Application of cefathiamidine and clavulanic acid combined preparation in tuberculosis treatment

Abstract

The invention relates to an application of a cefathiamidine and clavulanic acid combined preparation in tuberculosis treatment, cefathiamidine and clavulanic acid are combined for use, and the activity of resisting mycobacterium tuberculosis is remarkably improved. In-vitro experiments prove that the beta-lactamase inhibitor clavulanic acid remarkably improves the antituberculous effect of cefathiamidine, and the combined preparation shows remarkable advantages in the aspect of inhibiting the growth of mycobacterium tuberculosis and has a relatively strong inhibiting effect on drug-resistant strains. Meanwhile, the combined preparation shows relatively low cytotoxicity in an in-vitro experiment, and shows relatively good safety. In addition, the combined preparation has a potential application prospect in treatment of drug-resistant tuberculosis, and can effectively supplement the existing tuberculosis treatment scheme.

Description

Technical Field

[0001] The invention relates to the technical field of medicine, and in particular to application of a combined preparation of cefathiamidine and clavulanic acid in the treatment of tuberculosis. Background Art

[0002] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis, which poses a serious threat to human health. The main treatment for TB is to inhibit or kill Mycobacterium tuberculosis through the combined use of anti-tuberculosis drugs. However, with the deepening of TB treatment, some new challenges have gradually emerged.

[0003] Current problems in the development of tuberculosis treatment drugs and preparations include: 1. Drug resistance problem: The drug resistance of Mycobacterium tuberculosis has gradually increased, especially the emergence of multidrug resistance (MDR-TB) and extensively drug resistance (XDR-TB), which has significantly reduced the efficacy of existing drugs.

[0004] 2. Limitations of traditional drugs: Traditional first-line anti-tuberculosis drugs (such as isoniazid and rifampicin) show high toxic side effects in some cases and have limited killing effects on drug-resistant strains.

[0005] 3. Long treatment cycle: Tuberculosis treatment usually takes 6 months or longer, which not only increases the financial burden on patients, but also easily leads to treatment failure and the emergence of drug-resistant bacteria due to irregular use of drugs.

[0006] In the face of the above problems, researchers are constantly exploring new drugs and combined treatment options to improve the effectiveness of tuberculosis treatment. Among them, the combined use of cephalosporin antibiotics and β-lactamase inhibitors has gradually become a promising research direction.

[0007] Cefathiamidine: Cefathiamidine is a cephalosporin antibiotic with broad-spectrum antibacterial activity, especially against Gram-positive bacteria. It achieves its bactericidal effect by interfering with the synthesis of bacterial cell walls.

[0008] Clavulanic Acid: Clavulanic Acid is a β-lactamase inhibitor that can effectively inhibit the β-lactamase secreted by Mycobacterium tuberculosis, which is one of the main mechanisms for bacteria to resist β-lactam antibiotics.

[0009] To date, there are no reports on the quantitative study of the anti-tuberculosis activity of cefathiamidine combined with clavulanic acid. Summary of the invention

[0010] In view of the above problems, the purpose of the present invention is to propose: the use of cefathiamidine combined with clavulanic acid in the preparation of drugs and / or Mycobacterium tuberculosis inhibitors for preventing and / or treating tuberculosis, so as to provide a new option for the treatment of tuberculosis.

[0011] The invention provides use of cefathiamidine and clavulanic acid in preparing a medicine for preventing and / or treating tuberculosis.

[0012] In one embodiment, the tuberculosis includes pulmonary tuberculosis and extrapulmonary tuberculosis in humans or animals caused by Mycobacterium tuberculosis.

[0013] In one embodiment, the Mycobacterium tuberculosis is selected from: MtbH37Rv (ATCC 27294), MtbH37Ra (ATCC25177), BCG Tokyo strain (ATCC 35737), Mycobacterium marinum (BAA55), Mycobacterium smegmatis (MC2155), clinically isolated multidrug-resistant strains (Mtb28, Mtb 1731), Mycobacterium abscessus (ATCC 19977), Staphylococcus aureus (CMCC26003), Staphylococcus epidermidis (CMCC 26069), Streptococcus suis (SS2-1-F0) Escherichia coli (ATCC25922), Klebsiella pneumoniae (ATCC29213), Acinetobacter baumannii (ATCC19606), Pseudomonas aeruginosa (CMCC10104).

