CN113209090A - Application of methimazole in preparation of metallo-beta-lactamase inhibitor - Google Patents

Application of methimazole in preparation of metallo-beta-lactamase inhibitor Download PDF

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CN113209090A
CN113209090A CN202110371139.7A CN202110371139A CN113209090A CN 113209090 A CN113209090 A CN 113209090A CN 202110371139 A CN202110371139 A CN 202110371139A CN 113209090 A CN113209090 A CN 113209090A
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methimazole
beta
metallo
lactamase
bacteria
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CN113209090B (en
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姜志辉
石磊
张冰
何羡霞
袁进
杨琰
杨晨
关慧
蔡敏捷
蔡学究
陈丽丹
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Southern Theater Command General Hospital of PLA
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention belongs to the field of medicines, and discloses application of methimazole in serving as and/or preparing a metal beta-lactamase inhibitor. The invention discloses the application of methimazole and/or derivatives thereof as and/or in the preparation of a metallo-beta-lactamase inhibitor for the first time, wherein the methimazole and/or the derivatives thereof have good inhibition effect on the metallo-beta-lactamase, can protect antibiotics from being degraded by bacteria, improve the sensitivity of the bacteria to the antibiotics and reverse the drug resistance of the bacteria to the antibiotics; meanwhile, the methimazole and/or the derivatives thereof have good synergistic effect when being combined with antibiotics, and can be used as an adjuvant drug for inhibiting bacteria. And the normal clinical application dose of the methimazole has little toxic and side effects on human bodies, and the methimazole has been used as a medicine for treating hyperthyroidism clinically for many years and has good safety.

Description

Application of methimazole in preparation of metallo-beta-lactamase inhibitor
Technical Field
The invention belongs to the field of medicines, and particularly relates to application of methimazole in serving as and/or preparing a metal beta-lactamase inhibitor.
Background
Beta-lactam antibiotics become the most common clinical antibiotics due to the characteristics of high efficiency and low toxicity, and play an important role in controlling and treating gram-negative bacterial infection. However, long term unjustified use and even abuse of antibiotics has caused serious resistance to bacteria, which poses a serious threat to public health problems worldwide.
The predominant resistance mechanism of bacteria is the production of beta-lactamases, which hydrolyze the lactam ring of beta-lactam antibiotics to render the antibiotics ineffective, thereby conferring resistance to the antibiotics. Beta-lactamases can be classified into four classes a-D, based on their amino acid sequence, wherein the active site of class B beta-lactamases contains 1-2 zinc ions and are therefore also known as metallo beta-lactamases (MBLs).
Based on the amino acid sequence and the binding mode of zinc ions, MBLs are further divided into 3 subclasses, B1 class, B2 class and B3 class, while metallo-beta-lactamases of B1 class have a broader substrate spectrum than other subclasses of metallo-beta-lactamases and can hydrolyze almost all beta-lactam antibiotics, including carbapenem antibiotics known as the "last line of defense". The gene can not only rapidly carry out cross-strain propagation through plasmids, but also is very easy to generate variation due to the action of rearrangement of the plasmids, and the like, and can be integrated on the chromosome of bacteria to ensure that the heredity and the drug resistance of the bacteria are more stable. At present, no effective metallo-beta-lactamase inhibitor is available on the market clinically. Therefore, the development of metallo-beta-lactamase inhibitors is imminent.
Methimazole (MMI), also known as tazobactam, is a thiourea antithyroid agent. Because of the advantages of long half-life period, relatively low incidence rate of liver damage, wide application range, convenient taking, low price and the like, the medicine is clinically used as a first-line medicine for treating hyperthyroidism, and the methimazole is still clinically used as a first-choice medicine for treating hyperthyroidism until now. At present, the report of methimazole as a metallo beta-lactamase inhibitor is not available.
Disclosure of Invention
The first aspect of the invention aims to provide application of methimazole and/or derivatives thereof in serving as and/or preparing a metallo-beta-lactamase inhibitor.
