WO2023085741A1 - Composition for preventing or treating multiple organ failure caused by infection, comprising 3'-sialyllactose, 6'-sialyllactose, or salt thereof as active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing or treating multiple organ failure caused by infection, comprising 3'-sialyllactose, 6'-sialyllactose, or a salt thereof as an active ingredient. More particularly, as a result of administering 3'-sialyllactose or 6'-sialyllactose to an LPS-induced acute inflammatory mouse model, an increase in inflammatory cytokines in the blood of a mouse is suppressed, and damage to the liver and lungs is suppressed, thereby helping prevent, treat, and improve cytokine storm-related inflammation-related diseases.

Description

Composition for preventing or treating multiple organ failure caused by infection, containing 3'-sialyllactose, 6'-sialyllactose or a salt thereof as an active ingredient

The present invention relates to a composition for preventing or treating multiple organ failure caused by infection, comprising 3'-sialyllactose, 6'-sialyllactose or a salt thereof as an active ingredient.

Multiple organ dysfunction syndrome (MOF), or total organ failure, is a condition in which the body's organs fail to function properly, stop working, or become severely sluggish. There can be many causes, and it can occur when bacteria causing pneumonia, nephritis, laryngitis, etc. circulate throughout the body, when symptoms such as sepsis appear, or when immunity is weakened by taking antibiotics due to cancer. there is.

In particular, in the case of the coronavirus (COVID-19), which has caused more than 200 million confirmed cases and 4.55 million deaths worldwide until recently, high mortality rates are reported mainly in elderly patients aged 70 years or older with underlying diseases, but prior to infection Among young patients with no special underlying disease, a sudden critical condition and death occurred, and the “cytokine storm” caused by viral infection became a hot topic.

A cytokine storm is a large-scale inflammatory reaction caused by an excessive response of the body's immune system to attack a virus that has invaded from the outside and attacks normal cells. Cytokine release syndrome or hypercytokineemia (called hypercytoinemia). That is, excessive secretion of cytokines, which are immune substances, due to penetration of external viruses, etc. causes excessive fever, and protein modification may occur as human proteins are exposed to such high heat of 40 degrees or more. As a result, normal cells can be attacked by immune cells, and secondary damage of organ failure occurs in the process of destroying body tissues by causing an overreaction of the immune system.

Even during the Spanish flu, cytokine storm was pointed out as the cause of the very high mortality rate in healthy people aged 25 to 45 years old, and avian influenza (H5N1) also showed high mortality rate due to multiple organ failure caused by the cytokine storm. Similar symptoms have also been reported with the Ebola virus.

In Korea, when MERS infections spread in 2015, patients with deteriorating conditions appeared at a young age without underlying diseases, and cytokine storms and multiple organ failures began to be discussed. In the COVID-19 pandemic, some cytokine storms has been identified as a major cause of

Therefore, when a cytokine storm causing multiple organ failure progresses, prompt measures must be taken to stop it, and accordingly, research on the development of effective treatments capable of regulating the immune system and blocking specific causes of immune hyperreaction is urgently needed.

On the other hand, sialyllactose is a form in which sialic acid is attached to lactose (milk sugar), a major nutrient that mammalian mothers give to their offspring, and is one of the most abundant human milk oligosaccharides in colostrum of breast milk. It is a substance that helps brain development, cognitive improvement, and immune function. It has an immune protective effect against pathogens in newborns, and is known to inhibit the attachment and infection of bacteria and viruses such as influenza virus, HIV-1, and rotavirus. there is. It is also known to inhibit the binding of cholera toxin.

As prior art, Korean Patent Publication No. 10-2020-0023226 discloses a compound containing a polyvalent sialyl oligosaccharide residue and a composition for preventing or treating viral infectious diseases containing the compound as an active ingredient. It is different from compositions for preventing or treating cytokine storm-related inflammatory diseases containing '-sialyllactose, 6'-sialyllactose or a salt thereof as an active ingredient in terms of composition and effect.

Korean Patent Registration No. 2065575 discloses a core that inhibits the infection process of influenza virus by binding to hemagglutinin present on the surface of influenza virus, and a conjugate comprising sialic acid, sialyllactose or derivatives thereof bound to the surface thereof. And its use is described, but there are also differences from the configuration and effects of the present invention.

