WO2020033677A1 - Méthodes de diagnostic et de traitement d'implants infectés - Google Patents

Méthodes de diagnostic et de traitement d'implants infectés Download PDF

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Publication number
WO2020033677A1
WO2020033677A1 PCT/US2019/045693 US2019045693W WO2020033677A1 WO 2020033677 A1 WO2020033677 A1 WO 2020033677A1 US 2019045693 W US2019045693 W US 2019045693W WO 2020033677 A1 WO2020033677 A1 WO 2020033677A1
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Prior art keywords
biofilm
pca
implant
composition
patient
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PCT/US2019/045693
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English (en)
Inventor
Lanny Leo JOHNSON
Original Assignee
Johnson Lanny Leo
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Priority claimed from US16/058,909 external-priority patent/US10398664B2/en
Application filed by Johnson Lanny Leo filed Critical Johnson Lanny Leo
Priority to CA3106105A priority Critical patent/CA3106105A1/fr
Publication of WO2020033677A1 publication Critical patent/WO2020033677A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/11—Aldehydes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N7/00—Ultrasound therapy

Definitions

  • the present invention relates to compositions for use in methods for treating microbial infections on the surfaces of an implant or other surfaces near an implant in a patient.
  • the methods are useful for treating biofilm infections.
  • the methods include using minimally invasive techniques such as ultrasound for facilitating the detection of biofilm infections on an implant or other surface of a patient and destroying the infection by
  • compositions for use in methods for treating infections e.g., bacterial infections
  • the methods are useful for treating biofilm infections (e.g., biofilm bacterial infections).
  • the methods include the use of minimally invasive techniques for determining the presence of an infection on the implant and destroying the infection by administering an anthocyanin or an anthocyanidin or metabolites thereof (e.g., protocatechu ic acid or 2,4,6 trihydroxybenzaldehyde).
  • protocatechuic acid, 2,4,6- trihydroxybenzaldehyde, or a mixture or combination thereof, for use in a method of treating a biofilm bacterial infection on a surface of an implant or a surface of a patient suspected of having a biofilm bacterial infection is disclosed.
  • the method includes removing a biofilm-forming bacteria by a minimally invasive technique comprising needle aspiration or an application of ultrasound or both to determine a presence of a biofilm around or on the surface of the implant or surface of the patient, wherein the minimally invasive technique dislodges the removed biofilm-forming bacteria from the biofilm colony.
  • the method also includes inhibiting or destroying the biofilm colony and all biofilm-forming bacteria on or around the surface of the implant in the patient or surface of the patient by administering a composition comprising protocatechuic acid or 2,4,6-trihydroxybenzaldehyde or a mixture or combination thereof or administering pure crystals or a powder of
  • the ultrasound is applied at a frequency and power sufficient to dislodge the biofilm-forming bacteria off the surface of the implant in a patient or the surface of the patient. In some embodiments, the ultrasound is applied at least one of (i) prior to performing a first needle aspiration and (ii) after performing a first needle aspiration. In some
  • the minimally invasive technique is needle aspiration. In some embodiments, the minimally invasive technique is needle aspiration and application of ultrasound. In some embodiments, the ultrasound is applied prior to performing a first needle aspiration. In some embodiments, the ultrasound is applied after performing a first needle aspiration. In some embodiments, the surface of the patient is a closed wound, intact skin, or skin having a sinus track.
  • the implant is a medical device, a medical or surgical implant, total joint prosthesis, a catheter, a dental implant, or a heart or vascular graft.
  • a presence of the biofilm on the surface of the implant in the patient or the surface of the patient in previously negative tests for a biofilm-forming bacteria following needle aspiration requires application of ultrasound to dislodge a second portion of biofilm- forming bacteria from the biofilm colony for confirmation of the presence of the biofilm on the surface of the implant in the patient or surface of the patient.
  • the ultrasound is applied by a transcutaneous probe, external stimulation, or lithotripsy.
  • a presence of the biofilm on the surface is determined by a needle aspiration prior to the application of the composition or the crystals.
  • the biofilm comprises a biofilm-forming bacteria selected from Pseudomonas aeruginosa and Methicillin-resistant
  • the composition is a solution comprising protocatechuic acid or 2,4,6-trihydroxybenzaldehyde or a mixture or combination thereof. In some embodiments, the solution comprises about 0.25% to about 50% by weight of protocatechuic acid or 2,4,6- trihydroxybenzaldehyde or a mixture or combination thereof. In some embodiments, the solution comprises about 20% to about 30% by weight of protocatechuic acid or 2,4,6-trihydroxybenzaldehyde or a mixture or combination thereof.
  • the composition is a solution comprising protocatechuic acid or 2,4,6-trihydroxybenzaldehyde or a mixture or combination thereof and the solution comprises at least one of (i) about 0.25% to about 50% by weight of protocatechuic acid or 2,4,6- trihydroxybenzaldehyde or a mixture or combination thereof and (ii) about 20% to about 30% by weight of protocatechuic acid or 2,4,6- trihydroxybenzaldehyde or a mixture or combination thereof.
  • the composition, powder, or the crystals is administered once. In some embodiments, the composition, powder, or crystals is administered continuously, hourly, daily, weekly, or monthly.
  • ultrasound is applied to or near the surface of the implant or surface of the patient having the biofilm bacterial infection to dislodge the biofilm colony and facilitate release of a biofilm-forming bacteria from the surface.
  • an aspirate obtained from performing the needle aspiration is cultured to determine if any biofilm-forming bacteria are present, thereby indicating a presence of the biofilm bacterial infection on the surface of the implant or the surface of the patient.
  • an aspirate obtained from performing the needle aspiration is cultured to determine if any biofilm forming bacteria are present on the surface of the implant or the surface of the patient following administration of the
  • an aspirate obtained from performing the needle aspiration is cultured to determine if any biofilm-forming bacteria are present, thereby indicating a presence of the biofilm bacterial infection on the surface of the implant or the surface of the patient at least one of (i) prior to administration of the composition or the crystals and (ii) following
  • the ultrasound is applied at a frequency of about 10 kHz to about 60 kHz. In some embodiments, the ultrasound is applied at a power density of about 0.1 W/cm 2 to about 0.5 W/cm 2 In some embodiments, the ultrasound is applied using at least one of (i) a frequency of about 10 kHz to about 60 kHz and (ii) a power density of about 0.1 W/cm 2 to about 0.5 W/cm 2 . In some embodiments, the ultrasound is applied at a frequency of about 40 kHz and a power density of about 0.22 W/cm 2 . In some embodiments, the ultrasound is applied percutaneously and transcutaneously. In some embodiments, the surface is further washed with a physiologically compatible solution to facilitate release of a biofilm-forming bacteria. In some embodiments, the surface is washed with a composition comprising an anti- microbial peptide.
  • a method includes a) performing a needle aspiration to dislodge and remove a first portion of biofilm-forming bacteria from the biofilm colony; b) culturing the first portion of biofilm-forming bacteria to determine a presence of the biofilm-forming bacteria around or on the surface of the implant or surface of the patient; c) if the culture is negative, applying an ultrasound to dislodge and remove a second portion of biofilm- forming bacteria from the biofilm colony; d) if the culture is identified as positive, administering to a surrounding space in proximity of the surface of the implant or the surface of a patient at least one of (i) a solution comprising protocatechuic acid in an amount of about 0.25% to about 50% by weight, (ii) a solution comprising 2,4,6-trihydroxybenzaldehyde in an amount of about 0.25% to about 50% by weight, (iii) a solution comprising a mixture or combination of protocatechuic acid and 2,4,6-
  • a method includes a) performing a needle aspiration to determine a presence of a biofilm-forming bacteria around or on the surface of the implant or surface of the patient; b) culturing the biofilm-forming bacteria obtained from the needle aspiration to determine a presence of the biofilm- forming bacteria around or on the surface of the implant or surface of the patient; c) if the culture is negative, then an ultrasound is applied to dislodge the biofilm colony and biofilm-forming bacteria from the surface of the implant or the surface of the patient; d) if the culture is identified as positive, then administering to a surrounding space in proximity of the surface of the implant or the surface of a patient a solution comprising protocatechuic acid or 2,4,6- trihydroxybenzaldehyde or a mixture or combination thereof in an amount of about 0.25% to about 50% by weight or administering crystals of
  • the implant is a medical device, a medical or surgical implant, a dental implant, total joint prosthesis, a catheter, or a heart or vascular graft.
  • Figure 1 provides the minimum, maximum and optimum pH for growth of microorganisms. Acidic environments retard proliferation of various bacteria. Anthocyanins, anthocyanidins and main metabolites are unstable relative to basic pH; thus, anthocyanins, anthocyanidins and main metabolites thereof have the potential to lower the pH of wound tissue as well as any surfaces and act as bactericidal or bacteriostatic.
  • Figure 2 is the metabolic pathway of cyanidin-3-glucoside (C3G) and includes the chemical structures of cyanidin-3-glucoside and cyanidin and their metabolites.
  • Figure 3 is the chemical structure of Protocatechuic acid (PCA), a dihydroxybenzoic acid, a type of phenolic acid. It is a major metabolite of antioxidant polyphenols found in certain plants, including green tea.
  • PCA Protocatechuic acid
  • Figure 4 compares concentrations of C3G and PCA to determine optimal effective concentrations. Bacterial burdens for P. aeruginosa were compared after treatment with C3G or PCA at 48 and 96 hours. A
  • Figure 5 is a chart disclosing potential sources of PCA.
