Composition for inhibiting methicillin-resistant staphylococcus epidermidis and application thereof

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a composition for inhibiting methicillin-resistant staphylococcus epidermidis and application of the composition. The invention provides a composition. The composition comprises an adriamycin derivative and an acrolein derivative. Furthermore, the compound shows good inhibition or killing activity on drug-resistant staphylococcus. Experiments prove that doxorubicin activates excessive production of endogenous reactive oxygen species (ROS) of cells and cooperates with CA, MCA or OCA drugs targeting reductase or peroxidase to produce an open source interception type ROS storm and inhibit MRSE growth.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a composition for inhibiting methicillin-resistant Staphylococcus epidermidis and an application thereof. Background Art

[0002] The emergence of methicillin-resistant Staphylococcus epidermidis (MRSE) poses an increasing problem to human health. In immunocompromised and hospitalized patients, MRSE infections may lead to bacteremia and sepsis, causing difficult-to-treat infections. MRSE is considered resistant to β-lactam antibiotics, but MRSE strains are also resistant to many commonly used antibiotic groups, such as aminoglycosides, fluoroquinolones, macrolides, tetracyclines, and chloramphenicol. The antibiotic pool for treating MRSE is shrinking, and uncontrolled MRSE infections are becoming a pressing health problem.

[0003] Doxorubicin is a first-line anticancer drug currently used in clinical practice. It can directly insert into the DNA of cancer cells to cause DNA damage, and quickly activate the excessive production of cellular reactive oxygen species (ROS), mediating the initiation of suicide programs such as ferroptosis in cells, causing cell aging, apoptosis, and autophagy. Doxorubicin activates the excessive production of endogenous reactive oxygen species (ROS) in cells, and cooperates with drugs targeting reductases or peroxidases to create an open-source intercepted endogenous ROS storm, which can be designed to mediate ferroptosis of drug-resistant bacteria. However, a technical solution that cooperates with doxorubicin to kill drug-resistant bacteria has not yet been developed. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a composition that can effectively treat bacteremia and sepsis caused by Staphylococcus epidermidis, and overcome the problem of difficulty in treating infections caused by drug-resistant bacteria in clinical practice.

[0005] The first aspect of the present invention aims to provide a composition.

[0006] The second aspect of the present invention aims to provide a medicine.

[0007] The third aspect of the present invention aims to provide an application.

[0008] The fourth aspect of the present invention aims to provide a method.

[0009] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0010] The first aspect of the present invention provides a composition comprising doxorubicin and its derivatives, and acrolein derivatives.

[0011] Preferably, the doxorubicin and its derivatives include at least one of doxorubicin, epirubicin, daunorubicin, pirarubicin, or pharmaceutically acceptable salts thereof.

[0012] Preferably, the acrolein derivative comprises cinnamaldehyde, o-oxymethylcinnamaldehyde, p-oxymethylcinnamaldehyde, hydroxycinnamaldehyde, or pharmaceutically acceptable extracts thereof; the extracts include but are not limited to cinnamon oil (OCA) described in Example 1.

[0013] Preferably, the mass ratio of doxorubicin and its derivatives to acrolein derivatives is 1:(0.1-100).

[0014] Preferably, the mass ratio of the doxorubicin and its derivatives to the acrolein derivatives is 1:(0.5-50).

[0015] Preferably, the mass ratio of the doxorubicin and its derivatives to the acrolein derivatives is 1:(1-25).

[0016] Preferably, the mass ratio of the doxorubicin and its derivatives to the acrolein derivatives is

[0017] The second aspect of the present invention provides a medicine comprising the composition of the first aspect of the present invention.

[0018] Preferably, the medicament comprises pharmaceutically acceptable excipients.

[0019] Preferably, the pharmaceutically acceptable excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and carriers.

[0020] The third aspect of the present invention provides use of the composition of the first aspect of the present invention and / or the medicament of the second aspect of the present invention in the preparation of a product for treating bacterial infection.

[0021] Preferably, the bacteria comprises Staphylococcus.

[0022] Preferably, the Staphylococcus aureus comprises Staphylococcus epidermidis.

[0023] Preferably, the Staphylococcus aureus includes drug-resistant Staphylococcus aureus.

[0024] Preferably, the drug-resistant Staphylococcus includes methicillin-resistant Staphylococcus epidermidis.

[0025] The fourth aspect of the present invention provides a method for inhibiting bacterial proliferation in vitro for non-therapeutic purposes, comprising the step of using the composition of the first aspect of the present invention and / or the drug of the second aspect of the present invention.

