Local hemostatic agent containing mesenchymal stem cells

Abstract

To provide a local hemostatic agent having rapid and persistent hemostatic action, persistent anti-inflammatory action and tissue regeneration ability, and having little risk of infectious diseases.SOLUTION: The present invention relates to a local hemostatic agent containing mesenchymal stem cells as an active ingredient. The inventors have found that mesenchymal stem cells have a rapid hemostatic effect in addition to an anti-inflammatory effect and a tissue regeneration ability, thereby completing the invention. Since the effect of the local hemostatic agent of the invention lasts as long as the cells survive in the affected area, the persistence of the effect can be expected. It is effective for administration to patients suffering from chronic bleeding or inflammation caused thereby, particularly hemophilia patients.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a topical hemostatic agent containing mesenchymal stem cells.

Background Art

[0002] Bleeding due to trauma, internal bleeding in the body such as in the gastrointestinal tract and blood vessels, etc., may pose a life-threatening risk if there is a large amount of bleeding. Therefore, hemostatic treatment, especially hemostatic treatment in surgical operations, is essential. In the case of mild bleeding, hemostasis is achieved by the patient's own natural blood coagulation reaction. However, depending on the degree of injury, it may be difficult to achieve hemostasis even by using basic hemostatic methods such as compression, ligation, and suturing. Or there may be cases of bleeding from adhesion detachment sites or bleeding from the digestive organs.

[0003] By the way, mesenchymal stem cells are cells with pluripotency and self-renewal ability, and can differentiate into various cells including chondrocytes, osteocytes, muscle cells, adipocytes, etc. In the field of regenerative medicine, which has been in the spotlight in recent years, the use of mesenchymal stem cells has begun to be used for the treatment of body disorders that are impossible to cure or regenerate. In addition, therapeutic agents focusing on exosomes secreted by mesenchymal stem cells have also been developed. Specifically, focusing on the proliferation and differentiation ability containing mesenchymal stem cells, a bandage containing a material solution containing mesenchymal stem cells and a bioactive factor composition has been developed, which is said to have the effects of accelerating wound healing and repair and hemostasis and analgesia (for example, see Patent Document 1). In addition, an adhesion-promoting hemostatic film and vesicles containing exosomes secreted from mesenchymal stem cells (for example, see Patent Documents 2 to 3) are also known. On the other hand, there is also a report that a patient who received intravenous administration of adipose tissue-derived mesenchymal stem cells died of pulmonary thromboembolism (for example, see Non-Patent Document 1).

[0004]

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0006]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] Hemostatic treatment is very important to prevent excessive bleeding. However, hemostasis by suturing may be constricted when the damaged area is extensive. Currently available hemostatic agents include fibrin glue derived from human plasma components, those with oxidized cellulose as the main component, and those made from collagen as raw materials. However, they may take a long time to stop bleeding, and there are problems such as the fact that blood products such as fibrin glue are manufactured using the blood of an unspecified number of people, so the risk of infectious diseases cannot be denied. Therefore, the currently available hemostatic agents are currently required to be improved by both doctors and patients.

[0008] For promoting the regeneration and repair of damaged tissues, therapeutic agents containing mesenchymal stem cells or mesenchymal stem cell secretions have been developed. However, the dressing described in Patent Document 1 requires containing a material solution containing mesenchymal stem cells and a bioactive factor composition as an essential requirement. Comparative Example 2 with only mesenchymal stem cells is described as having a therapeutic effect inferior to that of the Example containing mesenchymal stem cells and an active factor composition. Therefore, it is presumed that the hemostatic effect of only mesenchymal stem cells is insufficient. Patent Document 2 describes the exosomes secreted by mesenchymal stem cells, and Patent Document 3 describes the hemostatic effect of vesicles derived from mesenchymal stem cells, without touching on the effects of mesenchymal stem cells.

[0009] Therefore, in the present invention, it is an object to provide a topical hemostatic agent having a rapid and persistent hemostatic action, a persistent anti-inflammatory action and tissue regeneration ability, and a low risk of infectious diseases.

Means for Solving the Problems

[0010] In order to solve the above problems, the present inventors focused on mesenchymal stem cells and examined their effectiveness in detail. As a result, it was found that mesenchymal stem cells have a rapid and persistent hemostatic effect in addition to a persistent anti-inflammatory action and tissue regeneration ability, leading to the completion of the present invention.

[0011] That is, the present invention has the following configurations. [1] A topical hemostatic agent containing mesenchymal stem cells as an active ingredient. [2] The topical hemostatic agent according to [1], wherein the mesenchymal stem cells are mesenchymal stem cells into which 7ND, a dominant negative inhibitor of CCL2, is introduced. [3] The topical hemostatic agent according to [1] or [2], which is in a liquid or sheet form. [4] The topical hemostatic agent according to [1] or [2], which is for chronic inflammation.

Effects of the Invention

[0012] The topical hemostatic agent of the present invention has a rapid and sustained hemostatic effect, a sustained anti-inflammatory effect, and tissue regeneration ability, and has a low risk of infectious diseases. In particular, since the topical hemostatic agent of the present invention uses cells, the effect can be expected to be sustained as long as the cells survive at the affected site. Therefore, it is effective for patients suffering from chronic bleeding and the resulting inflammation. In addition, since the topical hemostatic agent of the present invention coagulates blood by the extrinsic coagulation cascade, it is suitable for administration to hemophilia patients in whom the intrinsic blood coagulation cascade is less likely to progress. In addition, since the topical hemostatic agent of the present invention has tissue regeneration ability, it has a cartilage differentiation and regeneration effect on destroyed cartilage in the administration to hemophilic arthropathy patients. Furthermore, since the topical hemostatic agent of the present invention can be used by culturing the patient's own mesenchymal stem cells, the risks of rejection, infectious diseases, and carcinogenesis are also low.

