CN113262214A - Application of cinacalcet hydrochloride in preparation of antibacterial drugs - Google Patents
Application of cinacalcet hydrochloride in preparation of antibacterial drugs Download PDFInfo
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- CN113262214A CN113262214A CN202110269396.XA CN202110269396A CN113262214A CN 113262214 A CN113262214 A CN 113262214A CN 202110269396 A CN202110269396 A CN 202110269396A CN 113262214 A CN113262214 A CN 113262214A
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- cinacalcet hydrochloride
- drug
- staphylococcus aureus
- gram
- positive bacteria
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- QANQWUQOEJZMLL-PKLMIRHRSA-N cinacalcet hydrochloride Chemical compound Cl.N([C@H](C)C=1C2=CC=CC=C2C=CC=1)CCCC1=CC=CC(C(F)(F)F)=C1 QANQWUQOEJZMLL-PKLMIRHRSA-N 0.000 title claims abstract description 119
- 229960000478 cinacalcet hydrochloride Drugs 0.000 title claims abstract description 116
- 229940124350 antibacterial drug Drugs 0.000 title claims abstract description 13
- 238000002360 preparation method Methods 0.000 title abstract description 5
- 241000191967 Staphylococcus aureus Species 0.000 claims abstract description 73
- 239000003814 drug Substances 0.000 claims abstract description 70
- 229940079593 drug Drugs 0.000 claims abstract description 66
- 241000192125 Firmicutes Species 0.000 claims abstract description 50
- 239000003242 anti bacterial agent Substances 0.000 claims description 29
- 229960002227 clindamycin Drugs 0.000 claims description 27
- KDLRVYVGXIQJDK-AWPVFWJPSA-N clindamycin Chemical compound CN1C[C@H](CCC)C[C@H]1C(=O)N[C@H]([C@H](C)Cl)[C@@H]1[C@H](O)[C@H](O)[C@@H](O)[C@@H](SC)O1 KDLRVYVGXIQJDK-AWPVFWJPSA-N 0.000 claims description 27
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- 229940088710 antibiotic agent Drugs 0.000 claims description 20
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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
- A61K31/13—Amines
- A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
- A61K31/137—Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine or methadone
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- 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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- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Oncology (AREA)
- Emergency Medicine (AREA)
- Communicable Diseases (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The invention belongs to the technical field of medicines, and particularly relates to application of cinacalcet hydrochloride in preparation of antibacterial drugs. The invention provides application of cinacalcet hydrochloride in preparation of antibacterial drugs, and experimental results show that the cinacalcet hydrochloride has a bactericidal effect, the minimum inhibitory concentration to gram-positive bacteria is 4-14 mug/mL, and the bacteriostatic effect is strong. In addition, in a drug resistance development experiment, through 25 generations of domestication, staphylococcus aureus 29213 and MRSA (ATCC43300) do not generate drug resistance to cinacalcet hydrochloride, and drug-resistant strains can be avoided; cinacalcet hydrochloride has a curative effect on a mouse pneumonia model infected by staphylococcus aureus in vivo; the cinacalcet hydrochloride has curative effect on a mouse pneumonia model infected by staphylococcus aureus in vivo, and the used medicament dose does not generate toxicity on mouse liver, spleen and kidney tissues, so the safety is good.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to application of cinacalcet hydrochloride in preparation of antibacterial drugs.
Background
The advent of antibiotics has solved a major challenge in humans in the treatment of bacterial infections, however, current treatments for bacterial infections are suffering from the rapidly increasing challenge of multiple resistant strains, and large amounts of antibiotics also lose their powerful antimicrobial halo. Not only gram-negative bacteria produce multidrug-resistant (MDR) pathogens, but also gram-positive pathogens develop a large number of resistant bacteria, causing a major cause of bacterial infection-staphylococcus aureus, particularly methicillin-resistant staphylococcus aureus (MRSA), has become difficult to treat and causes severe infections.
Therefore, the search for drugs that inhibit drug-resistant bacteria is a problem that researchers in the field are urgently required to solve.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide the application of cinacalcet hydrochloride in preparing antibacterial drugs, wherein the cinacalcet hydrochloride can inhibit gram-positive bacteria including drug-resistant gram-positive bacteria, inhibit the formation of gram-positive bacteria biomembranes and eliminate the gram-positive bacteria biomembranes; the second objective of the present invention is to provide an antibacterial agent which has no tissue toxicity and is safe in a therapeutic dose.
The invention provides an application of cinacalcet hydrochloride in preparing antibacterial drugs.
Experimental results show that the cinacalcet hydrochloride has a bactericidal effect, the minimum inhibitory concentration to gram-positive bacteria is 4-14 mug/mL, and the bacteriostatic effect is strong. In addition, in a drug resistance development experiment, through 25 generations of domestication, staphylococcus aureus 29213 and MRSA (ATCC43300) do not generate drug resistance to cinacalcet hydrochloride, and drug-resistant strains can be avoided; cinacalcet hydrochloride has a curative effect on a mouse pneumonia model infected by staphylococcus aureus in vivo; the cinacalcet hydrochloride has curative effect on a mouse pneumonia model infected by staphylococcus aureus in vivo, and the used drug dose does not generate toxicity on mouse liver, spleen and kidney tissues, so the safety is good.