[0014] The present invention also provides an application of cefathiamidine and clavulanic acid in the preparation of a Mycobacterium tuberculosis inhibitor.

[0015] In one embodiment, the cefathiamidine and clavulanic acid have inhibitory activity against Mycobacterium tuberculosis in vitro.

[0016] In one embodiment, the Mycobacterium tuberculosis is selected from: MtbH37Rv (ATCC 27294), MtbH37Ra (ATCC25177), BCG Tokyo strain (ATCC 35737), Mycobacterium marinum (BAA55), Mycobacterium smegmatis (MC2155), clinically isolated multidrug-resistant strains (Mtb28, Mtb 1731), Mycobacterium abscessus (ATCC 19977), Staphylococcus aureus (CMCC26003), Staphylococcus epidermidis (CMCC 26069), Streptococcus suis (SS2-1-F0) Escherichia coli (ATCC25922), Klebsiella pneumoniae (ATCC29213), Acinetobacter baumannii (ATCC19606), Pseudomonas aeruginosa (CMCC10104).

[0017] In one embodiment, the minimum inhibitory concentration of cefathiamidine and clavulanic acid against a standard strain of Mycobacterium tuberculosis in vitro is 0.031 μg / mL.

[0018] In one embodiment, the cefathiamidine cellular safety of the Mycobacterium tuberculosis inhibitor is not less than 1000 μg / mL.

[0019] In one embodiment, the Mycobacterium tuberculosis inhibitor cefathiamidine is capable of destroying the cell morphology of Mycobacterium tuberculosis in a dose-dependent manner.

[0020] The beneficial effects of the present invention are: The present invention found that cefathiamidine has effective inhibitory activity against Mycobacterium tuberculosis, with a minimum inhibitory concentration (MIC) of 0.031 μg / mL for the standard strain H37Rv and 0.5 μg / mL for the attenuated strain H37Ra, and has little effect on Gram-negative bacteria. Furthermore, the β-lactamase inhibitor clavulanate can improve its anti-tuberculosis effect. After combined with clavulanic acid, the minimum inhibitory concentration of cefathiamidine against the attenuated strain H37Ra is 0.031 μg / mL, and its combined use effect is 16 times stronger than that of rifampicin alone (MIC0.5 μg / mL). Intracellular anti-tuberculosis Mycobacterium tuberculosis experiments show that clavulanic acid can increase the intracellular antibacterial activity of cefathiamidine (10 μg / mL, 5 μg / mL, 2.5 μg / mL) by 1 to 3 times. By observing the morphological changes of bacteria, cefathiamidine can destroy the morphological integrity of bacterial cells. In addition, the IC50 value of cefathiamidine on A549, MCF-7, Vero and Huh7 cells was greater than 1000 μg / mL, showing extremely low cytotoxicity. Therefore, cefathiamidine combined with clavulanic acid can be used as a candidate drug for the treatment of tuberculosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the structural formula of cefathiamidine of the present invention.

[0022] Figure 2 Schematic diagram of the CCK8 method of the present invention for determining the effect of cefathiamidine on different cell viabilities.

[0023] Figure 3 The schematic diagram of the chessboard method of the present invention is to detect the combined effect of cefathiamidine and clavulanic acid against Mycobacterium tuberculosis.

[0024] Figure 4 This is the result of the cefathiamidine combined with clavulanic acid of the present invention inhibiting the growth of MTB in macrophages.

[0025] Figure 5 The morphological changes of H37Ra after 7 days of cefathiamidine treatment were observed using scanning electron microscopy. DETAILED DESCRIPTION

[0026] In order to deepen the understanding of the present invention, the present invention will be further described in detail below in conjunction with examples. The examples are only used to explain the present invention and do not constitute a limitation on the protection scope of the present invention.

