A drug for the treatment of ischemic vascular disease

Abstract

The present invention relates to a therapeutic agent for ischemic angiopathy that is a medicinal composition for treating ischemic angiopathy, the medicinal composition including mesenchymal stem cells as an active ingredient, wherein the therapeutic agent for ischemic angiopathy is characterized by being administered to a patient for whom recanalization therapy of an occluded blood vessel has been ineffective and by the therapeutic effect of the therapeutic agent being improved.

Description

[Technical Field]

[0001] Related applications This specification includes the contents described in the specification of Japanese Patent Application No. 2020-195816 (filed November 26, 2020), which forms the basis of the priority claim of this application. Technical field This invention relates to a therapeutic agent for ischemic vascular disease, including mesenchymal stem cells. More specifically, it relates to a therapeutic agent for ischemic vascular disease that is administered to patients who do not respond to recanalization therapy for occluded blood vessels, and is characterized by improving the therapeutic effect. [Background technology]

[0002] Recanalization therapy for acute ischemic stroke is a standard treatment strategy for patients with occlusion or stenosis of cerebral arteries. However, despite an improvement in the success rate of recanalization to approximately 70%, 54% of patients still have unfavorable outcomes, with a modified Rankin Score (mRS) of 3-6 points at 90 days. Recent studies suggest that impaired cerebral microcirculation may be a major contributing factor to poor outcomes after major vessel reperfusion in acute ischemic stroke. Therefore, there is a need to develop additional treatment methods to improve microcirculation through the restoration of the microvascular system.

[0003] Mesenchymal stem cells (MSCs) are known to have protective effects on the brain (parenchyma and blood vessels). Administration of MSCs after cerebral infarction has been shown to reduce infarct volume and improve behavioral function, as confirmed using experimental infarct models (Non-Patent Documents 1-3, Patent Document 1). Furthermore, numerous cases of intravenous administration of MSCs have been performed to treat cerebral infarction patients, with improvements in motor function and the site of injury reported (Non-Patent Document 4, Patent Document 2).

[0004] The inventors have reported that the risk of cerebral hemorrhage can be reduced by combining recanalization therapy for occluded blood vessels with intravenous administration of mesenchymal stem cells (Patent Document 3). [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] WO2002 / 000849 [Patent Document 2] WO2009 / 002503 [Patent Document 3] WO2017 / 111153 issue [Non-patent literature]

[0006] [Non-Patent Document 1] Iihoshi S.et al.,Brain Res.2004;1007:1-9. [Non-Patent Document 2] Nomura T. et al.,Neuroscience.2005;136:161-169. [Non-Patent Document 3] Honma T. et al.,Exp.Neurol.2006;199:56-66. [Non-Patent Document 4] Honmou O. et al., Brain. 2011;134:1790-1807. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The objective of this invention is to improve the therapeutic effect of recanalization therapy in patients with ischemic diseases who do not achieve sufficient therapeutic effects. [Means for solving the problem]

[0008] The inventors compared the therapeutic effects of MSCs administered after stroke induction using two experimental models exhibiting identical behavioral symptoms and ischemic stroke volume, confirming that intravenous administration of MSCs enhances the therapeutic effect of recanalization therapy after middle cerebral artery occlusion (MCAO).

