WO2018154091A1 - Association of n-acetylcysteine and colistin for use in bacterial infections - Google Patents

Association of n-acetylcysteine and colistin for use in bacterial infections Download PDF

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Publication number
WO2018154091A1
WO2018154091A1 PCT/EP2018/054611 EP2018054611W WO2018154091A1 WO 2018154091 A1 WO2018154091 A1 WO 2018154091A1 EP 2018054611 W EP2018054611 W EP 2018054611W WO 2018154091 A1 WO2018154091 A1 WO 2018154091A1
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Prior art keywords
colistin
nac
maltophilia
association according
synergistic pharmacological
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PCT/EP2018/054611
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French (fr)
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Lucia PALLECCHI
Francesco SERGIO
Gian Maria Rossolini
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Zambon S.P.A.
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Priority to AU2018223985A priority Critical patent/AU2018223985B2/en
Priority to EP18707039.6A priority patent/EP3585372A1/en
Priority to JP2019545362A priority patent/JP7241687B2/en
Priority to CA3053441A priority patent/CA3053441A1/en
Priority to EA201991889A priority patent/EA201991889A1/en
Priority to BR112019017113A priority patent/BR112019017113A2/en
Priority to US16/487,227 priority patent/US11382948B2/en
Priority to CN201880012765.3A priority patent/CN110352053B/en
Publication of WO2018154091A1 publication Critical patent/WO2018154091A1/en
Priority to CONC2019/0009216A priority patent/CO2019009216A2/en
Priority to US17/664,232 priority patent/US20220370549A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/164—Amides, e.g. hydroxamic acids of a carboxylic acid with an aminoalcohol, e.g. ceramides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a synergistic pharmacological association of N- acetylcysteine (hereinafter NAC) and colistin for use in the treatment of bacterial infections caused by one or more pathogens selected from Stenotrophomonas maltophilia (S. maltophilia) and Acinetobacter baumannii (A. baumannii) strains.
  • NAC N- acetylcysteine
  • colistin for use in the treatment of bacterial infections caused by one or more pathogens selected from Stenotrophomonas maltophilia (S. maltophilia) and Acinetobacter baumannii (A. baumannii) strains.
  • NAC N- acetylcysteine
  • colistin for use in the treatment of bacterial infections caused by one or more pathogens selected from Stenotrophomonas maltophilia (S. maltophilia) and Acinetobacter baumannii (A
  • Colistin also termed polymixin E, a polymixin antibiotic produced by certain strains of Bacillus polymixa, consists of a cationic cyclic heptapeptide with a tripeptide side chain acylated at the N terminus by a fatty acid through an a-amide linkage (Reviews of Anti-Infective Agents CID 2005; 40: 1033-41).
  • colistin sulfate which is administered orally for bowel decontamination and topically as a powder for the treatment of bacterial skin infections
  • CMS colistimethate sodium
  • colistin methanesulfate pentasodium colistimethanesulfate
  • colistin sulfonyl methate parenteral (intravenous, intramuscular, aerosolized and intrathecal/intraventricular) therapy.
  • Colistin can be therefore administered as a prodrug in form of colistimethate sodium, which is readily idrolized to form sulfomethylated derivatives, as well as colistin sulfate, the active form of the drug.
  • Colistin has recently gained a crucial role for the treatment of various types of infections (e.g. pneumonia, bacteremia, urinary tract infections) caused by Gram- negative pathogens expressing a multidrug resistance phenotype (e.g. non-fermenting Gram-negative pathogens, and carbapenem-resistant enterobacteria).
  • infections e.g. pneumonia, bacteremia, urinary tract infections
  • Gram- negative pathogens e.g. non-fermenting Gram-negative pathogens, and carbapenem-resistant enterobacteria.
  • Pseudomonas aeruginosa P. aeruginosa
  • other multidrug resistant non-fermenting Gram-negative pathogens e.g. Acinetobacter baumannii, Stenotrophomonas maltophilia
  • Colistin represents a last-resort agent for the treatment of infections caused by multidrug resistant A. baumannii.
  • great concerns arise from the rising trend of resistance to colistin in A. baumannii, which often results in pan-drug resistance phenotypes (PDR), with no available treatment option left.
  • PDR pan-drug resistance phenotypes
  • Colistimethate sodium is a combination of the negatively charged molecular ion colistin sulphomethate with positive sodium ions.
  • Colistimethate sodium can be produced by a sulfomethylation reaction of colistin with formaldehyde followed by sodium bisulfite, which leads to the addition of a sulfomethyl group to the primary amines of colistin (Antimicrob. Agents Chemother. April 2003 vol. 47 no. 4 1364-1370).
  • Colistimethate sodium is commercially available under various brand names by different manufacturers throughout the world, for example as PromixinTM (Profile Pharma Limited).
  • the term "colistin” includes colistin and its pharmaceutically acceptable salts and/or prodrugs such as colistin sulfate, colistimethate sodium, colistin methane sulphate and colimycin or colomycin, all intended to refer to the bactericidal cyclopeptide antibiotic colystin or precursors thereof such as the prodrug colimycin which is converted to the active drug colistin.
  • the term "colistin” refers to colistin sulfate (in accordance with 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).
  • NAC is the acetylated precursor of both the amino acid L-cysteine and reduced glutathione (GSH).
  • GSH reduced glutathione
  • NAC can be typically considered a non-antibiotic drug, to which antibacterial and antibiofilm properties have been associated in some cases.
  • Parry and Neu found that NAC had the characteristics to inhibit the growth of both gram-positive and gram-negative bacteria, including Staphylococcus aureus, P. aeruginosa, Klebsiella pneumoniae and Enterobacter cloacae (Journal of Clinical Microbiology, Jan 1977, p.58-61).
  • NAC is commercially available under various brand names by different manufacturers throughout the world, for example as FluimucilTM (Zambon Spa).
  • S. maltophilia infections are commonly associated with respiratory tract diseases, in particular chronic respiratory tract diseases; for example, S. maltophilia infections can trigger pulmonary exacerbations of chronic obstructive lung disease (COPD), Cystic Fibrosis (CF) and bronchiectasis non CF.
  • COPD chronic obstructive lung disease
  • CF Cystic Fibrosis
  • bronchiectasis non CF bronchiectasis
  • S. maltophilia are characterized by a multidrug resistance phenotype and the ability to form biofilms. In virtue of these features, they are responsible for chronic lung colonization in individuals with weakened immune systems or chronic lung disease, particularly CF and bronchiectasis non CF, which may last several months or years and are difficult or impossible to be eradicated by current antibiotic treatment strategies.
  • A. baumannii is an important and difficult-to-treat pathogen, which can infect the respiratory tract of patients affected by respiratory tract diseases, including chronic respiratory tract diseases.
  • respiratory tract diseases including chronic respiratory tract diseases.
  • A. baumannii infections in patients with pneumonia, in particular, in ventilator- associated pneumonia (VAP) represents one of the most dreadful complications that occur in the critical care setting.
  • A. baumannii has been also identified in the CF population and in the population with bronchiectasis non CF.
