阅读说明：本技术 氨磷汀作为ndm-1抑制剂或抗生素保护剂的新用途 (New application of amifostine as NDM-1 inhibitor or antibiotic protective agent ) 是由 张秀英 王春花 李晓婷 于 2021-09-12 设计创作，主要内容包括：本发明公开了氨磷汀作为NDM-1抑制剂或抗生素保护剂的新用途,属于氨磷汀新医药用途领域。本发明通过NDM-1酶抑制试验、微量稀释棋盘法以及时间-杀菌曲线试验等发现氨磷汀能够显著抑制NDM-1的活性并恢复美罗培南对产NDM-1大肠杆菌的抗菌活性。由此,本发明确定氨磷汀可作为NDM-1抑制剂,将其与β-内酰胺类抗生素联合应用能够减少甚至消除NDM-1对β-内酰胺类抗生素的水解、恢复耐药菌对β-内酰胺类抗生素的敏感性。(The invention discloses a new application of amifostine as an NDM-1 inhibitor or an antibiotic protective agent, belonging to the field of new medical application of amifostine. According to the invention, through NDM-1 enzyme inhibition test, a micro dilution chessboard method, a time-sterilization curve test and the like, the amifostine is found to be capable of obviously inhibiting the activity of NDM-1 and recovering the antibacterial activity of meropenem on NDM-1-producing escherichia coli. Therefore, the invention determines that the amifostine can be used as an NDM-1 inhibitor, and the combined application of the amifostine and the beta-lactam antibiotics can reduce or even eliminate the hydrolysis of NDM-1 to the beta-lactam antibiotics and restore the sensitivity of drug-resistant bacteria to the beta-lactam antibiotics.)

1. The application of amifostine in preparing a new Delhi metallo-beta-lactamase-1 inhibitor.

2. The application of amifostine in preparing antibiotic protective agent.

3. Use according to claim 2, characterized in that: the antibiotic is a beta-lactam antibiotic.

4. Use according to claim 3, characterized in that: the beta-lactam antibiotics comprise carbapenems, cephalosporins or penicillins.

5. Use according to claim 1 or 2, characterized in that: the amifostine comprises a prototype, a pharmaceutically acceptable salt thereof or an amifostine-containing formulation.

6. A pharmaceutical composition for inhibiting pathogenic bacteria, comprising: a prophylactically or therapeutically effective amount of an antibiotic, an antibiotic protectant, and a pharmaceutically acceptable adjuvant or carrier; wherein the antibiotic protective agent is amifostine.

7. The pharmaceutical composition according to claim 6, wherein: the pathogenic bacteria are bacteria.

8. The pharmaceutical composition according to claim 7, wherein: the bacteria are gram-negative or positive pathogenic bacteria, preferably NDM-1 positive bacteria.

9. The pharmaceutical composition according to claim 6, wherein: the beta-lactam antibiotics comprise carbapenems, cephalosporins or penicillins.

10. The pharmaceutical composition according to claim 6 or 9, characterized in that: the beta-lactam antibiotic is meropenem.

Technical Field

The invention relates to a new pharmacological application of amifostine, in particular to a new pharmacological application of the amifostine as an NDM-1 inhibitor or an antibiotic protective agent, belonging to the field of new pharmacological activity of the amifostine.

Background

The beta-lactam antibiotics have strong bactericidal activity, low toxicity and wide adaptation diseases, and are important medicaments for treating infectious diseases caused by sensitive bacteria at present. Beta-lactam antibiotics include carbapenems, cephalosporins, penicillins, and the like, all of which structurally have a beta-lactam ring that exerts antibacterial activity. With the widespread use of beta-lactam antibiotics, more and more bacteria are developing beta-lactamase mediated resistance.

The production of beta-lactamase, which catalyzes the cleavage of the C-N bond at the beta-lactam ring in beta-lactam antibiotics to open the ring, leading to inactivation of the antibiotic, is one of the important mechanisms responsible for bacterial resistance. Based on amino acid sequence homology, beta-lactamase can be divided into serine-beta-lactamase and metallo-beta-lactamase, and the serine-beta-lactamase plays a catalytic role depending on serine of an active center and can be inhibited by clinically used antibiotics such as clavulanic acid, sulbactam, tazobactam and the like. The metal-beta-lactamase plays a catalytic role by depending on zinc ions of an active center, can hydrolyze all beta-lactam antibiotics including carbapenems, widely exists in various gram-negative and positive drug-resistant pathogenic bacteria, and has no effective inhibitor clinically.

