CN110302384B

CN110302384B - Composition containing dithiocarbamate and metal ion chelating agent and pharmaceutical application thereof

Info

Publication number: CN110302384B
Application number: CN201810583236.0A
Authority: CN
Inventors: 付再林; 宋必卫; 许海燕
Original assignee: Hangzhou Distanry Medical Technology Co ltd
Current assignee: Zhejiang Baidai Pharmaceutical Technology Co.,Ltd.
Priority date: 2018-03-20
Filing date: 2018-06-06
Publication date: 2022-12-16
Anticipated expiration: 2038-06-06
Also published as: CN115607535A; CN110302384A

Abstract

The invention relates to a pharmaceutical composition containing dithiocarbamic acid derivatives and a metal ion chelating agent, and application of the pharmaceutical composition in treating or preventing infection. The pharmaceutical composition can effectively reduce MIC of beta-lactam, fluoroquinolone and aminoglycoside antibiotics on multidrug-resistant or pan-drug-resistant pseudomonas aeruginosa, acinetobacter baumannii and klebsiella pneumoniae by 32 times or more in a synergistic way.

Description

Composition containing dithiocarbamate and metal ion chelating agent and pharmaceutical application thereof

Technical Field

The invention belongs to the field of medicines, relates to a pharmaceutical composition, and particularly relates to a composition containing dithiocarbamate and a metal ion chelating agent, and pharmaceutical application of the composition in anti-infective medicines.

Background

Bacterial resistance has become a major threat to public health safety since the use of antibiotics in the clinical treatment of bacterial infections has become an increasing problem. The World Health Organization (WHO) in 2017 divides 12 kinds of drug-resistant bacteria into three grades of 'critical', 'high' and 'medium' according to the mortality, drug-resistant situation, epidemiological tendency and the like of patients, wherein carbapenems-resistant acinetobacter baumannii, pseudomonas aeruginosa and enterobacteriaceae mainly including klebsiella pneumoniae are listed as 'critical' grades. The three pathogenic bacteria gradually show the trend of multi-drug resistance, pan-drug resistance and even full-drug resistance, and the clinically available treatment means are quite limited.

In recent years, it has been found that metal ions, particularly calcium, magnesium, iron, copper, zinc, manganese and the like, are closely related to bacterial resistance, and play important roles in the aspects of promoting the lateral propagation of bacterial resistance plasmids, promoting the proliferation and division of bacteria, promoting the invasion and adhesion, promoting the generation of biological envelope, hydrolyzing antibiotics, resisting oxidative damage and the like. The Jillian Orasnsa team found that calcium ions can control the expansion and contraction of pili through the binding state with PilY1 protein, affect the twitching movement of pathogenic bacteria, and are closely related to the invasion and adhesion of pathogenic bacteria (Orans J, johnson MD, coggan KA, et al. Crystal structure analysis machinery of Pseudomonas PilY1 as an invasive calcium-dependent regulator of bacterial surface mobility. Proc Natl Acad Sci USA 2010;107 (3): 1065-70). Calcium ions can make bacteria competent, and promote the lateral transfer of drug-resistant plasmids in the same or even different bacteria. In addition to calcium ions, zhukayue et al found that heavy metal ions such as copper, iron, zinc, etc. also enhanced the horizontal transfer of the multiple drug-resistant plasmid pYN1 in different species of bacteria (zhukayue. Study of the horizontal transfer ability of the multiple drug-resistant plasmid pYN1 in different species of bacteria [ D ]. University of south river, 2016). Poole K has also shown in its studies that heavy metal ions such as copper and zinc can promote bacterial resistance (Poole K. At the new of antibiotics and metals: the impact of Cu and Zn on antibiotic activity and resistance. Trends Microbiol.2017;25 (10): 820-32). In addition, ions such as calcium, magnesium and iron can remarkably promote the generation of biofilms of pathogenic bacteria such as pseudomonas aeruginosa, acinetobacter baumannii and klebsiella pneumoniae, and the biofilms can greatly increase the MIC. Wang YC et al found that after biofilm formation Acinetobacter baumannii had a significantly reduced sensitivity to antibiotics such as meropenem, imipenem, sulbactam, tigecycline, and polymyxin, the formation of biofilms generally increased the 5 antibiotics MBC by 50-200 times, up to 3200 times (Wang YC, kuo SC, yang YS, et al. Industrial or combined effects of meropenem, imipenem, sulbactam, colistin, and tigecycline, bioofdm embedded antibiotic and antibiotic industries. Antisense organisms 4670-6. In addition to hindering antibiotics, biofilm production may also facilitate the lateral spread of drug-resistant plasmids between bacteria. In addition, iron ions are the active center of iron sulfur proteins, which are important carriers for electron transport in the respiratory chain. Copper ions and manganese ions are also active centers of peroxide dismutase, and the high expression of the peroxide dismutase can relieve the oxidation damage in pathogenic bacteria. Metallo beta-lactamases contain one or two zinc ions at their active site, which promote nucleophilic attack of beta-lactams by polarizing water molecules, water-releasing all marketed lactam antibiotics outside aztreonam (Bounaga S, laws AP, galleni M, et al. The mechanism of catalysis and the inhibition of the Bacillus cereus zinc-dependent beta-lactam. Biochem J.1998;331 (Pt 3): 703-11). In the above-mentioned prior art documents, there is no indication that the combination of a plurality of the above-mentioned metal ions can inhibit the proliferation, invasion or development of drug resistance of bacteria.

Dithiocarbamic acid derivatives as flotation agents commonly used in mining industry can complex with metal zinc and copper ions to form a complex, and have the effects of resisting bacteria, viruses and tumor growth and metastasis, such as sodium diethyldithiocarbamate (DDTC) which is clinically used as an immunomodulator for treating AIDS (ZL 93100610). Zhang En et al found that dithiocarbamic acid derivatives can restore the sensitivity of NDM-1-producing Enterobacteriaceae to meropenem by complexing zinc ions in metallo-lactamase (CN 107184581A), but the patent did not make any corresponding study on metallo-lactamase-negative resistant bacteria as to whether dithiocarbamic acid derivatives could restore their sensitivity to lactam antibiotics. Further, it is not known from the patent whether or not a dithiocarbamate derivative can restore the sensitivity of drug-resistant bacteria to non-lactam antibiotics such as fluoroquinolones and aminoglycosides.

Polymethacrylic acids and polycarboxylic chelating agents are commonly used as corrosion and scale inhibitors for circulating water and boiler water, complexing agents for cyanide-free electroplating, chelating agents in textile printing and dyeing industry and oxygen bleaching stabilizers, wherein ethylene diamine tetraacetic acid calcium sodium is clinically used for treating heavy metal poisoning, and hydroxy ethylidene diphosphonic acid sodium can be used as a calcium regulator for treating tumor bone metastasis. The polymethine phosphonic acids are not applied in the aspects of antibiosis or auxiliary antibiosis. EDTA can be used for inhibiting the generation of the biofilm of pseudomonas aeruginosa and staphylococcus aureus directly or in combination with other medicines. The effect of complex formulation of ceftriaxone, sulbactam and EDTA on the treatment of multidrug-resistant Acinetobacter baumannii was found by Prache Sathe et al in a small sample size clinical trial to be comparable to the effective rate of the combination of Meropenem and polymyxin regimen (Sathe P, maddani S, kulkarni S, et al. Management of vehicle associated pulmonary with a new anti-inflammatory adjuvant entry (ceftriaxone + Sulbactam + diodate) -A non-associated with space vector. J Crcaro. 2017; 41. 145-149), and it was found that metal ion chelators such as EDTA have the effect of increasing the susceptibility of drug-resistant bacteria to lactam antibiotics.

In summary, the prior art shows that:

1) Not all metal ion chelators restore sensitivity to various classes of antibiotic drugs (e.g., beta-lactams, fluoroquinolones, aminoglycosides, and the like, the same applies below);

2) Whether all the dithiocarbamic acid derivatives can recover the sensitivity to various antibiotic drugs cannot be judged;

3) The dithiocarbamate derivatives can restore the sensitivity of metal beta-lactamase negative drug-resistant bacteria (such as pseudomonas aeruginosa, acinetobacter baumannii, klebsiella pneumoniae and the like, the same below) to various antibiotic drugs, and cannot be judged;

4) If the dithiocarbamate derivatives and the metal ion chelating agent are combined, whether the sensitivity of metal beta-lactamase negative drug-resistant bacteria to various antibiotic drugs can be recovered in a synergistic manner cannot be judged.

Therefore, how to find a suitable composition containing dithiocarbamate and metal ion chelating agent to solve the above-mentioned drawbacks of the prior art is a technical problem which needs to be solved urgently by those skilled in the art.

Disclosure of Invention

The present invention aims to provide compositions comprising dithiocarbamates and metal ion chelating agents and their pharmaceutical use against infection.

