WO2013058441A1 - Non-genetic antibiotic resistant inhibitor - Google Patents

Abstract

The present invention relates to a compound giving sensitivity against antibiotics to microorganisms having non-genetic antibiotic resistance; to a composition including the compound for inhibiting non-genetic antibiotic resistance; to antibiotic aids and antibiotics including the compound, a composition for the prevention or treatment of microbial infectious diseases, which includes the compound as an effective ingredient; to a method for killing non-genetic antibiotic resistant microorganisms including a step of treating non-genetic antibiotic resistant microorganisms with antibiotics and the compound; and to a method for treating microbial infectious diseases including a step of treating an individual suffering from diseases caused by an infection from non-genetic antibiotic resistant microorganisms by using the compound together with antibiotics. Since the compound of the present invention can prevent secondary infection by inhibiting non-genetic antibiotic resistance exhibited when various antibiotics are administered, the compound can be effectively used for the prevention or treatment of microbial infection using antibiotics.

Description

Non-genetic antibiotic resistance inhibitors

The present invention relates to a non-genetic antibiotic resistance inhibitor, and more particularly, the present invention relates to a compound capable of imparting sensitivity to antibiotics to a microorganism having a non-genetic antibiotic resistance, a composition for inhibiting a non-genetic antibiotic resistance comprising the compound, Antibiotic adjuvant comprising the compound, antibiotics and pharmaceutical compositions for preventing or treating microbial infectious diseases comprising the compound as an active ingredient, non-genetic antibiotic resistant microorganism comprising the step of treating the antibiotic and the compound to a non-genetic antibiotic resistant microorganism The present invention relates to a method for treating microbial infectious diseases comprising the step of treating the compound with an antibiotic in an individual caused by the killing method and the infection of the non-genetic antibiotic resistant microorganism.

An antibiotic is a generic term for a substance that kills or inhibits growth of microorganisms or bacteria, and generally means a metabolite produced by a microorganism that inhibits or kills the development of other microorganisms in a small amount. These antibiotics can be traced back to ancient Greek times, and can be found in propolis, extracts from herbs, etc., but scientific and systematic research was carried out after Fleming's discovery of penicillin in 1928. These antibiotics can be classified according to various criteria, and can be classified into bacteriostatic drugs that inhibit the growth of bacteria and bactericidal drugs that can kill the bacteria themselves based on their effects on the bacteria. In addition, according to chemical properties, antibacterial antibiotics such as penicillin and cephalosporin, antifungal antibiotics such as griseofulvin and fucidin, and macrolides extracted from bacteria and aminoglycosides (aminoglycoside) ), Tetracycline and chloramphenicol such as mycin antibiotics.

In addition, penicillin has fewer side effects than other antibiotics, but some bacteria produce enzymes that break down penicillin, and artificially synthesized methicillin (methicillin) or oxacillin (oxacillin) is used together. The world's most powerful antibiotic is vancomycin, which was developed in the 1950s when Staphylococcus aureus resistant to methicillin, which is an alternative to penicillin, was spread and developed to treat severe infections of Staphylococcus aureus. Has been used.

However, such vancomycin can not be used for a long time, mainly due to the emergence of antibiotic-resistant bacteria. Antibiotic-resistant bacteria refers to strains that have developed resistance to antibiotics due to genetic or non-genetic causes.Resistant strains of antibiotics have been newly developed and used since the emergence of resistant bacteria against penicillin in the 1960s. Problems that arise have emerged. That is, as with all microorganisms, pathogens are obtained by obtaining mutation resistance or antibiotic resistance genes by self-defense, and the frequency of expression of these resistant bacteria is increasing as the abuse and abuse of antibiotics increases.

