Antibacterial adjuvant containing a biphenyl derivative compound as an active ingredient and use thereof

Abstract

The present invention relates to an antibacterial adjuvant containing a biphenyl derivative compound as an active ingredient and various uses thereof. The compound of the present invention reduces the amount of polymyxin antibiotics, and the composition is applied to inhibit the proliferation of gram-negative bacteria, and shows an effect of inhibiting the growth and killing gram-negative bacteria by being used in combination with polymyxin antibiotics to increase the sensitivity of gram-negative bacteria to polymyxin antibiotics, can significantly reduce side effects such as nephrotoxicity, and can prevent or treat septicemia and septic shock caused by overuse of antibiotics.

Description

TECHNICAL FIELD

[0001] The present application relates to an antibacterial supplement containing a biphenyl derivative compound as an active ingredient, and various techniques using the same. BACKGROUND

[0002] The emergence and rise of antibiotic-resistant bacteria, which survive even when exposed to antibiotics, have caused a huge problem worldwide. As of 2018, 700,000 people died worldwide due to infection with antibiotic-resistant bacteria, and in Korea, 9,000 cases of infection and 3,600 deaths, with a mortality rate of about 40%, were caused by infection with five representative antibiotic-resistant bacteria (methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Acinetobacter baumannii (MDRA), multidrug-resistant Pseudomonas aeruginosa (MRPA), vancomycin-resistant enterococci (VRE), and carbapenem-resistant Enterobacteriaceae (CRE)) in one year. In addition, the total social cost caused by the medical expenses, nursing expenses, and productivity loss due to premature death of patients infected with antibiotic-resistant bacteria is estimated to be 550 billion won per year in Korea, and thus the spread of antibiotic resistance can cause economic and human health losses. In particular, due to the emergence of multi-drug resistant bacteria (called "superbugs") that are resistant to all existing antibiotics at the same time, the damage caused by antibiotic-resistant bacteria is expected to increase exponentially.

[0003] It is known that antibiotic-resistant bacteria acquire resistance to antibiotics by acquiring drug resistance-related genes of the corresponding mechanism in the following ways: ① producing antibiotic-degrading enzymes to inactivate antibiotics, or changing the structure of antibiotics through antibiotic-converting enzymes, ② activating antibiotic efflux by antibiotic influx inhibition / efflux pumps to reduce the intracellular antibiotic concentration; ③ changing target proteins that bind to antibiotics by mutation, etc. Antibiotic-resistant bacteria effectively resist antibiotics by mobilizing two or more drug resistance mechanisms, and it is known that the more mechanisms are mobilized, the higher the degree of resistance. Therefore, antibiotics against resistant strains should ① inhibit new bacterial targets that have not been known so far, or ② avoid the development of resistance by inhibiting various target groups.

[0004] To solve the problem of infection by antibiotic-resistant bacteria, new antibiotics need to be developed, but it is difficult to mine targets for new drug development, new drug development costs an average of about $800 million and takes at least 10 years, leading to difficulties. A more serious problem is that, although new antibiotics can be developed by mining new targets to avoid the currently known drug resistance mechanisms, drug-resistant bacteria can quickly appear. In fact, linezolid, which has a different mechanism of action from traditional antibiotics, was the only new target inhibitor approved in the 2000s, but drug-resistant bacteria have already appeared. Therefore, in terms of cost and time, it can be an effective strategy to develop a substance that can inhibit the drug resistance mechanisms acquired by antibiotic-resistant bacteria rather than developing a new antibiotic to improve the efficacy of existing antibiotics in response to antibiotic-resistant bacterial infections.

[0005] Therefore, there is a greater need to develop a technology to kill bacteria by increasing sensitivity to antibiotics while using antibiotics that cannot be treated due to drug resistance. SUMMARY

[0006] TECHNICAL PROBLEM

[0007] The present application has been made to solve the problems of the related art and provides an antibacterial supplement that improves the sensitivity of bacteria to antibiotics.

[0008] In addition, another object of the present application is to provide an antibacterial composition comprising the above antibacterial supplement and a polymyxin antibiotic as active ingredients.

[0009] In addition, another object of the present application is to provide a pharmaceutical composition for preventing or treating organ damage caused by sepsis or septic shock, comprising the above antibacterial supplement and a polymyxin antibiotic as active ingredients.

[0010] TECHNICAL SOLUTION

[0011] To achieve the above object, one aspect of the present application provides an antibacterial supplement comprising a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0012] [Chemical Formula 1]

[0013]

[0014] wherein X is a halogen atom, R1 and R2 are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.

[0015] In addition, another aspect of the present application provides an antibacterial composition comprising an antibacterial supplement and a polymyxin antibiotic as active ingredients.

[0016] Furthermore, another aspect of the present invention provides a pharmaceutical composition for preventing or treating organ damage caused by sepsis or septic shock, comprising the aforementioned antibacterial supplement and polymyxin antibiotics as active ingredients.

[0017] Beneficial effects

[0018] The antibacterial supplement of the present invention improves the sensitivity of Gram-negative bacteria to polymyxin antibiotics, thereby reducing the therapeutic dose of polymyxin antibiotics used to inhibit the growth of Gram-negative bacteria by up to 128 times.

[0019] Furthermore, when the antibacterial supplement of the present invention is administered in combination with polymyxin antibiotics, it induces a synergistic effect, thereby demonstrating the effect of inhibiting the growth of Gram-negative bacteria and killing Gram-negative bacteria.

[0020] Furthermore, according to the present invention, conventional side effects such as nephrotoxicity caused by excessive use of polymyxin can be significantly reduced, and sepsis and septic shock caused by excessive use of antibiotics can be prevented or treated.

[0021] The effects of the present invention are not limited to those described above, and those skilled in the art can clearly understand other effects not mentioned herein through the following description. Attached Figure Description

[0022] Figure 1 This is a graph showing the inhibitory effects of PA108 and its derivatives (PA108-1 to 14) and polymyxin B (PMB) on the bacterial growth of Acinetobacter baumannii strains by relative bacterial respiration rate.