In order to realize the purpose of the invention, the technical scheme is as follows:

the invention provides a pharmaceutical composition, which comprises the following active ingredients:

a) A dithiocarbamate derivative or a pharmaceutically acceptable salt selected from the group consisting of:

in the active ingredient a, the pharmaceutically acceptable salt is sodium salt;

b) A metal ion chelating agent selected from the group consisting of:

ethylenediaminetetramethylphosphinic acid salt (i), hexamethylenediaminetetramethylenephosphonic acid salt (ii), diethylenetriaminepentamethylenephosphonic acid salt (iii), aminotrimethylenephosphonic acid salt (iv), ethylenediamine dipehthalic acid salt (v), hydroxyethylidene diphosphonic acid salt (vi), 2-phosphonobutane-1, 2, 4-tricarboxylic acid salt (vii), ethylenediaminetetraacetic acid salt (viii), nitrilotriacetic acid salt (ix), diethylenetriaminepentaacetic acid salt (x), hydroxyethylethylenediaminetriacetic acid salt (xi), ethyleneglycol bis (2-aminoethyl ether) tetraacetic acid salt (xii), 1, 2-bis (2-aminophenoxy) ethane-N, N, N ', N' -tetraacetic acid salt (xiii);

in the active ingredient b, the metal ion chelating agent is sodium salt, potassium salt or ammonium salt.

The invention also provides a first pharmaceutical use of a pharmaceutical composition as defined in the preceding claim, namely for the preparation of an anti-infective medicament, in particular an antibacterial medicament, which pharmaceutical composition has an inhibitory effect on multidrug-resistant or pan-drug-resistant pseudomonas aeruginosa, acinetobacter baumannii or klebsiella pneumoniae.

In the pharmaceutical application, the molar ratio of the two active ingredients in the pharmaceutical composition provided by the invention is a: b = (100: 1) to 1: 100), preferably in a molar ratio of a: b = (9: 1) - (1: 9).

In addition, the invention also provides a second pharmaceutical application of the pharmaceutical composition as described in the right, namely the pharmaceutical composition can be combined with beta-lactam antibiotics, and has synergistic inhibition effect on multidrug-resistant or pan-resistant pseudomonas aeruginosa, acinetobacter baumannii or klebsiella pneumoniae.

Preferably, the beta-lactam antibiotics comprise carbapenems and cephalosporins, wherein the carbapenem antibiotic is meropenem, and the cephalosporins antibiotic is cefoperazone sulbactam.

More preferably, in the pharmaceutical application, the molar ratio of the two active ingredients in the pharmaceutical composition provided by the invention is a: b = (100: 1) to 1: 100), and the molar ratio is preferably a: b = (9: 1) - (1: 9).

In addition, the invention also provides a third pharmaceutical application of the pharmaceutical composition as described in the right, namely the pharmaceutical composition can be combined with aminoglycoside antibiotics, and has synergistic inhibition effect on multidrug-resistant or pan-resistant pseudomonas aeruginosa, acinetobacter baumannii or klebsiella pneumoniae.

Preferably, the aminoglycoside antibiotic is amikacin.

In addition, the invention also provides a fourth pharmaceutical application of the pharmaceutical composition as described in the right, namely the pharmaceutical composition is combined with fluoroquinolone antibiotics to achieve synergistic inhibition effect on multidrug-resistant or pan-resistant pseudomonas aeruginosa, acinetobacter baumannii or klebsiella pneumoniae.

Preferably, the fluoroquinolone antibiotic is levofloxacin.

It is known that the higher the drug resistance of pathogenic bacteria, the lower the sensitivity to antibiotics, and theoretically, the antibiotic effective for pathogenic bacteria with higher drug resistance is also effective for pathogenic bacteria with low drug resistance. The inventor selects 3 drug-resistant bacteria (1 each of pseudomonas aeruginosa, klebsiella pneumoniae and acinetobacter baumannii with highest drug resistance degree in test strains, and the detection result is negative through an EDTA-Meropenem double-paper diffusion method test, which indicates that the 3 drug-resistant bacteria do not produce metal beta-lactamase and produce KPC-2 enzyme through klebsiella pneumoniae in an identification test), and the drug-resistant conditions of the 3 drug-resistant bacteria are detected through a drug-sensitive test as follows: meropenem MIC is 128 mu g/ml, cefoperazone sulbactam MIC is 512 mu g/ml, amikacin is 512 mu g/ml, levofloxacin is 64 mu g/ml. The execution standard of 2017 version antibacterial drug sensitivity test (CLSI 2017-M100) indicates that when the concentrations of meropenem, cefoperazone sulbactam, amikacin and levofloxacin antibiotic are respectively less than or equal to 4 mu g/ml, less than or equal to 16 mu g/ml and less than or equal to 2 mu g/ml, the strain to be detected is defined to be sensitive to the antibiotic, so that the drug resistance of 3 selected drug-resistant bacteria is improved by at least 32 times compared with the sensitive strain.

The research idea of the invention is as follows:

firstly, the inventor tests the antibacterial effect of the 3 resistant bacteria by using dithiocarbamic acid derivatives (0.5 mmol/L) alone in combination with meropenem (4 mu g/ml) or cefoperazone sulbactam (16 mu g/ml) or amikacin (16 mu g/ml) or levofloxacin (2 mu g/ml), using metal ion chelating agents (0.5 mmol/L) alone in combination with antibiotics with the above concentrations, and using the composition (0.5 mmol/L +0.5 mmol/L) in combination with antibiotics with the above concentrations.

Secondly, preference is given to combinations of 9, namely 1/i, 1/ii, 1/iii, 11/i, 11/ii, 11/iii, 15/ii, 15/iii, 15/i, 15/ii and 15/iii, of the dithiocarbamate derivative compounds and of the metal ion chelating agents, considering the antibacterial effect of the combination of compositions in different proportions of meropenem (4. Mu.g/ml) or cefoperazone sulbactam (16. Mu.g/ml) or amikacin (16. Mu.g/ml) or levofloxacin (2. Mu.g/ml), and further preference is given to a preferred ratio of the two compounds in each composition.

Next, preference is given to using numbers 1,11,15 of the dithiocarbamate derivative compounds and numbers i, ii, iii of the metal ion chelating agents to examine the effect of 9 combinations 1/i, 1/ii, 1/iii, 11/i, 11/ii, 11/iii, 15/i, 15/ii, 15/iii, etc. on the MIC of meropenem, cefoperazone sulbactam, amikacin and levofloxacin.

Next, it is preferable that 1,11,15 in the dithiocarbamate derivative compound and i in the metal ion chelating agent are tested for the above 3 resistant bacteria, and it is examined whether the antibacterial efficacy of antibiotics such as meropenem, cefoperazone sulbactam, amikacin and levofloxacin can be synergistically increased between 1,11,15 and i.

Finally, to verify the effectiveness of the combination of the above antibiotics against many common clinically resistant bacteria, clinical multi-resistant or pan-resistant Pseudomonas aeruginosa (16 strains), acinetobacter baumannii (13 strains, 2 strains, producing metallo beta-lactamase) and Klebsiella pneumoniae (15 strains, 3 strains, producing KPC enzyme) were collected, and the antibacterial effectiveness of 3 combinations such as 1/i, 11/i, 15/i, etc. in combination with meropenem (4 μ g/ml), cefoperazone sulbactam (8 μ g/ml), amikacin (8 μ g/ml) or levofloxacin (2 μ g/ml) was examined.

In conclusion, the inventors found that the dithiocarbamate and the metal ion chelating agent have synergistic effect, which is mainly shown in the following aspects:

(1) the composition can restore the sensitivity of metal beta-lactamase-producing drug-resistant bacteria to lactam antibiotics, can restore the sensitivity of metal beta-lactamase-negative multi-drug-resistant or pan-drug-resistant bacteria such as pseudomonas aeruginosa, acinetobacter baumannii, klebsiella pneumoniae and other drug-resistant strains to carbapenems such as meropenem, third-generation cephalosporins and enzyme composite preparations such as cefoperazone sulbactam, and singly selects dithiocarbamic acid derivatives or metal ion chelating agents to be combined with the beta-lactam antibiotics (such as meropenem and cefoperazone sulbactam) to have no effect or poor effect (no clinical significance) on the metal beta-lactamase-negative drug-resistant bacteria;

(2) the composition not only can effectively and synergistically reduce the MIC (minimum inhibitory concentration) values of beta-lactam antibiotics (such as carbapenems, cephalosporins and enzyme composite preparations thereof) to multidrug-resistant or pan-resistant pseudomonas aeruginosa, acinetobacter baumannii and klebsiella pneumoniae, but also can reduce the MIC values of non-beta-lactam antibiotics such as fluoroquinolones (such as levofloxacin) and aminoglycosides (such as amikacin) to the drug-resistant bacteria, and the main drug resistance mechanisms of the two antibiotics are irrelevant to metallo beta-lactamase, which is not mentioned in the prior patents and documents and discovered by the inventor for the first time;

(3) the dithiocarbamic acid derivatives and the metal ion chelating agent in the composition are not simply added in action, have synergistic effect, and particularly have the synergistic effect on metal beta-lactamase negative drug-resistant bacteria, and the dosage of the dithiocarbamic acid derivatives and the metal ion chelating agent in the composition is greatly reduced compared with that of single use when the same antibacterial effect is achieved by combining the same dosage of antibiotics;

(4) the effective rate of the composition combined with carbapenems (such as meropenem), the third-generation cephalosporins and enzyme composite preparations thereof (such as cefoperazone sulbactam), fluoroquinolones (such as levofloxacin) or aminoglycosides (such as amikacin) to multi-drug resistant or pan-drug resistant pseudomonas aeruginosa is over 90 percent, the effective rate to acinetobacter baumannii is close to 100 percent, and the effective rate of the composition combined with dithiocarbamic acid derivatives or metal ion chelating agents to the 4 antibiotic drug resistant bacteria is about 30 percent in common and is far lower than that of the composition combined antibiotics;

(5) more surprisingly, the composition can restore the color change sensitivity of KPC enzyme Klebsiella pneumoniae to meropenem, cefoperazone sulbactam, levofloxacin and amikacin, while the single use of dithiocarbamate derivatives or metal ion chelating agents can not restore the sensitivity of KPC enzyme Klebsiella pneumoniae to the above four antibiotics even with a large dose (4-8 mmol/L), and has great clinical significance.