[0026] Preferably, the bacteria comprises Staphylococcus.

[0027] Preferably, the Staphylococcus aureus comprises Staphylococcus epidermidis.

[0028] Preferably, the Staphylococcus epidermidis includes methicillin-resistant Staphylococcus epidermidis.

[0029] The beneficial effects of the present invention are:

[0030] The present invention provides a composition, including a doxorubicin derivative and an acrolein derivative. The composition can show good inhibitory or killing activity against drug-resistant staphylococci. Experiments have confirmed that doxorubicin activates the excessive production of endogenous reactive oxygen species (ROS) in cells, synergistically targets reductase or peroxidase with cinnamaldehyde drugs, creates an open-source trapped ROS storm, and inhibits the growth of MRSE. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Cinnamon oil according to Example 1 of the present invention (OCA) The chemical composition of OCA was analyzed by GC-MS (Thermo Scientific Trace DSQ II) using a DB-5H column (30.0 m×0.25 mm, 0.25 μm film thickness) and helium as the carrier gas (0.8 mL / min).

[0032] Figure 2 Different concentrations of CA, MCA, and OCA assisted with doxorubicin hydrochloride to treat MRSE. OD 600 value.

[0033] Figure 3 The results showed that CA synergized with doxorubicin hydrochloride to mediate ROS and inhibit the growth of MRSE.

[0034] Figure 4 Growth curve was used to evaluate the inhibitory effect of CA synergistically with doxorubicin hydrochloride on MRSE.

[0035] Figure 5 This is a picture showing that OCA synergizes with doxorubicin hydrochloride to inhibit the adhesion of MRSE. MRSE dyed with CFDA-SE is green, and the more green it is, the more MRSE bacteria adhere to the HaCat cells. DETAILED DESCRIPTION

[0036] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0037] Example 1 Preparation of cinnamon oil

[0038] Cinnamon oil (OCA) was prepared from dried cinnamon branches according to the 2020 edition of the Chinese Pharmacopoeia, and the components were identified using GS-MS (Thermo Scientific Trace DSQ II). The results are as follows Figure 1 The cinnamon oil prepared in this example is a yellow clear liquid with a cinnamon-specific aroma, 86.179% cinnamaldehyde (CA), and 2.145% o-methoxycinnamaldehyde (MCA), meeting the quality standard of the Chinese Pharmacopoeia (cinnamaldehyde content>75%).

[0039] The standard products CA and MCA used in the present invention were purchased from Guangzhou McLean Reagent Company.

[0040] Example 2 MRSE drug sensitivity test

[0041] According to the Clinical and Laboratory Standards Institute 2017 (CLSI, 2017) guidelines, the MRSE (ATCC35984) resistance test was determined. The results are shown in Table 1. MRSE (ATCC35984) is resistant to amoxicillin, cephalexin, gentamicin sulfate, clarithromycin, and clindamycin hydrochloride. The MRSE strains in the implementation plan are confirmed to be resistant to antibiotics such as aminopenicillins, β-lactams, macrolides, aminoglycosides, and lincomycins. The MIC of vancomycin is 1.2μg / mL, and MRSE (ATCC35984) is sensitive to vancomycin. The MIC of CA inhibiting MRSE is 210μg / mL, the MIC of MCA inhibiting MRSE is 210μg / mL, the MIC of OCA inhibiting MRSE is 260μg / mL, and the MIC of doxorubicin hydrochloride is >100μg / mL. It was confirmed that when used alone, CA, MCA, and OCA had only moderate activity in inhibiting MRSE, and doxorubicin hydrochloride did not show antibiotic-level antibacterial effect against MRSE. C When the concentration of 0.5 μg / mL hydrogen peroxide was 0.5 μg / mL, CA inhibited the significant increase of MRSE's MIC, indicating that the reducing substance Vc has an antagonistic effect on the antibacterial activity of CA. In addition, MRSE cannot survive in 0.5 μg / mL hydrogen peroxide, indicating that oxidative stress can inhibit or kill MRSE.

