Application of cefathiamidine combined with clavulanic acid in the treatment of tuberculosis

Abstract

The present invention relates to an application of a combined preparation of cefathiamidine and clavulanic acid in the treatment of tuberculosis. Cefathiamidine and clavulanic acid are used in combination to significantly improve the activity against Mycobacterium tuberculosis. It is verified by in vitro experiments that the β-lactamase inhibitor clavulanic acid significantly improves the anti-tuberculosis effect of cefathiamidine. The combined preparation shows significant advantages in inhibiting the growth of Mycobacterium tuberculosis and has a strong inhibitory effect on drug-resistant strains. At the same time, the combined preparation shows lower cytotoxicity in in vitro experiments and shows better safety. In addition, the combined preparation has potential application prospects in the treatment of drug-resistant tuberculosis and can effectively supplement existing tuberculosis treatment regimens.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, 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, posing a serious threat to human health. The primary treatment for TB is a combination of anti-TB drugs designed to inhibit or kill M. tuberculosis. However, as TB treatment progresses, new challenges are emerging.

[0003] Current problems in tuberculosis treatment, namely drug and formulation research and development, include:

[0004] 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.

[0005] 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.

[0006] 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.

[0007] Faced with these challenges, researchers are constantly exploring new drugs and combination treatments 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.

[0008] Cefathiamidine: Cefathiamidine is a cephalosporin antibiotic with broad-spectrum antibacterial activity, particularly against Gram-positive bacteria. It achieves its bactericidal effect by interfering with bacterial cell wall synthesis.

[0009] 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 of bacteria to resist β-lactam antibiotics.

[0010] 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

[0011] In view of the above problems, the present invention aims to propose the use of cefathiamidine combined with clavulanic acid in the preparation of drugs and / or Mycobacterium tuberculosis inhibitors for the prevention and / or treatment of tuberculosis, thereby providing a new option for the treatment of tuberculosis.

[0012] The present invention provides use of cefathiamidine and clavulanic acid in preparing a medicine for preventing and / or treating tuberculosis.

[0013] In one embodiment, the tuberculosis includes pulmonary tuberculosis and extrapulmonary tuberculosis in humans or animals caused by Mycobacterium tuberculosis.

[0014] In one embodiment, the Mycobacterium tuberculosis is selected from the group consisting of: 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), and Pseudomonas aeruginosa (CMCC10104).

[0015] The present invention also provides an application of cefathiamidine and clavulanic acid in the preparation of a Mycobacterium tuberculosis inhibitor.

[0016] In one embodiment, the cefathiamidine and clavulanic acid have inhibitory activity against Mycobacterium tuberculosis in vitro.

[0017] In one embodiment, the Mycobacterium tuberculosis is selected from the group consisting of: 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), and Pseudomonas aeruginosa (CMCC10104).

[0018] 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.

[0019] In one embodiment, the Mycobacterium tuberculosis inhibitor has a cell safety of cefathiamidine of no less than 1000 μg / mL.

[0020] In one embodiment, the Mycobacterium tuberculosis inhibitor cefathiamidine is capable of destroying the cell morphology of Mycobacterium tuberculosis in a dose-dependent manner.

[0021] The beneficial effects of the present invention are:

[0022] The present study found that cefathiamidine exhibits potent inhibitory activity against Mycobacterium tuberculosis, with a minimum inhibitory concentration (MIC) of 0.031 μg / mL against the standard strain H37Rv and 0.5 μg / mL against the attenuated strain H37Ra. It exhibits minimal effect on Gram-negative bacteria. Furthermore, the β-lactamase inhibitor clavulanate can enhance its anti-tuberculosis activity. Combined use with clavulanic acid resulted in a minimum inhibitory concentration (MIC) of 0.031 μg / mL against the attenuated strain H37Ra, making the combined effect 16-fold greater than that of rifampicin alone (MIC 0.5 μg / mL). Intracellular anti-M. tuberculosis experiments demonstrated that clavulanic acid enhanced the intracellular antibacterial activity of cefathiamidine (10 μg / mL, 5 μg / mL, and 2.5 μg / mL) by 1-3 times. Observation of bacterial morphological changes revealed that cefathiamidine disrupted the morphological integrity of bacterial cells. Furthermore, the IC50 values ​​of cefathiamidine against A549, MCF-7, Vero, and Huh7 cells were greater than 1000 μg / mL, demonstrating extremely low cytotoxicity. Therefore, cefathiamidine combined with clavulanic acid could be a candidate drug for the treatment of tuberculosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the structural formula of cefathiamidine of the present invention.

