Use of combined compound medicine in treating pathogenic bacteria persistent infection

Abstract

Disclosed is use of a combined compound medicine in treating pathogenic bacteria persistent infection. Existing antibiotic monotherapy or combination therapy is usually difficult to kill pathogenic bacteria in the persister state, especially nutrient-insensitive persister bacteria, which often lead to a series of problems of long treatment period, frequent condition recurrence and deterioration, treatment failure, drug-resistant mutation, etc. At present, drugs capable of efficiently killing highly persistent pathogenic bacteria in a clinically applicable concentration range are still unavailable. The novel compound medicine of polymyxin and aminoglycoside antibiotics disclosed in the present disclosure without precedence is capable of destroying bacterial cell membranes in a short time within a relatively wide range of clinically achievable plasma concentrations, leading to rapid death of a large number of bacteria, but is safe for human-derived cells. Therefore, the compound medicine is expected to be applied to the clinical treatment of various acute or chronic infectious diseases caused by pathogenic bacteria in the persister state, shortening the treatment course, preventing the recurrence of infection recurrence, and slowing down the emergence of drug-resistant mutations.

Description

Application of a combined compound medicine in the treatment of persistent infection caused by pathogenic bacteria Technical Field

[0001] The present invention belongs to biomedical technology and relates to the use of a combined compound medicine in the treatment of persistent pathogenic bacterial infections, specifically the use of a combined compound medicine of polymyxin and aminoglycoside antibiotics in the treatment of persistent pathogenic bacterial infections. Background Art

[0002] The persistence of bacteria leads to the failure of nearly half of clinical antibiotic treatments. It is the main cause of chronic infection, recurrent disease attacks and deterioration. It can also accelerate the emergence of resistance mutations, further exacerbating the crisis of antibiotic resistance.

[0003] The mechanism of bacterial persistence has made some progress in the past two decades, but it is still controversial and has not been fully revealed. According to whether there are sufficient exogenous nutrients in the bacterial culture during antibiotic treatment, the reported in vitro persistence research models can be divided into two categories: (1) "Nutrient-insensitive" persistence. Even under conditions of abundant exogenous nutrients, bacterial cells have extremely strong tolerance to almost all types of bactericidal antibiotics and do not die even when killed by high-dose antibiotics. This phenomenon is called "nutrient-insensitive" persistence, which belongs to the true persistence and is the type of persistence that the present invention strives to overcome. (2) "Nutrient-sensitive" persistence / tolerance. This type of state, which is caused by the lack of nutrients in the culture, resulting in the inability of antibiotics to reach the target or the inability to produce sufficient reactive oxygen free radicals, resulting in very few cell deaths, was mistakenly considered to be persistence in the past. Its survival phenotype should actually be classified as tolerance according to the definition and mechanism, not true persistence. The "persistence" reported in most literature belongs to this type. This type of model usually involves directly adding antibiotics to the stationary phase culture of bacteria (without fresh exogenous nutrients), or treating the bacterial cells with antibiotics while they are starved in saline. The surviving bacteria can often be killed and eliminated in large numbers as long as there are sufficient nutrients.

[0004] Clinical anti-infective treatments lack effective drugs and methods to kill "nutrient-insensitive" persister pathogens. Current reports on drug-resistant persisters primarily derive from the aforementioned second type of "nutrient-sensitive" persistence / tolerance model. In 2011, Professor James Collins' group at the Massachusetts Institute of Technology reported that direct addition of glucose, fructose, or mannitol to stationary-phase LB liquid cultures of wild-type Escherichia coli enhanced the bactericidal activity of gentamicin. This effect was completely inhibited by the proton motive force inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP), indicating that gentamicin's intracellular uptake is dependent on proton motive force. Kim Lewis' group at Northeastern University reported that the antimicrobial peptide ADEP4 can persistently activate the proteolytic enzyme ClpP, thereby killing stationary-phase Escherichia coli cultures in vitro. Unfortunately, clinical trials of this compound failed. In 2023, Professor Fu Xinmiao's team reported that heat-treating Escherichia coli culture at 55°C for five minutes promoted the killing of persister bacteria by aminoglycosides. However, this method cannot be used to treat intra-tissue infections because most human cells cannot withstand heat shock. Furthermore, the proton motive force inhibitor CCCP can be combined with high concentrations of aminoglycosides (500 μg / mL), ampicillin, or ciprofloxacin to kill bacteria in the plateau phase. However, CCCP remains highly toxic to human and animal cells even at lower concentrations, making it unsuitable for clinical use. In summary, commonly used clinical antibiotic monotherapy fails to achieve effective therapeutic effects by killing persister bacteria, and there are no reports of combination antibiotics that can effectively kill persister bacteria at clinical blood concentrations.

[0005] Aminoglycoside antibiotics alone are ineffective against nutrient-insensitive persisters. Aminoglycosides are a well-known group of antibiotics with very similar structures and bactericidal mechanisms, comprising different functionally similar antibiotic molecules. Common aminoglycosides include kanamycin, streptomycin, amikacin, tobramycin, gentamycin, and netilmicin. These antibiotics generally have no killing effect on nutrient-insensitive persisters within clinical blood concentrations.

[0006] There are no reports on the effectiveness of polymyxins in combination with other bactericidal antibiotics in killing persister bacteria. Polymyxins are a well-known group of polypeptide antibiotics with very similar molecular structures and bactericidal mechanisms. They have inhibitory effects on most Gram-negative bacteria. Their structures typically differ only in the acyl group and two amino acids. Typical clinical agents include polymyxin B and polymyxin E, currently used primarily as second-line antibiotics.

[0007] In summary, finding new molecules or compound formulations of existing antibiotics that can quickly kill persister bacteria is of great significance for clinical anti-infection treatment, especially chronic infections and recurrent bacterial infections, and for improving public health. It also generates huge economic value in the field of biomedicine. Technical issues

[0008] Existing antibiotic monotherapy or combination therapy is usually difficult to kill pathogenic bacteria in a persister state, especially nutrient-insensitive persisters, which often lead to a series of problems such as long treatment cycles, repeated attacks and deterioration of the disease, treatment failure, and drug-resistant mutations. At present, there is no drug that can effectively kill high-persistent pathogens within a clinically applicable concentration range. The novel compound drug of polymyxin antibiotics and aminoglycoside antibiotics disclosed for the first time in the present invention can destroy bacterial cell membranes in a short time within a wide range of clinical blood drug concentrations, leading to the rapid death of a large number of bacteria, but is safe for human cells. Therefore, it is expected to be applied to the clinical treatment of various acute or chronic infectious diseases caused by persister pathogenic bacteria, shorten the course of treatment, prevent recurrence of infection, and slow down the generation of drug-resistant mutations. Technical Solutions

[0009] The present invention adopts the following technical solutions:

[0010] A use of a combined compound medicine in preparing a medicine for treating persistent infection caused by pathogenic bacteria, wherein the combined compound medicine comprises polymyxin and aminoglycoside antibiotics.

