Pharmaceutical composition for the prevention of restenosis within a stent

Abstract

The present invention relates to a pharmaceutical composition containing mesenchymal stem cells administered to a patient who has received a stent. Specifically, the present invention relates to a pharmaceutical composition for tissue repair and regeneration, comprising mesenchymal stem cells and characterized by being administered to a patient who has received a stent, and to the prevention of neointimal hyperplasia or restenosis within the stent by said pharmaceutical composition.

Description

Technology Field

[0001] Related applications

[0002] This specification includes the contents described in the specification of Japanese patent application No. 2019-020707 (filed February 7, 2019), which is the basis of the priority of this application.

[0003] Technology field

[0004] The present invention relates to a pharmaceutical composition comprising mesenchymal stem cells administered to a patient who has received a stent. The pharmaceutical composition of the present invention is administered to a patient who has received a stent to promote tissue repair and regeneration of the affected area and to prevent restenosis within the stent. Background Technology

[0005] In carotid artery stenting (CAS) or percutaneous transluminal angioplasty and stenting (PTAS) performed for the treatment of carotid or intracranial atherosclerosis, postoperative in-stent restenosis is a critical complication leading to the recurrence of stroke (cerebral infarction). While previous studies have reported a low risk of ipsilateral cerebral infarction recurrence after CAS, moderate to severe restenosis after PTAS occurs more frequently than after endarterectomy, and restenosis increases the risk of ipsilateral recurrent stroke and transient ischemic attack (TIA). Despite improvements in surgical technique, in-stent restenosis after PTAS for intracranial atherosclerosis has been reported as a serious complication, with an incidence rate of up to 31%. Stenosis results in stroke or TIA in 39% of patients. Generally, these findings emphasize the need to prevent in-stent restenosis after PTAS to prevent ipsilateral ischemic stroke or TIA and to improve clinical reversal after CAS and PTAS.

[0006] As a mechanism of restenosis within a stent, the possibility of neointimal hyperplasia induced by an inflammatory response to the stent struts can be cited (Non-patent Literature 1 and 2). In cardiology, drug-eluting stents (DES) have been developed to reduce the restenosis rate by preventing neointimal hyperplasia through the suppression of inflammatory responses. Although the use of DES has been attempted for purposes other than intracranial arterial disease, the pre-procedure complication rate remains high due to technical obstacles caused by stent stiffness and neurotoxicity. Therefore, the development of a new approach to inhibit neointimal hyperplasia is desired.

[0007] It has been reported that intravenous administration of bone marrow-derived mesenchymal stem cells (MSCs) reduces reperfusion impairment and infarct volume, thereby improving behavioral function in experimental stroke models (Patent Documents 1 to 5). In addition to the therapeutic mechanisms of MSCs, such as neuroprotection, neurogenesis, induction of axonal regeneration, angiogenesis, restoration of the blood-brain barrier, erythematous elastin, preservation of cortical junctions, neuroplasticity, and secretion of neurotrophic factors that lead to remote responses, a mechanism suggested by recent studies is the stabilization of the inflammatory microenvironment by the release of various anti-inflammatory mediators (Non-patent Documents 3 to 7) (Non-patent Document 8). Prior art literature

[0008] WO2002 / 000849 WO2009 / 034708 WO2018 / 034023 WO2018 / 034314 WO2017 / 188457

[0009] Dussaillant GR, et al: Small stent size and intimal hyperplasia contribute to restenosis: a volumetric intravascular ultrasound analysis. J Am Coll Cardiol 26:720-724, 1995Hoffmann R, et al: Patterns and mechanisms of in-stent restenosis. A serial intravascular ultrasound study. Circulation 94:1247-1254, 1996Lankford KL, et al: Intravenously delivered mesenchymal stem cell-derived exosomes target M2-type macrophages in the injured spinal cord. PLoS One 13:e0190358, 2018Nakazaki M, 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 127:917-926, 2017Prockop DJ, et al: Mesenchymal stem / stromal cells(MSCs): role as guardians of inflammation. Mol Ther 20:14-20, 2012Shi Y, et al: Immunoregulatory mechanisms of mesenchymal stem and stromal cells in inflammatory diseases.Nat Rev Nephrol, 2018Sasaki Y, et al: Synergic Effects of Rehabilitation and Intravenous Infusion of Mesenchymal Stem Cells After Stroke in Rats. Phys Ther 96:1791-1798, 2016. The problem to be solved

[0010] The objective of the present invention is to provide a pharmaceutical composition for tissue repair and regeneration that can be administered even to patients who have received a stent, and furthermore, to provide a novel method for preventing restenosis after PTAS and the recurrence of stroke or TIA associated therewith. means of solving the problem

[0011] The inventors hypothesized that intravenous administration of MSCs inhibits neointimal hyperplasia by suppressing the inflammatory response to implanted stent struts using a pig model. The inventors implanted two types of bare metal stents into miniature pigs, implanting one stent into the common carotid artery (CCA) corresponding to the human middle cerebral artery (MCA) and the other stent into the superficial cervical artery (SCA) corresponding to the human CCA, and performed image analysis using divascular angiography (DSA) and intravascular ultrasound (IVUS). In addition, they evaluated histological findings, including inflammatory changes around the stent struts, after MSC administration. Furthermore, they confirmed that MSCs can be administered to patients who have received stents, and that this can inhibit the progression of neointimal hyperplasia in the stented CCA and SCA.

[0012] The present invention is completed based on the above findings and relates to (1) to (15) below.

[0013] (1) A pharmaceutical composition for tissue repair and regeneration, comprising mesenchymal stem cells, characterized by being administered to a patient who has received a stent.