[0024] Figure 2 Schematic diagram of the CCK8 method of the present invention for determining the effects of cefathiamidine on different cell viabilities.

[0025] Figure 3 Schematic diagram of the checkerboard method of the present invention for detecting the combined effect of cefathiamidine and clavulanic acid against Mycobacterium tuberculosis.

[0026] Figure 4 This is the result of the cefathiamidine combined with clavulanic acid of the present invention inhibiting the growth of MTB in macrophages.

[0027] Figure 5 The morphological changes of H37Ra after 7 days of cefathiamidine treatment were observed using scanning electron microscopy. DETAILED DESCRIPTION

[0028] In order to deepen the understanding of the present invention, the present invention will be further described in detail below with reference to the examples. The examples are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0029] Example 1

[0030] 1. Research materials and testing methods:

[0031] 1.1 Drugs:

[0032] A high-throughput screening model for inhibitors at the Mycobacterium tuberculosis H37Rv (ATCC27294) cell level was used to screen 7285 samples of the bioactive compound library L4000 purchased from Shanghai Taosu Biotechnology Co., Ltd., and a cephalosporin antibiotic cefathiamidine was screened out to have anti-tuberculosis activity. The structural formula is as follows: Figure 1 shown.

[0033] Drugs were dissolved in dimethyl sulfoxide (DMSO) according to the instructions and stored in a refrigerator at -80°C to avoid repeated freeze-thaw cycles. Clavulanate potassium and amikacin were purchased from Sigma-Aldrich.

[0034] 1.2 Strains and Reagents

[0035] The strains used in this study were: MtbH37Rv (ATCC 27294), MtbH37Ra (ATCC 25177), 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 (CMCC 26003), Staphylococcus epidermidis (CMCC 26069), Streptococcus suis (SS2-1-F0), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 29213), Acinetobacter baumannii (ATCC 19606), and Pseudomonas aeruginosa (CMCC 10104). All mycobacteria were cultured in a 10% OADC-containing medium. Mycobacterium strains, except Mycobacterium marinum, were cultured in Middlebrook 7H9 liquid medium (7H9, Becton Dickinson Company, USA) containing 0.2% glycerol and 0.05% Tween 80 (BD, USA, 212351). Mycobacterium marinum was 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.

[0036] 1.3 Cell morphology determination

[0037] Mycobacterium tuberculosis was cultured to the logarithmic growth phase and diluted to an OD600 of 0.005. Cefathiamidine was then treated for 7 days at concentrations of 0, 0.125 μg / mL, 0.25 μg / mL, and 0.375 μg / mL. The cells were then fixed with 2.5% (vol / vol) glutaraldehyde for 12 hours at 4°C and 1% osmium tetroxide (OsO4) in 0.1 M PBS for 1 hour at 25°C. The cells were then rinsed with PBS three times for 15 minutes each. The samples were then immersed in 50%, 70%, 85%, and 95% ethanol for 15 minutes each, followed by dehydration in anhydrous ethanol and butanol for three 30-minute cycles. The samples were then dried and prepared using a gold / palladium alloy sputtering technique. The samples were examined using a Hitachi S3400N scanning electron microscope (SEM) at an accelerating voltage of 1.0 kV.

[0038] 2. Study on the antibacterial activity of cefathiamidine against mycobacteria

[0039] 2.1 Minimum Inhibitory Concentration (MIC) Determination:

[0040] When determining the minimum inhibitory concentration (MIC) of cefathiamidine against mycobacteria, the bacteria were first pre-cultured to the logarithmic growth phase (OD600 of 0.5-1), and then 5 × 10 6 The cells were inoculated at a concentration of 100 μL CFU / mL in a 96-well round-bottom microtiter plate. Compounds were added simultaneously using a 2-by-2 dilution method and incubated at 37°C for 7 days. Subsequently, 30 μL of 0.05% (w / v) resazurin containing 10% Tween 80 was added, and the cells were incubated at 37°C for 16 hours. The concentration in the last blue well in each row was considered the minimum inhibitory concentration (MIC). The experiment was repeated three times.

[0041] 2.2 Experimental results: Cefathiamidine has antibacterial activity against Mycobacterium tuberculosis.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] In addition to mycobacteria, preliminary susceptibility testing of cefathiamidine was conducted on a variety of pathogens, including Staphylococcus aureus, Staphylococcus aureus, and Streptococci. The results showed that cefathiamidine has strong efficacy, with antimicrobial activity (MIC values) ranging from 1 to 8 μg / mL (see Table 1 for details).

