A cationic main-chain poly (carbonate-imidazolium) potent against mycobacterium abscessus and other resistant bacteria in mice

Abstract

Disclosed herein is an oligomeric or polymeric material according to the repeating unit of formula I where: each x is 1, 2 or 3; Y- is a pharmaceutically acceptable anionic counterion; Z is selected from -S-S-, -O-C(O)-O-, -NH-C(O)-O-, -O-C(O)-NH-, and -NH-C(O)-NH-, and n represents the repeating unit of the oligomer or polymer, or a pharmaceutically acceptable solvate thereof.

Description

[0001] A CATIONIC MAIN-CHAIN POLY(CARBONATE-IMIDAZOLIUM) POTENT AGAINST MYCOBACTERIUM ABSCESSUS AND OTHER RESISTANT BACTERIA IN MICE

[0002] Field of Invention

[0003] The present invention generally relates to main-chain cationic polymers, and more particularly relates to cationic main-chain imidazolium-derived oligomeric or polymeric materials potent against mycobacteria, and Gram-negative and Gram-positive bacteria.

[0004] Background

[0005] The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0006] There are few treatment options for the eradication of recalcitrant antibiotic resistant pathogenic bacteria. At least two classes of such bacterial infections are in critical need of new treatment options. The incidence of infection due to non-tuberculosis mycobacteria (NTM) has recently been increasing, and in some developed countries has surpassed that of tuberculosis (TB). More than 190 NTM species have been identified that are ubiquitous in the soil and the environment. Some of them are opportunistic pathogens for humans. The incidence of NTM is currently up to 14.1 cases / 100,000 worldwide. Mycobacterium abscessus causes severe respiratory, skin and mucosal infections. M. abscessus has high intrinsic resistance to antibiotics, with very few treatment options. It has an impermeable cell wall due to the abundance of hydrophobic mycolic acids and secretion of multiple extracellular enzymes such as beta-lactamase, phosphotransferases, or acetyltransferases that inactivate antibiotics. Many antibiotics are bacteriostatic rather than bactericidal to M. abscessus. Clinically, there is no single antibiotic that can effectively treat M. abscessus lung infection.

[0007] The current treatment approach entails the prolonged use of combinations of at least three antibiotics, with treatment duration typically lasting at least 18-24 months. Further, current treatment yields mixed results, as the average rate of success of patients achieving eradication is only 45.6 %, and recurrence rates are approximately 30 % within one year of treatment. New treatment strategies for M. abscessus are urgently needed in view of the sub-optimal performance of current antibiotic treatments. Another class of dangerous bacteria is the ESKAPE group, which include both Gram-positive (Enterococcus faecium and Staphylococcus aureus) and Gram-negative (Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) bacteria. These pathogens are highly virulent and have become resistant to many common antibiotics, complicating therapy of their infections and leading to failures of modern surgical operations and chemotherapy interventions that require antibiotics-based infection prophylaxis. Alarmingly, some pathogens now resist even last-resort antibiotics such as colistin and carbapenems. Carbapenem-resistant ESKAPE top the “World Health Organisation (WHO) Bacterial Priority Pathogens List, 2024” which lists critical or high priority pathogens in dire need for new antibiotics. New treatment options are urgently needed for both ESKAPE pathogens and M. abscessus.

[0008] Cationic antimicrobial peptides (AMPs) play crucial roles across a wide range of organisms in defending against microbial pathogens and are considered promising alternatives to traditional antimicrobials. The cationic nature of these peptides is essential for their antimicrobial activity. Bacterial membranes are predominantly composed of negatively charged phospholipids, which attract the positively charged cationic peptides through electrostatic interactions. However, despite significant research progress, the translation of AMPs into clinical application has had limited success. Only five AMPs are in clinical use as antibiotic alternatives, out of more than 3000 AMPs catalogued in the Antimicrobial Peptide Database. Among the barriers to clinical application are their limited efficacy, toxicity to mammalian cells, and peptide instability. Polymers, with their diverse chemical backbone, degradability, and tunable cationic and hydrophobic properties, have been explored as synthetic mimics of cationic antimicrobial peptides, offering potential to overcome these challenges. Further, like antimicrobial peptides, they usually exhibit a low propensity to elicit antimicrobial resistance, and can be designed to eradicate persisters and biofilms. A common mechanism of killing is membrane disruption though this is not restrictive for new cationic polymers. The cationic property of cationic antimicrobial polymers is typically conferred by cationic side-chains, and in the last twenty years or so, many such side-chain cationic polymers have been designed and synthesized. However, none of them have been reported to exhibit efficacy against M. abscessus. Further, no single cationic polymer has shown broad-spectrum bactericidal activity against the ESKAPE bacteria and M. abscesses in in vivo models. In fact, the combination of the physiological, genomic, and biochemical differences between Gram-negative bacteria, Grampositive bacteria, and mycobacteria makes it very challenging to develop broad-spectrum antimicrobial agents that are effective against all these pathogens. Therefore, to overcome at least one of the aforementioned problems, there exists a need for new cationic main-chain poly(carbonate-imidazolium).

[0009] Summary of Invention

[0010] Aspects and embodiments of the invention are provided in the following numbered clauses.

[0011] 1. An oligomeric or polymeric material according to the repeating unit of formula I:

[0012]

[0013] where:

[0014] each x is 1, 2 or 3;

[0015] Y’ is a pharmaceutically acceptable anionic counterion;

[0016] Z is selected from -S-S-, -O-C(O)-O-, -NH-C(O)-O-, -O-C(O)-NH-, and -N-C(O)-N-, and n represents the repeating unit of the oligomer or polymer, or a pharmaceutically acceptable solvate thereof.

[0017] 2. The oligomeric or polymeric material according to Clause 1, wherein x is 1 or 2.

[0018] 3. The oligomeric or polymeric material according to Clause 2, wherein x is 1.

[0019] 4. The oligomeric or polymeric material according to any one of the preceding clauses, wherein Z is -O-C(O)-O-.

[0020] 5. The oligomeric or polymeric material according to any one of the preceding clauses, wherein Y' is selected from one or more of the group consisting of Cl-, Br, I", NO3“, BF4“, PF6“, and AcO, optionally wherein Y-is AcO-.

[0021] 6. The oligomeric or polymeric material according to Clause 1, wherein the oligomeric or polymeric material is selected from:

[0022]

[0023]

[0024] 7. The oligomeric or polymeric material according to Clause 6, wherein the oligomeric or

[0025]

[0026]

[0027] 8. The oligomeric or polymeric material according to Clause 7, wherein the oligomeric or

[0028]

[0029] optionally wherein the oligomeric or polymeric material has a number average molecular weight of from 2,000 to 4,000 g / mol, such as from 3,000 to 3,900 g / mol, such as 3,898 g / mol.

[0030] 9. The oligomeric or polymeric material according to any one of the preceding clauses, wherein the oligomeric or polymeric material has a number average molecular weight of from 1,000 to 20,000 g / mol, such as from 1,600 to 5,000 g / mol, such as from 2,000 to 4,000 g / mol, such as from 3,000 to 3,900 g / mol, such as 3,898 g / mol.

[0031] 10. A nanoparticle comprising:

[0032] an oligomeric or polymeric material as described in any one of Clauses 1 to 9; and an organic polyacid, wherein

[0033] the organic polyacid serves to crosslink the oligomeric or polymeric material to provide the nanoparticle.

[0034] 11. The nanoparticle according to Clause 10, wherein the organic polyacid is selected from one or more of the group consisting of tartaric acid, malic acid, succinic acid, phytic acid, and citric acid, optionally wherein the organic polyacid is citric acid.

[0035] 12. Use of an oligomeric or polymeric material as described in any one of Clauses 1 to 9, or a nanoparticle as described in Clause 10 or Clause 11 in medicine.

[0036] 13. Use of an oligomeric or polymeric material as described in any one of Clauses 1 to 9, ora nanoparticle as described in Clause 10 or Clause 11, in the manufacture of a medicament to treat a bacterial infection.

[0037] 14. An oligomeric or polymeric material as described in any one of Clauses 1 to 9, or a nanoparticle as described in Clause 10 or Clause 11 for use in the treatment of a bacterial infection.

[0038] 15. A method of treating a bacterial infection, the method comprising the steps of providing a pharmaceutically effective amount of an oligomeric or polymeric material as described in any one of Clauses 1 to 9, or a nanoparticle as described in Clause 10 or Clause 11 and administering it to a subject in need thereof.

[0039] 16. The use according to Clause 13, the material or nanoparticle for use according to Clause 14 or the method according to Clause 15, wherein the bacterial infection is selected from one or more of the group consisting of a wound infection, bacteremia or sepsis, a urinary tract infection, and a lung infection (e.g. a lung infection), optionally wherein the lung infection is caused by one of more of the group consisting of E. faecalis, and more particularly, M. abscessus, E. coli, K. pneumoniae, A. baumannii, P. aeruginosa, S. marcescens, P. mirabilis, and S. aureus. Drawings

[0040] FIG. 1 depicts the synthetic schemes of degradable linkers with different hydrophobicity and degradable groups.

[0041] FIG. 2 depicts (a) synthetic schemes of main-chain degradable polyimidazoliums via one-pot Poly-Radziszewski reaction, (b) Chemical structures of synthesized mainchain degradable polyimidazoliums with varying spacer hydrophobicity and degradable linkers.

[0042] FIG. 3 depicts1H nuclear magnetic resonance (NMR) of carbonate diamine monomer with 2-carbon spacing in D2O.

[0043] FIG. 4 depicts1H NMR of MCOP-1 in D2O.

[0044] FIG. 5 depicts in vitro biocompatibility of MCOP-1. (a) Cytotoxicity of MCOP-1 for 24 h, 48 h and 72 h against mouse embryonic fibroblast 3T3 cells, (b) Hemolysis of MCOP-1 at 10,000 µg / ml, 5 % rabbit blood was used.

[0045] FIG. 6 depicts killing kinetics of MCOP-1 against (a) M. abscessus, (b) Gram-positive bacteria S. aureus MRSA Lac, (c) Gram-negative bacteria P. aeruginosa PAO1 at different concentrations.

