Application of ebseoline in preparation of medicine for treating chronic wound infection diseases related to anti-gram positive bacterial biofilm

Abstract

The invention provides an application of ebseoline in preparation of a medicine for treating chronic wound infection diseases related to gram-positive bacterial biofilm resistance, and ebseoline can remove gram-positive bacterial biofilm by down-regulating arginine catabolism, so that ebseoline can be used for treating chronic wound infection diseases related to gram-positive bacterial biofilm resistance. The method has an obvious removal effect on biological membranes formed by MRSA laboratory mode strains, MRSA clinical isolated strains from different sources and gram positive bacteria such as enterococcus faecalis, enterococcus faecium and staphylococcus epidermidis. Therefore, the small molecule compound can be used as a medicine for removing a gram-positive bacterial biofilm and is used for clinical infection treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of compound applications, and particularly to the application of ebselen in the preparation of a drug for treating chronic wound infections associated with Gram-positive bacterial biofilms. Background Art

[0002] A bacterial biofilm is a bacterial community formed on the surface of an attachment by bacteria during their growth process to adapt to the living environment. Corresponding to the growth mode of planktonic cells, its structure includes bacteria and the extracellular matrix secreted by themselves, and it is a special growth mode of bacteria with protection. Bacterial biofilms widely exist in natural and industrial environments, and are easily formed on various surfaces such as indwelling medical devices and human tissues, which can cause chronic infections, medical implant infections, and recurrent infections.

[0003] Research has found that bacterial biofilms are closely related to various skin diseases such as chronic wounds, acne, and atopic dermatitis. When bacteria attach to a wound and reproduce, they are embedded in the extracellular matrix and form a biofilm that harms wound healing, and clinical manifestations such as redness, swelling, heat, and pain can be observed. However, due to the protection of the bacterial biofilm, various treatment measures, including antibiotic treatment, often fail to work effectively, resulting in the prolongation of wound healing.

[0004] In addition, biofilms formed on medical implants (including prosthetic joints, catheters, pacemakers, and dental implants) are considered the main cause of implant-related infections. Biofilm bacteria show higher tolerance to antibiotics (including antibiotics with a "persistent" phenotype), which makes these infections difficult to treat with systemic antibiotics alone.

[0005] Ebselen (ebselen), also known as ebselen, CAS No.: 60940-34-3, and its chemical structure is as Figure 1 shown. Ebselen is a newly chemically synthesized non-steroidal selenium-containing drug. However, there is no relevant report on whether ebselen can be used to remove Gram-positive bacterial biofilms.

[0006] Currently, microbial biofilm-related infections are a uniquely challenging problem that hinders the healing of 60% of chronic wounds and 6% of acute wounds, which may lead to serious socio-economic pressures. Currently, the clinical treatment of biofilm infections still heavily relies on antibiotics. However, the complexity of biofilms and their increased resistance to traditional antibiotics make the removal of biofilms full of challenges, and there is an urgent need for powerful new antibacterial agents. Summary of the Invention

[0007] To solve the above problems, the present invention aims to provide the use of ebselen in the preparation of drugs for treating chronic wound infections related to anti-Gram-positive bacterial biofilms, which can effectively remove biofilms of Gram-positive bacteria represented by Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium, etc., and significantly reduce the morbidity and mortality caused by Gram-positive bacterial infections. The technical solution of the present invention is as follows: The use of ebselen in the preparation of drugs for treating chronic wound infections related to anti-Gram-positive bacterial biofilms. Preferably, the Gram-positive bacteria include Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis or Enterococcus faecium or others.

[0008] More preferably, the chronic wound biofilm diseases caused by Staphylococcus aureus infection include: skin and soft tissue infections, abscesses or boils, implant-related osteomyelitis, cardiac device-related endocarditis, ventilator-associated pneumonia, catheter-related urinary tract infections and other indwelling medical device infections; the chronic wound biofilm diseases caused by Enterococcus faecalis infection include: skin and soft tissue infections, urinary tract infections, dental caries, endocarditis, bacteremia or others; The chronic wound biofilm diseases caused by Enterococcus faecium infection include: skin and soft tissue infections, urinary tract infections, peritonitis, endocarditis, bacteremia or others.

