EP2536406A2 - Bismuth-thiols comme antiseptiques destinés à des utilisations biomédicales, comprenant le traitement de biofilms bactériens et d'autres utilisations - Google Patents

Bismuth-thiols comme antiseptiques destinés à des utilisations biomédicales, comprenant le traitement de biofilms bactériens et d'autres utilisations

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Publication number
EP2536406A2
EP2536406A2 EP11740333A EP11740333A EP2536406A2 EP 2536406 A2 EP2536406 A2 EP 2536406A2 EP 11740333 A EP11740333 A EP 11740333A EP 11740333 A EP11740333 A EP 11740333A EP 2536406 A2 EP2536406 A2 EP 2536406A2
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EP
European Patent Office
Prior art keywords
antibiotic
bis
bismuth
compound
thiol
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP11740333A
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German (de)
English (en)
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EP2536406A4 (fr
Inventor
Brett Hugh James Baker
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Microbion Corp
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Microbion Corp
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Publication date
Priority claimed from PCT/US2010/023108 external-priority patent/WO2010091124A2/fr
Application filed by Microbion Corp filed Critical Microbion Corp
Publication of EP2536406A2 publication Critical patent/EP2536406A2/fr
Publication of EP2536406A4 publication Critical patent/EP2536406A4/fr
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/245—Bismuth; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N55/00—Biocides, pest repellants or attractants, or plant growth regulators, containing organic compounds containing elements other than carbon, hydrogen, halogen, oxygen, nitrogen and sulfur
    • A01N55/02—Biocides, pest repellants or attractants, or plant growth regulators, containing organic compounds containing elements other than carbon, hydrogen, halogen, oxygen, nitrogen and sulfur containing metal atoms
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/65—Tetracyclines
    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/14—Peptides containing saccharide radicals; Derivatives thereof, e.g. bleomycin, phleomycin, muramylpeptides or vancomycin
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    • 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
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    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/145—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic compounds
    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
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    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/02—Local antiseptics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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 presently disclosed invention embodiments relate to compositions and methods for the treatment of microbial infections.
  • the present embodiments relate to improved treatments for managing bacterial infections in epithelial tissues, including in wounds such as chronic wounds and acute wounds, and in clinical, personal healthcare, and other contexts, including treatment of bacterial biofilms and other conditions.
  • the complex series of coordinated cellular and molecular interactions that contribute to skin wound healing and responding to and resisting microbial infections and/or to healing or maintenance of bodily tissues may be adversely impacted by a variety of external factors, such as opportunistic and nosocomial infections (e.g., clinical regimens that can increase the risk of infection), local or systemic administration of antibiotics (which may influence cell growth, migration or other functions and can also select for antibiotic-resistant microbes), frequent wound dressing changes, open-air exposure of wounds to speed healing, the use of temporary artificial structural support matrix or scaffold materials, the possible need for
  • Wounds occur when the continuity between cells within a tissue, or between tissues, is disrupted, for instance, by physical, mechanical, biological, pathological and/or chemical forces ⁇ e.g., burns, dermal infections, puncture wounds, gunshot or shrapnel wounds, skin ulcers, radiation poisoning, malignancies, gangrene, autoimmune disease, immunodeficiency disease, respiratory insult such as by inhalation or infection, gastrointestinal insult such as by deleterious ingestion or infection, circulatory and hematologic disorders including clotting defects,) or other traumatic injuries, or the like.
  • forces e.g., burns, dermal infections, puncture wounds, gunshot or shrapnel wounds, skin ulcers, radiation poisoning, malignancies, gangrene, autoimmune disease, immunodeficiency disease, respiratory insult such as by inhalation or infection, gastrointestinal insult such as by deleterious ingestion or infection, circulatory and hematologic disorders including clotting defects,) or other traumatic injuries,
  • acute wounds such as may result from injury, trauma, surgical intervention, or other causes, typically lack underlying health deficits and heal rapidly, but may on occasion fail to do so due to the presence of an infection; rapidly forming bacterial biofilms have been described in acute wounds (e.g.,
  • Additional factors that may contribute to the development of chronic wounds include losses in mobility (e.g., that result in continued pressure being applied to a wound site), deficits of sensation or mental ability, inaccessibility of the wound site (e.g., in the respiratory or gastrointestinal tracts) and circulatory deficits. Infection at a chronic wound site may be detected by the clinical signs of skin redness, edema, pus formation and/or unpleasant odor, or other relevant, clinically accepted criteria.
  • Acute wounds that cannot heal properly may thus be present, and chronic wounds thus may develop, in higher organisms (including but not limited to humans and other mammals) when the host's immune system has been overwhelmed by bacterial infection of a wound site (e.g., an acute wound), creating permissive conditions for bacteria to invade and further destroy tissue.
  • a wound site e.g., an acute wound
  • chronic wounds are wounds that do not heal within three months, and instead of becoming smaller they tend to grow larger as the bacterial infiltration progresses.
  • Chronic wounds may become very painful and stressful for the patient when nearby nerves become damaged (neuropathy) as the wound progresses.
  • Chronic wounds may in some cases originate as acute wounds and thus may include, for example, gunshot or shrapnel wounds, burns, punctures, venous ulcers, pressure ulcers, diabetic ulcers, radiation poisoning, malignancies, dermal infections, gangrene, surgical wounds, diabetic foot ulcers, decubitis ulcers, venous leg ulcers, infected and/or biofilm-containing nonhealing surgical wounds, pyoderma gangrenosum, traumatic wounds, acute arterial insufficiency, necrotizing fasciitis, osteomyelitis (bone infection), and radiation injuries, such as osteoradionecrosis and soft tissue radionecrosis, or other types of wounds.
  • gunshot or shrapnel wounds burns, punctures, venous ulcers, pressure ulcers, diabetic ulcers, radiation poisoning, malignancies, dermal infections, gangrene, surgical wounds, diabetic foot ulcers, decubitis ulcers, venous leg ulcers, in
  • Venous ulcers for example, occur mostly in the legs, as a result of poor circulation (e.g., ischemia), malfunctioning valves of veins, or repeated physical trauma (e.g., repetitive injury).
  • Pressure ulcers may be present when local pressure that is exerted at or around a wound site is greater than blood pressure, for instance, such that poor circulation, paralysis, and/or bed sores may contribute to, or exacerbate, the chronic wound.
  • Diabetic ulcers may occur in individuals with diabetes mellitus, for example, persons in whom uncontrolled high blood sugar can contribute to a loss of feeling in the extremities, leading to repetitive injuries and/or neglect on the part of the individual to attend to injuries.
  • Factors that can complicate or otherwise influence clinical onset and outcome of chronic wounds include the subject's immunological status (e.g., immune suppression, pathologically (e.g., HIV- AIDS), radiotherapeutically or pharmacologically compromised immune system; age; stress); skin aging (including photochemical aging), and development and progression of biofilms within the wound.
  • immunological status e.g., immune suppression, pathologically (e.g., HIV- AIDS), radiotherapeutically or pharmacologically compromised immune system
  • age e.g., HIV- AIDS
  • radiotherapeutically or pharmacologically compromised immune system e.g., HIV- AIDS
  • pharmacologically compromised immune system e.g., HIV- AIDS
  • age e.g., HIV- AIDS
  • skin aging including photochemical aging
  • Wound-related injuries may be accompanied by lost or compromised organ function, shock, bleeding and/or thrombosis, cell death (e.g., necrosis and/or apoptosis), stress and/or microbial infection. Any or all of these events, and especially infection, can delay or prevent the effective tissue repair processes that are involved in wound healing. Hence, it can be important as early as possible in an individual who has sustained a wound to remove nonviable tissue from a wound site, a process referred to as debridement, and also to remove any foreign matter from the wound site, also referred to as wound cleansing.
  • Severe wounds, acute wounds, chronic wounds, burns, and ulcers can benefit from cellular wound dressings.
  • Several artificial skin products are available for nonhealing wounds or burns such as: Apligraft® (Norvartis), Demagraft®, Biobrane®, Transcyte® (Advance Tissue Science), Integra® Dermal Regeneration Template® (from Integra Life Sciences
  • antibiotics are not effective for the treatment of chronic wounds, and are generally not used unless an acute bacterial infection is present.
  • Current approaches include administration or application of antibiotics, but such remedies may promote the advent of antibiotic-resistant bacterial strains and/or may be ineffective against bacterial biofilms. It therefore may become especially important to use antiseptics when drug resistant bacteria (e.g., methicillin resistant Staphylococcus aureus, or MRSA) are detected.
  • drug resistant bacteria e.g., methicillin resistant Staphylococcus aureus, or MRSA
  • MRSA methicillin resistant Staphylococcus aureus
  • antiseptics may be toxic to host cells at the concentrations that may be needed to be effective against an established bacterial infection, and hence such antiseptics are unsuitable. This problem may be particularly acute in the case of efforts to clear infections from natural surfaces, including internal epithelial surfaces, such as respiratory (e.g., airway, nasopharyngeal and laryngeal paths, tracheal, pulmonary, bronchi, bronchioles, alveoli, etc.) or
  • gastrointestinal e.g., buccal, esophageal, gastric, intestinal, rectal, anal, etc.
  • tracts or other epithelial surfaces.
  • S. aureus including MRSA (Methicillin Resistant Staphylococcus aureus), Enterococci, E. coli, P. aeruginosa,
  • Streptococci and Acinetobacter baumannii. Some of these organisms exhibit an ability to survive on non-nutritive clinical surfaces for months. S. aureus, has been shown to be viable for four weeks on dry glass, and for between three and six months on dried blood and cotton fibers (Domenico et al., 1999 Infect.
  • Microbial biofilms are associated with substantially increased resistance to both disinfectants and antibiotics.
  • Biofilm morphology results when bacteria and/or fungi attach to surfaces. This attachment triggers an altered transcription of genes, resulting in the secretion of a remarkably resilient and difficult to penetrate polysaccharide matrix, protecting the microbes.
  • Biofilms are very resistant to the mammalian immune system, in addition to their very substantial resistance to antibiotics. Biofilms are very difficult to eradicate once they become established, so preventing biofilm formation is a very important clinical priority. Recent research has shown that open wounds can quickly become contaminated by biofilms. These microbial biofilms are thought to delay wound healing, and are very likely related to the establishment of serious wound infections.
  • epithelial tissues e.g., generally avascular epithelial surfaces that form barriers between an organism and its external environment, such as those found in skin and also found in the linings of respiratory and gastrointestinal tracts, glandular tissues, etc.
  • the skin is the largest body organ in humans and other higher vertebrates (e.g., mammals), protecting against environmental insults through its barrier function, mechanical strength and imperviousness to water.
  • skin provides a protective body covering that permits maintenance of physiological equilibria.
  • Skin architecture is well known. Briefly, epidermis, the skin outer layer, is covered by the stratum corneum, a protective layer of dead epidermal skin cells (e.g., keratinocytes) and extracellular connective tissue proteins. The epidermis undergoes a continual process of being sloughed off as it is replaced by new material pushed up from the underlying epidermal granular cell, spinous cell, and basal cell layers, where continuous cell division and protein synthesis produce new skin cells and skin proteins (e.g., keratin, collagen).
  • dead epidermal skin cells e.g., keratinocytes
  • extracellular connective tissue proteins e.g., extracellular connective tissue proteins.
  • the dermis lies underneath the epidermis, and is a site for the elaboration by dermal fibroblasts of connective tissue proteins (e.g., collagen, elastin, etc.) that assemble into extracellular matrix and fibrous structures that confer flexibility, strength and elasticity to the skin.
  • connective tissue proteins e.g., collagen, elastin, etc.
  • nerves, blood vessels, smooth muscle cells, hair follicles and sebaceous glands are also present in the dermis.
  • the skin As the body's first line of defense, the skin is a major target for clinical insults such as physical, mechanical, chemical and biological (e.g., xenobiotic, autoimmune) attack that can alter its structure and function.
  • the skin is also regarded as an important component of immunological defense of the organism.
  • resident white blood cells e.g., lymphocytes, macrophages, mast cells
  • epidermal dendritic (Langerhans) cells having potent antigen-presenting activity, which contribute to immunological protection.
  • Pigmented melanocytes in the basal layer absorb potentially harmful ultraviolet (UV) radiation.
  • Disruption of the skin presents undesirable risks to a subject, including those associated with opportunistic infections, incomplete or inappropriate tissue remodeling, scarring, impaired mobility, pain and/or other complications.
  • epithelial surfaces e.g., respiratory tract, gastrointestinal tract and glandular linings
  • Damaged or broken skin may result, for example, from wounds such as cuts, scrapes, abrasions, punctures, burns (including chemical burns), infections, temperature extremes, incisions (e.g., surgical incisions), trauma and other injuries. Efficient skin repair via wound healing is therefore clearly desirable in these and similar contexts.
  • Wound healing occurs in three dynamic and overlapping phases, beginning with the formation of a fibrin clot.
  • the clot provides a temporary shield and a reservoir of growth factors that attracts cells into the wound. It also serves as a provisional extracellular matrix (ECM) that the cells invade during repair. Intermingled with clot formation is the inflammatory phase, which is characterized by the infiltration of phagocytes and neutrophils into the wound, which clear the wound of debris and bacteria, while releasing growth factors that amplify the early healing response.
  • the process of restoring the denuded area is initiated in the proliferation phase of healing and is driven by
  • Keratinocytes are chemokines, cytokines, and proteases that have been secreted from the immune cells and are concentrated within the clot. Keratinocytes are chemokines, cytokines, and proteases that have been secreted from the immune cells and are concentrated within the clot. Keratinocytes are chemokines, cytokines, and proteases that have been secreted from the immune cells and are concentrated within the clot. Keratinocytes are chemokines, cytokines, and proteases that have been secreted from the immune cells and are concentrated within the clot. Keratinocytes are chemokines, cytokines, and proteases that have been secreted from the immune cells and are concentrated within the clot. Keratinocytes are chemokines, cytokines, and proteases that have been secreted from the immune cells and are concentrated within the clot. Keratinocytes are chemokines, cytokines, and proteases
  • the remodeling phase is the final phase of wound repair and it is carried out by the myofibroblasts, which facilitate connective tissue contraction, increase wound strength, and deposit the ECM that forms the scar (Martin, P. Wound Healing-Aiming for Perfect Skin Regeneration. Science 1997;4:75-80).
  • a number of natural products (e.g., antibiotics) and synthetic chemicals having antimicrobial, and in particular antibacterial, properties are known in the art and have been at least partially characterized by chemical structures and by antimicrobial effects, such as ability to kill microbes ("cidal” effects such as bacteriocidal properties), ability to halt or impair microbial growth (“static” effects such as bacteriostatic properties), or ability to interfere with microbial functions such as colonizing or infecting a site, bacterial secretion of exopolysaccharides and/or conversion from planktonic to biofilm populations or expansion of biofilm formation.
  • Antibiotics, disinfectants, antiseptics and the like are discussed, for example, in U.S. 6,582,719, including factors that influence the selection and use of such compositions, including, e.g., bacteriocidal or bacteriostatic potencies, effective concentrations, and risks of toxicity to host tissues.
  • Bismuth a group V metal, is an element that (like silver) possesses antimicrobial properties. Bismuth by itself may not be
  • preparations are able to inhibit biofilm formation.
  • BT compounds have proven activity against MRSA (methicillin resistant S. aureus), MRSE (methicillin resistant S. epidermidis),
  • Mycobacterium tuberculosis Mycobacterium avium, drug-resistant P.
  • aeruginosa enterotoxigenic E. coli, enterohemorrhagic E. coli, Klebsiella pneumoniae, Clostridium difficile, Heliobacter pylori, Legionella pneumophila, Enterococcus faecalis, Enterobacter cloacae, Salmonella typhimurium, Proteus vulgaris, Yersinia enterocolitica, Vibrio cholerae, and Shigella Flexneri
  • bismuth- thiol (BT) compounds may be used as antiseptic agents for use in the treatment of a wide variety of clinical infectious diseases and conditions and in personal healthcare, while also decreasing the costs incurred for the treatment of such infections, including savings that are realized by prevention or prophylaxis mediated at least in part by BTs.
  • formulations for treating tissues and/or surfaces that contain bacterial biofilms or bacteria related to biofilm formation which formulations comprise one or more BT compound and one or more antibiotic compound, as described herein, where according to non-limiting theory, appropriately selected combinations of BT compound(s) and antibiotic(s) based on the present disclosure provide heretofore unpredicted synergy in the antibacterial (including anti-biofilm) effects of such formulations, and/or unpredicted enhancing effects, for prevention, prophylaxis and/or therapeutically effective treatment against microbial infections including infections that contain bacterial biofilms.
  • unprecedented bismuth- thiol compositions comprising substantially monodisperse microparticulate suspensions, and methods for their synthesis and use.
  • a bismuth-thiol composition comprising a plurality of microparticles that comprise a bismuth-thiol (BT) compound, substantially all of said microparticles having a volumetric mean diameter of from about 0.4 ⁇ to about 5 ⁇ , wherein the BT compound comprises bismuth or a bismuth salt and a thiol-containing compound.
  • BT bismuth-thiol
  • a bismuth-thiol composition comprising a plurality of microparticles that comprise a bismuth-thiol (BT) compound, substantially all of said microparticles having a volumetric mean diameter of from about 0.4 ⁇ to about 5 ⁇ and being formed by a process that comprises (a) admixing, under conditions and for a time sufficient to obtain a solution that is substantially free of a solid precipitate, (i) an acidic aqueous solution that comprises a bismuth salt comprising bismuth at a concentration of at least 50 mM and that lacks a hydrophilic, polar or organic solubilizer, with (ii) ethanol in an amount sufficient to obtain an admixture that comprises at least about 5%, 10%, 15%, 20%, 25% or 30% ethanol by volume; and (b) adding to the admixture of (a) an ethanolic solution comprising a thiol-containing compound to obtain a reaction solution, wherein the thiol-containing compound is
  • the bismuth salt is Bi(N0 3 ) 3 .
  • the acidic aqueous solution comprises at least 5%, 10%, 15%, 20%, 22% or 22.5% bismuth by weight.
  • the acidic aqueous solution comprises at least 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5% nitric acid by weight.
  • the thiol-containing compound comprises one or more agents selected from 1 ,2-ethane dithiol, 2,3-dimercaptopropanol, pyrithione,
  • dithioerythritol 3,4-dimercaptotoluene, 2,3-butanedithiol, 1 ,3-propanedithiol, 2- hydroxypropane thiol, 1-mercapto-2-propanol, dithioerythritol, alpha-lipoic acid and dithiothreitol.
  • a method for preparing a bismuth-thiol composition that comprises a plurality of microparticles that comprise a bismuth-thiol (BT) compound, substantially all of said microparticles having a volumetric mean diameter of from about 0.4 ⁇ to about 5 ⁇ , said method comprising the steps of (a) admixing, under conditions and for a time sufficient to obtain a solution that is substantially free of a solid precipitate, (i) an acidic aqueous solution that comprises a bismuth salt comprising bismuth at a concentration of at least 50 mM and that lacks a hydrophilic, polar or organic solubilizer, with (ii) ethanol in an amount sufficient to obtain an admixture that comprises at least about 5%, 10%, 15%, 20%, 25% or 30% ethanol by volume; and (b) adding to the admixture of (a) an ethanolic solution comprising a thiol- containing compound to obtain a reaction solution, wherein the thio
  • the method further comprises recovering the precipitate to remove impurities.
  • the bismuth salt is Bi(NO3)3.
  • the acidic aqueous solution comprises at least 5%, 10%, 15%, 20%, 22% or 22.5% bismuth by weight.
  • the acidic aqueous solution comprises at least 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5% nitric acid by weight.
  • the thiol-containing compound comprises one or more agents selected from the group consisting of 1,2-ethane dithiol, 2,3- dimercaptopropanol, pyrithione, dithioerythritol, 3,4-dimercaptotoluene, 2,3- butanedithiol, 1 ,3-propanedithiol, 2-hydroxypropane thiol, 1 -mercapto-2- propanol, dithioerythritol, dithiothreitol, alpha-lipoic acid, methanethiol (CH 3 SH [m-mercaptan]), ethanethiol (C 2 H 5 SH [e- mercaptan]), 1-propanethiol (C 3 H7SH [n-P mercaptan]), 2-propanethiol (CH 3 CH(SH)CH 3 [2C 3 mercaptan]), butanethiol (C 4 H 9 SH
  • nanoparticles dodecanethiol functionalized silver nanoparticles, hexa(ethylene glycol)mono-11-(acetylthio)undecyl ether, mercaptosuccinic acid, methyl 3- mercaptopropionate, nanoTether BPA-HH, NanoThinksTM 18, NanoThinksTM 8, NanoThinksTM ACID11 , NanoThinksTM ACID16, NanoThinksTM ALC011 , NanoThinksTM THI08, octanethiol functionalized gold nanoparticles, PEG dithiol average M n 8,000, PEG dithiol average mol wt 1 ,500, PEG dithiol average mol wt 3,400, S-(11-bromoundecyl)thioacetate, S-(4- cyanobutyl)thioacetate, thiophenol, triethylene glycol mono-11- mercaptoundecyl ether, trimethylol
  • a method for protecting a natural surface including a biological tissue surface such as an epithelial tissue surface, against one or more of a bacterial pathogen, a fungal pathogen and a viral pathogen, comprising contacting the epithelial tissue surface with an effective amount of a BT composition under conditions and for a time sufficient for one or more of (i) prevention of infection of the surface by the bacterial, fungal or viral pathogen, (ii) inhibition of cell viability or cell growth of
  • the BT composition comprises a plurality of microparticles that comprise a bismuth-thiol (BT) compound, substantially all of said microparticles having a volumetric mean diameter of from about 0.4 ⁇ to about 5 ⁇ .
  • BT bismuth-thiol
  • the bacterial pathogen comprises at least one of (i) one or more gram-negative bacteria; (ii) one or more gram-positive bacteria; (iii) one or more antibiotic- sensitive bacteria; (iv) one or more antibiotic-resistant bacteria; (v) a bacterial pathogen that is selected from Staphylococcus aureus (S. aureus), MRSA (methicillin-resistant S. aureus), Staphylococcus epidermidis , MRSE
  • Enterobacter cloacae Salmonella typhimurium, Proteus vulgaris, Yersinia enterocolitica, Vibrio cholera, Shigella flexneri, vancomycin-resistant
  • VRE Enterococcus
  • Burkholderia cepacia complex Enterococcus
  • Francisella tularensis Enterococcus
  • Bacillus anthracis Yersinia pestis
  • Pseudomonas aeruginosa vancomycin- resistant enterococci
  • Streptococcus pneumonia penicillin-resistant
  • the bacterial pathogen exhibits antibiotic resistance. In certain embodiments the bacterial pathogen exhibits resistance to an antibiotic that is selected from methicillin, vancomycin, naficilin, gentamicin, ampicillin, chloramphenicol, doxycycline and tobramycin.
  • the natural surface comprises an oral/buccal cavity surface.
  • the natural surface comprises a biological surface such as bone, joint, muscle, ligament, or tendon.
  • the surface comprises an epithelial tissue surface that comprises a tissue that is selected from epidermis, dermis, respiratory tract, gastrointestinal tract and glandular linings.
  • the step of contacting is performed one or a plurality of times. In certain embodiments at least one step of contacting comprises one of spraying, irrigating, dipping and painting the natural surface. In certain embodiments at least one step of contacting comprises one of inhaling, ingesting and orally irrigating. In certain embodiments least one step of contacting comprises administering by a route that is selected from topically, intraperitoneally, orally, parenterally, intravenously, intraarterially,
  • transdermally sublingually, subcutaneously, intramuscularly, transbuccally, intranasally, via inhalation, intraoccularly, intraauricularly, intraventricularly, subcutaneously, intraadiposally, intraarticularly and intrathecally.
  • the BT composition comprises one or more BT compounds selected from the group consisting of BisBAL, BisEDT, Bis-dimercaprol, Bis- DTT, Bis-2-mercaptoethanol, Bis-DTE, Bis-Pyr, Bis-Ery, Bis-Tol, Bis-BDT, Bis- PDT, Bis-Pyr/Bal, Bis-Pyr/BDT, Bis-Pyr/EDT, Bis-Pyr/PDT, Bis-Pyr/Tol, Bis- Pyr/Ery, bismuth-1-mercapto-2-propanol, and Bis-EDT/2-hydroxy-1- propanethiol.