[0027] Embodiment 1 1. Research materials and testing methods: 1.1 Drugs: Using the inhibitor high-throughput screening model at the Mycobacterium tuberculosis H37Rv (ATCC27294) cell level, 7285 samples of the bioactive compound library L4000 purchased from Shanghai Taosu Biotechnology Co., Ltd. were screened, and a cephalosporin antibiotic cefathiamidine was screened out to have anti-tuberculosis activity, with the structural formula shown below: Figure 1 shown.

[0028] The drugs were dissolved in dimethyl sulfoxide (DMSO) according to the instructions and stored in a refrigerator at -80°C to avoid repeated freezing and thawing. Potassium clavulanate and amikacin were purchased from Sigma-Aldrich.

[0029] 1.2 Strains and reagents: The strains used in this study were: MtbH37Rv (ATCC 27294), MtbH37Ra (ATCC25177), BCG Tokyo strain (ATCC 35737), Mycobacterium marinum (BAA55), Mycobacterium smegmatis (MC2155), clinically isolated multidrug-resistant strains (Mtb28, Mtb 1731), Mycobacterium abscessus (ATCC 19977), Staphylococcus aureus (CMCC26003), Staphylococcus epidermidis (CMCC 26069), Streptococcus suis (SS2-1-F0), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC29213), Acinetobacter baumannii (ATCC19606), Pseudomonas aeruginosa (CMCC10104). All mycobacteria were cultured in 10% OADC medium. (BD, USA, 212351), 0.2% glycerol and 0.05% Tween 80 in Middlebrook 7H9 liquid medium (7H9, Becton Dickinson Company, USA). Except for Mycobacterium marinum, other mycobacteria were cultured in a 37°C incubator. Mycobacterium marinum was cultured in a 32°C incubator. The remaining strains were cultured in MH liquid medium at 37°C.

[0030] 1.3 Cell morphology determination Mycobacterium tuberculosis was cultured to the logarithmic growth phase and diluted to OD600 of 0.005 in culture medium. It was treated with 0, 0.125 μg / mL, 0.25 μg / mL and 0.375 μg / mL of cefathiamidine for 7 days, followed by fixation with 2.5% (volume / volume) glutaraldehyde at 4°C for 12 hours and 1% osmium tetroxide (OsO4) in 0.1 M PBS at 25°C for 1 hour. After that, it was rinsed with PBS three times, each for 15 minutes. The samples were immersed in 50%, 70%, 85% and 95% ethanol solutions for 15 minutes in sequence, and then dehydrated in anhydrous ethanol and butanol for 30 minutes three times. Finally, the samples were dried and prepared by gold / palladium alloy sputtering technology. The samples were detected using a S3400N scanning electron microscope (SEM) produced by Hitachi, Japan, with an accelerating voltage of 1.0 kV.

[0031] 2. Study on the antibacterial activity of cefathiamidine against mycobacteria 2.1 Minimum inhibitory concentration (MIC) determination: When determining the minimum inhibitory concentration (MIC) of ceftriaxone against mycobacteria, the bacteria were first pre-cultured to the logarithmic growth phase (OD600 of 0.5-1), and then 5 × 10 6 CFU / mL concentration was inoculated in a 96-well round-bottom microtiter plate (total volume of 100 μL), and the compound was added at the same time, and the compound was diluted by 2 dilution method, and incubated at 37°C for 7 days. Then 30 μL of 0.05% (w / v) resazurin containing 10% Tween 80 was added, and cultured at 37°C for 16 hours. The concentration of the last blue well in each row was regarded as the minimum inhibitory concentration (MIC). The experiment was repeated three times.

[0032] 2.2 Experimental results: Cefathiamidine has antibacterial activity against Mycobacterium tuberculosis.

[0033] The anti-tuberculosis compound cefathiamidine was screened from the compound library by high-throughput screening. The chemical structure of cefathiamidine is as follows: Figure 1 shown.

[0034] In order to evaluate the efficacy of cefathiamidine, its effect on mycobacteria was first tested. The results showed that cefathiamidine could significantly inhibit the growth of mycobacteria, especially Mycobacterium tuberculosis.