Drawings

FIG. 1 shows the effectiveness of the compositions 1/i, 11/i, 15/i in combination with MEM, CFP, AK or LFX anti-XDR-PA tests

FIG. 2 shows the effectiveness of the compositions 1/i, 11/i, 15/i in combination with MEM, CFP, AK or LFX anti-XDR-KP tests

FIG. 3 shows the effectiveness of the compositions 1/i, 11/i, 15/i in combination with MEM, CFP, AK or LFX anti-XDR-AB tests

Detailed Description

In order to make the technical solution of the present invention better understood by those skilled in the art, the technical solution in the present embodiment will be specifically described below with reference to the accompanying drawings in the present application. It should be noted that the following examples are only for illustrating the present invention and are not to be construed as limiting the present invention, and any modifications and changes made to the present invention within the spirit and scope of the claims are included in the scope of the present invention.

The raw materials, 15 dithiocarbamate derivatives (a) and 13 metal ion chelating agents (b), which are used in the following examples, were commercially available from Sigma or alatin reagent company, and the structures thereof were confirmed.

EXAMPLE 1 examination of the antibacterial Effect of a pharmaceutical composition comprising a and b having different compositions in combination with an antibiotic for three types of drug-resistant bacteria, pseudomonas aeruginosa, klebsiella pneumoniae, acinetobacter baumannii

[ Experimental materials ]

(1) The pharmaceutical composition comprises: 15 active ingredient a alone, 13 active ingredient b alone, 15 active ingredient a in combination with 13 active ingredient b;

(2) antibiotics: selecting different representative antibiotics, such as carbapenem antibiotic selected from meropenem, cephalosporin antibiotic selected from cefoperazone sulbactam, aminoglycoside selected from amikacin, and fluoroquinolone selected from levofloxacin;

(3) drug-resistant strains: 3 strains of drug-resistant bacteria with the highest drug-resistant degree are selected, wherein the strains are respectively 1 strain of pseudomonas aeruginosa, klebsiella pneumoniae and acinetobacter baumannii, and the three strains are all shown to be metal beta-lactamase negative through EDTA double-paper sheet synergy test.

[ purpose of experiment ]

The drug combination of which the a and the b are preferably combined is screened by considering the combination form of different components of the drug combination a and b, or only a or only b under three conditions and combining the antibacterial effect of different types of antibiotics on the metal beta-lactamase negative drug-resistant bacteria.

[ Experimental methods ]

Selecting 1 strain of each of drug-resistant strains pseudomonas aeruginosa, klebsiella pneumoniae and acinetobacter baumannii, and detecting the drug-resistant conditions of 3 strains of drug-resistant bacteria through a drug sensitivity test as follows: meropenem MIC>128 mu g/ml Cefoperazone sulbactam MIC>512 mug/ml amikacin>512 mu g/ml, and levofloxacin more than or equal to 64 mu g/ml. Taking the pathogenic bacteria in logarithmic growth phase, preparing the bacteria liquid into 0.5 McLeod turbidity, and continuously diluting 100 times (equivalent to 1-2 × 10 times) before use ⁶ CFU/ml) and added to a 96-well plate at 150 μ l per well.

Using 4 antibiotics with final concentration as control, namely meropenem (4 mu g/ml), cefoperazone sulbactam (16 mu g/ml), amikacin (16 mu g/ml) and levofloxacin (2 mu g/ml), selecting dithiocarbamic acid derivative (active component a, final concentration 0.5 mmol/L), metal ion chelating agent (active component b, final concentration 0.5 mmol/L) and pharmaceutical composition (active component a + active component b, final concentration 0.5mmol/L +0.5 mmol/L), respectively combining q with 4 antibiotics and incubating with the pathogenic bacteria, wherein the total volume is 200 mu L/hole, and measuring turbidity after 24 hours, to examine the antibacterial effect of the series combination on the 3 drug-resistant bacteria.

[ Experimental results ]

The results are shown in tables 1 to 12. In tables 1 to 12 below, compound i represents 15 active ingredients a, and compound ii represents 13 active ingredients b.

"-" indicates no bacterial growth was observed for the corresponding well clarification;

"+" indicates that the corresponding well has little bacterial growth and the turbidity is less than 1/10 of that of the control well;

"+ +" represents equal amount of growth in bacteria in corresponding wells, turbidity is 1/10-3/10 of control wells;

"+ + + + +" indicates that the corresponding well has a large amount of bacteria growth, and the turbidity is more than 3/10 of that of the control well;

"\" represents replacement with a broth containing only the corresponding concentration of antibiotic.

TABLE 1 antibacterial Effect of the combination of the compositions Meropenem on drug-resistant Pseudomonas aeruginosa

TABLE 2 antibacterial Effect of the combination of the composition with cefoperazone sulbactam on drug-resistant Pseudomonas aeruginosa

TABLE 3 antibacterial Effect of compositions in combination with amikacin on drug-resistant Pseudomonas aeruginosa

TABLE 4 antibacterial Effect of the combination of the compositions with levofloxacin on drug-resistant Pseudomonas aeruginosa

TABLE 5 antibacterial Effect of the combination of Meropenem on drug-resistant Klebsiella pneumoniae

TABLE 6 antibacterial Effect of the combination of Cefoperazone sulbactam with the composition on drug-resistant Klebsiella pneumoniae

TABLE 7 antibacterial Effect of the combination of Amikacin with drug resistant Klebsiella pneumoniae

TABLE 8 antibacterial Effect of the combination of levofloxacin and the composition on drug-resistant Klebsiella pneumoniae

TABLE 9 antibacterial Effect of the combination of Meropenem on drug-resistant Acinetobacter baumannii

TABLE 10 antibacterial Effect of the combination of Cefoperazone sulbactam with the drug-resistant Acinetobacter baumannii

TABLE 11 antibacterial Effect of the combination of compositions with Amikacin on drug-resistant Acinetobacter baumannii

TABLE 12 antibacterial Effect of the combination of levofloxacin and Acinetobacter baumannii

As is clear from tables 1 to 12, with respect to 3 types of metallo-beta-lactamase-negative pan-resistant bacteria such as Pseudomonas aeruginosa, klebsiella pneumoniae, and Acinetobacter baumannii, there are the following cases:

(1) active ingredients a and b are not used, and only meropenem, cefoperazone sulbactam, amikacin and levofloxacin are used, so that the bactericidal effect is not generated, and bacteria are proliferated in a large quantity.

(2) The use of dithiocarbamate derivatives (i.e. active ingredient a) or the use of metal ion chelating agents (i.e. active ingredient b) in combination with 4 antibiotics such as meropenem, cefoperazone sulbactam, amikacin, levofloxacin, is basically ineffective with a slight degree of bacteriostasis, but still belongs to the category of ineffective elimination of germs.

(3) The dithiocarbamic acid derivatives and the metal ion chelating agent are combined together for 195 times, and are combined with 4 antibiotics such as meropenem, cefoperazone sulbactam, amikacin and levofloxacin, so that bacteria can be completely eliminated or mostly eliminated, and the antibacterial effect is obviously superior to that of the single active component a or b; the majority of the a + b compositions are shown to significantly restore the sensitivity of Pseudomonas aeruginosa, klebsiella pneumoniae and Acinetobacter baumannii to antibiotics, while a or b alone is not effective.

(4) Among the metal ion chelating agents, the combination of the polymethine chelating agent with the dithiocarbamate derivative is superior to the combination of the polycarboxylic acid with the dithiocarbamate derivative in restoring the bacterial sensitivity, indicating that the polymethine chelating agent has a higher chelating index with the metal ion.

[ Experimental conclusion ]

Based on the results of the experiments in tables 1 to 12, the inventors selected 9 combinations 1/i, 1/ii, 1/iii, 11/i, 11/ii, 11/iii, 15/i, 15/ii, 15/iii as the preferred combination for the combination of active ingredients a + b.

Based on the above, the antibacterial effect of each antibiotic combined by the 9 compositions according to different proportions is further examined.

Example 2 examination experiment of antibacterial effects of a pharmaceutical composition comprising a and b in different proportions on three drug-resistant bacteria, namely Pseudomonas aeruginosa, klebsiella pneumoniae and Acinetobacter baumannii, in combination with antibiotic

[ Experimental materials ]

(1) The pharmaceutical composition comprises: 9 combinations of active ingredient a in combination with active ingredient b, 1/i, 1/ii, 1/iii, 11/i, 11/ii, 11/iii, 15/i, 15/ii, 15/iii;

[ EXPERIMENTAL OBJECTS ] of the present invention

On the basis of the 9 a + b combinations selected in example 1, a was examined for these 9 combinations: b, when the antibiotics with different types are combined to achieve the antibacterial effect on metal beta-lactamase negative drug-resistant bacteria, screening out a: b is the preferred ratio of the pharmaceutical composition.

