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WO2017027968A1 - Hybrid antibiotics-based adjuvants overcome resistance in pseudomonas aeruginosa - Google Patents

Hybrid antibiotics-based adjuvants overcome resistance in pseudomonas aeruginosa Download PDF

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Publication number
WO2017027968A1
WO2017027968A1 PCT/CA2016/050959 CA2016050959W WO2017027968A1 WO 2017027968 A1 WO2017027968 A1 WO 2017027968A1 CA 2016050959 W CA2016050959 W CA 2016050959W WO 2017027968 A1 WO2017027968 A1 WO 2017027968A1
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Prior art keywords
tobramycin
tether
aminoglycoside
compound
pump inhibitor
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French (fr)
Inventor
Frank Schweizer
Bala Kishan GORITYALA
Goutam GUCHHAIT
Xuan YANG
George G. Zhanel
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University of Manitoba
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University of Manitoba
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/453—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with oxygen as a ring hetero atom
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/7036—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin having at least one amino group directly attached to the carbocyclic ring, e.g. streptomycin, gentamycin, amikacin, validamycin, fortimicins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/21—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Pseudomonadaceae (F)

Definitions

  • Pseudomonas aeruginosa is the leading cause of nosocomial infections and chronic lung infections in cystic fibrosis patients with mortality rates ranging from 30-50%' 2 .
  • infections caused by P. aeruginosa are particularly difficult to treat as the organism is both intrinsically resistant and capable of acquiring resistance to most antibiotics 3 .
  • This is in large part the result of the low permeability of its outer membrane which is 12-100 times lower than that of E. coli and is caused by selective porins 4 .
  • the reduced uptake of antibiotic across the outer membrane in P. aeruginosa enables secondary adaptive resistance mechanisms to work more efficiently including efflux due to the intrinsic or induced expression of efflux pumps and antibiotic- modifying enzymes 3 .
  • Clinically useful anti-pseudomonal antibiotics are limited to select penicillins (eg. piperacillin tazobactam), cephalosporins (eg. ceftazidime), carbapenems (eg. imipenem), fluoroquinolones (eg. ciprofloxacin), aminoglycosides (eg. tobramycin) and colistin but resistance to these agents is steadily increasing with few novel anti-pseudomonal agents in clinical development 4 .
  • penicillins eg. piperacillin tazobactam
  • cephalosporins eg. ceftazidime
  • carbapenems eg. imipenem
  • fluoroquinolones eg. ciprofloxacin
  • aminoglycosides eg. tobramycin
  • colistin colistin
  • tobramycin and ciprofloxacin possess multiple modes of action and distinct uptake mechanisms.
  • Tobramycin's mode of action is concentration dependent. At low-concentrations ( ⁇ 4 ⁇ g/mL), tobramycin binds to the 30S ribosomal subunit, thereby leading to the disruption of protein synthesis while at higher concentrations (> 8 disruption of the outer membrane is observed 5 .
  • the antimicrobial action of the fluoroquinolones is mediated through the inhibition of two type II DNA topoisomerase enzymes, DNA gyrase and topoisomerase IV which play an essential role in DNA relaxation, partitioning replicated chromosomal DNA during cell division and decatenation reactions 6 .
  • tobramycin and ciprofloxacin also display different uptake mechanisms.
  • the uptake of aminoglycosides into Gram-negative bacteria involves interactions at sites at which divalent cations cross-bridge adjacent polyanionic lipopolysaccharide (LPS) that causes destabilization of the outer membrane and results in self-promoted uptake of the antibiotic or other extracellular molecules 7
  • LPS polyanionic lipopolysaccharide
  • uptake of fluoroquinolones in Gram-negative bacteria does not occur by self-promoted uptake, but rather involves diffusion across the cell membrane with uptake through the porin pathway 8 .
  • Multitargeting antibiotics are expected to limit the frequency of spontaneous resistance that can arise from mutation in the target gene 29 .
  • One approach to design multitargeting antibacterials involves the covalent attachment of two different pharmacophores that inhibit dissimilar targets in the bacterial cell generating hybrid antibiotics 14 .
  • EPIs Resistance-Nodulation-Division
  • perturbation or disruption of the proton motive force (PMF) which energize efflux pumps can also be used to block the function of RND pumps 36,37 .
  • PMF proton motive force
  • EPIs have been described 38 including 1 -(1 -naphthylmethyl)- piperazine (NMP) 39 , paroxetine (PAR) 38,40 and dibasic peptides like DBP 41 ( Figure 8) and for some, efficacy has been established in animal models of infection 42 .
  • NMP is a broad spectrum EPI which synergizes with multiple classes of antibiotics including tetracyclines, fluoroquinolones, macrolides, penicillins and rifampicin against certain clinically relevant Gram-negative pathogens like E.
  • PAR potentiates tetracyclines and fluoroquinolones in Gram-positive and Gram-negative bacteria excluding P. aeruginosa 40 .
  • EPIs such as DBP, NMP and PAR inhibit efflux of tetracycline and fluoroquinolone antibiotics in certain Gram-negative bacteria they are still subject to intrinsic resistance in organisms like P. aeruginosa.
  • a compound comprising an aminoglycoside connected to a fluoroquinolone or an efflux pump inhibitor.
  • a method of enhancing the anti-bacterial activity of an antibacterial compound comprising co-administering said antibacterial compound with a second compound comprising an aminoglycoside connected to a fluoroquinolone or efflux pump inhibitor.
  • a method of preparing a medicament for treatment of a bacterial infection comprising admixing a compound comprising an aminoglycoside connected to a fluoroquinolone or efflux pump inhibitor and a suitable excipient.
  • a method of determining if an agent of interest has antibacterial activity comprising:
  • a method of determining the effect of a compound comprising an aminoglycoside connected to a fluoroquinolone or an efflux pump inhibitor on the antibacterial activity of an agent of interest comprising:
  • a method of determining if an agent of interest has antibacterial activity comprising:
  • a method of potentiating a tetracycline antibiotic or mitomycin C comprising coadministering the tetracycline antibiotic or mitomycin C with a compound comprising tobramycin connected to a ciprofloxacin or moxifloxacin or an efflux pump inhibitor.
  • a compound comprising tobramycin connected to a ciprofloxacin or moxifloxacin or an efflux pump inhibitor, for potentiating a tetracycline antibiotic or mitomycin C.
  • FIG. 1 Structure of tobramycin-ciprofloxacin hybrids.
  • Compounds la-le are hybrids with a tether attached to the C-5 position of tobramycin while compounds 2 and 3 contain the tether at the C-2" or C-6" ⁇ respectively.
  • Compounds 4 and 5 are fragments of lead structure le.
  • FIC index were governed by the aliphatic chain leanth that connects tobramycin and ciprofloxacin among hybrids. High FIC indices of 4 and 5 showcase the importance of dual antibiotic role in the hybrid molecule (b) Potentiation of ciprofloxacin or moxifloxacin by le in MDR and XDR clinical isolates of P. aeruginosa strains as shown by the FIC index, (c) Enhanced Efficacy of combination of le with moxifloxacin in in vivo was demonstrated in Galleria mellonella infection model in a dose- dependent fashion.
  • the % luminiscence was calculated compared to no antibiotic control (assumed to be 100%). Each data point represents an average luciferase activity of the triplicates with +/- percentage error.
  • the IC50 values ( ⁇ ) tobramycin (0.0062 ⁇ 0.0002), le (8.05 ⁇ 0.57), 2 (0.97 ⁇ 0.05) and 3 (3.44 ⁇ 0.09) were obtained as described in biological methods, (c) Inhibition of P. aeruginosa DNA gyrase A activity in the presence of ciprofloxacin, le, 2 and 3.
  • FIG. 1 Overview of tobramycin-moxifloxacin hybrids used in this study. Hybrids differ in the linkage to tobramycin. In hybrid 10 moxifloxacin is linked via a C12-tether to the C-5 position of tobramycin while hybrids 20 and 30 are linked to the C-2" and C-6"-positions in tobramycin.
  • FIG. 5 Effects of hybrid 10 on bacterial killing and in vivo efficacy.
  • A Time-kill curves showing the effect of varying concentrations of hybrid 10 and MIC concentration of colistin (Col) on the viability of P. aeruginosa PAOl cells grown in MH broth. No colony-forming units (CFU) were found with 10 at 2 x MIC (blue) after 24 hours. Experiment was performed three times independently and each data point is an average of three determinations ⁇ SEM.
  • B Enhanced dose dependent efficacy of hybrid 10 in comparison with tobramycin (TOB) and moxifloxacin (MOX) in XDR P.
  • aeruginosa (#104354) over a period of 24 h was demonstrated in Galleria mellonella in vivo infection model.
  • Larvae infected with #104354 (106 CFU/mL) resulted in 73% killing after 24 h.
  • single dosage monotherapy 50 mg kg of moxifloxacin, or tobramycin or hybrid 10 resulted in 27%, 20% or 100% survival of the larvae, respectively, after 24 h. No killing was observed due to physical trauma when larvae were injected with equal volumes of PBS.
  • FIG. 6 Effects of hybrid 10 on the outer membrane. Concentration-dependent permeabilization of the outer membrane by tobramycin-moxifloxacin hybrid 10 is indicated by the accumulation of 1 -N-phenylnaphthylamine (NPN) in P. aeruginosa PAOl cells. 50 ⁇ g mL (medium plum), 32 ⁇ g mL (cyan), 16 ⁇ g mL (gold), or 8 ⁇ g/mL (pale lavender) of hybrid 10 were used, along with 5 ⁇ g/mL colistin (Col) (pale violet red) as positive control. Hybrid 10 was added at 120 seconds. Experiment was performed in triplicate and each data point is an average of three determinations ⁇ SEM. (B) Synergistic FIC index of hybrid 10 in combination with various classes of antibiotics against PAOl.
  • NPN -N-phenylnaphthylamine
  • FIG. 7 Protein translation activity, gyrase A activity and emergence of resistance.
  • A Inhibition of in vitro protein translation in E. coli S30 extract by tobramycin, moxifloxacin, hybrid 10 and 20. Protein translation assays were performed as described in methods section. The percentage luminescence was calculated compared to no antibiotic control (assumed to be 100%). Each data point represents an average luciferase activity of the triplicates with ⁇ percentage error. The IC50 values ( ⁇ ) for tobramycin (0.0062 ⁇ 0.0002), 10 (4.7 ⁇ 0.21), and 20 (0.13 ⁇ 0.003) were obtained.
  • B Inhibition of P.
  • FIG. 1 Structures of the efflux pump inhibitors (EPIs) NMP, PAR and DBP and tobramycin (TOBHinked EPI conjugates TOB -NMP (lOOa-f), TOB-PAR (200) and TOB-DBP (300).
  • EPIs efflux pump inhibitors
  • Figure 9 (a) Enhanced dose-dependent efficacy of a combination of conjugate lOOf and minocycline in XDR P. aeruginosa #101856 over a period of 24 h was demonstrated in a Galleria mellonella in vivo infection model. Combination therapy (37.5 mg/kg of lOOf + 37.5 mg/kg of minocycline) or (75 mg/kg of lOOf + 75 mg kg of minocycline) resulted in 10% or 77% survival of the larvae, respectively after 24 h.
  • FIG. 1 In- vivo studies: Synergy demonstration between mitomycin C (MYT-C) and adjuvant 10 in Galleria mellonella infection model infected with XDR P. aeruginosa strain # 262. Mitomycin C (10 mg/kg) provided low level of protection ( ⁇ 20%) while combination therapy of adjuvant 10 (10 mg kg) and Mitomycin C (10 mg/kg) produced 80% survival after 24 hours.
  • TOB tobramycin
  • a compound comprising an aminoglycoside connected to a fluoroquinolone or efflux pump inhibitor.
  • any suitable tether or linker known in the art may be used to connect the fluoroquinolone and the aminoglycoside provided that the tether is sufficiently flexible and sufficiently long that the aminoglycoside and the fluoroquinolone are able to function as discussed below. However, if the tether gets too long, some of the activity of the compound may be lost.
  • the tether or linker may be approximately 2-24 carbons long. As will be appreciated by one of skill in the art, this does not mean that the tether or linker necessarily needs to include 2-24 carbon molecules, merely that the tether or linker must be of the same approximate length as 2-24 carbon molecules.
  • the tether may comprise a length of 2-24, 3-24, 4-24, 5-24, 6-24, 2-23, 2-22, 2-21, 2-20, 2-19, 2-18, 3-23, 3-22, 3-21, 3-20, 3-19, 3-18, 4-23, 4-22, 4-21, 4- 20, 4-19, 4-18, 5-23, 5-22, 5-21, 5-20, 5-19, 5-18, 6-23, 6-22, 6-21, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12 carbon atoms.
  • the aminoglycoside is tobramycin.
  • the fluoroquinolone is ciprofloxacin or moxifloxacin.
  • the compound is tobramycin connected to ciprofloxacin or moxifloxacin or an efflux pump inhibitor.
  • the efflux pump inhibitor is selected from the group consisting of NMP, PAR and DBP.
  • the tobramycin is tethered to the ciprofloxacin or moxifloxacin or efflux pump inhibitor.
  • the tether is a flexible tether, as shown in Figure 1.
  • the tether may comprise 4-12 repeating alkyl units.
  • the tether may be connected to any suitable position on tobramycin, for example, a hydroxyl group.
  • the hydroxyl group may be for example, at the C-5 position, the C-4' position, the C-4" position, the C-2" position or the C- 6" position on tobramycin.
