KR20220073662A - Compositions for Treating Infectious Disease Comprising Exosome Derived from Stem Cell Treated with Thrombin - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating an infectious disease, comprising an exosome derived from thrombin-treated stem cells as an active ingredient, a pharmaceutical preparation containing the same, and a method for preparing the same. The present inventors specifically confirmed that exosomes isolated from thrombin-treated stem cells significantly reduced the expression of inflammatory cytokines under inflammatory conditions. It is expected to be usefully used in related fields for the improvement or treatment of various infectious diseases caused by the induced immune or inflammatory response.

Description

Compositions for Treating Infectious Disease Comprising Exosome Derived from Stem Cell Treated with Thrombin}

The present invention relates to a pharmaceutical composition for the prevention or treatment of an infectious disease, comprising an exosome derived from thrombin-treated stem cells as an active ingredient, a pharmaceutical preparation containing the same, and a manufacturing method thereof.

BACKGROUND ART Infectious diseases are diseases caused by infection with microorganisms such as bacteria, viruses, fungi, and parasites, and are subdivided into various diseases depending on the type of the microorganism to be infected. Microbial infections can affect a part of the body (localized infection) or the whole body (systemic infection), where local infections include, for example, abscesses and bladder infections, and severe systemic infections include sepsis or septic shock. Symptoms of infection may include fever, rapid pulse, rapid breathing, anxiety, and confusion, and most of the effects of infection are resolved when the infection is effectively treated.

Acute Respiratory Distress syndrome (ARDS), in which a severe infection such as sepsis occurs as one of the main causes, is when substances such as cytokines are secreted into the blood due to infection or damage to the body and when it reaches the lungs, severe Inflammation is caused and Research results have been reported that various inflammatory cytokines are involved in initiating and amplifying the inflammatory response in acute respiratory distress syndrome, and it is also known that the balance between inflammatory mediators and anti-inflammatory mediators plays an important role (Journal of the Korean Society of Internal Medicine, Vol. 68, No. 5, Vol. 537, 2005). The disease shows severe respiratory distress symptoms that cannot sustain life without a ventilator, usually several hours to several days after exposure to a causative agent. In general, for treatment, intensive care including mechanical ventilation is performed using a ventilator, which is not a fundamental treatment, but supports the patient's breathing for the time required for the patient's lungs to heal. However, at present, the mechanical ventilation method is almost the only one. In addition, if an infection is accompanied by antibiotic treatment, etc., but it is an intractable disease for which there are few effective treatments to date, so there is an urgent need to develop a treatment for this.

On the other hand, stem cells are known as cells involved in tissue regeneration, treatment and immune response with their pluripotency. Using these characteristics, mesenchymal stem cells are isolated and cultured from umbilical cord blood, bone marrow, etc. to develop various diseases and symptoms. Efforts have been made to develop it as a therapeutic agent for However, cell therapy using these stem cells itself has the potential of tumorogenicity due to DNA transfer, the possibility of vascular obstruction or myocardial infarction due to its large size, and rejection due to cell surface antigen during allogeneic transplantation. It has limitations such as a problem of reaction, a difficult manufacturing process, and a high production cost.

Exosomes are membrane-structured small vesicles (approximately 30-100 nm in diameter) secreted from various cells, and do not separate directly from the plasma membrane in the study through electron microscopy, but rather inside cells called multivesicular bodies (MVBs). It originates from a specific compartment and was observed to be released and secreted out of the cell. That is, when polycystic body and plasma membrane fusion occurs, vesicles are released into the extracellular environment, which is called exosomes. Although it is not clear by what molecular mechanism these exosomes are made, various types of immune cells including red blood cells, B-lymphocytes, T-lymphocytes, dendritic cells, platelets, macrophages, and tumor cells, It is known that stem cells also produce and secrete exosomes while they are alive.

In particular, stem cell-derived exosomes contain not only receptors and proteins, but also nuclear components, and are known to play a role in intercellular communication. In addition, the stem cell-derived exosomes contain relatively less animal serum than stem cells, so the risk of zoonosis caused by animal serum infection can also be excluded. Considering these characteristics of exosomes, cell therapy using exosomes is expected to be a new paradigm that can overcome the limitations of existing stem cell therapies.

As a result of research efforts by applying stem cell-derived exosomes to infectious diseases, Raw 264.7 cells were treated with lipopolysaccharide (LPS) to create an infectious environment, and stem cell-derived liquid treated with thrombin, LPS, and TNF, respectively. As a result of treating the sosome, in the case of the thrombin-treated stem cell-derived exosome-treated group, an excellent anti-inflammatory effect was confirmed by reducing the production of inflammatory cytokines, and the present invention was completed based on this.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for preventing or treating infectious diseases, comprising, as an active ingredient, a thrombin-treated stem cell-derived exosome.

