KR20170089766A - Betaglucan-peptide complexes, preparation method thereof, and pharmaceutical composition for use in preventing or treating inflammatory diseases containing the same as an active ingredient - Google Patents

Abstract

The present invention relates to a betaglucan-peptide complex, a preparation method of the same, and a pharmaceutical composition for preventing or treating inflammatory diseases comprising the same as an active ingredient. Specifically, shizophyllan (SPG) which is beta-1,3-glucan, is covalently bound to SH3 peptide originated from c-Src to form SPG-c-Src-SH3 peptide complex, and the pharmaceutical composition comprises the complex as an active ingredient for preventing or treating inflammatory diseases such as sepsis and colitis. The betaglucan-peptide complex according to the present invention significantly reduces the production of active oxygen (ROS) and inflammatory cytokines by targeting p47phox, and thus can be beneficially used as a pharmaceutical composition for preventing or treating ROS-related life-threatening inflammatory diseases such as sepsis and colitis. Further, the betaglucan-peptide complex according to the present invention can be beneficially used in the development of novel peptide-based drugs targeting p47phox.

Description

TECHNICAL FIELD The present invention relates to a beta-glucan-peptide complex, a method for producing the beta-glucan-peptide complex, and a pharmaceutical composition for preventing or treating an inflammatory disease containing the same as an active ingredient. as an active ingredient}

The present invention relates to a beta-glucan-peptide complex, a process for its preparation, and a pharmaceutical composition for the prophylaxis or treatment of inflammatory diseases containing it as an active ingredient. More particularly, the SPG-c-Src-SH3 peptide complex is prepared by covalently binding SH3 peptide derived from c-Src with Schizophyllan (SPG), which is a beta-1,3-glucan, And to a pharmaceutical composition for the prophylaxis or treatment of inflammatory diseases such as sepsis and colitis.

Protein or lipid alteration induced by NADPH oxidase (NOX) -independent reactive oxygen species (ROS) and oxidant has been shown to be associated with fatal inflammatory conditions such as sepsis and colitis It has to do with the tissue damage observed in reaction (Zhong Y et.al., Nature cell biology , 11 (4):.. 468-476, 2009; Gump JM et.al., Trends in molecular medicine, 13 (10 ): 443-448, 2007; Wadia JS et al . , Nature medicine., 10 (3): 310-315, 2004; Hotchkiss RS et al. , Journal of Immunology, 176 (9): 5471-5477 , 2006). Sepsis is characterized by a systemic inflammatory response to gram-negative bacterial infections. There is increasing evidence that ROS plays an important role in the pathological treatment of sepsis by modulating Toll-like receptor (TLR) 4 activation, immune cell activation and endothelial damage (Fang FC., Nature reviews Microbiology., 2 ): 820-832, 2004). Excessive ROS (superoxide and hydrogen peroxide) in intracellular and extracellular cells may cause acute hyper-inflammatory responses in phagocytes (macrophages and neutrophils) (Matsunaga K et al. , Nature cell biology., 11 (4): 385-396, 2009; El Gazzar M. et al. , Molecular and cellular biology. , 29 (7): 1959-1971, 2009). Host factors that modulate ROS levels in cells may play important modulators in the pathogenesis of sepsis (Xu J. et al ., Nature medicine., 15 (11): 1318-1321 , 2009; Jain A. et al. , Nature., 473 (7348): 484-488, 2011).

Ulcerative colitis (UC) is a recurrent inflammatory bowel disease (IBD) characterized by chronic and recurrent inflammation of the gastrointestinal tract. Pathogenic mechanisms of colitis are complex and involve interactions between genetic, host immune systems and environmental factors (Hubbard, WJ, et al ., Shock , Shock, 2005; 24 Suppl 1: 52-57, 2005; Cavaillon JM et al ., Scandinavian journal of infectious diseases, 35 (9): 535-544, 2003). One of the major factors in the development of colitis is inadequate mucosal immune response to intestinal microorganisms leading to mucosal tissue injury and chronic inflammation in IBD patients (Victor VM et al ., Current pharmaceutical design .. 11 (24 ): 3141-3158, 2005). In addition, excessive ROS production was observed in inflammatory mucosa of IBD patients (Hutchins NA et al ., Trends in molecular medicine., 20 (4): 224-233, 2014; Buchweitz JP et . Al., The Journal of pharmacology and experimental therapeutics, 323 (2): 675-683, 2007). These highly cytotoxic molecules may contribute to tissue damage in IBD and may result in cytotoxicity by non-phagocytes (e.g., intestinal epithelial cells) into the colonic mucosa of the IBD patient Molecules can be released. However, molecular pathways regulating ROS production through NOX enzymes in primary intestinal epithelial cells during acute and chronic inflammation are not well known.

The NOX complex is composed of two transmembrane proteins: flavocytochrome b components (gp91phox / NOX2 and p22phox) and four cytosolic proteins (p47phox, p67phox , p40phox, and Rac1 / 2) are present in the phagocyte (Lambeth JD et al ., Annual review of pathology, 9: 119-145, 2014). The activation of the cytoplasmic component is transferred to the transmembrane catalytic protein gp91phox / NOX2 to form a functional NOX complex (ROS), which is involved in cell signaling Secondary messenger, involved in several physiological and pathological processes. Many studies have shown that NOX-dependent ROS production (NOX-d) is involved in regulating TLR4 signaling, inflammatory response and disease pathogenesis ependent ROS generation), particularly colon epithelial cells express richly in NOX1, a homologue most closely related to NOX2 in terms of structure and function (Victor VM et al. ., Current pharmaceutical design, 11 ( 24):... 3141-3158, 2005; Hutchins NA et.al., Trends in molecular medicine .. 20 (4): 224-233, 2014) the enzyme is normal and pathogenic It is in intimate contact with bacteria and can play an important role in the local innate immune and inflammatory response in the intestine (Bruno BJ. et al. , Therapeutic delivery .. 4 (11): 1443-1467, 2013). Several studies have shown that pro-inflammatory cytokines, bacteria, and bacterial products, such as IL-18, IFN-γ and TNF-α, inhibit NOX expression in cultured intestinal epithelial cells in vitro And ROS generation can be stimulated. Activation of NOX requires the interaction with p22phox and its partners regulating it (NOXO1, p47phox homolog, NOX activator 1, p67phox homolog, Rac1 GTPase). NOXO1 and NOXA1 transcripts are also abundantly expressed in the colon. To date, it is unclear whether NOX expression and activity are related to pathological aspects such as sepsis and IBD.

Schizophyllan (SPG), which is a soluble form of β-glucan derived from Schizophyllum commune , is a β- (1-3), which forms a triple helix in a neutral solution. ) - glucan (β- (1-3) -glucan family) polysaccharide. When the alkaline solution of zypohilane (SPG) is neutralized, it is denatured to form a single chain and back to the original triple helix through hydrophobic and hydrogen bonding interactions. During these physicochemical interactions, the two main-chain glucose and one oligonucleotide (ODN) bases or peptides of β- (1-3) -glucan are the original triple helix Instead of going back, it forms a stoichiometric complex. Using this complex, the SPG-based drug delivery system is designed to deliver a functional ODN to targeted cells (Yang CS et al ., Cell host & microbe .., 11 (3): 277-289, 2012; Deretic V. et.al, Scientific American, 298 (5):... 74-81, 2008; Deretic V. et.al, Cell host & microbe, 5 (6): 527-549, 2009; Craik DJ et al ., Chemical biology & drug design., 81 (1): 136-147, 2013).

Dectin-1 has been identified as a pathogen pattern recognition receptor that binds to β-glucan such as SPG. Dectin-1 has been identified as a macrophage, dendritic cell Glucan receptors in antigen presenting cells (APCs) such as non-phagocytes (epithelial cells and endothelial cells) (Lee YS et al. , EMBO molecular medicine., 7 (5): 577-592, 2015). Thus, the complex will be recognized by the Dectin-1 of antigen presenting cells (APCs), and ODNs or peptides will be ingested by these cells. The engagement of deutin-1 by? -glucan induces phosphorylation of an immunoreceptor tyrosine-based activation motif-like sequence within the cytoplasmic domain. The association of C-Src or tyrosine kinase with CARD9 / BCL10 / MALT1 complex -NF-κB (CARD9 / BCL10 / MALT1 complex-nuclear factor-κB) pathway in dendritic cells and macrophages Induced assembly of scaffolds (Kim SD et al., Journal of Immunology, 185 (7): 4302-4310, 2010). However, to date, the role of SPG in ROS-induced immune responses has not been elucidated in detail.

The present inventors have identified that C-Src is an important regulator of NOX activity through physical and functional interaction with p47phox. In other words, C-Src competitively inhibited the binding of p47phox to other NOX subunits and effectively blocked the assembly of NOX complex (NOX complex). In addition, SH3-derived peptides from C-Src have been shown to bind to sipyo-pilan (SPG) to block NOX interactions, resulting in marked inhibition of reactive oxygen species (ROS) and inflammatory cytokine production. In addition, CLP (cecal ligation procedure) - polymicrobial sepsis and DSS (dextran sodium sulfate) -induced ulcerative colitis mouse in vivo Treatment of the animal model with the SPG / C-Src-SH3 peptide complex (SPG / C-Src-SH3 peptide complex) significantly reduces mortality by modulating the excessive production of ROS and inflammation, But also improved the pathological response. Taken together, the present inventors have found that C-Src plays an important role in NOX activity through direct binding with p47phox and can inhibit excessive inflammatory response.

Excessive inflammatory responses caused by intracellular reactive oxygen species (ROS) can cause a variety of diseases including sepsis and colitis, but the regulator of this process is still not well defined. Thus, the SPG-C-Src-SH3 peptide complex of the present invention can target or treat p47phox as a target, thereby effectively treating or treating a fatal inflammatory disease associated with ROS such as sepsis and colitis, Which is useful for the development of novel peptide-based drugs.