The second aspect of the invention aims to provide the application of methimazole and/or its derivatives in the preparation and/or application of drugs for improving the sensitivity of bacteria to antibiotics.
The third aspect of the invention aims to provide the application of the antibiotic and the methimazole and/or the derivative thereof in preparing the medicament for inhibiting the bacteria.
The fourth aspect of the present invention is directed to a combination drug comprising an antibiotic and methimazole and/or its derivative.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the invention, there is provided the use of methimazole and/or its derivatives as and/or in the preparation of a metallo-beta-lactamase inhibitor.
Methimazole is an antithyroid agent and has a molecular formula of C4H6N2S, the CAS number is 60-56-0, and the structural formula is shown as the formula (I).
Figure BDA0003009352000000021
Preferably, the derivative comprises a pharmaceutically acceptable salt, hydrate, solvate, polymorph, tautomer or prodrug of methimazole.
Preferably, the metallo-beta-lactamase is at least one of an IMP-7 type metallo-beta-lactamase, an NDM-1 type metallo-beta-lactamase and a VIM-2 type metallo-beta-lactamase; further preferably, the metallo-beta-lactamase is an NDM-type 1 metallo-beta-lactamase.
Preferably, the source of the metallo-beta-lactamase comprises extraction in nature or preparation from a genetically engineered strain.
In a second aspect of the present invention, there is provided the use of methimazole and/or its derivatives as and/or in the manufacture of a medicament for increasing the sensitivity of bacteria to antibiotics.
Methimazole is an antithyroid agent and has a molecular formula of C4H6N2S, the CAS number is 60-56-0, and the structural formula is shown as the formula (I).
Preferably, the derivative comprises a pharmaceutically acceptable salt, hydrate, solvate, polymorph, tautomer or prodrug of methimazole.
Preferably, the bacterium is a drug-resistant bacterium that expresses a metallo-beta-lactamase; further preferably, the bacteria are Escherichia coli (Escherichia coli), Klebsiella pneumoniae (Klebsiella pneumoniae), Pseudomonas aeruginosa (Pseudomonas aeruginosa).
Preferably, the antibiotic is a β -lactam antibiotic; further preferably, the antibiotic is at least one of penicillin, cephalosporin, cephamycin, thiomycin and carbapenem antibiotics; still further preferably, the antibiotic is at least one of meropenem, imipenem, ertapenem, cephalexin, cefuroxime, cefdinir, ceftriaxone, ceftazidime, ampicillin and amoxicillin.
Preferably, the metallo-beta-lactamase is at least one of an IMP-7 type metallo-beta-lactamase, an NDM-1 type metallo-beta-lactamase and a VIM-2 type metallo-beta-lactamase; further preferably, the metallo-beta-lactamase is at least one of an IMP-7 type metallo-beta-lactamase and an NDM-1 type metallo-beta-lactamase.
In a third aspect of the invention, there is provided the use of an antibiotic and methimazole and/or a derivative thereof in the manufacture of a medicament for inhibiting bacteria.
Methimazole is an antithyroid agent and has a molecular formula of C4H6N2S, the CAS number is 60-56-0, and the structural formula is shown as the formula (I).
Preferably, the derivative comprises a pharmaceutically acceptable salt, hydrate, solvate, polymorph, tautomer or prodrug of methimazole.
Preferably, the antibiotic is a β -lactam antibiotic; further preferably, the antibiotic is at least one of penicillin, cephalosporin, cephamycin, thiomycin and carbapenem antibiotics; still further preferably, the antibiotic is at least one of meropenem, imipenem, ertapenem, cephalexin, cefuroxime, cefdinir, ceftriaxone, ceftazidime, ampicillin and amoxicillin.
Preferably, the bacterium is a drug-resistant bacterium that expresses a metallo-beta-lactamase; further preferably, the bacteria are Escherichia coli (Escherichia coli), Klebsiella pneumoniae (Klebsiella pneumoniae), Pseudomonas aeruginosa (Pseudomonas aeruginosa).
