Antimicrobial combinations in treatment of pseudomonas aeruginosa biofilm associated infections

Abstract

The present invention relates to a synergistic combination of colistin and N-acetylcysteine (NAC) for use in the treatment and eradication of mature biofilm related infections caused by Pseudomonas aeruginosa. In particular, a synergistic combination of colistin and NAC is useful for treating and eradicating Pseudomonas aeruginosa infection in patients suffering from chronic pulmonary disease and recurrent Pseudomonas aeruginosa infection.

Description

Field of the Invention

[0001] The present invention relates to a synergistic combination of two known drugs for the treatment of mature biofilm-associated P. aeruginosa infections.

[0002] Prior art

[0003] According to the National Institutes of Health (NIH), approximately 65% ​​of all microbial infections and 80% of all chronic infections are associated with the presence of bacterial biofilms.

[0004] However, it has only recently been defined that bacterial biofilms represent a significant threat to public health, as they render pathogens less sensitive to antibiotics and to the host immune system.

[0005] In addition, biofilm-related infection is related to the deterioration of multiple infectious diseases caused by MDR (multi-drug resistant) bacteria, and the MDR bacteria are usually resistant to more recently developed antibiotics. In fact, the antibiotics currently used can reduce the number of bacteria in biofilm, but they cannot completely eradicate the bacteria in the protected environment (Fernandez Barat, 2017 et al. J. Cyst. Fibros. 16, 222-229 DOI: https: / / doi.org / 10.1016 / j.jcf.2016.08.005).

[0006] Extracellular polymeric substances (EPS) (hereafter biofilm) are made of proteins (<1-2%, including enzymes), DNA (<1%), polysaccharides (1-2%), RNA (<1%), and water (up to 97%) that allow the flow of nutrients within the biofilm matrix.

[0007] Biofilm formation is due to the activation of bacterial mechanisms and gene transcription that are unusual for the planktonic stage. In fact, when a bacterial cell transitions to the biofilm stage of growth, it undergoes a phenotypic switch in behavior, in which a large number of genes are differentially regulated and expressed (Blasi F. et al. Respiratory Medicine, 2016, 117: 190-197; Becker P. et al., Appl. Env. Microbiology, July 2001, 67(7): 2958-2965; Trubenova et al. Trends in microbiol, March 22, 2022, https: / / doi.org / 10.1016 / j.tim.2022.02.005).

[0008] In fact, biofilm-forming pathogens display characteristics that are different from those displayed by the same pathogens in the planktonic growth phase, including resistance or sensitivity to antibiotics. These differences explain why many antibiotics effective against planktonic (fast-growing) bacteria are ineffective against the same bacteria embedded in biofilms, where the embedded bacteria also have a reduced growth rate or are quiescent and therefore less sensitive to the mechanisms behind the activity of the most common antibiotics.

[0009] This may also explain why diagnostic methods may also fail to determine antibiotic susceptibility and the most appropriate antibiotic therapy in bacterial infections: diagnostic tests are usually performed on bacteria in the planktonic phase of growth, while infections are maintained in a latent state by biofilm-grown bacteria (Trubenova et al. 2022). Furthermore, until recently, biofilm-grown bacteria were studied using standard assays developed for planktonic bacteria, while the study of biofilm-grown bacteria in this phase required specific tests that, unfortunately, still lack good standardization.

[0010] See Blasi, 2016 (cited above, see Figure 1 ), biofilm formation is a multi-step process in which bacteria need to adhere to a substrate in a reversible manner, then their adhesion becomes irreversible, only then do they develop microcolonies, and later, they develop stable colonies with mature biofilms.

[0011] Pseudomonas aeruginosa has a high incidence in hospital-acquired infections and is associated with a high mortality rate. In addition, it is involved in a variety of persistent biofilm infections and is a major pathogen in chronic lung infections. Morse et al. (Frontiers in Immunology, 2021, 12: 1-15) reviewed the interaction between this pathogen in the growing biofilm stage and the human immune system, and Figure 1 A clear schematic diagram of the different maturation stages of biofilms is reported, including young biofilms and mature / old biofilms, each of which contains different cell populations and different characteristics. Typically, biofilm-related infections remain asymptomatic until the biofilm has reached the mature / old stage and begins to release planktonic cells or biofilm fragments (i.e., so-called septic emboli), which are responsible for the clinical manifestations of the disease. Conventional antibiotic therapy can usually kill planktonic cells and some biofilm-embedded cells released from the biofilm, "persister" cells (dormant cells that are insensitive to antibiotics due to a unique physiological state) remain within the biofilm and are ready to promote recurrence of infection.

[0012] Furthermore, it has been shown that polysaccharide components in P. aeruginosa, particularly rhamnolipids, act as powerful detergents, causing cell necrosis and neutrophil elimination, thus further shielding the pathogen from host immune responses (Jensen P. et al., Microbiology, 2007, 153: 1329-1338).

[0013] In the literature, controversial results have been reported regarding the supposed interaction between antibiotics and mucolytics such as NAC. In 1981, Roberts and Cole found that 2%-5% NAC exhibited antimicrobial activity against Pseudomonas aeruginosa and that low concentrations of NAC enhanced the effect of carbenicillin against Pseudomonas aeruginosa (Journal of Infection Vol. 3, No. 4, December 1981, pp. 353-359).

[0014] In 2016, the results of a study conducted by Landini et al. on the effect of high NAC concentrations on the activity of antibiotics against a range of respiratory pathogens demonstrated that high NAC concentrations did not interfere with the activity of the most commonly used antibiotics, while NAC impaired the activity of carbapenems (Landini et al. Antimicrob. Agents Chemother. December 2016, Vol. 60, No. 12, 7513-7517).

[0015] However, all the biological mechanisms induced by NAC have not yet been fully elucidated.

[0016] The strong synergistic activity of NAC and colistin against S. maltophilia and A. baumannii has been described by the same applicant in WO 2018 / 154091. Infections with these pathogens are usually harmless to healthy people, only cause severe pneumonia in immunocompromised patients, and are only rarely associated with fatal consequences.