Under these circumstances, the present inventors have made extensive efforts to develop a therapeutic agent for the treatment of inflammatory diseases such as sepsis and colitis, and found that C-Src binds to p47phox to produce active oxygen (ROS) and assembly of NOx complex And the C-Src SH3-derived peptide consisting of amino acids 91-108 and 121-140 of C-Src, and schizophyllan (β-1,3-glucan) , SPG) -containing SPG-C-Src-SH3 peptide complex. It has also been found that the complex inhibits the production of NOX subunit assembly and proinflammatory mediators and is applied to an animal model of sepsis or colitis mice, Inflammatory disease, and the therapeutic effect, thereby completing the present invention.

Zhong Y et al., Nature cell biology., 11 (4): 468-476, 2009 Gump JM et al., Trends in molecular medicine, 13 (10): 443-448, 2007 Wadia JS et al., Nature medicine., 10 (3): 310-315, 2004 Hotchkiss RS et al., Journal of Immunology, 176 (9): 5471-5477, 2006 Fang FC., Nature reviews Microbiology., 2 (10): 820-832, 2004 Matsunaga K et al., Nature cell biology., 11 (4): 385-396, 2009 El Gazzar M. et al., Molecular and cellular biology., 29 (7): 1959-1971, 2009 Xu J. et al., Nature medicine., 15 (11): 1318-1321, 2009 Jain A. et al., Nature., 473 (7348): 484-488, 2011 Hubbard WJ. et al., Shock., Shock. 2005; 24 Suppl 1: 52-57, 2005 Cavaillon JM. et al., Scandinavian journal of infectious diseases, 35 (9): 535-544, 2003 Victor VM. et al., Current pharmaceutical design .. 11 (24): 3141-3158, 2005 Hutchins NA. et al., Trends in molecular medicine, 20 (4): 224-233, 2014 Buchweitz JP. et al., The Journal of Pharmacology and Experimental Therapeutics, 323 (2): 675-683, 2007 Lambeth JD. et al., Annual review of pathology., 9: 119-145, 2014 Bruno BJ. et al., Therapeutic delivery .. 4 (11): 1443-1467, 2013 Yang CS. et al., Cell host & microbe., 11 (3): 277-289, 2012 Deretic V. et al., Scientific American., 298 (5): 74-81, 2008 Deretic V. et al., Cell host & microbe., 5 (6): 527-549, 2009 Craig DJ. et al., Chemical biology & drug design., 81 (1): 136-147, 2013 Kim SD. et al., Journal of immunology., 185 (7): 4302-4310, 2010

It is an object of the present invention to provide a beta glucan-peptide complex for the prevention or treatment of inflammatory diseases.

It is another object of the present invention to provide a method for producing the beta-glucan-peptide complex.

It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating an inflammatory disease comprising the beta-glucan-peptide complex as an active ingredient.

In order to achieve the above object, the present invention provides a beta-glucan-peptide complex, a process for producing the beta-glucan-peptide complex, and a pharmaceutical composition for preventing or treating an inflammatory disease containing the same. More specifically, SPG-c-Src-SH3 peptide complex is prepared by covalently binding SH3 peptides derived from c-Src with Schizophyllan (SPG), which is a beta-1,3-glucan, And a pharmaceutical composition for the prophylaxis or treatment of inflammatory diseases such as sepsis and colitis.

Hereinafter, the present invention will be described in detail.

In one embodiment, the present invention provides a pharmaceutical composition comprising (i) beta-1,3-glucan, and (ii) a beta-1,3- 2 < / RTI > for the prophylaxis or treatment of inflammatory diseases.

The term "beta-1,3-glucan" in the present invention refers to a polysaccharide polymerized with glucose centered on a beta-1,3 chemical bond and includes microorganisms such as mushroom, yeast, Beta-glucan derived from microorganisms produced by separation from cell walls or extracellular polysaccharides of plant cells, and plant beta-glucan extracted from cereal dietary fiber such as barley and oats. Specifically, beta-1,3-glucan is a polymeric polysaccharide substance that forms a beta-1,3-bond between glucose molecules, and includes, but not limited to, Schizophyllan (SPG) Zymosan, Curdlan AL, Scleroglucan, or Laminarin. ≪ RTI ID = 0.0 >

The Schizophyllan (SPG) is a soluble form of β-glucan derived from Schizophyllum commune and forms a triple helix in a neutral solution. It is a polysaccharide.

On the other hand, Zymosan is β-glucan derived from the cell wall of S. cerevisiae , Curdlan AL is β-glucan derived from Alcaligenes faecalis , Glucan, and Scleroglucan is Sclerotium < RTI ID = 0.0 > glutamic acid, and laminarin may be? -glucan derived from Laminaria digitata .

The term "peptide" in the present invention is an SH3 peptide derived from c-Src and consists of amino acids 91 to 108 (SEQ ID NO: 1: ALYDYESRTETDLSFKKG) and 121 to 140 (SEQ ID NO: 2: WWLAHSLSTGQTGYIPSNYV) of c-Src .

In one embodiment of the invention, the peptides are referred to as c-Src-SH-1 and c-Src-SH2, respectively, and either c-Src-SH-1 or c- Src-SH-1 and c-Src-SH2 were mixed.

The peptide may be labeled with a fluorescent substance at the N terminus, if necessary. The fluorescent substance may include, but not limited to, TAMRA (carboxytetramethylrhodamine), Alexafluor 647, TMR (tetramethylrhodamine), rhodamine green Rhodamine Green, Alexa fluor 488, YOYO1, EVOblue 50, phycoerythrin (PE), Texas Red, TR, tetramethylrhodamine isothiocyanate (TRITC) Fluorescein carboxylic acid (FCA), fluorescein thiourea (FTH), acethocycoumarin-3-1, fluorescein-5-1, fluorescein-6- 1, 2 ', 7'-dichlorofluorescein -5-1, 2', 7'-dichlorofluorescein-6-1, dehydrotetramethylosamine -4-1, tetra Tetramethylrhodamine-5-1, and tetramethylrhodamine-6-1. There.

The SH3 peptide derived from Schizophyllan (SPG), which is the beta-1,3-glucan, and the c-Src comprising the amino acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2, .

The chemical bond may be covalent bond or noncovalent bond, but not limited thereto, and the noncovalent bond may be mediated through any noncovalent bonding force including hydrogen bonding, electrostatic (ion) bonding and / or van der Waals attraction .

On the other hand, the SH3 peptide derived from c-Src consisting of the above-mentioned beta-1,3-glucan, Schizophyllan (SPG) and the amino acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2 is a covalently bonded complex And may be a covalently bonded complex indirectly through a linker.

In one embodiment of the present invention, succinic anhydride is added to convert a hydroxyl group of Schizophyllan (SPG) into a carboxyl group to introduce a carboxyl group (-COOH group) at the terminal of Schizophyllan (SPG) And the amino group of the SH3 peptide derived from the c-Src peptide consisting of the amino acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2 is covalently bonded to the carboxyl group (-COOH group) of the above-mentioned zypho-pyran (SPG) to form a betaglucan- (Fig. 8).

The covalent bond may be formed by adding a crosslinker. Examples of the crosslinking agent include 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC; (3-dimethylaminopropyl) carbodiimide, N-Hydroxy succinimide (NHS), glutaraldehyde, NHS ester, maleimide, pyridyl disulfide, hydrazide and alkoxy amines have.

In another aspect, the present invention provides a pharmaceutical composition for the prophylaxis or treatment of an inflammatory disease comprising a beta-glucan-peptide complex as an active ingredient.

The beta-glucan-peptide complex is a form in which SH3 peptide derived from c-Src comprising the amino acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2 is covalently bonded to sipofilane (SPG) , p47phox can be targeted to inhibit the production of reactive oxygen species (ROS) and the production of the inflammatory cytokines TNF-a, IL-6, IL-l [beta] or IL-18.

In one embodiment of the present invention, CLP (cecal ligation procedure) -based polymicrobial sepsis and DSS (dextran sodium sulfate) -induced ulcerative colitis mice are administered in vivo Treatment of the animal model with SPG / C-Src-SH3 peptide complex (SPG / C-Src-SH3 peptide complex) significantly reduces mortality by modulating the excessive production of ROS and proinflammatory cytokines, And also improved the pathological response in the model (FIGS. 11 and 12).

Accordingly, the composition comprising the SPG / C-Src-SH3 peptide complex according to the present invention has an effect of reducing the mortality of sepsis and colitis animal models and significantly inhibiting the production of ROS and inflammatory cytokines, Inflammatory diseases such as colitis can be prevented or treated.

Specifically, the inflammatory diseases include, but are not limited to, sepsis, septic shock, inflammatory bowel disease (IBD), peritonitis, nephritis, acute bronchitis, chronic bronchitis, osteoarthritis, Chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, acute lung injury, and broncho-pulmonary dysplasia.

The inflammatory bowel disease (IBD) may be Ulcerative colitis (UC) or Crohns disease, although not limited thereto.

The term "prevention" in the present invention means all the actions of inhibiting or delaying the onset of an inflammatory disease by the administration of the pharmaceutical composition of the present invention. By "treatment" Of all inflammatory diseases are treated or benefited.

The pharmaceutical compositions according to the present invention may further comprise suitable carriers, excipients or diluents conventionally used in the production of pharmaceutical compositions.

Examples of the carrier, excipient or diluent which can be used in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

The composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method.

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose sucrose), lactose, gelatin, and the like.

In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.

The amount of the composition to be used may vary depending on the age, sex, and body weight of the patient, but may be administered in an amount of 1 to 100 mg / kg once to several times per day.

In addition, the dosage of such a composition may be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

The composition can be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

In yet another aspect, the invention provides a method of preparing a beta glucan-peptide complex for the prevention or treatment of an inflammatory disease, comprising:

(a) dissolving beta-1,3-glucan in an organic solvent and adding a cyclic anhydride to convert the hydroxy group of beta-1,3-glucan to a carboxyl group , Introducing a carboxyl group (-COOH group) at the beta-1,3-glucan terminal; And

(b) binding an amino group of a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and / or 2 with the carboxyl group (-COOH group) of the beta-1,3-glucan of step (a) to form a betaglucan- step.