An object of the present invention is to provide a composition for preventing or treating multiple organ failure caused by infection, containing 3'-sialyllactose, 6'-sialyllactose or a salt thereof as an active ingredient.

In order to solve the above problems, the present invention provides a composition for preventing or treating multiple organ failure caused by infection, containing 3'-sialyllactose, 6'-sialyllactose or a salt thereof as an active ingredient.

The infections include bacterial infections, viral infections and other infectious diseases. The bacterial infection is Salmonella genus, Chlamydophila genus, Coxiella genus, Rickettsia genus, Borrelia genus, Bartonella genus, Chlamydia Genus, Legionella, Anaplasma, Ehrlichia, Rochalimaea, Brucella, Francisella, Neisseria ) genus, Nocardia genus, Rhodococcus genus, Staphylococcus genus, Mycoplasma genus, Mycobacterium genus, Yersinia genus, The infection may be caused by one or more bacteria selected from the group consisting of Escherichia genus, Enterococcus genus, Listeria genus, and Streptococcus genus.

In addition, the viral infections include influenza virus, influenza A virus subtype H1N1, avian influenza virus, rhinovirus, adenovirus, and coronavirus , Parainfluenza virus (parainfluenza virus), respiratory syncytial virus (respiratory syncytial virus), herpes virus (Herpesvirus, HSV), and may be an infection by any one or more viruses selected from the group consisting of hepatitis virus. In addition, the coronavirus (coronavirus) is severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome type 2 coronavirus (SARS-Cov-2), Middle East respiratory syndrome coronavirus (MERS-CoV), human corona virus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), human coronavirus NL63 (HCoV-NL63) and human coronavirus HKU1.

The other infectious diseases may be infections caused by protozoa or fungi, and include malaria, cryptosporiasis, cryptococcosis, candidiasis, amebiasis, toxoplasmosis, aspergillosis, giardiasis, pneumocystis infection and spores. It may be one or more selected from the group consisting of rotrichosis.

The multiple organ failure refers to a risk to life maintenance due to the deterioration or loss of multiple organ functions in a short period of time as an inflammatory response in the body is accelerated by the infection or the like. It is also called 'total organ failure', and in English it is called 'Multiple Organ Failure (MOF)', 'Multiple Organ Dysfunction Syndrome (MODS)', 'Total Organ Failure (TOF)', 'Multisystem Organ Failure (MSOF)', etc. The multiple organ failure includes heart failure, respiratory failure, renal failure, liver failure, and the like, and life-sustaining activities become extremely difficult, resulting in a very high probability of death.

The multiple organ failure may be caused by a cytokine storm induced by the infection. Cytokine storm, also called cytokine release syndrome or hypercytokineemia, is an excessive response of the body's immune system to fight viruses infiltrated from the outside and attacks even normal cells. It is a large-scale inflammatory response that occurs

The composition for preventing or treating inflammatory diseases comprising 3'-sialyllactose, 6'-sialyllactose or a salt thereof as an active ingredient of the present invention contains inflammatory cytokines TNF-α, IL-β and The increase of GM-CSF can be suppressed. In addition, the inflammatory response can be improved by reducing the levels of LDH, GPT, GOT, CPK, and BUN, which are increased by LPS.

The composition for preventing or treating inflammatory diseases comprising 3'-sialyllactose, 6'-sialyllactose or a salt thereof as an active ingredient of the present invention can reduce the degree of liver and lung tissue damage caused by LPS. can In particular, treatment with 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200) in liver tissue significantly reduced the activities of STAT1 and STAT3 increased by LPS, resulting in an excellent liver response in the acute inflammatory response caused by LPS. show a protective effect.

The present invention provides a composition for preventing or treating inflammatory diseases comprising 3'-sialyllactose, 6'-sialyllactose or a salt thereof as an active ingredient.