[0014] (2) A pharmaceutical composition described in (1) which is or was a patient having any of the following diseases selected from: atherosclerotic arteriosclerosis, ischemic heart disease including myocardial infarction, ischemic cerebrovascular disorder including cerebral infarction and transient ischemic attack (TIA), occlusive arteriosclerosis (ASO), Buerger's disease, arteriosclerotic lesions in blood vessels throughout the body, dissecting vascular lesions including dissecting aneurysms, and aneurysms.

[0015] (3) A pharmaceutical composition described in (1) or (2), wherein the stent is a carotid artery stent (CAS) or a percutaneous angioplasty stent (PTAS).

[0016] (4) 10 per single dose 6 A pharmaceutical composition described in any one of (1) to (3), characterized by administering two or more cells.

[0017] (5) A pharmaceutical composition described in any one of (1) to (4), wherein the mesenchymal stem cells are mesenchymal stem cells derived from bone marrow or blood.

[0018] (6) A pharmaceutical composition described in any one of (1) to (5), wherein the mesenchymal stem cells are mesenchymal stem cells derived from the patient's bone marrow or blood.

[0019] (7) A pharmaceutical composition described in any one of (1) to (6) that prevents restenosis within a stent (neointima hyperplasia within a stent).

[0020] (8) A pharmaceutical composition for preventing restenosis within a stent, comprising mesenchymal stem cells, and characterized by being administered to a patient who has received a stent.

[0021] (9) A pharmaceutical composition described in (8) which is or was a patient who is suffering from any of the following diseases selected from atheromatous arteriosclerosis, ischemic heart disease including myocardial infarction, ischemic cerebrovascular disorder including cerebral infarction and transient ischemic attack (TIA), occlusive arteriosclerosis (ASO), Buerger's disease, arteriosclerotic lesions in blood vessels throughout the body, dissecting vascular lesions including dissecting aneurysms, and aneurysms.

[0022] (10) A pharmaceutical composition described in (8) or (9) in which the stent is a carotid artery stent (CAS) or a percutaneous angioplasty stent (PTAS).

[0023] (11) 10 with a single administration 6 A pharmaceutical composition described in any one of (8) to (10), characterized by administering more than one cell.

[0024] (12) A pharmaceutical composition described in any one of (8) to (11), wherein the mesenchymal stem cells are derived from bone marrow or blood.

[0025] (13) A pharmaceutical composition described in any one of (8) to (12), wherein the mesenchymal stem cells are mesenchymal stem cells derived from the patient's bone marrow or blood.

[0026] (14) A pharmaceutical composition described in any one of (8) to (13) that prevents restenosis within a stent (neointima hyperplasia within a stent).

[0027] (15) A method for preventing restenosis within a stent, characterized by administering mesenchymal stem cells intravenously to a patient who has received a stent. Effects of the invention

[0028] According to the present invention, a pharmaceutical composition for tissue repair and regeneration that can be administered to patients who have received a stent is provided. The pharmaceutical composition of the present invention can prevent restenosis within the stent after PTAS and prevent the recurrence of stroke or the occurrence of TIA. It is known that mesenchymal stem cells have the effect of promoting the regeneration of damaged sites in ischemic vascular disorders such as myocardial infarction or cerebral infarction and improving motor function impairment. Furthermore, it is known that they have therapeutic effects on dementia, chronic cerebral infarction, chronic spinal cord injury, neurodegenerative diseases, mental disorders, and higher-order functional impairments due to the effect of promoting synapse formation and plasticity in the damaged site (lesion). Therefore, the pharmaceutical composition of the present invention can be expected to prevent restenosis within the stent and also have desirable effects on the above-mentioned symptoms. Brief explanation of the drawing

[0029] Figure 1 shows the experimental protocol of the example. (A) 14 days after stent implantation, miniature pigs were randomly divided into a placebo group and an MSC group, and either placebo or MSC (each 1.0 × 10⁶ 8 (B) Intravenous administration of cells. Disseminated angiography (DSA) and intravascular ultrasound (IVUS) were performed immediately before administration and on days 1, 7, and 28. (B) Angiography of the stent-indwelling vessel and the contralateral common carotid artery (control) after evaluation on day 28 (arrows indicate CCA and SCA). Figure 2 shows the results of angiography. CCA with CW (A: placebo group, B: MSC group), SCA with ML (C: placebo group, D: MSC group). The results of quantifying stenosis (stenosis rate) are shown in E and F (E: CCA with CW, F: SCA with ML). Scale bar = 4 mm (A, B), 1 mm (C, D). *p<0.05, **P<0.01, (CW: Carotid WALLSTENT (registered trademark), ML: Multi-Link 8 (registered trademark)). Figure 3 shows the IVUS results. CCA with CW (A: placebo group, B: MSC group), SCA with ML (C: placebo group, D: MSC group). The results of quantifying stenosis (stenosis rate) are shown in E and F (E: CCA with CW, F: SCA with ML). Scale bar = 2 mm (A, B), 1 mm (C, D). *p<0.05 Figure 4 shows the results of HE staining (low-magnification microscopic images). Control artery (A: placebo group, D: MSC group), CCA (B: placebo group, E: MSC group), SCA (C: placebo group, F: MSC group). Quantified results of stenosis (stenosis rate) are shown in G and H (G: CCA with CW, H: SCA with ML). Scale bar = 10 mm (A, B, D, E), 5 mm (C, F). *p<0.05, **P<0.01 Figure 5 shows the results of HE staining (high-magnification microscopic images). Control artery (A: placebo group, D: MSC group), CCA (B: placebo group, E: MSC group), SCA (C: placebo group, F: MSC group). Quantified results of stenosis (stenosis rate) are shown in G and H (G: CCA with CW, H: SCA with ML). Scale bar = 10 mm (A, D), 5 mm (B, C, E, F). **P<0.01 Specific details for implementing the invention

[0030] The present invention relates to a pharmaceutical composition for tissue repair and regeneration that can be administered to patients after stent placement.

