KR20200061242A - Use of mesenchymal stem cell having enhanced efficacy using NADPH oxidase inhibitors - Google Patents

Abstract

The present invention relates to function-enhanced mesenchymal stem cells and uses thereof. Function-enhanced mesenchymal stem cells according to an aspect have a blood vessel regeneration effect or a neuranagenesis effect, thereby being able to be usefully used in pharmaceutical compositions or cell therapeutic agents for treating or preventing various diseases. A composition for enhancing the function of mesenchymal stem cells contains an inhibitor of NADPH oxidizer.

Description

Use of mesenchymal stem cell having enhanced efficacy using NADPH oxidase inhibitors}

Reinforcement of functions through inhibition of NADPH oxidant relates to mesenchymal stem cells and uses thereof.

Mesenchymal stem cells, which have been highly anticipated as a new treatment for many incurable diseases based on excellent results in non-clinical studies, are administered as cells originally expected as stem cells through active research and clinical trials of the past 10 years. It is found that the main mechanism of action is to show the effect through secreting various active ingredients and gene regulatory substances rather than replacing the damaged tissue with differentiation, and the effect has been found to be less than expected.

Due to the problem of low efficacy in clinical application, various methods of enhancing mesenchymal stem cell function to enhance the effect of mesenchymal stem cells have been studied. The method that was mainly attempted was a method of introducing a gene for the purpose of enhancing a specific function into a cell, but there is a problem that the clinical application is limited due to the use of genetic manipulation itself and viral vectors. In order to overcome this problem, there is hypoxic preconditioning that creates a hypoxic environment when cultured as a method that is actually used to improve the function of mesenchymal stem cells, but the process advantages due to increased cell proliferation efficiency are obvious. However, there is no dramatic improvement in efficacy when administered, and further studies on how to strengthen the function are still needed.

One aspect provides a composition for enhancing the function of mesenchymal stem cells comprising an inhibitor of NADPH oxidizing agent.

Another aspect provides a method for producing a function-enhanced mesenchymal stem cell comprising passage-incubating mesenchymal stem cells in a medium containing an inhibitor of the NAPDH oxidant.

Another aspect provides a function-enhanced mesenchymal stem cell population.

Another aspect provides a pharmaceutical composition for the prevention or treatment of cerebral hemorrhage or ischemic brain disease comprising the function-enhanced mesenchymal stem cells.

One aspect provides a composition for enhancing the function of mesenchymal stem cells comprising an inhibitor of NADPH oxidizing agent.

The composition for enhancing the function of mesenchymal stem cells according to an aspect of the present invention suppresses oxidative damage by inhibiting NADPH oxidase (NADPH oxidase) in mesenchymal stem cells, prevents oxidative damage, and enhances function by exhibiting improved oxidative damage inhibitory effects. It can be used to obtain mesenchymal stem cells. The enhanced mesenchymal stem cells may be useful for the prevention or treatment of brain hemorrhage and/or ischemic brain disease.

The composition may be a medium composition.

The term "inhibitor of NADPH oxidant" herein is not limited as long as it inhibits the activity of NADP oxidant or inhibits free radicals through it. Specifically, the inhibitor of the NADPH oxidant is apocynin (1-(4-hydroxy-3-methoxyphenyl)-ethanone), trolox (trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2 -Carboxylic Acid, pyrrolidine dithiocarbamate (PDTC), glutathione (GSH), catalase, manganese-superoxide dismutase (MnSOD), vitamin E (Vitamin) E) and quercetin (Quercetin).

The aposinin is pocinum cannabinum ( pocynum cannabinum ), a compound that is used as an inhibitor of NADPH oxidant, an enzyme related to free radical formation. Therefore, it is known to inhibit the production of free radicals by inhibiting the NADPH oxidant when treating apocynin in cells.

The trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) is an analog of vitamin E and is soluble in water and may play a role in reducing oxidative stress or damage during treatment. have.

The inhibitor of the NADPH oxidant can enhance the mesenchymal stem cell neuronal cell death inhibition, neuroprotective effect, and hematoma inhibitory effect. The inhibitor of the NADPH oxidant can reduce the production of free radicals to prevent oxidative damage and, consequently, exhibit a higher oxidative damage inhibitory effect.

The concentration of the inhibitor of the NADPH oxidant can be selected by those skilled in the art within a range capable of inhibiting the NADPH oxidant. For example, it may be included at a concentration of about 10 μM to 200 μM, about 50 μM to 150 μM, or 100 μM. If the NADPH oxidant is less than about 10 μM, there may not be sufficient inhibitory effect of the NADPH oxidant.