These resistant bacteria are classified into two types, one is genetically resistant and the other is non-genetic resistant. First, genetically resistant microorganisms are genetically modified microorganisms that have been transformed to produce proteins that can cause resistance to antibiotics, such as enzymes that can degrade the treated antibiotics, resulting in genetic variation. it means. For example, beta-lactam antibiotics such as penicillin may be degraded by an enzyme called beta-lactamase, and microorganisms producing such beta-lactamase may be decomposed to beta-lactam antibiotics. Resistance can be shown (Korean Patent Publication No. 2010-0130176). Next, the non-genetic resistant bacteria, unlike the genetic cause, have not been identified with the exact cause, but most antibiotics die when antibiotics are treated, but always occur at a constant frequency (about 10 ^-5 to 10 ^-6 ). It means a resistant cell.

In contrast to genetically resistant bacteria that have been studied in more detail, non-genetic resistant bacteria are not known to involve at least direct genetic variation associated with antibiotics, although detailed studies have not been actively studied. In addition, it is known that non-genetic antibiotic-resistant bacteria of the same frequency are produced by inoculating and re-inoculating the resistant microorganisms that survived the antibiotic treatment and then treating the antibiotics again. Until now, non-genetic resistant bacteria have been identified by phenotypic variant. It is expected to occur. Therefore, such non-genetic resistant bacteria have been pointed out as the main cause of secondary infection, and studies to suppress the occurrence of such non-genetic resistant bacteria have been conducted.

For example, WO 2008/073444 discloses that the cause of the non-genetic resistant bacteria is due to the expression of the PhoU protein, and it is disclosed that the generation of the non-genetic resistant bacteria can be suppressed by inhibiting the expression of the protein. / 019736 discloses a method for removing non-genetic resistant bacteria by treating electrochemical treatments alone or in combination with antibiotics.

However, since it is difficult to assume that non-genetic resistant bacteria always express PhoU protein for antibiotics, and it is difficult to perform electrochemical treatments on patients, substantial research has yet to be made to prevent secondary infections caused by non-genetic resistant bacteria. It is not performed until now.

The inventors have identified compounds capable of inhibiting antibiotic resistance in microorganisms in which non-genetic antibiotic resistance occurs, and the compounds themselves do not exhibit antimicrobial activity against microorganisms, but when used in combination with antibiotics, inhibit non-genetic antibiotic resistance. Since it can be, it was confirmed that it can be utilized to effectively treat microbial infections by preventing secondary infection, and completed the present invention.

One object of the present invention is to provide a compound capable of giving microorganisms susceptibility to antibiotics.

Another object of the present invention is to provide a composition for inhibiting non-genetic antibiotic resistance comprising the compound.

Another object of the present invention is to provide an antibiotic adjuvant comprising the compound.

Still another object of the present invention is to provide a pharmaceutical composition for preventing or treating microbial infectious diseases comprising an antibiotic and the compound as an active ingredient.

It is still another object of the present invention to provide a method for killing a non-genetic antibiotic resistant microorganism comprising treating the antibiotic with the non-genetic antibiotic resistant microorganism.

Still another object of the present invention is to provide a method for treating a microbial infectious disease comprising treating the compound with an antibiotic in a subject caused by the infection of a non-genetic antibiotic resistant microorganism.

Compounds of the present invention can prevent secondary infection by inhibiting the non-genetic antibiotic resistance that appears when administered to a variety of antibiotics, it will be widely used to effectively prevent or treat microbial infections using antibiotics.

1A is a graph showing the frequency of antibiotic resistant bacteria in Escherichia coli treated with each antibiotic alone or mixed.

1B is a graph showing the change in the frequency of resistant bacteria of E. coli according to the treatment concentration of norfloxacin.

1C is a graph showing the change in the frequency of resistant bacteria of E. coli according to the treatment concentration of ampicillin.

1D is a graph showing the level of change in cfu / ml of E. coli treated with ampicillin alone, E. coli treated with ampicillin and again treated with ampicillin, and E. coli treated with ampicillin and again treated with norfloxacin to be.

Figure 2 is a schematic diagram showing the process of selecting a compound capable of imparting susceptibility to antibiotics to microorganisms having non-genetic antibiotic resistance of the present invention.

3A is a graph showing the antibiotic resistance inhibitory effect of Compound E04 over time.

3B is a graph showing the antibiotic resistance inhibitory effect of Compound C10 over time.