[0031] 1. Mesenchymal stem cells

[0032] "Mesenchymal stem cells" used in the pharmaceutical composition of the present invention are stem cells that have multipotential and self-replicating ability and exist in small amounts among the stromal cells of mesenchymal tissue, and are known to have the ability to differentiate into connective tissue cells such as bone cells, chondrocytes, and adipocytes, as well as into nerve cells or myocardial cells.

[0033] As a source for mesenchymal stem cells, they may be cells differentiated from ES cells or induced pluripotent stem cells (iPS cells), cells that have undergone differentiation, or cells isolated and proliferated from living organisms. Examples of living organisms include bone marrow, peripheral blood, umbilical cord blood, fetal embryos, and brains, but mesenchymal stem cells derived from bone marrow or blood, particularly bone marrow mesenchymal stem cells, and among them, human bone marrow mesenchymal stem cells are preferred. Bone marrow-derived mesenchymal stem cells have advantages such as 1) the potential for significant effects, 2) a low risk of side effects, 3) the potential for a sufficient supply of donor cells, 4) being a non-invasive treatment and allowing for autologous transplantation, 5) a low risk of infection, 6) no concern regarding immune rejection, 7) no ethical issues, 8) being socially acceptable, and 9) being easy to establish as a general medical practice. Furthermore, bone marrow transplantation is a treatment already used in clinical practice, and its safety has been confirmed. In addition, bone marrow-derived stem cells have high migration capabilities, allowing them to reach the target damaged tissue not only through local transplantation but also through intravenous administration, thereby enabling the expectation of therapeutic effects.

[0034] The cells may be derived from allogeneic cells or autologous cells, but autologous (derived from the patient's own cells) mesenchymal stem cells are preferred.

[0035] It is preferable that the mesenchymal stem cells used in the present invention be in an undifferentiated state. This is because cells in an undifferentiated state have a high proliferation rate and a high survival rate after in vivo introduction. An undifferentiated state can be confirmed, for example, by the absence of expression of the differentiation marker CD24. The inventors have also developed a method for obtaining such cells, the details of which are described in WO2009 / 002503.

[0036] In the above method developed by the inventors, cells isolated from bone marrow fluid, etc., under conditions where they do not substantially come into contact with an anticoagulant (heparin, etc.) are proliferated using a medium containing allogeneic serum (preferably autologous serum; human serum in a human pharmaceutical composition) and also containing no anticoagulant (heparin, etc.) or containing it at a very low concentration. "Containing no anticoagulant or containing it at a very low concentration" means not containing an effective amount of anticoagulant as an anticoagulant. Specifically, for example, in the case of heparin or its derivatives, the effective amount as an anticoagulant is typically about 20 to 40 μ / mL, but in the method developed by the inventor, by minimizing the amount added to the blood collection tube for sample collection in advance, the amount in the sample collected from the living organism is less than 5 U / mL, preferably less than 2 U / mL, more preferably less than 0.2 U / mL, and the amount present in the medium when culturing cells is less than 0.5 U / mL, preferably less than 0.2 U / mL, more preferably less than 0.02 U / mL relative to the volume of the medium (see WO2009 / 034708).

[0037] The cell density in the culture medium affects the properties of the cells and the direction of differentiation. In the case of mesenchymal stem cells, if the cell density in the medium exceeds 8,500 cells / cm², the properties of the cells change; therefore, it is desirable to subculture at a maximum of 8,500 cells / cm² or less, and more preferably, subculture at a point where it reaches 5,500 cells / cm² or more.

[0038] In the above method developed by the inventors, since a human serum-containing medium is used, considering the burden on the serum donor, it is desirable to exchange the medium as infrequently as possible, for example, at least once a week, more preferably once or twice a week.

[0039] The total number of cells in the culture is 10 8 Subculture is repeated until there are more than [number] cells. The required number of cells may vary depending on the purpose of use, but for example, the number of mesenchymal stem cells required for transplantation to treat ischemic brain diseases such as cerebral infarction is 10 7 One or more, 10 in the present invention 6 It is thought to be more than one. According to the method developed by the inventors, 10 in about 12 days 7 Canine mesenchymal stem cells can be obtained.

[0040] Proliferated mesenchymal stem cells may be preserved, if necessary, by methods such as cryopreservation (e.g., in an ultra-low temperature freezer at -152°C) until use. 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 may be suspended in a cryopreservation solution containing 20.5 mL of ordinary filtered 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, Cryoserve manufactured by Nipro Co., Ltd. may be used as DMSO, and low molecular weight dextran L strain manufactured by Otsuka Seiyaku may be used as dextran, but are not limited to these.

[0041] The quality and function of the mesenchymal stem cells prepared as described above may be confirmed by a) adding a cytokine to a culture containing the mesenchymal stem cells and confirming that the mesenchymal stem cells express CX3CL1, or by b) confirming that the mesenchymal stem cells express EGFR and / or ITGA4.

[0042] Examples of "inflammatory cytokines" to be used include TNF-α, INFγ, IL-1, IL-6, IL-8, IL-12, and IL-18; 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.

[0043] The above method may further include a process for confirming the presence of one or more selected from BDNF, VEGF, and HGF in a culture (without cytokine addition). In particular, it is important to confirm the presence of BDNF and / or VEGF, and it is most important to confirm the presence of BDNF.

