WO2014078801A1 - Methods and compositions comprising guanidines for treating biofilms - Google Patents

Methods and compositions comprising guanidines for treating biofilms Download PDF

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Publication number
WO2014078801A1
WO2014078801A1 PCT/US2013/070598 US2013070598W WO2014078801A1 WO 2014078801 A1 WO2014078801 A1 WO 2014078801A1 US 2013070598 W US2013070598 W US 2013070598W WO 2014078801 A1 WO2014078801 A1 WO 2014078801A1
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WIPO (PCT)
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composition
guanidine
biofilm
different
same
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PCT/US2013/070598
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French (fr)
Inventor
Jon Clardy
Thomas Böttcher
Richard Losick
Illana Kolodkin-Gal
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President And Fellows Of Harvard College
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Publication of WO2014078801A1 publication Critical patent/WO2014078801A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
    • A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
    • A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
    • A01N47/44—Guanidine; Derivatives thereof
    • 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
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • 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
    • 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

  • Bio films are communities of cells that settle and proliferate on surfaces and are covered by an exopolymer matrix. They are slow-growing and many are in the stationary phase of growth. A hallmark of biofilms is an extracellular matrix typically consisting of protein, exopolysaccharide and sometimes DNA, that holds the cells together in the community. They can be formed by most, if not all, pathogens. According to the CDC, 65% of ail infections in the United States are caused by biofilms that can be formed by common pathogens. Biofilms are also found in industrial settings, such as in drinking water distribution systems.
  • the method comprises contacting a surface with a composition comprising an effective amount of a guanidine, thereby treating, reduci g, inhibiting formation of the biofilm, or triggering disa ssembly of the biofilm.
  • the guanidine has three atoms separating amino or guanidine groups.
  • the amino groups are ionizable.
  • the amino groups are positively charged.
  • the guanidines are branched.
  • the guanidines are linear.
  • the guanidine has Formula (I) or Formula (II),
  • M is C(RiR2)-Y-C(R 3 R 4 ), meta-phenylene, or 1 ,3-Cs-C cycloalkane, where the meta- phenyiene or cycloalkane may be substituted by Rio or have one or more N heteroatoms in its ring; each Ri, R 2 , R 3 , R 4 , R5, and R is H, C1-C12 alkyl, Ci-C 12 alkenyl, C1-C12 alkynyl, Ci- C12 alkoxy, alkaryl, aryl, or heteroaryl, so that each Ri, R.
  • Compounds of Formulae (I) and (II) may be acyclic or cyclic. Compounds of Formulae (I) and (II) may be linear or branched,
  • Y is QRj e). In some embodiments, Y is NRj 0 .
  • M is meta-phenylene.
  • the meta- phenylene may be substituted with CrCg alkyl, Q Cg alkenyl, or C Cg alkynyl.
  • M is meta-phenylene and substituted with an N heteroatom on the ring such that M is 2,6 ⁇ pyridinediyl.
  • M is 1,3-cyclohexanediyl.
  • M is a 1,3-cyclohexanediyl substituted with an N heteroatom on the ring such that M is 2,6-piperidinediyl.
  • M is a 1 ,3-cyelohexanediy! substituted with three N heteroatoms on the ring such that M is l,3,5-triazine-2,4-diyl.
  • M is 1 ,3 cyclopentanediyl.
  • M is 1,3-cyclopentanediyl substituted with an N heteroatom on the ring such that M is 2,5-pyrrolidinediyi.
  • M is cyclopent- 1 ,3-ene- 1 ,4-diyl.
  • M is cyclopent- 1 ,3-ene- 1 ,4-diyl substituted with an N heteroatom on the ring such that M is 2,5-pyrroldiyl.
  • Ri is hydrogen. In some embodiments, R? is hydrogen. In some embodiments, R 3 is hydrogen. In some embodiments, R 4 is hydrogen. In some embodiments, R 5 is hydrogen. In some embodiments, Re is hydrogen. In some embodiments, R 7 is hydrogen. In certain embodiments, each of Ri, R 2 , R 3 , R , R 5 , and Re is H. In other embodiments, each of Ri, ! ⁇ . ⁇ . R. 3 , 4, R 5 , and Re is H or CI 1 ;
  • Rj and R3 covalently bond to each other to form a ring.
  • each of R , Rp is H, Ci-Ce alkyl, C3-C6 alkenyl, or C 3 ⁇ Ce alkynyl.
  • Rg is hydrogen. In some embodiments, Rg
  • each R 3 ⁇ 4 is the same. In some embodiments, each Rg is different.
  • R 9 is hydrogen.
  • RJQ is hydrogen.
  • x is 1. In some embodiments, x is 2. In certain embodiments, x is 3, In further embodiments, x is 4. In still further embodiments, x is 5, In other
  • x is 6.
  • Y is CH 2 , each of Rj, R 2 , R3, R4, R5, and Re is H and x is 1 , 2 or 3.
  • the guanidine is a tautomer of Formulae (! or (11).
  • a guanidine of Formula (11) may be represented by the Formulae:
  • the guanidine is a compound in Table 1.
  • the bacteria are Gram-negative or Gram-positive bacteria.
  • the bacteria are Bacillus, Staphylococcus, E. coli, or Pseudomonas bacteria.
  • the bacteria are mycobacteria.
  • the method comprises contacting a surface with a composition comprising an effective amount of a guanidine combined with a D-amino acid, thereby treating, reducing, inhibiting formation of the biofilm, or triggering disassembly of the biofilm.
  • compositions such as industrial, therapeutic or pharmaceutical compositions, comprising one or more guanidines.
  • the invention features compositions, such as therapeutic or pharmaceutical compositions, comprising one or more guanidines.
  • the one or more guanidines are combined with one or more D-amino acids.
  • the composition comprises at least one guanidine of Formulae (! or (II),
  • the guanidine has Formulae (! or (II) and the D-aniino acid is selected from the group consisting of D-alanine, D-cysteine, D-aspartic acid, D-glutamic acid, D-histidine, D-isoleucine, D-lysine, D-lexicine, D-asparagine, D-proline, D-glutamine, D-arginine, D-serine, D-threonine, D-valine, D-tryptophan, D-tyrosine, and a combination thereof, or the D-amino acid is a synergistic combination of two or more D-amino acids selected from the group consisting of D-alanine, D- cysteine, D-aspartic acid, D-glutamic acid, D-phenylalanine, D-histidine, D-isoleu
  • the D-amino acid is D-tyrosine or the combination of D-amino acids comprises D-tyrosine,
  • the composition further comprises one or more of D proline and D phenylalanine.
  • the composition further comprises one or more of D-leucine, D-tryptophan, and D-methionine.
  • the composition further comprises one or more of D-alanine, D-cysteine, D-aspartic acid, D- glutamic acid, D-phenylalanine, D-histidine, D-isoleucine, D-lysine, D-leucine, D-methionine, D-asparagine, D-proline, D-glutamine, D-arginine, D-serine, D-threonine, D-valine, D- tryptophan, and D-tyrosine.
  • Another aspect of this disclos ure is directed to methods of treating a biofilm-related disorder in a subject in need thereof!, the method comprising administering to the subject a composition comprising an effective amount of a guanidine.
  • the guanidine has Formulae (I) or ( ⁇ ).
  • the gtianidine is a compound in Table 1.
  • the method comprises administering to the subject a composition comprising a combination of at least one guanidine and at least one D-amino acid, thereby treating the biofilm-related disorder.
  • the guanidine has Formulae (I) or (II) and the D-amino acid is selected from the group consisting of D-alanine, D-eysteine, D-aspartic acid, D-glutamic acid, D-histidine, D-isoleucine, D-lysine, D-leucine, D-asparagine, D-proline, D-glutamine, D-arginine, D-serine, D-threonine, D- valine, D-tryptophan, D-tyrosine, and a combination thereof, or the D-amino acid is a synergistic combination of two or more D- amino acids selected from the group consisting of D-alanine, D-cysteine, D-as
  • the D-amino acid is D-tyrosine or the combination of D-amino acids comprises D-tyrosine.
  • the composition further comprises one or more of D-proline and D-phenylalanine.
  • the composition further comprises one or more of D-leucine, D-tryptophan, and D-methionine.
  • the composition further comprises one or more of D-alanine, D-cysteine, D-aspartic acid, D- glutamic acid, D-phenylalanine, D-histidine, D-isoleucine, D-lysine, D-leucine, D-methionine, D-asparagine, D-proline, D-glutamine, D-arginine, D-serine, D-threonine, D-valine, D- tryptophan, and D-tyrosine,
  • the composition is administered to a surface of the subject selected from the group of dermal and mucosal surfaces and combinations thereof.
  • the surface is an oral surface, a skin surface, a urinary tract surface, a vaginal tract surface, or a lung surface.
  • composition is administered to the subject via
  • the subject is a human.
  • the formation of a biofilm is inhibited. In other embodiments, a previously formed biofilm is disrupted.
  • the guanidine is administered at a concentration of about 0.1 iiM to about 100 ⁇ , for example, at a concentration of 0.1 iiM to 100 ⁇ , In certain embodiments, the D-amino acid combined with the guanidine is administered at a concentration of about 0.1 nM to about 100 ⁇ , for example, at a concentratior! of 0.1 nM to 100 ⁇ .
  • the biofilm-related disorder is selected from the group consisting of pneumonia, cystic fibrosis, otitis media, chronic obstructive pulmonary disease, and a urinary tract infection and combinations thereof.
  • the biofilm-related disorder is a medical device-related infection.
  • the biofilm-related disorder is a periodontal disease, such as gingivitis, periodontitis or breath malodor.
  • the biofilm-related disorder is caused by bacteria.
  • the bacteria are Gram-negative or Gram-positive bacteria.
  • the bacteria are of the genus ActinobaciUus, Acinetobacter, Aeromonas, Bordetella, Brevibaculus, Brucella, Bacteroides, Burkholderia, Borelia, Bacillus, Campylobacter, Capnocytophaga, Cardiobacterium, Citrobacter, Clostridium, Chlamydia, Eikenella, Enterobacter, Escherichia, Entembacter, Francisella, Fusobacterium, Flavobacterium, Haemophilus, Helicobacter, Kingella, Klebsiella, Legionella, Listeria, Leptospirae, Moraxella, Morganella, Mycoplasma, Mycobacterium, Neisseria, Pasteurella, Proteus, Prevotella, Plesiomonas, Pseudomonas, Providencia, Rickettsia, Stenotrophomonas, Staphylococcus, Streptococcus, Strept
  • Another aspect of this disclosure is directed to methods of treating, reducing, inhibiting biofilm formation by biofilm forming bacteria, or triggering biofilm disassembly on a biologically-related surface, the method comprising contacting a biological surface with a composition comprising an effective amount of a guanidine.
  • the guanidine has Formulae ( ⁇ ) or (II),
  • the composition and related methods include a combination of at least one guanidine and at least one D-amino acid, thereby treating, reducing or inhibiting formation of the biofilm.
  • the guanidine has Formulae (I) or (II) and the D-amino acid is selected from the group consisting of D-alanine, D-eysteine, D-aspartic acid, D- glutamic acid, D-histidine, D-isoleueiiie, D-lysine, D-leucine, D-aspara.gine, D-proline, D- glutamine, D-arginine, D-serine, D-threonine, D-valine, D -tryptophan, D-tyrosine, and a combination thereof, or the D-amino acid is a synergistic combination of two or more D-amino acids selected from, the group consisting of D-alanine, D-eystein
  • the composition comprises a guanidine and D-tyrosine.
  • the composition further comprises one or more of D-proline and D-phenylalanine.
  • the composition further comprises one or more of D-leucine, D- tryptophan, and D-methionine.
  • the composition in addition to D-tyrosine, the composition further comprises one or more of D-alanine, D-cysteine, D-aspartic acid,
  • D-glutamic acid D-phenylalanine, D-histidine, D-isoleucine, D-lysine, D-leucine, D-methionine, D-asparagine, D-proline, D-glutamine, D-arginine, D-serine, D-threonine, D-valine, and
  • the surface comprises a medical device, a wound dressing, a contact lens, or an oral device.
  • the medical device is selected from the group consisting of a clamp, forcep, scissors, skin hook, tubing, needle, retractor, scaler, drill, chisel, rasp, saw, catheter, orthopedic device, artificial heart valve, prosthetic joint, voice prosthetic, stent, shunt, pacemaker, surgical pin, respirator, ventilator, and an endoscope and combinations thereof.
  • the method further comprises administering a biocicle.
  • the biocide is an antibiotic.
  • the guanidine has Formula (I) or (II).
  • the guanidine has Formulae (I) or (II) or is selected from the group of compounds in Table 1, or is a combination one or more compounds in Table 1, the D-amino acid is selected from, the group consisting of D-alanine, D-cysteine, D-aspartic acid, D-glutamic acid, D-histidine, D-isoleucine, D-lysine, D-leucine, D-asparagine, D-proline, D-glutamine, D-arginine, D-serine, D-threonine, D-valine,
  • D-tryptophan, D-tyrosine, and a combination thereof or the combination of D-amino acids is a synergistic combination of two or more D-amino acids selected from the group consisting of D- alanine, D-cysteine, D-aspartic acid, D-glutamic acid, D-phenylalanine, D-histidine, D- isoleucine, D-lysine, D-leucine, D-methionine, D-asparagine, D-proline, D-glutamine, D- arginine, D-serine, D-threonine, D-valine, D-tryptophan, and D-tyrosine.
  • compositions comprising at least one guanidine, or at least o e guanidine combined with at least one D-aniino acid or a mixture of D-amino acids, in an amount effective to treat, reduce, inhibit biofilm formation, or trigger biofilm disassembly.
  • the guanidine has Formulae (I) or (II) or is selected from, the group of compounds in Table 1 , or is a combination one or more compounds in Table 1
  • the D-amino acid is selected from the group consisting of D-alanine, D-cysteine, D- aspartic acid, D-glutamic acid, D-histidine, D-isoleucine, D-lysine, D-leucine, D-asparagine, D- proline, D-glutamine, D-arginine, D-serine, D-fhreonine, D-valine, D-tryptophan, D-tyrosine, and a combination thereof or the combination of D-amino acids is a synergistic combination of two or more D-amino acids selected from the group consisting of D-alanine, D-cysteine, D- aspartic acid, D-glutamic acid, D-phenylalanine, D-histidine,
  • the composition comprises a guanidine and D-tyrosine
  • the guanidine has Formulae (I) or (II) or is selected from the group of compounds in Table 1 , or is a combination one or more compounds in Table 1 .
  • the composition further comprises one or more of D-proline and D-phenylalanine.
  • the composition further comprises one or more of D-leucine, D ⁇ tryptophan, and D-methionine.
  • the composition further comprises one or more of D-alanine, D-cysteine, D-aspartic acid, D-glutamic acid, D-phenylalanine, D- histidine, D-isoleucine, D-lysine, D-leucine, D-methionine, D-asparagine, D-proline, D- glutamine, D-arginine, D-serine, D-threonine, D-valine, D-tryptophan, and D-tyrosine.
  • any of the foregoing compositions can also comprise polyhexamethvlene biguanide, chlorhexidme, xylitol, triciosan, or chlorine dioxide.
  • any of the foregoing compositions can also comprise a pharmaceutically acceptable carrier.
  • the effective amount is an amount effective to treat or prevent, a biofilm-related disorder.
  • an effective amount comprises an amount effective to treat or prevent a biofilm on a surface.
  • the biofilm-related disorder is pneumonia, cystic fibrosis, otitis media, chronic obstructive pulmonary disease, or a urinary tract infection.
  • the biofilm-related disorder is a medical device- related infection.
  • the composition further comprises an agent suitable for application to the surface.
  • the composition is formulated as a wash solution, a dressing, a wound gel, or a synthetic tissue.
  • the composition is formulated as tablets, pills, troches, capsules, aerosol spray, solutions, suspensions, gels, pastes, creams, or foams.
  • the composition is formulated for parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, vaginal and rectal administration.
  • Another aspect of this disclosure is directed to biofilm. resistant medical devices, comprising a surface likely to contact a biological fluid and a guanidine.
  • the guanidine has Formulae (I) or (II) or is selected from the group of compounds in Table 1.
  • the medical device further comprises a D-amino acid, or a combination of D-amino acids, combined with the guanidine and coated on or impregnated into said surface.
  • the D-amino acid is selected from the group consisting of D-alanine, D-cysteine, D-aspartic acid, D-glutamic acid, D-histidine, D-isoleucine, D-lysine, D- leucine, D-asparagine, D ⁇ proline, D-glutamine, D-arginine, D-serine, D-threonine, D-valine, D- tryptoplian, D-tyrosine, and a combination thereof, or the combination of D-amino acids is in an amount effective to treat, reduce, or inhibit biofilrn formation, the combination of D-amino acids is a synergistic combination of two or more D-amino acids selected from the group consisting of D-alanine, D-cysteine, D-aspartic acid, D-glutamic acid, D-phenylalanine, D-histidine, D- isoleucine, D-lysine, D
  • the D-amino acid is D-tyrosine or the combination of D-amino acids comprises D-tyrosine,
  • the composition further comprises one or more of D proline and D phenylalanine.
  • the composition further comprises one or more of D-leucine, D-tryptophan, and D-methionine.
  • the composition further comprises one or more of D-alanine, D-cysteine, D-aspartic acid, D- glutamic acid, D-phenylalanine, D-histidine, D-isoleucine, D-lysine, D-leucine, D-methionine, D-asparagine, D-proline, D-glutamine, D-arginine, D-serine, D-threonine, D-valine, D- rryptophan, and D-tyrosine.
  • D-alanine D-cysteine
  • D-aspartic acid D- glutamic acid
  • D-phenylalanine D-histidine
  • D-isoleucine D-lysine
  • D-leucine D-methionine
  • D-asparagine D-proline
  • D-glutamine D-arginine
  • D-serine D-threonine
  • D-valine D-valine
  • the guanidine is formulated as a slow-release formulation. In certain embodiments, the guanidine combined with the D-amino acid is formulated as a slow- release formulation. In some embodiments, the surface is essentially free of L-amino acids. In further embodiments, the surface is essentially free of detergent. [0044] In some embodiments, the device is selected from one or more of clamp, forcep, scissors, skin hook, tubing, needle, retractor, scaler, drill, chisel, rasp, saw, catheter, orthopedic device, artificial heart valve, prosthetic joint, voice prosthetic, stent, shunt, pacemaker, surgical pin, respirator, ventilator and endoscope.
  • a further aspect of the instant disclosure is directed to potable liquids comprising a guanidine.
  • the guanidine has Formulae (I) or (II).
  • the guanidine is selected from the group of compounds in Table 1.
  • at, least one guanidine is combined with at least one D-amino acid or a combination of D-amino acids, at a concentration in the range of 0.000001 % to 0.1 %.
  • wherein the guanidine has Formulae (!
  • the D-amino acid is selected from the group consisting of D-alanine, D-cysteine, D-aspartic acid, D-glutamic acid, D-histidine, D-isoleucine, D- lysine, D-leucine, D-asparagine, D-proiine, D-glutamine, D-arginine, D-serine, D-threonine, D-valiiie, D-tryptophan, D-tyrosine, and a combination thereof, or the combination of D-amino acids is a synergistic combination of two or more D-amino acids selected from the group consisting of D-alanine, D-cysteine, D-aspartic acid, D-glutamic acid, D-phenylalanine, D- histidine, D ⁇ isoleucine, D-ly
  • the D-amino acid is D-tyrosine or the combination of D-amino acids comprises D-tyrosine,
  • the composition further comprises one or more of D proline and D phenylalanine.
  • the composition further comprises one or more of D-leucine, D-tryptophan, and D-methionine.
  • the composition further comprises one or more of D-alanine, D-cysteine, D-aspartic acid, D- glutamic acid, D-phenylalanine, D-histidine, D-isoleucine, D-lysine, D-leucine, D-methionine, D-asparagine, D-proline, D-glutamine, D-arginine, D-serine, D-threonine, D-valine, D- tryptophan, and D-tyrosine.
  • compositions resistant to bio film formation comprising a carrier and an effective amount of a guanidine or polyamine.
  • the carrier is pharmaceutically or cosmetically suitable base, a fluid base, or a polymeric binder.
  • the guanidine has Formulae (I) or (II).
  • the guanidine is selected from the group of compounds in Table 1.
  • the guanidine is combined with a D-ammo acid or a combination of D-amino acids, distributed in the base, thereby treating, reducing or inhibiting formation of the biofilm.
  • the D-amino acid is selected from the group consisting of D-alamne, D ⁇ cysteine, D-aspartic acid, D-glutamic acid, D-histidine, D-isoleucine, D-lysine, D-leucine, D- asparagine, D-proline, D-glutamine, D-arginine, D-serine, D-threonine, D-valine, D-tryptophan, D-tyrosine, and a combination thereof, or the combination of D-amino acids is a synergistic combination of two or more D-amino acids selected from the group consisting of D-alanine, D- cysteine, D-aspartic acid, D-giutaniic acid, D-phenylalanine, D-histidine, D-isoleucine, D-lysine, D-leucine, D-methionine, D-asparagine, D-proline, D-glut
  • Another aspect of this disclosure is directed to a coated article resistant to biofilm formation, wherein an article comprises a coating on at least one exposed surface and the coating comprises an amount of a guanidine effective to inhibit or reduce biofilm formation.
  • the guanidine has Formulae (I) or (IT).
  • the guanidine is selected from the group of compounds in Table 1 .
  • the guanidine is combined with a D-amino acid or a combination of D-amino acids, thereby treating, reducing or inhibiting formation of the biofilm.
  • the D-amino acid is selected from the group consisting of D-alanine, D-cysteine, D-aspartic acid, D-glutamic acid, D-histidine, D- isoleucine, D-lysine, D-leucine, D-asparagine, D-proline, D-glutamine, D-arginine, D-serine, D- threonine, D-valine, D-tryptophan, D-tyrosine, and a combination thereof, or the combination of D-amino a cids is a synergistic combination of two or more D-amino acids selected from the group consisting of D-alanine, D-cysteine, D-aspartic acid, D-glutamic acid, D-phenylalanine, D-histidine, D-isolexicine, D-lysine, D-leucine, D-methionine, D-asparagine, D-
  • the base is essentially free of the corresponding L-amino acid or L-amino acids relative to the D-amino acids or combination of D-amino acids,
  • the base is selected from a liquid, gel, paste, or powder.
  • the composition is selected from the group consisting of shampoos, bath additives, hair care preparations, soaps, lotions, creams, deodorants, skin-care preparations, cosmetic personal care preparations, intimate hygiene preparations, foot care preparations, light protective preparations, skin tanning preparations, insect repe Hants, antiperspirants, shaving preparations, hair removal preparations, fragrance preparations, dental care, denture care and mouth care preparations and combinations thereof.
  • Another aspect of the disclosure is directed to fatty acids salts of the guani dines and polyamines disclosed herein.
  • the fatty acid chain length of these salts ranges from C4 to C24.
  • the fatty acid chain length of these salts ranges from Ce to Ci6.
  • compositions comprising a guanidine or polyarnine disclosed herein, or a combination of one or more gxiani dines and one or more polyamines, and a carrier structure.
  • the guanidine or polyamione or combination of one or more guanidines and one or more polyamines is incorporated into the carrier structure.
  • the carrier structure is a micelle.
  • the micelle which may or may not be a mixed micelle comprising fatty acids of different chain lengths, comprises fatty acids having chain lengths ranging from C o to C 20 .
  • the micelle comprises fatty acids and other lipids.
  • the other lipids are tauroeholate or cholesterol.
  • the carrier structure is a liposome.
  • the liposome is unilamellar. In other embodiments, the liposome is multilamellar.
  • Another aspect of this disclosure is directed to using the fatty acid salts of the guanidines and polyamines disclosed herein in the methods, compositions, devices, liquids, and articles of this disclosure.
  • a further aspect of this disclosure is directed to using the
  • compositions comprising a carrier structure and a guanidme or polyamine, or combination of one or more guanidines and one or more polyamines, in the methods, compositions, devices, liquids, and articl es of this disclosure.
  • FIG. 1 A shows the inhibition of biofilm formation by compounds 6a, 7a, and 1 1 compared to the biofilm inhibition by norspermine and norspermidine.
  • FIG. I B shows the breakdown of preexisting biofilms within 12 hours by compound 7.
  • FIG. 2A shows the averate protonation of polyamines and representative guanidines.
  • FIG. 2B shows the classical, incorrect representation of protonated biguanidines.
  • FIG. 2C shows the correct protonation state of compound 1 1 .
  • FIG. 2D shows the cropped biguanidine moiety from crystal structure of compound 11.
  • FIGS. 3A and 3B shows the MBICs plotted against the average degree of protonation of polyamines and guani dines for B. subtilis and S, aureus., respectively.
  • FIG. 4 is a representation of the crystal structure of compound 2.
  • FIG. 5 is a representation of the crystal structure of compound 5.
  • FIG. 6 is a representation of the crystal structure of compound 1 1 .
  • FIGS. 7 A and 7B shows the inhibition of biofilm formation by compounds norspermidine micelle formulations A, B, and C.
  • alkyl refers to a hydrocarbon chain that may be a straight chain or branched chain.
  • the chain may contain an indicated number of carbon atoms.
  • Q-C 12 indicates that the group may have from 1 to 12 (inclusive) carbon atoms in it.
  • An alkyl group can be substituted or unsubstituted. When substituted, one or more carbon atoms may be replaced with a heteroatom such as N, O, or S.
  • alkoxy refers to a straight or branched chain saturated or unsaturated hydrocarbon containing at least one oxygen atom.
  • the chain may contain an indicated number of carbon atoms.
  • C 1 -C 12 alkoxy indicates that the group may have from 1 to 12 (inclusive) carbon atoms and at least, one oxygen atom.
  • Examples of a C 1 -C 12 alkoxy include, but are not limited to, methoxy, ethoxy, isopropoxy, butoxy, n-pentoxy, isopentoxy, neopentoxy, and hexoxy.
  • An alkoxy group can be substituted or unsubstituted. When substituted, one or more carbon atoms may be replaced with a heteroatom such as N, 0, or S.
  • alkenyl refers to a straight or branched chain hydrocarbon containing at least one carbon-carbon double bond.
  • the chain may contain an indicated number of carbon atoms.
  • “Ci-C] 2 alkenyl” indicates that the group may have from 1 to 12 (inclusive) carbon atoms and at least one carbon-carbon double bond.
  • Exemplary such groups include, but are not limited to, ethenyi (also called “vinyl”), allyl, propenyl, crotyl, 2-isopentenyl, ailenyl, butenyl, butadienyl, pentenyl, pentadienvi, 3(l,4-pentadienyl), hexenyl and hexadienvl.
  • ethenyi also called “vinyl”
  • allyl propenyl
  • crotyl 2-isopentenyl
  • ailenyl butenyl, butadienyl
  • pentenyl pentadienvi
  • 3(l,4-pentadienyl) pentenyl
  • pentadienvi 3(l,4-pentadienyl)
  • 3(l,4-pentadienyl) hexenyl and hexadienvl.
  • alkynyl refers to a straight or branched chain hydrocarbon radical containing at least one carbon-carbon triple bond.
  • the chain may contain an indicated number of carbon atoms.
  • C 2 -C] 2 alkynyl indicates that the group may have from 2 to 12 (inclusive) carbon atoms and at least one carbon-carbon triple bond.
  • exemplary such groups include, but are not limited to, ethynyl, propynyl and butynyl.
