KR102594908B1 - A composition for preventing, alleviating or treating a sepsis or septic shock - Google Patents

Abstract

The present invention relates to a composition for preventing, improving or treating sepsis or septic shock. When the compound of the present invention is administered in combination with interferon beta, it can exert a significant synergistic effect in the treatment of sepsis or septic shock.

Description

Composition for preventing, alleviating or treating a sepsis or septic shock {A composition for preventing, alleviating or treating a sepsis or septic shock}

The present invention relates to a composition for preventing, improving or treating sepsis or septic shock.

Bacterial infections and other powerful stimuli initiate an immune response that can cause systemic inflammation, or Systemic Inflammatory Response Syndrome (SIRS). Severe SIRS can result in varying degrees of fever, hypotoxemia, trachypnea, tachycardia, endothelial inflammation, myocardial dysfunction, hyperfusion, mental status abnormalities, vascular collapse, and ultimately It causes organ damage, such as multiple organ failure syndrome (MODS), which involves acute respiratory distress syndrome, coagulopathy, heart failure, renal failure, shock and/or coma.

Sepsis is defined as a situation in which infection is confirmed or suspected along with a systemic inflammatory response. Severe sepsis is defined as sepsis accompanied by organ dysfunction (hypotension, hypoxia, oliguria, metabolic acidosis, thrombocytopenia, and impaired consciousness). Septic shock is defined as severe sepsis in which blood pressure does not normalize even with fluid therapy or administration of vasopressors. Sepsis may progress to clinical stages of severe sepsis and ultimately septic shock. Clinical sepsis is broadly defined as a condition in which infiltration by microbial agents is associated with clinical symptoms of infection. Clinical symptoms of sepsis include (1) body temperature >38°C or <36°C; (2) heart rate > 90 beats per minute; (3) respiratory rate > 20 breaths per minute or _PaC0 < 32 mmHg; (4) White blood cell count >12000/cu mm, <4,000/cu mm, or >10% immature (band) form; (5) There may be organ dysfunction, hyperfusion, or high blood pressure.

When infection occurs, macrophages at the site of infection are activated and secrete TNF-α and IL6, which increases the release of plasma proteins into tissues, increases the migration of phagocytes and lymphocytes into tissues, and increases platelet adhesion to blood vessel walls. bring about In this way, local blood vessels are occluded and pathogens are concentrated at the site of infection. In particular, sepsis occurs as a systemic infection and is accompanied by severe vascular occlusion induced by TNF-α. Additionally, systemic release of TNF-α causes loss of plasma volume due to vasodilation and increased vascular permeability, resulting in shock. In septic shock, TNF-α further triggers disseminated intravascular coagulation (blood clotting), resulting in clot formation in small blood vessels and massive consumption of blood clotting proteins. Because the patient's blood loses its ability to clot, vital organs such as the kidneys, liver, heart and lungs are damaged due to failure of normal perfusion. The mortality rates of severe sepsis and septic shock are reported to be 25-30% and 40-70%, respectively.

In many cases of sepsis, E. coli is the pathogen, but other Gram-negative bacteria, such as Klebsiella , Enterobacter , Serratia , and Pseudomonas , can also be involved. The state can be initiated. Gram-positive microorganisms, such as Staphlococcus , and systemic viral and fungal infections also initiate sepsis in some cases.

The urogenital tract, gastrointestinal tract, and respiratory tract are the most common sites of infection resulting in sepsis. Other sepsis-related sites of infection include wounds, burns, and pelvic sites, and intravenous catheter sites.

Sepsis is understood to occur as a result of complex interactions between infectious agents and the host's immune, inflammatory, and coagulation systems. Both the degree of host response and the characteristics of the pathogen causing infection have a significant impact on the prognosis of sepsis. Organ failure observed in sepsis occurs when the host's response to the infectious bacteria is inadequate, and if the host's response to the infectious bacteria is excessively amplified, it can cause damage to the host's own organs. Based on this concept, antagonistic substances against proinflammatory cytokines such as TNF-α, IL1β, and IL6, which play a leading role in the host's inflammatory response, were attempted as treatments for sepsis, but most of them failed. Mechanical ventilation treatment, Activated protein C (activated protein C) administration and glucocorticoid treatment are currently being attempted, but various limitations are being pointed out. Therefore, there is a need for new treatments to prevent, improve, or treat sepsis and septic shock, for which no clear treatment has yet been developed despite its high mortality rate.

