CN117442739A - Application of combined compound medicine in treatment of pathogenic bacteria persistent infection - Google Patents

Abstract

The invention discloses an application of a combined compound medicine in the treatment of pathogenic bacteria persistent infection. The existing antibiotic single-drug treatment or combined drug treatment is generally difficult to kill pathogenic bacteria in a persistent state, particularly nutrition-insensitive persistent bacteria, which often cause a series of problems such as long treatment period, repeated attack and deterioration of illness state, treatment failure, drug resistance mutation and the like. At present, no medicine capable of efficiently killing high-retention pathogenic bacteria in a clinically applicable concentration range is known. The polymyxin and aminoglycoside antibiotic novel compound medicine disclosed by the invention can destroy bacterial cell membranes within a relatively wide range of clinical blood concentration and in a short time, so that a large number of bacteria die quickly, but is safe to human cells, thus being expected to be applied to clinical treatment of various acute or chronic infectious diseases caused by persistent pathogenic bacteria, shortening the course of treatment, preventing infection recurrence and slowing down drug-resistant mutation generation.

Description

Application of combined compound medicine in treatment of pathogenic bacteria persistent infection

Technical Field

The invention belongs to the biomedical technology, relates to application of a combined compound medicine in the treatment of pathogenic bacteria persistent infection, and in particular relates to application of a combined compound medicine of polymyxin and aminoglycoside antibiotics in the treatment of pathogenic bacteria persistent infection.

Background

The ability of bacteria to remain (Persistence) results in near half of clinical antibiotic treatment failures, which are the main causes of chronic infections, recurrent episodes and exacerbations, and also accelerates the development of Resistance mutations, further exacerbating the crisis of antibiotic Resistance.

The mechanism by which bacteria persist has evolved for nearly twenty years, and is still controversial and not fully revealed. Reported in vitro retention studies can be divided into two categories depending on whether sufficient exogenous nutrients are present in the bacterial culture at the time of antibiotic treatment: (1) "nutrient insensitive" persists. Bacterial cells have extremely strong tolerance to almost all types of bactericidal antibiotics even under the condition of rich exogenous nutrients, and do not die even if killed under the condition of high-dose antibiotics, and the phenomenon is called as 'nutrition insensitive' retention, belongs to the retention in the true sense, and is the retention type which the invention aims to overcome. (2) "nutrient-sensitive" persistence/tolerance. Such a state, in which the antibiotics cannot reach the target or generate a sufficient amount of active oxygen radicals due to the lack of nutrition in the culture and the death of cells is extremely small, has been mistakenly considered to be retained in the past, and the survival phenotype thereof is actually classified as tolerance according to definition and mechanism, but is not retained in the true sense. Most literature reports that "retention" is of this type. Such models typically add antibiotics directly to the platform culture of bacteria (without fresh exogenous nutrients) or treat bacterial cells with antibiotics when starved in physiological saline, and the surviving bacteria are often killed and eliminated in large amounts only under conditions of adequate nutrition.

Effective killing of clinical anti-infective therapy deficiencyA medicine and a means for retaining pathogenic bacteria in a nutrition-insensitive way. Current reports on retention fungus drug killing are mainly derived from the second type of "nutrition sensitive" retention/tolerance study model described above. Reported in 2011 by James Collins professor task group of the American university of hemp and university institute of technology, E.coli in wild typeEscherichia coli) Glucose, fructose or mannitol is directly added into the platform LB liquid culture, so that the sterilization effect of gentamicin can be promoted, and the effect can be completely inhibited by a proton motive force inhibitor, namely carbonyl cyanide m-chlorophenylhydrazone (CCCP), which indicates that the intracellular absorption of gentamicin depends on proton motive force. The Kim Lewis topic group of university of North east America reports that the antimicrobial peptide ADEP4 can continuously activate the proteolytic enzyme ClpP, thereby playing a role in killing E.coli plateau cultures in vitro; unfortunately, clinical trials of this compound failed. The professor 2023 paid new seedlings reported that heat treatment of E.coli plateau cultures at 55℃for five minutes promoted aminoglycosides to kill bacteria remaining in the plateau, a method which was not useful for the treatment of internal tissue infections due to the inability of most human cells to withstand heat shock. In addition, the proton motive force inhibitor CCCP can be combined with high-concentration aminoglycosides (500 mug/mL), ampicillin (Ampicillin) or Ciprofloxacin (Ciprofloxacin) to kill bacteria in the platform stage; however, CCCP is highly toxic to human and animal cells even at relatively low concentrations and thus cannot be used clinically. In a word, the single-drug treatment of the common clinical antibiotics can not obtain good curative effect by killing the infected persistant bacteria, and no report that the compound antibiotics can effectively kill the persistant bacteria at the clinical blood concentration has been found.

The aminoglycoside antibiotics alone cannot be effectively killed by the 'nutrition insensitive' persister. Aminoglycosides (Aminoglycosides) are a well known group of antibiotics, which are extremely similar in structure and bactericidal mechanism, comprising different antibiotic molecules with similar functions. Common aminoglycosides mainly include Kanamycin (Kanamycin), streptomycin (Streptomycin), amikacin (Amikacin), tobramycin (Tobramycin), gentamycin (Gentamicin), netilmicin (Netilmicin), and the like. These antibiotics generally have no killing effect on "nutrient insensitive" persisters over the clinical blood concentration range.

The combined use of polymyxin and other bactericidal antibiotics has not been reported to kill persisters. Polymyxin (Polymyxin) is a well-known group of polypeptide antibiotics with extremely similar molecular structures and bactericidal mechanisms, has an inhibitory effect on most gram-negative bacteria, is usually structurally different only from ester acyl groups and two amino acids, and is mainly used as a second-line antibiotic at present, and typical clinical medicines of Polymyxin B (Polymyxin B) and Polymyxin E (Polymyxin E) are mainly used.

In conclusion, a novel molecular or existing antibiotic compound formulation capable of rapidly killing the persisting bacteria is found, and the compound formulation has important significance for clinical anti-infective therapy, particularly chronic infection and recurrent bacterial infection diseases, and has great economic value in the field of biological medicine.

Disclosure of Invention

The existing antibiotic single-drug treatment or combined drug treatment is generally difficult to kill pathogenic bacteria in a persistent state, particularly nutrition-insensitive persistent bacteria, which often cause a series of problems such as long treatment period, repeated attack and deterioration of illness state, treatment failure, drug resistance mutation and the like. At present, no medicine capable of efficiently killing high-retention pathogenic bacteria in a clinically applicable concentration range is known. The polymyxin antibiotic and aminoglycoside antibiotic novel compound medicine disclosed by the invention can destroy bacterial cell membranes in a relatively wide range of clinical blood concentration and in a short time, so that a large number of bacteria die rapidly, but is safe to human cells, thus being expected to be applied to clinical treatment of various acute or chronic infectious diseases caused by persistent pathogenic bacteria, shortening the course of treatment, preventing infection recurrence and slowing down drug-resistant mutation generation.

The invention adopts the following technical scheme:

an application of a combined compound medicine in preparing a medicament for treating pathogenic bacteria persistent infection, wherein the combined compound medicine comprises polymyxin and aminoglycoside antibiotics.

An application of a combined compound medicine in preparing a medicament for inhibiting pathogenic bacteria retention, wherein the combined compound medicine comprises polymyxin and aminoglycoside antibiotics.

An application of a combined compound medicine in preparing a medicine for synergism and elimination of persistent pathogenic bacteria, wherein the combined compound medicine comprises polymyxin and aminoglycoside antibiotics. Further, the medicament also comprises other antibacterial agents.

