JP2015113290A - Drug efflux pump inhibitor and application thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel pharmaceutical or sterilization agent, effective for therapy and prevention of bacterial infection caused by multi-drug-resistant Pseudomonas aeruginosa (MDRP), etc.SOLUTION: Provided are: a bacterial efflux pump inhibitor of MexY of Pseudomonas aeruginosa or of a bacterium having high homology with MexY, the bacterial efflux pump inhibitor comprising patulin; an antibacterial agent to a bacterium having the efflux pump, the antibacterial agent comprising a combination of patulin and antibacterial drugs which may be a substrate of the efflux pump; a method for identifying multi-drug-resistant mechanism of pathogenic Pseudomonas aeruginosa strain, which is characterized by separating Pseudomonas aeruginosa strains from a patient infected by multi-drug-resistant Pseudomonas aeruginosa, respectively/independently and combinationally contacting the strains with patulin and antibacterial drugs which may be a substrate of MexY efflux pump of Pseudomonas aeruginosa, and deciding bacterial strains having synergistically inhibited growth in the case of having combination contact relative to the case of respective and independent contact, as strains having multi-drug-resistance mechanism by hyperfunction of MexY efflux pump; and the like.

Description

  The present invention relates to inhibitors of bacterial drug efflux pumps and uses thereof. More specifically, the present invention relates to inhibition of RND-type efflux pumps of bacteria comprising Patulin, Pseudomonas aeruginosa MexY, or bacteria having high homology with MexY and using aminoglycoside antibacterial drugs as efflux substrates The present invention also relates to an agent for preventing and treating infection of bacteria such as Pseudomonas aeruginosa (especially multidrug-resistant Pseudomonas aeruginosa), which is a combination of an agent, patulin, and an antibacterial agent discharged by the discharge pump.

Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) is a glucose non-fermenting Gram-negative bacilli and is resident in human skin, respiratory organs, gastrointestinal tract and the like. Although it is hardly pathogenic to healthy people, it causes refractory infections to susceptible hosts such as sick and elderly people with reduced immunity.
Multi-Drug Resistant Pseudomonas aeruginosa (MDRP) is a group of three types of carbapenem antibiotics, fluoroquinolone antibiotics, and aminoglycoside antibiotics that can be expected to have strong antibacterial activity against Pseudomonas aeruginosa. A strain that has acquired resistance to all antimicrobial agents. At present, the isolation rate of MDRP is estimated to be 1 to several percent in Japan. Once an infection has developed, there are few effective antibacterial agents, so it is difficult to treat and many cases die.

Pseudomonas aeruginosa is highly resistant and multidrug resistant by combining an endogenous resistance mechanism with an acquired resistance mechanism such as plasmid transmission from other resistant bacteria. Endogenous refers to a resistance mechanism based on a gene originally possessed on a chromosome, and refers to acquiring resistance by, for example, mutating the gene or its expression regulatory mechanism by continuous use of a specific antibacterial drug. Endogenous tolerance mechanisms include β-lactam drug-degrading enzyme overproduction, decreased drug permeability due to decreased expression of permeabilized outer membrane proteins, decreased affinity due to mutation of antimicrobial target protein, drug excretion There is an increase in pump function. Among them, the discharge pump discharges various antibacterial drugs, and is known to be greatly involved in multidrug resistance.
The most important of the Pseudomonas aeruginosa efflux pump is the RND (Resistance-Nodulation-cell Division) type, which is called three types of proteins (the RND efflux protein (RND) that exists in the inner membrane and is responsible for substrate recognition and transport, and the outer membrane) It is a multi-component efflux pump consisting of channel protein (OEP) and periplasmic protein (MFP) that binds both. There are at least 12 RND-type efflux pump operons in the genome of Pseudomonas aeruginosa. Of these, the mexAB - oprM operon ( mexA encodes MFP, mexB encodes RND, and oprM encodes the OEP component) is mainly expressed in wild strains, but is expressed in any operon. There is a control gene, and its expression may be induced by a substrate, etc., and in a resistant mutant, the suppression of the expression is released by the mutation of the control gene, and the production amount of the efflux pump may be constantly increased. MexXY-OprM (Non-Patent Document 1) (the mexXY operon does not have a unique OEP gene, and OprM is an OEP component) is highly expressed in most MDRPs and contributes to multidrug resistance. It became clear that it was important.

For Pseudomonas aeruginosa that has acquired multidrug resistance by enhancing the function of the drug efflux pump, if the concentration of the antibacterial drug in the microbial cell can be increased by inhibiting the efflux pump, the sensitivity to the antibacterial drug that has become resistant can be increased. It can be recovered and is effective in the treatment and prevention of MDRP infection.
To date, several efflux pump inhibitors for Pseudomonas aeruginosa have been reported (see, for example, Patent Documents 1 to 3), all of which target MexB, and report on drugs that inhibit MexY. There is nothing. Although the substrate specificity of each efflux pump is sweet and recognizes a wide variety of compounds, the substrate specificity varies depending on the RND protein. Aztreonam is specific to MexB, whereas aminoglycoside antibacterials Known to be specific for MexY (quinolone, macrolide, chloramphenicol, and tetracycline antibacterials are excreted by both MexB and MexY; . Therefore, in order to sufficiently inhibit the excretion of fluoroquinolone antibacterial agents, in addition to the MexB inhibitor, a MexY inhibitor is also required. Not practical. Aminoglycoside antibacterial drugs are one of the few antibacterial drugs that are not excreted by a plurality of pumps, and drugs that inhibit MexY can be expected as concomitant drugs of aminoglycoside drugs.

Patulin (Patulin), the genus Penicillium (Penicillium), Aspergillus (Aspergillus), Byssochlamys genus (Byssochlamys), is a mycotoxin variety of microorganisms produced belonging to Paecilomyces (Paecilomyces), known as mycotoxins apple . It is acutely toxic to many animal species, with an oral LD ₅₀ in rats and mice of 29-55 mg / kg, and the US Food and Drug Administration (FDA) has set the content in juice drinks to 50 μg / L. It is regulated (Non-Patent Document 4). In Japan, equivalent standards are established based on the Food Sanitation Law. Further, as subacute toxicity, gastrointestinal abnormalities, anorexia, weight loss, etc. have been reported, but no toxicity has been observed when administered to monkeys at 5 to 500 μg / kg / day (Non-patent Document 4).
Patulin has broad antibacterial activity against many bacteria and some fungi (see, for example, Non-Patent Document 5), and clinical trials for colds have been conducted before (Non-Patent Document 6). As shown above, since it may show toxicity at a high dose, it has not been considered as a medicine for a long time. In addition, regarding the antibacterial activity against Bordetella bronchiseptica, a synergistic effect by the combined use of rifampicin and bothromycin has been reported (Non-patent Document 7), but only a transcription or translation inhibition is suggested as a mechanism, and an efflux pump or Pseudomonas aeruginosa is suggested. Nothing is known about the effect on bacteria.

