KR102109744B1 - A composition comprising decursin derivative for preventing or treating sepsis or septic shock - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating sepsis or septic shock containing a decursin derivative represented by chemical formula 1 as an active component. The compound has an excellent effect of reducing the expression of HMGB1, a major mediator of sepsis, and particularly has an excellent effect of increasing a survival rate of an animal model in which sepsis is induced, thereby being able to be usefully used as a pharmaceutical composition for preventing or treating sepsis or septic shock.

Description

A composition comprising decursin derivative for preventing or treating sepsis or septic shock

The present invention relates to a composition for the prevention or treatment of sepsis or septic shock comprising a deckersin derivative.

Sepsis is a condition in which a microbial infection causes severe inflammatory reactions throughout the body. Fever symptoms of body temperature rising above 38 degrees, hypothermia falling below 36 degrees, respiratory rate increased to more than 24 times per minute (empty breathing), heart rate of more than 90 times per minute (tachycardia), increased or significantly decreased white blood cell counts on blood tests The case of having more than one symptom is called systemic inflammatory response syndrome, and is called sepsis when the systemic inflammatory response syndrome is caused by infection of a microorganism. Sepsis can potentially cause septic shock, which results in poor functioning of various organs in the body (heart, kidneys, liver, brain, lungs, etc.) and severe shock. In some cases, the path of infection of microorganisms is not well known, but appendicitis, otitis media, dermatitis, bedsores, lung disease, cholecystitis, pyelonephritis, osteomyelitis are known as the causes of sepsis.

To date, the underlying treatment for the treatment of sepsis has not been identified and is mainly treated with injections of antibiotics or antifungal agents. Treatment medication and duration may be determined depending on the type of microorganism or hemodialysis or transfusion may be performed depending on the patient's condition. When antibiotics and antifungal drugs are well received, septicemia may be cured, but patients may be difficult to treat, such as those infected with drug-resistant microorganisms, patients with weak immunity, or starting treatment too late.

The mortality rate of sepsis is very high, about 50 to 70%, and it is known worldwide that it accounts for a high proportion of the causes of death. Drotrecogin alfa (Xigris), the only FDA-approved treatment for sepsis, has been found to be ineffective, and there have been no serious sepsis treatments approved to date since it was removed from the market in 2011 (Ranieri, VM et. al., N Engl J Med., 366 (22), 2055-2064, 2012). Therefore, the development of a therapeutic agent for sepsis has significant clinical implications, and research has been actively conducted to develop a therapeutic agent for sepsis at home and abroad.

High mobility group box 1 (HMGB1), like histones, is one of the most important chromatin proteins and is expressed in vascular endothelium when there is vascular damage such as sepsis, monocytes, macrophage, and humans. It is secreted by human umbilical vein endothelial cells.

Receptor for advanced glycation end products (RAGE), TLR2 (Toll like receptor 2) and TLR4 (Toll like receptor 4) are known as receptors for HMGB1. The adhesion molecule (VCAM-1, ICAM-1, E-selectin) on endothelial cells is induced by the action of the receptor and HMGB1, which promotes inflammation through the recruitment of white blood cells (Bae, JS et al. , Blood, 118 (14), 3952-3959, 2011). It can be measured in most patients up to a week after the diagnosis of sepsis or septic shock, and the level of HMGB1 is associated with the degree of organ dysfunction (Gibot, S. et al., Intensive Care Med., 33 ( 8), 1347-1353, 2007; Sunden-Cullberg, J. et al., Crit Care Med., 33 (3), 564-573, 2005).

It is known that HMGB1 is detected in the serum of sepsis patients, and that serum levels of HMGB1 are significantly increased in patients with bad prognosis, and it is known that an increase in the concentration of HMGB1 in the plasma of severe sepsis patients is likely to result in death. HMGB1 is mainly used as a target factor for the prevention or treatment of (Lee, W. et al., Toxicol Appl Pharmacol., 262 (1), 91-98, 2012; Yang, EJ et al., J Cell Physiol. , 228 (5), 975-982, 2013; Wang, H. et al., Science, 285 (5425), 248-251, 1999).