The combined compound medicine plays a role in inhibiting the retention of pathogenic bacteria and is safe to human cells. As a matter of common sense, safety refers to pharmaceutically acceptable safety. The growth and metabolism of the human THP-1 cells treated by the compound polymyxin-aminoglycoside antibiotics are not obviously different from those of a normal growth control group without antibiotics, however, the CCCP of the existing respiratory chain proton motive force inhibitor obviously inhibits the growth and leads to cell death. Therefore, the peak blood concentration of the compound antibiotic in clinical sterilization is far lower than the test concentration, and the compound antibiotic can theoretically ensure the safety of human cells.

Preferably, the pathogenic bacteria are nutrient insensitive persisters. Bacterial cells have extremely strong tolerance to almost all types of bactericidal antibiotics even under the condition of rich exogenous nutrients, and do not die even if killed under the condition of high-dose antibiotics, and the phenomenon is called nutrition-insensitive retention and belongs to retention in a true sense. The clinical anti-infection treatment lacks medicaments and means for effectively killing the persistent pathogenic bacteria, and the current technology about the killing of the persistent bacteria medicaments mainly aims at nutrition sensitive persistent/tolerant, and the medication scheme aiming at nutrition insensitive persistent bacteria is fresh. The invention discloses a combined compound medicament of polymyxin and aminoglycoside antibiotics for the first time, which can rapidly kill a large amount of persistent tolerating bacteria of inactive metabolism, has the effect far superior to that of single antibiotic administration, and also has the effect of killing bacteria in an active metabolism growth state superior to that of single antibiotic, so that the invention has potential important application value in clinical anti-bacterial infection treatment, is expected to be used for treating bacterial infection diseases including pneumonia, blood flow infection, urinary tract infection, respiratory tract infection, intestinal tract infection and the like, and is also expected to be used for bacterial infection treatment of animals.

Therefore, the invention discloses application of a combined compound medicine in preparing anti-inflammatory or anti-infective medicines, wherein the combined compound medicine comprises polymyxin and aminoglycoside antibiotics.

In the present invention, the polymyxin antibiotics include polymyxin or salts, hydrates, solvates thereof; aminoglycoside antibiotics include aminoglycosides or salts, hydrates, solvates thereof.

In the invention, the aminoglycoside antibiotics are a large class of molecules with similar structures and functions, and comprise kanamycin, amikacin, tobramycin, gentamicin and streptomycin which are commonly used in clinic, and further comprise other aminoglycoside drug molecules with similar structures and functions.

In the present invention, polymyxins are a large class of molecules with similar structure and function, including polymyxin B, polymyxin E, and other molecules with similar structure and function.

In the combined compound medicine, the content of the aminoglycoside antibiotics is 0.01-99.9%, preferably 75-99%, more preferably 85-99%, and can be 86-99%, 87-99%, 88-99%, 89-99%, 90-99%, 91-99%, 92-99%, 93-99%, 94-99%, 95-99%, 96-99%, 97-99% and 98-99% by weight percent. The proportion is calculated according to the total weight of the polymyxin antibiotics and the aminoglycoside antibiotics being 100 percent, and other components such as a carrier are not included; if other carrier components are included, the equal proportion calculation is performed by weight.

In the invention, the active ingredients of the combined compound medicine are polymyxin antibiotics and aminoglycoside antibiotics, and further, the combined compound medicine can also comprise a carrier; as common knowledge, the active ingredients polymyxin and aminoglycoside antibiotics exist in the same carrier to form a compound medicine, namely the polymyxin-aminoglycoside compound antibiotics.

In the invention, pathogenic bacteria are in a platform culture period or an exponential growth period. The polymyxin-aminoglycoside compound antibiotics not only can kill the remaining bacteria of the culture in the stage of the platform stage with high efficiency, but also can kill the cells in the exponential growth phase with high efficiency, so that the survival level of the polymyxin-aminoglycoside compound antibiotics is reduced by 7 orders of magnitude, and the remaining level is reduced by 100 times. The polymyxin-aminoglycoside compound antibiotics can kill and remove bacterial cells with high efficiency whether they are in an active metabolic state during exponential growth or in a sustained state during slow or lag phase of metabolism.

In the invention, pathogenic bacteria comprise gram-negative bacteria and gram-positive bacteria, and the polymyxin-aminoglycoside compound antibiotics have 100 times lower retention and survival average compared with amikacin single-drug killing groups after killing different clinical isolate cultures of arbitrarily selected escherichia coli and klebsiella pneumoniae by the compound antibiotics; the retention level was reduced by at least 10-fold for clinical isolates of pseudomonas aeruginosa and staphylococcus aureus, a positive bacteria. These results indicate that the residual bacteria removal capacity of the polymyxin-aminoglycoside compound antibiotics is superior to that of the existing single antibiotics, and the polymyxin-aminoglycoside compound antibiotics have universality in gram-negative bacteria and gram-positive bacteria.

Compared with the prior art, the invention has the following advantages:

(1) The combined compound medicine of the polymyxin and the aminoglycoside antibiotics disclosed by the invention for the first time can quickly kill a large amount of persistent tolerating bacteria of inactive metabolism, the effect is far better than that of single antibiotic administration, and the effect of killing bacteria in the growth state of active metabolism is also better than that of single antibiotic; the combined compound of other antibiotics in the clinical concentration range and the combined compound of polymyxin and clinically common lactam or quinolone antibiotics have no effect of killing nutrition insensitive persisters in a large amount and rapidly; in the compound medicine, the combination indexes of the polymyxin B and the polymyxin E and aminoglycosides are 0.25 and 0.4 respectively, and the compound medicine has the function of rapidly destroying bacterial cell membranes, and the effects of destroying cell membranes and killing persistent bacteria are not achieved by the independent actions of polymyxin or aminoglycosides with the same concentration. Therefore, the compound medicine of the invention has potential important application value in clinical antibacterial infection treatment, is expected to be used for treating bacterial infection diseases including but not limited to pneumonia, blood flow infection, urinary tract infection, respiratory tract infection, intestinal tract infection and the like, and is also expected to be used for bacterial infection treatment of animals.

(2) The polymyxin and aminoglycoside antibiotic combined compound medicine disclosed by the invention has the advantages that the capability of clearing bacteria is widely effective in representative gram-negative bacteria and positive bacteria, and the application range in clinical anti-infection treatment and animal treatment is obviously not limited to the representative strain tested by the invention, so that the polymyxin and aminoglycoside antibiotic combined compound medicine is applicable to most pathogenic bacteria.

Drawings

FIG. 1 is a wild typeE.coliViability after killing of different types of bactericidal antibiotics; wherein a is 3 MIC kanamycin (mic=8 μg/mL), B is 20 MIC ciprofloxacin (mic=0.03 μg/mL), C is 20 MIC ampicillin (mic=6 μg/mL), D is 3 MIC polymyxin B (mic=0.3 μg/mL).

FIG. 2 is a schematic view ofhipA7The strain has high retention activity on different bactericidal antibiotics, and the wild strain is used as a control; wherein a is 3 MIC kanamycin, 3 MIC streptomycin, B is 20 MIC ciprofloxacin, 20 MIC ampicillin, C is 3 MIC polymyxin B, 3 MIC mitomycin C (mic=0.8 μg/mL).

FIG. 3 is a schematic view ofmetG2The strain has high retention activity on different bactericidal antibiotics, and the wild strain is used as a control; wherein A is 3 MIC kanamycin, 20 MIC ciprofloxacin and 20 MIC ampicillin, and B is 3 MIC polymyxin B and 3 MIC mitomycin C.