Bacterial biofilm refers to a mass of bacterial aggregated film-like material formed by bacteria adhering to a contact surface, secreting polysaccharide matrices, fibrin, lipoprotein, etc., surrounding themselves. Many antibiotics lack the ability to eradicate established biofilms. And the cinacalcet hydrochloride can inhibit the formation of gram-positive bacteria biomembrane and remove the gram-positive bacteria biomembrane when the concentration is more than 0.5 times MIC.
New uses of older drugs are becoming more and more important as a means of developing new drugs for the treatment of bacterial infections, and the known safety profiles, pharmacokinetic profiles, formulations, dosages and manufacturing processes of these drugs provide a convenient means for accelerating drug reuse. The new use of old medicine greatly reduces the research and development cost and research and development period of the medicine.
Oral cinacalcet hydrochloride is the first calcium mimetic approved for use in the treatment of secondary Hyperparathyroidism (HPT) in Chronic Kidney Disease (CKD) patients undergoing dialysis. Is also approved for the treatment of parathyroid cancer hypercalcemia in the united states and europe. This is a calcimimetic and has a novel mechanism of action, which directly regulates the major regulator of parathyroid hormone (PTH) secretion, namely the calcium sensitive receptor (CaSR) on parathyroid chief cells. Cinacalcet hydrochloride decreases circulating PTH levels by increasing CaSR sensitivity to extracellular calcium. Importantly, cinacalcet hydrochloride is well tolerated in clinical trials, with the majority of adverse events occurring from mild to moderate severity.
To date, no research report on the inhibition of gram-positive bacteria by cinacalcet hydrochloride has been discovered. The invention provides the application of clinical drug cinacalcet hydrochloride in preparing antibacterial drugs for the first time, the cinacalcet hydrochloride can thoroughly eliminate gram-positive bacteria and has relatively consistent minimum inhibitory concentration to various drug-resistant strains, so that the clinical application range of the cinacalcet hydrochloride is expanded, and the cinacalcet hydrochloride has the potential of developing drugs with antibacterial infection effects.
Preferably, the antibacterial agent is an anti-gram-positive bacteria agent.
Preferably, the gram-positive bacteria are selected from one or more of staphylococcus, streptococcus, enterococcus or bacillus.
Preferably, the staphylococcus is staphylococcus aureus and/or staphylococcus haemolyticus;
the streptococcus is streptococcus pneumoniae and/or streptococcus suis;
the enterococcus is enterococcus faecalis;
the bacillus is bacillus subtilis.
Experimental results show that the minimum inhibitory concentration of cinacalcet hydrochloride to streptococcus pneumoniae is 4 mug/mL, the minimum inhibitory concentration to streptococcus suis is 8 mug/mL, the minimum inhibitory concentration to bacillus subtilis is 8 mug/mL, the minimum inhibitory concentration to staphylococcus haemolyticus is 9.3 mug/mL, and the minimum inhibitory concentration to enterococcus faecalis is 10.7 mug/mL.
Preferably, the gram-positive bacteria are drug-resistant gram-positive bacteria and/or non-drug-resistant gram-positive bacteria;
the drug-resistant gram-positive bacteria are single-drug-resistant gram-positive bacteria and/or multi-drug-resistant gram-positive bacteria.
The minimum inhibitory concentration of the cinacalcet hydrochloride to the drug-resistant gram-positive bacteria is 8-14 mug/mL.
Preferably, the drug-resistant gram-positive bacteria are one or more of beta-lactam antibiotic-resistant gram-positive bacteria, aminoglycoside antibiotic-resistant gram-positive bacteria, quinolone antibiotic-resistant gram-positive bacteria, tetracycline antibiotic-resistant gram-positive bacteria, or lincomycin antibiotic-resistant gram-positive bacteria.
In the invention, the gram-positive bacteria resistant to the beta-lactam antibiotics comprise methicillin-resistant staphylococcus aureus and/or amoxicillin-resistant staphylococcus aureus;
the gram-positive bacteria resistant to aminoglycoside antibiotics comprise gentamicin-resistant staphylococcus aureus and/or amikacin-resistant staphylococcus aureus;
gram-positive bacteria resistant to quinolone antibiotics include ciprofloxacin-resistant staphylococcus aureus and/or levofloxacin staphylococcus aureus;
tetracycline-resistant gram-positive bacteria include tetracycline-resistant staphylococcus aureus;
gram-positive bacteria resistant to lincomycin antibiotics include clindamycin-resistant staphylococcus aureus.
The minimum inhibitory concentration of the cinacalcet hydrochloride to clinical multi-drug resistant staphylococcus aureus strains is 8-14 mug/mL.
The invention also provides an antibacterial medicament, which comprises: cinacalcet hydrochloride.
Preferably, the method further comprises the following steps: an antibiotic.
Preferably, the antibiotic is one or more than two of beta-lactam antibiotics, aminoglycoside antibiotics, tetracycline antibiotics or lincomycin antibiotics.
Preferably, the beta-lactam antibiotics are ampicillin and/or methicillin;
the aminoglycoside antibiotic is gentamicin;
the tetracycline antibiotic is tigecycline;
the lincomycin antibiotics are clindamycin.
Experimental results show that the combination of cinacalcet hydrochloride, clindamycin, ampicillin, gentamicin, clindamycin and methicillin has additive effect, and a new thought is provided for clinical treatment of bacterial infection, multiple drug resistance infection and severe infection.