In 8 months 2010, a new Delhi metallo-beta-lactamase-1 (NDM-1) which hydrolyzes carbapenem drugs is reported in a J.Lancet.Scent, and is called as 'super bacteria' because NDM-1-producing bacteria have wide drug resistance to cause difficult infection treatment. The blaNDM-1 gene is positioned on a plasmid, can be independently replicated outside chromosomes and can be horizontally transferred among different strains, so that strains which are originally sensitive to antibiotics can obtain drug resistance. NDM-1 is metal-beta-lactamase which is discovered in recent years and has the widest influence range and the most serious harm degree, the bacteria expressing NDM-1 almost show high drug resistance to all antibiotics, only tigecycline and polymyxin have certain inhibition effect on the antibiotics, and the continuously evolved mutant strains make clinical treatment harder. NDM-1 can hydrolyze beta-lactam antibiotics commonly used in clinic, and the inhibitor can inhibit the activity of NDM-1 enzyme, so that the beta-lactam antibiotics are protected, and the antibacterial effect of the beta-lactam antibiotics is recovered, therefore, the search for the inhibitor of NDM-1 is the most urgent requirement for inhibiting infection caused by 'super bacteria'.

Amifostine is a chemotherapy cell protective agent and a radiotherapy protective agent, is mainly used for the adjuvant therapy of various cancers clinically, can selectively protect normal tissue cells when killing cancer cells, can obviously reduce the toxicity of kidney, bone marrow, heart, ear and nervous system caused by chemotherapy drugs, has certain effect on preventing toxic and side effects of hematopoietic system, immune injury and the like caused by radiotherapy, and is also used for reducing infection related to neutropenia. Until now, no report of the application of amifostine to NDM-1 inhibitors is found at home and abroad.

Disclosure of Invention

The invention mainly aims to provide a new application of amifostine as an NDM-1 inhibitor or an antibiotic protective agent.

In order to achieve the above object, the technical solution adopted by the present invention comprises:

on the one hand, the invention discloses a new pharmacological application of amifostine as an NDM-1 inhibitor, namely, the hydrolysis of NDM-1 on beta-lactam antibiotics is inhibited, and the antibacterial activity of the beta-lactam antibiotics on NDM-1-carrying positive bacteria is recovered. Accordingly, amifostine can be used as an antibiotic protectant, especially as a protectant of beta-lactam antibiotics.

The invention provides a pharmaceutical composition for inhibiting pathogenic bacteria, which comprises an effective amount of antibiotics, an antibiotic protective agent and a pharmaceutically acceptable carrier or auxiliary material, wherein the antibiotics are preferably beta-lactam antibiotics; the antibiotic protective agent is amifostine.

The pharmaceutical composition for inhibiting pathogenic bacteria is prepared into clinically common preparations according to the conventional preparation method in the field, such as powder, granules, tablets, capsules, injections and the like, and is introduced into muscles, endothelium, subcutaneous tissues, veins and mucosal tissues by injection, oral administration, nasal drip, eye drop, physical or chemical mediated methods, or is mixed or coated by other substances and then is introduced into the body.

The carrier or the auxiliary material refers to a carrier or an auxiliary material which is conventional in the pharmaceutical field, such as: diluents, disintegrants, lubricants, excipients, binders, glidants, fillers, surfactants, and the like; in addition, other adjuvants such as flavoring agents and sweeteners may also be added to the pharmaceutical composition.

The diluent may be one or more ingredients that increase the weight and volume of the tablet; common diluents include lactose, starch, pregelatinized starch, microcrystalline cellulose, sorbitol, mannitol, and inorganic calcium salts. The most common of them are lactose, starch, microcrystalline cellulose.