[0023] Figure 2 This is a graph showing the effect of polymyxin B (PMB) treatment alone, PA108 treatment alone, or a combination of polymyxin B (PMB) and PA108 on the bacterial growth inhibition of Acinetobacter baumannii strains by relative bacterial respiration rate.

[0024] Figure 3 This is a graph showing the effect of polymyxin B (PMB) treatment alone, PA108 treatment alone, or a combination of polymyxin B (PMB) and PA108 treatment on cell death in Acinetobacter baumannii strains.

[0025] Figure 4 The results show that checkerboard analysis confirmed whether the combined treatment with polymyxin B (PMB) and PA108 produced a synergistic effect on Acinetobacter baumannii strains.

[0026] Figure 5These are photographs of bacterial morphological changes observed under a fluorescence microscope (top) and a scanning electron microscope (bottom) when treated with polymyxin B (PMB) alone, with PA108 alone, or with a combination of polymyxin B (PMB) and PA108.

[0027] Figure 6 The nephrotoxicity of polymyxin B (left) and PA108 (right) on human kidney cells is shown by renal cell survival rate.

[0028] Figure 7 The study showed the survival rates of mice in a mouse sepsis model infected with Acinetobacter baumannii strain after administration of polymyxin B (PMB) alone, PA108 alone, or a combination of polymyxin B (PMB) and PA108.

[0029] Figure 8 The results, obtained by checkerboard analysis, demonstrate whether the combined treatment with polymyxin B (PMB) and PA108 produced a synergistic effect on three strains of Klebsiella pneumoniae and three strains of Pseudomonas aeruginosa, which were resistant to polymyxin antibiotics.

[0030] Figure 9 The figures show the number of genes expressed that were increased (left) and decreased (right) when treated with polymyxin B (PMB) alone, PA108 alone, or a combination of polymyxin B (PMB) and PA108, compared to the control group.

[0031] Figure 10 The images show the functional information of genes with statistically significant increases or decreases in expression when treated with the combined polymyxin B (PMB) and PA108 compared to the control group (purple: group treated with the combined PMB and PA108, yellow: group treated with PMB, blue: group treated with PA108).

[0032] Figure 11 The changes in bacterial cell membrane permeability (left) and cytoplasmic membrane potential (right) are shown when Acinetobacter baumannii strains are treated with polymyxin B (PMB) alone, with PA108 alone, or with a combination of polymyxin B (PMB) and PA108. Detailed Implementation

[0033] The present invention will be described in detail below.

[0034] One aspect of the present invention provides an antibacterial supplement that improves the sensitivity of bacteria to antibiotics.

[0035] The antibacterial supplement of the present invention comprises a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:

[0036] [Chemical Formula 1]

[0037]

[0038] In chemical formula 1, X can be any halogen atom selected from the group consisting of F, Cl, Br and I, or X can be Cl.

[0039] In chemical formula 1, R1 and R2 can each be a hydrogen atom or an alkyl group independently.

[0040] Alkyl refers to a straight-chain or branched saturated aliphatic hydrocarbon group, which can be an alkyl group having 1 to 12, 1 to 10, 1 to 8, or 1 to 6 carbon atoms. Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc.

[0041] Furthermore, the alkyl group can be substituted or unsubstituted. When the alkyl group is substituted, the substituent can be substituted at any possible connection point. The substituent is preferably one or more independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl, and alkoxycarbonyl, but is not limited thereto.

[0042] "Pharmaceutically acceptable salt" refers to a salt prepared by conventional methods. Salts include inorganic acids and organic acids, and can be any one of the following groups, but are not limited to: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, malic acid, acetic acid, oxalic acid, tartaric acid, citric acid, lactic acid, furoic acid, succinic acid, maleic acid, salicylic acid, benzoic acid, phenylacetic acid, and mandelic acid.

[0043] "Pharmaceutically acceptable salts" also include salts formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, and zinc, as well as salts formed from bases such as ammonia, ethylenediamine, N-methylglutamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenylethylamine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane, and tetramethylammonium hydroxide. These salts can be prepared by standard procedures, such as the reaction of free acids with appropriate organic or inorganic bases. "Pharmaceutically acceptable salts" also include free acids, free bases, and zwitterionic forms.

[0044] The compound represented by Formula 1 can improve the sensitivity of antibiotic-resistant bacteria to antibiotics. Therefore, the compound represented by Formula 1 can be used as an active ingredient in antimicrobial supplements that can be used in combination with antibiotics to combat antibiotic-resistant bacteria.

[0045] "Antibacterial supplements," also known as "antibiotic supplements," refer to substances that do not directly kill bacteria but increase their sensitivity to antibiotics by inhibiting bacterial resistance and inducing intracellular accumulation of antibiotics.

[0046] "Antibiotics" generally refers to substances with antibacterial activity. Examples include, but are not limited to, penicillins; cephalosporins; monolactams and carbapenems, which are β-lactam antibiotics with a β-lactam ring as their basic structure; polymyxins that alter bacterial cell membrane permeability, thus exhibiting antibacterial activity; aminoglycosides that enter the cytoplasm through porins on the outer membrane of Gram-negative bacteria, thereby exhibiting antibacterial activity; macrolides that bind to the 50S of bacterial ribosomes, inhibiting protein synthesis to exhibit antibacterial activity; tetracyclines, which exhibit antibacterial activity against a variety of bacteria and are known as broad-spectrum antibiotics; glycopeptides that inhibit bacterial cell wall biosynthesis to exhibit antibacterial activity; lincomycins, isolated from *Streptomyces lincolnensis* and exhibiting good antibacterial activity against anaerobic bacteria; quinolones that interfere with bacterial DNA replication to exhibit antibacterial effects; and any one or more of the groups consisting of combinations thereof and their derivatives, especially polymyxin antibiotics.