  • Figure 6 is a table providing a summary of the effectiveness of certain anthocyanins, anthocyanidins and a metabolite, including bactericidal or bacteriostatic activity. During this test, the purity, concentrations and molecular weight of these test substances (compounds) were known. The carrier was water and the dose was accurately calculated. Delphinidin limited growth against C. perfringens, S. aureus, and MRSA. Pelargonidin limited growth of P. acnes, C. perfingens, S. aureus, MRSA, and S. pyogenes.
  • Cyanidin Cl was effective against C. difficile, C. perfringens, S. aureus ATCH 6538, S. aureus (MRSA) ATCH 33591 , S. mutans, and S. pyogenes.
  • a proprietary formulation of cyanidin-3-glucoside (approximately 28% C3G by weight) had limited effectiveness during this study (18-24 hours for aerobes; 48 hours for anaerobes (C. albicans and L case/); however, this C3G formulation, was effective against P. acnes, E. coli, MRSA, K. pneumoniae, and P. aeruginosa.
  • PCA Protocatechuic acid
  • PCA P. aeruginosa.
  • PCA was also effective on C. albicans, which is important considering its ability to form biofilms and difficulty in treating C. albicans when existing with a catheter or implant.
  • Figure 7 is a table summarizing in vitro test results of 2, 4, 6
  • Figure 8A is a photographic image illustrating in vitro test results of 2,4,6 TFIBA against P. aeruginosa.
  • Figure 8B is a photographic image illustrating in vitro test results of 2,4,6 trihydroxybenzaldehyde against
  • FIG. 8C is a photographic image illustrating in vitro test results of 2,4,6 TFIBA against P. acnes.
  • Figure 9A shows the results of a rodent back skin tape study where concentrations of PCA and C3G in a vehicle of water were utilized to determine effectiveness against P. aeruginosa skin infections.
  • Figure 9B shows the results of rodent back skin tape study where concentrations of PCA and C3G in a vehicle of water were utilized to determine effectiveness against P. aeruginosa skin infections.
  • Figure 10A shows the results of an additional rodent back skin tape study to determine effective dosages of PCA and C3G in a vehicle of water that would be bactericidal for P. aeruginosa.
  • Figure 10B shows the results of an additional rodent back skin tape study to determine effective dosages of PCA and C3G in a vehicle of water that would be bactericidal for
  • Figure 10C shows the results of an additional rodent back skin tape study to determine effective dosages of PCA and C3G in a vehicle of water that would be bactericidal for P. aeruginosa.
  • Figure 11 A shows the results of a rodent back skin study to determine the effects of PCA and C3G on the local growth hormones in untreated skin wounds of rodents.
  • a concentration of 25 mM PCA increased local growth hormone levels of IGF-1 at the site of the untreated skin wound.
  • Figure 11 B shows the results of a rodent back skin study to determine the effects of PCA and C3G on the local growth hormones in untreated skin wounds of rodents.
  • FIG. 11 C shows the results of a rodent back skin study to determine the effects of PCA and C3G on the local growth hormones in untreated skin wounds of rodents.
  • a concentration of 25 mM PCA increased local growth hormone levels of EGF at the site of the untreated skin wound.
  • Figure 12A is a photographic image of rodents treated with a topical solution of C3G (28%); at an acidic pH, this solution maintains a purple or red color and quickly metabolized at elevated pH levels, the C3G changes to a pink or even clear color.
  • the purple color of C3G remained on the rodent wound surface scar, thus indicating the pH remained acidic on the wound surface.
  • the C3G material on the surface was confirmed by subsequent histology.
  • Figure 12B is a photographic image of tissue from a study utilizing the homogenized wound tissue method used in this study, whereby the purple color indicates that the wound probably retained an acidic pH .
  • Figure 13A shows the IGF-1 response to 25 mM PCA in various environments, including tape stripped, tape stripped and infected with P. aeruginosa, tape stripped and treated with PCA, and tape stripped infected with P. aeruginosa, and PCA treated.
  • Figure 13B shows the TGF-b response to 25 mM PCA in various environments, including tape stripped, tape stripped and infected with P. aeruginosa, tape stripped and treated with PCA, and tape stripped infected with P. aeruginosa, and PCA treated.
  • Figure 13C shows the EGF response to 25 mM PCA in various environments, including tape stripped, tape stripped and infected with P. aeruginosa, tape stripped and treated with PCA, and tape stripped infected with P. aeruginosa, and PCA treated.
  • Figure 14 is a photographic image of a cross section of rodent skin.
  • Figure 15 is a photographic image of a cross section of rodent skin.
  • Figure 16 is a photographic image of a cross section of rodent skin.
  • Figure 17 is a photographic image of a cross section of rodent skin.
  • Figure 18 is a photographic image of a cross section of rodent skin.
  • Figure 19 is a photographic image of a cross section of rodent skin.
  • Figure 20 is a photographic image of a cross section of rodent skin.
  • Figure 21 is a photographic image of a cross section of rodent skin.
  • Figure 22 is a photographic image of a cross section of rodent skin.
  • Figure 23 is a photographic image of a cross section of rodent skin.
  • Figure 24 is a photographic image of a cross section of rodent skin.
  • Figure 25 is a photographic image of a cross section of rodent skin.
  • Figure 26 is a photographic image of a cross section of rodent skin.
  • Figure 27 is a photographic image of a cross section of rodent skin.
  • Figure 28 is a photographic image of a cross section of rodent skin.
  • Figure 29 is a photographic image of a cross section of rodent skin.
  • Figure 30 is a photographic image of a cross section of rodent skin.
  • Figure 31 is a photographic image of a cross section of rodent skin.
  • Figure 32 provides a chart studying the effectiveness of anthocyanin and anthocyanidin metabolites against various microbes, including P. acnes, C. difficile, E. coli 8739 and 43895, S. aureus 6538, 33591 , P. aeruginosa 9027, MRSA 51625 and Legionella 43662, methicillin resistant
  • MRSE staphylococcus epidermis
  • Figure 33 provides a chart summarize results of testing PCA against Pseudomonas biofilm.
  • Figure 34 provides a chart summarize results of testing PCA against Pseudomonas biofilm.
  • Figure 35 provides a chart summarize results of testing PCA against
  • Figure 36 provides a chart summarize results of testing PCA against MRSA biofilm.
  • Figure 37 provides a chart summarize results of testing PCA against Pseudomonas biofilm.
  • Figure 38 provides a chart summarize results of testing PCA against MRSA biofilm.
  • Figure 39 provides a chart of results of testing PCA against P. acnes on the skin.
  • Figure 40 shows the results for a single spray of 30% PCA in isopropyl alcohol on 10 million biofilms colonies of Pseudomonas aeruginosa.
  • Figure 41 shows the concentration of 30% has lesser effect on MRSA, but still 90%.
  • Figure 42 provides a composite of the results based only for no growth cultures following treatment by each solution. *Note that the 1 % PCA was not included.
  • Figure 43 provides the summation percentages of“no growth.”
  • the word“comprise” and variations of the word, such as“comprising” and“comprises,” means “including but not limited to,” and is not intended to exclude, for example, other components, extracts, additives, or steps. It is also contemplated that embodiments described as“comprising” components, the invention also includes those same inventions as embodiments“consisting of” or“consisting essentially of.”
  • Ranges can be expressed herein as“approximately” or from“about” one particular value, and/or to“about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value.
  • a weight percent of a reagent, component, or compound unless specifically stated to the contrary, is based on the total weight of the reagent, component, composition or formulation in which the reagent, component, or compound is included, according to its usual definition.
  • “reduction,” is meant decrease or lower a characteristic (e.g., inflammation, growth or viability of microorganisms, in particular biofilms).
  • promote or other forms of the word, such as“promoting,” is meant to induce a particular event or characteristic, or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur.
  • “Treat” or other forms of the word, such as“treating,”“treatment” or treated,” is used here to mean to administer a composition or to perform a method in order to induce, reduce, eliminate, and prevent a characteristic (e.g., inflammation, growth or viability of microbes). It is generally understood that treating involves providing an effective amount of the composition to the mammal or surface (e.g., near a surface of an implant or patient) for treatment.
  • an amount of a composition or of a compound as provided herein is meant an amount sufficient to provide the desired benefit, either a reduction or prevention of microorganism growth or survival including biofilm colonies. As disclosed herein, the exact amount required will vary from use to use depending on a variety of
  • processing parameters such as the type of surface, the type of microorganism to be treated, the surface size, the mode of delivery and the like.
  • vehicle carrier refers to the manner in which the reagents or compositions may be delivered, including as a liquid, an injectable formulation, a solution, suspension, dispersion, and oral compositions and the like.
  • growth factors include but are not limited to, fibroblast growth factor (FGF), FGF-1 , FGF-2, FGF-4, platelet- derived growth factor (PDGF), insulin-binding growth factor (IGF), IGF-1 , IGF- 2, epidermal growth factor (EGF), transforming growth factor (TGF), TGF-b, TGF-a, and collagen growth factors, and/or biologically active derivatives of these growth factors.
  • bacteriaicidal or“antimicrobial” is meant the ability to effect (e.g., eliminate, inhibit decrease, or prevent) microorganism growth, viability, and/or survival at any concentration. It also means to kill the microorganism. It is further meant to eliminate, inhibit, decrease, prevent, destroy, or kill a biofilm and/or biofilm-forming microorganisms.
  • bacteriostatic is meant the ability to effect (e.g., stabilize or prevent future growth or prevent new growth) microorganism growth at any concentration.
  • a bacteriostatic compound, agent or reagent does not eliminate or kill the bacteria.