A composition for preventing or treating inflammatory diseases containing 3'-sialyllactose, 6'-sialyllactose or a salt thereof as an active ingredient may be provided as a pharmaceutical composition. The 3'-sialyllactose or 6'-sialyllactose is preferably 0.001 to 50% by weight, more preferably 0.001 to 40% by weight, most preferably 0.001 to 30% by weight based on the total weight of the pharmaceutical composition. can be added as

The pharmaceutical composition is formulated in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, liquids, aerosols, external preparations, suppositories and sterile injection solutions according to conventional methods, respectively. can Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants, sweeteners, and acidulants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient, for example, 3'-sialyllactose or 6'-sialyllactose of the present invention. For example, it is prepared by mixing starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, preservatives, and acidulants are used. can be included Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspensions. As a base for the suppository, witepsol, macrogol, tween-61, cacao butter, laurin paper, glycerogeratin, and the like may be used.

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, sex, and weight of the subject to be treated, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the route of administration, and the prescriber's judgment. Determination of dosage based on these factors is within the level of those skilled in the art, and generally dosages range from 0.01 mg/kg/day to approximately 500 mg/kg/day. A preferred dose is 0.1 mg/kg/day to 200 mg/kg/day, and a more preferred dose is 1 mg/kg/day to 200 mg/kg/day. Administration may be administered once a day, or may be administered in several divided doses. The dosage is not intended to limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, livestock, and humans through various routes. All modes of administration are contemplated, eg oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine intrathecal or intracerebrovascular injection and dermal application. Since 3'-sialyllactose or 6'-sialyllactose of the present invention has little toxicity and side effects, it is a drug that can be safely used even when taken for a long period of time for preventive purposes.

The present invention provides a health functional food for preventing or improving inflammatory diseases, including 3'-sialyllactose, 6'-sialyllactose or a salt thereof, and food additives acceptable in food science.

In the health functional food, the content of 3'-sialyllactose or 6'-sialyllactose is preferably 0.001 to 50% by weight, more preferably 0.001 to 30% by weight, and most preferably 0.001 to 30% by weight based on the total weight of the total food. It may be added at 10% by weight.

The health functional food includes the form of tablets, capsules, pills or liquids, and foods to which the extract of the present invention can be added include, for example, various foods, beverages, gum, tea, vitamin complexes, health functional foods, etc.

The present invention relates to a composition for preventing or treating multiple organ failure caused by infection, comprising 3'-sialyllactose, 6'-sialyllactose or a salt thereof as an active ingredient, which is a cytokine induced by bacterial or viral infection. Inhibition and prevention of storm-induced aggravation can play an important role in improving morbidity and mortality.

1 is a schematic diagram showing an experimental plan using an LPS-induced acute hyperinflammation mouse model.

Figure 2 shows that 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200) in LPS-induced acute excess inflammation mouse model is TNF-α (A), IL-1β (B) and GM-CSF (C ) is a graph showing the effect on

Figure 3 is 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200) in LPS-induced acute excess inflammation mouse model LDH (A), GPT (B), GOT (C), CPK (D) And it is a graph showing the effect on BUN (E).

Figure 4 is a result showing the effect of 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200) on liver and lung tissue damage in LPS-induced acute excess inflammation mouse model. (A) Liver tissue micrograph, (B) Liver injury evaluation, (C) Lung tissue micrograph, (D) Lung injury evaluation.

Figure 5 is a result showing the effect of 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200) on immune cell invasion in the liver of an LPS-induced acute excess inflammation mouse model. (A) CD45 immunofluorescence staining in liver tissue, (B) CD45 fluorescence intensity evaluation in liver tissue (C) Evaluation of immune cell infiltration in liver tissue

Figure 6 is a result showing the effect of 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200) on immune cell invasion in the lungs of an LPS-induced acute hyperinflammatory mouse model. (A) Immunofluorescence staining picture of CD45 in lung tissue, (B) Evaluation of CD45 fluorescence intensity in lung tissue (C) Evaluation of immune cell infiltration in lung tissue

Figure 7 is a result showing the effect of 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200) on STAT3 activity in the liver and lungs of LPS-induced acute excess inflammation mouse model. (A) p-STAT3 immunofluorescence staining in liver tissue, (B) evaluation of p-STAT3 fluorescence intensity in liver tissue (C) p-STAT3 immunofluorescence staining image in lung tissue, (D) p-STAT3 fluorescence in lung tissue strength evaluation

Figure 8 is a result showing the effect of 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200) on the STAT1 / STAT3 activation mechanism in the liver and lungs of LPS-induced acute excess inflammation mouse model.