[0009] In other words, the present invention relates to the following (1) to (11). (1) A pharmaceutical composition for treating ischemic vascular disease, comprising mesenchymal stem cells as an active ingredient, to be administered to patients for whom recanalization therapy of occluded blood vessels is ineffective. (2) The pharmaceutical composition according to (1), wherein the recanalization therapy for the occluded vessel comprises one or more selected from the administration of a thrombolytic agent, a platelet aggregation inhibitor, and an anticoagulant, and the physical removal of the thrombus. (3) The pharmaceutical composition according to (1) or (2), wherein the ischemic vascular disorder is ischemic cerebrovascular disorder or myocardial infarction. Examples of ischemic cerebrovascular disorders include cerebral infarction and transient ischemic attack. Preferably, the ischemic cerebrovascular disorder is cerebral infarction. (4) A pharmaceutical composition according to any one of (1) to (3) for administration to acute to subacute ischemic vascular disorders. For example, the pharmaceutical composition of the present invention is administered to patients who have undergone recanalization therapy for an occluded vessel in the acute phase but who do not achieve the desired effect from the recanalization therapy. (5) A pharmaceutical composition according to any one of (1) to (4) for which a patient receives recanalization therapy for an occluded vessel in the acute phase of ischemic vascular disease, but the recanalization therapy is ineffective, and which is administered from the acute to subacute phase of ischemic vascular disease. (6) A pharmaceutical composition according to any one of (1) to (5) that improves the therapeutic effect of recanalization therapy in ischemic vascular disease. (7) The pharmaceutical composition according to (6), wherein the improvement in therapeutic effect is one or more selected from an increase in local cerebral blood flow, recovery of the microvascular system, and improvement in behavioral function. Alternatively, the improvement in therapeutic effect is an improvement in the function of the nervous system. (8) The pharmaceutical composition according to any one of (1) to (7), wherein the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells. Preferably, the mesenchymal stem cells are mesenchymal stem cells isolated from the bone marrow of the patient receiving the administration. (9) A method for treating ischemic vascular disease, characterized by administering a pharmaceutical composition containing mesenchymal stem cells to patients in whom recanalization therapy of occluded blood vessels is ineffective. (10) A method for improving the effect of reperfusion therapy in the treatment of ischemic vascular disorders, characterized by administering a pharmaceutical composition containing mesenchymal stem cells to a patient in whom the effect of reperfusion therapy of an occluded blood vessel cannot be obtained. (11) Use of mesenchymal stem cells in the manufacture of a pharmaceutical composition for treating ischemic vascular disorders, characterized in that the pharmaceutical composition is administered to a patient in whom the effect of reperfusion therapy of an occluded blood vessel cannot be obtained.

Advantages of the Invention

[0010] According to the present invention, in patients in whom the therapeutic effect of reperfusion therapy in ischemic diseases cannot be obtained (the effect is insufficient), by intravenously administering MSC, the therapeutic effect of reperfusion therapy can be improved. According to the present invention, an increase in regional cerebral blood flow and promotion of microvascular system recovery are expected, thereby leading to a better clinical outcome for patients who have received reperfusion therapy.

Brief Description of the Drawings

[0011] [Figure 1] Figure 1A shows an overview of the experimental protocol. Figures 1B - 1D show MRA images (B: MRA of uninjured rats, C: complete occlusion of the MCA on the 7th day after pMCAO induction, D: reperfusion on the 7th day after tMCAO induction). [Figure 2] Figure 2 shows the results of the treadmill load test. The graph shows the maximum speed at which the rats could run on the treadmill on the 7th, 8th, 11th, 14th, 21st, 28th, 35th, 42nd, and 49th days (**P < 0.01, *P < 0.05). [Figure 3] Figure 3 shows the results of MRI analysis of the ischemic lesion site. Figure 3A shows representative T2WI of four groups. Figure 3B shows the estimated ischemic lesion volume. [Figure 4] Figure 4 shows the results of ASL - MRI analysis. Figure 4A shows representative ASL images of four groups 7 days and 49 days after MCAO induction. Figure 4B shows the rCBF (regional cerebral blood flow) rate of each group on the 7th, 8th, 11th, 14th, 21st, 28th, 35th, 42nd, and 49th days (**P < 0.01, *P < 0.05). [Figure 5] Figure 5 shows the results of the three-dimensional analysis of capillaries. Figures 5A-5D show representative confocal microscopy images of the four experimental groups 49 days after MCAO induction. Figure 5E shows the ratio of capillary volume of the ischemic hemisphere (ipsilateral) to the contralateral control hemisphere (ipsilateral / contralateral) (**P<0.01, *P<0.05). [Modes for carrying out the invention]

[0012] 1. Drugs for treating ischemic vascular disease The present invention relates to a pharmaceutical composition for treating ischemic vascular disorders, including mesenchymal stem cells, characterized in that it is administered to patients who do not respond to recanalization therapy for occluded blood vessels (for example, administration of pharmaceuticals including thrombolytic agents, platelet aggregation inhibitors, and blood coagulation inhibitors, or physical removal of thrombi).

[0013] [Mesenchymal stem cells] The mesenchymal stem cells used in this invention are stem cells that exist in small amounts within the stromal cells of mesenchymal tissues and possess multipotency and self-renewal capabilities. They are known to differentiate not only into connective tissue cells such as osteocytes, chondrocytes, and adipocytes, but also into nerve cells and cardiomyocytes.

