Pharmaceutical composition for treating sepsis by Acinetobacter baumannii

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of sepsis-related diseases comprising aspartic acid as an active ingredient. By including aspartic acid as an active ingredient according to one aspect, the effect of inducing the secretion of inflammatory cytokines during immune tolerance or immunosuppression can be achieved, and through this, the survival rate of a sepsis animal model compared to an antibiotic administration group can be increased, thereby making it useful for the prevention and / or treatment of sepsis or systemic inflammatory response syndromes associated with immune tolerance or immunosuppression.

Description

Technology Field

[0001] This relates to a pharmaceutical composition for the prevention or treatment of sepsis, septic shock, or systemic inflammatory response syndrome. Background Technology

[0002] Sepsis is an acute, severe disease in which a patient becomes infected by causative agents such as bacteria, viruses, and fungi, suffers from a high fever, and can eventually die. At this time, there are two main causes leading to death: first, the immune function becomes overactive due to a so-called 'cytokine storm,' and this excessive autoimmune function destroys the body's own organs (hyperinflammation); and second, the immune function is not strong enough to suppress the proliferation of causative agents, causing them to spread throughout the blood and organs, resulting in organ failure (immune paralysis).

[0003] To date, no fundamental cure for sepsis has been identified. Sepsis does not respond well to conventional anti-inflammatory treatments, and even drotrecogin alfa (Xigris®, Engl. Ranieri, VM et al., 2012), the only drug to receive FDA approval, was withdrawn from the market because its therapeutic efficacy against sepsis was not clearly established during clinical trials. Currently, sepsis is primarily treated with intravenous antibiotics or antifungals, with the specific medication and duration determined by the type of microorganism. Additionally, hemodialysis or blood transfusions may be administered depending on the patient's condition. While sepsis can be completely cured if antibiotics and antifungals are effective, treatment becomes difficult and can lead to death in cases involving drug-resistant microorganisms, immunocompromised patients, or when treatment is started too late. In addition, despite advancements in various intensive care treatments, including antibiotic therapy (Anderson, RN2002; Andreu Ballester, JC et al., 2008; Angus, DC et al., 2001), severe sepsis and septic shock are the third leading cause of death in developed countries.

[0004] Therefore, it is necessary to diagnose and treat early symptoms of sepsis before they progress to severe sepsis or septic shock, and furthermore, there is a need to develop treatments for sepsis. The problem to be solved

[0005] One aspect provides a pharmaceutical composition for the prevention or treatment of sepsis or septic shock comprising aspartic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

[0006] Another aspect is that aspartic acid provides a complex linked to a peptide or antibody through a linker.

[0007] Another aspect is to provide a health functional food for the prevention or improvement of sepsis or septic shock containing aspartic acid or a food-grade salt thereof as an active ingredient. means of solving the problem

[0008] One aspect provides a pharmaceutical composition for the prevention or treatment of sepsis or septic shock comprising aspartic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

[0009] Another aspect provides a health functional food for the prevention or improvement of sepsis or septic shock comprising aspartic acid or a food-grade acceptable salt thereof as an active ingredient.

[0010] In this specification, the term "prevention" collectively refers to partially or completely delaying or preventing the onset or recurrence of a disease, disorder, or its associated symptoms, preventing the acquisition or reacquisition of a disease or disorder, or reducing the risk of acquiring a disease or disorder. Such prevention refers to any act of suppressing or delaying the occurrence of inflammation or inflammation-related diseases, disorders, or symptoms through the administration of a composition according to the present invention.

[0011] In this specification, the term "improvement" may mean any action that at least reduces parameters related to the condition being treated, such as the degree of symptoms.

[0012] In this specification, the term "treatment" refers to any act that improves or beneficially alters a disease, disorder, or its associated symptoms. For example, it includes the inhibition, alleviation, or elimination of the development of sepsis and related diseases.

[0014] In one embodiment, the aspartic acid may be L-aspartic acid or D-aspartic acid.

[0015] The above L-aspartic acid may be represented by the following chemical formula 1:

[0016] [Chemical Formula 1]

[0017] .

