WO2022150657A1 - Therapeutic thiazine dye compositions and methods of use - Google Patents

Therapeutic thiazine dye compositions and methods of use Download PDF

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Publication number
WO2022150657A1
WO2022150657A1 PCT/US2022/011718 US2022011718W WO2022150657A1 WO 2022150657 A1 WO2022150657 A1 WO 2022150657A1 US 2022011718 W US2022011718 W US 2022011718W WO 2022150657 A1 WO2022150657 A1 WO 2022150657A1
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Prior art keywords
therapeutic composition
wound
composition
phosphoric acid
infection
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PCT/US2022/011718
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French (fr)
Inventor
Alena McALLISTER
Original Assignee
AB BioInnovations, Inc.
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Publication date
Priority claimed from US17/242,185 external-priority patent/US20220168317A1/en
Application filed by AB BioInnovations, Inc. filed Critical AB BioInnovations, Inc.
Publication of WO2022150657A1 publication Critical patent/WO2022150657A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0043—Nose
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/5415—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with carbocyclic ring systems, e.g. phenothiazine, chlorpromazine, piroxicam
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/42—Phosphorus; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/19—Cosmetics or similar toiletry preparations characterised by the composition containing inorganic ingredients
    • A61K8/24—Phosphorous; Compounds thereof
    • A—HUMAN NECESSITIES
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    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
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    • A61K8/731—Cellulose; Quaternized cellulose derivatives
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    • A61K9/0056—Mouth soluble or dispersible forms; Suckable, eatable, chewable coherent forms; Forms rapidly disintegrating in the mouth; Lozenges; Lollipops; Bite capsules; Baked products; Baits or other oral forms for animals
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    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A61K9/0078—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy for inhalation via a nebulizer such as a jet nebulizer, ultrasonic nebulizer, e.g. in the form of aqueous drug solutions or dispersions
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    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
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    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
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    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
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    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
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    • A—HUMAN NECESSITIES
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
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    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
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    • A61L2300/442—Colorants, dyes

Definitions

  • the present invention relates to therapeutic thiazine dye compositions and methods of use and more particularly to bactericidal, antiviral, anti-inflammatory, and regenerative compositions including methylene blue and phosphoric acid and its salts.
  • nosocomial infections also known as nosocomial infections, are infections acquired in a hospital or other health care facility and are potentially caused by organisms that are resistant to antibiotics. Nosocomial infections represent the tenth major course of death in the United States. The annual economic impact of nosocomial infections is estimated to be in the range of 55-70 billion U.S. dollars.
  • compositions ameliorate the aforementioned shortcomings of current surgical procedures in a safe, non-toxic manner.
  • compositions comprising Dosage Forms. Uses and Methods
  • non-toxic therapeutic compositions and dosage forms formed from a mixture of a thiazine dye and phosphoric acid in aqueous solution. This mixture can be further processed into a variety of pharmaceutical and surgical dosage forms where the misture is either remains in aqueous solution or has been dried and will subsequently redissolve in water or body fluids.
  • Therapeutic compositions and dosage forms of the present disclosure exhibit surprising bactericidal, antiviral, anti-inflammatory, and regenerative properties, and stop bleeding on the site of application.
  • a therapeutic composition comprising 1.0 x 10 7 2.5 x 10 1 % (w/w) thiazine dye, and 1.0 x 10 5 2 0 (W/W) phosphoric acid.
  • the thiazine dye is methylene blue.
  • a method of treating a subject in need thereof comprising causing the administration of the therapeutic composition comprising 1.0 x 10 7 2.5 x 10 1 % (w/w) thiazine dye, and 1.0 x 10 5 2 0 (w/w) phosphoric acid.
  • the subject may be a human or an animal.
  • the present antimicrobial and anti-inflammatory composition facilitates a safe, non-toxic surgical procedure for amelioration of wound infection and inflammation, as well as enhances the ability to close a wound after a surgical procedure.
  • the composition increases bone density and new bone formation as well as promotes angiogenesis at the surgical site.
  • the antimicrobial composition comprises an aqueous solution of phosphoric acid (H3PO4) and an antimicrobial thiazine dye in a bioabsorbable sponge.
  • the phosphoric acid concentration in the sponge is in the range of about 0.1 percent to about 40 percent by weight of the solution depending on how the phosphoric acid-containing sponge is used.
  • the sponge for initial cleaning of a wound, contains phosphoric acid at a concentration of about 5 to about 40 percent by weight of the aqueous solution.
  • the sponge for applications when the bioabsorbable sponge is left in a closed wound, contains phosphoric acid in an amount in the range of about 0.1 percent by weight to about 5 percent by weight based on the weight of the solution. In various aspects, the sponge contains phosphoric acid in an amount in the range of about 0.1 percent by weight to about 2.5 percent by weight based on the weight of the solution. In various aspects, the sponge contains phosphoric acid in an amount in the range of about 0.75 to about 2.5 percent by weight of the solution
  • the bioabsorbable sponge contains the aqueous phosphoric acid solution in an amount in the range of about 5 to about 40 percent by weight, based on the weight of the bioabsorbable sponge.
  • antimicrobial thiazine dye is present in the aqueous phosphoric acid solution in an amount in the range of about 0.0001 percent by weight to about 1 percent by weight. In various aspects, antimicrobial thiazine dye is present in the aqueous phosphoric acid solution in an amount in the range of about 0.1 to about 1 percent by weight, based on the weight of the aqueous phosphoric acid solution. In various aspects, the thiazine dye is methylene blue.
  • the bioabsorbable sponge comprises gelatin. In various aspects, the bioabsorbable sponge comprises collagen. In various aspects, the aqueous solution contains a biocompatible thickening agent. In various aspects, the biocompatible thickening agent is carboxymethyl cellulose.
  • an antiviral textile material that can be converted into wearing apparel such as face masks, turtle necks, scarves, bandanas, and other partial or full body coverings is described.
  • the antiviral textile material has a hydrophilic fibrous substrate.
  • a thiazine dye such as methylene blue, and phosphoric acid.
  • the thiazine dye and phosphoric acid are present in equimolar amounts.
  • the thiazine dye is distributed in an amount in the range of 0.001 to about 1 grams per cubic centimeter of the substrate.
  • the phosphoric acid is distributed in an amount in the range of about 0.001 to about 1 grams per cubic centimeter of the substrate.
  • the patent or application file contains at least one drawing executed in color.
  • Fig. 1 shows the volume of tissue infection over time of rats infected in muscle and bone tissue with E. coli bacteria and treated once with a combination of methylene blue and phosphoric acid as described herein.
  • FIG. 2 shows the volume of tissue infection over time of rats infected in muscle and bone tissue with P. aeruginosa bacteria and treated once with a composition comprising methylene blue and phosphoric acid as described herein.
  • Fig. 3 A shows the volume of tissue infection over time of rats infected in muscle and bone tissue with MRSA bacteria and treated once with a composition comprising methylene blue and phosphoric acid as described herein.
  • Fig. 3B shows the concentration of C-reactive protein in the MRSA infected rats.
  • Fig. 4 shows the surgical site swelling, as assessed by hind leg humoral width near the site of infection by intramuscular injection of P.
  • Fig. 5A shows a histology image of soft tissue of a rat infected with MRSA and not further treated.
  • Fig. 5B shows a histology image of muscle tissue of a rat infected with MRSA for 48 hours and treated with a combination of 0.5% methylene blue and 35% phosphoric acid.
  • Fig. 5C shows a histology image of muscle tissue of a rat infected with MRSA and not further treated.
  • Fig. 5D shows a histology image of bone and soft tissue of a rat infected with MRSA for 48 hours and treated with a combination of 0.5% methylene blue and 35% phosphoric acid.
  • Fig. 6A shows an MRI image of an infection site in soft tissue of a rat 48 hours after injection of MRSA.
  • Fig. 6B shows an MRI image of the same infection site as in Fig. 6A at 10 days after treatment with a combination of 0.5% methylene blue and 35% phosphoric acid.
  • Fig. 6C shows an MRI image of the same infection site as in Fig. 6A at 30 days after treatment with a combination of 0.5% methylene blue and 35% phosphoric acid.
  • Fig. 7A shows a histomorphometry image of a young rat of a calcein and tetracycline stain showing new bone formation after bone injury and treatment with a combination of 0.15% (w/w) methylene blue and 0.33% (w/w) phosphoric acid.
  • Fig. 7B shows a histomorphometry image of an elderly rat of a calcein and tetracycline stain showing new bone formation after bone injury and treatment with a combination of methylene blue and phosphoric acid.
  • Fig. 8A shows a PET scan image of a rat after bone injury and no further treatment.
  • Fig. 8B shows a PET scan image of a rat after bone injury and treatment with a combination of 0.15% (w/w) methylene blue and 0.33% (w/w) phosphoric acid.
  • Fig. 9 shows a traditional timeline for a patient receiving a dental implant (top) and a timeline for a patient receiving a dental implant and being treated once with a with a combination of methylene blue and phosphoric acid (bottom).
  • Fig. 10 shows a perspective view of a bandage utilizing a bioabsorbable sponge in accordance with an embodiment of the invention.
  • FIG. 11 shows a partially exploded perspective view of a face mask having a fibrous intermediate layer impregnated with a combination of methylene blue and phosphoric acid.
  • the term “about” is used herein to mean approximately, roughly, around, or in the regions of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” can modify a numerical value above and below the stated value by a variance of, e.g., 10 percent, up or down (higher or lower). In some aspects of the disclosure, the term “about” encompasses a deviation from the recited value of between 0.001% and 10%, inclusive of the endpoints. In some aspects, the term “about” encompasses an increase from the recited value of between 0.001% and 10%, inclusive of the endpoints. In some aspects, the term “about” encompasses a decrease from the recited value of between 0.001% and 10%, inclusive of the endpoints.
  • the terms “effective amount” and “therapeutically effective amount” refer to an amount of a composition of the disclosure that provides beneficial or desired therapeutic and/or prophylactic results.
  • beneficial or desired results can include, for example, one or more results such as eliminating or reducing the risk, lessening the severity, or delaying the onset of an infection, inflammation or bone degeneration, its complications, and intermediate pathological phenotypes presenting during development of the infection or bone degeneration.
  • beneficial or desired results can include, for example, one or more clinical results such as decreasing one or more symptoms and pathological conditions resulting from or associated with the infection or bone degeneration, increasing the quality of life of those suffering from the infection or bone degeneration, decreasing the dose of other medications required to treat the infection or bone degeneration, enhancing the effect of other medication such as via targeting, delaying the progression of the infection or bone degeneration, and/or prolonging survival.
  • An effective amount can be, for example, an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly.
  • an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an effective amount may be considered in the context of administering one or more therapeutic agents.
  • An effective amount can be administered in one dosage or can be divided into multiple dosages, the total of such dosages being the effective amount.
  • an effective amount can be provided in two separate administrations over a period of time, that in aggregate, provide the effective amount of the composition described herein.
  • Thiazine dyes include methylene blue, azure A, azure B, toluidine blue 0, thiamine, and the like.
  • a preferred thiazine dye is methylene blue (3,7- bis(dimethylamine) - phenothiazin-5-ium chloride.
  • Minimum inhibitory concentration as used herein shall mean the lowest concentration of a chemical or combination of chemicals, e.g., methylene blue, phosphoric acid, and combinations thereof, which prevents visible growth of a bacteria.
  • post-op shall mean the time period after the second surgery (treatment) described in Example 20 herein.
  • post-op shall mean the time period after the second surgery (treatment) described in Example 21 herein.
  • post-infection shall mean the time period after the first surgery described in Example 20 herein.
  • post-infection shall mean the time period after the first surgery described in Example 21 herein.
  • post-infection time is essentially equivalent to the post-op time plus 48 hours (because the second surgery took place 48 hours after the first surgery).
  • post-infection time is essentially equivalent to the post-op time plus 72 hours (because the second surgery took place 72 hours after the first surgery).
  • association product refers to any molecular structure resulting from a combination of methylene blue and phosphoric acid and includes ionic molecular structures, weak electrostatically bonded complexes, and covalently bonded complexes, optionally including at least one additional molecule.
  • association with one another refers to a reaction of one molecule of methylene blue with one molecule of phosphoric acid to form an association product.
  • topical cream refers to an emulsion of oil and water that is applied to a body surface including a skin or mucous membrane and is either low viscosity and includes a lotion or high viscosity and includes a gel.
  • gel refers to a non-fluid colloidal network or polymer network that is expanded throughout its whole volume by a fluid.
  • a “gel” can contain a covalent polymer network, e.g., a network formed by cross-linking polymer chains or by nonlinear polymerization.
  • a “gel” can contain a polymer network formed through the physical aggregation of polymer chains, caused by hydrogen bonds, crystallization, helix formation, and/or complexation that result in regions of local order acting as the network junction points.
  • the term “gel” includes a hydrogel in which the swelling agent is water.
  • emulsion refers to a fluid colloidal system in which liquid droplets and/or liquid crystals are dispersed in a liquid.
  • the continuous phase can be an aqueous phase or an organic lipid, e.g., an oil.
  • spray refers to a liquid that is dispersed in the form of small droplets.
  • bioabsorbable refers to capable of being absorbed into living tissue.
  • bioabsorbable sponge refers to a porous absorbent biocompatible, water-insoluble, pliable, absorbent material that, when implanted in a human or other mammalian body, is absorbed by the body.
  • textile material refers to a material made of interlacing fibers.
  • hydrophilic fibrous substrate refers to a substrate made from fibers that has a tendency to mix with or be wetted by water.
  • non-woven cotton refers to a fabric-like material made from short and long fibers bonded together by chemical, mechanical, heat or solvent treatment.
  • water-repellant cotton layer refers to a cotton layer that has a hydrophobic coating such that it repels water intrusion into the layer.
  • bandage refers to a cloth material that is used to cover a wound and can be used together with a wound dressing or on its own.
  • wound dressing refers to a material that is in direct contact with a wound and can be a cloth, a foam, a film, a hydrocolloid, a hydrogel, an alginate or a collagen.
  • bone site refers to a location on or within a bone that is accessible from the outside of a body because of a surgery or penetrating bone fracture or a location on or within a bone that is accessible by introduction of a needle.
  • a “dental bone site” is a bone site that is located in the oral cavity and is accessible through the mouth.
  • wound refers to a location on a body of an injury to the body that involves laceration or breaking of a membrane, e.g., a skin, and damage to a surrounding tissue.
  • open wound refers to a wound that is in direct contact with the atmosphere surrounding the body.
  • chronic wound refers to a wound that has not healed within a time frame reasonable for the healing of a wound in the body location, e.g., within 10 days for a wound located on the skin.
  • a chronic wound includes an ulcer, a diabetic lesion, an eczema, acne vulgaris, a chronic surgical wound, and a chronic infectious wound.
  • contacting refers to physical interaction of one thing with another, e.g., of an aqueous solution with a body, a wound, or a bone.
  • placing into refers to the introduction of a material into an opening created by a wound or injury in a tissue
  • administering refers to an action that introduces a composition to a subject, either internally or externally, e.g., by infusion, injection, inhalation, or creation of a direct contact between the composition and the subject or a portion of the subject.
  • regenerating a bone refers to an activity that results in a generation of bone tissue in an area of the body where bone tissue had previously been lost due to injury or osteoporosis.
  • fractured bone refers to a bone whose physiological structure has been interrupted.
  • osteoporotic bone refers to a bone whose density has been diminished and whose mechanical strength is reduced due to the diminished bone density.
  • subject refers to a mammal, including an animal and a human.
  • compositions disclosed herein comprise one or more thiazine dyes and phosphoric acid, or salts thereof.
  • the thiazine dye is methylene blue. It was surprisingly found that therapeutic compositions comprising methylene blue and phosphoric acid work to produce bactericidal, antiviral, anti-inflammatory, and regenerative effects that are not observed in compositions containing one of either methylene blue or phosphoric acid, but not both.
  • compositions comprising methylene blue and phosphoric acid promote fast onset of cytogenesis, potent angiogenesis, complete cytological osteogenesis, promotion of physiological ossification, and mobilization of hydroxyapatite to the site of new bone growth, accelerate the extrinsic pathway of coagulation and control surgical bleeding.
  • the therapeutic compositions provided herein comprise a thiazine dye, phosphoric acid and/or an association product thereof. [0075] In some aspects, the therapeutic compositions comprise 1.0 x 10 7 -2.5 x 10 1 %
  • the therapeutic compositions comprise 1.0 x 10 6 — 1 x 10 4 % (w/w) thiazine dye and 1.0 x 10 4-1.0 x 10 2 % (w/w) phosphoric acid.
  • the therapeutic composition comprise a thiazine dye in the range of about 1.0 x 10 7 % (w/w) to about 1 % (w/w), about 5.0 x 10 7 % (w/w) to about 1 % (w/w), about 1.0 x 10 6 % (w/w) to about 1 % (w/w), about 5.0 x 10 6 % (w/w) to about 1 % (w/w), about 1.0 x 10 5 % (w/w) to about 1 % (w/w), about 5.0 x 10 5 % (w/w) to about 1 % (w/w), about 1.0 x 10 4 % (w/w) to about 1 % (w/w), about 5.0 x 10 4 % (w/w) to about 1 % (w/w), about 5.0 x 10 4 % (w/w) to about 1 % (w/w), about 0.001 % (w/w) to about 1 % (w/w), about
  • the therapeutic composition comprise a thiazine dye in the range of about 1.0 x 10 7 % (w/w) to about 1 % (w/w), and phosphoric acid in the range of about 1.0 x 10 5 % (w/w) to about 40 % (w/w), about 5.0 x 10 5 % (w/w) to about 37 % (w/w), about 1.0 x 10 4 % (w/w) to about 35 % (w/w), about 5.0 x 10 4 % (w/w) to about 30 % (w/w), about 0.001 % (w/w) to about 25 % (w/w), about 0.005 % (w/w) to about 20 % (w/w), about 0.01 % (w/w) to about 15 % (w/w), about 0.02 % (w/w) to about 10 %
  • the therapeutic composition comprise a thiazine dye in the range of about 0.01% (w/w) to about 3% (w/w), about 0.1% (w/w) to about 2% (w/w), about 0.3 % (w/w) to about 1.5 % (w/w), about 0.5% (w/w) to about 1 % (w/w) and phosphoric acid in the range of about 0.01% (w/w) to about 3% (w/w), about 0.1% (w/w) to about 2% (w/w), about 0.3 % (w/w) to about 1.5 % (w/w), about 0.5% (w/w) to about 1%
  • the thiazine dye is methylene blue.
  • association product is bis ammonium salt of formula I
  • the thiazine dye is blue, azure A, azure B, toluidine blue 0, thiazine, and the like.
  • the therapeutic compositions further comprise water as solvent.
  • the therapeutic compositions are in form of an aqueous solution.
  • the aqueous solution comprises a biocompatible thickening agent.
  • the thickening agent is sodium carboxymethyl cellulose.
  • therapeutic compositions comprising thiazine dye and phosphoric acid can be, e.g., a mouthwash, a nasal spray, a lozenge, an inhalable therapeutic composition, a topical cream, an emulsion, a gel, or lotion, or an acute or chronic wound treatment liquid, gel, spray.
  • the aqueous solution can be further processed to form a bandage or wound dressing.
  • wound dressing is a cloth, a foam, a film, a hydrocolloid, a hydrogel, an alginate or a collagen.
  • therapeutic compositions comprising thiazine dye and phosphoric acid can be used in first aid applications for open wounds as the therapeutic compositions provided herein are antibacterial, antiviral, antifungal, anti-parasitic and/or anti-inflammatory and stop bleeding at the site of application and promote healing.
  • therapeutic compositions comprising thiazine dye and phosphoric acid may be used in conjunction with wound dressings such as band aids, surgical sutures, wound gauzes, surgical sponges (e.g., bioabsorbable sponges), and the like.
  • wound dressings may be pretreated with therapeutic compositions comprising thiazine dye and phosphoric acid.
  • compositions comprising thiazine dye and phosphoric acid can be administered prophylactically in order to reduce the risk of infection by microbes, such as bacteria, viruses, fungi and parasites.
  • prophylactic administration of compositions comprising thiazine dye and phosphoric acid protects the airways (e.g., nasal passages, sinuses, bronchial tubes, and/or lungs) and/or mouth and/or throat of a subject from infection and inflammation as the composition has no cell toxicity. .
  • compositions comprising thiazine dye and phosphoric acid can be administered in order to treat microbial infection and/or inflammation of the airways, mouth and/or throat of a subject.
  • compositions comprising thiazine dye and phosphoric acid can be administered in order to promote regeneration of damaged tissue and/or a bone, increase bone density at the site of application, and induce new bone formation.
  • therapeutic compositions comprising thiazine dye and phosphoric acid may contain one or more essential oils, dried or fresh fruit particles, suspending agents, surfactants, emollients, emulsifiers, and/or cationic polymers, e.g., as disclosed in US Publication No. 2005/0031573, which is incorporated by reference herein in its entirety.
  • the present antimicrobial and anti-inflammatory bioabsorbable sponge composition can be used in conjunction with a wide variety of human and veterinary surgical interventions such as hemostasis, detection of bacterial infection, enhancement of soft tissue healing, enhancement of bone density and/or bone formation at the surgical site, enhancement of angiogenesis at the surgical site, and the like.
  • the present bioabsorbable sponge can be also used as a foam layer in a bandage or antibacterial and anti-inflammatory dressing for treatment of chronic, non-healing wounds such as chronic ulcers, diabetic lesions, and the like.
  • the present bioabsorbable sponge can be also used as first aid in remote or battlefield regions, as well as for post extraction wound care in dentistry, for bacterial detection in wounds, and the like.
  • the bioabsorbable sponge is non-immunogenic.
  • Illustrative bioabsorbable sponges can have a gelatin matrix, a collagen matrix, a poly-L-lactic acid matrix, a polyglycolic matrix, a poly(lactic-co-glycolic)acid matrix and the like. Particularly well suited for present purposes is a bioabsorbable sponge commercially available under the designation GEL-FOAM® from Pfizer, Inc. as a water- insoluble, porous, pliable product prepared from purified pork skin gelatin USP. [0094] In some aspects, the bioabsorbable sponge is impregnated with an aqueous phosphoric acid solution.