[0047] Polymyxin antibiotics are peptide antibiotics consisting of a large, positively charged ring, a heptapeptide ring, and three amino acid tails linked to aliphatic acids. Furthermore, polymyxin antibiotics are produced by the non-ribosomal peptide synthase system in Gram-negative bacteria (such as *Paenibacillus polymyxa*), possessing activity that binds to phospholipids present in the outer membrane of many Gram-negative bacteria, activating phospholipases to disrupt the cell membrane. Polymyxin antibiotics can be polymyxin A, polymyxin B, polymyxin C, polymyxin D, and polymyxin E (colistin), particularly B1 (C... 56 H 98 N 16 O 13 (molecular weight: 1203.49) and B2 (C 55 H 96 N 16 O 13 Polymyxin B or polymyxin E (molecular weight: 1189.47) are the main components. Polymyxin B and polymyxin E have the same structure, except that they differ from each other by an amino acid linked to the 6th position, which is phenylalanine (Phe) and leucine (Leu), respectively.

[0048] Bacteria can be included without limitation as long as they are membrane-bound Gram-negative bacteria. Specifically, bacteria can be pathogenic Gram-negative bacteria, such as *Escherichia* sp., *Acinetobacter* sp., *Pseudomonas* sp., and *Klebsiella* sp. Specifically, *Escherichia* genus includes, but is not limited to, *Escherichia coli*, *Escherichia albertii*, *Escherichia blattae*, *Escherichia fergusonii*, *Escherichia hermannii*, and *Escherichia vulneris*. The genus Acinetobacter includes, but is not limited to, Acinetobacter baumannii, Acinetobacter junii, Acinetobacter boissieri, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Acinetobacter nosocomialis, Acinetobacter schindleri, and Acinetobacter ursingii. The genus *Pseudomonas* includes, but is not limited to, *Pseudomonas aeruginosa*, *Pseudomonas fluorescens*, *Pseudomonas putida*, *Pseudomonas chlororaphis*, *Pseudomanas pertucinogena*, *Pseudomanas stutzeri*, and *Pseudomanas syringae*. The genus *Klebsiella* includes, but is not limited to, *Klebsiella pneumoniae*, *Klebsiella granulomatis*, *Klebsiella oxytoca*, and *Klebsiella terrigena*.

[0049] In particular, bacteria may exhibit resistance to antibiotics, especially to at least one of the antibiotics mentioned above. "Antibiotic-resistant bacteria" refers to bacteria that are no longer or almost unaffected by at least one previously effective antibiotic; that is, these bacteria are capable of resisting antibiotics that previously had effective antibacterial activity. Antibiotic-resistant bacteria can pass on their resistance to offspring. The mechanisms of antibiotic resistance are diverse. For example, resistance can manifest through the following mechanisms: impermeability mechanisms, physically preventing antibiotics from reaching the bacterial interior or site of action; efflux mechanisms, preventing effective amounts of antibiotics from reaching the bacterial interior or site of action by rapidly clearing antibiotics from within the bacteria; metabolic mechanisms, destroying antibiotics by converting them into harmless (or less harmful) compounds or compounds that are more easily excreted; or bypass mechanisms, using pathways other than inhibiting bacteria through antibiotics, or through bacterial target forms (such as enzymes) that are less sensitive to antibiotics or target-free bacteria.

[0050] In addition, antibiotic-resistant bacteria can be resistant to two or more antibiotics, and this is also known as multidrug-resistant bacteria. Multidrug-resistant bacteria include, for example, methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA), vancomycin-intermediate Staphylococcus aureus (VISA), vancomycin-resistant Enterococci (VRE), vancomycin-sensitive Enterococci (VSE), multidrug-resistant Pseudomonas aeruginosa (MRPA), carbapenem-resistant Pseudomonas aeruginosa (CRPA), carbapenem-resistant Acinetobacter baumannii (CRAB), and carbapenem-resistant Enterobacteriaceae (CRE), etc.

[0051] In specific examples of this invention, it has been demonstrated that when bacteria typically resistant to polymyxin antibiotics are treated in combination with polymyxin at a concentration in which the compound represented by Formula 1 itself does not possess antibacterial activity, the death of these resistant bacteria is further promoted. These results clearly demonstrate that the compound represented by Formula 1 can further enhance the susceptibility of antibiotic-resistant bacteria and can be used as an effective ingredient in antibacterial supplements, thereby restoring the antibacterial activity of antibiotics.

[0052] Another aspect of the present invention provides an antibacterial composition comprising an antibacterial supplement comprising a compound represented by chemical formula 1 as an active ingredient and an antibiotic as an active ingredient.

[0053] As described above, since the compound represented by chemical formula 1 of the present invention has the effect of improving the sensitivity of antibiotic-resistant bacteria to antibiotics, the above-mentioned antibacterial supplement is used in combination with antibiotics to restore the conventional antibacterial activity of antibiotics.

[0054] When the antibacterial composition of the present invention is applied to antibiotic-resistant bacteria, the expression levels of genes involved in zinc ion binding, same protein binding, and iron ion binding, as well as the expression levels of genes involved in peroxidase activity, can be significantly reduced, while the expression levels of genes involved in substance transport across the cell membrane, especially the expression levels of genes involved in ATPase-coupled sulfate transmembrane transporter activity, can be significantly increased.

[0055] The antibacterial composition of the present invention can alter the environment inside and outside the bacterial cell membrane, disrupt the integrity of the lipid bilayer region of the cell membrane, cause high depolarization of the cell membrane, thereby disrupting cytoplasmic homeostasis, reducing cell permeability, and leading to cell death.

[0056] Antibiotic-resistant bacteria, as described above, can be Gram-negative bacteria in particular.

[0057] In addition, antibiotics, as mentioned above, can be polymyxin antibiotics in particular.

[0058] Therefore, compared with the use of polymyxin antibiotics alone, the antibacterial composition of the present invention can more effectively improve the antibacterial activity of polymyxin antibiotics against polymyxin-resistant bacteria.

[0059] In a specific embodiment of the invention, when antibacterial supplements and polymyxin antibiotics are used in combination to treat bacteria resistant to polymyxin antibiotics, a synergistic effect is observed, thereby inhibiting bacterial growth and disrupting cell membranes to lead to cell death.

[0060] Furthermore, because the antibacterial composition of the present invention uses an antibacterial supplement in combination, sufficient antibacterial activity can be obtained without overdosing on polymyxin antibiotics. Therefore, side effects such as nephrotoxicity caused by overdosing on polymyxin antibiotics can be significantly reduced.