[0046] Table 1 Results of antibacterial activity assay of cefathiamidine in clinical isolates and standard strains

[0047] ;

[0048] However, the activity of cefathiamidine against Gram-negative bacteria is weak or negligible.

[0049] In addition, the minimum bactericidal concentration (MBC) of cefathiamidine was 8.0 μg / mL when tested on the H37Ra strain, indicating that it has significant antibacterial activity.

[0050] 2.3 Detection of the effect of cefathiamidine on cell viability

[0051] 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 supplemented with 10% fetal bovine serum (FBS).

[0052] 1×10 4 Cells in logarithmic growth were evenly distributed in each well of a microplate, and 100 μL of cell culture medium was added. The cells were incubated at 37°C and 5% CO₂ for 12 hours to allow attachment. After cell attachment, the supernatant was discarded, and the cells were incubated with various concentrations of cefathiamidine (0.4–1000 μg / mL) for 48 hours.

[0053] The control group was treated with cells containing the same concentration of DMSO and included in the experiment.

[0054] After incubation, 10 μL of CCK-8 reagent was added to each well and incubated at 37 °C for 2 hours. The absorbance was measured at 450 nm using a microplate reader (ThermoFisher, VLBLATD2). The cell viability (%) was calculated from three independent replicates. The CC50 value was calculated using a four-parameter logistic curve (GraphPad Prism 7.0) (as shown in Figure 2 for each group, n = 3).

[0055] 3 Synergistic antibacterial activity of cefathiamidine and clavulanic acid

[0056] 3.1 Determination of the synergy index of combined drugs by checkerboard method. The data of the MTB proliferation rate (100%) compared with the control group are shown in Table 2, and the checkerboard diagram is shown in Figure 3 as follows:

[0057] The synergy index (FICI) value of cefathiamidine and clavulanic acid was determined using the two-fold dilution method. 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.

[0058] Table 2 Data sheet for determining the synergy index of combined drugs by checkerboard method

[0059] ;

[0060] In the quantification process, the resazurin assay method was used. The FICI value was calculated by adding the quotient of the lowest inhibitory concentration (A and B respectively) in the compound row and the compound MIC (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.

[0061] Checkerboard method to detect the combination of cefathiamidine and clavulanic acid against Mycobacterium tuberculosis H37Ra. FICI = 0.31. The heatmap shows the average of three experimental replicates.

[0062] 3.2 Synergistic effect of cefathiamidine and clavulanate against Mycobacterium tuberculosis

[0063] Based on the antibacterial mechanism of ceftriaxone, the antibacterial effect of cefathiamidine can be enhanced by the use of β-lactamase inhibitors. Clavulanic acid is the most commonly used β-lactamase inhibitor, so the inhibitory effect of cefathiamidine on MTB was determined in combination with cefathiamidine. The relative growth at the end of the assay was evaluated, and the FICI (Filtration Index) was calculated. Checkerboard assay results showed that in the presence of clavulanic acid, the minimum inhibitory concentration (MIC) of cefathiamidine decreased from 0.5 μg / ml to 0.03 μg / ml, with an FICI value of 0.31, indicating that the combined use of clavulanic acid enhances the anti-TB activity of cefathiamidine.

[0064] 3.3 Determination of Mycobacterium tuberculosis-infected macrophages and CFU

[0065] One day before infection, 5 × 10 5 RAW264.7 cells were seeded at a high density in 6-well plates. 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 diluted appropriately for infection. Bacteria were added to the cells at an MOI of 10:1, and 1 ml of cell culture medium was added per well. After 5 hours of cell uptake, extracellular bacteria were removed by washing twice with PBS. 400 μg / mL of amikacin was added to the culture medium. After incubation at 37°C, 5% CO2 for 4 hours, the medium was discarded, and the cells were washed twice with PBS. The cells were then treated with 2.5 μg / mL, 5 μg / mL, or 10 μg / mL of cefathiamidine alone or in combination with 32 μg / mL clavulanic acid. After 24 hours, the cells were lysed using PBS containing 1% Triton X-100 (Sigma-Aldrich, 9036195), and the resulting 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. 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. To investigate the inhibitory effect of cefathiamidine combined with clavulanic acid on the growth of MTB in macrophages, RAW264.7 cells were treated with various concentrations of cefathiamidine alone or in combination with 32 μg / mL clavulanic acid. Colonies were counted 4 hours after infection with H37Ra. Data are means ± SD from three independent experiments. Analysis of variance (ANOVA) was performed.