In one embodiment of the present invention, Schizophyllan (SPG), a beta-1,3-glucan derived from Schizophyllum commune , is dissolved in an organic solvent and cyclic anhydride is added to form a carboxyl group (SPG) having a carboxyl terminal end and a SH3 peptide derived from c-Src composed of the amino acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2 The SPG / C-Src-SH3 peptide complex (SPG / C-Src-SH3 peptide complex) was prepared by covalently bonding an amine group (FIG.

The organic solvent includes, but is not limited to, dimethylsulfoxide (DMSO), methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, pyridine, N-dimethylformamide (DMF), 1,3-butylene glycol, propylene glycol, and mixed solvents thereof.

The cyclic anhydride may be, but is not limited to, succinic anhydride, maleic anhydride or glutaric anhydride.

The amino acid sequence of the SH3 peptide derived from c-Src is composed of amino acids 91 to 108 (SEQ ID NO: 1: ALYDYESRTETDLSFKKG) and 121 to 140 (SEQ ID NO: 2: WWLAHSLSTGQTGYIPSNYV) of c-Src.

(SEQ ID NO: 1) and SPG-c-Src-WWLAHSLSTGQTGYIPSNYV (SEQ ID NO: 2) in the SPG / C-Src-SH3 peptide complex Or more.

On the other hand, the step (b) may be carried out in the presence of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide NHS (N-Hydroxy succinimide).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

The betaglucan-peptide complex according to the present invention targets p47phox and significantly inhibits the production of reactive oxygen species (ROS) and inflammatory cytokines, and thus is useful as a pharmaceutical for the prevention or treatment of a fatal inflammatory disease associated with ROS such as sepsis and colitis May be useful as a composition. On the other hand, it can be useful for the development of new peptide-based drugs targeting p47phox.