[0035] The test results showed that the minimum inhibitory concentration (MIC) values ​​of cefathiamidine against H37Ra, H37Rv, BCG, Mycobacterium marinum and two clinical isolates were 0.5μg / mL, 0.031μg / mL, 0.5μg / mL, 0.5μg / mL, 0.15μg / mL and 2.5μg / mL, respectively.

[0036] In addition to mycobacteria, preliminary drug sensitivity tests of cefathiamidine were conducted on a variety of pathogens, including Staphylococcus aureus, Staphylococcus aureus, and Streptococcus. The results showed that cefathiamidine has a strong efficacy and has antimicrobial activity (MIC value) between 1 and 8 μg / mL, as shown in Table 1.

[0037] Table 1 Results of the antibacterial activity test of cefathiamidine in clinical isolates and standard strains ; However, the activity of cefathiamidine against Gram-negative bacteria is weak or negligible.

[0038] In addition, the minimum bactericidal concentration (MBC) of ceftriaxone was 8.0 μg / mL when the strain H37Ra was tested, indicating that it had significant antibacterial activity.

[0039] 2.3 Detection of the effect of cefathiamidine on cell viability To evaluate the cytotoxicity of cefathiamidine, the biological viability of African green monkey kidney cells (Vero), human lung cancer cells (A549), human hepatoma cells (HuH7), and human breast cancer cells (MCF-7) were determined using the CCK8 assay. The cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS).

[0040] 1×10 4 Cells in logarithmic growth state were evenly distributed in each microplate, and 100 μL of cell culture medium was added. Incubated at 37°C and 5% CO2 for 12 hours to promote their attachment to the plate. After the cells adhered, the supernatant was discarded and different concentrations of ceftriaxone (0.4-1000 μg / mL) were added and cultured for 48 hours.

[0041] The control group was treated with cells of equal concentration of DMSO and included in the experiment.

[0042] After the incubation, 10 μL of CCK-8 reagent was added to each well and incubated at 37°C for 2 h. The absorbance was measured at 450 nm using a microplate reader (ThermoFisher, VLBLATD2). The cell viability (%) was calculated by three independent repeat experiments. The CC50 value (e.g., Figure 2 As shown, n = 3 per group).

[0043] 3 Study on the synergistic antibacterial activity of cefathiamidine and clavulanic acid 3.1 The chessboard method was used to determine the synergistic index of combined drug use. The MTB increment rate (100%) compared with the control group is shown in Table 2. Figure 3As shown: The fractional inhibitory concentration index (FICI) value of cefathiamidine and clavulanic acid was determined using the two-fold dilution method. Mycobacterium tuberculosis (MTB) in the logarithmic growth phase was diluted to a final concentration of OD600 = 0.005 and inoculated into a 96-well plate at a volume of 100 μL / well. Each compound was added starting from 2×MIC and diluted 2-fold, and cultured at 37 °C for 5 days.

[0044] Table 2 Data table for determining the synergistic index of combined drugs by checkerboard method ; During the quantification process, the resazurin assay method was used. The FICI value was calculated by adding the quotient of the lowest inhibitory concentration in the compound row (A and B respectively) and the MIC of the compound (MICA and MICB respectively). By analyzing the FICI value, it can be concluded that there is complete synergy (FICI ≤ 0.5) or partial synergy (0.5 < FICI ≤ 0.75) or no significant effect (0.75 < FICI ≤ 2) or antagonism (FICI > 2) between cefathiamidine and the tested antibiotics.

[0045] Checkerboard assay for the combination of cefathiamidine and clavulanic acid against Mycobacterium tuberculosis H37Ra. FICI = 0.31. The heatmap shows the average of three experimental replicates.

[0046] 3.2 Synergistic anti-Mycobacterium tuberculosis effect of cefathiamidine and clavulanate: According to the antibacterial mechanism of cefathiamine, the antibacterial effect of cefathiamidine can be enhanced with a β-lactamase inhibitor. Clavulanic acid is the most commonly used β-lactamase inhibitor, so its inhibitory effect in combination with cefathiamidine on MTB was determined. After evaluation, the relative growth at the end of the determination was evaluated and the FICI was calculated. The results of the checkerboard test showed that in the presence of clavulanic acid, the minimum inhibitory concentration of cefathiamidine decreased from 0.5 μg / ml to 0.03 μg / ml, and the FICI value was 0.31, indicating that the combination of clavulanic acid can enhance the anti-tuberculosis effect of cefathiamidine.