Preferably, the metallo-beta-lactamase is at least one of an IMP-7 type metallo-beta-lactamase, an NDM-1 type metallo-beta-lactamase and a VIM-2 type metallo-beta-lactamase; further preferably, the metallo-beta-lactamase is at least one of an IMP-7 type metallo-beta-lactamase and an NDM-1 type metallo-beta-lactamase.
In a fourth aspect of the invention, there is provided a medicament comprising:
(1) (ii) an antibiotic; and
(2) methimazole and/or its derivatives.
Methimazole is an antithyroid agent and has a molecular formula of C4H6N2S, CAS number 60-56-0, structureThe formula is shown as a formula (I).
Preferably, the derivative comprises a pharmaceutically acceptable salt, hydrate, solvate, polymorph, tautomer or prodrug of methimazole.
Preferably, the antibiotic is a β -lactam antibiotic; further preferably, the antibiotic is at least one of penicillin, cephalosporin, cephamycin, thiomycin and carbapenem antibiotics; still further preferably, the antibiotic is at least one of meropenem, imipenem, ertapenem, cephalexin, cefuroxime, cefdinir, ceftriaxone, ceftazidime, ampicillin and amoxicillin.
Preferably, the medicament further comprises pharmaceutically acceptable auxiliary materials.
Preferably, the adjuvant comprises at least one of diluent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, surfactant, adsorption carrier, and lubricant.
Preferably, the formulation types of the drug include solid formulations, liquid formulations, and semi-solid formulations.
Preferably, the solid formulation includes tablets, granules, powders and capsules.
Preferably, the liquid formulation comprises an injection.
Preferably, the semi-solid formulation comprises an ointment and a cream.
The invention has the beneficial effects that:
the invention discloses the application of methimazole and/or derivatives thereof as and/or in the preparation of a metallo-beta-lactamase inhibitor for the first time, wherein the methimazole and/or the derivatives thereof have good inhibition effect on the metallo-beta-lactamase, can protect antibiotics from being degraded by bacteria, improve the sensitivity of the bacteria to the antibiotics and reverse the drug resistance of the bacteria to the antibiotics; meanwhile, the methimazole and/or the derivatives thereof have good synergistic effect when being combined with antibiotics, and can be used as a compound medicine for inhibiting bacteria. In addition, the methimazole has little toxic and side effects on human bodies under normal clinical dosage, and has good safety as a medicine for treating hyperthyroidism in clinic for many years.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. The materials, reagents and the like used in the present examples are commercially available reagents and materials unless otherwise specified.
Example 1 measurement of inhibitory Activity of methimazole on metallo beta-lactamase
The substrate is hydrolyzed by the enzyme to cause the decrease of the absorbance value, so that the degree of hydrolysis of the substrate can be characterized by the change of the absorbance, thereby judging the activity of the enzyme. The change in absorbance of the substrate after hydrolysis by metallo-beta-lactamase was measured at a wavelength of 300nm using meropenem (50. mu.M) as a reporter substrate. The concentration of metallo-beta-lactamase was 4nM, the buffer was 50mM HEPES (4-hydroxyethylpiperazineethanesulfonic acid), 0.1mM ZnSO4The pH is 7.2, the reaction temperature is 25 ℃, and the specific experimental method is as follows:
1. determination of inhibitory Activity of methimazole on IMP-7 type metallo beta-lactamase
(1) Methimazole was dissolved in HEPES buffer and formulated to different concentrations (0.1, 1, 10, 50, 100, 200, 300, 500 μ M, respectively) with three wells per concentration, 10 μ L of IMP-7 type metallo-beta-lactamase solution (final concentration of 4nM) was added and incubated at 25 ℃ for 15min to allow methimazole to bind to the enzyme well.
(2) The system is transferred into a quartz cuvette, 50 mu L of meropenem (the final concentration is 50 mu M) is added, the change of the absorbance value is rapidly measured, and the data is recorded.