Compared with the prior art, the invention has the beneficial effects that:
(1) the application of the cinacalcet hydrochloride in preparing the antibacterial drugs is that the applicant provides the application of the clinical drug cinacalcet hydrochloride in preparing the antibacterial drugs for the first time, the cinacalcet hydrochloride can thoroughly eliminate gram-positive bacteria and has consistent minimum inhibitory concentration to various drug-resistant strains, so that the clinical application range of the cinacalcet hydrochloride is expanded, and the cinacalcet hydrochloride has the drug development potential as the antibacterial infection effect.
(2) In the antibacterial drug, cinacalcet hydrochloride has the biofilm inhibiting and removing capacity of gram-positive bacteria, and the risk of generating immunity to antibiotics and an immune system due to the formation of a biofilm can be reduced. Importantly, after serial passage for 25 generations, cinacalcet hydrochloride did not develop drug resistance in staphylococcus aureus 29213 and MRSA (ATCC43300) strains, and drug resistant strains could be avoided.
(3) Cinacalcet hydrochloride is approved by drug supervision and management departments of multiple countries to be marketed in the world, has more reliable pharmacological safety, does not need to perform complex toxicology experiments, has good development and utilization prospects, has important practical significance for multi-drug-resistant gram-positive treatment, and provides a new drug choice for clinical treatment of multi-drug-resistant gram-positive bacterial infection.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a Minimum Inhibitory Concentration (MIC) chart of cinacalcet hydrochloride against gram-positive bacteria tested in example 1 of the present invention, wherein the bar charts correspond to the groups from top to bottom in sequence from left to right;
FIG. 2 is a graph showing the effect of different concentrations of cinacalcet hydrochloride on the growth of Staphylococcus aureus MRSA (ATCC43300) in example 1 of the present invention;
FIG. 3 is a graph of the Minimum Inhibitory Concentration (MIC) of cinacalcet hydrochloride against a drug-resistant strain of Staphylococcus aureus in example 2 of the present invention;
FIG. 4 is a plot of the Minimum Inhibitory Concentration (MIC) of cinacalcet hydrochloride against multiple drug resistant Staphylococcus aureus clinical strains in example 3 of the present invention;
FIG. 5 is a graph showing the results of the combination of cinacalcet hydrochloride with clindamycin, ampicillin, gentamicin, clindamycin, and methicillin, respectively, in example 4 of the present invention;
FIG. 6 is a graph of the time sterilization profile of cinacalcet hydrochloride against Staphylococcus aureus (29213) -sensitive strain in example 5 of the present invention;
FIG. 7 is a graph showing the inhibitory effect of cinacalcet hydrochloride on Staphylococcus aureus MRSA (ATCC43300) biofilms in example 6 of the present invention;
FIG. 8 is a graph showing the effect of cinacalcet hydrochloride on biofilm removal in example 7 of the present invention;
FIG. 9 is a graph showing the development of bacterial resistance to cinacalcet hydrochloride and clindamycin in example 8 of the present invention;
FIG. 10 is a graph showing the effect of cinacalcet hydrochloride on pneumonia models in example 9 of the present invention, wherein A is the survival curve of CNA pneumonia models treated, B is the change of bacterial load in lungs after CNA treatment, and C is the fresh tissue map and H & E staining map of lungs of control and treatment groups;
FIG. 11 is a graph of toxicity test results of cinacalcet hydrochloride on mouse organs, wherein A is an H & E staining graph of liver, spleen and kidney of control group and drug group mice, B is an AST result graph in blood of control group and drug group mice, and C is an ALT result graph in blood of control group and drug group mice;
in the figure, CNA represents cinacalcet hydrochloride, GEN represents gentamicin; CIP stands for ciprofloxacin; CLDM represents clindamycin; AMP stands for ampicillin and TIG for tigecycline.
Detailed Description
The invention provides an application of cinacalcet hydrochloride in preparing antibacterial drugs, wherein the cinacalcet hydrochloride can inhibit gram-positive bacteria including drug-resistant gram-positive bacteria, inhibit the formation of gram-positive bacteria biomembranes and eliminate the gram-positive bacteria biomembranes.
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the embodiment of the present invention, the information of the drugs, reagents and consumables used is as follows:
cinacalcet hydrochloride available from the ceramic company (Topscience); DMSO purchased from sika biotechnology, guangzhou; tryptone dry powder (cat # LP0042), yeast extract (cat # LP0021), Todd-Hewitt broth (cat # CM0189) were purchased from Sigma; tryptone soy broth dry powder (cat # 024051) was purchased from Kyoho microorganisms; sodium chloride was purchased from Guangzhou chemical; 7cm petri dishes were purchased from Jiete.
The culture medium was prepared as follows:
(1) TSB liquid medium: mixing 30g tryptone and soybean broth dry powder (cat 024051) with 1L distilled water, and sterilizing at 121 deg.C for 15 min.
(2) LB liquid medium: adding 10g tryptone dry powder, 5g yeast extract and 10g sodium chloride (15 g agar powder is required to be added into a solid culture medium) into 1L distilled water, mixing uniformly, and sterilizing for 30min to obtain the final product.
(3) THYE liquid medium: adding yeast extract dry powder 5g and Todd-Hewitt broth dry powder 36.4g into 1000mL water, mixing, adding 121 deg.C, and sterilizing for 15 min.
The instrument employs a biosafety cabinet, a microplate reader (Biotek), an ultraviolet spectrophotometer, and a thermostated incubator (Thermo).
For a further understanding of the invention, reference will now be made in detail to the following examples.
EXAMPLE 1 determination of the antibacterial Effect of cinacalcet hydrochloride
This example measures the antibacterial effect of cinacalcet hydrochloride.