3C is a graph showing the antibiotic resistance inhibitory effect of Compound H04 over time.

3D is a graph showing the antibiotic resistance inhibition effect according to the treatment concentration change of C10 and E04.

E of FIG. 3 is a photograph showing the anti-life property of the compound exhibiting the inhibitory effect.

4A is a graph showing the antibiotic effect of norfloxacin against E. coli over the treatment time of Compound C10.

4B is a graph showing the resistance inhibitory effect of each compound (C10 or E04) for various antibiotics or various strains.

4C is a graph showing the inhibitory effect of the compound of the present invention on the antibiotic resistant bacteria of Pseudomonas aeruginosa treated with norfloxacin.

According to one embodiment, the present invention provides a non-genetic antibiotic resistant microorganism, which is capable of giving antibiotics and susceptibility to antibiotics to the microorganism.

As used herein, the term "non-genetic antibiotic resistance" refers to resistance to antibiotics that appear without genetic factors such as microbial variation, with most microorganisms having a frequency of 10 ^-5 to 10 ^-6 for all antibiotics. This includes cells that have non-genetic antibiotic resistance. This non-genetic antibiotic resistance has been pointed out as a cause of secondary infection after treatment with antibiotics during the disease infection, and after the antibiotic effect disappeared, the cells grow again to increase the population of bacteria, and genetic variation It is considered to be an effect by phenotypic variant because it is not included at all.

As used herein, the term “antibiotic” refers to a compound or composition that reduces the viability of a microorganism or inhibits the growth or proliferation of a microorganism, with most antibiotics being derivatives of modified substances derived from mold. By “inhibiting growth or proliferation” is meant increasing the generation time (ie, the time required for bacterial cells to divide or double) by at least about two times. Preferred antibiotics are those that can increase the generation time by at least about 10 times (eg, at least about 100 times or even indefinitely as the whole cell dies). The antibiotics can be classified according to various criteria, usually according to the time of development or in terms of antimicrobial mechanism. For example, depending on the time of development, the first-generation antibiotics exhibiting a narrow antimicrobial range and acid instability, the second-generation antibiotics that are stable against acids, and extended to the gram-positive and negative bacteria, and even Pseudomonas aeruginosa or entero-negative bacteria It can be classified as a third-generation antibiotic that extends the antimicrobial range. In addition, in terms of antibacterial mechanism, it can be classified into beta-lactam antibiotics, micro-like antibiotics, tetracycline antibiotics, quinolone antibiotics, aminoglycoside antibiotics, glycopeptide antibiotics and sulfonamide antibiotics. The antibiotics used in the present invention include first- or third-generation antibiotics or betalactam antibiotics, microlyke antibiotics, tetracycline antibiotics, quinolone antibiotics, aminoglycoside antibiotics, glycopeptide antibiotics, and sulfonamides. It includes all antibiotics, and is not limited depending on the type of antibiotic.

As used herein, the term "sensitivity to antibiotics" means a property in which one microorganism reacts effectively with a specific antibiotic, resulting in its death. In the present invention, susceptibility to antibiotics means the property of killing by all antibiotics as defined above, rather than specific antibiotics such as beta-lactam antibiotics or quinolone antibiotics.

As used herein, the term "compound capable of imparting sensitivity to antibiotics" refers to a compound which gives one microorganism an effective response to a specific antibiotic and consequently dies. In the present invention, the compound is not particularly limited, but may be any one of the following formulas (I) to (IV), derivatives thereof, pharmaceutically acceptable salts thereof, combinations thereof, and the like.