  • BisBAL BisEDT
  • Bis-dimercaprol Bis- DTT
  • Bis-2-mercaptoethanol Bis-DTE
  • Bis-Pyr Bis-Ery
  • Bis-Tol Bis-BDT
  • Bis- PDT Bis-Pyr/Bal
  • Bis-Pyr/BDT Bis-Pyr/EDT
  • the bacterial pathogen exhibits antibiotic resistance.
  • the above described method further comprises contacting the natural surface with a synergizing antibiotic and/or with an enhancing antibiotic, simultaneously or sequentially and in any order with respect to the step of contacting the surface with the BT composition.
  • the synergizing and/or enhancing antibiotic comprises an antibiotic that is selected from an aminoglycoside antibiotic, a carbapenem antibiotic, a cephalosporin antibiotic, a fluoroquinolone antibiotic, a glycopeptide antibiotic, a lincosamide antibiotic, a penicillinase-resistant penicillin antibiotic, and an aminopenicillin antibiotic.
  • the synergizing and/or enhancing antibiotic is an aminoglycoside antibiotic that is selected from amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin,
  • paromomycin paromomycin, rhodostreptomycin, streptomycin, tobramycin and apramycin.
  • a method for overcoming antibiotic resistance comprising contacting the surface simultaneously or sequentially and in any order with an effective amount of (1) at least one bismuth-thiol (BT) composition and (2) at least one antibiotic that is enhanced by, and/or that is capable of acting synergistically with the at least one BT composition, under conditions and for a time sufficient for one or more of: (i) prevention of infection of the surface by the bacterial pathogen, (ii) inhibition of cell viability or cell growth of substantially all planktonic cells of the bacterial pathogen, (iii) inhibition of biofilm formation by the bacterial pathogen, and (iv) inhibition of biofilm viability or biofilm growth of substantially all biofilm
  • the bacterial pathogen comprises at least one of: (i) one or more gram-negative bacteria; (ii) one or more gram-positive bacteria; (iii) one or more antibiotic-sensitive bacteria; (iv) one or more antibiotic-resistant bacteria; (v) a bacterial pathogen that is selected from Staphylococcus aureus (S. aureus), MRSA (methicillin-resistant S. aureus), Staphylococcus
  • MRSE methicillin-resistant S. epidermidis
  • Mycobacterium tuberculosis Mycobacterium avium
  • Pseudomonas aeruginosa drug-resistant P. aeruginosa
  • Escherichia coli enterotoxigenic E. coli
  • enterohemorrhagic E enterohemorrhagic E.
  • Streptococcus pneumonia Escherichia coli, Burkhoideria cepacia, Bukholderia multivorans, Mycobacterium smegmatis and Acinetobacter baumannii.
  • the bacterial pathogen exhibits resistance to an antibiotic that is selected from methicillin, vancomycin, naficilin, gentamicin, ampicillin, chloramphenicol, doxycycline, tobramycin, clindamicin and gatifloxacin.
  • the natural surface comprises an oral/buccal cavity surface.
  • the natural surface comprises a biological surface such as bone, joint, muscle, ligament, or tendon.
  • the surface comprises a tissue that is selected from the group consisting of epidermis, dermis, respiratory tract, gastrointestinal tract and glandular linings.
  • the step of contacting is performed one or a plurality of times.
  • at least one step of contacting comprises one of spraying, irrigating, dipping and painting the surface.
  • at least one step of contacting comprises one of inhaling, ingesting and orally irrigating.
  • At least one step of contacting comprises administering by a route that is selected from topically, intraperitoneally, orally, parenterally, intravenously, intraarterially, transdermally, sublingually, subcutaneously, intramuscularly, transbuccally, intranasally, via inhalation, intraoccularly, intraauricularly, intraventricularly, subcutaneously, intraadiposally,
  • the BT composition comprises one or more BT compounds selected from BisBAL, BisEDT, Bis- dimercaprol, Bis-DTT, Bis-2-mercaptoethanol, Bis-DTE, Bis-Pyr, Bis-Ery, Bis- Tol, Bis-BDT, Bis-PDT, Bis-Pyr/Bal, Bis-Pyr/BDT, Bis-Pyr/EDT, Bis-Pyr/PDT, Bis-Pyr/Tol, Bis-Pyr/Ery, bismuth-1-mercapto-2-propanol, and Bis-EDT/2- hydroxy-1-propanethiol.
  • the synergizing and/or enhancing antibiotic comprises an antibiotic that is selected from clindamicin, gatifloxacin, an aminoglycoside antibiotic, a carbapenem antibiotic, a
  • cephalosporin antibiotic a fluoroquinolone antibiotic, a glycopeptide antibiotic, a lincosamide antibiotic, a penicillinase-resistant penicillin antibiotic, and an aminopenicillin antibiotic.
  • the synergizing and/or enhancing antibiotic is an aminoglycoside antibiotic that is selected from amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin,
  • paromomycin paromomycin, rhodostreptomycin, streptomycin, tobramycin and apramycin.
  • an antiseptic composition comprising (a) at least one BT compound; (b) at least one antibiotic compound that is enhanced by and/or is capable of acting
  • the BT compound is selected from BisBAL, BisEDT, Bis-dimercaprol, Bis-DTT, Bis-2-mercaptoethanol, Bis-DTE, Bis-Pyr, Bis-Ery, Bis-Tol, Bis-BDT, Bis-PDT, Bis-Pyr/Bal, Bis-Pyr/BDT, Bis-Pyr/EDT, Bis-Pyr/PDT, Bis-Pyr/Tol, Bis-Pyr/Ery, bismuth-1-mercapto-2-propanol, and Bis-EDT/2-hydroxy-1-propanethiol.
  • the BT composition comprises a plurality of microparticles that comprise a bismuth-thiol (BT) compound, substantially all of said
  • microparticles having a volumetric mean diameter of from about 0.4 ⁇ ⁇ to about 5 ⁇ .
  • the BT compound is selected from
  • the antibiotic compound comprises an antibiotic that is selected from methicillin, vancomycin, naficilin, gentamicin, ampicillin, chloramphenicol, doxycycline, tobramycin, clindamicin, gatifloxacin and an aminoglycoside antibiotic.
  • the aminoglycoside antibiotic is selected from amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, rhodostreptomycin,
  • aminoglycoside antibiotic is amikacin.
  • a method for treating a natural surface that supports or contains bacterial biofilm comprising (a) identifying a bacterial infection on or in the surface as comprising one of (i) gram positive bacteria, (ii) gram negative bacteria, and (iii) both (i) and (ii); and (b) administering a formulation that comprises one or more bismuth thiol (BT) compositions to the surface, wherein (i) if the bacterial infection comprises gram positive bacteria, then the formulation comprises therapeutically effective amounts of at least one BT compound and at least one antibiotic that is rifamycin, (ii) if the bacterial infection comprises gram negative bacteria, then the formulation comprises therapeutically effective amounts of at least one BT compound and amikacin, (iii) if the bacterial infection comprises both gram positive and gram negative bacteria, then the formulation comprises
  • the biofilm comprises one or a plurality of antibiotic-resistant bacteria.
  • treating the surface comprises at least one of: (i) eradicating the bacterial biofilm, (ii) reducing the bacterial biofilm, and (iii) impairing growth of the bacterial biofilm.
  • the BT composition comprises a plurality of microparticles that comprise a bismuth-thiol (BT) compound, substantially all of said microparticles having a volumetric mean diameter of from about 0.4 ⁇ to about 5 ⁇ .
  • Figure 1 shows surviving numbers (log CFU; colony forming units) from Pseudomonas aeruginosa colony biofilms grown for 24 hours on 10% tryptic soy agar (TSA) at 37°C, followed with indicated treatment for 18 hours.
  • Indicated antibiotic treatments are TOB, tobramycin 10X MIC; AMK, amikacin 100X MIC; IPM, imipenem 10X MIC; CEF, cefepime 10X MIC; CIP,
  • Figure 2 shows surviving numbers (log CFU) from
  • Indicated antibiotic treatments are Rifampicin, RIF 100X MIC; daptomycin, DAP 320X MIC; minocycline, MIN 100X MIC; ampicillin, AMC 10X MIC; vancomycin, VAN 10X MIC; Cpd 2B, compound 2B (Bis-BAL, 1 :1.5), Cpd 8-2, compound 8-2 (Bis-Pyr/BDT (1 :1/0.5).
  • Figure 3 shows scratch closure over time of keratinocytes exposed to biofilms. (*) Significantly different from control (P ⁇ 0.001).
  • Figure 4A and 4B show the subinhibitory BisEDT reversing antibiotic-resistance to several antibiotics. Effects of antibiotics with and without BisEDT (0.05 pg/ml) on a lawn of MRSA (Methicillin-resistant S. aureus) is shown. Panel A shows standard antibiotic-soaked discs alone, and Panel B shows discs combined with a BisEDT (BE).
  • Figure 5 shows the effect of BisEDT and antibiotics on biofilm formation.
  • S. epidermidis grown in TSB + 2% glucose in polystyrene plates for 48h at 37°C.
  • GM gentamicin
  • VM vancomycin
  • CZ cefazolin
  • NC nafcillin
  • RP rifampicin
  • Figure 7 is a bar graph showing the mean S. aureus bacteria levels detected on the bone and hardware samples from open fractures in an in vivo rat model following treatment with three BT formulations, Bis-EDT , MB-1 1 and MB-8-2 with or without Cefazolin antibiotic treatment. Standard errors of the mean are shown as error bars. Animals euthanized early are not excluded from the analysis, however samples from one animal in group 2 have been excluded due to gross contamination. DETAILED DESCRIPTION
  • BT bismuth-thiol
  • certain bismuth-thiol (BT) compounds as provided herein in certain embodiments including BT microparticles having a volumetric mean diameter of from about 0.4 ⁇ to about 5 ⁇
  • certain other BT compounds even if provided as microparticles
  • potent antiseptic, antibacterial and/or anti-biofilm activity against particular bacteria including bacteria associated with a number of clinically significant infections including infections that can comprise bacterial biofilms.
  • certain embodiments of the invention described herein relate to surprising advantages that are provided by novel bismuth-thiol (BT) compositions that, as disclosed herein, can be made in preparations that comprise a plurality of BT
  • microparticles that are substantially monodisperse with respect to particle size (e.g. , having volumetric mean diameter from about 0.4 ⁇ to about 5 pm).
  • the microparticulate BT is not provided as a component of a lipid vesicle or liposome such as a multilamellar phosphocholine-cholesterol liposome or other multilamellar or unilamellar liposomal vesicle.
  • antibacterial and anti-biofilm efficacies of certain antibiotics may be significantly enhanced (e.g., increased in a statistically significant manner) by treating the infection (e.g., by direct application on or in an infected site such as a natural surface) with one or more of these antibiotics in concert, simultaneously or sequentially and in either order, with a selected BT compound.
  • certain BT compounds may be significantly enhanced (e.g., increased in a statistically significant manner) by treating the infection (e.g., by direct application on or in an infected site such as a natural surface) with one or more of these antibiotics in concert, simultaneously or sequentially and in either order, with a selected BT compound.
  • the antibiotic and the BT compound may be administered simultaneously or sequentially and in either order, and it is noteworthy that the specific synergizing or enhancing combinations of one or more antibiotic and one or more BT compound as disclosed herein for treatment of a particular infection (e.g., a biofilm formed by gram-negative or gram-positive bacteria) did not exhibit predictable (e.g., merely additive) activities but instead acted in an unexpectedly synergistic or enhancing [e.g., supra-additive) fashion, as a function of the selected antibiotic, the selected BT compound and the specifically identified target bacteria.
  • a particular infection e.g., a biofilm formed by gram-negative or gram-positive bacteria
  • not every BT compound may synergize with, or be enhancing for, every antibiotic, and not every antibiotic may synergize with, or be enhancing for, every BT compound, such that antibiotic-BT synergy and BT-antibiotic enhancement generally are not predictable.
  • specific combinations of synergizing or enhancing antibiotic and BT compounds surprisingly confer potent antibacterial effects against particular bacteria, including in particular environments such as natural surfaces as described herein, and further including in certain situations antibacterial effects against biofilms formed by the particular bacteria.
  • certain BT-synergizing antibiotics are described herein, which includes an antibiotic that is capable of acting synergistically (FICI ⁇ 0.5) with at least one BT composition that comprises at least one BT compound as provided herein, where such synergy manifests as a detectable effect that is greater (i.e., in a statistically significant manner relative to an appropriate control condition) in magnitude than the effect that can be detected when the antibiotic is present but the BT compound is absent, and/or when the BT compound is present but the antibiotic is absent.
  • FICI ⁇ 0.5 synergistically
  • BT composition that comprises at least one BT compound as provided herein
  • certain BT-antibiotic combinations exhibit enhancement (0.5 ⁇ FICI ⁇ 1.0), where such enhancement manifests as a detectable effect that is greater (i.e., in a statistically significant manner relative to an appropriate control condition) in magnitude than the effect that can be detected when the antibiotic is present but the BT compound is absent, and/or when the BT compound is present but the antibiotic is absent.
  • Examples of such a detectable effect may in certain embodiments include (i) prevention of infection by a bacterial pathogen, (ii) inhibition of cell viability or cell growth of substantially all planktonic cells of a bacterial pathogen, (iii) inhibition of biofilm formation by a bacterial pathogen, and (iv) inhibition of biofilm viability or biofilm growth of substantially all biofilm-form cells of a bacterial pathogen, but the invention is not intended to be so limited, such that in other contemplated embodiments antibiotic-BT synergy may manifest as one or more detectable effects that may include alteration (e.g., a statistically significant increase or decrease) of one or more other clinically significant parameters, for example, the degree of resistance or sensitivity of a bacterial pathogen to one or more antibiotics or other drugs or chemical agents, the degree of resistance or sensitivity of a bacterial pathogen to one or more chemical, physical or mechanical conditions (e.g., pH, ionic strength,
  • a virus e.g., a virus, another bacterium, a biologically active polynucleotide, an immunocyte or an
  • immunocyte product such as an antibody, cytokine, chemokine, enzyme including degradative enzymes, membrane-disrupting protein, a free radical such as a reactive oxygen species, or the like).
  • immunocyte product such as an antibody, cytokine, chemokine, enzyme including degradative enzymes, membrane-disrupting protein, a free radical such as a reactive oxygen species, or the like.
  • synergy may be determined by determining an antibacterial effect such as those described herein using various concentrations of candidate agents (e.g., a BT and an antibiotic individually and in combination) to calculate a fractional inhibitory concentration index (FICI) and a fractional bactericidal concentration index (FBCI), according to Eliopoulos et al. (Eliopoulos and Moellering, (1996) Antimicrobial
  • candidate agents e.g., a BT and an antibiotic individually and in combination
  • FICI fractional inhibitory concentration index
  • FBCI fractional bactericidal concentration index
  • Synergy may be defined as an FICI or FBCI index of ⁇ 0.5, and antagonism at >4. (e.g., Odds, FC (2003) Synergy, antagonism, and what the chequerboard puts between them. Journal of Antimicrobial Chemotherapy 52:1). Synergy may also be defined
  • synergy may be defined as an effect that results from a combination of two drugs (e.g., an antibiotic and a BT composition) wherein the effect of the combination is greater (e.g., in a statistically significant manner) than it would be if the concentration of the second drug is replaced by the first drug.
  • two drugs e.g., an antibiotic and a BT composition
  • a combination of BT and antibiotic will be understood to synergize when a FICI value that is less than or equal to 0.5 is observed.
  • certain BT-antibiotic combinations may exhibit a FICI value between 0.5 and 1.0 that signifies a high potential for such synergy, and which may be observed using non-optimal concentrations of at least one BT and at least one antibiotic that exhibit unilateral or mutually enhanced cooperative antimicrobial efficacy.
  • Such an effect may also be referred to herein as "enhanced” antibiotic activity or “enhanced” BT activity.
  • Enhanced antibiotic and/or BT activity may be detected according to certain embodiments when the presence both (i) of at least one BT at a concentration that is less (in a statistically significant manner) than the characteristic minimum inhibitory concentration (MIC) for that BT for a given target microbe (e.g. , a given bacterial species or strain), and (ii) of at least one antibiotic at a concentration that is less (in a statistically significant manner) than the characteristic IC 50 (concentration that inhibits the growth of 50% of a microbial population; e.g., Soothill et al., 1992 J Antimicrob Chemother
  • BT-antibiotic combination results in enhanced (in a statistically significant manner) antimicrobial efficacy of the BT-antibiotic combination relative to the antimicrobial effect that would be observed if either antimicrobial agent (e.g. , the BT or the antibiotic) were used at the same concentration in the absence of the other antimicrobial agent (e.g. , the antibiotic or the BT).
  • antimicrobial agent e.g. , the BT or the antibiotic
  • "enhanced" antibiotic and/or BT activity is present when a FICI value that is less than or equal to 1.0, and greater than 0.5, is determined.
  • synergistic or enhanced antibiotic and/or BT activity may be determined according to methods known in the art, such as using Loewe additivity-based models (e.g. , FIC index, Greco model), or Bliss independence based models (e.g., non-parametric and semi-parametric models) or other methods described herein and known in the art (e.g.,
  • Certain other embodiments contemplate specific combinations of one or more antibiotic and one or more BT compound as disclosed herein that may exhibit synergizing or enhancing effects in vivo for treatment of a particular infection (e.g. , a biofilm formed by gram-negative or gram-positive bacteria), even where the BT compound(s) and antibiotic(s) did not exhibit predictable (e.g., merely additive) activities in vivo but instead acted in an unexpectedly synergistic or enhancing (e.g., supra-additive; or conferring an effect when two or more such agents are present in combination that is greater (e.g., in a statistically significant manner) than the effect that is obtained if the
  • a statistically significant reduction in bacterial counts observed post- treatment for the BT-antibiotic combination as compared to the antibiotic treatment or BT compound alone is an indication of synergizing or enhancing effects.
  • Statistical significance can be determined using methods well-known to the skilled person.
  • a reduction observed in this or other in vivo models by at least 5%, 10%, 20%, 30%, 40%, or 50% of bacterial counts observed in the injury post-treatment for the BT-antibiotic combination as compared to the antibiotic treatment or BT compound alone is considered an indication of synergizing or enhancing effects.
  • exemplary indicia of in vivo infections may be determined according to established methodologies that have been developed for quantification of the severity of the infection, such as a variety of wound scoring systems known to the skilled person (see e.g., scoring systems reviewed in European Wound Management Association (EWMA), Position Document:
  • EWMA European Wound Management Association
  • BT-antibiotic combinations include ASEPSIS (Wilson AP, J Hosp Infect 1995; 29(2): 81-86; Wilson et al., Lancet 1986; 1 : 311-13), the Victoria Wound Assessment Scale (Bailey IS, Karran SE, Toyn K, et al. BMJ 1992; 304: 469-71). See also, Horan TC, Gaynes P, Martone WJ, et al. , 1992 Infect Control Hosp Epidemiol 1992; 13: 606-08.
  • recognized clinical indicia of wound healing known to the skilled clinician may also be measured in the presence or absence of BT compounds and/or antibiotics, such as wound size, depth, granulation tissue condition, infection, etc. Accordingly, and based on the present disclosure, the skilled person will readily appreciate a variety of methods for determining whether a BT composition -antibiotic combination alters (e.g., increases or decreases in a statistically significant manner relative to appropriate controls) in vivo wound healing.
  • a wide variety of methods for treating microbially infected natural surfaces such as surfaces that support or contain bacterial biofilms, with an effective amount (e.g., in certain embodiments a therapeutically effective amount) of a composition or formulation that comprises one or more BT compounds and, optionally, one or more antibiotic compounds, such as one or more synergizing antibiotics, or one or more enhancing antibiotics, as provided herein.
  • an effective amount e.g., in certain embodiments a therapeutically effective amount
  • a composition or formulation that comprises one or more BT compounds and, optionally, one or more antibiotic compounds, such as one or more synergizing antibiotics, or one or more enhancing antibiotics, as provided herein.
  • compositions that comprise one or more BT compounds for use as antiseptics.
  • An antiseptic is a substance that kills or prevents the growth of microorganisms, and may be typically applied to living tissue, distinguishing the class from disinfectants, which are usually applied to inanimate objects (Goodman and Gilman's "The Pharmacological Basis of Therapeutics ", Seventh Edition, Gilman et al., editors, 1985, Macmillan Publishing Co., (hereafter, Goodman and Gilman”) pp. 959-960).
  • disinfectants which are usually applied to inanimate objects
  • antiseptics are ethyl alcohol and tincture of iodine.
  • Germicides include antiseptics that kill microbes such as microbial pathogens.
  • compositions that comprise one or more BT compounds and one or more antibiotic compound (e.g., a synergizing antibiotic and/or an enhancing antibiotic as provided herein).
  • antibiotics are known in the art and typically comprise a drug made from a compound produced by one species of microorganism to kill another species of microorganism, or a synthetic product having an identical or similar chemical structure and mechanism of action, e.g., a drug that destroys microorganisms within or on the body of a living organism, including such drug when applied topically.
  • an antibiotic may belong to one of the following classes: aminoglycosides, carbapenems, cephalosporins, fluoroquinolones, glycopeptide antibiotics, lincosamides (e.g., clindamycin), penicillinase-resistant penicillins, and aminopenicillins.
  • Antibiotics thus may include, but need not be limited to, oxacillin, piperacillin, cefuroxime, cefotaxime, cefepime, imipenem, aztreonam, streptomycin, tobramycin, tetracycline, minocycline, ciprofloxacin, levofloxacin, erythromycin, linezolid, phosphomycin, capreomycin, isoniazid, ansamycin, carbacephem, monobactam, nitrofuran, penicillin, quinolone, sulfonamide, Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin, Rifampin, Rifabutin,
  • tetracycline glycylcycline, methicillin, vancomycin, naficilin, gentamicin, ampicillin , chloramphenicol, doxycycline, tobramycin, amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin,
  • rhodostreptomycin streptomycin, tobramycin, apramycin, clindamicin, gatifloxacin, aminopenicillin, and others known to the art.
  • Compendia of these and other clinically useful antibiotics are available and known to those familiar with the art (e.g., Washington University School of Medicine, The Washington Manual of Medical Therapeutics (32 nd Ed.), 2007 Lippincott, Williams and Wilkins, Philadelphia, PA; Hauser, AL, Antibiotic Basics for Clinicians, 2007 Lippincott, Williams and Wilkins, Philadelphia, PA).
  • An exemplary class of antibiotics for use with one or more BT compounds in certain herein disclosed embodiments is the aminoglycoside class of antibiotics, which are reviewed in Edson RS, Terrell CL.
  • aminoglycosides Mayo Clin Proc. 1999 May; 74(5):519-28.
  • This class of antibiotics inhibits bacterial growth by impairing bacterial protein synthesis, through binding and inactivation of bacterial ribosomal subunits.
  • aminoglycosides also exhibit bacteriocidal effects through disruption of cell walls in gram-negative bacteria.
  • Aminoglycoside antibiotics include gentamicin, amikacin, streptomycin, and others, and are generally regarded as useful in the treatment of gram-negative bacteria, mycobacteria and other microbial pathogens, although cases of resistant strains have been reported.
  • the aminoglycosides are not absorbed through the digestive tract and so are not generally regarded as being amenable to oral formulations.
  • Amikacin for example, although often effective against gentamicin-resistant bacterial strains, is typically administered intravenously or intramuscularly, which can cause pain in the patient.
  • certain embodiments disclosed herein contemplate oral administration of a synergizing BT/antibiotic combination (e.g., where the antibiotic need not be limited to an aminoglycoside) for instance, for treatment of an epithelial tissue surface at one or more locations along the oral cavity, gastrointestinal tract/ alimentary canal.
  • a synergizing BT/antibiotic combination e.g., where the antibiotic need not be limited to an aminoglycoside
  • an epithelial tissue surface at one or more locations along the oral cavity, gastrointestinal tract/ alimentary canal.