  • A. baumannii infections with bacteremia, wound infections, urinary tract infections and meningitis has been also described. It can be noted that there has been a dramatic increase in the number of A. baumannii strains with resistance to multiple antibiotic classes and the multidrug-resistant nature of this pathogen represents a challenge in the treatment and control of nosocomial infections, with relatively limited treatment options.
  • nosocomial pneumonia due to multidrug resistant gram-negative bacteria comprising A. baumannii is among the most serious complications that occur in the intensive care unit (ICU) setting. Mortality, morbidity and health care costs are substantially increased by this type of infection.
  • ICU intensive care unit
  • the present inventors faced the problem of treating a bacterial infection caused by one or more pathogens selected from S. maltophilia and/or A. baumannii. After a long set of tests and experimentations, the present inventors have surprisingly found that the association of NAC and colistin has a synergistic antimicrobial effect against a consistent number of S. maltophilia and/or A. baumannii clinical isolates, even resulting in a bactericidal effect.
  • the association of NAC and colistin has also been found to have synergistic anti- biofilm activity in in vitro biofilm models of S. maltophilia and/or A. baumannii.
  • the anti-biofilm activity of the association of NAC and colistin against S. maltophilia and/or A. baumannii biofilms envisages a potential use also for both preventing and eradicating colonization by said pathogens, especially in the hospitalized patients affected by diseases including, for example, chronic bronchitis, emphysema, COPD and exacerbations, CF, bronchiectasis non CF, pneumonia, VAP, bacteremia, wound infections, urinary tract infections and meningitis.
  • colistin represents one of the last therapeutic options to fight Gram-negative bacteria, the appearance of colistin-resistant Gram-negative pathogens such as S. maltophilia and/or A. baumannii is of great concern.
  • the present inventors by concentrating their efforts in identifying an effective treatment able to restore susceptibility of said pathogens to the colistin treatment, have unexpectedly found that colistin effectiveness against colistin-resistant Gram- negative pathogens such as, e.g., A. baumannii and S. maltophilia can be surprisingly restored by administering colistin together with NAC.
  • colistin-resistant Gram- negative pathogens such as, e.g., A. baumannii and S. maltophilia
  • the MIC of all colistin resistant strains tested lowered at or below the susceptibility breackpoint in the presence of NAC 8 mg/ml.
  • Figure 1 Time kill curves. NAC alone (1.6 and 8 mg/ml), colistin alone (2 and 8 ⁇ g/ml and combination NAC + colistin (1.6 mg/ml + 2 ⁇ g/ml, 1.6 mg/ml + 8 ⁇ g/ml, 8 mg/ml + 2 ⁇ g/ml, 8 mg/ml + 8 ⁇ g/ml).
  • Figure 2 Time kill curves of S. maltophilia isolates with sub MIC concentrations of N-acetylcysteine (NAC 1.6, 3.2 and 8 mg/ml) and colistin (COL 2, 8 ⁇ g/ml) alone and in combination.
  • -x- Control; — O- Colistin 2 ⁇ g/ml; ⁇ Colistin 8 ⁇ g/ml;— : NAC 1.6 mg/ml; "" (dotted line) NAC 3.2 mg/ml; (dashed line) NAC 8 mg/ml; - ⁇ - : NAC 1.6 mg/ml + Colistin 2 ⁇ g/ml; - ⁇ - : NAC 1.6 mg/ml + Colistin 8 ⁇ g/ml; " # " (dotted line) NAC 3.2 mg/ml + Colistin 2 ⁇ g/ml; (dotted line) NAC 3.2 mg/ml +
  • FIG. 3 Antibiofilm activity of NAC, colistin and colistin/NAC combinations (sub- MIC concentrations) tested on a Cystic Fibrosis isolate of S. maltophilia. Biofilms were grown on Calgary Device (MBEC assay) for 48 hrs and challenged for 24 hours with NAC alone, Colistin alone at two different concentration: 2 ⁇ g/ml and 8 ⁇ g/ml and the combination of the two (NAC 8 mg/ml + Colistin 2 ⁇ g/ml and 8 ⁇ g/ml).
  • pathogens selected from S. maltophilia and A. baumannii strains
  • a bacterial infection associated with a respiratory tract disease particularly a chronic respiratory tract disease such as e.g., CF, bronchiectasis non CF and COPD.
  • a bacterial infection is caused by a pathogen selected from S. maltophilia and A. baumannii strains, either expressing a colistin susceptible or resistant phenotype; especially, a pathogen selected from S. maltophilia and A. baumannii strains, expressing a colistin resistant phenotype.
  • a bacterial infection caused by S. maltophilia strain can be detected, for example, in patients suffering from respiratory tract diseases, particularly chronic respiratory tract diseases such as, for example, in patients suffering from COPD, CF or bronchiectasis non CF, especially when these patients have weakened immune systems.
  • a bacterial infection caused by A. baumannii strain can be detected, for example, in patients suffering from pneumonia, VAP, bacteremia; wound infections, urinary tract infections, CF and bronchiectasis non CF, especially when these patients are hospitalized patients.
  • patients suffering of a bacterial infection caused by a pathogen selected from S. maltophilia and A. baumannii strains as described above are immunocompromised and/or hospitalized patients.
  • a bacterial infection caused by a pathogen selected from S. maltophilia and A. baumannii strains according to the present invention may be a biofilm-associated infection.
  • the terms “synergistic” and “synergistically” as applied to the effect of NAC and colistin used in association refer to a greater antibacterial effect obtained when the above- identified bacteria are treated by either NAC or colistin alone.
  • the effect of NAC and colistin used in association is greater than the simple addition of the effects of each agent administered alone, i.e. there is an effect which surpasses expectations based on additive effects.
  • NAC neuropeptide-susceptible S. maltophilia and colistin-susceptible A. baumannii strains, wherein NAC is able to prevent in vivo emergence of colistin resistance, during colistin treatment regimens.
  • NAC and colistin of the pharmacological association according to the present invention may be administered in either order, separately or concurrently, with overlapping or non-overlapping periods of administration, via the same or different modes of administration.
  • NAC and colistin can be concurrently administered in a single dosage form or alternatively NAC and colistin can be administered in separate dosage forms in either order, concomitantly or sequentially, with overlapping or non-overlapping periods of administration, via the same of different modes of administration.
  • the concurrent or separate administration of the pharmacological association of NAC and colistin has the effect of in inhibiting or suppressing the growth and/or killing a strain of a pathogen selected from S. maltophilia and A. baumannii strains, said effect being unexpectedly and surprisingly greater than what is seen when bacteria are contacted by either NAC or colistin alone.
  • the present inventors noticed that with an increase in the use of colistin to treat infections caused by multidrug resistant Gram-negative pathogens, resistance to colistin has been increasingly reported.
  • the present inventors strongly perceived the great need of an effective treatment against colistin-resistant Gram-negative pathogens such as, e.g., A. baumannii and S. maltophilia, able to restore susceptibility of said pathogens to the antibiotic treatment, or to prevent in vivo emergence of colistin resistance during colistin treatment regimens.
  • colistin-resistant Gram-negative pathogens such as, e.g., A. baumannii and S. maltophilia
  • colistin effectiveness against colistin-resistant Gram-negative pathogens such as, e.g., A. baumannii and S. maltophilia can be surprisingly restored by the administration of the association of NAC with colistin.