[0011] A use of a combined compound medicine in preparing a drug for inhibiting the retention of pathogenic bacteria, wherein the combined compound medicine comprises polymyxin and aminoglycoside antibiotics.

[0012] A combination drug is used in the preparation of a drug for enhancing the effectiveness of eliminating persistent pathogenic bacteria, wherein the combination drug comprises polymyxin and an aminoglycoside antibiotic and further comprises other antimicrobial agents.

[0013] The combined compound drug of the present invention exerts an inhibitory effect on the retention of pathogenic bacteria and is safe for human cells. As common sense, so-called safety refers to pharmaceutically recognized safety. The growth and metabolism of human THP-1 cells treated with the composite polymyxin-aminoglycoside antibiotics of the present invention were not significantly different from those of the normal growth control group without antibiotics. However, the existing respiratory chain proton motive force inhibitor CCCP significantly inhibited growth and caused cell death. Therefore, the peak blood concentration of the composite antibiotic of the present invention for sterilization in clinical use is much lower than the above-mentioned test concentration, and it can theoretically ensure safety for human cells.

[0014] Preferably, the pathogenic bacteria are nutrient-insensitive persisters. Even under conditions of abundant exogenous nutrients, bacterial cells have extremely strong tolerance to almost all types of bactericidal antibiotics, and they do not die even when killed by high-dose antibiotics. This phenomenon is called "nutrient-insensitive" persistence, which is a true persistence. Clinical anti-infection treatment lacks drugs and means to effectively kill persisting pathogens. Current technologies for killing persisters with drugs mainly target nutrient-sensitive persistence / tolerance, and there are few medication regimens for nutrient-insensitive persisters. The present invention discloses for the first time a combined drug of polymyxin (myxomycin) and aminoglycoside antibiotics, which can quickly and massively kill inactive metabolic persisters and tolerant bacteria, with an effect far superior to that of a single antibiotic. It is also better than a single antibiotic in killing bacteria in an actively metabolic growth state. Therefore, it is obvious that it has potential important application value in clinical anti-bacterial infection treatment and is expected to be used to treat bacterial infections including pneumonia, bloodstream infections, urinary tract infections, respiratory tract infections, intestinal infections, etc., and is also expected to be used to treat bacterial infections in animals.

[0015] Therefore, the present invention discloses an application of a combined compound medicine in the preparation of an anti-inflammatory or anti-infective medicine, wherein the combined compound medicine comprises polymyxin and an aminoglycoside antibiotic.

[0016] In the present invention, polymyxin antibiotics include polymyxin or its salts, hydrates, and solvates; aminoglycoside antibiotics include aminoglycoside or its salts, hydrates, and solvates.

[0017] In the present invention, aminoglycoside antibiotics are a large class of molecules with similar structures and functions. Aminoglycoside antibiotics include clinically commonly used kanamycin, amikacin, tobramycin, gentamicin, and streptomycin, as well as other aminoglycoside drug molecules with similar structures and functions.

[0018] In the present invention, polymyxins are a large class of molecules with similar structures and functions, including polymyxin B and polymyxin E, and also include other molecules with similar structures and functions.

[0019] In the present invention, the content of aminoglycoside antibiotics in the combined compound medicine is 0.01-99.9%, preferably 75-99%, more preferably 85-99%. In terms of weight percentage, it 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%. The above proportions are calculated based on the total weight of "polymyxin antibiotics and aminoglycoside antibiotics" as 100%, excluding other ingredients such as carriers; if other carrier ingredients are included, the corresponding proportions are calculated by weight.

[0020] In the present invention, the active ingredients of the combined compound medicine are polymyxin antibiotics and aminoglycoside antibiotics, and further, a carrier may be included; as common sense, the active ingredients polymyxin and aminoglycoside antibiotics are present in the same carrier to form a compound medicine, which is called a polymyxin-aminoglycoside compound antibiotic.

[0021] In the present invention, pathogenic bacteria are in the plateau or exponential growth phase. The polymyxin-aminoglycoside antibiotic combination not only effectively kills persister bacteria in plateau cultures, but also cells in the exponential growth phase, reducing their survival by seven orders of magnitude and their persistence by 100-fold. Regardless of whether the bacterial cells are in a metabolically active state of exponential growth or in a persister state of slow metabolism or stagnation, the polymyxin-aminoglycoside antibiotic combination effectively kills and eliminates them.

[0022] In the present invention, pathogenic bacteria include Gram-negative and Gram-positive bacteria. The polymyxin-aminoglycoside combination antibiotic of the present invention reduced the persistence of randomly selected clinical isolates of Escherichia coli and Klebsiella pneumoniae by 100-fold compared to the amikacin monotherapy group. For clinical isolates of Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus, the persistence was reduced by at least 10-fold. These results demonstrate that the polymyxin-aminoglycoside combination antibiotic has superior persistence-clearing capabilities compared to existing single antibiotics and is universally applicable to both Gram-negative and Gram-positive bacteria. Beneficial effects

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] (1) The combined compound drug of polymyxin (myxin) and aminoglycoside antibiotics disclosed for the first time in the present invention can quickly and massively kill inactive metabolic persister-tolerant bacteria, and its effect is far better than that of a single antibiotic. It is also better than a single antibiotic in killing bacteria in an active metabolic growth state. The combined compound drug of other types of antibiotics within the clinical concentration range, as well as the combined compound drug of polymyxin and commonly used clinical beta-lactam or quinolone antibiotics, have not been shown to have the effect of rapidly killing nutritionally insensitive persister bacteria in large quantities. In the compound drug of the present invention, the combined index of polymyxin B and polymyxin E with aminoglycosides is 0.25 and 0.4 respectively, and has the function of rapidly destroying bacterial cell membranes, while the same concentration of polymyxin or aminoglycoside alone cannot achieve the effect of destroying cell membranes and killing persister bacteria. Therefore, the compound drug of the present invention obviously has potential important application value in clinical anti-bacterial infection treatment, and is expected to be used to treat bacterial infections including but not limited to pneumonia, bloodstream infection, urinary tract infection, respiratory tract infection, intestinal infection and the like, and is also expected to be used to treat bacterial infections in animals.

[0025] (2) The polymyxin combined with aminoglycoside antibiotics disclosed in the present invention has the ability to eliminate bacteria in a wide range of representative Gram-negative and Gram-positive bacteria. Its application range in clinical anti-infection treatment and animal treatment is obviously not limited to the representative strains tested in the present invention, but should be applicable to the vast majority of pathogenic bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 shows the survival ability of wild-type E. coli after being killed by different types of bactericidal antibiotics; 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), and D is 3 MIC polymyxin B (MIC=0.3 μg / mL).

[0027] Figure 2 shows that the hipA7 strain has high persistence and survival against different bactericidal antibiotics, with the wild-type strain as the control; A represents 3 MIC kanamycin and 3 MIC streptomycin, B represents 20 MIC ciprofloxacin and 20 MIC ampicillin, and C represents 3 MIC polymyxin B and 3 MIC mitomycin C (MIC = 0.8 μg / mL).

