WO2022010263A1 - Pharmaceutical composition containing peptide-bound recombinant exosomes for preventing or treating inflammation-mediated diseases caused by rage overexpression - Google Patents

Abstract

The present invention relates to: a pharmaceutical composition containing peptide-bound recombinant exosomes for preventing or treating inflammation-mediated diseases caused by RAGE overexpression; and a pharmaceutical preparation comprising the composition. According to the present invention, exosomes bound to a RAGE-targeting peptide specifically bind to RAGE to inhibit RAGE-mediated signaling pathways, and thus exosomes themselves exhibit anti-inflammatory effects and treatment effects on inflammation-mediated diseases, and more synergistic effects could be achieved by encapsulating an appropriate treatment drug in the exosomes. Therefore, it is expected that exosomes according to the present invention can be developed into a therapeutic agent exhibiting excellent treatment effects on the inflammation-mediated diseases for which there is no non-cytotoxic, safe, economical, and effective treatment.

Description

Pharmaceutical composition for the prevention or treatment of inflammatory-mediated diseases caused by overexpression of RAGE comprising a peptide-coupled recombinant exosome

The present invention relates to a pharmaceutical composition for the prevention or treatment of an inflammatory-mediated disease caused by overexpression of RAGE comprising a recombinant exosome bound to a peptide, and a pharmaceutical formulation comprising the composition.

Receptor for advanced glycation endproducts (RAGE) is a cell membrane multi-receptor that binds to various types of ligands belonging to the immunoglobulin family. It is characterized as an intracellular product. A ligand that binds to RAGE is a substance in which glycosylation has occurred non-enzymatically in cells, and it is known that diseases mediated by RAGE vary depending on the type of ligand that binds to RAGE. RAGE expression level is low in the normal state, but when ligand binds to RAGE, cell activation is continuously induced in the cell by receptor-dependent signaling, and when the ligand concentration is high, RAGE expression on the cell membrane is upregulated. RAGE-dependent intracellular responses are sustained by this upregulation, and as a result, it is known to mediate various diseases such as diabetes, diabetic complications, amyloidosis, immune/inflammatory responses, chronic kidney disease, and cancer.

In relation to RAGE-mediated immune/inflammatory response, important ligands are known advanced glycation endproducts (AGE), S100/calgranulins, and amphoterin. The interaction between AGE and RAGE is well known to promote the activation of inflammation-related genes, and S100/calgranulins is a closely related family of calcium-binding polypeptides whose interaction with RAGE plays a major role in mediating the inflammatory response. It is known that it plays an important role in the initiation of an inflammatory response by activating -12 and TNF-α. In addition, since the level of RAGE is observed to be high in patient groups of various diseases such as diabetes patients, it has been reported that RAGE plays a key role in inducing inflammation in these diseases. For example, it has been reported that HMGB1, one of the ligands of RAGE, plays an important role in tissue damage and necrosis in ischemic diseases such as ischemic myocardial infarction. It is known that HMGB1 is secreted from the nucleus to the cytoplasm in a mouse model. In addition, RAGE is closely related to the pathophysiology of these diseases in sepsis, chronic kidney disease including diabetes, and acute respiratory distress syndrome, and is attracting attention as a therapeutic target for these diseases.

In this regard, in order to suppress the onset of various inflammatory-mediated diseases caused by RAGE, studies have been conducted to inhibit the RAGE-mediated signaling pathway by binding to RAGE. For example, prevention or treatment of myocarditis using sRAGE (soluble RAGE) lacking the transmembrane domain and cytoplasmic domain of RAGE (Korea Patent Publication No. 2013-0082474) and using an antibody that specifically binds to RAGE. treatment techniques were reported. However, the prior technologies are based on large proteins having a large molecular weight, and not only have relatively poor stability, but are all intended to inhibit only the signaling process of RAGE.

Therefore, it is necessary to develop an innovative therapeutic agent that can achieve a more synergistic therapeutic effect by not only inhibiting the RAGE-mediated signaling process by targeting RAGE, but also using therapeutic substances for the target disease.

Under the above background, the present inventors produced a recombinant exosome that can specifically bind to RAGE to inhibit the RAGE-mediated signaling pathway while encapsulating a therapeutic drug for a target disease, and the exosome is the It has been confirmed that a synergistic therapeutic effect can be achieved when a therapeutic drug is encapsulated while exhibiting an anti-inflammatory effect and a therapeutic effect on the inflammatory-mediated disease by itself, thereby completing the present invention.

Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory-mediated diseases caused by RAGE overexpression, comprising an exosome bound to a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient, and a pharmaceutical formulation comprising the composition intended to provide

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 is an inflammation-mediated disease caused by overexpression of RAGE (receptor for advanced glycation endproducts) comprising an exosome bound to a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient It provides a pharmaceutical composition for the prevention or treatment of.

In one embodiment of the present invention, the peptide may specifically bind to RAGE.

In another embodiment of the present invention, the exosome may be obtained by transfecting a cell with a plasmid vector encoding the peptide and then culturing the cell.

In another embodiment of the present invention, the culture may be made for 10 to 20 days.

In another embodiment of the present invention, the pharmaceutical composition may further include a drug.

In another embodiment of the present invention, the drug may be encapsulated inside the exosome.

In another embodiment of the present invention, the drug may be any one selected from the group consisting of compounds, biodrugs, nucleic acids, peptides, proteins, natural products, hormones, contrast agents, antibodies, and combinations thereof.

In another embodiment of the present invention, the inflammation-mediated disease caused by RAGE overexpression is acute lung injury, acute respiratory distress syndrome, pneumonia, asthma, ischemic brain disease, ischemic myocardial infarction, brain tumor, sepsis, diabetes and diabetic kidney disease. It may be any one selected from the group consisting of.

In addition, the present invention provides a pharmaceutical formulation for preventing or treating inflammation-mediated diseases caused by overexpression of receptor for advanced glycation endproducts (RAGE), comprising the composition.

In one embodiment of the present invention, the formulation may be in the form of an injection, injection, or spray.

In addition, the present invention provides a method for preventing or treating inflammation-mediated diseases caused by RAGE overexpression, comprising administering to an individual a pharmaceutical composition comprising an exosome bound to a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient provides

In addition, the present invention provides the use of the pharmaceutical composition for preventing or treating inflammation-mediated diseases caused by overexpression of RAGE.

According to the present invention, the exosomes to which RAGE-targeting peptides bind specifically bind to RAGE, thereby inhibiting the RAGE-mediated signaling pathway, thereby exhibiting anti-inflammatory effects and therapeutic effects on inflammatory-mediated diseases, and the exosomes A more synergistic therapeutic effect can be achieved by encapsulating an appropriate therapeutic drug in development is expected to be possible.

1 shows the structure and sequence of a plasmid DNA into which RBP-Lamp2b and hygromycin resistance genes are inserted.

Figure 2 is a result of Western blot to verify whether the cells transfected with the plasmid of Figure 1 to produce RBP-bound exosomes (RBP-exosomes) (Unmod-exo; general secreted from normal cells) exosome, RBP-exo: RBP-exosome).

3a is a result of measuring the size and zeta potential of RBP-exosomes according to the present invention by a dynamic light scattering method.

Figure 3b shows a scanning electron microscope (SEM) image of the general exosomes and RBP-exosomes according to the present invention.

Figure 4 is RBP-in order to evaluate the anti-inflammatory effect of the exosome according to the present invention, Raw264.7 cells were treated with LPS and the exosomes were treated with each concentration (1, 3, 5, 10, 20 μg). The results of measuring the concentration of TNF-α, a post-inflammatory factor, are shown.

Figure 5 is after the treatment of LPS into the trachea of a mouse model of acute lung injury 2 hours later, each material including RBP-exosomes was injected into the lungs, and bronchoalveolar lavage (BAL fluid) and lung tissue (Tissue) of the mouse were collected the next day. to measure the TNF-α and IL-1β concentrations (Control: negative control group, LPS alone: LPS alone treatment group, Curcumin only: curcumin alone treatment group, Unmod-exo-Cur: curcumin-encapsulated general exosome treatment group, RBP-exo: RBP-exosome-treated group alone, RBP-exo/Cur: Curcumin-encapsulated RBP-exosome-treated group).

6 is a result of H&E staining for each mouse-derived lung tissue collected in FIG. 5 .

7 is a normal exosome (Unmod-exo), RBP-exosome (RBP-exo) according to the present invention, and PEI25k, a material having a high positive charge, to the nerve cells, respectively, and measuring the cell viability It is the result of analyzing whether or not cytotoxicity is present.

8 is a result of measuring the expression level of RAGE through FACS after treating normal exosomes or RBP-exosomes to neurons cultured in a hypoxic environment.

