CN114099507B

CN114099507B - Application of antihistamine medicine in preparing antibacterial medicine

Info

Publication number: CN114099507B
Application number: CN202111677103.8A
Authority: CN
Inventors: 李秀云; 尹虹; 杨慧军; 范媛媛; 董芳地; 刘德泉; 周玉侠; 孙敏; 徐安然
Original assignee: Shandong Maternal And Child Health Hospital
Current assignee: Shandong Maternal And Child Health Hospital
Priority date: 2021-12-31
Filing date: 2021-12-31
Publication date: 2022-09-20
Anticipated expiration: 2041-12-31
Also published as: CN115154468A; CN115154468B; CN115252592B; CN114099507A; CN115252592A

Abstract

The invention relates to the technical field of medicines, in particular to application of antihistamine medicines in preparation of antibacterial medicines, wherein the antibacterial medicines comprise antihistamine medicines or a medicinal composition of antibiotics and antihistamine medicines, and the antibacterial medicines are selected from the following components: cyproheptadine hydrochloride, desloratadine or diphenhydramine hydrochloride; alternatively, a pharmaceutical composition of an antibiotic and an antihistamine comprises: the composition of epinastine hydrochloride and cefepime, the composition of epinastine hydrochloride and ampicillin sodium, the composition of desloratadine and mezlocillin sodium, the composition of diphenhydramine hydrochloride and cefepime, and the composition of olopatadine hydrochloride and cefepime. The invention discovers that various antihistamines known in the prior art have bacteriostatic action, and also discovers that various antihistamines can enhance the sensitivity of bacteria to antibiotics, thereby providing an effective solution for solving the widely-occurring bacterial drug resistance.

Description

Application of antihistamine medicine in preparing antibacterial medicine

Technical Field

The invention relates to the technical field of medicines, in particular to application of antihistamine medicines in preparation of antibacterial medicines.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

The prevalence of the multi-drug-resistant bacteria of children is wide, and large sample investigation shows that the incidence rate of the multi-drug-resistant bacteria is different from 8-45%; specimens mainly come from sputum, blood, pus, secretion and urine, and are common in ICU, newborn and respiratory department; in children infection, the clinically common gram-positive bacteria are staphylococcus aureus, streptococcus pneumoniae, coagulase-negative staphylococcus, enterococcus faecium and enterococcus faecalis respectively at the 5 th position before infection; common gram-negative bacteria mainly include Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa and Acinetobacter baumannii.

Antibacterial drugs have been developed for many years, resulting in the development of bacterial resistance due to their large and irregular use; antimicrobial drug resistance has become one of the major threats to human health; common resistance mechanisms include: production of drug-resistant enzymes, alteration of drug targets, decreased cell membrane permeability, efflux pump expression, and biofilm formation. The development of new antibacterial agents is a very slow process, much slower than the emergence rate of drug-resistant strains, and often encounters a number of obstacles. Re-developing the antibacterial effects of approved marketed drugs is a promising alternative strategy, with the potential to rapidly enter preclinical and clinical trials, reducing the cost and time of drug development.

The inventors found that, since the development of a new antibacterial drug is very difficult, it is an effective method to study a non-antibacterial drug having an antibacterial effect or to enhance the antibacterial activity of an existing antibacterial drug using a sensitizer.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide the application of antihistamine medicines in preparing antibacterial medicines and also provides the application of the antihistamine medicines as antibiotic sensitizers; the present inventors have found that various antihistamines known in the art have bacteriostatic effects, and have also found that various antihistamines can enhance the sensitivity of bacteria to antibiotics; provides an effective solution for solving the widely-occurring bacterial drug resistance.

Firstly, the invention provides the application of antihistamine medicines in preparing antibacterial medicines;

in a first aspect of the present invention, there is provided: the application of antihistamine medicines in preparing antibacterial medicines;

preferably, the antihistamine comprises: cyproheptadine hydrochloride, desloratadine and diphenhydramine hydrochloride;

in particular to an application of cyproheptadine hydrochloride in preparing multiple drug resistant acinetobacter baumannii drugs;

preferably, the effective concentration of the cyproheptadine hydrochloride for inhibiting the multiple drug-resistant acinetobacter baumannii is more than or equal to 8 mu g/mL, or more than or equal to 16 mu g/mL, or more than or equal to 32 mu g/mL, or more than or equal to 64 mu g/mL, or more than or equal to 128 mu g/mL, or more than or equal to 256 mu g/mL.

Or the application of desloratadine in preparing the anti-Klebsiella pneumoniae medicine;

preferably, the effective concentration of desloratadine for inhibiting Klebsiella pneumoniae is more than or equal to 128 mug/mL.

Or the diphenhydramine hydrochloride is applied to the preparation of the anti-staphylococcus aureus medicament;

preferably, the effective concentration of diphenhydramine hydrochloride for inhibiting staphylococcus aureus is more than or equal to 256 mug/mL.

The invention discovers for the first time that the antihistaminic drug cyproheptadine hydrochloride has obvious effect of resisting multiple drug resistant acinetobacter baumannii in vitro, and has good inhibition effect on the growth of acinetobacter baumannii strains (AB1, AB2, AB3 and AB 11);

the antihistamine drug desloratadine is also found to have the effect of resisting Klebsiella pneumoniae, and the desloratadine can obviously inhibit the growth of three experimental strains of Klebsiella pneumoniae KP37, KP40 and KP53 (p is less than 0.05) when the concentration is 128 mu g/mL;

the diphenhydramine hydrochloride serving as an antihistamine drug has the effect of resisting staphylococcus aureus and has a good inhibition effect on the growth of staphylococcus aureus (SA1, SA2 and SA3) strains.

Secondly, the invention provides the application of the antihistamine medicine as the antibiotic sensitizer;

in a second aspect of the invention, the use of an antihistamine as an antibiotic sensitiser is provided;

preferably, the antihistamine comprises epinastine hydrochloride, olopatadine hydrochloride, desloratadine, diphenhydramine hydrochloride, cyproheptadine hydrochloride;

preferably, the antibiotic comprises cefepime, ampicillin sodium, mezlocillin sodium, meropenem;

in particular to an application of epinastine hydrochloride, diphenhydramine hydrochloride or olopatadine hydrochloride as cefepime sensitizer;

or, the epinastine hydrochloride is used as the ampicillin sodium sensitizer;

or, the epinastine hydrochloride is used as the mezlocillin sodium sensitizer;

or the application of desloratadine as a mezlocillin sodium sensitizer;

or the application of desloratadine as an ampicillin sodium sensitizer;

or, the cyproheptadine hydrochloride is used as the meropenem sensitizer;

further, an application of epinastine hydrochloride and cefepime in preparation of a drug for resisting pseudomonas aeruginosa is provided;

preferably, the concentration ratio of the epinastine hydrochloride and cefepime in the combined application of the epinastine hydrochloride and the cefepime for inhibiting the pseudomonas aeruginosa is 1: 1 or 1: 2 or 1: 4 or 1: 8 or 1: 16.

or the diphenhydramine hydrochloride and cefepime are combined to prepare the medicine for resisting pseudomonas aeruginosa;

preferably, the concentration ratio of the diphenhydramine hydrochloride and cefepime in the combined application of the diphenhydramine hydrochloride and the cefepime for inhibiting pseudomonas aeruginosa is 1: 1 or 1: 2 or 1: 4 or 1: 8 or 1: 16.

or the application of olopatadine hydrochloride and cefepime in preparing the anti-pseudomonas aeruginosa medicine;

preferably, the concentration ratio of the olopatadine hydrochloride and cefepime in the combined application of inhibiting pseudomonas aeruginosa is 1: 1 or 1: 2 or 1: 4 or 1: 8 or 1: 16.

the research of the invention finds that epinastine hydrochloride, diphenhydramine hydrochloride and olopatadine hydrochloride can be used as the synergist of cefepime to enhance the antibacterial action of cefepime on multi-drug-resistant pseudomonas aeruginosa.

