmill speed and underwent the mNSS, including the beam-balance test, every 7 days from day 3 to day 45. In vivo T2WI images were also obtained after behavioral testing. Histological evaluation and in vivo DTI were then performed. Figure 2 shows the results of behavioral tests. In all behavioral tests, there were no significant differences between the groups on day 3 after MCAO or before treatment. On days 3, 10, and 17, rats were intravenously injected with vehicle or MSCs after behavioral testing. White arrows indicate vehicle administration, black arrows indicate standard-dose MSC administration, and white arrows with a horizontal line indicate high-dose MSC administration. The behavioral scores for each group are represented by white bars (vehicle), black bars (MSC-1), white bars with a horizontal line (HD-MSC), and white bars with a diagonal line (MSC-3). (*p <0.05, **p <0.01, ***p <0.001). A: Treadmill stress test. The maximum speed at which rats could run on a motorized treadmill was recorded at a 25° incline. The motorized treadmill was operated at a maximum speed of 100 m / min. The maximum velocity of rats in the MSC-3 group tended to be higher than that of the other groups from 17 days after the second MSC injection. Significant differences were observed between the groups on days 38 and 45. B: mNSSs. From 17 days after the second MSC injection, the scores of the MSC-3 group tended to be lower than those of the other groups. Significant differences were observed between the groups on day 45. C: Beam-balance test. Rats without impairment were scored 0, and rats that immediately fell off the beam were scored 6. Evaluation of hyperintense volumes on T2WI.A: Representative T2WI images obtained on days 3, 10, 17, 24, 31, 38, and 45 after MCAO in the four experimental groups (vehicle, MSC-1, HD-MSC, and MSC-3). B: Quantification of lesion volume assessed using T2WI. (p<0.05, **p<0.01, ***p<0.001). R = right, L = left. Scale bar = 5 mm. Figure 4 shows the thickness of the CC on day 45 after MCAO. A: Vehicle group (n = 9). B: MSC-1 group (n = 9). C: HD-MSC group (n = 9). D: MSC-3 group (n = 9). The white dashed line indicates the border of the CC. Scale bar = 100 μm. E: Quantification of CC thickness using Nissl staining. (*p <0.05, **p <0.01, ***p <0.001) Figure 5 shows quantification of CC area 45 days after MCAO. A: Vehicle group (n = 9). B: MSC-1 group (n = 9). C: HD-MSC group (n = 9). D: MSC-3 group (n = 9). Scale bar = 3 mm (A-D), 2 mm (E-H). I: Quantification of CC area using in vivo sagittal T2WI. (*p <0.05, **p <0.01, ***p <0.001) Figure 6 shows evaluation of all neural tracts passing through interhemispheric connections via the CC. A: Vehicle group (n = 9). B: MSC-1 group (n = 9). C: HD-MSC group (n = 9). D: MSC-3 group (n = 9). Scale bar = 3 mm. E: Quantification of the number of fiber tracts. (*p <0.05, **p <0.01, ***p <0.001) Figure 7 shows the correlation analysis between behavioral function and anatomical changes. A: A positive correlation between CC thickness and peak velocity (r = 0.72, R 2 In the treadmill stress test on day 45, an increase in CC thickness correlated with an increase in maximum velocity, as indicated by a positive correlation between CC thickness and maximum velocity (r = 0.70, R 2 = 0.49, p < 0.001). C: There was a positive correlation between CC thickness and maximum velocity (r = 0.71, R 2The increase in CC area correlated with the increase in maximum speed during the treadmill stress test on day 45, as shown by the positive correlation between CC thickness and area (r = 0.74, R 2 = 0.55, p < 0.001), the increase in CC thickness was correlated with the increase in CC area. E: The positive correlation between the number of lines and CC thickness (r = 0.70, R 2 The increase in the CC area was correlated with the increase in CC thickness, as shown by the positive correlation between CC thickness and area (r = 0.75, R 2 = 0.57, p < 0.001), the number of tracts correlated with an increase in CC area.

[0012] The present invention provides a pharmaceutical composition for treating acute or subacute cerebral infarction, which contains mesenchymal stem cells as an active ingredient, and is administered in a single dose of 0.3 x 10 7 ~2.0 x 10 8 The present invention relates to a pharmaceutical composition comprising mesenchymal stem cells, which is administered intravenously to a subject three or more times.

[0013] 1. Mesenchymal stem cells The "mesenchymal stem cells" used in the pharmaceutical composition of the present invention are stem cells that have pluripotency and self-renewal capabilities and are present in trace amounts among the interstitial cells of mesenchymal tissue. They are known to have the ability to differentiate not only into connective tissue cells such as bone cells, chondrocytes, and adipocytes, but also into nerve cells and cardiac muscle cells.

