KR20200015814A - Bismuth-thiols as antiseptics for biomedical uses, including treatment of bacterial biofilms and other uses - Google Patents

Abstract

To provide a formulation comprising a preservative formulation, a novel method for treating natural surfaces containing bacterial biofilms, including unexpected synergistic or enhancing effects between bismuth-thiol (BT) compounds and certain antibiotics Compositions and methods comprising homogeneous particulate suspensions are described. The BT compounds and BT compounds described, including the desired efficacy of certain such compositions for treating certain Gram-positive bacterial infections, and the apparent preferred efficacy of certain such compositions for treating certain Gram-negative bacterial infections. And previously unanticipated antibacterial and anti-microbial properties of antibiotic combinations are also described.

Description

BISMUTH-THIOLS AS ANTISEPTICS FOR BIOMEDICAL USES, INCLUDING TREATMENT OF BACTERIAL BIOFILMS AND OTHER USES}

[See before and after related application]

This application claims the benefit of PCT Application No. PCT / US2010 / 023108, filed February 3, 2010, and US Provisional Patent Application No. 61 / 373,188, filed August 12, 2010, each of which references The entirety of which is incorporated herein by reference.

[Technical field]

Inventive aspects currently described herein relate to compositions and methods for the treatment of microbial infections. In particular, the present invention provides for the management of bacterial infections in epithelial tissue, including wounds such as chronic wounds and acute wounds, and treatment of bacterial biofilms and other conditions in clinical, personal health care, and other contexts. For improved treatment.

Description of the related technology

A complex family of coordinated cellular and molecular interactions that contribute to skin wound healing, respond to microbial infections, are resistant to microbial infections, and / or generally contribute to the healing or maintenance of body tissues are opportunistic and intra-hospital infections (eg For example, clinical therapies that may increase the risk of infection), topical or systemic administration of antibiotics (which may affect cell growth, migration or other infections, and may also be chosen for antibiotic-resistant microorganisms), and common wounds Dressing changes, open-air exposure of wounds to accelerate healing, temporary artificial structural support matrix or scaffold material, possible need for dead tissue removal and / or infected or necrotic tissue And / or may be adversely affected by various external factors, such as repeated surgery to remove other factors. There.

Thus, wound healing continues to be a huge challenge for clinicians worldwide. Current treatments for refractory wounds are impractical and ineffective and often require a number of surgeries to close the wound. For example, Regranex ^® (Becaplemin, Ortho-McNeil Pharmaceutical, Inc., Sales: Ethicon, Inc., recombinant platelet-derived growth factor) is illustrated as one of the few available treatments for chronic wounds. However, it is expensive to produce and has limited clinical use.

Chronic and acute wounds and wound biofilms

A wound is one that has continuity between cells in tissues, or between tissues, for example, physical, mechanical, biological, pathological and / or chemical forces (eg, burns, skin infections, wounds, gunshot wounds or grenade, Circulatory and blood disorders including skin ulcers, radiation poisons, malignant cancers, gangrene, autoimmune diseases, immunodeficiency diseases, respiratory damage such as by inhalation or infection, gastrointestinal damage such as by harmful ingestion or infection, coagulation defects ) Or by other traumatic injuries.

Limited levels of bacterial contamination in the wound or "colonization" of the wound may not necessarily impede the process of wound healing, while the presence of a sufficient number of bacteria to subdue host immune defenses indicates that the bacterial biofilm Existing wounds or acute wounds or chronic wounds, such as bacterial growth, can lead to wound infections that proceed adversely to the host. Bryant and Nix, Acute and Chronic Wounds: Current Management Concepts, 2006 Mosby (Elsevier) , NY; Baronoski, Wound Care Essentials: Practical Principles (2nd Ed.), 2007 Lippincott, Williams and Wilkins, Philadelphia, PA. For example, acute wounds can result from injury, trauma, surgical operations, or other causes, and typically lack a potential health disorder and cure quickly but sometimes fail to perform due to the presence of an infection; Rapid formation of bacterial biofilms is described in acute wounds (eg WO / 2007/061942). Additional factors that may contribute to the progression of chronic wounds include mobility (eg, creating sustained pressure applied to the wound site), lack of sensory or mental power, inaccessibility of the wound site (eg respiratory or Gastrointestinal tract) and circulatory defects. Infections at chronic wound sites may be detected by skin redness, edema, pus formation and / or unpleasant odors or other related, clinically acceptable criteria.

Therefore, chronic wounds may be present in higher organisms (including humans and other mammals), as acute wounds may be present that cannot be adequately healed. , But not limited thereto, may be allowed to generate a proliferative state for the bacteria to invade and further destroy the tissue. In general, chronic wounds are wounds that do not heal within three months, and instead of becoming smaller, they tend to grow larger as bacterial infiltration progresses. Chronic wounds can be very painful and stressful for patients who have injured nearby nerves as they progress (neuropathy). These injuries affect 4 million Americans each year and cost about $ 9 billion for treatment. Individuals suffering from age are mostly over 60 years old.

Chronic wounds may originate as acute wounds in some cases, for example, gunshot or debris wounds, burns, ulcers, nerve ulcers, pressure ulcers, diabetic ulcers, radiation poisoning, malignant tumors, skin infections, ulcers, surgical wounds, Diabetic foot ulcers, pressure sores, venous leg ulcers, infected and / or biofilm-containing non-surgical non-surgical surgical wounds, plexus dermatosis, trauma wounds, acute arterial insufficiency, necrotizing fasciitis, osteomyelitis (bone infection), and radiation damage, for example For example, radiation osteonecrosis and soft tissue radiation necrosis, or other types of wounds. For example, venous ulcers may include poor circulation (eg, ischemia), malfunctioning valves of the veins, or repeated physical trauma (eg, repeated damage). Pressure ulcers may be present, for example, when the local pressure exerted at or around the wound is greater than the blood pressure, such that poor circulation, paralysis, and / or pressure sores may contribute to or exacerbate chronic wounds. Diabetic ulcers may cause an individual with diabetes mellitus, eg, uncontrolled high blood sugar, to contribute to loss of sensation in the limbs, resulting in repeated damage and / or neglect the individual's attention to damage in some cases. do. Factors that may be complex or that may affect the outcome of clinical onset and chronic wounds include, but are not limited to, the immunological state of the subject (eg, immunosuppression, pathological (eg, HIV-AIDS), radiotherapy). Or pharmacologically compromised immune system; age; stress); Skin aging (including photochemical aging), and the development and progression of wound biofilms. In the case of epithelial tissue in the respiratory and / or gastrointestinal tract, the difficulty in developing epithelial surface-cleaning fluid forces or localized microenvironments conducive to microbial survival may lead to clinical complications. Can be.

Wound-related injuries may be accompanied by lost or compromised term function, shock, bleeding and / or thrombosis, cell death (eg, necrosis and / or apoptosis), stress and / or microbial infection. Any or all of these phenomena and especially infections can delay or prevent the effective tissue repair process involved in wound healing. Thus, in the wounded individual, the non-viable tissue is removed from the wound site, a process referred to as dead tissue removal as early as possible, and also any foreign material from the wound site referred to as wound cleansing It can be important to remove it.

Severe wounds, acute wounds, chronic wounds, burns, and ulcers can be beneficial from cellular wound dressings. Apligraft ^® [manufactured by Novartis ^{(Norvartis)], Demagraft ®,} Biobrane ®, Transcyte ® [ manufactured by Advanced Tissue Sciences (Advance Tissue Science)], Integra ® Dermal Regeneration Template ® [ manufactured by Integra Life Scientific System Technology (Integra Life Sciences Technology)], and it is available on several products artificial skin wounds or burns do not heal, such as OrCel ^®. However, these products are not designed to focus on the problem of bacterial tissue infiltration and wound spreading.

Unfortunately, systemic antibiotics are not effective in the treatment of chronic wounds and are generally not used unless an acute bacterial infection is present. Current attempts include the administration or application of antibiotics, but such therapeutic agents may promote the emergence of antibiotic-resistant bacterial strains and / or may not be effective against bacterial biofilms. Thus, the use of preservatives may be particularly important when drug resistant bacteria (eg methicillin resistant Staphylococcus aureus or MRSA) are detected. While many preservatives are widely used, established bacterial populations or subpopulations may not respond to these agents, or any other currently available treatment. In addition, such preservatives are not suitable because many preservatives may be toxic to host cells at concentrations that may be required to be effective against established bacterial infections. The problem may be the respiratory system (e.g., airway, nasopharyngeal and laryngeal pathways, tracheal, lungs, bronchi, bronchioles, alveoli, etc.) or the stomach (e.g., in the cheeks, esophagus, stomach, intestine, rectum, anus, etc.). It may be particularly acute in the case of an effort to clear infection from natural epithelial surfaces, such as internal epithelial surfaces, such as ducts, or other epithelial surfaces.

Of particular concern is the release of free, single-cellularized (") cells into organized multicellular communities with markedly different susceptibility to environmental agents including behavioral patterns, gene expression, and antibiotics by intercellular attachment. Planktonic ") is an infection consisting of bacterial biofilms, which is a relatively recently recognized bacterial organization in which bacteria collect. Biofilms may employ biological defense mechanisms not found in planktonic bacteria, which may protect biofilm populations against antibiotics and host immune responses. Established biofilms can be prevented by a tissue-healing process.

A common microbiological contaminant that remains persistent and faces potentially harmful infections is S. a. A. aureus, for example, MRSA (Methicillin Resistant Staphylococcus aureus), Enterococcus, E. coli. E. coli, p. P. aeruginosa, Streptococcus, and Acinetobacter baumannii. Some of these organisms exhibit the ability to survive on non-nutritive clinical surfaces for the oral cavity. s. It has been found that S. aureus can survive for 4 weeks in dried glass and between 3 and 6 months in dried blood and cotton fibers (Domenico et al., 1999 Infect. Immun. 67: 664-669). this. E. coli and blood. P. aeruginosa has been shown to contain S. aureus in dried blood and cotton fibers. It was found to survive much longer than Aureus.

Microbial biofilms are associated with substantially increased resistance to both preservatives and antibiotics. Biofilm morphology is produced when bacteria and / or fungi adhere to the surface. This attachment initiates altered transcription of the gene, resulting in the secretion of a remarkably elastic and difficult to penetrate polysaccharide matrix to protect the microorganism. In addition to their very substantial resistance to antibiotics, biofilms are also very resistant to the mammalian immune system. Because biofilms are very difficult to eradicate once they are established, preventing biofilm formation is a very important clinical priority. Recent investigations have revealed that open wounds can be rapidly contaminated by biofilms. These microbial biofilms are believed to delay wound healing and are very similarly associated with the establishment of serious wound infections.

Current guidelines for protection for military wounds, for example, provide for violent and complete cleaning or irrigation and removal of dead tissue (see Blankenship CL, Guidelines for care of open combat casualty wounds, Fleet Operations and Support.US Bureau of Medicine and Surgery). Although this early intervention is important, it is not appropriate to prevent the progression of infection. Additional therapeutic steps are required to promote healing by taking after dead tissue removal and to reduce the chance of establishing wound infections and wound biofilms that are established by reducing microbial bio-burden.

Due to the complex nature of the wound trauma wound, the potential for infection is particularly high when considering the introduction of foreign objects and the introduction of other environmental contaminants. Both clustered and clinical environments (including humans in both of these environments) serve as potentially important sources of pathogenic microorganisms, especially for those suffering from open and / or complex wounds. Acute and chronic wounds, including surgical and colonic wounds, are already infected; Compromises the body's primary defense and barrier to skin. Thus, the wound exposes the body's interior (a humid and nourishing environment) to opportunistic and pathogenic infections. Many of these infections, particularly persistent wound infections, may be associated with biofilm formation, as evidenced in the case of chronic wounds (James et al., 2008). Wound infections in hospitals constitute one of the most common causes of infection in hospitals, and wounds in military and natural disaster environments are particularly sensitive to microbial contamination. Clustered wounds are typically associated with tissue damage, tend to be widespread and deep, external bodies can be introduced to interfere with local blood supply, fractures and burns, and to induce shock and compromised immune defenses. It can be susceptible to infection.

Skin structure and wound healing

Complete functional skin and other epithelial tissues (eg, those found in the skin and also those found in the inner walls of the respiratory and gastrointestinal tracts, gland tissues, etc., avascular epithelium that forms a barrier between the organism and its external environment) Maintenance of the surface) is important for the health and survival of humans and other animals. Skin is the largest body organ in humans and other higher vertebrates (eg mammals) that protects against environmental damage through its barrier function, mechanical strength and impermeability to water. As an important environmental interface, the skin provides a protective body covering that helps maintain physiological equilibrium.

Skin structure is well known. In summary, the epidermis, the outer skin layer, is covered with a stratum corneum, a protective layer of dead epithelial skin cells (eg, keratinocytes) and extracellular connective tissue proteins. The epithelium is replaced by a new material that is pushed up from the underlying epithelial granule cells, spiny cells, and basal cell layers, thus undergoing a continuous process of exfoliation, where continuous cell division and protein synthesis is achieved by new skin cells and skin proteins (e.g. For example, keratin, collagen). The dermis lies under the epidermis and is elaborated by dermal fibroblasts of connective tissue proteins (eg, collagen, elastin, etc.) assembled into an extracellular matrix and a fibrous structure that gives the skin flexibility, strength and elasticity. ) Site. The skin also contains nerves, blood vessels, smooth muscle cells, hair follicles and dermal oil glands.

As the body's first line of defense, the skin is a major target for clinical damage such as physical, mechanical, chemical and biological (eg, alien, autoimmune) attacks that can alter its structure and function. Skin is also considered an important component of the immunological defense of the organism. Leukocyte cells (eg lymphocytes, macrophages, mast cells) that migrate and remain in the skin and epithelial resin (Langerhans) cells with potent antigen-labeling activity, which contribute to immunological protection, can be found. . Pigmented melanocytes in the basal layer absorb potentially harmful ultraviolet (UV) radiation. Destruction of the skin presents undesirable risks to the subject, including those associated with opportunistic infections, incomplete or inappropriate tissue remodeling, scarring, impaired mobility, pain and / or other complications. Like skin, other epithelial surfaces (eg, respiratory tract, gastrointestinal tract and gland lining) have defined structural properties when healthy, so that infection or other destruction can represent a serious health risk.

Damaged or torn skin can result from, for example, cuts, abrasions, scratches, abrasions, cuts, burns (including chemical burns), infections, extreme temperatures, incisions (eg surgical incisions), trauma and other injuries. have. Thus, efficient skin recovery through wound healing may be clearly desirable in these and similar contexts.

Although the skin exhibits an incredible capacity for self-recovery after many types of damage, skin healing does not occur quickly enough and / or inappropriate cellular tissue damage mechanisms result in integration, barrier properties, mechanical strength, elasticity, flexibility, Or incompletely remodeled skin, which may lack other desirable properties of the intact skin. Thus, skin wound healing represents this related challenge, for example with respect to chronic wounds.

Wound healing occurs in three dynamic, overlapping phases that begin with the formation of fibrin coagulation. This coagulation provides a temporary shield and a reservoir of growth factors that attack the cells into the wound. It also serves as a transient extracellular matrix (ECM) in which cells invade during recovery. Mixed with coagulation formation is an inflammatory phase, which is characterized by infiltration of macrophages and neutrophils into the wound, which releases growth factors that amplify the early healing response, eliminating wound debris and bacteria. The process of restoring the exfoliated site is initiated by healing proliferation and is induced by chemokines, cytokines and proteases secreted from immune cells and concentrated in coagulum. Keratinocytes are stimulated to proliferate and migrate, forming a new layer of epithelium that covers the wound while wound angiogenesis delivers oxygen, nutrients, and inflammatory cells to the wounded site. The remodeling phase is the final phase of wound repair and is performed by myofibroblasts, which promote connective tissue contraction, increase wound strength and deposit ECMs that form scars.

Bismuth thiol- (BT) type preservative

Numerous natural products (eg antibiotics) and synthetic chemicals having antimicrobial and especially antibacterial properties are known in the art and have chemical structure and antimicrobial effects such as the ability to kill microorganisms (bacterial). "Sterilization" effects such as properties), the ability to stop or decay microbial growth ("static" effects such as bacteriostatic properties) or colonize or infect sites, bacterial secretion of exopolysaccharides and / or plankton Is characterized at least in part by the ability to disrupt microbial function such as conversion from to biofilm populations or expansion of biofilm formation. Antibiotics, disinfectants, preservatives and the like (bismuth-thiol or the like), including, for example, bactericidal or bacteriostatic efficacy, effective concentrations, and factors affecting the selection and use of such compositions, including risks of toxicity to host tissues. BT compounds) are discussed, for example, in US Pat. No. 6,582,719.

Bismuth, a Group 5 metal, is a silver-like element with antimicrobial properties. As bismuth itself may not be therapeutically useful and may exhibit certain inappropriate properties, it may instead be typically administered as a delivery means using complexing agents, carriers and / or other vehicles, the most common of which is Pepto Bismol ^® Wherein bismuth is combined (chelated) with the subsalicylate. Previous studies have provided exemplary bismuth thiol (BT) compounds in combination with certain thiol- (-SH, sulfhydryl) containing compounds, such as ethanol dithiol with bismuth, compared to other bismuth preparations currently available. It has been measured to improve the antimicrobial efficacy of bismuth. There are many thiol compounds that can be used to produce BT [see, eg, Domenico et al., 2001 Antimicrob. Agent. Chemotherap. 45 (5): 1417-1421, Domenico et al., 1997 Antimicrob. Agent. Chemother. 41 (8): 1697-1703, and US Re-registered Patent No. RE37,793, US Patent 6,248,371, US Patent 6,086,921, and US Patent 6,380,248; In addition, see, eg, US Pat. No. 6,582,719. Some of these agents can inhibit biofilm formation.

BT compounds are MRSA [methicillin resistant S. aureus. S. aureus], MRSE [methicillin resistant S. aureus]. S. epidermidis], Mycobacterium tuberculosis, Mycobacterium avium, drug-resistant blood. P. aeruginosa, enterotoxic. E. coli, intestinal bleeding Coli, Klebsiella pneumoniae, Clostridium difficile, Heliobacter pylori, Legionella pneumophila, Enterococcus faecalis Enterobacter cloacae, Salmonella typhimurium, Proteus vulgaris, Yersinia enterocolitica, Vibrio cholerae flex, and Sigel 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 monolithic virus type 1 (HSV-1) and HSV-2, and yeasts and fungi such as Candida albicans. BT roles also reduce bacterial pathogenicity, inhibit or kill a broad spectrum of antibiotic-resistant microorganisms (gram-positive and gram-negative), prevent biofilm formation, prevent septic shock, treat sepsis, and antibiotics that have already shown resistance There has been evidence of an increase in bacterial susceptibility to cancer. See, eg, Domenico et al., 2001 Agents Chemother. 45: 1417-1421; Domenico et al., 2000 Infect. Med. 17: 123-127; Domenico et al., 2003 Res. Adv. In Antimicrob. Agents & Chemother. 3: 79-85; 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.

Despite the availability of BT compounds for more than a decade, the effective selection of appropriate BT compounds for specific infectious disease adaptation remains a difficult goal to achieve, where the behavior of special BT against special microorganisms is unpredictable, Here the synergistic activity of special antibiotics and special BT against special microorganisms cannot be predicted, where the in vitro BT effect cannot always predict the BT effect in vivo, where the planktonic (single-cell) microorganism The BT effect on the population cannot predict the BT effect on the microbial community, such as bacteria organized into biofilms. In addition, limitations in solubility, tissue permeability, bioavailability, biodistribution, etc. hinder the ability to safely and effectively deliver clinical benefit in the case of some BT compounds. The invention described herein focuses on these needs and provides other related advantages.

A brief summary

For the first time, and without being bound by theory, according to certain embodiments described herein, bismuth-thiol (BT) compounds are used as preservatives for use in the treatment of a wide range of clinical infectious diseases and conditions and in personal healthcare. On the other hand, it also reduces the cost imposed on the treatment of such infections, including the savings realized by at least partially mediated prevention or prevention by BT.

Further, in certain embodiments, bacteria associated with biofilm formation (eg, bacteria that can form or otherwise promote biofilm), including one or more BT compounds and one or more antibiotic compounds, as described herein. Or formulations for treating tissues and / or surfaces containing bacterial biofilms are contemplated, where appropriately selected combinations of BT compound (s) and antibiotic (s) based on the present disclosure are in accordance with non-limiting theories. The antibacterial (including anti-biofilm) effects of the formulations and / or the unexpected improvement effects for the prevention, prevention and / or therapeutically effective treatment of microbial infections, including infections containing bacterial biofilms, have so far been Provides unexpected synergy

Also provided herein are unprecedented bismuth-thiol compositions comprising substantially monodisperse microparticulate suspensions, and methods and uses for their synthesis.

According to certain embodiments of the invention described herein, a bismuth-thiol composition comprising a plurality of microparticles comprising a bismuth-thiol (BT) compound having a volume average diameter of substantially all of the microparticles is from about 0.4 μm to about 5 μm Wherein a BT compound includes a bismuth or bismuth salt and a thiol-containing compound. In another embodiment substantially all of the particulates have a volume average diameter of from about 0.4 μm to about 5 μm and (a) for a time sufficient and under sufficient conditions to obtain a solution that is substantially free of solid precipitate, (i) at least 50 mM An acidic aqueous solution comprising a bismuth salt comprising a concentration of bismuth and lacking a hydrophilic, polar or organic solubilizer and (ii) at least about 5%, 10%, 15%, 20%, 25% or 30% by volume of ethanol Mixing with an amount of ethanol sufficient to obtain a mixture comprising; And (b) an ethanolic solution containing a thiol-containing compound present in the reaction solution in a molar ratio of about 1: 3 to about 3: 1 relative to bismuth in the mixture of step (a), the microparticles comprising the BT compound. There is provided a bismuth-thiol composition formed by a process comprising adding under sufficient conditions and for a sufficient time to form a precipitate to obtain a reaction solution. In certain embodiments, the bismuth salt is Bi (N0 ₃ ) ₃ . In certain embodiments, the acidic aqueous solution comprises at least 5 wt%, 10 wt%, 15 wt%, 20 wt%, 22 wt% 22.5 wt% bismuth. In certain embodiments, the acidic aqueous solution contains at least 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5% by weight nitric acid. Include. In certain embodiments, the thiol-containing compound is 1,2-ethane dithiol, 2,3-dimercaptopropanol, pyrithione, dithioerythritol, 3,4-dimercaptotoluene, 2,3-butanedithiol, 1 Or 3-propanedithiol, 2-hydroxypropane thiol, 1-mercapto-2-propanol, dithioerythritol, alpha-lipoic acid and dithiothritol.

In another embodiment, (a) a bismuth salt comprising bismuth at a concentration of at least 50 mM, under conditions and for sufficient time to obtain a solution substantially free of solid precipitate, and comprising a hydrophilic, polar or organic Mixing an acidic aqueous solution lacking a solubilizer with (ii) at least about 5%, 10%, 15%, 20%, 25% or 30% ethanol; And (b) an ethanolic solution comprising a thiol-containing compound present in the mixture of step (a) in a molar ratio of about 1: 3 to about 3: 1 relative to bismuth in the reaction mixture, the microparticles comprising the BT compound Bismuth-thiol (BT) having a volume average diameter of substantially all of the fine particles of about 0.4 μm to about 5 μm, including a condition sufficient to form a precipitate comprising and adding for a sufficient time to obtain a reaction solution. A method of making a bismuth-thiol composition comprising a plurality of microparticles comprising a compound is provided. In certain embodiments, the method further comprises recovering 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%, 10%, 15%, 20%, 22%, or 22.5% bismuth by weight. In certain embodiments, the acidic aqueous solution contains at least 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5% by weight nitric acid. Include. In certain embodiments, the thiol-containing compound is 1,2-ethane dithiol, 2,3-dimercaptopropanol, pyrithione, dithioerythritol, 3,4-dimercaptotoluene, 2,3-butanedithiol, 1 , 3-propanedithiol, 2-hydroxypropane thiol, 1-mercapto-2-propanol, dithioerythritol, dithiothritol, alpha-lipoic acid, methanethiol (CH ₃ SH [m-mercaptan]) , Ethanethiol (C ₂ H ₅ SH [e-mercaptan]), 1-propanethiol (C ₃ H ₇ SH [nP 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]), pentanethiol (C ₅ H- ₁₁ SH [pentyl mercaptan]), coenzyme A, lipoamide, glutathione, cysteine, cystine, 2-mercaptoethanol, dithiothritol, dithioerythritol, 2-mercaptoindole, transglutaminase , (11-mercaptodecyl) hexa (ethylene glycol), (11-mercaptodecyl) tetra (ethylene glycol), (11-mercaptodecyl) Tra (ethylene glycol) functionalized gold nanoparticles, 1,1 ', 4', 1 "-terphenyl-4-thiol, 1,11-undecandithiol, 1,16-hexadecanedithiol, 1,2- Ethanedithiol technical grade, 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 furum, 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-octanethiol, 1-pentadecanthiol, 1-pentanethiol, 1-propanethiol, 1-tetradecanethiol, 1- Tetradecanethiol furum, 1-undecanethiol, 11- (1H-pyrrol-1-yl) undecane-1-thiol, 11-amino-1-undecantthiol hydrochloride, 11-bro -1-undecanthiol, 11-mercapto-1-undecanol, 11-mercapto-1-undecanol, 11-mercaptodecanoic acid, 11-mercaptodecanoic acid, 11-mercaptoundecyl trifluoro Acetate, 11-mercaptodecylphosphate, 12-mercaptododecanoic acid, 12-mercaptododecanoic acid, 15-mercaptopentadecanoic acid, 16-mercaptohexadecanoic acid, 16-mercaptohexadecanoic acid, 1 H , 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-nonnafluoro-1-hexanethiol furum , 3- (dimethoxymethylsilyl) -1-propanethiol, 3-chloro-1-propanethiol, 3-mercapto-1-propanol, 3-mercapto-2-butanol, 3-mercapto- N -nonyl Propionamide, 3-mercaptopropionic acid, 3-mercaptopropyl-functionalized silica gel, 3-methyl-1-butanethiol, 4,4'-bis (mercaptomethyl) biphenyl, 4,4'-dimercapto Stilbene, 4- (6-mer Tohexyloxy) benzyl alcohol, 4-cyano-1-butanethiol, 4-mercapto-1-butanol, 6- (ferrocenyl) hexanethiol, 6-mercapto-1-hexanol, 6-mercaptohexane 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, decanthiol 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 ^™ 18, NanoThinks ^™ 8, NanoThinks ^™ ACID11, NanoThinks ^™ ACID16, NanoThinks ^™ ALCO11 , NanoThinks ^TM THIO8, octane thiol functionalized gold nanoparticles, PEG dithiol average Mn 8,000, PEG-dithiol-average molecular weight (mol wt) 1,500, PEG-dithiol-average molecular weight 3,400, S- (11- bromo moun Sil) thioacetate, S- (4-cyanobutyl) thioacetate, thiophenol, triethylene glycol mono-11-mercaptoundecyl ether, trimethylolpropane tris (3-mercaptopropionate), [11- (Methylcarbonylthio) undecyl] tetra (ethylene glycol), m -carbonan-9-thiol, p -terphenyl-4,4 "-dithiol, tert-dodecyl mercaptan, tert-nonyl mercaptan One or more agents selected from the group.

In other embodiments (i) prevention of surface infection by bacterial, fungal or viral pathogens, (ii) inhibition of cell viability or cell growth of substantially all planktonic cells of bacterial, fungal or viral pathogens, (iii) Sufficient time and under conditions sufficient for one or more of: inhibition of biofilm formation by bacterial, fungal or viral pathogens, and (iv) biofilm viability of bacteria, fungal or viral pathogens or inhibition of biofilm growth of substantially all biofilm-forming cells. While contacting an epithelial tissue surface with an effective amount of a BT composition comprising a plurality of particulates comprising a bismuth-thiol (BT) compound, wherein the volume average diameter of substantially all particulates is from about 0.4 μm to about 5 μm. Include biological tissue surfaces, such as epithelial tissue surfaces, for one or more of bacterial, fungal, and viral pathogens Is provided a method of protecting natural surface. In certain embodiments, bacterial pathogens include (i) one or more Gram-negative bacteria; (ii) one or more Gram-positive bacteria; (iii) one or more antibiotic-sensitive bacteria; (iv) one or more antibiotic-resistant bacteria; (v) Staphylococcus aureus [S. S. aureus], MRSA (methicillin-resistant, S. aureus), Staphylococcus epidermidis, MRSE (methicillin-resistant S. epidermidis), My Cobacterium tuberculosis, Mycobacterium avium, Pseudomonas aeruginosa, drug-resistant blood. Aeruginosa, Escherichia coli, Enterotoxin production. E. coli, intestinal bleeding E. coli, Klebsiella pneumoniae, Clostridium difficile, Heliobacter pylori, Legionella pneumophila, Enterococcus faecalis, Enterococcus faecalis Methicillin-sensitive Enterococcus faecalis, Enterobacter cloacae, Salmonella typhimurium, Proteus vulgaris, Yersica co. , Vibrio cholera, Shigella flexneri, Vancomycin-resistant Enterococcus (VRE), Burkholderia cepacia complex, Francisca tullarensis (Francisella tularensis), Bacillus anthracis, Yersinia pestis, Pseudomonas Pseudomonas aeruginosa, vancomycin-resistant enterococci, Streptococcus pneumonia, penicillin-resistant Streptococcus pneuumonia, E. coli, Escherichia At least one of the bacterial pathogens selected from Burkholderia cepacia, Bucolderia multivorans, Mycobacterium smegmatis and Acinetobacter baumannii It includes. In certain embodiments, bacterial pathogens exhibit antibiotic resistance. In certain embodiments, bacterial pathogens are resistant to antibiotics selected from methicillin, vancomycin, napicillin, gentamicin, ampicillin, chloramphenicol, doxycycline and tobramycin.

In certain embodiments, the natural surface comprises an oral / intranasal steel surface. In further embodiments, 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 epithelium, skin, respiratory tract, gastrointestinal tract, and gland lining.

In certain embodiments, the contacting step is performed once or multiple times. In certain embodiments, at least one step of contacting comprises one of spraying, perfusion, dipping and painting the natural surface. In certain embodiments, at least one contacting step comprises one of inhalation, digestion and oral perfusion. In certain embodiments, the at least one contacting step is inhaled topically, intraperitoneally, orally, parenterally, intravenously, intraarterally, percutaneously, sublingually, subcutaneously, intramuscularly, intraballally, intranasally, inhaled. Through, intraocular, intraatrial, intraventricular, subcutaneous, intrafatal, intraarticular and intradural routes. In certain embodiments, the BT composition comprises 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 at least one BT compound selected from the group consisting of bis-EDT / 2-hydroxy-1-propanethiol.

In certain embodiments, bacterial pathogens exhibit antibiotic resistance. In certain other embodiments the methods described above further comprise contacting the natural surface concurrently or continuously with synergistic antibiotics and / or enhancing antibiotics, and in any order with respect to contacting the surface with the BT composition. do. In certain embodiments, the synergistic and / or enhancing antibiotics include aminoglycoside antibiotics, carbapenem antibiotics, cephalosporin antibiotics, fluoroquinolone antibiotics, glycopeptide antibiotics, lincosamide antibiotics, penicillinase-resistant penicillin antibiotics, and Antibiotics selected from aminophenicillin antibiotics. In certain embodiments, the synergistic and / or enhancing antibiotics are amikacin, arbecasin, gentamicin, kanamycin, neomycin, netylmycin, paromomycin, rhostreptomycin, streptomycin, tobramycin, and apramycin. Aminoglycoside antibiotics selected from mycins.

In another aspect of the invention described herein, (i) prevention of surface infection by a bacterial pathogen, (ii) inhibition of cell viability or cell growth of substantially all planktonic cells of the bacterial pathogen, (iii) bacterial pathogens Under conditions sufficient for at least one of inhibiting biofilm formation by (i) biofilm viability of bacterial pathogens or inhibiting biofilm growth of substantially all biofilm-forming cells and for a sufficient time, the volume average diameter of substantially all of the With (1) at least one bismuth-thiol (BT) composition and (2) at least one BT composition comprising a plurality of particulates comprising a bismuth-thiol (BT) compound, from about 0.4 μm to about 5 μm Contacting the surface simultaneously or continuously and in any order with an effective amount of at least one antibiotic that can act synergistically and / or is thereby enhanced, A method of overcoming antibiotic resistance on a natural surface in which a bio-resistant bacterial pathogen is present (eg, at least one of the anti-bacterial effects of at least one antibiotic known to have an anti-bacterial effect against bacteria of the same bacterial species) In the case of bacterial pathogens that are resistant to, such pathogens are made sensitive to antibiotics. In certain embodiments, bacterial pathogens include (i) one or more Gram-negative bacteria; (ii) one or more Gram-positive bacteria; (iii) one or more antibiotic-sensitive bacteria; (iv) one or more antibiotic-resistant bacteria; (v) Staphylococcus aureus (S. aureus), MRSA (methicillin-resistant, S. aureus), Staphylococcus epidermidis, MRSE (methicillin-resistant S. epidermy) Dis), Mycobacterium tuberculosis, Mycobacterium avium, Pseudomonas aeruginosa, drug-resistant blood. Aeruginosa, Escherichia coli, Enterotoxin production. E. coli, intestinal bleeding E. coli, Klebsiella pneumoniae, Clostridium difficile, Helicobacter phyllori, Legionella pneumophila, Enterococcus paecalis, Methicillin-sensitive enterococcus paecalis, Enterobacter clocae, Salmonella typhimurium , Proteus vulgaris, Yersinia enterocytica, Vibrio cholera, Shigella flexneri, Vancomycin-Resistant Enterococcus (VRE), Burcolderia Sephacia Complex, Francisla Tularensis, Bacillus anthracis, Yersinia Festis, Pseudomonas aeruginosa, vancomycin-resistant enterococci, streptococcus pneumoniae, penicillin-resistant streptococcus pneumoniae, Escherichia coli, burcolderia sepacia, bucholderia motivorans, At least one bacterial pathogen selected from Mycobacterium smegmatis and Axinetobacter baumannini Include.