Also provides an application of epinastine hydrochloride combined with ampicillin sodium or epinastine hydrochloride combined with mezlocillin sodium in preparing medicines for resisting methicillin-resistant staphylococcus aureus;

preferably, the concentration ratio of the epinastine hydrochloride and the ampicillin sodium in the combined application for inhibiting the methicillin-resistant staphylococcus aureus is 32: 1;

or the epinastine hydrochloride is combined with mezlocillin sodium to prepare the medicine for resisting methicillin-resistant staphylococcus aureus;

preferably, the concentration ratio of the epinastine hydrochloride and the mezlocillin sodium in the combined application for inhibiting methicillin-resistant staphylococcus aureus is 32: 1;

the invention discovers for the first time that epinastine hydrochloride serving as an antihistamine can be used as an antibiotic sensitizer, particularly as a sensitizer of antibiotic ampicillin sodium or mezlocillin sodium, has the effect of promoting the antibiotic antibacterial effect, and particularly obtains a good technical effect in resisting methicillin-resistant staphylococcus aureus.

Or the application of desloratadine and mezlocillin sodium in preparing the anti-acinetobacter baumannii medicine;

preferably, the concentration ratio of the desloratadine to the mezlocillin sodium in the combined application of the desloratadine and the mezlocillin sodium for inhibiting acinetobacter baumannii is 1: 8;

or the application of desloratadine and ampicillin sodium in preparing the anti-acinetobacter baumannii medicine;

preferably, the concentration ratio of the desloratadine to the ampicillin sodium when used in combination for inhibiting acinetobacter baumannii is 8: 1;

the invention also discovers that the combined use of desloratadine and mezlocillin sodium or ampicillin sodium has partial synergistic effect or synergistic effect on the multi-drug-resistant acinetobacter baumannii.

Or the cyproheptadine hydrochloride and meropenem are combined to prepare the medicine for resisting the carbapenem-resistant Klebsiella pneumoniae;

preferably, the concentration ratio of the cyproheptadine hydrochloride and meropenem when combined application to the carbapenem-resistant Klebsiella pneumoniae is 16-32: 1, more preferably 32: 1 or 16: 1;

in a third aspect of the invention, there is provided an antibacterial medicament comprising an antihistamine, or a pharmaceutical composition of an antibiotic and an antihistamine;

in one or more embodiments, the antibacterial agent is selected from: cyproheptadine hydrochloride, desloratadine or diphenhydramine hydrochloride;

alternatively, the pharmaceutical composition of antihistamine and antibiotic comprises: a combination of epinastine hydrochloride and cefepime; a combination of diphenhydramine hydrochloride and cefepime; a combination of olopatadine hydrochloride and cefepime; a combination of epinastine hydrochloride and ampicillin sodium; a combination of epinastine hydrochloride and mezlocillin sodium; a combination of desloratadine and mezlocillin sodium; a combination of desloratadine and ampicillin sodium; a combination of cyproheptadine hydrochloride and meropenem;

in a fourth aspect of the present invention, a pharmaceutical preparation is provided, which comprises the above antibacterial agent and a pharmaceutically acceptable carrier or adjuvant.

The medicinal carrier or the auxiliary materials comprise one or more pharmaceutically or dietetically acceptable diluents, wetting agents, adhesives, disintegrating agents, lubricants, regulators and other auxiliary materials.

The dosage form of the pharmaceutical preparation is tablets, pills, sprays, capsules, granules, oral liquid, powder, syrup, injection, spray, suppositories and the like.

The specific embodiment of the invention has the following beneficial effects:

the invention discovers that the antihistamine cyproheptadine hydrochloride has good inhibition effect on the growth of acinetobacter baumannii (AB1, AB2, AB3 and AB 11);

the cyproheptadine hydrochloride can inhibit the growth of AB11 strain by more than 80% at a concentration of 64 mug/mL, and can completely inhibit the growth of AB11 strain at a concentration of 128 mug/mL. For the AB1 strain, cyproheptadine hydrochloride can inhibit the growth of the experimental strain by more than 80% at a concentration of 128. mu.g/mL, and can completely inhibit the growth of the experimental strain at a concentration of 256. mu.g/mL. For AB2 and AB3, cyproheptadine hydrochloride inhibited growth of 80% -100% of the experimental strains at a concentration of 128. mu.g/mL.

The invention also discovers that the antihistamine desloratadine has good inhibition effect on the growth of Klebsiella pneumoniae (KP37, KP40 and KP53) strains;

desloratadine significantly inhibited growth of three experimental strains at a concentration of 128. mu.g/mL (p < 0.05). The growth rate of KP37, KP40, KP53 can be reduced to below 2% (p < 0.01).

The invention also discovers that the antihistamine diphenhydramine hydrochloride has good inhibition effect on the growth of staphylococcus aureus (SA1, SA2 and SA3) strains;

the diphenhydramine hydrochloride can almost completely inhibit the growth of three staphylococcus aureus strains when the concentration is 256 mu g/mL, which shows that the bacteriostatic effect is excellent.

The invention also discovers that epinastine hydrochloride, diphenhydramine hydrochloride and olopatadine hydrochloride can be used as the synergist of cefepime to enhance the antibacterial action of cefepime on multi-drug-resistant pseudomonas aeruginosa;

the cefepime with the concentration of 0.03mM has no inhibition effect on the strain (p is more than 0.05), and the inhibition effect of the cefepime (p is less than 0.05) can be obviously enhanced when epinastine hydrochloride with the concentration of more than or equal to 0.03mM is added, or diphenhydramine hydrochloride with the concentration of more than or equal to 0.03mM is added, or olopatadine hydrochloride with the concentration of more than or equal to 0.03mM is added, so that the growth rate of the strain is reduced to be less than 20%.

The invention discovers that the combination of the epinastine hydrochloride serving as the antihistamine and the mezlocillin sodium or the ampicillin sodium has the synergistic effect of resisting methicillin-resistant staphylococcus aureus; the combination of epinastine hydrochloride and ampicillin sodium, or the combination of epinastine hydrochloride and mezlocillin sodium can almost completely inhibit the growth of methicillin-resistant staphylococcus aureus, and the FICI model is used as the evaluation standard, and the experimental result of the in vitro combination effect shows that the two drugs show synergistic effect on the methicillin-resistant staphylococcus aureus when being used in combination;

the epinastine hydrochloride (128 mu g/mL) and the mezlocillin sodium (4 mu g/mL) can almost completely inhibit the growth of methicillin-resistant staphylococcus aureus when being used together; epinastine hydrochloride (128. mu.g/mL) combined with ampicillin sodium (4. mu.g/mL) almost completely inhibited the growth of methicillin-resistant Staphylococcus aureus.