[0009] More preferably, the indwelling medical device infections include biofilm infections on the surface of prosthetic joints, catheters, pacemakers or dental implants.

[0010] More preferably, the application includes at least one of inhibiting biofilm formation and removing biofilms.

[0011] More preferably, the concentration of ebselen in the drug is 1-100 μg / L. More preferably, the ebselen includes ebselen and / or its pharmaceutically acceptable salts.

[0012] More preferably, the drug includes ebselen and at least one pharmaceutically acceptable excipient.

[0013] More preferably, the dosage form of the drug is an antibacterial dressing dosage form or an injection dosage form. The dosage form of the drug is an antibacterial dressing, an injection, a topical gel or a spray.

[0014] More preferably, the antibacterial dressing contains ebselen-loaded hydrogel, nanofiber membrane or medical gauze; The injection is an intravenous injection or a local lesion injection preparation.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention explores a new disease treatment field for the existing drug ebselen. Given that the safety and effectiveness of ebselen have been verified to a certain extent, we conduct in-depth research on this basis in order to discover its broader application potential. The present invention preliminarily evaluates the activity effect of ebselen in the biofilm clearance of Gram-positive bacteria. The highly pathogenic MRSA strain USA300 JE2 is selected as the representative strain for research. The results show that ebselen has the activity to clear the MRSA biofilm. In the urethral catheterization model and intramedullary implant model of C57BL / 6 male mice, compared with the control group, ebselen treatment can significantly clear the MRSA biofilm formed on the implant. Intraperitoneal injection of ebselen can clear 90% of the biofilm on the catheter surface and significantly improve the survival rate. In addition, ebselen has an obvious clearance effect on the biofilms formed by MRSA clinical strains from different sources and Gram-positive bacteria such as Enterococcus faecalis, Enterococcus faecium, and Staphylococcus epidermidis. Further research confirms that ebselen can down-regulate the expression level of arginine catabolism in MRSA clinical strains from different sources and Gram-positive bacteria such as Enterococcus faecalis, Enterococcus faecium, and Staphylococcus epidermidis, resulting in the accumulation of arginine in bacterial cells. Arginine is an important multifunctional amino acid. In addition to being a component of proteins, it can also serve as a carbon / nitrogen source and an ATP source to promote metabolism under specific circumstances. MRSA maintains a low level of arginine by highly expressing the arginine catabolic mobile element gene arc cluster, which is a key factor for the formation and maintenance of its biofilm. And our results show that ebselen treatment can significantly down-regulate MRSA arginine catabolism, thereby clearing the biofilm.

[0016] Ebselen shows excellent clearance effect on Gram-positive bacterial biofilms by down-regulating the expression level of arginine catabolism, providing new ideas and effective countermeasures for the development of new antibacterial drugs for biofilm clearance. Brief Description of the Drawings

[0017] Figure 1 is the molecular formula of ebselen; Figure 2 is the research result diagram of ebselen clearing MRSA biofilm on mouse urinary catheters; Figure 3 is the research result diagram of ebselen clearing the biofilm of MRSA JE2 strain at different gradient concentrations; Figure 4 is the research result diagram of ebselen clearing the biofilm of MRSA clinical isolated strains; Figure 5 is the research result diagram of ebselen clearing the biofilms of other Gram-positive bacteria; Figure 6 Figure showing the results of ebselen-induced arginine accumulation in MRSA JE2 strain Figure 7 Figure showing the results of ebselen-induced arginine accumulation in clinical isolates of MRSA Figure 8 Figure showing the results of ebselen-induced arginine accumulation in other Gram-positive bacteria Detailed implementation manners

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0019] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the range.