[0046] FIG. 7 depicts in vivo biocompatibility and antimicrobial activities against M. abscessus. (a) Bacteria count in the lungs before treatment and after daily intranasal dosage of DI water (UNT), 10 mg / kg MCOP-1, 10 mg / kg MCOP-1 nanoparticles (MCOP-1 NPs) in murine lung infection model induced by Mabs CIP104536 (R). Symbols represent the bacterial counts in mice, and the central lines represent the geometric mean of the individual counts (n = 5). Statistical analysis was done by one-way ANOVA followed by Dunnett test, P < 0.0001 (****). (b) Mouse body weight changes over 10 days treatment in Mabs CIP104536 (R) induced lung infection model.

[0047] FIG. 8 depicts the dynamic light scattering (DLS) size distribution of MCOP-1 nanoparticles.

[0048] FIG. 9 depicts in vivo biocompatibility and antimicrobial activities of MCOP-1 against Gramnegative and Gram-positive bacteria in lung infection model and peritonitis infection model, (a) Cartoon scheme illustrating lung infection model, (b) Bacteria count in the lungs after an intranasal dose of MCOP-1 at 30 mg / kg in K. pneumoniae ATCC 10031 and MRSA USA300 murine lung infection models. The mice were harvested at 24 h post infection. Symbols represent the bacterial counts in mice, and the central lines represent the geometric mean of the individual counts (n = 5). (c) Mouse body weight changes over one week after intranasal (IN) administration of 50 mg / kg MCOP-1. (d) Cartoon scheme illustrating peritonitis infection model, (e) Bacteria count in the intraperitoneal (IP) cavity, spleen (S), liver (L) and kidneys (K) after two intraperitoneal doses of 15 mg / kg MCOP-1 at 6 h apart in murine carbapenem-resistant E. coli BAA 2774 peritonitis infection model. The mice were harvested at 26 h post infection. Symbols represent the bacterial counts in mice, and the central lines represent the geometric mean of the individual counts (n = 5). (f) Survival of mice in murine carbapenem-resistant E. coli BAA 2774 peritonitis infection model treated by MCOP-1 or vehicle control.

[0049] FIG. 10 depicts bacteria count in IP fluid, blood, liver, spleen and kidneys of animals after intraperitoneal infection with carbapenem-resistant E. coli BAA 2774 before drug treatment.

[0050] FIG. 11 depicts in vivo acute, accumulated and long-term toxicity to liver and kidneys of MCOP-1, clinical biomarkers for liver and kidney functions: ALT, alanine transaminase; AST, aspartate transaminase; CRE, creatinine; BUN, blood urea nitrogen. P>0.05 for comparison of 1 day, 7-day treatment and at the end of test (14 day) to the pre-treatment group; Statistical analysis was performed using Prism 6 via one-way ANOVA analysis.

[0051] FIG. 12 depicts antimicrobial mechanism of MCOP-1 due to physical membrane disruption, (a) Field emission scanning electron microscopy (FE-SEM) images of E. coli without and with treatment with 4 x MIC MCOP-1. (b) Fluorescence microscopy of propidium iodide (PI) stained E. coli without and with treatment with 4 x MIC MCOP-1. Scale bar stands for 5 pm. (c) Fluorescence measurements of E. coli treated with MCOP-1 at different concentrations. Statistical analysis was done by one-way ANOVA followed by Dunnett test. The obtained P value is less than 0.0001, shown as **** in figure, (d) Cytoplasmic membrane potential dissipation of E. coli treated with MCOP-1 at different concentrations using 3,3’-Dipropylthiadicarbocyanine iodide (DiSC3(5)) as probe. Polymyxin B (PMB) was used as positive control.

[0052] FIG. 13 depicts resistance evolution profile of E. coli 8739 by serial passaging in the presence of MCOP-1. Ciprofloxacin was used as control.

[0053] FIG. 14 depicts antimicrobial mechanism of MCOP-1 due to DNA binding and breakage, (a) Isothermal titration calorimetry thermogram of MCOP-1 with bacterial DNA and its fitting curve and thermodynamic parameter, (b) Representative confocal images of E. coli MG1655 SMR14334 Gam-GFP strain with no drug treatment (untreated control, i), treatment of MCOP-1 at 16 µg / mL (ii), or treatment of DNA-targeting drug bleomycin at 20 µg / mL (positive control, iii) for 2 h in MHB at 37 °C. Foci formation indicates double-stranded DNA breaks, and arrows mark representative foci formed in MCOP-1 and bleomycin treated samples. Scale bar stands for 5 pm. (c) ROS detection of E. coli treated by MCOP-1 at different concentrations using a ROS-sensitive dye 2’,7’-dichlorofluorescein diacetate (DCFDA) as probe. Statistical analysis was done by one-way ANOVA followed by Dunnett test, P < 0.0001 (*“*); P = 0.0009(***); P > 0.05 (ns), (d) ROS quencher N-acetylcysteine (NAC) inhibits the killing kinetics of MCOP-1 against E. coli.

[0054] Description

[0055] It has been surprisingly found that the main-chain cationic imidazolium-derived oligomeric or polymeric materials, or nanoparticles disclosed herein are non-toxic to mammalian cells, and exhibit excellent broad-spectrum antimicrobial activity against pathogenic Gram-positive (Enterococcus faecium and Staphylococcus aureus) and Gram-negative (Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) bacteria.

[0056] Thus, in a first aspect of the invention, there is provided an oligomeric or polymeric material according to the repeating unit of formula I:

[0057]

[0058] where:

[0059] each x is 1, 2 or 3;

[0060] Y’ is a pharmaceutically acceptable anionic counterion;

[0061] Z is selected from -S-S-, -O-C(O)-O-, -NH-C(O)-O-, -O-C(O)-NH-, and -NH-C(O)-NH-, and n represents the repeating unit of the oligomer or polymer, or a pharmaceutically acceptable solvate thereof.

[0062] In embodiments herein, the word “comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components / features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of” or “consists essentially of’). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of” or the phrase “consists essentially of” or synonyms thereof and vice versa.

[0063] The phrase, “consists essentially of’ and its pseudonyms may be interpreted herein to refer to a material where minor impurities may be present. For example, the material may be greater than or equal to 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greater than 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.

[0064] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an oxygen carrier” includes mixtures of two or more such oxygen carriers, reference to “the catalyst” includes mixtures of two or more such catalysts, and the like.

[0065] For the avoidance of doubt, it is explicitly contemplated that where a number of numerical ranges related to the same feature are cited herein, that the end points for each range are intended to be combined in any order to provide further contemplated (and implicitly disclosed) ranges.

[0066] In some embodiments that may be mentioned herein, x may be 1 or 2. In further embodiments, x may be 1.

[0067] As will be appreciated, Z is generally symmetrical that is: -S-S-, -O-C(O)-O-, and -NH-C(O)-NH-. However, Z may be asymmetrical when it is used to form a carbamate linkage, i.e. -NH-C(O)-O- or -O-C(O)-NH-. It will be further appreciated by a person skilled in chemistry that the reaction conditions used herein to form said carbamate linkages may result in there being a mixture of both carbamate linkage types in the compounds disclosed herein. This may be as a statistical mixture or any other possible mixture. It will also be appreciated that selection of the synthetic conditions or starting materials may allow for one or the other carbamate linkages to be selected for and this may in turn allow for compounds that only contain one of the carbamate linkage types. Similarly, blocks of repeating units of one carbamate type alternating with the other carbamate type may be provided. In particular embodiments of the invention that may be mentioned herein, Z may be -O-C(O)-O-.

[0068] Y' may be any suitable pharmaceutically acceptable anionic counterion. Examples of pharmaceutically acceptable anionic counterions that may be mentioned herein include, but are not limited to Cl“, Br“, l“, NO3“, BF4“, PF6“, and AcO. For example, Y’ may be AcO.

[0069] In some embodiments that may be mentioned herein, the oligomeric or polymeric material may be selected from:

[0070]

[0071]

[0072] The oligomeric or polymeric materials of the invention may be prepared using any suitable polymerization procedure. Details of a general polymerization procedure that may be used is provided in Example 2 below.

[0073] While depicted having a specific carbamate linkage, as discussed above it will be appreciated that compounds (b), (f) and (i) may be formed having a specific carbamate linkage (e.g. that depicted; -NH-C(O)-O-), the opposite arrangement (i.e. -O-C(O)-NH-) or a mixture of both in the repeating units. As such, the structures provided are only provided for the sake of brevity.

[0074] Oligomeric or polymeric materials of the invention may be isolated from their reaction mixtures using conventional techniques (e.g. dialysis, etc.).

[0075] In some embodiments that may be mentioned herein, the oligomeric or polymeric material

[0076]

[0077]

[0078] The oligomeric or polymeric material may have any suitable number average molecular weight.

[0079] In some embodiments that may be mentioned herein, the oligomeric or polymeric material may be:

[0080]

[0081] . In such embodiments, the oligomeric or polymeric material may have a number average molecular weight of from 2,000 to 4,000 g / mol, such as from 3,000 to 3,900 g / mol, such as 3,898 g / mol.

[0082] In some embodiments that may be mentioned herein, the oligomeric or polymeric material may have a number average molecular weight of from 1,000 to 20,000 g / mol, such as from 1,600 to 5,000 g / mol, such as from 2,000 to 4,000 g / mol, such as from 3,000 to 3,900 g / mol, such as 3,898 g / mol.

[0083] As mentioned above, also encompassed by formula I are any solvates of the oligomeric or polymeric materials. Preferred solvates are solvates formed by the incorporation into the solid state structure (e.g. crystal structure) of the oligomeric or polymeric materials of the invention of molecules of a non-toxic pharmaceutically acceptable solvent (referred to below as the solvating solvent). Examples of such solvents include water, alcohols (such as ethanol, isopropanol and butanol) and dimethylsulfoxide. Solvates can be prepared by recrystallising the oligomeric or polymeric materials of the invention with a solvent or mixture of solvents containing the solvating solvent. Whether or not a solvate has been formed in any given instance can be determined by subjecting crystals of the oligomeric or polymeric material to analysis using well known and standard techniques such as thermogravimetric analysis (TGE), differential scanning calorimetry (DSC) and X-ray crystallography.

[0084] The solvates can be stoichiometric or non-stoichiometric solvates. Particularly preferred solvates are hydrates, and examples of hydrates include hemihydrates, monohydrates and dihydrates.