International Publication No. 2002/087589 Pamphlet International Publication No. 2005/113579 Pamphlet International Publication No. 2005/089738 Pamphlet

Masuda, N. et al., Antimicrob. Agents Chemother., 44 (12): 3322-3327 (2000) Morita, Y. et al., J. Gen. Appl.Microbiol., 47: 27-32 (2001) Li, X.-Z. and Nikaido, H., Drugs, 69 (12): 1555-1623 (2009) Puel, O. et al., Toxins, 2: 613-631 (2010) Wallen, L.L. et al., J. Antibiot., 33 (7): 767-769 (1980) Chalmers, I. and Clarke, M., Int. J. Epidemiol., 33 (2): 253-260 (2004) Dulaney, E.L. and Jacobsen, C.A., J. Antibiot., 40 (8): 1211-1212 (1987)

  An object of the present invention is to provide a novel drug or disinfectant effective for the treatment and prevention of Pseudomonas aeruginosa, especially MDRP infection.

In order to achieve the above object, the present inventors have focused on substances that can inhibit the MexXY-OprM efflux pump, focusing on the fact that amikacin, an aminoglycoside antibacterial agent, is an excretion substrate specific to MexY. As a result of the search, it was found that patulin, which has weak antibacterial activity against MDRP itself, markedly inhibits the growth of multiple MDRP strains in the presence of amikacin at a concentration that does not exhibit antibacterial activity. In order to verify that this growth-inhibiting effect is based on inhibition of the MexY efflux pump, amikacin (MexY-specific efflux substrate; membrane) was equidistantly spaced in four directions around the patulin-impregnated disk using the disk diffusion method. Discs impregnated with good permeability), aztreonam (MexB specific excretion substrate; better membrane permeability), erythromycin (more likely to be a substrate for MexY than MexB; slightly poor membrane permeability) or vancomycin (difficult membrane permeability) When the formation of a blocking circle was observed, patulin showed a remarkable synergistic effect with amikacin and a slight combined effect with erythromycin, but did not show a combined effect with aztreonam and vancomycin. Therefore, patulin inhibits the excretion of amikacin, which is a specific substrate of the efflux pump, by inhibiting the MexY efflux pump, not by inhibiting MexB or increasing membrane permeability, thereby reducing the concentration of amikacin in the microbial cells. It was thought that MDRP was changed to amikacin sensitivity by increasing to a level sufficient to exert antibacterial activity. Among Gram-negative bacteria other than Pseudomonas aeruginosa, bacteria having a high homology with MexY and having an RND-type efflux pump using an aminoglycoside antibacterial drug as an effluent substrate are known. Therefore, it was strongly suggested that patulin also inhibits these efflux pumps and can exert a synergistic antibacterial effect against the bacteria when used in combination with an aminoglycoside antibacterial agent or the like.
As a result of further studies based on these findings, the present inventors have completed the present invention.

That is, the present invention provides the following.
[1] A bacterial efflux pump inhibitor comprising patulin, wherein the efflux pump comprises a protein comprising an amino acid sequence having 80% or more similarity to the amino acid sequence represented by SEQ ID NO: 1. An inhibitor having an aminoglycoside antibacterial as a substrate.
[2] The inhibitor according to [1] above, wherein the bacterium is a gram-negative bacterium.
[3] The inhibitor according to the above [1], wherein the discharge pump is a Pseudomonas aeruginosa MexY discharge pump.
[4] An antibacterial agent against bacteria comprising a combination of patulin and an antibacterial agent capable of serving as a substrate for a bacterial efflux pump, wherein the efflux pump comprises 80% or more of the amino acid sequence represented by SEQ ID NO: 1. An antibacterial agent comprising a protein containing a similar amino acid sequence and having an aminoglycoside antibacterial as a substrate.
[5] Antibacterial agents that can serve as substrates for bacterial efflux pumps are quinolones, aminoglycosides, coumermycins, chloramphenicol, lipopeptides, glycylcycline, ketolides, macrolides, oxazolidinones, fosfomycin, streptogramins and tetracyclines. The antibacterial agent according to [4] above, which is one or more selected from the group consisting of:
[6] The antibacterial agent according to the above [4], wherein the antibacterial agent capable of serving as a substrate for a bacterial efflux pump is an aminoglycoside.
[7] The antibacterial agent according to [6] above, wherein the aminoglycoside is amikacin.
[8] The antibacterial agent according to any one of the above [4] to [7], which is used for treatment or prevention of bacterial infection or sterilization of bacteria.
[9] The agent according to any one of [1] to [8] above, wherein the bacterium is multidrug resistant Pseudomonas aeruginosa.
[10] The removal of the bacteria from the object, characterized by contacting the object contaminated with or possibly with the bacteria with patulin and an antibacterial agent capable of serving as a substrate for the efflux pump of the bacteria. A fungal method, wherein the efflux pump comprises a protein comprising an amino acid sequence having 80% or more similarity to the amino acid sequence represented by SEQ ID NO: 1, and uses an aminoglycoside antibacterial agent as a substrate ,Method.
[11] The Pseudomonas aeruginosa strain isolated from a patient infected with multidrug-resistant Pseudomonas aeruginosa is contacted with patulin and an antibacterial agent that can be a substrate for the Pseudomonas aeruginosa MexY efflux pump alone and in combination, In contrast to the case of contact with a single substance, a strain whose growth has been synergistically inhibited when contacted in combination is determined to be a strain having a multidrug resistance mechanism by enhancing the function of the MexY efflux pump. A method for identifying a multidrug resistance mechanism of a pathogenic Pseudomonas aeruginosa strain characterized by the above.

  Patulin can specifically inhibit Pseudomonas aeruginosa MexY efflux pumps and efflux pumps derived from other bacteria with high homology to MexY, so use in combination with antibacterial drugs that can be evacuated by these efflux pumps Thus, it exhibits a remarkable antibacterial action against bacteria such as Pseudomonas aeruginosa, especially MDRP. For example, existing aminoglycoside antibacterials such as amikacin and tobrushan were ineffective or could only be used at high doses for MDRP, but when used in combination with patulin, these existing drugs can be used at low doses It becomes. By using antibacterial drugs that already have resistant bacteria, it is also useful to prevent the emergence of resistant bacteria against polymyxin B and colistin, which are currently effective.