One object of the present invention is to provide a composition for preventing, improving, or treating sepsis or septic shock.

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

One embodiment of the present invention includes interferon beta; It relates to a composition for preventing, improving, or treating sepsis or septic shock, comprising as an active ingredient at least one selected from the group consisting of a compound represented by the following formula (1):

[Formula 1]

Other embodiments of the invention include interferon beta; and a composition for preventing, improving, or treating sepsis or septic shock, comprising the compound represented by Formula 1 above as an active ingredient.

The interferon beta of the present invention may include interferon beta 1a (IFN-β1a) or interferon beta 1b (IFN-β1b), which are two isoforms of interferon. Interferon beta 1a is a glycosylated protein produced from the Chinese hamster ovary (CHO) containing the human interferon beta gene and is composed of 166 amino acid residues and is 25 kD in size; interferon beta 1b is a protein composed of 165 amino acid residues produced from Escherichia coli. It lacks sugar, methionine residue at amino acid number 1, and cysteine residue number 17 is substituted with serine.

For the purpose of the present invention, the interferon beta may be interferon beta 1a or 1b, but is not limited thereto.

The compound represented by Formula 1 of the present invention is 3-carbamoyl-1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolane-2- 1] pyridin-1-ium (3-Carbamoyl-1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyridin-1-ium).

The interferon beta of the present invention; And the compound represented by Formula 1 can induce the expression of SIRT1 (silent mating type information regulation 2 homolog; sirtuin 1).

The SIRT1 of the present invention is a NAD+-dependent deacetylase and is known as an enzyme that regulates protein function by deacetylating lysine residues of various proteins (Ageing Res, Vol. 1 pages 313-326, (2002)), and NAD+ It is most similar to yeast Sir2, which has dependent class ² histone deacetylation activity. In particular, it cuts off acetyl groups attached to transcription factors such as Nuclear factor-kB and p53 and regulates their functions (Cancer Res, Vol.64 pages 7513-7525, (2004); Cell, Vol.107, pages 149-159 , (2001); Trends Endocrinol Metab, Vol.17 pages 186-191, (2006)). In addition, SIRT1 is involved in chromatin reorganization associated with gene repression, DNA damage response, and lifespan extension accompanying dietary restriction (Chen et al., Science 310, 1641, 2005). In other words, SIRT1, like Sir2 in yeast, reorganizes chromatin and suppresses gene expression through histone deacetylation, and induces deacetylation of various transcription factors related to cell growth, stress response, and endocrine regulation in addition to histone proteins. When the expression of SIRT1 is increased, the expression level of inflammatory cytokines is reduced and the expression level of cytokines that can suppress inflammation is increased, making it possible to very effectively treat sepsis or septic shock. For the purpose of the present invention, the interferon beta and the compound represented by Formula 1 increase the expression of SIRT1 when administered separately, and further increase the expression of SIRT1 when administered in combination compared to when administered alone. A significant synergistic effect can be exerted in the treatment of sepsis or septic shock.

The sepsis of the present invention is an inflammatory reaction caused by abnormal activation of the body's immune system when pathogenic gram-negative bacteria infect the body, lipopolysaccharide (LPS), a cell wall component, acts as a toxin, and spreads throughout the body. If it causes infection or the symptoms are severe, it may be accompanied by shock.

The septic shock of the present invention is sepsis accompanied by low blood pressure and abnormal perfusion, and sepsis causes life-threatening low blood pressure and organ failure. Therefore, when treating the causative sepsis, septic shock can also be prevented, improved, or It can be treated.

The composition of the present invention can be used as a pharmaceutical composition or food composition.

The "prevention" of the present invention may include without limitation any action that can block, suppress or delay symptoms caused by sepsis or septic shock using the composition of the present invention.

The “treatment” of the present invention may include, without limitation, any action that improves or benefits symptoms caused by sepsis or septic shock using the composition of the present invention.

The "improvement" of the present invention may include without limitation any action in which symptoms caused by sepsis or septic shock are improved or beneficially changed by using the composition of the present invention.