  • compositions and methods described herein as disinfectants refers to preparations that kill, or block the growth of, microbes on an external surface of an inanimate object.
  • a BT compound may be a composition that comprises bismuth or a bismuth salt and a thiol- (e.g., -SH, or sulfhydryl) containing compound, including those that are described (including their methods of preparation) in Domenico et al., 1997 Antimicrob. Agent.
  • a thiol- e.g., -SH, or sulfhydryl
  • the BT compound comprises bismuth in association with the thiol-containing compound via ionic bonding and/or as a coordination complex, while in some other embodiments bismuth may be associated with the thiol-containing compound via covalent bonding such as may be found in an organometallic compound.
  • BT compounds may also be prepared according to
  • an acidic aqueous bismuth solution that contains dissolved bismuth at a concentration of at least 50 mM, at least 100 mM, at least 150 mM, at least 200 mM, at least 250 mM, at least 300 mM, at least 350 mM, at least 400 mM, at least 500 mM, at least 600 mM, at least 700 mM, at least 800 mM, at least 900 mM or at least 1 M and that lacks a hydrophilic, polar or organic solubilizer is admixed with ethanol to obtain a first ethanolic solution, which is reacted with a second ethanolic solution comprising a thiol-containing compound to obtain a reaction solution, wherein the thiol- containing compound is present in the reaction solution at a molar ratio of from about 1 :3 to about 3:1 relative to the bismuth, under conditions
  • exemplary BTs include compound 1 B-1 , Bis-EDT
  • compositions comprising BT compounds may desirably yield compositions comprising BT compounds where such compositions have one or more desirable properties, including ease of large-scale production, improved product purity, uniformity or consistency (including uniformity in particle size), or other properties useful in the preparation and/or administration of the present topical formulations.
  • BT compositions prepared according to the methods described herein for the first time, exhibit an advantageous degree of homogeneity with respect to their occurrence as a substantially monodisperse suspension of microparticles each having a volumetric mean diameter (VMD) according to certain presently preferred embodiments of from about 0.4 ⁇ to about 5 Mm.
  • VMD volumetric mean diameter
  • MMD mass median diameter
  • MMAD mass median aerodynamic diameter
  • VMD, MMD and MMAD may be the same if environmental conditions are maintained, e.g., standard humidity.
  • VMD, MMD and MMAD measurements are considered to be under standard conditions such that descriptions of VMD, MMD and MMAD will be comparable.
  • dry powder particle size determinations in MMD, and MMAD are also considered comparable.
  • preferred embodiments relate to a substantially monodisperse suspension of BT-containing microparticles.
  • Generation of a defined BT particle size with limited geometric standard deviation may, for instance, optimize BT deposition, accessibility to desired target sites in or on a natural surface, and/or tolerability by a subject to whom the BT microparticles are administered.
  • Narrow GSD limits the number of particles outside the desired VMD or MMAD size range.
  • a liquid or aerosol suspension of microparticles containing one or more BT compounds disclosed herein having a VMD from about 0.5 microns to about 5 microns.
  • a liquid or aerosol suspension having a VMD or MMAD from about 0.7 microns to about 4.0 microns is provided.
  • a liquid or aerosol suspension having aVMD or MMAD from about 1.0 micron to about 3.0 microns is provided.
  • a liquid suspension comprising one or a plurality of BT compound particles of from about 0.1 to about 5.0 microns VMD, or of from about 0.1 , about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8 or about 0.9 microns to about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5 or about 8.0 microns, the particle comprising a BT compound prepared as described herein.
  • a BT preparation described for the first time herein which is "substantially"
  • a BT composition that comprises a BT compound in microparticulate form wherein "substantially" all of the microparticles have a volumetric mean diameter (VMD) within a specified range (e.g., from about 0.4 ⁇ to about 5 ⁇ ), includes those compositions in which at least 80%, 85%, 90%, 91 %, 92%, 93%, or 94%, more preferably at least 95%, 96%, 97%, 98%, 99% or more of the particles have a VMD that is within the recited size range.
  • VMD volumetric mean diameter
  • characterization in a manner that facilitates regulatory compliance according to one or more of pharmaceutical, formulary and cosmeceutical standards.
  • the herein described substantially monodisperse BT microparticles may advantageously be produced without the need for micronization, i.e., without the expensive and labor-intensive milling or supercritical fluid processing or other equipment and procedures that are typically used to generate microparticles (e.g., Martin et al. 2008 Adv. Drug Deliv. Rev. 60(3):339; Moribe et al., 2008 Adv. Drug Deliv. Rev. 60(3):328; Cape et al., 2008 Pharm. Res. 25(9):1967; Rasenack et al. 2004 Pharm. Dev. Technol. 9(1): 1-13).
  • the present embodiments offer beneficial effects of substantially uniform microparticulate preparations, including without limitation enhanced and substantially uniform solubilization properties, suitability for desired administration forms such as oral, inhaled or dermatological/ skin wound topical forms, increased bioavailability and other beneficial properties.
  • the BT compound microparticulate suspension can be administered as aqueous formulations, as suspensions or solutions in aqueous as well as organic solvents including halogenated hydrocarbon propellants, as dry powders, or in other forms as elaborated below, including preparations that contain wetting agents, surfactants, mineral oil or other ingredients or additives as may be known to those familiar with formulary, for example, to maintain individual microparticles in suspension.
  • Aqueous formulations may be aerosolized by liquid nebulizers employing, for instance, either hydraulic or ultrasonic atomization.
  • Propellant-based systems may use suitable pressurized dispensers.
  • Dry powders may use dry powder dispersion devices, which are capable of dispersing the BT-containing microparticles effectively. A desired particle size and distribution may be obtained by choosing an appropriate device.
  • a method for preparing a bismuth-thiol (BT) composition that comprises a plurality of microparticles that comprise a BT compound, substantially all of such microparticles having a volumetric mean diameter (VMD) of from about 0.1 to about 8 microns, and in certain preferred
  • embodiments from about 0.4 microns to about 5 microns.
  • the method comprises the steps of (a) admixing, under conditions and for a time sufficient to obtain a solution that is
  • an acidic aqueous solution that comprises a bismuth salt comprising bismuth at a concentration of at least 50 mM and that lacks a hydrophilic, polar or organic solubilizer, with (ii) ethanol in an amount sufficient to obtain an admixture that comprises at least about 5%, 10%, 15%, 20%, 25% or 30%, and preferably about 25% ethanol by volume; and (b) adding to the admixture of (a) an ethanolic solution comprising a thiol- containing compound to obtain a reaction solution, wherein the thiol-containing compound is present in the reaction solution at a molar ratio of from about 1 :3 to about 3:1 relative to the bismuth, under conditions and for a time sufficient for formation of a precipitate which comprises the BT compound.
  • the bismuth concentration in the acidic aqueous solution may be at least 100 mM, at least 150 mM, at least 200 mM, at least 250 mM, at least 300 mM, at least 350 mM, at least 400 mM, at least 500 mM, at least 600 mM, at least 700 mM, at least 800 mM, at least 900 mM or at least 1 M.
  • the acidic aqueous solution comprises at least 5%, 10%, 15%, 20%, 22% or 22.5% bismuth by weight.
  • the acidic aqueous solution may in certain preferred embodiments comprise at least 5% or more nitric acid by weight, and in certain other embodiments the acidic aqueous solution may comprise at least 0.5%, at least 1%, at least 1.5%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5% or at least 5% nitric acid by weight.
  • the thiol-containing compound may be any thiol-containing compound as described herein, and in certain embodiments may comprise one or more of 1 ,2-ethane dithiol, 2,3-dimercaptopropanol, pyrithione,
  • dithioerythritol 3,4-dimercaptotoluene, 2,3-butanedithiol, 1 ,3-propanedithiol, 2- hydroxypropane thiol, 1-mercapto-2-propanol, dithioerythritol and dithiothreitol.
  • exemplary thiol-containing compounds include alpha-lipoic acid, methanethiol (CH 3 SH [m-mercaptan]), ethanethiol (C 2 H 5 SH [e- mercaptan]), 1- propanethiol (C3H7SH [n-P mercaptan]), 2-Propanethiol (CH 3 CH(SH)CH 3 [2C 3 mercaptan]), butanethiol (C 4 H 9 SH ([n-butyl mercaptan]), tert-butyl mercaptan (C(CH 3 ) 3 SH [t-butyl mercaptan]), pentanethiols (C 5 HnSH [pentyl mercaptan]), coenzyme A, lipoamide, glutathione, cysteine, cystine, 2-mercaptoethanol, dithiothreitol, dithioerythritol, 2-mercaptoindole, transgluta
  • nanoparticles dodecanethiol functionalized silver nanoparticles, hexa(ethylene glycol)mono-11-(acetylthio)undecyl ether, mercaptosuccinic acid, methyl 3- mercaptopropionate, nanoTether BPA-HH, NanoThinks TM 18, NanoThinks TM 8, NanoThinksTM ACID11 , NanoThinksTM ACID16, NanoThinksTM ALC011 , NanoThinksTM THIO8, octanethiol functionalized gold nanoparticles, PEG dithiol average M n 8,000, PEG dithiol average mol wt 1 ,500, PEG dithiol average mol wt 3,400, S-(11-bromoundecyl)thioacetate, S-(4- cyanobutyl)thioacetate, thiophenol, triethylene glycol mono-11- mercaptoundecyl ether, trimethyl
  • reaction conditions including temperature, pH, reaction time, the use of stirring or agitation to dissolve solutes and procedures for collecting and washing precipitates, are described herein and employ techniques generally known in the art.
  • BT products are provided as microparticulate suspensions having substantially all
  • microparticles with VMD from about 0.4 to about 5 microns in certain preferred embodiments, and generally from about 0.1 microns to about 8 microns according to certain other embodiments.
  • bismuth is provided in an acidic aqueous solution that comprises a bismuth salt at a concentration of from at least about 50 mM to about 1 M, and nitric acid in an amount from at least about 0.5% to about 5% (w/w), and preferably less than 5% (weight/weight), and that lacks a hydrophilic, polar or organic solubilizer.
  • Hydrophilic, polar or organic solubilizers include propylene glycol (PG) and ethylene glycol (EG) and may also include any of a large number of known solubility enhancers, including polar solvents such as dioxane and dimethylsulfoxide (DMSO), polyols (including, e.g., PG and EG and also including polyethylene glycol (PEG), polypropyleneglycol (PPG), pentaerythritol and others), polyhydric alchohols such as glycerol and mannitol, and other agents.
  • polar solvents such as dioxane and dimethylsulfoxide (DMSO)
  • polyols including, e.g., PG and EG and also including polyethylene glycol (PEG), polypropyleneglycol (PPG), pentaerythritol and others
  • PEG polyethylene glycol
  • PPG polypropyleneglycol
  • DMF dimethylformamide
  • NMP N-methyl-2-pyrrolidone
  • solvents including those commonly used as hydrophilic, polar or organic solubilizers as provided herein, may be selected, for instance, based on the solvent polarity/ polarizability (SPP) scale value using the system of Catalan et al. (e.g., 1995 Liebigs Ann. 241 ; see also Catalan, 2001 In: Handbook of Solvents, Wypych (Ed.), Andrew Publ., NY, and references cited therein), according to which, for example, water has a SPP value of 0.962, toluene a SPP value of 0.655, and 2-propanol a SPP value of 0.848.
  • SPP solvent polarity/ polarizability
  • Solubility parameters may also include the interaction parameter C, Hildebrand solubility parameter d, or partial (Hansen) solubility parameters: ⁇ , 5h and 5d, describing the solvent's polarity, hydrogen bonding potential and dispersion force interaction potential, respectively.
  • the highest value for a solubility parameter that describes a solvent or co-solvent system in which the bismuth salt comprising bismuth will dissolve may provide a limitation for the aqueous solution that comprises the bismuth salt, for instance, according to the presently described method for preparing a microparticulate BT composition. For example, higher 5h vafues will have a greater hydrogen bonding ability and would therefore have a greater affinity for solvent molecules such as water. A higher value of maximum observed 6h for a solvent may therefore be preferred for situations where a more hydrophilic environment is desired.
  • BisEDT having the structure shown below in formula I may be prepared according to the following reaction scheme:
  • aqueous acidic bismuth solution such as a Bi(NO 3 ) 3 solution (e.g., 43% Bi(NO 3 )3 (w/w), 5% nitric acid (w/w), 52% water (w/w), available from Shepherd Chemical Co., Cincinnati, OH) with stirring, followed by slow addition of absolute ethanol (4 L).
  • a Bi(NO 3 ) 3 solution e.g., 43% Bi(NO 3 )3 (w/w), 5% nitric acid (w/w), 52% water (w/w), available from Shepherd Chemical Co., Cincinnati, OH
  • An ethanolic solution (1.56 L) of a thiol compound such as 1 ,2-ethanedithiol [ ⁇ 0.55 M] may be separately prepared by adding, to 1.5 L of absolute ethanol, 72.19 ml_ (0.863 moles) of 1 ,2-ethanedithiol using a 60 ml_ syringe, and then stirring for five minutes.
  • 1 ,2- ethanedithiol (CAS 540-63-6) and other thiol compounds are available from, e.g., Sigma-Aldrich, St. Louis, MO.
  • the ethanolic solution of the thiol compound may then be slowly added to the aqueous Bi(NO 3 )3 / HNO3 solution with stirring overnight to form a reaction solution.
  • the thiol-containing compound may be present in the reaction solution, according to certain preferred embodiments, at a molar ratio of from about 1 :3 to about 3:1 relative to the bismuth.
  • the formed product is allowed to settle as a precipitate comprising microparticles as described herein, which is then collected by filtration and washed sequentially with ethanol, water and acetone to obtain BisEDT as a yellow amorphous powdered solid.
  • the crude product may be redissolved in absolute ethanol with stirring, then filtered and washed sequentially with ethanol several times followed by acetone several times.
  • the washed powder may be triturated in 1 M NaOH (500ml_), filtered and washed sequentially with water, ethanol and acetone to afford purified microparticulate BisEDT.
  • bismuth inhibits the ability of bacteria to produce extracellular polymeric substances (EPS) such as bacterial exopolysaccharides, and this inhibition leads to impaired biofilm formation.
  • Bacteria are believed to employ the glue-like EPS for biofilm cohesion.
  • EPS may contribute to bacterial pathogenicity such as interference with wound healing.
  • bismuth alone is not therapeutically useful as an intervention agent, and is instead typically administered as part of a complex such as a BT.
  • Bismuth-thiols are thus a family of compositions that includes compounds that result from the chelation of bismuth with a thiol compound, and that exhibit dramatic improvement in the antimicrobial therapeutic efficacy of bismuth.
  • BTs exhibit remarkable anti-infective, anti-biofilm, and immunomodulatory effects.
  • Bismuth thiols are effective against a broad-spectrum of microorganisms, and are typically not affected by antibiotic-resistance.
  • BTs prevent biofilm formation at remarkably low (sub-inhibitory) concentrations, prevent many pathogenic characteristics of common wound pathogens at those same sub-inhibitory levels, can prevent septic shock in animal models, and may be synergistic with many antibiotics.
  • synergy in the antibacterial effects of one or more specified BT when combined with one or more specified antibiotic compound is not readily predictable based on profiles of separate antibiotic and BT effects against a particular bacterial type, but surprisingly may result from selection of particular BT-antibiotic combinations in view of the specific bacterial population, including identification of whether gram-negative or gram-positive (or both) bacteria are present.
  • antibiotics that synergize with certain BTs may include one or more of amikacin, ampicillin, aztreonam, cefazolin, cefepime, chloramphenicol, ciprofloxacin, clindamycin (or other lincosamide antibiotics), daptomycin (Cubicin®), doxycycline, gatifloxacin, gentamicin, imipenim, levofloxacin, linezolid (Zyvox®), minocycline, nafcilin, paromomycin, rifampin, sulphamethoxazole, tetracycline, tobramycin and vancomycin.
  • MRSA MRSA
  • compositions and/or methods in which may be included the combination of a BT compound and one or more antibiotics selected from amikacin, ampicillin, cefazolin, cefepime, chloramphenicol, ciprofloxacin, clindamycin (or another lincosamide antibiotic), daptomycin (Cubicin®),_doxycycline, gatifloxacin, gentamicin, imipenim, levofloxacin, linezolid (Zyvox®), minocycline, nafcilin, paromomycin, rifampin,
  • compositions and/or methods in which may be included the combination of a BT compound and one or more antibiotics from which expressly excluded may be one or more antibiotic selected from amikacin, ampicillin, cefazolin, cefepime, chloramphenicol, ciprofloxacin, clindamycin (or other lincosamides), daptomycin (Cubicin®), doxycycline, gatifloxacin, gentamicin, imipenim, levofloxacin, linezolid (Zyvox®), minocycline, nafcilin, paromomycin, rifampin, sulphamethoxazole, tobramycin and vancomycin.
  • antibiotics from which expressly excluded may be one or more antibiotic selected from amikacin, ampicillin, cefazolin, cefepime, chloramphenicol, ciprofloxacin, clindamycin (or other lincosamides), daptomycin (
  • gentamicin and tobramycin belong to the aminoglycoside class of antibiotics. Also expressly excluded from certain contemplated embodiments are certain compositions and methods described in Domenico et al., 2001 Agents Chemother. 45:1417-1421 ;
  • Domenico et al. 2000 Infect. Med. 17:123-127; Domenico et al., 2003 Res.
  • compositions and methods for treating a subject with a composition that comprises the herein described microparticulate BT and that optionally and in certain other embodiments also comprises a synergizing and/or an enhancing antibiotic.
  • promoting skin tissue repair (or other tissue repair, such as epithelial tissue, bone, joint, muscle tendon, or ligament repair) is contemplated.
  • promoting skin tissue or other epithelial tissue repair may comprise stimulating or disinhibiting one or more cellular wound repair activities selected from (i) epithelial cell (e.g.,
  • epithelial cell e.g., keratinocyte
  • dermal fibroblast growth e.g., keratinocyte
  • downregulation of epithelial cell e.g., keratinocyte
  • dermal fibroblast collagenase gelatinase or matrix
  • compositions and methods that relate to art accepted models for wound repair based on keratinocyte wound closure following a scratch wound.
  • compositions for treating a microbial infection on or in a natural surface for use according to the embodiments described herein may include in certain embodiments compositions that comprise bismuth-thiol (BT) compounds as described herein, and which may in certain distinct but related embodiments also include other compounds that are known in the art such as one or more antibiotic compounds as described herein.
  • BT compounds and methods for making them are disclosed herein and are also disclosed, for example, in Domenico et al. (1997 Antimicrob. Agent. Chemother. 41 (8): 1697- 1703; 2001 Antimicrob. Agent. Chemother. 45(5)1417-1421) and in U.S.
  • certain preferred BT compounds are those that contain bismuth or a bismuth salt ionically bonded to, or in a coordination complex with, a thiol- containing compound, such as a composition that comprises bismuth chelated to the thiol-containing compound, and certain other preferred BT compounds are those that contain bismuth or a bismuth salt in covalent bond linkage to the thiol-containing compound. Also preferred are substantially monodisperse microparticulate BT compositions as described herein.
  • methods for treating a natural surface comprising administering to the surface at least one microparticulate BT compound as described herein.
  • the method further comprises administering, simultaneously or sequentially and in either order, at least one antibiotic compound, which in certain preferred embodiments may be a synergizing antibiotic as described herein, and which in certain other preferred embodiments may be an enhancing antibiotic as described herein.
  • the antibiotic compound may be an
  • aminoglycoside antibiotic a carbapenem antibiotic, a cephalosporin antibiotic, a fluoroquinolone antibiotic, a glycopeptides antibiotic, a lincosamide antibiotic, a penicillinase-resistant penicillin antibiotic, or an aminopenicillin antibiotic.
  • a preferred therapeutically effective formulation may comprise a BT compound (e.g., BisEDT, bismuth: 1 ,2-ethanedithiol; BisPyr,
  • a BT compound e.g., BisEDT, bismuth: 1 ,2-ethanedithiol; BisPyr,
  • BT compound and linezolid Zyvox®, Pfizer, Inc., NY, NY
  • a BT compound e.g., BisEDT, bismuth:1 ,2- ethanedithiol; BisPyr, bismuth:pyrithione; BisEDT/Pyr, bismuth:1 ,2- ethanedithiol/pyrithione
  • ampicillin cefazolin, cefepime, chloramphenicol, clindamycin (or another lincosamide antibiotic), daptomycin (Cubicin®), doxycycline, gatifloxacin, gentamicin, imipenim, levofloxacin, linezolid (Zyvox®), nafcilin, paromomycin, rifampin, sulphamethoxazole, tobramycin and vancomycin.
  • BisEDT bismuth:1 ,2- ethanedithiol
  • a preferred therapeutically effective formulation may comprise a BT compound and amikacin.
  • Certain related embodiments contemplate treatment of an infection comprising gram negative bacteria with a BT compound and another antibiotic, such as another aminoglycoside antibiotic, which in certain embodiments is not gentamicin or tobramycin.
  • other related embodiments contemplate identifying one or more bacterial populations or subpopulations in or on a natural surface by the well known criterion of being gram positive or gram negative, according to methodologies that are familiar to those skilled in the medical microbiology art, as a step for selecting appropriate antibiotic compound(s) to include in a formulation to be administered according to the present methods.
  • compositions and methods may find use in the treatment of microbes (e.g., bacteria, viruses, yeast, molds and other fungi, microbial parasites, etc.) in a wide variety of contexts, typically by application or administration of the herein described compounds (e.g., one or more microparticulate BTs alone or in combination with one or more microbes (e.g., bacteria, viruses, yeast, molds and other fungi, microbial parasites, etc.) in a wide variety of contexts, typically by application or administration of the herein described compounds (e.g., one or more microparticulate BTs alone or in combination with one or more
  • Such natural surfaces include but are not limited to mammalian tissues (e.g., epithelia including skin, scalp, gastrointestinal tract lining, buccal cavity, etc.; endothelia, cell and tissue membranes such as peritoneal membrane, pericardial membrane, pleural membrane, periosteal membrane, meningeal membranes, sarcolemal membranes, and the like; cornea, sclera, mucous membranes, etc.; and other mammalian tissues such as teeth, bone, joint, tendon, ligament, muscle, heart, lung, kidney, liver, spleen, gall bladder, pancreas, bladder, nerve, etc.).
  • mammalian tissues e.g., epithelia including skin, scalp, gastrointestinal tract lining, buccal cavity, etc.
  • endothelia, cell and tissue membranes such as peritoneal membrane, pericardial membrane, pleural membrane, periosteal membrane, meningeal membranes, sarcolemal membranes, and the like
  • microparticulate antimicrobial agents described herein may be used to suppress microbial growth, reduce microbial infestation, reduce biofilm, prevent conversion of bacteria to biofilm, prevent or inhibit microbial infection and any other use described herein. These agents are also useful for a number of antiviral purposes, including prevention or inhibition of viral infection by herpes family viruses such as cytomegalovirus, herpes simplex virus Type 1 , and herpes simplex virus Type 2, and/or infection by other viruses.
  • herpes family viruses such as cytomegalovirus, herpes simplex virus Type 1 , and herpes simplex virus Type 2, and/or infection by other viruses.
  • the agents are useful for the prevention or inhibition of viral infection by a variety of viruses, such as, single stranded RNA viruses, single stranded DNA viruses, Rous sarcoma virus (RSV), hepatitis A virus, hepatitis B virus (HBV), Hepatitis C (HCV), Influenza viruses, west nile virus (WNV), Epstein-Barr virus (EBV), eastern equine encephalitis virus (EEEV), severe acute respiratory virus (SARS), human immunodeficiency virus (HIV), human papilloma virus (HPV), and human T cell lymphoma virus (HTLV).
