A pharmaceutical composition and uses thereof

Abstract

The application discloses a kind of pharmaceutical composition, and the pharmaceutical active ingredient of its drug includes desoximycin 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, with application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a pharmaceutical composition and its application, particularly to a composition using doxorinosine and meropenem as active materials and its application in the preparation of formulations for treating Klebsiella pneumoniae infection. Background Technology

[0002] Klebsiella pneumoniae is an opportunistic Gram-negative bacterium. It is commonly found in nosocomial infections and is one of the key pathogens monitored for nosocomial infections in clinical practice. With the widespread use of antibiotics, the problem of drug resistance in Klebsiella pneumoniae has become increasingly prominent, especially the emergence and spread of carbapenem-resistant Klebsiella pneumoniae (CRKP), making clinical treatment extremely difficult. CRKP was first reported by Mackenzie et al. in 1997, and 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 of the 21st century. In my country, CRKP was first reported by Wei et al. in 2006, and since then, the detection rate has continued to increase, making it one of the important drug-resistant bacteria threatening human health.

[0003] Currently, the development of carbapenem resistance in pathogens is mainly related to the production of carbapenemases, such as KPC-2 and NDM-5. Because carbapenemases can hydrolyze almost all β-lactam antibiotics, including meropenem, treatment of resistant bacterial infections becomes more difficult, leading to extremely high CRKP resistance, extremely high mortality, and global transmission. However, treatment options for CRKP are severely limited, resulting in a predicament in the treatment of CRKP infections.

[0004] Didanosine (abbreviated Did, 2',3'-dideoxyinosine, CAS No. 69655-05-6) is a purine nucleoside reverse transcriptase inhibitor that competitively inhibits the action of retroviruses on the natural substrate dideoxyadenosine triphosphate, stopping the elongation of the DNA chain and thus hindering the replication and proliferation of HIV virus. It is used to treat viral diseases.

[0005] Meropenem (abbreviated MEM, CAS number 119478-56-7) is a synthetic broad-spectrum carbapenem antibiotic that exerts its antibacterial effect by inhibiting bacterial cell wall synthesis. Meropenem can easily penetrate the cell walls of most Gram-positive and Gram-negative bacteria to reach its target, penicillin-binding protein (PBPS).

[0006] There are no reports of the combined use of norinosine and meropenem for the treatment of KPC-2 and NDM-5 positive carbapenem-resistant Klebsiella pneumoniae. Summary of the Invention

[0007] This invention is the first to discover the synergistic effect of didosorbide and meropenem. A checkerboard minimum inhibitory concentration (MIC) assay confirmed that didosorbide significantly enhances the antibacterial efficacy of meropenem, particularly against KPC-2 and NDM-5 positive carbapenem-resistant Klebsiella pneumoniae. Furthermore, in vitro bacterial growth curve experiments further validated the time-dependent bactericidal kinetics of this drug combination. This invention reveals that didosorbide can restore the antibacterial activity of meropenem against resistant strains and reverse bacterial resistance to meropenem while reducing the dosage required. This invention helps alleviate the increasingly serious problem of bacterial resistance and provides a new treatment strategy for the clinical treatment of Klebsiella pneumoniae infections, possessing significant application value.

[0008] To address the problems existing in the prior art, the first aspect of the present invention provides a pharmaceutical composition, wherein the pharmaceutical active ingredient of the pharmaceutical composition includes a first active substance and a second active substance;

[0009] The first active substance is any one or a combination of two of the following: desoxyinosine and pharmaceutical salts of desoxyinosine;

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

[0011] In some embodiments, the ratio of the first active substance to the second active substance is 2-50 μmol (e.g., any amount or a range between any two of the following: 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 said isosorbide inosine include toluenesulfonate, methanesulfonate, malate, acetate, citrate, malonate, tartrate, succinate, lactate, benzoate, ascorbate, α-ketoglutarate, α-glycerophosphate, hydrochloride, sulfate, nitrate, bicarbonate, carbonate, phosphate, hydrobromide, and hydroiodide.

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

[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 formulations for preventing, alleviating, treating, controlling, controlling contamination caused by Klebsiella pneumoniae, and / or inhibiting the proliferation of Klebsiella pneumoniae.

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

[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 formulation is selected from pharmaceuticals, food additives, feed additives, surgical skin disinfectants, and environmental disinfectants.

[0019] A third aspect of the present invention provides the use of doxorinosine and / or pharmaceutically acceptable salts of doxorinosine in the preparation of formulations for restoring the antibacterial activity 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. Attached Figure Description

[0021] Figure 1 The results of the minimum inhibitory concentration (MIC) test for doxorubicin combined with meropenem against KPC-2 positive Klebsiella pneumoniae M189-3 were shown using the broth microdilution checkerboard method.

[0022] Figure 2 The results of the minimum inhibitory concentration (MIC) test for doxorinosine combined with meropenem against NDM-5 positive Klebsiella pneumoniae 1LAN were presented using the broth microdilution checkerboard method.

[0023] Figure 3 Time-bactericidal curves of different treatment groups against KPC-2 positive Klebsiella pneumoniae M189-3.

[0024] Figure 4 Time-bactericidal curves of NDM-5 positive Klebsiella pneumoniae 1LAN for different treatment groups. Detailed Implementation

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

[0026] Materials and reagents used in this invention:

[0027] Meropenem was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

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

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

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

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

[0032] MHB broth medium: purchased from Beijing Luqiao Technology Co., Ltd., and prepared as follows: weigh 25.0g of dry medium powder, dissolve in 1L of distilled water, adjust the pH to 7.0±0.1, heat to boiling until completely dissolved, autoclave at 121℃ for 15min, cool and store for later use.

