CN116983388A - Composition for resisting drug-resistant gram-negative bacterial infection and application thereof - Google Patents

Composition for resisting drug-resistant gram-negative bacterial infection and application thereof Download PDF

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CN116983388A
CN116983388A CN202310918274.8A CN202310918274A CN116983388A CN 116983388 A CN116983388 A CN 116983388A CN 202310918274 A CN202310918274 A CN 202310918274A CN 116983388 A CN116983388 A CN 116983388A
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enantiomer
colistin
closantel
drug
resistant gram
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贺利民
丁同燕
郭泽宇
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South China Agricultural University
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    • A61K38/00—Medicinal preparations containing peptides
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    • A—HUMAN NECESSITIES
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    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • 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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Abstract

The invention belongs to the technical field of antibiosis, and discloses a composition for resisting drug-resistant gram-negative bacteria infection and application thereof. The composition is composed of a combination of a closantel enantiomer and colistin, wherein the closantel enantiomer is a closantel R-enantiomer or a closantel S-enantiomer. According to the invention, by constructing a mouse abscess model and carrying out combined injection administration on the chlorocyanioxazin enantiomer and colistin, the enantiomer and colistin are found to have good synergistic effect on the treatment of drug-resistant gram-negative bacteria, compared with the combination of two enantiomers and colistin and single drug, the bacterial load in the mouse abscess is reduced by at least 3-7 log CFU/g, and the drug effect of the enantiomer and colistin is obviously different in stereoselectivity, and the R-enantiomer is 4log CFU/g lower than the S-enantiomer.

Description

Composition for resisting drug-resistant gram-negative bacterial infection and application thereof
Technical Field
The invention belongs to the technical field of antibiosis, and in particular relates to a composition for resisting drug-resistant gram-negative bacterial infection and application thereof.
Background
Pseudomonas aeruginosa, also called Pseudomonas aeruginosa, is widely distributed on skin, intestinal tract and respiratory tract of natural and normal human and animal, and is one of the more common conditional pathogenic bacteria in clinic. Pseudomonas aeruginosa is an opportunistic infectious agent that causes immune damage, typically affecting the lungs and urinary tract, or causing burns, wounds and other blood infections, such as sepsis. Although rare, pseudomonas aeruginosa can also cause pneumonia. There is also a chance of infection of the cornea of the eye in situations where the contact lens is not completely cleaned. In many ventilator-associated pneumonia studies, pseudomonas aeruginosa is one of the bacteria that need to be sequestered. Pyocin, a blue-green toxic metabolite, is one of the causative agents of pseudomonas aeruginosa and can kill caenorhabditis elegans under oxidative stress, but studies have indicated that salicylic acid can inhibit the production of pyocin. 10% of the conditions in nosocomial infections are caused by Pseudomonas aeruginosa. In the absence of appropriate treatment, it is also one of the bacteria that causes dermatitis. It is also the most common bacterium responsible for burn infections.
Klebsiella pneumoniae is the most important bacterium in the genus Klebsiella of enterobacteriaceae (commonly known as Klebsiella pneumoniae), and the resulting diseases account for more than 95% of Klebsiella infections. When the disease resistance of the human body is reduced, the human body enters the lung through the respiratory tract to cause the fusion of large leaves or small leaves to be changed, and the above leaves are more common. Exudates in klebsiella pneumoniae lesions are sticky and heavy, causing leaf gaps to fall down. Bacteria have a capsule that, when grown and propagated within the alveoli, causes necrosis, liquefaction of tissue, and formation of single or multiple abscesses. Exudative or purulent effusion can occur when lesions involve the pleura and pericardium. Focal fibrous tissue hyperplasia is active and easy to be mechanized; cellulosic pleural effusions can develop adhesions early. In nosocomial-infected sepsis, klebsiella, pseudomonas aeruginosa, serratia and the like are important pathogenic bacteria, and the death rate is high.
Thus, rational antibiotic intervention remains an effective means of treating gram-negative bacterial infections today. Colistin is one of the important drugs for clinical treatment of pseudomonas aeruginosa infection. However, the widespread use of colistin has led to an increase in the detection rate of the colistin-resistant pseudomonas aeruginosa and klebsiella pneumoniae, which exhibit the characteristic of multidrug resistance to antibacterial agents, greatly limiting the antibacterial efficacy of colistin. By adopting a strategy of combining colistin and synergist medicines, the sensitivity of drug-resistant bacteria to colistin can be improved, and the development trend of bacterial drug resistance inhibited by the use amount of antibiotics can be greatly reduced.