  • viruses such as, single stranded RNA viruses, single stranded DNA viruses, Rous sarcoma virus (RSV), hepatitis A virus, hepatitis B virus (HBV), Hepatitis C (HCV), Influenza viruses, west nile virus (WNV), Epstein-Barr virus (EBV), eastern equine encepha
  • antimicrobial agents include, but are not limited to, treatment or prevention of bacterial infection, of tuberculosis, of fungal infections such as yeast and mold infections (for example, Candida (e.g., Candida albicans, Candida glabrata, C. parapsilosis, C. tropicalis, and C. dubliniensis) or
  • the agent is used at a dosage not generally lethal to bacteria but which is nonetheless sufficient to reduce protective
  • a microparticulate BT compound described herein or composition comprising the microparticulate BT
  • interspecies quorum sensing is related to biofilm formation. Certain agents that increase LuxS-dependent pathway or interspecies quorum sensing signal (see, e.g., U.S. Patent No. 7,427,408) contribute to controlling development and/or proliferation of a biofilm.
  • Exemplary agents include, by way of example, N-(3-oxododecanoyl)-L- homoserine lactone (OdDHL) blocking compounds and N-butyryl-L-homoserine lactone (BHL) analogs, either in combination or separately (see, e.g., U.S.
  • Patent No. 6,455,03 An oral hygiene composition
  • An oral hygiene composition comprising a
  • microparticulate BT compound and at least one anti-biofilm agent can be delivered locally for disruption and inhibition of bacterial biofilm and for treatment of periodontal disease (see, e.g., U.S. Patent No.6,726,898).
  • compositions Comprising Microparticulate Bismuth-Thiols and Uses for Oral Hygiene and for Treating Inflammation and Infection of the Mouth.
  • compositions comprising microparticulate BT
  • compositions are formulated for oral use and may be used in methods for preventing or reducing microbial growth in the mouth and for preventing and/or treating microbial infections and inflammation of the oral cavity. These compositions are therefore useful for preventing or treating (i.e., reducing or inhibiting development of, reducing the likelihood of occurrence or recurrence of) dental plaque, halitosis, periodontal disease, gingivitis, and other infections of the mouth.
  • the oral compositions comprising microparticulate BT compound may also be useful for preventing and/or controlling (i.e., slowing, retarding, inhibiting) biofilm development, disrupting a biofilm, or reducing the amount of biofilm present on an oral surface, particularly a tooth or gums.
  • Good oral hygiene is important not only for oral health, but for prevention of several chronic conditions. Controlling bacterial growth in the mouth may help lower risk of heart disease, preserve memory, and reduce the risk of infection and inflammation in other areas of the body. People with diabetes are at greater risk for developing severe gum problems, and reducing the risk of gingivitis by maintaining good oral health may help control blood sugar. Pregnant women may be more likely to experience gingivitis, and some research suggests a relationship between gum disease in pregnant women and delivery of preterm, low-birth-weight infants.
  • Bacteria are the primary etiologic agents in periodontal disease. More than 500 bacterial strains may be found in dental plaque (Kroes et al., Proc. Natl. Acad. Sci. USA 96:14547-52 (1999)). Bacteria have evolved to survive in the environment of the tooth surface, gingival epithelium, and oral cavity as biofilms, which contributes to the difficulty in treating periodontitis. Bactericidal agents as well as antibiotics that are currently used to treat such infections often do not kill all of offending organisms. Use of an agent that is ineffective against certain bacteria species may result in proliferation of resistant bacterial species. Moreover, these agents may cause unpleasant side effects, such allergic reactions, inflammation, and tooth discoloration.
  • Dental bacterial plaque is a biofilm that adheres tenaciously to tooth surfaces, restorations, and prosthetic appliances.
  • the primary means to control biofilms in the mouth is through mechanical cleaning (i.e., tootbrushing, flossing, etc.).
  • the tooth's surface is colonized predominantly by gram-positive facultative cocci, which are primarily streptococci species.
  • the bacteria excrete an extracellular slime layer that helps anchor the bacteria to the surface and provides protection for the attached bacteria.
  • Microcolony formation begins once the surface of the tooth has been covered with attached bacteria.
  • the biofilm grows primarily through cell division of adherent bacteria, rather than through the attachment of new bacteria. Doubling times of bacteria forming plaque are rapid in early development and slower in more mature biofilms.
  • Coaggregation occurs when bacterial colonizers subsequently adhere to bacteria already attached to the pellicle.
  • the result of coaggregation is the formation of a complex array of different bacteria linked to one another.
  • the gingival margin becomes inflamed and swollen. Inflammation may result in creation of a deepened gingival sulcus.
  • the biofilm extends into this subgingival region and flourishes in this protected environment, resulting in the formation of a mature subgingival plaque biofilm. Gingival inflammation does not appear until the biofilm changes from one composed largely of gram-positive bacteria to one containing gram- negative anaerobes.
  • a subgingival bacterial microcolony composed
  • Bacterial microcolonies protected within the biofilm are typically resistant to antibiotics (administered systemical!y), antiseptics or disinfectants (administered locally), and immune defenses.
  • Antibiotic doses that kill free- floating bacteria for example, need to be increased as much as 1 ,500 times to kill biofilm bacteria. At this high concentration, these antimicrobials tend to be toxic to the patient as well (see, e.g., Coghlan 1996, New Scientist 2045:32-6; Elder et al., 1995, Eye 9:102-9).
  • a microparticulate BT compound may be incorporated into oral hygiene compositions , such as but not limited to, toothpaste, mouthwash (i.e., mouth rinse), oral gels, dentifrice powders, oral sprays (including a spray dispersed by an oral inhaler), edible film, chewing gum, oral slurry, denture liquid cleaners, denture storage liquids, and dental floss, which may be routinely used by any subject.
  • a microparticulate BT compound may be incorporated into oral hygiene compositions that are used primarily by dental care professions, including for example, fluoride liquid treatments, cleaning compositions, buffing compositions, oral rinses, and dental floss.
  • present embodiments contemplate replacement of antimicrobials formulated with oral hygiene compositions, which are described in the art, with the presently described microparticulate BT compounds to provide the advantages disclosed herein, including the range of antimicrobial activities, solubility and bioavailability, anti-biofilm effects, non-toxicity, enhancement of antibiotic efficacies, and other properties as described herein.
  • a microparticulate BT compound may also be used for preventing or treating caries and/or inflammation (i.e., reducing the likelihood of occurrence or recurrence of caries and/or inflammation, respectively) by administering the microparticulate BT compound to the surface of the teeth.
  • a composition comprising a microparticulate BT compound may be a mucoadhesive
  • composition that is applied to the surface of a tooth and/or gum or oral mucous membrane may be in any form that adheres to some extent to a surface or that delivers a pharmaceutically effective amount of the active ingredient(s) to the desired surface.
  • a microparticulate BT compound can also be formulated to release slowly from the composition applied to the tooth.
  • the composition may be a gel (e.g., a hydrogel, thiomer, aerogel, or organogel) or liquid.
  • An organogel may comprise an organic solvent, lipoic acid, vegetable oil, or mineral oil.
  • Such gel or liquid coating formulations may be applied interior or exterior to an amalgam or composite or other restorative composition.
  • a slow-release composition may deliver a pharmaceutically effective amount of microparticulate BT compound for 1 , 2, 3, 4, 5, 6, or 7 (a week) days or for 2, 3, 4, 5, 6, 7 weeks, or 1 , 2, 3, 4, 5, or 6 months.
  • Such compositions can be prepared by a person skilled in the art using any number of methods known in the art.
  • antimicrobial compositions are provided for oral use that comprise
  • microparticulate BT compound and one or more additional antimicrobial compounds or agents.
  • additional antimicrobial compounds or agents are particularly useful.
  • compositions comprising s and a second antimicrobial agent that when administered in combination have enhanced or synergistic antimicrobial effects, as described herein.
  • an enhanced antimicrobial effect may be observed when a
  • microparticulate BT compound is administered together with an antimicrobial agent that chelates iron.
  • a microparticulate BT compound is formulated with an anti-inflammatory agent, compound, small molecule, or macromolecule (such as a peptide or polypeptide).
  • microparticulate BT compounds described herein may be formulated for oral use.
  • microparticulate BT compounds that are prepared with hydrophobic thiols ⁇ e.g., thiochlorophenol) may be used and which may exhibit greater capability than less hydrophobic BT compounds to adhere to teeth and tissues of the mouth.
  • BT compounds that have a net negative charge, such as those having a 1 :2 molar ratio (bismuth to thiol) may also have favorable adhesive properties.
  • the oral hygiene compositions comprising a microparticulate BT compound may further comprise one or more active ingredients and/or one or more orally suitable excipients or carriers.
  • the oral hygiene compositions may further comprise baking soda or another alkaline compound or substance. Because of the chemical and physical properties of baking soda, it has wide range of applications, including cleaning, deodorizing, and buffering. Baking soda neutralizes odors chemically, rather than masking or absorbing them. Baking soda can be combined with a microparticulate BT compound either as a mixture of powders, or dissolved or suspended in any one of the dentifrice powders, gels, pastes, and liquids described herein.
  • a microparticulate BT compound can be combined with other alkali metal bicarbonate or carbonate substances (e.g., potassium bicarbonate or calcium carbonate) that help maintain a desired alkaline pH and that also possess cleansing and deodorizing properties.
  • alkali metal bicarbonate or carbonate substances e.g., potassium bicarbonate or calcium carbonate
  • Oral hygiene compositions comprising a microparticulate BT compound may further comprise one or more of the following ingredients.
  • Antimicrobial agents for example, chlorhexidine; sanguinarine extract;
  • metronidazole metronidazole
  • quaternary ammonium compounds such as cetylpyridinium chloride
  • bis-guanides e.g., chlorhexidine digluconate, hexetidine, octenidine, alexidine
  • halogenated bisphenolic compounds e.g., 2,2' methylenebis-(4- chloro-6-bromophenol) or other phenolic antibacterial compounds
  • alkylhydroxybenzoate alkylhydroxybenzoate; cationic antimicrobial peptides; aminoglycosides;
  • quinolones lincosamides
  • penicillins cephalosporins, macrolides
  • tetracyclines other antibiotics known in the art
  • Coleus forskohlii essential oil silver or colloidal silver antimicrobials
  • tin- or copper-based antimicrobials Manuka oil; oregano; thyme; rosemary; or other herbal extracts; and grapefruit seed extract.
  • Anti-inflammatory or antioxidant agents for example, ibuprofen, flurbiprofen, aspirin, indomethacin, aloe vera, turmeric, olive leaf extract, cloves, panthenol, retinol, omega-3 fatty acids, gamma-linolenic acid (GLA), green tea, ginger, grape seed, etc.
  • Anti-caries agents for example, sodium- and stannous fluoride, aminefluorides, sodium monofluorophosphate, sodium
  • Plague buffers for example, urea, calcium lactate, calcium glycerophosphate, and strontium polyacrylates.
  • Vitamins for example, Vitamins A, C and E.
  • Plant extracts- Desensitizing agents for example, potassium citrate, potassium chloride, potassium tartrate, potassium bicarbonate, potassium oxalate, potassium nitrate, and strontium salts.
  • Anti-calculus agents for example, alkali-metal pyrophosphates, hypophosphite-containing polymers, organic phosphonates and phosphocitrates etc.
  • Biomolecules for example, bacteriocins,
  • bacteriophages for example, peppermint and spearmint oils, fennel, cinnamon, etc.
  • Proteinaceous materials for example, collagen. Preservatives. Opacifying agents. Coloring agents. pH- adjusting agents. Sweetening agents.
  • Pharmaceutically acceptable carriers for example, starch, sucrose, water or water/alcohol systems etc.
  • Surfactants for example, anionic, nonionic, cationic and zwitterionic or amphoteric surfactants, saponins from plant materials (see, e.g., U.S. Patent No.
  • Particulate abrasive materials for example, silicas, aluminas, calcium carbonates, dicalcium phosphates, calcium pyrophosphates, hydroxyapatites, trimetaphosphates, insoluble hexametaphosphates, agglomerated particulate abrasive materials, chalk, fine ground natural chalk and the like.
  • Humectants for example, glycerol, sorbitol, propyleneglycol, xylitol, lactitol etc.
  • Binders and thickeners for example, sodium carboxy methyl cellulose, hydroxyethyl cellulose (Natrosol®), xanthan gum, gum arabic, synthetic polymers (e.g., polyacrylates and carboxyvinyl polymers such as Carbopol®). Polymeric compounds that enhance the delivery of active ingredients such as antimicrobial agents. Buffers and salts to buffer the pH and ionic strength of the oral care composition. Bleaching agents: for example, peroxy compounds (e.g., potassium peroxydiphosphate). Effervescing systems: for example, sodium bicarbonate/citric acid systems. Color change systems. In particular embodiments, an abrasive is silica or fine ground natural chalk.
  • the oral hygiene compositions comprising a microparticulate BT compound that are formulated for use as a toothpaste may further comprise a humectant (for example, glycerol or sorbitol), a surface-active agent, binding agent, and/or a flavoring agent.
  • a humectant for example, glycerol or sorbitol
  • the toothpastes may also include a
  • oral hygiene compositions including toothpaste, have a pH between 7 and 7.5, between 7.5 and 8, between 8 and 8.5, or between 8.5 and 9, which may enhance the antimicrobial activity of the microparticulate BT compound.
  • the toothpaste compositions described herein may include one or more of chalk, dicalcium phosphate dihydrate, sorbitol, water, hydrated aluminum oxide, precipitated silica, sodium lauryl sulfate, sodium carboxymethyl cellulose, flavoring, sorbitan monooleate, sodium saccharin, tetrasodium pyrophosphate, methyl paraben, propyl paraben.
  • One or more coloring agents for example, FD&C Blue, can be employed if desired.
  • Other suitable ingredients that may be including in a toothpaste formulation are described in the art, for example, in U.S. Pat. No. 5,560,517.
  • the oral hygiene composition is a mouthspray and comprises a microparticulate BT compound, an alkaline buffer (e.g., potassium bicarbonate), an alcohol, a sweetener component, and a flavor system.
  • the flavor system may also have or more of the following: a flavorant, a humectant, a surfactant, a sweetener, and a colorant agent (see, e.g., U.S. Patent No. 6,579,513).
  • Surfactants described herein and known in the art for use in oral hygiene compositions may be anionic, nonionic, or amphoteric.
  • microparticulate BT-containing oral hygiene composition may be combined with additional active ingredients such as taurolidine and taurultam, which have been described in the art as useful for including in toothpastes, tooth gels, and mouthwashes for treating treat serious infections (see, e.g., United Kingdom Patent Application No., GB 1557163, U.S. Patent No. 6,488,912).
  • microparticulate BT can also be combined with one or more additional antimicrobial agents that when combined with microparticulate BT, the combination has additive or synergistic effects.
  • an oral hygiene composition described herein may further comprise at least one or more anti-biofilm agents for controlling biofilm development, disrupting a biofilm, or reducing the amount of biofilm.
  • interspecies quorum sensing is related to biofilm formation. Certain agents that increase LuxS-dependent pathway or interspecies quorum sensing signal (see, e.g., U.S. Patent No. 7,427,408) contribute to controlling development and/or proliferation of a biofilm.
  • Exemplary agents include, by way of example, N-(3-oxododecanoyl)-L- homoserine lactone (OdDHL) blocking compounds and N-butyryl-L-homoserine lactone (BHL) analogs, either in combination or separately (see, e.g., U.S.
  • Patent No. 6455031 An oral hygiene composition comprising a
  • microparticulate BT compound and at least one anti-biofilm agent can be delivered locally for disruption and inhibition of bacterial biofilm and for treatment of periodontal disease (see, e.g., U.S. Patent No.6,726,898).
  • An oral hygiene composition described herein may contain a sufficient amount of a microparticulate BT compound that effects substantial antimicrobial action during the time required for a normal tooth brushing, mouth rinsing, or flossing.
  • a microparticulate BT compound may be retained on oral surfaces (such as tooth, amalgam, composite, mucous membrane, gums).
  • a microparticulate BT compound retained on the teeth and gums after completion of brushing, rinsing, flossing, for example, may continue to provide extended anti-biofilm and anti-inflammatory action.
  • microparticulate BT compounds are slowly released from muco-adhesive polymers or other agents that contribute to retention of microparticulate BT compound on mucosal and tooth surfaces.
  • Microparticulate BTcompounds may be added to stable, viscous,
  • mucoadhesive aqueous compositions which may also be used for the prevention and treatment of ulcerative, inflammatory, and/or erosive disorders of mucous membranes and/or the delivery of pharmaceutically active
  • oral hygiene compositions comprising a microparticulate BT compound further comprise olive oil, which may enhance plaque removal.
  • olive oil in a product intended for oral hygiene, such as a toothpaste, a mouthwash, a spray, oral inhaler, or chewing gum, may contribute to elimination or reduction (a decrease) of bacterial plaque and/or to elimination or reduction (decrease of) in the numbers of bacteria present in the buccal cavity, thereby achieving a reduction in the occurrence of dental diseases (e.g., tooth decay, periodontal disease) and halitosis (see, e.g., U.S. Patent No. 7,074,391).
  • dental diseases e.g., tooth decay, periodontal disease
  • halitosis see, e.g., U.S. Patent No. 7,074,391.
  • an oral hygiene composition comprising a microparticulate BT compound may further comprise a mucosal disinfectant preparation for topical application in the mouth.
  • An oral hygiene composition may further comprise an aqueous slurry useful for cleaning the tongue and throat (see, e.g., U.S. Patent No. 6,861 ,049).
  • an oral hygiene composition comprising a microparticulate BT compound may further comprise at least one mint that is used for preventing (i.e., reducing the likelihood of occurrence) formation of a cavity (dental caries) or reducing the number of cavities.
  • CaviStat® Ortek Therapeutics, Inc., Roslyn Heights, NY
  • arginine and calcium which helps neutralize acid pH and promotes adherence of calcium to enamel surfaces.
  • the inclusion of mint in an oral hygiene composition comprising a microparticulate BT
  • a microparticulate BT compound may thus increase pH and enhance adherence of a microparticulate BT compound to oral surfaces.
  • compositions comprising Microparticulate Bismuth-Thiols
  • compositions comprising a microparticulate BT compound for preventing and/or treating microbial infections and inflammation resulting from an orthopedic procedure (e.g., orthopedic surgery, orthopedic therapy, arthroplasty (including two-step arthoplasty), orthodontic therapy).
  • the compositions comprising microparticulate BT compounds as described herein are therefore useful for preventing and/or treating (i.e., reducing or inhibiting development of, reducing the likelihood of occurrence or recurrence of) microbial infections of the skeleton and supporting structure (i.e., bones, joints, muscles, ligaments, tendons) such as osteomyelitis.
  • compositions described herein comprising a microparticulate BT compound may also be useful for preventing and/or controlling (i.e., slowing, retarding, inhibiting) biofilm development, disrupting a biofilm, or reducing the amount of biofilm present in a joint or on the surface of a bone, ligament, tendon, or tooth.
  • compositions described herein for orthopedic use that comprise a microparticulate BT compound may further comprise one or more additional antimicrobial compounds or agents. Particularly useful are the compositions comprising a microparticulate BT compound and a second antimicrobial agent that when administered in combination have enhanced or synergistic antimicrobial effects, as described herein. By way of an additional example, an enhanced antimicrobial effect may be observed when a
  • microparticulate BT compound is administered together with an antimicrobial agent that chelates iron.
  • a microparticulate BT compound is formulated with an anti-inflammatory agent, compound, small molecule, or macromolecule (such as a peptide or polypeptide).
  • compositions comprising a microparticulate BT compound may be combined with at least one other antimicrobial agent (i.e., a second, third, fourth, etc. antimicrobial agent) that when administered in combination have enhanced or synergistic antimicrobial effects (i.e., greater than an additive effect).
  • an enhanced antimicrobial effect may be observed when a microparticulate BT compound is administered together with an antimicrobial agent that chelates iron.
  • compositions comprising a microparticulate BT compound may be combined with at least one other antimicrobial agent and/or anti-inflammatory agent selected from the following: Antimicrobial agents: for example, chlorhexidine; sanguinarine extract; metronidazole; quaternary ammonium compounds (such as
  • cetylpyridinium chloride cetylpyridinium chloride
  • bis-guanides e.g., chlorhexidine digluconate, hexetidine, octenidine, alexidine
  • halogenated bisphenolic compounds e.g., 2,2' methylenebis-(4-chloro-6-bromophenol) or other phenolic antibacterial compounds
  • alkylhydroxybenzoate cationic antimicrobial peptides
  • Anti-inflammatory or antioxidant agents for example, ibuprofen, flurbiprofen, aspirin, indomethacin, aloe vera, turmeric, olive leaf extract, cloves, panthenol, retinol, omega-3 fatty acids, gamma- linolenic acid (GLA), green tea, ginger, grape seed, etc.
  • GLA gamma- linolenic acid
  • compositions comprising microparticulate BT compound may further comprise an antibiotic selected from clindamycin, vancomycin, daptomycin, cefazolin, gentamicin, tobramycin, metronidazole, cefaclor, ciprofloxacin, or other antimicrobial such as a quaternary ammonium compound (e.g., benzalkonium chloride, cetyl pyridinium chloride), an anti-microbial zeolite, alkali metal hydroxide, or an alkaline earth metal oxide.
  • an antibiotic selected from clindamycin, vancomycin, daptomycin, cefazolin, gentamicin, tobramycin, metronidazole, cefaclor, ciprofloxacin, or other antimicrobial such as a quaternary ammonium compound (e.g., benzalkonium chloride, cetyl pyridinium chloride), an anti-microbial zeolite, alkali metal hydroxide
  • compositions may optionally comprise one or more pharmaceutically suitable carriers (i.e., excipients), surfactants, buffers, diluents, and salts, and bleaching agents, which are described herein. Accordingly, these and certain of the related herein disclosed embodiments contemplate inclusion in such products and processes of the presently disclosed microparticulate BT compositions, which may include one or more microparticulate BT, and which may also optionally further include an antibiotic such as a synergizing or an enhancing antibiotic as described herein.
  • microparticulate BTs whether as individual BTs or BTs in which the bismuth moiety is replaced with a different Group V metal such as antimony (Sb) or arsenic (As), and/or as such BTs in combination with one or more antibiotic with which, as described herein, the BT exhibits synergizing or enhanced
  • microparticulate BTs in such formulations along with other components such as vitamins, minerals, amino acids, hydrocarbons including carbohydrates, fatty acids, oils, phytonutrients, teas, herbs or herbal extracts, and/or other nutritional or food products, may in certain embodiments result in the blockage or retardation of nutrient uptake by microbial populations in the gastrointestinal tract, in a manner that promotes increased (e.g., in a statistically significant manner relative to an appropriate control) bioavailability of the BT and optionally the antibiotic and/or of the additional nutritional component(s) to the host digestive tract.
  • other components such as vitamins, minerals, amino acids, hydrocarbons including carbohydrates, fatty acids, oils, phytonutrients, teas, herbs or herbal extracts, and/or other nutritional or food products
  • bioavailability of the BT and optionally of the antibiotic and/or of the additional nutritional component(s) to the host digestive tract may be decreased (e.g., in a statistically significant manner relative to an appropriate control).
  • Gl pathologic gastrointestinal
  • microparticuiate BT compound e.g., the microparticuiate BT compound, the antibiotic, or one or more particular nutrients
  • formulations for increasing or decreasing the Gl tract presence of one or more components may be prepared using the presently disclosed microparticuiate BTs (or AsT or SbT).
  • microparticuiate BT compounds in compositions for oral delivery to reduce fecal or digestive gas odors, for instance in patients who have undergone colostomy, and in other compositions for topical delivery to reduce underarm, foot or other body odors associated with topical microbial presence.
  • a number of skin and Gl tract microbial populations, including planktonic and biofilm bacteria, are susceptible to low concentrations of the herein described microparticuiate BT compounds, including such BT
  • certain embodiments contemplate orally delivered and topically delivered microparticuiate BT formulations to decrease (e.g., in a statistically significant manner relative to an appropriate control) populations of Gl-resident or skin-resident bacteria in a manner that reduces or alleviates the problem of unwanted odor.
  • Oral and topical pharmaceutical formulations are described below, such that these and related embodiments offer advantages associated with the present microparticuiate formulation of BT, such as compatible bioavailability and solubility properties and low toxicity; other factors that may influence the selection of antimicrobial compositions are described elsewhere herein and may also be found, e.g., in U.S. 6,582,719.
