Pharmaceutical composition containing mesenchymal stem cells for preventing in-stent restenosis

Abstract

The present invention relates to a pharmaceutical composition containing mesenchymal stem cells to be administered to a patient who has undergone stent implantation. Specifically, the present invention relates to: a pharmaceutical composition which is a pharmaceutical composition for tissue repair / regeneration characterized by containing mesenchymal stem cells and being administered to a patient who has undergone stent implantation; and the prevention of in-stent neointimal hyperplasia or in-stent restenosis by the pharmaceutical composition.

Description

Pharmaceutical composition for preventing restenosis within a stent

[0001] Related Applications This specification includes the contents described in the specification of Japanese Patent Application No. 2019-020707 (filed February 7, 2019), which is the basis for the priority of this application. Technical Field The present invention relates to a pharmaceutical composition comprising mesenchymal stem cells administered to a patient who has undergone stent placement. The pharmaceutical composition of the present invention is administered to a patient who has undergone stent placement to promote tissue repair and regeneration in the affected area and to prevent restenosis within the stent.

[0002] In carotid artery stenting (CAS) or percutaneous transluminal angioplasty and stenting (PTAS), performed to treat carotid artery or intracranial atherosclerosis, postoperative in-stent restenosis is a serious complication that can lead to recurrent stroke (cerebral infarction). While previous studies have reported a low risk of ipsilateral stroke recurrence after CAS, moderate to severe restenosis occurs more frequently after PTAS than after endarterectomy, and restenosis increases the risk of ipsilateral recurrent stroke and transient ischemic attack (TIA). Despite improvements in surgical techniques, in-stent restenosis after PTAS for intracranial atherosclerosis has also been reported as a serious complication, with an incidence of up to 31%. Stenosis results in stroke or TIA in 39% of patients. Overall, these findings highlight the need to prevent in-stent restenosis after PTAS in order to prevent ipsilateral ischemic stroke or TIA and to improve clinical outcomes after CAS and PTAS.

[0003] One possible mechanism for in-stent restenosis is neointimal hyperplasia induced by an inflammatory response to the stent struts (Non-Patent Documents 1 and 2). In cardiology, drug-eluting stents (DES) have been developed to reduce restenosis rates by preventing neointimal hyperplasia through suppression of the inflammatory response. Off-label use of DES for intracranial artery disease has also been attempted, but the rate of preoperative complications is high due to technical difficulties caused by stent rigidity and neurotoxicity. Therefore, the development of new approaches to inhibit neointimal hyperplasia is desired.

[0004] Intravenous administration of bone marrow-derived mesenchymal stem cells (MSCs) has been reported to reduce reperfusion injury and infarct volume, and improve behavioral function in experimental stroke models (Patent Documents 1-5). In addition to the therapeutic mechanisms of MSCs, such as neuroprotection, neurogenesis, axonal regeneration induction, angiogenesis, blood-brain barrier restoration, remyelination, preservation of cortical connections, and secretion of neurotrophic factors that lead to neuroplasticity and remote responses, recent studies suggest that another mechanism is the stabilization of the inflammatory microenvironment through the release of various anti-inflammatory mediators (Non-Patent Documents 3-7) (Non-Patent Document 8).

[0005] WO2002 / 000849 WO2009 / 034708 WO2018 / 034023 WO2018 / 034314 WO2017 / 188457

[0006] 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.

[0007] The object of the present invention is to provide a pharmaceutical composition for tissue repair and regeneration that can be administered to patients who have undergone stent placement, and furthermore, to provide a novel method for preventing restenosis after PTAS and the associated recurrence of stroke or TIA.

[0008] The inventors hypothesized that intravenous administration of MSCs would suppress neointimal hyperplasia by inhibiting the inflammatory response to transplanted stent struts using a pig model. They inserted two types of bare-metal stents into miniature pigs, transplanting one stent into the common carotid artery (CCA), corresponding to the human middle cerebral artery (MCA), and the other into the superficial carotid artery (SCA), corresponding to the human carotid carotid artery (CCA). Image analysis was performed using digital angiography (DSA) and intravascular ultrasound (IVUS). Furthermore, histological findings, including inflammatory changes around the stent struts after MSC administration, were evaluated. The inventors confirmed that MSCs could be administered to patients who had undergone stent placement, and that this could suppress the progression of neointimal hyperplasia in the stent-placed CCA and SCA.