  • antiseptic is meant an antimicrobial reagent or composition that is applied to any surface, including skin or tissue, and particulary described herein on or around an implant or other surface of a patient near the implant to effect (e.g., eliminate, inhibit, decrease or prevent) microorganism growth, viability, and/or survival at any concentration. It is further meant to eliminate, inhibit, decrease, prevent, destroy, or kill a biofilm and/or biofilm-forming microorganisms.
  • “disinfect” or other forms of the word, such as“disinfectant” or “disinfecting,” is meant decrease or lower a characteristic (e.g., eliminate, reduce, inhibit, decrease, or prevent) microorganism growth, viability or survival at any concentration.
  • sanitize or other forms of the word, such as“sanitizer” or “sanitizing,” is meant decrease or lower a characteristic (e.g., eliminate, reduce, inhibit, decrease, or prevent) microorganism growth, viability or survival at any concentration. It is generally understood that sanitizing involves providing an effective amount of the composition to any surface (e.g., a surface of an implant or other surface of a patient near the implant).
  • Sterilize it is meant to kill microbes on the article being sterilized. Sterilize and sterilization include cold sterilization methods.
  • Irvine, CA product means a cyanidin 3-glucoside anthocyanin extracted from black rice and containing approximately 28% cyanidin 3-glucoside by weight with an additional 5% other anthocyanins.
  • the present invention provides methods of diagnosing infected implants or other surfaces of a patient, compositions and uses for treating the infection on the surface of an inserted implant and other surfaces of a patient. More specifically, the methods and compositions described herein include the administration of an anthocyanin or an anthocyanidin or metabolites thereof, preferably protocatechu ic acid (referred to herein as“PCA”) and/or 2,4,6 trihydroxybenzaldehyde (referred to herein as“2,4,6 THBA”) to inhibit or completely destroy a biofilm and all biofilm-forming microorganisms on or around a surface of an implant or of the patient.
  • PCA protocatechu ic acid
  • 2,4,6 THBA 2,4,6 trihydroxybenzaldehyde
  • the methods and compositions are used for the treatment of mammals, including humans. As described above, prior to the invention described herein there existed a great need for methods and compositions for diagnosing and treating infected implants. Accordingly, the methods and compositions described herein are suitable for the diagnosis and treatment of mammals having an infection on or around the surface of an implant. Such animals include equine, canine, and feline. [0083] Some embodiments of the invention described herein include methods of diagnosing whether an implant or other surface of a patient is infected with an infectious microorganism. In particular, the methods are suitable for detecting biofilm infections. The methods of diagnosing an infected implant or other surface of a patient are minimally invasive and may be carried out completely in an outpatient setting.
  • Exemplary and non-limiting surfaces of the patient include a closed wound, intact skin, skin having a sinus track, or any surface of a patient (internal and external).
  • Exemplary and non-limiting examples of implants include medical devices, a medical or surgical implant, total joint prosthesis, dental implants, a catheter, or a heart or vascular graft.
  • the method for determining whether an implant is infected or surface of a patient is infected includes performing a needle aspiration biopsy to detect the presence of a bacterial infection on or around the surface of an implant.
  • a needle biopsy is performed by aseptically preparing the skin lying above the area of the biopsy.
  • a needle is inserted into the skin and into the tissue overlying the implant or other internal surface of a patient and interstitial fluid is aspirated from the area surrounding the implant or area of infection on the surface of a patient.
  • the biopsy may be guided by the use of additional imaging aids including fluoroscopic equipment, computed tomography, and or ultrasonography as is known in the art (see e.g., Yee et al., Journal of Orthopaedic Surgery, 21 , pp. 236-240 (2013)).
  • additional imaging aids including fluoroscopic equipment, computed tomography, and or ultrasonography as is known in the art (see e.g., Yee et al., Journal of Orthopaedic Surgery, 21 , pp. 236-240 (2013)).
  • a physiologically compatible solution is utilized to irrigate the area surrounding the implant.
  • physiologically compatible solutions are well known in the art and include saline and Ringer’s solution and the like. Irrigation of the surrounding tissue and implant may increase the likelihood of aspirating fluid containing an infectious microorganism.
  • movement of the tissue such as flexion, rotation, and adduction of joints can be used to facilitate the aspiration of an infectious microorganism.
  • Exemplary devices for performing the needle aspiration include a needle attached to a housing for aspirating fluid from an area surrounding the implant suspected of having an infection.
  • the aspiration device may include a connector for attaching a needle to the device and a plunger for extracting a sample of the joint fluid from the body of the patient through the needle into the housing of the device.
  • the length and diameter of the needle depends upon the location of implant relative to the surface of the skin, accessibility of the implant, and ease of obtaining an aspirate.
  • Suitable needles may be of a diameter ranging anywhere from 12 gauge to 30 gauge and from less than an inch in length to several inches in length as is necessary to reach the suspected area of infection or surface of an implant that is suspected to have an infection.
  • the resulting needle aspiration biopsy of tissue surrounding the surface of the implant is cultured to identify the presence of any infectious microorganisms.
  • the aspirate may be Gram stained to identify the presence of any bacteria microorganisms prior to culture.
  • the aspirate is cultured to determine a presence of an infection.
  • General bacteriological culturing methods are well known in the art.
  • aspirates can be inoculated on blood agar plates and grown for 24 to 48 hours under aerobic and anaerobic conditions. The presence of growth is monitored.
  • aspirates can be inoculated in a growth broth, which is monitored for turbidity.
  • the microorganism can be cultured for a day or for up to several weeks to maximize the possibility of detection. Prolonged culture periods can be more effective in identifying certain microorganisms including, for example, Propionibacterium, Bacilli, and Peptostreptococcus species.
  • blood cultures may be utilized to aid in the detection of slow growing microorganisms.
  • anthocyanin or anthocyanidin or metabolite thereof e.g., PCA or 2,4,6 THBA
  • diagnosis of infection of an implant includes performing additional diagnostic assays, in addition to performing a minimally invasive needle aspiration biopsy technique. These include serologic tests including sedimentation rate (ESR) determination, C-reactive protein levels, white blood cell count, and neutrophil percentage.
  • ESR sedimentation rate
  • C-reactive protein levels C-reactive protein levels
  • neutrophil percentage C-reactive protein levels
  • genetic analysis for the detection of microorganism genetic material may be performed using polymerase chain reaction (PCR) detection methods.
  • Other clinical elements include, loss of function, onset of pain in the implant area, sinus tract, persistant wound drainage, or fever that may be indicative of an infection.
  • the application of ultrasound is utilized to facilitate release of an infectious microorganism in situ.
  • the application of ultrasound is described herein as particularly useful for detecting a biofilm infection present on or around an implant or other surface of a patient.
  • biofilm infections it is not always possible to obtain a positive detection for the presence of pathogenic microorganisms because the microorganisms are in a quiescent non-growth stage and are enveloped in a thick extracellular matrix.
  • the application of ultrasound disrupts the structure of the extracellular matrix of the biofilm such that a biofilm-forming microorganism can be detected by any of the
  • Ultrasound techniques for disrupting biofilms from implant materials in vitro is known in the art ( see Granick et al., Eplasty 17(e13), pp. 128-134, 2017 and Trampuz et al., NEJM Z51 , pp. 654-663, 2007). These techniques have not been contemplated for disrupting a biofilm infection on an infected implant in situ or other surface of a patient in vivo. As first described herein, ultrasound energy can be applied in vivo to facilitate the release of any biofilm-forming microorganisms present on an infected implant or other surface of a patient.
  • lithotripsy Conventionally, ultrasound energy has been utilized for moving and ablating kidney stones through the technique of shock wave lithotripsy (see e.g., U.S. Patent Nos. 5,496,306, 6,770,039 and 6,123,679).
  • Suitable devices for performing lithotripsy include electrohydraulic, piezoelectric, and electromagnetic lithotripters, such as those available from Dornier Med Tech, Xi Xin Medical Instruments Co. Ltd, and Tissue Regeneration Technologies (see also e.g., Connors et al., J Urol. 191 (1 ), 2014).
  • the frequency, pressure, focal width, shock wave rate, and power density of the lithotripter is selected such that a biofilm is disrupted, while minimizing potential damage to surrounding tissues of the patient.
  • the application of ultrasound energy can be utilized in a focussed manner to exert a biocidal effect to the infectious microorganisms and biofilm colonies. In this way, the method of applying ultrasound energy through a lithotripter aids in the sanitization of an infected implant.
  • the frequency of the oscillating acoustic pressure wave is from about 1 kHz to about 60 kHz. In some embodiments, the frequency is about 10 kHz to about 40 kHz. In some embodiments, the frequency is about 1 kHz, about 5 kHz, about 10 kHz, about 15 kHz, about 20 kHz, about 25 kHz, about 30 kHz, about 35 kHz, about 40 kHz, about 45 kHz, about 50 kHz, about 55 kHz, or about 60 kHz.
  • the pressure generated by the lithotripter is about 5 MPa to about 160 MPa. In some embodiments, the pressure is about 10 MPa to about 40 MPa. In some embodiments, the pressure is about 1 MPa, about 5 MPa, about 10 MPa, about 15 MPa, about 20 MPa, about 25 MPa, about 30 MPa, about 35 MPa, about 40 MPa, about 50 MPa, about 60 MPa, about 70 MPa, about 80 MPa, about 90 MPa, about 100 MPa, about 120 MPa, about 140 MPa, or about 160 MPa.
  • the spatial distribution of acoustic energy or focal width is from about 1 mm to about 30 mm. In some embodiments, the focal width is about 5 mm to about 15 mm. In some embodiments, the focal width about 1 mm, about 5 mm, about 10 mm, about 15 mm, about 20 mm, about 25 mm, or about 30 mm.