(3) Calculating the inhibition rate of methimazole with different concentrations on IMP-7 type metallo beta-lactamase, plotting the concentration of the compound on the inhibition rate, and calculating by fitting a curve to obtain IC50The value is obtained.
2. Determination of NDM-1 type metallo beta-lactamase inhibitory activity of methimazole
(1) Methimazole was dissolved in HEPES buffer and formulated to different concentrations (0.1, 1, 10, 50, 100, 200, 300, 500 μ M, respectively) with three wells per concentration, 10 μ L of NDM-1 metallo-beta-lactamase solution (final concentration of 4nM) was added, and incubation was carried out at 25 ℃ for 15min to allow methimazole to bind well to the enzyme.
(2) The system is transferred into a quartz cuvette, 50 mu L of meropenem (the final concentration is 50 mu M) is added, the change of the absorbance value is rapidly measured, and the data is recorded.
(3) Calculating the inhibition rate of methimazole with different concentrations on NDM-1 type metallo beta-lactamase, drawing the inhibition rate by the concentration of the compound, and calculating by fitting a curve to obtain IC50The value is obtained.
3. Determination of inhibitory Activity of methimazole on VIM-2 type metallo beta-lactamase
(1) Methimazole was dissolved in HEPES buffer and formulated to different concentrations (0.1, 1, 10, 50, 100, 200, 300, 500 μ M each) with three wells, 10 μ L of VIM-2 metallo-beta-lactamase solution (final concentration 4nM) was added, and incubation was carried out at 25 ℃ for 15min to allow methimazole to bind to the enzyme sufficiently.
(2) The system is transferred into a quartz cuvette, 50 mu L of meropenem (the final concentration is 50 mu M) is added, the change of the absorbance value is rapidly measured, and the data is recorded.
(3) Calculating the inhibition rate of methimazole with different concentrations on VIM-2 type metallo beta-lactamase, drawing the inhibition rate by the concentration of the compound, and calculating by fitting a curve to obtain IC50The value is obtained.
The results of the inhibitory activity of methimazole on metallic beta-lactamase of IMP-7 type, NDM-1 type and VIM-2 type are shown in Table 1: methimazole has inhibitory effect on IMP-7 type and NDM-1 type metallo beta-lactamase, especially on NDM-1 type metallo beta-lactamase, and IC5098.84 + -12.14 μ M and 57.16 + -7.02 μ M Ki.
TABLE 1 Emamectin IC for metallic beta-lactamases of IMP-7, NDM-1 and VIM-2 type50(. mu.M) andKi(μM)
Figure BDA0003009352000000051
example 2 evaluation of the Effect of Mesoxazole in combination with Meropenem in inhibiting the production of MBLs drug-resistant bacteria
The Minimum Inhibitory Concentration (MIC) of the combination of methimazole and meropenem on MBLs drug-resistant strains is determined by adopting a trace broth dilution method. The strains for producing MBLs used in the experiment are the genetically engineered bacteria E.coli BL21(DE3)/pET28a-IMP-7, E.coli BL21(DE3)/pET28a-VIM-2 and E.coli BL21(DE3)/pET28a-NDM-1, which are purchased from Shanghai Biotech Co., Ltd.
FICI is used to judge the interaction of two drugs when used in combination and is defined according to the following equationA+FICB＝CA/MICA+CB/MICBWherein MICAAnd MICBMIC values for Compounds A and B, respectively, alone, and CAAnd CBIs the drug concentration of compound a and B in the effective combination. If the FICI is less than or equal to 0.5, the two medicines are considered to have synergistic action, if the FICI is more than 0.5 and less than or equal to 4, the two medicines are considered to have weak synergistic action or no related action, and if the FICI is more than or equal to 4, the two medicines are considered to have antagonistic action. The smaller the FICI, the stronger the drug synergy.