[0013] In this specification, "topical" refers to a preparation applied to the surface or gap of an object, that is, application to the internal surface or external surface or gap of an object. The surface refers to the epithelium of humans and non-human mammals described below, the epidermis covering the body surface such as the skin and nails, the epithelium constituting the mucosa of the luminal organs, the urethra, etc. It also includes surgical cut end faces and the surfaces of incision sites. In addition, the topical hemostatic agent of the present invention includes administration to a gap. Specifically, it includes administration into the joint cavity. Since the topical hemostatic agent of the present invention delivers the active ingredient to the affected area through the surface of the target site such as the epithelium, it does not pass through blood vessels and does not cause thrombus by ADSC administration as in Non-Patent Document 1.

Brief Description of the Drawings

[0014]

Figure 1

Figure 2

Figure 3

Figure 4

Mode for Carrying Out the Invention

[0015] Hereinafter, embodiments of the present invention will be described in detail, but the scope of the present invention is not limited to these embodiments. In the present specification, "contained as an active ingredient" means containing a mesenchymal stem cell in an effective amount for treatment. <Mesenchymal stem cell>

[0016] Mesenchymal stem cells (hereinafter sometimes referred to as "MSCs") are somatic stem cells derived from mesenchyme (mesodermal tissue) and have the ability to differentiate belonging to the mesenchymal system. Their characteristics vary depending on the tissue to be collected, and they are called adipose tissue-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and dental pulp-derived mesenchymal stem cells for each tissue collected. In the present invention, adipose tissue-derived mesenchymal stem cells or bone marrow-derived mesenchymal stem cells are preferred from the viewpoints of promoting blood coagulation and ease of preparation. The biological origin of any of the cells is not particularly limited, and includes mammals other than humans (including pet animals, livestock, and laboratory animals. Specifically, for example, monkeys, pigs, cows, horses, goats, sheep, dogs, cats, mice, rats, guinea pigs, hamsters, etc.).

[0017] In the present invention, adipose tissue-derived mesenchymal stem cells (hereinafter sometimes referred to as "ADSCs") are a type of biological tissue composed of adipocytes, and bone marrow-derived mesenchymal stem cells (hereinafter sometimes referred to as "BMSCs") refer to somatic stem cells found in bone marrow. As long as either type of cell maintains pluripotency, cells obtained by culturing (including subculture) such somatic stem cells can be either ADSCs or BMSCs. Usually, these mesenchymal stem cells are prepared in an "isolated state" as cells constituting a cell population (including cells other than mesenchymal stem cells), starting from adipose tissue (in the case of ADSCs) or bone marrow (in the case of BMSCs) isolated from a living body. Here, the "isolated state" means a state removed from its original environment (i.e., a state of constituting a part of a living body), that is, a state existing in a state different from its original existence state by artificial manipulation.

[0018] The preparation of mesenchymal stem cells in the present invention may follow conventional methods. Mesenchymal stem cells are widely used for various purposes, and those skilled in the art can easily prepare them with reference to literature and textbooks. It is also possible to use cells distributed from public cell banks or commercially available cells. Hereinafter, as an example of a method for preparing mesenchymal stem cells, a method for preparing ADSCs (an example) will be described. <Method for Preparing Adipose Tissue-Derived Mesenchymal Stem Cells>

[0019] ADSCs are prepared through processes such as the separation, washing, concentration, and culture of stem cells from adipose tissue. The method for preparing ADSCs is not particularly limited. For example, ADSCs can be prepared according to known methods (Fraser JK et al. (2006), Fat tissue: an underappreciated source of stem cells for biotechnology. Trends in Biotechnology; Apr.24(4):150-4. Epub 2006 Feb 20. Review.; Zuk PA et al. (2002), Human adipose tissue is a source of multipotent stem cells. Molecular Biology of the Cell; Dec;13(12):4279-95.; Zuk PA et al. (2001), Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Engineering; Apr;7(2):211-28. etc. are for reference). In addition, devices for preparing ADSCs from adipose tissue (for example, the Celution (registered trademark) device (Cytori Therapeutics, Inc., San Diego, USA)) are also commercially available, and you may choose to prepare ADSCs using such devices. When using such devices, a cell population containing ADSCs can be separated from adipose tissue (K. Lin. et al. Cytotherapy (2008) Vol. 10, No. 4, 417-426). Hereinafter, specific examples of the method for preparing ADSCs will be shown. (1) Preparation of cell population from adipose tissue

[0020] Adipose tissue is collected from humans and non-human mammals by means such as excision and aspiration. The non-human mammal may be any of the animals described above. Also, the age and gender of the organism are not particularly limited. In order to avoid the problem of immune rejection, it is preferable to collect adipose tissue (autologous adipose tissue) from the same individual as the recipient. However, it does not prevent the use of adipose tissue (allogeneic) from the same species of animal or adipose tissue from a different species of animal. In particular, by culturing adipose tissue collected from the same individual as the recipient, the risk of infection can be reduced, and the biocompatibility at the time of administration is also good.