  • the shock wave rate is about 10 shock waves per minute to about 200 shock waves per minute. In some embodiments, the shock wave rate is about 20 shock waves per minute to about 80 shock waves per minute. In some embodiments, the shock wave rate is about 10 shock waves per minute, about 20 shock waves per minute, about 30 shock waves per minute, about 35 shock waves per minute, about 40 shock waves per minute, about 45 shock waves per minute, about 50 shock waves per minute, about 55 shock waves per minute, about 60 shock waves per minute, about 80 shock waves per minute, about 100 shock waves per minute, about 120 shock waves per minute, about 140 shock waves per minute, about
  • the power density is from about 0.1 W/cm 2 to about 25,000 W/cm 2 . In some embodiments, the power density is from about 1 W/cm 2 to about 10,000 W/cm 2 . In some embodiments, the power density is from about 20 W/cm 2 to about 5,000 W/cm 2 . In some embodiments, the power density is from about 100 W/cm 2 to about 3,000 W/cm 2 .
  • the ultrasound may be applied prior to performing a needle aspiration biopsy. Any microorganisms present in a biofilm can be loosened to increase the likelihood of detection of the biofilm-forming microorganism. Alternatively, the ultrasound may be applied following a first unsuccessful needle aspiration biopsy to increase the likelihood that any biofilm-forming bacteria are detected. Thus, in some embodiments, ultrasound is applied prior to performing a needle aspiration biopsy. In some other embodiments, ultrasound is applied after a first needle aspiration biopsy is performed.
  • the methods described herein include determining if an infection, including a biofilm infection, is present on or around the surface of the implant or other surface of a patient.
  • the methods described herein further include administering an effective amount of an anthocyanin or an anthocyanidin or metabolites thereof, preferably PCA and 2,4,6 THBA as an anti-microbial agent.
  • PCA was also effective on C. albicans, which is important considering its ability to form biofilms and difficulty in treating C. albicans when existing within a catheter or an implant.
  • FIG 32 it is shown that PCA is effective against C. difficile, P. acnes 6919, E. coli 8739 and 43895, S. aureus 6538,
  • S. aureus 33591 P. aeruginosa 9027, methicillin resistant staphylococcus epidermis (MRSE), including MRSE ATCC 51625, and Legionella 43662, and others.
  • Figure 32 shows that 2,4,6 THBA is effective against E. coli 8739 and 43895, S. aureus 6538, S. aureus 33591 , P. aeruginosa 9027, methicillin resistant staphylococcus epidermis (MRSE), including MRSE ATCC 51625, and Legionella 43662, and others.
  • MRSE methicillin resistant staphylococcus epidermis
  • Biofilms are comprised of bacteria that form colonies and produce a surrounding matrix film to protect themselves.
  • the biofilm forming bacteria can form colonies that attach to foreign bodies, each other and tissues.
  • the bacteria aggregate in clusters and are surrounded by extracellular polymer matrix.
  • the biofilms are hard to destroy and therefore kill the underlying bacteria and provide the basis for much of the antibiotic resistance that has developed.
  • the formation of a biofilm is a two-step process: 1. adherence of cells to a foreign body surface; and 2. accumulation of cells to form multilayered cell clusters.
  • PCA was able to stop the formation of a biofilm as well as kill bacterial in already formed biofilms.
  • the biofilms tested were Pseudomonas aeruginosa ATCC 700888 and Staphylococcus aureus ATCC 33591 (MRSA). The tests are described in more details in Examples 7 and 8 and Figures 35 and 36.
  • the implant or other surface of a patient is treated with an effective amount of an anthocyanin or
  • anthocyanidin or a metabolite thereof in situ Exemplary and non-limiting anthocyanins include cyanidin-3-glucoside or delphinidin-3-glucoside, cyanidin-3-galactoside, and pelargonidin-3-galactoside. Exemplary and non- limiting anthocyanidins include cyanidin, delphinidin, pelargonidin, malvidin and petunidin.
  • the implant or other surface of a patient is treated with a metabolite of anthocyanins and anthocyanidins including PCA, 2,4,6 THBA, and combinations thereof.
  • the anthocyanin or anthocyanidin or metabolite thereof, including PCA or 2,4,6 THBA may be administered as pure crystals or as part of a composition described herein.
  • are methods of prophylactically treating a preoperative skin incision site comprising administering an anthocyanin, an anthocyanidin or a metabolite thereof to a patient in need of such treatment an effective amount of the anthocyanin or anthocyanidin compound wherein microbial growth is prevented or reduced prior to any surgery involving the installation of an implant.
  • a method of disinfecting a surface of an implant or other surface of a patient includes contacting said surface with an anthocyanin, an anthocyanidin and/or a metabolite thereof in an effective amount of the anthocyanin, anthocyanidin and/or metabolite compound wherein microbial growth is prevented, reduced or eliminated.
  • the microbial growth that is reduced includes infections and biofilm infections on the surface of an implant or other surface of a patient that may be an endogenous or exogenous source, including but not limited to MRSA, P. acnes, S. aureus, P. aeruginosa, E. coli, S. epidermidis,
  • the effective amount of the anthocyanin or anthocyanidin or metabolite thereof, including PCA or 2,4,6 THBA or a combination thereof is from about 1 pg to about 1000 mg. In some embodiments, the effective amount of the anthocyanin or anthocyanidin or metabolite thereof, including PCA or 2,4,6 THBA or a combination thereof is from about 1 pg to about 1000 mg. In some
  • the effective amount is from about 1 pg to about 500 mg. In some embodiments, the effective amount is from about 1 pg to about 50 mg. In some embodiments, the effective amount is from about 1 pg to about 10 mg. In some embodiments, the effective amount is from about 1 pg to about 1000 pg. In some embodiments, the effective amount is from about 1 pg to about 500 pg. In some embodiments, the effective amount is from about 1 pg to about 50 pg.
  • the anthocyanin, anthocyanidin, or metabolite thereof is incorporated as pure crystals in an implantable material, such as a mesh including titanium or stainless steel.
  • the crystals are applied to the metal where there is surface configuration that provides for housing of the crystal on the surface. In this way, the crystals remain in place in crystal form until activated when subject to fluid common to the mammalian body.
  • the anthocyanin, anthocyanidin, or metabolite thereof can be present as a polymorphous, semi-crystalline, hydrate, amorphous or polyamorphous forms.
  • the crystals of the anthocyanin or anthocyanidin or metabolite thereof may be micronized using known micronization techniques known in the art.
  • the micronized crystals may be useful for embedding upon a surface of to enhance the dissolution of the compound when provided in a solution or when administered in vivo to a patient.
  • the crystals have a mean particle distribution of ⁇ 1000 pm, ⁇ 500 pm, ⁇ 400 pm, ⁇ 300 pm, ⁇ 200 pm, ⁇ 100 pm, ⁇ 50 pm, or even ⁇ 10 pm.
  • the particles may have a“d90”, “d50”, and“d10” distribution of the forgoing sizes, meaning that (90%, 50%, or 10%, respectively) of particle sizes are less than a specified size or size range.
  • d90 ⁇ 90 pm means that 90% of the particle sizes within a distribution of particles are less than or equal to 90 pm.
  • the anthocyanin, anthocyanidin, or metabolite thereof is substantially pure.
  • the compounds are at least 75% to greater than 99% pure.
  • the compounds are at least 90%, 95%, 99%, or 99.99% pure as assessed by techniques routine in the art, such as high performance liquid chromatography (HPLC).
  • the anthocyanin, anthocyanidin, or metabolite thereof is provided as a pharmaceutically acceptable salt form.
  • salts forms are those formed by, for example, contacting a free base of the compound with a suitable acid in a suitable solvent under suitable conditions that will form an acid addition salt.
  • the salt form includes alkali metal salts such as, for example, sodium or potassium salts, alkaline earth metal salts such as, for example, calcium and magnesium salts, and salts with organic or inorganic acid such as, for example,
  • hydrochloric acid hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, citric acid, formic acid, maleic acid, succinic acid, tartaric acid,
  • the anthocyanin, anthocyanidin or metabolite thereof may be administered to treat the infected implant or other surface of a patient by methods commonly used in the art for the treatment of infected implants with antimicrobial compounds. These include, but are not limited to, topical adminstration above the area of infection, transdermal administration above the area of infection, enteric administration (e.g., oral or rectal), sublingual administration, or other parenteral injection, including epidermal injection, intravenous injection, subcutaneous injection, intra muscular injection, intra- articular injection, or other injection to administer the anthocyanin or anthocyanidin in proximity to the infected implant or other infected surface of a patient.
  • enteric administration e.g., oral or rectal
  • sublingual administration e.g., sublingual administration
  • parenteral injection including epidermal injection, intravenous injection, subcutaneous injection, intra muscular injection, intra- articular injection, or other injection to administer the anthocyanin or anthocyani
  • the anthocyanin, anthocyanidin, or metabolite thereof is administered to a patient having an infected implant or other surface of a patient at least one time.
  • the anthocyanin, anthocyanidin, or metabolite thereof is administered at a set frequency.
  • the frequency of administration may be once a week, twice a week, three times a week, four times a week, five times a week, six times a week, daily, twice daily, three times daily, four times daily, or hourly.
  • the anthocyanin, anthocyanidin, or metabolite thereof may be administered continuously through infusion or irrigation of the infected surface of a patient or implant. This type of administration is possible because the anthocyanin or anthocyanidin or metabolites thereof described herein are safe and with little to no demonstrated local or systemic toxicity.
  • the anthocyanin is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • anthocyanidin, or metabolite thereof is administered prophylactically to a patient receiving an implant to prevent or reduce the likelihood of an infection.