[0014] Sources of mesenchymal stem cells include cells induced to differentiate from embryonic stem cells or induced pluripotent stem cells (iPS cells), established cell lines, and cells isolated and expanded from living organisms. In living organisms, bone marrow, peripheral blood, umbilical cord blood, fetal embryos, and brain are examples. However, bone marrow or blood-derived mesenchymal stem cells, particularly bone marrow mesenchymal stem cells, are preferred. Bone marrow-derived mesenchymal stem cells offer the following advantages: 1) significant efficacy, 2) low risk of side effects, 3) sufficient donor cell supply, 4) non-invasive autotransplantation, 5) low risk of infection, 6) no risk of immune rejection, 7) no ethical issues, 8) social acceptance, and 9) widespread adoption as a standard medical treatment. Furthermore, bone marrow transplantation is already a clinically used treatment with a proven safety profile. Furthermore, bone marrow-derived stem cells have high migratory properties, allowing them to reach the target damaged tissue via intravenous administration as well as local transplantation, resulting in therapeutic efficacy.

[0015] The species of mesenchymal stem cells is not particularly limited and can be appropriately selected depending on the recipient. For human transplantation, primate-derived cells, particularly monkey- or human-derived cells, are preferred, with human-derived cells being more preferred.

[0016] Mesenchymal stem cells may be derived from allogeneic cells or autologous cells, but from the standpoint of safety, autologous cell-derived mesenchymal stem cells (derived from the patient's own cells) are preferred, and from the standpoint of convenience, allogeneic cell-derived mesenchymal stem cells are preferred.

[0017] The mesenchymal stem cells used in the present invention are preferably in an undifferentiated state. This is because cells in an undifferentiated state have a high proliferation rate and a high survival rate after introduction into the body. The undifferentiated state can be confirmed, for example, by the absence of expression of CD24, a differentiation marker. The inventors have also developed a method for obtaining such cells, the details of which are described in WO2009 / 034708.

[0018] In the method developed by the inventors, cells isolated from bone marrow fluid or the like under conditions substantially free of contact with anticoagulants (e.g., heparin) are grown in a medium containing allogeneic serum (preferably autologous serum; in the case of pharmaceutical compositions for humans, human serum) and containing no or very low concentrations of anticoagulants (e.g., heparin). "Containing no or very low concentrations of anticoagulants" means that the anticoagulant is not contained in an effective amount. Specifically, for example, the effective amount of heparin or its derivatives is typically approximately 20-40 μg / mL. However, in the method developed by the inventors, by minimizing the amount of heparin added to the blood collection tube beforehand, the amount in the sample collected from the living body is less than 5 U / mL, preferably less than 2 U / mL, and more preferably less than 0.2 U / mL. During cell culture, the amount present in the medium is less than 0.5 U / mL, preferably less than 0.2 U / mL, and more preferably less than 0.02 U / mL per volume of medium (see WO2009 / 034708).

[0019] The density of cells in the culture medium affects the properties and direction of differentiation of the cells. In the case of mesenchymal stem cells, a cell density of 8,500 cells / cm 2 If the number exceeds this, the properties of the cells will change, so the maximum number is 8,500 cells / cm 2 It is preferable to subculture at a density of 5,500 cells / cm or less, more preferably 5,500 cells / cm 2 Once this is reached, the cells are subcultured.

[0020] Since the method developed by the inventors uses a human serum-containing medium, it is desirable to change the medium as infrequently as possible, taking into consideration the burden on serum donors; for example, the medium is changed at least once a week, more preferably once or twice a week.

[0021] The culture was performed with a total cell count of 10 8 Subculture is repeated until the number of cells reaches 10 or more. The number of cells required varies depending on the purpose of use. For example, the number of mesenchymal stem cells required for transplantation to treat ischemic brain diseases such as cerebral infarction is 10 or more. 7 More than 10 in this invention 6According to the method developed by the inventors, it is thought that 10 7 mesenchymal stem cells can be obtained.

[0022] The expanded mesenchymal stem cells may be stored, as needed, by techniques such as cryopreservation (for example, in a deep freezer at -152°C) until use. In particular, in the present invention, in which the cells are administered multiple times, it is preferable to divide the MSCs prepared by a single bone marrow aspiration and store them frozen until administration.

[0023] For cryopreservation of MSCs, a medium containing serum (preferably human serum, more preferably autologous serum), dextran, and DMSO (medium for mammalian cells such as RPMI) is used as the cryopreservation solution. For example, cells can be suspended in a cryopreservation solution containing 20.5 mL of standard filter-sterilized RPMI, 20.5 mL of autologous serum collected from a patient, 5 mL of dextran, and 5 mL of DMSO, and then cryopreserved at -150°C. For example, DMSO can be cryoprotectant (DMSO) manufactured by Nipro Corporation, and dextran can be low molecular weight dextran L injection manufactured by Otsuka Pharmaceutical, but these are not limiting examples.

