WO2013096823A1 - Cationic peptides and use of such peptides for inhibiting exotoxin production - Google Patents
Cationic peptides and use of such peptides for inhibiting exotoxin production Download PDFInfo
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- WO2013096823A1 WO2013096823A1 PCT/US2012/071345 US2012071345W WO2013096823A1 WO 2013096823 A1 WO2013096823 A1 WO 2013096823A1 US 2012071345 W US2012071345 W US 2012071345W WO 2013096823 A1 WO2013096823 A1 WO 2013096823A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4723—Cationic antimicrobial peptides, e.g. defensins
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
- A61L15/32—Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/42—Use of materials characterised by their function or physical properties
- A61L15/46—Deodorants or malodour counteractants, e.g. to inhibit the formation of ammonia or bacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L17/00—Materials for surgical sutures or for ligaturing blood vessels ; Materials for prostheses or catheters
- A61L17/005—Materials for surgical sutures or for ligaturing blood vessels ; Materials for prostheses or catheters containing a biologically active substance, e.g. a medicament or a biocide
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L17/00—Materials for surgical sutures or for ligaturing blood vessels ; Materials for prostheses or catheters
- A61L17/14—Post-treatment to improve physical properties
- A61L17/145—Coating
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L28/00—Materials for colostomy devices
- A61L28/0007—Materials for colostomy devices containing macromolecular materials
- A61L28/0023—Polypeptides; Proteins; Degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L28/00—Materials for colostomy devices
- A61L28/0034—Use of materials characterised by their function or physical properties
- A61L28/0038—Medicaments; Biocides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L28/00—Materials for colostomy devices
- A61L28/0061—Materials for coating
- A61L28/0069—Macromolecular materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
- A61L29/04—Macromolecular materials
- A61L29/044—Proteins; Polypeptides; Degradation products thereof
- A61L29/048—Other specific proteins or polypeptides not covered by A61L29/045 - A61L29/047
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F13/00—Bandages or dressings; Absorbent pads
- A61F13/00051—Accessories for dressings
- A61F13/00063—Accessories for dressings comprising medicaments or additives, e.g. odor control, PH control, debriding, antimicrobic
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F13/00—Bandages or dressings; Absorbent pads
- A61F13/15—Absorbent pads, e.g. sanitary towels, swabs or tampons for external or internal application to the body; Supporting or fastening means therefor; Tampon applicators
- A61F13/84—Accessories, not otherwise provided for, for absorbent pads
- A61F13/8405—Additives, e.g. for odour, disinfectant or pH control
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- This invention relates to inhibition of bacterial exotoxin production, and more particularly to cationic peptides and use of such peptides to inhibit bacterial exotoxin production without substantially inhibiting bacterial growth.
- Staphylococcus aureus is a gram-positive bacterium that is a significant cause of disease throughout the world. The organism is ubiquitous, with estimates of almost 40% of humans being colonized on mucosal surfaces. See, Lowy, N Engl J Med 339:520-32 (1988); and McCormick, et al, Annu Rev Microbiol 55:77-104 (2001).
- the illnesses caused by the organism range from relatively benign infections such as furuncles and soft tissue abscesses, to life-threatening illnesses such as toxic shock syndrome (TSS), sepsis, and infective endocarditis.
- TSS toxic shock syndrome
- S. aureus causes diseases primarily through production of a large number of virulence factors, both cell surface- expressed and secreted.
- TSS toxin-1 TSS toxin-1
- TSST-1 superantigen TSS toxin-1
- TSST-1 is the cause of menstrual TSS, a condition typically associated with healthy women who are using certain tampons and who have vaginal colonization with S. aureus.
- Schlievert Lancet 1 : 1 149-50 (1986); Schlievert, et al, J Clin Microbiol, 42:2875-6 (2004).
- TSST-1 is the cause of up to 50% of non- menstrual TSS, with most such cases being associated with upper respiratory tract infections; most of the rest of non-menstrual TSS is associated with the superantigens staphylococcal enterotoxins B and C.
- Superantigens cause serious human illnesses by causing massive cytokine production, resulting in an acute-onset illness characterized by fever and vomiting and diarrhea (flu-like symptoms), hypotension, a sunburn-like rash, peeling of the skin upon recovery, and a variable multi-organ component.
- the cationic peptides described herein can be used to inhibit exotoxin production, and can be included in compositions containing a medical or hygienic device.
- a medical or hygienic device For example, all or part of the surface of the medical or hygienic device can be coated with or impregnated with one or more peptides described herein.
- the peptides are non-toxic, non-inflammatory and non-irritating, and suitable for treating vertebrate subjects or for coating or impregnating on medical or hygienic devices that are to be used with vertebrate subjects.
- this document features peptides that are from 10 to 50 amino acids in length (e.g., 18 to 30 amino acids in length), wherein the percentage of positively charged amino acids above the number of negatively charged amino acids is 15% or higher (e.g., 18% or higher, 25% or higher, 50% or higher, 70% or higher, or 90% or higher).
- the bacterial exotoxin can be from a bacterial genus selected from the group consisting of Staphylococci, Neisseriae,
- Streptococci Chlamydia trachomatis, Treponemae, Haemophilus, Bordetellae,
- Gardnerella vaginalis Bacillus, Clostridium, Escherichia coli, Vibrio cholerae,
- Salmonella Shigellaa, Mycobacteria, Francisella, Yersinia, Burkholderia,
- the positively charged amino acids can include one or more of lysine, a lysine analog, arginine, an arginine analog, histidine, or a histidine analog.
- the peptide can have the amino acid sequence
- this document features a composition comprising one or more peptides and a pharmaceutically acceptable carrier, wherein each peptide is a peptide described above, as well as a method of inhibiting exotoxin production that includes contacting a vertebrate subject with such a composition.
- the composition can be formulated for subcutaneous administration, intramuscular administration, oral administration, topical administration, or vaginal administration.
- composition that includes a medical or hygienic device (e.g., a vaginal tampon, surgical suture, a surgical bandage, a surgical dressing, an osmotic pump, or ostomy device); and any one or more of the peptides described above. All or part of the device can be impregnated, or all or part of the surface of the device can be coated, with the one or more peptides.
- a medical or hygienic device e.g., a vaginal tampon, surgical suture, a surgical bandage, a surgical dressing, an osmotic pump, or ostomy device
- a medical or hygienic device e.g., a vaginal tampon, surgical suture, a surgical bandage, a surgical dressing, an osmotic pump, or ostomy device
- All or part of the device can be impregnated, or all or part of the surface of the device can be coated, with
- This document also features a method of inhibiting exotoxin production.
- the method includes delivering one or more peptides described above to a tissue or organ of a vertebrate subject, wherein the tissue or organ is, or is in risk of being, infected with a bacterium.
- cationic peptides compositions containing one or more cationic peptides, articles of manufacture including such compositions, and methods of using the cationic peptides for inhibiting the production of bacterial exotoxins.
- the cationic peptides have TSST-1 inhibition activity, independent of antimicrobial activity, and are non-inflammatory and non- irritating.
- the cationic peptides described herein are believed to inhibit signal transduction through two component systems, similar to the a-globin chain of hemoglobin, with the consequence being reduction of TSST-1 production without inhibition of bacterial growth. Inhibition of exotoxin production by S.
- aureus may result in failure of the organism to colonize human surfaces and thus facilitate prevention of S. aureus illnesses.
- the cationic peptides described herein represent a class of molecules that can interfere with S. aureus function, despite lacking antimicrobial effects.