  • the patient is also prophylactically monitored using the diagnostic methods described herein to identify an infection prior to a clinical manifestation of the infection.
  • the anthocyanin, anthocyanidin, or metabolite thereof is administered with a second pharmaceutical agent.
  • second pharmaceutical agents include additional anthocyanin or anthocyanidin compounds or metabolites thereof, antibiotics, analgesics, and anti-inflammatory agents.
  • the therapeutic effective dose may vary depending on a wide variety of factors. For instance, the dose may vary depending on the formulation, method of application of the therapeutic reagent or combination with other reagents, or compositions, compounds or combination of compositions or compounds to the infected implant or surface of a patient.
  • Compositions may vary depending on the formulation, method of application of the therapeutic reagent or combination with other reagents, or compositions, compounds or combination of compositions or compounds to the infected implant or surface of a patient.
  • compositions for treating infected implants or other infected surfaces of a patient.
  • the disclosed antimicrobial reagents and compositions can be used to eliminate, reduce, and/or prevent microorganism growth, viability, or survival.
  • the compositions including an anthocyanin or anthocyanidin or metabolite thereof in an effective amount to eliminate, reduce, and/or prevent microorganism growth, viability, or survival of a biofilm causing an infection of an implant or other surface of a patient.
  • anthocyanidin metabolite is isolated reagents.
  • the present composition includes PCA,
  • the present invention provides for a
  • compositions for treating an infected implant or other surface of a patient including: a) an anthocyanin; b) anthocyanidin; c) a metabolite of an anthocyanin or anthocyanidin such as C3G, PCA, 2,4,6 THBA, vanillic and hippuric acid.
  • the present composition includes PCA, 2,4,6 THBA, or a combination thereof.
  • compositions include those that can be applied to or near the site of the infected implant or infected surface of a patient.
  • the composition comprising anthocyanin or anthocyanidin or metabolite thereof may be in the form of a liquid solution, suspension, a dispersion, or an emulsion.
  • the anthocyanin, anthocyanidin, or metabolite thereof is about 0.5% to about 90% by weight of the composition. In some embodiments, the anthocyanin, anthocyanidin, or metabolite thereof is about 0.5% to about 70% by weight of the composition. In some embodiments, the anthocyanin, anthocyanidin, or metabolite thereof is about 0.5% to about 50% by weight of the composition. In some embodiments, the anthocyanin, anthocyanidin, or metabolite thereof is about 0.5% to about 30% by weight of the composition. In some embodiments, the anthocyanin, anthocyanidin, or metabolite thereof is about 0.5% to about 20% by weight of the composition.
  • the anthocyanin, anthocyanidin, or metabolite thereof is about 5% to about 30% by weight of the composition. In some embodiments, the anthocyanin, anthocyanidin, or metabolite thereof is about 5% to about 30% by weight of the composition. In some
  • the anthocyanin, anthocyanidin, or metabolite thereof is about 0.5% to about 20% by weight of the composition. In some embodiments, the anthocyanin or anthocyanidin or metabolite thereof is about 0.5%, about 1 %, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90% by weight of the composition.
  • the anthocyanins or anthocyanidins and metabolites thereof is provided in concentrations of about 10 to 200 mM. In other embodiments, anthocyanins, or anthocyanidins, or metabolites thereof are provided in any recited composition or method of use in a range of between 20 to 200 mM. In yet other embodiments, the anthocyanin, anthocyanidin, or metabolites thereof provided in any recited composition or method of use is provided in a range of between 20 to 100 mM.
  • the anthocyanin, anthocyanidin, or metabolites thereof provided in any recited composition or method of use is provided in a range of between 50 to 100 mM. In yet other embodiments, the anthocyanin, anthocyanidin, or metabolites thereof provided in any recited composition or method of use is provided in a range of between 20 to 50 mM.
  • the anthocyanin, anthocyanidin, or metabolite thereof is in the form of crystals that are embedded into a composition.
  • compositions in which the crystals are embedded include a cloth or a mesh, such as titanium or stainless steel.
  • the anthocyanin, anthocyanidin, or metabolite thereof is in the form of a powder.
  • a powder form is contemplated for further use in a composition described herein (e.g., to be dissolved or suspended therein) or imbedded into a composition.
  • the disclosed composition can be selected from anthocyanins, anthocyanidins, metabolites of anthocyanin and anthocyanidin compounds, or a combination thereof.
  • the anthocyanin can be selected from cyanidin-3-glucoside or delphinidin-3-glucoside, cyanidin-3-galactoside, and pelargonidin-3-galactoside and combinations thereof.
  • the anthocyanidins can be selected from cyanidin, delphinidin, pelargonidin, malvidin, petunidin, and combinations thereof.
  • metabolites of anthocyanins and anthocyanidins can be selected from PCA and 2,4,6 THBA and combinations thereof.
  • the anthocyanin, anthocyanidin, or metabolite thereof is in a solution. In some embodiments, the metabolite of the
  • anthocyanin or anthocyanidin is PCA or 2,4,6 THBA.
  • the metabolite of the anthocyanin or anthocyanidin is PCA.
  • the metabolite of the anthocyanin or anthocyanidin is
  • the anthocyanin, anthocyanidin, or a mixture or combination thereof is provided in a composition including a carrier that includes 50% to 91 % isopropyl alcohol.
  • a carrier that includes 50% to 91 % isopropyl alcohol.
  • 2,4,6 THBA or a mixture or combination thereof is provided in a composition including a carrier that includes 50% to 91 % isopropyl alcohol.
  • PCA is provided in a composition including a carrier that includes 50% to 91 % isopropyl alcohol.
  • 2,4,6 THBA is provided in a composition including a carrier that includes 50% to 91 % isopropyl alcohol.
  • compositions described herein are suitable for routes of
  • administration including oral, injection, intravenous, subcutaneous, epidermal, topical, sublingual, buccal, inhalation, intradermal, subcutaneous, intra articular, soft tissue, and cutaneous.
  • Oral administration of the compositions of this disclosure may include a liquid or semisolid form, tablet, pill, capsule, powder, or gel.
  • oral administration will be in a liquid composition.
  • compositions including a liquid pharmaceutically inert carrier such as water may be considered for oral administration.
  • a liquid pharmaceutically inert carrier such as water
  • Other pharmaceutically compatible liquids or semisolids may also be used. The use of such liquids and
  • semisolids and manufacturing of tablet, pill, powder, or gel compositions is well known to those of skill in the art.
  • the injectable compositions be in liquid or semi-liquid form.
  • Other pharmaceutically compatible liquids or semi-liquids may also be used.
  • the use of such liquids and semi-liquids is well known to those of skill in the art.
  • the composition is formulated as a topical composition. More preferable, the vehicle of the topical composition delivery is in the form of a liquid, salve, soap, spray, foam, cream, emollient, gel, ointment, balm or transdermal patch.
  • the compositions can be in the form of an aqueous solution.
  • the compositions disclosed herein are in the form of a liquid, gel, suspension, dispersion, solid, emulsion, aerosol, for example, powders, tablets, capsules, pills, liquids, suspensions, dispersions or emulsions.
  • the compositions disclosed herein can be in the form suitable for dilutions.
  • the compositions can be in the form of a powder, cream, paste, gel or solid that can be reconstituted.
  • the antimicrobial composition can also include at least one additive selected independently from a carrier, a diluent, an adjuvant, a solubilizing agent, a suspending agent, a filler, a surfactant, a secondary antimicrobial agent, a preservative, a viscosity modifier, a thixotropy modifier, a wetting agent, an emulsifier, or any combinations thereof.
  • the disclosed antimicrobial composition can further comprise at least one surfactant selected from a cationic surfactant, an anionic surfactant, a non-ionic surfactant, and an amphoteric surfactant.
  • the disclosed antimicrobial and/or pharmaceutical compositions may further comprise medicament is selected from the group consisting of anesthetic agents, cleansers, antiseptic agents, scar reducing agents, immunostimulating agents, antiviral agents, antikeratolytic agents, anti-inflammatory agents, antifungal agents, antihistamine agents, antibacterial agents, bioadhesive agents, inhibitors of prostaglandin synthesis, antioxidants, and mixtures thereof.
  • medicament is selected from the group consisting of anesthetic agents, cleansers, antiseptic agents, scar reducing agents, immunostimulating agents, antiviral agents, antikeratolytic agents, anti-inflammatory agents, antifungal agents, antihistamine agents, antibacterial agents, bioadhesive agents, inhibitors of prostaglandin synthesis, antioxidants, and mixtures thereof.
  • the disclosed antimicrobial compositions can optionally include one or more additives such as carriers, adjuvants, solubilizing agents, suspending agents, diluents, surfactants, other antimicrobial agents, preservatives, fillers, wetting agents, antifoaming agents, emulsifiers, and additives designed to affect the viscosity or ability of the composition to adhere to and/or aid in the treatment of an infected implant or other surface of a patient.
  • additives such as carriers, adjuvants, solubilizing agents, suspending agents, diluents, surfactants, other antimicrobial agents, preservatives, fillers, wetting agents, antifoaming agents, emulsifiers, and additives designed to affect the viscosity or ability of the composition to adhere to and/or aid in the treatment of an infected implant or other surface of a patient.
  • compositions including the selected active components, including the anthocyanins or anthocyanidins and metabolites thereof, are without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained.
  • the antimicrobial compositions disclosed herein can further comprise a carrier.
  • carrier means a compound
  • composition substance, or structure that, when in combination with a compound or composition disclosed herein, facilitates preparation
  • “Pharmaceutically acceptable carrier” means a compound, composition, substance, or structure that is useful in neither preparing a pharmaceutical composition which is generally safe, non-toxic, and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary as well as human pharmaceutical use.