[0021] Examples of adipose tissue include subcutaneous fat, visceral fat, intramuscular fat, and intermuscular fat. Among these, subcutaneous fat can be collected very easily under local anesthesia, so the burden on the donor during collection is small, and it can be said to be a preferable cell source. Usually, one type of adipose tissue is used, but it is also possible to use two or more types of adipose tissue in combination. Also, adipose tissue collected in multiple times (not necessarily the same type of adipose tissue) can be mixed and used for subsequent operations. The amount of adipose tissue collected can be determined in consideration of the type of donor, the type of tissue, or the amount of ADSC required, and is, for example, about 0.5 to 500 g. When using a human as the donor, it is preferable to limit the amount collected at one time to about 10 to 20 g or less in consideration of the burden on the donor. The collected adipose tissue is subjected to the following enzyme treatment after removing the attached blood components and fragmenting it as necessary. Note that the blood components can be removed by washing the adipose tissue in an appropriate buffer or culture solution.

[0022] Enzymatic treatment is performed by digesting adipose tissue with enzymes such as collagenase, trypsin, dispase, etc. Such enzymatic treatment may be carried out according to methods and conditions known to those skilled in the art (for example, refer to R.I. Freshney, Culture of Animal Cells: A Manual of Basic Technique, 4th Edition, A John Wiley & Sones Inc., Publication). The cell population obtained by the above enzymatic treatment includes somatic stem cells, endothelial cells, stromal cells, hematopoietic cells, and / or their progenitor cells, etc. The types and ratios of cells constituting the cell population depend on the origin and type of the adipose tissue used. (2) Obtaining a sedimented cell population (SVF fraction: stromal vascular fractions)

[0023] The cell population is subsequently subjected to centrifugation. The sediment obtained by centrifugation is collected as a sedimented cell population (also referred to as the "SVF fraction" in this specification). The conditions of centrifugation vary depending on the type and amount of cells, but are, for example, 1 to 10 minutes at 800 to 1500 rpm. Incidentally, prior to centrifugation, it is preferable to subject the cell population after enzymatic treatment to filtration or the like to remove undigested tissue and the like contained therein.

[0024] The "SVF fraction" obtained here contains ADSCs. Incidentally, the types and ratios of cells constituting the SVF fraction depend on the origin and type of the adipose tissue used, the conditions of enzymatic treatment, and the like. Also, the characteristics of the SVF fraction are shown in the pamphlet of International Publication No. 2006 / 006692A1. (3) Selective culture and cell recovery of adherent cells (ADSCs)

[0025] The SVF fraction contains, in addition to ADSCs, other cell components (endothelial cells, stromal cells, hematopoietic cells, their progenitor cells, etc.). Therefore, in one aspect of the present invention, the following selective culture is performed to remove unnecessary cell components from the SVF fraction. And the cells obtained as a result are used in the present invention as ADSCs.

[0026] First, after suspending the SVF fraction in an appropriate medium, it is seeded in a culture dish and cultured overnight. Floating cells (non-adherent cells) are removed by changing the medium. Thereafter, the culture is continued while appropriately changing the medium (for example, once every 2 to 4 days). Subculture is performed as necessary. The number of passages is not particularly limited, but from the viewpoint of maintaining pluripotency and proliferation ability, it is not preferable to repeat subculture excessively (it is preferable to limit it to about 5 passages). In addition, as the medium for culture, a normal medium for animal cell culture can be used. For example, Dulbecco's modified Eagle's Medium (DMEM) (Nissui Pharmaceutical Co., Ltd., etc.), α-MEM (Dainippon Pharmaceutical Co., Ltd., etc.), DMEM:Ham's F12 mixed medium (1:1) (Dainippon Pharmaceutical Co., Ltd., etc.), Ham's F12 medium (Dainippon Pharmaceutical Co., Ltd., etc.), MCDB201 medium (Functional Peptide Research Institute), etc. can be used. A medium supplemented with serum (fetal bovine serum, human serum, sheep serum, etc.) or a serum substitute (such as Knockout serum replacement (KSR)) may be used. The addition amount of the serum or serum substitute can be set, for example, within the range of 5% (v / v) to 30% (v / v).

[0027] By the above operations, adherent cells selectively survive and proliferate. Subsequently, the proliferated cells are collected. The collection operation may follow a conventional method. For example, the cells after enzyme treatment (trypsin or dispase treatment) can be easily collected by detaching them with a cell scraper or pipette. By using the collected cells (ADSC), a cell population containing ADSC with high purity can be prepared. Also, when sheet culture is performed using a commercially available temperature-sensitive culture dish, etc., it is also possible to collect the cells in a sheet form without enzyme treatment. Specifically, a method of culturing mesenchymal stem cells in a temperature-sensitive culture dish or a temperature-responsive incubator until confluent to form a cell sheet and detaching the cell sheet by lowering the temperature can be mentioned. For example, 1×10 3 ~100×10 3 cells / cm 2 are seeded in a 6 cm diameter petri dish and cultured at 37°C, 5% CO 2When incubated in the presence of [substance], it becomes confluent in 5 to 10 days. 10×10 3 ~1000×10 3 cells / cm 2 It is preferably cultured until this state is reached. The topical hemostatic agent of the invention is preferably used in the form of a sheet. When using the cell sheet, it is preferable because a matrix that may have an adverse effect in the body is not required when used as a sheet-shaped topical hemostatic agent. In addition, since adhesive proteins such as extracellular matrix are maintained at the bottom of the cell sheet, it is preferable because the cell sheet can adhere to the affected tissue when used as a sheet-shaped topical hemostatic agent. (4) Low-serum culture (selective culture in low-serum medium) and cell recovery

[0028] In one embodiment of the present invention, the following low-serum culture is performed instead of or after the operation of (3) above. And the cells obtained as a result are used in the present invention as ADSC.