  • An alkynyl group can be substituted or unsubstituted. When substituted, one or more carbon atoms may be replaced with a heteroatom such as N, O, or S.
  • cycloalkane refers to saturated carbon ring systems having from. 3 to 20 carbons.
  • examples of cycloalkanes include, but are not limited to, cyclopropane, cyc!obutane, cyclopentane, cyclohexane, cycloheptane, and cyclooctane.
  • a cycloalkane can be substituted or unsubstituted. When substituted, one or more carbon atoms may be replaced with a heteroatom such as IN, O, or S.
  • aryl refers to cyclic aromatic carbon ring systems containing from 6 to 18 carbons.
  • Examples of an aryl group include, but are not limited to, phenyl, naphthyl, anthracenyl, tetracenyl, and phenanthrenyl.
  • An aryl group can be unsubstituted or substituted. When substituted, one or more carbon atoms may be replaced with a heteroatom such as N, O, or S.
  • aralkyl refers to an alkyl group where an H has been replaced with an aryl group.
  • An aralkyl group may be unsubstituted or it may be substituted on the hydrocarbon chain or the aryl ring. When substituted, one or more carbon atoms may be replaced with an N, O, or S.
  • heteroaryl refers to mono and hicyciic aromatic groups of 4 to 10 atoms containing at least one heteroatom. Heteroatom as used in the term heteroaryl refers to oxygen, sulfur and nitrogen, A heteroaryl group can be unsubstituted or substituted.
  • tautomer refers to isomers of compounds that readily interconvert due to the phenomenon wherein a hydrogen atom or proton of one atom of a molecule shifts to another atom and is accompanied by a switch of single bond and adjacent double bond.
  • disorder is a disturbance or derangement that affects the normal function of the body of a subject.
  • a disease is a pathological condition of an organ, a body part, or a system resulting from various causes, such as infection, genetic defect, or environmental stress that is characterized by an identifiable group of symptoms.
  • a disorder or disease can refer to a biofilm-related disorder that is characterized by a disease-related gro wth of bacteria in that a bion lm is established.
  • prevent refer herein to the inhibition of the development or onset of a biofilm or of a biofilm-related disorder or the prevention of the recurrence, onset, or development of one or more indications or symptoms of a biofilm or of a biofilm-related disorder on a surface or in a subject resulting from the administration of a composition described herein (e.g., a prophylactic or therapeutic composition), or the administration of a combination of therapies (e.g., a combination of prophylactic or therapeutic compositions).
  • a composition described herein e.g., a prophylactic or therapeutic composition
  • a combination of therapies e.g., a combination of prophylactic or therapeutic compositions.
  • treat refers to administering a composition described herein in an amount, manner (e.g., schedule of administration), and/or mode (e.g., route of administration), effective to improve a disorder or a symptom thereof, or to prevent or slow the progression of a disorder or a symptom thereof.
  • a composition described herein in an amount, manner (e.g., schedule of administration), and/or mode (e.g., route of administration), effective to improve a disorder or a symptom thereof, or to prevent or slow the progression of a disorder or a symptom thereof.
  • This can be evidenced by, e.g., an improvement in a parameter associated with a biofilm or with a biofilm-related disorder or an indication or symptom thereof, e.g., to a statistically significant degree or to a degree detectable to one skilled in the art.
  • An effective amount, manner, or mode can vary depending on the surface, application, and/or subject and may be tailored to the surface, application, and/or subject.
  • a treatment can prevent or slow deterioration resulting from a biofilm or from a biofilm-related disorder or an indication or symptom thereof on an effected surface or in an affected or diagnosed subject,
  • a "micel le” is defined as a particular type of molecular assembly in which amphipathic molecules are arranged in a approximately spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydro philic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.
  • a liposome refers to a vesicle composed of lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior.
  • Exampl es of other guanidines include the amino acid arginine, the organic soluble base triazabicyclodecene, and the naturally produced neurotoxin saxitocin.
  • This disclosure is directed to the surprising discovery that certain guanidines inhibit biofilm formation and/or trigger disassembly of existing biofilms.
  • This disclosure is based, at least in part, on the discover ⁇ ' that guanidines present in conditioned medium from mature biofilms inhibit biofilm formation and trigger the disassembly of existing biofilms.
  • the biofilm-inhibiting effect of the compounds in Table 2 was specific in that closely related guanidines, compounds I and 2 (differing only the number of methylene groups between the amino or guanidine groups), exhibited little activity.
  • Guanidines discovered to be particularly suitable for use as biofilm inhibitors include guanidines comprising positively charged or ionizable amino or guanidine groups separated by three atoms.
  • exopolysaccharide Norspermidine and a certain class of polyamines interact with the e opolysaccharide by binding to negatively charged or neutral sugars via hydrogen bonding.
  • the class of guanidines disclosed herein also have similar structure-activity relationship where the activity depends on the charge of the amino and guanidine groups and certain spacing between the amino or guanidine groups.
  • Exopolysaccharides often contain negatively charged residues (e.g. uronic acid) or neutral sugars with polar groups (e.g. poly-N-acetylglucosamine).
  • polar groups e.g. poly-N-acetylglucosamine.
  • binding of the guanidines to negatively charged structures of the exopolysaccharide would neutralize the charge and collapse the interwoven meshwork leading to biofilm disruption. Accordingly, it is believed that, the structure and the charge of the amino and guanidine groups of the guanidines disclosed herein allow these certain guanidines to interact with the exopolysaccharide to cause the biofilm to disassemble.
  • the biofilm-inhibiting effect of the guanidines is not limited to B. subi is.
  • the compounds in Table 2 were tested on inhibition of biofilm formation against B. subtilis, which as a natural producer of norspermidine served as a model, and S, aureus as a related pathogenic species with high clinical relevance.
  • MBICs for S. aureus are based on 75% reduction of biofiim formation, b free base, c chloride, ''sulfate salts, *incomplete inhibition.
  • Example 17 and FIG. IB in addition to preventing biofiim. formation, the compounds were also able to trigger disassembly of existing biofilms.
  • the counter ion of the amines and guanidmes also has an effect on activity.
  • the free base of norspermidine was threefold more active than the chloride salt which was again about threefold more active in comparison to the free base for B, subiilis (Table 3). Therefore, free bases of selected compounds were generated and compared with the corresponding salts.
  • subtilis the free base of biofilm inhibiting compounds was more or at least equally active as the salt, while for S. aureus there was no clear trend.
  • Solubility products ( s ; determined as described in Example 18) indicate no correlation with the activity of the compounds and suggest that bioavaiiablitiy and delivery into the biofilm matrix may be relevant parameters (Table 7).
  • the charge of the amino and guanidines groups is also an important factor for inhibiting biofilm formation and triggering biofilm disassembly.
  • the pKa values of selected compounds were determined, as explained in Example 19. The results (shown in Table 7 after Example 19) show that the more effective guanidines have highly ionizable amino and guanidine groups and higher pKa values. This ionizable feature of the guanidines further supports the theory that the guanidines target the exopo lysacc haride .
  • the class of guanidines disclosed herein were developed as highly active agents for the prevention of biofilm formation that also were able to break down existing biofilms and may find application for medicinal purposes.
  • the charge and spacing between positively charged groups are important factors for the mechanism and activity of the compounds.
  • the activity of the disclosed biofilm inhibitors is in the lower range of what has been reported in the literature for compounds that, inhibit, the formation of biofilms without adversely affecting bacterial growth.
  • compositions such as therapeutic or pharmaceutical compositions, comprising one or more guaniclines.
  • the guanidine has at least three amino or guanidine groups separated by three atoms either in a. straight chain or cyclic molecule.
  • the guanidine has Formula (I) or Formula (II),
  • Y is ⁇ ⁇ , ⁇ ). O, S, NH, or NR I 0 ;
  • M is C(RiR.2)-Y-C(R.3R4), meta-phenylene, or 1 ,3-Cs-C 7 cycloalkane, where the meta- phenylene or cycloalkane may be substituted by Rio or have one or more N heteroatoms in its
  • the guanidine is a compound in Table 1.
  • compositions of the present, disclosure include a compound from Table ! , or a combination of one or more compounds from Table I ,
  • guanidines can inhibit biofilm formation in cell populations, and in particular, in biofilm-forming bacteria.
  • guanidines 6 and 7 significantly retard the formation of biofilm in bacteria! colonies such as Staphylococcus aureus and Bacillus subtilis.
  • Guanidines have been demonstrated to reduce biofilm-forming activity by measuring the OD595 of cells that adhere to the surface as a measure of biofilm formation.
  • guanidines can disrupt established biofilms. Even after bacteria have established a biofilm, contact of the biofilm with a solution containing a guanidine results in the dismption and disassembly of the pellicle.
  • guanidines can disrupt pellicles formed by bacterial colonies such as Bacillus subtilis. See, e.g., FIG. 1 and Example 17. Guanidines have been demonstrated to reduce biofilm-forming activity by measuring the OD595 of cells in free medium and compared to the OD595 in the residual pellicle.
  • a hiofilm-related disorder present in a subject in need thereof is treated by administering to the subject a composition having an effective amount of a guanidine, or a. pharmaceutically acceptable salt, or derivative thereof, thereby treating the biofilm-related disorder.
  • a guanidine can be administered at a concentration of 0.1 nM to 100 ⁇ , e.g. , 1 nM to 10 ⁇ , 5 nM to 5 ⁇ , or 10 nM to 1 ⁇ , In other embodiments, a guanidine can be administered at a concentration of about 0.1 nM to about 100 ⁇ , e.g. , about 1 nM to about 10 ⁇ , about 5 nM to about 5 ⁇ , or about 10 nM to about 1 ⁇ .
  • Exemplary guanidines found to be particularly effective in inhibiting or treating biofi!m formation include compounds in Table 1. Those compounds can be used, for example, at concentrations of less than 1 niM, or less than 500 ⁇ . ⁇ or less than 1 0 ⁇ , or at a concentration of 0.1 nM to 100 ⁇ , e.g., 1 M to 10 ⁇ , 5 nM to 5 ⁇ , or 10 nM to 1 ⁇ .
  • Polyamines are small organic compounds found in most cel ls. Polyamines putrescine (5 ,4-diaminobutane), spermidine (1 ,8-diamino-4-azaoctane) and spermine (l , 12-diamino-4,9- diazaoctane) are required in micromolar to miUimolar concentrations to support a wide variety of cellular functions. Depletion of polyamines can result in disruption of cellular functions and can cause cytotoxicity. For example, spermidine and spermine promote biofilm formation in some bacteria. Certain polyamines inhibit biofilm formation and/or disassemble existing biofilms.
  • Polyamines present in conditioned medium from mature biofilms inhibit biofilm formation and trigger the disassembly of existing biofilms. It was discovered that the biofiim- inhibiting effect of norspermidine was specific in that a closely related polyaniine, spermidine (differing only by an extra methylene group), exhibited little activity. Similarly, another polyaniine, norspermine, was also active in biofilm inhibition whereas its close relative spermine (once again, having an extra methylene) was inactive. These discoveries led to the development of the polyamines described herein that can inhibit biofilm formation and trigger biofilm disassembly. Polyamines discovered to be particularly suitable for use as biofilm inhibitors include polyamines comprising propylamine units and whose amino units are ionizable.
  • a guanidine and a D-amino acid can act synergistically to inhibit biofilm formation or trigger biofilm disassembly
  • guanidines and D-amino acids inhibit biofilm formation and trigger biofilm disassembly by different mechanisms.
  • a guanidine can be co administered with an amino acid, and in particular with a D-amino acid, to inhibit biofilm formation or trigger biofilm disassembly.
  • guanidines and D-amino acids work result in synergism between the guanidine and D-amino acid and, in some embodiments, lower amounts of guanidines and D-amino acids are used to inhibit biofilm formation and/or trigger biofilm disassembly.
  • Standard amino acids can exist in either of two optical isomers, called L- or D-amino acids, which are mirror images of each other. While L-amino acids represent, the vast majority of amino acids found in proteins, D-amino acids are components of the peptidoglycan cell walls of bacteria.
  • the D-amino acids described herein are capable of penetrating biofilms on living and non-living surfaces, of preventing the adhesion of bacteria to surfaces and any further build-up of the biofilm, of detaching such biofilm and/or inhibiting the further growth of the biofilm- forming micro-organisms in the biological matrix, or of killing such micro-organisms.
  • D-amino acids are known in the art and can be prepared using known techniques. Exemplary methods include, e.g., those described in U.S. Pub!. No. 20090203091 . D-amino acids are also commercially available (e.g., from Sigma Chemicals, St. Louis, Mo.).
  • Any D-amino acid can be used in the methods described herein, including without limitation D-alanine, D-cysteine, D-aspartic acid, D-glutamic acid, D-phenylalanine, D-histidine, D-isoleucine, D- lysine, D-ieucine, D-methionine, D-asparagine, D-proline, D-glutamine, D- arginine, D-serine, D-threonine, D-valine, D-tryptophan, or D-tyrosine.
  • a D-amino acid can be used alone or in combination with other D-amino acids.
  • D-amino acids are used in combination.
  • D-tyrosine, D-leucine, D-methionine, or D-tryptophan, either alone or in combination are used in the methods described herein.
  • D-tyrosine, D ⁇ pro!ine and D-phenylalanine are used in the methods described herein.
  • a D-amino acid combined with a guamdine or po!yarnme can be administered at a concentration of 0.1 nM to 100 ⁇ , e.g., 1 nM to 10 ⁇ , 5 nM to 5 ⁇ , or 10 nM to 1 ⁇ .
  • a D-amino acid can be administered at a concentration of about 0, 1 nM to about 500 ⁇ , ⁇ , e.g., about 1 nM to about 50 ⁇ , ⁇ , about 5 nM to about 5 ⁇ , ⁇ , or about 10 nM: to about 1 ⁇ .
  • D-tyrosine An exemplary D-amino acid found to be particular ⁇ ' effective in inhibiting or treating biofilm formation when combined with a guanidine or polyamine includes D-tyrosine.
  • D-tyrosine can be used, for example, as concentrations of less than 1 mM, or less than 100 ⁇ or less than 10 ⁇ , or at a concentration of 0.1 nM to 100 ⁇ , e.g., 1 nM to 10 ⁇ , 5 nM to 5 ⁇ , or 10 nM to 1 ⁇ ,
  • D-tyrosine is used in combination with one or more of D-proline and D-phenylalanine. In some embodiments, D-tyrosine is used in combination with one or more of D-leucine, D-tryptophan, and D-metliionine.
  • the combinations of D-tyrosine with one or more of D-proline, D-phenylalanine, D-leucine, D-tryptophan, and D-methionine can be synergistic and can be effective in inhibiting or treating biofilm formation at total D-amino acid concentrations of 10 ⁇ or less, e.g., about 1 nM to about 50 ⁇ , ⁇ , about 5 nM to about 5 ⁇ , or about 10 nM to about 1 ⁇ , ⁇ , or at a concentration of 0.1 nM to 100 ⁇ , e.g., 1 nM to 10 ⁇ , 5 nM to 5 ⁇ , or 10 nM to l ⁇ .
  • the combinations of guanidines or polyamines and D-amino acids are equimoiar. In some embodiments, the combinations of D-amino acids are equimoiar. In other embodiments, the combinations of D-amino acids are not in equimoiar amounts.
  • the composition is essentially free of L-amino acids.
  • the composition comprises less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 1%, less than about 0.5%>, less than about 0,25%, less than about 0.1%, less than about 0.05%, less than about 0.025%, less than about 0.01%, less than about 0.005%, less than about 0.0025%, less than about 0.001%, or less, of L-amino acids.
  • the composition comprises less than 30%, less than 20%, less than 10%, less than 5%, less than 1%, less than 0.5%, less than 0.25%, less than 0.1%, less than 0.05%, less than 0.025%, less than 0.01 %, less than 0.005%, less than 0.0025%, less than 0.001% of L- amino acids.
  • the percentage of L-amino acid is relati ve to the corresponding D-amino acid.
  • a racemic mixture of L- amino acid and D- amino acid contains 50 % L-amino acid. 0105]
  • the composition is essentially free of detergent.
  • the composition comprises, less than about 30 wt %, less than about 20 wt %, less than about 10 wt %, less than about 5 wt %, less than about 1 wt %, less than about 0.5 wt %, less than about 0.25 wt %, less than about 0.1 wt %, less than about 0.05 wt %, less than about 0.025 wt %, less than about, 0.01 wt %, less than about 0.005 wt %, less than about, 0.0025 wt %, less than about 0.001 wt %, or less, of a detergent.
  • the composition comprises, relative to the overall composition, less than about 30 wt %, less than 20 wt %, less than 10 wt %, less than 5 wt %, less than 1 wt %, less than 0.5 wt %, less than 0.25 wt %, less than 0, 1 wt %, less than 0.05 wt %, less than 0.025 wt %, less than 0.01 wt %, less than 0.005 wt %, less than 0.0025 wt %, less than 0.001 wt % of a detergent.
  • the surfactant will interact with the active agent, which could greatly affect the agent's efficacy.
  • it can be necessary to screen agents effectiveness relative to anionic surfactants, cationic surfactants, non-ionic surfactants and zwitter ionic surfactants as a screening to determine if the presence of the surfactant type alters the efficacy. Reducing or eliminating detergents, can increase the efficacy of the compositions and/or reduce formulation complications .
  • the composition is essentially free of both detergent and L- amino acids.
  • Biofilm-associated bacteria are protected from environmental insults, such as antibiotics (Bryers, Biotechnol. Bioeng. 100: 1 (2008)).
  • biofiims age, nutrients become limiting, waste products accumulate, and it is advantageous for the biofilm-associated bacteria to return to a planktonic existence (Karatan, et al, Microbiol. Mol. Biol. Rev. 73:310 (2009)).
  • biofiims have a finite lifetime, characterized by eventual disassembly.
  • Gram-negative bacteria, Gram-positive bacteria, and mycobacteria in addition to other unicellular organisms, can produce biofiims.
  • Bacterial biofiims are surface-attached communities of cells that are encased within an extracellular polysaccharide matrix produced by the colonizing cells.
  • Biofilm development occurs by a series of programmed steps, which include initial attachment to a surface, formation of three-dimensional microcolonies, and the subsequent development of a mature biofilm. The more deeply a ceil is located within a biofilm (such as, the closer the cell is to the solid surface to which the biofilm is attached to, thus being more shielded and protected by the bulk of the biofilm matrix), the more metabolically inactive the cells are.
  • a biofilm also is made up of various and diverse non-cellular components and can include, but are not limited to carbohydrates (simple and complex), lipids, proteins (including polypeptides), and lipid complexes of sugars and proteins
  • a biofilm may include an integrated community of two or more bacteria species (polymicrobic biofiims), or predominantly one specific bacterium.
  • the biofilm can allow bacteria to exist in a dormant state for a certain amount of time until suitable growth conditions arise thus offering the microorganism a selective advantage to ensure its survival.
  • this selection can pose serious threats to human health in that biofilms have been observed to be involved in about 65% of human bacterial infections (Smith, Adv. Dmg Deliv. Rev. 57: 1539-1550 (2005); Hail-Stoodley, et ai, Nat. Rev. Microbiol. 2:95- 108 (2004)).
  • biofilms can also affect a wide variety of biological, medical, and processing operations,
  • biofilms are formed by biofilm-forming bacteria.
  • the bacteria can be a gram negative bacterial species or a gram positive bacterial species.
  • Nonlimiting examples of such bacteria include a member of the genus Actinobacillus (such as Actinobacillus actinomycetemcomitans), a m ember of the genus Acinetobacter (such as Acinetobacter baumannii), a member of the genus Aeromonas, a member of the genus Bordetella (such as Bordetella pertussis, Bordetella bronchiseptica, or Bordetella parapertussis), a member of the genus Brevibacillus, a member of the genus Brucella, a member of the genus Bacteroides (such as Bacteroides fragilis), a member of the genus Burkholderia (such as Burkholderia cepacia or Burkholderia pseudomallei), a member of the genus Borelia (such as Borelia burgdorferi), a member of the genus Bacillus (such as Bacillus anthracis or Bacill
  • a member of the genus Prevotella a member of the genus Plesiomonas (such as Plesiomonas shigelloides), a member of the genus Pseudomonas (such as Pseudomonas aeruginosa), a member of the genus Providencia, a member of the genus Rickettsia (such as Rickettsia rickettsii or Rickettsia typhi), a member of the genus Stenotrophomonas (such as Stenolrophomonas maltophila), a member of the genus Staphylococcus (such as Staphylococcus aureus or Staphylococcus epidermidis), a member of the genus Streptococcus (such as Streptococcus viridans, Streptococcus pyogenes (group A), Streptococcus
  • Salmonella typhimurium Salmonella typhimurium
  • a. member of the genus Serratia such as Serratia marcescens
  • a member of the genus Shigella a member of the genus Spirillum (such as Spirillum minus)
  • a. member of the genus Treponema such as Treponema pallidum
  • a member of the genus Veillonella a member of the genus Vibrio (such as Vibrio cholerae.
  • Vibrio parahemolyticus or Vibrio vulnificus
  • a member of the genus Yersinia such as Yersinia enterocolitica, Yersinia pestis, or Yersinia pseudotuberculosis
  • a member of the genus Xanthomonas such as Xanthomonas maltophilia
  • Bacillus subtilis forms architecturally complex communities on semisolid surfaces and thick pellicles at the air/liquid interface of standing cultures (Lopez, et al , FEMS Microbiol Rev. 33: 152 (2009); Aguilar, et al, Curr. Opin. Microbiol. 10:638 (2007); Vlamakis, et al. , Genes Dev. 22:945 (2008); Branda, et al , Proc. Natl. Acad. Sci. USA 98: 1 1621 (2001)).
  • Bopez, et al FEMS Microbiol Rev. 33: 152 (2009)
  • Aguilar, et al Curr. Opin. Microbiol. 10:638 (2007)
  • Vlamakis, et al. Genes Dev. 22:945 (2008)
  • Branda, et al Proc. Natl. Acad. Sci. USA 98: 1 1621 (2001)
  • subtilis biofilms consist of long chains of cells held together by an extracellular matrix consisting of an exopolysaccharide and amyloid fibers composed of the protein TasA (Branda, el al, Proc. Natl. Acad. Sci. USA 98: 11621 (2001 ); Branda, et al, Mol. Microbiol. 59: 1229 (2006); Romero, et al... Proc. Natl. Acad. Sci. USA (2010, in press)).
  • the protein TasA Branda, el al, Proc. Natl. Acad. Sci. USA 98: 11621 (2001 ); Branda, et al, Mol. Microbiol. 59: 1229 (2006); Romero, et al... Proc. Natl. Acad. Sci. USA (2010, in press)).
  • exopolysaccharide is produced by enzymes encoded by the epsA-0 operon ("eps operon”) and the TasA protein is encoded by the promoter-distal gene of the yqxM-sipW-tasA operon (“yqxM operon”) (Chu, et al, Mol. Microbiol. 59: 1216 (2006)).
  • Biofilm-producing bacteria e.g., a species described herein, can be found in a live subject, in vitro, or on a surface, as described herein.
  • guanidines and polyamines described herein, or a combination of one or more guanidines and one or more polyamines can be formulated so that they are incorporated into carrier structures comprising lipids including, but not limited to, micelles, liposomes, and lipid particles. Such formulations can be even more potent, than the fatty acid salts alone and provide a convenient, way for formulation and application. As described in Examples 15 and 16, various micelle formulations were prepared and tested. Table 5 shows the MBICs for the various norspermidine micelle formulations.
  • the micelles formulated with fatty acids and norspermidine are even more potent than the fatty acid salts of norspermidine alone. Accordingly, formulations comprising carrier structures and one or more of the guanidines or polyamines disclosed herein, or a combination of one or more of the guanidines and one or more of the polyamines disclosed herein, would also have increased potency. Therefore, in some instances, one or more guanidines or polyamines, or a combination of one or more guanidines and one or more polyamines, can be incorporated into a micelle. In some instances, the micelles, including mixed micelles having fatty acids of different chain lengths, can be formulated with fatty acids ranging from C6 to C 2 o in chain length.
  • Carrier structures also include lipid particles comprising one or more charged lipids,
  • a complex of nucleic acid and lipid particles can be referred to as an association complex.
  • An association complex of nucleic acid and lipid particle may be a liposome, a nanoparticle, an ion pair, a lipoplex, or a combination thereof.
  • Lipoplexes are composed of charged lipid biiayers sandwiched between DNA layers, as described, e.g., in Feigner, Scientific American.
  • Lipid particles include, but are not limited to, liposomes. Liposomes may have one or more lipid membranes. Many different types of liposomes exist and many liposomes can be used with the guanidines and polyamines of this disclosure.
  • a liposome can be used with a guanidine or polyamine so long as the liposome can be formulated with the guanidines and/or polyamines to make a stable formulation.
  • the liposomes may be unilamellar or multilamellar.
  • the lipid particles of the present invention may further comprise one or more additional lipids and/or other components such as cholesterol.
  • Other lipids may be included in the liposome compositions of the present invention for a variety of purposes, such as to prevent lipid oxidation or to attach ligands onto the liposome surface. Any of a number of lipids may be present in liposomes of the present invention, including amphipathic, neutral, catio ic, and anionic lipids. Such lipids can be used alone or in combination. Specific examples of additional lipid components that may be present are described below.
  • Additional components that may be present in a lipid particle of the present invention include bilayer stabilizing components such as polyamide oligomers (see, e.g., U.S. Patent No. 6,320,017), peptides, proteins, detergents, lipid-derivatives, such as PEG coupled to
  • the lipid particles include one or more of a second amino lipid or charged lipid, a neutral lipid, and a sterol.
  • Neutral lipids when present in the lipid particle, can be any of a number of lipid species which exist either in an uncharged or neutral zwitterionic form at physiological pH.
  • lipids include, for example phosphocholines (PC), phospbatidylethanolarmnes (PE), phosphatidylserines (PS), cardiolipins, diacylphosphatidylcholine,
  • the neutral lipid component is a lipid having two acyl groups, (i.e., diacylphosphatidylcholine and diacylphosphatidylethanolamine).
  • Lipids having a variety of acyl chain groups of varying chain length and degree of saturation are available or may be isolated or synthesized by well-known techniques.
  • lipids containing saturated fatty acids with carbon chain lengths in the range of Cio to C 2 0 are used, in another group of embodiments, lipids with mono or ⁇ unsaturated fatty acids with carbon chain lengths in the range of C 10 to C 20 are used. Additionally, lipids having mixtures of saturated and unsaturated fatty acid chains can be used.
  • the neutral lipids used in the present invention are DOPE, DSPC, POPC, DPPC or any related
  • the neutral lipids useful in the present invention may also be composed of sphingomyelin, dihydrosphingomyeline, or phospholipids with other head gro ps, such as serine and inositol.
  • the sterol component of the lipid mixture when present, can be any of those sterols conventionally used in the field of liposome, lipid vesicle or lipid particle preparation.
  • An exemplary sterol is cholesterol.
  • protonatable lipids which carry a net positive charge at about physiological pH, in addition to those specifically described above, may also be included in lipid particles of the present invention.
  • Such protonatable lipids include, but are not limited to, N,N ⁇ dioleyl ⁇ N,N- dimethylammonium chloride (“DODAC”); N-(2,3- dioleyloxy)propyl-N,N-N-triethylammonium chloride (“DOTMA”); N,N-distearyl-N,N- dimethvlammonium bromide (“DDAB”); N-(2,3- dioleoyloxy)propyi)-N,N,N- trimethylammonium chloride (“DOTAP”); l,2-Dioieyloxy-3- trimethylaminopropane chloride salt (“DOTAP.
