Use of muc1 inhibitors in the preparation of medicaments for the prevention or treatment of bacterial infections

Abstract

The application relates to application of a MUC1 inhibitor in preparation of a medicine for preventing and treating bacterial infection, and particularly relates to application of the MUC1 inhibitor in preparation of a preparation for improving sensitivity of antibiotic treatment. By applying the MUC1 inhibitor, the sensitivity of antibiotic treatment of bacterial infection can be improved, the antibiotic dosage can be reduced by 50%, and a new strategy for optimizing the antibiotic dosage of bacterial infection is provided. In the application and the medicine composition, the MUC1 inhibitor has good cell compatibility and no obvious cytotoxicity, and has a good application prospect in the biomedical field.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, and in particular to the application of MUC1 inhibitor in the preparation of a drug for preventing and / or treating bacterial infection. BACKGROUND

[0002] Bacterial infection is a serious threat to human health and is one of the main causes of human death worldwide. With the passage of time, bacteria continue to evolve, and the abuse of antibiotics leads to the rapid development of bacterial resistance. The common method is to develop new antibacterial drugs, however, it takes at least 10-12 years from research and development to clinical application of new antibacterial drugs, while it only takes about 2 years for bacteria to develop resistance.

[0003] Therefore, it is urgent to develop new antibacterial drugs and new strategies for anti-infection. SUMMARY

[0004] The purpose of the present application is to provide the application of MUC1 inhibitor in the preparation of a drug for preventing or treating bacterial infection. The MUC1 inhibitor can reduce the adhesion force in the process of bacterial-cell interaction. In the process of antibiotic prevention or treatment of bacterial infection, the addition of MUC1 inhibitor can significantly improve the sensitivity of antibiotic treatment, thereby reducing the amount of antibiotic and the development of drug resistance.

[0005] To this end, in a first aspect, the present application provides the application of MUC1 inhibitor for the preparation of a formulation for improving the sensitivity of antibiotic treatment.

[0006] In some embodiments, the MUC1 inhibitor is selected from GO-203TFA.

[0007] In some embodiments, the antibiotic comprises at least one selected from the group consisting of cephalosporins, quinolones, fluoroquinolones, penicillins, lactamase inhibitors, carbapenems, monobactams, macrolides, lincosamides, glycopeptides, rifampicins, oxazolidinones, tetracyclines, aminoglycosides, streptomycetes, and sulfonamides.

[0008] In some embodiments, the antibiotic comprises a quinolone antibiotic, in particular at least one selected from the group consisting of ciprofloxacin, enoxacin, gatifloxacin, grepafloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, sparfloxacin, trovafloxacin, hydroxybenzoic acid, gemifloxacin, or perfloxacin.

[0009] In a second aspect of the present application, the application of MUC1 inhibitor in the preparation of a drug for preventing and / or treating bacterial infection is provided.

[0010] In some embodiments, the MUC1 inhibitor is selected from GO-203TFA.

[0011] In some embodiments, the bacteria comprises at least one selected from the group consisting of gram-negative bacteria, gram-positive bacteria.

[0012] In some embodiments, the bacteria comprises at least one selected from the group consisting of Staphylococcus aureus, Escherichia coli, Streptococcus (e.g., Pneumococcus), Mycobacterium tuberculosis, Klebsiella, Serratia, and Proteus.

[0013] In some embodiments, the MUC1 inhibitor is present in the medicament at an effective dose concentration.

[0014] In some embodiments, the medicament further comprises an antibiotic.

[0015] In some embodiments, the antibiotic comprises at least one selected from the group consisting of cephalosporins, quinolones, fluoroquinolones, penicillins, lactamase inhibitors, carbapenems, monobactams, macrolides, lincosamides, glycopeptides, rifampins, oxazolidinones, tetracyclines, aminoglycosides, streptomycetes, and sulfonamides.

[0016] In some embodiments, the antibiotic comprises a quinolone antibiotic, specifically at least one selected from the group consisting of ciprofloxacin, enoxacin, gatifloxacin, grepafloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, sparfloxacin, trovafloxacin, hydroxyphenylcarboxylic acid, gemifloxacin, or perfloxacin.

[0017] In a third aspect, the present application provides a use of a MUC1 inhibitor in combination with an antibiotic in the manufacture of a medicament for preventing and / or treating a bacterial infection.

[0018] In some embodiments, the MUC1 inhibitor is selected from GO-203 TFA.

[0019] In some embodiments, the antibiotic comprises at least one selected from the group consisting of cephalosporins, quinolones, fluoroquinolones, penicillins, lactamase inhibitors, carbapenems, monobactams, macrolides, lincosamides, glycopeptides, rifampins, oxazolidinones, tetracyclines, aminoglycosides, streptomycetes, and sulfonamides.

