WO2012137164A1 - Compositions antimicrobiennes, compositions antibiofilms et leurs utilisations - Google Patents

Compositions antimicrobiennes, compositions antibiofilms et leurs utilisations Download PDF

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Publication number
WO2012137164A1
WO2012137164A1 PCT/IB2012/051690 IB2012051690W WO2012137164A1 WO 2012137164 A1 WO2012137164 A1 WO 2012137164A1 IB 2012051690 W IB2012051690 W IB 2012051690W WO 2012137164 A1 WO2012137164 A1 WO 2012137164A1
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Prior art keywords
compound
biofilm
substrate
living tissue
composition
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PCT/IB2012/051690
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English (en)
Inventor
Shlomo Nedvetzki
Doron Steinberg
Morris Srebnik
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Yissum Research Development Co of Hebrew University of Jerusalem
BiolineRx Ltd
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Yissum Research Development Co of Hebrew University of Jerusalem
BiolineRx Ltd
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Publication of WO2012137164A1 publication Critical patent/WO2012137164A1/fr
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    • 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
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/69—Boron compounds
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention in some embodiments thereof, relates to pharmacology, and, more particularly, but not exclusively, to treatments which are useful in inhibiting bacterial growth and/or in preventing, reducing and/or interfering with biofilm formation.
  • Antibiotics which are also referred to herein and in the art as antibacterial or antimicrobial agents, are natural substances of relatively small size in molecular terms, which are typically released by bacteria or fungi. These natural substances, as well as derivatives and/or modifications thereof, are used for many years as medications for treating infections caused by bacteria. However, over the decades, almost all the prominent infection-causing bacterial strains have developed resistance to antibiotics.
  • Biofilms are a multicellular high density ecological environment of mostly bacteria and/or fungi and their secretions, but it is not considered a multicellular organism per se. Once a microorganism attach to a surface it undergoes a series of changes, the most obvious of which is the excretion of a slimy material consisting mostly of extra-cellular polysaccharides (EPS).
  • EPS extra-cellular polysaccharides
  • EPS soluble microbiological products
  • SMP soluble microbiological products
  • NOM natural organic matter
  • biofilms are also dynamic hydrogels which capriciously move, detach and reform on a wide variety of environmental or engineered surfaces.
  • biofouling can be defined as the unspecific adsorption of biological material onto surfaces upon their immersion in a fluid.
  • EPS secreted by bacteria and other colonizing microorganisms envelope and anchor them to the substrate thereby altering the local surface chemistry which can stimulate further growth such as the recruitment and settlement of microorganisms.
  • Biofouling via biofilm formation causes the deterioration in the microbiological quality of water by inducing biocorrosion termed microbiologically influenced corrosion and biofouling of piping, membranes, containers and reservoirs.
  • Biofilms also interrupt the flow of ions and water to and from the substrate surface by acting as a diffusion barrier.
  • the reduction of localized oxygen by cathodic reactions within the electrolyte can accelerate the corrosion of a metallic substrate by creating a differential aeration concentration cell.
  • the corrosion and weathering caused by biofilm can lead to considerable damage to heat exchangers, unexpected corrosion of stainless steel, and premature destruction of membranes, and many other technological, industrial and homestead aliments.
  • Streptococcus mutans is a cardinal member of the oral biofilm associated with dental caries while Candida albicans is associated with oral candidiasis.
  • Quorum sensing which is effected by secreting small molecules termed auto inducers (AFs) into their environment. Quorum sensing takes place especially in biofilms were the microbes are at close proximity to one another. This phenomenon affects many physiological and metabolic pathways of bacteria, including the formation of biofilms and antibacterial resistance.
  • Inter species QS may affect microbes' physiology and virulence properties resulting in enhanced virulent properties of biofilms.
  • Small peptides, AI-2 (furanosyl borate diester), AI-1 (N-acylhomoserine lactones) and C-AI (Cholera AI) have been shown to act as signal molecules in QS in many types of bacteria, including oral bacteria.
  • Eukaryotic cells such as fungi have also been shown to communicate with each other by producing signal molecules (AIs), however, the AIs of bacteria and fungi differ chemically; production of farnesol by C. albicans at high cell densities is the first QS system which has been discovered in eukaryotes.
  • Farnesol has been identified as QS agent that blocks the morphological transition from yeast to the filament form and affects bio film formation in C. albicans. The mechanism by which farnesol is sensed by C. albicans is not yet known.
  • Farnesoic acid and tyrosol were also shown to posses AI properties in C. albicans.
  • Eukaryotes seem to have evolved efficient mechanisms to manipulate bacterial QS and thereby protect themselves from pathogenic bacterial attack and competition [Hogan, D.A., 2006, Eukaryot Cell, 5, 613-9].
  • QSIs quorum sensing inhibitors
  • the eukaryotic host may be simultaneously conversing with a variety of different bacterial strains that it encounters in its natural habitat, potentially encouraging the beneficial ones and antagonizing the harmful strains.
  • certain bacteria have evolved mechanisms to fine-tune gene regulation of eukaryotic with their QS signals [Hogan D.A. et al, 2004, Mol. Microbiol, 54, 1212-23]. This eukaryote-bacterial cross talk could be exploited to model manipulative techniques that interfere with bacterial QS.
  • Boron containing compounds have received increasing attention as therapeutic agents over the past few years as technology in organic synthesis has expanded to include this atom. Boron containing compounds have been shown to have various biological activities, including an antibacterial activity [Bailey et al., Antimicrobial Agents and Chemotherapy, (1980), 17, 549-553].
  • U.S. Patent Nos. 6,075,014 and 6,184,363 disclose that a number of phenyl boronic acids are effective against bacteria resistant to beta-lactam antibiotics. These compounds, or pharmaceutically acceptable salts thereof, are antibacterial by themselves, although at higher concentrations than beta-lactam antibiotics.
  • WO 2005/021559 discloses a novel family of oxazaborolidines which can act as antibacterial agents, particularly in preventing or reducing biofilm formation.
  • the present inventors have uncovered that 4-phenylboronic acid and/or an ester thereof can be efficiently used, either alone or in combination with an anti-microbial agent (e.g., an antibiotic), in inhibiting a growth of various bacterial strains and importantly, in preventing or reducing biofilm formation.
  • an anti-microbial agent e.g., an antibiotic
  • composition comprising a compound having general Formula A':
  • R 3 and R 4 are each independently selected from the group consisting of hydrogen, alkyl, aryl and cycloalkyl, or, alternatively, Ri and R 2 are joined together so as to form a ring, an antimicrobial agent and a pharmaceutically acceptable carrier.
  • R 3 and R4 are joined together so as to form a ring.
  • the ring is selected from the group consisting of 5-, 6-, 7-, 8-, 9- and 10-membered ring.
  • the ring comprises a nitrogen atom.
  • the ring comprises a N- methyldiethylamine moiety.
  • R 3 and R 4 are each hydrogen.
  • the composition is packaged in a packaging material and identified in print, in or on the packaging material, for use in inhibiting a growth of a pathogenic microorganism in or on a substrate.
  • the substrate is a living tissue, the composition being identified for use in the treatment of a medical condition associated with the pathogenic microorganism in a subject comprising the living tissue.
  • the composition is packaged in a packaging material and identified in print, in or on the packaging material, for use in reducing or preventing the formation of a biofilm and/or disrupting a biofilm in or on a substrate.
  • the substrate is a living tissue
  • the composition being identified for use in the treatment of a medical condition in which reducing or preventing the formation of the biofilm and/or disrupting the biofilm in the living tissue is beneficial.
  • the compound and the antimicrobial agent act in synergy.
  • a method of inhibiting a growth of a pathogenic microorganism in or on a substrate comprising co-contacting the substrate with an antimicrobial agent and with antimicrobial effective amount of a compound having Formula A', as described herein, an antibacterial agent and a pharmaceutically acceptable carrier.
  • the substrate is a living tissue
  • the method being for treating a medical condition associated with the pathogenic microorganism in a subject comprising the living tissue.
  • a method of reducing or preventing the formation of a biofilm and/or disrupting a biofilm in or on a substrate comprising co-contacting the substrate with an antimicrobial agent and an antimicrobial effective amount of a compound having Formula A', as described herein.
  • the substrate is a living tissue
  • the method being for treating a medical condition in which reducing or preventing the formation of the biofilm and/or disrupting the biofilm in the living tissue is beneficial.
  • the compound and the antimicrobial agent act in synergy.
  • the substrate is a living tissue and the product is a medicament for treating a medical condition associated with the pathogenic microorganism in a subject comprising the living tissue.
  • the substrate is a living tissue and the product is a medicament for treating a medical condition in which reducing or preventing the formation of the biofilm and/or disrupting the biofilm in the living tissue is beneficial.
  • the compound and the antimicrobial agent act in synergy.
  • an article comprising a substrate, a compound having general Formula A', as described herein, and an antimicrobial agent, each being incorporated in or on the substrate.
  • the antimicrobial agent is an antibacterial agent.
  • the antibacterial agent is a non beta-lactam antibacterial agent.
  • the antibacterial agent is selected from the group consisting of gentamicin and tobramycin.
  • Ri and R 2 are each independently selected from the group consisting of alkyl, aryl and cycloalkyl, or, alternatively, Ri and R 2 are joined together so as to form a ring.
  • Ri and R 2 are joined together so as to form a ring.
  • the ring is selected from the group consisting of 5-, 6-, 7-, 8-, 9- and 10-membered ring.
  • the ring comprises a nitrogen atom.
  • the ring comprises a N- methyldiethylamine moiety.
  • the composition is packaged in a packaging material and identified in print, in or on the packaging material, for use in inhibiting a growth of a pathogenic microorganism in or on a substrate.
  • the substrate is selected from the group consisting of a living tissue and an inanimate object.
  • the substrate is a living tissue
  • the composition being a pharmaceutical composition which further comprises a pharmaceutically acceptable carrier.
  • the composition is packaged in a packaging material and identified in print, in or on the packaging material, for use in the treatment of a medical condition associated with a pathogenic microorganism in a subject comprising the living tissue.
  • composition is an antibiofilm composition.
  • the composition is packaged in a packaging material and identified in print, in or on the packaging material, for use in reducing or preventing the formation of a biofilm and/or disrupting a biofilm in or on a substrate.
  • the substrate is selected from the group consisting of a living tissue and an inanimate object.
  • the substrate is a living tissue
  • the composition being identified for use in the treatment of a medical condition in which reducing or preventing the formation of the biofilm and/or disrupting the biofilm in the living tissue is beneficial.
  • the composition is for use in combination with an additional active agent.
  • the composition further comprises an additional active agent.
  • the additional active agent is an antimicrobial agent.
  • the compound and the antimicrobial agent act in synergy.
  • a method of inhibiting a growth of a pathogenic microorganism in or on a substrate comprising contacting the substrate with an antimicrobial effective amount of a compound having a general Formula A, as described herein.
  • the substrate is selected from the group consisting of a living tissue and an inanimate object.
  • the substrate is a living tissue
  • the method being for use in the treatment of a medical condition associated with the pathogenic microorganism in a subject comprising the living tissue.
  • a method of reducing or preventing the formation of a biofilm and/or disrupting a biofilm in or on a substrate comprising contacting the substrate with an antimicrobial effective amount of a compound having general Formula A, as described herein.
  • the substrate is selected from the group consisting of a living tissue and an inanimate object.
  • the substrate is a living tissue
  • the method being for treating a medical condition in which reducing or preventing the formation of the biofilm and/or disrupting the biofilm in the living tissue is beneficial.
  • the method further comprises contacting the substrate with an additional active agent.
  • the additional active agent is an antimicrobial agent.
  • the compound and the antimicrobial agent act in synergy.
  • a use of a compound having general Formula A, as described herein, in the manufacture of a product for inhibiting a growth of a pathogenic microorganism in or on a substrate is provided.
  • the substrate is selected from the group consisting of a living tissue and an inanimate object.
  • the substrate is a living tissue
  • the compound is being for use in the treatment of a medical condition associated with the pathogenic microorganism in a subject comprising the living tissue.
  • the substrate is a living tissue and the product is a medicament for treating a medical condition associated with the pathogenic microorganism in a subject comprising the living tissue.
  • a compound having general Formula A, as described herein for use in a method of reducing or preventing the formation of a biofilm and/or disrupting a biofilm in or on a substrate.
  • the substrate is selected from the group consisting of a living tissue and an inanimate object.
  • the substrate is a living tissue, the compound being for use in a method of treating a medical condition in which reducing or preventing the formation of the biofilm and/or disrupting the biofilm in the living tissue is beneficial.
  • the substrate is a living tissue and the product is a medicament for treating a medical condition in which reducing or preventing the formation of the biofilm and/or disrupting the biofilm in the living tissue is beneficial.
  • the compound as described herein is for use in combination with an additional active agent.
  • the product is for use in combination with an additional active agent.
  • the product further comprises an additional active agent.
  • the additional active agent is an antimicrobial agent.
  • the compound and the antimicrobial agent act in synergy.
  • an article comprising a substrate and a compound having general Formula A, as described herein, incorporated in or on the substrate.
  • the article further comprises an antimicrobial agent being incorporated in or on the substrate.
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, abrogating, substantially inhibiting or slowing the propagation of at least one cause of a (medical) condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
  • a subject in need thereof as used herein describes a human, or mammal, who has bacterial infection or is at the risk of developing microbial infection (i.e., predisposed).
  • microbial infection encompasses diseases and conditions resulting from or associated with presence of pathogenic microorganism in a subject.