[0085] For a more detailed discussion of solvates and the methods used to make and characterise them, see Bryn et al., Solid-State Chemistry of Drugs, Second Edition, published by SSCI, Inc of West Lafayette, IN, USA, 1999, ISBN 0-967-06710-3.

[0086] In a second aspect of the invention, there is provided a nanoparticle comprising:

[0087] an oligomeric or polymeric material as described in the first aspect of the invention; and

[0088] an organic polyacid, wherein

[0089] the organic polyacid serves to crosslink the oligomeric or polymeric material to provide the nanoparticle.

[0090] Any suitable organic polyacid may be used. Examples of organic polyacids that may be mentioned herein include, but are not limited to tartaric acid, malic acid, succinic acid, phytic acid, and citric acid. For example, the organic polyacid may be citric acid.

[0091] When used herein, the term “nanoparticle” is intended to refer to particles that have an average hydrodynamic diameter of from 0.1 to 2,000 nm. In more particular embodiments of the invention that may be disclosed herein, the nanoparticle may have an average hydrodynamic diameter of from 20 to 100 nm, such as from 25 to 75 nm, such as from 30 to 50 nm, such as about 42 nm. For the avoidance of doubt, it is explicitly contemplated that where a number of numerical ranges related to the same feature are cited herein, that the end points for each range are intended to be combined in any order to provide further contemplated (and implicitly disclosed) ranges.

[0092] Any suitable method may be used to determine the hydrodynamic diameter of the nanoparticle. For example, dynamic light scattering (DLS) may be used to determine the hydrodynamic diameter of the nanoparticle. Details of the DLS technique are provided in Example 5 below. In a third aspect of the invention, there is provided use of an oligomeric or polymeric material as described in the first aspect of the invention, or a nanoparticle as described in the second aspect of the invention in medicine.

[0093] In a fourth aspect of the invention, there is provided use of an oligomeric or polymeric material as described in the first aspect of the invention, or a nanoparticle as described in the second aspect of the invention, in the manufacture of a medicament to treat a bacterial infection.

[0094] In a fifth aspect of the invention, there is provided an oligomeric or polymeric material as described in the first aspect of the invention, or a nanoparticle as described in the second aspect of the invention for use in the treatment of a bacterial infection.

[0095] In a sixth aspect of the invention, there is provided a method of treating a bacterial infection, the method comprising the steps of providing a pharmaceutically effective amount of an oligomeric or polymeric material as described in the first aspect of the invention, or a nanoparticle as described in the second aspect of the invention and administering it to a subject in need thereof.

[0096] A non-limiting list of bacteria that may be susceptible to the oligomer or polymeric materials, or nanoparticles of the invention include: Acidothermus cellulyticus, Acinetobacter baumannii, Actinomyces odontolyticus, Alkaliphilus metalliredigens, Alkaliphilus oremlandii, Arthrobacter aurescens, Bacillus amyloliquefaciens, Bacillus clausii, Bacillus halodurans, Bacillus licheniformis, Bacillus pumilus, Bacillus subtilis, Bifidobacterium adolescentis, Bifidiobacterium longum, Burkholderia thailandensis, Caldicellulosiruptor saccharolyticus, Carboxydothermus hydrogenoformans, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium botulinum, Clostridium cellulolyticum, Clostridium difficile, Clostridium kluyveri, Clostridium leptum, Clostridium novyi, Clostridium perfringens, Clostridium tetani, Clostridium thermocellum, Corynebacterium diphtheriae, Corynebacterium efficiens, Corynebacterium glutamicum, Corynebacterium jeikeium, Corynebacterium urealyticum, Desulfitobacterium hafniense, Desulfotomaculum reducens, Enterobacter cloacae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Eubacterium ventriosum, Exiguobacterium sibiricum, Fingoldia magna, Geobacillus kaustophilus, Geobacillus thermodenitrificans, Janibacter sp., Kineococcus radiotolerans, Klebsiella pneumoniae, Lactobacillus fermentum, Listeria monocytogenes, Listeria innocua, Listeria welshimeri, Moorella thermoacetica, Mycobacterium abscessus, Mycobacterium avium, Mycobacterium bovis, Mycobacterium gilvum, Mycobacterium leprae, Mycobacterium paratuberculosis, Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycobacterium vanbaalenii, Nocardioides sp., Nocardia farcinica, Oceanobacillus iheyensis, Pelotomaculum thermopropionicum, Proteus mirabilis, Pseudomonas aeruginosa, Rhodococcus sp., Saccharopolyspora erythraea, Serratia marcescens, coagulase-negative Staphylococcus species, Staphylococcus aureus, methicillin resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, methicillin resistant Staphylococcus epidermidis (MRSE), Streptococcus agalactiae, Streptococcus gordonii, Streptococcus mitis, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus sanguinis, Streptococcus suis, Streptococcus uberis, Streptomyces avermitilis, Streptomyces coelicolor, Thermoanaerobacter ethanolicus, Thermoanaerobacter tengcongensis, and combinations thereof. Specific bacteria that may be mentioned herein are discussed in the examples below.

[0097] The term “bacterial infection” covers any disease or condition caused by a bacterium in or on a subject. Examples of bacterial infections include, but are not limited to a wound infection, bacteremia or sepsis, a urinary tract infection, and a lung infection. For example, the bacterial infection may be a lung infection. For example, the lung infection may be caused by one of more of the group consisting of Enterococcus faecalis (E. faecalis), and more particularly, Mycobacterium abscessus (M. abscessus), Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumonia), Acinetobacter baumannii (A. baumannii), Pseudomonas aeruginosa (P. aeruginosa), Serratia marcescens (S. marcescens), Proteus mirabilis (P. mirabilis), and Staphylococcus aureus (S. aureus).

[0098] For the avoidance of doubt, in the context of the present invention, the term “treatment” includes references to therapeutic or palliative treatment of patients in need of such treatment, as well as to the prophylactic treatment and / or diagnosis of patients which are susceptible to the relevant disease states.

[0099] The terms “patient’ and “patients” include references to mammalian (e g. human) patients. As used herein the terms "subject" or "patient" are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human. In some embodiments, the subject is a subject in need of treatment or a subject with a disease or disorder. However, in other embodiments, the subject can be a normal subject. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered. The term “effective amount” refers to an amount of an oligomeric or polymeric material, or a nanoparticle, which confers a therapeutic effect on the treated patient (e.g. sufficient to treat or prevent the disease). The effect may be objective (i.e. measurable by some test or marker) or subjective (i.e. the subject gives an indication of or feels an effect).

[0100] As noted above, the oligomeric or polymeric materials, and nanoparticles of the invention may be used in the treatment of bacterial infections. Thus, there is also provided a pharmaceutical composition comprising the oligomeric or polymeric materials, or nanoparticles of the invention and one or both of a pharmaceutically acceptable adjuvant and carrier.

[0101] Oligomeric or polymeric materials, or nanoparticles of the invention may be administered by any suitable route, but may particularly be administered orally, intravenously, intramuscularly, cutaneously, subcutaneously, transmucosally (e g. sublingually or buccally), rectally, transdermally, nasally, pulmonarily (e.g. tracheally or bronchially), topically, by any other parenteral route, in the form of a pharmaceutical preparation comprising the oligomeric or polymeric material, or nanoparticle in a pharmaceutically acceptable dosage form. Particular modes of administration that may be mentioned include oral, intravenous, cutaneous, subcutaneous, nasal, intramuscular or intraperitoneal administration.

[0102] Oligomeric or polymeric materials, or nanoparticles of the invention will generally be administered as a pharmaceutical formulation in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, which may be selected with due regard to the intended route of administration and standard pharmaceutical practice. Such pharmaceutically acceptable carriers may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use. Suitable pharmaceutical formulations may be found in, for example, Remington The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995). For parenteral administration, a parenterally acceptable aqueous solution may be employed, which is pyrogen free and has requisite pH, isotonicity, and stability. Suitable solutions will be well known to the skilled person, with numerous methods being described in the literature. A brief review of methods of drug delivery may also be found in e.g. Langer, Science (1990) 249, 1527.

[0103] Otherwise, the preparation of suitable formulations may be achieved routinely by the skilled person using routine techniques and / or in accordance with standard and / or accepted pharmaceutical practice. The amount of the oligomeric or polymeric materials, or nanoparticles of the invention in any pharmaceutical formulation used in accordance with the present invention will depend on various factors, such as the severity of the condition to be treated, the particular patient to be treated, as well as the oligomeric or polymeric materials, or nanoparticles which is / are employed. In any event, the amount of oligomeric or polymeric materials, or nanoparticles in the formulation may be determined routinely by the skilled person.

[0104] For example, a solid oral composition such as a tablet or capsule may contain from 1 to 99 % (w / w) active ingredient; from 0 to 99% (w / w) diluent or filler; from 0 to 20% (w / w) of a disintegrant; from 0 to 5% (w / w) of a lubricant; from 0 to 5% (w / w) of a flow aid; from 0 to 50% (w / w) of a granulating agent or binder; from 0 to 5% (w / w) of an antioxidant; and from 0 to 5% (w / w) of a pigment. A controlled release tablet may in addition contain from 0 to 90 % (w / w) of a release-controlling polymer.

[0105] A parenteral formulation (such as a solution or suspension for injection or a solution for infusion) may contain from 1 to 50 % (w / w) active ingredient; and from 50% (w / w) to 99% (w / w) of a liquid or semisolid carrier or vehicle (e g. a solvent such as water); and 0-20% (w / w) of one or more other excipients such as buffering agents, antioxidants, suspension stabilisers, tonicity adjusting agents and preservatives.

[0106] Depending on the disorder, and the patient, to be treated, as well as the route of administration, oligomeric or polymeric materials, or nanoparticles may be administered at varying therapeutically effective doses to a patient in need thereof.

[0107] However, the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the mammal over a reasonable timeframe. One skilled in the art will recognize that the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the potency of the specific oligomeric or polymeric material, or nanoparticle, the age, condition, body weight, sex and response of the patient to be treated, and the stage / severity of the disease.

[0108] Administration may be continuous or intermittent (e.g. by bolus injection). The dosage may also be determined by the timing and frequency of administration. In the case of oral or parenteral administration the dosage can vary from about 0.01 mg to about 1000 mg per day of a oligomeric or polymeric material, or nanoparticle.