  • An exemplary BT compound, BisEDT has been applied (50 uL of a 1 mg/mL solution in DMSO) to the axillary area in human test subjects and shown to neutralize body odors for two to three days.
  • a mixture of BisEDT in talcum powder applied to the feet of a human test subject substantially reduced foot odor.
  • Laboratory mice fed 1 mg/kg BisEDT orally twice daily for five days exhibited 90% reductions in the number of fecal flora.
  • Related embodiments also contemplate a generally useful deodorant for any thiol-containing solution that emits odors (e.g., fish oils such as salmon oil), comprising a
  • microparticulate BT preparation as described herein that is made with an excess of bismuth, and that can be added to the thiol-containing solution as an odor quenching agent.
  • the resulting mixture retains the antimicrobial properties of the microparticulate BT.
  • solvents such as other biological source oils or butters, for instance, hemp oil, tea tree oil, shea butter, flax seed oil, fish oils, and in certain embodiments such oils as may have an independent or synergistic anti-inflammatory and/or pain- reducing and/or other beneficial physiologic effect.
  • compositions containing the microparticulate BT compounds disclosed herein; in certain such embodiments the pharmaceutical composition may further comprise one or more antibiotics such as an antibiotic with which the BT compound exhibits a synergizing or enhancing effect as described herein.
  • a composition comprising one or more such microparticulate BT compounds in a pharmaceutically acceptable carrier, excipient or diluent and in a therapeutic amount, as disclosed herein, when administered to an animal, preferably a mammal, most preferably a human patient.
  • microparticulate BT compounds or their pharmaceutically acceptable salts, in pure form or in an appropriate
  • compositions can be carried out via any of the accepted modes of administration of agents for serving similar utilities.
  • the pharmaceutical compositions can be prepared by combining a microparticulate BT compound with an appropriate pharmaceutically acceptable carrier, diluent or excipient, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols.
  • Typical routes of administering such pharmaceutical compositions include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, rectal, vaginal, and intranasal.
  • parenteral as used herein includes
  • compositions are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient.
  • Compositions that will be administered to a subject or patient take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a compound in aerosol form may hold a plurality of dosage units.
  • Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The
  • composition to be administered will, in any event, contain a therapeutically effective amount of a compound, or a pharmaceutically acceptable salt thereof, for treatment of a disease or condition of interest in accordance with the teachings herein.
  • compositions useful herein also contain a pharmaceutically acceptable carrier, including any suitable diluent or excipient, which includes any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • a pharmaceutically acceptable carrier including any suitable diluent or excipient, which includes any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • acceptable carriers include, but are not limited to, liquids, such as water, saline, glycerol and ethanol, and the like. A thorough discussion of pharmaceutically acceptable carriers, diluents, and other excipients is presented in
  • a pharmaceutical composition may be in the form of a solid or liquid.
  • the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form.
  • the carrier(s) may be liquid, with the compositions being, for example, an oral syrup, injectable liquid or an aerosol, which is useful in, for example, inhalatory administration.
  • the pharmaceutical composition When intended for oral administration, the pharmaceutical composition is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.
  • the pharmaceutical composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form.
  • Such a solid composition will typically contain one or more inert diluents or edible carriers.
  • binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin
  • excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like
  • lubricants such as magnesium stearate or Sterotex
  • glidants such as colloidal silicon dioxide
  • sweetening agents such as honey, sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent.
  • the pharmaceutical composition when in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.
  • a liquid carrier such as polyethylene glycol or oil.
  • the pharmaceutical composition may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension.
  • the liquid may be for oral administration or for delivery by injection, as two examples.
  • preferred composition contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer.
  • a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.
  • the liquid pharmaceutical compositions may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben;
  • sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents
  • antibacterial agents such as benzyl alcohol or methyl paraben
  • antioxidants such as ascorbic acid or sodium bisulfite
  • chelating agents such as ethylenediaminetetraacetic acid
  • buffers such as acetates, citrates or
  • parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • Physiological saline is a preferred adjuvant.
  • An injectable pharmaceutical composition is preferably sterile.
  • a liquid pharmaceutical composition intended for either parenteral or oral administration should contain an amount of a microparticulate BT compound such that a suitable dosage will be obtained. Typically, this amount is at least 0.01% of a microparticulate BT compound in the composition. When intended for oral administration, this amount may be varied to be between 0.1 and about 70% of the weight of the composition.
  • Preferred oral pharmaceutical compositions contain between about 4% and about 50% of the BT compound.
  • Preferred pharmaceutical compositions and preparations according to the present invention are prepared so that a parenteral dosage unit contains between 0.01 to 10% by weight of the microparticulate BT compound prior to dilution.
  • the pharmaceutical composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base.
  • the base may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, shea butter, tea tree oil, flax seed oil, hemp oil or other plant or vegetable oils including those known to have anti-inflammatory and/or anti-pain or other beneficial effects, salmon oil or other fish oils including those known to have anti-inflammatory and/or anti-pain or other beneficial effects, diluents such as water and alcohol, and emulsifiers and stabilizers.
  • Thickening agents may be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device.
  • Topical formulations may contain a concentration of the microparticulate BT compound from about 0.1 to about 10% w/v (weight per unit volume).
  • the pharmaceutical composition may be intended for rectal administration, in the form, for example, of a suppository, which will melt in the rectum and release the drug.
  • the composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient.
  • bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.
  • the pharmaceutical composition may include various materials, which modify the physical form of a solid or liquid dosage unit.
  • the composition may include materials that form a coating shell around the active ingredients.
  • the materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents.
  • the active ingredients may be encased in a gelatin capsule.
  • the pharmaceutical composition in solid or liquid form may include an agent that binds to the microparticulate BT compound and thereby assists in the delivery of the compound. Suitable agents that may act in this capacity include a monoclonal or polyclonal antibody, a protein or a liposome. Certain contemplated embodiments, however, expressly exclude the inclusion of a liposome in the pharmaceutical composition.
  • the pharmaceutical composition may consist of dosage units that can be administered as an aerosol.
  • aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols of the microparticulate BT compounds may be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the active ingredient(s).
  • Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like, which together may form a kit.
  • activators e.g., acoustic pressure regulators
  • valves e.g., a valve
  • subcontainers e.g., a syrene-maleic anhydride-semiconductors
  • compositions may be prepared by methodology well known in the pharmaceutical art. For example, a
  • composition intended to be administered by injection can be prepared by combining a compound of the invention with sterile, distilled water so as to form a solution.
  • a surfactant may be added to facilitate the formation of a homogeneous solution or suspension.
  • Surfactants are compounds that non-covalently interact with the compound of the invention so as to facilitate dissolution or homogeneous suspension of the compound in the aqueous delivery system.
  • microparticulate BT compounds are administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific compound employed; the metabolic stability and length of action of the compound; the age, body weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy.
  • a therapeutically effective daily dose is (for a 70 kg mammal) from about 0.001 mg/kg (i.e.
  • a therapeutically effective dose is (for a 70 kg mammal) from about 0.01 mg/kg (i.e., 7 mg) to about 50 mg/kg (i.e., 3.5 g); more preferably a therapeutically effective dose is (for a 70 kg mammal) from about 1 mg/kg (i.e., 70 mg) to about 25 mg/kg (i.e., 1.75 g).
  • the total dose required for each treatment can be administered by multiple doses or in a single dose over the course of the day, if desired.
  • microparticulate BT compounds and/or compositions can be any vertebrate animal, such as mammals.
  • the preferred recipients are mammals of the Orders Primate (including humans, apes and monkeys), Arteriodactyla (including horses, goats, cows, sheep, pigs), Rodenta (including mice, rats, rabbits, and hamsters), and Carnivora (including cats, and dogs).
  • the preferred recipients are turkeys, chickens and other members of the same order. The most preferred recipients are humans.
  • a microparticulate BT-containing pharmaceutical composition for topical applications, it is preferred to administer an effective amount of a microparticulate BT-containing pharmaceutical composition to a target area, e.g., skin surfaces, mucous membranes, and the like.
  • This amount will generally range from about 0.0001 mg to about 1 g of a BT compound per application, depending upon the area to be treated, whether the use is diagnostic, prophylactic or therapeutic, the severity of the symptoms, and the nature of the topical vehicle employed.
  • a preferred topical preparation is an ointment, wherein about 0.001 to about 50 mg of active ingredient is used per cc of ointment base.
  • the pharmaceutical composition can be formulated as transdermal compositions or transdermal delivery devices ("patches"). Such compositions include, for example, a backing, active compound reservoir, a control membrane, liner and contact adhesive. Such transdermal patches may be used to provide continuous pulsatile, or on demand delivery of the compounds of the present invention as desired.
  • microparticulate BT compositions can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.
  • Controlled release drug delivery systems include osmotic pump systems and dissolutional systems containing polymer-coated reservoirs or drug-polymer matrix formulations. Examples of controlled release systems are given in U.S. Pat. Nos. 3,845,770 and 4,326,525 and in P. J. Kuzma et al, Regional
  • microparticulate BT compositions can also be delivered through intra-nasal drug delivery systems for local, systemic, and nose-to-brain medical therapies.
  • Controlled Particle Dispersion (CPD)TM technology traditional nasal spray bottles, inhalers or nebulizers are known by those skilled in the art to provide effective local and systemic delivery of drugs by targeting the olfactory region and paranasal sinuses.
  • the invention also relates in certain embodiments to an intravaginal shell or core drug delivery device suitable for administration to the human or animal female.
  • the device may be comprised of the active pharmaceutical ingredient in a polymer matrix, surrounded by a sheath, and capable of releasing the compound in a substantially zero order pattern on a daily basis similar to devices used to apply testosterone as described in WO 98/50016.
  • compositions and methods may also find use in the treatment of acute and chronic wounds and wound biofilms, including, for example, as burn creams, as topicals for the treatment of existing wounds including those described herein, for prevention of chronic wounds, for treatment of MRSA skin infections, and for other related indications as disclosed herein and as will be apparent to the skilled person in view of the present disclosure.
  • Non-limiting examples of bacteria against which the herein described compositions and methods may find beneficial use include Staphylococcus aureus (S. aureus), MRSA (methicillin-resistant S. aureus), Staphylococcus epidermidis , MRSE (methicillin-resistant S. epidermidis), Mycobacterium tuberculosis, Mycobacterium avium, Pseudomonas aeruginosa, drug-resistant P. aeruginosa, Escherichia coli, enterotoxigenic E. coli, enterohemorrhagic E.
  • Enterobacter cloacae Salmonella typhimurium, Proteus vulgaris, Yersinia enterocolitica, Vibrio cholera, Shigella flexneri, vancomycin-resistant
  • VRE Enterococcus
  • Burkholderia cepacia complex Enterococcus
  • Francisella tularensis Enterococcus
  • Bacillus anthracis Yersinia pestis
  • Pseudomonas aeruginosa vancomycin- sensitive and vancomycin-resistant enterococci
  • methicillin-sensitive and methicillin-resistant staphylococci e.g., S. aureus , S.
  • Acinetobacter baumannii Staphylococcus haemolyticus, Staphylococcus hominis, Enterococcus faecium, Streptococcus pyogenes, Streptococcus agalactiae, Bacillus anthracis, Klebsiella pneumonia, Proteus mirabilis, Proteus vulgaris, Yersinia enterocolytica, Stenotrophomonas maltophilia, Streptococcus pneumonia, penicillin-resistant Streptococcus pneumonia, Burkholderia cepacia, Bukholderia multivorans, Mycobacterium smegmatis and E. cloacae.
  • Certain embodiments relate to methods, compositions and kits for treating an acute or chronic wound or a wound biofilm in a subject, which may comprise promoting skin tissue repair in the subject, or for altering one or more cellular wound repair activity in a cell or plurality of cells.
  • a cell generally indicates a single cell, whereas a plurality of cells indicates more than one cell.
  • the cells may comprise a tissue, organ or entire organism. Furthermore, the cell or cells may be located in vivo, in vitro, or ex vivo. Maintaining cell, tissue and organ cultures are routine procedures for one of skill in the art, the conditions and media for which can be easily ascertained. (See, for example, Freshney, Culture of Animal Cells: A Manual of Basic Technique, Wiley-Liss 5 th Ed. (2005); Davis, Basic Cell Culture, Oxford University Press 2 nd Ed. (2002)).
  • certain embodiments relate to methods for treating an acute or chronic wound or a wound biofilm in a subject that comprises administering to the subject a therapeutically effective amount of a composition comprising a BT compound as described herein for use in such method (e.g., as provided in the form of a plurality of substantially
  • a BT compound such as BisEDT or BisBAL or other compounds presented in Table 1 herein, or any other BT agent such as those described in Domenico et al. (1997 Antimicrob. Agent. Chemother. 41 :1697; 2001 Antimicrob. Agent. Chemother. 45:1421) and/or in U.S. RE37,793, U.S. 6,248,371 , U.S. 6,086,921 , and U.S. 6,380,248 and/or as prepared according to the methods disclosed herein.
  • a BT compound such as BisEDT or BisBAL or other compounds presented in Table 1 herein, or any other BT agent such as those described in Domenico et al. (1997 Antimicrob. Agent. Chemother. 41 :1697; 2001 Antimicrob. Agent. Chemother. 45:1421) and/or in U.S. RE37,793, U.S. 6,248,371 , U.S. 6,086,921 , and U.S. 6,380,248 and
  • Certain other embodiments relate to methods that comprise contacting any natural surface with a composition comprising one or more of the herein described microparticulate BT compounds, where such step of contacting may comprise one or more of directly applying, coating, dipping, irrigating, spraying, painting or otherwise bringing the BT composition into contact with the natural surface.
  • the step of administering to a subject such as a human or other mammalian subject may be performed by any means known to the art, for example, topically (including via direct administration to skin or to any epithelial tissue surface, including such surfaces as may be present in glandular tissues or in the respiratory and/or gastrointestinal tracts), vaginally, intraperitoneally, orally, parenterally, intravenously, intraarterially, transdermal ⁇ , sublingually, subcutaneously, intramuscularly, transbuccally, intranasally, via inhalation, intraoccularly, subcutaneously, intraadiposally, intraarticularly or intrathecally.
  • topically including via direct administration to skin or to any epithelial tissue surface, including such surfaces as may be present in glandular tissues or in the respiratory and/or gastrointestinal tracts
  • vaginally intraperitoneally, orally, parenterally, intravenously, intraarterially, transdermal ⁇ , sublingually, subcutaneously, intramuscularly, transbuccally
  • administering may be performed topically, where pharmaceutical excipients or carriers for topical use are described herein and known in the art.
  • certain invention embodiments described herein relate to topical formulations of the described BT compounds (e.g., BisEDT and/or BisBAL), which formulations may in certain further embodiments comprise one or more antibiotic compounds as described herein, for instance, amikacin, ampicillin, cefazolin, cefepime, chloramphenicol, ciprofloxacin, clindamycin (or another lincosamide antibiotic), daptomycin (Cubicin®), doxycycline, gatifloxacin, gentamicin, imipenim, levofloxacin, linezolid (Zyvox®), minocycline, nafcilin, paromomycin, rifampin, sulphamethoxazole, tobramycin and vancomycin; or a carbapenem antibiotic, a cephalosporin antibiotic, a fluoroquinolone antibiotic, a glycopeptide antibiotic, a lincosamide antibiotic, a penicillin
  • streptomycin streptomycin, tobramycin or apramycin, and/or a lipopeptide antibiotic such as daptomycin (Cubicin®), or an oxazolidinone antibiotic such as linezolid
  • BT compound(s) and optionally one or more antibiotics
  • a pharmaceutically acceptable carrier e.g., a pharmaceutically acceptable carrier, excipient or diluent and in a therapeutic amount, as disclosed herein, when administered topically to an animal, preferably a mammal, and most preferably a human, and in particularly preferred embodiments, a human having an acute or chronic wound or a wound that contains a bacterial infection which may be biofilm-related (e.g., in which bacteria capable of promoting biofilm formation may be present but a biofilm is not yet detectable) or that contains a bacterial infection such as a biofilm or other bacterial presence.
  • biofilm-related e.g., in which bacteria capable of promoting biofilm formation may be present but a biofilm is not yet detectable
  • a bacterial infection such as a biofilm or other bacterial presence.
  • Topical administration of the BT compounds described herein, or their pharmaceutically acceptable salts, in pure form or in an appropriate pharmaceutical composition can be carried out via any of the accepted modes of topical administration of agents for serving similar utilities.
  • Topical application or administration of a composition includes, in preferred
  • composition e.g., a topical formulation
  • epithelial tissue surface e.g., respiratory tract, gastrointestinal tract and/or glandular epithelial linings
  • undergoing treatment which may be at one or more localized or widely distributed skin and/or other epithelial tissue surface sites and which may generally refer to contacting the topical formulation with an acute or chronic wound site that is surrounded by intact stratum corneum or epidermis but need not be so limited; for instance, certain embodiments contemplate as a topical application the administration of a topical formulation described herein to injured, abraded or damaged skin, or skin of a subject undergoing surgery, such that contact of the topical formulation may take place not only with stratum corneum or epidermis but also with skin granular cell, spinous cell, and/or basal cell layers, and/or with dermal or underlying tissues, for example, as may accompany certain types of wound repair or wound healing or other skin tissue remodeling.
  • Such skin tissue repair may therefore comprise, in certain preferred embodiments, dermal wound healing, as may be desirable, for example, in preventing or ameliorating an acute chronic wound or a wound biofilm or, as another example, in preventing or ameliorating skin wound dehiscence, or in improving, accelerating or otherwise enhancing dermal wound healing when an acute or chronic wound and/or skin wound dehiscence may be present.
  • Certain other embodiments that contemplate topical administration to an epithelial tissue surface present in respiratory tract, gastrointestinal tract and/or glandular linings similarly may comprise administration of the topical formulation by an appropriate route as will be known in the art for delivering a topical preparation as provided hereih, to one or more epithelial tissue surfaces present in respiratory (e.g., airway, nasopharyngeal and laryngeal paths, tracheal, pulmonary, bronchi, bronchioles, alveoli, etc.) and/or gastrointestinal (e.g., buccal, esophageal, gastric, intestinal, rectal, anal, etc.) tracts, and/or other epithelial surfaces.
  • respiratory e.g., airway, nasopharyngeal and laryngeal paths, tracheal, pulmonary, bronchi, bronchioles, alveoli, etc.
  • gastrointestinal e.g., buccal, esophageal, gas
  • topical administration may comprise direct application into an open wound.
  • an open fracture or other open wound may include a break in the skin that may expose additional underlying tissues to the external environment in a manner that renders them susceptible to microbial infection.
  • topical administration may be by direct contact of the herein described BT composition with such damaged skin and/or another epithelial surface and/or with other tissues, such as, for instance, connective tissues including muscle, ligaments, tendons, bones, circulatory tissues such as blood vessels, associated nerve tissues, and any other organs that may be exposed in such open wounds.
  • connective tissues including muscle, ligaments, tendons, bones, circulatory tissues
  • blood vessels, associated nerve tissues and any other organs that may be exposed in such open wounds.
  • other tissues that may be exposed, and hence for which such direct contact is contemplated include kidney, bladder, liver, pancreas, and any other tissue or organ that may be so detrimentally exposed to opportunistic infection in relation to an open wound.
  • the topical formulations may be prepared by combining the described BT compound (e.g., comprising a compound described in U.S. RE37.793, U.S. 6,248,371 , U.S. 6,086,921 , and/or U.S. 6,380,248 and/or prepared according to the present disclosure such as the herein described microparticulate BT suspensions), and in certain related embodiments by combining one or more desired antibiotics (e.g., an
  • aminoglycoside antibiotic such as amikacin
  • an appropriate pharmaceutically acceptable carrier, diluent or excipient for use in a topical formulation preparation, and may be formulated into preparations in solid, semi-solid, gel, cream, colloid, suspension or liquid or other topically applied forms, such as powders, granules, ointments, solutions, washes, gels, pastes, plasters, paints, bioadhesives, microsphere suspensions, and aerosol sprays.
  • compositions of these and related embodiments are formulated so as to allow the active ingredients contained therein, and in particularly preferred embodiments the herein described BT compound(s) alone or in combination with one or more desired antibiotics (e.g., a carbapenem antibiotic, a cephalosporin antibiotic, a fluoroquinolone antibiotic, a glycopeptide antibiotic, a lincosamide antibiotic, a penicillinase-resistant penicillin antibiotic, and an aminopenicillin antibiotic, or an aminoglycoside antibiotic such as amikacin, or rifamycin) which may be applied simultaneously or sequentially and in either order, to be bioavailable upon topical administration of the formulation containing the BT compound(s) and/or antibiotic composition(s) to an acute or chronic wound and optionally to surrounding skin of a subject, such as a mammal, including a human, and in certain preferred embodiments a human patient having an acute or chronic wound, or being at increased risk for having, an acute or chronic wound or a wound biofilm or wound dehiscence
  • Certain embodiments disclosed herein contemplate topical administration of a BT compound and of an antibiotic, including administration that may be simultaneous or sequential and in either order, but the invention is not intended to be so limited and in other embodiments expressly contemplates a distinct route of administration for the BT compound relative to the route of administration of the antibiotic.
  • the antibiotic may be administered orally, intravenously, or by any other route of administration as described herein, while the BT compound may be
  • the BT compound may be administered topically as provided herein, while the antibiotic may be simultaneously or sequentially (and in any order) administered by a distinct route, such as orally, intravenously, transdermal ⁇ , subcutaneously, intramuscularly and/or by any other route of administration.
  • the topical formulations described herein deliver a therapeutically effective amount of the antiseptic or wound-healing agent(s) (and optionally the antibiotic(s)) to the wound site, for instance, to skin cells such as dermal fibroblasts.
  • Preferred formulations may be contacted with a desired site such as a topical wound site, a chronic wound, an epithelial tissue surface or other intended site of administration by spraying, irrigating, dipping and/or painting; such formulations therefore may exhibit ready permeability into the skin, as can be determined according to any of a number of established methodologies known to the art for testing the skin permeability of a drug composition (see, e.g., Wagner et al., 2002 J. Invest. Dermatol.
  • compositions, and formulations comprising such compositions, that will be administered to the skin of a subject or patient may in certain embodiments take the form of one or more dosage units, where for example, a liquid-filled capsule or ampule may contain a single dosage unit, and a container of a topical formulation as described herein in aerosol form may hold a plurality of dosage units.
  • a liquid-filled capsule or ampule may contain a single dosage unit
  • a container of a topical formulation as described herein in aerosol form may hold a plurality of dosage units.
  • Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000).
  • an antiseptic and/or wound healing-promoting compound as provided herein e.g., a BT compound
  • a pharmaceutically acceptable salt thereof in accordance with the present teachings.
  • the present topical formulations may take any of a wide variety of forms, and include, for example, creams, lotions, solutions, sprays, gels, ointments, pastes or the like, and/or may be prepared so as to contain liposomes, micelles, and/or microspheres. See, e.g., U.S. Patent No. 7,205,003.
  • creams as is well known in the arts of pharmaceutical and cosmeceutical formulation, are viscous liquids or semisolid emulsions, either oil-in-water or water-in-oil.
  • Cream bases are water-washable, and contain an oil phase, an emulsifier, and an aqueous phase.
  • the oil phase also called the "internal” phase, is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol.
  • the aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant.
  • the emulsifier in a cream formulation is generally a nonionic, anionic, cationic or amphoteric surfactant.
  • Lotions which are preferred for delivery of cosmetic agents, are preparations to be applied to the skin surface without friction, and are typically liquid or semi-liquid preparations in which solid particles, including the active agent, are present in a water or alcohol base. Lotions are usually suspensions of solids, and preferably comprise a liquid oily emulsion of the oil-in-water type.
  • Lotions are preferred formulations herein for treating large body areas, because of the ease of applying a more fluid composition. It is generally preferred that the insoluble matter in a lotion be finely divided. Lotions will typically contain suspending agents to produce better dispersions as well as compounds useful for localizing and holding the active agent in contact with the skin, e.g., methylcellulose, sodium carboxymethyl-cellulose, or the like.
  • Solutions are homogeneous mixtures prepared by dissolving one or more chemical substances (solutes) in a liquid such that the molecules of the dissolved substance are dispersed among those of the solvent.