[0020] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. In case of conflict with any incorporated literature, the content of this specification shall prevail.

[0021] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and embodiments of the present invention are only exemplary.

[0022] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0023] The Gram-positive bacterial strains used in the following examples include: Methicillin-resistant Staphylococcus aureus MRSA JE2 strain and Staphylococcus epidermidis Staphylococcus epidermidisATCC 12228 is preserved in our laboratory, Enterococcus faecalis Enterococcus faecalis ATCC 29212 and Enterococcus faecium Enterococcus faecalis ATCC 700221 was purchased from Beijing Baocang Biotechnology Co., Ltd. Clinical methicillin-resistant Staphylococcus aureus strains (MRSA 131, MRSA996, MRSA 602 and MRSA738) were obtained from the Second People's Hospital of Yichang (Yichang, China).

[0024] The above strains are preserved in the research group of Zou Lili in the School of Basic Medicine, China Three Gorges University. Among them, the MRSA USA300 JE2 strain was used in Examples 1-2. Example 1 Ebselen can remove MRSA biofilm on urinary catheters of mice Twelve female C57BL / 6 mice were pre-fed for three days and divided into 2 groups in separate cages: Control group (6 mice), 15 mg / Kg ebselen group (6 mice). They were fasted for 12 h before modeling. Infection was carried out on Day 0: After anesthetizing the mice with 400 mg / mL chloral hydrate, the mice were placed on their backs. A 1 mL syringe was used to aspirate 50 μ μL of MRSA JE2 bacterial solution, connected to a 32-gauge drop glue needle, and the needle was connected to a PE10 catheter. After exhausting the air bubbles, the perineal area around the urethra was cleaned with iodophor. Then, the end of the stylet with a 23 mm long tube was pushed into the urethral orifice until the front end entered the bladder. The catheter was pushed forward until the shorter part was pushed away from the handle, so that the 2 mm short catheter was freely placed in the bladder cavity. The long catheter was gently pulled out, and the urethral orifice was disinfected again. Administration was carried out on Day 1 and Day 3 after modeling: The Control group was intraperitoneally injected with the ebselen solvent DMSO; the 15 mg / Kg ebselen group was intraperitoneally injected with 15 mg / kg ebselen. Blood was taken from the eyeballs on Day 2 and Day 4, and the mice were sacrificed by cervical dislocation. The liver, spleen, kidney, bladder and catheter implants were aseptically removed. A part of each organ was taken with a scalpel and placed in an embedding box, soaked in tissue fixative for subsequent histopathological examination, and the other part was placed in an Ep tube for detecting the bacterial load in the tissue. The catheter implants were placed in the electron microscope fixative and sent for examination by scanning electron microscope to observe the formation of catheter biofilm.

[0025] The results are as Figure 2 shown. 2(A) is the survival rate curve for statistically analyzing the survival rate of experimental mice, indicating that ebselen can effectively improve the survival rate of MRSA-infected mice. 2(B) is the SEM scanning electron micrograph taken after the catheter formed a film, indicating that ebselen has an excellent effect on removing the MRSA biofilm on the urinary catheters in the bodies of mice. Example 2 Ebselen can remove the biofilm of MRSA JE2 strain on catheters in vitro Cultivate the MRSA JE2 strain until the OD of the bacterial liquid reaches the logarithmic growth phase 600 nm Adjust it to 0.4, and group it into vancomycin / ebselen at concentrations of 10 - 100 μg / mL. After adding drugs with concentration gradients, inoculate the bacterial liquid containing different concentrations of drugs into a 96-well plate, with 5 replicates in each group and 200 μL in each well. Place the 96-well plate in a constant temperature incubator at 37°C. After culturing for 16 h, add crystal violet for staining, and measure the OD on an enzyme-linked immunosorbent assay (ELISA) reader 595 nm . Use the time point as the abscissa and the absorbance value as the ordinate to plot the growth curve. The results are as Figure 3 shown. It is found that 50 μg / mL of ebselen can effectively remove the MRSA biofilm, and the effect of ebselen is significantly better than that of vancomycin