 In certain embodiments, bacterial pathogens are resistant to antibiotics selected from methicillin, vancomycin, napicillin, gentamicin, ampicillin, chloramphenicol, doxycycline, tobramycin, clindamycin and gatifloxacin.

In certain embodiments, the natural surface comprises an oral / intranasal steel surface. In further embodiments, the natural surface comprises a biological surface, such as a bone, joint, muscle, ligament or tendon.

In certain embodiments, the surface comprises tissue selected from the group consisting of epithelium, skin, respiratory tract, gastrointestinal tract and gland lining. In certain embodiments, the contacting step is performed once or multiple times. In certain embodiments, at least one step of contacting comprises one of spraying, perfusion, dipping and painting the natural surface. In certain other embodiments, the at least one contacting step comprises one of inhalation, digestion and oral perfusion. In certain embodiments, the at least one contacting step is inhalation topically, intraperitoneally, orally, parenterally, intravenously, intraarterally, percutaneously, sublingually, subcutaneously, intramuscularly, intraballally, intranasally, inhaled. Via, intraocular, intraatrial, intraventricular, subcutaneous, intrafatal, intraarticular and intradural routes. In certain embodiments, the BT composition comprises 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 at least one BT compound selected from the group consisting of bis-EDT / 2-hydroxy-1-propanethiol. In certain embodiments, the synergistic and / or enhancing antibiotics include clindamycin, gatifloxacin, aminoglycoside antibiotics, capbafenem antibiotics, cephalosporin antibiotics, fluoroquinolone antibiotics, glycopeptide antibiotics, lincosamide antibiotics, Penicillinase-resistant penicillin antibiotics, and aminopenicillin antibiotics. In certain embodiments, the synergistic and / or enhancing antibiotics are amikacin, arbecasin, gentamicin, kanamycin, neomycin, netylmycin, paromomycin, rhostreptomycin, streptomycin, tobramycin, and apramycin. Aminoglycoside antibiotics selected from mycins.

Returning to another embodiment, (a) at least one BT compound; (b) at least one antibiotic compound that can be enhanced and / or synergistically acted upon by a BT compound; And (c) a pharmaceutically acceptable carrier or excipient comprising a topical carrier. 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 particulates comprising bismuth-thiol (BT) compounds wherein substantially all of the particulates have a volume average diameter of about 0.4 μm to about 5 μm. In certain embodiments, the BT compound is selected from BisEDT and BisBAL. In certain embodiments, the antibiotic compound comprises an antibiotic selected from methicillin, vancomycin, napicillin, gentamicin, ampicillin, chloramphenicol, doxycycline, tobramycin, clindamycin, gatifloxacin, and aminoglycoside antibiotics. In certain embodiments the aminoglycoside antibiotics are selected from amikacin, arbecasin, gentamicin, kanamycin, neomycin, netylmycin, paromomycin, rhostreptomycin, streptomycin, tobramycin and apramycin. In certain embodiments the aminoglycoside antibiotic is amikacin.

In certain other embodiments, identifying as comprising (a) one or both of (i) Gram-positive bacteria, (ii) Gram-negative bacteria, and (iii) both (i) and (ii) on or within the surface; And (b) treating the surface by administering to the surface a formulation comprising at least one bismuth thiol (BT) composition, wherein a method of treating a natural surface supporting or containing bacterial biofilm is provided, wherein (i ) If the bacterial infection comprises gram positive bacteria, the formulation comprises a therapeutically effective amount of at least one BT compound and at least one antibiotic that is rifamycin, and (ii) if the bacterial infection comprises gram negative bacteria, Is a therapeutically effective amount of at least one BT compound and amikacin, and (iii) if the bacterial infection comprises both Gram-positive and Gram-negative bacteria, the formulation may contain a therapeutically effective amount of one or multiple BT compounds, Lipa. Mycin and amikacin.

In certain embodiments, the biofilm comprises one or multiple antibiotic-resistant bacteria. In certain embodiments the treatment of the surface comprises (i) eradication of bacterial biofilms; at least one of (ii) reducing bacterial biofilm and (iii) impairing growth of bacterial biofilm. In certain embodiments, the BT composition comprises a plurality of particulates comprising a bismuth-thiol (BT) compound wherein the volume average diameter of substantially all of the particulates is from about 0.4 μm to about 5 μm.

These and other aspects of the disclosure described in aspects of the invention will be apparent with reference to the following detailed description and the accompanying drawings. U.S. Patent, U.S. Patent Application Publications, US patent applications, foreign patents, foreign patent applications, and non-patent publications are all incorporated herein by reference in their entirety, as if each were incorporated individually. Aspects and aspects of the present invention may optionally be modified to use the concepts of various patents, patent applications, and publications to still provide further aspects.

details

A particular aspect of the invention described herein is that certain bismuth-thiol (BT) compounds (volume average diameters) as provided herein, rather than certain other bismuth-thiol (BT) compounds, even In certain embodiments, including BT microparticles that are between about 0.4 μm and about 5 μm) potent preservatives for special bacteria, including bacteria associated with a number of clinically significant infections, including infections that may include bacterial biofilms It is based on the surprising finding that it exhibited antibacterial and / or anti-microbial activity.

Unexpectedly, not all BT compounds are uniformly effective against these bacteria in a predictable fashion, but instead showed different efficacy depending on the target bacterial species. In particular and as described herein, certain BT compounds (preferably including BT microparticles having a volume average diameter of about 0.4 μm to about 5 μm) exhibit higher efficacy against Gram-negative bacteria, but Other BT compounds (including BT microparticles, preferably with a volume average diameter of about 0.4 μm to about 5 μm), have developed a clinically relevant strategy for the management of bacterial infections, including bacterial biofilm infections, according to a non-limiting theory. In a way that can be provided for the first time, it has been found to show greater efficacy against Gram-positive bacteria.

In addition, and as described in more detail below, certain aspects of the invention described herein relate to particle size (eg, a volume average diameter of about 0.4 μm to about 5 μm), as described herein. Can be prepared into a formulation comprising a plurality of BT microparticles that are substantially monodisperse. In certain of these and related embodiments, the micronized BT is not provided as a component of liquid vesicles or liposomes, such as multilayer lamellar phosphocholine-cholesterol liposomes or other multilayer lamellar or monolamellar liposome vesicles.

As also described herein, with respect to certain embodiments, the antibacterial and anti-microbial efficacy of certain antibiotics, which are antibiotics that have already been found to lack potent therapeutic effects on such bacterial infections, can cause the infection to affect one of these antibiotics. It has been found that treatment can be significantly improved (eg, increased in a statistically significant manner) by treating with the above and selected BT compounds simultaneously or sequentially and in any order. In ways that could not have been predicted prior to the present disclosure, certain BT compounds may be combined with certain antibiotics to elevate as provided herein with respect to antibacterial and / or anti-microbial activity against certain bacterial species or bacterial strains. Combinations of actions or enhancements may be provided. As described in more detail below, the unexpected properties of these combinations indicate that certain BT / antibiotic combinations act synergistically or exhibit enhancement against certain bacteria, while certain other BT / antibiotic combinations are synergistic. Or by observation that they failed to exhibit enhanced antibacterial and / or anti-microbial activity.

According to these and related embodiments, the antibiotics and BT compounds may be administered simultaneously or continuously and in any order, and for the treatment of special infections (eg, biofilms formed by Gram-negative or Gram-positive bacteria). Specific synergistic or enhanced combinations of one or more antibiotics and one or more BT compounds as described herein did not show predictable (eg, only additional) activity, but instead selected antibiotics, selected BT compounds, and specifically identified target bacteria. It is noteworthy that, as a function of, it acted in an unexpected synergistic or enhanced (eg, super-additive) form.

For example, in the manner of description and in a non-limiting manner, as described herein, with respect to a variety of natural surfaces that have been effectively or efficaciously microbially infected, and with respect to improved substantially monodisperse particulate BT formulations. Either or both of the compound and the specific BT compound may exert limited antibacterial effects when used alone against a particular bacterial strain or species, but combinations of both antibiotic compounds and BT compounds may be potent antimicrobial agents against the same bacterial strain or species. Exhibits a bacterial effect, which, when used alone, is larger than the simple sum of the effects of each compound (with statistical significance) and, according to a non-limiting theory, the BT's and / or BT's effectiveness in antibiotic efficacy. Antibiotic-BT synergistic effects (eg FICI ≤ 0.5) or enhancement effects (eg 0.5) of antibiotics in efficacy <FICI ≤ 1.0). Thus, but not all, BT compounds may be elevated or enhanced with all antibiotics, but not all, antibiotics are elevated or enhanced with all BT compounds, such that antibiotic-BT synergy and BT-antibiotic enhancement are generally unpredictable. not. Instead, and in accordance with certain embodiments described herein, the special combination of synergistic or enhanced antibiotics and BT compounds surprisingly includes a special environment, such as a natural surface, as described herein, and, in certain circumstances, biofilms formed by special bacteria. It provides a strong antibacterial effect against special bacteria, further comprising an antibacterial effect against.

That is, certain BT- synergistic antibiotics are described herein, which include antibiotics that can act synergistically (FICI <0.5) with at least one BT composition comprising at least one BT compound as provided herein Where the synergistic effect is greater in scale than the effect that can be detected in the presence of antibiotics but in the absence of a BT compound and / or in the absence of a BT compound but in the absence of antibiotics (ie, statistically in relation to appropriate control conditions). In a significant way). Similarly, certain BT-antibiotic combinations exhibit enhancement (0.5 <FICI <1.0), where such enhancement is in the presence of antibiotics but in the absence of BT compounds and / or in the presence of BT compounds but in the absence of antibiotics. If a larger detectable effect (ie, in a statistically significant manner with respect to appropriate control conditions) than a detectable effect.

Examples of such detectable effects include, in certain embodiments, (i) preventing infection by bacterial pathogens, (ii) inhibiting cell survival or cell growth of substantially all planktonic cells of bacterial pathogens, and (iii) by bacterial pathogens. Inhibition of biofilm formation, and (iv) biofilm viability of bacterial pathogens or inhibition of biofilm growth of substantially all biofilm-forming cells, but the present invention is not intended to be so limited, and in other contemplated embodiments, antibiotics A synergistic effect of BT is one or more other clinically significant parameters, such as the degree of resistance or sensitivity of bacterial pathogens to one or more antibiotics or other drugs or chemicals, one or more chemical, physical or mechanical conditions (eg, For example, the degree of resistance or sensitivity of bacterial pathogens to pH, ionic strength, temperature, pressure, and / or one or more biologics (eg For example, bacterial alterations to viruses, other bacteria, biologically active polynucleotides, immune cells or antibodies, cytokines, chemokines, enzymes including degrading enzymes, membrane-destructive proteins, free radicals such as reactive oxygen species, etc. Alterations (eg, statistically significant increases or decreases) in the degree of resistance or sensitivity of a source.

Those familiar with the art are aware that the effect of a special agent on the structure, function and / or activity of a bacterial population, as provided herein, is such that the effect of a synergistic or enhanced antibiotic-BT combination is free of one component of the combination. If more than a simple sum of the observed effects can be measured for the purpose of identifying antibiotic-BT synergy or enhancement present [see, for example, Coico et al. (Eds.), Current Protocols in Microbiology, 2008, John Wiley & Sons, Hoboken, NJ; Schwalbe et al., Antimicrobial Susceptibility Testing Protocols, 2007, CRC Press, Boca Raton, FL. These and various other criteria will be recognized.

For example, in certain embodiments, synergism can be achieved by measuring antibacterial effects, such as those described herein, using varying concentrations of candidate agents (eg, individually and in combination with BT and antibiotics). Eliopoulos et al., Eliopoulos and Moellering, (1996) Antimicrobial combinations. In Antibiotics in Laboratory Medicine (Lorian, V., Ed.), Pp. 330-96, Williams and Wilkins, Baltimore, MD, USA, can be determined by calculating the fractional inhibitory concentration index (FICI) and fractional bactericidal concentration index (FBCI). Synergy can be defined as a FICI or FBCI index of ≦ 0.5, and antagonism of> 4. See, for example, Odds, FC (2003) Synergy, antagonism, and what the chequerboard puts between them. Journal of Antimicrobial Chemotherapy 52: 1]. Synergism also typically results in a ≧ 4 fold reduction in antibiotic concentration, or alternatively, for example in Hollander et al. It can be defined as fractional inhibitory concentration as described in (1998 Antimicrob. Agents Chemother. 42: 744.) In certain embodiments, synergy is from a combination of two drugs (eg, antibiotics and BT compositions). It can be defined as the effect produced, wherein the effect of the combination is greater than the effect when the concentration of the second drug is replaced by the first drug (eg, in a statistically significant manner).

Thus, as described herein and in certain preferred embodiments, the combination of BT and antibiotic will be understood to be synergistic when a FICI value of 0.5 or less is observed (Odds, 2003). As also described herein, according to certain other preferred embodiments and non-limiting theories, certain BT-antibiotic combinations may exhibit a FICI value of between 0.5 and 1.0, indicating high efficacy for this synergy. It is described that this can be observed using a non-optimal concentration of at least one BT and at least one antibiotic that alone or mutually exhibits enhanced cooperative antimicrobial efficacy. This effect may also be referred to herein as "enhanced" antibiotic activity or "enhanced" BT activity.

The enhanced antibiotic and / or BT activity is at least at a concentration that is (i) below the characteristic minimum inhibitory concentration (MIC) of BT (in a statistically significant manner) for a given target microorganism (eg, a given bacterial species or strain). One BT, and (ii) a characteristic IC ₅₀ (concentration that inhibits growth of 50% of the microbial population; see, eg, Soothill et al., 1992 J Antimicrob Chemother 29 (2): 137) (statistical In a significant manner) and / or at the concentration below the biofilm-preventive concentration (BPC) of the antibiotic for a given target microorganism, the presence of both antimicrobials (eg BT or antibiotics) ) Improves (in a statistically significant) antimicrobial effect of the BT-antibiotic combination compared to the antimicrobial effects that can be observed when used at the same concentration in the absence of other antimicrobial agents (e.g. antibiotics or BT). When creating a can be detected in accordance with a particular embodiment. In preferred embodiments, the "enhanced" antibiotics and / BT is active, if there is more than 1.0 and less than 0.5 FICI value, as measured.

As will be appreciated by those skilled in the art based on the present disclosure, in certain embodiments, synergistic or enhanced antibiotic and / or BT activity may be achieved by a Loewe additivity-based model [eg, FIC index, Greco model], or Bliss independence based model (e.g., non-parametric and semi-parameter models) or described herein and known in the art (e.g., For example, Meletiadis et al., 2005 Medical Mycology 43: 133-152) can be measured according to methods known in the art, such as using other methods. Thus, exemplary methods for measuring synergistic or enhanced antibiotic and / or BT activity are described, for example, in Meletiadis et al., 2002 Rev Med Microbiol 13: 101-117; White et al., 1996 Antimicrob Agents Chemother 40: 1914-1918; Mouton et al., 1999 Antimicrob Agents Chemother 43: 2473-2478.

Certain other embodiments include those in which the BT compound (s) and antibiotic (s) did not exhibit predictable (eg, simply additional) activity in vivo, but instead included selected antibiotics, selected BT compounds, and infections thereof. At least one function of a specifically identified target bacterial species, in an unexpected synergistic or enhancement mode (eg, when two or more such agents are replaced when the concentration of the second agent is replaced by the first agent) Special infections (eg, gram-negative or gram-positive), even when present in combinations greater (eg, in a statistically significant manner), even when they are effective or hyper-additive Of one or more antibiotics and one or more BT compounds as described herein, which may have a synergistic or enhanced effect in vivo for the treatment of biofilms formed by bacteria). Consider the transfer of the combination. Thus, depending on these and related embodiments, the FICI or FBCI values (which are measured in vitro) may not be readily available in certain in vivo situations but instead the BT-antibiotic synergistic or enhancing effect is a quantifiable matrix of infection. Can be measured in the manner provided by

For example, in one embodiment as in the in vivo open fracture Rattus norvegicus femur critical defect model, as described in Example 11, antibiotic therapy or BT compound alone A statistically significant decrease in the number of bacteria after observed treatment for the compared BT-antibiotic combination is an index of synergistic or enhancing effect. Statistical significance can be measured using methods well known to those skilled in the art. In certain other embodiments, at least 5%, 10%, 20%, 30%, 40%, or 50% of the number of bacteria observed in injury following treatment with a BT-antibiotic combination compared to antibiotic treatment or a BT compound alone The reduction observed in these or other in vivo models up to is considered an index of synergistic or enhancing effect.

Other exemplary indices of in vivo infection can be measured according to established methodologies developed for the quantification of the severity of infection, such as various wound scoring systems known to those skilled in the art. European Wound Management Association (EWMA), Position Document: Identifying criteria for wound infection. Scoring system considered in London: MEP Ltd, 2005]. Illustrative wound scoring systems that can be used in assessing the synergistic or enhancing activity of a BT-antibiotic combination as described herein are described in ASEPSIS [Wilson AP, J Hosp Infect 1995; 29 (2): 81-86; Wilson et al., Lancet 1986; 1: 311-13, Southampton Wound Assessment Scale (Bailey Bailey, Karran SE, Toyn K, et al. BMJ 1992; 304: 469-71). Horan TC, Gaynes P, Martone WJ, et al., 1992 Infect Control Hosp Epidemiol 1992; 13: 606-08. In addition, recognized clinical indices of wound healing known to the skilled person, such as wound size, depth, granulated tissue condition, infection, and the like, can be measured in the presence or absence of BT compounds and / or antibiotics. Thus, and based on the present disclosure, those skilled in the art will appreciate that the BT composition-antibiotic combination alters wound healing in vivo (e.g., increases or decreases in a statistically significant manner relative to an appropriate control). Will be readily appreciated.

In terms of these and related embodiments, an effective amount of a composition or formulation comprising one or more BT compounds and, optionally, one or more antibiotic compounds, such as one or more synergistic antibiotics, or one or more enhanced antibiotics, as provided herein (eg, For example, various methods are provided herein for treating a microbially infected natural surface, such as a surface that supports or contains a bacterial biofilm in a specific embodiment. Based on the present disclosure, it will be apparent that certain antibiotics may be contemplated for use in the treatment of a given type of infection, based on the present disclosure, wherein such antibiotics may be used to treat the same type of infection in a person familiar with the art. It has already been considered ineffective.

Thus, certain embodiments contemplate compositions comprising one or more BT compounds for use as preservatives. Preservatives are substances that kill or prevent the growth of microorganisms and are typically applied to living tissues to distinguish these classes from disinfectants that are generally applied to non-living subjects. Goodman and Gilman's "The Pharmacological Basis of Therapeutics" ", Seventh Edition, Gilman et al., Editors, 1985, Macmillan Publishing Co., hereinafter Goodman and Gilman" pp. 959-960]. Typical examples of preservatives are tinctures of ethyl alcohol and iodine ( germanium contains preservatives that kill microorganisms, such as microbial pathogens.

Certain embodiments described herein can contemplate compositions comprising one or more BT compounds and one or more antibiotic compounds (eg, synergistic antibiotics and / or enhanced antibiotics as provided herein). Antibiotics are known in the art and are typically drugs made from compounds produced by one species of microorganism to kill other species of microorganisms, or synthetic products having the same or similar chemical structure and mechanism of action, for example And drugs that destroy microorganisms in the body or in the body of a living organism, including such drugs when applied topically. Among the embodiments described herein, antibiotics may belong to one of the following classes: aminoglycosides, carbapenems, cephalosporins, fluoroquinolones, glycopeptide antibiotics, lincosamide (eg, clinda) Superstition), penicillinase-resistant penicillin, and aminopenicillin. Thus, antibiotics are oxacillin, piperacillin, cepuroxime, cytotaxime, cefepime, imipenem, aztreonam, streptomycin, tobramycin, tetracycline, minocycline, ciprofloxacin, levofloxacin, erythromycin, linezolid, phospho Formycin, capreomycin, isoniazid, ansamycin, carbacefem, monobactam, nitrofuran, penicillin, quinolone, sulfonamide, clofazimin, daphson, capremycin, cycloserine, etabutol, ethionamide, isoniazid , Pyrazineamide, rifampicin, rifampin, rifabutin, rifaptine, streptomycin, arsphenamine, chloramphenicol, phosphomycin, fusidic acid, linezolid, metronidazole, mupyrosine, platensimacin, quinupristin, dalpopristin , Rifaximin, thiamphenicol, tinidazole, aminoglycoside, beta-lactam, penicillin, cephalosporin, car Bafenem, fluroquinolone, ketolide, lincosamide, macrolide, oxazolidinone, streptogamine, sulfonamide, tetracycline, glycylcycline, methicillin, vancomycin, napicillin, gentamicin, ampicillin, chloramphenicol , Doxycycline, tobramycin, amikacin, arbecasin, gentamicin, kanamycin, neomycin, netylmycin, paromomycin, rhostreptomycin, streptomycin, tobramycin, apramycin, clindamycin, Gatifloxacin, aminopenicillin, and others known in the art. A summary of these and other clinically useful antibiotics is available in the art and known to those familiar with the art. See, for example, Washington University School of Medicine, The Washington Manual of Medical Therapeutics (32nd Ed.) , 2007 Lippincott, Williams and Wilkins, Philadelphia, PA; Hauser, AL, Antibiotic Basics for Clinicians, 2007 Lippincott, Williams and Wilkins, Philadelphia, PA].

Exemplary classes of antibiotics for use with one or more BT compounds in certain embodiments described herein are described in Edson RS, Terrell CL. The aminoglycosides. Mayo Clin Proc. 1999 May; 74 (5): 519-28, the aminoglycoside class of antibiotics discussed. This class of antibiotics inhibits bacterial growth by impairing bacterial protein synthesis through binding and inactivation of bacterial ribosomal subunits. In addition to these bacteriostatic properties, aminoglycosides also exhibit bacteriostatic effects through the destruction of cell walls in Gram-negative bacteria.

Aminoglycoside antibiotics include gentamicin, amikacin, streptomycin, and others and, although classes of resistant strains have been reported, are generally useful for the treatment of gram-negative bacteria, mycobacteria and other microbial pathogens. Is considered. Since aminoglycosides are not absorbed through the digestive tract, they are generally not considered to be able to be treated with oral formulations. For example, amikacin, which is often effective against gentamicin-resistant bacterial strains, is typically administered intravenously or intramuscularly, which can cause pain in a patient. In addition, toxicity associated with aminoglycoside antibiotics such as amikacin can lead to kidney damage and / or irreversible hearing loss.

Notwithstanding these properties, certain embodiments described herein may be used to provide elevated BT / antibiotic combinations (e.g. , Oral administration, if the antibiotic need not be limited to aminoglycosides. In addition, in certain other embodiments, the use of the compositions and methods described herein as antiseptics may be contemplated as referring to agents that kill or block the growth of microorganisms on the outer surface of an inanimate subject.

As also described elsewhere herein, the BT compounds are bismuth or bismuth salts and thiol- (eg -SH, or sulfhydryl) containing compounds, for example, see Domenico et al., 1997. Antimicrob. Agent. Chemother. 41 (8): 1697-1703, Domenico et al., 2001 Antimicob. Agent. Chemother. 45 (5): 1417-1421, and US Re-registered Patent No. RE37,793, US Patent 6,248,371, US Patent 6,086,921, and US Patent 6,380,248; It may also be a composition comprising, for example, those described in US Pat. No. 6,582,719, including methods for their preparation. However, certain embodiments are not limited and other BT compounds including bismuth or bismuth salts and thiol-containing compounds may be contemplated. Thiol-containing compounds 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 ionic bonding and / or as a coordination complex, but in some other embodiments bismuth is via a covalent bond, as can be found in organometallic compounds However, certain contemplated embodiments specifically exclude BT compounds, which are organometallic compounds such as compounds in which bismuth is found in covalent bonds to organic moieties.

Exemplary BT compounds are shown in Table 1:

TABLE 1

Exemplary BT Compounds

Based on the known tendency of bismuth to form trivalent complexes with sulfur containing compounds and the stereochemical ratios of the reactants used, the atomic ratios for single bismuth atoms for comparison are shown. The atomic ratio shown may not be the correct molecular formulation for all species in a given formulation. The number in parentheses is one (or more) thiol agent (eg Bi: thiol1 / thiol2) "CPD", the ratio of bismuth to the compound.

BT compounds for use in certain presently described embodiments are described by established procedures [see, eg, US Re-registered Patent RE37,793, US Patent 6,248,371, US Patent 6,086,921 and US Patent 6,380,248; Domenico et al., 1997 Antimicrob. Agent. Chemother. 41 (8): 1697-1703, Domenico et al., 2001 Antimicob. Agent. Chemother. 45 (5): 1417-1421 and in certain other embodiments BT compounds may also be prepared according to the methods described herein. Accordingly, certain preferred embodiments contemplate the synthetic methods described herein for preparing BT compounds and in particular for obtaining BT compounds in substantially monodisperse particulate form, wherein the concentration of dissolved bismuth is at least 50 mM, at least 100. mM, at least 150 mM, at least 200 mM, at least 250 mM, at least 300 mM, at least 350 mM, at least 400 mM, at least 500 mM, at least 600 mM, at least 700 mM, at least 800 mM, at least 900 mM or at least 1 M An acidic aqueous bismuth solution lacking a hydrophobic polar or organic solubilizing agent is mixed with ethanol to obtain a first ethanolic solution, which is sufficient to form conditions and times (e.g. Concentration, solvent strength, temperature, pH, mixing and / or pressure, etc., as described herein and as would be appreciated by one of skill in the art based on the present disclosure. Conditions) to react with a second ethanolic solution containing a thiol-containing compound to obtain a reaction solution in which the thiol-containing compound is present in the reaction solution in a molar ratio of about 1: 3 to about 3: 1 relative to bismuth. .

Thus, exemplary BTs include Compound 1B-1, bis-EDT (bismuth-1,2-ethane dithiol, reactant at 1: 1); Compound 1B-2, bis-EDT (1: 1.5); Compound 1B-3, bis-EDT (1: 1.5); Compound 1C, bis-EDT (soluble Bi formulation, 1: 1.5); Compound 2A, bis-Bal (bismuth-British anti-Lewisite (bismuth-dimercapprolol, 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-butanedithiol, 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, propane thiol (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 / Ery (1: 1 / 0.5); Compound 15, bis-EDT / 2-hydroxy, propane thiol (1: 1/1) (see, eg, Table 1).

Without wishing to be bound by theory, the process for preparing a BT compound as described herein, comprising preparing or obtaining an acidic liquid aqueous solution comprising bismuth, such as an aqueous nitric acid solution comprising bismuth nitrate, in certain preferred embodiments is preferably BT. A composition comprising a compound can be obtained, wherein the composition is useful for mass production ease, improved product purity, uniformity or consistency (including uniformity in particle size), or for the preparation and / or administration of the present topical formulations. Have different characteristics.

In a particular embodiment, BT compositions prepared according to the methods described herein relate to their occurrence as substantially monodisperse suspensions of particulates having a volume average diameter (VMD) of about 0.4 μm to about 5 μm, in accordance with certain presently preferred embodiments. To show an advantageous degree of homogeneity. Measurement of particle size may be referred to as volume average diameter (VMD), mass median diameter (MMD), or mass median aerodynamic diameter (MMAD). These measurements can be made, for example, by impact (MMD and MMAD) or laser (VMD) characterization. In the case of liquid particles, VMD, MMD and MMAD may be the same if environmental conditions are maintained, for example at standard humidity. However, if humidity is not maintained, MMD and MMAD measurements will be less than VMD due to dehydration during impactor measurements. For the purposes of this description, the description of VMD, MMD and MMAD will be compared by considering that VMD, MMD and MMAD measurements are under standard conditions. Similarly, dry powder particle size measurements in MMD and MMAD are also considered comparable.

As described herein, preferred embodiments relate to a substantially monodisperse suspension of BT-containing particulates. The generation of defined BT particle sizes with limited geometrical standard deviation (GSD) is, for example, BT deposition, access to the target site of interest in or on the natural surface and / or tolerance by the subject to which the BT microparticles are administered. ) Can be optimized. Narrow GSD limits the number of particles outside the desired VDM or MMAD size range.

In one embodiment, a liquid or aerosol suspension of particulates containing one or more BT compounds described herein is provided wherein the VMD is from about 0.5 microns to about 5 microns. In another embodiment, a liquid or aerosol suspension having about 0.7 microns to about 4.0 microns VMP or MMAD is provided. In another embodiment, a liquid or aerosol suspension having a VMD or MMAD of about 1.0 to about 3.0 microns is provided. In certain other preferred embodiments, about 0.1 to about 5.0 micron VMD, 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 micron to about 1.0, about 1.5, A BT compound as described herein of 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 There is provided a liquid suspension comprising one or more of the BT compound particles comprising.

Thus, and in certain preferred embodiments, a BT composition comprising a BT agent, as described herein, substantially in the form of "substantially" monodispersed, e. The mean diameter (VMD) is, for example, within a defined range of about 0.4 μm to about 5 μm) of at least 80%, 85%, 90%, 91%, 92%, 93%, or 94% of the particles. And more preferably at least 95%, 96%, 97%, 98%, 99% or more include compositions having VMDs in the size ranges mentioned.

These and related properties of the BT compositions prepared according to the synthetic methods described herein allow for lower cost and ease of production than BT described above, and compliance with one or more pharmaceutical, formulation and cosmetic cosmetic standards. In an unprecedented advantage, including uniformity in the composition that can tolerate its characterization.

Additionally or alternatively, the substantially monodisperse BT microparticles described herein can be used without the need for micronization, ie other devices and processes typically used to produce microparticles, or expensive and labor-intensive grinding or supercritical It can be advantageously produced in the absence of fluid processing. See, for example, Martin et al. 2008 Adv. Drug Deliv. Rev. 60 (3): 339; Moribe et al., 2008 Adv. Drug Deliv. Rev. 60 (3): 328; Cape et al., 2008 Pharm. Res. 25 (9): 1967; Rasenack et al. 2004 Pharm. Dev. Technol. 9 (1): 1-13]. Thus, the present embodiments include improved and substantially uniform solubilization properties, suitability for the desired dosage form, such as oral, inhalation or dermatological / dermal wound topical form, increased bioavailability and other advantageous properties, It provides the beneficial effect of a substantially uniform particulate formulation, but not limited to this.

BT compound particulate suspensions are available to those familiar with the formulation, as aqueous formulations, as suspensions or solutions in aqueous and also organic solvents containing halogenated hydrocarbon propellants as anhydrous powders, or for example to maintain individual particulates in suspension. It may be administered in other forms as described below, including additives as may be known or preparations containing other ingredients, wetting agents, surfactants or mineral oils. Aqueous formulations can be aerosolized, for example, with a liquid nebulizer using hydraulic or ultrasonic granularity. Propellant-based systems may use suitable pressure dispensers. Anhydrous powder can use the anhydrous powder dispersion apparatus which can disperse | distribute BT-containing microparticles | fine-particles effectively. The desired particle size and distribution can be obtained by selecting the appropriate device.

As also shown above, according to certain embodiments substantially all of the particulates have a volume average diameter (VMD) of from about 0.1 to about 8 microns, and in certain preferred embodiments, including a BT compound, from about 0.4 microns to about 5 microns Also provided herein is a method of making a bismuth-thiol (BT) composition comprising a plurality of particulates.

In general terms, the process comprises (a) a bismuth salt comprising bismuth at a concentration of at least 50 mM and for sufficient time to obtain a solution that is substantially free of solid precipitates and is hydrophilic, polar Or an acidic aqueous solution lacking an organic solubilizer, (ii) at least about 5%, 10%, 15%, 20%, 25% or 30%, and preferably about 25% by volume of ethanol Mixing with a sufficient amount of ethanol to obtain a mixture comprising; And (b) adding to the mixture of step (a) an ethanolic solution comprising a thiol-containing compound under conditions and for a sufficient time to form a precipitate comprising the BT compound, so that the thiol-containing compound is reacted with the reaction solution. Obtaining a reaction solution present in the molar ratio of about 1: 3 to about 3: 1 relative to bismuth.