  • the solution may contain other pharmaceutically acceptable and/or cosmeceutically acceptable chemicals to buffer, stabilize or preserve the solute.
  • solvents used in preparing solutions are ethanol, water, propylene glycol or any other pharmaceutically acceptable and/or cosmeceutically acceptable vehicles.
  • Gels are semisolid, suspension-type systems.
  • Single-phase gels contain organic macromolecules distributed substantially uniformly throughout the carrier liquid, which is typically aqueous, but also, preferably, contain an alcohol, and, optionally, an oil.
  • organic macromolecules i.e., gelling agents, may be chemically crosslinked polymers such as crosslinked acrylic acid polymers, for instance, the "carbomer” family of polymers, e.g.,
  • carboxypolyalkylenes that may be obtained commercially under the Carbopol® trademark.
  • hydrophilic polymers such as polyethylene oxides, polyoxyethylene-polyoxypropylene copolymers and polyvinylalcohol; cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and methyl cellulose; gums such as tragacanth and xanthan gum; sodium alginate; and gelatin.
  • dispersing agents such as alcohol or glycerin can be added, or the gelling agent can be dispersed by trituration, mechanical mixing or stirring, or combinations thereof.
  • Ointments are semisolid preparations that are typically based on petrolatum or other petroleum derivatives.
  • the specific ointment base to be used is one that will provide for a number of desirable characteristics, e.g., emolliency or the like.
  • an ointment base should be inert, stable, nonirritating, and nonsensitizing. As explained in Remington: The Science and Practice of Pharmacy, 19th Ed.
  • ointment bases may be grouped in four classes: oleaginous bases; emulsifiable bases;
  • Oleaginous ointment bases include, for example, vegetable oils, fats obtained from animals, and semisolid hydrocarbons obtained from petroleum.
  • Emulsifiable ointment bases also known as absorbent ointment bases, contain little or no water and include, for example, hydroxystearin sulfate, anhydrous lanolin, and hydrophilic petrolatum.
  • Emulsion ointment bases are either water-in-oil (W/O) emulsions or oil-in-water (O/W) emulsions, and include, for example, cetyl alcohol, glyceryl
  • Preferred water-soluble ointment bases are prepared from polyethylene glycols of varying molecular weight (see, e.g., Remington, Id.).
  • Pastes are semisolid dosage forms in which the active agent is suspended in a suitable base. Depending on the nature of the base, pastes are divided between fatty pastes or those made from single-phase aqueous gels.
  • the base in a fatty paste is generally petrolatum or hydrophilic petrolatum or the like.
  • the pastes made from single-phase aqueous gels generally
  • Formulations may also be prepared with liposomes, micelles, and microspheres.
  • Liposomes are microscopic vesicles having one (unilamellar) or a plurality (multilamellar) of lipid walls comprising a lipid bilayer, and, in the present context, may encapsulate and/or have adsorbed to their lipid
  • membranous surfaces one or more components of the topical formulations herein described, such as the antiseptic, wound healing/skin tissue/ epithelial tissue repair-promoting compounds (e.g., microparticulate BT compounds, optionally along with one or more antibiotics) or certain carriers or excipients.
  • Liposomal preparations herein include cationic (positively charged), anionic (negatively charged), and neutral preparations.
  • Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are available under the tradename Lipofectin® (GIBCO BRL, Grand Island, N.Y.).
  • anionic and neutral liposomes are readily available as well, e.g., from Avanti Polar Lipids (Birmingham, AL), or can be easily prepared using readily available materials.
  • Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine,
  • dioleoylphosphatidyl choline DOPC
  • DOPG dioleoylphosphatidyl glycerol
  • DOPE dioleoylphoshatidyl ethanolamine
  • Micelles are known in the art as comprised of surfactant molecules arranged so that their polar headgroups form an outer spherical shell, while the hydrophobic, hydrocarbon chains are oriented towards the center of the sphere, forming a core. Micelles form in an aqueous solution containing surfactant at a high enough concentration so that micelles naturally result.
  • Surfactants useful for forming micelles include, but are not limited to, potassium laurate, sodium octane sulfonate, sodium decane sulfonate, sodium dodecane sulfonate, sodium lauryl sulfate, docusate sodium,
  • decyltrimethylammonium bromide dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, tetradecyltrimethyl-ammonium chloride, dodecylammonium chloride, polyoxyl-8 dodecyl ether, polyoxyl-12 dodecyl ether, nonoxynol 10, and nonoxynol 30.
  • microspheres similarly, may be incorporated into the presently described topical formulations. Like liposomes and micelles, microspheres essentially encapsulate one or more components of the present formulations. They are generally, but not necessarily, formed from lipids, preferably charged lipids such as phospholipids. Preparation of lipidic microspheres is well known in the art.
  • solvents including relatively small amounts of alcohol, may be used to solubilize certain topical formulations.
  • solvents including relatively small amounts of alcohol, may be used to solubilize certain
  • formulation components It may be desirable, for certain topical formulations or in cases of particularly severe skin injury such as a post-surgical acute or chronic wound or post-surgical dermal wound dehiscence, to include in the topical formulation an added skin permeation enhancer in the formulation.
  • Suitable enhancers include, but are not limited to, ethers such as diethylene glycol monoethyl ether (available commercially as Transcutol®) and diethylene glycol monomethyl ether; surfactants such as sodium laurate, sodium lauryl sulfate, cetyltrimethylammonium bromide, benzalkonium chloride, Poloxamer® (231 , 182, 184), Tween® (20, 40, 60, 80), and lecithin (U.S. Pat. No.
  • ethers such as diethylene glycol monoethyl ether (available commercially as Transcutol®) and diethylene glycol monomethyl ether
  • surfactants such as sodium laurate, sodium lauryl sulfate, cetyltrimethylammonium bromide, benzalkonium chloride, Poloxamer® (231 , 182, 184), Tween® (20, 40, 60, 80), and lecithin (U.S. Pat. No.
  • alcohols such as ethanol, propanol, octanol, benzyl alcohol, and the like; polyethylene glycol and esters thereof such as polyethylene glycol monolaurate (PEGML; see, e.g., U.S. Pat. No. 4,568,343); amides and other nitrogenous compounds such as urea, dimethylacetamide (DMA),
  • ethanolamine, diethanolamine, and triethanolamine ethanolamine, diethanolamine, and triethanolamine; terpenes; alkanones; and organic acids, particularly citric acid and succinic acid.
  • Azone® and sulfoxides such as DMSO and Ci 0 MSO may also be used, but are less preferred.
  • Most preferred skin permeation enhancers are those lipophilic co- enhancers typically referred to as "plasticizing" enhancers, i.e., enhancers that have a molecular weight in the range of about 150 to 1000 daltons, an aqueous solubility of less than about 1 wt %, preferably less than about 0.5 wt %, and most preferably less than about 0.2 wt %.
  • the Hildebrand solubility parameter of plasticizing enhancers is in the range of about 2.5 to about 10, preferably in the range of about 5 to about 10.
  • Preferred lipophilic enhancers are fatty esters, fatty alcohols, and fatty ethers.
  • fatty acid esters examples include methyl laurate, ethyl oleate, propylene glycol monolaurate, propylene glycerol dilaurate, glycerol monolaurate, glycerol monooleate, isopropyl n-decanoate, and octyldodecyl myristate.
  • Fatty alcohols include, for example, stearyl alcohol and oleyl alcohol
  • fatty ethers include compounds wherein a diol or triol, preferably a C 2 -C4 alkane diol or triol, are substituted with one or two fatty ether substituents. Additional skin permeation enhancers will be known to those of ordinary skill in the art of topical drug delivery, and/or are described in the relevant literature. See, e.g.,
  • additives may be included in the topical formulations according to certain embodiments of the present invention, in addition to those identified above. These include, but are not limited to, antioxidants, astringents, perfumes, preservatives, emollients, pigments, dyes, humectants, propellants, and sunscreen agents, as well as other classes of materials whose presence may be cosmetically, medicinally or otherwise desirable. Typical examples of optional additives for inclusion in the
  • preservatives such as sorbate; solvents such as isopropanol and propylene glycol; astringents such as menthol and ethanol; emollients such as
  • polyalkylene methyl glucosides humectants such as glycerine; emulsifiers such as glycerol stearate, PEG-100 stearate, polyglyceryl-3 hydroxylauryl ether, and polysorbate 60; sorbitol and other polyhydroxyalcohols such as polyethylene glycol; sunscreen agents such as octyl methoxyl cinnamate (available commercially as Parsol MCX) and butyl methoxy benzoylmethane (available under the tradename Parsol 1789); antioxidants such as ascorbic acid (vitamin C), ct-tocopherol (Vitamin E), ⁇ -tocopherol , ⁇ -tocopherol, ⁇ -tocopherol, ⁇ - tocopherol , ⁇ 1 -tocopherol, ⁇ 2 - ⁇ , ⁇ -tocopherol , and retinol (vitamin A); essential oils, ceramides,
  • perhydrosqualene mineral oils, synthetic oils, silicone oils or waxes (e.g., cyclomethicone and dimethicone), fluorinated oils (generally
  • perfluoropolyethers examples include fatty alcohols (e.g., cetyl alcohol), and waxes (e.g., beeswax, carnauba wax, and paraffin wax); skin-feel modifiers; and thickeners and structurants such as swelling clays and cross-linked carboxypolyalkylenes that may be obtained commercially under the Carbopol® trademark.
  • fatty alcohols e.g., cetyl alcohol
  • waxes e.g., beeswax, carnauba wax, and paraffin wax
  • skin-feel modifiers e.g., beeswax, carnauba wax, and paraffin wax
  • thickeners and structurants such as swelling clays and cross-linked carboxypolyalkylenes that may be obtained commercially under the Carbopol® trademark.
  • conditioners and moisturizing agents include, by way of example, pyrrolidine carboxylic acid and amino acids; organic antimicrobial agents such as 2,4,4 , -trichloro-2-hydroxy diphenyl ether (triclosan) and benzoic acid; anti-inflammatory agents such as acetylsalicylic acid and glycyrrhetinic acid; anti-seborrhoeic agents such as.
  • cosmeceutically active agents may be present, for example, a-hydroxyacids, a-ketoacids, polymeric hydroxyacids, moisturizers, collagen, marine extracts, and antioxidants such as ascorbic acid (vitamin C), a-tocopherol (Vitamin E) or other tocopherols such as those described above, and retinol (vitamin A), and/or cosmetically acceptable salts, esters, amides, or other derivatives thereof.
  • Additional cosmetic agents include those that are capable of improving oxygen supply in skin tissue, as described, for example, in WO 94/00098 and WO 94/00109. Sunscreens may also be included.
  • Other embodiments may include a variety of non-carcinogenic, non-irritating healing materials that facilitate treatment with the formulations of certain embodiments of the invention.
  • healing materials may include nutrients, minerals, vitamins, electrolytes, enzymes, herbs, plant extracts, honey, glandular or animal extracts, or safe therapeutic agents that may be added to the formulation to facilitate dermal healing.
  • the amounts of these various additives are those conventionally used in the cosmetics field, and range, for example, from about 0.01 % to about 20% of the total weight of the topical formulation.
  • the formulations of certain embodiments of the invention may also include conventional additives such as opacifiers, fragrance, colorant, gelling agents, thickening agents, stabilizers, surfactants, and the like.
  • Other agents may also be added, such as antimicrobial agents, to prevent spoilage upon storage, i.e., to inhibit growth of microbes such as yeasts and molds.
  • Suitable antimicrobial agents are typically selected from methyl and propyl esters of p-hydroxybenzoic acid (e.g., methyl and propyl paraben), sodium benzoate, sorbic acid, imidurea, and combinations thereof.
  • the formulations may also contain irritation-mitigating additives to minimize or eliminate the possibility of skin irritation or skin damage resulting from the anti-infective acute or chronic wound healing and skin tissue repair-promoting compound to be administered, or from other components of the composition.
  • Suitable irritation- mitigating additives include, for example: a-tocopherol ; monoamine oxidase inhibitors, particularly phenyl alcohols such as 2-phenyl-1-ethanol; glycerin; salicylates; ascorbates; ionophores such as monensin; amphiphilic amines; ammonium chloride; N-acetylcysteine; capsaicin; and chioroquine.
  • the irritation-mitigating additive if present, may be incorporated into the topical formulation at a concentration effective to mitigate irritation or skin damage, typically representing not more than about 20 wt %, more typically not more than about 5 wt %, of the formulation.
  • the topical formulations may also contain, in addition to the antiseptic/ wound healing/ anti-biofilm/ skin tissue repair-promoting compound (e.g., a BT compound, preferably as substantially homogeneous microparticles as provided herein, and optionally in combination with one or more synergizing antibiotics as described herein), a therapeutically effective amount of one or more additional pharmacologically active agents suitable for topical
  • the antiseptic/ wound healing/ anti-biofilm/ skin tissue repair-promoting compound e.g., a BT compound, preferably as substantially homogeneous microparticles as provided herein, and optionally in combination with one or more synergizing antibiotics as described herein
  • Such agents may include an asymmetrical lamellar aggregate consisting of phospholipids and oxygen-loaded fluorocarbon or a fluorocarbon compound mixture, which are capable of improving oxygen supply in skin tissue, as described, for example, in International Patent Publication Nos. WO 94/00098 and WO 94/00109.
  • Suitable pharmacologically active agents that may be
  • incorporated into the present topical formulations and thus topically applied may include but are not limited to, the following: agents that improve or eradicate pigmented or non-pigmented age spots, keratoses, and wrinkles; antimicrobial agents; antibacterial agents; antipruritic and antixerotic agents; antiinflammatory agents; local anesthetics and analgesics; corticosteroids; retinoids (e.g., retinoic acid; vitamins; hormones; and antimetabolites.
  • agents that improve or eradicate pigmented or non-pigmented age spots, keratoses, and wrinkles may include but are not limited to, the following: agents that improve or eradicate pigmented or non-pigmented age spots, keratoses, and wrinkles; antimicrobial agents; antibacterial agents; antipruritic and antixerotic agents; antiinflammatory agents; local anesthetics and analgesics; corticosteroids; retinoids (e.g., retinoic acid; vitamins; hormones; and antimetabolites.
  • topical pharmacologically active agents include acyclovir, amphotericins, chlorhexidine, clotrimazole, ketoconazole, econazole, miconazole, metronidazole, minocycline, nystatin, neomycin, kanamycin, phenytoin, para-amino benzoic acid esters, octyl methoxycinnamate, octyl salicylate, oxybenzone, dioxybenzone, tocopherol, tocopheryl acetate, selenium sulfide, zinc pyrithione, diphenhydramine, pramoxine, lidocaine, procaine, erythromycin, tetracycline, clindamycin, crotamiton, hydroquinone and its monomethyl and benzyl ethers, naproxen, ibuprofen, cromolyn, retinoic acid, retinol,
  • betamethasone dipropionate triamcinolone acetonide, fluocinonide, clobetasol propionate, minoxidil, dipyridamole, diphenylhydantoin, benzoyl peroxide, and 5-fluorouracil.
  • an antibiotic such as a carbapenem antibiotic, a cephalosporin antibiotic, a fluoroquinolone antibiotic, a glycopeptide antibiotic, a lincosamide antibiotic, a penicillinase-resistant penicillin antibiotic, an aminopenicillin antibiotic, or an aminoglycoside antibiotic such as amikacin.
  • a pharmacologically acceptable carrier may also be incorporated in the topical formulation of certain present embodiments and may be any carrier conventionally used in the art.
  • examples include water, lower alcohols, higher alcohols, honey, polyhydric alcohols, monosaccharides, disaccharides, polysaccharides, sugar alcohols such as, for example, glycols (2-carbon), glycerols (3-carbon), erythritols and threitols (4-carbon), arabitols, xylitols and ribitols (5-carbon), mannitols, sorbitols, dulcitols and iditols (6-carbon), isomaltols, maltitols, lactitols and polyglycitols, hydrocarbon oils, fats and oils, waxes, fatty acids, silicone oils, nonionic surfactants, ionic surfactants, silicone surfactants, and water-based mixtures and emul
  • Topical formulation embodiments of the present invention may be applied regularly to whatever acute or chronic wound site (e.g., the wound itself and surrounding tissue, including surrounding tissue that appears unaffected by infection or otherwise normal or healthy) or skin area or other epithelial tissue surface (e.g., gastrointestinal tract, respiratory tract, glandular tissue) requires treatment with the frequency and in the amount necessary to achieve the desired results.
  • wound site e.g., the wound itself and surrounding tissue, including surrounding tissue that appears unaffected by infection or otherwise normal or healthy
  • epithelial tissue surface e.g., gastrointestinal tract, respiratory tract, glandular tissue
  • the frequency of treatment depends on the nature of the skin (or other epithelial tissue) condition (e.g., an acute or chronic wound or other skin wound such as may be found in dehiscence that results from a surgical incision, or other types of skin wounds), the degree of damage or deterioration of the skin (or other tissue), the responsiveness of the user's skin (or other tissue), the strength of the active ingredients (e.g., the herein described wound- healing/ antiseptic/ anti-biofilm/ skin tissue repair-promoting compounds such as a BT compound and optionally one or more additional pharmaceutically active ingredients, such as an antibiotic, e.g., amikacin or other antibiotic) in the particular embodiment, the effectiveness of the vehicle used to deliver the active ingredients into the appropriate layer of the skin (or other epithelial surface-containing tissue), the ease with which the formula is removed by physical contact with bandages or other dressings or clothing, or its removal by sweat or other intrinsic or extrinsic fluids, and the convenience to the subject's or patient's activity level
  • Typical concentrations of active substances such as the BT compound antiseptic/ anti-biofilm/ wound-healing/ skin tissue repair-promoting compositions described herein can range, for example, from about 0.001 -30% by weight based on the total weight of the composition, to about 0.01 -5.0%, and more preferably to about 0.1 -2.0%.
  • active substances such as the BT compound antiseptic/ anti-biofilm/ wound-healing/ skin tissue repair-promoting compositions described herein can range, for example, from about 0.001 -30% by weight based on the total weight of the composition, to about 0.01 -5.0%, and more preferably to about 0.1 -2.0%.
  • active substances such as the BT compound antiseptic/ anti-biofilm/ wound-healing/ skin tissue repair-promoting compositions described herein can range, for example, from about 0.001 -30% by weight based on the total weight of the composition, to about 0.01 -5.0%, and more preferably to about 0.1 -2.0%.
  • compositions of these embodiments of the present invention may be applied to an acute or chronic wound and/or to the skin at a rate equal to from about 1 .0 mg/cm 2 of skin to about 20.0 mg/cm 2 of skin.
  • topical formulations include, but are not limited to, aerosols, alcohols, anhydrous bases (such as lipsticks and powders), aqeuous solutions, creams, emulsions (including either water-in-oil or oil-in-water emulsions), fats, foams, gels, hydro-alcoholic solutions, liposomes, lotions, microemulsions, ointments, oils, organic solvents, polyols, polymers, powders, salts, silicone derivatives, and waxes.
  • Topical formulations may include, for example, chelating agents, conditioning agents, emollients, excipients, humectants, protective agents, thickening agents, or UV absorbing agents.
  • chelating agents for example, chelating agents, conditioning agents, emollients, excipients, humectants, protective agents, thickening agents, or UV absorbing agents.
  • Chelating agents may be optionally included in topical formulations, and may be selected from any agent that is suitable for use in a cosmetic composition, and may include any natural or synthetic chemical which has the ability to bind divalent cationic metals such as Ca 2+ , Mn 2+ , or Mg 2+ .
  • Examples of chelating agents include, but are not limited to EDTA, disodium EDTA, EGTA, citric acid, and dicarboxylic acids.
  • Conditioning agents may also be optionally included in topical formulations.
  • skin conditioning agents include, but are not limited to, acetyl cysteine, N-acetyl dihydrosphingosine, acrylates/behenyl
  • acrylate/dimethicone acrylate copolymer adenosine, adenosine cyclic phosphate, adensosine phosphate, adenosine triphosphate, alanine, albumen, algae extract, allantoin and deriviatives, aloe barbadensis extracts, aluminum PCA, amyloglucosidase, arbutin, arginine, azulene, bromelain, buttermilk powder, butylene glycol, caffeine, calcium gluconate, capsaicin, carbocysteine, carnosine, beta-carotene, casein, catalase, cephalins, ceramides, chamomilla recutita (matricaria) flower extract, cholecalciferol, cholesteryl esters, coco- betaine, coenzyme A, corn starch modified, crystallins, cycloethoxymethicone, cysteine DNA, cytochrome C,
  • Topical formulations may also optionally include one or more emollients, examples of which include, but are not limited to, acetylated lanolin, acetylated lanolin alcohol, acrylates/Ci 0- 3o alkyl acrylate crosspolymer, acrylates copolymer, alanine, algae extract, aloe barbadensis extract or gel, althea officinalis extract, aluminum starch octenylsuccinate, aluminum stearate, apricot (prunus armeniaca) kernel oil, arginine, arginine aspartate, arnica montana extract, ascorbic acid, ascorbyl palmitate, aspartic acid, avocado (persea gratissima) oil, barium sulfate, barrier sphingolipids, butyl alcohol, beeswax, be
  • hexacaprylate/hexacaprate DMDM hydantoin, DNA, erythritol, ethoxydiglycol, ethyl linoleate, eucalyptus globulus oil, evening primrose (Oenothera biennis) oil, fatty acids, fructose, gelatin, geranium maculatum oil, glucosamine, glucose glutamate, glutamic acid, glycereth-26, glycerin, glycerol, glyceryl distearate, glyceryl hydroxystearate, glyceryl laurate, glyceryl linoleate, glyceryl myristate, glyceryl oleate, glyceryl stearate, glyceryl stearate SE, glycine, glycol stearate, glycol stearate SE, glycosaminoglycans, grape (
  • polysorbate 80 polysorbate 85, potassium myristate, potassium palmitate, potassium sorbate, potassium stearate, propylene glycol, propylene glycol dicaprylate/dicaprate, propylene glycol dioctanoate, propylene glycol
  • RNA rosemary (rosmarinus officinalis) oil, rose oil, safflower (carthamus tinctorius) oil, sage (salvia officinalis) oil, salicylic acid, sandalwood (santalum album) oil, serine, serum protein, sesame (sesamum indicum) oil, shea butter (butyrospermum parkii), silk powder, sodium chondroitin sulfate, sodium DNA, sodium hyaluronate, sodium lactate, sodium palmitate, sodium PCA, sodium polyglutamate, sodium stearate, soluble collagen, sorbic
  • stearoxytrimethylsilane stearyl alcohol, stearyl glycyrrhetinate, stearyl heptanoate, stearyl stearate, sunflower (helianthus annuus) seed oil, sweet almond (prunus amygdalus dulcis) oil, synthetic beeswax, tocopherol, tocopheryl acetate, tocopheryl linoleate, tribehenin, tridecyl neopentanoate, tridecyl stearate, triethanolamine, tristearin, urea, vegetable oil, water, waxes, wheat (triticum vulgare) germ oil, and ylang ylang (cananga odorata) oil.
  • a topical formulation may contain a suitable excipient, which typically should have a high affinity for the skin, be well tolerated, stable, and yield a consistency that allows for easy utilization.
  • Suitable topical excipients and vehicles can be routinely selected for a particular use by those skilled in the art, and especially with reference to one of many standard texts in the art, such as Remington's Pharmaceutical Sciences, Vol. 18, Mack Publishing Co., Easton, Pa. (1990), in particular Chapter 87.
  • one or more humectants are also included in the topical formulation. Examples of humectants include, but are not limited to, amino acids,
  • chondroitin sulfate diglycerin, erythritol, fructose, glucose, glycerin, glycerol, glycol, 1 ,2,6-hexanet ol, honey, hyaluronic acid, hydrogenated honey, hydrogenated starch hydrolysate, inositol, lactitol, maltitol, maltose, mannitol, natural moisturization factor, PEG-15 butanediol, polyglyceryl sorbitol, salts of pyrollidone carboxylic acid, potassium PCA, propylene glycol, sodium
  • glucuronate sodium PCA, sorbitol, sucrose, trehalose, urea, and xylitol.