The test strain information of this example is as follows:
streptococcus pneumoniae (s.pneumoniae D39) was purchased from china type culture collection; streptococcus suis (BM407), Staphylococcus aureus (S.aureus Newman), Methicillin-resistant Staphylococcus aureus (MRSA) (ATCC43300) were purchased from the China general microbiological culture Collection center; enterococcus faecalis (179521), staphylococcus haemolyticus (179595), from southern medical university clinical laboratory, belonging to multi-drug resistant strains, strain resistance information is given in table 3; bacillus subtilis 2508 is from the guangdong province collection of microorganisms; coli (BW25113), pseudomonas aeruginosa (p.aer μ ginosa ATCC9027) among gram-negative bacteria were from the american type culture collection bank.
In this example, three single clones of each strain were assayed for Minimum Inhibitory Concentration (MIC).
1. Experimental procedure
1.1 determination of the MIC of the test strain by cinacalcet hydrochloride.
Streptococcus pneumoniae was inoculated into 0.5% THYE liquid medium at 5% for overnight activation, and transferred to fresh medium at 5% for the next day when OD is reached600Taking out the culture medium to about 0.6, adding 5 mu L of bacterial liquid into a 48-hole plate, diluting the 10mg/mL cinacalcet hydrochloride mother liquor, setting the working concentration range to be 1-12 mu g/mL, performing three biological repetitions of each concentration, supplementing the culture medium to 500 mu L of each hole by using 0.5% THYE (Tetramethylbenzidine), culturing, and placing the culture plate in 5% CO2And culturing for 12 hours in a constant temperature incubator at 37 ℃.
Streptococcus suis, inoculated at 1% inoculum size into 0.5% THYE broth overnight for activation. The next day, the culture was again transferred to fresh medium in an amount of 1%. And after the culture is finished to the logarithmic median, adding 5 mu L of bacterial liquid into a cell plate, diluting the 10mg/mL cinacalcet hydrochloride mother liquor, setting the working concentration range to be 1-12 mu g/mL, and performing three biological repetitions of each concentration by taking 2 mu g/mL as a concentration gradient. Then, the culture was carried out by supplementing 500. mu.L with 0.5% THYE solution, and the plate was placed in 5% CO2And culturing for 12 hours in a constant temperature incubator at 37 ℃.
Inoculating Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa to fresh LB liquid culture medium at an inoculum size of 1% respectively, activating overnight, and inoculating to fresh culture medium at an inoculum size of 1% the next day for culture to OD600And when the concentration reaches about 0.6, adding 5 mu L of bacterial liquid into a 48-pore plate, diluting the 10mg/mL cinacalcet hydrochloride mother liquor, setting the working concentration range to be 1-14 mu g/mL, and performing three biological repetitions of each concentration by taking 2 mu g/mL as a concentration gradient. Then, the whole culture process was carried out in a 200rpm, 37 ℃ incubator for 12 hours with LB liquid medium supplemented to 500. mu.L.
Staphylococcus aureus, enterococcus faecalis and hemolytic staphylococcus are inoculated into TSB liquid culture medium according to the inoculation amount of 1% for overnight activation, and are transferred to fresh culture medium for culture once the next day according to the amount of 1%. And after the culture is finished to the logarithmic median, adding 5 mu L of bacterial liquid into a cell plate, diluting the 10mg/mL cinacalcet hydrochloride mother liquor, setting the working concentration range to be 4-14 mu g/mL, and performing three biological repetitions of each concentration by taking 2 mu g/mL as a concentration gradient. Then, the plate was cultured in a TSB medium supplemented to 500. mu.L, and the plate was incubated at 37 ℃ in a thermostat incubator at 200rpm for 12 hours.
In this example, the enzyme-linked immunosorbent assay was used to measure the concentration of the bacterial liquid at a wavelength of 600nm, and the absorbance OD600<The lowest drug concentration at which no bacterial growth was visible to the naked eye in the 0.1 well was designated as the MIC, which in turn gave the MIC of cinacalcet hydrochloride against the gram-positive bacteria described above, and DMSO was used as a negative control.
1.2 growth Effect of cinacalcet hydrochloride on Staphylococcus aureus MRSA (ATCC43300)
Staphylococcus aureus MRSA (ATCC43300) was inoculated into TSB liquid medium at an inoculum size of 1% for overnight activation, and was transferred to fresh medium at an inoculum size of 1% the next day. After the culture period reaches the logarithmic phase, 5. mu.L of the bacterial solution is taken and added to the cell plate, and meanwhile, the 10mg/mL cinacalcet hydrochloride mother solution is diluted into 1 × MIC, 3/4 × MIC, 1/2 × MIC, 1/4 × MIC and 1/8 × MIC, each concentration is three biological replicates, and 1% DMSO is used as a negative control of growth influence. The plates were then incubated with TSB medium supplemented to 500. mu.L and tested for bacterial growth on a microplate reader EPOCH2TC (Biotek). The program used a kinetic assay model, 37 ℃, 280rpm, every 15min for 12h incubation. Data derivation using Graphpad analysis.