FIG. 4 is a diagram ofhipA7The strain has high retention and survival ability for combined killing of different antibiotics, and the wild strain is used as a control; wherein A is 20 MIC ciprofloxacin+20 MIC ampicillin; b is 20 MIC ciprofloxacin+3 MIC kanamycin, C is 20 MIC ciprofloxacin+3 MIC streptomycin, D is 20 MIC ciprofloxacin+3 MIC polymyxin B, E is 20 MIC ciprofloxacin+3 MIC polymyxin E, F is 20 MIC ciprofloxacin+3 MIC mitomycin C, and G is 20 MIC ampicillin+3 MIC kanamycin.

FIG. 5 is a schematic view of a displaymetG2Group of strains against different antibioticsThe synthetic killing has high retention activity, and the wild strain is used as a control; wherein A is 20 MIC ciprofloxacin+20 MIC ampicillin, B is 20 MIC ciprofloxacin+3 MIC streptomycin, C is 20 MIC ciprofloxacin+3 MIC polymyxin B, D is 20 MIC ciprofloxacin+3 MIC polymyxin E, E is 20 MIC ciprofloxacin+3 MIC mitomycin C, F is 20 MIC ampicillin+3 MIC kanamycin.

FIG. 6 shows high-efficiency killing of polymyxin B-kanamycin in combinationhipA7High retention bacteria, wherein A is a compound drug of kanamycin (final concentration of 20 mug/mL) -polymyxin B (0.9 mug/mL), and the control group is treated by single drug with 20 mug/mL kanamycin or 0.9 mug/mL polymyxin B; kanamycin in the compound medicine of B is 20 mug/mL, and polymyxin B is 0.003, 0.03, 0.3 or 0.6 mug/mL respectively; alternatively, the compound contains 8. Mu.g/mL kanamycin and 0.45. Mu.g/mL polymyxin B; the compound antibiotics of C contain 20 mug/mL kanamycin and 1 mug/mL polymyxin E, and the control group carries out single-drug killing by using 20 mug/mL kanamycin or 1 mug/mL polymyxin E; the compound medicine of D contains 30 mug/mL of ampicillin and 0.9 mug/mL of polymyxin B, 0.15 mug/mL of ciprofloxacin and 0.9 mug/mL of polymyxin B, and the control group carries out single-medicine killing by using 30 mug/mL of ampicillin, 0.15 mug/mL of ciprofloxacin or 0.9 mug/mL of polymyxin B.

FIG. 7 shows high-efficiency killing of polymyxin B-kanamycin in combinationmetG2High retention bacteria and wild typeE.coliThe remaining bacteria, wherein A isE. coli metG2Strain B was wild type and a control group was single treated with 2.5 MIC kanamycin, 3 MIC polymyxin B using a combination antibiotic containing 3 MIC polymyxin B and 2.5 MIC kanamycin.

FIG. 8 shows wild type in the index phase of efficient killing of polymyxin B-kanamycin in combinationE.coliThe bacteria are maintained, the experimental group contains compound antibiotics of 3 MIC polymyxin B and 2.5 MIC kanamycin, and the control group is single-drug killing.

FIG. 9 shows the high killing effect of polymyxin B and aminoglycosides in multiple compoundshipA7High retention bacteria, wherein the compound antibiotic of A contains 0.9 mug/mL of polymyxin B and 16 mug/mL of amikacin, 0.9 mug/mL of polymyxin B and 16 mug/mL of streptomycin,the compound antibiotics of B contain 0.9 mug/mL of polymyxin B and 16 mug/mL of tobramycin, the compound antibiotics of C contain 0.9 mug/mL of polymyxin B and 12 mug/mL of gentamicin, and the concentration of aminoglycoside molecules in the compound antibiotics of D is uniformly 6 mug/mL.

FIG. 10 shows the high killing effect of polymyxin B and aminoglycosides in multiple compoundsmetG2High retention bacteria, wherein the compound antibiotics of A contain 0.9 mu g/mL of polymyxin B and 6 mu g/mL of amikacin, the compound antibiotics of B contain 0.9 mu g/mL of polymyxin B and 6 mu g/mL of streptomycin, the compound antibiotics of C contain 0.9 mu g/mL of polymyxin B and 6 mu g/mL of tobramycin, the compound antibiotics of D contain 0.9 mu g/mL of polymyxin B and 6 mu g/mL of gentamicin, and single medicines are used as controls.

FIG. 11 shows that polymyxin B and aminoglycosides are highly effective against wild type antibioticsE. coliThe bacterium is maintained, wherein the compound antibiotics of A contain 0.9 mu g/mL of polymyxin B and 6 mu g/mL of amikacin, the compound antibiotics of B contain 0.9 mu g/mL of polymyxin B and 6 mu g/mL of streptomycin, the compound antibiotics of C contain 0.9 mu g/mL of polymyxin B and 6 mu g/mL of tobramycin, the compound antibiotics of D contain 0.9 mu g/mL of polymyxin B and 12 mu g/mL of gentamicin, and the single medicine is used as a control.

FIG. 12 shows that the polymyxin-aminoglycoside compound antibiotic has a killing effect on the retention of clinical pathogenic bacteria, wherein A is Klebsiella pneumoniae clinical isolate 9030, B is Klebsiella pneumoniae clinical isolate 9182, C is Escherichia coli clinical isolate 0005, D is Escherichia coli clinical isolate 0011, E is Pseudomonas aeruginosa clinical isolate 1128, and F is Staphylococcus aureus clinical isolate 1115.

FIG. 13 is a graph showing the effects of polymyxin B-kanamycin on a combination antibiotichipA7The cell membrane potential was lost, wherein A was a compound antibiotic containing 0.9. Mu.g/mL polymyxin B and 20. Mu.g/mL kanamycin, B was 20. Mu.g/mL kanamycin alone, and C was 0.9. Mu.g/mL polymyxin B.

FIG. 14 is a graph showing the results of polymyxin B-kanamycin hipA7Cell membrane rupture, where a is 2 hours and B is 4 hours.

FIG. 15 is a graph showing the effects of polymyxin B-kanamycin on a combination antibiotichipA7Cell membrane disruptionSplit, wherein a is a compound antibiotic containing 0.9 μg/mL polymyxin B and 20 μg/mL kanamycin, B is 20 μg/mL kanamycin alone, and C is 0.9 μg/mL polymyxin B.

FIG. 16 is an evaluation of safety of polymyxin-aminoglycoside compound antibiotics against human cells.

Detailed Description

The invention discloses application of a polymyxin antibiotic and aminoglycoside antibiotic combined compound medicine in pathogenic bacteria inhibition and killing, especially in preparation of a medicament for treating persistent infection of pathogenic bacteria, and particularly the pathogenic bacteria are 'nutrition insensitive' persistent bacteria. Wherein the polymyxin antibiotic comprises polymyxin or a salt, hydrate, solvate thereof; aminoglycoside antibiotics include aminoglycosides or salts, hydrates, solvates thereof.

In the invention, the active ingredients of the medicine are polymyxin antibiotics and aminoglycoside antibiotics, and the medicine further comprises a carrier.

In view of the fact that polymyxin is a large class of molecules with similar structures and functions, the polymyxin component range of the combined compound medicament disclosed by the invention is not limited to polymyxin B and polymyxin E, but also comprises other molecules with similar structures and functions. The content ratio of the polymyxin antibiotics in the finished polymyxin-aminoglycoside compound antibiotics is 0.001-95%, so that the applicable concentration range of the polymyxin antibiotics in the sterilization process reaches 0.001-200 mug/mL.

