JP6685991B2 - Bismuth-thiol as a disinfectant for biomedical use, including treatment of bacterial biofilms and other uses - Google Patents

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of PCT Application No. PCT / US2010 / 023108, filed February 3, 2010, and US Provisional Application No. 61 / 373,188, filed August 12, 2010. , Each of which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION Embodiments of the invention disclosed herein relate to compositions and methods for the treatment of microbial infections. In particular, embodiments of the invention include treatment of bacterial biofilms and other conditions, in wounds such as chronic and acute wounds, and in epithelial tissue in clinical personal health care and other aspects. It relates to improved treatments for controlling bacterial infections.

Description of Related Art A complex series of coordinated intramolecular and intermolecular interactions that contribute to cutaneous wound healing and response and resistance to microbial infections, and / or to the healing or retention of body tissues are commonly found in, for example, opportunistic infections and nosocomial infections. Infection (eg, clinical regimens that may increase the risk of infection), local or systemic administration of antibiotics (may affect cell development, migration or other functions, and may also be selected for antibiotic resistant microorganisms) Yes, frequent wound dressing changes, open air exposure of wounds to accelerate healing, use of temporary artificial structural support matrix or scaffold material, debridement of infected or necrotic tissue by debridement and / or repeated surgery To be adversely affected by various external factors, such as the potential need for things and / or other factors. There is a match.

  Thus, wound healing has long been a daunting challenge for clinicians around the world. Current treatments of refractory wounds are impractical and ineffective, often requiring multiple surgeries to close the wound. For example, Regranex® (becaplermine, a recombinant platelet-derived growth factor available from Ortho-McNeil Pharmaceuticals, Inc., Ethicon, Inc.) is one of the few available treatments for chronic wounds. , Costly to manufacture and have limited clinical utility.

Chronic and Acute Wounds and Wound Biofilms Continuity between cells within tissues, or continuity between tissues, may include, for example, physical, mechanical, biological, pathological and / or chemical forces (eg, burns, Dermal infection, stab wound, wound with gun or shrapnel, skin ulcer, radioactive poisoning, malignant tumor, gangrene, autoimmune disease, immunodeficiency disease, respiratory injury due to inhalation or infection, ingestion or infection of dangerous substances, etc. A wound occurs when it is destroyed by gastrointestinal injury due to cerebral injury, circulation and hematopoietic disorders such as inability to coagulate), or other traumatic injury.

A slight level of bacterial contamination in the wound, or "colonization" of the wound, does not necessarily interfere with the process of wound healing, but in the presence of sufficient numbers of bacteria to overwhelm the host's immune defenses, acute wounds or It can result in chronic wounds or wounds in which bacterial biofilms are present, such as wound infections where bacterial development leads to progressive host injury. Bryant and Nix, Acute and Chronic Wounds :;: (. 2 nd Ed) Current Management Concepts, 2006 Mosby (Elsevier), NY Baronski, Wound Care Essentials Practical Principles, 2007 Lippincott, Williams and Wilkins, Philadelphia, PA). For example, an acute wound, which may result from injury, trauma, surgical intervention, or other causes, typically heals rapidly with no underlying health deficiency, but in some cases an infection is present. Sometimes this is not the case, and the rapid formation of bacterial biofilms has been described in acute wounds (eg WO / 2007/061942). Additional factors that can contribute to the development of chronic wounds include lack of mobility (eg, which exerts continuous pressure on the wound site), loss of sensory or mental capacity, inaccessibility of the wound site (eg, Respiratory or gastrointestinal tract), and circulatory defects. Infection at the site of chronic wounds may be detected by skin redness, edema, pus formation and / or unpleasant odors, or other relevant clinically accepted criteria.

  Thus, when the host's immune system is overwhelmed by bacterial infection of the wound site (eg, acute wounds) and permissive conditions arise for the bacteria to enter the tissue and be further destroyed, higher organisms (non-limiting). In humans and other mammals, there may be acute wounds that do not heal properly and thus chronic damage may occur. Chronic wounds are generally wounds that do not heal within 3 months and tend to grow larger as bacterial invasion progresses, instead of becoming smaller. Chronic wounds may cause greater pain and stress to the patient as nearby nerves become impaired (neuropathy) as the wound progresses. These wounds affect 4 million Americans each year and cost approximately $ 9 billion to treat. Most affected people are over the age of 60.

  Chronic wounds can sometimes occur as acute wounds, for example, handgun or shrapnel wounds, burns, puncture wounds, venous ulcers, pressure ulcers, diabetic ulcers, radiation poisoning, malignant tumors, dermal infections. , Gangrene, surgical wound, diabetic leg ulcer, pressure ulcer, venous leg ulcer, infectious and / or biofilm containing refractory surgical wound, gangrenous pyoderma, traumatic wound, acute arterial insufficiency, necrotizing fasciitis, It can include osteomyelitis (bone infection), radioactive injury such as radiation osteonecrosis and soft tissue radiation necrosis, or other types of wounds. For example, venous ulcers most often occur in the lower extremities due to restricted circulation (eg, ischemia), venous valve dysfunction, or repetitive physical trauma (eg, recurrent injury). For example, if local pressure applied at or near the wound site is greater than blood pressure, pressure ulcers may occur, resulting in impaired circulation, paralysis, and / or bed sores causing chronic damage. , Or may make it worse. Diabetic ulcers may occur in diabetic patients, for example, uncontrolled hyperglycemia resulting in loss of sensation in the extremities, repeated damage to the patient's body parts, and / or unnoticed damage. Factors that complicate or otherwise affect the clinical signs and outcomes of chronic wounds include the patient's immune status (eg, immunosuppression, pathology (eg, HIV-AIDS), radiation therapy, or Immune system damaged by drugs; age; stress); skin aging (such as photochemical aging), and the development and progression of biofilms in wounds. In the case of epithelial tissue in the respiratory and / or gastrointestinal tract, inaccessibility, occlusion, difficulty in generating fluid forces to wash epithelial surfaces, or creation of a local microenvironment that induces microbial survival, May cause clinical problems.

  Wound-related damage may be accompanied by loss or impairment of organ function, shock, bleeding and / or thrombus, cell death (eg necrosis and / or apoptosis), stress and / or microbial infection. Any or all of these events, particularly infections, can delay or interfere with the effective tissue repair processes involved in wound healing. Therefore, in individuals with persistent wounds, removing non-viable tissue from the wound site (a process called debridement) and any foreign material from the wound site (also known as wound cleansing) as soon as possible. Called) can be important.

  Severe wounds, acute wounds, chronic wounds, burns, and ulcers can benefit from cell wound dressings. Multiple artificial skin products, for example, Apprigraft® (Norvartis), Demagraft®, Biobrane®, Transscience® (Advance Tissue Science), Integra® DermalRegistration Registry (Registered). ) (Integra Life Sciences Technology), and OrCel® are available for refractory wounds or burns. However, these products are not designed to address the problems of bacterial tissue invasion and wound spread.

  Unfortunately, systemic antibiotics are not effective in treating chronic wounds and are generally not used unless an acute bacterial infection is present. Current approaches include the administration or application of antibiotics, but such rescue methods may promote the emergence of antibiotic resistant strains and / or may be ineffective against bacterial biofilms. . Therefore, it may be particularly important to use antiseptics when drug resistant bacteria (eg methicillin resistant Staphylococcus aureus or MRSA) are detected. Although there are many widely used antiseptics, established bacterial populations or subpopulations may not respond to these agents, or any other currently available treatment. In addition, multiple antiseptics can be toxic to host cells at concentrations required to be effective against established bacterial infections, which makes such antiseptics inadequate. This problem may be due to internal epithelial surfaces such as the respiratory tract (eg, respiratory tract, nasopharynx and larynx, trachea, lungs, bronchi, bronchioles, alveoli) or gastrointestinal tract (eg, oral cavity, esophagus, stomach, intestines, It can be particularly acute in an attempt to clear infections from natural surfaces, including the rectum, anus, etc.) or other epithelial surfaces.

  Of particular concern are infectious agents composed of bacterial biofilms, which are relatively recently recognized bacterial tissues, in which free-floating single-cell (plankton) bacteria have markedly different behavioral patterns and gene expression due to cell-cell adhesion. , And organized multicellular consortia (biofilms) that are sensitive to environmental drugs such as antibiotics. Biofilms may develop biological defense mechanisms not found in planktonic bacteria, which can protect the biofilm consortium from antibiotics and host immune responses. Established biofilms can stop the tissue healing process.

  Common microbiological contaminants in persistent and potentially toxic infections include S. cerevisiae, including MRSA (methicillin-resistant Staphylococcus aureus). Aureus, Enterococcus, E. Coli, P. Aeruginosa, Streptococcus, and Acinetobacter baumannii. Some of these organisms show the ability to survive on non-nutritional clinical surfaces for several months. S. Aureus has been shown to be viable on dry glass for 4 weeks and on dry blood and cotton fibers for 3-6 months (Domenico et al., 1999 Infect. Immun. 67: 664-669). . E. Coli and P. Both aeruginosa and S. aeruginosa were tested on dry blood and cotton fibers. It was shown to survive longer than Aureus (previous work).

  Microbial biofilms are associated with a substantial increase in resistance to both fungicides and antibiotics. The biofilm morphology occurs when bacteria and / or fungi attach to the surface. This attachment causes alterations in gene transcription, resulting in secretions that are remarkably active and difficult to penetrate the polysaccharide matrix to protect the microorganism. In addition to having very substantial resistance to antibiotics, biofilms are also very resistant to the mammalian immune system. Prevention of biofilm formation is a very important clinical priority, as biofilms are very difficult to eradicate once established. Recent studies have shown that open wounds can be rapidly contaminated with biofilms. These microbial biofilms are believed to delay wound healing and are likely associated with the establishment of severe wound infections.

  For example, current guidelines for military wound care specify energetic and complete irrigation and debridement (Blankenship CL, Guideline for care of open wounds, Leaf Operation Operations. (Bureau of Medicine and Surgery). This initial intervention is important, but not sufficient to prevent the onset of infection. Additional therapeutic steps are required to undergo subsequent debridement to promote healing and reduce the microbial bioburden, thereby reducing the chance of wound infection and colonization of wound biofilms.

  Due to the complex nature of military trauma wounds, the likelihood of infection is great and the introduction of foreign matter and other environmental pollutants is particularly considered. Both military and clinical environments (including humans present in both of these environments) are important for both potentially pathogenic microorganisms, especially for those suffering from open and / or complex wounds. Acts as a reliable source. Acute and chronic wounds, including surgical wounds and military wounds, already damage the skin, the body's primary defense and barrier to infection. Wounds thus expose the interior of the body (the moist and nutritive environment) to opportunistic and pathogenic infections. Many of these infections, especially persistent wound infections, may be involved in biofilm formation, as shown in the example of chronic wounds (James et al., 2008). Wound infections in hospital facilities are one of the most common causes of nosocomial infections, and wounds obtained in military and natural disaster environments are particularly susceptible to microbial contamination. Military wounds are typically associated with tissue damage, tend to be extensive and deep, can introduce foreign bodies and interfere with local blood supply, can be associated with fractures and burns, and can cause shock. Military wounds are vulnerable to infection, as they can cause infections and impair immune defense.

Skin structure and wound healing Skin and other epithelial tissues that are intact and functioning (eg, found on the generally avascular epithelial surface that forms a barrier between an organism and its external environment, such as skin, and breathing). Maintenance of the organs and lining of the gastrointestinal tract, glandular tissue, etc.) is critical to the health and survival of humans and other animals. The skin is the largest body organ of humans and other higher vertebrates (eg mammals) and protects against environmental damage through barrier function, mechanical strength and water impermeability. As a critical environmental interface, the skin provides a protective body covering, allowing maintenance of physiological equilibrium.

  The structure of skin is well known. Briefly, the epidermis, the outer layer of skin, is covered by a protective layer of stratum corneum, the inactive epidermal skin cells (eg, keratinocytes) and extracellular connective tissue proteins. When the epidermis is replaced by new material pushed up from the underlying epidermal granule cells, spinous cells, and basal cell layers, it undergoes a continuous process of molting, with continuous cell division and protein synthesis resulting in new skin cells and skin proteins ( For example, keratin, collagen). The dermis, which lies below the epidermis, assimilates connective tissue proteins (eg, collagen, elastin, etc.) by dermal fibroblasts and assembles the extracellular matrix and fibrous structure to give the skin flexibility, strength and elasticity. It is a part to be given. Within the dermis are also nerves, blood vessels, smooth muscle cells, hair follicles and fat glands.

  As the body's first line of defense, the skin is an important target for clinical injuries such as physical, mechanical, chemical and biological (eg xenobiotic, autoimmune) attacks that can change structure and function. is there. The skin is also regarded as an important component of the immune defense of the organism. In the skin, migratory and resident leukocytes (eg lymphocytes, macrophages, mast cells) and epidermal dendritic (Langerhans) cells, which have a strong antigen-presenting activity and contribute to immunological defense, can be found. Colored melanocytes in the basal layer absorb potentially harmful ultraviolet (UV) radiation. Destruction of the skin confers an inappropriate risk to the subject, including risks associated with opportunistic infections, incomplete and inappropriate tissue remodeling, scarring, mobility disorders, pain and / or other disorders. Like the skin, other epithelial surfaces (eg, the respiratory tract, gastrointestinal tract, and lining of the glands) have defined structural attributes during normal life, so that infection or other disruption poses a significant health risk. There is.

  Skin damage or destruction due to wounds such as cuts, abrasions, abrasions, stabs, burns (including chemical burns), infections, extreme temperatures, incisions (eg surgical incisions), trauma and other injuries There are cases. Therefore, efficient skin repair by wound healing is clearly desirable in these and similar aspects.

  Although the skin naturally exhibits significant self-repairing abilities after many types of injury, skin healing does not occur rapidly enough and / or is poorly remodeled due to inadequate cellular repair mechanisms. There are still many aspects in which the resulting skin can result in loss of integrity, barrier properties, mechanical strength, elasticity, softness, or other desirable properties in intact skin. Wound healing of the skin thus presents such associated challenges, for example in the context of chronic wounds.

  Wound healing occurs in three dynamic and overlapping periods, beginning with the formation of fibrin clots. The fibrin clot provides a temporary shield and reservoir for growth factors to attract cells into the wound. It also acts as a temporary extracellular matrix (ECM), which cells enter during repair. Overlapped with fibrin clot formation is the inflammatory phase, where the infiltration of phagocytes and neutrophils into the wound releases growth factors that amplify the early healing response while clearing wound debris and bacteria. It is characterized by doing. The process of regenerating the exfoliated area is initiated during the proliferative phase of healing, engineered by chemokines, cytokines and proteases secreted by immune cells, which are concentrated within the fibrin clot. Keratinocytes are stimulated to proliferate and migrate to form a new epithelial layer over the wound, while wound angiogenesis delivers oxygen, nutrients, and inflammatory cells to the wound area. The remodeling phase, the final phase of wound repair, is carried out by myofibroblasts, which promote connective tissue contraction, increase wound strength, and attach ECM to form scars. (Martin, P. Wound Healing-Aiming for Perfect Skin Generation. Science 1997; 4: 75-80).

Bismuth Thiol (BT) Based Disinfectants Natural products (eg, antibiotics) and synthetic chemicals with antimicrobial, in particular antibacterial, properties are known in the art and are , Antimicrobial effects, such as the ability to kill microorganisms (“killing” effects such as bactericidal), the ability to stop or damage the growth of microorganisms (“static” effects such as bacteriostatic), or microbial function, such as at a site , At least in part, characterized by their ability to interfere with colonization or infection of the plant, secretion of exopolysaccharides by bacteria, and / or conversion of plankton populations to biofilm populations or expansion of biofilm formation. Antibiotics, fungicides, disinfectants, etc., including factors that influence the selection and use of such compositions, such as bactericidal or bacteriostatic capacity, effective concentrations, and risk of toxicity to host tissues (bismuth-thiol, etc. Or a BT compound) is described, for example, in US Pat. S. 6,582,719.

  Bismuth is a Group V metal, an element with antimicrobial properties (similar to silver). Bismuth alone may not be therapeutically useful and may exhibit certain inadequate properties and therefore may be administered by delivery in complexing agents, carriers and / or other vehicles instead, The most common example is Pepto Bismol® in which bismuth is combined (chelated) with hyposalicylate. Combining certain thiol (-SH, sulfhydryl) containing compounds, such as ethanedithiol, and bismuth to provide exemplary bismuth thiol (BT) compounds provides bismuth compounds compared to other currently available bismuth preparations. Previous studies have determined that antimicrobial potency is improved. There are many thiol compounds that can be used to produce BT (eg, Domenico et al., 2001 Antimicrob. Agent. Chemotherap. 45 (5): 1417-1421, Domenico et al., 1997 Antimicron. 41 (8): 1697-1703, and US RE 37,793, US 6,248,371, 6,086,921, and 6,380,248. , See also US 6,582,719), several of these preparations are capable of inhibiting biofilm formation.

  BT compounds are MRSA (methicillin resistant S. aureus), MRSE (methicillin resistant S. epidermidis), Mycobacterium tuberculosis, Mycobacterium avium, drug resistant P. Aeruginosa, enterotoxigenic E. E. coli, enterohemorrhagic E. Coli, Klebsiella pneumoniae, Clostridium difficile, Helicobacter pylori, Legionella pneumophila, Enterococcus faecalis, Enterobacter cloacae, Salmonella typhimurium, Proteus vulgaris, Yersinia enterocolichica, Vibrio correle, and Activity against Shigella flexneri has been demonstrated (Domenico et al., 1997 Antimicrob. Agents Chemother. 41: 1697-1703). There is also evidence of activity against cytomegalovirus, herpes simplex virus type 1 (HSV-1) and HSV-2, and yeast and fungi such as Candida albicans. The role of BT is to reduce bacterial virulence, inhibit or kill microorganisms resistant to broad-spectrum antibiotics (Gram-positive and Gram-negative bacteria), prevent biofilm formation, and prevent septic shock. However, it has also been demonstrated in treating sepsis and increasing bacterial susceptibility to previously resistant antibiotics (eg, Domenico et al., 2001 Agent. Chemother. 45: 1417-1421; Domenico et al. , 2000 Infect. Med. 17: 123-127; Antimicrob. Agents & Chemother. 3: 79-85, Domenico et al., 2003 Res. Adv .; Domenico et al., 1997 Ant. Microb. Agent. Chemother. 41 (8): 1697-1703; Domenico et al., 1999 Infect. Immun. 67: 664-669; Huang et al. 1999 J Antimicrob. Chemiother 60; Chem., 2003 J Antimicrob. Chemother. 52: 915-919; Wu et al., 2002 Am J Respir Cell Mol Biol. 26: 731-738).

  Despite the availability of BT compounds for well over 10 years, the effective selection of appropriate BT compounds for the indication of a particular infectious disease remains an elusive target, and a specific target for specific microorganisms. The behavior of BT is unpredictable, the synergistic activity of a particular BT and a particular antibiotic against a particular microorganism cannot be predicted, and the effect of BT in vitro usually predicts the effect of BT in vivo. First, the BT effect on plankton (single cell) microbial populations cannot be a predictor of the BT effect on microbial consortia, eg, bacteria that are organized into biofilms. In addition, limitations such as solubility, tissue permeability, bioavailability, and biodistribution may interfere with the ability of some BT compounds to safely and effectively deliver clinical benefit. There is. Embodiments of the invention disclosed herein address these needs and present other related benefits.

Brief Summary Although not first disclosed herein and wishing to be bound by theory, according to certain embodiments described herein, a bismuth-thiol (BT) compound is It can be used as a disinfectant for the treatment of a wide variety of clinically infectious diseases and conditions, as well as in personal health care, such as the savings performed by prevention or protection mediated at least in part by BT, such as It also reduces the cost of treating the infection.

  Also in certain embodiments, a bacterial biofilm or a bacterium associated with biofilm formation (eg, biofilm forming or biofilm forming) comprising one or more BT compounds and one or more antibiotic compounds described herein. Formulations for treating tissues and / or surfaces, including bacteria, which may otherwise be promoted) are contemplated and, according to non-limiting theory, with a BT compound appropriately selected based on the present disclosure. The combination with antibiotics has previously predicted unexpected synergistic effects on the antibacterial (including anti-biofilm) effects of such combinations, and / or the prevention, protection and prevention of microbial infections such as those involving bacterial biofilms. And / or provide an unexpected enhancement effect for therapeutically effective treatment.

  Also, provided herein for the first time is an unprecedented bismuth-thiol composition comprising a substantially monodisperse particulate suspension, and methods of synthesizing and using the same.

According to certain embodiments of the invention described herein, there is thus provided a bismuth-thiol composition comprising a plurality of microparticles comprising a bismuth-thiol (BT) compound, wherein substantially all of the microparticles are provided. Has a volume average diameter of about 0.4 μm to about 5 μm, and the BT compound comprises bismuth or a bismuth salt and a thiol-containing compound. In another embodiment, there is provided a bismuth-thiol composition comprising a plurality of microparticles comprising a bismuth-thiol (BT) compound, wherein substantially all of the microparticles have a volume average diameter of about 0.4 μm to about 5 μm. Hydrophilic, polar or organic, comprising: (a) a bismuth salt with a concentration of at least 50 mM bismuth, under conditions and for a time (a) sufficient to obtain a solution substantially free of solid precipitate. An acidic aqueous solution free of a soluble solubilizer is sufficient to obtain a mixture containing (ii) at least about 5%, 10%, 15%, 20%, 25% or 30% ethanol by volume. In a molar ratio of about 1: 3 to about 3: 1 to bismuth under conditions and for a time sufficient to form a precipitate containing (b) particulates containing the BT compound. The ethanolic solution containing the thiol-containing compounds present in 応溶 solution was added to the blend of (a), to obtain a reaction solution, it is formed by a method comprising. In certain embodiments, the bismuth salt is, Bi _{(NO 3) 3.} In certain embodiments, the acidic aqueous solution comprises at least 5 wt%, 10 wt%, 15 wt%, 20 wt%, 22 wt% or 22.5 wt% bismuth. In certain embodiments, the acidic aqueous solution is at least 0.5% by weight, 1% by weight, 1.5% by weight, 2% by weight, 2.5% by weight, 3% by weight. It contains 3.5%, 4%, 4.5% or 5% by weight nitric acid. In certain embodiments, the thiol-containing compound is 1,2-ethanedithiol, 2,3-dimercaptopropanol, pyrithione, dithioerythritol, 3,4-dimercaptotoluene, 2,3-butanedithiol, 1,3- It contains one or more agents selected from propanedithiol, 2-hydroxypropanethiol, 1-mercapto-2-propanol, α-lipoic acid, and dithiothreitol.

In another embodiment, a method of preparing a bismuth-thiol composition comprising a plurality of microparticles comprising a bismuth-thiol (BT) compound is provided, wherein substantially all of the microparticles are from about 0.4 μm to about 5 μm. (I) a bismuth salt containing bismuth at a concentration of at least 50 mM under conditions and time such that the method has a volume average diameter and (a) is sufficient to obtain a solution substantially free of solid precipitate. (Ii) at least about 5% by volume, 10% by volume, 15% by volume, 20% by volume, 25% by volume or 30% by volume of ethanol, containing an acidic aqueous solution containing no hydrophilic, polar or organic solubilizer. Admixing with ethanol in an amount sufficient to obtain an admixture containing; and (b) conditions and time sufficient to form a precipitate containing particulates containing the BT compound, relative to bismuth. About 1: 3 to about 3: ethanolic solution containing the thiol-containing compound present in the reaction solution in a molar ratio was added to the admixture of (a), comprising the step of obtaining a reaction solution. In certain embodiments, the method further comprises collecting the precipitate to remove impurities. In certain embodiments, the bismuth salt is, Bi _{(NO 3) 3.} In certain embodiments, the acidic aqueous solution comprises at least 5 wt%, 10 wt%, 15 wt%, 20 wt%, 22 wt% or 22.5 wt% bismuth. In certain embodiments, the acidic aqueous solution is at least 0.5% by weight, 1% by weight, 1.5% by weight, 2% by weight, 2.5% by weight, 3% by weight. It contains 3.5%, 4%, 4.5% or 5% by weight nitric acid. In certain embodiments, the thiol-containing compound is 1,2-ethanedithiol, 2,3-dimercaptopropanol, pyrithione, dithioerythritol, 3,4-dimercaptotoluene, 2,3-butanedithiol, 1,3- Propanedithiol, 2-hydroxypropanethiol, 1-mercapto-2-propanol, dithiothreitol, α-lipoic acid, methanethiol (CH ₃ SH [m-mercaptan]), ethanethiol (C ₂ H ₅ SH [e- Mercaptan]), 1-propanethiol (C ₃ H ₇ SH [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]), pentane thiol (C ₅ H ₁₁ SH [pentyl mercaptan]), coenzyme A, lipoamide, glutathione, cysteine, cystine, 2-mercaptoethanol, dithiothreitol, 2-mercaptoindole, Transglutaminase, (11-mercaptoundecyl) hexa (ethylene glycol), (11-mercaptoundecyl) tetra (ethylene glycol), (11-mercaptoundecyl) tetra (ethylene glycol) functionalized gold nanoparticles, 1, 1 ′, 4 ′, 1 ″ -terphenyl-4-thiol, 1,11-undecanedithiol, 1,16-hexadecanedithiol, technical grade 1,2-ethanedithiol, 1,3-propanedithiol, 1,4-benzenedimethane All, 1,4-butanedithiol, 1,4-butanedithiol diacetate, 1,5-pentanedithiol, 1,6-hexanedithiol, 1,8-octanedithiol, 1,9-nonanedithiol, adamantanethiol, 1-butanethiol, 1-decanethiol, 1-dodecanethiol, 1-heptanethiol, 1-heptanethiol plum (purum), 1-hexadecanethiol, 1-hexanethiol, 1-mercapto (triethylene glycol), 1- Mercapto (triethylene glycol) methyl ether functionalized gold nanoparticles, 1-mercapto-2-propanol, 1-nonanethiol, 1-octadecanethiol, 1-octanethiol, 1-pentadecanethiol, 1-pentanethiol, 1- Propanethiol, 1- Tetradecanethiol, 1-tetradecanethiol plum, 1-undecanethiol, 11- (1H-pyrrol-1-yl) undecane-1-thiol, 11-amino-1-undecanethiol hydrochloride, 11-bromo-1-undecanethiol , 11-mercapto-1-undecanol, 11-mercaptoundecanoic acid, 11-mercaptoundecyl trifluoroacetic acid salt, 11-mercaptoundecylphosphoric acid, 12-mercaptododecanoic acid, 15-mercaptopentadecanoic acid, 16-mercaptohexadecanoic acid. 1H, 1H, 2H, 2H-perfluorodecanethiol, 2,2 '-(ethylenedioxy) diethanethiol, 2,3-butanedithiol, 2-butanethiol, 2-ethylhexanethiol, 2-methyl-1 -Propanethiol, 2 -Methyl-2-propanethiol, 2-phenylethanethiol, 3,3,4,4,5,5,6,6,6-nonafluoro-1-hexanethiol plum, 3- (dimethoxymethylsilyl) -1- Propanethiol, 3-chloro-1-propanethiol, 3-mercapto-1-propanol, 3-mercapto-2-butanol, 3-mercapto-N-nonylpropionamide, 3-mercaptopropionic acid, 3-mercaptopropyl functional group Silica gel, 3-methyl-1-butanethiol, 4,4′-bis (mercaptomethyl) biphenyl, 4,4′-dimercaptostilbene, 4- (6-mercaptohexyloxy) benzyl alcohol, 4-cyano-1 -Butanethiol, 4-mercapto-1-butanol, 6- (ferrocenyl) hexaneti , 6-mercapto-1-hexanol, 6-mercaptohexanoic acid, 8-mercapto-1-octanol, 8-mercaptooctanoic acid, 9-mercapto-1-nonanol, biphenyl-4,4′-dithiol, 3- Butyl mercaptopropionate, copper (I) 1-butanethiolate, cyclohexanethiol, cyclopentanethiol, decanethiol functionalized silver nanoparticles, dodecanethiol functionalized gold nanoparticles, dodecanethiol functionalized silver nanoparticles, hexa (Ethylene glycol) mono-11- (acetylthio) undecyl ether, mercaptosuccinic acid, methyl 3-mercaptopropionate, NanoTether BPA-HH, NanoThinks (trademark) 18, NanoThinks (trademark) 8, NanoThinks (trademark). ACID11, NanoThinks (TM) ACID16, NanoThinks (TM) ALCO11, NanoThinks (TM) THIO8, octane thiol functionalized gold nanoparticles, PEG dithiol average _M n 8,000, PEG dithiol average molecular weight 1,500, PEG dithiol average molecular weight 3,400, S- (11-bromoundecyl) thioacetate, S- (4-cyanobutyl) thioacetate, thiophenol, triethylene glycol mono-11-mercaptoundecyl ether, trimethylolpropane tris (3-mercaptopropionate) ), [11- (methylcarbonylthio) undecyl] tetra (ethylene glycol), m-carborane-9-thiol, p-terphenyl-4,4 ″ -dithiol, ter. - dodecyl mercaptan, one or more agents selected from the group consisting of tert- nonyl mercaptan.

  In another embodiment, (i) prevention of natural surface infection by bacterial, fungal or viral pathogens, (ii) inhibition of cell viability or cell development of substantially all planktonic cells of bacterial, fungal or viral pathogens. , (Iii) inhibition of biofilm formation by bacterial, fungal or viral pathogens, and (iv) inhibition of biofilm viability or biofilm development of substantially all biofilm-type cells of bacterial, fungal or viral pathogens, 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 the above, wherein the BT composition comprises a plurality of microparticles comprising a bismuth-thiol (BT) compound. Of the bacterial, fungal and viral pathogens, wherein substantially all of the microparticles have a volume average diameter of about 0.4 μm to about 5 μm. Chino against one or more, a method of protecting natural surface, including biological tissue surface, such as epithelial tissue surface is provided. In certain embodiments, the bacterial pathogen is (i) one or more Gram-negative bacteria; (ii) one or more Gram-positive bacteria; (iii) one or more antibiotic-susceptible bacteria; (iv) one or more. (V) Staphylococcus aureus (S. aureus), MRSA (methicillin-resistant S. aureus), Staphylococcus epidermidis, MRSE (methicillin-resistant S. epidermidis), Mycobacterium tuberculosis Cis, Mycobacterium avium, Pseudomonas aeruginosa, drug resistant P. Aeruginosa, Escherichia coli, enterotoxigenic E. E. coli, enterohemorrhagic E. Koli, Klebsiella pneumoniae, Clostridium difficile, Helicobacter pylori, Legionella pneumophila, Enterococcus faecalis, methicillin-susceptible Enterococcus faecalis, Enterobacter cloacae, Salmonella typhimurium, Proteus vulgaris, and Elsinia enterocoli. , Vibrio cholerae, Shigella flexneri, Vancomycin-resistant Enterococcus (VRE), Burkholderia cepacia, Francisella tularensis, Bacillus anthracis, Yersinia pestis, Pseudomonas aeruginosa, Vancomycin-resistant Enterococcus pneumoniae, Streptococcus, Streptococcus, Streptococcus, Streptococcus, Streptococcus, Streptococcus Resistant Streptococcus pneumoniae, Escherichia Coli, including Burkholderia cepacia, Burkholderia multi borane scan, bacterial pathogens selected from Mycobacterium smegmatis and Acinetobacter Baumani, at least one of. In certain embodiments, the bacterial pathogen is antibiotic resistant. In a particular embodiment, the bacterial pathogen is resistant to an antibiotic selected from methicillin, vancomycin, nafcillin, gentamicin, ampicillin, chloramphenicol, doxycycline and tobramycin.

  In certain embodiments, natural surfaces include buccal / oral surfaces. In a further embodiment, the natural surface comprises a biological surface, such as a bone, joint, muscle, ligament, or tendon.

  In certain embodiments, the surface comprises an epithelial tissue surface comprising a tissue selected from the epidermis, dermis, respiratory tract, gastrointestinal tract and lining of glands.

  In certain embodiments, the contacting step is performed one or more times. In certain embodiments, the at least one contacting step comprises one of spraying, irrigating, dipping and applying the natural surface. In certain embodiments, the at least one contacting step comprises one of inhalation, ingestion and oral irrigation. In certain embodiments, at least one contacting step comprises topical, intraperitoneal, oral, parenteral, intravenous, intraarterial, transdermal, sublingual, subcutaneous, intramuscular, buccal, intranasal, inhalation, ocular. Administered by a route selected from internal, intraaural, intraventricular, intrafatty, intraarticular and intrathecal. In certain embodiments, the BT composition is 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- It contains at least one BT compound selected from the group consisting of propanol and Bis-EDT / 2-hydroxy-1-propanethiol.

  In certain embodiments, the bacterial pathogen is antibiotic resistant. In another particular embodiment, the method comprises the step of contacting the natural surface with a synergizing antibiotic and / or enhancing agent simultaneously or sequentially in any order with respect to the step of contacting the natural surface with the BT composition. The method further includes contacting with an antibiotic. In certain embodiments, the synergistic and / or potentiating antibiotics are aminoglycoside antibiotics, carbapenem antibiotics, cephalosporin antibiotics, fluoroquinolone antibiotics, glycopeptide antibiotics, lincosamide antibiotics. Includes antibiotics selected from antibiotics, penicillinase resistant penicillin antibiotics, and aminopenicillin antibiotics. In certain embodiments, the synergistic and / or potentiating antibiotic is an aminoglycoside antibiotic selected from amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmycin, paromomycin, rhodostreptomycin, streptomycin, tobramycin and apramycin. Is.