  • the preferred attachment site for ciprofloxacin is the secondary amino group of the piperazine ring in ciprofloxacin as other attachment at other positions may interfere with the mode of action of ciprofloxacin.
  • a method of treating a bacterial infection in an individual in need of such treatment comprising administering to said individual an effective amount of a compound comprising an aminoglycoside connected to a fluoroquinolone or efflux pump inhibitor.
  • the aminoglycoside is connected to the fluoroquinolone via a tether, as discussed above.
  • the aminoglycoside is tobramycin.
  • the fluoroquinolone is ciprofloxacin or moxifloxacin.
  • the efflux pump inhibitor is selected from the group consisting of NMP, PAR and DBP.
  • the compound is tobramycin connected to ciprofloxacin or moxifloxacin or the efflux pump inhibitor, as discussed above.
  • the tobramycin is tethered to the ciprofloxacin, moxifloxacin or efflux pump inhibitor.
  • any suitable tether or linker known in the art may be used to connect the tobramycin and the ciprofloxacin, moxifloxacin or efflux pump inhibitor provided that the tether is sufficiently flexible and sufficiently long that the tobramycin and the ciprofloxacin, moxifloxacin or efflux pump inhibitor are able to function as discussed below.
  • the agent of interest is attached to the aminoglycoside by a tether.
  • the tether may be a flexible tether.
  • the tether may be selected from the group consisting of but by no means limited to a flexible alkyl linker, an unsaturated alkyl linker, an ethylene glycol linker or an aromatic linker.
  • the tether may comprise a length of 2-24, 3-24, 4-24, 5-24, 6-24, 2-23, 2-22, 2-21, 2-20, 2-19, 2-18, 3-23, 3-22, 3-21, 3-20, 3-19, 3-18, 4-23, 4-22, 4-21, 4-20, 4-19, 4-18, 5- 23, 5-22, 5-21, 5-20, 5-19, 5-18, 6-23, 6-22, 6-21, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12 carbon atoms.
  • the aminoglycoside is tobramycin.
  • the tether is a flexible tether, as shown in Figure 1.
  • the tether may comprise 4-12 repeating alkyl units.
  • the tether may be connected to any suitable position on tobramycin, for example, a hydroxyl group.
  • the hydroxyl group may be for example, at the C-5 position, the C-4' position, the C-4" position, the C-2" position or the C-6" position of the tobramycin.
  • the bacterial infection is caused by a pathogenic gram-negative bacteria.
  • the pathogenic gram-negative bacteria may be for example but by no means limited to E. coli, Acetinobacter, Salmonella, Shigella, Enterobacteriaceae, Pseudomonas, Moraxella, Heliobacter, Stenotrophomonas, Bdellovibrio, Legionella and the like.
  • the pathogenic gram- negative bacteria is P. aeruginosa, E. coli or Acetinobacter baumannii.
  • the P. aeruginosa strain is an extremely drug-resistant strain of P. aeruginosa.
  • an "effective amount” is an amount that is sufficient to accomplish at least one of the following: reduction in the severity of the symptoms associated with the bacterial infection; and reduction in the CFU per ml of bacteria in a sample taken from the individual. As discussed herein, this may be accomplished by increasing or enhancing the anti-bacterial activity of another compound. For example, while not wishing to be bound to a particular theory or hypothesis, the inventors believe that the compounds described herein enhance the permeability of the gram-negative outer membrane and thereby in effect act as a delivery system. As such, in these embodiments, an “effective amount” is an amount that is sufficient to enhance the permeability of the outer membrane of the infective bacteria so that an antibacterial agent can enter the bacterial cell. As will be appreciated by one of skill in the art, this means that a wide variety of compounds that have bacteria targets but cannot cross the outer membrane on their own can now be considered to be anti-bacterial agents when used in combination with the compounds of the invention.
  • the compound may be co-administered with a second antibiotic.
  • antibiotics and antibacterial agents including but by no means limited to fluoroquinolones, beta- lactams (penicillins, cephalosporins (ceftazidimes and the like), carbapenems, monobactams), rifampicin, vancomycin, trimethoprim, trimethoprim plus sulfamethoxazole, colistin, polymyxin, chloramphenicol, novobiocin, and macrolides (erythromycin and the like).
  • antibiotics and antibacterial agents including but by no means limited to fluoroquinolones, beta- lactams (penicillins, cephalosporins (ceftazidimes and the like), carbapenems, monobactams), rifampicin, vancomycin, trimethoprim, trimethoprim plus sulfamethoxazole, colistin, polymyxin, chloramphenicol, novobiocin, and macro
  • antibiotic agents such as fluoroquinolones (ciprofloxacin, moxifloxacin, levofloxacin and the like), clindamycin, streptogramins, sulfonamides and monobactams.
  • a method of enhancing the antibacterial activity of an antibacterial compound comprising co-administering said antibacterial compound with a second compound comprising an aminoglycoside connected to a fluoroquinolone or efflux pump inhibitor as discussed above.
  • the aminoglycoside is connected to the fluoroquinolone via a tether as discussed above.
  • the aminoglycoside is tobramycin.
  • the fluoroquinolone is ciprofloxacin or moxifloxacin.
  • the efflux pump inhibitor is selected from the group consisting of MP, PAR and DBP.
  • the compound is tobramycin connected to ciprofloxacin, moxifloxacin or an efflux pump inhibitor.
  • the tobramycin is tethered to the ciprofloxacin as discussed above.
  • any suitable tether or linker known in the art may be used to connect the tobramycin and the ciprofloxacin, moxifloxacin and efflux pump inhibitor provided that the tether is sufficiently flexible and sufficiently long that the tobramycin and the ciprofloxacin, moxifloxacin or efflux pump inhibitor are able to function as discussed herein.
  • an effective amount of the second compound is co-administered with the antibiotic compound.
  • an "effective amount” is an amount that is sufficient to at least increase the effectiveness of the anti-bacterial compound, for example, such that less of the first compound is required or the time between administrations is increased or the severity of the bacterial infection is reduced to a greater extent than on administration of the anti-bacterial compound alone.
  • Other methods for detenrdning an effective amount of the compound to be administered to the individual will be apparent to one of skill in the art and may depend on many other factors, for example, the age and general condition of the individual and the severity of the infection and/or symptoms associated with the infection.
  • a compound comprising tobramycin connected to a ciprofloxacin or moxifloxacin or an efflux pump inhibitor to potentiate tetracycline antibiotics or mitomycin C.
  • the efflux pump inhibitor is selected from the group consisting of NMP,
  • tetracycline antibiotics are well known to those of skill in the art and as such suitable tetracycline antibiotics that can be used in this aspect of the invention will be well known to those of skill in the art.
  • the tetracycline antibiotic may be for example but by no means limited to minocycline, tigecycline, doxycycline and the like.
  • any suitable tether or linker known in the art may be used to connect tobramycin to the ciprofloxacin, moxifloxacin or efflux pump inhibitor provided that the tether is sufficiently flexible and sufficiently long that tobramycin and the ciprofloxacin or moxifloxacin or an efflux pump inhibitor are able to function as discussed below. However, if the tether gets too long, some of the activity of the compound may be lost.
  • the tether or linker may be approximately 2-24 carbons long. As will be appreciated by one of skill in the art, this does not mean that the tether or linker necessarily needs to include 2-24 carbon molecules, merely that the tether or linker must be of the same approximate length as 2-24 carbon molecules.
  • the tether is a flexible tether.
  • the tether may be selected from the group consisting of but by no means limited to a flexible alkyl linker, an unsaturated alkyl linker, an ethylene glycol linker or an aromatic linker.
  • the tether may comprise a length of 2-24, 3-24, 4-24, 5-24, 6-24, 2-23, 2-22, 2-21, 2-20, 2-19, 2-18, 3-23, 3-22, 3-21, 3-20, 3-19, 3-18, 4-23, 4-22, 4-21, 4- 20, 4-19, 4-18, 5-23, 5-22, 5-21, 5-20, 5-19, 5-18, 6-23, 6-22, 6-21, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12 carbon atoms.
  • the tether may comprise 4-12 repeating alkyl units.
  • the tether may be connected to any suitable position on tobramycin, for example, a hydroxyl group.
  • the hydroxyl group may be for example, at the C-5 position, the C-4' position, the C-4" position, the C-2' ' position or the C-6" position on tobramycin.
  • a compound comprising an aminoglycoside connected to a fluoroquinolone as discussed above for enhancing the antibacterial activity of an agent.
  • the agent does not necessarily have antibacterial properties on its own and may need to be co-administered with the compound in order to enter the bacterial cell.
  • the agent may have antibacterial properties on its own but these antibacterial properties may be enhanced by increased entry of the agent into the bacterial cell by the membrane permeability enhancing activity of the agent.
  • a method of preparing a medicament for treatment of a bacterial infection comprising admixing a compound comprising an aminoglycoside connected to a fluoroquinolone or an efflux pump inhibitor and a suitable excipient.
  • the compound may be formulated for co-administration with an agent having anti-bacterial properties either alone or when administered in combination with the compound, as discussed herein.
  • the aminoglycoside is tobramycin.
  • the fluoroquinolone is ciprofloxacin or moxifloxacin, as discussed herein.
  • the efflux pump inhibitor is selected from the group consisting of NMP, PAR andDBP.
  • the compounds described herein serve as adjuvants and can potentiate other classes of antibiotics and antibacterial agents.
  • this includes antibacterial agents which may not have antibacterial activity on their own due to an inability to cross the outer membrane of the bacterial target.
  • many known and unknown compounds can interact with validated antibacterial targets such as for example but by no means limited to enzymes, RNA, DNA, proteins, and the like, but possess no or poor antibacterial activity because they cannot cross the bacterial membranes in gram-negative organisms.
  • one function of the tobramycin- ciprofloxacin hybrids is to enhance the permeability of the outer membrane in P. aeruginosa. This facilitates cell penetration and may convert non antibacterial agents into antibacterial ones. As such the adjuvants would serve as a delivery system.
  • a method of determining if an agent of interest has antibacterial activity comprising: co-adniinistering the agent of interest with a com ound comprising an aminoglycoside connected to a fluoroquinolone or efflux pump inhibitor to a bacterium of interest; and
  • the agent of interest may be any compound thought, proposed or hypothesized to have an antibacterial effect once inside the cell of a gram-negative bacterium.
  • any suitable gram-negative bacterium may be used for the screening process.
  • the compound and the agent of interest may be administered to a population or culture of gram-negative bacterial cells and survival may be determined by comparing survival of bacteria treated with the agent of interest and the compound to survival of bacteria treated only with the agent of interest.
  • a method of determining the effect of a compound comprising an aminoglycoside connected to a fluoroquinolone or efflux pump inhibitor on the antibacterial activity of an agent of interest comprising:
  • the effect of the compounds of the invention on any agent, whether it has antibacterial properties, that is, bactericidal (killing of bacteria) or bacteriostatic (inhibition of growth) on its own or not, can be determined.
  • Hybrid le was selected for further combination studies against a panel of 8 clinical P. aeruginosa isolates including six MD (non-susceptible or resistant to > 3 chemically unrelated anti- pseudomonal classes), and six extremely-drug resistant (XDR) (non-susceptible or resistant to > 5 chemically unrelated anti-pseudomonal classes).
  • the panel also included two colistin-non-susceptible or resistant P. aeruginosa strains.
  • the fluoroquinolone-resistant isolates contained a single mutation (T83-I) in GyrA which was absent in the colistin-non-susceptible or resistant strains.
  • aeruginosa strains we observed additive effects or weaker synergistic effects (FIC index ⁇ 0.31 - 0.75) but still observed 16-fold fold potentiation of moxifloxacin and 2- to 8-fold potentiation of ciprofloxacin at 1 ⁇ 4 MIC of hybrid le.
  • the reduced synergistic effects or additive effects of le against colistin-resistant P. aeruginosa likely reflects a reduced self-promoted uptake caused by transfer of cationic 4-amino-4-deoxy-l-arabinose to lipid A in these organisms 14 .
  • Combination studies of moxifloxacin with various membrane-active agents confirmed that the observed synergistic effects of le were not the result of nonspecific membranolytic effect. This is in accordance with the observed low hemolytic activity of le which resulted in ⁇ 10% hemolytic activity at 1000 ⁇ g mL and low cytotoxic properties against cancer cell lines.
  • hybrid antibiotics consisting of tobramycin and ciprofloxacin are powerful multimodal adjuvants that can restore antibacterial activity and enhance efficacy with fluoroquinolones in MDR and XDR P. aeruginosa.
  • the strong synergistic effects of le against P. aeruginosa are also observed with other classes of antibiotics except carbapenems and aminoglycosides.
  • aeruginosa PA01 (b) is less prone to efflux than 2 nd and 3 rd generation fluoroquinolone antibiotics and (c) interacts as a hydrophobic fluoroquinolone more strongly with the cytoplasmic membrane than ciprofloxacin.
  • the secondary amino function in moxifloxacin was selected for point of modification as alkylation of this function retains potent antibacterial activity against bacterial pathogens 32 .
  • a C-12 tether length was selected based on previous results obtained with the tobramycin-ciprofloxacin hybrid antibiotics that demonstrated that this tether length is optimal for outer membrane penetration and synergism with other classes of antibiotics in P. aeruginosa 11 , We also prepared hybrids 20 and 30 bearing a tether at the C-2"- and C-6 "-positions to study how the nature of the tobramycin linkage affects the antibacterial activity.