[0009] The present invention was completed based on the above findings and relates to the following (1) to (15). (1) A pharmaceutical composition for tissue repair and regeneration, comprising mesenchymal stem cells, characterized in that it is administered to a patient who has undergone stent placement. (2) The pharmaceutical composition according to (1), wherein the patient is suffering from or has suffered from any of the following diseases selected from atherosclerosis, ischemic heart disease including myocardial infarction, ischemic cerebrovascular disease including cerebral infarction and transient ischemic attack (TIA), obstructive arteriosclerosis (ASO), Buerger's disease, atherosclerotic lesions in blood vessels throughout the body, vascular dissecting lesions including dissecting aneurysms, and aneurysms. (3) The pharmaceutical composition according to (1) or (2), wherein the stent placement is carotid artery stent placement (CAS) or percutaneous transluminal angioplasty and stent placement (PTAS). (4) With a single administration, 10 6 A pharmaceutical composition according to any one of (1) to (3), characterized in that one or more cells are administered. (5) A pharmaceutical composition according to any one of (1) to (4), wherein the mesenchymal stem cells are mesenchymal stem cells derived from bone marrow or blood. (6) A pharmaceutical composition according to any one of (1) to (5), wherein the mesenchymal stem cells are mesenchymal stem cells derived from the bone marrow or blood of the patient. (7) A pharmaceutical composition according to any one of (1) to (6) that prevents in-stent restenosis (neointimal hyperplasia within the stent). (8) A pharmaceutical composition for preventing in-stent restenosis, comprising mesenchymal stem cells, and characterized in that it is administered to a patient who has undergone stent placement. (9) The pharmaceutical composition according to (8), wherein the patient is suffering from or has suffered from any of the following diseases selected from atherosclerosis, ischemic heart disease including myocardial infarction, ischemic cerebrovascular disease including cerebral infarction and transient ischemic attack (TIA), arteriosclerosis obliterans (ASO), Buerger's disease, atherosclerotic lesions in blood vessels throughout the body, vascular dissecting lesions including dissecting aneurysms, and aneurysms. (10) The pharmaceutical composition according to (8) or (9), wherein the stent placement is carotid artery stent placement (CAS) or percutaneous transluminal angioplasty and stent placement (PTAS). (11) In a single dose, 10 6A pharmaceutical composition according to any one of (8) to (10), characterized in that one or more cells are administered. (12) A pharmaceutical composition according to any one of (8) to (11), wherein the mesenchymal stem cells are mesenchymal stem cells derived from bone marrow or blood. (13) A pharmaceutical composition according to any one of (8) to (12), wherein the mesenchymal stem cells are mesenchymal stem cells derived from the bone marrow or blood of the patient. (14) A pharmaceutical composition according to any one of (8) to (13), for preventing in-stent restenosis (neointimal hyperplasia within the stent). (15) A method for preventing in-stent restenosis, characterized in that mesenchymal stem cells are administered intravenously to a patient who has undergone stent placement.

[0010] The present invention provides a pharmaceutical composition for tissue repair and regeneration that can be administered to patients who have undergone stent placement. The pharmaceutical composition of the present invention can prevent in-stent restenosis after PTAS and prevent stroke recurrence and the occurrence of TIA. Mesenchymal stem cells are known to promote regeneration of damaged areas in ischemic vascular disorders such as myocardial infarction and cerebral infarction, and to improve motor function impairment. Furthermore, they are known to have therapeutic effects on dementia, chronic cerebral infarction, chronic spinal cord injury, neurodegenerative diseases, mental illnesses, and higher-order cognitive dysfunction due to their synapse formation and plasticity-promoting effects at the damaged area (lesion). Therefore, the pharmaceutical composition of the present invention is expected to prevent in-stent restenosis and also have favorable effects on the above-mentioned symptoms.

[0011] Figure 1 shows the experimental protocol for 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 (1.0 x 10) was administered. 8Cells were administered intravenously. Angiography (DSA) and intravascular ultrasound (IVUS) were performed immediately before administration and on days 1, 7, and 28. (B) Angiography of the stented 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 implanted (A: placebo group, B: MSC group), SCA with ML implanted (C: placebo group, D: MSC group). The results of quantifying stenosis (stenosis rate) are shown in E and F (E: CCA with CW implanted, F: SCA with ML implanted). Scale bars = 4 mm (A, B), 1 mm (C, D). * p<0.05, ** P<0.01, (CW: Carotid WALLSTENT®, ML: Multi-Link 8®). Figure 3 shows the results of IVUS. CCA with CW implanted (A: placebo group, B: MSC group), SCA with ML implanted (C: placebo group, D: MSC group). The quantified stenosis (stenosis rate) is shown in E and F (E: CCA with CW implanted, F: SCA with ML implanted). Scale bars = 2 mm (A, B), 1 mm (C, D). * p < 0.05. Figure 4 shows the results of HE staining (low magnification microscopy image). Control artery (A: placebo group, D: MSC group), CCA (B: placebo group, E: MSC group), SCA (C: placebo group, F: MSC group). The quantified stenosis (stenosis rate) is shown in G and H (G: CCA with CW implanted, H: SCA with ML implanted). Scale bars = 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). The quantified results of stenosis (stenosis rate) are shown in G and H (G: CCA with CW implantation, H: SCA with ML implantation). Scale bars = 10 mm (A, D), 5 mm (B, C, E, F). ** P<0.01

[0012] This invention relates to a pharmaceutical composition for tissue repair and regeneration that can be administered to patients after stent placement.

