Pharmaceutical composition and application thereof

Abstract

The invention discloses a pharmaceutical composition. The active pharmaceutical ingredients of the pharmaceutical composition comprise dihydroxyinosine and meropenem. The composition can synergistically and efficiently inhibit klebsiella pneumoniae, especially KPC-2 positive carbapenem-resistant klebsiella pneumoniae and NDM-5 positive carbapenem-resistant klebsiella pneumoniae, and has an application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biological medicine, and relates to a pharmaceutical composition and its application, in particular to a composition with didanosine and meropenem as active materials and its application in the preparation of a preparation for treating Klebsiella pneumoniae infection. Background Art

[0002] Klebsiella pneumoniae is a conditional pathogenic Gram-negative bacterium. It is commonly found in nosocomial infections and is one of the key pathogens monitored clinically in hospitals. With the widespread use of antibiotics, the problem of drug resistance of Klebsiella pneumoniae has become increasingly prominent. In particular, the emergence and spread of Carbapenem-resistant Klebsiella pneumoniae (CRKP) have made clinical treatment extremely difficult. In 1997, MACKENZIE et al. first reported CRKP. From 2002 to 2015, the detection rate of CRKP in the United States soared from less than 0.1% to 24.6%. The United States has listed CRKP as one of the most threatening pathogens in the 21st century. In China, in 2006, WEI et al. first reported CRKP, and the detection rate has been increasing since then, becoming one of the important drug-resistant bacteria threatening human health currently.

[0003] Currently, the formation of pathogen carbapenem resistance is mainly related to the production of carbapenemases, such as carbapenemases KPC-2 and NDM-5. Since carbapenemases can hydrolyze almost all β-lactam antibiotics including meropenem, it makes the treatment of drug-resistant bacterial infections more difficult, resulting in extremely high drug resistance, high fatality rate and global spread of CRKP. However, the treatment regimens for CRKP are too limited, thus leading to a dilemma in the treatment of CRKP infections.

[0004] Didanosine (abbreviated as Did, 2’,3’-dideoxyinosine, CAS No. 69655-05-6) is a purine nucleoside reverse transcriptase inhibitor, which competitively inhibits the action of retroviruses on the natural substrate adenosine 5'-triphosphate, terminates the elongation of DNA chains, and thus hinders the replication and proliferation of HIV viruses, and is used to treat viral diseases.

[0005] Meropenem (abbreviated as MEM, CAS No. 119478-56-7) is a synthetic broad-spectrum carbapenem antibiotic, which exerts antibacterial effects by inhibiting the synthesis of bacterial cell walls. Meropenem easily penetrates the cell walls of most Gram-positive and negative bacteria to reach its target penicillin-binding proteins (PBPs).

[0006] There has been no report on the combined use of didanosine and meropenem for the treatment of Klebsiella pneumoniae resistant to carbapenem with KPC-2 and NDM-5 positive. Summary of the Invention

[0007] The present invention has first discovered the synergistic effect of didanosine and meropenem. Through the checkerboard minimum inhibitory concentration test, it is confirmed that didanosine can significantly enhance the antibacterial efficacy of meropenem, especially when against Klebsiella pneumoniae resistant to carbapenem with KPC-2 and NDM-5 positive. In addition, the present invention also uses the in vitro bacterial growth curve experiment to further verify the time-dependent bactericidal kinetic characteristics of this drug combination. The present invention reveals that didanosine can restore the antibacterial effect of meropenem on drug-resistant strains, and while reducing the dosage of meropenem, reverse the drug resistance of bacteria to meropenem. The present invention helps to alleviate the increasingly serious problem of bacterial drug resistance and provides a new treatment strategy for the clinical treatment of Klebsiella pneumoniae infectious diseases, having important application value.

[0008] To solve the problems existing in the prior art, the first aspect of the present invention provides a pharmaceutical composition, and the pharmaceutically active ingredients of the pharmaceutical composition include a first active substance and a second active substance;

[0009] The first active substance is any one or a combination of two of didanosine and a pharmaceutical salt of didanosine;

[0010] The second active substance is any one, a combination of any two or a combination of three of meropenem, a pharmaceutical salt of meropenem, and a hydrate of meropenem.