FIG. 1 is a graph showing the results of Schizophyllan (SPG) blocking TLR4-ROS signal through inhibition of p47phox activation according to an embodiment of the present invention. After pretreatment with SPG at various concentrations (0, 1, 10, or 100 μg / ml) for 1 hour, the cells were incubated for 30 minutes, (B and F) for 18 hours, (C) D) for 4 hours, (E) 2 hours or (G right) for 15 minutes with LPS (100 ng / ml). (A) NADPH oxidase (NOX) activity of BMDM (bone marrow-derived macrophage) was confirmed by LPS stimulation in the presence of SPG (0, 1, 10, 100 ㎍ / ㎖). DHE and CM-H ₂ DCFDA fluorescence intensities were determined to detect O ^2- and H ₂ O ₂ production of BMDM, respectively (right). (B) Culture supernatant was collected and assayed for cytokine production by ELISA assay (enzyme-linked immunosorbent assay). (C, F and G) BMDM and then phosphorylated forms of MAPK (p42 / 44, p38 and SAPK / JNK), phosphorylated total forms of IκB-α (C), gp91phox, p22phox, Phosphorylated forms of p47phox and p67phox (F), p47phox (S304, S345, S359 and S370) (G) were confirmed by immunoblotting (IB) and actin was used as an internal control. (D) Co-transfection of pRL-TK Renilla plasmid (pRL-TK Renilla plasmid) for NF-κB reporter plasmid and normalization into RAW264.7 cells. . To perform the luciferase assay, cells were stimulated with LPS in the presence of zymogen (SPG) two days later. (E) Immunofluorescence microscopy was used to identify p65 (green) in BMDM, where the nuclei were stained with DAPI (blue), a DNA-intercalating dye.
FIG. 2 is a graph showing the results of measurement of the concentration of beta-1, 3-glucan, Zymosan, Curdlan AL, Scleroglucan, Effects of Laminarin on NOX (NADPH oxidase) activity and inflammatory cytokines TNF-α, IL-6 and IL-10 production in LPS-induced macrophages Fig. Zymosan, Curdlan AL, and Scrooglucan, which are beta-1,3-glucan at various concentrations (0, 1, 10 and 100 占 퐂 / Scleroglucan and Laminarin were pretreated with BMDM (Bone Marrow-derived Macrophage) for 1 hour or (B) for 18 hours and stimulated with LPS (100 ng / ml) for 30 minutes. (A) NADPH oxidase (NOX) activity of bone marrow-derived macrophage (BMDM) was confirmed by stimulation with LPS in the presence of beta-1,3-glucan (0, 1, 10, 100 ㎍ / ml). (B) Culture supernatant was collected and assayed for cytokine production by ELISA assay (enzyme-linked immunosorbent assay).
Figure 3 shows the results of p47phox interaction with C-Src and regulation of NOX (NADPH oxidase) assembly according to one embodiment of the present invention Fig. (A) The p47phox complex was treated with Flag-p47phox-expressing (expressed) THP-1, which had been treated with Schizophyllan (SPG; 10 g / ml) for 1 hour and stimulated with LPS for 30 minutes Lt; / RTI > Immunoprecipitation (IP) analysis of the p47phox complex was performed using an α-FLAG antibody conjugated with agarose beads and analyzed by mass spectrometry analysis. (B and C) THP-1 cells pretreated with Schizophyllan (SPG; 10 占 퐂 / ml) were treated with LPS (100 ng / ml) and stimulated for 15-30 minutes. After the cells were harvested, immunoprecipitation (IP) was performed on p47phox and phosphorylated forms of C-Src and p47phox (S304, S345, S359 and S370) (B) or gp91phox, p22phox , p47phox and p67phox (C) were confirmed by performing immunoblot (IB). (D) Binding mapping schematically shows the structure of p47phox and C-Src (above). 293T cells were treated with V5-C-Src or a mutant thereof (ΔSH2-KD, ΔKD) with a control vector flag -47 phox or 47 phox truncated mutant (ΔSH3-PRR, ΔAIR-PRR, ΔPRR) were co-transfected. The cells underwent co-immunoprecipitation (co-IP) with antibodies against V5 or a flag and then underwent immunoblot (IB) with a flag or antibody to V5 (Bottom, left). 293T cells were co-transfected with AU1-p67phox or AU1-p40phox with flag-p47phox increasing the amount of V5-C-Src and immunized with antibodies against the flag After immunoprecipitation (IP) was performed, the immunoblot (IB) was carried out with the antibody against the flag (Flag), AU1, V5 (lower right). Actin was used as an intrinsic control.
Figure 4 shows the results of C-Src interaction with p47phox and inhibition of binding with p67phox and p40phox according to one embodiment of the present invention. (A) p47phox and C-Src structures. (B) 293T cells were co-transfected with AU1-p67phox or AU1-p40phox co-transfected with flag-p47phox increasing the amount of V5-C-Src and immunized with antibodies against AU1 Immunoprecipitation (IP) was performed. Immunoblot (IB) was then performed with the antibody against Flag (Flag), AU1, V5, and actin was used as an internal control.
FIG. 5 is a graph showing the results of the prophylactic or therapeutic effects of systemic sepsis and colitis caused by treatment with Schizophyllan (SPG) according to an embodiment of the present invention. (A) Schematic diagram for constructing a CLP-guided animal model treated with zymogen (SPG) or a vehicle as a control (above). The survival rate of CLP-induced mice treated with orally administered 20 mg / kg of zyphofilane (SPG) per mouse was monitored for 7 days and the mortality rate was measured for 10 mice per group (below). (B) Hematoxylin-eosin staining results (left) representative of lung and spleen obtained 18 hours after induction of CLP in 5 mice per group (histopathology score) section) from the H & E staining results (right). (C) schematic diagram for constructing a 5% DSS-induced colitis animal model treated with zymogen (SPG) or Vehicle as a control (above). The survival rate of DSS-induced mice treated with zymogen (SPG) or Vehicle was monitored for 25 days. (Log-rank test) as compared to control mice. (D) Weight change of DSS-induced mice treated with zymogen (SPG) or Vehicle. (E) The clinical parameters of the proposed colitis animal model, such as weight loss, fecal concentration, and colonic hemorrhage, are shown in graph form. (H) (Hematoxylin-eosin) staining results (left) of the colon obtained on day 6 after DSS treatment in 5 mice per group (left). Histopathology scores are shown as middle and distal And from the H & E staining results of the colon section of the site (right).
FIG. 6 is a graph showing the results of the prophylactic or therapeutic effects of systemic sepsis and colitis caused by treatment with Schizophyllan (SPG) according to an embodiment of the present invention. (A) The body weight of mice treated with orally administered 20 mg / kg of zymoglobulin (SPG) per mouse or mice treated with vehicle as a control group was measured for 4 weeks (left). In addition, cytokine secretion levels were measured (right) in the serum obtained from the treatment with shisoplan (SPG) or control as a vehicle for 2 weeks. (B) The morphological differences of the spleen of CLP-induced mice treated with zymogen (SPG) or vehicle as a control were compared. Here, the UN is a test group that has not processed anything. (C) The survival rate of DSS-induced mice treated with Shizo pilana (SPG) or vehicle (Vehicle) was observed for 25 days (left) and weight change (right). (Left) and difference in length (right) of the colon of DSS-induced mice treated with Drosophila DPG (SPG) or vehicle as a control.
FIG. 7 is a graph showing a result of synthesis of a peptide coupled with SPG according to an embodiment of the present invention. FIG. (A) On the road showing the sequence alignment of C-Src, p67phox and p47phox, the boxes represent SH3-1 peptide and SH3-2 peptide of the present invention. (B) SPG (Schizophyllan), SGP / SA, Schizophyllan / succinic anhydride / 1-Ethyl-3 (3-dimethylaminopropyl) NHS) and SPG / SA / NHS / SH3 (Schizophyllan / succinic anhydride / 1-Ethyl-3 (3-dimethyl aminopropyl) carbodiimide (EDC) and N-Hydroxy succinimide Src-SH3 peptide complex). (C) SPG (Schizophyllan), SGP / SA (Schizophyllan / succinic anhydride) and SPG / SA / NHS / SH3 (Schizophyllan / succinic anhydride / 1-Ethyl- The results of ¹ H NMR spectra of the succinimide (NHS) / SH3 peptide (i.e., SPG / C-Src-SH3 peptide complex) are shown. (D) peptide of the present invention. Here, std is an abbreviation of standard. SH3-1 peptide or SH-2 peptide / SPG is a SH3 peptide conjugated SPG conjugated with SPG, SPG is dissolved in DMSO at a concentration of 1 mg / It is a Schizohpyllan. (E) Immunofluorescence micrographs of Raw264.7 cells against TAMRA-C-Src-SH3 peptides labeled with TAMRA. Immunofluorescence in Raw264.7 cells was performed by labeling C-Src-SH3 with TAMRA. Raw264.7 cells were treated with SPG / TAMRA-C-Src-SH3 peptide complex (10 μg / ml) for 1 hour and the intake of SPG / TAMRA-C-Src-SH3 peptide complexes was shown.
FIG. 8 is a graph showing the results of a process for preparing a beta-glucan-peptide complex by linking SPG (Schizohpyllan), which is beta-1,3-glucan, with SH3 peptide according to an embodiment of the present invention Fig. Specifically, the biological binding of SPG to functional peptides by cross linking using anhydride is illustrated and depicted.
FIG. 9 is a graph showing the results of blocking the TLR4-ROS signaling by the SPG / C-Src-SH3 peptide complex according to an embodiment of the present invention. (A) 293T cells were co-transfected with flag-p47phox for 12 hours with AU1-p67phox (left) or AU1-p40phox (right) (Scramble) peptide complex, SPG-SH3-1 peptide complex, SPG-SH3-2 peptide complex, or SPG-SH3-1 and SH3-2 peptide complexes as a control group for 6 hours Respectively. Immunoprecipitation (IP) was then performed with the antibody against the flag, followed by immunoblot (IB) with the Flag (Flag), the antibody against AU1. Actin was used as an intrinsic control. (Scramble) peptide complex, SPG-SH3-1 peptide complex, SPG-SH3-2 (SEQ ID NO: 2), which is a control group, and (B) flag (flag) -p47phox-expressing THP- Peptide complex, or the SPG-SH3-1 and SH3-2 peptide complexes were pretreated for 1 hour and stimulated with LPS (100 ng / ml) for 30 minutes. The cells were then harvested and immunoprecipitation (IP) was performed with the antibody against the flag and immunoblot (IB) as an antibody against p67phox, p40phox, p22phox, gp91phox or Flag (Flag) . Actin was used as an intrinsic control. (Scramble) peptide complex, SPG-SH3-1 peptide complex, SPG-SH3-2 (Raw264.7-Flag-p47phox) Peptide complexes or SPG-SH3-1 and SH3-2 peptide complexes and stimulated with LPS (100 ng / ml), followed by measurement of NOX (NADPH oxidase) activity. (D) A pRL-TK Renilla plasmid for NF-κB reporter plasmid and pRL-TK Renilla plasmid for normalization was cloned into Flag-p47phox-expressing Raw264.7 cells (Raw264 Gt; 7-Flag-p47phox). ≪ / RTI > After 2 days, the cells were treated with the SPG-SC (Scramble) peptide complex, the SPG-SH3-1 peptide complex, the SPG-SH3-2 peptide complex or the SPG-SH3-1 and SH3-2 peptide complexes , And stimulated with LPS, followed by luciferase assay to measure NK-κB activity. (Scramble) peptide complex, SPG-SH3-1 peptide complex, SPG-SH3-2 (E) flag (flag) -p47phox-expressing Raw264.7 cells (Raw264.7-Flag- p47phox) Peptide complex, or the SPG-SH3-1 and SH3-2 peptide complexes were pretreated for 1 hour and stimulated with LPS for 18 hours. The culture supernatant was then harvested and the enzyme immunoassay (ELISA) was performed to measure the amount of cytokine secreted.
FIG. 10 is a graph showing the effect of the SPG / C-Src-SH3 peptide complex on TLR (Toll-like receptor) and NLRP3 inflammation-mediated NOX assembly and ROS-mediated (ROS mediated pro-inflammatory signaling). ≪ Desc / Clms Page number 2 > SH3-1 peptide complex, SPG-SH3-2 peptide complex, or SPG-SH3-1 and SH3-1 peptide complexes (1, 5, 10 쨉 g / ml) in Raw264.7 and THP- 2 peptide complexes were cultured for 72 hours and MTT assays were performed to measure cell viability. (B) 293T cells were co-transfected with AU1-p67phox for 12 hours in a flag-p47phox, and treated with SPG-1 as a control (1, 5, 10 / / The SC (Scramble) peptide complex, the SPG-SH3-1 peptide complex, the SPG-SH3-2 peptide complex, or the SPG-SH3-1 and SH3-2 peptide complexes were treated for 6 hours. Immunoprecipitation (IP) was then performed with the antibody against the flag, followed by immunoblot (IB) with the Flag (Flag), the antibody against AU1. Actin was used as an intrinsic control. (Scramble) peptide complex, SPG-SH3-1 peptide complex, SPG-SH3-2 (Raw264.7-Flag-p47phox) Peptide complex, or the SPG-SH3-1 and SH3-2 peptide complexes were pretreated for 1 hour and stimulated with LPS (100 ng / ml) for 30 minutes. The cells were then harvested and immunoprecipitation (IP) was performed with the antibody against the flag and immunoblot (IB) as an antibody against p67phox, p40phox, p22phox, gp91phox or Flag (Flag) . Actin was used as an intrinsic control. (D) Treatment of Raw264.7 cells with the control SPG-SC (Scramble) peptide complex, SPG-SH3-1 peptide complex, SPG-SH3-2 peptide complex or SPG-SH3-1 and SH3-2 peptide complex And stimulated with LPS (100 ng / ml), and the activity of NOX (NADPH oxidase) was measured. (LPS-primed J774A.1-Flag-p47phox), which was stimulated for 4 hours with (E) LPS (100 ng / ml) ) Peptide complex, the SPG-SH3-1 peptide complex, the SPG-SH3-2 peptide complex, or the SPG-SH3-1 and SH3-2 peptide complexes were stimulated with Dextran Sulfate Sodium (DSS) for 18 hours. The culture supernatant was then harvested and the enzyme immunoassay (ELISA) was performed to measure the amount of cytokine secreted.
FIG. 11 is a graph showing the therapeutic effect of cecal ligation and puncture-induced polymicrobial sepsis according to the SPG / C-Src-SH3 peptide complex treatment according to an embodiment of the present invention. (A) a control group, SPG-SC (Scramble) peptide, SPG-SH3-1 peptide, SPG-SH3-2 peptide, or SPG-SH3-1 and SH3-2 peptide, This is a schematic diagram for model building (above). SPG-SC3 (Scramble) peptide, SPG-SH3-1 peptide, SPG-SH3-2 peptide, or SPG-SH3-1 and SH3-2 peptide at a dose of 10 mg / kg per mouse, PBS as a negative control Survival of peritoneal injected CLP-induced mice was observed for 7 days and mortality was measured for 20 mice per group (below). (Log-rank test) as compared to CLP-induced mice in which the control (control) SPG-SC (Scramble) peptide was administered. (D, n = 6), serum cytokine levels (C, n = 5), typical lung and spleen H & E staining (D, n = 5), splenocytes (E and F) used for immunoblot (IB) and bacterial counts (G, n = 8) were used as control SPG-SC (Scramble) peptide, CLP- , SPG-SH3-2 peptide, or SPG-SH3-1 and SH3-2 peptide, and PBS as a negative control group at 20 hours. (Scramble) peptide, SPG-SH3-1 peptide, SPG-SH3-2 peptide, or SPG-SH3-1 and SH3-2 peptide after CLP induction in 5 mice per group as a negative control (Hematoxylin-eosin) staining results (left) of lung and spleen obtained after 20 hours of treatment with PBS. The histopathology score was derived from the H & E staining results of the lung sections (Right). (E) Splenocytes were used to perform immunoblot (IB) and the expression levels of iNOS and COX-2 in spleen cells were confirmed and shown. Actin was used as an intrinsic control. (F) After harvesting the spleen cells, immunoprecipitation (IP) was performed with an antibody against p47phox, followed by immunoblotting (IB) with antibodies against p67phox, p40phox or p47phox. Actin was used as an intrinsic control. (G) The number of bacteria in the blood (left) and peritoneal fluid (middle) was measured and shown. In addition, the direct bacterial killing effect of the SPG / C-Src-SH3 peptide complex on E. coli DH5α on LB broth plates was measured and the number of surviving colonies was counted (right).
FIG. 12 shows the results of treatment of DSS (dextran sodium sulfate) -induced ulcerative colitis according to the SPG / C-Src-SH3 peptide complex treatment according to an embodiment of the present invention. (A) SPG-SC (Scramble) peptide, SPG-SH3-1 peptide, SPG-SH3-2 peptide, or SPG-SH3-1 and SH3-2 peptide as a control group and 5% DSS - Guideline for establishing an animal model of induced colitis (above). SPG-SC3 (Scramble) peptide, SPG-SH3-1 peptide, SPG-SH3-2 peptide, or SPG-SH3-1 and SH3-2 peptide at a dose of 10 mg / kg per mouse, PBS as a negative control Survival of peritoneal injected CLP-induced mice was monitored for 25 days and mortality was measured for 20 mice per group (below). (Log-rank test) as compared to DSS-induced mice receiving the control SPG-SC (Scramble) peptide. (B) Body weight changes of DSS-induced mice (n = 15) treated with SPG-SC (Scramble) peptide or SPG-SH3-1 and SH3-2 peptide complexes as controls were measured. (C) The clinical parameters of the proposed colitis mouse model (n = 15), such as weight loss, fecal concentration, colon bleeding, are graphically represented. (D, I), NO (NADPH oxidase) activity in colon homogenates (D, n = 6), cytokine levels (E, n = 5), and colon homogenates Colon homogenates (H and I) used for myeloperoxidase activity (F, n = 5), representative colon H & E staining (G, n = 5), immunoblot (IB) , SPG-SC3 (Scramble) peptide, SPG-SH3-1 peptide, SPG-SH3-2 peptide, or SPG-SH3-1 and SH3-2 peptide as a control group and PBS (UN; untreated) 6 days later. (Scramble) peptide, SPG-SH3-1 peptide, SPG-SH3-2 peptide, or SPG-SH3-1 and SH3-2 peptide after induction of DSS in 5 mice per group (G) The results of H & E (Hematoxylin-eosin) staining of the colon obtained on the 6th day after treatment with PBS were obtained. The histopathology score was derived from the H & E staining results of the colon section. (H and I) colon homogenates were precipitated with trichloroacetic acid to concentrate the proteins. Subsequently, immunoblot (IB) was performed (H) to confirm the expression levels of IL-1β, IL-18 and caspase-1 proteins and immunoprecipitation was performed with antibodies against p47phox. IP), followed by immunoblotting (IB) with antibodies to p67phox, p40phox or p47phox (I). Actin was used as an intrinsic control.

Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example  1: Preparation of experimental materials and experimental methods

Example  1-1: Cell preparation

The mouse macrophage cell line RAW264.7 (ATCC TIB-71; American Type Culture Collection) and HEK293T (ATCC-11268) cells were treated with 10% fetal bovine serum (FBS), sodium pyruvate ), Nonessential amino acids (NAA), penicillin G (100 IU / ml), and streptomycin (100 ug / ml) were inoculated in DMEM medium (Invitrogen). THP-1 (ATCC TIB-202) cells, a human monocytic cell line, were cultured using RPMI 1640-GlutaMAX ^™ medium containing 10% FBS. Transient transfection injection (transient transfection) are lipofectamine ^{® 3000 (Lipofectamine ® 3000; Invitrogen} ) according to the manufacturer's manual was performed using, or calcium phosphate (calcium phosphate) (Clontech). Stable cell lines of THP-1 and Raw264.7 were generated with 2 ug / ml of puromycin using the standard selection protocol.

Example  1-2: Reagent preparation

LPS ( Escherichia coli O111: B4), Zymosan (cell wall preparation of S. cerevisiae ), curdlan AL (curdlan AL ) obtained from Alcaligenes faecalis, sclerotium Scleroglucan obtained from Sclerotium rolfsii and Laminarin obtained from Laminaria digitata were purchased from Invivogen and were purchased from Schizophyllum commune ( Schizophyllum commune ) SPG) was obtained from QueGen Biotech. Succinic anhydride, DMSO, EDC (1-Ethyl-3- (3-dimethyl aminopropyl) carbodiimide) and NHS (N-Hydroxy succinimide) were purchased from Sigma-Aldrich Korea Respectively. Nanopure water and 25 mM MES (2- ( N- morpholino) ethanesulfonic acid buffer, pH 5.0) were used to synthesize SPG (Schizophyllan) -SH3 peptides.

Example  1-3: Construction of plasmid ( plasmid  construction)

Plasmids encoding NF-κB luciferase reporter plasmids encoding the full-length of p47phox (Flag-p47phox) are described in Jain A et al . , Nature protocols., 7 (3): 445-452 (2012) and Rice TW et al ., Critical care medicine., 38 (8): 1685-1694 (2010). Plasmids encoding other regions of p47phox (ΔSH3-PRR, ΔAIR-PRR, ΔPRR), p67phox, p40phox, cC-src and cC-src (ΔSH2-KD, ΔKD) and subcloning between Afl II and Xba I sites in pEF-IRES. The transient and stable expression in all mammalian cells was derived from the pEF-IRES-Puro expression vector (pEF-IRES-Puro expression vector). All constructs were sequenced using an ABI PRISM 377 automatic DNA sequencer to verify 100% homology with the original sequence.

Example  1-4: Immunoprecipitation  immune Blot  analysis

Immunoprecipitation and immunoblot assays were performed in accordance with Jain A et al ., Nature protocols. 7 (3): 445-452 (2012) and Rice TW et al ., Critical care medicine. 38 8): 1685-1694 (2010). Antibody binding was visualized using chemiluminescence (ECL: Millipore) and detected by Vilber chemiluminescence analyzer (Fusion SL 3; Vilber Lourmat).

Example  1-5: Peptides peptide preparation

C-Src-SH3 peptides were synthesized commercially and purified in acetate salt form to avoid abnormal reactions in pre-clinical studies (Peptron, Korea). The amino acid sequences of the peptides used in the present invention are shown in Table 1 below. On the other hand, C-Src-SH3 peptide was labeled with Fluorophore TAMRA at the N-terminus as needed.

Peptides (peptide) Amino acid sequence
(amino acid sequence, from N to C) SEQ ID NO: Src-SH3-1 (aa 91-108) ALYDYESRTETDLSFKKG One Src-SH3-2 (aa 121-140) WWLAHSLSTGQTGYIPSNYV 2 Src-SH3-1-Scramble ETRKESFYLKDSTAGYDL 3 Src-SH3-2-Scramble NGTHSSGVLQSLITYPWAWY 4

Example  1-6: At the end - COOH group  Have SPG ( COOH -terminated SPG ) synthesis

The synthesis was carried out according to a modified protocol reported in Hsu CC et al ., Biochemical pharmacology., 85 (3): 385-395 (2013). SPG (5 mg, 0.011 mM) was dissolved in DMSO for 6 hours with continuous mild agitation. A succinic anhydride solution (0.1 mM in DMSO) was added dropwise to the SPG (Schizophyllan) solution. The solution mixture was stirred at 50 < 0 > C for 12 hours and then the mixture was transferred to a dialysis membrane (MWCO: 6-8 kDa, Spectra / Por., USA). Dialysis was performed using deionized (DI) water for 3 days. Finally, the mixture was lyophilized and the remaining product was in the form of a sponge-like fiber. The presence of COOH and C = O on the surface was analyzed by FT-IR and ¹ H-NMR spectroscopy.

Example  1-7: At the end NHS  Have SPG ( NHS -terminated SPG ) synthesis

The carboxyl group terminal SPG (Schizophyllan) can react with an NHS functional group. SPG / SA (COOH-SPG, 5 mg) and EDC (10 mg; 0.1 mM) were dissolved in 5 ml of DMSO. Activation of cross-linked COOH-SPG with an EDC crosslinker was followed by the addition of NHS (10 mg; 0.2 mM) to the solution and the mixture was stirred at room temperature for 24 hours. The resulting mixture was then dialyzed for 3 days and lyophilized. The purified sample was in the form of a sticky gel. Fully lyophilized SPG / SA / EDC was again analyzed by FT-IR and ¹ H-NMR spectroscopy.

Example  1-8: Peptides and Combined SPG (peptide conjugated SPG ) Synthesis of

First, each of the peptides, Src-SH3-1 (aa 91-108) and Src-SH3-2 (aa 121-140) (4.5 mg) was dissolved in DMSO with stirring. SPG / SA (COOH-SPG, 5 mg) was dissolved in 1 ml of DMSO and adjusted to pH 6 with 4 ml of MES buffer. EDC (20 mg) and NHS (10 mg) were then added to the solution mixture and stirred at 4 < 0 > C for 24 hours. Finally, dialysis was performed using deionized (DI) water for three days with periodic replacement of the buffer solution (diaysate) every 6 hours. The reaction mixture was then lyophilized and stored at-20 C until use.

Example  1-9: Fluorescently labeled Peptides and Combined SPG (fluorescence tagged peptide conjugated SPG)

SPG / SA (COOG-SPG, 5 mg) was dissolved in 1 ml of DMSO and adjusted to pH 6 with 4 ml of MES buffer. EDC (10 mg) and NHS (10 mg) were then added to the solution mixture and reacted for 1 hour. Subsequently, fluorescently tagged peptide (1 mg) was added to the mixture and stored at 4 [deg.] C for 12 hours in the dark. Next, the lyophilized reaction mixture was lyophilized and the concentration was analyzed by integrating the absorbance spectra curve in the range of 250 to 450 nm.

Example  1-10: Characterization (characterization method)

The functional groups of the product were analyzed by FTIR (Fourier Transform IR) and ¹ H-NMR (Breker ARX 500 MHz spectrometer). The SPF / peptide characteristics were measured by the aabsorbance spectra of 200 μl of the SPG / peptide aliquot of the tagged-peptide (Synergy Mix, Bioteck Inc.). ). The absorbance spectrum of the peptide was integrated for each sample to calculate its concentration (the area under the curve between 250 and 450 nm).

Example  1-11: NOX ( NADPH oxidase ) Activity measurement

The intracellular superoxide production was measured by lucigenin-bis-N-methylacridinium nitrate-ECL method (Wadia JS et al ., Nature medicine., 10 (3): 310-315, 2004; Jaine et al. , Nature protocols., 7 (3): 445-452, 2012).

Example  1-12: Sepsis and colitis mouse animal model construction

A sepsis mouse model induced by CLP (cecal ligation and puncture) and an acute colitis mouse model induced by DSS (dextran sodium sulfate) were prepared using a 6-week-old C57BL / 6 female mouse (Samtako, Korea) Opal SM et al. , Jama ., 309 (11): 1154-1162 (2013) and Hotchkiss RS et al. , Journal of Immunology., 176 (9): 5471-5477 (2006). All animal related procedures were reviewed and approved by Hanyang University Animal Protection Agency and Usage Committee.

Example  1-13: Clinical score and histology

Clinical scores of colitis were measured daily for body weight, occult blood and total bleeding loss and fecal concentration during the period of colitis induction. Clinical scores were measured by two skilled investigators who were unaware of the treatment group (Huang et al ., International immunopharmacology ., 28 (1): 444-449, 2015). The spleen, lung, and colon of the mouse were fixed with 10% formalin and embedded in paraffin for immunohistochemistry of the tissue section. The paraffin section was cut to a thickness of 4 μm and H & E (hematoxylin and eosin) staining was performed. The histopathologic score was obtained from Osuchowski MF et al. , Journal of Immunology, 177 (3): 1967-74 (2006) and Liu W et al. , Cell research., 25 (6): 691-706 (2015).