[0014] Sources of mesenchymal stem cells include bone marrow, peripheral blood, umbilical cord blood, fetal embryos, brain, dental pulp, and bone, but bone marrow-derived mesenchymal stem cells (bone marrow mesenchymal stem cells), especially human bone marrow mesenchymal stem cells, are preferred. Bone marrow-derived mesenchymal stem cells have several advantages, including: 1) the potential for significant efficacy, 2) a low risk of side effects, 3) the expectation of a sufficient supply of donor cells, 4) the possibility of autologous transplantation as a non-invasive treatment, 5) a low risk of infection, 6) no concern about immune rejection, 7) no ethical issues, 8) social acceptance, and 9) the ease with which it can become widely established as a common medical treatment. Furthermore, bone marrow transplantation is already a treatment used in clinical practice, and its safety has been confirmed. In addition, bone marrow-derived stem cells have high migratory potential, and can reach the target damaged tissue not only through local transplantation but also through intravenous administration, allowing for expected therapeutic effects.

[0015] The cells may be cells differentiated from ES cells or induced pluripotent stem cells (such as iPS cells), established cell lines, or cells isolated and proliferated from living organisms. The cells may be allogeneic or autologous, but autologous (derived from the patient's own cells) mesenchymal stem cells are preferred.

[0016] The mesenchymal stem cells used in this invention are preferably in an undifferentiated state. This is because undifferentiated cells have a high proliferation rate and survival rate after introduction into vivo. The inventors have also developed a method for obtaining such cells, and the details are described in WO2009 / 002503.

[0017] In the method developed by the inventors, cells separated from bone marrow fluid or the like under conditions that do not substantially come into contact with an anticoagulant (such as heparin) are grown in a culture medium that contains human serum (preferably autologous serum) and does not contain an anticoagulant (such as heparin) or contains it in a very low concentration.

[0018] The cell density in the culture medium affects the properties and differentiation direction of the cells. In the case of mesenchymal stem cells, a cell density of 8,500 cells / cm³ in the culture medium is important. 2 If the number exceeds this, the properties of the cells will change, so the maximum is 8,500 cells / cm³. 2 Subculturing is preferably carried out at the following rate, more preferably at 5,500 cells / cm². 2 Once the above stage is reached, subculture should be initiated.

[0019] Since the method developed by the inventors uses a culture medium containing human serum, it is desirable to minimize the number of culture medium changes, taking into consideration the burden on serum donors. For example, the culture medium should be changed at least once a week, more preferably once or twice a week.

[0020] The culture was performed when the total number of cells was 10 8 The cell culture is repeatedly passed through until more than 10 cells are obtained. The required number of cells may vary depending on the intended use, but for example, the number of mesenchymal stem cells required for transplantation to treat ischemic brain diseases such as cerebral infarction is 10. 7It is considered to be 10 or more. According to the method developed by the inventors, 10 7 mesenchymal stem cells can be obtained in about 12 days.

[0021] The proliferated MSCs can be stored by methods such as cryopreservation (for example, in a deep freezer at -152 °C) until use as needed. For 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 the cryopreservation solution. For example, cells can be suspended in a cryopreservation solution containing 20.5 mL of normal filter-sterilized RPMI, 20.5 mL of autologous serum collected from a patient, 5 mL of dextran, and 5 mL of DMSO and cryopreserved at -150 °C. For example, as DMSO, Cryoprotective Solution (DMSO) manufactured by Nipro Corporation can be used, and as dextran, Low Molecular Dextran L manufactured by Otsuka Pharmaceutical Co., Ltd. can be used, but it is not limited thereto.

[0022] In the pharmaceutical composition of the present invention, the larger the number of MSCs contained, the more preferable it is. However, considering the administration timing to the subject and the time required for culturing, it is practical that it is the minimum amount showing an effect. Therefore, in a preferred embodiment of the pharmaceutical composition of the present invention, the number of mesenchymal stem cells is 10 7 or more, preferably 5×10 7 or more, more preferably 10 8 or more, still more preferably 5×10 8 or more.

[0023] The pharmaceutical product of the present invention is preferably a parenteral administration preparation, more preferably a parenteral systemic administration preparation, and particularly an intravenous administration preparation. Suitable dosage forms for parenteral administration include injectable preparations such as solution-type injectables, suspension-type injectables, emulsion-type injectables, and injections prepared at the time of use, as well as grafts. The parenteral administration preparation is in the form of an aqueous or non-aqueous isotonic sterile solution or suspension, and is formulated into an appropriate unit dose form by appropriately combining pharmacologically acceptable carriers or media, specifically sterile water or physiological saline, culture media (especially culture media used for mammalian cell culture such as RPMI), physiological buffers such as PBS, vegetable oil, emulsifiers, suspending agents, surfactants, stabilizers, excipients, vehicles, preservatives, binders, etc.