[0029] In low-serum culture, the SVF fraction (when this step is performed after (3), the cells recovered in (3) are used) is cultured under low-serum conditions to selectively proliferate the target somatic stem cells (i.e., ADSC). Since only a small amount of serum is used in the low-serum culture method, it becomes possible to use the serum of the subject (patient) to whom the cell preparation of the present invention is administered. That is, culture using autologous serum becomes possible. By using autologous serum, a cell preparation that excludes heterogeneous animal materials during the manufacturing process, has high safety, and can be expected to have a high therapeutic effect is provided. The "low-serum conditions" here refer to conditions in which the medium contains 5% or less serum. Preferably, cell culture is performed in a culture solution containing 2% (V / V) or less serum. More preferably, cell culture is performed in a culture solution containing 2% (V / V) or less serum and 1 to 100 ng / ml of fibroblast growth factor-2 (bFGF). A serum-free medium may also be used.

[0030] The serum is not limited to fetal bovine serum, and human serum, sheep serum, etc. can be used. Preferably, human serum is used, and more preferably, the serum of the subject to which the cell preparation of the present invention is applied (i.e., autologous serum) is used. As for the medium, a medium for normal animal cell culture can be used on the condition that the amount of serum contained during use is low. For example, Dulbecco's modified Eagle's Medium (DMEM) (Nissui Pharmaceutical Co., Ltd., etc.), α-MEM (Dainippon Pharmaceutical Co., Ltd., etc.), DMEM:Ham's F12 mixed medium (1:1) (Dainippon Pharmaceutical Co., Ltd., etc.), Ham's F12 medium (Dainippon Pharmaceutical Co., Ltd., etc.), MCDB201 medium (Functional Peptide Research Institute), etc. can be used.

[0031] By culturing in the above method, somatic stem cells (ADSCs) can be selectively proliferated. In addition, since the somatic stem cells (ADSCs) that proliferate under the above culture conditions have high proliferation activity, the number of cells required for the present invention can be easily prepared by subculture. Incidentally, International Publication No. 2006 / 006692A1 pamphlet shows the characteristics of cells that selectively proliferate by culturing the SVF fraction in low serum.

[0032] Subsequently, the cells selectively proliferated by the above low serum culture are collected. The collection operation may be performed in the same manner as in the case of (3) above. By using the collected cells (ADSCs), a cell preparation containing ADSCs with high purity can be obtained.

[0033] In the above method, cells grown by low-serum culture of the SVF fraction will be used. However, it is also possible to use, as ADSCs, cells grown by directly culturing (without centrifugation to obtain the SVF fraction) a cell population obtained from adipose tissue in low-serum culture. That is, in one aspect of the present invention, cells that proliferate when a cell population obtained from adipose tissue is cultured in low-serum culture are used as ADSCs. Further, instead of somatic stem cells obtained by selective culture (the above (3) and (4)), the SVF fraction (containing adipose tissue-derived mesenchymal stem cells) may be used as it is. Here, "used as it is" means used in the present invention without undergoing selective culture. <Method for preparing mesenchymal stem cells into which the 7ND gene has been introduced>

[0034] The local hemostatic agent of the present invention may contain mesenchymal stem cells into which the 7ND gene has been introduced (hereinafter, sometimes referred to as "7ND-MSC"). 7ND is a deletion mutant of CC chemokine ligand (hereinafter, sometimes referred to as "CCL2") secreted from mesenchymal stem cells.

[0035] It is considered that CCL2 strongly induces the migration and activation of macrophages, which play a central role in the inflammatory response, by binding to its receptor CCR2. Since 7ND-MSC has been transduced with the 7ND gene, which is a deletion mutant of CCL2, it functions as a dominant negative inhibitor of CCL2 and strongly inhibits macrophage migration.

[0036] The following describes the method for adjusting 7ND-MSC. The FLAG tag (3 = C-terminal) deletion mutant CCL2 (7ND) is recloned from the 7ND pCDNA3 expression vector 16 into a lentiviral vector using the unique sites NheI and XbaI sites. The envelope coding plasmid pLP / VSVG, the packaging plasmid pCMVdR8.91, and pBGJR-EGFP-7ND or pBGJR-EGFP are transiently transfected into 293T cells by the lipofection method using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). The virus particles secreted into the supernatant are collected to prepare a vector stock.

[0037] 7ND-MSC can be prepared by incubating MSC with the vector stock at 37 °C in the presence of polybrene 4 μg / mL (Sigma-Aldrich, St.).

[0038] The introduction efficiency of 7ND can be confirmed by incubating MSC with the vector stock in the presence of polybrene 4 μg / mL (Sigma-Aldrich, St.) and performing fluorescence-activated cell sorter analysis of EGFP expression. As a control, intact-MSC (MSC without gene transfer) is used. When a lentivirus is used as the vector, the gene transfer efficiency is preferably 90% - 100%, more preferably 95 - 100%, and even more preferably 98 - 100%. <Mechanism of Action of the Local Hemostatic Agent of the Present Invention> (1) Hemostasis Mechanism

[0039] Wound healing generally progresses towards healing through a hemostasis phase, an inflammation phase, a proliferation phase, and a remodeling phase. In the hemostasis phase, platelets and fibrin form a blood clot to occlude the damaged site and stop bleeding. Subsequently, vasoactive substances and inflammatory substances such as histamine are released from the damaged tissue, attracting inflammatory cells such as neutrophils, monocytes, and macrophages to the damaged site. These inflammatory cells process bacteria and foreign particles in the wound, promoting wound cleansing. This period is called the inflammation phase. As wound cleansing progresses, fibroblasts migrate from the periphery to reconstruct the extracellular matrix. It then proceeds with angiogenesis and collagen deposition, leading to the formation of granulation tissue. When collagen production is sufficient and a systematic vascular network is formed, the wound surface is covered with skin, and the physical strength increases. This period is called the remodeling phase.