  • DODAC N,N ⁇ dioleyl ⁇ N,N- dimethylammonium chloride
  • DOTMA N-(2,3- di
  • lipids can be used, including, but not limited to, LIPOFECTiN (including DOTMA and DOPE, available from GiBCO/BRL), and LIPOFECTAMINE (comprising DOSPA and DOPE, available from GIBCO/BRL).
  • LIPOFECTiN including DOTMA and DOPE, available from GiBCO/BRL
  • LIPOFECTAMINE comprising DOSPA and DOPE, available from GIBCO/BRL
  • Anionic lipids suitable for use in lipid particles include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N- dodecanoyl phosphatidylethanoloamine, N-succinyl phosphatidylethanolamine, N- glutaryl phosphatidylethanolamine, lysylphosphatidylgiycerol, and other anionic modifying groups joined to neutral lipids.
  • amphipathic lipids are included in lipid particles of the present invention.
  • “Amphipathic lipids” refer to any suitable material, wherein the hydrophobic portion of the lipid material orients into a hydrophobic phase, while the hydrophilic portion orients toward the aqueous phase.
  • Such compounds include, but are not limited to,
  • phospholipids include sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidyiserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatdylcholine,
  • lysophosphatidylcholine lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, or dilinoleoylphosphatidylcholine.
  • Other phosphorus-lacking compounds such as sphingolipids, glycosphingolipid families, diacylgiycerols, and ⁇ -acyloxyacids, can also be used. Additionally, such amphipathic lipids can be readily mixed with other lipids, such as triglycerides and sterols.
  • the lipid particle can include a lipid selected to reduce aggregation of lipid particles during formation, which may result from steric stabilization of particles which prevents charge-induced aggregation during formation.
  • PEG polyethylene glycol
  • PAO polyarmde oligomers
  • compositions of the invention ATTA-lipids are described, e.g. , in U.S. Patent No, 6,320,017, and PEG-lipid conjugates are described, e.g., in U.S. Patent Nos. 5,820,873, 5,534,499 and 5,885,613.
  • concentration of the lipid component selected to reduce aggregation is about 1 to 15% (by mole percent of lipids).
  • PEG-modified lipids (or lipid- polyoxyethylene conjugates) that are useful in the present invention can have a variety of "anchoring" lipid portions to secure the PEG portion to the surface of the lipid vesicle.
  • PEG- modified lipids examples include PEG-modified phosphatidylethanolamine and phosphatide acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20) which are described in USSN 08/486,214, PEG-modified dialkylamines and PEG-modified 1,2- diacyloxypropan-3-amines. Particularly preferred are PEG-modified diacylgiycerols and dialkylgiycerols. [0129] In embodiments where a sterically-large moiety such as PEG or ATTA are conjugated to a lipid anchor, the selection of the lipid anchor depends on what type of association the conjugate is to have with the lipid particle. It is understood that mPEG
  • the PEG-modified lipid may be formulated to be rapidly lost from the nucleic acid-lipid particle in vivo and hence the PEG-modified lipid will possess relatively short lipid anchors.
  • t e nucleic acid-lipid particle may exhibit a longer plasma circulation lifetime and hence the PEG-modified lipid will possess relatively longer lipid anchors.
  • Micelles for use with the guanidines and polyamines described herein may be formed by mixing an aqueous solution of one or more guanidines, one or more polyamines, or a combination of one or more guanidines and one or more polyamines, and micelle forming compounds.
  • Micelle forming compounds to form micelles for use with the guanidines and polyamines of this disclosure include fatty acids having chain length from C 6 to C20. The micelle forming fatty acids may be added at the same time or after the guanidine or poly amine.
  • Emulsifying agents such as bile salts (e.g., sodium taurocholate) may be added in a buffered solution to formulate micelles.
  • Micelles will form when the concentration of the fatty acids is greater than the critical micelle concentration (CMC). Once the CMC is met, the fatty acids will form a micelle that contains a guanidine or polyamine.
  • CMC critical micelle concentration
  • the fatty acids will form a micelle that contains a guanidine or polyamine.
  • a guanidine or polyamine, or a combination is incorporated into a liposome, the aqueous portion within the liposome contains the guanidine or polyamine, or combination.
  • Commercially available liposomes can used or liposomes can be prepared using known techniques.
  • liposomes can be prepared using dipalmitoyl phosphatidyl choline (DPPC) or dipalmitoyl phosphatidyl glycerol (DPPG) and cholesterol in buffered aqueous solutions followed by extrusion.
  • DPPC dipalmitoyl phosphatidyl choline
  • DPPG dipalmitoyl phosphatidyl glycerol
  • cholesterol in buffered aqueous solutions followed by extrusion.
  • the guanidmes and polyarchies may be added during or after the liposome preparation process.
  • guanidmes and D-amino acids described herein can be incorporated into pharmaceutical compositions.
  • the guanidmes and D-amino acids can be incorporated into pharmaceutical compositions as pharmaceutically acceptable salts or derivatives.
  • Such compositions typically include a guanidine, or a guanidine and a D-amino acid, and a pharmaceutically acceptable carrier.
  • a "pharmaceutically acceptable carrier” means a carrier that can be administered to a subject together with a guanidine or with a guanidine and a D-amino acid described herein, which does not destroy the pharmacological activity thereof.
  • Pharmaceutically acceptable carriers include, e.g., solvents, binders, dispersion media, coatings, preservatives, colorants, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.
  • salts includes, but is not limited to, water- soluble and water-insoluble salts, such as the acetate, amsonate (4,4-diarninostilbene-2,2- disulfonate), benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camsyiate, carbonate, chloride, citrate, clavulariate, ⁇ hydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate
  • phosphate/diphosphate picrate, polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate, suhacetate, succinate, sulfate, sulfosaliculate, suxamate, tannate, tartrate, teoelate, tosylate, triethiodide, and valerate salts.
  • the D-amino acids may also be in the form of esters or derivatives.
  • suitable esters include formates, acetates, propionates, butyrates, isobutyrates, pentanoates, crotonates, and benzoates.
  • Some pharmaceutically acceptable derivatives include a chemical group which increases aqueous solubility.
  • Non-limiting examples of pharmaceutically acceptable carriers that can be used include poiy(ethylene-co-vinyl acetate), PVA, partially hydrolyzed poly(ethylene-co-vinyl acetate), poly(ethylene-co-vinyi acetate-co-vinyl alcohol), a cross-linked poly(ethylene-co-vinyl acetate), a cross-linked partially hydrolyzed poly(ethylene-co- vinyl acetate), a cross-linked poly(ethylene-co-vinyl acetate-co-vinyl alcohol), poly-D,L-lactic acid, poly-L-lactic acid, polyglycolic acid, PGA , copolymers of lactic acid and glycolic acid (PLGA), polycaprolactone, polyvalerolactone, poly (anhydrides), copolymers of polycaprolactone with polyethylene glycol, copolymers of polylactic acid with polyethylene glycol, polyethylene glycol; and combinations and blends thereof.
  • PVA partially hydrolyzed poly(ethylene-co-vin
  • Other carriers include, e.g., an aqueous gelatin, an aqueous protein, a polymeric carrier, a cross-linking agent, or a combination thereof.
  • the carrier is a matrix.
  • the carrier includes water, a pharmaceutically acceptable buffer salt, a pharmaceutically acceptable buffer solution, a pharmaceutically acceptable antioxidant, ascorbic acid, one or more low molecular weight pharmaceutically acceptable polypeptides, a peptide comprising about 2 to about 10 amino acid residues, one or more pharmaceutically acceptable proteins, one or more pharmaceutically acceptable amino acids, an essential-to-human amino acid, one or more pharmaceutically acceptable carbohydrates, one or more pharmaceuticaily aceeptable carbohydrate-derived materials, a non-reducing sugar, glucose, sucrose, sorbitol, trehalose, mannitol, maltodextrin, dextrins, cyclodextrin, a pharmaceutically acceptable chelating agent, EDTA, DTPA, a
  • a pharmaceutical composition containing a guanidine, or a combination of a guanidine and a D-amino acid can be formulated to be compatible with its intended route of administration as known by those of ordinary skill in the art.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, vaginal and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition can be sterile and can be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be accomplished by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin (see, e.g., Remington: The Science and Practice of
  • Sterile injectable solutions can be prepared by incorporating a guanidine, or combination of a guanidine and a D-amino acid, in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the methods of preparation include, without limitation, vacuum drying and freeze- drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier.
  • a guanidine, or a combination of a guanidine and a D-amino a cid can be incorporated with excipients and used in the form of tablets, pills, troches, or capsules, e.g. , gelatin capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • a guanidine for administration by inhalation, can be delivered in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, but, are not limited to, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accompl ished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into, e.g., ointments, salves, gels, or creams as generally known in the art.
  • a guanidine for treatment of acute or chronic wounds, can be formulated as a dressing, a wash solution, gel, or a synthetic tissue.
  • a biofilm can form on an oral surface (such as teeth, tongue, back of throat, and the like). These bio films can be associated with day-to-day bacterial activity of natural flora located in such environments, but can also be associated with oral-related disease(s), such as periodontal disease (for example, gingivitis or periodontitis), breath malodor, or dental caries.
  • periodontitis a common form of periodontal disease, is believed to be caused by a small group of Gram-negative bacteria present on the tooth root surfaces as biofilms, in particular,
  • Porphyromonas gingivalis Bacieroides forsyihus and Actinobacillus actinomycetemcomitans, with the latter found mostly in cases of juvenile periodontitis.
  • Other bacteria which may be involved in periodontal disease include T. denticola, T. socranskii, F. nucleatum, and P.
  • the guanidine, or combination of a guanidine and D-amino acid, for use on oral surfaces can be formulated as a gum, paste (such as toothpaste), which can then be directly applied to the biofilm of such a surface in a subject.
  • the paste formulation can further comprise an abrasive.
  • a guanidine, or combination of a guanidine and D-amino acid can also exist as a gel formulation or in liquid formulation.
  • the guanidine, or combination of a guanidine and D- amino acid can be formulated as a mouthwash that can directly come into contact with the biofilm on the oral surface of a. subject.
  • a guanidine, or combination of a. guanidine and D-amino acid can be formulated as a polymer film or platelet (e.g., as a slow-release formulation) for treating or preventing oral conditions.
  • the guanidine, or combination of a guanidine and D-amino acid , of the present invention may be used for adjunctive antimicrobial therapy for periodontitis and applied directly to a tooth or between teeth in the form of a chip.
  • the oral care compositions of the present invention may be in various forms including therapeutic rinses, especially mouth rinses; dentifrices such as toothpastes, tooth gels, and tooth powders; non-abrasive gels; mouth sprays; mousse; foams; chewing gums, lozenges and breath mints; drinking water additives; dental solutions and irrigation fluids; and dental implements such as dental floss and tape.
  • the dental implement can be impregnated fibers including dental floss or tape, chips, strips, films and polymer fibers.
  • an oral composition can contain from about 0.01 % to about 15 % by weight, e.g., 0.01 % to 15 % by weight, based on the total weight of the composition, of one or more guanidine, or combination of a guanidine and D-amino acid, and orally tolerable adjuvants.
  • an oral composition includes 10 % by weight, sorbitol, 10 % by weight glycerol, 1 5 % by weight ethanol, 15 % by weight propylene glycol, 0.5 % by weight sodium lauryl sulfate, 0.25 % by weight sodium methylcocyl taurate, 0.25 % by weight polyoxypropylene/polyoxyethylene block copolymer, 0.50 % by weight peppermint flavouring, 0.1 to 0.5 % by weight of one or more guanidine, or combination of a guanidine and D-amino acid, and 48.6 % by weight water.
  • An oral composition can be, for example, in the form of a gel, a paste, a cream or an aqueous preparation (mouthwash).
  • the oral composition can also comprise compounds that release fluoride ions which are effective against the formation of caries, for example inorganic fluoride salts, e.g. sodium, potassium, ammonium or calcium fluoride, or organic fluoride salts, e.g. amine fluorides, which are known under the trade name OLAFLUOR.
  • Oral compositions can further comprise compounds known in the art to be "orally acceptable carriers," which as used herein means conventional additives in oral care compositions including but not limited to fluoride ion sources, anti-calculus or anti-tartar agents, buffers, abrasives such as silica, bleaching agents such as peroxide sources, alkali metal bicarbonate salts, thickening materials, humectants, water, surfactants, titanium dioxide, flavor system, sweetening agents, xylitol, coloring agents, and mixtures thereof.
  • fluoride ion sources such as anti-calculus or anti-tartar agents
  • buffers such as silica
  • bleaching agents such as peroxide sources, alkali metal bicarbonate salts
  • thickening materials such as peroxide sources, alkali metal bicarbonate salts
  • humectants water
  • surfactants titanium dioxide
  • sweetening agents xylitol
  • coloring agents and mixtures thereof.
  • these carriers may be included at levels typically from about 50% to about 99%, preferably from about 70% to about 98%, and more preferably from about 90% to about 95%, by weight of the oral composition.
  • the choice of a carrier to be used is basically determined by the way the composition is to be introduced into the oral cavity.
  • the oral compositions are in the form of dentifrices, such as toothpastes, tooth gels and tooth powders.
  • Components of such toothpaste and tooth gels general ly include one or more of a dental abrasive (from about 6% to about 50%), a surfactant (from about 0.5% to about 10%), a thickening agent (from about 0.1 % to about 5%), a humectant (from about 10% to about 55%), a flavoring agent (from about 0.04% to about 2%), a sweetening agent (from about 0.1 % to about 3%), a coloring agent (from about 0.01% to about 0.5%) and water (from about 2% to about 45%).
  • a dental abrasive from about 6% to about 50%
  • a surfactant from about 0.5% to about 10%
  • a thickening agent from about 0.1 % to about 5%
  • a humectant from about 10% to about 55%)
  • a flavoring agent from about 0.04% to about 2%
  • a sweetening agent from about 0.1 % to about 3%
  • a coloring agent from about 0.01%
  • Such toothpaste or tooth gel may also include one or more of an anticaries agent (from about 0,05% to about 0.3% as fluoride ion) and an anticalcuius agent (from about 0.1% to about 13%). Tooth powders contain substantially ail non-liquid
  • compositions are liquid products, including mouthwashes or rinses, mouth sprays, dental solutions and irrigation fluids.
  • Components of such mouthwashes and mouth sprays typically include one or more of water (from about 45% to about 95%), ethanol (from about 0% to about 25%), a humectant (from about 0% to about 50%), a surfactant (from about 0.01% to about 7%), a flavoring agent (from about 0.04% to about 2%), a sweetening agent (from about 0.1% to about 3%), and a coloring agent (from about 0.001% to about 0.5%).
  • Such mouthwashes and mouth sprays may also include one or more of an anticaries agent (from about 0.05% to about 0.3% as fluoride ion) and an anticalculus agent (from about 0.1 % to about 3%).
  • Components of dental solutions generally include one or more of water (from about 90% to about 99%), preservative (from about 0,01 % to about 0.5%), thickening agent (from 0% to about 5%), flavoring agent (from about 0.04% to about 2%), sweetening agent (from about 0.1% to about 3%), and surfactant (from 0% to about 5%).
  • compositions containing a guanidine, or combination of a guanidine and D-amino acid can also be prepared in the form of suppositories (e.g. , with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g. , with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the composition is essentially free of detergent.
  • a detergent can contribute to the toxicity of a composition.
  • the composition comprises less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 1 %, less than about 0.5%, less than about 0.25%, less than about 0.1 %, less than about 0.05%, less than about 0.025%, less than about 0.01%, less than about 0.005%, less than about 0.0025%, less than about 0,001 %, or less, of a detergent, e.g., less than 30%, less than 20%, less than 10%, less than 5%, less than 1 %, less than 0.5%, less than 0.25%, less than about 0.1 %, less than 0.05%, less than 0.025%, less than 0.01 %, less than 0.005%, less than about 0.0025%, less than 0.001%, of a detergent.
  • compositions can be prepared with a carrier that protects the guanidine, or combination of a guanidine and D-amino acid, against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems (as described, e.g., in Tan, et ah, Pharm, Res. 24:2297-2308, 2007).
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate,
  • polyanhydrides polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations are apparent to those skilled in the art.
  • the materials can also be obtained commercially (e.g., from Alza Corp., Mountain View, CA).
  • Liposomal suspensions (including liposomes targeted to particular cells with monoclonal antibodies to cell surface antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, e.g. , as described in U.S. Pat. No.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. , for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effecti ve in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED50. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to normal cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies generally within a range of circulating concentrations that include the EL1 ⁇ 2 with little or no toxicity.
  • the dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the route of administration utilized for any compound used in the methods described herein, the
  • therapeutically effective dose can be estimated initially from cell culture assays.
  • A. dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 5 .3 (i.e., the concentration of the test compound which achie ves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography. Information for preparing and testing such compositions are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy, 21st edition, Lippincott Williams & Wilkins, Gennaro, ed. (2006)).
  • about 0.0005 uM guanidine to about 50 uM guanidine is administered, e.g., about 0.001 uM guanidine to about 25 ⁇ guanidine, about 0.002 uM guanidine to about 10 uM guanidine, about 0.003 uM guanidine to about 5 uM guanidine, about 0.004 uM guanidine to about 1 uM guanidine, about 0.005 uM guanidine to about 0.5 uM guanidine, about 0.05 ⁇ , ⁇ guanidine to about 0.1 ⁇ , ⁇ guanidine, or about 0.02 ⁇ , ⁇ guanidine to about 0.1 ⁇ guanidine, e.g., .0005 uM guanidine to 50 ⁇ guanidine is administered, 0.005 ⁇ , ⁇ guanidine to 25 ⁇ guanidine, 0.002 ⁇ guanidine to 10 ⁇
  • ⁇ D-amino acid to about 50 ⁇ , ⁇ D- amino acid is administered, e.g., about 0.001 ⁇ D-amino acid to about 25 ⁇ D-amino acid, about 0.002 ⁇ D-amino acid to about 10 ⁇ D-amino acid, about 0.003 ⁇ D-amino acid to about 5 ⁇ D-amino acid, about 0.004 ⁇ D-amino acid to about 1 ⁇ D-amino acid, about 0.005 ⁇ D-amino acid to about 0.5 ⁇ D-amino acid, about, 0.01 ⁇ D-amino acid to about 0.1 ⁇ D-amino acid, or about 0.02 ⁇ D-amino acid to about 0.1 ⁇ D-amino acid, e.g., .0005 ⁇ , ⁇ D-amino acid to 50 ⁇ D-amino acid is administered, 0.001 ⁇ ,
  • a D-amino acid is administered at nanomolar concentrations, e.g., at about 5 nM, at about 10 nM, at about 15 nM, at about 20 nM, at about 25 nM, at about 30 uM, at about 50 nM, or more, or preferably at 5 nM, at 10 nM, at 15 nM, at 20 nM, at 25 nM, at 30 nM, or at 50 nm.
  • a therapeutically effective amount or dosage of a guanidine, or combination of a guanidine and D-amino acid can range from about, 0.001 mg/kg body weight, to about 100 mg/kg body weight, e.g., from about 0.01 mg/kg body weight to about 50 mg/kg body weight, from about 0.025 mg/kg body weight to about 25 mg/kg body weight, from about 0.1 mg/kg body weight to about 20 mg/kg body weight, from about 0.25 mg/kg body weight to about 20 mg/kg body weight, from about 0,5 mg/kg body weight to about 20 mg/kg body weight, from about 0.5 mg/kg body weight to about 10 mg/kg body weight, from about 1 mg kg body weight to about 10 mg/kg body weight, or about 5 mg/kg body weight, or preferably 0.001 mg/kg body weight to 100 nig/kg body weight, e.g., from 0.01 mg/kg body weight to 50 mg/kg body weight, from 0.025 mg/kg body weight
  • a physician will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present.
  • treatment of a subject with a therapeutically effective amount of a guanidine, or combination of a guanidine and D-amino acid can include a single treatment or a series of treatments.
  • a subject is treated with a guanidine, or combination of a guanidine and D-amino acid, in the range of between about 0.06 mg to about 120 nig, one time per week for between about 1 to 10 weeks, alternatively between 2 to 8 w r eeks, between about 3 to 7 weeks, or for about 4, 5, or 6 weeks, or preferably between 0.06 mg to 120 mg, one time per week for between 1 to 10 weeks, alternatively between 2 to 8 weeks, between 3 to 7 weeks, or for 4, 5, or 6 weeks.
  • the effective dosage of a guanidine, or combination of a guanidine and D-amino acid, used for treatment may increase or decrease over the course of a particular treatment.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • instructions for administration A person of ordinary skill in the art will appreciate that the pharmaceutical compositions described herein can be formulated as single-dose vials.
  • Treatment of a subject with a therapeutically effecti ve amount of a guanidine-, or combination of a guanidine and D-amino acid-, containing pharmaceutical composition described herein can be a single treatment, continuous treatment, or a series of treatments divided into multiple doses.
  • the treatment can include a single administration, continuous administration, or periodic administration over one or more years. Chronic, long-term administration can be indicated in some cases.
  • each formulation is administered in an amount sufficient to suppress or reduce or eliminate a deleterious effect or a symptom of a. biofilm-related disorder or condition described herein.
  • Guanidines, or combinations of guanidines and D-amino acids are suitable as antibiofilm active substances in personal care preparations, for example shampoos, bath additives, hair care preparations, liquid and solid soaps (based on synthetic surfactants and salts of saturated and/or unsaturated fatty acids), lotions and creams, deodorants, other aqueous or alcoholic solutions, e.g. cleansing solutions for the skin, moist cleaning cloths, oils or powders.
  • Fropionibacterium acnes which is the predominant microorganism occurring in acne, may reside in biofilms.
  • guanidines, or combinations of guanidines and D-amino acids are particularly suitable for personal care compositions for use in controlling acne.
  • the invention accordingly relates also to personal care preparations comprising one or more guanidine, or a combination of a guanidine and D-amino acid, described herein and cosmetically tolerable carriers or adjuvants,
  • guanidines, or combinations of guanidines and D-amino acids, described herein are also suitable for imparting antibiofilm properties to a range of formulations used in personal care.
  • Personal care preparations can contain from about, 0.01% to about 15 % by weight, for example, from about 0.1% to about 10 % by weight, or 0.01% to 15 % by weight, for example, from 0.1 % to 10 % by weight, based on the total weight of the preparation, of one or more guanidine, or combination of a guanidine and D-amino acid, and cosmetically tolerable adjuvants.
  • such preparation can include, in addition to one or more guanidines, or combinations of guanidines and D-amino acids, further constituents, for example sequestering agents, colourings, perfume oils, thickening or solidifying agents (consistency regulators), emollients, UV-absorbers, skin protective agents, antioxidants, additives that improve the mechanical properties, such as dicarboxylic acids and/or aluminium, zinc, calcium or magnesium salts of C14-C22 fatty acids, and, optionally,
  • the anti-acne composition comprising a guanidine, or a combination of a guanidine and D-amino acid, can further comprise at least one antimicrobial agent.
  • the antimicrobial agent is an antibiotic.
  • the antibiotic may be selected from, the group consisting of tobramycin, clindamycin, ciprofloxacin, tetracyclines, rifampin, triclosan, oxfloxacin, macrolides, penicillins, cephalosporins, amoxicillia ⁇ lavulante,
  • the present invention provides a method for controlling acne, comprising administering to a subject afflicted with acne an effective amount of an anti-acne composition comprising one or more guanidines, or combinations of guanidines and D-amino acids, wherein the amount of the guanidine, or combination of a guanidine and D-amino acid, in the anti-acne composition is sufficient to prevent, reduce, inhibit or remove a biofilm.
  • Personal care preparations can be in the form of a water-in-oil or oil-in-water emulsion, an alcoholic or alcohol-containing formulation, a vesicular dispersion of an ionic or non-ionic ampiphilic lipid, a gel, a solid stick or an aerosol formulation.
  • the cosmetically tolerable adjuvant contains preferably from about 5 % to about 50 % of an oil phase, from about 5 % to about 20 % of an emulsifier and from about 30 % to 90 % water, or 5 % to 50 % of an oil phase, from 5 % to 20 % of an emulsifier and from 30 % to 90 % water.
  • the oil phase can comprise any oil suitable for cosmetic formulations, for example one or more hydrocarbon oils, a wax, a natural oil, a silicone oil, a fatty acid ester or a fatty alcohol.
  • Preferred mono- or poly-ols are ethanol, isopropanol, propylene glycol, hexylene glycol, glycerol and sorbitol.
  • Cosmetic formulations described herein are used in various fields. Such preparations include, without limitation, for example: skin-care preparations, e.g. skin-washing and cleansing preparations in the form of tablet-form or liquid soaps, synthetic detergents or washing pastes, bath preparations, e.g. liquid (foam baths, milks, shower preparations) or solid bath preparations, e.g. bath cubes and bath salts; skin-care preparations, e.g. skin emulsions, multi-emulsions or skin oils; cosmetic personal care preparations, e.g. facial make-up in the form of day creams or powder creams, face powder (loose or pressed), rouge or cream makeup, eye-care preparations, e.g.
  • skin-care preparations e.g. skin-washing and cleansing preparations in the form of tablet-form or liquid soaps, synthetic detergents or washing pastes
  • bath preparations e.g. liquid (foam baths, milks, shower preparations) or solid bath preparation
  • eye shadow preparations mascaras, eyeliners, eye creams or eye-fix creams
  • lip-care preparations e.g. lipsticks, lip gloss, lip contour pencils, nail-care preparations, such as nail varnish, nail varnish removers, nail hardeners or cuticle removers
  • intimate hygiene preparations e.g. intimate washing lotions or intimate sprays
  • foot-care preparations e.g.
  • foot baths foot powders, foot creams or foot balsams, special deodorants and antiperspirants or callus-removing preparations
  • light-protective preparations such as sun milks, lotions, creams or oils, sunblocks or tropicals, pre-tanning preparations or after-sun preparations
  • skin-tanning preparations e.g. self-tanning creams
  • depigmenting preparations e.g. preparations for bleaching the skin or skin- lightening preparations
  • insect-repellents e.g.
  • insect-repellent oils lotions, sprays or sticks
  • deodorants such as deodorant sprays, pump-action sprays, deodorant gels, sticks or roll-ons
  • antiperspirants e.g. antiperspirant sticks, creams or rol l-ons
  • preparations for cleansing and caring for blemished skin e.g. synthetic detergents (solid or liquid), peeling or scrub preparations or peeling masks
  • shaving preparations e.g.
  • fragrance preparations e.g. fragrances (eau de Cologne, eau de toilette, eau de perfume, perfume de toilette, perfume), perfume oils or perfume creams
  • dental care denture-care and mouth-care preparations, e.g. toothpastes, gel toothpastes, tooth powders, mouthwash concentrates, anti-plaque mouthwashes, denture cleaners or denture fixatives
  • cosmetic hair-treatment preparations e.g. hair-washing preparations in the form of shampoos and conditioners, hair-care preparations, e.g.
  • pretreatment preparations hair tonics, styling creams, styling gels, pomades, hair rinses, treatment packs, intensive hair treatments, hair-structuring preparations, e.g. hair- waving preparations for permanent waves (hot wave, mild wave, cold wave), hair- straightening preparations, liquid hair-setting preparations, hair foams, hairsprays, bleaching preparations, e.g. hydrogen peroxide solutions, lightening shampoos, bleaching creams, bleaching powders, bleaching pastes or oils, temporary, semipermanent or permanent hair colorants, preparations containing self-oxidising dyes, or natural hair colorants, such as henna or camomile.