(A) Single antibacterial activity of patulin (2.5-20 μg) and various antibacterial drugs (50 μg), and (B) Amikacin (96 μg / mL) and patulin (2.5-20 μg) or various antibacterial drugs (50 μg) It is a figure which shows the antibacterial activity by combined use. The numbers in the disk indicate the diameter of the blocking circle (mm). P: Patulin, AMK: Amikacin. (C) Antibacterial activity against combination of erythromycin (64 μg / mL) and patulin (2.5-20 μg) or various antibacterial drugs (50 μg) against multidrug-resistant Pseudomonas aeruginosa MDRP No. 74 strain, and (D) chloram It is a figure which shows the antibacterial activity by combined use of a phenicol (128 microgram / mL), patulin (2.5-20 micrograms), or various antimicrobial agents (50 micrograms). The numbers in the disk indicate the diameter of the blocking circle (mm). P: Patulin, EM: Erythromycin, CP: Chloramphenicol. It is a figure which shows the antibacterial activity by combined use with (E) tetracycline (64 microgram / mL), patulin (2.5-20 micrograms), or various antimicrobial agents (50 micrograms) with respect to multidrug resistant Pseudomonas aeruginosa MDRP No. 74 strain. The numbers in the disk indicate the diameter of the blocking circle (mm). P: Patulin, TC: Tetracycline. (A) single antibacterial activity of patulin (2.5-20 μg) and various antibacterial drugs (50 μg) against multidrug-resistant Pseudomonas aeruginosa MDRP No. 50 strain, and (B) amikacin (48 μg / mL) and patulin (2.5 -20 μg) or a combination of various antibacterial drugs (50 μg). The numbers in the disk indicate the diameter of the blocking circle (mm). P: Patulin, AMK: Amikacin. (C) Antibacterial activity by combined use of erythromycin (64 μg / mL) and patulin (2.5-20 μg) or various antibacterial drugs (50 μg) against multi-drug resistant Pseudomonas aeruginosa MDRP No. 50 strain, and (D) chloram It is a figure which shows the antibacterial activity by combined use of a phenicol (128 microgram / mL), patulin (2.5-20 micrograms), or various antimicrobial agents (50 micrograms). The numbers in the disk indicate the diameter of the blocking circle (mm). P: Patulin, EM: Erythromycin, CP: Chloramphenicol. It is a figure which shows the antibacterial activity by combined use with (E) tetracycline (64 microgram / mL), patulin (2.5-20 micrograms), or various antimicrobial agents (50 micrograms) with respect to multidrug resistant Pseudomonas aeruginosa MDRP No. 50 strain. The numbers in the disk indicate the diameter of the blocking circle (mm). P: Patulin, TC: Tetracycline. It is a figure which shows that the combined use effect of patulin and amikacin is due to inhibition of the MexXY-OprM efflux pump by patulin. Sample: Culture extract of patulin producing strain PSTF0045 (20 μL), AMK: amikacin (50 μg), AZT: aztreonam (50 μg), EM: erythromycin (50 μg), VCM: vancomycin (50 μg).

The present invention provides a bacterial efflux pump inhibitor (hereinafter also referred to as the inhibitor of the present invention) comprising patulin.
The “bacterial efflux pump (hereinafter also referred to as the efflux pump of the present invention)” that can be inhibited by the inhibitor of the present invention is a Pseudomonas aeruginosa PAO1 strain MexY protein consisting of the amino acid sequence represented by SEQ ID NO: 1, 80 It is an RND-type efflux pump consisting of a protein with at least% similarity and using an aminoglycoside antibacterial as a substrate. The MexXY-OprM efflux pump system combines MexY as an RND component that functions as a substrate recognition and transport body in the multi-component efflux pump system of Pseudomonas aeruginosa, and an REP component and an OEP component that is an outer membrane channel. It is an evacuation pump containing MexX as an MFP component that is a periplasmic protein. The OEP component can be selected as appropriate, but usually, OprM having a large production amount is often the OEP component. In addition, the “substrate” of the discharge pump means a substance that can be recognized by an RND component such as MexY and discharged from the discharge pump.

Amino acid sequence “similarity” refers to an optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably the algorithm is sequenced for optimal alignment). The ratio of the same amino acid residues and similar amino acid residues to all overlapping amino acid residues in the case of introduction of a gap into one or both of the above. "Similar amino acids" means amino acids that are similar in physicochemical properties, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn) ), Basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), amino acids with small side chains (Gly, Ala, Ser, Thr, Met), etc. Examples include amino acids classified into groups. It is expected that substitution with such similar amino acids will not change the phenotype of the protein (ie, is a conservative amino acid substitution). Specific examples of conservative amino acid substitutions are well known in the art and are described in various literature (see, for example, Bowie et al., Science, 247: 1306-1310 (1990)).
The similarity of amino acid sequences in the present specification was determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; allow gaps; matrix = BLOSUM62; filtering) = OFF), when the amino acid sequence represented by SEQ ID NO: 1 and other amino acid sequences are aligned, the similar amino acid residues (positive: in the case of the same amino acid, Ser and Thr, Ser and Ala, Ser and Asn, Asn and Asp, Asn and His, Asp and Glu, Glu and Gln, Glu and Lys, Gln and Arg, Gln and Lys, His and Tyr, Arg and Lys, Met and Ile, Met and Leu, and Met Val, Ile and Leu, Ile and Val, Leu and Val, Phe and Tyr, Phe and Trp, Tyr and Trp matching) ratio (%) can be calculated.

The amino acid sequence of the MexY protein derived from Pseudomonas aeruginosa PAO1 represented by SEQ ID NO: 1 is registered in the NCBI database as Accession No. NP_250708. The MexY protein is highly conserved between each P. aeruginosa strain and most strains show 99% similarity and identity in the BLAST analysis. In addition to Pseudomonas aeruginosa, there are bacteria that express a drug efflux pump corresponding to MexY. For example, as with Pseudomonas aeruginosa, Achromobacter sp. (Eg A. xylosoxidans , A. baumannii ) and various Burkholderia sp. (Eg B. pseudomallei , B. cepacia complex) A drug efflux pump with approximately 80% similarity and approximately 70% identity to Pseudomonas aeruginosa MexY (eg A. xylosoxidans AxyY (Accession No. EGP45231; 74% identity, 86% similarity) , A. baumannii AdeB , B. pseudomallei (Accession No. YP_006274748; 70% identity, 83% similarity) and B. cepacia complex AmrB, etc.) It is known to be one of the resistance mechanisms against glycoside antibacterial drugs. Therefore, not only Pseudomonas aeruginosa MexY but also a high homology with MexY (ie 80% or more amino acid similarity and / or 70% or more amino acid identity), and a substrate for an antibacterial drug similar to MexY A drug efflux pump possessed by other bacteria having specificity (particularly, using an aminoglycoside as an efflux substrate) is also included in the evacuation pump of the present invention.