[0024] The quality and function of the mesenchymal stem cells prepared as described above may be confirmed by: a) adding cytokines to a culture containing the mesenchymal stem cells and confirming that the mesenchymal stem cells express CX3CL1; or b) confirming that the mesenchymal stem cells express EGFR and / or ITGA4 (preferably, 90% or more of the cells express EGFR and / or ITGA4) without the addition of cytokines.

[0025] The "inflammatory cytokines" used include TNF-α, INFγ, IL-1, IL-6, IL-8, IL-12, and IL-18, and among these, it is preferable to include TNF-α, INFγ, and IL-6, and it is more preferable to use a mixture of TNF-α, INFγ, and IL-6.

[0026] The confirmation of quality and function may further include a step of confirming that the mesenchymal stem cells express one or more selected from BDNF, VEGF, and HGF in the absence of added cytokines. It is particularly important to confirm the presence of BDNF and / or VEGF, and it is most important to confirm the presence of BDNF.

[0027] If mesenchymal stem cells express CX3CL1 upon the addition of inflammatory cytokines, the mesenchymal stem cells are expected to have excellent inflammation-modulating (immunomodulating) properties. If 90% or more of the mesenchymal stem cells express EGFR and / or ITGA4, the mesenchymal stem cells are expected to have excellent accumulation ability at the site of injury. Furthermore, if any of trophic factors such as BDNF, VEGF, and HGF is present in the culture medium, the mesenchymal stem cells are expected to contain mesenchymal stem cells with high neuroprotective properties. In particular, the presence of BDNF and / or VEGF, especially BDNF, can be an important indicator of MSCs with high neuroprotective properties. Mesenchymal stem cells secrete BDNF, VEGF, and / or HGF even without stimulation, and secretory ability can be confirmed by evaluating secretion from unstimulated cells or by evaluating secretion from cells stimulated with inflammatory cytokines.

[0028] It is preferable to use expression at the protein level rather than the gene level as an indicator for the expression of the above-mentioned CX3CL1, EGFR, ITGA4, BDNF, VEGF, and HGF. In the case of cell surface proteins such as EGFR and ITGA4, it is preferable to measure using flow cytometry (FCM) for its simplicity and sensitivity, while in the case of secreted proteins such as CX3CL1, BDNF, VEGF, and HGF, it is preferable to use a bead assay for its simplicity and sensitivity.

[0029] 2. Cerebral infarction

[0030] Cerebral infarction is a pathological condition in which cerebral ischemia occurs due to occlusion or stenosis of cerebral arteries, resulting in necrosis or a state close to necrosis of brain tissue. The disease stages of cerebral infarction can be broadly divided into acute, subacute, and chronic phases, but since the distinction between the acute and subacute phases is not clear, in this specification both are collectively referred to as "acute or subacute cerebral infarction."

[0031] The "chronic phase" of cerebral infarction refers to a period in which symptoms are stable, recovery is difficult, and the disease continues to progress slowly. Chronic cerebral infarction is generally considered to occur six months or more after onset in humans (Caplan LR, "Caplan's Stroke - A Clinical Approach," 5th ed., CAMBRIDGE UNIVERSITY PRESS, 2016), and six weeks or more in rats. Therefore, in humans, "acute or subacute cerebral infarction" usually refers to cerebral infarction occurring within six months of onset, while "chronic cerebral infarction" usually refers to cerebral infarction occurring six months or more after onset.

[0032] 3. Pharmaceutical Composition of the Present Invention The pharmaceutical composition of the present invention contains mesenchymal stem cells (MSCs) and is a pharmaceutical composition for treating acute or subacute cerebral infarction.

[0033] MSCs have neuroprotective properties, and it is known that in acute and subacute cerebral infarction, intravenous administration of MSCs reduces infarct volume and improves behavioral function due to this protective effect. Meanwhile, in chronic cerebral infarction, MSCs promote brain plasticity (neuroplasticity), enabling recovery of motor and brain function. The inventors have found that in acute or subacute cerebral infarction, multiple administrations of MSCs exert a greater neuroprotective effect than single administrations, thereby promoting recovery of motor and brain function. Furthermore, they have found that multiple administrations induce brain plasticity (neuroplasticity), even in the acute or subacute phases, enabling greater improvement in motor and brain function.

[0034] As used herein, the term "neuroprotective effect" refers to the effect of protecting neurons by inhibiting or slowing the progression of neurodegeneration. MSCs exert this neuroprotective effect by secreting various neuroprotective factors.