[0109] In any event, the medical practitioner, or other skilled person, will be able to determine routinely the actual dosage, which will be most suitable for an individual patient. The above-mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

[0110] Advantages of the present invention may include the following, which may or may not be described elsewhere herein.

[0111] The aspects of the invention described herein (e g. the above-mentioned oligomeric or polymeric materials, nanoparticles, methods and uses) may have the advantage that, in the treatment of the conditions described herein, they may be more convenient for the physician and / or patient than, be more efficacious than, be less toxic than, have better selectivity over, have a broader range of activity than, be more potent than, produce fewer side effects than, or may have other useful pharmacological properties over, similar oligomeric or polymeric materials, nanoparticles, methods (treatments) or uses known in the prior art for use in the treatment of those conditions or otherwise.

[0112] Other oligomeric or polymeric materials, and nanoparticles of the invention may be prepared in accordance with techniques that are well known to those skilled in the art, for example as described hereinafter in the Examples section.

[0113] The oligomeric or polymeric materials, and nanoparticles of the invention exhibit a pronounced antimicrobial action, especially against Gram-positive (Enterococcus faecium and Staphylococcus aureus) and Gram-negative (Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) bacteria. They are therefore also suitable in the treatment of a wound infection, bacteremia or sepsis, a urinary tract infection, and a lung infection, as described hereinbefore.

[0114] Further aspects and embodiments of the invention will now be discussed by reference to the following non-limiting examples.

[0115] Examples Materials

[0116] All commercially available chemical compounds were purchased from Sigma-Aldrich, Alfa-Aesar, Tokyo Chemical Industry, Cambridge Isotope Laboratories or Aik Moh Paints & Chemicals. Anhydrous toluene was obtained from distillation with sodium under argon. Merck 60 F254 pre-coated silica gel plate was used in the analytical thin-layer chromatography (TLC).

[0117]

[0118] Visualization was performed under a UV lamp, potassium permanganate stain or iodine stain or ninhydrin stain. Merck silica gel 60 was used for column chromatography. Proton nuclear magnetic resonance (1H NMR) spectra were measured with a Bruker Avance DPX-300 spectrometer at 300MHz or 400MHz.1H chemical shift was recorded relative to HOD (4.79 ppm). Agilent gel permeation chromatography (GPC) system was employed to measure the molecular weight (Mn) and molecular weight distribution (Đ) of polymers. The system was operated at 40 °C using a solution mixture of methanol, acetic acid, sodium acetate and water as the mobile phase. Narrow dispersion polyethylene glycol (PEG) or pullulan standards were used as references.

[0119] Example 1. General procedure for preparation of degradable monomers (FIG. 1)

[0120] In a 250 mL round-bottom flask fitted with a dry N2inlet and magnetic stirrer, dry toluene (150 mL) was added. Then, 1,1'-carbonyldiimidazole (CDI) (0.03 mol) was added, followed by the starting materials SM (0.01 mol) and NaOH (0.003 mol). The resulting mixture was heated at 60 °C with stirring overnight. For the synthesis of carbamate linkers, CDI (0.03 mol) and SM (0.03 mol) were premixed with NaOH (0.003 mol) and stirred for 4 hours. Subsequently, the Boc-protected amine monomers were added, and the reaction was allowed to continue overnight under the same conditions. Throughout the reaction, a clear solution formed and was observed. After completion of the reaction, the mixture was cooled to room temperature. The solution was then concentrated under reduced pressure, dissolved in dichloromethane (DCM), and washed three times with water. The solution was further dried using anhydrous Na2SO4 and concentrated under reduced pressure. The resulting crude product was then subjected to purification by column chromatography to yield purified Boc-protected degradable monomers 2.

[0121] The purified intermediate compound 2 was dissolved in dry DCM, and trifluoroacetic acid (TFA) was added at a 1:1 ratio. The mixture was stirred at room temperature for 18 hours. Finally, the resulting degradable monomers 3 were obtained after concentrating the mixture under reduced pressure. Example 2. General procedure for polymerization

[0122] A first mixture of glyoxal 40 wt% (0.4 mmol) and formaldehyde 37 wt% (0.4 mmol) in glacial acetic acid (AcOH) and tetrahydrofuran (THF) (2:1) at 0 °C (ice water) was prepared. A second solution mixture of degradable diamine (0.32 mmol) in AcOH and THF (2:1) at 0 °C (ice water) was also prepared. The first mixture was added dropwise to the second mixture over 3 mins at 0 °C (ice water). Then the reaction mixture was allowed to warm to room temperature and then reacted at 100 °C for 2 hours. The final reaction mixture was directly transferred into a 1000-Dalton cut-off dialysis membrane (Repligen, USA) and dialyzed against 5 L acidified water at 0 °C (ice water) for 3 days.

[0123] Example 3. Synthesis and characterization of degradable polymers

[0124] Mammalian cell toxicity assay

[0125] Cytotoxicity: A colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to measure the cytotoxicity of the polymers (T. Mosmann, J. Immunol. Methods 1983, 65(1-2), 55-63). Briefly, the cultured cell lines were harvested from the flask by trypsinization treatment and the harvested cells were transferred into a 50 ml centrifugation tube. The cell concentration was adjusted to a density of 2 x104cells / ml. 200 pl cell solutions were pipetted into a 96-well collagen-treated tissue culture plate. The plate was incubated (37 °C, 5% CO2 and humidity) for 24 h without agitation. Polymer stock solution was prepared in sterilized PBS at 10 mg / ml and diluted to desired concentrations in DMEM complete medium. The polymer in DMEM solution was pipetted into the 96-well plate with seeded cells. Wells without compounds were used as controls. The plate was incubated with 5% CO2 and humidity at 37°C without agitation for 24, 48 or 72 hours. After that, the culture medium containing the compounds was removed and the cells were rinsed twice with PBS. MTT (200 pl of 5mg / ml) was pipetted into each well and the plate was incubated with 5% CO2 and humidity at 37 °C for 4 hours. The MTT was discarded and dimethyl sulfoxide (DMSO) was pipetted into each well and the plate shaken (at 150rpm) until the intracellular formazan crystals were completely solubilized. A microplate spectrophotometer was used to record the absorbance of each well at 570 nm. The percentage cell viability was calculated by the following formula:

[0126] % Cell viability= (Average abs of treated cells) / (Average abs of controls) *100%

[0127] Hemolytic activity test: In view of its exceptionally low toxicity toward 3T3 fibroblasts, the hemolytic activity of MCOP-1 was assessed at a single, very high, MCOP-1 concentration, 10mg / ml. Rabbit red blood cells were collected by centrifugation at 3,000 rpm for 5 mins, followed by washing four times with PBS. The concentration of red blood cells was adjusted to 5% vol / vol in PBS solution. Polymer solution at a concentration of 20 mg / ml was prepared and 50 pl of this polymer solution (or the same volume of positive or negative control solutions) and 50 pl of erythrocyte suspension was pipetted into wells of a 96-well plate. 0.1% Triton X-100, which lyses all the red blood cells, was used as positive control and PBS was added as negative control. The plate was incubated at 37 °C under mild shaking at 150 rpm for 1 h. The plate was then subjected to centrifugation at 3000 rpm for 10 mins. 50 pl supernatant in each well was moved to another 96-well plate with 50 pl PBS in each well. A microplate spectrophotometer was used to record the absorbance of each well at 540 nm. The percentage hemolysis was obtained from the below formula:

[0128] Percentage hemolysis= [(Oβ– On) / (OP– On)] x 100% where Oβis the reading of the polymers, Onis the reading of the negative control (PBS), and Opis the reading of positive control (0.1% Triton X-100).

[0129] Results and discussion

[0130] Different degradable linkers such as carbonate (O), carbamate (A), urea (U) and disulfide (SS), as well as degradable linkers having different hydrophobicity made from 4, 6, or 8 carbon atoms (FIG. 2) were synthesized. As an illustration of the monomer and polymer synthesis, we shall describe the process for the MCOPs. Degradable carbonate diamine linkers were first prepared via a 1,1,1-carbonyldiimidazole (CDI) coupling reaction with M-Boc-ethanolamine in the presence of a catalytic amount of NaOH (S. P. Rannard & N. J. Davis, Org. Lett. 1999, 7(6), 933 - 936). Subsequently, tert- Butyl oxy carbonyl (Boc) groups were removed via trifluoroacetic acid (TFA) treatment (FIGS. 2 and 3). The synthesis of MCOPs was carried out through a one-pot Poly-Radziszewski reaction. This involved mixing aqueous formaldehyde and aqueous glyoxal solution with freshly prepared carbonate diamine monomers in the presence of an acid catalyst (FIG. 2) (J.-P. Lindner, Macromolecules 2016, 49(6), 2046-2053). The molecular weights of the polymers were controlled by adjusting various reaction parameters, including the stoichiometric ratios of diamine to aldehyde / glyoxal, the concentration of starting materials, reaction temperature and reaction time. Under optimized conditions, employing a stoichiometric ratio of carbonate diamine to aldehyde of 0.8: 1 and 0.4 M aldehyde concentration, the reaction conducted at 100 °C for 2 hours produced MCOPs with a high molecular weight (Mn) of 3898 g / mol (Table 1). The presence of proton peaks at 8.93 ppm and 7.53 ppm confirmed the formation of imidazolium rings in MCOPs. (FIG. 4).

[0131] Table 1. Summary of properties and antimicrobial efficacy of degradable antimicrobial main-chain polyimidazoliums with different degradable linkers. No. Samples MnD MIC (pg / ml) Cytotoxicity (g / mol)

[0132] ' S. PAO1bCC50(pg / ml)caureus3

[0133] 1 MCOP-1 3898 1.7 8 8 >1024 2 MCOP-2 6036 1.7 8 8 512

[0134] 3 MCOP-3 17295 2.3 8 8 <32

[0135] 4 MCAP-1 1641 1.1 >128 >128 >1024 5 MCAP-2 2408 1.2 >128 >128 >1024 6 MCAP-3 3880 1.9 32 32 32

[0136] 7 MCUP-1 2302 1.1 >128 >128 >1024 8 MCUP-2 2863 1.1 >128 >128 512

[0137] 9 MCUP-3 4722 1.4 64 32 128

[0138] 10 MCSSP N.A. N.A. 16 8 >1024aS. aureus 29213.

[0139] bP. aeruginosa PA01.

[0140] cCell line used was 3T3.