[0013] 1. Mesenchymal Stem Cells The "mesenchymal stem cells" used in the pharmaceutical composition of the present invention are stem cells that exist in trace 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] The source of mesenchymal stem cells can be cells differentiated from ES cells or induced pluripotent stem cells (iPS cells), established cell lines, or cells isolated and proliferated from living organisms. In living organisms, examples include bone marrow, peripheral blood, umbilical cord blood, fetal embryos, and brain, but bone marrow or blood-derived mesenchymal stem cells, especially bone marrow mesenchymal stem cells, and among these, human bone marrow mesenchymal stem cells, are preferred. Bone marrow-derived mesenchymal stem cells have several advantages, including: 1) the expectation of significant effects, 2) a low risk of side effects, 3) the expectation of a sufficient supply of donor cells, 4) it is a non-invasive treatment and autologous transplantation is possible, 5) a low risk of infection, 6) no concern about immune rejection, 7) no ethical issues, 8) social acceptance, and 9) it is likely to become widely established as a common medical treatment. 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 migratory properties 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 derived from allogeneic cells or autologous cells, but autologous mesenchymal stem cells (derived from the patient's own 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 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, 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 where they do not substantially contact an anticoagulant (such as heparin) are grown using a medium containing homologous serum (preferably autologous serum; human serum for human pharmaceutical compositions) and not containing an anticoagulant (such as heparin) or containing it at an extremely low concentration. "Not containing an anticoagulant or containing it at an extremely low concentration" means not containing an effective amount of the anticoagulant as an anticoagulant. Specifically, for example, in the case of heparin or its derivatives, the effective amount as an anticoagulant is usually about 20 - 40 μ / mL, but in the method developed by the inventors, by minimizing the amount added to the blood collection tube for sample collection in advance, the amount in the sample collected from the living body is less than 5 U / mL, preferably less than 2 U / mL, more preferably less than 0.2 U / mL, and the amount present in the medium when culturing the cells is less than 0.5 U / mL, preferably less than 0.2 U / mL, more preferably less than 0.02 U / mL with respect to the volume of the medium (see WO2009 / 034708).

[0018] The density of cells in the medium affects the properties and differentiation direction of the cells. In the case of mesenchymal stem cells, if the cell density in the medium exceeds 8,500 cells / cm 2 exceeds, the properties of the cells will change, so it is preferably subcultured at a maximum of 8,500 cells / cm 2 or less, and more preferably subcultured when it reaches 5,500 cells / cm 2 or more.

[0019] Since the inventors' developed method uses a medium containing human serum, considering the burden on the serum donor, it is desirable that the medium exchange be performed as few times as possible. For example, the medium is exchanged at least once a week, more preferably once to twice a week.

[0020] The culture is repeatedly subcultured until the total number of cells reaches 10 8 or more. The required number of cells can vary depending on the purpose of use. For example, the number of mesenchymal stem cells required for transplantation for the treatment of ischemic brain diseases such as cerebral infarction is 10 7 or more, and in the present invention, 10 6It is thought that there are more than 10. According to the method developed by the inventors, 10 can be produced in about 12 days. 7 Individual mesenchymal stem cells can be obtained.

[0021] The proliferated mesenchymal stem cells may be stored, if necessary, by cryopreservation or other methods (e.g., in a deep freezer at -152°C) until use. For cryopreservation, a culture medium containing serum (preferably human serum, more preferably autologous serum), dextran, and DMSO (such as a mammalian cell medium like RPMI) is used as the cryopreservation solution. For example, cells can be suspended in a cryopreservation solution containing 20.5 mL of normally filtered and sterile RPMI, 20.5 mL of autologous serum collected from the patient, 5 mL of dextran, and 5 mL of DMSO, and then cryopreserved at -150°C. For example, Nipro Corporation's Cryoserve can be used as DMSO, and Otsuka Pharmaceutical's Low Molecular Weight Dextran L Injection can be used as dextran, but these are not the only options.

[0022] The quality and function of the mesenchymal stem cells prepared as described above may be confirmed by a) adding cytokines to the 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.

[0023] Examples of "inflammatory cytokines" to be used include TNF-α, INFγ, IL-1, IL-6, IL-8, IL-12, and IL-18, with TNF-α, INFγ, and IL-6 being preferred, and a mixture of TNF-α, INFγ, and IL-6 being even more preferred.

[0024] The above method may further include a step of confirming the presence of one or more selected from BDNF, VEGF, and HGF in the culture (without added cytokines). In particular, it is important to confirm the presence of BDNF and / or VEGF, and confirming the presence of BDNF is the most important.

[0025] If mesenchymal stem cells express CX3CL1 upon the addition of inflammatory cytokines, these mesenchymal stem cells can be expected to have superior anti-inflammatory (immunomodulatory) effects. Furthermore, if more than 90% of mesenchymal stem cells express EGFR and / or ITGA4, these mesenchymal stem cells can be expected to have superior accumulation ability at injury sites. In addition, if any of the nutritional factors such as BDNF, VEGF, and HGF are present in the culture medium, it can be expected that the medium contains mesenchymal stem cells with high neuroprotective effects. In particular, the presence of BDNF and / or VEGF, and especially BDNF, can be important indicators of MSCs with high neuroprotective effects. Mesenchymal stem cells secrete BDNF, VEGF, and / or HGF even without stimulation, but secretory capacity can be confirmed by evaluating secretion from unstimulated cells or by evaluating secretion from cells after stimulation with inflammatory cytokines.