- superantigens such as TSST-1
- TSST-1 are critical to the ability of S. aureus to cause serious illnesses such as necrotizing pneumonia (see, e.g., Schlievert, J Infect Dis, 200:676-8 (2009); and Strandberg, et ah, J Infect Dis, 202: 1690-7 (2010)).
- agents that inhibit superantigen production would be expected to prevent the ability of S. aureus to establish infections.
- the methods described herein can be used to inhibit production of exotoxins from numerous bacterial genera, including bacteria associated with the use of medical and/or hygienic devices.
- the peptides described herein can inhibit production of exotoxin produced from bacteria of the following genera:
- Staphylococci e.g., S. aureus, S. intermedius, S. epidermidis, and other coagulase negative Staphylococci
- Neisseriae e.g., N. gonorrheae and N. meningitidis
- Streptococci e.g., Group A Streptococcus (e.g., S. pyogenes), Group B Streptococcus (e.g., S. agalactiae), Group C Streptococcus, Group G Streptococcus, S. pneumoniae, and viridans Streptococci
- Chlamydia e.g., C.
- Treponemae e.g., T. pallidum, T. permur, and T. cerateum
- Haemophilus bacteria e.g., H. ducreyi, H. influenzae, and H. aegyptius
- Bordetellae e.g., B. pertussis, B. parapertussis, and B. bronchiseptica
- Gardnerella e.g., G. vaginalis
- Bacillus e.g., B. anthracis and B. cereus
- Clostridium e.g., C. perfringens, C. septicum, C. novyi, and C.
- Escherichia e.g., E. coli
- Vibrio e.g., V. cholera
- Salmonella e.g., S. enteriditis, S. typhimurium, and S. typhi
- Shigella bacteria Mycobacteria
- Francisella bacteria e.g., Yersinia bacteria (e.g., Y. pestis)
- Burkholderia bacteria Pseudomonas bacteria, and Brucella bacteria.
- peptides described herein can inhibit production of bacterial exotoxins such as TSST-1, Staphylococcal alpha, beta, gamma, and delta hemolysins, Streptococcal pyrogenic exotoxins (SPEs), streptolysin O, streptolysin S,
- bacterial exotoxins such as TSST-1, Staphylococcal alpha, beta, gamma, and delta hemolysins
- SPEs Streptococcal pyrogenic exotoxins
- streptolysin O streptolysin S
- Staphylococcal enterotoxins SEs; such as SEA, SEB, SEC, or SEE
- SEs Staphylococcal enterotoxins
- A-B toxins diphtheria exotoxin
- cholera exotoxin cholera exotoxin
- pertussis exotoxin shiga toxin, shiga-like toxin, anthrax (B.
- anthracis toxin
- botulinal exotoxin botulinal exotoxin
- tetanus exotoxin tracheal cytotoxin
- Helicobacter toxins alpha toxin (lecithinase), kappa toxin (collagenase), mu toxin (hyaluronidase), leukocidin, elastase and other proteases, a Panton Valentine leukocidin component (S or F component), a porin, Listeria monocytogenes cytolysin, aerolysin, Bacillus anthracis protective antigen, and nucleases.
- Cationic peptides described herein are 10 to 50 amino acids in length, e.g., 12 to 48, 15 to 45, 15 to 40, 15 to 35, 18 to 30, or 20 to 30 amino acids in length, wherein the percentage (calculated with respect to the total number of amino acids) of positively charged amino acids above the number of negatively charged amino acids is 15% or higher (e.g., 18% or higher, 25% or higher, 50% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher).
- a peptide is 40 amino acids in length and contains 8 negatively charged amino acids, there would have to be 14 or more positively charged amino acids in the peptide to be a "cationic peptide.”
- a peptide is 40 amino acids in length and contains only 2 negatively charged amino acids in a peptide, there would have to be only 8 or more positively charged amino acids in order for it to be a "cationic peptide.”
- the percentage of positively charged amino acids is assessed at a pH ranging from 4.0 to 8.0 (e.g., at physiological pH).
- Positively charged amino acids include lysine or an analog thereof, arginine or an analog thereof, or histidine or an analog thereof.
- Analogs of lysine, arginine and histidine include, for example, homolysine, ornithine, diaminobutyric acid, diaminopimelic acid, diaminopropionic acid and homoarginine as well as trimethylysine and trimethylornithine, 4-aminopiperidine-4-carboxylic acid, 4-amino- 1 - carbamimidoylpiperidine-4-carboxylic acid, and 4-guanidinophenylalanine.
- Cationic peptides described herein can include ⁇ -, ⁇ -, and/or ⁇ -amino acids, natural or unnatural amino acids, and amino acids of D- and/or L-stereochemistry, and can be synthesized on a solid phase and purified to >95% purity (e.g., by high performance liquid chromatography (HPLC)).
- Stereochemistry of amino acids may be designated by preceding the name or abbreviation with the designation "D” or “d” or “L” or “1” as appropriate.
- a cationic peptide contains all D- enantiomers. In other embodiments, the cationic peptide contains both D and L enantiomers.
- Amino acids with many different protecting groups e.g., Boc
- appropriate for use in the solid phase synthesis of polypeptides are commercially available.
- natural amino acid refers to one of the twenty most common occurring amino acids. Natural amino acids are referred to herein by their standard one letter abbreviation.
- non-natural amino acid or “non-natural” refers to any derivative of a natural amino acid including D forms, and ⁇ and ⁇ amino acid derivatives.
- Non-limiting examples of non-natural amino acids or amino acid derivatives that can be incorporated into a cationic peptide include the following (common abbreviations in parentheses): ⁇ -Alanine ( ⁇ -Ala), ⁇ - Aminobutyric Acid (GABA), 2-Aminobutyric Acid (2 -Abu), a,P-Dehydro-2- aminobutyric Acid ( ⁇ -Abu), ⁇ -aminobutyric acid ( ⁇ -Abu), 1 -Aminocyclopropane- 1 - carboxylic Acid (ACPC), Aminoisobutyric Acid (Aib), 2-Amino-thiazoline-4- carboxylic Acid, 5-Aminovaleric Acid (5-Ava), 6-Aminohexanoic Acid (6-Ahx), 8- Aminooctanoic Acid (8-Aoc), 11 -Aminoundecanoic Acid (1 1-Aun), 12- Aminododecanoic Acid (12-Ado),
- Aminooxyacetic Acid (Aoa), 2-Aminotetraline-2-carboxylic Acid (Ate), 4-Amino-5- cyclohexyl-3-hydroxypentanoic Acid (ACHPA), para-Aminophenylalanine (4-NH2- Phe), Biphenylalanine (Bip), ara-Bromophenylalanine (4-Br-Phe), ortho- Chlorophenylalanine (2-Cl-Phe), meto-Chlorophenylalanine (3-Cl-Phe), ara- Chlorophenylalanine (4-Cl-Phe), meto-Chlorotyrosine (3-Cl-Tyr), para- Benzoylphenylalanine (Bpa), tert-Butylglycine (Tie), Cyclohexylalanine (Cha), Cyclohexylglycine (Chg), Diaminopimelic acid (DAP), 2,3-Diaminopropionic
- compositions that include one or more cationic peptides described herein.
- a composition can include two, three, four or more peptides.
- the compositions can be formulated for any route of administration by mixing the one or more peptides them with any one or more of a variety of pharmaceutically acceptable carriers, emulsifiers, excipients, and/or stabilizers known in the art [Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. Ed. 1980].
- Acceptable carriers, emulsifiers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include: buffers, such as phosphate, citrate, and other non-toxic organic acids; antioxidants such as ascorbic acid; low molecular weight (less than 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such
- polyvinylpyrrolidone amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugar alcohols such as mannitol, or sorbitol; salt- forming counterions such as sodium; and/or nonionic surfactants such as Tween, Pluronics, or PEG.