[ Experimental methods ]

The antibiotics were selected from meropenem (4 μ g/ml), cefoperazone sulbactam (16 μ g/ml), amikacin (16 μ g/ml), levofloxacin (2 μ g/ml), the pharmaceutical compositions a/b were selected from the 9 combinations 1/i, 1/ii, 1/iii, 11/i, 11/ii, 11/iii, 15/i, 15/ii, 15/iii, the ratio of a to b being 20.

[ Experimental results ]

The results are shown in tables 13 to 24. In tables 13 to 24 below, the compositions are constituted as a/b, with 20 indicating the presence of only active ingredient a and 0 indicating the presence of only active ingredient b.

"-" indicates no bacterial growth was observed in the corresponding well;

"+" indicates turbid bacterial growth in the corresponding well.

TABLE 13 combination of different proportions of the composition with meropenem (4. Mu.g/ml) resistance to pan-drug resistant Pseudomonas aeruginosa

TABLE 14 combination of different proportions of cefoperazone sulbactam (16. Mu.g/ml) in the pan-resistant Pseudomonas aeruginosa test

TABLE 15 combination of compositions in different proportions with amikacin (16. Mu.g/ml) resistance to pan-drug resistant Pseudomonas aeruginosa

TABLE 16 combination of various proportions of levofloxacin (2. Mu.g/ml) in combination with the test for the resistance to pan-drug resistant Pseudomonas aeruginosa

TABLE 17 combination of different proportions of the compositions in combination with meropenem (4. Mu.g/ml) pan-resistant Klebsiella pneumoniae assay

TABLE 18 combination of cefoperazone sulbactam (16. Mu.g/ml) in different proportions for pan-resistant Klebsiella pneumoniae

TABLE 19 combination of Amikacin (16. Mu.g/ml) in different proportions for pan-resistant Klebsiella pneumoniae test

TABLE 20 combination of various compositions in different proportions with anti-pan-drug resistant Klebsiella pneumoniae test (2. Mu.g/ml)

TABLE 21 combination of different proportions of the composition with meropenem (4. Mu.g/ml) pan-resistant A.baumannii test

TABLE 22 combination of different proportions of cefoperazone sulbactam (16. Mu.g/ml) pan-resistant A.baumannii test

TABLE 23 combination of Amikacin (16. Mu.g/ml) with different proportions of the composition for pan-resistant A.baumannii assay

TABLE 24 combination of various ratios of levofloxacin (2. Mu.g/ml) with anti-pan-drug resistant A. Baumannii test

As is clear from tables 13 to 24, there are several cases of the 3 metallo-beta-lactamase-negative pan-resistant bacteria such as Pseudomonas aeruginosa, klebsiella pneumoniae, and Acinetobacter baumannii:

(1) the use of dithiocarbamate derivatives (i.e. active ingredient a) or the use of metal ion chelating agents (i.e. active ingredient b) in combination with 4 antibiotics such as meropenem, cefoperazone sulbactam, amikacin, levofloxacin, did not effectively inhibit or kill pathogenic bacteria.

(2) After the composition (a + b) in a proper proportion range is incubated with the antibiotics and pathogenic bacteria for 24 hours, no bacteria grow in the corresponding hole, and the culture solution is clear and transparent. Different compositions, different antibiotics to be used in combination and different strains of the subject have an influence on the preferred ratio of the compositions.

As can be seen from Table 13, the preferred ratios of the different compositions (1/i, 1/ii, 1/iii, 11/i, 11/ii, 11/iii, 15/i, 15/ii and 15/iii) in combination with meropenem resistant Pseudomonas aeruginosa were 3/17 to 19/1, 1/9 to 19/1, 3/17 to 19/1 and 3/17 to 19/1, respectively;

as can be seen from Table 14, the preferred ratios of the different combinations (1/i, 1/ii, 1/iii, 11/i, 11/ii, 11/iii, 15/i, 15/ii and 15/iii) in combination with cefoperazone sulbactam anti-pan-resistant Pseudomonas aeruginosa are 3/17-19/1, 3/17-9/1, 1/9-19/1, 3/17-9/1 and 3/17-9/1, respectively;

as can be seen from Table 15, the preferred ratios of the different compositions (1/i, 1/ii, 1/iii, 11/i, 11/ii, 11/iii, 15/i, 15/ii and 15/iii) in combination with amikacin resistant Pseudomonas aeruginosa were 3/17 to 19/1, 3/17 to 1/9, 3/17 to 9/1, 1/9 to 19/1, 3/17 to 9/1 and 3/17 to 9/1, respectively;

as can be seen from Table 16, the preferred ratios of the various combinations (1/i, 1/ii, 1/iii, 11/i, 11/ii, 11/iii, 15/i, 15/ii and 15/iii) of levofloxacin anti-flooding resistant Pseudomonas aeruginosa are, respectively, from 1/9 to 19/1, from 3/17 to 9/1, from 1/9 to 19/1, from 1/9 to 9/1 and from 1/9 to 19/1;

as can be seen from Table 17, the preferred ratios of the different compositions (1/i, 1/ii, 1/iii, 11/i, 11/ii, 11/iii, 15/i, 15/ii and 15/iii) in combination with the meropenem anti-pan-resistant Klebsiella pneumoniae were 1/19 to 9/1, 3/17 to 17/3, 1/9 to 17/3, 1/19 to 9/1, 1/9 to 4/1, 1/19 to 9/1, 3/17 to 17/3 and 1/9 to 9/1, respectively;

as can be seen from Table 18, the preferred ratios of the different combinations (1/i, 1/ii, 1/iii, 11/i, 11/ii, 11/iii, 15/i, 15/ii and 15/iii) in combination with cefoperazone sulbactam anti-pan-resistant Klebsiella pneumoniae are 1/19-9/1, 1/9-9/1, 1/19-19/1, 3/17-9/1 and 1/9-9/1, respectively;

as can be seen from Table 19, the preferred ratios of the different compositions (1/i, 1/ii, 1/iii, 11/i, 11/ii, 11/iii, 15/i, 15/ii and 15/iii) in combination with amikacin resistant Klebsiella pneumoniae were 1/19 to 19/1, 1/9 to 9/1, 1/19 to 19/1, 1/9 to 19/1, 1/19 to 19/1, 1/9 to 9/1 and 1/9 to 19/1, respectively;

as can be seen from Table 20, the preferred ratios of the different compositions (1/i, 1/ii, 1/iii, 11/i, 11/ii, 11/iii, 15/i, 15/ii and 15/iii) in combination with levofloxacin anti-pan-resistant Klebsiella pneumoniae were from 1/19 to 9/1, from 3/17 to 9/1, respectively,

1/9-17/3, 1/9-19/1, 1/9-17/3, 1/19-9/1, 3/17-17/3 and 1/9-9/1;

as can be seen from Table 21, the preferred ratios of the different combinations (1/i, 1/ii, 1/iii, 11/i, 11/ii, 11/iii, 15/i, 15/ii and 15/iii) of Meropenem resistant A.baumannii are, respectively, from 1/9 to 19/1, from 1/9 to 9/1, from 1/19 to 19/1, from 1/9 to 9/1 and from 1/9 to 19/1;

as can be seen from Table 22, the preferred ratios of the different combinations (1/i, 1/ii, 1/iii, 11/i, 11/ii, 11/iii, 15/i, 15/ii and 15/iii) in combination with cefoperazone sulbactam anti-pan-resistant A.baumannii are 19/1 to 9/1, 3/17 to 9/1, 1/9 to 9/1, 1/19 to 19/1, 1/9 to 19/1, 1/19 to 9/1, 1/9 to 9/1 and 1/9 to 9/1, respectively;

as can be seen from Table 23, the preferred ratios of the different compositions (1/i, 1/ii, 1/iii, 11/i, 11/ii, 11/iii, 15/i, 15/ii and 15/iii) in combination with amikacin resistant A.baumannii were from 1/19 to 19/1, from 1/9 to 9/1, respectively,

1/9-9/1, 1/19-19/1, 1/9-9/1 and 1/19-19/1;

as can be seen from Table 24, the preferred ratios of the different compositions (1/i, 1/ii, 1/iii, 11/i, 11/ii, 11/iii, 15/i, 15/ii and 15/iii) in combination with levofloxacin anti-pan-resistant A.baumannii are from 1/9 to 19/1, from 3/17 to 9/1, respectively,

1/9-17/3, 1/19-19/1, 1/9-9/1, 1/9-19/1, 1/9-9/1 and 1/9-9/1;

[ Experimental conclusion ]

In summary, the effective ratio (molar ratio) of the dithiocarbamate derivative to the metal ion chelating agent is generally between 1 and 19, preferably between 1/9 and 9/1, and the combination of the above antibiotics with different ratios in this range can effectively kill or inhibit pathogenic microorganisms.