Patulin (Patulin) used in the present invention, Penicillium (Penicillium), Aspergillus (Aspergillus), Byssochlamys sp (Byssochlamys), a mycotoxin various microorganisms produced belonging to Paecilomyces (Paecilomyces), the following structural formula (IUPAC name: 4-hydroxy-4H-furo [3,2-c] pyran-2 (6H) -one).

Specific microorganisms that produce patulin include, for example, P. carneum , P. clavigerum , P. concentricum , P. coprobium , P. dipodomyicola , P. expansum , P. glandicola , P. gladioli , P , belonging to the genus Penicillium. . griseofulvum, P. marinum, P . paneum, P. sclerotigenum, P. vulpinum, Aspergillus clavatus belonging to the genus Aspergillus, a. giganteus, a. longivesica , Byssochlamys belonging to Byssochlamys genus. nivea, Paecilomyces saturatus etc. belonging to Paecilomyces For example, but not limited to. Of these, P. expansum , the main pathogen of apple mold poison, can be easily separated from apple fruit. Patulin can be obtained by, for example, cultivating the above patulin-producing bacteria in an appropriate culture solution (eg, YES culture solution (2% yeast extract, 15% sucrose), etc.) at 25 ° C., and concentrating the resulting culture supernatant. Then, extract with chloroform and then with ethyl acetate, combine the extracts and isolate and crystallize by the method of Harwig et al. (Can. Inst. Food Sci. Technol. J., 6: 22-25 (1973)). Can be manufactured. Patulin can also be chemically synthesized by converting arabinose into a protected ketone, followed by a Wittig condensation reaction, followed by an acid-catalyzed cyclization reaction and a dehydration reaction. Alternatively, commercially available patulin (eg, Sigma-Aldrich, product number P1639) may be purchased and used.
In the present specification, patulin may be used in the sense of encompassing analogs thereof in addition to the compound represented by the above structural formula. The analog here is not particularly limited as long as it can specifically inhibit the efflux pump of the present invention, and examples thereof include esters (eg, acetate ester, benzoate ester) and the like, which are metabolized in the animal body. Further, a prodrug type compound that exhibits the above inhibitory activity may also be included therein. Alternatively, precursors in patulin biosynthesis (eg, ascradiol, neopatulin) are also included in the patulin analog. These precursors are, for example, an enzyme gene that catalyzes a reaction for converting a desired precursor into patulin or other precursors existing in a patulin biosynthetic enzyme gene cluster on the patulin-producing bacterial chromosome by a conventional method. It can be purified by a well-known technique such as HPLC from the culture supernatant obtained by deficient culture of the defective strain.

Patulin has antibacterial activity against Pseudomonas aeruginosa, especially MDRP, although it is weak itself (minimum growth inhibitory concentration MIC = 128 μg / mL). Inhibiting the pump suppresses MexY-mediated excretion of aminoglycoside antibacterials, which are MexY excretion substrates that are also added alone at concentrations that do not exhibit antibacterial effects, and reduce the intracellular concentration of these antibacterials Can be maintained at a level sufficient for the
As a result, patulin and aminoglycoside antibacterial drugs are the only drugs that currently have antibacterial effects against unlicensed MDRPs in Japan, even at concentrations that do not exhibit antibacterial effects alone. Antimicrobial effect can be expected against MDRP instead of colistin.
Therefore, patulin can be administered by administration to bacteria infected with antibacterial drugs excreted by the evacuation pump of the present invention such as MexY, preferably Gram-negative pathogens including Pseudomonas aeruginosa, especially MDRP infected patients. Through the specific inhibition of the efflux pump of the present invention, making the bacteria (high) sensitive (detolerant) to the antibacterial agent and treating bacterial infections by administering low doses of existing drugs to enable. That is, patulin is useful as a sensitivity enhancing agent or an imparting agent (detolerizing agent) for the antibacterial agent that is the effluent substrate of the evacuation pump of the present invention.

  Patulin can be used as it is or according to a method known per se, and mixed with a pharmacologically acceptable carrier, such as tablets (including sugar-coated tablets and film-coated tablets), powders, granules, capsules (soft capsules). Including), orally disintegrating tablets, liquids, injections, suppositories, sustained-release agents, patches, etc., orally or parenterally (eg, topical, rectal, intravenous administration, etc.) it can.

The content of patulin in the inhibitor of the present invention is not particularly limited, and is about 0.01 to 100% by weight of the whole preparation.
There are no particular limitations on the dose and frequency of administration of the inhibitor, and the appropriate dose and frequency of administration depending on the type and severity of the microbial infection, the presence or absence of the underlying disease, the patient's age, weight, etc. Can be selected. For example, when orally administered as an agent for enhancing or imparting antimicrobial susceptibility to patients with MDRP infection, about 0.005 to about 5 mg / kg / day as patulin for adults, preferably about 0.01 to about 0.5 ng / kg / day can be administered once a day or divided into 2-3 times a day.

