Use of derazantinib for the manufacture of a medicament for the treatment of gram-positive bacterial infections

Abstract

The application provides a use of Derazantinib, CAS No. 1234356-69-4, for preparing an anti-Gram-positive bacterial infection drug. The technical scheme of the application discloses a new medical use of Derazantinib. The technical scheme of the application discloses a new medical use of Derazantinib. Derazantinib shows better antibacterial activity on Staphylococcus aureus, can significantly inhibit the formation of a biofilm, and can penetrate a mature biofilm to effectively kill bacteria in the mature biofilm. Derazantinib shows more significant bactericidal activity than vancomycin. In addition, Derazantinib shows strong inhibitory effect on MRSA, MSSA, Enterococcus faecalis and other clinically drug-resistant bacteria.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to the application of Derazantinib in the preparation of a drug for resisting gram-positive bacterial infection. BACKGROUND

[0002] Gram-positive bacteria (including Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis and Enterococcus faecium, etc.) are common pathogenic bacteria of community infection and nosocomial infection. Gram-positive bacteria show inherent resistance and acquired resistance to antibiotics. With the extensive use of antibacterial drugs, in recent years, there have been an increasing number of reports of gram-positive bacteria resistant to top antibiotics (including vancomycin, linezolid, daptomycin, etc.), which has brought severe challenges to clinical anti-infection treatment. For example, the detection rate of methicillin-resistant Staphylococcus aureus (MRSA) is rising, and it is in a high priority position in the list of WHO's "key pathogens" of antibiotic resistance. In addition, another difficult problem in the treatment of common gram-positive bacteria is that a high proportion of gram-positive bacteria are prone to form biofilms, which is one of the important reasons for the poor effect of clinical treatment of gram-positive bacterial infection. The formation of biofilm can reduce the sensitivity to antibacterial drugs and escape the attack and phagocytosis of host immune cells. When the concentration of antibiotics decreases, bacteria will proliferate to refill the biofilm and fall off into the surrounding tissue and blood, leading to recurrence, causing chronic infection and delayed healing. Therefore, developing new drugs for resisting Staphylococcus aureus infection that can both inhibit bacterial growth and inhibit biofilm formation has become one of the current research hotspots. SUMMARY

[0003] In view of the above technical problems, the present application discloses the application of Derazantinib (Azeliragon) in the preparation of a drug for resisting gram-positive bacterial infection. Derazantinib has high activity in resisting the growth of gram-positive bacteria and resisting biofilm.

[0004] To this end, the technical solution adopted by the present application is as follows:

[0005] The application of Derazantinib in the preparation of a drug for resisting gram-positive bacterial infection, wherein the Derazantinib has a CAS number of 1234356-69-4; and the Derazantinib has the effects of inhibiting the growth and biofilm formation of gram-positive bacteria.

[0006] The structure of the Derazantinib is shown in formula (1):

[0007]

[0008] Derazantinib, a potent FGFR1-3 kinase inhibitor, also has activity against colony-stimulating factor-1-receptor (CSF1R) and vascular endothelial growth factor receptor-2 (VEGFR2), suggesting a potential differential role in treating iCCA patients. Derazantinib has shown clinically meaningful efficacy and durable objective responses, which support the therapeutic potential of Derazantinib in previously treated iCCA patients harboring FGFR2 fusions / rearrangements. The clinical safety of Derazantinib is well controlled, and the incidence of drug-related hand-foot syndrome, stomatitis, retinal and nail toxicity is low compared with FGFR inhibitor drugs. These findings support the need for increased molecular analysis of patients with cholangiocarcinoma. However, so far there has been no report on the antibacterial activity of Derazantinib compound.

[0009] Through a large number of experimental studies, it is found that Derazantinib shows antibacterial activity and antibiofilm activity against gram-positive bacteria. Through the influence of Derazantinib on the growth curve of Staphylococcus aureus planktonic bacteria, the bactericidal curve and the proton motive force of the plasma membrane, it is found that Derazantinib has certain antibacterial activity against clinically isolated Staphylococcus aureus, and the MIC is between 6.25 μM and 25 μM. In addition, Derazantinib shows strong inhibitory effect on MRSA, MSSA, Enterococcus faecalis and other clinically resistant bacteria. Bactericidal activity is an important determinant of predicting the results of clinical antibacterial therapy, and Derazantinib shows more significant bactericidal activity than vancomycin, especially for MSSA. In addition, the formation of Staphylococcus aureus biofilm is an important virulence factor leading to chronic infection, and only a few traditional antibiotics are effective against biofilm. Derazantinib has a strong inhibitory effect on the formation of Staphylococcus aureus biofilm, and more importantly, Derazantinib can also penetrate mature biofilm and kill Staphylococcus aureus in mature biofilm. These results show that Derazantinib has potential application value in clinical Staphylococcus aureus anti-infection therapy.