  • colistin susceptibility can be restored by NAC concentrations possibly achievable by topical administration.
  • the MIC of all colistin resistant strains tested lowered at or below the susceptibility breakpoint in the presence of NAC 8 mg/ml.
  • the association of NAC and colistin may be administered to the patient in the form of one or more pharmaceutical formulations.
  • the pharmaceutical formulations of the present invention comprise NAC and/or colistin together with a carrier suitable for pharmaceutical use, consisting of one or more excipients.
  • the pharmaceutical formulations of the invention may comprise both NAC and colistin together with a carrier suitable for pharmaceutical use, consisting of one or more excipients, i.e. pharmaceutical formulations for concurrent administration of both NAC and colistin; or the pharmaceutical formulations of the invention may comprise NAC together with a carrier suitable for pharmaceutical use, consisting of one or more excipients; or the pharmaceutical formulations of the invention may comprise colistin together with a carrier suitable for pharmaceutical use, consisting of one or more excipients, i.e. separate pharmaceutical formulations for sequential or concomitant administration of NAC and colistin.
  • the term "excipient” comprises any inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • carrier comprises any substance suitable as a vehicle for delivering NAC and/or colistin to a suitable in vivo or in vitro site.
  • compositions comprising NAC and/or colistin that are suitable to be administered to a subject.
  • Acceptable methods for preparing the pharmaceutical formulations according to the invention are well known to a person skilled in the art.
  • the terms “individual” “subject” and “patient” are used interchangeably to refer to a member of mammalian specie, preferably a human, which is afflicted with a particular disease, disorder or condition.
  • antibacterial means reducing the harmful effects of bacteria by inhibiting, suppressing the growth and/or killing them.
  • antibacterial agents includes NAC and colistin.
  • bacteria means having a destructive killing action upon bacteria.
  • bacterial infection refers to any situation in which the presence of a microbial population(s) is damaging to a host mammal.
  • an individual is “suffering" from a microbial infection when excessive numbers of a microbial population are present in or on an individual's body, or when the effects of the presence of a microbial population(s) is damaging the cells or other tissue of an individual.
  • bacterial infection refers to an infection caused by a strain of bacteria for which the use of a synergistic pharmacological association of NAC and colistin disclosed herein is appropriate.
  • the terms “treat”, “treating” and “treatment” refer to a diminution, a decrease, a limitation, or a mitigation of the degree, intensity, extent of a bacterial infection or its related disease conditions and symptoms caused by a strain of S. maltophilia and/or A. baumanii, that is achieved by a reduction, inhibition or suppression of growth, replication, and/or propagation, or death or destruction of said bacteria, on or in the subject.
  • the term "pharmacological association” refers to either a fixed combination of NAC and colistin in one unit dosage form, a non-fixed combination or a kit of parts for the combined administration where NAC and colistin, as defined above, may be administered simultaneously, independently at the same time or separately within time intervals that allow the combination partners to show a synergistic effect.
  • compositions of the present invention including oral, parenteral (subcutaneous, intramuscular or intravenous) and inhalation route.
  • oral or “orally” refer to the introduction into the body by mouth whereby absorption occurs in one or more of the following areas of the body: the mouth, stomach, small intestine, and the small blood vessels of the oral mucosa.
  • Non-limiting examples of pharmaceutical formulations according to the present invention for oral administration include, for example, tablets, coated tablets, granulates, pills, capsules, liquids, gels, syrups, suspensions, and the like, for oral ingestion by an individual.
  • Suitable carriers for oral administration are well known in the art.
  • compositions according to the invention may be formulated for example in aqueous solutions such as in physiologically compatible buffers or physiological salt buffer.
  • Formulations for injection may be presented in unit dosage forms, for example, in ampoules, or in multi-dose containers with, optionally, an added preservative.
  • compositions according to the invention may be formulated in solutions, suspensions and dry powder and delivered by using conventional means, so that optimal quantities of a suitable range of particle sizes are provided to the patient.
  • the pharmaceutical formulations according to the invention may be preferably administered to the respiratory tract.
  • the present invention also provides aerosol pharmaceutical formulations comprising NAC and/or colistin.
  • compositions of the present invention may be manufactured in conventional manners, following processes well known in the art.
  • the amount of NAC and colistin for use according to the present invention may vary depending on the administration route, the selected kind of composition, the individual characteristics of the patient, the duration of the treatment and the nature of concurrent therapies.
  • the synergistic effective amount of the pharmacological association of NAC and colistin can produce a diminution, a decrease, a limitation, or a mitigation of the degree, intensity, extent of a bacterial infection or its related symptoms caused by a strain of A. baumannii and/or S. maltophilia.
  • the amount of NAC and colistin sufficient to have a synergistic effect on a bacterial infection caused by a strain of A. baumannii and/or S. maltophilia may vary, for example, in view of the physical characteristics of the patient, the severity of the subject's symptoms, the form of the infection, the identity of the bacteria, the formulations and the means used for administering the drug. The specific dose for a given subject is usually set by the judgement of the attending physician.
  • an effective amount of colistin may be between about 0.075 million units and 12 million units (i.e. between about 6mg and 960 mg), preferably between about 0.5 million units and 12 million units (i.e. between about 40 mg and 960 mg); the effective amount of NAC may vary between 100 and 5800 mg, preferably between 100 and 4600 mg, to be administered in a single dose or in more repeated doses.
  • a dose may be administered all at once or slowly over a period of time, such as with an i.v. or by inhalation administration.
  • dose unit dose
  • dose dosage
  • effective dose refers to physically discrete units that contain a predetermined quantity of active ingredient calculated to produce a desired therapeutic effect.
  • a single dose is thus a predetermined quantity of colistin or NAC that is administered to a patient.
  • compositions, route of administration, and dosage can be chosen by the individual physician in view of the patient's condition. Treatment should be continued for as long as required to receive the benefit of the invention.
  • a determination of a synergistic interaction between NAC and colistin may be based on the results obtained from the assays described here below.
  • ⁇ FIC total fractional inhibitory concentration
  • the MIC of NAC was 32 mg/ml for two isolates, and > 32 mg/ml for the remaining ones. Synergism between colistin and NAC was observed in checkerboard assays with all tested isolates. In particular, a restoration of colistin susceptibility (i.e. MIC ⁇ 2 ⁇ g/ml) was observed with 100% and 52% of tested strains in the presence of NAC 4 mg/ml and NAC 2 mg/ml, respectively.
  • time-kill assays were also performed according to CLSI guidelines with two selected strains (i.e. Z165 and Z167, Col R ).
  • biofilm eradication test on the two isolates was also carried out as follows: biofilms were grown in the NUNC TSP lid system (MB EC assay), for seven days in daily refreshed CAMHB (static conditions). Preformed biofilms were then exposed to two colistin concentrations (i.e. 2 and 8 ⁇ g/ml), and three NAC concentrations (i.e. 1.6, 3.2 and 8 mg/ml), alone and in combination, for 24 hours. After exposure, biofilms were disrupted by sonication and mean viable cell count per peg (log CFU/peg) was determined (detection limit 1.3 log CFU/peg). Data were obtained in at least two independent experiments, with at least six replicates per condition per experiment.