Examples of the pharmacologically acceptable carrier that can be used in the production of the inhibitor of the present invention include various organic or inorganic carrier substances that are commonly used as pharmaceutical materials. For example, excipients, lubricants in solid formulations, Examples thereof include binders, disintegrants, water-soluble polymers, basic inorganic salts; solvents, solubilizers, suspending agents, isotonic agents, buffering agents, soothing agents, etc. in liquid preparations. Further, if necessary, additives such as ordinary preservatives, antioxidants, colorants, sweeteners, sour agents, foaming agents, and fragrances can be used.
Examples of the excipient include lactose, sucrose, D-mannitol, starch, corn starch, crystalline cellulose, light anhydrous silicic acid, titanium oxide and the like.
Examples of the lubricant include magnesium stearate, sucrose fatty acid ester, polyethylene glycol, talc, stearic acid and the like.
Examples of the binder include hydroxypropylcellulose, hydroxypropylmethylcellulose, crystalline cellulose, starch, polyvinylpyrrolidone, gum arabic powder, gelatin, pullulan, and low-substituted hydroxypropylcellulose.
Disintegrants include (1) crospovidone, (2) croscarmellose sodium (FMC-Asahi Kasei), carmellose calcium (Gotoku Pharmaceutical) and other super disintegrants, (3) carboxymethyl starch sodium ( Examples include Matsutani Chemical Co., Ltd.), (4) Low-substituted hydroxypropyl cellulose (eg, Shin-Etsu Chemical Co., Ltd.), and (5) Corn starch. The “crospovidone” is crosslinked with a chemical name of 1-ethenyl-2-pyrrolidinone homopolymer, including those called polyvinylpolypyrrolidone (PVPP) and 1-vinyl-2-pyrrolidinone homopolymer. Examples of the polymer may include any of the following polymers, and specific examples include Kollidon CL (manufactured by BASF), Polyplastidon XL (manufactured by ISP), Polyplastidone XL-10 (manufactured by ISP), and Polyplastidone. INF-10 (manufactured by ISP).
Examples of the water-soluble polymer include ethanol-soluble water-soluble polymers [eg, cellulose derivatives such as hydroxypropyl cellulose (hereinafter sometimes referred to as HPC), polyvinylpyrrolidone, etc.], ethanol-insoluble water-soluble polymers [eg, , Hydroxypropylmethylcellulose (hereinafter sometimes referred to as HPMC), cellulose derivatives such as methylcellulose and sodium carboxymethylcellulose, sodium polyacrylate, polyvinyl alcohol, sodium alginate, guar gum and the like].
Examples of the basic inorganic salt include basic inorganic salts of sodium, potassium, magnesium and / or calcium. Preferred is a basic inorganic salt of magnesium and / or calcium. More preferred is a basic inorganic salt of magnesium. Examples of the basic inorganic salt of sodium include sodium carbonate, sodium hydrogen carbonate, disodium hydrogen phosphate and the like. Examples of the basic inorganic salt of potassium include potassium carbonate and potassium hydrogen carbonate. Examples of the basic inorganic salt of magnesium include heavy magnesium carbonate, magnesium carbonate, magnesium oxide, magnesium hydroxide, magnesium metasilicate aluminate, magnesium silicate, magnesium aluminate, synthetic hydrotalcite [Mg ₆ Al ₂ ( OH) ₁₆ · CO ₃ · 4H ₂ O] and alumina hydroxide / magnesium, preferably heavy magnesium carbonate, magnesium carbonate, magnesium oxide, magnesium hydroxide and the like. Examples of the basic inorganic salt of calcium include precipitated calcium carbonate and calcium hydroxide.
Examples of the solvent include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil, olive oil and the like.
Examples of the solubilizer include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like.
Examples of the suspending agent include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate; for example, polyvinyl alcohol, polyvinylpyrrolidone And hydrophilic polymers such as sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, and the like.
Examples of the isotonic agent include glucose, D-sorbitol, sodium chloride, glycerin, D-mannitol and the like.
Examples of the buffer include buffer solutions of phosphate, acetate, carbonate, citrate and the like.
Examples of soothing agents include benzyl alcohol.
Examples of the preservative include p-hydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, and the like.
Examples of the antioxidant include sulfite, ascorbic acid, α-tocopherol and the like.
Examples of the colorant include edible pigments such as edible yellow No. 5, edible red No. 2 and edible blue No. 2; edible lake pigments and red peppers.
Examples of the sweetener include saccharin sodium, dipotassium glycyrrhizin, aspartame, stevia, thaumatin and the like.
Examples of the sour agent include citric acid (anhydrous citric acid), tartaric acid, malic acid and the like.
Examples of the foaming agent include sodium bicarbonate.
As a fragrance | flavor, any of a synthetic material and a natural product may be sufficient, for example, lemon, lime, orange, menthol, strawberry, etc. are mentioned.

  Patulin is compression-molded according to a method known per se, for example, by adding the above-mentioned carrier such as an excipient, a disintegrant, a binder or a lubricant, and then, if necessary, taste masking, enteric or long-lasting. For the purpose, an oral preparation can be obtained by coating by a method known per se. In the case of an enteric preparation, an intermediate layer may be provided between the enteric layer and the drug-containing layer by a method known per se for the purpose of separating both layers.

  For example, when patulin is used as an orally disintegrating tablet, for example, a core containing crystalline cellulose and lactose is coated with patulin and, if necessary, a basic inorganic salt, and further coated with a coating layer containing a water-soluble polymer. The resulting composition is coated with an enteric coating layer containing polyethylene glycol, then coated with an enteric coating layer containing triethyl citrate, further coated with an enteric coating layer containing polyethylene glycol, and finally coated with mannitol. Thus, fine particles can be obtained, and the resulting fine particles and additives can be mixed and manufactured by a molding method.

  Examples of the enteric coating layer include cellulose acetate phthalate (CAP), hydroxypropylmethylcellulose phthalate, hydroxymethylcellulose acetate succinate, and a methacrylic acid copolymer [for example, Eudragit L30D-55 (trade name; Laem Co., Ltd., Kollicoat MAE30DP (trade name; manufactured by BASF), Polykid PA30 (trade name; manufactured by Sanyo Kasei Co., Ltd.), etc.], water-based enteric polymer bases such as carboxymethyl ethyl cellulose and shellac; methacrylic acid copolymer [For example, Eudragit NE30D (trade name), Eudragit RL30D (trade name), Eudragit RS30D (trade name), etc.], etc .; water-soluble polymers; triethyl citrate, polyethylene glycol, acetylated monoglyceride, triacetin A kind of plasticizer such as castor oil or It includes a layer formed of such a mixture species or more.

  Examples of the additive include water-soluble sugar alcohols (for example, sorbitol, mannitol, maltitol, reduced starch saccharified product, xylitol, reduced palatinose, erythritol, etc.), crystalline cellulose (for example, Theorus KG 801, Avicel PH 101, Avicel PH 102, Avicel PH 301, Avicel PH 302, Avicel RC-591 (crystalline cellulose / carmellose sodium), etc., low-substituted hydroxypropyl cellulose (for example, LH-22, LH-32, LH-23, LH- 33 (Shin-Etsu Chemical Co., Ltd.) and mixtures thereof, etc.), binders, acidulants, foaming agents, sweeteners, fragrances, lubricants, colorants, stabilizers, excipients, disintegration Agents are also used.

As described above, patulin functions as a sensitization enhancing or imparting agent (detolerizing agent) of a bacterium resistant to an antibacterial agent that is an effluent substrate of the evacuation pump of the present invention. Therefore, the present invention also provides an antibacterial agent against the bacteria (hereinafter also referred to as a concomitant agent of the present invention), which is a combination of patulin and an antibacterial agent that can serve as a substrate for the bacterial efflux pump. The concomitant drug of the present invention can be used for any strain as long as it is a bacterium that expresses the efflux pump of the present invention described above. However, the function of the efflux pump is enhanced (for example, in the case of Pseudomonas aeruginosa, mexX − It is particularly useful against Pseudomonas aeruginosa that has acquired multidrug resistance by including mutations in the mexY operon regulatory gene and increased expression of the MexY efflux pump adapted to growth stress.