  In another embodiment of the invention described herein, (i) prevention of infection by bacterial pathogens of the natural surface where antibiotic resistant pathogens are present, (ii) substantially all plankton of bacterial pathogens. Inhibition of cell viability or cell development of sex cells, (iii) inhibition of biofilm formation by bacterial pathogens, and (iv) biofilm viability or biofilm development of substantially all biofilm-type cells of bacterial pathogens. Inhibiting conditions and time sufficient for one or more of the following: a natural surface on which an antibiotic resistant pathogen is present is treated with an effective amount of (1) at least one bismuth-thiol (BT) composition and ( 2) at least one antibiotic that is potentiated by and / or capable of acting synergistically with at least one BT composition, either simultaneously or sequentially. Wherein the BT composition comprises a plurality of microparticles comprising a bismuth-thiol (BT) compound, wherein substantially all of the microparticles have a volume average diameter of about 0.4 μm to about 5 μm. And thereby overcomes antibiotic resistance on the surface of epithelial tissue, overcomes antibiotic resistance on the natural surface where an antibiotic resistant bacterial pathogen is present (eg having an antibacterial effect against bacteria of the same species) For bacterial pathogens that are resistant to at least one antibacterial effect of at least one antibiotic known in the art). In certain embodiments, the bacterial pathogen is (i) one or more Gram-negative bacteria; (ii) one or more Gram-positive bacteria; (iii) one or more antibiotic-susceptible bacteria; (iv) one or more. (V) Staphylococcus aureus (S. aureus), MRSA (methicillin-resistant S. aureus), Staphylococcus epidermidis, MRSE (methicillin-resistant S. epidermidis), Mycobacterium tuberculosis Cis, Mycobacterium avium, Pseudomonas aeruginosa, drug resistant P. Aeruginosa, Escherichia coli, enterotoxigenic E. E. coli, enterohemorrhagic E. Koli, Klebsiella pneumoniae, Clostridium difficile, Helicobacter pylori, Legionella pneumophila, Enterococcus faecalis, methicillin-susceptible Enterococcus faecalis, Enterobacter cloacae, Salmonella typhimurium, Proteus vulgaris, and Elsinia enterocoli. , Vibrio cholerae, Shigella flexneri, Vancomycin-resistant Enterococcus (VRE), Burkholderia cepacia, Francisella tularensis, Bacillus anthracis, Yersinia pestis, Pseudomonas aeruginosa, Vancomycin-resistant Enterococcus pneumoniae, Streptococcus, Streptococcus, Streptococcus, Streptococcus, Streptococcus, Streptococcus Resistant Streptococcus pneumoniae, Escherichia Coli, including Burkholderia cepacia, Burkholderia multi borane scan, bacterial pathogens selected from Mycobacterium smegmatis and Acinetobacter Baumani, at least one of.

  In a particular embodiment, the bacterial pathogen is resistant to an antibiotic selected from methicillin, vancomycin, nafcillin, gentamicin, ampicillin, chloramphenicol, doxycycline, tobramycin, clindamycin and gatifloxacin.

  In certain embodiments, natural surfaces include buccal / oral surfaces. In a further embodiment, the natural surface comprises a biological surface, such as a bone, joint, muscle, ligament, or tendon.

  In certain embodiments, the surface comprises a tissue selected from the group consisting of epidermis, dermis, respiratory tract, gastrointestinal tract and lining of glands. In certain embodiments, the contacting step is performed one or more times. In certain embodiments, the at least one contacting step comprises one of spraying, irrigating, dipping and applying the surface. In another particular embodiment, the at least one contacting step comprises one of inhalation, ingestion and oral irrigation. In certain embodiments, at least one contacting step comprises topical, intraperitoneal, oral, parenteral, intravenous, intraarterial, transdermal, sublingual, subcutaneous, intramuscular, buccal, intranasal, inhalation, ocular. Administered by a route selected from internal, intraaural, intraventricular, intrafatty, intraarticular and intrathecal. In certain embodiments, the BT composition is 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- It contains at least one BT compound selected from the group consisting of propanol and Bis-EDT / 2-hydroxy-1-propanethiol. In certain embodiments, the synergistic and / or potentiating antibiotics are clindamycin, gatifloxacin, aminoglycoside antibiotics, carbapenem antibiotics, cephalosporin antibiotics, fluoroquinolone antibiotics, It includes an antibiotic selected from glycopeptide antibiotics, lincosamide antibiotics, penicillinase resistant penicillin antibiotics, and aminopenicillin antibiotics. In certain embodiments, the synergistic and / or potentiating antibiotic is an aminoglycoside antibiotic selected from amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmycin, paromomycin, rhodostreptomycin, streptomycin, tobramycin and apramycin. Is.

  In another embodiment, (a) at least one BT compound; (b) at least one antibiotic compound potentiated by and / or capable of acting synergistically with it; and (C) Disinfectant compositions are provided that include a pharmaceutically acceptable excipient or carrier, including a carrier for topical use. In certain embodiments, the BT compound is 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. In certain embodiments, the BT composition comprises a plurality of microparticles comprising a bismuth-thiol (BT) compound, wherein substantially all of the microparticles have a volume average diameter of about 0.4 μm to about 5 μm. In a particular embodiment, the BT compound is selected from BisEDT and BisBAL. In certain embodiments, the antibiotic compound comprises an antibiotic selected from methicillin, vancomycin, nafcillin, gentamicin, ampicillin, chloramphenicol, doxycycline, tobramycin, clindamycin, gatifloxacin and aminoglycoside antibiotics. . In a particular embodiment, the aminoglycoside antibiotic is selected from amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, rhodostreptomycin, streptomycin, tobramycin and apramycin. In certain embodiments, the aminoglycoside antibiotic is amikacin.

  In other particular embodiments, (a) the bacterial infection on or in the surface comprises: (i) Gram-positive bacteria, (ii) Gram-negative bacteria, and (iii) (i) and (ii), A), and (b) administering to the surface a formulation comprising one or more bismuth thiol (BT) compositions, thereby treating the surface. Provided is a method of treating a natural surface supporting or containing a film, wherein (i) when the bacterial infection comprises Gram positive bacteria, the combination comprises at least one BT compound and at least one rifamycin. And (ii) when the bacterial infection comprises Gram-negative bacteria, the combination comprises a therapeutically effective amount of at least one BT compound and amikacin, (Iii) Bacterial infection There comprising When including both Gram-positive and Gram-negative bacteria, compounding agents BT compounds, one or more therapeutically effective amount of a rifamycin and amikacin.

  In certain embodiments, the biofilm comprises one or more antibiotic resistant bacteria. In certain embodiments, treating the surface comprises: (i) eradicating the bacterial biofilm, (ii) reducing the bacterial biofilm, and (iii) suppressing the growth of the bacterial biofilm. At least one of them is included. In certain embodiments, the BT composition comprises a plurality of microparticles comprising a bismuth-thiol (BT) compound, wherein substantially all of the microparticles have a volume average diameter of about 0.4 μm to about 5 μm.

  These and other aspects of the embodiments of the invention described herein will be apparent upon reference to the following detailed description and the accompanying drawings. U. S. RE37, 793, U.S.P. S. 6,248,371, U.S.P. S. 6,086,921, and U.S.P. S. US patents and US patent application publications, US patent applications, foreign patents, foreign patent applications and non-patent issuances referenced herein and / or listed in the application data sheets, including US Pat. No. 6,380,248. All of the articles are incorporated herein by reference in their entirety as if each were individually incorporated. Aspects and embodiments of the invention may be modified as needed and various patents, applications and publication concepts may be used to provide further embodiments.

Brief description of multiple appearances in the drawing
Viable counts (logCFU; colony forming units) of Pseudomonas aeruginosa colony biofilms obtained after growth on 10% trypsin soy agar (TSA) for 24 hours at 37 ° C followed by the indicated treatment for 18 hours. Indicates. The indicated antibiotic treatments are TOB, tobramycin 10xMIC; AMC, amikacin, 100xMIC; IPM, imipenem 10xMIC; CEF, cefepime 10xMIC; CIP, ciprofloxacin, 100xMIC; Cpd 2B. , Compound 2B (Bis-BAL, 1: 1.5). (MIC; minimum inhibitory concentration, eg, the lowest concentration that prevents bacterial growth). Shows viable counts (logCFU) of Staphylococcus aureus colony biofilms obtained after 24 hours of growth on 10% trypsin soy agar followed by the indicated treatment. The indicated antibiotic treatments were rifamycin, RIF 100 × MIC; daptomycin, DAP 320 × MIC; minocycline, MIN 100 × MIC; ampicillin, AMP 10 × MIC; vancomycin, VAN 10 × MIC; Cpd 2B, compound 2B (compound 2B ( Bis-BAL, 1: 1.5), Cpd8-2, compound 8-2 (Bis-Pyr / BDT, 1: 1 / 0.5). Figure 9 shows scratch closure of keratinocytes exposed to biofilm over time. ( ^* ) Significantly different from control (P <0.001). Figure 4 shows the sub-inhibited BisEDT that suppresses antibiotic resistance to multiple antibiotics. Figure 4 shows the effect of antibiotics with or without BisEDT on the lawn of MRSA (methicillin resistant S. aureus). Panel A shows only standard antibiotic soaked discs. [GM = gentamicin, CZ = cefazoline, FEP = cefepime, IPM = imipenem, SAM = ampicillin / sulbactam], LVX = levofloxacin. Figure 4 shows the sub-inhibited BisEDT that suppresses antibiotic resistance to multiple antibiotics. Figure 4 shows the effect of antibiotics with or without BisEDT on the lawn of MRSA (methicillin resistant S. aureus). Panel B shows a disc mixed with BisEDT (BE). [GM = gentamicin, CZ = cefazoline, FEP = cefepime, IPM = imipenem, SAM = ampicillin / sulbactam], LVX = levofloxacin. Figure 3 shows the effect of BisEDT and antibiotics on biofilm formation. S. Epidermidis was grown in TSB + 2% glucose in polystyrene plates at 37 ° C. for 48 hours. Gatifloxacin (GF), Clindamycin (CM), Minocycline (MC), Gentamicin (GM), Vancomycin (VM), Cefazolin (CZ), Nafcillin (NC), and Rifampicin (RP). Results were expressed as mean variability of BPC (2-fold serial dilution step) with 0.25 μM BisEDT (n = 3). S. cerevisiae grown in TSB + 2% glucose for 48 hours at 37 ° C. 3 shows the effect of BisEDT and antibiotics on the development of Epidermidis. Results are expressed as the average percent change of MIC (dilution step) with increasing BisEDT (n = 3). See legend to Figure 5 for antibiotic definitions. Detected in bone and device samples of open fractures in an in vivo rat model with or without cefazolin antibiotic treatment in addition to three BT formulations, Bis-EDT, MB-11 and MB-8-2 S. It is a bar graph which shows an aureus average level. The standard error of the average value is shown as an error bar. Prematurely euthanized animals were not excluded from the analysis, but a sample of one animal in Group 2 was excluded due to significant contamination.

  Certain embodiments of the invention disclosed herein provide that certain bismuth-thiol (BT) compounds provided herein have BT microparticles having a volume average diameter of (about 0.4 μm to about 5 μm. In certain embodiments, including), a strong disinfectant, antibacterial and / or anti-bio agent against certain bacteria, including bacteria associated with multiple clinically significant infections, including infections that may include bacterial biofilms. It is based on the surprising finding that it exhibits film activity but not other specific BT compounds (even if provided as microparticles).

  Contrary to expectations, not all BT compounds are uniformly effective against such bacteria in a predictable manner, but instead show different potencies depending on the target bacterial species. In particular, as described herein, a specific BT compound can be provided by a non-limiting theory that can for the first time provide clinically relevant strategies for the control of bacterial infectious agents, including bacterial biofilm infectious agents. It has been found that (preferably comprising BT microparticles having a volume average diameter of about 0.4 μm to about 5 μm) exhibits greater efficacy against Gram-negative bacteria, but other specific BT compounds (preferably Containing BT microparticles having a volume average diameter of about 0.4 μm to about 5 μm) was found to exhibit greater efficacy against Gram-positive bacteria.

  In addition, as described in more detail below, certain embodiments of the invention described herein have particle sizes (eg, from about 0.4 μm to about 5 μm) as disclosed herein. It relates to the surprising advantage offered by the novel bismuth-thiol (BT) compositions that can be produced in a preparation comprising a plurality of BT microparticles that are substantially monodisperse with respect to (having a volume average diameter). In certain of these and related embodiments, the particulate 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 vesicles.

  Also disclosed herein, for certain embodiments, certain antibiotic antibacterial and anti-biofilms previously found to lack a potent therapeutic effect against such bacterial infections. Efficacy by treating the infectious agent with one or more of these antibiotics and the selected BT compound either cooperatively, simultaneously or sequentially in any order (eg, the site of infection such as a natural surface). It has been discovered that it can be significantly enhanced (eg, increased in a statistically significant manner) by direct application to or on the surface of. In an approach that could not have been predicted prior to the disclosure of the present invention, certain BT compounds have been described herein in combination with certain antibiotics for antimicrobial and / or antibiofilm activity against certain species or strains. The provided synergistic or enhancing combinations can be provided. Certain BT / antibiotics / combinations act synergistically or potentiate against certain bacteria, while other particular BT / antibiotics / combinations have such synergistic or potent antimicrobial and The observation that // failed to exhibit anti-biofilm activity substantiates the unexpected properties of such combinations, which are described in more detail below.

  According to these and related embodiments, the antibiotic and the BT compound may be administered simultaneously or sequentially in either order, and may be administered by a particular infectious agent (eg, formed by Gram-negative or Gram-positive bacteria). Specific synergistic or enhancing combinations of one or more antibiotics disclosed herein with one or more BT compounds for treating a biofilm) are predictable (eg, simply Does not exhibit additive activity and, instead, is unexpectedly synergistic or enhancing (eg, superphase) as a function of the selected antibiotic, the selected BT compound and the specifically identified target bacterium. It is noteworthy that it worked in an additive manner.

  For example, and by way of illustration and not limitation, in the context of a wide variety of actual or potentially microbially infected natural surfaces, and further in the context of improved substantially monodisperse particulate BT formulations. And / or a specific BT compound disclosed in U.S.A. may exert a slight antibacterial effect when used alone against a specific strain or species, The combination of both compound and BT compound is more potent (with statistical significance) on the same strain or species on a larger scale than the simple sum of the effects of each compound when used alone. Anti-BT synergistic effect of BT on the efficacy of antibiotics, and / or of antibiotics on the efficacy of BT, according to non-limiting theory. (E.g., FICI ≦ 0.5) is believed to reflect or enhancing effect (e.g. 0.5 <FICI ≦ 1.0). Therefore, each BT compound is not synergistic or potentiating with respect to each antibiotic, and each antibiotic is not synergistic or potentiating with respect to each BT compound. -BT synergism and BT-antibiotic potentiation are generally not predictable. Instead, according to certain embodiments disclosed herein, certain combinations of synergistic or potentiating antibiotics and BT compounds surprisingly have particular properties such as the natural surfaces described herein. Confers a strong antibacterial effect on specific bacteria, such as an antibacterial effect on biofilms formed by specific bacteria in the environment, and more particularly in specific circumstances.

  That is, certain BT containing antibiotics capable of acting synergistically with at least one BT composition comprising at least one BT compound provided herein (FICI ≦ 0.5). Antibiotics that synergize with are described herein, and such synergistic properties are found in the presence of the antibiotic but not the BT compound, and / or in the presence of the BT compound but not the antibiotic. In some cases, it is manifested as a detectable effect on a larger scale than that which can be detected (ie in a statistically significant manner with respect to the appropriate control conditions). Similarly, certain BT-antibiotic combinations show potentiation (0.5 <FICI <1.0), such potentiation in the presence of antibiotic but no BT compound, and And / or in the presence of the BT compound but no antibiotic, manifests as a detectable effect on a larger scale than that detectable (ie, in a statistically significant manner with respect to the appropriate control conditions).

  Examples of such detectable effects are, in certain embodiments, (i) prevention of infection by bacterial pathogens, (ii) inhibition of cell viability or cell development of substantially all planktonic cells of bacterial pathogens. , (Iii) inhibition of biofilm formation by bacterial pathogens, and (iv) inhibition of biofilm viability or biofilm development of substantially all biofilm-type cells of bacterial pathogens, the invention comprising In other contemplated embodiments, without limitation, antibiotic-BT synergy is one or more other clinically important parameters, such as one or more antibiotics or other drugs or chemistries. Degree of resistance or susceptibility of the bacterial pathogen to the agent, resistance of the bacterial pathogen to one or more chemical, physical or mechanical conditions (eg pH, ionic strength, temperature, pressure) The degree of sex and susceptibility, and / or one or more biological agents (eg, viruses, other bacteria, biologically active polynucleotides, immune cells or immune cell products such as antibodies, cytokines, chemokines, degrading enzymes) One or more detectable that may include a change (eg, a statistically significant increase or decrease) in the degree of resistance or susceptibility of the bacterial pathogen to an enzyme, a membrane-disrupting protein, a free radical, such as a reactive oxygen species, May be exhibited as an effect.

  Antibiotics present when the effect of a synergistic or potentiating antibiotic-BT combination, as provided herein, exceeds the simple sum of the effects observed in the absence of one component of the combination. Those of skill in the art will understand these and various other criteria by which the effect of a particular drug on structure, function and / or activity of a bacterial population may be determined in order to ascertain substance-BT synergism or potentiation. Wax (for example, Coico et al. (Eds.), Current Protocols in Microbiology, 2008, John Wiley & Sons, Hobken, NJ; Schwalbe et al., Antispicus Antibiotic). 007, CRC Press, Boca Raton, FL).

  For example, in certain embodiments, antibacterial effects as described herein are measured using various concentrations of candidate agents (eg, BT and antibiotics alone or in combination) to determine Eliopoulos et al. (Antibiotics). in Laboratory Medicine (Lorian, V., Ed.), pp. 330-96, Williams and Willkins, Baltimore, MD, Eliopoulos and Moellering, iris part (1996) Part of the Eliopoulos and Moellering, iris (b)). Synergy may be determined by calculating the bactericidal concentration index (FBCI). Synergy may be defined as a FICI or FBCI index of 0.5 or less, and antagonism of more than 4 (eg, Odds, FC (2003) Synergy, antagonism, and what the chequerboard pour between thoenthe. Antibiological Chemotherapy 52: 1). Synergy is conventionally defined as a 4-fold or greater reduction in antibiotic concentration, or using the partial inhibitory concentration (FIC), as described, for example, by Hollander et al. (1998 Antimicrob. Agents Chemother. 42: 744). You may. In certain embodiments, synergy is obtained with a combination of two drugs (eg, an antibiotic and a BT composition) and is greater than when the concentration of the second drug is replaced by the first drug ( It may be defined as the effect of the combination (eg, in a statistically significant manner).

  Thus, as described herein, in certain preferred embodiments, the combination of BT and antibiotics will be understood to be synergistic when a FICI value of 0.05 or less is observed (Odds, 2003). As also described herein, in certain other preferred embodiments, and according to non-limiting theory, certain BT-antibiotic combinations represent such a high synergistic potential. It is disclosed that FICI values of 0.5 to 1.0 can be exhibited, the synergistic properties of which are non-optimal of at least one BT and at least one antibiotic showing unilateral or mutually enhanced cooperative antimicrobial efficacy. It may be observed using concentration. Such effects may be referred to herein as "enhancing" antibiotic activity or "enhancing" BT activity.

According to a particular embodiment, (i) a concentration (in a statistically significant manner) lower than the characteristic minimum inhibitory concentration (MIC) of BT for a given target microorganism (eg a given strain or strain). At least one BT of (ii) lower than (ii) a characteristic IC ₅₀ (a concentration that inhibits the development of 50% of the microbial population; eg, Soothill et al., 1992 J Antimicrob Chemother 29 (2): 137). Both in a statistically significant manner) and / or at least one antibiotic at a concentration lower than the biofilm prophylactic concentration (BPC) of the antibiotic for a given target microorganism, either Of an antimicrobial agent (eg, BT or antibiotic) at the same concentration in the absence of the other antimicrobial agent (eg, antibiotic or BT). For anti-microbial effect is, when the result in high (in a statistically significant technique) BT- antibiotic antimicrobial efficacy of substances and combinations, enhancing antibiotics and / or BT activity can be detected. In a preferred embodiment, "enhancing" antibiotic and / or BT activity is present when FICI values below 1.0 and above 0.5 are measured.

  As will be appreciated by those of skill in the art based on the present disclosure, in certain embodiments, the synergistic or potentiating antibiotic and / or BT activity is determined by methods known in the art, eg, to the additive properties of Loewe. -Based models (eg, FIC index, Greco model), or Bliss independence-based models (eg, nonparametric and semiparametric models) or other methods known in the art described herein (eg, Meletiadis et al., 2005 Medical Mycology 43: 133-152). Thus, exemplary methods of measuring synergistic or potentiating antibiotic and / or BT activity are described, for example, in Meletiadis et al. , 2005 Medical Mycology 43: 133-152 and the references cited therein (also Meletiadis et al., 2002 Rev Med Microbial 13: 101-117; White et al. 1914-1918; Mouton et al., 1999 Antimicrob Agents Chemother 43: 2473- 2478).

  Another particular embodiment is that the BT compound and the antibiotic do not show predictable (simply additive) activity in vivo, and instead the selected antibiotic, the selected BT compound, and the infectious agent are selected. Unexpectedly synergistic or enhancing (eg, super-additive; or two or more such agents are present in combination as a function of one or more specifically identified target bacterial species, including , Even when acting in a manner that provides a greater effect (eg, in a statistically significant manner) than that obtained by replacing the concentration of the second drug with the first drug, One or more antibiotics disclosed herein that may exhibit synergistic or potentiating effects in vivo for the treatment of infectious agents, such as biofilms formed by Gram-negative or Gram-positive bacteria. One or more substances It contemplates specific combination of the BT compound. Therefore, according to these and related embodiments, in certain in vivo situations, FICI or FBCI values (as determined in vitro) may not be immediately available, instead the synergistic or potentiating effect of BT-antibiotics It will be appreciated that may be determined in a manner that is derived from a quantifiable measure of infectivity.

  For example, in one embodiment, such as the rat murine femoral critical defect model of in vivo open fracture described in Example 11, observed after treatment with BT-antibiotic combination as compared to antibiotic treatment or BT compound alone. A statistically significant reduction in bacterial count is an indicator of synergistic or enhancing effect. Statistical significance can be determined using methods well known to those of skill in the art. In other particular embodiments, at least 5%, 10%, 20%, 30% of the number of bacteria observed in the lesion after treatment with the BT-antibiotic combination is compared to antibiotic treatment or BT compound alone. A reduction of 40%, or 50%, observed in this or other in vivo models is considered indicative of synergistic or potentiating effect.

  Other exemplary indicators of in vivo infection are established methodologies developed to quantify the severity of infection, such as various wound scoring systems known to those of skill in the art (eg, European Wound Management Association (EWMA), Position Document: Identifying criterion for wound infection. London: See MEP Ltd, 2005 Scoring System). An exemplary wound scoring system that may be used in assessing the synergistic or enhancing activity of the BT-antibiotic combination described herein is ASESIS (Wilson AP, J Hosp Infect 1995; 29 (2). : 81-86; Wilson et al., Lancet 1986; 1: 311-13), the Southampton Wound Assessment Scale (Bailey IS, Karran SE, Toyn K, et al. BM-46: 71-); 304: 1992; . Horan TC, Gaynes P, Martone WJ, et al. , 1992 Infect Control Hosp Epidemiol 1992; 13: 606-08. In addition, recognized clinical indicators of wound healing known to the skilled clinician, such as wound size, depth, granulation tissue status, infections, etc., in the presence or absence of BT compounds and / or antibiotics. You may measure with. Therefore, based on the disclosure of the present invention, to determine whether a BT composition-antibiotic combination alters in vivo wound healing (eg, increases or decreases in a statistically significant manner relative to appropriate controls). Various methods will immediately be appreciated by those skilled in the art.

  In view of these and related embodiments, a microbially infected natural surface, eg, a surface bearing or containing a bacterial biofilm, is provided with one or more BT compounds and optionally one or more antibiotic compounds, eg, the present specification. Treated with an effective amount (eg, in certain embodiments, a therapeutically effective amount) of a composition or combination comprising one or more synergistic antibiotics or one or more potentiating antibiotics provided in the publication. A wide variety of ways to do so are provided herein. Based on the present disclosure, it is understood that certain antibiotics previously considered ineffective by a person of ordinary skill in the art against a given type of infection are contemplated herein for use in treating the same type of infection. I want to.

  Thus, certain embodiments contemplate compositions containing one or more BT compounds for use as antiseptics. An antiseptic is a substance that kills or interferes with the growth of microorganisms and is typically applied to living tissue to distinguish it from fungicides that are normally applied to inanimate subjects. Goodman and Gilman's "The Pharmacologic Basis of Therapeutics", Seventh Edition, Gilman et al., Ed., Ed. Common examples of antiseptics are ethyl alcohol and tincture of iodine. Bactericides include disinfectants that kill microbes such as microbial pathogens.

Certain embodiments described herein include one or more BT compounds and one or more antibiotic compounds (eg, the synergistic and / or potentiating antibiotics provided herein). Compositions that include may be contemplated. Antibiotics are known in the art and are typically drugs produced from compounds that are produced by one species of microorganism to kill another species of microorganism, or the same or similar chemical structure and mechanism of action. A drug produced from a synthetic product having, for example, a drug that destroys microorganisms in or on living organisms when applied topically. In particular, the embodiments disclosed herein are those in which the antibiotic may belong to one of the following classes: aminoglycosides, carbapenems, cephalosporins, fluoroquinolones, glycopeptide antibiotics, lincosamides (eg, , Clindamycin), penicillinase-resistant penicillin, and aminopenicillin. Thus, antibiotics include, but are not limited to, oxacillin, piperacillin, cefuroxime, cefotaxime, cefepime, imipenem, aztreonam, streptomycin, tobramycin, tetracycline, minocycline, ciprofloxacin, levofloxacin, erythromycin, linezolidomycin, fosfomycin, capromycin. , Isoniazid, ansamycin, carbacephem, monobactam, nitrofuran, penicillin, quinolone, sulfonamide, clofazimine, dapson, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin, rifampin, rifabutin, rifampin, streptomycin, streptomycin, streptomycin. Namin, chloramphenicol, phospho Isin, fusidic acid, linezolid, metronidazole, mupirocin, platensimycin, quinupristin, dalfopristin, rifaximin, thiamphenicol, tinidazole, aminoglycoside, β-lactam, penicillin, cephalosporins, carbapenems, fluroquinolones, ketolides, lincosamides, macrolides. Oxazolidinone, streptogramin, sulfonamide, tetracycline, glycylcycline, methicillin, vancomycin, nafcillin, gentamicin, ampicillin, chloramphenicol, doxycycline, tobramycin, amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmycin, paromomycin, rhodomycin. Streptomycin, tobrama Mention may be made of isin, apramycin, clindamycin, gatigloxacin, aminopenicillin, and others known in the art. Introduction of these and other clinically useful antibiotics are available and known to those skilled in 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 PA, Philadel.

  Exemplary classes of antibiotics for use with one or more BT compounds in certain disclosed embodiments are described by Edson RS, Terrell CL. The aminoglycosides. Mayo Clin Proc. 1999 May; 74 (5): 519-28, which is an aminoglycoside class of antibiotics. This class of antibiotics inhibits bacterial growth by damaging bacterial protein synthesis through binding and inactivation of bacterial ribosomal subunits. In addition to such bacteriostatic properties, aminoglycosides also show a bactericidal effect through the disruption of the cell wall of Gram-negative bacteria.

  Aminoglycoside antibiotics include gentamicin, amikacin, streptomycin, etc., which are generally considered useful for the treatment of Gram-negative bacteria, mycobacteria and other microbial pathogens, although a classification of resistant strains has been reported. Aminoglycosides are not absorbed through the digestive tract and are therefore not generally considered applicable to oral formulations. Amikacin, for example, is often effective against gentamicin-resistant strains, but is typically administered intravenously or intramuscularly and can induce pain in patients. In addition, the toxicity associated with aminoglycoside antibiotics such as amikacin can lead to kidney damage and / or irreversible hearing loss.

  Despite these properties, certain embodiments disclosed herein are synergistic, for example, for treating epithelial tissue surfaces at one or more locations along the oral cavity, gastrointestinal / gastrointestinal tract. Oral administration of BT / antibiotic combinations is contemplated (eg, antibiotics need not be limited to aminoglycosides). Also contemplated in other particular embodiments are herein described as fungicides, which refer to preparations that kill or block the growth of outer surface microorganisms of inanimate subjects. The use of the provided compositions and methods.

  As also described elsewhere in this specification, BT compounds can be prepared according to Domenico et al. , 1997 Antimicrob. Agent. Chemother. 41 (8): 1697-1703, Domenico et al. , 2001 Antimicrob. Agent. Chemother. 45 (5): 1417-1421, and U.S.P. S RE37,793, U.S.S. S. No. 6,248,371, No. 6,086,921, and No. 6,380,248 (see also US Pat. No. 6,582,719) (including preparation methods). It may also be a composition comprising a bismuth or bismuth salt and a thiol (eg, -SH, or sulfhydryl) containing compound. However, particular embodiments are not so limited and may contemplate other BT compounds including bismuth or bismuth salts and thiol-containing compounds. The thiol-containing compound may contain 1, 2, 3, 4, 5, 6 or more thiol (eg, -SH) groups. In a preferred embodiment, the BT compound comprises bismuth associated with a thiol-containing compound via an ionic bond and / or as a coordination complex, although in some other embodiments the bismuth is in an organometallic compound. May be associated with the thiol-containing compound via a covalent bond, as can be found in. However, certain contemplated embodiments explicitly exclude BT compounds, where the organometallic compound is a compound in which bismuth is found in a covalent linkage to the organic moiety.

＊ 硫黄含有化合物と三価錯体を形成するために用いられる反応体の化学量論比およびビスマスの公知の傾向に基づき、比較のために単一ビスマス原子に対する原子比を示す。示された原子比は、所定の調製物中の全ての化学種の正確な分子式でない場合がある。カッコ内の数字は、１種（またはそれを超える）チオール剤に対するビスマスの比である（例えば、Ｂｉ：チオール１／チオール２）。「ＣＰＤ」：化合物。 Exemplary BT compounds are shown in Table 1.

* Based on the stoichiometric ratio of the reactants used to form the trivalent complex with the sulfur-containing compound and the known tendency of bismuth, the atomic ratio to a single bismuth atom is shown for comparison. The atomic ratios shown may not be the exact molecular formula for all species in a given preparation. Numbers in parentheses are ratios of bismuth to one (or more) thiol agent (eg, Bi: thiol 1 / thiol 2). "CPD": Compound.

  The BT compounds used in certain of the embodiments disclosed herein may be prepared according to established procedures (eg, US RE 37,793, US 6,248,371, 6,086, 921, and No. 6,380,248; Domenico et al., 1997 Antimicrob. Agent. Chemother. 41 (8): 1697-1703, Domenico et al., 2001 Antimicrob. Agent 5: Chem. 1417-1421), and in other particular embodiments the BT compound may be prepared according to the methodologies described herein. Thus, certain preferred embodiments include conditions and times sufficient to form a precipitate that includes particulates that include a BT compound (e.g., concentrations described in this specification and understood by one of skill in the art based on this disclosure, solvent). Conditions such as strength, temperature, pH, mixing and / or pressure), dissolved bismuth 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 An aqueous solution of acidic bismuth containing 600 mM, at least 700 mM, at least 800 mM, at least 900 mM, or at least 1 M and free of hydrophilic, polar or organic solubilizer is mixed with ethanol to prepare the first ethanol. A solution is obtained and reacted with a second ethanolic solution containing a thiol-containing compound present in the reaction solution in a molar ratio to bismuth of about 1: 3 to about 3: 1 to obtain a reaction solution, BT Contemplated are the synthetic methods described herein for preparing the compounds, particularly for obtaining the BT compounds in substantially monodisperse, particulate form.

  Thus, as an exemplary BT, Compound 1B-1, Bis-EDT (1: 1 bismuth-1,2-ethanedithiol reactant); Compound 1B-2, Bis-EDT (1: 1.5); Compound 1B -3, Bis-EDT (1: 1.5); Compound 1C, Bis-EDT (soluble Bi preparation, 1: 1.5); Compound 2A, Bis-Bal (bismuth-UK anti-lewisite (bismuth-di). Mercaprol, bismuth-2,3-dimercaptopropanol), 1: 1); Compound 2B, Bis-Bal (1: 1.5); Compound 3A Bis-Pyr (bismuth pyrithione, 1: 1.5); Compound 3B, Bis-Pyr (1: 3); Compound 4, Bis-Ery (bismuth dithioerythritol, 1: 1.5); Compound 5, Bis-Tol (bismuth- 3,4-dimercaptotoluene, 1: 1.5); Compound 6, Bis-BDT (bismuth-2,3-butanediol, 1: 1.5); Compound 7, Bis-PDT (bismuth-1,3) -Propanedithiol, 1: 1.5); Compound 8-1 Bis-Pyr / BDT (1: 1/1); Compound 8-2, Bis-Pyr / BDT (1: 1 / 0.5); Compound 9 , Bis-2-hydroxy, propanethiol (bismuth-1-mercapto-2-propanol, 1: 3); Compound 10, Bis-Pyr / Bal (1: 1 / 0.5); Compound 11, Bis-Pyr / EDT (1: 1 / 0.5); Compound 12 Bis-Pyr / Tol (1: 1 / 0.5); Compound 13, Bis-Pyr / PDT (1: 1 / 0.5); Compound 14 Bis- Pyr / ry (1: 1 / 0.5); compound 15, Bis-EDT / 2- hydroxy propane thiol (1: 1/1) and the like (e.g., see Table 1).

  While not wishing to be bound by theory, in certain preferred embodiments, it may include preparing or obtaining an acidic aqueous liquid solution containing bismuth, such as an aqueous nitric acid solution containing bismuth nitrate, BT. The methods of the present disclosure for preparing compounds may, if desired, be facilitated by mass production, improved product purity, homogeneity or consistency (including particle size uniformity), or topical formulations of the present invention. It is believed that a composition comprising a BT compound can be obtained that has one or more desired properties, including other properties useful in the preparation and / or administration of

  In certain embodiments, the BT compositions prepared by the method described herein for the first time each have a volume average diameter (VMD) of about 0.4 μm to about 5 μm, according to certain presently preferred embodiments. It has been discovered that it exhibits advantageous homogeneity in relation to their appearance as substantially monodisperse particulate suspensions. The measured particle size can be referred to as the volume mean diameter (VMD), mass median diameter (MMD), or aerodynamic mass median diameter (MMAD). These measurements may be performed, for example, by impaction (MMD and MMAD) or laser (VMD) characterization. In the case of liquid particles, VMD, MMD and MMAD can be the same, provided that environmental conditions such as normal humidity are maintained. However, if humidity is not maintained, MMD and MMAD measurements will be smaller than VMD due to dehydration during impactor measurements. For purposes of this specification, VMD, MMD and MMAD measurements are considered to be under standard conditions, so the description of VMD, MMD and MMAD would be equivalent. Similarly, dry powder particle size measurements on MMD and MMAD are considered equivalent.

  Preferred embodiments, as described herein, relate to a substantially monodisperse suspension of BT-containing microparticles. Producing a defined BT particle size with a small geometric standard deviation (GSD) produces, for example, BT attachment, accessibility to desired target sites within or on the natural surface, and / or BT microparticles. Tolerability may be optimized depending on the subject receiving The narrow GSD limits the number of particles outside the desired VMD or MMAD size range.

  In one embodiment, a particulate liquid or aerosol suspension having a VMD of about 0.5 microns to about 5 microns is provided that contains one or more BT compounds disclosed herein. In another embodiment, a liquid or aerosol suspension having a VMD or MMAD of about 0.7 micron to about 4.0 micron is provided. In another embodiment, a liquid or aerosol suspension having a VMD or MMAD of about 1.0 micron to about 3.0 microns is provided. In other particular embodiments, the VMD is from about 0.1 to about 5.0 microns, or 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 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 as described herein. Provided is a liquid suspension containing one or more BT compound particles prepared as described above.