[0018] In another embodiment, the composition may further include ornithine.

[0019] In one embodiment, the ornithine may be L-ornithine or D-ornithine.

[0020] The above L-ornithine may be represented by the following chemical formula 2.

[0021] [Chemical Formula 2]

[0022] .

[0024] In one embodiment, the aspartic acid, the ornithine, or aspartic acid and ornithine may be administered as a single active ingredient for the prevention or treatment of sepsis or septic shock. For example, the composition may consist of (i) aspartic acid or an acceptable salt thereof and an acceptable excipient or carrier, or (ii) aspartic acid or an acceptable salt thereof, ornithine or an acceptable salt thereof and an acceptable excipient or carrier.

[0026] In this specification, the term "sepsis" refers to the human body's immune response to invading pathogens, specifically systemic inflammatory response syndrome (SIRS) caused by the rapid overproduction of inflammatory cytokines, also known as a "cytokine storm." Despite advancements in healthcare, the incidence and mortality rates of sepsis are on the rise, with mortality reaching approximately 30%. During the onset of sepsis, the initial immune response is mediated by innate immune cells such as macrophages and neutrophils. Macrophages produce pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α, which reduce the invading pathogens; however, the overproduction of inflammatory cytokines leads to systemic inflammation and multi-organ damage.

[0027] The major causative bacteria of the above-mentioned sepsis are Gram-negative and Gram-positive bacteria, from the group consisting of Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Shigella sonnei, Staphylococcus aureus, Streptococcus pyogenes, Bacillus cereus, Clostridium perfringens, Enterococcus faecalis, Salmonella enteritidis, and Campylobacter jejuni It may be one or more selected.

[0028] In one embodiment, the sepsis or septic shock may be a disease accompanied by immunosuppression or immune paralysis.

[0029] Aspartic acid and / or ornithine according to one embodiment can restore reduced endotoxin resistance in sepsis patients. Aspartic acid and / or ornithine according to one embodiment may induce the secretion of inflammatory cytokines during immune tolerance or immunosuppression. Therefore, aspartic acid and / or ornithine according to one embodiment may be useful for the prevention or treatment of sepsis.

[0030] In one embodiment, the reduced endotoxin resistance may be present in peripheral blood mononuclear cells (PMBCs) or splenocytes. Accordingly, the aspartic acid and / or ornithine may be administered after confirmation of reduced endotoxin resistance in a sepsis patient or biological sample. Accordingly, the aspartic acid according to one embodiment may be administered to a sepsis patient with reduced endotoxin resistance.

[0031] In this specification, the term “biological sample” may be a sample isolated from an individual having sepsis or at risk of developing sepsis, or cultured cells. The biological sample may be blood, plasma, serum, tissue, urine, mucus, saliva, tears, sputum, cerebrospinal fluid, pleural fluid, nipple aspirate, lymph fluid, airway fluid, serous fluid, genitourinary fluid, breast milk, lymphatic fluid, semen, cerebrospinal fluid, fluid within organ systems, ascites, cystic tumor fluid, amniotic fluid, tissue, spleen, splenocytes, splenocyte tissue, or a combination thereof. If the biological sample is blood or plasma, since blood or plasma, which is easy to collect, is used as a sample, the individual's organ tissues are not removed, allowing for convenient analysis without placing a burden on the individual. The cells may be cells isolated from the individual or cultured cells. In one embodiment, the biological sample may be peripheral blood mononuclear cells (PMBC) or splenocytes.

[0032] In this specification, the term "pharmaceutical composition" may refer to a molecule or compound that imparts several beneficial effects upon administration to a subject. The beneficial effects may include enabling diagnostic decisions; improving a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder, or disease; and generally addressing a disease, symptom, disorder, or pathological condition.

[0033] The above-mentioned object may be a mammal, for example, a human, cow, horse, pig, dog, sheep, goat, or cat.

[0034] The above pharmaceutical composition may be formulated into a preparation selected from the group consisting of tablets, soft or hard capsules, pills, powders, suspensions, syrups, injections, and granules.