In view of the fact that the aminoglycoside antibiotics are a large class of molecules with similar structures and functions, the combined compound application range disclosed by the invention is not limited to kanamycin, amikacin, tobramycin, gentamicin, streptomycin and netilmicin which are commonly used in clinic, and the aminoglycoside antibiotics also comprise other aminoglycoside drug molecules with similar structures and functions. The content of aminoglycosides in the finished compound medicine is 0.01-99.5%, so that the applicable concentration range of the aminoglycosides in the sterilization process reaches 0.01-200 mug/mL.

The two proportions (content ratio) are calculated according to the total weight of the polymyxin antibiotics and the aminoglycoside antibiotics being 100 percent, and other components such as a carrier are not included; if other carrier components are included, the equal proportion calculation is performed by weight.

As a matter of common knowledge, the term salt is a pharmaceutically acceptable salt, referring to a disclosed compound or derivative, modified by preparing an acid or base salt thereof. Examples of pharmaceutically acceptable salts include: basic groups such as inorganic or organic acid salts of amines, acidic groups such as alkali metal or organic salts of carboxylic acids, conventional non-toxic salts or quaternary ammonium salts. Conventional non-toxic salts include mineral acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric and nitric acids; or salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, and the like. Pharmaceutically acceptable salts can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. In some cases, such salts may be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of both; preferred organic solvents are diethyl ether, ethyl acetate, ethanol, isopropanol or acetonitrile. As a matter of common sense, the active ingredient includes all pharmaceutically acceptable salts, all hydrates and/or solvates of the compound. Certain functional groups, such as hydroxyl, amino, etc., form complexes and/or coordination compounds with water and/or various solvents in various physical forms of the compound.

As a matter of common sense, the term carrier is a pharmaceutically acceptable carrier, is art-recognized, and refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, which participates in carrying or transporting the active ingredient. Some examples of materials that may be used as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; diols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; water; isotonic saline; ringer's solution; ethanol; phosphate buffer solution; other non-toxic compatible substances used in pharmaceutical formulations.

The mode of administration of the combination compound of the present invention is not limited and includes oral, intravenous, intramuscular, subcutaneous, transdermal, inhalation, buccal, ocular, sublingual, vaginal, rectal, etc. The combination partners of the present invention can be administered in unit dosage forms and/or formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. Exemplary forms of oral administration include tablets, capsules, elixirs, syrups and the like. External routes include intravenous, intra-arterial, intraperitoneal, epidural, intra-urethral, intrasternal, intramuscular, and subcutaneous, as well as any other art-recognized route of parenteral administration. Modes of administration include needle (including microneedle) syringes, needleless syringes, and infusion techniques, as well as any other parenteral mode of administration recognized in the art. Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffers and may also be formulated as sterile nonaqueous solutions or in dry form for use in combination with a suitable vehicle such as sterile pyrogen-free water. Preparation of parenteral formulations under sterile conditions, such as by lyophilization, can be readily accomplished using standard pharmaceutical techniques well known to those skilled in the art.

The dosage of the combination of the present invention is conventional and may be selected according to the method of administration, the condition to be treated, the severity of the condition, whether the condition is to be treated or prevented, and the age, weight and health of the person to be treated. In the present invention, the co-administration of the polymyxin and aminoglycoside antibiotic may be performed simultaneously by any suitable means, unlike prior sequential, separate or single pharmaceutical formulations.

The specific medicines adopted by the invention are existing products, the specific preparation and experimental method are conventional technologies, the test is laboratory prior art, the test strain is mainly a commercially available standard strain, and the individually adopted clinical isolate is any hospital-available infectious bacteria isolate.

The proportion of cells retained in bacterial cultures is generally higher in the plateau phase of nutrient deficiency. To find out the model of the bacterium Escherichia coliE.coli) The retention capacity of most bactericidal antibiotic types is that BW25113 standard strains with the background of escherichia coli K12 are cultivated in a commercial conventional LB liquid culture medium at 37 ℃ to a plateau, and then are inoculated into a fresh LB liquid culture medium containing a multiple of Minimum Inhibitory Concentration (MIC) antibiotics according to the ratio of 1:20 for killing, the residual antibiotics are sampled after different times, the residual antibiotics are removed by centrifugation twice, LB agar plates without antibiotics are coated after ten times gradient dilution in physiological saline, the number of surviving colonies (Colony forming unit, CFU) is counted after two days of cultivation, the initial CFU before dosing is used as a control, and the survival rate and the retention level of the bacteria are calculated, in particular, a conventional experiment method and the test reference of other bacteria are adopted. The antibiotics used are of the classical bactericidal type comprising: beta-lactams (blocking cell wall synthesis, such as ampicillin), fluoroquinolones (causing double strand DNA breaks, such as ciprofloxacin), aminoglycosides (acting on ribosomes, causing protein translation errors, such as kanamycin), polymyxins (interfering with cell membrane function, such as polymyxin B), mitomycins (causing double strand breaks by DNA interchain GC bridging), such as mitomycin C; the drug dose is at a concentration that kills sensitive bacteria, typically a known multiple MIC, and the specific concentration is calculated from the MIC and the multiple.

Fluorescence microscopy and flow cytometry detect the signal of a fluorescent substance associated with the bacterial cell, according to the standard procedures and detection modes of the instrument or reagent.

The above is a general method for the bacterial experiment of the present invention, and is also a literature-based method for analyzing the level of bacterial retention. The antibiotics adopted by the invention are all common clinical antibiotics; experimental results show that for the persistent bacteria, the existing antibiotic single-drug treatment has no good sterilization effect, and no report on compound drug treatment of the persistent bacteria is found.

1. The antibiotics are treated for a long time at high concentration, and can not effectively kill 'nutrition insensitive' persistent bacteria in wild type escherichia coli groups.

A small fraction of cells in a wild type e.coli plateau culture population have high persistence and when diluted into nutrient-rich medium for killing, even after high concentration of bactericidal antibiotic treatment, this small fraction of cells survive by remaining resistant to killing by existing clinically common antibiotics, and is therefore referred to as "nutrient-insensitive" persistence.

Wild typeE.coli BW25113 plateau cultures were treated with kanamycin (24. Mu.g/mL, 3 MIC) in excess of the peak plasma concentration (20. Mu.g/mL) for 10 hours, with 10-fold reduction in viability, maintaining the level of retention at 0.001%; similarly, 10 h was treated with ciprofloxacin (0.6. Mu.g/mL, 20 MIC), ampicillin (120. Mu.g/mL, 20 MIC) or polymyxin B (0.9. Mu.g/mL, 3 MIC), and the retention level of the culture was 0.001% (see FIG. 1). The results indicate that the wild type E. coliAbout one ten thousandth of the cells in the plateau culture are in a 'nutrition insensitive' retention state, and when the nutrition exists, the cells survive after being treated for a long time by high concentration of different kinds of common clinical sterilization antibiotics, and can recover growth and reproduction after the antibiotics are withdrawn.