  • the aqueous phosphoric acid solution constitutes about 0.1 to about 40 percent by weight of the aqueous solution. In some aspects, the aqueous phosphoric acid solution constitutes about 0.1 to about 2.5 percent by weight of the aqueous solution. In some aspects, the aqueous phosphoric acid solution constitutes about 5 to about 40 percent by weight of the sponge. In some aspects, the aqueous phosphoric acid solution constitutes about 10 to about 30 percent by weight of the sponge.
  • the aqueous phosphoric acid solution contains a thiazine dye, such as methylene blue, azure A, azure B, toluidine blue 0, thianine, and the like.
  • a preferred thiazine dye is methylene blue (3,7-bis(dimethylamine)-phenothiazin-5-ium chloride).
  • the thiazine dye especially methylene blue, exerts anti microbial activities.
  • the bioabsorbable sponge is impregnated with an aqueous solution comprising a thiazine dye and phosphoric acid.
  • the bioabsorbable sponge is impregnated with an aqueous solution comprising methylene blue and phosphoric acid.
  • the thiazine dye is present in the aqueous phosphoric acid solution of the bioresorbable composition in an amount in the range of about 0.0001 to about 1 weight percent, based on the weight of the solution. In various aspects, the thiazine dye is present in the aqueous phosphoric acid solution of the bioresorbable composition in an amount in the range of about 0.1 to about 1 weight percent, based on the weight of the solution. In various aspects, the thiazine dye is present in the aqueous phosphoric acid solution of the bioresorbable composition in an amount in the range of about 0.01 to about 0.1 weight percent, based on the weight of the solution.
  • the thiazine dye is released from the bioabsorbable sponge to a surrounding tissue as the bioresorbable sponge is liquefied and absorbed by the surrounding tissue.
  • the thiazine dye serves as a color indicator for bacteria present in a wound because the bacteria will be stained dark or black by the thiazine dye.
  • tissues containing bacteria can be surgically removed before a surgical procedure is performed and/or before a wound is closed.
  • methylene blue is present in the aqueous phosphoric acid solution of the bioresorbable composition in an amount in the range of about 0.0001 to about 1 weight percent, based on the weight of the solution.
  • methylene blue is present in the aqueous phosphoric acid solution of the bioresorbable composition in an amount in the range of about 0.1 to about 1 weight percent, based on the weight of the solution. In some aspects, methylene blue is present in the aqueous phosphoric acid solution of the bioresorbable composition in an amount in the range of about 0.01 to about 0.1 weight percent, based on the weight of the solution. In some aspects, methylene blue is released from the bioabsorbable sponge to a surrounding tissue as the bioresorbable sponge is liquefied and absorbed by the surrounding tissue,
  • the bioresobable sponge comprises gelatin.
  • the bioresobable sponge comprises collagen.
  • the bioresobable sponge comprises poly-L-lactic acid. In some aspects, the bioresobable sponge comprises poly-glycolic acid. In some aspects, the bioresobable sponge comprises poly(lactic-co-glycolic)acid,
  • compositions disclosed herein are used for wound pretreatment.
  • a biocompatible thickener can be added to an aqueous acid solution and the phosphoric acid content in the solution can be in the range of about 20 to about 40 weight percent, based on the weight of the solution.
  • a thickened aqueous phosphoric acid solution has a viscosity of about 100 to about 2000 contipoises (cp).
  • the thickener is sodium carboxymethyl cellulose having an average degree of substitution of about 21 % to about 33%.
  • the term "apparel” means textile wearing apparel positionable over the mouth and nasal passages of a human, e.g., a mask, a turtle neck, a scarf, a bandana, and the like, as well as a full or partial body covering.
  • the term "textile” means a fibrous artifact made by weaving, felting or crocheting natural fibers, synthetic fibers, and mixtures thereof.
  • a textile material suitable as a substrate for the thiazine dye and phosphoric acid is hydrophilic and fibrous, and sufficiently air permeable or porous to permit a human to breathe therethrough.
  • the textile material can be woven material or a non-woven material.
  • Preferred are non-woven materials made of cotton, cellulose, polypropylene, and the like, having a density of about 20 to 25 grams per square meter, and filtering particles having a size of 1 micron and larger.
  • the textile material filters particles having a size of 0.3 microns and larger.
  • the amount of thiazine dye distributed within the fibrous substrate of the textile material is in the range of about 0.001 to about 1 grams per cubic centimeter (cm 3 ) of the substrate. In various aspects, the amount of thiazine dye distributed within the fibrous substrate of the textile material is in the range of about 0.1 to about 0.5 grams per cubic centimeter of the substrate.
  • the amount of phosphoric acid distributed within the fibrous substrate of the textile material is in the range of 0.001 to about 1 grams per cubic centimeter of the substrate.
  • an equimolar amount of thiazine dye and phosphoric acid is present in the fibrous substrate.
  • the thiazine dye may be methylene blue.
  • face mask 10 has an outer layer 12 made of water repellant cotton, an intermediate layer 14 made of non-woven cotton, an inner layer 16 made of silk. Intermediate layer 14 is hydrophilic and provides a fibrous substrate into which methylene blue and phosphoric acid are distributed. Aperture 18 in outer layer 12 is provided for attachment of an appropriate securing strap.
  • the antiviral textile material is moistened by exhaled moisture. In some instances, especially when ambient humidity is relatively low, it is desirable to moisten the facial covering. This can be achieved by applying a mist or spray of water to the facial covering prior to use.
  • an antiviral solution containing a thiazine dye and phosphoric acid can be applied to the facial covering prior to use or at intervals during use, depending on ambient conditions.
  • the antiviral solution suitable for this purpose contains about 0.1 to about 5 weight percent of phosphoric acid based on the weight of the antiviral solution. In various aspects, the antiviral solution contains about 0.75 to about 2.5 weight percent based on the weight of the antiviral solution.
  • the antiviral thiazine dye is present in the aqueous solution in an amount in the range of about 0.01 to about 1 weight percent based on the weight of the antiviral solution.
  • the textile material comprises a hydrophilic fibrous substrate that is located between an outer water-repellant cotton layer and an inner silk layer.
  • the outer water-repellant cotton layer retains exhaled moisture.
  • composition comprising methylene blue, phosphoric acid and its association product induce a fast onset of cytogenesis, potent angiogenesis, complete osteogenesis, physiological ossification, mobilization of hydroxyapatite to a site of new bone formation, acceleration of the extrinsic pathway of coagulation, stimulation of bone and soft tissue bio regeneration and control of bleeding.
  • the above effects were observed after single administration of a composition comprising methylene blue, phosphoric acid and its association product to a wound or surgery site.
  • compositions described herein can be used for the treatment of bacterial, viral, fungal, and parasitic infections, for the treatment of a wound, for regenerating bone, and for prevention of an infection, e.g., a respiratory infection.
  • the therapeutic compositions are used for the treatment of a bacterial infection.
  • the therapeutic composition is used for the treatment of an acute infection or a chronic infection.
  • the infection is an infection of a body surface, e.g., a skin or mucous membrane.
  • the infection is an infection of a surgical site.
  • the infection is an infection of a fractured bone.
  • the infection is an infection of the respiratory system.
  • the infection is a bacterial infection and is caused by is infected with Acinetobacter baumannii, Actinomyces sp., Actinobacillus actinomycetemcomitans, Aeromonas sp., Anaplasma phagocytophilum, Alcaligenes xylosoxidans, Actinobacillus actinomycetemcomitans, Bacillus sp., Bacteroides sp., Bartonella sp.
  • Bifidobacterium sp. Bordetella sp., Borrelia sp., Brucella sp, Burkholderia sp., Campylobacter sp., Capnocytophaga sp., Cardiobacterium hominis, Chlamydia trachomatis, Chlamydophila pneumonia, Chlamydophila psittaci, Citrobacter sp.
  • the infection is a viral infection and is caused by an Adenovirus
  • Bocavirus Coronavirus, Enterovirus, Metapneumovirus, Parainfluenza virus 1-4, Respiratory Syncytial Virus A and B, Rhinovirus, Herpesvirus, Varicella zoster virus, Epstein-Barr Virus, Influenza, human Parechovirus, Measles virus, Middle East Respiratory Syndrome coronavirus (MERS CoV), Mumps virus, or Severe Acute Respiratory Syndrome Coronavirus (SARS CoV).
  • MERS CoV Middle East Respiratory Syndrome coronavirus
  • SARS CoV Severe Acute Respiratory Syndrome Coronavirus
  • the infection is a fungal infection and is caused by Candida albicans, C. glabrata, C. dubliniensis, C. krusei, C. parapsilosis, C.tropicalis, C. orthopsilosis, C. guilliermondii, C. rugosa, C. auris, C. lusitaniae, Aspergillus fumigatus, A. flavus, A. terreus, A. niger, A. candidus, A. clavatus, or A. ochraceus.
  • the infection is a parasitic infection and is caused by
  • the therapeutic composition is administered by direct contact of a body surface, a surgical site and/or a fractured bone.
  • the therapeutic composition is administered by direct contact of a body surface, a surgical site and/or a fractured bone with an aqueous solution of the therapeutic composition.
  • the therapeutic composition is administered by direct contact of a body surface, a surgical site and/or a fractured bone with a bioabsorbable composition. In some aspects, the therapeutic composition is administered by direct contact of a body surface, a surgical site and/or a fractured bone with a sponge containing an aqueous solution of the therapeutic composition.
  • the therapeutic composition is administered by direct contact of a body surface, a surgical site and/or a fractured bone with a textile material containing an aqueous solution of the therapeutic composition.
  • the therapeutic composition is administered as a lotion, gel, topical cream, spray, spray.
  • the therapeutic composition is injected into an infected tissue or organ. In some aspects, the therapeutic composition is injected into an infected surgical site and/or a fractured bone.
  • the therapeutic composition is administered by inhalation.
  • the therapeutic composition is administered at a concentration of methylene blue of 1.0 x 10 7 — 1.0 % (w/w), and a concentration of phosphoric acid of 1.0 x 10 5 -2.0 % (w/w).
  • the therapeutic composition is administered at a concentration of methylene blue of 1.0 x 10 7 0.25 % (w/w), and a concentration of phosphoric acid of 1.0 x 10 5 -2.0 % (w/w).
  • the therapeutic composition is administered as an aqueous solution with a concentration of methylene blue of 1.0 x 10 7 — 1.0 % (w/w), and a concentration of phosphoric acid of 1.0 x 10 5 -2.0 % (w/w).
  • the therapeutic composition is administered as an aqueous solution with a concentration of methylene blue of 1.0 x 10 7 -0.25 % (w/w), and a concentration of phosphoric acid of 1.0 x 10 5 -2.0 % (w/w).
  • the therapeutic composition is administered as a topical cream with a concentration of methylene blue of 1.0 x 10 7 — 1.0 % (w/w), and a concentration of phosphoric acid of 1.0 x 10 5 -2.0 % (w/w).
  • the therapeutic composition is administered as a topical cream with a concentration of methylene blue of 1.0 x 10 7 -0.25 % (w/w), and a concentration of phosphoric acid of 1.0 x 10 5 -2.0 % (w/w).
  • the therapeutic composition is administered as a gel with a concentration of methylene blue of 1.0 x 10 7 — 1.0 % (w/w), and a concentration of phosphoric acid of 1.0 x 10 5 -2.0 % (w/w).
  • the therapeutic composition is administered as a gel with a concentration of methylene blue of 1.0 x 10 7 -0.25 % (w/w), and a concentration of phosphoric acid of 1.0 x 10 5 -2.0 % (w/w).
  • the therapeutic composition is administered as a mouthwash with a concentration of methylene blue of 1.0 x 10 7 — 1.0 % (w/w), and a concentration of phosphoric acid of 1.0 x 10 5 -2.0 % (w/w).
  • the therapeutic composition is administered as a mouthwash with a concentration of methylene blue of 1.0 x 10 7 -0.25 % (w/w), and a concentration of phosphoric acid of 1.0 x 10 5 -2.0 % (w/w).
  • the therapeutic composition is administered as a spray with a concentration of methylene blue of 1.0 x 10 7 — 1.0 % (w/w), and a concentration of phosphoric acid of 1.0 x 10 5 -2.0 % (w/w).
  • the therapeutic composition is administered as a spray with a concentration of methylene blue of 1.0 x 10 7 -0.25 % (w/w), and a concentration of phosphoric acid of 1.0 x 10 5 -2.0 % (w/w).
  • the therapeutic composition is administered as an inhalant with a concentration of methylene blue of 1.0 x 10 7 — 1.0 % (w/w), and a concentration of phosphoric acid of 1.0 x 10 5 -2.0 % (w/w).
  • the therapeutic composition is administered as an inhalant with a concentration of methylene blue of 1.0 x 10 7 -0.25 % (w/w), and a concentration of phosphoric acid of 1.0 x 10 5 -2.0 % (w/w).
  • the therapeutic composition is administered as a bioabsorbable composition with a concentration of methylene blue of 1.0 x 10 7 — 1.0 % (w/w), and a concentration of phosphoric acid of 1.0 x 10 5 -2.0 % (w/w).
  • the therapeutic composition is administered as bioabsorbable composition with a concentration of methylene blue of 1.0 x 10 7 -0.25 % (w/w), and a concentration of phosphoric acid of 1.0 x 10 5 -2.0 % (w/w).
  • the therapeutic composition is administered as a bioabsorbable composition with a concentration of phosphoric acid in the range of about 0.1 percent to about 40 percent by weight.
  • the therapeutic composition is administered as a bioabsorbable composition with a concentration of phosphoric acid in the range of about 0.1 percent to about 40 percent by weight and a concentration of methylene blue in the range of about 0.0001 to about 1 percent by weight.
  • the therapeutic composition is administered as a bioabsorbable composition with a concentration of phosphoric acid in an amount in the range of about 0.1 to about 1 percent by weight and a concentration of methylene blue in an amount in the range of about 0.0001 percent by weight to about 1 percent by weight,
  • the therapeutic composition is administered as a textile material with a concentration of methylene blue in an amount in the range of 0.001 to about 1 grams per cubic centimeter of the substrate of the textile material and phosphoric acid in an amount in the range of about 0.001 to about 1 grams per cubic centimeter of the substrate of the textile material.
  • a therapeutic formulation in accordance with aspects of the invention is prepared as follows.
  • Methylene Blue Solution A is prepared by diluting 85 m ⁇ of a 1% (w/v) methylene blue solution in water to a total volume of 1000 ml, resulting in a Methylene Blue Solution A concentration of 8.5 x 10 5 % (w/v).
  • Phosphoric Acid Solution A is prepared by diluting 1 g of 85% (w/w) phosphoric acid in water to a total volume of 850 ml, resulting in a Phosphoric Acid Solution A concentration of 1.0 x 10 1 % (w/v).
  • Therapeutic Formulation #1 is created by combining 86.6 g of Methylene Blue
  • the resulting composition comprises methylene blue at a final concentration of approximately 7.4 x 10
  • composition has bactericidal, antiviral, regenerative, and anti-inflammatory properties in human and animal subjects.
  • a therapeutic formulation in accordance with aspects of the invention is prepared as follows.
  • Methylene Blue Solution B is prepared by diluting 3.0 m ⁇ of a 1% (w/v) methylene blue solution in water to a total volume of 1000 ml, resulting in a Methylene Blue Solution B concentration of 3.0 x 10 6 % (w/v).
  • Phosphoric Acid Solution A is prepared by diluting 1 g of 85% (w/w) phosphoric acid in water to a total volume of 850 ml, resulting in a Phosphoric Acid Solution A concentration of 1.0 x 10 1 % (w/v).
  • Therapeutic Formulation #2 is created by combining 86.6 g of Methylene Blue
  • the resulting composition comprises methylene blue at a final concentration of approximately 2.6 x 10
  • Example 2 6 % (w/w) and phosphoric acid at a final concentration of approximately 2.0 x 10 3 % (w/w).
  • This composition has the same constituents as in Example 1, but the concentration of methylene blue is much lower than in Example 1. Even when the concentration of methylene blue has the concentrations of this Example 2, we have found that this composition has bactericidal, antiviral, regenerative, and anti-inflammatory properties in human and animal subjects.
  • Methylene Blue Solution A was prepared by diluting 85 m ⁇ of a 1% (w/v) methylene blue solution in water to a total volume of 1000 ml, resulting in a Methylene Blue Solution A concentration of 8.5 x 10 5 % (w/v).
  • Phosphoric Acid Solution A was prepared by diluting 1 g of 85% (w/w) phosphoric acid in water to a total volume of 850 ml, resulting in a Phosphoric Acid Solution A concentration of 1.0 x 10 1 % (w/v).
  • Mouthwash #1 was created by first combining the materials of Phase A, shown in
  • Phase A mixture was then combined with the material of Phase B, shown in Table 1, below.
  • the resulting mouthwash comprises methylene blue at a final concentration of approximately 7.4 x 10 5 % (w/w) and phosphoric acid at a final concentration of approximately 2.0 x 10 3 % (w/w).
  • composition has bactericidal, antiviral, regenerative, and anti-inflammatory properties in human and animal subjects.
  • Methylene Blue Solution B was prepared by diluting 3.0 m ⁇ of a 1% (w/v) methylene blue solution in water to a total volume of 1000 ml, resulting in a Methylene Blue Solution B concentration of 3.0 x 10 6 % (w/v).
  • Phosphoric Acid Solution A was prepared by diluting 1 g of 85% (w/w) phosphoric acid in water to a total volume of 850 ml, resulting in a Phosphoric Acid Solution A concentration of 1.0 x 10 1 % (w/v).
  • Mouthwash #2 was created by first combining the materials of Phase A shown in
  • Phase A mixture was then combined with the material of Phase B, shown in Table 2, below.
  • the resulting mouthwash comprises methylene blue at a final concentration of approximately 2.6 x 10 6 % (w/w) and phosphoric acid at a final concentration of approximately 2.0 x 10 3 % (w/w).
  • Example 4 we have found that this composition has bactericidal, antiviral, regenerative, and anti-inflammatory properties in human and animal subjects.
  • a therapeutic nasal spray in accordance with aspects of the invention was prepared as follows. [0169] Methylene Blue Solution A was prepared by diluting 85 m ⁇ of a 1% (w/v) methylene blue solution in water to a total volume of 1000 ml, resulting in a Methylene Blue Solution A concentration of 8.5 x 10 5 % (w/v).
  • Phosphoric Acid Solution A was prepared by diluting 1 g of 85% (w/w) phosphoric acid in water to a total volume of 850 ml, resulting in a Phosphoric Acid Solution A concentration of 1.0 x 10 1 % (w/v).
  • the therapeutic nasal spray was created by first combining the materials of Phase
  • Phase A shown in Table 3, below.
  • the materials of Phase B shown below in Table 3, were combined.
  • the Phase A mixture was combined with the Phase B mixture.
  • the resulting nasal spray comprises methylene blue at a final concentration of approximately 4.2 x 10 6 % (w/w) and phosphoric acid at a final concentration of approximately 3.0 x 10 3 % (w/w).
  • this composition has bactericidal, antiviral, regenerative, and anti-inflammatory properties in human and animal subjects.
  • Example 6 Use of Therapeutic Formulation #1 to Reduce the Number of Microorganisms in a Patient’s Mouth During Oral Procedures
  • Example 7 To reduce the number of microorganisms, including viruses, in a patient’s mouth during oral procedures, the patient is administered the therapeutic formulation of Example
  • Example 7 Use of Therapeutic Formulation #2 to Reduce the Number of Microorganisms in a Patient’s Mouth During Oral Procedures
  • Example 8 Use of Mouthwash #1 to Reduce the Number of Microorganisms in a Patient’s Mouth During Oral Procedures
  • the patient is administered the mouthwash of Example 3 just prior to an oral procedure, thereby decreasing the risk of infection for the patient and decreasing the risk of cross infection to health care staff.
  • Example 9 Use of Mouthwash #2 to Reduce the Number of Microorganisms in a Patient’s Mouth During Oral Procedures
  • the patient is administered the mouthwash of Example 4 just prior to an oral procedure, thereby decreasing the risk of infection for the patient and decreasing the risk of cross infection to health care staff.
  • a human subject administers the nasal spray formulation of Example 5 into each nostril.
  • Example 5 0.1-1.0 ml of the nasal spray formulation of Example 5 into each nostril at least once per day.
  • the nasal spray is administered using nasal spray devices well-known in the art.
  • the nasal spray formulation coats the lining of the nasal passages, sinuses, and other proximal airway tissues, exerting bactericidal and antiviral effects on these membranes and tissues.
  • the formulation reduces inflammation-induced secretion and opens air passages.
  • Example 11 Use of Nasal Spray to Prophylactically Reduce the Risk of Infection
  • a human subject administers the nasal spray formulation of Example 5 into each nostril.
  • the human subject administers 0.1-1.0 ml of the nasal spray formulation of Example 5 into each nostril at least once per day as needed.
  • the nasal spray is administered using nasal spray devices well-known in the art.
  • the nasal spray formulation coats the lining of the nasal passages, sinuses, and other proximal airway tissues, exerting bactericidal and antiviral effects on these membranes and tissues.
  • Example 12 Use of Nasal Spray to Treat Infection
  • the nasal spray formulation of Example 5 is administered into each nostril.
  • the human subject administers 0.1-1.0 ml of the nasal spray formulation of Example 5 into each nostril at least once per day post-infection for up to four weeks.
  • the nasal spray is administered using nasal spray devices well-known in the art.
  • the nasal spray formulation coats the lining of the nasal passages, sinuses, and other proximal airway tissues, exerting bactericidal, antiviral, anti-inflammatory, and regenerative effects on these membranes and tissues.
  • Example 13 Use of Inhaled Formulation to Prophylactically Reduce the Risk of Infection
  • a human subject inhales the nasal spray formulation of Example 5.
  • Example 5 0.1-1.0 ml of the nasal spray formulation of Example 5 at least once per day as needed using inhaler or nebulizer devices well-known in the art.
  • the nasal spray formulation coats the lining of the bronchial tubes and lungs, exerting bactericidal and antiviral effects on these membranes and tissues.
  • Example 5 In order to treat a human subject having a bacterial and/or viral infection of the bronchial tubes or lungs, the nasal spray formulation of Example 5 is inhaled by the human subject.
  • the human subject inhales 0.1-1.0 ml of the nasal spray formulation of Example 5 at least once per day post-infection for up to four weeks using inhaler or nebulizer devices well-known in the art.