[0035] As used herein, "brain plasticity" refers to the phenomenon in which neurons and brain circuits create an optimal processing system based on the environment and needs, and undamaged areas function to compensate for the function of damaged areas. Specifically, multiple administrations of MSCs promote the reconstruction of neural circuits through synapse formation, thereby promoting brain plasticity and promoting functional improvement in acute or subacute cerebral infarction. As used herein, "brain plasticity" is used interchangeably with "neuroplasticity."

[0036] The lower limit of the single dose (the number of MSCs contained in one intravenous administration) of the pharmaceutical composition of the present invention is about 0.3 x 10 7 cells, preferably about 0.5 x 10 7 pcs, more preferably about 10 7 , more preferably about 0.3 x 10 8 pieces, particularly preferably about 0.5 x 10 8 The upper limit of a single dose of the pharmaceutical composition of the present invention is about 2.5 x 10 8 pieces, preferably about 2.0 x 10 8 pieces, more preferably about 1.5 x 10 8 There are individuals.

[0037] A single dose of the pharmaceutical composition of the present invention is typically about 0.3 x 10 7 ~Approx. 2.0 x 10 8 pieces, preferably about 0.5 x 10 7 ~Approx. 2.0 x 10 8 pcs, more preferably about 10 7 ~Approx. 2.0 x 10 8 , more preferably about 0.3 x 10 8 ~Approx. 2.0 x 10 8 pcs, particularly preferably about 0.5 x 10 8 ~Approx. 2.0 x 10 8 There are individuals.

[0038] As used herein, the term "about" refers to a value that varies by plus or minus 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1% of the reference value. Preferably, the term "about" refers to a range of plus or minus 10%, 5%, or 1%, of the reference value.

[0039] The pharmaceutical composition of the present invention is administered to a subject with acute or subacute cerebral infarction three or more times, preferably three to six times, more preferably three or four times.

[0040] Since the "acute or subacute phase" of human cerebral infarction lasts up to about 6 months from the onset, the administration interval of the pharmaceutical composition of the invention is about 15 to about 30 days, preferably about 20 to about 30 days.

[0041] The pharmaceutical composition of the present invention is administered to a subject parenterally, preferably intravenously. Dosage forms suitable for intravenous administration include injections such as solution injections, suspension injections, emulsion injections, and injections prepared just before use.

[0042] Preferably, the pharmaceutical composition of the present invention is in the form of an aqueous or non-aqueous isotonic sterile solution or suspension, and is formulated into an appropriate unit dosage form by appropriately combining, for example, pharmacologically acceptable carriers or vehicles, specifically, sterile water, physiological saline, culture media (particularly media used for culturing mammalian cells, such as RPMI), physiological buffer solutions such as PBS, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, excipients, vehicles, preservatives, binders, etc.

[0043] Examples of aqueous solutions for injection include physiological saline, culture media, physiological buffer solutions such as PBS, isotonic solutions containing glucose or other auxiliary agents, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, and these may be used in combination with an appropriate solubilizing agent, such as alcohol, specifically ethanol, polyalcohol, propylene glycol, polyethylene glycol, or a nonionic surfactant, such as polysorbate 80 or HCO-50.

[0044] The pharmaceutical composition of the present invention may contain the above-mentioned cryopreservation solution, serum (preferably human serum, more preferably autologous serum), dextran, and a medium (medium for mammalian cells such as RPMI) containing DMSO.

[0045] A unit dosage form (e.g., a bag) of the pharmaceutical composition contains a single dose of MSCs as described above, but may contain slightly more than the single dose described above so that the required number of MSCs can be administered to the patient.

[0046] The pharmaceutical composition of the present invention, when administered multiple times, increases the thickness and area of ​​the corpus callosum compared to a single administration, thereby improving motor function. The pharmaceutical composition of the present invention, when administered multiple times, enhances neuroprotective effects and promotes brain plasticity (neuroplasticity) compared to a single administration, thereby promoting improvement of motor function and brain function.

[0047] The present invention will be specifically explained below with reference to examples, but the present invention is not limited to these examples.

[0048] 1. Materials and Methods 1.1 Animals The use of animals in this study was approved by the Sapporo Medical University Animal Care and Use Committee. All procedures were performed in accordance with institutional guidelines.