[0141] N.A.: not available due to no signal obtained from GPC measurement.

[0142] The other polymers, main-chain cationic carbamate-co-imidazolium-derived polymer (MCAPs), main-chain cationic urea-co-imidazolium-derived polymer (MCUPs) and main-chain cationic dithiol(SS)-coimidazolium-derived polymer (MCSSP) (FIG. 2b), were similarly prepared. They were synthesized using similar CDI coupling reactions and TFA deprotection (FIG. 2). The Poly-Radziszewski reactions were conducted using freshly prepared degradable linkers (FIG. 2a). We also prepared these main-chain co-polyimidazoliums with different hydrophobicity, using 4, 6, or 8 carbon atoms flanking the cleavable group in the degradable linkers. The molecular weights of the copolymers and their chemical structures were characterized using gel permeation chromatography (GPC) (Table 1) and nuclear magnetic resonance (NMR) spectroscopy, respectively.

[0143] Urea diamine monomer with 2-carbon spacing

[0144] 1H NMR (400 MHz, D2O) δ 3.36 (s, 4H), 3.03 (t, 4H). Carbamate diamine monomer with 2-carbon spacing

[0145] 1H NMR (400 MHz, D2O) δ 3.24 (m, 6H), 4.27 (d, 2H).

[0146] Carbonate diamine monomer with 3-carbon spacing

[0147] 1H NMR (400 MHz, D2O) δ 1.96 (t, 2H), 2.06 (d, 2H), 4.15 (d, 2H).

[0148] Urea diamine monomer with 3-carbon spacing

[0149] 1H NMR (400 MHz, D2O) δ 1.71 (t, 4H), 2.99 (dt, 8H).

[0150] Carbamate diamine monomer with 3-carbon spacing

[0151] 1H NMR (400 MHz, D2O) δ 1.79 (dt, 4H), 2.99 (m, 6H), 4.00 (d, 2H).

[0152] Carbonate diamine monomer with 4-carbon

[0153]

[0154] 1H NMR (400 MHz, D2O) δ 1.53 (t, 8H), 2.81 (s, 4H), 3.96 (t, 4H).

[0155] Urea diamine monomer with 4-carbon spacing

[0156] 1H NMR (400 MHz, D2O) δ 1.14 (m, 8H), 2.59 (d, 8H).

[0157] Carbamate diamine monomer with 4-carbon spacing

[0158] 1H NMR (400 MHz, D2O) δ 1.56 (m, 8H), 2.59 (m, 8H).

[0159] MCOP-2

[0160] 1H NMR (400 MHz, D2O) δ 2.21 (b, 4H), 4.20 (b, 8H), 7.50 (b, 2H), 8.85 (b, 1H).

[0161] MCOP-3

[0162] 1H NMR (400 MHz, D2O) 5 1.50-2.00 (b, 8H), 4.00–4.20 (b, 8H), 7.50 (b, 2H), 8.85 (b, 1H).

[0163] MCAP-1

[0164] 1H NMR (400 MHz, D2O) δ 3.40 (b, 2H), 4.00–4.50 (b, 6H), 7.50 (b, 2H), 8.85 (b, 1H).

[0165] MCAP-2

[0166] 1H NMR (400 MHz, D2O) 6 1.50-2.50 (b, 4H), 3.50–4.50 (b, 8H), 7.50 (b, 2H), 8.85 (b, 1H).

[0167] MCAP-3

[0168] 1H NMR (400 MHz, D2O) 5 1.00-2.00 (b, 8H), 3.00–4.50 (b, 8H), 7.50 (b, 2H), 8.85 (b, 1H). MCUP-1

[0169] 1H NMR (400 MHz, D2O) δ 3.80–4.50 (b, 8H), 7.50 (b, 2H), 8.85 (b, 1H).

[0170] MCUP-2

[0171] 1H NMR (400 MHz, D2O) δ 1.00–4.50 (b, 12H), 7.50 (b, 2H), 8.85 (b, 1H).

[0172] MCUP-3

[0173]

[0174] 1H NMR (400 MHz, D2O) δ 1.00–4.50 (b, 16H), 7.50 (b, 2H), 8.85 (b, 1H).

[0175] MCSSP

[0176] 1H NMR (400 MHz, D2O) δ 3.10 (b, 4H), 4.50 (b, 4H), 7.50 (b, 2H), 8.85 (b, 1H).

[0177] Example 4. MCOP-1 is bactericidal against antibiotic-resistant bacteria and M. abscessus and is not cytotoxic

[0178] MIC test

[0179] MIC determination for M. abscessus followed a reported protocol (R. Sorayah et al., ACS Infectious Diseases 2019, 5(12), 2055-2060). Briefly, 200 pl of mycobacteria culture (OD6000.005) was added into 96-well flat bottom plates. Then, 2 pl of drugs at varying concentrations were dispensed into the wells, and the plates were incubated for 2 days at 32 °C for the M. abscessus wildtype. The optical density at 600 nm was measured using a BioTek Cytation 3 Cell Imaging Multiple-mode reader. MIC50, defined as the concentration of the drug inhibiting at least 50% of bacterial growth, was calculated using GraphPad Prism 10.2 software.

[0180] MIC determination for the other strains was following the published protocol (A. Luther et al., Nature 2019, 576(7787), 452-458). The MIC values of the polymers against the tested microorganisms were measured in 96-well plates according to CLSI guidelines. Polymer stock solutions of 10 mg / ml were prepared in sterilized deionized water (DI water). Mueller Hinton Broth (MHB) (BD, Difco™) (50 pl) was added into the wells of a 96-well plate and used to dilute the polymers by two-fold series dilution. 50 pl of bacterial suspension was then pipetted into each well and the plates were incubated at 37 °C for 18 hours. Bacterial growth was measured by checking the absorbance at 600nm (OD600) on a Tecan Spark 10M or by visual observation of the turbidity. The lowest concentration that led to a reduction in bacterial growth by more than 90%, or the absence of a defined bacterial pellet, was defined as the MIC value.

[0181] Time-killing assay Kill kinetics assays for M. abscessus also followed a reported protocol (C. Dupont et al., Antimicrob. Agents Chemother. 2017, 61(11)). M. abscessus cultures were adjusted to OD6000.005 and dispensed in 24-well plates. Drugs at specified concentrations were added into the respective wells, and the plates were incubated at 32 °C. Bacterial viability at specified time points was determined by CFU enumeration on LB agar plates.

[0182] Kill kinetics assay for the other strain followed the published protocol (L. L. Ling et al., Nature 2015, 577(7535), 455-459). Bacteria cells were grown, diluted, and aliquoted into 96 well plates as described for the MIC assay, and then mixed with 50 pl volume of medium containing polymer at 1×, 2× and 4× MIC. The plates were sealed and incubated at 37 °C with shaking at 200 rpm.

[0183] At 0, 0.5, 1, 2, 3, 4, 6 and 24 h post-inoculation, each well to be sampled was thoroughly mixed with a multi-channel pipette and 20 pl of sample was removed, serially diluted in sterile phosphate buffered saline (PBS), plated on LB agar plates, and incubated at 37 °C for 12 hours. Colonies were counted to determine the CFU / mL at each time point.

[0184] Results and discussion

[0185] For initial screening of various synthesized degradable copolyimidazoliums, the antimicrobial efficacy against representative rapidly growing Gram-positive S. aureus 29213 and Gramnegative P. aeruginosa PAO1 bacteria was assessed by determining their minimum inhibitory concentrations (MICs). Notably, MCOP-1 exhibited antimicrobial activity against both S. aureus 29213 and P. aeruginosa PAO1 at an MIC of 8 pg / ml (Table 1). Increasing the hydrophobicity in MCOPs (i.e. MCOP-2 and MCOP-3 compared to MCOP-1) did not enhance antimicrobial potency (Table 1). Altering the degradable linkers from carbonate (in MCOPs) to carbamate (in MCAPs), urea (in MCUPs), and dithiol (in MCSSP) impaired the antimicrobial activities (Table 1).

[0186] The mammalian cell biocompatibility of the degradable copolyimidazoliums was evaluated with mouse embryonic 3T3 fibroblasts. A standard MTT assay was conducted to measure the metabolic activity of live cells in vitro, with biocompatibility quantified by calculating half maximal cytotoxicity concentration (CC50) values (Table 1). Remarkably, MCOP-1 did not induce toxicity in mammalian cells even at the highest concentration tested (CC50> 1024 µg / ml) (Table 1). In contrast to their hydrophobicity-independent antimicrobial potency, MCOP toxicity towards mammalian cells increased with hydrophobicity (Table 1). MCOP-1 exhibited no long-term (72 h) toxicity, even at the highest concentration at 1024 µg / ml (FIG. 5a). Moreover, MCOP-1 displayed negligible hemolysis, even at the highest tested concentration of 10,000 µg / ml (FIG. 5b).

[0187] Of the ten copolymers tested, MCOP-1 exhibited the best combination of MIC and CC50 values and was further evaluated for its antimicrobial efficacy. MCOP-1 maintained robust antimicrobial activity against various drug-resistant strains. The polymer was highly potent (with MIC of 6 pg / ml) against M. abscessus, as well as carbapenem-resistant and colistinresistant E. coli strains, carbapenem-resistant Gram-negative bacteria of K. pneumoniae, A. baumannii, P. aeruginosa, and methicillin-resistant Gram-positive of S. aureus (with MICs of 4-32 pg / ml) (Table 2). Interestingly, MCOP-1 was also potent against bacteria intrinsically resistant to colistin due their less anionic cell envelope, such as Serratia marcescens and Proteus mirabilis (Table 2), which is indicative of an antimicrobial mechanism of action distinct from that of traditional cationic antimicrobial polymers.

[0188] Table 2. MICs of MCOP-1 against M. abscessus and a panel of antibiotic-resistant Grampositive bacteria and Gram-negative bacteria.

[0189] MICs (pg / ml)

[0190] Strains - MCOP-1 Colistin

[0191] NTM

[0192] M. abscessus CIP104536 6 N. A.