[0017] Charrier et al. in: "Cysteamine a novel mucoactive antimicrobial & antibiofilm agent for the treatment of cystic fibrosis” Orphanet Journal of Rare Diseases 2014 9:189; doi:10.1186 / s13023-014-0189-2) disclosed the activity of cysteamine in increasing the sensitivity of Pseudomonas aeruginosa to tobramycin. He focused on the activity of this mucolytic agent, focusing on the viscoelastic properties of cysteamine on DNA and mucin (two major components of mucus), the effect on preventing biofilm formation, and the killing effect of cysteamine.

[0018] Pseudomonas aeruginosa is involved in a variety of persistent biofilm infections. In chronic lung patients, Pseudomonas aeruginosa infection is associated with frequent exacerbations and higher mortality (Malhotra et al. 2019 Clin. Microbiol. Rev. 32 (3): e00138-18; Parkins et al. Clin Microbiol Rev 31 (4): e00019-18). In addition, the activation of host innate and adaptive immunity leads to a chronic inflammatory state and causes extensive tissue damage, profoundly affecting lung function. This makes effective treatment of the pathogen extremely urgent, especially for these patients.

[0019] Of note, in patients with cystic fibrosis or other chronic respiratory diseases, colistin is often used as a last resort treatment for infections caused by this pathogen or other multidrug-resistant non-fermenting Gram-negative pathogens (e.g., Acinetobacter baumannii, Stenotrophomonas maltophilia), thus also increasing the risk of development and spread of resistance to this antibiotic between pathogens belonging to different strains.

[0020] Therefore, given the spread of multidrug-resistant phenotypes and the low efficacy of new-generation antibiotics over the past few years, new therapeutic options that can overcome and circumvent the immune escape mechanisms of bacterial pathogens, especially the major pathogen Pseudomonas aeruginosa, are extremely urgent. SUMMARY OF THE INVENTION

[0022] The present invention relates to the use of a synergistic combination of colistin and N-acetylcysteine ​​(NAC) for treating bacterial infections caused by the pathogen Pseudomonas aeruginosa, wherein the pathogen is in the biofilm stage of growth. Preferably, the bacterial infection is associated with respiratory diseases such as chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), non-cystic fibrosis bronchiectasis (NCFB), ventilator-associated pneumonia (VAP) and exacerbations thereof.

[0023] The combination is particularly useful against Pseudomonas aeruginosa infections where the pathogen has a multidrug resistant (MDR) or colistin resistant (CSTR) phenotype, such as, for example, in hospital associated infections (HAIs) or healthcare associated infections (HCAIs).

[0024] The combination of colistin and NAC is synergistic when the two drugs are administered simultaneously, even if not together, sequentially or separately, by the same or different routes of administration, with or without overlapping administration periods. According to a preferred embodiment, colistin is administered by inhalation route.

[0025] A further object of the invention is a kit of parts comprising a vial of colistin as powder and a vial of NAC aqueous solution or NAC as tablets or granules, optionally in combination with a diluting device, for the joint administration of a synergistic combination of colistin and NAC.

[0026] Methods of treatment with a synergistic combination of colistin and NAC for treating Pseudomonas aeruginosa infections, wherein the pathogen is in the biofilm stage of growth, and methods of treatment for eradicating such bacterial infections in a subject in need thereof, are also embodiments encompassed by the present invention.

[0027] BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 . Antibiofilm activity of N-acetylcysteine ​​(NAC) 8000 mg / L (grey squares), colistin 2, 4 and 8 mg / L (open squares with light grey, dark grey and black borders, respectively), and combinations of NAC 8000 mg / L with colistin 2, 4 and 8 mg / L (grey squares with light grey, dark grey and black borders, respectively) against Pseudomonas aeruginosa Z154 from CF (mucoid and colistin-susceptible). Filled circles indicate biofilms in the absence of NAC and / or colistin (i.e., control). The solid black line above the symbols indicates the median value. The x-axis was set at the detection limit (i.e., 20 cfu / peg). Statistical significance was determined using the Kruskal-Wallis test with Dunn's correction. ****, p<0.0001. cfu: colony forming unit.

[0029] Figure 2 .Antibiofilm activity of N-acetylcysteine ​​(NAC) 8000 mg / L (grey squares), colistin 8 mg / L (open squares), and NAC / colistin combination (grey squares with black borders) against P. aeruginosa PAO1 (A) and P. aeruginosa Z34 from CF (B), both of which are non-mucoid and colistin-susceptible. Filled circles represent biofilms in the absence of NAC and / or colistin (i.e., controls). The solid black line above the symbols represents the median value. The x-axis was set at the detection limit (i.e., 20 cfu / peg). Statistical significance was determined using the Kruskal-Wallis test with Dunn's correction. ***, p<0.001; ****, p<0.0001. cfu: colony forming unit.

[0030] Figure 3 .Antibiofilm activity of N-acetylcysteine ​​(NAC) 8000 mg / L (grey squares), colistin 8 mg / L (open squares), and NAC / colistin combination (grey squares with black borders) against Pseudomonas aeruginosa M19 (A) and Pseudomonas aeruginosa M25 (B) (both from CF, mucoid and colistin-susceptible). Filled circles represent biofilms in the absence of NAC and / or colistin (i.e., controls). The solid black line above the symbols represents the median value. The x-axis was set at the detection limit (i.e., 20 cfu / peg). Statistical significance was determined using the Kruskal-Wallis test with Dunn's correction. **, p<0.01; ***, p<0.001; ****, p<0.0001. cfu: colony forming unit.

[0031] Figure 4 .Antibiofilm activity of N-acetylcysteine ​​(NAC) 8000 mg / L (grey squares), colistin 32 mg / L (open squares), and NAC / colistin combination (grey squares with black borders) against Pseudomonas aeruginosa FC238 from CF (non-mucoid and colistin-susceptible). Filled circles represent biofilms in the absence of NAC and / or colistin (i.e., control). The solid black line above the symbols represents the median value. The x-axis was set at the detection limit (i.e., 20 cfu / peg). Statistical significance was determined using the Kruskal-Wallis test with Dunn's correction. ****, p<0.0001. cfu: colony forming unit.