  Thus, in certain preferred embodiments, BT preparations first described herein that are “substantially” monodisperse, eg, “substantially” all of the microparticles, are within a specific range (eg, about 0). A BT composition comprising a particulate form of a BT compound having a volume average diameter (VMD) of 0.4 .mu.m to about 5 .mu.m) is at least 80%, 85%, 90%, 91%, 92%, 93%, or 94% of the particles. %, More preferably at least 95%, 96%, 97%, 98%, 99%, or more, having those compositions having a VMD within the quoted size range.

  These and related properties of BT compositions prepared by the synthetic methods described herein allow characterization in a manner that facilitates regulatory compliance in accordance with one or more pharmaceutical, prescription and cosmeceutical standards. Presents unprecedented advantages over previously described BTs, such as lower manufacturing cost and ease of manufacture, and homogeneity within the composition.

  Additionally or alternatively, the substantially monodisperse BT microparticles described herein advantageously do not require micronization, ie expensive and labor intensive milling or supercritical fluid processing, or microparticles. May be produced without the use of other equipment and procedures typically used to produce (eg, 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. 1-13). Thus, embodiments of the present invention include, without limitation, enhanced substantially homogeneous solubilizing properties, suitability for a desired dosage form, such as oral, inhalation or topical form for dermatological / skin wounds, It presents the beneficial effects of a substantially homogeneous particulate preparation, including a high degree of bioavailability and other beneficial properties.

  Particulate suspensions of BT compounds can be, for example, wetting agents, surfactants, mineral oils, or other ingredients or additives that will be known to those skilled in the art of formulation, for holding the individual microparticles in suspension. As an aqueous formulation, such as a formulation containing, as a suspension or solution in an aqueous solvent and an organic solvent such as a halogenated hydrocarbon propellant, as a dry powder, or in other forms detailed below. be able to. Aqueous formulations may be aerosolized with a liquid nebulizer using, for example, either hydraulic or ultrasonic atomization. Propellant-based systems may use suitable pressure dispensers. As the dry powder, a dry powder dispersion device capable of effectively dispersing the BT-containing fine particles may be used. The desired particle size and distribution may be obtained by selecting the appropriate device.

  As also mentioned above, also provided herein by certain embodiments is a method of preparing a BT composition comprising a plurality of microparticles comprising a bismuth-thiol (BT) compound, such as Substantially all of the fine particles have a volume average diameter (VMD) of about 0.1 to about 8 microns, and in certain preferred embodiments about 0.4 microns to about 5 microns.

  In general conditions, the method comprises: (a) a bismuth salt containing bismuth at a concentration of at least 50 mM for a time and for a time sufficient to obtain a solution substantially free of solid precipitate; An acidic aqueous solution free of organic, polar or organic solubilizers (ii) admixed with a sufficient amount of ethanol to obtain at least about 5% by volume, 10% by volume, 15% by volume, 20% by volume, 25% by volume. Or obtaining a blend containing 30% by volume, preferably about 25% by volume of ethanol; and (b) about 1: 3 to bismuth under conditions and for a time sufficient to form a precipitate containing the BT compound. Adding an ethanolic solution containing a thiol-containing compound present in the reaction solution in a molar ratio of about 3: 1 to the mixture of (a) to obtain a reaction solution.

In certain preferred embodiments, the bismuth salts may be Bi (NO _{3) 3,} but the bismuth may be provided in other forms may be understood by the present disclosure. In certain embodiments, the bismuth concentration in the acidic aqueous solution is 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 It may be at least 1M. In certain embodiments, the acidic aqueous solution comprises at least 5 wt%, 10 wt%, 15 wt%, 20 wt%, 22 wt% or 22.5 wt% bismuth. The acidic aqueous solution may comprise at least 5% by weight or more nitric acid in certain preferred embodiments, and in other specific embodiments the acidic aqueous solution is at least 0.5% by weight, at least 1% by weight. At least 1.5% by weight, at least 2% by weight, at least 2.5% by weight, at least 3% by weight, at least 3.5% by weight, at least 4% by weight, at least 4.5% by weight or at least 5% by weight nitric acid. May be included.

The thiol-containing compound may be any thiol-containing compound described herein, and in certain embodiments, 1,2-ethanedithiol, 2,3-dimercaptopropanol, pyrithione, dithioerythritol, Contains at least one of 3,4-dimercaptotoluene, 2,3-butanedithiol, 1,3-propanedithiol, 2-hydroxypropanethiol, 1-mercapto-2-propanol, and dithiothreitol. Good. Other exemplary thiol-containing compounds include α-lipoic acid, methanethiol (CH ₃ SH [m-mercaptan]), ethanethiol (C ₂ H ₅ SH [e-mercaptan]), 1-propanethiol (C ₃ H ₇ SH [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]), pentane thiol (C ₅ H ₁₁ SH [pentyl mercaptan]), coenzyme A, lipoamide, glutathione, cysteine, cystine, 2-mercaptoethanol, dithiothrey. Thor, dithioerythritol, 2-mercaptoindole, trans Luminase and any of the following thiol compounds available from Sigma-Aldrich (St. Louis, Mo.) include: (11-mercaptoundecyl) hexa (ethylene glycol), (11-mercaptoundecyl) tetra (ethylene glycol). , (11-Mercaptoundecyl) tetra (ethylene glycol) functionalized gold nanoparticles, 1,1 ′, 4 ′, 1 ″ -terphenyl-4-thiol, 1,11-undecanedithiol, 1,16- Hexadecanedithiol, technical grade 1,2-ethanedithiol, 1,3-propanedithiol, 1,4-benzenedimethanethiol, 1,4-butanedithiol, 1,4-butanedithiol diacetate, 1, 5-pentanedithiol, 1,6-hexanedithiol, 1,8 Octanedithiol, 1,9-nonanedithiol, adamantanethiol, 1-butanethiol, 1-decanethiol, 1-dodecanethiol, 1-heptanethiol, 1-heptanethiol plum, 1-hexadecanethiol, 1-hexanethiol, 1 -Mercapto (triethylene glycol), 1-mercapto (triethylene glycol) methyl ether functionalized gold nanoparticles, 1-mercapto-2-propanol, 1-nonanethiol, 1-octadecanethiol, 1-octanethiol, 1- Pentadecanethiol, 1-pentanethiol, 1-propanethiol, 1-tetradecanethiol, 1-tetradecanethiol plum, 1-undecanethiol, 11- (1H-pyrrol-1-yl) undecane-1-thiol, 11-ami No-1-undecanethiol hydrochloride, 11-bromo-1-undecanethiol, 11-mercapto-1-undecanol, 11-mercaptoundecanoic acid, 11-mercaptoundecyltrifluoroacetate, 11-mercaptoundecylphosphate, 12-mercaptododecanoic acid, 15-mercaptopentadecanoic acid, 16-mercaptohexadecanoic acid, 1H, 1H, 2H, 2H-perfluorodecanethiol, 2,2 '-(ethylenedioxy) diethanethiol, 2,3-butanedithiol , 2-butanethiol, 2-ethylhexanethiol, 2-methyl-1-propanethiol, 2-methyl-2-propanethiol, 2-phenylethanethiol, 3,3,4,4,5,5,6,6. 6,6-Nonafluoro-1-hexanethiol plum, 3- (di (Methoxymethylsilyl) -1-propanethiol, 3-chloro-1-propanethiol, 3-mercapto-1-propanol, 3-mercapto-2-butanol, 3-mercapto-N-nonylpropionamide, 3-mercaptopropionic acid , 3-mercaptopropyl functionalized silica gel, 3-methyl-1-butanethiol, 4,4'-bis (mercaptomethyl) biphenyl, 4,4'-dimercaptostilbene, 4- (6-mercaptohexyloxy) benzyl. Alcohol, 4-cyano-1-butanethiol, 4-mercapto-1-butanol, 6- (ferrocenyl) hexanethiol, 6-mercapto-1-hexanol, 6-mercaptohexanoic acid, 8-mercapto-1-octanol, 8 -Mercaptooctanoic acid, 9-mercapto-1 Nonanol, biphenyl-4,4'-dithiol, butyl 3-mercaptopropionate, copper (I) 1-butanethiolate, cyclohexanethiol, cyclopentanethiol, decanethiol functionalized silver nanoparticles, dodecanethiol functionalized gold Nanoparticles, dodecanethiol functionalized silver nanoparticles, hexa (ethylene glycol) mono-11- (acetylthio) undecyl ether, mercaptosuccinic acid, methyl 3-mercaptopropionate, NanoTether BPA-HH, NanoThink (TM) 18, NanoThinks (trademark) 8, NanoThinks (trademark) ACID11, NanoThinks (trademark) ACID16, NanoThinks (trademark) ALCO11, NanoThinks (trademark) THIO8, octane All functionalized gold nanoparticles, PEG dithiol average _M n 8,000, PEG dithiol average molecular weight 1,500, PEG dithiol average molecular weight 3,400, S- (11- bromo undecyl) thioacetate, S- (4-cyanobutyl ) Thioacetate, thiophenol, triethylene glycol mono-11-mercaptoundecyl ether, trimethylolpropane tris (3-mercaptopropionate), [11- (methylcarbonylthio) undecyl] tetra (ethylene glycol), m-carborane -9-thiol, p-terphenyl-4,4 ''-dithiol, tert-dodecyl mercaptan and tert-nonyl mercaptan.

  Exemplary reaction conditions, such as temperature, pH, reaction time, the use of stirring or agitation to dissolve the solute, and procedures for collecting and washing the precipitate are described herein and are generally described in the art. A known technique is used.

  Unlike the previously described methodologies for making BT compounds, according to the method of the present invention for preparing BT, the BT product is from about 0.4 micron to about about 0.4 micron in certain preferred embodiments. 5 micron, and according to another particular embodiment, is provided as a microparticle suspension with substantially all microparticles having a VMD of generally about 0.1 micron to about 8 microns. Further unlike previous approaches, according to embodiments of the present invention, the bismuth is at a concentration of at least about 50 mM to about 1 M bismuth salt, and at least about 0.5% (w / w) to about 5% (w). / W), preferably provided in an acidic aqueous solution containing nitric acid in an amount of less than 5% (weight / weight) and free of hydrophilic, polar or organic solubilizers.

  In this regard, the method of the present invention provides that bismuth is not water soluble at 50 μM (eg, US RE 37793) and that bismuth is unstable in water (eg, Kuvshinova et al., 2009 Russ. J. Chem. 54 (11): 1816), surprising and expected given the generally recognized art that bismuth becomes unstable in aqueous nitric acid unless a hydrophilic, polar or organic solubilizer is present. Present outside interests. For example, in all of the most authoritative descriptions of BT preparation methodologies (eg, Domenico et al., 1997 Antimicrob. Agents. Chemother. 41: 1697; U.S. 6,380,248; U.S. RE 37,793. No. US Pat. No. 6,248,371), the hydrophilic solubilizer propylene glycol is required to dissolve bismuth nitrate, and the bismuth concentration of the solution prepared for reaction with thiols is 15 mM. Well below, which limits the available modes of production of BT compounds.

  In contrast, the present disclosure does not require hydrophilic, polar or organic solubilizers to dissolve bismuth, and surprisingly higher concentrations are achieved. Hydrophilic, polar or organic solubilizers include propylene glycol (PG) and ethylene glycol (EG), polar solvents such as dioxane and dimethylene sulfoxide (DMSO), polyols (eg polyethylene glycol (PEG), polypropylene. It may include any of a number of known solubility enhancers, including glycols (PPG), including pentaerythritol), polyhydric alcohols such as glycerol and mannitol, and other agents. Other highly polar, water-miscible organic substances include dimethyl sulfoxide (DMSO), dimethylformamide (DMF) and NMP (N-methyl-2-pyrrolidone).

  Thus, solvents such as those commonly used as hydrophilic, polar or organic solubilizers provided herein include, for example, Catalan et al. (Eg, 1995 Liebigs Ann. 241; Handbook of Solvent, Wypych (Ed.), (See also Catalan, 2001 in Andrew Publ., NY, and references cited therein), which may be selected based on solvent polarity / polarizability (SPP) scale values. It will be understood by those skilled in the art that, for example, water has an SPP value of 0.962, toluene has an SPP value of 0.655, and 2-propanol has an SPP value of 0.848. A method for determining the SPP value of a solvent based on the UV measurement of a 2-N, N-dimethyl-7-nitrofluorene / 2-fluoro-7-nitrofluorene probe / homomorph pair has been described ( Catalan et al., 1995).

  Solvents having either the desired SPP value based on the solubility characteristics of the particular BT composition (either as a pure single component solvent or a solvent mixture of 2, 3, 4, or more solvents; solvent blends As for sex, see, for example, Godfrey 1972 Chem. Technology. According to a particularly preferred embodiment of the method step, no hydrophilic, polar or organic solubilizer is required to dissolve the bismuth.

  The solubility parameter may include the interaction parameter C, the Hildebrand solubility parameter d, or the partial (Hansen) solubility parameter: δp, δh and δd, respectively, which represent solvent polarity, hydrogen bonding capacity and dispersive force interaction capacity. . In certain embodiments, the highest value of the solubility parameter that describes the solvent or co-solvent system in which the bismuth salt, including bismuth, dissolves is determined by, for example, the method of preparing the particulate BT composition described herein. A limit on the aqueous solution to include may be provided. For example, higher δh values have greater hydrogen bonding capacity and therefore greater affinity for solvent molecules such as water. Therefore, in situations where a more hydrophilic environment is desired, higher values of the maximum observed solvent δh may be preferred.

By way of non-limiting example, BisEDT having the structure shown in Formula I below may be prepared by the following reaction scheme:

Briefly, by way of non-limiting example, an excess of 5% aqueous HNO ₃ (11.4 L) at room temperature is added to a solution of Bi (NO ₃ ) ₃ (eg 43% Bi (NO ₃ ) ₃ (w / w ) 5% nitric acid (w / w), 52% water (w / w), 0.331 L (about 0.575 mol) of an acidic bismuth aqueous solution such as Shepherd Chemical Co., Cincinnati, OH). Absolute ethanol (4 L) may be slowly added in addition. Separately, 72.19 mL (0.863 mol) of 1,2-ethanedithiol was added to 1.5 L of absolute ethanol using a 60 mL syringe, and then stirred for 5 minutes to prepare 1,2-ethanedithiol [~ 0. An ethanolic solution (1.56 L) of a thiol compound such as .55M] may be prepared. 1,2-ethanedithiol (CAS 540-63-6) and other thiol compounds are described, for example, in Sigma-Aldrich, St. Available from Louis, MO. Then, the ethanolic solution of the thiol compound was slowly added to the aqueous _{Bi (NO 3) 3 / HNO} 3 solution, stirred overnight, the reaction solution may be to form. The thiol-containing compound may be present in the reaction solution in a molar ratio to bismuth of from about 1: 3 to about 3: 1, according to certain preferred embodiments. The product formed was precipitated as a precipitate containing the microparticles described herein, then collected by filtration and washed successively with ethanol, water and acetone to give BisEDT as a yellow amorphous powder solid. obtain. The crude product may be redissolved in absolute alcohol with stirring, then filtered and washed successively with ethanol several times and then acetone several times. The washed powder may be triturated in 1 M NaOH (500 mL), filtered and washed subsequently with water, ethanol and acetone to give purified particulate BisEDT.

  According to a non-limiting theory, bismuth inhibits the ability of bacteria to produce extracellular polymeric substances (EPS), such as extracellular polysaccharides, which leads to impaired biofilm formation. Bacteria are believed to use glue-like EPS for biofilm adhesion. Depending on the nature of the infectious agent, biofilm formation and EPS assimilation may contribute to bacterial pathogenicity such as impeding wound healing. However, bismuth alone is not therapeutically useful as an intervention agent and is instead typically administered as part of a complex such as BT. Thus bismuth-thiol (BT) is a family of compositions containing compounds that result from chelation of bismuth with thiol compounds and that show dramatic improvements in the antibacterial therapeutic efficacy of bismuth. BT exhibits significant anti-infective, anti-biofilm and immunomodulatory effects. Bismuth-thiol is effective against a wide range of microorganisms and is typically unaffected by antibiotic resistance. BT can prevent biofilm formation at significantly lower (sub-inhibitory) concentrations, prevent many pathogenic features of common wound pathogens at the same sub-inhibitory level, and prevent septic shock in animal models, It can be synergistic with many antibiotics.

  As described herein, such synergy in the antibacterial effect of one or more specific BT when combined with one or more specific antibiotic compound is due to the distinct antibiotics for a particular bacterial type. And cannot be immediately predicted based on the profile of the BT effect, but surprisingly it is not possible to detect specific Gram-negative or Gram-positive bacteria (or both) in consideration of specific bacterial populations, such as confirmation of the presence. It may be obtained by selecting a BT-antibiotic / combination. For example, as disclosed herein, certain BT synergistic antibiotics include amikacin, ampicillin, aztreonam, cefazolin, cefepime, chloramphenicol, ciprofloxacin, clindamycin (or other lincosamide antibiotics. Substance), daptomycin (Cubicin®), doxycycline, gatifloxacin, gentamicin, imipenim, levofloxacin, linezolid (Zyvox®), minocycline, nafcillin, paromomycin, rifampin, sulfamethoxazole, tetracycline, tobracyclin, tobramycin, tobramycin. And one or more of vancomycin. MRSAs that have little or no individual susceptibility to, for example, gentamicin, cefazoline, cefepime, sphamethoxazole, imipenim or levofloxacin are exposed to these antibiotics in the presence of the BT compound BisEDT. In vitro studies have shown to show significant sensitivity to one of these. Thus, specific embodiments contemplated herein include BT compounds and amikacin, ampicillin, cefazoline, cefepime, chloramphenicol, ciprofloxacin, clindamycin (or other lincosamide antibiotics), daptomycin. (Cubicin®), doxycycline, gatifloxacin, gentamicin, imipenim, levofloxacin, linezolid (Zyvox®), minocycline, nafcillin, paromomycin, rifampin, sulfamethoxazole, tobramycin and vancomycin. Compositions and / or methods that may include combinations with one or more antibiotics are explicitly contemplated, and other specific embodiments contemplated herein include BT compounds and amikacin, Picillin, aztreonam, cefazolin, cefepime, chloramphenicol, ciprofloxacin, clindamycin (or other lincosamides), daptomycin (Cubicin®), doxycycline, gatifloxacin, gentamicin, imipenim, levofloxacin, One or more antibiotics that can be explicitly excluded from one or more antibiotics selected from linezolid (Zyvox®), minocycline, nafcillin, paromomycin, rifampin, sulfamethoxazole, tobramycin and vancomycin; Compositions and / or methods that may include combinations of Note here that gentamicin and tobramycin belong to the aminoglycosides of antibiotics. Also explicitly excluded from certain contemplated embodiments are Domenico et al. , 2001 Agent. Chemother. 45 (5): 1417-1421; Domenico et al. , 2000 Infect. Med. 17: 123-127; Antimicrob. Agents & Chemother. 3: 79-85, Domenico et al. , 2003 Res. Adv. Domenico et al. , 1997 Antimicrob. Agents Chemother. 41 (8): 1697-1703; Domenico et al. , 1999 Infect. Immun. 67: 664-669; Huang et al. 1999 J Antimicrob. Chemother. 44: 601-605; Veloira et al. , 2003 J Antimicrob. Chemother. 52: 915-919; Wu et al. , 2002 Am J Respir Cell Mol Biol. 26: 731-738; Halwani et al. , 2008 Int. J Pharmaceut. 358: 278; Halwani et al. , 2009 Int. J. Pharmaceut. 373: 141-146, and it is noted that none of these publications teach or suggest the monodisperse particulate BT compositions disclosed herein. I want to.

  Thus, as described herein, in certain preferred embodiments, the particulate BTs described herein are included, and optionally, in other specific embodiments, also synergistic and / or potentiating antibiotics. Compositions comprising and compositions and methods for treating a subject are provided. Those of ordinary skill in the relevant art will recognize, based on the disclosure of the present invention, the appropriate clinical aspects and circumstances in which such treatment may be desirable, the criteria for which are, inter alia, surgical, military surgical. , Dermatological, traumatic, geriatric, cardiovascular, metabolic disorders (eg diabetes, obesity, etc.), infections and inflammations (epithelial lining of respiratory or gastrointestinal tract, or other such as glandular tissue) (E.g., epithelial tissue surface), and related medical specialties and subspecialty areas, and other medical technology areas.

  Therefore, in certain embodiments disclosed herein and known in the art, promoting skin tissue repair (or repair of other tissues such as epithelial tissue, bone, joints, muscles, tendons, or ligaments). Is contemplated. In certain embodiments, promoting skin tissue or other epithelial tissue repair includes (i) migration of epithelial cells (eg, keratinocytes) or dermal fibroblasts, (ii) epithelial cells (eg, keratinocytes). Or development of dermal fibroblasts, (iii) down-regulation of collagenase, gelatinase or matrix metalloproteinase activity of epithelial cells (eg keratinocytes) or dermal fibroblasts, (iv) extracellular matrix of dermal fibroblasts It may comprise stimulating or derepressing one or more intracellular wound repair activities selected from protein deposition, and (v) induction or enhancement of dermal angiogenesis. Methodologies for identifying and characterizing such intracellular wound repair activity have been described and therefore include the wound tissue repair promoting compounds disclosed herein, eg, the BT agents described herein. The effect of the composition on these and related activities can be determined immediately based on this disclosure and without undue experimentation. For example, disclosed herein are compositions and methods relating to art-accepted models for wound repair based on keratinocyte wound closure following scratches.

  Preferred compositions for treating microbial infections on or in natural surfaces used in accordance with the embodiments described herein include, in certain embodiments, bismuth-thiol (BT) compounds described herein. The composition may comprise a compound comprising other compounds known in the art, such as one or more antibiotic compounds described herein, in different but related specific embodiments. Good. BT compounds and methods of making them are disclosed herein, for example, in Domenico et al. (1997 Antimicrob. Agent. Chemother. 41 (8): 1697-1703; 2001 Antimicrob. Agent. Chemother. 45 (5): 1417-1421) and U.S.S.N. S RE37,793, U.S.S. S. 6,248,371, 6,086,921, and 6,380,248. As also mentioned above, certain preferred BT compounds are those containing bismuth or bismuth salts ionically bound to or in coordination complexes with thiol-containing compounds, such as bismuth chelated to thiol-containing compounds. Other specific preferred BT compounds are those containing bismuth or a bismuth salt in a covalent bond to a thiol-containing compound. Also preferred are the substantially monodisperse particulate BT compositions described herein. Any of the compounds described herein for the first time having use in the compositions and methods for past treatment of bacterial infections or for facilitating the treatment of natural surfaces described herein. The characterization in other aspects also did not predict that the methods of the invention using such compounds would have the beneficial effects described herein.

Thus, according to a preferred embodiment, there is provided a method of treating a natural surface, comprising administering to the natural surface at least one particulate BT compound as described herein. In certain embodiments, the method can result in the synergistic antibiotics described herein in certain preferred embodiments, and in other particular embodiments described herein. It further comprises administering at least one antibiotic compound, which may be a potentiating antibiotic, either simultaneously or sequentially in any order. Antibiotic compounds include aminoglycoside antibiotics, carbapenem antibiotics, cephalosporin antibiotics, fluoroquinolone antibiotics, glycopeptide antibiotics, lincosamide antibiotics, penicillinase resistant penicillin antibiotics, or aminopenicillin antibiotics. It may be an antibiotic. Clinically useful antibiotics are discussed elsewhere herein, for example, 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.

  As described herein, in certain embodiments, in the case of bacterial infections including Gram-positive bacteria, a preferred therapeutically effective combination is a BT compound (eg, BisEDT, bismuth; 1,2- Ethanedithiol; BisPyr, bismuth: pyrithione; BisEDT / Pyr, bismuth: 1,2-ethanedithiol / pyrithione) and rifamycin, or BT compound and daptomycin (Cubicin®, Cubist Pharmaceuticals, Lxington, MA), or BT. Compounds and linezolid (Zyvox®, Pfizer, Inc., NY, NY), or BT compounds (eg, BisEDT, bismuth; 1,2-ethanedithiol; BisPyr, bismuth: pyrithione; BisEDT / P r, bismuth: 1,2-ethanedithiol / pyrithione) and ampicillin, cefazoline, cefepime, chloramphenicol, clindamycin (or other lincosamide antibiotics), daptomycin (Cubicin®), doxycycline, gatifur. To the unexpected discovery that it could include one or more of: xacin, gentamicin, imipenim, levofloxacin, linezolid (Zyvox®), nafcillin, paromomycin, rifampin, sulfamethoxazole, tobramycin and vancomycin. Comes from.

  As also described herein, certain embodiments provide the unexpected benefit that in the case of bacterial infections including Gram-negative bacteria, a preferred therapeutically effective combination may include a BT compound and amikacin. Originating from discovery. Certain related embodiments contemplate treating an infectious agent, including Gram-negative bacteria, with a BT compound and another antibiotic, eg, another aminoglycoside antibiotic that is not gentamicin or tobramycin in certain embodiments. Thus, in view of these embodiments, other related embodiments are of the art of medical methodology as a step of selecting appropriate antibiotic compounds for inclusion in the formulations administered by the methods of the present invention. It is contemplated to identify one or more bacterial populations or subpopulations within or on a natural surface by well-known criteria that are Gram positive or Gram negative, by methodologies well known to those of skill in the art.

  The compositions and methods described herein typically utilize compounds described herein (eg, with only one or more particulate BT alone, or one or more synergistic agents disclosed herein). And / or in combination with potentiating antibiotics), applied to or administered to microbial sites, such as the presence of microorganisms on or within a natural surface, to provide microorganisms in various aspects (eg, bacteria, viruses, yeasts, molds and It may find application in the treatment of other fungi, microbial parasites, etc.). Such natural surfaces include, but are not limited to, mammalian tissues (eg, skin, scalp, lining of the gastrointestinal tract, epithelium such as the oral cavity; endothelium, cells and tissue membranes such as peritoneum, pericardium, periosteum, periosteum, marrow). Membranes, muscle fibrous membranes, etc .; corneas, sclera, mucous membranes, and other mammalian tissues such as teeth, bones, joints, tendons, ligaments, muscles, hearts, lungs, kidneys, liver, spleen, gallbladder, pancreas, Bladder, nerves, etc.).

  The particulate antimicrobial agents described herein are for inhibiting microbial growth, for reducing microbial parasitics, for reducing biofilms, for preventing bacterial to biofilm conversion, and for microbial infections. May be used for prevention or inhibition of, and for any of the other uses described herein. These agents have multiple antiviral purposes, including the prevention or inhibition of viral infections by herpes virus, such as cytomegalovirus, herpes simplex type 1, and herpes simplex type 2, and / or infection by other viruses. Is also useful. In this connection, the agents are different viruses, such as single-stranded RNA viruses, single-stranded DNA viruses, Rous sarcoma virus (RSA), hepatitis A virus, hepatitis B virus (HBV), hepatitis C. Virus (HCV), Influenza virus, West Nile virus (WNV), Epstein-Barr virus (EBV), Eastern equine encephalitis virus (EEEV), Severe acute respiratory virus (SARS), Human immunodeficiency virus (HIV), Human papilloma It is useful for the prevention or inhibition of viral infections by viruses (HPV) and human T-cell lymphoma virus (HTLV).

  Other internal and topical pharmaceutical uses of the antimicrobial agents described herein include, but are not limited to, bacterial infections, tuberculosis, fungal infections such as yeast and mold infections (e.g., Candida (e.g., Candida). Genus (eg, Candida albicans, Candida glabrata, C. parapsilosis, C. tropicalis, and C. dubriniensis) or cryptococcus or other fungi), treatment of Helicobacter pylori infection, and peptic ulcer disease or Prevention includes, in one embodiment, the agent at a dose that is not generally lethal to bacteria, yet is sufficient to reduce the protective polysaccharide coating that otherwise resists the natural immune response. Thus, this technique is used for human symbiotic microorganisms (eg, normal intestinal flora) Without damage to the extent that can be a case of using antibiotics is believed to assist in eradication of bacterial infection through the immune system.

  By way of example, and not limitation, specific contemplated embodiments are described herein.

  In certain embodiments, a particulate BT compound (or composition comprising a particulate BT compound) described herein is used to control biofilm development, disrupt biofilm, or reduce the amount of biofilm. May be combined with at least one or more anti-biofilm agents. As understood in the art, interspecies quorum sensing is involved in biofilm formation. Certain agents that increase LuxS-dependent pathways or interspecific quorum sensing signals (see, eg, US Pat. No. 7,427,408) contribute to the control of biofilm development and / or proliferation. Exemplary agents include, for example, N- (3-oxododecanoyl) -L-homoserine lactone (OdDHL) blocking compounds and N-butyryl-L-homoserine lactone (BHL) analogs, either in combination or separately. (See, eg, US Pat. No. 6,455,031). An oral hygiene composition comprising a particulate BT compound and at least one anti-biofilm agent can be delivered locally for disruption and inhibition of bacterial biofilms and for the treatment of periodontal disease (eg, US See Japanese Patent No. 6,726,898).

Compositions Comprising Particulate Bismuth-Thiol, and Uses for Oral Hygiene and for Treating Oral Inflammation and Infections In another embodiment, compositions comprising particulate BT compounds are formulated for oral use, It may be used in a method for preventing or reducing microbial growth in the mouth and for preventing and / or treating microbial infections and inflammation of the oral cavity. Therefore, these compositions are intended to prevent or treat plaque, halitosis, periodontal disease, gingivitis, and other infections of the mouth (ie, to reduce or prevent their onset, and to prevent their occurrence or recurrence). Useful (to reduce the likelihood). Oral compositions comprising particulate BT compounds prevent and / or control (ie, slow, delay, inhibit) biofilm development, disrupt biofilms, or oral surfaces, particularly teeth or It can also be useful in reducing the amount of biofilm present on the gums.

  Entrapped food particles, poor oral hygiene and poor oral health, and inadequate cleaning of full dentures can promote microbial development on the teeth, around the gums, and on the tongue. The continued development of microorganisms and the presence of caries can result in halitosis, plaque (ie, biofilm formed by microbial colonization), gingivitis, and inflammation. Without proper oral care (eg, brushing, flossing), more severe infections, such as periodontal disease and jaw infection, can result.

  Good oral hygiene is important not only for oral health but also for the prevention of multiple chronic conditions. Controlling bacterial growth in the mouth can help reduce the risk of heart disease, maintain memory, and reduce the risk of infection and inflammation in other areas of the body. People with diabetes are at greater risk of developing severe gum problems and maintaining good oral health to reduce the risk of gingivitis can help control blood glucose levels. Pregnant women are more likely to suffer from gingivitis and several studies have suggested a link between gum disease in pregnant women and delivery of preterm, low birth weight infants.

  Bacteria are the major etiological agents in periodontal disease. Over 500 strains may be found in dental plaque (Kroes et al., Proc. Natl. Acad. Sci. USA 96: 14547-52 (1999)). Bacteria have evolved to survive as biofilms on tooth surfaces, gingival epithelium, and oral environment, which makes periodontitis treatment difficult. The bactericides and antibiotics currently used to treat such infections often do not kill all of the problem organisms. The use of drugs that are ineffective against certain strains can result in the growth of resistant strains. Moreover, these agents may induce unwanted side effects, such allergic reactions, inflammation, and tooth discoloration.

  Bacterial plaque on teeth is a biofilm that adheres firmly to tooth surfaces, restorations and prostheses. The main means of controlling biofilm in the mouth is by mechanical cleaning (ie, brushing, flossing, etc.). If not subjected to such a wash, within 2 days thereafter, the tooth surface is predominantly stained by facultative Gram-positive cocci, which is predominantly Streptococcus. Bacteria secrete an extracellular mucus layer that helps the bacteria fix to the surface and protects the attached bacteria. Microcolony formation begins when the tooth surface is covered with attached bacteria. Biofilms grow primarily by cell division of adherent bacteria rather than attachment of new bacteria. The doubling time of plaque formed by bacteria is rapid in early development and slow in more mature biofilms.

  Co-aggregation occurs when the engrafting bacteria continue to adhere to bacteria that have already adhered to the pellicle. The result of coaggregation is the formation of complex arrays of different bacteria linked together. A few days after uninterrupted plaque formation, the gingival margin becomes inflamed and swollen. Inflammation may form deep gingival sulcus. The biofilm spreads to this subgingival region and proliferates in this protected environment, forming a mature subgingival plaque biofilm. Gingival inflammation is not observed until the biofilm changes from predominantly Gram-positive to Gram-negative anaerobic bacteria. Subgingival bacterial microcolonies, composed primarily of Gram-negative anaerobic bacteria, become established in the gingival sulcus within 3-12 weeks after the initiation of plaque formation on the gingival margin. Currently, most of the bacterial species suspected of having periodontal pathogens are anaerobic Gram-negative bacteria.

  Bacterial microcolonies protected within biofilms are typically resistant to antibiotics (systemic administration), antiseptics or fungicides (topical administration), and immune defense. For example, the antibiotic dose that kills airborne bacteria should be increased by a factor of 1500 over the biofilm killing dose. At this high concentration, these antimicrobial agents tend to be toxic to patients as well (see, eg, Coghlan 1996, New Scientist 2045: 32-6; Elder et al., 1995, Eye 9: 102-9). .

  Careful and frequent physical removal of bacterial plaque biofilms is the most effective means of eliminating and controlling plaque. However, subgingival plaque in the pocket cannot be reached by brush, floss, or mouth rinse. Therefore, frequent periodontal debridement of the subgingival root surface by a dental hygienist or dentist is an essential element in the prevention and treatment of periodontitis.

  In certain embodiments, the particulate BT compound is an oral hygiene composition that can be routinely used by any subject, such as, but not limited to, toothpaste, mouthwash (i.e., mouthrinse), oral gel, toothpaste, oral cavity. It may be incorporated into a spray (such as a spray dispersed by an inhaler), an edible film, a chewing gum, a mouth slurry, a complete denture cleansing solution, a complete denture preservation solution, and dental floss. Particulate BT compounds may be incorporated into oral hygiene compositions primarily used by dental care professionals including, for example, liquid fluorine treatments, cleaning compositions, buffing compositions, mouth rinses, and dental floss. Embodiments of the present invention replace antimicrobial agents formulated with oral hygiene compositions described in the art with the particulate BT compounds described herein to provide antimicrobial activity, solubility and bioavailability. It is contemplated to provide the benefits disclosed herein, including range of degrees, anti-biofilm effect, non-toxicity, enhanced antibiotic efficacy, as well as other properties described herein.