[0024] Examples of aqueous solutions for injection include physiological saline, culture medium, physiological buffers such as PBS, isotonic solutions containing glucose or other adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride. These may also be used in combination with appropriate solubilizers, such as alcohol, specifically ethanol, polyalcohol, propylene glycol, polyethylene glycol, or nonionic surfactants such as polysorbate 80 and HCO-50.

[0025] [Ischemic vascular disease] The pharmaceutical product of the present invention is used to treat ischemic vascular disorders. Ischemic vascular disorders refer to conditions in which local vascular damage (e.g., degeneration, occlusion) occurs due to a decrease in arterial blood flow, and examples include ischemic cerebrovascular disease and ischemic heart disease.

[0026] Ischemic cerebrovascular disease: The pharmaceutical product of the present invention is used to treat ischemic cerebrovascular disease. As mentioned above, MSCs are known to have protective effects on the brain (parenchyma and blood vessels) and are already used in the treatment of ischemic cerebrovascular disease such as cerebral infarction by intravenous administration.

[0027] Examples of ischemic cerebrovascular disorders include cerebral infarction (e.g., atherothrombotic cerebral infarction, cerebral thrombosis, cerebral embolism, lacunar infarction, BAD (Branch Atheromatous Disease), Trousseau syndrome, blood coagulation disorders, arterial dissection, venous infarction, vasculitis, antiphospholipid antibody syndrome), and transient ischemic attack (TIA).

[0028] Ischemic heart disease: The pharmaceutical product of the present invention can also be used to treat ischemic heart diseases such as myocardial infarction. Myocardial infarction is a condition in which blockage or narrowing occurs in the coronary arteries that supply oxygen and nutrients to the heart, reducing blood flow and causing the heart muscle to become ischemic and die.

[0029] [Recanalization therapy for occluded blood vessels] The pharmaceutical formulation of the present invention is administered to patients with ischemic vascular disease who do not respond to recanalization therapy for occluded vessels. As mentioned above, recanalization therapy is a standard treatment strategy for ischemic vascular disease, and while some patients experience dramatic improvement in symptoms immediately after recanalization, others do not respond. In the present invention, "not responding to recanalization therapy for occluded vessels" does not mean that there is no effect at all, but rather refers to patients whose clinical symptoms have not recovered sufficiently. For example, it targets patients whose improvement rate after recanalization therapy is insufficient when evaluated using an index of daily living activities such as the Modified Rankin Scale (mRS) or a neurological symptom scale such as the NIH stroke scale. The pharmaceutical composition of the present invention is used for such patients who do not respond to recanalization therapy and improves the effectiveness of recanalization therapy.

[0030] Recanalization therapy for occluded blood vessels is not particularly limited and may include, for example, the administration of medications including thrombolytic agents, platelet aggregation inhibitors, and blood coagulation inhibitors, or the physical removal of the thrombus.

[0031] Thrombolytic agents: In ischemic vascular disorders (especially acute or hyperacute ischemic cerebrovascular disorders and myocardial infarction), the first-line treatment is to reopen the occluded vessel by thrombolysis or physical removal of the thrombus to prevent necrosis due to ischemia. Examples of thrombolytic agents include urokinase, prourokinase, tissue plasminogen activator (t-PA), nasalprase, and streptokinase.

[0032] Platelet aggregation inhibitors (antiplatelet drugs): Platelet aggregation inhibitors (antiplatelet drugs) can prevent thrombus formation by inhibiting platelet aggregation. Examples of platelet aggregation inhibitors include, but are not limited to, aspirin, clopidogrel, cilostazol, and ticlopidine.

[0033] Blood coagulation inhibitors (anticoagulants): Anticoagulants prevent thrombus formation by inhibiting the function of clotting factors. Examples of anticoagulants include, but are not limited to, heparin, low molecular weight heparin, argatroban, danaparoid sodium, dalteparin, nadroparin, bemiparin, fondaparinux, and antithrombin agents such as argatroban.

[0034] Physical removal of blood clots: Physical removal methods for thrombi include, but are not limited to, mechanical thrombectomy via endovascular surgery and carotid endarterectomy. Other methods include bypass surgery, stent placement, balloon angioplasty, aspiration therapy, and restoring blood flow through ultrasound or other means.

[0035] The timing of administration of the pharmaceutical product of the present invention is not particularly limited as long as it is after recanalization therapy of the occluded vessel, but it is administered to patients with ischemic vascular disease in the acute or subacute phase, preferably from the acute to subacute phase. Typically, it is preferable that the pharmaceutical composition of the present invention, containing MSCs, be administered if the recanalization therapy of the occluded vessel is not effective in the acute phase of ischemic vascular disease, and then in the acute to subacute phase.