The invention also discovers that the combined use of desloratadine and mezlocillin sodium or ampicillin sodium has partial synergistic effect or synergistic effect on the multiple drug-resistant acinetobacter baumannii;

when 2 mu g/mL of desloratadine and 16 mu g/mL of mezlocillin sodium are used together, the growth of multiple drug-resistant acinetobacter baumannii can be inhibited;

32 mu g/mL of desloratadine and 4 mu g/mL of mezlocillin sodium; when the combination is used, the growth of multiple drug-resistant acinetobacter baumannii can be inhibited.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a graph showing the effect of different concentrations of cyproheptadine hydrochloride on the growth rate of multiple drug resistant A.baumannii in example 1;

wherein, the graph A shows the influence on the growth rate of the multiple drug-resistant Acinetobacter baumannii AB1 strain;

FIG. B is a graph showing the effect on growth rate of multiple drug-resistant A.baumannii AB2 strains;

FIG. C is the effect on growth rate of multiple drug-resistant A.baumannii AB3 strain;

FIG. D is the effect on growth rate of multiple drug-resistant A.baumannii AB11 strain;

FIG. 2 is a graph showing the effect of different concentrations of chlorpheniramine maleate on the growth rate of multiple drug resistant Acinetobacter baumannii in example 1;

wherein, the graph A shows the effect on the growth rate of multiple drug-resistant Acinetobacter baumannii AB1 strains;

FIG. 3 is a graph showing the effect of different concentrations of cetirizine on the growth rate of multiple drug resistant A.baumannii in example 1;

FIG. 4 is a graph showing the effect of desloratadine on the growth rate of Klebsiella pneumoniae in example 2;

wherein, the graph A shows the influence on the growth rate of Klebsiella pneumoniae KP37 strain;

FIG. B is a graph showing the effect on the growth rate of Klebsiella pneumoniae KP40 strain;

FIG. C is the effect on the growth rate of Klebsiella pneumoniae KP53 strain;

FIG. 5 is a graph showing the effect of cetirizine on the growth rate of Klebsiella pneumoniae in example 2;

FIG. C is the effect on the growth rate of Klebsiella pneumoniae KP53 strain;

FIG. 6 is a graph showing the effect of chlorpheniramine maleate on the growth rate of Klebsiella pneumoniae in example 2;

FIG. C is the effect on the growth rate of Klebsiella pneumoniae KP53 strain;

FIG. 7 is a graph showing the effect of clemastine fumarate on the growth rate of Klebsiella pneumoniae in example 2;

FIG. C is the effect on the growth rate of Klebsiella pneumoniae KP53 strain;

FIG. 8 is a graph of the effect of various concentrations of diphenhydramine hydrochloride on the growth rate of Staphylococcus aureus in example 3;

wherein panel A is the effect on the growth rate of Staphylococcus aureus SA1 strain;

panel B is the effect of growth rate of Staphylococcus aureus SA2 strain;

panel C is the effect of growth rate of Staphylococcus aureus SA3 strain;

FIG. 9 is a graph of the effect of varying concentrations of chlorpheniramine maleate on the growth rate of Staphylococcus aureus in example 3;

panel B is the effect of growth rate of Staphylococcus aureus SA2 strain;

panel C is the effect of growth rate of Staphylococcus aureus SA3 strain;

FIG. 10 is a graph of the effect of different concentrations of cetirizine on the growth rate of Staphylococcus aureus in example 3;

panel B is the effect of growth rate of Staphylococcus aureus SA2 strain;

panel C is the effect of growth rate of Staphylococcus aureus SA3 strain;

FIG. 11 shows that epinastine hydrochloride enhances the bactericidal effect of cefepime on P.aeruginosa in example 4;

FIG. 12 is a graph showing that diphenhydramine hydrochloride enhances the bactericidal effect of cefepime on Pseudomonas aeruginosa in example 4;

FIG. 13 shows that olopatadine hydrochloride enhances the bactericidal effect of cefepime on Pseudomonas aeruginosa in example 4;

FIG. 14 shows that desloratadine enhances the bactericidal effect of cefepime on P.aeruginosa in example 4.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

The invention will be further explained and illustrated with reference to specific examples.

Example 1

Static action determination of antihistaminic cyproheptadine hydrochloride for resisting multidrug-resistant acinetobacter baumannii

Experimental strains

In the course of the experiment, 4 strains (AB1, AB2, AB3 and AB11) were obtained from clinical laboratory in hospitals, and have been identified as Acinetobacter baumannii, and the sensitivity of the Acinetobacter baumannii to clinically common antibiotics was determined (see Table 1). The strains used for the tests were stored in a refrigerator at-80 ℃ and subcultured on MHA solid medium at least twice before use.

TABLE 1 determination of the susceptibility of the test strains to clinically customary antibiotics

R: resisant, stands for the resistance of the strain to this antibiotic.

Drugs and primary agents

Cyproheptadine hydrochloride, Dalian biotech Limited (Liaoning Dalian); chlorpheniramine maleate, Dalian biotech Limited (Liaoning Dalian); cetirizine, Dalian biotech Limited (Liaoning Dalian); mhb broth medium CAMHB broth), Qingdao high-tech industrial garden haibo biotechnology limited (Shandong Qingdao); MHA agar medium, haibo biotechnology limited, high-tech industrial garden of Qingdao (Shandong Qingdao); physiological saline, Shanghai Aladdin Biotechnology GmbH (Shanghai);

Method

preparation of bacterial liquid

Unfreezing acinetobacter baumannii stored at the temperature of minus 20 ℃ at room temperature, inoculating the acinetobacter baumannii to an MHA agar culture medium, culturing for 18-20h at the temperature of 35 ℃, re-inoculating single well-developed colony, and culturing for 18-20h at the temperature of 35 ℃ to ensure that the strain is in a logarithmic phase. Selecting several bacterial colonies, preparing into bacterial suspension with normal saline, adjusting the turbidity of the sample tube to be consistent with that of 0.5 McBt tube, and the concentration of the bacterial liquid is about 1.5 × 10 ⁸ CFU/mL, diluted with CAMHB broth to final working concentration of the bacterial liquidIs 5 x 10 ⁵ CFU/mL, final viable count for concentration verification.

Preparation of mother liquor of medicine

The appropriate pharmaceutical solvent is selected according to the guidelines of the American society for clinical and laboratory standards (CLSI M100-S30) and the instructions of the reagent manufacturer. The cyproheptadine hydrochloride is dissolved in absolute ethyl alcohol to prepare solution with the concentration of 20480 mug/mL, and the chlorpheniramine maleate and the cetirizine maleate are both dissolved in water to prepare solution with the concentration of 20480 mug/mL. All drugs were stored in a freezer at-20 ℃ until use. The medicine is naturally thawed and thawed in a refrigerator at 4 deg.C before each use.

Determination of action of cyproheptadine hydrochloride against acinetobacter baumannii

According to the broth microdilution method of CLSIM100-S30, the liquid medicine is diluted by CAMHB broth to 2-fold working concentration, the concentration range for screening cyproheptadine hydrochloride is 0-256 mug/mL, and the concentration range for screening chlorpheniramine maleate and cetirizine is 0-512 mug/mL. Sucking 100 μ L of the drug in the order of low to high concentration, adding into 96-well plate, and adding 100 μ L of bacterial liquid (5 × 10) into each well ⁵ CFU/mL), where positive growth control wells contained only the inoculum without drug and blank control wells contained only CAMHB broth. The dosed 96-well plates were incubated for 18h at 35 ℃ in an incubator according to the requirements of the CLSIM100-S30 protocol, the results were observed and the bacterial growth rate was recorded. The growth rate of each well was measured and recorded using a microplate reader. The Minimum Inhibitory Concentration (MIC), i.e., the lowest concentration of drug that completely inhibits bacterial growth in the microdilution wells; MIC ₈₀ The lowest drug concentration to inhibit 80% of bacterial growth (compared to growth control) in the microdilution wells.