[0042] Table 1: MRSE Drug Sensitivity Test (ATCC35984)

[0043]

[0044] Example 3: CA, MCA, and OCA Synergistically with Doxorubicin Hydrochloride against MRSE

[0045] According to the guidelines of the Clinical and Laboratory Standards Institute 2017 (CLSI, 2017), the broth dilution checkerboard method was used to determine the efficacy of CA synergistically with doxorubicin hydrochloride against MRSE. The MRSE bacteria in the logarithmic growth phase were resuspended and diluted to 2×10 6 CFU / mL with sterilized CAMHB broth medium. 100 μL of the MRSE bacterial suspension was taken for each well and inoculated on a 96-well plate, and treated with different concentrations of CA and doxorubicin hydrochloride for 24 hours. 0.1% DMSO was used as a vehicle control. After incubation at 37 °C for 24 h, the OD 600 value was measured with an enzyme-linked immunosorbent assay reader. The experimental methods for MCA and OCA synergistically with doxorubicin hydrochloride against MRSE were the same as those for CA. The OD 600 results are as follows Figure 2 shown. The FICI results were calculated according to Formula 1 and are shown in Table 2. The experimental results showed that when the FICI value of CA synergistically with doxorubicin hydrochloride was 0.115, the FICI value of MCA synergistically with doxorubicin hydrochloride was 0.118, and the FICI value of OCA synergistically with doxorubicin hydrochloride was 0.162. The FICI values of CA, MCA, and OCA synergistically with doxorubicin hydrochloride against MRSE were all < 0.5. The results indicated that CA, MCA, and OCA had a significant synergistic effect with doxorubicin hydrochloride against MRSE.

[0046] Formula 1: FICI = MIC A药联用 / MIC A药单用 +MIC B药联用 / MIC B药单用 .

[0047] Table 2: FICI Values of Synergistic Anti-MRSE (MRSE, ATCC35984)

[0048]

[0049] Among them, the MIC of doxorubicin hydrochloride > 100.0 μg / mL was calculated as 100 μg / mL. FICI ≤ 0.5 was determined as a synergistic effect, 0.5 < FICI < 1 was determined as a synergistic enhancement effect, and FICI = 1 was determined as an additive effect. + indicates the use of two substances together.

[0050] Example 5: CA Synergistically with Doxorubicin Hydrochloride to Create an Open-Source Intercepting ROS Storm to Inhibit the Growth of MRSE

[0051] The CM-H2DCFDA staining method described in the literature was used to determine the ROS level in bacteria after MCA treatment (Dongetal., 2015). After treating the bacteria with the specified concentration of the antimicrobial composition, the cell pellets were collected and stained with 2μM CM-H2DCFDA at 37°C for 45min. 0.1% DMSO was used as a control. After washing with HBSS, the cell suspension was analyzed by flow cytometry and quantified by median fluorescence intensity (MFI). The results are shown in Figure 3 As shown. Compared with the control group, when MRSE was treated with 20.0μg / mL CA or 2μg / mL doxorubicin hydrochloride, the intracellular ROS increased. When MRSE was treated with the antibacterial composition of 20.0μg / mL CA and 2μg / mL doxorubicin hydrochloride, the intracellular ROS level of bacteria increased significantly. The experiment confirmed that doxorubicin activated the excessive production of endogenous reactive oxygen species (ROS) in cells, synergistically targeted reductase or peroxidase CA drugs, created an open source intercepted ROS storm, and inhibited the growth of MRSE.

[0052] The growth curve was used to evaluate the inhibitory effect of CA on MRSE in combination with doxorubicin hydrochloride. MRSE in the logarithmic phase was diluted to ~10 6 CFU / mL, treated with (10.0μg / mL CA + 1μg / mL doxorubicin hydrochloride) and (20.0μg / mL CA + 2μg / mL doxorubicin hydrochloride), 0.1% DMSO as control. Incubated at 37°C, the bacterial growth was monitored within 0-24h to evaluate the anti-MRSE activity of the antibacterial composition, and the OD was measured every 2h. 600 The results are as follows Figure 4 As shown. The control group began to increase after 2 hours of incubation, indicating that the drug-loaded control did not affect the proliferation of MRSE (Kerk et al., 2018). In the presence of the antibacterial composition (10.0 μg / mL CA + 1 μg / mL doxorubicin hydrochloride), the OD at 8 hours 600 However, during the whole experiment, in the presence of the antibacterial composition (20.0 μg / mL CA + 2 μg / mL doxorubicin hydrochloride), OD 600 There was no significant increase. This showed that CA synergized with doxorubicin hydrochloride to inhibit MRSE in a dose-dependent manner.