[0040] In this hemostasis phase, prothrombin is activated to thrombin, which acts on fibrinogen to ultimately form fibrin, resulting in blood gelation. If thrombin formation is insufficient, a sufficient hemostatic effect cannot be obtained. On the other hand, since thrombin has a significant inflammatory effect, a balance in thrombin formation is important.

[0041] There are two types of systems for activating prothrombin: the intrinsic coagulation system that starts with the activation of factor XII of blood coagulation and the extrinsic coagulation system that starts with tissue factor. Hemophilia is a blood disorder in which the intrinsic coagulation reaction does not proceed well.

[0042] The inventors of the present invention focused on tissue factor that initiates the extrinsic coagulation reaction. The extrinsic coagulation reaction begins when tissue factor (hereinafter sometimes referred to as "TF") forms a complex with activated coagulation factor VII and activates coagulation factor X. TF is usually highly expressed in fibroblasts outside blood vessels and the like, and when blood flows out due to vascular injury, it immediately binds to activated coagulation factors in the blood and is said to initiate the coagulation cascade reaction. Since TF is highly expressed on the surface of mesenchymal stem cells, the inventors focused on this and completed the present invention. That is, if the local hemostatic agent of the present invention is administered, the blood coagulation cascade can be rapidly initiated and has a rapid hemostatic effect. In addition, it does not increase the amount of thrombin that induces inflammation. It is particularly effective for hemostasis in hemophilia patients who have deficiencies or qualitative abnormalities in blood coagulation factors VIII and IX required for the intrinsic coagulation reaction.

[0043] In addition, since the local hemostatic agent of the present invention uses cells, it is also an advantageous effect that the effect persists as long as the cells survive. Since adipose-derived mesenchymal stem cells are known to survive for about one week to 10 days, it is presumed that the local hemostatic agent of the present invention also has a persistent effect during that period. This is particularly advantageous for the treatment of hemophilia. The hemostatic treatment method for hemophilia is generally the "on-demand therapy" that supplements the deficient coagulation factor at the time of bleeding. However, the in vivo half-lives of blood coagulation factors VIII and IX are 8 to 12 hours and 16 to 20 hours, respectively, and the in vivo half-lives are not necessarily long. Therefore, when using currently commercially available hemophilia preparations, whether plasma-derived or recombinant, it is necessary to administer them multiple times for bleeding events. Depending on the degree of bleeding, systemic administration of coagulation factors is required for about 1 day to 7 days, so patients have to endure frequent hospital visits and the pain of frequent injections. In that regard, if the local hemostatic agent of the present invention is used, it is presumed that the cells will survive and the effect will persist for one week to 10 days, so the burden on the patient can be reduced. (2) Inflammatory suppression mechanism

[0044] Mesenchymal stem cells have an anti-inflammatory effect. However, it is said that they cannot significantly regulate the function of macrophages, which are inflammatory cells.

[0045] A more detailed explanation is as follows. Mesenchymal stem cells suppress the proliferation of cells responsible for immune responses such as T cells and B cells. In the aforementioned inflammatory period, mesenchymal stem cells change the migrated macrophages from the M1 type that enhances the inflammatory response to the M2 type that suppresses the inflammatory response, and terminate the inflammatory response. However, compared with T cells, B cells, etc., the degree of regulation of the macrophage function of mesenchymal stem cells is not considered to be large.

[0046] In addition, if inflammatory cells such as macrophages migrate and infiltrate excessively during the inflammatory period, inflammatory cytokines such as reactive oxygen species and proteases produced by macrophages may cause secondary damage to host cells. As a result, further inflammation may be caused during the inflammatory period, and chronic inflammation may persist because it cannot escape from the cycle. For example, in patients with hemophilic arthropathy, bleeding activates macrophages and angiogenesis is likely to occur. However, the blood vessels newly formed during inflammation are thin and fragile, so they may burst and bleed even with a slight impact. Further bleeding causes further macrophages to migrate, so the inflammation inside the joint is chronicized by repeating inflammation and repair.

[0047] Therefore, while there is a need to administer drugs with a stronger anti-inflammatory effect, especially to patients suffering from chronic inflammation, the inventors focused on MSCs transfected with the 7ND gene. Since the cells inhibit the migration of inflammatory cells such as macrophages, they exhibit a further anti-inflammatory effect.

[0048] As described above, 7ND is a deletion mutant of CCL2, and 7ND-MSC functions as a dominant negative inhibitor of CCL2. Since it strongly inhibits the migration of macrophages, it has a further anti-inflammatory effect. It is suitable for administration to patients suffering from chronic inflammation, such as keloid and hemophilia patients. <Application target of hemostatic agent>

[0049] The topical hemostatic agent of the present invention can be used for various purposes for the purpose of topical hemostasis in humans and mammals other than humans described above. For example, it can be used as a hemostatic agent for wound sites, a dental hemostatic agent such as a hemostatic agent for tooth extraction, a hemostatic agent for organ transplantation / tissue transplantation, a surgical hemostatic agent, a surgical hemostatic agent, a nail hemostatic agent, and a hemostatic agent for anastomosis sites.

[0050] In addition, since the topical hemostatic agent of the present invention has a continuous hemostatic effect, an anti-inflammatory effect, and tissue regeneration ability, it can be used for patients suffering from chronic bleeding and the resulting inflammation. It is particularly suitable for administration to keloid patients and hemophilia patients. That is, the topical hemostatic agent of the present invention can be used for chronic inflammation.