"Stem cell" as used herein refers to a cell that can develop into any tissue. There are two basic features, first of all, self-renewal that makes itself by repeated division, and multipotency that can differentiate into cells with specific functions according to the environment.

As used herein, "mesenchymal stem cell (MSC)" is a multipotent stem having the ability to differentiate into various mesodermal cells, including bone, cartilage, fat, and muscle cells, or ectodermal cells such as nerve cells. Cells. The mesenchymal stem cells may include mesenchymal stem cells of mammals, for example animals including humans.

The mesenchymal stem cells may be derived from one or more selected from the group consisting of umbilical cord, umbilical cord, cord blood, placenta, fat, and bone marrow.

The umbilical cord may mean a line that connects the mother and the belly so that the mammalian fetus can grow in the placenta, and generally has three blood vessels surrounded by Wharton's Jelly, that is, two belly arteries and It may mean a tissue composed of one umbilical vein.

The placenta is a structure that mediates material exchange necessary for the survival and growth of the fetus between the fetus and the mother. It may mean that a part of the tabernacle, which is attached to the inner wall of the mother's uterus and is connected to the fetus by the umbilical cord and wraps the fetus outside, is formed by bonding to the mother's endometrium. Placental-derived mesenchymal stem cells may have better proliferative power or replication ability than fat-derived mesenchymal stem cells.

The fat may be derived from subcutaneous adipose tissue, bone marrow adipose tissue, mesenteric adipose tissue, gastrointestinal adipose tissue, or posterior peritoneal adipose tissue, and the adipose tissue-derived mesenchymal stem cells are capable of differentiating into various tissue cells and cultured. It may mean a cell having a property of growing on the surface of the container.

Separation of the mesenchymal stem cells can be carried out in a manner obvious to those skilled in the art, for example, Pittenger et al. (Science 284: 143, 1997) and van et al. (J. Clin. Invest., 58: 699, 1976).

Enhancing the function of the mesenchymal stem cells may be to inhibit the production of free radicals in the mesenchymal stem cells. Oxygen free radicals (reactive oxygen species) are oxygen free radicals that are generated in various metabolic processes and attack biological tissues and damage cells as oxygen that enters the body during the breathing process is used for oxidation. do.

NADPH oxidant is an enzyme that produces free radicals in the body, and most other free radical generating enzymes produce free radicals after being damaged by free radicals, whereas NADPH oxidant has the function of first producing free radicals.

Another aspect provides a method for producing a function-enhanced mesenchymal stem cell comprising passage-incubating mesenchymal stem cells in a medium containing an inhibitor of the NAPDH oxidant.

The inhibitor of the NAPDH oxidant and the enhancement of the functions of mesenchymal stem cells are as described above.

The medium is DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, DMEM/F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10), DMEM/F- 12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12), α-MEM (α-Minimal essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Isocove's Modified Dulbecco's Medium), and KnockOut DMEM It may be one or more selected.

The medium can be supplemented with additives. Generally, it may contain neutral buffers (e.g. phosphate and/or high concentration bicarbonate) and protein nutrients (e.g. serum, such as FBS, serum substitutes, albumin, or essential and non-essential amino acids, such as glutamine) in isotonic solutions. Furthermore, lipids (fatty acid, cholesterol, HDL or LDL extracts of serum) and other components found in most preservative media of this kind (such as insulin or transferrin, nucleosides or nucleotides, pyruvate, any ionized form or salt) Sugars, such as glucose, selenium, glucocorticoids, such as hydrocortisone and/or reducing agents, such as β-mercaptoethanol).

The passage culture may be performed for a time required for the mesenchymal stem cells to be doubling after treating the NADPH oxidant inhibitor. In one embodiment, the mesenchymal stem cells 2.5 x 10 ⁶ cells may be performed for 72 hours to double, but is not limited thereto.

In the method for manufacturing a mesenchymal stem cell with enhanced function according to an embodiment, the number of passages of the passage culture is not particularly limited, and the number of passages may be appropriately selected according to the number of desired proliferating cells. Typically, it may be at least 1 passage, at least 10 passages, for example, by performing 1 to 20 passages and 3 to 15 passages, it is possible to obtain a clinically necessary number of accumulated proliferative cells.

The function-enhanced mesenchymal stem cells prepared by the above method may have a neuroprotective effect, an inhibitory effect of hematoma, and an inhibitory effect on neuronal cell death.

Another aspect provides a functionally enhanced mesenchymal stem cell population prepared by passage culturing mesenchymal stem cells in a medium containing an inhibitor of the activity of an NAPDH oxidant.