[0044] If mesenchymal stem cells express CX3CL1 upon the addition of inflammatory cytokines, it can be expected that said mesenchymal stem cells have excellent inflammatory modulatory activity (immune modulatory activity), and if more than 90% of the mesenchymal stem cells express EGFR and / or ITGA4, it can be expected that said mesenchymal stem cells have excellent aggregation ability at the site of injury. In addition, if any of the trophic factors such as BDNF, VEGF, and HGF are present in the medium, it can be expected that the medium contains mesenchymal stem cells with high neuroprotective activity, and among them, the presence of BDNF and / or VEGF, particularly the presence of BDNF, can be an important indicator of MSCs with high neuroprotective activity. Mesenchymal stem cells secrete BDNF, VEGF, and / or HGF even without stimulation, and the secretory ability can be confirmed by evaluating secretion from unstimulated cells or by evaluating secretion from cells after stimulation with inflammatory cytokines.

[0045] It is preferable to use expression at the protein level rather than the gene level as an indicator for the expression of the above CX3CL1, EGFR, ITGA4, BDNF, VEGF, and HGF. For cell surface proteins such as EGFR and ITGA4, it is preferable to measure using flow cytometry (FCM) for convenience and sensitivity, and for secreted proteins such as CX3CL1, BDNF, VEGF, and HGF, it is preferable to use a bead assay for convenience and sensitivity.

[0046] 2. Pharmaceutical composition of the present invention

[0047] The pharmaceutical composition of the present invention is a pharmaceutical composition for tissue repair and regeneration administered to a patient who has received a stent. "Intensive restenosis" refers to restenosis that occurs due to neointimal hyperplasia within an indwelling stent after PTAS. Typically, restenosis occurs 3 to 6 months after stent placement and is a serious complication that leads to a recurrence of cerebral infarction or a recurrent ischemic attack. The pharmaceutical composition of the present invention is a pharmaceutical composition for preventing this intwelling restenosis.

[0048] "Pharmaceuticals for tissue repair and regeneration" means pharmaceuticals intended to aid in the repair of damaged areas or to aid in the repair of aged areas. Tissues are not specifically limited, but examples may include nervous system tissues including the brain and spinal cord, kidneys, pancreas, liver, intestines, stomach, digestive organs, lungs, heart, spleen, blood vessels, blood, skin, bone, cartilage, teeth, and the prostate. Specifically, diseases or disorders subject to pharmaceuticals for tissue repair and regeneration include, but are not limited to, kidney disorders, liver disorders, pancreatic disorders including diabetes mellitus, prostate enlargement, hyperlipidemia, higher-order brain function disorders including aphasia and dementia, post-resuscitation encephalopathy, ischemic heart diseases including angina pectoris and myocardial infarction, ischemic cerebrovascular disorders such as cerebral infarction and arteriosclerosis, and spinal cord injuries (see WO2009 / 034708).

[0049] Although it is preferable to have a larger number of mesenchymal stem cells included in the pharmaceutical composition of the present invention, considering the timing of administration to the patient and the time required for culture, it is practical to have the minimum amount that produces an effect. Therefore, in a preferred embodiment of the pharmaceutical composition of the present invention, the number of mesenchymal stem cells is 10 6 At least 10, preferably 5×10 6 One or more, more preferably 10 7 At least one, more preferably 5×10 7 One or more, more preferably 108 At least one, more preferably 5×10 8 It is more than one.

[0050] The pharmaceutical composition of the present invention is preferably a parenteral administration formulation, more preferably a parenteral systemic administration formulation, particularly an intravenous administration formulation. Formulations suitable for parenteral administration include injectables such as solution-based injectables, suspension-based injectables, emulsion-based injectables, and injectables prepared on the spot, or grafts. The parenteral administration formulation 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, for example, a pharmacologically acceptable carrier or medium, specifically sterile water or physiological saline, a culture medium (particularly a medium used for culturing mammalian cells such as RPMI), a physiological buffer such as PBS, vegetable oil, an emulsifier, a suspending agent, a surfactant, a stabilizer, an excipient, a vehicle, a preservative, a binder, etc. In addition, it may be maintained using a drug-eluting stent (DES).

[0051] As an aqueous solution for injection, examples include physiological saline, a culture medium, a physiological buffer such as PBS, an isotonic solution containing glucose or other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, etc., and may be used in combination with a suitable solubilizing adjuvant, such as an alcohol, specifically ethanol, polyalcohol, propylene glycol, polyethylene glycol, or a nonionic surfactant, such as polysorbate 80, HCO-50, etc.

[0052] The first administration of the pharmaceutical composition of the present invention is preferably within 180 days after PTAS, more preferably 0 to 90 days, more preferably 0 to 60 days, and even more preferably 0 to 30 days, but is not limited thereto.

[0053] 3. Targets for administration of the pharmaceutical composition of the present invention

[0054] The pharmaceutical composition of the present invention is administered to a patient who has received stent placement. “Stent placement” is a treatment in which a stent is inserted, placed, and retained inside a blood vessel to reopen a narrowed artery from within the blood vessel, to treat dissecting lesions such as aortic dissection, or to reinforce a blood vessel in an aneurysm. Methods for stent placement include carotid artery stent placement (CAS) and percutaneous angioplasty and stent placement (PTAS), but are not limited thereto.

[0055] CAS is a treatment method that involves placing a stent in the narrowed area of ​​the carotid artery and expanding the blood vessel. PTAS is a treatment method that involves inserting a balloon catheter into the blood vessel, inflating the balloon at the narrowed or occluded area to expand it, and then placing a stent to expand the blood vessel. The present invention is intended for all patients who have received stent placement and are at risk of restenosis within the stent.