Example  1-14: Miscellaneous procedures

Antibody assay, MTT assay, protein purification, mass spectrometry, luciferase assay, sandwich ELISA, confocal analysis, ROS measurement, bacterial count, MPO assay, and the like were performed according to a conventional method known in the art.

Example  1-15: Statistical analysis

Results from independent experiments (mean ± SD) were analyzed using a two-tailed Student's t-test. Differences were considered significant at p <0.05. For survival comparison, results were graphed and analyzed according to the Kaplan-Meier survival analysis, the product-limit method, using log-rank (Mantel-Cox) GraphPad Software).

Example  2: Experimental results

Example  2-1: The schizophyllan (SPG) p47phox Dephosphoric acid Through dephosphorylation, reactive oxygen species, ROS ) And inflammation. &Lt; / RTI &gt;

Beta-glucan is known to inhibit the LPS-induced inflammatory response in vitro and in vivo (Cohen J., Nature., 420 (6917): 885-891, 2002; Balk RA, Critical care clinics , 16 (2):.. 179-192, 2000; Dombrovskiy VY et.al., Critical care medicine, 35 (5):... 1244-1250, 2007; Martin GS et .al. , The New England Journal of Medicine. , 348 (16): 1546-1554, 2003). However, little is known about the intracellular regulatory mechanism of beta-glucan in macrophages. Identifying potential anti-inflammatory agents for Lipopolysaccharide / TLR4 (Toll-like receptor 4) stimulation, based on reactive oxygen species (ROS) and inflammation formation The inventors of the present invention firstly identified several beta-glucan forms obtained from several different sources, namely, schizophyllan (SPG), Zymosan, Curdlan AL, Experiments have confirmed whether glucan (Scleroglucan) and laminarin inhibit NOX (NADPH oxidase) activity and the production of inflammatory cytokines, TNF-α, IL-6 and IL-10 1, A, B, and 2).

As a result, Schizophyllan (SPG), a water-soluble β-glucan form of Schizophyllum commune , was found to be effective in NOX (NADPH oxidase) activity and activity in LPS-stimulated macrophages (Reactive Oxygen Species (ROS) and inflammation) (Fig. 1A and Fig. 1B).

Activation of mitogen-activated protein kinase (MAPK) / NF-κB signaling is essential in the response of macrophages to pro-inflammatory stimuli such as LPS and cytokines (Gay NJ et al ., Annual review of biochemistry., 76: 141-165, 2007). Thus, the present inventors have confirmed that Schizophyllan (SPG) affects MAPK / NK-κB signaling cascades in LPS-treated cells.

As a result, phosphorylation of p38 and ERK1 / 2 in a dose-dependent manner by pre-treatment of LPS-stimulated cells with Schizophyllan (SPG) (Down-regulation), but did not affect JNK / SAPK (FIG. 1C). In addition, it was confirmed that the degradation and phosphorylation of IκB-α were significantly inhibited by BMDM (bone marrow derived macrophage) pretreated with epidermal growth factor (SPG) in a concentration-dependent manner. In this regard, LPS-induced NF-κB luciferase promoter activity and nuclear translocation of NK-κB p65 were observed in BMG treated with zymogen (SPG) ) And also remarkably decreased (D in Fig. 1 and E in Fig. 1).

The production of NOX (NADPH oxidase) and reactive oxygen species (ROS) are regulated by the expression of NOX subunits (Lambeth JD., Nature Reviews Immunology. 4 (3): 181-189, 2004) Phosphorylation of several serine residues located in the C-terminal region (Ser-303 to Ser-379) of the p47phox regulatory subunit of Lambeth JD et al. , Annual review of pathology., 9: 119-145, 2014), the present inventors have determined whether LPS-induced NOX expression is regulated by zymogen (SPG) And confirmed through experiments.

As a result, the expression of LPS-induced NOX subunits in macrophages cultured by treatment of epidermal growth factor (SPG) with various concentrations (0, 1, 10, 100 ㎍ / (F in FIG. 1), phosphorylation of p47phox S345, S359, and S370 was completely inhibited by pretreatment with sipyoilane (SPG) (G in FIG. 1). Therefore, the above results show that Schizophyllan (SPG), a water-soluble β-glucan form of Schizophyllum commune , has been shown to inhibit p47phox activation and ROS-stimulated macrophage activation in LPS-stimulated macrophages, And negative regulation of inflammatory signaling (ROS-inflammatory signaling).

Example  2-2: p47phox  In macrophages, C- With Src  Interaction and regulation of NOX (NADPH oxidase) assembly.

To characterize the role of p47phox in TLR4 signaling mediated by schizophyllan (SPG), the present inventors have determined whether p47phox interacts with molecules involved in the regulation of reactive oxygen species (ROS) Were confirmed through experiments. Co-immunoprecipitation (co-IP) analysis of the p47phox complex by pretreatment with zyphopyran (SPG) was performed in LPS-stimulated cells. The purified p47phox complex was analyzed by mass spectrometry analysis. As a result, it was confirmed that C-Src, a 60-kDa protein, was associated with p47phox (FIG. 3A). The dephosphorylation of p47phox S345, S359 and S370 occurs during LPS stimulation in the presence of zyphopyran (SPG), which strongly interacts with endogenous p47phox, although C-Src is transient (15-30 minutes) And was confirmed by endogenous co-immunoprecipitation (co-IP) analysis (Fig. 3B). Interestingly, it was confirmed that the interaction of p47phox with gp91phox, p22phox, p67phox and p40phox was significantly inhibited after binding to C-Src in macrophages (Fig. 3B and Fig. 3C). These results indicate that C-Src can act as an immunomodulator by inhibiting the assembly of a p47phox-mediated NADPH oxidase complex.

Various truncated mutants of p47phox and C-src (van der Vliet A., Free radical biology & medicine., 44 (6): 938-955 (2008); Yang CS et al . , Journal of Immunology, 182 (6): 3696-3705 (2009)) confirmed that PX of p47phox is required for interaction with SH3 of C-Src in HEK 293T cells 3 D and FIG. 4). As the amount of C-Src was increased, a decrease in the interaction between p47phox and p67phox or p40phox in HEK 293T cells was confirmed by a competition assay using a C-Src construct (see D and 4).

These results suggest that C-Src acts as a negative regulator essential for ROS-mediated inflammatory responses through the binding of SH3 to the PX domain of p47phox and TLR4 The binding of SH3 to the PX domain of p47phox enhances the capacity of C-Src to compete with other NOXs (NADPH oxidase complex) for Toll-like receptor 4) signaling, schizophyllan, SPG).

Example  2-3: Effect of schizophyllan (SPG) in mouse animal models in systemic sepsis and colitis

Schizophyllan (SPG) To determine if it exhibits a protective effect against mouse animal models from septic shock due to polymicrobial peritonitis and sepsis, the present inventors have routinely performed studies on the pathogenesis of fatal human bacterial infection-induced systemic inflammation A cecal ligation and puncture mouse animal model similar to the human polymicrobial infection-induced systemic inflammatory response syndrome was used (Yang CS et al ., Cell host & microbe., 11 (3): 264- 276, 2012). First, in vivo The cytotoxicity of zyphopyran (SPG) was measured in the culture medium. There was no significant difference in body weight reduction and systemic inflammation between the group administered with orally administered shingo pill (SPG) for 28 days and the vehicle as a control (Fig. 6A). Next, we demonstrated the protective effect of zymogen (SPG) on mortality due to CLP in mouse animal models. Notably, in the case of CLP (cecal ligation and puncture) -induced mice, which had been pre-treated orally with zymoglobulin (SPG) at a dose of 20 mg / kg per mouse for 3 weeks, 70% Lt; RTI ID = 0.0 &gt; CLP &lt; / RTI &gt; induction. That is, it was confirmed that the mortality rate of CLP-induced mice pretreated with Shizo pilana (SPG) was remarkably reduced (Fig. 5A). On the other hand, the survival rate of CLP-induced mice was significantly reduced in the vehicle-treated control group.

Also, in accordance with the mortality results, H & E (Hematoxylin and Eosin) staining for spleen and lung tissue of CLP-induced mice treated with vehicle as a control group resulted in alveolar wall ) Showed severe pulmonary inflammation and hepatocyte (eosinophilia and pyknosis increase) and necrosis of splenocytes (karyorrhexis), both of which were thickened. However, CLP-induced mice treated with zymogen (SPG) significantly reduced such changes as severe lung inflammation and hepatocyte and splenocyte necrosis (Fig. 5B, left). Semiquantitative scoring of these histologic parameters can be used to detect not only the number and distribution of inflammatory cells in the tissue but also the number of inflammatory cells in the bronchiolar epithelium, Including non-inflammatory changes. In splenomegaly induced by CLP, the severity of sepsis in CLP-induced mice treated with zymogen (SPG) was significantly lower compared to vehicle-treated control CLP-induced mice (B in Fig. 5, right, and Fig. 6). From the above results, it can be seen that sipyoilan (SPG) plays a protective role in CLP-induced sepsis.

In addition, progenitor pilran (schizophyllan, SPG) is in vivo (DSS) (dextran sodium sulfate) -induced acute colitis mouse model was used to confirm further experimental results on whether it could inhibit gastrointestinal inflammation induced by DSS (dextran sodium sulfate). The DSS model has been shown to recite many clinical observations associated with human ulcerative colitis (UC) (Yang CS et al ., Cell host & microbe., 11 (3): 277- 89, 2012; Deretic V. et.al., Scientific American, 298 (5):.. 74-81, 2008; Deretic V. et.al., Cell host & microbe, 5 (6): 527-549, 2009). For the construction of acute colitis mouse model, mice were treated with 3% or 5% DSS for 6 days and the survival rate was evaluated for 25 days. Under these conditions, DSS-induced mice pretreated with zymogen (SPG) showed a significantly higher survival rate compared to DSS-treated mice treated with vehicle as a control (FIG. 5C and FIG. 6C). Consistent with the survival rate results, DSS-induced mice pretreated with zyphopyran (SPG) were associated with weight loss and clinical characteristics associated with the enhancement of gastrointestinal disease (consistnecy and rectal bleeding) And significantly increased the length of the colon compared to the DSS-induced control mice treated with vehicle (FIG. 5D, FIG. 5E, FIG. 6C and FIG. 6, D). This clinical evaluation was confirmed by histological examination of representative colon sections (Shibutani ST. Et al ., Nature immunology., 16 (10): 1014-1024, 2015). Semiquantitative scoring of these histologic parameters can be used to detect enterocyte loss, crypt inflammation, infiltration of neutrophils with lamina propria mononuclear cells, And elements such as epithelial hyperplasia. The severity of colitis in DSS-induced mice treated with zymogen (SPG) was found to be significantly lower compared to vehicle-treated control DSS-induced mice (Fig. 5F). This standard pathological test in DSS-induced mice indicated that the pathological changes during colitis induction were greatly improved by zymogen (SPG).