In recent years, the agricultural rural department has developed a 'veterinary antibacterial agent use reduction test point action' in a national range, and calls the cultivation end to use veterinary antibacterial agents normally, reasonably, scientifically and cautiously. Under the trend of 'resistance reduction and resistance limitation' of the breeding end, the realization of 'resistance reduction and efficacy enhancement' becomes a new challenge while ensuring the clinical curative effect of the antibacterial drug. Among them, the "old medicine" with "synergy" is found from the approved medicines, and the antibacterial effect is improved by compatibility with the existing important antibacterial medicines, so that the susceptibility to multi-drug resistant pathogenic bacteria is restored, which is one of the important strategies for realizing the substitution of antibiotics and solving the problem of bacterial drug resistance in the current veterinary clinic. The ability of racemates of many chiral drugs such as chlorocyanioxazin to "synergistically enhance" has been found, whereas the combination of its single enantiomer with colistin has little interest in the treatment of bacterial infections. At present, chiral colistin synergists are mostly applied in the form of racemates, and no single enantiomer is applied as a colistin synergist.
Disclosure of Invention
In order to solve the above-mentioned disadvantages and drawbacks of the prior art, an object of the present invention is to provide a composition for combating drug-resistant gram-negative bacterial infections.
It is a further object of the present invention to provide the use of the above composition against drug resistant gram negative bacterial infection.
The aim of the invention is achieved by the following technical scheme:
a composition for combating a drug-resistant gram-negative bacterial infection, said composition comprising a combination of a closantel enantiomer and a colistin, said closantel enantiomer being either the closantel R-enantiomer or the closantel S-enantiomer.
Preferably, the drug-resistant gram-negative bacteria are pseudomonas aeruginosa, klebsiella pneumoniae, escherichia coli or salmonella; the mass ratio of the cyaniodate enantiomer to the colistin is (0.1-100): 1.
Preferably, when the drug-resistant gram-negative bacterium is pseudomonas aeruginosa, the mass ratio of the chlorocyaniosaliamine R-enantiomer to colistin is (0.1-50) 1, and the mass ratio of the chlorocyaniosaliamine S-enantiomer to colistin is (0.2-100) 1; the mass concentration of the R-enantiomer of the cyaniodate is 0.05-5 mg/mL, and the mass concentration of the S-enantiomer of the cyaniodate is 0.1-10 mg/mL; the mass concentration of the colistin is 0.05-5 mg/mL.
More preferably, when the drug-resistant gram-negative bacterium is pseudomonas aeruginosa, the mass ratio of the R-enantiomer of the closantel to the colistin is (0.1-25) 1, and the mass ratio of the S-enantiomer of the closantel to the colistin is (0.2-25) 1; the mass concentration of the R-enantiomer of the cyaniodate is 0.07-3 mg/mL, and the mass concentration of the S-enantiomer of the cyaniodate is 0.2-5 mg/mL; the mass concentration of the colistin is 0.08-3 mg/mL.
Further preferably, when the drug-resistant gram-negative bacterium is Pseudomonas aeruginosa, the mass ratio of the R-enantiomer of the closantel to the colistin is (0.5-1): 1, and the mass ratio of the S-enantiomer of the closantel to the colistin is (0.5-2): 1; the mass concentration of the R-enantiomer of the cyaniodate is 0.1-0.5 mg/mL, and the mass concentration of the S-enantiomer of the cyaniodate is 0.5-1 mg/mL; the mass concentration of the colistin is 0.2-1 mg/mL.
Preferably, when the drug-resistant gram-negative bacterium is klebsiella pneumoniae, the mass ratio of the R-enantiomer of the closantel to the colistin is (0.2-30) 1, and the mass ratio of the S-enantiomer of the closantel to the colistin is (0.3-50) 1; the mass concentration of the R-enantiomer of the cyaniodate is 0.05-3 mg/mL, and the mass concentration of the S-enantiomer of the cyaniodate is 0.1-5 mg/mL; the mass concentration of the colistin is 0.1-3 mg/mL.
More preferably, when the drug-resistant gram-negative bacterium is klebsiella pneumoniae, the mass ratio of the R-enantiomer of the closantel to the colistin is (0.4-5) 1, and the mass ratio of the S-enantiomer of the closantel to the colistin is (0.4-15) 1; the mass concentration of the R-enantiomer of the cyaniodate is 0.1-1 mg/mL, and the mass concentration of the S-enantiomer of the cyaniodate is 0.3-3 mg/mL; the mass concentration of the colistin is 0.2-1.2 mg/mL.
Further preferably, when the drug-resistant gram-negative bacterium is klebsiella pneumoniae, the mass ratio of the chlorocyanioside R-enantiomer to colistin is (0.5-3): 1, and the mass ratio of the chlorocyanioside S-enantiomer to colistin is (0.5-2): 1; the mass concentration of the R-enantiomer of the closantel is 0.4-0.8 mg/mL, and the mass concentration of the S-enantiomer of the closantel is 0.5-1 mg/mL; the mass concentration of the colistin is 0.3-1 mg/mL.