  • Results showed a remarkable antibiofilm synergistic activity of combinations including colistin 8 ⁇ g/ml (i.e. representing 0.25 x and 0.06 x MIC for Z165 and Z167, respectively), with a marked reduction of viable biofilm cells also observed in the presence of the lower NAC concentration tested (i.e. 1.6 mg/ml).
  • colistin 2 and 8 ⁇ g/ml, representing concentrations achievable in serum and epithelial lining fluid - ELF, respectively
  • NAC 1.6 and 8 mg/ml, likely achievable in ELF by topical administration
  • Colistin MICs ranged from 0.5 ⁇ g/ml to 128 ⁇ g/ml (MIC50, 16 ⁇ g/ml; MIC90, 64 ⁇ g/ml).
  • the MIC of NAC was 16 mg/ml for nine isolates, >32 mg/ml for one isolate, and 32 mg/ml for the remaining ones. Synergism between colistin and NAC was observed in checkerboard assays with all tested isolates.
  • Time-kill curves confirmed the synergy observed by checkerboard assays, demonstrating a bactericidal effect of colistin/NAC combinations at sub-MIC concentrations ( Figure 2 A and B).
  • the antibiofilm activity of colistin/NAC combinations was investigated using the MBEC assay, the reference procedure for biofilm susceptibility testing (. Mature S. maltophilia (48 -hours old) were challenged for 24 hours with NAC and colistin, either alone or in combination. Viable cell count of challenged biofilms was compared to controls in order to assess the antibiofilm activity of the tested drugs and drugs/combinations.
  • NAC 8 mg/ml plus colistin 2 ⁇ g/ml accounted for a reduction of more than 3 log colony forming units (CFU)/peg compared to controls ( Figure 3).

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Abstract

The present invention relates to a synergistic pharmacological association of NAC and colistin for use in the treatment of a bacterial infection caused by one or more pathogens selected from S. maltophilia and A. baumannii strains, in particular a bacterial infection associated with a respiratory tract disease, such as a chronic respiratory tract disease comprising CF, bronchiectasis non CF and COPD.

Description

ASSOCIATION OF N- ACETYLCYSTEINE AND COLISTIN FOR USE IN BACTERIAL INFECTIONS FIELD OF THE INVENTION
The present invention relates to a synergistic pharmacological association of N- acetylcysteine (hereinafter NAC) and colistin for use in the treatment of bacterial infections caused by one or more pathogens selected from Stenotrophomonas maltophilia (S. maltophilia) and Acinetobacter baumannii (A. baumannii) strains. In particular, the present invention relates to a synergistic pharmacological association of NAC and colistin for use in the treatment of a disease associated with bacterial infections caused by one or more pathogens selected from S. maltophilia and A. baumannii strains, particularly for use in the treatment of a respiratory tract disease.
BACKGROUND OF THE INVENTION
Colistin, also termed polymixin E, a polymixin antibiotic produced by certain strains of Bacillus polymixa, consists of a cationic cyclic heptapeptide with a tripeptide side chain acylated at the N terminus by a fatty acid through an a-amide linkage (Reviews of Anti-Infective Agents CID 2005; 40: 1033-41).
Two different forms of colistin are available for clinical use: colistin sulfate which is administered orally for bowel decontamination and topically as a powder for the treatment of bacterial skin infections, and colistimethate sodium (CMS) (also called colistin methanesulfate, pentasodium colistimethanesulfate, and colistin sulfonyl methate) for parenteral (intravenous, intramuscular, aerosolized and intrathecal/intraventricular) therapy.
Colistin can be therefore administered as a prodrug in form of colistimethate sodium, which is readily idrolized to form sulfomethylated derivatives, as well as colistin sulfate, the active form of the drug.
Colistin has recently gained a crucial role for the treatment of various types of infections (e.g. pneumonia, bacteremia, urinary tract infections) caused by Gram- negative pathogens expressing a multidrug resistance phenotype (e.g. non-fermenting Gram-negative pathogens, and carbapenem-resistant enterobacteria).
In patients affected by cystic fibrosis or other chronic respiratory diseases, it is commonly used to treat infections caused by Pseudomonas aeruginosa (P. aeruginosa) or other multidrug resistant non-fermenting Gram-negative pathogens (e.g. Acinetobacter baumannii, Stenotrophomonas maltophilia).
Colistin represents a last-resort agent for the treatment of infections caused by multidrug resistant A. baumannii. In this perspective, great concerns arise from the rising trend of resistance to colistin in A. baumannii, which often results in pan-drug resistance phenotypes (PDR), with no available treatment option left.
Treatment of S. maltophilia infections is complicated by intrinsic multidrug resistance. In case the first line treatment based on trimethoprim- sulphamethoxazole is not suitable (for patient intolerance or acquired resistance), colistin is among the second line options, with the advantage of being also administrable by nebulization. However, increasing colistin resistance rates in S. maltophilia have been documented, representing a matter of great concern.
Colistimethate sodium is a combination of the negatively charged molecular ion colistin sulphomethate with positive sodium ions.
Colistimethate sodium can be produced by a sulfomethylation reaction of colistin with formaldehyde followed by sodium bisulfite, which leads to the addition of a sulfomethyl group to the primary amines of colistin (Antimicrob. Agents Chemother. April 2003 vol. 47 no. 4 1364-1370).
Colistimethate sodium is commercially available under various brand names by different manufacturers throughout the world, for example as PromixinTM (Profile Pharma Limited).
According to the present invention the term "colistin" includes colistin and its pharmaceutically acceptable salts and/or prodrugs such as colistin sulfate, colistimethate sodium, colistin methane sulphate and colimycin or colomycin, all intended to refer to the bactericidal cyclopeptide antibiotic colystin or precursors thereof such as the prodrug colimycin which is converted to the active drug colistin. In a particular aspect, for in vitro susceptibility testing in the experimental part of the present invention the term "colistin" refers to colistin sulfate (in accordance with 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).
NAC is the acetylated precursor of both 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 who have viscid or thickened airway mucus for a range of chronic respiratory illnesses, including chronic bronchitis, emphysema, COPD and exacerbations, cystic fibrosis, bronchiectasis, as an antidote due to acetaminophen overdose and as a potential treatment of diseases characterized by free radical, oxidant damage.
NAC can be typically considered a non-antibiotic drug, to which antibacterial and antibiofilm properties have been associated in some cases. For example, in 1977, Parry and Neu found that NAC had the characteristics to inhibit the growth of both gram-positive and gram-negative bacteria, including Staphylococcus aureus, P. aeruginosa, Klebsiella pneumoniae and Enterobacter cloacae (Journal of Clinical Microbiology, Jan 1977, p.58-61).
In the literature, controversial results have been reported regarding the interference of antibiotics and mucolytic agents such as NAC. In 1981, Roberts and Cole found that 2%-5% of NAC exhibited antimicrobial activity against P. aeruginosa and that the effect of the carbenicillin on P. aeruginosa was augmented by low concentrations of NAC (Journal of Infection Volume 3, Issue 4, December 1981, Pages 353-359). In 2016, the result of a study carried out by Landini et al. on the effect of high NAC concentrations on antibiotic activity against a collection of respiratory pathogens, demonstrated that high NAC concentrations do not interfere with the activity of the most commonly used antibiotics, whereas NAC compromised the activity of carbapenems (Antimicrob. Agents Chemother. December 2016 vol. 60 no. 12 7513- 7517).