Taken together, these results indicate that epidermal filament (SPG) plays a protective role in polymicrobial sepsis and acute colitis. In other words, it was found that Shijo filan (SPG) has a preventive and therapeutic effect on polymicrobial sepsis and acute colitis.

Example  2-4: SPG / C- Src -SH3 Peptides  Complex SPG / C- Src -SH3 peptide complex)

Based drug delivery system based on schizophyllan (SPG), which is beta-1, 3-glucan (beta-1,3-glucan) the was designed to deliver the targeted (targeted) cells (Padkin A. et.al., Critical care medicine , 31 (9):... 2332-2338, 2003; Vincent JL et.al., Jama, 274 ( 8: 639-644, 1995; Janeway CA. et al., Annual review of immunology., 20: 197-216, 2002). Accordingly, the inventors have discovered that a conserved residue of p67phox and p47phox can be used to inhibit the interaction of p47phox with C-Src as a formation of a complex comprising schizophyllan (SPG) SH3 of the C-Src sequence was used (Fig. 7A and Table 1). The SPG / C-Src-SH3 peptide complex was prepared as described in Examples 1-5 to 1-10 above (Fig. 8). A series of crosslinking steps is required to conjugate the SPG and the peptide. Succinic anhydride (SA) (a form dissolved in DMSO) has converted the hydroxyl group of the SPG surface to carboxylic acid groups. After that, carboxylic acid terminated SPG having a carboxylic acid at the terminal was converted to a C═O bond at ~ 1723 cm ^-1 , which is not present in a conventional SPG (naive SPG), using FT-IR spectroscopy (Fig. 7B). In addition, ¹ H-NMR (300 MHz, DMSOD6) results confirmed the presence of carboxylic acid on the SPG surface, demonstrating an important resonance signal corresponding to 2.9 ppm of the succinic anhydride proton in SPG. In addition, the ¹ H-NMR spectrum of SPG / C-Src-SH3 shows characteristic shifts of 3.6 ppm and 6.9 to 7.5 ppm for the protons of tryptophan (Tryptophan, Trp; W) in SPG / c-Src-SH3 gave. On the other hand, cross-linking of functionalized SPG with carboxylic acid and crosslinking agent EDC / NHC formed amide bonds (FIG. 7C). Thereafter, analysis using FT-IR spectroscopy revealed an amide group at 1550 cm ^-1 . Thus, it has been demonstrated that the binding motif for the peptide has successfully conjugated with the crosslinker. Analysis of the final product, SPG / C-Src-SH3 (SPG / SA / NHS / SH3) using FT-IR spectroscopy showed that the final product was not only C = O, NC = O peak , The presence of other peaks involved in aromatic C = C stretch in tyrosine (Try; Y) and / or tryptophan (Tryptophan, Trp; W) B). The presence of the peptide in the mixture for the final product was also confirmed by UV-Vis Spectroscopy analysis (FIG. 7D). On the other hand, it was indirectly confirmed that SPG-conjugated peptide successfully conjugated by performing 4-15% gradient native gel electrophoresis. Further, immunofluorescence was performed to confirm that the SPG-SH3 peptide complexes were taken up by macrophages. As a result, it was confirmed that Raw267.4 cells more effectively ingested the SPG / TAMRA-C-Src-SH3 peptide complexes than the TAMRA-C-Src-SH3 peptide alone . Therefore, it was found from the above results that schizophyllan (SPG) plays an important role in the intracellular delivery of C-Src-SH3 peptide to macrophages.

Example  2-5: SPG / C- Src -SH3 Peptides  The complex NOX ( NADPH oxidase ) -Negative effect on NOX-mediated signaling.

The effect of schizophyllan (SPG) -SH3 peptides on pharmacological and biological profiles was investigated more specifically. First, the cytotoxicity of the peptide was measured in order to exclude the possibility of the inhibition effect from the cell growth inhibition. Cytotoxicity of Raw 264.7 cells and THP-1 cells treated with various concentrations of peptides (1, 5, 10 ㎍ / ml) and measured for cytotoxicity did not show any significant difference in the cell growth by the peptides, And did not show cytotoxicity by the peptides (Fig. 10A). Next, the possibility of whether the SH3-peptide affects the NOX (NADPH oxidase) assembly is experimentally confirmed. The interaction between p47phox-p67phox or p40phox during HEK 293T cells treated with SPG-SH3-1 peptide complex, SPG-SH3-2 peptide complex, SPG-SH3-1 and SH3-2 peptide complex and incubated for 6 hours . On the other hand, SPG-SC (Scramble) peptide complex used as a control and SPG alone treatment did not block the interaction between p47phox-p67phox or p40phox (Fig. 9A). In particular, it was confirmed that the interaction between p47phox-p67phox and p40phox was not blocked by the SH3 peptide alone, which was not conjugated with SPG. Thus, it was found that the intracellular delivery of C-Src-SH3 peptide was ineffective without SPG binding (FIG. 10B).

Following microbial infection or TLR activation, the NOX (NADPH oxidase) subunit is periodically activated by immune cells (e.g., macrophages and monocytes) and non-immune cells For example, endothelial cells and epithelial cells, and activates the cells to promote the production of reactive oxygen species (ROS) and inflammatory cytokines from them (van der Vliet A. et al .., Free radical biology & medicine , 44 (6): 938-955, 2008). In order to confirm whether the SPG-SH3 peptide complex of the present invention blocked TLR4-ROS signaling, SPP-SC (SEQ ID NO: 1), which is a control group for THP-1-Flag-p47phox cells and Raw264.7- Scramble peptide complex, SPG-SH3-1 peptide complex, SPG-SH3-2 peptide complex or SPG-SH3-1 and SH3-2 peptide complexes at various concentrations (1, 5, 10 / / And stimulated with LPS / TLR4 for 30 min. Then, immunoprecipitation (IP) was performed on Flag-p47phox complex and immunoblot (IB) was performed with various antibodies against p67phox, p40phox, p22phox and gp91phox. As a result, treatment with SPG-SH3 peptide significantly inhibited p47phox-mediated NADPH oxidase interaction and NOX assembly. In contrast, p47phox-mediated -NOX interaction and NOX assembly were not inhibited by the SPG-SC (Scramble) peptide complex used as a control (FIG. 9B and FIG. 10C). In addition, when SPG-SH3 peptide was treated, LPS-induced superoxide anion production, NOX activity, and NF-κB luciferase (NF-κB- luciferase promoter activity was significantly inhibited in a concentration-dependent manner (Fig. 9C, 9D and 10D). NOX signaling (NADPH oxidase signaling) is known to regulate the expression of pro-inflammatory cytokines (Gay NJ et al., Annual review of biochemistry., 76: 141-165, 2007 van der Vliet A. et al. , Free radical biology & medicine, 44 (6): 938-955, 2008). Therefore, in order to confirm whether the SPG-SH3 peptide complex of the present invention regulates the expression of pro-inflammatory cytokine, SPG-SC (Scramble) peptide complex, which is a control group in Raw 264.7 cell or J744A.1 cell, , SPG-SH3-1 peptide complex, SPG-SH3-2 peptide complex, or SPG-SH3-1 and SH3-2 peptide complexes at various concentrations (1, 5, 10 / / NLRP3 inflammatory modulators (nucleotide-binding domain, leucine-rich repeats containing family, pyrin domain-containing-3 inflammasome) inducers were stimulated. Then supernatant was collected and the amount of cytokine expressed was analyzed. As a result, compared to the case of treatment with SPG-SC (Scramble) peptide used as a control group, when treated with SPG-SH3 peptide, proinflammatory cytokines such as TNF-α, IL-6, IL-1β and IL- It was confirmed that production of pro-inflammatory cytokine was significantly inhibited (FIG. 9E and FIG. 10E). These results indicate that the SPG / C-Src-SH3 peptide complex specifically regulates ROS mediated pro-inflammatory signaling induced by the TLR4 and NLRP3 inflammatory regulatory complex (NLRP3 inflammasome) Can be used.

Example  2-6: Therapeutic effect of SPG / C-Src-SH3 peptide complex in mouse model of systemic sepsis mice

In order to confirm that the SPG / C-Src-SH3 peptide complex of the present invention shows a protective effect on an animal model of CLP (cecal ligation and puncture) -induced polymicrobial sepsis mouse, CLP The effect of SPG / C-Src-SH3 peptide complex on the mortality rate was tested. Specifically, SPG / C-Src-SH3-1 peptide, SPG / C-Src-SH3-2 peptide, or SPG / C-Src-SH3-1 peptide at a dose of 10 mg / And SH3-2 peptide were injected intraperitoneally three times at 3, 6 and 12 hours, and the survival rate of CLP-induced mice was confirmed. As a result, it was confirmed that 40% of the CLP-induced mice treated with the SPG-SH3-1 peptide or the SPG-SH3-2 peptide were protected from death due to CLP induction. Specifically, in the case of CLP-induced mice in which the SPG / C-Src-SH3-1 and SH3-2 peptides were intraperitoneally administered at a dose of 10 mg / kg per mouse, 80% of the mice were protected from death due to CLP induction Respectively. That is, it was confirmed that the mortality of the CLP-induced mouse treated with the SPG / C-Src-SH3 peptide complex was remarkably reduced (FIG. 11A). Also, consistent with the mortality results, the NOX activity and the serum levels of serum (in the spleen of the CLP-induced mice treated with the SPG / C-Src-SH3 peptide complexes as compared to the vehicle treated vehicle as the control group serum concentrations of pro-inflammatory cytokines such as TNF-α, IL-6, IL-1β and IL-18 were significantly reduced. However, the concentrations of IL-10 and IL-4 did not show a significant difference between the CLP-induced mouse group treated with the SPG / C-Src-SH3 peptide complex and the CLP-induced mouse group treated with vehicle as a control (Fig. 11B and Fig. 11C). H & E (Hematoxylin and Eosin) staining was performed on the spleen and lung tissues of CLP-induced mice treated with SPG / C-Src-SH3 peptide complex and CLP-induced mice treated with vehicle as a control group And semiquantitative scoring of histologic parameters was measured based on this. As a result, in the case of CLP-induced mice treated with the SPG / C-Src-SH3 peptide complex as compared to vehicle-treated control CLP-induced mice, the infiltration of immune cells was reduced, Spleen and pulmonary tissue were reduced, and iNOS and COX-2 protein were expressed at low levels in the spleen (FIG. 11D and FIG. 11E).