The application of the composition for resisting drug-resistant gram-negative bacteria infection in preparing animal antibacterial agents.
Preferably, the composition is used for injection in an amount of 10 to 30. Mu.L/20 to 30g.
Compared with the prior art, the invention has the following beneficial effects:
1. according to the invention, the enantiomer of the cyaniotidil is applied to the antibacterial synergist of the colistin for the first time, and the fact that the enantiomer of the cyaniotidil (R-enantiomer or S-enantiomer) has good gain effect on the activity of drug-resistant gram-negative bacteria (such as pseudomonas aeruginosa and klebsiella pneumoniae) of the colistin is found, and the composition has synergistic treatment effect on the infection of drug-resistant gram-negative bacteria (such as pseudomonas aeruginosa and klebsiella pneumoniae), and the drug effects of the two enantiomers are different, so that the accurate drug administration of the cyaniotidil is facilitated.
2. According to the invention, by constructing a mouse abscess model and using the combination of the chlorocyanidoxiosamine and the colistin for injection administration, the combination of the chlorocyanidoxiosamine enantiomer and the colistin has a good synergistic effect on the treatment of drug-resistant gram-negative bacteria, the drug effects of the two enantiomers are obviously different, compared with the combination of the two enantiomers and the colistin, the amount of bacteria in the abscess is at least reduced by 3-7 log CFU/g, the drug effects of the two enantiomers are obviously different in stereoselectivity, and the R-enantiomer is 4log CFU/g lower than the S-enantiomer.
Drawings
FIG. 1 shows the effect of a single drug of closantel R-enantiomer, S-enantiomer, colistin on the amount of mouse abscess bacteria in example 1.
FIG. 2 is a graph showing the effect of a single drug of closantel R-enantiomer, S-enantiomer, colistin on mouse abscess volume according to example 1.
FIG. 3 shows the effect of the R-enantiomer of iohexosamine in combination with colistin or the S-enantiomer in combination with colistin on the amount of mouse abscess bacteria according to example 2.
FIG. 4 is a graph showing the effect of the R-enantiomer of iohexosamine in combination with colistin or the S-enantiomer in combination with colistin on mouse abscess volume according to example 2.
FIG. 5 shows the effect of the R-enantiomer of cyaniodate with colistin or the S-enantiomer with colistin on the amount of mouse abscess bacteria in example 3.
FIG. 6 is a graph showing the effect of the R-enantiomer of cyaniodate with colistin or the S-enantiomer with colistin on mouse abscess volume according to example 3.
Detailed Description
The present invention is further illustrated below in conjunction with specific examples, but should not be construed as limiting the invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Example 1
Establishment of a mouse abscess model, and adaptability characteristics of pseudomonas aeruginosa PA02 strain in the abscess model under the single drug action of the chlorocyanioxazin enantiomer and colistin.
1. Test materials: 24 female mice, kunming female mice, were purchased from Hunan Stokes Levoda laboratory animal Co., ltd, and were weighing 25.+ -.3 g,1mL of sterile syringes.
2. Preparation work before test: the skin toxicity test was performed prior to subcutaneous injection by dissolving 10mg/kg body weight of the R-enantiomer of closantel or the S-enantiomer of closantel with 5mg/kg body weight of colistin in 0.5% dimethyl sulfoxide (DMSO), respectively.
The PA02 strain is inoculated on an LB agar plate and cultured to a proper size.
3. Evaluation of the single drug effect of the R-enantiomer of the iohexosamine, the S-enantiomer of the iohexosamine and colistin in mice.
(1) Inoculating the tested PA02 strain into a 50mL sterile centrifuge tube filled with 10mLMH broth, placing the sterile centrifuge tube into a shaking table at 37 ℃ for 180rpm incubation until the sterile centrifuge tube is in the logarithmic phase, and taking out the centrifuge tube; bacterial cells were washed 2 times with sterile physiological saline (0.9% NaCl) before injection and resuspended to 1X 10 7 CFU/mL。
(2) And (3) injecting the PA02 bacteria (50 mu L) obtained in the step (1) into the skin shaving area of the mice to establish a thigh abscess model of the mice.
(3) Control group: only bacteria are used for counteracting the toxin, and after 1h of counteracting the toxin, the single medicine group is injected.
(4) Mice with thigh abscess were injected with 25 μl of drug, respectively: 1mg/mL of R-enantiomer single drug group (equivalent to 1mg/kg body weight of the R-enantiomer of closantel per mouse); 1mg/mL of S-enantiomer single drug group (equivalent to 1mg/kg body weight of the S-enantiomer of the closantel per mouse); 1mg/mL colistin single dose group (equivalent to 1mg/kg body weight colistin per mouse).