[0056] Patients often have underlying diseases such as atherosclerosis, ischemic heart disease including myocardial infarction, ischemic cerebrovascular disorders including cerebral infarction and transient ischemic attack (TIA), occlusive arteriosclerosis (ASO), Buerger's disease, arteriosclerotic lesions in blood vessels throughout the body, dissecting vascular lesions including dissecting aneurysms, and aneurysms.

[0057] [Ischemic Cerebrovascular Disorder]

[0058] Examples of ischemic cerebrovascular disorders include cerebral infarction (e.g., atheromatous thrombotic 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).

[0059] [Ischemic Heart Disease]

[0060] Ischemic heart disease refers to a condition in which blood fails to reach the myocardium due to the narrowing or occlusion of the coronary arteries caused by factors such as arteriosclerosis; an example is myocardial infarction. Myocardial infarction refers to a condition in which occlusion or narrowing occurs in the coronary arteries that supply oxygen and nutrients to the heart, reducing blood flow and causing the myocardium to become ischemic and undergo necrosis.

[0061] [Atheropathic arteriosclerosis]

[0062] Atherosclerosis refers to the hardening of arteries due to lipid abnormalities, diabetes, high blood pressure, smoking, lack of exercise, etc., and ultimately results in the blockage of blood flow in the arteries, causing cerebral infarction or myocardial infarction.

[0063] [Vascular dissociative lesions]

[0064] A vascular dissecting lesion refers to a pathological condition in which, such as a dissecting aneurysm, the media of the intima, media, and adventitia that make up the artery wall becomes weakened, causing the intima and adventitia to dissipate, resulting in the blood vessel at the site of the lesion consisting only of the adventitia, making it prone to rupture.

[0065] [aneurism]

[0066] An aneurysm refers to a pathological condition in which the wall of an artery in the affected area expands thinly and becomes cyst-like, such as a cerebral aneurysm, aortic aneurysm, or abdominal aortic aneurysm.

[0067] 4. Effects of the pharmaceutical composition of the present invention

[0068] The pharmaceutical composition of the present invention can prevent restenosis within a stent by alleviating inflammatory responses and inhibiting neointimal hyperplasia. By doing so, the risk of stroke recurrence or recurrent ischemic attack associated with restenosis within a stent can be reduced, thereby enabling safe stent placement.

[0069] As described above, mesenchymal stem cells are known to have effects such as neuroprotection, neurogenesis, induction of axonal regeneration, angiogenesis, restoration of the blood-brain barrier, erythroplasia, preservation of cortical junctions, neuroplasticity, and secretion of neurotrophic factors that induce remote responses. In relation to these effects, the inventors have confirmed that the administration of mesenchymal stem cells promotes the regeneration of damaged sites in ischemic vascular disorders such as myocardial infarction or cerebral infarction and improves motor function impairments. Furthermore, it has been confirmed that the administration of mesenchymal stem cells can treat dementia, chronic cerebral infarction, chronic spinal cord injury, neurodegenerative diseases, mental disorders, and higher-order functional impairments through the effects of promoting synapse formation and plasticity at the damaged site (lesion). Therefore, the pharmaceutical composition of the present invention is expected to prevent restenosis within the stent and also exert desirable effects on the aforementioned underlying diseases that the patient has or has had.

[0070] Examples

[0071] 1. Materials and Methods

[0072] (1) Preparation of human mesenchymal stem cells

[0073] Human MSCs were prepared according to the method. Specifically, MSCs (P2) were purchased from Lonza (Walkersville, MD), diluted in Dulbecco modified Eagle medium, supplemented with 10% FBS (Life Technologies), 2 mM L-glutamine (Sigma-Aldrich), and 100 U / ml penicillin-streptomycin (Sigma-Aldrich), seeded in 150 mm tissue culture dishes (IWAKI), and incubated for several days at 37°C under a 5% CO2 humidified atmosphere. When the cells had nearly reached fusion, adhered cells were detached using trypsin-EDTA solution (Mediatech Inc.), and 1 × 10⁶ cells were prepared. 4Subcultured at a cell / ml level. In this way, MSCs were produced at a relatively short culture period (subculture: 4 times) at a rate of 1×10⁶ 8 Cells were proliferated. The proliferated MSCs were isolated, diluted with a preservation solution [8 ml of RPMI (Life Technologies), 8 ml of autologous serum obtained from each animal, 2 ml of low molecular weight dextran L (Otsuka), and 2 ml of dimethyl sulfoxide (Nipro)] on the day of stent insertion, and stored in a freezer (-80°C) (Sanyo) until use. A preservation solution not containing MSCs was prepared as a placebo. On the day of administration, the preservation solution containing the cryopreserved MSCs was thawed in a 37°C water bath at the bedside of the burn treatment room. 1.0 × 10⁶ 8 Canine cells (survival rate; 99.6±0.5%) or a placebo were administered intravenously to each animal. The phenotype of the surface antigen on the MSCs was CD34 - , CD45 - , CD73 + and CD105 + was.

[0074] (2) Experimental protocol

[0075] The experimental protocol is shown in Figure 1. SPF adult minipigs (Gottingen Minipig, Oriental Yeast Co., Tokyo, Japan) weighing 30 to 38 kg were pretreated with 100 mg / day of aspirin and 15 mg / day of lansoprazole 7 days prior to stent insertion. On the day of stent insertion, two types of stents were inserted: Carotid WALLSTENT (registered trademark) (Boston Scientific, Natick, MA) and Multi-Link 8 (registered trademark) (Abbott Vascular Company, Abbott Park, IL). 14 days after stent insertion, following angiography (DAS) and intravascular ultrasound (IVUS), all pigs were administered a placebo or MSC (1.0 × 10⁻¹⁰ each) via the right ear vein under general anesthesia. 8Cells) were administered intravenously, and miniature pigs were randomly divided into two experimental groups: a placebo group and an MSC group. Starting one day before the administration of the placebo or MSC, all pigs were administered cyclosporine A (10 mg / kg, oral) daily.