Formula I: 4- (4- (3-chlorophenyl) piperazin-1-yl) piperidin-3-yl 2-naphthoate (A10)

Formula II: 3- (4- (4-methoxyphenyl) piperazin-1-yl) piperidin-4-yl biphenyl-4-carboxylate (C10)

Formula III: (E) -2-nitro-5-styrylfuran (E04)

Formula IV: 1- (3- (bis (4-fluorophenyl) methoxy) phenyl) piperazine (H04)

According to one embodiment of the present invention, in order to select a compound capable of removing a microorganism that exhibits non-genetic antibiotic resistance, the present inventors may treat ampicillin with E. coli and then add various compounds to reduce the resistance to ampicillin. Compounds were screened (Example 2). As a result, four compounds were selected: 4- (4- (3-chlorophenyl) piperazin-1-yl) piperidin-3-yl 2-naphthoate (A10), 3- (4- (4- methoxyphenyl) piperazin-1-yl) piperidin-4-yl biphenyl-4-carboxylate (C10), (E) -2-nitro-5-styrylfuran (E04) or 1- (3- (bis (4-fluorophenyl) methoxy ) phenyl) piperazine (H04).

The selected compound itself shows little antibacterial activity to Escherichia coli, but when treated with antibiotics such as ampicillin or norfloxacin, it has been shown to exhibit the effect of inhibiting antibiotic resistance, thereby improving the effect of antibiotics (execution) Example 3). In addition, the selected compounds showed the same antibiotic resistance inhibitory effect against other antibiotics such as levofloxacin, cyprofloxacin, and the like, and showed the same antibiotic resistance inhibitory effect against Pseudomonas sp. (Example 4).

Thus, the compounds of the present invention can inhibit antibiotic resistance occurring in most microorganisms, and this resistance inhibitory effect can be applied to most antibiotics, thereby preventing secondary infection due to non-genetic antibiotic resistance generated in microorganisms. It is anticipated that the present invention was first identified by the present inventors that this secondary infection prevention effect can be universally applied to most microorganisms and most antibiotics.

According to another embodiment, the present invention provides a composition for inhibiting non-genetic antibiotic resistance or an antibiotic adjuvant comprising the compound.

As described above, the compound provided in the present invention can inhibit antibiotic resistance occurring in most microorganisms, and this resistance inhibitory effect can be applied to most antibiotics, and thus antibiotic resistance using the composition containing the compound. It is possible to effectively remove the remaining microorganisms.

The composition may be included alone or in combination with a compound provided in the present invention, and may be used in combination with a desired antibiotic to remove microorganisms in a desired environment.

In addition, preferably in order to induce effective killing of the microorganism, it may be provided in the form of a composition comprising the compound and a known antibiotic, the compound may be included alone or in combination, the antibiotic is also known beta lactam Antibiotics, microlye antibiotics, tetracycline antibiotics, quinolone antibiotics, aminoglycoside antibiotics, glycopeptide antibiotics and sulfonamide antibiotics may be included alone or in combination.

In addition, to counteract microorganisms with hereditary antibiotic resistance, an inhibitor to hereditary antibiotic resistance may further be included. For example, it may further include a beta lactamase inhibitor for killing microorganisms having hereditary antibiotic resistance to betagramtak antibiotic.

According to another embodiment, the present invention provides a pharmaceutical composition for the prophylaxis or treatment of microbial infectious diseases comprising the compound and an antibiotic as an active ingredient.

As used herein, the term "microbial infectious disease" means a disease caused by pathogenic microorganisms parasitic to a subject. The microbial infectious disease used in the present invention is a type of microorganism such as prokaryotic or eukaryotic, components of pathogenic substances such as microbial toxins or extracellular toxins, sole or combined causes by infection of microorganisms. The type of pathogenesis mechanism, cystitis or sepsis is not limited by the location of the onset, etc., but is preferably cholera caused by cholera bacteria, bacterial erythritis caused by erythritis, whooping cough caused by pertussis, typhoid caused by typhoid bacteria, or larynx caused by diphtheria bacteria. Diphtheria and non-diphtheria, visceral plague and pulmonary plague caused by plague bacteria, scarlet fever caused by hemolytic streptococci, isolated, sepsis and dermatitis, tuberculosis caused by Mycobacterium tuberculosis, arthritis tuberculosis, Bacterial food poisoning caused by renal tuberculosis and tuberculous meningitis, Salmonella and enteritis Vibrio and the like.