The pharmaceutical composition of the present invention is not limited to these, but can be formulated and used in the form of oral dosage forms such as powders, granules, capsules, tablets, and aqueous suspensions, external preparations, suppositories, and sterile injection solutions according to conventional methods. You can. Preferably, the pharmaceutical composition is for intratracheal administration or inhalation administration; Alternatively, it may be formulated to be used as an injection, but is not limited thereto. For the purpose of the present invention, the active ingredient may be formulated for inhalation administration so that it can reach the target organ in a yield suitable for prevention or treatment, but is not limited thereto.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. For oral administration, the pharmaceutically acceptable carrier may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavorings, etc., and for injections, buffers, preservatives, Analgesics, solubilizers, isotonic agents, stabilizers, etc. may be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives, etc. may be used. The dosage form of the pharmaceutical composition of the present invention can be prepared in various ways by mixing it with a pharmaceutically acceptable carrier as described above. For example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be manufactured in the form of unit dose ampoules or multiple doses. there is. In addition, it can be formulated as a solution, suspension, tablet, capsule, sustained-release preparation, etc.

Examples of carriers, excipients and diluents suitable for the formulation of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate. , cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil can be used. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. may be additionally included.

The route of administration of the pharmaceutical composition of the present invention is not limited to these, but is oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, Includes sublingual or rectal areas. It may be administered orally or parenterally, and preferably orally, but is not limited thereto.

The parenteral of the present invention includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention can also be administered in the form of a suppository for rectal administration.

The pharmaceutical composition of the present invention may vary depending on several factors, including the activity of the specific compound used, age, body weight, general health, gender, diet, administration time, administration route, excretion rate, drug formulation, and the severity of the specific disease to be prevented or treated. It may vary, and the dosage of the pharmaceutical composition may vary depending on the patient's condition, weight, degree of disease, drug form, route and period of administration, but may be appropriately selected by a person skilled in the art, and may be administered at 0.0001 to 50 mg/day. It can be administered in kg or 0.001 to 50 mg/kg. Administration may be administered once a day, or may be administered several times. The above dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention can be formulated into pills, dragees, capsules, solutions, gels, syrups, slurries, and suspensions.

When the food composition of the present invention is manufactured in the form of a beverage, there are no particular limitations other than including the food composition in the indicated ratio, and various flavoring agents or natural carbohydrates can be contained as additional ingredients like ordinary beverages. . Specifically, natural carbohydrates include monosaccharides such as glucose, disaccharides such as fructose, common sugars such as polysaccharides such as sucrose, dextrin, and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. It can be included. The flavoring agent may be a natural flavoring agent (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and a synthetic flavoring agent (saccharin, aspartame, etc.).

The food composition of the present invention contains various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, It may further include pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated drinks, etc.

The ingredients included in the food composition of the present invention can be used independently or in combination. The ratio of the additive does not correspond to a key element of the present invention, but may be selected in the range of 0.1 to about 50 parts by weight per 100 parts by weight of the food composition of the present invention, but is not limited thereto.

The cosmetic composition of the present invention includes lotion, nutritional lotion, nutritional essence, massage cream, beauty bath water additive, body lotion, body milk, bath oil, baby oil, baby powder, shower gel, shower cream, sunscreen lotion, and sunscreen cream. , suntan cream, skin lotion, skin cream, sunscreen cosmetics, cleansing milk, hair loss cosmetics, face and body lotion, face and body cream, skin whitening cream, hand lotion, hair lotion, cosmetic cream, jasmine oil, bath. Manufactured in the form of soap, liquid soap, beauty soap, shampoo, hand sanitizer (hand cleaner), non-medical medicated soap, cream soap, facial wash, body cleanser, scalp cleanser, hair rinse, cosmetic soap, teeth whitening gel, toothpaste, etc. It can be. The composition of the present invention may further include solvents commonly used in the production of cosmetic compositions, appropriate carriers, excipients, or diluents.

The present invention relates to a composition for preventing, improving or treating sepsis or septic shock. When the compound of the present invention is administered in combination with interferon beta, it can exert a significant synergistic effect in the treatment of sepsis or septic shock.