The function-enhanced mesenchymal stem cells are as described above.

Another aspect provides a cell therapeutic agent, pharmaceutical composition or formulation comprising the function-enhanced mesenchymal stem cells, a cell population thereof, or a culture medium thereof as an active ingredient.

Another aspect provides the use of the above-enhanced mesenchymal stem cells, cell populations, or cultures thereof for use in the manufacture of cell therapy agents, pharmaceutical compositions or formulations.

The pharmaceutical composition may be a pharmaceutical composition for preventing or treating cerebral hemorrhage and/or ischemic brain disease.

The term cerebral hemorrhage (cerebral hemorrhage) in the present specification is a cerebrovascular disorder caused by bleeding of the cerebral blood vessels, also called cerebral hemorrhage.

The ischemic brain disease may be selected from the group consisting of stroke, stroke, stroke, cerebral infarction, head injury, Alzheimer's disease, vascular dementia, Creutzfeldt-Jakob disease, coma and shock brain injury, but is not limited thereto. As used herein, "ischemic stroke" or "stroke" may mean a disease caused by necrosis of brain tissue or cells by reducing cerebral blood flow for a period of time or more, and "cerebral infarction" )".

Another aspect is to treat a disease, for example, cerebral hemorrhage and/or ischemic brain disease, comprising administering the functionally enhanced mesenchymal stem cells, a population of cells, or a culture thereof to an individual in need thereof. Provide a way to prevent.

The function-enhanced mesenchymal stem cells are as described above.

The function-enhanced mesenchymal stem cells reduce oxidative damage by reducing the production of free radicals, thereby suppressing neuroprotective effect, neuronal cell death inhibitory effect, and hematoma increase, as well as for treating brain hemorrhage and ischemic brain disease. It can be usefully used in cell therapeutic agents or pharmaceutical compositions.

The dosage of the cell therapeutic agent or pharmaceutical composition according to one embodiment is 1.0 X 10 ³ to 1.0 X 10 ¹⁰ cells/kg (body weight) or individual, or 1.0 X 10 ⁷ to 1.0 X based on the enhanced mesenchymal stem cells 10 ⁸ cells/kg (body weight) or individual. However, the dosage may be variously prescribed by factors such as the formulation method, the administration method, the patient's age, weight, sex, morbidity, food, administration time, administration route, excretion rate, and response sensitivity, and those skilled in the art Taking these factors into consideration, the dosage can be appropriately adjusted. The number of times of administration may be one or two or more times within the range of clinically acceptable side effects, and the administration site may be administered at one place or two or more places. For animals other than humans, the dose is the same as the human dose per kg or per individual, or, for example, by the volume ratio (e.g., average value) of the target animal and human organ (heart). The amount converted can be administered. As a target animal for treatment according to an embodiment, humans and other target mammals can be exemplified, and specifically, humans, monkeys, mice, rats, rabbits, sheep, cows, dogs, horses, pigs, etc. Is included.

The cell therapeutic agent or pharmaceutical composition according to an embodiment may include the above-mentioned mesenchymal stem cells with enhanced function and a pharmaceutically acceptable carrier and/or additive as an active ingredient. Examples include sterile water, physiological saline, conventional buffers (phosphoric acid, citric acid, other organic acids, etc.), stabilizers, salts, antioxidants (ascorbic acid, etc.), surfactants, suspending agents, isotonic agents, or preservatives. can do. For topical administration, it is also preferable to combine with organic substances such as biopolymers, inorganic substances such as hydroxyapatite, specifically collagen matrix, polylactic acid polymer or copolymer, polyethylene glycol polymer or copolymer, and chemical derivatives thereof. . When the cell therapeutic agent or pharmaceutical composition according to one embodiment is formulated into a formulation suitable for injection, the cell aggregate may be dissolved in a pharmaceutically acceptable carrier or frozen in a dissolved solution.

The mesenchymal stem cells with enhanced function according to one embodiment are various types that are strengthened, treated, or replaced by engraftment, transplantation, or injection of a desired cell cluster, for example, a stem cell or a derived cell cluster, of a body tissue or organ. Can be used in treatment protocols. The function-enhanced mesenchymal stem cells can replace or enhance existing tissues to become new or altered tissues or to be combined with biological tissues or structures.

The cell therapeutic agent or pharmaceutical composition according to one embodiment, if necessary according to the administration method or formulation, suspending agent, dissolution aid, stabilizer, isotonic agent, preservative, anti-adsorption agent, surfactant, diluent, excipient, pH adjuster, It may contain a painless agent, a buffer, a reducing agent, an antioxidant, and the like. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, 19th ed., 1995.