Next, the present inventors confirmed through experiments whether or not the SPG / C-Src-SH3 peptide complex has pharmacological activity in vivo . In the search for therapeutic agents to treat fatal inflammatory diseases, it is important to detect the NOX protein binding profile and activity in vivo when evaluating new peptides. In agreement with the in vitro and in vivo results shown in Figures 9B, 10C and 11B, the NOX protein binding profile (NOX) in the spleen of CLP-induced mice treated with the SPG / C-Src-SH3 peptide complex protein binding profile and activity of SPG / C-Src-SH3 peptides did not affect the degree of protein expression in splenocytes, but the binding of p47phox to p67phox or p40phox was markedly reduced by SPG / C-Src-SH3 peptide treatment (F in Fig. 11). On the other hand, CLP-induced mortality is positively associated with bacterial colony forming units (CFU) in peripheral blood and peritoneal fluid (Matsunaga K. et. al., Nature cell biology., 11 (4): 385-96, 2009), confirming that the SPG / C-Src-SH3 peptide of the present invention also improved bacterial elimination. Treatment of CLP-induced mice with SPG / C-Src-SH3 peptide resulted in a significant decrease in bacterial colony forming units (CFU) in peripheral blood and peritoneal fluid Respectively. In particular, treatment with SPG-SC (Scramble) peptide, a control group, did not kill the bacteria, but a direct bactericidal effect was observed when SPG / C-Src-SH3 peptide was used G in Fig. 11).

Therefore, it was confirmed from the above results that the SPG / C-Src-SH3 peptide complex had a therapeutic effect of alleviating the CLP-induced septicemia. Thus, the SPG / C- And thus can be used as a therapeutic agent for intractable inflammatory diseases.

Example  2-7: In colitis mouse animal models, SPG / C- Src -SH3 Peptic Therapeutic effect

In order to confirm that the SPG / C-Src-SH3 peptide complex of the present invention shows a protective effect against a mouse model of DSS (dextran sodium sulfate) -induced acute colitis mouse, first, The therapeutic effect of the SPG / C-Src-SH3 peptide complex on the mortality due to colitis was verified. Specifically, SPG / C-Src-SH3-1 peptide, SPG / C-Src-SH3-2 peptide, or SPG / C-Src-SH3-1 peptide at a dose of 10 mg / And SH3-2 peptide were intraperitoneally injected 5 times, and the survival rate of DSS-induced mice was confirmed. As a result, it was confirmed that 65% of the CLP-induced mice treated with the SPG / C-Src-SH3-1 and SH3-2 peptide complexes were protected from death due to CLP induction. That is, it was confirmed that the mortality of DSS-induced mice treated with the SPG / C-Src-SH3 peptide complexes was remarkably reduced (FIG. 12A). In addition, the DSS-induced mice treated with the SPG / C-Src-SH3-1 and SH3-2 peptide complexes compared with those treated with the SPG-SC (Scramble) peptide used as the control group, IL-6, IL-6, and IL-6 levels in weight loss and clinical characteristics (consistnecy and rectal bleeding), NOX activity, and colon homogenates associated with the enhancement of gastrointestinal disease (Fig. 12B to Fig. 12F) that the concentration of pro-inflammatory cytokines such as IL-1 [beta] and IL-18 and MPO (myeloperoxidase) activity were markedly decreased. CLP-induced mice treated with SPG / C-Src-SH3 peptide complex and H & E (Hematoxylin and Eosin) staining were performed on the colon tissue of DSS-induced mice treated with SPG-SC (Scramble) Based on this, semiquantitative scoring of histologic parameters was measured. As a result, the severity of colitis was reduced in DSS-induced mice treated with the SPG / C-Src-SH3 peptide complex as compared to the control DSS-induced mice treated with SPG-SC (Scramble) IL-1β, IL-18 and caspase-1 (G in FIG. 12 and H in FIG. 12).

Next, the present inventors confirmed through experiments whether or not the SPG / C-Src-SH3 peptide complex has pharmacological activity in vivo . Detecting the NOX protein binding profile and activity in vivo when evaluating new peptides is important in the search for therapeutic agents to treat fatal inflammatory diseases. Consistent with the in vivo results shown in Fig. 12D, the NOX protein binding profile and activity of the DSS-induced mouse colon treated with the SPG / C-Src-SH3 peptide complex , But the binding of p47phox to p67phox or p40phox was markedly reduced by treatment with SPG / C-Src-SH3 peptide (I of FIG. 12), although it did not affect the degree of protein expression in the colon.

Therefore, it was confirmed from the above results that the SPG / C-Src-SH3 peptide complex had a therapeutic effect of alleviating the DSS-induced colitis, and the SPG / C-Src-SH3 peptide complex of the present invention, It can be said that it can be usefully used as a therapeutic agent for inflammatory diseases such as inflammation.

<110> Industry-University Cooperation Foundation Hanyang University ERICA Campus <120> Betaglucan-peptide complexes, preparation method thereof, and          pharmaceutical composition for use in preventing or treating          inflammatory diseases containing the same active ingredient <130> P16U22D0509 <150> KR 10-2016-0009128 <151> 2016-01-26 <160> 4 <170> KoPatentin 3.0 <210> 1 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Src-SH3-1 (aa 91-108) <400> 1 Ala Leu Tyr Asp Tyr Glu Ser Arg Thr Glu Thr Asp Leu Ser Phe Lys   1 5 10 15 Lys Gly         <210> 2 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Src-SH3-2 (aa 121-140) <400> 2 Trp Trp Leu Ala His Ser Leu Ser Thr Gly Gln Thr Gly Tyr Ile Pro   1 5 10 15 Ser Asn Tyr Val              20 <210> 3 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Src-SH3-1-Scramble <400> 3 Glu Thr Arg Lys Glu Ser Phe Tyr Leu Lys Asp Ser Thr Ala Gly Tyr   1 5 10 15 Asp Leu         <210> 4 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Src-SH3-2-Scramble <400> 4 Asn Gly Thr His Ser Ser Gly Val Leu Gln Ser Leu Ile Thr Tyr Pro   1 5 10 15 Trp Ala Trp Tyr              20

Claims (13)

(I) beta-1,3-glucan, and (ii) a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and / or 2 which is linked to the beta-1,3- Beta glucan-peptide complex for the prevention or treatment of inflammatory disease.
The beta-glucan-peptide complex according to claim 1, wherein the beta-1,3-glucan is schizophyllan (SPG).
2. The beta-glucan-peptide complex according to claim 1, wherein the beta-1,3-glucan and the peptide consisting of the amino acid sequence of SEQ ID NO: 1 and / or 2 are bound to each other by chemical bonding.
4. The method according to claim 3, wherein the beta-1,3-glucan is schizophyllan (SPG), and the binding is selected from the group consisting of a carboxyl group (-COOH group) introduced at the beta-1,3- (-NH2 group). &Lt; / RTI &gt;
A pharmaceutical composition for preventing or treating an inflammatory disease comprising the beta-glucan-peptide complex according to any one of claims 1 to 4 as an active ingredient.
6. The method of claim 5, wherein the beta-glucan-peptide complex targets p47phox and inhibits production of reactive oxygen species (ROS) and inhibits the production of inflammatory cytokines TNF-a, IL-6, IL- &Lt; / RTI &gt;
6. The method of claim 5, wherein the inflammatory disease is selected from the group consisting of sepsis, septic shock, inflammatory bowel disease (IBD), peritonitis, nephritis, acute bronchitis, chronic bronchitis, osteoarthritis, wherein the composition is any one selected from the group consisting of chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, acute lung injury, and broncho-pulmonary dysplasia.
The pharmaceutical composition according to claim 7, wherein the inflammatory bowel disease (IBD) is Ulcerative colitis (UC) or Crohns disease.
(a) dissolving beta-1,3-glucan in an organic solvent and adding a cyclic anhydride to convert the hydroxy group of beta-1,3-glucan to a carboxyl group , Introducing a carboxyl group (-COOH group) at the beta-1,3-glucan terminal; And
(b) binding an amino group of a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and / or 2 with the carboxyl group (-COOH group) of the beta-1,3-glucan of step (a) to form a betaglucan- Peptide complex for the prophylaxis or treatment of an inflammatory disease.
10. The method of claim 9, wherein the beta-1,3-glucan is schizophyllan (SPG).
10. The process of claim 9, wherein the organic solvent of step (a) is selected from the group consisting of dimethylsulfoxide (DMSO), methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, Wherein the solvent is selected from the group consisting of pyridine, N, N-dimethylformamide (DMF), 1,3-butylene glycol, propylene glycol and mixed solvents thereof.
10. The method of claim 9, wherein the cyclic anhydride of step (a) is succinic anhydride, maleic anhydride or glutaric anhydride.
10. The method of claim 9, wherein step (b) comprises reacting 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) Wherein the reaction is carried out by a reaction between N-hydroxysuccinimide (NHS).

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