The specific experimental method is as follows:
1. evaluation of effect of combination of methimazole and meropenem in inhibiting production of IMP-7 type drug-resistant bacteria
(1) Inoculating a strain (E.coli BL21(DE3)/pET28a-IMP-7 strain) preserved at ultralow temperature into a sterile LB solid culture medium under the aseptic operating condition, putting the strain into a constant temperature incubator at 37 ℃ for overnight culture, picking a single colony, transferring the single colony into 3mL of LB liquid culture medium (containing 50mg/mL of kanamycin), and culturing the single colony in the constant temperature incubator at 37 ℃ until the logarithmic growth phase to obtain a bacterial suspension; adjusting the concentration of the bacterial liquid to 0.5 McLeod concentration by a McLeod turbidimeter, diluting the LB liquid culture medium by 100 times, and obtaining the bacterial number of about 1 × 106CFU/mL。
(2) Adding 100 mu L of LB liquid culture medium into the 2 nd to 12 th rows of the 96-well plate, adding 100 mu L of meropenem solution (256 mu g/mL) or inhibitor (methimazole, 256 mu g/mL) into the 1 st row, sucking 100 mu L of the liquid medicine in the 2 nd row after fully mixing uniformly, adding the liquid medicine into the 3 rd row, mixing uniformly again, sequentially diluting the liquid medicine by the multiple dilution method to obtain the medicine with the concentration of 0.0625-128 mu g/mL; 100 μ L of diluted bacterial solution was added to each well, and the MIC of IMP-7-producing drug-resistant bacteria was determined using meropenem or methimazole alone, three wells per concentration.
(3) Jointly diluting the liquid medicine on a 96-well plate according to the horizontal direction and the vertical direction, and carrying out gradient dilution on meropenem in the horizontal row, wherein the method is the same as the step 2, but the volumes of the added LB liquid culture medium and the meropenem are both 50 mu L (the final concentration of the meropenem is 0.0625-128 mu g/mL), and 50 mu L of inhibitor (methimazole) with different concentrations diluted in a multiple ratio in advance is added in the vertical row, and the final concentration is 2-128 mu g/mL; 100 mu L of diluted bacteria liquid is added into each hole, so as to determine the MIC of the combination of the meropenem and the methimazole inhibitor (methimazole) on IMP-7 type drug-resistant bacteria, and three multiple holes are arranged at each concentration.
(4) Each set of experiments was set up with three parallel controls: taking Escherichia coli ATCC25922 as a quality control standard, taking captopril as a positive control, and simultaneously arranging a sterile hole and a drug-free hole; the 96-well plate was incubated in a 37 ℃ incubator for 24 hours, and the results were observed and MIC values were recorded.
2. Evaluation of effect of combination of methimazole and meropenem on inhibiting generation of VIM-2 type drug-resistant bacteria
(1) Inoculating a strain (E.coli BL21(DE3)/pET28a-VIM-2 strain) preserved at ultralow temperature into a sterile LB solid culture medium under the aseptic operating condition, placing the strain in a constant temperature incubator at 37 ℃ for overnight culture, picking a single colony, transferring the single colony into 3mL of LB liquid culture medium (containing 50mg/mL of kanamycin), and culturing the single colony in the constant temperature incubator at 37 ℃ until the logarithmic growth phase to obtain a bacterial suspension; adjusting the concentration of the bacterial liquid to 0.5 McLeod concentration by a McLeod turbidimeter, diluting the LB liquid culture medium by 100 times, and obtaining the bacterial number of about 1 × 106CFU/mL。
(2) Adding 100 mu L of LB liquid culture medium into the 2 nd to 12 th rows of the 96-well plate, adding 100 mu L of meropenem solution (256 mu g/mL) or inhibitor (methimazole, 256 mu g/mL) into the 1 st row, sucking 100 mu L of the liquid medicine in the 2 nd row after fully mixing uniformly, adding the liquid medicine into the 3 rd row, mixing uniformly again, sequentially diluting the liquid medicine by the multiple dilution method to obtain the medicine with the concentration of 0.0625-128 mu g/mL; 100 μ L of diluted bacterial solution was added to each well, and the MIC of VIM-2-producing drug-resistant bacteria with meropenem or methimazole alone was determined, three wells per concentration.