  • FIG. 1 is a comparative bar plot presenting the results of the optical density measurements (blue) and bacterial count (light blue) of methicillin resistant Staphylococcus aureus US A300 strain with different concentrations of Compound K;
  • FIG. 2 is a comparative bar plot presenting the results of the optical density measurements (orange) and bacterial count (yellow) of Pseudomonas aeruginosa ATCC27312 strain with different concentrations of Compound K;
  • FIG. 3 is a bar graph presenting methicillin resistant Staphylococcus aureus
  • FIG. 4 is a bar graph presenting the Pseudomonas aeruginosa ATCC27312 strain inhibition zones by different concentrations of Compound K, measured in cm 2 using planimetry method;
  • FIGs. 5A-B are comparative bar plots presenting the results of the optical density measurements of methicillin resistant Staphylococcus aureus US A300 strain in the presence of different concentrations of Compound K (yellow bars) and Compound F (light blue bars) (FIG. 5A) and of different concentrations of Gentamicin (FIG. 5B);
  • FIGs. 6A-B are comparative bar plots presenting the results of the optical density measurements of Pseudomonas aeruginosa ATCC27312 strain with different concentrations of Compound K (orange bars) and Compound F (blue bars) (FIG. 6A) and of different concentrations of Tobramycin (FIG. 6B);
  • FIGs. 7A-D present representative photographs of wounds induced in tested pigs during, before and after treatments (following inoculation with bacterial strains), as well as throughout the assessment days, wherein gauze in all treatment groups were in place and moist on day 1 showing a slight adherence to the wound bed (FIG. 7A), while re- injury was not observed (FIG. 7B), and wherein wound fluid was observed from all treatment groups after 24 hours biofilm formation in wounds assigned to biofilm elimination assessment (FIG. 7C and Figure 7D);
  • FIGs. 8A-B present photographs of a wound induced in a tested pig and infected with methicillin resistant Staphylococcus aureus (FIG. 8B), and treated within 20 minutes after inoculation with vehicle (FIG. 8A);
  • FIG. 9 is a bar graph showing methicillin resistant Staphylococcus aureus USA300 biofilm inhibition in the presence of 100 mg/ml (light blue bars), 40 mg/ml (yellow bars), and 10 mg/ml (green bars) of Compound K (denoted EDP-5), of vehicle only (purple bars) and of mupirocin (blue bars) control treatments, compared to control untreated wounds (orange bars);
  • FIG. 10 is a bar graph showing inhibition of biofilm of methicillin resistant S. aureus US A300 in the presence of various concentrations of Compound K (denoted EDP-5) and control treatments following 24 hours (Day 1) of a first treatment (1 Rx) (light blue bars) and following additional 24 hours (Day 2) of a second treatment (2 Rx);
  • FIGs. 11A-B are bar graphs showing bacterial counts of methicillin resistant Staphylococcus aureus US A300 biofilm in wounds (light blue bars) compared with bacterial count in gauze (red bars), 24 hours (Day 1) after one treatment application (1 Rx) of various concentrations of Compound K (denoted EDP-5) and control treatments (FIG. 11 A), and bacterial counts of S. aureus USA300 in wounds (yellow bars) compared with bacterial count in gauze (orange bars) 24 hours (Day 2) after a second treatment application (2 Rx) of various concentrations of Compound K (denoted EDP- 5) and control treatments (FIG. 1 IB);
  • FIG. 12 is a bar graph showing elimination of methicillin resistant
  • FIG. 13 is a bar graph showing bacterial count of methicillin resistant Staphylococcus aureus US A300 after 24 hours of biofilm formation in wounds (different color bars), compared with bacterial count in gauze (green bars), following one treatment with various concentrations of Compound K (denoted EDP-5) and control treatments;
  • FIG. 14 is a bar graph showing inhibition of Pseudomonas aeruginosa ATCC 27312 bio film formation after one application (Day 1, 1 Rx) and two applications (Day 2, 2 Rx) of treatment with 40 mg/ml (light blue bars), 10 mg/ml (yellow bars), 4 mg/ml (green bars) of Compound K (denoted EDP-5) and vehicle (purple bars), silver sulfadiazine (blue bars) and control (brown bars) treatments;
  • FIG. 15 is bar graph comparing inhibition of Pseudomonas aeruginosa ATCC 27312 bio film after one application (Day 1, 1 Rx; light blue bars) and two applications (Day 2, 2 Rx; yellow bars) of treatment with various concentrations of Compound K (denoted EDP-5) and control treatments;
  • FIGs. 16A-B are bar graphs showing bacterial count of Pseudomonas aeruginosa ATCC 27312 in wounds (light blue bars), compared with bacterial count in gauze (red bars), 24 hours (Day 1) after one treatment application (1 Rx) (FIG. 16A) and bacterial count of Pseudomonas aeruginosa ATCC 27312 in wounds (yellow bars), compared with bacterial count in gauze (orange bars), additional 24 hours (Day 2) after second treatment application (2 Rx) (FIG. 16B) of treatment with various concentrations of Compound K (denote EDP-5) and control treatments;
  • FIG. 17 is a bar graph showing elimination of Pseudomonas aeruginosa ATCC 27312 bio film after 24 hours of bio film formation, following treatment of various concentrations of Compound K (denoted EDP-5) and control treatments;
  • FIG. 18 is a bar graph showing bacterial count of Pseudomonas aeruginosa ATCC 27312 after 24 hours of bio film formation in wounds (different color bars), compared with bacterial count in gauze (green bars), following one treatment with various concentrations of Compound K (denoted EDP-5) and control treatments;
  • FIGs. 19A-B present a bar graph showing inhibition of bio film formation Pseudomonas aeruginosa ATCC27312 after one treatment with BL 6031 (Compound F) 10 mg/ml (light blue bars) and 30 mg/ml (yellow bars), with BL 6031 (Compound K) in 30 % PEG vehicle, 20 mg/ml (grey blue bars) and 60 mg/ml (pink bars), of BL 6030 (Compound K) in 30 % PG vehicle, 20 mg/ml (dark yellow bars) and 60 mg/ml (lavender bars), with Tobramycin 100 ⁇ g/ml (purple bars) and 50 ⁇ g/ml (blue bars) and in untreated wound (brown bars) (FIG.
  • Compound F 10 mg/ml
  • 30 mg/ml grey blue bars
  • 60 mg/ml pink bars
  • FIGs. 20A-B present bar graphs showing inhibition of bio film formation of
  • FIGs. 21A-B present bar graphs showing inhibition of methicillin resistant Staphylococcus aureus (MRSA) biofilm formation 24 hours after application of treatment with 75 mg/ml (pink bars) and 37.5 mg/ml (blue bars) of BL 6031 (Compound F), with Gentamicin 200 ⁇ g/ml (green bars) and 100 ⁇ g/ml (yellow bars), with 150 mg/ml (red bars) and 75 mg/ml (light blue bars) of BL 6030 (Compound K) in 30 % PG vehicle, with 150 mg/ml (bright green bars) and 75 mg/ml (brown bars) of BL 6030 (Compound K) in 30 % PEG vehicle, with Mupirocin (purple bars) and in untreated wound (sea green bars) (FIG.
  • MRSA methicillin resistant Staphylococcus aureus
  • FIGs. 22A-B present bar graphs showing elimination of methicillin resistant Staphylococcus aureus (MRSA) US A300 biofilm 24 hours after application of treatment with 75 mg/ml (plum bars) and 37.5 mg/ml (blue bars) of BL 6031 (Compound F), with Gentamicin 200 ⁇ g/ml (green bars) and 100 ⁇ g/ml (yellow bars), with 150 mg/ml (red bars) and 75 mg/ml (light blue bars) of BL 6030 (Compound K) in 30 % PG vehicle, with 150 mg/ml (bright green bars) and 75 mg/ml (brown bars) of BL 6030 (Compound K) in 30 % PEG vehicle, with Mupirocin (purple bars) and in untreated wound (sea green bars) (FIG.
  • MRSA methicillin resistant Staphylococcus aureus
  • FIGs. 23A-B present bar graphs showing inhibition of Pseudomonas aeruginosa ATCC27312 biofilm formation 24 hours after application of treatment with 10 mg/ml (plum bars) and 30 mg/ml (blue bars) of BL 6031 (Compound F), with 20 mg/ml (green bars) and 60 mg/ml (yellow bars) of BL 6030 (Compound K), with Tobramycin 200 ⁇ g/ml (red bars), 100 ⁇ g/ml (light blue bars) and 50 ⁇ g/ml (bright green bars), with 30 % PEG vehicle (brown bars), with Silver Sulfadiazine (purple bars) and of untreated wound (sea green bars) (FIG.
  • FIGs. 24A-B present bar graphs showing elimination of Pseudomonas aeruginosa ATCC27312 biofilm 24 hours after application of treatment with 10 mg/ml (plum bars) and 30 mg/ml (blue bars) of BL 6031 (Compound F), with 20 mg/ml (green bars) and 60 mg/ml (yellow bars) of BL 6030 (Compound K), with Tobramycin 200 ⁇ g/ml (red bars), 100 ⁇ g/ml (light blue bars) and 50 ⁇ g/ml (bright green bars), with 30 % PEG vehicle (brown bars), with Silver Sulfadiazine (purple bars) and of untreated wound (sea green bars) (FIG.
  • FIGs. 25A-B present bar graphs showing inhibition of methicillin resistant Staphylococcus aureus (MRSA) biofilm formation 24 hours after application of treatment with Gentamicin 25 ⁇ g/ml (plum bars), 37.5 mg/ml (blue bars) BL 6031 (Compound F), with Gentamicin 50 ⁇ g/ml (green bars), with 75 mg/ml (yellow bars) BL 6031 (Compound F), with a combined therapy of Gentamicin 25 ⁇ g/ml and 37.5 mg/ml BL 6031 (Compound F) (rose bars), with Gentamicin 100 ⁇ / ⁇ 1 (bright green bars), with 30 % PEG vehicle (brown bars), with Mupirocin (purple bars) and in untreated wound (sea green bars) (FIG.
  • MRSA methicillin resistant Staphylococcus aureus
  • FIGs. 26A-B present bar graphs showing elimination of methicillin resistant Staphylococcus aureus (MRSA) US A300 biofilm 24 hours after application of treatment with Gentamicin 25 ⁇ g/ml (plum bars), 37.5 mg/ml (blue bars) BL 6031 (Compound F), with Gentamicin 50 ⁇ g/ml (green bars), with 75 mg/ml (yellow bars) BL 6031 (Compound F), with a combined therapy of Gentamicin 25 ⁇ g/ml and 37.5 mg/ml BL 6031 (Compound F) (rose bars), with Gentamicin 100 ⁇ g/ml (bright green bars), with 30 % PEG vehicle (brown bars), with Mupirocin (purple bars) and in untreated wound (sea green bars) (FIG.
  • MRSA methicillin resistant Staphylococcus aureus
  • FIGs. 27A-B present bar graphs showing inhibition of Pseudomonas aeruginosa ATCC27312 biofilm formation 24 hours after application of treatment with Tobramycin 25 ⁇ g/ml (plum bars), 10 mg/ml (blue bars) BL 6031 (Compound F), with Tobramycin 50 ⁇ g/ml (green bars), with 30 mg/ml (yellow bars) BL 6031 (Compound F), with a combined therapy of Tobramycin 25 ⁇ g/ml and 10 mg/ml BL 6031 (Compound F) (rose bars), with a combined therapy of Tobramycin 50 ⁇ g/ml and 30 mg/ml BL 6031 (Compound F) (light blue bars), with Tobramycin 100 ⁇ g/ml (bright green bars), with 30 % PEG vehicle (brown bars), with Silver Sulfadiazine (purple bars) and in untreated wound (sea green bars) (FIG.
  • FIGs. 28A-B present bar graphs showing elimination of Pseudomonas aeruginosa ATCC27312 bio film 24 hours after application of treatment with Tobramycin 25 ⁇ g/ml (plum bars), 10 mg/ml (blue bars) BL 6031 (Compound F), with Tobramycin 50 ⁇ g/ml (green bars), with 30 mg/ml (yellow bars) BL 6031 (Compound F), with a combined therapy of Tobramycin 25 ⁇ g/ml and 10 mg/ml BL 6031 (Compound F) (rose bars), with a combined therapy of Tobramycin 50 ⁇ g/ml and 30 mg/ml BL 6031 (Compound F) (light blue bars), with Tobramycin 100 ⁇ g/ml (bright green bars), with 30 % PEG vehicle (brown bars), with Silver Sulfadiazine (purple bars) and in untreated wound (sea green bars) (FIG.
  • the present invention in some embodiments thereof, relates to pharmacology, and, more particularly, but not exclusively, to treatments which are useful in inhibiting bacterial growth and/or in preventing, reducing and/or interfering with biofilm formation.
  • U.S. Patent No. 6,448,238 teaches 4-fluorophenylboronic acid and other phenylboronic acids and their use as beta-lactamase inhibitors.
  • the compounds described in this patent are taught to be used in combination with beta-lactam antibiotics for treatment of infections of bacteria which are resistant to beta-lactam antibiotics.
  • the present inventors have uncovered that exposing pathogenic bacteria to 4- fluorophenylboronic acid (designated herein as Compound F or BL 6031), combined with anti-bacterial agents which are not beta-lactam antibiotics ("non beta-lactam antibiotics”) resulted in surprisingly strong inhibition of bacterial growth.
  • esters of 4-fluorophenylboronic acid can perform similarly to 4-fluorophenylboronic acid.
  • the present inventors have further uncovered that 4-phenylboronic acid and/or esterified derivatives thereof are effective as antimicrobial and/or anti-biofilm agents when used in combination with other antimicrobial agents, showing mostly synergistic activity effect.
  • 4-Fluorophenylboronic acid is also referred to herein as Compound F or BL 6031 and has the following structure:
  • Ri and R 2 are each independently selected from the group consisting of alkyl, aryl and cycloalkyl, or, alternatively, Ri and R 2 are joined together so as to form a ring.
  • alkyl describes a saturated aliphatic hydrocarbon including straight chain and branched chain groups.