Decursin and decursinol angelate are present in Korean native beetles, tribes, and body herbs. These are anti-tumor effect, bacterial growth suppression effect, circulatory system disease improvement effect, metabolic enzyme suppression effect, anti-inflammatory effect, antioxidant effect, cognitive improvement promotion effect, skin elasticity improvement effect, skin wound treatment effect, skin whitening effect (other than hand chooyoung, medicine Journal, 53 (6), 303-313, 2009; Korean Registered Patent No. 10-1575897; Korean Registered Patent No. 10-0379191; Korean Patent Publication No. 10-2011-0087803) .

On the other hand, as a prior art related to the composition for the treatment of sepsis of deckersine derivatives, the prior paper [Jung, JS et al., Korean J Physiol Pharmacol., 12 (2), 79-81, 2008] treats sepsis of sepsis The effect was disclosed, Korean Patent Publication No. 10-2002-0029462 discloses the effect of preventing and treating septic shock of dekersinol, and Korean Registered Patent No. 10-1215379 has an anti-inflammatory effect comprising deckersinol derivatives. It was started.

However, there has been no previous report confirming the effect of treating sepsis for the deckercin derivative compound represented by Formula 1 as in the present invention, and in particular, confirming that the composition has an excellent effect of treating sepsis compared to deckersinol.

Korean Registered Patent No. 10-1575897, cosmetic or pharmaceutical composition containing decursin as an active ingredient, registered on December 02, 2015. Korean Patent Registration No. 10-0379191, New Use of Deckercin as Melanin Synthesis Inhibitor, Registered on March 26, 2003. Korean Registered Patent No. 10-1215379, Pharmaceutical Composition Containing Deckersinol Derivative, Registered on December 18, 2012. Korean Patent Publication No. 10-2002-0029462, a composition for preventing and treating septic shock, including deckersinol, published on April 19, 2002. Korean Patent Publication No. 10-2011-0087803, a skin wound treatment agent containing deckersin and deckersinol angelate, published on August 03, 2011.

Chu-Young Son, et al. Angelica gigas Nakai Extract for New Drug Development Decursin and Decursinol Angelate Pharmacological Action, Journal of Pharmacy, 53 (6), 303-313, 2009. Bae, J. S. et al., Activated protein C inhibits high mobility group box 1 signaling in endothelial cells, Blood, 118 (14), 3952-3959, 2011. Bae, J. S. et al., Transforming growth factor β-induced protein promotes severe vascular inflammatory responses, Am J Respir Crit Care Med., 189 (7), 779-786, 2014. Gibot, S. et al., High-mobility group box 1 protein plasma concentrations during septic shock, Intensive Care Med., 33 (8), 1347-1353, 2007. Jung, B. et al., Anti-septic effects of dabrafenib on HMGB1-mediated inflammatory responses, BMB Rep., 49 (4), 214-219, 2016. Jung, B. et al., Suppressive effects of three diketopiperazines from marine-derived bacteria on TGFBIp-mediated septic responses in human endothelial cells and mice, Arch Pharm Res., 39 (6), 843-854, 2016. Jung, J. S. et al., Protective effect of decursinol on mouse models of sepsis: enhancement of interleukin-10, Korean J Physiol Pharmacol., 12 (2), 79-81, 2008. Kim, J. et al., ROS homeostasis and metabolism: a critical liaison for cancer therapy, Exp Mol Med., 48 (11), e269, 2016. Lee, W. et al., Barrier protective effects of withaferin A in HMGB1-induced inflammatory responses in both cellular and animal models, Toxicol Appl Pharmacol., 262 (1), 91-98, 2012. Ranieri, V. M. et al., Drotrecogin alfa (activated) in adults with septic shock, N Engl J Med., 366 (22), 2055-2064, 2012. Sunden-Cullberg, J. et al., Persistent elevation of high mobility group box-1 protein (HMGB1) in patients with severe sepsis and septic shock, Crit Care Med., 33 (3), 564-573, 2005. Wang, H. et al., HMG-1 as a late mediator of endotoxin lethality in mice, Science, 285 (5425), 248-251, 1999. Wang, H. et al., Cholinergic agonists inhibit HMGB1 release and improve survival in experimental sepsis, Nat Med., 10 (11), 1216-1221, 2004. Yang, E. J. et al., Barrier protective effects of rosmarinic acid on HMGB1-induced inflammatory responses in vitro and in vivo, J Cell Physiol., 228 (5), 975-982, 2013.