In addition, the pharmaceutical composition may further include an inhibitor for the above-described genetic antibiotic resistance in order to improve the prevention or treatment effect of the microbial infectious disease.

In addition, the pharmaceutical composition may further comprise a pharmaceutically acceptable diluent, excipient or carrier. The composition comprising a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid form preparations for oral administration include tablet pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or lactose in one or more compounds. ) And gelatin. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups, and include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Can be. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The composition is any one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilizers and suppositories. It may have a formulation.

According to another embodiment, the present invention provides a microorganism comprising treating said compound with an antibiotic to give said microorganism susceptibility to antibiotics to an individual caused by a disease caused by infection with a non-genetic antibiotic resistant microorganism. Provided are methods of treating infectious diseases.

In order to carry out the treatment method, the composition of the present invention may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" of the present invention refers to a disease at a reasonable benefit / risk ratio applicable to medical treatment. Means sufficient to be treated, and effective dose levels are factors, including individual type and severity, age, sex, activity of the drug, sensitivity to the drug, time of administration, route and rate of administration, duration of treatment, drug used concurrently. And other factors well known in the medical arts. In order to carry out the treatment method, the composition of the present invention may be administered individually or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered in a single or multiple times. . In consideration of all the above factors, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects.

According to another embodiment for achieving the above object, the present invention provides a method for killing a non-genetic antibiotic resistant microorganism comprising the step of treating the antibiotic and the compound to a non-genetic antibiotic resistant microorganism.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention more specifically, but the scope of the present invention is not limited by these examples.

Example 1: Confirmation of non-genetic antibiotic resistance

In order to confirm non-genetic antibiotic resistance, E. coli was treated with antibiotics, and the frequency of antibiotic-resistant bacteria was measured and compared.

Specifically, E. coli was inoculated into 50 ml LB medium and cultured to a cell concentration of about 10 ⁸ cells / ml, and the cultures were dispensed in 3 ml portions of the culture vessel, and ampicillin (0 to 0), which is a beta-lactam antibiotic. 500 µg / ml) and Norfloxacin (0-10 µg / ml), a Quinolones family of antibiotics, were treated separately or together, and then the frequency of E. coli resistant to each antibiotic was compared (A in FIG. 1). . 1A is a graph showing the frequency of antibiotic resistant bacteria in Escherichia coli treated with each antibiotic alone or mixed. As shown in FIG. 1A, even when each antibiotic was used alone or in combination, it was confirmed that resistant bacteria appeared at a frequency of 10 ⁻⁵ or less.

In addition, the frequency change of the strains resistant to each antibiotic was measured according to the concentration change of each antibiotic (B and C of FIG. 1). 1B is a graph showing the change in the frequency of resistant bacteria of E. coli according to the treatment concentration of norfloxacin, Figure 1 C is a graph showing the change in the frequency of resistant bacteria of E. coli according to the treatment concentration of ampicillin. As shown in B and C of Figure 1, even if increasing the treatment concentration of each antibiotic was found to be resistant bacteria at a frequency of 10 ^-5 to 10 ^-6 .

Finally, after treatment with E. coli Ampicillin, and then treated with ampicillin or norfloxacin again, the level of change in cfu / ml of E. coli over time was measured (D in Fig. 1). 1D is a graph showing the level of change in cfu / ml of E. coli treated with ampicillin alone, E. coli treated with ampicillin and again treated with ampicillin, and E. coli treated with ampicillin and again treated with norfloxacin to be. As shown in D of FIG. 1, it was confirmed that a certain level of E. coli remained even after treatment with antibiotics.

Therefore, even if the antibiotic alone or mixed treatment, it was confirmed that the strain resistant to the antibiotic survives.

Example 2: Selection of compounds capable of imparting antibiotic sensitivity to microorganisms having non-genetic antibiotic resistance

Escherichia coli was cultured to a cell concentration of about 10 ⁸ cells / ml in an LB medium, and 200 µl of the culture solution was divided into 96-well plates, respectively, and then ampicillin (100 µg / ml) alone (control) or ampicillin and about 6800 compounds. Various compounds selected from the library consisting of (25μM) were treated together (experimental group) and incubated for 9 hours at 37 ℃. After the incubation was completed, 10 μl of the culture was dispensed into an LB plate medium and cultured, and the colonies of the experimental group treated with the antibiotic and the compound were screened and selected when the colonies were smaller than the colonies of the control group treated with the antibiotic alone. It was. As a result, 52 compounds could be selected first.