[0010] As a further improvement of the present application, the gram-positive bacteria are at least one of Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Staphylococcus epidermidis or Streptococcus pneumoniae.

[0011] As a further improvement of the present application, the concentration of Derazantinib in the treatment system is not less than 6.25 μM.

[0012] As a further improvement of the present application, the drug is a pharmaceutical composition or preparation.

[0013] The present application also discloses application of Derazantinib in preparation of a coating for inhibiting gram-positive bacteria, the coating being used for the surface of a medical device, the Derazantinib having a CAS number of 1234356-69-4 and a structural formula as shown in formula (1); the Derazantinib has the effect of inhibiting the growth and biofilm formation of gram-positive bacteria.

[0014] As a further improvement of the present application, in the coating, the concentration of the Derazantinib is no less than 6.25 μM.

[0015] The present application also discloses application of Derazantinib in preparation of an antibacterial agent against gram-positive bacteria, the Derazantinib having a CAS number of 1234356-69-4 and a structural formula as shown in formula (1); the Derazantinib has the effect of inhibiting the growth and biofilm formation of gram-positive bacteria.

[0016] Compared with the prior art, the present application has the beneficial effects that:

[0017] The technical scheme of the present application discloses a new medical use of Derazantinib, the Derazantinib shows antibacterial activity and bactericidal activity against gram-positive bacteria such as Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis and Enterococcus faecium, the MIC 50 and the MIC 90 are 12.5 μM and 25 μM, the Derazantinib has a significant inhibitory effect on the growth of Staphylococcus aureus (MRSA) at a concentration of 1 / 2 MIC; in addition, the Derazantinib can inhibit the formation of Staphylococcus aureus biofilm in a dose-dependent manner, and can kill bacteria in mature biofilm, and achieves the bacteriostatic effect by changing the permeability of Staphylococcus aureus cell membrane and inducing peroxide stress. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is a result graph of the growth inhibition effect of Derazantinib on Staphylococcus aureus and Enterococcus faecalis planktonic bacteria according to an embodiment of the present application; wherein A-C are Staphylococcus aureus ATCC29213, SA113 and YUSA139 respectively, and D-F are Enterococcus faecalis 16C166, 16C51 and OG1RF respectively.

[0019] Figure 2Figure is the result graph of the growth inhibition of Staphylococcus aureus and Enterococcus faecalis by Derazantinib according to the embodiment of the present application; wherein A is the 24-hour killing curve of Derazantinib on CHS101 (MSSA); B is the 6-hour killing curve of Derazantinib on CHS101 (MSSA); C is the 6-hour killing curve of Derazantinib on YUSA145 (MRSA); D is the 24-hour killing curve of Derazantinib on 16C51.

[0020] Figure 3 Figure is the experimental result graph of Derazantinib inhibiting the formation of Staphylococcus aureus biofilm according to the embodiment of the present application; wherein A is the OD600 value of MRSA after treatment with different concentrations of Derazantinib, B is the OD600 value of MRSA after treatment with different concentrations of Derazantinib, C is the OD570 value of 1% crystal violet staining of MRSA after treatment with different concentrations of Derazantinib, and D is the OD570 value of 1% crystal violet staining of MSSA after treatment with different concentrations of Derazantinib.

[0021] Figure 4 Figure is the laser confocal graph of 1 / 2×MIC Derazantinib on Staphylococcus aureus according to the embodiment of the present application; wherein a) is the 3D graph of biofilm formed by Staphylococcus aureus YUSA145, b) is the 3D graph of biofilm formed by YUSA145 treated with 1 / 2×MIC Derazantinib, c) is the cross-sectional graph of biofilm formed by Staphylococcus aureus YUSA145, and d) is the cross-sectional graph of biofilm after YUSA145 is treated with 1 / 2×MIC Derazantinib.

[0022] Figure 5 Figure is the result of the PI fluorescence value of Derazantinib slowly rising with the concentration of the drug according to the embodiment of the present application.

[0023] Figure 6 Figure is the result of Derazantinib showing a gradient of hyperpolarization with increasing drug concentration according to the embodiment of the present application.