[0049] 1.2 Preparation of MSCs. MSCs were cultured as previously described (Takemura et al., supra). Specifically, bone marrow was obtained from the femurs of 35 adult male Sprague-Dawley (SD) rats. MSCs obtained from one donor were sufficient for approximately two "single" injections of MSCs. The bone marrow was supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U / mL penicillin, and 0.1 mg / mL streptomycin and cultured for 7 days (5% CO2, 37°C). When the culture reached nearly 100% confluence, adherent cells were detached with trypsin-ethylenediaminetetraacetic acid solution and 1 × 10 cells were cultured. 4 MSCs were used in this study after two passages. Previous surface antigen analysis revealed that they expressed cluster of differentiation (CD45) - , CD73 + , CD90 + , CD106 - The phenotype of MSCs was revealed. MSCs could be induced to develop into mesenchymal lineages such as osteocytes, adipocytes, and chondrocytes.

[0050] 1.3 Cerebral ischemia model A rat model of permanent middle cerebral artery occlusion (MCAO) was used as a cerebral infarction model. Unilateral MCAO was induced using a previously reported intravascular occlusion method. Male SD adult rats (9 weeks old, n = 36) were anesthetized by intraperitoneal (IP) injection of ketamine (75 mg / kg) and xylazine (10 mg / kg). A 25.0 mm 3-0 Monosof TM A monofilament nylon suture was heated with a flame, curled, and advanced from the external carotid artery into the lumen of the internal carotid artery until it occluded the origin of the MCA. The suture was left in place for permanent occlusion, and the incision was closed.

[0051] 1.4 Experimental Protocol The experimental protocol is shown in Figure 1. After induction of permanent MCAO, T2-weighted MR images (T2WI) were acquired on day 3 to assess the cerebral infarction volume. The total volume of the basal ganglia and cerebral cortex was insufficient (350 mm). 3 Fourteen rats with a mean age of less than 18 years were excluded. Rats that underwent MCAO were then randomly assigned to one of four experimental groups: 1) rats that received an intravenous injection of vehicle alone 3 days after MCAO (vehicle group [n = 9], 1 mL of fresh DMEM, no cells); 2) rats that received a standard single intravenous injection of MSCs 3 days after MCAO (MSC-1 group [n = 9], 1 × 10 cells in 1 mL of fresh DMEM). 6 3) rats that received a single intravenous injection of high-dose (HD) MSCs on day 3 after MCAO (HD-MSC group [n = 9], 3 × 10 cells in 1 mL of fresh DMEM) 6 cells); and 4) rats intravenously injected with MSCs three times on days 3, 10, and 17 after MCAO (MSC-3 group [n = 9], 1 × 10 cells in 1 mL of fresh DMEM). 6 The remaining cells were stained with 0.4% trypan blue immediately after treatment to confirm high cell viability (>99%).

[0052] Because this study was not an autologous transplant, cyclosporine A was used to avoid potential immune reactions. All rats received daily injections of cyclosporine A (10 mg / kg, IP) starting the day before MSC or vehicle administration after MCAO induction. Behavioral testing was performed weekly from day 3 to day 45 after MCAO induction, with MSC or vehicle administration occurring one day after the behavioral testing. After in vivo T2WI was obtained on day 45, the animals were euthanized for histological and ex vivo DTI analyses.

[0053] 1.5 Statistical Analysis All statistical analyses were performed using JMP Pro 17 (SAS Institute, Cary, NC, USA). Continuous data were assessed for normality using the Shapiro-Wilk test. Normally distributed continuous data were analyzed using one-way ANOVA. If significant, subgroup comparisons were performed using the Tukey-Kramer test. Continuous data that did not pass the normality test were compared using the Kruskal-Wallis test. If significant, subgroup comparisons were performed using the Steel-Dwass test. Furthermore, behavioral and functional improvements in each group were analyzed using one-way repeated measures ANOVA. Correlations between treadmill stress testing, CC thickness, CC area, and number of DTI tracts were assessed using Pearson's correlation coefficient. Data are expressed as mean ± standard error of the mean (SEM). Differences were considered statistically significant at p < 0.05.

[0054] 2. Results: 2.1 Behavioral analysis Behavioral performance was evaluated in the experimental groups (vehicle group, n = 9; MSC-1 group, n = 9; HD-MSC group, n = 9; MSC-3 group, n = 9), and the treadmill stress test, mNSS, and beam-balance test (part of the mNSS) were performed every 7 days from day 3 to day 45 after MCAO induction.

[0055] In the treadmill stress test (Figure 2A), the maximum running speed of the rats was recorded. There was no difference in speed between the groups on day 3. The vehicle group showed spontaneous recovery on day 24 and continued to plateau until day 45, the end of the test period. Rats in the MSC-1 and HD-MSC groups became faster than the vehicle group from day 38 onwards. Rats in the MSC-1 and HD-MSC groups performed similarly at each time point throughout the test period. Rats in the MSC-3 group were faster than the vehicle group from day 31 onwards and faster than both the MSC-1 and HD-MSC groups from day 38 onwards.