  • the alkyl group has 1 to 20 carbon atoms. Whenever a numerical range; e.g., " 1-20", is stated herein, it implies that the group, in this case the alkyl group, may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms.
  • the alkyl is a medium size alkyl having 1 to 10 carbon atoms.
  • the alkyl is a lower alkyl having 1 to 4 carbon atoms.
  • the alkyl group may be substituted or unsubstituted.
  • Substituted alkyl may have one or more substituents, whereby each substituent group can independently be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halide, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, sulfonamide, C-carboxylate, O- carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, N-carbamate, O-carbamate, C-amide, N-amide, guanyl, guanidine and hydrazine, as defined herein.
  • substituent group can independently be, for example, hydroxyalkyl, trihaloalkyl, cycl
  • the alkyl group can be an end group, wherein it is attached only to the respective oxygen atom in Formula A, or a linking group, which is connected to the respective oxygen moiety and further to one or more other moieties (e.g., in cases where Ri and R 2 form together a ring).
  • cycloalkyl describes an all-carbon monocyclic or fused ring (i.e., rings which share an adjacent pair of carbon atoms) group where one or more of the rings does not have a completely conjugated pi-electron system.
  • the cycloalkyl group may be substituted or unsubstituted.
  • Substituted cycloalkyl may have one or more substituents, whereby each substituent group can independently be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halide, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, sulfonamide, C-carboxylate, O- carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, N-carbamate, O-carbamate, C-amide, N-amide, guanyl, guanidine and hydrazine, as defined herein.
  • substituent group can independently be, for example, hydroxyalkyl, trihaloalkyl
  • the cycloalkyl group can be an end group, wherein it is attached only to the respective oxygen atom in Formula A, or a linking group, which is connected to the respective oxygen moiety and further to one or more other moieties (e.g., in cases where Ri and R 2 form together a ring) at one or more positions thereof.
  • aryl describes an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi-electron system.
  • the aryl group may be substituted or unsubstituted.
  • Substituted aryl may have one or more substituents, whereby each substituent group can independently be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halide, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, sulfonamide, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, N-carbamate, O-carbamate, C-amide, N-amide, guanyl, guanidine and hydrazine, as defined herein.
  • substituent group can independently be, for example, hydroxyalkyl, trihaloalkyl,
  • the aryl group can be an end group, wherein it is attached only to the respective oxygen atom in Formula A, or a linking group, which is connected to the respective oxygen moiety and further to one or more other moieties (e.g., in cases where Ri and R 2 form together a ring) at one or more positions thereof.
  • Ri and R 2 are joined together so as to form a ring with the -0-B-O- skeleton of the boronic acid.
  • the ring can comprise from 4 to 12 atoms, or from 5 to 10 atoms, and thus can be a 5-, 6-, 7-, 8-, 9- or 10-membered ring.
  • the ring is a monocylic ring.
  • a ring formed by Ri and R 2 is naturally a heterocylic ring since it includes at least one boron atom and 2 oxygen atoms derived from the boronic acid skeleton.
  • the ring formed by Ri and R 2 includes a moiety that bridges the two oxygen atoms of the boronic acid of which the compounds disclosed herein are esters.
  • Ri and R 2 can be joined so as form a bridging moiety which bridges the two oxygen atoms of the boronic acid of which the compounds disclosed herein are esters.
  • This bridging moiety can be, for example, a hydrocarbon chain, optionally substituted, and optionally interrupted by one or more heteroatoms such as, but not limited to, nitrogen (NR'), -O- or -S-.
  • hydrocarbon chain describes a moiety having a backbone chain that is comprised of carbon atoms, which can be unsubstituted (namely, have hydrogens attached to the carbon atoms) or substituted by various substituents, as described hereinabove for an alkyl.
  • a hydrocarbon moiety can therefore be comprised of one or more alkyls, one or more cycloalkyls and/or one or more aryls, as these are defined herein, wherein each can be substituted or unsubstituted.
  • the hydrocarbon chain is 2 to 8 carbon atoms in length, and can thus be 2, 3, 4, 5, 6, 7 or 8 carbon atom in length.
  • the ring comprises an alkylene chain, namely, a hydrocarbon chain as described herein, comprised of one or more alkyls.
  • the alkylene chain can be linked via one carbon atom to one oxygen of the boronic acid skeleton and via another carbon to the other oxygen of the boronic acid skeleton.
  • the alkylene chain is 2 to 8 carbon atoms in length, and can thus be 2, 3, 4, 5, 6, 7 or 8 carbon atom in length.
  • the hydrocarbon chain comprises one or more heteroatoms, as described herein.
  • Such hydrocarbon chains can be comprised of an alkylene chain, as described herein, which is interrupted by one or more heteroatoms.
  • such hydrocarbon chains can comprise one or more of a heteroalicyclic moiety or a heteroaryl moiety, whereby a heteroatom of such moieties forms a part of the hydrocarbon chain.
  • the ring comprises a hydrocarbon as described herein, which is interrupted by one or more nitrogen atoms, such that the ring formed by Ri and R 2 in Formula A comprises one or more nitrogen atoms.
  • the ring comprises an alkylene chain, as described herein, interrupted by one or more nitrogen atoms.
  • the ring comprises a N-methyldiethylamine moiety, bridging the two oxygen atoms in Formula A.
  • Ri is methyl and R 2 is ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl;
  • Ri is ethyl and R 2 is ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl;
  • Ri is propyl and R 2 is ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl;
  • Ri is butyl and R 2 is ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl;
  • Ri is pentyl and R 2 is ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl;
  • Ri is hexyl and R 2 is ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl;
  • Ri is heptyl and R 2 is ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl;
  • Ri is octyl and R 2 is ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl;
  • Ri is nonyl and R 2 is ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl;
  • Ri is decyl and R 2 is ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl; Ri is cyclopropyl and R 2 is ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl;
  • Ri is cyclobutyl and R 2 is ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl;
  • Ri is cyclopentyl and R 2 is ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl;
  • Ri is cyclohexyl and R 2 is ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl;
  • Ri is cycloheptyl and R 2 is ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl;
  • Ri is any other cycloalkyl and R 2 is ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl;
  • Ri is cyclopropyl and R 2 is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl or any other cycloalkyl;
  • Ri is cyclobutyl and R 2 is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl or any other cycloalkyl;
  • Ri is cyclopentyl and R 2 is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl or any other cycloalkyl;
  • Ri is cyclohexyl and R 2 is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl or any other cycloalkyl;
  • Ri is cycloheptyl and R 2 is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl or any other cycloalkyl;
  • Ri is cyclooctyl and R 2 is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl or any other cycloalkyl;
  • Ri is phenyl, naphthalenyl or any other aryl and R 2 is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl or any other cycloalkyl;
  • Ri is phenyl, naphthalenyl or any other aryl and R 2 is ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl;
  • Ri is phenyl, naphthalenyl or any other aryl and R 2 is phenyl, naphthalenyl or any other aryl, including any combination thereof.
  • Additional exemplary compounds have Formula A as described herein, wherein Ri, O, B, O and R 2 form a ring such that Ri and R 2 form together any of the following bridging moieties (in a biradical form thereof, which is also referred to herein as a linking moiety):
  • R 1 2 a moiety having the formula: R 1 2 , wherein Ri and R 2 are each independently an alkyl linking moiety, a cycloalkyl linking moiety or an aryl linking moiety, as defined herein, and Ra is hydrogen, alkyl, cycloalkyl or aryl, as defined herein, such as, but not limited to, the following moieties:
  • N-methylethaneamine N-dimethylamine, N-methylpropaneamine, N- methylbutaneamine, N-methylpentaneamine, N-methylhexanamine, N-diethylamine, N- ethylpropaneamine, N-ethylbutaneamine, N-ethylpentaneamine, N-ethylhexanamine, N- dipropylamine, N-propylbutaneamine, N-propylpentaneamine, N-propylhexanamine, N- dibutylamine, N-butylpentaneamine, N-butylhexanamine, N-methylpropaneamine, N- methylbutaneamine, N-methylpentaneamine, N-methylhexanamine, whereby in each of the above the nitrogen can be substituted by an alkyl, cycloalkyl or aryl, as defined herein; and a moiety having the formula: wherein Ri and
  • R 2 are each independently an alkyl linking moiety, a cycloalkyl linking moiety or an aryl linking moiety, as defined herein.
  • the bridging moiety comprises an N-methyldiethylamine moiety.
  • Ri, O, B, O and R 2 form a 1,3,6,2-dioxazaborocane ring, such as 6-methyl- 1,3,6,2-dioxazaborocane ring.
  • an exemplary ester compound according to some embodiments of the present invention is Compound K, as described in the Examples section that follows.
  • heteroaryl describes a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system.
  • heteroaryl groups include pyrrole, furane, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine.
  • the heteroaryl group may be substituted or unsubstituted.
  • Substituted heteroaryl may have one or more substituents, whereby each substituent group can independently be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halide, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, sulfonamide, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, O-carbamate, N-carbamate, C-amide, N-amide, guanyl, guanidine and hydrazine.
  • substituent group can independently be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl
  • the heteroaryl group can be an end group, as this phrase is defined hereinabove, where it is attached to a single adjacent atom, or a linking group, as this phrase is defined hereinabove, connecting two or more moieties at two or more positions thereof.
  • Representative examples are pyridine, pyrrole, oxazole, indole, purine and the like.
  • heteroalicyclic describes a monocyclic or fused ring group having in the ring(s) one or more atoms such as nitrogen, oxygen and sulfur.
  • the rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi-electron system.
  • the heteroalicyclic may be substituted or unsubstituted.
  • Substituted heteroalicyclic may have one or more substituents, whereby each substituent group can independently be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halide, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, sulfonamide, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, O-carbamate, N-carbamate, C-amide, N-amide, guanyl, guanidine and hydrazine.
  • substituent group can independently be, for example, hydroxyalkyl, trihaloalkyl, cycloalky
  • the heteroalicyclic group can be an end group, as this phrase is defined hereinabove, where it is attached to a single adjacent atom, or a linking group, as this phrase is defined hereinabove, connecting two or more moieties at two or more positions thereof.
  • Representative examples are piperidine, piperazine, tetrahydrofurane, tetrahydropyrane, morpholino and the like.
  • amine describes both a -NR'R” group and a -NR- group, wherein R' and R" are each independently hydrogen, alkyl, cycloalkyl, aryl, as these terms are defined hereinbelow.
  • the amine group can therefore be a primary amine, where both R' and R" are hydrogen, a secondary amine, where R' is hydrogen and R" is alkyl, cycloalkyl or aryl, or a tertiary amine, where each of R' and R" is independently alkyl, cycloalkyl or aryl.
  • R' and R" can each independently be hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halide, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, sulfonamide, carbonyl, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, N-carbamate, O-carbamate, C- amide, N-amide, guanyl, guanidine and hydrazine.
  • amine is used herein to describe a -NR'R" group in cases where the amine is an end group, as defined hereinunder, and is used herein to describe a -NR'- group in cases where the amine is a linking group.
  • hydroxyl describes a -OH group.
  • alkoxy describes both an -O-alkyl and an -O-cycloalkyl group, as defined herein.
  • aryloxy describes both an -O-aryl and an -O-heteroaryl group, as defined herein.
  • thiohydroxy describes a -SH group.
  • thioalkoxy describes both a -S-alkyl group, and a -S-cycloalkyl group, as defined herein.
  • thioaryloxy describes both a -S-aryl and a -S-heteroaryl group, as defined herein.
  • cyano describes a -C ⁇ N group.
  • nitro describes an -N0 2 group.
  • halide and "halo” describes fluorine, chlorine, bromine or iodine.
  • haloalkyl describes an alkyl group as defined above, further substituted by one or more halide.
  • dithiosulfide refers to a -S-SR' end group or a -S-S- linking group, as these phrases are defined hereinabove, where R' is as defined herein.
  • phosphinyl describes a -PR'R" end group or a -PR'- linking group, as these phrases are defined hereinabove, with R' and R" as defined hereinabove.
  • peroxo describes an -O-OR' end group or an -O-O- linking group, as these phrases are defined hereinabove, with R' as defined hereinabove.
  • thiourea which is also referred to herein as “thioureido”, describes a
  • hydrozine describes a -NR'-NR"R" ' end group or a -NR'-NR"- linking group, as these phrases are defined hereinabove, with R', R", and R'" as defined herein.
  • R3 and R 4 are each independently selected from the group consisting of hydrogen, alkyl, aryl and cycloalkyl, or, alternatively, R3 and R 4 are joined together so as to form a ring.
  • R3 and R 4 are each independently selected from the group consisting alkyl, aryl and cycloalkyl, or, alternatively, R3 and R 4 are joined together so as to form a ring, and are as described herein for Ri and R 2 , in Formula A.
  • At least one of R3 and R 4 is hydrogen and the other is as described herein for Ri and R 2 (when Ri and R 2 do not form a ring).
  • each of R 3 and R4 is hydrogen, such that the compound having Formula A' is 4-fluorophenylboronic acid.
  • Embodiments of the present invention in which a compound having Formula A' is Compound F relate to a combined use of Compound F and an additional active agent (e.g., an antibacterial agent), as described herein.
  • an additional active agent e.g., an antibacterial agent
  • an exemplary ester of 4- fluorophenylboronic acid, Compound K exhibits an antimicrobial activity, and was further found to prevent or reduce bio films both ex-vivo and in-vivo, and that these activities can be enhanced synergistically in combination with antibacterial agents.
  • 4-fluorophenylboronic esters as described herein can be used effectively both in antimicrobial compositions, and in compositions for preventing, reducing and eliminating biofilms both ex-vivo and in-vivo, and that these activities can be enhanced synergistically in combination with antibacterial agents.
  • Compound F 4-fluorophenylboronic acid
  • Compound F can be used effectively both in antimicrobial compositions, and in compositions for preventing, reducing and eliminating biofilms both ex-vivo and in- vivo, and that these activities can be enhanced, additively and even synergistically, in combination with antibacterial agents.