- amino acids such as glycine, glutamine, asparagine, arginine, or lysine
- monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans chelating agents such as EDTA
- sugar alcohols such as mannitol, or sorbitol
- salt- forming counterions such as sodium
- nonionic surfactants such as Tween, Pluronics, or PEG.
- an aqueous gel e.g., containing 96.3% H 2 0, 2.7% Natrosol 250 HX Pharm Hydroxyethylcellulose (Hercules Inc.), 0.85% NaCl, 0.1% sorbic acid, and 0-0.02% caramel color by weight; pH is adjusted to 4.4 with sodium hydroxide
- the peptides can be a component of a cream or solution to be applied topically to an infected area or to an area at risk of being infected, optionally in combination with any known non-toxic delivery agent and/or penetrant.
- compositions described herein can be administered orally or by intravenous infusion, or injected subcutaneous ly, intramuscularly, intrathecally, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratracheally, or intrapulmonarily.
- the dosage required depends on the choice of the route of administration; the nature of the formulation; the nature of the patient's condition; the subject's size, weight, surface area, age, and sex; other drugs being administered; and the judgment of the attending physician. Suitable dosages are in the range of 0.001-100.0 mg kg. Wide variations in the needed dosage are to be expected in view of the differing efficiencies of various routes of administration.
- a suitable delivery vehicle e.g., polymeric microparticles or implantable devices
- Encapsulation of the peptide in a suitable delivery vehicle may increase the efficiency of delivery, particularly for oral delivery.
- compositions described herein include one or more cationic peptides and a medical or hygienic device.
- a "medical or hygienic device” is a device that is inserted into a bodily canal of a vertebrate subject, inserted into a bodily cavity of a vertebrate subject, or applied to a tissue or organ of a vertebrate animal for the purpose of: (a) wound protection; (b) preventing or reducing unwanted, or overcoming restricted, release from the body of the vertebrate subject of a bodily fluid, bodily secretion, or excreta (e.g., blood, menses, urine, lymphatic fluid, cerebrospinal fluid, semen, saliva, vaginal secretions, mucus, or feces); (c) delivering a drug or some other therapeutic or prophylactic agent to a subject; (d) replacing absent or supplementing defective organ functions; or (e) maintaining the patency of a bodily canal (e.g., a blood
- Devices of interest include, without limitation: rectal devices such as suppositories, enemas, and catheters; nasal, tracheal, or esophageal delivery devices; vaginal devices such as vaginal tampons and contraceptive devices (e.g., diaphragms or intrauterine devices (IUDs)); venous, arterial, intracranial and other needles, catheters and stents; renal dialysis accesses; surgical bandages, sutures, or dressings; ostomy devices; incontinence devices;
- rectal devices such as suppositories, enemas, and catheters
- nasal, tracheal, or esophageal delivery devices vaginal devices such as vaginal tampons and contraceptive devices (e.g., diaphragms or intrauterine devices (IUDs)); venous, arterial, intracranial and other needles, catheters and stents; renal dialysis accesses;
- osmotic pumps e.g., mini osmotic pumps
- body cavity e.g., the peritoneal cavity
- the peptides and the device can be provided separately. Thus, they can be provided in the form of a kit or article of manufacture, optionally also containing packaging materials.
- the device and the one or more peptides can optionally be in separate containers.
- the peptide(s) in such compositions can be formulated as described above. Generally, however, the peptide(s) and device will be provided in a combined form. Thus, all or part of the device can be impregnated, or all or part of the surface of the device can be coated, with the one or more peptides.
- applying a device to a vertebrate subject means inserting all or part of the device into a bodily canal (e.g., a vagina) or bodily cavity (e.g., a peritoneum or pleural cavity) of the subject or placing all or part of the device in a touching relationship with the surface or within the body of a tissue or organ of the subject.
- a bodily canal e.g., a vagina
- bodily cavity e.g., a peritoneum or pleural cavity
- the document also provides methods of using a composition that includes one or more peptides and a device.
- the devices and peptide(s) can be used separately or combined prior to use. If used separately, the peptide can be administered by any of the methods described above and the device applied to the vertebrate subject as described above. Where the device and peptides are provided in a combined form, the composition is applied in same manner as the device alone.
- the subject can be a vertebrate, for example: a mammal such as a human, non-human primate (e.g., monkey), mouse, rat, hamster, gerbil, guinea pig, cow, sheep, goat, horse, pig, rabbit, dog, or cat; or a bird such as a chicken, turkey, canary, eagle, or hawk.
- a composition described herein that contains one or more peptides can be delivered to a tissue or organ of a vertebrate subject such as a human, that is, or is at risk of being, infected with a bacterium to inhibit exotoxin production in the subject.
- exotoxin production can be inhibited by inserting, applying, or implanting a medical or hygienic device in the subject that includes one or more peptides described herein (e.g., a medical or hygienic device that is impregnated, or where all or part of the surface of the device is coated, with the one or more peptides).
- a medical or hygienic device in the subject that includes one or more peptides described herein (e.g., a medical or hygienic device that is impregnated, or where all or part of the surface of the device is coated, with the one or more peptides).
- compositions described herein can be combined with packaging material and sold as articles of manufacture or kits. Components and methods for producing articles of manufactures are well known.
- the articles of manufacture may combine one or more therapeutic compositions described herein.
- the articles of manufacture may further include sterile water, pharmaceutical carriers, buffers, antibodies, indicator molecules, and/or other useful reagents for detecting microbial diseases.
- Instructions describing how a composition or vaccine is effective for preventing the incidence of an infection, preventing the occurrence of the clinical signs of an infection, ameliorating the clinical signs of an infection, lowering the risk of the clinical signs of an infection, lowering the occurrence of the clinical signs of an infection and/or reducing the spread of infections may be included in such kits.
- a composition described herein may be provided in a pre-packaged form in quantities sufficient for a single administration.
- S. aureus MN8 a methicillin susceptible (MSSA) clinical isolate from a menstrual TSS patient and representative of 75% of menstrual TSS isolates (See Schlievert, et al, J Infect Dis 143:509-16 (1981)), was used for all of the experiments described herein.
- MN8 was cultured overnight in Todd Hewitt (Difco laboratories, Detroit, MI). The next day, the organism was diluted in fresh Todd Hewitt broth to approximately 10 4 colony-forming units (CFU)/mL for inoculation. The organism was cultured for 9 hours in the presence of potential exotoxin- inhibitory peptides with shaking (200 revolutions/mm),
- Exotoxin-inhibiting peptides Peptides for use in all studies were synthesized and purified to homogeneity by the University of Minnesota Microchemicai Facility, except for defensin peptides (human neutrophil peptide- ! (H P-1 ) and human neutrophil peptide-2 (HNP-2)), which were purchased from Sigma-Aldrich (St. Louis, MO). All peptides were diluted into culture media to concentrations ranging from 5.0 to ⁇ 5 x lO "6 g/mL. The sequences of the peptides are shown in Table 1, with the percentage of positive residues shown in parentheses.
- MN8 was cultured for 9 h with shaking (200 revolutions/min) in the presence of peptides in volumes of 2 mL culture per tube. After incubation, a sample of each culture was used for plate-count determination of colony-forming units/ml and a sample was used for TSST-1 quantification as described by Schlievert, et al,
- Each sample was resuspended in distilled water (50 ⁇ ) and clarified by centrifugation (14,000 x g, 5 min) and then added to 50 ⁇ sodium-dodecyl sulfate polyacryiamide gel electrophoresis (SDS-PAGE) sample buffer (final volume l/10 m original culture volume). Twenty microliters of each sample were electrophoresed on SDS-PAGE gels and transblotted onto PVDF membranes; control samples of purified TSST-1 w r ere treated comparably, ranging from 10 .g/lane to 0.1 ug/lane.