[0047] 3.3 Determination of Mycobacterium tuberculosis-infected macrophages and colony-forming units (CFUs) One day before infection, at 5×10 per well 5RAW264.7 was inoculated on a 6-well plate at a cell density of 1.5 μg / mL. Mycobacterium tuberculosis in the logarithmic growth phase was centrifuged at 2000 g for 5 minutes and washed twice with PBS. The bacteria were resuspended in DMEM medium, the OD600 was recorded, and the cells were appropriately diluted for infection. Bacteria were added to infect cells at an MOI of 10:1, and 1 ml / well of cell culture medium was added. After 5 hours of cell uptake, extracellular bacteria were washed twice with PBS to remove them, and 400 μg / mL of amikacin was added to the culture medium. After incubation for 4 hours at 37°C and 5% CO2, the culture medium was discarded and washed twice with PBS. The cells were then treated with 2.5 μg / mL, 5 μg / mL, 10 μg / mL of cefathiamidine alone or in combination with 32 μg / mL of clavulanic acid. After 24 hours, the cells were lysed with PBS containing 1% Triton X-100 (Sigma-Aldrich, 9036195), and the lysate was serially diluted on Middlebrook 7H10 agar plates containing 10% OADC and 0.5% glycerol and incubated at 37°C for 2 to 3 weeks. The bacterial colonies were then counted and the number of colonies (CFU) in each dilution was estimated. The results were shown in Table 1. Figure 4 As shown, **p<0.01; ***p<0.001; ****p<0.0001. In the experiment of inhibiting the growth of MTB in macrophages by cefathiamidine combined with clavulanic acid, RAW264.7 cells were treated with different concentrations of cefathiamidine alone or in combination with clavulanic acid 32 μg / mL, and colony counts were performed 4 hours after H37Ra infection. Data are the means ± SDs of three independent experiments. Analysis of variance (ANOVA) was performed.

[0048] 3.4 Clavulanic acid can enhance the intracellular antibacterial activity of cefathiamidine: The present invention continues to study the in vitro inhibitory ability of cefathiamidine on the intracellular growth of mycobacteria. After H37Ra infected RAW264.7 cells (infection multiplier of 10:1) for 5 hours, extracellular bacteria were treated with 400μg / mL amikacin. RAW264.7 cells were then treated with DMSO, 2.5, 5 and 10μg / mL cefathiamidine alone or in combination with 32μg / mL clavulanic acid, and colony counts were performed 24 hours after treatment. The results showed that cefathiamidine alone reduced the number of intracellular bacterial CFUs, while combined with clavulanic acid, it inhibited bacterial growth to a greater extent. These data indicate that β-lactamase inhibitors may enhance the antibacterial ability of cefathiamidine in cells.

[0049] 3.5 Cell viability is not affected by cefathiamidine: After observing the synergistic effect of antibiotics in the infection model, we were interested in exploring whether the tested concentrations had any cytotoxicity to cells. We analyzed a variety of cell lines treated with different concentrations of cefathiamidine, including human tumor cells A549, Huh7, MCF-7, and normal monkey kidney cells Vero. Cefathiamidine was dissolved in DMSO at concentrations ranging from 0.4μg / mL to 1000μg / mL. The results of the CCK-8 assay showed that the viability of all cells decreased slightly during the 48-hour culture, indicating that cefathiamidine was still non-cytotoxic even at a high concentration of 1000μg / mL.

[0050] 3.6 Cefathiamidine can cause morphological changes in Mycobacterium tuberculosis We used scanning electron microscopy to analyze the effects of different concentrations of cefathiamidine (0.25× MIC, 0.5× MIC and 0.75× MIC, where MIC = 0.5 μg / mL) on the morphological changes of MtbH37Ra. Figure 5 The morphological characteristics of Mycobacterium tuberculosis are shown. Scanning electron microscopy images show that cefathiamidine can destroy the cell morphology of Mycobacterium tuberculosis in a dose-dependent manner.

