CN115518063B - Application of melatonin in preparation of medicines for inhibiting tigecycline-resistant bacteria - Google Patents
Application of melatonin in preparation of medicines for inhibiting tigecycline-resistant bacteria Download PDFInfo
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- 229960004089 tigecycline Drugs 0.000 title claims abstract description 63
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- DRLFMBDRBRZALE-UHFFFAOYSA-N melatonin Chemical compound COC1=CC=C2NC=C(CCNC(C)=O)C2=C1 DRLFMBDRBRZALE-UHFFFAOYSA-N 0.000 title claims abstract description 52
- 229960003987 melatonin Drugs 0.000 title claims abstract description 52
- 239000003814 drug Substances 0.000 title claims abstract description 40
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- WTXBRZCVLDTWLP-UHFFFAOYSA-N 5-fluoro-1H-indole-2-carboxylic acid Chemical compound FC1=CC=C2NC(C(=O)O)=CC2=C1 WTXBRZCVLDTWLP-UHFFFAOYSA-N 0.000 description 1
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- 201000008225 Klebsiella pneumonia Diseases 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
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- XZWYZXLIPXDOLR-UHFFFAOYSA-N metformin Chemical compound CN(C)C(=N)NC(N)=N XZWYZXLIPXDOLR-UHFFFAOYSA-N 0.000 description 1
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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
- A61K31/40—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
- A61K31/403—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 carbocyclic rings, e.g. carbazole
- A61K31/404—Indoles, e.g. pindolol
- A61K31/4045—Indole-alkylamines; Amides thereof, e.g. serotonin, melatonin
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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
- A61K31/65—Tetracyclines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to application of melatonin in preparation of a medicine for inhibiting tigecycline-resistant bacteria. The invention provides an application of melatonin in preparing a drug for inhibiting tigecycline-resistant bacteria. The application provided by the invention reveals that the melatonin and tigecycline have synergistic effect when being combined, can obviously improve the antibacterial effect on drug-resistant bacteria, and has better effect than that of each drug-resistant bacteria when being independently used. The invention provides a direction for researching reversion of drug-resistant bacteria by combined administration of endogenous substances and antibiotics.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an application of melatonin in preparing a medicine for inhibiting tigecycline drug-resistant bacteria mediated by a TmexCD1-ToprJ1 efflux pump.
Background
Melatonin is an endogenous substance widely found in animals, plants, bacteria, etc., synthesized from the essential amino acid tryptophan molecule through a series of biochemical reactions. Melatonin is mainly synthesized and secreted by pine cone body in human body under normal light and dark condition, and can transmit circadian cycle signal to human body. Melatonin is also a good antioxidant, can remove free radicals of organisms, and plays an important role in resisting oxidative damage in organisms.
Tigecycline is the first new glycylcycline antibiotic approved for clinical treatment, and is also a third generation tetracycline antibiotic developed following minocycline, and was approved by the FDA in the united states for entry into the united states market in 2005. Tigecycline has a broad antibacterial spectrum and shows good antibacterial activity against most gram-negative bacteria (except for Proteus and Pseudomonas aeruginosa) and gram-positive bacteria. Tigecycline has been considered one of the "last line of defense" for the treatment of multiple resistant gram-negative bacterial infections. As a glycylcycline antibiotic with broad-spectrum antibacterial activity, tigecycline can overcome tetracycline resistance mediated by active efflux pumps or ribosome protection in enterobacteriaceae. However, with the wide clinical application of tigecycline, especially the increase of CRKP, reports on tigecycline-resistant enterobacteria are increasing. The RND efflux pump gene cluster, tmexCD1-toprJ1, encoded by a plasmid was first reported in 2020, which resulted in strains resistant to tetracyclines (including tigecycline and elavastin) and reduced susceptibility to a variety of clinically important antibacterial agents.
Finding an adjuvant capable of inhibiting the function of the TmexCD1-ToprJ1 efflux pump to be combined with tigecycline, and increasing the concentration of tigecycline in bacteria to restore the sensitivity of the strain to tigecycline is a feasible and effective strategy. Many researchers have found substances having the function of inhibiting an efflux pump by chemical synthesis or screening from natural products, and their use in combination with antibacterial agents in vitro tests can significantly enhance the activity of antibacterial agents. However, the existing efflux pump inhibitors are difficult to be applied to clinic due to the problems of safety, specificity and the like, so the prior art still searches for new safe and effective efflux pump inhibitors.