[0026] For the expression of CX3CL1, EGFR, ITGA4, BDNF, VEGF, and HGF, it is preferable to use protein-level expression as an indicator rather than gene-level expression. For cell surface proteins such as EGFR and ITGA4, flow cytometry (FCM) is preferred for its simplicity and sensitivity, while for secreted proteins such as CX3CL1, BDNF, VEGF, and HGF, a bead assay is preferred for its simplicity and sensitivity.

[0027] 2. Pharmaceutical Composition of the Present Invention The pharmaceutical composition of the present invention is a tissue repair and regeneration pharmaceutical composition administered to patients who have undergone stent placement. "Intrastent restenosis" refers to restenosis that occurs after PTAS due to neointimal hyperplasia within the implanted stent. Typically, restenosis occurs 3 to 6 months after stent placement and is a serious complication that can lead to recurrence of cerebral infarction or ischemic recurrence. The pharmaceutical composition of the present invention is a pharmaceutical composition for preventing this intrastent restenosis.

[0028] "Tissue repair and regeneration drugs" refer to drugs that assist in the repair of damaged areas or areas that have aged due to aging. The tissues are not particularly limited, but examples include nervous system tissues including the brain and spinal cord, kidneys, pancreas, liver, intestines, stomach, digestive organs, lungs, heart, spleen, blood vessels, blood, skin, bones, cartilage, teeth, and prostate gland. Diseases and disorders that can be treated with tissue repair and regeneration drugs include, but are not limited to, kidney disorders, liver disorders, pancreatic disorders including diabetes, benign prostatic hyperplasia, hyperlipidemia, higher brain dysfunction including aphasia and dementia, post-resuscitation encephalopathy, ischemic heart disease including angina pectoris and myocardial infarction, ischemic cerebrovascular disorders such as cerebral infarction and arteriosclerosis, and spinal cord injury (see WO2009 / 034708).

[0029] While a higher number of mesenchymal stem cells is preferable in the pharmaceutical composition of the present invention, it is practical to use the minimum amount necessary to show efficacy, taking into account the timing of administration to the patient and the time required for culture. Therefore, in a preferred embodiment of the pharmaceutical composition of the present invention, the number of mesenchymal stem cells is 10 6 One or more, preferably 5 × 10 6 More than 10 comfortable 7 More than one, comfortable 5x10 7 More than 10 comfortable 8 One or more, more preferably 5 × 10 8 There are more than one.

[0030] The pharmaceutical composition of the present invention is preferably a parenteral administration preparation, more preferably a parenteral systemic administration preparation, particularly an intravenous administration preparation. Dosage forms suitable for parenteral administration include injections such as solution injections, suspension injections, emulsion injections, injections prepared at the time of use, and grafts. The preparation for parenteral administration is in the form of an aqueous or non-aqueous isotonic sterile solution or suspension. For example, it is formulated into an appropriate unit dosage form by appropriately combining a pharmaceutically acceptable carrier or medium, specifically, sterile water, physiological saline, culture medium (especially the culture medium 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. It may also be held in a drug eluting stent (DES) or the like.

[0031] Examples of the aqueous solution for injection include physiological buffer solutions such as physiological saline, culture medium, and PBS, isotonic solutions containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, etc. It may be used in combination with a suitable solubilizing agent, such as alcohol, specifically ethanol, polyalcohol, propylene glycol, polyethylene glycol, and nonionic surfactants, such as polysorbate 80, HCO-50, etc.

[0032] The first administration of the pharmaceutical composition of the present invention is preferably within 180 days after PTAS, more preferably from 0 day to 90 days, more preferably from 0 day to 60 days, and even more preferably from 0 day to 30 days, but is not limited thereto.

[0033] 3. Administration target of the pharmaceutical composition of the present invention The pharmaceutical composition of the present invention is administered to patients who have received stent implantation. "Stent implantation" is a treatment in which a stent is inserted, placed, and retained inside a blood vessel in order to reopen a stenosed artery, to treat dissection lesions such as aortic dissection, or to reinforce a blood vessel in an aneurysm. Methods of stent implantation include carotid artery stenting (CAS) and percutaneous transluminal angioplasty and stenting (PTAS), etc., but are not limited thereto.

[0034] CAS is a treatment method that involves implanting a stent at the stenotic site of the carotid artery to dilate the blood vessel. PTAS is a treatment method that involves inserting a balloon catheter into the blood vessel, inflating the balloon at the stenotic or occluded site to dilate it, and then implanting a stent to dilate the blood vessel. The present invention targets all patients who have received stent implantation and are at risk of in-stent restenosis.

[0035] Patients often have, as primary diseases, atherosclerotic diseases such as atherosclerosis, ischemic heart diseases including myocardial infarction, ischemic cerebrovascular diseases including cerebral infarction and transient ischemic attack (TIA), arteriosclerosis obliterans (ASO), Buerger's disease, atherosclerotic lesions in blood vessels throughout the body, dissecting vascular lesions including dissecting aneurysm, and aneurysm, etc.