[0036] The pharmaceutical product of the present invention can improve the therapeutic effect of recanalization therapy when used in patients who do not respond to recanalization therapy for occluded vessels. For example, administration of MSCs promotes an increase in local cerebral blood flow, recovery of the microvascular system, and / or improvement of behavioral function. In patients with ischemic vascular disease, a clinical problem is that many do not recover neurological function even after receiving recanalization therapy. Administration of MSCs improves this neurological function and enables better clinical outcomes for patients who have undergone recanalization therapy for occluded vessels (e.g., endovascular thrombectomy for large vessel occlusion).

[0037] 2. Treatment methods for ischemic vascular disease The present invention provides a method for treating ischemic vascular disorders, characterized by administering a pharmaceutical composition containing MSCs to patients who do not respond to recanalization therapy for occluded blood vessels (such as administration of pharmaceuticals containing thrombolytic agents, platelet aggregation inhibitors, or blood coagulation inhibitors, or physical removal of thrombi).

[0038] The inventors have already confirmed that MSC administration reduces the risk of cerebral hemorrhage by protecting the vascular endothelium and suppressing endothelial damage, thereby enabling safe recanalization therapy of occluded vessels (e.g., thrombolytic therapy or physical removal of thrombi). In this invention, it has been confirmed that in patients who do not respond to recanalization therapy, intravenous administration of MSCs helps improve microcirculation, leading to increased local cerebral blood flow and recovery of the microvascular system. These effects, combined with the tissue regeneration and repair effects of MSCs, improve motor function, promote healing of infarcted lesions, and enable more effective treatment of ischemic vascular disease. [Examples]

[0039] 1. Overview The following hypothesis was tested: intravenous administration of mesenchymal stem cells (MSCs) enhances the therapeutic effect of recanalization therapy after middle cerebral artery occlusion (MCAO) in rats. Two experimental models exhibiting identical behavioral symptoms and ischemic stroke volume, regardless of whether recanalization therapy was performed, were used. MSCs were administered 7 days after stroke induction, and the therapeutic effects were compared.

[0040] [Test Group] (1) pMCAO (pMCAO) + vehicle administration (2) tMCAO (tMCAO) + vehicle administration (3) pMCAO + MSC administration (4) tMCAO + MSC administration - pMCAO (Permanent Middle Cerebral Artery Occlusion) -tMCAO (transient middle cerebral artery occlusion): occlusion of the middle cerebral artery for 110 minutes.

[0041] [Evaluation criteria] Behavioral function, ischemic volume, and regional cerebral blood flow (rCBF) were recorded during the experiment. Histological evaluation of the microvascular system was also performed.

[0042] 2. Materials and Methods [Preparation of MSCs from rat bone marrow] MSCs were prepared based on previously reported information (Nakazaki et al, Neuroscience 408:361-377, 2019). Specifically, bone marrow obtained from the femur of adult Wistar rats (6-8 weeks old) was diluted to 15 ml in Dulbecco's modified Eagle medium (DMEM), and 10% heat-inactivated fetal bovine serum, 2 mM l-glutamine, 100 U / ml penicillin, and 0.1 mg / ml streptomycin were added. The culture was incubated at 37°C for 3 days in a humidified atmosphere containing 5% CO2. When the culture reached near confluence, adherent cells were detached using trypsin-ethylenediaminetetraacetic acid solution (Millipore Sigma) and 1 × 10⁶ cells were removed. 4 The cells were subcultured in cells / ml medium. In this experiment, cultured MSCs after 3 passages were used. Phenotypic analysis of surface antigens was performed, and the MSCs were found to be CD45 - CD73 + CD90 + , and CD106 - I confirmed that this was the case.

[0043] [Cerebral ischemia model] Following previous reports, permanent middle cerebral artery occlusion (pMCAO) and transient middle cerebral artery occlusion (tMCAO) were induced. Adult male SD rats (280-330g; n=72) were anesthetized by intraperitoneal injection of ketamine (75 mg / kg) and xylazine (10 mg / kg). This study employed a suture-based occlusion / recanalization model. For pMCAO, 20.0-24.0 mm long 3-0 surgical MONOSOF sutures (Medtronic) were inserted from the external carotid artery into the lumen of the internal carotid artery until the origin of the MCA was blocked, with the tip rounded using a flame (Nagahama et al, Brain Res 1695:37-44, 2018; Namioka et al, J Neurosurg:1-8, 2018). In the case of tMCAO, the same surgical MONOSOF suture (Medtronic), with its tip rounded with a flame, was inserted from the external carotid artery into the lumen of the internal carotid artery until the origin of the MCA was blocked for 110 minutes. Recanalization was performed 110 minutes after MCAO by removing the suture. Physiological variables (rectal temperature, blood pH, pO2, pCO2, and blood pressure) were maintained within normal ranges during surgery in all animals, and there were no statistically significant differences between groups.