The “antibacterial agent that can serve as a substrate for the evacuation pump of the present invention” is an antibacterial agent that is excreted from the cells by the evacuation pump of the present invention, even if it is a substrate specific to the evacuation pump. May be a typical substrate. Preferably, it is an antibacterial agent having higher selectivity for the evacuation pump of the present invention than other drug evacuation pumps (for example, in the case of Pseudomonas aeruginosa, MexAB-OprM, MexCD-OprJ, etc.), more preferably the evacuation pump of the present invention. It is a specific antibacterial drug. Antibacterial agents specific to the efflux pump of the present invention include, for example, aminoglycoside antibacterial agents (for example, amikacin (AMK), tobramycin (TOB), streptomycin (SM), neomycin (NM), kanamycin (KM), Gentamicin (GM) and the like. Non-exhaust pump specific antibacterials include, for example, quinolone antibacterials (eg ciprofloxacin (CPFX), levofloxacin (LVFX), nalidixic acid (NA), pyromidine acid (PA), pipemidine acid ( PPA), sinoxacin (CNX), norfloxacin (NFLX), ofloxacin (OFLX), enoxacin (ENX), tosufloxacin (TFLX), sparfloxacin (SPFX), etc.), macrolide antibacterials (eg erythromycin (EM)) , Oleandomycin (OM), spiramycin (SPM), clarithromycin (CAM), etc., tetracycline antibiotics (eg, tetracycline (TC), chlortetracycline (CTC), oxytetracycline (OTC), minocycline (MINO) ), Lincomycin (LCM), chloramphenocol (CP), carbenicillin (CBPC) and sulbenicillin (SBPC), most penicillin antibacterials (eg, Most cephem antibacterial drugs except cefrosin (CFS) and ceftazidime (CAZ)) (cillin G (PCG), cloxacillin (MCIPC), nafcillin (NFPC), amoxiline (AMPC), piperacillin (PIPC), ampicillin (ABPC), etc. (Examples: cefamandole (CMD), cefuroxime (CXM), cefoperazone (CPZ), cefotaxime (CTX), ceftidixime (CZX), ceftriazone (CTRX), cefpirom (CPR), cefepime (CFPM), cefozopran (CZOP), Carbapenem antibacterial drugs (eg, meropenem (MEPM), doripenem (DRPM), etc.), excluding cefoseris (CFSL), cefoxitin (CFX), etc., imipenem (IPM), etc. ), Sulfamethoxazal (SMX) and the like, and rifampicin (RFP).
On the other hand, antibacterial agents that are not discharged by the discharge pump of the present invention include some β-lactam antibacterial agents specific to MexB of Pseudomonas aeruginosa (eg, carbenicillin (CBPC), sulbenicillin (SBPC), ceftazidime (CAZ) , Moxalactam (LMOX), aztreonam (AZT), etc.), as well as novobiocin (NV), cefsulosin (CFS), and flomoxef (FMOS).
Carbapenem antibacterials, fluoroquinolone antibacterials and aminoglycoside antibacterials are inherently antibacterials against Pseudomonas aeruginosa, and amino acids such as amikacin specific to MexY efflux pumps. Glycoside antibacterial agents are particularly preferably exemplified as the combined antibacterial agents with patulin.

  The antibacterial agent that can serve as a substrate for the efflux pump of the present invention is mixed with patulin according to a method known per se, and a single pharmaceutical composition (for example, tablet, powder, granule, capsule (including soft capsule), liquid, injection Suppositories, suppositories, sustained-release agents, etc.) may be formulated into a combination, ie, combined, or each may be formulated separately and administered to the same subject simultaneously or with a time lag. The dosage of antibacterial drugs varies depending on the subject of administration, administration route, symptoms, etc., and also varies depending on the degree of selectivity of the antibacterial efflux pump. In the infectious disease which becomes, it can be used by the dosage normally used.

  The concomitant drug of the present invention is a bacterium having the evacuation pump of the present invention, preferably Gram-negative pathogens such as Pseudomonas aeruginosa, especially MDRP infections (eg, respiratory infections such as pneumonia, liver / biliary system infections, Wound infection, pyelonephritis / complex urinary tract infection, tonsillitis, bacterial otitis media / sinusitis, skin / soft tissue infection, sepsis, endocarditis, peritonitis, pleurisy, nucleus pulpitis, osteomyelitis, etc. ) In addition to being used for the treatment of patients, it can be administered to asymptomatic patients admitted to hospitals where nosocomial infections of the bacteria have occurred (for patients undergoing ventilator management, etc.) Since contact infection can also occur, it can also be administered to healthcare professionals as a contact infection prevention), facilities, equipment, etc. in facilities where nosocomial infections have occurred (for example, taps, sinks, ice makers, humidifiers, nebulizers) In humid environments such as endoscopes and ventilators For disinfection of equipment / equipment, contaminated disinfectant, etc.) and for prevention of nosocomial infections, for example, in the form of liquids, sprays, antibacterial sheets, etc. And can be applied to contaminated or potentially contaminated sites.

The present invention also provides an antibacterial agent (preferably a MexY-specific antibacterial agent such as an aminoglycoside antibacterial agent) that can be used as a substrate for patulin and Pseudomonas aeruginosa MexY efflux pumps. Antibacterial drugs) in combination with each other alone and in combination with each other, the strains that have been synergistically inhibited when contacted in combination with each other are the MexY efflux pumps. A method for identifying a multidrug resistance mechanism of a pathogenic Pseudomonas aeruginosa strain, characterized in that it is determined that the strain has a multidrug resistance mechanism due to enhancement of the function of.
Isolation of pathogens from patients with MDRP infection can be achieved by plating sputum, pus, urine, feces, blood, ascites, pleural effusion, meninges, etc. collected from the patient on, for example, NAC agar medium (Nissui). Although it can be performed using as an index the formation of a colony that produces a fluorescent substance, other methods well known in the art may be used. A colony of Pseudomonas aeruginosa is picked up and subjected to liquid culture to prepare a sample culture solution.
The antibacterial activity test can be performed, for example, by a disk diffusion method, a micro liquid dilution method, or a combination thereof. For example, a clinical isolate, the test subject, is seeded on a solid medium containing no drug and a solid medium containing either patulin or an antibacterial drug discharged by the MexY discharge pump. Place a disk impregnated with patulin or the antibacterial agent, and in the latter, place a disk impregnated with the drug not contained in the solid medium, incubate for an appropriate period of time, and prevent growth growth around each disk When the diameter of the inhibition is synergistically increased in the latter plate, the subject has a multidrug resistance mechanism by enhancing the function of the MexY efflux pump, so patulin and MexY It can be predicted that a treatment protocol in combination with antimicrobials excreted by the drainage pump will be effective. At this time, the concentration of the drug contained in the solid medium and the amount of the drug impregnated in the disk can be appropriately selected based on the MIC value determined in advance by the micro liquid dilution method.
Alternatively, if the method shown in Example 2 described later is used, the multidrug resistance mechanism of clinical isolates can be analyzed more strictly and multilaterally.