An object of the present invention is to provide a composition for the prevention or treatment of sepsis or septic shock comprising a deckersine derivative represented by Formula 1.

The present invention relates to a pharmaceutical composition for the prevention or treatment of sepsis or septic shock comprising the compound represented by Formula 1 as an active ingredient.

[Formula 1]

The compound is included in the present invention without limitation, as long as it is a method that can be prepared according to a conventional method in the art, as well as a pharmaceutically acceptable salt.

In addition, the compound is excellent in inhibiting the expression of HMGB1 (high mobility group box 1).

The pharmaceutical composition according to the present invention can be formulated in a suitable form together with a commonly used pharmaceutically acceptable carrier. “Pharmaceutically acceptable” refers to a composition that is physiologically acceptable and does not cause allergic reactions or similar reactions, such as gastrointestinal disorders, dizziness, etc., when administered to humans. In addition, the composition may be formulated and used in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., according to a conventional method.

Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl paraoxybenzoate, propyl paraoxybenzoate, talc, magnesium stearate, and mineral oil may be included, but is not limited thereto. In the case of formulation, it is prepared using diluents or excipients such as fillers, stabilizers, binders, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient in the compounds of the present invention, for example, starch, microcrystalline cellulose, sucrose or lactose, It is prepared by mixing low-substituted hydroxypropyl cellulose and hypromellose. Also, in addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, intravenous solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used, various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, can be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin, glycerol, gelatin, etc. may be used. Adjuvants such as sterile and / or preservatives, stabilizers, hydrating or emulsifying accelerators, salts and / or buffers for osmotic pressure control, and other treatments of the compound or a pharmaceutically acceptable salt thereof for formulation into a formulation for parenteral administration It can be prepared as a solution or suspension by mixing in water with a useful substance, and it can be prepared as an ampoule or vial unit dosage form.

The pharmaceutical composition comprising the compound of the present invention as an active ingredient can be administered to various mammals, such as rats, livestock, and humans. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dura mater or intracranial injection. The dosage is the age, gender, weight of the subject to be treated, the specific disease or pathology to be treated, the severity of the disease or pathology, the time of administration, the route of administration, the absorption, distribution and excretion rate of the drug, the type of other drug used and the prescriber's It will depend on the judgment. Dosage determination based on these factors is within the level of those skilled in the art, and dosages generally range from 0.01 mg / kg / day to approximately 2000 mg / kg / day. A more preferred dosage is 1 mg / kg / day to 500 mg / kg / day. The administration may be administered once a day, or may be divided into several times. The above dosage does not limit the scope of the present invention in any way.

In another aspect, the present invention provides a health functional food for preventing or improving sepsis or septic shock comprising a compound represented by Formula 1 and a food-acceptable food supplement additive. The health functional food refers to food manufactured or processed using ingredients or ingredients having useful functionality, and includes, for example, health supplements, functional foods, nutritional supplements, and supplements.

The compound may be added in an amount of preferably 0.001% to 50% by weight, more preferably 0.001% to 30% by weight, and most preferably 0.001% to 10% by weight relative to the total weight of the dietary supplement. have. The health functional food of the present invention includes tablets, capsules, pills or liquids, and foods to which the compounds of the present invention can be added include, for example, various foods, beverages, gums, teas, vitamin complexes, etc. There is this.