  • the nasal spray formulation coats the lining of the bronchial tubes and lungs, exerting bactericidal, antiviral, anti-inflammatory, and regenerative effects on these membranes and tissues.
  • Example 15 Use of Therapeutic Skin Cream to Cleanse and Regenerate Skin Tissue
  • a topical cream comprising methylene blue at a final concentration of approximately 7.4 x 10 5 % (w/w) and phosphoric acid at a final concentration of approximately 2.0 x 10 3 % (w/w) is applied to the skin at the area of interest.
  • the topical cream is similar to the formulation of Example 1, but comprises lipids and other topical cream components well-known in the art. The topical cream is applied to the area of interest at least once per day as needed.
  • the topical cream comprises methylene blue at a final concentration of approximately 2.6 x 10 6 % (w/w) and phosphoric acid at a final concentration of approximately 2.0 x 10 3 %.
  • the topical cream is similar to the formulation of Example 2, but comprises lipids and other topical cream components well-known in the art. The topical cream is applied to the area of interest at least once per day as needed.
  • Example 16 Use of Therapeutic Skin Cream to Treat Inflammation and/or Infection of the Skin
  • a topical cream comprising methylene blue at a final concentration of approximately 7.4 x 10 5 % (w/w) and phosphoric acid at a final concentration of approximately 2.0 x 10 3 % (w/w) is applied to the skin at the area of interest.
  • the topical cream is similar to the formulation of Example 1, but comprises lipids and other cream components well-known in the art. The topical cream is applied to the area of interest at least once per day as needed.
  • the topical cream comprises methylene blue at a final concentration of approximately 2.6 x 10 6 % (w/w) and phosphoric acid at a final concentration of approximately 2.0 x 10 3 %.
  • the topical cream is similar to the formulation of Example 2, but comprises lipids and other topical cream components well-known in the art. The topical cream is applied to the area of interest at least once per day as needed.
  • a human subject dissolves one lozenge slowly in the mouth, repeating as needed.
  • the lozenge comprises methylene blue at a final concentration of approximately 7.4 x 10 5 % (w/w) and phosphoric acid at a final concentration of approximately 2.0 x 10 3 % (w/w).
  • the lozenge may further comprise sugars, starches, and flavorings.
  • the lozenge comprises methylene blue at a final concentration of approximately 2.6 x 10 6 % (w/w) and phosphoric acid at a final concentration of approximately 2.0 x 10 3 % (w/w).
  • the dissolved lozenge coats the lining of the mouth and throat, exerting bactericidal and antiviral effects on these membranes and tissues.
  • a human subject dissolves one lozenge slowly in the mouth, repeating at least once a day as needed.
  • the lozenge comprises methylene blue at a final concentration of approximately 7.4 x 10 5 % (w/w) and phosphoric acid at a final concentration of approximately 2.0 x 10 3 % (w/w). Methylene blue also provides a numbing effect.
  • the lozenge may further comprise sugars, starches, and flavorings.
  • the lozenge comprises methylene blue at a final concentration of approximately 2.6 x 10 6 % (w/w) and phosphoric acid at a final concentration of approximately 2.0 x 10 3 % (w/w).
  • the dissolved lozenge coats the lining of the mouth and throat, exerting bactericidal, antiviral, anti-inflammatory, and regenerative effects on these membranes and tissues.
  • Example 19 Minimum Inhibitory Concentrations (MIC) of Phosphoric Acid and Methylene Blue on Select Bacterial Species
  • a 2-fold serial dilution series was generated consisting of 24 members having a phosphoric acid concentration ranging from 85% to 1.0132789612 x 10 5 %.
  • a 2-fold serial dilution series was generated consisting of 24 members having a methylene blue concentration ranging from 1% to 1.1920929 x 10 7 %.
  • Each dilution series was prepared using Cation-Adjusted Muller Hinton broth (CAMHB).
  • 0.1 ml of each dilution in order from most concentrated to least concentrated, was placed into a separate well of a 96-well tissue-culture plate.
  • Select bacterial species were diluted in CAMHB so that 0.1 ml contained approximately 1 x 10 6 bacteria per ml.
  • 0.1 ml of diluted bacterial suspension was added to each well containing the phosphoric acid or methylene blue. The tissue-culture plates were incubated at 37°C for 24 hours.
  • 0.1 ml of each dilution in order from most concentrated to least concentrated, was placed into an individual well of a 96-well tissue-culture plate.
  • Select bacterial species were diluted in CAMHB so that 0.1 ml contained approximately 1 x 10 6 bacteria per ml.
  • 0.1 ml of diluted bacterial suspension was added to each well containing the phosphoric acid and methylene blue. The tissue-culture plates were incubated at 37°C for 24 hours.
  • CLSI Clinical and Laboratory Standard Institute
  • M7-A6 Approved Standard M7-A6. 6. Clinical and Laboratory Standards Institute; Wayne, PA: 2006. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically.
  • Example 20 In Vivo Testing of the Bactericidal/Antibacterial Properties of Methylene Blue, Phosphoric Acid, and a Mixture of Phosphoric Acid and Methylene Blue in Rats
  • treatment group 5 except for treatment group 5 and the treatments shown in Table 6 were applied onto the entire area of the open wound for the 5 minutes. Treatment solutions were prepared using sterile water. After five minutes, the entire wound area was rinsed with sterile water.
  • the infected tissue area was stained blue by the methylene blue, the stained tissue was surgically removed and the site was rinsed one more time with sterile water until completely stain-free.
  • the wound was closed with internal resorbable stiches and skin staples. Animals received 1 ml saline solution subcutaneously and 0.03 mg/kg of buprenorphine for pain. Buprenorphine was then administered at 0.02 mg/kg every 12 hours for the next 48 hours. Anti-inflammatory pain killers were avoided.
  • the infected tissue area appeared “curled” and was distinctively different from the healthy tissues surrounding it. Healthy tissue did not appear to be affected by the 0.33% phosphoric acid solution. The observed curled tissues were surgically removed. The site was rinsed again with sterile water and the wound was closed with internal resorbable stiches and skin staples. Animals received 1 ml saline solution subcutaneously and 0.03 mg/kg of buprenorphine for pain. Buprenorphine was then administered at 0.02 mg/kg every 12 hours for the next 48 hours. Anti-inflammatory pain killers were avoided.
  • the humoral width measurements of the infected hind leg post-op and phosphoric acid treatment was about 29 mm at 24 hours and about 26 mm at 72 hours post-op. (Fig.
  • the humoral width measurements of the infected hind leg was about 33 mm 24 hours post-op. and about 30 mm 72 hours post-op. (Fig. 4)
  • the negative control group received no treatment after being infected at the first surgery and did not undergo the second surgery. 48 hours after the first surgery, animals received 1 ml saline solution subcutaneously and 0.03 mg/kg of buprenorphine for pain. Buprenorphine was then administered at 0.02 mg/kg every 12 hours for the next 48 hours. Anti-inflammatory pain killers were avoided.
  • the humoral width measurements of the infected hind leg were taken 72 hours post-infection (equivalent to 24 hours post-op for the other treatment groups) and 120 hours post-infection (equivalent to 72 hours post-op for the other treatment groups). At 72 hours post-infection, a humoral width of 38 mm was measured at the infection site. At 120 hours post-infection, a humoral width measurement of 47 mm was measured at the infection site.
  • animals of treatment group 3 displayed the fastest healing, anti inflammatory, and regeneration rate.
  • the wound was closed externally and internally with the healthy tissues.
  • Animals in treatment group 2 showed the second-best healing.
  • Animals in treatment group 1 displayed significant irritation of the tissues, even after being rinsed out thoroughly after the application of the 0.015% methylene blue solution.
  • the treatment group 1 wound was not healing well and showed signs of inflammation.
  • Animals of treatment group 4 showed signs of inflammation and internal healing was delayed.
  • treatment using a solution containing both 0.33% phosphoric acid and 0.015% methylene blue was superior at reducing swelling, as assessed by measuring humoral width near the infection site, compared to treatment using a 0.015% methylene blue solution alone (treatment group 1) or a 0.33% phosphoric acid solution alone (treatment group 2).
  • FIG. 5 The photographs of Fig. 5 are from the low dose treatment (0.015% methylene blue and 0.33% phosphoric acid) with higher doses resulting in faster wound healing and faster reduction of inflammation.
  • the rats were infected with the MRS A (Staphylococcus aureus ATCC BAA- 1762) at a concentration of 4 x 10 8 per ml. The rats were sedated. A small incision was made at a femur site and a slow rotor drill was used to create a dehiscence/trauma of about 0.5 mm depth in the femoral bone, which is equivalent to about half the thickness of the cortical bone of the femur. MRSA bacteria (four microliters) were introduced into the bone and surrounding soft tissues. The wounds were then closed with internal stitches and skin clips. All rats showed severe signs of infection after 72 hours - swelling and cyanotic color of this site, ruffled fur, loss of weight, hunched posture, decreased mobility. An MRI scan showed change in soft tissue at the infected site.
  • MRS A Staphylococcus aureus ATCC BAA- 1762
  • the initial treatment was performed by using a composition comprising 37% phosphoric acid gel and methylene blue carried by a bioabsorbable sponge (GELFOAM®) applied onto the entire area of the wound for five minutes. After five minutes, the sponge was removed. The wound was rinsed out with saline solution. The wound was not bleeding. The bacteria present were stained black visually demarcating the line of the infected tissue, which was surgically removed. The residual black stained tissues were removed by using the ultrasound machine with the smooth up to avoid tissue damage. The ultrasound waves cleaned the deepest layers of the tissue and restored a fresh blood flow. A bioabsorbable sponge (GELFOAM®, Pfizer, Inc.) containing an aqueous solution of phosphoric acid and methylene blue was inserted into the wounds and the wounds were closed.
  • GELFOAM® bioabsorbable sponge
  • Rats in Group #1 received bioabsorbable sponge soaked with an aqueous solution containing 0.5 weight percent phosphoric acid and 1 weight percent methylene blue. The wounds were then closed with internal resorbable stitches and skin clips.
  • Rats in Group #2 received bioabsorbable sponge soaked with an aqueous solution containing 2 weight percent phosphoric acid and 1 weight percent methylene blue. The wounds were then closed with internal resorbable stitches and skin clips.
  • Rats in Group #3 received a bioabsorbable sponge soaked with an aqueous solution containing 5 weight percent phosphoric acid and 1 weight percent methylene blue. The wounds were then closed with internal resorbable stitches and skin clips.
  • Rats in Group #4 received a bioabsorbable sponge soaked with a aqueous solution containing 25 weight percent phosphoric acid and 1 weight percent methylene blue. The wounds were then closed with internal resorbable stitches and skin clips.
  • Groups # l-#3 showed rapid healing. A healthy change in fur, mobility, reduction of swelling and redness of the wound site were observed.
  • the rats started to gain weight in a continuous manner, at an average of about 2 to 3 grams per day.
  • rats fur had restored their initial healthy shine and condition, rats did not display any signs of pain or disease, were rearing using both legs.
  • the wound site was healed within 30 days post-infection.
  • the MRL CT scan and cell histology results showed no signs of infection and inflammation on the site. All evaluations showed presence of the new healthy tissue formed, new bone formation and bone density increase.
  • the MRI scan done on the tenth post-operative day showed that infected site volume had decreased by 62% and new tissues were formed.
  • Rats in Group #4 showed signs of secondary infection starting on Day 3 post-op.
  • Example 22 In Vivo Study of MRSA InfectionHealthy Sprague Dawley female rats (Charles River Laboratories, Wilmington, MA) 6 months of age (four groups, five per group), were used for study. The rats were infected with the MRSA (Staphylococcus aureus ATCC BAA-1762) at a concentration of 4 x 10 8 per ml. The rats were sedated, MRSA bacteria (four microliters) were injected into muscle and bone tissue. All rats showed severe signs of infection after 48 hours - swelling and cyanotic color of this site, ruffled fur, loss of weight, hunched posture, decreased mobility. An MRI scan showed change in soft tissue at the infected site (Fig. 5A and 5B).
  • MRSA Staphylococcus aureus ATCC BAA-1762
  • Example 23 In Vivo Bone Regeneration
  • PET scan images of the bone of rats treated with a combination of methylene blue and phosphoric acid and untreated control shows that the bone density in the treated rats was substantially higher than in the untreated rats (Fig. 7).
  • an implant can be placed immediately at the tooth extraction surgery together with bone grafting tissue and a crown can be placed about 6 months later (Fig. 8, lower timeline).
  • composition comprising methylene blue and phosphoric acid as described herein promotes a fast onset of cytogenesis, potent angiogenesis complete cytological osteogenesis, promotion of physiological ossification, and mobilization of hydroxyapatite to the site of new bone growth,
  • a therapeutic composition comprising:
  • a method of treating a subject in need thereof comprising: causing the administration of the therapeutic composition of any one of the preceding claims, wherein the subject is selected from the group consisting of a human and an animal.
  • a bioabsorbable antimicrobial and anti-inflammatory composition comprising: a bioabsorbable water-insoluble sponge containing an aqueous solution of phosphoric acid and a thiazine dye, wherein phosphoric acid concentration is in the range of about 0.1 to about 40 percent by weight of the aqueous solution.
  • bioabsorbable composition in accordance with claim P8 wherein the phosphoric acid concentration is about 0.1 to about 2.5 percent by weight of the aqueous solution.
  • bioabsorbable composition in accordance with claim P8 wherein the phosphoric acid concentration is about 5 to about 40 percent by weight of the aqueous solution.
  • PI 1 The bioabsorbable composition in accordance with claim P8, wherein the thiazine dye methylene blue.
  • bioabsorbable composition in accordance with claim P10 wherein methylene blue is present in the aqueous phosphoric acid solution in an amount in the range of about 0.0001 percent by weight to about 1 percent by weight, based on the weight of the aqueous phosphoric acid solution.
  • biocompatible composition in accordance with claim P10 wherein methylene blue is present in the aqueous phosphoric acid solution in an amount in the range of about 0.1 to about 1 percent by weight of the aqueous phosphoric acid solution.
  • PI 6 The bioabsorbable composition in accordance with claim P8, wherein the sponge contains about 5 to about 40 percent by weight of the aqueous phosphoric acid solution, based on the weight of the bioabsorbable sponge.
  • PI 7 The bioabsorbable sponge in accordance with claim P8, wherein the aqueous solution contains a biocompatible thickening agent.
  • bioabsorbable sponge in accordance with claim P17 wherein the thickening agent is sodium carboxymethyl cellulose.
  • An antiviral textile material comprising: a hydrophilic fibrous substrate; an antiviral thiazine dye; and phosphoric acid; the hydrophilic fibrous substrate having porosity sufficient to permit a human to breathe therethrough, having said thiazine dye distributed therewithin in an amount in the range of 0.001 to about 1 grams per cubic centimeter of said substrate, and having phosphoric acid distributed therewith in an amount in the range of about 0.001 to about 1 grams per cubic centimeter of said substrate.

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Abstract

Therapeutic bactericidal, antiviral, anti-inflammatory, antifungal, blood clotting, and regenerative compositions comprising thiazine dye and phosphoric acid.

Description

THERAPEUTIC THIAZINE DYE COMPOSITIONS AND METHODS OF USE
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Application No. 17/242,185 filed April 27,
2021, and claims priority to U.S. Provisional Application No. 63/134,945 filed January 7, 2021, each of which is incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0002] The present invention relates to therapeutic thiazine dye compositions and methods of use and more particularly to bactericidal, antiviral, anti-inflammatory, and regenerative compositions including methylene blue and phosphoric acid and its salts.
BACKGROUND ART
[0003] Hospital-acquired infections, also known as nosocomial infections, are infections acquired in a hospital or other health care facility and are potentially caused by organisms that are resistant to antibiotics. Nosocomial infections represent the tenth major course of death in the United States. The annual economic impact of nosocomial infections is estimated to be in the range of 55-70 billion U.S. dollars.
[0004] No effective treatment is currently available for cases where antibiotic treatment of wounds with bacterial infections is not effective. In addition, bacteria present in an open wound are not visible to the eye. Thus, during treatment of wounds, usually a substantial amount of adjacent tissue is removed in order to minimize the likelihood of infection. Also, current surgical debridement procedures are associated with post operative pain and swelling, do not permit an immediate reconstruction of the surgical procedure site, and require massive amounts of antibiotics for a lengthy period of time.
[0005] Osteoporotic bone and joint replacement in elderly patients is more difficult due to bone osteoporosis. Connective/supportive implants and plates do not hold in place as well due to relatively softer bone structure. In addition, an osteoporotic bone heals more slowly. In case of a fracture, it can take several months for the fracture to heal. [0006] The present compositions ameliorate the aforementioned shortcomings of current surgical procedures in a safe, non-toxic manner.
[0007] In addition, inhalation and exhalation of air contaminated by a harmful virus or other pathogens is a common route for infection of humans. Face masks and other protective gear provide a barrier to the transmission of such pathogens.
[0008] There is an ongoing need to improve these barriers.
SUMMARY
[0009] The present compositions ameliorate the aforementioned shortcomings of current surgical procedures in a safe, non-toxic manner.
Compositions. Dosage Forms. Uses and Methods
[0010] Provided are non-toxic therapeutic compositions and dosage forms formed from a mixture of a thiazine dye and phosphoric acid in aqueous solution. This mixture can be further processed into a variety of pharmaceutical and surgical dosage forms where the misture is either remains in aqueous solution or has been dried and will subsequently redissolve in water or body fluids. Therapeutic compositions and dosage forms of the present disclosure exhibit surprising bactericidal, antiviral, anti-inflammatory, and regenerative properties, and stop bleeding on the site of application.
[0011] Also provided are pharmaceutical uses and methods of treatment that employ non toxic therapeutic compositions and dosage forms of the present disclosure.
[0012] In accordance with one embodiment of the invention, there is provided a therapeutic composition comprising 1.0 x 107 2.5 x 10 1 % (w/w) thiazine dye, and 1.0 x 105 2 0 (W/W) phosphoric acid. In various aspects, the thiazine dye is methylene blue.
[0013] In accordance with another embodiment of the invention, there is provided a method of treating a subject in need thereof, the method comprising causing the administration of the therapeutic composition comprising 1.0 x 107 2.5 x 10 1 % (w/w) thiazine dye, and 1.0 x 105 2 0 (w/w) phosphoric acid. The subject may be a human or an animal. Bioabsorbable Sponge
[0014] The present antimicrobial and anti-inflammatory composition facilitates a safe, non-toxic surgical procedure for amelioration of wound infection and inflammation, as well as enhances the ability to close a wound after a surgical procedure. In cases of bone fracture, the composition increases bone density and new bone formation as well as promotes angiogenesis at the surgical site.
[0015] In accordance with one embodiment of the invention, the antimicrobial composition comprises an aqueous solution of phosphoric acid (H3PO4) and an antimicrobial thiazine dye in a bioabsorbable sponge. In various aspects, the phosphoric acid concentration in the sponge is in the range of about 0.1 percent to about 40 percent by weight of the solution depending on how the phosphoric acid-containing sponge is used.
[0016] In various aspects, for initial cleaning of a wound, the sponge contains phosphoric acid at a concentration of about 5 to about 40 percent by weight of the aqueous solution.
In various aspects, for applications when the bioabsorbable sponge is left in a closed wound, the sponge contains phosphoric acid in an amount in the range of about 0.1 percent by weight to about 5 percent by weight based on the weight of the solution. In various aspects, the sponge contains phosphoric acid in an amount in the range of about 0.1 percent by weight to about 2.5 percent by weight based on the weight of the solution. In various aspects, the sponge contains phosphoric acid in an amount in the range of about 0.75 to about 2.5 percent by weight of the solution
[0017] In various aspects, the bioabsorbable sponge contains the aqueous phosphoric acid solution in an amount in the range of about 5 to about 40 percent by weight, based on the weight of the bioabsorbable sponge.
[0018] In various aspects, antimicrobial thiazine dye is present in the aqueous phosphoric acid solution in an amount in the range of about 0.0001 percent by weight to about 1 percent by weight. In various aspects, antimicrobial thiazine dye is present in the aqueous phosphoric acid solution in an amount in the range of about 0.1 to about 1 percent by weight, based on the weight of the aqueous phosphoric acid solution. In various aspects, the thiazine dye is methylene blue.
[0019] In various aspects, the bioabsorbable sponge comprises gelatin. In various aspects, the bioabsorbable sponge comprises collagen. In various aspects, the aqueous solution contains a biocompatible thickening agent. In various aspects, the biocompatible thickening agent is carboxymethyl cellulose.
Antiviral Textile
[0020] In accordance with another embodiment of the invention, an antiviral textile material that can be converted into wearing apparel such as face masks, turtle necks, scarves, bandanas, and other partial or full body coverings is described. The antiviral textile material has a hydrophilic fibrous substrate. Distributed within the fibrous substrate is a thiazine dye, such as methylene blue, and phosphoric acid. In various aspects, the thiazine dye and phosphoric acid are present in equimolar amounts. In various aspects, the thiazine dye is distributed in an amount in the range of 0.001 to about 1 grams per cubic centimeter of the substrate. In various aspects, the phosphoric acid is distributed in an amount in the range of about 0.001 to about 1 grams per cubic centimeter of the substrate.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] The patent or application file contains at least one drawing executed in color.
Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
[0022] The foregoing features of will be more readily understood by reference to the following detailed description, taken with reference to the accompanying drawings, in which:
[0023] Fig. 1 shows the volume of tissue infection over time of rats infected in muscle and bone tissue with E. coli bacteria and treated once with a combination of methylene blue and phosphoric acid as described herein.
[0024] Fig. 2 shows the volume of tissue infection over time of rats infected in muscle and bone tissue with P. aeruginosa bacteria and treated once with a composition comprising methylene blue and phosphoric acid as described herein.