The disintegrant can be one or more of crosslinked polyvinylpyrrolidone (with a total weight ratio of 2-6%), crosslinked sodium carboxymethylcellulose (with a total weight ratio of 2-6%), alginic acid (with a total weight ratio of 2-5%), and microcrystalline cellulose (with a total weight ratio of 5-15%). Wherein the preferred ratio is crosslinked polyvinylpyrrolidone (2-7% by weight) and crosslinked sodium carboxymethylcellulose (2-6% by weight). Most preferably crosslinked polyvinylpyrrolidone (in a ratio of 2-6% by weight relative to the total weight).

The lubricant comprises one or a mixture of stearic acid, sodium stearate, magnesium stearate, calcium stearate, polyethylene glycol, talcum powder and hydrogenated vegetable oil. Magnesium stearate is most preferred. The amount of the lubricant is in the range of 0.10 to 1% (by total weight), and is generally 0.25 to 0.75%, and preferably 0.5 to 0.7%.

The binder may be one or more ingredients that facilitate granulation. It may be starch slurry (10-30% by weight of the total binder), hydroxypropyl methylcellulose (2-5% by weight of the total binder), polyvinylpyrrolidone (2-20% by weight of the total binder), preferably ethanol aqueous solution of polyvinylpyrrolidone, and most preferably 50% ethanol aqueous solution of polyvinylpyrrolidone.

The glidant can be one or a mixture of more of superfine silica gel powder, talcum powder and magnesium trisilicate.

The surfactant may be one or more components that improve wetting and increase drug dissolution. Sodium lauryl sulfate is often used (the usual range is 0.2-6% by weight, based on the total weight).

The amifostine described in the present invention includes prototypes, pharmaceutically acceptable salts or formulations containing amifostine.

The representative drug of the beta-lactam antibiotic in the invention is meropenem, and the molecular formula is as follows: c₁₇H₂₅N₃O₅S, molecular weight is: 383.5.

the NDM-1 enzyme is a recombinant NDM-1 enzyme extracted from nature or prepared by using genetic engineering bacteria.

The "pathogenic bacteria" in the present invention are preferably gram-negative or gram-positive pathogenic bacteria, more preferably NDM-1 positive bacteria.

Amifostine, alternative name: amifostine and ethiophosphoric acid, and the chemical name is 2- (3-amino propylamino) -ethanethiol phosphate. The molecular formula is as follows: c₅H₁₅N₂O₃PS, molecular weight: 214.2, white crystalline powder or freeze-dried block, has slight odor, melting point 160-:

according to the invention, through NDM-1 enzyme inhibition test, enzyme inhibition rate and half inhibition concentration determination, chessboard method determination minimum inhibitory concentration, time-sterilization curve method and the like, the amifostine is found to be capable of inhibiting the activity of NDM-1 and recovering the antibacterial activity of meropenem on drug-resistant bacteria carrying NDM-1, and the amifostine can be used for treating NDM-1 positive bacteria infectious diseases and has wide medical application.

Detailed description of the invention

The invention firstly uses the crystal structure of NDM-1 (PDB: 4EY2) in a protein database as a target protein, applies computer-aided drug design software GLIDE and MAESTRO, and calculates the binding free energy of the binding part of amifostine and NDM-1 ligand by adopting a molecular docking method. Molecular docking results show that the binding free energy of the docking product is less than-10.0 Kcal/mol, and the amifostine is tightly bound in an active region of NDM-1 centered on zinc ions, so the amifostine is considered as a candidate compound with potential NDM-1 inhibition effect.

On the basis, the invention further detects the inhibitory activity of the amifostine on NDM-1 enzyme, and the detection result shows that the amifostine can inhibit the activity of the NDM-1 enzyme in a dose-dependent manner, the maximum inhibition rate is 82.3 percent, and the IC50 is 25.4 +/-0.6 mu M.

The test result of the minimum inhibitory concentration shows that the amifostine has no inhibitory effect when used alone, and the combined use of the amifostine and the meropenem can reduce the MIC value of the meropenem to NDM-1 positive escherichia coli by 16 times. The FIC index shows that the combination of amifostine and meropenem has obvious synergistic effect on inhibiting the strain producing NDM-1.

According to the test result of the time-sterilization curve, the amifostine combined with meropenem can obviously inhibit the growth of NDM-1 positive escherichia coli.