The present invention relates to a pharmaceutical composition for the prevention or treatment of sepsis or septic shock comprising the deckersine derivative represented by Formula 1 as an active ingredient. The compound has an excellent effect of reducing the expression of HMGB1, a major mediator of sepsis, and in particular has an excellent effect of increasing the survival rate of the animal model in which sepsis is induced, and thus can be usefully used as a pharmaceutical composition for the prevention or treatment of sepsis or septic shock. have.

1 is a result of confirming the cell viability according to the concentration of the compound 1 of the present invention in human umbilical vein endothelial cells.
Figure 2A is a result of confirming the protein expression level of HMGB1 receptors (TLR2, TLR4 and RAGE) according to the concentration of compound 1 of the present invention in human umbilical vein endothelial cells treated with HMGB1 by ELISA, Figure 2B is HMGB1 treated This is a result of confirming the protein expression level of the adhesion molecules (VCAM-1, ICAM-1 and E-selectin) according to the treatment of each concentration of Compound 1 of the present invention in human umbilical vein endothelial cells by ELISA.
3 is a result of confirming HMGB1 protein expression according to the concentration-specific treatment of the present invention in the animal model of sepsis induced by CLP surgery.
Figure 4 is a result confirming the survival rate according to the concentration of the compound 1 of the present invention in the animal model of sepsis induced by CLP surgery.
5 is a result confirming the numerical changes of AST, ALT, creatinine, BUN and LDH according to the treatment of Compound 1 of the present invention in an animal model in which sepsis is induced by CLP surgery.

Hereinafter, a preferred embodiment 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 contents introduced herein are thorough and complete, and are provided to sufficiently convey the spirit of the present invention to those skilled in the art.

<Example 1. Preparation of deckersine derivatives>

(S)-(+)-Deckersinol (0.406mmol, 1eq) dissolved in anhydrous dichloromethane is trans-3- (3-pyridyl) acrylic acid (0.609mmol, 1.5eq), 1-ethyl-3- ( Mixed with 3-dimethylaminopropyl) carbodiimide (EDC, 0.812mmol, 2eq) and 4-dimethylaminopyridine (4-DMAP, 0.162mmol, 0.4eq) and stirred at room temperature for 12 hours. The mixture obtained from the above process was concentrated under reduced pressure, and the residue was purified by flash silica gel column chromatography to obtain (7S)-(+)-3- (3-pyridyl) -acrylic acid of the following Chemical Formula 1, 8,8-dimethyl- 2-oxo-6,7-dihydro-2H, 8Hpyrano [3,2-g] chromen-7-yl-ester ((7S)-(+)-3- (3-Pyridyl) -acrylic acid , 8,8-dimethyl-2-oxo-6,7-dihydro-2H, 8Hpyrano [3,2-g] chromen-7-yl-ester, compound 1), and its physicochemical data are as follows. .

[Formula 1]

Yield 96.7%, white solid, mp: 105 ° C, Rf = 0.24 (1: 1 n-hexane-ethyl acetate);

+ 48.5 (c = 3, CHCl₃);

+ 48.5 (c = 3, CHCl ₃ );

¹ H NMR (400 MHz, CDCl ₃ ): δ _H 8.72 (1H, d, J = 2.2 Hz, H-5 '), 8.61 (1H, dd, J = 1.5, 4.9 Hz, H-7'), 7.82 (1H, d, J = 8.4 Hz, H-9 '), 7.67 (1H, d, J = 16.0 Hz, H-3'), 7.59 (1H, d, J = 9.6 Hz, H-4), 7.33 (1H, dd, J = 4.8, 8.0 Hz, H-8 '), 7.18 (1H, s, H-5), 6.84 (1H, s, H-10), 6.50 (1H, d, J = 16.4 Hz , H-2 ′), 6.24 (1H, d, J = 9.6 Hz, H-3), 5.22 (1H, t, J = 4.8 Hz, H-7), 3.26 (1H, dd, J = 4.8, 17.2 Hz, H-6a), 2.95 (1H, dd, J = 4.8, 17.2 Hz, H-6b), 1.44 (3H, s, CH ₃ -8), 1.40 (3H, s, CH ₃ -8);