NAC is commercially available under various brand names by different manufacturers throughout the world, for example as FluimucilTM (Zambon Spa). S. maltophilia infections are commonly associated with respiratory tract diseases, in particular chronic respiratory tract diseases; for example, S. maltophilia infections can trigger pulmonary exacerbations of chronic obstructive lung disease (COPD), Cystic Fibrosis (CF) and bronchiectasis non CF.
S. maltophilia infections occur principally, but not exclusively, in debilitated and immunosuppressed individuals.
S. maltophilia are characterized by a multidrug resistance phenotype and the ability to form biofilms. In virtue of these features, they are responsible for chronic lung colonization in individuals with weakened immune systems or chronic lung disease, particularly CF and bronchiectasis non CF, which may last several months or years and are difficult or impossible to be eradicated by current antibiotic treatment strategies.
As S. maltophilia, A. baumannii is an important and difficult-to-treat pathogen, which can infect the respiratory tract of patients affected by respiratory tract diseases, including chronic respiratory tract diseases. For example, the presence of A. baumannii infections in patients with pneumonia, in particular, in ventilator- associated pneumonia (VAP), represents one of the most dreadful complications that occur in the critical care setting.
A. baumannii has been also identified in the CF population and in the population with bronchiectasis non CF.
The association of A. baumannii infections with bacteremia, wound infections, urinary tract infections and meningitis has been also described. It can be noted that there has been a dramatic increase in the number of A. baumannii strains with resistance to multiple antibiotic classes and the multidrug-resistant nature of this pathogen represents a challenge in the treatment and control of nosocomial infections, with relatively limited treatment options. For example, nosocomial pneumonia due to multidrug resistant gram-negative bacteria comprising A. baumannii, is among the most serious complications that occur in the intensive care unit (ICU) setting. Mortality, morbidity and health care costs are substantially increased by this type of infection. Despite a significant amount of time and energy has been devoted to studying effective treatments for dealing successfully with bacterial infections, especially of the airway and/or lung, caused by a range of pathogens including emerging pathogens such as S. maltophilia and A. baumannii, there is substantially lack of good therapeutic options and hence there remains a need for better treatments of said bacterial infections, in particular those associated with chronic lung diseases such as, for example, CF, bronchiectasis non CF and COPD.
SUMMARY OF THE INVENTION
Accordingly, the present inventors faced the problem of treating a bacterial infection caused by one or more pathogens selected from S. maltophilia and/or A. baumannii. After a long set of tests and experimentations, the present inventors have surprisingly found that the association of NAC and colistin has a synergistic antimicrobial effect against a consistent number of S. maltophilia and/or A. baumannii clinical isolates, even resulting in a bactericidal effect.
The association of NAC and colistin has also been found to have synergistic anti- biofilm activity in in vitro biofilm models of S. maltophilia and/or A. baumannii. The anti-biofilm activity of the association of NAC and colistin against S. maltophilia and/or A. baumannii biofilms envisages a potential use also for both preventing and eradicating colonization by said pathogens, especially in the hospitalized patients affected by diseases including, for example, chronic bronchitis, emphysema, COPD and exacerbations, CF, bronchiectasis non CF, pneumonia, VAP, bacteremia, wound infections, urinary tract infections and meningitis.
More in detail, the present inventors noticed that with an increase in the use of colistin to treat infections caused by multidrug resistant Gram-negative pathogens, resistance to colistin has been increasingly reported.
Since colistin represents one of the last therapeutic options to fight Gram-negative bacteria, the appearance of colistin-resistant Gram-negative pathogens such as S. maltophilia and/or A. baumannii is of great concern.
The present inventors, by concentrating their efforts in identifying an effective treatment able to restore susceptibility of said pathogens to the colistin treatment, have unexpectedly found that colistin effectiveness against colistin-resistant Gram- negative pathogens such as, e.g., A. baumannii and S. maltophilia can be surprisingly restored by administering colistin together with NAC. In particular, the MIC of all colistin resistant strains tested lowered at or below the susceptibility breackpoint in the presence of NAC 8 mg/ml.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Time kill curves. NAC alone (1.6 and 8 mg/ml), colistin alone (2 and 8 μg/ml and combination NAC + colistin (1.6 mg/ml + 2 μg/ml, 1.6 mg/ml + 8 μg/ml, 8 mg/ml + 2 μg/ml, 8 mg/ml + 8 μg/ml). Symbols: : Control; (dotted line): NAC 1.6 mg/ml;— : NAC 8 mg/ml; -O- Colistin 2 μg/ml; -□- Colistin 8 μg/ml; O (dotted line) NAC 1.6 mg/ml + Colistin 2 μg/ml; □ (dotted line): NAC
1.6 mg/ml + Colistin 8 μg/ml; — O- : NAC 8 mg/ml + Colistin 2 μg/ml; — Ώ~ : NAC 8 mg/ml + Colistin 8 μg/ml.
Figure 2. Time kill curves of S. maltophilia isolates with sub MIC concentrations of N-acetylcysteine (NAC 1.6, 3.2 and 8 mg/ml) and colistin (COL 2, 8 μg/ml) alone and in combination. A) STX: trimethoprim- sulphametoxazole resistant S. maltophilia strain; B) S. maltophilia CF: strain from a Cistic Fibrosis patient. Symbols: -x- : Control;— O- Colistin 2 μg/ml; ~Ώ~ Colistin 8 μg/ml;— : NAC 1.6 mg/ml; "" (dotted line) NAC 3.2 mg/ml; (dashed line) NAC 8 mg/ml; -·- : NAC 1.6 mg/ml + Colistin 2 μg/ml; -■- : NAC 1.6 mg/ml + Colistin 8 μg/ml; "# " (dotted line) NAC 3.2 mg/ml + Colistin 2 μg/ml; (dotted line) NAC 3.2 mg/ml +
Colistin 8 μg/ml; - - ·- - (dashed line): NAC 8 mg/ml + Colistin 2 μg/ml; - -■- - (dashed line) : NAC 8 mg/ml + Colistin 8 μg/ml.
Figure 3. Antibiofilm activity of NAC, colistin and colistin/NAC combinations (sub- MIC concentrations) tested on a Cystic Fibrosis isolate of S. maltophilia. Biofilms were grown on Calgary Device (MBEC assay) for 48 hrs and challenged for 24 hours with NAC alone, Colistin alone at two different concentration: 2 μg/ml and 8 μg/ml and the combination of the two (NAC 8 mg/ml + Colistin 2 μg/ml and 8 μg/ml).
DETAILED DESCRIPTION OF THE INVENTION
It is therefore a first aspect of the present invention a synergistic pharmacological association of NAC and colistin for use in inhibiting or suppressing the growth and/or killing a strain of a pathogen selected from S. maltophilia and A. baumannii strains.
It is another aspect of the present invention a synergistic pharmacological association of NAC and colistin for use in the treatment of a bacterial infection caused by one or more pathogens selected from S. maltophilia and A. baumannii strains, in particular a bacterial infection associated with a respiratory tract disease, particularly a chronic respiratory tract disease such as e.g., CF, bronchiectasis non CF and COPD.