Another object of the present invention is to provide a pharmaceutical formulation for the treatment of infectious diseases, comprising the pharmaceutical composition.

In addition, it is another object of the present invention to provide a method for preparing the pharmaceutical composition.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention provides a pharmaceutical composition for preventing or treating infectious diseases, comprising thrombin-treated stem cell-derived exosomes as an active ingredient.

In one embodiment of the present invention, the stem cells are from the group consisting of mesenchymal stem cells, human tissue-derived mesenchymal stromal cells, human tissue-derived mesenchymal stem cells, multipotent stem cells, and amnion epithelial cells. may be selected.

In another embodiment of the present invention, the mesenchymal stem cells may be derived from umbilical cord, umbilical cord blood, bone marrow, fat, muscle, skin, amniotic membrane, placenta or other tissues.

In another embodiment of the present invention, the infectious disease may be selected from the group consisting of infectious lung disease, sepsis and systemic inflammatory response syndrome.

In another embodiment of the present invention, the infectious lung disease may be acute respiratory distress syndrome (ARDS).

In another embodiment of the present invention, the pharmaceutical composition may further include an auxiliary component selected from the group consisting of a culture medium, cytokines, growth factors and genes.

In addition, the present invention provides a pharmaceutical formulation for treating an infectious disease, comprising the pharmaceutical composition.

In one embodiment of the present invention, the pharmaceutical formulation may be in the form of injections, injections, sprays, liquids, or patches.

In addition, the present invention provides a method for preparing the pharmaceutical composition, comprising the following steps.

(a) treating thrombin after culturing stem cells;

(b) separating the exosomes from the culture medium of step (a); and

(c) preparing a composition containing the exosome isolated in step (b) as an active ingredient.

In one embodiment of the present invention, the thrombin of step (a) may be contained in the medium at a concentration of 1-1000 unit/ml.

In another embodiment of the present invention, the exosomes of step (b) may be separated by performing centrifugation at 3,000-100,000 g for 10 minutes to 5 hours.

In addition, the present invention provides a method for preventing or treating an infectious disease, comprising administering the pharmaceutical composition to a subject.

In addition, the present invention provides the use of the pharmaceutical composition for preventing or treating infectious diseases.

The present inventors specifically confirmed that exosomes isolated from thrombin-treated stem cells significantly reduced the expression of inflammatory cytokines in the inflammatory condition compared to the case of untreated thrombin or treated with other substances. The thrombin-treated stem cell-derived exosome according to the present invention is expected to be usefully used in related fields for the improvement or treatment of various infectious diseases caused by an immune response or an inflammatory response induced through infection.

Figure 1, RAW 264.7 cells were treated with LPS, and then exosomes isolated from untreated stem cells (naive), exosomes isolated from stem cells treated with LPS, TNF and thrombin, respectively, were treated with the cells This is the result of measuring the concentrations of inflammatory cytokines, TNF-α and IL-1β, in the cell culture medium.
Figure 2, in an animal model of infectious disease, saline administration group (control group, ARDS) and thrombin-treated stem cell-derived exosome administration group (ARDS-exo), inflammatory cytokines (TNF-α, IL-1β, IL- 6) This is the result of measuring the concentration.

The present inventors have confirmed the excellent anti-inflammatory effect of thrombin-treated stem cell-derived exosomes in an infectious environment, based on this, completed the present invention.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating infectious diseases, comprising, as an active ingredient, a thrombin-treated stem cell-derived exosome.

As used herein, the term “thrombin” is a proteolytic enzyme involved in blood coagulation, and serves to catalyze a reaction that hydrolyzes soluble fibrinogen in blood to change it into insoluble fibrin. In the present invention, by treating the stem cells, it exhibits the function of effectively improving the therapeutic effect of the exosomes isolated from the stem cells for the treatment of infectious diseases.

As used herein, the term “stem cell” refers to an 'undifferentiated cell' that has differentiation ability but has not yet differentiated, which can differentiate into two or more different types of cells while having self-replicating ability. cells that have the ability to The stem cells of the present invention may be autologous or allogeneic stem cells, and may be derived from any type of animal, including humans and non-human mammals.