Disclosure of Invention
In order to solve the problems, the invention provides a technical scheme for the application of melatonin in preparing a drug for inhibiting tigecycline-resistant bacteria. The application and related research provided by the invention show that after melatonin and tigecycline are combined, the activity of the klebsiella pneumoniae carrying the tigecycline resistance of the TmexCD1-ToprJ1 efflux pump can be synergistically inhibited through various mechanisms.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an application of melatonin in preparing a drug for inhibiting tigecycline drug-resistant bacteria mediated by a TmexCD1-ToprJ1 efflux pump.
The invention also provides application of melatonin in preparing a medicament for treating infection caused by tigecycline drug-resistant bacteria mediated by a TmexCD1-ToprJ1 efflux pump.
The invention also provides application of melatonin combined with tigecycline in preparing a medicament for treating drug-resistant bacteria infection.
The invention also provides application of melatonin and tigecycline in preparation of medicines for killing tigecycline-resistant klebsiella pneumoniae (Klebsiella pneumonia). In the present invention, the klebsiella pneumoniae is preferably tigecycline-resistant klebsiella pneumoniae carrying a TmexCD1-ToprJ1 efflux pump.
The invention also provides application of melatonin and tigecycline in preparation of medicines for inhibiting formation of bacterial biofilm.
The invention also provides a melatonin combined with tigecycline for use in the preparation of a composition for disrupting bacterial outer membrane integrity and/or bacterial outer membrane permeability and/or PMFIs used in the medicine.
The invention provides an antibacterial pharmaceutical composition, which comprises tetracycline medicines of tigecycline and melatonin.
Preferably, the dosage of the melatonin is more than or equal to 4mg/mL; the dosage of tigecycline is more than or equal to 4 mug/mL.
Preferably, the weight ratio of the melatonin to the tigecycline is (1000-2000): 1-4; further preferably 2000 (1-4).
In order to eliminate the drug resistance of tigecycline, the invention screens out a combination with an inhibiting effect on tigecycline drug-resistant klebsiella pneumoniae through a trace broth chessboard method experiment. The result shows that the melatonin and the tigecycline have remarkable inhibition effect on tigecycline resistant bacteria in vitro when being combined, and the effect is superior to that of each single drug, so that the tigecycline and the melatonin have remarkable synergistic effect when being combined. The relative dosage of each substance in the composition is greatly reduced, thereby reducing possible adverse reactions and side effects, improving the medication safety, and increasing the compliance of patients to take, so as to ensure the curative effect. The invention provides a new research thought and research and development direction for solving the drug resistance problem that tigecycline is becoming serious.
Drawings
FIG. 1 is a graph showing the FIC index profile of a drug-resistant Klebsiella pneumoniae against tigecycline in combination with melatonin;
FIG. 2 is a graph showing the growth of Klebsiella pneumoniae in combination with melatonin;
FIG. 3 shows the fluorescence intensity of rhodamine under melatonin action;
fig. 4 shows the membrane potential of klebsiella pneumoniae under melatonin action.
Detailed Description
The following description refers to reagents or apparatus used without reference to specific techniques or conditions, and are carried out under conventional experimental conditions, and without a specific instruction from the reagent company, under conditions suggested by the instruction. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
1. Bacterial species and antibiotics
Tmexcd1-ToprJ1 efflux pump-mediated tigecycline resistant klebsiella pneumoniae.
Quality control strain E.coli (ATCC 25922) (purchased from China veterinary medicine institute).
Melatonin, tigecycline, are commercially available, both pure.
2. Chessboard method drug sensitivity experiment
The checkerboard microdilution method is adopted to combine 2, 1/2, 1/4, 1/8 and 1/16MIC concentrations of the corresponding strains of each drug on a 96-well reaction plate according to columns and rows respectively, and the inoculum size is 1 multiplied by 10 5 CFU/mL, observed after incubation at 37℃for 18-20 h, was repeated at least three times per group.