[0024] Figure 7 Figure is the result of the hemolytic activity analysis of Derazantinib on human red blood cells according to the embodiment of the present application. DETAILED DESCRIPTION

[0025] The preferred embodiments of the present application are further described in detail below.

[0026] Embodiment 1

[0027] The MIC values of Derazantinib on S. aureus, S. epidermidis, E. faecalis and E. faecium were determined by micro-broth dilution method, and the specific steps included:

[0028] The turbidity of the overnight culture was adjusted to 0.5 McFarland (bacterial amount was about 1.0-1.5 x 10 8 The bacterial solution was diluted 1:100 with CAMHB medium and then added to a 96-well plate, 12 wells per row. Ten gradient wells (200, 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, and 0.39 μM) of drugs were set, 200 uL of the above bacterial solution was added to the 11th well as a positive control, and 200 uL of CAMHB medium was added to the 12th well as a negative control. The MIC value determination culture conditions and time of each antibacterial drug were performed according to the CLSI guidelines. After 18 hours of culture at 37°C, the results were observed. The drug concentration well in which the bacterial solution precipitate could not be seen by the naked eye was calculated as the MIC value.

[0029] The MIC value results are shown in Table 1. It can be seen that Derazantinib has better antibacterial activity on various gram-positive bacteria, and the MIC values are mainly distributed between 6.25 μM and 25 μM.

[0030] Table 1 MIC values of Derazantinib on gram-positive bacteria

[0031]

[0032] Note: MRSA: methicillin-resistant S. aureus; MSSA: methicillin-sensitive S. aureus; E. faecalis: Enterococcus faecalis; S. epidermidis: Staphylococcus epidermidis; E. faecium: Enterococcus faecium; n: the number of strains measured.

[0033] Example 2

[0034] Effect of Derazantinib on the growth of S. aureus and E. faecalis.

[0035] To verify whether Derazantinib can inhibit the growth of S. aureus, we used different concentrations of Derazantinib to treat different S. aureus and E. faecalis, and detected the OD value at different time points. The specific steps are as follows: S. aureus or E. faecalis was cultured overnight, and the overnight culture was diluted 1000 times with TSB medium and then added to a 96-well plate. Different concentrations (1 / 16x, 1 / 8x, 1 / 4x, 1 / 2x and 1x) of Derazantinib were added, and then the plate was placed in a full-automatic growth curve analyzer. The absorbance (OD 600 ) value at 600 nm wavelength was measured every 1 hour to detect the content of planktonic bacteria in the culture supernatant, the incubation temperature was 37°C, and the growth curve was drawn. Among them, the treated S. aureus was ATCC29213, SA113 and YUSA139, and the E. faecalis was 16C166, 16C51 and OG1RF.

[0036] The obtained growth curve is shown in Figure 1 It can be seen that at 1 / 2x MIC concentration, Derazantinib can delay the growth of part of S. aureus and E. faecalis planktonic bacteria, but does not affect the maximum growth level. At 1x MIC concentration, Derazantinib shows complete inhibition effect on the growth of all S. aureus and E. faecalis. These results preliminarily show that Derazantinib has the potential to be used as an anti-Gram-positive bacterial (especially for MRSA) infection drug.

[0037] Example 3

[0038] To study the time and dose-dependent effect of Derazantinib on the antibacterial activity of Gram-positive bacteria, and compare the activity with that of antibiotics Daptomycin and Vancomycin, this example uses Derazantinib to perform a killing curve analysis on S. aureus and E. faecalis.

[0039] The YUSA145, CHS101 and 16C51 bacterial solution at the logarithmic phase (OD 600 = 0.5) was diluted 100 times, and then was placed in a shaking incubator at 200 rpm after being treated with (1x, 2x, 4x MIC) Derazantinib, (4 μg / mL) Daptomycin and (16 μg / mL) Vancomycin, respectively. Subsequently, samples were collected at 0, 2, 4, 6 and 24 hours, respectively, and were serially diluted with sterile saline, and then were plated on TSB plates and incubated at 37°C. After 24 hours, the colony count was performed. The colony count was expressed as CFU / mL.

[0040] The killing curve is shown in Figure 2As shown, Derazantinib showed bactericidal effect on methicillin-sensitive Staphylococcus aureus (MSSA) CHS101 at 2xMIC, and on methicillin-resistant Staphylococcus aureus (MRSA) YUSA145 at 4xMIC, with the bacterial count reduced to the lower limit of detection within 24 hours, stronger than 16 μg / mL vancomycin and 4 μg / mL daptomycin. Derazantinib also showed significant bactericidal effect on Enterococcus faecalis clinical strain 16C51 at 4xMIC.