¹³ C NMR (100 MHz, CDCl ₃ ): δ _C 165.6 (C-1 '), 161.2 (C-2), 156.3 (C-9a), 154.2 (C-10a), 151.2 (C-5'), 149.8 (C-7 ′), 143.1 (C-4), 142.1 (C-3 ′), 134.2 (C-9 ′), 129.8 (C-4 ′), 128.7 (C-5), 123.7 (C- 8 ′), 119.5 (C-2 ′), 115.5 (C-5a), 113.4 (C-3), 112.9 (C-4a), 104.8 (C-10), 76.5 (C-8), 70.5 (C -7), 27.8 (C-6), 24.9 (CH ₃ -8), 23.4 (CH ₃ -8);

IT-TOF / MS: m / z = 378.1325 [M + H] ⁺ , 400.1129 [M + Na] ⁺ .

<Example 2. Cytotoxicity confirmation>

MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazoliumbromide) on the cytotoxicity of the treatment of dekersin derivatives represented by Chemical Formula 1 (compound 1) in human umbilical vein endothelial cells It was confirmed by an assay method.

First, human umbilical vein endothelial cells (Primary HUVECs, Cambrex Bioscience, Charles City, IA) were published in a previous article [Jung, B. et al., BMB Rep., 49 (4), 214-219, 2016; Kim, J. et al., Exp Mol Med., 48 (11), e269, 2016], and was used for experiments by passage 3 ~ 5 times.

Next, the subcultured cells were dispensed at 5 × 10 ³ cells per well in a 96-well plate and cultured for 24 hours, followed by washing with a new medium and displaying Compound 1 represented by Formula 1 prepared in Example 1 (( 7S)-(+)-3- (3-pyridyl) -acrylic acid, 8,8-dimethyl-2-oxo-6,7-dihydro-2H, 8H pyrano [3,2-g] chromen- 7-day-ester) was treated for 48 hours by concentration (0, 10, 20 and 50 μM). After 48 hours, the cells were washed and then reacted for 4 hours by adding 100 µl 1mg / ml MTT solution. Then, 150 μl of DMSO was added to dissolve the formazanitis produced in the cells, and absorbance was measured at 540 nm with a Microplate reader (Tecan Austria GmbH, Austria). The cell viability was calculated from the absorbance measurement results and is shown in FIG. 1.

Referring to FIG. 1, when treating the human umbilical vein endothelial cell with compound 1, a deckersine derivative represented by Chemical Formula 1, it is a substance that does not exhibit cytotoxicity by exhibiting a cell survival rate equivalent to that of a control group (untreated group) regardless of concentration. I could confirm.

<Example 3. Confirmation of antiseptic effect in human umbilical vein endothelial cells>

Cell surface marker receptors (RAGE, TLR2 and TLR4) and cell adhesion to the anti-septic effect of treatment with dekersin derivatives represented by Chemical Formula 1 (compound 1) in human umbilical vein endothelial cells treated with HMGB1 and induced sepsis It was confirmed by changes in protein expression of molecules (VCAM-1, ICAM-1 and E-selectin).

Receptor for advanced glycation end products (RAGE), a receptor for HMGB1, toll-like receptor 2 (TLL2) and protein-like expression level of TLR4 (Toll like receptor 4), and VCAM-, an adhesion molecule induced by HMGB1 and receptor interaction The expression levels of proteins of 1 (vascular cell adhesion molecule-1), ICAM-1 (intercellular adhesion molecule-1) and E-selectin are described in the literature [Jung, B. et al., BMB Rep., 49 (4), 214-219, 2016; Jung, B. et al., Arch Pharm Res., 39 (6), 843-854, 2016].

When the human umbilical vein endothelial cells passaged in Example 2 form a monolayer, HMGB1 (1 µg / ml) is 16 hours (RAGE, TLR2, TLR4, VCAM-1 and ICAM-1) or 22 hours (E -selectin). HMGB1 was treated with the compound 1 of the present invention in concentration-treated cells (0, 10, 20 μM), and then fixed with 1% (w / v) paraformaldehyde.