The cell therapeutic agent or pharmaceutical composition according to one embodiment is formulated by using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by those skilled in the art to which the present invention pertains. It can be manufactured in unit dose form or can be prepared by incorporating into a multi-dose container. The formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in powder, granule, tablet or capsule form. In addition, cell therapy products can be formulated into injectable formulations. In this case, known conventional ingredients for formulation can be used, and can be formulated in a conventional manner.

Functional enhancement mesenchymal stem cells according to one aspect has a blood vessel regeneration effect, or a neuroregeneration effect, it can be usefully used in pharmaceutical compositions or cell therapeutics for the treatment or prevention of various diseases.

Figure 1 is the placebo-treated group (placebo), rats with enhanced function of mesenchymal stem cells (NOX inhibition-MSC), and control mesenchymal stem cells with rats (MSC) administered hematopoietic induction at 24 hours after hematoma size It is a picture showing.
FIG. 2 shows hematoma size at 24 hours after inducing brain hemorrhage in the placebo-treated group (placebo), rats with enhanced mesenchymal stem cells (NOX inhibition-MSC), and rats with control mesenchymal stem cells (MSC). It is a graph showing the results of the measurement.
Figure 3 is a placebo-treated group (placebo), rats administered with enhanced mesenchymal stem cells (NOX inhibition-MSC), and control mesenchymal stem cells administered rats (MSC) around 24 hours after hematoma induced brain hemorrhage It is a micrograph of a nerve cell.
Figure 4 is a placebo-treated group (placebo), rats administered with enhanced mesenchymal stem cells (NOX inhibition-MSC), and control mesenchymal stem cells administered rats (MSC) around 24 hours after hematoma induced brain hemorrhage It is a graph measuring the degree of neuronal degeneration.

It will be described in more detail through the following examples. However, these examples are intended to illustrate one or more embodiments by way of example, and the scope of the present invention is not limited to these examples.

Example 1. Preparation of mesenchymal stem cells with enhanced function

Mesenchymal stem cells with enhanced function through the treatment of NADPH oxidant activity inhibitors were produced. Specifically, the mesenchymal stem cells derived from human placenta were isolated. The separated 2x10 ⁶ to 3x10 ⁶ mesenchymal stem cells were placed in a 175T flask, placed in 35 ml medium to which 100 μM of NADPH oxidant activity inhibitor Apocynin was added, and cultured for 72 hours. After the culture, the medium was discarded, and the cells were washed with phosphate buffered saline, and mesenchymal stem cells were separated to prepare enhanced mesenchymal stem cells.

Experimental Example 1. Confirmation of the therapeutic effect of intramembrane bleeding of mesenchymal stem cells with enhanced function

In order to confirm the therapeutic effect of intra-parenchymal bleeding of the mesenchymal stem cells with enhanced function prepared in Example 1, it was performed as follows. Intra-ventricular hemorrhage models were prepared using rats, and the mesenchymal stem cells that were not enhanced with NADPH were intravenously administered. Hematoma size at 24 hours after inducing brain hemorrhage was compared and evaluated.

Figure 1 is the placebo-treated group (placebo), rats administered with enhanced mesenchymal stem cells (NOX inhibition-MSC), and control mesenchymal stem cells administered rats (MSC), hematoma size 24 hours after inducing brain hemorrhage It is a picture showing.

FIG. 2 shows hematoma size at 24 hours after inducing brain hemorrhage in the placebo-treated group (placebo), rats with enhanced mesenchymal stem cells (NOX inhibition-MSC), and rats with control mesenchymal stem cells (MSC). It is a graph showing the results of the measurement.

As shown in Figures 1 and 2, both the rats to which the function-enhanced mesenchymal stem cells were administered (NOX inhibition-MSC) and the rats to which the control mesenchymal stem cells were administered (MSC) had significantly hematoma size compared to the placebo-administered group. Suppressed. Therefore, it was confirmed that the mesenchymal stem cells suppress the hematoma size and have the therapeutic effect of intrahepatic bleeding.

In addition, the rats to which the function-enhanced mesenchymal stem cells were administered were significantly inhibited in hematoma size compared to the rats to which the control mesenchymal stem cells were administered. Therefore, it was confirmed that mesenchymal stem cells treated with NADPH oxidant activity inhibitors on mesenchymal stem cells had an enhanced therapeutic effect on brain hemorrhage.