9 is a result of measuring changes in cerebral blood flow over time through laser Doppler analysis to fabricate an ischemic stroke model by inducing middle cerebral artery occlusion in a mouse and to confirm whether the model was successfully created.

10 is a result of analyzing the expression level of TNF-α by performing immunohistochemical staining using brain tissue sections prepared by processing each substance in the ischemic stroke mouse model of FIG. 9 and removing the brain 24 hours later ( Control: Tissue from a negative control mouse, MCAO: Tissue from an ischemic stroke mouse, Naked-AMO: Tissue from a mouse administered with anti-miRNA oligonucleotide not encapsulated in exosomes, Unmod-EXO: Tissue from a mouse treated with general exosomes encapsulated in AMO , RBP-EXO: AMO-encapsulated RBP-exosome-administered mouse tissue).

11 is a result of confirming the expression of TNF-α when 3 days have elapsed after performing the experiment of FIG. 10 .

12 is a result of analyzing the RAGE expression level by performing immunohistochemical staining in the ischemic stroke mouse model of FIG. 9 .

13 is a result of analyzing the expression level of Bcl-2 by performing immunohistochemical staining using the same brain tissue section as in FIG. 10 .

14 is a result of TUNEL assay using the same brain tissue slice as in FIG. 10 .

15 is a result of confirming the effect of reducing cerebral infarction after 3 days have elapsed after each substance was treated in the ischemic stroke mouse model of FIG. 9 .

The present inventors are able to specifically bind to RAGE to inhibit the RAGE-mediated signaling pathway and to encapsulate various therapeutic drugs for target diseases. Its excellent anti-inflammatory and therapeutic effects on inflammatory-mediated diseases were confirmed.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating inflammation-mediated diseases caused by RAGE (receptor for advanced glycation endproducts) overexpression, comprising an exosome bound to a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

In addition, the present invention provides a pharmaceutical formulation for preventing or treating inflammation-mediated diseases caused by RAGE overexpression, comprising the pharmaceutical composition.

As used herein, the term “prevention” refers to any action of suppressing or delaying the onset of an inflammatory-mediated disease caused by RAGE overexpression 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 inflammatory-mediated disease caused by overexpression of RAGE are improved or beneficially changed by administration of the pharmaceutical composition according to the present invention.

In the present invention, the peptide is composed of the amino acid sequence of SEQ ID NO: 1, and has a function of specifically binding to RAGE expressed in a cell membrane. In this case, the peptide is 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95%, 96%, 97%, 98%, 99 of the amino acid sequence of SEQ ID NO: 1, respectively. It may include an amino acid sequence having more than % sequence homology.

In the present invention, the term "exosome" refers to a small vesicle with a membrane structure secreted from various cells, and is a vesicle that is released into the extracellular environment due to the fusion of the polycystic body with the plasma membrane. The exosomes include both those naturally secreted from cells or artificially secreted.

In the present invention, the exosome may be obtained by culturing the cell after transfecting the cell with a plasmid vector encoding the peptide consisting of the amino acid sequence of SEQ ID NO: 1. Preferably, the plasmid vector may include RBP-Lamp2b and a hygromycin resistance gene.

The method for transfecting the cell with the plasmid vector is not particularly limited, and one skilled in the art can selectively apply a method known in the art. Preferably, in the present invention, the transfection was carried out using a liposome. .

According to the type of cell, those skilled in the art can set appropriate culture conditions by a method known in the art, and the culture period may be 10 to 20 days, preferably 10 to 18 days, more preferably Preferably, it can be done for 12 to 16 days, most preferably 14 days, but it can be appropriately adjusted by those skilled in the art depending on the type of cell and culture conditions.

The pharmaceutical composition according to the present invention may further include a drug (drug) for the target disease to be treated, wherein the drug may be encapsulated inside the exosome to which the peptide according to the present invention is bound.

The drug is a compound drug (chemical drug), biodrug (biodrug), nucleic acid drug (nucleic acid drug), peptide drug (peptide drug), protein drug (protein drug), natural product drug (natural product drug), hormone (hormone) , a contrast agent, an antibody, and a combination thereof may be any one selected from the group consisting of.

The "bio-drug" refers to various biopharmaceuticals such as (original) biologics, biogenerics, biobetters, and biosuperiors. The bio-drug refers to any drug manufactured, secreted, or semi-synthesized from a biological origin, and includes, but is not limited to, vaccines, blood products, antigens, cell products, gene therapy products, stem cells, and the like.