[0056] The mNSS assessment (Figure 2B) revealed no significant differences in scores on day 3 among all groups. The vehicle group showed spontaneous recovery on day 24 and continued to plateau until the end of the study period on day 45. The scores of the MSC-1 and HD-MSC groups were lower than those of the vehicle group on days 38 and 45, but the scores of the MSC-1 and HD-MSC groups remained similar at each time point throughout the study period. The scores of the MSC-3 group were lower than those of the vehicle group from day 17 onward and were lower than those of both the MSC-1 and HD-MSC groups on days 24, 31, and 45. Repeated-measures ANOVA showed a significant effect for the MSC-3 group after day 38 [F(1, 8) = 10.0, p < 0.05], indicating that only the MSC-3 group did not plateau and continued to improve from day 38 to day 45. The other groups (vehicle, MSC-1, and HD-MSC) showed no significant differences between days 38 and 45 by repeated-measures ANOVA, suggesting that these groups plateaued and did not improve after day 38. There was also no significant difference in scores on day 3 in the beam-balance test (Figure 2C). The vehicle group showed spontaneous recovery until day 17 and continued to plateau until day 45, the end of the study period. The scores of both the MSC-1 and HD-MSC groups became lower than those of the vehicle group from day 38 onward. The scores of the MSC-1 and HD-MSC groups remained similar at each time point throughout the study period. The scores of the MSC-3 group were lower than those of the vehicle group from day 24 onward, and were lower in both the MSC-1 and HD-MSC groups at day 45. Repeated measures ANOVA showed a significant effect for the MSC-3 group after 38 days [F(1, 8) = 6.4, p < 0.05], indicating that only the MSC-3 group continued to improve from day 38 to day 45. The other groups (vehicle, MSC-1, HD-MSC) were not significantly different from day 38 to day 45 by repeated measures ANOVA, suggesting that the other groups plateaued after day 38 and did not improve.

[0057] 2.2 Ischemic Lesion Volume by MR Imaging Analysis In vivo MRI was used to estimate the ischemic lesion volume in each group (vehicle group, n = 9; MSC-1 group, n = 9; HD-MSC group, n = 9; MSC-3 group, n = 9). T2WI images were acquired on days 3, 10, 17, 24, 31, 38, and 45 after MCAO induction. T2WI images on day 3 confirmed no significant differences in cerebral infarction volume between groups. Representative images at the caudate-occipital complex level are shown in Figure 3A. Lesion volume in each group was calculated by analyzing the high-intensity areas on T2WI images acquired throughout the cerebrum (Figure 3B). In the vehicle group, the high-intensity areas on T2WI naturally decreased until day 38 and remained stable from day 38 to day 45. From day 31 onward, the volumes of the MSC-1 and HD-MSC groups decreased more than those of the vehicle group. The hyperintense volume on T2WI in the MSC-1 and HD-MSC groups was similar at each time point during the study. The MSC-3 group showed a statistically significant reduction in ischemic lesion volume from day 17 compared to the vehicle group, and from day 24 compared to both the MSC-1 and HD-MSC groups.

[0058] 2.3 CC Thickness by Nissl Staining The thickness of the CC at the bregma-0.26 mm level was examined by Nissl staining (Figure 4E). The CC in the MSC-3 group (Figure 4D, n = 9) was the thickest among all groups, while the CC in the MSC-1 group (Figure 4B, n = 9) and the HD-MSC group (Figure 4C, n = 9) were thicker than the CC in the vehicle group (Figure 4D, n = 9). There was no difference in CC thickness between the MSC-1 and HD-MSC groups.

[0059] 2.4 CC Area by Sagittal T2WI The CC area in the central brain was examined using ex vivo T2WI (Figure 5I). The CC area in the MSC-3 group (Figures 5D and 5H, n = 9) was the largest among all groups, while the CC areas in the MSC-1 group (Figures 5B and 5F, n = 9) and HD-MSC group (Figures 5C and 5G, n = 9) were larger than that in the vehicle group (Figures 5A and 5E, n = 9). There was no difference in CC area between the MSC-1 and HD-MSC groups.

[0060] 2.5 In vivo DTI analysis. Ex vivo DTI tractography was used to evaluate interhemispheric nerve tracts via the CC in the vehicle (Fig. 6A; n = 9), MSC-1 (Fig. 6B; n = 9), HD-MSC (Fig. 6C; n = 9), and MSC-3 (Fig. 6D; n = 9) groups 45 days after MCAO. Quantitative analysis of DTI tractography sequences showed that the number of fibers was highest in the MSC-3 group, and higher in the MSC-1 and HD-MSC groups than in the vehicle group. There was no significant difference in the number of tracts between MSC-1 and HD-MSC.