[0035] The above pharmaceutical composition may be intended for oral or parenteral administration. Preparations for oral administration include tablets, soft or hard capsules, pills, powders, suspensions, syrups, injections, and granules, and these preparations may be prepared by mixing one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients. Preparations for parenteral administration may be creams, lotions, ointments, ointments, liquids, aerosols, fluid extracts, elixirs, infusions, sachets, patches, or injections.

[0036] The above pharmaceutical composition may include excipients, for example, conventional fillers, extenders, binders, disintegrants, anticoagulants, lubricants, wetting agents, pH adjusters, nutrients, vitamins, electrolytes, alginic acid and its salts, pectic acid and its salts, protective colloids, glycerin, flavorings, emulsifiers, or preservatives.

[0037] The above pharmaceutical composition may be prepared by additionally including one or more pharmaceutically acceptable excipients or carriers. The pharmaceutically acceptable excipients or carriers may be saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Additionally, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets. Furthermore, the composition may be preferably formulated according to each disease or component by appropriate methods in the art.

[0038] The above aspartic acid and / or ornithine is present in an amount of 0.0001 wt% to 99.0 wt% based on the total weight of the composition, for example, 0.01 wt% to 60 wt%, 0.01 wt% to 40 wt%, 0.01 wt% to 30 wt%, 0.01 wt% to 20 wt%, 0.01 wt% to 10 wt%, 0.01 wt% to 5 wt%, 0.05 wt% to 60 wt%, 0.05 wt% to 40 wt%, 0.05 wt% to 30 wt%, 0.05 wt% to 20 wt%, 0.05 wt% to 10 wt%, 0.05 wt% to 5 wt%, 0.1 wt% to 60 wt%, 0.1 wt% to 40 wt%, 0.1 wt% to 30 wt%, 0.1 wt%. It may be included in an amount of 20% by weight, 0.1% by weight to 10% by weight, 0.1% by weight to 5% by weight, 5% by weight to 20% by weight, 5% by weight to 18% by weight, 6% by weight to 15% by weight, 8% by weight to 15% by weight, or 7% by weight to 10% by weight.

[0039] In this specification, the term "health functional food" refers to a food manufactured or processed for the purpose of health supplementation using specific ingredients as raw materials or by methods such as extraction, concentration, purification, or mixing of specific ingredients contained in food raw materials, and refers to a food designed and processed to fully exert bio-regulatory functions on the body, such as biological defense, regulation of biological rhythms, and prevention and recovery from disease, through said ingredients. The above-mentioned health functional food composition can perform functions related to the prevention and improvement of sepsis and related diseases.

[0040] There are no specific restrictions on the types of the above-mentioned foods. Examples of foods to which the above-mentioned substance may be added include formulations selected from the group consisting of powders, granules, tablets, capsules, pills, gels, jellies, suspensions, emulsions, syrups, tea bags, infused teas, and health drinks, and include all health foods in the conventional sense.

[0041] The above health drink composition may include various sweeteners, flavorings, or natural carbohydrates as additional ingredients, as in ordinary beverages. The sweetener may be a natural sweetener or a synthetic sweetener. The natural sweetener may be taumatin or stevia extract, and the synthetic sweetener may be saccharin or aspartame.

[0042] The health beverage composition of the present invention may include various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. The aforementioned natural carbohydrates may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin. The proportion of the natural carbohydrates may generally be about 0.01 to 10 g, preferably about 0.01 to 0.1 g, per 100 ml of the composition of the present invention.

[0043] The above-mentioned health functional food may include food-grade acceptable food additives and may include a suitable carrier commonly used in the manufacture of health functional foods.