[0051] In particular, the topical hemostatic agent of the present invention coagulates blood by the extrinsic coagulation reaction cascade with TF as the initiator without passing through the intrinsic coagulation reaction cascade related to blood coagulation factor VIII (hemophilia A) or factor IX (hemophilia B). Therefore, a sufficient hemostatic effect can be obtained even when administered to hemophilia patients.

[0052] Incidentally, as one of the complications frequently seen in hemophilia A and B, there is hemophilic arthropathy in which repeated joint bleeding causes pain and limitation of the range of motion. Hemophilic arthropathy is caused by bleeding in the joint, but since the inside of the joint is narrow, even a small amount of bleeding causes pressure and strong pain. Repeated bleeding makes it easier for blood to accumulate in the joint, and the components contained in the blood may have an adverse effect on the synovium and cartilage. When synovitis is caused, bleeding becomes even easier. In this way, hemophilic arthropathy causes joint deformation or joint contracture (the range of motion of the joint becomes narrow and it becomes difficult to move), causing impairment of joint function and significant reduction in QOL.

[0053] Since the topical hemostatic agent of the present invention has a sustained hemostatic effect, anti-inflammatory effect, and tissue regeneration ability, it can suppress bleeding in the joint, relieve inflammation such as synovitis that has developed, and further has a cartilage regeneration effect for damaged cartilage. In severe cases such as when the joint is deformed due to hemophilic arthropathy, treatment methods such as surgical operations and rehabilitation have been taken so far. However, the topical hemostatic agent of the present invention is also advantageous in that it is minimally invasive and has less burden on patients compared to these treatment methods. <Method for manufacturing hemostatic agent and dosage form>

[0054] The topical hemostatic agent of the present invention contains the aforementioned MSC. If necessary, it can be formulated by mixing with non-toxic and inert pharmaceutically acceptable excipients, such as solid, semi-solid, or liquid diluents, dispersants, fillers, and carriers. Furthermore, within a range that does not impair the effects of the present invention, stabilizers, preservatives, pH adjusters, binders, disintegrants, surfactants, lubricants, fluidity promoters, flavoring agents, coloring agents, fragrance preservatives, media, physiological saline, and drugs having other medicinal effects may be included as additives. It may also contain an adhesive material for fixing on the skin, or an outer coating for protecting the wound from external influences or minimizing blood outflow.

[0055] The topical hemostatic agent of the present invention is preferably used by directly attaching it to the bleeding site. There is no limitation on its form, but a liquid or sheet form is preferred for application to the bleeding site.

[0056] The liquid hemostatic agent is preferably used by making MSC into an aqueous solution and spraying it onto the application site with a spray or the like. Since it adheres well to the irregular uneven surface peculiar to the wound, it can achieve good hemostasis. It is particularly suitable for places where it is difficult to use a sheet hemostatic agent, such as deep inside the surgical site or narrow places. When administered into the joint, it is preferable to locally administer the liquid hemostatic agent into the joint. The topical hemostatic agent of the present invention has a rapid blood coagulation reaction, and furthermore, since cells are used, as long as the cells survive, it has a hemostatic effect, anti-inflammatory effect, and tissue regeneration ability for a certain period after administration.

[0057] The sheet-shaped hemostatic agent preferably contains the mesenchymal stem cells of the present invention on one side of a solid surface, an object, or another solid medium such as a strip or a film. A medical material that can be decomposed in vivo can be molded into a sheet shape, and a sheet in which MSCs are immobilized thereon or a sheet in which the cells themselves are formed into a sheet shape can be used. Since the sheet-shaped hemostatic agent can be applied to the affected area for compression hemostasis and compression closure, it is particularly effective for bleeding sites with jetting or exudative bleeding.

[0058] In addition, for commercially available hemostatic agents in which a fibrin membrane is formed by the reaction of fibrinogen and thrombin and has a hemostatic effect, since the molecular weight of thrombin is small, if it enters the blood vessel through an open wound, a thrombus may be formed in the blood vessel, and the blood flow at the site where the thrombus is formed may be blocked, so there is a risk of myocardial infarction or pulmonary embolism. On the other hand, since the local hemostatic agent of the present invention contains mesenchymal stem cells, considering their size, it is considered that the cells will not enter the blood vessel from the wound against the hydrostatic pressure in a short time. <Dosage of the hemostatic agent>

[0059] In the case of the liquid local hemostatic agent of the present invention, it is preferable that MSCs are contained in the cell suspension at 5×10 5 ~20×10 5 cells / mL. In the case of a sheet shape, it is preferable to use a cell sheet containing MSCs at 10×10 3 ~1000×10 3 cells / cm 2 or a sheet in which cells are attached to one side of a solid medium so as to have the above cell number. The dosage can be appropriately adjusted in consideration of the purpose of use, the target disease, the gender, age, weight of the application target, the condition of the affected area, etc. For example, in the case of a liquid local hemostatic agent, it is 50 cells / kg to 1×10 6It is preferable that the MSC be adjusted to be administered at

[0060] When administering the topical hemostatic agent of the present invention, it may be administered in combination with other therapeutic agents. In order to obtain a stronger hemostatic effect, blood coagulation factors may be administered. The combined administration means administering at the same time as the administration of the therapeutic agent for hemophilic arthropathy of the present invention, or before or after the administration of the therapeutic agent for hemophilic arthropathy of the present invention. Alternatively, the topical hemostatic agent of the present invention and the above other therapeutic agents can be mixed to form a single preparation.