[0061] 2.6 Correlation analysis between anatomical changes and behavioral function Pearson's correlation analysis was performed to evaluate the relationship between behavioral function and anatomical changes. The maximum treadmill speed on day 45 was used as an index of behavioral function. Pearson's correlation analysis revealed a significant positive correlation between the maximum treadmill speed and the number of tracts on DTI (Figure 7A, r = 0.72, n = 36, p < 0.0001, R 2 = 0.52), CC thickness as determined by Nissl staining (Figure 7B, r = 0.70, n = 36, p < 0.0001, R 2 = 0.49) and CC area on T2WI (Fig. 7C, r = 0.71, n = 36, p < 0.0001, R 2 = 0.51). Furthermore, a positive correlation was observed between anatomical changes, CC thickness, and CC area on T2WI (Fig. 7D, r = 0.74, n = 36, p < 0.0001, R 2 = 0.55), the number of tracts on DTI, and CC thickness on Nissl staining (Figure 7E, r = 0.70, n = 36, p < 0.0001, R 2 = 0.49), the number of tracts on DTI and the CC area on T2WI (Fig. 7F, r = 0.75, n = 36, p < 0.0001, R 2 A positive correlation was found between the two (= 0.57).

[0062] Furthermore, linear regression analysis (Figure 7A: y = 18.22 × + 1656.51, Figure 7B: y = 1.51 × + 204.09, Figure 7C: y = 0.012 × + 2.80) suggested a correlation between the degree of functional improvement and anatomical changes (i.e., tract number, CC thickness, and CC area on DTI). Furthermore, linear regression analysis (Figure 7D: y = 92.94 × - 35.82) suggested a correlation with CC thickness and CC area (Figure 7E: y = 8.19 × + 319.70, Figure 7F: y = 1097.53 × - 1154.67), suggesting that an increase in the number of tracts in the CC region is associated with increases in CC thickness and area.

[0063] 3. Discussion: This study demonstrated that multiple MSC administration (MSC-3), administered three times at weekly intervals, had a greater therapeutic effect than single administration in terms of both functional recovery and structural changes in the acute phase of cerebral infarction. A single administration of standard dose (MSC-1) and a single administration of high-dose (HD-MSC) resulted in comparable therapeutic effects, revealing the existence of a ceiling effect for the number of MSCs administered per MSC administration. The multiple-administration (MSC-3) group, which showed superior functional outcomes, administered the same total number of cells as the single-administration HD-MSC group. Interestingly, the behavioral scores of rats in the single-administration MSC groups (MSC-1, HD-MSC) plateaued within a few weeks of administration. However, rats in the MSC-3 group showed significant functional improvement, which persisted until the end of the study period. This significant recovery pattern was confirmed by repeated-measures ANOVA of the mNSS and beam-balance test. Therefore, multiple administration of MSCs may enable greater functional recovery compared with a single administration of MSCs during the acute phase of cerebral ischemia. These data suggest that the therapeutic effect may be enhanced by the temporal distribution of MSC administration, rather than simply the number of MSCs administered.

[0064] In terms of mechanisms, MSCs orchestrate processes such as neuroprotection, stabilization of disrupted blood-brain barrier, angiogenesis, remyelination of demyelinated axons, axon regeneration, and induction of neuroplasticity. In this study, as shown by in vivo MRI, after intravenous administration of MSCs, the MSC-3 group (1 × 10 63 x cells: 3 x 10 total 6 In the HD-MSC group (3 × 10 6 A significant reduction in the hyperintense volume on T2WI was observed compared with a single high-dose bolus (3 × 10 cells). 6 The total number of cells was 1 x 10 6 3 x cells: 3 x 10 total 6 This may explain the enhanced neuroprotective effect of multiple MSC administration in the acute phase, despite the fact that the number of MSCs administered was the same. However, in the chronic phase, multiple administrations did not effectively induce neuroprotection. No changes were observed in the T2WI hyperintense volume in the chronic phase of cerebral infarction after MSC administration. Therefore, multiple administrations of MSCs with higher neuroprotective properties may be effective in the treatment of acute cerebral ischemia. These preclinical results, which show that temporally dispersed MSC administration is more effective than a single standard administration, should be taken into consideration in clinical settings.