(5) The abscess was monitored daily, and after 3 days of bacterial injection, the length (L) and width (W) of the abscess were measured for each mouse, and the abscess volume (V) was calculated as: v= (pi/6) ×l×w 2 。
(6) Skin abscesses (containing effusion) were removed 3 days after bacterial infection, homogenized with 1mL of sterile physiological saline for 5 minutes, and bacterial counts were determined by serial dilution. To obtain the residual bacterial load of PA02 in the target tissue after treatment.
FIG. 1 shows the effect of the R-enantiomer, S-enantiomer, colistin on the amount of mouse abscess bacteria after single drug treatment of example 1. Where MDL represents a median line of data. FIG. 2 is a schematic diagram of the R-enantiomer, S-enantiomer, and the viscosity of the iodate-binding agent of example 1Effects of mouse abscess volume following single drug treatment with mycomycin. As can be seen from fig. 1 and 2, the mice 10 were given 7 CFU/mL and 50 mu L of PA02 bacteria can realize effective modeling of rat thigh abscess. After injection of 1mg/kg body weight of the single drug of the R-enantiomer of the cyaniodate and the S-enantiomer of the cyaniodate, the abscess volumes are 420mm respectively 3 And 430mm 3 With the control group (437 mm) 3 ) Very close together. The abscess volume after 1mg/kg of body weight colistin single drug injection is slightly reduced compared with the control group, but still exceeds 380mm 3 . The bacteria numbers in the infected tissues of the control and single drug groups were comparable after injection (about 10 7 CFU), resulting in a larger abscess volume, the results indicate that the injected single drug group was ineffective.
Example 2
Establishment of a mouse abscess model, and adaptability of the pseudomonas aeruginosa PA05 strain under the combined action of the chlorocyanioxazin and the colistin in the abscess model.
1. Test materials: hunan Stokes Lemonda laboratory animal Co.Ltd.purchased 18 female mice Kunming, weighing 25+ -3 g,1mL sterile syringes.
2. Preparation work before test: the skin toxicity test was performed prior to subcutaneous injection by dissolving 10mg/kg body weight of the R-enantiomer of closantel or the S-enantiomer of closantel with 5mg/kg body weight of colistin in 0.5% DMSO, respectively. The PA05 strain is inoculated on an LB agar plate and cultured to a proper size.
3. Comparison of the efficacy of the R-enantiomer of closantel with colistin, the S-enantiomer of closantel and colistin in vivo in mice.
(1) Inoculating the tested PA05 strain into a 50mL sterile centrifuge tube filled with 10mLMH broth, placing the sterile centrifuge tube into a shaking table at 37 ℃ for 180rpm incubation until the sterile centrifuge tube is in a logarithmic growth phase, and taking out the centrifuge tube; bacterial cells were washed 2 times with sterile physiological saline (0.9% NaCl) before injection and resuspended to 1X 10 7 CFU/mL。
(2) Taking the bacteria (50 mu L) in the step (1) to inject into a skin shaving area of a mouse, and establishing a thigh abscess model of the mouse.
(3) Control group: only bacteria are used for counteracting the toxin, and after 1h of counteracting the toxin, the drug of the combined group is injected.
(4) Mice with thigh abscess were injected with 25 μl of drug, respectively: 0.05mg/mL of R-enantiomer+0.5 mg/mL of colistin combination (equivalent to the administration of a composition of 0.05mg/kg of the body weight of the R-enantiomer of closantel and 0.5mg/kg of the body weight of colistin per mouse, the mass ratio of the R-enantiomer of closantel to colistin being 0.1:1); 0.05mg/mL of S-enantiomer+0.5 mg/mL of colistin combination (equivalent to administration of a composition of 0.05mg/kg body weight of closantel S-enantiomer and 0.5mg/kg body weight of colistin per mouse, the mass ratio of closantel S-enantiomer to colistin being 0.2:1).
(5) The abscess of the thighs of the mice was monitored daily, and after 3 days of bacterial injection, the length (L) and width (W) of the abscess were measured for each mouse, and the abscess volume (V) was calculated as: v= (pi/6) ×l×w 2 。
(6) Skin abscesses (containing effusion) were removed 3 days after bacterial infection, homogenized with 1mL of sterile physiological saline for 5 minutes, and bacterial counts were determined by serial dilution. To obtain the residual bacterial load of PA05 in the target tissue after treatment.