9 is a SARS-CoV-2 spike pseudotype virus assay result of 6'-sialyllactose (GCV200).

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the disclosure herein is provided so that it will be thorough and complete, and will fully convey the spirit of the invention to those skilled in the art.

<Experimental Example 1. Checking the effect of 3'-sialyllactose or 6'-sialyllactose on cytokine expression in LPS-induced acute excessive inflammation mouse model>

In order to confirm the protective effect of 3'-sialyllactose or 6'-sialyllactose from acute inflammatory organ damage, 3'-sialyllactose or 6'-sialyllactose organ damage using LPS-induced acute excess inflammation mouse model The protective efficacy was evaluated.

ICR mice were treated with 10 mg/kg LPS by intraperitoneal injection (I.P. injection) for 24 hours to construct an acute inflammation model. Six experimental groups were divided as shown in Table 1, and 7 to 8 ICR mice were assigned to each experimental group. 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200) was administered intraperitoneally (IP) at a dose of 100 mg/kg, respectively, 2 hours before inducing acute excessive inflammation by LPS. The timetable for administration of 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200) and LPS treatment is shown in FIG. 1 .

group process number of mice One Vehicle 7 2 GCV100 100 mg/kg 8 3 GCV200 100 mg/kg 8 4 LPS 10 mg/kg 8 5 LPS + GCV100 8 6 LPS + GCV200 8

After 24 hours of LPS treatment, blood was collected, and secretion amounts of TNF-α (A), IL-1β (B), and GM-CSF in serum were confirmed through ELISA analysis and are shown in FIG. 2 . The amount of each cytokine was expressed as a relative value to the control group treated only with the vehicle (Vehicle, V).

IL-1β is a key cytokine that regulates inflammatory and immune responses. It activates signal transduction pathways such as NF-kB and MAPK inside the cell, thereby suppressing inflammatory cytokines such as TNF-α and GM-CSF and inflammatory and immune responses. It induces the expression of various proteins involved. In addition, TNF-α is a pro-inflammatory cytokine mainly secreted by macrophages and is involved in the regulation of immune cells, cell proliferation, and differentiation. Another inflammatory cytokine affected by IL-1β, GM-CSF, exerts important cytokine effects by regenerating the production of granulocytes and macrophages.

As shown in Figure 2, the level of TNF-α in serum was increased by more than 5500% by LPS, and by 100 mg/kg of 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200) each The level of TNF-α in serum showed a decreasing trend. Serum secretion of IL-1β and GM-CSF was also rapidly increased by LPS, and the increased cytokines were decreased by 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200). In particular, 3'-sialyllactose (GCV100) treatment significantly reduced both serum levels of IL-1β and GM-CSF, which were increased by LPS induction, and 6'-sialyllactose (GCV200) was also involved in LPS induction. significantly decreased the serum level of GM-CSF increased by

As shown in the above results, it was confirmed that administration of 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200) reduced TNF-α, IL-1β, and GM-CSF increased by LPS. .

<Experimental Example 2. Confirmation of the effect of 3'-sialyllactose or 6'-sialyllactose on long-term damage index in LPS-induced acute excessive inflammation mouse model>

Glutamine oxaloacetate transaminase (GOT), glutamine pyruvate transaminase (Glutamic Pyrubic Transaminase (GPT), creatine phosphokinase (CPK), blood urea nitrogen (BUN), and lactate dehydrogenase (LDH) were confirmed through biochemical analysis, which are shown in FIG. 3 .

Blood lactate dehydrogenase (LDH) is contained in many tissue cells and is often highly active in malignant tumors, liver diseases, heart diseases, etc., so it is used for analysis for disease screening. Glutamate Oxaloacetate Transaminase (GOT) and Glutamic Pyrubic Transaminase (GPT) are enzymes that transfer amino acids from the blood to the liver as glutamate. It leaks into the blood and can be used as an indicator of liver damage. In addition, Creatine Phosphokinase (CPK), found in various tissues and cell types, can analyze muscle tissue damage, and blood urea nitrogen (BUN), a nitrogen element produced in the body during protein breakdown. It means a number and is an indicator of kidney damage.