In certain preferred embodiments, the bismuth salt may be Bi (N0 ₃ ) ₃ , but it will be appreciated that bismuth may also be provided in other forms in accordance with 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 at least 1M. In certain embodiments, the acidic aqueous solution comprises at least 5%, 10%, 15%, 20%, 22%, or 22.5% by weight bismuth. In certain preferred embodiments the acidic aqueous solution comprises at least 5 wt% or more nitric acid, and in certain other embodiments the acidic aqueous solution is at least 0.5 wt%, at least 1 wt%, at least 1.5 wt%, at least 2 wt%, at least 2.5 wt% , At least 3 wt%, at least 3.5 wt%, at least 4 wt%, at least 4.5 wt% or at least 5 wt% nitric acid.

The thiol-containing compound may be any thiol-containing compound as described herein, and in certain embodiments, 1,2-ethane dithiol, 2,3-dimercaptopropanol, pyrithione, dithioerythritol, 3, At least one of 4-dimercaptotoluene, 2,3-butanedithiol, 1,3-propanedithiol, 2-hydroxypropane thiol, 1-mercapto-2-propanol, dithioerythritol and dithiothritol It may include. Other exemplary thiol-containing compounds include alpha-lipoic acid, methanethiol (CH ₃ SH [m-mercaptan]), ethanethiol (C ₂ H ₅ SH [e-mercaptan]), 1-propanethiol (C ₃ H ₇ SH [nP 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]), pentanethiol (C ₅ H ₁₁ SH [pentyl mercaptan]), coenzyme A, lipoamide, glutathione, cysteine, cystine, 2-mer In the following thiol compounds available from captoethanol, dithiothreitol, dithioerythritol, 2-mercaptoindole, transglutaminase, and Sigma-Aldrich (St. Louis, MO) Which includes: (11-mercaptodecyl) hexa (ethylene glycol), (11-mercaptodecyl) tetra (ethylene glycol), (11-mercaptodecyl) tetra (ethylene glycol) functionalized gold nano Particles, 1,1 ', 4', 1 "-terphenyl-4-thiol, 1,11-unde Dithiol, 1,16-hexadecanedithiol, 1,2-ethanedithiol technical grade, 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 -Decanthiol, 1-dodecanethiol, 1-heptane thiol, 1-heptane thiol furum, 1-hexadecanethiol, 1-hexanethiol, 1-mercapto- (triethylene glycol), 1-mercapto- (tri Ethylene glycol) methyl ether functionalized gold nanoparticles, 1-mercapto-2-propanol, 1-nonanthiol, 1-octadecanethiol, 1-octanethiol, 1-octanethiol, 1-pentadecanthiol, 1- Pentanethiol, 1-propanethiol, 1-tetradecanethiol, 1-tetradecanethiol furum, 1-undecantiol, 11- (1H-pyrrol-1-yl) undecane-1-thiol, 11-amino-unde Canthiol hydrochloride, 11-bromo-1-undecanthiol, 11-mercapto-1-undecanol, 11-mercapto-1-undecanol, 11-mercaptodecanoic acid, 11- Mercaptodecanoic acid, 11-mercaptodecyl trifluoroacetate, 11-mercaptodecyl phosphate, 12-mercaptododecanoic acid, 12-mercaptododecanoic acid, 15-mercaptopentadecanoic acid, 16-mercapto Hexadecanoic acid, 16-mercaptohexadecanoic acid, 1H, 1H, 2H, 2H-perfluorodecanethiol, 2,2 '-(ethylenedioxy) diethanethiol, 2,3-butanedithiol, 2- Butanethiol, 2-ethylhexanethiol, 2-methyl-1-propanediol, 2-methyl-2-propanethiol, 2-phenylethanethiol, 3,3,4,4,5,5,6,6,6 -Nonafluoro-1-hexanethiol furum, 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-functionalized silica gel, 3-methyl-1-butanethiol, 4,4'-bis (mercaptomethyl) ratio Phenyl, 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-mercaptoonanoic acid, 9-mercapto-1-nonanol, biphenyl-4, 4'-dithiol, butyl 3-mercaptopropionate, copper (I) 1-butanethiolate, cyclohexanethiol, cyclopentanethiol, decanthiol functionalized silver nanoparticles, dodecanethiol functionalized gold nanoparticles, Dodecanethiol functionalized silver nanoparticles, hexa (ethylene glycol) mono-11- (acetylthio) undecyl ether, mercaptosuccinic acid, methyl 3-mercaptopropionate, nanoterter BPA-HH, NanoThinks ^TM 18, NanoThinks ^TM 8, NanoThinks ^TM , ACID11, NanoThinks ^TM ACID16, NanoThinks ^TM ALC011, NanoThinks ^TM THIO8, Octanethiol functionalized gold nanoparticles, PEG dithiol average Mn 8,000, PEG dithiol average molecular weight 1,500, PEG dithiol average molecular weight 3,400, S -(11-bromodecyl) thioacetate, S- (4-cyanobutyl) thioacetate, thiophenol, triethylene glycol mono-11-mercapto Decyl 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 are described herein, including temperature, pH, reaction time, stirring or agitation to dissolve the solute, and the process for collecting and washing the precipitate, using techniques commonly known in the art. do.

Unlike the methods described above for producing BT compounds, according to the present method for producing BT, the BT product generally has a VMD of about 0.4 to about 5 microns in certain preferred embodiments and generally about It is provided as a particulate suspension with substantially all particulates ranging from 0.1 micron to about 8 microns. In addition, contrary to the foregoing attempts, bismuth according to this embodiment has a bismuth salt at a concentration of at least about 50 mM to about 1 M, and at least about 0.5% to about 5% (w / w) and preferably 5% (weight). / Weight), provided in an acidic aqueous solution containing nitric acid in an amount less than hydrophilic, polar or organic solubilizer.

In this regard, the process of the present invention is that the bismuth is not 50 μM water soluble (e.g., US Re-registered Patent No. RE37793), and the bismuth is unstable in water [see, for example, Kuvshinova et al., 2009 Russ. J Inorg. Chem 54 (11): 1816), bismuth offers surprising and unpredictable advantages in terms of generally accepted teachings in the art that are unstable even in nitric acid solutions when no hydrophilic, polar or organic solubilizers are present. For example, techniques for defining BT formulation methods (eg, Domenico et al., 1997 Antimicrob. Agents. Chemother. 41: 1697; US Pat. No. 6,380,248; US Re-registered Patent RE37793; US Pat. 6,248,371), hydrophilic solubilizing agent propylene glycol is needed to dissolve bismuth nitrate, and the bismuth concentration of the solution prepared for reaction with thiol is less than 15 mM, thus making available production aspects for BT compounds. Restrict.

In contrast, according to the present disclosure, there is no need for hydrophilic, polar or organic solubilizers to dissolve bismuth, but higher concentrations are surprisingly achieved. Hydrophilic, polar or organic solubilizers include propylene glycol (PG) and ethylene glycol (EG) and also include dioxane and dimethylsulfoxide (DMSO), polyols [eg, PG and EG and also polyethylene glycol (PEG ), Polar solvents such as polypropylene glycol (PPG), pentaerythritol and others, polyhydric alcohols such as glycerol and mannitol, and many known soluble enhancers, including other agents. Other high polarity water-miscible organic materials include dimethylsulfoxide (DMSO), dimethylformamide (DMF) and NMP (N-methyl-2-pyrrolidone).

Thus, solvents, including those commonly used as hydrophilic, polar or organic solubilizers as provided herein, have, for example, a polarization rate (SPP) value of water of 0.962, an SPP value of toluene of 0.655, Catalan et al. (See, eg, 1995 Liebigs Ann. 241; See also: Catalan, 2001 In: Handbook of Solvents, Wypych (Ed.), Andrew Publ., NY, and references cited therein] for selection based on solvent polarity / polarization (SPP) scale values. Can be. Determining the SPP value of a solvent based on UV measurement of 2-N, N-dimethyl-7-nitrofluorene / 2-fluoro-7-nitrofluorene probe / homomorph pair A method is described (Catalan et al., 1995).

Solvents with the desired SPP value based on the solubility (solubility) properties of the special BT composition (whether as a pure single component solvent or as a solvent mixture of two, three, four or more solvents; for solvent miscibility For example, see Godfrey 1972 Chem. Technol. 2: 359), according to certain preferred embodiments of the synthetic method steps described herein, as shown above, a hydrophilic, polar or organic solubilizer dissolves bismuth. Although not required to make it easy, it can be readily identified by those familiar with the art in terms of this disclosure.

Solubility parameters can also be described in part by the interaction parameters C, Hildebrand solubility parameter d, or partial [Hansen, describing the polarity, hydrogen bond potential and dispersibility interaction potential of the solvent, respectively. )] Solubility parameters: δp, δh and δd. In certain embodiments, the maximum value for the solubility parameter describing the solvent or co-solvent system in which the bismuth salt comprising bismuth will be dissolved is determined, for example, according to the methods currently described for preparing particulate BT compositions. It may provide a restriction on the aqueous solution that contains. For example, a higher 5δ value will have greater hydrogen bonding capacity and therefore a greater affinity for solvent molecules such as water. Thus, higher values of 6δ maximum observed for solvents may be desirable in situations where a more hydrophilic environment is required.

As a non-limiting example, BisEDTs having the structure of Formula I shown below can be prepared according to the following schemes:

In summary, and as a non-limiting illustrative example, 0.331 L (about 0.575 moles) of aqueous acidic bismuth solution, such as Bi (NO ₃ ), in excess (11.4 L) of 5% aqueous HNO ₃ at room temperature ₃ solution [eg 43% Bi (NO ₃ ) ₃ (w / w), 5% nitric acid (w / w), 52% water (w / w), Shepherd Chemical Company, Cincinnati, Ohio Commercially available from Chemical Co.] may be added slowly with stirring, and then anhydrous ethanol (4 L) may be added slowly. Thiol compound, for example, an ethanolic solution (1.56 L) of 1,2-ethanedithiol [˜0.55 M] was added to 1.5 L of absolute ethanol, 72.19 mL (0.863 moles) of 1,2-ethanedithiol. It can be prepared by adding 60 mL of syringe and stirring for 5 minutes. 1,2-ethanedithiol (CAS 540-63-6) and other thiol compounds are commercially available, for example, from Sigma-Aldrich, St. Louis, Missouri. The ethanol solution of the thiol compound is added to Bi (NO ₃ ) ₃ / HNO ₃ aqueous solution with stirring overnight to form a reaction solution. The thiol-containing compound may be present in the reaction solution in a molar ratio of about 1: 3 to about 3: 1 relative to bismuth, according to certain preferred embodiments. The product formed is allowed to precipitate as a precipitate comprising microparticles as described herein, then collected by filtration and washed successively with ethanol, water and acetone to give BisEDT as a yellow amorphous powder solid. The crude product is redissolved with stirring in anhydrous ethanol, filtered and washed several times with ethanol and several times with acetone. The washed powder can be triturated in 1M NaOH (500 mL), then filtered and washed successively with water, ethanol and acetone to obtain purified finely divided BisEDT.

In accordance with non-limiting theory, bismuth inhibits the bacteria's ability to produce extracellular polymeric materials (EPS), such as bacterial exopolysaccharides, which inhibition leads to impaired biofilm formation. Bacteria are believed to use glue-like EPS for biofilm attachment. Depending on the nature of the infection, biofilm formation and the synthesis of EPS cannot contribute to bacterial pathogenicity such as interference with wound healing. However, bismuth alone is not therapeutically useful as an interventional agent, and instead is typically administered as part of a complex such as BT. Thus, bismuth-thiol (BT) is a family of compositions comprising compounds resulting from the chelation of bismuth and thiol compounds and comprising compounds that exhibit significant improvement in the antimicrobial therapeutic efficacy of bismuth. BT exhibits significant anti-infective, anti-biofilm, and immunomodulatory effects. Bismuth-thiol (BT) is effective against a broad spectrum of microorganisms and is typically not affected by antibiotic-resistant. BT can prevent biofilm formation at significantly lower (below inhibitory) concentrations, prevent many pathogenic properties of common wound pathogens at levels below the same inhibitory concentration, prevent septic shock in animal models, and May be synergistic.

As described herein, this synergy in the antibacterial effects of one or more prescribed BT when combined with one or more defined antibiotic compounds is readily based on the profile of the separate antibiotic and BT effects for the particular bacterial type. Although unpredictable, it may surprisingly be generated from the selection of a specific BT-antibiotic combination in terms of a particular bacterial population, including the identification of the presence of Gram-negative or Gram-positive (or both) bacteria. For example, as described herein, antibiotics that synergize with certain BT include amikacin, ampicillin, aztreonam, cefazoline, cefepime, chloramphenicol, ciprofloxacin, clindamycin (or other lincosamide antibiotics), Daphtomycin (Cubicin ^® ), doxycycline, gatifloxacin, gentamicin, imipenim, levofloxacin, linezolid ^® , minocycline, napcillin, paromomycin, rifampin, sulfamemethazole, tetracycline, tophal And at least one of bramycin and vancomycin. In vitro studies have shown, for example, that MRSA, which is poor or not fully sensitive to individual gentamicin, cefazoline, cefepime, sulfamemethazole, imipenim or levofloxacin, is exposed to antibiotics in the presence of BT compound BisEDT. It was demonstrated that it showed significant sensitivity to either of the antibiotics. Thus, certain embodiments contemplated herein are BT compounds and amikacin, ampicillin, cefazoline, cefepime, chloramphenicol, ciprofloxacin, clindamycin (or other lincosamide antibiotics), daphtomycin (Cubicin ^® ), doxycycline One or more antibiotics selected from gatifloxacin, gentamicin, imipenim, levofloxacin, linezolid (Zyvox ^® ), minocycline, naphcillin, paromomycin, rifampin, sulfamemethazole, tetracycline, tobramycin and vancomycin While other compositions and / or methods contemplated herein may be included, certain other embodiments contemplated herein include BT compounds and amikacin, ampicillin, cefazoline, ce, which are expressly excluded from one or more antibiotics therefrom. pepim, chloramphenicol, ciprofloxacin, clean the myth (or other Linkoping four mid antibiotic), the neoplasm? azithromycin (Cubicin ^®), doxycycline, Gatti Rock reaper, gentamicin myth, already fetched s, levofloxacin, linezolid (Zyvox ^®), minocycline, naphthyl cylinder, wave our erythromycin, rifampin, sulfamic methoxy Sasol, tetracycline, tobramycin, and combinations of one or more antibiotic selected from vancomycin Contemplated compositions and / or methods may be included. In this connection it is noted that gentamicin and tobramycin belong to the aminoglycoside class of antibiotics. See also, Domenico et al., 2001 Agents Chemother. 45: 1417-1421; Domenico et al., 2000 Infect. Med. 17: 123-127; Domenico et al., 2003 Res. Adv. In Antimicrob. Agents & Chemother. 3: 79-85; 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. Certain compositions and methods described in 373: 141-146 are expressly excluded from certain contemplated embodiments; It will be noted here that none of these publications teach or suggest a monodisperse particulate BT composition described herein.

Thus and as described herein, in certain preferred embodiments, a composition for treating a subject with a composition also comprising the particulate BT described herein and optionally and in certain other embodiments synergistic and / or enhancing antibiotics. And a method are provided. Those familiar with the relevant field of knowledge may, on the basis of this in-flight, find appropriate clinical contexts and situations in which such treatment may be desirable, in particular, for example, surgery, advanced surgery, dermatology, traumatic medicine, senile, cardiovascular, metabolism. Diseases (eg, diabetes, obesity, etc.), infections and inflammation (including the epithelial lining of the respiratory or gastrointestinal tract, or other epithelial tissue surfaces such as in gland tissue), and other related medical specificities and subspecialties Will recognize established standards in the medical field.

Accordingly, it will be appreciated that in certain embodiments as described herein and known in the art, promotion of skin tissue repair (or other tissue repair, such as epithelial tissue, bone, joint, muscle tendon, or joint repair) is considered. In certain embodiments, the promotion of skin tissue or other epithelial tissue recovery may include (i) epithelial cells (eg keratinocytes) or skin fibroblast migration, (ii) epithelial cells (eg keratinocytes) or skin fibroblasts. Growth, (iii) down regulation of epithelial cells (eg, keratinocytes) or dermal fibroblast collagenase, gelatinase or matrix, (iv) dermal fibroblast extracellular matrix protein deposition, and (v) cutaneous blood vessels Stimulating or depressing one or more cell wound repair activities selected from induction or reinforcement of formation. Methods of identifying and characterizing such cellular wound repair activity include the effects of compositions comprising wound tissue repair-promoting compounds described herein on these and related activities, eg, BT agents as described herein. It has been described based on the description so that it can be measured easily and without undue experimentation. For example, compositions and methods are described that relate to models accepted in the art for wound recovery based on keratinocyte wound closure after scratch wounds.

Preferred compositions for treating microbial infections on or within natural surfaces for use in accordance with embodiments described herein comprise, in certain embodiments, bismuth-thiol (BT) compounds as described herein and include certain obvious but related In an aspect, it may include a composition comprising other compounds known in the art, such as one or more antibiotic compounds as described herein. BT compounds and methods of making them are described herein and are described, for example, in Domenico et al. (1997 Antimicrob. Agent. Chemother. 41 (8): 1697-1703; 2001 Antimicrob. Agent. Chemother. 45 (5) 1417-1421) and US Re-registered Patents RE37,793, US Patent 6,248,371, United States Patent 6,086,921, and US Patent 6,380,248. As also noted above, certain preferred BT compounds are those containing bismuth or bismuth salts that are ionically bonded to or coordinated with thiol-containing compounds, such as compositions comprising bismuth chelated to thiol-containing compounds. And certain other preferred BT compounds are those containing bismuth or bismuth salts in a covalent linking state to the thiol-containing compound. Also preferred are substantially monodisperse particulate BT compositions as described herein. From previous efforts to treat bacterial infections or from prior characterization in other contexts of any of the compounds described herein for the first time as having use in compositions and methods for facilitating the treatment of natural surfaces described herein. Again, it was not possible to predict whether the methods herein using these compounds could have the beneficial effects described herein.

Thus, according to a preferred embodiment, a method for treating a natural surface is provided, including administering at least one particulate BT compound described herein to a surface. In certain embodiments, the method further comprises at least one antibiotic compound that may be a synergistic antibiotic as described herein in certain preferred embodiments, and may be a potentiating antibiotic as described herein in certain other preferred embodiments. And administering simultaneously or sequentially and in any order. The antibiotic compound may be an aminoglycoside antibiotic, carbapenem antibiotic, cephalosporin antibiotic, fluoroquinolone antibiotic, glycopeptide antibiotic, lincosamide antibiotic, penicillinase-resistant penicillin antibiotic, or aminopenicillin antibiotic. Clinically useful antibiotics are also discussed herein and are described, for example, in Washington University School of Medicine, The Washington Manual of Medical Therapeutics (32 ^nd Ed.), 2007 Lippincott, Williams and Wilkins , Philadelphia, PA; and in Hauser, AL, Antibiotic Basics for Clinicians, 2007 Lippincott, Williams and Wilkins, Philadelphia, PA.

As described herein, certain embodiments provide that, in the case of bacterial infections, including Gram positive bacteria, preferred therapeutically effective formulations include BT compounds (eg, BisEDT, bismuth: 1,2-ethanedithiol; BisPyr, Bismuth: pyrithione; BisEDT / Pyr, bismuth 1,2-ethanedithiol / pyrithione) and rifamycin, or BT compounds and doptomycin (e.g. Cubicin ^® from Cubicin Pharmaceuticals) ), Lexington, Mass.], Or BT compound and linezolide [Zyvox ^® , manufactured by Pfizer, Inc., New York, NY], or BT compound (e.g. , BisEDT, bismuth: 1,2-ethanedithiol; BisPyr, bismuth: pyrithione; BisEDT / Pyr, bismuth: 1,2-ethanedithiol / pyrithione) and one or more ampicillins, cefazoline, cefepime, chloramphenicol, Clindamycin (or other lincosamide antibiotics), daphtomycin (Cubicin ^® ), doxycycline, gatifloxacin, gentamicin, imipenim, levofloxazine, Zyvox ^® , napcillin, paromomycin, rifampin, sulfametoxazole, tobramycin and vancomycin It originates from an unexpected discovery that it may involve abnormalities.

As also described herein, certain embodiments originate from the unpredictable finding that, in the case of bacterial infections comprising Gram negative bacteria, the preferred therapeutically effective formulation may comprise BT compounds and amikacin. Certain related embodiments contemplate treating infections comprising Gram negative bacteria with BT compounds and other antibiotics, such as other aminoglycoside antibiotics, in certain embodiments that are not gentamicin or tobramycin. Thus and in terms of these embodiments, another related aspect is a step for selecting the appropriate antibiotic compound (s) for inclusion in a formulation to be administered according to the present method, according to methods familiar to those skilled in the medical microbiology, Gram positive or It is contemplated to identify one or more bacterial populations or subpopulations in or on natural surfaces by well known criteria of Gram negative.

The compositions and methods described herein typically comprise a compound described herein (eg, with one or more particulate BT alone or with one or more synergistic and / or enhancing antibiotics as described herein) on a natural surface. Or by application or administration to a microbial site, such as the presence of microorganisms within a natural surface, in a broad context can find use in the treatment of microorganisms (eg, bacteria, viruses, yeasts, fungi and other fungi, microbial parasites, etc.). Such natural surfaces include mammalian tissues (e.g., skin, scalp, gastrointestinal lining, buccal cavity, etc .; endothelial, peritoneal, pericardial, pleural, periosteal, meninge, myofiber, etc. membranes, corneas, sclera, Mucous membranes and the like; and other mammalian tissues such as teeth, bones, joints, tendons, ligaments, muscles, heart, lungs, kidneys, liver, spleen, gallbladder, pancreas, bladder, nerves, and the like).

The particulate antimicrobial agents described herein are intended to inhibit microbial growth, reduce microbial prevalence, reduce biofilms, prevent the conversion of bacteria to biofilms, prevent or inhibit microbial infections and any other described herein. It can be used for the purpose. These agents are also used to prevent viral infections by herpes family viruses such as cytomegalovirus, herpes monovirus virus type 1, and herpes family viruses such as herpes monovirus virus type 2 and / or infection by other viruses. Or for many antiviral purposes including inhibition. In this regard, the preparations include single-stranded RNA virus, single-stranded DNA virus, Rous sarcoma virus (RSV), 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 virus (HPV), and human T cell lymphoma virus (HTLV).

Other internal and external pharmaceutical uses of the antimicrobial agents described herein include bacterial infections, tuberculosis, yeast and fungi [eg, Candida (eg Candida albicans, Candida glabrata). Fungi, such as C. parapsilosis, C. tropicalis, and C. dubliniensis or Cryptococcus or other fungi infections. Including, but not limited to, treatment of infections, Helicobacter pylori infections, and peptic ulcer disease In one embodiment, the agent is not generally lethal to bacteria, but nevertheless also natural. It is used at a dose sufficient to reduce the protective polysaccharide coating that can withstand the immune response of the subject. It is believed that the secondary mediated elimination-up of microbial infection if not harmful to the immune system (for example, a bunch of normal intestinal bacteria, etc.).

In a non-limiting manner, the specific contemplated aspects are now described.

In certain embodiments, the particulate BT compounds (or compositions comprising the particulate BT compounds) described herein can be combined with at least one anti-biofilm agent to modulate biofilm development, disrupt biofilm, or reduce the amount of biofilm. have. As is understood in the art, interspecies quorum sensing is associated with biofilm formation. Certain agents that increase the LuxS-dependent pathway or interspecies quorum sensing signal (see, eg, US Pat. No. 7,427,408) contribute to regulating the development and / or proliferation of mycelia. Exemplary agents include, for example, N- (3-oxododecanoyl) -L-homoserine lactone (OdDHL) blocking compounds and N-butyryl-L-homoserine lactone (BHL) analogs alone or in combination (See, eg, US Pat. No. 6,455,031). Oral hygiene compositions comprising the particulate BT compound and at least one anti-biofilm can be delivered topically for the destruction and inhibition of bacterial biofilms, and for the treatment of periodontal disease (see, eg, US Pat. No. 6,726,898). number).

A composition comprising particulate bismuth-thiol and for oral hygiene and for treating inflammation and infections of the oral cavity .

In another embodiment, the composition comprising the particulate BT compound is formulated for oral use and can be used in a method for preventing or reducing microbial growth in the oral cavity and for preventing and / or treating microbial infection and inflammation in the oral cavity. Thus, these compositions prevent or treat plaque, bad breath, oral inflammation, periodontal disease, gingivitis, and other infections of the oral cavity (ie, plaque, bad breath, inflammation of the oral cavity, periodontal disease, gingivitis, and other infections of the oral cavity). Reducing or inhibiting the development of or reducing the likelihood of its occurrence or recurrence). Oral compositions comprising the particulate BT compounds also prevent and / or control (ie, slow, retard, or inhibit) biofilm development, destroy biofilms, or prevent biofilms from being present on oral surfaces, especially teeth or gums. It may be useful to reduce the amount.

Trapped food particles, poor oral hygiene and poor oral health, and improper cleaning of dentures can promote microbial growth between the teeth, around the gums, and over the tongue. Continuous microbial growth and the presence of tooth decay can produce bad breath, plaque (ie, biofilms formed by colonization of microorganisms), gingivitis and inflammation. In the absence of proper oral protection (eg, gargle, floss), more serious infections may follow, such as periodontal disease and infection of the lower jaw.

Good oral hygiene is important not only for oral health, but also for the prevention of serious chronic conditions. Regulation of bacterial growth in the oral cavity may help to lower the risk of heart disease, preserve memory and reduce the risk of infection and inflammation in other parts of the body. People with diabetes are at greater risk of developing serious gum problems, so reducing your risk of gingivitis by maintaining good oral health can help control your blood sugar. Pregnant women may be more likely to experience gingivitis, and some researchers suggest a relationship between gum disease and the delivery of premature, low birth weight infants.

Bacteria are the major pathogens in periodontal disease. More than 500 bacterial strains can be found in plaque (Kroes et al., Proc. Natl. Acad. Sci. USA 96: 14547-52 (1999). Bacteria have evolved to survive as biofilms in the tooth surface, gum epithelium, and oral environment, which contributes to the difficulty in treating periodontitis. Fungicides and antibiotics currently used to treat such infections often do not kill all of the organisms in question. The use of agents that are ineffective against certain bacterial species can result in the propagation of resistant bacterial species. Moreover, these agents can cause unpleasant side effects such as allergic reactions, inflammation and tooth discoloration.

Bacterial plaque (plaque) is a biofilm that adheres continuously to the oral surface, restoration and prosthesis. The main means for controlling the biofilm in the oral cavity is through mechanical cleaning (ie brushing, flossing, etc.). Within the first two days after this cleaning is not taken, the surface of the tooth is colonized by predominantly Gram-positive facultative cocci, which are predominantly Streptococcus species. The bacterium secretes an extracellular mucus layer that helps fix the bacterium on its surface and provides protection against attached bacteria. Micro colony formation is initiated when the surface of the oral cavity is covered with adhered bacteria. The biofilm is covered not by the attachment of new bacteria, but mainly through cell division of the attached bacteria. The doubling time of plaque-forming bacteria is early in development and slower in more mature biofilms.

Aggregation occurs when a bacterial colonizer subsequently adheres to bacteria already attached to the thin film. The result of aggregation is the formation of a complex arrangement of different bacteria that are linked together. After several days of undisturbed plaque formation, the area around the gums becomes inflammatory and swells. Inflammation can result in the production of deeply gingival fissures. The biofilm extends into these subgingival areas and thrives in this protected environment, resulting in the formation of mature subgingival plaque biofilms. Gum inflammation does not appear until the biofilm changes from one consisting primarily of gram-positive bacteria to one containing gram-negative anaerobes. The subgingival bacterial microcolon, consisting mainly of Gram-negative anaerobic bacteria, is established in the gum sulcus between 3 and 12 weeks. Most bacterial species currently suspected as periodontitis pathogens are anaerobic, gram-negative bacteria.

Protected bacterial microcolons in biofilms are typically resistant to antibiotics (systemically administered), preservatives or disinfectants (topically administered), and immune defenses. Antibiotic doses that kill free-floating bacteria need to be increased by 1,500 times, for example, to kill biofilm bacteria. At these high concentrations, these antimicrobial agents also tend to be toxic to patients (see, eg, Coghlan 1996, New Scientist 2045: 32-6; Elder et al., 1995, Eye 9: 102-9).

Diligent and frequent physical removal of bacterial plaque biofilms is the most effective means of removing and controlling plaques. However, subgingival plaque in gum pockets cannot be reached by brushing, flossing, or mouthwash. Therefore, frequent periodontal debridement of the subgingival root surface by a dental hygienist or dentist is an essential requirement for the prevention and treatment of periodontitis.

In certain embodiments, the particulate particulate BT compound can be used as a toothpaste, mouthwash (ie, mouthwash), oral gel, toothpaste powder, oral spray (including spray dispersed by oral inhaler), which may be conventionally used by certain subjects, It may be incorporated into oral care compositions including, but not limited to, edible films, chewing gum, oral slurries, denture cleaners, denture stocks, and dental floss. The particulate BT compounds can be incorporated into oral hygiene compositions primarily used by dentist practitioners, including, for example, fluoride liquid treatments, cleaning compositions, buffer compositions, oral cleaning solutions, and dental floss. This aspect provides the benefits described herein to provide the benefits described herein, including a wide range of antimicrobial activity, solubility and bioavailability, anti-bactericidal effects, non-toxicity, enhancement of antibiotic efficacy, and other properties as described herein. It is contemplated to replace the antimicrobial agent formulated with the oral care composition described in the particulate BT compound described herein.

The particulate BT compound can also be used to prevent or treat tooth decay and / or inflammation (ie, reduce the likelihood of occurrence or relapse, respectively) by administering the particulate BT compound to the surface of the tooth. A composition comprising a particulate BT compound, which may be a mucoadhesive composition applied to the surface of a tooth and / or gum or oral mucosa, adheres to the surface to some extent or delivers a pharmaceutically effective amount of the active ingredient (s) to the required surface. It may be in a specific form. The particulate BT compound may also be formulated to release slowly from the composition applied to the tooth. For example, the composition may be a gel (eg, hydrogel, thiomer, aerogel or organogel) or liquid. The organogel may comprise an organic solvent, lipoic acid, vegetable oil or inorganic oil. Such gel or liquid coating formulations may be applied internally or externally to amalgam or complex or other prosthetic compositions. Sustained release compositions contain an effective amount of the particulate BT compound for 1, 2, 3, 4, 5, 6, or 7 (1 week) days or for 2, 3, 4, 5, 6, 7 weeks, or 1, 2, It can be delivered for 3, 4, 5, or 6 months. Such compositions can be prepared by one skilled in the art using any method known in the art.

In certain other embodiments and as described herein, an antimicrobial composition is provided for oral use comprising a particulate BT compound and one or more additional antimicrobial compounds or agents. Particularly useful are compositions comprising s and a second antimicrobial agent that has an enhanced or synergistic antimicrobial effect as described herein when administered together. As an example, an improved antimicrobial effect can be observed when the particulate BT compound is administered with an antimicrobial agent that chelates iron. In another particular embodiment, 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 can be formulated for oral use. In certain embodiments, particulate BT compounds prepared with hydrophobic thiols (eg, thiochlorophenol) may be used, which may exhibit greater capacity than hydrophobic BT compounds that hardly adhere to teeth and tissues of the oral cavity. BT compounds having a net negative charge, such as a molar ratio of 1: 2 (bismuth to thiol), may also have good adhesion properties.

Oral hygiene compositions comprising 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 care 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, deodorization and buffering. Baking soda chemically neutralizes rather than masks or absorbs odors. Baking soda may be combined as a mixture of particulate BT compounds and powders, or dissolved or suspended in any one of the toothpaste powders, gels, pastes, and liquids described herein. In other embodiments, the particulate BT compound may be combined with other alkali metal bicarbonates or carbonate materials (eg potassium bicarbonate or calcium carbonate) that maintain the desired alkali pH and also have cleaning and deodorizing properties.

The oral care composition comprising the particulate BT compound may also include one or more of the following ingredients.

Antimicrobial agents : for example chlorhexidine; Sanginarin Extract; Metronidazole; Quaternary ammonium compounds (eg, cetylpyridinium chloride); Bis-guanides (eg, chlorhexidine digluconate, hexetidine, octenidine, alexidine); Halogenated bisphenolic compounds (e.g., 2,2 'methylenebis- (4-chloro-6-bromophenol) or other phenolic antibacterial compounds; alkylhydroxybenzoates; cationic antimicrobial peptides; aminoglycoses Seeds; quinolone; lincosamide; penicillin; cephalosporins, macrolides; tetracyclines; other antibiotics known in the art; Coleus forskohlii essential oils; silver or colloids Sex silver antimicrobials; tin- or copper-based antimicrobials; Manuka oil; oregano; thyme; rosemary; or other herbal extracts; and grape seed extracts.