[0036] [Ischemic cerebrovascular disease] Examples of ischemic cerebrovascular diseases include, for example, cerebral infarction (such as atherosclerotic thrombotic cerebral infarction, cerebral thrombosis, cerebral embolism, lacunar infarction, BAD (Branch Atheromatous Disease), Trousseau's syndrome, blood coagulation disorder, arterial dissection, venous infarction, vasculitis, antiphospholipid antibody syndrome), and transient ischemic attack (TIA), etc.

[0037] [Ischemic heart disease] Ischemic heart disease is a disease caused by stenosis or occlusion of the coronary artery due to causes such as arteriosclerosis, resulting in the inability of blood to reach the myocardium. For example, myocardial infarction can be cited. Myocardial infarction refers to a state in which occlusion or stenosis occurs in the coronary blood vessels that supply oxygen and nutrients to the heart, the blood flow decreases, and the myocardium becomes ischemic and necrotic.

[0038] [Atherosclerosis] Atherosclerosis means that the artery hardens due to lipid abnormalities, diabetes, hypertension, smoking, lack of exercise, etc., and ultimately causes cerebral infarction, myocardial infarction, etc. as a result of the blood flow in the artery being blocked.

[0039] [Dissecting vascular lesion] Dissecting vascular lesion refers to a pathological condition in which, among the intima, media, and adventitia that make up the arterial wall, such as dissecting aneurysm, the media becomes fragile and weakened, and the intima and adventitia dissociate. As a result, the blood vessel at the lesion site consists only of the adventitia and is in a state where it is likely to rupture.

[0040] [Aneurysm] An aneurysm is a condition in which the wall of an artery in a diseased area becomes thin and bulges, forming a swollen mass, such as a cerebral aneurysm, aortic aneurysm, or abdominal aortic aneurysm.

[0041] 4. Effects of the Pharmaceutical Composition of the Present Invention The pharmaceutical composition of the present invention can prevent in-stent restenosis by reducing inflammatory responses and inhibiting neointima-hyperplasia. This reduces the risk of stroke recurrence and ischemic recurrence associated with in-stent restenosis, thereby enabling safe stent placement.

[0042] As mentioned above, mesenchymal stem cells are known to have effects such as neuroprotection, neurogenesis, axonal regeneration induction, angiogenesis, blood-brain barrier restoration, remyelination, preservation of cortical connections, and secretion of neurotrophic factors that bring about neuroplasticity and remote responses. In connection with these effects, the inventors have confirmed that administration of mesenchymal stem cells promotes regeneration of damaged sites in ischemic vascular disorders such as myocardial infarction and cerebral infarction, and improves motor function impairment. Furthermore, they have confirmed that administration of mesenchymal stem cells can treat dementia, chronic cerebral infarction, chronic spinal cord injury, neurodegenerative diseases, mental illnesses, and higher-order cognitive dysfunction by promoting synapse formation and plasticity at the site of injury (lesion). Therefore, the pharmaceutical composition of the present invention is expected to prevent in-stent restenosis and also exert favorable effects on the above-mentioned underlying diseases that the patient has or has had.

[0043] 1. Materials and Methods (1) Preparation of Human Mesenchymal Stem Cells Human MSCs were prepared according to previously reported methods. Specifically, MSCs (P2) were purchased from Lonza (Walkersville, MD), diluted in Dulbecco's modified Eagle medium, and 10% FBS (Life Technologies), 2 mM L-glutamine (Sigma-Aldrich), and 100 U / ml penicillin-streptomycin (Sigma-Aldrich) were added. The cells were then seeded in 150 mm tissue culture dishes (IWAKI) and incubated at 37°C for several days under a humidified atmosphere of 5% CO2. When the cells reached near confluence, adherent cells were detached with trypsin-EDTA solution (Mediatech Inc.) and 1 × 10⁶ cells were removed. 4The cells were subcultured at a rate of cells / ml. Thus, the MSCs were cultured for a relatively short period (subculture: 4 times) to obtain 1 × 10⁶ cells. 8 The cells were grown to the cellular level. The grown MSCs were isolated and diluted on the day of stent insertion 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)], and stored in a freezer (-80°C) (Sanyo) until use. A preservation solution without MSCs was prepared as a placebo. On the day of administration, the preservation solution containing the frozen MSCs was thawed in a 37°C water bath at the bedside in the image-guided treatment room. 1.0 × 10 8 Each animal was intravenously administered either individual cells (viability: 99.6 ± 0.5%) or a placebo. The phenotype of the surface antigen on the MSC was CD34. - CD45 - CD73 + , and CD105 + That was the case.

[0044] (2) Experimental Protocol The experimental protocol is shown in Figure 1. SPF adult miniature pigs weighing 30-38 kg (Gottingen Minipig, Oriental Yeast Co., Tokyo, Japan) were pre-treated with 100 mg / day of aspirin and 15 mg / day of lansoprazole 7 days before stent insertion. On the day of stent insertion, two types of stents were inserted: Carotid WALLSTENT® (Boston Scientific, Natick, MA) and Multi-Link 8® (Abbott Vascular Company, Abbott Park, IL). Fourteen days after stent insertion, after angiography (DAS) and intravascular ultrasound (IVUS), all pigs were given either placebo or MSC (1.0 × 10¹⁶ each) via the right ear vein under general anesthesia. 8 Miniature cells were administered intravenously, and the pigs were randomly divided into two experimental groups: a placebo group and an MSC group. Starting one day before placebo or MSC administration, all pigs were administered cyclosporine A (10 mg / kg, orally) daily.