[0028] Figure 3 shows that the metG2 strain has high persistence and survival against different bactericidal antibiotics, with the wild-type strain as the control; A represents 3 MIC kanamycin, 20 MIC ciprofloxacin, and 20 MIC ampicillin, and B represents 3 MIC polymyxin B and 3 MIC mitomycin C.

[0029] Figure 4 shows that the hipA7 strain has high persistence and survival ability against the combination of different types of antibiotics, and the wild-type strain is the control; 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.

[0030] Figure 5 shows that the metG2 strain has high persistence survival against the combination of different types of antibiotics, and the wild-type strain is the control; 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, and F is 20 MIC ampicillin + 3 MIC kanamycin.

[0031] Figure 6 shows that the combination of polymyxin B and kanamycin effectively kills hipA7 high-persistence bacteria. A is a combination of kanamycin (final concentration 20 μg / mL) and polymyxin B (0.9 μg / mL), and the control group is treated with 20 μg / mL kanamycin or 0.9 μg / mL polymyxin B as a single drug. B's combination contains 20 μg / mL kanamycin and 0.003, 0.03, 0.3, or 0.6 μg / mL polymyxin B, respectively; or, the combination contains 8 μg / mL kanamycin and 0.45 μg / mL polymyxin B. C's combination antibiotics contain 20 μg / mL kanamycin and 1 μg / mL polymyxin E, and the control group is treated with 20 μg / mL kanamycin or 1 μg / mL polymyxin E as a single drug. D's combination antibiotics contain 30 μg / mL ampicillin and 0.9 μg / mL polymyxin B, 0.15 μg / mL ciprofloxacin, and 0.9 μg / mL polymyxin B, while the control groups were treated with 30 μg / mL ampicillin, 0.15 μg / mL ciprofloxacin, or 0.9 μg / mL polymyxin B for single-drug killing.

[0032] Figure 7 shows that the polymyxin B-kanamycin combination drug is highly effective in killing metG2 high-persistent bacteria and wild-type E. coli persisters. A is the E. coli metG2 strain, and B is the wild type. A combination antibiotic containing 3 MIC polymyxin B and 2.5 MIC kanamycin was used, and the control group was treated with 2.5 MIC kanamycin and 3 MIC polymyxin B as single drugs.

[0033] Figure 8 shows that the combination of polymyxin B and kanamycin is highly effective in killing wild-type E. coli persisters in the exponential phase. The experimental group contained 3 MIC polymyxin B and 2.5 MIC kanamycin combination antibiotics, while the control group was killed by single drugs.

[0034] Figure 9 shows that multiple combination antibiotics of polymyxin B and aminoglycosides are highly effective in killing hipA7 high-persistence bacteria. The combination antibiotics in A contain 0.9 μg / mL polymyxin B and 16 μg / mL amikacin, 0.9 μg / mL polymyxin B and 16 μg / mL streptomycin, the combination antibiotics in B contain 0.9 μg / mL polymyxin B and 16 μg / mL tobramycin, the combination antibiotics in C contain 0.9 μg / mL polymyxin B and 12 μg / mL gentamicin, and the aminoglycoside molecular concentration in the combination antibiotics in D is uniformly 6 μg / mL.

[0035] Figure 10 shows that multiple combination antibiotics of polymyxin B and aminoglycosides are highly effective in killing metG2 high-persistence bacteria. The combination antibiotic A contains 0.9 μg / mL polymyxin B and 6 μg / mL amikacin, the combination antibiotic B contains 0.9 μg / mL polymyxin B and 6 μg / mL streptomycin, the combination antibiotic C contains 0.9 μg / mL polymyxin B and 6 μg / mL tobramycin, and the combination antibiotic D contains 0.9 μg / mL polymyxin B and 6 μg / mL gentamicin. Single drugs were used as controls.

[0036] Figure 11 shows that multiple combination antibiotics of polymyxin B and aminoglycosides are highly effective in killing wild-type E. coli persisters. The combination antibiotic A contains 0.9 μg / mL polymyxin B and 6 μg / mL amikacin, the combination antibiotic B contains 0.9 μg / mL polymyxin B and 6 μg / mL streptomycin, the combination antibiotic C contains 0.9 μg / mL polymyxin B and 6 μg / mL tobramycin, and the combination antibiotic D contains 0.9 μg / mL polymyxin B and 12 μg / mL gentamicin. Single drugs were used as controls.

[0037] Figure 12 shows the killing effect of polymyxin-aminoglycoside combination antibiotics on clinical pathogenic bacteria persisters, where 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.

[0038] Figure 13 shows the loss of hipA7 cell membrane potential caused by polymyxin B-kanamycin combination antibiotics, where A is a combination 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.

[0039] FIG14 shows the rupture of hipA7 cell membrane caused by polymyxin B-kanamycin combination antibiotics, where A represents 2 hours and B represents 4 hours.

[0040] Figure 15 shows that polymyxin B-kanamycin combination antibiotics cause hipA7 cell membrane rupture, where A is a combination 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.

[0041] FIG16 is a safety assessment of polymyxin-aminoglycoside combination antibiotics on human cells. Modes for Carrying Out the Invention

[0042] This invention discloses for the first time a combination of a polymyxin antibiotic and an aminoglycoside antibiotic for inhibiting and killing pathogenic bacteria, particularly for the preparation of a drug for treating persistent pathogenic bacterial infections, particularly "nutrient-insensitive" persisters. The polymyxin antibiotics include polymyxin or its salts, hydrates, or solvates; and the aminoglycoside antibiotics include aminoglycosides or their salts, hydrates, or solvates.

[0043] In the present invention, the active ingredients of the drug are polymyxin antibiotics and aminoglycoside antibiotics, and further include a carrier.

[0044] Given that polymyxins represent a broad class of molecules with similar structures and functions, the polymyxin components of the combination drug disclosed herein are not limited to polymyxin B and polymyxin E but also encompass other molecules with similar structures and functions. The polymyxin antibiotics comprise 0.001 to 95% of the finished polymyxin-aminoglycoside combination antibiotic, resulting in a suitable concentration range of 0.001 to 200 μg / mL during bactericidal activity.

[0045] Given that aminoglycoside antibiotics represent a broad class of molecules with similar structures and functions, the scope of the combined medication disclosed herein is not limited to the commonly used clinically available aminoglycosides kanamycin, amikacin, tobramycin, gentamicin, streptomycin, and netilmicin, but should also include other aminoglycoside drug molecules with similar structures and functions. The aminoglycoside content in the finished compound medication ranges from 0.01 to 99.5%, resulting in an applicable concentration range of 0.01 to 200 μg / mL during the bactericidal process.

[0046] The above two proportions (content ratios) are calculated based on the total weight of "polymyxin antibiotics and aminoglycoside antibiotics" as 100%, excluding other ingredients such as carriers; if other carrier ingredients are included, the corresponding proportions shall be calculated by weight.