  • hybrids 10, 20 and 30 were tested for antibacterial activity by determining the minimal inhibitory concentration (MIC) against select Gram-negative and Gram-positive bacteria (Table 1).
  • MIC minimal inhibitory concentration
  • MRSA methicillin-resistant Staphylococcus aureus
  • the promising antipseudomonal properties of hybrid 10 provided the rationale to study the antibacterial activity of 10 against a select panel of MDR and XDR P.
  • aeruginosa isolates obtained from multiple Canadian hospitals which are resistant to tetracyclines (doxycycline), cephalosporins (cefepime), carbapenems (meropenem and imipenem), fluoroquinolones (ciprofloxacin and moxifloxacin) and aminoglycosides (tobramycin and gentamicin) but remain susceptible to colistin.
  • aeruginosa PAOl at 2x MIC concentration over a 24 hour time period (Fig. 5A).
  • hybrid 10 causes ⁇ 2.5% hemolysis of human erythrocytes at 1000 ⁇ g/mL and shows low cytotoxicity CC 50 » 30 ⁇ M against cancer cell lines.
  • aeruginosa strain #104354 resistant to all' classes of antipseudomonal agents except colistin
  • injection of 10 or tobramycin or moxifloxacin at 2h post infection a single dose of moxifloxacin (50 mg/kg) or tobramycin (50 mg/kg) or no drug resulted in 27% or 20% or 27% survival of the larvae at 24 h, respectively mdicating that both antibiotics provide insufficient protection.
  • single dose therapy (50 mg kg) of hybrid 10 resulted in 100% survival after 24 h (Fig. 5B).
  • hybrid 10 strongly synergizes with the fluoroquinolones ciprofloxacin and moxifloxacin against a panel of MDR and XDR P. aeruginosa isolates.
  • ciprofloxacin- susceptible MIC - 1 ⁇ g mL "1
  • CLSI breakpoints were reached for 6/6 ciprofloxacin-resistant, MDR, XDR or PDR P. aeruginosa isolates at 1 ⁇ 4 MIC of hybrid 10 ( ⁇ 2 ⁇ g/mL "1 ).
  • hybrids 10 and 20 showed a > 750-fold and >20-fold reduction in activity when compared to tobramycin (Fig. 7A).
  • hybrid 10 was a 20-fold less potent inhibitor of gyrase A when compared to moxifloxacin while hybrids 20 and 30 displayed equipotency than moxifloxacin (Fig. 7B).
  • aeruginosa PAOl was exposed to subinhibitory (MIC/2) concentrations of moxifloxacin, tobramycin and hybrid 10 during 24 successive subcultures (Fig. 7C).
  • MIC/2 subinhibitory concentrations of moxifloxacin, tobramycin and hybrid 10 during 24 successive subcultures
  • hybrids 20 and 30 displayed weak antibacterial activity.
  • the potent antipseudomonal properties of 10 cannot be rationalized by inhibition of protein translation or topoisomerase IV activity as this hybrid displays the weakest enzyme inhibitory activity against these enzymes among the three hybrids studied, but rather reflect other modes of action. Instead, hybrid 10 exerts its antipseudomonal effects by affecting the outer membrane integrity as supported by the TEM-study, NPN-permeability assay, PI staining and high synergy with outer membrane impermeable antibiotics.
  • hybrid 10 exerts pleiotropic effects by affecting the integrity of both outer- and inner membranes in P. aeruginosa.
  • the effects on the outer membrane result in enhanced cell penetration while the effects on the cytoplasmic membrane result in membrane depolarization that perturb the PMF.
  • the PMF plays a necessary role in excretion of proteins, toxic metabolites and efflux of antibiotics multimodal antibacterial effects of hybrid 10 are expected.
  • hybrid 10 possesses a low likelihood of resistance development when compared to its individual antibiotic components tobramycin and moxifloxacin.
  • the antibacterial activity using the minimal inhibitory concentration (MIC) of TOB-NMP conjugates lOOa-f were evaluated against a panel of clinically relevant pathogens but none of the conjugates demonstrated potent Gram-positive (MIC > 8 ⁇ /mL) or Gram-negative (MIC > 32 activity.
  • MIC minimal inhibitory concentration
  • FIC fractional inhibitory concentration
  • aeruginosa isolates in the presence of conjugates lOOf, 200 and 300 at a fixed concentration ( ⁇ 8 ⁇ g/mL, ⁇ 0.25 x MIC). These results show that all three conjugates lower the MIC of minocycline from 8-256 fold against the 8 selected MDR or XDR P. aeruginosa isolates. Importantly, in 96% of cases, conjugates 100f, 200 and 300 at a concentration of ⁇ 8 ⁇ g/mL ⁇ ⁇ 0.25 X MIC reached minocycline susceptibility (MIC ⁇ 1 ⁇ g/mL) against the 8-selected MDR or XDR P. aeruginosa isolates (Table 5).
  • conjugate 300 causes ⁇ 5% hemolysis of ovine erythrocytes at 1000 ⁇ g/mL and show low cytotoxicity (CC50 > 30 ⁇ ) against cancer cell lines while increased toxicity was noted for conjugate 300.
  • Tolerability studies in G. mellonella using a dosage of 200 mg/kg of lOOf or 200 showed no toxic effects up to 96 hours while a dose of 100 mg/kg of 300 resulted in 100% killing of the larvae after 24 hours.
  • the toxicity of conjugate 300 in the larvae prevented further use of this compound in the insect model.
  • Efficacy studies were performed by infecting the larvae with a lethal dose of (1.0 x 10 5 CFUs) of XDR P.
  • aeruginosa strain #101856 followed by injection of the drug combination 2 h post infection.
  • Monotherapy with a single dose (75 mg/kg) of minocycline or lOOf (75 mg/kg) or NMP (75 mg/kg) resulted in 100% killing of the larvae within 24 hours indicating that monotherapy was not able to provide protection of the larvae.
  • combination therapy (37.5 mg/kg lOOf + 37.5 mg/kg minocycline or 75 mg/kg lOOf + 75 mg/kg minocycline) resulted in 10% or 77% survival of the larvae, respectively, after 24 hours (Figure 9a).
  • efficacy was seen for conjugate 200.
  • single dose combination therapy 75 mg 200 + 75 mg minocycline
  • single dose monotherapy with minocycline 75 mg/kg
  • conjugate 200 75 mg/kg
  • conjugates lOOf permeabilizes the outer membrane of PAOl in a dose-dependent manner using the NPN (1-N-phenylnapthylamine) assay ( Figure 9b) 18 . Similar dose-dependent permeability was seen for conjugates 200 and 300. Next, we assessed whether the combination of conjugates lOOf, 200 or 300 with outer membrane impermeable antibiotics are synergistic in P. aeruginosa PAOl .
  • Mitomycin C is an antitumor antibiotic which crosslinks DNA. As seen in Table 7 we observed strong synergy of hybrids le and 10 with Mitomycin C. Furthermore, we also could demonstrate this effect in vivo using the Galleria mellonella infection model (Figure 11).
  • E.coli CAN-ICU 61714 (GEN-R) 4 64 64 ⁇ 0.25 8
  • Table 3B Effect of fixed concentration of ciprofloxacin ( ⁇ 4 ⁇ g mL “1 ; ⁇ 1/4 MIC) or moxifloxacin ( ⁇ 4 ⁇ mL "1 ; ⁇ 1/4 MIC) on MIC of hybrid 10

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Abstract

Adjuvants which rescue antibiotics against multidrug-resistant (MDR) organisms are a promising combination strategy to overcome bacterial resistance. While the combination of β-lactam β-lactamase inhibitor antibiotic has been successful in restoring antibacterial efficacy the use of adjuvants to restore fluoroquinolone efficacy in MDR pathogens has been challenging. We describe here adjuvants comprising an aminoglycoside connected to a fluoroquinolone or an efflux pump inhibitor which restore antibacterial in vitro activity and enhance in vivo efficacy of fluoroquinolones and tetracycline antibiotics against MDR and extremely-drug resistant (XDR) Pseudomonas aeruginosa.

Description

HYBRID ANTIBIOTICS-BASED ADJUVANTS OVERCOME RESISTANCE IN PSEUDOMONAS
AERUGINOSA
PRIOR APPLICATION INFORMATION
The instant application claims the benefit of US Provisional Patent Application, filed August 14, 2015, serial number 62/205,234, entitled 'HYBRID ANTIBIOTICS -BASED ADJUVANTS OVERCOME RESISTANCE IS PSEUDOMONAS AEURGINOSA AND ENHANCE FLUOROQUINOLONE EFFICACY" and US Provisional Patent Application 62/329,274, filed April 29, 2016, entitled "HYBRID ANTIBIOTIC OVERCOMES RESISTANCE IN P. AERUGINOSA BY ENHANCING OUTER MEMBRANE PENETRATION AND REDUCING EFFLUX", the contents of both of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
Pseudomonas aeruginosa is the leading cause of nosocomial infections and chronic lung infections in cystic fibrosis patients with mortality rates ranging from 30-50%''2. Among Gram- negative pathogens, infections caused by P. aeruginosa are particularly difficult to treat as the organism is both intrinsically resistant and capable of acquiring resistance to most antibiotics3. This is in large part the result of the low permeability of its outer membrane which is 12-100 times lower than that of E. coli and is caused by selective porins4. The reduced uptake of antibiotic across the outer membrane in P. aeruginosa enables secondary adaptive resistance mechanisms to work more efficiently including efflux due to the intrinsic or induced expression of efflux pumps and antibiotic- modifying enzymes3. Clinically useful anti-pseudomonal antibiotics are limited to select penicillins (eg. piperacillin tazobactam), cephalosporins (eg. ceftazidime), carbapenems (eg. imipenem), fluoroquinolones (eg. ciprofloxacin), aminoglycosides (eg. tobramycin) and colistin but resistance to these agents is steadily increasing with few novel anti-pseudomonal agents in clinical development4. Among the aminoglycosides and fluoroquinolones tobramycin and ciprofloxacin are the most potent agents in the respective classes against P. aeruginosa. Both tobramycin and ciprofloxacin possess multiple modes of action and distinct uptake mechanisms. Tobramycin's mode of action is concentration dependent. At low-concentrations (< 4 μg/mL), tobramycin binds to the 30S ribosomal subunit, thereby leading to the disruption of protein synthesis while at higher concentrations (> 8 disruption of the outer membrane is observed5. In contrast, the antimicrobial action of the fluoroquinolones is mediated through the inhibition of two type II DNA topoisomerase enzymes, DNA gyrase and topoisomerase IV which play an essential role in DNA relaxation, partitioning replicated chromosomal DNA during cell division and decatenation reactions6. Besides their distinct modes of action, tobramycin and ciprofloxacin also display different uptake mechanisms. The uptake of aminoglycosides into Gram-negative bacteria involves interactions at sites at which divalent cations cross-bridge adjacent polyanionic lipopolysaccharide (LPS) that causes destabilization of the outer membrane and results in self-promoted uptake of the antibiotic or other extracellular molecules7 In contrast, uptake of fluoroquinolones in Gram-negative bacteria does not occur by self-promoted uptake, but rather involves diffusion across the cell membrane with uptake through the porin pathway8.
The distinct uptake pathways of tobramycin and ciprofloxacin provide an opportunity to optimize the transport of fluoroquinolone antibiotics across the outer membrane. We hypothesized that appending a tobramycin-based vector to ciprofloxacin will enable the fluoroquinolone (such as ciprofloxacin or moxifloxacin) to penetrate the outer membrane of P. aeruginosa via self-promoted uptake. Ideally this approach should be combined with original uptake mechanisms of ciprofloxacin to maximize penetration across the outer membrane.
Two strategies, multicomponent antibiotic adjuvants26*27,28 and single component-based antibacterial polypharmacology28 are currently investigated to combat bacterial resistance. Both strategies exploit multiple modes of action. There is strong evidence that antibacterials need to interact with multiple targets and induce pleiotropic effects on the bacterial cell in order to be successful antibiotics as observed for β-lactams, fluoroquinolones and aminoglycosides28,29. Multitargeting antibiotics are expected to limit the frequency of spontaneous resistance that can arise from mutation in the target gene29. One approach to design multitargeting antibacterials involves the covalent attachment of two different pharmacophores that inhibit dissimilar targets in the bacterial cell generating hybrid antibiotics14. Over the past 40 years more than 25 hybrid antibiotics containing fluoroquinolone-, aminoquinolone-, vancomycin-, rifamycin-, oxazolidinone, β-lactam and aminoglycoside-moieties have been described. Despite their composition of at least one pharmacophore entailing excellent inherent Gram-negative activity, all clinically-evaluated hybrid antibiotics are devoid of potent Gram-negative activity. The generally observed poor Gram-negative activity of hybrid antibiotics against Gram-negative bacteria and P. aeruginosa in particular has been attributed to reduced penetration of the outer membrane due to increased molecular weight11,29'30.