[0044] In addition to the above, the composition may include various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Other compositions may include fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages. These ingredients may be used independently or in combination. Although the proportion of these additives is not critical, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

[0045] In this specification, pharmaceutically and / or food-engineerally acceptable salts may be, but are not limited to, acid addition salts or basic salts formed by pharmaceutically and / or food-engineerally acceptable free acids. Acid addition salts may be prepared by conventional methods, for example, by dissolving aspartic acid and / or ornithine derivatives in an excess amount of aqueous acid solution and precipitating the salt using a water-miscible organic solvent, for example, methanol, ethanol, acetone, or acetonitrile. An equal molar amount of the compound and an acid or alcohol (e.g., glycol monomethyl ether) in water may be heated, and then the mixture may be dried by evaporation or the precipitated salt may be filtered by suction.

[0046] Acid addition salts are obtained from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromide, hydroiodide, nitrous acid, or phosphoric acid, and non-toxic organic acids such as aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates and alkandioates, aromatic acids, and aliphatic and aromatic sulfonic acids. Salts that are pharmaceutically / food engineering non-toxic include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, iodide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, malieate, buty-1,4-dioate, hexane-1,6-dioate, benzoates, chlorobenzoates, methyl benzoates, dinitrobenzoates, hydroxybenzoates, and methoxybenzoates. Includes phthalates, terephthalates, benzenesulfonates, toluenesulfonates, chlorobenzenesulfonates, xylenesulfonates, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, or mandelate.

[0048] Another aspect provides a complex in which aspartic acid is linked to at least one peptide or antibody (e.g., via a linker), e.g., an antibody-drug conjugate (ADC).

[0049] In this specification, the term "Antibody-Drug Conjugate (ADC)" refers to a form in which a drug (e.g., aspartic acid) and an antibody are chemically linked without reducing the biological activity of the antibody and the drug (e.g., aspartic acid). In the present invention, the antibody-drug conjugate refers to a form in which a drug (e.g., aspartic acid) is bound to an amino acid residue at the N-terminus of the heavy chain and / or light chain of an antibody, specifically a form in which a drug (e.g., aspartic acid) is bound to an α-amine group at the N-terminus of the heavy chain and / or light chain of an antibody.

[0050] Examples of reactive groups capable of reacting with the α-amine group to crosslink in this specification include, as long as they can react with the α-amine group at the N-terminus of the heavy or light chain of the antibody to crosslink, the type thereof is not particularly limited and includes all types known in the art that react with amine groups. Examples may be any one of isothiocyanate, isocyanates, acyl azide, NHS ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imidoester, carbodiimide, anhydride, and fluorophenyl ester, but are not limited thereto.

[0051] The above antibody may refer to a protein that specifically recognizes a cell's receptor or target protein, or specifically binds to a cell's receptor or target protein.

[0052] The protein that specifically binds to the receptor or target protein of the cell may be any one selected from the group consisting of antibodies, antigen-binding fragments, affibodies, diabodies, and aptamers.

[0053] The above peptide may refer to a molecule composed of at least two amino acid units. The above peptide may include a cell-permeable peptide.

[0054] The above linker refers to a molecule capable of linking the above aspartic acid to the above antibody or protein. Exemplarily, the payload may be a compound, peptide, polypeptide, and / or protein, but is not limited thereto. Amino acid sequences suitable for the above peptide linker are known in the art. Other flexible linkers are known in the art, for example, G and S linkers that add amino acid residues such as T and A to maintain flexibility as well as polar amino acid residues to improve water solubility. Accordingly, in one embodiment, the linker may be a flexible linker comprising G, S, and / or T residues. The above linker is (G p S s ) n and (S p G s ) n It may have a general formula selected from, wherein, independently, p is an integer from 1 to 10, s is 0 or an integer from 0 to 10, p + s is an integer less than or equal to 20, and n is an integer from 1 to 20. In one embodiment, the linker may include an Fc-linking peptide.

[0055] Compound linkers are C1~C 20 Alkylene group, C6~C 30 Aryllene group or C3~C 30It may be a compound containing a heteroarylene group. As used in this specification for heteroarylene groups, the term hetero means that one or more of the carbon atoms constituting these aromatic or alicyclic rings, for example, 1 to 5 carbon atoms, are substituted with one or more heteroatoms selected from the group consisting of N, O, S, P and combinations thereof.