[0044] [Experimental protocol (Figure 1A)] Seven days after the establishment of pMCAO and tMCAO, T2-weighted images (T2WI) were acquired and the initial infarct volume was evaluated. The initial infarct volume on day 7, immediately before MSC or vehicle administration, was the baseline (300 ± 60 mm). 3 Animals that did not meet the following criteria were excluded from the experiment (inclusion rate was approximately 40%). Rats that received pMCAO and tMCAO were randomized into four groups. In Group 1 (n=18), pMCAO rats were intravenously administered a vehicle (1 ml of fresh DMEM, without cells). In Group 2 (n=19), tMCAO rats were intravenously administered a vehicle (1 ml of fresh DMEM, without cells). In Group 3 (n=18), pMCAO rats were administered MSCs (1.0 × 10⁶ each). 6 1 ml of DMEM containing MSCs (cells) was administered intravenously. In group 4 (n=17), tMCAO rats were given MSCs (1.0 × 10⁶ each). 6One ml of DMEM containing cells was administered intravenously. Intravenous administration was performed via the left femoral vein. All rats were administered cyclosporine A (10 mg / kg, ip) daily. All rats underwent a treadmill load test and infarct volume measurement using MRI. For rats selected from each group, rCBF was measured using ASL-MRI and histological evaluation was performed by three-dimensional analysis of capillaries.

[0045] [Treadmill stress test] The treadmill load test was conducted in accordance with previously reported (Nagahama et al. 2018, and (Nakazaki et al. J Neurosurg 127:917-926, 2017). Specifically, rats were made to run on a treadmill (Muromachi Inc.) set to a speed of 20 m / min and an incline of 20° for 20 minutes twice a week before induction of pMCAO or tMCAO. Rats that could not run at 70 m / min were excluded. Three trials were performed with a cutoff time of 180 seconds after induction of pMCAO or tMCAO. The maximum speed that rats could run on the treadmill was recorded on day 7 after induction of pMCAO or tMCAO (immediately before administration of MSC or vehicle), and on days 8, 11, 14, 21, 28, 35, 42, and 49 (1, 4, 7, 14, 21, 28, 35, and 42 days after administration of MSC or vehicle, respectively).

[0046] [MRI research] MRI measurements were performed using a 7-T MRI scanner (70 / 16 PharmaScan, Bruker Biospin MRI GmbH, Ettlingen, Germany). Rats were anesthetized by intraperitoneal administration of ketamine (75 mg / kg) and xylazine (10 mg / kg).

[0047] Three-dimensional time-of-flight magnetic resonance angiography (MRA) images were acquired, and complete occlusion of the MCA by pMCAO (Figure 1C) and recanalization of tMCAO (Figure 1D) were confirmed 7 days after pMCAO or tMCAO induction (immediately before MSC or vehicle administration). Figure 1B shows the MRA images of intact animals.

[0048] T2WI was acquired to measure infarct volume. As previously reported, ischemic volume was calculated from T2WI using ImageJ software (version 1.52, NIH) (Nagahama et al 2018, cited above). Then, the high signal areas were summed and multiplied by the slice thickness and the gap between slices to calculate the lesion volume. MR images were acquired 7 days after pMCAO or tMCAO induction (immediately before MSC or vehicle administration), and 8, 11, 14, 21, 28, 35, 42, and 49 days (1, 4, 7, 14, 21, 28, 35, and 42 days after MSC or vehicle administration, respectively).

[0049] [ASL] CBF images were acquired using continuous ASL via single-shot spin-echo echo-planer imaging (EPI). CBF maps were calculated using in-house code in Matlab (The Math Works Inc.). CBF was calculated in mL / 100g / min using Buxton's common dynamic patency model. Following previous reports, Bregma-0.4mm coronal slices were selected for quantification of cerebral blood flow (Nakazaki et al. 2017, cited above). Regions of interest (ROIs) were placed in the ischemic cortex and the non-infarcted hemisphere. rCBF derived from ASL for each ROI was quantified using ImageJ software (NIH). The rCBF ratio was calculated based on the rCBF of the ischemic cortex divided by the rCBF of the non-infarcted hemisphere.