[0026] Example 3: Ebselen can remove the MRSA clinical strain biofilm on an in vitro catheter Cultivate the clinically isolated MRSA strains (MRSA 131, MRSA996, MRSA 602, and MRSA738) until the OD of the bacterial liquid reaches the logarithmic growth phase 600 nm Adjust it to 0.4, and group it into DMSO / 40 μ μg / mL of ebselen, inoculate it into a 12-well plate, with 3 replicates in each group. Place an 8-mm PE10 catheter in each well and culture it at 37°C for 12 - 16 h to form a biofilm on the catheter surface. Use sterile forceps to remove the catheter, rinse it 3 times in PBS to wash away the planktonic bacteria on the surface, cut a part and place it in 1 mL of electron microscope fixative for submission for scanning electron microscopy (SEM); accurately measure the length of another part and then put it into 1 mL of PBS

[0027] Take out the catheter in the electron microscope fixative and rinse it 2 times with PBS (10 min each time). Dehydration: sequentially place it in 50%, 70%, and 90% ethanol for dehydration for 15 min, and then place it in absolute ethanol for dehydration 2 times (15 min each time); replacement: replace it once with absolute ethanol: tert-butanol (1:1) and pure tert-butanol (15 min each time); drying: dry the sample through a critical point dryer Quorum K850 for 4 h; sputter coating: stick the sample on the SEM sample stage and coat a metal film on the sample surface with an ion sputtering instrument HITACHI MC1000; finally, observe and take pictures with a scanning electron microscope HITACHI Regulus 8100

[0028] The results are as Figure 4 shown. The first row is the MIC result diagram, and the second row is the laser confocal microscope scanning result diagram. The results all show that ebselen can effectively remove the biofilms of clinically isolated MRSA strains from different sources

[0029] Example 4: Ebselen can remove the biofilms of other Gram-positive bacteria on an in vitro catheter Enterococcus faecalis Enterococcus faecalis ATCC 29212, Enterococcus faecium Enterococcus faecalis ATCC 700221 and Staphylococcus epidermidis Staphylococcus epidermidis were cultured to the logarithmic growth phase until the OD of the bacterial liquid 600 nm was adjusted to 0.4, grouped into DMSO / ebselen, inoculated on 8-well chamber coverslips, incubated at 37 °C for 18 - 24 h, then taken out, washed with PBS to remove planktonic bacteria, the biofilm at the bottom of the wells was stained with a bacterial viability / toxicity detection kit, and then the clearance of ebselen on the biofilm of MRSA clinical strains and other strains was measured using a laser scanning confocal microscope. The results are as Figure 5 shown. Ebselen can effectively remove the biofilms of Enterococcus faecalis, Enterococcus faecium, and Staphylococcus epidermidis.

[0030] Example 5 Ebselen causes arginine accumulation in MRSA JE2 strain The MRSA JE2 strain was cultured to the logarithmic growth phase, and the OD of the bacterial liquid was adjusted with a liquid medium 600 nm to 0.4, mixed with DMSO or ebselen, incubated in a 37 °C constant temperature incubator respectively, centrifuged at 4 °C, 8000 rpm for 5 min, the bacterial cell precipitate was collected, the supernatant was removed, the bacterial cells were washed repeatedly with pre-cooled PBS 3 times, the bacterial cell precipitate was resuspended with double-distilled water and sonicated on ice for 15 min to release intracellular amino acids and their derivatives. The protein concentration was measured by the BCA method, and the total protein concentration of the sample was obtained through the regression equation and the standard curve drawn.