[0193] Gram-negative

[0194] E. coli MG 1899 8 2

[0195] E. coli ECOR 8 2

[0196] E. coli BAA-2774 8 2

[0197] E. co / / NMT1833 8 4

[0198] K. pneumonia KPNR 8 2

[0199] K. pneumonia SGH10 8 2

[0200] A. baumannii AB-1 16 2

[0201] A. baumannii BAA 2803 16 4

[0202] P. aeruginosa PAO1 8 2

[0203] P. aeruginosa PAER 32 2 P. aeruginosa BAA-2797 32 2

[0204] S. marcescens ATCC 13880 16 >128

[0205] P. mirabilis ATCC 7002 32 >128

[0206] Gram-positive

[0207] S. aureus ATCC 29213 8 32

[0208] S. aureus MRSA USA300 8 32

[0209] S. aureus MRSA Lac 4 64

[0210] Note: MIC for NTM is MIC50; MIC for the other strains is MIC90.

[0211] We measured the killing kinetics in M. abscessus cells by MCOP-1. The copolymer MCOP-1 exhibited bactericidal activity, unlike many antibiotics that only show bacteriostatic activity. 4 × MIC was more effective than 2 × MIC dosing (FIG. 6a). However, the killing occurred mainly at early time points following the drug exposure, and the number of viable cells recovered gradually over time (FIG. 6a). This was the case even at higher concentration (FIG. 6a and 4 × MIC). The regrowth might be due to the presence of persister cells within the bacterial population. Persister cells are a small subset of dormant cells that are inherently tolerant to antimicrobial agents. After MCOP-1 degraded in the media, these cells can reinitiate growth, leading to the observed re-growth in vitro. With representative Gram-positive MRSA and Gram-negative bacteria PAO1, the killing kinetics of MCOP-1 against these pathogens revealed rapid eradication, completely eliminating both bacterial strains at 2 to 4-fold the MIC within 1 h (FIGS. 6b-c).

[0212] Example 5. In vivo therapeutic potential against M. abscessus and ESKAPE pathogens

[0213] MCOP-1 nanoparticle preparations

[0214] MCOP-1 nanoparticles were synthesized using a nanocomplex formation approach following established procedures (M. Chai et al., Adv. Healthc. Mater. 2020, 9(3), 1901542; and J. A. Finbloom et al., Sci. Adv. 2023, 9(3), eade8039). Stock solutions of MCOP-1 (20 mg / ml) and citric acid (160 mg / ml) were prepared. 1 ml of MCOP-1 stock solution was vigorously vortexed for 3 minutes with an equal volume of citric acid stock solution at 0 °C, resulting in the formation of MCOP-1 nanoparticles. The particle size was determined using dynamic light scattering (DLS).

[0215] DLS measurement Malvern Zetasizer Nano was used for dynamic light scattering measurements to estimate particle sizes.

[0216] In vivo efficacy in a murine NTM lung infection model

[0217] The in vivo efficacy of the drugs against NTM was assessed in mice using a modified lung infection model (S. Zhang etal., Antimicrob. Agents Chemother. 2020, 64(8), e00236-20). All animal experiments were approved by the Nanyang Technological University Institutional Animal Care and Use Committee, under the IACUC protocol A21039. 7-week-old BALB / c mice were purchased from InVivos (Singapore), and immunosuppression was achieved through intraperitoneal injection of cyclophosphamide one day prior to the infection (100 mg / kg), as well as 4 days (100 mg / kg) and 8 days (75 mg / kg) post infection (p.i.). The bacterial inoculum was prepared as follows: M. abscessus CIP104536 (rough) culture was grown at 37 °C to exponential phase in Middlebrook 7H9 broth medium supplemented with 0.05% Tween 80 and 10% Albumin-Dextrose-Saline (ADS), pelleted, washed, resuspended in PBS containing 0.025% Tween 80 to a final concentration of ~1 x108CFU / ml, and passed through a 27-gauge needle a few times to break up any small clumps. All mice were infected intranasally with 30 pl of this inoculum and divided randomly into groups of five animals. Starting on day 2 p.i. for a total of 10 days, the mice were treated daily with various drugs (10 mg / kg via intranasal delivery) or drug vehicle controls. 5 mice were sacrificed on day 2 p.i. before drug treatment to determine bacteria amount prior to treatment. The remaining mice were sacrificed on day 12 p.i. Lungs were harvested, homogenized in PBS containing 0.025% Tween 80, and serial dilutions were plated on 7H11 agar and incubated at 37 °C for CFU enumeration.

[0218] In vivo efficacy in Gram-negative and Gram-positive bacteria infection models

[0219] Lung infection model: 5-to-6-week female ICR Mice were immunosuppressed by intraperitoneal administration of 150 mg / kg cyclophosphamide 4 days before infection and 100 mg / kg cyclophosphamide 1 day before infection. Bacterial cultures of K. pneumoniae ATCC 10031 or MRSA USA300 were prepared and used to infect the mice. 40 µL of ~1×1010CFU / mL of MRSA USA300 or ~1×108CFU / ml of K. pneumoniae ATCC 10031 were administered via the intranasal route to introduce the infection. After 2 hours of infection, a single dosage of MCOP-1 was administered via the intranasal route. Mice were euthanized at indicated time points (24 hours) by CO2 asphyxiation followed by cervical dislocation. The lung was recovered and stored in ice-cold PBS and was homogenized using a high-throughput tissue homogenizer (Omni). The bacterial load in the lungs was quantified by plating and CFU counting of serially diluted samples on LB agar plates. Intraperitoneal infection model: Bacterial cultures of E. coli BAA 2774 were prepared and used to infect mice. 200 pl of ~1x106CFU / ml of E. coli BAA 2774 in 5% mucin were injected into the intraperitoneal cavity to introduce the infection. After 2 hours of infection, two dosages of MCOP-1, 6 hours apart, were injected into the intraperitoneal cavity. PBS was used as a control. Mice in the antibacterial efficacy arm of the study were euthanized at indicated time points (26 hours) by CO2 asphyxiation followed by cervical dislocation. The intraperitoneal fluid, kidney, spleen and liver were recovered and stored in ice-cold PBS. Organs were homogenized using a high-throughput tissue homogenizer (Omni). The bacteria present in the organs and abdomen were quantified by plating and CFU counting of serially diluted samples on LB agar plates. Mice in the survival arm of the study were monitored for 7 days. A five-point body condition score, based on body weight, body temperature, respiration, motility and posture, was used to determine the clinical endpoint for mice. Mice were euthanized upon reaching those endpoints in accordance with the Nanyang Technological University Institutional Care and Use Committee (IACUC) guidelines.

[0220] All studies and protocols were approved by the Nanyang Technological University IACUC under the approved protocols A19001 and A20029.

[0221] In vivo toxicity assessment

[0222] A single dose of 50 mg / kg of polymer was administered by the intranasal route to 5 to 6-week-old female ICR mice. The body weight of the mice was measured and recorded. The mice were observed in detail for any indications of toxicity effect within the first six hours after administration, and daily for a period of 7 days. All the animals were weighed and visual observations for mortality, behavioral pattern, changes in physical appearance, injury, pain and signs of illness were made daily during the study period.

[0223] To measure drug hepatotoxicity and nephrotoxicity, the mice were repetitively injected with MCOP-1 at dosage of 15 mg / kg / day for 7 days via intraperitoneal route. Biomarkers related to liver (ALT and AST) and kidney (BUN and CRE) function were determined before treatment and after 1 and 7 days’ repetitive injection as well as at the end of the study (Day 14).

[0224] All studies and protocols were approved by the Nanyang Technological University IACUC under the approved protocols: A20029 and A19001.

[0225] Results and discussion

[0226] Encouraged by the promising in vitro antimicrobial potency and biocompatibility of MCOP-1, we assessed its in vivo potency against M. abscessus. We used a murine lung infection model of M. abscessus, to explore the in vivo efficacy of MCOP-1. However, MCOP-1 exhibited no statistically significant in vivo efficacy on M. abscessus infection (FIG. 7a). We hypothesized that nanoformulation may help better lung distribution and retention. We then formulated MCOP-1 into nanoparticles (NPs) via nanocomplexation with citric acid (M. Chai et al., Adv. Healthcare Mater. 2020, 9(3), 1901542; and J. A. Finbloom et al., Sei. Adv. 2023, 9(3), eade8039). The size of the MCOP-1 NPs (~42 nm, FIG. 8) was within the optimal range for lung disease management.

[0227] Significantly, MCOP-1 NPs, administered at a daily dose of 10 mg / kg-day x 10 days, led to 96.5 % of reduction in M. abscessus burden in the lungs of infected animals (FIG. 7a). As the unformulated MCOP-1 did not significantly reduce the bacterial burden compared to the vehicle control, the nanoparticle formulation of the MCOP-1 agent appears to be a critical factor for efficacy against M. abscessus. We also measured the body weights of mice receiving MCOP-1 nanoparticles and they remained within a safe range (FIG. 7b), supporting the therapeutic potential of MCOP-1 nanoparticles.

[0228] Further, we evaluated MCOP-1 against K. pneumoniae and MRSA in an in vivo model of neutropenic lung infection (FIG. 9a) (L. L. Ling etal., Nature 2015, 577(7535), 455-459; and P. A. Smith etal., Nature 2018, 567(7722), 189-194). K. pneumoniae, which is known to cause bacterial pneumonia, was used to establish the lung infections. At 2 h post-infection, the mice were treated with 30 mg / kg MCOP-1 given by intranasal administration. The treatment led to a reduction of more than 99 % of bacterial count in the lungs (FIG. 9b). S. aureus, another bacterium associated with nosocomial lung infections, was also tested, using the MRSA USA300 strain. Intranasal administration of single dose of MCOP-1 at 30 mg / kg reduced the bacterial load in the lungs by 99.8 % (FIG. 9b). We also evaluated the toxicity of MCOP-1 administered via the intranasal route at higher than therapeutic dose. MCOP-1 did not cause apparent toxicity in mice after a single bolus administration of as much as 50 mg / kg via the intranasal (IN) route. Throughout the 7-day monitoring period, all mice survived and exhibited normal activity. Visual inspection did not reveal any signs illness or lethargy. The body weight of all mice showed a slight decrease on the first day, but subsequently rapidly recover so that the mice showed steady weight gain afterwards (FIG. 9c), suggesting that the degradation products of MCOP-1 are not toxic to mice.