Figure 1 shows the manufacturing process of vascular endothelial cell-specific SIRT1 deletion mice according to an embodiment of the present invention and the confirmation of inhibition of SIRT1 expression in vascular endothelial cells. Specifically, exploration of mice carrying the floxed SIRT1 gene and Cre recombinase (Tek-Cre) at a position downstream of the Tek promoter through PCR (A), mice carrying the Tek-Cre gene and both alleles of the floxed SIRT1 gene. It was confirmed that SIRT1 expression was lost in vascular endothelial cells (B). where: WT; Normal mouse (wild type) and KO; SIRT1 gene deletion mouse (knockout).
Figure 2 shows nicotinamide, NMN (nicotinamide mononucleotide), known to increase intracellular NAD concentration according to an embodiment of the present invention. The structure of NR (nicotinamide riboside) and its effect on the survival rate of sepsis-induced mice when administered in combination with interferon beta are shown. Specifically, the structure of the compound that increases the NAD concentration is shown, and the change in survival rate of septic mice is shown after coadministration of interferon beta and each nicotinamide (B), NMN (C), or NR (D). .
Figure 3 shows mouse yolk sac endothelial cells (MYSECs) treated with interferon beta (IFN-β) at different concentrations from 0 to 1000 U/ml for 24 hours according to an embodiment of the present invention (A). and (B) treated with 1000 U/ml over time.
Figure 4 shows the change in survival rate of septic mice measured according to the combined administration of interferon beta and NR in macrophage- or vascular endothelial cell-specific SIRT1 gene-deleted mice according to an embodiment of the present invention.
Figure 5 shows the results of measuring the concentration of TNF-α and IL6 in the blood through ELISA method to determine the effect on the secretion amount of inflammatory cytokines in septic mice by combined administration of interferon beta and NR according to an embodiment of the present invention. It is shown.
Figure 6 shows the results of confirming the effect on organ damage in septic mice caused by the administration of interferon beta and NR through hematoxylin-eosin staining of tissue sections of organs removed from septic mice treated with interferon beta or NR. will be.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Experimental method

[Experimental Method 1] laboratory animals

Male C57BL/6 mice weighing 20 to 24 g and 7-8 weeks old were purchased from Nara Biotech (Seoul, Korea). SIRT1 loxP/loxP mice (B6.129-SirtatmqYgu/J) and Tek-Cre mice (B6.Cg-Tg(Tek-cre)1Ywa/J) were purchased from Jackson Laboratory (Maine, USA). Mice were raised under constant temperature conditions of 20-24°C, illuminated for 12 hours, light blocked for 12 hours, and fed ad libitum with regular feed. This experiment was conducted in accordance with the rules of the Chonbuk National University Animal Experiment Ethics Committee.

[Experiment Method 2] Sepsis animal model

Sepsis was induced by cecal ligation and puncture (CLP). The mouse was anesthetized with 500 ㎕ of ketamine, 120 ㎕ of rompun, and 500 ㎕ of phosphate buffered saline (PBS), an incision was made in the mid-abdominal cavity, the cecum was exposed to the outside, and the end of the ileocecal valve was tied with a 6.0-silk suture. It was ligated with (silk suture), punctured once using an 18-gauge needle, and a 1-2 mm drop of feces was squeezed out and returned to the peritoneal cavity, and the laparotomy site was sutured with 4.0-silk. Here, a group of animals whose cecum was exposed but returned to the abdominal cavity without ligation or puncture was used as a sham control animal model.

[Experimental Method 3] drug injection

In the case of the control group, PBS was injected into the mouse abdominal cavity twice at 6 and 18 hours after CLP was performed as in Experimental Method 2 above. Meanwhile, in the experimental group, 0.4 μg of interferon beta (IFN-β, Bio-Techne, USA) and 9.4 μmole of nicotinamide and NMN (nicotinamide mononucleotide, SigmaAldrich, USA) were administered at 6 and 18 hours after CLP. Alternatively, NR (nicotinamide riboside, SigmaAldrich, USA) was dissolved in PBS and injected into the mouse abdominal cavity.