[0032] Figure 5.Antibiofilm activity of N-acetylcysteine ​​(NAC) 8000 mg / L (grey squares), colistin 64 mg / L (open squares), and NAC / colistin combination (grey squares with black borders) against Pseudomonas aeruginosa Z154 (A) and Pseudomonas aeruginosa Z34 (B) in the ASM biofilm model. Filled circles represent biofilms in the absence of NAC and / or colistin (i.e., control). The solid black line above the symbols represents the median value. The x-axis is set at the detection limit (i.e., 100 cfu / mL). Statistical significance was determined using the Kruskal-Wallis test with Dunn's correction. **, p<0.01; ***, p<0.001; ****, p<0.0001. cfu: colony forming unit.

[0033] Figure 6 Kill curves showing the differential effects of anaerobic (A) and aerobic (B) conditions on planktonic cultures of exponentially growing Pseudomonas aeruginosa Z154 treated with N-acetylcysteine ​​(NAC) 8000 mg / L (grey squares), colistin 0.25 mg / L (open squares), and NAC / colistin combination (grey squares with black borders). Filled circles represent planktonic cultures in the absence of NAC and / or colistin (i.e., controls). Median values ​​with ranges are plotted. Dashed line indicates limit of detection (i.e., 17 cfu / mL). cfu: colony forming unit. DETAILED DESCRIPTION OF THE INVENTION

[0035] According to the present invention, the term "colistin", also known as polymyxin E, consists of a cationic cyclic heptapeptide with a tripeptide side chain acylated at the N-terminus by a fatty acid via an α-amide bond. In the following, the term colistin includes colistin and its pharmaceutically acceptable salts and / or prodrugs such as colistin sulfate, colistin sodium methanesulfonate, colistin methane sulphate and colimycin or colomycin, all of which are intended to refer to the bactericidal cyclic peptide antibiotic colistin or its precursors, such as the prodrug colomycin that is converted into the active drug colistin.

[0036] Two different forms of colistin are available for clinical use: colistin sulfate, which is administered orally for intestinal decontamination and topically as a powder for the treatment of bacterial skin infections, and colistin sodium methanesulfonate (CMS) (also known as colistin methanesulfate, pentasodium colistimethanesulfate, and colistin sulfonyl methate) for parenteral (intravenous, intramuscular, nebulized, and intrathecal / intraventricular) therapy. Colistin can thus be administered as a prodrug in the form of colistin sodium methanesulfonate (colistin sodium methanesulfonate is readily hydrolyzed to form a sulfomethylated derivative), as well as colistin sulfate, the active form of the drug. In a specific aspect, for the in vitro susceptibility testing in the experimental part of the present invention, the term "colistin" refers to colistin sulfate (according to the international guidelines for antimicrobial susceptibility testing provided by the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST)).

[0037] Colistin has recently gained a crucial role in treating various types of infections (e.g. pneumonia, bacteremia, urinary tract infections) caused by Gram-negative pathogens expressing a multidrug-resistant phenotype (e.g. non-fermenting Gram-negative pathogens and carbapenem-resistant enterobacteria) and represents one of the last treatment options for Gram-negative bacteria (including Pseudomonas aeruginosa). Therefore, the emergence of colistin-resistant Pseudomonas aeruginosa strains with elevated minimum inhibitory concentrations (MICs) is of great concern. In fact, very high amounts of colistin are known to be highly toxic to the host, and this limits its use, even when colistin would be the only option.

[0038] N-acetylcysteine, commonly abbreviated as NAC, is an acetylated precursor of the amino acid L-cysteine ​​and reduced glutathione (GSH). Historically, it has been used as a mucolytic agent with antioxidant and anti-inflammatory properties in patients with viscous or thickened airway mucus for a range of chronic respiratory diseases including chronic bronchitis, emphysema, COPD and exacerbations, cystic fibrosis, bronchiectasis, as an antidote due to acetaminophen overdose, and as a potential treatment for diseases characterized by free radical, oxidative damage. NAC is commercially available worldwide by different manufacturers under various brand names, for example as FluimucilTM (Zambon Spa).

[0039] From the perspective of developing more effective treatment options for MDR pathogens, which would avoid the use of extremely high (and toxic) antibiotic concentrations required to eradicate bacterial infections in biofilms, the synergy between colistin and NAC should be of great interest, firstly because NAC is not an antibiotic, and secondly because the molecule is not toxic, or is toxic only at extremely high concentrations. Therefore, its synergy in combination with colistin allows the use of lower antibiotic concentrations, but the lower antibiotic concentrations are effective against bacteria embedded in biofilms, even with an MDR phenotype.

[0040] Representing a first embodiment of the present invention is the use of a synergistic combination of colistin and NAC for the disruption of already formed biofilms of P. aeruginosa and bactericidal activity against P. aeruginosa cells already in the biofilm stage of growth, enclosed in mature biofilms.

[0041] "Growing biofilm stage" or "biofilm stage" refers to the mature stage of biofilm development. It is characterized by cell division, production of extracellular matrix and the spread of the final cells. This stage is also identified as the "mature biofilm" stage. In vitro assays are often used to distinguish biofilm-related infections, such as crystal violet staining, metabolic or molecular assays, growth on peg-lids (such as the Calgary Biofilm Device (CBD)) on microtiter plates, determination of the minimum biofilm eradication concentration (MBEC) (both described in Harrison JJ et al. (Nat Protoc, 2010; 5: 1236-54)), biofilm ring test (BRT), biofilm sensitivity test, or optical imaging of biopsies, growth of biofilm bacteria on nutrient plate agar after diffusion of planktonic bacteria.

[0042] However, these assays do not distinguish between the several stages of biofilm formation.

[0043] In vivo, biofilm-associated infections are associated with recurrence of infection.