2. Results of the experiment
Referring to FIG. 1 and Table 1, FIG. 1 is a graph showing the Minimum Inhibitory Concentration (MIC) of cinacalcet hydrochloride against enterococcus faecalis-179521, Staphylococcus haemolyticus-179595, Streptococcus suis BM407, Streptococcus pneumoniae D39, Bacillus subtilis 2508, MRSA (ATCC43300) and Staphylococcus aureus Newman in example 1 of the present invention, and Table 1 is the MIC value (measured for 12 hours) of cinacalcet hydrochloride against the gram-positive bacteria, Escherichia coli BW25113 and Pseudomonas aeruginosa ATCC9027 in example 1 of the present invention. The results show that the cinacalcet hydrochloride has an inhibition effect on the gram-positive bacterial infection and can inhibit various gram-positive bacterial infections; however, cinacalcet hydrochloride has a weak inhibitory effect on gram-negative bacteria.
FIG. 2 is a graph showing the effect of cinacalcet hydrochloride at various concentrations on the growth of Staphylococcus aureus MRSA (ATCC43300) in example 1 of the present invention. The results show that cinacalcet hydrochloride can completely inhibit the growth of MRSA (ATCC43300) at the MIC of MRSA (ATCC 43300).
TABLE 1 Minimum Inhibitory Concentration (MIC) of cinacalcet hydrochloride in example 1 of the present invention against the test strain (12h) of this example
Example 2 determination of the Minimum Inhibitory Concentration (MIC) of cinacalcet hydrochloride against a Single drug resistant Strain of Staphylococcus aureus
The test strain information of this example is as follows:
sensitive strain of Staphylococcus aureus (29213) was from the China general microbiological culture Collection center; the staphylococcus aureus single-resistant clindamycin strain (29213), the staphylococcus aureus single-resistant gentamicin strain (29213) and the staphylococcus aureus single-resistant ciprofloxacin strain (29213) are obtained by domesticating sensitive strains of the staphylococcus aureus (29213) through sub-inhibitory concentration.
The domestication mode of the staphylococcus aureus single-resistant clindamycin strain (29213), the staphylococcus aureus single-resistant gentamicin strain (29213) and the staphylococcus aureus single-resistant ciprofloxacin strain (29213) is as follows: respectively measuring the Minimum Inhibitory Concentrations (MIC) of clindamycin, gentamicin and ciprofloxacin to primary sensitive strains of staphylococcus aureus (29213); adding the determined amount of 1/2MIC into a TSB culture medium containing 1% of primary bacteria, performing acclimation culture at 37 ℃ and 200rpm for 12h, determining new MIC, performing acclimation culture at the determined 1/2MIC, and repeating the steps until the drug resistance times of the three antibiotics to staphylococcus aureus are improved by more than 40 times.
1. Experimental procedure
Selecting single methicillin-resistant Staphylococcus aureus strain MRSA (ATCC43300), single clindamycin-resistant Staphylococcus aureus strain (29213), single gentamycin-resistant Staphylococcus aureus strain (29213) and single resistance ringThree monoclonal stocks of a staphylococcus aureus strain of propifloxacin (29213) were inoculated at 1% inoculum size into TSB broth for overnight activation. The next day, the culture was again transferred to fresh TSB medium in an amount of 1%. When OD is reached600And when the concentration reaches about 0.8, adding 5 mu L of bacterial liquid into a cell plate, diluting the 10mg/mL cinacalcet hydrochloride mother liquor, and setting the working concentration range to be 4-14 mu g/mL by taking 2mg/mL as a concentration gradient. Then supplementing 500 μ L with TSB culture medium, culturing at 37 deg.C and 200rpm constant temperature incubator for 12h, measuring the concentration of the cultured bacteria solution with microplate reader at wavelength of 600nm, and measuring the light absorption value OD600<The lowest drug concentration at which no bacterial growth was seen with the naked eye in the 0.1 well was designated as MIC, and the MIC of cinacalcet hydrochloride against drug-resistant Staphylococcus aureus was determined, and DMSO was used as a negative control.
2. Results of the experiment
Referring to fig. 3 and table 2, fig. 3 is a graph showing the Minimum Inhibitory Concentration (MIC) of cinacalcet hydrochloride against staphylococcus aureus-resistant strains in example 2 of the present invention, and table 2 is a MIC value (measurement time 12h) of cinacalcet hydrochloride against staphylococcus aureus-resistant strains in example 2 of the present invention. The result shows that the cinacalcet hydrochloride can inhibit the growth of staphylococcus aureus single-drug-resistant strain at the concentration of 10-11.3 mu g/mL.
TABLE 2 Minimum Inhibitory Concentration (MIC) of cinacalcet hydrochloride against Staphylococcus aureus resistant strain (12h) in example 2 of the present invention
Example 3 determination of Minimum Inhibitory Concentration (MIC) of cinacalcet hydrochloride against clinically isolated multidrug-resistant Staphylococcus aureus
The test strains of this example were clinically isolated multidrug-resistant staphylococcus aureus (168272, 168023, 166471, 166534, 166138, 168293, 900624, 168205, 179634, 179148, 179475, 179459, 178425, 178524, 178360) from southern university of medical sciences clinical. The above numbering is the systematic numbering of the corresponding strains in southern medical university clinical laboratory, and the resistance profiles of the relevant strains are shown in table 3.