Anti-inflammatory or antioxidants : for example, ibuprofen, flurbiprofen, aspirin, indomethacin, aloe vera, turmeric, olive leaf extract, cloves, panthenol, retinol, omega-3 fatty acids, gamma-linolenic acid (GLA), green tea, ginger, grape seeds, etc.

Anti-cavities : for example sodium- and tin fluorides, amine fluorides, sodium monofluorophosphates, sodium monofluorophosphates, sodium trimetaphosphate, zinc citrate or other zinc agents, and casein.

Plaque buffers such as urea, calcium lactate, calcium glycerophosphate, and strontium polyacrylate.

Vitamins : for example, vitamins A, C and E.

Plant extracts. Desensitizers : for example potassium citrate, potassium chloride, potassium tartrate, potassium bicarbonate, potassium oxalate, potassium nitrate, and strontium salts.

Anti-stone agents : for example alkali-metal pyrophosphates, hypophosphite-containing polymers, organic phosphonates and phosphotates, and the like.

Biomolecules : for example, bacteriocins, bacteriophages, antibodies, bacteria and the like.

Flavoring agents : for example peppermint and spearmint oils, fennel, cinnamon and the like.

Proteinaceous substance : for example, collagen.

Preservative .

Whitening agent. coloring agent. pH-regulator. Sweetener .

Pharmaceutically acceptable carriers : for example starch, sucrose, water or water / alcohol systems and the like.

Surfactants : For example, anionic, nonionic, cationic and zwitterionic or amphoteric surfactants, saponins from plant materials (see, eg, US Pat. No. 6,485,711).

Particulate Abrasives : For example, silica, alumina, calcium carbonate, dicalcium phosphate, calcium pyrophosphate, hydroxyapatite, trimetaphosphate, insoluble hexametaphosphate, aggregated particulate abrasive, chalk, finely divided natural chalk, etc. .

Wetting Agents : For example, glycerol, sorbitol, propylene glycol, xylitol, lactitol and the like.

Binders and thickeners : for example sodium carboxy methyl cellulose, hydroxyethyl cellulose (Natrosol ^® ), xanthan gum, gum arabic, synthetic polymers (e.g. polyacrylates and carboxyvinyl polymers, e.g. 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 oral care compositions.

Bleach : For example, peroxy compounds (eg potassium peroxydiphosphate).

Boiling system: for example, sodium bicarbonate / citric acid system.

Color changing system .

In a particular embodiment, the abrasive is silica or finely divided chalk.

Oral hygiene compositions comprising particulate BT compounds formulated for use as dentifrices may also include humectants (eg, glycerol or sorbitol), surfactants, binders and / or flavoring agents. Toothpastes may also include sweetening, whitening, preservatives and antimicrobial agents. The pH of dentifrices and other compositions for oral use is typically between pH 5.5 and 8.5. In certain embodiments, oral care compositions comprising toothpastes have a pH between 7 and 7.5, between 7.5 and 8, between 8 and 8.5, or between 8.5 and 9, which can enhance the antimicrobial activity of the particulate BT compound. Toothpaste compositions described herein include one or more chalk, dicalcium phosphate dihydrate, sorbitol, water, hydrated aluminum oxide, precipitated silica, sodium lauryl sulfate, sodium carboxymethyl cellulose, flavors, sorbitan monooleate, sodium saccharin , Sodium tetrapyrophosphate, methyl paraben, propyl paraben. One or more colorants, for example FD & C Blue, can be used if desired. Other suitable ingredients that may be included in dentifrice compositions are described in the art, for example in US Pat. No. 5,560,517.

In another particular embodiment, the oral care composition is an oral nebulizer and comprises a particulate BT compound, an alkali buffer (eg potassium bicarbonate), an alcohol, a sweetener component, and a flavor system. Flavor systems may also have one or more of the following ingredients: flavoring agents, wetting agents, surfactants, sweeteners and coloring agents (see, eg, US Pat. No. 6,579,513). Surface-active agents described herein and known in the art for use in oral care compositions may be anionic, nonionic or amphoteric.

In another embodiment, the particulate BT-containing oral hygiene composition may be combined with additional active ingredients such as taurolidine and taurultam as described in the art as useful for inclusion in toothpastes, tooth gels and mouthwashes to treat serious infections. (See GB 1557163, US Pat. No. 6,488,912). As described herein, particulate BT can also be combined with one or more additional antimicrobial agents where the combination has an additive or synergistic effect when combined with particulate BT.

In still other particular embodiments, the oral care compositions described herein may also further include at least one anti-biofilm agent for controlling biofilm development, disrupting biofilm, or reducing the amount of biofilm. As understood in the art, interspecies quorum sensing is associated with biofilm formation. Certain agents that increase the LuxS-dependent pathway or interspecies quorum sensing signal (see, eg, US Pat. No. 7,427,408) contribute to controlling the development and / or proliferation of biofilms. Exemplary agents include, for example, N- (3-oxododecanoyl) -L-homoserine lactone (OdDHL) blocking compounds and N-butyryl-L-homoserine lactone (BHL) analogs together or alone ( See, for example, US Pat. No. 6,533,503. Oral hygiene compositions comprising the particulate BT compound and at least one anti-bial agent can be topically delivered for the destruction and inhibition of bacterial biofilms and the treatment of periodontal disease (see, eg, US Pat. No. 6,726,898).

The oral care compositions described herein may contain an amount of particulate BT compound sufficient to exert substantial antimicrobial action during the time required for normal brushing, mouthwashing or flossing. As described herein, the particulate BT compound may be retained on the oral surface (eg, teeth, amalgam, complexes, mucous membranes, gums). Particulate BT compounds retained in the teeth and gums after brushing, cleaning, flossing are complete, for example, can provide extended anti-bacterium and anti-inflammatory action.

In another embodiment, the particulate BT compound is released slowly from the mucoadhesive polymer or other agent that contributes to retention of the particulate BT compound at the mucosa and dental surface. Particulate BT compounds are stable, which can also be used for delivery to the mucosal surface or to the systemic circulation for the prevention and treatment of ulcers, inflammation and / or erosive disorders of the mucosa and / or for the topical treatment of pharmaceutically active compounds. , Viscous, viscous, aqueous compositions may be added (see, eg, US Pat. No. 7,547,433).

In another embodiment, the oral care composition comprising the particulate BT compound further comprises olive oil which can improve plaque removal. Using olive oil in products intended for oral hygiene, such as toothpaste, mouthwash, spray, oral inhaler or chewing gum, eliminates or reduces (decreases) bacterial plaques and / or eliminates many bacteria present in the oral cavity. Or by contributing to the reduction (decrease thereof), which may contribute to achieving a reduction in the occurrence of dental diseases (eg, caries, periodontal disease) and bad breath (see, eg, US Pat. No. 7,074,391).

In another embodiment, the oral care composition comprising the particulate BT compound may further comprise a mucosal disinfectant formulation for topical application in the oral cavity. The oral care composition may also further comprise an aqueous slurry useful for cleaning the tongue and larynx (see, eg, US Pat. No. 6,861,049). In still other embodiments, the oral care composition comprising the particulate BT compound also includes at least one mint used to prevent the formation of cavities (cavities) (i.e. reduce the tendency to develop) or to reduce the number of cavities. (mint) may be further included. One such mint, called CaviStat ^® (Ortec Terapactics, Inc., Rosslyn Heights, NY), provides arginine and calcium to promote the adhesion of calcium to enamel surfaces and to help neutralize acidic pH. It contains. Thus, the incorporation of mint in an oral care composition comprising the particulate BT compound can enhance the adhesion of the particulate BT compound to the oral surface and increase the pH.

A composition comprising particulate bismuth-thiol formulated for orthopedic use .

In a particular embodiment, prevent and / or treat microbial infections and inflammation resulting from orthopedic surgery (eg, orthopedic surgery, orthopedic therapy, arthroplasty (including two-stage arthroplasty), orthodontic therapy) A method of using a composition comprising a particulate BT compound is provided. Compositions comprising particulate BT compounds as described herein prevent and / or treat (ie, reduce their development) of microbial infections of the skeletal and support structures (ie, bones, joints, muscles, ligaments, tendons), such as osteomyelitis. Or inhibit, or reduce the likelihood of its appearance or recurrence). Compositions described herein comprising particulate BT compounds also prevent and / or control (ie, slow, retard, inhibit) biofilm development, destroy biofilms, intraarticular or bone, ligament, tendons or It may be useful to reduce the amount of biofilm present on the surface of a tooth.

Compositions described herein for orthopedic use comprising particulate BT compounds may also further include one or more additional antimicrobial compounds or agents. Particularly useful are compositions comprising a particulate BT compound and a second bivalent antimicrobial agent, as described herein, having an enhanced or synergistic antimicrobial effect as described herein. As a further example, an improved antimicrobial effect can be observed when the particulate BT compound is administered with an antimicrobial agent that chelates iron. In another particular embodiment, the particulate BT compound is formulated with an anti-inflammatory agent, compound, small molecule, or macromolecule (eg, peptide or polypeptide).

Compositions comprising particulate BT compounds may have at least one other antimicrobial agent (ie, antimicrobial agents such as second, third, fourth, etc.) that, when administered together, have an enhanced or synergistic antimicrobial effect (ie, greater than an additive effect). It can be combined with. As an example, an improved antimicrobial effect can be observed when the particulate BT compound is administered with an antimicrobial agent that chelates iron. In a particular embodiment, the composition comprising the particulate BT compound can be combined with at least one other antimicrobial and / or anti-inflammatory agent selected from:

Antimicrobial agents : for example chlorhexidine; Sanginarin Extract; Metronidazole; Quaternary ammonium compounds (eg, cetylpyridinium chloride); Bis-guanides (eg, chlorhexidine digluconate, hexetidine, octenidine, alexidine); Halogenated bisphenolic compounds (e.g., 2,2 'methylenebis- (4-chloro-6-bromophenol) or other phenolic antibacterial compounds; alkylhydroxybenzoates; cationic antimicrobial peptides; aminoglycoses Seeds; quinolone; lincosamide; penicillin; cephalosporins, macrolides; tetracyclines; other antibiotics known in the art; cholus forskoliy essential oils; silver or colloidal silver microbial agents; tin- or copper-based Antimicrobial agents; manuka oil; oregano; thyme; lobmari; or other herbal extracts; and grape seed extracts.

Anti-inflammatory or antioxidants : for example, ibuprofen, flurbiprofen, aspirin, indomethacin, aloe vera, turmeric, olive leaf extract, cloves, panthenol, retinol, omega-3 fatty acids, gamma-linolenic acid (GLA) , Green tea, ginger, grape seed etc. In a particular embodiment, the composition comprising the particulate BT compound may also contain clindamycin, vancomycin, daphtomycin, cefazoline, gentamicin, tobramycin, metronidazole, cefachlor, ciprofloxacin, or quaternary ammonium compounds (eg, Other antimicrobial agents such as benzalkonium chloride, cetyl pyridinium chloride), anti-microbial zeolites, alkali metal hydroxides or alkaline earth metal oxides. The composition may optionally include one or more pharmaceutically suitable carriers (ie, excipients), surfactants, buffers, diluents, and salts, and bleaches described herein. Accordingly, these and certain related embodiments described herein may include one or more particulate BTs, and may also optionally further include an antibiotic such as a synergistic or enhanced antibiotic as described herein. Consider incorporation into these products and processes.

Biological, biomedical and other uses for particulate BT .

Certain other embodiments are BT in which individual BT or bismuth residues are substituted with different Group V metals, such as antimony (Sb) or arsenic (As), in oral ingested nutritional formulations, and / or as described herein. Along with this is the use of the particulate BT described herein, regardless of whether it is BT in combination with one or more antibiotics that exhibit synergistic or enhanced antimicrobial activity.

In accordance with non-limiting theory, such formulations may be combined with other ingredients such as hydrocarbons, fatty acids, oils, phytonutrients, teas, herbs or herbal extracts, and / or other nutrients or foodstuffs, including vitamins, minerals, amino acids, carbohydrates. Incorporation of particulate BT in a particular embodiment results in increased bioavailability (eg, in a statistically significant manner with respect to appropriate controls) of BT and optionally antibiotics and / or additional nutritional ingredient (s) to the host digestive tract. In a manner that facilitates, it blocks or delays the absorption of nutrients by microbial populations in the gastrointestinal tract. In certain other embodiments, hydrocarbons, fatty acids, oils, phytonutrients, teas, herbs or herbal extracts and / or other nutrients including special vitamins, minerals, amino acids, carbohydrates contained in oral particulate BT (or AsT or SbT) formulations Or by altering the food product, the bioavailability of BT and optionally antibiotics and / or additional nutritional ingredient (s) to the host digestive tract can be lowered (eg, in a statistically significant manner with respect to appropriate controls).

For example, if pathological gastrointestinal (GI) tract infections are present, administering a particulate BT formulation that prevents intestinal absorption of the BT compounds to make them bioavailable in the GI tract to exert antimicrobial effects on infectious pathogens. It may be desirable. Those skilled in the art are aware of a number of vitamins, minerals, amino acids, carbohydrate-containing hydrocarbons, fatty acids, oils, phytonutrients, teas, herbs or herbal extracts and / or other nutrients or foodstuffs that promote or prevent GI tract absorption. By recognizing, formulations for increasing or decreasing the GI tract presence of one or more components (eg, particulate BT compounds, antibiotics, or one or more special nutrients) are prepared using the currently described particulate BT (or AsT or SbT). You can do it.

Certain other embodiments provided herein provide compositions for oral delivery to reduce fecal or digestive gas odors, for example, in patients undergoing colonic ejaculation, and to reduce armpits, feet or other body odors associated with the presence of topical microorganisms. For the incorporation of the particulate BT compounds currently described in other compositions for topical delivery. Many skin and GI vascular microbial populations, including planktonic and biofilm bacteria, have a low concentration of the particulate BT compounds described herein that include such BT compounds when present in combination with ameliorating or synergistic antibiotics as described herein. It is sensitive to.

Thus, certain embodiments are orally delivered to reduce (eg, in a statistically significant manner to appropriate controls) a population of GI-residual or skin-residual bacteria in a manner that reduces or alleviates unwanted odor problems. And topically delivered particulate BT formulations. Oral and topical pharmaceutical formulations have the advantage that these and related embodiments relate to current particulate formulations of BT such as miscible bioavailability and solubility characteristics and low toxicity; It is described below to provide other factors that can affect the selection of antimicrobial compositions also described herein and can be found, for example, in US Pat. No. 6,582,719.

An exemplary BT compound, BisEDT, has been applied to the axillary site (50 μL 1 mg / mL solution in DMSO) of human test subjects and has been found to neutralize body odor for 2-3 days. The mixture of BisEDT as a talc powder applied to the feet of human test subjects substantially reduced foot odor. Laboratory mice fed orally twice daily with 1 mg / kg BisEDT showed a 90% reduction in the number of fecal bacteria herds. A related aspect is also any thiol-emitting odor, including particulate BT formulations as described herein, which may be prepared with excess bismuth and added to the thiol-containing solution as an odor quenching agent. Consider generally useful deodorants for containing solutions (eg fish oil such as salmon oil). The resulting mixture retains the antimicrobial properties of particulate BT. Other contemplated applications include other biological source oils or butters such as hemp oil, tea tree oil, shea butter, flaxseed oil, fish oils, and independent or synergistic anti-inflammatory and / or Or include such oils in certain embodiments as they may have pain-reducing and / or other beneficial physiological effects.

Pharmaceutical Compositions and Administration

Certain embodiments also relate to pharmaceutical compositions containing the particulate BT compounds described herein; In certain such embodiments, the pharmaceutical composition may further comprise one or more antibiotics, such as antibiotics, wherein the BT compound exhibits a synergistic or enhanced effect as described herein. In one embodiment, when administered to an animal, preferably a mammal, most preferably a human patient, one or more such particulates in therapeutic amounts and in a pharmaceutically acceptable carrier, excipient or diluent as described herein A composition is provided comprising a BT compound.

Administration of the particulate BT compounds, or their pharmaceutically acceptable salts, in neat form or as a suitable pharmaceutical composition, can be carried out via any of the acceptable models of administration of the formulations to provide similar uses. Pharmaceutical compositions can be prepared by combining the particulate BT compounds with suitable pharmaceutically acceptable carriers, diluents or excipients, and may be prepared in solid, semi-solid, liquid or gaseous form, such as tablets, capsules, powders, It can be formulated into granules, ointments, solutions, suppositories, injections, inhalants, gels, microemulsions and aerosols. Representative routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, rectal, vaginal and intranasal. As used herein, the term parenteral includes subcutaneous injection, intravenous, intramuscular, intrasternal injection or infusion techniques. The pharmaceutical composition is formulated such that the active ingredient contained therein becomes available upon administration of the composition to a patient. The composition to be administered to a subject or patient takes the form of one or more dosage units, where, for example, the tablet may be a single dosage unit and the container of the compound in aerosol form may maintain multiple dosage units. Practical methods for preparing such dosage forms are known or will be familiar to those skilled in the art (eg, The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000)). The composition to be administered will in any case contain a therapeutically effective amount of a compound, or a pharmaceutically acceptable salt thereof, for the treatment of the desired disease or condition in accordance with the teachings herein. Pharmaceutical compositions useful herein also include any pharmaceutical agent that does not itself induce the production of antibodies that are harmful to the individual receiving the composition, and includes any suitable diluent or excipient that can be administered without undue toxicity. And a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, but are not limited to, liquids such as water, saline, glycerol, ethanol and the like. A complete discussion of pharmaceutically acceptable carriers, diluents and other excipients is provided in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. current edition).

The pharmaceutical composition may be in the form of a solid or a liquid. In one aspect, the carrier (s) are granulated so that the composition is, for example, in tablet or powder form. The carrier (s) can be, for example, a liquid that is a composition useful for inhaled administration, for example oral syrup, injectable liquid or aerosol.

When intended for oral administration, the pharmaceutical composition is preferably in solid or liquid form, wherein the semi-solid, semi-liquid, suspension and gel forms are included in the forms contemplated herein as solid or liquid.

As a solid composition for oral administration, the pharmaceutical composition may be formulated in powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or similar form. Such solid compositions will 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, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin; Excipients such as starch, lactose or dextrins, disintegrants such as alginic acid, sodium alginate, primogel, corn starch and the like; Lubricants such as magnesium stearate or sterotex; Glidants such as colloidal silicon dioxide; Sweetening agents such as honey, sugar or saccharin; Flavoring agents such as peppermint, methyl salicylate or orange flavor; And colorants.

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

The pharmaceutical composition may be in the form of a liquid, for example in the form of elixir, syrup, liquid, emulsion or suspension. The liquid can be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred compositions contain, in addition to the compositions of the present invention, one or more of sweetening, preservatives, dyes / colorants, and flavor enhancers. In compositions intended to be administered by injection, one or more of surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents may be included.

Liquid pharmaceutical compositions may contain one or more of the following adjuvants, regardless of whether they are in solution, suspensions or other similar forms: sterile diluents such as water for injection, saline solutions, preferably physiological saline, Ringer's solution, isotonic Fixed oils such as synthetic sodium or diglycerides, which may be provided as a solvent or suspension medium, polyethylene glycol, glycerin, propylene glycol or other solvents; Antibacterial agents such as benzyl alcohol or methyl parabens; Antioxidants such as ascorbic acid or sodium hydrogen sulfite, chelating agents such as ethylenediaminetetraacetic acid; Buffers such as acetate, citrate or phosphate and agents for controlling isotonicity such as sodium chloride or dextrose. Parenteral preparations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred excipient. Injectable pharmaceutical compositions are preferably sterile.

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

The pharmaceutical composition may be intended for topical administration, in which case the carrier may suitably comprise a liquid, suspension, ointment or gel base. For example, the substrate may include one or more of the following: petroleum, lanolin, polyethylene glycol, beeswax, inorganic oils, shea butter, tea tree oil, flaxseed oil, hemp oil, or anti-inflammatory and / or Other plant or vegetable oils, including those known to have anti-pain or other beneficial effects, salmon oils or anti-inflammatory and / or other fish oils, including those known to have anti-pain or other beneficial effects, water and alcohols Diluents such as, and emulsifiers and stabilizers. Thickeners may be present in the pharmaceutical compositions for topical administration. If intended for transdermal administration, the composition may comprise a transdermal patch or iontophoresis device. Topical formulations may contain a concentration of the particulate BT compound of about 0.1 to about 10% w / v (weight per unit volume).

The pharmaceutical composition may be intended for melting rectally to release the drug, for example rectal administration in the form of suppositories. The composition for rectal administration may contain an oily base as a suitable nonirritating excipient. Such substrates include, but are not limited to, lanolin, cocoa butter and polyethylene glycol.

The pharmaceutical composition may comprise various substances, which modify the physical form of the solid or liquid dosage unit. For example, the composition may include a material that forms a coating shell around the active ingredient. The material forming the coating shell is typically inert and can be selected, for example, from sugars, shellac, and other enteric coatings. Alternatively, the active ingredient may be sealed in a gelatin capsule.

Pharmaceutical compositions in solid or liquid form can include agents that assist in the delivery of the compound by binding to the particulate BT compound. Suitable agents that can act at such doses include monoclonal or polyclonal antibodies, proteins or liposomes. However, certain contemplated embodiments exclude the incorporation of liposomes into the pharmaceutical composition.

The pharmaceutical composition may be in dosage units that may be administered as an aerosol. The term aerosol is used to refer to a variety of systems ranging from colloidal properties to systems consisting of pressurized packages. Delivery can be by liquefied or compressed gas or by a suitable pump system that disperses the active ingredient. Aerosols of the particulate BT compounds can deliver the active ingredient (s) by being delivered in a single phase, two-phase or three-phase system. Delivery of the aerosol includes the necessary containers, activators, valves, subcontainers, and the like, which together may form a kit. One skilled in the art can determine the desired aerosol without undue experimentation.

Pharmaceutical compositions can be prepared by methods well known in the art of pharmacy. For example, pharmaceutical compositions intended to be administered by injection can be prepared by combining the compounds of the invention with sterile, distilled water to form a liquid. Surfactants may be added to promote the formation of a homogeneous liquid or suspending agent. Surfactants are compounds that promote dissolution or homogeneous suspension of a compound in an aqueous delivery system by non-covalently interacting with a compound of the present invention.

The particulate BT compounds, or pharmaceutically acceptable salts thereof, described herein may be selected from the activity of the specific compound employed; Metabolic stability and length of action of the compound; The age, body weight, general health, sex and diet of the patient; Mode and time of administration; Discharge rate; Drug combinations; The severity of the particular disease or condition; And a therapeutically effective amount that will vary depending upon various factors, including the subject being treated. Generally, a therapeutically effective daily dose is (from 70 kg of mammals) about 0.001 mg / kg (ie 0.07 mg) to about 100 mg / kg (ie 7.0 g); Preferably the therapeutically effective dose is (for 70 kg mammals) about 0.01 mg / kg (ie 7 mg) to about 50 mg / kg (ie 3.5 g); More preferably the therapeutic dosage (for 70 kg mammals) is about 1 mg / kg (ie 70 mg) to about 25 mg / kg (ie 1.75 g).

The range of effective dosages provided herein is not intended to limit the preferred dosage ranges. However, the most preferred dose will be adjusted to the individual subject, as understood and measurable by one skilled in the art. See, eg, Berkowe et al., Eds., The Merck Manual, 16th edition, Merck and Co. Rahway, NJ, 1992; Goodman etna., Eds., Goodman and Oilman's The Pharmacological Basis of Therapeutics, 10th 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, Pharmacology, Little, Brown and Co., Boston, (1985); Osolci al., Eds., Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Co., Easton, PA (1990); Katzung, Basic and Clinical Pharmacology, Appleton and Lange, Norwalk, CT (1992).

The total dose required for each treatment may be administered in multiple or single doses, if desired, over one day. In general, treatment is initiated at lower doses that are below the optimal dose of the compound. Thereafter, the dose is increased in small increments until the optimum effect is achieved under the circumstances. Diagnostic pharmaceutical compounds or compositions may be administered alone or in combination with other diagnostic agents and / or pathologies, or agents directed to other symptoms of pathology. The recipient of administration of the particulate BT compound and / or composition can be any vertebrate, such as a mammal. Among mammals, preferred recipients are primates (including humans, apes and monkeys), cows (including horses, goats, cattle, sheep and pigs), rodents (including mice, rats, rabbits and hamsters), and carnivores (cats and Dogs). Among algae, preferred recipients are turkey, chicken and many other identical necks. The most preferred recipient is human.

For topical application, it is desirable to administer an effective amount of the particulate BT-containing pharmaceutical composition to a target site, such as the skin surface, mucous membranes, and the like. The amount will generally range from about 0.0001 mg to about 1 g of BT compound per application, depending on the site to be treated, the severity of the symptoms, and the nature of the topical vehicle used, regardless of whether the use is diagnosed, prevented or treated. Preferred topical formulations are ointments, wherein from about 0.001 to about 50 mg of the active ingredient is used per cc of ointment base. Pharmaceutical compositions can be formulated as transdermal compositions or transdermal delivery devices (“patches”). Such compositions include, for example, backings, active ingredient reservoirs, control membranes, liners, and contact adhesives. Such transdermal patches can be used to provide continuous beats or, if desired, upon delivery of the compounds of the present invention.

The particulate BT composition can be formulated to provide rapid, sustained or delayed release of the active ingredient after administration to a patient by using procedures known in the art. Controlled release drug delivery systems include osmotic pump systems and dissolution systems containing polymer-coated reservoirs or drug-polymer matrix formulations. Examples of controlled release systems are provided in US Pat. Nos. 3,845,770 and 4,326,525 and PJ Kuzma et al, Regional Anesthesia 22 (6): 543-551 (1997), all of which are incorporated herein by reference. It is incorporated into.

The particulate BT composition can also be delivered via an intranasal drug delivery system for topical, systemic and nose-to-brain medical therapy. Controlled particle dispersion (CPD) ^™ technology, traditional nasal spray bottles, inhalers or nebulizers provide effective local and systemic delivery of drugs by targeting the olfactory area and paranasal sinuses.

The invention also relates to an intravaginal shell or core drug delivery device suitable for administration to a human or animal female in certain embodiments. The device may consist of the active pharmaceutical ingredient in a polymer matrix surrounded by a coating and compound in substantially zero order form on a daily basis similar to the device used for application to testosterone as described in WO 98/50016. Can emit.

Current methods for ocular delivery include topical administration (eye drop), subconjunctival injection, perocular injection, intravitreal injection, surgical implantation and iontophoresis (using small currents to transport ionized drugs into and through body tissues). It includes. One skilled in the art can combine the most suitable excipient with a compound for safe and effective intraocular administration.

The most suitable route will depend on the nature and severity of the condition being treated. Those skilled in the art will also be familiar with methods of administration (oral, intravenous, inhalation, subcutaneous, rectal, etc.), dosage forms, suitable pharmaceutical excipients and other matters related to the delivery of a compound to a subject in need thereof.

The presently disclosed compositions and methods also include, for example, burn creams, for the treatment of existing wounds, including those described herein, for the prevention of chronic wounds, for the treatment of MRSA skin infections, and as described herein. And other related prescription topical agents that will be familiar to the skilled artisan in the context of the present disclosure, for use in the treatment of acute and chronic wounds and wound biofilms.

Non-limiting examples of bacteria in which the compositions and methods described herein may find advantageous uses in accordance with certain embodiments as described herein include Staphylococcus aureus (S. aureus), MRSA (methicillin -Resistant, S. aureus), Staphylococcus epidermidis, MRSE (methicillin-resistant S. epidermidis), Mycobacterium tuberculosis, Mycobacterium avium, Pseudomonas ae Luginosa, drug-resistant blood. Aeruginosa, Escherichia coli, Enterotoxin production. E. coli, intestinal bleeding E. coli, Klebsiella pneumoniae, Clostridium difficile, Helicobacter phyllori, Legionella pneumophila, Enterococcus paecalis, Methicillin-sensitive enterococcus paecalis, Enterobacter clocae, Salmonella typhimurium , Proteus vulgaris, Yersinia enterocytica, Vibrio cholera, Shigella flexneri, Vancomycin-tolerant enterococcus (VRE), Burcolderia Sephacia complex, Francisla Tularensis, Bacillus anthracis, Yersinia Festis, Pseudomonas aeruginosa, vancomycin-sensitive enterokoki [see, e. E. faecalis, E. E. faecium], methicillin-sensitive and methicillin-resistant staphylocoki [eg, S. a. Aureus, S. S. epidermidis and Axinetobacter baumannini, Staphylococcus haemolyticus, Staphylococcus hominis, Enterococcus faecium ), Streptococcus pyogenes, Streptococcus agalactiae, Bacillus anthracis, Klebsiella pneumonia, Proteus mirabilis mirabilis, Proteus vulgaris, Yersinia enterocolytica, Stenotrophomonas maltophilia, Streptococcus pneumonia, and penicillincus-resistant streptococcus Pneumoniae, Burkholderia cepacia, Burkholderia multivorans, Mycobacterium smeg Tees (Mycobacterium smegmatis) and the. E. cloacae.

Implementation of certain embodiments of the present invention, unless specifically indicated otherwise, will employ routine methods of microbiology, molecular biology, biochemistry, cell biology, virology and immunology techniques within the skill of the art, with reference to some of which It is made below for the purpose of explanation. Such techniques are explained fully in the literature. See, eg, Sambrook, et al. Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DAW Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984).

Unless the content otherwise requires, throughout this specification and claims, the terms “comprises” and variations thereof, such as “comprises” and “comprising,” include, but are not limited to, open. Should be considered in a comprehensive sense.

Throughout this specification, "an aspect" or "an aspect" or "an aspect" means that a particular feature, structure, and characteristic described in connection with the aspects is included in at least one aspect of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. In addition, special features, structures or properties may be combined in any suitable manner in one or more embodiments.

Certain embodiments include methods, compositions, and methods for treating acute or chronic wounds or wound biofilms in a subject or altering one or more cell wound repair activities in a cell or multiple cells, which may include promoting skin tissue repair in the subject and Relates to a kit. Cells generally represent a single cell, but many cells represent one or more cells. Cells can include tissues, organs or whole organisms. In addition, the cell or cells may be located in vivo, in vitro or ex vivo. Maintaining cell, tissue and organ cultures is a routine procedure of those skilled in the art, and conditions and media for this can be easily estimated. See, eg, Freshney, Culture of Animal Cells: A Manual of Basic Technique, Wiley-Liss 5th Ed. (2005); Davis, Basic Cell Culture, Oxford University Press 2nd Ed. (2002)].

As described herein, certain embodiments are provided as a BT compound as described herein for use in a method of treating an acute or chronic wound or wound biofilm (eg, in the form of a number of substantially monodisperse particulates). ) And optionally antibiotic compounds as described herein for use in such methods in certain further embodiments, such as BT compounds such as BisEDT or BisBAL or other compounds shown in Table 1 herein, or by reference. Domenico et al. (1997 Antimicrob. Agent. Chemother. 41: 1697; 2001 Antimicrob. Agent. Chemother. 45: 1421) and / or US Re-registered Patents RE37,793, US Patent 6,248,371, US Patent 6,086,921, and United States Acute or chronic wounds in a subject, including administering to the subject a therapeutically effective amount of a composition also comprising those described in patent 6,380,248 and / or any other BT agent as prepared according to the methods described herein. Or to a method for treating a wound biofilm. Certain other embodiments relate to a method comprising contacting any natural surface with a composition comprising one or more of the particulate BT compounds described herein, wherein the contacting step comprises direct application, coating, dipping, irrigating, Spraying, painting or otherwise contacting the BT composition with a natural surface.

The step of administering to a subject, such as a human or other mammalian subject, can be any means known in the art, for example, as it may be present locally (skin or glandular tissue or respiratory and / or gastrointestinal tract). Direct administration to any other epithelial tissue surface including the same surface), intravaginally, intraperitoneally, orally, parenterally, intravenously, intraarterally, percutaneously, sublingually, subcutaneously, intramuscularly, Internally, intranasally, through inhalation, intraocularly, subcutaneously, fatty, intraarterial or intradural.

In a preferred embodiment, administration can be carried out topically, wherein topical pharmaceutical excipients or carriers are described herein and known in the art.