(3) Jointly diluting the liquid medicine on a 96-well plate according to the horizontal direction and the vertical direction, and carrying out gradient dilution on meropenem in the horizontal row, wherein the method is the same as the step 2, but the volumes of the added LB liquid culture medium and the meropenem are both 50 mu L (the final concentration of the meropenem is 0.0625-128 mu g/mL), and 50 mu L of inhibitor (methimazole) with different concentrations diluted in a multiple ratio in advance is added in the vertical row, and the final concentration is 2-128 mu g/mL; 100 mu L of diluted bacteria liquid is added into each hole, so as to determine the MIC of the combination of the meropenem and the methimazole inhibitor (methimazole) to the VIM-2 type drug-resistant bacteria, and three multiple holes are arranged at each concentration.
(4) Each set of experiments was set up with three parallel controls: taking Escherichia coli ATCC25922 as a quality control standard, taking captopril as a positive control, and simultaneously arranging a sterile hole and a drug-free hole; the 96-well plate was incubated in a 37 ℃ incubator for 24 hours, and the results were observed and MIC values were recorded.
3. Evaluation of effect of combination of methimazole and meropenem in inhibiting generation of NDM-1 type drug-resistant bacteria
(1) Inoculating a strain (E.coli BL21(DE3)/pET28a-NDM-1 strain) preserved at ultralow temperature into a sterile LB solid culture medium under the aseptic operating condition, placing the strain in a constant temperature incubator at 37 ℃ for overnight culture, picking a single colony, transferring the single colony into 3mL of LB liquid culture medium (containing 50mg/mL of kanamycin), and culturing the single colony in the constant temperature incubator at 37 ℃ until the logarithmic growth phase to obtain a bacterial suspension; adjusting the concentration of the bacterial liquid to 0.5 McLeod concentration by a McLeod turbidimeter, diluting the LB liquid culture medium by 100 times, and obtaining the bacterial number of about 1 × 106CFU/mL。
(2) Adding 100 mu L of LB liquid culture medium into the 2 nd to 12 th rows of the 96-well plate, adding 100 mu L of meropenem solution (256 mu g/mL) or inhibitor (methimazole, 256 mu g/mL) into the 1 st row, sucking 100 mu L of the liquid medicine in the 2 nd row after fully mixing uniformly, adding the liquid medicine into the 3 rd row, mixing uniformly again, sequentially diluting the liquid medicine by the multiple dilution method to obtain the medicine with the concentration of 0.0625-128 mu g/mL; 100 μ L of diluted bacterial solution was added to each well, and the MIC of NDM-1-type MBLs drug-resistant bacteria was determined using Meropenem or methimazole alone, three wells per concentration.
(3) Jointly diluting the liquid medicine on a 96-well plate according to the horizontal direction and the vertical direction, and carrying out gradient dilution on meropenem in the horizontal row, wherein the method is the same as the step 2, but the volumes of the added LB liquid culture medium and the meropenem are both 50 mu L (the final concentration of the meropenem is 0.0625-128 mu g/mL), and 50 mu L of inhibitor (methimazole) with different concentrations diluted in a multiple ratio in advance is added in the vertical row, and the final concentration is 2-128 mu g/mL; 100 mu L of diluted bacteria liquid is added into each hole, so as to determine the MIC of the meropenem and the methimazole inhibitor (methimazole) combined for NDM-1 type MBLs drug-resistant bacteria, and three multiple holes are arranged at each concentration.
(4) Each set of experiments was set up with three parallel controls: taking Escherichia coli ATCC25922 as a quality control standard, taking captopril as a positive control, and simultaneously arranging a sterile hole and a drug-free hole; the 96-well plate was incubated in a 37 ℃ incubator for 24 hours, and the results were observed and MIC values were recorded.