[0044] The final stage of mature biofilm development, the disintegration and spread of bacteria, plays an important role in determining the persistence and recurrence of biofilm-associated infections in patients with a history of P. aeruginosa infection, particularly those with cystic fibrosis and other chronic lung diseases (e.g., chronic obstructive pulmonary disease and non-CF bronchiectasis). Indeed, P. aeruginosa, a prevalent infection in these patients, once established in the CF airways, develops into a chronic infection that often persists indefinitely, contributing to a decline in lung function, frequent exacerbations, and higher mortality.

[0045] The results behind the present invention provide evidence that the present combination of colistin and NAC is effective against Pseudomonas aeruginosa enclosed in an already formed mature biofilm (according to the definitions and schematics provided in, for example, Moser et al. Front. Immunol. 12:625597 (doi: 10.3389 / fimmu.2021.625597) (see in particular) through a suitable and specific assay developed for bacteria in the biofilm stage. Figure 1 )) have synergistic anti-biofilm activity, thus avoiding recurrence of infection.

[0046] According to the present invention, the term "anti-biofilm" refers to the inhibition or suppression of biofilm persistence or maintenance of bacteria that have been enclosed in a biofilm. For the purposes of the present invention, the activity of NAC against "mature biofilm" refers to the eradication or reduction of Pseudomonas aeruginosa growth when the Pseudomonas aeruginosa is enclosed in an already formed (pre-formed) biofilm.

[0047] This condition is related to the in vitro conditions disclosed in this example, where the biofilm is allowed to grow in a suitable medium (such as CAMHB at 35°C under static conditions) for at least 24 hours before the already formed (preformed) biofilm is exposed to NAC and an antibiotic, i.e. colistin, alone or in combination.

[0048] In other words, and without being limited by a particular scientific theory, the observed synergistic antibiofilm activity could be due to different mechanisms, such as increased permeability of antibiotics in extracellular polymeric substances (EPS, such as biofilms), direct synergistic inhibitory activity on biofilm formation, inhibition of bacterial growth and the consequent reduction or maintenance of biofilms that entrap bacteria, and enhancement of the bactericidal activity of each drug.

[0049] Thus, the present invention also relates to, and preferably relates to, a synergistic pharmacological combination of colistin and N-acetylcysteine ​​(NAC) to eradicate or reduce in vitro growth of Pseudomonas aeruginosa, wherein the Pseudomonas aeruginosa have been encapsulated or embedded in a mature biofilm.

[0050] Chronic infection caused by P. aeruginosa is associated with adaptive changes of the pathogen in the lungs of chronic patients, such as CF patients. Transformation to a mucoid phenotype, shift to a biofilm mode of growth, and acquisition of antibiotic resistance are frequently observed, which are caused by cumulative exposure to antibiotics during treatment and lead to ineffective antibiotic therapy and thus worse clinical outcomes.

[0051] Therefore, the present synergistic combination is particularly useful for patients suffering from chronic lung diseases and Pseudomonas aeruginosa infection to combat their exacerbations and relapses.

[0052] More generally, the combination according to the invention may represent the best therapeutic option for eradicating Pseudomonas aeruginosa infection and reducing lung decline in patients with chronic lung diseases (e.g., those with cystic fibrosis, chronic obstructive pulmonary disease, and non-CF bronchiectasis). In addition, the invention may be used in ventilator-associated pneumonia (VAP).

[0053] In fact, without being limited by a specific scientific theory, it is believed that the ability of the combination of colistin and NAC according to the present invention to destroy the biofilm of Pseudomonas aeruginosa can increase the sensitivity of the pathogen to antibiotics. At the same time, the inflammatory state induced by bacteria and also by some biofilm components is also alleviated, thus improving lung function in a different way. Prior to the current results, to the best of our knowledge, no data on the Pseudomonas aeruginosa biofilm model were available.

[0054] Therefore, the observed synergistic antibiofilm activity according to the present invention may result from different mechanisms, which act simultaneously or not and are only partially elucidated, such as increased permeability of antibiotics in extracellular polymers (EPS, e.g. biofilms), direct synergistic inhibitory activity on biofilm formation, or direct inhibition of bacterial growth.

[0055] In fact, even the mechanisms behind the biological activity of NAC have not been fully elucidated and are likely pleiotropic.

[0056] According to the present invention, the terms "synergistic" and "synergistically" applied to the effect of colistin and NAC used in combination (whether simultaneously or sequentially) refer to a greater antibacterial effect obtained relative to the treatment of the above-identified bacteria by colistin or NAC alone. This synergistic effect can be clearly seen in the figures attached to the present application, in which the control treatment (colistin or NAC alone) is always included in the determination. In general, the effect of colistin and NAC used in combination (whether simultaneously or sequentially) is greater than the simple addition of the effects of each agent applied alone, i.e. there is an effect that exceeds the expected effect based on an additive effect. Statistical methods (such as those described in more detail in the experimental part) can be used in the art to determine synergistic or simple additive effects.

[0057] The synergistic combination between colistin and NAC has an impact on multidrug resistance (MDR) and colistin resistance (CST) R ) strains, and could also consistently reduce the colistin MIC in colistin-resistant Pseudomonas aeruginosa strains. This is extremely important for antibiotics such as colistin that are toxic at very high doses.

[0058] The observed synergy was common to different P. aeruginosa isolates, as determined by standard methods described in more detail in the experimental section, and was independent of the bacterial mucoid or non-mucoid phenotype, genotype (e.g. ST-type or O-type), and antibiotic sensitivity or resistance pattern. Given the great variability in responses between isolates belonging to the same pathogen type (e.g., (Aiyer A. et al. Antibiotics 2021, 10, 1176. https: / / doi.org / 10.3390 / antibiotics10101176 ), the bacterial populations embedded in the biofilm environment are highly heterogeneous and, last but not least, even within the same isolate, differences in biological responses between the planktonic and biofilm phases of growth are extremely significant.

[0059] The present synergistic combination of colistin and NAC is therefore proposed to be particularly useful for recurrent P. aeruginosa infections or recurrence of infections (usually due to MDR isolates), as well as for the eradication of said infections, since the synergistic combination is able to affect bacteria embedded in mature biofilms.