  • antibacterial refers to a property of a substance (e.g., a compound or a composition) that can effect a parameter of microorganism, as defined herein, including death, eradication, elimination, reduction in number, reduction of growth rate, inhibition of growth, change in population distribution of one or more species of microbial life forms.
  • This term encompasses antibacterial agents, which are also referred to herein as antibiotics.
  • an antimicrobial substance e.g., a compound or a composition
  • an antimicrobial substance can be manifested, for example, by reducing or increasing adhesion of the microorganism to the substrate; by effecting enzymatic activity; and/or by effecting the viability of the microorganism (e.g., bacteria), as further discussed herein.
  • a composition which includes, as an active ingredient, a compound having general Formula A as described herein and optionally a carrier.
  • composition which includes, as an active ingredient, a compound having general Formula A' as described herein (e.g., Compound F), an additional active agent (e.g., an antimicrobial or antibacterial agent), as described herein, and optionally a carrier.
  • a compound having general Formula A' as described herein e.g., Compound F
  • an additional active agent e.g., an antimicrobial or antibacterial agent
  • compositions are referred to herein as an antimicrobial or an antibacterial composition.
  • the composition is packaged in a packaging material and is identified in print, in or on the packaging material, for use in inhibiting a growth of a pathogenic microorganism in or on a substrate.
  • Microorganisms can exist on living tissues or on non-living, organic or inorganic substrates, referred to herein an inanimate objects or substrates, and constitute a prevalent mode of microbial life in natural, industrial and medical settings.
  • a substrate as defined herein encompasses living tissues and inanimate objects.
  • living tissue is meant to encompass any part of a living organism, a bodily site or a living organ.
  • the phrase "bodily site” includes any organ, tissue, membrane, cavity, blood vessel, tract, biological surface or muscle, which contacting therewith (e.g., delivering thereto or applying thereon) the compound disclosed herein is beneficial.
  • Exemplary bodily sites include, but are not limited to, the skin, a dermal layer, the scalp, an eye, an ear, a mouth, a throat, a stomach, a small intestines tissue, a large intestines tissue, a kidney, a pancreas, a liver, the digestive system, the respiratory tract, a bone marrow tissue, a mucosal membrane, a nasal membrane, the blood system, a blood vessel, a muscle, a pulmonary cavity, an artery, a vein, a capillary, a heart, a heart cavity, a male or female reproductive organ and any visceral organ or cavity.
  • living tissue encompasses also samples of a living organism or subject, namely a human or an animal, which have been removed from the organism and maintained viable for any purpose, and encompasses the living subject itself as a whole, e.g., a plant, a human or an animal (e.g., a mammal).
  • the phrase "inanimate object” is meant to encompass any surface of an object which may harbor a microorganism, such as, but not limited to, an implantable medical device such as a gastric or duodenal sleeve, a topical medical device such as a wound dressing, a subcutaneous medical device such as a subcutaneous injection port, a percutaneous medical device such as a catheter, a syringe needle or an endoscopic device, a vessel, a tube, a lid, a wrap, a package, a work surface or area, a warehouse, a package and the like, as is further described hereinafter in the context of "substrate”.
  • the phrase "inhibiting the growth of a microorganism” refers to an effect of a compound, a composition or a combination of a compound with another active agent, which stops and/or reverses the propagation of a microorganism, such that at least one cell or a culture thereof is no longer multiplying or growing and/or is killed as a result of coming in contact with the compound, the composition or the combination of compounds.
  • the effect of inhibiting the growth thereof is oftentimes beneficial.
  • the antimicrobial composition is packaged in a packaging material and is identified in print, in or on the packaging material for use in inhibiting a growth of a microorganism in or on inanimate objects, as discussed herein.
  • a composition may be in a form of, for example, solution, paste, liquid, spray or powder.
  • the composition when the substrate is a living tissue, the composition is a pharmaceutical composition.
  • a pharmaceutical composition which comprises a compound having general Formula A as described herein and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition which comprises a compound having general Formula A' as described herein (e.g., Compound F), an additional active agent (e.g., an antimicrobial or antibacterial agent) and a pharmaceutically acceptable carrier.
  • a compound having general Formula A' as described herein e.g., Compound F
  • an additional active agent e.g., an antimicrobial or antibacterial agent
  • pharmaceutical composition refers to a preparation of any boronic acid or ester compound as described herein, with other chemical components such as pharmaceutically acceptable and suitable carriers and excipients, and optionally with additional active agents, such as another antimicrobial agent.
  • additional active agents such as another antimicrobial agent.
  • the purpose of a pharmaceutical composition is to facilitate administration of the compound (or combination of compounds) to a subject, or optionally to facilitate its application (contacting) in or on an inanimate object.
  • pharmaceutically acceptable carrier refers to a carrier or a diluent that does not cause significant irritation to an organism and does not inhibit the distribution, therapeutic properties or otherwise does not abrogate the biological activity and properties of the administered or applied compound.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration or application of a drug.
  • compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more pharmaceutically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredient(s) into preparations which can be used pharmaceutically.
  • Proper formulation is dependent upon the route of administration chosen.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (see e.g., Fingl et al, 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p. l).
  • the pharmaceutical composition may be formulated for administration in either one or more of routes depending on whether local or systemic treatment or administration is of choice, and on the area to be treated. Administration may be done orally, by inhalation, or parenterally, for example by intravenous drip or intraperitoneal, subcutaneous, intramuscular or intravenous injection, or topically (including transdermally, ophtalmically, vaginally, rectally, intranasally).
  • the pharmaceutical composition is formulated as a wound dressing, using methods known in the art.
  • compositions to be administered or otherwise applied will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
  • compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA (the U.S. Food and Drug Administration) approved kit, which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may, for example, comprise metal or plastic foil, such as, but not limited to a blister pack or a pressurized container (for inhalation).
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions for human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S.
  • compositions comprising active ingredient(s) according to embodiments of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of a particular medical condition, disease or disorder, as is detailed herein.
  • the pharmaceutical composition presented herein is packaged in a packaging material and identified in print, in or on the packaging material, for use in inhibiting a growth of a pathogenic microorganism in a subject in need thereof.
  • the product is a medicament.
  • the term “medicament” is used interchangeably with the phrase “pharmaceutical composition”.
  • the product is also referred to herein as an article or article-of-manufacture.
  • a compound having general Formula A as described herein for use in a method of inhibiting a growth of a pathogenic microorganism in or on a substrate, as defined herein.
  • the compound having general Formula A as described herein is for use in a method of inhibiting a growth of a pathogenic microorganism in a subject which comprises the living tissue.
  • Compound F for use in a method of inhibiting a growth of a pathogenic microorganism in or on a substrate, as defined herein, wherein the compound having general Formula A' is used in combination with an additional active agent (e.g., antimicrobial agent or antibacterial agent).
  • an additional active agent e.g., antimicrobial agent or antibacterial agent.
  • the compound having general Formula A' as described herein is for use in a method of inhibiting a growth of a pathogenic microorganism in a subject which comprises the living tissue, wherein the compound having general Formula A' is used in combination with an additional active agent (e.g., antimicrobial agent or antibacterial agent).
  • an additional active agent e.g., antimicrobial agent or antibacterial agent.
  • a method of inhibiting a growth of a pathogenic microorganism in or on a substrate which is effected by contacting the substrate with an antimicrobial effective amount of a compound having general Formula A as described herein.
  • a method of inhibiting a growth of a pathogenic microorganism in or on a substrate which is effected by co-contacting, as described herein, the substrate with an antimicrobial effective amount of a compound having general Formula A' as described herein (e.g., Compound F) and with an additional active agent (e.g., an antimicrobial or antibacterial agent).
  • a compound having general Formula A' as described herein e.g., Compound F
  • an additional active agent e.g., an antimicrobial or antibacterial agent
  • an antimicrobial effective amount describes an amount of an antimicrobial agent which will effect one or more parameters of a microorganism, including death, eradication, elimination, reduction in number, reduction of growth rate, inhibition of growth, change in population distribution of one or more species of microbial life forms, as described herein.
  • an antimicrobial effective amount is an amount that reduces to some extent the population of a microorganism in or on a substrate.
  • an antimicrobial effective amount is an amount that reduces by at least 10 %, 20 %, 30 %, 40 %, 50 % or to a higher extent, the population of a microorganism in or on a substrate.
  • reduction in the population of a microorganism in or on a substrate is determined by measuring the number of colony forming units of the microorganism grown upon contacting an antimicrobial agent and comparing it to the number of colony forming units of the microorganism when grown without an antimicrobial agent.
  • a substrate can be a living tissue or an inanimate object, as these terms are defined hereinabove.
  • the method when the substrate is a living tissue, such as a bodily site of a subject, the method is effected by administering to the subject an antimicrobial effective amount of a compound having general Formula A as described herein.
  • the administration can be effected orally, rectally, intravenously, topically, intranasally, intradermally, transdermally, subcutaneously, intramuscularly, intrperitoneally or by intrathecal catheter.
  • a compound having general Formula A or A' as described herein can be administered either per se or as a part of a pharmaceutical composition as described herein.
  • the mode of administration is selected to suite the medical condition which is being treated. For example, in treating a systemic infection where rapid distribution of the therapeutic agent(s) is needed, drug(s) are typically administered orally or intravenously. When treating a local infection, the drug is administered locally, topically, transdermally, subcutaneously or intramuscularly.
  • an exemplary method of inhibiting a growth of a pathogenic microorganism in a subject is effected topically by applying a composition containing the active compound (a compound having general Formula A as described herein) onto a wound.
  • the mode of inhibiting a growth of a pathogenic microorganism in or on the substrate is effected typically by dipping, spraying, coating, or otherwise applying the active compound(s) or a composition comprising the same, as described herein, in or on the substrate.
  • a catheter for prolonged percutaneous use can be coated with a composition containing a compound having general Formula A as described herein so as to inhibit the growth of a pathogenic microorganism therein or thereon.
  • a catheter for prolonged percutaneous use can also be coated with a composition containing a compound having general Formula A' as described herein (e.g., Compound F) and an additional active agent (e.g., antimicrobial or antibacterial agent) so as to inhibit the growth of a pathogenic microorganism therein or thereon.
  • additional active agent e.g., antimicrobial or antibacterial agent
  • Other medical devices can be similarly coated, as is detailed hereinbelow.
  • pathogenic microorganism is used to describe any microorganism which can cause a disease or infection in a higher organism, such as humans or any animals grown for commercial or recreational purposes, fish, poultry, insects (e.g., bees) and mammals.
  • a pathogenic microorganism is one that causes diseases and adverse effects in humans, hence, a pathogenic microorganism in the context of embodiments of the present invention is regarded as a cause of a medical condition associated therewith.
  • the pathogenic microorganism may belong to any family of organisms such as, but not limited to, prokaryotic organisms, eubacterium, archaebacterium, eukaryotic organisms, yeast, fungi, algae, protozoa, and other microscopic parasites.
  • Compound K was found to be a highly efficient agent against a wide spectrum of bacteria, including Gram-negative bacteria and Gram-positive bacteria, when used either alone or in combination with an antibiotic.
  • Compound F was found to be a highly efficient agent against a wide spectrum of bacteria, including Gram-negative bacteria and Gram-positive bacteria, when used either alone or in combination with an antibiotic.
  • the phrase "pathogenic microorganism” refers to a bacterium (or a bacterial strain).
  • bacteria refers to all prokaryotic organisms, including those within all of the phyla in the Kingdom Procaryotae. It is intended that these terms encompass all microorganisms considered to be bacteria including Mycoplasma, Chlamydia, Actinomyces, Streptomyces, and Rickettsia. All forms of bacteria are included within this definition including cocci, bacilli, spirochetes, spheroplasts, protoplasts, etc. Also included within these terms are prokaryotic organisms that are Gram-negative or Gram-positive.
  • Gram-negative and “Gram- positive” refer to staining patterns with the Gram-staining process, which is well known in the art. (See e.g., Finegold and Martin, Diagnostic Microbiology, 6th Ed., CV Mosby St. Louis, pp. 13-15 (1982)).
  • Gram-positive bacteria are bacteria that retain the primary dye used in the Gram stain, causing the stained cells to generally appear dark blue to purple under the microscope.
  • Gram-negative bacteria do not retain the primary dye used in the Gram stain, but are stained by the counterstain. Thus, Gram- negative bacteria generally appear red.
  • bacteria are continuously cultured.
  • bacteria are uncultured and existing in their natural environment (e.g., at the site of a wound or infection) or obtained from patient tissues (e.g., via a biopsy). Bacteria may exhibit pathological growth or proliferation.
  • Non-limiting examples of bacteria include bacteria of a genus selected from the group including Salmonella, Shigella, Escherichia, Enterobacter, Serratia, Proteus, Yersinia, Citrobacter, Edwardsiella, Providencia, Klebsiella, Hafnia, Ewingella, Kluyvera, Morganella, Planococcus, Stomatococcus, Micrococcus, Staphylococcus, Vibrio, Aeromonas, Plessiomonas, Haemophilus, Actinobacillus, Pasteurella, Mycoplasma, Ureaplasma, Rickettsia, Coxiella, Rochalimaea, Ehrlichia, Streptococcus, Enter ococcus, Aerococcus, Gemella, Lactococcus, Leuconostoc, Pedicoccus, Bacillus, Corymb acterium, Arcanobacterium, Actinomyces, Rhodococcus, Listeria
  • the pathogenic bacteria are of one or more of the following species: Acinetobacter baumanii, Belicobacter pylori, Burkholderia multivorans, Campylobacter jejuni, Deinococcus radiodurans , E.
  • the bacteria are of the species Staphylococcus aureus.