Results

As can be seen from FIG. 1, cyproheptadine hydrochloride significantly inhibited the growth of AB1 and AB2 (p <0.001) at both 128. mu.g/mL and 256. mu.g/mL, as compared to the control group without drug; as can be seen from FIG. 1, cyproheptadine hydrochloride significantly inhibited the growth of AB3 and AB11 (p <0.001) at concentrations of 8. mu.g/mL, 16. mu.g/mL, 32. mu.g/mL, 64. mu.g/mL, 128. mu.g/mL, and 256. mu.g/mL, as compared to the control group without drug. As can be seen from fig. 2 and 3, chlorpheniramine maleate or cetirizine maleate had no bacteriostatic effect on 4 strains.

Example 2

Inhibition of klebsiella pneumoniae by desloratadine as an antihistamine

Experimental strains

Clinically isolated Klebsiella pneumoniae (KP37, KP40, KP53) were collected, and all strains were correctly identified and tested for susceptibility to clinically common antibiotics.

Preparation of bacterial suspension

3-5 colonies were picked from MHA plates that had been purified and cultured overnight and inoculated into sterile 0.9% sodium chloride solution. The suspension was adjusted to 0.5 McLeod turbidity (about 1X 10) using a McLeod tube ⁸ CFU/mL), and then diluted 100-fold (about 1X 10) with calcium-adjusted MH broth medium ⁶ CFU/mL)。

Preparation of pharmaceutical solutions

The stock solutions of desloratadine, chlorpheniramine maleate, cetirizine and clemastine fumarate were formulated to concentrations of 40960. mu.g/mL, 20480. mu.g/mL and 10240. mu.g/mL, respectively, as required by the American society for clinical laboratory standardization standards.

Broth dilution method

The drug was diluted in multiple proportions in calcium-adjusted MH broth medium to different dilution concentrations. 100 μ L of each concentration of drug solution was added to the set wells of a sterile 96-well plate, and 100 μ L of diluted bacterial solution was added to each well, while negative and medium controls were performed. Culturing at 37 ℃ for 16-18 h, observing the result and recording the bacterial growth rate corresponding to each liquid medicine.

Results of the experiment

Drug sensitive results of the experimental strains

The results are shown in Table 2. Therefore, KP40 and KP53 in the three experimental strains are multidrug-resistant Klebsiella pneumoniae.

TABLE 2 drug sensitivity results of the experimental strains to clinically common antibiotics

S: sensitive (S); i: intermediate sensitivity (I); r: resistance (R).

Effect of antihistamine at various concentrations on growth rates of Klebsiella pneumoniae

TABLE 3 influence of desloratadine on the growth rate of Klebsiella pneumoniae

And (4) surface note: the growth rate (%) in the table is represented as the average of the growth rates in three experiments. + -. standard deviation. The statistical control group for each desloratadine concentration was a growth control group without drug intervention. Statistical analysis using the Dunnett method in the one-way ANOVA analysis, p <0.001 was considered as statistically different.

TABLE 4 Effect of cetirizine on the growth rate of Klebsiella pneumoniae

And (4) surface note: the growth rate (%) in the table is represented as the average of the growth rates in three experiments. + -. standard deviation. Each statistical control group of cetirizine concentrations was a growth control group without drug intervention. Statistical analysis using the Dunnett method in the one-way ANOVA analysis, p <0.001 was considered as statistically different.

TABLE 5 influence of chlorpheniramine maleate on the growth rate of Klebsiella pneumoniae

And (4) surface note: the growth rate (%) in the table is represented as the average of the growth rates in three experiments. + -. standard deviation. Each statistical control group of chlorpheniramine maleate concentration was a growth control group without drug intervention. Statistical analysis using the Dunnett method in the one-way ANOVA analysis, p <0.001 was considered as statistically different.

TABLE 6 influence of clemastine fumarate on the growth rate of Klebsiella pneumoniae

And (4) surface note: the growth rate (%) in the table is represented as the average of the growth rates in three experiments. + -. standard deviation. The statistical control group for each concentration of clemastine fumarate was a growth control group without drug intervention. Statistical analysis using the Dunnett method in the one-way ANOVA analysis, p <0.001 was considered as statistically different.

As can be seen from Table 3 and FIG. 4, when compared with the control group without desloratadine, the desloratadine can significantly inhibit the growth of three experimental strains (p <0.001) at a concentration of 128 μ g/mL, and the growth rate of KP37, KP40 and KP53 can be reduced to below 2%. As can be seen from tables 4-6 and FIGS. 5-7, cetirizine, chlorpheniramine maleate, and clemastine fumarate all had no inhibitory effect on the test strains (p > 0.001).

Example 3

Determination of influence of diphenhydramine hydrochloride on growth of staphylococcus aureus

Experimental Material

The raw material medicines of diphenhydramine hydrochloride, chlorpheniramine maleate and cetirizine are purchased from Dalian Meiren biotechnology limited company; 3 strains of staphylococcus aureus (SA1, SA2 and SA3) are separated from inpatient specimens; McLeod Boehringer is available from Beijing Tianan Union technologies, Inc.; the calm MH broth medium and the MHA medium were purchased from Qingdao Hi-Tech Industrial park Haibo Biotech Ltd.

Preparation of staphylococcus aureus liquid and experimental medicine stock solution

5-6 single cells were picked from bacterial plates that had been isolated, purified and freshly cultured overnightAnd (5) inoculating the bacterial colony in an MHA culture medium for enrichment for 16 h. Preparing bacterial suspension with physiological saline, adjusting the turbidity of the sample tube to be consistent with that of 0.5 McBt tube, wherein the concentration of bacterial liquid is about 1.5 × 10 ⁸ CFU/mL, diluted with calcium-adjusted MH broth to give final working concentration of 5X 10 ⁵ CFU/mL. Sterile water is used for respectively preparing diphenhydramine hydrochloride, chlorpheniramine maleate and cetirizine with the concentration of 20480 mu g/mL as storage solution.

Test tube double dilution method for determining inhibition effect of different drugs on staphylococcus aureus

Different medicines are respectively diluted into 2 concentrations by times in a calcium-adjusted MH broth culture medium, and the concentrations of diphenhydramine hydrochloride are as follows: 128 μ g/mL and 256 μ g/mL, chlorpheniramine maleate concentrations were: 512 μ g/mL and 1024 μ g/mL, cetirizine concentrations were: 512. mu.g/mL and 1024. mu.g/mL. Respectively adding 100 mu L of the prepared medicines with different concentrations into different holes of a 96-hole plate; then using a pipettor to adjust the concentration to 5X 10 ⁵ Adding 100 mu L of CFU/mL geotrichum liquid into the hole; as a growth control group, 100. mu.L of calcium-adjusted MH broth and 100. mu.L of the bacterial solution were added to 1 well, and as a "medium background control", 200. mu.L of calcium-adjusted MH broth alone was added to 1 well. The 96-well plate was incubated overnight (18h) at 37 ℃ and the OD at a wavelength of 600nm was measured with a microplate reader and the growth rate of each group was further calculated.