[0056] Peptides that bind to aspartic acid may consist of amino acids having neutral or hydrophobic side chains and amino acids having positively charged side chains in the biological environment.

[0057] The aspartic acid moiety and the peptide moiety are connected via amide bonds. The aspartic acid moiety and the peptide moiety constituting the aspartic acid derivative administered to the body can be separated into their respective moiety by peptide-degrading enzymes, etc. Accordingly, the aspartic acid derivative maintains a stable structure in vitro, while being degraded into the aspartic acid moiety in vivo, it can exhibit the physiological activity characteristic of aspartic acid. Effects of the invention

[0058] By including aspartic acid as an active ingredient according to one aspect, it is possible to achieve the effect of inducing the secretion of inflammatory cytokines during immune tolerance or immunosuppression, and thereby increase the survival rate of a sepsis animal model compared to an antibiotic administration group, which can be usefully applied to the prevention and / or treatment of sepsis or systemic inflammatory response syndromes associated with immune tolerance or immunosuppression. Brief explanation of the drawing

[0059] Figure 1 is a graph showing the concentration of TNF-α following treatment with L-ornithine L-aspartate (LOLA) in human monocyte cell lines derived from patients with acute mononuclear leukemia in which immune paralysis was induced by LPS; LOLA: L-ornithine L-aspartate. Figure 2 is a graph showing the survival rate of rats treated with L-ornithine L-aspartic acid (LOLA) in a septic rat model. Figure 3 is a graph showing the level of TNF-α following aspartic acid treatment in human mononuclear cell lines derived from patients with acute mononuclear leukemia whose immune paralysis was induced by LPS. Figure 4 is a graph showing the concentration of TNF-α following treatment with GOT siRNA or / and aspartic acid in human macrophage cell lines with an induced immune hyperactivity state; GOT1: glutamic-oxaloacetic transaminase 1. Specific details for implementing the invention

[0060] Preferred embodiments are presented below to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the invention and do not limit the scope of the invention. Since the embodiments are subject to various modifications, they are not limited to the embodiments disclosed below but can be implemented in various forms.

[0062] Experimental Example 1. Immunostimulatory effect of L-ornithine L-aspartate (LOLA) in immune-paralyzed human monocytes - In vitro ( in vitro )

[0063] To confirm the immune-enhancing (immune-boosting) effect of aspartic acid on an immune paralysis (immune weakening) state induced by two administrations of LPS (Lipopolysaccharide), an immune paralysis state was induced in human monocyte cell lines (THP-1) by administering LPS twice, and the concentration of the inflammatory cytokine TNF-α was measured in groups treated with or untreated with L-ornithine L-aspartate (LOLA).

[0064] Specifically, human monocyte cell lines (THP-1) derived from patients with acute mononuclear leukemia were placed in a 48-well plate at a ratio of 1 x 10⁶ 5 The cells were aliquoted. To induce an immunoparalysis (immunodeficiency) state, LPS was administered in two stages. The aliquoted cells were treated with the first LPS and washed after 4 hours. Subsequently, after a 16-hour resting period without exposure to LPS, the second LPS was administered. After inducing an immunoparalysis (immunodeficiency) state with the second LPS, the concentration of TNF-α was measured after 4 hours. The LPS concentration was 10 ng / ml.

[0065] At this time, the control group consisted of a control group that was not treated with LPS and an LPS group that was treated only with LPS and not with LOLA, and the experimental group consisted of groups administered LOLA at doses of 0.1g, 0.05g (500mg / ml), and 0.01g (100mg / ml), respectively. The concentration of TNF-α was analyzed for each group, and the results are shown in Table 1 and Figure 1.

[0066] Group TNF-α Standard deviation n control group 0.00 0.00 3 LPS 51.05 1.02 3 LOLA 0.1g 65.45 3.86 3 LOLA 0.05g(500mg / ml) 89.52 8.11 3 LOLA 0.01g(100mg / ml) 83.82 1.32 3

[0067] Figure 1 is a graph showing the concentration of TNF-α following LOLA treatment in human mononuclear cell lines derived from patients with acute mononuclear leukemia whose immune paralysis was induced by LPS.