[0061] In specific examples of the present invention, it has been demonstrated that when polymyxin antibiotics that cause nephrotoxicity are used in combination with the antibacterial supplement of the present invention, nephrotoxicity is not caused in animal models of sepsis, and survival rate is improved.

[0062] Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of organ damage caused by sepsis or septic shock, the composition comprising an antibacterial supplement comprising a compound of formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient, and an antibiotic as an active ingredient.

[0063] Antibiotics, as mentioned above, can be polymyxins. Sepsis or septic shock can be caused by Gram-negative bacteria, and there are no restrictions on the type of bacteria, as long as they are resistant to antibiotics; for example, they can be Escherichia spp. and / or Acinetobacter spp.

[0064] Sepsis is a condition characterized by a severe systemic inflammatory response caused by a microbial infection. When two or more of the following symptoms are present: fever or hypothermia, rapid breathing (tachypnea), rapid heart rate (tachycardia), and an elevated or significantly decreased white blood cell count in blood tests, it is called systemic inflammatory response syndrome (SIRS). If SIRS is caused by a microbial infection, it is called sepsis. Pathogens continuously or intermittently enter the bloodstream from the site of infection and colonize various organs and tissues to form lesions, manifesting as severe systemic symptoms. Sepsis is more likely to affect the frail, elderly, and debilitated. Sepsis can potentially lead to septic shock. In severe cases of sepsis, the function of various organs (such as the heart, kidneys, liver, brain, and lungs) declines, leading to a state of shock.

[0065] In specific examples of the present invention, it has been demonstrated that the survival rate of animal models of sepsis has been improved and the number of bacteria (colony-forming units, CFU) infecting organs including the liver, lungs, kidneys, and spleen has been significantly reduced. Therefore, the composition of the antimicrobial supplement and polymyxin antibiotics of the present invention can be used for the prevention or treatment of sepsis.

[0066] "Prevention" refers to all effects of suppressing or delaying the onset of sepsis or septic shock by applying the composition according to the invention.

[0067] "Treatment" refers to all actions that improve or beneficially alter symptoms associated with sepsis and conditions associated with multiple organ dysfunction syndrome (e.g., fever of varying degrees, hypoxemia, reflex tachycardia, endotheliitis, myocardial infarction, altitude sickness, altered mental status, vascular failure and organ damage, acute respiratory distress syndrome, coagulopathy, heart failure, renal failure, shock and / or coma, etc.).

[0068] Furthermore, the present invention can also inhibit organ damage caused by sepsis. The pharmaceutical compositions of the present invention can prevent or treat sepsis by inhibiting organ damage caused by sepsis. The organ refers to an organ damaged by sepsis, and the damaged organ inhibited by the compositions of the present invention is not limited; for example, it can be at least one selected from the group consisting of the liver, kidney, and lung.

[0069] The pharmaceutical compositions according to the present invention can be formulated by adding non-toxic and pharmaceutically acceptable carriers, enhancers, excipients, etc., according to common methods. For example, they can be formulated into oral or parenteral formulations, such as tablets, capsules, lozenges, liquids, and suspensions.

[0070] In addition, excipients that can be used in the pharmaceutical compositions according to the present invention may also include sweeteners, binders, solubilizers, solvents, wetting agents, emulsifiers, tensioning agents, adsorbents, disintegrants, antioxidants, preservatives, lubricants, fillers, flavorings, etc., for example, lactose, glucose, sucrose, mannitol, sorbitol, cellulose, glycine, silicon dioxide, talc, stearic acid, glyceryl stearate, magnesium stearate, magnesium aluminum silicate, starch, gelatin, carrageenan, alginate, sodium alginate, methylcellulose, sodium carboxymethyl cellulose, agar, water, ethanol, polyethylene glycol, polyvinylpyrrolidone, sodium chloride, calcium chloride, orange flavoring, strawberry flavoring, vanilla flavoring, etc.

[0071] The concentration level of the compounds of the present invention can be in the range of 0.1 wt% to 95 wt%, that is, relative to the total weight of the pharmaceutical composition, which is sufficient to obtain the desired effect.

[0072] The pharmaceutical compositions of the present invention can be administered to mammals, such as rats, mice, livestock, and humans, via various routes. All routes of administration are permissible, including transdermal, oral, rectal, intravenous, intraperitoneal, intramuscular, subcutaneous, endometrial, or intraventricular injection, with any oral or intravenous route of administration being preferred, but not limited thereto.

[0073] Administration may include one or more active ingredients having the same or similar functions. For administration, one or more pharmaceutically acceptable carriers may be further included. Pharmaceutically acceptable carriers may be physiological saline, sterile water, Ringer's solution, buffered physiological saline, glucose solution, maltodextrin solution, glycerol, ethanol, and mixtures thereof, and other commonly used additives such as antioxidants, buffers, and antibacterial agents may be added if necessary. Furthermore, by further adding diluents, dispersants, surfactants, binders, and lubricants, the compounds according to the invention can be readily formulated into various forms, for example, into injectable formulations such as aqueous solutions, suspensions and emulsions, powders, tablets, capsules, pills, granules, or injections.

[0074] The amount of the compound varies within a certain range depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and disease severity. The daily dose of the compound of the present invention is 0.0001 to 100 mg / kg, preferably 0.001 to 30 mg / kg, administered once or multiple times daily. Furthermore, the administration period can range from one day to two months, but is not limited, until a disease prevention or treatment effect is observed.

[0075] Because the antibacterial supplement of the present invention increases the sensitivity of Gram-negative bacteria to polymyxin antibiotics, the treatment amount of polymyxin antibiotics used to inhibit the growth of Gram-negative bacteria can be reduced by up to 128 times. This significantly reduces traditional side effects such as nephrotoxicity caused by excessive polymyxin administration and shows excellent bacterial growth inhibition and killing effects. As a result, the antibacterial activity of polymyxin antibiotics against polymyxin-resistant Gram-negative bacteria can be restored. Therefore, the present invention has the effect of using traditional antibiotics as is without the need to develop new antibiotics separately.