  The particulate BT compound is for preventing or treating caries and / or inflammation by administering the particulate BT compound to the surface of the tooth (ie, to reduce the likelihood of developing or recurring caries and / or inflammation, respectively). May be used. The composition comprising the particulate BT compound may be a mucoadhesive composition applied to the surface of teeth and / or gums or oral mucosa, in any form that adheres to some degree, or in a pharmaceutically effective amount. It may be in any form that delivers the active ingredient of (1) to the desired surface. The particulate BT compound may also be formulated to be slowly released from the composition applied to the tooth. For example, the composition may be a gel (eg, hydrogel, thiomer, aerogel, or organogel) or a liquid. The organogel may include organic solvents, lipoic acid, vegetable oils, or mineral oils. Such gel or liquid coating formulations may be applied to the interior or exterior of the amalgam or composite or other restorative composition. The sustained release composition provides a pharmaceutically effective amount of the particulate BT compound for 1, 2, 3, 4, 5, 6, or 7 days (1 week), or 2, 3, 4, 5, 6, 7 It may be delivered weekly or for 1, 2, 3, 4, 5, or 6 months. Such compositions can be prepared by one of ordinary skill in the art using any number of methods known in the art.

  In other particular embodiments, an antimicrobial composition comprising a particulate BT compound and one or more additional antimicrobial compounds or agents, as described herein, is provided for oral use. Particularly useful are compositions comprising a second antimicrobial agent which, when administered in combination, have a potentiating or synergistic antimicrobial effect as described herein. By way of example, a potentiating antimicrobial effect may be observed when the particulate BT compound is administered with an iron chelating antimicrobial agent. In other particular embodiments, the particulate BT compound is formulated with an anti-inflammatory agent, compound, small molecule, or macromolecule (eg, peptide or polypeptide).

  Any of the particulate BT compounds described herein may be formulated for oral use. In certain embodiments, a particulate BT compound prepared with a hydrophobic thiol (eg, thiochlorophenol) may be used, which has a greater ability to adhere to tooth and mouth tissues than less hydrophobic BT compounds. It may be shown large. BT compounds with a net negative charge, such as those with a molar ratio of 1: 2 (bismuth to thiol), may have suitable adhesion properties.

  Oral hygiene compositions containing the particulate BT compound may further comprise one or more active ingredients and / or one or more orally suitable excipients or carriers. In one embodiment, the oral hygiene composition may further comprise baking soda or other alkaline compound or substance. Due to the chemical and physical properties of baking soda, it has a wide range of applications including cleaning, deodorizing, and buffering. Baking soda chemically neutralizes rather than masks or absorbs odors. Baking soda can be combined with the particulate BT compound as a powder mixture or can be dissolved or suspended in any one of the toothpastes, gels, pastes and liquids described herein. In other embodiments, the particulate BT compound is combined with other alkali metal bicarbonate or carbonate materials (eg, potassium bicarbonate or calcium carbonate) that help maintain the desired alkaline pH and also have cleaning and deodorizing properties. be able to.

Oral hygiene compositions containing particulate BT compounds may further include one or more of the following ingredients.
Antimicrobial agents: for example, chlorhexidine: sanguinarine extract, metronidazole, quaternary ammonium compounds (for example cetylpyridinium chloride); bisguanides (for example chlorhexidine digluconate, hexetidine, octenidine, alexidine); halogenated bisphenol compounds (for example , 2,2'-Methylenebis- (4-chloro-6-bromophenol) or other phenolic antibacterial compounds; alkyl hydroxybenzoates; cationic antimicrobial peptides; aminoglycosides; quinolones; lincosamides; penicillins: cephalosporins; macrolides Tetracycline; other antibiotics known in the art; Coleus forskoli essential oils; silver or silver colloid antimicrobials; tin or copper based Antimicrobial agents; manuka oil; oregano; thyme; rosemary; or other herbal extracts; and grapefruit seed extract.
Anti-inflammatory or antioxidant: for example, ibuprofen, flurbiprofen, aspirin, indomethacin, aloe vera, turmeric, olive leaf extract, clove, panthenol, retinol, ω-3 fatty acid, γ-linolenic acid (GLA), green tea. , Ginger, grape seed, etc. Anti-cavity agents: for example sodium and stannous fluoride, amine fluorides, sodium monofluorophosphate, sodium trimetaphosphate, zinc citrate or other zinc agents, and casein. Plaque buffer: for example, urea, calcium lactate, calcium glycerophosphate, and strontium polyacrylate. Vitamins: For example, vitamins A, C and E. Plant extract. Desensitizing agents: For example, potassium citrate, potassium chloride, potassium tartrate, potassium bicarbonate, potassium oxalate, potassium nitrate, and strontium salts. Anticalculus agents: for example, alkali metal pyrophosphates, hypophosphite-containing polymers, organic phosphonates and phosphocitrates. Biomolecule: For example, bacteriocin, bacteriophage, antibody, enzyme, etc. Flavoring agents: for example peppermint and spearmint oil, fennel, cinnamon and the like. Proteinaceous material: eg collagen. Preservative. Opacifier. Colorant. pH adjuster. Sweetener. Pharmaceutically acceptable carriers such as starch, sucrose, water or water / alcohol systems. Surfactants: for example, anionic, nonionic, cationic and zwitterionic or amphoteric surfactants, saponins derived from plant materials (see, for example, US Pat. No. 6,485,711). Particle abrasives: for example, silica, alumina, calcium carbonate, dicalcium phosphate, calcium pyrophosphate, hydroxyapatite, trimetaphosphate, insoluble hexametaphosphate, agglomerated particle abrasives, chalk, finely ground natural chalk and the like. Hygroscopic agents: for example, glycerol, sorbitol, propylene glycol, xylitol, lactitol and the like. Binders and thickeners: eg sodium carboxymethylcellulose, hydroxyethylcellulose (Natrosol®), xanthan gum, gum arabic, synthetic polymers (eg polyacrylates and carboxyvinyl polymers, eg Carbopol®). Polymeric compounds that enhance the delivery of active ingredients such as antimicrobial agents. Buffers and salts that buffer the pH and ionic strength of the oral care composition. Bleach: For example, a peroxy compound (eg potassium peroxydiphosphate). Firing system: For example, sodium bicarbonate / citric acid system. Discoloration system. In certain embodiments, the abrasive is silica or finely ground natural chalk.

  An oral hygiene composition comprising a particulate BT compound formulated for use as a toothpaste further comprises a hygroscopic agent (eg glycerol or sorbitol), a surfactant, a binder, and / or a flavoring agent. Good. Toothpastes may contain sweetening agents, whitening agents, preservatives and antimicrobial agents. The pH of compositions for toothpaste and other oral uses is typically pH 5.5-8.5. In certain embodiments, the oral hygiene compositions, including toothpaste, have a pH of 7-7.5, 7.5-8, 8-8.5, or 8.5-9 and have a particulate BT. The antimicrobial activity of the compound can be enhanced. The toothpaste compositions described herein include chalk, dicalcium phosphate dihydrate, sorbitol, water, hydrated aluminum oxide, precipitated silica, sodium lauryl sulfate, sodium carboxymethyl cellulose, flavoring agents, monoolein. Acid sorbitan, sodium saccharin, tetrasodium pyrophosphate, methylparaben, propylparaben. If desired, one or more colorants such as FD & C Blue can be used. Other suitable ingredients that may be included in toothpaste formulations are described in the art, eg, in US Pat. No. 5,560,517.

  In one particular embodiment, the oral hygiene composition is a mouth spray and comprises particulate BT compound, an alkaline buffer (eg potassium bicarbonate), alcohol, a sweetening ingredient, and a flavoring system. The flavoring system may have one or more of the following: flavoring agents, hygroscopic agents, surfactants, sweeteners, and colorants (see, eg, US Pat. No. 6,579,513). ). Surfactants described herein and known in the art for use in oral hygiene compositions may be anionic, nonionic or amphoteric.

  In another embodiment, oral hygiene compositions containing particulate BT have been described in the art to be useful for inclusion in toothpastes, tooth gels, mouthwashes, and the like to treat severe infections. It may also be combined with further active ingredients such as taurolidine and taurultam (see for example British Patent Application No. GB 1557163, US Pat. No. 6,488,912). As described herein, the particulate BT can also be combined with one or more additional antimicrobial agents, which when combined with the particulate BT, the combination has an additive or synergistic effect.

  In yet another specific embodiment, the oral hygiene compositions described herein are at least one for controlling biofilm production, disrupting biofilm, or reducing the amount of biofilm. Or more anti-biofilm agents may be further included. As understood in the art, interspecies quorum sensing is involved in biofilm formation. Certain agents that increase LuxS-dependent pathways or interspecific quorum sensing signals (see, eg, US Pat. No. 7,427,408) contribute to the control of biofilm development and / or proliferation. Exemplary agents include, for example, N- (3-oxododecanoyl) -L-homoserine lactone (OdDHL) blocking compounds and N-butyryl-L-homoserine lactone (BHL) analogs, either in combination or separately. (See, eg, US Pat. No. 6,455,031). An oral hygiene composition comprising a particulate BT compound and at least one anti-biofilm agent can be delivered locally for disruption and inhibition of bacterial biofilms, and treatment of periodontal disease (eg, US See Japanese Patent No. 6,726,898).

  The oral hygiene compositions described herein also contain a sufficient amount of particulate BT compound to perform a substantial antimicrobial effect in the time required for normal tooth brushing, mouth rinse, or flossing. Good. As described herein, the particulate BT compound may be retained on the oral surface (eg, teeth, amalgams, composites, mucous membranes, gums). For example, the particulate BT compound retained on the teeth and gums after completion of brushing, rinsing, flossing may continue to provide long term anti-biofilm and anti-inflammatory effects.

  In another embodiment, the particulate BT compound is slowly released from a mucoadhesive polymer or other agent that contributes to retention of the particulate BT compound on the mucosal or tooth surface. The particulate BT compound may be added to a stable viscous mucoadhesive aqueous composition for the prevention and treatment of mucosal ulcerative, inflammatory and / or erosive disorders, and / or topical treatment. It may be used for delivery of pharmaceutically active compounds to mucosal surfaces or transfer into the systemic circulation (see, eg, US Pat. No. 7,547,433).

  In another embodiment, the oral hygiene composition comprising the particulate BT compound further comprises olive oil, which may enhance plaque removal. The use of olive oil in products intended for oral hygiene, such as toothpaste, mouthwash, spray, buccal inhalers, or chewing gum, eliminates or reduces (reduces) bacterial plaque and / or It contributes to the elimination or reduction (reduction) of the number of bacteria present, which can lead to a reduction of tooth disease (eg caries, periodontal disease) and halitosis (eg US Pat. No. 7,074). , 391).

  In other embodiments, the oral hygiene composition comprising the particulate BT compound may further comprise a mucosal germicidal preparation for topical application in the mouth. The oral hygiene composition may further comprise an aqueous slurry useful for cleaning the tongue and throat (eg US Pat. No. 6,861,049). In yet another embodiment, the oral hygiene composition comprising the particulate BT compound is used at least to prevent the formation of (cavities) cavities (ie, reduce the likelihood of development) or reduce the number of cavities. It may further contain one mint. One such mint, called CaviStat® (Ortek Therapeutics, Inc., Roslyn Heights, NY), contains arginine and calcium to help neutralize acidic pH and to attach calcium to the enamel surface. Promote. Inclusion of mint in an oral hygiene composition comprising particulate BT compound can thus raise the pH and enhance adhesion of particulate BT compound to the oral surface.

Compositions Comprising Particulate Bismuth-Thiol Formulated for Orthopedic Use In certain embodiments, orthopedic procedures (eg, orthopedic surgery, orthopedic treatment, arthroplasty (including two-stage arthroplasty). ), Orthodontic treatment), and a method of using a composition comprising a particulate BT compound for preventing and / or treating microbial infections and inflammation. Compositions comprising the particulate BT compounds described herein therefore prevent and / or treat microbial infections of the skeletal and supporting structures (ie bones, joints, muscles, ligaments, tendons), such as osteomyelitis. (Ie, reduce or prevent its onset, reduce the likelihood of its onset or recurrence). The compositions described herein that include a particulate BT compound prevent and / or control (ie, slow, delay, inhibit) the development of biofilms, disrupt biofilms, or intra-articularly, or It may also be useful in reducing the amount of biofilm present on the surface of bones, ligaments, tendons or teeth.

  The compositions described herein for orthopedic use that include a particulate BT compound may further include one or more additional antimicrobial compounds or agents. Particularly useful are compositions comprising a particulate BT compound and a second antimicrobial agent that, when administered in combination, have the potentiating or synergistic antimicrobial effect described herein. As an additional example, enhanced antimicrobial effects can be observed when the particulate BT compound is administered with an iron-chelating antimicrobial agent. In other particular embodiments, the particulate BT compound is formulated with an anti-inflammatory agent, compound, small molecule, or macromolecule (eg, peptide or polypeptide).

  The composition comprising the particulate BT compound, when administered in combination, has at least one other antimicrobial agent (ie, a second, that has a potentiating or synergistic antimicrobial effect (ie, greater than an additive effect). Third, fourth, etc. antimicrobial agents). By way of example, enhanced microbial effects can be observed when the particulate BT compound is administered with an iron-chelating antimicrobial agent. In certain embodiments, the composition comprising the particulate BT compound may be combined with at least one other antimicrobial and / or anti-inflammatory agent selected from: antimicrobial agent: eg chlorhexidine. : Sanguinarine extract, metronidazole, quaternary ammonium compound (for example, cetylpyridinium chloride); Bisguanide (for example, chlorhexidine digluconate, hexetidine, octenidine, alexidine); halogenated bisphenol compound (for example, 2,2'-methylenebis) -(4-chloro-6-bromophenol) or other phenolic antibacterial compounds; alkyl hydroxybenzoates; cationic antimicrobial peptides; aminoglycosides; quinolones; lincosamides; penicillins: cephalosporins; macros Tetracycline; other antibiotics known in the art; essential oils of Coleus forskoli; silver or silver colloid antimicrobial agents; tin or copper based antimicrobial agents; manuka oil; oregano; thyme; rosemary Or other herbal extracts; and grapefruit seed extracts Anti-inflammatory or anti-oxidants: eg ibuprofen, flurbiprofen, aspirin, indomethacin, aloe vera, turmeric, olive leaf extract, cloves, panthenol, retinol, ω-. 3 fatty acids, γ-linolenic acid (GLA), green tea, ginger, grape seed, etc. In certain embodiments, the composition comprising the particulate BT compound is clindamycin, vancomycin, daptomycin, cefazoline, gentamicin, tobucin. Antibiotics selected from mycin, metronidazole, cefaclor, ciprofloxacin, or other antimicrobial agents such as quaternary ammonium compounds (eg benzalkonium chloride, cetylpyridinium chloride), antimicrobial zeolites, alkalis The composition may further comprise a metal hydroxide, or an alkaline earth metal oxide, the composition of which comprises one or more pharmaceutically suitable carriers (ie, excipients) described herein. Surfactants, buffers, diluents, and salts, and bleaching agents may optionally be included, and thus these and certain of the related embodiments disclosed herein include one or more microparticles. BT, and optionally further antibiotics such as the synergistic or potentiating antibiotics described herein. Particulate BT compositions disclosed herein contemplates the inclusion and processes to such products.

Biological, Biomedical, and Other Uses of Particulate BT Other specific embodiments include BT alone, or bismuth moieties substituted with different Group V metals, such as antimony (Sb) or arsenic (As). BT, and / or, as described herein, such BT in combination with one or more antibiotics in which BT exhibits synergistic or enhanced antimicrobial activity in an orally ingested nutritional formulation. Any of the following are contemplated for use of the particulate BT described herein.

  Non-limiting theory involves vitamins, minerals, amino acids, hydrocarbons including carbohydrates, fatty acids, oils, phytonutrients, tea, herbs or herbal extracts, and / or other ingredients such as other nutritional or food products. The inclusion of particulate BT in such formulations may, in certain embodiments, increase the bioavailability of BT and optionally antibiotics and / or additional nutritional components to the digestive tract of the host ( For example, an approach that promotes (in a statistically significant manner relative to an appropriate control) can result in a block or delay of nutrient uptake by a microbial population in the gastrointestinal tract. In certain other embodiments, certain vitamins, minerals, amino acids, carbohydrate-containing hydrocarbons, fatty acids, oils, phytonutrients, teas, herbs or herbal extracts contained in oral particulate BT (or AsT or SbT) formulations. And / or altering other nutritional or food products may reduce the bioavailability of BT and optionally antibiotics and / or additional nutritional components to the digestive tract of the host ( (Eg, in a statistically significant manner relative to appropriate controls).

  For example, in the presence of a pathological gastrointestinal (GI) tract infection, the BT compound may be retained to retain its bioavailability within the GI tract in order to exert an antimicrobial effect against infectious pathogens. It may be desirable to administer a particulate BT formulation that interferes with intestinal absorption. Those of ordinary skill in the art are aware of multiple vitamins, minerals, amino acids, hydrocarbons including carbohydrates, fatty acids, oils, phytonutrients, teas, herbs and / or herbal extracts that promote or interfere with GI tract absorption of nutrients. A formulation that increases or decreases the presence of one or more components of the GI tract (eg, a particulate BT compound, an antibiotic, or one or more specific nutrients) is provided with a particulate BT (or It can be prepared using AsT or SbT).

  Other particular embodiments provided herein are in compositions for oral delivery to reduce the odor of faeces or digestive gases, or in the presence of local microbes, for example in patients undergoing colostomy. It is contemplated that the armchairs associated with the invention include particulate BT compounds disclosed herein in other topical delivery compositions to reduce foot or other body odor. Multiple skin and GI tract microbial populations, including planktonic and biofilm bacteria, when present with the enhancing or synergistic antibiotics described herein, the particulate BT described herein. It is sensitive to low concentrations of such particulate BT compounds, including compounds.

  Thus, certain embodiments reduce the population of GI or skin resident bacteria in a manner that reduces or alleviates the problem of inappropriate odor (eg, statistically significant relative to appropriate controls). Microparticle BT formulations for oral and topical delivery are contemplated. Oral and topical pharmaceutical formulations are described below, and these and related embodiments provide advantages associated with the particulate BT formulations of the present invention, such as compatible bioavailability and solubility characteristics, and Other factors that present low toxicity and may influence the selection of antimicrobial compositions are described elsewhere in this specification, eg, US Pat. S. It can also be found in 6,582,719.

  Application of the exemplary BT compound, BisEDT, to the axillary region in human test subjects (50 μL of a 1 mg / mL solution in DMSO) was shown to neutralize body odor for 2-3 days. The BisEDT mixture in talc powder applied to human tested paws substantially reduced leg odor. Experimental mice orally ingested 1 mg / kg BisEDT twice a day for 5 days showed a 90% reduction in fecal flora counts. Any odor-breathing thiol-containing solution (eg, salmon), including the particulate BT preparations described herein, which is produced with excess bismuth and can be added as a deodorant to the thiol-containing solution. Commonly useful deodorants for fish oils (such as oils of) are also contemplated. The resulting mixture retains the antimicrobial properties of particulate BT. Other contemplated applications include solvents such as oils or butters of other biological sources, such as hemp oil, tea tree oil, shea butter, linseed oil, fish oil, and in certain embodiments, Mention may be made of oils which may have independent or synergistic anti-inflammatory and / or pain reducing properties and / or other beneficial physiological effects.

Pharmaceutical Compositions and Administration Certain embodiments also relate to pharmaceutical compositions containing a particulate BT compound disclosed herein, in certain such embodiments, the pharmaceutical composition comprises a BT compound herein. It may further comprise one or more antibiotics, such as those antibiotics that exhibit synergistic or potentiating effects as described in the text. In one embodiment, one or more such particulate BT compounds are pharmaceutically acceptable as disclosed herein when administered to an animal, preferably a mammal, most preferably a human patient. Compositions containing a pharmaceutical amount in a carrier, excipient or diluent are provided.

  Administration of the particulate BT compound or a pharmaceutically acceptable salt, either in pure form or in a suitable pharmaceutical composition, is carried out via any of the accepted modes of administration for agents that serve similar purposes. You can The pharmaceutical composition can be prepared by combining the particulate BT compound with a suitable pharmaceutically acceptable carrier, diluent or excipient, and tablets, capsules, powders, granules, ointments, solutions, suppositories, The preparations may be formulated in solid, semi-solid, liquid or gaseous forms such as injectable solutions, inhalants, gels, microspheres and aerosols. Typical routes of administering such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, rectal, vaginal, and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrathecal injections or infusion techniques. The pharmaceutical composition is formulated such that the active ingredients contained therein will be bioavailable upon administration of the composition to a patient. The composition to be administered to a subject or patient is in the form of one or more dosage units, for example a tablet may be a single dosage unit and a container of the compound in aerosol form may be a multiple dosage unit. May be held. The actual methods for preparing such dosage forms are known or apparent to those of skill in the art, see, for example, The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and 2000). I want to. The composition to be administered will, in any event, contain a pharmaceutically effective amount of the compound, or pharmaceutically acceptable salt thereof, for the treatment of the disease or condition of interest in accordance with the teachings herein.

  The pharmaceutical compositions useful herein also contain a pharmaceutically acceptable carrier, including any diluents or excipients and as such are harmful to the individual to whom the composition is administered. Includes any pharmaceutical agent that can be administered without producing antibodies and without undue toxicity. Pharmaceutically acceptable carriers include, but are not limited to, liquids such as water, saline, glycerol and ethanol. A thorough discussion of pharmaceutically acceptable carriers, diluents, and other excipients is given in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ, latest edition).

  The pharmaceutical composition may be in solid or liquid form. In one aspect, the composition is in tablet or powder form, for example, because the carrier is a particle. The carrier may be a liquid and the composition may be, for example, an oral syrup, injectable solution, or an aerosol, eg useful for inhaled administration.

  When intended for oral administration, the pharmaceutical composition is preferably in either solid or liquid form, and semi-solid, semi-liquid, suspension and gel forms, as either solid or liquid, herein. Included in the forms considered in the book.

  The pharmaceutical composition as a solid composition for oral administration may be mixed in the form of powder, granules, compressed tablets, pills, capsules, chewing gum, wafers and the like. Such solid compositions typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrin; disintegrants such as alginic acid, Sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; lubricants such as colloidal silicon dioxide; sweeteners such as honey, sucrose or saccharin; flavoring agents such as peppermint, methyl salicylate or orange flavors; And colorants.

  When the pharmaceutical composition is in the form of a capsule, such as a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.

  The pharmaceutical composition may be in liquid form, for example in the form of elixirs, syrups, solutions, emulsions or suspensions. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred compositions contain, in addition to the compounds of this invention, one or more of a sweetening agent, preservative, dye / colorant, and flavor enhancer. Compositions intended for administration by injection may contain one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffering agent, stabilizing agent and isotonicity agent.

  Liquid pharmaceutical compositions, whether in the form of solutions, suspensions, etc., may contain one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, Preferably saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides, polyethylene glycols, glycerin, propylene glycol or other solvents that can act as solvents or suspending media; 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 phosphates, and tonicity adjusting agents such as sodium chloride. Or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is the preferred adjuvant. Injectable pharmaceutical compositions are preferably sterile.

  Liquid pharmaceutical compositions intended for either parenteral or oral administration should contain an amount of particulate BT compound such that a suitable dosage will be obtained. Typically this amount is at least 0.01% particulate BT compound in the composition. When intended for oral administration, this amount may vary from 0.1 to about 70% by weight of the composition. Preferred oral pharmaceutical compositions contain about 4% to about 50% BT compound. Preferred pharmaceutical compositions and preparations according to the present invention are prepared such that a parenteral dosage unit contains 0.01-10% by weight of particulate BT compound prior to dilution.

  The pharmaceutical composition may be intended for topical administration, in which case the carrier may optionally comprise a solution, emulsion, ointment or gel base. The base may include, for example, one or more of the following: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, shea butter, tea tree oil, linseed oil, hemp oil, or anti-inflammatory. And / or other vegetable or vegetable oils, including those known to have anti-painful or other beneficial effects, salmon oil, or having anti-inflammatory and / or anti-painful or other beneficial effects Other fish oils, including those known in the art, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickeners may be present in the pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoretic device. The topical formulation may contain a particulate BT compound concentration of about 0.1 to about 10% w / v (weight per unit volume).

  The pharmaceutical composition may be intended for rectal administration, eg in the form of suppositories, which melt in the rectum and release the drug. Compositions for rectal administration may contain an oily base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter, and polyethylene glycol.

  The pharmaceutical composition may include various materials that modify the physical form of a solid or liquid dosage unit. For example, the composition may include materials that form a coating shell around the active ingredients. The material forming the coating shell is typically inert and may be selected, for example, from sugar, shellac, and other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule.

  The solid or liquid form of the pharmaceutical composition may include an agent that is attached to the particulate BT compound, thereby helping to deliver the compound. Suitable agents that can act in this capacity include monoclonal or polyclonal antibodies, proteins or liposomes. However, certain contemplated embodiments explicitly exclude the inclusion of liposomes within the pharmaceutical composition.

  The pharmaceutical composition may be made up of dosage units that can be administered as an aerosol. The term aerosol is used to describe a variety of systems, from those that are colloidal to those that consist of a pressurized container. Delivery may be by a liquefied or compressed gas that dispenses the active ingredient, or by a suitable pump system. Aerosols of particulate BT compound may be delivered in a single-phase, two-phase, or three-phase system to deliver the active ingredient. Aerosol delivery includes necessary containers, activators, valves, sub-containers, etc., which together may form a kit. One of ordinary skill in the art may determine the preferred aerosol without undue experimentation.

  The pharmaceutical composition may be prepared by methodologies well known in the pharmaceutical industry. For example, a pharmaceutical composition intended for administration by injection can be prepared by mixing a compound of the invention with sterile distilled water to form a solution. Surfactants may be added to facilitate the formation of homogeneous solutions or suspensions. Surfactants are compounds that interact non-covalently with the compounds of this invention to facilitate dissolution or homogeneous suspension of the compound in an aqueous delivery system.

  The particulate BT compound described herein, or a pharmaceutically acceptable salt thereof, is administered in a therapeutically effective amount, which amount is the activity of the specific compound used; the metabolic stability of the compound. Sex and duration of action; patient age, weight, general health, sex and diet; mode of administration and time; excretion rate; drug combination; severity of a particular disorder or condition; and treatment the subject is receiving. It varies depending on various factors including the first. Generally, a therapeutically effective daily dose will be (in a 70 kg mammal) from about 0.001 mg / kg (ie 0.07 mg) to about 100 mg / kg (ie 7.0 g); preferably therapeutically effective. An amount of about 0.01 mg / kg (ie, 7 mg) to about 50 mg / kg (ie, 3.5 g) (for a 70 kg mammal); more preferably a therapeutically effective dose is (of 70 kg). In mammals, it is from about 1 mg / kg (ie 70 mg) to about 25 mg / kg (ie 1.75 g).

The effective dose ranges provided herein are not limiting and represent preferred dose ranges. However, the most preferred dosage will be understood by one of ordinary skill in the related art, as can be determined, so that adapt to each subject (e.g., Berkow et al., Eds, The Merk Manual 16 th edition, Merk and Co., Rahway, N.J., 1992 ;. Goodmanetna eds, Goodman and Cilman's The Pharmacological Basis of Therapeutics, 10 th edition, Pergamon Press, Inc., Elmsford, N.Y, (2001);. Avery 's Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics, 3rd edition, ADIS Press, LTD., Williams and Wilkins, Baltimore, MD. (1987), Ebadi, Pharma, Res., 1985. ^{Pharmaceutical Sciences, 18 th edition, Mack} Publishing Co., Easton, PA (1990); Katzung, see Basic and Clinical Pharmacology, Appleton and Lange , Norwalk, CT (1992)).

  If desired, the total dose required for each treatment can be administered by repeated or single doses within a day. Generally, treatment is 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 circumstance is reached. The diagnostic pharmaceutical compound or composition can be administered alone or in combination with other diagnostic agents and / or pharmaceutical agents for its pathology or for other conditions of its pathology. The recipient of administration of the particulate BT compound and / or composition can be any vertebrate, eg, a mammal. Preferred recipients among mammals are primates (human, ape, monkey, etc.), Artiodactyla (horse, goat, cow, sheep, pig, etc.), rodents (mouse, rat, rabbit, and hamster, etc.) , And carnivorous mammals such as cats and dogs. Among birds, the preferred recipients are turkeys, chickens and other animals of the same eye. The most preferred recipient is human.

  For topical application, it is preferable to administer an effective amount of a particulate BT-containing pharmaceutical composition to a target area, such as the skin surface, mucous membranes and the like. This amount will generally range from about 0.0001 mg BT compound per application, depending on the area to be treated, whether diagnostic, prophylactic or therapeutic, the severity of the condition and the nature of the topical vehicle used. It will be in the range of about 1 g. A preferred topical preparation is an ointment, in which case about 0.001 to about 50 mg of active ingredient are used per cc of ointment base. The pharmaceutical composition can be formulated as a transdermal composition or a transdermal delivery device (“patch”). Such compositions include, for example, a support, a reservoir of active compound, a control membrane, a liner and a contact adhesive. Such transdermal patches may be used to provide continuous, pulsatile, or on-demand delivery of the compounds of the present invention if desired.

  The particulate BT composition can be formulated to provide a rapid, sustained, or delayed release of the active ingredient after administration to the patient by using 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 P.P. J. Kuzma et al, Regional Anesthesia 22 (6): 543-551 (1997), all of which are incorporated herein by reference.

  Particulate BT compositions can also be delivered via intranasal drug delivery systems for topical, systemic, and nose-to-brain medical therapies. Controlled Particle Dispersion (CPD) ™ technology, traditional intranasal spray bottles, inhalers or nebulizers can provide effective local and systemic delivery of drugs by targeting the olfactory region and sinuses. , Are known to those skilled in the art.

  The invention also relates, in certain embodiments, to an intravaginal shell or core type drug delivery device suitable for administration to a human or animal female. The device may be comprised of a pharmaceutically active ingredient within a polymer matrix and surrounded by a sheath, as described in WO 98/5016, at a daily number similar to the device used to apply testosterone. , It may be possible to release the compound in a substantially zero order pattern.

  Current intraocular delivery methods include topical administration (eye drops), subconjunctival injection, periocular injection, intravitreal injection, surgical implantation and iontophoresis (using small currents into and into body tissues. Transporting the ionized drug via). The person skilled in the art will combine the best suited excipients with a compound that is safe and effective for intraocular administration.

  The most suitable route will depend on the nature and severity of the condition being treated. The person skilled in the art is also familiar with the determination of the mode of administration (oral, intravenous, inhalation, subcutaneous, rectal, etc.), the mode of administration, the appropriate pharmaceutical excipients and other things related to the delivery of the compound to the subject in need. Would have done.

  The compositions and methods described herein are for treating MRSA skin infections, for example, as a cream for burns, as a topical agent to treat pre-existing wounds as described herein, to prevent chronic wounds. For use in the treatment of acute and chronic wounds and wound biofilms, and for other related indications disclosed herein and apparent to those of skill in the art in view of the present disclosure. Good.

  Non-limiting examples of bacteria for which the compositions and methods described herein may find beneficial use according to certain embodiments described herein are 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. E. coli, enterohemorrhagic E. Koli, Klebsiella pneumoniae, Clostridium difficile, Helicobacter pylori, Legionella pneumophila, Enterococcus faecalis, methicillin-susceptible Enterococcus faecalis, Enterobacter cloacae, Salmonella typhimurium, Proteus vulgaris, and Elsinia enterocoli. , Vibrio cholere, Shigella flexneri, Vancomycin-resistant Enterococcus (VRE), Burkholderia cepacia, Francisella tularensis, Bacillus anthracis, Yersinia pestis, Pseudomonas aeruginosa, Vancomycin-sensitive and vancomycin-resistant Enterococcus (eg, E. faecalis, E. faecium), methicillin sensitivity and Syringe-resistant Staphylococcus (eg, S. aureus, S. epidermidis) and Acinetobacter baumannii, Staphylococcus haemolyticus, Staphylococcus hominis, Enterococcus faecium, Streptococcus pyogenes, Streptococcus agalactia, Bacillus, Bacillus Klebsiella pneumoniae, Proteus mirabilis, Proteus bulgaris, Yersinia enterocolitica, Stenotrophomonas maltophilia, Streptococcus pneumoniae, Penicillin-resistant Streptococcus pneumoniae, Berkhorderia cepacia, Berkhorderia multivolans, Mycobacterium smegmatis and E. Black fly is mentioned.

  The practice of certain embodiments of the invention is within the skill of the art, unless otherwise stated to the contrary, conventional methods of microbiology, molecular biology, biochemistry, cell biology, virology and immunology techniques, And, for the purposes of illustration, use several references provided below. Such techniques are explained fully in the literature. For example, Sambrook, et al. , Molecular Cloning: A Laboratory Manual (2nd Edition, 1989): Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait ed., 1984); eds., 1984); Animal Cell Culture (R. Freshney ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984).

  Unless the context requires otherwise, throughout the specification and claims the language “comprise” and variations thereof, such as “comprises” and “comprising”, It has an open, inclusive meaning and must be construed as "including but not limited to."

  References to “one embodiment” or “embodiment” or “aspect” throughout this specification may refer to at least one feature, structure or characteristic described in connection with that embodiment of at least one of the invention. It is meant to be included in the embodiment. The appearances of the phrase "one embodiment" or "an embodiment" in various places throughout this specification are not necessarily all referring to that embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner in one or more embodiments.

Certain embodiments may include promoting skin tissue repair in a subject, for treating acute or chronic wounds or wound biofilms in a subject, or one or more cells in a cell or cells. Methods, compositions and kits for altering wound repair activity. A cell generally refers to a single cell, but multiple cells refer to more than one cell. Cells may include tissues, organs or whole organisms. Further, the cell or cells may be placed in vivo, in vitro, or ex vivo. Maintaining cell, tissue and organ cultures is a routine procedure for those of skill in the art, the conditions and media of which can be readily ascertained (eg Freshney, Culture of Animal Cells: A Manual of Basic Technique). ^{, Wiley-Liss 5 th Ed (} 2005);. Davis, see Basic Cell Culture, Oxford University Press 2 nd Ed (2002))..

  As disclosed herein, certain embodiments relate to methods of treating acute or chronic wounds or wound biofilms in a subject, the methods described herein being used in such methods. Antibiotics described herein comprising a BT compound (eg, provided in the form of a plurality of substantially monodisperse microparticles), optionally in certain further embodiments, for use in such a method. Administering to the subject a composition that also includes a substance compound in a therapeutically effective amount, eg, the BT compound is BisEDT or BisBAL, or another compound shown in Table 1 herein, or Domenico et al. 1997 Antimicrob. Agent. Chemother. 41: 1697; 2001 Antimicrob. Age. .. T Chemother 45: 1421) and / or U. S RE37,793, U.S.S. S. With any other BT agent described in US Pat. Nos. 6,248,371, 6,086,921, and 6,380,248 and / or prepared by the methods disclosed herein. is there. Other particular embodiments relate to a method comprising contacting any natural surface with a composition comprising one or more of the particulate BT compounds described herein, the step of such contacting being direct. Application, coating, dipping, irrigation, spraying, application or other methods of 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 by any means known in the art, such as topical (skin or any epithelial tissue surface, such as glandular tissue or respiratory tract and / or gastrointestinal tract). (Including direct administration to surfaces that may be present in the duct), vaginal, intraperitoneal, oral, parenteral, intravenous, intraarterial, transdermal, sublingual, subcutaneous, intramuscular, buccal, intranasal, inhalation , Intraocular, intrafatty, intraarticular or intrathecal.