[0053] Example 6 OCA cooperates with doxorubicin hydrochloride to inhibit the adhesion of MRSE

[0054] Dissolve OCA and doxorubicin hydrochloride with 4 times the mass of DMSO, and then dilute with CAMHB broth medium to a certain concentration of antibacterial composition. After sufficient vortexing, take 100 μL in a 15mL centrifuge tube to make the final antibacterial composition concentration (20.0 μg / mL LOCA + 2.0 μg / mL doxorubicin hydrochloride), (40.0 μg / mL LOCA + 4.0 μg / mL doxorubicin hydrochloride), and each concentration is 3 technical parallels. Take MRSE bacteria in the logarithmic growth period, resuspend them with CAMHB broth medium, take an appropriate amount and put it in the above 15mL centrifuge tube, quantify it with CAMHB broth medium to 10mL, and the bacterial concentration is 2.0×10 7 CFU / mL. The same concentration of DMSO was used as a control and cultured in a 37°C water bath constant temperature shaker at 180 rpm for 4 h.

[0055] Bacterial staining: Quantify the bacteria treated with the antibacterial composition and the control group to 5.0 mL (OD600 value 0.2), centrifuge at 4000g for 10 min after bacterial counting, and discard the supernatant. Add PBS liquid to resuspend, wash and centrifuge at 4000g for 10 min, discard the supernatant, and repeat washing twice. Resuspend with 1.0 mL of PBS, add 2 μmol of CFDA-SE for staining, mix gently, and incubate at 37°C for 40 min. Centrifuge at 4000g for 10 min, remove the supernatant, add 1% FBS / HBSS liquid to wash 3 times, quantify to 1 mL, and set aside.

[0056] At the predetermined time, HaCaT cells were plated at 5 × 10 5 The cells were inoculated at a ratio of 1 / 4 cells per well (12-well plate), cultured in DMEM double-antibody culture medium containing 10 v / v% FBS at 37°C and 5% CO2 for 24 hours until fusion, and matured for another 24 hours before inoculating bacteria. Wash twice with 1v / v% FBS / HBSS liquid, discard the supernatant, add a certain amount of CFDA-SE to dye MRSE bacteria, and incubate at 37°C for 2 hours. After incubation, gently wash 3 times with 1v / v% FBS / HBSS solution and store in 1v / v% FBS / HBSS liquid. Subsequently, an integrated cell imager was used to directly observe the adhesion of MRSE to cells. The green fluorescence channel suitable for CFDA-SE dye was selected to record the changes in the number of MRSE adhesion to HaCaT cells after drug treatment of bacteria. The results are shown in the figure. Figure 5 As shown, green represents MRSE bacteria adhered to HaCaT cells. The results show that the antibacterial composition of OCA and doxorubicin hydrochloride significantly inhibits the adhesion of MRSE to Hacat cells and reduces the erosion of MRSE to HaCat cells. It confirms that OCA synergizes with doxorubicin hydrochloride to inhibit the toxic phenotype of MRSE adhesion to host cells.

Claims

1. A composition comprising doxorubicin and its derivatives, and acrolein derivatives.

2. The composition according to claim 1, characterized in that: The doxorubicin and its derivatives include at least one of doxorubicin, epirubicin, daunorubicin, pirarubicin, or their pharmaceutically acceptable salts.

3. The composition according to claim 1, characterized in that: The acrolein derivatives include cinnamaldehyde, o-oxymethylcinnamaldehyde, p-oxymethylcinnamaldehyde, hydroxycinnamaldehyde, or pharmaceutically acceptable extracts thereof.

4. The composition according to claim 1, characterized in that: The mass ratio of the adriamycin and its derivatives to the acrolein derivatives is 1:(0.1-100).

5. A medicine comprising the composition according to any one of claims 1 to 4.

6. The drug according to claim 6 is characterized in that: The drug includes pharmaceutically acceptable excipients.

7. Use of the composition according to any one of claims 1 to 4 and / or the drug according to any one of claims 5 to 6 in the preparation of a product for treating bacterial infection.

8. The use according to claim 7, characterized in that: The bacteria include Staphylococcus; Preferably, the Staphylococcus aureus comprises Staphylococcus epidermidis.

9. The use according to claim 8, characterized in that: The staphylococci include drug-resistant staphylococci; Preferably, the drug-resistant Staphylococcus includes methicillin-resistant Staphylococcus epidermidis.

10. A method for inhibiting bacterial proliferation in vitro for non-therapeutic purposes, comprising the step of using the composition according to any one of claims 1 to 4, and / or the drug according to any one of claims 5 to 6; Preferably, the bacteria include Staphylococcus; Preferably, the Staphylococcus includes Staphylococcus epidermidis; Preferably, the Staphylococcus epidermidis includes methicillin-resistant Staphylococcus epidermidis.

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