As shown in Figure 3, LPS administration significantly increased the concentrations of LDH, GOT, GPT, CPK and BUN, and 100 mg/day of 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200), respectively. The levels of CPK and BUN were significantly reduced by kg treatment. In addition, GPT levels were significantly reduced by 3'-sialyllactose (GCV100) treatment, and LDH and GOT levels were significantly decreased by 6'-sialyllactose (GCV200) treatment. In addition, in all experimental groups that did not show statistical significance, the organ damage index increased by LPS induction tended to decrease by 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200).

<Experimental Example 3. Analysis of tissue damage through H&E staining>

After sacrificing the animals in Experimental Example 1, tissue damage was analyzed through H&E staining of liver and lung tissues excised. The extracted liver and lung tissues were fixed with 4% formalin, the tissues were fixed using paraffin through a dehydration process, and then the paraffin-embedded tissues were cut into 5 μm thickness through a tissue cutter. The cut tissues were subjected to paraffin removal using xylene and dehydration using ethanol, followed by intra-tissue cell nuclei staining using hematoxylin reagent and intra-tissue cytoplasmic staining using eosin Y. After completion of staining, the degree of tissue damage was confirmed through a microscope, and organ damage was evaluated according to criteria, and the results are shown in FIG. 4 .

The degree of damage to liver tissue is 0 when it is in a normal state without any abnormalities, 1 when there is a wound in the tissue due to inflammation, such as fatty liver, and 1 such as liver lesion due to many wounds and invasion of neutrophil granulocytes, an immune cell into the tissue. It was judged as stage 2 if it was visible, and stage 3 or higher if necrosis was seen due to the formation of cell wall collapse due to many wounds and intracellular damage in tissues such as liver cirrhosis.

The degree of damage to the lung tissue is grade 1 when there is small damage due to inflammation, grade 2 when lung tissue collapse due to inflammation is seen, grade 3 when alveolar septum thickening and proliferation of alveolar cells appear, and lungs caused by inflammation of the lungs. Structural collapse due to enteritis and pulmonary nodule was evaluated as 4 levels.

As shown in Figures 4A and 4B, in the group administered with LPS alone, liver damage was evaluated in 2 to 3 stages, and 3'-sialyllactose (GCV100) or 6'-sialyllactose (before induction of inflammation by LPS) GCV200) at 100 mg/kg each, the degree of liver damage was reduced by one level for both substances.

In addition, as shown in FIGS. 4C and 4D, in the group administered with LPS alone, alveolar thickness was increased while structural collapse was observed, and the degree of lung damage was evaluated in 3 to 4 stages, and 3'-sialyl When lactose (GCV100) or 6'-sialyllactose (GCV200) was treated at 100 mg/kg each, no structural collapse of the lungs was observed due to LPS treatment, and the thickening of the alveoli was observed, suggesting that the degree of lung damage was reduced. It was confirmed that the reduction in 1 to 2 steps.

As a result of this experiment, in the LPS-induced inflammatory model, liver and lung damage through acute inflammatory response was reduced by administration of 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200). It was confirmed that Ryllactose (GCV100) or 6'-Sialyllactose (GCV200) has an effect of protecting the liver and lungs from damage, and in particular, it was confirmed that they have excellent activity in the recovery of liver tissue.

<Experimental Example 4. CD45 immunofluorescence staining technique>

Immunofluorescence staining technique is a test that can confirm the position of the antigen in the sample by labeling the antibody with fluorescence, reacting with the antigen in the cut tissue, and then exposing the fluorescence. The effect of 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200) on CD45 expression was confirmed through immunofluorescence staining of CD45 in liver and lung tissues of experimental animals obtained in Example 1. .

As an immune cell regulator in acute and chronic inflammatory responses, CD45 directly reacts with T cells or B cells and regulates the signal transduction mechanism of inflammatory cells. It is also known to serve as a marker that increases the inflammatory response of macrophages by the STAT1 signaling pathway through interferon gamma.