  In a preferred embodiment, the administration may be performed topically and pharmaceutical excipients or carriers for topical use are described herein and are known in the art.

  As mentioned previously, certain embodiments of the invention described herein relate to topical combinations of the described BT compounds (eg, BisEDT and / or BisBAL), the combination comprising In further embodiments, one or more antibiotic compounds described herein, such as amikacin, ampicillin, cefazolin, cefepime, chloramphenicol, ciprofloxacin, clindamycin (or other lincosamides. Antibiotics), daptomycin (Cubicin®), doxycycline, gatifloxacin, gentamicin, imipenim, levofloxacin, linezolid (Zyvox®), minocycline, nafcillin, paromomycin, rifampin, sulfamethoxazole, tobramycin. Or vancomycin; or carbapenem antibiotics, cephalosporin antibiotics, fluoroquinolone antibiotics, glycopeptide antibiotics, lincosamide antibiotics, penicillinase-resistant penicillin antibiotics, and / or aminopenicillin antibiotics and / or Or an aminoglycoside antibiotic such as amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmycin, paromomycin, rhodostreptomycin, streptomycin, tobramycin or apramycin, and / or a lipopeptide antibiotic such as daptomycin (Cubicin®), Alternatively, it may contain an oxazolidinone antibiotic such as linezolid (Zyvox (registered trademark)). These and related formulations may relate to animals, preferably mammals, most preferably humans, in a particularly preferred embodiment humans with acute or chronic wounds, or biofilms (eg biofilm formation). There may be bacteria that can promote but biofilms are not yet detectable) When administered topically to humans with wounds containing bacterial infections or wounds with bacterial infections or the presence of bacteria such as biofilms Comprising a BT compound (and optionally one or more antibiotics) in a therapeutic amount in a pharmaceutically acceptable carrier, excipient or diluent, as disclosed herein. Good.

  Topical administration of the BT compounds described herein, or pharmaceutically acceptable salts thereof, in pure form or in a suitable pharmaceutical composition, is any of the accepted modes of topical administration of drugs for similar uses. Can be carried out through Topical application or administration of the composition, in a preferred embodiment, involves applying the composition (eg, topical formulation) to the skin and / or other epithelial tissue surface (eg, respiratory tract, gastrointestinal tract and / or the like) of the subject to be treated. Or gland epithelial lining), which may be one or more localized or widely distributed skin and / or other epithelial tissue surface sites, typically topical formulations. May be, but need not be limited to, contacting an acute or chronic wound site surrounded by an intact stratum corneum or epidermis, for example, certain embodiments include, as topical application, an injury, abrasion or injury. It is contemplated that the topical combination described herein will be administered to the skin injured, or to the skin of a subject who has undergone surgery, where contact of the topical combination results in the stratum corneum or epidermal layer. As well as in skin granule cells, spiny cells, and / or basal cell layers, such as with certain types of wound repair or wound healing or other skin tissue remodeling, and / or in the dermis or underlying tissue. You may break.

  Such skin tissue repair is therefore, in certain preferred embodiments, for example preventing or ameliorating acute or chronic wounds or wound biofilms, or, as another example, preventing or ameliorating dehiscence of skin wounds. Or, it may include dermal wound healing, which may be desirable in improving, promoting or otherwise enhancing dermal wound healing where acute or chronic wounds and / or cutaneous wound tears may be present. Other particular embodiments contemplated for topical administration to the surface of epithelial tissue present in the respiratory tract, gastrointestinal tract, and / or glandular epithelial lining similarly provide the topical preparations provided herein to the respiratory tract. Present in the duct (eg, respiratory tract, nasopharyngeal and laryngeal route, trachea, lung, bronchus, bronchiole, alveoli, etc.) and / or gastrointestinal tract (eg, oral cavity, esophagus, stomach, intestine, rectum, anus, etc.) 1 It may include administration of a topical formulation by any suitable route known in the art for delivery to one or more epithelial tissues, and / or other epithelial surfaces.

  According to certain contemplated embodiments, topical administration may include direct application to open wounds. For example, open fractures or other open wounds may include lacerations in the skin that may expose additional underlying tissue to the external environment in a manner that sensitizes microbial infections. Such a situation is not uncommon in military wounds of certain types of acute trauma, including, for example, type III (severe) open fractures. According to these and related embodiments, topical administration may involve administration of the BT compositions described herein to such impaired skin and / or other epithelial surfaces and / or other tissues, such as muscle, Even by direct contact with connective tissue such as ligaments, tendons, bones, circulatory tissue such as blood vessels, related nerve tissue, and any other organ that may be exposed in such open wounds Good. Examples of other tissues that may be exposed, i.e., where such direct contact is intended, may be exposed to opportunistic infections associated with open wounds in the kidney, bladder, liver, pancreas, and such adverse conditions. Includes any other tissue or organ.

  Topical formulations (e.g. pharmaceutical compositions) are described as BT compounds (e.g. RE37,793, US 6,248,371, 6,086,921 and / or 6,). No. 380,248, and / or compounds prepared according to the present disclosure, such as the particulate BT suspensions described herein). In certain embodiments, one or more desired antibiotics (eg, aminoglycoside antibiotics such as amikacin) are separately or together with a BT compound suitable for use in a suitable pharmaceutically acceptable formulation. It may be prepared by combining with a suitable carrier, diluent or excipient and may be a solid, semi-solid, gel, cream, colloid, suspension or liquid, or other Where application forms, such as powders, granules, ointments, solutions, wash, gels, pastes, plasters, liniments, bioadhesives, microsphere suspensions, and the like in an aerosol spray, may be incorporated into the preparation.

  The pharmaceutical compositions of these and related embodiments are formulated so that the active ingredients can be included in the compositions, and in certain preferred embodiments, the BT compounds described herein, alone or in combination. The above desired antibiotics (for example, carbapenem antibiotics, cephalosporin antibiotics, fluoroquinolone antibiotics, glycopeptide antibiotics, lincosamide antibiotics, penicillinase-resistant penicillin antibiotics, and aminopenicillin antibiotics) Substance, or an aminoglycoside antibiotic, such as amikacin, or rifamycin), which is administered to a subject, such as a mammal, such as a human, in certain preferred embodiments, a human patient having acute or chronic wounds, or Acute or chronic wound or wound biofilm Or combination of BT compound and / or antibiotic compound to acute or chronic wounds and optionally surrounding skin of human patients at high risk of wound dehiscence (eg obese and / or diabetic individuals) May be applied in any order, either simultaneously or sequentially, so as to be bioavailable upon topical administration. Although certain embodiments disclosed herein contemplate topical administration of BT compounds and antibiotics, such as administration which may be in either order, either simultaneously or sequentially, the invention is not limited thereto. However, in other embodiments, a separate BT compound administration route is explicitly contemplated with respect to the antibiotic administration route. That is, the antibiotic may be administered orally, intravenously, or by any other route of administration described herein, and the BT compound is administered by a route independent of the route used for the antibiotic. May be. As a non-limiting example, the BT compound may be administered topically as provided herein, and the antibiotic may be administered simultaneously or sequentially (and in any order) by different routes, eg orally, It may be administered intravenously, transdermally, subcutaneously, intramuscularly, and / or by any other route of administration.

  The topical formulations described herein may be applied by spraying, irrigating, dipping and / or applying a therapeutically effective amount of an antiseptic or wound healing agent (and optionally an antibiotic) to the wound site, For example, delivery to skin cells such as dermal fibroblasts. The preferred formulation may be contacted with a desired site, such as a topical wound site, a chronic wound, an epithelial tissue surface or other site intended for administration, such a formulation therefore being capable of skin penetration of the drug composition. It may exhibit immediate permeability into the skin, which may be determined by any of a number of established methodologies known in the art for examining sex (eg, Wagner et al., 2002 J. Invest. Dermatol. 118: 540 and references cited therein; Bronaugh et al., 1985 J. Pharm. Sci. 74:64; Bosman et al., 1998 J. Pharm. Biomed. Anal. 17: 493-499; al., 1996 J. Pharm. Bi. Med. Anal. 1996 14: 1015-23; Bonferoni et al., 1999 Pharm. Dev. Technol. 4: 45-53; 59, Franz, Instrumentation and methodology for in vitro skin diffusion cells for method abbreviated employment, Transderm Controlled Constraction (CMS), Transdermall Controlled, Constraction (C). el Dekker, New York, 1987, Tojo in the 127-158, Design and calibration of in vitro permeation apparatus; Dermatological Formulations: Percutaneous absorption, Marcel Dekker, New York, 1983, Barry in the 234-295, Methods for studying percutaneous absorption reference).

  Compositions to be administered to the skin of a subject or patient, and formulations containing such compositions may, in certain embodiments, be in the form of one or more dosage units, for example liquid filled capsules or ampoules. , A single dosage unit may be included, and a container of the topical formulation described herein in aerosol form may hold multiple dosage units. The actual methods for preparing such dosage forms are known or apparent to those of skill in the art, see, for example, The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and 2000). I want to. The composition or formulation to be administered is, in any event, an antiseptic and / or a healing-promoting compound (eg, a BT compound) provided herein in accordance with the teachings of the present invention, or a pharmaceutically acceptable form thereof. Contains a therapeutically effective amount of salt.

  As mentioned above, the topical formulations of the present invention may take any of a wide variety of forms including, for example, creams, lotions, solutions, sprays, gels, ointments, pastes and / or liposomes. , Micelles and / or microspheres. See, eg, US Pat. No. 7,205,003. Creams, for example, are mucilages or semisolid emulsions, either oil-in-water or water-in-oil, as are well known in the pharmaceutical and cosmetic formulation arts. The cream base is washable with water and contains an oil phase, an emulsifier and an aqueous phase. The oil phase, also called the "internal" phase, is generally composed of petrolatum and a fatty alcohol such as cetyl alcohol or stearyl alcohol. The water phase is usually, but not necessarily, greater than the oil phase in volume and generally contains a hygroscopic agent. Emulsifiers in cream formulations are generally nonionic, anionic, cationic or amphoteric surfactants.

  A preferred lotion for cosmetic delivery is a formulation that is applied to the skin surface without rubbing, typically a liquid or semi-liquid formulation in which solid particles containing the active agent are present in a water or alcohol base. Is. Lotions are usually solid suspensions, and include liquid oily emulsions, preferably oil-in-water. Lotions are the preferred formulation herein for treating large body surface areas because of the ease of applying the highly fluid composition. It is generally preferred that the insoluble material in the lotion be micronized. Lotions typically contain a suspending agent, which, in addition to the suspending agent that produces a better dispersion, is useful for localizing the active ingredient and retaining it in contact with the skin. It contains compounds such as methylcellulose, sodium carboxymethylcellulose and the like.

  A solution is a homogenous mixture prepared by dissolving one or more chemical substances (solutes) in a liquid and dispersing the dissolved molecules of the substance in the solvent molecules. The solution may contain other pharmaceutically-acceptable and / or cosmetically-acceptable chemicals to buffer, stabilize or store the solute. Common examples of solvents used to prepare solutions are ethanol, water, propylene glycol or any other pharmaceutically acceptable and / or cosmetically acceptable vehicle.

  Gels are semisolid, suspension-type systems. Single-phase gels are typically aqueous, but preferably contain alcohols, and organic macromolecules distributed substantially homogeneously throughout the carrier liquid, which also contains any oil. Preferred "organic macromolecules" or gelling agents may be chemically cross-linked polymers such as cross-linked acrylic acid polymers such as "carbomers" of polymers such as carboxypolyalkylenes, which are Carbopol®. (Trademark). Also preferred in certain embodiments are hydrophilic polymers such as polyethylene oxide, polyoxyethylene-polyoxypropylene copolymers, and polyvinyl alcohol; cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, hydroxy. It may also be propylmethylcellulose phthalate, and methylcellulose; gums such as tragacanth and xanthan gum; sodium alginate; and gelatin. To prepare a homogeneous gel, a dispersant such as alcohol or glycerin can be added and the gelling agent can be dispersed by milling, mechanical mixing or stirring, or a combination thereof.

  Ointments, which are also well known in the art, are semisolid preparations typically based on petrolatum or other petroleum derivatives. The particular ointment base used is one that provides a plurality of desired characteristics, such as emollient properties, as will be appreciated by those skilled in the art. The ointment base, like other carriers or vehicles, must be inert, stable, non-irritating, and non-sensitizing. Remington: The Science Base and Practice of Pharmacy, 19th Ed (Easton, Pa .: Mack Publishing Co., 1995), p1399-1404, ointment bases are classified into four classes; Agents; emulsion bases; and water-soluble bases. Oily ointment bases include, for example, vegetable oils, fats obtained from animals, and semi-solid hydrocarbons obtained from petroleum. Emulsifying ointment bases, also known as absorbable ointment bases, contain little or no water and include, for example, hydroxystearic 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 monostearate, lanolin, and stearic acid. Preferred water-soluble ointment bases are prepared from polyethylene glycols of various molecular weights (see, eg, Remington, supra).

  Pastes are semisolid dosage forms in which the active agent is suspended in a suitable base. The paste is fractionated into a fat paste or a paste made of a single-phase aqueous gel, depending on the nature of the base. The base in the fat paste is generally petrolatum or hydrophilic petrolatum or the like. Pastes made from single-phase aqueous gels generally incorporate carboxymethyl cellulose or the like as a base.

  Formulations may be prepared using liposomes, micelles and microspheres. Liposomes are microscopic vesicles that have one (unilamellar) or multiple (multilamellar) lipid wall containing a lipid bilayer, and is used herein as one of the topical formulations described herein. The above components, such as antiseptics, wound healing / skin tissue / epithelial tissue repair-promoting compounds (eg, particulate BT compounds, optionally with one or more antibiotics) or specific carriers or excipients, may be added to a lipid membrane. It may be encapsulated on the surface and / or adsorbed on the surface of the lipid membrane. The liposomal preparations herein include cationic (positively charged), anionic (negatively charged), and neutral preparations. Anionic liposomes are readily available. For example, N [1- (2,3-dioleyloxy) propyl] -N, N, N-triethylammonium (DOTMA) liposome is commercially available under the trade name Lipofectin (registered trademark) (GIBCO BRL, Grand Island, NY. ) Available as. Similarly, anionic and neutral liposomes are readily available, eg, from Avanti Polar Lipids (Birmingham, AL), or can be readily prepared using readily available materials. Such materials include phosphatidylcholine, cholesterol, phosphatidylethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE), among others. These materials can also be mixed with DOTMA in suitable ratios. Methods for producing liposomes using these materials are well known in the art.

  Micelles consist of surfactant molecules arranged so that the polar head groups form the outer spherical shell and the hydrophobic hydrocarbon chains are oriented towards the center of the sphere to form the core. Are known in the art. The micelles are naturally obtained because they form in an aqueous solution containing a sufficiently high concentration of surfactant. Surfactants useful in forming micelles include, but are not limited to, potassium laurate, sodium octane sulfonate, sodium decane sulfonate, sodium dodecane sulfonate, sodium lauryl sulfate, sodium doxate, decyl trimethyl ammonium bromide. , Dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, tetradecyltrimethylammonium chloride, dodecylammonium chloride, polyoxy-8 dodecyl ether, polyoxy-12 dodecyl ether, nonoxynol 10, and nonoxynol 30.

  Similarly, microspheres may be incorporated into the topical formulations described herein. Like liposomes and micelles, microspheres essentially encapsulate one or more components of the formulations of the present invention. 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.

Various additives known to those skilled in the art may be included in the topical formulation. For example, a solvent containing a relatively small amount of alcohol may be used to solubilize a particular formulation ingredient. In certain topical formulations, or especially in the case of severe skin damage, such as postoperative acute or chronic wounds or postoperative dermal wound dehiscence, including a skin penetration enhancer added to the topical formulation May be desirable. Examples of suitable enhancers include, but are not limited to, ethers such as diethylene glycol monoethyl ether (commercially available as Transcutol®) and diethylene glycol monomethyl ether; surfactants such as sodium laurate, sodium lauryl sulfate, cetyltrimethyl. Ammonium bromide, benzalkonium chloride, Poloxamer® (231, 182, 184), Tween® (20, 40, 60, 80), and lecithin (US Pat. No. 4,783,450); Alcohols such as ethanol, propanol, octanol, benzyl alcohol and the like; polyethylene glycol and its esters such as polyethylene glycol monolaurate (PEGML; eg US Pat. No. 4). 568,343); amides and other nitrogen compounds such as urea, dimethylacetamide (DMA), dimethylformamide (DMF), 2-pyrrolidone, 1-methyl-2-pyrrolidone, ethanolamine, diethanolamine, and triethanolamine. Terpenes; alkanones; and organic acids, especially citric acid and succinic acid. Azone® and sulfoxides such as DMSO and C ₁₀ MSO may be used, but are less preferred.

The most preferred skin penetration enhancers are those lipophilic co-enhancers, typically referred to as "plasticizing" enhancers, i.e. molecular weights in the range of about 150-1000 Daltons, about 1% by weight. Less than, preferably less than about 0.5% by weight, and most preferably less than about 0.2% by weight of the water-soluble enhancer. The Hildebrand solubility parameter of the plasticizer is in the range of about 2.5 to about 10, preferably in the range of about 5 to about 10. Preferred lipophilicity enhancers are fatty esters, fatty alcohols, and fatty ethers. Specific and most preferred examples of fatty acid esters include methyl laurate, ethyl oleate, propylene glycol monolaurate, propylene glycerol dilaurate, glycerol monolaurate, glycerol monooleate, isopropyl n-decanoate, and octyldodecyl. There is a millistart. The fatty alcohols include, for example, stearyl alcohol and oleyl alcohol, as the fatty ethers, diols or triols, preferably substituted with C ₂ -C ₄ alkane diols or triols are one or two fatty ether substituent There are compounds. Additional skin penetration enhancers are known to those of ordinary skill in the art of topical drug delivery and / or are described in the relevant literature. For example, Percutaneous Penetration Enhancers eds. Smith et al. (CRC Press, Boca Raton, FL, 1995).

In addition to those identified above, various other additives may be included in the topical formulations according to certain embodiments of the invention. These include, but are not limited to, antioxidants, astringents, fragrances, preservatives, emollients, pigments, dyes, hygroscopic agents, propellants, and sunscreens, and their presence as cosmetics, as medicines, Or other types of materials that are otherwise desirable. Typical examples of optional additives included in the formulations of certain embodiments of the present invention are: preservatives such as sorbate; solvents such as isopropanol and propylene glycol; astringents such as Menthol and ethanol; emollients such as polyalkylene methyl glucosides; hygroscopic agents such as glycerin; emulsifiers such as glycerol stearate, PEG-100 stearate, polyglyceryl-3 hydroxylauryl ether, and polysorbate 60; sorbitol and other polyhydroxy alcohols. , Eg polyethylene glycol; sunscreens, such as octyl methoxycinnamate (commercially available as Parsol MCX) and butyl methoxybenzoyl methane (commercially available as Parsol 1789). Antioxidants such as ascorbic acid (vitamin C), alpha-tocopherol (vitamin E), beta-tocopherol, .gamma.-tocopherol, .delta.-tocopherol, .epsilon.-tocopherol, zeta ₁ - tocopherol, zeta ₂ - tocopherol, .eta. tocopherol, And retinol (vitamin A); essential oils, ceramides, essential fatty acids, mineral oils, humectants and other surfactants, such as the PLURONIC® series of hydrophilic polymers available from BASF (Mt Olive, NJ), vegetable oils ( For example, soybean oil, palm oil, liquid fractions of shea butter, sunflower oil), animal oils (eg perhydrosqualene), mineral oils, synthetic oils, silicone oils or waxes (eg cyclomethicone and dimethicone), fluorinated Oil (generally Perfu Oropolyethers), fatty alcohols (eg cetyl alcohol), and waxes (eg beeswax, carnauba wax, and paraffin wax); skin-feel modifiers; and thickeners and structures. ), For example, swelling clays and crosslinked carboxypolyalkylenes which may be obtained commercially under the trade name Carbopol®.

  Other additives include materials that tone the skin by delaying the loss of water content in the skin (especially the upper layers of the skin within the stratum corneum) to maintain flexibility and / or protect the skin. Beneficial drugs are included. Such conditioners and humectants include, by way of example, pyrrolidine carboxylic acids and amino acids; organic antimicrobial agents such as 2,4,4'-trichloro-2-hydroxydiphenyl ether (triclosan) and benzoic acid; anti-inflammatory agents such as Acetylsalicylic acid and glycyrrhetinic acid; antiseborrheic agents such as retinoic acid; vasodilators such as nicotinic acid; melanogenesis inhibitors such as kojic acid; and mixtures thereof. Other cosmetically active agents which are preferably included are, for example, α-hydroxy acids, α-keto acids, polymeric hydroxy acids, humectants, collagen, marine extracts, and antioxidants such as ascorbic acid (vitamin C). ), Α-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. You may. Additional cosmetic agents include, for example, those described in WO94 / 00098 and WO94 / 00109 that are capable of improving the oxygen supply within skin tissue. Sunscreen agents may be included.

  Other embodiments may include various non-carcinogenic, non-irritating healing materials that facilitate treatment with the formulations of certain embodiments of the invention. Such healing materials include nutrients, minerals, vitamins, electrolytes, enzymes, herbs, plant extracts, honey, gland or animal extracts, or safe therapeutic agents that may be added to the combination to promote dermal healing. You may stay. The amounts of these various additives are those conventionally used in the cosmetic field, for example in the range from about 0.01% to about 20% of the total weight of the topical formulation.

  The formulations of certain embodiments of the present invention may include conventional additives such as opacifying agents, fragrances, colorants, gelling agents, thickeners, stabilizers, surfactants and the like. Other agents, such as antimicrobial agents, may be added to prevent spoilage during storage, ie, to inhibit the growth of microorganisms such as yeast and mold. Suitable antimicrobial agents are typically selected from the methyl and propyl esters of p-hydroxybenzoic acid (eg methyl and propylparaben), sodium benzoate, sorbic acid, imidourea, and combinations thereof. The combination is a stimulant alleviating agent that minimizes or eliminates the possibility of skin irritation or skin damage resulting from the anti-infective acute or chronic wound healing and skin tissue repair-promoting compounds administered, or from other components of the composition. You may contain the additive. Suitable stimulant additives include α-tocopherol; monoamine oxidase inhibitors, in particular phenyl alcohols such as 2-phenyl-1-ethanol; glycerin; salicylates; ascorbates; ionophores such as monensin; amphiphiles. Amines; ammonium chloride; N-acetyl cysteine; capsaicin; and chloroquine. The irritant additive, if present, is typically effective in alleviating irritation or skin damage, which represents less than about 20% by weight of the formulation, more typically less than about 5%. It may be incorporated in the topical formulation at a concentration.

  The topical formulation may include a disinfectant / wound healing / anti-biofilm / skin tissue repair-promoting compound (eg, a BT compound, preferably as a substantially homogeneous microparticle provided herein, and optionally herein. In addition to the one or more synergistic antibiotics mentioned), it may contain a therapeutically effective amount of one or more additional pharmacologically active agents suitable for topical administration. Such agents include phospholipids capable of improving oxygenation in skin tissue and oxygenated fluorocarbons or fluorocarbon compounds, for example as described in WO 94/00098 and WO 94/00109. It may also contain asymmetric lamellar agglomerates consisting of a mixture.

  Suitable pharmacologically active agents that may be incorporated into the topical formulations of the present invention, ie, topically applied, may include without limitation: pigmented or unpigmented age spots, horns. Anti-microbial agents; anti-microbial agents, anti-itch and anti-dryness agents; anti-inflammatory agents; local anesthetics and analgesics; corticosteroids; retinoids (eg retinoic acid; vitamins, Hormones, and antimetabolites). Some examples of topical pharmacologically active agents are acyclovir, amphotericin, chlorhexidine, clotrimazole, ketoconazole, econazole, miconazole, metronidazole, minocycline, nystatin, neomycin, kanamycin, phenytoin, p-aminobenzoic acid ester. , Octyl methoxycinnamate, octyl salicylate, oxybenzone, dioxybenzone, tocopherol, tocopheryl acetate, selenium sulfide, zinc pyrithione, diphenhydramine, paramoxine, lidocaine, procaine, erythromycin, tetracycline, clindamycin, crotamiton and its monomethyl and benzyl ethers. , Naproxen, ibuprofen, cromolyn, retinoic acid, retinol Retinyl palmitate, retinyl acetate, coal tar, griseofulvin, estradiol, hydrocortisone, hydrocortisone 21-acetic acid, hydrocortisone 17-valeric acid, hydrocortisone 17-butyric acid, progesterone, betamethasone valeric acid, betamethasone dipropionate, triamcinolone acetonide, clobectonidone. Mention may be made of propionic acid, minoxidil, dipyridamole, diphenylhydantoin, benzoyl peroxide, and 5-fluorouracil. As also mentioned above, certain embodiments include antibiotics such as carbapenem antibiotics, cephalosporin antibiotics, fluoroquinolone antibiotics, glycopeptide antibiotics, lincosamide antibiotics, penicillinase resistant penicillin. Incorporation into a combination drug of a system antibiotic, an aminopenicillin antibiotic, or an aminoglycoside antibiotic, such as amikacin, is contemplated.

  A pharmaceutically acceptable carrier may be incorporated into the particular topical formulation of the present embodiments and may be any carrier conventionally used in the art. Examples are water, lower alcohols, higher alcohols, honey, polyhydric alcohols, monosaccharides, disaccharides, polysaccharides, sugar alcohols such as glycol (2-carbon), glycerol (3-carbon), erythritol and threitol. (4-carbon), arabitol, xylitol and ribitol (5-carbon), mannitol, sorbitol, dulcitol and iditol (6-carbon), isomaltol, maltitol, lactitol and polyglycitol, hydrocarbon oil, fat and wax, Included are aqueous and emulsion based mixtures of fatty acids, silicone oils, nonionic surfactants, ionic surfactants, silicone surfactants, and such carriers.

  Embodiments of the topical formulations of the present invention affect any acute or chronic wound site (e.g., the wound itself and surrounding tissues, e.g., infection) that requires treatment at the frequency and amount necessary to achieve the desired result. Regular application to peripheral tissues that may not have been received or appear to be other normal or healthy) or skin areas or other epithelial tissue surfaces (eg, gastrointestinal tract, respiratory tract, glandular tissue) Good. The frequency of treatment depends on the condition of the skin (or epithelium) (eg other acute or chronic wounds, or other skin wounds that may be found in dehiscence caused by surgical resection, or other types of skin wounds), skin ( Degree of injury or deterioration of other tissues), responsiveness of the user's skin (or other tissues), strength of the active ingredient in certain embodiments (eg, wound healing / disinfection / disinfection as described herein). Anti-biofilm / skin tissue repair-promoting compounds such as BT compounds and optionally one or more additional pharmaceutically active ingredients such as antibiotics such as amikacin or other antibiotics, skin (or other epithelial surface containing). The combination is removed by the efficacy of the vehicle used to deliver the active ingredient to the appropriate layer of tissue), physical contact with bandages or other dressings or clothing. Ease of being removed by perspiration or other intrinsic or extrinsic fluids, as well as the ease of the activity level or lifestyle of a subject or patient.

Typical concentrations of antiseptic / anti-biofilm / wound healing / skin tissue repair-promoting compositions of active agents, such as the BT compounds described herein, are 0.001 to 30 based on the total weight of the composition, for example. It may range from wt% to about 0.01 to 5.0%, more preferably about 0.1 to 2.0%. As a representative example, compositions of these embodiments of the invention may be applied to acute or chronic wounds and / or skin at a rate of about 1.0 mg / cm ² skin to about 20.0 mg / cm ² skin. Good. Representative examples of topical formulations include, but are not limited to, aerosols, alcohols, anhydrous bases (eg lipsticks and powders), aqueous solutions, creams, emulsions (including either water-in-oil or oil-in-water emulsions). , Fats, foams, gels, hydroalcoholic 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, protectants, thickeners, or UV absorbers. One of ordinary skill in the art will appreciate that formulations other than those listed may be used in embodiments of the invention.

A chelating agent may optionally be included in the topical formulation, may be selected from any agent suitable for use in cosmetic compositions, and may include a ^cation such as Ca ²⁺ , Mn ²⁺ , or Mg ^2+. It may include any natural or synthetic chemical agent that has the ability to bind a cation metal. Examples of chelating agents include, but are not limited to, EDTA, disodium EDTA, EGTA, citric acid, and dicarboxylic acids.

  Conditioning agents may also optionally be included in the topical formulation. Examples of skin conditioning agents include, but are not limited to, acetyl cysteine, N-acetyl dihydrosphingosine, acrylate / behenyl acrylate / dimethicone acrylate copolymer, adenosine, cyclic adenosine monophosphate, adenosine monophosphate, adenosine triphosphate. Phosphoric acid, alanine, albumen, seaweed extract, allantoin and derivatives, aloe barbadensis extract, aluminum PCA, amyloglucosidase, arbutin, arginine, azulene, bromelain, powdered buttermilk, butylene glycol, caffeine, calcium gluconate, Capsaicin, carbocysteine, carnosine, β-carotene, casein, catalase, cephalin, ceramide, chamomile recutita (matricaria) flower extract, cholecalci Ferrol, cholesteryl ester, cocobetaine, coenzyme A, processed corn starch, crystallin, cycloethoxymethicone, cysteine DNA, cytochrome C, daltside, dextran sulfate, dimethicone copolyol, dimethylsilanol hyaluronate, DNA, elastin, elastin amino acid, epithelium Growth factor, ergocalciferol, ergosterol, ethylhexyl PCA, fibronectin, folic acid, gelatin, gliadin, β-glucan, glucose, glycine, glycogen, glycolipid, glycoprotein, glycosaminoglycan, glycosphingolipid, horseradish peroxidase, Hydrogenated proteins, protein hydrolysates, jojoba oil, keratins, keratin amino acids, and kinetin, lactofe , Lanosterol, lauryl PCA, lecithin, linoleic acid, linolenic acid, lipase, lysine, lysozyme, malt extract, maltodextrin, melanin, methionine, inorganic salt, niacin, niacinamide, oat amino acid, oryzanol, palmitoyl hydrolyzed protein, bread Creatine, papain, PEG, pepsin, phospholipid, phytosterol, placental enzyme, placental lipid, pyridoxal 5-phosphate, quercetin, resorcinol acetate, riboflavin, RNA, saccharomyces lysate extract, silk amino acid, sphingolipid, stearamidopropyl Betaine, stearyl palmitate, tocopherol, tocopheryl acetate, tocopheryl linoleate, ubiquinone, Vitis vinichella (grape) seed oil, wheat amyloid Acid, xanthan gum, and zinc gluconate. As will be readily appreciated by those skilled in the art, skin conditioning agents other than those listed above may be combined with the disclosed compositions or the formulations provided thereby.

The topical formulation may optionally include one or more emollients, including, but not limited to, acetylated lanolin, acetylated lanolin alcohol, acrylate / _C10-30 alkyl acrylate crosspolymers, Acrylate copolymer, Alanine, Seaweed extract, Aloe barbadensis extract or gel, Altea officinalis extract, Starch octenyl succinate aluminum, Aluminum stearate, Apricot (Prunus Armeniaca) kernel oil, Arginine, Arginine aspartate, Arnica Montana extract, ascorbic acid, ascorbyl palmitate, aspartic acid, avocado (Persia gratissima) oil, barium sulfate, barrier sphingolipid, butyl alcohol, beeswax, bees Nyl alcohol, β-sitosterol, BHT, birch (Vetula alba) bark extract, borage (borago officinalis) extract, 2-bromo-2-nitropropane-1,3-diol, nagicida (luscus acureatus) extract, Butylene glycol, calendula officinalis extract, calendula officinalis oil, calendula (euphorbia serifera) wax, canola oil, caprylic / capric triglyceride, cardamom (eletaria cardamomum) oil, carnauba (copernicia serifera) wax, carrageenan (Condrus crispus), carrot (Daucus carlotta sativa) oil, castor (ricinus communis) oil, ceramide, ceresin, ceteareth-5, ceteareth-12, ceteareth-20, Cetearyl cutarate, ceteth-20, ceteth-24, cetyl acetate, cetyl octanoate, cetyl palmitate, chamomile (Antemis nobilis) oil, cholesterol, cholesterol ester, cholesteryl hydroxystearate, citric acid, salvia (salvia sclarea) Oil, cocoa (theobroma cacao) butter, coco-caprylic / capric acid, coconut (Cocos nucifera) oil, collagen, collagen amino acid, corn (Gee Mace) oil, fatty acid, decyl oleate, dextrin, diazolidinyl urea, dimethy Corn copolyol, dimethiconol, dioctyl adipate, dioctyl succinate, dipentaerythrityl hexacaprylate / hexacaprate, DMDM hydantoin, DNA, erythritol, ethoxydiglycol , Ethyl linoleate, eucalyptus globulus oil, evening primrose (Oenocera biennis) oil, fatty acid, fructose, gelatin, wild geranium oil, glucosamine, glucose glutamate, glutamic acid, glyceres-26, glycerin, glycerol, glyceryl distearate, hydroxystearin Glyceryl acid salt, glyceryl laurate, glyceryl linoleate, glyceryl myristate, glyceryl oleate, glyceryl stearate, glyceryl stearate SE, glycine, glycol stearate, glycol stearate SE, glycosaminoglycan, grape (Vitis vinichella) Seed oil, hazel (Corirus americana) nut oil, hazel (Corirus averana) nut oil, hexylene glycol, honey, hyal Lonic acid, hybrid safflower (Casamas tinctorias) oil, hydrogenated castor oil, hydrogenated cocoglyceride, hydrogenated coconut oil, hydrogenated lanolin, hydrogenated lecithin, hydrogenated palm glyceride, hydrogenated palm kernel oil, hydrogenated large Soybean oil, hydrogenated tallow glyceride, hydrogenated vegetable oil, hydrolyzed collagen, hydrolyzed elastin, hydrolyzed glycosaminoglycan, hydrolyzed keratin, hydrolyzed soy protein, hydroxylated lanolin, hydroxyproline, imidazolidinyl urea, iodopropynyl butyl carbamate , Isocetyl stearate, stearoyl isocetyl stearate, isodecyl oleate, isopropyl isostearate, isopropyl lanolate, isopropyl myristate, isopropyl palmitate, isopropyl stearate , Isostearamide DEA, isostearic acid, isostearyl lactate, isostearyl neopentanoate, jasmine (Jasminum officinale) oil, jojoba (Vaxus chinensis) oil, kelp, kukui (Aleurites molcana) nut oil, lactamide MEA, lanes- 16, lanes-10 acetate, lanolin, lanolin, lanolin alcohol, lanolin alcohol, lanolin oil, lanolin wax, lavender (lavandula angustifolia) oil, lecithin, lemon (citrus medica limonum) oil, linoleic acid, linolenic acid, macadamia・ Ternifolia nut oil, magnesium stearate, magnesium sulfate, maltitol, cattle millet (camomira recutita) oil, methylglucose sesquistearate, methylsilanol PCA, microcrystalline wax, ore Product oil, mink oil, Mortierella oil, myristyl lactate, myristyl myristate, myristyl propionate, dicaprylic / dicaprate neopentyl glycol, octyldodecanol, octyldodecyl myristate, octyldodecyl stearoylstearate, octyl hydroxystearate, Octyl palmitate, octyl salicylate, octyl stearate, oleic acid, olive (olea europaea) oil, orange (citrus aurantium dulcis) oil, palm (elaeis guiensis) oil, palmitic acid, pantethine, panthenol, bread Tenyl ethyl ether, paraffin, PCA, peach (Prunus persica) kernel oil, peanut (Arakis hippogae) oil, PEG-8 C12 18 ester, PEG-15 coca , PEG-150 distearic acid, PEG-60 glyceryl isostearate, PEG-5 glyceryl stearate, PEG-30 glyceryl stearate, PEG-7 hydrogenated castor oil, PEG-40 hydrogenated castor oil, PEG-60 hydrogenated Castor oil, PEG-20 methyl glucose sesquistearate, PEG-40 sorbitan peroleate, PEG-5 soy sterol, PEG-10 soy sterol, PEG-2 stearate, PEG-8 stearate, PEG-20 stearate, stearin Acid PEG-32, stearic acid PEG-40, stearic acid PEG-50, stearic acid PEG-100, stearic acid PEG-150, pentadecalactone, peppermint (mentapiperita) oil, petrolatum, phospholipids, polyamino sugar condensate , Jii Polyglyceryl-3 stearate, polyquaternium-24, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, polysorbate 85, potassium myristate, potassium palmitate, potassium sorbate, potassium stearate, propylene glycol, dicaprylic acid / dicapric acid. Propylene glycol, propylene glycol dioctanoate, propylene glycol dipelargonate, propylene glycol laurate, propylene glycol stearate, SE propylene glycol stearate, PVP, pyridoxine dipalmitate, quaternium-15, quaternium-18 hectorite, quaternium-22, Retinol, retinyl palmitate, rice (Oriza sativa) nuka oil, RNA Rosemary (Losmarinus officinalis) oil, rose oil, safflower (Casamas tinctorias) oil, sage (Salvia officinalis) oil, salicylic acid, sandalwood (Santalum album) oil, serine, serum protein, sesame (sesamum (Indica) oil, shea butter (butyrospermum / parky), silk powder, sodium chondroitin sulfate, sodium DNA, sodium hyaluronate, sodium lactate, sodium palmitate, sodium PCA, sodium polyglutamate, sodium stearate, water-soluble collagen, sorbin Acid, sorbitan laurate, sorbitan oleate, sorbitan palmitate, sorbitan sesquioleate, sorbitan stearate, sorbitol, soybean (glycine soya) oil, sphingolipid Squalane, squalene, stearamide stearate MEA, stearic acid, stearoxydimethicone, 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, tribehenine, tridecyl neopentanoate, tridecyl stearate, triethanolamine, tristearin, urea, vegetable oil, water, wax, wheat (Triticum vulgare) ) Germ oil, and ylang-ylang (Cananga odorata) oil.