Most cells in E.coli plateau cultures are in a "nutrient-sensitive" retention/tolerance state. Unlike the small portion of the "nutrient insensitive" retentive bacteria described above,E.colimost cells in the BW25113 platform culture can not reach a target or can not generate enough active oxygen free radicals under the condition that part of key nutrients without exogenous nutrients are deleted or exogenous nutrients are completely deleted, so that the antibiotics can be in a state of extremely few death; however, when diluted toThe plateau cultures can be killed largely and rapidly by various bactericidal antibiotics common in clinic after being placed in nutrient-rich medium and treated with antibiotics (see fig. 1). Most of these cells that survive the nutrient starvation but are killed in the presence of external nutrients have been mistakenly considered persisters in the past, and their survival phenotype characteristics are actually classified as tolerance by definition and are not truly persisting. Thus, retention referred to hereinafter in this disclosure refers to "nutrient insensitive" retention.

FIG. 1 is a wild typeE.coliViability after killing of different classes of bactericidal antibiotics, wherein A isE.coliThe platform culture 1:20 of BW25113 wild strain was inoculated into fresh LB liquid medium, 3 MIC kanamycin (MIC=8. Mu.g/mL) was immediately added, 10 h was cultured by shaking at 37℃and 160 rpm, samples were taken at different time points, the drug was washed off, ten-fold gradient dilution in physiological saline was performed, spotted onto LB agar plates, and viable cell Count (CFU) was measured after 2 days of culture (d); B. c, D the conditions for killing were as a, 20 MIC ciprofloxacin (mic=0.03 μg/mL), 20 MIC ampicillin (mic=6 μg/mL) or 3 MIC polymyxin B (mic=0.3 μg/mL), respectively. Each number was obtained from at least three independent experiments, error bars representing mean ± standard deviation.

2. E.coli hipA7AndmetG2the strain has a high level of retention and about 10% of cells in its plateau culture resist single drug killing by any of five broad classes of bactericidal antibiotics.

E.coli hipA7AndmetG2as two typical high-retention strains, both can be obtained by purchase or self-construction according to literature for further evaluation of the killing effect of antibiotics on high-retention cells.hipA7The strain obtained by evolutionary screening in 1983 is the first recognized high-retention cell strain and is often used for studying the retention formation mechanism. methionyl-tRNA synthetase gene reported in 2012 metG2Mutant strains are obtained by screening transposon libraries and have the same characteristics as those of the mutant strainshipA7The strains have similar high retention force.

hipA7About 10% of cell pairs in strain plateau culturesThe five types of bactericidal antibiotics have strong retention and can resist single-drug killing of any type of antibiotics.hipA7The cell culture in the plateau stage is subjected to killing treatment of 10 h in fresh LB medium containing 3 MIC kanamycin, and the retention level is maintained at 10% compared with the wild typeE. coliIncreased by 10,000 times, and similarly,hipA7the survival levels of the plateau cultures were maintained at about 10% after killing with 20 MIC ciprofloxacin, 20 MIC ampicillin, 3 MIC polymyxin B or 3 MIC mitomycin C (see fig. 2). FIG. 2 is a schematic view ofhipA7The strain has high retention activity on different bactericidal antibiotics, wherein A isE.coli hipA7The plateau cultures of strain and wild type strain were diluted 1:20 to fresh LB liquid medium, immediately added with 3 MIC kanamycin, cultured at 37℃with shaking at 160 rpm for 10 h, sampled at regular time, and the viable cell count was determined as in the condition of FIG. 1A; B. conditions for C are as in fig. 1 a, and the antibiotics used for killing are 3 MIC streptomycin, 20 MIC ciprofloxacin, 20 MIC ampicillin, 3 MIC polymyxin B or 3 MIC mitomycin C (mic=0.8 μg/mL), respectively. Each number was obtained from at least three independent experiments, error bars representing mean ± standard deviation.

metG2About 1-10% of cells in the plateau cultures of the strain have strong retention of all five classes of bactericidal antibiotics. And (3) withhipA7Similarly to this, the process is carried out,metG2the survival levels of the cell cultures at plateau stage after killing 10 h with different antibiotic single drugs, including 20 MIC ciprofloxacin, 20 MIC ampicillin, 3 MIC streptomycin, 3 MIC polymyxin B or 3 MIC mitomycin C, were maintained at 1-10% (fig. 3). FIG. 3 is a schematic view ofmetG2The strain has high retention activity on different bactericidal antibiotics; wherein A, B isE.coli metG2The platform cultures 1:20 of the strain and the wild type strain were inoculated into fresh LB liquid medium, and 3 MIC kanamycin, 20 MIC ciprofloxacin, 20 MIC ampicillin, 3 MIC polymyxin B or 3 MIC mitomycin C were added, respectively, and the culture was performed under the conditions of FIG. 1A, and the number of surviving cells treated for various times was measured. Each number was obtained from at least three independent experiments, error bars representing mean ± standard deviation.

The above results show that,E.coli hipA7andmetG2compared with the wild strain, the level of the 'nutrition insensitive' persistent bacteria in the strain is increased by thousands times, and even under the condition of rich exogenous nutrients, any single-drug treatment of the common sterilization antibiotics (including aminoglycosides and polymyxins) in clinic cannot effectively kill the persistent bacterial cells.

3. Sequential combination of bactericidal antibiotics does not effectively kill wild typeE.coliAndhipA7、metG2the remaining cells of the strain.

To examine the effect of combined killing of antibiotic single drug sequential treatment on the viability of persisters, wild typeE.coliAndhipA7、metG2the platform culture of the strain was treated with one antibiotic under conditions sufficient for exogenous nutrients 5 h, washed to remove the antibiotic, and continuously killed again with another antibiotic of a different target type 5 h to determine survival.

hipA7Immediately after 5 h of the strain culture is killed by 20 MIC ciprofloxacin, the strain culture is replaced by another target type of antibiotics for further killing, such as 20 MIC ampicillin, 3 MIC kanamycin, 3 MIC streptomycin, 3 MIC polymyxin B/polymyxin E (Colistin) and 3 MIC mitomycin C, and the cell retention level is maintained at 3-10%; also, the process of the present invention is,hipA7the culture is killed by 20 MIC ampicillin in advance by 5 h, and then killed by 3 MIC kanamycin by 5 h, and the cell retention level is still maintained at 3-10%; see fig. 4. However, wild typeE.coliThe culture, which is killed by the combination of the above-mentioned various antibiotics, has a retention level as low as 0.001-0.0001%. FIG. 4 is a diagram ofhipA7The strain has high retention and survival ability for combined killing of different antibiotics, and the wild strain is used as a control; wherein A is E.coli hipA7The platform cultures of strain and wild type strain were diluted 1:20 to fresh LB liquid medium, immediately added with 20 MIC ciprofloxacin, incubated at 37℃with shaking at 160 rpm for 5 h, centrifuged to collect the cells, washed 2 times to remove the drug, resuspended in an equal volume of fresh LB medium containing 20 MIC ampicillin, continued to incubate and kill 5 h, and surviving cells were assayed as conditions in FIG. 1AA number; the conditions of B to G are as A, and the types and concentrations of antibiotics of different killing combinations are 20 MIC ciprofloxacin, 3 MIC kanamycin, 3 MIC streptomycin, 3 MIC polymyxin B/E, 3 MIC mitomycin C, and 20 MIC ampicillin, respectively. Each number was obtained from at least three independent experiments, error bars representing mean ± standard deviation.

metG2The strain culture is combined and killed by single-drug sequential treatment of different antibiotics, and then is presented withhipA7Similar high retention. For example, after 20 MIC ciprofloxacin killing 5 h, killing was continued with 20 MIC ampicillin, 3 MIC streptomycin, or 3 MIC polymyxin B for 5 h, and the retention level of cells was maintained at 1-5% (fig. 5). FIG. 5 is a schematic view of a displaymetG2The strain has high retention and survival ability for combined killing of different antibiotics, and the wild strain is used as a control; wherein A is E.coli metG2The platform cultures of the strain and the wild strain are diluted to fresh LB liquid medium according to the ratio of 1:20, 20 MIC ciprofloxacin is immediately added, shaking culture is carried out at 37 ℃ and 160 rpm for 5 h, cells are collected by centrifugation, medicines are removed by washing for 2 times, an equal volume of fresh LB medium containing 20 MIC ampicillin is added for resuspension, culture is continued and killing is carried out for 5 h, and the number of surviving cells is measured according to the condition of A in figure 1; the conditions of B to F are as A, and the types and concentrations of antibiotics of different killing combinations are 20 MIC ciprofloxacin, 3 MIC kanamycin, 3 MIC streptomycin, 3 MIC polymyxin B/E, 3 MIC mitomycin C and 20 MIC ampicillin respectively. Each number was obtained from at least three independent experiments, error bars representing mean ± standard deviation.