Results

As shown in the figure 8, the influence of experimental medicines with different concentrations on the growth rate of staphylococcus aureus shows that diphenhydramine hydrochloride almost completely inhibits the growth of 3 staphylococcus aureus strains (p is less than 0.001) when the concentration is 256 mu g/mL, which indicates that the bacteriostatic effect is excellent. As can be seen from FIGS. 9 and 10, chlorpheniramine maleate and cetirizine had no inhibitory effect on chlorpheniramine maleate and cetirizine (p > 0.001).

Example 4

Antihistamine for enhancing antibacterial effect of cephalosporin drugs on pseudomonas aeruginosa

Experimental strains

The strain is as follows: pseudomonas aeruginosa strain 1.

Strains obtained from the clinical laboratory in hospitals during this experiment, which have been identified as Pseudomonas aeruginosa, were tested for their sensitivity to clinically common antibiotics (see Table 7). According to the result of drug sensitivity, the strain is changed into a multi-drug resistant strain. All the strains for the test were stored in a refrigerator at-80 ℃ and subcultured on CN agar plates, minimum three times, before use.

TABLE 7 determination of the susceptibility of the test strains to clinically customary antibiotics

Drugs and principal agents

Epinastine hydrochloride, Dalian Melam Biotechnology Ltd (Liaoning Dalian); diphenhydramine hydrochloride, Dalian Melam Biotechnology GmbH (Liaoning Dalian); olopatadine hydrochloride, Dalian biotech GmbH (Liaoning Dalian); cefepime, Dalian Meilan Biotechnology Ltd (Liaoning Dalian); calm MH broth culture medium, haibo biotechnology limited, high-tech industrial garden of Qingdao (Shandong Qingdao); CN agar plate, haibo biotechnology limited, high tech industrial garden of Qingdao (Qingdao, Shandong); phosphate Buffered Saline (PBS), Dalian America Biotechnology Limited (Dalong Liaoning).

Method

Preparation of drug stock solution

Preparation of a drug storage solution: the appropriate pharmaceutical solvent is selected according to the guidelines of the American society for clinical and laboratory standards (CLSI M100-S30) and the instructions of the reagent manufacturer. The epinastine hydrochloride and cefepime are dissolved in sterile distilled water to prepare 48mM solution, and the solution is stored in a refrigerator at the temperature of 20 ℃ below zero for later use. The medicine is naturally thawed and thawed in a refrigerator at 4 deg.C before each use.

Preparation of bacterial liquid

Taking out the preserved strain from a refrigerator at-80 deg.C, placing in a constant temperature box at 37 deg.C for natural dissolution, selecting appropriate amount of bacteria liquid with bacteria ring, inoculating onto CN agar plate, and culturing in constant temperature incubator at 37 deg.C for 18 h. Then, single colonies with good growth are selected and inoculated on CN agar plates again for subculture, and the culture is carried out for 18h at 37 ℃. The culture is subcultured at least three times, so that the method can be used for experimental research. Selecting appropriate amount of colony with good growth from CN agar plate, dissolving in sterile test tube containing sterile PBS solution, mixing with vortex mixer, comparing with Chinese bacteria turbidity standard tube, and adjusting the concentration of experimental bacteria liquid to 0.5 McLee unit (1.5 × 10) ⁸ CFU/mL), diluted with sterile calcium-adjusted MH broth to give final working concentration of 10 ⁶ CFU/mL。

Dilution of medicinal solutions

Determination of the antibacterial action of drugs

Drugs and bacteria liquid are added into a 96-well plate, and each group of culture system is as follows:

experiments were performed in 96-well plates, with the drugs diluted twice in minute amounts, and only 100. mu.L of calcium-adjusted MH broth plus 100. mu.L of bacterial suspension was added without drug (control group) as a growth control group. Adding 100 mu L of cefepime solution with corresponding concentration and 100 mu L of bacterial solution into the cefepime group; 50 mu L of cefepime solution with corresponding concentration, 50 mu L of antihistamine medicine solution with corresponding concentration and 100 mu L of bacteria liquid are added into the combined medicine group.

The culture systems of the above groups are cultured at 35 ℃ for 18h, and the OD value of each group is read by a microplate reader, and the growth rate of each group is further calculated.

Results

The growth rate of pseudomonas aeruginosa in 96-well plates after drug intervention is shown in figures 11-14. As can be seen from FIG. 11, compared with the growth control group, cefepime with a concentration of 0.03mM alone has no inhibitory effect on the strain (p is more than 0.05), and the antibacterial effect of cefepime (p is less than 0.05) can be remarkably enhanced by adding epinastine hydrochloride with a concentration of more than or equal to 0.03mM, so that the growth rate of the strain is reduced to less than 20%. As can be seen from FIG. 12, compared with the growth control group, cefepime with a concentration of 0.03mM alone has no inhibitory effect on the strain (p > 0.05), and when diphenhydramine hydrochloride with a concentration of more than or equal to 0.03mM is added, the bacteriostatic effect of cefepime (p <0.05) can be remarkably enhanced, and the growth rate of the strain is reduced to below 20%. As can be seen from FIG. 13, compared with the growth control group, cefepime with a concentration of 0.03mM alone has no inhibitory effect on the strain (p > 0.05), and when olopatadine hydrochloride with a concentration of more than or equal to 0.03mM is added, the bacteriostatic effect of cefepime (p <0.05) can be remarkably enhanced, and the growth rate of the strain is reduced to below 20%. As can be seen from FIG. 14, there was no synergistic effect (p > 0.05) of desloratadine on cefepime at any dose in this study.

Therefore, epinastine hydrochloride, diphenhydramine hydrochloride and olopatadine hydrochloride can be used as synergists of cefepime to enhance the antibacterial action of cefepime on multidrug-resistant pseudomonas aeruginosa.

Example 5

Anti-bacterial experiments with antihistamines separately combined with different antibiotics:

the experimental strains are methicillin-resistant staphylococcus aureus 10011808 and acinetobacter baumannii 619008; strains were obtained from the clinical laboratory in hospitals during the course of the experiment and have been correctly identified. The strains used for the tests were stored in a refrigerator at-80 ℃ and subcultured on MHA solid medium at least twice before use.

Preparation of drugs and main reagents: epinastine hydrochloride, cyproheptadine hydrochloride, mezlocillin sodium, ampicillin sodium, ticarcillin, piperacillin, calmodulin (MH) broth (CAMHB), MHA agar medium, Phosphate Buffered Saline (PBS).

Preparation of CAMHB broth: weighing 8.80g of CAMHB powder, placing into a 500mL glass bottle, adding distilled water, stirring to dissolve, autoclaving at 121 deg.C for 30min, cooling, and storing in a 4 deg.C refrigerator for use.

Preparation of MHA agar medium: weighing 16.80g MHA powder, placing into 500mL conical flask, adding distilled water, stirring to dissolve, autoclaving at 121 deg.C for 30min, cooling, and storing in 4 deg.C refrigerator for use.

Preparation of a drug storage solution: the appropriate pharmaceutical solvent is selected according to the guidelines of the American society for clinical and laboratory standards (CLSI M100-S30) and the instructions of the reagent manufacturer. The epinastine hydrochloride and the ampicillin sodium are dissolved in sterile distilled water to prepare solutions with the concentration of 20480 mu g/mL, and the solutions are stored in a refrigerator with the temperature of-20 ℃ for use. The medicine is naturally thawed and thawed in a refrigerator at 4 deg.C before each use.