One other approach is the use of small molecule-based adjuvants capable of overcoming (intrinsic) resistance in P. aeruginosa26,27,35. Examples of antibacterial adjuvants include β-lactamase inhibitors which prevent inactivation of β-lactam antibiotics, membrane permeabilizers which destabilize the outer membrane in bacteria and efflux pump inhibitors (EPIs). So far only β-lactamase inhibitors have been approved as adjuvants for clinical use34. EPIs block the function of efflux pumps in Gram-negative bacteria by competing with the antibiotic binding site or by perturbation of the outer membrane channel or assembly of the tripartite protein complex of Resistance-Nodulation-Division (RND) pumps36. Alternatively, perturbation or disruption of the proton motive force (PMF) which energize efflux pumps can also be used to block the function of RND pumps36,37. Several EPIs have been described38 including 1 -(1 -naphthylmethyl)- piperazine (NMP)39, paroxetine (PAR)38,40 and dibasic peptides like DBP41 (Figure 8) and for some, efficacy has been established in animal models of infection42. NMP is a broad spectrum EPI which synergizes with multiple classes of antibiotics including tetracyclines, fluoroquinolones, macrolides, penicillins and rifampicin against certain clinically relevant Gram-negative pathogens like E. coli, Acinetobacter baumannii and Klebsiella pneumoniae but not P. aeruginosa34'*1 '. PAR potentiates tetracyclines and fluoroquinolones in Gram-positive and Gram-negative bacteria excluding P. aeruginosa40. Although, EPIs such as DBP, NMP and PAR inhibit efflux of tetracycline and fluoroquinolone antibiotics in certain Gram-negative bacteria they are still subject to intrinsic resistance in organisms like P. aeruginosa.
SUMMARY OF THE INVENTION
According to an aspect of the invention, there is provided a compound comprising an aminoglycoside connected to a fluoroquinolone or an efflux pump inhibitor.
According to a further aspect of the invention, there is provided a method of treating a bacterial infection in an individual in need of such treatment comprising administering to said individual an effective amount of a compound comprising an aminoglycoside connected to a fluoroquinolone or an efflux pump inhibitor.
According to yet another aspect of the invention, there is provided a method of enhancing the anti-bacterial activity of an antibacterial compound comprising co-administering said antibacterial compound with a second compound comprising an aminoglycoside connected to a fluoroquinolone or efflux pump inhibitor.
According to a still further aspect of the invention, there is provided use of a compound comprising an aminoglycoside connected to a fluoroquinolone as discussed above for enhancing the antibacterial activity of an agent.
According to another aspect of the invention, there is provided a method of preparing a medicament for treatment of a bacterial infection comprising admixing a compound comprising an aminoglycoside connected to a fluoroquinolone or efflux pump inhibitor and a suitable excipient.
According to a yet further aspect of the invention, there is provided a method of determining if an agent of interest has antibacterial activity comprising:
co-administering the agent of interest with a compound comprising an aminoglycoside connected to a fluoroquinolone or efflux pump inhibitor to a bacterium of interest; and
determining survival of the bacterium.
According to another aspect of the invention, there is provided a method of determining the effect of a compound comprising an aminoglycoside connected to a fluoroquinolone or an efflux pump inhibitor on the antibacterial activity of an agent of interest comprising:
co-administering the agent of interest with the compound to a bacterium of interest; and determining survival of the bacterium,
According to a yet further aspect of the invention, there is provided a method of determining if an agent of interest has antibacterial activity comprising:
attaching the agent of interest to an aminoglycoside;
administering the attached agent of interest and aminoglycoside to a bacterium; and determining survival of the bacterium.
According to another aspect of the invention, there is provided a method of potentiating a tetracycline antibiotic or mitomycin C comprising coadministering the tetracycline antibiotic or mitomycin C with a compound comprising tobramycin connected to a ciprofloxacin or moxifloxacin or an efflux pump inhibitor.
According to a still further aspect of the invention, there is provided use of a compound comprising tobramycin connected to a ciprofloxacin or moxifloxacin or an efflux pump inhibitor, for potentiating a tetracycline antibiotic or mitomycin C.
DESCRIPTION OF THE FIGURES
Figure 1. Structure of tobramycin-ciprofloxacin hybrids. Compounds la-le are hybrids with a tether attached to the C-5 position of tobramycin while compounds 2 and 3 contain the tether at the C-2" or C-6"} respectively. Compounds 4 and 5 are fragments of lead structure le.
Figure 2. Hybrid antibiotics synergize with fluoroquinolone antibiotics in P. aeruginosa in vitro and in vivo, (a) Potentiation of moxifloxacin by different compounds in P. aeruginosa PAOl.
FIC index were governed by the aliphatic chain leanth that connects tobramycin and ciprofloxacin among hybrids. High FIC indices of 4 and 5 showcase the importance of dual antibiotic role in the hybrid molecule (b) Potentiation of ciprofloxacin or moxifloxacin by le in MDR and XDR clinical isolates of P. aeruginosa strains as shown by the FIC index, (c) Enhanced Efficacy of combination of le with moxifloxacin in in vivo was demonstrated in Galleria mellonella infection model in a dose- dependent fashion. Monotherapy with moxifloxacin (50 mg kg-1) and le (50 mg kg-1 ) resulted in 100% death of the larvae after 24 h; combination of both moxifloxacin and hybrid l e under the similar dosage conditions induced 100% survival of the larvae after 24 hours (d) Long-term survival of combination therapy consisting of various dosages of moxifloxacin and hybrid le.
Figure 3. Mechanism of synergy for hybrid le. (a) Permeabilization of outer membrane by hybrid compound le was measured by accumulation of 1 -N-phenylnaphthylamine (NPN) in PAOl cells. Permeabilization caused by l e is a concentration-dependant effect 50 μg mL (blue), 16 μg mL (tan), 8 μg mL (purple). Colistin 5 μg/mL (black) was used as positive control; NPN was added at 120 seconds (b) Inhibition of in vitro protein translation using E. coli S30 extract by tobramycin, le, 2 and 3. Protein translation assays were performed as described in biological methods (supporting information, 3.9). The % luminiscence was calculated compared to no antibiotic control (assumed to be 100%). Each data point represents an average luciferase activity of the triplicates with +/- percentage error. The IC50 values (μΜ) tobramycin (0.0062 ± 0.0002), le (8.05 ± 0.57), 2 (0.97 ± 0.05) and 3 (3.44 ± 0.09) were obtained as described in biological methods, (c) Inhibition of P. aeruginosa DNA gyrase A activity in the presence of ciprofloxacin, le, 2 and 3. Inhibition of DNA gyrase is plotted against concentrations of the compounds and each data point represents an average value of band (supercoiled) intensities from two independent sets of reactions (supporting information, appendix 1). The percentage of supercoiled DNA was measured compared to the no antibiotic control (assumed to be 100%). The IC50 values (μΜ) of ciprofloxacin (2.4 ± 0.08), le (0.5 ± 0.01 ), 2 (0.7 ± 0.03) and 3 (0.8 ± 0.02) were obtained as discussed in biological methods section (supporting information 3.10). (d) Synergistic effects of l e with various classes of antibiotics using FIC index.
Figure 4. Overview of tobramycin-moxifloxacin hybrids used in this study. Hybrids differ in the linkage to tobramycin. In hybrid 10 moxifloxacin is linked via a C12-tether to the C-5 position of tobramycin while hybrids 20 and 30 are linked to the C-2" and C-6"-positions in tobramycin.
Figure 5. Effects of hybrid 10 on bacterial killing and in vivo efficacy. (A) Time-kill curves showing the effect of varying concentrations of hybrid 10 and MIC concentration of colistin (Col) on the viability of P. aeruginosa PAOl cells grown in MH broth. No colony-forming units (CFU) were found with 10 at 2 x MIC (blue) after 24 hours. Experiment was performed three times independently and each data point is an average of three determinations ± SEM. (B) Enhanced dose dependent efficacy of hybrid 10 in comparison with tobramycin (TOB) and moxifloxacin (MOX) in XDR P. aeruginosa (#104354) over a period of 24 h was demonstrated in Galleria mellonella in vivo infection model. Larvae infected with #104354 (106 CFU/mL) resulted in 73% killing after 24 h. In contrast, single dosage monotherapy (50 mg kg) of moxifloxacin, or tobramycin or hybrid 10 resulted in 27%, 20% or 100% survival of the larvae, respectively, after 24 h. No killing was observed due to physical trauma when larvae were injected with equal volumes of PBS. Two independent experiments were conducted for each dosage of antibiotic/hybrid, where each experiment involved 15 worms (n = 30). Significant differences between 0 and 24 h are indicated by * (p value < 0.05).
Figure 6. (A) Effects of hybrid 10 on the outer membrane. Concentration-dependent permeabilization of the outer membrane by tobramycin-moxifloxacin hybrid 10 is indicated by the accumulation of 1 -N-phenylnaphthylamine (NPN) in P. aeruginosa PAOl cells. 50 μg mL (medium plum), 32 μg mL (cyan), 16 μg mL (gold), or 8 μg/mL (pale lavender) of hybrid 10 were used, along with 5 μg/mL colistin (Col) (pale violet red) as positive control. Hybrid 10 was added at 120 seconds. Experiment was performed in triplicate and each data point is an average of three determinations ± SEM. (B) Synergistic FIC index of hybrid 10 in combination with various classes of antibiotics against PAOl.
Figure 7. Protein translation activity, gyrase A activity and emergence of resistance. (A) Inhibition of in vitro protein translation in E. coli S30 extract by tobramycin, moxifloxacin, hybrid 10 and 20. Protein translation assays were performed as described in methods section. The percentage luminescence was calculated compared to no antibiotic control (assumed to be 100%). Each data point represents an average luciferase activity of the triplicates with ± percentage error. The IC50 values (μΜ) for tobramycin (0.0062 ± 0.0002), 10 (4.7 ± 0.21), and 20 (0.13 ± 0.003) were obtained. (B) Inhibition of P. aeruginosa DNA gyrase A activity in the presence of moxifloxacin, hybrid 10, 20 or 30. Each data point represents an average value of band (supercoiled) intensities from two independent sets of reactions (see Appendix in the supporting information). The percentage of supercoiled DNA was measured compared to the no-antibiotic control (assumed to be 100%). IC50 values (μΜ) for moxifloxacin (2.6 ± 0.02), 10 (54.7 ± 2.9), 20 (2.1 ± 0.08) and 30 (2.6 ± 0.14) were calculated. (C) Emergence of resistance studies in P. aeruginosa after 25 serial passages in the presence of tobramycin, moxifloxacin and hybrid 10: Col = colistin, TOB = tobramycin, MOX - moxifloxacin.
Figure 8. Structures of the efflux pump inhibitors (EPIs) NMP, PAR and DBP and tobramycin (TOBHinked EPI conjugates TOB -NMP (lOOa-f), TOB-PAR (200) and TOB-DBP (300).
Figure 9. (a) Enhanced dose-dependent efficacy of a combination of conjugate lOOf and minocycline in XDR P. aeruginosa #101856 over a period of 24 h was demonstrated in a Galleria mellonella in vivo infection model. Combination therapy (37.5 mg/kg of lOOf + 37.5 mg/kg of minocycline) or (75 mg/kg of lOOf + 75 mg kg of minocycline) resulted in 10% or 77% survival of the larvae, respectively after 24 h. In contrast, monotherapy using a single dosage of lOOf (75 mg/kg), minocycline (75 mg/kg), NMP (75 mg/kg) or no treatment resulted in 100% killing of the larvae at < 24 h. Each experiment involved usage of 15 larvae from different batches; in total 2 experiments were performed per one dosage of antibiotic/hybrid (n = 30). Significant difference between 0 and 24 h indicated by * (P value < 0.05). (b) Permeabilization of outer membrane by conjugate lOOf was measured by accumulation of 1-N-phenylnaphthylamine (NPN) in PAOl cells. Permeabilization caused by lOOf is a concentration-dependent effect. Triton X 100 (1%) and EDTA (10 mM) were used as positive controls, (c) Synergistic effects of conjugates lOOf (TOB- MP), 200 (TOB-PAR) and 300 (TOB-DBP) in combination with outer membrane impermeable antibiotics (novobiocin, rifampicin, vancomycin, erythromycin, chloramphenicol and trimethoprim) in P. aeruginosa PAOl .
Figure 10. Single-dose therapy of minocycline (ΜΓΝΟ) and le combination in Galleria mellonella infection model ; ΜΓΝΟ = Minocycline; CIP = Ciprofloxacin; TOB = Tobramycin using a XDR P. aeruginosa strain PA262
Figure 1 1. In- vivo studies: Synergy demonstration between mitomycin C (MYT-C) and adjuvant 10 in Galleria mellonella infection model infected with XDR P. aeruginosa strain # 262. Mitomycin C (10 mg/kg) provided low level of protection (< 20%) while combination therapy of adjuvant 10 (10 mg kg) and Mitomycin C (10 mg/kg) produced 80% survival after 24 hours.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference.
Adjuvants which rescue antibiotics against multidrug-resistant (MDR) organisms are a promising combination strategy to overcome bacterial resistance. While the combination of -lactam/ - lactamase inhibitor antibiotic has been successful in restoring antibacterial efficacy the use of adjuvants to restore fluoroquinolone efficacy in MDR pathogens has been challenging. We describe here adjuvants which restore antibacterial in vitro activity and enhance in vivo efficacy of fluoroquinolones against MDR and extremely-drug resistant (XDR) Pseudomonas aeruginosa.
Moreover, we demonstrate that the potent antipseudomonal properties of the hybrid can be synergistically potentiated with other classes of antibiotics including fluoroquinolones thereby lowering the absolute minimal inhibitory concentration (MIC) of the hybrid to < 1 μg/nϊL"1 against a panel of
MDR, XDR isolates from Canadian hospitals while at the same time reaching susceptible CLSI breakpoints for fluoroquinolone antibiotics. Furthermore, to reduce intrinsic resistance, we decided on a strategy that utilizes a tobramycin (TOB) vector to deliver the EPI through the outer membrane of P. aeruginosa. It was anticipated that joining an amphiphilic TOB-based vector to an EPI would facilitate penetration through the outer membrane of P. aeruginosa by the self-promoted uptake of aminoglycosides44 and amphiphilic aminoglycosides10'31'45'46. In addition, the resultant TOB-EPI conjugate was expected to reduce efflux as aminoglycosides are considered to be poor substrates for most efflux pumps in P. aeruginosa11 except the MexXY-OprM pump47.