In a particular aspect, a bacterial infection is caused by a pathogen selected from S. maltophilia and A. baumannii strains, either expressing a colistin susceptible or resistant phenotype; especially, a pathogen selected from S. maltophilia and A. baumannii strains, expressing a colistin resistant phenotype.
A bacterial infection caused by S. maltophilia strain can be detected, for example, in patients suffering from respiratory tract diseases, particularly chronic respiratory tract diseases such as, for example, in patients suffering from COPD, CF or bronchiectasis non CF, especially when these patients have weakened immune systems.
A bacterial infection caused by A. baumannii strain can be detected, for example, in patients suffering from pneumonia, VAP, bacteremia; wound infections, urinary tract infections, CF and bronchiectasis non CF, especially when these patients are hospitalized patients.
In a more particular aspect of the present invention, patients suffering of a bacterial infection caused by a pathogen selected from S. maltophilia and A. baumannii strains as described above are immunocompromised and/or hospitalized patients.
A bacterial infection caused by a pathogen selected from S. maltophilia and A. baumannii strains according to the present invention may be a biofilm-associated infection.
The above aspects are based on the observation that there is a synergistic interaction between NAC and colistin, which provides a significant antibacterial effect.
According to the present invention the terms "synergistic" and "synergistically" as applied to the effect of NAC and colistin used in association (whether simultaneously or sequentially) refer to a greater antibacterial effect obtained when the above- identified bacteria are treated by either NAC or colistin alone. In some embodiments, the effect of NAC and colistin used in association (whether simultaneously or sequentially) is greater than the simple addition of the effects of each agent administered alone, i.e. there is an effect which surpasses expectations based on additive effects.
It is a further aspect of the present invention a synergistic pharmacological association of NAC and colistin for use in the treatment of a bacterial infection caused by one or more pathogens selected from colistin-resistant S. maltophilia and colistin-resistant A. baumannii strains, wherein NAC is able to restore susceptibility of said pathogens to colistin.
It is a further aspect of the present invention a pharmacological association of NAC and colistin for use in the treatment of a bacterial infection caused by one or more pathogens selected from colistin-susceptible S. maltophilia and colistin-susceptible A. baumannii strains, wherein NAC is able to prevent in vivo emergence of colistin resistance, during colistin treatment regimens.
NAC and colistin of the pharmacological association according to the present invention may be administered in either order, separately or concurrently, with overlapping or non-overlapping periods of administration, via the same or different modes of administration.
For example, NAC and colistin can be concurrently administered in a single dosage form or alternatively NAC and colistin can be administered in separate dosage forms in either order, concomitantly or sequentially, with overlapping or non-overlapping periods of administration, via the same of different modes of administration.
The concurrent or separate administration of the pharmacological association of NAC and colistin has the effect of in inhibiting or suppressing the growth and/or killing a strain of a pathogen selected from S. maltophilia and A. baumannii strains, said effect being unexpectedly and surprisingly greater than what is seen when bacteria are contacted by either NAC or colistin alone. In detail, the present inventors noticed that with an increase in the use of colistin to treat infections caused by multidrug resistant Gram-negative pathogens, resistance to colistin has been increasingly reported.
Since colistin represents one of the last antibiotic resorts to fight Gram-negative bacteria, the appearance of colistin-resistant Gram-negative pathogens is of great concern.
The present inventors strongly perceived the great need of an effective treatment against colistin-resistant Gram-negative pathogens such as, e.g., A. baumannii and S. maltophilia, able to restore susceptibility of said pathogens to the antibiotic treatment, or to prevent in vivo emergence of colistin resistance during colistin treatment regimens.
The present inventors have found that colistin effectiveness against colistin-resistant Gram-negative pathogens such as, e.g., A. baumannii and S. maltophilia can be surprisingly restored by the administration of the association of NAC with colistin. In particular, colistin susceptibility can be restored by NAC concentrations possibly achievable by topical administration. In particular, the MIC of all colistin resistant strains tested lowered at or below the susceptibility breakpoint in the presence of NAC 8 mg/ml.
The association of NAC and colistin may be administered to the patient in the form of one or more pharmaceutical formulations.
The pharmaceutical formulations of the present invention comprise NAC and/or colistin together with a carrier suitable for pharmaceutical use, consisting of one or more excipients. For example, the pharmaceutical formulations of the invention may comprise both NAC and colistin together with a carrier suitable for pharmaceutical use, consisting of one or more excipients, i.e. pharmaceutical formulations for concurrent administration of both NAC and colistin; or the pharmaceutical formulations of the invention may comprise NAC together with a carrier suitable for pharmaceutical use, consisting of one or more excipients; or the pharmaceutical formulations of the invention may comprise colistin together with a carrier suitable for pharmaceutical use, consisting of one or more excipients, i.e. separate pharmaceutical formulations for sequential or concomitant administration of NAC and colistin.
According to the present invention, the term "excipient" comprises any inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
According to the present invention, the term "carrier" comprises any substance suitable as a vehicle for delivering NAC and/or colistin to a suitable in vivo or in vitro site.
Pharmaceutically acceptable excipients are those compounds well known to a skilled person in the art that can be used to produce formulations comprising NAC and/or colistin that are suitable to be administered to a subject. Acceptable methods for preparing the pharmaceutical formulations according to the invention are well known to a person skilled in the art.
It is a further object of the present invention a method of treating a bacterial infection caused by a pathogen selected from S. maltophilia and A. baumannii strains in a subject in need thereof, which comprises administering an association comprising NAC and colistin, wherein the association has a synergistic antibacterial effect. It is a further object of the present invention a method of treating a bacterial infection caused by a pathogen selected from S. maltophilia and A. baumannii strains in a subject in need thereof, which comprises concurrently administering an association comprising NAC and colistin, wherein the association has a synergistic antibacterial effect.
It is a further another object of the present invention a method of treating a bacterial infection caused by a pathogen selected from S. maltophilia and A. baumannii strains in a subject in need thereof, which comprises separately administering an association comprising NAC and colistin, wherein the association has a synergistic antibacterial effect.
According to the present invention, the terms "individual" "subject" and "patient" are used interchangeably to refer to a member of mammalian specie, preferably a human, which is afflicted with a particular disease, disorder or condition. According to the present invention the term "antibacterial" means reducing the harmful effects of bacteria by inhibiting, suppressing the growth and/or killing them. According to the present invention the term "antibacterial agents" includes NAC and colistin.
According to the present invention the term "bactericidal" means having a destructive killing action upon bacteria.
According to the present invention, "bacterial infection" refers to any situation in which the presence of a microbial population(s) is damaging to a host mammal. Thus, an individual is "suffering" from a microbial infection when excessive numbers of a microbial population are present in or on an individual's body, or when the effects of the presence of a microbial population(s) is damaging the cells or other tissue of an individual. In particular, "bacterial infection" refers to an infection caused by a strain of bacteria for which the use of a synergistic pharmacological association of NAC and colistin disclosed herein is appropriate.