[0011] In some embodiments, the dosage ratio of the first active substance to the second active substance is 2-50 μmol (for example, any dosage or the range between any two dosages among 2 μmol, 3 μmol, 4 μmol, 5 μmol, 6 μmol, 7 μmol, 8 μmol, 9 μmol, 10 μmol, 11 μmol, 12 μmol, 13 μmol, 14 μmol, 15 μmol, 16 μmol, 17 μmol, 18 μmol, 19 μmol, 20 μmol, 25 μmol, 30 μmol, 35 μmol, 40 μmol, 45 μmol, 50 μmol): 1 mg.

[0012] In some embodiments, the pharmaceutical salts of didanosine include tosylate, mesylate, malate, acetate, citrate, malonate, tartrate, succinate, lactate, benzoate, ascorbate, α-ketoglutarate, α-glycerophosphate, hydrochloride, sulfate, nitrate, bicarbonate, carbonate, phosphate, hydrobromide, and hydroiodide of didanosine.

[0013] The medicinal salts of meropenem include toluenesulfonate, mesylate, malate, acetate, citrate, malonate, tartrate, succinate, lactate, benzoate, ascorbate, α-ketoglutarate, α-glycerophosphate, hydrochloride, sulfate, nitrate, hydrogencarbonate, carbonate, phosphate, hydrobromide and hydroiodide of meropenem.

[0014] In some embodiments, the pharmaceutical composition further contains pharmaceutical excipients.

[0015] The second aspect of the present invention provides the use of the pharmaceutical composition described in the first aspect of the present invention in the preparation of a preparation for preventing a disease caused by Klebsiella, alleviating a disease caused by Klebsiella, treating a disease caused by Klebsiella, controlling a disease caused by Klebsiella, controlling a contamination caused by Klebsiella and / or inhibiting the proliferation of Klebsiella.

[0016] In some embodiments, the Klebsiella is Klebsiella pneumoniae producing carbapenemase.

[0017] In some embodiments, the Klebsiella pneumoniae is selected from KPC-2 positive carbapenem-resistant Klebsiella pneumoniae and NDM-5 positive carbapenem-resistant Klebsiella pneumoniae.

[0018] In some embodiments, the preparation is selected from drugs, food additives, feed additives, skin disinfectants for surgery, and disinfectants for environmental disinfection.

[0019] The third aspect of the present invention provides the use of didanosine and / or the medicinal salt of didanosine in the preparation of a preparation for restoring the antibacterial effect of meropenem against drug-resistant strains.

[0020] In some embodiments, the drug-resistant bacteria are selected from KPC-2 positive carbapenem-resistant Klebsiella pneumoniae and NDM-5 positive carbapenem-resistant Klebsiella pneumoniae. Description of the Drawings

[0021] Figure 1 Shows the test results of the minimum inhibitory concentration of didanosine combined with meropenem against KPC-2 positive Klebsiella pneumoniae M189-3 detected by the broth microdilution checkerboard method.

[0022] Figure 2 Shows the test results of the minimum inhibitory concentration of didanosine combined with meropenem against NDM-5 positive Klebsiella pneumoniae 1LAN detected by the broth microdilution checkerboard method.

[0023] Figure 3 Is the time-kill curve of different treatment groups against KPC-2 positive Klebsiella pneumoniae M189-3.

[0024] Figure 4 Time-kill curves of different treatment groups against NDM-5 positive Klebsiella pneumoniae 1LAN. Specific implementation manners

[0025] To make the objectives, technical solutions and advantages of the present invention clearer, the following will further describe the embodiments of the present invention in detail with reference to the accompanying drawings.

[0026] Materials and reagents used in the present invention:

[0027] Meropenem was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0028] Didanosine (Did) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0029] The KPC-2 positive carbapenem-resistant Klebsiella pneumoniae M189-3 strain is a wild strain isolated from the laboratory of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0030] The NDM-5 positive carbapenem-resistant Klebsiella pneumoniae 1LAN strain was provided by Jiangsu Academy of Agricultural Sciences.