In the present invention, the drug may be preferably a hydrophobic anti-inflammatory agent, a hydrophobic natural drug, a micronucleic acid drug, a hydrophobic anticancer agent, or an anti-inflammatory peptide, but is not limited thereto.

In the present invention, the inflammation-mediated disease by RAGE overexpression is acute lung injury, acute respiratory distress syndrome, pneumonia, asthma, ischemic brain disease, ischemic myocardial infarction, brain tumor, sepsis, diabetes and diabetic kidney disease in the group consisting of It may be any one selected, but is not limited thereto.

The present inventors confirmed the specificity of the peptide-bound exosomes according to the present invention to RAGE through specific examples and the therapeutic effect of anti-inflammatory and inflammatory-mediated diseases through this.

In one embodiment of the present invention, by transfecting cells with a plasmid vector encoding the peptide and culturing the cells, an exosome in which a peptide (RBP) specifically binding to RAGE is expressed on the surface was obtained, Its physical properties were confirmed (see Example 1).

In another embodiment of the present invention, after treating Raw264.7 cells with LPS that induces inflammation, the exosomes bound to RBP according to the present invention are treated for each concentration, and the exosome itself also targets RAGE to have an anti-inflammatory effect. was confirmed to represent (see Example 2).

In another embodiment of the present invention, in order to evaluate whether the RBP-coupled exosome according to the present invention actually has a therapeutic effect on acute lung injury, which is a type of inflammation-mediated disease caused by overexpression of RAGE, acute lung injury mouse After injecting the exosomes into the lungs of the model, bronchoalveolar lavage fluid and lung tissue were collected to analyze the therapeutic effect. As a result, it was confirmed that a significant therapeutic effect was shown in the group in which the exosome was injected alone, and moreover, it was confirmed that a higher therapeutic effect appeared in the group treated by encapsulating curcumin, an anti-inflammatory natural drug, in the exosome ( see Example 3).

In another embodiment of the present invention, the therapeutic effect of the RBP-coupled exosome according to the present invention in ischemic stroke as another example of an inflammatory-mediated disease caused by overexpression of RAGE was verified. Specifically, it was confirmed that the exosomes did not induce cytotoxicity even in sensitive neurons as a result of treating the neurons with the RBP-coupled exosomes. It was found that the expression of RAGE was significantly reduced when the exosome was treated (see Example 4-1). Furthermore, as a result of verifying the ischemic stroke therapeutic effect of the exosome according to the present invention through histological analysis using an ischemic stroke mouse model, it was confirmed that an excellent therapeutic effect was obtained by encapsulating a drug in the exosome and treating it (Example 4) -2).

From the results of the examples of the present invention, the exosomes bound to RBP according to the present invention do not induce cytotoxicity and have anti-inflammatory effects and treatment of inflammatory-mediated diseases by themselves through high specificity for RAGE-expressing cells. It can be seen that a synergistic therapeutic effect can be achieved when the drug is encapsulated inside the exosome and treated together.

The pharmaceutical composition according to the present invention includes an exosome bound to a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient, and may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is commonly used in formulation, and includes, but is not limited to, saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and the like. 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, etc., pills, capsules, granules or tablets. 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 is not particularly limited in formulation, but may be formulated as an injection, injection, spray, inhalation, or external preparation for skin.

The pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intravenously, subcutaneously, intraperitoneally, intranasally, inhaled or applied topically) according to a desired method, and the dosage may vary depending on the patient's condition. and body weight, disease severity, drug form, administration route and time, but 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 may be administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. 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 per 1 kg of body weight, preferably 0.01 to 100 mg, may be administered daily or every other day, or divided into 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.

As another aspect of the present invention, the present invention provides a method for preventing or treating inflammation-mediated diseases by RAGE overexpression, comprising administering the pharmaceutical composition to an individual.

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 inflammation-mediated diseases caused by overexpression of RAGE.

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. RBP-exosome production and characterization

The present inventors tried to prepare an exosome (hereinafter, RBP-exosome) to which a RAGE-binding peptide (RBP) specifically binds to the surface, and evaluate its efficacy.