[0025] Fig. 3 A shows the volume of tissue infection over time of rats infected in muscle and bone tissue with MRSA bacteria and treated once with a composition comprising methylene blue and phosphoric acid as described herein. Fig. 3B shows the concentration of C-reactive protein in the MRSA infected rats. [0026] Fig. 4 shows the surgical site swelling, as assessed by hind leg humoral width near the site of infection by intramuscular injection of P. aeruginosa in rats treated only with debridement (Treatment Group 4), treated with 0.33% (w/w) phosphoric acid (Treatment Group 2), treated with 0.15% (w/w) methylene blue (Treatment Group 1) and treated with a combination of 0.15% (w/w) methylene blue and 0.33% (w/w) phosphoric acid (Treatment Group 3)
[0027] Fig. 5A shows a histology image of soft tissue of a rat infected with MRSA and not further treated. Fig. 5B shows a histology image of muscle tissue of a rat infected with MRSA for 48 hours and treated with a combination of 0.5% methylene blue and 35% phosphoric acid. Fig. 5C shows a histology image of muscle tissue of a rat infected with MRSA and not further treated. Fig. 5D shows a histology image of bone and soft tissue of a rat infected with MRSA for 48 hours and treated with a combination of 0.5% methylene blue and 35% phosphoric acid.
[0028] Fig. 6A shows an MRI image of an infection site in soft tissue of a rat 48 hours after injection of MRSA. Fig. 6B shows an MRI image of the same infection site as in Fig. 6A at 10 days after treatment with a combination of 0.5% methylene blue and 35% phosphoric acid. Fig. 6C shows an MRI image of the same infection site as in Fig. 6A at 30 days after treatment with a combination of 0.5% methylene blue and 35% phosphoric acid.
[0029] Fig. 7A shows a histomorphometry image of a young rat of a calcein and tetracycline stain showing new bone formation after bone injury and treatment with a combination of 0.15% (w/w) methylene blue and 0.33% (w/w) phosphoric acid. Fig. 7B shows a histomorphometry image of an elderly rat of a calcein and tetracycline stain showing new bone formation after bone injury and treatment with a combination of methylene blue and phosphoric acid.
[0030] Fig. 8A shows a PET scan image of a rat after bone injury and no further treatment. Fig. 8B shows a PET scan image of a rat after bone injury and treatment with a combination of 0.15% (w/w) methylene blue and 0.33% (w/w) phosphoric acid.
[0031] Fig. 9 shows a traditional timeline for a patient receiving a dental implant (top) and a timeline for a patient receiving a dental implant and being treated once with a with a combination of methylene blue and phosphoric acid (bottom). [0032] Fig. 10 shows a perspective view of a bandage utilizing a bioabsorbable sponge in accordance with an embodiment of the invention.
[0033] Fig. 11 shows a partially exploded perspective view of a face mask having a fibrous intermediate layer impregnated with a combination of methylene blue and phosphoric acid.
DETAILED DESCRIPTION
I. Definitions
[0034] As used in this description and the accompanying claims, the following terms shall have the meanings indicated, unless the context otherwise requires:
[0035] The term “about” is used herein to mean approximately, roughly, around, or in the regions of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” can modify a numerical value above and below the stated value by a variance of, e.g., 10 percent, up or down (higher or lower). In some aspects of the disclosure, the term “about” encompasses a deviation from the recited value of between 0.001% and 10%, inclusive of the endpoints. In some aspects, the term “about” encompasses an increase from the recited value of between 0.001% and 10%, inclusive of the endpoints. In some aspects, the term “about” encompasses a decrease from the recited value of between 0.001% and 10%, inclusive of the endpoints.
[0036] Units, prefixes, and symbols are denoted in their Systeme International de Unites
(SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Where a range of values is recited, it is to be understood that each intervening integer value, and each fraction thereof, between the recited upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range can independently be included in or excluded from the range, and each range where either, neither or both limits are included is also encompassed within the disclosure. Thus, ranges recited herein are understood to be shorthand for all of the values within the range, inclusive of the recited endpoints. For example, a range of 1 to 10 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. [0037] Where a value is explicitly recited, it is to be understood that values which are about the same quantity or amount as the recited value are also within the scope of the disclosure. Where a combination is disclosed, each sub-combination of the elements of that combination is also specifically disclosed and is within the scope of the disclosure. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element of a disclosure is disclosed as having a plurality of alternatives, examples of that disclosure in which each alternative is excluded singly or in any combination with the other alternatives are also hereby disclosed; more than one element of a disclosure can have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.
[0038] As used herein, the terms “effective amount” and “therapeutically effective amount” refer to an amount of a composition of the disclosure that provides beneficial or desired therapeutic and/or prophylactic results. For prophylactic use, beneficial or desired results can include, for example, one or more results such as eliminating or reducing the risk, lessening the severity, or delaying the onset of an infection, inflammation or bone degeneration, its complications, and intermediate pathological phenotypes presenting during development of the infection or bone degeneration. For therapeutic use, beneficial or desired results can include, for example, one or more clinical results such as decreasing one or more symptoms and pathological conditions resulting from or associated with the infection or bone degeneration, increasing the quality of life of those suffering from the infection or bone degeneration, decreasing the dose of other medications required to treat the infection or bone degeneration, enhancing the effect of other medication such as via targeting, delaying the progression of the infection or bone degeneration, and/or prolonging survival. An effective amount can be, for example, an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly. As it is understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an effective amount may be considered in the context of administering one or more therapeutic agents. An effective amount can be administered in one dosage or can be divided into multiple dosages, the total of such dosages being the effective amount. For example, an effective amount can be provided in two separate administrations over a period of time, that in aggregate, provide the effective amount of the composition described herein.
[0039] Thiazine dyes include methylene blue, azure A, azure B, toluidine blue 0, thiamine, and the like. A preferred thiazine dye is methylene blue (3,7- bis(dimethylamine) - phenothiazin-5-ium chloride.
[0040] Minimum inhibitory concentration (“MIC”) as used herein shall mean the lowest concentration of a chemical or combination of chemicals, e.g., methylene blue, phosphoric acid, and combinations thereof, which prevents visible growth of a bacteria.
[0041] As used herein with respect to Example 20, “post-op,” and the like, shall mean the time period after the second surgery (treatment) described in Example 20 herein. As used herein with respect to Example 21, “post-op,” and the like, shall mean the time period after the second surgery (treatment) described in Example 21 herein.
[0042] As used herein with respect to Example 20, “post-infection,” and the like, shall mean the time period after the first surgery described in Example 20 herein. As used herein with respect to Example 21, “post-infection,” and the like, shall mean the time period after the first surgery described in Example 21 herein. For animals that have been treated via the second surgery described in Example 20 herein, post-infection time is essentially equivalent to the post-op time plus 48 hours (because the second surgery took place 48 hours after the first surgery). For animals that have been treated via the second surgery described in Example 21 herein, post-infection time is essentially equivalent to the post-op time plus 72 hours (because the second surgery took place 72 hours after the first surgery).
[0043] As used herein “association product” refers to any molecular structure resulting from a combination of methylene blue and phosphoric acid and includes ionic molecular structures, weak electrostatically bonded complexes, and covalently bonded complexes, optionally including at least one additional molecule.
[0044] As used herein “associated with one another” refers to a reaction of one molecule of methylene blue with one molecule of phosphoric acid to form an association product.
[0045] As used herein “topical cream” refers to an emulsion of oil and water that is applied to a body surface including a skin or mucous membrane and is either low viscosity and includes a lotion or high viscosity and includes a gel. [0046] As used herein “gel” refers to a non-fluid colloidal network or polymer network that is expanded throughout its whole volume by a fluid. A “gel” can contain a covalent polymer network, e.g., a network formed by cross-linking polymer chains or by nonlinear polymerization. A “gel” can contain a polymer network formed through the physical aggregation of polymer chains, caused by hydrogen bonds, crystallization, helix formation, and/or complexation that result in regions of local order acting as the network junction points. The term “gel” includes a hydrogel in which the swelling agent is water.
[0047] As used herein “emulsion” refers to a fluid colloidal system in which liquid droplets and/or liquid crystals are dispersed in a liquid. In an emulsion the continuous phase can be an aqueous phase or an organic lipid, e.g., an oil.
[0048] As use herein “spray” refers to a liquid that is dispersed in the form of small droplets.
[0049] As used herein “bioabsorbable” refers to capable of being absorbed into living tissue.
[0050] As used herein “bioabsorbable sponge” refers to a porous absorbent biocompatible, water-insoluble, pliable, absorbent material that, when implanted in a human or other mammalian body, is absorbed by the body.
[0051] As used herein “textile material” refers to a material made of interlacing fibers.
[0052] As used herein “hydrophilic fibrous substrate” refers to a substrate made from fibers that has a tendency to mix with or be wetted by water.
[0053] As used herein “non-woven cotton” refers to a fabric-like material made from short and long fibers bonded together by chemical, mechanical, heat or solvent treatment.
[0054] A used herein “water-repellant cotton layer” refers to a cotton layer that has a hydrophobic coating such that it repels water intrusion into the layer.
[0055] As used herein “bandage” refers to a cloth material that is used to cover a wound and can be used together with a wound dressing or on its own.
[0056] As used herein “wound dressing” refers to a material that is in direct contact with a wound and can be a cloth, a foam, a film, a hydrocolloid, a hydrogel, an alginate or a collagen.
[0057] As used herein “bone site” refers to a location on or within a bone that is accessible from the outside of a body because of a surgery or penetrating bone fracture or a location on or within a bone that is accessible by introduction of a needle. [0058] As used herein a “dental bone site” is a bone site that is located in the oral cavity and is accessible through the mouth.
[0059] As used herein, “wound” refers to a location on a body of an injury to the body that involves laceration or breaking of a membrane, e.g., a skin, and damage to a surrounding tissue.
[0060] As used herein “open wound” refers to a wound that is in direct contact with the atmosphere surrounding the body.
[0061] As used herein “chronic wound” refers to a wound that has not healed within a time frame reasonable for the healing of a wound in the body location, e.g., within 10 days for a wound located on the skin. A chronic wound includes an ulcer, a diabetic lesion, an eczema, acne vulgaris, a chronic surgical wound, and a chronic infectious wound.
[0062] As used herein “contacting” refers to physical interaction of one thing with another, e.g., of an aqueous solution with a body, a wound, or a bone.
[0063] As used herein “placing into” refers to the introduction of a material into an opening created by a wound or injury in a tissue
[0064] As used herein “administering” refers to an action that introduces a composition to a subject, either internally or externally, e.g., by infusion, injection, inhalation, or creation of a direct contact between the composition and the subject or a portion of the subject.
[0065] As used herein “regenerating a bone” refers to an activity that results in a generation of bone tissue in an area of the body where bone tissue had previously been lost due to injury or osteoporosis.
[0066] As used herein “fractured bone” refers to a bone whose physiological structure has been interrupted.
[0067] As sued herein “osteoporotic bone” refers to a bone whose density has been diminished and whose mechanical strength is reduced due to the diminished bone density.
[0068] As used herein “’’subject” refers to a mammal, including an animal and a human.
II. Therapeutic Compositions
[0069] Provided are non-toxic therapeutic compositions exhibiting surprising bactericidal, antiviral, anti-inflammatory, and regenerative properties, and which stop bleeding on the site of application. [0070] Therapeutic compositions disclosed herein comprise one or more thiazine dyes and phosphoric acid, or salts thereof. In various aspects, the thiazine dye is methylene blue. It was surprisingly found that therapeutic compositions comprising methylene blue and phosphoric acid work to produce bactericidal, antiviral, anti-inflammatory, and regenerative effects that are not observed in compositions containing one of either methylene blue or phosphoric acid, but not both.
[0071] The combination of phosphoric acid on the methylene blue forms a bis ammonium salt (shown below) which, without wanting to be bound by theory, appears to be a chemo- specific agent that has antibacterial/antimicrobial /antifungal properties. However, a bis ammonium salt of methylene blue and phosphoric acid also appears to block (downregulate) pro-inflammatory cytokines by an unknown mechanism. The synergistic effects of these two independent mechanisms of actions may promote the mechanisms of bone /tissue repair in the novel treatment provided herein. ocsx
Figure imgf000012_0001
N Phosphoric acid H3N ci I H2P JO4¾ COl I
Methylene Blue
3-N3, N3-dimethylamino-7-aminophenthiazininium 3-N3, N3-dimethylamino-phenthiazininium chloride chloride-7-ammonium phosphate
[0072] It is important to note that neither phosphoric acid nor methylene blue have the demonstrated types of antibacterial/antimicrobial /antifungal/anti-parasitic/anti- inflammatory and/or regenerative effects when used as single agents but rather the combination of both is required to make the active pharmaceutical ingredient (API).
[0073] Without wanting to be bound by theory it is hypothesized that the therapeutic compositions comprising methylene blue and phosphoric acid promote fast onset of cytogenesis, potent angiogenesis, complete cytological osteogenesis, promotion of physiological ossification, and mobilization of hydroxyapatite to the site of new bone growth, accelerate the extrinsic pathway of coagulation and control surgical bleeding.
II. A Dosage Forms
[0074] In some aspects, the therapeutic compositions provided herein comprise a thiazine dye, phosphoric acid and/or an association product thereof. [0075] In some aspects, the therapeutic compositions comprise 1.0 x 107-2.5 x 10 1 %
(w/w) thiazine dye and 1.0 x 105-2.0% (w/w) phosphoric acid. In various aspects, the therapeutic compositions comprise 1.0 x 106— 1 x 104 % (w/w) thiazine dye and 1.0 x 10 4-1.0 x 102 % (w/w) phosphoric acid.
[0076] In some aspects, the therapeutic composition comprise a thiazine dye in the range of about 1.0 x 107 % (w/w) to about 1 % (w/w), about 5.0 x 107 % (w/w) to about 1 % (w/w), about 1.0 x 106 % (w/w) to about 1 % (w/w), about 5.0 x 106 % (w/w) to about 1 % (w/w), about 1.0 x 105 % (w/w) to about 1 % (w/w), about 5.0 x 105 % (w/w) to about 1 % (w/w), about 1.0 x 104 % (w/w) to about 1 % (w/w), about 5.0 x 104 % (w/w) to about 1 % (w/w), about 0.001 % (w/w) to about 1 % (w/w), about 0.005 % (w/w) to about 1 % (w/w), about 0.01 % (w/w) to about 1 % (w/w), about 0.02 % (w/w) to about 1 % (w/w), about 0.03 % (w/w) to about 1 % (w/w), about 0.04 % (w/w) to about 1 % (w/w), about 0.05 % (w/w) to about 1 % (w/w), about 0.06 % (w/w) to about 1 % (w/w), about 0.07 % (w/w) to about 1 % (w/w), about 0.08 % (w/w) to about 1 % (w/w), about 0.09 % (w/w) to about 1 % (w/w), about 0.1 % (w/w) to about 1 % (w/w), about 0.11 % (w/w) to about 1 % (w/w), about 0.12 % (w/w) to about 1 % (w/w), about 0.13 % (w/w) to about 1 % (w/w), about 0.14 % (w/w) to about 1 % (w/w), about 0.15 % (w/w) to about 1 %
(w/w), about 0.16% (w/w) to about 1 % (w/w), about 0.17% (w/w) to about 1 % (w/w), about 0.18 % (w/w) to about 1 % (w/w), about 0.19 % (w/w) to about 1 % (w/w), about 0.2 % (w/w) to about 1 % (w/w), about 0.21 % (w/w) to about 1 % (w/w), about 0.22 % (w/w) to about 1 % (w/w), about 0.23 % (w/w) to about 1 % (w/w), about 0.24 % (w/w) to about 1 % (w/w), about 0.25 % (w/w) to about 1 % (w/w), about 0.26 % (w/w) to about 1 % (w/w), about 0.27 % (w/w) to about 1 % (w/w), about 0.28 % (w/w) to about 1 %
(w/w), about 0.29 % (w/w) to about 1 % (w/w), about 0.30 % (w/w) to about 1 % (w/w), about 0.31 % (w/w) to about 1 % (w/w), about 0.32 % (w/w) to about 1 % (w/w), about 0.33 % (w/w) to about 1 % (w/w), about 0.34 % (w/w) to about 1 % (w/w), about 0.35 % (w/w) to about 1 % (w/w), about 0.36 % (w/w) to about 1 % (w/w), about 0.37 % (w/w) to about 1 % (w/w), about 0.38 % (w/w) to about 1 % (w/w), about 0.39 % (w/w) to about 1 % (w/w), about 0.40 % (w/w) to about 1 % (w/w), about 0.41 % (w/w) to about 1 %
(w/w), about 0.42 % (w/w) to about 1 % (w/w), about 0.43 % (w/w) to about 1 % (w/w), about 0.44 % (w/w) to about 1 % (w/w), about 0.45 % (w/w) to about 1 % (w/w), about 0.46 % (w/w) to about 1 % (w/w), about 0.47 % (w/w) to about 1 % (w/w), about 0.48 % (w/w) to about 1 % (w/w), about 0.49 % (w/w) to about 1 % (w/w), about 0.50 % (w/w) to about 1 % (w/w), about 0.51 % (w/w) to about 1 % (w/w), about 0.52 % (w/w) to about 1 % (w/w), about 0.53 % (w/w) to about 1 % (w/w), about 0.54 % (w/w) to about 1 %
(w/w), about 0.55 % (w/w) to about 1 % (w/w), about 0.56 % (w/w) to about 1 % (w/w), about 0.57 % (w/w) to about 1 % (w/w), about 0.58 % (w/w) to about 1 % (w/w), about 0.59 % (w/w) to about 1 % (w/w), about 0.60 % (w/w) to about 1 % (w/w), about 0.61 % (w/w) to about 1 % (w/w), about 0.62 % (w/w) to about 1 % (w/w), about 0.63 % (w/w) to about 1 % (w/w), about 0.64 % (w/w) to about 1 % (w/w), about 0.65 % (w/w) to about 1 % (w/w), about 0.66 % (w/w) to about 1 % (w/w), about 0.67 % (w/w) to about 1 %
(w/w), about 0.68 % (w/w) to about 1 % (w/w), about 0.69 % (w/w) to about 1 % (w/w), about 0.70 % (w/w) to about 1 % (w/w), about 0.71 % (w/w) to about 1 % (w/w), about 0.72 % (w/w) to about 1 % (w/w), about 0.73 % (w/w) to about 1 % (w/w), about 0.74 % (w/w) to about 1 % (w/w), about 0.75 % (w/w) to about 1 % (w/w), about 0.76 % (w/w) to about 1 % (w/w), about 0.77 % (w/w) to about 1 % (w/w), about 0.78 % (w/w) to about 1 % (w/w), about 0.79 % (w/w) to about 1 % (w/w), about 0.80 % (w/w) to about 1 %
(w/w), about 0.81 % (w/w) to about 1 % (w/w), about 0.82 % (w/w) to about 1 % (w/w), about 0.83 % (w/w) to about 1 % (w/w), about 0.84 % (w/w) to about 1 % (w/w), about 0.85 % (w/w) to about 1 % (w/w), about 0.86 % (w/w) to about 1 % (w/w), about 0.87 % (w/w) to about 1 % (w/w), about 0.88 % (w/w) to about 1 % (w/w), about 0.89 % (w/w) to about 1 % (w/w), about 0.90 % (w/w) to about 1 % (w/w), about 0.91 % (w/w) to about 1 % (w/w), about 0.92 % (w/w) to about 1 % (w/w), about 0.93 % (w/w) to about 1 %
(w/w), about 0.94 % (w/w) to about 1 % (w/w), about 0.95 % (w/w) to about 1 % (w/w), about 0.96 % (w/w) to about 1 % (w/w), about 0.97 % (w/w) to about 1 % (w/w), about 0.98 % (w/w) to about 1 % (w/w), or about 0.99 % (w/w) to about 1 % (w/w) and phosphoric acid in the range of about 1.0 x 10 5 % (w/w) to about 40 % (w/w), about 5.0 x 105 % (w/w) to about 40 % (w/w), about 1.0 x 104 % (w/w) to about 40 % (w/w), about 5.0 x 104 % (w/w) to about 40 % (w/w), about 0.001 % (w/w) to about 40 % (w/w), about 0.005 % (w/w) to about 40 % (w/w), about 0.01 % (w/w) to about 40 % (w/w), about 0.02 % (w/w) to about 40 % (w/w), about 0.03 % (w/w) to about 40 % (w/w), about 0.04 % (w/w) to about 1 % (w/w), about 0.05 % (w/w) to about 40 % (w/w), about 0.06 % (w/w) to about 40 % (w/w), about 0.07 % (w/w) to about 140% (w/w), about 0.08 % (w/w) to about 40 % (w/w), about 0.09 % (w/w) to about 40 % (w/w), about 0.1 % (w/w) to about 40 % (w/w), about 0.11 % (w/w) to about 40 % (w/w), about 0.12 % (w/w) to about 40 % (w/w), about 0.13 % (w/w) to about 40 % (w/w), about 0.14 % (w/w) to about 40 %
(w/w), about 0.15 % (w/w) to about 40 % (w/w), about 0.16% (w/w) to about 40 %
(w/w), about 0.17 % (w/w) to about 40 % (w/w), about 0.18 % (w/w) to about 40 %
(w/w), about 0.19 % (w/w) to about 40 % (w/w), about 0.2 % (w/w) to about 40 % (w/w), about 0.21 % (w/w) to about 40 % (w/w), about 0.22 % (w/w) to about 40 % (w/w), about 0.23 % (w/w) to about 40 % (w/w), about 0.24 % (w/w) to about 40 % (w/w), about 0.25 % (w/w) to about 40 % (w/w), about 0.26 % (w/w) to about 40 % (w/w), about 0.27 % (w/w) to about 40 % (w/w), about 0.28 % (w/w) to about 40 % (w/w), about 0.29 % (w/w) to about 40 % (w/w), about 0.30 % (w/w) to about 40 % (w/w), about 0.31 % (w/w) to about 40 % (w/w), about 0.32 % (w/w) to about 40 % (w/w), about 0.33 % (w/w) to about 40 % (w/w), about 0.34 % (w/w) to about 40 % (w/w), about 0.35 % (w/w) to about 40 % (w/w), about 0.36 % (w/w) to about 40 % (w/w), about 0.37 % (w/w) to about 40 %
(w/w), about 0.38 % (w/w) to about 40 % (w/w), about 0.39 % (w/w) to about 40 %
(w/w), about 0.40 % (w/w) to about 40% (w/w), about 0.41 % (w/w) to about 40 % (w/w), about 0.42 % (w/w) to about 40 % (w/w), about 0.43 % (w/w) to about 40 % (w/w), about 0.44 % (w/w) to about 40 % (w/w), about 0.45 % (w/w) to about 40 % (w/w), about 0.46 % (w/w) to about 40 % (w/w), about 0.47 % (w/w) to about 40 % (w/w), about 0.48 % (w/w) to about 40 % (w/w), about 0.49 % (w/w) to about 40 % (w/w), about 0.50 % (w/w) to about 40 % (w/w), about 0.51 % (w/w) to about 40 % (w/w), about 0.52 % (w/w) to about 40 % (w/w), about 0.53 % (w/w) to about 40 % (w/w), about 0.54 % (w/w) to about 40 % (w/w), about 0.55 % (w/w) to about 40 % (w/w), about 0.56 % (w/w) to about 40 % (w/w), about 0.57 % (w/w) to about 40 % (w/w), about 0.58 % (w/w) to about 40 %
(w/w), about 0.59 % (w/w) to about 40 % (w/w), about 0.60 % (w/w) to about 40 %
(w/w), about 0.61 % (w/w) to about 40 % (w/w), about 0.62 % (w/w) to about 40 %
(w/w), about 0.63 % (w/w) to about 40 % (w/w), about 0.64 % (w/w) to about 40 % (w/w), about 0.65 % (w/w) to about 40 % (w/w), about 0.66 % (w/w) to about 40 %
(w/w), about 0.67 % (w/w) to about 40 % (w/w), about 0.68 % (w/w) to about 40 %
(w/w), about 0.69 % (w/w) to about 40 % (w/w), about 0.70 % (w/w) to about 40 %
(w/w), about 0.71 % (w/w) to about 40 % (w/w), about 0.72 % (w/w) to about 40 %
(w/w), about 0.73 % (w/w) to about 40 % (w/w), about 0.74 % (w/w) to about 40 %
(w/w), about 0.75 % (w/w) to about 40 % (w/w), about 0.76 % (w/w) to about 40 % (w/w), about 0.77 % (w/w) to about 40 % (w/w), about 0.78 % (w/w) to about 40 %
(w/w), about 0.79 % (w/w) to about 40 % (w/w), about 0.80 % (w/w) to about 40 %
(w/w), about 0.81 % (w/w) to about 40 % (w/w), about 0.82 % (w/w) to about 40 %
(w/w), about 0.83 % (w/w) to about 40 % (w/w), about 0.84 % (w/w) to about 40 %
(w/w), about 0.85 % (w/w) to about 40 % (w/w), about 0.86 % (w/w) to about 40 %
(w/w), about 0.87 % (w/w) to about 40 % (w/w), about 0.88 % (w/w) to about 40 %
(w/w), about 0.89 % (w/w) to about 40 % (w/w), about 0.90 % (w/w) to about 40 %
(w/w), about 0.91 % (w/w) to about 40 % (w/w), about 0.92 % (w/w) to about 40 %
(w/w), about 0.93 % (w/w) to about 40 % (w/w), about 0.94 % (w/w) to about 40 %
(w/w), about 0.95 % (w/w) to about 40 % (w/w), about 0.96 % (w/w) to about 40 %
(w/w), about 0.97 % (w/w) to about 40 % (w/w), about 0.98 % (w/w) to about 40 %
(w/w), about 0.99 % (w/w) to about 40 % (w/w) or any combinations thereof.