Then, after removing the paraformaldehyde and washing the cells three times, the antibody (TLR2 antibody: A-9, TLR4 antibody: H-80, RAGE antibody: A-9, Santa Cruz, CA; VCAM-1, ICAM-1 , E-selectin, 1:50 each, Temecula, CA), and reacted at 37 ° C and 5% CO ₂ for 1 hour. After the reaction, the cells were washed and then treated with peroxidase-conjugated antimouse IgG antibody (Sigma, Saint Louis, MO) for 1 hour. After washing three times and reacting with an o-phenylenediamene substrate (o-phenylenediamene substrate, Sigma, St. Louis, MO), absorbance at 490 nm was measured. Changes in protein expression confirmed from absorbance measurements are shown in FIGS. 2A and 2B.

2A and 2B, when HMGB1 is treated to induce septicemia, human umbilical vein endothelial cells of the present invention are treated with the expression of TLR2, TLR4 and RAGE, receptors of HMGB1, and the interaction of HMGB1 with receptors when Compound 1 of the present invention is treated. It was confirmed that the expression of the induced adhesion molecules VCAM-1, ICAM-1 and E-selection was inhibited in a concentration-dependent manner.

From this, the present invention represented by the formula (1) derivatives (compound 1) HMGB1 is treated with HMGB1 in human umbilical vein endothelial cells, the effect of suppressing the receptor expression of HMGB1 is excellent, and HMGB1 and the action of the receptor Since the inhibitory effect is excellent, it was found that it can be usefully used as a composition for the treatment of sepsis.

<Example 4. Confirmation of anti-septic effect in animal model induced by sepsis>

In the animal model in which sepsis was induced by CLP surgery, the anti-septic effect of treatment with the dekersin derivative represented by Chemical Formula 1 (Compound 1) was changed in HMGB1 protein expression, survival rate, histopathology, organ damage, and thyroid hormone change. It was confirmed by.

Example 4-1. Confirmation of HMGB1 expression change

First, in order to induce septicemia in an animal model, the prior literature [Wang, H. et al., Nat Med., 10 (11), 1216-1221, 2004; Bae, JS et al., Am J Respir Crit Care Med., 189 (7), 779-786, 2014] was used for the CLP surgery (cecal ligation and puncture operation). It was handled with the approval of the committee (IRB No. KNU 2017-102).

Male C57BL / 6 mice (6-7 weeks of age, weight 27 g, Orient Bio Co., Sungnam, Republic of Korea) had a temperature of 20-25 ° C, humidity of 40-45%, and day / night at 12-hour intervals Acclimatized for 12 days. Subsequently, a 2 cm midline incision was performed on the anesthetized mouse to expose the intestine adjacent to the appendix, and the 5 mm region from the end of the appendix was tightly tied with a 3.0-silk suture and 22-gauge. A hole was made with a needle. Subsequently, the cecum was gently squeezed to extrude a small amount of excrement from the hole site, placed back into the peritoneal cavity, and then the open abdomen was closed with a 4.0-silk suture. At this time, in the case of the control group (sham, sham-operated mice), the appendix was exposed, but it was placed into the abdominal cavity without performing the step of binding and puncture. Each mouse was exposed to the symptoms of sepsis 24 hours after CLP surgery, showing symptoms such as shivering, bristled hair, or weakness.

After 12 hours of CLP surgery, Compound 1 represented by Formula 1 of the present invention was injected intravenously (0, 0.14, 0.28 and 0.56 mg / kg) by concentration, and after 24 hours of CLP surgery, mouse blood was collected and serum samples were collected. Got

To confirm the expression level of HMGB1 by ELISA (competitive enzyme-linked immunosorbent assays) using the serum sample obtained from the above process, 96-well flat plastic microtiter plates (Corning, NY, USA) 0.02% sodium aza It was coated with HMGB1 protein (Abnova, Taipei, Taiwan) dissolved in 20mM carbonate / bicarbonate buffer (pH 9.6) containing sodium azide for 4 nights at night. Then, the plate was washed three times with PBS-T (PBS-0.05% [w / w] Tween 20), and then stored at 4 ° C.