The operation steps are as follows: firstly, preparing melatonin and tigecycline into 8, 4, 2, 1/2 and 1/4 xMIC concentrations by using MH broth according to a double dilution method, then sequentially adding 50 mu.L of tigecycline with diluted concentration into the first 6 rows of 1-6 rows of 96-well plates, sequentially adding 50 mu.L of melatonin with diluted concentration into the first 6 rows of 1-6 rows, and finally combining to obtain the final drug concentration of 2-1/16 MIC. Line 7 is tigecyclineThe single drug controls, 8 th column of melatonin, were added with 100. Mu.L of the diluted drug solution. Then 100. Mu.L of diluted bacterial liquid was added to each well, and the 7 th row of intersecting holes were positive controls of the bacterial liquid group. And the calculation result of the FICI is used as the judgment basis of the combined drug susceptibility test. Fici=fic a +FIC b ＝MIC ab /MIC a +MIC ba /MIC b Wherein MIC a ，MIC b MIC values for a and b, respectively, when administered alone ab MIC (MIC) ba MIC values for drug a and drug b when combined are the MIC values for drug a and drug b, respectively.
Evaluation criteria for drug synergy: FICI is less than or equal to 0.5, and the synergism is achieved; FICI is less than or equal to 1 and is 0.5, and the addition effect is achieved; fici is 1 to less than or equal to 2, and has no influence; FICI >2, antagonism.
The experimental results are shown in fig. 1, and as can be seen from fig. 1, melatonin and tigecycline have synergistic antibacterial effect by screening with a micro-broth dilution method. The 16 clinically isolated Klebsiella pneumoniae strains carrying a TmexCD1-ToprJ1 efflux pump were subjected to a combination drug sensitization of melatonin and tigecycline. The results show that: under the combined action of melatonin and tigecycline, FIC values of 16 Klebsiella pneumoniae strains are all less than or equal to 0.5, and good synergistic effect is shown.
3. Growth curve drawing experiment
According to the following steps of 1:1000 the preserved tigecycline-resistant klebsiella pneumoniae was inoculated in 1mL of LB broth and cultured overnight, and then culture 1:1000 was diluted into 20mLLB broth and incubated at 37℃and 180rpm for 4h.
The test is divided into four groups, namely a Contral group, a TIG (1/4 MIC) group, a MEL (1/4 MIC) group and a TIG (1/4 MIC) +MEL (1/4 MIC) group, and each group takes 2mL of activated bacterial liquid. Wherein the control group (i.e., the Contral group) is not treated, the tigecycline group (i.e., the TIG group) is added with tigecycline having a final concentration of 4 μg/mL, the melatonin group (i.e., the MEL group) is added with melatonin having a final concentration of 4mg/mL, and the combination group (i.e., the TIG (1/4 MIC) +MEL group) is added with tigecycline having a final concentration of 4 μg/mL and melatonin having a final concentration of 4 mg/mL.
The four groups of centrifuge tubes are put into a shaking table at 37 ℃ for incubation, and sampling is carried out at time points of 0h, 4h, 8h and 24h respectivelyCounting is performed. The method comprises the following steps: 6 EP tubes of 2mL are taken and numbered 10 in sequence -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 To each of the 6 EP tubes, 900. Mu.L of sterile PBS was added, and 100. Mu.L of the bacterial suspension was sampled at the above time point and added to 10 -1 In the tube, the mixture is diluted to 10 times -6 Tube, 20. Mu.L of each dilution of the bacterial solution was then dropped on LB plate, and the solution was uniformly coated with a disposable coating rod, and the coating was repeated for 3 times.
After culturing in an incubator at 37℃for 24 hours with inversion, plates with colony numbers between 30 and 300 were counted. And (3) data processing: the average colony count on the three plates of the same dilution is calculated, and the bacterial liquid concentration is calculated according to the average colony count. Stock concentration (CFU/mL) =average colony count x dilution x 50.
The experimental results are shown in fig. 2, and the time sterilization curve experiment shows that the melatonin and tigecycline can be used together to remarkably slow down the growth of bacteria. The combination of melatonin and tigecycline produced a log less bacterial growth after 24 hours than the control and melatonin groups 10 X 3, shows that melatonin in combination with tigecycline can significantly reduce bacterial growth.