[0077] In some aspects, the therapeutic composition comprise a thiazine dye in the range of about 1.0 x 107 % (w/w) to about 1 % (w/w), and phosphoric acid in the range of about 1.0 x 105 % (w/w) to about 40 % (w/w), about 5.0 x 105 % (w/w) to about 37 % (w/w), about 1.0 x 104 % (w/w) to about 35 % (w/w), about 5.0 x 104 % (w/w) to about 30 % (w/w), about 0.001 % (w/w) to about 25 % (w/w), about 0.005 % (w/w) to about 20 % (w/w), about 0.01 % (w/w) to about 15 % (w/w), about 0.02 % (w/w) to about 10 %
(w/w), about 0.03 % (w/w) to about 5 % (w/w), about 0.04 % (w/w) to about 2 % (w/w), about 0.05 % (w/w) to about 1 % (w/w), about 0.06 % (w/w) to about 0.5 % (w/w), about 0.07 % (w/w) to about 0.2 % (w/w), about 0.08 % (w/w) to about 0.1 % (w/w) or any combinations thereof.
[0078] In some aspects, the therapeutic composition comprise a thiazine dye in the range of about 0.01% (w/w) to about 3% (w/w), about 0.1% (w/w) to about 2% (w/w), about 0.3 % (w/w) to about 1.5 % (w/w), about 0.5% (w/w) to about 1 % (w/w) and phosphoric acid in the range of about 0.01% (w/w) to about 3% (w/w), about 0.1% (w/w) to about 2% (w/w), about 0.3 % (w/w) to about 1.5 % (w/w), about 0.5% (w/w) to about 1%
(w/w), or any combinations thereof. In some aspects, the thiazine dye is methylene blue.
[0079] In some aspects, the association product is bis ammonium salt of formula I
Figure imgf000017_0001
3-N3,N3 -dimethylamino-phenthiazininium chloride-7-ammonium phosphate
Formula L
[0080] In some aspects, the thiazine dye is blue, azure A, azure B, toluidine blue 0, thiazine, and the like.
[0081] In some aspects, the therapeutic compositions further comprise water as solvent.
[0082] In some aspects, the therapeutic compositions are in form of an aqueous solution.
[0083] In some aspects, the aqueous solution comprises a biocompatible thickening agent. In some aspects the thickening agent is sodium carboxymethyl cellulose.
[0084] In some aspects, therapeutic compositions comprising thiazine dye and phosphoric acid can be, e.g., a mouthwash, a nasal spray, a lozenge, an inhalable therapeutic composition, a topical cream, an emulsion, a gel, or lotion, or an acute or chronic wound treatment liquid, gel, spray.
[0085] In another aspect, the aqueous solution can be further processed to form a bandage or wound dressing. In some aspects, wound dressing is a cloth, a foam, a film, a hydrocolloid, a hydrogel, an alginate or a collagen.
[0086] .In some aspects, therapeutic compositions comprising thiazine dye and phosphoric acid can be used in first aid applications for open wounds as the therapeutic compositions provided herein are antibacterial, antiviral, antifungal, anti-parasitic and/or anti-inflammatory and stop bleeding at the site of application and promote healing. For example, therapeutic compositions comprising thiazine dye and phosphoric acid may be used in conjunction with wound dressings such as band aids, surgical sutures, wound gauzes, surgical sponges (e.g., bioabsorbable sponges), and the like. In some aspects wound dressings may be pretreated with therapeutic compositions comprising thiazine dye and phosphoric acid.
[0087] In some aspects, therapeutic compositions comprising thiazine dye and phosphoric acid can be administered prophylactically in order to reduce the risk of infection by microbes, such as bacteria, viruses, fungi and parasites. In some aspects, prophylactic administration of compositions comprising thiazine dye and phosphoric acid protects the airways (e.g., nasal passages, sinuses, bronchial tubes, and/or lungs) and/or mouth and/or throat of a subject from infection and inflammation as the composition has no cell toxicity. .
[0088] In some aspects, therapeutic compositions comprising thiazine dye and phosphoric acid can be administered in order to treat microbial infection and/or inflammation of the airways, mouth and/or throat of a subject.
[0089] In some aspects, therapeutic compositions comprising thiazine dye and phosphoric acid can be administered in order to promote regeneration of damaged tissue and/or a bone, increase bone density at the site of application, and induce new bone formation.
[0090] In some aspects, therapeutic compositions comprising thiazine dye and phosphoric acid may contain one or more essential oils, dried or fresh fruit particles, suspending agents, surfactants, emollients, emulsifiers, and/or cationic polymers, e.g., as disclosed in US Publication No. 2005/0031573, which is incorporated by reference herein in its entirety.
III. Bioabsorbable Sponges
[0091] The present antimicrobial and anti-inflammatory bioabsorbable sponge composition can be used in conjunction with a wide variety of human and veterinary surgical interventions such as hemostasis, detection of bacterial infection, enhancement of soft tissue healing, enhancement of bone density and/or bone formation at the surgical site, enhancement of angiogenesis at the surgical site, and the like. The present bioabsorbable sponge can be also used as a foam layer in a bandage or antibacterial and anti-inflammatory dressing for treatment of chronic, non-healing wounds such as chronic ulcers, diabetic lesions, and the like. The present bioabsorbable sponge can be also used as first aid in remote or battlefield regions, as well as for post extraction wound care in dentistry, for bacterial detection in wounds, and the like.
[0092] In various aspects, the bioabsorbable sponge is non-immunogenic.
[0093] Illustrative bioabsorbable sponges can have a gelatin matrix, a collagen matrix, a poly-L-lactic acid matrix, a polyglycolic matrix, a poly(lactic-co-glycolic)acid matrix and the like. Particularly well suited for present purposes is a bioabsorbable sponge commercially available under the designation GEL-FOAM® from Pfizer, Inc. as a water- insoluble, porous, pliable product prepared from purified pork skin gelatin USP. [0094] In some aspects, the bioabsorbable sponge is impregnated with an aqueous phosphoric acid solution. In some aspects, the aqueous phosphoric acid solution constitutes about 0.1 to about 40 percent by weight of the aqueous solution. In some aspects, the aqueous phosphoric acid solution constitutes about 0.1 to about 2.5 percent by weight of the aqueous solution. In some aspects, the aqueous phosphoric acid solution constitutes about 5 to about 40 percent by weight of the sponge. In some aspects, the aqueous phosphoric acid solution constitutes about 10 to about 30 percent by weight of the sponge.
[0095] In some aspects, the aqueous phosphoric acid solution contains a thiazine dye, such as methylene blue, azure A, azure B, toluidine blue 0, thianine, and the like. A preferred thiazine dye is methylene blue (3,7-bis(dimethylamine)-phenothiazin-5-ium chloride). In some aspects, the thiazine dye, especially methylene blue, exerts anti microbial activities.
[0096] In some aspects, the bioabsorbable sponge is impregnated with an aqueous solution comprising a thiazine dye and phosphoric acid.
[0097] In some aspects, the bioabsorbable sponge is impregnated with an aqueous solution comprising methylene blue and phosphoric acid.
[0098] In various aspects, the thiazine dye is present in the aqueous phosphoric acid solution of the bioresorbable composition in an amount in the range of about 0.0001 to about 1 weight percent, based on the weight of the solution. In various aspects, the thiazine dye is present in the aqueous phosphoric acid solution of the bioresorbable composition in an amount in the range of about 0.1 to about 1 weight percent, based on the weight of the solution. In various aspects, the thiazine dye is present in the aqueous phosphoric acid solution of the bioresorbable composition in an amount in the range of about 0.01 to about 0.1 weight percent, based on the weight of the solution. In some aspects, the thiazine dye is released from the bioabsorbable sponge to a surrounding tissue as the bioresorbable sponge is liquefied and absorbed by the surrounding tissue. In some aspects, the thiazine dye serves as a color indicator for bacteria present in a wound because the bacteria will be stained dark or black by the thiazine dye. In some aspects, tissues containing bacteria can be surgically removed before a surgical procedure is performed and/or before a wound is closed. [0099] In some aspects, methylene blue is present in the aqueous phosphoric acid solution of the bioresorbable composition in an amount in the range of about 0.0001 to about 1 weight percent, based on the weight of the solution. In some aspects, methylene blue is present in the aqueous phosphoric acid solution of the bioresorbable composition in an amount in the range of about 0.1 to about 1 weight percent, based on the weight of the solution. In some aspects, methylene blue is present in the aqueous phosphoric acid solution of the bioresorbable composition in an amount in the range of about 0.01 to about 0.1 weight percent, based on the weight of the solution. In some aspects, methylene blue is released from the bioabsorbable sponge to a surrounding tissue as the bioresorbable sponge is liquefied and absorbed by the surrounding tissue,
[0100] In some aspects, the bioresobable sponge comprises gelatin.
[0101] In some aspects, the bioresobable sponge comprises collagen.
[0102] In some aspects, the bioresobable sponge comprises poly-L-lactic acid. In some aspects, the bioresobable sponge comprises poly-glycolic acid. In some aspects, the bioresobable sponge comprises poly(lactic-co-glycolic)acid,
[0103] In some aspects, the compositions disclosed herein are used for wound pretreatment. In some aspects, a biocompatible thickener can be added to an aqueous acid solution and the phosphoric acid content in the solution can be in the range of about 20 to about 40 weight percent, based on the weight of the solution. In various aspects, a thickened aqueous phosphoric acid solution has a viscosity of about 100 to about 2000 contipoises (cp). In various aspects, the thickener is sodium carboxymethyl cellulose having an average degree of substitution of about 21 % to about 33%.
IV. Textile Materials
[0104] As used herein, the term "apparel" means textile wearing apparel positionable over the mouth and nasal passages of a human, e.g., a mask, a turtle neck, a scarf, a bandana, and the like, as well as a full or partial body covering.
[0105] As used herein, the term "textile" means a fibrous artifact made by weaving, felting or crocheting natural fibers, synthetic fibers, and mixtures thereof.
[0106] A textile material suitable as a substrate for the thiazine dye and phosphoric acid is hydrophilic and fibrous, and sufficiently air permeable or porous to permit a human to breathe therethrough. The textile material can be woven material or a non-woven material. Preferred are non-woven materials made of cotton, cellulose, polypropylene, and the like, having a density of about 20 to 25 grams per square meter, and filtering particles having a size of 1 micron and larger. In various aspects, the textile material filters particles having a size of 0.3 microns and larger.
[0107] In various aspects, the amount of thiazine dye distributed within the fibrous substrate of the textile material is in the range of about 0.001 to about 1 grams per cubic centimeter (cm3) of the substrate. In various aspects, the amount of thiazine dye distributed within the fibrous substrate of the textile material is in the range of about 0.1 to about 0.5 grams per cubic centimeter of the substrate.
[0108] In various aspects, the amount of phosphoric acid distributed within the fibrous substrate of the textile material is in the range of 0.001 to about 1 grams per cubic centimeter of the substrate.
[0109] In various aspects, an equimolar amount of thiazine dye and phosphoric acid is present in the fibrous substrate. The thiazine dye may be methylene blue.
[0110] Referring to Fig. 10, face mask 10 has an outer layer 12 made of water repellant cotton, an intermediate layer 14 made of non-woven cotton, an inner layer 16 made of silk. Intermediate layer 14 is hydrophilic and provides a fibrous substrate into which methylene blue and phosphoric acid are distributed. Aperture 18 in outer layer 12 is provided for attachment of an appropriate securing strap.
[0111] During use on a facial covering, the antiviral textile material is moistened by exhaled moisture. In some instances, especially when ambient humidity is relatively low, it is desirable to moisten the facial covering. This can be achieved by applying a mist or spray of water to the facial covering prior to use. Alternatively, an antiviral solution containing a thiazine dye and phosphoric acid can be applied to the facial covering prior to use or at intervals during use, depending on ambient conditions. In various aspects, the antiviral solution suitable for this purpose contains about 0.1 to about 5 weight percent of phosphoric acid based on the weight of the antiviral solution. In various aspects, the antiviral solution contains about 0.75 to about 2.5 weight percent based on the weight of the antiviral solution. In various aspects, the antiviral thiazine dye is present in the aqueous solution in an amount in the range of about 0.01 to about 1 weight percent based on the weight of the antiviral solution. [0112] In some aspects, the textile material comprises a hydrophilic fibrous substrate that is located between an outer water-repellant cotton layer and an inner silk layer. In some aspects, the outer water-repellant cotton layer retains exhaled moisture.
[0113] Without wanting to be bound by theory it is hypothesized that the composition comprising methylene blue, phosphoric acid and its association product induce a fast onset of cytogenesis, potent angiogenesis, complete osteogenesis, physiological ossification, mobilization of hydroxyapatite to a site of new bone formation, acceleration of the extrinsic pathway of coagulation, stimulation of bone and soft tissue bio regeneration and control of bleeding. Importantly, the above effects were observed after single administration of a composition comprising methylene blue, phosphoric acid and its association product to a wound or surgery site.
V. Use and Methods of Treatment Aspects
[0114] The therapeutic compositions described herein can be used for the treatment of bacterial, viral, fungal, and parasitic infections, for the treatment of a wound, for regenerating bone, and for prevention of an infection, e.g., a respiratory infection.
[0115] In some aspects, the therapeutic compositions are used for the treatment of a bacterial infection. In some aspects, the therapeutic composition is used for the treatment of an acute infection or a chronic infection. In some aspects, the infection is an infection of a body surface, e.g., a skin or mucous membrane. In some aspects, the infection is an infection of a surgical site. In some aspects, the infection is an infection of a fractured bone. In some aspects, the infection is an infection of the respiratory system.
[0116] In some aspects, the infection is a bacterial infection and is caused by is infected with Acinetobacter baumannii, Actinomyces sp., Actinobacillus actinomycetemcomitans, Aeromonas sp., Anaplasma phagocytophilum, Alcaligenes xylosoxidans, Actinobacillus actinomycetemcomitans, Bacillus sp., Bacteroides sp., Bartonella sp. Bifidobacterium sp., Bordetella sp., Borrelia sp., Brucella sp, Burkholderia sp., Campylobacter sp., Capnocytophaga sp., Cardiobacterium hominis, Chlamydia trachomatis, Chlamydophila pneumonia, Chlamydophila psittaci, Citrobacter sp. Coxiella burnetii, Corynebacterium sp., Clostridium sp., Eikenella corrodens, Enterobacter sp., Enterococcus sp., Ehrlichia sp., Erysipelothrix rhusiopathiae, Eubacterium sp., Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Gemella morbillorum, Haemophilus sp, Helicobacter sp., Kingella kingii, Klebsiella sp., Lactobacillus sp., Listeria monocytogenes, Leptospira interrogans, Legionella pneumophila, Leptospira interrogans, Peptostreptococcus sp., Moraxella catarrhalis, Morganella sp., Mobiluncus sp., Micrococcus sp., Mycobacterium sp., Mycoplasm sp., Nocardia sp., Neisseria sp., Pasteurella multocida, Plesiomonas shigelloides, Prevotella sp., Prevotella intermedia, Porphyromonas sp., Prevotella melaninogenica, Proteus sp., Providencia sp., Pseudomonas aeruginosa, Propionibacterium acnes, Rhodococcus equi, Salmonella sp., Serratia sp, Shigella sp., Staphylococcus sp., Streptococcus sp., Spirillum minus, Streptobacillus moniliformi, Treponema sp., Tropheryma whippelii, Ureaplasma urealyticum, Veillonella sp., Vibrio sp., Yersinia sp. or Xanthomonas maltophilia.
[0117] In some aspects, the infection is a viral infection and is caused by an Adenovirus,
Bocavirus, Coronavirus, Enterovirus, Metapneumovirus, Parainfluenza virus 1-4, Respiratory Syncytial Virus A and B, Rhinovirus, Herpesvirus, Varicella zoster virus, Epstein-Barr Virus, Influenza, human Parechovirus, Measles virus, Middle East Respiratory Syndrome coronavirus (MERS CoV), Mumps virus, or Severe Acute Respiratory Syndrome Coronavirus (SARS CoV).
[0118] In some aspects, the infection is a fungal infection and is caused by Candida albicans, C. glabrata, C. dubliniensis, C. krusei, C. parapsilosis, C.tropicalis, C. orthopsilosis, C. guilliermondii, C. rugosa, C. auris, C. lusitaniae, Aspergillus fumigatus, A. flavus, A. terreus, A. niger, A. candidus, A. clavatus, or A. ochraceus.
[0119] In some aspects, the infection is a parasitic infection and is caused by
Trypanosoma, Leishmania, Toxoplasma, Eimeria, Neospora, Cyclospora and Cryptosporidia families. Parasites that cause Microsporidial infections, Malaria, visceral leishmaniasis often known as kalaazar, African sleeping sickness, toxoplasmosis, giardiasis and Chagas' disease , Plasmodium vivax, Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Trypanosoma protoza, Entamoeba histolytica, Trichomonas vaginalis, Giardia lamblia, Trypanosoma brucei gambiense, Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania major, Leishmania tropica, Leishmania aethiopica, Leishmania infantum, Leishmania braziliensis, Leishmania mexicana, Leishmania amazonensis, Leishmania donovani-Leishmania infantum complex, Cryptosporidium parvum, Toxoplasma gondii, Encephalitozoon species, Nosema species and Septata intestinalis. [0120] In some aspects, the methods of treatment described herein comprise administration of a therapeutic composition comprising methylene blue, phosphoric acid and/or an association product thereof to a subject in need thereof.
[0121] In some aspects, the therapeutic composition is administered by direct contact of a body surface, a surgical site and/or a fractured bone.
[0122] In some aspects, the therapeutic composition is administered by direct contact of a body surface, a surgical site and/or a fractured bone with an aqueous solution of the therapeutic composition.
[0123] In some aspects, the therapeutic composition is administered by direct contact of a body surface, a surgical site and/or a fractured bone with a bioabsorbable composition. In some aspects, the therapeutic composition is administered by direct contact of a body surface, a surgical site and/or a fractured bone with a sponge containing an aqueous solution of the therapeutic composition.
[0124] In some aspects, the therapeutic composition is administered by direct contact of a body surface, a surgical site and/or a fractured bone with a textile material containing an aqueous solution of the therapeutic composition.
[0125] In some aspects, the therapeutic composition is administered as a lotion, gel, topical cream, spray, spray.
[0126] In some aspects, the therapeutic composition is injected into an infected tissue or organ. In some aspects, the therapeutic composition is injected into an infected surgical site and/or a fractured bone.
[0127] In some aspects, the therapeutic composition is administered by inhalation.
[0128] In some aspects, the therapeutic composition is administered at a concentration of methylene blue of 1.0 x 107— 1.0 % (w/w), and a concentration of phosphoric acid of 1.0 x 105-2.0 % (w/w).
[0129] In some aspects, the therapeutic composition is administered at a concentration of methylene blue of 1.0 x 107 0.25 % (w/w), and a concentration of phosphoric acid of 1.0 x 105-2.0 % (w/w).
[0130] In some aspects, the therapeutic composition is administered as an aqueous solution with a concentration of methylene blue of 1.0 x 107— 1.0 % (w/w), and a concentration of phosphoric acid of 1.0 x 105-2.0 % (w/w). [0131] In some aspects, the therapeutic composition is administered as an aqueous solution with a concentration of methylene blue of 1.0 x 107-0.25 % (w/w), and a concentration of phosphoric acid of 1.0 x 105-2.0 % (w/w).