The discovery that linking an amphiphilic tobramycin vector to MP, PAR or DBP generates adjuvants which reduce the intrinsic resistance barrier in P. aeruginosa and restore antibacterial activity and efficacy in minocycline against MDR and XDR P. aeruginosa isolates, opens up opportunities to develop novel combination therapies against one of the most challenging pathogens. Moreover, as efflux pumps are ubiquitous in Gram-negative bacteria and present a major challenge to drug development for all antibiotics, our study suggests that TOB-EPI conjugates may also enhance the efficacy of other classes of antibiotics against MDR P. aeruginosa pathogens but also against other intrinsically resistant Gram-negative pathogens.
Specifically, in one aspect of the invention, there is provided a compound comprising an aminoglycoside connected to a fluoroquinolone or efflux pump inhibitor.
As will be appreciated by one of skill in the art, any suitable tether or linker known in the art may be used to connect the fluoroquinolone and the aminoglycoside provided that the tether is sufficiently flexible and sufficiently long that the aminoglycoside and the fluoroquinolone are able to function as discussed below. However, if the tether gets too long, some of the activity of the compound may be lost.
For example, the tether or linker may be approximately 2-24 carbons long. As will be appreciated by one of skill in the art, this does not mean that the tether or linker necessarily needs to include 2-24 carbon molecules, merely that the tether or linker must be of the same approximate length as 2-24 carbon molecules.
In some embodiments, the tether is a flexible tether. The tether may be selected from the group consisting of but by no means limited to a flexible alkyl linker, an unsaturated alkyl linker, an ethylene glycol linker or an aromatic linker. For example, the tether may comprise a length of 2-24, 3-24, 4-24, 5-24, 6-24, 2-23, 2-22, 2-21, 2-20, 2-19, 2-18, 3-23, 3-22, 3-21, 3-20, 3-19, 3-18, 4-23, 4-22, 4-21, 4- 20, 4-19, 4-18, 5-23, 5-22, 5-21, 5-20, 5-19, 5-18, 6-23, 6-22, 6-21, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12 carbon atoms.
In some embodiments, the aminoglycoside is tobramycin. In some embodiments, the fluoroquinolone is ciprofloxacin or moxifloxacin.
In yet other embodiments, the compound is tobramycin connected to ciprofloxacin or moxifloxacin or an efflux pump inhibitor.
In some embodiments, the efflux pump inhibitor is selected from the group consisting of NMP, PAR and DBP.
In some embodiments, the tobramycin is tethered to the ciprofloxacin or moxifloxacin or efflux pump inhibitor.
As will be appreciated by one of skill in the art, any suitable tether or linker known in the art may be used to connect the tobramycin and the ciprofloxacin provided that the tether is sufficiently flexible and sufficiently long that the tobramycin and the ciprofloxacin, moxifloxacin or efflux pump inhibitor are able to function as discussed herein.
In some embodiments, the tether is a flexible tether, as shown in Figure 1. For example, the tether may comprise 4-12 repeating alkyl units.
As discussed herein, the tether may be connected to any suitable position on tobramycin, for example, a hydroxyl group. The hydroxyl group may be for example, at the C-5 position, the C-4' position, the C-4" position, the C-2" position or the C- 6" position on tobramycin.
In some embodiments, the preferred attachment site for ciprofloxacin is the secondary amino group of the piperazine ring in ciprofloxacin as other attachment at other positions may interfere with the mode of action of ciprofloxacin.
According to another aspect of the invention, there is provided a method of treating a bacterial infection in an individual in need of such treatment comprising administering to said individual an effective amount of a compound comprising an aminoglycoside connected to a fluoroquinolone or efflux pump inhibitor.
In some embodiments, the aminoglycoside is connected to the fluoroquinolone via a tether, as discussed above.
In some embodiments, the aminoglycoside is tobramycin.
In some embodiments, the fluoroquinolone is ciprofloxacin or moxifloxacin.
In some embodiments, the efflux pump inhibitor is selected from the group consisting of NMP, PAR and DBP.
In yet other embodiments, the compound is tobramycin connected to ciprofloxacin or moxifloxacin or the efflux pump inhibitor, as discussed above.
In some embodiments, the tobramycin is tethered to the ciprofloxacin, moxifloxacin or efflux pump inhibitor. As will be appreciated by one of skill in the art, any suitable tether or linker known in the art may be used to connect the tobramycin and the ciprofloxacin, moxifloxacin or efflux pump inhibitor provided that the tether is sufficiently flexible and sufficiently long that the tobramycin and the ciprofloxacin, moxifloxacin or efflux pump inhibitor are able to function as discussed below.
In another aspect of the invention, there is provided a method of deterrnining if an agent of interest has antibacterial activity comprising:
attaching the agent of interest to an aminoglycoside;
administering the attached agent of interest and aminoglycoside to a bacterium; and determining survival of the bacterium.
In some embodiments, the agent of interest is attached to the aminoglycoside by a tether.
The tether may be a flexible tether. The tether may be selected from the group consisting of but by no means limited to a flexible alkyl linker, an unsaturated alkyl linker, an ethylene glycol linker or an aromatic linker. For example, the tether may comprise a length of 2-24, 3-24, 4-24, 5-24, 6-24, 2-23, 2-22, 2-21, 2-20, 2-19, 2-18, 3-23, 3-22, 3-21, 3-20, 3-19, 3-18, 4-23, 4-22, 4-21, 4-20, 4-19, 4-18, 5- 23, 5-22, 5-21, 5-20, 5-19, 5-18, 6-23, 6-22, 6-21, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12 carbon atoms.
In some embodiments, the aminoglycoside is tobramycin.
In some embodiments, the tether is a flexible tether, as shown in Figure 1. For example, the tether may comprise 4-12 repeating alkyl units.
As discussed herein, the tether may be connected to any suitable position on tobramycin, for example, a hydroxyl group. The hydroxyl group may be for example, at the C-5 position, the C-4' position, the C-4" position, the C-2" position or the C-6" position of the tobramycin.
In some embodiments, the bacterial infection is caused by a pathogenic gram-negative bacteria. The pathogenic gram-negative bacteria may be for example but by no means limited to E. coli, Acetinobacter, Salmonella, Shigella, Enterobacteriaceae, Pseudomonas, Moraxella, Heliobacter, Stenotrophomonas, Bdellovibrio, Legionella and the like. In some embodiments, the pathogenic gram- negative bacteria is P. aeruginosa, E. coli or Acetinobacter baumannii. In some embodiments, the P. aeruginosa strain is an extremely drug-resistant strain of P. aeruginosa.
As will be appreciated by one of skill in the art, an "effective amount" is an amount that is sufficient to accomplish at least one of the following: reduction in the severity of the symptoms associated with the bacterial infection; and reduction in the CFU per ml of bacteria in a sample taken from the individual. As discussed herein, this may be accomplished by increasing or enhancing the anti-bacterial activity of another compound. For example, while not wishing to be bound to a particular theory or hypothesis, the inventors believe that the compounds described herein enhance the permeability of the gram-negative outer membrane and thereby in effect act as a delivery system. As such, in these embodiments, an "effective amount" is an amount that is sufficient to enhance the permeability of the outer membrane of the infective bacteria so that an antibacterial agent can enter the bacterial cell. As will be appreciated by one of skill in the art, this means that a wide variety of compounds that have bacteria targets but cannot cross the outer membrane on their own can now be considered to be anti-bacterial agents when used in combination with the compounds of the invention.
Other methods for determining an effective amount of the compound to be administered to the individual will be apparent to one of skill in the art and may depend on many other factors, for example, the age and general condition of the individual and the severity of the infection and/or symptoms associated with the infection.
As discussed herein, the compound may be co-administered with a second antibiotic.
As shown herein, synergy is achieved on coadministration of the compound with a variety of antibiotics and antibacterial agents, including but by no means limited to fluoroquinolones, beta- lactams (penicillins, cephalosporins (ceftazidimes and the like), carbapenems, monobactams), rifampicin, vancomycin, trimethoprim, trimethoprim plus sulfamethoxazole, colistin, polymyxin, chloramphenicol, novobiocin, and macrolides (erythromycin and the like).
Accordingly, given the variety of different agents for which synergy has been established, it is believed to be a sound prediction that the compound will act as an adjuvant or will otherwise enhance the activity of an even wider variety of antibiotic agents known in the art, for example, but by no means limited to antimicrobial peptides, quinolone antibiotics such as fluoroquinolones (ciprofloxacin, moxifloxacin, levofloxacin and the like), clindamycin, streptogramins, sulfonamides and monobactams.
In another embodiment of the invention, there is provided a method of enhancing the antibacterial activity of an antibacterial compound comprising co-administering said antibacterial compound with a second compound comprising an aminoglycoside connected to a fluoroquinolone or efflux pump inhibitor as discussed above.
In some embodiments, the aminoglycoside is connected to the fluoroquinolone via a tether as discussed above.
In some embodiments, the aminoglycoside is tobramycin.
In some embodiments, the fluoroquinolone is ciprofloxacin or moxifloxacin.
In some embodiments, the efflux pump inhibitor is selected from the group consisting of MP, PAR and DBP.
In yet other embodiments, the compound is tobramycin connected to ciprofloxacin, moxifloxacin or an efflux pump inhibitor.
In some embodiments, the tobramycin is tethered to the ciprofloxacin as discussed above.
As will be appreciated by one of skill in the art, any suitable tether or linker known in the art may be used to connect the tobramycin and the ciprofloxacin, moxifloxacin and efflux pump inhibitor provided that the tether is sufficiently flexible and sufficiently long that the tobramycin and the ciprofloxacin, moxifloxacin or efflux pump inhibitor are able to function as discussed herein.
Preferably, an effective amount of the second compound is co-administered with the antibiotic compound.
As will be appreciated by one of skill in the art, in these embodiments, an "effective amount" is an amount that is sufficient to at least increase the effectiveness of the anti-bacterial compound, for example, such that less of the first compound is required or the time between administrations is increased or the severity of the bacterial infection is reduced to a greater extent than on administration of the anti-bacterial compound alone. Other methods for detenrdning an effective amount of the compound to be administered to the individual will be apparent to one of skill in the art and may depend on many other factors, for example, the age and general condition of the individual and the severity of the infection and/or symptoms associated with the infection.
In other embodiments, there is provided a compound comprising tobramycin connected to a ciprofloxacin or moxifloxacin or an efflux pump inhibitor to potentiate tetracycline antibiotics or mitomycin C.
In some embodiments, the efflux pump inhibitor is selected from the group consisting of NMP,
PAR and DBP.
It is of note that tetracycline antibiotics are well known to those of skill in the art and as such suitable tetracycline antibiotics that can be used in this aspect of the invention will be well known to those of skill in the art. For example, the tetracycline antibiotic may be for example but by no means limited to minocycline, tigecycline, doxycycline and the like.
As will be appreciated by one of skill in the art, any suitable tether or linker known in the art may be used to connect tobramycin to the ciprofloxacin, moxifloxacin or efflux pump inhibitor provided that the tether is sufficiently flexible and sufficiently long that tobramycin and the ciprofloxacin or moxifloxacin or an efflux pump inhibitor are able to function as discussed below. However, if the tether gets too long, some of the activity of the compound may be lost.
For example, the tether or linker may be approximately 2-24 carbons long. As will be appreciated by one of skill in the art, this does not mean that the tether or linker necessarily needs to include 2-24 carbon molecules, merely that the tether or linker must be of the same approximate length as 2-24 carbon molecules.
In some embodiments, the tether is a flexible tether. The tether may be selected from the group consisting of but by no means limited to a flexible alkyl linker, an unsaturated alkyl linker, an ethylene glycol linker or an aromatic linker. For example, the tether may comprise a length of 2-24, 3-24, 4-24, 5-24, 6-24, 2-23, 2-22, 2-21, 2-20, 2-19, 2-18, 3-23, 3-22, 3-21, 3-20, 3-19, 3-18, 4-23, 4-22, 4-21, 4- 20, 4-19, 4-18, 5-23, 5-22, 5-21, 5-20, 5-19, 5-18, 6-23, 6-22, 6-21, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12 carbon atoms.
In some embodiments, the tether may comprise 4-12 repeating alkyl units.
As discussed herein, the tether may be connected to any suitable position on tobramycin, for example, a hydroxyl group. The hydroxyl group may be for example, at the C-5 position, the C-4' position, the C-4" position, the C-2' ' position or the C-6" position on tobramycin. In another embodiment of the invention, there is provided use of a compound comprising an aminoglycoside connected to a fluoroquinolone as discussed above for enhancing the antibacterial activity of an agent. As discussed herein, it is important to note that the agent does not necessarily have antibacterial properties on its own and may need to be co-administered with the compound in order to enter the bacterial cell. Alternatively, the agent may have antibacterial properties on its own but these antibacterial properties may be enhanced by increased entry of the agent into the bacterial cell by the membrane permeability enhancing activity of the agent.
According to another aspect of the invention, there is provided a method of preparing a medicament for treatment of a bacterial infection comprising admixing a compound comprising an aminoglycoside connected to a fluoroquinolone or an efflux pump inhibitor and a suitable excipient. The compound may be formulated for co-administration with an agent having anti-bacterial properties either alone or when administered in combination with the compound, as discussed herein.