[0076] Another aspect of the present invention provides a method for preventing, improving or treating organ damage caused by sepsis or septic shock, comprising: administering to an individual an antimicrobial supplement comprising a compound of formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient; and a polymyxin antibiotic.

[0077] Another aspect of the invention provides the use of an antimicrobial supplement in the preparation of a medicament for the prevention, improvement or treatment of organ damage caused by sepsis or septic shock, said antimicrobial supplement comprising a compound of chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient; and a polymyxin antibiotic.

[0078] The best way to implement an invention

[0079] The present invention will be described in detail below through embodiments and experimental examples. However, the following embodiments and experimental examples are merely illustrative of the present invention and do not limit the description of the present invention in any way.

[0080] Example 1: Selection of compounds to improve bacterial sensitivity to polymyxin antibiotics

[0081] 1-1. Select compound PA108

[0082] In this invention, a substance capable of acting in conjunction with polymyxins to kill polymyxin-resistant bacteria was screened from a variety of compounds in the compound library of the Korea Institute of Chemical Technology by measuring cellular respiration rate. As the bacteria used in this experiment, Acinetobacter baumannii colistin resistance 357 (hereinafter referred to as R357 strain), isolated from patients with sepsis infection at Yonsei University Hospital, was provided and used. This strain is both polymyxin-resistant and multidrug-resistant.

[0083] Specifically, on LB agar plates where bacteria had already grown, a colony was inoculated into 3 ml of LB broth and incubated overnight at 37°C and 220 rpm for 16 hours. Then, 50 ml of a 1 / 1000 dilution culture medium solution was prepared in a 250 ml flask, to which 0.5% triphenyltetrazolium chloride (TTC) and 16 μg / ml polymyxin B (PMB) were added and mixed. The solution was then aliquoted into 96-well plates at 198 μl per well. Finally, each different candidate compound was adjusted to a concentration of 5 μM and aliquoted into each well at 2 μl, then mixed. The plates were observed using a phenotypic microarray at 37°C for 24 hours, and the OD values ​​were measured again using a multi-functional microplate reader for comparison.

[0084] As a result, a compound with the activity of improving the sensitivity of bacteria to polymyxin antibiotics (the compound of chemical formula 2 below) was screened out and named PA108 for subsequent experiments.

[0085] [Chemical Formula 2]

[0086]

[0087] 1-2. Confirmation of the activity of PA108 derivatives as antibacterial supplements

[0088] To determine the structural correlations of the PA108 compounds' activities, 14 PA108 derivatives were prepared (see Table 1), and the synergistic effect of PMB with these derivatives was verified by measuring bacterial respiration rates. The PA108 derivatives were either provided by the compound library of the Korea Institute of Chemical Technology or synthesized directly.

[0089] Specifically, on LB agar plates where bacteria had already grown, a colony was inoculated into 3 ml of LB broth and incubated overnight at 37°C and 220 rpm for 16 hours. Then, 50 ml of a 1 / 1000 dilution of culture medium was prepared in a 250 ml flask, to which 0.5% triphenyltetrazolium chloride (TTC) and 16 μg / ml polymyxin B (PMB) were added and mixed. The solution was then aliquoted into 96-well plates at 198 μl per well. Finally, each different candidate compound was adjusted to a concentration of 5 μM and aliquoted into each well at 2 μl, then mixed. The plates were observed using a phenotypic microarray at 37°C for 24 hours, and the OD values ​​were measured again using a multi-functional microplate reader for comparison.

[0090] The results showed that, apart from the original PA108, the derivatives did not exhibit any significant effect in killing strain R357. Figure 1 ).

[0091] Table 1

[0092]

[0093]

[0094] Example 2: Confirmation of the antibacterial growth effect of PA108

[0095] To confirm the antibacterial activity of PA108, the cellular respiration rate was measured using the method described in Example 1, thereby confirming the bacterial growth inhibition effect.

[0096] First, a colony of bacteria was inoculated into 3 ml of LB broth on an LB agar plate containing the bacteria and incubated overnight at 220 rpm at 37°C for 16 hours. Then, 50 ml of a 1 / 1000 dilution of culture medium was prepared in a 250 ml flask, and 0.5% triphenyltetrazolium chloride (TTC) was added and mixed. The solution was then aliquoted into 96-well plates at 198 μl per well. 16 μg / mL PMB and 5 μM PA108 were each aliquoted at 2 μl per well, adjusting the concentrations of PMB (16 μg / mL) and PA108 (5 μM) to three wells. Each treatment was repeated three times, then the samples were mixed, observed at 37°C for 24 hours using a phenotypic microarray, and the OD values ​​were measured again using a multi-functional microplate reader. The results were then compared.

[0097] As a result, since the minimum inhibitory concentration (MIC) of strain R357 against PMB was 64 μg / mL, it was confirmed that the low concentration of 16 μg / mL had no inhibitory effect on cell growth. Figure 2(Black). Furthermore, it was confirmed that PA108 (a developed polymyxin-based antibacterial supplement) did not inhibit autologous cell growth at a concentration of 5 μM. Figure 2 (blue). However, when treated with both PMB and PA108 simultaneously, cellular respiration was confirmed to be effectively inhibited. Figure 2 ,red).

[0098] Example 3: Confirmation of the cell death-increasing effect of PA108

[0099] Antibiotics primarily function in two ways: one is as an inhibitory agent, which suppresses bacterial growth without killing the bacteria; the other is as a bactericidal agent, which kills the bacteria. To verify whether treatment with PA108 in combination with polymyxin actually increased the bactericidal effect, bacterial viability tests were conducted.

[0100] First, a colony was inoculated into 3 ml of LB broth on an LB agar plate and incubated overnight at 37°C and 220 rpm for 16 hours. Then, 1% of the culture was diluted into a 250 ml Erlenmeyer flask and incubated until OD = 0.5. 3 ml of the growth medium was aliquoted into three 15 ml round culture tubes to prepare and distribute three treatment groups: one containing 16 μg / mL PMB and 5 μM PA108, and the other containing PMB (16 μg / mL) and PA108 (5 μM). 100 μl of each treatment group was spread onto each of the three LB agar plates at 1, 4, 7, and 12 hours, and incubated at 37°C and 220 rpm to confirm colony-forming units (CFU).