[0132] In some aspects, the therapeutic composition is administered as a topical cream with a concentration of methylene blue of 1.0 x 107— 1.0 % (w/w), and a concentration of phosphoric acid of 1.0 x 105-2.0 % (w/w).
[0133] In some aspects, the therapeutic composition is administered as a topical cream with a concentration of methylene blue of 1.0 x 107-0.25 % (w/w), and a concentration of phosphoric acid of 1.0 x 105-2.0 % (w/w).
[0134] In some aspects, the therapeutic composition is administered as a gel with a concentration of methylene blue of 1.0 x 107— 1.0 % (w/w), and a concentration of phosphoric acid of 1.0 x 105-2.0 % (w/w).
[0135] In some aspects, the therapeutic composition is administered as a gel with a concentration of methylene blue of 1.0 x 107-0.25 % (w/w), and a concentration of phosphoric acid of 1.0 x 105-2.0 % (w/w).
[0136] In some aspects, the therapeutic composition is administered as a mouthwash with a concentration of methylene blue of 1.0 x 107— 1.0 % (w/w), and a concentration of phosphoric acid of 1.0 x 105-2.0 % (w/w).
[0137] In some aspects, the therapeutic composition is administered as a mouthwash with a concentration of methylene blue of 1.0 x 107-0.25 % (w/w), and a concentration of phosphoric acid of 1.0 x 105-2.0 % (w/w).
[0138] In some aspects, the therapeutic composition is administered as a spray with a concentration of methylene blue of 1.0 x 107— 1.0 % (w/w), and a concentration of phosphoric acid of 1.0 x 105-2.0 % (w/w).
[0139] In some aspects, the therapeutic composition is administered as a spray with a concentration of methylene blue of 1.0 x 107-0.25 % (w/w), and a concentration of phosphoric acid of 1.0 x 105-2.0 % (w/w).
[0140] In some aspects, the therapeutic composition is administered as an inhalant with a concentration of methylene blue of 1.0 x 107— 1.0 % (w/w), and a concentration of phosphoric acid of 1.0 x 105-2.0 % (w/w). [0141] In some aspects, the therapeutic composition is administered as an inhalant with a concentration of methylene blue of 1.0 x 107-0.25 % (w/w), and a concentration of phosphoric acid of 1.0 x 105-2.0 % (w/w).
[0142] In some aspects, the therapeutic composition is administered as a bioabsorbable composition with a concentration of methylene blue of 1.0 x 107— 1.0 % (w/w), and a concentration of phosphoric acid of 1.0 x 105-2.0 % (w/w).
[0143] In some aspects, the therapeutic composition is administered as bioabsorbable composition with a concentration of methylene blue of 1.0 x 107-0.25 % (w/w), and a concentration of phosphoric acid of 1.0 x 105-2.0 % (w/w).
[0144] In some aspects, the therapeutic composition is administered as a bioabsorbable composition with a concentration of phosphoric acid in the range of about 0.1 percent to about 40 percent by weight.
[0145] In some aspects, the therapeutic composition is administered as a bioabsorbable composition with a concentration of phosphoric acid in the range of about 0.1 percent to about 40 percent by weight and a concentration of methylene blue in the range of about 0.0001 to about 1 percent by weight.
[0146] In some aspects, the therapeutic composition is administered as a bioabsorbable composition with a concentration of phosphoric acid in an amount in the range of about 0.1 to about 1 percent by weight and a concentration of methylene blue in an amount in the range of about 0.0001 percent by weight to about 1 percent by weight,
[0147] In some aspects, the therapeutic composition is administered as a textile material with a concentration of methylene blue in an amount in the range of 0.001 to about 1 grams per cubic centimeter of the substrate of the textile material and phosphoric acid in an amount in the range of about 0.001 to about 1 grams per cubic centimeter of the substrate of the textile material.
Example 1: Therapeutic Formulation #1
[0148] A therapeutic formulation in accordance with aspects of the invention is prepared as follows.
[0149] Methylene Blue Solution A is prepared by diluting 85 mΐ of a 1% (w/v) methylene blue solution in water to a total volume of 1000 ml, resulting in a Methylene Blue Solution A concentration of 8.5 x 105 % (w/v). [0150] Phosphoric Acid Solution A is prepared by diluting 1 g of 85% (w/w) phosphoric acid in water to a total volume of 850 ml, resulting in a Phosphoric Acid Solution A concentration of 1.0 x 10 1 % (w/v).
[0151] Therapeutic Formulation #1 is created by combining 86.6 g of Methylene Blue
Solution A, 0.2 g of Phosphoric Acid Solution A, and 13.2 g of water. The resulting composition comprises methylene blue at a final concentration of approximately 7.4 x 10
5 % (w/w) and phosphoric acid at a final concentration of approximately 2.0 x 103 % (w/w). We have found that this composition has bactericidal, antiviral, regenerative, and anti-inflammatory properties in human and animal subjects.
Example 2: Therapeutic Formulation #2
[0152] A therapeutic formulation in accordance with aspects of the invention is prepared as follows.
[0153] Methylene Blue Solution B is prepared by diluting 3.0 mΐ of a 1% (w/v) methylene blue solution in water to a total volume of 1000 ml, resulting in a Methylene Blue Solution B concentration of 3.0 x 106 % (w/v).
[0154] Phosphoric Acid Solution A is prepared by diluting 1 g of 85% (w/w) phosphoric acid in water to a total volume of 850 ml, resulting in a Phosphoric Acid Solution A concentration of 1.0 x 10 1 % (w/v).
[0155] Therapeutic Formulation #2 is created by combining 86.6 g of Methylene Blue
Solution B, 0.2 g of Phosphoric Acid Solution A, and 13.2 g of water. The resulting composition comprises methylene blue at a final concentration of approximately 2.6 x 10
6 % (w/w) and phosphoric acid at a final concentration of approximately 2.0 x 103 % (w/w). This composition has the same constituents as in Example 1, but the concentration of methylene blue is much lower than in Example 1. Even when the concentration of methylene blue has the concentrations of this Example 2, we have found that this composition has bactericidal, antiviral, regenerative, and anti-inflammatory properties in human and animal subjects.
Example 3: Mouthwash Formulation #1
[0156] An oral hygiene mouthwash in accordance with aspects of the invention was prepared as follows. [0157] Methylene Blue Solution A was prepared by diluting 85 mΐ of a 1% (w/v) methylene blue solution in water to a total volume of 1000 ml, resulting in a Methylene Blue Solution A concentration of 8.5 x 105 % (w/v).
[0158] Phosphoric Acid Solution A was prepared by diluting 1 g of 85% (w/w) phosphoric acid in water to a total volume of 850 ml, resulting in a Phosphoric Acid Solution A concentration of 1.0 x 10 1 % (w/v).
[0159] Mouthwash #1 was created by first combining the materials of Phase A, shown in
Table 1, below. The Phase A mixture was then combined with the material of Phase B, shown in Table 1, below.
Table 1: Mouthwash #! Formulation
Figure imgf000028_0001
[0160] The resulting mouthwash comprises methylene blue at a final concentration of approximately 7.4 x 105 % (w/w) and phosphoric acid at a final concentration of approximately 2.0 x 103 % (w/w).
[0161] We have found that this composition has bactericidal, antiviral, regenerative, and anti-inflammatory properties in human and animal subjects.
Example 4: Mouthwash Formulation #2
[0162] An oral hygiene mouthwash in accordance with aspects of the invention was prepared as follows. [0163] Methylene Blue Solution B was prepared by diluting 3.0 mΐ of a 1% (w/v) methylene blue solution in water to a total volume of 1000 ml, resulting in a Methylene Blue Solution B concentration of 3.0 x 106 % (w/v).
[0164] Phosphoric Acid Solution A was prepared by diluting 1 g of 85% (w/w) phosphoric acid in water to a total volume of 850 ml, resulting in a Phosphoric Acid Solution A concentration of 1.0 x 10 1 % (w/v).
[0165] Mouthwash #2 was created by first combining the materials of Phase A shown in
Table 2, below. The Phase A mixture was then combined with the material of Phase B, shown in Table 2, below.
Table 2: Mouthwash #2 Formulation
Figure imgf000029_0001
[0166] The resulting mouthwash comprises methylene blue at a final concentration of approximately 2.6 x 106 % (w/w) and phosphoric acid at a final concentration of approximately 2.0 x 103 % (w/w).
[0167] Even when the concentration of methylene blue has the concentrations of this
Example 4, we have found that this composition has bactericidal, antiviral, regenerative, and anti-inflammatory properties in human and animal subjects.
Example 5: Nasal Spray Formulation
[0168] A therapeutic nasal spray in accordance with aspects of the invention was prepared as follows. [0169] Methylene Blue Solution A was prepared by diluting 85 mΐ of a 1% (w/v) methylene blue solution in water to a total volume of 1000 ml, resulting in a Methylene Blue Solution A concentration of 8.5 x 105 % (w/v).
[0170] Phosphoric Acid Solution A was prepared by diluting 1 g of 85% (w/w) phosphoric acid in water to a total volume of 850 ml, resulting in a Phosphoric Acid Solution A concentration of 1.0 x 10 1 % (w/v).
[0171] The therapeutic nasal spray was created by first combining the materials of Phase
A, shown in Table 3, below. Next, the materials of Phase B, shown below in Table 3, were combined. Finally, the Phase A mixture was combined with the Phase B mixture.
Table 3: Nasal Spray Formulation
Figure imgf000030_0001
[0172] The resulting nasal spray comprises methylene blue at a final concentration of approximately 4.2 x 106 % (w/w) and phosphoric acid at a final concentration of approximately 3.0 x 103 % (w/w). [0173] We have found that this composition has bactericidal, antiviral, regenerative, and anti-inflammatory properties in human and animal subjects.
Example 6: Use of Therapeutic Formulation #1 to Reduce the Number of Microorganisms in a Patient’s Mouth During Oral Procedures
[0174] To reduce the number of microorganisms, including viruses, in a patient’s mouth during oral procedures, the patient is administered the therapeutic formulation of Example
1 just prior to an oral procedure, thereby decreasing the risk of infection for the patient and decreasing the risk of cross infection to health care staff. It reduces periodontal infections and gingival swelling (gingivitis) induced by oral pathogens.
[0175] Just prior to an oral procedure, the patient vigorously swishes 10 ml of the therapeutic formulation of Example 1 around their mouth for 1 minute and then spits it out.
Example 7: Use of Therapeutic Formulation #2 to Reduce the Number of Microorganisms in a Patient’s Mouth During Oral Procedures
[0176] To reduce the number of microorganisms, including viruses, in a patient’s mouth during oral procedures, the patient is administered the therapeutic formulation of Example
2 just prior to an oral procedure, thereby decreasing the risk of infection for the patient and decreasing the risk of cross infection to health care staff. It should be used immediately after the treatment to avoid post-treatment bleeding, inflammation, swelling and pain, and to reduce the time of the healing.
[0177] Just prior to an oral procedure, the patient vigorously swishes 10 ml of the therapeutic formulation of Example 2 around their mouth for 1 minute and then spits it out.
Example 8: Use of Mouthwash #1 to Reduce the Number of Microorganisms in a Patient’s Mouth During Oral Procedures
[0178] To reduce the number of microorganisms, including viruses, in a patient’s mouth during oral procedures, the patient is administered the mouthwash of Example 3 just prior to an oral procedure, thereby decreasing the risk of infection for the patient and decreasing the risk of cross infection to health care staff.
[0179] Just prior to an oral procedure, the patient vigorously swishes 10 ml of the mouthwash of Example 3 around their mouth for 1 minute and then spits it out. Example 9: Use of Mouthwash #2 to Reduce the Number of Microorganisms in a Patient’s Mouth During Oral Procedures
[0180] To reduce the number of microorganisms, including viruses, in a patient’s mouth during oral procedures, the patient is administered the mouthwash of Example 4 just prior to an oral procedure, thereby decreasing the risk of infection for the patient and decreasing the risk of cross infection to health care staff.
[0181] Just prior to an oral procedure, the patient vigorously swishes 10 ml of the mouthwash of Example 4 around their mouth for 1 minute and then spits it out.
Example 10: Use of Nasal Spray to Prophylactically Reduce the Risk of Infection
[0182] In order to prophylactically reduce the risk of infection, for example, during a pandemic, a human subject administers the nasal spray formulation of Example 5 into each nostril.
[0183] To reduce the risk of bacterial and viral infection, the human subject administers
0.1-1.0 ml of the nasal spray formulation of Example 5 into each nostril at least once per day. The nasal spray is administered using nasal spray devices well-known in the art.
The nasal spray formulation coats the lining of the nasal passages, sinuses, and other proximal airway tissues, exerting bactericidal and antiviral effects on these membranes and tissues. The formulation reduces inflammation-induced secretion and opens air passages.
Example 11 : Use of Nasal Spray to Prophylactically Reduce the Risk of Infection
[0184] In order to prophylactically reduce the risk of infection, for example, during a pandemic, a human subject administers the nasal spray formulation of Example 5 into each nostril.
[0185] To reduce the risk of bacterial and/or viral infection, the human subject administers 0.1-1.0 ml of the nasal spray formulation of Example 5 into each nostril at least once per day as needed. The nasal spray is administered using nasal spray devices well-known in the art. The nasal spray formulation coats the lining of the nasal passages, sinuses, and other proximal airway tissues, exerting bactericidal and antiviral effects on these membranes and tissues. Example 12: Use of Nasal Spray to Treat Infection
[0186] In order to treat a human subject having a bacterial and/or viral infection of the nasal passages or upper airways, the nasal spray formulation of Example 5 is administered into each nostril.
[0187] To treat a human subject having a bacterial and/or viral infection, the human subject administers 0.1-1.0 ml of the nasal spray formulation of Example 5 into each nostril at least once per day post-infection for up to four weeks. The nasal spray is administered using nasal spray devices well-known in the art. The nasal spray formulation coats the lining of the nasal passages, sinuses, and other proximal airway tissues, exerting bactericidal, antiviral, anti-inflammatory, and regenerative effects on these membranes and tissues.
Example 13: Use of Inhaled Formulation to Prophylactically Reduce the Risk of Infection
[0188] In order to prophylactically reduce the risk of infection, for example, during a pandemic, a human subject inhales the nasal spray formulation of Example 5.
[0189] To reduce the risk of bacterial and/or viral infection, the human subject inhales
0.1-1.0 ml of the nasal spray formulation of Example 5 at least once per day as needed using inhaler or nebulizer devices well-known in the art. The nasal spray formulation coats the lining of the bronchial tubes and lungs, exerting bactericidal and antiviral effects on these membranes and tissues.
Example 14: Use of Inhaled Formulation Spray to Treat Infection
[0190] In order to treat a human subject having a bacterial and/or viral infection of the bronchial tubes or lungs, the nasal spray formulation of Example 5 is inhaled by the human subject.
[0191] To treat a human subject having a bacterial and/or viral infection, the human subject inhales 0.1-1.0 ml of the nasal spray formulation of Example 5 at least once per day post-infection for up to four weeks using inhaler or nebulizer devices well-known in the art. The nasal spray formulation coats the lining of the bronchial tubes and lungs, exerting bactericidal, antiviral, anti-inflammatory, and regenerative effects on these membranes and tissues. Example 15: Use of Therapeutic Skin Cream to Cleanse and Regenerate Skin Tissue
[0192] In order to cleanse (sterilize) and regenerate the skin of a human or animal subject, a topical cream comprising methylene blue at a final concentration of approximately 7.4 x 105 % (w/w) and phosphoric acid at a final concentration of approximately 2.0 x 103 % (w/w) is applied to the skin at the area of interest. The topical cream is similar to the formulation of Example 1, but comprises lipids and other topical cream components well-known in the art. The topical cream is applied to the area of interest at least once per day as needed.
[0193] Alternatively, the topical cream comprises methylene blue at a final concentration of approximately 2.6 x 106 % (w/w) and phosphoric acid at a final concentration of approximately 2.0 x 103 %. The topical cream is similar to the formulation of Example 2, but comprises lipids and other topical cream components well-known in the art. The topical cream is applied to the area of interest at least once per day as needed.
Example 16: Use of Therapeutic Skin Cream to Treat Inflammation and/or Infection of the Skin
[0194] In order to treat inflammation and/or infection and/or open acute and non-healing wounds (e.g., diabetics and bed sores) of the skin of a human or animal subject, a topical cream comprising methylene blue at a final concentration of approximately 7.4 x 105 % (w/w) and phosphoric acid at a final concentration of approximately 2.0 x 103 % (w/w) is applied to the skin at the area of interest. The topical cream is similar to the formulation of Example 1, but comprises lipids and other cream components well-known in the art. The topical cream is applied to the area of interest at least once per day as needed.
[0195] Alternatively, the topical cream comprises methylene blue at a final concentration of approximately 2.6 x 106 % (w/w) and phosphoric acid at a final concentration of approximately 2.0 x 103 %. The topical cream is similar to the formulation of Example 2, but comprises lipids and other topical cream components well-known in the art. The topical cream is applied to the area of interest at least once per day as needed.
Example 17: Use of Therapeutic Lozenge to Prophylactically Reduce the Risk of Infection
[0196] In order to prophylactically reduce the risk of infection, for example, during a pandemic, a human subject dissolves one lozenge slowly in the mouth, repeating as needed. The lozenge comprises methylene blue at a final concentration of approximately 7.4 x 105 % (w/w) and phosphoric acid at a final concentration of approximately 2.0 x 10 3 % (w/w). The lozenge may further comprise sugars, starches, and flavorings.
[0197] Alternatively, the lozenge comprises methylene blue at a final concentration of approximately 2.6 x 106 % (w/w) and phosphoric acid at a final concentration of approximately 2.0 x 103 % (w/w).
[0198] The dissolved lozenge coats the lining of the mouth and throat, exerting bactericidal and antiviral effects on these membranes and tissues.
Example 18: Use of Therapeutic Lozenge to Prophylactically Reduce the Risk of Infection
[0199] In order to treat a human subject having a bacterial and/or viral infection of the mouth or throat, a human subject dissolves one lozenge slowly in the mouth, repeating at least once a day as needed. The lozenge comprises methylene blue at a final concentration of approximately 7.4 x 105 % (w/w) and phosphoric acid at a final concentration of approximately 2.0 x 103 % (w/w). Methylene blue also provides a numbing effect. The lozenge may further comprise sugars, starches, and flavorings.
[0200] Alternatively, the lozenge comprises methylene blue at a final concentration of approximately 2.6 x 106 % (w/w) and phosphoric acid at a final concentration of approximately 2.0 x 103 % (w/w).
[0201] The dissolved lozenge coats the lining of the mouth and throat, exerting bactericidal, antiviral, anti-inflammatory, and regenerative effects on these membranes and tissues.
Example 19: Minimum Inhibitory Concentrations (MIC) of Phosphoric Acid and Methylene Blue on Select Bacterial Species
[0202] Starting with an 85% phosphoric acid solution, a 2-fold serial dilution series was generated consisting of 24 members having a phosphoric acid concentration ranging from 85% to 1.0132789612 x 105%. Similarly, starting with a 1% methylene blue solution, a 2-fold serial dilution series was generated consisting of 24 members having a methylene blue concentration ranging from 1% to 1.1920929 x 107%. Each dilution series was prepared using Cation-Adjusted Muller Hinton broth (CAMHB).
[0203] For each dilution series, 0.1 ml of each dilution, in order from most concentrated to least concentrated, was placed into a separate well of a 96-well tissue-culture plate. Select bacterial species were diluted in CAMHB so that 0.1 ml contained approximately 1 x 106 bacteria per ml. Next, 0.1 ml of diluted bacterial suspension was added to each well containing the phosphoric acid or methylene blue. The tissue-culture plates were incubated at 37°C for 24 hours.
[0204] After 24 hours, each well was visually assessed using a light microscope for bacterial growth. For each compound, the dilution having the lowest concentration capable of inhibiting bacterial growth was defined as the MIC. These results are presented in Table 4, below, the percentages being the final concentration of the compound in the 0.2 ml of total liquid in each well.
Table 4: MIC of Methylene Blue and Phosphoric Acid
Figure imgf000036_0001
[0205] In a separate experiment, equal volumes of 85% phosphoric acid and 1% methylene blue were mixed together (1 : 1). Starting with this mixture, a 2-fold serial dilution series was generated using CAMHB consisting of 24 members having a phosphoric acid/methylene blue concentration ranging from 42.5%/0.5% to 5.066394806 x 106%/5.9604645 x 108%, respectively.
[0206] 0.1 ml of each dilution, in order from most concentrated to least concentrated, was placed into an individual well of a 96-well tissue-culture plate. Select bacterial species were diluted in CAMHB so that 0.1 ml contained approximately 1 x 106 bacteria per ml. Next, 0.1 ml of diluted bacterial suspension was added to each well containing the phosphoric acid and methylene blue. The tissue-culture plates were incubated at 37°C for 24 hours.
[0207] After 24 hours, each well was visually assessed using a light microscope for bacterial growth. The dilution with the lowest concentrations capable of inhibiting bacterial growth was defined as the MIC. These results are presented in Table 5, below, the percentages being the final concentration of each compound in the 0.2 ml of total liquid in each well. Table 5: MIC of a Phosphoric Acid/Methylene Blue Mixture
Figure imgf000037_0001
[0208] The protocol, above, was a modification of the microbroth dilution assay of the
Clinical and Laboratory Standard Institute (CLSI), which is incorporated by reference herein in its entirety. CLSI. Approved Standard M7-A6. 6. Clinical and Laboratory Standards Institute; Wayne, PA: 2006. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically.
Example 20: In Vivo Testing of the Bactericidal/Antibacterial Properties of Methylene Blue, Phosphoric Acid, and a Mixture of Phosphoric Acid and Methylene Blue in Rats
[0209] To assess the ability of a mixture of phosphoric acid and methylene blue to prevent infection and promote healing, in vivo experiments were performed on rats. For these experiments, two surgeries were performed on each rat tested.
[0210] In the first surgery, the rear right thigh of each animal was infected by intramuscular injection of 0.2 ml of Pseudomonas aeruginosa ATCC 27853 at a concentration of 3.8 x 105 per ml. Animals were left to develop the infection for 48 hours. All animals developed inflammation (swelling, change of the skin color (bluish, purple)) at the injection site and were limping on the infected leg.