  • hair-structuring preparations e.g. hair- waving preparations for permanent waves (hot wave, mild wave, cold wave), hair- straightening preparations, liquid hair-setting preparations, hair foams, hairsprays, bleaching preparations, e.g. hydrogen peroxide solutions, lightening shampoos, bleaching creams, bleaching powder
  • An exemplary soap has, for example, the following composition: 0.01 to 5 % by weight of one or more guanidines, or combinations of guanidines and D-amino acids, 0.3 to 1 % by weight titanium dioxide, 1 to 10 % by weight stearic acid, soap base ad 100 %, e.g. a sodium salt of tallow fatty acid or coconut fatty acid, or glycerol.
  • An exemplary shampoo has, for example, the following composition: 0,01 to 5 % by weight of one or more guanidines, or combination of a guanidine and D-amino acid, 12.0 % by weight sodium laureth-2-sulfate, 4.0 % by weight cocamidopropyl betaine, 3.0 % by weight NaCl and water ad 100 %.
  • An exemplary deodorant has, for example, the following composition: 0.01 to 5 % by weight of one or more guanidines, or combination of a guanidine and D-amino acid, 60 % by weight ethanol, 0.3 % by weight perfume oil, and water ad 100 %.
  • a pharmaceutical composition comprising a guanidine, or combination of a guanidine and D-amino acid, is administered to prevent or reduce biofilm formation on a biologically relevant surface or substrate.
  • a biofilm can affect, the surface of a lung (such as the lung of a subject with pneumonia, cystic fibrosis, or COPD), such as epithelial cells of the lung,
  • the surface is a biologically relevant surface is a surface that is likely to contact a biological fluid, e.g., a liquid component of a subject such as blood, serum, sputum, lacrimal secretions, semen, urine, vaginal secretions, and tissue samples and the like.
  • a biological fluid e.g., a liquid component of a subject such as blood, serum, sputum, lacrimal secretions, semen, urine, vaginal secretions, and tissue samples and the like.
  • the biologically relevant surface can be a component of a medical device, instrument, or implant.
  • Nonlimiting examples include clamps, forceps, scissors, skin hooks, tubing (such as endotracheal or gastrointestinal tubes), needles, retractors, scalers, drills, chisels, rasps, saws, catheters including indwelling catheter (such as urinary catheters, vascular catheters, peritoneal dialysis catheter, central venous catheters), catheter components (such as needles, Leur-Lok connectors, needleless connectors), orthopedic devices, artificial heart valves, prosthetic joints, voice prostheses, stents, shunts, pacemakers, surgical pins, respirators, ventilators, and endoscopes.
  • the present invention is particularly well-suited to substantially reduce the risk of biofilm accumulation on the surfaces of a medical device adapted for prolonged term
  • a medical device utilized for a prolonged period of time may implanted for a period longer than 24 hours, such as a week.
  • a subject can be administered a guanidine, or combination of a guanidine and D-amino acid, prior to, during, or after implantation/insertion of a medical device, catheter, stent, prosthesis, and the like, or application of a wound dressing.
  • the wound dressing includes an antimicrobial, such as silver. Treatment before or after implantation can take place immediately before or after the implantation or several hours before or after implantation, or at, a time or times that the skilled physician deems appropriate.
  • a guanidine, or combination of a guanidine and D-amino acid can be applied to a surface by any known means, such as by covering, coating, contacting, associating with, filling, or loading the surface with a therapeutic amount of a guanidine, or combination of a guanidine and D-amino acid.
  • a guanidine, or combination of a guanidine and D- amino acid is directly affixing to a surface by either spraying the surface with a
  • polymer/guanidine, or a polymer/combination of a guanidine and D-amino acid, film by dipping the surface into a polymer/guanidine solution, or a polymer/combination of a guanidine and D- amino acid solution, or by other covalent or noncovalent means.
  • the surface is coated with a substance (such as a hydrogel) that absorbs the guanidine, or combination of a guanidine and D-amino acid.
  • the composition can be a coating or a film.
  • one or more guanidine, or combination of a guanidine and D-amino acid, described herein can be part of a composition which also comprises a binder.
  • the binder may be any polymer or oligomer compatible with the present antibiofilms.
  • the binder may be in the form of a polymer or oligomer prior to preparation of the antibiofilm composition, or may form by polymerization during or after preparation, including after application to the substrate. In certain applications, such as certain coating applications, it will be desirable to crosslink the oligomer or polymer of the antibiofiim composition after application.
  • binder includes materials such as glycols, oils, waxes and surfactants commercially used in the pharmaceutical and personal care industries. It is preferred that materials that are Generally Regarded as Safe (G.R.A.S.) be used.
  • the binder is the thermoplastic polymer matrix used to prepare the film.
  • the composition is a coating, it may be applied as a liquid solution or suspension, a paste, gel, oil or the coating composition may be a solid, for example a powder coating which is subsequently cured by heat, UV light or other method.
  • the binder can be comprised of any polymer used in coating formulations or film preparation.
  • the binder is a thermoset, thermoplastic, elastomeric, inherently crosslinkecl or crosslmked polymer.
  • Thermoset, thermoplastic, elastomeric, inherently crosslinked or crosslmked polymers include polyolefin, polyamide, polyurethane, polyacrylate, polyaerylamide, polycarbonate, polystyrene, polyvinyl acetates, polyvinyl alcohols, polyester, halogenated vinyl polymers such as PVC, natural and synthetic rubbers, alkyd resins, epoxy resins, unsaturated polyesters, unsaturated polyamides, polyimides, silicon containing and carbamate polymers, ftuorraated polymers, crosslinkable acrylic resins derived from substituted acrylic esters, e.g. from epoxy acrylates, urethane acrylates or polyester acrylates.
  • the polymers may also be blends and copolymers of the preceding chemistries.
  • Biocompatible coating polymers such as, poly[-alkoxyalkanoate-co-3- hydroxyalkenoate] (PHAE) polyesters, Geiger, et. al, Polymer Bulletin 52, 65-70 (2004), can also serve as binders in the present invention.
  • Alkyd resins, polyesters, polyurethanes, epoxy resins, silicone containing polymers, polyacrylates, polyacrylamides, fluorinated polymers and polymers of vinyl acetate, vinyl alcohol and vinyl amine are non-limiting examples of common coating binders useful in the present invention.
  • Other known coating binders are part of the present disclosure.
  • Coatings can be crosslinked with, for example, melamine resins, urea resins, isocyanates, isocyanurates, polyisocyanates, epoxy resins, anhydrides, poly acids and amines, with or without accelerators.
  • the compositions described herein can be, for example, a coating applied to a surface which is exposed to conditions favorable for bi ⁇ accumulation. The presence of one or more guanidines, or combinations of guanidines and D-amino acids, described herein in said coating can prevent the adherence of organisms to the surface.
  • the coating may be solvent borne or aqueous.
  • Aqueous coatings are typically considered more environmentally friendly.
  • the coating can be an aqueous dispersion of one or more guanidines, or combinations of guanidines and D-amino acids, described herein and a binder or a water based coating or paint.
  • the coating can comprise an aqueous dispersion of one or more guanidines, or combinations of guanidines and D-amino acids, and an acrylic, methaeryiic or acrylamide polymers or co-polymers or a poly[-alkoxyalkanoate-co-3-hydroxyalkenoate] polyester.
  • the coating composition can be applied to a surface by any conventional means including spin coating, dip coating, spray coating, draw down, or by brush, roller or other applicator. A drying or curing period can be performed.
  • Coating or film thickness can vary depending on the application and can readily be determined by one skilled in the art after limited testing.
  • a composition described herein can be in the form of a protective laminate film.
  • Such a film can comprise thermoset, thermoplastic, elastomeric, or crosslinked polymers.
  • polymers examples include, but are not limited to, polyolefin, polyamide, polyurethane, polyacrylate, polyaerylamide, polycarbonate, polystyrene, polyvinyl acetates, polyvinyl alcohols, polyester, halogenated vinyl polymers such as PVC, natural and synthetic rubbers, alk d resins, epoxy resins, unsaturated polyesters, unsaturated polyamides, polyimides, fiuorinated polymers, silicon containing and carbamate polymers.
  • the polymers can also be blends and copolymers of the preceding chemistries.
  • composition described herein when a composition described herein is a preformed film, it can be applied to a surface by, for example, the use of an adhesive, or co-extruded onto the surface. It can also be mechanically affixed via fasteners which may require the use of a sealant or caulk wherein the esters of the instant invention may also be advantageously employed.
  • a plastic film can also be applied with heat which includes calendaring, melt applications and shrink wrapping.
  • a guanidine-containing composition can include other additives such as antioxidants, UV absorbers, hindered amines, phosphites or phosphonites, henzofuran-2-ones, thiosynergists, polyamide stabilizers, metal stearates, nucleating agents, fillers, reinforcing agents, lubricants, ernu!sifiers, dyes, pigments, dispersants, other optical brighteners, flame retardants, antistatic agents, blowing agents and the like, such as the materials listed below, or mixtures thereof.
  • additives such as antioxidants, UV absorbers, hindered amines, phosphites or phosphonites, henzofuran-2-ones, thiosynergists, polyamide stabilizers, metal stearates, nucleating agents, fillers, reinforcing agents, lubricants, ernu!sifiers, dyes, pigments, dispersants, other optical brighteners, flame
  • Plastic-based medical devices that benefit from the present method include, but are not limited to, plastics articles used in the field of medicine, e.g. dressing materials, syringes, catheters etc., so-called “medical devices", gloves and mattresses.
  • plastics are polypropylene, polyethylene, PVC, POM, polysulfones, polyethersulfones, polystyrenics, polyamides, polyurethanes, polyesters, polycarbonate, polyaerylics and methaerylies, polybutadienes, thermoplastic polyolefins, ionomers, unsaturated polyesters and blends of polymer resins including ABS, SAN and PC/ABS.
  • the guanidines, or combinations of guanidines and D-amino acids, especially in low concentrations, can be safely used even in applications where ingestion is possible, such as reusable water bottles or drinking fountains where a biofilm may develop.
  • the surfaces of such water transport devices can be rinsed with a formulation containing one or more guanidines, or combinations of guanidines and D-amino acids, described herein, or low levels of one or more guanidines, or combinations of guanidines and D-amino acids, can be introduced into the water that passes through the containers of conduits.
  • about 0.0001% or less or up to about 1%, typically less than about 0.1% by weight of one or more guanidines, or combinations of guanidines and D-amino acids may be introduced into such water.
  • very small amounts are effective in many circumstances and concentrations of about 0.000001% to about, 0.1%, for example, about 0.000001 % to about 0.01 %, or about 0.000001% to about 0.001 %, or 0.000001% to 0.1%, 0.000001% to 0.01 %, or 0.000001 % to 0.005 %, can be used in such applications.
  • guanidines When used in a coating or film, small amounts of one or more guanidines, or combinations of guanidines and D-amino acids, can be present for short term use, for example, one use, seasonal or disposable items, especially those applications which involve possible human contact, splints, catheters, tubing, dental equipment etc.
  • about 0.001% or less up to about 5% for example up to about 3% or about 2%, or preferably 0.001% or less up to 5%, up to 3% or 2% by weight of one or more amino acids may be used in such coatings or films.
  • guanidines Given the high activity of the instant guanidines, or combinations of guani dines and D-amino acids, very small amounts are effective in many circumstances and concentrations of about 0.0001% to about 1%, for example, about 0.0001 % to about 0.5%, or about 0.0001% to about 0.01 % can be used in coating applications, or preferably 0.0001% to 1%, 0.0001 % to 0.5%, or 0.0001% to 0.01 % by weight of one or more guanidines, or combinations of guanidines and D- amino acids.
  • guanidines, or combinations of guanidines and D-amino acids can be used, for example about 0.0001 % to about 3%, for example about 0.005 % up to about 1% one or more guanidines, or combinations of guanidines and D-amino acids, can be used, or preferably, 0.00001% to 10%, 0.0001% to 3 0.001% up to 1 % by weight one or more guanidines, or combinations of guanidines and D-amino acids, can be used, in situations in which the guanidines, or combinations of guanidines and D-amino acids, are impregnated into the surface of an already prepared molded article or fiber, the actual amount of a guanidine, or combination of a guanidine and D-amino-acid, present at, the surface can depend on the substrate material, the formulation of the
  • guanidines Given the high activity of the instant guanidines, or combinations of guanidines and D-amino acids, very small amounts are effective in many circumstances, and concentrations of about 0.0001 % to about 5 %, for example, about 0.0005 % to about 0.1%, or about 0.0001% to about 0.01 % can be used in plastics, or preferably 0.0001 % to 1 %, 0.0001% to 0.1%, or 0.0001% to 0.01 % by weight of one or more amino acids can by used..
  • Inhibition or reduction in a biofiim by treatment with a guanidine, or combination of a guanidine and D-amino acid can be measured using techniques well established in the art. These techniques enable one to assess bacterial attachment by measuring the staining of the adherent biomass, to view microbes in vivo using microscopy methods; or to monitor cell death in the biofilm in response to toxic agents.
  • the biofilm can be reduced with respect to the surface area covered by the biofilm, thickness, and consistency (for example, the integrity of the biofilm).
  • biofilm assays include microtiter plate biofilm. assays, fluorescence-based biofilm.
  • Such assays can be used to measure the activity of a D-amino acid on the disruption or the inhibition of formation of a biofilm (Lew, et aL, (2000) Curr. Med. Chem. 7(6):663-72; Werner, et aL, (2006) Brief Funct. Genomic Froteomic 5(l):32-6).
  • treatment can be assayed by measuring the growth of bacteria and/or can be quantified by measuring the density of a biofilm-forming bacteria in a biological sample.
  • biological samples include blood , serum, sputum, lacrimal secretions, semen, urine, vaginal secretions, and tissue samples.
  • the reduction in the growth of bacteria can also be measured by chest X-rays or by a pulmonary function test (PFT) (for example, spirometry or forced expiratory volume (FEVi)).
  • PFT pulmonary function test
  • FEVi forced expiratory volume
  • the presence or growth of biofilm-producing bacteria can be measured by detecting the presence of antigens of biofilm-producing bacteria in a biological sample, such as those described above.
  • an antibody to S. pneumoniae components can be used to assay colonization/infection in a subject afflicted with a biofilm-related condition or disorder, such as by assaying the presence of Streptococcus antigens in a biological sample.
  • Such antibodies can be generated according to methods well established in the art or can be obtained commercially (for example, from Abeam, Cambridge, MA; Cell Sciences Canton, MA; Novus Biologicals, Littleton, CO; or GeneTex, San Antonio, TX).
  • Appropriate therapies for the treatment of biofilm-re fated disorders with a guanidine, or combination of a guanidine and D-amino acid can be determined using techniques well established in the art. For example, animal models using mammals can be used to assess the efficacy of treatment with guanidines, or combinations of guanidines and D-amino acids.
  • Non- limiting examples include implanting polymer beads, e.g., polymethylmethacrylate (PMMA) beads loaded with the guanidines, or combinations of guanidines and D-amino acids, in rats and assessing their ability to prevent biofilms.) polymethylmethacrylate (PM MA) beads in rats and catheters in rabbits have been used as animal models for biofilm formation for Staph aureus. See, e.g., Anguita-Alonzo, et ah, Antimicrobial Agents and Chemotherapy, July 2007, p. 2594- 2596, and Beenken, et al. J ournal o f B acterio 1 o y , July 2004, p. 4665-4684, which are hereby incorporated in their entirety by reference. .
  • PMMA polymethylmethacrylate
  • PM MA polymethylmethacrylate
  • Biofilms are understood, very generally, to be aggregations of living and dead microorganisms, especially bacteria, that adhere to living and non-living surfaces, together with their metabolites in the form of extracellular polymeric substances (EPS matrix), e.g. polysaccharides.
  • EPS matrix extracellular polymeric substances
  • the activity of antibiofiim substances that normally exhibit a pronounced growth-inhibiting or lethal action with respect to planktonic cells may be greatly reduced with respect to
  • microorganisms that are organized in biofilms, for example because of inadequate penetration of the active substance into the biological matrix.
  • a guanidine, or combination of a guanidine and D-amino acid can be administered alone or in combination with a second agent, e.g., a biocide, an antibiotic, or an antimicrobial agent, to treat a biofiim or to prevent the formation of a biofilm.
  • a second agent e.g., a biocide, an antibiotic, or an antimicrobial agent
  • An antibiotic can be co-administerecl with the guanidine, or combination of a guanidine and D-amino acid, either sequentially or simultaneously.
  • any of the compositions described herein can be formulated to include one or more guanidines, or combinations of giianidines and D-amino acids, and one or more second agents.
  • the antibiotic can be any compound known to one of ordinary skill in the art that can inhibit the growth of, or kill, bacteria.
  • Useful, non-limiting examples of antibiotics include lineosamides (clindomycin); chloramphenicols; tetracyclines (such as Tetracycline,
  • Neomycin beta-lactams (such as penicillins, cephalosporins, Imipenem, Aztreonam);
  • glycopeptide antibiotics such as vancomycin
  • polypeptide antibiotics such as bacitracin
  • macrolides erythromycins
  • amphotericins such as Sulfanilamide
  • Nitrofurantoin Phenazopyridine; trimethoprim; rifampicins; metronidazoles; cefazolins;
  • Lincomycin Spectinomycin; mupirocins; quinolones (such as Nalidixic Acid, Cinoxacin, Norfloxacin, Ciprofloxacin, Perfloxacin, Ofloxacin, Enoxacin, Fleroxacin, Levofloxacin);
  • novobiocins novobiocins
  • polymixins such as Carbenicillin
  • antipseudomonals such as Carbenicillin
  • antibiotics are commercially available, e.g., from Daiichi Sankyo, Inc.
  • biocides include biguanide, chiorliexidine, triclosan, chlorine dioxide, and the like.
  • antimicrobial agents include, but are not limited to, Pyrithiones, especially the zinc complex (ZPT); Octopirox®; Dimethyklimethyloi Hydantoin (Glydant®); Methyichloroisofhiazolinoiie/methylisothiazolinone ( athon CG®); Sodium Sulfite; Sodium Bisulfite; Imidazolidinyl Urea (Germail 115®, Diazolidinyl Urea (Germaill II®); Benzyl Alcohol; 2-Bronio-2-nitropropane ⁇ l ,3-diol (Bronopol®); Formalin (formaldehyde);
  • Methyldibromo Tiitrile Glutaronitrile ( l,2-Dibromo-2,4-dicyanobutane or Tektamer®);
  • Glutaraldehyde 5 ⁇ hro " ⁇ no-5-nitro ⁇ l ,3-dioxane (Bronidox®); Phenethyl Alcohol; o- Phenylphenol/sodium o-phenyi "" phenoi; Sodium Hydroxymethyiglycinate (Suttocide A®); Polymethoxy Bicyclic Oxazolidine (Nuosept C®); Dimethoxane; Thimersal; Dichlorobenzyl Alcohol; Captan; Chlorphenenesin; Dichiorophene; Chlorbutanol; Glyceryl Laurate;
  • Cosmocil® poly(hexamefhyienebiguanide) hydrochloride
  • silver compounds such as organic silver salts ir anorganic silver salts, silver chloride including formulations thereof such as JM Acticare® and micronized silver particles.
  • Methods and treatments using guanidines, or combinations of gua idines and D- amino acids include inhibiting or preventing the formation of biofilm, even or especially without inhibiting organism growth, and alos the disruption of a biofilm once formed.
  • a guanidine, or combination of a guanidine and D-amino acid can be used to treat, biofilm -related disorders in a subject by administering to the subject an effective amount of guanidine, or combination of a guanidine and D-amino acid, that reduces biofilm formation in the subject.
  • a reduction in bacterial growth is indicative of the reduction in, or inhibition of, biofilm production in the subject.
  • a guanidine or combination of a guanidine and D-amino acid, can inhibit or reduce biofilm formation by diminishing adherence of biofilm- forming bacteria to a surface or by increasing bacterial death.
  • This therapeutic approach can be useful for the treatment of biofilm-related disorders or conditions, or medical device-related infections associated with the formation of micro bial biofilms.
  • Non-limiting examples of biofilm-related disorders include otitis media, prostatitis, cystitis, bronchiectasis, bacterial endocarditis, osteomyelitis, dental caries, periodontal disease, infectious kidney stones, acne, Legionnaire's disease, chronic obstructive pulmonary disease (COPD), and cystic fibrosis.
  • COPD chronic obstructive pulmonary disease
  • subjects with cystic fibrosis display an accumulation of biofilm in the lungs and digestive tract.
  • Subjects afflicted with COPD such as emphysema and chronic bronchitis, display a. characteristic inflammation of the airways wherein airflow through such airways, and subsequently out of the lungs, is chronically obstructed.
  • Bio film-related disorders can also e compass infections derived from
  • medical device-related infections such as infections from biliary stents, orthopedic implant infections, and catheter-related infections (kidney, vascular, peritoneal).
  • An infection can also originate from sites where the integrity of the skin and'Or soft tissue has been compromised.
  • Non-limiting examples include dermatitis, ulcers from peripheral vascular disease, a burn injury, and trauma.
  • a Gram-positive bacterium such as S. pneumoniae, can cause opportunistic infections in such tissues.
  • the ability of S. pneumoniae to infect burn wound sites e.g., is enhanced due to the breakdown of the skin, burn- related immune defects, and antibiotic selection.
  • a subject is treated.
  • a subject can be a mammal including, but not limited to, a. primate (e.g., a monkey, such as a cynomolgous monkey, a chimpanzee, and a human).
  • a subject can be a non-human animal such as a bird (e.g., a. quail, chicken, or turkey), a farm animal (e.g., a cow, goat, horse, pig, or sheep), a pet (e.g., a cat, dog, or guinea pig, rat, or mouse), or laboratory animal (e.g. , an animal model for a disorder).
  • Non -limiting representative subjects can be a human infant, a pre-adolescent child, an adolescent, an adult, or a senior/elderly adult.
  • a subject in need of treatment can be one afflicted with one or more of the infections or disorders described herein.
  • the subject is at risk of developing a biofilm on or in a biologically relevant surface, or already has developed such a biofilm.
  • Such a subject at risk can be a candidate for treatment with a guanidine, or combination of a guanidine and D-amino acid, in order to inhibit the development or onset of a biofilm- production-related disorder/condition or prevent the recurrence, onset, or development of one or more symptoms of a. biofilm-related disorder or condition.
  • Such a subject can be harboring an immature biofilm that is clinically evident or detectable to the skilled artisan, but that has not yet fully formed.
  • a subject at risk of developing a biofilm can also be one in which implantation of an indwelling device, such as a medical device, is scheduled.
  • the risk of developing a biofilm can also be due to a propensity of developing a biofilm-related disease (such as the presence of a channel transporter mutation associated with cystic fibrosis).
  • a biofilm-related disorder can be at an early stage, e.g., no bacterial infection and/or biofilm formation is yet detected.
  • the methods described herein can be used to prevent biofilm formation in the airways of a cystic fibrosis patient.
  • a cystic fibrosis patient Such a patient can be treated while free of bacterial infection of the airways or upon detection of a bacterial infection.
  • the methods described herein can be used to prevent biofilm formation in superficial wounds of a patient. Such wounds can include burns. In some examples, the methods described herein can be used to prevent biofilm formation in patients with diabetic leg syndrome.
  • Room temperature denotes a temperature from the range of 20-25°C.
  • Free base (2h) xh e su if ate sa lt (200 mg, 0.659 mmol) was dissolved in 10 mL H 2 0 and a solution of ⁇ a (03 ⁇ 4, ⁇ nig, 0.659 mmol) in 6 mL H O was added. Precipitation was completed at 4°C, the suspension was filtered through celite and lyophilized The residue was extracted with hot methanol and filtered through celite giving the free base in quantitative yield. The free base was well soluble in hot methanol, the sulfate salt and barium salts were insoluble.
  • Example 3 Preparation of l-l -A ⁇ 'V'-diisopropylguaiiidinyiethaae-l-yll-l '-fethane-l,!-
  • Norspermidine (700 uL, 5 mmol) was a "" e " to a solution of S-Methylisothiourea 1 ⁇ 2 H-SO. ⁇ 2505 mg, 18 mmol) in 7 mL H clamp0 and stirred for 3 days at 105°C. A white precipitate formed after standing overnight at 4°C. The precipitate was sucked off and washed with ice cold H ⁇ O followed by ethanol. Reerystallization from H O afforded the sesquisulfate hydrate of the product (1275 mg, 67%) as white solid.
  • Octanoic acid salt Free base 5b (13.7 mg, 0.0635 mol) was mixed with 635 uL of 300 mM octanoic acid in H20/ethanol (2: 1 vol ) to give a lOOmM stock solution of the octanoic acid salt.
  • Norspermidine (1399 ,u,L, 10 mmol) was dissolved in 7 mL ethanoi, 1.5 mL of concentrated hydrochloric acid were added whereupon a white precipitate formed, C anamide (1640 mg, 39 mmol) was added and the reaction mixture was refluxed for 36.5 hours and let to cool. The solvent was removed resulting in a highly viscous mass which crystallized after 8 days at room temperature. The crystalline material was washed with isopropanol and ice cold ethanoi.
  • Free base ⁇ £fe xhe chloride salt (350 mg, 0.91 mmol) was dissolved in 10 raL H 2 0 and a suspension of A 2 SO « m I» 1 -36 mmol) was added i 6 mL H 2 0. After sonication, precipitation was completed at 4°C, the suspension was filtered through celite and a solution of Ba.(OH) 2 (234 mg, 1.36 mmol) in 5 mL H . Q was added. Precipitation was completed at 4 C C and the suspension was filtered through celite and lyophilized. The residue was extracted with hot methanol and filtered through celite giving the free base in quantitative yield. The free base was well soluble in hot methanol, the sulfate salt, the silver and barium salts were insoluble.
  • Octanoic acid salt Free base 6b (20.6 mg, 0.0802 mmol) was mixed with 802 ⁇ iL of 300 raM octanoic acid in H20/ethanol (2: 1 vol ) to give a 100 raM stoclv of the octanoic acid salt
  • Free base Qfc The su3fate sa [ t 2 oo mg, 0.41 1 mmol) was dissolved in 10 itiL H 2 0 and a solution of * a (QH)., 041 mg, 0,822 mmol) in 6 mL H O was added. Precipitation was completed at 4°C, the suspension was filtered through celite and lyophilized. The residue was extracted with hot methanol and filtered through celite giving the free base in quantitative yield. The free base was well soluble in hot methanol, the sulfate salt and barium salts w r ere insoluble.
  • Octanoie acid salt Free base 7b (12.5 mg, 0.0458 mmol) was mixed with 458 iL of 400 mM octanoie acid in H2Q/ethanol (2: 1 vol) to give a 100 niM stock of the octanoie acid salt.
  • Free base (lib): The chloride salt (350 mg, 1.00 mmol) was dissolved i 10 mL ⁇ 2 ⁇ 3 and a suspension of Ag ? SO (313 mg, 1.00 mmol) was added in 10 mL H.Q. After sonication, precipitation was completed at 4°C and a solution of Ba(OH) ;) (172 mg, 1.00 mmol) in 5 mL Hiker0 was added. Precipitation was completed at 4°C, 5 ml, ethanol were added and the suspension was filtered through celite and lyophilized. The residue was extracted with hot ethanol and filtered through celite giving the free base in 33% yield. The free base was well soluble in hot ethanol, the sulfate salt, the silver and barium salts were insoluble.