[0060] "Recurrence" of an infection refers to an infection caused by the same pathogen after a successful treatment regimen. "Eradication" refers to the clearance of the pathogen and recovery of the patient after a given period of time.

[0061] This synergistic combination can also be used for nosocomial infection or hospital-acquired infection (HAI), that is, infection obtained in a hospital or other healthcare institution. In order to emphasize both hospital and non-hospital environments, it is sometimes replaced by healthcare-associated infection (HAI or HCAI). Such infection can be obtained in a hospital, nursing home, rehabilitation facility, outpatient clinic or other clinical environment. MDR bacteria, particularly MDR Pseudomonas aeruginosa bacteria are usually the cause of HAI or HCAI and are associated with a high mortality rate.

[0062] Colistin and NAC of the synergistic combination according to the present invention may be administered sequentially, separately or simultaneously, with or without overlapping administration periods and by the same or different administration routes.

[0063] For example, colistin and NAC may be administered simultaneously in a single dose or in separate dosage forms, sequentially, concomitantly or sequentially, by the same or different routes of administration, with or without overlapping administration times.

[0064] Thus, the synergistic combination of colistin and NAC may be administered to a patient in the form of a single or separate pharmaceutical compositions.

[0065] The pharmaceutical composition of the invention may comprise colistin and NAC with a carrier suitable for pharmaceutical use and / or one or more excipients for simultaneous administration of colistin and NAC. Alternatively, the pharmaceutical preparation of the invention may comprise NAC with a carrier suitable for pharmaceutical use and / or one or more excipients, and separately, the colistin preparation may comprise a carrier suitable for pharmaceutical use and / or one or more excipients. Separate pharmaceutical preparations for sequential or concomitant administration of colistin and NAC may provide a synergistic combination according to the invention.

[0066] According to the present invention, the term "excipient" includes any inert substance added to a pharmaceutical composition to further facilitate administration of the active ingredient.

[0067] According to the present invention, the term "carrier" includes any substance suitable as a vehicle for delivering colistin and / or NAC to a suitable in vivo or in vitro site.

[0068] The composition (or formulation) comprising the synergistic combination of the present invention may be administered by any suitable route of administration: oral, parenteral (subcutaneous, intramuscular, intraperitoneal or intravenous) or inhalation, inhalation being the preferred route of administration.

[0069] According to the present invention, the term "oral" or "orally" refers to introduction into the body via the oral cavity, whereby absorption occurs in one or more of the following areas of the human body: oral cavity, stomach, small intestine and small blood vessels of the oral mucosa.

[0070] Non-limiting examples of pharmaceutical preparations according to the present invention for oral administration include, for example, tablets, coated tablets, granules, pills, capsules, liquids, gels, syrups, suspensions, etc., for oral administration by an individual. Suitable carriers for oral administration are well known in the art.

[0071] For parenteral administration, the pharmaceutical composition according to the invention can be formulated into, for example, aqueous solutions, such as physiologically compatible buffers or physiological saline buffers. Preparations for injection can be provided in sterile unit dosage form, for example in ampoules or in multiple dose containers with optionally added preservatives.

[0072] For administration by inhalation route, the pharmaceutical preparation according to the present invention may be formulated as a solution, suspension or dry powder of a suitable particle size range and delivered by using conventional methods to provide the optimal amount to the patient.

[0073] According to a particularly preferred embodiment of the present invention, the above-identified infections are treated by inhalation route. In fact, administration by inhalation allows high drug concentrations to be achieved in the epithelial lining fluid (ELF) while minimizing systemic toxicity. In particular, colistin is preferably administered by inhalation route.

[0074] Alternatively, the combination of colistin and NAC may be provided by administering one drug (i.e. colistin) by the inhalation route and the other drug systemically (i.e. orally or parenterally). A drug combination in which colistin is administered by the inhalation route and NAC is administered (by inhalation, orally or parenterally) is particularly preferred.

[0075] By inhalation route, colistin is preferably colistin sodium methanesulfonate (CMS) in a sterile saline composition suitable for administration by inhalation, nebulization or aerosol spray in an amount of CMS equivalent to at least 20 mg CBA / day, and Pseudomonas aeruginosa infections.

[0076] The following conversion table for colistin base activity (CBA), international units and amounts (mg) is provided ("European Medicines Agency completes review of polymyxin-based medicines", December 16, 2014):

[0077]

[0078]

[0079] Preferably, the composition for inhalation is prepared extemporaneously at the time of use from a suitable dose of CMS in powder form. Suitable compositions contain CMS in an amount of at least 30-35 mg CBA / mL to 60-70 mg CBA / mL and are administered twice daily. According to a preferred embodiment, CMS is prepared at a concentration of at least 30-35 mg CBA in 1 mL of a solution suitable for inhalation, and about one third (0.3 mL) is delivered by inhalation through a suitable inhalation device, delivering about 10 mg CBA per inhalation.

[0080] Sterile saline aqueous solutions of colistin sodium methanesulfonate, which are usually used for dispersion as powders, preferably contain sodium chloride (NaCl) at a concentration of 0.4% to 0.9% w / v in sterile water for injection (WFI) or a suitable physiological sterile buffer solution. The preferred final NaCl concentration in the solution for inhalation containing CMS for use in treatment according to the present invention is 0.4% to 0.5% w / v, even more preferably 0.45% w / v.

[0081] As mentioned above, the CMS composition is preferably prepared extemporaneously, ie, at the time of use, or if stored at 2 to 8°C, within 24 hours.

[0082] According to one embodiment, the amount of colistin and NAC sufficient to have a synergistic effect on bacterial infections caused by biofilm-forming Pseudomonas aeruginosa strains can vary, for example, taking into account the patient's physical characteristics, such as weight, severity of the subject's symptoms, form, nature and bacterial load of the infection, or the formulation and method used to administer the drug. The specific dosage for a given subject is generally set by the judgment of the attending physician.