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

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

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

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

[0037] According to the CLSI 2024 standard, the minimum inhibitory concentrations (MICs) of noroxyinosine and meropenem against carbapenem-resistant Klebsiella pneumoniae strains M189-3 and 1LAN were determined using the broth microdilution method. The specific steps are as follows:

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

[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 of 0.22 μm) to obtain the meropenem stock solution.

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

[0041] 4. Take a 96-well microplate and inoculate each well with 100 μL of drug solution (made by diluting and mixing doxorubicin and meropenem in a certain proportion) and 100 μL of Klebsiella pneumoniae dilution.

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

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

[0044] 5. After completing step 4, incubate the 96-well plate at 37°C for 16–18 hours.

[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 doxorinosine 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 criterion was that when FICI ≤ 0.5, the two substances were considered to have a synergistic effect. The experiment was repeated three times, and the results were averaged.

[0048] The test results for strain M189-3 are as follows: Figure 1 As shown. From Figure 1 As can be seen, the MIC of isosorbide alone for strain M189-3 is >500 μM, and the MIC of meropenem alone is 64 μg / mL. The FICI of isosorbide and meropenem combined against KPC-2 positive Klebsiella pneumoniae strain M189-3 is <0.05 (calculated by star-shaped pore concentration), which meets the synergistic judgment criteria, indicating that the combination of the two has a synergistic antibacterial effect against strain M189-3.

[0049] The test results of 1 LAN strain are as follows Figure 2 As shown. From Figure 2 As can be seen, the MIC of 1LAN strain alone with isosorbide is 500 μM, and the MIC of meropenem alone is 128 μg / mL. The FICI of isosorbide and meropenem combined against NDM-5 positive Klebsiella pneumoniae 1LAN strain is 0.125 (calculated by star-shaped well concentration), which meets the synergistic judgment criteria, indicating that the combination of the two has a synergistic antibacterial effect against 1LAN strain.

[0050] Example 2: Time-bacterial killing curve of carbapenem-resistant Klebsiella pneumoniae using a combination of deltamethasone and meropenem.

[0051] 1. KPC-2 positive carbapenem-resistant Klebsiella pneumoniae and NDM-5 positive carbapenem-resistant Klebsiella pneumoniae bacterial suspensions were separately transferred to 15 mL of LB broth at a 1:1000 volume ratio and incubated at 37°C and 200 rpm until OD = 0.6. The cultures were then diluted 100-fold with LB broth to obtain a bacterial dilution with a concentration of approximately 1 × 10⁻⁶. 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 tube, and randomly divide the 12 test tubes into 4 groups: the isosorbide group, the meropenem group, the combined group, and the 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 dosage are as follows:

[0054] Desoxyinosine group: final concentration of desoxyinosine 160 μM.

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

[0056] Combined group: final concentration of desoxyinosine 160 μM; final concentration of meropenem 8 μg / ml.

[0057] Control group: No treatment.

[0058] Then, at 0h, 2h, 4h, 6h, 10h and 24h, 100μL of culture medium was spread onto LB solid culture plates and incubated overnight for colony counting.

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

[0060] Desoxyinosine group: final concentration of desoxyinosine 160 μM.

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

[0062] Combined group: final concentration of desoxyinosine 160 μM; final concentration of meropenem 32 μg / ml.

[0063] Control group: No treatment.

[0064] Then, at 0h, 2h, 4h, 6h, 10h and 24h, 100μL of culture medium was spread onto LB solid culture plates and incubated overnight for colony counting.

[0065] 3. After completing step 2, plot the time-sterilization curve with the culture time as the x-axis and the log10 value of the number of colonies per mL of the reaction system as the y-axis. The result judgment criteria are: at the 24-hour time point, if the number of colonies in the combined group decreases by ≥2log10 compared with the single drug group, it is judged as synergistic.

[0066] The test results for strain M189-3 are as follows: Figure 3 As shown, compared with the meropenem group and the isosorbide group treated alone, the bacterial colony count in the combined treatment group decreased by 6 log10 after 24 h, which met the synergistic judgment criteria, indicating that the combined treatment of isosorbide and meropenem has a synergistic effect on KPC-2 positive carbapenem-resistant Klebsiella pneumoniae.

[0067] The test results of 1 LAN strain are as follows Figure 4 As shown, compared with the meropenem group and the isosorbide group treated alone, the bacterial colony count in the combined treatment group decreased by 6 log10 after 24 hours, meeting the synergistic criteria. This synergistic effect indicates that the combined treatment with isosorbide and meropenem has a synergistic effect against NDM-5 positive carbapenem-resistant Klebsiella pneumoniae.

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

[0069] As is known from common technical knowledge, this invention can be implemented through other embodiments that do not depart from its spirit or essential characteristics. Therefore, the disclosed embodiments described above are merely illustrative in all respects and are not the only ones. All modifications within the scope of this invention or its equivalents are included in this invention.

Claims

1. Use of a composition in the preparation of an agent for treating diseases caused by Klebsiella pneumoniae and / or inhibiting the proliferation of Klebsiella pneumoniae; The Klebsiella pneumoniae strain was selected from KPC-2 positive carbapenem-resistant Klebsiella pneumoniae. The pharmaceutically active ingredients of the composition include doxorinosine and meropenem; The ratio of the amount of desoxyinosine to the amount of meropenem is 160 μmol: 8 mg.

2. Use according to claim 1, wherein The formulation is selected from drugs, surgical skin disinfectants, and environmental disinfectants.

3. The use according to claim 1, wherein The composition also contains pharmaceutical excipients.

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