- SDS-PAGE sodium-dodecyl sulfate polyacryiamide gel electrophoresis
- HNPs were weakly anti-staphyiococcai. At 5 ⁇ g/mL, both HNP-1 and HNP-2 inhibited MN8 growth by approximately 1 log; at all lower HNP doses, no antimicrobial activity was observed. In contrast, HNP-1 and HNP-2 both were highly inhibitory of production of TSST-1, with complete inhibition at doses as low as 10 "3 ⁇ ' ⁇ (Table 2).
- HNP-1 and HNP-2 effect on growth and TSST-1 production of S. aureus MN8.
- EXAMPLE 3 Hemoglobin a-chain peptides
- the ⁇ -globin chain of human hemoglobin inhibits TSST-1 production without affecting S. aureus growth. See, Schlievert, et a!., Biochemistry 46: 14349-58 (2007).
- Hbg-1 (see Table 1) was identified from a scan of the ⁇ -globin chain for cationic peptides. Hbg-1 contains 18% positive charges. Hbg-1 inhibited TSST-1 production completely at 5.0 and 5 x 10 "1 ⁇ g/mL but lost activity at lower peptide concentrations. No antimicrobial activity was observed with Hbg-1. See Table 3. Table 3
- Hbg-1 peptide was scrambled to produce Hbg-2, which was assayed for activity.
- Hbg-2 was not antimicrobial at any concentration tested, but the peptide was comparably active as Hbg-1 for inhibition of TSST-1 production (Table 3).
- Hbg-3 The activity of Hbg- 1 also was evaluated when immobilized on agarose beads (referred to as Hbg-3). It was hypothesized that if charge was the principal determinant of activity, the immobilized peptide would retain its TSST-1 inhibition activity. Hbg-3 was not antimicrobial but retained TSST-1 inhibition activity. See Table 3. This is important since it shows that the peptides can be added to implanted devices and retain TSST-1 inhibition activity.
- Hbg-1 peptides were modified to increase the number of positively charged amino acids and tested for exotoxin inhibition and antimicrobial activity.
- the modified peptides contained no positively charged amino acids (SP-3), 50% positively charged amino acids (SP-2), or 70% positively charged amino acid residues (SP-1).
- SP-3 no positively charged amino acids
- SP-2 50% positively charged amino acids
- SP-1 70% positively charged amino acid residues
- SP-3 peptide (no positively charged amino acids) lacked antimicrobial and TSST-1 inhibition activity (Table 4).
- SP-1 and SP-2 the highly positively charged peptides (SP-1 and SP-2) showed the greatest TSST-1 inhibition activity compared to all other peptides tested, without affecting bacterial growth (Table 4).
- SP-1 (70% + ) inhibited TSST-1 production by > 50% at concentrations of 5 x 10 " 5 ⁇ g/mL or greater. This represents a 3 -log improvement in TSST-1 inhibition compared to the original Hbg-1 peptide shown in Table 3.
- each of the SP-2 (50% + charge) and SP-1 (70% + charge) peptides was assessed in rabbits as follows. Three rabbits/group were challenged vaginally with 200 ⁇ /day of 1.0 ⁇ g/mL of peptide (SP-2 or SP-1) or PBS, all of which were formulated in an aqueous gel (96.3% H 2 0, 2.7% Natrosol 250 HX Pharm Hydroxyethylcellulose (Hercules Inc.), 0.85% NaCl, 0.1% sorbic acid, and 0-0.02% caramel color by weight; pH is adjusted to 4.4 with sodium hydroxide). The animals were examined after 7 days for signs of inflammation and vaginal irritation as induced by the peptides. No irritation or inflammation was observed, indicating the peptides did not show toxicity.
Abstract
Cationic peptides and use of such peptides to inhibit bacterial exotoxin production without substantially inhibiting bacterial growth are described.
Description
CATIONIC PEPTIDES AND USE OF SUCH PEPTIDES FOR INHIBITING EXOTOXIN PRODUCTION
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application No.
61/578,570, filed December 21, 201 1. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.
TECHNICAL FIELD
This invention relates to inhibition of bacterial exotoxin production, and more particularly to cationic peptides and use of such peptides to inhibit bacterial exotoxin production without substantially inhibiting bacterial growth.
BACKGROUND
Staphylococcus aureus is a gram-positive bacterium that is a significant cause of disease throughout the world. The organism is ubiquitous, with estimates of almost 40% of humans being colonized on mucosal surfaces. See, Lowy, N Engl J Med 339:520-32 (1988); and McCormick, et al, Annu Rev Microbiol 55:77-104 (2001). The illnesses caused by the organism range from relatively benign infections such as furuncles and soft tissue abscesses, to life-threatening illnesses such as toxic shock syndrome (TSS), sepsis, and infective endocarditis. S. aureus causes diseases primarily through production of a large number of virulence factors, both cell surface- expressed and secreted. One of the major secreted exotoxins is the superantigen TSS toxin-1 (TSST-1). See, Bergdoll, et al, Lancet 1 : 1017-21 (1981); Bergdoll and Schlievert, Lancet ± ' 691 (1984); and Schlievert, et al. , J Infect Dis 143 :509-16 (1981). TSST-1 is the cause of menstrual TSS, a condition typically associated with healthy women who are using certain tampons and who have vaginal colonization with S. aureus. See Schlievert, Lancet 1 : 1 149-50 (1986); Schlievert, et al, J Clin Microbiol, 42:2875-6 (2004). Additionally, TSST-1 is the cause of up to 50% of non- menstrual TSS, with most such cases being associated with upper respiratory tract infections; most of the rest of non-menstrual TSS is associated with the superantigens staphylococcal enterotoxins B and C. Superantigens cause serious human illnesses by
causing massive cytokine production, resulting in an acute-onset illness characterized by fever and vomiting and diarrhea (flu-like symptoms), hypotension, a sunburn-like rash, peeling of the skin upon recovery, and a variable multi-organ component. See, Davis, et al, N Engl J Med, 303 : 1429-35 (1980); Marrack and Kappler, Science, 248:705-11 (1990); and Shands, et al, NEngl JMed 303 : 1436-42 (1980).
SUMMARY
This document is based on the discovery of cationic peptides that can inhibit bacterial exotoxin production without substantially inhibiting bacterial growth. The cationic peptides described herein can be used to inhibit exotoxin production, and can be included in compositions containing a medical or hygienic device. For example, all or part of the surface of the medical or hygienic device can be coated with or impregnated with one or more peptides described herein. The peptides are non-toxic, non-inflammatory and non-irritating, and suitable for treating vertebrate subjects or for coating or impregnating on medical or hygienic devices that are to be used with vertebrate subjects.
In one aspect, this document features peptides that are from 10 to 50 amino acids in length (e.g., 18 to 30 amino acids in length), wherein the percentage of positively charged amino acids above the number of negatively charged amino acids is 15% or higher (e.g., 18% or higher, 25% or higher, 50% or higher, 70% or higher, or 90% or higher). Such peptides inhibit bacterial exotoxin production without substantially inhibiting bacterial growth. The bacterial exotoxin can be from a bacterial genus selected from the group consisting of Staphylococci, Neisseriae,
Streptococci, Chlamydia trachomatis, Treponemae, Haemophilus, Bordetellae,
Gardnerella vaginalis, Bacillus, Clostridium, Escherichia coli, Vibrio cholerae,
Salmonella, Shigellaa, Mycobacteria, Francisella, Yersinia, Burkholderia,
Pseudomonas, and Brucella. The positively charged amino acids can include one or more of lysine, a lysine analog, arginine, an arginine analog, histidine, or a histidine analog. The peptide can have the amino acid sequence
SFPTTKTYFPHFDLSHGSAQVK (SEQ ID NO: 3),
STKPFFYFLHTQVKASPTSHDG (SEQ ID NO:4),
SKPKKKKYKPHKKKSHKSAKKK (SEQ ID NO: 5), or
SKPKKKTYKPHKDLSHGSAKKK (SEQ ID NO:6).