  • the carrier maybe aqueous or oily.
  • the carrier may further include suitable pH modifying agents including alkali metal salts, such as sodium or potassium hydroxide, or acidic
  • oxidation stabilizers e.g., sodium bisulfite or preservatives, e.g., benzyl alcohol, may advantageously be incorporated into the compositions.
  • the antimicrobial compositions disclosed herein can also comprise adjuvants such as preserving, wetting, emulsifying, suspending agents, and dispensing agents. Prevention of the action of other microorganisms can be accomplished by various antifungal agents, for example, parabens, chlorobutanol, phenol, and the like.
  • Suitable suspending agents can include, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,
  • microcrystalline cellulose aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
  • compositions can also comprise
  • solubilizing agents and emulsifiers as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl alcohol, benzyl benzoate, propyleneglycol, 1 ,3-butyleneglycol, dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofur fury 1 alcohol, polyethyleneglycols and fatty acid esters of sorbitan or mixtures of these substances, and the like.
  • the additives can be present in the disclosed compositions in any amount for the individual anthocyanin, anthocyanidin or metabolite thereof compound components.
  • the composition may further be in the form of a gel or include an in situ gel-forming agent that results in the release of the anthocyanin, anthocyanidin or metabolite thereof over an extended period.
  • extended release gel- forming compositions are generally known in the art (see e.g.,
  • kits for diagnosing and treating an infected implant or surface of a patient with a therapeutically effective amount of an anthocyanin or anthocyanidin or metabolite thereof may include a minimally invasive sampling device for detecting the presence of an infectious or pathological microorganism, such as a sterile wrapped needle and syringe.
  • the kit further includes an anthocyanin, anthocyanidin or metabolite thereof in an amount effective for the treatment of an infected implant or surface of a patient.
  • the anthocyanin, anthocyanidin, or metabolite thereof can be provided in a liquid or gellable composition described herein.
  • the anthocyanin, anthocyanidin, or metabolite thereof can be provided as a crystal present in a vial that can be further prepared as a liquid composition or embedded in an implantable material.
  • the amount of the anthocyanin, anthocyanidin, or metabolite thereof present in the kit may range from 1 mg to greater than or equal to 10,000 mg.
  • the kit may also include culturing devices including culture plates, tubes, and growth media as known in the art.
  • the kit may further include instructions for diagnosing and treating a patient suspected of having an infection of an implant or other surface according to the invention.
  • compositions and methods provided are exemplary and are not intended to limit the scope of any of the specified embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in any and all variations or iterations.
  • the scope of the compositions, formulations, methods, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences herein described. The examples below describe non-limiting examples of the inventive embodiments of the invention.
  • Example 1 Use of in vitro studies for antimicrobial susceptibility testing of anthocyanins, anthocyanidins, or metabolites and compounds thereof
  • the control sample was amoxicillin, an antimicrobial with very effective broad-spectrum antibiotic properties.
  • Samples included delphinidin, pelargonidin, cyanidin Cl, 28% cyanindin-3-glucoside (C3G), protocatechuic acid (PCA) and 2,4,6 trihydroxybenzaldehyde (2,4,6 THBA).
  • each plate was examined.
  • the diameters of the zones of complete inhibition were measured, including the diameter of the disc. Zones were measured to the nearest millimeter, using sliding calipers. The size of the zones of inhibition was interpreted by referring to NCCLS standard.
  • Results were interpreted as follows: Nl was no inhibition of growth under the test sample, I was inhibition of growth under the test sample, NZ indicated no zone of inhibition surrounding the test sample, and CZ indicated a clear zone of inhibition surrounding the sample and zone width in millimeters. See figure 6 for complete results.
  • the testing samples had bactericidal and bacteriostatic activity against many of the organisms.
  • P. acnes an organism that is very difficult to treat, often requiring multiple current antibiotics for effective treatment, was susceptible to both C3G and PCA. Indeed, both of these test samples were bactericidal against P. acnes.
  • PCA was also effective against Staphlococcus aureus ATCC 33591 , known as Methicillin Resistant Staph aureus (MRSA), Staphlococcus epidermidis ATCC 51625, known as Methicillin Resistant Staph epidermidis (MRSE), E. coli 8739 and 43895, and Legionella 43662.
  • MRSA Methicillin Resistant Staph aureus
  • MRSE Methicillin Resistant Staph epidermidis
  • E. coli 8739 and 43895 E. coli 8739 and 43895
  • Legionella 43662 Legionella 43662.
  • Pseudomonas aeruginosa a common pathogen in wounds, especially burns, as well as chronic lung infections. Amoxicillin, the control sample, had no effect on P. aeruginosa. Similarly, Candida albicans, frequently a co pathogen in wounds, was susceptible to PCA.
  • Pseudomonas aeruginosa a common pathogen in wounds, especially burns. Amoxicillin, the control sample, had no effect on P. aeruginosa. Similarly, Candida albicans, frequently a co pathogen in wounds, was susceptible to PCA.
  • the present invention provides advantages over the prior art, including providing anthocyanin, anthocyanidin, their metabolites or combinations thereof to a wound to provide a reduction or elimination of bacteria. It is contemplated that the invention will also find use in the treatment of surfaces, including medical devices and medical implants, to reduce or eliminate bacteria.
  • Example 2 Use of mouse model to determine dose levels and intervals of test samples.
  • mice had back skin tape stripped and the stripped site (wound) was infected with P. aeruginosa (ACTA 9027).
  • the test reagents were applied topically in an aqueous solution on the stripped site at two hours and daily for four days.
  • Cyanidin 3-glucoside (C3G), an anthocyanin, and its main metabolite PCA were formulated and tested at several doses.
  • the aqueous carrier was water.
  • the C3G formulation included 50 mM, 100 mM and 200 mM dose concentrations.
  • the PCA formulation included at 50, 100 and
  • Results were collected from the mice at day five. Both C3G and PCA decreased the bacterial burden; however, none were statistically significant. See figure 9A. There was a trend towards a decreasing concentration of PCA, with 50 mM being the most effective. The most effective dose of C3G was 100 mM. It is contemplated that because C3G degrades to PCA in this
  • test results may indicate that C3G was not being tested alone, but rather was a combination of C3G and its metabolites, including a combination of C3G and PCA as the effective agents.
  • Example 3 Use of mouse model to further determine effective dose levels and dose intervals of test samples.
  • mice had back skin tape stripped and the stripped site (wound) was infected with P. aeruginosa (ACTA 27853).
  • the test reagents were applied topically in an aqueous solution on the stripped site at two hours and daily on day 1 , 2 and 3.
  • C3G, an anthocyanin and its main metabolite PCA were formulated and tested at several doses.
  • the aqueous carrier was water.
  • the C3G formulation included 100 mM and 200 mM dose concentrations and the PCA formulation included 25 and 50 mM dose concentrations.
  • Results were collected from the mice at day two and four. Both C3G and PCA decreased the bacterial burden at 48 and 96 hours. (See Figure 9B). The most significant decrease of bacteria was observed at 25 mM of and 100 and 200 mM of C3G. Although PCA at 25 mM reduced the bacterial burden at both time periods, its activity was statistically significant at 48 hours. C3G at both 100 mM and 200 mM significantly reduced the bacterial burden at 48 and 96 hours.
  • Example 4 Use of a mouse model for wound healing.
  • mice were shaved but unstrapped and uninfected (normal rodent skin).
  • the test reagents were applied topically in an aqueous solution on the unstripped site at two hours and daily on day 1 , 2 and 3.
  • Example 5 Use of mouse model to determine isolated effect of 25 mM solution of PCA in various environments.
  • the PCA test reagent was applied topically in an aqueous solution on the stripped site at two hours and 24 hours.
  • the testing reagents consisted of and PCA formulated at one dose, 25mM, in an aqueous solution.
  • Levels of IGF-1 , TGF-b, and EGF levels in the skin tissue at 48 hours were measured by ELISA. There were two control groups; the stripped skin and the stripped skin and infected. Results:
  • the infected stripped skin showed the highest level with IGF-1 (statistically significant) and TGF-b. This is representative of tissue response to injury and infection; similarly, the EGF response was very inconsistent compared to the other two growth hormones.
  • EGF response levels were different than either IGF-1 or TGF-b. They were highest in the stripped and uninfected wound and lowest in the stripped, infected and treated wound. Therefore, the treatment optimized the amount of hormone production compared to the untreated infection. This is beneficial to limit scarring while promoting healing over the controls.
  • PCA at 25 mM acts on stripped and infected mice skin and optimizes the IGF- 1 production and optimizes the local growth hormones.
  • Example 6 Use of mice to establish wound promoting effect of compositions.
  • Control Group 1 three mice with only tape stripped wounds on the back. These mice were not infected or treated. The skin was harvested at time zero, 2 and 48 hours for histology examination.
  • Control Group 2 three had tape stripped wounds and infection. Tissue submitted at 2 and 48 hours for histological examination.
  • mice had skin stripped wounds and infection. Treatment varied by reagent and dosage. Testing reagents included PCA at 25 and 50 mM and C3G at 100 and 200 mM.
  • Pseudomonas aeruginosa procured from American Type Culture Collection, Manassas, VA was used to infect the experimental groups of mice. The organism was grown overnight at 37°C at ambient atmosphere trypticase soy agar plates supplemented with 5% sheep blood cells. The culture will be aseptically swabbed and transferred to tubes of trypticase soy broth. The optical density will be determined at 600 nm. The cultures will be diluted to provide an inoculum of approximately 9.0 logio CFU per mouse in a volume of 100 pL. Inoculum count was estimated before inoculation by optical density and confirmed after inoculation by dilution and back count.