On the other hand, the same experiment as described above, except for treating each of the first 52 selected compounds to E. coli each divided into the 96-well plate, to obtain each colony, the size of the colony Compounds that did not shrink were reselected. In other words, 9 compounds which did not have antibiotic ability against Escherichia coli by the compound itself were secondary screened.

Of the nine selected compounds, the final selection of compounds capable of reducing the frequency of resistant bacteria to 10% or less compared to the frequency of resistant bacteria remaining when ampicillin alone was treated (FIG. 2). Figure 2 is a schematic diagram showing the process of selecting a compound capable of imparting susceptibility to antibiotics to microorganisms having non-genetic antibiotic resistance of the present invention. As shown in Figure 2, four compounds (C10, H04, A10 and E04) were finally selected, and their specific names are as follows: 4- (4- (3-chlorophenyl) piperazin-1-yl) piperidin- 3-yl 2-naphthoate (A10), 3- (4- (4-methoxyphenyl) piperazin-1-yl) piperidin-4-yl biphenyl-4-carboxylate (C10), (E) -2-nitro-5- styrylfuran (E04) and 1- (3- (bis (4-fluorophenyl) methoxy) phenyl) piperazine (H04)

Structural formulas of the selected compounds are shown in the following formulas (I) to (IV):

Formula I: 4- (4- (3-chlorophenyl) piperazin-1-yl) piperidin-3-yl 2-naphthoate (A10)

Formula II: 3- (4- (4-methoxyphenyl) piperazin-1-yl) piperidin-4-yl biphenyl-4-carboxylate (C10)

Formula III: (E) -2-nitro-5-styrylfuran (E04)

Formula IV: 1- (3- (bis (4-fluorophenyl) methoxy) phenyl) piperazine (H04)

Example 3 Inhibitory Effects of Selected Compounds on Non-Dielectric Antibiotic Resistance

In Example 2, it was intended to confirm whether the compound selected for non-genetic antibiotic resistance inhibitory effect.

To this end, a time dependent killing assay was performed using C10, H04 or E04, which can be obtained in large quantities by the inventors of the four selected compounds.

Specifically, E. coli was inoculated into 50 ml LB medium and cultured to a cell concentration of about 10 ⁸ cells / ml. The cultures were dispensed in 3 ml portions of the culture vessel, and Norfloxacin (5 µg), a quinolones-based antibiotic, was used. / ML) was incubated at 37 ° C. for 9 hours with or without C10, H04 or E04 (25 μM each) alone or in combination. After the incubation was completed, 10 μl of the culture was dispensed into an LB plate medium and cultured, and the generated colonies were counted every given time, and the counted colonies were measured in terms of cfu / ml (FIGS. 3A to 3C). .

First, FIG. 3A is a graph showing the antibiotic resistance inhibitory effect of Compound E04 over time. As shown in FIG. 3A, E. coli and antibiotic-treated E. coli treated with only the compound showed substantially similar levels of cfu / ml, whereas E. coli treated with norfloxacin alone or with E04 was significantly lower. It was confirmed to represent the level of cfu / ml.

Next, FIG. 3B is a graph showing the antibiotic resistance inhibitory effect of Compound C10 over time. As shown in FIG. 3B, E. coli and antibiotic-treated E. coli showed the same level of cfu / ml, but E. coli treated with norfloxacin alone or with C10 was significantly lower. It was confirmed that cfu / ml of.

3C is a graph showing the antibiotic resistance inhibitory effect of Compound H04 over time. As shown in FIG. 3C, E. coli and antibiotic-treated E. coli showed the same level of cfu / ml, but E. coli treated with norfloxacin alone or with H04 was significantly lower. It was confirmed that cfu / ml of.