[0083] However, for example, the effective amount of colistin may be about 75,000 units to 12 million units (i.e., about 6 mg to 960 mg), preferably about 500,000 units to 12 million units (i.e., about 40 mg to 960 mg), to be administered in a single dose or in multiple repeated doses; the effective amount of NAC may vary from 300 mg / day to 4.800 mg / day, to be administered in a single dose or in multiple repeated doses.

[0084] NAC is preferably administered in the pharmaceutical form of tablets, liquid compositions for oral or inhalation use, and / or granules at a dosage of 600 mg / day to 3000 mg / day.

[0085] The inhalation route is preferred for treating infections of the upper and lower respiratory tract, such as those occurring in patients with cystic fibrosis and other chronic lung diseases (e.g., chronic obstructive pulmonary disease and non-CF bronchiectasis) or in ventilator associated pneumonia (VAP). This route of administration can be selected by determining the active dose of NAC and / or colistin available in the epithelial lining fluid (ELF) following inhalation.

[0086] A further object of the invention is represented by a method for treating bacterial infections caused by Pseudomonas aeruginosa and exacerbations thereof in a subject in need thereof, which method comprises the simultaneous administration of a combination of colistin and NAC, wherein said combination has a synergistic activity against bacteria enclosed in an established or mature biofilm.

[0087] A further object of the present invention is a method of treating a bacterial infection caused by Pseudomonas aeruginosa in a subject in need thereof, the method comprising separate administration of colistin and NAC, wherein the combination of the two has synergistic activity against bacteria enclosed in mature biofilms.

[0088] Further embodiments of the invention are represented by methods for treating Pseudomonas aeruginosa infection in a subject experiencing recurrent Pseudomonas aeruginosa infection, particularly infection with an MDR or ColR isolate.The subjects and specific conditions have been described in detail above.

[0089] According to the present invention, the terms "individual," "subject," and "patient" are used interchangeably to refer to a member of a mammalian species, preferably a human, who suffers from a particular disease, disorder or condition.

[0090] In summary, the synergistic combination of colistin and N-acetylcysteine ​​(NAC) is proposed herein for the treatment and eradication of biofilm-associated infections caused by "hard-to-treat" Pseudomonas aeruginosa, more preferably, for the treatment and eradication of Pseudomonas aeruginosa infections in patients with chronic lung disease and recurrent Pseudomonas aeruginosa infections.

[0091] The present invention will be described in more detail in the following experimental section, where it is noteworthy that results were obtained with clinical isolates of Pseudomonas aeruginosa from different types of infections and the results were tested in assays specifically designed to detect activity against biofilm-embedded cells. Differently, many, if not all, prior art studies were conducted with reference to a Pseudomonas aeruginosa strain (PAO1), and most were conducted under planktonic culture conditions. In fact, methods for studying antibiotic susceptibility of bacteria embedded in biofilms have only recently been adopted and standardized. They are based on resting cultures grown under static conditions.

[0092] Experimental Section

[0093] Example 1. Characterization of Pseudomonas aeruginosa strains

[0094] Fifteen clinical isolates were from CF, one MDR clinical isolate from respiratory tract infection (RTI) in the intensive care unit (ICU), and the reference strain Pseudomonas aeruginosa PAO1 (Table 1). Identification was performed by MALDI-TOF MS (Bruker, Shimadzu). Antimicrobial susceptibility was determined using the reference broth microdilution method (CLSIMethods2018). Whole genome sequencing of clinical isolates was performed using the Illumina (San Diego, CA, USA) MiSeq platform using a 2×150 bp paired-end method. SPAdes (Bankevich et al. 2012 J Comput Biol 19 (5): 455-77.) was used to assemble the original reads, and the draft genome was used to determine MLST-type and O-type on the Oxford PubMLST website (https: / / pubmlst.org / ) (Jolley et al. 2018 Wellcome Open Res 3: 124) and on the Center for Genomic Epidemiology website (https: / / cge.cbs.dtu.dk / services / PAst-1.0 / ) (Thrane JCM 2016). By combining the results from Illumina with the results obtained using the Oxford Nanopore Technologies (Oxford, UK) MinION platform, the complete genome of Pseudomonas aeruginosa Z154 was obtained and assembled from scratch using Unicycler v0.4.4.

[0095] The characteristics of P. aeruginosa isolates in this study are summarized in Table 1 .

[0096] PAO1 represents a reference strain (Stover CK et al., Nature, 2000; 406:959-976).

[0097] Table 1. Characterization of Pseudomonas aeruginosa strains

[0098]

[0099]

[0100] CST, colistin; NAC, N-acetylcysteine.

[0101] a CF, cystic fibrosis, RTI ICU , respiratory tract infections in the intensive care unit (ICU).

[0102] bAccording to the MLST Pasteur protocol.

[0103] c CB R , resistant to carbapenems (imipenem and meropenem); CP R , resistant to cephems (ceftazidime and cefepime); FQ R , resistant to fluoroquinolones (ciprofloxacin); AG R , resistant to aminoglycosides (amikacin, gentamicin), CST R , resistant to colistin.

[0104] Example 2. Antibiofilm activity of NAC and colistin.

[0105] In the present invention, the Calgary biofilm device (also referred to as the Nunc-TSP cover system in the experimental section) is the method selected to test the in vitro anti-biofilm activity of colistin and NAC alone or in combination. NAC stock solution (100g / L) was prepared immediately before use. NAC powder (Zambon, Bresso, Italy) was dissolved in sterile distilled water, the pH was adjusted to 6.5-6.8 with NaOH 10M, and the solution was filtered through a 0.22-μm membrane filter. Unless otherwise stated, all experiments were carried out in cation-regulated Mueller-Hinton broth (CAMHB) (Becton Dickinson, Milan, Italy), starting from appropriately concentrated culture media to avoid broth dilution when using NAC solutions.

[0106] Colistin Colistin stock solution (10 mg / L) was prepared immediately before use by dissolving colistin powder (Colistin sulfate, AppliChem, Darmstadt, Germany) in sterile distilled water.