  • testing reagents were topically applied at 2 and 24 hours with 100 uL of fluid spread over the wound.
  • Thickness The thickness of the dermal layer was observed.
  • Hair Follicles The hair follicles and the layer of surrounding cells were observed. Hair follicles presence is critically important to skin wound healing. (Gharzi A, Reynolds AJ, Jahoda CA. Plasticity of hair follicle dermal cells in wound healing and induction. Exp Dermatol. 2003 Apr; 12 (2): 126-36). The dermal sheath surrounding the hair follicle has the progenitor cells for contributing fibroblasts for wound healing. (Johada CA, Reynolds AJ. Hair follicle dermal sheath cells: unsung participants in wound healing. Lancet. 2001 Oct 27; 358(9291 ): 1445-8).
  • Vascularity Vascularity was observed, but an assessment of angiogenesis was not performed on the 48 hour material since new vascularity takes three to twelve days to develop. (Busuioc CJ, et al. Phases of cutaneous angiogenesis process in experimental third-degree skin burns: histological and immunohistochemical study. Rom J Morphol Embryol. 2013; 54(1 ):163-710.)
  • Inflammation The presence of cellular infiltration was observed and its location.
  • Skin Thickness The thickness of the skin was estimated related to the uninfected, untreated wound. This depth was estimated on the uniform histology photomicrographs from the surface to the muscle layer. Results:
  • CONTROL GROUP 1 Uninfected and untreated.
  • Time Zero (See figures 14-15) At time zero following the wound stripping there was cellular covering of the surface. The dermal layer was not thickened. The hair follicles have a single cellular lining. There was minimal vascularity and no inflammation. The depth of the tissue was considered zero for future bench mark 0+.
  • CONTROL GROUP 2 Infected and untreated.
  • the experimental model provided evidence of a histological contrast between the control and experimental groups.
  • Control Group 2 that was wound stripped and infected showed a clear contrast to the uninfected Control Group 1.
  • This histological condition provided clear contrast to the treatment groups. All treatment groups by comparison showed healing response with multiple layer cellular
  • PCA at a concentration of 25 mM also showed collagen layer formation between the epidermis and dermis.
  • Example 7 PCA’s effect on Pseudomonas aeruginosa ATCC 700888 and Staphylococcus aureus ATCC 33591 (MRS A) biofilms
  • the inventor has shown that a composition comprising PCA was able to stop the growth of a biofilm formation as well as destroy already formed biofilms.
  • the biofilms tested were Pseudomonas aeruginosa ATCC 700888 and Staphylococcus aureus ATCC 33591 (MRSA).
  • MRSA Staphylococcus aureus ATCC 33591
  • the following amounts were tested on a polyester cloth and sintered 316 stainless steel mesh: The cloth was a piece cut from a polyester pillow case. The cloth was soaked in the PCA solution and air dried for 24 hours. The cloth was dried when tested.
  • PCA crystals imbedded vehicle: 5-ply sintered 40 micron mesh, 1 -1
  • Staphylococcus aureus ATCC 33591 (MRSA) were placed in reactors and allowed to grow and form biofilms. Then cloths and metal were treated by coating with PCA solutions and then were left to dry. Two sets of the stainless steel mesh had crystals imbedded into the mesh to replicate placement into a mesh or coated joint implant.
  • a standard ASTM E-2647 drip flow biofilm reactor was used to grow a biofilm and the treated surfaces (as well as the control) were placed into the reactors and the biofilm was allowed to grow for about 6 hours. The samples received a continuous nutrient flow for an additional time period for about 48 hours to promote a steady growth rate of the biofilm.
  • CFU Colony forming unites
  • the invention also provides a method of blocking initial attachment of the bacteria to the implant and therefor preventing growth/development of a biofilm on an implant.
  • an anthocyanin, anthocyanidin or metabolite thereof depends upon the species of bacterial to be eradicated. It was discovered that a 10% concentration of PCA was not effective on Pseudomonas aeruginosa (see figure 33), whereas a 20% concentration was effective (see figure 34).
  • mice Female BALB/c mice, ordered from Harlan and weighing 17-19 g, were acclimated to housing conditions and handled in accordance with AUP number TP-18-13. The animals were acclimated for 4 days prior to bacterial challenge. Only animals deemed healthy and fully immunocompetent were included in this study. Cages were prepared with 2 mice per cage.
  • mice were fed Teklad Global Rodent Diet (Harlan) and water ad libitum. Mice were housed in static cages with Teklad 1/8” corn cob bedding inside bioBubble® Clean Rooms that provide H.E.P.A filtered air into the bubble environment at 100 complete air changes per hour. All treatments and infectious challenges were carried out in the bioBubble® environment. The environment was controlled to a temperature range of 74° ⁇ 4°F and a humidity range of 30-70%. Treatment groups were identified by cage card. All procedures carried out in this experiment were conducted in compliance with all the laws, regulations and guidelines of the National Institutes of Health and with the approval of the TransPharm Animal Care and Use Committee.
  • each mouse was anesthetized in an Isoflurane induction chamber and the lesion site was cleared of hair. An area of approximately 2.0 cm x 2.0 cm of skin on the dorsal area of each mouse was cleared through use of the depilatory agent Nair®.
  • TS agar plates supplemented with 5% sheep blood cells.
  • the culture was aseptically swabbed and transferred to tubes of TS broth and allowed to grow for 72 hours.
  • the cultures were diluted to provide challenge inoculum of approximately 6.0-7.0 log10 CFU per 50 pL in PBS.
  • the final CFU count from the challenge suspension determined that 6.0 Iog10 CFU per mouse were delivered.
  • test treatment PCA
  • PCA test treatment
  • test article preparations were administered topically at 2, 24, 48, and 72 hours following the bacterial challenge. While untreated mice were harvested at 2 hours post infection, CFU burden was not detected. However at 96 hours post infection, the CFU burden rose from 6.0 Iog10 to 6.65 Iog10, indicating a successful inoculation.
  • mice were humanely euthanized and skin was aseptically removed from the infection site. Skin samples were placed in homogenation vials with 2.0 ml_ PBS, weighed and homogenized using a mini-bead beater. Homogenate was serially diluted and plated anaerobically on TSA agar plates for enumeration of colony forming units per gram of skin tissue.
  • Example 10 Antibiotic testing with PCA or 2,4,6 THBA using propylene glycol
  • PPG was placed on a paper disc and then either PCA or 2,4,6 THBA was applied.
  • the paper disc was then placed on colonies of various bacterial in a Petri dish. At a certain uniform time they were inspected and classified in the following categories:
  • NZ no accompanying zone of inhibition
  • CZ clear zone of inhibition surrounding the sample and zone measured in millimeters (mm).
  • Example 11 PC A to sterilize/disinfect human skin study
  • Phase 1 The active test reagent was topically applied 1.54% PCA in sterile water to the anterior shoulder region. This 1.54% solution of PCA in water was used effectively in our prior animal wound studies.
  • the controls were Chloraprep (2% Chlorhexidine in 70% isopropyl alcohol) and Betadine (9.0% to 12.0% available iodine in water). Cultures were taken before application and 20 minutes after application. The initial harvest was by a surface swab. Application was by soaked sponge, without force or scrubbing. The second harvest was performed with the back edge of a sterile knife blade scraping with pressure in attempt to maximize the harvest from the deeper sebaceous glands and hair follicles. The specimens were placed in culture media.
  • Phase 2 included eleven medical students and was same method as Phase 1. However the PCA vehicle was changed to 70% isopropyl alcohol. This allowed a higher concentration of PCA than possible in sterile water, 10%.
  • Phase 2a The 70% isopropyl alcohol vehicle was tested for its bactericidal properties. All cultures that were negative or markedly reduced with PCA topical solution were examined for exact nature of the index bacteria and the post treatment cultures that showed no or minimal growth. In this way it was learned what specific bacterial strains PCA could eliminate or reduce.
  • Phase 1 showed the aqueous solution of 1.54% PCA to be partially effective as compared to the controls.
  • phase 1 of the Loma Linda Studies
  • G02231 Phase II This study involved 11 human subjects. There were 5 males and 6 females. The ages were 23-33 years. There were two reagents. The control was 70% isopropyl alcohol.
  • the PCA source was a phytochemical extract from Nanjing Zelang Medical Technology Co. LTD. This source was chosen due to markedly reduced cost of goods compared to that which is biochemically manufactured.
  • the experimental dose was 9 +/- % PCA in 70% isopropyl alcohol. 10 grams of PCA was placed in 100 ml of isopropyl alcohol. The isopropyl alcohol allowed for a greater dose of PCA than water (allowed more PCA to be dissolved). The initial harvest was by a surface swab.
  • Phase 2 results with 9+% solution of PCA were compared to similar test Phase 1 and this showed this to comparable to Betadine in effectiveness, but not with Chloraprep, which killed all the bacterial colonies.
  • Phase 2a showed that 70% isopropyl alcohol (IPA) alone had few antibacterial properties. Of course sterile water used in Phase 1 had no antibacterial properties, therefore the testing was on the effectiveness of the PCA.
  • IPA isopropyl alcohol
  • the first and 2nd method of harvesting differed.
  • the 2nd was accomplished with pressure wiping with the back end of a sterile scalpel so as to maximize the harvest even from sub surface sweat gland and hair follicles.
  • Phase 3 Another test was conducted where the PCA was dissolved in 15 ml propylene glycol.
  • the PPH is a skin penetration enhancer and assists in dissolving PCA.