[0101] As a result, similar to the results for cellular respiration rate, single treatments with PMB or PA108 did not show any effect in killing the R357b strain. Figure 3 (Black, blue). However, it was confirmed that in the treatment group using both PMB and PA108, cell death was caused ( Figure 3 (Red). In addition, it was confirmed that, based on 14 hours, the bactericidal effect of the simultaneous treatment group was 107 times that of the control group and the single treatment group, and the final bactericidal effect was 99.99999%.

[0102] Example 4: Confirmation of the synergistic antibacterial effect of polymyxin antibiotics and PA108

[0103] To determine whether PA108 would exhibit a synergistic effect when combined with polymyxin antibiotics, a checkerboard test was conducted, and the fractionated inhibitory concentration index (FICI) was determined based on the test results.

[0104] First, inoculate 3-4 colonies onto LB agar plates into 4-5 ml of LB broth and incubate overnight at 37°C and 220 rpm for 16 hours. Then, prepare 5 ml of a 1 / 10000 dilution of culture medium in 15 ml round culture tubes and incubate at 37°C and 220 rpm until the OD value reaches 0.08-0.1. Next, dilute the solution to 1 / 20 and aliquot 196 μl into each well of a 96-well plate. Allocate 2 μl each of PA108 and PMB to each well, at concentrations of 0-160 μM and 0-64 μg / ml, respectively. After mixing, incubate at 37°C for 18 hours and measure the OD value using a multi-functional microplate reader.

[0105] Next, to confirm the synergistic effect, the fractionated inhibitory concentration (FIC) index was calculated using a standard equation. This index assesses the effect of the combination of a compound and an antibiotic on efficacy by comparing the MIC value with the MIC value of the compound alone. The standard equation is: Combination of compound A alone / MIC of combination of compound A and compound B alone / MIC of compound B = FIC A + FIC B = FIC index. MIC and FICI values ​​are shown in Table 2.

[0106] It is confirmed that when the FICI value is below 0.5, the effect is synergistic; when the FICI value is between 0.5 and 4.0, the effect is moderate; and when the FICI value is above 4, the effect is antagonistic.

[0107] Table 2

[0108]

[0109] The results confirmed that, since the FICI values ​​of PA108 and PMB were both 0.093, the killing effect on drug-resistant bacteria was synergistic. Figure 4 ).

[0110] Example 5: Confirmation of the morphological cell death effect of polymyxin antibiotics and PA108

[0111] To confirm the morphological changes of bacteria when PA108 was combined with polymyxin antibiotics, the morphological changes were observed using fluorescence microscopy and scanning electron microscopy.

[0112] First, a colony was inoculated into 3 ml of LB broth on an LB agar plate and incubated overnight at 37°C and 220 rpm for 16 hours. Then, 1% of the medium was diluted in a 250 ml Erlenmeyer flask and incubated until OD = 0.5. Cells were then treated with 16 μg / ml PMB and 5 μM PA108, respectively, and incubated for 9 hours. The cells were centrifuged at 8,000 × g for 10 minutes at 4°C, washed three times, and then resuspended in PBS. 3 ml of the cell suspension was fixed and observed using a scanning electron microscope. The LIVE / DEAD BacLight bacterial viability kit (Cat#.L7007, Invitrogen, Waltham, Massachusetts, USA) was used to add SYTO9 (67mM, 3μL), which fluoresces green when it binds to the DNA and RNA of live cells, and propidium iodide (PI) (1.67mM, 3μL), which fluoresces red when it binds to the DNA and RNA of dead cells. The final volume was 3ml for each sample. The samples were then incubated in a dark room at room temperature for 15 hours and observed under a fluorescence microscope.

[0113] As a result, green fluorescence was observed in the single-treatment groups (PMB or PA08) under fluorescence microscopy, confirming that cells did not die as in the control group. However, red fluorescence was observed in the treatment groups treated with both PMB and PA108, thus confirming cell death. Figure 5 (See the image above).

[0114] Furthermore, when observed using a scanning electron microscope, it was confirmed that, like the control group, the cells treated with either PMB or PA08 alone showed no damage to the cell surface and retained their intact morphology. However, it was confirmed that in the treatment group treated with both PMB and PA108, cells shrank, and intracellular substances leaked out, resulting in disruption of cell shape and structure. Figure 5 (See the image below).

[0115] Example 6: Confirmation of nephrotoxicity of PA108

[0116] Because polymyxin antibiotics are known to be nephrotoxic, human-derived kidney cells (ACHN) are used. The nephrotoxicity of PMB and PA108 was determined using CRL-1611.

[0117] First, use 2×10 4 ACHN kidney cells were cultured at 1 cell / well for 24 hours, and the medium was replaced with a mixture of PMB or PA108 in all wells. Cell viability was assessed for cytotoxicity by measuring cell titer glo as the ratio of ATP at each time point to that at 0 hours after treatment with PMB or PA108 for 0, 24, 48, 72, and 96 hours.

[0118] The results confirmed that, under PMB conditions, renal cell viability began to decrease from a concentration of 50 μg / mL. Figure 6 (Left figure). For polymyxin-resistant strain R357, nephrotoxicity was expected at concentrations of 64 μg / ml, which would demonstrate therapeutic efficacy in R357 infections, given the minimum inhibitory concentration of polymyxin is 64 μg / ml. However, in the case of PA108, no nephrotoxicity was observed at a working concentration of 5 μM, and even at higher concentrations, renal cell survival was shown to be above 80%, unlike PMB (…). Figure 6 (See right figure). Therefore, when treated with PMB and PA108 simultaneously, even a concentration of 16 μg / mL of PMB showed bactericidal activity against polymyxin-resistant strain R357. Thus, it is expected that no nephrotoxicity will be observed when treating with the combination of PMB and PA108.