TABLE 3 drug resistance of the relevant strains (MIC units:. mu.g/mL)
1. Experimental procedure
Selecting clinically separated multiple drug-resistant staphylococcus aureus monoclonal, and preserving the seeds at-80 ℃ for later use. Activating clinical bacteria liquid, inoculating to TSB liquid culture medium according to the inoculation amount of 1% for overnight activation. The next day, the culture was again transferred to fresh TSB medium in an amount of 1%. When OD is reached600And when the concentration reaches about 0.8, adding 5 mu L of bacterial liquid into a cell plate, diluting the 10mg/mL cinacalcet hydrochloride mother liquor, and setting the working concentration range to be 4-14 mu g/mL by taking 2 mu g/mL as a concentration gradient. Then supplementing 500 μ L with TSB culture medium, culturing at 37 deg.C for 12h in 200rpm shaking table, measuring the concentration of the cultured bacteria liquid with microplate reader at wavelength of 600nm, and measuring the light absorption value OD600<The lowest drug concentration at which no bacterial growth was seen with the naked eye in the 0.1 well was designated as the MIC, which in turn gave the MIC of cinacalcet hydrochloride against the multi-antibiotic resistant Staphylococcus aureus, and DMSO was used as a negative control.
2. Results of the experiment
The results are shown in fig. 4 and table 4, where fig. 4 is a graph of the Minimum Inhibitory Concentration (MIC) of cinacalcet hydrochloride against multiple drug-resistant staphylococcus aureus clinical strains in example 3 of the present invention, and table 4 is a MIC value (measurement time is 12h) of cinacalcet hydrochloride against multiple drug-resistant staphylococcus aureus in example 3 of the present invention. The result shows that the cinacalcet hydrochloride has an inhibiting effect on the clinical drug-resistant bacteria of gram-positive bacteria by 8-14 mu g/mL, and can inhibit the growth of the clinical drug-resistant bacteria of the gram-positive bacteria.
TABLE 4 Minimum Inhibitory Concentration (MIC) of cinacalcet hydrochloride against multidrug resistant Staphylococcus aureus in example 3 of the present invention
Example 4 determination of the Effect of cinacalcet hydrochloride in combination with antibiotics
1. Experimental procedure
In this example, five different antibiotics were selected for combined administration with cinacalcet hydrochloride, namely β -lactam antibiotics Ampicillin (ampicilin, AMP), aminoglycoside antibiotics Gentamicin (GEN), tetracycline antibiotics Tigecycline (TIG), lincomycin antibiotics Clindamycin (cldamycin, CLDM), and β -lactam antibiotics Methicillin (MET). The MICs of five antibiotics to Staphylococcus aureus MRSA (ATCC43300) were first tested, and were determined in the same manner as in example 1. And then five different types of antibiotics are respectively used in combination with the cinacalcet hydrochloride, namely, the combined result of the antibiotics and the cinacalcet hydrochloride under different concentration ratios is determined in a 96-well plate by using a chessboard test method. Adding antibiotics into a pore plate, supplementing the antibiotics to 198 mu L by using a TSB culture medium, adding 2 mu L of bacterial liquid transferred to an OD 600-0.8 bacterial liquid, culturing for 12h in a constant-temperature incubator at 37 ℃ at 200rpm, measuring the concentration of the cultured bacterial liquid by using an enzyme-labeling instrument under the condition of the wavelength of 600nm, verifying three biological compound pores in a 48-pore plate by using a drug combination corresponding to a pore with an absorbance OD600 of less than 0.1, and judging the interaction by calculating a part of bacteriostatic concentration index (FIC).
Wherein, FIC is the MIC of the first drug when the drugs are combined, the MIC of the first drug when the drugs are singly applied, the MIC of the second drug when the drugs are combined and the MIC of the second drug when the drugs are singly applied, and FIC indexes are less than or equal to 0.5, 0.5-1, 1-2 and 2 respectively represent synergy, addition, irrelevance and antagonism.
2. Results of the experiment
FIG. 5 shows the results of the combination of cinacalcet hydrochloride with clindamycin, ampicillin, gentamicin, clindamycin and methicillin in example 4 of the present invention. The results show that the combination of cinacalcet hydrochloride, clindamycin, ampicillin, gentamicin, clindamycin and methicillin has additive effect, and a new idea is provided for clinical treatment of bacterial infection, multiple drug resistance infection and severe infection.
It should be noted that cinacalcet hydrochloride can also be used in combination with more than two antibiotics selected from clindamycin, ampicillin, gentamicin, clindamycin, or methicillin.
EXAMPLE 5 determination of the time Sterilization Curve of cinacalcet hydrochloride against Staphylococcus aureus (29213) -sensitive Strain
1. Experimental procedure
In this example, DMSO was used as a negative control group, and AMP was used as a positive control group.
After the staphylococcus aureus (29213) sensitive strain is cultured in the TSB culture medium overnight, 1 percent of the sensitive strain is transferred to a fresh TSB culture medium and cultured to OD600After 0.515, cells were diluted 1:100 to 2 × 10 in TSB medium6CFU/mL, DMSO, AMP (1 × MIC, 2 × MIC), cinacalcet hydrochloride CNA (1 × MIC, 2 × MIC) were added, and incubated at 37 ℃ and 200rpm, serially diluted at the indicated time points, spread on agar plates, and cultured at 37 ℃ for 16 hours to determine the number of viable cells (CFU/mL).
2. Results of the experiment
Referring to fig. 6, fig. 6 is a graph showing the time sterilization of cinacalcet hydrochloride against staphylococcus aureus (29213) -sensitive strain in example 5 of the present invention. DMSO was used as a negative control, and AMP (1 × MIC, 2 × MIC) was used as a positive control. The dotted line represents 99.99% of the bactericidal effect. The result shows that the cinacalcet hydrochloride can effectively kill the staphylococcus aureus 29213 and has stronger action effect than AMP.