  • composition of matter was created when 20 grams of PCA was placed in 85 ml 70% isopropyl alcohol making a concentration of PCA
  • the index subject #18 aerobic growth was too numerous to count. There were 2 different species; Staphylococcus epidermidis / hominis and Micrococcus luteus.
  • the Subject #18’s post PCA treatment aerobic culture, #20 showed 2 colonies; S. capitis and S. epidermidis. For reasons unknown, neither of these species was identified in the index culture, and both are considered non-pathogens.
  • the pre-PCA treatment index culture on subject #37 had colonies too numerous to count (TNTC) with heavy growth. The colony species were reported as one; Staphylococcus capitus.
  • the Subject #37’s post PCA treatment aerobic culture #39, showed one (1 ) colony growth, but not the former S. capitus, but was identified Staphylococcus epidermidis as the residual. As in prior phases, the residual growth was most often a non-pathogen on aerobic culture.
  • the one post PCA culture that showed a bacterial colony culture was subject #37 with post PCA treatment anaerobic culture being #39. There were 2 two colonies of same species; Staphylococcus epidermidis. There were no Propionibacterium acnes colonies.
  • Phase 3 showed the effectiveness of 17%+/- PCA in a composition of matter that had skin penetration properties; propylene glycol and essence of peppermint oil.
  • G0241 ⁇ Method The variations in materials and methods in this phase were based upon results of the previous phases. They included changes in the solution, the method of dissolving the PCA, the timing of skin application, plus using two applications to simulate the present day recommendations of surgical skin preparation.
  • the index pre-treatment culture average colony counts of the 4 groups were similar.
  • #8 pre PCA treatment anaerobic culture had 8 colony count but zero at 20 and 60 minutes. The interest was to learn what bacterial were eliminated. There were two species; S. capitis and S. epidermidis. #12 post PCA at 60 minutes showed 1 spreader colony on the plate (SPR) 50 colonies. This contrasted with the pre-treatment of 28 colonies. The species found in #12 aerobic were Klebsiella pneumonia/oxytoca and Micrococcus leuteus. These are not common pathogens. #14 pre PCA anaerobic showed 115 colonies. The species were Propionibacterium acnes, S. lugdunensis, and Kocuria varians. There was no growth at 20 minutes showing that potential pathogen P. acnes was eliminated.
  • S. epidermidis. #21 was the post PCA at 20 minutes and showed 1 anaerobic colony of P. acnes, a reduction from 39 colonies.
  • #32 was pre Chloraprep with aerobic colony count of 59 with the following species: Micrococcus luteus/lylae, Micrococcus luteus, and Kocuria kristinae. There was no colony growth at 20 or 60 minutes. #32 was pre Chloraprep anaerobic with 17 colonies with the following species: S. epidermidis and Gemella
  • Species Analysis The pre-treatment species were predominately saprotrophic or commensal organisms. Propionibacterium acnes was identified in two subjects. After PCA treatment the specific species analysis showed predominately saprotrophic or commensal organisms. The potential pathogen Propionibacterium acnes was identified with 19 colonies pre-PCA treatment in #14 but showed no growth at 20 minutes after PCA treatment. P. acnes was identified pre-PCA treatment in #19 with 19 colonies and post treatment at 20 minutes there was one remaining colony of P. acnes.
  • the questions proposed in the purpose of this proof of principle pilot study were answered in the affirmative.
  • the optimal dose of PCA to act as a human skin disinfectant is greater than 10%.
  • There is a facilitating delivery vehicle which is at least 70% isopropyl alcohol, which allows higher concentrations of PCA to go into solution than water, propylene glycol, and/or essential oils alone.
  • the results were optimized by the addition of known skin penetration enhancers; propylene glycol and essence of peppermint oil.
  • P. acnes normally resides deep in the skin’s hair follicles and or sebaceous glands. Therefore, non-penetration common commercial disinfectants are not effective.
  • PCA in one or more of these vehicles provided a broad-spectrum disinfectant effect comparable to existing commercial products; isopropyl alcohol, Chloraprep® and Betadine®.
  • the other method is to establish clear superiority to an existing product. Both were considered in this study.
  • the various PCA solutions showed a non-inferiority status to 70% Isopropyl alcohol, Betadine® and Chloraprep®.
  • the PCA solutions in this study showed superiority to all but Chloraprep®.
  • the materials and methods were modified in attempt to improve the results of the test reagent PCA.
  • the vehicle was changed from water to isopropyl alcohol to facilitate an increase dose of PCA.
  • the vehicle was change to include reagents known to have skin penetration properties as well as antibiotic properties; i.e. propylene glycol and essence of peppermint oil.
  • Phase III the best PCA results in both aerobic and anaerobic cultures were obtained with a mixture of 17% PCA, propylene glycol and essence of peppermint oil.
  • the anaerobic cultured bacteria are those likely to be below the skin surface and perhaps were affected by the addition of the skin penetrator enhancing reagents.
  • PCA effectiveness was demonstrated in Phase I, even with the low dose of a 1.24 % aqueous solution. This aqueous solution of PCA decreased the colonies of Propionibacterium acnes that were too numerous to count down to about to 1 and 5 colonies on same subject after treatment. Solutions of PCA at 10% or higher removed all pathogens, including P. acnes, but not all bacteria.
  • Chloraprep® results in the literature since there were many variables in methods and timing of harvesting. There is not an apparent explanation for the differing results at 20 minutes in Phase I and IV in our study. Yet in Phase IV Chloraprep® was better than 20% PCA in 91 % isopropyl alcohol and 5 ml of essence of peppermint oil except at 20-minute aerobic cultures, which were sterile with the PCA solution. Note that isopropyl alcohol was not in Phase IV solution.
  • anthocyanins and anthocyanidins or combinations of anthocyanins, anthocyanidins or their metabolites that are bactericidal or antimicrobial was determined by conducting in vitro testing described above.
  • Anthocyanidins that were tested at 100 mM (44.938 grams per liter) with less than one milliliter per dose included delphinidin, pelargonidin, and cyanidin Cl and cyanidin-3-glucoside.
  • Protocatechuic acid and 2, 4, 6 trihydroxybenzaldehyde, the anthocyanidin metabolites, were also tested at the same concentrations. Referring to FIGS. 6-8, delphinidin limited growth against C.
  • P. aeruginosa S. aureus 6538 and 33591 (MRSA); it also was effective against A. pullulans, ATCC 15233, a fungi.
  • CFU colony forming units
  • bacteria have a range of pH at which growth is optimized, and most bacteria are more viable at basic pH ranges.
  • anthocyanins, anthocyanidins and their metabolites also have an acidic pH and have the potential to have bactericidal or bacteriostatic modes of action.
  • C3G and PCA reagents have an acidic pH, their bactericidal or bacteriostatic mode of action is by direct contact with the bacteria.
  • Examples of common local growth hormones related to skin wound healing include Epidermal growth factor (EGF), Insulin-like growth factor-1 (IGF-1 ) and Transforming Growth Factor -Beta (TGF-b).
  • EGF Epidermal growth factor
  • IGF-1 Insulin-like growth factor-1
  • TGF-b Transforming Growth Factor -Beta
  • Epidermal growth factor or EGF is a growth factor that stimulates cell growth, proliferation, and differentiation by binding to its receptor EGFR.
  • IGF-1 is important in skin repair by stimulating keratinocyte proliferation and migration as well as collagen production by fibroblasts. Its expression is important during wound healing such that retarded healing has been correlated with reduced IGF-1 levels. While local administration of IGF-1 to wound sites enhanced wound closure and stimulated granulation tissue formation, increased IGF-1 receptor expression was reported in chronic wounds and in hypertrophic scars.
  • IGF-1 stimulation was associated with increased invasive capacity of keloid fibroblasts.
  • Systemic delivery of IGF-1 also caused hyperglycemia, electrolyte imbalance, and edema. Therefore, it is desirable to have slightly elevated but not over elevated IGF-1 by a treatment modality.
  • TGF-b also is important in skin would healing; however, it is considered a pro- fibrotic growth factor and increased levels of TGF-b or prolonged presence has been identified as causing hypertrophic scaring.
  • FIG. 11 tests were performed on rodent skin to explore the effects of PCA on the local growth hormones in rodent skin.
  • a concentration of 25 mM PCA increased local growth hormone levels at the site of the untreated skin wound.
  • figure 11 demonstrates that a single reagent or compound would optimize local growth hormones to promote healing without scarring.
  • Approximately 25 mM PCA was the optimal reagent and dose.
  • optimization is possible using the compositions of the present invention.
  • all three local growth hormones were lowered in the simulated clinical pathological environment (stripped and infected); however, the lowering of these hormones was not to the extent of absences.

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Abstract

La présente invention concerne l'acide protocatéchuique, le 2,4,6-trihydroxybenzaldéhyde ou un mélange ou une combinaison associés, destinés à être utilisés dans une méthode de traitement d'infections microbiennes sur les surfaces d'un implant ou d'autres surfaces à proximité d'un implant chez un patient. En particulier, les méthodes sont utiles pour traiter des infections par biofilm. Les méthodes comprennent l'utilisation de techniques minimalement invasives, notamment l'utilisation d'ultrasons en vue de faciliter la détection d'infections par biofilm sur l'implant ou une autre surface chez un patient et d'éliminer l'infection par administration d'une anthocyanine ou d'une anthocyanidine ou d'un métabolite associé. En particulier, l'invention concerne l'acide protocatéchuique (RCA) ou le 2,4,6-trihydroxybenzaldéhyde (2,4,6 THBA) destiné à être utilisé dans le traitement d'un implant infecté ou d'une autre surface chez un patient.
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