  • the bacteria are of the species Staphylococcus epidermidis
  • a wide-spectrum activity range is oftentimes indicative of activity in inhibiting the growth of eukaryotic microorganisms such as fungi, as well as prokaryotic microorganisms such as bacteria.
  • pathogenic fungi against which a compound having general Formula A as described herein can be efficiently used according to the present embodiments include, without limitation, fungi of the genus Absidia: Absidia corymbifera; genus Ajellomyces: Ajellomyces capsulatus, Ajellomyces dermatitidis; genus Arthroderma: Arthroderma benhamiae, Arthroderma fulvum, Arthroderma gypseum, Arthroderma incurvatum, Arthroderma otae, Arthroderma vanbreuseghemii; genus Aspergillus: Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger; genus Blastomyces: Blastomyces dermatitidis; genus Candida: Candida albicans, Candida glabrata, Candida guilliermondii, Candida krusei, Candida parapsilosis, Candida tropicalis, Candida pell
  • pathogenic parasites and protozoa against which a compound having general Formula A as described herein may be used according to the present embodiments include, without limitation, various types of amoeba, Leishmania spp, Plasmodium falciparum Trypanosoma cruzi (causing Chagas' disease), Trypanosoma bucei (causing "sleeping sickness"), Plasmodium vivax (causing malaria), Cryptosporidium parvum (causing cryptosporidiosis), Cyclospora cayetanensis, Giardia lamblia (causing giardiasis) and many others.
  • Resistance of microorganism to antimicrobial agents is the ability of a microorganism to withstand the antimicrobial effects of any given agents (antibiotics).
  • the antimicrobial action of any given agent is putting an environmental pressure on the target (and also non-targeted) microorganisms.
  • the microorganisms which have a mutation that will allow it to survive will live on to reproduce. These newly evolved strain(s) will then pass this trait to their offspring, which will constitute a fully resistant generation.
  • an exemplary compound having general Formula A as described herein designated herein as Compound K, was shown to be effective against methicillin resistant Staphylococcus aureus (MRSA).
  • Compound F as described herein was shown to be effective against methicillin resistant Staphylococcus aureus (MRSA).
  • Both exemplary Compounds K and F were shown to be effective when used either alone or in combination with an antibiotic. Since this strain is associated with severe medical conditions, the use of a compound having general Formula A or A' as described herein may be one of the means to combat this and other antibiotic resistant pathogens and ameliorate or cure the medical conditions associated therewith.
  • Non-limiting examples of conventional antibacterial agents include, but are not limited to, gentamicin, ampicillin, amikacin (AK), cefazolin, ceftriaxone, clindamycin, cephalothin, ciprofloxacin, chloramphenicol, ceftazidime (CAZ), cefepime (CPE), erythromycin, trimethoprim/sulfamethoxazole (T/S), gatifloxacin, piperacillin/tazobactam (P/T), aztreonam (AZT), imipenem, levofloxacin, penicillin, oxacillin, nitrofurantoin, linezolid, moxifloxacin, meropenem (MER), tobramycin (TO), ciprofloxacin (CP), tetracycline, vancomycin, rifampin synercid, streptomycin Synergy, colistin (CT) and chloramphenicol
  • antimicrobial agent examples include polyene-based antifungal agents such as amphotericin, amphotericin B, nystatin and pimaricin, amphotericin B liposomal formulations (AmBisome, Abelcet, Amphocil), azole-based antifungal agents such as fluconazole, itraconazole and ketoconazole, allylamine- or morpholine-based antifungal agents such as allylamines (naftifine, terbinafme), and antimetabolite-based antifungal agents such as 5-fluorocytosine, and fungal cell wall inhibitor such as echinocandins like caspofungin, micafungin and anidulafungin.
  • polyene-based antifungal agents such as amphotericin, amphotericin B, nystatin and pimaricin, amphotericin B liposomal formulations (AmBisome, Abelcet, Amphocil)
  • the pharmaceutical composition described herein is packaged in a packaging material and identified in print, in or on the packaging material, for use in the treatment of a medical condition associated with the pathogenic microorganism in a subject in need thereof.
  • the medicament as described herein for inhibiting a growth of a pathogenic microorganism in a subject presented hereinabove is being for use in the treatment of a medical condition associated with the pathogenic microorganism in a subject in need thereof.
  • a compound having general Formula A as described herein is for use in a method of treating a medical condition associated with the pathogenic microorganism in a subject in need thereof.
  • the method as described herein is being for treating a medical condition associated with the pathogenic microorganism in a subject in need thereof.
  • a compound having general Formula A' as described herein e.g., Compound F
  • an additional active agent e.g., an antimicrobial or antibacterial agent
  • the method as described herein is being for treating a medical condition associated with the pathogenic microorganism in a subject in need thereof.
  • a subject in need thereof describes a subject that has one or more tissues, organs or any bodily site, infected by the pathogenic microorganism.
  • the subject is an animal, preferably a mammal, more preferably a human being.
  • the term "associated" in the context embodiments of the present invention means that at least one adverse manifestation of the medical condition is caused by a pathogenic microorganism.
  • the phrase "medical condition associated with a pathogenic microorganism” therefore encompasses medical conditions of which the microorganism may be the primary cause of the medical condition or a secondary effect of the main medical condition(s).
  • Medical conditions associated with a pathogenic microorganism include infections, infestation, contaminations and transmissions by or of pathogenic microorganisms such as those described herein.
  • a disease causing infection is the invasion into the tissues of a plant or an animal by pathogenic microorganisms.
  • the invasion of body tissues by parasitic worms and other higher pathogenic organisms such as lice is oftentimes referred to as infestation.
  • Invading organisms such as bacteria typically produce toxins that damage host tissues and interfere with normal metabolism; some toxins are actually enzymes that break down host tissues. Other bacterial substances may inflict their damage by destroying the host's phagocytes, rendering the body more susceptible to infections by other pathogenic microorganisms. Substances produced by many invading organisms cause allergic sensitivity in the host. Infections may be spread via respiratory droplets, direct contact, contaminated food, or vectors, such as insects. They can also be transmitted sexually and from mother to fetus.
  • Examples of medical conditions and diseases caused by bacterial infections include, without limitation, actinomycosis, anthrax, aspergillosis, bacteremia, bacterial skin diseases, bartonella infections, botulism, brucellosis, burkholderia infections, Campylobacter infections, candidiasis, cat-scratch disease, chlamydia infections, cholera, Clostridium infections, coccidioidomycosis, cryptococcosis, dermatomycoses, diphtheria, ehrlichiosis, epidemic louse borne typhus, Escherichia coli infections, fusobacterium infections, gangrene, general infections, general mycoses, gonorrhea, gram-negative bacterial infections, gram-positive bacterial infections, histoplasmosis, impetigo, klebsiella infections, legionellosis
  • Fungal infections or mycoses are classified depending on the degree of tissue involvement and mode of entry into the host. Main classes are superficial, subcutaneous, systemic and opportunistic infections.
  • Non-limiting examples of medical conditions associated with fungi or other eukaryotes include superficial mycoses infections, "ringworm” or “tinea”, candidiasis or “thrush”, subcutaneous mycoses, sporotrichosis, systemic mycoses, histoplasmosis, blastomycosis, coccidiomycosis, paracoccidiodomycosis, aspergillosis, systemic candidosis and cryptococcosis and Pneumocystis.
  • Medical conditions associated with pathogenic parasites and protozoa which may be treatable by a compound having general Formula A as described herein, include, without limitation, acanthamoeba infection, African trypanosomiasis (sleeping sickness), alveolar echinococcosis, amebiasis (entamoeba histolytica infection), American trypanosomiasis (Chaga's disease), ancylostoma infection (hookworm infection, cutaneous larva migrans, CLM), angiostrongylus infection (angiostrongyliasis), angiostrongyliasis (angiostrongylus infection), anisakis infection (anisakiasis), anisakiasis (anisakis infection), ascariasis (intestinal roundworms), babesia infection (babesiosis), babesiosis (babesia infection), balantidiasis (balantidium infection), balantidium infection), balantidium infection
  • ebiasis echinococcosis
  • elephantiasis filamentariasis, lymphatic filariasis
  • endolimax nana infection Entamoeba coli infection, Entamoeba dispar infection, Entamoeba heartmanni infection, Entamoeba histolytica infection (amebiasis), Entamoeba polecki infection, Enterobiasis (pinworm infection), fasciola infection (fascioliasis), fascioliasis (fasciola infection), fasciolopsiasis (fasciolopsis infection), fasciolopsis infection (fasciolopsiasis), filariasis (lymphatic filariasis, elephantiasis), foodborne diseases, giardiasis (giardia infection, "beaver fever”), gnathostoma infection (gnathost
  • biofilm refers to an aggregate of living cells which are stuck to each other and/or immobilized onto a surface as colonies.
  • the cells are frequently embedded within a self-secreted matrix of extracellular polymeric substance (EPS), also referred to as “slime”, which is a polymeric sticky mixture of nucleic acids, proteins and polysaccharides.
  • EPS extracellular polymeric substance
  • the living cells forming a biofilm can be cells of a unicellular microorganism (prokaryotes, archaea, bacteria, eukaryotes, protists, fungi, algae, euglena, protozoan, dinoflagellates, apicomplexa, trypanosomes, amoebae and the likes), or cells of multicellular organisms in which case the biofilm can be regarded as a colony of cells (like in the case of the unicellular organisms) or as a lower form of a tissue.
  • a unicellular microorganism prokaryotes, archaea, bacteria, eukaryotes, protists, fungi, algae, euglena, protozoan, dinoflagellates, apicomplexa, trypanosomes, amoebae and the likes
  • the biofilm can be regarded as a colony of cells (like in the case of the unicellular organisms)
  • the cells are of microorganism origins, and the biofilm is a biofilm of microorganisms, such as bacteria and fungi.
  • the cells of a microorganism growing in a biofilm are physiologically distinct from cells in the "planktonic form" of the same organism, which by contrast, are single-cells that may float or swim in a liquid medium.
  • Biofilms can go though several life-cycle steps which include initial attachment, irreversible attachment, one or more maturation stages, and dispersion.
  • anti-biofilm formation activity or "anti-quorum sensing activity”, as these equivalent terms are used herein interchangeably, refer to the capacity of a substance to effect the prevention of formation of a biofilm of bacterial, fungal and/or other cells; and/or to effect a disruption and/or the eradication of an established and/or matured biofilm of bacterial, fungal and/or other cells; and/or to effect a reduction in the rate of buildup of a biofilm of bacterial, fungal and/or other cells on a surface of a substrate.
  • anti-biofilm formation compound/composition/agent refers to a substance having an anti-biofilm formation activity, as defined herein.
  • ester compounds described herein can be used in methods and compositions which are directed at anti-biofilm formation purposes, as detailed hereinbelow.
  • the acid compound as described herein can also be used in methods and compositions which are directed at anti-biofilm formation purposes, as detailed hereinbelow.
  • the activity of preventing or reducing the formation of a biofilm, and the activity of disrupting a biofilm which has been established before treatment may be achieved by identical or different ABF agents.
  • the prevention or reducing of forming a biofilm assumes that the biofilm has not yet been formed, and hence the presence of the ABF agent is required also in cases where no biofilm is present or detected.
  • the biofilm has already been formed and the disruption thereof is desirable; thus in these cases the ABF agent according to embodiments of the present invention, may be introduced before, during or after the detection of presence of the biofilm.
  • preventing in the context of the formation of a biofilm, indicates that the formation of a biofilm is essentially nullified or is reduced by at least 20 % of the appearance of the biofilm in a comparable situation lacking the presence of the ABF agent. Alternatively, preventing means a reduction to at least 15 %, 10 % or 5 % of the appearance of the biofilm in a comparable situation lacking the presence of the ABF agent. Methods for determining a level of appearance of a biofilm are known in the art.
  • disrupting in the context of the formation of a biofilm, indicates that the mass of a biofilm is reduced to at least 20 % of its mass prior to the introduction of the ABF agent presented herein.
  • disrupting means a reduction in the mass of the biofilm to at least 30 %, 40 % or 50 % of its original mass prior to the introduction of the ABF agent.
  • disrupting a biofilm, or reducing a biofilm mass results in converting at least a portion of the biofilm (e.g., at least 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 % and even 100 %) into planktonic cells that formed the biofilm.
  • an anti-biofilm composition which comprises a compound having general Formula A as described herein, and can optionally further comprise a carrier.
  • an anti-biofilm composition which comprises a compound having general Formula A' as described herein (e.g., Compound F), and an additional active agent (e.g., an antimicrobial or antibacterial agent), and can optionally further comprise a carrier.
  • a compound having general Formula A' as described herein e.g., Compound F
  • an additional active agent e.g., an antimicrobial or antibacterial agent
  • a composition as presented herein is packaged in a packaging material and identified in print, in or on the packaging material, for use in reducing or preventing the formation of a bio film and/or disrupting a biofilm in or on a substrate, as described herein.
  • Biofilms may form on living tissues or form on non-living, organic or inorganic substrates, referred to herein an inanimate objects or substrates, and constitute a prevalent mode of microbial life in natural, industrial and medical settings.
  • the substrate can be a living tissue or an inanimate object, as described herein.
  • a composition comprising a compound having general Formula A or A' as described herein is referred to as a pharmaceutical composition, as described herein.
  • the pharmaceutical composition presented herein is packaged in a packaging material and identified in print, in or on the packaging material, for use in reducing or preventing the formation of a biofilm and/or disrupting a biofilm in the living tissue or in a subject in need thereof, which comprises said living tissue, as described hereinabove.
  • the pharmaceutical composition is identified for use in the treatment of a medical condition in which reducing or preventing the formation of the biofilm and/or disrupting the biofilm in the living tissue is beneficial.