[0211] In the second surgery, under sedation, the infected area was surgically opened
(except for treatment group 5) and the treatments shown in Table 6 were applied onto the entire area of the open wound for the 5 minutes. Treatment solutions were prepared using sterile water. After five minutes, the entire wound area was rinsed with sterile water.
Table 6: Treatment Groups
Figure imgf000037_0002
[0212] In conveying information regarding these experiments for its use in the preparation of U.S. Application No. 17/242,185, filed April 27, 2021, to which the present application claims priority, an inadvertent error was made with respect to the treatment conditions for treatment group 1 and treatment group 3. These errors have been corrected herein.
[0213] To assess the degree of swelling (an indicator of inflammation) in the infected hind legs of rats in each treatment group, calipers were used to measure the humoral width of the infected hind leg. Measurements for rats in treatment groups that underwent the second surgery were taken as close to the infected site as possible. Prior to infection, the weight of the rats of each treatment group averaged 230 g and the rats had an average humoral width of 12 mm in the hind leg that was to be infected. A humoral width of 12 mm was used as a baseline measurement indicative of a healthy hind leg having little or no inflammation.
Treatment Group 1
[0214] Following infection, 0.015% methylene blue was applied to the entire open surgical site for 5 minutes, and the surgical site was rinsed with sterile water. The application of methylene blue stopped the bleeding at the surgical site.
[0215] The infected tissue area was stained blue by the methylene blue, the stained tissue was surgically removed and the site was rinsed one more time with sterile water until completely stain-free. The wound was closed with internal resorbable stiches and skin staples. Animals received 1 ml saline solution subcutaneously and 0.03 mg/kg of buprenorphine for pain. Buprenorphine was then administered at 0.02 mg/kg every 12 hours for the next 48 hours. Anti-inflammatory pain killers were avoided.
[0216] At 48 hours post-op, the animals developed a higher degree of inflammation in general compared to the negative control (treatment group 5). Animals treated with 0.015% methylene blue solution showed fur loss, fluctuation of the infected site, and increased limping. Animals treated with methylene blue were the most lethargic compared to all other treatment groups (general signs of illness). Furthermore, the limping on the infected leg increased.
[0217] At 72 hours post-op. the opened surgical site did not close as would be expected in healthy healing tissue, the surgical area showed inflamed tissue and the surgical area and adjacent tissue was swollen. [0218] To assess the degree of swelling at the site of infection, humoral width measurements of the infected hind leg were taken 24 hours post-op and 72 hours post-op. The right hind leg of rats treated with a 0.015% methylene blue solution had an average humoral width of about 20 mm 24 hours post-opand of about 19 mm 72 hours post-op. (Fig. 4).
Treatment Group 2
[0219] Following infection, 0.33% phosphoric acid was applied to the entire open surgical site for 5 minutes; the surgical site was rinsed with sterile water and the bleeding stopped.
[0220] As exemplified by the tissue being pointed to by the yellow arrow in Fig. 4B, the infected tissue area appeared “curled” and was distinctively different from the healthy tissues surrounding it. Healthy tissue did not appear to be affected by the 0.33% phosphoric acid solution. The observed curled tissues were surgically removed. The site was rinsed again with sterile water and the wound was closed with internal resorbable stiches and skin staples. Animals received 1 ml saline solution subcutaneously and 0.03 mg/kg of buprenorphine for pain. Buprenorphine was then administered at 0.02 mg/kg every 12 hours for the next 48 hours. Anti-inflammatory pain killers were avoided.
[0221] At 48 hours post-op. and treatment with the 0.33% phosphoric acid solution, animals showed a partial reduction in swelling. The site appeared bluish and wound healing margins were not closing-up as would be expected in healthy tissue healing. Fur growth was noticeable, but not to the degree expected in healthy animals. Animals were active and limping was reduced.
[0222] At 72 hours post-op. the opened surgical site showed a reduction in swelling and internal wound tissues showed healing. The tissues adjacent to the wound appeared healthy.
[0223] The humoral width measurements of the infected hind leg post-op and phosphoric acid treatment was about 29 mm at 24 hours and about 26 mm at 72 hours post-op. (Fig.
4)·
Treatment Group 3
[0224] Following infection, a combination of 0.33% phosphoric acid and 0.015% methylene blue was applied to the entire open surgical site for 5 minutes and the surgical site was rinsed with sterile water. The bleeding did not stop entirely (approximately 95% of the bleeding was stopped).
[0225] The infected tissue area is stained blue and also appeared “curled.” Only stained and curled tissues were surgically removed. The site was rinsed again with sterile water until completely stain-free and the wound was closed with internal resorbable stiches and skin staples. Animals received 1 ml saline solution subcutaneously and 0.03 mg/kg of buprenorphine for pain. Buprenorphine was then administered at 0.02 mg/kg every 12 hours for the next 48 hours. Anti-inflammatory pain killers were avoided.
[0226] At 48 hours post-op. and treatment with the solution containing both 0.33% phosphoric acid and 0.015% methylene blue, animals showed a noticeable reduction in swelling and bluish color. Fur growth appeared as would be expected in a healthy animal. Limping was noticeably reduced and animals were lively and rearing, putting full weight on the hind leg that had undergone surgery.
[0227] At 72 hours post-op. and treatment with the solution containing both 0.33% phosphoric acid and 0.015% methylene blue, the wound appeared filled with healthy tissues. All adjacent tissues appeared perfectly healthy. The humoral width measurements of the infected hind leg of rats treated with a solution containing both 0.33% phosphoric acid and 0.015% methylene blue were about 17 mm 24 hours post-op. and about 12 mm 72 hours post-op. (Fig. 4)
Treatment Group 4
[0228] Since there were no indicators of the extent of infected tissues (because no methylene blue was used), a much larger area of the tissues were resected, leaving a noticeable tissue dehiscence. After debridement, the wound was rinsed with sterile water. The wound continued bleeding throughout, even after the application of sutures. Pressure using a sterile cotton gauze was applied to stop the bleeding. The wound was closed with internal resorbable stiches and skin staples. Animals received 1 ml saline solution subcutaneously and 0.03 mg/kg of buprenorphine for pain. Buprenorphine was then administered at 0.02 mg/kg every 12 hours for the next 48 hours. Anti-inflammatory pain killers were avoided.
[0229] At 48 hours post-op. treatment with conventional debridement, the wound healing appeared healthy. [0230] At 72 hours post-op. the surgical site was not closing up and was oozing. The surrounding tissues appeared to be dark in color (chronic inflammation).
[0231] The humoral width measurements of the infected hind leg was about 33 mm 24 hours post-op. and about 30 mm 72 hours post-op. (Fig. 4)
Treatment Group 5
[0232] The negative control group, received no treatment after being infected at the first surgery and did not undergo the second surgery. 48 hours after the first surgery, animals received 1 ml saline solution subcutaneously and 0.03 mg/kg of buprenorphine for pain. Buprenorphine was then administered at 0.02 mg/kg every 12 hours for the next 48 hours. Anti-inflammatory pain killers were avoided.
[0233] At 120 hours post-infection the opened infected site showed infected tissues that were swollen and appeared a different color than the healthy tissue.
[0234] The humoral width measurements of the infected hind leg were taken 72 hours post-infection (equivalent to 24 hours post-op for the other treatment groups) and 120 hours post-infection (equivalent to 72 hours post-op for the other treatment groups). At 72 hours post-infection, a humoral width of 38 mm was measured at the infection site. At 120 hours post-infection, a humoral width measurement of 47 mm was measured at the infection site.
[0235] An external inspection at 72 Hours Post-Op (120 Hours after Infection)
[0236] showed that animals from treatment group 3 (treated with the solution both 0.33% phosphoric acid and 0.015 methylene blue) had a considerable reduction of swelling that was practically reduced to the natural, healthy shape of the thigh. The animal from treatment group 4 (treated with conventional debridement) showed swelling of the surgical site. The animal from treatment group 5 (no treatment negative control) had the most swelling of the infected site.
[0237] In conclusion, animals of treatment group 3 displayed the fastest healing, anti inflammatory, and regeneration rate. At 72 hours post-op, the wound was closed externally and internally with the healthy tissues. Animals in treatment group 2 showed the second-best healing. Animals in treatment group 1, displayed significant irritation of the tissues, even after being rinsed out thoroughly after the application of the 0.015% methylene blue solution. At 72 hours post-op, the treatment group 1 wound was not healing well and showed signs of inflammation. Animals of treatment group 4 showed signs of inflammation and internal healing was delayed.
[0238] As shown in Fig. 5, treatment using a solution containing both 0.33% phosphoric acid and 0.015% methylene blue (treatment group 3) was superior at reducing swelling, as assessed by measuring humoral width near the infection site, compared to treatment using a 0.015% methylene blue solution alone (treatment group 1) or a 0.33% phosphoric acid solution alone (treatment group 2).
[0239] Interestingly, the low pH of the treatment solutions used for treatment group 2 and treatment group 3 did not appear to affect cells toxically, but to the contrary, appeared to facilitate faster regeneration and healing.
[0240] The photographs of Fig. 5 are from the low dose treatment (0.015% methylene blue and 0.33% phosphoric acid) with higher doses resulting in faster wound healing and faster reduction of inflammation.
Example 21: In Vivo Studies using Bioabsorbable Sponges
[0241] Healthy Sprague Dawley female rats (Charles River Laboratories, Wilmington,
MA) 6 months of age (four groups, five per group), were used for study. The rats were infected with the MRS A (Staphylococcus aureus ATCC BAA- 1762) at a concentration of 4 x 108 per ml. The rats were sedated. A small incision was made at a femur site and a slow rotor drill was used to create a dehiscence/trauma of about 0.5 mm depth in the femoral bone, which is equivalent to about half the thickness of the cortical bone of the femur. MRSA bacteria (four microliters) were introduced into the bone and surrounding soft tissues. The wounds were then closed with internal stitches and skin clips. All rats showed severe signs of infection after 72 hours - swelling and cyanotic color of this site, ruffled fur, loss of weight, hunched posture, decreased mobility. An MRI scan showed change in soft tissue at the infected site.
[0242] A second surgery/treatment was performed 72 hours post infection. Rats were sedated and outer skin was cleaned with iodine. Skin staples were removed and the wounds reopened. Infected sites showed purulent exudate, granulation of tissue, and excessive bleeding. Most of the granulated tissue and oxidate were removed with a curette. Tissue samples were taken for a histological evaluation.
[0243] The initial treatment was performed by using a composition comprising 37% phosphoric acid gel and methylene blue carried by a bioabsorbable sponge (GELFOAM®) applied onto the entire area of the wound for five minutes. After five minutes, the sponge was removed. The wound was rinsed out with saline solution. The wound was not bleeding. The bacteria present were stained black visually demarcating the line of the infected tissue, which was surgically removed. The residual black stained tissues were removed by using the ultrasound machine with the smooth up to avoid tissue damage. The ultrasound waves cleaned the deepest layers of the tissue and restored a fresh blood flow. A bioabsorbable sponge (GELFOAM®, Pfizer, Inc.) containing an aqueous solution of phosphoric acid and methylene blue was inserted into the wounds and the wounds were closed.
[0244] The treated tissues had stopped bleeding and the infected sites were clearly delineated since methylene blue has an affinity to bind and stain bacteria. Residual infected tissues were surgically removed. An ultrasound machine with saline/water irrigation was used for final cleaning of tissue and to stimulate blood flow.
[0245] Rats in Group #1 received bioabsorbable sponge soaked with an aqueous solution containing 0.5 weight percent phosphoric acid and 1 weight percent methylene blue. The wounds were then closed with internal resorbable stitches and skin clips.
[0246] Rats in Group #2 received bioabsorbable sponge soaked with an aqueous solution containing 2 weight percent phosphoric acid and 1 weight percent methylene blue. The wounds were then closed with internal resorbable stitches and skin clips.
[0247] Rats in Group #3 received a bioabsorbable sponge soaked with an aqueous solution containing 5 weight percent phosphoric acid and 1 weight percent methylene blue. The wounds were then closed with internal resorbable stitches and skin clips.
[0248] Rats in Group #4 received a bioabsorbable sponge soaked with a aqueous solution containing 25 weight percent phosphoric acid and 1 weight percent methylene blue. The wounds were then closed with internal resorbable stitches and skin clips.
[0249] All four groups of rats showed signs of recovery in the next 24 hours. Rats in
Groups # l-#3 showed rapid healing. A healthy change in fur, mobility, reduction of swelling and redness of the wound site were observed. On Day 3 post-op, the rats started to gain weight in a continuous manner, at an average of about 2 to 3 grams per day. On Day 3 post-op rats fur had restored their initial healthy shine and condition, rats did not display any signs of pain or disease, were rearing using both legs. The wound site was healed within 30 days post-infection. The MRL CT scan and cell histology results showed no signs of infection and inflammation on the site. All evaluations showed presence of the new healthy tissue formed, new bone formation and bone density increase. The MRI scan done on the tenth post-operative day showed that infected site volume had decreased by 62% and new tissues were formed.
[0250] Rats in Group #4 showed signs of secondary infection starting on Day 3 post-op.
These rats began to rip at the skin clips, the wounds were opened, and a purulent oxidate was observed. MRI scan showed a reduction of the MRSA created tissue destruction and the presence of new, superficial tissue damage was observed as well. The rats in Group #4 were euthanized, and pathology revealed soft tissue inflammation around partially disintegrated bioabsorbable sponge.
[0251] Example 22: In Vivo Study of MRSA InfectionHealthy Sprague Dawley female rats (Charles River Laboratories, Wilmington, MA) 6 months of age (four groups, five per group), were used for study. The rats were infected with the MRSA (Staphylococcus aureus ATCC BAA-1762) at a concentration of 4 x 108 per ml. The rats were sedated, MRSA bacteria (four microliters) were injected into muscle and bone tissue. All rats showed severe signs of infection after 48 hours - swelling and cyanotic color of this site, ruffled fur, loss of weight, hunched posture, decreased mobility. An MRI scan showed change in soft tissue at the infected site (Fig. 5A and 5B).
[0252] At 48 hours after MRSA infection, rats were sedated. The infected wound area was opened and treated one time with a combination of 0.5% methylene blue and 35% phosphoric acid in a gel present in a bioabsorbable sponge by contacting the sponge to the entire area of the wound for five minutes. After five minutes, the sponge was removed. The wound was rinsed out with saline solution and the incision closed. At 10 and 30 days post treatment, tissue samples were taken for a histological evaluation and MRI images were recorded. The histology demonstrated no signs of infection and/or inflammation at the infection site and complete healing of the infection 30 days after treatment with the sponge (Fig. 5B and 5C). The MRI images demonstrated a reduction in an infectious defect 10 days after treatment and no residual infection remaining at 30 days after treatment with the combination of 0.5% methylene blue and 35% phosphoric acid in a gel present in a bioabsorbable sponge. Example 23 : In Vivo Bone Regeneration
[0253] Healthy Sprague Dawley female rats (Charles River Laboratories, Wilmington,
MA) 6 months of age and three years of age were used to test bone regeneration using the compositions described herein. A small incision was made at a femur site and a slow rotor drill was used to create a trauma of about 0.5 mm depth in the femoral bone, which is equivalent to about half the thickness of the cortical bone of the femur. The incisions were close and the rats observed for new bone formation at the trauma sites. Histomorphometric images with calcein and tetracycline stains showed new bone formation in both, the young (6 months) and the elderly (3 years) rat in a similar fashion (Fig. 6).
[0254] PET scan images of the bone of rats treated with a combination of methylene blue and phosphoric acid and untreated control shows that the bone density in the treated rats was substantially higher than in the untreated rats (Fig. 7).
Example 24: Dental Implants in Patients
[0255] Traditionally, a dental implant procedure takes up to 2 years form the extraction of a decayed or infected tooth to the crown placement on an implant (Fig. 8, upper timeline)
[0256] In contrast, when the tooth extraction site is treated once with a combination of methylene blue and phosphoric acid, an implant can be placed immediately at the tooth extraction surgery together with bone grafting tissue and a crown can be placed about 6 months later (Fig. 8, lower timeline).
[0257] Without wanting to be bound by theory, it is hypothesized that a composition comprising methylene blue and phosphoric acid as described herein promotes a fast onset of cytogenesis, potent angiogenesis complete cytological osteogenesis, promotion of physiological ossification, and mobilization of hydroxyapatite to the site of new bone growth,
[0258] Various aspects of the present invention may be characterized by the potential claims listed in the paragraphs following this paragraph (and before the actual claims provided at the end of this application). These potential claims form a part of the written description of this application. Accordingly, subject matter of the following potential claims may be presented as actual claims in later proceedings involving this application or any application claiming priority based on this application. Inclusion of such potential claims should not be construed to mean that the actual claims do not cover the subject matter of the potential claims. Thus, a decision to not present these potential claims in later proceedings should not be construed as a donation of the subject matter to the public.
[0259] Without limitation, potential subject matter that may be claimed (prefaced with the letter “P” so as to avoid confusion with the actual claims presented below) includes:
PI. A therapeutic composition comprising:
1.0 x 107-2.5 X 10 1 % (w/w) thiazine dye, and
1.0 x 105-2.0% (W/W) phosphoric acid.
P2. The therapeutic composition of claim PI, wherein the thiazine dye is methylene blue.
P3. The therapeutic composition of any one of claims P1-P2, wherein the therapeutic composition is a mouthwash.
P4. The therapeutic composition of any one of claims P1-P2, wherein the therapeutic composition is a nasal spray.
P5. The therapeutic composition of any one of claims P1-P2, wherein the therapeutic composition is a topical cream.
P6. The therapeutic composition of any one of claims P1-P2, wherein the therapeutic composition is a lozenge.
P7. A method of treating a subject in need thereof, the method comprising: causing the administration of the therapeutic composition of any one of the preceding claims, wherein the subject is selected from the group consisting of a human and an animal.
P8. A bioabsorbable antimicrobial and anti-inflammatory composition comprising: a bioabsorbable water-insoluble sponge containing an aqueous solution of phosphoric acid and a thiazine dye, wherein phosphoric acid concentration is in the range of about 0.1 to about 40 percent by weight of the aqueous solution.
P9. The bioabsorbable composition in accordance with claim P8, wherein the phosphoric acid concentration is about 0.1 to about 2.5 percent by weight of the aqueous solution.
P10. The bioabsorbable composition in accordance with claim P8, wherein the phosphoric acid concentration is about 5 to about 40 percent by weight of the aqueous solution. PI 1. The bioabsorbable composition in accordance with claim P8, wherein the thiazine dye methylene blue.
P12. The bioabsorbable composition in accordance with claim P10, wherein methylene blue is present in the aqueous phosphoric acid solution in an amount in the range of about 0.0001 percent by weight to about 1 percent by weight, based on the weight of the aqueous phosphoric acid solution.
P13. The biocompatible composition in accordance with claim P10, wherein methylene blue is present in the aqueous phosphoric acid solution in an amount in the range of about 0.1 to about 1 percent by weight of the aqueous phosphoric acid solution.
P14. The bioabsorbable composition in accordance with claim P8, wherein the sponge comprises gelatin.
PI 5. The bioabsorbable composition in accordance with claim P8, wherein the sponge comprises collagen.
PI 6. The bioabsorbable composition in accordance with claim P8, wherein the sponge contains about 5 to about 40 percent by weight of the aqueous phosphoric acid solution, based on the weight of the bioabsorbable sponge.
PI 7. The bioabsorbable sponge in accordance with claim P8, wherein the aqueous solution contains a biocompatible thickening agent.
P18. The bioabsorbable sponge in accordance with claim P17, wherein the thickening agent is sodium carboxymethyl cellulose.
PI 9. An antiviral textile material comprising: a hydrophilic fibrous substrate; an antiviral thiazine dye; and phosphoric acid; the hydrophilic fibrous substrate having porosity sufficient to permit a human to breathe therethrough, having said thiazine dye distributed therewithin in an amount in the range of 0.001 to about 1 grams per cubic centimeter of said substrate, and having phosphoric acid distributed therewith in an amount in the range of about 0.001 to about 1 grams per cubic centimeter of said substrate.
P20. The antiviral textile material in accordance with claim PI 9 wherein the antiviral thiazine dye is methylene blue. P21. The antiviral textile material in accordance with claim P20 wherein the methylene blue and the phosphoric acid are present in equimolar amounts.
[0260] The aspects of the invention described above are intended to be merely exemplary; numerous variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to be within the scope of the present invention as defined in any appended claims.

Claims

WHAT IS CLAIMED IS:
1. A therapeutic composition, comprising a thiazine dye, phosphoric acid and/or an association product thereof, wherein the composition comprises:
1.0 x 107— 1.0 % (w/w) thiazine dye, and 1.0 x 105-2.0 % (w/w) phosphoric acid.
2. The therapeutic composition of claim 1 comprising:
1.0 x 107-2.5 X 10 1 % (w/w) thiazine dye, and 1.0 x 105-2.0 % (w/w) phosphoric acid.
3. The therapeutic composition of claim 1 or claim 2, wherein the thiazine dye is methylene blue.
4. The therapeutic composition of any one of claims 1, 2 or 3, wherein the association product is bis ammonium salt of formula I
Figure imgf000049_0001
3-N3,N3 -dimethylamino-phenthiazininium chloride-7-ammonium phosphate
Formula I.
5. The therapeutic composition of any one of claims 1-4, wherein the therapeutic composition further comprises water as a solvent.
6. The therapeutic composition of any one of claims 1-4, wherein the composition is in the form of an aqueous solution.
7. The therapeutic composition of claim 6, wherein the aqueous solution comprises a biocompatible thickening agent.
8. The therapeutic composition of claim 7, wherein the thickening agent is sodium carboxymethyl cellulose.
9. The therapeutic composition of any one of claims 1-8, wherein the therapeutic composition is a mouthwash.
10. The therapeutic composition of any one of claims 1-8, wherein the therapeutic composition is a nasal spray.
11. The therapeutic composition of any one of claims 1-8, wherein the therapeutic composition is a topical cream.
12. The therapeutic composition of any one of claims 1-8, wherein the therapeutic composition is a gel.
13. The therapeutic composition of any one of claims 1-8, wherein the therapeutic composition is an emulsion.
14. The therapeutic composition of any of claims 1-8, wherein the composition is a wound dressing.
15. The therapeutic composition of claim 14, wherein the wound dressing is selected from the group consisting of a cloth, a foam, a film, a hydrocolloid, a hydrogel, an alginate and a collagen.