EXAMPLE 3 Effect of pharmaceutical composition consisting of different Components a and b on MIC of 4 antibiotics

[ Experimental materials ]

(1) The pharmaceutical composition comprises: 9 combinations of active ingredient a and active ingredient b, 1/i, 1/ii, 1/iii, 11/i, 11/ii, 11/iii, 15/i, 15/ii, 15/iii;

(3) drug-resistant strains: 3 strains of drug-resistant bacteria with the highest drug-resistant degree are selected, 1 strain of each of pseudomonas aeruginosa, klebsiella pneumoniae and acinetobacter baumannii, and the three strains are shown to be negative to metal beta-lactamase through an EDTA double-paper sheet synergistic test.

[ purpose of experiment ]

Based on the 9 a + b combinations selected in example 1, the effect of these 9 combinations on the MICs of different types of antibiotics was examined.

[ Experimental methods ]

The assay was performed in 96-well plates and divided into four groups, i.e., dithiocarbamate derivative (-)/metal ion chelator (-)/antibiotic (+) group, dithiocarbamate derivative (+)/metal ion chelator (-)/antibiotic (+) group, dithiocarbamate derivative (-)/metal ion chelator (+)/antibiotic (+) group, and dithiocarbamate derivative (+)/metal ion chelator (+)/antibiotic (+) group, with two rows for each group. Diluting 4 antibiotics in 96 micro-porous plate, adding dithiocarbamic acid derivatives or metal ion chelating agent, supplementing (-) part with blank culture solution, and adding drug-resistant bacteria. The final concentration of the meropenem in the 1 st hole is 128 mu g/ml, the final concentration of the cefoperazone sodium and sulbactam sodium in the 1 st hole is 512 mu g/ml, the final concentration of the amikacin in the 1 st hole is 512 mu g/ml, the final concentration of the levofloxacin in the 1 st hole is 64 mu g/ml, the final concentrations of the dithiocarbamic acid derivative and the metal ion chelating agent are both 0.5mmol/L, the final concentration of the bacterial liquid is about 106CFU/ml, and the incubation time is 24 hours.

[ Experimental results ]

The results are shown in tables 25 to 28, and in tables 25 to 28 described below, "KP (KPC-producing enzyme)" means KPC-producing Klebsiella pneumoniae, "XDR-PA" means pan-resistant Pseudomonas aeruginosa, and "XDR-AB" means pan-resistant Acinetobacter baumannii.

TABLE 25 Effect of dithiocarbamate derivatives, metal ion chelators and compositions thereof on the MIC of meropenem

	1(-)/i(-)/MEM(+)	1(+)/i(-)/MEM(+)	1(-)/i(+)/MEM(+)	1(+)/i(+)/MEM(+)
					KP (producing KPC enzyme)	＞128	＞128	＞128	2
XDR-PA	＞128	64	32	2
					XDR-AB	＞128	128	32	＜0.125
	1(-)/ii(-)/MEM(+)	1(+)/ii(-)/MEM(+)	1(-)/ii(+)/MEM(+)	1(+)/ii(+)/MEM(+)
					KP (producing KPC enzyme)	＞128	＞128	＞128	4
XDR-PA	＞128	64	64	4
					XDR-AB	＞128	128	64	0.25
	1(-)/iii(-)/MEM(+)	1(+)/iii(-)/MEM(+)	1(-)/iii(+)/MEM(+)	1(+)/iii(+)/MEM(+)
					KP (producing KPC enzyme)	＞128	＞128	＞128	4
XDR-PA	＞128	64	32	4
					XDR-AB	＞128	128	16	0.25
	11(-)/i(-)/MEM(+)	11(+)/i(-)/MEM(+)	11(-)/i(+)/MEM(+)	11(+)/i(+)/MEM(+)
					KP (producing KPC enzyme)	＞128	＞128	＞128	2
XDR-PA	＞128	64	32	2
					XDR-AB	＞128	64	32	＜0.125
	11(-)/ii(-)/MEM(+)	11(+)/ii(-)/MEM(+)	11(-)/ii(+)/MEM(+)	11(+)/ii(+)/MEM(+)
					KP (producing KPC enzyme)	＞128	＞128	＞128	8
XDR-PA	＞128	64	64	4
					XDR-AB	＞128	64	64	0.5
	11(-)/iii(-)/MEM(+)	11(+)/iii(-)/MEM(+)	11(-)/iii(+)/MEM(+)	11(+)/iii(+)/MEM(+)
					KP (producing KPC enzyme)	＞128	＞128	＞128	2
XDR-PA	＞128	64	32	4
					XDR-AB	＞128	64	16	0.25
	15(-)/i(-)/MEM(+)	15(+)/i(-)/MEM(+)	15(-)/i(+)/MEM(+)	15(+)/i(+)/MEM(+)
					KP (producing KPC enzyme)	＞128	128	＞128	1
XDR-PA	＞128	＞128	32	2
					XDR-AB	＞128	64	32	0.25
	15(-)/ii(-)/MEM(+)	15(+)/ii(-)/MEM(+)	15(-)/ii(+)/MEM(+)	15(+)/ii(+)/MEM(+)
					KP (producing KPC enzyme)	＞128	128	＞128	4
XDR-PA	＞128	＞128	64	4
					XDR-AB	＞128	64	64	0.5
	15(-)/iii(-)/MEM(+)	15(+)/iii(-)/MEM(+)	15(-)/iii(+)/MEM(+)	15(+)/iii(+)/MEM(+)
					KP (producing KPC enzyme)	＞128	128	＞128	8
XDR-PA	＞128	＞128	32	4
					XDR-AB	＞128	64	16	0.5

TABLE 26 Effect of dithiocarbamate derivatives, metal ion chelators and compositions thereof on the MIC of cefoperazone sulbactam

	1(-)/i(-)/CFP(+)	1(+)/i(-)/CFP(+)	1(-)/i(+)/CFP(+)	1(+)/i(+)/CFP(+)
					KP (producing KPC enzyme)	＞512	＞512	＞512	4
XDR-PA	＞512	＞512	128	2
					XDR-AB	＞512	128	128	0.25
	1(-)/ii(-)/CFP(+)	1(+)/ii(-)/CFP(+)	1(-)/ii(+)/CFP(+)	1(+)/ii(+)/CFP(+)
					KP (producing KPC enzyme)	＞512	＞512	＞512	4
XDR-PA	＞512	＞512	128	4
					XDR-AB	＞512	128	256	0.5
	1(-)/iii(-)/CFP(+)	1(+)/iii(-)/CFP(+)	1(-)/iii(+)/CFP(+)	1(+)/iii(+)/CFP(+)
					KP (producing KPC enzyme)	＞512	＞512	＞512	4
XDR-PA	＞512	＞512	128	4
					XDR-AB	＞512	128	256	0.5
	11(-)/i(-)/CFP(+)	11(+)/i(-)/CFP(+)	11(-)/i(+)/CFP(+)	11(+)/i(+)/CFP(+)
					KP (producing KPC enzyme)	＞512	＞512	＞512	2
XDR-PA	＞512	256	128	2
					XDR-AB	＞512	128	128	＜0.125
	11(-)/ii(-)/CFP(+)	11(+)/ii(-)/CFP(+)	11(-)/ii(+)/CFP(+)	11(+)/ii(+)/CFP(+)
					KP (producing KPC enzyme)	＞512	＞512	＞512	4
XDR-PA	＞512	256	128	4
					XDR-AB	＞512	128	256	1
	11(-)/iii(-)/CFP(+)	11(+)/iii(-)/CFP(+)	11(-)/iii(+)/CFP(+)	11(+)/iii(+)/CFP(+)
					KP (producing KPC enzyme)	＞512	＞512	＞512	4
XDR-PA	＞512	256	128	4
					XDR-AB	＞512	128	256	1
	15(-)/i(-)/CFP(+)	15(+)/i(-)/CFP(+)	15(-)/i(+)/CFP(+)	15(+)/i(+)/CFP(+)
					KP (producing KPC enzyme)	＞512	256	＞512	2
XDR-PA	＞512	256	128	2
					XDR-AB	＞512	128	128	＜0.125
	15(-)/ii(-)/CFP(+)	15(+)/ii(-)/CFP(+)	15(-)/ii(+)/CFP(+)	15(+)/ii(+)/CFP(+)
					KP (producing KPC enzyme)	＞512	256	＞512	4
XDR-PA	＞512	256	128	2
					XDR-AB	＞512	128	256	1
	15(-)/iii(-)/CFP(+)	15(+)/iii(-)/CFP(+)	15(-)/iii(+)/CFP(+)	15(+)/iii(+)/CFP(+)
					KP (producing KPC enzyme)	＞512	256	＞512	4
XDR-PA	＞512	256	128	4
					XDR-AB	＞512	128	256	0.25

TABLE 27 Effect of dithiocarbamate derivatives, metal ion chelators and compositions thereof on amikacin MIC