In the present invention, the stem cells of the present invention include embryonic stem cells or adult stem cells, preferably adult stem cells. The adult stem cells may be mesenchymal stem cells, human tissue-derived mesenchymal stromal cells, human tissue-derived mesenchymal stem cells or pluripotent stem cells, preferably mesenchymal stem cells, but limited thereto it's not going to be

The mesenchymal stem cells may be mesenchymal stem cells derived from umbilical cord, umbilical cord blood, bone marrow, fat, muscle, skin, amniotic membrane, placenta and other tissues, but is not limited thereto.

As used herein, the term “exosome” refers to a small (approximately 30-100 nm diameter) vesicle with a membrane structure secreted from various cells, and the fusion of the polycystic body with the plasma membrane takes place in the extracellular environment vesicles that are released into The exosomes include those that are naturally secreted, or those that are artificially secreted. Because exosome-based therapeutics are cell-free, they do not contain DNA, so there is little risk of carcinogenesis, and there is no cell surface antigen, so there is no problem of transplant rejection, so they are safe and off-the-shelf products. Since drug development is possible, there is an advantage in reducing the manufacturing cost.

As used herein, the term “prevention” refers to any action that suppresses or delays the onset of an infectious disease by administration of the pharmaceutical composition according to the present invention.

As used herein, the term “treatment” refers to any action in which symptoms for an infectious disease are improved or beneficially changed by administration of the pharmaceutical composition according to the present invention.

In the present invention, "infectious disease" includes all diseases caused by microbial infection such as bacteria, viruses, fungi and parasites, and preferably may be a disease caused by an immune response and an inflammatory response through infection, , More preferably, it may be selected from the group consisting of infectious lung disease, sepsis and systemic inflammatory response syndrome, and the infectious lung disease may be most preferably acute respiratory distress syndrome.

The Acute Respiratory Distress Syndrome (ARDS) is an intractable disease for which there is little treatment, and is mainly caused by serious infection or pneumonia, and may also be caused by physical injury or other organ problems. Acute Respiratory Distress Syndrome reduces blood oxygen levels and releases certain cytokines secreted by damaged lung cells and white blood cells into the bloodstream, which can lead to inflammation and complications in other organs.

The sepsis is a systemic response to microbial infection, and the cause of sepsis / septic shock is the frequency of several bacteria, particularly E. coli , Pseudomonas aeruginosa , Klebsiella pneumoniae , etc. Gram-negative bacteria is known to be high. In order to pathophysiologically remove the invading infectious bacteria from the living body, all immune systems such as various blood cells and endothelial cells as well as bone marrow are activated, and major organs are damaged by many mediators such as cytokines secreted in this process. has been reported to be

The systemic inflammatory response syndrome refers to a clinical condition in which two or more of fever, temperature drop, rapid pulse, rapid respiration, and abnormally high white blood cell count exist simultaneously among symptoms caused by inflammation.

The pharmaceutical composition of the present invention may further include an auxiliary component selected from the group consisting of a culture medium, cytokines, growth factors and genes.

The pharmaceutical composition of the present invention may be used alone or in combination with methods using other therapeutic agents for the treatment of infectious diseases.

The pharmaceutical composition of the present invention includes an exosome derived from thrombin-treated stem cells as an active ingredient, and may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is commonly used in the formulation, and includes saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, etc., but is limited thereto. It does not, and may further include other conventional additives, such as antioxidants and buffers, if necessary. In addition, diluents, dispersants, surfactants, binders, lubricants, etc. may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, pill, capsule, granule or tablet. Regarding suitable pharmaceutically acceptable carriers and formulations, formulations can be preferably made according to each component using the method disclosed in Remington's literature.

The pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) according to a desired method, and the dosage may vary depending on the condition and weight of the patient, and the disease. Although it varies depending on the degree, drug form, administration route and time, it may be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat or diagnose a disease at a reasonable benefit/risk ratio applicable to medical treatment or diagnosis, and the effective dose level is determined by the patient's disease type, severity, drug activity, sensitivity to drugs, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs, and other factors well-known in the medical field. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, weight, absorption of the active ingredient into the body, inactivation rate and excretion rate, disease type, and drugs used in combination, in general 0.001 to 150 mg, preferably 0.01 to 100 mg per 1 kg of body weight, may be administered daily or every other day, or may be administered in divided doses 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, the severity of obesity, sex, weight, age, etc., the dosage is not intended to limit the scope of the present invention in any way.

In addition, the present invention provides a pharmaceutical formulation for treating an infectious disease, comprising the pharmaceutical composition.

The pharmaceutical formulation may be in the form of an injection, injection, spray, liquid, or patch formulation, but is not limited thereto, and those skilled in the art may select and use a formulation suitable for the treatment of the corresponding infectious disease.

