Medical application of copper salt in preparation of medicine for relieving photolysis of tigecycline

Abstract

The invention provides medical application of copper salt with medical value in preparation of a medicine for relieving photolysis of tigecycline. A minimum inhibitory concentration detection test, a sterilization curve detection, a cell protection test and a mouse staphylococcus aureus skin infection treatment effect experiment prove that the copper salt represented by copper gluconate significantly enhances the treatment effect of tigecycline as an external medicine. The photolysis effect of tigecycline is reduced by adding the copper salt, so that the antibacterial effect of tigecycline under any illumination condition is enhanced. The concentration of the used copper salt is far lower than the antibacterial effect of the copper salt. Therefore, the copper salt represented by the copper gluconate can be used for new drug development. The discovery of the application has very important clinical application value, can greatly promote the medication spectrum and indications of tigecycline as an externally applied drug, can solve the problem that no externally applied antibacterial drug is available for skin or soft tissue infection of various complex drug-resistant bacteria clinically, and can generate great economic benefits at the same time.

Description

Technical Field

[0001] The invention relates to the medical use of copper salt in preparing a drug for alleviating the photolysis of tigecycline, and belongs to the field of medical pharmacy. Background Art

[0002] All salts whose cations are copper ions are collectively referred to as copper salts, in which the valence of copper ions is +2. Copper salts are widely used in industrial production, medical and health care, and environmental disinfection. High concentrations of copper ions have a good killing effect on pathogenic microorganisms. At the same time, copper ions are essential trace elements for the body, and play an important role in maintaining the body's hematopoietic function, iron ion metabolism, the activity of multiple cell enzymes, maintaining the integrity of the central nervous system, and promoting the health of bones, blood vessels, and skin. The copper content in adults can reach 50-120 mg, with the highest content in the liver, brain, kidneys, and heart. Some copper ion salts such as copper gluconate, amino acid complex copper, copper sulfate, and copper peptides can be used to treat various diseases in humans or animals. For example, hydrated copper sulfate is the main component of the traditional Chinese medicine gallstones. Gallstones can be taken orally or externally as a traditional Chinese medicine, and have the effects of vomiting, astringency, detoxification, astringency, and erosion. Copper gluconate is a health product with the effects and functions of anti-oxidation, improving anemia, caring for the skin, treating osteoporosis, and relieving hypertension. Amino acid complex copper is a nutritional feed additive that can meet the copper needs of animals. Therefore, further development of the medical use of copper salts is of great significance.

[0003] Tetracycline antibiotics play an important role in clinical treatment and animal husbandry. Among them, tigecycline has attracted extensive attention as the "last line of defense" for the treatment of complex resistant Gram-negative and Gram-positive bacterial infections. 29 H 39 N 5 O 8 ), is a broad-spectrum glycyl cyclic peptide antibiotic that can only be administered intravenously at present, and belongs to the third generation of tetracycline antibiotics. Tigecycline has a good therapeutic effect on complicated intraperitoneal infections, complicated skin and soft tissue infections, and community-acquired pneumonia caused by pathogens including methicillin-resistant Staphylococcus aureus, vancomycin-resistant Staphylococcus aureus, Escherichia coli, Enterococcus, Klebsiella pneumoniae, Clostridium perfringens, and Streptococcus microcarbazine. However, the potential toxicity and extremely significant photolysis of tigecycline in vivo seriously restrict its preparation into different dosage forms for clinical treatment. Therefore, if the photolysis of tigecycline can be effectively solved (the photolysis rate of tigecycline can be reduced or its photolysis pathway can be changed), the clinical use range of tigecycline will be greatly promoted. For example, it can be prepared into external-use dosage forms such as ointments, sprays, emulsions or hydrogels for the treatment of complicated skin and soft tissue infections, which will significantly reduce its potential in vivo accumulation toxicity. Summary of the invention

[0004] The molecular structure of tigecycline is as follows:

[0005]

[0006] Tigecycline molecular formula: C 29 H 39 N 5 O 8 , molecular weight: 585.649

[0007] The present invention finds that copper salts represented by copper gluconate can alleviate or change the photolysis of tigecycline.

[0008] The present invention finds that copper salts represented by copper gluconate can enhance the antibacterial activity of tigecycline under light conditions.

[0009] The present invention finds that copper salts represented by copper gluconate combined with tigecycline have a good therapeutic effect on mouse skin infection mediated by Staphylococcus aureus.

[0010] The present invention also provides the use of copper salts represented by copper gluconate in the preparation of medicines for treating pathogenic bacteria infections.