As indicated above, certain embodiments of the invention described herein relate to topical formulations of the described BT compounds (eg, BisEDT and / or BisBAL), which formulations are described herein in certain further embodiments. One or more antibiotic compounds, such as amikacin, ampicillin, cefazoline, cefepime, chloramphenicol, ciprofloxacin, clindamycin (or other lincosamide antibiotics), daphtomycin (Cubicin ^® ), doxycycline, gatifloc Reaper, gentamicin, imipenim, levofloxacin, linezolid (Zyvox ^® ), minocycline, napcillin, paromomycin, rifampin, sulfamomethazole, tobramycin and vancomycin; Or carbapenem antibiotics, cephalosporin antibiotics, fluoroquinolone antibiotics, glycopeptide antibiotics, lincosamide antibiotics, penicillinase-resistant penicillin antibiotics, and / or aminopenicillin antibiotics, and / or aminoglycoside antibiotics, for example , Amikacin, arbecasin, gentamicin, kanamycin, neomycin, netylmycin, paromomycin, rhodospreptomycin, streptomycin, tobramycin or apramycin, and / or lipopeptide antibiotics, for example For example daphtomycin (Cubicin ^® ), or oxazolidinone antibiotics such as linezolid (Zyvox ^® ). These and related formulations, when topically administered to animals, preferably mammals and most preferably humans, BT compound (s) (and optionally one) in a pharmaceutically acceptable carrier, excipient or diluent as described herein Antibiotics) in a therapeutic amount, and in particularly preferred embodiments, a human may be biofilm-related (eg, where bacteria may be present that can promote biofilm formation, but biofilms may still be undetectable). Have a wound or acute or chronic wound that contains a bacterial infection or that contains a bacterial infection such as a biofilm or other bacterial presence.

Topical administration of the BT compounds, or pharmaceutically acceptable salts thereof, described herein, either in pure form or as a suitable pharmaceutical composition, can be carried out via any of the accepted modes of topical administration of the formulation to provide similar uses. Topical application or administration of the composition, in a preferred embodiment, involves the composition (eg, topical formulation) of the skin and / or other epithelial tissue surface (eg, the respiratory tract, gastrointestinal tract and / or gland epithelial endothelium) of the subject being treated. Direct contact, which may be present in one or more localized or widely distributed skin and / or other epithelial tissue surface areas and is generally not limited to, surrounded by the complete stratum corneum or epidermis. Contact with an acute or chronic wound site; For example, certain embodiments are topical application whereby contact of the topical formulation can occur only in the stratum corneum or epithelium by administering the topical formulation described herein to the damaged, flaky or injured skin, or to the skin of a subject under surgery. May occur with skin granule cells, strabismus cells and / or basal cell layers, and / or with skin or underlying tissues, for example, as they may be accompanied by certain types of wound healing or wound healing or other skin tissue remodeling. Can be.

Thus, such skin tissue repair is, in certain preferred embodiments, for example in preventing or alleviating sudden chronic wounds or wound biofilms, or in another example, in preventing or alleviating skin wound dehiscence, or acute Or skin wound healing as chronic wounds and / or skin wound dehiscation may be desirable in improving, accelerating or otherwise improving skin wound healing. Certain other embodiments contemplating topical administration to the surface of epithelial tissue present in the respiratory tract similarly include topical formulations as provided herein in the respiratory (eg, airway, nasopharyngeal and laryngeal pathways, organs, lungs, Bronchial, bronchioles, alveoli, etc.) and / or one or more epithelial tissue surfaces present in the gastrointestinal tract (eg, buccal, esophagus, stomach, intestine, rectum, anus, etc.) and / or other epithelial surfaces for delivery. Administration of the topical formulation by any suitable route known in the art.

According to certain contemplated embodiments, topical administration may comprise applying directly into an open wound. For example, open fractures or other open wounds may include intradermal rupture that may expose additional underlying tissue to the external environment in a manner that makes them sensitive to microbial infection. This situation is common for certain types of acute traumatic cluster wounds, including, for example, type III (severe) open fractures. According to these and related embodiments, topical administration may be performed by subjecting the BT composition described herein to such damaged skin and / or other epithelial surfaces and / or other tissues such as circulatory tissue such as muscles, ligaments, tendons, bones, blood vessels, Direct contact with associated neural tissue and any other organ that may be exposed to such open wounds. Examples of other tissues that may be exposed, and thus such direct contact may be considered are the kidneys, bladder, liver, pancreas, and any other tissues or organs that may be adversely exposed to opportunistic infections with respect to open wounds. It includes.

Topical formulations (e.g., pharmaceutical compositions) include the described BT compounds (e.g., US Re-registered Patent No. RE37,793, US Patent 6,248,371, US Patent 6,086,921, and / or US Patent 6,380,248 Combinations of compounds prepared according to the present disclosure, such as the particulate BT suspensions described in and / or described herein, and in certain related embodiments one or more desired antibiotics (eg, amino such as amikacin). Glycoside antibiotics) can be prepared independently or in combination with BT compounds, in combination with appropriate pharmaceutically acceptable carriers, diluents or excipients for use in topical formulation formulations, and may be prepared as solid, semi-solid, gel, cream, Colloids, suspensions or liquids or other topically applied forms, for example powders, granules, ointments, solutions, cleaning agents, gels, pastes, plasters, paints , Bioadhesives, microspheres, suspensions, and aerosol sprays.

The pharmaceutical compositions of these and related embodiments may comprise the active ingredient contained therein and, in particularly preferred embodiments, the BT compound (s) described herein, alone or concurrently, continuously and in any order, with one or more desired antibiotics ( For example, carbapenem antibiotics, cephalosporin antibiotics, fluoroquinolone antibiotics, glycopeptide antibiotics, lincosamide antibiotics, penicillinase-resistant penicillin antibiotics, and aminopenicillin antibiotics, or amikacin or rifamycin Formulated with an aminoglycoside antibiotic), thereby causing acute or chronic wounds of formulations containing BT compound (s) and / or antibiotic composition (s) and, optionally, subjects, such as mammals, including humans, and certain preferred Having an acute or chronic wound in an embodiment or an acute or chronic wound or wound biofilm or Bioavailability is achieved upon topical administration to the surrounding skin of a person at increased risk of having a wound dehiscence (eg, diabetic and / or diabetic individuals). While certain embodiments described herein contemplate topical administration of BT compounds and antibiotics, including administration that may be administered simultaneously or sequentially and in any order, the invention is not intended to be limited thereto and in other embodiments administration of antibiotics Expressly contemplating the specific route of administration of the BT compound for the route. Thus, the antibiotic may be administered orally, intravenously, or by any other route of administration described herein, but the BT compound may be administered by a route separate from the route used for antibiotics. By way of non-limiting, illustrative example, BT compounds may be administered topically as provided herein, while antibiotics may be administered by clear routes such as oral, intravenous, transdermal, subcutaneous, intramuscular and / or any other route of administration. May be administered simultaneously or sequentially (and in any order).

The topical formulations described herein deliver a therapeutically effective amount of a therapeutically effective amount of a preservative or wound-healing agent (s) (and optionally antibiotic (s)) to a wound site, for example skin cells such as dermal fibroblasts. . Preferred formulations may be contacted with a site of interest such as a topical wound site, chronic wound, epithelial tissue surface or other intended site of administration by spraying, perfusion dipping and / or painting; Such formulations may be prepared by a number of established methods known in the art for testing the skin penetrating ability of drug compositions. See, eg, Wagner et al., 2002 J. Invest. Dermatol. 118: 540, and references cited therein; Bronaugh et al., 1985 J. Pharm. Sci. 74:64; Bosnian et al., 1998 J. Pharm. Biomed. Anal. 17: 493-499; Bosnian et al., 1996 J. Pharm Biomed Anal. 1996 14: 1015-23; Bonferoni et al., 1999 Pharm. Dev. Technol. 4: 45-53; Frantz, Instrumentation and methodology for in vitro skin diffusion cells in methodology for skin absorption. In: Methods for Skin Absorption (Kemppainen & Reifenrath, Eds), CRC Press, Florida, 1990, pp. 35-59; Tojo, Design and calibration of in vitro permeation apparatus. In: Transdermal Controlled Systemic Medications (Chien YW, Ed), Marcel Dekker, New York, 1987, 127-158; Barry, Methods for studying percutaneous absorption. In: Dermatological Formulations: Percutaneous absorption, Marcel Dekker, New York, 1983, 234-295], which may indicate an easy penetration into the skin.

Compositions, and formulations comprising such compositions, to be administered to the skin of a subject or patient may take the form of one or more dosage units in certain embodiments, wherein the liquid filled capsules or ampoules may contain a single dosage unit, and Containers of topical formulations, as described herein, in aerosol form, can hold multiple dosage units. Actual methods of preparing such dosage forms are known or will be apparent to those skilled in the art (eg, The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000)). The composition or formulation to be administered, in any case as provided herein, contains a therapeutically effective amount of a preservative and / or wound healing promoting compound (eg, a BT compound), or a pharmaceutically acceptable salt thereof, in accordance with the teachings of the present invention. something to do.

As noted above, the topical formulations of the present invention may take any of a wide variety of forms, including, for example, creams, lotions, liquids, sprays, gels, ointments, pastes, and / or liposomes, Micelles, and / or contain microspheres (see, eg, US Pat. No. 7,205,003). For example, as is well known in the art of pharmaceutical and medicinal cosmetic formulations, creams are oil-in-water or oil-in-oil viscous liquids. Or semisolid emulsions. Cream bases are water-washable and contain an oil phase, an emulsifier and an aqueous phase. The oil phase, also referred to as the "inner" phase, generally consists of petroleum and fatty acids such as cetyl or stearyl alcohol. The aqueous phase generally exceeds the oil phase in volume, although not necessarily, and generally contains a humectant. Emulsifiers in cream formulations are generally nonionic, anionic, cationic or amphoteric surfactants.

Preferred lotions for the delivery of cosmetic preparations are formulations to be applied to the skin surface without friction and are typically liquid or semi-liquid formulations in which solid particles comprising the active agent are present in a water or alcohol base. Lotions are generally suspensions of solids and preferably include liquid oily emulsions of the oil-in-water type. Lotions are the preferred formulations herein for the treatment of large body parts due to the ease of application of more fluid compositions. It is generally preferred that the insoluble material is finely divided in the lotion. Lotions will typically contain suspending agents to obtain better dispersions and compounds useful for localizing and maintaining the active agent upon contact with the skin, such as methylcellulose, sodium carboxymethyl-cellulose, and the like.

Liquid formulations are homogeneous mixtures prepared by dissolving one or more chemicals (solutes) in a liquid so that molecules of the dissolved substance are dispersed in the molecules of the solvent. The solution may contain other pharmaceutically acceptable and / or pharmaceutically cosmetically acceptable chemicals to buffer, stabilize or preserve the solute. General examples of solvents used to prepare liquids are ethanol, water, propylene glycol or any other pharmaceutically acceptable and / or pharmaceutically cosmetically acceptable vehicle.

Gels are semisolid, suspension-type systems. Single-phase gels typically contain an organic macromolecule that is substantially uniformly dispersed throughout the carrier liquid, which is typically aqueous but also preferably alcohol, and optionally oil. Preferred "organic macromolecules", ie gelling agents, are commercially available under chemically crosslinked acrylic acid polymers, such as crosslinked acrylic acid polymers, for example the "Carbomer" family of polymers, for example under the Carbopol ^® brand name. Carboxypolyalkylene which may be used. Also in certain embodiments hydrophilic polymers such as polyethylene oxide, polyoxyethylene-polyoxypropylene copolymers and polyvinyl alcohol; Polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and methyl cellulose; Gums such as tragacanth and xanthan gum; Sodium alginate; And gelatin. To prepare a monoform gel, a dispersant such as alcohol or glycerin can be added, or the gelling agent can be dispersed by grinding, mechanical mixing or stirring, or a combination thereof.

Ointments also well known in the art are typically semisolid preparations based on petroleum or other petroleum derivatives. As will be familiar to those skilled in the art, the particular ointment base to be used will provide a number of desirable features, such as emolliency and the like. By using other carriers or vehicles, the ointment substrate must be inert, stable, nonirritating and nonsensitizing. Remington: The Science and Practice of Pharmacy, 19th Ed. As described in (Easton, Pa .: Mack Publishing Co., 1995), at pages 1399-1404, ointment substrates are classified into four classes: oil-based substrates; Emulsifiable substrates; Latex substrates; And water-soluble substrates. The oleaginous ointment base includes, for example, vegetable oils, fats obtained from animals, and semisolid hydrocarbons obtained from petroleum. Emulsifiable ointment substrates, also known as adsorbent ointment substrates, contain little or no water and include, for example, hydroxystearin sulfate, lanolin anhydrous, and hydrophilic petroleum. Latex ointment bases are oil-in-water (W / O) emulsions or oil-in-oil (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, for example, Remington, Id.).

Pastes are semisolid dosage forms in which the active agent is suspended in a suitable substrate. Depending on the nature of the substrate, the paste is dispensed between those prepared from fatty pastes or single-phase aqueous gels. Substrates in fatty pastes are generally petroleum or hydrophilic petroleum and the like. Pastes prepared from single-phase aqueous gels generally incorporate carboxymethylcellulose or the like as a substrate.

Formulations can also be prepared with liposomes, micelles, and microspheres. Liposomes are microscopic vesicles having a liquid wall of one (monolamela) or multiple (polylamellar) comprising a lipid bilayer, wherein in the context of these liquid membrane surfaces one or more components of the topical formulations described herein, e.g. For example, antiseptic, wound healing / skin tissue / epithelial tissue repair-promoting compounds (eg, particulate BT compounds, optionally with one or more antibiotics) or certain carriers or excipients may be encapsulated and / or adsorbed. Liposomal formulations herein include cationic (positively charged), anionic (negally charged), and neutral agents. Cationic liposomes are readily available. For example, N [1-2,3-dioleyloxy) propyl] -N, N, N-triethylammonium (DOTMA) liposomes are trademarked Lipofectin ^® [GIBCO BRL, New York State Available in Grand Iceland. Similarly, anionic and neutral liposomes can also be prepared using, for example, Avanti Polar Lipids (Birmingham, Alab.) And also readily available or readily available materials. Such materials include, among others, phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidyl ethanolamine (DOPE). These materials can also be mixed with DOTMA in an appropriate ratio. Methods for preparing liposomes using these materials are well known in the art.

While micelles are composed of arranged surfactant molecules, their polar headgroups form an outer spherical shell, while hydrophobic hydrocarbon chains are oriented towards the center of the sphere, forming a core. Known in The micelles are naturally formed by forming in an aqueous solution containing a surfactant at a sufficient high concentration. Useful surfactants for forming micelles include potassium laurate, sodium octane sulfonate, sodium decane sulfonate, sodium dodecane sulfonate, sodium lauryl sulfate, docusate sodium decyltrimethylammonium bromide, dodecyltrimethylammonium bromide, tetradecyltrimethyl Ammonium bromide, tetradecyltrimethyl-ammonium chloride, dodecylammonium chloride, polyoxyl-8 dodecyl ether, polyoxyl-12 dodecyl ether, nonoxynol 10, and nonoxynol 30.

Similarly, microspheres can be incorporated into the topical formulations currently described. Similar to liposomes and micelles, the microspheres essentially enclose one or more components of the formulation. These are generally formed from, but not necessarily, lipids, preferably charged lipids such as phospholipids. The preparation of lipidic microspheres is well known in the art.

Various additives as known to those skilled in the art can also be included in the topical formulations. For example, a solvent comprising a relatively small amount of alcohol can be used to dissolve certain formulation components. In the case of a particular topical formulation or particularly severe skin damage such as postoperative acute or chronic wounds or post-surgical skin wound dehiscence, it may be desirable to include an added skin penetration enhancer in the formulation. Examples of suitable enhancers include ethers such as diethylene glycol monoethyl ether (commercially available as Transcutol ^® ) and diethylene glycol monomethyl ether; Sodium laurate, sodium lauryl sulfate, cetyltrimethylammonium bromide, benzalkonium chloride, Poloxamer ^® (231, 182, 184), Tween ^® (20, 40, 60, 80), and lecithin (US Pat. No. 4,783,450) Surfactants such as; Alcohols such as ethanol, propanol, octanol, benzyl alcohol and the like; Polyethylene glycol and esters thereof, such as polyethylene glycol monolaurate (PEGML; see, eg, US Pat. No. 4,568,343); Amides and other nitrogenous compounds such as urea, dimethylacetamide (DMA), dimethylformamide (DMF), 2-pyrrolidone, 1-methyl-2-pyrrolidone, ethanolamine, diethanolamine and triethanolamine; Terpenes; Alkanones; And organic acids, in particular citric acid and succinic acid. Azone ^® and sulfoxides such as DMSO and C ₁₀ MSO can also be used, but are rarely preferred.

Most preferred skin penetration enhancers are lipophilic co-enhances typically referred to as "plasticization" enhancers, ie, molecular weights of about 150 to 1000 Daltons, water solubility of less than about 1% by weight, preferably about Less than 0.5% by weight, and most preferably less than about 0.2% by weight. The Hildebrand solubility parameter of the plasticizer enhancer is in the range of about 2.5 to about 10, preferably in the range of about 5 to about 10. Preferred lipophilic enhancers are fatty esters, fatty alcohols, and fatty ethers. Examples of special and most preferred fatty acid esters are methyllaurate, ethyllaurate, propylene glycol monolaurate, propylene glycerol dilaurate, glycerol monolaurate, glycerol monooleate, isopropyl n-decanoate, and octyldode It contains a yarn myristate. Fatty alcohols include, for example, stearyl alcohol and oleyl alcohol, while fatty ethers are diols or triols, preferably C ₂ -C ₄ alkane diols or triols with one or two fatty ether substituents It includes a compound substituted with. Additional skin penetration enhancers will be known to those of ordinary skill in the art of topical drug delivery and / or described in, eg, Percutaneous Penetration Enhancers, eds. Smith et al. (CRC Press, Boca Raton, FL, 1995).

Various other additives may be included in the topical formulations according to certain embodiments of the present invention, in addition to those defined above. These include antioxidants, astringents, perfumes, preservatives, emollients, pigments, dyes, humectants, propellants and sunscreens, and also other classes of substances whose presence may be cosmetically, medicamentally or also desirable. Including, but not limited to. Representative examples of any additives for incorporation into the formulations of certain embodiments of the invention are as follows: preservatives such as sorbate; Solvents such as isopropanol and propylene glycol; Astringents such as menthol and ethanol; Emollients such as polyalkylene methyl glucoside; Humectants, such as glycerin; Emulsifiers such as glycerol stearate, PEG-100 stearate, polyglyceryl-3 hydroxylauryl ether, and polysorbate 60; Sorbitol and other polyhydroxyalcohols such as polyethylene glycol; Sunscreens such as octyl methoxyl cinnamate (commercially available as Parsol MCX) and butyl methoxy benzoylmethane (available under the trademark Parsol 1789); Ascorbic acid (vitamin C), α-tocopherol (vitamin E), β-tocopherol, γ-tocopherol, δ-tocopherol, ε-tocopherol, ζ ₁ -tocopherol, ζ ₂ -tocopherol, η-tocopherol, and retinol (vitamin A) Antioxidants); Essential oils, ceramides, essential fatty acids, mineral oil, wetting agents, and, other surface active agents, for example, BASF (BASF), PLURONIC ^® series, vegetable oil of a hydrophilic polymer, available from (NJ Mount Olive material) (e.g., Soybean oil, palm oil, liquid fractions of shea butter, sunflower oil), animal oils (eg perhydrosqualene), inorganic oils, synthetic oils, silicone oils or waxes (eg cyclomethicone and dimethicone) Fluorinated oils (generally perfluoropolyethers), fatty alcohols (eg cetyl alcohol), and waxes (eg beeswax, carnauba wax, and paraffin wax); Skin-feel modifiers; And thickeners and structurants such as swellable clays and cross-linked carboxypolyalkylenes which are commercially obtainable under the Carbopol ^® brand name.

Other additives include beneficial agents such as substances that maintain skin (especially the upper layer of skin in the stratum corneum) and condition it by delaying the decrease in its water content and / or protect the skin. Such conditioners and wetting agents include, for example, phylloridine 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; Anti-seborrhea agents such as retinoic acid; Vasodilators such as nicotinic acid; Melanogenesis inhibitors such as kojic acid; And mixtures thereof. Others that advantageously include medicinal cosmetically active agents such as α-hydroxy acids, α-keto acids, polymeric hydroxy acids, wetting agents, collagen, marine extracts and ascorbic acid (vitamin C), There may be antioxidants such as 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. . Additional cosmetic preparations include those that can improve oxygen supply to skin tissue, as described, for example, in WO 94/00098 and WO 94/00109. Sunscreens may also be included.

Other embodiments may include various non-carcinogenic non-irritating healing substances that facilitate treatment with the formulations of certain embodiments of the present invention. Such healing substances may include nutrients, minerals, vitamins, electrolytes, enzymes, herbs, plant extracts, honey, spring or animal extracts, or safe therapeutics that can be added to the formulation to promote skin healing. The amounts of these various additives are those conventionally used in the cosmetic field and are, for example, in the range of about 0.01% to about 20% of the total weight of the topical formulation.

Formulations of certain embodiments of the present invention may also include conventional additives such as whitening agents, perfumes, colorants, gelling agents, thickeners, stabilizers, surfactants and the like. Other agents, such as antimicrobial agents, may also be added to prevent rot on storage, ie to inhibit the growth of microorganisms such as yeast and mold. Suitable antimicrobial agents are typically selected from p-hydroxybenzoic acid (eg, methyl and propyl parabens), sodium benzoate, sorbic acid, imidurea, and combinations thereof. The formulations also minimize or eliminate the potential for skin irritation or skin damage generated from anti-infectious acute or chronic wound healing and skin tissue repair-promoting compounds to be administered by containing irritation-releasing additives, or from other components of the composition. can do. Suitable stimulation-releasing additives include, for example, α-tocopherol; Monoamine oxidase inhibitors, especially phenyl alcohols such as 2-phenyl-1-ethanol; glycerin; Salicylates; Ascorbate; Ion permeable carriers such as monensin; Amphoteric amines; Ammonium chloride; N-acetylcysteine; Capsaicin; And kioroquines. Stimulation-releasing additives, when present, may be incorporated at concentrations effective to alleviate irritation or skin damage in topical formulations, which typically represent up to about 20%, more typically up to about 5%, by weight of the formulation.

Topical formulations may also be used as antiseptic / wound healing / anti-microbial / skin tissue repair-promoting compounds (eg, preferably as substantially homogeneous microparticles provided herein and optionally with one or more synergistic antibiotics as described herein). Combined BT compounds), together with one or more additional pharmacologically active agents suitable for topical administration in a therapeutically effective amount. Such agents may be used as phospholipids and oxygen-loaded fluorocarbons, which may improve oxygen supply into skin tissue, as described, for example, in International Patent Publications WO 94/00098 and WO 94/00109. Asymmetric lamellar aggregates consisting of a mixture of fluorocarbon compounds.

Suitable pharmacologically active agents that can be topically applied by incorporation into the topical formulations of the invention include, but are not limited to: improving or eradicating pigmented or non-pigmented blotch, keratosis, and wrinkles Formulations; Antimicrobial agents; Antibacterial agents; Anti-itch and anti-drying agents; Anti-inflammatory agents; Local anesthetics and analgesics; Corticosteroids; Retinol (eg, retinoic acid); vitamin; hormone; And anti-metabolic agents, but are not limited thereto. Some examples of topical pharmacologically active agents include acylclovir, amphotericin, chlorhexidine, clotrimazole, ketoconazole, echonazol, myconazole, metronidazole, minocycline, nystatin, neomycin, kanamycin, phenoitoin, para- Amino benzoic acid esters, octyl methoxycinnamate, octyl salicylate, oxybenzone, dioxybenzone, tocopherol, tocopheryl acetate, selenium sulfide, zinc pyrithione, diphenhydramine, pramoxin, lidocaine, procaine, erythromycin, Tetracycline, clindamycin, crotamitone, hydroquinone and its monomethyl and benzyl ethers, natroxen, ibuprofen, chromoline, retinoic acid, retinol, retinyl palmitate, retinyl acetate, coal tar, gly Theofulvin, estradiol, hydrocortisone, hydrocortisone 21-acetate, hydrocortisone 17-baler Nitrate, hydrocortisone 17-butyrate, progesterone, betamethasone valerate, betamethasone dipropionate, triamcinolone acetonide, fluorinide, clobetasol propionate, minoxidil, dipyridamole, diphenylhydantoin, benzoyl peroxide , And 5-fluorouracil. As also shown above, certain embodiments include carbapenem antibiotic, cephalosporin antibiotic, fluoroquinolone antibiotic, glycopeptide antibiotic, lincosamide antibiotic, penicillinase-resistant penicillin antibiotic, aminopenicillin antibiotic, or amikacinaceae Consider incorporation into the formulation of antibiotics, such as aminoglycoside antibiotics.

Pharmacologically acceptable carriers can also be incorporated into certain topical formulations of the present embodiments and can be certain carriers commonly used in the art. Examples include water, lower alcohols, higher alcohols, honey, polyhydric alcohols, monosaccharides, disaccharides, polysaccharides, sugar alcohols such as glycols (2-carbons), glycerol (3-carbons), erythritol and traceols (4- Carbon), arabitol, xylitol and ribitol (5-carbon), mannitol, sorbitol, dulcitol and iditol (6-carbon), isomalthol, maltitol, lactinol and polyglycitol, hydrocarbon oils, fats And oils, waxes, fatty acids, silicone oils, nonionic surfactants, ionic surfactants, silicone surfactants, and water-based and emulsion-based mixtures of such carriers.

Topical formulation embodiments of the invention can be applied regularly or anywhere on the acute or chronic wound site (e.g., surrounding tissue including normal or healthy peripheral tissue that is deemed or otherwise unaffected by the wound) or the skin site or other Epithelial tissue surfaces (eg, gastrointestinal tract, respiratory tract, gland tissue) require treatment using the number and amount of times necessary to achieve the desired result. The number of treatments can be attributed to the nature of the skin (or other epithelial tissue) condition (eg, other skin wounds or other types of skin wounds, such as those found in acute or chronic wounds or dehiscence resulting from surgical incisions), user Reactivity of the skin (or other tissue), in particular embodiments, with the strength of the active ingredient (e.g., BT compounds and optionally one or more additional pharmaceutically active ingredients such as antibiotics such as amikacin or other antibiotics) Such as wound-healing / preservative / anti-biofilm / skin tissue repair-promoting compounds) described herein, the effectiveness of the vehicle used to deliver the active ingredient into the appropriate layer of skin (or other epithelial surface-containing tissue), Ease removed by physical contact with a bandage or other dressing or garment, or removal thereof by sweat or other inherent or external fluid, and a subject Depends on the convenience of the activity level or lifestyle of the patient.

Representative concentrations of active substances, such as the BT compound antiseptic / anti-biofilm / wound-healing / skin tissue repair-promoting compositions described herein, for example, from about 0.001 to 30% by weight based on the total weight of the composition 0.01 to 5.0%, and more preferably up to about 0.1 to 2.0%. As one representative example, the compositions of this aspect of the invention may be applied to acute or chronic wounds and / or skin at a ratio equal to about 1.0 mg / cm ^{2 of} skin to about 20.0 mg / cm ² of skin. Representative examples of topical formulations include aerosols, alcohols, anhydrous bases (e.g. lipsticks or powders), aqueous solutions, creams, emulsions (oil-in-water or water-in-oil emulsions), fats, blowing agents , Gels, hydro-alcohol solutions, liposomes, lotions, microemulsions, ointments, oils, organic solvents, polyols, polymer preparations, powders, salt preparations, silicone derivatives, and waxes Do not. Topical formulations may include, for example, chelating agents, conditioners, emollients, excipients, wetting agents, protective agents, thickeners, or UV absorbers. Those skilled in the art will appreciate that formulations other than those listed may be used in aspects of the present invention.

Chelating agents may be optionally included in topical formulations and may be selected from any of the formulations suitable for use in cosmetic compositions, and have any ability to bind divalent cation metals such as Ca ²⁺ , Mn ²⁺ , or Mg ^2+. It may include natural or synthetic chemicals. Examples of chelating agents include, but are not limited to, EDTA, disodium EDTA, EGTA, citric acid, and dicarboxylic acid.

Conditioners may also optionally be included in the topical formulations. Examples of skin conditioners include acetyl cysteine, N-acetyl dichlorosphingosine, acrylate / behenyl acrylate / dimethicone acrylate copolymers, adenosine, adenosine cyclic phosphate, adenosine phosphate, adenosine triphosphate, alanine, albumen, Algae extracts, allantoin and derivatives, aloe barbadensis extract, aluminum PCA, amyloglucosidase, arbutin, arginine, azulene, bromelain, buttermilk powder, butylene glycol, caffeine, calcium gluconate, capsaicin, Carbocysteine, carnosine, beta-carotene, casein, catalase, cephalin, ceramide, chamomilla recutits (matricaria) flower extract, cholecalciferol, cholesteryl ester, coco Betaine, coenzyme A, modified corn starch, crystals, cycloethoxymethicone, cysteine DNA, cytochrome C, Rutoside, dextran sulfate, dimethicone copolyol, dimethylsilanol hyaluronate, DNA, elastin, elastin amino acid, epidermal growth factor, ergocalciferol, ergosterol, ethylhexyl PCA, fibronectin, folic acid, gelatin, glatin Liadine, beta-glucan, glucose, glycine, glycogen, glycolipid, glycoprotein, glycosaminogen glycan, glycosphingolipid, horseradish peroxidase, hydrogenated protein, hydrolyzed protein, jojoba oil, Keratin, keratin amino acids, and kinetin, lactoferrin, lanosterol, lauryl PCA, lecithin, linoleic acid, linolenic acid, lipase, lysine, lysozyme, malt extract, maltodextrin, melanin, methionine, inorganic salts, niacin, niacinamide, oat amino acids, Oryzanol, palmitoyl hydrolyzed protein, pancreatin, papain, PEG, pepsin, phospholipids, phytosterols, placental enzymes, platelet lipids, Lidoxal 5-phosphate, quercetin, resorcinol acetate, riboflavin, RNA, saccharomyces lysate extract, silk amino acid, sphingolipids, stearamidopropyl betaine, stearyl palmitate, tocopherol, tocopheryl acetate, toco Peryl linoleate, ubiquinone, bitis vinifera (grape) seed oil, wheat amino acids, xanthan gum, and zinc gluconate. Skin conditioners other than those listed above can be combined with the compositions or formulations provided herein as will be readily appreciated by those skilled in the art.