  In some embodiments, topical formulations may contain suitable excipients, which typically have a high affinity for the skin and are well tolerated, It must produce a consistency that is stable and readily available. Suitable topical excipients and vehicles can be found by those skilled in the art, in particular Remington's Pharmaceutical Sciences, Vol. 18, Mack Publishing Co. , Easton, Pa. With reference to one of the many standard textbooks in the art, such as in particular Chapter 87 of (1990), one can make routine choices for a particular application. Optionally, one or more hygroscopic agents are also included in the topical formulation. Examples of hygroscopic agents include, without limitation, amino acids, chondroitin sulfate, diglycerin, erythritol, fructose, glucose, glycerin, glycerol, glycols, 1,2,6-hexanetriol, honey, hyaluronic acid, hydrogenated honey, Hydrogenated starch hydrolyzate, inositol, lactitol, maltitol, maltose, mannitol, natural moisturizing factor, PEG-15 butanediol, polyglyceryl sorbitol, pyrrolidone carboxylate, potassium PCA, propylene glycol, sodium glucuronate, sodium PCA, sorbitol, Examples include sucrose, trehalose, urea, and xylitol.

  Certain embodiments contemplate topical formulations containing one or more additional skin protectants. Examples of skin protectants include, but are not limited to, seaweed extract, allantoin, aluminum hydroxide, aluminum sulfate, betaine, camellia sinensis leaf extract, cerebroside, dimethicone, glucuronolactone, glycerin, kaolin, lanolin, malt extract, minerals. Mention may be made of oil, petrolatum, potassium gluconate, and talc. It will be immediately appreciated by those skilled in the art that skin protectants other than those listed above may also be combined with the disclosed compositions of the present invention or the formulations provided thereby.

  Surfactants may also be included in certain topical formulations contemplated herein, if desired, and may include cationic, anionic, zwitterionic or nonionic surfactants, or those. Can be selected from any natural or synthetic surfactant suitable for use in a cosmetic composition, such as a mixture of (Rosen, M., "Surfactants and Interfacial Phenomena," Second Edition, John Wiley & Sons, Sons, Wheat & Sons). , 1988, Chapter 1p431). Examples of cationic surfactants include, but are not limited to, DMDAO or other amine oxides, long chain primary amines, diamines, polyamines and their salts, quaternary ammonium salts, polyoxyethylenated salts. Mention may be made of long-chain amines, as well as quaternized polyoxyethylenated long-chain amines. Examples of anionic surfactants include, but are not limited to, SDS; carboxylic acid salts (eg soaps); sulfonic acid salts, sulfuric acid salts, phosphoric acid and polyphosphate esters; alkyl phosphates; monoalkyl phosphates. And phthalate (MAP); and salts of perfluorocarboxylic acids. Examples of zwitterionic surfactants are cocamidopropyl hydroxysultaine (CAPHS) and others that are pH sensitive and require special attention in designing the optimum pH of the combination (i.e. , Alkylaminopropionic acid, imidazoline carboxylate, and betaine) or those that are not pH sensitive (eg, sulfobetaine, sultaine). Examples of nonionic surfactants include, but are not limited to, alkylphenol ethoxylates, alcohol ethoxylates, polyoxyethylenated polyoxypropylene glycols, polyoxyethylenated mercaptans, long chain carboxylic acid esters, alkanols. Mention may be made of amides, tertiary acetylene glycols, polyoxyethylenated silicones, N-alkylpyrrolidones, and alkylpolyglycosidases. Wetting agents, mineral oils or other surfactants, such as nonionic detergents or agents such as one or more of the PLURONIC® series (BASF, Mt Olive, NJ), for example, also by non-limiting theory, It may be included to prevent aggregation of BT particles in the particle suspension. Any combination of surfactants is acceptable. Certain embodiments include at least one anionic surfactant and at least one cationic surfactant that are compatible, ie, do not form a complex that appreciably precipitates upon mixing, or It may comprise at least one cationic surfactant and at least one zwitterionic surfactant.

  Examples of thickeners that may also be present in certain topical formulations include, but are not limited to, acrylamide copolymers, agarose, amylopectin, bentonite, calcium alginate, carboxymethyl cellulose calcium, carbomer, carboxymethyl chitin, cellulose gum, dextrin. , Gelatin, hydrogenated tallow, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, magnesium alginate, methyl cellulose, microcrystalline cellulose, pectin, various PEGs, polyacrylic acid, polymethacrylic acid, polyvinyl alcohol, various PPGs, sodium acrylate copolymers, sodium carrageenan, xanthan gum, and yeast β-glucan It is below. Thickeners other than those listed above may be used in embodiments of the invention.

  According to certain embodiments contemplated herein, the topical formulation may include one or more sunscreens or UV absorbers. If ultraviolet (UVA and UVB) absorption properties are desired, such agents include, for example, benzophenone, benzophenone-1, benzophenone-2, benzophenone-3, benzophenone-4, benzophenone-5, benzophenone-6, benzophenone-7, Benzophenone-8, benzophenone-9, benzophenone-10, benzophenone-11, benzophenone-12, benzyl salicylate, butyl PABA, cinnamic acid ester, cinoxate, DEA-methoxycinnamic acid, diisopropyl methylcinnamic acid, ethyldihydroxypropyl PABA, diisopropylcinnamic acid Ethyl, ethyl methoxycinnamate, ethyl PABA, ethyl urocanate, glyceryl octanoate dimethoxycinnamate, glyceryl PABA, glyco salicylate , Homosalate, p- methoxycinnamic acid isoamyl, titanium, zinc, zirconium, silicon, oxides of manganese and cerium, PABA, PABA esters, Parsol 1789, and isopropylbenzyl salicylate benzyl, and may include mixtures thereof. Those skilled in the art will appreciate that sunscreens and UV absorbers or protectants other than those listed may be used in certain embodiments of the invention.

  The topical formulations disclosed herein are typically effective at pH values between about 2.5 and about 10.0. Preferably, the pH of the composition is at or near the following pH range: 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. ~ 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. One of ordinary skill in the art may add suitable pH adjusting ingredients to the compositions of the present invention to adjust the pH to an acceptable range. If it is recognized that the pH may vary from its original value depending on the composition of the compounding agent and the storage conditions, the specific pH with "about" is the pH actually measured at any given time. One of ordinary skill in the art will understand that it includes formulations that can be raised or lowered by 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or 0.1 pH units or less than Ah

  Creams, lotions, gels, ointments, pastes, etc. may be spread over the affected surface and gently rubbed in. The solution may be applied in the same manner, but more typically will be applied using a dropper, swab etc. and will be applied carefully to the affected area. This application regimen will depend on many factors that can be readily determined, such as the severity of the wound and the responsiveness to the initial treatment, but will usually include continuous one or more daily applications. Let's do it. One of ordinary skill in the art can immediately determine the appropriate amount of the combination to be administered, the method of administration and the rate of repetition. Generally, the formulations of these and related embodiments of the invention will be applied in a range from 1 or 2 or more times per week to 1, 2, 3, 4 or more times per day. Is contemplated.

  As also discussed above, the topical formulations thus useful herein also contain a pharmaceutically acceptable carrier, including any suitable diluents or excipients, and as such The composition can be administered without undue toxicity, including any pharmaceutical agent that is not harmful to the subject to whom it is administered. Pharmaceutically acceptable carriers include, but are not limited to, liquids such as water, saline, glycerol and ethanol, viscosity enhancing agents (e.g. balsam fir resin) or film forming agents such as collodion or nitrocellulose solutions. Can also be included. A thorough discussion of pharmaceutically acceptable carriers, diluents, and other excipients is given in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ, latest edition).

  When the topical formulation is in the form of a gel- or liquid-filled capsule, such as a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil. Liquid pharmaceutical compositions of particular embodiments of the invention, whether in solution, suspension or other similar form, may include one or more of the following: sterile diluents such as water for injection. , Saline solutions, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic monoglycerides or diglycerides which can serve as a solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol. Or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; additional antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate Or phosphates, and tonicity agents such as sodium chloride Or dextrose.

  For topical administration, the carrier may suitably comprise a solution, emulsion, ointment or gel base. Bases may include, for example, one or more of the following: petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents such as water and alcohols, and emulsifiers and stabilizers. Thickeners may be present in pharmaceutical or cosmetic compositions for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device. Topical formulations may include a concentration of the compound of a particular embodiment of the invention from about 0.1 to about 10% w / v (weight / unit volume). Topical formulations may be provided in the form of creams, lotions, solutions, sprays, gels, ointments, pastes and / or containing liposomes, micelles, microspheres and / or other microparticulate or nanoparticulate delivery elements. May be. Topical formulations include time-release or sustained release particles or pellets that can be administered directly to the wound site, such as sustained release ethylene vinyl acetate polymer (eg, Elvax® 40, Aldrich, Milwaukee, WI) pellets. It may be provided in the form.

  The topical formulation may include an agent that binds to the skin tissue repair-promoting compound, thereby assisting delivery to skin epithelial cells (eg, keratinocytes) and / or fibroblasts. Suitable agents capable of acting in this capacity include inclusion agents such as cyclodextrins; other agents may include proteins or liposomes.

  Topical formulations of certain embodiments of the invention may be provided in the form of dosage units that can be administered as an aerosol. The term aerosol is used to indicate a variety of systems ranging from colloidal to systems consisting of pressurized packaging. Delivery may be by a liquefied or compressed gas or by a suitable pump system that disperses the active ingredient. Aerosols of the compounds of certain embodiments of this invention may be delivered in a single phase, biphasic, or triphasic system to deliver the active ingredient (s). Aerosol delivery includes necessary containers, activators, valves, sub-containers, etc., which may together form a kit. One of ordinary skill in the art may determine the preferred aerosol for delivery of the topical formulation to the skin or wound site without undue experimentation.

  Topical formulations may be prepared by methodology well known in the pharmaceutical art. For example, a pharmaceutical composition intended to be administered as a spray, wash or rinse to the wound site or skin may include a solution of the BT disinfectant / wound healing / anti-biofilm / skin tissue repair-promoting compounds described herein. It can be prepared by admixing with sterile distilled water to form. Surfactants may be added to facilitate the formation of homogeneous solutions or suspensions. Surfactants are compounds that interact non-covalently with antioxidant active compounds to facilitate dissolution or homogeneous suspension of the compound in an aqueous delivery system.

  The BT disinfectant / wound healing / anti-biofilm / skin tissue repair-promoting compound, or pharmaceutically acceptable salt thereof, used in the topical formulation is administered in a therapeutically effective amount, , The nature of the wound site (if relevant), the activity of the specific BT compound used (such as with or without aminoglycoside antibiotics, eg, a combination of antibiotics such as amikacin); metabolic stability and action of the compound Duration; subject's age, weight, general health, sex, skin type, immune status and diet; mode of administration and time; excretion rate; drug combination; specific skin wound severity for which skin tissue repair is desirable; and Varies depending on various factors, including the treatment the subject is receiving. Generally, a therapeutically effective daily dose will be (in a 70 kg mammal) from about 0.001 mg / kg (ie 0.07 mg) to about 100 mg / kg (ie 7.0 g); preferably therapeutically effective. A dose of about 0.01 mg / kg (ie, 7 mg) to about 50 mg / kg (ie, 3.5 g) (for a 70 kg mammal); more preferably a therapeutically effective dose is (of 70 kg). In mammals, it is from about 1 mg / kg (ie 70 mg) to about 25 mg / kg (ie 1.75 g).

The effective dose ranges provided herein are not limiting and represent preferred dose ranges. However, the most preferred dosage will be understood by one of ordinary skill in the related art, as may be determined, will be adapted to each target (e.g., Berkow et al., Eds. , The Merk Manual 16 th edition, Merk and Co., Rahway, N.J., 1992 ;... Goodman et al, eds, Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10 th edition, Pergamon Press, Inc., Elmsford, N.Y, ( 2001); Avery's Drug Treatment: Principles and Practice of Clinical Pharmacology a. d Therapeutics, 3rd edition, ADIS Press, Ltd., Williams and Wilkins, Baltimore, MD. (1987), Ebadi, Pharmacy, Little, Bol. ^{s Pharmaceutical Sciences, 18 th edition,} Mack Publishing Co., Easton, PA (1990); Katzung, Basic and Clinical Pharmacology, Appleton and Lange, Norwalk, CT (1992) reference).

  If desired, the total dose required for each treatment can be administered by repeated or single doses during the day. Certain preferred embodiments contemplate a single application of the topical formulation per day. Generally, in clear 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 circumstance is reached.

  The topical combination may be administered alone or in combination with other treatments and / or pharmaceutical agents directed at skin wounds or at other related symptoms or etiologic agents. For example, as mentioned above, the topical formulation may further comprise retinoic acid. As another example, a topical formulation may include one or more of the skin tissue repair promoting compounds described herein, or two or more such compounds having different cell wound repair activities. You may stay.

  The recipient of the topical formulations described herein can be any vertebrate, eg, a mammal. Preferred recipients among mammals are primates (human, ape, monkey, etc.), Artiodactyla (horse, goat, cow, sheep, pig, etc.), rodents (mouse, rat, rabbit, and hamster, etc.) , And carnivorous mammals such as cats and dogs. Among birds, the preferred recipients are turkeys, chickens and other animals of the same eye. The most preferred recipient is a human, and particularly preferred is a human with an acute or chronic wound, or a wound containing a biofilm.

  For topical application, according to the embodiments described herein, an effective amount of a pharmaceutical composition comprising a BT compound disinfectant / wound healing / anti-biofilm / skin tissue repair-promoting compound is administered to a target area, for example. It is preferred to administer to cutaneous wounds, such as acute or chronic wounds, and / or areas at risk of skin (eg, wound dehiscence). This amount will generally depend on the area to be treated, the severity of the condition (or past or planned surgical resection), and the nature of the local vehicle used, and the particular embodiment of the invention per application. Of a compound of about 0.0001 mg to about 1 g. Preferred topical preparations are ointments or sustained release pellets, in which case from about 0.001 to about 50 mg of active ingredient are used per cc of ointment base or pellet suspension. The pharmaceutical composition can be formulated as a transdermal composition or a transdermal delivery device (“patch”). Such compositions include, for example, a support, an active compound reservoir, a control membrane, a liner and a contact adhesive. Such transdermal patches may, if desired, be used to provide continuous, pulsatile, or on-demand delivery of the compounds of the present invention.

  The compositions of certain embodiments can be formulated to provide rapid, sustained, or delayed release of the active ingredient after administration to the patient by using 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 P.P. J. Kuzma et al, Regional Anesthesia 22 (3): 543-551 (1997), all of which are incorporated herein by reference.

  The most suitable route will depend on the nature and severity of the condition being treated. Those skilled in the art will appreciate that topical administration methods (spray, cream, open application, closed dressings, dipping, washing, etc.), dosage forms, suitable pharmaceutical excipients, and compounds to the subject in need thereof. He is also familiar with determining other matters related to delivery.

  Throughout this specification, the language “comprise”, “comprises” and “comprising” includes the referenced step or element, or steps or elements, unless otherwise necessary. However, it is to be understood that it does not exclude any other step or element or steps or elements. "Consisting of" is meant to include and be limited to what follows the idiom "consisting of". That is, the phrase "consisting of" indicates that the listed element is required or required and that no other element can be present. “Consisting essentially of” is meant to include any element listed after the phrase and that does not interfere with or contribute to the activity or action specified in the disclosure for the listed element. Limited to elements. That is, the phrase "consisting essentially of" means that the listed elements are required or required, but that other elements are not required, depending on whether they affect the activity or action of the listed elements. , May or may not be present.

  In this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless otherwise specified. The term “about” or “approximately” as used in certain embodiments herein, when present in front of a numerical value, is that value plus or minus 5%, 6%, 7%, 8% or 9%. Indicates the range of. In other embodiments, the term “about” or “approximately”, when present before a numerical value, indicates the range plus or minus 10%, 11%, 12%, 13% or 14%. In yet another embodiment, the term "about" or "approximately", when present before a numerical value, ranges from the value plus or minus 15%, 16%, 17%, 18%, 19% or 20%. Indicates.

  The following examples are given by way of illustration and not limitation.

Example 1
Preparation of BT Compound The following BT compound was prepared according to the method of Domenico et al. (US RE37,793, US 6,248,371, 6,086,921, 6,380,248). Or as microparticles according to the synthetic protocol described below for BisEDT. Shown are the atomic ratios for a single bismuth atom, by comparison, based on the theoretical ratios of the reactants used to form the trivalent complex with the sulfur-containing compound and the known propensity of bismuth. Numbers in parentheses are ratios of bismuth to one (or several) thiol agent (eg Bi: thiol 1 / thiol 2; see also Table 1).
1) CPD 1B-1 Bis-EDT (1: 1) BiC ₂ H ₄ S ₂
2) CPD 1B-2 Bis-EDT (1: 1.5) BiC ₃ H ₆ S ₃
3) CPD 1B-3 Bis-EDT (1: 1.5) BiC ₃ H ₆ S ₃
4) CPD 1C Bis-EDT (soluble Bi preparation) (1: 1.5) BiC ₃ H ₆ S ₃
5) CPD 2A Bis-Bal (1: 1) BiC ₃ H ₆ S ₂ O
6) CPD 2B Bis-Bal (1: 1.5) BiC _4.5 H ₉ O _1.5 S ₃
7) CPD 3A Bis-Pyr (1: 1.5) BiC _7.5 H ₆ N _1.5 O _1.5 S _1.5
8) CPD 3B Bis-Pyr (1: 3) BiC ₁₅ H ₁₂ N ₃ O ₃ S ₃
9) CPD 4 Bis-Ery (1: 1.5) BiC ₆ H ₁₂ O ₃ S ₃
10) CPD 5 Bis-Tol (1: 1.5) BiC _10.5 H ₉ S ₃
11) CPD 6 Bis-BDT (1: 1.5) BiC ₆ H ₁₂ S ₃
12) CPD 7 Bis-PDT (1: 1.5) BiC _4.5 H ₉ S ₃
13) CPD 8-1 Bis-Pyr / BDT (1: 1/1)
14) CPD 8-2 Bis-Pyr / BDT (1: 1 / 0.5)
15) CPD 9 Bis-2 hydroxy, propanethiol (1: 3)
16) CPD 10 Bis-Pyr / Bal (1: 1 / 0.5)
17) CPD 11 Bis-Pyr / EDT (1: 1 / 0.5)
18) CPD 12 Bis-Pyr / Tol (1: 1 / 0.5)
19) CPD 13 Bis-Pyr / PDT (1: 1 / 0.5)
20) CPD 14 Bis-Pyr / Ery (1: 1 / 0.5)
21) CPD 15 Bis-EDT / 2 hydroxy, propanethiol (1: 1/1)

  Particulate bismuth-1,2-ethanedithiol (Bis-EDT, soluble bismuth preparation) was prepared as follows:

To an excess of room temperature 5% aqueous HNO ₃ (11.4 L) in 15 L polypropylene carboy, aqueous Bi (NO ₃ ) ₃ solution (43% Bi (NO ₃ ) ₃ (w / w), 5% nitric acid ( w / w), 52% (w / w) water, Shepherd Chemical Co., Cincinnati, OH, product number 2362; δ-1.6 g / mL) 0.331 L (-0.575 mol) with gentle stirring. To the mixture, anhydrous ethanol (4 L) was slowly added. A small amount of white precipitate formed, but dissolved with continuous stirring. Separately, using a 60 mL syringe, 72.19 mL (0.863 mol) of 1,2-ethanedithiol (CAS 540-63-6) was added to 1.5 L of absolute ethanol, followed by stirring for 5 minutes. An ethanolic solution of 1,2-ethanedithiol (~ 1.56L, ~ 0.55M) was prepared. Thereafter, the 1,2-ethanedithiol / EtOH reagents over aqueous _{Bi (NO 3) 3 / HNO} 3 solution for 5 hours with gentle dropwise and stirred overnight continuously. The product formed was allowed to settle as a precipitate for approximately 15 minutes, after which the filtrate was removed using a peristaltic pump at 300 mL / min. Then, the product was recovered by filtering through a filter paper in a 15 cm diameter Buchner funnel, and successively washed with ethanol, USP water and acetone at a volume of 500 mL each three times, and BisEDT (694.51 g / mol) was yellow amorphous. Obtained as a fine powder solid. The product was placed in a 500 mL brown glass bottle and dehydrated with CaCl ₂ under high vacuum for 48 hours. The recovered material (yield ~ 200g) released a odor characteristic of thiols. The crude product was redissolved in 750 mL of absolute ethanol, stirred for 30 minutes, then filtered, washed 3 times with 50 mL of ethanol, 2 times with 50 mL of acetone, followed by 500 mL of acetone again. The rewashed powder was triturated in 1 M NaOH (500 mL), filtered and washed 3 times with 220 ml of water, 2 times with 50 mL of ethanol and once with 400 mL of acetone to give 156.74 g of purified BisEDT. . The next batch, prepared in essentially the same manner, gave a yield of about 78.91%.

The product was previously shown by analysis of data from ¹ H and ¹³ C nuclear magnetic resonance (NMR), infrared spectroscopy (IR), ultraviolet spectroscopy (UV), mass spectroscopy (MS) and elemental analysis. It was characterized to have the structure of Formula I An HPLC method was developed to determine the chemical purity of BisEDT, whereby samples were prepared in DMSO (0.5 mg / mL). Λ _max was measured by scanning a solution of BisEDT in DMSO at 190-600 nm. Waters (Millipore Corp., Milford, MA) with a UV detector monitoring at 265 nm (λ _max ) and a YMC Pack PVC Sil NP (inner diameter 250 × 4.6 mm) analytical column (Waters) with an injection volume of 2 μL. Using a model 2695 chromatograph, isocratic HPLC elution at 1 mL / min with 0.1% formic acid in acetone: water (9: 1) as the mobile phase was performed at ambient temperature to detect a single peak. , The chemical purity was 100 ± 01%. Elemental analysis was consistent with the structure of formula (I).

  The dry particulate material was characterized to characterize the particle size. Briefly, the microparticles were resuspended in 2% Pluronic® F-68 (BASF, Mt. Olive, NJ) and the suspension sonicated in a standard water bath sonicator for 10 minutes. After that, analysis was performed using a Nanosizer / Zetasizer Nano-S particle analyzer (model ZEN1600 (without zeta potential measurement capability), Malverm Instruments, Worcestershire, UK) according to the manufacturer's recommendations. The combined data from the two measurements show that the particles have a monomodal distribution with all detectable events occurring between 0.6 and 4 microns volume mean diameter (VMD) with a peak at about 1.3 microns. Had a VMD. On the contrary, when BisEDT was prepared by the method of the prior art (Domenico et al., 1997 Antimicrob. Agents Chemother. 41 (8): 1697-1703), most of the particles were heterodisperse and significantly large in size, resulting in VMD. It became impossible to characterize based on.

Example 2
Colony Biofilm Model of Chronic Wound Infection: Inhibition by BT Compounds Bacteria present in chronic wounds essentially follow the method described (Anderl et al., 2003 Antimicrob Agents Chemother) to adopt the biofilm lifestyle. 47: 1251-56; Walters et al., 2003 Antimicrob Agents Chemother 47: 317; Wentland et al., 1996 Biotchnol. Prog. 12: 316; Zheng et al., Ic), ic. For the effect on bacterial cell survival using biofilm, BT was applied to biofilm. I inspected it.

  Briefly, colony biofilms were grown on 10% trypsin soy agar for 24 hours and transferred to Mueller Hinton plates with treatment. After the treatment, the biofilm was dispersed in peptone water containing 2% w / v glutathione (BT neutralization), serially diluted with peptone water, and then spotted on a counting plate. Two bacteria isolated from chronic wounds were used separately in generating colony biofilms for testing. These were the Gram-negative strain of Pseudomonas aeruginosa and the gram-positive methicillin-resistant Staphylococcus aureus (MRSA).

  Bacterial biofilm colonies were treated essentially as described (Anderl et al., 2003 Antimicrob Agents Chemother 47: 1251-56; Walters et al., 2003 Antimicrob Agents Chemolter 19. Prog. 12: 316; Zheng et al., 2002 Antimicrob Agents Chemother 46: 900), grown on microporous membranes on agar plates. Colony biofilms exhibit many of the well-known properties of other biofilm models, for example, they were composed of densely aggregated cells in a highly hydrated matrix. As reported elsewhere (Brown et al., J Surg Res 56: 562; Millward et al., 1989 Microbios 58: 155; Dutch et al., 1995 J Pharm Pharmacol 47: 1094; Trower. Med Microbiol 46;: 425), it was observed that bacteria in colony biofilms also showed significantly reduced antimicrobial susceptibility upon quantification in the higher performance in vitro biofilm reactors. Colony biofilms developed immediately and reproducibly in large numbers. According to a non-limiting theory, this colony biofilm model shares some of the characteristics of infected wounds, with bacteria being aerial with biofilm and nutrients supplied from below a minimal amount of fluid. Grew at the interface. Various sources of nutrients, such as blood agar, which is believed to mimic in vivo trophic conditions, were used to culture colony biofilms.

Colony biofilms were prepared by inoculating 5 μl spots of planktonic bacterial liquid medium onto 25 mm diameter polycarbonate filter membranes. Prior to inoculation, the membrane was sterilized by exposing the membrane to UV light for 10 minutes per side. The inoculum was grown overnight in 37 ° C. bacterial medium, diluted with fresh medium to an optical density of 0.1 at 600 nm and then attached to the membrane. The membrane was then placed on an agar plate containing growth medium. Then, the plate was covered, and the plate was inverted and placed in a 37 ° C. incubator. Every 24 hours, membranes and colony biofilms were transferred to new plates using sterile forceps. Colony biofilms were used in experiments, typically 48 hours after development, at which time there were approximately 10 ⁹ bacteria per membrane. The colony biofilm method has been successfully used to culture a wide variety of single-species and mixed-species biofilms.

To measure susceptibility to antimicrobial agents (eg, BT compounds such as BT compound combinations; antibiotics; and BT compound-antibiotic combinations), colony biofilms supplemented with candidate antimicrobial agents. It was transferred to agar plate. Colony biofilms were transferred daily to new treatment plates when the duration of exposure to antimicrobial treatment exceeded 24 hours. At the end of the treatment period, the colony biofilm was placed in a tube containing 10 ml of buffer and vortexed for 1-2 minutes to disperse the biofilm. In some cases, the sample had to be briefly treated with a tissue homogenizer to break cell aggregates. The resulting cell suspension was then serially diluted and allowed to stand and the surviving bacteria were enumerated and reported as colony forming units (CFU) per unit area. Survival data was analyzed using the log ₁₀ transform.

  For each type of bacterial biofilm colony culture (Pseudomonas aeruginosa, PA; methicillin-resistant Staphylococcus aureus, MRSA or SA), 5 antibiotics and 13 BT compounds were tested. Antimicrobial agents tested for PA include BisEDT and BTs referred to herein as Compounds 2B, 4, 5, 6, 8-2, 9, 10, 11 and 15 (see Table 1) and antibiotics. Tobramycin, amikacin, imipenim, cefazolin, and ciprofloxacin. Antimicrobial agents tested against SA include BisEDT and BTs designated as compounds 2B, 4, 5, 6, 8-2, 9, 10 and 11 (see Table 1), and the antibiotics rifampicin, daptomycin, minocycline, It contained ampicillin and vancomycin. Antibiotics were tested at concentrations approximately 10-400 times the minimum inhibitory concentration (MIC) according to established microbiological methodologies, as described above in the "Brief Description of the Drawings".

  Seven BT compounds showed significant effects on PA bacterial survival at the concentrations tested, and two BT compounds demonstrated significant effects on MRSA survival at the concentrations tested; bacterial survival Representative results showing the effect of BT on bis compounds are shown in Figure 1 for BisEDT and BT compound 2B (tested against PA) and in Figure 2 for BT compounds 2B and 8-2 (tested against SA). And both are relative to the effect of the indicated antibiotic. As also shown in FIGS. 1 and 2, the inclusion of the indicated BT compound in combination with the indicated antibiotic has a synergistic effect, thereby reducing the ability of the bacteria to survive the antibiotic alone or the BT compound. Enhanced compared to either antibacterial effect alone. The effect of Compound 15 (BisEDT / 2-hydroxy, propanethiol (1: 1/1)) at a concentration of 80 μg / mL in the PA survival assay was obtained using the combination of 1600 μg / mL AMK + 80 μg / mL BisEDT (FIG. It showed an effect (not shown) comparable to that of 1).

Example 3
Inhibition of Chronic Wound Infection by Drip Flow Biofilm Model BT Compounds Drip flow biofilms are an approved and reliable model for forming bacterial biofilms and testing the effects of candidate antimicrobial compounds on them. is there. The drip flow biofilm forms on the coupons (substrates) in the grooves of the drip flow reactor. Many different types of materials can be used as substrates for bacterial biofilm formation, such as frosted glass microscope slides. The nutrient liquid medium is added to the cell chamber of the drip flow bioreactor by dropwise addition near the top, then the medium is placed in the chamber, followed by a 10 ° C. ramp to the length of the coupon.

Growing a biofilm in a drip flow bioreactor, with a BT compound alone or in combination with an antibiotic compound, and / or with an antibiotic compound alone or in combination with another antibacterial agent including a BT compound Or other conventional or candidate treatments for chronic wounds. The BT compound is thus characterized for its effect on bacterial biofilm in the drip flow reactor. Biofilms in drip flow reactors are prepared according to established methodologies (eg, Stewart et al., 2001 J Appl Microbiol. 91: 525; Xu et al., 1998 Appl. Environ. Microbiol. 64: 4035). . This design involves culturing biofilms on tilted polystyrene coupons in a covered chamber. An exemplary medium is 5% v / v adult donor bovine serum (pH 6.8) that mimics high serum protein and very low iron concentrations, similar to in vivo biofilm growth conditions such as chronic wounds. supplemented, 1 g / l _{glucose, 0.5g / l NH 4 NO 3} , 0.25g / l KCl, 0.25g / l KH 2 PO 4, containing _{0.25g / l MgSO 4 · 7H 2} O There is. The medium is added drop-wise (5 ml / hr) onto 4 coupons contained in 4 separate parallel chambers, each 10 cm x 1.9 cm and 1.9 cm deep. The reactor, divided into chambers, was assembled from polysulfone plastic. Each chamber is fitted with an individual removable plastic lid that can be tightly closed. The biofilm reactor is placed in a 37 ° C. incubator and warmed by passing the bacterial cell culture through an aluminum heating bath held in the incubator. This method reproduces the antibiotic-resistant phenotype observed within certain biofilms, while mimicking the environment of low fluid shear and its closeness to the air interface features of chronic wounds It provides continuous recruitment and is compatible with numerous analytical methods that characterize and monitor the efficacy of introduced candidate antimicrobial regimens. The drip flow reactor has been successfully used for culturing a wide variety of pure and mixed-species biofilms. Biofilms are typically allowed to grow for 2-5 days prior to application of the antimicrobial agent.

  To measure the effect of an anti-biofilm agent on a biofilm grown in a drip flow reactor, a fluid stream passing over the biofilm is treated with a desired treatment formulation (eg, one or more BT compounds and / or Or modified or supplemented with one or more antibiotics, or controls, and / or other candidate agents). The flow is allowed to continue for the specified treatment period. The treated biofilm coupon is then briefly removed from the reactor and the biofilm scraped into a beaker containing 10 ml of buffer. Samples are briefly treated with a tissue homogenizer (typically 30 seconds to 1 minute) to disperse bacterial aggregates. Serially dilute the suspension and enumerate viable microorganisms according to standard microbiological methodology.