4. Rhodamine B efflux test
The tigecycline-resistant overnight culture of klebsiella pneumoniae was washed twice with PBS and the concentration OD was adjusted with PBS 600 Rhodamine B was then added at a final concentration of 20 μm, incubated at 37 ℃ with shaking at 200rpm for 30min to reach absorption equilibrium. Then ice-bath for 10min, the absorption of rhodamine B is stopped. Cells were collected by centrifugation at 4000rpm for 5min, washed three times with PBS, and resuspended in 2mL of 1% glucose in PBS. Then, melatonin liquid medicines with different concentrations are respectively added for treatment and are incubated for 30min at 37 ℃ with shaking. The bacterial liquid after the metformin treatment is centrifuged for 10min at 4000rpm, 200 mu L of supernatant is taken in a 96-well black ELISA plate, and the fluorescence intensity is measured by a multifunctional ELISA. The relevant parameters were set as follows: excitation wavelength 540nm and emission wavelength 625nm. Experiments were repeated 3 times and significant differences between groups were analyzed by one-way ANOVA using SPSS19.0 software.
To examine the effect of melatonin on the function of the efflux pump TmexCD1-ToprJ1, the efflux of the efflux pump substrate rhodamine was measured separately. The discharge of rhodamine is shown in fig. 3, and the fluorescent intensity of rhodamine is obviously reduced under the treatment of melatonin with the concentration of 1mg/mL, 2mg/mL and 4mg/mL, which shows that the discharge of rhodamine by a TmexCD1-ToprJ1 discharge pump can be reduced by melatonin.
5. PMF assay
The RND efflux pump uses proton power as an energy source to drive the efflux of the substrate, and thus uses the fluorescent probe disec 3 (5) to measure the proton power of melatonin-treated klebsiella pneumoniae KP 10.
With fluorescent probes DiSC 3 (5) The proton kinetics of melatonin-treated klebsiella pneumoniae was determined. The bacterial liquid cultured to the logarithmic phase was inoculated into 5mL of LB broth and cultured at 37℃for 8 hours at 200 rpm. The cultures were then washed 3 times with PBS and DiSC was added at a final concentration of 2. Mu.M 3 (5) Incubation was carried out at 37℃for 30min at 200rpm, 180. Mu.L of the sample was added to a 96-well black ELISA plate and fluorescence was measured. The fluorescence intensity was measured every 5min for a total of 75min. Melatonin (0, 5mg/mL, 10mg/mL, and 20 mg/mL) was added at different concentrations in 20. Mu.L in the dark at 10 min. The excitation wavelength was set at 622nm and the emission wavelength at 670nm. 3 replicate wells were set up in parallel for each sample, and significant differences were analyzed by one-way ANOVA using SPSS19.0 software.
The results are shown in FIG. 4 and indicate that melatonin selectively disrupts membrane potential in PMFThereby affecting the energy source of the pump and causing the pump to function impaired.
In conclusion, melatonin and tigecycline have a synergistic effect on Tmexcd1-ToprJ1 positive klebsiella pneumoniae, and the action mechanism of the melatonin and tigecycline mainly influences PMF of bacteria, so that energy sources of an efflux pump are blocked, and a substrate tigecycline cannot be excreted by Tmexcd1-ToprJ1, so that accumulation of tigecycline in the bacteria is increased, and bacterial death is finally caused. This study shows that melatonin is a very promising antibiotic adjuvant, which can be used in combination with tigecycline to combat the tigecycline resistance problem caused by the novel RND efflux pump TmexCD1-ToprJ 1. At the same time, more preclinical studies are necessary to fully explore the therapeutic potential of this drug combination.
The foregoing is merely a preferred embodiment of the invention, and it should be noted that modifications and enhancements can be made to the inventors without departing from the principles of the present invention, which modifications and enhancements are also considered to be within the scope of the present invention.
Claims (2)
1. The application of melatonin and tigecycline in preparing medicaments for killing tigecycline-resistant klebsiella pneumoniae.
2. The use according to claim 1, wherein the tigecycline resistant klebsiella pneumoniae comprises a tigecycline resistant klebsiella pneumoniae mediated by a tmaxcd 1-ToprJ1 efflux pump.
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