	1(-)/i(-)/AK(+)	1(+)/i(-)/AK(+)	1(-)/i(+)/AK(+)	1(+)/i(+)/AK(+)
					KP (producing KPC enzyme)	＞512	＞512	128	4
XDR-PA	＞512	256	64	4
					XDR-AB	＞512	256	64	0.125
	1(-)/ii(-)/AK(+)	1(+)/ii(-)/AK(+)	1(-)/ii(+)/AK(+)	1(+)/ii(+)/AK(+)
					KP (producing KPC enzyme)	＞512	＞512	128	8
XDR-PA	＞512	256	128	4
					XDR-AB	＞512	256	64	0.25
	1(-)/iii(-)/AK(+)	1(+)/iii(-)/AK(+)	1(-)/iii(+)/AK(+)	1(+)/iii(+)/AK(+)
					KP (producing KPC enzyme)	＞512	＞512	128	8
XDR-PA	＞512	256	64	8
					XDR-AB	＞512	256	64	0.5
	11(-)/i(-)/AK(+)	11(+)/i(-)/AK(+)	11(-)/i(+)/AK(+)	11(+)/i(+)/AK(+)
					KP (producing KPC enzyme)	＞512	＞512	128	4
XDR-PA	＞512	256	64	4
					XDR-AB	＞512	128	64	＜0.125
	11(-)/ii(-)/AK(+)	11(+)/ii(-)/AK(+)	11(-)/ii(+)/AK(+)	11(+)/ii(+)/AK(+)
					KP (producing KPC enzyme)	＞512	＞512	128	8
XDR-PA	＞512	256	128	4
					XDR-AB	＞512	128	64	0.25
	11(-)/iii(-)/AK(+)	11(+)/iii(-)/AK(+)	11(-)/iii(+)/AK(+)	11(+)/iii(+)/AK(+)
					KP (producing KPC enzyme)	＞512	＞512	128	4
XDR-PA	＞512	256	64	4
					XDR-AB	＞512	128	64	0.5
	15(-)/i(-)/AK(+)	15(+)/i(-)/AK(+)	15(-)/i(+)/AK(+)	15(+)/i(+)/AK(+)
					KP (producing KPC enzyme)	＞512	512	128	4
XDR-PA	＞512	256	64	2
					XDR-AB	＞512	128	64	＜0.125
	15(-)/ii(-)/AK(+)	15(+)/ii(-)/AK(+)	15(-)/ii(+)/AK(+)	15(+)/ii(+)/AK(+)
					KP (producing KPC enzyme)	＞512	512	128	4
XDR-PA	＞512	256	128	4
					XDR-AB	＞512	128	64	0.25
	15(-)/iii(-)/AK(+)	15(+)/iii(-)/AK(+)	15(-)/iii(+)/AK(+)	15(+)/iii(+)/AK(+)
					KP (producing KPC enzyme)	＞512	512	128	8
XDR-PA	＞512	256	64	4
					XDR-AB	＞512	128	64	1

TABLE 28 Effect of dithiocarbamate derivatives, metal ion chelators, and compositions thereof on MIC of levofloxacin

	1(-)/i(-)/LFX(+)	1(+)/i(-)/LFX(+)	1(-)/i(+)/LFX(+)	1(+)/i(+)/LFK(+)
					KP (producing KPC enzyme)	＞64	32	32	0.5
XDR-PA	64	64	16	1
					XDR-AB	＞64	16	16	0.125
	1(-)/ii(-)/LFX(+)	1(+)/ii(-)/LFX(+)	1(-)/ii(+)/LFX(+)	1(+)/ii(+)/LFK(+)
					KP (producing KPC enzyme)	＞64	32	32	1
XDR-PA	64	64	16	1
					XDR-AB	＞64	16	16	0.25
	1(-)/iii(-)/LFX(+)	1(+)/iii(-)/LFX(+)	1(-)/iii(+)/LFX(+)	1(+)/iii(+)/LFK(+)
					KP (producing KPC enzyme)	＞64	32	16	0.5
XDR-PA	64	64	16	1
					XDR-AB	＞64	16	16	0.125
	11(-)/i(-)/LFX(+)	11(+)/i(-)/LFX(+)	11(-)/i(+)/LFX(+)	11(+)/i(+)/LFX(+)
					KP (producing KPC enzyme)	＞64	32	32	0.5
XDR-PA	64	32	16	1
					XDR-AB	＞64	16	16	0.125
	11(-)/ii(-)/LFX(+)	11(+)/ii(-)/LFX(+)	11(-)/ii(+)/LFX(+)	11(+)/ii(+)/LFX(+)
					KP (producing KPC enzyme)	＞64	32	32	2
XDR-PA	64	32	16	2
					XDR-AB	＞64	16	16	0.125
	11(-)/iii(-)/LFX(+)	11(+)/iii(-)/LFX(+)	11(-)/iii(+)/LFX(+)	11(+)/iii(+)/LFX(+)
					KP (product of KP)KPC enzyme)	＞64	32	16	2
XDR-PA	64	32	16	2
					XDR-AB	＞64	16	16	0.5
	15(-)/i(-)/LFX(+)	15(+)/i(-)/LFX(+)	15(-)/i(+)/LFX(+)	15(+)/i(+)/LFX(+)
					KP (producing KPC enzyme)	＞64	32	32	0.5
XDR-PA	64	32	16	1
					XDR-AB	＞64	16	16	0.5
	15(-)/ii(-)/LFX(+)	15(+)/ii(-)/LFX(+)	15(-)/ii(+)/LFX(+)	15(+)/ii(+)/LFX(+)
					KP (producing KPC enzyme)	＞64	32	32	1
XDR-PA	64	32	16	1
					XDR-AB	＞64	16	16	0.5
	15(-)/iii(-)/LFX(+)	15(+)/iii(-)/LFX(+)	15(-)/iii(+)/LFX(+)	15(+)/iii(+)/LFX(+)
					KP (producing KPC enzyme)	＞64	32	16	1
XDR-PA	64	32	16	1
					XDR-AB	＞64	16	16	0.5

As can be seen from tables 25 to 28,

(1) the drug resistance of the 3 strains of drug-resistant bacteria is 32 times of that of sensitive bacteria, and even if meropenem (128 mu g/ml), cefoperazone sulbactam (512 mu g/ml), amikacin (512 mu g/ml) and levofloxacin (64 mu g/ml) are used in high dose, the pathogenic bacteria cannot be effectively eliminated.

(2) The single use of the dithiocarbamate derivative or the single use of the metal ion chelating agent can reduce the MIC value of the antibiotic to 1/2-1/4 of the original value, but the concentration of the antibiotic is still far greater than the concentration which can be achieved by blood, so the single use of the dithiocarbamate derivative or the metal ion chelating agent in combination with the antibiotic has little clinical significance for treating the 3 kinds of drug-resistant bacteria.

(3) When the 9 a + b compositions selected based on the example 1 are combined with antibiotics, the MIC value of the antibiotics can be reduced remarkably, the MIC value is generally reduced by more than 32 times, the MIC value can be reduced by more than 1000 times at most, and the clinical treatment significance is achieved (the clinical average blood concentration is far greater than the MIC value of the antibiotics at the moment).

[ Experimental conclusion ]

The test shows that the pharmaceutical composition a + b provided by the invention not only can reduce the MIC of lactam antibiotics, but also has similar effects on fluoroquinolone antibiotics and aminoglycoside antibiotics, and the effects are relatively more excellent in 3 combinations of 1/i, 11/i and 15/i.

Furthermore, it is known that, because of different pharmaceutical mechanisms, both fluoroquinolone antibiotics and aminoglycoside antibiotics cannot be decomposed by beta-lactamase (containing metal beta-lactamase), the pharmaceutical composition provided by the invention can restore the sensitivity effect of fluoroquinolone antibiotics and aminoglycoside antibiotics, belongs to the innovative discovery of the inventor, and can not be referred to and inspired from the fact that the pharmaceutical composition provided by the invention is effective on beta-lactamase antibiotics.

EXAMPLE 4 investigation of the sensitivity of a pharmaceutical composition consisting of different components a and b to antibiotics by a synergistic pan-resistant bacterium

[ Experimental materials ]

(1) The pharmaceutical composition comprises: 3 combinations of active component a and active component b, which are 1/i, 11/i and 15/i;

[ EXPERIMENTAL OBJECTS ] of the present invention

Based on the 3 a + b combinations selected in example 4, the 3 combinations were examined for synergistic antibacterial efficacy against different types of antibiotics.

[ Experimental methods ]

Taking the 3 strains of the drug-resistant bacteria in the logarithmic growth phase, preparing the bacteria liquid into 0.5 McLeod turbidity, continuously diluting by 100 times (corresponding to 1-2 multiplied by 106 bacteria number/ml) with a culture solution containing meropenem (4 mu g/ml) or cefoperazone sulbactam (8 mu g/ml) or amikacin (8 mu g/ml) or levofloxacin (2 mu g/ml) before use, adding 96-well plates into each well by 150 mu L, then respectively adding the dithiocarbamic acid derivatives 1,11,15 and the metal ion chelating agent i which are diluted by times, wherein the corresponding final concentrations are respectively 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 and 0.063mmol/L, measuring the turbidity after 24 hours, and calculating the MIC value through software.