In the present invention, through specific examples, it was confirmed that thrombin-treated stem cell-derived exosomes have an excellent therapeutic effect on infectious diseases.

In one embodiment of the present invention, the umbilical cord blood-derived mesenchymal stem cells were treated with thrombin and cultured, and then the exosomes were separated by centrifugation (see Example 1).

In another embodiment of the present invention, in order to evaluate the therapeutic effect of the exosomes on infectious diseases, the mouse-derived macrophages, Raw 264.7 cells, were treated with LPS, and then the exosomes isolated from untreated stem cells, each After treating the cells with exosomes isolated from LPS, TNF and thrombin-treated stem cells, the concentration of inflammatory cytokines TNF-α and IL-1β in the cell culture medium was measured. As a result, thrombin-treated stem cell-derived exo It was confirmed that only the concentration of the two cytokines was significantly reduced only in the group treated with the moth (see Example 2-1).

In addition, in the infectious disease animal model, the thrombin-treated stem cell-derived exosome-administered group (ARDS-exo), compared to the control group (saline-administered group), had inflammatory cytokine concentrations such as TNF-α, IL-1β, and IL-6. was found to decrease statistically significantly (see Example 2-2).

Accordingly, as another aspect of the present invention, the present invention comprises the steps of (a) treating thrombin after culturing stem cells; (b) separating the exosomes from the culture medium of step (a); And (c) provides a method for preparing the pharmaceutical composition comprising the step of preparing a composition containing the exosome isolated in step (b) as an active ingredient.

In the present invention, the concentration of thrombin treatment is not particularly limited as long as it is a concentration suitable for enhancing the efficacy of stem cells/exosomes, but it is preferably contained in the medium at a concentration of 1-1000 unit/ml.

There is no limitation in the method of isolating exosomes in the present invention, for example, in culture, centrifugation, ultra-high-speed centrifugation, filtration by a filter, gel filtration chromatography, pre-flow electrophoresis, capillary electrophoresis, using a polymer Separation may be performed using a method such as separation or a combination thereof, and preferably centrifugation/ultracentrifugation may be used. At this time, centrifugation/ultracentrifugation is preferably performed for 10 minutes to 5 hours at 4 ℃, 3,000-100,000 g.

The medium used for cell culture in the present invention contains essential elements for the growth and proliferation of cells, such as sugar, amino acids, various nutrients, serum, growth factors, and minerals. means a mixture for the growth and propagation of The medium that can be used in the present invention is DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI1640, 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 Commercially prepared media or artificially synthesized media, such as, but not limited thereto.

As another aspect of the present invention, the present invention provides a method for preventing or treating an infectious disease comprising administering to an individual a pharmaceutical composition comprising an exosome derived from thrombin-treated stem cells as an active ingredient.

In the present invention, "individual" means a subject in need of treatment for a disease, and more specifically, human or non-human primates, mice, rats, dogs, cats, horses and cattle. means mammals.

In addition, the present invention provides the use of the pharmaceutical composition for preventing or treating infectious diseases.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Induction of exosome secretion by thrombin treatment in stem cells

Human umbilical cord blood-derived mesenchymal stem cells (3×10 ⁵ cells) were aliquoted in a 100 mm culture dish (orange scientific cat# 4450200) and cultured for 1 week. After confirming that the cells were saturated and proliferated in the culture dish, it was replaced with a serum-free culture medium (MEM alpha media) diluted with 50 unit/ml thrombin (REYON Pharmaceutical. Co., LTD), and cultured again for 6 hours. . Thereafter, the culture solution was divided into centrifuge tubes and centrifuged at 4° C. and 6,000 rpm for 30 minutes, and the supernatant was transferred to a new tube to remove cell debris. Again, the supernatant was ultracentrifuged at 4° C. and 100,000 rpm for 2 to 4 hours, and then the supernatant was further removed to obtain exosomes.

Example 2. Confirmation of anti-inflammatory and anti-infectious disease effects by thrombin-treated stem cell-derived exosomes

The present inventors conducted the following experiment to find out whether thrombin-treated stem cell-derived exosomes can treat diseases induced by infection, such as acute respiratory distress syndrome (ARDS), through anti-inflammatory effects.

2-1. in vitro Experiment

After culturing mouse-derived macrophages, RAW 264.7 cells, the cells were activated by treatment with lipopolysaccharide (LPS) in the membrane of Gram-negative bacteria, which is one of the external immune inducers. Subsequently, after treatment with exosomes isolated from untreated stem cells, or exosomes isolated from stem cells treated with LPS, TNF and thrombin, respectively, a representative inflammation-inducing cytokine, TNF-α (tumor necrosis) Factor-α) and IL-1β (Interleukin 1 beta) concentrations in the culture medium were measured.