[0041] Example 4

[0042] Effect of Derazantinib on Staphylococcus aureus biofilm formation experiment.

[0043] Biofilm formation is a difficulty in anti-infective treatment, therefore, the effectiveness of a drug must be monitored for its ability to inhibit biofilm. In this embodiment, Staphylococcus aureus strain SA113 (MSSA) was used to study the anti-planktonic and anti-biofilm activity of Derazantinib. The biofilm was semi-quantitatively analyzed by crystal violet staining, repeated 3 times, with 3 replicate wells each time, and the average value was taken as the final test result. The operation steps are briefly described as follows: the overnight bacterial solution was diluted 1000 times in TSBG medium containing 2% glucose in a 96-well plate, 3 replicate wells per group, different sub-inhibitory concentrations of Derazantinib were added, and blank medium without bacterial solution was used as negative control, and solvent DMSO was used as positive control. After incubation at 37°C for 24 hours, the absorbance at 600 nm was measured on a microplate reader. The culture solution was gently aspirated and washed with sterile PBS for 3 times, and dried at room temperature. Methanol fixation for 15 minutes, 1% crystal violet staining for 15 minutes, washing with sterile water for 3 times until the control wells were colorless, and drying at room temperature. Add 200 μL of absolute ethanol per well to dissolve, shake for 1 minute, and measure the absorbance at 570 nm on a microplate reader.

[0044] The results are shown in Figure 3 As shown, when Derazantinib was at 1 / 2xMIC, it showed a more significant inhibitory effect on the biofilm formed by Staphylococcus aureus strain.

[0045] The present embodiment further observes the influence of Derazantinib on mature biofilm by using laser confocal microscope, and the steps are as follows: the biofilm is constructed by using glass dish method, and the change of live bacteria in the biofilm is observed by laser confocal microscope, and the steps are as follows: the Staphylococcus aureus clinical MSSA strain SA113 is inoculated in the culture dish with glass inlay at the bottom, and then is placed in a tin foil box, wrapped and placed at 37°C for 24 hours, washed with sterile 0.9% NaCl for 3 times, then Derazantinib with different concentrations is added into the TSBG medium for continuous culture for 24 hours, washed with sterile PBS, then Live / Dead fluorescent dye is added, and the staining is carried out at room temperature for 30 minutes, and then observed and photographed under laser confocal microscope. Live / Dead fluorescent dye contains two different nucleic acid dyes, which can quickly distinguish live bacteria with complete plasma membrane from dead bacteria with incomplete plasma membrane. SYTO9 green fluorescent nucleic acid dye can stain both live and dead bacteria, and iodinated propyl iodide (PI) will increase the fluorescence intensity and show red color only when it enters the bacteria and binds to nucleic acid through the damaged cell membrane. The results are shown in Figure 4 Fig. 6, SYTO9 and PI can stain live cells and dead cells to green and red respectively. When the concentration of Derazantinib is 1 / 2 x MIC, the proportion of dead cells (red) is significantly increased compared with the control group, indicating that Derazantinib can reduce the number of bacteria in mature biofilm. In summary, Derazantinib has better antibiofilm activity.

[0046] Example 5

[0047] Membrane permeability assay and cytoplasmic membrane potential assay experiment.

[0048] The logarithmic phase Staphylococcus aureus SA113 cells are adjusted to OD 600 = 0.05, and incubated with PI solution. The suspension is treated with different concentrations of Derazantinib (final concentration is 1 x, 2 x and 4 x MIC) and vancomycin (4 μg / mL, 8 μg / mL), and 0.1% DMSO and 0.1% Triton as controls, and the fluorescence intensity (excitation wavelength is 504 nm, and emission wavelength is 523 nm) is continuously monitored. The measurement of membrane potential is referred to the previous literature, and the logarithmic phase Staphylococcus aureus CHS101 cells are adjusted to OD 600= 0.05, the above logarithmic cell suspension and 2 μΜ DiBAC4(3) were suspended for 5 minutes with 0.1% DMSO and 0.1% Triton as controls. After adding Derazantinib at different concentrations, the fluorescence intensity was monitored at an excitation wavelength of 622 nm and an emission wavelength of 670 nm, and the results are shown in Figure 5 and Figure 6 .