[0011] Positive effects of the present invention

[0012] The present invention provides a new medical use of copper salts represented by copper gluconate in the preparation of drugs for relieving the photolysis of tigecycline, and discloses that copper salts represented by copper gluconate can significantly enhance the antibacterial activity of tigecycline under light conditions. Compared with the treatment of tigecycline alone, the present invention significantly enhances the therapeutic effect of tigecycline as an external medicine for skin or soft tissue infections after adding copper salts represented by copper gluconate, effectively expanding the pharmaceutical dosage form of tigecycline, which will greatly improve the clinical application range of tigecycline and provide a safer and more reliable pharmaceutical preparation for the treatment of skin or soft tissue infections caused by complex drug-resistant pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the time-killing curve of copper gluconate combined with tigecycline against Escherichia coli and Staphylococcus aureus under light conditions;

[0014] Figure 2 Copper gluconate combined with tigecycline against Staphylococcus aureus colonization in mouse skin infection. DETAILED DESCRIPTION

[0015] Through minimum inhibitory concentration detection tests, time-kill curve tests, cell protection tests, and treatment tests on Staphylococcus aureus skin infections in mice, it was confirmed that copper salts represented by copper gluconate significantly enhance the therapeutic effect of tigecycline as an external medication.

[0016] 1. Minimum inhibitory concentration test

[0017] The antibacterial activity of copper salts (including copper gluconate, copper sulfate, gallstones, amino acid complex copper and chlorophyll copper), tigecycline alone and the combination of the two against Escherichia coli, Acinetobacter baumannii, Staphylococcus aureus and Streptococcus was tested in a sterile 96-well microplate by the chessboard method, and the MIC values ​​of the two alone and in combination were determined, and the system inhibition index (FIC) was calculated. FIC = MIC (antibiotic combination) / MIC (antibiotic alone) + MIC (copper salt combination) / MIC (copper salt alone), the results are shown in Tables 1 and 2.

[0018] Table 1. MIC values ​​of tigecycline against Staphylococcus aureus and Escherichia coli under illumination and non-illumination conditions

[0019]

[0020] Table 2. MIC values ​​and FIC calculation results of different copper salts combined with tigecycline against different bacteria under light conditions

[0021]

[0022] Note: FIC<0.5 indicates that the two have synergistic effect.

[0023] The results are shown in Table 1. There is a significant difference in the MIC values ​​of tigecycline against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus under illumination and non-illumination conditions, that is, illumination causes photolysis of tigecycline. This indicates that the photolysis of tigecycline greatly limits its clinical use as an external medicine.

[0024] The results are shown in Table 2. The MICs of different copper salts, copper gluconate, copper sulfate, Chinese medicine gallstones, amino acid complex copper and chlorophyll copper against Escherichia coli, Acinetobacter baumannii, Salmonella, Staphylococcus epidermidis, Staphylococcus aureus and Streptococcus are all ≥2.048mg / mL. That is, the concentration used in this project (16μg / mL) is far lower than its inhibitory concentration. Copper salts can significantly improve the antibacterial activity of tigecycline under light conditions (MIC values ​​are reduced by 16-64 times), indicating that different forms of copper salts have excellent synergistic antibacterial effects with tigecycline under light conditions.

[0025] 2. Time-kill curve detection of copper gluconate combined with tigecycline

[0026] The overnight cultured Staphylococcus aureus USA300 and Escherichia coli ZJ487 were cultured in the culture medium until the logarithmic growth phase, and the bacterial count was adjusted to 1×10 8 CFUs / mL(OD 600nm=0.1) for later use. The following treatments were performed: the positive control group was not treated with any treatment, the antibiotic group was treated with tigecycline alone, the copper gluconate group was treated with copper gluconate, and the combination group was treated with tigecycline and copper gluconate. The bacterial solution was adjusted so that the concentration of the bacterial solution in each test tube was 5×10 5 CFUs / mL. Then shake the centrifuge tube to mix well and dispense into 96-well plates. Continue culturing under light conditions, take the bacterial solution in the corresponding wells at each time point, dilute it in multiples, count it on the plate, and draw a time-killing curve.

[0027] The results are attached Figure 1 As shown in Figure 2, neither tigecycline nor copper gluconate alone could completely kill Staphylococcus aureus in the culture medium under light conditions (see Figure 2). Figure 1 A) or Escherichia coli (attached Figure 1 B), and the combination of the two completely killed all the bacteria in the culture medium within 6 hours, indicating that the two have a significant synergistic bactericidal effect.