The liver and lung tissues obtained in Example 3 were fixed in paraffin, cut into 5 μm, removed from paraffin, and dehydrated, and reacted with CD45 antibody for 2 hours. After the reaction, a fluorescent secondary antibody was reacted, and after the reaction was completed, the nucleus was stained with DAPI, and the fluorescence intensity and the number of infiltrating immune cells were calculated, and the results are shown in FIGS. 5 and 6 .

In the liver tissue, as shown in Figures 5A and 5B, the expression of CD45 was increased by more than 3 times compared to the control group (Vehicle) in the LPS-induced hyperinflammatory liver, and 3'-sialyllactose (GCV100) or 6'-sial When each 100 mg/kg of relactose (GCV200) was treated, it was confirmed that the expression of CD45 was reduced by half. In addition, as shown in Figure 5C, the number of immune cells infiltrated into the liver tissue compared to the control group (Vehicle) in the LPS-induced hyperinflammation liver increased more than twice as compared to the control group (Vehicle), and 3'-sialyl When lactose (GCV100) or 6'-sialyllactose (GCV200) was treated at 100 mg/kg each, a significant decrease was confirmed. In the case of analyzing the number of infiltrated immune cells in liver and lung tissue, hematoxylin and eosin-stained tissue photographs were analyzed. Immune cells were identified through morphological differences in hematoxylin-stained nuclei in histological photographs, and the number of immune cells infiltrating each tissue was analyzed, and the data in the graph was expressed as a fold difference from the control group.

In addition, as shown in FIGS. 6A and 6B in lung tissue, the expression of CD45, which had been increased by LPS, was reduced by nearly 1/2-fold by GCV100 and GCV200 treatment, respectively, and as shown in FIG. 6C, the infiltrated lung tissue The number of immune cells was also significantly decreased in both GCV100 and GCV 200 treated groups.

<Experimental Example 5. Confirmation of STAT3 activity in tissue through immunofluorescence staining>

STAT3 (Signal transducer and activator of transcription 3) activity is one of the immune mechanisms through LPS-induced TLR4 (Toll like receptor 4) activity. TLR4 activated by LPS induces gene expression of interferons, interleukins, and cytokines by regulating the activity of a transcription factor called NF-κB through activation of Myd88. activate the receptor. Interferon receptors are among the activated receptors, and the receptors activated by interferon gamma increase the activity of STAT3. Activated STAT3 enters the nucleus and increases the expression of immune response substances as a transcription factor.

In order to confirm the mechanism by which 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200) inhibits the inflammatory response in an LPS-induced hyperinflammatory animal model, the present inventors investigated intra-tissue through immunofluorescence staining technique. STAT3 expression was confirmed.

Immunofluorescence staining of STAT3 in the liver and lung tissues of experimental animals obtained in the same manner as in Example 1 was performed by modifying the method of Example 4 to confirm STAT3 expression in tissues, and the results are shown in FIG. 7 .

As shown in Figures 7A and 7B, LPS increased STAT3 expression in the liver by more than 3-fold compared to the negative control group, and upon pretreatment with 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200) , reduced the STAT3 expression increased by LPS by about 50%.

As shown in FIGS. 7C and 7D in lung tissue, STAT3 expression, which was increased by LPS, was significantly decreased by 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200).

<Experimental Example 6. Confirmation of STAT1/3 protein expression in tissue through Western blot>

In order to confirm the mechanism by which 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200) inhibits the inflammatory response in LPS-induced hyperinflammatory animal models, STAT1/3 protein expression was examined through Western blot. Confirmed.

The liver and lungs extracted in the same manner as in Example 1 were stored at -70 ° C, and then 6 mg tissue was dissolved using 2X SDS buffer, and the dissolved tissue was loaded on an SDS-PAGE gel, followed by anti-STAT1 and STAT3 antibodies. Western blotting was performed using and shown in FIG. 8 .

STAT1 is an immune mechanism-related factor together with STAT3, and is activated through a sub-mechanism of interleukin-6 or a sub-mechanism of TLR4, and is known to regulate the expression of genes related to immune response by binding to NF-kB.