First, in order to prepare the RBP-exosome, HEK293T cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% Fetal Bovine Serum (FBS) (v/v) at 37° C. and 5% CO ₂ conditions. . Next, the cultured cells were dispensed in a 175mm ² wide cell culture flask, and when the cultured cells were grown to about 70% of the culture area, the medium was replaced with DMEM not containing FBS, and then Lipofectamine 2000 (Invitrogen, Carlsbad, CA) was used. A plasmid vector into which RBP-Lamp2b and hygromycin resistance genes were inserted was transfected and introduced into the cells. The structure and RBP sequence (SEQ ID NO: 1) of the plasmid vector used above are illustrated in FIG. 1 . After 4 hours of transfection, the medium was replaced with DMEM containing 10% FBS and 200 μg/ml of hygromycin, followed by culture for 2 weeks. After that, the surviving cells were divided into several flasks and cultured, and as shown in FIG. 2 by performing western blot, RBP-exosome (RBP-exo) was expressed through the expression of the HA tag linked to RBP. production was confirmed. Subsequently, the culture medium (DMEM) of the RBP-exosome-secreting cell line prepared according to the above procedure contains a large amount of the exosomes, so the exoEasy Maxi kit (product of Qiagen) is used to purify the exosomes from the culture medium, and the The concentration was quantitatively determined.

Furthermore, in order to analyze the physical properties of the RBP-exosome prepared above, dynamic light scattering measurement was performed to confirm the size and surface charge in the receptor of the nano-sized material. As a result, as shown in Fig. 3a, the size of the RBP-exosome was 31.62 nm ± 13.67, and it was confirmed that it had a negative charge of -8.19 mV ± 2.50. It can be seen that the above results are similar to the physical properties of general exosomes (Unmod-exo), and RBP-exosomes (RBP-exo) were visually inspected through a scanning electron microscope (SEM) as shown in FIG. 3b . also confirmed.

Example 2. Confirmation of anti-inflammatory effect of RBP-exosomes

In the RBP-exosome according to the present invention, a peptide binding to RAGE is present on the surface of the exosome, and the RAGE plays an important role in the immune/inflammatory response, and the interaction with the ligand is IL-12 and TNF- It is known that it plays an important role in the initiation of an inflammatory response by activating α. Therefore, it was attempted to investigate whether the RBP-exosome of the present invention can induce an anti-inflammatory effect by specifically binding to RAGE and inhibiting the RAGE-mediated signaling pathway.

To this end, Raw264.7 cells were treated with LPS (lipopolysaccharide) to induce an inflammatory response, and the concentration of TNF-α was measured after treatment with RBP-exosomes (RBP-exo) by concentration. As a result, as shown in FIG. 4 , RBP-exosome itself did not induce cytotoxicity at a level similar to that of the negative control group, and compared to the group treated with only LPS, RBP-exosome itself. It was confirmed that the concentration of TNF-α decreased in the group treated with. These results suggest that RBP on the RBP-exosome surface targets and specifically binds to RAGE-expressing cells and inhibits its activity, thereby inducing an anti-inflammatory effect through reduction of NF-κb.

Example 3. Confirmation of Acute Lung Injury Treatment Effect of RBP-Exosome

The present inventors tried to evaluate whether the RBP-exosome according to the present invention actually has a therapeutic effect on a disease in which an inflammatory response is caused by overexpression of RAGE in vivo. To this end, as an example of the disease, an animal model of acute lung injury was prepared and an experiment was conducted to confirm the therapeutic effect. Acute lung injury is conventionally treated by artificially injecting air into the alveoli through tracheal intubation, and various drugs and systems for delivering them have been developed to lower the fatality rate, but limitations such as efficiency, toxicity, and lack of specificity for inflammatory cells Therefore, it is difficult to treat.

Specifically, the present inventors treated 20 μg of LPS into the trachea of male BALB/c mice having an average body weight of 21 g, and then injected RBP-exosomes into the lungs by intratracheal injection 2 hours later. At this time, in order to evaluate not only the RBP-exosome alone, but also the therapeutic effect when a therapeutic substance is encapsulated in the exosome, a hydrophobic natural drug curcumin, which is known to have a strong anti-inflammatory effect, is encapsulated RBP-exosome. (RBP-exo/Cur) and curcumin-encapsulated normal exosomes (Unmod-exo/Cur) were each injected under the same conditions. The next day, ELISA was performed after collecting bronchoalveolar lavage fluid (BAL fluid) and lung tissue from the mice injected with each of the above substances.