[0107] Determination: First, the biofilm sensitivity test was performed using the Nunc-TSP cap system (Thermo Fisher Scientific, Waltham, MA, the United States), as previously described (Harrison JJ et al., Nat Protoc, 2010; 5: 1236-54). In short, the biofilm was grown in CAMHB at 35 ° C under static conditions for 24 hours. The preformed biofilm was then exposed to NAC 8000 mg / L and colistin (colistin sulfate, AppliChem, Darmstadt, Germany) 2 to 32 mg / L alone and in combination. Colistin concentration was selected according to the preliminary results of the anti-biofilm sensitivity test (performed by using the biofilm assay reported above) and the colistin MIC of each strain (for colistin-sensitive strains, the colistin MIC value was 1-2 mg / L; for colistin-resistant strains, the MIC value was between 4 and 512 mg / L). After 24 hours of exposure (i.e., 35°C, static conditions), the biofilms were washed twice with 200 μL phosphate buffered saline (PBS) (Sigma Aldrich, Milan, Italy) to remove loosely adherent bacteria, and adherent cells were removed from the pegs by sonication for 30 minutes (Elma Transsonic T 460, Singen, Germany) in 200 μL of tryptic soy broth (TSB) (Oxoid, Milan, Italy) (i.e., recovery medium) supplemented with 1% Tween 20 (Sigma Aldrich). The median of cfu per peg (cfu / peg) was then determined by plating 10 μL of appropriately diluted recovery medium on tryptic soy agar (TSA) (Oxoid) and incubating at 35°C for 24 hours (detection limit of 20 cfu / peg). After 48 hours of incubation, colony counts were also double checked.

[0108] Data from this biofilm model were obtained in at least three independent experiments, each with at least twelve replicates per condition.

[0109] Statistical analysis of biofilm susceptibility assays was performed using GraphPad Prism version 8.0 (San Diego, CA, USA). Multiple comparison tests were performed by Kruskal-Wallis test with Dunn's correction. RNA-seq statistical analysis was performed using SeqManNGen v17.3 software tool.

[0110] Results: Pseudomonas aeruginosa Z154 (a mucoid, MDR, colistin-susceptible isolate from CF) was first used to test the potential antibiofilm synergistic effect of NAC 8000 mg / L plus different colistin concentrations. Figure 1As shown in , relevant synergistic effects were already observed at colistin 2 mg / L (i.e., the colistin MIC of the test isolate), with a dose-dependent effect as colistin concentration increased, and complete eradication of the biofilm in the case of the combination of NAC 8000 mg / L plus colistin 8 mg / L ( Figure 1 ).

[0111] The remaining sixteen isolates were initially tested with a combination of NAC 8000 mg / L plus colistin 8 mg / L. To test for synergy, the concentration of colistin was varied against biofilm-forming bacteria that were either highly sensitive (n=7) or particularly resistant (n=2) to colistin. Figures 2 to 4 ). Overall, relevant synergistic effects of the NAC / colistin combination were observed in the case of all tested isolates, including the three colistin-resistant isolates. Notably, an isolate (FC237) had a very high MIC to colistin, which was reduced to 1 / 64 in the presence of NAC.

[0112] In summary, these data demonstrate that NAC enhances the activity of colistin against preformed biofilms of colistin-sensitive and colistin-resistant Pseudomonas aeruginosa isolates from CF, regardless of mucoid / non-mucoid phenotype, resistance pattern, and ST- and O-type. The results of this study are consistent with the previously observed anti-biofilm synergy of the NAC / colistin combination against colistin-sensitive and colistin-resistant strains of Acinetobacter baumannii and Stenotrophomonas maltophilia (Pollini et al. 2018JAntimicrob Chemother, 73:2388-2395, Ciacci et al. 2019, Antibiotics 8:101).

[0113] Example 3. Activity of NAC / colistin combination in artificial sputum medium (ASM) biofilm model

[0114] Methods: The potential anti-biofilm synergy of the NAC / colistin combination was further investigated using an in vitro ASM biofilm model (Kirchner et al. 2012 Immunology 64: e3857) to simulate P. aeruginosa biofilm conditions within CF mucus. Two selected strains from CF (P. aeruginosa Z154 and Z34) that exhibited different characteristics (i.e., mucoid / non-mucoid phenotype, antimicrobial susceptibility pattern, MLST-type, O-type) (Table 1) were studied. Briefly, biofilms were grown in ASM in 24-well tissue culture treated plates at 35°C under static conditions for 72 hours. The preformed biofilms were then exposed to NAC 8000 mg / L and colistin 64 mg / L alone and in combination. Preliminary experiments with lower colistin concentrations (i.e., 2 to 32 mg / L) did not show significant synergistic antibiofilm activity, while higher colistin concentrations (i.e., >64 mg / L) resulted in eradication of biofilm cultures even in the absence of NAC (data not shown). After 24 hours of exposure (i.e., 35° C., static conditions), bacterial biofilms were disrupted by 30 minutes of sonication followed by manual pipetting, and the median of cfu per mL (cfu / mL) was determined following the same protocol described for the Nunc-TSP lid assay. Data from this biofilm model were obtained in at least three independent experiments, each with at least twelve replicates per condition.

[0115] Results: Two strains of P. aeruginosa from CF that exhibited different phenotypes were selected for susceptibility determination using the ASM biofilm model: P. aeruginosa Z34 (non-mucoid, MDR, ST17, O1) and P. aeruginosa Z154 (mucoid, MDR, ST412, O6). Biofilms were grown in ASM to mimic the environmental conditions of P. aeruginosa biofilms experienced in CF mucus. Preformed biofilms were then challenged with a NAC / colistin combination in the same medium.

[0116] like Figure 5 As shown in Figure , a significant synergistic effect of the combination of NAC 8000 mg / L and colistin 64 mg / L was observed in both strains ( Figure 5). Compared to experiments performed with the Nunc-TSP cap system, the concentration of colistin that allowed synergy to be observed was much higher (i.e., 32xMIC), likely due to the strong ionic interactions of colistin with ASM components (e.g., extracellular DNA, mucins) (Huang et al. 2015 Antimicr Agents Chemioter, 59:5925-5931). In fact, preliminary experiments with lower colistin concentrations did not show colistin antibiofilm activity or synergy with NAC (data not shown). Furthermore, the antibiofilm activity of NAC alone against P. aeruginosa Z154 observed in the Nunc-TSP cap system was not observed in the ASM model ( Figure 5 ), demonstrating that the efficacy of NAC alone against preformed P. aeruginosa biofilms may be limited in vivo.