In some embodiments, the aminoglycoside is tobramycin.
In some embodiments, the fluoroquinolone is ciprofloxacin or moxifloxacin, as discussed herein.
In some embodiments, the efflux pump inhibitor is selected from the group consisting of NMP, PAR andDBP.
As discussed herein, the compounds described herein, for example, the tobramycin- ciprofloxacin and tobramycin-EPI hybrids, serve as adjuvants and can potentiate other classes of antibiotics and antibacterial agents.
As will be appreciated by one of skill in the art, this includes antibacterial agents which may not have antibacterial activity on their own due to an inability to cross the outer membrane of the bacterial target. For instance, many known and unknown compounds can interact with validated antibacterial targets such as for example but by no means limited to enzymes, RNA, DNA, proteins, and the like, but possess no or poor antibacterial activity because they cannot cross the bacterial membranes in gram-negative organisms. As shown in Figure 3a, one function of the tobramycin- ciprofloxacin hybrids is to enhance the permeability of the outer membrane in P. aeruginosa. This facilitates cell penetration and may convert non antibacterial agents into antibacterial ones. As such the adjuvants would serve as a delivery system.
According to another aspect of the invention, there is provided a method of determining if an agent of interest has antibacterial activity comprising: co-adniinistering the agent of interest with a com ound comprising an aminoglycoside connected to a fluoroquinolone or efflux pump inhibitor to a bacterium of interest; and
determining survival or growth inhibition of the bacterium.
In some embodiments, the aminoglycoside is tobramycin and the fluoroquinolone is ciprofloxacin, as discussed herein.
As will be appreciated by one of skill in the art and as discussed herein, the agent of interest may be any compound thought, proposed or hypothesized to have an antibacterial effect once inside the cell of a gram-negative bacterium.
As discussed above, any suitable gram-negative bacterium may be used for the screening process.
Furthermore, the compound and the agent of interest may be administered to a population or culture of gram-negative bacterial cells and survival may be determined by comparing survival of bacteria treated with the agent of interest and the compound to survival of bacteria treated only with the agent of interest.
According to another aspect of the invention, there is provided a method of determining the effect of a compound comprising an aminoglycoside connected to a fluoroquinolone or efflux pump inhibitor on the antibacterial activity of an agent of interest comprising:
co-administering the agent of interest with the compound to a bacterium of interest; and deterrnining survival of the bacterium.
As will be appreciated by one of skill in the art, in these embodiments, the effect of the compounds of the invention on any agent, whether it has antibacterial properties, that is, bactericidal (killing of bacteria) or bacteriostatic (inhibition of growth) on its own or not, can be determined.
To probe this hypothesis we prepared tobramycin-ciprofloxacin hybrids la, lb, lc, Id and le in which the two drugs are conjoined by a flexible alkyl tether (Figure 1). The design of tobramycin-ciprofloxacin hybrids was guided by previous structure activity studies. The C-5 position of tobramycin was selected for attachment of the tether as this position retains antibacterial activity in Gram-negative bacteria9. The secondary amino group of the piperazine ring in ciprofloxacin was selected as the point of attachment because it does not interfere with gyrase A and topoisomerase IV activity in E. coli and enhances Gram-negative uptake6'11. The hybrids were tested for antibacterial activity by determining the minimal inhibitory concentration (MIC) against select Gram-negative and Gram-positive bacteria4. All hybrids displayed weak antibacterial activity (MIC > 16 μg/mL) with the exception of E. coli ATCC 25922. In addition, we noticed that le displayed antibacterial activity (MIC = 4 μg/mL) against a wild-type P. aeruginosa strain.
To assess whether transport of the fluoroquinolone antibiotics proceeds by multiple uptake mechanisms, we determined using P. aeruginosa PAOl whether combinations of hybrids la-le with moxifloxacin (Figure 2a) or levofloxacin were additive or synergistic using the fractional inhibitory concentration (FIC) index12 as a measure of the interaction between two antimicrobial agents. FIC indices of 1 , < 0.5 and > 4 indicate no interaction, synergy and antagonism, respectively13. The results indicate that hybrid le demonstrated optimal synergy (FIC index < 0.077 - < 0.139) on growth inhibition. Synergism was also observed with lb, lc and Id but not with l , tobramycin, ciprofloxacin, hybrid fragment tobramycin-C-12 (3) and hybrid fragment ciprofloxacin-C12 (4).
Hybrid le was selected for further combination studies against a panel of 8 clinical P. aeruginosa isolates including six MD (non-susceptible or resistant to > 3 chemically unrelated anti- pseudomonal classes), and six extremely-drug resistant (XDR) (non-susceptible or resistant to > 5 chemically unrelated anti-pseudomonal classes). The panel also included two colistin-non-susceptible or resistant P. aeruginosa strains. The fluoroquinolone-resistant isolates contained a single mutation (T83-I) in GyrA which was absent in the colistin-non-susceptible or resistant strains. We determined the FIC index of le in combination with moxifloxacin or ciprofloxacin against six colistin-susceptible but XDR P. aeruginosa strains. The results indicated that le displayed strong synergy with FIC indices < 0.03 - < 0.28 against these pathogens (Figure 2b). As a control, combination studies of moxifloxacin or ciprofloxacin with tobramycin, indicated no synergistic effects against XDR P. aeruginosa strains. In the case of the colistin-resistant P. aeruginosa strains we observed additive effects or weaker synergistic effects (FIC index < 0.31 - 0.75) but still observed 16-fold fold potentiation of moxifloxacin and 2- to 8-fold potentiation of ciprofloxacin at ¼ MIC of hybrid le. The reduced synergistic effects or additive effects of le against colistin-resistant P. aeruginosa likely reflects a reduced self-promoted uptake caused by transfer of cationic 4-amino-4-deoxy-l-arabinose to lipid A in these organisms14. Combination studies of moxifloxacin with various membrane-active agents confirmed that the observed synergistic effects of le were not the result of nonspecific membranolytic effect. This is in accordance with the observed low hemolytic activity of le which resulted in < 10% hemolytic activity at 1000 μg mL and low cytotoxic properties against cancer cell lines.
In order to gain insight into the structural requirements responsible for the observed potentiating effect of hybrid le we also prepared two other tobramycin-C12-ciprofioxacin hybrids, the C-2"- and C-6''-tobramycm-linked ciprofloxacin hybrids 2 and 3 (Figure 1). Combination studies on four XDR P. aeruginosa strains confirmed that hybrids 2 and 3 were also able to synergize with moxifloxacin and ciprofloxacin for some XDR strains suggesting that hybrid le displays superior adjuvant properties.
To demonstrate that the synergistic effects of hybrid le on growth inhibition of P. aeruginosa translates into a measurable in vivo effect we selected the Galleria mellonella infection model. Galleria mellonella larvae is an established in vivo model to study the efficacy of antimicrobial monotherapy15'16 and combination therapy against MDR P. aeruginosa17" . We determined (a) the maximal tolerated dose of le by the larvae to be 150 mg/kg and (b) determined the lethal injection dose of P. aeruginosa #101856 CFUs (1.0 x 103) that resulted in 100% killing of the larvae within 24 hours. Efficacy studies (monotherapy) using a dosage of le (50 mg/kg, 75 mg/kg or 100 mg/kg) or moxifloxacin (50 mg/kg, 75 mg kg or 100 mg/kg) resulted in 100% killing of the larvae at < 36 hours indicating that monotherapy was not able to provide protection for the larvae past 36 hours. In contrast, single dosage (100 mg kg) of a 1 :1 mass ratio consisting of moxifloxacin and hybrid le were able to protect 100% for a 24 hour, 60% for a 36 hour, 23% for a 48 hour and 20% of the larvae for a 96 hour period, respectively (Figure 2c). A slightly improved endpoint of 26% survival for a period of 96 hours could be achieved by using a double dosage (additional 100 mg/kg le + 100 mg/kg moxifloxacin after 24 hours) therapy.
To gain an insight into the protective function of le we performed a series of mechanistic studies using P. aeruginosa PAOl. First, we demonstrated that le permeabiUzed the outer membrane of P. aeruginsosa in a dose-dependent manner using the NPN (1-N-phenylnaphtylamine) assay19'20 (Figure 3 a). Whereas, we did not observe significant depolarization of the cytoplasmic membrane as determined by using a depolarization assay21. We also assessed whether the hybrids retain their original modes of action. In the protein translation assay hybrids 2, 3 and le showed 156-fold, 554-fold and 1290-fold reduction in activity when compared to tobramycin (Figure 3b). In contrast hybrids le, 2 and 3 were 3-5 fold better inhibitors of DNA gyrase A (Figure 3c) and 2-3 fold better inhibitors of topoisomerase IV compared to ciprofloxacin.
The increased permeability of the outer membrane in PAOl induced by hybrid le is consistent with the strong synergy with outer membrane impermeable antibiotics including rifampicin (FIC index < 0.04), novobiocin (FIC index 0.06), trimethoprim (FIC index 0.06), erythromycin (FIC index 0.07) and chloramphenicol (FIC index 0.07). In addition, we also were able to observe strong synergistic effects with polar antibiotics including minocycline (FIC index 0.15) and ceftazidime (FIC index 0.18) but also with colistin (FIC index 0.15). In contrast, hybrid le was unable to demonstrate synergy with gentamicin (FIC index 2) and meropenem (FIC index 2) (Figure 3d). Combination studies of le with moxifloxacin against other Gram-negative organisms (E. coli and Acinetobacter baumannii) indicate reduced synergistic effects (FIC index <0.25 - <0.36) when compared to P. aeruginosa while hybrids 2 or 3 showed additive effects.
This study demonstrates that hybrid antibiotics consisting of tobramycin and ciprofloxacin are powerful multimodal adjuvants that can restore antibacterial activity and enhance efficacy with fluoroquinolones in MDR and XDR P. aeruginosa. The strong synergistic effects of le against P. aeruginosa are also observed with other classes of antibiotics except carbapenems and aminoglycosides.
The design of tobramycin-moxifioxacin hybrids 10, 20 and 30 was guided by previous structure activity studies (Fig. 4). Initially, the C-5 hydroxyl position of tobramycin in hybrid 10 was selected for attachment of the tether since this position retains antibacterial activity in Gram-negative bacteria9,10. The 4th generation fluoroquinolone moxifloxacin was selected as second pharmacophore as it possesses (a) potent antipseudomonal activity (MIC = 1 μg mL"1) against wild-type P. aeruginosa PA01; (b) is less prone to efflux than 2nd and 3 rd generation fluoroquinolone antibiotics and (c) interacts as a hydrophobic fluoroquinolone more strongly with the cytoplasmic membrane than ciprofloxacin. The secondary amino function in moxifloxacin was selected for point of modification as alkylation of this function retains potent antibacterial activity against bacterial pathogens32. A C-12 tether length was selected based on previous results obtained with the tobramycin-ciprofloxacin hybrid antibiotics that demonstrated that this tether length is optimal for outer membrane penetration and synergism with other classes of antibiotics in P. aeruginosa11 , We also prepared hybrids 20 and 30 bearing a tether at the C-2"- and C-6 "-positions to study how the nature of the tobramycin linkage affects the antibacterial activity.
The hybrids 10, 20 and 30 were tested for antibacterial activity by determining the minimal inhibitory concentration (MIC) against select Gram-negative and Gram-positive bacteria (Table 1). Among hybrids 10, 20 and 30, hybrid 10 showed consistently the highest antibacterial activity and displayed good activity (MIC = 1 μg mL"1) against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA). Of special consideration is the potent activity of 10 against three P. aeruginosa strains (MIC = 4-8 μ^ηιΐ,) including two gentamicin-resistant P. aeruginosa strains. Interestingly, hybrid 10 (MW = 1217.5) displays comparable antipseudomonal activity against these three P. aeruginosa strains than ciprofloxacin (MW = 367.8) when the changes in molecular weight are factored in. The promising antipseudomonal properties of hybrid 10 provided the rationale to study the antibacterial activity of 10 against a select panel of MDR and XDR P. aeruginosa isolates obtained from multiple Canadian hospitals which are resistant to tetracyclines (doxycycline), cephalosporins (cefepime), carbapenems (meropenem and imipenem), fluoroquinolones (ciprofloxacin and moxifloxacin) and aminoglycosides (tobramycin and gentamicin) but remain susceptible to colistin. We also included two PDR P. aeruginosa strains (#91433 and #101243) resistant to all known antipseudomonal agents including colistin (Table 2). Our results demonstrate that hybrid 10 possesses potent activity (MIC = 1-8 μ g mL) against a panel of 8 MDR, XDR and PDR P. aeruginosa isolates. In comparison, hybrids 20 and 30 in which the linker is attached to a different position in tobramycin are > 8-times less active than hybrid 10 indicating that the positioning of the tether in tobramycin is crucial for potent antibacterial activity. Similar MIC ranges (2 < MIC < 8 μ g/mL) of 10 were also seen against a panel of 12 fluoroquinolone-resistant (ciprofloxacin-resistant and moxifloxacin- resistant) but tobramycin-susceptible MDR P. aeruginosa isolates from multiple Canadian intensive care units indicating potent activity against diverse P. aeruginosa phenotypes (Table 1). After demonstrating that hybrid 10 possesses potent and selective antipseudomonal activity against diverse MDR and XDR P. aeruginosa pathogens, we then studied the killing kinetics of P. aeruginosa PA01 and observed complete eradication of P. aeruginosa PAOl at 2x MIC concentration over a 24 hour time period (Fig. 5A). Using the Galleria melonella model system, we determined that hybrid 10 causes < 2.5% hemolysis of human erythrocytes at 1000 μ g/mL and shows low cytotoxicity CC50 » 30 μ M against cancer cell lines. We assessed the tolerability of 10 in Galleria mellonella and did not see any toxic effects up to the maximal dose of 600 mg/kg over a period of 96 hours. Efficacy studies were performed by infecting the larvae with a dose (106 CFUs) of XDR P. aeruginosa strain #104354 (resistant to all' classes of antipseudomonal agents except colistin) followed by injection of 10 or tobramycin or moxifloxacin at 2h post infection. Therapy with a single dose of moxifloxacin (50 mg/kg) or tobramycin (50 mg/kg) or no drug resulted in 27% or 20% or 27% survival of the larvae at 24 h, respectively mdicating that both antibiotics provide insufficient protection. In contrast, single dose therapy (50 mg kg) of hybrid 10 resulted in 100% survival after 24 h (Fig. 5B). We also observed enhanced long-term survival effects upon treatment with hybrid 10. For instance, treatment of the infected larvae with a single dose (75 mg/kg) of moxifloxacin, or tobramycin or 10 resulted in 7%, 0% or 20% survival after a 96 hour period. Survival rate of the larvae upon therapy with 10 was dose- dependent.