  • a use of a compound having general Formula A as described herein in the manufacture of a product for reducing or preventing the formation of a biofilm and/or disrupting a biofilm in or on a substrate there is provided a use of a compound having general Formula A' as described herein (e.g., Compound F) in the manufacture of a product for reducing or preventing the formation of a biofilm and/or disrupting a biofilm in or on a substrate, wherein the medicament further comprises an additional active agent or is for use in combination with an additional active agent (e.g., an antimicrobial or antibacterial agent).
  • an additional active agent e.g., an antimicrobial or antibacterial agent
  • the product is a medicament for reducing or preventing the formation of a biofilm and/or disrupting a biofilm in the living tissue or in a subject in need thereof, which comprises said living tissue, as described hereinabove.
  • the medicament is for the treatment of a medical condition in which reducing or preventing the formation of the biofilm and/or disrupting the biofilm in the living tissue is beneficial.
  • a compound having general Formula A as described herein for use in a method of reducing or preventing the formation of a biofilm and/or disrupting a biofilm in or on a substrate.
  • a compound having general Formula A' as described herein for use, in combination with an additional active agent (e.g., an antimicrobial or antibacterial agent) in a method of reducing or preventing the formation of a biofilm and/or disrupting a biofilm in or on a substrate.
  • an additional active agent e.g., an antimicrobial or antibacterial agent
  • a compound having general Formula A or A' as described herein is for use in a method of reducing or preventing the formation of a biofilm and/or disrupting a biofilm the living tissue or in a subject in need thereof, which comprises said living tissue, as described hereinabove.
  • a compound having general Formula A or A' as described herein is identified for use in the treatment of a medical condition in which reducing or preventing the formation of the biofilm and/or disrupting the biofilm in the living tissue is beneficial.
  • a method of reducing or preventing the formation of a biofilm and/or disrupting a biofilm in or on a substrate which is effected by contacting the substrate with an antimicrobial effective amount of a compound having general Formula A as described herein.
  • a method of reducing or preventing the formation of a biofilm and/or disrupting a biofilm in or on a substrate which is effected by co-contacting the substrate with an antimicrobial effective amount of a compound having general Formula A' as described herein (e.g., Compound F) and with an additional active agent (e.g., an antimicrobial or antibacterial agent).
  • a compound having general Formula A' as described herein e.g., Compound F
  • an additional active agent e.g., an antimicrobial or antibacterial agent
  • the phrase "antimicrobial effective amount” describes an amount of an anti-biofilm ingredient which will reduce, prevent and/or disrupt, at least to some extent, the formation of a biofilm of a microorganism, as described herein, in or on a substrate harboring the microorganism.
  • the method when the substrate is a living tissue, such as a bodily site of a subject, the method is effected by administering to the subject an antimicrobial effective amount of a compound having general Formula A or A' as described herein.
  • the administration can be effected orally, rectally, intravenously, topically, intranasally, intradermally, transdermally, subcutaneously, intramuscularly, intrperitoneally or by intrathecal catheter.
  • a compound having general Formula A or A' as described herein can administered either per se or as a part of a pharmaceutical composition as described herein.
  • the method is being for treating a medical condition in which reducing or preventing the formation of the biofilm and/or disrupting the biofilm in the living tissue is beneficial.
  • the mode of administration in the context of reducing, preventing and/or disrupting a biofilm formation is similar to the mode of administration in the context of inhibiting the growth of a microorganism.
  • an exemplary method of reducing, preventing and/or disrupting a biofilm in a subject is effected topically by applying a composition containing the active ingredient(s) on a wound.
  • This method is also effective in reducing, preventing and/or disrupting a biofilm formation on the wound dressing used to treat the wound.
  • streptococcal infections which is the basis for the intense virulence of many streptococcal species.
  • the mode of reducing, preventing and/or disrupting a biofilm formation in or on the substrate is effected typically by dipping, spraying, coating, or otherwise applying a formulation containing the active ingredient(s) in or on the substrate.
  • a catheter for prolonged percutaneous use can be coated with a formulation containing a compound having general Formula A or A' as described herein so as to reduce, prevent and/or disrupt, at least to some extent, the formation of a biofilm therein or thereon.
  • the anti-biofilm composition as described herein is used to reduce, prevent and/or disrupt, at least to some extent, the formation of a biofilm in a medical device by coating or impregnating the device with an amount of the composition capable of reducing, preventing and/or disrupting formation of the biofilm.
  • Medical device includes any material or device that is used on, in, or through a subject's body, for example, in the course of medical treatment (e.g., for a disease or injury).
  • Medical devices include, but are not limited to, such items as medical implants, wound care devices, drug delivery devices, and body cavity and personal protection devices.
  • the medical implants include, but are not limited to, urinary catheters, intravascular catheters, dialysis shunts, wound drain tubes, skin sutures, vascular grafts, implantable meshes, intraocular devices, heart valves, and the like.
  • Wound care devices include, but are not limited to, general wound dressings, biologic graft materials, tape closures and dressings, and surgical incise drapes.
  • Drug delivery devices include, but are not limited to, needles, drug delivery skin patches, drug delivery mucosal patches and medical sponges.
  • Body cavity and personal protection devices include, but are not limited to, tampons, sponges, surgical and examination gloves, and toothbrushes.
  • birth control devices include, but are not limited to, intrauterine devices (IUDs), diaphragms and condoms. Coating or impregnating the medical device with the anti-biofilm composition as described herein may be effected via various techniques known in the art (see, for example, in US Pat. Nos.
  • the antimicrobial and ABF activity of 4-fluorophenylboronic acid (Compound F) and of an exemplary ester compound having general Formula A as described herein (Compound K) has been shown to be enhanced when acting in the presence of another active agent or bioactive agent, such as another antimicrobial agent (e.g., an antibiotic).
  • another antimicrobial agent e.g., an antibiotic
  • the antimicrobial and ABF activity of various antimicrobial agents was shown to be enhanced when acting in the presence of 4-fluorophenylboronic acid and of an exemplary ester compound having general Formula A as described herein.
  • any of the compounds, compositions or medicaments presented herein may be used in combination with an additional active agent, or alternatively, the compositions or medicaments as presented herein may include an additional active agent.
  • antimicrobial compositions for compounds having general Formula A, which comprise a compound having general Formula A' as described herein and an additional active agent, as described herein.
  • the additional active agent e.g., an additional antimicrobial agent
  • the additional active agent is not an ingredient of the composition or medicament containing a compound having general Formula A' as described herein, it can be held as a separate composition in a separate container and packaged together with the container holding the composition containing a compound having general Formula A' as described herein, or it can be packaged separately.
  • the methods as described herein are effected by co-contacting the substrate with a compound having general Formula A' as described herein and an additional active agent as described herein.
  • the additional agent can be administered to or otherwise contacted with the substrate, concomitantly, concurrently, simultaneously, consecutively or sequentially with a compound having general Formula A' as described herein.
  • composition which comprises both a compound having general Formula A' as described herein and an additional active agent, and optionally further comprises a carrier.
  • the composition can be an antimicrobial composition, an anti-bio film composition or a pharmaceutical composition, as described herein.
  • a method as described herein which is effected by co-contacting the substrate with a compound having general Formula A' as described herein and with an additional active agent.
  • a method as described herein is effected by co- administering to a subject in need thereof a compound having general Formula A' as described herein and an additional active agent.
  • Co-contacting and co-administering can be effected concomitantly, concurrently, sequentially or consecutively.
  • a method of inhibiting a growth of a pathogenic microorganism in a subject which is effected by co- administering to a subject in need thereof an synergistically effecting amount of a compound having general Formula A' as described herein and an antimicrobial agent.
  • a method of reducing or preventing the formation of a biofilm and/or disrupting a biofilm in or on a substrate which is effected co- contacting the substrate with an antimicrobial effective amount of a compound having general Formula A' as described herein and an antimicrobial agent.
  • the additional active agent is an antimicrobial agent, and according to some embodiments, the additional antimicrobial agent acts in synergy with a compound having general Formula A' as described herein in inhibiting a growth of a microorganism and/or within the ABF activity exhibited thereby.
  • a synergy between two antimicrobial agents may be determined by methods well known in the art. An exemplary method is demonstrated in Example 2 in the Examples section that follows.
  • the antimicrobial agent is an antibacterial agent (an antibiotic).
  • an antibacterial agents which are suitable for use in the context of these embodiments of the present invention include, without limitations, amikacin, amoxicillin, ampicillin, azithromycin, Aztreonam, cefepime, cefonicid, cefotetan, ceftazidine, cephalosporin, cephamycin, Chloramphenicol, chlortetracycline, ciprofloxacin, clarithromycin, clindamycin, colistin, cycloserine, dalfopristin, doxycycline, ephalothin, erythromycin, gentamicin, kanamycin, levofloxacin, lincosamide, linezolid, meropenem, moxifloxacin, mupirocin, neomycin, oxytetracycline, piperacillin, penicillin, quinupristin
  • the additional antimicrobial agent used together with a compound having general Formula A' as described herein is other than a beta-lactam antibiotic agent, or is a non beta-lactam antimicrobial agent.
  • Beta-lactam antimicrobial/antibacterial agents are those agents having a
  • azetidin-2-one motif, and include, without limitation, amoxicillin, ampicillin, azlocillin, aztreonam (Azactam), benzathine penicillin, benzylpenicillin (penicillin G), carbenicillin, cefaclor, cefamandole, cefazolin, cefepime, cefixime, cefotaxime, cefotetan, cefoxitin, cefpirome, cefpodoxime (ATDOX-200), ceftazidime, ceftriaxone, cefuroxime, cephalexin, cephalothin, clavulanic acid, cloxacillin, dicloxacillin, doripenem, ertapenem, faropenem, flucloxacillin, imipenem, meropenem, methicillin, mezlocillin, nafcillin, nocardicin A, oxacillin, penicillin, phenoxymethylpen
  • non beta-lactam antimicrobial agent refers to a bacteriostatic or a bactericidic (antibiotic) agent which is not of the beta-lactam class of antibiotics or one not having a "azetidin-2-one" motif.
  • non beta-lactam antibacterial agent include, without limitation, mafenide, cephalothin, vancomycin, cefazolin, imipenemen, clyndamycin, synercid, erythromycin, tetracycline, ciprofloxacin, tigecycline and ertapenem, as well as aminoglycoside antibiotics such as, but not limited to, amikacin, apramycin, arbekacin, butirosin, dibekacin, fortimycin, G- 418, gentamicin, hygromycin, donkacin, dibekacin, netlmicin, istamycin, isepamycin, kanamycin, lividomycin, neamine, neomycin, paromomycin, ribostamycin, sisomycin, spectinomycin, streptomycin and tobramycin.
  • aminoglycoside antibiotics such as, but not limited to, amikacin, apramycin,
  • the compound having Formula A' is 4-fluorophenylboronic acid (Compound F, BL 6031) and the additional active agent is Tobramycin.
  • the compound having Formula A' is Compound K (BL 6030) and the additional active agent is gentamicin.
  • the compound having Formula A' is Compound K (BL 6030) and the additional active agent is Tobramycin.
  • Compounds having Formula A or A' as described herein, either alone or in combination with an additional active agent can be applied on or in various substrates, for exhibiting antimicrobial or anti-biofouling effect, as described herein.
  • an additional active agent e.g., an antimicrobial agent
  • substrate as used herein, is as described hereinabove, and further refers to any surface, structure, product or material which can support, harbor or promote the growth of a microorganism.
  • Non-limiting examples include the inner walls of a storage container that is routinely treated with anti-microbial preferably anti-fungal agents, a soil and/or soil enrichment supplements, any agricultural product or crop such as wood, fiber, fruit, vegetable, flower, extract, horticultural crop and any other processed or unprocessed agricultural product or crop which are produced from organic origins such living plants or animals, a cosmetic product, a building, warehouse, compartment, container or transport vehicle, a dye or a paint and any other materials and industrial compounds used for which require protection of their surfaces against microbes, moulds and fungi attacks, such as, for example, construction materials.
  • Such products include, for example, food products, agricultural products, cosmetic products and many more. Due to their effect in reducing the load of microorganisms, the compounds described herein can be utilized as a preservative in such products.
  • the substrate is a medical device or any other device which is intended for contacting a living tissue, as defined herein.
  • a wound dressing having a compound represented by Formula A or A' as described herein, either alone or in combination with an additional active agent (e.g., an antimicrobial agent).
  • an additional active agent e.g., an antimicrobial agent
  • a method of reducing the load of a pathogenic microorganism in or on a substrate the method being effected by contacting the substrate an antimicrobial effective amount of a compound having Formula A or A' as described herein, optionally in combination with an additional antimicrobial agent.
  • reducing the load refers to a decrease in the number of the microorganism(s), or to a decrease in the rate of their growth or both in the substrate as compared to a non-treated substrate.
  • Staphylococcus aureus MRSA; strain USA 400
  • Staphylococcus epidermidis Strain ATCC 35984
  • Pseudomonas aeruginosa Strain ATCC 27853
  • MIC Minimum inhibitory concentration
  • MEC minimum biofilm eliminating concentration
  • MIC and MBEC values for each antibacterial agent (antibiotic), the tested compound and combination thereof are determined according to the procedure described by Ceri et al. (Methods Enzymol 337:377-85, 2001) using SENSITITRE ® Gram Positive and Gram Negative MIC Plates (TREC Diagnostics Systems, Cleveland, OH) and BiofilmTM test panels (Inovotech Inc., Edmonton, AB Canada).
  • the combined treatment with Compound F and the tested antibiotic is considered synergic when the resulting FIC value is equal to or lower than 0.5.
  • Tables 1 and 2 below show the effect of Compound F in combination with clinical non beta-lactam antibiotics on the growth of methicillin-resistant Staphylococcus aureus.
  • the combinations of Compound F with Cephalothin, Vancomycin, Cefazolin, Imipenemen, Clyndamycin, Synercid, Erythromycin, or Tetracycline resulted in synergic inhibition of bacterial growth.