[0031] The above two carbapenem-resistant isolates were identified as Klebsiella pneumoniae by 16S rDNA. The drug susceptibility test showed that both strains were resistant to meropenem (MIC>16mg / L). The results of drug resistance gene detection showed that they carried the carbapenemase resistance genes KPC-2 gene and NDM-5 gene respectively.

[0032] MHB broth medium: Purchased from Beijing Land Bridge Technology Co., Ltd. The preparation method is as follows: Weigh 25.0g of the medium powder, dissolve it in 1L of distilled water, adjust the pH to 7.0±0.1, heat and boil until completely dissolved, sterilize at 121℃ for 15 minutes, and store it for later use after cooling.

[0033] LB broth medium: Weigh 10.0g of tryptone, 5.0g of yeast extract powder, and 10.0g of sodium chloride, dissolve them in 1000mL of distilled water, adjust the pH to 7.0±0.1, sterilize at 121℃ for 15 minutes, and store it at room temperature for later use after cooling.

[0034] LB agar: Weigh 10.0g of tryptone, 5.0g of yeast extract powder, 10.0g of sodium chloride, and 15.0g of agar powder, dissolve them in 1000mL of distilled water, adjust the pH to 7.0±0.1, sterilize at 121℃ for 15 minutes. When the agar temperature drops to 60℃, pour it into a disposable sterile petri dish and store it at room temperature after cooling.

[0035] PBS buffer: Weigh 8 g of NaCl, 0.2 g of KCl, 1.44 g of NaH2PO4 and 0.24 g of KH2PO4, dissolve them in 800 mL of distilled water, adjust the pH value to 7.2 with HCl, and make up the volume to 1 L with distilled water. Autoclave at 121 °C for 15 minutes and store at room temperature after cooling.

[0036] Example 1: Determination of the synergistic effect of didanosine and meropenem

[0037] According to the method of the CLSI standard 2024 edition, the broth microdilution method was used to determine the minimum inhibitory concentration (MIC) of didanosine and meropenem against carbapenem-resistant Klebsiella pneumoniae strains M189-3 and 1LAN. The specific steps are as follows:

[0038] 1. The bacterial suspensions of strains M189-3 and 1LAN in the logarithmic growth phase were adjusted to a turbidity of 0.6 McFarland with a McFarland turbidimeter, and then diluted 100-fold with MHB broth medium to obtain a bacterial suspension with a concentration of approximately 1×10 6 CFU / mL.

[0039] 2. Weigh an appropriate amount of meropenem powder, dissolve it in deionized water to obtain a meropenem solution with a concentration of 10240 μg / mL, and then filter it through a sterile filter membrane (pore size 0.22 μm) to obtain the meropenem stock solution.

[0040] 3. Weigh an appropriate amount of didanosine powder, dissolve it in dimethyl sulfoxide (DMSO) to obtain a didanosine solution with a concentration of 20 mM.

[0041] 4. Take a 96-well microplate, and inoculate 100 μL of the drug solution (diluted and mixed in proportion by didanosine and meropenem) and 100 μL of the diluted Klebsiella pneumoniae suspension into each well.

[0042] For the carbapenem-resistant Klebsiella pneumoniae strain M189-3 positive for KPC-2, in each row from bottom to top, the final concentration of meropenem in each well is 0 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL, 128 μg / mL from left to right; in each column from right to left, the final concentration of didanosine in each well is 0 μM, 7.8125 μM, 15.625 μM, 31.2 μM, 62.5 μM, 125 μM, 250 μM, 500 μM from bottom to top.

[0043] For the NDM-5 positive carbapenem-resistant Klebsiella pneumoniae strain 1LAN, in each row from bottom to top, the final concentration of meropenem in each well is 0 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL, 128 μg / mL, 256 μg / mL, 512 μg / mL in sequence; in each column from right to left, the final concentration of didanosine in each well is 0 μM, 7.8125 μM, 15.625 μM, 31.25 μM, 62.5 μM, 125 μM, 250 μM, 500 μM in sequence.

[0044] 5. Incubate the 96-well plate after completing step 4 at 37 °C for 16 - 18 h.