Whether there was a synergistic effect between the dithiocarbamate derivative 1,11,15 and the metal ion chelator i in increasing bacterial sensitivity was determined by the microchart method. And adding 50 mu l of chelating agents i with different concentrations of 1/64MIC, 1/32MIC, 1/16MIC, 1/8MIC, 1/4MIC, 1/2MIC, 1MIC and 2MIC into each hole of the A-H row of the 96-well plate in sequence. 50 mu l of dithiocarbamic acid derivatives with different concentrations of 1/128MIC, 1/64MIC, 1/32MIC, 1/16MIC, 1/8MIC, 1/4MIC, 1/2MIC, 1MIC, 2MIC and 4MIC are added into each hole of the 1 st to 10 th vertical columns of the 96-hole plate respectively. Then, 100. Mu.l of the bacterial suspension was added to each well at a concentration of 3X 106 CFU/mL. Culturing in a constant temperature incubator at 37 deg.C for 24h, and determining the lowest drug concentration without germ growth as MIC. The culture broths involved in the test all contained the corresponding concentrations of antibiotics. The antibacterial effect of the medicine is evaluated by calculating part of antibacterial concentration index (FICI), the FICI is not more than 0.5 and is synergistic effect, the FICI is more than 0.5 and is not more than 1 and is additive effect, the FICI is more than 1 and is not more than 2 and is irrelevant effect, and the FICI is more than 2 and is antagonistic effect. The calculation formula is as follows:

FICI = (MICa combination/single drug MICa) + (MICb combination/single drug MICb). In the formula (I), the compound is shown in the specification,

"MICa combination" means the minimum inhibitory concentration of the dithiocarbamate derivative when the dithiocarbamate derivative is used in combination with a metal ion chelating agent;

"MICb combination" represents the minimum inhibitory concentration of the metal ion chelating agent in the combination of the metal ion chelating agent with the dithiocarbamate derivative;

"MICa single drug" means the minimum inhibitory concentration of the dithiocarbamate derivative alone in combination with the antibiotic at the concentrations described above;

"MICb single drug" represents the minimum inhibitory concentration when a metal ion chelator is used alone in combination with the antibiotic at the concentrations described above.

[ Experimental results ]

The results are shown in tables 29 to 31.

TABLE 29 test for the synergistic increase of antibiotic susceptibility of pan-resistant bacteria of active ingredient a1 and active ingredient bi

TABLE 30 test for increasing antibiotic susceptibility of pan-resistant bacteria synergistically with active ingredient a11 and active ingredient bi

TABLE 31 test for the synergistic increase in the susceptibility of pan-resistant bacteria to antibiotics of active ingredient a15 and active ingredient bi

[ Experimental results ]

As can be seen from tables 29 to 31, at the concentrations of meropenem (4. Mu.g/ml), cefoperazone sulbactam (8. Mu.g/ml), amikacin (8. Mu.g/ml) and levofloxacin (2. Mu.g/ml), the sensitivity of the drug-resistant bacteria to the antibiotics at the above concentrations can be restored only by increasing the dose of the dithiocarbamate derivative (i.e., active ingredient a) 1,11,15 or the metal ion chelating agent (i.e., active ingredient a) i alone to 4 to 8mmol/L, whereas the combination of the dithiocarbamate derivative and the metal ion chelating agent can restore the sensitivity of the drug-resistant strain to the antibiotics only at a lower dose.

[ Experimental conclusion ]

From the FICI values in tables 29 to 31, it is clear that the dithiocarbamate derivatives 1,11 and 15, when used in combination with the metal ion chelating agent i, have a significant synergistic effect on 4 antibiotics in terms of increasing bacterial susceptibility, and the FICI values are all less than 0.5, generally between 0.06 and 0.25, so that the two classes of compounds in the composition are not simply added in terms of promoting antibiotic synergism or increasing bacterial susceptibility.

Example 5 investigation of resistance status of different types of pan-resistant strains

[ purpose of experiment ]

In order to further verify the antibacterial effect of the composition combined with antibiotics such as meropenem, cefoperazone sulbactam, amikacin, levofloxacin and the like, multidrug-resistant or pan-resistant pseudomonas aeruginosa XDR-PA (16 strains), acinetobacter baumannii XDR-AB (13 strains, wherein the strains produce metal beta-lactamase 2 strains) and klebsiella pneumoniae XDR-KP (15 strains, wherein the strains produce metal beta-lactamase 3 strains and produce KPC enzyme 7 strains) are additionally collected clinically; and selecting dithiocarbamic acid derivatives 1,11 and 15 and a metal ion chelating agent i, and investigating 7 conditions of 1,11,15, i, 1/i, 11/i and 15/i combined with meropenem, cefoperazone sulbactam, amikacin and levofloxacin to obtain 28 combined modes with the antibacterial effective rate on the 44 multi-drug-resistant or pan-drug-resistant pathogenic bacteria.

[ Experimental methods ]

Taking the above 44 pathogenic bacteria in logarithmic growth phase, preparing bacterial liquid into 0.5 McLeod turbidity, continuously diluting 100 times (equivalent to 1-2 × 106 bacteria/ml) before use, adding 150 μ L of 96-well plate per well, then adding 50 μ L of corresponding antibiotics or compositions containing antibiotics, wherein the final concentration of meropenem, cefoperazone sulbactam, amikacin and levofloxacin is 4 μ g/ml, 8 μ g/ml and 2 μ g/ml, the final concentration of dithiocarbamic acid derivatives 1,11 and 15 and the metal ion chelating agent i is 0.5mmol/L, incubating for 24 hours, and visually inspecting to determine the effectiveness by clarification and transparency.

[ Experimental results ]

The results of the drug resistance of Pseudomonas aeruginosa XDR-PA (16 strains), acinetobacter baumannii XDR-AB (13 strains) and Klebsiella pneumoniae XDR-KP (15 strains) are shown in tables 32-34.

TABLE 32 drug resistance of the 16 multidrug-resistant or pan-resistant Pseudomonas aeruginosa strains

XDR-PA numbering

MEM

IPM

FEP

CAZ

PRL

ATM

TIM

PIP

CFP

TOB

AK

CN

LFX

CIP

PB

1

R

I

R

S

R

S

2

R

I

R

S

R

S

3

R

S

R

S

4

R

S

R

5

S

R

S

R

S

6

S

R

I

R

S

7

R

S

R

S

R

S

8

S

R

S

9

R

I

R

S

10

R

S

R

S

11

S

R

S

R

S

R

S

12

R

S

R

S

13

R

S

R

S

14

R

S

R

15

R

S

16

R

I

R

S

TABLE 33 drug resistance of multidrug-resistant or pan-resistant A.baumannii strains

XDR-AB numbering

MEM

IPM

FEP

CAZ

TIM

PIP

CFP

CRO

SAM

AK

CN

CIP

LFX

MH

PB

RL

1

R

I

R

I

S

R

S

R

2

R

S

R

3

R

I

R

S

R

4

R

S

R

5

S

R

S

R

S

R

6

R

S

7

R

S

R

S

8

R

S

R

9

R

S

R

S

10

R

S

R

S

R

11

R

S

12

R

S

I

13

S

R

S

R

I

R

S

R

S

TABLE 34 drug resistance of multidrug-resistant or pan-resistant Klebsiella pneumoniae

XDR-KP numbering

MEM

IPM

FEP

CAZ

TIM

PIP

CFP

CRO

FOX

SAM

AK

CN

CIP

LFX

MH

RL

1

R

S

R

2

R

S

R

3

S

R

S

R

S

R

S

R

4

R

S

R

5

S

R

S

6

R

7

R

S

R

8

R

9

R

S

R

10

R

S

R

11

R

S

R

12

S

R

13

R

S

R

14

R

S

R

15

R

In tables 31 to 34, R: drug resistance; i: an intermediary; s: sensitivity;

MEM: meropenem; IPM: imipenem; FEP: cefepime; CAZ: ceftazidime; TIM: ticarcillin/clavulanic acid; PIP: piperacillin/tazobactam; CPF: cefoperazone/sulbactam; CRO: ceftriaxone; FOX: cefoxitin; SAM: ampicillin/sulbactam; AK: amikacin; CN: gentamicin; CIP: ciprofloxacin; LFX: levofloxacin; MH: minocycline; PB: polymyxin; RL: compound sulfamethoxazole.

In addition, the antibacterial efficacy results of the pharmaceutical composition of the present invention are shown in fig. 1 to 3. In FIGS. 1 to 3, "Con" indicates the use of only meropenem (4. Mu.g/ml), cefoperazone sulbactam (8. Mu.g/ml), amikacin (8. Mu.g/ml), levofloxacin (2. Mu.g/ml) groups; "1", "11", "15" and "i" represent the group of antibiotics of dithiocarbamate derivatives 1,11,15 and metal ion chelator i, respectively, in combination with the concentrations indicated above; "1/i", "11/i" and "15/i" represent combinations of the compositions with the above concentrations of antibiotics.

[ Experimental conclusion ]

From the results of fig. 1 to fig. 3, it can be known that meropenem, cefoperazone sulbactam, amikacin, and levofloxacin respectively combine with 3 pharmaceutical compositions (i.e. 1/i, 11/i, and 15/i) provided by the present invention to effectively kill pathogenic bacteria, the effective rate of 44 resistant bacteria in total tested 3 types is above 90%, and the effective rate of partial compositions combined with antibiotics is close to 100%.

However, the antibacterial effectiveness of 4 antibiotics such as meropenem, cefoperazone sulbactam, amikacin and levofloxacin is generally about 30% by using dithiocarbamic acid derivatives (such as No. 1,11, 15) alone or using metal ion chelating agents (such as No. i) in combination, which is far lower than the effectiveness of the combination antibiotics.

Therefore, the pharmaceutical composition provided by the invention is also shown to have remarkable synergistic effect on different pan-drug resistant strains, namely pseudomonas aeruginosa, acinetobacter baumannii and klebsiella pneumoniae.