[0229] We also evaluated our compound for combating bacterial peritonitis, an abdominal infection that can rapidly progress to sepsis associated with high morbidity. E. coli is the predominant pathogen responsible for peritonitis. Carbapenem-resistant E. coli infections have higher mortality rates than infections caused by carbapenem-sensitive E. coli strains. Carbapenem-resistant E. coli induced peritonitis was first established in the mice (FIG. 9d). Then, intraperitoneal administration of MCOP-1 at 15 mg / kg-dose x 2 doses reduced the bacterial load by more than 99.9999 % in the intraperitoneal cavity and by more than 99.9 % in the tested internal organs (spleen, liver, and kidneys) (FIG. 9e). Moreover, 100 % of the mice receiving this drug regimen survived up to 7 days, whereas all untreated control animals succumbed from infection 1-2 days post-infection (FIG. 9f). The E. coli results indicate the curative potential of the drug even in the presence of an established infection. In this model, the infection had disseminated to the bloodstream and distant organs prior to drug treatment (FIG. 10), underscoring the therapeutic promise of MCOP-1. Moreover, MCOP-1 administered at a daily dose of 15 mg / kg-day-dose x 7 doses caused no significant change in biomarkers for hepatotoxicity and nephrotoxicity compared to untreated mice, suggesting that the agent caused no acute, accumulated or long-term toxicity to liver and kidneys (FIG. 11).

[0230] These comprehensive in vivo evaluations demonstrate the potential of MCOP-1 as a non-toxic therapeutic agent, particularly in the treatment of challenging infections with M. abscessus and colistin- / carbapenem-resistant ESKAPE pathogens that cause serious lung infections and bacterial peritonitis.

[0231] Example 6. MCOP-1 permeabilizes bacterial membranes and targets DNA

[0232] Resistance evolution assay

[0233] The resistance evolution study was performed following a published protocol (L. L. Ling et al., Nature 2015, 517(7535), 455). 5x105CFU / ml log-phase E. coli 8739 was incubated together with 0.5x, 1x, 2xMIC polymers or antibiotic control in 96-well plates (final well volume of 100 pl) at 37 °C under shaking at 220 rpm. After 24 to 48 hours incubation, bacterial turbidity was checked using a Tecan Spark 10M. The wells with the highest concentration of drugs that allowed bacteria growth were used as inoculum stock for the next passage. Bacteria were passaged 24 times.

[0234] Field emission scanning electron microscopy (FE-SEM) imaging

[0235] An overnight culture of E. coli was prepared by inoculating 3 colonies into MHB and incubating them overnight at 37°C. A 1:1000 dilution of the overnight culture was transferred into fresh MHB and grown for 4 hours at 37 °C to reach the log phase (OD600nm= 0.2). A bacterial suspension of 2 x 106CFU / mL was then incubated with an equal volume of polymer solution at 37 °C for 2 hours, while a bacterial suspension with PBS served as the blank control. After incubation, the bacteria were washed with PBS, fixed overnight with 4% paraformaldehyde, and then washed again with PBS. The samples were dehydrated using a graded ethanol series (20%-99.7%) and dried in air before being characterized using FE-SEM on a Verios G4 instrument from FEI.

[0236] PI staining assay

[0237] The PI staining assay was performed following the reported protocol (A. Sabnis et al., eLife 2021,10, e65836). Mid-log phase bacteria were harvested by centrifugation, washed three times with PBS buffer, and resuspended to a bacterial density of 108CFU / ml in different concentrations of MCOP-1 or PBS buffer alone. MCOP-1 was removed by centrifugation after incubation for 1 hour at 37 °C. 100 pl bacteria in PBS suspension was pipetted into a 96-well plate (Black, Corning Costar) and incubated with 100 pl PI dye solution (30 pM) for 10 mins. The fluorescence signal was subsequently measured (TECAN fluorescence spectrometer) at an excitation wavelength of 535 nm and an emission wavelength of 617 nm. For microscopy imaging, Pl-stained bacteria were transferred to a polylysine-coated Petri dish (MatTek Corporation) and imaged using a Zeiss LSM800 confocal microscope.

[0238] Membrane permeabilization assay

[0239] The membrane permeabilization experiment using 3,3’-Dipropylthiadicarbocyanine iodide DiSCs(5) as the probe was performed following a reported protocol (L. Ejim etal., Nat. Chem. Biol. 2011, 7(6), 348-350). Mid-log phase bacteria were harvested by centrifugation, washed three times with 5 mM HEPES buffer containing 20 mM glucose and 0.1 M KCI (pH 7.8) and diluted to a bacterial density of 107CFU / ml. 0.2 mM EDTA (pH 8.0) was used to increase the permeability of the outer membrane of bacteria. In a 96-well black plate, DiSCs(5) solution was added to 190 pl bacteria suspension to a final concentration of 1 pM. The DiSCs(5) dye was allowed to gradually quench at 37 °C. 10 pl of MCOP-1 at different concentrations in HEPES buffer solution or HEPES buffer only was added into the bacterial solution. Changes in fluorescence due to the disruption of the cytoplasmic membrane were recorded using a spectrometer (TECAN fluorescence spectrometer) at an excitation wavelength of 622 nm and an emission wavelength of 670 nm.

[0240] Isothermal titration calorimetry (ITC)

[0241] All ITC experiments were performed at 37 °C with PEAQ-ITC (MicroCai Malvern). Both the polymers and DNA were dispersed in PBS buffer at pH = 7.4. 2 pl volumes of DNA solution were titrated into 300 pl polymer solution in the sample cell at 750 rpm stirring speed with a reference power of 10 pcal / s. Every experiment consisted of 19 injections at 150 s intervals. The background signals of DNA or polymer to PBS buffer were measured and subtracted during data analysis to exclude the sample dilution heat. The thermodynamic parameters were obtained by fitting the data to one set binding model using the software in the system (MicroCai PEAQ-ITC Analysis).

[0242] Intracellular double-stranded DNA breakage assay

[0243] To monitor MCOP-1 effects in causing intracellular dsDNA breaks, E. coli MG1655 Gam-GFP strain (SMR14334) was cultured and imaged after drug treatment following a reported method (M. Yong etal., Antimicrob. Agents Chemother. 2023, 67(5), e00355-23; T. Nagarajan et al., Res. Microbiol. 2023, 774(8), 104136; C. Shee et al., eLife 2013, 2, e01222; and P. Belenky et al., Cell Rep. 2015, 73(5), 968-980). Briefly, overnight culture of SMR14334 was diluted 100-fold in MHB and incubated at 37 °C, 150 rpm for 1 hour before the addition of doxycycline at 20 ng / mL to induce the expression of Gam-GFP protein for another 1 hour. Cultures were then treated with bleomycin (20 pg / mL, positive control) or MCOP-1 (16 pg / mL) at 37 °C for 2 hours. After drug treatment, bacteria cells were fixed with 4% paraformaldehyde for 30 mins. Samples were then analyzed on FV3000 Olympus Confocal Microscope for foci formation.

[0244] Intracellular reactive oxygen species (ROS) assay

[0245] The ROS generation was measured using a ROS-sensitive dye, 2’,7’-dichlorofluorescein diacetate (DCFDA), following the published protocol (M. Zhou et al., Angew. Chem. Int. Ed.

[0246] 2020, 59(16), 6412-6419; and D. Zhang etal., Adv. Sci. 2022, 9(14), 2104871). Mid-log phase bacteria were harvested by centrifugation, washed three times with PBS and resuspended to a bacterial density of 109CFU / ml in PBS. DCFDA solution was added to the bacteria suspension to a final concentration of 20 pM and incubated at 37 °C. The bacteria were harvested by centrifugation and washed three times after 30 min incubation. 190 pl bacteria suspension was pipetted into a black 96-well plate (Corning Costar), then 10 pl MCOP-1 or PBS was added into the bacterial solution at the desired concentrations. The fluorescence signal was immediately measured (TECAN fluorescence spectrometer) at an excitation wavelength of 488 nm and an emission wavelength of 530 nm.

[0247] Results and discussion

[0248] Since positively charged antimicrobial polymers usually interact with and disrupt the negatively charged bacteria membrane, we first used field emission scanning electron microscope (FE-SEM) to observe the morphology of E. coli treated with MCOP-1. In the untreated control group, most bacteria displayed intact and smooth membrane surfaces (FIG. 12a). In contrast, treatment with MCOP-1 resulted in wrinkling and even lysis of bacterial membrane (FIG. 12a), indicating that bacterial membrane is one of the targets of MCOP-1. To further confirm the membrane lysis effect of MCOP-1, we conducted a propidium iodide (PI) assay, which selectively stains bacteria with damaged membranes (W. Zhong et al., Proc. Natl. Acad. Sci. USA 2020, 777(49), 31376-31385). MCOP-1 significantly increases the PI signal in bacteria compared to untreated control (FIGS. 12b-c), corroborating the membrane activity of MCOP-1. We also used 3,3’-dipropylthiadicarbocyanine iodide (DiSC3(5)), a cytoplasmic membrane permeabilization probe, to assess the integrity of the plasma membrane. The DiSCs(5) dye accumulates in polarized bacterial plasma membranes where its fluorescence is selfquenched. Disruption of the bacterial plasma membrane leads to collapse of the transmembrane potential and release of fluorescent DiSC3(5). MCOP-1 triggered increased DiSCs(5) fluorescence intensity in tested bacteria (FIG. 12d). The membrane lysis effect of MCOP-1 was also corroborated by the lack of resistance evolution in E. coli when repetitively challenged with the compound (FIG. 13).

[0249] Notably, MCOP-1's membrane perturbation effects were weaker than polymyxin control (FIG.

[0250] 12d), but MCOP-1 exhibits good potency against polymyxin (colistin)-resistant bacteria (Table 2). We hypothesized that the antimicrobial activities of MCOP-1 may have contributions from additional killing mechanisms beyond membrane perturbation. We observed that MCOP-1 exhibited a strong binding affinity to bacterial DNA (KA = 7.1 * 10® M’1, FIG. 14a), suggesting that it may also kill bacteria via interaction with DNA, potentially interfering with the transcription process. To explore these questions, we imaged MCOP-1-induced intracellular DNA damage in E. coli Gam-GFP strain (C. Shee et al., eLife 2013, 2, e01222.), which is an engineered strain designed to identify intracellular double-stranded DNA (dsDNA) breaks through GFP foci formation. MCOP-1 treatment indeed resulted in many GFP foci observed through confocal microscopy (FIG. 14b), indicating interaction with and disruption of intracellular DNA by MCOP-1. Damage to DNA has been associated with the triggering of a spontaneous SOS response in bacteria, leading to the production of ROS. An excess of ROS can trigger a bacterial death program. We measured intracellular ROS generation using the ROS-sensitive dye 2’, 7’-dichlorofluorescin diacetate (DCFDA) and observed a significant increase in ROS signals in MCOP-1 -treated bacteria compared to untreated controls (FIG.