[0076] Dissection angiography (DSA) and IVUS were performed immediately after the administration of placebo or MSC, and on days 1, 7, and 28. After evaluation on day 28, the PTAS-treated vessel and the contralateral common carotid artery (CCA), which served as the control artery, were excised for histological analysis. Physiological parameters were monitored during the procedure of subsequent tests, including PTAS, DSA, and IVUS (Table 1). Experiments using blood samples prior to the above procedure were also performed on days 0, 1, 7, and 28 after stent placement following the administration of placebo or MSC (Table 2).

[0077]

[0078] Table 1: Physiological parameters

[0079]

[0080] Table 2: Blood Sample Data

[0081] (3) Stent insertion (PTAS)

[0082] Stent placement was performed under general anesthesia. Animals were fasted for 12 hours prior to surgery. Anesthesia was induced by intramuscular injection of ketamine (10 mg / kg), dexmedetomidine hydrochloride (0.03 mg / kg), and midazolam hydrochloride (0.18 mg / kg), and maintained by inhaling isoflurane (0.5-5%) through intubation to minimize pain. During surgery, Ringer's lactic acid solution (5-10 ml / kg / hour) was administered intravenously.

[0083] Stent placement was performed using a single angiography system (Infinix Celeve-i; Toshiba Medical System, Tokyo, Japan). A 5-F sheathless guiding catheter was inserted into the right femoral artery by ultrasound-guided puncture. Subsequently, the 5-F sheathless guiding catheter was advanced and positioned in the right CCA (arrow in Fig. 1B) as a human carotid artery model. Pre-dilation was performed using a 5.0 mm balloon catheter to damage the intima. A Carotid WALLSTENT (registered trademark) (10×24 mm) was deployed in the CCA, and post-dilation was performed using a 5.0 mm balloon catheter. After stent deployment, full device dilation was confirmed by IVUS. Subsequently, a guiding catheter was positioned in the superficial carotid artery (SCA, head in arrow in Fig. 1B) as a human cerebral artery model. Contrast imaging was performed using a 3.0 mm balloon catheter, MULTI-LINK 8 (registered trademark) (3.0 × 23 mm) was deployed into the artery, and pre-dilation was performed using a 3.0 mm balloon catheter. This was confirmed by IVUS.

[0084] All mini pigs were administered 2,000 to 3,000 units of heparin intravenously, and an active coagulation time of 250 to 300 seconds was maintained. The animals were placed on a hot water recirculating heating pad. Physiological parameters, including heart rate (HR), systolic arterial blood pressure (sBP) and diastolic arterial blood pressure (dBP), body temperature (BT), and oxygen saturation (SaO2), were continuously measured via an esophageal probe.

[0085] (4) Evaluation of angiographic findings

[0086] Angiographic images were obtained using a single angiography system. This procedure was performed under general anesthesia. A 4-F sheathless guiding catheter was inserted into the femoral artery by ultrasound-guided puncture. Subsequently, the 4-F sheathless guiding catheter was advanced and positioned within the stent-indwelling artery, and contrast agent was administered selectively. Quantitative analysis of the initial and follow-up angiographies was performed using the NIH Image J plug-in "Neuro J" length analysis tool (version 1.39, National Institutes of Health, Bethesda, Maryland). Stenosis rate is defined as the percentage of neointimal thickness at the center of the stent: (1 - lumen diameter / stent diameter) × 100. All initial and follow-up measurements were performed in an anterior-posterior projection.

[0087] (5) Evaluation of IVUS findings

[0088] After acquiring angiographic images, IVUS images were obtained using an IVUS catheter (TVC Imaging System, MC8, InfraRedx Inc.). Using a 4-F sheathless guiding catheter, a 0.014-inch guide wire was advanced to the repositioning of the stent, and a 3.2F rapid exchange catheter was inserted over the wire. After administering 200 µg of nitroglycerin, IVUS images were obtained. The stenosis rate is defined as the percentage of neointimal thickness in the center of the stent: (1 - lumen diameter / stent diameter) × 100.

[0089] (6) Histological findings

[0090] At the endpoint, the animal was prepared for histological analysis (Fig. 1A). Cardiac perfusion was performed with physiological saline and 0.1 M phosphate buffer (5 liters). Subsequently, the vessel containing the stent and the contralateral CCA (as a control artery) were removed, and the tissue was fixed with 4% paraformaldehyde. This vessel was embedded in a resin mixture of 2-hydroxyethyl methacrylate and methyl methacrylate monomers. Sections of the center of the stent or normal vessel were cut with a cemented tungsten carbide knife (RM2245, Leica, Germany) and stained with hematoxylin eosin (New Histology Science Laboratory Corporation, Tokyo, Japan). The sections were evaluated using an optical microscope (BX51, Olympus).

[0091] Histological analysis including neointimal area and inflammation score (%) was performed. The neointimal area was measured around the stent and on the intimal surface. The ratio of the neointimal area was defined as neointimal area / stent area × 100. The inflammation score was used to evaluate the degree and density of inflammatory infiltration in individual struts. The evaluation was defined as follows: 0: no inflammatory cells around the strut; 1: mild non-circular lymphoid proliferative infiltration around the strut; 2: localized, moderate to high-density cell aggregates non-circularly surrounding the strut; 3: high-density lymphoid proliferative cell infiltration around the strut.