[0047] As is common sense, the term salt is a pharmaceutically acceptable salt and refers to a disclosed compound or derivative modified by preparing an acid or base salt thereof. Examples of pharmaceutically acceptable salts include: inorganic or organic acid salts of basic groups such as amines, alkali metal or organic salts of acidic groups such as carboxylic acids, conventional non-toxic salts or quaternary ammonium salts. Conventional non-toxic salts include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid; 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 and isethionic acid. Pharmaceutically acceptable salts can be synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods. In some cases, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or in an organic solvent, or in a mixture of the two; preferred organic solvents are ether, ethyl acetate, ethanol, isopropanol, or acetonitrile. As a general rule, the active ingredient includes all pharmaceutically acceptable salts, all hydrates, and / or solvates of the compound. Certain functional groups, such as hydroxyl, amino, and the like, form complexes and / or coordination compounds with water and / or various solvents in various physical forms of the compound.

[0048] As a general knowledge, the term carrier is a pharmaceutically acceptable carrier, which is recognized in the art 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 can 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 carboxymethylcellulose, ethylcellulose, 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; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; water; isotonic saline; Ringer's solution; ethanol; phosphate buffered saline; and other nontoxic compatible substances used in pharmaceutical formulations.

[0049] The combination drug of the present invention may be administered in any manner, including oral, intravenous, intramuscular, subcutaneous, transdermal, inhalation, oral, ophthalmic, sublingual, vaginal, and rectal administration. The combination drug of the present invention may be administered in unit dosage forms and / or formulations containing conventional, non-toxic, pharmaceutically acceptable carriers, adjuvants, and vehicles. Exemplary forms for oral administration include tablets, capsules, elixirs, syrups, and the like. External administration routes include intravenous, intraarterial, intraperitoneal, epidural, intraurethral, ​​intrasternal, intramuscular, and subcutaneous administration, as well as any other art-recognized parenteral administration routes. Administration methods include needle (including microneedle) syringes, needle-free syringes, and infusion techniques, as well as any other art-recognized parenteral administration methods. Parenteral formulations are typically aqueous solutions that may contain excipients such as salts, carbohydrates, and buffers. They may also be formulated as sterile non-aqueous solutions or dried forms for use in combination with a suitable vehicle (e.g., sterile, pyrogen-free water). The preparation of parenteral formulations under sterile conditions, for example, by lyophilization, can readily be accomplished using standard pharmaceutical techniques well known to those skilled in the art.

[0050] The dosage of the combined drug of the present invention is conventional and can be selected based on 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 polymyxin and aminoglycoside antibiotics can be carried out simultaneously by any suitable means, as opposed to the conventional sequential, separate, or single pharmaceutical formulation administration.

[0051] The specific drugs used in the present invention are existing products, the specific preparation and experimental methods are conventional technologies, the testing is based on existing laboratory technology, the test strains are mainly commercially available standard strains, and the clinical isolates used individually are infectious bacterial isolates available in any hospital.

[0052] The proportion of persister cells in bacterial cultures is typically higher during the nutrient-deficient plateau phase. To investigate the ability of the model bacterium Escherichia coli (E. coli) to retain most bactericidal antibiotics, the BW25113 standard strain of the E. coli K12 background was cultured in commercially available LB liquid medium at 37°C until the plateau phase. The strain was then inoculated at a ratio of 1:20 into fresh LB liquid medium containing multiple minimum inhibitory concentrations (MICs) of antibiotics for killing. After different treatment times, samples were collected and centrifuged twice to remove residual antibiotics. The culture was then diluted tenfold in saline and plated on antibiotic-free LB agar plates. After two days of incubation, the number of surviving colony forming units (CFU) was counted. The initial CFU before drug addition was used as a control to calculate bacterial survival and persistence. This protocol was followed by standard experimental methods, and testing of other bacteria can be similar. The antibiotics used are classic bactericidal antibiotics, including: β-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), and mitomycins (causing double-strand breaks through GC bridges between DNA chains, such as mitomycin C). The drug dosage adopts the concentration that can kill sensitive bacteria, usually a multiple of the well-known MIC. The specific concentration can be calculated based on the MIC and the multiple.

[0053] Fluorescence microscopy and flow cytometry are used to detect bacterial intracellular fluorescent substance signals according to the standard procedures and detection methods of the instruments or reagents.

[0054] The above is the general method for bacterial testing used in the present invention and is also a common analytical method used in the literature to analyze bacterial persistence. The antibiotics used in the present invention are all commonly used in clinical practice. The experimental results show that existing monotherapy antibiotics are ineffective against persisting bacteria, and there are no reports of combined antibiotics for the treatment of persisting bacteria.

[0055] 1. High concentrations of antibiotics and long-term treatment cannot effectively kill the "nutrient-insensitive" persisters in the wild-type Escherichia coli population.

[0056] A small number of cells in the stationary phase culture population of wild-type Escherichia coli have high persistence capacity. When diluted into nutrient-rich culture medium for killing, even after treatment with high concentrations of bactericidal antibiotics, this small number of cells can survive by persisting to resist the killing of existing common clinical antibiotics, and are therefore called "nutrient-insensitive" persisters.

[0057] Treatment of wild-type E. coli BW25113 cultures at stationary phase with kanamycin (24 μg / mL, 3 MICs) for 10 hours, exceeding the peak plasma concentration of 20 μg / mL, reduced viability 100,000-fold, with persister survival remaining at 0.001%. Similarly, treatment with ciprofloxacin (0.6 μg / mL, 20 MICs), ampicillin (120 μg / mL, 20 MICs), or polymyxin B (0.9 μg / mL, 3 MICs) for 10 hours resulted in persister levels of 0.001% (see Figure 1). These results indicate that approximately one in 100,000 cells in stationary phase cultures of wild-type E. coli is in a "nutrient-insensitive" persister state. These cells survive high-concentration, prolonged treatment with various commonly used clinical bactericidal antibiotics in the presence of nutrients through persistence and can resume growth and reproduction after antibiotic withdrawal.

[0058] The vast majority of cells in stationary-phase Escherichia coli cultures are in a "nutrient-sensitive" persister / tolerance state. Unlike the small number of "nutrient-insensitive" persisters mentioned above, the vast majority of cells in stationary-phase E. coli BW25113 cultures exhibit minimal mortality when depleted of key nutrients or completely lacking exogenous nutrients, preventing antibiotics from reaching their targets or producing sufficient reactive oxygen species (ROS). However, when diluted into nutrient-rich medium and treated with antibiotics, stationary-phase cultures are rapidly and extensively killed by various common clinical bactericidal antibiotics (see Figure 1). These cells, which survive nutrient starvation but are killed in the presence of external nutrients, were previously mistakenly identified as persisters. Their survival phenotype, by definition, is actually characterized as tolerance, not true persisters. Therefore, the term "persisters" referred to herein herein generally refers to "nutrient-insensitive" persisters.