[0020] In some embodiments, the antibiotic comprises a quinolone antibiotic, specifically at least one selected from the group consisting of ciprofloxacin, enoxacin, gatifloxacin, grepafloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, sparfloxacin, trovafloxacin, hydroxyphenylcarboxylic acid, gemifloxacin, or perfloxacin.

[0021] In some embodiments, the bacteria include at least one selected from the group consisting of Gram-negative bacteria and Gram-positive bacteria.

[0022] In some embodiments, the bacteria include at least one selected from the group consisting of Staphylococcus aureus, Escherichia coli, Streptococcus (e.g., Pneumococcus), Mycobacterium tuberculosis, Klebsiella pneumoniae, Serratia marcescens, and Proteus.

[0023] In a fourth aspect, the present invention provides a pharmaceutical composition comprising a MUC1 inhibitor and an antibiotic.

[0024] In some embodiments, the MUC1 inhibitor is selected from GO-203TFA.

[0025] In some embodiments, the concentration of the MUC1 inhibitor is 2.5–10 μM; for example, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM, 9.5 μM, 10 μM, etc.

[0026] In some embodiments, the antibiotic comprises at least one selected from the group consisting of cephalosporins, quinolones, fluoroquinolones, penicillins, lactamase inhibitors, carbapenems, monobactams, macrolides, lincosamides, glycopeptides, rifampin, oxazolidinones, tetracyclines, aminoglycosides, gramineous plants, and sulfonamides.

[0027] In some embodiments, the antibiotics include quinolone antibiotics, specifically including at least one selected from the group consisting of ciprofloxacin, enoxacin, gatifloxacin, grappafloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, sparfloxacin, trovafloxacin, hydroxybenzoic acid, gemifloxacin, or perfluorofloxacin.

[0028] In some embodiments, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0029] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0030] This invention provides related applications of MUC1 inhibitors, including the use of MUC1 inhibitors in the preparation of formulations that improve the sensitivity to antibiotic treatment, the use of MUC1 inhibitors in the preparation of drugs for the prevention and / or treatment of bacterial infections, and the use of MUC1 inhibitors in combination with antibiotics in the preparation of drugs for the prevention and / or treatment of bacterial infections. This invention also provides pharmaceutical compositions comprising MUC1 and antibiotics.

[0031] The application explores the regulation of MUC1 inhibitor on the adhesion of host cells and bacteria by constructing a physical force biological interaction experiment system between bacteria and cells, and verifies that MUC1 protein plays a role of adhesion target between host cells and bacteria. The application provides a theoretical basis and an original explanation for the above-mentioned application and product of the MUC1 inhibitor.

[0032] The application also verifies the effect of the MUC1 inhibitor on reducing cell-bacteria adhesion and improving the sensitivity of antibiotic treatment of bacterial infection through in vitro experiments. The application of the MUC1 inhibitor can reduce the antibiotic dosage by about 50%, and provides a new strategy for optimizing the antibiotic dosage for bacterial infection.

[0033] The MUC1 inhibitor in the above-mentioned application and pharmaceutical composition has good cell compatibility and no obvious cytotoxicity, and has a good application prospect in the biomedical field. BRIEF DESCRIPTION OF DRAWINGS

[0034] Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The accompanying drawings are merely illustrative and are not considered to be limiting on the present application. In the drawings:

[0035] Figure 1 : Schematic diagram of the principle of MUC1 inhibitor regulating bacterial-host cell interaction, wherein (A) is a schematic diagram of the process of infection between bacteria and host cells through movement, adhesion, internalization and transmission, wherein bacterial adhesion is the primary key step of bacterial infection; (B) is a schematic diagram of the relationship between MUC1 inhibitor, cell-bacteria adhesion and bacterial infection amount and a representative fitting curve of adhesion.

[0036] Figure 2 : Characterization results of MUC1 inhibitor on bacterial-host cell interaction, wherein (A) is a confocal imaging diagram of cells co-incubated with GO-203TFA; (B) is a quantitative result of the expression level of MUC1 protein in cells co-incubated with GO-203TFA; (C) is a cell viability detection result of cells treated with different concentrations of GO-203TFA; (D) is a confocal imaging diagram of cells infected with Staphylococcus aureus after treated with GO-203TFA; (E) is a result of detecting the total amount of bacteria adhering to and infecting cells by detecting the green fluorescence signal of Staphylococcus aureus in cells by flow cytometry after cells are infected with Staphylococcus aureus after treated with GO-203TFA; (F) is a result of counting the Staphylococcus aureus adhering to cells after cells are infected with Staphylococcus aureus after treated with GO-203TFA.