As a result, as shown in FIG. 5 , in the case of mice injected with RBP-exo/cur, the concentrations of TNF-α and IL-1β, which are inflammation-inducing factors in both lung tissue and bronchoalveolar lavage, were the same as in normal mice not treated with LPS. decreased to a similar level. In addition, it was found that even when only RBP-exosomes in which curcumin is not encapsulated, a significant anti-inflammatory effect was exhibited. Moreover, the above results could also be confirmed through the histological analysis results of hematoxylin & eosin (H&E) staining of the lung tissue of FIG. 6 . These results show that RBP-exosome itself, which is not encapsulated with a therapeutic substance, exhibits a significant therapeutic effect on acute lung injury, and that when curcumin, an anti-inflammatory substance, is encapsulated (RBP-exo/cur), the treatment drug is loaded. A higher synergistic therapeutic effect can be achieved, suggesting that it can contribute to the treatment and survival of patients with acute lung injury without an effective treatment.

Example 4. Confirmation of stroke therapeutic effect of RBP-exosome

4-1. Analysis of changes in cytotoxicity and RAGE expression on neurons

The present inventors tried to confirm the RBP-exosome-mediated therapeutic effect in acute lung injury and other diseases in which an inflammatory response due to overexpression of RAGE appears. Ischemic stroke, which requires the development of a therapeutic agent specific to damaged tissue due to its short time period, was selected as a target disease and the experiment was conducted.

First, in order to determine whether the RBP-exosome according to the present invention induces cytotoxicity in very sensitive neurons, RBP-exosome (RBP-Exo), normal exosome (Unmod-Exo) and very high (+ ) Cell viability was measured after each treatment with charged PEI25k on neurons. As a result, as shown in FIG. 7 , it was confirmed that the RBP-exosome according to the present invention had very low cytotoxicity.

Furthermore, for ischemic conditions similar to stroke, _{neurons cultured in 1% oxygen (O 2} ) for 24 hours were treated with the two types of exosomes, and then the expression level of RAGE was analyzed by FACS. As a result, as can be seen in FIG. 8 , the expression of RAGE was significantly increased under hypoxic conditions (Control-Hypoxia) compared to normal conditions (Control-Normoxia), whereas RBP-exo in neurons cultured under hypoxic conditions It was confirmed that the expression of RAGE was significantly reduced when the moth was treated. These results could indirectly predict that RBP-exosomes induce inhibition of inflammation-related signaling pathways related to NF-κb under RAGE through the family.

4-2. Analysis of Stroke Treatment Efficacy of RBP-Exosomes in Animal Models

Therefore, based on the above results, an ischemic stroke animal model was prepared according to the following method, and the actual therapeutic effect of RBP-exosomes was investigated. Specifically, in order to induce ischemic stroke by inducing Middle Cerebral Artery occlusion (MCAo) in mice, a head capable of blocking blood vessels by melting the tip of a nylon suture was prepared in a certain size, and after appropriate anesthesia, an external carotid The suture was inserted about 1.8 to 2 cm through the artery. After finishing the suture insertion part and the overall bleeding part using a surgical clip and thread, the mice were allowed to rest at a constant temperature, and then the suture was removed after 1 hour to induce reperfusion to produce a model very similar to an actual stroke. At this time, laser doppler analysis was performed to confirm that the ischemic stroke animal model was successfully produced as shown in FIG. 9 . After processing each material, the brain was extracted 24 hours later, and tissue sections were prepared to analyze the therapeutic effect. At this time, the material is anti-microRNA oligonucleotide (Anti-miRNA oligonucleotide) alone (Naked-AMO), the AMO is encapsulated in the general exosome (Unmod-EXO), AMO is encapsulated RBP- exosome (RBP-EXO) ) were treated respectively and the results were compared.

First, immunohistochemical staining was performed to analyze the expression level of TNF-α, an inflammation inducing factor, in the brain tissue of stroke model mice. As a result, as shown in FIG. 10 , it was confirmed that the expression of TNF-α was significantly reduced in the case of the group treated with the RBP-exosome in which the therapeutic nucleic acid was encapsulated. Although the expression of TNF-α was decreased to a high level even in the case of a normal exosome (Unmod-EXO) in which RBP was not expressed, the RBP-exosome according to the present invention specifically acts on cells in a hypoxic state in which RAGE is expressed. Therefore, it was judged to exhibit a higher anti-inflammatory effect. As shown in FIG. 11 , the remarkable anti-inflammatory effect of the RBP-exosomes as described above was confirmed that the expression of TNF-α was maintained low even after 3 days had elapsed after the treatment.