[0045] Angiography (DSA) and IVUS were performed immediately after placebo or MSC administration, and on days 1, 7, and 28. After evaluation on day 28, the PTAS-treated vessels and the contralateral common carotid artery (CCA), which served as the control artery, were excised for histological analysis. Physiological parameters were monitored during the follow-up study procedures, including PTAS, DSA, and IVUS (Table 1). Experiments using blood samples prior to the aforementioned procedures were also performed on days 0, 1, 7, and 28 after placebo or MSC administration and stent placement (Table 2).

[0046] Table 1: Physiological parameters

[0047] Table 2: Blood sample data

[0048] (3) Stent placement (PTAS) Stent placement was performed under general anesthesia. The animals were fasted for 12 hours prior to the 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 anesthesia was maintained by inhalation of isoflurane (0.5-5%) via intubation to minimize pain. During the surgery, lactated Ringer's solution (5-10 ml / kg / hour) was administered intravenously.

[0049] 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. Next, the 5-F sheathless guiding catheter was advanced and placed in the right carotid artery accelerae (CCA) (Figure 1B, arrow) as a model of the human carotid artery. Predilation was performed using a 5.0 mm balloon catheter to damage the intima layer. A Carotid WALLSTENT® (10 × 24 mm) was deployed in the CCA, and postdilation was performed using a 5.0 mm balloon catheter. After stent deployment, complete device expansion was confirmed by IVUS. Next, a guiding catheter was placed in the superficial carotid artery (SCA, Figure 1B, head of arrow) as a model of the human cerebral artery. Angiography was performed using a 3.0 mm balloon catheter. A MULTI-LINK8® (registered trademark) (3.0 × 23 mm) was deployed into the artery, and predation was performed using the 3.0 mm balloon catheter. Confirmation was made by IVUS.

[0050] All miniature pigs were intravenously administered 2000–3000 units of heparin to maintain an activated clotting time of 250–300 seconds. The animals were placed on a warm water recirculation heating pad. Physiological parameters such as 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.

[0051] (4) Evaluation of angiographic findings 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. The 4-F sheathless guiding catheter was then advanced and positioned within the stent-placed artery, and contrast agent was selectively administered. Quantitative analysis of initial and follow-up angiography was performed using the NIH Image J plug-in "Neuro J" length analysis tool (version 1.39, National Institutes of Health, Bethesda, Maryland). The stenosis rate was defined as the percentage of neointimal thickness in the central part of the stent: (1 - lumen diameter / stent diameter) × 100. All initial and follow-up measurements were performed in anterior-posterior projection.

[0052] (5) Evaluation of IVUS findings After obtaining angiographic images, IVUS images were obtained using an IVUS catheter (TVC Imaging System, MC8, InfraRedx Inc.). A 0.014-inch guidewire was advanced distal to the stent using a 4-F sheathless guiding catheter, and a 3.2F rapid exchange catheter was inserted onto the wire. After administration of 200 μg nitroglycerin, IVUS images were obtained. The stenosis rate is defined as the percentage of neointima thickness in the central part of the stent: (1 - lumen diameter / stent diameter) × 100.

[0053] (6) Histological findings At the endpoint, the animals were prepared for histological analysis (Figure 1A). Cardiac perfusion was performed with physiological saline and 0.1 M phosphate buffer (5 liters). The stented vessel and the contralateral CCA (as a control artery) were then removed, and the tissue was fixed with 4% paraformaldehyde. This container was embedded in a mixed resin of 2-hydroxyethyl methacrylate and methyl methacrylate monomer. Sections of the central part 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 a light microscope (BX51, Olympus).

[0054] Histological analysis was performed, including neointimal area and inflammation score (%). Neointimal area was measured around the stent and on the intima. The percentage of neointimal area was defined as neointimal area / stent area × 100. The inflammation score was used to assess the degree and density of inflammatory infiltration in individual struts. The evaluation was as follows: 0: no inflammatory cells around the strut, 1: mild non-circular lymphoid tissue proliferative infiltration around the strut, 2: localized, moderate to dense cell aggregates surrounding the strut in a non-circular shape, 3: dense lymphoid cell infiltration around the strut.

[0055] (7) Statistical Analysis All statistical analyses were performed using JMP 12.2 on Windows (SAS Institute Inc., Cary, NC). To compare multiple stenosis rates measured by angiography and IVUS images, repeated measures analysis of variance (ANOVA) was performed followed by the Sidak post hoc test. Differences between groups were analyzed using the Student t-test. Error bars represent the mean ± SD. A p-value < 0.050 was considered statistically significant.