And (3) sterilizing the articles:

sterilizing a glass instrument: sterilizing turbiditubes, beakers, various test tubes and other glass culture dishes with bacteria under high pressure (121 ℃,30min) before experiments, washing, cleaning, naturally drying, completely wrapping with newspaper, sterilizing in a dry heat sterilization box, and sterilizing at 160 ℃ for 2h for later use.

Gun head sterilization: inserting the gun head into the gun head box, tightly wrapping the gun head box with newspaper, placing into an autoclave for sterilization (121 ℃,30min), and placing into a biological safety cabinet for later use.

And (3) sterilizing other articles: after each experiment, waste such as gun heads, cleaning cloths and the like or bacteria-carrying articles with bacteria liquid are put into an autoclave for autoclaving (121 ℃ for 30min), and then are treated. Before each test, the test pieces were subjected to UV sterilization in a biosafety cabinet for about 30 min.

Determination of MIC value of antibacterial agent:

the Minimum Inhibitory Concentration (MIC) of the test strain with the antibacterial agent was measured by the broth microdilution method according to the CLSI (document M100-S30) standard.

Preparing a bacterial liquid:

taking out the preserved strain from a refrigerator at-80 deg.C, placing in a constant temperature box at 37 deg.C for natural dissolution, selecting appropriate amount of bacteria liquid with bacteria ring, inoculating onto MHA solid culture medium, and culturing in constant temperature incubator at 37 deg.C for 18 h. Then, a single colony which grows well is selected and inoculated on the MHA solid culture medium again for subculture, and the culture is carried out for 18h at 37 ℃. The culture is subcultured at least three times, so that the method can be used for experimental research. Selecting appropriate amount of colony with good growth from MHA solid culture medium by using a bacteria-extracting ring, dissolving in a sterile test tube filled with sterile PBS solution, and mixing in vortexMixing the mixture on a mixer by oscillation, comparing with Chinese bacteria turbidity standard tube, and adjusting the concentration of experimental bacteria liquid to 0.5 McLett unit (1.5 × 10) ⁸ CFU/mL), diluted with sterile CAMHB broth to give a final working concentration of 10 ⁶ CFU/mL。

Diluting the liquid medicine:

the dilution method of each drug is the same, and a two-fold micro-dilution method is adopted. Serial dilutions were made in test tubes to make drug solutions of different concentrations 2 times higher than the working concentration in 96-well plates for future use.

Detection of drug combination effect:

the chessboard method is adopted to research the in vitro effect of the combination of the two medicines on the strains. Sequentially sucking 50 mu L of antibiotic (mezlocillin sodium, ampicillin sodium, ticarcillin and piperacillin) solution according to the concentration of the liquid medicine from low to high, and sequentially adding the solution into the 2 nd to 11 th columns of a sterile 96-pore plate; absorbing 50 mu L of epinastine hydrochloride or cyproheptadine hydrochloride solution according to the sequence of the concentration of the liquid medicine from low to high, and sequentially adding the epinastine hydrochloride or cyproheptadine hydrochloride solution into 7-1 rows (the last row of holes is not added) of a sterile 96-well plate; and sucking 100 mu L of prepared bacterial liquid and adding the bacterial liquid into all the holes in the 1-11 columns. All wells in column 12 were used as a control of medium, without drug, plus only 200 μ L of CAMHB broth; the first and last column 2-11 wells were supplemented with 50 μ lcamthb broth. H1 wells served as drug-free bacterial growth controls. After the liquid medicine and the bacterial liquid are added, the 96-well plate is placed in a constant-temperature incubator at 37 ℃ for culturing for 18 hours, and the experimental result is observed and recorded. MIC refers to the lowest drug concentration that completely inhibits bacterial growth in the microdilution wells. Each experiment was repeated three times.

Evaluation method of drug combination:

experiment FICI model is applied to explain and evaluate the interaction result of the medicine. The FICI model is obtained according to the Loewe Addivity (LA) theory, which considers that the drug and the drug cannot interact with each other, so that the concentration points of the drug which can produce the same effect when being used singly and used together are compared, and the FICI model is one of the most common combined drug sensitivity evaluation methods which are internationally accepted at present. When the model is used for judging the interaction of the medicines, part of bacteriostatic concentration needs to be calculated firstlyFractional Inhibition Control Index (FICI). FICI is the sum of FICs of drug A and drug B (FIC) _A +FIC _B ) The formula is as follows: FICI ═ FIC _A +FIC _B The results show that the medicine is (MIC of A medicine when the medicines are combined/MIC of A medicine when the medicines are used alone) + (MIC of B medicine when the medicines are combined/MIC of B medicine when the medicines are used alone). Interpretation of the results the evaluation criteria were as follows: FICI less than or equal to 0.5 indicates that the two medicines have synergistic effect; 0.5<FICI less than or equal to 1 indicates that the two medicines have additive effect; 1<FICI less than or equal to 2 indicates that the two medicines have no interaction; FICI>2, the two drugs have antagonistic action.

The in vitro antibacterial action of the drug combination is as follows:

the experiment uses a chessboard method to determine the in vitro combined action of different drug combinations on methicillin-resistant staphylococcus aureus 10011808 and acinetobacter baumannii 619008, the growth rate distribution of the drug combination in a 96-well plate after drug drying is shown in tables 8-15, the drug combined action results according to tables 8-15 are collated into table 16, and the in vitro combined action experiment results are shown in table 16.

As can be seen from tables 8 and 16, epinastine hydrochloride (128. mu.g/mL) combined with mezlocillin sodium (4. mu.g/mL) almost completely inhibited the growth of methicillin-resistant Staphylococcus aureus;

as can be seen from tables 9 and 16, epinastine hydrochloride (128. mu.g/mL) combined with ampicillin sodium (4. mu.g/mL) almost completely inhibited the growth of methicillin-resistant Staphylococcus aureus;

as can be seen from tables 10-11 and 16, the combination of epinastine hydrochloride with ticarcillin or piperacillin had no synergistic effect on methicillin-resistant Staphylococcus aureus;

as can be seen from tables 12-13 and 16, cyproheptadine hydrochloride and mezlocillin sodium or ampicillin sodium have no synergistic effect on methicillin-resistant Staphylococcus aureus;

as can be seen from tables 14-15 and 16, epinastine hydrochloride in combination with mezlocillin sodium or ampicillin sodium did not have a synergistic effect on Acinetobacter baumannii.

TABLE 8 Effect of epinastine hydrochloride in combination with mezlocillin sodium on the growth rate of methicillin-resistant Staphylococcus aureus

The underlined wells represent the wells corresponding to the optimal concentration combination for the combined antibacterial action of the two drugs

TABLE 9 Effect of epinastine hydrochloride in combination with ampicillin sodium on the growth rate of methicillin-resistant Staphylococcus aureus

The underlined wells represent the wells corresponding to the optimal concentration combination for the combination of two drugs

TABLE 10 Effect of epinastine hydrochloride in combination with ticarcillin on the growth rate of methicillin-resistant Staphylococcus aureus

TABLE 11 Effect of epinastine hydrochloride in combination with piperacillin on the growth rate of methicillin-resistant Staphylococcus aureus

TABLE 12 influence of cyproheptadine hydrochloride in combination with mezlocillin sodium on the growth rate of methicillin-resistant Staphylococcus aureus

TABLE 13 influence of cyproheptadine hydrochloride in combination with ampicillin sodium on the growth rate of methicillin-resistant Staphylococcus aureus

TABLE 14 Effect of epinastine hydrochloride in combination with mezlocillin sodium on Acinetobacter baumannii growth rate

TABLE 15 Effect of epinastine hydrochloride in combination with ampicillin sodium on Acinetobacter baumannii growth rate

TABLE 16 Combined in vitro Effect of epinastine hydrochloride and mezlocillin sodium on methicillin-resistant Staphylococcus aureus

^a 10011808 is the strain number of methicillin-resistant Staphylococcus aureus; 619008 is the strain number of Acinetobacter baumannii;

^b MIC _A represents the MIC of epinastine hydrochloride alone;

^c C _A table MIC of epinastine hydrochloride when used in combination;

^d MIC _B represents the MIC of mezlocillin sodium alone;

^e C _B table MIC of mezlocillin sodium when two drugs are used in combination;

^f FICI less than or equal to 0.5 indicates that the two medicines have synergistic effect; 0.5<FICI less than or equal to 1 indicates that the two medicines have additive effect; 1<FICI less than or equal to 2 indicates that the two medicines have no interaction; FICI>2, the two drugs have antagonistic action.