The above results indicate that retention phenomena (especially nutrient insensitive retention) with broad resistance to all bactericidal antibiotics are present in cultures of different strains, such as wild typeE.coliAnd to the process for preparing the samehipA7、metG2Mutant strains, which are likely to have stress-retaining ability similar to spores or spores, which are resistant to almost all bactericidal antibiotic species currently in common use in the market, survive long-term treatment with high concentrations of antibiotics, and kill even if combined in turns against different target species The sample can survive due to the retention, which is a difficult problem that the clinical anti-infective therapy cannot overcome until now.

Example 1

The compound medicine of polymyxin and aminoglycoside antibiotics can kill various remaining bacteria in the clinical blood concentration range with high efficiency.

Under the experimental conditions of clinical blood concentration or lower than clinical blood concentration, different antibiotic compounds obtained by combining polymyxin and various aminoglycoside molecules can quickly kill various tested persistent bacteria in a large amount, so that the purpose of quickly eliminating the persistent bacteria in a large amount in a short time is achieved. The peak of the clinical blood drug concentration of kanamycin is known to be about 18-20. Mu.g/mL, which is a concentration forE.coliBW25113 is approximately 2.5 MIC; the clinical administration guiding amount of the polymyxin is 2 mg/kg, and the average blood concentration is about 2.8 mug/mL (range of 0.68-4.88 mug/mL).

The polymyxin-aminoglycoside compound antibiotics adopted in the experiment are pre-mixed to prepare compound medicines with different proportions, and then the compound medicines are used, so that the synergistic effect of the two medicinal components is optimal; for in vitro experiments, the two medicines can be added into the same culture for killing according to a certain proportion. In the drug concentrations referred to in the examples, mL was calculated as medium.

Referring to FIG. 6, the compound drug, polymyxin B-kanamycin, kills efficientlyhipA7High retention bacteria, wherein A isE.coli hipA7The strain platform culture was diluted 1:20 to fresh LB medium, kanamycin (final concentration 20. Mu.g/mL) -polymyxin B (0.9. Mu.g/mL) was immediately added as a compound, the control group was treated with 20. Mu.g/mL kanamycin or 0.9. Mu.g/mL polymyxin B for single drug treatment, shaking culture was performed at 37℃and 160 rpm for 10 h, and the cell viability at different killing times was determined according to the conditions of FIG. 1A; b conditions are as shown in A, wherein kanamycin in the compound medicine is 20 mug/mL, and polymyxin B is 0.003, 0.03, 0.3 or 0.6 mug/mL respectively; alternatively, the compound contains 8. Mu.g/mL kanamycin and 0.45. Mu.g/mL polymyxin B; c conditions were as in A, the compound antibiotic contained 20. Mu.g/mL kanamycin and 1. Mu.g/mL polymyxin E (also known as kriging), and the control group used 20. Mu.g/mL kanaSingle-drug killing is carried out on the mycin or 1 mug/mL polymyxin E; under the condition of A, the compound medicine contains 30 mug/mL of ampicillin and 0.9 mug/mL of polymyxin B, 0.15 mug/mL of ciprofloxacin and 0.9 mug/mL of polymyxin B respectively, and the control group carries out single medicine killing by using 30 mug/mL of ampicillin, 0.15 mug/mL of ciprofloxacin or 0.9 mug/mL of polymyxin B respectively. Each number was obtained from at least three independent experiments, error bars representing mean ± standard deviation. hipA7The strain platform culture is diluted into a fresh LB culture medium, and immediately compound medicines of polymyxin B-kanamycin are added for killing, wherein the concentrations of the compound medicines are respectively 0.9 mug/mL (3 MIC) and 20 mug/mL (2.5 MIC);hipA7the survival rate of the remaining bacteria is rapidly reduced by 7 orders of magnitude, and the remaining level is as low as 10 ^-7 ～10 ^-8 At least one million-fold reduction compared to 2.5 MIC kanamycin or 3 MIC polymyxin B single drug treated control group (5-10% survival retention level); 2.5 MIC kanamycin is compounded with lower concentration polymyxin B (0.01, 0.1, 1 and 2 MIC) respectively, after killing,hipA7the retention survival level of the plant is reduced by 10 to 10000 times; similarly, polymyxin B, kanamycin, and 1 MIC (8. Mu.g/mL) were reduced to 1.5 MIC (0.45. Mu.g/mL) and 1 MIC in the combination,hipA7the retention level of (c) decreases by more than a factor of 10. Therefore, the polymyxin B and kanamycin are compounded, and can be killed rapidly and massively within a wide concentration range which can be achieved by clinical blood concentrationhipA7High retention of bacteria. The compound medicine of polymyxin E and kanamycin can kill persistant bacteria rapidly. 2.5 MIC kanamycin and 1.0 mug/mL (10 MIC) polymyxin E,hipA7the retention level after killing was reduced by at least 10000 times. Compared with a single-drug killing control group, the compound drug of polymyxin B and ampicillin or ciprofloxacin, hipA7The retention level of the steel is not changed significantly, and is still kept about 5 to 10 percent; the results show that the technical effect is better than that of single medicine without combined administration or any compound medicine, especially the combined action of polymyxin and beta-lactam which damages cell walls or quinolone antibiotics which cause DNA fragmentation has no synergistic effect in the aspect of persistent bacteria elimination, and also shows that the polymyxin and aminoglycoside compound medicine are mutually increasedThe phenomenon of effectively and rapidly killing the remaining bacteria is unexpected.

To illustrate the bactericidal synergy of polymyxins and aminoglycosides, conventional methods calculate their synergy index (i.e., partial inhibitory concentration index, FICI for short, less than or equal to 0.5 indicates significant synergy). The FICI value for kanamycin and polymyxin B complex was 0.25 and for polymyxin E complex was 0.4. Therefore, the polymyxin and the aminoglycoside antibiotics form a compound medicine, which not only can kill the persistent bacteria rapidly, but also can reduce the concentration of the medicine required by inhibiting the bacterial growth respectively through the synergistic effect.

Example two

The polymyxin-aminoglycoside compound antibiotics can kill rapidly and largelymetG2High retention bacteria and wild type E. coliLow levels of retention bacteria.