FIG. 3 shows the effect of the R-enantiomer of iohexosamine in combination with colistin or the S-enantiomer in combination with colistin on the amount of mouse abscess bacteria according to example 2. Where MDL represents a median line of data. FIG. 4 is a graph showing the effect of the R-enantiomer of iohexosamine in combination with colistin or the S-enantiomer in combination with colistin on mouse abscess volume according to example 2. As can be seen from fig. 3 and 4, the mice 10 were given 7 CFU/mL and 50 mu L of PA05 bacteria can realize effective modeling of rat thigh abscess. The combination of the R-enantiomer of the cyaniotidea or the S-enantiomer of the cyaniotidea and colistin has remarkable synergic antibacterial effect on the strain. After treatment with 0.05mg/kg body weight of the R-enantiomer of cyaniodate-1 +0.5mg/kg body weight of colistin, no abscess appears at the skin lesions of the mice (volume=0 mm) 3 ). Whereas after treatment with 0.1mg/kg body weight of the S-enantiomer of cyaniodate-I-L +0.5mg/kg body weight of colistin, the growth of abscess was significantly inhibited with a volume of 38.3mm 3 Indicating that the abscess is not completely healed. Abscess volume is positively correlated with CFU. Compared with the control group, the weight of the chlorcyaniodides is 0.1mg/kgThe bacterial load of the salix S-enantiomer +0.5mg/kg body weight colistin treated group was reduced by 3 orders of magnitude, approximately 10 4 CFU. This suggests that the combination limits abscess growth by inhibiting bacterial proliferation. The bacterial load on the skin of mice after treatment with 0.05mg/kg body weight of the R-enantiomer of closantel plus 0.5mg/kg body weight of colistin was 0CFU, indicating that the abscess had healed. The results show that the R-enantiomer and the S-enantiomer of the cyaniodate have remarkable drug resistance reversing effects in vivo, and can effectively treat drug resistance pseudomonas aeruginosa infection after being combined with colistin. The difference in the potency of the two enantiomers is evident, the bacterial loads of the R-enantiomer and the S-enantiomer in combination with colistin treatment groups differ by 4 orders of magnitude.
Example 3
Establishment of a mouse abscess model, and adaptability of pseudomonas aeruginosa TRPA179 strain in the abscess model under the combined action of the chlorocyanioxazin and the colistin.
1. Test materials: hunan Stokes Lemonda laboratory animal Co.Ltd.purchased 18 female mice Kunming, weighing 25+ -3 g,1mL sterile syringes.
2. Preparation work before test: the skin toxicity test was performed prior to subcutaneous injection by dissolving 10mg/kg body weight of the R-enantiomer of closantel or the S-enantiomer of closantel with 5mg/kg body weight of colistin in 0.5% DMSO, respectively. The PA09 strain was inoculated on LB agar plates and cultured to a proper size.
3. Comparison of the efficacy of the combination of the R-enantiomer of closantel and colistin, the S-enantiomer of closantel and colistin in mice.
(1) Inoculating the test TRPA179 strain into a 50mL sterile centrifuge tube filled with 10mLMH broth, placing into a shaking table at 37 ℃ for 180rpm incubation until the logarithmic phase of growth, and taking out the centrifuge tube; bacterial cells were washed 2 times with sterile physiological saline (0.9% NaCl) before injection and resuspended to 1X 10 7 CFU/mL。
(2) Injecting the TRPA179 bacteria (50 mu L) in the step (1) into the skin shaving area of the mice to establish a thigh abscess model of the mice.
(3) Control group: only bacterial challenge is performed. After 1h of detoxification, the drug injection treatment of the combination group is carried out.
(4) Mice with thigh abscess were injected with 25 μl of drug, respectively: 3mg/mL of R-enantiomer+0.1 mg/mL of colistin combination (equivalent to 3mg/kg of a composition of the body weight of the R-enantiomer of the closantel and 0.1mg/kg of the body weight of colistin per mouse, the mass ratio of the R-enantiomer of the closantel to the colistin being 30:1); 8mg/mL of S-enantiomer+0.1 mg/mL of colistin combination (corresponding to the administration of 8mg/kg of a composition of the body weight of the S-enantiomer of the closantel and 0.1mg/kg of the body weight of colistin per mouse, the mass ratio of the S-enantiomer of the closantel to the colistin being 80:1).
(5) The abscess of the thighs of the mice was monitored daily, and after 3 days of bacterial injection, the length (L) and width (W) of the abscess were measured for each mouse, and the abscess volume (V) was calculated as: v= (pi/6) ×l×w 2 。
(6) Skin abscesses (containing effusion) were removed 3 days after bacterial infection, homogenized with 1mL of sterile physiological saline for 5 minutes, and bacterial counts were determined by serial dilution. To obtain residual bacterial amounts of TRPA179 in the target tissue after injection.