[0049] PI dye is a kind of cell nucleus staining reagent that can stain DNA, commonly used for apoptosis detection, which is a bromide ethidium analogue that releases red fluorescence after embedding double-stranded DNA. Although it cannot pass through the membrane of living cells, it can pass through the damaged cell membrane to stain the nucleus, so it is used to detect late apoptosis and necrotic cells (the difference between early apoptosis cells and late apoptosis cells and necrotic cells is the membrane permeability). The membrane permeability of late apoptosis and necrotic cells will increase, which will give the nucleic acid stain (PI) that cannot enter the nucleus of living cells the opportunity to enter the cell and bind to DNA. By Figure 5 It can be seen that the PI fluorescence value of Derazantinib increases with the concentration of the drug, but the increase is not significant.

[0050] DiBAC4(3) is a lipophilic anion fluorescent dye for detecting cell membrane potential. It has no fluorescence by itself, but emits fluorescence after binding to the protein in the cytoplasm. The increase in intracellular fluorescence intensity of DiBAC4(3) entering the cell indicates that the cell is depolarized; on the contrary, the decrease in intracellular fluorescence intensity indicates that the cell is hyperpolarized. As shown in Figure 6 , after being treated with Derazantinib, methicillin-sensitive Staphylococcus aureus (MSSA) CHS101 was hyperpolarized with the increase of drug concentration.

[0051] From the data of this example, it can be seen that Derazantinib achieves bacteriostatic effect by changing the membrane permeability of Staphylococcus aureus and inducing peroxide stress.

[0052] Example 6

[0053] Derazantinib on red blood cell hemolysis test.

[0054] Fresh human red blood cells (RBCs) were washed with PBS and then resuspended in 4% PBS, 100 ml was added to a round bottom 96-well polystyrene microtiter plate. Then, with Triton X-100 as a positive control, the concentration of Derazantinib was diluted by 2 times successively starting from 400 μg / mL. Then, the mixture was incubated at 37℃ with shaking at 60 rpm for 1 hour. After incubation, the 96-well plate was centrifuged at 1000 g for 3 minutes, and 100 μl of supernatant was taken from each well and transferred to a new 96-well polystyrene plate. The absorbance was measured at A450.

[0055] The results are shown in Figure 7 As shown in

[0056] The data processing and image drawing of the above examples were performed by using GraphPad Prism 8.0 software. P<0.05 was considered to have statistical difference.

[0057] The above is a further detailed description of the present application in combination with specific preferred embodiments, and the specific implementation of the present application cannot be limited to these descriptions. For ordinary skilled persons in the art to which the present application belongs, a number of simple deductions or substitutions can be made without departing from the concept of the present application, and all of them should be considered to fall within the protection scope of the present application.

Claims

1. Use of derazantinib for the manufacture of a medicament against a Gram-positive bacterial infection, characterized in that: The Derazantinib has a CAS number of 1234356-69-4; the Derazantinib has an effect of inhibiting growth of gram-positive bacteria; the gram-positive bacteria are at least one of Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, and Staphylococcus epidermidis.

2. Use of Derazantinib for the manufacture of a medicament for the treatment of a Gram-positive bacterial infection according to claim 1, characterized in that: The Derazantinib has a concentration of not less than 6.25 μM in the treatment system.

3. Use of Derazantinib for the manufacture of a medicament for the treatment of a Gram-positive bacterial infection according to claim 1, characterized in that: The medicine is an injection, a tablet, a pill, a capsule, a suspension, a granule, a spray, or an emulsion.

4. Use of derazantinib for the manufacture of a coating for inhibiting gram-positive bacteria, characterized in that: The coating is used on the surface of a medical device; the Derazantinib has a CAS number of 1234356-69-4; the Derazantinib has an effect of inhibiting growth of gram-positive bacteria; the gram-positive bacteria are at least one of Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, and Staphylococcus epidermidis.

5. Use of Derazantinib for the preparation of a coating for the inhibition of Gram-positive bacteria according to claim 4, characterized in that: The Derazantinib has a concentration of not less than 6.25 μM in the coating.

6. Use of derazantinib for the manufacture of an antibacterial agent against Gram-positive bacteria, characterized in that: The Derazantinib has a CAS number of 1234356-69-4; the Derazantinib has an effect of inhibiting growth of gram-positive bacteria; the gram-positive bacteria are at least one of Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, and Staphylococcus epidermidis.

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