[0117] Taken together, these data demonstrate that the antibiofilm synergistic effect of the NAC / colistin combination against P. aeruginosa strains from CF is also retained under environmental conditions that mimic CF mucus. This data is particularly promising for clinical applications.

[0118] Example 4. Time-kill assay of NAC / colistin combination on planktonic cultures grown under aerobic and anaerobic conditions.

[0119] Time-kill assays of the NAC / colistin combination were performed with planktonic cultures of P. aeruginosa Z154 under aerobic and anaerobic conditions.

[0120] Method: according to CLSI guide (CLSI methods M26A), colistin-sensitive strain Z154 was used to carry out time-killing assay. Under aerobic and anaerobic conditions, colistin 0.25 mg / L alone and in combination with NAC 8000 mg / L was tested. We decided to use this colistin concentration because higher concentrations caused planktonic cultures to be eradicated (data not shown). The culture medium (CAMHB) for obtaining anoxic cultures was placed under an anaerobic atmosphere by using AnaeroGen test kit (Oxoid) before use for 3 days and during the entire experiment. Kill curves were carried out in borosilicate glass bottles that were 20 mL CAMHB in final volume. When exposed for 0, 2, 4, 8 and 24 hours, ten times of dilution were plated on TSA, and the quantity of cfu / mL (detection limit was 17 cfu / mL) was determined. Data were obtained from at least four independent experiments, and each experiment had two replicates per condition.

[0121] Results: The results showed that the combination of colistin 0.25 mg / L (1 / 8 MIC) and NAC 8000 mg / L had a significant bactericidal effect on planktonic cultures grown under anaerobic conditions ( Figure 6 In particular, eradication of the starting inoculum was achieved after 24 hours of growth. On the other hand, in all experiments, identical cultures grown in the presence of oxygen were not affected by the activity of the combinations tested, demonstrating the influence of growth conditions on the sensitivity of P. aeruginosa to the NAC / colistin combination ( Figure 6 ). As reported previously, anaerobic cultures are more sensitive to killing by colistin than aerobic cultures, and this may be particularly relevant to P. aeruginosa biofilms in CF mucus, where biofilm cells grow under anoxic conditions that are related not only to the location of the bacteria within the biofilm (i.e., anoxic conditions in deeper layers) but also to the strong O2 depletion caused by polymorphonuclear leukocytes (PMNs), dictating that the entire biofilm grows in the absence of aerobic respiration.

[0122] In summary, these results show that there is synergy between NAC and colistin for planktonic cultures of colistin-sensitive Pseudomonas aeruginosa strains grown in the absence of oxygen. This is also the first evidence of such synergy, which is not evident in the same cultures grown in the presence of oxygen. Taken together, these data will support the synergy of the NAC / colistin combination observed in mature biofilms, in which most bacterial cells are embedded in the anaerobic niche.

Claims

1. A synergistic pharmacological combination of colistin and N-acetylcysteine ​​(NAC) for use in the treatment of bacterial infections caused by the pathogen Pseudomonas aeruginosa, wherein the pathogen is in the mature biofilm stage of growth.

2. The combination for use according to claim 1, wherein the bacterial infection is associated with a respiratory disease.

3. The combination for use according to claim 2, wherein the respiratory disease is a chronic respiratory disease selected from the group consisting of chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), non-cystic fibrosis bronchiectasis (NCFB) and exacerbations thereof.

4. The combination for use according to claim 2, wherein the respiratory disease is ventilator associated pneumonia (VAP).

5. The combination for use according to any one of claims 1 to 4, wherein the Pseudomonas aeruginosa pathogen has at least one of the following characteristics: multidrug resistance (MDR) or colistin resistance (CST R ) phenotype.

6. The combination for use according to claim 1, wherein the bacterial infection is a recurrent infection.

7. The combination for use according to any one of claims 1 to 6, wherein the bacterial infection is a hospital-associated infection (HAI) or a healthcare-associated infection (HCAI).

8. The combination for use according to any one of claims 1 to 7, wherein colistin and NAC are administered sequentially, separately or simultaneously by the same or different administration routes, with or without overlapping administration time periods.

9. The combination for use according to claim 8, wherein colistin and NAC are administered by inhalation route.

10. A kit of parts comprising a vial of colistin as powder and NAC as powder, tablet or aqueous composition, optionally in combination with a diluent device, for the joint administration of said synergistic combination of colistin and NAC.

11. A kit of parts according to claim 10, wherein the colistin is diluted in an aqueous composition for administration by inhalation route.

12. The synergistic pharmacological combination of colistin and N-acetylcysteine ​​(NAC) for use according to claim 1, wherein the treatment comprises eradication of a bacterial infection caused by the pathogen Pseudomonas aeruginosa, and wherein the pathogen is in a mature biofilm stage of growth.

13. A synergistic pharmacological combination of colistin and N-acetylcysteine ​​(NAC) for use in treating bacterial infections caused by the pathogen Pseudomonas aeruginosa, wherein the pathogen is encapsulated in a mature biofilm.

14. A synergistic pharmacological combination of colistin and N-acetylcysteine ​​(NAC) for use in the eradication of Pseudomonas aeruginosa in vitro, wherein the Pseudomonas aeruginosa are encapsulated in a mature biofilm.

15. Use of said synergistic pharmacological combination of colistin and NAC for the preparation of an anti-biofilm medicament for the treatment of Gram-negative infections caused by Pseudomonas aeruginosa, wherein said pathogen is in the mature biofilm stage of growth.

16. The use according to claim 13, wherein the treatment comprises eradication of a bacterial infection caused by Pseudomonas aeruginosa, wherein the pathogen is in a mature biofilm stage of growth.

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