As shown in Figure 8, in the control group (Veh) and 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200) alone treatment group, there was no change in the activity of STAT1 and STAT3 in liver tissue, but simultaneously with LPS Both STAT1 and STAT3 activities were increased in the treatment group. However, when pre-treated with 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200), it was confirmed that both the activities of STAT1 and STAT3 increased by LPS were suppressed. In addition, LPS treatment increased the activity of both STAT1 and STAT3 in both lung tissue and lung tissue, and when 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200) was treated, LPS-induced The activities of both STAT1 and STAT3 were reduced. In particular, a more excellent inhibitory effect was confirmed in the 6'-sialyllactose (GCV200) treatment group.

Through these results, the activity of the STAT1 / STAT3 mechanism increased by LPS in liver and lung tissue, 3'-sialyllactose (GCV100) or 6 It is shown that the treatment of '-sialyllactose (GCV200) reduces and has an anti-inflammatory effect in a hyperinflammatory model. In particular, treatment with 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200) in liver tissue significantly reduced the activities of STAT1 and STAT3 increased by LPS, thereby protecting the liver in the acute inflammatory response caused by LPS. It was confirmed that the effect was excellent.

Excessive immune response due to acute inflammation causes damage to various tissues such as liver, lung, and kidney as well as inflammatory diseases such as arthritis and atopy. Inflammatory cells are activated. Macrophages activated by LPS secrete pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β. As it binds to the receptor, the receptor is activated and activates its own inflammatory mechanisms. As these inflammatory mechanisms secrete inflammatory cytokines again, the concentration of inflammatory cytokines becomes excessively high as a positive feedback.

The present invention confirmed the effect of 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200) in an animal model with acute immunity induced by administration of LPS.

The degree of damage to each tissue increased by at least 1.6 times to 4 times or more by LPS administration, and all were significantly reduced by 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200) administration. In addition, the liver showed damage to the degree of liver cirrhosis due to LPS administration, and diseases such as pulmonary nodules and pneumonitis were observed in the lungs, but 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200) administration Both liver and lung damage were significantly reduced. In particular, in the case of the liver, when 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200) was administered, LPS treatment reduced the cirrhosis-like appearance to a slight inflammation such as fatty liver. In the case of LPS treatment, all lung structures collapsed, and when 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200) was administered to alveoli proliferated by inflammation, lung damage was significantly reduced.

As for the above effects, 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200) treatment inhibited liver and lung damage in acute inflammation caused by LPS administration, suppressed inflammatory mechanisms, and overlooked and Infiltration of inflammatory cells in lung tissue was suppressed. In addition, it was confirmed that 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200) significantly inhibited the expression of STAT3, which was increased by 2-3 times in liver and lung tissues by LPS, respectively. It was found that lactose (GCV100) or 6'-sialyllactose (GCV200) can reduce the expression of pro-inflammatory cytokines through inhibition of STAT1/STAT3 activity.

<Experimental Example 7. in vitro SARS-CoV-2 Spike pseudotype virus assay>

SARS-CoV-2 Spike pseudotype virus assay was performed to determine whether 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200) could inhibit the expression of the spike protein of SARS-CoV-2. .

100 copy number of VSV-G, SARS-CoV-2 spike (WT) and SARS-CoV-2 spike (D614G) pseudotype viruses were treated with 6'-sialyllactose (GCV200) at a concentration of 10-80 mM to induce luciferase Activity was measured and shown in FIG. 9 .

As shown in Figure 9, it was confirmed that 6'-sialyllactose (GCV200) inhibits the entry into cells of SARS-CoV-2 Wild type and D614G mutant pseudotype virus in a concentration-dependent manner.

<Formulation Example 1. Pharmaceutical formulation>

Formulation Example 1-1. manufacture of tablets

200 mg of 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200) of the present invention was mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. After adding 10% gelatin solution to this mixture, it was pulverized and passed through a 14 mesh sieve. It was dried, and a mixture obtained by adding 160 g of potato starch, 50 g of active agent, and 5 g of magnesium stearate was made into tablets.