In another aspect, this document features a composition comprising one or more peptides and a pharmaceutically acceptable carrier, wherein each peptide is a peptide described above, as well as a method of inhibiting exotoxin production that includes contacting a vertebrate subject with such a composition. The composition can be formulated for subcutaneous administration, intramuscular administration, oral administration, topical administration, or vaginal administration.
This document also features a composition that includes a medical or hygienic device (e.g., a vaginal tampon, surgical suture, a surgical bandage, a surgical dressing, an osmotic pump, or ostomy device); and any one or more of the peptides described above. All or part of the device can be impregnated, or all or part of the surface of the device can be coated, with the one or more peptides.
This document also features a method of inhibiting exotoxin production. The method includes delivering one or more peptides described above to a tissue or organ of a vertebrate subject, wherein the tissue or organ is, or is in risk of being, infected with a bacterium.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the exemplary methods and materials are described below. All publications, patent applications, patents, Genbank® Accession Nos, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present application, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
DETAILED DESCRIPTION
This document features cationic peptides, compositions containing one or more cationic peptides, articles of manufacture including such compositions, and methods of using the cationic peptides for inhibiting the production of bacterial exotoxins. As set forth in the Examples, the cationic peptides have TSST-1 inhibition activity, independent of antimicrobial activity, and are non-inflammatory and non-
irritating. Without being bound to a particular mechanism, the cationic peptides described herein are believed to inhibit signal transduction through two component systems, similar to the a-globin chain of hemoglobin, with the consequence being reduction of TSST-1 production without inhibition of bacterial growth. Inhibition of exotoxin production by S. aureus may result in failure of the organism to colonize human surfaces and thus facilitate prevention of S. aureus illnesses. The cationic peptides described herein represent a class of molecules that can interfere with S. aureus function, despite lacking antimicrobial effects. Furthermore, superantigens such as TSST-1, are critical to the ability of S. aureus to cause serious illnesses such as necrotizing pneumonia (see, e.g., Schlievert, J Infect Dis, 200:676-8 (2009); and Strandberg, et ah, J Infect Dis, 202: 1690-7 (2010)). As such, agents that inhibit superantigen production would be expected to prevent the ability of S. aureus to establish infections.
The methods described herein can be used to inhibit production of exotoxins from numerous bacterial genera, including bacteria associated with the use of medical and/or hygienic devices. For example, the peptides described herein can inhibit production of exotoxin produced from bacteria of the following genera:
Staphylococci (e.g., S. aureus, S. intermedius, S. epidermidis, and other coagulase negative Staphylococci), Neisseriae (e.g., N. gonorrheae and N. meningitidis), Streptococci (e.g., Group A Streptococcus (e.g., S. pyogenes), Group B Streptococcus (e.g., S. agalactiae), Group C Streptococcus, Group G Streptococcus, S. pneumoniae, and viridans Streptococci), Chlamydia (e.g., C. trachomatis, Treponemae (e.g., T. pallidum, T. pertenue, and T. cerateum), Haemophilus bacteria (e.g., H. ducreyi, H. influenzae, and H. aegyptius), Bordetellae (e.g., B. pertussis, B. parapertussis, and B. bronchiseptica), Gardnerella (e.g., G. vaginalis), Bacillus (e.g., B. anthracis and B. cereus), Clostridium (e.g., C. perfringens, C. septicum, C. novyi, and C. tetani), Escherichia (e.g., E. coli), Vibrio (e.g., V. cholera), Salmonella (e.g., S. enteriditis, S. typhimurium, and S. typhi), Shigella bacteria, Mycobacteria, Francisella bacteria, Yersinia bacteria (e.g., Y. pestis), Burkholderia bacteria, Pseudomonas bacteria, and Brucella bacteria.
For example, peptides described herein can inhibit production of bacterial exotoxins such as TSST-1, Staphylococcal alpha, beta, gamma, and delta hemolysins,
Streptococcal pyrogenic exotoxins (SPEs), streptolysin O, streptolysin S,
Staphylococcal enterotoxins (SEs; such as SEA, SEB, SEC, or SEE), A-B toxins, diphtheria exotoxin, cholera exotoxin, pertussis exotoxin, shiga toxin, shiga-like toxin, anthrax (B. anthracis) toxin, botulinal exotoxin, tetanus exotoxin, tracheal cytotoxin, Helicobacter toxins, alpha toxin (lecithinase), kappa toxin (collagenase), mu toxin (hyaluronidase), leukocidin, elastase and other proteases, a Panton Valentine leukocidin component (S or F component), a porin, Listeria monocytogenes cytolysin, aerolysin, Bacillus anthracis protective antigen, and nucleases.
Cationic peptides described herein are 10 to 50 amino acids in length, e.g., 12 to 48, 15 to 45, 15 to 40, 15 to 35, 18 to 30, or 20 to 30 amino acids in length, wherein the percentage (calculated with respect to the total number of amino acids) of positively charged amino acids above the number of negatively charged amino acids is 15% or higher (e.g., 18% or higher, 25% or higher, 50% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher). For example, if a peptide is 40 amino acids in length and contains 8 negatively charged amino acids, there would have to be 14 or more positively charged amino acids in the peptide to be a "cationic peptide." As another example, if a peptide is 40 amino acids in length and contains only 2 negatively charged amino acids in a peptide, there would have to be only 8 or more positively charged amino acids in order for it to be a "cationic peptide." Typically, the percentage of positively charged amino acids is assessed at a pH ranging from 4.0 to 8.0 (e.g., at physiological pH). Positively charged amino acids include lysine or an analog thereof, arginine or an analog thereof, or histidine or an analog thereof. Analogs of lysine, arginine and histidine include, for example, homolysine, ornithine, diaminobutyric acid, diaminopimelic acid, diaminopropionic acid and homoarginine as well as trimethylysine and trimethylornithine, 4-aminopiperidine-4-carboxylic acid, 4-amino- 1 - carbamimidoylpiperidine-4-carboxylic acid, and 4-guanidinophenylalanine.
Cationic peptides described herein can include α-, β-, and/or γ-amino acids, natural or unnatural amino acids, and amino acids of D- and/or L-stereochemistry, and can be synthesized on a solid phase and purified to >95% purity (e.g., by high performance liquid chromatography (HPLC)). Stereochemistry of amino acids may be designated by preceding the name or abbreviation with the designation "D" or "d"
or "L" or "1" as appropriate. In some embodiments, a cationic peptide contains all D- enantiomers. In other embodiments, the cationic peptide contains both D and L enantiomers. Amino acids with many different protecting groups (e.g., Boc) appropriate for use in the solid phase synthesis of polypeptides are commercially available.