[0059] Figure 1 shows the survival of wild-type E. coli after exposure to different bactericidal antibiotics. A plateau culture of wild-type E. coli BW25113 was inoculated at a ratio of 1:20 into fresh LB liquid medium. Immediately, 3 MIC kanamycin (MIC = 8 μg / mL) was added and cultured at 37°C with shaking at 160 rpm for 10 h. Samples were collected at different time points, washed to remove the drug, and then serially diluted tenfold in saline. The samples were then spotted on LB agar plates and the number of viable cells (CFU) was determined after 2 days of culture. The conditions in B, C, and D were similar to those in A, except that the antibiotics used for the bactericidal effects were 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 value is obtained from at least three independent experiments, and error bars represent the mean ± standard deviation.

[0060] 2. E. coli hipA7 and metG2 strains have high levels of persistence, and approximately 10% of the cells in their stationary phase cultures can resist killing by a single drug from any of the five major classes of bactericidal antibiotics.

[0061] E. coli hipA7 and metG2 are two typical high-persister strains. Both can be purchased or constructed according to literature to further evaluate the effectiveness of antibiotics against high-persister cells. The hipA7 strain, obtained through evolutionary selection in 1983, is the first recognized high-persister cell strain and is frequently used to study the mechanisms of persistence. A metG2 mutant of the methionyl-tRNA synthetase gene, reported in 2012, was obtained through transposon library screening and exhibits similar high-persister activity to the hipA7 strain.

[0062] Approximately 10% of hipA7 cells in stationary-phase cultures showed strong persistence against all five bactericidal antibiotics, resisting single-drug killing by any of these classes. After a 10-hour killing treatment in fresh LB medium containing 3 MIC kanamycin, the persistence of hipA7 cells remained at 10%, a 10,000-fold increase compared to wild-type E. coli. Similarly, survival remained at approximately 10% after treatment with 20 MIC ciprofloxacin, 20 MIC ampicillin, 3 MIC polymyxin B, or 3 MIC mitomycin C (see Figure 2). Figure 2 shows the high persistence of hipA7 against different bactericidal antibiotics. Figure A shows stationary-phase cultures of E. coli hipA7 and wild-type strains diluted 1:20 into fresh LB liquid medium. 3 MIC kanamycin was immediately added and cultured at 37°C with shaking at 160 rpm for 10 h. Samples were taken periodically to determine the number of viable cells, as described in Figure 1A. Figures B and C show the same conditions as in Figure 1A, with the killing antibiotics being 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 value is obtained from at least three independent experiments, and error bars represent the mean ± standard deviation.

[0063] Approximately 1–10% of cells in stationary-phase cultures of the metG2 strain exhibited strong persistence against all five bactericidal antibiotics. Similar to hipA7, after 10 h of treatment with various single-agent antibiotics, including 20 MIC ciprofloxacin, 20 MIC ampicillin, 3 MIC streptomycin, 3 MIC polymyxin B, or 3 MIC mitomycin C, the survival rate of metG2 cells remained at 1–10% (Figure 3). Figure 3 demonstrates the high persistence of the metG2 strain against various bactericidal antibiotics. A and B represent stationary-phase cultures of E. coli metG2 and wild-type strains inoculated at a 1:20 ratio into fresh LB liquid medium and supplemented with 3 MIC kanamycin, 20 MIC ciprofloxacin, 20 MIC ampicillin, 3 MIC polymyxin B, or 3 MIC mitomycin C, respectively. The cultures were cultured under the conditions described in Figure 1A, and the number of surviving cells was determined at different treatment times. Each value was obtained from at least three independent experiments, and the error bars represent the mean ± standard deviation.

[0064] The above results show that compared with the wild-type strain, the level of "nutrient-insensitive" persisters in the stationary phase culture of E. coli hipA7 and metG2 strains increased by thousands of times. Even under conditions of abundant exogenous nutrients, any commonly used bactericidal antibiotics (including aminoglycosides and polymyxins) in clinical practice cannot effectively kill the persister bacterial cells with single-drug treatment.

[0065] 3. The sequential combination of bactericidal antibiotics was unable to effectively kill the persister cells of wild-type E. coli and hipA7 and metG2 strains.

[0066] To investigate the effects of combined killing by sequential antibiotic treatment on the viability of persister bacteria, stationary phase cultures of wild-type E. coli and hipA7 and metG2 strains were treated with one antibiotic for 5 h under conditions of sufficient exogenous nutrients. The antibiotic was then washed away and replaced with another antibiotic of a different target type for another 5 h, and the survival rate was measured.

[0067] When hipA7 cultures were treated with 20 MIC ciprofloxacin for 5 hours and then immediately switched to another target antibiotic, such as 20 MIC ampicillin, 3 MIC kanamycin, 3 MIC streptomycin, 3 MIC polymyxin B / polymyxin E (colistin), or 3 MIC mitomycin C, the cell survival level remained at 3-10%. Similarly, when hipA7 cultures were pretreated with 20 MIC ampicillin for 5 hours and then switched to 3 MIC kanamycin for 5 hours, the cell survival level remained at 3-10% (see Figure 4). However, in wild-type E. coli cultures, the survival level was as low as 0.001-0.0001% after being treated with various antibiotic combinations. Figure 4 shows the high persistence of hipA7 against different antibiotic combinations, with the wild-type strain serving as the control. Figure A shows stationary phase cultures of E. coli hipA7 and wild-type strains diluted 1:20 into fresh LB medium. 20 MIC ciprofloxacin was immediately added and cultured at 37°C with shaking at 160 rpm for 5 h. Cells were harvested by centrifugation, washed twice to remove the drug, and resuspended in an equal volume of fresh LB medium containing 20 MIC ampicillin. Culture and killing were continued for another 5 h. The number of viable cells was determined under the conditions described in Figure 1 (A). Conditions in Figures B to G are similar to those in Figure A, except that the antibiotic combinations used were 20 MIC ciprofloxacin, 3 MIC kanamycin, 3 MIC streptomycin, 3 MIC polymyxin B / E, 3 MIC mitomycin C, and 20 MIC ampicillin, respectively. Each value is obtained from at least three independent experiments, and error bars represent the mean ± standard deviation.

[0068] MetG2 cultures treated with a combination of different antibiotics exhibited high persistence rates similar to hipA7. For example, after 5 hours of killing with 20 MIC ciprofloxacin, followed by 5 hours of killing with 20 MIC ampicillin, 3 MIC streptomycin, or 3 MIC polymyxin B, the persistence of cells remained at 1–5% (Figure 5). Figure 5 shows the high persistence of the metG2 strain in response to different antibiotic combinations, with the wild-type strain serving as the control. Figure A shows stationary phase cultures of E. coli metG2 and wild-type strains diluted 1:20 into fresh LB medium. 20 MIC ciprofloxacin was immediately added and cultured at 37°C with shaking at 160 rpm for 5 h. Cells were harvested by centrifugation, washed twice to remove the drug, and resuspended in an equal volume of fresh LB medium containing 20 MIC ampicillin. Culture and killing were continued for another 5 h. The number of viable cells was determined under the conditions described in Figure 1 (A). Conditions in Figures B to F are similar to those in Figure A, except that the antibiotic combinations used were 20 MIC ciprofloxacin, 3 MIC kanamycin, 3 MIC streptomycin, 3 MIC polymyxin B / E, 3 MIC mitomycin C, and 20 MIC ampicillin, respectively. Each value is obtained from at least three independent experiments, and error bars represent the mean ± standard deviation.