In order to confirm whether the effect of reducing RAGE expression confirmed in FIG. 8 also occurs in the ischemic stroke animal model, the expression of RAGE was confirmed through immunohistochemical staining. As a result, as shown in FIG. 12 , it was confirmed that the RBP-exosome-treated mouse group of the present invention significantly decreased the expression of the RAGE factor compared to other experimental groups.

In addition, the expression level of Bcl-2, which acts with mitochondria as an intrinsic pathway in relation to apoptosis and has a significant influence in determining the survival and death of cells, was observed by immunohistochemical staining. As shown in FIG. 13, RBP- It was confirmed that Bcl-2 was expressed at the highest level in the group (RBP-EXO) treated by encapsulating the therapeutic nucleic acid in the exosome. Moreover, in the TUNEL assay test result of FIG. 14 using DNA nodule formation that occurs during cell death, it was confirmed that the group using RBP-exosome had the most reduced BrdU signal due to the most excellent therapeutic effect and low toxicity.

Overall, RBP-exosomes show low toxicity and high specificity for RAGE-expressing cells due to RBP expressed on the surface of exosomes, and thus can effectively target RAGE, which is frequently expressed in inflammatory diseases. In addition, since the exosome itself exhibits an effect of inhibiting RAGE, an anti-inflammatory effect of inhibiting various inflammatory responses related to NF-Kb can be induced. Through this, it was found that there is no suitable treatment for acute lung injury, stroke, etc., and it can be used for therapeutic purposes for diseases related to inflammation, and a higher level of therapeutic effect can be achieved by encapsulating the therapeutic drug inside the exosome. .

4-3. Analysis of cerebral infarction treatment efficacy of RBP-exosomes

prepared in Example 4-2 After administration of Unmod-Exo, RBP-Exo and PEI25k to an ischemic stroke animal model (MCAO control), the actual cerebral infarction treatment efficacy was confirmed. Specifically, after 3 days have elapsed after administration of the above substances and exosomes, when a specific cerebral infarction improvement effect is confirmed, as shown in FIG. 15 , the RBP-exosome (RBP-Exo) of the present invention has the most remarkable effect It was confirmed that cerebral infarction could be improved.

The description of the present invention described 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. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

According to the present invention, the exosomes to which RAGE-targeting peptides bind specifically bind to RAGE, thereby inhibiting the RAGE-mediated signaling pathway, thereby exhibiting anti-inflammatory effects and therapeutic effects on inflammatory-mediated diseases, and the exosomes A more synergistic therapeutic effect can be achieved by encapsulating a therapeutic drug suitable for expected to be used.

Claims (9)

1. A pharmaceutical composition for preventing or treating inflammation-mediated diseases caused by overexpression of RAGE (receptor for advanced glycation endproducts), comprising, as an active ingredient, an exosome bound to a peptide consisting of the amino acid sequence of SEQ ID NO: 1.
2. The method of claim 1,

   The peptide is characterized in that it specifically binds to RAGE, a pharmaceutical composition.
3. According to claim 1,

   The exosome is a pharmaceutical composition, characterized in that obtained by culturing the cell after transfecting the cell with a plasmid vector encoding the peptide.
4. 4. The method of claim 3,

   The culture is characterized in that made for 10 to 20 days, the pharmaceutical composition.
5. According to claim 1,

   The pharmaceutical composition, characterized in that it further comprises a drug, pharmaceutical composition.
6. 6. The method of claim 5,

   The drug is characterized in that it is encapsulated inside the exosome, a pharmaceutical composition.
7. 7. The method of claim 6,

   The drug is any one selected from the group consisting of compounds, biodrugs, nucleic acids, peptides, proteins, natural products, hormones, contrast agents, antibodies, and combinations thereof, a pharmaceutical composition.
8. According to claim 1,

   The inflammation-mediated disease caused by RAGE overexpression is selected from the group consisting of acute lung injury, acute respiratory distress syndrome, pneumonia, asthma, ischemic brain disease, ischemic myocardial infarction, brain tumor, sepsis, diabetes, and diabetic kidney disease. Characterized in the pharmaceutical composition.
9. According to claim 1,

   The composition is a pharmaceutical composition, characterized in that it is formulated in the form of injection, injection or spray.

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