Example 6 inhibition of biofilm by cinacalcet hydrochloride
1. Experimental procedure
Staphylococcus aureus MRSA (ATCC43300) was activated overnight, transferred at 1%, OD600 ═ 0.8, and then inoculated in a 1% amount into a well plate containing 1% DMSO, cinacalcet hcl CNA (0.5 xmic, 1 xmic, 2 xmic, 4 xmic), four biological replicates per gradient, incubated at 37 ℃ for 24h, supernatant aspirated, washed with PBS three times, dried at 37 ℃ for 3h, then dried at room temperature, stained with 0.2% crystal violet for 10min, after removal of excess crystal violet, washed with PBS three times, the remaining biofilm was dissolved with 30% acetic acid, diluted 1:4, and absorbance was measured at 570nm with a plate reader.
2. Results of the experiment
FIG. 7 is a graph showing the effect of cinacalcet hydrochloride on the biofilm inhibition by Staphylococcus aureus MRSA (ATCC43300) in example 6 of the present invention. The results showed that cinacalcet hydrochloride (0.5 × MIC) had a significant difference in the ability to inhibit the formation of the MRSA (ATCC43300) biofilm in staphylococcus aureus compared to DMSO, and cinacalcet hydrochloride (1 × MIC, 2 × MIC, 4 × MIC) had a very significant ability to inhibit the formation of the MRSA (ATCC43300) biofilm.
Example 7 determination of the biofilm removing ability of cinacalcet hydrochloride
1. Experimental procedure
Staphylococcus aureus 29213 or MRSA (ATCC43300) was activated overnight, transferred to a 96-well plate (200. mu.L system) or a 48-well plate (500. mu.L system) at 1%, filmed in an incubator at 37 ℃ for 24 hours, then the supernatant was removed, the suspended cells were washed off with PBS, 1% DMSO diluted with TSB, cinacalcet HCl CNA (0.5 × MIC, 1 × MIC, 2 × MIC, 4 × MIC) were added, four biological replicates per gradient were added, the supernatant was aspirated after incubation in the incubator at 37 ℃ for 24 hours, the biofilm was washed with PBS, dried at 37 ℃ for 3 hours and at room temperature, stained with 0.2% crystal violet for 10min, the excess crystal violet was removed, washed three times with PBS, the remaining biofilm was dissolved with 30% acetic acid, diluted at 1:5, and the absorbance was measured at 570nm with a microplate reader.
2. Results of the experiment
Please refer to fig. 8, which is a graph showing the effect of cinacalcet hydrochloride on removing biofilm in example 7 of the present invention. The results show that cinacalcet hydrochloride (0.5 × MIC, 1 × MIC, 2 × MIC, 4 × MIC) has significant differences in the removal capacity of staphylococcus aureus biofilms compared to the blank DMSO.
Example 8 testing of cinacalcet hydrochloride for development of resistance to bacteria
1. Experimental procedure
To examine the development of drug resistance to bacteria by serial passages, exponentially growing staphylococcus aureus 29213 and MRSA (ATCC43300) were diluted 1% into TSB medium (500 μ L per well) containing cinacalcet hcl CNA at different drug concentrations. The bacteria were cultured at 37 ℃ and 200rpm for 12h to obtain new MICs, 10. mu.L of the bacteria were transferred to fresh TSB medium (1mL) in 1/2MIC wells, cultured at 37 ℃ and 200rpm until after the exponential growth phase, and then diluted 1% to a new concentration gradient for the next generation of MIC determination. Repeating the steps, and carrying out passage once every 12 h. This series of passages was repeated for 25 passages. And CLDM acclimatized staphylococcus aureus 29213 was used as a positive control and dimethylsulfoxide (1% DMSO) was used as a growth control. MIC shifts are calculated by dividing the MIC of the corresponding generation by the initial MIC of the first day.
2. Results of the experiment
Results referring to fig. 9, a graph of the development of bacterial resistance to cinacalcet hydrochloride and clindamycin according to example 8 of the present invention is shown. The results show that staphylococcus aureus 29213 and MRSA (ATCC43300) did not develop resistance to CNA (cinacalcet hydrochloride) by acclimation for 25 generations, while CLDM (clindamycin) developed higher resistance to CLDM by acclimation for 25 generations of staphylococcus aureus 29213, which increased MIC by 40-fold compared to primary.
Example 9 testing the therapeutic Effect of cinacalcet hydrochloride on model pneumonia
1. Experimental procedure
14 Balb/c mice (Beijing Huafukang biotech GmbH) with the age of 4-5 weeks are randomly divided into an infection control group (7) and a drug group (7), and 40 mu L of staphylococcus aureus clinical strain 166138 bacterial liquid (4 multiplied by 10) is respectively dripped into the left nasal cavity of the mice9CFU/mouse), 4 hours later, each of the drug group mice was gavaged with 20mg/kg cinacalcet hydrochloride, and the control group was givenSolvent (10% DMSO + 40% PEG300+ 5% tween-80+ 45% PBS). Mice were kept upright for 1min after inoculation to ensure adequate uptake by the mice. Then, gavage with 10mg/kg CNA or solvent once a day, observing the behavior of the mice, recording the mortality and body weight, and H-treating the surviving mice lungs on the sixth day&E, staining the section for observation, and taking a part to record the lung bacteria load.