[0037] Figure 3 Effect of MUC1 inhibitor on bacterial-host cell adhesion, wherein (A) is a schematic diagram of single bacterial-cell adhesion force measurement based on fluid force microscopy; (B) is a picture of fluid force microscopy probe; (C) is a comparison of adhesion force of cells without MUC1 inhibitor treatment, cells with MUC1 inhibitor treatment, respectively, to Staphylococcus aureus;

[0038] Figure 4 Effect of MUC1 inhibitor on bacterial antibiotic sensitivity, wherein (A) is survival rate of bacteria treated with different concentrations of GO-203TFA; (B) is a picture of colony counting of Staphylococcus aureus on infected cells after GO-203TFA treatment and addition of different concentrations of ciprofloxacin; (C) is statistical data of colony counting results in (B). DETAILED DESCRIPTION

[0039] Exemplary embodiments of the present disclosure will be described in greater detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it is understood that the present disclosure can be embodied in various forms without being limited by the embodiments set forth herein. Rather, these embodiments are provided so that the present disclosure will be thoroughly and completely understood, and will fully convey the scope of the present disclosure to those skilled in the art.

[0040] In some embodiments of the present disclosure, there is provided use of a MUC1 inhibitor for the manufacture of a medicament for increasing sensitivity to antibiotic treatment.

[0041] In some embodiments, there is provided use of a MUC1 inhibitor for the manufacture of a medicament for preventing and / or treating bacterial infection.

[0042] In some embodiments, there is provided use of a MUC1 inhibitor in combination with an antibiotic for the manufacture of a medicament for preventing and / or treating bacterial infection.

[0043] In some embodiments, there is provided a pharmaceutical composition comprising a MUC1 inhibitor and an antibiotic.

[0044] In some embodiments, the MUC1 inhibitor is selected from GO-203TFA, which is a fully D-amino acid peptide consisting of a poly-R transduction domain linked to a CQCRRKN motif that binds to the cytoplasmic tail of MUC1 and blocks MUC1 homodimerization; and induces reactive oxygen species production and loss of mitochondrial transmembrane potential. GO-203TFA is available through commercial channels.

[0045] According to an embodiment of the present application, the MUC1 inhibitor can significantly reduce the adhesion infection amount of bacteria to cells in the process of cell-bacteria interaction, thereby improving the antibiotic sensitivity of bacterial infection.

[0046] According to an embodiment of the present application, the MUC1 inhibitor can regulate the protein expression of cell surface MUC1, significantly reduce the adhesion between bacteria and cells, and indicate that the MUC1 mucin can be used as a new antibacterial target for blocking the initial adhesion of host-oriented bacteria.

[0047] As shown in Figure 1 , Figure 1 is a schematic diagram of the principle of the MUC1 inhibitor regulating the interaction between bacteria and host cells provided by the embodiment of the present application. The present application clarifies the relationship between the MUC1 protein, the adhesion between bacteria and cells, and bacterial infection by constructing a physical force biological interaction experimental system between bacteria and cells, thereby explaining the principle that the MUC1 inhibitor can be used to improve the antibiotic sensitivity of bacterial infection.

[0048] In an embodiment of the present application, the characterization method of the MUC1 inhibitor regulating cell-bacteria adhesion is as follows: IEC-6 cells are inoculated in a well plate and cultured for 12 hours to be completely adhered, and a culture medium containing the MUC1 inhibitor GO-203TFA is added for 1 hour as an experimental group; before the experiment, a silicon nitride cantilever is selected for testing and calibrated according to the size of the bacteria and the measurement object; during the experiment, a single live bacterium to be captured is searched in the liquid environment of the interaction between the bacteria and the cells; then the fluid force microscope probe capturing the single bacterium is moved close to the cell surface at a preset speed, and when the preset force is reached, it is stopped for a preset time, and then the probe is returned to obtain the adhesion force curve data between the bacteria and the cells; based on the obtained adhesion force curve data between the bacteria and the cells, the adhesion force calculation formula is used to calculate the adhesion force between the bacteria and the cells.

[0049] The inventive concept of the present application includes, as a breakthrough point from the perspective of biomechanics, discovering the MUC1 protein as a new antibacterial target for blocking the initial adhesion of host-oriented bacteria, based on the force-biochemical changes mediated by the MUC1 protein, proposing the effect of the MUC1 inhibitor on improving the antibiotic treatment sensitivity, and screening out GO-203TFA.