[0066] 3.4 Clavulanic acid can enhance the intracellular antibacterial activity of cefathiamidine:

[0067] The present invention further investigated the in vitro inhibitory effect of cefathiamidine on the intracellular growth of mycobacteria. Five hours after H37Ra infection of RAW264.7 cells (multiplicity of infection 10:1), extracellular bacteria were treated with 400 μg / mL amikacin. RAW264.7 cells were then treated with DMSO, 2.5, 5, and 10 μg / mL cefathiamidine alone, or in combination with 32 μg / mL clavulanic acid. Colony counts were performed 24 hours after treatment. The results showed that cefathiamidine alone reduced the number of intracellular bacterial CFUs, while cefathiamidine combined with clavulanic acid resulted in a greater inhibition of bacterial growth. These data suggest that β-lactamase inhibitors may enhance the intracellular antibacterial activity of cefathiamidine.

[0068] 3.5 Cell viability is not affected by cefathiamidine:

[0069] After observing synergistic effects between antibiotics in infection models, we were interested in investigating whether the concentrations tested exhibited any cytotoxicity. We analyzed various cell lines treated with varying concentrations of cefathiamidine, including human tumor cells A549, Huh7, and MCF-7, and normal monkey kidney Vero cells. Cefathiamidine was dissolved in DMSO at concentrations ranging from 0.4 μg / mL to 1000 μg / mL. CCK-8 assays revealed a slight decrease in viability across all cells over the 48-hour culture period, demonstrating that cefathiamidine was non-cytotoxic even at a high concentration of 1000 μg / mL.

[0070] 3.6 Cefathiamidine can cause morphological changes in Mycobacterium tuberculosis

[0071] 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.

[0072] 4 Conclusion

[0073] This study used a high-throughput model to screen the anti-tuberculosis drug cefathiamidine (a cephalosporin) from a compound library. The efficacy of cefathiamidine against selected mycobacterial strains, including two clinically isolated multidrug-resistant Mtb strains and other strains, was investigated. The results demonstrated that cefathiamidine exhibited significant inhibitory activity against most mycobacterial strains.

[0074] According to a widely accepted hypothesis, an MBC / MIC ratio ≤ 4 indicates a compound's bactericidal activity, while an MBC / MIC ratio > 4 indicates a compound's bacteriostatic activity. The present study showed that cefathiamidine had an MIC of 0.5 μg / mL and an MBC of 8 μg / mL against H37Ra, 16 times the MIC, demonstrating significant antibacterial activity. The present study found that cefathiamidine alone exhibited comparable inhibition against Mycobacterium tuberculosis as several first-line anti-TB drugs; however, when used in combination with the β-lactamase inhibitor clavulanic acid, its efficacy exceeded that of standard anti-TB drugs.

[0075] The present study found that cefathiamidine can specifically inhibit mycobacteria, primarily by inhibiting the assembly of peptidoglycan and disrupting the integrity of bacterial structure. The β-lactam inhibitor clavulanic acid can enhance the anti-tuberculosis effect of cefathiamidine in vitro and even in cells, even exceeding the potency of the classic anti-tuberculosis drug rifampicin. These findings suggest 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 against more clinically single-drug resistant, multidrug resistant, and even XDR strains, and evaluate the therapeutic effect of this combination in mouse models of acute MTB infection and persistent infection, in order to accumulate data for the ultimate clinical advancement of this approach.

[0076] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended 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 pulmonary tuberculosis and extrapulmonary tuberculosis in humans or animals 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; The tuberculosis includes pulmonary tuberculosis and extrapulmonary tuberculosis in humans or animals caused by Mycobacterium tuberculosis; the Mycobacterium tuberculosis is selected from: standard strain MtbH37Rv (ATCC 27294) and attenuated strain MtbH37Ra (ATCC25177).

4. Application of cefathiamidine and clavulanic acid in the preparation of Mycobacterium tuberculosis inhibitors; The Mycobacterium tuberculosis is selected from: MtbH37Rv (ATCC 27294), MtbH37Ra (ATCC25177).

5. The use according to claim 4, characterized in that: The cefathiamidine and clavulanic acid have inhibitory activity against tuberculosis bacteria in vitro.

6. The use according to claim 4, characterized in that: The minimum inhibitory concentration of the cefathiamidine and clavulanic acid to the standard strain of Mycobacterium tuberculosis MtbH37Rv (ATCC 27294) in vitro is 0.03 μg / mL.

7. The use according to claim 5, characterized in that: The cefathiamidine cell safety of the Mycobacterium tuberculosis inhibitor is not less than 1000 μg / mL.

8. The use according to claim 5, 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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