To gain insight into the protective function of 10, we performed a series of mechanistic studies with P. aeruginosa PAOl. First, we demonstrated that 10 permeabilizes the outer membrane of P. aeruginosa in a dose-dependent manner using the NPN (1-N-phenylnapthylamine) assay (Fig. 6A)20. We also assessed whether the combination of 10 with other classes of antibiotics were additive or synergistic by using the fractional inhibitory concentration (FIC) index as a measure of the interaction between two antibacterial agents12. We observed strong synergy of 10 with outer membrane impermeable agents interacting with intracellular targets including novobiocin (FIC index 0.12), rifampicin (FIC index 0.15), vancomycin (FIC index - 0.26) and erythromycin (FIC index = 0.26) indicating that hybrid 10 enhances cellular uptake of these agents into P. aeruginosa. In addition, we also observed strong synergy with other classes of antibiotics including chloramphenicol (FIC index 0.15), minocycline (FIC index 0.15), moxifloxacin (FIC index - 0.18), trimethoprim (FIC index = 0.25), ceftazidime (FIC index = 0.37) and colistin (FIC index = 0.37) but not with meropenem (FIC index = 2), tobramycin (FIC index > 2) and gentamicin (FIC = 5) against P. aeruginosa PAOl (Figure 6B). Importantly, hybrid 10 strongly synergizes with the fluoroquinolones ciprofloxacin and moxifloxacin against a panel of MDR and XDR P. aeruginosa isolates. For instance, ciprofloxacin- susceptible (MIC - 1 μg mL"1) CLSI breakpoints were reached for 6/6 ciprofloxacin-resistant, MDR, XDR or PDR P. aeruginosa isolates at ¼ MIC of hybrid 10 (< 2 μg/mL"1). In comparison, the same susceptible breakpoints were reached for moxifloxacin in 5/6 moxifloxacin-resistant, MDR, XDR or PDR isolates indicating that hybrid 10 strongly synergizes with both clinically used fluoroquinolone antibiotics (Table 3A). At the same time the presence of ciprofloxacin or moxifloxacin at or below their respective ¼ MIC (< 4 μg mL"1) synergizes hybrid 10 to lower its MIC < 1 Dg/mL"1 for the 6 tested MDR, XDR and PDR P. aeruginosa isolates (Table 3B).
Next, we assessed whether the hybrids retain their original modes of action. In a protein translation assay hybrids 10 and 20 showed a > 750-fold and >20-fold reduction in activity when compared to tobramycin (Fig. 7A). Similarly, hybrid 10 was a 20-fold less potent inhibitor of gyrase A when compared to moxifloxacin while hybrids 20 and 30 displayed equipotency than moxifloxacin (Fig. 7B). These studies indicate that the predominant, original antibacterial effects of tobramycin and moxifloxacin are greatly reduced in hybrid 10. Next we studied the potential of hybrid 10 to select for resistance. We used a procedure of selective pressure in which P. aeruginosa PAOl was exposed to subinhibitory (MIC/2) concentrations of moxifloxacin, tobramycin and hybrid 10 during 24 successive subcultures (Fig. 7C). As can be seen from the data, the relative MIC values of tobramycin, moxifloxacin and hybrid 10 increased by 512-, 16- and 2-fold respectively against PAOl indicating that hybrid 10 strongly delays resistance development when compared to moxifloxacin and tobramycin over the 25 day period of this ex eriment.
Furthermore, except for hybrid 10, hybrids 20 and 30 displayed weak antibacterial activity. The potent antipseudomonal properties of 10 cannot be rationalized by inhibition of protein translation or topoisomerase IV activity as this hybrid displays the weakest enzyme inhibitory activity against these enzymes among the three hybrids studied, but rather reflect other modes of action. Instead, hybrid 10 exerts its antipseudomonal effects by affecting the outer membrane integrity as supported by the TEM-study, NPN-permeability assay, PI staining and high synergy with outer membrane impermeable antibiotics. In addition, besides its effect on the outer membrane, hybrid 10 also affects the state of the cytoplasmic membrane by depolarizing the cytoplasmic membrane in a concentration- dependent manner and reducing swimming motility at sub MIC concentration. Combination studies of 10 with the aminoglycoside gentamicin are antagonistic suggesting that hybrid 10 affects the electrical component of the proton motive force in P. aeruginosa.
Our study demonstrates that hybrid 10 exerts pleiotropic effects by affecting the integrity of both outer- and inner membranes in P. aeruginosa. The effects on the outer membrane result in enhanced cell penetration while the effects on the cytoplasmic membrane result in membrane depolarization that perturb the PMF. As the PMF plays a necessary role in excretion of proteins, toxic metabolites and efflux of antibiotics multimodal antibacterial effects of hybrid 10 are expected. Besides displaying potent antipseudomonal properties against MDR, XDR and PDR P. aeruginosa isolates the activity of 10 can be synergized with other classes of antibiotics including fluoroquinolone antibiotics thereby reaching ciprofloxacin-susceptible (MIC = 1 μg mL"1) CLSI breakpoints while at the same time reducing the MIC of 10 to (< mL"1) against MDR, XDR and PDR P. aeruginosa isolates. Lastly, hybrid 10 possesses a low likelihood of resistance development when compared to its individual antibiotic components tobramycin and moxifloxacin.
We also prepared a series of TOB-NMP conjugates lOOa-f differing in the length of the tether conjoining TOB and NMP (Figure 8). NMP was selected as it does not potentiate efflux-prone antibiotics against P. aeruginosa but instead inhibits efflux pumps in E. coli and A. baumannii38. The absence of EPI function in P. aeruginosa by NMP is likely the result of intrinsic resistance. The secondary amino function of the piperazine ring in NMP was linked to the C-5 osition in tobramycin as this position was expected to promote outer membrane penetration in TOB-NMP conjugates 100a- f31. The antibacterial activity using the minimal inhibitory concentration (MIC) of TOB-NMP conjugates lOOa-f were evaluated against a panel of clinically relevant pathogens but none of the conjugates demonstrated potent Gram-positive (MIC > 8 μ /mL) or Gram-negative (MIC > 32 activity. We then assessed the adjuvant functions of lOOa-f by using the fractional inhibitory concentration (FIC) index12 as a measure of the interaction between two agents. We performed combination studies of lOOa-f with the tetracycline antibiotic minocycline which has been shown to be a substrate of P. aeruginosa RND efflux pumps38. We observed synergistic effects of conjugates lOOb-f with minocycline (FIC index 0.13 - 0.28) in P. aeruginosa PAOl but no synergistic effects with 100a (FIC index > 0.5) as well as tobramycin (FIC index = 1.06), indicating that a tether > 2 carbon atoms is required for synergy (Table 4). No synergy was observed with a combination of NMP and minocycline (FIC index > 1 .0). These results■ indicate that TOB-NMP conjugates can overcome the intrinsic resistance of the pump inhibitor NMP in P. aeruginosa PAOl . Although TOB-NMP conjugates lOOd-f show comparable FIC indices, adjuvant lOOf required the lowest concentration to achieve optimal synergy with minocycline (FIC index = 0.19). For instance, a fixed concentration of lOOf (8 μ^ηιΐ, (7.2 μΜ)) achieved a 16-fold reduction of the MIC of minocycline (MIC = 8 g mL reduced to 0.5 μg/mL) against P. aeruginosa PAOl (Table 4). We also prepared TOB-PAR conjugate 200 and TOB- DBP conjugate 300 both bearing a C12 tether and explored their synergistic effects with minocycline. Similarly to TOB-NMP l OOf, both TOB-PAR conjugate 200 (FIC index = 0.19) and TOB-DBP conjugate 300 (FIC index = 0.09) demonstrated strong synergism with minocycline against P. aeruginosa PAOl (Table 4). No synergy was observed with paroxetine (FIC index = 1) while strong synergy was observed with the P. aeruginosa-active pump inhibitor DBP (FIC index = 0.13) as shown in Table 1. Comparing the adjuvant properties of DBP with TOB-DBP (300) to potentiate minocycline shows that TOB-DBP shows enhanced potency than DBP. For instance, using a fixed concentration (8 μg/mL) of DBP (12.1 μΜ) or TOB-DBP (5.8 μΜ) resulted in 8- or 64-fold reduction in MIC of minocycline, respectively indicating that the presence of a TOB vector in DBP enhances the adjuvant properties (Table 4). Besides potentiating minocycline, we also demonstrated that adjuvants lOOf, 200 and 300 strongly synergized (0.13 < FIC index < 0.25) with other members of the tetracycline class of antibiotics including doxycycline and tigecycline.
Next we assessed the effect of the TOB-EPI conjugates lOOf, 200 and 300 in combination with minocycHne against a panel of 8 clinical P. aeruginosa isolates including six MDR (non-susceptible or resistant to > 3 chemically unrelated anti-pseudomonal classes), and six extremely-drug resistant (XDR) (non-susceptible or resistant to > 5 chemically unrelated anti-pseudomonal classes, obtained from different Canadian hospitals. The panel also included two colistin-non-susceptible or resistant P. aeruginosa strains. The results indicate that all three TOB-EPI conjugates demonstrated strong synergistic effects (0.02 < FIC index < 0.38) with minocycline against the selected panel of MDR and XD P. aeruginosa isolates (Table 5). The following FIC index ranges for the TOB-EPI conjugates against the selected 8 MDR or XDR P. aeruginosa isolates were observed: TOB-NMP (0.02 < FIC index < 0.28); TOB-PAR (0.02 < FIC index < 0.38) and TOB-DBP (0.03 < FIC index < 0.31) as shown in Table 5. We also measured the absolute MIC of minocycline against the 8 MDR/XDR P. aeruginosa isolates in the presence of conjugates lOOf, 200 and 300 at a fixed concentration (< 8 μg/mL, < 0.25 x MIC). These results show that all three conjugates lower the MIC of minocycline from 8-256 fold against the 8 selected MDR or XDR P. aeruginosa isolates. Importantly, in 96% of cases, conjugates 100f, 200 and 300 at a concentration of < 8 μg/mL} < 0.25 XMIC reached minocycline susceptibility (MIC < 1 μg/mL) against the 8-selected MDR or XDR P. aeruginosa isolates (Table 5).
In Galleria mellonella larvae infection model studies, we determined that conjugates lOOf and
200 cause < 5% hemolysis of ovine erythrocytes at 1000 μg/mL and show low cytotoxicity (CC50 > 30 μΜ) against cancer cell lines while increased toxicity was noted for conjugate 300. Tolerability studies in G. mellonella using a dosage of 200 mg/kg of lOOf or 200 showed no toxic effects up to 96 hours while a dose of 100 mg/kg of 300 resulted in 100% killing of the larvae after 24 hours. The toxicity of conjugate 300 in the larvae prevented further use of this compound in the insect model. Efficacy studies were performed by infecting the larvae with a lethal dose of (1.0 x 105 CFUs) of XDR P. aeruginosa strain #101856 followed by injection of the drug combination 2 h post infection. Monotherapy with a single dose (75 mg/kg) of minocycline or lOOf (75 mg/kg) or NMP (75 mg/kg) resulted in 100% killing of the larvae within 24 hours indicating that monotherapy was not able to provide protection of the larvae. In contrast, combination therapy (37.5 mg/kg lOOf + 37.5 mg/kg minocycline or 75 mg/kg lOOf + 75 mg/kg minocycline) resulted in 10% or 77% survival of the larvae, respectively, after 24 hours (Figure 9a). Similarly, efficacy was seen for conjugate 200. For instance, single dose combination therapy (75 mg 200 + 75 mg minocycline) resulted in 56% survival of the larvae while single dose monotherapy with minocycline (75 mg/kg) or conjugate 200 (75 mg/kg) resulted in 100% killing after 24 hours. These results indicate that combinations of minocycline/1OOf and minocycline/200 possess therapeutic potential.