  • Octanoic acid salt Free base 1 lb (12.8 mg, 0.0464 mmol) was mixed with 464 ⁇ of 200 mM octanoic acid in H20/ethanol (2: 1 vol) to give a 100 mM stock of the octanoic acid salt.
  • Example J 2 Preparation of l-(2-(2-IniinoimidazoHdiii-l-vi)ethvnguaiiidine sulfate (12)
  • norspermidme 140 ⁇ ,, 1.0 mmol was added. The solvent was evaporated and the residue washed twice with 10 mL hexanes and 30 mL ethyl acetate (yield 663 mg, 91%).
  • Palmitic acid (769 rng, 3.0 nimol) was dissolved in 10 mL ethanol and norspermidine (140 ⁇ ,, 1.0 mmol) was added. The solvent was evaporated and the residue washed twice with 10 mL hexanes and 30 mL ethyl acetate (yield 827 mg, 92%).
  • Myristic acid (411 mg, 1.8 mmol) was dissolved in 20 mL ethanol and norspermidine (84 ⁇ ., 0.6 mmol) was added. The solvent was evaporated and the residue washed twice with 10 mL hex arte then sucked off and washed with 30 mL ethyl acetate (yield 431 mg, 88%).
  • B. subtilis strain NCBI 3610 a wild strain of B. subtilis, which is capable of forming robust biofilms. 5
  • Staphylococcus aureus SCOl was obtained from the Kolter lab collection. 2
  • B. subtilis strain NCBI 3610 and S. aureus SCOl were isolated as a single colonies growing overnight at 37°C on solid LB medium.
  • Biofilm inhibition assay B. subtilis strain NCBI 3610 was grown for 2 hours with shaking at 37°C, then diluted 1 : 1000 in either 3 mi/well (12 well plates, polystyrene, VWR) or 1.5 mi/well (24 wells plates, polystyrene, VWR) of defined biofilm medium MSgg (pH 7.0). Cells were grown without shaking for 3 days at 25°C. For pH experiments bacteria, were additionally grown in MSgg at pH 6.0 and 8.0. Floating biofilms (pellicles) ⁇ , subtilis were examined optically.
  • S. aureus strain SCOl was grown overnight with shaking at 37°C, then diluted 1 : 100 in 1 ml/well of TSB medium, 0.5% glucose, 3% NaCl (pH 7.0). Cells were grown without shaking for 24 hours at, 37°C. For pH experiments bacteria were additionally grown in MSgg at pH 6.0 and 8.0. Submerged biofilms of S. aureus were observed and quantified by crystal violet staining as below.
  • FIG. 1A shows the results of contacting a preexisting biofilm with compound 7a
  • FIGS. 7A-B Inhibition of biofilm formation by different norspermidine micelle formulations is shown in FIGS. 7A-B.
  • the charge of the compounds is also an important property for binding with the exopolysaecharide.
  • guanidine groups in the related structure 2 significantly increase the third pKa value to 6.3, the central amino group remains non-protonated (101) at physiological pH values, which was confirmed by X-ray structure analysis (FIG. 4). It could remain inactive in bioassays at pH 7 due to either its charge or structure.
  • guanidine groups on the scaffold of norsperrnidine or norsperniine increased all individual pKa values compared to the corresponding polyamines (FIG. 2A) causing the average degree of protonation to rise which is in line with the increased activity of 6 over norsperrnidine and 7 over norsperniine.
  • 5 was active but did not display increased activity over norsperrnidine, despite higher degree of protonation.
  • compound 5 may be a critical factor, which was confirmed by a novel method for the delivery of compounds into the biofiim.
  • Norsperniine carries on average 3.3 positive charges at pH 7 corresponding to about 30% fully charged (1111) molecules, while the majority is only triple charged (1110 or 1101) and will likely populate predominantly the protonation micro-state (1101) with one non-charged secondary amine, 14, 16, 17.
  • the protonation mico-state (1101) would not comply with the triple charged motif (111) and may contribute to the higher activity of norsperrnidine over norsperniine,
  • the active compound 11 exists at maximum in a double protonated form which is due to high pKa values virtually the only relevant species until pH 8.5.
  • the structure and protonation ofbiguanides is in the literature frequently misrepresented as reported by Bharatam et al., who showed by theoretical studies that the central nitrogen ofbiguanides usually carries hydrogen neither in neutral nor in charged states (FIG. 2B and C). This N is rather partially negatively charged while the positi ve charge is delocahzed between the terminal nitrogens of any biguanide.
  • the crystal stracture of protonated 11 confirmed these results (FIG. 2D and FIG. 6), making 11 analogous to the (1111) motif of fully charged compound 7, which explains its activity.
  • Table 8 Crystal data, and data on collection and refinement.

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Abstract

Methods of treating or reducing biofilms, treating a biofilm-related disorder, triggering biofilm disassembly, and preventing biofilm formation using guanidines is described.

Description

METHODS AND COMPOSITIONS COMPRISING GUANIDINES FOR TREATING BIOFILMS
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional Application No. 61/727,447, filed November 16, 2052 and U.S. Provisional Application No. 61 /777,267, filed March 12, 2013, the contents of which are hereby incorporated by reference.
STATEMENT OF GOVERNMENT RIGHTS
[0002] This invention was made with Government, support under National Institutes of
Health awards GM086258 and HG005824. The Government has certain rights in the invention.
BACKGROUND
[0003] Bio films are communities of cells that settle and proliferate on surfaces and are covered by an exopolymer matrix. They are slow-growing and many are in the stationary phase of growth. A hallmark of biofilms is an extracellular matrix typically consisting of protein, exopolysaccharide and sometimes DNA, that holds the cells together in the community. They can be formed by most, if not all, pathogens. According to the CDC, 65% of ail infections in the United States are caused by biofilms that can be formed by common pathogens. Biofilms are also found in industrial settings, such as in drinking water distribution systems.
SUMMARY
[0004] It has been discovered that certain guanidines inhibit biofilm formation and trigger biofilm disassembly. Aspects of this disclosure feature methods of treating, reducing, inhibiting biofilm formation by bacteria, and triggering biofilm disassembly, in some embodiments, the method comprises contacting a surface with a composition comprising an effective amount of a guanidine, thereby treating, reduci g, inhibiting formation of the biofilm, or triggering disa ssembly of the biofilm.
[ooos; It has been discovered that the structure of certain guanidines and partially gua.nidinyla.ted polyamines contributes to the ability of those compounds to inhibit biofilm formation and trigger disassembly of a biofilm.. In certain embodiments, the guanidine has three atoms separating amino or guanidine groups. In certain embodiments, the amino groups are ionizable. In certain embodiments, the amino groups are positively charged. In some embodiments, the guanidines are branched. In some embodiments, the guanidines are linear. In certain embodiments, the guanidine has Formula (I) or Formula (II),
Figure imgf000003_0001
or a base, salt, hydrate, or tautomer thereof, wherein
? Y is Ci'RsRe), O. S, NH, or NR] 0;
M is C(RiR2)-Y-C(R3R4), meta-phenylene, or 1 ,3-Cs-C cycloalkane, where the meta- phenyiene or cycloalkane may be substituted by Rio or have one or more N heteroatoms in its ring; each Ri, R2, R3, R4, R5, and R is H, C1-C12 alkyl, Ci-C12 alkenyl, C1-C12 alkynyl, Ci- C12 alkoxy, alkaryl, aryl, or heteroaryl, so that each Ri, R.2, R3, R4, R5, and Re may be the same or different; each R7, R9, and Rio is H, Ci-Cie alkyl, C3-C16 alkenyl, C3-C16 alkynyl, aryl, heteroaryl, or C7-C22 aralkyl, so that each R7, R9, and Rio may be the same or different; each R is H or -C(=NR7)-NR7H, so that each R8 may be the same or different; and each x an integer from. 1 to 6, inclusive.
[0006] Compounds of Formulae (I) and (II) may be acyclic or cyclic. Compounds of Formulae (I) and (II) may be linear or branched,
[0007] In certain embodiments, Y is QRj e). In some embodiments, Y is NRj0.
[0008] In some embedments, M is meta-phenylene. In some embodiments, the meta- phenylene may be substituted with CrCg alkyl, Q Cg alkenyl, or C Cg alkynyl. In some embodiments, M is meta-phenylene and substituted with an N heteroatom on the ring such that M is 2,6~pyridinediyl. In other embodiments, M is 1,3-cyclohexanediyl. In still other embodiments, M is a 1,3-cyclohexanediyl substituted with an N heteroatom on the ring such that M is 2,6-piperidinediyl. In other embodiments, M is a 1 ,3-cyelohexanediy! substituted with three N heteroatoms on the ring such that M is l,3,5-triazine-2,4-diyl. In other embodiments, M is 1 ,3 cyclopentanediyl. In other embodiments, M is 1,3-cyclopentanediyl substituted with an N heteroatom on the ring such that M is 2,5-pyrrolidinediyi. In other embodiments, M is cyclopent- 1 ,3-ene- 1 ,4-diyl. In other embodiments, M is cyclopent- 1 ,3-ene- 1 ,4-diyl substituted with an N heteroatom on the ring such that M is 2,5-pyrroldiyl.
[0009] In some embodiments, Ri is hydrogen. In some embodiments, R? is hydrogen. In some embodiments, R3 is hydrogen. In some embodiments, R4 is hydrogen. In some embodiments, R5 is hydrogen. In some embodiments, Re is hydrogen. In some embodiments, R7 is hydrogen. In certain embodiments, each of Ri, R2, R3, R , R5, and Re is H. In other embodiments, each of Ri, !<.··. R.3, 4, R5, and Re is H or CI 1 ;
[0010] In some embodiments, Rj and R3 covalently bond to each other to form a ring.
In some embodiments, each of R , Rp is H, Ci-Ce alkyl, C3-C6 alkenyl, or C3~Ce alkynyl.
[0011] In some embodiments, Rg is hydrogen. In some embodiments, Rg
is -C(= R7)- R7H. In some embodiments, each R¾ is the same. In some embodiments, each Rg is different.
[0012] In some embodiments, R9 is hydrogen. In some embodiments, RJQ is hydrogen.
[0013] In some embodiments, x is 1. In some embodiments, x is 2. In certain embodiments, x is 3, In further embodiments, x is 4. In still further embodiments, x is 5, In other
embodiments, x is 6.
In accordance with certain embodiments, Y is CH2, each of Rj, R2, R3, R4, R5, and Re is H and x is 1 , 2 or 3. [0014] In some embodiments, the guanidine is a tautomer of Formulae (!) or (11). For example, in some embodiments, a guanidine of Formula (11) may be represented by the Formulae:
Figure imgf000006_0001
[0015] In some embodiments, the guanidine is a compound in Table 1.
Table 1
Figure imgf000006_0002
Figure imgf000007_0001
[0016] In some embodiments, the bacteria are Gram-negative or Gram-positive bacteria. In particular embodiments, the bacteria are Bacillus, Staphylococcus, E. coli, or Pseudomonas bacteria. In some embodiments, the bacteria are mycobacteria.
[0017] In certain embodiments, the method comprises contacting a surface with a composition comprising an effective amount of a guanidine combined with a D-amino acid, thereby treating, reducing, inhibiting formation of the biofilm, or triggering disassembly of the biofilm.
[0018] In other aspects, this disclosure features compositions, such as industrial, therapeutic or pharmaceutical compositions, comprising one or more guanidines. [0019] In other aspects, the invention features compositions, such as therapeutic or pharmaceutical compositions, comprising one or more guanidines. In some embodiments, the one or more guanidines are combined with one or more D-amino acids. In some embodiments, the composition comprises at least one guanidine of Formulae (!) or (II), In some embodiments, the guanidine has Formulae (!) or (II) and the D-aniino acid is selected from the group consisting of D-alanine, D-cysteine, D-aspartic acid, D-glutamic acid, D-histidine, D-isoleucine, D-lysine, D-lexicine, D-asparagine, D-proline, D-glutamine, D-arginine, D-serine, D-threonine, D-valine, D-tryptophan, D-tyrosine, and a combination thereof, or the D-amino acid is a synergistic combination of two or more D-amino acids selected from the group consisting of D-alanine, D- cysteine, D-aspartic acid, D-glutamic acid, D-phenylalanine, D-histidine, D-isoleucine, D-lysine, D-leucine, D-methionine, D-asparagine, D-proline, D-glutamine, D-arginine, D-serine, D- threonine, D-valine, D-tryptophan, and D-tyrosine.
[0020] In some embodiments, the D-amino acid is D-tyrosine or the combination of D-amino acids comprises D-tyrosine, In other embodiments, the composition further comprises one or more of D proline and D phenylalanine. In other embodiments, the composition further comprises one or more of D-leucine, D-tryptophan, and D-methionine. In some embodiments, the composition further comprises one or more of D-alanine, D-cysteine, D-aspartic acid, D- glutamic acid, D-phenylalanine, D-histidine, D-isoleucine, D-lysine, D-leucine, D-methionine, D-asparagine, D-proline, D-glutamine, D-arginine, D-serine, D-threonine, D-valine, D- tryptophan, and D-tyrosine.
[0021] Another aspect of this disclos ure is directed to methods of treating a biofilm-related disorder in a subject in need thereof!, the method comprising administering to the subject a composition comprising an effective amount of a guanidine. In some embodiments, the guanidine has Formulae (I) or (Π). In other embodiments, the gtianidine is a compound in Table 1.
[0022] In some embodiments, the method comprises administering to the subject a composition comprising a combination of at least one guanidine and at least one D-amino acid, thereby treating the biofilm-related disorder. In some embodiments, the guanidine has Formulae (I) or (II) and the D-amino acid is selected from the group consisting of D-alanine, D-eysteine, D-aspartic acid, D-glutamic acid, D-histidine, D-isoleucine, D-lysine, D-leucine, D-asparagine, D-proline, D-glutamine, D-arginine, D-serine, D-threonine, D- valine, D-tryptophan, D-tyrosine, and a combination thereof, or the D-amino acid is a synergistic combination of two or more D- amino acids selected from the group consisting of D-alanine, D-cysteine, D-aspartic acid, D- glutamic acid, D-phenylalanine, D-histidine, D-iso leucine, D-lysine, D-leucine, D-methionine, D-asparagine, D-proline, D-glutamine, D-arginine, D-serine, D-threonine, D-valine, D- tryptophan, and D-tyrosine.
[0023] In some embodiments, the D-amino acid is D-tyrosine or the combination of D-amino acids comprises D-tyrosine. In other embodiments, the composition further comprises one or more of D-proline and D-phenylalanine. In other embodiments, the composition further comprises one or more of D-leucine, D-tryptophan, and D-methionine. In some embodiments, the composition further comprises one or more of D-alanine, D-cysteine, D-aspartic acid, D- glutamic acid, D-phenylalanine, D-histidine, D-isoleucine, D-lysine, D-leucine, D-methionine, D-asparagine, D-proline, D-glutamine, D-arginine, D-serine, D-threonine, D-valine, D- tryptophan, and D-tyrosine,
[0024] In some embodiments, the composition is administered to a surface of the subject selected from the group of dermal and mucosal surfaces and combinations thereof. In other embodiments, the surface is an oral surface, a skin surface, a urinary tract surface, a vaginal tract surface, or a lung surface.
[0025] In some embodiments, the composition is administered to the subject via
subcutaneous, intra-muscular, intra-peritoneal, intravenous, oral, nasal, or topical administration, and a combination thereof.
[0026| In some embodiments, the subject is a human.
[0027] In some embodiments, the formation of a biofilm is inhibited. In other embodiments, a previously formed biofilm is disrupted.
[0028] In some embodiments, the guanidine is administered at a concentration of about 0.1 iiM to about 100 μΜ, for example, at a concentration of 0.1 iiM to 100 μΜ, In certain embodiments, the D-amino acid combined with the guanidine is administered at a concentration of about 0.1 nM to about 100 μΜ, for example, at a concentratior! of 0.1 nM to 100 μΜ.
[0029] In further embodiments, the biofilm-related disorder is selected from the group consisting of pneumonia, cystic fibrosis, otitis media, chronic obstructive pulmonary disease, and a urinary tract infection and combinations thereof. In other embodiments, the biofilm-related disorder is a medical device-related infection. In further embodiments, the biofilm-related disorder is a periodontal disease, such as gingivitis, periodontitis or breath malodor. In still further embodiments, the biofilm-related disorder is caused by bacteria. In some embodiments, the bacteria are Gram-negative or Gram-positive bacteria. In still other embodiments, the bacteria, are of the genus ActinobaciUus, Acinetobacter, Aeromonas, Bordetella, Brevibaculus, Brucella, Bacteroides, Burkholderia, Borelia, Bacillus, Campylobacter, Capnocytophaga, Cardiobacterium, Citrobacter, Clostridium, Chlamydia, Eikenella, Enterobacter, Escherichia, Entembacter, Francisella, Fusobacterium, Flavobacterium, Haemophilus, Helicobacter, Kingella, Klebsiella, Legionella, Listeria, Leptospirae, Moraxella, Morganella, Mycoplasma, Mycobacterium, Neisseria, Pasteurella, Proteus, Prevotella, Plesiomonas, Pseudomonas, Providencia, Rickettsia, Stenotrophomonas, Staphylococcus, Streptococcus, Streptomyces, Salmonella, Serratia, Shigella, Spirillum, Treponema, Veillonella, Vibrio, Yersinia, or
Xanthomonas .
[0030] Another aspect of this disclosure is directed to methods of treating, reducing, inhibiting biofilm formation by biofilm forming bacteria, or triggering biofilm disassembly on a biologically-related surface, the method comprising contacting a biological surface with a composition comprising an effective amount of a guanidine. In certain embodiments, the guanidine has Formulae (Ϊ) or (II),
[0031] In certain embodiments, the composition and related methods include a combination of at least one guanidine and at least one D-amino acid, thereby treating, reducing or inhibiting formation of the biofilm. In some embodiments, the guanidine has Formulae (I) or (II) and the D-amino acid is selected from the group consisting of D-alanine, D-eysteine, D-aspartic acid, D- glutamic acid, D-histidine, D-isoleueiiie, D-lysine, D-leucine, D-aspara.gine, D-proline, D- glutamine, D-arginine, D-serine, D-threonine, D-valine, D -tryptophan, D-tyrosine, and a combination thereof, or the D-amino acid is a synergistic combination of two or more D-amino acids selected from, the group consisting of D-alanine, D-eysteine, D-aspartic acid, D-glutamic acid, D-phenyialanine, D-histidine, D-isoleucine, D-lysine, D-leucine, D-methionine,
D-asparagine, D-proline, D-ghrtamine, D-arginine, D-serine, D-threonine, D-valine,
D-tryptophan, and D-tyrosine.
[0032] In some embodiments of the foregoing methods, the composition comprises a guanidine and D-tyrosine. In addition to D-tyrosine, in some embodiments, the composition further comprises one or more of D-proline and D-phenylalanine. in still other embodiments, in addition to D-tyrosine, the composition further comprises one or more of D-leucine, D- tryptophan, and D-methionine. In still further embodiments, in addition to D-tyrosine, the composition further comprises one or more of D-alanine, D-cysteine, D-aspartic acid,
D-glutamic acid, D-phenylalanine, D-histidine, D-isoleucine, D-lysine, D-leucine, D-methionine, D-asparagine, D-proline, D-glutamine, D-arginine, D-serine, D-threonine, D-valine, and
D-tryptophan.
[0033] In some embodiments, the surface comprises a medical device, a wound dressing, a contact lens, or an oral device. In other embodiments, the medical device is selected from the group consisting of a clamp, forcep, scissors, skin hook, tubing, needle, retractor, scaler, drill, chisel, rasp, saw, catheter, orthopedic device, artificial heart valve, prosthetic joint, voice prosthetic, stent, shunt, pacemaker, surgical pin, respirator, ventilator, and an endoscope and combinations thereof.
[0034] In some embodiments of any of the foregoing methods, the method further comprises administering a biocicle. In some embodiments, the biocide is an antibiotic.
[0035] Another aspect of this disclosure is directed to an oral composition comprising an orally acceptable carrier and an effective amount of a guanidine. In some embodiments, the guanidine has Formula (I) or (II). In certain embodiments, the guanidine has Formulae (I) or (II) or is selected from the group of compounds in Table 1, or is a combination one or more compounds in Table 1, the D-amino acid is selected from, the group consisting of D-alanine, D-cysteine, D-aspartic acid, D-glutamic acid, D-histidine, D-isoleucine, D-lysine, D-leucine, D-asparagine, D-proline, D-glutamine, D-arginine, D-serine, D-threonine, D-valine,
D-tryptophan, D-tyrosine, and a combination thereof or the combination of D-amino acids is a synergistic combination of two or more D-amino acids selected from the group consisting of D- alanine, D-cysteine, D-aspartic acid, D-glutamic acid, D-phenylalanine, D-histidine, D- isoleucine, D-lysine, D-leucine, D-methionine, D-asparagine, D-proline, D-glutamine, D- arginine, D-serine, D-threonine, D-valine, D-tryptophan, and D-tyrosine.
(0036'j Yet another aspect of the invention is directed to compositions comprising at least one guanidine, or at least o e guanidine combined with at least one D-aniino acid or a mixture of D-amino acids, in an amount effective to treat, reduce, inhibit biofilm formation, or trigger biofilm disassembly. In certain embodiments, the guanidine has Formulae (I) or (II) or is selected from, the group of compounds in Table 1 , or is a combination one or more compounds in Table 1 , the D-amino acid is selected from the group consisting of D-alanine, D-cysteine, D- aspartic acid, D-glutamic acid, D-histidine, D-isoleucine, D-lysine, D-leucine, D-asparagine, D- proline, D-glutamine, D-arginine, D-serine, D-fhreonine, D-valine, D-tryptophan, D-tyrosine, and a combination thereof or the combination of D-amino acids is a synergistic combination of two or more D-amino acids selected from the group consisting of D-alanine, D-cysteine, D- aspartic acid, D-glutamic acid, D-phenylalanine, D-histidine, D-isoleucine, D-lysine, D-leucine, D-methionine, D-asparagine, D-proline, D-giutamine, D-arginine, D-serine, D-threonine, D- valine, D-tryptophan, and D-tyrosine.
[0037] In some embodiments, the composition comprises a guanidine and D-tyrosine, In certain embodiments, the guanidine has Formulae (I) or (II) or is selected from the group of compounds in Table 1 , or is a combination one or more compounds in Table 1 . In other embodiments the composition further comprises one or more of D-proline and D-phenylalanine. In still other embodiments, the composition further comprises one or more of D-leucine, D~ tryptophan, and D-methionine. In further embodiments, the composition further comprises one or more of D-alanine, D-cysteine, D-aspartic acid, D-glutamic acid, D-phenylalanine, D- histidine, D-isoleucine, D-lysine, D-leucine, D-methionine, D-asparagine, D-proline, D- glutamine, D-arginine, D-serine, D-threonine, D-valine, D-tryptophan, and D-tyrosine.
[0038] In some embodiments, any of the foregoing compositions can also comprise polyhexamethvlene biguanide, chlorhexidme, xylitol, triciosan, or chlorine dioxide. In other embodiments, any of the foregoing compositions can also comprise a pharmaceutically acceptable carrier. In still other embodiments of any the foregoing compositions, the effective amount is an amount effective to treat or prevent, a biofilm-related disorder. In some
embodiments, an effective amount, comprises an amount effective to treat or prevent a biofilm on a surface.
[0039] In yet other embodiments of any the foregoing compositions, the biofilm-related disorder is pneumonia, cystic fibrosis, otitis media, chronic obstructive pulmonary disease, or a urinary tract infection. In some embodiments, the biofilm-related disorder is a medical device- related infection.
[0040] In some embodiments of any of the foregoing compositions, the composition further comprises an agent suitable for application to the surface. In other embodiments of any of the foregoing compositions, the composition is formulated as a wash solution, a dressing, a wound gel, or a synthetic tissue. In further embodiments, the composition is formulated as tablets, pills, troches, capsules, aerosol spray, solutions, suspensions, gels, pastes, creams, or foams. In some embodiments, the composition is formulated for parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, vaginal and rectal administration.
[0041] Another aspect of this disclosure is directed to biofilm. resistant medical devices, comprising a surface likely to contact a biological fluid and a guanidine. In certain embodiments, the guanidine has Formulae (I) or (II) or is selected from the group of compounds in Table 1. in some embodiments, the medical device further comprises a D-amino acid, or a combination of D-amino acids, combined with the guanidine and coated on or impregnated into said surface. In some embodiments, the D-amino acid is selected from the group consisting of D-alanine, D-cysteine, D-aspartic acid, D-glutamic acid, D-histidine, D-isoleucine, D-lysine, D- leucine, D-asparagine, D~proline, D-glutamine, D-arginine, D-serine, D-threonine, D-valine, D- tryptoplian, D-tyrosine, and a combination thereof, or the combination of D-amino acids is in an amount effective to treat, reduce, or inhibit biofilrn formation, the combination of D-amino acids is a synergistic combination of two or more D-amino acids selected from the group consisting of D-alanine, D-cysteine, D-aspartic acid, D-glutamic acid, D-phenylalanine, D-histidine, D- isoleucine, D-lysine, D-leucine, D-methionine, D-asparagine, D-proline, D-glutamine, D- arginine, D-serine, D-threonine, D-valine, D-tryptophan, and D-tyrosine.
[0042] In some embodiments, the D-amino acid is D-tyrosine or the combination of D-amino acids comprises D-tyrosine, In other embodiments, the composition further comprises one or more of D proline and D phenylalanine. In other embodiments, the composition further comprises one or more of D-leucine, D-tryptophan, and D-methionine. In some embodiments, the composition further comprises one or more of D-alanine, D-cysteine, D-aspartic acid, D- glutamic acid, D-phenylalanine, D-histidine, D-isoleucine, D-lysine, D-leucine, D-methionine, D-asparagine, D-proline, D-glutamine, D-arginine, D-serine, D-threonine, D-valine, D- rryptophan, and D-tyrosine.
[0043] In some embodiments, the guanidine is formulated as a slow-release formulation. In certain embodiments, the guanidine combined with the D-amino acid is formulated as a slow- release formulation. In some embodiments, the surface is essentially free of L-amino acids. In further embodiments, the surface is essentially free of detergent. [0044] In some embodiments, the device is selected from one or more of clamp, forcep, scissors, skin hook, tubing, needle, retractor, scaler, drill, chisel, rasp, saw, catheter, orthopedic device, artificial heart valve, prosthetic joint, voice prosthetic, stent, shunt, pacemaker, surgical pin, respirator, ventilator and endoscope.
[0045] A further aspect of the instant disclosure is directed to potable liquids comprising a guanidine. In some embodiments, the guanidine has Formulae (I) or (II). In some embodiments, the guanidine is selected from the group of compounds in Table 1. In other embodiments at, least one guanidine is combined with at least one D-amino acid or a combination of D-amino acids, at a concentration in the range of 0.000001 % to 0.1 %. In some embodiments, wherein the guanidine has Formulae (! ) or (II) or is selected from the group of compounds in Table 1 , or is a combination one or more compounds in Table 1, the D-amino acid is selected from the group consisting of D-alanine, D-cysteine, D-aspartic acid, D-glutamic acid, D-histidine, D-isoleucine, D- lysine, D-leucine, D-asparagine, D-proiine, D-glutamine, D-arginine, D-serine, D-threonine, D-valiiie, D-tryptophan, D-tyrosine, and a combination thereof, or the combination of D-amino acids is a synergistic combination of two or more D-amino acids selected from the group consisting of D-alanine, D-cysteine, D-aspartic acid, D-glutamic acid, D-phenylalanine, D- histidine, D~isoleucine, D-lysine, D-leucine, D~methionine, D-asparagine, D-proline, D- glutamine, D-arginine, D-serine, D-threonine, D-valine, D-tryptophan, and D-tyrosine.