According to the present invention, the terms "treat", "treating" and "treatment" refer to a diminution, a decrease, a limitation, or a mitigation of the degree, intensity, extent of a bacterial infection or its related disease conditions and symptoms caused by a strain of S. maltophilia and/or A. baumanii, that is achieved by a reduction, inhibition or suppression of growth, replication, and/or propagation, or death or destruction of said bacteria, on or in the subject.
According to the present invention, the term "pharmacological association" refers to either a fixed combination of NAC and colistin in one unit dosage form, a non-fixed combination or a kit of parts for the combined administration where NAC and colistin, as defined above, may be administered simultaneously, independently at the same time or separately within time intervals that allow the combination partners to show a synergistic effect.
Any suitable route of administration may be used for the compositions of the present invention, including oral, parenteral (subcutaneous, intramuscular or intravenous) and inhalation route.
According to the present invention, the terms "oral" or "orally" refer to the introduction into the body by mouth whereby absorption occurs in one or more of the following areas of the body: the mouth, stomach, small intestine, and the small blood vessels of the oral mucosa.
Non-limiting examples of pharmaceutical formulations according to the present invention for oral administration include, for example, tablets, coated tablets, granulates, pills, capsules, liquids, gels, syrups, suspensions, and the like, for oral ingestion by an individual. Suitable carriers for oral administration are well known in the art.
For parenteral administration, pharmaceutical formulations according to the invention may be formulated for example in aqueous solutions such as in physiologically compatible buffers or physiological salt buffer. Formulations for injection may be presented in unit dosage forms, for example, in ampoules, or in multi-dose containers with, optionally, an added preservative.
For administration by inhalation route, pharmaceutical formulations according to the invention may be formulated in solutions, suspensions and dry powder and delivered by using conventional means, so that optimal quantities of a suitable range of particle sizes are provided to the patient.
The pharmaceutical formulations according to the invention may be preferably administered to the respiratory tract. Thus, the present invention also provides aerosol pharmaceutical formulations comprising NAC and/or colistin.
Pharmaceutical compositions of the present invention may be manufactured in conventional manners, following processes well known in the art.
The amount of NAC and colistin for use according to the present invention may vary depending on the administration route, the selected kind of composition, the individual characteristics of the patient, the duration of the treatment and the nature of concurrent therapies.
For example, the synergistic effective amount of the pharmacological association of NAC and colistin can produce a diminution, a decrease, a limitation, or a mitigation of the degree, intensity, extent of a bacterial infection or its related symptoms caused by a strain of A. baumannii and/or S. maltophilia. According to one embodiment, the amount of NAC and colistin sufficient to have a synergistic effect on a bacterial infection caused by a strain of A. baumannii and/or S. maltophilia may vary, for example, in view of the physical characteristics of the patient, the severity of the subject's symptoms, the form of the infection, the identity of the bacteria, the formulations and the means used for administering the drug. The specific dose for a given subject is usually set by the judgement of the attending physician.
However, as an example, an effective amount of colistin may be between about 0.075 million units and 12 million units (i.e. between about 6mg and 960 mg), preferably between about 0.5 million units and 12 million units (i.e. between about 40 mg and 960 mg); the effective amount of NAC may vary between 100 and 5800 mg, preferably between 100 and 4600 mg, to be administered in a single dose or in more repeated doses.
Depending of the means of administration a dose may be administered all at once or slowly over a period of time, such as with an i.v. or by inhalation administration. According to the present invention, the term "dose", "unit dose", "dosage", "effective dose" and related terms refer to physically discrete units that contain a predetermined quantity of active ingredient calculated to produce a desired therapeutic effect. A single dose is thus a predetermined quantity of colistin or NAC that is administered to a patient.
The exact composition, route of administration, and dosage can be chosen by the individual physician in view of the patient's condition. Treatment should be continued for as long as required to receive the benefit of the invention.
A determination of a synergistic interaction between NAC and colistin may be based on the results obtained from the assays described here below.
In vitro antimicrobial synergism between NAC and colistin was determined following EUCAST guidelines. In particular, the total fractional inhibitory concentration (∑FIC) was calculated as follows:∑FIC equals FIC of agent A plus FIC of agent B, where the FIC of agent A or B is the minimum inhibitory concentration (MIC) of agent A or B in the presence of the other divided by the MIC of agent A or B alone. Synergism was defined as a∑FIC value of <0.5.
Example 1
Activity of NAC in association with colistin against A. baumannii
Seven colistin resistant A. baumannii clinical isolates were included in the study (colistin MIC range 16-256 μ^πιΐ, MIC50=64 μ^πιΐ, MIC90=128 μ^πιΐ). Classic checkerboard assays were used to investigate potential synergism between colistin (range 0.25-256 μg/ml) and NAC (range 0.5-32 mg/ml). Fraction inhibitory concentration indices (FICIs) were calculated, and synergism was defined as FICI values < 0.5. Synergy between colistin and NAC was also confirmed by time-kill assays performed with one isolate, using two different concentrations of colistin (2 and 8 μg/ml, representing concentrations achievable in serum and epithelial lining fluid - ELF, respectively) and NAC (1.6 and 8 mg/ml, likely achievable in ELF by topical administration).
The MIC of NAC was 32 mg/ml for two isolates, and > 32 mg/ml for the remaining ones. Synergism between colistin and NAC was observed in checkerboard assays with all tested isolates. In particular, a restoration of colistin susceptibility (i.e. MIC < 2 μg/ml) was observed with 100% and 52% of tested strains in the presence of NAC 4 mg/ml and NAC 2 mg/ml, respectively. Time-kill curves confirmed the synergy observed by checkerboard assays, demonstrating a bactericidal effect of colistin/NAC combinations at sub-MIC concentrations (Figure 1 shows the curves obtained with isolate N50, with NAC MIC = >32 mg/ml and Colistin MIC = 16 μ-g ml).
In order to further investigate the synergism of colistin/NAC combinations against colistin-resistant A. baumannii strains, time-kill assays were also performed according to CLSI guidelines with two selected strains (i.e. Z165 and Z167, ColR).
Two colistin concentrations (i.e. 2 and 8 μg/ml), and three NAC concentrations (i.e.
1.6, 3.2 and 8 mg/ml) were tested alone and in combination with colistin, with determination of viable cells performed after 0, 2, 4, 6, 8, 24 and 48 hours of exposure (detection limit 25 CFU/ml). Data were obtained in two independent experiments, with two replicates per condition per experiment and show a dose- dependent potentiation of colistin activity by NAC. In particular, a complete eradication of the starting inocula was achieved with combinations including NAC 8 mg/ml (i.e. absence of regrowth after 48 hours of incubation).
A biofilm eradication test on the two isolates was also carried out as follows: biofilms were grown in the NUNC TSP lid system (MB EC assay), for seven days in daily refreshed CAMHB (static conditions). Preformed biofilms were then exposed to two colistin concentrations (i.e. 2 and 8 μg/ml), and three NAC concentrations (i.e. 1.6, 3.2 and 8 mg/ml), alone and in combination, for 24 hours. After exposure, biofilms were disrupted by sonication and mean viable cell count per peg (log CFU/peg) was determined (detection limit 1.3 log CFU/peg). Data were obtained in at least two independent experiments, with at least six replicates per condition per experiment.