[0045] 6. After completing step 5, calculate the MIC value and the fractional inhibitory concentration index (FICI) to determine whether there is a synergistic effect between didanosine and meropenem.

[0046] FICI = MIC 美罗培南联合 / MIC 美罗培南单用 +MIC 去羟肌苷联合 / MIC 去羟肌苷单用

[0047] The judgment criterion is: when FICI ≤ 0.5, it is judged that there is a synergistic effect between the two. The experiment is repeated three times and the results are averaged.

[0048] The measurement results of strain M189 - 3 are as Figure 1 shown. It can be seen from Figure 1 that for strain M189 - 3, the MIC of didanosine alone > 500 μM, the MIC of meropenem alone = 64 μg / mL, and the FICI of the combination of didanosine and meropenem against KPC-2 positive Klebsiella pneumoniae strain M189 - 3 < 0.05 (calculated based on the concentration of the star-shaped wells), meeting the synergistic judgment criterion, indicating that the combination of the two has a synergistic antibacterial effect on strain M189 - 3.

[0049] The measurement results of strain 1LAN are as Figure 2 shown. It can be seen from Figure 2 that for strain 1LAN, the MIC of didanosine alone = 500 μM, the MIC of meropenem alone = 128 μg / mL, and the FICI of the combination of didanosine and meropenem against NDM-5 positive Klebsiella pneumoniae strain 1LAN = 0.125 (calculated based on the concentration of the star-shaped wells), meeting the synergistic judgment criterion, indicating that the combination of the two has a synergistic antibacterial effect on strain 1LAN.

[0050] Example 2: Time-kill curve of didanosine combined with meropenem against carbapenem-resistant Klebsiella pneumoniae

[0051] 1. Transfer 15 mL of the bacterial suspensions of KPC-2 positive carbapenem-resistant Klebsiella pneumoniae and NDM-5 positive carbapenem-resistant Klebsiella pneumoniae respectively according to a volume ratio of 1:1000. The culture medium is LB broth medium. Culture at 37 °C with 200 rpm until OD = 0.6, and then dilute with LB broth medium by 100 times in volume to obtain a bacterial dilution, and the concentration of the bacteria is about 1×10 6 CFU / mL.

[0052] 2. For each strain, take 12 test tubes, add 2 mL of MH broth medium and 5 mL of bacterial suspension to each test tube. Randomly divide the 12 test tubes into 4 groups: didanosine group, meropenem group, combination group and control group, with 3 test tubes in each group, and perform the following treatments:

[0053] For KPC-2 positive carbapenem-resistant Klebsiella pneumoniae, the grouping and dosages are as follows:

[0054] Didanosine group: The final concentration of didanosine is 160 μM.

[0055] Meropenem group: The final concentration of meropenem is 8 μg / ml.

[0056] Combination group: The final concentration of didanosine is 160 μM; the final concentration of meropenem is 8 μg / ml.

[0057] Control group: No treatment.

[0058] Then, take 100 μL of the culture solution at 0 h, 2 h, 4 h, 6 h, 10 h and 24 h respectively and spread it on the LB solid culture plate, and culture overnight for colony counting.

[0059] For NDM-5 positive carbapenem-resistant Klebsiella pneumoniae, the grouping and dosages are as follows:

[0060] Didanosine group: The final concentration of didanosine is 160 μM.

[0061] Meropenem group: The final concentration of meropenem is 32 μg / ml.

[0062] Combination group: The final concentration of didanosine is 160 μM; the final concentration of meropenem is 32 μg / ml.

[0063] Control group: No treatment.

[0064] Then, take 100 μL of the culture solution at 0 h, 2 h, 4 h, 6 h, 10 h and 24 h respectively and spread it on the LB solid culture plate, and culture overnight for colony counting.

[0065] 3. After completing Step 2, plot a time-kill curve with the culture time as the abscissa and the log10 value of the number of colonies per mL of the reaction system as the ordinate. The result determination criterion: At the 24-hour time point, if the decrease in the number of colonies in the combination group is ≥2 log10 compared to the group treated with a single drug alone, it is determined as synergistic.