Example 4
Inhibition of Keratinocyte Scratch Repair by Wound Biofilms: Biofilm Inhibition by BT Compounds This example modifies the established in vitro keratinocyte scratch model of wound healing to demonstrate biofilm-related wound pathology. And to reach models with relevance to wound healing, particularly acute or chronic wounds or the wound-containing biofilms described herein. The keratinocyte scratch model for the effects of chronic wound biofilms allows the cultivation of mammalian (eg human) keratinocyte and bacterial biofilm populations to proceed in separate chambers in fluid contact with each other, Allows evaluation of the effect of conditions that affect the effect of soluble components assimilated by biofilms on keratinocyte wound healing events.

  Neonatal human foreskin cells are cultured as monolayers in treated plastic dishes in which controlled "wounds" or scratches are formed by mechanical means (e.g., sterile scalpel, razor, By physical disruption of the monophase, such as scratching the essentially linear cell-free zone between the regions of the monolayer using a suitable tool such as a cell scraper, tweezers, or other instrument). The in vitro keratinocyte monolayer model system is known to undergo structural and functional processes of cells in response to wound events in a manner that mimics wound healing in vivo. According to the embodiments disclosed herein, the effect of the presence of bacterial biofilms on such processes, for example on wound healing time, was observed and selected in these and related embodiments. The effect of the presence of candidate antimicrobial (eg, antibacterial and antibiofilm) treatments is also evaluated.

  Wound keratinocyte monolayers cultured in the presence of biofilms were investigated by morphological, biochemical, molecular genetic, cytophysiological and other parameters to show that the introduction of BT compounds was It is determined whether the destructive effects of the film are altered (eg, increased or decreased in a statistically significant manner relative to the appropriate control). To assess the effect of such treatments on the effects of biofilms on the model wound healing process after examining the toxicity of each BT compound treatment, wounds were first designed with each BT compound alone, and with BT compound. Expose to the combination.

  In an exemplary embodiment, a membrane (eg, a TransWell membrane insert, etc.) retained in a previous tissue culture well and in fluid communication with a keratinocyte monolayer that is scratched to initiate the wound healing process. Incubate day 3 biofilms above. Biofilms cultured from genuine acute or chronic wounds are contemplated for use in these and related embodiments.

  Thus, an in vitro system was developed to evaluate the effect of soluble biofilm components on human keratinocyte migration and proliferation. The system separates biofilm and keratinocytes with a dialysis membrane. Keratinocytes were cultured from neonatal foreskin as previously described (Fleckman et al., 1997 J Invest. Dermatol. 109: 36; Piepkorn et al., 1987 J Invest. Dermatol. 88: 215-219). Develop as a confluent monolayer on glass coverslips. The monolayer of keratinocytes can then be scratched to produce a "wound" of uniform width, after which the cell repair process can be monitored (eg 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). The chamber is then assembled using aseptic technique by placing the artificial wound in the bottom of a sterile double-sided chamber. Keratinocyte growth medium (EpiLife) with or without antibiotics and / or bismuth-thiol is filled on both sides of the chamber. The uninoculated system is used as a control.

  The system is inoculated with bacteria isolated from the wound and incubated in static conditions for 2 hours to allow the bacteria to adhere to the surface in the upper chamber. Following the attachment period, liquid media flow is initiated in the upper chamber to remove non-attached cells. Thereafter, the non-adherent cells are washed out to continue the flow of medium at a rate that minimizes the growth of plactonic cells in the upper chamber. After an incubation time in the range of 6-48 hours, the system (keratinocyte monolayer on coverslips and bacterial biofilm on membrane substrate) is disassembled and coverslips removed and analyzed. In a related embodiment, the mature biofilm is allowed to grow in the upper chamber prior to assembly of the chamber. In other related embodiments, at least one indicator of scratch healing is altered (eg, statistically relative to an appropriate control), eg, the time elapsed while wound repair occurs or other signs of wound repair. Agent or combination of agents (for increasing or decreasing in a significant manner) to a keratinocyte scratch repair, such as a BT compound or a potential synergistic BT compound + antibiotic combination ( For example, BT compounds provided herein, such as BT provided in particulate form, as well as amikacin, ampicillin, cefazoline, cefepime, chloramphenicol, ciprofloxacin, clindamycin (or other lincosamide antibiotics. ), Daptomycin (Cubicin®), doxycycline, gatifloxacin Biofilms and scratches to determine the effects of gentamicin, imipenim, levofloxacin, linezolid (Zyvox®), minocycline, nafcillin, paromomycin, rifampin, sulfamethoxazole, tobramycin and vancomycin). Separate co-cultures of wounded keratinocyte monolayers are performed in the absence or presence of one or more BT compounds, optionally with or without one or more antibiotics (eg, 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).

Example 5
Inhibition of Keratinocyte Scratch Repair by Wound Biofilms Isolated human keratinocytes were cultured on glass coverslips to create scratches according to the methodology described in Example 4 above. Wounded cultures were maintained on membrane supports in fluid communication with keratinocyte cultures, under culture conditions, alone or in the presence of co-cultured biofilms. The time interval of scratch closure was then measured while keratinocyte cell development and / or migration reestablished the keratinocyte monolayer across the scratch zone. FIG. 3 shows the effect of the presence of the biofilm in fluid communication (but not in direct contact) on the healing time of scratched keratinocyte monolayers.

  Thus, in certain embodiments, a scratched cell (eg, keratinocyte or fibroblast) monolayer is cultured in the presence of a bacterial biofilm with or without a candidate anti-biofilm agent. Evaluating the index of healing of the scratched cell monolayer in the absence and presence of the candidate anti-biofilm agent, wherein at least one index of healing is facilitated. A drug (eg, a BT compound such as a substantially monodisperse BT microparticle suspension described herein) alone or an antibiotic, eg, amikacin, ampicillin, cefazoline, cefepime, chloramphenicol, ciproff Loxacin, clindamycin, daptomycin (Cubicin®), doxycycline, gatifloxacin, gentamysis Acute, chronic wounds , Or a method of identifying an agent that treats a chronic wound, identified as a suitable agent to treat a wound comprising a biofilm.

Example 6
Synergistic Bismuth-Thiol (BT) Antibiotic Combination This example illustrates the combination of one or more bismuth-thiol compounds with one or more antibiotics against various bacterial species and strains, including multiple antibiotic resistant strains. An example of a demonstrated synergistic effect of the combination is shown.

  Materials and Methods susceptibility testing, NCCLS protocol (National Committee for Clinical Laboratory Standards (1997) Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically:... Approved Standard M7-A2 and Informational Supplement M100-S10 NCCLS, Wayne, PA , USA) and by broth dilution in 96-well tissue culture plates (Nalge Nunc International, Denmark).

Briefly, an overnight bacterial culture was utilized to prepare a 0.5 McFarland standard suspension that was further 1:50 in cation-conditioned Mueller-Hinton broth medium (BBL, Cockeysville, MD, USA). Diluted (˜2 × 10 ⁶ cfu / ml). BT (prepared as described above) and antibiotics were added in increasing concentrations to keep the final volume constant at 0.2 mL. Cultures were incubated at 37 ° C. for 24 hours and turbidity assessed by absorbance at 630 nm using an ELISA plate reader (Biotek Instruments, Winooski, VT, USA) according to the manufacturer's recommendations. The minimum inhibitory concentration (MIC) was expressed as the lowest drug concentration that inhibited growth for 24 hours. Viable bacterial counts (cfu / mL) were determined by standard plating on nutrient agar. The minimum bactericidal concentration (MBC) was expressed as the drug concentration that reduced the initial viability by 99.9% at 24 hours after inoculation.

  The checkerboard method was used to assess the activity of antimicrobial combinations. The partial inhibitory concentration index (FICI) and the partial bactericidal concentration index (FBCI) were calculated using Eliopoulos et al. Eliopoulos and Meollering, (1996) Antimicrobiological combinations.). Synergy was defined as an FICI or FBCI index of 0.5 or less, no interaction above 0.5 and 4 or less, and antagonisticity above 4 (Odds, FC (2003) Synergy, antagonism). , And what the cherubord puts between tem. Journal of Antimicrobial Chemotherapy 52: 1). As before, a 4-fold or greater reduction in antibiotic concentration was also defined as synergism.

ＢＥ＝０．２μｇ／ｍｌ ＢｉｓＥＤＴ；菌株は、Ｗｉｎｔｈｒｏｐ−Ｕｎｉｖｅｒｓｉｔｙ Ｈｏｓｐｉｔａｌ， Ｍｉｎｅｏｌａ， ＮＹのＣｌｉｎｉｃａｌ Ｍｉｃｒｏｂｉｏｌｏｇｙ Ｌａｂｏｒａｔｏｒｙから得た。ナフシリンは、Ｓｉｇｍａ（Ｓｔ． Ｌｏｕｉｓ， ＭＯ）から得た。

ＢＥ＝０．２μｇ／ｍｌ ＢｉｓＥＤＴ；菌株は、Ｗｉｎｔｈｒｏｐ−Ｕｎｉｖｅｒｓｉｔｙ Ｈｏｓｐｉｔａｌ， Ｍｉｎｅｏｌａ， ＮＹのＣｌｉｎｉｃａｌ Ｍｉｃｒｏｂｉｏｌｏｇｙ Ｌａｂｏｒａｔｏｒｙから得た。ナフシリンは、Ｓｉｇｍａから得た。

ＢＥ＝０．２μｇ／ｍｌ ＢｉｓＥＤＴ；菌株Ｓ２４４６−３は、Ｗｉｎｔｈｒｏｐ−Ｕｎｉｖｅｒｓｉｔｙ Ｈｏｓｐｉｔａｌ， Ｍｉｎｅｏｌａ， ＮＹのＣｌｉｎｉｃａｌ Ｍｉｃｒｏｂｉｏｌｏｇｙ Ｌａｂｏｒａｔｏｒｙから得た。抗体は、Ｓｉｇｍａから得た。

ＧＭ＝ゲンタマイシン；菌株Ｓ２４００−１は、Ｗｉｎｔｈｒｏｐ−Ｕｎｉｖｅｒｓｉｔｙ Ｈｏｓｐｉｔａｌ， Ｍｉｎｅｏｌａ， ＮＹのＣｌｉｎｉｃａｌ Ｍｉｃｒｏｂｉｏｌｏｇｙ Ｌａｂｏｒａｔｏｒｙから得た。ゲンタマイシンは、ＷｉｎｔｈｒｏｐのＰｈａｒｍａｃｙ Ｄｅｐａｒｔｍｅｎｔから得た。太字は相乗性。

μｇ／ｍｌでのデータ；菌株Ｓ２４００−１は、Ｗｉｎｔｈｒｏｐ−Ｕｎｉｖｅｒｓｉｔｙ Ｈｏｓｐｉｔａｌ， Ｍｉｎｅｏｌａ， ＮＹのＣｌｉｎｉｃａｌ Ｍｉｃｒｏｂｉｏｌｏｇｙ Ｌａｂｏｒａｔｏｒｙから得た。抗生物質は、ＷｉｎｔｈｒｏｐのＰｈａｒｍａｃｙ Ｄｅｐａｒｔｍｅｎｔから得た。

μｇ／ｍｌでのデータ；菌株Ｓ２４００−１は、Ｗｉｎｔｈｒｏｐ−Ｕｎｉｖｅｒｓｉｔｙ Ｈｏｓｐｉｔａｌ， Ｍｉｎｅｏｌａ， ＮＹのＣｌｉｎｉｃａｌ Ｍｉｃｒｏｂｉｏｌｏｇｙ Ｌａｂｏｒａｔｏｒｙから得た。抗生物質は、ＷｉｎｔｈｒｏｐのＰｈａｒｍａｃｙ Ｄｅｐａｒｔｍｅｎｔから得た。

μｇ／ｍｌでのデータ；菌株Ｓ２４００−１は、Ｗｉｎｔｈｒｏｐ−Ｕｎｉｖｅｒｓｉｔｙ Ｈｏｓｐｉｔａｌ， Ｍｉｎｅｏｌａ， ＮＹのＣｌｉｎｉｃａｌ Ｍｉｃｒｏｂｉｏｌｏｇｙ Ｌａｂｏｒａｔｏｒｙから得た。抗生物質は、ＷｉｎｔｈｒｏｐのＰｈａｒｍａｃｙ Ｄｅｐａｒｔｍｅｎｔから得た。

μｇ／ｍｌでのデータ；抗生物質は、Ｗｉｎｔｈｒｏｐ−Ｕｎｉｖｅｒｓｉｔｙ Ｈｏｓｐｉｔａｌ， Ｍｉｎｅｏｌａ， ＮＹのＰｈａｒｍａｃｙ Ｄｅｐａｒｔｍｅｎｔから得た。

ＡＢ＝抗生物質；ＣＭ＝クロラムフェニコール；ＡＭ＝アンピシリン；ＢＥ＝０．３μｇ／ｍｌのＢｉｓＥＤＴ、菌株は、Ｄｅｐａｒｔｍｅｎｔ ｏｆ Ｍｏｌｅｃｕｌａｒ Ｇｅｎｅｔｉｃｓ ａｎｄ Ｃｅｌｌ Ｂｉｏｌｏｇｙ，Ｔｈｅ Ｕｎｉｖｅｒｓｉｉｔｙ ｏｆ Ｃｈｉｃａｇｏ， Ｃｈｉｃａｇｏ，ＩＬのＤｒ． ＭＪ Ｃａｓａｄａｂａｎの研究室から得た。抗生物質は、Ｗｉｎｔｈｒｏｐ−Ｕｎｉｖｅｒｓｉｔｙ Ｈｏｓｐｉｔａｌ， Ｍｉｎｅｏｌａ， ＮＹのＰｈａｒｍａｃｙ Ｄｅｐａｒｔｍｅｎｔから得た。

ＤＯＸ＝ドキシサイクリン；ＢＥ＝０．３μｇ／ｍｌのＢｉｓＥＤＴ；菌株は、Ｄｅｐａｒｔｍｅｎｔ ｏｆ Ｂａｃｔｅｒｉｏｌｏｇｙ， Ｔｈｅ Ｕｎｉｖｅｒｓｉｔｙ ｏｆ Ｂｒｉｓｔｏｌ， Ｂｒｉｓｔｏｌ， ＵＫのＤｒ．Ｉ Ｃｈｏｐｒａの研究室から得た。抗生物質は、Ｗｉｎｔｈｒｏｐ−Ｕｎｉｖｅｒｓｉｔｙ Ｈｏｓｐｉｔａｌ， Ｍｉｎｅｏｌａ， ＮＹのＰｈａｒｍａｃｙ Ｄｅｐａｒｔｍｅｎｔから得た。

Ａｇｒ＝アミノグリコシド耐性；ＮＮ＝トブラマイシン；ＰＡ＝シュードモナス・エルギノーサ；ＢＥ＝ＢｉｓＥＤＴ、０．３μｇ／ｍｌ；菌株は、Ｄｅｐａｒｔｍｅｎｔ ｏｆ Ｍｉｃｒｏｂｉｏｌｏｇｙ ａｎｄ Ｉｍｍｕｎｏｌｏｇｙ， Ｑｕｅｅｎｓ Ｕｎｉｖｅｒｓｉｔｙ， Ｏｎｔａｒｉｏ， ＣＮのＤｒ． Ｋ． Ｐｏｏｌｅの研究室から得た。トブラマイシンは、Ｗｉｎｔｈｒｏｐ−Ｕｎｉｖｅｒｓｉｔｙ Ｈｏｓｐｉｔａｌ， Ｍｉｎｅｏｌａ， ＮＹのＰｈａｒｍａｃｙ Ｄｅｐａｒｔｍｅｎｔから得た。

ＮＮ＝トブラマイシン；ＢＥ＝ＢｉｓＥＤＴ、０．４μｇ／ｍｌ；菌株は、Ｄｅｐａｒｔｍｅｎｔ ｏｆ Ｐｅｄｉａｔｒｉｃｓ ａｎｄ Ｃｏｍｍｕｎｉｃａｂｌｅ Ｄｉｓｅａｓｅ， Ｕｎｉｖｅｒｓｉｔｙ ｏｆ Ｍｉｃｈｉｇａｎ， Ａｎｎ Ａｒｂｏｒ， ＭＩのＤｒ． Ｊ． Ｊ． ＬｉＰｕｍａの研究室から；そして同じくＶｅｌｏｉｒａ．他，２００３により得た。トブラマイシンは、Ｗｉｎｔｈｒｏｐ−Ｕｎｉｖｅｒｓｉｔｙ Ｈｏｓｐｉｔａｌ， Ｍｉｎｅｏｌａ， ＮＹのＰｈａｒｍａｃｙ Ｄｅｐａｒｔｍｅｎｔから得た。

ＮＮ＝トブラマイシン；ＢＥ＝ＢｉｓＥＤＴ、０．４μｇ／ｍｌ；菌株は、Ｄｅｐａｒｔｍｅｎｔ ｏｆ Ｐｅｄｉａｔｒｉｃｓ ａｎｄ Ｃｏｍｍｕｎｉｃａｂｌｅ Ｄｉｓｅａｓｅ， Ｕｎｉｖｅｒｓｉｔｙ ｏｆ Ｍｉｃｈｉｇａｎ， Ａｎｎ Ａｒｂｏｒ， ＭＩのＤｒ． Ｊ． Ｊ． ＬｉＰｕｍａの研究室から；そして同じくＶｅｌｏｉｒａ．他，２００３により得た。トブラマイシンは、Ｗｉｎｔｈｒｏｐ−Ｕｎｉｖｅｒｓｉｔｙ Ｈｏｓｐｉｔａｌ， Ｍｉｎｅｏｌａ， ＮＹのＰｈａｒｍａｃｙ Ｄｅｐａｒｔｍｅｎｔから得た。

ＮＮ＝トブラマイシン；ＢＥ＝ＢｉｓＥＤＴ、０．８μｇ／ｍｌ；Ｌｉｐｏ−ＢＥ−ＮＮ＝リポソームＢＥ−ＮＮ；菌株は、Ｄｅｐａｒｔｍｅｎｔ ｏｆ Ｃｈｅｍｉｓｔｒｙ ａｎｄ Ｂｉｏｃｈｅｍｉｓｔｒｙ， Ｌａｕｒｅｎｔｉａｎ Ｕｎｉｖｅｒｓｉｔｙ， Ｏｎｔａｒｉｏ， ＣＮのＤｒ． Ａ． Ｏｍｒｉの研究室から得た（Ｍ株は、粘液様Ｂ．セパシア；ＰＡ＝Ｐ．エルギノーサ；ＳＡ＝Ｓ．アウレウス）。トブラマイシンは、Ｗｉｎｔｈｒｏｐ−Ｕｎｉｖｅｒｓｉｔｙ Ｈｏｓｐｉｔａｌ， Ｍｉｎｅｏｌａ， ＮＹのＰｈａｒｍａｃｙ Ｄｅｐａｒｔｍｅｎｔから得た。

ＮＮ＝トブラマイシン；ＢＥ＝ＢｉｓＥＤＴ、０．８μｇ／ｍｌ；Ｌｉｐｏ−ＢＥ−ＮＮ＝リポソームＢＥ−ＮＮ；菌株は、Ｄｅｐａｒｔｍｅｎｔ ｏｆ Ｃｈｅｍｉｓｔｒｙ ａｎｄ Ｂｉｏｃｈｅｍｉｓｔｒｙ， Ｌａｕｒｅｎｔｉａｎ Ｕｎｉｖｅｒｓｉｔｙ， Ｏｎｔａｒｉｏ， ＣＮのＤｒ． Ａ． Ｏｍｒｉの研究室から得た（Ｍ株は、粘液様Ｂ．セパシア；ＰＡ＝Ｐ．エルギノーサ；ＳＡ＝Ｓ．アウレウス）。トブラマイシンは、Ｗｉｎｔｈｒｏｐ−Ｕｎｉｖｅｒｓｉｔｙ Ｈｏｓｐｉｔａｌ， Ｍｉｎｅｏｌａ， ＮＹのＰｈａｒｍａｃｙ Ｄｅｐａｒｔｍｅｎｔから得た。

ＢＥ＝ＢｉｓＥＤＴ；ＮａＰＹＲ＝ナトリウムピリチオン；化学薬品は、Ｓｉｇｍａ Ａｌｄｒｉｃｈから得た。太字は相乗性。示された菌株は、Ａｍｅｒｉｃａｎ Ｔｙｐｅ Ｃｕｌｔｕｒｅ Ｃｏｌｌｅｃｔｉｏｎ （ＡＴＣＣ， Ｍａｎａｓｓａｓ， ＶＡ）からのものであった。 The results are shown in Tables 2-17.

BE = 0.2 μg / ml BisEDT; strains were obtained from Clinical Microbiology Laboratory of Winthrop-University Hospital, Mineola, NY. Nafcillin was obtained from Sigma (St. Louis, MO).

BE = 0.2 μg / ml BisEDT; strains were obtained from Clinical Microbiology Laboratory of Winthrop-University Hospital, Mineola, NY. Nafcillin was obtained from Sigma.

BE = 0.2 μg / ml BisEDT; strain S2446-3 was obtained from Clinical Microbiology Laboratory of Winthrop-University Hospital, Mineola, NY. Antibodies were obtained from Sigma.

GM = gentamicin; Strain S2400-1 was obtained from Clinical Microbiology Laboratory of Winthrop-University Hospital, Mineola, NY. Gentamicin was obtained from Pharmacy Department of Winthrop. Bold is synergistic.

Data at μg / ml; Strain S2400-1 was obtained from Clinical Microbiology Laboratory at Winthrop-University Hospital, Mineola, NY. Antibiotics were obtained from Pharmacy Department, Winthrop.

Data at μg / ml; Strain S2400-1 was obtained from Clinical Microbiology Laboratory at Winthrop-University Hospital, Mineola, NY. Antibiotics were obtained from Pharmacy Department, Winthrop.

Data at μg / ml; Strain S2400-1 was obtained from Clinical Microbiology Laboratory at Winthrop-University Hospital, Mineola, NY. Antibiotics were obtained from Pharmacy Department, Winthrop.

Data in μg / ml; Antibiotics were obtained from Pharmacy Department, Winthrop-University Hospital, Mineola, NY.

AB = antibiotic; CM = chloramphenicol; AM = ampicillin; BE = 0.3 μg / ml of BisEDT, strain is Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago. Obtained from MJ Casadaban's laboratory. Antibiotics were obtained from the Pharmacy Department of Winthrop-University Hospital, Mineola, NY.

DOX = doxycycline; BE = 0.3 μg / ml of BisEDT; strains are Department of Bacteriology, The University of Bristol, Bristol, UK Dr. I Chopra's laboratory. Antibiotics were obtained from the Pharmacy Department of Winthrop-University Hospital, Mineola, NY.

Agr = Aminoglycoside resistance; NN = Tobramycin; PA = Pseudomonas aeruginosa; BE = BisEDT, 0.3 μg / ml; Strains are Department of Microbiology and Immunology, Queens University, Inc. K. Obtained from Poole's laboratory. Tobramycin was obtained from the Pharmacy Department of Winthrop-University Hospital, Mineola, NY.

NN = Tobramycin; BE = BisEDT, 0.4 μg / ml; J. J. From the LiPuma lab; and also Veloira. Et al., 2003. Tobramycin was obtained from the Pharmacy Department of Winthrop-University Hospital, Mineola, NY.

NN = Tobramycin; BE = BisEDT, 0.4 μg / ml; J. J. From the LiPuma lab; and also Veloira. Et al., 2003. Tobramycin was obtained from the Pharmacy Department of Winthrop-University Hospital, Mineola, NY.

BE = BisEDT, 0.8 μg / ml; Lipo-BE-NN = liposome BE-NN; strains are Department of Chemistry and Biochemistry, Laurentian University, CN. A. Obtained from Omri's laboratory (M strain is mucoid B. cepacia; PA = P. Aeruginosa; SA = S. Aureus). Tobramycin was obtained from the Pharmacy Department of Winthrop-University Hospital, Mineola, NY.

BE = BisEDT, 0.8 μg / ml; Lipo-BE-NN = liposome BE-NN; strains are Department of Chemistry and Biochemistry, Laurentian University, CN. A. Obtained from Omri's laboratory (M strain is mucoid B. cepacia; PA = P. Aeruginosa; SA = S. Aureus). Tobramycin was obtained from the Pharmacy Department of Winthrop-University Hospital, Mineola, NY.

BE = BisEDT; NaPYR = sodium pyrithione; chemicals were obtained from Sigma Aldrich. Bold is synergistic. The strains shown were from the American Type Culture Collection (ATCC, Manassas, VA).

Example 7
Comparison of the effects of bismuth-thiol (BT) and antibiotics on Gram-positive and Gram-negative bacteria including antibiotic resistant strains In this example, the in vitro activity of BisEDT and the comparative agent was compared to the Gram positive and It was evaluated against multiple clinical isolates of negative bacteria.

Materials and Methods Test compounds and test concentration ranges are as follows: BisEDT (Domenico et al 1997; Domenico et al., Antimicrob. Agents. Chemother. 45 (5): 1417-1421, and Example 1). 16-0.015 [mu] g / mL; Linezolid (ChemPacifica Inc., # 35710), 64-0.06 [mu] g / mL; Daptomycin (Cubist Pharmaceuticals # MCB2007), 32-0.03 [mu] g / ml and 16-0.015 [mu] g / mL; Vancomycin (Sigma-Aldrich, St. Louis, MO, # V2002), 64-0.06 μg / mL; Ceftazidime (Sigma # C3809), 6 4-0.06 μg / mL and 32-0.03 μg / mL; imipenem (USP, NJ, # 1337809), 16-0.015 μg / mL and 8-0.008 μg / mL; ciprofloxacin (US Pharmacopoeia, # IOC265), 32-0.03 μg / mL and 4-0.004 μg / mL; gentamicin (Sigma # G3632) 32-0.03 μg / mL and 16-0.015 μg / mL. All test substances except gentamicin were dissolved in DMSO and gentamicin was dissolved in water. Stock solutions were prepared at 40 times the highest concentration in the test plate. The final concentration of DMSO in the test system was 2.5%.

Organisms Organisms were obtained from the following clinical laboratories: CHP, Clarian Health Partners, Indianapolis, IN; UCLA, University of California, California, K. L. Cener, Public Health Research Institute Tuberculosis Center, New York, NY; ATCC, American Type Culture Collection, Manassas, VA; Mt. Sina. , Mount Sinai Hospital, New York, NY; UCSF, University of California San Francisco General Hospital, San Francisco, CA; Bronson Hospital, Bronson Methodist Hospital, Kalamazoo, MI; isolates for quality management, American Type Culture Collection (ATCC , Manassas, VA). Organisms were streaked for isolation on the agar medium appropriate for each organism. Colonies were swabbed from isolation plates and placed in suspension in a suitable broth containing cryoprotectant. The suspension was aliquoted into cold storage vials and kept at -80 ° C. Abbreviations: BisEDT, bismuth-1,2-ethanedithiol; LZD, linezolid; DAP, daptomycin; VA, vancomycin; CAZ, ceftazidime; IPM, imipenem; CIP, ciprofloxacin; GM, gentamicin; MSSA, methicillin-sensitive staphylo. Coccus aureus; Quality control strain of CLSI QC, Clinical and Laboratory Standards Institute; MRSA, methicillin-resistant Staphylococcus aureus; CA-MRSA, community-acquired methicillin-resistant Staphylococcus aureusus;・ Epidermidis; MRSE, methicillin-resistant Staphylococcus epidermidis; VSE, vancomycin sensitivity Enterococcus.

Isolates from freezing vials in the appropriate medium (Trypticase Soy Agar (Becton-Dickinson, Sparks, MD) for most organisms) or Trypticase Soy Agar for Streptococcus + 5% Sheep blood (Clevelentic, Scl.). Streaked up. The plates were incubated overnight at 35 ° C. Quality control organisms were included. The medium used for the MIC assay was Mueller Hinton II Broth (MHB II-Becton Dickinson, # 212322) for most organisms. MHBII was supplemented with 2% lysed horse blood (Cleveland Scientific Lot # H13913) to adapt to the development of Streptococcus pyogenes and Streptococcus agalactiae. Media was prepared at 102.5% of normal weight to offset the dilution caused by adding 5 μL of drug solution to each well of the microdilution panel. In addition, testing with daptomycin supplemented the medium with an additional 25 mg / L Ca ²⁺ .

  MIC assay, Clinical and Laboratory Standards Institute (Clinical and Laboratory Standards Institute Methods for Dilution Antimicrobial Susceptibyity Tests for Bacteria That Grow Aerobically;.. Approved Standard-Seventh Edition Clinical and Laboratory Standards Institute document M7-A7 [ISBN 1-56238- 587-9]. Clinical and Laboratory Standards Instit. ute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2006) and performed serial dilution and liquid transfer using an automatic liquid handler. Automatic liquid handlers included the Multidrop 384 (Labsystems, Helsinki, Finland), Biomek 2000 and Multimek 96 (Beckman Coulter, Fullerton CA). Wells in columns 2-12 of a standard 96-well microdilution plate (Falcon 3918) were filled with Multidrop 384, 150 μL of water for DMSO or gentamicin. Drug (300 μL) was dispensed into these plates in the appropriate row, column 1. These became mother plates in the preparation of test plates (daughter plates). Biomek 2000 completed the line transfer to the 11th row of the mother plate. The wells in row 12 contained no drug and were growth control wells in the daughter plate. 185 μL of the appropriate test medium (described above) was added to the daughter plates using a Multidrop 384. Daughter plates were prepared on a Multimek 96 instrument by transferring 5 μL of drug solution from each well of the mother plate to each corresponding well of each daughter plate in one step.

  A standard inoculum for each organism was prepared by the CLSI method (ISBN 1-56238-587-9, cited above). The suspension was prepared in MHB to equal the turbidity of 0.5 McFarland standard. The suspension was diluted 1: 9 with broth suitable for the organism. The inoculum of each organism was dispensed into vertically divided sterile reservoirs (Beckman Coulter) and used to inoculate the plates using a Biomek 2000. The daughter plate was placed on the working surface of the Biomek 2000, which was turned over so that the inoculation was from low drug concentration to high drug concentration. Biomek 2000 delivered 10 μL of the standardized inoculum to each well. This resulted in a final cell concentration of approximately 5 x 105 colony forming units / mL in the daughter plate. Thus, the wells of the daughter plate finally contained 185 μL broth, 5 μL drug solution, and 10 μL bacterial inoculum. The plates were stacked in triplicate, the top plates were capped, placed in plastic bags and most of the isolates were incubated at 37 ° C for approximately 18 hours. Plates of Streptococcus were read after 20 hours of incubation. The microplate was viewed from the bottom using a plate viewer. For each test medium, uninoculated soluble control plates were observed for evidence of drug precipitation. The MIC was read and recorded as the lowest drug concentration that inhibited the visual development of the organism.

Results All commercially available drugs were soluble at all tested concentrations in both media. BisEDT showed a trace amount of precipitate at 32 μg / mL, but did not affect the MIC readings because the inhibitory concentrations of all tested organisms were well below that concentration. On each assay day, the appropriate quality control strain was included in the MIC assay. The MIC values obtained from these strains are used as appropriate for the published quality control ranges for each drug (Clinical and Laboratory Standards Institute 1-Performance Standards Bibliographic Assistance Stimulation of the Organism Standards Institute of the United States of America). -653-0] .Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2008).

  On each assay day, the appropriate quality control strain was included in the MIC assay. The MIC values obtained from these strains are appropriately used for the quality control ranges published for each drug. -653-0]). Of the 141 types of quality control strains for which the quality control range was published, 140 types (99.3%) were within the specified range. One exception is S. Imipenem against Aureus strain 29213, one value obtained in one run was one dilution below the published QC range (0.008 μg / mL or less). All other quality control results during the implementation were within the specified quality control range.

  BisEDT is a methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant S. It demonstrated potent activity against Aureus (MRSA) and community-acquired MRSA (CA-MRSA), inhibited all tested strains at 1 μg / mL and below, and had a MIC90 value of 0 in all three organism groups. It was 0.5 μg / mL. BisEDT showed greater activity than linezolid and vancomycin, and comparable activity to daptomycin. Imipenem was more potent against MSSA than BisEDT (MIC90 = 0.03 μg / mL). However, while MRSA and CAMRSA were resistant to imipenem, BisEDT demonstrated activity comparable to that shown with MSSA. BisEDT was highly active against methicillin-sensitive and methicillin-resistant Staphylococcus epidermidis (MSSE and MRSE) with MIC90 of 0.12 and 025 μg / mL, respectively. BisEDT was more active against MSSE than any of the other agents tested except imipenem. BisEDT was the most active drug tested for MRSE.

  BisEDT demonstrated comparable activity to daptomycin, vancomycin, and imipenem against vancomycin-sensitive Enterococcus faecalis (VSEfc) with a MIC90 value of 2 μg / mL. Importantly, BisEDT was the most active drug tested against vancomycin resistant Enterococcus faecalis (VREfc) with a MIC90 value of 1 μg / mL.

  BisEDT was highly active against vancomycin-sensitive Enterococcus faecium (VSEfm), had a MIC90 value of 2 μg / mL, its activity was similar to or similar to daptomycin, and was 1 dilution higher than vancomycin. .. BisEDT and linezolid were the most active agents tested against vancomycin-resistant Enterococcus faecium (VREfm), each demonstrating a MIC90 value of 2 μg / mL. The activity of BisEDT against Streptococcus pyogenes (MIC90 value of 0.5 μg / mL) was comparable to vancomycin, greater than linezolid, and slightly lower than daptomycin and ceftazidime. The compound inhibited all tested strains below 0.5 μg / mL. In these studies, the strain at least susceptible to BisEDT was Streptococcus agalactiae, and the observed MIC90 value was 16 μg / mL. BisEDT was less active than all tested drugs except gentamicin.

  The activity of BisEDT and the comparative substance against the included Gram-negative bacteria demonstrated the efficacy of BisEDT against Acinetobacter baumannii (MIC90 value of 2 μg / mL), making BisEDT the most active compound tested. MICs were elevated in a significant number of test isolates for comparison agents, with MIC 90 values out of the range for these agents. BisEDT is a potent inhibitor of Escherichia coli and inhibited all strains below 2 μg / mL (MIC90 = 2 μg / mL). The compound was less active than imipenem but more active than ceftazidime, ciprofloxacin and gentamicin. BisEDT also demonstrated activity against Klebsiella pneumoniae with a MIC90 value of 8 μg / mL, comparable to imipenem. The relatively high MIC90 values exhibited by imipenem, ceftazidime, ciprofloxacin, and gentamicin indicated that this is a highly antibiotic resistant group of organisms. BisEDT was the most active compound tested against Pseudomonas aeruginosa with a MIC90 value of 4 μg / mL. There was a high level of resistance to the comparator in this group of test isolates.