Topical formulations may also optionally include one or more emollients, examples of which include acetylated lanolin, acetylated lanolin alcohols, acrylates / C _10-30 alkyl acrylate crosspolymers, acrylate copolymers, alanine, algae extracts , Aloe barbadensis extract or gel, altea officinalis extract, aluminum starch octenylsuccinate, aluminum stearate, apricot [prunus armeniaca] seed oil, arginine, arginine aspa Latex, arnica montana extract, ascorbic acid, ascorbyl palmitate, aspartic acid, avocado [(persea gratissima] oil, barium sulfate, barrier sphingolipids, butyl alcohol , Beeswax, behenyl alcohol, beta-sitosterol, BHT, birch [betula alba] bark extract, borage [purple] Borago officinalis] extract, 2-bromo-2-nitropropane-1,3-diol, butcherbroom [ruscus aculeatus] extract, butylene glycol , Calendula Offalilis Extract, Calendula Popinalis Oil, Candelia [euphorbia cerifera] Wax, Canola Oil, Caprylic / Capric Triglycerides, Cardamom [Eletaria Cardamomium] (elettaria cardamomum)] oil, carnauba [copernicia cerifera] wax, carrageenan [chondrus crispus], carrot [daucus carota saliva] Oils, castors [ricinus communis] oils, ceramides, ceresin, cetheareth-5, ceteareth-12, ceteareth-20, cetearyl octanoate, cetetsu-20, Ceteth-24, Cetyl Acetate, Cetyl Octanoate, Cetyl Palmitate , Chamomile [anthemis nobilis] oil, cholesterol, cholesterol ester, cholesteryl hydroxystearate, citric acid, clary [salvia sclarea] oil, cocoa [theobroma cacao (theobroma cacao)] butter, coco-caprylate / caprate, coconut [cocos nucifera] oil, collagen amino acids, corn [zea mays] oil, fatty acids, decyl oleate, dextrin, Diazolidinyl urea, dimethicone copolyol, dimethiconol, dioctyl adipate, dioctyl succinate, dipentaerythryl hexacaprylate / hexacaprate, DMDM hydantoin, DNA, erythritol, ethoxydi Glycol, Ethyl Linoleate, Eucalyptus globulus Oil, Evening Braided [oenothera biennis] Oil, Fatty Acids, Truck Tose, Gelatin, Geranium Macul Tomb oil, glucosamine, glucose glutamate, glutamic acid, glycerin-26, glycerin, glycerol, glyceryl distearate, glyceryl hydroxystearate, glyceryl laurate, glyceryl linoleate, glyceryl myristate, glyceryl oleate Eight, Glyceryl Stearate, Glyceryl Stearate SE, Glycine, Glycol Stearate, Glycol Stearate SE, Glycosaminoglycans, Grapes [vitis vinifera] Seed Oil, Hazel [Corylus americana (corylus americana)] nut oil, hazelnut [corylus avellana] nut oil, hexylene glycol, honey, hyaluronic acid, safflower [carthamus tinctorius] oil, hydrogenation Castor oil, hydrogenated coco-glycerides, hydrogenated coconut oil, hydrogenated lanolin, hydrogenated lecithin, Hydrogenated palm glycerides, hydrogenated palm kernel oil, hydrogenated soybean oil, hydrogenated resin glycerides, hydrogenated vegetable oils, hydrolyzed collagen, hydrolyzed elastin, hydrolyzed glycosaminoglycans, hydrolyzed Keratin, hydrolyzed soy protein, hydroxylated lanolin, hydroxyproline, imidazolidinyl urea, iodopropynyl butylcarbamate, isocetyl stearate, isocetyl stearoyl stearate, isodecyl oleate Isopropyl isostearate, isopropyl ranolate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, isostearamide DEA, isostearic acid, isostearyl lactate, isostearyl neopentanoate, Jasmine [jasminum officinale] oil, jojoba [northus chinensis (buxus) chinensis] oil, kelp, kukui [aleurites moluccana] nut oil, lactamide MEA, laneth-16, laneth-10 acetate, lanolin, lanolin acid, lanolin alcohol , Lanolin oil, lanolin wax, lavender [lavandula angustifolia] oil, lecithin, lemon [citrus medica limonum] oil, lenoleic acid, linolenic acid, macadamia ternifolia Nut Oil, Magnesium Stearate, Magnesium Sulfate, Maltitol, Matricaria [chamomilla recutita] Oil, Methyl Glucose Sequistearate, Methyl Silanol PCA, Microcrystalline Wax, Inorganic Oil, Mink Oil , Mortierella oil, myristyl lactate, myristyl myristate, myristyl propionate, neopentyl glycol dicaprylate / dicaprate, octyldodecanol, octyl Decyl myristate, octyldodecyl stearoyl stearate, octyl hydroxystearate, octyl palmitate, octyl salicylate, octyl stearate, oleic acid, olive [olea europaea] oil, orange [ Citrus aurantium dulcis] oil, palm [elaeis guineensis] oil, palmitic acid, panthetin, panthenol, panthenyl ethyl ether, paraffin, PCA, peach [prunus] Prunus persica] seed oil, peanut [arachis hypogaea] oil, PEG-8 C12 18 ester, PEG-15 cocamine, PEG-150 distearate, 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 Set Quistearate, P EG-40 sorbitan peroleate, PEG-5 soy sterol, PEG-10 soy sterol, PEG-2 stearate, PEG-8 stearate, PEG-20 stearate, PEG-32 stearate, PEG-40 stearate , PEG-50 stearate, PEG-100 stearate, PEG-150 stearate, pentadecaractone, peppermint [mentha piperita] oil, petroleum, phospholipids, polyamine sugar condensates, polyglyceryl-3 di Isostearate, polyquaternium-24, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, polysorbate 85, potassium myristate, potassium palmitate, potassium sorbate, potassium stearate , Propylene glycol, propylene glycol dicaprylate / dicaprate, propylene glycol dioctanoate, propylene glycol difellargonate, propylene glycol laurate, propylene glycol stearate, Rholene glycol stearate SE, PVP, pyridoxine dipalmitate, quaternium-15, quaternium-18 hectorite, quaternium-22, retinol, retinyl palmitate, rice [oryza sativa] Barn oil, RNA, rosemary [rosmarinus officinalis] oil, rose oil, safflower [carthamus tinctorius] oil, sage [Salvia opusinalis (oil) salvia officinalis] oil, salicylic acid, sandalwood oil [santalum album] oil, serine, cerum protein, sesame [sesamum indicum] oil, shea butter [butyrospermum karkyi parkii)], silk powder, sodium chondroitin sulfate, sodium DNA, sodium hyaluronate, sodium lactate, sodium palmitate, sodium PCA, sodium polyglutamate, sodium stearate, soluble collagen, sorbic acid, sorbitan laurate, Sorbitan oleate, sorbitan palmitate, sorbitan sesquinoleate, sorbitan stearate, sorbitol soybean [glycine soja] oil, sphingolipids, squalene, squalene, stearamide MEA-stearate, stearates Acid, stearoxy dimethicone, stearoxytrimethylsilane, stearyl alcohol, stearyl glycyrrhetinate, stearyl heptanoate, stearyl stearate, sunflower [helianthus annuus] seed oil, thinning Prunus amygdalus dulcis oil, synthetic beeswax, tocopherol, tocopheryl acetate, tocopheryl linoleate, tribehenin, tridecyl neopentanoate, tridecyl stearate, triethanolamine , Tristearin, urea, vegetable oils, water, waxes, wheat [triticum vulgare] sprout oil, and ylang ylang) [cananga odorata] oils, including but not limited to.

In some embodiments, topical formulations may contain suitable excipients that are highly affinity to skin, good tolerant, stable and readily available for consistency. Suitable topical excipients and vehicles are for special use by those skilled in the art, and in particular, see Remington's Pharmaceutical Sciences, Vol. 18, Mack Publishing Co., Easton, Pa. (1990), in particular Chapter 87, which may be commonly selected by reference to one of the many standard textbooks in the art. Optionally one or more wetting agents are also included in the topical formulations. Examples of humectants include amino acids, chondroitin sulfate, diglycerin, erythritol, fructose, glucose, glycerin, glycerol, glycol, 1,2,6-hexanetriol, honey, hyaluronic acid, hydrogenated honey, hydrogenated starch hydrolysate, Inositol, lactitol, maltitol, maltose, mannitol, natural wetting factor, PEG-15 butanediol, polyglyceryl sorbitol, salts of pyrrolidone carboxylic acid, potassium PCA, propylene glycol, sodium gluconate, sodium PCA, sorbitol, shoe Cross, trehalose, urea and xylitol.

Certain embodiments contemplate topical formulations containing one or more additional skin protectants. Examples of skin protectants include algae extract, allantoin, aluminum hydroxide, aluminum sulfate, betaine, camellia leaf extract, cerebroside, dimethicone, glucuronolactone, glycerin, kaolin, lanolin, malt extract, inorganic oil, petroleum It may include, but is not limited to, potassium gluconate and talc.

Surfactants may also be included in certain topical formulations contemplated herein and suitable for use in cosmetic compositions such as cationic, anionic, zwitterionic, or non-ionic surfactants, or mixtures thereof. Surface active agents (see Rosen, M., "Surfactants and Interfacial Phenomena," Second Edition, John Wiley & Sons, New York, 1988, Chapter 1, pages 4 31). Examples of cationic surfactants include DMDAO or other amine oxides, long chain primary amines, diamines and polyamines and salts thereof, quaternary ammonium salts, polyoxyethylenated long chain amines, and quaternized polyoxyethylenated long chain amines. Including, but not limited to. Examples of anionic surfactants include SDS; Salts of carboxylic acids (eg soaps); Salts of sulfonic acid, salts of sulfuric acid, phosphoric acid and polyphosphoric acid esters; Alkyl phosphates; Monoalkyl phosphate (MAP); And salts of perfluorocarboxylic acids. Examples of zwitterionic surfactants are commoamidopropyl hydroxide (CAPHS) and others that are pH-sensitive and require special attention in designing the proper pH of the formulation (ie, alkylaminopropionic acid, already Dazoline carboxylates and betaines) or those that are not pH-sensitive (eg, sulfobetaines, sultaines). Examples of non-ionic surfactants include alkylphenol ethoxylates, alcohol ethoxylates, polyoxyethylenated polyoxypropylene glycols, polyoxyethylenated mercaptans, long chain carboxylic acid esters, alconolamides, tert acetylenic glycols. , Polyoxyethylenated silicones, N-alkylpyrrolidones, and alkylpolyglycosidases. Ionic detergent or, PLURONICS ^® Series [manufactured - a wetting agent, a mineral oil or non-: The other surface-active agent such as a preparation such as one or more members of BASF (BASF), NJ, Mount Olive material) In addition, for example and non-limiting In theory it can be included to prevent aggregation of BT particulates in the particulate suspension. Any combination of surfactants is suitable. Certain embodiments include at least one anionic and one cationic surfactant, or at least one cationic and one zwitterionic surfactant that is miscible, i.e., does not form a complex that precipitates noticeably upon mixing. It may include.

Examples of thickeners that may also be present in certain topical formulations include acyllamide copolymer, agarose, amylopectin, betonite, calcium alginate, calcium carboxymethyl cellulose, carbomer, carboxymethyl chitin, cellulose gum, dextrin, gelatin, Hydrogenated resins, hydroxytailcellulose, hydroxypropylcellulose, hydroxypropyl starch, magnesium alginate, methylcellulose, microcrystalline cellulose, pectin, various PEGs, polyacrylic acid, polymethacrylic acid, polyvinyl alcohol, various PPG , Sodium acrylate copolymer, sodium carrageenan, xanthan gum, and yeast beta glucan. Thickeners other than those listed above may also be used in embodiments of the present 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 required, such agents are, for example, benzophenone, benzophenone-1, benzophenone-2, benzophenone-3, benzophenone-4, benzophenone-5, benzo Phenone-6, benzophenone-7, benzophenone-8, benzophenone-9, benzophenone-10, benzophenone-11, benzophenone-12, benzyl salicylate, butyl PABA, cinnamate ester, synoxate, DEA -Methoxycinnamate, diisopropyl methyl cinnamate, ethyl dihydroxypropyl PABA, ethyl diisopropylcinnamate, ethyl methoxycinnamate, ethyl PABA, ethyl urocanate, glyceryl octanoate dimethoxycinnamate , Glyceryl PABA, glycol salicylate, homosalate, isoamyl p-methoxycinnamate, titanium, zinc, zirconium, silicon, manganese and oxides of cerium, PABA, PABA esters, Parsol 1789, And isopropylbenzyl salicylate, and mixtures thereof. Can be. Those skilled in the art will appreciate that sunscreens and UV absorbers or protective agents other than those listed may be used in certain embodiments of the present invention.

Topical formulations described 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 To about pH 10, about pH 3 to about pH 9, about pH 3 to about pH 8, and about pH 3 to about pH 8.5. Most preferably, the pH is about pH 7 to about pH 8. One skilled in the art can adjust the pH to an acceptable range by adding an appropriate pH adjusting component to the composition of the present invention. “About” defined pH includes formulations in which the actual measured pH at any time provided to a person skilled in the art may be below or above a prescribed value of no more than 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or 0.1 pH units. It will be appreciated that formulation formulation and storage conditions can be created by shifting the pH from its original value.

Creams, lotions, gels, ointments, pastes and the like can be sprayed or gently rubbed onto the affected surface. The solution may be applied in the same way, but more commonly will be applied with a dropper, swab, etc., and will be applied carefully to the affected area. The application regimen will depend on a number of factors that can be readily measured, such as the severity of the wound and its responsiveness to the initial treatment, but will generally include one or more applications per day on a progression basis. One skilled in the art can readily determine the optimal amount of dosage form to be administered, the method of administration and the repetition rate. In general, formulations of these and related embodiments of the present invention will be applied in a range of once or twice a week or more to once, twice, three times, four or more times daily.

As also discussed above, topical formulations useful herein are pharmaceuticals that include any suitable diluent or excipient, including any pharmaceutical formulation that can be administered without undue toxicity to itself, the receptor receiving the composition. It contains an acceptable carrier. Pharmaceutically acceptable carriers include, but are not limited to, liquids such as water, saline, glycerol, ethanol, and the like, and also form films such as viscosity enhancers (e.g. balsam fir resin) or collidion or nitrocellulose solutions. It may include the agent. A complete discussion of pharmaceutically acceptable carriers, diluents and other excipients is given in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. current edition).

If the topical formulation is in the form of a gel- or liquid-filled capsule, for example a gelatin capsule, it may contain, in addition to substances of this type, a liquid carrier such as polyethylene glycol or oil. Liquid pharmaceutical compositions of certain embodiments of the present invention may comprise one or more of the following, whether they are liquids, suspensions or other similar forms: water for injection, saline solution, preferably physiological saline, Ringer's solution, Sterile diluents such as isotonic sodium chloride, synthetic mono or diglycerides, polyethylene glycol, glycerin, propylene glycol or other solvents, which may serve as a solvent or suspending medium; Antibacterial agents such as benzyl alcohol or methyl parabens; Additional antioxidants such as ascorbic acid or sodium hydrogen sulfite; Chelating agents such as ethylenediaminetetraacetic acid (EDTA); Buffers such as acetate, citrate or phosphate, and stiffness modifiers such as sodium chloride or dextrose.

For topical administration, the carrier may suitably include a solution, emulsion, ointment or gel base. For example, such substrates may include one or more of the following: diluents such as petroleum, lanolin, polyethylene glycol, beeswax, inorganic oils, 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 comprise a transdermal patch or iontophoretic device. Topical formulations may contain a compound of certain embodiments of the invention at a concentration of about 0.1 to about 10% w / v (weight per unit volume). Topical formulations may be provided in the form of creams, lotions, solutions, sprays, gels, ointments, pastes, and / or contain liposomes, micelles, microspheres and / or other particulate or nanoparticle delivery components. Topical formulations may also be administered time-release or sustained-release particles or pellets that can be administered directly to the wound site, for example, sustained-release ethylene vinyl acetate polymers (eg, Elvax ^® 40, manufactured by Aldrich, Milwaukee, WI.

Topical formulations may include agents that bind to skin tissue repair-promoting compounds and thus assist in their delivery to skin epithelial cells (eg keratinocytes) and / or fibroblasts. Suitable agents that can act at this dose include clathrating agents such as cyclodextrins, and other agents can include proteins or liposomes.

Topical formulations of certain embodiments of the invention may also be provided in dosage unit form, which may be administered as an aerosol. The term aerosol is used to refer to various systems ranging from those of colloidal properties to systems consisting of pressurized packages. Delivery can be understood by a liquefied or compressed gas or by a suitable pump system for dispensing the active ingredient. Aerosols of the compounds of certain embodiments of the invention may deliver the active ingredient (s) by delivery to a single phase, two-phase, or three phase system. Delivery of the aerosol includes the necessary containers, activators, valves, small containers, and the like, which together may form a kit. One skilled in the art can determine a preferred aerosol for delivering the topical formulation to the skin or wound area without undue testing.

Topical formulations can be prepared by methods well known in the art of pharmacy. For example, a pharmaceutical composition intended to be administered by spraying, washing or cleaning the wound site or skin may be prepared by sterile distilled water with a BT preservative / wound-healing / anti-biofilm / skin tissue repair-promoting compound as described herein. It can be prepared by forming a solution in combination with. Surfactants may be added to promote the formation of a homogeneous solution or suspension. Surfactants interact non-covalently with antioxidant active compounds to promote dissolution or homogeneous suspension of the compounds in the aqueous delivery system.

The BT preservative / wound-healing / anti-biofilm / skin tissue repair-promoting compound for use in topical formulations or pharmaceutically acceptable salts thereof is administered in a therapeutically effective amount, the amount of which is characterized by the nature of the wound site (if relevant). The activity of specific BT compounds used (incorporation or absence from the formation of antibiotics such as aminoglycoside antibiotics, for example amikacin); Metabolic safety and length of action of the compound; The age, body weight, general health, sex, skin type, immune status, and diet of the subject; Mode and time of administration; Discharge rate; Drug combinations; Severity of special skin wounds requiring skin tissue repair; And the therapy the subject is undergoing. Generally, a therapeutically effective daily dose is (from 70 kg of mammals) about 0.001 mg / kg (ie 0.07 mg) to about 100 mg / kg (ie 7.0 g); Preferably the therapeutically effective dose is (for 70 kg mammals) about 0.01 mg / kg (ie 7 mg) to about 50 mg / kg (ie 3.5 g); More preferably the therapeutically effective dosage (for 70 kg mammals) is about 1 mg / kg (ie 70 mg) to about 25 mg / kg (ie 1.75 g).

The range of effective dosages provided herein represents a preferred range of dosages and is not intended to be limited thereto. However, the most preferred dose will be adjusted according to the individual subject, as can be understood and measured by those skilled in the art. See, eg, Berkow et al., Eds., The Merck Manual, 16th edition, Merck and Co., Rahway, NJ. , 1992; Goodman et al., Eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th 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, Pharmacology, Little, Brown and Co., Boston, (1985); Osolci al., Eds., Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Co., Easton, PA (1990); Katzung, Basic and Clinical Pharmacology, Appleton and Lange, Norwalk, CT (1992).

The total dose required for each treatment may be administered in multiple doses or in a single dose, if desired. Certain preferred embodiments contemplate a single application of topical formulations per day. In general, and in clear embodiments, treatment may commence at a lower dose that is below the optimal dose of the compound. Thereafter, the dose is increased in small increments until the optimum effect is reached under the circumstances.

Topical formulations may be administered alone or in combination with other treatments and / or agents directed to skin wounds or directed to other related symptoms or etiology factors. For example, and as indicated above, the topical formulation may further comprise retinoic acid. As another example, the topical formulation can include one or more skin tissue repair-promoting compounds described herein, or can include two or more such compounds with different cell wound repair activity.

Recipients of the topical formulations described herein can be any vertebrate, eg, a mammal. Among mammals, preferred recipients are primate necks (including humans, apes and monkeys), cows (including horses, goats, cattle, sheep and pigs), rodents (including mice, rats, rabbits and hamsters), and carnivores (cats and Dogs). Among algae, preferred recipients are turkey, chicken and many other identical necks. The most preferred recipient is a human and a person with a biofilm or one or more acute or chronic wounds.

For topical application, an effective amount of a pharmaceutical composition comprising a BT compound antiseptic / wound-healing / anti-biofilm / skin tissue repair-promoting compound according to an embodiment described herein is administered to a target site, eg, an acute or chronic wound. It is desirable to administer to skin wounds such as, and / or to risky areas of the skin (eg, wound dehiscence). Such amounts generally range from about 0.0001 mg to about 1 g of a compound of a particular embodiment of the invention per application, depending on the site to be treated, the severity of the wound (or past or contemplated surgical incision), and the nature of the topical vehicle used. Can be. Preferred topical formulations are ointments or sustained release pellets, wherein from about 0.001 to about 50 mg of the active ingredient are used per cc of ointment base or pellet suspending agent. Pharmaceutical compositions can be formulated as transdermal compositions or transdermal delivery devices (“patches”). Such compositions include, for example, backing, active compound reservoirs, control membranes, liners, and contact adhesives. Such transdermal patches can be used to provide continuous beats or, if desired, upon delivery of the compounds of the present invention.

Compositions of certain embodiments may be formulated to provide rapid, sustained or delayed release of the active ingredient after administration to a patient by using procedures known in the art. Controlled release drug delivery systems include osmotic pump systems and dissolution systems containing polymer-coated reservoirs or drug-polymer matrix formulations. Examples of controlled release systems are provided in US Pat. Nos. 3,845,770 and 4,326,525 and in PJ Kuzma et al, Regional Anesthesia 22 (6): 543-551 (1997), all of which are incorporated herein by reference. It is incorporated into.

The most suitable route will depend on the nature and severity of the condition being treated. Those skilled in the art will also determine topical administration methods (spraying, creaming, open application, closed dressing, dipping, washing, etc.), dosage forms, suitable pharmaceutical excipients, and other problems related to the delivery of a compound to a subject in need thereof. be used to.

Throughout the specification, unless the content otherwise requires, the words “comprise,” and “comprising” include the stated step or component or the inclusion of steps or groups of ingredients, but no other step. Or will not exclude a component or steps or group of components. "Consisting of" means including and limited to the phrase "consisting of". Thus, the phrase “consisting of” indicates that the components listed are required or mandatory and other components may not be present. “Consisting essentially of” means including any ingredient listed after the phrase and is limited to other ingredients that do not interfere with or contribute to the activity or action defined in the description of the listed ingredient. Thus, the phrase “consisting essentially of” indicates that the listed ingredients are required or mandatory, but other ingredients are not required and may or may not be present depending on whether or not they affect the activity or action of the listed ingredients. .

In this specification and the appended claims, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. As used herein, in a particular embodiment, the term "about" or "approximately" refers to a value ± 5%, 6%, 7%, 8% or 9% when present before a numerical value. In other embodiments, the term “about” or “approximately” refers to a range of values ± 10%, 11%, 12%, 13%, or 14% when present before a figure. In still other embodiments, the term “about” or “ "Approximately" indicates a range of ± 15%, 16%, 17%, 18%, 19% or 20% when present before the figure.

Bismuth-thiol compositions according to the present invention comprising a plurality of microparticles comprising a bismuth-thiol (BT) compound having a volume average diameter of substantially all of the microparticles all have a biofilm formation, biofilm viability It can be usefully used to inhibit biofilm growth, bacterial cell viability or bacterial cell growth.

Brief description of some perspectives of the drawings
FIG. 1 shows the number of survival from Pseudomonas aeruginosa colony biofilms grown for 24 hours at 37 ° C. on 10% tryptic soy agar (TSA) followed by 18 hours of treatment (log CFU; colony). Forming unit). Antibiotic treatment shown is TOB, tobramycin 10X MIC; AMK, amikacin 100X MIC; IPM, imipenem 10X MIC; CEF, cefepime 10X MIC; CIP, ciprofloxacin 100X MIC; Cpd 2B, Compound 2B (bis-BAL, 1: 1.5). (MIC; minimum inhibitory concentration, eg, the lowest concentration that prevents bacterial growth).
FIG. 2 shows the number of survival (log CFU) from Staphylococcus aureus colony biomass following the indicated treatments after 24 hours growth on 10% trimsin soybean agar. Antibiotic treatment shown is rifampicin, RIF 100X MIC; Daphtomycin, DAP 320X MIC; Minocycline, MIN 100X MIC; Ampicillin, AMC 10X MIC; Vancomycin, VAN 10X MIC; Cpd 2B, Compound 2B (bis-BAL, 1: 1.5), Cpd 8-2, Compound 8-2 (bis-Pyr / BDT (1: 1 / 0.5).
3 shows scratch closure over time of keratinocytes exposed to the biofilm. (*) Significantly different from control (P <0.001).
4A and 4B show anti-inhibitory BisEDT reversal antibiotic-resistance for several antibiotics. The effect of antibiotics in the presence and absence of BisEDT (0.05 μg / ml) is shown in the field of MRSA (methicillin-resistant S. aureus). Panel A shows only standard antibiotic-immersed discs and Panel B shows discs combined with BisEDT (BE). [GM = gentamicin, CZ = cefazoline, FEP = cefepime, IPM = imfenim, SAM = ampicillin / sulbactam, LVX = levofloxacin.
5 shows the effect of BisEDT and antibiotics on biofilm formation. s. Epidermidis was grown for 48 hours at 37 ° C. in TSB + 2% Glucose in polystyrene plates. Gatifloxacin (GF), clindamycin (CM), minocycline (MC), gentamicin (GM), vancomycin (VM), cefazoline (CZ), naphcillin (NC), and rifampicin (RP). Results are expressed as mean change in BPC (serial 2-fold dilution steps) at 0.25 μM BisEDT (n = 3).
FIG. 6 shows S. grown for 48 hours at 37 ° C. in TSB and 2% glucose. The effect of BisEDT and antibiotics on growth of epidermidis. Results are shown as mean change in MIC (dilution step) with increasing BisEDT (n = 3). See the legend in FIG. 5 for antibiotic definitions.
FIG. 7 shows bone and hardware samples from open fractures in an in vivo rat model after treatment with three BT formulations, bis-EDT, MB-11 and MB-8-2, with or without cefazoline antibiotic treatment. Mean S detected in Bar graph showing Aureus bacterial levels. The standard error of the mean is represented by error bars. Early euthanized animals were not excluded from the analysis, but samples from one animal in group 2 were excluded due to significant contamination.

The following examples are presented for illustrative purposes and are not limiting.

Example

Example 1: Preparation of BT Compound

The following BT compounds were prepared according to methods such as Domenico et al. (See US Re-registered Patent No. RE37,793, US Patent 6,248,371, US Patent 6,086,921, US Patent 6,380,248) or as described below for BisEDT. As microparticles according to the prepared synthesis protocol. For comparison, the atomic ratio for a single bismuth atom is shown, based on the known tendency of bismuth to form trivalent complexes with sulfur containing compounds and the stoichiometric ratio of the reactants used. The number in parentheses is the ratio of bismuth to one (or more) thiol agent (eg Bi: thiol1 / thiol2; 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 Manufacture) (1: 1.5) BiC ₃ H ₆ S ₃

5) CPD 2A Bis-Bal (1: 1) BiC ₃ H ₆ S ₂ 0

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-2hydroxy, propane thiol (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 / 2hydroxy, Propane Thiol (1: 1/1)

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

0.331 L (˜0.575 moles) of Bi (N0 ₃ ) ₃ aqueous solution [43% Bi (N0 ₃ ) ₃ (w) in excess (11.4 L) of 5% aqueous HNO ₃ in 15 L of polypropylene carboy at room temperature / w), 5% nitric acid (w / w), 52% water (w / w), Product No. 2362 of Shepherd Chemical Co., Cincinnati, Ohio; δ-1.6 g / mL] was added dropwise while stirring, followed by the slow addition of anhydrous ethanol (4 L). Some white precipitate formed was dissolved by continuous stirring. Ethanolic solution of 1,2-ethanedithiol (CAS 540-63-6) (~ 1.56 L, -0.55 M) was added to 1.5 L of absolute ethanol, 72.19 mL (0.863 moles) of 1,2-ethanedithiol Was prepared separately by adding with a 60 mL syringe and stirring for 5 minutes. Thereafter, 1,2-ethanedithiol / EtOH reagent was slowly added dropwise while continuously stirring overnight to an aqueous solution of Bi (NO ₃ ) ₃ / HNO ₃ over 5 hours. The product formed was allowed to settle into the precipitate for about 15 minutes and then the filtrate was removed using a peristaltic pump at 300 mL / min. The product is then collected by filtration on fine filter paper in a 15-cm diameter Buchner funnel and washed three times in succession with 500-mL volumes of ethanol, USP water, and acetone, respectively, to give a yellow amorphous powder solid. BisEDT (694.51 gm / mole) was obtained. The product was placed in 500 mL amber glass jar and dried over CaCl ₂ under high vacuum for 48 hours. The recovered material (yield ˜200 g) removed the thiol-characteristic odor. The crude product was redissolved in 750 mL of absolute ethanol, stirred for 30 minutes, then filtered, washed successively with 3 x 50 mL ethanol, 2 x 50 mL acetone and washed again with 500 mL acetone. The rewashed powder was triturated in 1M NaOH (500 mL), filtered and washed with 3 x 220 mL of water, 2 x 50 mL of ethanol, and 1 x 400 mL of acetone to yield 156.74 gm of purified BisEDT. It was. Subsequent batches produced essentially in the same manner produced yields of about 78-91%.

The product is characterized as having the structure shown above in Formula I by data analysis from ¹ H and ³ C nuclear magnetic resonance (NMR), infrared spectroscopy (IR), ultraviolet spectroscopy (UV), mass spectrometry (MS) and elemental analysis. It was. An HPLC method was developed to measure the chemical purity of BisEDT in which samples were prepared in DMSO (0.5 mg / mL). λ _max was measured by scanning a solution of BisEDT in DMSO between 190 and 600 nm. YMC Pack PVC Sil NP, 5 μm, 250 × 4.6 mm internal diameter analytical column (Waters) using a UV detector to monitor isocratic HPLC elution at 1 mL / min at 265 nm (λ _max ), 2 μm injection volume at ambient temperature Waters (Millipore, Milford, MA) on a Model 2695 chromatograph performed in a mobile phase of 0.1% formic acid in acetonitrile: water (9: 1) and 100 ± 0.1% A single peak was detected that reflects chemical purity. Elemental analysis is consistent with the structural formula (I).

The dried particulate material was characterized to evaluate particle size properties. In summary, the particles are resuspended in 2% Pluronic ^® F-68 (BASF, Mount Olive, NJ) and the suspension is nanosized in a water bath sonicator for 10 minutes. Standard setting prior to analysis using Zetasizer Nano-S Particle Analyzer (Model ZEN 1600 (Zeta-potentiometric capabilityless), Manufacturer of Melbourne Instruments, Worcestershire, UK, according to manufacturer's recommendations) It was sonicated. From the edited data of the two measurements, the particulates exhibited a single phase distribution in which all detectable events in the volume mean diameter (VMD) were between about 0.6 microns and 4 microns and the peak VMD was about 1.3 microns. In contrast, when BisEDT was prepared according to the preceding method (Domenico et al., 1997 Antimicrob. Agents Chemother. 41 (8): 1697-1703), most of the particles were heterodisperse and based on VMD. Significantly larger size, excluding their characterization.

Example 2: Colony Biofilm Model of Chronic Wound Infection

Inhibition by BT Compound

Since bacteria present in chronic wounds adopt a biofilm lifestyle, BT has been described by methods described in Anderl et al., 2003 Antimicrob Agents Chemother 47 ': 125-56; Walters et al., 2003 Antimicrob Agents Chemother 47: 317; Wentland et al., 1996 Biotchnol. Prog. 12: 316; Biofilms were tested for their effect on bacterial cell survival using biofilms essentially prepared according to Zheng et al., 2002 Antimicrob Agents Chemother 46: 900.

In summary, colony biofilms were grown for 24 hours on 10% tryptic soy agar and transferred to Mueller Hinton plates containing treatment. After treatment, biofilms were dispersed into peptone water containing 2% w / v glutathione (neutralizing BT) and serially diluted into peptone water before spotting on the plates for counting. Two bacteria isolated from chronic wounds were used separately for production of colony biofilms for testing. These were Gram-negative bacterial strains Pseudomonas aeruginosa, and Gram-positive methicillin resistant Staphylococcus aureus (MRSA).

Bacterial biofilm colonies are described in Anderl et al., 2003 Antimicrob Agents Chemother 47: 1251-56; Walters et al. , 2003 Antimicrob Agents Chemother 47: 317; Wentland et al., 1996 Biotchnol. Prog. 12: 316; Zheng et al., 2002 Antimicrob Agents Chemother 46: 900, were grown on top of the microporous membrane essentially remaining on the agar plate. Colony biofilms exhibited many familiar features of other biofilm models, for example, they consisted of cells densely packed in highly hydrated matrices. See also Brown et al., J Surg Res 56: 562; Millward et al, 1989 Microbios 58: 155; Sutch et al., 1995 J Pharm Pharmacol 47: 1094; As reported in Thrower et al., 1997 J Med Microbiol 46: 425, it was observed that colony biofilm bacteria exhibited the same significantly reduced anti-microbial susceptibility, which is quantified in a more sophisticated manner in in vitro biofilm reactors. It became. Colony biofilms were produced in large numbers easily and reproducibly. According to a non-limiting theory, this colony biofilm model shared some of the characteristics of infected wounds; The bacteria grew at the interface of nutrients and air supplied below the biofilm and from minimal fluid flow. Various nutrient sources were used to culture colony biofilms containing blood agar, which are believed to mimic nutrient conditions in vivo.

Colony biofilms were prepared by inoculating a 5 μl spot of planktonic bacterial liquid culture into a 25 mm diameter polycarbonate filter membrane. The membrane was sterilized by exposure to ultraviolet light for 10 minutes per side before inoculation. Inoculum was grown overnight at 37 ° C. in bacterial medium and diluted to an optical density of 0.1 at 600 nm in fresh medium before being deposited on the membrane. Thereafter, the membrane was placed on agar plate containing growth medium. The plate was then covered and placed inverted in the incubator at 37 ° C. Every 24 hours, the membranes and colony biofilms were transferred to fresh plates using sterile forceps. The waxy biofilm is typically used for experiments after 48 hours of growth, at which time there were about 10 ⁹ bacteria per membrane. The colony biofilm method was used successfully to cultivate a wide range of single and mixed species biofilms.

To determine sensitivity to antimicrobial agents (e.g., BT compounds; antibiotics; and BT compounds containing a combination of BT compound-antibiotic combinations), colony biofilms were agar supplemented with candidate antimicrobial treatment agent (s). Transfer to plate. If the duration of exposure to antimicrobial treatment exceeded 24 hours, colony biofilms were transferred to fresh treatment plates daily. 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, it was necessary to simply process the sample with a tissue homogenizer to destroy cell aggregates. The resulting cell suspension was subsequently serially diluted and plated to enumerate viable bacteria and reported as colony forming units (CFU) per unit area. Survival data were analyzed using the log ₁₀ log transformed (log ₁₀ transformation).

Five antibiotics and 13 BT compounds were tested for each type of bacterial biofilm colony cultures (Summonas aeruginosa, PA; methicillin resistant Staphylococcus aureus, MRSA or SA). . Antimicrobial agents tested for PA are herein BisEDT and Compounds 2B, 4, 5, 6, 8-2, 9, 10, 11 and 15 (see Table 1), and antibiotics tobramycin, amikacin, already BT, referred to as penim, cefazoline, and ciprofloxacin. Antimicrobial agents tested for SA are BisEDT and BT referred to herein as Compounds 2B, 4, 5, 6, 8-2, 9, 10 and 11 (see Table 1), and antibiotics, rifampicin, daphtomycin, Minocycline, ampicillin and vancomycin. As described above under “Simple Description of Drawings”, antibiotics were tested at concentrations of about 10-400 times the minimum inhibitory concentration (MIC) according to established microbiology methods.