The term "natural" or "naturally occurring" amino acid refers to one of the twenty most common occurring amino acids. Natural amino acids are referred to herein by their standard one letter abbreviation. The term "non-natural amino acid" or "non-natural" refers to any derivative of a natural amino acid including D forms, and β and γ amino acid derivatives. Non-limiting examples of non-natural amino acids or amino acid derivatives that can be incorporated into a cationic peptide include the following (common abbreviations in parentheses): β-Alanine (β-Ala), γ- Aminobutyric Acid (GABA), 2-Aminobutyric Acid (2 -Abu), a,P-Dehydro-2- aminobutyric Acid (Δ-Abu), γ-aminobutyric acid (γ-Abu), 1 -Aminocyclopropane- 1 - carboxylic Acid (ACPC), Aminoisobutyric Acid (Aib), 2-Amino-thiazoline-4- carboxylic Acid, 5-Aminovaleric Acid (5-Ava), 6-Aminohexanoic Acid (6-Ahx), 8- Aminooctanoic Acid (8-Aoc), 11 -Aminoundecanoic Acid (1 1-Aun), 12- Aminododecanoic Acid (12-Ado), 2-Aminobenzoic Acid (2-Abz), 3-Aminobenzoic Acid (3-Abz, also known as meto-aminobenzoic acid (mABA)), 4-Aminobenzoic Acid (4-Abz), 4-Amino-3-hydroxy-6-methylheptanoic Acid (Statine, Sta),
Aminooxyacetic Acid (Aoa), 2-Aminotetraline-2-carboxylic Acid (Ate), 4-Amino-5- cyclohexyl-3-hydroxypentanoic Acid (ACHPA), para-Aminophenylalanine (4-NH2- Phe), Biphenylalanine (Bip), ara-Bromophenylalanine (4-Br-Phe), ortho- Chlorophenylalanine (2-Cl-Phe), meto-Chlorophenylalanine (3-Cl-Phe), ara- Chlorophenylalanine (4-Cl-Phe), meto-Chlorotyrosine (3-Cl-Tyr), para- Benzoylphenylalanine (Bpa), tert-Butylglycine (Tie), Cyclohexylalanine (Cha), Cyclohexylglycine (Chg), Diaminopimelic acid (DAP), 2,3-Diaminopropionic Acid (Dpr), 2,4-Diaminobutyric Acid (Dbu), 3,4-Dichlorophenylalanine (3,4-C12-Phe), 3,4- Diflurorphenylalanine (3,4-F2-Phe), 3,5-Diiodotyrosine (3,5-I2-Tyr), ortho- Fluorophenylalanine (2-F-Phe), meto-Fluorophenylalanine (3-F-Phe), ara- Fluorophenylalanine (4-F-Phe), meto-fluorotyrosine (3-F-Tyr), Homoarginine (Har), Homolysine (Hly), Homoserine (Hse), Homophenylalanine (Hfe), Homotyrosine
(Htyr), 5-Hydroxytryptophan (5-OH-Trp), Hydroxyproline (Hyp), para- Iodophenylalanine (4-I-Phe), 3-Iodotyrosine (3-I-Tyr), Indoline-2-carboxylic Acid (Idc), Isonipecotic Acid (Inp), meto-methyltyrosine (3-Me-Tyr), 1-Naphthylalanine (1-Nal), 2 Naphthylalanine (2-Nal), ara-Nitrophenylalanine (4-N02-Phe), 3- Nitrotyrosine (3-N02-Tyr), Norleucine (Me), Norvaline (Nva), Ornithine (Orn), ort/zo-Phosphotyrosine (H2P03-Tyr), Octahydroindole-2-carboxylic Acid (Oic), Penicillamine (Pen), Pentafluorophenylalanine (F5-Phe), Phenylglycine (Phg), Pipecolic Acid (Pip), Propargylglycine (Pra), Pyroglutamic Acid (pGlu), Sarcosine (Sar), Tetrahydroisoquinoline-3-carboxylic Acid (Tic), Thiazolidine-4-carboxylic Acid (Thioproline, Th), trimethy lysine, trimethylornithine, 4-aminopiperidine-4- carboxylic acid, 4-amino-l-carbamimidoylpiperidine-4-carboxylic acid, and 4- guanidinophenylalanine. It is noted that certain amino acids, e.g., hydroxyproline, that are classified as a non-natural amino acid herein, may be found in nature within a certain organism or a particular protein.
Compositions
This document also features compositions that include one or more cationic peptides described herein. For example, a composition can include two, three, four or more peptides. The compositions can be formulated for any route of administration by mixing the one or more peptides them with any one or more of a variety of pharmaceutically acceptable carriers, emulsifiers, excipients, and/or stabilizers known in the art [Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. Ed. 1980]. Acceptable carriers, emulsifiers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include: buffers, such as phosphate, citrate, and other non-toxic organic acids; antioxidants such as ascorbic acid; low molecular weight (less than 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such
polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugar alcohols such as mannitol, or sorbitol; salt- forming counterions such as sodium; and/or nonionic surfactants such as Tween, Pluronics, or PEG. In some embodiments, an aqueous gel
(e.g., containing 96.3% H20, 2.7% Natrosol 250 HX Pharm Hydroxyethylcellulose (Hercules Inc.), 0.85% NaCl, 0.1% sorbic acid, and 0-0.02% caramel color by weight; pH is adjusted to 4.4 with sodium hydroxide) can be used as an emulsifying agent for the peptides. In some embodiments, the peptides can be a component of a cream or solution to be applied topically to an infected area or to an area at risk of being infected, optionally in combination with any known non-toxic delivery agent and/or penetrant.
The compositions described herein can be administered orally or by intravenous infusion, or injected subcutaneous ly, intramuscularly, intrathecally, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratracheally, or intrapulmonarily. The dosage required depends on the choice of the route of administration; the nature of the formulation; the nature of the patient's condition; the subject's size, weight, surface area, age, and sex; other drugs being administered; and the judgment of the attending physician. Suitable dosages are in the range of 0.001-100.0 mg kg. Wide variations in the needed dosage are to be expected in view of the differing efficiencies of various routes of administration. Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art. Encapsulation of the peptide in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) may increase the efficiency of delivery, particularly for oral delivery.
Other compositions described herein include one or more cationic peptides and a medical or hygienic device. As used herein, a "medical or hygienic device" is a device that is inserted into a bodily canal of a vertebrate subject, inserted into a bodily cavity of a vertebrate subject, or applied to a tissue or organ of a vertebrate animal for the purpose of: (a) wound protection; (b) preventing or reducing unwanted, or overcoming restricted, release from the body of the vertebrate subject of a bodily fluid, bodily secretion, or excreta (e.g., blood, menses, urine, lymphatic fluid, cerebrospinal fluid, semen, saliva, vaginal secretions, mucus, or feces); (c) delivering a drug or some other therapeutic or prophylactic agent to a subject; (d) replacing absent or supplementing defective organ functions; or (e) maintaining the patency of a bodily canal (e.g., a blood vessel). Devices of interest include, without limitation: rectal devices such as suppositories, enemas, and catheters; nasal, tracheal, or
esophageal delivery devices; vaginal devices such as vaginal tampons and contraceptive devices (e.g., diaphragms or intrauterine devices (IUDs)); venous, arterial, intracranial and other needles, catheters and stents; renal dialysis accesses; surgical bandages, sutures, or dressings; ostomy devices; incontinence devices;
natural and synthetic implantable tissue matrices (see, for example, U.S. Patent No. 5,885,829); pace makers and pace maker wires and leads; synthetic and natural prostheses such as hip and knee prostheses and heart valves; osmotic pumps (e.g., mini osmotic pumps) that are implanted in body cavity (e.g., the peritoneal cavity) and provide slow delivery of a drug or some other therapeutic or prophylactic agent.