Formulation Example 1-2. Preparation of injection solution

100 mg of 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200) of the present invention, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. This solution was bottled and sterilized by heating at 20° C. for 30 minutes.

<Formulation Example 2. Manufacture of health functional food>

Formulation Example 2-1. Manufacture of health functional food

2g of 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200) of the present invention, suitable amount of vitamin mixture, vitamin A acetate 70μg, vitamin E 1.0mg, vitamin B1 0.13mg, vitamin B2 0.15mg, vitamins B6 0.5mg, Vitamin B12 0.2μg, Vitamin C 10mg, Biotin 10μg, Nicotinamide 1.7mg, Folic Acid 50μg, Calcium Pantothenate 0.5mg, Mineral Mixture Appropriate amount, Ferrous Sulfate 1.75mg, Zinc Oxide 0.82mg, Magnesium Carbonate 25.3mg, monobasic potassium phosphate 15mg, dibasic calcium phosphate 55mg, potassium citrate 90mg, calcium carbonate 100mg, magnesium chloride 24.8mg was prepared into granules, but it can be prepared by transforming into various formulations depending on the use. . In addition, the composition ratio of the vitamin and mineral mixture may be arbitrarily modified, and it may be prepared by mixing the above components according to a conventional health functional food manufacturing method.

Formulation example 2-2. Manufacture of health functional beverages

1 g of 3'-sialyllactose (GCV100) or 6'-sialyllactose (GCV200) of the present invention, 0.1 g of citric acid, 100 g of fructooligosaccharide, and 900 g of purified water were mixed and stirred, heated, filtered, The beverage was prepared by sterilization and refrigeration.

Claims (10)

A composition for preventing or treating multiple organ failure caused by infection, comprising sialyllactose or a salt thereof as an active ingredient. According to claim 1, The sialyllactose, A composition for preventing or treating multiple organ failure caused by infection, characterized in that it is 3'-sialyllactose or 6'-sialyllactose. According to claim 1, The infection is Salmonella genus, Chlamydophila genus, Coxiella genus, Rickettsia genus, Borrelia genus, Bartonella genus, Chlamydia genus , Legionella, Anaplasma, Ehrlichia, Rochalimaea, Brucella, Francisella, Neisseria Genus, Nocardia, Rhodococcus, Staphylococcus, Mycoplasma, Mycobacterium, Yersinia, S Multiple organ failure caused by infection characterized by infection by any one or more bacteria selected from the group consisting of Escherichia, Enterococcus, Listeria, and Streptococcus A composition for the prevention or treatment of According to claim 1, The infections include influenza virus, influenza A virus subtype H1N1, avian influenza virus, rhinovirus, adenovirus, coronavirus, para Influenza virus (parainfluenza virus), respiratory syncytial virus (respiratory syncytial virus), herpes virus (Herpesvirus, HSV), and multiple organs caused by infection, characterized in that infection by any one or more viruses selected from the group consisting of hepatitis virus A composition for preventing or treating insufficiency. According to claim 4, The coronaviruses include severe acute respiratory syndrome coronavirus (SARS-CoV), type 2 severe acute respiratory syndrome coronavirus (SARS-Cov-2), Middle East respiratory syndrome coronavirus (MERS-CoV), and human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), human coronavirus NL63 (HCoV-NL63) and human coronavirus HKU1. A composition for preventing or treating multiple organ failure caused by According to claim 1, The organ failure is a composition for preventing or treating multiple organ failure caused by infection, characterized in that it comprises at least one selected from the group consisting of heart failure, respiratory failure, liver failure, lung failure, and renal failure. According to claim 1, The composition for preventing or treating multiple organ failure caused by infection, characterized in that the composition suppresses the increase in inflammatory cytokines caused by infection. According to claim 1, The composition for preventing or treating multiple organ failure caused by infection, characterized in that the composition suppresses the increase in LDH, GPT, GOT, CPK or BUN caused by infection. A composition for preventing or treating inflammatory diseases comprising sialyllactose or a salt thereof as an active ingredient. A health functional food for preventing or improving inflammatory diseases containing sialyllactose or a salt thereof as an active ingredient.

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