[0031] LC-MS analysis was performed using an Agilent ultra-high performance liquid chromatography triple quadrupole mass spectrometer 1290 Infinity II + 6470B. Finally, the amino acid and polyamine content (ng / mg protein) per mg of protein in the bacterial lysate was calculated based on the total protein of the sample.

[0032] The results are as Figure 6 shown. After treatment with ebselen, the arginine content of MRSA further accumulated and increased with the extension of the treatment time from 1 h to 2 h. At the same time, it was found that the amounts of other related metabolites related to arginine metabolism also changed. For example, the contents of catabolic products such as ornithine and proline decreased, indicating that ebselen treatment inhibited the catabolism of arginine in MRSA, resulting in an increase in the intracellular arginine content. It is known that arginine is an important multifunctional amino acid. In addition to being a component of proteins, it can also promote metabolism as a carbon / nitrogen source and an ATP source under specific circumstances. MRSA promotes metabolism by highly expressing the arginine catabolic mobile element gene arcClusters, which maintain a low level of arginine, are key factors in the formation and maintenance of biofilms. Our results show that ebselen treatment can downregulate arginine catabolism in MRSA, thereby clearing biofilms.

[0033] Example 6 Ebselen causes arginine accumulation in clinical isolates of MRSA Clinical isolates of MRSA (MRSA 131, MRSA996, MRSA 602, and MRSA738) were cultured to the logarithmic growth phase, and the bacterial suspension was adjusted to OD 600 nm to 0.4 with a liquid medium. After adding DMSO or ebselen and mixing, the mixture was incubated in a 37 °C constant temperature incubator. Then, it was centrifuged at 8000 rpm for 5 min at 4 °C, and the cell pellet was collected. The supernatant was removed, and the cell pellet was washed repeatedly with pre-cooled PBS three times. The cell pellet was resuspended in double-distilled water and sonicated on ice for 15 min to release intracellular amino acids and their derivatives. The protein concentration was determined by the BCA method, and the total protein concentration of the sample was obtained by the regression equation and the standard curve drawn.

[0034] LC-MS analysis was performed using an Agilent ultra-high performance liquid chromatography triple quadrupole mass spectrometer 1290 Infinity II + 6470B. Finally, the amino acid and polyamine content (ng / mg protein) per mg of protein in the bacterial lysate was calculated based on the total protein of the sample.

[0035] The results are as Figure 7 shown. After treatment with ebselen, arginine also accumulated in the clinical strains of MRSA, while the contents of ornithine and proline decreased, indicating that ebselen treatment can also inhibit the arginine catabolism of clinical strains of MRSA, resulting in an increase in the intracellular arginine content. After treatment with ebselen in clinical strains of MRSA, we observed a downregulation of the arginine catabolism level in MRSA. It is confirmed that ebselen clears biofilms by inhibiting arginine catabolism, which is not only applicable to the laboratory model strain MRSA JE2 but also widely applicable to clinical isolates of MRSA.

[0036] Example 7 Ebselen causes arginine accumulation in other Gram-positive bacteria Enterococcus faecalis Enterococcus faecalis ATCC 29212, Enterococcus faecium Enterococcus faecalis ATCC 700221, and Staphylococcus epidermidis Staphylococcus epidermidis ATCC 12228 were cultured to the logarithmic growth phase, and the bacterial suspension was adjusted to OD 600 nmTo 0.4, after adding DMSO or ebselen and mixing evenly, place it in a 37 °C constant temperature incubator for incubation respectively. Then, centrifuge at 4 °C and 8000 rpm for 5 min, collect the bacterial cell precipitate, discard the supernatant, wash the bacterial cells repeatedly with pre-cooled PBS for 3 times, resuspend the bacterial cell precipitate with double-distilled water and sonicate on ice for 15 min to release intracellular amino acids and their derivatives. The protein concentration was determined by the BCA method, and the total protein concentration of the sample was obtained through the regression equation and the plotted standard curve.