In a 96-well plate, mouse serum obtained in the above process and anti-HMGB1 antibody (Abnova, diluted 1: 1000 in PBS-T) were pre-incubated at 37 ° C for 90 minutes, and then transferred to a plate coated with HMGB1 and transferred to room temperature. Was reacted for 30 minutes. Afterwards, the plate was washed 3 times with PBS-T, then peroxidase-conjugated anti-rabbit IgG antibodies, diluted 1: 2000 in PBS-T for 90 minutes at room temperature, Amersham Pharmacia Biotech , Piscataway, NJ). After 90 minutes, the plate was washed 3 times with PBS-T, and 200 μl of substrate solution (100 μg / ml o -phenylenediamine & 0.003% H ₂ O ₂ ) was treated in a dark room temperature room for 60 minutes. After 60 minutes, 50 µl of 8N and H ₂ SO ₄ was added to terminate the reaction, and the absorbance was confirmed at 490 nm.

Referring to FIG. 3, when the compound 1 represented by Chemical Formula 1 was treated in the mouse inducing sepsis, the effect of suppressing the expression of HMGB1 was excellent in a concentration-dependent manner. Therefore, it was found that the deckercin derivative compound of the present invention can be usefully used as a composition for the treatment of sepsis.

Example 4-2. Confirmation of change in survival rate

CLP surgery was performed by the method of Example 4-1, and compound 1 represented by Chemical Formula 1 was injected intravenously by concentration (0.28 and 0.56 mg / kg) at 12 hours and 50 hours after CLP surgery. After performing CLP surgery, the survival rate was confirmed at intervals of 6 hours from 140 hours (Kaplan-Meier survival analysis), and is shown in FIG. 4.

Referring to FIG. 4, when the compound 1 represented by Chemical Formula 1 is administered at a concentration of 0.28 mg / kg or 0.56 mg / kg to the septic-induced mouse, it is 140 hours compared to the untreated group (only CLP surgery is performed). Even after this, it was confirmed that the survival rate increased to 30-50%.

In particular, the present inventors injected compound 1 represented by Chemical Formula 1 intraperitoneally into the animal model in which sepsis was induced by CLP surgery before performing the experiment and intraperitoneally injected, thereby surviving from CLP surgery to 140 hours. As a result, as a result, it was confirmed that the survival rate of the compound of the present invention compared to decursinol increased by 15 times or more. Therefore, in the subsequent animal experiments, the anti-septic effect was confirmed by intravenous injection of Compound 1 after CLP surgery.

Example 4-3. Check for organ damage and thyroid hormone changes

CLP surgery was performed in the method of Example 4-1 to obtain blood of an animal model in which sepsis was induced, and aspartate transaminase (AST), alanine transaminase (ALT), creatinine, blood urea nitrogen (BUN) in mouse plasma, and The concentration of lactate dehydrogenase (LDH) was analyzed (commercial assay kits, Pointe Scientific, Lincoln Park, MI) and is shown in FIG. 5.

Referring to FIG. 5, when the compound 1 represented by Formula 1 of the present invention is administered to the septic-induced mouse, the levels of liver damage markers ALT and AST, kidney damage markers creatinine and BUN, and tissue damage markers LDH are reduced. It was confirmed that long-term damage caused by sepsis was suppressed.

From these results, it was found that the deckersin derivative represented by Chemical Formula 1 (Compound 1) of the present invention can be usefully used as a composition for treating sepsis and septic shock.

<Formulation Example 1. Preparation of tablets>

Compound 1 ((7S)-(+)-3- (3-pyridyl) -acrylic acid, 8,8-dimethyl-2-oxo-6,7-dihydro-2H, 8H pyrano [3,2] of the present invention -g] chromen-7-yl-ester) 20g was mixed with 175.9g lactose, 180g potato starch and 32g colloidal silicic acid. After adding 10% gelatin solution to this mixture, it was ground and passed through a 14 mesh sieve. This mixture was dried, and a mixture obtained by adding potato starch 160g, talc 50g, and magnesium stearate 5g was purified.