Seven BT compounds showed a significant effect on PA bacterial survival at the tested concentrations; two BT compounds demonstrated a pronounced effect on MRSA survival at the tested concentrations; Representative results showing BT effects in bacterial survival are shown in FIG. 1 for BisEDT and BT Compound 2B (tested for PA) and in FIG. 2 for BT Compound 2B and 8-2 (tested for SA), In both cases, the effects are shown in comparison with the indicated antibiotics. As also shown in FIGS. 1 and 2, inclusion of the indicated BT compound with the indicated antibiotic produced a synergistic effect, whereby reduced bacterial viability was improved over the anti-bacterial effect of the antibiotic alone or the BT compound alone. In the PA survival assay, compound 15 [bis-EDT / 2hydroxy, propane thiol (1: 1/1)] was used to produce a combination of 1600 μg / mL AMK and 80 μg / mL BisEDT at a concentration of 80 μg / mL. It showed an effect comparable to that obtained using (not shown) (FIG. 1).

Example 3 Drip Flow Model of Chronic Wound Infection

Inhibition by BT Compound

Droplet flow biofilms represent an accepted authentication model in the art for forming and testing the effects of candidate anti-bacterial compounds on bacterial biofilms. Droplet flow biofilms are produced in coupons (substrates) placed in the channels of a drop flow reactor. Many different types of materials can be used as substrates for bacterial biofilm formation, including translucent glass microscope slides. The nutrient liquid medium is introduced into a drip flow bioreactor cell chamber by dropping into a chamber near the top and then down the slope at 10 degrees to flow along the length of the coupon.

The biofilm grows in a drip flow bioreactor and is individually or together with the BT compound and / or separately with the antibiotic compound or with other antibiotics comprising the BT compound or with other conventional or candidate treatments for chronic wounds. Exposure Thus, BT compounds are characterized for their effect on bacterial biofilms in a drop-flow reactor. Biofilms in drop-flow reactors are prepared according to established methods (e.g. Stewart et al., 2001 J Appl Microbiol. 91: 525; Xu et al., 1998 Appl. Environ. Microbiol. 64: 4035). The design involves incubating the biofilms on the inclined polystyrene coupons in the covered chamber. Exemplary culture medium is 1 g / l supplemented with 5% v / v adult donor bovine serum (ph 6.8) that mimics iron limited conditions rich in serum proteins similar to biofilm growth conditions in vivo, such as in chronic wounds. Glucose, 0.5 g / l NH ₄ NO ₃ , 0.25 g / l KCl, 0.25 g / l KH ₂ P0 ₄ , 0.25 g / l MgSO ₄ -7H ₂ 0. The medium was flowed drop-wise (50 ml / h) over four coupons contained in four separate parallel chambers each measuring a depth of 10 cm x 1.9 cm x 1.9 cm. The chambered reactor is made from polysulfone plastic. Each chamber is equipped with a separate removable plastic lid that can be strongly sealed. The biofilm reactor is contained in an incubator at 37 ° C. and the bacterial cell culture medium is warmed by passing through an aluminum heat sink held in the incubator. The method regenerates the antibiotic resistance phenotype observed in certain biofilms, provides a continuous supplementation of nutrients while simulating the proximity to the low fluid shear environment and air interface characteristics of chronic wounds, and the effects of the introduced candidate antibacterial therapy. It is miscible with many analytical methods for characterizing and monitoring them. A drop-flow reactor was successfully used to incubate a wide variety of pure, mixed-species biofilms. Biofilms are typically grown for 2 to 5 days before applying the antimicrobial agent.

In order to determine the effect of the anti-microbial agent on the biofilm grown in a drop-flow reactor, the desired therapeutic formulation (eg, one or more BT compounds and / or one or more antibiotics, or Control, and / or other candidate formulations). Flow continues for the defined treatment period. The treated biofilm coupon is then simply removed from the reactor and the biofilm is scraped into a beaker containing 10 ml of buffer. Samples are simply processed using a tissue homogenizer (typically for 30 seconds to 1 minute) to disperse bacterial aggregates. Serially dilute and plate the suspension to list viable microorganisms according to standard microbiological methods.

Example 4: Wound Biofilm Inhibition of Keratinocyte Scratch Recovery

Biofilm inhibition by BT compound

This example provides an established in vitro keratinocyte scratch model of wound healing in order to reach biofilms associated with biofilm-associated wound pathology and wound healing, and in particular wounds containing acute or chronic wounds or biofilms as described herein. Describe the variation of. According to the keratinocyte scratch model of the effect of chronic wound biofilms, the culture of mammalian (eg human) keratinocytes and bacterial biofilm populations is carried out in separate chambers in contact with each other in keratocyte wound healing. In addition, the effects of the soluble components elaborately produced by the biofilm and the conditions affecting the effect should be evaluated.

Neonatal human epidermal cells are cultured as monolayers in treated plastic dishes, where controlled "wounds" or scratches in monolayers are mechanical means (eg, sterile scalpels, razors, cell scrapers). through physical disruption of the monolayer, such as by scraping linear cell-free zones essentially between areas of the monolayer using suitable tools such as scrapers, forceps, or other tools. In vitro keratinocyte monolayer model systems are known to undergo cellular structural and functional processes in response to wounding in a manner that simulates wound healing in vivo. In accordance with embodiments described herein, the effect of the presence of bacterial biofilms on such processes, for example, the healing time of scratches, is observed, and in these and related embodiments, the effect is also dependent on the selected candidate antimicrobial (eg, anti Bacterial and antimicrobial membranes).

Wounded keratinocyte monolayers cultured in the presence of biofilms are tested according to morphological, biochemical, molecular genetic, cytophysiological and other parameters to determine whether the introduction of BT compounds alters the damaging effects of biofilms (eg, For example, increase or decrease in a statistically significant manner relative to the appropriate control. The wounds were first exposed to each BT compound alone and the considered combination of BT compounds to test the toxicity of each BT compound treatment before evaluating the effect of this treatment on the biofilm effect on the model wound healing process.

In an exemplary embodiment, a 3-day biofilm is maintained on the tissue culture well (eg, a TransWell membrane insert, etc.) and through a scraped keratinocyte monolayer to initiate the wound healing process. Incubate by in fluid communication. Biofilms cultured outside certified acute or chronic wounds are contemplated for use in these and related embodiments.

Thus, an in vitro system was developed to assess the effect of soluble biofilm components on migration and proliferation of human keratinocytes. The system uses dialysis membranes to separate biofilms and keratinocytes. Keratinocytes were incubated from the newborn epidermis and glass covered as described previously (Fleckman et al., 1997 J Invest. Dermatol. 109: 36; Piepkorn et al., 1987 J Invest. Dermatol. 88: 215-219). Grow as a coherent monolayer on the slip. The keratinocyte monolayer can then be scraped to obtain a "wound" with a uniform width, followed by monitoring the cell repair process (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). Thereafter, the artificial wound is placed at the bottom of the sterile double-sided chamber and the chamber is assembled using aseptic technique. Both sides of the chamber were filled with keratinocyte growth medium (EpiLife) in the presence or absence of antibiotics and / or bismuth-thiol. Uninoculated system was used as a control.

The system is inoculated with bacteria isolated from the wound and incubated at rest for 2 hours to allow bacteria to adhere to the surface of the upper chamber. After the attachment period, liquid medium flow is initiated in the upper chamber to remove unattached cells. Thereafter, the flow of the medium is continued by washing unattached cells at a rate that minimizes the growth of planktonic cells in the upper chamber. After an incubation period ranging from 6 to 48 hours, the system (keratin monolayer on the coverslip and bacterial biofilm on the membrane substrate) is digested and analyzed by removing the cover slip. In a related embodiment, mature biofilms are grown before assembling the chamber in the upper chamber. In other related embodiments, separate co-culture of biofilms and scraped-cut keratinocyte monolayers is carried out with or without the presence of one or more BT compounds, optionally including or excluding one or more antibiotics, thereby repairing the keratinocytes of the scratched wound. For example, a candidate agent, such as a BT compound, or a potent synergistic BT compound-and-antibiotic combination [eg, a BT compound provided herein, such as BT provided in particulate form, and one or more amicacin, ampicillin, Cefazoline, cefepime, chloramphenicol, ciprofloxacin, clindamycin (or other lincosamide antibiotics), daphtomycin (Cubicin ^® ), doxycycline, gatifloxacin, gentamicin, imipenim, levofloxacin, linezolid (Zyvox) ^®), minocycline, and naphthyl published, our wave erythromycin, rifampin, measure the effectiveness of sulfamic methoxy Sasol, tobramycin, and vancomycin], for example, be I am modifying (e.g., increasing or decreasing at least one index of scratch wound healing, such as increasing or decreasing in a statistically significant manner relative to an appropriate control), such as the time elapsed for wound recovery or other wound-recovery index. Or identify a combination of agents (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: Wound Biofilm Inhibition of Keratinocyte Scratch Recovery

The isolated human keratinocytes were cultured on glass coverslips to scratch-wound according to the method described above in Example 4. Wounded cultures were maintained in the presence of co-cultured biofilms on membrane support in fluid communication with keratinocyte cultures or under culture conditions alone. The scratch closure time interval was measured while keratinocyte cell growth and / or migration reestablished the keratinocyte monolayer on the scratch zone. 3 illustrates the effect of the presence of fluid communication (but not in direct contact) of the biofilm on the healing time of the scraped keratinocyte monolayer.

Thus, in certain embodiments cultured in the presence of bacterial biofilm with or without candidate anti-biofilm agents presenting a scratch-wound cell (eg keratinocyte or fibroblast) monolayer; A method of identifying an agent for treating chronic wounds is contemplated, including evaluating the index of healing of scratch-damaged cell monolayers in the absence and presence of a candidate anti-bial agent, wherein at least one index of healing Agents to promote [e.g., substantially monodisperse BT microparticle suspensions as described herein alone or amikacin, ampicillin, cefazoline, cefepime, chloramphenicol, ciprofloxacin, clindamycin, daphtomycin (Cubicin ^® ), Doxycycline, gatifloxacin, gentamicin, imipenim, levofloxacin, linezolid (Zyvox ^® ), minocycline, napcillin, paromomycin, rifampin, sulfamomethoxazole, tobramycin and vancomycin] Synergistic combination with the same antibiotic] is identified as a suitable agent for treating acute or chronic wounds or wounds containing biofilms.

Example 6: synergistic bismuth-thiol (BT) -antibiotic combination

This example shows an example of a proven synergistic effect by the combination of one or more bismuth-thiol compounds and one or more antibiotics against various bacterial species and bacterial strains, including several antibiotic-resistant bacteria.

Materials and methods. Sensitivity studies were carried out in NCCLS protocols (National Committee for Clinical Laboratory Standards.) By broth dilution in 96-well tissue culture plates (Nalge Nunc International, Denmark). (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].

In summary, overnight McFarland standard suspensions were prepared using overnight bacterial cultures, which were cationic-controlled Müller-Hinton broth medium [BBL, Cockisville, MD, USA. Material] further diluted 1:50 (˜2 × 10 ⁶ cfu / mL). BT (prepared as described above) and antibiotics were added while maintaining the final volume fixed at 0.2 mL in incremental concentrations. Cultures were incubated for 24 hours at 37 ° C. and turbidity was assessed by absorption at 630 nm using an ELISA plate reader (Biotek Instruments, Winuskey, Vermont, USA). The minimum inhibitory concentration (MIC) is expressed as the lowest drug concentration that inhibits growth for 24 hours. The number of viable bacteria (cfu / mL) was determined by standard plating on nutrient agar. Minimum bactericidal concentration (MBC) was expressed as the concentration of drug that reduced initial viability by 99.9% after 24 hours of incubation.

The checkerboard method was used to assess the activity of antimicrobial combinations. Fractional inhibitory concentration index (FICI) and fractional bactericidal concentration index (FBCI) are described in Eliopoulos et al., Eliopoulos and Moellering, (1996) Antimicrobial combinations. In Antibiotics in Laboratory Medicine (Lorian, V., Ed.), Pp. 330-96, Williams and Wilkins, Baltimore, MD, USA. Synergy was defined as a FICI or FBCI index of ≦ 0.5, no interaction at> 0.5 to 4 and antagonism at> 4. See Odds, FC (2003) Synergy, antagonism, and what the chequerboard puts between them. Journal of Antimicrobial Chemotherapy 52: 1]. Synergy was also commonly defined as ≧ 4-fold reduction in antibiotic concentration.

The results are shown in Tables 2 to 17.

Example 7: Comparative Bismuth-Thiol (BT) and Antibiotic Effects on Gram-positive and Gram-negative Bacteria Including Antibiotic-Resistant Bacterial Strains

Bacterial strains

In vitro activity of BisEDT and comparative formulations in this Example was evaluated for a number of clinical isolates of Gram-positive and Gram-negative bacteria involved in skin and soft tissue infection.

Materials and methods. Test compounds and test concentration ranges were as follows: BisEDT [Domenico et al., 1997; Domenico et al., Antimicrob. Agents Chemother. 45 (5): 1417-1421. And Example 1, 16-0.015 μg / mL; Linezolid (manufactured by ChemPacifica Inc., # 35710), 64-0.06 μg / mL; Daptomycin (Cubist Pharmaceuticals # MCB2007), 32-0.03 μg / mL and 16-0.015 μg / mL; Vancomycin (manufactured by Sigma-Aldrich, St. Louis, Missouri, # V2002), 64-0.06 μg / mL; Ceftazidime (Sigma # C3809), 64-0.06 μg / mL and 32-0.03 μg / mL; Imipenem (United States Pharmacopeia, NJ, # 1337809) 16-0.015 μg / mL and 8-0.008 μg / mL; Ciprofloxacin (United States Pharmacopea, # 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 products except gentamicin were dissolved in DMSO; Gentamicin was dissolved in water. Stock solutions were prepared at 40-fold maximum concentrations in test plates. The final concentration of DMSO in the test system was 2.5%.

organism. Test organisms were obtained from the following clinical laboratories: CHP, Clarian Health Partners, Indianapolis, Indiana; UCLA, University of California Los Angeles Medical Center, University of California Los Angeles Medical Center, Los Angeles, CA; GR Micro, London, UK; PHRI TB Center, Public Health Research Institute Tuberculosis Center, New York, NY; ATCC, American Type Culture Collection, Manassas, VA; Mt Sinai Hosp., Mount Sinai Hospital, New York, NY; UCSF, University of California San Francisco General Hospital, San Francisco, CA; Branson Hospital, Branson Methodist Hospital, Kalamazoo, Michigan; Quality control isolates were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The organisms were streaked to separate on the appropriate agar medium for each organism. The colonies were picked up from a separation plate with cotton swabs and placed in suspension in an appropriate broth containing cryoprotectant. The suspension was aliquoted into cryogenic vials and maintained at -80 ° C.

Abbreviations are as follows: BisEDT, bismuth-1,2-ethanedithiol; LZD, linezolide; DAP, daphtomycin; VA, vancomycin; CAZ, ceftazidime; IPM, imipenem; CIP, ciprofloxacin; GM, gentamicin; MSSA, methicillin-sensitive Staphylococcus aureus; CLSI QC, clinical and laboratory standard organ quality control strains; MRSA, methicillin-resistant Staphylococcus aureus; CA-MRSA, co-acquired methicillin-resistant Staphylococcus aureus; MSSE, methicillin-sensitive Staphylococcus epiderimides; MRSE, methicillin-resistant Staphylococcus epiderimides; VSE, vancomycin-sensitive enterococcus.

Isolates from frozen vials Appropriate medium: tryticase soybean agar for most organisms (Becton-Dickinson, Sparks, MD) or trypticase soybean agar for Streptococcus and 5 Streaking over% sheep blood (Cleveland Scientific, Bath, Ohio). 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-manufactured by Becton Dickinson, # 212322) for most organisms Horse blood 2% digested to MHB II Supplemented by Cleveland Scientific Lot # H13913 provided growth to Streptococcus piogenes and Streptococcus agalactia Media was prepared at 102.5% normal weight to each well of the microdilution panel. The resulting dilution was offset by the addition of 5 μL of drug solution, and for the test with daptomycin, the medium was supplemented with an additional 25 mg / L Ca ²⁺ .

* The MIC assay method follows the procedures described in the Clinical and Laboratory Standards Institute (see Clinical and Laboratory Standards Institute.Methods for Dilution Antimicrobial Susceptibility 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 Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2006) using an automated liquid handler. Serial dilution and liquid delivery. Automated liquid handlers include the Multidrop 384 (manufactured by Labsystems, Helsinki, Finland), Biomek 2000, and Multimek 96 (Beckman Coulter, CA). Main fullerton material. Wells of columns 2-12 of a standard 96-well microdilution plate (Falcon 3918) were charged with 150 μL of DMSO or gentamicin water on a multidrop 384. Drug (300 μL) was dispersed in column 1 of the appropriate row in the plate.

These come from the parent plate from which the test plate (daughter plate) was made. Biomec 2000 was completed in series via column 11 in the parent plate. The wells of column 12 contained no drug and were organism growth control wells in daughter plates. Daughter plates were loaded using Multidrop 384 with 185 μL of appropriate test medium (as described above). Daughter plates were prepared on a Multimec 96 device that delivered 5 μL of drug solution in one step from each well of the parent plate to each corresponding well of each daughter plate.

Standardized inoculum of each organism was prepared per CLSI method (ISBN 1-56238-587-9, cited above). Suspensions were prepared in the same way as turbidity of 0.5 McFarland standard in MHB. The suspension was diluted 1: 9 in broth appropriate for the organism. Inoculum for each organism was dispensed into sterile reservoirs (Beckman coulter) divided by length and inoculated into plates using Biomec 2000. The daughter plate was placed on an inverted Biomec 2000 working surface to allow inoculation from low to high concentrations of drug. Biomex 2000 delivered 10 μL of standardized inoculum into each well. This yielded a final cell concentration in the daughter plate of about 5 × 10 ⁵ colony-forming units / mL. Thus, the wells of the daughter plates ultimately contained 185 μL broth, 5 μL drug solution, and 10 μL bacterial inoculum. The plates were stacked three heights, covered with a top plate, placed in a plastic bag and incubated for approximately 18 hours at 35 ° C. for most of the separator. Streptococcus plates were read after 20 hours of incubation. Microplates were observed from the bottom using a plate observer. For each of the test media, soluble control plates without inoculation were observed for evidence of drug precipitation. The MIC was read and recorded as the lowest concentration of drug that inhibited the visible growth of the organism.

result. All commercial drugs were soluble at both test concentrations in both media. BisEDT showed traces of precipitate at 32 μg / mL, but the MIC readout was unaffected because the inhibitory concentrations for all organisms tested were below that concentration. On each assay day, the appropriate quality control strain (s) were included in the MIC assay. The MIC values derived for these strains were appropriately determined from the quality control ranges published for each formulation (see Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Eighteenth Informational Supplement.CLSI document M100-S18 [ISBN 1 -56238-653-0] .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 (s) were included in the MIC assay. The MIC values derived from these strains are appropriately determined from the published quality control ranges for each formulation (see Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Eighteenth Informational Supplement.CLSI document M100-S18 [ISBN 1-)). 56238-653-0]). Of the 141 values for the quality control strain for which the quality control range was published, 140 (99.3%) were within the defined range. One exception is S. It was imipenem for Aureus 29213, which yielded one value (≦ 0.008 μg / mL) in a single run, one dilution below the published QC range. All other quality control results in this run were within the defined quality control range.

BisEDT is a methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant S. aureus. Strong activity was demonstrated for both aureus (MRSA) and community-acquired MRSA (CA-MRSA), with 1 μg / mL MIC90 value of 0.5 μg / mL for all three groups of organisms. All strains tested below mL were inhibited. BisEDT was greater than the activities of linezolid and vancomycin and showed the same activity as that of daphtomycin. Imipenem was more potent than BisEDT for MSSA (MIC90 = 0.03 μg / mL). However, MRSA and CAMRSA were resistant to imipenem but BisEDT demonstrated the same activity as shown for MSSA. BisEDT was highly active against methicillin-sensitive and methicillin-resistant Staphylococcus epidermidis (MSSE and MRSE), with values of 0.12 and 0.25 μg / mL, respectively. BisEDT was more active against MSSE than any of the other agents tested except imipenem. BisEDT was the most active formulation (maximum active formulation) tested for MRSE.

BisEDT demonstrated the same activity as that of daphtomycin, vancomycin and imipenem at a MIC90 value of 2 μg / mL against vancomycin-sensitive enterococcus paecalis (VSEfc). Significantly, BisEDT was the largest active agent tested against vancomycin-resistant enterococcus paecalis (VREfc) at a MIC90 value of 1 μg / mL.

BisEDT was very active against vancomycin-sensitive enterococcus faesium (VSEfm) with a MIC90 value of 2 μg / mL; Its activity was the same or similar to that of daphtomycin and was 1-diluted higher than that of vancomycin. BisEDT and linezolid were the largest active agents tested against vancomycin-resistant enterococcus faesium (VREfm), each demonstrating a MIC90 value of 2 μg / mL. The activity of BisEDT on Streptococcus pyogenes (MIC90 value of 0.5 μg / mL) was the same as that of vancomycin, larger than that of linezolid, and slightly smaller than that of daphtomycin and ceftazidime. Compounds inhibited all tested strains at 0.5 μg / mL or less. In these studies, the least sensitive species for BisEDT was Streptococcus agalactiae, where the MIC90 value observed was 16 μg / mL. BisEDT was less active than all the agents tested except gentamicin.

The activity of BisEDT and nonfactors on the included Gram-negative bacteria demonstrated BisEDT potency (MIC90 value of 2 μg / mL) against Axinetobacter Baumannni, making it the most active compound tested by BisEDT. Elevated MICs of a significant number of test isolates for the comparator formulations produced off-scale MIC90 values for these formulations. BisEDT was a potent inhibitor of Escherichia coli that inhibited all strains at 2 μg / mL or less (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 equal to that of imipenem. Relatively high MIC90 values were inhibited by imipenem, ceftazidime, ciprofloxacin, and gentamicin, indicating that this is a highly antibiotic-resistant group of organisms. BisEDT was the largest active compound tested for Pseudomonas aeruginosa at a MIC90 value of 4 μg / mL. There was a high level of resistance to the comparator formulation for this group of test isolates.

In summary, BisEDT has demonstrated a broad spectrum of efficacy against multiple clinical isolates representing multiple species, including species commonly included in acute and chronic skin and skin structure infections in humans. The activity of BisEDT and major comparator preparations was evaluated for 723 clinical isolates of Gram-positive and Gram-negative bacteria. BT compounds demonstrated broad spectrum activity, and for many test organisms in this study, BisEDT was the largest active compound tested in terms of anti-bacterial activity. BisEDT is MSSA, MRSA, CA-MRSA, MSSE, MRSE, and S. a. Maximum activity against piogenes, where the MIC90 value was 0.5 μg / mL or less. Strong active is also VSEfc, VREfc.VSEfm, VREfm, A. Baumaniyi, Lee. Coli, and blood. Proven for aeruginosa, where the MIC90 value was in the range of 1-4 μg / mL. The observed MIC90 value is K. Pneumoniae (MIC90 = 8 μg / mL), and S. This was the case with S. agalactiae (MIC90 = 16 μg / mL).

Example 8: Particulate BT-antibiotic Enhancement and Synergistic Activity

This example shows that particulate bismuth thiol (BT) promotes antibiotic activity through enhanced and / or synergistic interactions.

The main complex factor in treating infection is the emergence of bacterial resistance to antibiotics. s. Epidermides (MRSE) and S. a. Methicillin resistance in aureus (MRSA) actually reflects multiple drug resistance and makes it very difficult to eradicate these pathogens. However, no Staphylococcus from the hundreds of strains tested showed resistance to BT. In addition, at sub-MIC concentrations, BT reduced resistance to several important antibiotics.

Staphylococcus aureus . A graphical description of the antibiotic-resensitizing effect of subMIC bismuth ethanedithiol (BisEDT) on MRSA is provided (FIG. 4), which includes gentamicin, cefazoline, cefepime, imipenim, sulfamemethazole, and levofloxacin It shows enhanced antibiotic action of several classes of antibiotics. Thus, BisEDT nonspecifically enhanced the activity of the best antibiotics.

Broth dilution antimicrobial sensitivity studies were performed on 12 MRSA strains using several antibiotics combined with subMIC levels of BisEDT (Table 18). Both biofilm-resistant concentrations (BPC) and minimum inhibitory concentrations (MIC) were measured in special biofilm culture media (BHIG / X). MIC and BPC for gentamicin and cefazoline were reduced by subMIC BisEDT (BisEDT MIC, 0.2-0.4 μg / ml), but not below the breakpoint for sensitivity. subMIC BisEDT enhanced the sensitivity of MRSA to gatifloxacin and cefepime near the breakpoint for sensitivity. These strains were already sensitive to vancomycin, but significantly more so in the presence of subMIC BisEDT. In general, MIC and BPC were reduced two to five times with 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. MIC and BPC calculated at μg / ml are mean ± standard deviation from at least three tests. The right column lists the standard MICs for antibiotic sensitivity (S) and resistance (R).

Broth dilution studies of cefepime-resistant MRSA isolates are shown in Table 19. BisEDT significantly increased the inhibitory activity of cefepime in 11 of 12 isolates at 0.1 μg / ml. In this particular study, the data showed a synergy between BisEDT and cefepime (FIC <0.5), with many isolates at breakpoints for sensitivity.

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

The results for the combination study with naphcillin or gentamicin are shown in Table 20. BisEDT (0.2 μg / ml) when combined with naphcillin reduced MIC90 for naphcillin by four-fold for MRSA (FIC, 0.74). When combined with gentamicin, BisEDT reduced MIC90 for gentamicin by more than 10-fold for MRSA (FIC, 0.6). BT reversed the resistance of all four gentamicin-resistant isolates tested for clinically relevant concentrations (Domenico et al., 2002). MICs for these antimicrobials have been substantially reduced, especially for gentamicin. The broth used in these studies was tryticase soy broth (TSB) containing 2% glucose, with results similar to those observed in Muller-Hinton II broth fortified with 1% sheep blood.

Staphylococcus epidermidis. The activity of most classes of antibiotics was enhanced in the presence of BisEDT. In connection with BPC, clindamycin and gatifloxacin are combined with S. bisEDT. It showed significantly more antimicrobial membrane activity against Epidermides (FIG. 5). In different respects, BPCs for clindamycin, gatifloxacin and gentamicin decreased 50-fold, 10-fold and 4-fold, respectively, in the presence of subMIC BisEDT.

Only modest reductions in antimicrobial concentrations (BPC) were noted for minocycline, vancomycin and cefazoline, whereas rifampicin and naphcillin were not affected at 0.05 μg / ml BisEDT. At 0.1 μg / ml BisEDT, no biofilm was detected regardless of the antibiotic used, meaning no antagonism occurred. The BisEDT concentration was S. It was close to MIC in the case of Epidermidis (Domenico et al., 2003) (FIG. 5).

With regard to growth inhibition, seven of the eight antibiotics tested were tested for S. aureus. Significant improvement was found in the presence of 0.1 μg / ml (0.5 μM) BisEDT for epidermides (FIG. 6). MIC changes were most pronounced for clindamycin and gentamicin, followed by vancomycin, cefazoline, minocycline, gatifloxacin and naphcillin, and rifampicin was not affected. Among the antibiotics, this strain was resistant to (NC, CZ, GM, CM) and only cefazoline resistance was reversed to BisEDT to clinically relevant levels.

s. The minimum bacterial concentration (MBC) for most antibiotics tested for epidermidis was slightly reduced to subMIC BisEDT. Gentamicin showed maximum reduction in MBC (4-16 folds) followed by cefazoline (4-5 folds), vancomycin and naphcillin (3-4 folds), minocycline and gatifloxacin (2 folds) To 3 fold), clindamycin and rifampicin MBC remained largely unaffected. Clindamycin is a bacteriostatic agent, which explains its lack of bacterial activity. Sephazoline resistance has been reversed with respect to MBC (Domenico et al., 2003). This effect was additive.

The efficacy of antimicrobial agents has also been demonstrated in vivo in transplant infected rat models (Table 21). BisEDT levels as low as 0.1 μg / ml were also resistant to S. aureus for 7 days. It was possible to promote the prevention of Epidermidis biofilm.

As summarized in Table 21, implants 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. Physiological solutions (1 ml) containing MS and MR at 2 × 10 ⁷ cfu / ml were inoculated onto the implant surface using a tuberculin syringe. All implants were explanted 7 days after implantation and sonicated in sterile saline solution for 5 minutes to remove adherent bacteria. Quantification of viable bacteria was obtained by culturing dilutions on blood agar plates. The detection limit was approximately 10 cfu / cm ² .

^a group each had 15 animals; MS, methicillin-sensitive S. Epidermides; MR, methicillin-resistant S. Epidermidis

^b Dacron graft segment impregnated with 0.1 mg / l BT, 10 mg / l RIP, 10 mg / l rifampin

^c Statistically significant when compared with control group MS and MR

^d Statistically significant when compared to the MS3 group

^e Statistically significant when compared to MR1, MR2, and MR3 groups

Gram-negative bacteria. Tobramycin activity against resistant Pseudomonas aeruginosa was several-fold improved with subMIC BisEDT (Table 22). In this trial, MIC was defined in more detail as IC ₂₄ .

blood. Resistant strains of aeruginosa were cultured in Müller-Hilton II broth at 37 ° C. in the presence of tobramycin (NN) and BisEDT (BE; 0.33 μg / ml). MIC was measured as antibiotic concentration which inhibited growth for 24 ± 1 h.

For tobramycin-resistant burcolderia sefacia, 0.4 μg / ml BisEDT made 7 of 10 isolates sensitive tobramycin (mean FIC; 0.48) and reduced MIC ₉₀ by 10-fold (Table 23). . Both MIC and MBC of tobramycin were significantly reduced by subMIC BisEDT to achievable levels for 50 clinical bourcholdera sefacia isolates (Veloira et al., 2003). BisEDT and tobramycin in liposome form are blood. High synergy with aeruginosa has been demonstrated (Halwani et al., 2008; Halwani et al., 2009).

Chloramphenicol and ampicillin resistant Escherichia coli were made sensitive to these drugs by addition of subMIC BisEDT (Table 24).

this. Resistant strains of E. coli were cultured in Mueller-Hinton II broth in the presence of chloramphenicol (CM) or ampicillin (AMP) and BisEDT alone or in combination (BE; 0.33 μg / ml) at 37 ° C. It was. MIC was measured as antibiotic concentration which inhibited growth for 24 ± 1 h.

Tetracycline resistant Escherichia coli was made resistant to doxycycline by addition of subMIC BisEDT (Table 25). The combination showed synergy against the TET M and TET D strains (FIC <0.5) and had an additive effect on the TET A and TET B strains.

this. Resistant strains of E. coli were cultured in Müller-Hinton II broth at 37 ° C. in the presence of doxycycline (DOX) and BisEDT alone or in combination (BE; 0.33 μg / ml). MIC was measured as antibiotic concentration which inhibited growth for 24 ± 1 h.

References:

Domenico P, R O'Leary, BA Cunha. 1992. Differential effect of bismuth and salicylate compounds on antibiotic sensitivity of Pseudomonas aeruginosa. Eur J Clin Microbiol Infec Dis 1 1: 170-175; Domenico P, D Parikh, BA Cunha. 1994. bismuth modulation of antibiotic activity against gastrointestinal bacterial pathogens. Med Microbiol Lett 3: 1 14-119; Domenico P, Kazzaz JA, Davis JM, Niederman MS. 2002.Subinhibitory bismuth ethandithiol (BisEDT) sensitizes resistant Staphylococcus aureus to nafcillin or gentamicin. Annual Meeting, ASM, Salt Lake City, UT; Domenico P, Kazzaz JA, Davis JM. 2003. Combating antibiotic resistance with bismuth-thiols. Research Advances in Antimicrob Agents Chemother 3: 79-85; Domenico P, E Gurzenda, A Giacometti, O Cirioni, R Ghiselli, F Orlando, M Korem, V Saba, G Scalise, N Balaban. 2004. BisEDT and RIP act in synergy to prevent graft infections by resistant staphylococci. Peptides 25: 2047-2053; Halwani M, Blomme S, Suntres ZE, Alipour M, Azghani AO, Kumar A, Omri A. 2008. Liposomal bismuth-ethandithiol formulation enhances antimicrobial activity of tobramycin. Intl J Pharmaceut 358: 278-84; Halwani M, Hebert S, Suntres ZE, Lafrenie RM, Azghani AO, Omri A. 2009. bismuth-thiol incorporation enhances biological activities of liposomal tobramycin against bacterial biofilm and quorum sensing molecules production by Pseudomonas aeruginosa. 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.