  In summary, BisEDT exhibits broad spectrum capability against multiple clinical isolates representing multiple bacterial species, including those commonly associated with acute and chronic skin and skin structure infections in humans. The activity of BisEDT and key comparators was evaluated against 723 clinical isolates of Gram positive and Gram negative bacteria. The BT compound demonstrated broad spectrum activity, and for many of the tested organisms in this study, BisEDT was the most active compound tested for antibacterial activity. BisEDT includes MSSA, MRSA, CA-MRSA, MSSE, MRSE and S. It was most active against pyogenes and had a MIC90 value of 0.5 μg / mL or less. The potent activity is VSEfc, VREfc, VSEfm, VREfm, A. Bowmany, E. Coli, and P. Also demonstrated on aeruginosa, MIC90 values were in the range of 1-4 μg / mL. The MIC90 value is K. Pneumoniae (MIC90 = 8 μg / mL) and S. Observed with agalactiae (MIC90 = 16 μg / mL).

Example 8
Enhanced and Synergistic Activity of Particulate BT-Antibiotics This example demonstrates that particulate bismuth-thiol (BT) promotes antibiotic activity through potentiating and / or synergistic interactions.

  A major complication in treating infections is the emergence of bacterial antibiotic resistance. S. Epidermidis (MRSE) and S. Methicillin resistance in Aureus (MRSA) indeed represents multidrug resistance, which makes eradication of these pathogens very difficult. However, the hundreds of strains Staphylococcus genus tested did not show resistance to BT. In addition, subMIC concentrations of BT reduced resistance to several important antibiotics.

  Providing a graphical representation of the antibiotic anti-sensitizing effects of bismuth ethanedithiol (BisEDT) of subMIC against Staphylococcus aureus MRSA (Fig. 4), gentamicin, cefazoline, cefepime, imipenem, sulphamene Enhanced antibiotic action is shown by multiple classes of antibiotics, including methoxazole and levofloxacin. That is, BisEDT nonspecifically enhanced the activity of most antibiotics.

１２のＭＲＳＡ臨床単離物をＢＨＩＧ／Ｘ中で発育させて、０〜０．１μｇ／ｍＬ ＢｉｓＥＤＴの存在下で抗生物質の系列希釈物に暴露した。μｇ／ｍＬで計算されたＭＩＣおよびＢＰＣは、少なくとも３回のトライアルから得た平均±標準偏差である。右側の列は、抗生物質感受性（Ｓ）および耐性（Ｒ）に関する標準ＭＩＣを列挙している。 Antimicrobial susceptibility testing by broth dilution was performed on 12 MRSA strains with multiple antibiotics in combination with subMIC levels of BisEDT. Both biofilm preventive concentration (BPC) and minimum inhibitory concentration (MIC) were measured in a special biofilm medium (BHIG / X). Gentamicin and cefazolin MIC and BPC were reduced by subMIC BisEDT (BisEDT MIC, 0.2-0.4 μg / mL), but not below the sensitivity breakpoint. SubMIC BisEDT enhanced MRSA susceptibility to gatifloxacin and cefepime to near the breakpoint of sensitivity. These strains were already sensitive to vancomycin, but became more prominent in the presence of subMIC BisEDT. In general, MIC and BPC were reduced 2-5 fold by subMIC BisEDT.

Twelve MRSA clinical isolates were grown in BHIG / X and exposed to serial dilutions of antibiotics in the presence of 0-0.1 μg / mL BisEDT. MICs and BPCs calculated in μg / mL are mean ± standard deviation from at least 3 trials. The right column lists standard MICs for antibiotic sensitivity (S) and resistance (R).

１２種のセフェピム耐性ＭＲＳＡを、ｓｕｂＭＩＣのＢｉｓＥＤＴと組み合わせたセフェピムへの感受性について、ポリスチレンプレート内のＢＨＩＧ／Ｘ培地で３７℃で４８時間検査した。 Broth dilution studies of cefepime resistant MRSA isolates are shown in Table 19. BisEDT at 0.1 μg / mL significantly enhanced the inhibitory activity of cefepime in 11 out of 12 isolates. In this particular study, the data showed synergy between BisEDT and cefepime (FIC <0.5), with many of the isolates at break points of susceptibility.

Twelve cefepime-resistant MRSA were tested in BHIG / X medium in polystyrene plates for 48 hours at 37 ° C. for sensitivity of subMIC to cefepime in combination with BisEDT.

ＮＡＦまたはＧＭはμｇ／ｍＬを表わし；ＢＥは０．２μｇ／ｍＬ The results of the combination tests with nafcillin or gentamicin are shown in Table 20. BisEDT (0.2 μg / mL) in combination with nafcillin reduced the MIC90 of nafcillin over MRSA by more than 4-fold (FIC, 0.74). BisEDT in combination with gentamicin reduced gentamicin MIC90 by more than 10-fold over MRSA (FIC, 0.6). BT suppressed the resistance of all four tested gentamicin resistant isolates to clinically relevant concentrations [Domenico et al. , 2002]. The MIC of these antimicrobial agents was substantially reduced, especially for gentamicin. The broth used in these studies was Trypticase Soy Broth (TSB) containing 2% glucose, with results similar to those found in Mueller-Hinton II broth supplemented with 1% sheep blood.

NAF or GM represents μg / mL; BE is 0.2 μg / mL

  Most classes of activity of Staphylococcus epidermidis antibiotics were enhanced in the presence of BisEDT. With respect to BPC, clindamycin and gatifloxacin were combined with S. cerevisiae when combined with BisEDT. It showed significantly higher anti-biofilm activity against Epidermidis (Fig. 5). Stated differently, the clindamycin, gatifloxacin and gentamicin BPCs were reduced 50-fold, 10-fold and 4-fold respectively in the presence of subMIC BisEDT.

  Slight reductions in biofilm prophylactic concentration (BPC) were seen with minocycline, vancomycin, and cefazolin, whereas rifampicin and nafcillin were still unaffected at 0.05 μg / mL BisEDT. With 0.1 μg / mL BisEDT, no biofilm was detected irrespective of the antibiotic used, indicating that no antagonism occurred. This BisEDT concentration is S. It was similar to the Epidermidis MIC [Domenico et al. , 2003] (see FIG. 5).

  Regarding stunting, 7 of the 8 antibiotics tested were S. It was significantly enhanced in the presence of 0.1 μg / mL (0.5 μM) BisEDT versus Epidermidis (FIG. 6). The changes in MIC were most pronounced for clindamycin and gentamicin, followed by vancomycin, cefazoline, minocycline, gatifloxacin and nafcillin, with rifampicin unaffected. These strains were resistant to antibiotics (NC, CZ, GM, CM) and only cefazolin resistance was suppressed by BisEDT to clinically relevant levels.

  S. The minimum bactericidal concentration (MBC) of most antibiotics tested against Epidermidis was slightly reduced by SubMIC's BisEDT. Gentamicin showed a maximal reduction in MBC (4x to 16x) followed by cefazolin (4x to 5x), vancomycin and nafcillin (3x to 4x), minocycline and gatifloxacin (2x to 3x). ), The clindamycin and rifampicin MBCs were still largely unaffected. It is explained that clindamycin is a bacteriostat and has no bactericidal activity. Cefazolin resistance was suppressed for MBC [Domenico et al. , 2003]. These effects were additive.

  The efficacy of antimicrobial agents was also demonstrated in an in vivo rat graft infection model (Table 21). At low levels of 0.1 μg / mL BisEDT, 7 days of resistant S. It was possible to promote the prevention of Epidermidis biofilm.

^ａ 各群は動物１５匹であった。ＭＳ、メチシリン感受性Ｓ．エピデルミディス；ＭＲ、メチシリン耐性Ｓ．エピデルミディス
^ｂ ダクロン移植片に０．１ｍｇ／ｌのＢＴ、１０ｍｇ／ｌのＲＩＰ、１０ｍｇ／ｌのリファンピンを含浸させた。
^ｃ 対照群のＭＳおよびＭＲと比較した場合に統計学的に有意
^ｄ ＭＳ３群と比較した場合に統計学的に有意
^ｅ ＭＲ１群、ＭＲ２群およびＭＲ３群と比較した場合に統計学的に有意 Grafts impregnated with 0.1 μg / mL BisEDT, 10 μg / mL RIP and 10 μg / mL rifampin alone or in combination were implanted subcutaneously into rats as summarized in Table 21. A physiological solution (1 ml) containing 2 × 10 ⁷ cfu / ml MS and MR strains was inoculated to the surface of the implant using a tuberculin syringe. All implants were removed 7 days after implantation and sonicated for 5 minutes in sterile saline to remove adherent bacteria. The quantification of viable bacteria was obtained by culturing the dilutions on blood agar plates. The detection limit was approximately 10 cfu / cm ² .

^a Each group consisted of 15 animals. MS, methicillin sensitive S. Epidermidis; MR, methicillin resistant S. Epidermidis
^b Dacron implants were impregnated with 0.1 mg / l BT, 10 mg / l RIP, 10 mg / l rifampin.
^c statistically significant when compared to MS and MR in control group
^d statistically significant when compared to the MS3 group
^e statistically significant when compared to MR1, MR2 and MR3 groups

Ｐ．エルギノーサの耐性株を、トブラマイシン（ＮＮ）およびＢｉｓＥＤＴ（ＢＥ；０．３３μｇ／ｍｌ）の存在下、３７℃のＭｕｅｌｌｅｒ−Ｈｉｎｔｏｎ ＩＩブロス中で培養した。ＭＩＣを、２４±１時間の発育を阻害する抗生物質濃度として測定した。 The activity of tobramycin against Gram-negative resistant Pseudomonas aeruginosa was enhanced several-fold by BisEDT of subMIC (Table 22). In these trials, the MIC was more accurately defined as IC ₂₄ .

P. A resistant strain of Aeruginosa was cultivated in Mueller-Hinton II broth at 37 ° C. in the presence of tobramycin (NN) and BisEDT (BE; 0.33 μg / ml). The MIC was measured as the concentration of antibiotic that inhibited growth for 24 ± 1 hours.

^ａ ０．４μｇ／ｍｌのＢｉｓＥＤＴにより阻害された３種の菌株は、更なる試験から除外した。
０．５以下のＦＩＣ指数は、相乗性を示し、０．５を超え１．０未満のＦＩＣは、増強性を示す。 Against tobramycin resistant Burkholderia cepacia, 0.4 μg / mL BisEDT sensitized 7 of 10 isolates to tobramycin (mean FIC; 0.48) and reduced MIC ₉₀ by 10-fold. (Table 23). Both tobramycin MICs and MBCs were significantly reduced by subMIC BisEDT to feasible levels for 50 clinical Burkholderia cepacia isolates [Veloria et al. , 2003]. Liposomal forms of BisEDT and tobramycin are described in P. It has a high degree of synergism demonstrated against aeruginosa (Halwani et al., 2008; Halwani et al., 2009).

^a Three strains inhibited by 0.4 μg / ml BisEDT were excluded from further study.
An FIC index of 0.5 or less indicates synergism, and an FIC of more than 0.5 and less than 1.0 indicates enhancement.

Ｅ．コリの耐性株を、単独または組み合わせでのクロラムフェニコール（ＣＭ）またはアンピシリン（ＡＭＰ）およびＢｉｓＥＤＴの存在下、３７℃のＭｕｅｌｌｅｒ−Ｈｉｎｔｏｎ ＩＩブロス中で培養した（ＢＥ：０．３３μｇ／ｍｌ）。ＭＩＣを、２４±１時間の発育を阻害した抗生物質濃度として測定した。 Chloramphenicol and ampicillin resistant Escherichia coli were sensitized to these drugs by the addition of SubMIC BisEDT (Table 24).

E. Resistant strains of E. coli were cultivated 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 μg / ml). . The MIC was measured as the antibiotic concentration that inhibited growth for 24 ± 1 hours.

Ｅ．コリの耐性株を、単独または組み合わせでのドキシサイクリン（ＤＯＸ）およびＢｉｓＥＤＴの存在下、３７℃のＭｕｅｌｌｅｒ−Ｈｉｎｔｏｎ ＩＩブロス中で培養した（ＢＥ：０．３３μｇ／ｍｌ）。ＭＩＣを、２４±１時間の発育を阻害した抗生物質濃度として測定した。 Tetracycline-resistant Escherichia coli was sensitized to tetracycline by addition of SubMIC BisEDT (Table 25). The combination showed synergism for the TET M and TET D strains (FIC ≦ 0.5) and an additive effect for the TET A and TET B strains.

E. 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 μg / ml). The MIC was measured as the antibiotic concentration that inhibited growth for 24 ± 1 hours.

Reference Material Domenico P, R O'Leary, BA Cunha. 1992. Differential effect of bismuth and salicylate compounds on antibiotic sensitivity of Pseudomanas aeruginosa. Eur J Clin Microbiol INF Dis 11: 170-175; Domenico P, D Parikh, BA Cunha. 1994. Bismuth modulation of antibiotic activity against gastrointestinal bacterial pathogens. Med Microbiol Lett 3: 114-119; Domenico P, Kazzaz JA, Davis JM, Niederman MS. 2002. Subinhibit bismuth ethandithiol (BisEDT) sensitizes resist Staphylococcus aureus to nafcillin or gentamicin. Annual Meeting, ASM, Salt Lake City, UT; Domenico P, Kazzaz JA, Davis JM. 2003. Combining antibiotic resistance with bismuth-thiol. Research Advances in Antimicrob Agents Chemother 3: 79-85; 2004. BisEDT and RIP act in synergy to present graft grafts by resist Staphylococci. Peptides 25: 2047-2053; Halwani M, Bloome S, Suntres ZE, Alipour M, Azghani AO, Kumar A, Omri A .; 2008. Liposomal bismuth-ethanedithiol formation enhancements antimicrobiological activity of tobramycin. Intl J Pharmaceut 358: 278-84; Halwani M, Hebert S, Suntres ZE, Lafrenie RM, Azzhani AO, Omri A .; 2009. Bismuth-thiol corporation enhancement biological activities of liposomal tobramycin against biological biofilm and urine bolus inguture morphules. Int J Pharmaceut 373: 141-6; Veloira WG, Gurzenda EM, Domenico P, Davis JM, Kazzaz JA. 2003. Synergy of tobramycin and bismuth thiols against Burkholderia cepacia. J Antimicrob Chemother 52: 915-919.

ＳＡ、スタフィロコッカス・アウレウス；ＭＲＳＡ、メチシリン耐性スタフィロコッカス・アウレウス；ＥＦｃ、エンテロコッカス・フェカーリス；ＳＰ、ストレプトコッカス・ニューモニエ；ＰＲＳＰ、ペニシリン耐性ストレプトコッカス・ニューモニエ；ＥＣ、エシェリヒア・コリ；ＫＰ、クレブシエラ・ニューモニエ；ＰＡ、シュードモナス・エルギノーサ；Ｂｃｅｐ、バークホルデリア・セパシア；Ｂｍｕｌｔ、バークホルデリア・マルチボランス；Ａｂａｕ、アシネトバクター・バウマニー；Ｍｓｍｅｇ、マイコバクテリウム・スメグマチス Example 9
Enhancement and Synergistic Activity of Particulate BT-Antibiotics This example demonstrates that the particulate bismuth thiol, BisEDT, has antibiotic activity through enhanced and / or synergistic interactions with specific antibiotics for specific microbial target organisms. Indicates that it promotes The single point data for each combination shown in Table 26 was made essentially according to the method used in Example 8.

SA, Staphylococcus aureus; MRSA, methicillin-resistant Staphylococcus aureus; EFc, Enterococcus faecalis; SP, Streptococcus pneumoniae; PRSP, penicillin-resistant Streptococcus pneumoniae; EC, Escherichia coli; PA, Pseudomonas aeruginosa; Bcep, Burkholderia cepacia; Bmult, Burkholderia multiborans; Abau, Acinetobacter baumannii; Msmeg, Mycobacterium smegmatis

Example 10
Enhancement and synergistic activity of particulate BT-antibiotics Testing the effect of particulate BisEDT and four BisEDT analogs prepared as described above in combination with other agents on representative strains of multiple Gram-negative pathogens did. Utilizing a modification of the general test method, synergism (FICI ≦ 0.5), enhancement (0.5 <FICI ≦ 1.0) of the partial inhibitory concentration (FIC) and FIC index (FICI) used. ), Antagonism (FICI> 4.0) and no significance (1.0 <FICI ≦ 4.0) were determined (V Lorian. Williams and Wikins, Baltimore, MD, Antibiotics in Laboratory Medicinal Medicine). Edition, pp. 432-492, Elipoulos G and R Moellering. 1991. Antimicrobiological combinations .; Odds, 2003 J. Antimicrob. Chemother. 52 (1): 1. The FIC index was determined using the checkerboard technique and used for this test.

  Stock solutions of all test substances were prepared in appropriate solvent at 40 times the final target concentration. All test substances were solutions under these conditions. The final drug concentration in the FIC assay plate was set so that the MIC values for each drug for each tested organism were pooled, unless the strain was totally resistant to the test drug. The tested concentration ranges are shown in Table 27. Test organisms were initially obtained from clinical sources or from the American Type Culture Collection. Upon receipt, isolates were streaked on Tryptic Soy Agar II (TSA). Colonies were harvested from these plates and cell suspensions were prepared in the appropriate broth growth medium containing cryoprotectant. The aliquot was then frozen at -80 ° C. Frozen seeds of organisms tested in a given assay were thawed, streaked on TSA plates for isolation and incubated at 35 ° C. All organisms were examined on the Mueller-Hinton II Broth (Beckton Dickinson, lot no. 9044411). Broth was prepared at 1.05 times normal weight / volume to offset the 5% volume of drug in the final test plate.

  The Final Inhibitory Concentration (MIC) value was measured as before using a broth microdilution method for aerobic bacteria (Clinical and Laboratory Standards Institute (CLSI). Applied Standard-Eight Edition. CLSI document M07-A8 [ISBN 1-56238-689-1]. e, Pennsylvania 19087-1898 USA, 2009).

  FIC values were measured using the previously described broth microdilution method (Sweeney et al., 2003 Antimicrob. Agents Chemother. 47 (6): 1902-1906). Serial dilutions and liquid transfers were performed using automated liquid handlers (Multidrop 384, Labsystems, Helsinki, Finland; Biomek 2000 and Multimek 96, Beckman Coulter, Fullerton CA) to prepare the test plates.

  The Multidrop 384 was used to fill the appropriate wells of a standard 96-well microdilution plate (Falcon 3918) with 150 μL of the appropriate solvent, columns 2-12. 300 μL of each second test agent was added to each well in the first row of the plate. These plates were used to prepare "mother plates" of drug that provided serial dilutions for combination plates of drug. Using Biomek 2000, 150 μL of each second drug solution (40-fold) was transferred from the wells in the first row of the mother plate to generate 11 2-fold dilution series. Mother plates of Bis-EDT (and analogs) were serially diluted from top to bottom using a multichannel pipette. Combine two mother plates, one for each second drug and one for Bis-EDT (or analog), and transfer the same volume (using a multi-channel pipette) to the drug combination plate This formed a "checkerboard" pattern. Rows H and 12 each contained serial dilutions of one drug alone to measure MIC.

To the “daughter plate”, 180 μL of test medium was added using Multidrop 384. Then, using a Multimek 96, 10 μL of the drug solution was transferred from each well of the drug combination mother plate to each corresponding well of the daughter plate in one step. Finally, the daughter plate was inoculated with the test organism. A standardized inoculum for each organism was prepared according to published guidelines (CLSI, 2009). For all isolates, the inoculum of each organism was dispensed into vertically divided sterile reservoirs (Beckman Coulter) and the plates were inoculated with a Biomek 2000. The instrument delivered 10 μL of the standardized inoculum to each well to give a final cell concentration in the daughter plate of approximately 5 × 10 ⁵ colony forming units / mL.

  The test format produced an 8 × 12 checkerboard that tested each compound alone (columns 12 and 8) and in combination drug concentrations in various ratios. All organism plates were stacked in triplicate, the top plate was capped, placed in a plastic bag and incubated at 35 ° C for approximately 20 hours. After incubation, the microplate was removed from the incubator and viewed from the bottom using the ScienceWare plate viewer. On the prepared reading sheet, the MIC of drug 1 (row H), the MIC of drug 2 (column 12) and the boundary wells with and without development were entered.

FIC was measured by the formula: (MIC of compound 1 in combination / MIC of compound 1 alone) + (MIC of compound 2 in combination / MIC of compound 2 alone) using the Excel program. The FICI for the checkerboard was calculated from each FIC by the formula: (FIC ₁ + FIC ₂ + ... FIC _n ) / n, where n = number of each well per plate for which FIC is calculated. If a single drug produced MIC results that were out of range, the next highest concentration was used as the MIC value in the FIC calculation.

^１ＭＩＣ、最小阻止濃度
^２ＦＩＣＩ、部分阻止濃度指数

^１ＭＩＣ、最小阻止濃度
^２ＦＩＣＩ、部分阻止濃度指数

^１ＭＩＣ、最小阻止濃度
^２ＦＩＣＩ、部分阻止濃度指数

^１ＭＩＣ、最小阻止濃度
^２ＦＩＣＩ、部分阻止濃度指数

^１ＭＩＣ、最小阻止濃度
^２ＦＩＣＩ、部分阻止濃度指数

^１ＭＩＣ、最小阻止濃度
^２ＦＩＣＩ、部分阻止濃度指数

^１ＭＩＣ、最小阻止濃度
^２ＦＩＣＩ、部分阻止濃度指数

^１ＭＩＣ、最小阻止濃度
^２ＦＩＣＩ、部分阻止濃度指数

^１ＭＩＣ、最小阻止濃度
^２ＦＩＣＩ、部分阻止濃度指数

^１ＭＩＣ、最小阻止濃度
^２ＦＩＣＩ、部分阻止濃度指数

^１ＭＩＣ、最小阻止濃度
^２ＦＩＣＩ、部分阻止濃度指数

^１ＭＩＣ、最小阻止濃度
^２ＦＩＣＩ、部分阻止濃度指数

^１ＭＩＣ、最小阻止濃度
^２ＦＩＣＩ、部分阻止濃度指数

^１ＭＩＣ、最小阻止濃度
^２ＦＩＣＩ、部分阻止濃度指数

^１ＭＩＣ、最小阻止濃度
^２ＦＩＣＩ、部分阻止濃度指数

^１ＭＩＣ、最小阻止濃度
^２ＦＩＣＩ、部分阻止濃度指数

^１ＭＩＣ、最小阻止濃度
^２ＦＩＣＩ、部分阻止濃度指数

^１ＭＩＣ、最小阻止濃度
^２ＦＩＣＩ、部分阻止濃度指数

^１ＭＩＣ、最小阻止濃度
^２ＦＩＣＩ、部分阻止濃度指数

^１ＭＩＣ、最小阻止濃度
^２ＦＩＣＩ、部分阻止濃度指数 Particulate Bis-EDT, four particulate BT analogs, and all other drugs (and drug combinations) were soluble at all final test concentrations. The measured MIC and FICI values are shown in the table below.

¹ MIC, minimum inhibitory concentration
² FICI, partial inhibitory concentration index

¹ MIC, minimum inhibitory concentration
² FICI, partial inhibitory concentration index

¹ MIC, minimum inhibitory concentration
² FICI, partial inhibitory concentration index

¹ MIC, minimum inhibitory concentration
² FICI, partial inhibitory concentration index

¹ MIC, minimum inhibitory concentration
² FICI, partial inhibitory concentration index

¹ MIC, minimum inhibitory concentration
² FICI, partial inhibitory concentration index

¹ MIC, minimum inhibitory concentration
² FICI, partial inhibitory concentration index

¹ MIC, minimum inhibitory concentration
² FICI, partial inhibitory concentration index

¹ MIC, minimum inhibitory concentration
² FICI, partial inhibitory concentration index

¹ MIC, minimum inhibitory concentration
² FICI, partial inhibitory concentration index

¹ MIC, minimum inhibitory concentration
² FICI, partial inhibitory concentration index

¹ MIC, minimum inhibitory concentration
² FICI, partial inhibitory concentration index

¹ MIC, minimum inhibitory concentration
² FICI, partial inhibitory concentration index

¹ MIC, minimum inhibitory concentration
² FICI, partial inhibitory concentration index

¹ MIC, minimum inhibitory concentration
² FICI, partial inhibitory concentration index

¹ MIC, minimum inhibitory concentration
² FICI, partial inhibitory concentration index

¹ MIC, minimum inhibitory concentration
² FICI, partial inhibitory concentration index

¹ MIC, minimum inhibitory concentration
² FICI, partial inhibitory concentration index

¹ MIC, minimum inhibitory concentration
² FICI, partial inhibitory concentration index

¹ MIC, minimum inhibitory concentration
² FICI, partial inhibitory concentration index

Example 11
Effect of Bismuth-Thiol on Infection in Critically Femoral Bone Defects of Rats Current Care Standards for Open Fractures are Irigation, Debridement and Antibiotics, which Infect Bacterial Load in Wounds It is intended to be reduced to a degree. Despite these procedures, infections complicate up to 75% of severe tibial open fractures from war. Interestingly, although early infections often result from Gram-negative bacteria, late infections involved in healing problems and cleavage are due to Gram-positive infections, mostly Staphylococcus species (Johnson 2007).

  S. One of the reasons why Aureus is resistant to standard treatments is its ability to form biofilms. Bacteria within the biofilm can withstand concentrations of antimicrobial compounds that kill similar organisms in the culture medium (Costerton 1987).

  The purpose of this study was to determine if BT alone or in combination with antibiotics would reduce infection in a contaminated open fracture model. The contaminated rat femoral critical defect model is a well-accepted model and was used in the experiments described in this example. This model is a standardized model for comparing various potential treatments and their effects in terms of reducing infection and / or improving healing.

  The compounds (CPD) CPD-8-2 (bismuth-pyrithione / butanedithiol; Table 1) and CPD-11 (bismuth pyrithione / ethanedithiol; Table 1) have a different spectrum of activity than Bis-EDT. , But two BIS-Bis analogs that showed in vitro potency against biofilm-secreting bacteria.

Three BT formulations: Bis-EDT, CPD-11 and CPD-8-2 (see Table 1) with or without tobramycin and vancomycin in polymethylmethacrylate (PMMA) cement bead vehicle. When used, in vitro S. It showed an inhibitory effect on the Aureus strain. Three formulations of particulate BT were prepared in the clinically useful hydrogel form described herein. These BTs were tested in suspension in gels at a concentration of 5 mg / ml- ¹ which was found to be the appropriate concentration for gel delivery. The gel formulation matched the contours of the wound and did not need to be removed after application.

  Two treatment objectives were utilized, BT alone with the first objective and BT with the systemic antibiotic (ABx) in the second objective.

(A) BT alone S. Six hours after inoculation with Aureus, the necrotic tissue of the wound was removed, ligated with physiological saline, and 1 ml of BT gel was inserted into the defect.

(B) BT and systemic antibiotics (ABx)
S. Six hours after inoculation with Aureus, debridement of the wound was removed, ligated with saline and 1 ml of added BT gel was inserted into the defect. The antibiotic used was cefazoline at a dose equivalent to 5 mg kg ⁻¹ delivered by subcutaneous injection twice daily for a total of 3 days after injury. The first dose was administered immediately before debridement. Past data suggested that this dose may reduce bacterial levels from about 10 ⁶ to about 10 ⁴ and therefore measure the relative effects of different BT.

(C) Control S. Six hours after inoculation with Aureus, the necrotic tissue of the wound was removed and irrigated with saline. Control animals were also treated with cefazolin by the regimen described above.

procedure:
The procedure for the in vivo rat injury model was performed as described by Chen et al. (2002 J. Orthop. Res. 20: 142; 2005 J. Orthop. Res. 23: 816; 2006 J. Bone Joint Surg. Am. 88. : 1510; 2007 J. Orthop. Trauma 21: 693). Rats were anesthetized and prepared for surgery. A 3 cm incision was made on the anterolateral surface of the femoral shaft to expose it. The periosteum and attached muscle were detached from the bone. A polyacetyl plate (27 x 4 x 4 mm) was placed on the anterolateral surface of the femur. The plate was pre-drilled and passed through a 0.9 mm diameter twisted Kirschner wire. The substrates of these plates were shaped to match the contours of the femoral shaft. Pilot holes were excised through both femoral cortical bone using the plate as a template and twisted Kirschner wires were inserted through the plate and femur. The 6 mm spacing indentations on the plate served as guides for bone removal. In an attempt to prevent thermal damage, defects were created using a small vibrating saw while cooling the tissue with continuous irrigation.

For each of a plurality of groups of 10 S. Aureus 1 × 10 ⁵ CFU were inoculated and BT was treated 6 hours post-inoculation alone or in combination with antibiotics as previously described. The groups were as follows: Bis-EDT gel; MB-8-2 gel; Bis-EDT gel; Bis-EDT gel &Abx; MB-11 gel &Abx; MB-8-2 gel &Abx; control (Abx only). ).

  Animals were euthanized 14 days after surgery and bones and devices were sent for microbiological analysis, the results of which are shown in FIG.

  Based on the power analysis, 10 animals per group would provide 80% power to detect a 25% difference between the treated and control groups. In this case, the predicted standard deviation is 35% and α is 0.05.

  As shown in FIG. 7, when combined with Bis-EDT, MB-11 and MB-8-2, the antibiotic activity of cefazolin was enhanced compared to cefazolin or any Bis compound alone, and S of damaged bone was reduced. ． Reduced Aureus infection. Cefazolin in combination with MB-11 and MB-8-2 showed higher antibiotic activity compared to cefazolin alone, and the S. It has been shown to reduce Aureus infection. Bis-EDT was not considered to affect cefazolin activity in this capacity.

References:
Costester JW, Cheng KJ, Geesey GG, et al. Basic Biofilms in Nature and Disease. Ann Rev Microbiol. 1987; 41: 435-64.
Domenico P, Baldassarri L, Schoch PE, Kaehler K, Sasatsu M, Cunha BA. Activities of Bismuth Thiols again Staphylococci and Staphylococcal Biofilms. Antimicrob Agents and Chemother. 2001; 45 (5): 1417-21.
Halwani M, Bloome S, Suntres ZE, et al. Liposomal bismuth-ethanedithol formation enhancements antimicrobiological activity of tobramycin. Int J Pharm. 2008; 358: 278-84.
Johnson EN, Burns TC, Hayda RA, Hospital DR, Murray CK. Infection complications of open type III tibial fractions among comba casualties. Clin Infect Dis. 2007; 45 (4): 409-415.

Other Cited Documents and Related Documents Domenico et al. , Canadian J .; Microbiol. 31: 472-78 (1985); Domenico et al. , Reduction of capsular polysaccharides and potentiation of aminoglycoside inhibition in gram-negative bacteria with bismuth subsylates. J Antimicrob Chemo 1991; 28: 801-810; Domenico et al. , Infection 20: 66-72 (1992); Domenico et al. , Infect. Immun. 62: 4495-99 (1994); Domenico et al. , J. Antimicrol. Chemother. 38: 1031-40 (1996); Domenico et al. , Enhancement of bismuth antibacterial activity with lipophilic thiol chelators. Antimicrob Agents Chemother 1997; 41: 1697-703; Domenico et al. , Surface antiexposure by bismuth-dimercapsuppression of Klebsiella pneumoniae capsular polysaccharide. Infect Immun 67: 664-669 (1999); Domenico et al. , 2000. The potential of bismuth-thiols for treatment and prevention of infection. Infect Med 17: 123-127; Domenico et al. , Activities of bismuth thiols againstaff staphylococci and staphylococcal biofilms. Antimicrob Agents Chemother 2001; 45: 1417-21; Domenico et al. , Combining antibiotic resistance with bismuth-thiols. Resaerch Advances in Antimicrob Agents Chemother 2003; 3: 79-85; Domenico et al. , Reduction of capsular polysaccharides and potentiation of aminoglycoside inhibition in gram-negative bacteria with bismuth subsylates. J Antimicrob Chemo 1991; 28: 801-810; Domenico et al. , BisEDT and RIP act in synergy to prevent graft graft infections by resistive staphylococci. Peptides 2004. 25: 2047-53; Domenico et al. , 2005. Pyriteion enhanced antimicrobial activity of bismuth. Antibiotics for Clinicians 9: 291-297; U.S. Patent No. 6,582,719; U.S. Patent RE37,793, U.S. Patents 6,248,371, 6,086,921, 6,380,248. Nos. 6,582,719, 6,380,248, and 6,875,453.

  The various embodiments described above can be combined to provide further embodiments. All of the US patents, US patent application publications, US patent applications, foreign patents, foreign patent applications and non-patent publications referenced herein and / or listed in the application data sheet are hereby incorporated by reference in their entirety. Incorporated into the book. Aspects of the embodiments can be refined as needed to provide additional embodiments using various patent, application and publication concepts.

These and other changes can be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed as limiting the claims to the particular embodiments disclosed or claimed, as such. The claims are to be construed to cover all possible embodiments as well as the full scope of equivalents to which they are entitled.

Claims (16)

A bismuth-thiol composition comprising a plurality of fine particles containing a bismuth-thiol (BT) compound, wherein substantially all of the fine particles have a volume average diameter of 0.4 μm to 5 μm, and the BT compound is Bismuth-thiol composition comprising bismuth or a bismuth salt associated with 1,2-ethanedithiol, or bismuth covalently bound to 1,2-ethanedithiol , via an ionic bond or as a coordination complex . The bismuth-thiol composition according to claim 1 , wherein the BT compound comprises bismuth covalently bonded to 1,2-ethanedithiol. The bismuth-thiol composition according to claim 1 or 2 , wherein the BT compound is BisEDT. The composition of claim 1, wherein the microparticles have a VMD of 0.7 μm to 4 μm. The composition of claim 4, wherein the microparticles have a VMD of 1.0 μm to 3 μm. The composition of claim 1, wherein at least 80% or more of all microparticles have a VMD of 0.4 μm to 5 μm. 7. The composition of claim 6, wherein at least 95% or more of all microparticles have a VMD of 0.4 [mu] m to 5 [mu] m. The composition of claim 1, wherein the microparticles have a peak VMD of about 1.3 microns. The composition according to claim 8, wherein the size distribution of the fine particles is a monomodal distribution. The composition of claim 1, wherein the composition further comprises methyl cellulose, sodium chloride, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, or polysorbate 85. The composition of claim 1, wherein the composition is a suspension. The composition of claim 1, wherein the composition is an aerosol suspension. The composition of claim 3, wherein at least 80% or more of all the microparticles have a VMD of 0.4 μm to 5 μm. The composition of claim 3, wherein at least 95% or more of all the microparticles have a VMD of 0.4 μm to 5 μm. The composition according to claim 3, wherein the composition is a suspension. The composition of claim 3, wherein the composition is an aerosol suspension.

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