[0069] The above results indicate that the persistence phenomenon (especially nutrient-insensitive persistence) that has a broad resistance to all bactericidal antibiotics exists in cultures of different strains, such as wild-type E. coli and its hipA7 and metG2 mutant strains. This type of pan-antibiotic persisting bacterial cells is likely to have a persistence resistance similar to that of spores or spores, and has super strong resistance to almost all common types of bactericidal antibiotics on the market. They can still survive long-term treatment with high-concentration antibiotics. Even if antibiotics targeting different target types are killed in combination in turn, they can still survive due to persistence. This is a difficult problem that has not been overcome in clinical anti-infection treatment so far.

[0070] Example 1

[0071] The combination drug of polymyxin and aminoglycoside antibiotics can effectively kill various persistent bacteria within the clinical blood concentration range.

[0072] At clinical blood concentrations or below, different antibiotic combinations derived from polymyxins combined with various aminoglycoside molecules were able to rapidly and extensively kill various persister bacteria tested, achieving rapid and substantial elimination of persisters within a short period of time. The peak clinical blood concentration of kanamycin is approximately 18–20 μg / mL, which is approximately 2.5 MICs against E. coli BW25113. The recommended clinical dosage for polymyxins is 2 mg / kg, with a mean blood concentration of approximately 2.8 μg / mL (range, 0.68–4.88 μg / mL).

[0073] The polymyxin-aminoglycoside combination antibiotic used in the experiment was premixed in different ratios to optimize the synergistic effect of the two drug components. For in vitro experiments, the two drugs can also be added simultaneously to the same culture medium in a specific ratio for killing. In the examples, drug concentrations in mL are based on the culture medium.

[0074] See Figure 6, which shows that the polymyxin B-kanamycin combination drug efficiently kills hipA7 high-persistence bacteria. A is a stationary phase culture of E. coli hipA7 strain diluted 1:20 into fresh LB medium, and a combination of kanamycin (final concentration 20 μg / mL) and polymyxin B (0.9 μg / mL) is immediately added. The control group is treated with 20 μg / mL kanamycin or 0.9 μg / mL polymyxin B alone, and cultured at 37°C and 160 rpm for 10 h. The number of cell survivals at different killing times is determined according to the conditions of A in Figure 1; the conditions of B are the same as those of A, except that the kanamycin in the combination drug is 20 μg / mL and the polymyxin B is 0.003, 0.03, 0.3 or 0.6 μg / mL respectively; alternatively, the combination contains 8 μg / mL kanamycin and 0.45 μg / mL polymyxin B; the conditions of C are the same as those of A, except that the combination antibiotics contain 20 The control group received either 20 μg / mL kanamycin or 1 μg / mL polymyxin E (also known as Christine) for single-drug killing. Conditions in D were similar to those in A, except that the combination drug contained 30 μg / mL ampicillin and 0.9 μg / mL polymyxin B, or 0.15 μg / mL ciprofloxacin and 0.9 μg / mL polymyxin B, respectively. Control groups received either 30 μg / mL ampicillin, 0.15 μg / mL ciprofloxacin, or 0.9 μg / mL polymyxin B for single-drug killing. Each value was obtained from at least three independent experiments, and error bars represent the mean ± standard deviation. The hipA7 culture at stationary phase was diluted into fresh LB medium and immediately killed by the addition of a combination of polymyxin B and kanamycin at concentrations of 0.9 μg / mL (3 MIC) and 20 μg / mL (2.5 MIC), respectively. The survival rate of hipA7 persisters rapidly decreased by 7 orders of magnitude, with the persistence level as low as 10 -7 ~10 -8Compared to controls treated with either 2.5 MIC kanamycin or 3 MIC polymyxin B alone (persistence levels of 5-10%), this was at least a million-fold lower. Combinations of 2.5 MIC kanamycin with lower concentrations of polymyxin B (0.01, 0.1, 1, and 2 MIC) reduced hipA7 persistence by 10- to 10,000-fold. Similarly, reducing the polymyxin B and kanamycin concentrations in the combination to 1.5 MIC (0.45 μg / mL) and 1 MIC (8 μg / mL) decreased hipA7 persistence by more than 10-fold. Therefore, the combination of polymyxin B and kanamycin can rapidly and extensively kill high-persistent hipA7 bacteria across a wide range of clinically achievable blood concentrations. Combinations of polymyxin E and kanamycin also rapidly kill persisters. Combining 2.5 MIC kanamycin with 1.0 μg / mL (10 MIC) polymyxin B reduced hipA7 persistence by at least 10,000-fold after killing. Combinations of polymyxin B with ampicillin or ciprofloxacin did not significantly alter hipA7 persistence compared with controls killed by either drug alone, remaining at approximately 5-10%. These results suggest that not all combinations or combinations of drugs can achieve superior results compared to any single drug alone. In particular, the combination of polymyxin B with cell wall-damaging β-lactams or DNA-breaking quinolones showed no synergistic effect in eliminating persisters. This also suggests that the synergistic, rapid killing of persisters by polymyxin B and aminoglycoside combinations can be unpredictable.

[0075] To demonstrate the synergistic effect of polymyxins and aminoglycosides, their synergy index (i.e., fractional inhibitory concentration index, or FICI) was calculated using conventional methods; a value ≤ 0.5 indicates significant synergistic effect. The FICI value for the combination of kanamycin and polymyxin B was 0.25, while that for the combination with polymyxin E was 0.4. Therefore, polymyxins combined with aminoglycosides not only rapidly kill persister bacteria but also, through synergistic effects, reduce the concentrations of each drug required to inhibit bacterial growth.

[0076] Example 2

[0077] The polymyxin-aminoglycoside combination antibiotic can quickly and massively kill metG2 high-persistent bacteria and wild-type E. coli low-level persister bacteria.

[0078] See Figure 7, which shows that the polymyxin B-kanamycin combination drug effectively kills metG2 high-persistence bacteria and wild-type E. coli persisters, where A is the E. coli metG2 strain and B is the wild type; the stationary phase culture of the strain was diluted 1:20 into fresh LB medium, and the combination antibiotic containing 3 MIC polymyxin B and 2.5 MIC kanamycin was immediately added. The control group was treated with 2.5 MIC kanamycin and 3 MIC polymyxin B alone. The number of surviving cells at different killing times was measured under the conditions of Figure 1A. After the stationary phase culture of the metG2 strain was killed by the polymyxin B-kanamycin combination antibiotic, the survival rate was rapidly reduced by 7 orders of magnitude, and the persistence level was reduced by 10 2 ~10 6 Similarly, the persistence level of wild-type E. coli after being killed by the above-mentioned combination antibiotics decreased by nearly a thousand-fold, from 0.001% treated with a single antibiotic to 0.000001%. These results indicate that the polymyxin-aminoglycoside combination antibiotic can effectively kill E. coli persisters induced by different factors and at different survival levels.