[0050] [Three-dimensional analysis of capillaries] Microvascular recovery was analyzed with some modifications to previously reported findings (Komatsu et al. Brain Res 1334:84-92, 2010). Rats were anesthetized and intravenously perfused with FITC-lectin (1.6 mg / kg, MilliporeSigma). The brains were promptly excised and fixed with 4% paraformaldehyde at 4°C for 48 hours. Coronary vibratome sections (100 μm) were analyzed using a laser scanning confocal imaging system (LSM780 ELYRA S.1 system, Zeiss). Sections (just below the olfactory sulcus, 3 mm to the right of the midline, and 0.5 mm deep from the cortical surface) were scanned using a confocal microscope. Vascular volume was measured in three dimensions using ImageJ software (NIH). Capillary volume is expressed as the ratio of the ischemic hemisphere (ipsilateral) to the contralateral control hemisphere.

[0051] [Statistical analysis] All statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University), the graphical user interface for R (The R Foundation for Statistics Computing). Kruskal-Wallis tests and Steel-Dwass post-hoc tests were used for multiple comparisons. Data are presented as mean ± standard error (SEM). Differences were considered statistically significant at p<0.05.

[0052] 3.Results [Behavioral function] The maximum speed rats could run on a treadmill was recorded. Seven days after MCAO, no significant difference was observed among the four groups in the maximum speed achieved before administration of MSC or vehicle (p=0.475: Figure 2). The maximum speed of Group 4 (tMCAO+MSC: n=17) was higher than that of Group 3 (pMCAO+MSC; n=18) on days 1, 4, 21, 28, 35, 42, and 49 after pMCAO or tMCAO induction. These data suggest that administered MSCs enhance the therapeutic effect of recanalization therapy in cerebral ischemia.

[0053] MSC showed therapeutic effects in treadmill stress tests after tMCAO (group 4) compared to vehicle treatment (group 2: n=19) on days 14, 21, 28, 35, 42, and 49. In the pMCAO model, MSC administration (group 3) showed higher efficacy than vehicle administration (group 1: n=18) on days 35, 42, and 49 after MCAO induction. The data are summarized in Figure 2. As described above, MSC was effective in both the tMCAO and pMCAO models, but a greater effect was observed in the tMCAO model.

[0054] [Ischemic lesion volume measured by MRI] Ischemic lesion volume was estimated for four groups using in vivo MRI of the same animals used in behavioral studies. Representative T2WIs for the four groups at nine time points (days 7, 8, 11, 14, 21, 28, 35, 42, and 49 after MCAO induction; days 1, 4, 7, 14, 21, 28, 35, and 42 after MSC or vehicle administration) are shown in Figure 3A. No difference in initial stroke volume was observed between groups based on T2WIs obtained 7 days after MCAO induction and before MSC or vehicle administration (p=0.705). In all groups, the estimated lesion volume gradually decreased over the course of 49 days after MCAO induction (42 days after MSC or vehicle administration) (Figure 3B). There were no significant differences in estimated ischemic volume between groups.

[0055] [Arterial spin labeling (ASL), Magnetic resonance imaging (MRI)] To evaluate rCBF, quantitative hemodynamic analysis of ischemic brain tissue was performed using CBF maps obtained from ASL (see Methods and Figure 4). Representative ASL images of the four groups at 7 and 49 days after MCAO induction are shown in Figure 4A.

[0056] Seven days after MCAO induction (immediately before MSC or vehicle injection), the rCBF rate was higher in the transient occlusion group (Group 2: tMCAO + vehicle; n=11, and Group 4: tMCAO + MSC; n=11). The rCBF rate was higher in the permanent occlusion group (Group 1: pMCAO + vehicle; n=9, and Group 3: pMCAO + MSC; n=9). However, on day 49, the rCBF rate in Group 2, which received transient occlusion, decreased significantly, reaching the same level as Group 3, which received permanent occlusion and MSC treatment. Therefore, the rCBF rate after transient occlusion is significantly reduced without MSC administration (Group 2).

[0057] The ratio of regional cerebral blood flow between the two models (pMCAO and tMCAO) was substantially the same on day 7 (groups 1 and 2 were nearly identical, and groups 3 and 4 were nearly identical). However, the rCBF rate in the MSC-administered group was higher than that of the vehicle group on days 42 and 49 after pMCAO or tMCAO induction (group 1 < group 3 < group 2 < group 4). The rCBF rate in group 4 was also higher than that of group 3 on days 42 and 49 after pMCAO or tMCAO induction. In summary, transient occlusion treated with intravenous administration of MSCs 7 days after lesion induction (group 4) showed the greatest therapeutic effect among the experimental groups. The data are shown in Figure 4B.