[Experiment Method 4] Production of macrophage and vascular endothelial cell specific SIRT1 gene deletion mouse model

Macrophage-specific SIRT1 gene deletion mice were prepared according to the method reported by Hah et al. (PLOS one, 9(2), e87733, 2014). To obtain vascular endothelial cell-specific SIRT1 gene deletion mice, SIRT1 loxP/loxP mice (B6.129-SirtatmqYgu/J) and Tek-Cre mice (B6.Cg-Tg(Tek-cre)1Ywa/J) were crossed. Primers used for genotyping are shown in Table 1 below. Here, SIRT1 loxP/loxP mice were used as control mice.

gene name sequence number direction base sequence Sirt1-loxP SEQ ID NO: 1 Sense GGT TGA CTT AGG TCT TGT CTG SEQ ID NO: 2 Antisense CGT CCC TTG TAA TGT TTC CC Tek-Cre SEQ ID NO: 3 Sense GCG GTC TGG CAG TAA AAA CTA TC SEQ ID NO: 4 antisense GTG AAA CAG CAT TGC TGT CAC TT Internal Control SEQ ID NO: 5 Sense CTA GGC CAC AGA ATT GAA AGA TCT SEQ ID NO: 6 antisense GTA GGT GGA AAT TCT AGC ATC ATC C

As shown in Figures 1 (A) and (B), as a result of producing vascular endothelial cell-specific SIRT1 deletion mice, it was confirmed that SIRT1 was expressed in normal (WT) mice, but SIRT1 was not expressed in deleted mice (K/O). I was able to.

[Experiment Method 5] Isolation of Vascular Endothelial Cells from the Lung

After cutting the mouse lung into small pieces with scissors, 3 mg/ml collagenase II (Sigma, USA) was added, reacted at 37°C for 45 minutes, and shaken every 15 minutes to ensure proper reaction. The reaction solution was passed through a 21 gauge eight times to soften into a single cell suspension, filtered through a 40 μm nylon mesh, and serum was added to stop the reaction. Cells were centrifuged at 300 The reaction solution was centrifuged at 300 did.

[Experiment Method 6] Culture of Mouse Yolk Sac Endothelial Cells (MYSECs)

Mouse yolk sac endothelial cells were purchased from Cell Biologics, INC (Chicago, USA) and cultured in DMEM medium containing 10% fetal bovine serum (FBS), 0.2% glutamax, and 0.5% penicillin/streptomycin. Cultured at 4°C in 5% CO ₂ . After 1 to 2 subcultures, the cells were reinoculated into 6-well plates when the culture dish was 80-90% full. Here, in order to confirm the effect of interferon beta, when treating the drug, the cells were cultured for 24 hours in a serum-free medium from which FBS had been removed in advance, and then treated with interferon beta.

[Experiment Method 7] immunoblot

Protein extract (MPER™, ThermoFisher Scientific, USA) supplemented with 4% protease inhibitor cocktail (cOmplete™, Roche, Switzerland) and 10% phosphatase inhibitor (PhosSTOP™, Roche, Switzerland) were added to the cells. Cells were lysed for an hour. Afterwards, the supernatant was obtained after centrifugation at 12000 rpm at 4°C for 10 minutes, and the amount of protein was measured. Then, 20 μg of protein was subjected to 7.5% SDS-polyacrylamide (sodium dodecyl sulfate-polyacrylamide) gel electrophoresis. After electrophoresis, the proteins in the gel were transferred to a PVDF (polyvinylidene fluoride) membrane at 4℃, 80~120V for 120-150 minutes, and then incubated with 3% skim milk and 0.1% Tris Buffered Saline-Tween 20 (TBS-Tween) for 1 hour at room temperature. 20, pH 7.4) to prevent non-specific antibody binding. Specific primary antibodies, such as anti-SIRT1 (Millipore Sigma, USA) or anti-βactin, were reacted for 12 hours and washed three times for 5 minutes each with 0.1% TBS-Tween 20 solution. The secondary antibody specific to the primary antigen was reacted for 40 minutes using horseradish peroxidase combined with anti-Mouse IgG and anti-Rabbit IgG, and washed four times for 5 minutes each with TBS-Tween 20 solution. Finally, it was confirmed by color development according to the method described in the instructions for the ECL kit (Amersham Pharmacia Biotech, Uppsala, Sweden).