Example 6

Determination of effect of desloratadine and antibiotic combined synergistic anti-acinetobacter baumannii

Test strains

Acinetobacter baumannii (strain No. 619008) was isolated from clinical patient test specimens and identified as multiple drug-resistant Acinetobacter baumannii strains according to drug sensitivity measurements, and the test strains were stored in a freezer containing 20% glycerol and placed in a refrigerator at-80 ℃.

Primary reagent

Desloratadine (purity > 98%), mezlocillin sodium (purity > 98%), ampicillin sodium (purity > 98%), ceftazidime (purity > 98%) and cefotiam (purity > 98%) were purchased from Dalian Melam Biotechnology Ltd and stored at normal temperature in dark. The calcium-adjusted MH broth culture medium and the MHA agar culture medium are purchased from Qingdao high-tech industrial park Haibo biotechnology, Inc.

Preparation of bacterial liquid and medicinal solution

Preparing bacterial liquid: the cryopreserved strains were thawed at room temperature, streaked onto MHA agar plates, and incubated overnight in a 37 ℃ incubator. After the colonies are formed, a single colony on the plate is selected and inoculated into 10mL of fresh calcium-adjusted MH broth culture medium, and then the broth is placed into a constant temperature shaking table at 37 ℃ and is shaken for 12-18 h at the speed of 170r/min until the bacteria liquid is turbid. The concentration of the bacterial liquid is adjusted to 10 by using a McLeod turbidimeter ⁶ CFU/mL。

Preparing a raw medicine solution: dissolving desloratadine in DMSO to prepare a raw solution with the concentration of 20480 mug/mL; mezlocillin sodium and ampicillin sodium are dissolved in sterile distilled water to prepare original solution with the concentration of 20480 mu g/mL. All the original solutions were stored in a refrigerator at-20 deg.C for use, and the drug solutions were thawed naturally in a refrigerator at 4 deg.C before each use.

Combined susceptibility testing

A series of two-fold dilutions of each drug solution were made in calcium-adjusted MH broth to make a series of solutions with gradient concentrations. Sequentially adding 50 mu L of mezlocillin sodium with different concentrations into 2-11 columns of a sterile 96-well plate, sequentially adding 50 mu L of desloratadine with different concentrations into 1-6 rows of the sterile 96-well plate, adding 100 mu L of bacterial suspension into all the wells, and supplementing the well with less than 200 mu L of MH broth to 200 mu L. As a medium negative control, 200 μ L of calmodulin MH broth was added to all wells in column 12. After mixing well, the 96-well plate was transferred to a 37 ℃ incubator for 18h for 3 replicates.

In the laboratory, the Fractional Inhibition Concentration Index (FICI) is used as the basis for judging the effect of the combined drug susceptibility test:

FICI＝C _A /MIC _A +C _B /MIC _B

in the formula: MIC _A And MIC _B A, B is the MIC value of the drug alone; c _A And C _B Is A, B twoMIC values for each drug combination.

The effect of the combined drug susceptibility test can be judged according to the range of FICI: the FICI is less than or equal to 0.5, which is a synergistic effect; 0.5< FICI ≤ 1 is partial synergism (additive effect); 1< FICI < 2 as an irrelevant action; FICI >2 is antagonistic.

Results of the experiment

TABLE 17 Effect of Desloratadine in combination with mezlocillin sodium on growth rate of multiple drug resistant Acinetobacter baumannii

The combination of the two drugs at the bold and underlined marks provides the optimal drug concentration against the bacterium

TABLE 18 influence of Desloratadine in combination with ampicillin sodium on growth rate of multiple drug resistant A.baumannii

TABLE 19 Effect of desloratadine in combination with ceftazidime on growth rate of multidrug resistant Acinetobacter baumannii

TABLE 20 Effect of desloratadine in combination with cefotiam on the growth rate of multidrug resistant Acinetobacter baumannii

TABLE 21 evaluation of the Effect of drug combinations

^a MIC _A MIC representing loratadine alone;

^b C _A table MIC of desloratadine for the combination of two drugs;

^c MIC _B represents MIC of single antibiotics (mezlocillin sodium, ampicillin sodium, ceftazidime and cefotiam);

^d C _B the MIC of antibiotics (mezlocillin sodium, ampicillin sodium, ceftazidime and cefotiam) when the two drugs are combined is shown.

Conclusion of the experiment

The combination of the desloratadine and the mezlocillin sodium or the ampicillin sodium has partial synergistic effect or synergistic effect on the multi-drug-resistant acinetobacter baumannii, and the combination of the desloratadine and the ceftazidime or the cefotiam shows independent effect on the multi-drug-resistant acinetobacter baumannii.

Example 7

Determination of action of cyproheptadine hydrochloride for enhancing resistance of meropenem to carbapenem-resistant Klebsiella pneumoniae

Materials and methods

1. Material

1.1 test strains

The strains in the course of this experiment were obtained from the clinical laboratory of hospitals and have been identified and resistant to carbapenems of klebsiella pneumoniae. All strains used in the test were stored in a-80 ℃ freezer and subcultured on MHA agar plates a minimum of three times before use.

1.2 drugs with Primary Agents

Cyproheptadine, Dalian biotechnology, Inc. (Dalian Liaoning); meropenem, Dalian biotechnology limited (Liaoning Dalian); calm MH broth culture medium, haibo biotechnology limited, high-tech industrial garden of Qingdao (Shandong Qingdao); MHA agar plates, haibo biotechnology limited, high tech industrial garden of Qingdao (Shandong Qingdao); phosphate Buffered Saline (PBS), Dalian America Biotechnology Limited (Dalong Liaoning).

2. Method for producing a composite material

2.1 preparation of drug stock solution

Preparation of a drug storage solution: the appropriate pharmaceutical solvent is selected according to the guidelines of the American society for clinical and laboratory standards (CLSI M100-S30) and the instructions of the reagent manufacturer. The cyproheptadine hydrochloride and meropenem are respectively dissolved in absolute ethyl alcohol and sterile distilled water to prepare raw solutions with the concentration of 20480 mu g/mL, and the raw solutions are stored in a refrigerator with the temperature of-20 ℃ for use. The medicine is naturally thawed and thawed in a refrigerator at 4 deg.C before each use.