[0046] In some embodiments, the D-amino acid is D-tyrosine or the combination of D-amino acids comprises D-tyrosine, In other embodiments, the composition further comprises one or more of D proline and D phenylalanine. In other embodiments, the composition further comprises one or more of D-leucine, D-tryptophan, and D-methionine. In some embodiments, the composition further comprises one or more of D-alanine, D-cysteine, D-aspartic acid, D- glutamic acid, D-phenylalanine, D-histidine, D-isoleucine, D-lysine, D-leucine, D-methionine, D-asparagine, D-proline, D-glutamine, D-arginine, D-serine, D-threonine, D-valine, D- tryptophan, and D-tyrosine.
[0047] Another aspect of this disclosure is directed to compositions resistant to bio film formation, comprising a carrier and an effective amount of a guanidine or polyamine. In some embodiments, the carrier is pharmaceutically or cosmetically suitable base, a fluid base, or a polymeric binder. In some embodiments, the guanidine has Formulae (I) or (II). In some embodiments, the guanidine is selected from the group of compounds in Table 1. In certain embodiments, the guanidine is combined with a D-ammo acid or a combination of D-amino acids, distributed in the base, thereby treating, reducing or inhibiting formation of the biofilm. In some embodiments, the D-amino acid is selected from the group consisting of D-alamne, D~ cysteine, D-aspartic acid, D-glutamic acid, D-histidine, D-isoleucine, D-lysine, D-leucine, D- asparagine, D-proline, D-glutamine, D-arginine, D-serine, D-threonine, D-valine, D-tryptophan, D-tyrosine, and a combination thereof, or the combination of D-amino acids is a synergistic combination of two or more D-amino acids selected from the group consisting of D-alanine, D- cysteine, D-aspartic acid, D-giutaniic acid, D-phenylalanine, D-histidine, D-isoleucine, D-lysine, D-leucine, D-methionine, D-asparagine, D-proline, D-glutamine, D-arginine, D-serine, D- threonine, D-valine, D-tryptophan, and D-tyrosine.
[0048] Another aspect of this disclosure is directed to a coated article resistant to biofilm formation, wherein an article comprises a coating on at least one exposed surface and the coating comprises an amount of a guanidine effective to inhibit or reduce biofilm formation. In some embodiments, the guanidine has Formulae (I) or (IT). In some embodiments, the guanidine is selected from the group of compounds in Table 1 . In certain embodiments, the guanidine is combined with a D-amino acid or a combination of D-amino acids, thereby treating, reducing or inhibiting formation of the biofilm. In some embodiments, the D-amino acid is selected from the group consisting of D-alanine, D-cysteine, D-aspartic acid, D-glutamic acid, D-histidine, D- isoleucine, D-lysine, D-leucine, D-asparagine, D-proline, D-glutamine, D-arginine, D-serine, D- threonine, D-valine, D-tryptophan, D-tyrosine, and a combination thereof, or the combination of D-amino a cids is a synergistic combination of two or more D-amino acids selected from the group consisting of D-alanine, D-cysteine, D-aspartic acid, D-glutamic acid, D-phenylalanine, D-histidine, D-isolexicine, D-lysine, D-leucine, D-methionine, D-asparagine, D-proline, D- glutamine, D-arginine, D-serine, D-threonine, D-valine, D-tryptophan, and D-tyrosine.
[0049] In some embodiments of the methods and compositions of this disclosure, the base is essentially free of the corresponding L-amino acid or L-amino acids relative to the D-amino acids or combination of D-amino acids,
[0050] In some embodiments, the base is selected from a liquid, gel, paste, or powder. In further embodiments, the composition is selected from the group consisting of shampoos, bath additives, hair care preparations, soaps, lotions, creams, deodorants, skin-care preparations, cosmetic personal care preparations, intimate hygiene preparations, foot care preparations, light protective preparations, skin tanning preparations, insect repe Hants, antiperspirants, shaving preparations, hair removal preparations, fragrance preparations, dental care, denture care and mouth care preparations and combinations thereof.
Another aspect of the disclosure is directed to fatty acids salts of the guani dines and polyamines disclosed herein. In some embodiments, the fatty acid chain length of these salts ranges from C4 to C24. In some embodiments, the fatty acid chain length of these salts ranges from Ce to Ci6.
[0051] Yet another aspect of this disclosure is directed to compositions comprising a guanidine or polyarnine disclosed herein, or a combination of one or more gxiani dines and one or more polyamines, and a carrier structure. In some embodiments, the guanidine or polyamione or combination of one or more guanidines and one or more polyamines is incorporated into the carrier structure. In some embodiments, the carrier structure is a micelle. In some embodiments, the micelle, which may or may not be a mixed micelle comprising fatty acids of different chain lengths, comprises fatty acids having chain lengths ranging from Co to C20. In some
embodiments, the micelle comprises fatty acids and other lipids. In some embodiments, the other lipids are tauroeholate or cholesterol.
[0052] In some embodiments, the carrier structure is a liposome. In some embodiments, the liposome is unilamellar. In other embodiments, the liposome is multilamellar.
[0053] Another aspect of this disclosure is directed to using the fatty acid salts of the guanidines and polyamines disclosed herein in the methods, compositions, devices, liquids, and articles of this disclosure. A further aspect of this disclosure is directed to using the
compositions comprising a carrier structure and a guanidme or polyamine, or combination of one or more guanidines and one or more polyamines, in the methods, compositions, devices, liquids, and articl es of this disclosure.
BRIEF DESCRIPTION OF THE FIGURES
[0054] The following figures are presented for the purpose of illustration only, and are not intended to be limiting,
[0055] FIG. 1 A shows the inhibition of biofilm formation by compounds 6a, 7a, and 1 1 compared to the biofilm inhibition by norspermine and norspermidine. FIG. I B shows the breakdown of preexisting biofilms within 12 hours by compound 7.
[0056] FIG. 2A shows the averate protonation of polyamines and representative guanidines. FIG. 2B shows the classical, incorrect representation of protonated biguanidines. FIG. 2C shows the correct protonation state of compound 1 1 . FIG. 2D shows the cropped biguanidine moiety from crystal structure of compound 11.
[0057] FIGS. 3A and 3B shows the MBICs plotted against the average degree of protonation of polyamines and guani dines for B. subtilis and S, aureus., respectively.
[0058] FIG. 4 is a representation of the crystal structure of compound 2.
[0059] FIG. 5 is a representation of the crystal structure of compound 5.
[0060] FIG. 6 is a representation of the crystal structure of compound 1 1 .
FIGS. 7 A and 7B shows the inhibition of biofilm formation by compounds norspermidine micelle formulations A, B, and C.
DETAILED DESCRIPTION
[0061] 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, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present
specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
[0062] Other features and advantages of the invention will be apparent, from the following detailed description, and from, the claims. As will be apparent to one of skill in the art, specific features and embodiments described herein can be combined with any other feature or embodiment.
Definitions
[0063] The term "alkyl" refers to a hydrocarbon chain that may be a straight chain or branched chain. The chain may contain an indicated number of carbon atoms. For example, Q-C12 indicates that the group may have from 1 to 12 (inclusive) carbon atoms in it. An alkyl group can be substituted or unsubstituted. When substituted, one or more carbon atoms may be replaced with a heteroatom such as N, O, or S.
[0064] The term "alkoxy" refers to a straight or branched chain saturated or unsaturated hydrocarbon containing at least one oxygen atom. The chain may contain an indicated number of carbon atoms. For example, "C1-C12 alkoxy" indicates that the group may have from 1 to 12 (inclusive) carbon atoms and at least, one oxygen atom. Examples of a C1-C12 alkoxy include, but are not limited to, methoxy, ethoxy, isopropoxy, butoxy, n-pentoxy, isopentoxy, neopentoxy, and hexoxy. An alkoxy group can be substituted or unsubstituted. When substituted, one or more carbon atoms may be replaced with a heteroatom such as N, 0, or S.
[0065] The term "alkenyl" refers to a straight or branched chain hydrocarbon containing at least one carbon-carbon double bond. The chain may contain an indicated number of carbon atoms. For example, "Ci-C]2 alkenyl" indicates that the group may have from 1 to 12 (inclusive) carbon atoms and at least one carbon-carbon double bond. Exemplary such groups include, but are not limited to, ethenyi (also called "vinyl"), allyl, propenyl, crotyl, 2-isopentenyl, ailenyl, butenyl, butadienyl, pentenyl, pentadienvi, 3(l,4-pentadienyl), hexenyl and hexadienvl. When the indicated number of carbon atoms is 1, then the Ci alkenyl is double bonded to a carbon. An alkenyl group can be substituted or unsubstituted. When substituted, one or more carbon atoms may be replaced with a heteroatom such as N, O, or S.
[0066] The term "alkynyl" refers to a straight or branched chain hydrocarbon radical containing at least one carbon-carbon triple bond. The chain may contain an indicated number of carbon atoms. For example, "C2-C]2 alkynyl" indicates that the group may have from 2 to 12 (inclusive) carbon atoms and at least one carbon-carbon triple bond. Exemplary such groups include, but are not limited to, ethynyl, propynyl and butynyl. An alkynyl group can be substituted or unsubstituted. When substituted, one or more carbon atoms may be replaced with a heteroatom such as N, O, or S.
[0067] The term "cycloalkane" refers to saturated carbon ring systems having from. 3 to 20 carbons. Examples of cycloalkanes include, but are not limited to, cyclopropane, cyc!obutane, cyclopentane, cyclohexane, cycloheptane, and cyclooctane. A cycloalkane can be substituted or unsubstituted. When substituted, one or more carbon atoms may be replaced with a heteroatom such as IN, O, or S.
[0068 The term "aryl" refers to cyclic aromatic carbon ring systems containing from 6 to 18 carbons. Examples of an aryl group include, but are not limited to, phenyl, naphthyl, anthracenyl, tetracenyl, and phenanthrenyl. An aryl group can be unsubstituted or substituted. When substituted, one or more carbon atoms may be replaced with a heteroatom such as N, O, or S.
[0069] The term "aralkyl" refers to an alkyl group where an H has been replaced with an aryl group. An aralkyl group may be unsubstituted or it may be substituted on the hydrocarbon chain or the aryl ring. When substituted, one or more carbon atoms may be replaced with an N, O, or S. [0070] The term "heteroaryl" refers to mono and hicyciic aromatic groups of 4 to 10 atoms containing at least one heteroatom. Heteroatom as used in the term heteroaryl refers to oxygen, sulfur and nitrogen, A heteroaryl group can be unsubstituted or substituted.
[0071] The term "tautomer" refers to isomers of compounds that readily interconvert due to the phenomenon wherein a hydrogen atom or proton of one atom of a molecule shifts to another atom and is accompanied by a switch of single bond and adjacent double bond.
[0072] The terms "disorder", "disease", and "condition" are used herein interchangeably for a. condition in a subject. A disorder is a disturbance or derangement that affects the normal function of the body of a subject. A disease is a pathological condition of an organ, a body part, or a system resulting from various causes, such as infection, genetic defect, or environmental stress that is characterized by an identifiable group of symptoms. A disorder or disease can refer to a biofilm-related disorder that is characterized by a disease-related gro wth of bacteria in that a bion lm is established.
[0073] The terms "prevent," "preventing," and "prevention" refer herein to the inhibition of the development or onset of a biofilm or of a biofilm-related disorder or the prevention of the recurrence, onset, or development of one or more indications or symptoms of a biofilm or of a biofilm-related disorder on a surface or in a subject resulting from the administration of a composition described herein (e.g., a prophylactic or therapeutic composition), or the administration of a combination of therapies (e.g., a combination of prophylactic or therapeutic compositions).
[0074] As used herein, "treat", "treating" or "treatment" refers to administering a composition described herein in an amount, manner (e.g., schedule of administration), and/or mode (e.g., route of administration), effective to improve a disorder or a symptom thereof, or to prevent or slow the progression of a disorder or a symptom thereof. This can be evidenced by, e.g., an improvement in a parameter associated with a biofilm or with a biofilm-related disorder or an indication or symptom thereof, e.g., to a statistically significant degree or to a degree detectable to one skilled in the art. An effective amount, manner, or mode can vary depending on the surface, application, and/or subject and may be tailored to the surface, application, and/or subject. By preventing or slowing progression of a biofilm or of a biofilm-related disorder or an indication or symptom thereof, a treatment can prevent or slow deterioration resulting from a biofilm or from a biofilm-related disorder or an indication or symptom thereof on an effected surface or in an affected or diagnosed subject,
[0075] As sed herein, a "micel le" is defined as a particular type of molecular assembly in which amphipathic molecules are arranged in a approximately spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydro philic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.
(0076] As used herein, a "liposome" refers to a vesicle composed of lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior.
G aiiidisies
[0077] Generally, guanidines are a group of alkaline organic compounds that have a general common functional group of (R'R"N)(R"'R""N)C=NRs. The simplest guanidine has the formula (H2N)(H2N)C=NH and is a normal product of protein metabolism in humans. It is commonly used in the organic synthesis of plastics, resins, and explosives. Exampl es of other guanidines include the amino acid arginine, the organic soluble base triazabicyclodecene, and the naturally produced neurotoxin saxitocin.
[0078] This disclosure is directed to the surprising discovery that certain guanidines inhibit biofilm formation and/or trigger disassembly of existing biofilms. This disclosure is based, at least in part, on the discover}' that guanidines present in conditioned medium from mature biofilms inhibit biofilm formation and trigger the disassembly of existing biofilms. As shown in the Examples, it was discovered that the biofilm-inhibiting effect of the compounds in Table 2 was specific in that closely related guanidines, compounds I and 2 (differing only the number of methylene groups between the amino or guanidine groups), exhibited little activity. These discoveries, coupl ed with the results shown in the Examples, led to the development of the guanidines described herein that can inhibit biofilm formation and trigger biofilm disassembly. Guanidines discovered to be particularly suitable for use as biofilm inhibitors include guanidines comprising positively charged or ionizable amino or guanidine groups separated by three atoms.
[0079] Without being bound by theory, it is believed that the guanidines act to disrupt or inhibit biofilm by targeting the exopolysaccharide. There are several pieces of evidence, which are shown in the Examples, that support this. First, it was previously discovered that norspermidine and certain polyamines disrupt or inhibit biofilms by targeting the
exopolysaccharide, Norspermidine and a certain class of polyamines interact with the e opolysaccharide by binding to negatively charged or neutral sugars via hydrogen bonding. The class of guanidines disclosed herein also have similar structure-activity relationship where the activity depends on the charge of the amino and guanidine groups and certain spacing between the amino or guanidine groups.
[0080] Exopolysaccharides often contain negatively charged residues (e.g. uronic acid) or neutral sugars with polar groups (e.g. poly-N-acetylglucosamine). Without being bound by theory, it is possible that exopolysaccharide polymers form an interwoven meshwork in the matrix that helps hold cells together and that condensation of the polymers in response to norspermidiiie weakens the meshwork and causes release of polymers. Binding of the guanidines to negatively charged structures of the exopolysaccharide would neutralize the charge and collapse the interwoven meshwork leading to biofilm disruption. Accordingly, it is believed that, the structure and the charge of the amino and guanidine groups of the guanidines disclosed herein allow these certain guanidines to interact with the exopolysaccharide to cause the biofilm to disassemble.
[0081] Remarkably, the biofilm-inhibiting effect of the guanidines is not limited to B. subi is. As described in Example 16, the compounds in Table 2 were tested on inhibition of biofilm formation against B. subtilis, which as a natural producer of norspermidine served as a model, and S, aureus as a related pathogenic species with high clinical relevance.
Table 2
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
|0082] The results are shown in FIG. 1 A and the minimum biofiim inhibitory concentrations (MBICs) of the compounds tested in Example 16 are provided in Table 3. Some of the compounds exhibited remarkable activity for inhibition of biofilms with 5-19 times the activity on B. subtilis and more than 8 times the activity for S. aureus when compared to norspermidine.
Table 3. Minimal biofiim inhibitory concentrations (MBIC) for all compounds.
MBIC (uM) at pH 7.0
Compound
.5. subiilis S. aureus d
base 37.5±12.5 175+25
Norspermidine sulfate 500 250
chloride 150 500
Norspermine base 200 200
Di e thylene tri ami ne base > 1000 >1000
Biguanide chloride > 1000 >1000
Spermidine base 1000 1000
1 sulfate > 1000 >1000
sulfate >1000 750
b base 1000 1000 (50*)
3 chloride >1000 750
4 sulfate >1000 50
„ a sulfate 500 75
b base 375+125 400
chloride 10* >1000 (500*)
b base 10 50
sulfate 5 55+15
b base 2 250
sulfate 600 300
b base >1000 100
9 sulfate 100 500
10 formate 30 20+10
ί ί a chloride 30 300
11 b base 7±3 750+250
12 sulfate >1000 >1000
"MBICs for S. aureus are based on 75% reduction of biofiim formation, bfree base, cchloride, ''sulfate salts, *incomplete inhibition.
[0083] Furthermore, as also seen in Example 17 and FIG. IB, in addition to preventing biofiim. formation, the compounds were also able to trigger disassembly of existing biofilms.
[0084] Suprisingly, the counter ion of the amines and guanidmes also has an effect on activity. For instance, the free base of norspermidine was threefold more active than the chloride salt which was again about threefold more active in comparison to the free base for B, subiilis (Table 3). Therefore, free bases of selected compounds were generated and compared with the corresponding salts. For ?, subtilis, the free base of biofilm inhibiting compounds was more or at least equally active as the salt, while for S. aureus there was no clear trend. Solubility products ( s; determined as described in Example 18) indicate no correlation with the activity of the compounds and suggest that bioavaiiablitiy and delivery into the biofilm matrix may be relevant parameters (Table 7).
[0085] In addition to the structural and spatial properties of the guanidines, the charge of the amino and guanidines groups is also an important factor for inhibiting biofilm formation and triggering biofilm disassembly. To investigate the correlation between this important factor and the activity of the compounds, the pKa values of selected compounds were determined, as explained in Example 19. The results (shown in Table 7 after Example 19) show that the more effective guanidines have highly ionizable amino and guanidine groups and higher pKa values. This ionizable feature of the guanidines further supports the theory that the guanidines target the exopo lysacc haride .
[0086] The class of guanidines disclosed herein were developed as highly active agents for the prevention of biofilm formation that also were able to break down existing biofilms and may find application for medicinal purposes. The charge and spacing between positively charged groups are important factors for the mechanism and activity of the compounds. Furthermore, the activity of the disclosed biofilm inhibitors is in the lower range of what has been reported in the literature for compounds that, inhibit, the formation of biofilms without adversely affecting bacterial growth.
[0087] The guanidines described in this application were developed to interact with the e opolysaccharides of biofilms and prevent, biofilm formation by medically and industrially important mi croorgani sms . [0088] In some aspects, this disclosure features compositions, such as therapeutic or pharmaceutical compositions, comprising one or more guaniclines. in certain embodiments, the guanidine has at least three amino or guanidine groups separated by three atoms either in a. straight chain or cyclic molecule. In certain embodiments, the guanidine has Formula (I) or Formula (II),
Figure imgf000031_0001
or a base, salt, hydrate, or tautomer thereof, wherein
Y is Π Κ ,Κ ). O, S, NH, or NRI 0;
M is C(RiR.2)-Y-C(R.3R4), meta-phenylene, or 1 ,3-Cs-C7 cycloalkane, where the meta- phenylene or cycloalkane may be substituted by Rio or have one or more N heteroatoms in its
each i, R2, R3, R4, R5, and ¾ is H, Ci-C] ? alkyl, C1-C12 alkenyl, Ci-Ci2 alkynyl, Ci- Cj2 alkoxy, alkaryl, aryl, or heteroaryl, so that each Rj , R2, R3, R4, R5, and Re may be the same or different; each R7, R¾>, and R10 is H, C]-C]6 alkyl, C C , alkenyl, C3-C16 alkynyl, aryl, heteroaryl, or C7-C22 aralkyl, so that each R7, R9, and R10 may be the same or different; each R8 is H or -C(=NR7)-NR7H, so that each R8 may be the same or different; and each x an integer from 1 to 6, inclusive.
[0089] In some embodiments, the guanidine is a compound in Table 1.
[0090] In some embodiments, compositions of the present, disclosure include a compound from Table ! , or a combination of one or more compounds from Table I ,
[0091] In one or more embodiments, guanidines can inhibit biofilm formation in cell populations, and in particular, in biofilm-forming bacteria. By way of example, guanidines 6 and 7 significantly retard the formation of biofilm in bacteria! colonies such as Staphylococcus aureus and Bacillus subtilis. Guanidines have been demonstrated to reduce biofilm-forming activity by measuring the OD595 of cells that adhere to the surface as a measure of biofilm formation.
[0092] In one or more embodiments, guanidines can disrupt established biofilms. Even after bacteria have established a biofilm, contact of the biofilm with a solution containing a guanidine results in the dismption and disassembly of the pellicle. By way of example, guanidines can disrupt pellicles formed by bacterial colonies such as Bacillus subtilis. See, e.g., FIG. 1 and Example 17. Guanidines have been demonstrated to reduce biofilm-forming activity by measuring the OD595 of cells in free medium and compared to the OD595 in the residual pellicle.
[0093] In one aspect of this disclosure, a hiofilm-related disorder present in a subject in need thereof is treated by administering to the subject a composition having an effective amount of a guanidine, or a. pharmaceutically acceptable salt, or derivative thereof, thereby treating the biofilm-related disorder. A guanidine can be administered at a concentration of 0.1 nM to 100 μΜ, e.g. , 1 nM to 10 μΜ, 5 nM to 5 μΜ, or 10 nM to 1 μΜ, In other embodiments, a guanidine can be administered at a concentration of about 0.1 nM to about 100 μΜ, e.g. , about 1 nM to about 10 μΜ, about 5 nM to about 5 μΜ, or about 10 nM to about 1 μΜ.
[0094] Exemplary guanidines found to be particularly effective in inhibiting or treating biofi!m formation include compounds in Table 1. Those compounds can be used, for example, at concentrations of less than 1 niM, or less than 500 μ.Μ or less than 1 0 μΜ, or at a concentration of 0.1 nM to 100 μΜ, e.g., 1 M to 10 μΜ, 5 nM to 5 μΜ, or 10 nM to 1 μΜ.
Po!y amiss es
[0095] Polyamines are small organic compounds found in most cel ls. Polyamines putrescine (5 ,4-diaminobutane), spermidine (1 ,8-diamino-4-azaoctane) and spermine (l , 12-diamino-4,9- diazaoctane) are required in micromolar to miUimolar concentrations to support a wide variety of cellular functions. Depletion of polyamines can result in disruption of cellular functions and can cause cytotoxicity. For example, spermidine and spermine promote biofilm formation in some bacteria. Certain polyamines inhibit biofilm formation and/or disassemble existing biofilms.
[0096] Polyamines present in conditioned medium from mature biofilms inhibit biofilm formation and trigger the disassembly of existing biofilms. It was discovered that the biofiim- inhibiting effect of norspermidine was specific in that a closely related polyaniine, spermidine (differing only by an extra methylene group), exhibited little activity. Similarly, another polyaniine, norspermine, was also active in biofilm inhibition whereas its close relative spermine (once again, having an extra methylene) was inactive. These discoveries led to the development of the polyamines described herein that can inhibit biofilm formation and trigger biofilm disassembly. Polyamines discovered to be particularly suitable for use as biofilm inhibitors include polyamines comprising propylamine units and whose amino units are ionizable.
Polyamines suitable for use as biofilm inhibitors are described in U.S. Patent Application No.: 14/070,858, filed November 4, 2013, corresponding to PCT/US2012/036662, published as WO 2012/151554, the contents of which are hereby incorporated by reference.
Guanidiaes said Ammo Acids
[0097] It has been surprisingly discovered that a guanidine and a D-amino acid can act synergistically to inhibit biofilm formation or trigger biofilm disassembly, it has been discovered that guanidines and D-amino acids inhibit biofilm formation and trigger biofilm disassembly by different mechanisms. Thus, in one or more embodiments, a guanidine can be co administered with an amino acid, and in particular with a D-amino acid, to inhibit biofilm formation or trigger biofilm disassembly. The different mechanisms by which the guanidines and D-amino acids work result in synergism between the guanidine and D-amino acid and, in some embodiments, lower amounts of guanidines and D-amino acids are used to inhibit biofilm formation and/or trigger biofilm disassembly.
[0098] Standard amino acids can exist in either of two optical isomers, called L- or D-amino acids, which are mirror images of each other. While L-amino acids represent, the vast majority of amino acids found in proteins, D-amino acids are components of the peptidoglycan cell walls of bacteria. The D-amino acids described herein are capable of penetrating biofilms on living and non-living surfaces, of preventing the adhesion of bacteria to surfaces and any further build-up of the biofilm, of detaching such biofilm and/or inhibiting the further growth of the biofilm- forming micro-organisms in the biological matrix, or of killing such micro-organisms. D-amino acids are known in the art and can be prepared using known techniques. Exemplary methods include, e.g., those described in U.S. Pub!. No. 20090203091 . D-amino acids are also commercially available (e.g., from Sigma Chemicals, St. Louis, Mo.).
[0099] Any D-amino acid can be used in the methods described herein, including without limitation D-alanine, D-cysteine, D-aspartic acid, D-glutamic acid, D-phenylalanine, D-histidine, D-isoleucine, D- lysine, D-ieucine, D-methionine, D-asparagine, D-proline, D-glutamine, D- arginine, D-serine, D-threonine, D-valine, D-tryptophan, or D-tyrosine. A D-amino acid can be used alone or in combination with other D-amino acids. In exemplary methods, 2, 3, 4, 5, 6, or more D-amino acids are used in combination. Preferably, D-tyrosine, D-leucine, D-methionine, or D-tryptophan, either alone or in combination, are used in the methods described herein. In other preferred embodiments, D-tyrosine, D~pro!ine and D-phenylalanine, either alone or in combination, are used in the methods described herein.
[0100] A D-amino acid combined with a guamdine or po!yarnme can be administered at a concentration of 0.1 nM to 100 μΜ, e.g., 1 nM to 10 μΜ, 5 nM to 5 μΜ, or 10 nM to 1 μΜ. In other embodiments, a D-amino acid can be administered at a concentration of about 0, 1 nM to about 500 μ,Μ, e.g., about 1 nM to about 50 μ,Μ, about 5 nM to about 5 μ,Μ, or about 10 nM: to about 1 μΜ.