[Experiment Method 8] ELISA measurement of TNF-α and IL6

Sepsis was induced as described in Experimental Method 2, and blood collected from the facial vein of the mouse was centrifuged 18 hours later, and serum TNF-α and IL6 concentrations were measured using the TNF-α and IL6 ELISA kit (Koma Biotechnology, Korea). It was measured using the method suggested by the manufacturer.

[Experiment Method 9] Hematoxylin and Eosin staining

To observe changes in mouse kidney, lung, and liver tissues before and after inducing sepsis and after drug treatment, the organs were fixed with 10% paraformaldehyde at 4°C for 24 hours. The tissue of the fixed organ was embedded in paraffin and sectioned at a size of 5 μm. The sectioned tissues were deparaffinized with xylene, hydrolyzed at different ethanol concentrations (100, 90, 80, and 70%) for 5 minutes each, and then stained with hematoxylin solution (Sigma) for 3 minutes. The stained tissue was washed with water and stained again for 3 minutes using an eosin solution. After staining was completed, the tissue was washed well in running water, dehydrated using ethanol for 5 minutes at each concentration (70, 80, 90, and 100%), washed using xylene, and then encapsulated. Stained tissues were observed under an optical microscope.

[Experiment Method 10] Inhibition of SIRT1 expression in yolk sac endothelial cells

To investigate the role of SIRT1 in endothelial cell permeability, the expression of SIRT1 in yolk sac endothelial cells (MYSECs) was suppressed using RNAi. SIRT1 siRNA and control siRNA, Stealth siSIRT1 (siRNA ID MSS234959) and Stealth siControl (siRNA ID 462001), were purchased from Thermo Fisher Scientific, Inc. Stealth siSIRT1 (50 nM) and Stealth siControl (50 nM) were transfected into yolk sac endothelial cells (MYSECs) using Lipofectamine® RNAiMAX (Thermo Fisher Scientific, Inc, USA) according to the manufacturer's instructions. 24 hours after transformation, immunoblot was performed to measure the degree of inhibition of SIRT1 expression.

[Experiment Method 11] Statistical processing

Data were expressed as mean ± standard deviation, and P<0.05 was considered significant by Student's t-test.

Experiment result

[Experiment Result 1] Survival rate analysis according to combined treatment of interferon beta and our compound in mouse sepsis model

In a mouse sepsis model induced through cecal ligation and puncture (CLP) surgery as described in Experimental Method 2, combined administration of interferon beta and nicotinamide, NMN or NR as described in Experimental Method 3 Changes in survival rate were confirmed and shown in Figure 2.

As shown in Figure 2, the survival rate of mice 4 days after administering interferon beta to mice with induced sepsis was 40%. In addition, the sepsis treatment effect of interferon beta was not increased when nicotinamide or NMN was administered in combination. However, when NR was administered in combination, the effectiveness of treating sepsis caused by interferon beta was 60%, which was about 20% higher than when administered alone.

The above results show that among nicotinamide, NMN, and NR, which are known to increase the amount of NAD in cells, only NR can treat sepsis and increase survival rate when administered in combination with interferon beta. Therefore, the combined administration of interferon beta and NR significantly lowers the mortality rate of mice due to sepsis and can be effectively used to prevent or treat sepsis or septic shock.

[Experiment Result 2] Measurement of SIRT1 expression level in yolk sac endothelial cells after interferon beta treatment

Yolk sac endothelial cells (MYSECs) were treated with different concentrations of interferon beta (0, 100, 200, 400, 1000 units/ml) for 24 hours, and the expression level of SIRT1 was compared and confirmed through immunoblot. Additionally, 1000 units/ml of interferon beta was treated over time, and SIRT1 expression levels were compared through immunoblot. The immunoblot results are shown in Figures 3 (A) and (B).

As shown in Figures 3 (A) and (B), when yolk sac endothelial cells (MYSECs) were treated with interferon beta, SIRT1 protein expression level increased in a concentration-dependent manner (A), treated with 1000 units/ml of interferon. After 2 hours of cold storage, the expression level of SIRT1 protein began to increase (B).