[0051] 4 Conclusion In this study, a high-throughput model was used to screen the anti-tuberculosis drug cefathiamidine (a cephalosporin) from a compound library and the efficacy was studied against selected mycobacterial strains, including two clinically isolated multidrug-resistant Mtb strains and other strains. The experiment showed that CE showed significant inhibitory activity against most mycobacteria.

[0052] According to the widely accepted hypothesis, when the MBC / MIC ratio is ≤4, it indicates that the compound has a bactericidal effect; and when the MBC / MIC ratio is >4, it indicates that the compound has an antibacterial effect. The results of the present invention show that the MIC value of cefathiamidine against H37Ra is 0.5μg / mL, and the MBC value is 8μg / mL, which is 16 times the MIC, and has significant antibacterial activity. The present invention found that the inhibition of Mycobacterium tuberculosis by cefathiamidine alone is comparable to that of several first-line anti-tuberculosis drugs; and when used in combination with the β-lactamase inhibitor clavulanic acid, its efficacy exceeds that of standard anti-tuberculosis drugs.

[0053] The present invention found that cefathiamidine can specifically inhibit mycobacteria, mainly by inhibiting the assembly of peptidoglycan and destroying the integrity of the bacterial structure to exert its antibacterial effect. The β-lactam inhibitor clavulanic acid can enhance the anti-tuberculosis effect of cefathiamidine in vitro and even in cells, even exceeding the intensity of action of the classic anti-tuberculosis drug rifampicin. These research results show that the combination of cefathiamidine and clavulanic acid may become a candidate for the treatment of tuberculosis. Subsequent studies will further evaluate the inhibitory activity of this combination on more clinical single-drug resistance, multidrug resistance and even XDR strains, and evaluate the therapeutic effect of this combination on the acute infection MTB model and the persistent infection model in mice, in order to accumulate data for the ultimate promotion of this regimen to the clinic.

[0054] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. Use of cefathiamidine in the preparation of a medicament for preventing and / or treating tuberculosis, characterized in that: The tuberculosis includes human or animal pulmonary tuberculosis and extrapulmonary tuberculosis caused by Mycobacterium tuberculosis; the Mycobacterium tuberculosis is selected from: standard strain MtbH37Rv (ATCC 27294) and attenuated strain MtbH37Ra (ATCC25177).

2. The use according to claim 1, characterized in that: The minimum inhibitory concentration (MIC) values ​​of cefathiamidine against the standard strain MtbH37Rv (ATCC27294) and the attenuated strain MtbH37Ra (ATCC25177) were 0.5μg / mL and 0.031μg / mL, respectively.

3. Use of cefathiamidine and clavulanic acid in the preparation of a medicament for preventing and / or treating tuberculosis.

4. The use according to claim 3, characterized in that: The tuberculosis includes human or animal pulmonary tuberculosis and extrapulmonary tuberculosis caused by Mycobacterium tuberculosis; the Mycobacterium tuberculosis is selected from: standard strain MtbH37Rv (ATCC 27294) and attenuated strain MtbH37Ra (ATCC25177).

5. Application of cefathiamidine and clavulanic acid in the preparation of Mycobacterium tuberculosis inhibitors.

6. The use according to claim 5, characterized in that: The cefathiamidine and clavulanic acid have inhibitory activity against tuberculosis bacteria in vitro.

7. The use according to claim 6, characterized in that: The Mycobacterium tuberculosis is selected from: MtbH37Rv (ATCC27294), MtbH37Ra (ATCC25177).

8. The use according to claim 7, characterized in that: The minimum inhibitory concentration of the cefathiamidine and clavulanic acid against the standard strain of Mycobacterium tuberculosis MtbH37Rv (ATCC 27294) in vitro is 0.03 μg / mL.

9. The use according to claim 6, characterized in that: The cefathiamidine cell safety of the Mycobacterium tuberculosis inhibitor is not less than 1000 μg / mL.

10. The use according to claim 6, characterized in that: The Mycobacterium tuberculosis inhibitor cefathiamidine can destroy the cell morphology of Mycobacterium tuberculosis in a dose-dependent manner.

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