  • MIC Minimum Inhibitory Concentration
  • Antibacterial-agent combinations (compound F combined with clinical antibiotics) at minimal concentrations for inhibiting methicillin-resistant Staphylococcus aureus USA
  • MIC Minimum Inhibitory Concentration
  • FIC [(lowest MIC of compound F in combination) / (lowest MIC of compound F as single agent)] + [(lowest MIC of antibiotic in combination) / [(lowest MIC of antibiotic as single agent)].
  • MIC Minimum Inhibitory Concentration
  • Antibacterial-agent combinations (compound F combined with clinical antibiotics) at minimal concentrations for inhibiting Psuedomonas aeruginosa ATCC 27853 (sorted by FIC)
  • FIC [(lowest MIC of compound F in combination) / (lowest MIC of compound F as single agent)] + [(lowest MIC of antibiotic in combination) / [(lowest MIC of antibiotic as single agent)].
  • Phenyloxaboronidines are synthesized by reacting a respective phenylboronic acid with an equimolar amount of N-methyldiethanolamine in an organic solvent (e.g., toluene), optionally in the presence of a dehydrating agent (e.g., molecular sieves).
  • an organic solvent e.g., toluene
  • a dehydrating agent e.g., molecular sieves
  • Compound K is also referred to as BL-6030 or EDP-5.
  • the obtained viscous oil was solidified to a white solid upon standing on the bench at 20 °C for several days. This solid was filtered under vacuum and washed with petroleum ether several times. The solid was dried on a sintered funnel, then transferred to a vial and dried for 24 hours in a desiccator connected to a diaphragm pump, so as to afford Compound K as a white solid powder (14.9 grams, 94 % yield).
  • QSI quorum sensing inhibitors
  • the bioFILM PATM developed by InnovotechTM Inc., Canada, is a broth dilution antimicrobial susceptibility panel test was designed for use in determining antimicrobial agent susceptibility of both planktonic and biofilm Pseudomonas aeruginosa. This test has a panel of various antimicrobial agents which are diluted in recovery buffer at categorical breakpoint concentrations defined by Clinical and Laboratory Standard InstituteTM (CLSI).
  • CLSI Clinical and Laboratory Standard Institute
  • Panel wells were inoculated with planktonic and biofilm Pseudomonas aeruginosa using a 95 peg inoculation lid. Panels and pegged lids were then incubated at 35 °C for a minimum of 16 hours. Planktonic susceptibility and resistance was determined by measuring inhibition and growth in the presence of antimicrobial agents after 16-24 hours incubation at 35 °C. The pegged lid containing the biofilm bacteria that have been exposed to the antimicrobial agents was placed in a recovery media. Biofilm susceptibility and resistance was determined by measuring inhibition and growth after incubation for additional 16-24 hours at 35 °C.
  • the Gram-positive test panel was designed for use in determining antimicrobial agent susceptibility of both planktonic and bio films of Gram-positive organisms.
  • This broth dilution antimicrobial susceptibility test had various antimicrobial agents which are diluted in recovery buffer at categorical breakpoint concentrations defined by Clinical and Laboratory Standard InstituteTM (CLSI).
  • Panel wells were inoculated with planktonic and biofilm of clinically significant Gram-positive organisms of choice using a 96 peg inoculation lid. Panels and pegged lids were then incubated at 35 °C for a minimum of 16 hours. Planktonic susceptibility and resistance was determined by measuring inhibition and growth in the presence of antimicrobial agents after 16-24 hours incubation at 35 °C. The pegged lid containing the biofilm bacteria that have been exposed to the antimicrobial agents was placed in a recovery media. Biofilm susceptibility and resistance was determined by measuring inhibition and growth after incubation for additional 16-24 hours at 35 °C.
  • Stock solutions were prepared by either weighing each test compound or by weighing the vial containing the test compound; dissolving the entire amount of the compound in a known volume of 100 % DMSO and weighting the empty vial to get the amount dissolved in the DMSO.
  • the working concentrations of the compounds were made to be higher than the test concentration because the compounds were diluted when the inoculum was added.
  • the compounds for the Gram-negative panels (A) were setup as follows:
  • Compound A 0.1524 gram was dissolved in 24.764 ml of cation-adjusted Mueller-Hinton broth (CAMHB) with 2 % DMSO
  • Compound B 0.1517 gram was dissolved in 24.651 ml of cation-adjusted Mueller-Hinton broth (CAMHB) with 2 % DMSO.
  • Compound B was pre-dissolved in 100 % DMSO making a 5.5 ml solution of 0.1022 mg/ ⁇ of Compound B in DMSO. To make 25 ml of the working solution 1.505 ⁇ of the Compound B solution was added to 23.495 ml CAMHB.
  • Compound K was pre-dissolved in 100 % DMSO making a 3 ml solution of 0.3181 mg/ ⁇ of Compound K in DMSO. To make 25 ml of the working solution 484 ⁇ of the Compound K solution was added to 24.516 ml CAMHB.
  • Compound A 0.1195 gram was dissolved in 25.021 ml of CAMHB with 2 % DMSO.
  • Compound K was pre-dissolved in 100 % DMSO making a 3 ml solution of 0.3181 mg/ ⁇ of Compound K in DMSO. To make 25 ml of the working solution 375 ⁇ of the Compound K solution was added to 24.625 ml CAMHB.
  • Compound A 0.1108 gram was dissolved in 23.199 ml of CAMHB with 2 % DMSO.
  • Compound B was pre-dissolved in 100 % DMSO making a 5.5 ml solution of 0.1022 mg/ ⁇ of compound B in DMSO. To make 25 ml of the working solution 1.168 ⁇ of the Compound B solution was added to 23.832 ml CAMHB.
  • Compound K was pre-dissolved in 100 % DMSO making a 3 ml solution of 0.3181 mg/ ⁇ of Compound K in DMSO. To make 25 ml of the working solution 375 ⁇ of the Compound K solution was added to 24.625 ml CAMHB.
  • Each working solution of compound was used to make the 4 dilutions by serially diluting in 10 fold steps. From the working solution 2.5 ml was taken and mixed with 22.5 ml CAMHB, 2.5 ml was then removed from the second tube and mixed with 22.5 ml in the third tube. This was repeated until the fourth dilution, giving the five 10-fold dilutions. These dilutions end up being 4, 0.4, 0.04, 0.004 and 0.0004 gram/L after the antibiotics and organism are added.
  • antibiotics were tested either alone or in combination with the tested compounds, as described hereinabove: Gentamicin (GM), Amikacin (AK), Ceftazidime (CAZ), Cefepime (CPE), trimethoprim/sulfamethoxazole (T/S), piperacillin tazobactam (P/T), Aztreonam (AZT), Meropenem (MER), Tobramycin (TO), Ciprofloxacin (CP), Colistin (CT) and Chloramphenicol (ChA).
  • Gentamicin GM
  • Amikacin AK
  • Ceftazidime CAZ
  • Cefepime Cefepime
  • T/S trimethoprim/sulfamethoxazole
  • P/T piperacillin tazobactam
  • Aztreonam Aztreonam
  • MER Meropenem
  • TO Ciprofloxacin
  • CCT Colistin
  • ChA Chloram
  • Samples containing an antibiotic and a tested compound were prepared by mixing a sample of the tested compound with the antibiotic so as to afford the indicated concentration.
  • Staphylococcus epidermidis ATCC 35984
  • Pseudomonas aeruginosa ATCC 27853
  • the first sub-cultures of the bacterial organisms listed above were streaked out on TSA.
  • the organisms were incubated at 35 °C for 24 hours. Following growth the plate(s) were wrapped in parafilm at 4 °C.
  • a second sub-culture was streaked out on TSA and incubated at 35 °C for 24 hours. The second sub-cultures were used within 24 hours starting from the time it was first removed from incubation.
  • an inoculum in 3 ml sterile water that matches a 0.5 McFarland Standard (1.5 x 108 cells per ml) in a glass test tube using a sterile cotton swab was created.
  • This solution was diluted 250 x 100 ml CAMHB.
  • the diluted organism was gently stirred by swirling the flask to achieve uniform mixing of the organism.
  • One sample (100 ⁇ ) of the diluted organism was used for an inoculum check by serially diluting and spot plating on Tyrptic Soy Agar (TSA).
  • TSA Tyrptic Soy Agar
  • Gram-negative panels 20 ⁇ of sterile CAMHB was added to well HI 2.
  • Gram-positive panels 20 ⁇ of sterile CAMHB was added to well Al .
  • the device was placed on an orbital in a humidified incubator at 35 0 C for 24 hours set at 110 rpm. Following incubation the peg lid was rinsed by dipping it in a 96 well microtitre plate where each well contained 200 ⁇ of sterile saline for 1 minute. The peg lid was inserted into a recovery plate where each well contained 200 ⁇ of CAMHB. The device was incubated at 35 °C for 24 hours.
  • the challenge plate was incubated at 35 °C for 24 hours and read visually to determine MIC values (minimum inhibitory concentration) for each of the tested compounds for each organism shed from the biofilm during the challenge incubation.
  • MIC is defined as the minimum concentration of a compound that inhibits growth of the organism.
  • the recovery plate was incubated at 35 °C for 24 hours and read visually to determine MBEC values.
  • a microtiter plate reader was used to obtain optical density measurements at 630 nm (OD630). Clear wells (OD630 ⁇ 0.1) were evidence of biofilm eradication.
  • the MBEC is defined as the minimum concentration of a compound that inhibits growth of the biofilm.
  • MIC results were determined following the 24 hour incubation from the bioFILM PA and Gram+VE panels using the plate reader. To determine the minimum inhibitory concentration (MIC) values, turbidity (visually) in the wells of the challenge plate were looked for. Alternatively, a microtiter plate reader was used to obtain optical density measurements at 630 nm (OD 630 ). The MIC is defined as the minimum concentration of the tested antibacterial agent that inhibits growth of the organism. Clear wells (OD 6 3o ⁇ 0.1) were evidence of inhibition following a suitable period of incubation.
  • MBC value represents the lowest concentration which kills 99.9 % of the microorganism population. Results were determined following the 24 hour incubation from the Test panels using the plate reader. To determine the minimum bactericidal concentration (MBC) values, turbidity (visually) in the wells of the challenge plate was looked for. Alternatively, a microtiter plate reader was used to obtain optical density measurements at 630 nm (OD 6 3o).
  • MBEC recovery panels using the plate reader to determine the minimum biofilm eradication concentration (MBEC) values. Turbidity (visually) in the wells of the recovery plate was looked for. Alternatively, a microtiter plate reader was used to obtain optical density measurements at 630 nm (OD 6 3o). Clear wells (OD 6 3o ⁇ 0.1) were evidence of biofilm eradication.
  • the MBEC is defined as the minimum concentration of the antibacterial agent that inhibits growth of the biofilm.
  • fractional inhibitory concentration (FIC) index is most frequently used to define or to describe drug interactions, it has some limitations when used for drugs against filamentous fungi (molds) and other biofilm- forming microbial life forms. These limitations include observer's bias in the determination of the MIC and lack of agreement on the endpoints (MIC-0, MIC-1 and MIC-2 or >95 %, >75 % and >50 % growth inhibition, respectively) when studying drug combinations. Furthermore, statistical analysis and comparisons are oftentimes difficult to make and standardize.
  • FIC fractional inhibitory concentration
  • the MIC was determined for each test compound at each antibiotic concentration (i.e., at a particular antibiotic concentration, used as MICx c , the corresponding concentration of test compound which was the minimum compound concentration to result in bacterial growth inhibition at that antibiotic concentration was determined, and used as MIC Xc ).
  • the FIC index was calculated using the MICs determined in both (c) and (d). MICs less than or equal to the value selected in (c) or (d) indicate that the MIC value selected was the lowest concentration tested. MICs greater than the value selected in (c) or (d) indicate that there was growth at the highest concentration tested.
  • the lowest FIC index calculated for each compound-antibiotic combination was selected as the final FIC value to be tabulated.
  • Table 7-10 presents the Minimum Inhibitory Concentration (MIC) of antibacterial agents (tested compounds and clinical antibiotics) obtained for Pseudomonas aeruginosa ATCC 27853.
  • MIC Minimum Inhibitory Concentration
  • Table 8 presents the Antibacterial-agent combinations (test compounds combined with clinical antibiotics) at minimal concentrations for inhibiting Pseudomonas aeruginosa ATCC 27853 (sorted by FIC).
  • Table 9 below presents the Minimum Inhibitory Concentration (MIC) of antibacterial agents (tested compounds and clinical antibiotics) obtained for to Staphylococcus epidermidis ATCC 35984.
  • Table 10 presents the Antibacterial-agent combinations (test compounds combined with clinical antibiotics) at minimal concentrations for inhibiting Staphylococcus epidermidis ATCC 35984 (sorted by FIC)
  • Staphylococcus aureus MRSA; strain USA 400
  • Staphylococcus epidermidis Strain ATCC 35984
  • Pseudomonas aeruginosa strain ATCC 27853
  • Ciprofloxacin Gentamicin, Tetracycline, Tobramycin and Vancomycin. Determination of Minimum Inhibitory Concentration (MIC):
  • MIC values for each antibacterial agent (antibiotic), the tested compound and combination thereof were determined according to the procedure described by Ceri et al. (Methods Enzymol 337:377-85, 2001) using SENSITITRE ® Gram Positive and Gram Negative MIC Plates (TREC Diagnostics Systems, Cleveland, OH) and Bio filmTM test panels (Inovotech Inc., Edmonton, AB Canada).