[0058] [Three-dimensional analysis of capillaries] Three-dimensional analysis of the capillaries in the lesion was performed to determine the extent of the microvascular system. Representative confocal microscopy images of the four experimental groups 49 days after MCAO induction are shown in Figures 5A-5D. The MSC-administered groups showed a higher ratio (ipsilateral / contralateral) at 49 days after MCAO induction in both tMCAO (group 4; N=7 vs. group 2; N=9) and pMCAO (group 3; N=6 vs. group 1; N=5). The ratio in group 4 was larger than that in group 3. The ratio in group 2 was not statistically different from that of group 3. This suggests that in rats that did not show improvement at 7 days after recanalization, recanalization alone is insufficient to maintain the microvascular system without MSC administration. In summary, transient occlusion treated with MSCs (group 4) showed the greatest microvascular recovery among the four groups. The results are shown in Figure 5E.

[0059] 4. Discussion This experiment demonstrated that intravenous administration of MSCs enhances the therapeutic effect of recanalization stroke treatment. Behavioral function scores from treadmill stress tests, rCBF assessed by ASL, and FITC were also evaluated. + Microvascular recovery, as measured by 3D analysis of capillaries, was higher with MSC treatment after tMCAO than with MSC treatment after pMCAO. No significant differences were observed in assessed ischemic volume among the four groups. Therefore, MSC administration may enhance the therapeutic effect of MCAO recanalization therapy.

[0060] Recent advances in endovascular thrombectomy provide clinical benefits to most patients with acute cerebral ischemia caused by large vessel occlusion, although some patients do not respond adequately. This study analyzed the effects of MSC administration in pMCAO and tMCAO (occlusion duration: 110 minutes) using a model system that presented similar conditions during the post-ischemic trial period (7 to 49 days). Similar conditions were defined based on the evaluation of ischemic stroke volume and behavioral function between the pMCAO and tMCAO vehicle groups. Since MSCs or vehicles were administered to each group 7 days after pMCAO or tMCAO induction, this study can be considered a comparison of the therapeutic effects of MSC administration on subacute pMCAO and tMCAO. Both rCBF and the degree of microvascular recovery were greater in the tMCAO+MSC group than in the pMCAO+MSC group. While MSC administration showed therapeutic effects on both pMCAO and tMCAO, the combination of recanalization and MSC administration may yield better results. The results of this experiment confirmed that the insufficient therapeutic effect of recanalization therapy (where the patient's condition did not change from pMCAO on day 7 after recanalization) can be improved by administering MSCs.

[0061] Cell therapy using microsclerosing cells (MSCs) is being investigated for many neurological diseases, including cerebral infarction. Endovascular thrombectomy is currently an established treatment for cerebral infarction. Patients who have undergone acute recanalization therapy may receive MSC administration in clinical practice in the future. The promotion of both increased rCBF and microvascular recovery may contribute to better clinical outcomes for patients who have undergone endovascular thrombectomy due to vascular occlusion. [Industrial applicability]

[0062] According to the present invention, the therapeutic effect of recanalization therapy for occluded blood vessels (for example, administration of pharmaceuticals including thrombolytic agents, platelet aggregation inhibitors, and blood coagulation inhibitors, or physical removal of thrombi) is improved, leading to better clinical outcomes for patients.

[0063] All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. A pharmaceutical composition for treating ischemic vascular disease, comprising mesenchymal stem cells as an active ingredient, which is administered intravenously in the subacute phase of ischemic vascular disease to patients who have received recanalization therapy for occluded vessels in the acute phase of ischemic vascular disease but have not achieved the desired effect of the recanalization therapy. A pharmaceutical composition wherein the ischemic vascular disorder is ischemic cerebrovascular disorder.

2. The pharmaceutical composition according to claim 1, wherein the recanalization therapy for an occluded blood vessel comprises one or more selected from the administration of a thrombolytic agent, a platelet aggregation inhibitor, and a blood coagulation inhibitor, and the physical removal of the thrombus.

3. A pharmaceutical composition according to claim 1 or 2, which improves the therapeutic effect of recanalization therapy in ischemic vascular disease.

4. The pharmaceutical composition according to claim 3, wherein the improvement in therapeutic effect is one or more selected from an increase in local cerebral blood flow, recovery of the microvascular system, and improvement in behavioral function.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells.

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