Experimental Example  2. Enhanced Mesenchyme  Confirm the effect of stem cell suppression of neuronal cell death around hematoma

In order to confirm the effect of inhibiting nerve damage of the mesenchymal stem cells with enhanced function prepared in Example 1, the following was performed. Intraventricular hemorrhage models were prepared using rats, and the mesenchymal stem cells that were not enhanced with NADPH were intravenously administered. Nerve cells around hematoma were measured 24 hours after inducing brain hemorrhage.

Figure 3 is a placebo-treated group (placebo), rats administered with enhanced mesenchymal stem cells (NOX inhibition-MSC), and control mesenchymal stem cells administered rats (MSC) around 24 hours after hematoma induced brain hemorrhage It is a micrograph of a nerve cell.

Figure 4 is a placebo-treated group (placebo), rats administered with enhanced mesenchymal stem cells (NOX inhibition-MSC), and control mesenchymal stem cells administered rats (MSC) around 24 hours after hematoma induced brain hemorrhage It is a graph measuring the degree of neuronal degeneration.

As shown in Figures 3 and 4, both the rats that received the enhanced mesenchymal stem cells (NOX inhibition-MSC) and the rats that received the control mesenchymal stem cells (MSC) were compared with the placebo-administered group of neurons around the hematoma. The degree of degeneration was significantly suppressed. Therefore, it was confirmed that the mesenchymal stem cells have an effect of inhibiting neuronal cell death.

In addition, the rats to which the function-enhanced mesenchymal stem cells were administered suppressed the degree of degeneration of nerve cells around the hematoma compared to the rats to which the control mesenchymal stem cells were administered. Therefore, it was confirmed that the mesenchymal stem cells treated with the inhibitor of NADPH oxidant activity on the mesenchymal stem cells have enhanced neuroprotective effect.

Claims (13)

A composition for enhancing the function of mesenchymal stem cells comprising an inhibitor of NADPH oxidant. The method according to claim 1, wherein the inhibitor of the NADPH oxidant is aposinin (apocynin, 1-(4-hydroxy-3-methoxyphenyl)-ethanone), trolox (trolox, 6-hydroxy-2,5,7,8-tetramethylchroman -2-Carboxylic Acid, pyrrolidine dithiocarbamate (PDTC), glutathione (GSH), catalase, manganese-superoxide dismutase (MnSOD), manganese superoxide dismutase (MnSOD), Vitamin E (Vitamin E) and quercetin (Quercetin) consisting of at least one member selected from the group consisting of. The composition of claim 1, wherein the inhibitor of the NADPH oxidant is included in a concentration of 10 to 200 μM. The composition according to claim 1, wherein the mesenchymal stem cells are derived from one or more selected from the group consisting of umbilical cord, umbilical cord, cord blood, placenta, fat, and bone marrow. The method according to claim 1, wherein the enhancement of the mesenchymal stem cell function is to inhibit the production of free radicals in mesenchymal stem cells. A method for producing a mesenchymal stem cell with enhanced function, comprising sub-culturing the mesenchymal stem cells in a medium containing an inhibitor of a NAPDH oxidant. The method according to claim 6, The medium is DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, DMEM/F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10) , DMEM/F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12), α-MEM (α-Minimal essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Isocove's Modified Dulbecco's Medium), and KnockOut A method comprising one or more selected from the group consisting of DMEM. The method according to claim 6, wherein the culture is carried out for 72 hours. The method according to claim 6, wherein the inhibitor of the NADPH oxidant is aposinin (apocynin, 1-(4-hydroxy-3-methoxyphenyl)-ethanone), trolox (trolox, 6-hydroxy-2,5,7,8-tetramethylchroman -2-Carboxylic Acid, pyrrolidine dithiocarbamate (PDTC), glutathione (GSH), catalase, manganese-superoxide dismutase (MnSOD), manganese superoxide dismutase (MnSOD), Method of one or more selected from the group consisting of vitamin E and quercetin. A population of functionally enhanced mesenchymal stem cells prepared by passage culturing mesenchymal stem cells in a medium containing an inhibitor of a NAPDH oxidant. A pharmaceutical composition for the prevention or treatment of brain hemorrhage comprising mesenchymal stem cells cultured in a medium containing an activity inhibitor of NADPH oxidant. A pharmaceutical composition for the prevention or treatment of ischemic brain disease comprising mesenchymal stem cells cultured in a medium containing an activity inhibitor of NADPH oxidant. The pharmaceutical composition of claim 12, wherein the ischemic brain disease is at least one selected from the group consisting of stroke, stroke, stroke, cerebral infarction, head injury, Alzheimer's disease, vascular dementia, Creutzfeldt-Jakob disease, coma and shock brain injury. .

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