[0028] 3. Protective effect of copper gluconate combined with tigecycline on A459 cells

[0029] A549 cells were plated in a 96-well plate and cultured for 12 hours to allow them to adhere to the wall. Methicillin-resistant Staphylococcus aureus USA300 (bacteria: cells = 50:1) was added to each well, and different combinations of copper gluconate and tigecycline were added. The cells were cultured for another 6 hours under light conditions, and the supernatant was collected by centrifugation. The release of lactate dehydrogenase (LDH) in the supernatant was detected and the cell survival rate was calculated.

[0030] Table 3. The toxicity of copper gluconate combined with tigecycline in inhibiting Staphylococcus aureus against A549 cells

[0031]

[0032] The results are shown in Table 3. Under light conditions, copper gluconate or tigecycline alone could not effectively protect cells from Staphylococcus aureus-mediated cell damage (the protection rate was less than 50.00%), while the protection rate of the two combined was as high as 98.45%. This indicates that under light conditions, copper salts represented by copper gluconate can exert a synergistic antibacterial effect with tigecycline at the cellular level.

[0033] 4. Experimental therapeutic study of Staphylococcus aureus skin infection in mice

[0034] BALB / c mice (female, 18-22g) were pierced with a hole puncher after removing the fur on the back to establish skin injury wounds, and then the cultured Staphylococcus aureus USA300 was evenly dripped on the wounds, once every 12 hours, for 3 consecutive times to establish a Staphylococcus aureus suppurative infection model. After the model was successfully established, hydrogels containing: tigecycline alone, copper gluconate alone, and a combination of the two were prepared, and a hydrogel without any drug was used as a positive control. After the mice were grouped, the hydrogels with different treatments were applied to the wounds of each mouse, and the wounds were wrapped with transparent 3M tape and applied once every 24 hours. The skin healing of mice in each group was recorded separately, and the mice were killed 72 hours after treatment to take the skin tissue from the wound for colony colonization detection.

[0035] The results are attached Figure 2 As shown, compared with the positive control group and tigecycline or copper gluconate alone, the combined group significantly reduced the colonization of Staphylococcus aureus at the infected wounds of mice. The wounds of mice infected with Staphylococcus aureus in the combined group were dry and healed well, while the wounds of mice in the positive control group, tigecycline alone group or copper gluconate group had varying degrees of suppuration, poor healing effects or even worsened. This shows that copper gluconate can significantly improve the therapeutic effect of tigecycline on Staphylococcus aureus skin infections in experimental animals under light conditions.

[0036] 5. Summary

[0037] From the test results of the above different levels (molecular level, cellular level and experimental animal level), it can be obtained that copper salts with medicinal value represented by copper gluconate can alleviate the photolysis rate of tigecycline or change the photolysis pathway of tigecycline, thereby significantly improving the antibacterial effect of tigecycline under light conditions, and the antibacterial effect is wide-ranging, and can significantly kill a variety of pathogens including Escherichia coli, Salmonella, Acinetobacter baumannii, Staphylococcus aureus and Streptococcus. Thereby protecting cells from damage caused by bacterial infection, and further reducing the colonization of Staphylococcus aureus skin infection test mice and promoting the healing of Staphylococcus aureus infection wounds. This discovery of the present application has very important clinical application value, which will greatly promote the use spectrum and indications of tigecycline as an external medicine, and will solve the dilemma of no external antibacterial drugs available for skin or soft tissue infections of various complex drug-resistant bacteria in clinical practice, and will also produce great economic benefits.

Claims

1. The medical use of copper salts in the preparation of drugs for alleviating the photolysis of tigecycline.

2. The use according to claim 1, characterized in that The copper salts include all 2+ copper ion salts with medicinal value, including but not limited to copper gluconate, copper sulfate (the traditional Chinese medicine copper sulfate as the main component), amino acid complex copper, chlorophyll copper and copper peptide.

3. The use according to claim 1, characterized in that The mitigation of the photolysis of tigecycline may be to reduce its photolysis rate or change the photolysis pathway so that the degradation product still has antibacterial effect.

4. The use according to claim 1, characterized in that The described alleviation of the photolysis of tigecycline can further enhance the antibacterial effect of tigecycline under light conditions, and the antibacterial effects include but are not limited to Escherichia coli, Acinetobacter baumannii, Salmonella, Staphylococcus epidermidis, Staphylococcus aureus and Streptococcus, and can effectively treat infections caused by the above pathogens.

5. The use according to claim 1, characterized in that The copper salt is a pharmaceutically acceptable carrier, such as ointment, spray, tincture and hydrogel.

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