Results showed a remarkable antibiofilm synergistic activity of combinations including colistin 8 μg/ml (i.e. representing 0.25 x and 0.06 x MIC for Z165 and Z167, respectively), with a marked reduction of viable biofilm cells also observed in the presence of the lower NAC concentration tested (i.e. 1.6 mg/ml).
Example 2
Activity of NAC in association with colistin against S. maltophilia
Twenty S. maltophilia clinical isolates were tested, including also four isolates from patients affected by cystic fibrosis (CF). Two isolates showed resistance to trimethoprim-sulphamethoxazole. Synergism between colistin (range 0.25-256 μg/ml) and NAC (range 0.5-32 mg/ml) was investigated by classic checkerboard assays. Fraction inhibitory concentration indices (FICIs) were calculated, and synergism was defined as FICI values < 0.5. Time-kill assays were performed with two clinical isolates (including one strain resistant to trimethoprim- sulphamethoxazole, and one from CF). For this purpose, two different concentrations of colistin (2 and 8 μg/ml, representing concentrations achievable in serum and epithelial lining fluid - ELF, respectively) and NAC (1.6 and 8 mg/ml, likely achievable in ELF by topical administration) were tested alone and in combination. Colistin MICs ranged from 0.5 μg/ml to 128 μg/ml (MIC50, 16 μg/ml; MIC90, 64 μg/ml). The MIC of NAC was 16 mg/ml for nine isolates, >32 mg/ml for one isolate, and 32 mg/ml for the remaining ones. Synergism between colistin and NAC was observed in checkerboard assays with all tested isolates. In particular, NAC 8 mg/ml and 2 mg/ml lowered colistin MIC to < 2 μg/ml (the susceptibility breakpoint for A. baumannii and P. aeruginosa) for 100% and 47% of isolates with colistin MIC>2 μg/ml (n=17), respectively. Time-kill curves confirmed the synergy observed by checkerboard assays, demonstrating a bactericidal effect of colistin/NAC combinations at sub-MIC concentrations (Figure 2 A and B).
The antibiofilm activity of colistin/NAC combinations was investigated using the MBEC assay, the reference procedure for biofilm susceptibility testing (. Mature S. maltophilia (48 -hours old) were challenged for 24 hours with NAC and colistin, either alone or in combination. Viable cell count of challenged biofilms was compared to controls in order to assess the antibiofilm activity of the tested drugs and drugs/combinations.
Sub-MIC colistin/NAC combinations were also found to exert a relevant antibiofilm activity against mature S. maltophilia biofilms. In particular, NAC 8 mg/ml plus colistin 2 μg/ml accounted for a reduction of more than 3 log colony forming units (CFU)/peg compared to controls (Figure 3).
The results show that MIC of all colistin resistant strains tested (i.e. S. maltophilia, n=17; A. baumannii, n=7) lowered at or below the susceptibility breakpoint in the presence of NAC 8 mg/ml.

Claims

1) A synergistic pharmacological association of N-acetylcysteine (NAC) and colistin for use in the treatment of a bacterial infection caused by one or more pathogens selected from S. maltophilia and A. baumannii strains.
2) A synergistic pharmacological association according to claim 1, wherein pathogens selected from S. maltophilia and A. baumannii strains express a colistin resistant phenotype.
3) A synergistic pharmacological association according to claim 1 or 2, wherein the infection is a biofilm-associated infection.
4) A synergistic pharmacological association according to anyone of claims 1 to 3, wherein the bacterial infection is detected in patients with a respiratory tract disease.
5) A synergistic pharmacological association according to claim 4, wherein the respiratory tract disease is a chronic respiratory tract disease comprising Cystic Fibrosis (CF), bronchiectasis non CF and COPD.
6) A synergistic pharmacological association according to claim 1, wherein a bacterial infection caused by caused by S. maltophilia strain is detected in patients suffering from a respiratory tract disease. 7) A synergistic pharmacological association according to claim 1, wherein a bacterial infection caused by A. baumannii strain is detected in patients suffering from a respiratory tract disease.
8) A synergistic pharmacological association according to claim 4, wherein the patients are immunocompromised and/or hospitalized patients. 9) A synergistic pharmacological association of NAC and colistin for use in restoring susceptibility to antibiotic treatment in colistin-resistant S. maltophilia and colistin-resistant A. baumannii strains after bacterial infection caused by said one or more pathogens.
10) A synergistic pharmacological association according to claim 1, wherein NAC is able to prevent in vivo emergence of colistin resistance, during colistin treatment regimens.
11) A synergistic pharmacological association according to anyone of claims 1 to
10, wherein NAC and colistin are administered in either order, separately or concurrently, with overlapping or non-overlapping periods of administration. 12) A synergistic pharmacological association according to claim 11, wherein NAC and colistin are administered via the same or of different administration routes .
13) A synergistic pharmacological association according to anyone of claims 1 to
11, wherein NAC and colistin are concurrently administered in a single dosage form.
14) A synergistic pharmacological association according to anyone of claims 1 to 11, wherein NAC and colistin are administered in separate dosage forms in either order, concomitantly or sequentially, with overlapping or non-overlapping periods of administration.
15) A synergistic pharmacological association according to claim 14, wherein NAC and colistin are administered via the same of different modes of administration.
16) NAC for use in restoring susceptibility to antibiotic treatment in colistin-resistant S. maltophilia and colistin-resistant A. baumannii strains during bacterial infection caused by said one or more pathogens. 17) NAC for use in disrupting biofilm formation in colistin-resistant S. maltophilia and colistin-resistant A. baumannii strains during bacterial infection caused by said one or more pathogens.
PCT/EP2018/054611 2017-02-27 2018-02-26 Association of n-acetylcysteine and colistin for use in bacterial infections WO2018154091A1 (en)

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AU2018223985A AU2018223985B2 (en) 2017-02-27 2018-02-26 Association of n-acetylcysteine and colistin for use in bacterial infections
EP18707039.6A EP3585372A1 (en) 2017-02-27 2018-02-26 Association of n-acetylcysteine and colistin for use in bacterial infections
JP2019545362A JP7241687B2 (en) 2017-02-27 2018-02-26 Combination of N-acetylcysteine and colistin for use in bacterial infections
CA3053441A CA3053441A1 (en) 2017-02-27 2018-02-26 Association of n-acetylcysteine and colistin for use in bacterial infections
EA201991889A EA201991889A1 (en) 2017-02-27 2018-02-26 N-ACETYLCYSTEINE ASSOCIATE AND COLISTINE FOR USE IN BACTERIAL INFECTIONS
BR112019017113A BR112019017113A2 (en) 2017-02-27 2018-02-26 synergistic pharmacological association of n-acetylcysteine and colistin, and, n-acetylcysteine.
US16/487,227 US11382948B2 (en) 2017-02-27 2018-02-26 Association of N-acetylcysteine and colistin for use in bacterial infections
CN201880012765.3A CN110352053B (en) 2017-02-27 2018-02-26 Combination of N-acetylcysteine and polymyxin E for bacterial infections
CONC2019/0009216A CO2019009216A2 (en) 2017-02-27 2019-08-27 Association of n-acetylcysteine and colistin for use in bacterial infections
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