Example 9: Particulate BT-antibiotic Enhancement and Synergistic Activity

This example shows that micronized bismuth thiol BisEDT promotes antibiotic activity through enhanced and / or synergistic interactions with specific antibiotics for specific microbial target organisms. Single-point data for each of the combinations shown in Table 26 were generated essentially according to the method used in Example 8.

SA, Staphylococcus aureus; MRSA, methicillin-resistant Staphylococcus aureus; E Fc, Enterococcus paecalis; SP, Streptococcus pneumoniae; PRSP, penicillin-resistant Streptococcus pneumoniae; EC, Escherichia coli; KP, Klebsiella pneumoniae; PA, Pseudomonas aeruginosa; Bcep, Burcholderia Sephacia; Bmult, Burcolderia multiborance; Abau, Axinetobacter Baumannini; Msmeg, Mycobacterium smegmatis.

Example 10 Micronized BT-Antibiotic Enhancement and Synergistic Activity

The effects of micronized bis-EDT and four bis-EDT analogs and other agents prepared as described above on representative strains of seven Gram-negative pathogenic bacteria were tested. Use as a synergy (FICI ≤ 0.5), enhancer (0.5 <FICI ≤ 1.0), antagonist (FICI> 4.0), and fractional inhibitory concentration (FIC) and FIC index (FICI) using variations of common laboratory methods Indifference (1.0 <FICI <4.0) was measured (Eliopoulos G and R Moellering. 1991. Antimicrobial combination. In Antibiotics in Laboratory Medicine, Third Edition, edited by V Lorian. Williams and Wilkins, Baltimore, MD, pp. 432-492; Odds, 2003 J. Antimicrob. Chemother. 52 (1): 1]. The FIC index was measured using the checkerboard technique and used in the study.

Stock solutions of all test products were prepared at 40 × final target concentration in appropriate solvent. All test products were in solution under these conditions. Final drug concentrations in the FIC assay plates were set in parentheses with the MIC values of each formulation for each test organism, unless the strain was overall resistant to the test formulation. The concentration ranges tested are shown in Table 27. Test organisms have recently been provided by clinical sources or American type culture collections. Upon receipt, the isolates were streaked on trypsin soybean agar II (TSA). Colonies were harvested from these plates and cell suspensions were prepared in a suitable broth growth medium containing cryoprotectant. Aliquots were then frozen at -80 ° C. Frozen seeds of the organisms to be tested in the assays provided were thawed, streaked to separate on TSA plates and incubated at 35 ° C. All organisms were tested in Muller Hinton II broth (Becton Dickinson, lot no. 9044411). Broth was prepared at 1.05 × normal weight / volume to offset 5% by volume of drug in the final test plate.

Minimum inhibitory concentration (MIC) values were previously determined using Broth microdilution for aerobic bacteria. Clinical and Laboratory Standards Institute (CLSI). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard-Eighth Edition. CLSI document M07-A8 [ISBN 1-56238-689-1]. Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2009].

FIC values were measured using the Broth microdilution method described previously (Sweeney et al., 2003 Antimicrob. Agents Chemother. 47 (6): 1902-1906. To prepare the test plates, an automated liquid handler (Multidrop 384, Labsystems, Helsinki, Finland; Biomex 2000 and Multimex 96, Beckman Coulter, Fullerton, Calif.) Was used. Serial dilution and liquid delivery.

Appropriate wells of a standard 96-well microdilution plate (Falcon 3918) were charged using MultiGrop 384 in columns 2-12 with 150 μL of appropriate solvent. 300 microliters of each second test drug was added to each well in column 1 of the plate. These plates were used to prepare drug “parent plates” and provide them for a series of drug dilutions for drug combination plates. Eleven 2-fold serial dilutions were prepared by transferring 150 μL of each second drug solution (40 ×) from the wells in column 1 of the parent plate using Biomec 2000. Parent plates of Bis-EDT (and analogues) were serially diluted by hand using a multichannel pipette from top to bottom. “Checkerboard” pattern by delivering equal volumes (using multi-channel pipettes) to the drug combination plate by combining the two parent plates, one for each second drug and one for Bis-EDT (or analog) Formed. Column H and column 12 each contained serial dilutions of one of the formulations for the determination of MIC.

“Daughter Plate” was loaded into 180 μL of test medium using Multidrop 384. Subsequently, 10 μL of drug solution was transferred from each well of the drug combination parent plate to each corresponding well of the daughter plate using Multimec 96. Finally, daughter plates were inoculated with test organisms. Standardized inoculum of each organism was prepared according to known guidelines (CLSI, 2009). For all isolates, the inoculum for each organism was dispensed into sterile reservoirs divided by length (Beckman Coulter) and inoculated onto plates using Biomec 2000. The device delivered 10 μL of standardized inoculum to each well to obtain a final cell concentration in the daughter plate of approximately 5 × 10 ⁵ colony-forming-units / mL.

The test form produced an 8 × 12 checker plate where each compound was tested alone (column 12 and column H) and in combination at various ratios of drug concentration. All organism plates were stacked three heights, covered with a lid over the top plate, placed in a plastic bag and incubated at 35 ° C. for approximately 20 hours. After incubation, the microplates were removed from the incubator and viewed from the bottom using a ScienceWare plate viewer. The prepared reading sheets were marked against the walls of the MIC of drug 1 (column H), the MIC of drug 2 (column 12) and the growth-no growh interface.

FIC was measured using the Excel program according to the following formula: (MIC of compound 1 combined / MIC of compound 1 alone) + (MIC of compound 2 combined / MIC of compound 2 alone). FICI for checkerboard was calculated from the individual FICs from the formula: (FIC ₁ + FIC ₂ + ... FIC _n ) / n, where n is the number of individual wells per plate for which FIC was calculated. For example, when off-scale MIC results were obtained with the formulation alone, the following maximum concentrations were used as MIC values in the FIC calculations.

Particulate Bis-EDT, four particulate BT analogs, and all other formulations (and combinations of formulations) were soluble at all final test concentrations. The measured MIC and FICI values are shown in the tables below.

Example 11 Effect of Bismuth Thiol on Infection in the Ratt Norvegicus Femur Major Defect

Current standards of protection against open fractures are perfusion, dead tissue removal and antibiotics; This is intended to reduce bacterial load in the wound to the point where infection does not occur. Despite this treatment, infection still complicates up to 75% of severe multiple open tibial fractures. Interestingly, although early infections are often caused by Gram-negative bacteria, terminal infections associated with healing problems and amputation are due to Gram-positive infections, often Staphylococcus spp. (Johnson 2007).

s. One of the reasons why Aureus is resistant to standard treatment is their ability to form biofilms. Bacteria in the biofilm may be resistant to the concentration of antimicrobial compounds capable of killing similar organisms in the culture medium (Costerton 1987).

The purpose of the study was to determine whether BT would reduce infection in themselves or in open fracture models contaminated with antibiotics. The contaminated rat femur major defect model is a well-accepted model and was used for the experiments described in this example. The model provides a standardized model for comparing the various possible treatments and their effects in reducing infection and / or improving healing.

Compound (CPD) CPD-8-2 (bismuth pyrithione / butanedithiol; Table 1) and CPD-11 (bismuth pyrithione / ethanedithiol; Table 1) have different activity spectra than Bis-EDT, Two analogs of BIS-Bis that have been shown to be potent against biofilm secreting bacteria in vitro.

Three BT formulations, Bis-EDT, CPD-11 and CPD-8-2 (see Table 1), show the presence of tobramycin and vancomycin in polymethyl methacrylate (PMMA) cement bead vehicle and If used in the absence of S. The inhibitory effect was demonstrated against the Aureus strain. Three formulations of particulate BT were produced in the form of clinically useful hydrogel gels as described herein. These BTs were tested by suspending in gel at a concentration of 5 mg / ml ⁻¹ found to be the appropriate concentration for gel delivery. The gel formulation was tailored to the wound contour and did not require removal after application.

Two treatment arms were used: in the first arm, BT was used alone; BT was used with systemic antibiotics (ABx) in the second arm.

(a) BT only

s. Six hours after inoculation of Aererus, the wounds were debrided, perfused with saline and 1 ml of BT gel was inserted into the defect.

(b) BT with systemic antibody (ABx).

s. Six hours after inoculation with Aureus, the wound was debrided, perfused with brine and added 1 ml of BT gel added to insert into the defect. The antibiotic used was cefazoline at a dose equivalent to 5 mgKg ⁻¹ delivered via subcutaneous-injection twice daily for a total of three days after injury. The first dose was administered just before descaling. The foregoing data suggest that the dose produces a reduction in bacterial levels of about 10 ⁶ to about 10 ⁴ and still allows the relative effects of the different BTs to be measured.

(c) control

s. Six hours after inoculation with Aureus, the wounds were debrided and irrigated with saline. Control animals were also treated with cefazoline following the therapy described above.

process:

Procedures for in vivo rat injury models are described in 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, exposing the anterior side of the femoral trunk through a 3-cm incision, removing the periosteum and attached muscle from the bone polyacetyl plate (27 x 4 x 4 mm). The plate was predrilled to accommodate 0.9-mm diameter stranded Kirschner wire, and the base of these plates was formed to contour the thigh body. A pilot hole was drilled through the plate as a template through both the cortex of the thigh and the Kirschner wire strand was inserted through the plate and the thigh Notch 6 mm away from the plate provided as a guide for bone removal. (C) using a small vibrating top (oscillating saw) is cooled by a continuous infusion in an effort to avoid damage while the tissue heat to produce a defect.

1 x 10 ⁵ CFU S in 10 male groups each. Aureus was inoculated and treated 6 hours after inoculation as described above with BT alone or with antibiotics. The groups are as follows: Bis-EDT gel; MB-11 gel; MB-8-2 gel; Bis-EDT gel &Abx; MB-11 gel &Abx; MB-8-2 gel &Abx; Control group (Abx only).

Animals are euthanized 14 days after surgery and bone and hardware are sent for microbiological analysis, the results of which are shown in FIG. 7.

Based on force analysis, 10 animals per group were given 80% force to detect 25% difference between treatment and control groups. This is done with a predicted standard deviation of 35% and an alpha of 0.05.

As shown in FIG. 7, in combination with Bis-EDT, MB-11, and MB-8-2, cefazoline antibiotic activity was improved compared to either cefazoline or Bis compound alone, resulting in S. Reduced aureus infection. Sephazoline in combination with MB-11 and MB-8-2 exhibited improved antibiotic activity compared to cellzoline alone, resulting in S. aureus detected in hardware. Reduced aureus infection. Bis-EDT has not been shown to affect cefazoline in this capacity.

References:

Costerton JW, Cheng KJ, Geesey GG, et al. Bacterial Biofilms in Nature and Disease. Ann Rev Microbiol. 1987; 41 : 435-64.

Domenico P, Baldassarri L, Schoch PE, Kaehler K, m Sasatsu M, Cunha BA. Activities of Bismuth Thiols against Staphylococci and Staphyloccocal Biofilms. Antimicrob Agents and Chemother. 2001; 45 (5) : 1417-21.

Halwani M. Blomme S, Suntres ZE, et al. Liposomal bismuth-ethanedithol formulation enhances antimicrobial activity of tobramycin. Int J Pharm. 2008; 358 : 279-84.

Johnson EN, Burns TC, Hayda RA, Hospenthal DR, Murray CK. Infection complications of open type III tibial fractures among combat casualties. Clin Infect Dis. 2007; 45 (4) : 409-415.

Other citations and related literature

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 subsalicylate. 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 antigen exposure by bismuth-dimercaprol suppression 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 against staphylococci and staphylococcal biofilms. Antimicrob Angens Chemother 2001; 45: 1417-21; Domenico et al., Combating antibiotic resistance with bismuth-thiols. Research Advances in Antimicrob Agents Chemother 2003; 3: 79-85; Domenico et al., Reduction of capsular polysaccharides and otentiation of aminoglycoside inhibition in gram-negative bacterial with bismuth subsalicylate. J Antimicrob Chemo 1991; 28: 801-810; Domenico et al., BisEDT and RIP act in synergy to prevent graft infections by resistant staphylococci. Peptides 2004; 25: 2047-53; domenico et al., 2005. Pyrithione enhanced antimicrobial activity of bismuth. Antibiotics for Clinicians 9: 291-297; US Patent No. 6,582,719; US Re-registered Patent No. RE37,793; US Patent No. 6,248,371; US Patent No. 6,086,921; US Patent No. 6,380,248; US Patent No. 6,582,719; US Patent No. 6,380,248; US Patent No. 6,875,453.

The various aspects described above can be combined to provide further aspects. US patents, US patent application publications, US patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in these specifications and / or listed in the application bibliography are all incorporated herein by reference in their entirety. Aspects of the aspects can be modified as necessary to still provide additional aspects by using the concepts of various patents, patent applications, and publications.

These and other changes may be made to the aspects in view of the above detailed description. In general, in the following claims, the terms used should not be considered to limit the claims to the specific embodiments described in the specification and claims, but together with the full scope of equivalents to which such claims are entitled. It should be construed to include all possible aspects. Accordingly, the claims are not limited by the disclosure.

Claims (47)

A bismuth-thiol (BT) compound comprising a bismuth or bismuth salt and a thiol-containing compound, wherein the volumetric mean diameter of substantially all of the microparticles is from about 0.4 μm to about 5 μm Bismuth-thiol composition comprising a plurality of fine particles. (a) under conditions sufficient to yield a solution substantially free of solid precipitate and for a sufficient time, (i) a bismuth salt comprising bismuth at a concentration of at least 50 mM and lacking a hydrophilic, polar or organic solubilizer Mixing the acidic aqueous solution with (ii) an amount of ethanol sufficient to yield a mixture comprising about 25 volume percent ethanol; And
(b) to the mixture of step (a) an ethanolic solution containing a thiol-containing compound present in the reaction solution in a molar ratio of about 1: 3 to about 3: 1 relative to bismuth in the reaction solution comprising the BT compound The volume average diameter of substantially all of the fine particles formed by the process comprising adding under sufficient conditions and for a sufficient time to form a precipitate comprising fine particles to obtain a reaction solution is from about 0.4 μm to about 5 μm. And a bismuth-thiol composition comprising a plurality of fine particles comprising a bismuth-thiol (BT) compound. The bismuth-thiol composition of claim 2, wherein the bismuth salt is Bi (N0 ₃ ) ₃ . A bismuth-thiol composition according to claim 2, wherein said acidic aqueous solution comprises at least 5%, 10%, 15%, 20%, 22% or 22.5% by weight bismuth. The method of claim 2, wherein the acidic aqueous solution is at least 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5% by weight. Bismuth-thiol composition comprising nitric acid. The thiol-containing compound of claim 2, wherein the thiol-containing compound is 1,2-ethane dithiol, 2,3-dimercaptopropanol, pyrithione, dithioerythritol, 3,4-dimercaptotoluene, 2,3-butanedithiol , 1,3-propanedithiol, 2-hydroxypropane thiol, 1-mercapto-2-propanol, dithioerythritol, alpha-lipoic acid, dithiothritol, methanethiol (CH ₃ SH [m-mercaptan ]), Ethanethiol (C ₂ H ₅ SH [e-mercaptan]), 1-propanethiol (C ₃ H ₇ SH [nP 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]), pentanethiol ( C ₅ H ₁₁ SH [pentyl mercaptan]), coenzyme A, lipoamide, glutathione, cysteine, cystine, 2-mercaptoethanol, dithiothritol, dithioerythritol, 2-mercaptoindole, transglutamina Agent, (11-mercaptodecyl) hexa (ethylene glycol), (11-mercaptodecyl) tetra (ethylene glycol), (11-mercaptodecyl) Tra (ethylene glycol) functionalized gold nanoparticles, 1,1 ', 4', 1 "-terphenyl-4-thiol, 1,11-undecandithiol, 1,16-hexadecanedithiol, 1,2- Ethanedithiol technical grade, 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 furum, 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-octanethiol, 1-pentadecanthiol, 1-pentanethiol, 1-propanethiol, 1-tetradecanethiol, 1- Tetradecanethiol furum, 1-undecanethiol, 11- ( 1H -pyrrole-1-yl) undecane-1-thiol, 11-amino-1-undecantthiol hydrochloride, 11-bro MO-1-Undecanthiol, 11-Mercapto-1-Undecanol, 11-Mercapto-1-Undecanol, 11-Mercaptodecanoic Acid, 11-Mercaptodecanoic Acid, 11-Mercaptoundecyl Trifluor Loacetate, 11-mercaptodedecyl phosphate, 12-mercaptododecanoic acid, 12-mercaptododecanoic acid, 15-mercaptopentadecanoic acid, 16-mercaptohexadecanoic acid, 16-mercaptohexadecanoic acid, 1 H , 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-nonnafluoro-1-hexanethiol Furum, 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-functionalized silica gel, 3-methyl-1-butanethiol, 4,4'-bis (mercaptomethyl) biphenyl, 4,4'-dimer Captostilbene, 4- (6-mer Tohexyloxy) benzyl alcohol, 4-cyano-1-butanethiol, 4-mercapto-1-butanol, 6- (ferrocenyl) hexanethiol, 6-mercapto-1-hexanol, 6-mercaptohexane 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, decanthiol functionalized silver nanoparticles, dodecanethiol functionalized gold nanoparticles, dodecanethiol functionalized silver nanoparticles, hexa (ethylene glycol) mono-11 - (acetylthio) undecyl ether, mercapto toseok acid, methyl 3-mercaptopropionate, nano stator (nanoTether) BPA-HH, NanoThinks ^TM 18, 8 NanoThinks ^{TM, TM} NanoThinks ACID11, NanoThinks ACID16 ^{TM, TM} NanoThinks ALCO11 , NanoThinks ^TM THIO8, Octanethiol 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-bromodecyl) tee Oacetate, S- (4-cyanobutyl) thioacetate, thiophenol, triethylene glycol mono-11-mercaptodecyl ether, trimethylolpropane tris (3-mercaptopropionate), [11- (methyl Carbonylthio) undecyl] tetra (ethylene glycol), m-carbonan-9-thiol, p-terphenyl-4,4 "-dithiol, tert-dodecyl mercaptan, tert-nonyl mercaptan Bismuth-thiol composition comprising at least one agent selected. (a) under conditions sufficient for obtaining a solution substantially free of solid precipitate and for a sufficient time, (i) a bismuth salt comprising bismuth at a concentration of at least 50 mM and comprising a hydrophilic, polar or organic solubilizer Mixing the acidic aqueous solution lacking (ii) with ethanol in an amount sufficient to yield a mixture comprising about 25 volume percent ethanol; And
(b) to the mixture of step (a) an ethanolic solution comprising a thiol-containing compound present in a molar ratio of about 1: 3 to about 3: 1 relative to bismuth in the reaction solution, Bismuth-thiol (BT), wherein the volume average diameter of substantially all of the microparticles is from about 0.4 μm to about 5 μm, including adding under sufficient conditions and for a sufficient time to form a precipitate comprising A method of making a bismuth-thiol composition comprising a plurality of microparticles comprising a compound. 8. The method of claim 7, further comprising recovering the precipitate to remove impurities. 8. The method of claim 7, wherein said bismuth salt is Bi (NO ₃ ) ₃ . 8. The method of claim 7, wherein said acidic aqueous solution comprises at least 5 wt%, 10 wt%, 15 wt%, 20 wt%, 22 wt% or 22.5 wt% bismuth. The method of claim 7, wherein the acidic aqueous solution is at least 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5% by weight. Method of containing nitric acid. The thiol-containing compound according to claim 7, wherein the thiol-containing compound is 1,2-ethane dithiol, 2,3-dimercaptopropanol, pyrithione, dithioerythritol, 3,4-dimercaptotoluene, 2,3-butanediti Ol, 1,3-propanedithiol, 2-hydroxypropane thiol, 1-mercapto-2-propanol, dithioerythritol, dithiothritol, alpha-lipoic acid, methanethiol (CH ₃ SH [m-mer Captan]), ethanethiol (C ₂ H ₅ SH [e-mercaptan]), 1-propanethiol (C ₃ H ₇ SH [nP 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]), pentanethiol (C ₅ H ₁₁ SH [pentyl mercaptan]), coenzyme A, lipoamide, glutathione, cysteine, cystine, 2-mercaptoethanol, dithiothritol, dithioerythritol, 2-mercaptoindole, transgluta Minase, (11-mercaptodecyl) hexa (ethylene glycol), (11-mercaptodecyl) tetra (ethylene glycol), (11-mercaptodecyl) Tra (ethylene glycol) functionalized gold nanoparticles, 1,1 ', 4', 1 "-terphenyl-4-thiol, 1,11-undecandithiol, 1,16-hexadecanedithiol, 1,2- Ethanedithiol technical grade, 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 furum, 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-octanethiol, 1-pentadecanthiol, 1-pentanethiol, 1-propanethiol, 1-tetradecanethiol, 1- Tetradecanethiol furum, 1-undecanethiol, 11- (1H-pyrrol-1-yl) undecane-1-thiol, 11-amino-1-undecantthiol hydrochloride, 11-bro -1-undecanthiol, 11-mercapto-1-undecanol, 11-mercapto-1-undecanol, 11-mercaptodecanoic acid, 11-mercaptodecanoic acid, 11-mercaptoundecyl trifluoro Acetate, 11-mercaptodecylphosphate, 12-mercaptododecanoic acid, 12-mercaptododecanoic acid, 15-mercaptopentadecanoic acid, 16-mercaptohexadecanoic 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 furum, 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-functionalized silica gel, 3-methyl-1-butanethiol, 4,4'-bis (mercaptomethyl) biphenyl, 4,4'-dimercaptostilbene, 4 -(6-mercaptohex Oxy) 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, decanthiol 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 ^TM 18, NanoThinks ^TM 8, NanoThinks ^TM ACID11, NanoThinks ^TM ACID16, NanoThinks ^TM ALC011, NanoThinks ^TM THI08, Octanethiol 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) thioace Tate, S- (4-cyanobutyl) thioacetate, thiophenol, triethylene glycol mono-11-mercaptoundecyl ether, trimethylolpropane tris (3-mercaptopropionate), [11- (methylcarbo Nylthio) Undecyl] tetra (ethylene glycol), m-carborane-9-thiol, p-terphenyl-4,4 "-dithiol, tert-dodecyl mercaptan, tert-nonyl mercaptan A method comprising one or more agents. (i) prevention of surface infection by bacterial, fungal or viral pathogens,
(ii) inhibiting cell viability or cell growth of substantially all planktonic cells of bacterial, fungal or viral pathogens,
(iii) inhibition of biofilm formation by bacterial, fungal or viral pathogens, and
(iv) a volume average diameter of substantially all microparticles of from about 0.4 μm to about 5 under conditions sufficient for one or more of the biofilm viability or suppression of biofilm growth of substantially all biofilm-forming cells of a bacterial, fungal or viral pathogen. A natural surface is exposed to at least one of bacterial pathogens, fungal pathogens and viral pathogens, including contacting the natural surface with an effective amount of a BT composition comprising a plurality of particulates comprising a bismuth-thiol (BT) compound, which is μm. How to protect. The method of claim 13,
The viral pathogen is selected from (i) one or more Gram-negative bacteria;
(ii) one or more Gram-positive bacteria;
(iii) one or more antibiotic-sensitive bacteria;
(iv) one or more antibiotic-resistant bacteria;
(v) Staphylococcus aureus [S. S. aureus], MRSA (methicillin-resistant, S. aureus), Staphylococcus epidermidis, MRSE (methicillin-resistant S. epidermidis), My Cobacterium tuberculosis, Mycobacterium avium, Pseudomonas aeruginosa, drug-resistant blood. Aeruginosa, Escherichia coli, Enterotoxin production. Coli, intestinal bleeding E. coli, Klebsiella pneumoniae, Clostridium difficile, Heliobacter pylori, Legionella pneumophila, Enterococcus faecalis, Enterococcus faecalis Methicillin-sensitive Enterococcus faecalis, Enterobacter cloacae, Salmonella typhimurium, Proteus vulgaris, Yersica co. , Vibrio cholera, Shigella flexneri, Vancomycin-resistant Enterococcus (VRE), Burkholderia cepacia complex, Francisca tullarensis (Francisella tularensis), Bacillus anthracis, Yersinia pestis, Pseudomonas Pseudomonas aeruginosa, Streptococcus pneumonia, Penicillin-resistant Streptococcus pneuumonia, Escherichia coli, Burkholderia cepacia, Bucoldecia cepacia A method comprising at least one bacterial pathogen selected from the group consisting of Bukholderia multivorans, Mycobacterium smegmatis and Acinetobacter baumannii. The method of claim 13, wherein said bacterial pathogen is antibiotic resistant. The method of claim 13, wherein said bacterial pathogen is resistant to antibiotics selected from the group consisting of methicillin, vancomycin, napicillin, gentamicin, ampicillin, chloramphenicol, doxycycline, and tobramycin. The method of claim 13, wherein the surface comprises an epithelial tissue surface selected from epithelium, skin, respiratory tract, gastrointestinal tract, and gland lining. The method of claim 13, wherein said contacting step is performed once or multiple times. 19. The method of claim 18, wherein at least one step of contacting comprises one of spraying, perfusion, dipping and painting the natural surface. The method of claim 18, wherein the at least one contacting step comprises one of inhalation, digestion and oral perfusion. 19. The method of claim 18, wherein at least one contacting step is performed locally, intraperitoneally, orally, parenterally, intravenously, intraarterally, percutaneously, sublingually, subcutaneously, intramuscularly, intranasally, intranasally. And administering to the subject by inhalation, intraocularly, atrium, intraventricularly, subcutaneously, adipose, intraarticular and intradural. The composition of claim 13, wherein 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 A propanol, and at least one BT compound selected from the group consisting of bis-EDT / 2-hydroxy-1-propanthiol. The method of claim 13, wherein said bacterial pathogen is antibiotic resistant. 24. The method of any of claims 13 to 23, wherein the surface is associated with at least one of (i) a synergistic antibiotic and (ii) a cooperative antimicrobial potentiating antibiotic, and contacting the surface with a BT composition. Contacting simultaneously or sequentially and in any order. The method of claim 24, wherein the synergistic antibiotic or cooperative antimicrobial potentiating antibiotic is an aminoglycoside antibiotic, carbapenem antibiotic, cephalosporin antibiotic, fluoroquinolone antibiotic, glycopeptide antibiotic, lincosamide antibiotic, A penicillinase-resistant penicillin antibiotic and an aminopenicillin antibiotic comprising a antibiotic selected from the group consisting of antibiotics. The method of claim 25, wherein the synergistic antibiotic or the cooperative antimicrobial potentiating antibiotic is amikacin, arbecasin, gentamicin, kanamycin, neomycin, netylmycin, paromomycin, rhostreptomycin, streptoto The aminoglycoside antibiotic selected from the group consisting of mycin, tobramycin and apramycin. (i) prevention of infection of epithelial tissue surfaces by bacterial pathogens,
(ii) inhibition of cell viability or cell growth of substantially all planktonic cells of bacterial pathogens,
(iii) inhibition of biofilm formation by bacterial pathogens, and
(iv) under conditions sufficient for at least one of the biofilm viability or suppression of biofilm growth of substantially all biofilm-forming cells of the bacterial pathogen, and for a sufficient time, the volume average diameter of substantially all of the microparticles is from about 0.4 microns to about 5 microns. Can synergize or synergize with (1) at least one bismuth-thiol (BT) composition and (2) at least one BT composition, comprising a plurality of particulates comprising a bismuth-thiol (BT) compound A method of overcoming antibiotic resistance on a natural surface in which antibiotic-resistant bacterial pathogens are present, comprising contacting the epithelial tissue surface with an effective amount of at least one antibiotic present at the same time or continuously and in any order. The method of claim 27, wherein the bacterial pathogen is Staphylococcus aureus (S. aureus), MRSA (methicillin-resistant, S. aureus), Staphylococcus epidermidis, MRSE (methi) Cilin-resistant S. epidermidis), Mycobacterium tuberculosis, Mycobacterium avium, Pseudomonas aeruginosa, drug-resistant blood. Aeruginosa, Escherichia coli, Enterotoxin production. E. coli, intestinal bleeding E. coli, Klebsiella pneumoniae, Clostridium difficile, Helicobacter phyllori, Legionella pneumophila, Enterococcus paecalis, Methicillin-sensitive enterococcus paecalis, Enterobacter clocae, Salmonella typhimurium , Proteus vulgaris, Yersinia enterocytica, Vibrio cholera, Shigella flexneri, Vancomycin-tolerant enterococcus (VRE), Burcolderia Sephacia complex, Francisla Tularensis, Bacillus anthracis, Yersinia Festis, Pseudomonas aeruginosa, Streptococcus pneumoniae, Penicillin-resistant Streptococcus pneumoniae, Escherichia coli, Burcolderia Sephacia, Bucoldria multiborans, Mycobacterium smeg Method selected from the group consisting of matis and axinetobacter baumannini. The method of claim 27, wherein said bacterial pathogen is resistant to antibiotics selected from the group consisting of methicillin, vancomycin, napicillin, gentamicin, ampicillin, chloramphenicol, doxycycline, tobramycin, clindamycin and gatifloxacin. Way. The method of claim 27, wherein said surface comprises an epithelial surface of a tissue selected from the group consisting of epithelium, skin, respiratory tract, gastrointestinal tract, and gland lining. The method of claim 27, wherein said contacting step is performed once or multiple times. 32. The method of claim 31, wherein at least one step of contacting comprises one of spraying, perfusion, dipping and painting the natural surface. The method of claim 31, wherein the at least one contacting step comprises one of inhalation, digestion and oral perfusion. The method of claim 31, wherein at least one contacting step is performed locally, intraperitoneally, orally, parenterally, intravenously, intraarterally, percutaneously, sublingually, subcutaneously, intramuscularly, intracranially, intranasally. And administering to the subject by inhalation, intraocularly, atrium, intraventricularly, subcutaneously, adipose, intraarticular and intradural. The composition of claim 27 wherein 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 A propanol, and at least one BT compound selected from the group consisting of bis-EDT / 2-hydroxy-1-propanthiol. 36. The method of claim 35, wherein the synergistic or enhancing antibiotic is clindamycin, gatifloxacin, aminoglycoside antibiotics, carbapenem antibiotics, cephalosporin antibiotics, fluoroquinolone antibiotics, penicillinase-resistant penicillin antibiotics, and A method comprising an antibiotic selected from the group consisting of aminophenicillin antibiotics. 37. The method of claim 36, wherein the synergistic or enhancing antibiotics are amikacin, arbecasin, gentamicin, kanamycin, neomycin, netylmycin, paromomycin, rhostreptomycin, streptomycin, tobramycin and suba. The aminoglycoside antibiotic selected from the group consisting of pramycin. (a) at least one BT composition comprising a plurality of microparticles comprising a bismuth-thiol (BT) compound having a volume average diameter of substantially all of the microparticles; And (b) at least one antibiotic compound that can synergize with, or enhance with, a BT compound. The method of claim 38, wherein 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 A composition selected from the group consisting of -propanol, and bis-EDT / 2-hydroxy-1-propanthiol. The composition of claim 38, wherein the BT compound is selected from the group consisting of BisEDT and BisBAL. The antibiotic according to claim 38, wherein the antibiotic compound is selected from methicillin, vancomycin, napicillin, gentamicin, ampicillin, chloramphenicol, doxycycline, tobramycin, clindamycin, gatifloxacin, cefazoline and aminoglycoside antibiotics. Composition comprising a. 42. The method of claim 41, wherein the aminoglycoside antibiotics are amikacin, arbecasin, gentamicin, kanamycin, neomycin, netylmycin, paromomycin, rhostreptomycin, streptomycin, tobramycin and apramycin. Composition selected from the group consisting of. 42. The composition of claim 41, wherein said aminoglycoside antibiotic is amikacin. (a) identifying a bacterial infection on or within the natural surface as comprising one of (i) Gram positive bacteria, (ii) Gram negative bacteria, and (iii) both (i) and (ii); And
(b) treating a natural surface containing bacterial biofilm, comprising administering a formulation comprising at least one bismuth thiol (BT) composition to the natural surface, wherein (i) the bacterial infection is Gram If it comprises a positive bacterium, the formulation comprises an effective amount of at least one BT compound and at least one antibiotic that is rifamycin, and (ii) if the bacterial infection comprises gram negative bacteria, the formulation contains at least one effective amount of Wherein the formulation comprises a therapeutically effective amount of one or multiple BT compounds, rifamycin and amikacin, if the bacterial infection comprises both gram positive and gram negative bacteria. Method of treating natural surfaces containing biofilms. 45. The method of claim 44, wherein said bacterial infection comprises one or a plurality of antibiotic-resistant bacteria. 46. The method of claim 45, wherein the treatment comprises (i) eradication of bacterial biofilms; at least one of (ii) reducing bacterial biofilm and (iii) impairing growth of bacterial biofilm. 47. The plurality of microparticles according to any one of claims 44 to 46, wherein the BT composition comprises a bismuth-thiol (BT) compound wherein the volume average diameter of substantially all of the microparticles is from about 0.4 microns to about 5 microns. How to include.

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