[0065] Although it is difficult to distinguish between the effects of neuroprotection and neuroplasticity induction in this acute study, several lines of evidence suggest that neuroplasticity induction may also be at play. While the behavioral scores of rats in the MSC-1 and HD-MSC groups plateaued several weeks after administration, rats in the MSC-3 group showed significant functional improvement, which continued until the end of the study period. Repeated-measures ANOVA revealed a statistically significant difference in the MSC-3 group but not in the MSC-1 and HD-MSC groups. In a previous study of chronic stroke, the CC in the MSC-3 group was thicker, as observed in Nissl-stained sections, and more extensive in DTI visualization. This suggests that the CC was not simply "protected" by MSCs, but rather that its size increased, suggesting increased axonal content. Regarding induced neuroplasticity, accumulating evidence from our study indicates the following: (1) MSC administration leads to enhanced neuronal connectivity between the ischemic and non-ischemic cerebral hemispheres. In an acute cerebral ischemia model, administered MSCs activated the non-infarcted cortex, which projects from the contralateral infarcted cortex via interhemispheric cortical connections (corpus callosum). Functional MRI also demonstrated that MSC administration in a cerebral infarction model improved motor function and bilateral cortical hyperactivity. (2) We report a case series in which MSCs were administered to patients with chronic brain injury. In this case, DTI revealed enhanced inter-cortical connectivity, suggesting a potential therapeutic mechanism. (3) In a rat model of neonatal hypoxia-ischemia, MSC administration improved functional outcomes and induced brain tissue growth in the unaffected hemisphere. Furthermore, in severe perinatal brain injury, MSC administration increased the amount of remaining (non-ischemic) tissue in the contralateral hemisphere, the number of neurons, GABAergic cells, and cortical synapses, and improved cognitive function. (4) We demonstrated that MSC administration improved cognitive dysfunction in a whole-brain cerebral small vessel disease model. (5) In a spinal cord injury model, induced neuroplasticity was observed, and we found that administered MSCs could activate dormant neural circuits.This was also evident from the enhanced axonal projections between the dorsal corticospinal tract and the lateral navicular nucleus both rostral and caudal to the lesioned nucleus. These results suggest that MSC administration may increase the diameter of thin, pre-existing axons, which are difficult to detect using conventional tracing methods, and improve motor neuron conduction below the SCI site. (6) In the case of SCI, administered MSCs may activate distant sites, including the motor cortex. We identified 15 coding genes in the motor cortex of rats administered MSCs and vehicle after SCI, suggesting that a common gene signature in these distant neural tissues may trigger further downstream gene expression to restore neuronal function after MSC administration. (7) In our previous studies using chronic models of cerebral infarction and spinal cord injury, the therapeutic effect was strongest with multiple administrations. These therapeutic mechanisms may act through the promotion of neuroplasticity.

[0066] In summary, both neuroprotection, as evidenced by the reduction of hyperintensity volume on T2WI, and the induction of neuroplasticity, as evidenced by the continuous functional improvement after multiple MSC administrations, may be effective during the acute phase of ischemia. Therefore, multiple intravenous administration of MSCs may overcome the limited results of a single administration of MSCs. Cell-based therapies using MSCs are being investigated as treatments for various central nervous system disorders. The results presented here suggest that multiple intravenous infusions of MSCs may be effective in clinical protocols for acute cerebral ischemia and other diseases.

[0067] Since the progression of experimental cerebral infarction in rats is faster than that in humans, the "acute phase" in the examples may correspond to the "acute or subacute phase" of human cerebral infarction. Furthermore, based on the body weight, the number of cells administered to humans is estimated to be about 200 times the number of cells administered to rats. Therefore, the standard dose of 1 × 10 6 In humans, the number of cells is approximately 2 × 10 8 cells, and for safety reasons, the figure for humans is approximately 0.3 × 10 8 ~Approx. 2×10 8 It is estimated that the cell level is appropriate.

[0068] 4. Conclusions Our results demonstrate that multiple systemic administration of MSCs provides greater beneficial effects than a single administration of MSCs during the acute phase of MCAO through enhanced neuroprotection and induction of neuroplasticity.

[0069] According to the present invention, a greater functional improvement can be achieved in a method for treating acute or subacute cerebral infarction using mesenchymal stem cells.

[0070] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A pharmaceutical composition for treating acute or subacute cerebral infarction, comprising mesenchymal stem cells as an active ingredient, wherein a single dose is 0.3 x 10 7 ~2.0 x 10 8 The pharmaceutical composition, characterized in that it contains mesenchymal stem cells and is administered intravenously to a subject three or more times.

2. The pharmaceutical composition according to claim 1, characterized in that it is administered 3 to 6 times.

3. The pharmaceutical composition according to claim 1, characterized in that it is administered 3 to 4 times.

4. Single dose 0.3 x 10 8 ~2.0 x 10 8 The pharmaceutical composition of claim 1 , comprising mesenchymal stem cells.

5. The pharmaceutical composition according to claim 1, wherein the administration interval is 15 to 30 days.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the mesenchymal stem cells are blood- or bone marrow-derived mesenchymal stem cells.

7. The pharmaceutical composition according to claim 6, wherein the mesenchymal stem cells are mesenchymal stem cells derived from human blood or bone marrow.

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