[0068] As shown in Figure 1, when LPS was treated twice, the TNF-α secretion was very low at approximately 50 pg / ml, but when LOLA was administered, the TNF-α secretion increased. This indicates that the administration of LOLA has an immune-enhancing (increasing) effect.

[0070] Experimental Example 2. Confirmation of therapeutic effects by L-ornithine L-aspartate (LOLA) in a rat sepsis model - In vivo ( in vivo )

[0071] To confirm the therapeutic effect of aspartic acid on sepsis, LOLA (Hepa-Merz, Hanwha Pharma) was administered to a sepsis-induced rat model, and the survival rates of the rats were compared.

[0072] Specifically, male rats (Sprague-Dawley, Koatech) weighing 280–320 g were purchased and used as experimental models. Starting 7 days prior to the experiment, the rats were acclimatized to their surroundings by feeding them food and water in a rearing room controlled at an indoor temperature of 20–24°C and humidity of 40–60%, while alternating between bright and dark lighting every 12 hours. Short anesthesia was induced in the rats using isoflurane, and Zoletil (50 mg / kg) and Xylazine (10 mg / kg) were administered intramuscularly. Feces obtained from the cecum of a fecal donor rat were diluted with 5% dextrose saline at a ratio of 1:3 and thoroughly mixed. After making a 5 mm incision in the peritoneum of the rats to be septic induced, the fecal slurry and 5% dextrose saline were injected into the abdominal cavity according to the animal's weight. Additionally, the antibiotic Prefenem (25 mg / kg) was administered subcutaneously, and 5% glucose saline (30 mg / kg) was administered subcutaneously for fluid replacement. A model inducing secondary infection was created by re-injecting a fecal slurry 48 hours after sepsis induction. Likewise, the antibiotic Prefenem (25 mg / kg) was administered subcutaneously, and 5% glucose saline (30 mg / kg) was administered subcutaneously for fluid replacement.

[0073] The antibiotic prefenem (25 mg / kg) was administered subcutaneously to all groups at 0, 7, 24, 31, 48, 55, 72, and 79 hours. The control group (Control, n=16) was administered D-sorbitol (0.01 g / 0.1 ml sterile distilled water, 0.033 g / kg) diluted in 0.4 ml of normal saline, and the experimental group (HM, n=16) was administered L-ornithine L-aspartic acid (Hepamels 0.1 ml, 0.166 g / kg) diluted in 0.4 ml of normal saline. D-sorbitol or L-ornithine L-aspartate (LOLA) was administered to the control and experimental groups, respectively, by intravenous injection at 24 and 48 hours after sepsis induction, and by intraperitoneal injection at 31, 55, 72, and 79 hours. Subsequently, the survival rates of the rats in the experimental and control groups were analyzed, and the results are shown in Table 2 and Figure 2.

[0074] 0d 2d 4d 6d 8d 10d 12d 14d Control 16 16 6 4 2 2 2 2 HM 16 16 11 11 7 7 6 6

[0075] Figure 2 is a graph showing the survival rate of rats treated with L-ornithine L-aspartic acid (LOLA) in a sepsis rat model. As shown in Figure 2, it was confirmed that the survival rate (37.5%) of the experimental group administered LOLA (Hepa-Merz) containing aspartic acid was higher than the survival rate (12.5%) of the control group in the sepsis rat model.

[0076] Based on the above results, it was confirmed that L-ornithine L-aspartic acid (LOLA) has therapeutic effects on sepsis or systemic inflammatory response syndrome.

[0078] Experimental Example 3. Immunostimulatory effect of aspartic acid in immune-paralyzed human monocyte cells - In vitro ( in vitro )

[0079] To confirm the immune-enhancing (immune-increasing) effect of administering aspartic acid alone, a state of immune paralysis was induced in human monocyte cell lines (THP-1) by administering LPS twice, and the concentration of the inflammatory cytokine TNF-α was measured in groups treated with or not treated with aspartic acid at different concentrations. Cells dispensed in the same manner as in Experimental Example 1 were treated with LPS once, and after a resting period, LPS was administered twice to induce a state of immune paralysis (immune weakening), after which the level of TNF-α was measured 4 hours later. The concentration of LPS administered was 10 ng / ml.