On the other hand, the antibiotic resistance inhibitory effect of the compounds C10 and E04 showing excellent resistance inhibitory effect was compared for each treatment concentration of the compound (D in Fig. 3). 3D is a graph showing the antibiotic resistance inhibition effect according to the treatment concentration change of C10 and E04. As shown in D of FIG. 3, both compounds showed an effect of inhibiting antibiotic resistance in proportion to the treatment concentration, and it was confirmed that C10 exhibited an effect of inhibiting antibiotic resistance superior to E04.

In addition, it was confirmed whether the compounds C10 and E04, which exhibit the excellent resistance inhibitory effect, exhibit antimicrobial properties on their own. Specifically, the cultured Escherichia coli was sequentially diluted 10-fold (10 ² to 10 ⁸ cells / ml), and only the compounds were added to each of them to confirm whether the compounds exhibited their own anti-bioactivity (E of FIG. 3). . Each compound was treated for 9 hours at a concentration of 25 μM. Figure 3 E is a photograph showing the anti-life activity of the compound exhibiting a resistance inhibitory effect, None represents E. coli treated with nothing, C10 represents E. coli treated with Compound C10, E04 represents E. coli treated with Compound E04 . As shown in E of FIG. 3, C10 showed no antibiosis against E. coli, but E04 showed a certain level of anti-bioactivity against E. coli. However, it was confirmed that the E. coli at the concentration carried out in the present invention does not exhibit any antibiosis.

Therefore, all of the selected compounds do not exhibit antimicrobial properties by themselves, but when used with antibiotics, it was found that they have an effect of inhibiting antibiotic resistance.

Example 4 Confirmation of Scalability of Non-Dielectric Antibiotic Inhibitory Effect

Example 4-1: Comparison of Resistance Inhibitory Effect According to Treatment Time of Compound

In order to compare the inhibitory effect of non-genetic antibiotic resistance according to the compound treatment time, the following experiment was performed.

Specifically, E. coli was inoculated into 50 ml LB medium and cultured to a cell concentration of about 10 ⁸ cells / ml. The cultures were dispensed in 3 ml portions of the culture vessel, and Norfloxacin (5 µg), a quinolones-based antibiotic, was used. / Ml) was treated to confirm the production of non-genetic antibiotic resistance strains, C10 (5μM) was added alone and incubated at 37 ℃. Then, using the treated Escherichia coli, a time dependent killing assay was performed over time (FIG. 4A). 4A is a graph showing the antibiotic effect of norfloxacin against E. coli over the treatment time of Compound C10.

As shown in Fig. 4A, after treatment of the present invention even after sufficient resistance to E. coli treated with norfloxacin, it was confirmed that the generated resistance is lost. In addition, the compound shows an antibiotic resistance inhibitory effect irrespective of its treatment time, but it was found that the faster the treatment time, the more effectively inhibits antibiotic resistance.

Example 4-2 Inhibitory Effects of Non-Genetic Antibiotic Resistance on Other Antibiotics and Other Strains

The present inventors tried to determine whether the non-genetic antibiotic resistance inhibitory effect of the compound identified in Example 3 can be applied to other antibiotics or other strains as well.

Specifically, Escherichia coli is cultured to a cell concentration of about 10 ⁸ cells / ml in the LB medium, and the culture is dispensed in 3 ml into the culture vessel, quinolones-based levofloxacin (5 µg / ml) or cyprofloxacin (5 μg / ml) alone or in combination with compound (C10 or E04, 25 μM) and incubated, and then the frequency of E. coli resistant to each antibiotic was measured (FIG. 4B). In addition, Pseudomonas aeruginosa, one of the Gram-negative bacteria similar to Escherichia coli, was cultured to a cell concentration of about 10 ⁸ cells / ml in MHB medium, and the culture was dispensed into 3 ml of the culture vessel. Fluxacin (5 μg / ml) alone or in combination with a compound (C10 or E04, 25 μM) was incubated and then the frequency of strains resistant to each antibiotic was measured (FIG. 4B).