[0037] LC-MS analysis was performed using an Agilent ultra-high performance liquid chromatography triple quadrupole mass spectrometer 1290 Infinity II + 6470B. Finally, the amino acid and polyamine content (ng / mg protein) per mg of protein in the bacterial lysate was calculated based on the total protein of the sample.

[0038] The results are as Figure 8 shown. After treatment with ebselen, arginine also accumulated and increased in Staphylococcus epidermidis, Enterococcus faecalis, and Enterococcus faecium, while the contents of ornithine and proline decreased, indicating that ebselen treatment can also inhibit the catabolism of arginine in other Gram-positive bacteria such as Staphylococcus epidermidis, Enterococcus faecalis, and Enterococcus faecium, resulting in an increase in the intracellular arginine content. After treatment with ebselen, we observed a down-regulation of the arginine catabolism level in MRSA in Staphylococcus epidermidis, Enterococcus faecalis, and Enterococcus faecium. It was confirmed that ebselen clears biofilms by inhibiting arginine catabolism, which is not only applicable to MRSA strains but also widely applicable to other Gram-positive strains.

[0039] The technical solution of the present invention is explained by the above embodiments, but the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above specific embodiments to be implemented. Any improvements made by those skilled in the art based on the present invention, or equivalent substitutions of the materials selected for the present invention, etc., all fall within the protection scope of the patent.

Claims

1. Application of ebselen in the preparation of drugs for treating chronic wound infection diseases associated with Gram-positive bacterial biofilm.

2. The use of ebselen according to claim 1 in the preparation of a drug for treating chronic wound infection diseases associated with Gram-positive bacterial biofilm, characterized in that: Gram-positive bacteria include Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis or Enterococcus faecium or others.

3. The use of ebselen according to claim 2 in the preparation of a drug for treating chronic wound infection diseases associated with Gram-positive bacterial biofilm, characterized in that: The chronic wound biofilm diseases caused by Staphylococcus aureus and Staphylococcus epidermidis infection include: skin and soft tissue infection, abscess or furuncle, implant-related osteomyelitis, cardiac device-related endocarditis, ventilator-associated pneumonia, catheter-related urinary tract infection and other indwelling medical device infections; the chronic wound biofilm diseases caused by Enterococcus faecalis infection include: skin and soft tissue infection, urinary tract infection, dental caries, endocarditis, bacteremia or others; The chronic wound biofilm diseases caused by Enterococcus faecium infection include: skin and soft tissue infection, urinary tract infection, peritonitis, endocarditis, bacteremia or others.

4. The use of ebselen according to claim 3 in the preparation of a drug for treating chronic wound infection diseases associated with Gram-positive bacterial biofilms, characterized in that: The indwelling medical device infection includes biofilm infection on the surface of prosthetic joints, catheters, pacemakers or dental implants.

5. The use of ebselen according to claim 1 in the preparation of a drug for treating chronic wound infection diseases associated with Gram-positive bacterial biofilm, characterized in that: The application includes at least one of inhibiting biofilm formation and removing biofilm.

6. The use of ebselen in the preparation of a drug for treating chronic wound infection diseases associated with Gram-positive bacterial biofilm according to claim 1, characterized in that: The concentration of ebselen in the drug is 1-100 μg / L.

7. The use according to any one of claims 2 to 6, characterized in that: The ebselen includes ebselen and / or a pharmaceutically acceptable salt thereof.

8. The use according to any one of claims 2 to 6, characterized in that: The drug comprises ebselen and at least one pharmaceutically acceptable excipient.

9. The use according to any one of claims 2 to 6, characterized in that: The dosage form of the drug is an antibacterial dressing dosage form or an injection dosage form. The dosage form of the drug is an antibacterial dressing, injection, local external gel or spray.

10. The use according to claim 9, characterized in that: The antibacterial dressing comprises a hydrogel, a nanofiber membrane or a medical gauze loaded with ebselen; The injection is a preparation for intravenous injection or local intralesional injection.

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