[0079] Polymyxin B-aminoglycoside combination antibiotics can not only effectively kill persisters in stationary phase cultures, but also effectively kill sensitive cells in the exponential growth phase and "nutrient-insensitive" persisters, reducing their survival levels by 7 orders of magnitude and their persistence levels by 100-fold. See Figure 8, which shows the effective killing of wild-type E. coli persisters by a polymyxin B-kanamycin combination drug. Wild-type E. coli stationary phase cultures were inoculated into fresh LB liquid medium at a ratio of 1:200 and cultured with shaking until the exponential phase (OD 600 =0.3), a compound antibiotic containing 3 MIC polymyxin B and 2.5 MIC kanamycin was immediately added, or single drug killing was performed, and the number of surviving cells at different time points was determined. Each value was obtained from at least three independent experiments, and the error bars represent the mean ± standard deviation. The results show that regardless of whether the bacterial cells are in a metabolically active state during exponential growth or in a persister state with slow metabolism or stagnant period, the polymyxin-aminoglycoside compound antibiotic can effectively kill and eliminate them. Among wild-type cells, some cells are nutrient-insensitive persisters, accounting for about 0.001%; ​​these persisters can also be killed by the compound drug of the present invention, reducing the persistence level by about 100 times.

[0080] Example 3

[0081] Polymyxin is combined with various common aminoglycoside molecules to form a compound, which has similar high efficiency in killing and clearing persistent bacteria.

[0082] After confirming the synergistic bactericidal effect of the kanamycin-polymyxin combination, the team further investigated the ability of other aminoglycosides, including streptomycin, amikacin, tobramycin, and gentamicin, to eliminate persister bacteria when combined with polymyxins. Although the peak blood concentrations of most of these aminoglycosides can exceed 16 μg / mL, the concentration used in the tests was no higher than 16 μg / mL, with polymyxin B used at a concentration of 0.9 μg / mL. In theory, higher concentrations of these antibiotics should result in greater bacterial killing.

[0083] See Figure 9 , which shows that multiple combination antibiotics of polymyxin B and aminoglycosides efficiently kill hipA7 high-persistence bacteria. The stationary phase culture of E. coli hipA7 strain was diluted 1:20 into fresh LB medium and immediately added with antibiotics for killing. The combination antibiotics in A contained 0.9 μg / mL polymyxin B and 16 μg / mL amikacin, 0.9 μg / mL polymyxin B and 16 μg / mL streptomycin, the combination antibiotics in B contained 0.9 μg / mL polymyxin B and 16 μg / mL tobramycin, the combination antibiotics in C contained 0.9 μg / mL polymyxin B and 12 μg / mL gentamicin, and the aminoglycoside molecule concentration in the combination antibiotics in D was uniformly 6 μg / mL; the control group was treated with single drug killing, and the cell survival number at different time points was determined according to condition A in Figure 1 . Each value was obtained from at least three independent experiments, and the error bars represent the mean ± standard deviation. After polymyxin was combined with 16 μg / mL streptomycin, amikacin, tobramycin or 12 μg / mL gentamicin, the viability of hipA7 plateau culture decreased by 10 6 ~10 7 When the aminoglycoside concentration in the above-mentioned combination antibiotics was reduced to 6 μg / mL, the persistence survival level of hipA7 plateau cultures was reduced to 0.001-0.0001%, which was only about one order of magnitude higher than the survival level achieved by killing with the combination drug containing 16 μg / mL aminoglycoside.

[0084] See Figure 10, which shows the high efficiency of multiple compound antibiotics of polymyxin B and aminoglycosides in killing metG2 high-persistence bacteria. Among them, A to D are the conditions of E. coli metG2 strain culture, drug concentration of compound antibiotics, killing treatment, survival determination, etc., all of which are referenced to Figure 9C and Figure 9D. Each value was obtained from three independent experiments, and the error bars represent the mean ± standard deviation. After killing with polymyxin B and 6 μg / mL of the aforementioned aminoglycosides, the persister survival level of metG2 plateau cultures was reduced to 0.0001–0.00001%. Similarly, after killing with the combination antibiotics, the persister survival level of wild-type E. coli also decreased to 0.0001–0.00001%. Figure 11 shows the high efficiency of polymyxin B and aminoglycoside combination antibiotics in killing wild-type E. coli persisters. A to D represent the culture conditions of wild-type E. coli BW25113 strain, and the drug concentrations of the combination antibiotics, killing treatments, and survival assays are all similar to those in Figures 9C and 9D. Each value is obtained from at least three independent experiments, and error bars represent the mean ± standard deviation. In summary, all clinically common aminoglycoside combinations with polymyxin B effectively kill and eliminate persisters.

[0085] Example 4

[0086] Polymyxin-aminoglycoside combination antibiotics have a broad range of persister-killing abilities against both Gram-negative and Gram-positive bacteria.

[0087] The compound antibiotic of the present invention has a broad ability to eliminate persisters of both Gram-negative and Gram-positive pathogens. Several typical clinical pathogens were selected for killing. Gram-negative bacteria included clinical strains of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, and Gram-positive bacteria included clinical strains of Staphylococcus aureus. A representative compound antibiotic contains 0.9 μg / mL polymyxin B and 16 μg / mL amikacin. See Figure 12 for the killing effect of a polymyxin-aminoglycoside compound antibiotic on persisters of clinical pathogens. Cell culture, killing treatment, and survival assay conditions were similar to those in Figure 1A. A representative antibiotic combination contained 0.9 μg / mL polymyxin B and 16 μg / mL amikacin. A represents K. pneumoniae clinical isolate 9030, B represents K. pneumoniae clinical isolate 9182, C represents E. coli clinical isolate 0005, D represents E. coli clinical isolate 0011, E represents P. aeruginosa clinical isolate 1128, and F represents S. aureus clinical isolate 1115. Each value is obtained from three independent experiments, and error bars represent the mean ± standard deviation. The results showed that after killing with the combination antibiotic, the persistence of different E. coli and K. pneumoniae clinical isolates decreased by 100- to 100,000-fold compared to that in the amikacin monotherapy group. The persistence of P. aeruginosa and S. aureus decreased by 10-fold after treatment with the combination antibiotic compared to treatment with either drug alone. These results indicate that the polymyxin-aminoglycoside combination antibiotics have better ability to eliminate persister bacteria than single antibiotics and are universally applicable to both Gram-negative and Gram-positive bacteria.