[0092] (7) Statistical analysis

[0093] All statistical analyses were performed using JMP12.2 on Windows (SAS Institute Inc., Cary, NC). To compare multiple stenosis rates measured from angiographic and IVUS images, a repeated measures analysis of variance (ANOVA) was followed by a Sidak post hoc test. Differences between groups were analyzed using Student's t-test. Error bars represent mean ± SD. A p-value < 0.050 was considered statistically significant.

[0094] 2. Results

[0095] Ten animals underwent insertion of Carotid WALLSTENT (registered trademark) into the CCA and transplantation of Multi Link 8 (registered trademark) into the SCA. All animals survived after treatment and remained healthy until the end point of this study.

[0096] (1) Angiographic findings

[0097] Since occlusion of one SCA containing Multi Link 8 (registered trademark) was confirmed by angiography prior to the administration of placebo or MSC on Day 0, 10 CCAs containing Carotid WALLSTENT (registered trademark) (placebo; n=5, MSC; n=5) and 9 SCAs containing Multi Link 8 (registered trademark) were analyzed. There was no significant difference in the diameters of the CCAs and SCAs prior to stent insertion between the placebo and MSC groups (CCA; placebo: 4.82±0.09 mm, MSC: 4.82±0.22 mm, p=0.699; SCA; placebo: 2.83±0.38 mm, MSC: 2.68±0.16 mm, p=0.424). Before the administration of placebo or MSC on day 0, there was no difference in the stenosis rates of CCA and SCA, respectively (CCA, Fig. 2A, B, E, placebo: 8.4±3.7%, MSC: 8.8±1.7%, p=0.878; SCA, Fig. 2C, D, F, placebo: 6.8±2.3%, MSC: 6.8±2.7%, p=0.995).

[0098] Although there was no statistical difference in stenosis rates after the administration of placebo or MSC, progressive stenosis due to the placement of CCA and SCA stents was observed in both the placebo and MSC groups on days 1 and 7 (Fig. 2). On day 28 after the administration of placebo or MSC, the stenosis rate of the MSC group was lower than that of the placebo group for both CCA and SCA (CCA, Fig. 2 A, B, E; placebo: 35.9±8.0%, MSC: 34.6±2.7%, p=0.028; SCA, Fig. 2 C, D, F; placebo: 24.2±3.7%, MSC: 16.8±4.2%, p=0.044). By angiographic analysis, it was clear that intravenous administration of MSCs prevents neointimal hyperplasia induced by stent implantation in CCA and SCA.

[0099] (2) IVUS findings

[0100] Ten CCAs (placebo; N=5, MSC; n=5) receiving Carotid WALLSTENT (registered trademark) and nine SCAs (placebo; n=4, MSC; N=5) receiving Multi Link 8 (registered trademark) were analyzed by IVUS. On Day 0, prior to the administration of placebo or MSC, there was no significant difference in the stenosis rates of CCA and SCA (CCA, Fig. 3A, B, E; placebo: 8.8±2.1%, MSC: 9.0±2.7%, p=0.892; SCA, Fig. 3C, D, F; placebo: 7.0±2.2%, MSC: 7.4±2.2%, p=0.857).

[0101] Although there was no statistical difference in the stenosis rates between the two groups on days 1 and 7 after the administration of placebo or MSC, on day 28 after the administration of placebo or MSC, the stenosis rates of stent-implanted CCA and SCA in the MSC group were significantly lower than in the placebo group (CCA, Fig. 3 A, B, E, placebo: 27.6±6.1%, MSC: 18.7±4.1%, p=0.892; SCA, Fig. 3 C, D, F, placebo: 19.0±3.3%, MSC: 12.0±3.2%, p=0.025). IVUS analysis demonstrated that intravenous administration of MSC prevents neointimal hyperplasia induced by stent insertion.

[0102] (3) Histological findings

[0103] On day 28 after administration of placebo or MSC, 10 CCA sites receiving Carotid WALLSTENT (registered trademark) (placebo; n=5, MSC; n=5) and 9 SCA sites receiving Multi Link 8 (registered trademark) (placebo; n=4, MSC; n=5) were analyzed. In the control arteries obtained from contralateral CCAs, neointimal hyperplasia was not observed in either the placebo or MSC administration groups (Fig. 4A, D). In CCAs treated with Carotid WALLSTENT (registered trademark), endothelial hyperplasia was observed in the placebo group by hematoxylin-eosin (HE) staining, whereas endothelial hyperplasia was low in the MSC group (Fig. 4 B, E, G: placebo: 49.4±10.3%, MSC: 38.0±3.8%, p=0.483). The same results were observed in 9 SCAs treated with Multi Link 8 (registered trademark) (Fig. 4 C, F, H: placebo: 49.4±9.3%, MSC: 33.1±4.3%, p=0.010).

[0104] High-magnification HE staining images were used to analyze the infiltration of inflammatory cells in the arterial wall. In the control artery, no inflammatory cells were observed in the arterial wall in both the placebo group and the MSC group (Fig. 5A, D). However, in both Carotid WALLSTENT (registered trademark) (Fig. 5B) and Multi Link 8 (registered trademark) (Fig. 5C), numerous infiltrating inflammatory cells were observed around the struts in the placebo group. However, in the MSC group, the extent of infiltrating inflammatory cells around the struts was less than in the placebo group for both Carotid WALLSTENT (registered trademark) (Fig. 5E) and Multi Link 8 (registered trademark) (Fig. 5F). As a result of quantitative analysis, the average inflammation scores of CCA and SCA in the MSC administration group were significantly lower compared to the placebo administration group (CCA, Fig. 5 B, E, G; placebo: 1.83±0.18, MSC: 1.39±0.14, p=0.002; SCA, Fig. 5 C, F, H; placebo: 1.89±0.22, MSC: 1.40±0.15, p=0.005). Thus, through histological analysis, it was demonstrated that intravenous administration of MSC prevents neointimal hyperplasia and the infiltration of inflammatory cells into Carotid WALLSTENT (registered trademark) and Multi Link 8 (registered trademark).