[0066] The determination results of strain M189-3 are as Figure 3 shown. Compared with the individual treatments of the meropenem group and the didanosine group, the number of bacterial colonies in the combination group decreased by 6 log10 after 24 hours of treatment, meeting the synergistic determination criterion, indicating that the combination treatment of didanosine and meropenem has a synergistic effect on KPC-2-positive carbapenem-resistant Klebsiella pneumoniae.

[0067] The determination results of strain 1LAN are as Figure 4 shown. Compared with the individual treatments of the meropenem group and the didanosine group, the number of bacterial colonies in the combination group decreased by 6 log10 after 24 hours of treatment, meeting the synergistic determination criterion. Meeting the synergistic determination criterion indicates that the combination treatment of didanosine and meropenem has a synergistic effect on NDM-5-positive carbapenem-resistant Klebsiella pneumoniae.

[0068] Thus, it can be seen that the combination of didanosine and meropenem has a strong synergistic antibacterial effect on both KPC-2-positive and NDM-5-positive Klebsiella pneumoniae.

[0069] As is known by common technical knowledge, the present invention can be implemented by other embodiments that do not depart from its spiritual essence or essential features. Therefore, the above-disclosed embodiments are illustrative in all aspects and not exclusive. All changes within the scope of the present invention or equivalent to the present invention are encompassed by the present invention.

Claims

1. A pharmaceutical composition, wherein the pharmaceutically active ingredients of the pharmaceutical composition include a first active substance and a second active substance; The first active substance is any one or a combination of two of didanosine and pharmaceutically acceptable salts of didanosine; The second active substance is any one, any combination of two, or a combination of three of meropenem, pharmaceutically acceptable salts of meropenem, and hydrates of meropenem.

2. The pharmaceutical composition according to claim 1, wherein The dosage ratio of the first active substance to the second active substance is 2 - 50 μmol: 1 mg.

3. The pharmaceutical composition according to claim 1 or 2, characterized in that, The pharmaceutically acceptable salts of didanosine include tosylate, mesylate, malate, acetate, citrate, malonate, tartrate, succinate, lactate, benzoate, ascorbate, α-ketoglutarate, α-glycerophosphate, hydrochloride, sulfate, nitrate, bicarbonate, carbonate, phosphate, hydrobromide, and hydroiodide of didanosine. The pharmaceutically acceptable salts of meropenem include tosylate, mesylate, malate, acetate, citrate, malonate, tartrate, succinate, lactate, benzoate, ascorbate, α-ketoglutarate, α-glycerophosphate, hydrochloride, sulfate, nitrate, bicarbonate, carbonate, phosphate, hydrobromide, and hydroiodide of meropenem.

4. The pharmaceutical composition according to any one of claims 1-3, characterized in that, The pharmaceutical composition further contains pharmaceutical excipients.

5. Use of the pharmaceutical composition according to any one of claims 1 - 4 in the preparation of a preparation for preventing a disease caused by Klebsiella, alleviating a disease caused by Klebsiella, treating a disease caused by Klebsiella, controlling a disease caused by Klebsiella, controlling contamination caused by Klebsiella, and / or inhibiting the proliferation of Klebsiella.

6. The application as claimed in claim 5, wherein The Klebsiella is Klebsiella pneumoniae producing carbapenemase.

7. The application according to claim 5, characterized in that, The Klebsiella pneumoniae is selected from KPC-2 positive carbapenem-resistant Klebsiella pneumoniae and NDM-5 positive carbapenem-resistant Klebsiella pneumoniae.

8. The application according to claim 5, characterized in that, The preparation is selected from pharmaceuticals, food additives, feed additives, surgical skin germicides, and environmental disinfection germicides.

9. Use of didanosine and / or pharmaceutically acceptable salts of didanosine in the preparation of a preparation for restoring the antibacterial effect of meropenem against drug-resistant strains.

10. The application according to claim 9, characterized in that, The drug-resistant strains are selected from KPC-2 positive carbapenem-resistant Klebsiella pneumoniae and NDM-5 positive carbapenem-resistant Klebsiella pneumoniae.

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