Referring to FIG. 7, the compound drug, polymyxin B-kanamycin, kills efficientlymetG2High retention bacteria and wild typeE.coliThe remaining bacteria, wherein A isE.coli metG2Strain B is wild type; the strain plateau cultures were diluted 1:20 to fresh LB medium, compound antibiotics containing 3 MIC polymyxin B and 2.5 MIC kanamycin were immediately added, the control group was treated with 2.5 MIC kanamycin, 3 MIC polymyxin B for single drug treatment, and the cell viability at different killing times was determined according to the conditions of FIG. 1A.metG2After the strain is killed by polymyxin B-kanamycin compound antibiotics, the survival rate is rapidly reduced by 7 orders of magnitude, and the retention level is reduced by 10 ² ～10 ⁶ Doubling; similarly, wild typeE.coliThe retention level of the compound antibiotic is reduced by nearly thousand times, namely from 0.001% to 0.000001% of the single antibiotic treatment. These results indicate that different factors induce and are at different survival levelsE.coliThe compound antibiotics of the colistin-aminoglycoside can kill the colistin-aminoglycoside with high efficiency.

The polymyxin-aminoglycoside compound antibiotics not only can kill the remaining bacteria of the culture in the stage of the stage, but also can kill the sensitive cells in the exponential phase and the remaining bacteria in the non-sensitive state, so that the bacteria survive in water The level also decreases by 7 orders of magnitude, the retention level decreases by 100 times, see FIG. 8, which is a compound drug of polymyxin B-kanamycin which kills wild type with high efficiencyE.coliStay fungus, wild typeE.coliThe plateau cultures were inoculated at 1:200 into fresh LB liquid medium and shake-cultured to exponential phase (OD ₆₀₀ =0.3), compound antibiotics containing 3 MIC polymyxin B and 2.5 MIC kanamycin were immediately added, or single drug killing was performed, and cell survival numbers at different time points were determined. Each number was obtained from at least three independent experiments, error bars representing mean ± standard deviation. The results demonstrate that polymyxin-aminoglycoside compound antibiotics kill and eliminate bacterial cells with high efficiency, whether they are in an active metabolic state during exponential growth or in a sustained state during slow or lag phase of metabolism. Of the wild-type cells, some are of the nutrition-insensitive retentions, about 0.001%; the retaining bacteria can be killed by the compound medicine of the invention, and the retaining level is reduced by about 100 times.

Example III

The polymyxin and each common aminoglycoside molecule are combined and compounded, and the polymyxin and each common aminoglycoside molecule have similar high-efficiency killing and clearing capacities on the remaining bacteria.

After the synergistic sterilization function of kanamycin-polymyxin compound antibiotics is clarified, the remaining bacteria removal function after the compound of other aminoglycoside molecules and polymyxin is further measured, wherein the remaining bacteria removal function comprises streptomycin, amikacin, tobramycin and gentamicin. Although the peak concentration of these aminoglycosides in blood can be mostly higher than 16. Mu.g/mL, the concentration used in the test is not higher than 16. Mu.g/mL, and polymyxin B is used at a concentration of 0.9. Mu.g/mL. Theoretically, the higher the concentration of these antibiotics, the better the bacteria killing effect.

Referring to FIG. 9, the polymyxin B and aminoglycoside compound antibiotics are killed efficientlyhipA7The bacteria retention rate is high,E.coli hipA7the strain platform culture is diluted to a fresh LB culture medium according to a ratio of 1:20, and immediately antibiotic killing is added, wherein the compound antibiotic of A contains 0.9 mug/mL polymyxin B and 16 mug/mL amikacin, 0.9 mug/mL polymyxin B and 16 mug/mL streptomycin, and the compound antibiotic of B contains0.9 Mu g/mL of polymyxin B and 16 mu g/mL of tobramycin, wherein the compound antibiotics of C contain 0.9 mu g/mL of polymyxin B and 12 mu g/mL of gentamicin, and the concentration of aminoglycoside molecules in the compound antibiotics of D is uniformly 6 mu g/mL; the control group was a single drug kill treatment and cell viability at various time points was determined according to condition a in fig. 1, each value being obtained from at least three independent experiments, error bars representing mean ± standard deviation. After the polymyxin and 16 mug/mL streptomycin, amikacin or 12 mug/mL gentamycin are killed by the compound medicament,hipA7viability of the plateau cultures was reduced 10 ⁶ ～10 ⁷ The retention survival level is as low as 0.0001-0.00001%. The concentration of aminoglycoside in the compound antibiotics is reduced to 6 mug/mL,hipA7the survival level of the culture in the stage of the platform is as low as 0.001-0.0001%, which is only increased by about one order of magnitude compared with that obtained by killing compound medicines containing 16 mug/mL aminoglycosides.

Referring to FIG. 10, the polymyxin B and aminoglycoside compound antibiotics are killed with high efficiencymetG2High retention bacteria, wherein A to D areE.coli metG2Conditions such as strain culture, drug concentration of compound antibiotics, killing treatment, survival measurement and the like are referred to as C in FIG. 9 and D in FIG. 9, each value is obtained by three independent experiments, and error bars represent mean value.+ -. Standard deviation. After the polymyxin and 6 mug/mL of the aminoglycoside compound antibiotics are killed,metG2the retention and activity level of the culture in the stage of the platform is reduced to 0.0001-0.00001%; similarly, after killing by the compound antibiotic, wild typeE.coliThe survival level of the colistin is also reduced to 0.0001-0.00001%, see figure 11, and wild type is killed efficiently by multiple compound antibiotics of polymyxin B and aminoglycosidesE.coliThe remaining bacteria, wherein A to D are wild typeE.coliConditions such as BW25113 strain culture, drug concentration of compound antibiotics, killing treatment, survival measurement and the like are referred to as C in FIG. 9 and D in FIG. 9, each numerical value is obtained by at least three independent experiments, and error bars represent mean value.+ -. Standard deviation. In conclusion, all the compound antibiotics of clinical common aminoglycoside molecules and polymyxin have high-efficiency killing and clearing effects on persisters.

Example IV

The polymyxin-aminoglycoside compound antibiotics have wide persistent bacteria killing capability on gram-negative bacteria and gram-positive bacteria.

The compound antibiotic has wide capability of retaining bacteria for gram-negative and positive pathogenic bacteria, and several clinically common typical pathogenic bacteria are selected for killing, wherein the gram-negative bacteria comprise escherichia coli clinical strains, klebsiella pneumoniae clinical strains and pseudomonas aeruginosa clinical strains, and the gram-positive bacteria are staphylococcus aureus clinical strains; a representative compound antibiotic contains 0.9 μg/mL polymyxin B and 16 μg/mL amikacin. Referring to fig. 12, the polymyxin-aminoglycoside compound antibiotic has the effect of killing clinical pathogenic bacteria retention bacteria. Referring to FIG. 1A for cell culture, killing treatment, survival assay, etc., a representative compound antibiotic contains 0.9 μg/mL polymyxin B and 16 μg/mL amikacin, wherein A is Klebsiella pneumoniae clinical isolate 9030, B is Klebsiella pneumoniae clinical isolate 9182, C is Escherichia coli clinical isolate 0005, D is Escherichia coli clinical isolate 0011, E is Pseudomonas aeruginosa clinical isolate 1128, F is Staphylococcus aureus clinical isolate 1115; each value was obtained from three independent experiments and error bars represent mean ± standard deviation. The result shows that compared with amikacin single-drug killing groups, the retention level of the clinical isolate cultures of different escherichia coli and klebsiella pneumoniae is reduced by 100-100000 times after the clinical isolate cultures are killed by the compound antibiotics; the retention level of pseudomonas aeruginosa and staphylococcus aureus is reduced by 10 times compared with single drug treatment after the effect of compound antibiotics. These results indicate that the colistin-aminoglycoside complex antibiotics have better retentive bacteria clearance than single antibiotics, and have universality in gram-negative bacteria and positive bacteria.