[0105] 3. Discussion

[0106] In this study, we tested the hypothesis that MSCs inhibit neointimal hyperplasia by alleviating the inflammatory response to stent struts using miniature pig CCAs and SCAs. The diameters of the miniature pig CCAs and SCAs are similar to those of human CCAs and MCAs, respectively. Bilateral angiography and IVUS analysis revealed that intravenous administration of MSCs reduced the degree of stenosis within the stent in both the CCA containing Carotid WALLSTENT (registered trademark) and the SCA containing Multi Link 8 (registered trademark). Furthermore, histological examination demonstrated that the inflammatory response around the stent struts was inhibited in the MSC administration group compared to the placebo group in both the CCA and SCA containing the stents.

[0107] In this study, neointimal proliferation was observed in stent-implanted CCAs and SCAs in all animals, but the thickness of the neointimal layer was thinner in the MSC administration group than in the placebo administration group (Fig. 4). It has been reported that inflammatory responses after stent implantation contribute to neointimal formation, leading to restenosis within the stent. Inflammatory factors such as interleukin (IL)-1 and tumor necrosis factor α (TNF-α), produced by infiltrating monocytes and macrophages, play an important role in early inflammatory responses and neointimal proliferation after stent implantation. Specifically, the secretion of IL-1 and TNF-α, which leads to an increase in Matrix Metalloproteinase-9 (MMP-9), continues for at least 3 months after stent placement, and the activation of MMP-9 contributes to the degradation of several components of the extracellular matrix, altering molecular expression in the inner wall of the vessel where the stent is placed, and causing neointimal hyperplasia and restenosis within the stent. Therefore, by reducing the inflammatory response around the stent strut and preventing the activation of MMP-9, restenosis within the stent can be inhibited.

[0108] In previous studies by the inventors, it was shown that intravenous administration of MSCs inhibits the activation of MMP-9. MSCs secrete anti-inflammatory factors such as TNF-α stimulating gene / protein 6 (TSG-6), which inhibits MMP-9 activation, and transforming growth factor beta (TGF-β1), which inhibits MMP-9 activation, through the IL-1 and TNF-α cascades. Indeed, by ELISA analysis, the human MSCs used in the inventors' study secreted TGF-β1 in vitro (93.34±12.65 PG / 1.0×10⁻⁶). 4 Cells, N=3). In addition, recent studies have shown that when MSCs are exposed to an inflammatory environment, they can modulate the immune response through the release of various mediators, including immune-controlling molecules, exosomes, and chemokines. In short, intravenous administration of MSCs may inhibit the inflammatory response around the stent strut and lead to the prevention of neointimal hyperplasia and restenosis within the stent.

[0109] Atherosclerotic lesions in major cerebral or carotid arteries are a cause of stroke and account for approximately 5 to 20 percent of ischemic strokes. In these patients with neurological lesions, stent placement is performed after the onset of stroke to prevent subsequent neurological lesions and recurrent ipsilateral stroke. Intravenous administration of MSCs is considered a novel treatment for ischemic stroke. It is believed that ischemic stroke patients who have undergone CAS or PTAS can also be treated in the future by intravenous administration of MSCs. Intravenous administration of MSCs can improve neurological symptoms and prevent stenosis within the stent and recurrent stroke. This study aims to promote this protocol, which can improve the functional return of stroke patients with implanted stents.

[0110] Intravenous administration of MSCs is known as a medicine for tissue repair and regeneration in patients with acute spinal cord injury, but its effects on patients with stents were unknown. From the results of this study, it was confirmed that there are no adverse effects of intravenous MSC administration on patients with stents, and on the contrary, it has an effect of inhibiting endovascular restenosis. This makes intravenous administration of MSCs possible for patients with acute spinal cord injury who have stents. Industrial applicability

[0111] According to the present invention, it is possible to administer a pharmaceutical composition for tissue repair and regeneration containing MSCs to patients who have received a stent. The pharmaceutical composition containing MSCs can prevent the progression of neointimal hyperplasia within the blood vessel in which the stent is received, thereby preventing restenosis after PTAS.

[0112] All publications, patents, and patent applications cited in this specification are incorporated herein by reference as is.

Claims

Claim 1 A pharmaceutical composition for preventing restenosis within a stent, comprising mesenchymal stem cells derived from bone marrow or blood, administered intravenously to a patient who has received a stent, and 0.5 × 10⁶ per single administration 8 A pharmaceutical composition characterized by administering two or more cells. Claim 2 A pharmaceutical composition according to claim 1, wherein the patient is or was a patient having any disease selected from atheromatous arteriosclerosis, ischemic heart disease including myocardial infarction, ischemic cerebrovascular disorder including cerebral infarction and transient ischemic attack (TIA), occlusive arteriosclerosis (ASO), Buerger's disease, arteriosclerotic lesions in blood vessels throughout the body, dissecting vascular lesions including dissecting aneurysms, and aneurysms. Claim 3 A pharmaceutical composition according to claim 1, wherein the stent is a carotid artery stent (CAS) or a percutaneous angioplasty stent (PTAS). Claim 4 In any one of paragraphs 1 to 3, 10 with a single administration 8 A pharmaceutical composition characterized by administering two or more cells. Claim 5 A pharmaceutical composition according to claim 4, wherein the mesenchymal stem cells are mesenchymal stem cells derived from the patient's bone marrow or blood. Claim 6 delete Claim 7 delete Claim 8 delete

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