Examples

[0061] The present invention will be specifically described by the following examples, but the present invention is not limited thereto. In the examples, adipose tissue-derived mesenchymal stem cells (ADSC) and bone marrow-derived mesenchymal stem cells (BMSC) were used. Sometimes the general term "MSC" is used for both. <Preparation of ADSC>

[0062] ADSC was established from C57BL / 6N mice by the method described in Nakao N., et al., “Adipose tissue-derived mesenchymal stem cells facilitate hematopoiesis in vitro and in vivo: advantages over bone marrow-derived mesenchymal stem cells.” Am J Pathol 2010, 177:547-554. ​<Preparation of BMSCs>

[0063] BMSCs were established from C57BL / 6N mice by the method described in Nakao N., et al., “Adipose tissue-derived mesenchymal stem cells facilitate hematopoiesis in vitro and in vivo: advantages over bone marrow-derived mesenchymal stem cells.” Am J Pathol 2010, 177:547-554.

[0064] Cells at passages 5 - 15 were used for all. The mouse macrophage cell line RAW264.7 was purchased from the American Type Culture Collection (Manassas, VA) and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). <Preparation of 7ND-MSCs>

[0065] The FLAG-tag (3 = C-terminal) deletion mutant CCL2(7ND) was recloned from the 7ND pCDNA3 expression vector 16 into the lentiviral vector (pBGJR-EGFP; a gift from Dr. Stefano Rivella, Cornell University, New York, NY) using the unique site NheI and XbaI sites. An empty pBGJR EGFP vector was used as a control. The envelope coding plasmid pLP / VSVG, the packaging plasmid pCMVdR8.91, and pBGJR-EGFP-7ND or pBGJR-EGFP were transiently transfected into 293T cells by the lipofection method using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Viral particles secreted into the supernatant were collected to prepare vector stocks.

[0066] Figure 1(A) shows the main domain structure of the pBGJR-EGFP-7ND-FLAG vector. The FLAG-tag (3 = C-terminus) deletion mutant (7ND) of CCL2 was recloned from the 7ND pCDNA3 expression vector into a lentiviral vector, and EGFP (pBGJR-EGFP) was expressed under the control of the elongation factor-1 promoter (EF1). In Figure 1(A), LTR represents the long terminal repeat, PGK represents the phosphoglycerate kinase promoter, and WPRE represents the woodchuck hepatitis virus post-transcriptional control element.

[0067] The previously prepared ADSCs and BMSCs were incubated with the vector stock at 37°C in the presence of polybrene 4 μg / mL (Sigma-Aldrich, St.). The transduction efficiency of 7ND was confirmed by fluorescence-activated cell sorter analysis of EGFP expression. As a control, intact-ADSCs or intact-BMSCs (where intact indicates no gene introduction) were used. The results are shown in Figures 1(B) and (C). The transduction efficiency of 7ND into both ADSCs and BMSCs was 100%. <Characteristic evaluation>

[0068] (1) Measurement of the blood coagulation ability of MSCs The blood coagulation time was measured by the method used. Specifically, 100 μL of RPMI1640 or ADSCs (1×10 5 cells) were added to 50 μL of FVIII-deficient plasma as plasma equivalent to hemophilia A patients and FIX-deficient plasma as plasma equivalent to hemophilia B patients (both manufactured by George King Bio-Medical Inc., purchased from Cosmo Bio Co., Ltd.), and heated at 37°C for 2 minutes. Next, 100 μL of 20 mM calcium chloride solution was added to initiate the coagulation reaction. Then, it was gently shaken by hand, and the time until the fluidity decreased (visually, it became cloudy) was measured.

[0069] Also, 50 μL of human plasma (manufactured by George King Bio-Medical Inc., purchased from Cosmo Bio Co., Ltd.) was added to BMSCs (1×10 5Cells were added, and the blood coagulation time of BMSCs was measured by the same method as above.

[0070] As a control for ADSCs or BMSCs, RPMI-1640 medium (Gibco), a cell culture medium, was used. For the control of ADSCs, Normal Plasma (human normal plasma, manufactured by George King Bio-Medical Inc., purchased from Cosmo Bio Co., Ltd.) was used.

[0071] The results of the blood coagulation time when ADSCs were added and the results when BMSCs were added are shown in Table 1 and Figure 2. Figure 2(A) is a graph of the results in Table 1. As is clear from Table 1 and Figure 2(A), it was confirmed that in the plasma of hemophilia patients, the addition of ADSCs significantly shortened the coagulation time.

[0072] Figure 2(B) shows the results of measuring the blood coagulation time when BMSCs were added. It can be seen that the blood coagulation time becomes shorter depending on the number of added cells.

[0073]

Table 1

[0074] The cell proliferation abilities of BMSCs and 7ND-BMSCs in vitro were measured using a colorimetric assay method. Specifically, BMSCs (2000 cells per well) were seeded in a 96-well plate. After culturing for 72 hours, 10 μL of Tetra Color One reagent was added to each well, and the absorbance at 450 nm was measured 4 hours later. The proliferation rate was calculated as the OD value of 7ND-BMSC / the OD value of intact-BMSC × 100. Four repeated measurements were performed three times, and the average SD of a representative experiment among the three times is shown.

[0075] The results are shown in Fig. 3(A). 7ND-BMSC had an excellent cell proliferation ability similar to BMSC. Also, regarding the cell proliferation ability, no influence of 7ND lentivirus introduction was confirmed in 7ND-BMSC. (3) Measurement of tissue regeneration ability

[0076] Intact BMSC (non-transduced MSC), control (cont)-BMSC, and 7ND-BMSC were exposed to an adipocyte differentiation inducer (R&D Systems, Minneapolis, MN) for 14 days or an osteocyte differentiation inducer (R&D Systems) for 21 days. Here, Intact BMSC is BMSC that has not been manipulated at all (no gene introduction), and control-BMSC refers to BMSC into which only lentivirus has been introduced.