2.2 preparation of bacterial liquid

Taking out the preserved strain from a refrigerator at-80 deg.C, placing in a constant temperature box at 37 deg.C for natural dissolution, selecting appropriate amount of bacteria liquid with bacteria ring, inoculating onto MHA agar plate, and culturing in constant temperature incubator at 37 deg.C for 18 h. Then, single colonies with good growth are selected and inoculated on MHA agar plates again for subculture, and the culture is carried out for 18h at 37 ℃. The culture is subcultured at least three times, so that the method can be used for experimental research. Selecting appropriate amount of colony with good growth from CN agar plate, dissolving in sterile test tube containing sterile PBS solution, mixing with vortex mixer, comparing with Chinese bacteria turbidity standard tube, and adjusting the concentration of experimental bacteria liquid to 0.5 McLee unit (1.5 × 10) ⁸ CFU/mL), diluted with sterile calcium-adjusted MH broth to give a final working concentration of 5 × 10 ⁵ CFU/mL。

2.3 drug combination assay

This example uses a 96-well round bottom drug sensitive plate. Firstly, adding the medicine working solution with the final concentration of 2 times/4 times into medicine sensitive holes with the concentration of 50 mu L/100 mu L per hole, and then adding the prepared standard bacterial suspension into the medicine sensitive holes with the medicine liquid with the concentration of 100 mu L per hole. Growth control wells were filled with 100. mu.L of bacterial suspension and 100. mu.L of medium, the last well being a negative control well (blank control) with medium only. And (3) supplementing the rest holes of the drug sensitive plate with a culture medium to 200 mu L, placing the drug sensitive plate in a constant temperature incubator after the preparation is finished, culturing for 18 hours at 35 ℃, and reading the growth amount of bacteria in each hole by a visual method. The growth status of each well was compared to the growth status of growth control wells and the growth score in each well was recorded.

Second, result in

1. Antibacterial effect of cyproheptadine hydrochloride and meropenem combined application

The amount of Klebsiella pneumoniae grown in 96-well plates is shown in tables 26 to 27. As can be seen from Table 26, compared with the growth control group, 4. mu.g/mL meropenem alone or 128. mu.g/mL cyproheptadine hydrochloride alone had no inhibitory effect on the strain 310492, but 4. mu.g/mL meropenem in combination with 128. mu.g/mL cyproheptadine hydrochloride completely inhibited the growth of the strain 310492. As can be seen from Table 27, compared with the growth control group, 8. mu.g/mL meropenem alone or 128. mu.g/mL cyproheptadine hydrochloride alone had no inhibitory effect on the strain 313342, but 8. mu.g/mL meropenem in combination with 128. mu.g/mL cyproheptadine hydrochloride completely inhibited the growth of the strain 313342.

TABLE 22 Effect of cyproheptadine hydrochloride in combination with meropenem on the growth rate of carbapenem-resistant Klebsiella pneumoniae 310492

Setting the growth rate of bacteria in the growth control holes to be 100 percent as 5 minutes, and setting the growth rate of the holes consistent with the growth conditions to be 5 minutes; score 4 represents about 80% longer bacteria; score 3 represents about 60% longer bacteria; score 2 represents about 40% longer bacteria; score 1 represents about 20% of the bacteria; score 0 indicates no growth. The Minimum Inhibitory Concentration (MIC) should be judged to be 80% or more of the drug concentration in the wells with inhibited growth (score of 1 or less).

Underline marks the holes represent the best effect of the combination of the two drugs.

TABLE 23 Effect of cyproheptadine hydrochloride in combination with meropenem on the growth rate of carbapenem-resistant Klebsiella pneumoniae 313342

Setting the growth rate of bacteria in the growth control holes to be 100 percent as 5 minutes, and setting the growth rate of the holes consistent with the growth conditions to be 5 minutes; score 4 represents about 80% longer bacteria; score 3 represents about 60% longer bacteria; score 2 represents about 40% of the growth of the bacteria; score 1 represents about 20% longer bacteria; score 0 indicates no growth. The Minimum Inhibitory Concentration (MIC) should be judged to be 80% or more of the drug concentration in the wells with inhibited growth (score of 1 or less).

Underlined wells represent the best effect of the combination of the two drugs.

2. Evaluation of drug combination

Experiment FICI model is applied to explain and evaluate the interaction result of the medicine. The FICI model is obtained according to the Loewe Addivity (LA) theory, which considers that the drug and the drug cannot interact with each other, so that the concentration points of the drug which can produce the same effect when being used singly and used together are compared, and the FICI model is one of the most common combined drug sensitivity evaluation methods which are internationally accepted at present. When the model is applied to judge the interaction of the drugs, a Fractional Inhibitory Concentration Index (FICI) is calculated. The FICI values were calculated using experimental data observed with the naked eye. FICI is the sum of FICs for drug A and drug B (FICA + FICB), and the formula is as follows: FICI ═ FIC _A +FIC _B (combined MIC of A drug/MIC of A drug alone) + (combined MIC of B drug/MI of B drug alone)C) .1. the Interpretation of the results the evaluation criteria were as follows: FICI less than or equal to 0.5 indicates that the two medicines have synergistic effect; 0.5<FICI less than or equal to 1 indicates that the two medicines have partial synergistic action; 1<FICI less than or equal to 2 indicates that the two medicines have no interaction; FICI>2, the two drugs have antagonistic action.

Table 24 is prepared from the best effects of the combinations in tables 22 and 23.

TABLE 24 evaluation of the Effect of cyproheptadine hydrochloride in combination with meropenem against carbapenem-resistant Klebsiella pneumoniae

^a MIC _A Represents the MIC of meropenem when used alone;

^b C _A table MIC of meropenem when the two drugs are combined;

^c MIC _B represents the MIC of cyproheptadine hydrochloride when used alone;

^d C _B table MIC of cyproheptadine hydrochloride for the combination of the two drugs.

As can be seen from Table 24, the effect of the combination of drugs is as follows:

the combination of 4 mu g/mL meropenem and 128 mu g/mL cyproheptadine hydrochloride has synergistic inhibition effect on the strain 310492, and the combination of 8 mu g/mL meropenem and 128 mu g/mL cyproheptadine hydrochloride has synergistic inhibition effect on the strain 313342.

In conclusion, the cyproheptadine hydrochloride can enhance the bacteriostatic action of meropenem on carbapenem-resistant Klebsiella pneumoniae

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of cyproheptadine hydrochloride in preparation of multiple drug-resistant acinetobacter baumannii drugs.

2. The use of claim 1, wherein the cyproheptadine hydrochloride is effective at a concentration of 8 μ g/mL or more to inhibit multiple drug resistant acinetobacter baumannii.

3. The use of claim 2, wherein the cyproheptadine hydrochloride is effective at a concentration of 16 μ g/mL or more to inhibit multiple drug resistant acinetobacter baumannii.

4. The use of claim 3, wherein the cyproheptadine hydrochloride is effective at a concentration of greater than or equal to 32 μ g/mL for inhibiting multiple drug resistant Acinetobacter baumannii.

5. The use of claim 4, wherein the cyproheptadine hydrochloride is effective at a concentration of greater than or equal to 64 μ g/mL for inhibiting multiple drug resistant Acinetobacter baumannii.

6. The use of claim 5, wherein the cyproheptadine hydrochloride is effective at a concentration of 128 μ g/mL or more for inhibiting multiple drug resistant Acinetobacter baumannii.

7. The use of claim 6, wherein the cyproheptadine hydrochloride is effective at a concentration of greater than or equal to 256 μ g/mL for inhibiting multiple drug resistant Acinetobacter baumannii.