[0050] According to an embodiment of the present application, by applying the MUC1 inhibitor, the antibiotic dosage can be reduced by about 50%, thereby providing a new strategy for optimizing the antibiotic dosage for bacterial infection.

[0051] According to the embodiment of the present application, the MUC1 inhibitor is used to improve the antibiotic treatment sensitivity, has good cell compatibility, no obvious cytotoxicity, inhibits bacterial infection by blocking the initial adhesion of bacteria in a host-oriented manner, and has a potential application prospect in the biomedical field.

[0052] In the embodiments of the present application, the conditions of cell culture are 37℃, 5% CO2, unless otherwise specified.

[0053] Embodiment 1

[0054] In this embodiment, the law of MUC1 inhibitor regulating bacterial adhesion and infection of cells is explored. In this embodiment, rat intestinal crypt epithelial cells (IEC-6) are selected as the model host cells, which can form a polarized monolayer, so they are often used to study the infection behavior of bacteria in host cells. In addition, Staphylococcus aureus (S. aureus ATCC29213) is selected as the model pathogenic bacteria, which is the most common pathogenic bacteria in clinical practice. S. aureus is pre-transfected with plasmid pSC19-GFP (erythromycin resistance) to express green fluorescent protein (GFP). The MUC1 inhibitor used in this embodiment is GO-203TFA, which is purchased from MedChemExpress (MCE).

[0055] The specific steps are as follows:

[0056] First, IEC-6 cells are inoculated into a 12-well plate at a concentration of 1×10 6 cells / well to form a monolayer of epithelial cells. Then, GO-203TFA-containing medium is added and incubated for 1 hour, and the concentration of GO-203TFA in the medium is set to the following gradient: control group: 0 (i.e. no GO-203TFA), experimental group: 2.5 μM, 5 μM, 7.5 μM, 10 μM; then washed twice with PBS, and then S. aureus is added for infection for 2 hours to obtain the test sample.

[0057] Detection and analysis:

[0058] (1) Laser confocal imaging and cell viability detection are performed on the cells after 1 hour of GO-203TFA addition and incubation.

[0059] Before imaging, DAPI is used to restain the cell nucleus, and GO-203TFA is subjected to immunofluorescence staining. The confocal imaging results are shown in Figure 2 (A), where Control represents the control group, and 2.5 μM GO-203TFA represents the experimental group treated with 2.5 μM GO-203TFA, wherein blue indicates the cell nucleus, and green indicates the MUC1 protein. Figure 2(B) is the quantification result of MUC1 protein expression level in IEC-6 cells. Figure 2 (C) is the cell viability detection result after treating IEC-6 cells with different concentrations of GO-203 TFA.

[0060] (ii) The following tests were performed on the test sample obtained after 2 hours of S. aureus infection:

[0061] 1. Flow cytometry was used to analyze the total amount of bacteria adhering to infected cells

[0062] The cells were digested with 0.25% (w / v) trypsin and then resuspended in 1 mL of PBS buffer containing 3% FBS. After filtering the cells through a 70 μm filter, the green fluorescent signal of the bacteria in the cells was detected using a flow cytometer to analyze the total amount of bacteria adhering to infected cells. The results are shown in Figure 2 (E).

[0063] 2. Colony counting method was used to detect the total amount of bacteria adhering to cells

[0064] All cells were lysed by supplementing the basal medium with 0.1% BSA and 0.1% Triton X-100 to release the adherent bacteria. The lysate was serially diluted and plated on agar plates, which were incubated overnight at 37°C and subjected to colony counting. The results are shown in Figure 2 (F).

[0065] 3. Laser confocal fluorescence microscopy was used to detect the distribution of bacteria adhering to host cells

[0066] The cells were fixed with 4% paraformaldehyde for 20 minutes and permeabilized with 0.1% (v / v) Triton X-100 for 3-5 minutes. Actin Red TM 555 Ready Probes TM was used to label F-actin cytoskeleton, DAPI was used to counterstain the cell nucleus, and bacteria stably expressed green fluorescent protein. The fluorescence distribution of bacteria adhering to host cells was photographed by Nikon A1 software and analyzed by Image J software. The results are shown in Figure 2 (D), where red indicates F-actin, blue indicates the cell nucleus, and green indicates S. aureus.