[0251] 14c). Importantly, the addition of 5 mM of the ROS quencher / V-acetylcysteine (NAC) to MCOP-1 treated bacteria substantially inhibited ROS generation, reducing it to levels similar to the untreated control (FIG. 14c). Moreover, the addition of 5 mM NAC dramatically reduced MCOP-1's killing kinetics (FIG. 14d). These findings support the inference that DNA binding contributes to the antimicrobial activity of MCOP-1. The lack of correlation between ROS levels and bacterial killing time course may be attributed to MCOP-1's dual mechanism of killing, where rapid membrane lysis leads to rapid bacterial elimination.

[0252] General Discussion In summary, MCOP-1 employs dual mechanisms to kill bacteria, which may explain its superior broad-spectrum antimicrobial activity compared to other cationic antimicrobial polymers that rely solely on membrane lysis (Z. Si et al., Chem. Sci. 2022, 73(2), 345-364). The multiplicity of kill mechanisms, and the specifics of these mechanisms (membrane perturbation and DNA damage), may account for the observed absence of resistance evolution and broad-spectrum antimicrobial activity.

[0253] In this study, we screened various degradable linkers to synthesize main-chain cationic imidazolium-derived copolymers via a simple and cost-effective one-pot Poly-Radziszewski reaction (J.-P. Lindner, Macromolecules 2016, 49(6), 2046-2053). We found that carbonate-containing polyimidazolium, called “main-chain carbonate-co-imidazolium-derived polymer (MCOP)”, has superior in vitro antibacterial and biocompatibility properties. Furthermore, we validated the in vivo MCOP-1's broad-spectrum bactericidal efficacy. MCOP-1, when formulated with citric acid, achieves efficacy against M. abscessus in a murine model of lung infection. MCOP-1 also demonstrated broad-spectrum antibacterial efficacy against Gramnegative and Gram-positive bacteria in multiple murine infection models. We also found that MCOP-1 showed no noticeable acute, accumulated, or long-term in vivo toxicity.

[0254] MCOP-1 rapidly reduces M. abscessus bacterial load in the lungs in a murine model, achieving 96.5 % reduction of the bacteria with 10 days of single-agent therapy at the dose of 10 mg / kgday. The standard of care antibiotic clarithromycin commonly used to treat NTM infections requires a dosing of 250 mg / kg-day x 10 days and achieves 90 % log reduction (U. S. Ganapathy etal., Antimicrob. Agents Chemother. 2021, 65(5)). NTM infections are clinically difficult to treat and there is no single drug that can eradicate NTM infections due to their extreme resistance to almost all classes of antibiotics. Cationic polymers, including MCOP-1, kill bacteria by membrane disruption; resistance towards this physical mechanism, as opposed to a specific target mechanism, evolves less readily. Moreover, MCOP-1 can translocate across the bacterial envelope to bind and damage DNA, leading to cell death. The dual mechanisms of MCOP-1 and the nature of these mechanisms hinder the evolution of resistance.

[0255] Toxicity is a common problem in antimicrobial applications of cationic polymers. Herein, we overcome the toxicity problem by exploring a range of degradable linkers; one of these, carbonate, resulted in a cationic polymer with excellent efficacy against M. abscessus and other bacteria. Though M. abscessus secretes various enzymes, it does not secrete enzymes for degrading carbonates, though carbonates are subjected to hydrolysis. We hypothesize that the carbonate linker stability can be tuned by the hydrophobicity of the adjoining alkyl group so that the cationic poly(carbonate) is transiently stable to exert short-term bactericidal effect but also sufficiently labile to degrade into nonactive small molecules over longer time periods, thereby reducing its toxicity. This range of properties permitted the identification of an optimal linker chemistry and linker size with the desired combination of low toxicity to eukaryotic cells and good antibacterial efficacy. MCOP-1 is stable enough to remain intact for a time sufficient to penetrate and kill bacteria but on longer time scales, it degrades into short cationic fragments, which are not toxic to mammalian cells. For the M. abscessus induced lung infection model, we formulated MCOP-1 into nanoparticles. Current therapeutic regimens often fail due to intrinsic drug resistance of the M. abscessus bacterium, as well as limited lung penetration of most antibiotics. The nanoparticle system was necessary for in vivo efficacy of MCOP-1 against M. abscessus.

[0256] The fully degradable cationic main-chain MCOP-1 disclosed herein has broad-spectrum antimicrobial activity against pathogenic Gram-negative and Gram-positive bacteria, as well as M. abscessus, which is superior to all reported cationic antimicrobial polymers with pendent cationic groups (S. Shabani et al., Nat. Rev. Bioeng. 2024, 2, 343-361; Y. Wu et al., Prog. Polym. Sci. 2023, 141, 101679; M. Haktaniyan & M. Bradley, Chem. Soc. Rev. 2022, 51(20), 8584-8611; K. Jung etal., Adv. Mater. 2022, 34(2), 2105063; and J. Portelinha et al., Chem. Rev. 2021, 121(4), 2648-2712). MCOP-Ts broad-spectrum activities may be attributed to its dual mechanisms of action: targeting both bacterial membrane and DNA, which are common to all these bacteria. Recently, Bai etal. demonstrated that main-chain cationic oligoamidines and oligoguanidines exert broad-spectrum antimicrobial activities by targeting bacterial membranes and DNA, which further supports our mechanism hypothesis (Z. Chen et al., Biomaterials 2021, 275, 120858; and S. Bai etal., Sci. Adv. 2021, 7(5), eabc9917). However, the antimicrobial efficacy of oligoamidines and oligoguanidines was translated only into in vivo skin infection models against either Gram-negative bacteria or Gram-positive bacteria, which may be due to the compounds’ toxicity to mammalian cells and fouling in in vivo systemic environments. The fully degradable nature of MCOP-1 contributes to its superior biocompatibility both in vitro and in vivo while the delocalized charges in imidazolium rings make MCOP-1 inherently more resistant to fouling in the complex in vivo systemic environment.

[0257] The incorporation of a degradable carbonate linker and fouling-resistant imidazolium into each repeat unit of MCOP-1 contributes significantly to its broad-spectrum in vivo systemic bactericidal activities against mycobacteria, and Gram-negative and Gram-positive bacteria. These findings underscore the potential of MCOP-1 as a promising therapeutic antimicrobial agent for combatting NTM infections, as well as carbapenem and colistin-resistant bacteria.

Claims

1. Claims1. An oligomeric or polymeric material according to the repeating unit of formula I:

4. 6.where:7.each x is 1, 2 or 3;8.Y- is a pharmaceutically acceptable anionic counterion;9.Z is selected from -S-S-, -O-C(O)-O-, -NH-C(O)-O-, -O-C(O)-NH-, and -NH-C(O)-NH-, and n represents the repeating unit of the oligomer or polymer, or a pharmaceutically acceptable solvate thereof.

2. The oligomeric or polymeric material according to Claim 1, wherein x is 1 or 2.

3. The oligomeric or polymeric material according to Claim 2, wherein x is 1.

4. The oligomeric or polymeric material according to any one of the preceding claims, wherein Z is -O-C(O)-O-.

5. The oligomeric or polymeric material according to any one of the preceding claims, wherein Y_is selected from one or more of the group consisting of Cl-, Br, I", NO3“, BF4“, PF6", and AcO, optionally wherein Y- is AcO-.

6. The oligomeric or polymeric material according to Claim 1, wherein the oligomeric or16.

18.

20.

7. The oligomeric or polymeric material according to Claim 6, wherein the oligomeric or23.

25.

8. The oligomeric or polymeric material according to Claim 7, wherein the oligomeric or polymeric material is:

29.

30. , optionally wherein the oligomeric or polymeric material has a number average molecular weight of from 2,000 to 4,000 g / mol, such as from 3,000 to 3,900 g / mol, such as 3,898 g / mol.

9. The oligomeric or polymeric material according to any one of the preceding claims, wherein the oligomeric or polymeric material has a number average molecular weight of from 1,000 to 20,000 g / mol, such as from 1,600 to 5,000 g / mol, such as from 2,000 to 4,000 g / mol, such as from 3,000 to 3,900 g / mol, such as 3,898 g / mol.

10. A nanoparticle comprising:33.an oligomeric or polymeric material as described in any one of Claims 1 to 9; and an organic polyacid, wherein34.the organic polyacid serves to crosslink the oligomeric or polymeric material to provide the nanoparticle.

11. The nanoparticle according to Claim 10, wherein the organic polyacid is selected from one or more of the group consisting of tartaric acid, malic acid, succinic acid, phytic acid, and citric acid, optionally wherein the organic polyacid is citric acid.

12. Use of an oligomeric or polymeric material as described in any one of Claims 1 to 9, or a nanoparticle as described in Claim 10 or Claim 11 in medicine.

13. Use of an oligomeric or polymeric material as described in any one of Claims 1 to 9, or a nanoparticle as described in Claim 10 or Claim 11, in the manufacture of a medicament to treat a bacterial infection.

14. An oligomeric or polymeric material as described in any one of Claims 1 to 9, or a nanoparticle as described in Claim 10 or Claim 11 for use in the treatment of a bacterial infection.

15. A method of treating a bacterial infection, the method comprising the steps of providing a pharmaceutically effective amount of an oligomeric or polymeric material as described in any one of Claims 1 to 9, or a nanoparticle as described in Claim 10 or Claim 11 and administering it to a subject in need thereof.

16. The use according to Claim 13, the material or nanoparticle for use according to Claim 14 or the method according to Claim 15, wherein the bacterial infection is selected from one or more of the group consisting of a wound infection, bacteremia or sepsis, a urinary tract infection, and a lung infection (e.g. a lung infection), optionally wherein the lung infection is caused by one of more of the group consisting of E. faecalis, and more particularly, M. abscessus, E. coli, K. pneumoniae, A. baumannii, P. aeruginosa, S. marcescens, P. mirabilis, and S. aureus.

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