As a result, as can be seen in FIG. 1 , the concentration of TNF-α and IL-1β in the culture medium was increased in the control group (NC) in which RAW 264.7 cells were treated with only LPS. In contrast, in the case of the group treated with the stem cell-derived exosomes without any treatment (naive), and the group treated with the stem cell-derived exosomes treated with LPS or TNF, the cytokines in the culture medium compared to the control, respectively their concentration decreased. In addition, in particular, in the case of exosomes derived from stem cells treated with thrombin, it was confirmed that the production amount of the two cytokines was statistically significantly decreased.

2-2. in vivo Experiment (infectious disease animal model)

In order to prepare an infectious disease animal model (ARDS mouse model), rumpun and ketamine saline were diluted and administered intraperitoneally to mice (16 to 24 weeks old) to anesthetize, then a catheter was intubated into the airway, and E. coli ( E. coli ) 0.05 cc was administered into the airway using a 1ml syringe.

After E. coli administration, the saline administration group (ARDS) and the thrombin-treated stem cell-derived exosome (hereinafter referred to as exosome) administration group (ARDS-exo) were divided, respectively, saline and exosomes were administered, 24 hours later, ifran solution After sacrificing under respiratory anesthesia, the mouse was sheared to reveal the airway from the neck, and then a catheter was inserted into the airway and fixed with a suture. After filling the lung with saline using a syringe, BAL (Bronchoalveolar Lavage) Fluid was carefully collected from the lung. The collected BAL fluid was stored frozen at -70°C until biochemical measurement of the supernatant from which cells and bacteria were removed by centrifugation.

After slowly dissolving BAL fluid stored at -70°C at 4°C, the concentration of inflammatory cytokines (TNF-α, IL-1β, IL-6) was measured using a multiplex cytokine kit according to the manufacturer's protocol.

As a result, as can be seen in Figure 2, in the infectious disease mouse model, in the exosome administration group (ARDS-exo) than in the saline administration group (ARDS), the concentration of inflammatory cytokines such as TNF-α, IL-1β, IL-6 was It was found that the decrease was statistically significant (t-test).

Through the above results, it can be seen that exosomes derived from thrombin-treated stem cells have excellent anti-inflammatory and infectious disease treatment effects, which are caused by inflammation caused by the immune response through the anti-inflammatory effect of the exosomes. To demonstrate that it can be used for the treatment of various diseases, especially infectious diseases.

The description of the present invention stated above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (11)

A pharmaceutical composition for preventing or treating an infectious disease, comprising, as an active ingredient, an exosome derived from thrombin-treated stem cells.
According to claim 1,
The stem cells are mesenchymal stem cells, human tissue-derived mesenchymal stromal cells, human tissue-derived mesenchymal stem cells, pluripotent stem cells and amnion epithelial cells, characterized in that selected from the group consisting of, pharmaceutical enemy composition.
3. The method of claim 2,
The mesenchymal stem cells are characterized in that derived from umbilical cord, umbilical cord blood, bone marrow, fat, muscle, skin, amniotic membrane or placenta, a pharmaceutical composition.
According to claim 1,
The infectious disease is characterized in that selected from the group consisting of infectious lung disease, sepsis and systemic inflammatory response syndrome, the pharmaceutical composition.
5. The method of claim 4,
The infectious lung disease is acute respiratory distress syndrome (ARDS), characterized in that the pharmaceutical composition.
According to claim 1,
The pharmaceutical composition is characterized in that it further comprises an auxiliary component selected from the group consisting of a culture medium, cytokines, growth factors and genes.
A pharmaceutical formulation for treating an infectious disease, comprising the composition of claim 1.
8. The method of claim 7,
The pharmaceutical formulation is an injection formulation, injection formulation, spray formulation, liquid formulation or patch formulation, characterized in that the pharmaceutical formulation.
A method for preparing the pharmaceutical composition of claim 1, comprising the steps of:
(a) treating thrombin after culturing stem cells;
(b) separating the exosomes from the culture medium of step (a); and
(c) preparing a composition containing the exosome isolated in step (b) as an active ingredient.
10. The method of claim 9,
Thrombin of step (a) is characterized in that it is contained in the medium at a concentration of 1-1000 unit / ml, the manufacturing method.
10. The method of claim 9,
The exosome of step (b) is characterized in that the separation by performing centrifugation at 3,000-100,000 g for 10 minutes to 5 hours, the production method.

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