In these compositions, the peptides and the device can be provided separately. Thus, they can be provided in the form of a kit or article of manufacture, optionally also containing packaging materials. The device and the one or more peptides can optionally be in separate containers. In the kit or article of manufacture there can optionally be instructions (e.g., on the packing materials or in a package insert) on how to apply the device and administer the peptide(s). The peptide(s) in such compositions can be formulated as described above. Generally, however, the peptide(s) and device will be provided in a combined form. Thus, all or part of the device can be impregnated, or all or part of the surface of the device can be coated, with the one or more peptides.
As used herein, "applying a device" to a vertebrate subject means inserting all or part of the device into a bodily canal (e.g., a vagina) or bodily cavity (e.g., a peritoneum or pleural cavity) of the subject or placing all or part of the device in a touching relationship with the surface or within the body of a tissue or organ of the subject.
The document also provides methods of using a composition that includes one or more peptides and a device. The devices and peptide(s) can be used separately or combined prior to use. If used separately, the peptide can be administered by any of the methods described above and the device applied to the vertebrate subject as described above. Where the device and peptides are provided in a combined form, the composition is applied in same manner as the device alone.
In any of the above described methods, the subject can be a vertebrate, for example: a mammal such as a human, non-human primate (e.g., monkey), mouse, rat,
hamster, gerbil, guinea pig, cow, sheep, goat, horse, pig, rabbit, dog, or cat; or a bird such as a chicken, turkey, canary, eagle, or hawk. For example, a composition described herein that contains one or more peptides can be delivered to a tissue or organ of a vertebrate subject such as a human, that is, or is at risk of being, infected with a bacterium to inhibit exotoxin production in the subject. In some embodiments, exotoxin production can be inhibited by inserting, applying, or implanting a medical or hygienic device in the subject that includes one or more peptides described herein (e.g., a medical or hygienic device that is impregnated, or where all or part of the surface of the device is coated, with the one or more peptides).
Articles of manufacture
Compositions described herein can be combined with packaging material and sold as articles of manufacture or kits. Components and methods for producing articles of manufactures are well known. The articles of manufacture may combine one or more therapeutic compositions described herein. In addition, the articles of manufacture may further include sterile water, pharmaceutical carriers, buffers, antibodies, indicator molecules, and/or other useful reagents for detecting microbial diseases. Instructions describing how a composition or vaccine is effective for preventing the incidence of an infection, preventing the occurrence of the clinical signs of an infection, ameliorating the clinical signs of an infection, lowering the risk of the clinical signs of an infection, lowering the occurrence of the clinical signs of an infection and/or reducing the spread of infections may be included in such kits. A composition described herein may be provided in a pre-packaged form in quantities sufficient for a single administration.
The following are examples of the practice of the invention. They are not to be construed as limiting the scope of the invention in any way.
EXAMPLES EXAMPLE 1 - Materials and Methods
S. aureus MN8, a methicillin susceptible (MSSA) clinical isolate from a menstrual TSS patient and representative of 75% of menstrual TSS isolates (See Schlievert, et al, J Infect Dis 143:509-16 (1981)), was used for all of the experiments
described herein. The organism was maintained in the laboratory in the lyophilized state. For experimentation, MN8 was cultured overnight in Todd Hewitt (Difco laboratories, Detroit, MI). The next day, the organism was diluted in fresh Todd Hewitt broth to approximately 104 colony-forming units (CFU)/mL for inoculation. The organism was cultured for 9 hours in the presence of potential exotoxin- inhibitory peptides with shaking (200 revolutions/mm),
Exotoxin-inhibiting peptides. Peptides for use in all studies were synthesized and purified to homogeneity by the University of Minnesota Microchemicai Facility, except for defensin peptides (human neutrophil peptide- ! (H P-1 ) and human neutrophil peptide-2 (HNP-2)), which were purchased from Sigma-Aldrich (St. Louis, MO). All peptides were diluted into culture media to concentrations ranging from 5.0 to <5 x lO"6 g/mL. The sequences of the peptides are shown in Table 1, with the percentage of positive residues shown in parentheses.
TABLE 1
Peptides tested for exotoxin-inhibition activity
Peptide effects on MN8 and TSST-1 production. For all studies, S. aureus
MN8 was cultured for 9 h with shaking (200 revolutions/min) in the presence of peptides in volumes of 2 mL culture per tube. After incubation, a sample of each culture was used for plate-count determination of colony-forming units/ml and a sample was used for TSST-1 quantification as described by Schlievert, et al,
Biochemistty 46: 14349-58 (2007). Briefly for TSST-I determination, I mL of each sample was treated overnight with 4 volumes of absolute ethanol to precipitate all
measurable TSST-1. Subsequently, the precipitate from each culture was collected by centrifugation (1000 x g, 10 min), the ethanol poured off, and the sample dried for 30 min under a laminar flow hood. Each sample was resuspended in distilled water (50 μΕ) and clarified by centrifugation (14,000 x g, 5 min) and then added to 50 μΕ sodium-dodecyl sulfate polyacryiamide gel electrophoresis (SDS-PAGE) sample buffer (final volume l/10m original culture volume). Twenty microliters of each sample were electrophoresed on SDS-PAGE gels and transblotted onto PVDF membranes; control samples of purified TSST-1 wrere treated comparably, ranging from 10 .g/lane to 0.1 ug/lane. Subsequently, Western immunoblots were developed with hyperimmune antibodies against TSST-1, then alkaline phosphatase-conjugated anti-rabbit IgG (Sigma-Aldrich), and finally substrate. The color reactions were visualized by using N1H program TmageJ for quantitative comparison to purified TSST-1 samples. The standard curve generated from purified TSST-1 typically gave R~ values of greater than 0.95, consistent with the reliability of this technique to be used quantitatively.
EXAMPLE 2 - HNP-1 and HNP-2
As shown in Table 2, HNPs were weakly anti-staphyiococcai. At 5 μg/mL, both HNP-1 and HNP-2 inhibited MN8 growth by approximately 1 log; at all lower HNP doses, no antimicrobial activity was observed. In contrast, HNP-1 and HNP-2 both were highly inhibitory of production of TSST-1, with complete inhibition at doses as low as 10"3 μ^'ηιΕ (Table 2).
TABLE 2
HNP-1 and HNP-2 effect on growth and TSST-1 production of S. aureus MN8.
EXAMPLE 3 - Hemoglobin a-chain peptides The α-globin chain of human hemoglobin inhibits TSST-1 production without affecting S. aureus growth. See, Schlievert, et a!., Biochemistry 46: 14349-58 (2007). Hbg-1 (see Table 1) was identified from a scan of the α-globin chain for cationic peptides. Hbg-1 contains 18% positive charges. Hbg-1 inhibited TSST-1 production completely at 5.0 and 5 x 10"1 μg/mL but lost activity at lower peptide concentrations. No antimicrobial activity was observed with Hbg-1. See Table 3. Table 3
Hemoglobin (Hbg) peptide effects on S. aureus MN8 growth and TSST-1 production
The Hbg-1 peptide was scrambled to produce Hbg-2, which was assayed for activity. Hbg-2 was not antimicrobial at any concentration tested, but the peptide was comparably active as Hbg-1 for inhibition of TSST-1 production (Table 3). These data suggest that the exotoxin inhibition activity of the Hbg peptides was related to charge properties.
The activity of Hbg- 1 also was evaluated when immobilized on agarose beads (referred to as Hbg-3). It was hypothesized that if charge was the principal determinant of activity, the immobilized peptide would retain its TSST-1 inhibition activity. Hbg-3 was not antimicrobial but retained TSST-1 inhibition activity. See Table 3. This is important since it shows that the peptides can be added to implanted devices and retain TSST-1 inhibition activity.