4B is a graph showing the resistance inhibitory effect of each compound (C10 or E04) for various antibiotics or various strains.

As shown in Fig. 4B, the compound of the present invention exhibits a resistance inhibitory effect to other antibiotics of the quinolones family other than norfloxacin (levofloxacin or cyprofloxacin), and to other strains of the Gram-negative bacteria family other than Escherichia coli. It also confirmed that the antibiotic resistance inhibitory effect.

Therefore, the compounds showing the antibiotic resistance inhibitory effect of the present invention was found to exhibit the same antibiotic resistance inhibitory effect against various antibiotics and various strains.

Example 4-3 Determination of Non-Dielectric Antibiotic Resistance of Compounds in the Stop Phase of Microorganisms

The following experiments were conducted to determine whether the compounds can inhibit non-genetic antibiotic resistance even in stationary phases other than the logarithmic phase of bacteria.

Specifically, E. coli is cultured to a cell concentration of about 10 ¹⁰ cells / ㎖ in LB medium, washed with PBS and diluted to a cell concentration of about 10 ⁸ cells / ㎖ in fresh LB medium and the dilution to the culture vessel Dispense 3 ml each and treat and incubate quinolones-based norfloxacin (5 μg / ml) alone or in combination with a compound (C10 or E04, 25 μM), Frequency was measured (FIG. 4C). 4C is a graph showing the inhibitory effect of the compound of the present invention on the antibiotic resistant bacteria of E. coli treated with norfloxacin. As shown in Figure 4C, it was confirmed that the compound can inhibit the non-genetic antibiotic resistance even in the stationary phase other than the logarithmic growth phase of the bacteria.

Claims (10)

A composition for inhibiting non-genetic antibiotic resistance, comprising at least one compound selected from the group consisting of any one compound of Formulas I to IV, derivatives thereof, and combinations thereof: [Formula I]

[Formula II]

[Formula III]

[Formula IV]

An antibiotic adjuvant comprising at least one compound selected from the group consisting of a compound of any of Formulas I-IV, derivatives thereof, and combinations thereof: [Formula I]

[Formula II]

[Formula III]

[Formula IV]

A pharmaceutical composition for preventing or treating microbial infectious diseases comprising an antibiotic and at least one compound selected from the group consisting of a compound of any one of Formulas I to IV, derivatives thereof, and combinations thereof: [Formula I]

[Formula II]

[Formula III]

[Formula IV]

The method according to any one of claims 1 to 3, The antibiotic is selected from the group consisting of beta-lactam antibiotics, micro-like antibiotics, tetracycline antibiotics, quinolone antibiotics, aminoglycoside antibiotics, glycopeptide antibiotics, sulfonamide antibiotics and combinations thereof Composition. The method of claim 3, The composition further comprises an inhibitor for hereditary antibiotic resistance. The method of claim 3, The composition further comprises a pharmaceutically acceptable diluent, excipient or carrier. At least one compound selected from the group consisting of a compound of any one of Formulas I to IV, derivatives thereof, and combinations thereof, which can impart antibiotic sensitivity to the non-genetic antibiotic resistant microorganisms; Method of killing non-genetic antibiotic resistant microorganisms comprising the steps of: [Formula I]

[Formula II]

[Formula III]

[Formula IV]

The method of claim 7, wherein The antibiotic is selected from the group consisting of beta-lactam antibiotics, micro-like antibiotics, tetracycline antibiotics, quinolone antibiotics, aminoglycoside antibiotics, glycopeptide antibiotics, sulfonamide antibiotics and combinations thereof . The method of claim 7, wherein And treating the inhibitor to hereditary antibiotic resistance. Composed of a compound of any one of formulas (I) to (IV), derivatives thereof, and combinations thereof capable of conferring antibiotic susceptibility to the microorganism together with antibiotics in a subject caused by an infection by a non-genetic antibiotic resistant microorganism A method of treating a microbial infectious disease comprising treating at least one compound selected from the group: [Formula I]

[Formula II]

[Formula III]

[Formula IV]

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