[0119] Example 7: Confirmation of survival rate after PA108 treatment in a mouse sepsis model

[0120] In a mouse model of infection with polymyxin-resistant strain R357, we investigated whether simultaneous treatment with PA108 and PMB was effective in treating sepsis caused by R357 infection.

[0121] Specifically, 5×10 7 R357 strain was intraperitoneally injected into 6-week-old C57BL / 6 mice to establish a mouse model of R357 infection-induced sepsis. The mice were then intraperitoneally injected with 100 μg / kg dose of PMB and 60 μg / kg dose of PA108, either alone or in combination, to determine the survival rate of the mice.

[0122] The results confirmed that all mice died within 42 hours in the control group, the PMB single-treatment group, and the PA108 single-treatment group. However, in the group treated with both PMB and PA108, the mice survived for up to 168 hours without showing any pathological symptoms. Figure 7 Furthermore, as a result of autopsy of mice in the group treated with both PMB and PA108, no R357 strain was detected in the mice's organs, thus confirming that simultaneous treatment with PMB and PA108 completely eliminated the R357 strain.

[0123] Example 8: Confirmation of the efficacy of the antimicrobial supplement in other species of polymyxin-resistant bacteria.

[0124] To determine whether there was a synergistic effect when PA108 and polymyxin antibiotics were used simultaneously to treat polymyxin-resistant strains other than strain R357, a checkerboard test was conducted, and the fractionated inhibitory concentration index (FICI) was determined based on the results.

[0125] First, on LB agar plates containing the bacteria (Klebsiella pneumoniae SCH530, SCH740, SCH777; Pseudomonas aeruginosa SMC-U9, SMC-U10, SMC-U11), 3 to 4 colonies were inoculated into 4 to 5 ml of LB broth and incubated overnight at 37°C and 220 rpm for 16 hours. The next day, 5 ml of medium diluted to 1 / 10000 was incubated in 15 ml round culture tubes at 37°C and 220 rpm until the OD value reached 0.08–0.1. The culture was then diluted to 1 / 20 and aliquoted into 96-well plates at 196 μl each. PA108 and PMB were aliquoted into each well at concentrations of 0–80 μM and 0–128 μg / mL, respectively. After mixing, the plates were incubated at 37°C for 18 hours, and the OD values ​​were measured using a multi-functional microplate reader.

[0126] Next, the FICI, MIC, and FICI values ​​were calculated using the same method as in Example 4, as shown in Table 3 below:

[0127] Table 3

[0128]

[0129] As a result, since the FICI values ​​of PA108 and PMB were below 0.5 (below 0.5 is the reference value for synergistic effect among all strains), it was confirmed that the effect of PA108 and PMB in killing other Gram-negative bacteria (Klebsiella pneumoniae SCH530, SCH740, SCH777, Pseudomonas aeruginosa SMC-U9, SMC-U10, SMC-U11) was a synergistic effect of PA108 and PMB. Figure 8 ).

[0130] Example 9 confirmed the changes in gene expression and the mechanism of action by simultaneously treating the patient with polymyxin antibiotics and PA108.

[0131] Transcriptome analysis confirmed that when PA108 was used in combination with the polymyxin antibiotic PMB to treat drug-resistant bacteria, the expression of the main genes responsible for the death of residual drug-resistant bacteria changed.

[0132] First, inoculate one colony into 3 ml of LB broth on an LB agar plate and incubate overnight at 37°C and 220 rpm for 16 hours. Then, dilute 1% of the culture medium into a 250 ml Erlenmeyer flask and incubate until OD = 0.5. Aliquot 5 ml of the culture medium into three 15 ml round culture tubes and treat them with 16 μg / ml PMB and 5 μM PA108, respectively, and 16 μg / ml PMB and 5 μM PA108, respectively, and incubate at 37°C and 220 rpm for 1 hour. Then, wash the bacteria three times with PBS, centrifuge at 4°C and 8,000 × g for 10 minutes, and store at -80°C.

[0133] Total RNA was extracted using the TruSeq Stranded Total RNA Sample Preparation Kit (including Ribo-ZeroH / M / R), and 150 bp cDNA fragments were synthesized to prepare a library for RNA sequencing. The quality of the synthesized cDNA library was assessed using an Agilent 2100 BioAnalyzer (Agilent, CA, USA). After amplifying the denatured template clusters, the DNA fragments were paired and sequenced at both ends using an Illumina Novaseq 6000 (Illumina, CA, USA). For transcriptome data analysis, reads were filtered, and the filtered reads were mapped to a reference genome using the STAR v.2.4.0b aligner. Subsequently, to measure gene expression levels, gene expression was quantified using the KAPA Library Quantification Kit (Kapa Biosystems, MA, USA) according to the manufacturer's library quantification protocol.

[0134] To identify differentially expressed genes (DEGs), Cufflinks v2.1.1 was used, gene expression levels were measured using a gene annotation database, and gene expression level count data were generated using HTSeq-count v0.6.1p1. In the generated data, genes with q values ​​less than 0.05 were identified as differentially expressed genes using the TCC R package.

[0135] Gene ontology (GO) analysis was performed on genes identified as differentially expressed genes (DEGs), classifying them into three categories: biological processes, molecular functions, and cellular components, and providing information on gene function.

[0136] As a result, significant gene changes were observed in all PMB single-treatment groups, PA108 single-treatment groups, and PMB and PA108 simultaneous treatment groups compared with the control group. Figure 9Furthermore, gene ontology (GO) analysis confirmed that when PMB and PA108 were used simultaneously, the expression levels of genes involved in zinc ion binding, monosomy kinase binding, and iron ion binding, as well as genes involved in peroxidase activity, may have significantly decreased, while the expression levels of genes involved in transmembrane transport, particularly those involved in ATPase-coupled sulfate transmembrane transporter activity, significantly increased. Figure 10 ).

[0137] It is speculated that when treated with low doses of the antibiotic PMB, a physical and chemical environment is created that allows for easier penetration of external substances. Furthermore, because PA108 can more easily penetrate into the space surrounding the cytoplasm, the expression of genes related to the cell membrane’s functions of protein oxidation and the transport of various nutrients is reduced, thus exhibiting a cell death effect.