  • Table 11 presents the Minimum Inhibitory Concentration (MIC) of the tested compounds (Compound F, Compound K) and the known non-beta lactam antibacterial agents (clinical antibiotics) provided to bacterial strains Staphylococcus aureus strain USA 400 (MRSA), Staphilococcus epidermitis ATCC 35984 (S. epidermitis) and Psuedomonas aeruginosa ATCC 27853 (P. aeruginosa)
  • MRSA Staphylococcus aureus strain USA 400
  • S. epidermitis Staphilococcus epidermitis ATCC 35984
  • Psuedomonas aeruginosa ATCC 27853 P. aeruginosa
  • Ciprofloxacin MRSA 4 ⁇ / ⁇ 1
  • Tobramycin P. aeruginosa 24 ⁇ g/ml
  • Table 12 below shows the effect of Compound F combined with clinical non beta-lactam antibiotics on the clinical bacterial strains tested (sorted by FIC). As shown therein, the combination of Compound F with Gentamicin, Ciprofloxacin, Tetracycline, Tobramycin or Vancomycin resulted in synergic inhibition of bacterial growth.
  • S. epidermidis 0.0000028 Tetracycline 0.25 0.50
  • SY P. aeruginosa 0.00028 Tobramycin 0.25 0.005
  • FIC [(lowest MIC of compound F in combination) / (lowest MIC of compound F as single agent)] + [(lowest MIC of antibiotic in combination) / [(lowest MIC of antibiotic as single agent)].
  • Table 13 below shows the effect of Compound K combined with clinical non beta-lactam antibiotics on the clinical bacterial strains tested (sorted by FIC). As shown therein, the combination of Compound K with Gentamicin, Ciprofloxacin, Tetracycline, Tobramycin or Vancomycin resulted in synergic inhibition of bacterial growth.
  • FIC [(lowest MIC of compound K in combination) / (lowest MIC of compound K as single agent)] + [(lowest MIC of antibiotic in combination) / [(lowest MIC of antibiotic as single agent)].
  • the bacterial strains were methicillin resistant Staphylococcus aureus (MRSA: USA300) and Pseudomonas aeruginosa (PA: ATCC27312).
  • MRSA methicillin resistant Staphylococcus aureus
  • PA Pseudomonas aeruginosa
  • All challenge inoculum suspensions were prepared by swabbing a 3-cm diameter area of the growth from a culture plate into 4.5 ml of sterile water, providing a suspension consisting of approximately 10 10 colony forming units/ml (CFU/ml).
  • MIC Minimum Inhibitory Concentration
  • the tube dilution test is the standard method for determining levels of resistance to a compound (antibiotic). Dilutions of the antibiotic were made in a liquid inoculum with a known number of organisms after overnight incubation. The lowest concentration (highest dilution) of antibiotic preventing appearance of turbidity was considered to be the minimum inhibitory concentration (MIC). At this dilution the antibiotic is bacteriostatic.
  • test tubes Fourteen (14) sterile, capped test tubes were labeled 1 through 14. All steps were carried out using aseptic technique.
  • test tubes All of the test tubes were incubated for 24 hours at 37 °C. After the incubation period, the tubes were examined for visible signs of bacterial growth, seen as turbidity in the suspension and were mixed by pipetting, and 200 ⁇ were deposited into 3 wells on a 96 wells plate. The optical density (OD) of the solution was measured in a spectrophotometer (BioRad Laboratories Inc.). As compared to the control diluted culture suspension, the without growth (no turbidity) was determined to be the minimum inhibitory concentration (MIC) for each strain of bacteria.
  • MIC minimum inhibitory concentration
  • MCC Minimum Bactericidal Concentration
  • the contents of the tubes were also subcultured onto antibiotic free medium (TSA) to examine bacterial growth (MBC).
  • TSA antibiotic free medium
  • MSC bacterial growth
  • Figure 1 presents a bar graph presenting the results of the optical density measurements and bacterial count of methicillin resistant Staphylococcus aureus USA300 strain with different concentrations of Compound K (denoted as EDP-5).
  • Figure 2 is a bar graph presenting the results of the optical density measurements and bacterial count of Pseudomonas aeruginosa ATCC27312 strain (PA) with different concentrations of Compound K.
  • Figure 3 is bar graph presenting methicillin resistant Staphylococcus aureus US A300 strain inhibition zones by various concentrations of Compound K measured in cm 2 using planimetry method.
  • Figure 4 is a bar graph presenting the Pseudomonas aeruginosa ATCC27312 bacteria growth inhibition zones by various concentrations of Compound K, measured in cm 2 using planimetry method.
  • Inhibition zone assay showed that 10 mg/ml Compound K had the largest zone of bacteria growth inhibition of methicillin resistant Staphylococcus aureus US A300 and Pseudomonas aeruginosa ATCC27312 (5.33 and 21.08 cm 2 , respectively) compared to any treatment. This concentration resulted in better inhibition zone than silver sulfadiazine when was tested against P. aeruginosa.
  • the bacterial strains were methicillin resistant Staphylococcus aureus (MRSA: USA300) and Pseudomonas aeruginosa (PA: ATCC27312).
  • the freeze-dried bacteria culture was recovered per ATCC standard recovering protocol. All challenge inoculum suspensions were prepared by swabbing a 3-cm diameter area of the growth from a culture plate into 4.5 ml of sterile water, providing a suspension consisting of approximately 10 10 colony forming units/ml (CFU/ml). Two (2) ml of this suspension were diluted into 150 ml of sterile tryptic soy broth (TSB), making the inoculum suspension approximately 10 6 CFU/ml. The concentration was confirmed using historical optical density measurements.
  • TTB sterile tryptic soy broth
  • the tube dilution test is the standard method for determining levels of resistance to a compound (antibiotic). Dilutions of the antibiotic were made in a liquid inoculum with a known number of organisms after overnight incubation. The lowest concentration (highest dilution) of antibiotic preventing appearance of turbidity was considered to be the minimum inhibitory concentration (MIC). At this dilution the antibiotic is bacteriostatic.
  • test tubes Fourteen (14) sterile, capped test tubes were labeled 1 through 14. All steps were carried out using aseptic technique.
  • 1.0 ml of vehicle was added to the tube number thirteen of Compound F or Compound K, or the tube number eight of the antibiotics.
  • 1.0 ml of sterile TSB was added to the tube number fourteen of Compound F or Compound K, or the tube number nine of the antibiotics.
  • test tubes All of the test tubes were incubated for 24 hours at 37 °C. After the incubation period, the tubes were examined for visible signs of bacterial growth, seen as turbidity in the suspension and were mixed by pipetting, and 200 ⁇ were deposited into 3 wells on a 96 wells plate. The optical density (OD) of the solution was measured in a spectrophotometer (BioRad Laboratories Inc.). As compared to the control diluted culture suspension, the without growth (no turbidity) was determined to be the minimum inhibitory concentration (MIC) for each strain of bacteria.
  • MIC minimum inhibitory concentration
  • Figure 5A is a bar graph presenting the results of the optical density measurements and bacterial count of methicillin resistant Staphylococcus aureus US A300 strain with different concentrations of Compound K and Compound F.
  • Figure 5B is a bar graph presenting the results of the optical density measurements and bacterial count of methicillin resistant Staphylococcus aureus US A300 strain with different concentrations of Gentamicin.
  • Figure 6A is a bar graph presenting the results of the optical density measurements and bacterial count of Pseudomonas aeruginosa ATCC27312 strain (PA) with different concentrations of Compound K and Compound F.
  • PA Pseudomonas aeruginosa ATCC27312 strain
  • Figure 6B is a bar graph presenting the results of the optical density measurements and bacterial count of Pseudomonas aeruginosa ATCC27312 strain (PA) with different concentrations of Tobramycin.
  • PA Pseudomonas aeruginosa ATCC27312 strain
  • Compound K also denoted EDP-5
  • MRSA meticillin resistant Staphylococcus aureus
  • Pseudomonas aeruginosa was tested on meticillin resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa in vivo.
  • Swine were used as experimental research animal since their skin is morphologically and biochemically similar to human skin.
  • Two animals were studied: one (1) animal with S. aureus and another animal with P. aeruginosa.
  • the young female specific pathogen free (SPF: Looper Farms, North Carolina) pigs weighing 35-40 kg were kept in-house for at least one week prior to initiating the experiment.
  • the animals were fed a basal diet ad libitum and housed in animal facilities (American Association for Accreditation of Laboratory Animal accredited) with controlled temperature (19-21 °C) and lights (12 hour/12 hour LD).
  • MRSA Methicillin Resistant Staphylococcus aureus
  • PA Pseudomonas aeruginosa
  • E. Mupirocin (as positive control to methicillin resistant S. aureus; cream was applied by adding approximately between 150 - 200 ⁇ to the top of the wound and covering with sterile gauze (2 x 2 inch 4 ply thick)
  • the wounds were inoculated with the appropriate bacterial strain as described in hereinbelow.
  • the wounds were divided into six groups of nine wounds each. Six wounds were treated on day 0 within 20 minutes of inoculation to determine the biofilm inhibition.
  • Treatments A, B, C and D were effected by adding 1.5 ml of the tested formulation to completely saturate a sterile gauze pad 2 x 2 inch in size and 4 ply thick (CurityTM Tyco Healthcare Group, Mansfield, MA). Gauze saturation with the formulation helped maintaining the formulation in direct contact with the wounds. Each saturated gauze pad was put on each wound, and covered with a polyurethane film dressing.
  • Wounds that were treated with positive controls received about 150 - 200 ⁇ of material which was sufficient to completely cover the wound area and surrounding skin.
  • Positive controls were spread out gently with a sterile Teflon spatula and covered with a sterile gauze pad and redressed with a polyurethane film dressing.
  • the challenge inoculum suspension was prepared by scraping the overnight growth from a culture plate into 5 ml of normal saline, so as to provide a suspension concentration of approximately 10 10 colony forming units/ml (CFU/ml) for each bacteria. Serial dilutions were made until a concentration of 10 6 CFU/ml was achieved.
  • the inoculum was vortexed and 25 ml of the suspension were inoculated into each wound.
  • serial dilutions of the suspension were plated onto culture media to quantify the exact concentration of viable organisms used for this experiment.
  • the gauze materials from all three wounds were placed in a sterile 50 ml polyurethane tube. Five (5) ml of all purpose neutralizer solution was added, vortexed and serially diluted for quantitation using spiral plater method described above. After removal of gauze, three wounds for each treatment group were recovered on day 1 after first treatment application (within 20 minutes after inoculation). The remaining six wounds for each treatment group were recovered on day 2 after second treatment application and after 24 hours bio film formation and first treatment application.
  • a sterile surgical steel cylinder (22 mm inside diameter) was placed around the wound area.
  • One (1) ml of all purpose neutralizer solution was drawn into the cylinder and the site was scrubbed with a sterile Teflon spatula for 30 seconds.
  • Oxicillin Resistance Screening Agar was used to isolate MRSA USA 300 from the wounds and a selective media for Pseudomonas aeruginosa (Pseudomonas Agar with CN supplement) was used to quantify Pseudomonas aeruginosa present in the suspension. All plates were incubated aerobically overnight (16-24 hours) at 37 °C, after which the number of viable colonies were counted.
  • Figures 7A-D present photographs of wounds during, before and after treatments, as well as throughout the assessment days, wherein gauze in all treatment groups were in place and moist on day 1 showing a slight adherence to the wound bed (Figure 7A), while re-injury was not observed (Figure 7B), and wherein wound fluid was observed from all treatment groups after 24 hours bio film formation in wounds assigned to bio film elimination assessment (Figure 7C and Figure 7D).
  • Figures 8A-B present photographs of wounds infected with methicillin resistant Staphylococcus aureus and treated within 20 minutes after inoculation with vehicle ( Figure 8 A) and untreated wounds (Figure 8B).
  • log CFU/ml log CFU/ml
  • MRSA methicillin resistant Staphylococcus aureus
  • PA Pseudomonas aeruginosa
  • Figure 9 is a bar graph showing methicillin resistant Staphylococcus aureus US A300 bio film inhibition after treatment application.
  • Figure 10 is a bar graph presenting data obtained for inhibition of biofilm of methicillin resistant S. aureus US A300 after treatment application.
  • wounds treated with mupirocin had S. aureus log CFU/ml counts at day 1 and 2 recovery times of 5.12 ⁇ 0.79 and 4.42 ⁇ 0.18, respectively.
  • Compound K reduced S. aureus counts (0.65 log CFU/ml) compared with day 1 after first treatment. Similar bacterial reduction was found with mupirocin (positive control) when this treatment was applied for 2 days (0.71 log CFU/ml).
  • Treatment with either 40 mg/ml or 5 mg/ml Compound K and vehicle increased MRSA count as compared to same treatments on day 1.
  • Figures 11 A-B are bar graphs presenting the data obtained for biofilm inhibition of methicillin resistant Staphylococcus aureus US A300 strain, compared with bacterial count in gauze, after 1 treatment application ( Figure 11 A), and biofilm inhibition of S. aureus USA300, compared with bacterial count in gauze, after 2 treatment applications ( Figure 11B).
  • Figure 12 is a bar graph presenting the data obtained for elimination of methicillin resistant Staphylococcus aureus US A300 biofilm after 24 hours of biofilm formation, followed by treatment application.

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Abstract

L'invention concerne des procédés, des objets et des compositions utilisant de l'acide 4-fluorophénylboronique ou des esters de celui-ci, présentant les formules A ou A' telles que décrites dans le présent document, en combinaison avec un agent actif supplémentaire, pour inhiber la croissance d'un microorganisme pathogène et/ou pour prévenir et/ou réduire la formation de biofilms microbiens et/ou pour briser les biofilms microbiens dans des tissus vivants ou des objets inanimés. En outre, des composés représentés par la formule générale A, telle que décrite dans le présent document, qui montrent une activité antimicrobienne et anti-formation de biofilms, ainsi que des procédés, des objets et des compositions les utilisant sont décrits.
PCT/IB2012/051690 2011-04-07 2012-04-05 Compositions antimicrobiennes, compositions antibiofilms et leurs utilisations Ceased WO2012137164A1 (fr)

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