The results of the antibacterial activity of meropenem in combination with methimazole against drug-resistant bacteria expressing NDM-1, VIM-2 or IMP-1 are shown in Table 2: the inhibitor (methimazole, captopril) can improve the antibacterial activity of meropenem: when the concentration of the inhibitor is 128 mug/mL, the inhibitor is used together with meropenem, the bacteriostatic effect of the meropenem on various MBLs drug-resistant bacteria can be improved, and compared with the single use of the meropenem, the combined use can effectively reduce the MIC value of the meropenem on the drug-resistant strains by 8 times at most, and the effect is better than that of captopril.
The FICI values for MBLs-expressing drug-resistant bacteria in combination with methimazole or captopril are shown in Table 3: the FICI value of the methimazole and the Meropenem used together for the drug-resistant bacteria expressing MBLs is less than or equal to 0.5, which shows that the methimazole and the Meropenem have good synergistic effect; the FICI value of the captopril and the meropenem which are used together for the MBLs-expressing drug-resistant bacteria is larger than that of methimazole, and the synergistic effect of the captopril and the meropenem is not as good as that of the methimazole and the meropenem.
The above results show that: when the composition is used with meropenem, the methimazole has effective synergistic antibacterial activity on various drug-resistant bacteria expressing metallo-beta-lactamase, which indicates that the methimazole can be used as an MBLs inhibitor, can reverse the drug resistance of carbapenem drug-resistant bacteria, effectively protects the meropenem from being hydrolyzed by MBLs, and improves the antibacterial activity of the meropenem on the MBLs drug-resistant bacteria. Therefore, the methimazole can be used as a metallo beta-lactamase inhibitor to be prepared into a composite preparation with beta-lactam antibiotics.
TABLE 2 MIC (μ g/mL) for MBLs drug-resistant bacteria expressed in combination with methimazole or captopril and meropenem
Figure BDA0003009352000000081
TABLE 3 synergistic antimicrobial index (FICI) of methimazole or captopril in combination with meropenem against MBLs-expressing drug-resistant bacteria
Figure BDA0003009352000000082
Figure BDA0003009352000000091
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (10)

1. Application of methimazole and/or its derivative in serving as and/or preparing metallo beta-lactamase inhibitor.
2. Use according to claim 1, characterized in that:
the metallo-beta-lactamase is at least one of IMP-7 type metallo-beta-lactamase, NDM-1 type metallo-beta-lactamase and VIM-2 type metallo-beta-lactamase;
preferably, the derivative comprises a pharmaceutically acceptable salt, hydrate, solvate, polymorph, tautomer or prodrug of methimazole.
3. The use of methimazole and/or its derivatives as and/or in the preparation of a medicament for increasing the sensitivity of bacteria to antibiotics.
4. Use according to claim 3, characterized in that:
the antibiotic is beta-lactam antibiotic.
5. Use according to claim 4, characterized in that:
the bacterium is a drug-resistant bacterium expressing metallo-beta-lactamase;
preferably, the derivative comprises a pharmaceutically acceptable salt, hydrate, solvate, polymorph, tautomer or prodrug of methimazole.
6. Use of an antibiotic and methimazole and/or a derivative thereof in the manufacture of a medicament for inhibiting bacteria.
7. Use according to claim 6, characterized in that:
the antibiotic is beta-lactam antibiotic.
8. Use according to claim 7, characterized in that:
the bacterium is a drug-resistant bacterium expressing metallo-beta-lactamase;
preferably, the derivative comprises a pharmaceutically acceptable salt, hydrate, solvate, polymorph, tautomer or prodrug of methimazole.
9. A medicament, comprising:
(1) (ii) an antibiotic; and
(2) methimazole and/or its derivatives.
10. The medicament of claim 9, wherein:
the antibiotic is beta-lactam antibiotic.
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