[0101] An exemplary D-amino acid found to be particular}}' effective in inhibiting or treating biofilm formation when combined with a guanidine or polyamine includes D-tyrosine. in some embodiments, D-tyrosine can be used, for example, as concentrations of less than 1 mM, or less than 100 μΜ or less than 10 μΜ, or at a concentration of 0.1 nM to 100 μΜ, e.g., 1 nM to 10 μΜ, 5 nM to 5 μΜ, or 10 nM to 1 μΜ,
[0102] In other embodiments, D-tyrosine is used in combination with one or more of D-proline and D-phenylalanine. In some embodiments, D-tyrosine is used in combination with one or more of D-leucine, D-tryptophan, and D-metliionine. The combinations of D-tyrosine with one or more of D-proline, D-phenylalanine, D-leucine, D-tryptophan, and D-methionine can be synergistic and can be effective in inhibiting or treating biofilm formation at total D-amino acid concentrations of 10 μΜ or less, e.g., about 1 nM to about 50 μ,Μ, about 5 nM to about 5 μΜ, or about 10 nM to about 1 μ,Μ, or at a concentration of 0.1 nM to 100 μΜ, e.g., 1 nM to 10 μΜ, 5 nM to 5 μΜ, or 10 nM to l μΜ.
[0103] In some embodiments, the combinations of guanidines or polyamines and D-amino acids are equimoiar. In some embodiments, the combinations of D-amino acids are equimoiar. In other embodiments, the combinations of D-amino acids are not in equimoiar amounts.
[0104] In some embodiments, the composition is essentially free of L-amino acids. For example, the composition comprises less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 1%, less than about 0.5%>, less than about 0,25%, less than about 0.1%, less than about 0.05%, less than about 0.025%, less than about 0.01%, less than about 0.005%, less than about 0.0025%, less than about 0.001%, or less, of L-amino acids. In other embodiments, the composition comprises less than 30%, less than 20%, less than 10%, less than 5%, less than 1%, less than 0.5%, less than 0.25%, less than 0.1%, less than 0.05%, less than 0.025%, less than 0.01 %, less than 0.005%, less than 0.0025%, less than 0.001% of L- amino acids. In preferred embodiments, the percentage of L-amino acid is relati ve to the corresponding D-amino acid. By way of example, a racemic mixture of L- amino acid and D- amino acid contains 50 % L-amino acid. 0105] In some embodiments, the composition is essentially free of detergent. For example, the composition comprises, less than about 30 wt %, less than about 20 wt %, less than about 10 wt %, less than about 5 wt %, less than about 1 wt %, less than about 0.5 wt %, less than about 0.25 wt %, less than about 0.1 wt %, less than about 0.05 wt %, less than about 0.025 wt %, less than about, 0.01 wt %, less than about 0.005 wt %, less than about, 0.0025 wt %, less than about 0.001 wt %, or less, of a detergent. In other embodiments, the composition comprises, relative to the overall composition, less than about 30 wt %, less than 20 wt %, less than 10 wt %, less than 5 wt %, less than 1 wt %, less than 0.5 wt %, less than 0.25 wt %, less than 0, 1 wt %, less than 0.05 wt %, less than 0.025 wt %, less than 0.01 wt %, less than 0.005 wt %, less than 0.0025 wt %, less than 0.001 wt % of a detergent. Many times in formulations containing detergents, e.g., surfactants, the surfactant will interact with the active agent, which could greatly affect the agent's efficacy. In some embodiments, it can be necessary to screen agents effectiveness relative to anionic surfactants, cationic surfactants, non-ionic surfactants and zwitter ionic surfactants as a screening to determine if the presence of the surfactant type alters the efficacy. Reducing or eliminating detergents, can increase the efficacy of the compositions and/or reduce formulation complications .
[0106] In other embodiments, the composition is essentially free of both detergent and L- amino acids.
Biofilms
[0107] Most bacteria can form complex, matrix-containing multicellular communities known as biofilms (O'Toole, et al, Annu. Rev. Microbiol. 54:49 (2000); Lopez, et al, FEMS Microbiol. Rev. 33: 152 (2009); aratan, et al, Microbiol. Mol. Biol. Rev. 73:310 (2009}).
Biofilm-associated bacteria are protected from environmental insults, such as antibiotics (Bryers, Biotechnol. Bioeng. 100: 1 (2008)). However, as biofiims age, nutrients become limiting, waste products accumulate, and it is advantageous for the biofilm-associated bacteria to return to a planktonic existence (Karatan, et al, Microbiol. Mol. Biol. Rev. 73:310 (2009)). Thus, biofiims have a finite lifetime, characterized by eventual disassembly.
[0108] Gram-negative bacteria, Gram-positive bacteria, and mycobacteria, in addition to other unicellular organisms, can produce biofiims. Bacterial biofiims are surface-attached communities of cells that are encased within an extracellular polysaccharide matrix produced by the colonizing cells. Biofilm development occurs by a series of programmed steps, which include initial attachment to a surface, formation of three-dimensional microcolonies, and the subsequent development of a mature biofilm. The more deeply a ceil is located within a biofilm (such as, the closer the cell is to the solid surface to which the biofilm is attached to, thus being more shielded and protected by the bulk of the biofilm matrix), the more metabolically inactive the cells are. The consequences of this physiologic variation and gradient create a collection of bacterial communities where there is an efficient system established whereby microorganisms have diverse functional traits. A biofilm also is made up of various and diverse non-cellular components and can include, but are not limited to carbohydrates (simple and complex), lipids, proteins (including polypeptides), and lipid complexes of sugars and proteins
(lipopolysaccharid.es and lipoproteins), A biofilm may include an integrated community of two or more bacteria species (polymicrobic biofiims), or predominantly one specific bacterium.
[0109] The biofilm can allow bacteria to exist in a dormant state for a certain amount of time until suitable growth conditions arise thus offering the microorganism a selective advantage to ensure its survival. However, this selection can pose serious threats to human health in that biofilms have been observed to be involved in about 65% of human bacterial infections (Smith, Adv. Dmg Deliv. Rev. 57: 1539-1550 (2005); Hail-Stoodley, et ai, Nat. Rev. Microbiol. 2:95- 108 (2004)).
[0110] As described herein, biofilms can also affect a wide variety of biological, medical, and processing operations,
Biofilm-For^
[0111] The methods described herein can be used to prevent or delay the formation of, and/or treat, biofilms. in exemplary methods, the biofilms are formed by biofilm-forming bacteria.. The bacteria can be a gram negative bacterial species or a gram positive bacterial species. Nonlimiting examples of such bacteria include a member of the genus Actinobacillus (such as Actinobacillus actinomycetemcomitans), a m ember of the genus Acinetobacter (such as Acinetobacter baumannii), a member of the genus Aeromonas, a member of the genus Bordetella (such as Bordetella pertussis, Bordetella bronchiseptica, or Bordetella parapertussis), a member of the genus Brevibacillus, a member of the genus Brucella, a member of the genus Bacteroides (such as Bacteroides fragilis), a member of the genus Burkholderia (such as Burkholderia cepacia or Burkholderia pseudomallei), a member of the genus Borelia (such as Borelia burgdorferi), a member of the genus Bacillus (such as Bacillus anthracis or Bacillus subtilis), a member of the genus Campylobacter (such as Campylobacter jejuni), a member of the genus Capnocytophaga, a member of the genus Cardiobacterium (such as Cardiobacterium hominis), a member of the genus Citrobacter, a member of the genus Clostridium (such as Clostridium telani or Clostridium difficile), a member of the genus Chlamydia (such as Chlamydia trachomatis, Chlamydia pneumoniae, or Chlamydia psijfaci), a member of the genus Eikenella (such as Eikenella corrodens), a member of the genus Enterobacter, a member of the genus Escherichia (such as Escherichia coll), a member of the genus Francisella (such as Francisella tularensis), a member of the genus Fusohacterium, a member of the genus Flavobacterium, a member of the genus Haemophilus (such as Haemophilus ducreyi or Haemophilus influenzae), a member of the genus Helicobacter (such as Helicobacter pylori), a member of the genus Kingella (such as Kingella kingae), a member of the genus Klebsiella (such as Klebsiella pneumoniae), a member of the genus Legionella (such as Legionella pneumophila), a member of the genus Listeria (such as Listeria monocytogenes), a member of the genus Leptospirae, a member of the genus Moraxella (such as Moraxella catarrhalis), a member of the genus Morganella, a member of the genus Mycoplasma (such as Mycoplasma hominis or Mycoplasma pneumoniae), a member of the genus Mycobacterium (such as Mycobacterium tuberculosis or Mycobacterium leprae), a member of the genus Neisseria (such as Neisseria gonorrhoeae or Neisseria meningitidis), a member of the genus Pasteurella (such as Pasteur ella multocida), a member of the genus Proteus (such as Proteus vulgaris or Proteus mirablis), a. member of the genus Prevotella, a member of the genus Plesiomonas (such as Plesiomonas shigelloides), a member of the genus Pseudomonas (such as Pseudomonas aeruginosa), a member of the genus Providencia, a member of the genus Rickettsia (such as Rickettsia rickettsii or Rickettsia typhi), a member of the genus Stenotrophomonas (such as Stenolrophomonas maltophila), a member of the genus Staphylococcus (such as Staphylococcus aureus or Staphylococcus epidermidis), a member of the genus Streptococcus (such as Streptococcus viridans, Streptococcus pyogenes (group A), Streptococcus agalactiae (group B), Streptococcus bovis, or Streptococcus pneumoniae), a member of the genus Streptomyces (such as Streptomyces hygroscopicus), a member of the genus Salmonella (such as Salmonella enteriditis. Salmonella typhi, or
Salmonella typhimurium), a. member of the genus Serratia (such as Serratia marcescens), a member of the genus Shigella, a member of the genus Spirillum (such as Spirillum minus), a. member of the genus Treponema (such as Treponema pallidum), a member of the genus Veillonella, a member of the genus Vibrio (such as Vibrio cholerae. Vibrio parahemolyticus , or Vibrio vulnificus), a member of the genus Yersinia (such as Yersinia enterocolitica, Yersinia pestis, or Yersinia pseudotuberculosis), and a member of the genus Xanthomonas (such as Xanthomonas maltophilia).
[0112] Specifically, Bacillus subtilis forms architecturally complex communities on semisolid surfaces and thick pellicles at the air/liquid interface of standing cultures (Lopez, et al , FEMS Microbiol Rev. 33: 152 (2009); Aguilar, et al, Curr. Opin. Microbiol. 10:638 (2007); Vlamakis, et al. , Genes Dev. 22:945 (2008); Branda, et al , Proc. Natl. Acad. Sci. USA 98: 1 1621 (2001)). B. subtilis biofilms consist of long chains of cells held together by an extracellular matrix consisting of an exopolysaccharide and amyloid fibers composed of the protein TasA (Branda, el al, Proc. Natl. Acad. Sci. USA 98: 11621 (2001 ); Branda, et al, Mol. Microbiol. 59: 1229 (2006); Romero, et al... Proc. Natl. Acad. Sci. USA (2010, in press)). The
exopolysaccharide is produced by enzymes encoded by the epsA-0 operon ("eps operon") and the TasA protein is encoded by the promoter-distal gene of the yqxM-sipW-tasA operon ("yqxM operon") (Chu, et al, Mol. Microbiol. 59: 1216 (2006)).
(0113] Biofilm-producing bacteria, e.g., a species described herein, can be found in a live subject, in vitro, or on a surface, as described herein.
(0114] It has now been surprisingly discovered that short and medium chain fatty acid salts of norspermidine showed increased activity compared to norspermidine alone. This discovery has been exploited to develop formulations of guanidines and polyamines, including fatty acid salts, micelles, and liposomes, with increased activity. The formulation of the guanidines and poiyamines as fatty acid salts may be accomplished, in some embodiments, by using fatty acids having chain lengths ranging from C4 to C20. In other embodiments, fatty acids having chain lengths ranging from C4 to C8 are used. As described below in Examples 15 and 16, certain fatty acid salts of some of the guanidines and poiyamines were made and tested. The MBICs of those fatty acid salt s are shown below in Table 4,
Table : Minimal bio film inhibitory e OBcentratioiis (M BIC) for fat ty acid salts of poiyamines and guanidines
Compound MBIC (μ .Μ ) at pH 7.0
B. subtil is
base 37.5±12.5
sulfate 500
chloride 150
Norspermidine hexanoate 15±5
octanoate
dodecanoate >200
palmitate >200
stearate >200
Hexanoic acid >200
Octanoic acid 2500
Dodecanoic acid >200
Palmitic acid >200
Stearic acid >200
5 a sulfate 500
b base 375±125
c octanoate 5
6 a chloride 10*
b base 10
c octanoate 10 "J a sulfate 5
b base 7
c octanoate <5
1 1 a chloride 30
b base 7±3
c octanoate 10
[0115] As can be seen in Table 4, the compounds formulated as fatty acid salts exhibit increased activity when compared to the sulfates or chlorides and free bases. For example, Compound 5, which was previously only moderately active, had increased activity as an octanoic acid salt that was 100-fold more potent than the sulfate and 70-fold more potent than the free base. Accordingly, these results demonstrate the increased activity of the fatty acid salts of the guanidines and polyamines described herein when compared to the guanidines and polyamines themselves. 0116] This disclosure is also directed to further exploitation of the discovery of the efficacy of fatty acid salts of guanidines and polyamines to develop additional formulations. The guanidines and polyamines described herein, or a combination of one or more guanidines and one or more polyamines, can be formulated so that they are incorporated into carrier structures comprising lipids including, but not limited to, micelles, liposomes, and lipid particles. Such formulations can be even more potent, than the fatty acid salts alone and provide a convenient, way for formulation and application. As described in Examples 15 and 16, various micelle formulations were prepared and tested. Table 5 shows the MBICs for the various norspermidine micelle formulations.
Table S: MBICs for norspermidine micelle formulations*
MB!C (juM) at pH 7.0 |
1 Formulation
B. subiiiis
base 37.5±12.5
Norspermidine sulfate 500
chloride 150
9:1 Tetradecanoic acid : Norspermidine
15
tetradecanoate (formulation C)
1 :1 Tetradecanoic acid : Norspermidine
10±2
tetradecanoate
Norspermidine dodecanoate
5±3
(formulation A)
Norspermidine tetradecanoate
10
(formulation B)
*formuiated in 0.9% saline and 10 mM sodium taurocholate
[0117] As can be seen, the micelles formulated with fatty acids and norspermidine are even more potent than the fatty acid salts of norspermidine alone. Accordingly, formulations comprising carrier structures and one or more of the guanidines or polyamines disclosed herein, or a combination of one or more of the guanidines and one or more of the polyamines disclosed herein, would also have increased potency. Therefore, in some instances, one or more guanidines or polyamines, or a combination of one or more guanidines and one or more polyamines, can be incorporated into a micelle. In some instances, the micelles, including mixed micelles having fatty acids of different chain lengths, can be formulated with fatty acids ranging from C6 to C2o in chain length.
[0118] Carrier structures also include lipid particles comprising one or more charged lipids, A complex of nucleic acid and lipid particles can be referred to as an association complex. An association complex of nucleic acid and lipid particle may be a liposome, a nanoparticle, an ion pair, a lipoplex, or a combination thereof. Lipoplexes are composed of charged lipid biiayers sandwiched between DNA layers, as described, e.g., in Feigner, Scientific American. Lipid particles include, but are not limited to, liposomes. Liposomes may have one or more lipid membranes. Many different types of liposomes exist and many liposomes can be used with the guanidines and polyamines of this disclosure. A liposome can be used with a guanidine or polyamine so long as the liposome can be formulated with the guanidines and/or polyamines to make a stable formulation. The liposomes may be unilamellar or multilamellar.
[0119] The lipid particles of the present invention may further comprise one or more additional lipids and/or other components such as cholesterol. Other lipids may be included in the liposome compositions of the present invention for a variety of purposes, such as to prevent lipid oxidation or to attach ligands onto the liposome surface. Any of a number of lipids may be present in liposomes of the present invention, including amphipathic, neutral, catio ic, and anionic lipids. Such lipids can be used alone or in combination. Specific examples of additional lipid components that may be present are described below.
[0120] Additional components that may be present in a lipid particle of the present invention include bilayer stabilizing components such as polyamide oligomers (see, e.g., U.S. Patent No. 6,320,017), peptides, proteins, detergents, lipid-derivatives, such as PEG coupled to
phosphatidylethanolamine and PEG conjugated to ceramides (see, e.g., U.S. Patent No,
5,885,613).
[0121] In particular embodiments, the lipid particles include one or more of a second amino lipid or charged lipid, a neutral lipid, and a sterol.
[0122] Neutral lipids, when present in the lipid particle, can be any of a number of lipid species which exist either in an uncharged or neutral zwitterionic form at physiological pH. Such lipids include, for example phosphocholines (PC), phospbatidylethanolarmnes (PE), phosphatidylserines (PS), cardiolipins, diacylphosphatidylcholine,
diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and eerebrosides. The selection of neutral lipids for use in the particles described herein is generally guided by consideration of, e.g., liposome size and stability of the liposomes in conjunction with the guanidines and polyamines. Preferably, the neutral lipid component is a lipid having two acyl groups, (i.e., diacylphosphatidylcholine and diacylphosphatidylethanolamine). Lipids having a variety of acyl chain groups of varying chain length and degree of saturation are available or may be isolated or synthesized by well-known techniques. In one group of embodiments, lipids containing saturated fatty acids with carbon chain lengths in the range of Cio to C20 are used, in another group of embodiments, lipids with mono or ^unsaturated fatty acids with carbon chain lengths in the range of C10 to C20 are used. Additionally, lipids having mixtures of saturated and unsaturated fatty acid chains can be used. Preferably, the neutral lipids used in the present invention are DOPE, DSPC, POPC, DPPC or any related
phosphatidylcholine. The neutral lipids useful in the present invention may also be composed of sphingomyelin, dihydrosphingomyeline, or phospholipids with other head gro ps, such as serine and inositol.
[0123] The sterol component of the lipid mixture, when present, can be any of those sterols conventionally used in the field of liposome, lipid vesicle or lipid particle preparation. An exemplary sterol is cholesterol.
[0124] Other protonatable lipids, which carry a net positive charge at about physiological pH, in addition to those specifically described above, may also be included in lipid particles of the present invention. Such protonatable lipids include, but are not limited to, N,N~dioleyl~N,N- dimethylammonium chloride ("DODAC"); N-(2,3- dioleyloxy)propyl-N,N-N-triethylammonium chloride ("DOTMA"); N,N-distearyl-N,N- dimethvlammonium bromide ("DDAB"); N-(2,3- dioleoyloxy)propyi)-N,N,N- trimethylammonium chloride ("DOTAP"); l,2-Dioieyloxy-3- trimethylaminopropane chloride salt ("DOTAP. CI"); 3P-(N-(N',N'-dimethylaminoethane)- carbamoyijcholesterol ("DC-Choi"), N-(l-(2,3-dioleyloxy)propyl)-N-2- (spermineearboxamido)efhyi)-N,N- dimethylammonium trifluor acetate ("DOSPA"), dioctadecylamidoglycyl carboxyspermine ("DOGS"), l,2-dileoyl-sn-3-phosphoethanolamine ("DOPE"), 1 ,2- dioleoyl-3-dimethyiammonium propane ("DODAP"), N, N-dimethyl-2,3- dioleyloxy propylamine ("DODMA"), and N-(l,2-dimyristyloxyprop-3-yl)-N,N- dimethyl-N- hydroxyetbyl ammonium bromide ("D RIE"). Additionally, a number of commercial preparations of lipids can be used, including, but not limited to, LIPOFECTiN (including DOTMA and DOPE, available from GiBCO/BRL), and LIPOFECTAMINE (comprising DOSPA and DOPE, available from GIBCO/BRL).
[0125] Anionic lipids suitable for use in lipid particles include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N- dodecanoyl phosphatidylethanoloamine, N-succinyl phosphatidylethanolamine, N- glutaryl phosphatidylethanolamine, lysylphosphatidylgiycerol, and other anionic modifying groups joined to neutral lipids.
[0126] In numerous embodiments, amphipathic lipids are included in lipid particles of the present invention. "Amphipathic lipids" refer to any suitable material, wherein the hydrophobic portion of the lipid material orients into a hydrophobic phase, while the hydrophilic portion orients toward the aqueous phase. Such compounds include, but are not limited to,
phospholipids, aminolipids, and sphingoiipids. Representative phospholipids include sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidyiserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatdylcholine,
lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, or dilinoleoylphosphatidylcholine. Other phosphorus-lacking compounds, such as sphingolipids, glycosphingolipid families, diacylgiycerols, and β-acyloxyacids, can also be used. Additionally, such amphipathic lipids can be readily mixed with other lipids, such as triglycerides and sterols.
[0127] In some cases, the lipid particle can include a lipid selected to reduce aggregation of lipid particles during formation, which may result from steric stabilization of particles which prevents charge-induced aggregation during formation.
[0128] Examples of lipids that reduce aggregation of particles during formation include polyethylene glycol (PEG)-modified lipids, monosialoganglioside Gml, and polyarmde oligomers ("PAO") such as (described in U.S. Pat. No. 6,320,017). Other compounds with uncharged, hydrophilic, steric -barrier moieties, which prevent aggregation during formulation, like PEG, Gml or ATTA, can also be coupled to lipids for use as in the methods and
compositions of the invention. ATTA-lipids are described, e.g. , in U.S. Patent No, 6,320,017, and PEG-lipid conjugates are described, e.g., in U.S. Patent Nos. 5,820,873, 5,534,499 and 5,885,613. Typically, the concentration of the lipid component selected to reduce aggregation is about 1 to 15% (by mole percent of lipids). Specific examples of PEG-modified lipids (or lipid- polyoxyethylene conjugates) that are useful in the present invention can have a variety of "anchoring" lipid portions to secure the PEG portion to the surface of the lipid vesicle.
Examples of suitable PEG- modified lipids include PEG-modified phosphatidylethanolamine and phosphatide acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20) which are described in USSN 08/486,214, PEG-modified dialkylamines and PEG-modified 1,2- diacyloxypropan-3-amines. Particularly preferred are PEG-modified diacylgiycerols and dialkylgiycerols. [0129] In embodiments where a sterically-large moiety such as PEG or ATTA are conjugated to a lipid anchor, the selection of the lipid anchor depends on what type of association the conjugate is to have with the lipid particle. It is understood that mPEG
(mw2000)-diastearoylphosphatidylemanolamine (PEG-DSPE) will remain associated with a liposome until the particle is cleared from circulation, possibly a matter of days. Other conjugates, such as PEG~CerC20 have similar staying capacity. PEG- CerC14, however, rapidly exchanges out of the formulation upon exposure to serum, with a Ti/2 less than 60 mins. in some assays. As illustrated in U.S. Pat. Application SN 08/486,214, at least three characteristics influence the rate of exchange: length of acyl chain, saturation of acyl chain, and size of the steric-barrier head group. Compounds having suitable variations of these features can be useful in some aspects of this present disclosure. For some therapeutic applications, the PEG-modified lipid may be formulated to be rapidly lost from the nucleic acid-lipid particle in vivo and hence the PEG-modified lipid will possess relatively short lipid anchors. In other therapeutic applications, t e nucleic acid-lipid particle may exhibit a longer plasma circulation lifetime and hence the PEG-modified lipid will possess relatively longer lipid anchors.
[0130] Micelles for use with the guanidines and polyamines described herein may be formed by mixing an aqueous solution of one or more guanidines, one or more polyamines, or a combination of one or more guanidines and one or more polyamines, and micelle forming compounds. Micelle forming compounds to form micelles for use with the guanidines and polyamines of this disclosure include fatty acids having chain length from C6 to C20. The micelle forming fatty acids may be added at the same time or after the guanidine or poly amine.
Emulsifying agents such as bile salts (e.g., sodium taurocholate) may be added in a buffered solution to formulate micelles. Micelles will form when the concentration of the fatty acids is greater than the critical micelle concentration (CMC). Once the CMC is met, the fatty acids will form a micelle that contains a guanidine or polyamine. [0131] When a guanidine or polyamine, or a combination, is incorporated into a liposome, the aqueous portion within the liposome contains the guanidine or polyamine, or combination. Commercially available liposomes can used or liposomes can be prepared using known techniques. For example, liposomes can be prepared using dipalmitoyl phosphatidyl choline (DPPC) or dipalmitoyl phosphatidyl glycerol (DPPG) and cholesterol in buffered aqueous solutions followed by extrusion. The guanidmes and polyarchies may be added during or after the liposome preparation process.
[0132] In instances where a guanidine, or a combination of a guanidine and a D-amino acid, is to be administered to a subject, the guanidmes and D-amino acids described herein can be incorporated into pharmaceutical compositions. The guanidmes and D-amino acids can be incorporated into pharmaceutical compositions as pharmaceutically acceptable salts or derivatives. Such compositions typically include a guanidine, or a guanidine and a D-amino acid, and a pharmaceutically acceptable carrier. As used herein, a "pharmaceutically acceptable carrier" means a carrier that can be administered to a subject together with a guanidine or with a guanidine and a D-amino acid described herein, which does not destroy the pharmacological activity thereof. Pharmaceutically acceptable carriers include, e.g., solvents, binders, dispersion media, coatings, preservatives, colorants, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.
[0133] The term "pharmaceutically acceptable salts" includes, but is not limited to, water- soluble and water-insoluble salts, such as the acetate, amsonate (4,4-diarninostilbene-2,2- disulfonate), benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camsyiate, carbonate, chloride, citrate, clavulariate, ^hydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, maiate, nialeate, mandelate, mesylate, methylbromide, methyinitrate, methylsulfate, mueate, napsylate, nitrate, N methylgiucamine ammonium salt, 3-hydroxy-2-naphthoate, oleate, oxalate, palniitate, pamoate (l ,l-metheiie-bis-2-hydroxy-3-naphthoate, einbonate), pantothenate,
phosphate/diphosphate, picrate, polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate, suhacetate, succinate, sulfate, sulfosaliculate, suxamate, tannate, tartrate, teoelate, tosylate, triethiodide, and valerate salts.
[0134] The D-amino acids may also be in the form of esters or derivatives. Examples of suitable esters include formates, acetates, propionates, butyrates, isobutyrates, pentanoates, crotonates, and benzoates. Some pharmaceutically acceptable derivatives include a chemical group which increases aqueous solubility.
[0ί35| Non-limiting examples of pharmaceutically acceptable carriers that can be used include poiy(ethylene-co-vinyl acetate), PVA, partially hydrolyzed poly(ethylene-co-vinyl acetate), poly(ethylene-co-vinyi acetate-co-vinyl alcohol), a cross-linked poly(ethylene-co-vinyl acetate), a cross-linked partially hydrolyzed poly(ethylene-co- vinyl acetate), a cross-linked poly(ethylene-co-vinyl acetate-co-vinyl alcohol), poly-D,L-lactic acid, poly-L-lactic acid, polyglycolic acid, PGA , copolymers of lactic acid and glycolic acid (PLGA), polycaprolactone, polyvalerolactone, poly (anhydrides), copolymers of polycaprolactone with polyethylene glycol, copolymers of polylactic acid with polyethylene glycol, polyethylene glycol; and combinations and blends thereof.
[0136] Other carriers include, e.g., an aqueous gelatin, an aqueous protein, a polymeric carrier, a cross-linking agent, or a combination thereof. In other instances, the carrier is a matrix. In yet another instances, the carrier includes water, a pharmaceutically acceptable buffer salt, a pharmaceutically acceptable buffer solution, a pharmaceutically acceptable antioxidant, ascorbic acid, one or more low molecular weight pharmaceutically acceptable polypeptides, a peptide comprising about 2 to about 10 amino acid residues, one or more pharmaceutically acceptable proteins, one or more pharmaceutically acceptable amino acids, an essential-to-human amino acid, one or more pharmaceutically acceptable carbohydrates, one or more pharmaceuticaily aceeptable carbohydrate-derived materials, a non-reducing sugar, glucose, sucrose, sorbitol, trehalose, mannitol, maltodextrin, dextrins, cyclodextrin, a pharmaceutically acceptable chelating agent, EDTA, DTPA, a chelating agent for a divalent metal ion, a chelating agent for a trivalent metal ion, glutathione, pharmaceutically acceptable nonspecific serum albumin, and/or combinations thereof.