Drawings

FIG. 1 is a diagram of the combination mode of the active area of an amifostine-NDM-1 composite system.

FIG. 2 is a time sterilization curve of amifostine in combination with meropenem on NDM-1 positive E.coli.

Detailed Description

The invention is further described below in conjunction with specific embodiments, the advantages and features of which will become apparent from the description. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.

Test example 1 molecular docking test of amifostine and target protein NDM-1

The experiment uses the crystal structure of NDM-1 (PDB: 4EY2) in a protein database as a target protein, applies computer-aided drug design software GLIDE and MAESTRO, and calculates the binding free energy of the binding part of the amifostine and the NDM-1 ligand by adopting a molecular docking method.

Molecular docking results found that the binding free energy of the docking product is less than-10.0 Kcal/mol, and that the amifostine is tightly bound in the active region of NDM-1 centered on zinc ions (figure 1), so the amifostine is considered as a candidate compound with potential NDM-1 inhibition effect.

Experimental example 2 expression of NDM-1 protein and isolation and purification

The gene sequence of blaNDM-1 is inserted into pET32 (alpha) plasmid through EcoRI and Xho I enzyme cutting sites to construct pET32 (alpha) -NDM-1 recombinant plasmid, and DNA sequencing verifies that the blaNDM-1 gene has no mutation in the connection process.

The recombinant plasmid is transferred into escherichia coli BL21(DE3) competent cells, screening is carried out through an ampicillin plate, a monoclonal colony is selected and inoculated into 5mL LB liquid culture medium, shaking is carried out at 37 ℃ and 180rpm overnight, 50% glycerol and bacterial liquid are mixed according to the proportion of 1:1, and the engineering bacteria E.coli BL21(DE3) -pET32 (alpha) -NDM-1 is preserved at-80 ℃.

Culturing the engineering bacteria in LB culture medium containing ampicillin at 37 deg.C and 180rpm to logarithmic growth phase with OD value of 0.6-0.8, inducing with isopropyl-beta-D-thiogalactoside (IPTG) with final concentration of 1mM at 37 deg.C for 4.5h, and centrifuging at 4 deg.C to collect bacteria.

The collected bacteria were resuspended in phosphate buffer (PBS, pH 8.0), the bacterial suspension was disrupted by an ultrasonic cell disruptor in ice bath, the bacterial lysate was centrifuged, the supernatant was collected and passed through a Ni-NTA His tag affinity column, and NDM-1 protein was isolated and purified by gradient elution using 0, 10, 20, 40, and 250mM imidazole. And finally, carrying out dialysis on the NDM-1 protein for 36h by using a dialysis bag with the molecular cut-off of 10KD, desalting, concentrating by using an ultrafiltration tube with the molecular cut-off of 10KD, and detecting an NDM-1 protein expression and purification result by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) imprinting to obtain the NDM-1 recombinant protein with the purity of more than 90%.

Test example 3 inhibition of NDM-1 enzyme Activity by Amifostine

The enzyme inhibition activity reaction system comprises 120 mu M meropenem serving as a substrate, 10mM 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES, pH 8.0) serving as a buffer solution, 3.0U NDM-1 enzyme and amifostine solutions with different concentration gradients. Incubating for 15min at 30 ℃, and detecting the enzyme activity by utilizing the 295nm wavelength of an enzyme-labeling instrument. Meanwhile, EDTA is used as a positive control, and DMSO is used as a negative control. The blank contains neither inhibitor nor enzyme as a floor for the system. Reactions were performed in 96-well plates, each reaction provided 3 replicate wells.

The specific process is as follows:

firstly, preparing a buffer solution for NDM-1 enzyme into a solution with the concentration of 3.0U, and incubating the solution at 30 ℃ for 10min to ensure that Zn is formed²⁺Fully occupy the active center; dissolving amifostine in buffer solution to prepare mother solution with the concentration of 100mM, then diluting the mother solution in a gradient manner, adding the diluted mother solution into NDM-1 enzyme, and incubating for 10min at 30 ℃ to ensure that the amifostine and the enzyme are fully combined; adding 50 mu L of meropenem into a 96-pore plate reaction system, placing the mixture into an enzyme labeling instrument, oscillating and uniformly mixing the mixture, and incubating the mixture for 15min at the temperature of 30 ℃ to detect the 295nm ultraviolet absorption change. Calculating the inhibition rate of the amifostine with different concentrations on NDM-1 enzyme hydrolysis substrates, wherein the calculation formula of the inhibition rate is as follows:

inhibition (%). ratio (1-amifostine sample enzyme reaction rate/average enzyme reaction rate of negative control wells) × 100%

And calculating the half inhibition concentration IC50 value of the amifostine to NDM-1.