Because of the TSST-1 inhibition activity of the above tested peptides, and data suggesting the activity resulted from the presence of positively charged amino acids, Hbg-1 peptides were modified to increase the number of positively charged amino acids and tested for exotoxin inhibition and antimicrobial activity. The modified peptides contained no positively charged amino acids (SP-3), 50% positively charged amino acids (SP-2), or 70% positively charged amino acid residues (SP-1). The sequences of SP-1, SP-2, and SP-3 are presented in Table 1.
The SP-3 peptide (no positively charged amino acids) lacked antimicrobial and TSST-1 inhibition activity (Table 4). In contrast, the highly positively charged peptides (SP-1 and SP-2) showed the greatest TSST-1 inhibition activity compared to all other peptides tested, without affecting bacterial growth (Table 4). SP-1 (70%+) inhibited TSST-1 production by > 50% at concentrations of 5 x 10" 5 μg/mL or greater. This represents a 3 -log improvement in TSST-1 inhibition compared to the original Hbg-1 peptide shown in Table 3.
Table 4
EXAMPLE 4 -Lack of Toxicity of Synthetic Peptides
The toxicity of each of the SP-2 (50% + charge) and SP-1 (70% + charge) peptides was assessed in rabbits as follows. Three rabbits/group were challenged vaginally with 200 μΐ/day of 1.0 μg/mL of peptide (SP-2 or SP-1) or PBS, all of which were formulated in an aqueous gel (96.3% H20, 2.7% Natrosol 250 HX Pharm Hydroxyethylcellulose (Hercules Inc.), 0.85% NaCl, 0.1% sorbic acid, and 0-0.02% caramel color by weight; pH is adjusted to 4.4 with sodium hydroxide). The animals were examined after 7 days for signs of inflammation and vaginal irritation as induced by the peptides. No irritation or inflammation was observed, indicating the peptides did not show toxicity.
OTHER EMBODIMENTS
While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
Claims
1. An isolated peptide that inhibits bacterial exotoxin production without
substantially inhibiting bacterial growth, wherein said peptide is from 10 to 50 amino acids in length, and wherein the percentage of positively charged amino acids above the number of negatively charged amino acids is 15% or higher.
2. The peptide of claim 1, wherein said bacterial exotoxin is from a bacterial genus selected from the group consisting of Staphylococci, Neisseriae, Streptococci, Chlamydia trachomatis, Treponemae, Haemophilus, Bordetellae, Gardnerella vaginalis, Bacillus, Clostridium, Escherichia coli, Vibrio cholerae, Salmonella, Shigellas, Mycobacteria, Francisella, Yersinia, Burkholderia, Pseudomonas, and Brucella.
3. The peptide of claim 1 or claim 2, wherein said peptide is from 18 to 30 amino acids in length.
4. The peptide of any one of claims 1-3, wherein the percentage of positively
charged amino acids above the number of negatively charged amino acids is 18% or higher.
5. The peptide of any one of claims 1-3, wherein the percentage of positively
charged amino acids above the number of negatively charged amino acids is 25% or higher.
6. The peptide of any one of claims 1-3, wherein the percentage of positively
charged amino acids above the number of negatively charged amino acids is 50% or higher.
7. The peptide of any one of claims 1-3, wherein the percentage of positively
charged amino acids above the number of negatively charged amino acids is 70% or higher.
8. The peptide of any one of claims 1-7, wherein said positively charged amino acids comprise one or more of lysine, a lysine analog, arginine, an arginine analog, histidine, or a histidine analog.
9. The peptide of any one of claims 1-3, wherein said peptide has the amino acid sequence SFPTTKTYFPHFDLSHGSAQVK (SEQ ID NO: 3),
STKPFFYFLHTQVKASPTSHDG (SEQ ID NO: 4), SKPKKKKYKPHKKKSHKSAKKK (SEQ ID NO: 5), or
SKPKKKTYKPHKDLSHGSAKKK (SEQ ID NO: 6).
10. A composition comprising one or more peptides and a pharmaceutically
acceptable carrier, wherein each peptide is a peptide of any one of claims 1-9.
1 1. The composition of claim 10, wherein said composition is formulated for
subcutaneous administration.
12. The composition of claim 10, wherein said composition is formulated for
intramuscular administration.
13. The composition of claim 10, wherein said composition is formulated for oral administration.
14. The composition of claim 10, wherein said composition is formulated for topical administration.
15. The composition of claim 10, wherein said composition is formulated for vaginal administration.
16. A composition comprising a medical or hygienic device; and the peptide of any one of claims 1-9.
17. The composition of claim 16, wherein all or part of the device is impregnated, or all or part of the surface of the device is coated, with the one or more peptides.
18. The composition of claim 16 or claim 17, wherein said device is a vaginal
tampon.
19. The composition of claim 16 or claim 17, wherein said device is a surgical suture, a surgical bandage, a surgical dressing, or an osmotic pump.
20. The composition of claim 16 or claim 17, wherein said device is an ostomy
device.
21. A method of inhibiting exotoxin production, said method comprising delivering said peptide of any one of claims 1-9 to a tissue or organ of a vertebrate subject, wherein said tissue or organ is, or is in risk of being, infected with a bacterium.
22. A method of inhibiting exotoxin production, said method comprising contacting a vertebrate subject with the composition of any one of claims 16-20.
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CN201280069162.XA CN104093734A (en) | 2011-12-21 | 2012-12-21 | Cationic peptides and use of such peptides for inhibiting exotoxin production |
EP12816208.8A EP2794645A1 (en) | 2011-12-21 | 2012-12-21 | Cationic peptides and use of such peptides for inhibiting exotoxin production |
CA2859720A CA2859720A1 (en) | 2011-12-21 | 2012-12-21 | Cationic peptides and use of such peptides for inhibiting exotoxin production |
US14/367,116 US9187529B2 (en) | 2011-12-21 | 2012-12-21 | Cationic peptides and use of such peptides for inhibiting exotoxin production |
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CN107467347A (en) * | 2017-07-31 | 2017-12-15 | 兰溪市酉泽饲料技术服务有限公司 | The preparation method of Penaeus Vannmei growth-promoting feed |
CN107568500A (en) * | 2017-07-31 | 2018-01-12 | 兰溪市酉泽饲料技术服务有限公司 | The preparation method of Penaeus Vannmei children phase feed |
CN107549453A (en) * | 2017-07-31 | 2018-01-09 | 兰溪市酉泽饲料技术服务有限公司 | Cherish the preparation method of ovum phase loach feed |
CN107467413A (en) * | 2017-07-31 | 2017-12-15 | 兰溪市酉泽饲料技术服务有限公司 | The preparation method of loach scrod feed |
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CN107348126A (en) * | 2017-07-31 | 2017-11-17 | 浦江县协盈动物饲料技术开发有限公司 | Cherish ovum phase loach feed |
CN107439843A (en) * | 2017-07-31 | 2017-12-08 | 浦江县协盈动物饲料技术开发有限公司 | Oogenesis phase penaeus vannamei boone feed |
CN107581401A (en) * | 2017-08-15 | 2018-01-16 | 兰溪市酉泽饲料技术服务有限公司 | Prawn children phase fermentative feedstuff of microbe |
CN107549508A (en) * | 2017-08-15 | 2018-01-09 | 兰溪市酉泽饲料技术服务有限公司 | Promote the preparation method of the feed of prawn husking |
CN107494991A (en) * | 2017-08-15 | 2017-12-22 | 兰溪市酉泽饲料技术服务有限公司 | The preparation method of prawn phagostimulant |
WO2020214518A1 (en) * | 2019-04-18 | 2020-10-22 | Arcuate Therapeutics, Inc. | Methods of treating renal conditions using peptides that improve mitochondrial function |
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