  • Certain embodiments contemplate topical formulations containing one or more additional skin protective agent.
  • skin protective agents may include, but are not limited to, algae extract, allantoin, aluminum hydroxide, aluminum sulfate, betaine, camellia sinensis leaf extract,
  • Surfactants may also desirably be included in certain topical formulations contemplated herein, and can be selected from any natural or synthetic surfactants suitable for use in cosmetic compositions, such as cationic, anionic, zwitterionic, or non-ionic surfactants, or mixtures thereof.
  • any natural or synthetic surfactants suitable for use in cosmetic compositions such as cationic, anionic, zwitterionic, or non-ionic surfactants, or mixtures thereof.
  • cationic surfactants may include, but are not limited to, DMDAO or other amine oxides, long-chain primary amines, diamines and polyamines and their salts, quaternary ammonium salts, polyoxyethylenated long-chain amines, and quaternized polyoxyethylenated long-chain amines.
  • anionic surfactants may include, but are not limited to, SDS; salts of carboxylic acids (e.g., soaps); salts of sulfonic acids, salts of sulfuric acid, phosphoric and polyphosphoric acid esters; alkylphosphates; monoalkyi phosphate (MAP); and salts of perfluorocarboxylic acids.
  • zwitterionic surfactants may include, but are not limited to, cocoamidopropyl hydroxysultaine (CAPHS) and others which are pH-sensitive and require special care in designing the appropriate pH of the formula (i.e., alkylaminopropionic acids, imidazoline carboxylates, and betaines) or those which are not pH-sensitive (e.g., sulfobetaines, sultaines).
  • non-ionic surfactants may include, but are not limited to, alkylphenol ethoxylates, alcohol ethoxylates,
  • polyoxyethylenated polyoxypropylene glycols polyoxyethylenated mercaptans, long-chain carboxylic acid esters, alkonolamides, tertiary acetylenic glycols, polyoxyethylenated silicones, N-alkylpyrrolidones, and alkylpolyglycosidases.
  • Wetting agents, mineral oil or other surfactants such as non-ionic detergents or agents such as one or more members of the PLURONICS® series (BASF, Mt. Olive, NJ) may also be included, for example and according to non-limiting theory, to discourage aggregation of BT microparticles within the PLURONICS® series (BASF, Mt. Olive, NJ) may also be included, for example and according to non-limiting theory, to discourage aggregation of BT microparticles within the
  • microparticulate suspension Any combination of surfactants is acceptable. Certain embodiments may include at least one anionic and one cationic surfactant, or at least one cationic and one zwitterionic surfactant which are compatible, i.e., do not form complexes which precipitate appreciably when mixed.
  • thickening agents examples include, but are not limited to, acrylamides copolymer, agarose, amylopectin, bentonite, calcium alginate, calcium carboxymethyl cellulose, carbomer, carboxymethyl chitin, cellulose gum, dextrin, gelatin, hydrogenated tallow, hydroxytheylcellulose, hydroxypropylcellulose,
  • hydroxpropyl starch magnesium alginate, methylcellulose, microcrystalline cellulose, pectin, various PEG'S, polyacrylic acid, polymethacrylic acid, polyvinyl alcohol, various PPG's, sodium acrylates copolymer, sodium carrageenan, xanthan gum, and yeast beta-glucan.
  • Thickening agents other than those listed above may also be used in embodiments of this invention.
  • a topical formulation may comprise one or more sunscreening or UV absorbing agents.
  • sunscreening or UV absorbing agents may include, for example, benzophenone, benzophenone-1 ,
  • Topical formulations disclosed herein are typically effective at pH values between about 2.5 and about 10.0.
  • the pH of the composition is typically effective at pH values between about 2.5 and about 10.0.
  • composition is at or about the following pH ranges: about pH 5.5 to about pH 8.5, about pH 5 to about pH 10, about pH 5 to about pH 9, about pH 5 to about pH 8, about pH 3 to about pH 10, about pH 3 to about pH 9, about pH 3 to about pH 8, and about pH 3 to about pH 8.5. Most preferably, the pH is about pH 7 to about pH 8.
  • pH adjusting ingredients to the compositions of the present invention to adjust the pH to an acceptable range. "About" a specified pH is understood by those familiar with the art to include formulations in which at any given time the actual measured pH may be less or more than the specified value by no more than 0.7, 0.6, 0.5, 0.4. , 0.3, 0.2 or 0.1 pH units, where it is recognized that
  • formulation composition and storage conditions may result in drifting of pH from an original value.
  • a cream, lotion, gel, ointment, paste or the like may be spread on the affected surface and gently rubbed in.
  • a solution may be applied in the same way, but more typically will be applied with a dropper, swab, or the like, and carefully applied to the affected areas.
  • the application regimen will depend on a number of factors that may readily be determined, such as the severity of the wound and its responsiveness to initial treatment, but will normally involve one or more applications per day on an ongoing basis.
  • One of ordinary skill may readily determine the optimum amount of the formulation to be administered, administration methodologies and repetition rates. In general, it is contemplated that the formulations of these and related embodiments of the invention will be applied in the range of once or twice or more weekly up to once, twice, thrice, four times or more daily.
  • topical formulations useful herein thus also contain a pharmaceutically acceptable carrier, including any suitable diluent or excipient, which includes any pharmaceutical agent that does not itself harm the subject receiving the composition, and which may be
  • Pharmaceutically acceptable carriers include, but are not limited to, liquids, such as water, saline, glycerol and ethanol, and the like, and may also include viscosity enhancers (e.g., balsam fir resin) or film-formers such as colloidion or nitrocellulose solutions.
  • viscosity enhancers e.g., balsam fir resin
  • film-formers such as colloidion or nitrocellulose solutions.
  • the topical formulation when in the form of a gel- or liquid-filled capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.
  • a liquid carrier such as polyethylene glycol or oil.
  • the liquid pharmaceutical compositions of certain embodiments of the invention may include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben;
  • antioxidants such as ascorbic acid or sodium bisulfite
  • chelating agents such as ethylenediaminetetraacetic acid (EDTA)
  • buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the carrier may suitably comprise a solution, emulsion, ointment or gel base.
  • the base may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers.
  • Thickening agents may be present in a pharmaceutical or cosmeceutical composition for topical administration. If intended for
  • the composition may include a transdermal patch or iontophoresis device.
  • Topical formulations may contain a concentration of the compound of certain embodiments of the invention from about 0.1 to about 10% w/v (weight per unit volume).
  • a topical formulation may be provided in the form of a cream, lotion, solution, spray, gel, ointment, paste or the like, and/or may contain liposomes, micelles, microspheres and/or other microparticle or nanoparticle delivery elements.
  • a topical formulation may also be provided in the form of time-release or sustained release particles or pellets, for example, slow-release ethylene vinyl acetate polymer (e.g., Elvax®40, Aldrich,
  • the topical formulation may include an agent that binds to the skin tissue repair-promoting compound and thereby assists in its delivery to skin epithelial cells (e.g., keratinocytes) and/or fibroblasts.
  • Suitable agents that may act in this capacity include clathrating agents such as cyclodextrins; other agents may include a protein or a liposome.
  • the topical formulation of certain embodiments of the invention may also be provided in the form of dosage units that can be administered as an aerosol.
  • aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols of compounds of certain embodiments of the invention may be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like, which together may form a kit. One skilled in the art, without undue experimentation may determine preferred aerosols for delivering topical formulations to the skin or to a wound site.
  • the topical formulations may be prepared by methodology well known in the pharmaceutical art.
  • a pharmaceutical composition intended to be administered to a wound site or to the skin as a spray, wash or rinse can be prepared by combining a BT antiseptic/ wound-healing/ anti- biofilm/ skin tissue repair-promoting compound as described herein with sterile, distilled water so as to form a solution.
  • a surfactant may be added to facilitate the formation of a homogeneous solution or suspension.
  • Surfactants are compounds that non-covalently interact with the antioxidant active compound so as to facilitate dissolution or homogeneous suspension of the compound in the aqueous delivery system.
  • the BT antiseptic/ wound-healing/ anti-biofilm/ skin tissue repair- promoting compounds for use in topical formulations, or their pharmaceutically acceptable salts, are administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the nature of the wound site (where relevant), the activity of the specific BT compound employed (including the inclusion or absence from the formulation of an antibiotic, such as an aminoglycoside antibiotic, e.g., amikacin); the metabolic stability and length of action of the compound; the age, body weight, general health, sex, skin type, immune status and diet of the subject; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular skin wound for which skin tissue repair is desired; and the subject undergoing therapy.
  • an antibiotic such as an aminoglycoside antibiotic, e.g., amikacin
  • a therapeutically effective daily dose is (for a 70 kg mammal) from about 0.001 mg/kg (i.e., 0.07 mg) to about 100 mg/kg (i.e., 7.0 g); preferably a therapeutically effective dose is (for a 70 kg mammal) from about 0.01 mg/kg (i.e., 7 mg) to about 50 mg/kg (i.e., 3.5 g); more preferably a therapeutically effective dose is (for a 70 kg mammal) from about 1 mg/kg (i.e., 70 mg) to about 25 mg/kg (i.e., 1.75 g).
  • the total dose required for each treatment can be administered by multiple doses or in a single dose over the course of the day, if desired. Certain preferred embodiments contemplate a single application of the topical formulation per day. Generally, and in distinct embodiments, treatment may be initiated with smaller dosages, which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached.
  • the topical formulation can be administered alone or in
  • topical formulation may further comprise retinoic acid.
  • topical formulation may comprise one or more skin tissue repair-promoting compounds described herein, or may comprise two or more such compounds having different cellular wound repair activities.
  • the recipients of the topical formulations described herein can be any vertebrate animal, such as mammals.
  • the preferred recipients are mammals of the Orders Primate (including humans, apes and monkeys), Arteriodactyla (including horses, goats, cows, sheep, pigs), Rodenta (including mice, rats, rabbits, and hamsters), and Carnivora (including cats, and dogs).
  • the preferred recipients are turkeys, chickens and other members of the same order.
  • the most preferred recipients are humans, and particularly preferred are humans having one or more acute or chronic wounds or wounds that contain biofilms.
  • a pharmaceutical composition comprising a BT compound antiseptic/ wound-healing/ anti-biofilm/ skin tissue repair-promoting compound according to the herein described embodiments, to a target area, e.g., a skin wound such as an acute or chronic wound, and/or an at-risk area (e.g., for wound
  • compositions can be formulated as transdermal compositions or transdermal delivery devices ("patches"). Such compositions include, for example, a backing, active compound reservoir, a control membrane, liner and contact adhesive.
  • transdermal patches may be used to provide continuous pulsatile, or on demand delivery of the compounds of the present invention as desired.
  • the compositions of certain embodiments can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.
  • Controlled release drug delivery systems include osmotic pump systems and dissolutional systems containing polymer-coated reservoirs or drug-polymer matrix formulations. Examples of controlled release systems are given in U.S. Pat. Nos. 3,845,770 and 4,326,525 and in P. J. Kuzma et al, Regional
  • the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 5%, 6%, 7%, 8% or 9%. In other embodiments, the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 10%, 11%, 12%, 13% or 14%. In yet other embodiments, the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 15%, 16%, 17%, 18%, 19% or 20%.
  • BT compounds were prepared either according to the methods of Domenico et al. (U.S. RE37,793, U.S.6,248,371 , U.S.
  • An ethanolic solution (-1.56 L, -0.55 M) of 1,2-ethanedithiol (CAS 540- 63-6) was separately prepared by adding, to 1.5 L of absolute ethanol, 72.19 mL (0.863 moles) of 1 ,2-ethanedithiol using a 60 mL syringe, and then stirring for five minutes.
  • the 1,2-ethanedithiol/ EtOH reagent was then slowly added by dropwise addition over the course of five hours to the aqueous Bi(NO 3 ) 3 / HNO 3 solution, with continued stirring overnight.
  • the formed product was allowed to settle as a precipitate for approximately 15 minutes, after which the filtrate was removed at 300 mL/min using a peristaltic pump.
  • the product was then collected by filtration on fine filter paper in a 15-cm diameter Buchner funnel, and washed sequentially with three, 500-mL volumes each of ethanol, USP water, and acetone to obtain BisEDT (694.51 gm/ mole) as a yellow amorphous powdered solid.
  • the product was placed in a 500 mL amber glass bottle and dried over CaCl 2 under high vacuum for 48 hours. Recovered material (yield -200 g) gave off a thiol-characteristic odor.
  • the crude product was redissolved in 750 mL of absolute ethanol, stirred for 30 min, then filtered and washed sequentially with 3 x 50 mL ethanol, 2 x 50 mL acetone, and washed again with 500 mL of acetone.
  • the rewashed powder was triturated in 1M NaOH (500 mL), filtered and washed with 3 x 220 mL water, 2 x 50 mL ethanol, and 1 x 400 mL acetone to afford 156.74 gm of purified BisEDT.
  • the product was characterized as having the structure shown above in formula I by analysis of data from 1 H and 3 C nuclear magnetic resonance (NMR), infrared spectroscopy (IR), ultraviolet spectroscopy (UV), mass spectrometry (MS) and elemental analysis.
  • NMR nuclear magnetic resonance
  • IR infrared spectroscopy
  • UV ultraviolet spectroscopy
  • MS mass spectrometry
  • An HPLC method was developed to determine chemical purity of BisEDT whereby the sample was prepared in DMSO (0.5mg/mL). The A max was determined by scanning a solution of BisEDT in DMSO between 190 and 600nm.
  • the dried particulate matter was characterized to assess the particle size properties. Briefly, microparticles were resuspended in 2%
  • Pluronic® F-68 BASF, Mt. Olive, J
  • the suspension was sonicated for 10 minutes in a water bath sonicator at standard setting prior to analysis using a Nanosizer/Zetasizer Nano-S particle analyzer (model ZEN 1600 (without zeta- potential measuring capacity), Malvern Instruments, Worcestershire, UK) according to the manufacturer's recommendations.
  • a Nanosizer/Zetasizer Nano-S particle analyzer model ZEN 1600 (without zeta- potential measuring capacity), Malvern Instruments, Worcestershire, UK
  • VMD volumetric mean diameter
  • the majority of particles were heterodisperse and of significantly larger size, precluding their characterization on the basis of VMD.
  • colony biofilms were grown on 10% tryptic soy agar for 24 hours, and transferred to Mueller Hinton plates containing treatments. After treatment the biofilms were dispersed into peptone water containing 2% w/v glutathione (neutralizes the BT), and serially diluted into peptone water before being spotted onto plates for counting. Two bacteria isolated from chronic wounds were used separately in the production of colony biofilms for testing. These were Pseudomonas aeruginosa, a gram negative bacterial strain, and Methicillin Resistant Staphylococcus aureus (MRSA), which is gram positive.
  • MRSA Methicillin Resistant Staphylococcus aureus
  • Colony biofilms were prepared by inoculating 5 ⁇ spots of planktonic bacterial liquid cultures onto a 25 mm diameter polycarbonate filter membrane. The membranes were sterilized prior to inoculation, by exposure to ultraviolet light for 0 min per side. The inocula were grown overnight in bacterial medium at 37°C and diluted in fresh medium to an optical density of 0.1 at 600 nm prior to deposition on the membrane. The membranes were then placed on the agar plate containing growth medium. The plates were then covered and placed, inverted, in an incubator at 37°C. Every 24 h, the membrane and colony biofilm were transferred, using sterile forceps, to a fresh plate. Colony biofilms were typically used for experimentation after 48 hours of growth, at which time there were approximately 10 9 bacteria per membrane. The colony biofilm method was successfully employed to culture a wide variety of single species and mixed species biofilms.
  • colony biofilms were transferred to agar plates supplemented with the candidate antimicrobial treatment agent(s). Where the duration of exposure to antimicrobial treatment exceeded 24 hours, the colony biofilms were moved to fresh treatment plates daily. At the end of the treatment period, the colony biofilms were placed in tubes containing 10 ml of buffer and vortexed for 1 -2 min to disperse the biofilm. In some cases, it was necessary to briefly process the sample with a tissue homogenizer to break up cell aggregates. The resulting cell suspensions were then serially diluted and plated to enumerate surviving bacteria, which were reported as colony forming units (CFU) per unit area. Survival data were analyzed using log
  • BT compounds were tested for each type of bacterial biofilm colony cultures (Pseudomonas aeruginosa, PA; methicilin resistant Staphylococcus aureus, MRSA or SA) five antibiotics and thirteen BT compounds were tested.
  • Antimicrobial agents tested against PA included the BTs referred to herein as BisEDT and Compounds 2B, 4, 5, 6, 8-2, 9, 10, 11 and 15 (see Table 1), and the antibiotics tobramycin, amikacin, imipenim, cefazolin, and ciprofloxacin.
  • Antimicrobial agents tested against SA included the BTs referred to herein as BisEDT and Compounds 2B, 4, 5, 6, 8-2, 9, 10 and 1 1 (see Table 1), and the antibiotics rifampicin, daptomycin, minocycline, ampicillin, and vancomycin. As described above under “brief descriptions of the drawings", antibiotics were tested at
  • MIC concentrations
  • Drip flow biofilms represent an art accepted authentic model for forming, and testing the effect of candidate anti-bacterial compounds against, bacterial biofilms.
  • Drip flow biofilms are produced on coupons (substrates) placed in the channels of a drip flow reactor. Many different types of materials can be used as the substrate for bacterial biofilm formation, including frosted glass microscope slides. Nutritive liquid media enters the drip flow bioreactor cell chamber by dripping into the chamber near the top, and then flows the length of a coupon down a 10 degree slope.
  • Biofilms are grown in drip flow bioreactors and exposed to BT compounds individually or in combinations and/or to antibiotic compounds individually or in combinations with other antibacterial agents, including BT compounds, or to other conventional or candidate treatments for chronic wounds. BT compounds are thus characterized for their effects on bacterial biofilms in the drip-flow reactor.
  • Biofilms in the drip-flow reactor are prepared according to established methodologies (e.g., Stewart et al., 2001 J Appl Microbiol. 91 :525; Xu et al., 1998 Appl. Environ. Microbiol. 64:4035). This design involves cultivating biofilms on inclined polystyrene coupons in a covered chamber.
  • An exemplary culture medium contains 1 g/l glucose, 0.5 g/l NH 4 NO 3 , 0.25g/l KCI, 0.25 g/l KH 2 P0 4 , 0.25 g/l MgS0 4 -7H 2 0, supplemented with 5% v/v adult donor bovine serum (ph 6.8) that mimics serum protein-rich, iron limited conditions that are similar to biofilm growth conditions in vivo, such as in chronic wounds.
  • This medium flows drop-wise (50ml/h) over four coupons contained in four separate parallel chambers, each of which measures 10cm x 1.9cm by 1.9cm deep.
  • the chambered reactor is fabricated from polysulfone plastic.
  • the biofilm reactor is contained in an incubator at 37° C, and bacterial cell culture medium is warmed by passing it through an aluminum heat sink kept in the incubator. This method reproduces the antibiotic tolerant phenotype observed in certain biofilms, mimics the low fluid shear environment and proximity to an air interface characteristic of a chronic wound while providing continual replenishment of nutrients, and is compatible with a number of analytical methods for characterizing and monitoring the effects of introduced candidate antibacterial regimens.
  • the drip-flow reactor has been successfully employed to culture a wide variety of pure and mixed- species biofilms. Biofilms are typically grown for two to five days prior to application of antimicrobial agents.
  • the fluid stream passing over the biofilm is amended or supplemented with the desired treatment formulation (e.g., one or more BT compounds and/or one or more antibiotics, or controls, and/or other candidate agents).
  • the desired treatment formulation e.g., one or more BT compounds and/or one or more antibiotics, or controls, and/or other candidate agents.
  • Flow is continued for the specified treatment period.
  • the treated biofilm coupon is then briefly removed from the reactor and the biofilm is scraped into a beaker containing 10 ml of buffer.
  • This sample is briefly processed (typically 30s to 1 min) with a tissue homogenizer to disperse bacterial aggregates.
  • the suspension is serially diluted and plated to enumerate surviving microorganisms according to standard microbiological methodologies.
  • This Example describes a modification of established in vitro keratinocyte scratch models of wound healing, to arrive at a model having relevance to biofilm-associated wound pathology and wound healing, and in particular to acute or chronic wounds or wounds containing biofilms as described herein.
  • cultivation of mammalian (e.g., human) keratinocytes and bacterial biofilm populations proceeds in separate chambers that are in fluid contact with one another, to permit assessment of the effects of conditions that influence the effects, of soluble components elaborated by biofilms, on keratinocyte wound healing events.
  • Newborn human foreskin cells are cultured as monolayers in treated plastic dishes, in which monolayers a controlled "wound" or scratch is formed by mechanical means (e.g., through physical disruption of the
  • Wounded keratinocyte monolayers cultured in the presence of biofilms are examined according to morphological, biochemical, molecular genetic, cell physiologic and other parameters to determine whether
  • BT comopunds alters (e.g., increases or decreases in a statistically significant manner relative to appropriate controls) the damaging effects of the biofilms. Wounds are first exposed to each BT compound alone, and to contemplated combinations of BT compounds, in order to test the toxicity of each BT compound treatment prior to assessing the effects of such treatments on biofilm influences toward the model wound healing process.
  • a three-day biofilm is cultured on a membrane (e.g., a TransWell membrane insert or the like) that is maintained in a tissue culture well above, and in fluid communication with, a keratinocyte monolayer that is scratched to initiate the wound healing process.
  • a membrane e.g., a TransWell membrane insert or the like
  • Biofilms cultured out of authentic acute or chronic wounds are contemplated for use in these and related embodiments.
  • an in vitro system for evaluating soluble biofilm component effects on migration and proliferation of human keratinocytes.
  • the system separates the biofilm and keratinocytes using a dialysis membrane. Keratinocytes are cultured from newborn foreskin as previously described (Fleckman et al., 1997 J Invest. Dermatol. 109:36;
  • the system is inoculated with wound-isolated bacteria and incubated in static conditions for two hours to enable bacterial attachment to surfaces in the upper chambers. Following the attachment period, liquid medium flow is initiated in the upper chamber to remove unattached cells. Flow of medium is then continued at a rate that minimizes the growth of planktonic cells within the upper chamber, by washout of unattached cells. After incubation periods ranging from 6 to 48 hours, the systems (keratinocyte monolayers on coverslips and bacterial biofilm on membrane substrate) are disassembled and the cover slips removed and analyzed.
  • the systems keratinocyte monolayers on coverslips and bacterial biofilm on membrane substrate
  • mature biofilms are grown in the upper chamber prior to assembling the chamber.
  • the separate co- culturing of biofilms and scratch-wounded keratinocyte monolayers is conducted in the absence and presence of one or more BT compounds, optionally with the inclusion or exclusion of one or more antibiotics, in order to determine effects of candidate agents such as BT compounds, or of potentially synergizing BT compound-plus-antibiotic combinations (e.g., a BT compound as provided herein such as a BT that is provided in microparticulate form, and one or more of amikacin, ampicillin, cefazolin, cefepime, chloramphenicol, ciprofloxacin, clindamycin (or another lincoasamide antibiotic), daptomycin (Cubicin®),_doxycycline, gatifloxacin, gentamicin, imipenim, levofloxacin, linezolid (Zyvox®), minocycline,
  • keratinocyte repair of the scratch wound e.g., to identify an agent or combination of agents that alters ⁇ e.g., increases or decreases in a statistically significant manner relative to appropriate controls
  • at least one indicator of scratch wound healing such as the time elapsing for wound repair to take place or other wound-repair indicia (e.g., Tao et al., 2007 PLoS ONE 2:e697; Buth et al. 2007 Eur. J Cell Biol. 86:747; Phan et al. 2000 Ann. Acad. Med. Singapore 29:27).
  • Isolated human keratinocytes were cultured on glass coverslips and scratch-wounded according to methodologies described above in Example 4. Wounded cultures were maintained under culture conditions alone or in the presence of a co-cultured biofilm on a membrane support in fluid
  • Figure 3 illustrates the effect that the presence in fluid communication (but without direct contact) of biofilms had on the healing time of scratched keratinocyte monolayers.