<Formulation Example 2. Preparation of capsules>

Compound 1 ((7S)-(+)-3- (3-pyridyl) -acrylic acid, 8,8-dimethyl-2-oxo-6,7-dihydro-2H, 8H pyrano [3,2] of the present invention -g] chromen-7-yl-ester) 100 mg, corn starch 100 mg, lactose 100 mg and magnesium stearate 2 mg are mixed and the above ingredients are mixed according to a conventional capsule preparation method and filled into a gelatin capsule. To prepare a capsule.

<Formulation Example 3. Preparation of injection>

Compound 1 ((7S)-(+)-3- (3-pyridyl) -acrylic acid, 8,8-dimethyl-2-oxo-6,7-dihydro-2H, 8H pyrano [3,2] of the present invention -g] chromen-7-yl-ester) 1 g, sodium chloride 0.6 g and ascorbic acid 0.1 g were dissolved in distilled water to make 100 ml. The solution was placed in a bottle and sterilized by heating at 20 ° C for 30 minutes.

<Formulation Example 4. Preparation of health functional food>

Compound 1 ((7S)-(+)-3- (3-pyridyl) -acrylic acid, 8,8-dimethyl-2-oxo-6,7-dihydro-2H, 8H pyrano [3,2] of the present invention -g] chromen-7-yl-ester) 20g, vitamin mixture proper amount, vitamin A acetate 70㎍, vitamin E 1.0mg, vitamin B1 0.13mg, vitamin B2 0.15mg, vitamin B6 0.5mg, vitamin B12 0.2㎍, vitamin C 10mg, biotin 10µg, nicotinic acid amide 1.7mg, folic acid 50µg, calcium pantothenate 0.5mg, mineral mixture amount, ferrous sulfate 1.75mg, zinc oxide 0.82mg, magnesium carbonate 25.3mg, potassium phosphate 15mg, 55 mg of dibasic calcium phosphate, 90 mg of potassium citrate, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride were mixed into granules, but can be prepared by modifying them into various formulations depending on the application. In addition, the composition ratio of the above-mentioned vitamin and mineral mixture may be arbitrarily modified, and the above ingredients may be mixed and prepared according to a conventional health functional food manufacturing method.

<Formulation Example 5. Preparation of health functional beverage>

Compound 1 ((7S)-(+)-3- (3-pyridyl) -acrylic acid, 8,8-dimethyl-2-oxo-6,7-dihydro-2H, 8H pyrano [3,2] of the present invention -g] chromen-7-yl-ester) 1g, citric acid 0.1g, fructooligosaccharide 100g, purified water 900g were mixed and stirred, heated, filtered, sterilized and refrigerated according to a conventional beverage preparation method to prepare a beverage.

Claims (6)

A pharmaceutical composition for the prevention or treatment of sepsis or septic shock comprising the compound represented by Formula 1 as an active ingredient.
[Formula 1]

According to claim 1,
The compound is a pharmaceutical composition for the prevention or treatment of sepsis or septic shock, characterized by inhibiting the expression of HMGB1 (high mobility group box 1). According to claim 1,
The composition is a pharmaceutical composition for the prevention or treatment of sepsis or septic shock, characterized in that it is formulated into a pharmaceutical dosage form by adding a pharmaceutically acceptable carrier, excipient or diluent. A health functional food for the prevention or improvement of sepsis or septic shock comprising the compound represented by Formula 1 as an active ingredient.
[Formula 1]

The method of claim 4,
The compound is a health functional food for prevention or improvement of sepsis or septic shock, characterized by inhibiting the expression of high mobility group box 1 (HMGB1). The method of claim 4,
The formulation of the health functional food is a health functional food for preventing or improving sepsis or septic shock, characterized in that it is selected from the group consisting of tablets, capsules, pills or liquids.

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