[0056] 2. Results Ten animals underwent insertion of Carotid WALLSTENT® into the CCA and transplantation of Multi Link8® into the SCA. All animals survived the procedure and remained healthy until the endpoint of this study.

[0057] (1) Angiographic findings Angiography performed on day 0 before administration of placebo or MSC confirmed occlusion of one SCA where Multi Link 8® was placed. Therefore, 10 CCAs where Carotid WALLSTENT® was placed (placebo; n = 5, MSC; n = 5) and 9 SCAs where Multi Link 8® was placed (placebo; n = 4, MSC; n = 5) were analyzed. There was no significant difference in the diameter of CCAs and SCAs before stent insertion between the placebo group and the MSC group (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). On day 0, before administration of placebo or MSC, there was no difference in the stenosis rates of the CCA and SCA, respectively (CCA, Figures 2A, B, E, placebo: 8.4±3.7%, MSC: 8.8±1.7%, p = 0.878; SCA, Figures 2C, D, F, placebo: 6.8±2.3%, MSC: 6.8±2.7%, p = 0.995).

[0058] After administration of placebo or MSC, there was no statistically significant difference in the stenosis rate. However, progressive stent-induced stenosis in the CCA and SCA was observed in both the placebo and MSC groups on days 1 and 7 (Figure 2). On day 28 after administration of placebo or MSC, the stenosis rate in the MSC-administered group was lower than that in the placebo group in both the CCA and SCA (CCA, Figure 2A, B, E, placebo: 35.9±8.0, MSC: 34.6±2.7, p = 0.028; SCA, Figure 2C, D, F, placebo: 24.2±3.7%, MSC: 16.8±4.2%, p = 0.044). Angiographic analysis revealed that intravenous administration of MSC prevented neointimal hyperplasia induced by stent implantation in the CCA and SCA.

[0059] (2) IVUS findings Ten carotid ductal septa (CCAs) implanted with Carotid WALLSTENT® (placebo; N = 5, MSC; n = 5), and nine scapular septa (SCAs) implanted with Multi Link8® (placebo; n = 4, MSC; N = 5) were analyzed by IVUS. On day 0, before placebo or MSC administration, there was no significant difference in the stenosis rate of CCAs and SCAs (CCA, Figures 3A, B, E, placebo: 8.8±2.1%, MSC: 9.0±2.7%, p = 0.892; SCA, Figures 3C, D, F, placebo: 7.0±2.2%, MSC: 7.4±2.2%, p = 0.857).

[0060] There were no statistically significant differences in stenosis rates between the two groups on days 1 and 7 after placebo or MSC administration. However, on day 28 after placebo or MSC administration, the stenosis rates of stent-placed CCA and SCA were significantly lower in the MSC-administered group than in the placebo-administered group (CCA, Figures 3A, B, E, placebo: 27.6±6.1%, MSC: 18.7±4.1%, p = 0.892; SCA, Figures 3C, D, F, placebo: 19.0±3.3%, MSC: 12.0±3.2%, p = 0.025). IVUS analysis demonstrated that intravenous administration of MSCs prevented neointimal hyperplasia induced by stent insertion.

[0061] (3) Histological findings On day 28 after administration of placebo or MSC, 10 carotid artery acicular occlusions (CCAs) implanted with Carotid WALLSTENT® (placebo; n = 5, MSC; n = 5) and 9 small artery acicular occlusions (SCAs) implanted with Multi Link 8® (placebo; n = 4, MSC; n = 5) were analyzed. In control arteries obtained from contralateral CCAs, neointimal hyperplasia was not observed in either the placebo or MSC groups (Figure 4A, D). In CCAs implanted with Carotid WALLSTENT®, neointimal hyperplasia was observed in the placebo group by hematoxylin-eosin (HE) staining, but was low in the MSC group (Figure 4B, E, G: placebo: 49.4±10.3%, MSC: 38.0±3.8%, p = 0.483). Similar results were observed in nine SCAs implanted with Multi Link 8® (Figure 4C, F, H: Placebo: 49.4±9.3%, MSC: 33.1±4.3%, p = 0.010).

[0062] High-magnification HE-stained images were used to analyze inflammatory cell infiltration in the arterial wall. In the control arteries, no inflammatory cells were observed in the arterial wall in either the placebo or MSC group (Figure 5A, D). However, in both Carotid WALLSTENT® (Figure 5B) and Multi Link 8® (Figure 5C), numerous peristral inflammatory cells were observed in the placebo group. However, in the MSC group, the degree of peristral inflammatory cell infiltration was less than in the placebo group in both Carotid WALLSTENT® (Figure 5E) and Multi Link 8® (Figure 5F). Quantitative analysis revealed that the mean inflammation scores for CCA and SCA in the MSC-treated group were significantly lower than in the placebo-treated group (CCA, Figures 5B, E, G, Placebo: 1.83±0.18, MSC: 1.39±0.14, p = 0.002; SCA, Figures 5C, F, H, Placebo: 1.89±0.22, MSC: 1.40±0.15, p = 0.005). Thus, histological analysis demonstrated that intravenous administration of MSCs prevented neointima-intimal hyperplasia and the infiltration of inflammatory cells into Carotid WALLSTENT® and Multi Link8®.