[0067] Example 2

[0068] This example further verifies the biomechanical mechanism of MUC1 inhibitors regulating cell-bacteria interaction force. In this example, single bacteria-cell adhesion force was analyzed by fluid force microscopy, as follows:

[0069] IEC-6 cells were first seeded into 12-well plates at a concentration of 1 x 10 6 cells / well and incubated for 12 hours to completely adhere. Then, 5 μM of MUC1 inhibitor GO-203 TFA was added to the culture medium for 1 hour as the experimental group (a control group without GO-203 TFA was also set up). Before the experiment, all the culture medium and buffer used were pre-filtered with a 0.22 μm filter. According to the size of the bacteria and the measured object, a silicon nitride cantilever was selected for testing and calibrated. During the experiment, single live bacteria to be captured were searched in the liquid environment where the bacteria and cells interacted. Negative pressure was applied by using a fluid force microscope pressure controller to make the pinhole of the probe tip suck the bacteria, and a fluid force microscope probe capturing single bacteria was obtained. Then, the fluid force microscope probe capturing single bacteria was moved towards the cell surface at a preset speed, stopped for a preset time when a preset force was reached, and then returned to the probe to obtain the adhesion force curve data between the bacteria and the cells. Based on the obtained adhesion force curve data between the bacteria and the cells, the adhesion force between the bacteria and the cells was calculated by using the adhesion force calculation formula:

[0070] F = USK

[0071] wherein U(v) is the voltage difference of the probe when it is affected by the adhesion force to the initial state, S(m / v) is the sensitivity of the probe, and k (N / m) is the spring constant.

[0072] The statistical chart of the calculation results of the adhesion force is shown in FIG. 16C, wherein Control represents the control group and MUC1 represents the experimental group. It can be seen that the adhesion force between the cells and the bacteria is significantly reduced by adding the MUC1 inhibitor. Figure 3

[0073] Example 3

[0074] In this example, the effect of the MUC1 inhibitor on the sensitivity of bacteria to antibiotic treatment was evaluated, and the specific steps were as follows:

[0075] A cell-bacteria interaction system was provided: IEC-6 cells were seeded into LB broth medium in 12-well plates at a concentration of 1 x 10 5 cells / well and incubated for 12 hours to completely adhere, and then 1.0 x 10 6 ​CFUs of S. aureus. Experimental group, before adding antibiotics, the following pretreatment: to the cell-bacteria interaction system, adding 5 μM of GO-203 TFA; control group, no pretreatment. Then, to the cell-bacteria interaction system, adding different concentrations of antibiotic ciprofloxacin, after 2 hours of incubation, washed twice with PBS, and then all cells were lysed to release the adherent colonization of bacteria by supplementing the basic medium containing 0.1% BSA and 0.1% Triton X-100. The lysed suspension was serially diluted and plated on agar plates, placed in a 37°C incubator overnight and photographed (results as shown in Figure 4 (B), in which GO-203 TFA represents the experimental group, and Control represents the control group) and colony counting (statistical results as shown in Figure 4 (C), in which GO-203 TFA represents the experimental group, and Control represents the control group). Thus, the effect of MUC1 inhibitor GO-203 TFA on the sensitivity of bacterial antibiotic treatment was determined. It can be seen that by adding GO-203 TFA, the sensitivity of antibiotic treatment when the bacteria infect the cells can be significantly improved.

[0076] In addition, the present embodiment also provides Figure 4 (A) to illustrate that GO-203 TFA itself has little killing effect on bacteria. The experimental method is to add gradient concentrations of GO-203 TFA to 1.0 x 10 6 CFUs of S. aureus in MH broth, after 18 hours of incubation, the proportion of surviving bacteria was detected. According to Figure 4 (A), GO-203 TFA has little killing effect on bacteria, and the reason why it can improve the sensitivity of bacterial antibiotic treatment is mainly due to the reduction of bacterial adhesion to cells.

[0077] The above is only the preferred specific embodiment of the present application, but the protection scope of the present application is not limited to this, any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. Use of a MUC1 inhibitor for the preparation of a medicament for the prevention and / or treatment of a bacterial infection, characterized in that, The MUC1 inhibitor is selected from GO-203 TFA; the drug further comprises ciprofloxacin; and the bacteria comprise Staphylococcus aureus.

2. Use according to claim 1, wherein The MUC1 inhibitor is present in the drug at an effective dose concentration.

3. Use of a MUC1 inhibitor in combination with an antibiotic for the manufacture of a medicament for the prevention and / or treatment of a bacterial infection, characterized in that, The MUC1 inhibitor is selected from GO-203 TFA; the antibiotic comprises ciprofloxacin; and the bacteria comprise Staphylococcus aureus.

4. A pharmaceutical composition, characterized by, The drug comprises a MUC1 inhibitor selected from GO-203 TFA and an antibiotic comprising ciprofloxacin.

5. The pharmaceutical composition of claim 4, wherein The concentration of the MUC1 inhibitor is 2.5-10 μM.

6. The pharmaceutical composition of claim 4, wherein The pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

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