[0077] Lipid deposition was evaluated by Oil Red O staining for the amount of vacuoles rich in intracellular lipids resulting from adipocyte differentiation. Osteogenic differentiation was evaluated by von Kossa staining for the degree of calcium deposition. The results are shown in Fig. 3(B). The upper panel shows lipid deposition, and the lower panel shows calcium deposition. As a result, 7ND-BMSC had the ability to differentiate into the adipogenic and osteogenic lineages similar to intact BMSC. Also, regarding the differentiation ability, no influence of 7ND lentivirus introduction was confirmed in 7ND-BMSC. (4) Measurement of 7ND secretion amount from 7ND-BMSC

[0078] BMSC or 7ND-BMSC (2000 cells per well) were seeded in a 96-well plate and cultured for 72 hours. 7ND was purified from the culture supernatant of 7ND-BMSC using the ANTI-FLAG M2 affinity gel system (Sigma-Aldrich). The 7ND content was measured using a specific human CCL2 enzyme-linked immunosorbent assay (eBioscience Inc.). The results are shown in Fig. 3(C). It was revealed that 7ND-BMSC secretes a large amount of 7ND. (5) Migration assay

[0079] The inhibitory effect of purified 7ND on the CCL2-CCR2 axis was evaluated using a transwell assay. Specifically, macrophage-like CCR2-expressing RAW264.7 cells were serum-starved overnight in DMEM supplemented with 1% FBS, and then seeded in the upper chamber of a cell culture insert with an 8-μm pore size (BD Biosciences, San Jose, CA) (5×10 5 cells in DMEM supplemented with 1% FBS per well). Recombinant mouse CCL2 (final concentration 10 ng / mL; PeproTech, Rocky Hill, NJ) and purified 7ND at various concentrations (final concentration 0 - 1000 ng / mL) were added to the lower chamber, and the cells were allowed to migrate for 4 hours. The membrane was fixed with 4% paraformaldehyde and stained with Giemsa. Four fields were randomly selected, and the migrated cells were counted using an optical microscope. The mean ± SD of a representative experiment out of three performed in quadruplicate is shown in Fig. 3(D). As a result, 7ND inhibited CCL2-induced migration of RAW264.7 cells in a dose-dependent manner. (6) Pharmacokinetics of 7ND-BMSC

[0080] To confirm the in vivo survival period of 7ND-BMSC, its pharmacokinetics were evaluated. Male C57BL / 6J mice, 6 - 10 weeks old, were purchased from Central Laboratory Animal. Cont-BMSC, 7ND-BMSC (5×10 5 cells in 200 μL of PBS) or the same volume of PBS was administered via the tail vein. Plasma was then collected periodically, and the 7ND content in the plasma was evaluated using a specific human CCL2 enzyme-linked immunosorbent assay (eBioscience Inc.). The experiment was performed using 5 mice per subgroup, and the mean and SD of the plasma CCL2 concentration were calculated. The same experiment was performed twice in total, and similar results were obtained. The results are shown in Fig. 3(E).

[0081] From the results in Figure 3, it can be seen that BMSCs and 7ND-BMSCs have cell proliferation ability and tissue regeneration ability (Figure 3(A)(B)). In particular, 7ND-BMSCs released a large amount of 7ND (Figure 3(C)), and 7ND inhibited the migration of macrophage-like CCR2-expressing RAW264.7 cells (Figure 3(D)). Therefore, it is speculated that 7ND has an effect of suppressing inflammation caused by excessive macrophage migration. Also, as is clear from Figure 3(E), the blood level of 7ND was the highest on the second day after injection and was not detected on the 11th day. From this, it was found that in vivo, the number of 7ND-BMSCs decreased over time. In other words, it can be inferred that 7ND-BMSCs survived for about 10 days after administration, and during that time, they had a hemostatic effect, an anti-inflammatory effect, and tissue regeneration ability. <Effect on hemophilic arthropathy>

[0082] C57 / B6;129S-F8 tm1Kaz Mice with hemophilia A of / J were purchased from Jackson Laboratory and bred in the facility. A 30G needle was punctured into the right knee of the mice to cause bleeding, thereby creating a hemophilic arthropathy model. 100,000 cells / 10 μL of ADSCs and 7ND-ADSCs were respectively injected into the joints of these model mice, and as a control, 10 μL of RPMI was injected. The administration was only once, and each model was evaluated with n = 5. Since the affected tissue swells when inflammation occurs, the treatment of arthritis is evaluated by the reduction of the diameter of the inflamed joint. In the present invention, the diameter of the right knee joint of the mice was measured with calipers, and the diameter of the joint was confirmed over time based on the joint diameter before puncture.

[0083] The results are shown in Figure 4. When ADSCs or 7ND-ADSCs were administered, the difference in joint diameter was significantly smaller. The anti-inflammatory effect and hemostatic effect of ADSCs and 7ND-ADSCs were equivalent, and both effects continued for 7 days.

Claims

**Claim 1** A topical hemostatic agent containing mesenchymal stem cells as an active ingredient. **Claim 2** The topical hemostatic agent according to claim 1, wherein the mesenchymal stem cells are mesenchymal stem cells into which 7ND, a dominant negative inhibitor of CCL2, is introduced. **Claim 3** The topical hemostatic agent according to claim 1 or 2, which is in a liquid or sheet form. **Claim 4** The topical hemostatic agent according to claim 1 or 2, which is for chronic inflammation.

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