16. The therapeutic composition of any one of claims 1-8, wherein the therapeutic composition is a lozenge.
17. A bioabsorbable composition comprising: a bioabsorbable water-insoluble sponge containing an aqueous solution of phosphoric acid and a thiazine dye, wherein phosphoric acid concentration is in the range of about 0.1 to about 40 percent by weight of the aqueous solution.
18. The bioabsorbable composition of claim 17, wherein the phosphoric acid concentration is about 0.1 to about 2.5 percent by weight of the aqueous solution.
19. The bioabsorbable composition in accordance of claim 17, wherein the phosphoric acid concentration is about 5 to about 40 percent by weight of the aqueous solution.
20. The bioabsorbable composition of any one of claims 17-19, wherein the thiazine dye is methylene blue.
21. The bioabsorbable composition of claim 20, wherein methylene blue is present in the aqueous phosphoric acid solution in an amount in the range of about 0.0001 percent by weight to about 1 percent by weight, based on the weight of the aqueous phosphoric acid solution.
22. The bioabsorbable composition of claim 20 or 21, wherein methylene blue is present in the aqueous phosphoric acid solution in an amount in the range of about 0.1 to about 1 percent by weight of the aqueous phosphoric acid solution.
23. The bioabsorbable composition of any one of claims 17-22, wherein the sponge comprises gelatin.
24. The bioabsorbable composition of any one of claims 17-22, wherein the sponge comprises collagen.
25. The bioabsorbable composition of any one of claims 17-22, wherein the sponge comprises poly-L-lactic acid.
26. The bioabsorbable composition of claim 17, wherein the sponge contains about 5 to about 40 percent by weight of the aqueous phosphoric acid solution, based on the weight of the bioabsorbable sponge.
27. The bioabsorbable composition of any one of claims 17-26, wherein the aqueous solution comprises a biocompatible thickening agent.
28. The bioabsorbable composition of claim 27, wherein the thickening agent is sodium carboxymethyl cellulose.
29. The bioabsorbable composition of any one of claims 17-28, wherein ions of the thiazine dye and the phosphoric acid are free or associated with one another.
30. A textile material comprising: a hydrophilic fibrous substrate; a thiazine dye; and phosphoric acid; the hydrophilic fibrous substrate having porosity sufficient to permit a human to breathe therethrough, having the thiazine dye distributed therewithin in an amount in the range of 0.001 to about 1 grams per cubic centimeter of the substrate, and having phosphoric acid distributed therewith in an amount in the range of about 0.001 to about 1 grams per cubic centimeter of the substrate.
31. The textile material of claim 30, wherein the thiazine dye is methylene blue.
32. The textile material of claim 30 wherein the methylene blue and the phosphoric acid are present in equimolar amounts.
33. The textile material of any one of claims 30-32, wherein the hydrophilic fibrous substrate is made of non-woven cotton.
34. The textile material of any one of claims 30-33, wherein the hydrophilic fibrous substrate is located between an outer water-repellant cotton layer and an inner silk layer.
35. The textile material of claim 34, wherein the outer water-repellant cotton layer retains exhaled moisture.
36. The textile material any one of claims 30-35, wherein ions of the thiazine dye and the phosphoric acid are free or associated with one another.
37. The textile material of any of claims 30-36, wherein the hydrophilic fibrous substrate, the thiazine dye and the phosphoric acid can be in form of a bandage.
38. A therapeutic composition comprising a thiazine dye, phosphoric acid and/or an association product thereof for use in a method of treating a wound.
39. The therapeutic composition of claim 38 for use in a method of treating a wound, wherein the therapeutic composition is an aqueous solution.
40. The therapeutic composition of claim 38 for use in a method of treating a wound, wherein the therapeutic composition is a gel.
41. The therapeutic composition of claim 38 for use in a method of treating a wound, wherein the therapeutic composition is an emulsion.
42. The therapeutic composition of claim 38 for use in a method of treating a wound, wherein the therapeutic composition is a wound dressing.
43. The therapeutic composition of claim 38 for use in a method of treating a wound, wherein the wound is a bone site other than a dental bone site.
44. The therapeutic composition of claim 43 for use in a method of treating a wound, wherein the therapeutic composition is an aqueous solution and is administered to the bone site by contacting the bone site with the aqueous solution.
45. The therapeutic composition of claim 43 for use in a method of treating a wound, wherein the therapeutic composition is contained in a bioabsorbable water-insoluble sponge and the therapeutic composition is administered to the bone site by contacting the bone site with the sponge.
46. The therapeutic composition of claims 38-42 for use in a method of treating a wound, wherein the wound is an open wound.
47. The therapeutic composition of claim 46 for use in a method of treating a wound, wherein the therapeutic composition is an aqueous solution and is administered to the wound by contacting the wound with the aqueous solution.
48. The therapeutic composition of claim 46 for use in a method of treating a wound, wherein the therapeutic composition is contained in a bioabsorbable water-insoluble sponge and the therapeutic composition is administered to the wound by contacting the wound with the sponge.
49. The therapeutic composition of claim 46 for use in a method of treating a wound, wherein the therapeutic composition is contained in a bioabsorbable water-insoluble sponge and the therapeutic composition is administered to the open wound by placing the sponge into the open wound.
50. The therapeutic composition of claim 38 for use in a method of treating a wound, wherein the wound is a chronic wound.
51. The therapeutic composition of claim 50 for use in a method of treating a wound, wherein the therapeutic composition is an aqueous solution and is administered to the chronic wound by contacting the chronic wound with the aqueous solution.
52. The therapeutic composition of claims 50 or 51 for use in a method of treating a wound, wherein the chronic wound is an ulcer, a diabetic lesion, an eczema, acne vulgaris, a chronic surgical wound, or a chronic infectious wound.
53. The therapeutic composition of claim 52 for use in a method of treating a wound, wherein the chronic infectious wound is infected with Acinetobacter baumannii, Actinomyces sp., Actinobacillus actinomycetemcomitans, Aeromonas sp., Anaplasma phagocytophilum, Alcaligenes xylosoxidans, Actinobacillus actinomycetemcomitans, Bacillus sp., Bacteroides sp., Bartonella sp. Bifidobacterium sp., Bordetella sp., Borrelia sp., Brucella sp, Burkholderia sp., Campylobacter sp., Capnocytophaga sp., Cardiobacterium hominis, Chlamydia trachomatis, Chlamydophila pneumonia, Chlamydophila psittaci, Citrobacter sp. Coxiella burnetii, Corynebacterium sp., Clostridium sp., Eikenella corrodens, Enterobacter sp., Enterococcus sp., Ehrlichia sp., Erysipelothrix rhusiopathiae, Eubacterium sp., Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Gemella morbillorum, Haemophilus sp, Helicobacter sp., Kingella kingii, Klebsiella sp., Lactobacillus sp., Listeria monocytogenes, Leptospira interrogans, Legionella pneumophila, Leptospira interrogans, Peptostreptococcus sp., Moraxella catarrhalis, Morganella sp., Mobiluncus sp., Micrococcus sp., Mycobacterium sp., Mycoplasm sp., Nocardia sp., Neisseria sp., Pasteurella multocida, Plesiomonas shigelloides, Prevotella sp., Prevotella intermedia, Porphyromonas sp., Prevotella melaninogenica, Proteus sp., Providencia sp., Pseudomonas aeruginosa, Propionibacterium acnes, Rhodococcus equi, Salmonella sp., Serratia sp, Shigella sp., Staphylococcus sp., Streptococcus sp., Spirillum minus, Streptobacillus moniliformi, Treponema sp., Tropheryma whippelii, Lireaplasma urealyticum, Veillonella sp., Vibrio sp., Yersinia sp. or Xanthomonas maltophilia.
54. A therapeutic composition comprising a thiazine dye, phosphoric acid and/or an association product thereof for use in a method of treating an infection.
55. The therapeutic composition of claim 54 for use in a method of treating an infection, wherein the infection is a bacterial infection, a viral infection, a fungal infection or a parasitic infection.
56. The therapeutic composition of claim 55 for use in a method of treating an infection, wherein the bacterial infection is an infection with gram-positive bacteria, gram-negative bacteria, or antibiotic-resistant strains.
57. The therapeutic composition of claim 55 for use in a method of treating an infection, wherein the bacterial infection is osteomyelitis, cellulitis, folliculitis, impetigo, boils, or bacterial vaginosis.
58. The therapeutic composition of claim 55 for use in a method of treating an infection, wherein the bacterial infection is an Acinetobacter baumannii, Actinomyces sp., Actinobacillus actinomycetemcomitans, Aeromonas sp., Anaplasma phagocytophilum, Alcaligenes xylosoxidans, Actinobacillus actinomycetemcomitans, Bacillus sp., Bacteroides sp., Bartonella sp. Bifidobacterium sp., Bordetella sp., Borrelia sp., Brucella sp, Burkholderia sp., Campylobacter sp., Capnocytophaga sp., Cardiobacterium hominis, Chlamydia trachomatis, Chlamydophila pneumonia, Chlamydophila psittaci, Citrobacter sp. Coxiella burnetii, Corynebacterium sp., Clostridium sp., Eikenella corrodens, Enterobacter sp., Enterococcus sp., Ehrlichia sp., Erysipelothrix rhusiopathiae, Eubacterium sp., Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Gemella morbillorum, Haemophilus sp, Helicobacter sp., Kingella kingii, Klebsiella sp., Lactobacillus sp., Listeria monocytogenes, Leptospira interrogans, Legionella pneumophila, Leptospira interrogans, Peptostreptococcus sp., Moraxella catarrhalis, Morganella sp., Mobiluncus sp., Micrococcus sp., Mycobacterium sp., Mycoplasm sp., Nocardia sp., Neisseria sp., Pasteurella multocida, Plesiomonas shigelloides, Prevotella sp., Prevotella intermedia, Porphyromonas sp., Prevotella melaninogenica, Proteus sp., Providencia sp., Pseudomonas aeruginosa, Propionibacterium acnes, Rhodococcus equi, Salmonella sp., Serratia sp, Shigella sp., Staphylococcus sp., Streptococcus sp., Spirillum minus, Streptobacillus moniliformi, Treponema sp., Tropheryma whippelii, Lireaplasma urealyticum, Veillonella sp., Vibrio sp., Yersinia sp. or Xanthomonas maltophilia infection.
59. The therapeutic composition of claim 55 for use in a method of treating an infection, wherein the infection is a viral infection.
60. The therapeutic composition of claim 59 for use in a method of treating an infection, wherein the viral infection is an Adenovirus, Bocavirus, Coronavirus, Enterovirus, Metapneumovirus, Parainfluenza virus 1-4, Respiratory Syncytial Virus A and B, Rhinovirus, Herpesvirus, Varicella zoster virus, Epstein-Barr Virus, Influenza, human Parechovirus, Measles virus, Middle East Respiratory Syndrome coronavirus (MERS CoV), Mumps virus, or Severe Acute Respiratory Syndrome Coronavirus (SARS CoV) infection.
61. The therapeutic composition of any one of claims 54-60 for use in a method of treating an infection, wherein the therapeutic composition is an aqueous solution.
62. The therapeutic composition of any one of claims 54-61 for use in a method of treating an infection, wherein the therapeutic composition is contained in a textile material.
63. The therapeutic composition of claim 62, wherein the textile material comprises: a hydrophilic fibrous substrate; a thiazine dye; and phosphoric acid; the hydrophilic fibrous substrate having porosity sufficient to permit a human to breathe therethrough, having the thiazine dye distributed therewithin in an amount in the range of 0.001 to about 1 grams per cubic centimeter of said substrate, and having phosphoric acid distributed therewith in an amount in the range of about 0.001 to about 1 grams per cubic centimeter of the substrate.
64. The therapeutic composition of any one of claims 54-61 for use in a method of treating an infection, wherein the infection is a wound and the therapeutic composition is contained in a wound dressing.
65. The therapeutic composition of any one of claims 54-61 for use in a method of treating an infection, wherein the therapeutic composition is contained in a spray.
66. The therapeutic composition of any one of claims 54-61 for use in a method of treating an infection, wherein the therapeutic composition is contained in a gel.
67. The therapeutic composition of any one of claims 54-61 for use in a method of treating an infection, wherein the therapeutic composition is contained in an emulsion.
68. The therapeutic composition of any one of claims 38-61 or 64-66 comprising:
1.0 x 107-2.5 X 10 1 % (w/w) thiazine dye, and 1.0 x 105-2.0 % (w/w) phosphoric acid.
69. A therapeutic composition comprising a thiazine dye, phosphoric acid and/or an association product thereof for use in a method of regenerating a bone.
70. The therapeutic composition of claim 69 for use in a method of regenerating a bone, wherein the bone is a fractured bone or an osteoporotic bone.
71. The therapeutic composition of claims 69 or 70 for use in a method of regenerating a bone, wherein the therapeutic composition is an aqueous solution and is administered to the bone by contacting the bone with the aqueous solution.
72. The therapeutic composition of claim 69 for use in a method of regenerating a bone, wherein the aqueous solution comprises a thickening agent.
73. The therapeutic composition of claim 72, wherein the thickening agent is sodium carboxymethyl cellulose.
74. The therapeutic composition of claims 69 or 70 for use in a method of regenerating a bone, wherein the therapeutic composition is contained in a bioabsorbable water- insoluble sponge and the therapeutic composition is administered to the bone by contacting the bone with the sponge.
75. A method of treating a wound in a subject in need thereof comprising administering to the wound a therapeutically effective amount of a composition comprising a thiazine dye, phosphoric acid and/or an association product thereof.
76. The method of claim 75, wherein the wound is a bone site other than a dental bone site.
77. The method of claim 76, wherein the therapeutic composition is an aqueous solution and is administered to the bone site by contacting the bone site with the aqueous solution.
78. The method of claim 76, wherein the therapeutic composition is contained in a bioabsorbable water-insoluble sponge and the therapeutic composition is administered to the bone site by contacting the bone site with the sponge.
79. The method of claim 75, wherein the wound is an open wound.
80. The method of claim 79, wherein the composition is an aqueous solution and is administered to the wound by contacting the wound with the aqueous solution.
81. The method of claim 80, wherein the aqueous solution comprises a biocompatible thickening agent.
82. The therapeutic composition of claim 81, wherein the thickening agent is sodium carboxymethyl cellulose.
83. The method of claim 76, wherein the composition is associated with a bioabsorbable water-insoluble sponge and the composition is administered to the bone site by contacting the bone site with the sponge.
84. The method of claim 79, wherein the composition is associated with a bioabsorbable water-insoluble sponge and the composition is administered to the open wound by placing the sponge into the open wound.
85. The method of claim 75, wherein the wound is a chronic wound.
86. The method of claim 85, wherein the composition is an aqueous solution and is administered to the chronic wound by contacting the chronic wound with the aqueous solution.
87. The method of claims 85 or 86, wherein the chronic wound is an ulcer, a diabetic lesion, an eczema, acne vulgaris, a chronic surgical wound, or a chronic infectious wound.
88. The method of claim 87, wherein the chronic infectious wound is infected with Acinetobacter baumannii, Actinomyces sp., Actinobacillus actinomycetemcomitans, Aeromonas sp., Anaplasma phagocytophilum, Alcaligenes xylosoxidans, Actinobacillus actinomycetemcomitans, Bacillus sp., Bacteroides sp., Bartonella sp. Bifidobacterium sp., Bordetella sp., Borrelia sp., Brucella sp, Burkholderia sp., Campylobacter sp., Capnocytophaga sp., Cardiobacterium hominis, Chlamydia trachomatis, Chlamydophila pneumonia, Chlamydophila psittaci, Citrobacter sp. Coxiella burnetii, Corynebacterium sp., Clostridium sp., Eikenella corrodens, Enterobacter sp., Enterococcus sp., Ehrlichia sp., Erysipelothrix rhusiopathiae, Eubacterium sp., Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Gemella morbillorum, Haemophilus sp, Helicobacter sp., Kingella kingii, Klebsiella sp., Lactobacillus sp., Listeria monocytogenes, Leptospira interrogans, Legionella pneumophila, Leptospira interrogans, Peptostreptococcus sp., Moraxella catarrhalis, Morganella sp., Mobiluncus sp., Micrococcus sp., Mycobacterium sp., Mycoplasm sp., Nocardia sp., Neisseria sp., Pasteurella multocida, Plesiomonas shigelloides, Prevotella sp., Prevotella intermedia, Porphyromonas sp., Prevotella melaninogenica, Proteus sp., Providencia sp., Pseudomonas aeruginosa, Propionibacterium acnes, Rhodococcus equi, Salmonella sp., Serratia sp, Shigella sp., Staphylococcus sp., Streptococcus sp., Spirillum minus, Streptobacillus moniliformi, Treponema sp., Tropheryma whippelii, Ureaplasma urealyticum, Veillonella sp., Vibrio sp., Yersinia sp. or Xanthomonas maltophilia.
89. A method of treating an infection in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a composition comprising a thiazine dye, phosphoric acid and/or an association product thereof.
90. The method of claim 89, wherein the infection is a bacterial infection, a viral infection, a fungal infection or a parasitic infection.
91. The method of claim 90, wherein the bacterial infection is an infection with gram-positive bacteria, gram-negative bacteria, and antibiotic-resistant strains.
92. The method of claim 91, wherein the bacterial infection is osteomyelitis, cellulitis, folliculitis, impetigo, boils, or bacterial vaginosis.
93. The method of claim 90, wherein the bacterial infection is an Acinetobacter baumannii, Actinomyces sp., Actinobacillus actinomycetemcomitans, Aeromonas sp., Anaplasma phagocytophilum, Alcaligenes xylosoxidans, Actinobacillus actinomycetemcomitans, Bacillus sp., Bacteroides sp., Bartonella sp. Bifidobacterium sp., Bordetella sp., Borrelia sp., Brucella sp, Burkholderia sp., Campylobacter sp., Capnocytophaga sp., Cardiobacterium hominis, Chlamydia trachomatis, Chlamydophila pneumonia, Chlamydophila psittaci, Citrobacter sp. Coxiella burnetii, Corynebacterium sp., Clostridium sp., Eikenella corrodens, Enterobacter sp., Enterococcus sp., Ehrlichia sp., Erysipelothrix rhusiopathiae, Eubacterium sp., Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Gemella morbillorum, Haemophilus sp, Helicobacter sp., Kingella kingii, Klebsiella sp., Lactobacillus sp., Listeria monocytogenes, Leptospira interrogans, Legionella pneumophila, Leptospira interrogans, Peptostreptococcus sp., Moraxella catarrhalis, Morganella sp., Mobiluncus sp., Micrococcus sp., Mycobacterium sp., Mycoplasm sp., Nocardia sp., Neisseria sp., Pasteurella multocida, Plesiomonas shigelloides, Prevotella sp., Prevotella intermedia, Porphyromonas sp., Prevotella melaninogenica, Proteus sp., Providencia sp., Pseudomonas aeruginosa,
Propionibacterium acnes, Rhodococcus equi, Salmonella sp., Serratia sp, Shigella sp., Staphylococcus sp., Streptococcus sp., Spirillum minus, Streptobacillus moniliformi, Treponema sp., Tropheryma whippelii, Ureaplasma urealyticum, Veillonella sp., Vibrio sp., Yersinia sp. or Xanthomonas maltophilia infection.
94. The method of claim 90, wherein the infection is a viral infection.
95. The method of claim 94, wherein the viral infection is an Adenovirus, Bocavirus,
Coronavirus, Enterovirus, Metapneumovirus, Parainfluenza virus 1-4, Respiratory Syncytial Virus A and B, Rhinovirus, Herpesvirus, Varicella zoster virus, Epstein-Barr Virus, Influenza, human Parechovirus, Measles virus, Middle East Respiratory Syndrome coronavirus (MERS CoV), Mumps virus, or Severe Acute Respiratory Syndrome Coronavirus (SARS CoV) infection.
96. The method of any one of claims 89-95, wherein the composition is aqueous.
97. The method of claim 96, wherein the aqueous composition comprises a thickening agent.
98. The method of claim 97, wherein the thickening agent is carboxymethyl cellulose.
99. The method of claims 89-96, wherein the composition is contained in a spray.
100. The method of claims 89-96, wherein the composition is contained in a gel.
101. The method of claims 89-96, wherein the composition is contained in an emulsion.
102. The method of any one of claims 89-101, wherein the composition comprises:
1.0 x 107— 1.0 % (w/w) thiazine dye, and
1.0 x 105-2.0 % (w/w) phosphoric acid.
103. The method of claims 89 or 90, wherein the composition is contained in a textile material.
104. The method of claim 103, wherein the textile material comprises: a hydrophilic fibrous substrate; a thiazine dye; and phosphoric acid; the hydrophilic fibrous substrate having porosity sufficient to permit a human to breathe therethrough, having the thiazine dye distributed therewithin in an amount in the range of 0.001 to about 1 grams per cubic centimeter of said substrate, and having phosphoric acid distributed therewith in an amount in the range of about 0.001 to about 1 grams per cubic centimeter of the substrate.
105. The method of claim 104, wherein the thiazine dye is methylene blue.
106. The method of claim 105, wherein the methylene blue and the phosphoric acid are present in equimolar amounts.
107. The method of one of claims 104-106, wherein the hydrophilic fibrous substrate is made of non- woven cotton.
108. The method of any of claims 86-107, wherein the method comprises contacting an infected area of the subject with the composition.
109. A method of regenerating a bone in a subject, the method comprising administering to the bone of the subject a therapeutically effective amount of a composition comprising a thiazine dye, phosphoric acid and/or an association product thereof.
110. The method of claim 109, wherein the bone is a fractured bone or an osteoporotic bone.
111. The method of claims 109 or 110, wherein the method comprises contacting the bone of the subject with the composition.
112. The method of claims 109-111, wherein the therapeutic composition is an aqueous solution and is administered to the bone by contacting the bone with the aqueous solution.
113. The method of claims 109-111, wherein the therapeutic composition is contained in a bioabsorbable water-insoluble sponge and the therapeutic composition is administered to the bone by contacting the bone with the sponge.
114. The method of any one of claims 109-113, wherein the composition comprises:
1.0 x 107-2.5 X 10 1 % (w/w) thiazine dye, and 1.0 x 105-2.0 % (w/w) phosphoric acid.
PCT/US2022/011718 2021-01-07 2022-01-07 Therapeutic thiazine dye compositions and methods of use WO2022150657A1 (en)

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