FIG. 5 shows the effect of the R-enantiomer of cyaniodate with colistin or the S-enantiomer with colistin on the amount of mouse abscess bacteria in example 3. Where MDL represents a median line of data. FIG. 6 is a graph showing the effect of the R-enantiomer of cyaniodate with colistin or the S-enantiomer with colistin on mouse abscess volume according to example 3. As can be seen from fig. 5 and 6, the mice 10 were given 7 CFU/mL, 50 mu L TRPA179 bacteria can realize effective modeling of rat thigh abscess. The combination of the R-enantiomer of the cyaniotidea or the S-enantiomer of the cyaniotidea and colistin has remarkable synergic antibacterial effect on the strain. After treatment with 3mg/kg of the R-enantiomer of closantel plus 0.1mg/kg of colistin or 8mg/kg of the S-enantiomer of closantel plus 0.1mg/kg of colistin, no abscess was observed in the lesions of the mice (volume=0 mm) 3 ) And the bacterial load on the skin of the mice was 0CFU. This suggests that the combination limits the growth of abscesses by inhibiting bacterial proliferation, the R-enantiomer and S-enantiomer of closantelThe construct has obvious drug resistance reversing effect in vivo, and can effectively treat drug resistance pseudomonas aeruginosa infection after being combined with colistin.
Example 4
Establishment of a mouse abscess model, and adaptability of Klebsiella pneumoniae K83 strain in the abscess model under the combined action of the chlorocyanioxazin and the colistin.
1. Test materials: hunan Stokes Lemonda laboratory animal Co.Ltd.purchased 18 female mice Kunming, weighing 25+ -3 g,1mL sterile syringes.
2. Preparation work before test: the skin toxicity test was performed prior to subcutaneous injection by dissolving 10mg/kg body weight of the R-enantiomer of closantel or the S-enantiomer of closantel with 5mg/kg body weight of colistin in 0.5% DMSO, respectively. The K83 strain is inoculated on an LB agar plate and cultured to a proper size.
3. Comparison of the efficacy of the combination of the R-enantiomer of closantel and colistin, the S-enantiomer of closantel and colistin in mice.
(1) Inoculating the tested K83 strain into a 50mL sterile centrifuge tube filled with 10mLMH broth, placing the sterile centrifuge tube into a shaking table at 37 ℃ for 180rpm incubation until the sterile centrifuge tube is in the logarithmic phase, and taking out the centrifuge tube; bacterial cells were washed 2 times with sterile physiological saline (0.9% NaCl) before injection and resuspended to 1X 10 7 CFU/mL。
(2) And (3) injecting the K83 bacteria (50 mu L) obtained in the step (1) into the skin shaving area of the mice to establish a thigh abscess model of the mice.
(3) Control group: only bacterial challenge is performed. After 1h of detoxification, the drug of the combination group is injected.
(4) Mice with thigh abscess were injected with 25 μl of drug, respectively: r-enantiomer + colistin combination (0.2 mg/mL +0.8mg/mL, equivalent to administration of a composition of 0.2mg/kg body weight of the R-enantiomer of closantel and 0.8mg/kg body weight of colistin per mouse, the mass ratio of the R-enantiomer of closantel to colistin being 0.25:1); 0.4mg/mL of S-enantiomer+0.8 mg/mL of colistin combination (equivalent to administration of a composition of 0.4mg/kg body weight of closantel S-enantiomer and 0.8mg/kg body weight of colistin per mouse, the mass ratio of closantel S-enantiomer to colistin being 0.5:1).
(5) The abscess of the thighs of the mice was monitored daily, and after 3 days of bacterial injection, the length (L) and width (W) of the abscess were measured for each mouse, and the abscess volume (V) was calculated as: v= (pi/6) ×l×w 2 。
(6) Skin abscesses (containing effusion) were removed 3 days after bacterial infection, homogenized with 1mL of sterile physiological saline for 5 minutes, and bacterial counts were determined by serial dilution to obtain residual bacterial amounts of K83 in the target tissue after treatment.
The experimental results showed that 10 was given to mice 7 The CFU/mL and 50 mu L of K83 bacteria can realize effective modeling of the thigh abscess of the mice. The combination of the R-enantiomer of the closantel and the colistin or the combination of the S-enantiomer of the closantel and the colistin has remarkable synergistic antibacterial effect on the strain. After treatment with 0.2mg/kg body weight of the R-enantiomer of cyaniodate-1 +0.8mg/kg body weight of colistin, no abscess appears at the skin lesions of the mice (volume=0 mm) 3 ). Whereas after treatment with 0.4mg/kg body weight of the S-enantiomer of cyaniodate-I-L +0.8mg/kg body weight of colistin, the growth of abscess was significantly inhibited with a volume of 18.5mm 3 Indicating that the abscess is not completely healed. Abscess volume is positively correlated with CFU. Compared with the control group, the bacterial load of the 0.4mg/kg body weight of the S-enantiomer of the cyaniodate/I/O+0.8 mg/kg body weight of the colistin-treated group is reduced by 4 orders of magnitude, about 10 3 CFU. This suggests that the combination limits abscess growth by inhibiting bacterial proliferation. The bacterial load on the skin of mice after treatment with 0.2mg/kg body weight of the R-enantiomer of closantel plus 0.8mg/kg body weight of colistin was 0CFU, indicating that the abscess had healed. These observations indicate that the R-enantiomer and S-enantiomer of the cyaniodate have significant reversal drug resistance in vivo, and can effectively treat drug-resistant klebsiella pneumoniae infection after being combined with colistin. The difference in the potency of the two enantiomers is evident, with the bacterial load of the R-enantiomer in combination with colistin or the S-enantiomer in combination with colistin treatment group differing by 3 orders of magnitude.