The detection result shows that the amifostine can inhibit the activity of NDM-1 enzyme in a dose-dependent manner, the maximum inhibition rate is 82.3%, and the IC50 is 25.4 +/-0.6 mu M.

Test example 4 test for minimum inhibitory concentration

2 mu.L of NDM-1 positive Escherichia coli is inoculated into 5mL of LB culture medium, and the culture is carried out at 37 ℃ and 180rpm shaking for activation for about 12h until the bacteria are in the late logarithmic phase. Taking the bacterial liquid into MH broth culture medium, adjusting the concentration of the bacterial liquid to 5 × 10⁶CFU/mL, adopting trace broth dilution method to respectively carry out drug sensitivity tests of amifostine and meropenem (concentration gradient is 1-2048 mu g/mL), and meanwhile, setting bacterial suspension without meropenem as a positive control and MH broth as a negative control. After the 96-well plate is cultured for 24h at 37 ℃, MIC results are read and are all operated in parallel for 3 times, and the MIC value of the medicine is the medicine concentration of a clear well in front of a turbid well in the 96-well plate.

Taking the activated and cultured bacterial liquid, matching a 96-pore plate by adopting a chessboard method, simultaneously detecting 77 possible concentration combinations, adding amifostine and meropenem with different concentration combinations, placing the 96-pore plate in a constant-temperature biochemical incubator at 37 ℃ for incubation for 24h, carrying out an antibacterial activity experiment of combining the amifostine and the meropenem and resisting NDM-1 positive bacteria, and determining the minimum inhibitory concentration MIC value of the combined application of the amifostine and the meropenem. And simultaneously setting a negative control group and a positive control group, performing parallel operation for 3 times, and calculating a part of antibacterial concentration index FIC value according to the MIC value by the following calculation method:

FIC ═ MIC (combined meropenem)/MIC (single meropenem) + MIC (combined amifostine)/MIC (single amifostine)

When FICI is less than or equal to 0.5, the FICI and the FICI act synergistically; 0.5< FICI is less than or equal to 4, and the two are unrelated; FICI >4, both antagonistic.

TABLE 1 MIC and FIC values of Meropenem combined amifostine for NDM-1 expressing positive E.coli

The test result of the minimum inhibitory concentration shows that the amifostine has no inhibitory effect when used alone, and the combined use of the amifostine and the meropenem can reduce the MIC value of the meropenem to NDM-1 positive escherichia coli by 16 times. The FIC index shows that the combination of amifostine and meropenem has obvious synergistic effect on inhibiting the strain producing NDM-1.

Test example 5 time-Sterilization Curve test

Selecting single colony from pure culture plate of NDM-1 positive Escherichia coli, inoculating in MH broth culture medium at 37 deg.C, culturing for 6-8 hr, collecting bacterial culture, and adjusting bacterial liquid concentration to 5 × 10 with sterile physiological saline by McLee turbidimeter⁷CFU/mL, and 10-fold dilution in sterile MH broth to a final concentration of 10⁶CFU/mL。

An antibiotic-free blank control group, a 4 mu g/mL meropenem and 64 mu g/mL amifostine combination group are arranged. The test group and the control group were cultured at 37 ℃ under the same concentration of bacterial solution, a quantitative culture solution was taken out from each group at 0, 1, 3, 5, 7, 9, and 11 hours, and transferred to the corresponding agar medium, and after culturing at 37 ℃ for 18 to 24 hours, the number of colonies was counted, and a time-sterilization curve was plotted with the logarithm of the number of colonies as the ordinate and the culture time as the abscissa (FIG. 2).

According to the time-sterilization curve shown in fig. 2, amifostine combined with meropenem can obviously inhibit the growth of NDM-1 positive escherichia coli.