  The present invention also provides a kit for identification of the multidrug resistance mechanism of the pathogenic Pseudomonas aeruginosa strain. The kit contains at least an antibacterial agent (preferably a MexY-specific antibacterial agent such as an aminoglycoside antibacterial agent) that can serve as a substrate for patulin and MexY efflux pump, Circular filter paper used for disk diffusion method, antibacterial agent for treatment of MDRP infection for evaluation of antibacterial activity by combination (polymyxin B (PL-B), colistin (CL), AZT / AMK combination etc.) may be included. Alternatively, in a kit for analyzing a multidrug resistance mechanism more strictly and multilaterally, in addition to the above-described configuration, an antibacterial agent specific to MexB (eg, AZT etc.), a non-specific MexY / MexB antibacterial agent ( For example: EM), an antibacterial agent (eg, VCM) that becomes resistant when the membrane permeability decreases may be further included.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to these in any meaning.

Example 1 Combined effect of patulin and MexY excretion substrate on MDRP (1) Combined effect of patulin and amikacin
Compared with Pseudomonas standard strain Pseudomonas aeruginosa PAO1 (Amikacin MIC = 1.25 μg / mL) as MDRP, the expression of the mexB gene and mexY gene is enhanced, and the oprD gene encoding the outer membrane permeation pore protein MDRP No. 74 (Amicacin MIC = 256 μg / mL) and MDRP No. 50 (Amicacin MIC = 128 μg / mL) were used (provided by BML, Inc.). MDRP No. 74 added to Mueller Hinton solid medium (AppliChem) supplemented with antibiotics or with 3/8 amicacin (MDRP No. 74: 96 μg / mL; MDRP No. 50: 48 μg / mL). Alternatively, MDRP No. 50 is seeded, and a plate impregnated with patulin (P; 2.5, 5, 10, 20 μg) or various antibacterial agents 50 μg is placed on each plate and cultured at 37 ° C. for 18 hours. The growth inhibition circle diameter formed around was measured. As a result, patulin showed weak antibacterial activity at 20 μg against any MDRP strain (FIGS. 1-1A and 2-1A). In amikacin-added plates, patulin inhibited MDRP growth in a dose-dependent manner. Even with 2.5 to 10 μg, which did not inhibit the growth of the antibiotic-free plate at all, the growth of MDRP was inhibited as much as or more than 50 μg of polymyxin B (PL-B) (FIGS. 1-1B and 2-1B).
From the above, it was revealed that a synergistic antibacterial effect against MDRP can be obtained by using patulin and amikacin in combination.

(2) Combined effects of patulin and erythromycin
MDRP No. 74 (MIC of erythromycin = 512 μg / mL) and MDRP No. 50 (MIC of erythromycin = 256 μg / mL) were used as MDRP. MDRP No. 74 or MDRP No. to Mueller Hinton solid medium (AppliChem) supplemented with 64 μg / mL erythromycin (MDRP No. 74: 1/8 concentration of MIC, MDRP No. 50: 1/4 concentration of MIC) 50 seeded, put on each plate a disk impregnated with patulin (P; 2.5, 5, 10, 20μg) or various antibacterial drugs 50μg, and incubate at 37 ℃ for 18 hours, forming around each disk The measured growth inhibition circle diameter was measured. As a result, in the erythromycin-added plate, patulin inhibited the growth of MDRP in a dose-dependent manner. Even with 5-10 μg which did not inhibit the growth at all on the antibiotic-free plate (FIGS. 1-1A and 2-1A), the growth of MDRP was inhibited (FIGS. 1-2C and 2-2C).
From the above, it was revealed that antimicrobial activity against MDRP is enhanced by using patulin and erythromycin in combination.

(3) Combined effects of patulin and chloramphenicol
MDRP No. 74 (chloramphenicol MIC = 256 μg / mL) and MDRP No. 50 (chloramphenicol MIC = 256 μg / mL) were used as MDRP. Alternatively, MDRP No. 74 or MDRP No. 50 is seeded on Mueller Hinton solid medium (AppliChem) supplemented with chloramphenicol 128 μg / mL at a concentration of 1/2 of MIC, and patulin (P; 2.5; , 5, 10, 20 μg) or a disc impregnated with 50 μg of various antibacterial agents, and cultured at 37 ° C. for 18 hours, and the growth inhibition circle diameter formed around each disc was measured. As a result, in the chloramphenicol-added plate, patulin inhibited the development of MDRP in a dose-dependent manner. Even with 10 μg that did not inhibit growth on the antibiotic-free plates (FIGS. 1-1A and 2-1A), the growth of MDRP was inhibited (FIGS. 1-2D and 2-2D).
From the above, it was revealed that antimicrobial activity against MDRP is enhanced by using patulin and chloramphenicol in combination.

(4) Combined effect of patulin and tetracycline
MDRP No. 74 (MIC of tetracycline = 128 μg / mL) and MDRP No. 50 (MIC of tetracycline = 128 μg / mL) were used as MDRP. MDRP No. 74 or MDRP No. 50 is seeded on Mueller Hinton solid medium (AppliChem) supplemented with 64 μg / mL of tetracycline at 1/2 concentration of MIC, and patulin (P; 2.5, 5, 10) 20 μg) or discs impregnated with 50 μg of various antibacterial agents were placed, cultured at 37 ° C. for 18 hours, and the growth inhibition circle diameter formed around each disc was measured. As a result, in the tetracycline-added plate, patulin inhibited the growth of MDRP in a dose-dependent manner. Even with 10 μg which did not inhibit growth on the antibiotic-free plate (FIGS. 1-1A and 2-1A), the growth of MDRP was inhibited (FIGS. 1-3E and 2-3E).
From the above, it has been clarified that antimicrobial activity against MDRP is enhanced by using patulin and tetracycline in combination.