[0080] At this time, the control group consisted of a control group not treated with LPS and an LPS group treated only with LPS and not aspartic acid, and the experimental group consisted of groups administered aspartic acid at doses of 5 mM, 10 mM, or 20 mM, respectively. The levels of TNF-α were analyzed for each group, and the results are shown in Table 3 and Figure 3.

[0081] Group TNF-α control group 0.00 LPS 116.69 Aspartic acid 5mM 160.83 Aspartic acid 10mM 149.79 Aspartic acid 20mM 162.90

[0082] Figure 3 is a graph showing the level of TNF-α following aspartic acid treatment in human mononuclear cell lines derived from patients with acute mononuclear leukemia whose immune paralysis was induced by LPS.

[0083] As shown in Figure 3, after treatment with secondary LPS, the TNF-α concentration showed a low immune response (endotoxin tolerance), but when aspartic acid was treated, the level of TNF-α increased, showing an immune-enhancing effect.

[0085] Experimental Example 4. Immunostimulatory effect of aspartic acid in immune-hyperinduced human macrophages - In vitro ( in vitro )

[0086] Immunosuppression was confirmed by treating human macrophages (M1) with induced immune hyperactivity with siRNA that inhibits GOT1 (glutamic-oxaloacetic transaminase 1), an enzyme involved in the production of aspartic acid, and then treating them with aspartic acid to verify the immune-enhancing effect of aspartic acid.

[0087] Specifically, human monocyte cell line (THP-1) at 1.5 x 10⁶ in a 24-well plate 6 The experimental groups were divided into the M0 group, in which no drug was applied to THP-1; the M1 group, in which THP-1 was treated with LPS at a concentration of 100 ng / ml, IFN-γ at 20 ng / ml, and GM-CSF (Granulocyte-macrophage colony-stimulating factor) at a concentration of 25 ng / ml for 24 hours to induce a strong inflammatory response and differentiate into M1; the siRNA (GOT1) group, in which siRNA was additionally treated as an inhibitor of GOT1, an enzyme involved in the production of aspartic acid; and the groups in which aspartic acid was additionally treated at concentrations of 5, 10, and 20 mM, respectively, which were classified into the siRNA + aspartic acid 5 mM group, siRNA + aspartic acid 10 mM group, and siRNA + aspartic acid 20 mM group. The degree of immune activity for each group was analyzed by measuring the concentration of TNF-α, and the results are shown in Table 4 and Figure 4.

[0088] Group TNF-α Standard deviation N M0 518.44 6.25 2 M1 7121.88 143.77 3 siRNA(GOT1) 778.13 38.27 3 siRNA + 5mM aspartic acid 3875.73 38.07 3 siRNA + 10mM aspartic acid 4252.29 116.86 3 siRNA + 20mM aspartic acid 4495.52 88.69 3

[0089] Figure 4 is a graph showing the concentration of TNF-α following treatment with GOT siRNA and / or aspartic acid in human macrophage cell lines with an induced immune hyperactivity state.

[0090] As shown in Figure 4, it was confirmed that the concentration of TNF-α, which was increased in the M1 group with induced immune hyperactivity, decreased upon treatment with siRNA of GOT1, an enzyme involved in the production of aspartic acid. The immune-enhancing effect of aspartic acid was confirmed by the fact that the concentration of TNF-α increased again when aspartic acid was treated to immune levels suppressed by GOT1 siRNA.

[0092] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

Claim 1 A pharmaceutical composition for the prevention or treatment of sepsis comprising aspartic acid and ornithine as active ingredients, wherein the aspartic acid restores reduced endotoxin resistance in sepsis patients, the ornithine is represented by the following chemical formula 2, and the sepsis is caused by Acinetobacter baumannii: [Chemical Formula 2] .

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