Example 5
Establishment of a mouse abscess model, and adaptability of Klebsiella pneumoniae K83 strain in the abscess model under the combined action of the chlorocyanioxazin and the colistin.
1. Test materials: hunan Stokes Lemonda laboratory animal Co.Ltd.purchased 18 female mice Kunming, weighing 25+ -3 g,1mL sterile syringes.
2. Preparation work before test: the skin toxicity test was performed prior to subcutaneous injection by dissolving 10mg/kg body weight of the R-enantiomer of closantel or the S-enantiomer of closantel with 5mg/kg body weight of colistin in 0.5% DMSO, respectively. The K83 strain is inoculated on an LB agar plate and cultured to a proper size.
3. Comparison of the efficacy of the combination of the R-enantiomer of closantel and colistin, the S-enantiomer of closantel and colistin in mice.
(1) Inoculating the tested K83 strain into a 50mL sterile centrifuge tube filled with 10mLMH broth, placing the sterile centrifuge tube into a shaking table at 37 ℃ for 180rpm incubation until the sterile centrifuge tube is in the logarithmic phase, and taking out the centrifuge tube; bacterial cells were washed 2 times with sterile physiological saline (0.9% NaCl) before injection and resuspended to 1X 10 7 CFU/mL。
(2) And (3) injecting the K83 bacteria (50 mu L) obtained in the step (1) into the skin shaving area of the mice to establish a thigh abscess model of the mice.
(3) Control group: only bacteria are used for counteracting the toxin, and after 1h of counteracting the toxin, the drug of the combined group is injected.
(4) Mice with thigh abscess were injected with 25 μl of drug, respectively: r-enantiomer + colistin combination (0.2 mg/mL +0.1mg/mL, equivalent to the administration of 2mg/kg body weight of a combination of the R-enantiomer of closantel and 0.1mg/kg body weight of colistin per mouse, the mass ratio of the R-enantiomer of closantel to colistin being 20:1); 0.4mg/mL of S-enantiomer+0.1 mg/mL of colistin combination (equivalent to the administration of a composition of 4mg/kg body weight of closantel S-enantiomer and 0.1mg/kg body weight of colistin per mouse, the mass ratio of closantel S-enantiomer to colistin being 40:1).
(5) The abscess of the thighs of the mice was monitored daily, and after 3 days of bacterial injection, the length (L) and width (W) of the abscess were measured for each mouse, and the abscess volume (V) was calculated as: v=(π/6)×L×W 2 。
(6) Skin abscesses (containing effusion) were removed 3 days after bacterial infection, homogenized with 1mL of sterile physiological saline for 5 minutes, and bacterial counts were determined by serial dilution. To obtain residual bacterial load of K83 in the target tissue after treatment.
The experimental results showed that 10 was given to mice 7 The CFU/mL and 50 mu L of K83 bacteria can realize effective modeling of the thigh abscess of the mice. The combination of the R-enantiomer of the closantel and the colistin or the combination of the S-enantiomer of the closantel and the colistin has remarkable synergistic antibacterial effect on the strain. After treatment with 2mg/kg of the R-enantiomer of closantel plus 0.1mg/kg of colistin or 4mg/kg of the S-enantiomer of closantel plus 0.1mg/kg of colistin, no abscess was observed in the lesions of the mice (volume=0 mm) 3 ) And the bacterial load on the skin of the mice was 0CFU. This shows that the combination drug can limit the growth of abscesses by inhibiting bacterial proliferation, and the R-enantiomer and S-enantiomer of the closantel have remarkable drug resistance reversing effects in vivo, and can effectively treat drug resistance pseudomonas aeruginosa infection after being combined with colistin.
In summary, according to the invention, by constructing a mouse abscess model and using the combination of the chlorocyanioxazin and the colistin for injection administration, the combination of the chlorocyanioxazin enantiomer and the colistin has a good synergistic effect on the treatment of drug-resistant gram-negative bacteria, the drug effect of the two enantiomers is obviously different, compared with the combination of the two with the colistin and the single drug, the amount of bacteria in the abscess is at least reduced by 3-7 log CFU/g (CFU/absess), the drug effect of the two is obviously different in stereoselectivity, and the R-enantiomer is 4log CFU/g lower than the S-enantiomer.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (10)

1. A composition for combating a drug-resistant gram-negative bacterial infection, said composition comprising a combination of a closantel enantiomer and a colistin, said closantel enantiomer being a closantel R-enantiomer or a closantel S-enantiomer.