Example 2 Confirmation of Mechanism of Patulin / Amikacin Combined Effect In order to elucidate the mechanism of the combined effect of patulin and amikacin on the growth of MDRP observed in Example 1, patulin (patulin-producing bacteria) was analyzed using a disk diffusion method. Centering on a disk impregnated with PSTF0045 culture extract (20 μL), amikacin (AMK; MexY specific efflux substrate with good membrane permeability), aztreonam (AZT; MexB specific efflux substrate) at equal intervals in four directions Good membrane permeability), erythromycin (EM; can be a substrate for both MexB and MexY, but MexY is more selective than MexB. Slightly poor membrane permeability) or vancomycin (VCM; membrane permeability) 50 μg The impregnated disk was placed and cultured, and formation of a blocking circle was observed. As a result, AZT formed a concentric circle concentrically from the center of the disk, whereas in AMK, the area where growth was inhibited was biased closer to the patulin-impregnated disk (FIG. 3). Even in the case of EM having higher selectivity for MexY than MexB, the inhibition circle was slightly inclined toward the patulin-impregnated disk, but the combined effect with patulin was not significant (FIG. 3). In the case of VCM with poor membrane permeability, no inhibition circle was formed, and no combined effect was observed (FIG. 3). Since the combined effect with VCM and EM having poor membrane permeability was absent or weak, it was considered that the combined effect of patulin with amikacin was not due to increased membrane permeability by patulin.
In summary, the combined effect of patulin and amikacin on the antibacterial activity against MDRP is due to the inhibitory action of patulin on the MexY efflux pump, which suppresses the excretion of amikacin outside the cell, and the intracellular concentration of amikacin is antibacterial. It was concluded that it was maintained to a degree sufficient to exert Therefore, it was considered that patulin has an antibacterial activity enhancing effect by combination with not only amikacin but also any antibacterial drug that can be excreted by the MexY excretion pump.

Example 3 Combined effect of patulin and amikacin against Pseudomonas aeruginosa and MDRP As test bacteria, Pseudomonas aeruginosa PAO1 (ATCC 15962), multidrug resistant Pseudomonas aeruginosa strain MDRP NCGM2.S1 (Antimicrob. Agents Chemother., 49 (2005) 3734-3742), PAO1-derived mexAB mexCD - oprJ mexEF - oprN- deficient strain YM34 (FEMS Microbiol. Lett., 202 (2001) 139-143) was used. The concentration of patulin was set to a concentration equal to or lower than the MIC (0 to 64 μg / ml) for each test strain, and the MIC of amikacin in the presence of patulin was tested. The MIC of amikacin alone was 64 μg / ml against MDRP NCGM2.S1, but it decreased to 16 μg / ml in the presence of patulin (64 μg / ml). The MIC of amikacin alone for the PAO1 strain was 2 μg / ml, but decreased to 0.5 μg / ml in the presence of patulin (8 μg / ml). Also for the YM34 strain, the MIC of 4 μg / ml with amikacin alone was reduced to 1 μg / ml in the presence of patulin (2 μg / ml). That is, patulin enhanced the antibacterial action of amikacin against any Pseudomonas aeruginosa (Table 1).

  While the invention has been described with emphasis on preferred embodiments, it will be apparent to those skilled in the art that the preferred embodiments can be modified. The present invention contemplates that the present invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the appended claims.

  The contents of all publications, including the patents and patent application specifications mentioned herein, are hereby incorporated by reference herein to the same extent as if all were expressly cited. .

  According to the present invention, patulin, an inhibitor of the efflux pump of Pseudomonas aeruginosa MexY or other bacteria having high homology thereto, the patulin, which is mainly composed of patulin, and the efflux pump of the present invention Provided is a bacterium having the evacuation pump, preferably a gram-negative pathogen such as Pseudomonas aeruginosa, particularly an MDRP infectious agent for treating or preventing infection, and a sterilizing agent for the bacterium, in combination with an antibacterial agent excreted by . Since patulin has been toxic to humans, it has not been considered for pharmaceutical use so far, but at least this technology can be put into practical use at least for the sterilization of hospital equipment and instruments that can be a source of nosocomial infections. Is possible. In addition, since the antibacterial activity of the concomitant drug is exerted even under sufficiently low dose conditions with no side effects on humans, it is promising as a therapeutic drug (medicine) for MDRP infection.

Claims (11)

  A bacterial efflux pump inhibitor comprising patulin, wherein the efflux pump comprises a protein comprising an amino acid sequence of 80% or more similarity to the amino acid sequence represented by SEQ ID NO: 1, Inhibitors that use carbohydrate antibacterials as substrates.   The inhibitor according to claim 1, wherein the bacterium is a gram-negative bacterium.   The inhibitor according to claim 1, wherein the evacuation pump is a Pseudomonas aeruginosa MexY evacuation pump.   An antibacterial agent against bacteria comprising a combination of patulin and an antibacterial agent capable of serving as a substrate for a bacterial efflux pump, wherein the efflux pump has at least 80% similarity to the amino acid sequence represented by SEQ ID NO: 1. An antibacterial agent comprising a protein containing an amino acid sequence having an aminoglycoside antibacterial agent as a substrate.   Antibacterials that can be substrates for bacterial efflux pumps are from the group consisting of quinolones, aminoglycosides, coumermycin, chloramphenicol, lipopeptides, glycylcycline, ketolide, macrolides, oxazolidinones, fosfomycin, streptogramin and tetracyclines. The antibacterial agent of Claim 4 which is 1 or more types selected.   The antibacterial agent according to claim 4, wherein the antibacterial agent capable of serving as a substrate for a bacterial efflux pump is an aminoglycoside.   The antibacterial agent according to claim 6, wherein the aminoglycoside is amikacin.   The antibacterial agent according to any one of claims 4 to 7, which is used for treatment or prevention of bacterial infections or bacteria elimination.   The agent according to any one of claims 1 to 8, wherein the bacterium is multidrug-resistant Pseudomonas aeruginosa.   An object of the present invention is to disinfect a bacterium from the object, the method comprising contacting an object contaminated or possibly with a bacterium with patulin and an antibacterial agent capable of serving as a substrate for the efflux pump of the bacterium. The efflux pump is a protein comprising an amino acid sequence having 80% or more similarity to the amino acid sequence represented by SEQ ID NO: 1, and using an aminoglycoside antibacterial agent as a substrate.   The Pseudomonas aeruginosa strain isolated from a patient infected with multidrug-resistant Pseudomonas aeruginosa is contacted with patulin and an antibacterial agent that can be a substrate for Pseudomonas aeruginosa MexY efflux pump alone and in combination, respectively. It is characterized in that a strain whose growth has been synergistically inhibited when contacted in combination is determined to be a strain having a multidrug resistance mechanism by enhancing the function of the MexY efflux pump. And a method for identifying a multidrug resistance mechanism of a pathogenic Pseudomonas aeruginosa strain.