[0063] 3. Discussion This study tested the hypothesis that MSCs inhibit neointimal hyperplasia by reducing the inflammatory response to stent struts, using CCAs and SCAs in miniature pigs. The diameters of the miniature pig CCAs and SCAs were similar to those of human CCAs and MCAs, respectively. Angiography and IVUS analysis of both showed that intravenous administration of MSCs reduced the degree of in-stent stenosis in both CCAs with Carotid WALLSTENT® and SCAs with Multi Link 8®. Furthermore, histological examination demonstrated that the inflammatory response around the stent struts was inhibited in the MSC-administered group compared to the placebo group in both stent-implanted CCAs and SCAs.

[0064] In this study, neointimal hyperplasia was observed in stent-carcinoma (CCA) and sacral carcinoma (SCA) in all animals, but the thickness of the neointimal layer was thinner in the MSC-treated group than in the placebo-treated group (Figure 4). It has been reported that the inflammatory response after stent placement contributes to neointimal formation and causes in-stent restenosis. Inflammatory factors such as interleukin (IL)-1 and tumor necrosis factor α (TNF-α) produced by infiltrating monocytes and macrophages play important roles in the early inflammatory response and neointimal hyperplasia after stent placement. Specifically, the secretion of IL-1 and TNF-α following an increase in Matrix Metalloproteinase-9 (MMP-9) continues for at least 3 months after stent placement, and MMP-9 activation contributes to the degradation of several components of the extracellular matrix, altering molecular expression in the vascular wall where the stent is placed, leading to neointimal hyperplasia and in-stent restenosis. Therefore, reducing the inflammatory response around the stent strut and preventing MMP-9 activation may inhibit in-stent restenosis.

[0065] Our previous research showed that intravenous administration of MSCs suppresses MMP-9 activation. MSCs secrete anti-inflammatory factors such as TNF-α-stimulated gene / protein 6 (TSG-6), which inhibits MMP-9 activation, and transforming growth factor beta (TGF-β1), which suppresses MMP-9 activation, via the IL-1 and TNF-α cascade. In fact, ELISA analysis showed that the human MSCs used in our study secreted TGF-β1 in vitro (93.34±12.65 PG / 1.0×10⁶). 4 (Cells, N = 3). Furthermore, 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 immunosuppressive molecules, exosomes, and chemokines. In short, intravenous administration of MSCs may inhibit the inflammatory response around stent struts, potentially leading to the prevention of neointima-hyperplasia and in-stent restenosis.

[0066] Atherosclerotic lesions in the major cerebral and carotid arteries contribute to stroke, accounting for approximately 5 to 20 percent of ischemic strokes. In these patients with neurological lesions, stent placement is performed after stroke to prevent further neurological complications and recurrent ipsilateral stroke. Intravenous administration of mesenchymal stents (MSCs) is considered a new treatment option for ischemic stroke. Patients with ischemic stroke who have undergone CAS or PTAS may also be treated with intravenous MSCs in the future. Intravenous MSCs can improve neurological symptoms and prevent in-stent stenosis and recurrent stroke. This study encourages this protocol, which may improve functional outcomes in stroke patients who have undergone stent placement.

[0067] Intravenous administration of medicinal stem cells (MSCs) is known as a tissue repair and regeneration drug for patients with acute spinal cord injury, but its effects on patients with stents were unknown. This study confirmed that intravenous administration of MSCs did not have adverse effects on patients with stents, and conversely, it had an effect of inhibiting intravascular restenosis. This makes intravenous administration of MSCs possible for patients with acute spinal cord injury who have undergone stent placement.

[0068] The present invention makes it possible to administer a tissue repair and regeneration pharmaceutical composition containing MSCs to patients who have undergone stent placement. The pharmaceutical composition containing MSCs can prevent the progression of neointimal hyperplasia in the stented blood vessel and prevent restenosis after PTAS.

[0069] All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. A pharmaceutical composition for tissue repair and regeneration, comprising mesenchymal stem cells, which is administered to a patient who has undergone stent placement.

2. The pharmaceutical composition according to claim 1, wherein the patient is suffering from or has suffered from any disease selected from atherosclerosis, ischemic heart disease including myocardial infarction, ischemic cerebrovascular disease including cerebral infarction and transient ischemic attack (TIA), arteriosclerosis obliterans (ASO), Buerger's disease, atherosclerotic lesions in blood vessels throughout the body, vascular dissecting lesions including dissecting aneurysms, and aneurysms.

3. The pharmaceutical composition according to claim 1 or 2, wherein the stent placement is carotid artery stent placement (CAS) or percutaneous transluminal angioplasty and stent placement (PTAS).

4. 10 doses per dose 6 The pharmaceutical composition according to any one of claims 1 to 3, wherein at least one cell is administered.

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

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

7. A pharmaceutical composition according to any one of claims 1 to 6, which prevents in-stent restenosis.

8. A pharmaceutical composition for preventing in-stent restenosis, comprising mesenchymal stem cells, the pharmaceutical composition being administered to a patient who has undergone stent placement.

Images