Claims

1. An anti-infective pharmaceutical composition comprising the following active ingredients:

b) A metal ion chelating agent selected from the group consisting of:

ethylenediamine tetramethylene phosphonate (i), hexamethylenediamine tetramethylmethylene phosphonate (ii), diethylenetriamine pentamethylene phosphonate (iii);

when the active components a and b in the pharmaceutical composition are in any molar ratio, the pharmaceutical composition is combined with the aminoglycoside antibiotic amikacin, and has an inhibition effect on pseudomonas aeruginosa negative by multidrug-resistant or pan-resistant metallo-beta-lactamase:

a1: bi = (18; or

a1: bii = (17; or

a1: b iii = (17; or

a11: bi = (18; or

a11: bii = (18; or

a11: b iii = (18; or

a15: bi = (18; or

a15: bii = (17; or

a15：bⅲ＝(17:3)～(4:16)。

2. An anti-infective pharmaceutical composition comprising the following active ingredients:

b) A metal ion chelating agent selected from the group consisting of:

when the active components a and b in the pharmaceutical composition are in any molar ratio as follows, the pharmaceutical composition is combined with the aminoglycoside antibiotic amikacin, and has an inhibition effect on multidrug-resistant or pan-resistant metal beta-lactamase negative klebsiella pneumoniae:

a1: bi = (18; or

a1: bii = (17; or

a1: b iii = (17; or

a11: bi = (18; or

a11: bii = (18; or

a11: b iii = (18; or

a15: bi = (18; or

a15: bii = (17; or

a15：bⅲ＝(18:2)～(3:17)。

3. An anti-infective pharmaceutical composition comprising the following active ingredients:

b) A metal ion chelating agent selected from the group consisting of:

ethylenediamine tetramethylene phosphonate (i), hexamethylenediamine tetramethylene phosphonate (ii), diethylenetriamine pentamethylene phosphonate (iii);

when the active components a and b in the pharmaceutical composition are in any one of the following molar ratios, the pharmaceutical composition is combined with the aminoglycoside antibiotic amikacin, and has an inhibition effect on multi-drug resistant or pan-drug resistant metal beta-lactamase negative acinetobacter baumannii:

a1: bi = (18; or

a1: bii = (17; or

a1: b iii = (17; or

a11: bi = (18; or

a11: bii = (18; or

a11: b iii = (18; or

a15: bi = (18; or

a15: bii = (17; or

a15：bⅲ＝(18:2)～(2:18)。

4. An anti-infective pharmaceutical composition comprising the following active ingredients:

b) A metal ion chelating agent selected from the group consisting of:

when the active components a and b in the pharmaceutical composition are in any molar ratio as follows, the pharmaceutical composition is combined with quinolone antibiotic levofloxacin to inhibit multi-drug resistant or pan-drug resistant metal beta-lactamase negative pseudomonas aeruginosa:

a1: bi = (18; or

a1: bii = (17; or

a1: b iii = (17; or

a11: bi = (18; or

a11: bii = (18); or

a11: b iii = (18; or

a15: bi = (18; or

a15: bii = (17; or

a15：bⅲ＝(18:2)～(3:17)。

5. An anti-infective pharmaceutical composition comprising the following active ingredients:

b) A metal ion chelating agent selected from the group consisting of:

when the active components a and b in the pharmaceutical composition are in any molar ratio as follows, the pharmaceutical composition is combined with quinolone antibiotic levofloxacin, and has an inhibiting effect on multidrug-resistant or pan-resistant metal beta-lactamase negative klebsiella pneumoniae:

a1: bi = (17; or

a1: bii = (17; or

a1: b iii = (16); or

a11: bi = (17; or

a11: bii = (16); or

a11: b iii = (17; or

a15: bi = (17; or

a15: bii = (16); or

a15：bⅲ＝(17:3)～(3:17)。

6. An anti-infective pharmaceutical composition comprising the following active ingredients:

b) A metal ion chelating agent selected from the group consisting of:

when the active ingredients a and b in the pharmaceutical composition are in any of the following molar ratios, the pharmaceutical composition is combined with quinolone antibiotic levofloxacin, and has an inhibition effect on multi-drug resistant or pan-drug resistant metal beta-lactamase negative acinetobacter baumannii:

a1: bi = (18; or

a1: bii = (17; or

a1: b iii = (16); or

a11: bi = (18; or

a11: bii = (17; or

a11: b iii = (17; or

a15: bi = (18; or

a15: bii = (17; or

a15：bⅲ＝(17:3)～(3:17)。

7. An anti-infective pharmaceutical composition comprising the following active ingredients:

b) A metal ion chelating agent selected from the group consisting of:

when the active ingredients a and b in the pharmaceutical composition are in any molar ratio as follows, the pharmaceutical composition is combined with the cephalosporin antibiotic cefoperazone sulbactam, and has an inhibiting effect on multi-drug resistant or pan-drug resistant metal beta-lactamase negative pseudomonas aeruginosa:

a1: bi = (18; or

a1: bii = (17; or

a1: b iii = (17; or

a11: bi = (18; or

a11: bii = (17; or

a11: b iii = (18; or

a15: bi = (18; or

a15: bii = (17; or

a15：bⅲ＝(17:3)～(4:16)。

8. An anti-infective pharmaceutical composition comprising the following active ingredients:

b) A metal ion chelating agent selected from the group consisting of:

when the active components a and b in the pharmaceutical composition are in any molar ratio as follows, the pharmaceutical composition is combined with cephalosporin antibiotics cefoperazone sulbactam, and has an inhibitory effect on multidrug-resistant or pan-resistant metallo-beta-lactamase negative klebsiella pneumoniae:

a1: bi = (17; or

a1: bii = (17; or

a1: b iii = (17; or

a11: bi = (18; or

a11: bii = (17; or

a11: b iii = (17; or

a15: bi = (18; or

a15: bii = (17; or

a15：bⅲ＝(17:3)～(3:17)。

9. An anti-infective pharmaceutical composition comprising the following active ingredients:

b) A metal ion chelating agent selected from the group consisting of:

when the active ingredients a and b in the pharmaceutical composition are in any one of the following molar ratios, the pharmaceutical composition is combined with cephalosporin antibiotics cefoperazone sulbactam, and has an inhibitory effect on multi-drug resistant or pan-drug resistant metallo-beta-lactamase negative acinetobacter baumannii:

a1: bi = (17; or

a1: bii = (17; or

a1: b iii = (17; or

a11: bi = (18; or

a11: bii = (18); or

a11: b iii = (18; or

a15: bi = (17; or

a15: bii = (17; or

a15：bⅲ＝(17:3)～(3:17)。

10. An anti-infective pharmaceutical composition comprising the following active ingredients:

b) A metal ion chelating agent selected from the group consisting of:

when the active components a and b in the pharmaceutical composition are in any one of the following molar ratios, the pharmaceutical composition is combined with carbapenem antibiotic meropenem, and has an inhibiting effect on multidrug-resistant or pan-resistant metallo-beta-lactamase negative pseudomonas aeruginosa:

a1: bi = (18; or

a1: bii = (18; or

a1: b iii = (18; or

a11: bi = (19; or

a11: bii = (18; or

a11: b iii = (18; or

a15: bi = (18; or

a15: bii = (18; or

a15：bⅲ＝(18:2)～(4:16)。

11. An anti-infective pharmaceutical composition comprising the following active ingredients:

b) A metal ion chelating agent selected from the group consisting of:

when the active components a and b in the pharmaceutical composition are in any one of the following molar ratios, the pharmaceutical composition is combined with carbapenem antibiotic meropenem, and has an inhibiting effect on multidrug-resistant or pan-resistant metallo-beta-lactamase negative klebsiella pneumoniae:

a1: bi = (17; or

a1: bii = (16; or

a1: b iii = (16; or

a11: bi = (17; or

a11: bii = (15; or

a11: b iii = (17; or

a15: bi = (17; or

a15: bii = (16); or

a15：bⅲ＝(17:3)～(3:17)。

12. An anti-infective pharmaceutical composition comprising the following active ingredients:

b) A metal ion chelating agent selected from the group consisting of:

when the active components a and b in the pharmaceutical composition are in any molar ratio, the pharmaceutical composition is combined with the carbapenem antibiotic meropenem, and has an inhibiting effect on multi-drug resistant or pan-drug resistant metal beta-lactamase negative acinetobacter baumannii:

a1: bi = (18; or

a1: bii = (17; or

a1: b iii = (17; or

a11: bi = (18; or

a11: bii = (17; or

a11: b iii = (18; or

a15: bi = (17; or

a15: bii = (17; or

a15：bⅲ＝(17:3)～(2:18)。

13. The application of a pharmaceutical composition in preparing an anti-infective medicament is characterized in that the pharmaceutical composition comprises the following active ingredients:

b) A metal ion chelating agent selected from the group consisting of:

ethylenediaminetetramethylenephosphonate;

the pharmaceutical composition can synergistically increase the sensitivity of the following antibiotics to multidrug-resistant or pan-resistant metallo-beta-lactamase negative pseudomonas aeruginosa, acinetobacter baumannii or klebsiella pneumoniae: meropenem, cefoperazone sulbactam, amikacin, levofloxacin.