To gain insight into the protective function of the conjugates, we performed a series of mechanistic studies with P. aeruginosa PAOl . We demonstrated that conjugate lOOf permeabilizes the outer membrane of PAOl in a dose-dependent manner using the NPN (1-N-phenylnapthylamine) assay (Figure 9b)18. Similar dose-dependent permeability was seen for conjugates 200 and 300. Next, we assessed whether the combination of conjugates lOOf, 200 or 300 with outer membrane impermeable antibiotics are synergistic in P. aeruginosa PAOl . For all three conjugates we observed strong synergy: with rifampicin (FIC index < 0.05), novobiocin (FIC index < 0.1), chloramphenicol (FIC index < 0.1), trimethoprim (FIC index < 0.1), vancomycin (FIC index < 0.15), erythromycin (FIC index < 0.2) indicating that the conjugates enhance cellular uptake of these antibiotics into P. aeruginosa PAOl (Figure 9c). We also tested the synergistic effects of hybrids le and 10 to potentiate the cancer drug Mitomycin C. Previous studies have shown that the antibacterial effect of Mitomycin C against P. aeruginosa is compromised by efflux. Mitomycin C is an antitumor antibiotic which crosslinks DNA. As seen in Table 7 we observed strong synergy of hybrids le and 10 with Mitomycin C. Furthermore, we also could demonstrate this effect in vivo using the Galleria mellonella infection model (Figure 11).
Finally, as shown in Table 6 and Figure 10 the hybrids comprising tobramycin connected to ciprofloxacin or moxifloxacin or an efflux pump inhibitor can be used to specifically potentiate tetracycline antibiotics and mitomycin C, as discussed herein.
While the preferred embodiments of the invention have been described above, it will be recognized and understood that various modifications may be made therein, and the appended claims are intended to cover all such modifications which may fall within the spirit and scope of the invention.
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Table 1 Antibacterial activity of hybrids 10, 20 and 30, ciprofloxacin (CIP) and tobramycin (TOB) against Gram-positive and Gram-negative pathogens.
MIC (n.g/mL)
Control organism 10 20 30 CIP TOB
S.aureus ATCC29213 1 4 16 <0.25 <0.25
MRSA ATCC 33592 1 8 16 <0.25 <0.25
MSSE CANWARD-2008 81388 2 2 8 <0.25 <0.25
MRSE CAN-ICU 61589 (CAZ >32) 16 8 8 128 1
E.faecalis ATCC 29212 4 32 32 1 8
E. faecium ATCC 27270 16 16 16 8 8
S.pneumoniae ATCC 4961 1 32 64 1 2
E.coli ATCC 25922 0.5 4 16 <0.25 0.5
E.coli CAN-ICU 61714 (GEN-R) 4 64 64 <0.25 8
E.coli CAN-ICU 63074 (AMK 32) 4 32 64 <0.25 8
E.coli CANWARD-2011 97615 128 128 128 256 128
P.aeruginosa ATCC 27853 4 16 64 1 0.5
P.aeruginosa CAN-ICU 62308 (GEN-R) 8 128 32 2 16
P.aeruginosa2 CANWARD-2011 96846 4 >128 64 4 256
S. maltophilia CAN-ICU 62584 32 >128 >128 32 >512
A. baumannii CAN-ICU 63169 16 128 >128 2 32
K.pneumoniae ΑΊ 'CC 13883 1 4 16 <0.25 <0.25
'(GEN-R, TOB-R, CIP-R) aac(3')»a 2(GEN-R, TOB-R) CIP = Ciprofloxacin TOB = Tobramycin GEN = Gentamicin AMK = Amikacin Table 2 Antibacterial activity g□□ IX1) of hybrids 10, 20 and 30 and select classes of antibiotics against MDR, XDR and PDR P. aeruginosa isolates.
Figure imgf000028_0001
A> P = Pseudomonas aeruginosa; DOX - Doxycycline (tetracycline); CPM = Cefepime (cephalosporin); MER = Meropenem (penem); DOR = Doripenem (penem); CIP = Ciprofloxacin (fluoroquinolone); MOX = Moxifloxacin (fluoroquinolone); GEN = Gentamicin (aminoglycoside); TOB = Tobramycin (aminoglycoside); COL = colistin; ND = not determined
Table 3A Effect of fixed concentration of hybrid 10 (< 2 μg□ D"1; < 1/4 MIC) on MIC of ciprofloxacin (CIP) or moxifloxacin (MOX)
Figure imgf000029_0001
Clinical PA isolate CIP Absolute MIC1 MOX Absolute MIC2
PA259-96918 256 <1 512 <1
PA260-97103 16 1 32 1
PA262-101856 16 1 64 2
PA264- 104354 16 1 32 1
91433* 8 0.25 8 0.12
101243* 2 0.06 8 0.06
1 MIC of ciprofloxacin when < ¼ MIC (< 2 μ§/τη1,) of 10 was used.
MIC of moxifloxacin when < ¼ MIC (< 2 μ την) of 10 was used.
s PDR strain resistant to all anti-pseudomonal classes including colistin
Table 3B: Effect of fixed concentration of ciprofloxacin (< 4 μg mL"1; < 1/4 MIC) or moxifloxacin (< 4 μ mL"1; < 1/4 MIC) on MIC of hybrid 10
Clinical PA isolate 10 Absolute MIC1 Absolute MIC2
PA259-96918 2 < 0.5 <1
PA260-97103 1 < 0.25 0.25
PA262-101856 4 < 0.5 <1
PA264-104354 4 < 0.5 < 1
91433* 8 < 1 < 1
101243 8 < 1 < 1
1 MIC of 10 when < ¼ MIC (< 4 μg/mL) of ciprofloxacin was used.
2 MIC of 10 when < ¼ MIC (< 4 g/mL) of moxifloxcin was used.
* PDR strain resistant to all anti-pseudomonal classes including colistin Table 4. Combination studies of TOB-EPIs (lOOa-lOOf, 200 or 300), EPIs (NMP, PAR and DBP) and tobramycin (TOB) with minocycline (MIN) against P. aeruginosa PAOl strain
Figure imgf000030_0001
of corresponding adjuvant. N/A: not applicable.
Table 5. Combination studies of TOB-EPIs (100f, 200 or 300) with minocycline against MDR/XDR P. aeruginosa clinical isolates
Figure imgf000031_0001
Table 6: Potentiation of minocycline with adjuvants le and 10 against MDR/XDR clinical P. aeruginosa isolates
Figure imgf000032_0001
Table 7: Potentiation of Mitomycin C with adjuvants le and 10 in wild and clinical MDR/XDR clinical isolates
Entry Strain Adjuvant FIC
Index
1 PAOl le 0.09
2 262 le O.07
3 259 le <0.12
4 264 le O.09
5 101885 le <0.18
6 100036 le <0.12
7 91433 le 0.37
8 101243 le <0.37
9 PAOl 10 0.37
10 262 10 0.18
11 259 10 0.37
12 264 10 0.28
13 101885 10 0.18
14 100036 10 <0.15
15 91433 10 0.37
16 101243 10 0.31

Claims

1. A compound comprising an aminoglycoside connected to a fluoroquinolone or an efflux pump inhibitor.
2. The compound according to claim 1 wherein the aminoglycoside is connected to the fluoroquinolone or the efflux pump inhibitor via a tether.
3. The compound according to claim 2 wherein the tether is a flexible tether.
4. The compound according to claim 2 wherein the tether is a repeating alkyl tether.
5. The compound according to claim 2 wherein the tether has a length of 2-24 carbons.
6. The compound according to claim 1 wherein the aminoglycoside is tobramycin.
7. The compound according to claim 1 wherein the fluoroquinolone is ciprofloxacin or moxifloxacin
8. The compound according to claim 1 wherein the efflux pump inhibitor is selected from the group consisting of MP, PAR and DBP.
9. The compound according to claim 6 wherein the tobramycin is connected at the C-5 position, the C-4' position, the C-4" position, the C-2" position or the C-6" position of the tobramycin.
10. A method of treating a bacterial infection in an individual in need of such treatment comprising administering to said individual an effective amount of a compound comprising an aminoglycoside connected to a fluoroquinolone or an efflux pump inhibitor.
11. The method according to claim 10 wherein the aminoglycoside is connected to the fluoroquinolone or the efflux pump inhibitor via a tether.
12. The method according to claim 11 wherein the tether is a flexible tether.
13. The method according to claim 11 wherein the tether is a repeating alkyl tether.
14. The method according to claim 11 wherein the tether has a length of 2-24 carbons.
15. The method according to claim 10 wherein the aminoglycoside is tobramycin.
16. The method according to claim 10 wherein the fluoroquinolone is ciprofloxacin or moxifloxacin.
17. The method according to claim 10 wherein the efflux pump inhibitor is selected from the group consisting of NMP, PAR and DBP.
18. The method according to claim 15 wherein the tobramycin is connected at the C-5 position, the C-4' position, the C-4" position, the C-2" position or the C-6" position position of the tobramycin.
19. The method according to claim 10 wherein the bacterial infection is caused by a pathogenic gram-negative bacteria.
20. The method according to claim 1 wherein the pathogenic gram-negative bacteria is P. aeruginosa.
21. The method according to claim 20 wherein the P. aeruginosa strain is an extremely drug-resistant strain of P. aeruginosa.
22 The method according to claim 10 wherein the compound is co-administered with a second antibiotic.
23. A method of enhancing the anti-bacterial activity of an antibacterial compound comprising coadministering said antibacterial compound with a second compound comprising an aminoglycoside connected to a fluoroquinolone or an efflux pump inhibitor.
24. The method according to claim 23 wherein the aminoglycoside is connected to the fluoroquinolone or the efflux pump inhibitor via a tether.
25. The method according to claim 24 wherein the tether is a flexible tether.
26. The method according to claim 24 wherein the tether is a repeating alkyl tether.
27. The method according to claim 24 wherein the tether comprises a length of 2-24 carbons.
28. The method according to claim 23 wherein the aminoglycoside is tobramycin.
29. The method according to claim 23 wherein the fluoroquinolone is ciprofloxacin or moxifloxacin
30. The method according to claim 23 wherein the efflux pump inhibitor is selected from the group consisting of NMP, PAR and DBP.
31. The method according to claim 28 wherein the tobramycin is connected at the C-5 position, the C-4' position, the C-4" position, the C-2" position or the C-6" position.
32. Use of a compound comprising an aminoglycoside connected to a fluoroquinolone or an efflux pump inhibitor for enhancing the antibacterial activity of an agent.
33. The use according to claim 32 wherein the aminoglycoside is tobramycin and the fluoroquinolone is ciprofloxacin or moxifloxacin
34. The use according to claim 32 wherein the amino glycoside is tobramycin and the efflux pump inhibitor is selected from the group consisting of NMP, PAR and DBP.
35. A method of preparing a medicament for treatment of a bacterial infection comprising admixing a compound comprising an aminoglycoside connected to a fluoroquinolone or an efflux pump inhibitor and a suitable excipient.
36. The method according to claim 35 wherein the aminoglycoside is tobramycin.
37. The method according to claim 35 wherein the fluoroquinolone is ciprofloxacin or rnoxifloxaicn
38. The method according to claim 35 wherein the efflux pump inhibitor is selected from the group consisting of MP, PAR and DBP.
39. A method of determining the effect of a compound comprising an aminoglycoside connected to a fluoroquinolone on the antibacterial activity of an agent of interest comprising:
co-administering the agent of interest with the compound to a bacterium of interest; and determining survival of the bacterium.
40. The method according to claim 39 wherein the aminoglycoside is tobramycin and the fluoroquinolone is ciprofloxacin or moxifloxacin
41. The method according to claim 39 wherein the aminoglycoside is tobramycin and the efflux pump inhibitor is selected from the group consisting of NMP, PAR and DBP.
42. a method of determining if an agent of interest has antibacterial activity comprising: attaching the agent of interest to an aminoglycoside;
administering the attached agent of interest and aminoglycoside to a bacterium; and determining survival of the bacterium.
43. The method according to claim 42 wherein the agent of interest is attached to the aminoglycoside by a tether.
44. The method according to claim 42 wherein the aminoglycoside is tobramycin.
45. The method according to claim 42 wherein the tether may be connected to a hydroxyl group at the C-5 position, the C-4' position, the C-4" position, the C-2" position or the C-6" position of the tobramycin.
46. A method of potentiating a tetracycline antibiotic or mitomycin C comprising coadministering the tetracycline antibiotic or mitomycin C with a compound comprising tobramycin connected to a ciprofloxacin or moxifloxacin or an efflux pump inhibitor.
47. The method according to claim 46 wherein the efflux pump inhibitor is selected from the group consisting of NMP, PAR and DBP.
48. The method according to claim 46 wherein the tobramycin is connected to the ciprofloxacin or the moxifloxacin or the efflux pump inhibitor by a tether or linker that is approximately 2-24 carbons long.
49. The method according to claim 48 wherein the tether is connected to a hydroxyl group, at the C-5 position, the C-4' position, the C-4" position, the C-2" position or the C-6" position on the tobramycin.
50. Use of a compound comprising tobramycin connected to a ciprofloxacin or moxifloxacin or an efflux pump inhibitor, for potentiating a tetracycline antibiotic or mitomycin C.
51, The use according to claim 50 wherein the efflux pump inhibitor is selected from the group consisting of NMP, PAR and DBP.
52. The use according to claim 50 wherein the tobramycin is connected to the ciprofloxacin or the moxifloxacin or the efflux pump inhibitor by a tether or linker that is approximately 2-24 carbons long.
53. The use according to claim 52 wherein the tether is connected to a hydroxyl group, at the C-5 position, the C-4' position, the C-4" position, the C-2" position or the C-6" position on the tobramycin.
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