2. Results of the experiment
Fig. 10 is a graph showing the effect of cinacalcet hydrochloride on pneumonia models in example 9 of the present invention, wherein a is the survival curve of CNA pneumonia models, B is the change of bacterial load in the lungs after CNA treatment, and C is the fresh tissue map and H & E staining map of the lungs of the control group and the treatment group. The result shows that after the pneumonia model mouse is administrated for five days, the cinacalcet hydrochloride can prolong the survival time of the mouse and improve the survival rate, the survival rate of a control group is only 14.2%, and the survival rate of an administration group is 42.8%. And the lung bacterial load of pneumonia model mice is obviously reduced after the administration. Tissue H & E staining shows that lung inflammatory cell infiltration, alveolar wall thickening and alveolar bleeding of mice without drug treatment obviously reduce lung inflammation in vivo after the mice are treated by cinacalcet hydrochloride.
Example 10 testing of toxicity of cinacalcet hydrochloride on mouse organs
1. Experimental procedure
10 4-5 week old Balb/c mice (Beijing Hua Fukang Biotechnology GmbH) were randomly assigned to control groups (5) and drug groups (5), each of the drug group mice on the first day was gavaged with 20mg/kg cinacalcet hydrochloride, and the control groups were given solvent (10% DMSO + 40% PEG300+ 5% tween-80+ 45% PBS). Mice were kept upright for 1min after inoculation to ensure adequate uptake by the mice. The mice were then gavaged once daily at 10mg/kg CNA or solvent and observed for behavior for 7 consecutive days followed by 5 days. H & E stained sections were observed for liver, spleen and kidney, and the enzymatic activities of liver function indices AST and ALT in blood were detected.
2. Results of the experiment
Fig. 11 shows toxicity test results of cinacalcet hydrochloride on mouse organs, wherein a is H & E staining patterns of liver, spleen and kidney of control and drug mice, B is AST result pattern in blood of control and drug mice, and C is ALT result pattern in blood of control and drug mice. The results show that the administration mode of the cinacalcet hydrochloride has no toxicity to tissues of liver, spleen and kidney, and the liver function indexes of glutamic-pyruvic transaminase ALT and glutamic-oxalacetic transaminase AST have normal enzyme activity and have no significant difference with a control group.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. Application of cinacalcet hydrochloride in preparing antibacterial drugs.
2. The use according to claim 1, wherein the antibacterial agent is an anti-gram-positive agent.
3. The use according to claim 2, wherein the gram-positive bacteria are selected from one or more of staphylococci, streptococci, enterococci or bacilli.
4. Use according to claim 3, wherein the staphylococci are Staphylococcus aureus and/or Staphylococcus haemolyticus;
the streptococcus is streptococcus pneumoniae and/or streptococcus suis;
the enterococcus is enterococcus faecalis;
the bacillus is bacillus subtilis.
5. Use according to claim 2, wherein the gram-positive bacterium is a drug-resistant gram-positive bacterium and/or a non-drug-resistant gram-positive bacterium;
the drug-resistant gram-positive bacteria are single-drug-resistant gram-positive bacteria and/or multi-drug-resistant gram-positive bacteria.
6. The use according to claim 5, wherein the drug-resistant gram-positive bacteria are selected from one or more of beta-lactam antibiotic-resistant gram-positive bacteria, aminoglycoside antibiotic-resistant gram-positive bacteria, quinolone antibiotic-resistant gram-positive bacteria, tetracycline antibiotic-resistant gram-positive bacteria, or lincomycin antibiotic-resistant gram-positive bacteria.
7. An antibacterial agent, comprising: cinacalcet hydrochloride.
8. The antibacterial agent according to claim 7, further comprising: an antibiotic.
9. The antibacterial agent according to claim 8, wherein the antibiotic is one or more selected from the group consisting of β -lactam antibiotics, aminoglycoside antibiotics, tetracycline antibiotics, and lincomycin antibiotics.
10. The antibacterial agent according to claim 9, wherein the β -lactam antibiotic is ampicillin and/or methicillin;
the aminoglycoside antibiotic is gentamicin;
the tetracycline antibiotic is tigecycline;
the lincomycin antibiotics are clindamycin.
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| CN114617886A (en) * | 2022-03-10 | 2022-06-14 | 中国医学科学院医药生物技术研究所 | Antibacterial use of compounds and pharmaceutically acceptable salts thereof |
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|---|---|---|---|---|
| WO2008075173A2 (en) * | 2006-12-15 | 2008-06-26 | Ruprecht-Karls-Universität Heidelberg | Methods for treating podocyte-related disorders |
| CN111840264A (en) * | 2020-08-14 | 2020-10-30 | 华中科技大学协和深圳医院 | Use of cisacana for inhibiting biological activity of gram-positive bacteria |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008075173A2 (en) * | 2006-12-15 | 2008-06-26 | Ruprecht-Karls-Universität Heidelberg | Methods for treating podocyte-related disorders |
| CN111840264A (en) * | 2020-08-14 | 2020-10-30 | 华中科技大学协和深圳医院 | Use of cisacana for inhibiting biological activity of gram-positive bacteria |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114617886A (en) * | 2022-03-10 | 2022-06-14 | 中国医学科学院医药生物技术研究所 | Antibacterial use of compounds and pharmaceutically acceptable salts thereof |
| CN114617886B (en) * | 2022-03-10 | 2023-09-26 | 中国医学科学院医药生物技术研究所 | Compound and antibacterial application of pharmaceutically acceptable salt thereof |
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