[0088] Example 5

[0089] Using a combination of 0.9 μg / mL polymyxin B and 20 μg / mL kanamycin as an example, cell membrane damage during antibiotic stress in hipA7 cultures was measured. See Figure 13 , which shows the loss of hipA7 cell membrane potential caused by the polymyxin B-kanamycin combination. A represents the combination of 0.9 μg / mL polymyxin B and 20 μg / mL kanamycin, B represents 20 μg / mL kanamycin alone, and C represents 0.9 μg / mL polymyxin B. E. coli hipA7 strain stationary phase culture was inoculated into fresh LB liquid medium at a ratio of 1:20, and a compound antibiotic containing a final concentration of 20 μg / mL kanamycin and 0.9 μg / mL polymyxin B was immediately added. Parallel samples were treated with the corresponding concentrations of single drugs and cultured at 37 ℃ and 160 rpm for 8 hours. DiSC3(5) was added at a final concentration of 2.5 μM 10 minutes before different sampling time points. The drugs and unbound dyes were washed to remove them, and the cell fluorescence intensity was detected by flow cytometry. 100,000 cells were analyzed for each sample. The results of three independent experiments were basically the same, and representative data from any one of the experiments were randomly selected for plotting. During the compound antibiotic treatment, when hipA7 cells were labeled with the membrane potential characterization fluorescent dye DiSC3(5), the DiSC3(5) signal also increased rapidly along with the rapid death of hipA7 cells, indicating a rapid decrease in the cell membrane potential. Even after a long period of time, the killing of hipA7 cells by kanamycin or polymyxin B alone did not cause a significant increase in the DiSC3(5) fluorescence signal. Propidium iodide (PI) was further used to measure cell membrane damage. As shown in Figures 14 and 15 , after killing by the polymyxin B-kanamycin combination antibiotic, the PI fluorescence signal increased rapidly along with the rapid death of cells in the hipA7 culture, indicating that the cell membrane had ruptured. After 2 h of treatment with kanamycin or polymyxin B alone, no increase in PI fluorescence signal was observed in the vast majority of cells, and fluorescence signals were detected in very few cells only after 4 h.

[0090] Figure 14 shows hipA7 cell membrane disruption caused by the polymyxin B-kanamycin combination, with A representing 2 hours and B representing 4 hours. As in Figure 12, stationary-phase E. coli hipA7 cultures were diluted into fresh medium and immediately treated with the polymyxin B-kanamycin combination for killing. Parallel samples were treated with the corresponding concentrations of the individual drugs. Samples were collected at 0, 2, and 4 hours. PI dye was added 10 minutes before sampling to a final concentration of 5 μM. After washing with saline to remove the drug and extracellular dye, cell fluorescence intensity was measured by flow cytometry. 100,000 cells were analyzed for each sample. Results from three independent experiments were essentially identical; representative data from any one of these experiments are plotted. Figure 15 shows hipA7 cell membrane disruption caused by a polymyxin B-kanamycin combination. A represents a combination containing 0.9 μg / mL polymyxin B and 20 μg / mL kanamycin, B represents 20 μg / mL kanamycin alone, and C represents 0.9 μg / mL polymyxin B. E. coli hipA7 cell culture, antibiotic killing, and PI staining parameters and conditions were the same as those in Figure 13. Samples at different time points were washed with saline, and single-cell fluorescence intensity was measured using fluorescence microscopy. Results from three independent experiments were essentially identical. Multiple fields of view were analyzed for each sample, and representative data from one of these experiments are plotted. Percentage data represent the proportion of dead cells with PI fluorescence relative to total cells. Statistical analysis was performed on 1,000 cells from multiple fields of view.

[0091] Example 6

[0092] To assess the safety of the combination antibiotic on human cells, the final concentrations of the two components of the combination antibiotic were increased to several times the bactericidal concentrations determined above. Representative combination antibiotics contained 6 μg / mL polymyxin B and 80 μg / mL kanamycin or 80 μg / mL amikacin. Their effects on the growth and metabolism of the human cell line THP-1 were investigated. Cell cultures without compound addition served as blank controls, and those supplemented with the respiratory chain proton motive force inhibitor CCCP served as positive controls. See Figure 16 for a safety assessment of the polymyxin-aminoglycoside combination antibiotic on human cells. THP-1 cells were seeded into 48-well plates and cultured at 37°C in a 5% CO2 incubator for 24 hours. The cell density per well was 4 to 5 × 10 5 cell / mL, compound antibiotics or single antibiotics with concentrations higher than blood concentrations were added, including 80 μg / mL kanamycin, 80 μg / mL amikacin, and 6 μg / mL polymyxin B. Parallel samples were added with 20 μg / mL CCCP as a control. The cultures were continued for 10 h. CCK-8 reagent was added at different times and the cultures were continued for 1 h. The OD was measured using a microplate reader. 450The effects of high-concentration antibiotics on human cell growth were investigated using a toxicity test. Each value was obtained from at least three independent experiments, and error bars represent mean ± standard deviation. The results showed that THP-1 cells treated with the two combined antibiotics showed no significant differences in growth and metabolism compared to a control group that grew normally without antibiotics. However, CCCP (20 μg / mL) significantly inhibited growth and caused cell death. Therefore, the peak blood concentration of the combined antibiotics used clinically to kill bacteria is far lower than the concentration tested in this toxicity test, theoretically ensuring safety for human cells.

[0093] No antibiotics, whether used alone or in combination, have been reported to kill nutrient-insensitive persisters at clinically applicable concentrations. The compound antibiotics of the present invention effectively kill nutrient-insensitive persisters regardless of their genotype or their initial persistence level (e.g., only 0.001% for the wild type, 5-10% for hipA7 / metG2), demonstrating the unexpected technical benefits of the compound antibiotics.

Claims

1. Use of a combined compound drug in the preparation of a therapeutic drug for treating persistent infection of pathogenic bacteria or a drug for inhibiting persistent pathogenic bacteria, characterized in that, The combined compound drug comprises a polymyxin antibiotic and an aminoglycoside antibiotic.

2. The application according to claim 1, characterized in that, The combined compound drug is safe for human cells.

3. Use of a combined compound drug in the preparation of a drug for synergistically clearing persistent pathogenic bacteria, characterized in that, The combined compound drug comprises a polymyxin antibiotic and an aminoglycoside antibiotic.

4. The application according to claim 3, wherein The drug further comprises other antibacterial agents.

5. The application according to claim 1 or 3, characterized in that The pathogenic bacterium is a nutrient-insensitive persister bacterium; the pathogenic bacterium includes Gram-negative bacteria and Gram-positive bacteria.

6. The application according to claim 1 or 3, characterized in that In the combined compound drug, the weight content ratio of the aminoglycoside antibiotic is 0.01-99.5%.

7. The application according to claim 1 or 3, characterized in that, The polymyxin antibiotic includes polymyxin or its salt, hydrate, solvate; the aminoglycoside antibiotic includes aminoglycoside or its salt, hydrate, solvate.

8. The application according to claim 7, wherein The polymyxin antibiotic includes polymyxin B or polymyxin E.

9. The application according to claim 7, characterized in that, The aminoglycoside antibiotic includes kanamycin, streptomycin, amikacin, tobramycin, gentamicin, netilmicin or other similar molecules.

10. Use of a combined compound drug in the preparation of an anti-inflammatory or anti-infective drug, characterized in that, The combined compound drug comprises a polymyxin antibiotic and an aminoglycoside antibiotic.

Images