[0138] Furthermore, it is speculated that superoxide (a reactive oxygen species that exhibits a high mutation rate and growth defects and acts as a toxin and hydrogen peroxide) induces intracellular oxidative stress, leading to a decrease in the expression of genes related to the activity of reactive oxygen species that regulate enzymes to control the response, thereby disrupting cellular homeostasis and thus exhibiting a cell death effect.

[0139] Furthermore, it is speculated that the expression of genes related to iron ion binding and protein breakdown, as well as genes related to phenylacetic acid catabolism, is suppressed, thereby inhibiting the synthesis of sideropores. PMB and PA108, which cross the cell membrane, bind to certain proteins and DNA, reducing metabolism and exhibiting cell death effects.

[0140] The antibacterial composition of the present invention uses a low concentration of antibiotic (PMBB) that has no direct effect on bacteria to improve cell membrane fluidity. Bacterial reactive oxygen species are overproduced through a substance (PA108) that enters the cytoplasm, disrupting cell homeostasis, thereby reducing cell permeability and leading to cell death.

[0141] Example 10 confirmed changes in the bacterial membrane by using polymyxin antibiotics and PA108 simultaneously.

[0142] Polymyxin E (colistin) specifically binds to the phospholipids and lipopolysaccharide (LPS) of the outer membrane of Gram-negative bacteria, exhibiting bactericidal activity. However, colistin-resistant bacteria exhibit resistance due to altered LPS levels, leading to decreased affinity between colistin and the bacterial outer membrane. Therefore, this study aims to confirm whether the addition of PA108 alters bacterial transport and permeability.

[0143] Specifically, for sample preparation, bacterial cultures grown for 16 hours were washed and resuspended in 0.01M PBS at pH 7.4. In the same buffer solution, the absorbance of the bacterial suspension at 600 nm was normalized to 0.5, and PI9 (cat. P1304MP, Thermo Fisher Scientific, Waltham, Massachusetts, USA) was added to a final concentration of 1.67 μM. After incubation at 37°C for 30 minutes, 198 μl of fluorescently labeled bacterial cells, along with 16 μg / ml PMB and 2 μl of 5 μM PA108, were added to a 96-well plate. After another 30 minutes of incubation, fluorescence was measured using a multi-functional microplate reader within the fluorescence measurement wavelength range of 535 nm excitation and 615 nm emission to measure membrane permeability.

[0144] The cell membrane potential was resuspended in 5 mM HEPES (pH 7.0, +5 mM glucose), and the potential-sensitive pigment DiSC3(5) (3,3'-dipropylthiadicocyanine iodide) (50 μM, Cat.D306, Invitrogen, Carlsbad, California) was added. The cells were incubated for 30 minutes, and then the fluorescence was measured using a multifunctional microplate reader to measure the membrane potential.

[0145] The results confirmed that combined treatment with PMB and PA108 altered the bacterial cell membrane potential. Figure 11 (See right figure), but it does not affect the overall membrane permeability ( Figure 11 (Left image).

[0146] The results showed that low-dose PMB treatment did not change the membrane permeability that leads to bacterial death, but rather created a physicochemical environment that made lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria unstable, making it easier for foreign substances to enter. At the same time, the treated PA108 entered the cell membrane, forming ion channels or pores in the cell membrane, causing high membrane depolarization.

[0147] The results showed that simultaneous treatment with PA108 as an antibacterial supplement to PMB caused damage to the lipid bilayer region of the bacterial cell membrane, leading to bacterial death.

Claims

1. Use as an active ingredient in the preparation of a medicament as an antibacterial supplement to improve bacterial susceptibility to polymyxin antibiotics, represented by the following chemical formula 1. in, The polymyxin antibiotics are polymyxin B or polymyxin E: [Chemical Formula 1] in, X is a halogen atom. R1 is a hydrogen atom, and R2 is an alkyl group having 1 to 3 carbon atoms.

2. The use according to claim 1, wherein, X is a chlorine atom, and R2 is a methyl group.

3. The use according to claim 1, wherein, The bacteria in question are Gram-negative.

4. The use according to claim 3, wherein, The Gram-negative bacteria are Escherichia sp., Acinetobacter sp., Pseudomonas sp., or Klebsiella sp.

5. The use according to claim 4, wherein, The Escherichia genus is selected from at least one of the group consisting of Escherichia coli, Escherichia albertii, Escherichia blattae, Escherichia fergusonii, Escherichia hermannii, and Escherichia vulneris; The Acinetobacter genus is selected from at least one of the following groups: Acinetobacter baumannii, Acinetobacter junii, Acinetobacter boissieri, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Acinetobacter nosocomialis, Acinetobacter schindleri, and Acinetobacter ursingii. The genus *Pseudomonas* is selected from at least one of the group consisting of *Pseudomonas aeruginosa*, *Pseudomonas fluorescens*, *Pseudomonas putida*, *Pseudomonas chlororaphis*, *Pseudomanas pertucinogena*, *Pseudomanas stutzeri*, and *Pseudomanas syringae*. The Klebsiella genus is selected from at least one of the group consisting of Klebsiella pneumoniae, Klebsiella granulomatis, Klebsiella oxytoca, and Klebsiella terrigena.

6. The use according to claim 3, wherein, The Gram-negative bacteria are polymyxin-resistant or multidrug-resistant bacteria.

7. Use of the antimicrobial supplement and polymyxin antibiotic as described in claim 1 in the preparation of antimicrobial compositions.

8. The use according to claim 7, wherein, The composition causes cell death in Gram-negative bacteria.

9. The use according to claim 7, wherein, The composition depolarizes the cell membrane of Gram-negative bacteria to disrupt the cell membrane.

10. Use of the antimicrobial supplement and polymyxin antibiotic as described in claim 1 in the preparation of a pharmaceutical composition for the prevention or treatment of organ damage caused by sepsis or septic shock.

11. The use according to claim 10, wherein, The sepsis or septic shock is caused by bacterial infection with Escherichia coli or Acinetobacter spp.

12. The use according to claim 10, wherein, The organ is selected from at least one of the group consisting of the liver, kidneys, and lungs.

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