[Experiment Result 3] Analysis of sepsis mouse survival rate following combined administration of interferon beta and NR in macrophage- or vascular endothelial cell-specific SIRT1 gene deletion mice

The survival rate of sepsis mice was measured in response to combined administration of interferon beta and NR in macrophage- or vascular endothelial cell-specific SIRT1 gene deletion mice described in Experimental Methods 2 to 4, and the results are shown in Figures 4 (A) to (C). It was.

As shown in Figure 4 (A) to (C), control mice in which SIRT1 is normally expressed developed sepsis in 40% of mice when administered interferon beta alone, 20% when administered only NR, and 60% when administered in combination. The survival rate showed that not only did interferon beta and NR each show an effect in treating sepsis, but it was also confirmed that when administered in combination, there was a significant synergistic effect in treating sepsis (A). On the other hand, not only did interferon beta and NR each show no sepsis treatment effect in macrophage- or vascular endothelial cell-specific SIRT1 gene deletion mice, but also no significant synergistic effect was confirmed in treating sepsis even when administered in combination (B and C) .

The above results show that SIRT1 expression in macrophages and vascular endothelial cells is directly related to the protective action of sepsis by interferon beta and NR.

[Experiment Result 4] Effect of administration of interferon beta and NR on the secretion amount of inflammatory cytokines in septic mice

In a mouse sepsis model induced through cecal ligation and CLP surgery as described in Experimental Methods 2 and 3, changes in inflammatory cytokines in serum following combined administration of interferon beta and NR were measured 18 hours after sepsis induction using ELISA method. This is shown in Figure 5.

As shown in Figure 5, when interferon beta or NR was administered to mice with induced sepsis, it was confirmed that the blood concentration of TNF-α and IL6, which are inflammatory cytokines increased due to sepsis, was reduced. Furthermore, it was confirmed that when interferon beta and NR are administered in combination, there is a synergistic effect in reducing the expression levels of all inflammatory cytokines.

From the above results, it can be seen that the combined administration of interferon beta and NR has a significant synergistic effect in reducing the blood concentration level of inflammatory cytokines caused by sepsis, and can very effectively prevent or treat sepsis or septic shock.

[Experiment Result 5] Effect of administration of interferon beta and NR on organ damage in septic mice

In a mouse sepsis model induced through cecal ligation and CLP surgery as described in Experimental Methods 2 and 3, changes in organ damage according to the administration of interferon beta and NR were measured 18 hours after sepsis induction, and are shown in Figure 6.

As shown in Figure 6, when sepsis was induced in mice through cecal ligation and CLP surgery, edema was observed in the kidney due to invasion of inflammatory cells into the renal cortex and medulla, and renal tubules, and in the lung, alveolar walls thickened. Lung tissue damage and inflammatory cell infiltration were observed, as well as hepatocyte edema and inflammatory cell infiltration.

However, it was confirmed that when interferon beta and NR were administered in combination to mice with induced sepsis, edema and inflammatory cell infiltration caused by sepsis were reduced. The above results show that the combined administration of interferon beta and NR can effectively prevent or treat sepsis or septic shock by suppressing organ damage caused by sepsis, that is, organ edema and inflammatory cell infiltration.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred implementation examples and do not limit the scope of the present invention. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

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Claims (10)

interferon beta (IFN-beta); and a pharmaceutical composition for preventing or treating sepsis or septic shock comprising a compound represented by the following formula (1) as an active ingredient:
[Formula 1]
delete According to paragraph 1,
A pharmaceutical composition wherein the interferon beta is interferon beta 1a or 1b. According to paragraph 1,
The interferon beta; And the compound represented by Formula 1 is a pharmaceutical composition that induces the expression of SIRT1 (silent mating type information regulation 2 homolog; sirtuin 1). According to paragraph 1,
The pharmaceutical composition reduces the blood concentration of inflammatory cytokines. interferon beta (IFN-beta); And a food composition for preventing or improving sepsis or septic shock comprising a compound represented by the following formula (1) as an active ingredient:
[Formula 1]
delete According to clause 6,
A food composition wherein the interferon beta is interferon beta 1a or 1b. According to clause 6,
The interferon beta; And the compound represented by Formula 1 is a food composition that induces the expression of SIRT1 (silent mating type information regulation 2 homolog; sirtuin 1). According to clause 6,
The food composition reduces the blood concentration of inflammatory cytokines.