  • a method of identifying an agent for treating a chronic wound comprising culturing a scratch-wounded cell (e.g., keratinocyte or fibroblast) monolayer in the presence of a bacterial biofilm with and without a candidate anti-biofilm agent being present; and assessing an indicator of healing of the scratch- wounded cell monolayer in the absence and presence of the candidate anti- biofilm agent, wherein an agent (e.g., a BT compound such as a substantially monodisperse BT microparticle suspension as described herein, alone or in synergizing combination with an antibiotic, such as one or more of amikacin, ampicillin, cefazolin, cefepime, chloramphenicol, ciprofloxacin, clindamycin, daptomycin (Cubicin®),_doxycycline, gatifloxacin, gentamicin, imipenim, levofloxacin, linezolid (Zyvox®), min
  • an agent e.g.,
  • This example shows instances of demonstrated synergizing effects by combinations of one or more bismuth-thiol compounds and one or more antibiotics against a variety of bacterial species and bacterial strains, including several antibiotic-resistant bacteria.
  • McFarland standard suspensions were used to prepare 0.5 McFarland standard suspensions, which were further diluted 1 :50 ( ⁇ 2 10 6 cfu/mL) in cation-adjusted Mueller-Hinton broth medium (BBL, Cockeysville, MD, USA).
  • BTs prepared as described above
  • antibiotics were added at incremental concentrations, keeping the final volume constant at 0.2 mL.
  • the Minimum Inhibitory Concentration (MIC) was expressed as the lowest drug concentration inhibiting growth for 24 h. Viable bacterial counts (cfu/mL) were determined by standard plating on nutrient agar. The Minimal Bactericidal Concentrations (MBC) was expressed as the concentration of drug that reduced initial viability by 99.9% at 24 h of incubation.
  • the checkerboard method was used to assess the activity of antimicrobial combinations.
  • the fractional inhibitory concentration index (FICI) and the fractional bactericidal concentration index (FBCI) were calculated, according to Eliopoulos et al. (Eliopoulos and Moellering, (1996) Antimicrobial combinations. In Antibiotics in Laboratory Medicine (Lorian, V., Ed.), pp. 330- 96, Williams and Wilkins, Baltimore, MD, USA).
  • Synergy was defined as an FICI or FBCI index of ⁇ 0.5, no interaction at >0.5-4 and antagonism at >4 (Odds, FC (2003) Synergy, antagonism, and what the chequerboard puts between them. Journal of Antimicrobial Chemotherapy 52:1).
  • Synergy was also defined conventionally as ⁇ 4-fold decrease in antibiotic concentration.
  • BE 0.2 Mg/ml BisEDT; Bacterial strains were obtained from the Clinical Microbiology Laboratory at Winthrop-University Hospital, Mineola, NY. Nafcillin obtained from Sigma.
  • GM gentamicin
  • Strain S2400-1 was obtained from the Clinical Microbiology Laboratory at Winthrop-University Hospital, Mineola, NY.
  • Gentamicin was obtained from the Pharmacy Department at Winthrop; synergy in bold
  • DOX doxycycline
  • BE BisEDT at 0.3 pg/ml
  • Strains were obtained from the laboratory of Dr. I Chopra, Department of Bacteriology, The University of Bristol, Bristol, UK. Antibiotics were obtained from the Pharmacy Department at Winthrop- University Hospital, Mineola, NY. TABLE 12
  • Agr aminoglycoside resistant
  • NN tobramycin
  • PA Pseudomonas aeruginosa
  • BE BisEDT, 0.3 pg/ml
  • Strains were obtained from the laboratory of Dr. K. Poole, Department of Microbiology and Immunology, Queens University, Ontario, CN. Tobramycin was obtained from the Pharmacy Department at Winthrop-University Hospital, Mineola, NY.
  • NN Tobramycin
  • BE BisEDT, 0.4 Mg/ml
  • Tobramycin was obtained from the Pharmacy Department at Winthrop-University Hospital, Mineola, NY. TABLE 14
  • NN Tobramycin
  • BE BisEDT, 0.4 Mg/ml
  • Strains were obtained from the laboratory of Dr. J.J. LiPuma, Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, Ml; also Veloira et al. 2003.
  • Tobramycin was obtained from the Pharmacy Department at Winthrop-Unive Hospital, Mineola, NY.
  • NN Tobramycin
  • BE BisEDT, 0.8 Mg/ml
  • Lipo-BE-NN liposomal BE-NN
  • NN Tobramycin
  • BE BisEDT, 0.8 Mg/ml
  • Lipo-BE-NN liposomal BE-NN
  • BACTERIAL STRAINS In this example the in vitro activities of BisEDT and comparator agents were assessed against multiple clinical isolates of Gram-positive and - negative bacteria that are responsible for skin and soft tissue infections.
  • Test compounds and test concentration ranges were as follows: BisEDT (Domenico et al., 1997; Domenico et al., Antimicrob. Agents Chemother. 45(5):1417-1421. and Example 1), 16-0.015 pg/mL; linezolid (Chem Pacifica Inc., #35710), 64-0.06 pg/mL; Daptomycin (Cubist Pharmaceuticals #MCB2007), 32-0.03 pg/mL and 16-0.015 pg/mL; vancomycin (Sigma-Aldrich, St. Louis, MO, # V2002), 64-0.06 pg/mL;
  • ceftazidime (Sigma #C3809), 64-0.06 pg/mL and 32-0.03 pg/mL; imipenem (United States Pharmacopeia, NJ, #1337809) 16-0.015 pg/mL and 8-0.008 pg/mL; ciprofloxacin (United States Pharmacopeia, # IOC265), 32-0.03 pg/mL and 4-0.004 pg/mL; gentamicin (Sigma #G3632) 32-0.03 pg/mL and 16-0.015 pg/mL. All test articles, except gentamicin, were dissolved in DMSO;
  • gentamicin was dissolved in water. Stock solutions were prepared at 40-fold the highest concentration in the test plate. The final concentration of DMSO in the test system was 2.5%.
  • Test organisms The test organisms were obtained from clinical laboratories as follows: CHP, Clarian Health Partners, Indianapolis, IN; UCLA, University of California Los Angeles Medical Center, Los Angeles, CA; GR Micro, London, UK; PHRI TB Center, Public Health Research Institute
  • Tuberculosis Center New York, NY; ATCC, American Type Culture Collection, Manassas, VA; Mt Sinai Hosp., Mount Sinai Hospital, New York, NY; UCSF, University of California San Francisco General Hospital, San Francisco, CA; Branson Hospital, Branson Cincinnati Hospital, Kalamazoo, Ml; quality control isolates were from the American Type Culture Collection (ATCC, Manassas, VA). Organisms were streaked for isolation on agar medium appropriate to each organism. Colonies were picked by swab from the isolation plates and put into suspension in appropriate broth containing a cryoprotectant. The
  • suspensions were aliquoted into cryogenic vials and maintained at -80°C.
  • BisEDT bismuth-1 ,2-ethanedithiol
  • LZD linezolid
  • DAP daptomycin
  • VA vancomycin
  • CAZ ceftazidime
  • IPM imipenem
  • CIP ciprofloxacin
  • GM gentamicin
  • MSSA methicillin-susceptible Staphylococcus aureus
  • CLSI QC Clinical and Laboratory Standards Institute quality control strain
  • MRSA methicillin-resistant Staphylococcus aureus
  • CA-MRSA community-acquired methicillin-resistant Staphylococcus aureus
  • MSSE methicillin-susceptible Staphylococcus epidermidis
  • MRSE methicillin-resistant Staphylococcus epidermidis
  • VSE vancomycin-susceptible Enterococcus.
  • the isolates were streaked from the frozen vials onto appropriate medium: Trypticase Soy Agar (Becton-Dickinson, Sparks, MD) for most organisms or Trypticase Soy Agar plus 5% sheep blood (Cleveland Scientific, Bath, OH) for streptococci. The plates were incubated overnight at 35°C.
  • the medium employed for the MIC assay was Mueller Hinton II Broth (MHB II- Becton Dickinson, # 212322) for most of the organisms.
  • MHB II was supplemented with 2% lysed horse blood (Cleveland Scientific Lot # H13913) to accommodate the growth of
  • Streptococcus pyogenes and Streptococcus agalactiae were prepared at 102.5% normal weight to offset the dilution created by the addition of 5 pL drug solution to each well of the microdilution panels. In addition, for tests with daptomycin, the medium was supplemented with an additional 25mg/L Ca 2+ .
  • Automated liquid handlers included the Multidrop 384 (Labsystems, Helsinki, Finland), Biomek 2000 and Multimek 96 (Beckman Coulter, Fullerton CA).
  • the wells of Columns 2-12 of standard 96-well microdilution plates (Falcon 3918) were filled with 150pL of DMSO or water for gentamicin on the Multidrop 384.
  • the drugs 300 pL were dispensed into Column 1 of the appropriate row in these plates.
  • the Biomek 2000 completed serial transfers through Column 11 in the mother plates.
  • the wells of Column 12 contained no drug and were the organism growth control wells in the daughter plates.
  • the daughter plates were loaded with 185 pL of the appropriate test media
  • the daughter plates were prepared on the Multimek 96 instrument which transferred 5 pL of drug solution from each well of a mother plate to each corresponding well of each daughter plate in a single step.
  • Standardized inoculum of each organism was prepared per CLSI methods (ISBN 1-56238-587-9, cited supra). Suspensions were prepared in MHB to equal the turbidity of a 0.5 McFarland standard. The suspensions were diluted 1 :9 in broth appropriate to the organism. The inoculum for each organism was dispensed into sterile reservoirs divided by length (Beckman Coulter), and the Biomek 2000 was used to inoculate the plates. Daughter plates were placed on the Biomek 2000 work surface reversed so that inoculation took place from low to high drug concentration. The Biomek 2000 delivered 10 pL of standardized inoculum into each well.
  • the wells of the daughter plates ultimately contained 185 pL of broth, 5 pL of drug solution, and 10 pL of bacterial inoculum. Plates were stacked 3 high, covered with a lid on the top plate, placed in plastic bags, and incubated at 35°C for approximately 18 hours for most of the isolates. The Streptococcus plates were read after 20 hours incubation. The microplates were viewed from the bottom using a plate viewer. For each of the test media, an uninoculated solubility control plate was observed for evidence of drug precipitation. The MIC was read and recorded as the lowest concentration of drug that inhibited visible growth of the organism.
  • BisEDT demonstrated potent activity against both methicillin- susceptible Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), and community-acquired MRSA (CA-MRSA), inhibiting all strains tested at 1 pg/mL or less with an MIC90 values of 0.5 pg/mL for all three organism groups.
  • BisEDT exhibited activity greater than that of linezolid and vancomycin and equivalent to that of daptomycin.
  • MRSA and CAMRSA were resistant to imipenem while BisEDT demonstrated activity equivalent to that shown for MSSA.
  • BisEDT was highly-active against methicillin-susceptible and methicillin-resistant Staphylococcus epidermidis (MSSE and MRSE), with MIC90 values of 0.12 and 0.25 pg/mL, respectively. BisEDT was more active against MSSE than any of the other agents tested except imipenem. BisEDT was the most active agent tested against MRSE.
  • BisEDT demonstrated activity equivalent to that of daptomycin, vancomycin, and imipenem against vancomycin-susceptible Enterococcus faecalis (VSEfc) with an MIC90 value of 2pg/mL.
  • VSEfc vancomycin-susceptible Enterococcus faecalis
  • VREfc vancomycin-resistant Enterococcus faecalis
  • VSEfm Enterococcus faecium
  • VREfm vancomycin-resistant Enterococcus faecium
  • comparator agents resulted in off-scale MIC90 values for these agents.
  • BisEDT demonstrated broad-spectrum potency against multiple clinical isolates representing multiple species, including species commonly involved in acute and chronic skin and skin structure infections in humans.
  • the activity of BisEDT and key comparator agents was evaluated against 723 clinical isolates of Gram-positive and Gram-negative bacteria.
  • the BT compound demonstrated broad spectrum activity, and for a number of the test organisms in this study, BisEDT was the most active compound tested in terms of anti-bacterial activity.
  • BisEDT was most active against MSSA, MRSA, CA-MRSA, MSSE, MRSE, and S. pyogenes, where the MIC90 value was 0.5 pg/mL or less. Potent activity was also demonstrated for VSEfc, VREfc.VSEfm, VREfm, A.
  • MIC90 8 pg/mL
  • S. agalactiae 16 pg/mL
  • This example shows that microparticulate bismuth thiols (BTs) promote antibiotic activity through enhancing and/or synergizing interactions.
  • BTs microparticulate bismuth thiols
  • MRSE Methicillin resistance in S. epidermidis
  • MRSA S. aureus
  • BTs at subinhibitory (subMIC) concentrations reduced resistance to several important antibiotics.
  • MIC 45 ⁇ 32 32 ⁇ 28 37 ⁇ 24 9.3 ⁇ 6.1 ⁇ 8 >32 12 MRSA clinical isolates were grown in BHIG/X and exposed to serial dilutions of antibiotics in the presence of 0-0.1 pg/ml BisEDT.
  • the MIC and BPC, calculated in pg/ml, are the means ⁇ standard deviations from at least three trials.
  • the right hand column lists the Standard MIC for antibiotic senstivity (S) and resistance (R)
  • MRSA Nafcillin or Gentamicin + BisEDT Synergy
  • Staphylococcus epidermidis The activities of most classes of antibiotic were promoted in the presence of BisEDT.
  • BisEDT Bisethylcholine
  • clindamycin and gatifloxacin showed significantly more antibiofilm activity against S. epidermidis when combined with BisEDT ( Figure 5).
  • the BPC for clindamycin, gatifloxacin and gentamicin were reduced 50-fold, 10-fold and 4-fold, respectively, in the presence of subMIC BisEDT.
  • MBC Minimum bactericidal concentration
  • Gentamicin showed the greatest reduction in MBC (4- to 16-fold), followed by cefazolin (4- to 5-fold), vancomycin and nafcillin (3- to 4-fold), minocycline and gatifloxacin (2- to 3-fold), while clindamycin and rifampicin MBC remained largely unaffected.
  • Clindamycin is a bacteriostatic agent, which explains its lack of bactericidal activity. Cefazolin resistance was reversed with respect to the MBC [Domenico et al., 2003]. These effects were additive.
  • BisEDT, 10 ⁇ g/ml RIP and 10 ⁇ g/ml rifampin, alone or combined were implanted s.c. into rats.
  • Physiological solution (1 ml) containing the MS and MR strains at 2x10 7 cfu/ml was inoculated onto the graft surface using a tuberculin syringe. All grafts were explanted at 7 days following implantation and sonicated for 5 minutes in sterile saline solution to remove the adherent bacteria. Quantitation of viable bacteria was obtained by culturing dilutions on blood agar plates. The limit of detection was approximately 10 cfu/cm 2 .
  • FIG Index ⁇ 0.5 indicates synergy: FICI >0.5 and ⁇ 1.0 indicates enhancement.
  • Resistant strains of E.coli were cultured in Mueller-Hinton II broth at 37°C in the presence of chloramphenicol (CM) or ampicillin (AMP) and BisEDT alone or in combination (BE; 0.33 pg/ml). The MIC was determined as the antibiotic concentration that inhibited growth for 24 ⁇ 1 h.
  • CM chloramphenicol
  • AMP ampicillin
  • BisEDT BisEDT alone or in combination
  • Tetracycline resistant Escherichia coli were made sensitive to doxycycline by the addition of subMIC BisEDT (Table 25). The combination exhibited synergy against the TET M and TET D strains (FIC ⁇ 0.5), with additive effects against the TET A and TET B strains.
  • Resistant strains of E.coli were cultured in Mueller-Hinton II broth at 37°C in the presence of doxycycline (DOX) and BisEDT alone or in combination (BE; 0.33 Mg/ml). The MIC was determined as the antibiotic concentration that inhibited growth for 24 ⁇ 1 h.
  • microparticulate bismuth thiol BisEDT promotes antibiotic activity through enhancing and/or synergizing interactions with specific antibiotics against specific microbial target organisms.
  • Single-point data for each indicated combination in Table 26 were generated essentially according to the methods used in Example 8.
  • Staphylococcus aureus E Fc, Enterococcus faecalis
  • SP Streptococcus pneumoniae
  • PRSP penicillin-resistant Streptococcus pneumoniae
  • EC Escherichia coli
  • KP Klebsiella pneumoniae
  • PA Pseudomonas aeruginosa
  • Beep Burkholderia cepacia
  • Bmult Bukholderia multivorans
  • Acinetobacter baumanii Msmeg, Mycobacterium smegmatis.
  • Bis-EDT analogs prepared as described above, and other agents against representative strains of several Gram-negative pathogenic bacteria were tested.
  • a modification of a common laboratory method was used to determine synergism (FICI ⁇ 0.5), enhancement (0.5 ⁇ FICI ⁇ 1.0), antagonism (FICI > 4.0) and indifference (1.0 ⁇ FICI ⁇ 4.0) used fractional inhibitory concentrations (FICs) and FIC indices (FICI) (Eliopoulos G and R Moellering. 1991 .
  • test articles Stock solutions of all test articles were prepared at 40X the final target concentration in the appropriate solvent. All test articles were in solution under these conditions. The final drug concentrations in the FIC assay plates were set to bracket the MIC value of each agent for each test organism, unless the strain was totally resistant to the test agent. The concentration ranges tested are displayed in Table 27.
  • the test organisms were originally received from clinical sources, or from the American Type Culture Collection. Upon receipt, the isolates were streaked onto Tryptic Soy Agar II (TSA). Colonies were harvested from these plates and a cell suspension was prepared in an appropriate broth growth medium containing cryoprotectant. Aliquots were then frozen at -80°C.
  • the frozen seeds of the organisms to be tested in a given assay were thawed, streaked for isolation onto TSA plates, and incubated at 35°C. All organisms were tested in Mueller Hinton II Broth (Becton Dickinson, Lot No.9044411 ). The broth was prepared at 1.05X normal weight/volume to offset the 5 % volume of the drugs in the final test plates.
  • MIC values were previously determined using the broth microdilution method for aerobic bacteria (Clinical and Laboratory Standards Institute (CLSI). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard— Eighth Edition. CLSI document M07-A8 [ISBN 1-56238-689-1 ]. Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2009.).
  • FIC values were determined using a broth microdilution method previously described (Sweeney et al., 2003 Antimicrob. Agents Chemother. 47(6):1902-1906). To prepare the test plates, automated liquid handlers (Multidrop 384, Labsystems, Helsinki, Finland; Biomek 2000 and Multimek 96, Beckman Coulter, Fullerton CA) were used to conduct serial dilutions and liquid transfers.
  • the "daughter plates" were loaded with 180 ⁇ _ of test medium using the Multidrop 384. Then, the Multimek 96 was used to transfer 10 ⁇ _ of drug solution from each well of the drug combination mother plate to each corresponding well of the daughter plate in a single step. Finally, the daughter plates were inoculated with test organism. Standardized inoculum of each organism was prepared per published guidelines (CLSI, 2009). For all isolates, the inoculum for each organism was dispensed into sterile reservoirs divided by length (Beckman Coulter), and the Biomek 2000 was used to inoculate the plates. The instrument delivered 10 ⁇ _ of standardized inoculum into each well to yield a final cell concentration in the daughter plates of approximately 5 x 10 5 colony-forming-units/mL
  • test format resulted in the creation of an 8 x 12 checkerboard where each compound was tested alone (Column 12 and Row H) and in combination at varying ratios of drug concentration. All organism plates were stacked three high, covered with a lid on the top plate, placed in plastic bags, and incubated at 35°C for approximately 20 hours. Following incubation, the microplates were removed from the incubators and viewed from the bottom using a ScienceWare plate viewer. Prepared reading sheets were marked for the MIC of drug 1 (row H), the MIC of drug 2 (column 12) and the wells of the growth-no growth interface.
  • Microparticulate Bis-EDT the four microparticulate BT analogs, and all of the other agents (and combinations of agents) were soluble at all final test concentrations.
  • the aim of this study was to determine whether BTs will reduce infection in a contaminated open fracture model either on their own or with antibiotics.
  • the contaminated rat femur critical defect model is a well-accepted model and was used for the experiments described in this Example. This model offers a standardized model for comparing various possible treatments and their effects on reducing infection and/or improving healing.
  • CPD-8-2 bismuth pyrithione/ butanedithiol; Table 1
  • CPD-11 bismuth pyrithione/ ethanedithiol; Table 1
  • the three BT formulations, Bis-EDT, CPD-11 and CPD-8-2 demonstrated inhibitory effects on S. aureus strains in vitro when used with and without Tobramicin and Vancomycin in a Poly Methyl Methacrylate (PMMA) cement bead vehicle.
  • Three formulations of microparticulate BTs were produced in a clinically useful hydrogel gel form as described herein. These BTs were tested suspended in a gel at a concentration of 5mg/ml "1 as has been found to be an appropriate concentration for gel delivery. The gel formulations conformed to the wound contours, and did not require removal following application.
  • BT was used singularly; in the second arm BT was used in conjunction with a systemic antibiotic (ABx).
  • ABx systemic antibiotic
  • the rats were anesthetized and prepped for surgery.
  • the rats were anesthetized and prepped for surgery.
  • anterolateral aspect of the femoral shaft was exposed through a 3-cm incision.
  • the periosteum and attached muscle was stripped from the bone.
  • a polyacetyl plate (27 x 4 x 4 mm) was placed on the anterolateral surface of the femur.
  • the plates were predrilled to accept 0.9-mm diameter threaded Kirschner wires.
  • the bases of these plates were formed to fit the contour of the femoral shaft. Pilot holes were drilled through both cortices of the femur using the plate as a template and threaded Kirschner wire was inserted through the plate and femur.
  • the notches that were 6 mm apart on the plate served as a guide for bone removal.
  • a small oscillating saw was used to create the defect while the tissue was cooled by continuous irrigation in an effort to prevent thermal damage.
  • Cefazolin antibiotic activity was enhanced as compared to Cefazolin or any of the Bis compounds alone to reduce S. aureus infection of injured bone.
  • Cefazolin in combination with MB-11 and MB-8-2 showed enhanced antibiotic activity as compared to Cefazolin alone to reduce S. aureus infection detected on hardware.
  • Bis-EDT did not appear to affect Cefazolin activity in this capacity.

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Abstract

La présente invention concerne des compositions et des procédés, comprenant de nouvelles suspensions microparticulaires homogènes, qui sont destinés à traiter des surfaces naturelles contenant des biofilms bactériens, comprenant des effets synergétiques ou amplificateurs insoupçonnés entre des composés bismuth-thiol (BT) et certains antibiotiques, pour fournir des formulations comprenant des formulations antiseptiques. L'invention concerne également les propriétés antibactériennes et anti-biofilm précédemment inattendues des composés BT et des combinaisons composé BT-plus-antibiotique décrites, comprenant les efficacités préférentielles de certaines de telles compositions pour traiter certaines infections par des bactéries gram-positives, et les efficacités préférentielles distinctes de certaines de telles compositions pour traiter certaines infections par des bactéries gram-négatives.
EP11740333.7A 2010-02-03 2011-02-03 Bismuth-thiols comme antiseptiques destinés à des utilisations biomédicales, comprenant le traitement de biofilms bactériens et d'autres utilisations Pending EP2536406A4 (fr)

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EP2844295A1 (fr) 2012-05-03 2015-03-11 Kala Pharmaceuticals, Inc. Nanoparticules pharmaceutiques permettant un transport muqueux amélioré
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US9827191B2 (en) 2012-05-03 2017-11-28 The Johns Hopkins University Compositions and methods for ophthalmic and/or other applications
WO2014052973A1 (fr) * 2012-09-28 2014-04-03 Stelo Technologies Procédés de fabrication de nanoparticules d'argent et leurs applications
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CA2788669C (fr) 2021-05-25
EP2536406A4 (fr) 2014-04-09
MX346409B (es) 2017-03-17
AU2011212941B2 (en) 2016-03-03
CN107308186A (zh) 2017-11-03
WO2011097347A2 (fr) 2011-08-11
JP2013518895A (ja) 2013-05-23
AU2011212941A1 (en) 2012-09-27
CN103079557A (zh) 2013-05-01

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