2. The composition of claim 1, wherein the resistant gram negative bacteria is pseudomonas aeruginosa, klebsiella pneumoniae, escherichia coli, or salmonella; the mass ratio of the cyaniodate enantiomer to the colistin is (0.1-100): 1.
3. The composition for resisting drug-resistant gram-negative bacterial infection according to claim 2, wherein when the drug-resistant gram-negative bacterium is pseudomonas aeruginosa, the mass ratio of the R-enantiomer of the closantel to the colistin is (0.1-50): 1, and the mass ratio of the S-enantiomer of the closantel to the colistin is (0.2-100): 1; the mass concentration of the R-enantiomer of the cyaniodate is 0.05-5 mg/mL, and the mass concentration of the S-enantiomer of the cyaniodate is 0.1-10 mg/mL; the mass concentration of the colistin is 0.05-5 mg/mL.
4. A composition for combating a drug resistant gram negative bacterial infection according to claim 3 wherein when said drug resistant gram negative bacteria is pseudomonas aeruginosa, the mass ratio of said R-enantiomer of closantel to colistin is (0.1-25): 1 and the mass ratio of S-enantiomer of closantel to colistin is (0.2-25): 1; the mass concentration of the R-enantiomer of the cyaniodate is 0.07-3 mg/mL, and the mass concentration of the S-enantiomer of the cyaniodate is 0.2-5 mg/mL; the mass concentration of the colistin is 0.08-3 mg/mL.
5. The composition for resisting a drug-resistant gram-negative bacterial infection according to claim 4, wherein when the drug-resistant gram-negative bacterium is pseudomonas aeruginosa, the mass ratio of the R-enantiomer of the closantel to the colistin is (0.5-1): 1, and the mass ratio of the S-enantiomer of the closantel to the colistin is (0.5-2): 1; the mass concentration of the R-enantiomer of the cyaniodate is 0.1-0.5 mg/mL, and the mass concentration of the S-enantiomer of the cyaniodate is 0.5-1 mg/mL; the concentration of the colistin is 0.2-1 mg/mL.
6. The composition for resisting a drug-resistant gram-negative bacterial infection according to claim 2, wherein when the drug-resistant gram-negative bacterium is klebsiella pneumoniae, the mass ratio of the R-enantiomer of the closantel to the colistin is (0.2 to 30): 1, and the mass ratio of the S-enantiomer of the closantel to the colistin is (0.3 to 50): 1; the mass concentration of the R-enantiomer of the cyaniodate is 0.05-3 mg/mL, and the mass concentration of the S-enantiomer of the cyaniodate is 0.1-5 mg/mL; the mass concentration of the colistin is 0.1-3 mg/mL.
7. The composition for resisting a drug-resistant gram-negative bacterial infection according to claim 6, wherein when the drug-resistant gram-negative bacterium is klebsiella pneumoniae, the mass ratio of the R-enantiomer of the closantel to the colistin is (0.4 to 5): 1, and the mass ratio of the S-enantiomer of the closantel to the colistin is (0.4 to 15): 1; the mass concentration of the R-enantiomer of the cyaniodate is 0.1-1 mg/mL, and the mass concentration of the S-enantiomer of the cyaniodate is 0.3-3 mg/mL; the mass concentration of the colistin is 0.2-1.2 mg/mL.
8. The composition for resisting a drug-resistant gram-negative bacterial infection according to claim 7, wherein when the drug-resistant gram-negative bacterium is klebsiella pneumoniae, the mass ratio of the R-enantiomer of the closantel to the colistin is (0.5-3): 1, and the mass ratio of the S-enantiomer of the closantel to the colistin is (0.5-2): 1; the mass concentration of the R-enantiomer of the closantel is 0.4-0.8 mg/mL, and the mass concentration of the S-enantiomer of the closantel is 0.5-1 mg/mL; the mass concentration of the colistin is 0.3-1 mg/mL.
9. Use of a composition according to claims 1-8 for combating resistant gram negative bacterial infections in the preparation of an animal antibacterial agent.
10. Use of a composition against drug resistant gram negative bacterial infection according to claim 9 for the preparation of an animal antibacterial agent, wherein the composition is for injection in an amount of 10 to 30 μl/20 to 30g.
CN202310918274.8A 2023-07-25 2023-07-25 Composition for resisting drug-resistant gram-negative bacterial infection and application thereof Pending CN116983388A (en)

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