Example five

Represented by a compound antibiotic containing 0.9. Mu.g/mL polymyxin B and 20. Mu.g/mL kanamycin, forhipA7Cell membrane damage during culture stress antibiotic killing was measured. Referring to FIG. 13, the polymyxin B-kanamycin complex antibiotic is responsible forhipA7Loss of cell membrane potential, wherein A is 0.9 μg/mL polymyxin B and 20. Mu.g/mL kanamycin, B is 20. Mu.g/mL kanamycin alone, and C is 0.9. Mu.g/mL polymyxin B.E.coli hipA7The strain platform culture was inoculated to fresh LB liquid medium at a ratio of 1:20, compound antibiotics containing kanamycin at a final concentration of 20. Mu.g/mL and polymyxin B at a final concentration of 0.9. Mu.g/mL were immediately added, parallel samples were treated with single drugs at the corresponding concentrations, 8.5. Mu.M DiSC3 (5) was added at a final concentration of 160 rpm for shaking culture at 37℃for 8 h, the drugs and unbound dye were washed off, the cell fluorescence intensity was measured by flow cytometry, 100,000 cells were analyzed for each sample, the results of three independent experiments were substantially identical, and representative data of one of the experiments were plotted. In the compound antibiotic treatment process, when the membrane potential is used for representing the fluorescent dye DiSC3 (5) for marking, the membrane potential is accompanied with hipA7The rapid death of the cells, the disec 3 (5) signal also rapidly increased, indicating a rapid decrease in the membrane potential of the cells; single drug killing of kanamycin or polymyxin B did not result in significant enhancement of the fluorescent signal of disec 3 (5) even after prolonged periods. Further Propidium Iodide (PI) was used to determine cell membrane damage, see figures 14 and 15, following killing by polymyxin B-kanamycin compound antibioticshipA7The rapid death of cells in culture, the rapid enhancement of PI fluorescent signal, indicates that rupture of cell membrane occurred; after kanamycin or polymyxin B single drug treatment of 2 h, most cells did not see PI fluorescence signal increase, and few cells could detect fluorescence signal after 4 h.

FIG. 14 is a graph showing the results of polymyxin B-kanamycinhipA7Cell membrane rupture, where a is 2 hours and B is 4 hours. As in the case of the condition of figure 12,E.coli hipA7the culture in the plateau stage is diluted to a fresh culture medium, polymyxin B-kanamycin compound antibiotics are immediately added for killing, parallel samples are treated by single medicines with corresponding concentrations, 0, 2 and 4 h samples are taken, 5 mu M PI dye with final concentration is added 10 min before taking the samples, after the medicines and extracellular dye are removed by washing with normal saline, the fluorescence intensity of cells is detected by a flow cytometer, and each sample is analyzed for 100,000 cells. The results of the three independent experiments are basically the same, and the representative of one of the experiments is taken The data are plotted. FIG. 15 is a graph showing the effects of polymyxin B-kanamycin on a combination antibiotichipA7Cell membrane disruption, wherein A is a compound antibiotic containing 0.9 μg/mL polymyxin B and 20 μg/mL kanamycin, B is 20 μg/mL kanamycin alone, C is 0.9 μg/mL polymyxin B, wherein,E.coli hipA7parameters and conditions of cell culture, antibiotic killing, PI staining and the like are all carried out according to FIG. 13, and after samples at different time points are washed by normal saline, the fluorescence intensity of cells is detected at the single cell level by a fluorescence microscope. The results of the three independent experiments are basically the same, each sample is analyzed for multiple fields, and representative data of one experiment is plotted; the percentage data represent the proportion of dead cells containing PI fluorescence to total cells, optionally obtained by statistical analysis of 1,000 cells in multiple fields.

Example six

To evaluate the safety of the compound antibiotics on human cells, the final concentrations of the two components in the compound antibiotics were increased to several times the above-described measured bactericidal concentrations, and the representative compound antibiotics contained 6 μg/mL polymyxin B and 80 μg/mL kanamycin or 80 μg/mL amikacin, and their effects on the growth and metabolism of human cell line THP-1 were examined, with cell cultures without compounds as blank controls, and with the addition of respiratory chain proton motive inhibitor CCCP as positive controls. Referring to fig. 16, the safety evaluation of polymyxin-aminoglycoside compound antibiotics on human cells is shown. THP-1 cells were seeded in 48-well plates at 5% CO ₂ Culturing in incubator at 37 deg.c for 24 h and cell density of 4-5×10 ⁵ cell/mL, adding compound antibiotic or single antibiotic with higher concentration than blood, wherein the concentration is 80 μg/mL kanamycin, 80 μg/mL amikacin, 6 μg/mL polymyxin B, parallel sample adding 20 μg/mL CCCP as control, continuing to culture 10 h, adding CCK-8 reagent at different time, continuing to culture 1 h, and measuring OD with enzyme marker instrument ₄₅₀ Values, examine the effect of high concentration antibiotics on human cell growth; each value was obtained from at least three independent experiments, and the error bars represent mean.+ -. Standard deviation, which indicated that THP-1 cells treated with the two above-described complex antibiotics were positive for growth and metabolism and no antibioticsThere was no significant difference in the normally grown control group, however CCCP (20. Mu.g/mL) significantly inhibited growth and resulted in cell death. Therefore, the peak blood concentration of the compound antibiotic for sterilization in clinical use is far lower than the current toxicity test concentration, and the safety of human cells can be ensured theoretically.

No antibiotics are reported in the prior art, whether administered alone, in combination or at clinically useful concentrations to kill nutritionally insensitive persisters. The nutrition-insensitive persisting bacteria caused by any genotype can be used, and the original persisting level can be high or low (for example, the wild type is only 0.001%, but not high) hipA7/metG25-10 percent of the compound antibiotics can effectively kill the antibiotics; this demonstrates the unexpected technical effect achieved by the compound antibiotics of the present invention.

Claims (10)

1. The application of the combined compound medicine in preparing a pathogenic bacteria retention infection treatment medicine or a pathogenic bacteria retention inhibition medicine is characterized in that the combined compound medicine comprises polymyxin antibiotics and aminoglycoside antibiotics.

2. The use according to claim 1, wherein the combination is safe for cells of human origin.

3. The application of the combined compound medicine in preparing the medicine for synergism and elimination of persistent pathogenic bacteria is characterized in that the combined compound medicine comprises polymyxin antibiotics and aminoglycoside antibiotics.

4. The use according to claim 3, wherein the medicament further comprises an additional antibacterial agent.

5. Use according to claim 1 or 3, wherein the pathogenic bacteria are nutrient insensitive persisters; pathogenic bacteria include gram-negative bacteria and gram-positive bacteria.

6. The use according to claim 1 or 3, wherein the weight content of aminoglycoside antibiotics in the combined compound medicine is 0.01-99.5%.

7. Use according to claim 1 or 3, wherein the polymyxin antibiotic comprises polymyxin or a salt, hydrate, solvate thereof; the aminoglycoside antibiotics comprise aminoglycoside or salts, hydrates and solvates thereof.

8. The use according to claim 7, wherein the polymyxin antibiotic comprises polymyxin B or polymyxin E.

9. The use according to claim 7, wherein the aminoglycoside antibiotic comprises kanamycin, streptomycin, amikacin, tobramycin, gentamicin, netilmicin or other similar molecules.

10. The application of a combined compound medicine in preparing anti-inflammatory or anti-infective medicines is characterized in that the combined compound medicine comprises polymyxin antibiotics and aminoglycoside antibiotics.