ASSOCIATION OF N-ACETYLCYSTEINE AND COLISTIN FOR USE IN BACTERIAL INFECTIONS

Abstract

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, in particular a bacterial infection associated with a respiratory tract disease, such as a chronic respiratory tract disease comprising cystic fibrosis (CF), bronchiectasis non CF and chronic obstructive pulmonary disease COPD is described.

Claims

1. A method of restoring susceptibility to antibiotic treatment in colistin-resistant S. maltophilia and colistin-resistant A. baumannii strain during or after bacterial infection caused by said one or more pathogens in patients in need thereof, said method comprising administering to said patients a pharmaceutical effective amount of a synergistic pharmacological association consisting of N-acetylcysteine (NAC), colistin and a carrier.

2. The method according to claim 1, wherein the infection is a biofilm-associated infection.

3. The method according to claim 1, wherein the infection is detected in patients with a respiratory tract disease.

4. The method according to claim 3, wherein the respiratory tract disease is a chronic respiratory tract disease comprising Cystic Fibrosis (CF), bronchiectasis non CF and chronic obstructive pulmonary disease (COPD).

5. The method according to claim 1, wherein said bacterial infection caused by S. maltophilia strain is detected in patients suffering from a respiratory tract disease.

6. The method according to claim 1, wherein the bacterial infection caused by A. baumannii strain is detected in patients suffering from a respiratory tract disease.

7. The method according to claim 1, wherein the patients are immunocompromized and/or hospitalized patients.

8. The method according to claim 1, wherein NAC and colistin are administered in either order, separately or concurrently, with overlapping or non-overlapping periods of administration.

9. The method according to claim 8, wherein NAC and colistin are administered via the same or different administration routes.

10. The method according to claim 1, wherein NAC and colistin are concurrently administered in a single dosage form.

11. The method according to claim 1, wherein NAC and colistin are administered in separate dosage forms in either order, concomitantly or sequentially, with overlapping or non-overlapping periods of administration.

12. A method of disrupting biofilm formation in colistin-resistant S. maltophilia and colistin-resistant A. baumannii strains during bacterial infection caused by said one or more pathogens in patients in need thereof, said method comprising: administering to said patients a pharmaceutical composition consisting of a pharmaceutical effective amount of NAC together with a carrier.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG. 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; Colistin 2 μg/ml; Colistin 8 μg/ml; ⋅⋅⋅⋅⋅◯⋅⋅⋅⋅⋅ (dotted line) NAC 1.6 mg/ml+Colistin 2 μg/ml; ⋅⋅⋅⋅⋅□⋅⋅⋅⋅⋅ (dotted line): NAC 1.6 mg/ml+Colistin 8 μg/ml; : NAC 8 mg/ml+Colistin 2 μg/ml; : NAC 8 mg/ml+Colistin 8 μg/ml.

[0034] FIG. 2A). 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. STX: trimethoprim-sulphametoxazole resistant S. maltophilia strain. Symbols: -×- : Control; —◯ Colistin 2 μg/ml; —□— 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.

[0035] FIG. 2B) 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. S. maltophilia CF: strain from a Cistic Fibrosis patient. Symbols: -×-: Control; —◯— Colistin 2 μg/ml; —□— 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.

[0036] 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 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

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] The above aspects are based on the observation that there is a synergistic interaction between NAC and colistin, which provides a significant antibacterial effect.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] The present inventors have found that colistin effectiveness against colistin-resistant

[0055] 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.

[0056] 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.

[0057] The association of NAC and colistin may be administered to the patient in the form of one or more pharmaceutical formulations.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] According to the present invention the term “bactericidal” means having a destructive killing action upon bacteria.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] Pharmaceutical compositions of the present invention may be manufactured in conventional manners, following processes well known in the art.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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 6 mg 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] A determination of a synergistic interaction between NAC and colistin may be based on the results obtained from the assays described here below.

[0086] In vitro antimicrobial synergism between NAC and colistin was determined following EUCAST guidelines. In particular, the total fractional inhibitory concentration (ΣFIC) was calculated as follows: ΣFIC 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 ΣFIC value of <0.5.

EXAMPLE 1

[0087] Activity of NAC in Association with Colistin Against A. baumannii

[0088] Seven colistin resistant A. baumannii clinical isolates were included in the study (colistin MIC range 16-256 μg/ml, MIC50=64 MIC90=128 μg/ml). 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).

[0089] 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 (FIG. 1 shows the curves obtained with isolate N50, with NAC MIC=>32 mg/ml and Colistin MIC=16 μg/ml).

[0090] 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, Col.sup.R). 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).

[0091] A biofilm eradication test on the two isolates was also carried out as follows: biofilms were grown in the NUNC TSP lid system (MBEC assay), for seven days in daily refreshed CAIVIHB (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.

[0092] Results showed a remarkable antibiofilm synergistic activity of combinations including colistin 8 μg/ml (i.e. representing 0.25× and 0.06×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

[0093] Activity of NAC in Association with Colistin Against S. maltophilia

[0094] 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.

[0095] 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 (FIG. 2A and FIG. 2B).

[0096] 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.

[0097] 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 (FIG. 3).

[0098] 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.