[0137] A pharmaceutical composition containing a guanidine, or a combination of a guanidine and a D-amino acid, can be formulated to be compatible with its intended route of administration as known by those of ordinary skill in the art. Nonlimiting examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, vaginal and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. [0138] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition can be sterile and can be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of
microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. It may be desirable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be accomplished by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin (see, e.g., Remington: The Science and Practice of
Pharmacy, 21 st edition, Lippincott Wil liams & Wilkins, Gennaro, ed. (2006)).
[0139] Sterile injectable solutions can be prepared by incorporating a guanidine, or combination of a guanidine and a D-amino acid, in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation include, without limitation, vacuum drying and freeze- drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
[0140] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, a guanidine, or a combination of a guanidine and a D-amino a cid, can be incorporated with excipients and used in the form of tablets, pills, troches, or capsules, e.g. , gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
[0141] For administration by inhalation, a guanidine, or a combination of a guanidine and a D-amino acid, can be delivered in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
[0142] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, but, are not limited to, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accompl ished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into, e.g., ointments, salves, gels, or creams as generally known in the art.
[0143] For treatment of acute or chronic wounds, a guanidine, or a combination of a guanidine and a D-amino acid, can be formulated as a dressing, a wash solution, gel, or a synthetic tissue.
[0144] A biofilm can form on an oral surface (such as teeth, tongue, back of throat, and the like). These bio films can be associated with day-to-day bacterial activity of natural flora located in such environments, but can also be associated with oral-related disease(s), such as periodontal disease (for example, gingivitis or periodontitis), breath malodor, or dental caries. By example, periodontitis, a common form of periodontal disease, is believed to be caused by a small group of Gram-negative bacteria present on the tooth root surfaces as biofilms, in particular,
Porphyromonas gingivalis, Bacieroides forsyihus and Actinobacillus actinomycetemcomitans, with the latter found mostly in cases of juvenile periodontitis. Other bacteria which may be involved in periodontal disease include T. denticola, T. socranskii, F. nucleatum, and P.
intermedia, L. acidophilus, L. cased, A. viscosus, S. sobrinus, S sanguis, S. viridans, and
S. mutans. Application of a guanidine, or a combination of a guanidine and a D-amino acid, onto such oral surfaces can inhibit or prevent bacterial biofilm formation. Generally, application onto such oral surfaces will be via a product which, in the ordinary course of usage, is not
intentionally swallowed for purposes of systemic administration but is rather retained in the oral cavity for a time sufficient to contact substantially all of the dental surfaces and/or oral tissues. The guanidine, or combination of a guanidine and D-amino acid, for use on oral surfaces can be formulated as a gum, paste (such as toothpaste), which can then be directly applied to the biofilm of such a surface in a subject. The paste formulation can further comprise an abrasive. A guanidine, or combination of a guanidine and D-amino acid, can also exist as a gel formulation or in liquid formulation. For example, the guanidine, or combination of a guanidine and D- amino acid, can be formulated as a mouthwash that can directly come into contact with the biofilm on the oral surface of a. subject. Additionally, a guanidine, or combination of a. guanidine and D-amino acid, can be formulated as a polymer film or platelet (e.g., as a slow-release formulation) for treating or preventing oral conditions. In one embodiment, the guanidine, or combination of a guanidine and D-amino acid , of the present invention may be used for adjunctive antimicrobial therapy for periodontitis and applied directly to a tooth or between teeth in the form of a chip. The oral care compositions of the present invention may be in various forms including therapeutic rinses, especially mouth rinses; dentifrices such as toothpastes, tooth gels, and tooth powders; non-abrasive gels; mouth sprays; mousse; foams; chewing gums, lozenges and breath mints; drinking water additives; dental solutions and irrigation fluids; and dental implements such as dental floss and tape. The dental implement can be impregnated fibers including dental floss or tape, chips, strips, films and polymer fibers.
[0145] For example, an oral composition can contain from about 0.01 % to about 15 % by weight, e.g., 0.01 % to 15 % by weight, based on the total weight of the composition, of one or more guanidine, or combination of a guanidine and D-amino acid, and orally tolerable adjuvants. One nonlimiting example of an oral composition includes 10 % by weight, sorbitol, 10 % by weight glycerol, 1 5 % by weight ethanol, 15 % by weight propylene glycol, 0.5 % by weight sodium lauryl sulfate, 0.25 % by weight sodium methylcocyl taurate, 0.25 % by weight polyoxypropylene/polyoxyethylene block copolymer, 0.50 % by weight peppermint flavouring, 0.1 to 0.5 % by weight of one or more guanidine, or combination of a guanidine and D-amino acid, and 48.6 % by weight water.
[0146] An oral composition can be, for example, in the form of a gel, a paste, a cream or an aqueous preparation (mouthwash). The oral composition can also comprise compounds that release fluoride ions which are effective against the formation of caries, for example inorganic fluoride salts, e.g. sodium, potassium, ammonium or calcium fluoride, or organic fluoride salts, e.g. amine fluorides, which are known under the trade name OLAFLUOR. Oral compositions can further comprise compounds known in the art to be "orally acceptable carriers," which as used herein means conventional additives in oral care compositions including but not limited to fluoride ion sources, anti-calculus or anti-tartar agents, buffers, abrasives such as silica, bleaching agents such as peroxide sources, alkali metal bicarbonate salts, thickening materials, humectants, water, surfactants, titanium dioxide, flavor system, sweetening agents, xylitol, coloring agents, and mixtures thereof. Such materials are well known in the art and are readily chosen by one skilled in the art based on the physical, aesthetic and performance properties desired for the compositions being prepared. These carriers may be included at levels typically from about 50% to about 99%, preferably from about 70% to about 98%, and more preferably from about 90% to about 95%, by weight of the oral composition. The choice of a carrier to be used is basically determined by the way the composition is to be introduced into the oral cavity. In one preferred embodiment, the oral compositions are in the form of dentifrices, such as toothpastes, tooth gels and tooth powders. Components of such toothpaste and tooth gels general ly include one or more of a dental abrasive (from about 6% to about 50%), a surfactant (from about 0.5% to about 10%), a thickening agent (from about 0.1 % to about 5%), a humectant (from about 10% to about 55%), a flavoring agent (from about 0.04% to about 2%), a sweetening agent (from about 0.1 % to about 3%), a coloring agent (from about 0.01% to about 0.5%) and water (from about 2% to about 45%). Such toothpaste or tooth gel may also include one or more of an anticaries agent (from about 0,05% to about 0.3% as fluoride ion) and an anticalcuius agent (from about 0.1% to about 13%). Tooth powders contain substantially ail non-liquid
components. Other preferred oral care compositions are liquid products, including mouthwashes or rinses, mouth sprays, dental solutions and irrigation fluids. Components of such mouthwashes and mouth sprays typically include one or more of water (from about 45% to about 95%), ethanol (from about 0% to about 25%), a humectant (from about 0% to about 50%), a surfactant (from about 0.01% to about 7%), a flavoring agent (from about 0.04% to about 2%), a sweetening agent (from about 0.1% to about 3%), and a coloring agent (from about 0.001% to about 0.5%). Such mouthwashes and mouth sprays may also include one or more of an anticaries agent (from about 0.05% to about 0.3% as fluoride ion) and an anticalculus agent (from about 0.1 % to about 3%). Components of dental solutions generally include one or more of water (from about 90% to about 99%), preservative (from about 0,01 % to about 0.5%), thickening agent (from 0% to about 5%), flavoring agent (from about 0.04% to about 2%), sweetening agent (from about 0.1% to about 3%), and surfactant (from 0% to about 5%).
[0147] The pharmaceutical compositions containing a guanidine, or combination of a guanidine and D-amino acid, can also be prepared in the form of suppositories (e.g. , with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
[0148] In some embodiments, the composition is essentially free of detergent. In some instances, a detergent can contribute to the toxicity of a composition. For example, the composition comprises less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 1 %, less than about 0.5%, less than about 0.25%, less than about 0.1 %, less than about 0.05%, less than about 0.025%, less than about 0.01%, less than about 0.005%, less than about 0.0025%, less than about 0,001 %, or less, of a detergent, e.g., less than 30%, less than 20%, less than 10%, less than 5%, less than 1 %, less than 0.5%, less than 0.25%, less than about 0.1 %, less than 0.05%, less than 0.025%, less than 0.01 %, less than 0.005%, less than about 0.0025%, less than 0.001%, of a detergent. [0149] Some pharmaceutical compositions can be prepared with a carrier that protects the guanidine, or combination of a guanidine and D-amino acid, against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems (as described, e.g., in Tan, et ah, Pharm, Res. 24:2297-2308, 2007).
Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations are apparent to those skilled in the art. The materials can also be obtained commercially (e.g., from Alza Corp., Mountain View, CA). Liposomal suspensions (including liposomes targeted to particular cells with monoclonal antibodies to cell surface antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, e.g. , as described in U.S. Pat. No.
4,522,81 1 .
[0150] It may be advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
[0151] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. , for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effecti ve in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to normal cells and, thereby, reduce side effects.
[0152] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies generally within a range of circulating concentrations that include the EL½ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods described herein, the
therapeutically effective dose can be estimated initially from cell culture assays. A. dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC5.3 (i.e., the concentration of the test compound which achie ves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography. Information for preparing and testing such compositions are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy, 21st edition, Lippincott Williams & Wilkins, Gennaro, ed. (2006)).
[0153] In some instances, about 0.0005 uM guanidine to about 50 uM guanidine is administered, e.g., about 0.001 uM guanidine to about 25 μΜ guanidine, about 0.002 uM guanidine to about 10 uM guanidine, about 0.003 uM guanidine to about 5 uM guanidine, about 0.004 uM guanidine to about 1 uM guanidine, about 0.005 uM guanidine to about 0.5 uM guanidine, about 0.05 μ,Μ guanidine to about 0.1 μ,Μ guanidine, or about 0.02 μ,Μ guanidine to about 0.1 μΜ guanidine, e.g., .0005 uM guanidine to 50 μ guanidine is administered, 0.005 μ,Μ guanidine to 25 μΜ guanidine, 0.002 μΜ guanidine to 10 μΜ guanidine, 0.003 μΜ guanidine to 5 μΜ guanidine, 0.004 μΜ guanidine to 1 μΜ guanidine, 0.005 μ,Μ guanidine to 0.5 μΜ guanidine, 0.01 μ,Μ guanidine to 0.1 μ.Μ guanidine, or 0.02 μ,Μ guanidine to 0.1 μ,Μ guanidine.
[0154] When combined with a guanidine, about 0.0005 μΜ D-amino acid to about 50 μ,Μ D- amino acid is administered, e.g., about 0.001 μΜ D-amino acid to about 25 μΜ D-amino acid, about 0.002 μΜ D-amino acid to about 10 μΜ D-amino acid, about 0.003 μΜ D-amino acid to about 5 μΜ D-amino acid, about 0.004 μΜ D-amino acid to about 1 μΜ D-amino acid, about 0.005 μΜ D-amino acid to about 0.5 μΜ D-amino acid, about, 0.01 μΜ D-amino acid to about 0.1 μΜ D-amino acid, or about 0.02 μΜ D-amino acid to about 0.1 μΜ D-amino acid, e.g., .0005 μ,Μ D-amino acid to 50 μΜ D-amino acid is administered, 0.001 μ,Μ D-amino acid to 25 μ D- ami o acid, 0.002 μΜ D-amino acid to 10 μ D-amino acid, 0.003 μ,Μ D-amino acid to 5 μΜ D-amino acid, 0.004 μΜ D-amino acid to 1 μΜ D-amino acid, 0.005 μΜ D-amino acid to 0.5 μΜ D-amino acid, 0.01 μΜ D-amino acid to 0.1 μΜ D-amino acid, or 0.02 μΜ D-amino acid to 0.1 μΜ D-amino acid. Preferably, a D-amino acid is administered at nanomolar concentrations, e.g., at about 5 nM, at about 10 nM, at about 15 nM, at about 20 nM, at about 25 nM, at about 30 uM, at about 50 nM, or more, or preferably at 5 nM, at 10 nM, at 15 nM, at 20 nM, at 25 nM, at 30 nM, or at 50 nm.
[0155] In other instances, a therapeutically effective amount or dosage of a guanidine, or combination of a guanidine and D-amino acid, can range from about, 0.001 mg/kg body weight, to about 100 mg/kg body weight, e.g., from about 0.01 mg/kg body weight to about 50 mg/kg body weight, from about 0.025 mg/kg body weight to about 25 mg/kg body weight, from about 0.1 mg/kg body weight to about 20 mg/kg body weight, from about 0.25 mg/kg body weight to about 20 mg/kg body weight, from about 0,5 mg/kg body weight to about 20 mg/kg body weight, from about 0.5 mg/kg body weight to about 10 mg/kg body weight, from about 1 mg kg body weight to about 10 mg/kg body weight, or about 5 mg/kg body weight, or preferably 0.001 mg/kg body weight to 100 nig/kg body weight, e.g., from 0.01 mg/kg body weight to 50 mg/kg body weight, from 0.025 mg/kg body weight to 25 mg/kg body weight, from 0.1 mg/kg body weight to 20 mg/kg body weight, from 0.25 mg/kg body weight to 20 mg/kg body weight, from 0.5 mg/kg body weight to 20 mg/kg body weight, from 0.5 mg/kg body weight to 10 mg/kg body weight, from 1 mg/kg body weight to 10 mg/kg body weight, or 5 mg/kg body weight,
[0156] A physician will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a guanidine, or combination of a guanidine and D-amino acid, can include a single treatment or a series of treatments. In one example, a subject is treated with a guanidine, or combination of a guanidine and D-amino acid, in the range of between about 0.06 mg to about 120 nig, one time per week for between about 1 to 10 weeks, alternatively between 2 to 8 wreeks, between about 3 to 7 weeks, or for about 4, 5, or 6 weeks, or preferably between 0.06 mg to 120 mg, one time per week for between 1 to 10 weeks, alternatively between 2 to 8 weeks, between 3 to 7 weeks, or for 4, 5, or 6 weeks. It will also be appreciated that the effective dosage of a guanidine, or combination of a guanidine and D-amino acid, used for treatment, may increase or decrease over the course of a particular treatment.
[0157] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. A person of ordinary skill in the art will appreciate that the pharmaceutical compositions described herein can be formulated as single-dose vials.
[0158] Treatment of a subject with a therapeutically effecti ve amount of a guanidine-, or combination of a guanidine and D-amino acid-, containing pharmaceutical composition described herein can be a single treatment, continuous treatment, or a series of treatments divided into multiple doses. The treatment can include a single administration, continuous administration, or periodic administration over one or more years. Chronic, long-term administration can be indicated in some cases. Generally, each formulation is administered in an amount sufficient to suppress or reduce or eliminate a deleterious effect or a symptom of a. biofilm-related disorder or condition described herein.
[0159] Guanidines, or combinations of guanidines and D-amino acids, are suitable as antibiofilm active substances in personal care preparations, for example shampoos, bath additives, hair care preparations, liquid and solid soaps (based on synthetic surfactants and salts of saturated and/or unsaturated fatty acids), lotions and creams, deodorants, other aqueous or alcoholic solutions, e.g. cleansing solutions for the skin, moist cleaning cloths, oils or powders. Fropionibacterium acnes, which is the predominant microorganism occurring in acne, may reside in biofilms. Thus, guanidines, or combinations of guanidines and D-amino acids, are particularly suitable for personal care compositions for use in controlling acne. The invention accordingly relates also to personal care preparations comprising one or more guanidine, or a combination of a guanidine and D-amino acid, described herein and cosmetically tolerable carriers or adjuvants,
[0160] The guanidines, or combinations of guanidines and D-amino acids, described herein are also suitable for imparting antibiofilm properties to a range of formulations used in personal care. Personal care preparations can contain from about, 0.01% to about 15 % by weight, for example, from about 0.1% to about 10 % by weight, or 0.01% to 15 % by weight, for example, from 0.1 % to 10 % by weight, based on the total weight of the preparation, of one or more guanidine, or combination of a guanidine and D-amino acid, and cosmetically tolerable adjuvants. Depending on the form of the personal care preparation, such preparation can include, in addition to one or more guanidines, or combinations of guanidines and D-amino acids, further constituents, for example sequestering agents, colourings, perfume oils, thickening or solidifying agents (consistency regulators), emollients, UV-absorbers, skin protective agents, antioxidants, additives that improve the mechanical properties, such as dicarboxylic acids and/or aluminium, zinc, calcium or magnesium salts of C14-C22 fatty acids, and, optionally,
preservatives.
[0161] In one embodiment, the anti-acne composition comprising a guanidine, or a combination of a guanidine and D-amino acid, can further comprise at least one antimicrobial agent. Preferably, the antimicrobial agent is an antibiotic. The antibiotic may be selected from, the group consisting of tobramycin, clindamycin, ciprofloxacin, tetracyclines, rifampin, triclosan, oxfloxacin, macrolides, penicillins, cephalosporins, amoxicillia^lavulante,
quinupristin/dalfopristin, amoxicillin/sulbactum, metronidazole, fluoroquinolones, quinolones, ketoiides, or aminoglycosides. The present invention provides a method for controlling acne, comprising administering to a subject afflicted with acne an effective amount of an anti-acne composition comprising one or more guanidines, or combinations of guanidines and D-amino acids, wherein the amount of the guanidine, or combination of a guanidine and D-amino acid, in the anti-acne composition is sufficient to prevent, reduce, inhibit or remove a biofilm.
[0162] Personal care preparations can be in the form of a water-in-oil or oil-in-water emulsion, an alcoholic or alcohol-containing formulation, a vesicular dispersion of an ionic or non-ionic ampiphilic lipid, a gel, a solid stick or an aerosol formulation. As a water-in-oil or oil- in-water emulsion, the cosmetically tolerable adjuvant, contains preferably from about 5 % to about 50 % of an oil phase, from about 5 % to about 20 % of an emulsifier and from about 30 % to 90 % water, or 5 % to 50 % of an oil phase, from 5 % to 20 % of an emulsifier and from 30 % to 90 % water. The oil phase can comprise any oil suitable for cosmetic formulations, for example one or more hydrocarbon oils, a wax, a natural oil, a silicone oil, a fatty acid ester or a fatty alcohol. Preferred mono- or poly-ols are ethanol, isopropanol, propylene glycol, hexylene glycol, glycerol and sorbitol.
[0163] Cosmetic formulations described herein are used in various fields. Such preparations include, without limitation, for example: skin-care preparations, e.g. skin-washing and cleansing preparations in the form of tablet-form or liquid soaps, synthetic detergents or washing pastes, bath preparations, e.g. liquid (foam baths, milks, shower preparations) or solid bath preparations, e.g. bath cubes and bath salts; skin-care preparations, e.g. skin emulsions, multi-emulsions or skin oils; cosmetic personal care preparations, e.g. facial make-up in the form of day creams or powder creams, face powder (loose or pressed), rouge or cream makeup, eye-care preparations, e.g. eye shadow preparations, mascaras, eyeliners, eye creams or eye-fix creams; lip-care preparations, e.g. lipsticks, lip gloss, lip contour pencils, nail-care preparations, such as nail varnish, nail varnish removers, nail hardeners or cuticle removers; intimate hygiene preparations, e.g. intimate washing lotions or intimate sprays; foot-care preparations, e.g. foot baths, foot powders, foot creams or foot balsams, special deodorants and antiperspirants or callus-removing preparations; light-protective preparations, such as sun milks, lotions, creams or oils, sunblocks or tropicals, pre-tanning preparations or after-sun preparations; skin-tanning preparations, e.g. self-tanning creams; depigmenting preparations, e.g. preparations for bleaching the skin or skin- lightening preparations; insect-repellents, e.g. insect-repellent oils, lotions, sprays or sticks; deodorants, such as deodorant sprays, pump-action sprays, deodorant gels, sticks or roll-ons; antiperspirants, e.g. antiperspirant sticks, creams or rol l-ons; preparations for cleansing and caring for blemished skin, e.g. synthetic detergents (solid or liquid), peeling or scrub preparations or peeling masks; hair-removal preparations in chemical form (depilation), e.g. hair-removing powders, liquid hair-removing preparations, cream- or paste-form, hair-removing preparations, hair-removing preparations in gel form or aerosol foams; shaving preparations, e.g. shaving soap, foaming shaving creams, non-foaming shaving creams, foams and gels, preshave preparations for dry shaving, aftershaves or aftershave lotions; fragrance preparations, e.g. fragrances (eau de Cologne, eau de toilette, eau de parfum, parfum de toilette, perfume), perfume oils or perfume creams; dental care, denture-care and mouth-care preparations, e.g. toothpastes, gel toothpastes, tooth powders, mouthwash concentrates, anti-plaque mouthwashes, denture cleaners or denture fixatives; cosmetic hair-treatment preparations, e.g. hair-washing preparations in the form of shampoos and conditioners, hair-care preparations, e.g. pretreatment preparations, hair tonics, styling creams, styling gels, pomades, hair rinses, treatment packs, intensive hair treatments, hair-structuring preparations, e.g. hair- waving preparations for permanent waves (hot wave, mild wave, cold wave), hair- straightening preparations, liquid hair-setting preparations, hair foams, hairsprays, bleaching preparations, e.g. hydrogen peroxide solutions, lightening shampoos, bleaching creams, bleaching powders, bleaching pastes or oils, temporary, semipermanent or permanent hair colorants, preparations containing self-oxidising dyes, or natural hair colorants, such as henna or camomile.
[0164] The following represent noniimiting examples of various formulations that can be prepared containing one or more guanidines, or combinations of guanidines and D-amino acids, A wide variety of similar formulations are known in the art into which one or more guanidines, or combinations of guanidines and D-amino acids, can readily be incorporated at various concentrations.
[0165] An exemplary soap has, for example, the following composition: 0.01 to 5 % by weight of one or more guanidines, or combinations of guanidines and D-amino acids, 0.3 to 1 % by weight titanium dioxide, 1 to 10 % by weight stearic acid, soap base ad 100 %, e.g. a sodium salt of tallow fatty acid or coconut fatty acid, or glycerol.
[0166] An exemplary shampoo has, for example, the following composition: 0,01 to 5 % by weight of one or more guanidines, or combination of a guanidine and D-amino acid, 12.0 % by weight sodium laureth-2-sulfate, 4.0 % by weight cocamidopropyl betaine, 3.0 % by weight NaCl and water ad 100 %.
[0167] An exemplary deodorant has, for example, the following composition: 0.01 to 5 % by weight of one or more guanidines, or combination of a guanidine and D-amino acid, 60 % by weight ethanol, 0.3 % by weight perfume oil, and water ad 100 %. |Ό168] In some instances, a pharmaceutical composition comprising a guanidine, or combination of a guanidine and D-amino acid, is administered to prevent or reduce biofilm formation on a biologically relevant surface or substrate. These surfaces include, but are not limited to, an epithelial or mucosal surface of the respirator}' tract, lungs, the oral cavity, the alimentary and vaginal tracts, in the ear or the surface of the eye, and the urinary tract. For example, a biofilm can affect, the surface of a lung (such as the lung of a subject with pneumonia, cystic fibrosis, or COPD), such as epithelial cells of the lung,
[01 9] In certain embodiments, the surface is a biologically relevant surface is a surface that is likely to contact a biological fluid, e.g., a liquid component of a subject such as blood, serum, sputum, lacrimal secretions, semen, urine, vaginal secretions, and tissue samples and the like. The biologically relevant surface can be a component of a medical device, instrument, or implant. Nonlimiting examples include clamps, forceps, scissors, skin hooks, tubing (such as endotracheal or gastrointestinal tubes), needles, retractors, scalers, drills, chisels, rasps, saws, catheters including indwelling catheter (such as urinary catheters, vascular catheters, peritoneal dialysis catheter, central venous catheters), catheter components (such as needles, Leur-Lok connectors, needleless connectors), orthopedic devices, artificial heart valves, prosthetic joints, voice prostheses, stents, shunts, pacemakers, surgical pins, respirators, ventilators, and endoscopes. The present invention is particularly well-suited to substantially reduce the risk of biofilm accumulation on the surfaces of a medical device adapted for prolonged term
implantation, wherein the medical device is intended to remain implanted for a relatively long period of from about 30 days to about 12 months or longer, and the resultant likelihood of premature failure of the device due to encrustation and occlusion by such biofilm. However, such encrustation may occur on medical devices after shorter periods of time, such as 30 days or less, as well, which would also be understood to be devices for prolonged term implantation. For example, in certain embodiments, a medical device utilized for a prolonged period of time may implanted for a period longer than 24 hours, such as a week.
[0170] In certain instances, a subject can be administered a guanidine, or combination of a guanidine and D-amino acid, prior to, during, or after implantation/insertion of a medical device, catheter, stent, prosthesis, and the like, or application of a wound dressing. In some instances, the wound dressing includes an antimicrobial, such as silver. Treatment before or after implantation can take place immediately before or after the implantation or several hours before or after implantation, or at, a time or times that the skilled physician deems appropriate.
[0171] A guanidine, or combination of a guanidine and D-amino acid, can be applied to a surface by any known means, such as by covering, coating, contacting, associating with, filling, or loading the surface with a therapeutic amount of a guanidine, or combination of a guanidine and D-amino acid. In specific examples, a guanidine, or combination of a guanidine and D- amino acid, is directly affixing to a surface by either spraying the surface with a
polymer/guanidine, or a polymer/combination of a guanidine and D-amino acid, film, by dipping the surface into a polymer/guanidine solution, or a polymer/combination of a guanidine and D- amino acid solution, or by other covalent or noncovalent means. In other instances, the surface is coated with a substance (such as a hydrogel) that absorbs the guanidine, or combination of a guanidine and D-amino acid.
[0172] The composition can be a coating or a film. When applied as a part of a film or coating, one or more guanidine, or combination of a guanidine and D-amino acid, described herein can be part of a composition which also comprises a binder. The binder may be any polymer or oligomer compatible with the present antibiofilms. The binder may be in the form of a polymer or oligomer prior to preparation of the antibiofilm composition, or may form by polymerization during or after preparation, including after application to the substrate. In certain applications, such as certain coating applications, it will be desirable to crosslink the oligomer or polymer of the antibiofiim composition after application. The term "binder" as used herein includes materials such as glycols, oils, waxes and surfactants commercially used in the pharmaceutical and personal care industries. It is preferred that materials that are Generally Regarded as Safe (G.R.A.S.) be used.
[0173] When the composition is a thermoplastic film which is applied to a surface, for example, by the use of an adhesive or by melt applications including calendaring and co- extrusion, the binder is the thermoplastic polymer matrix used to prepare the film. When the composition is a coating, it may be applied as a liquid solution or suspension, a paste, gel, oil or the coating composition may be a solid, for example a powder coating which is subsequently cured by heat, UV light or other method.
[0174] As the composition of the in vention may be a coating or a film, the binder can be comprised of any polymer used in coating formulations or film preparation. For example, the binder is a thermoset, thermoplastic, elastomeric, inherently crosslinkecl or crosslmked polymer. Thermoset, thermoplastic, elastomeric, inherently crosslinked or crosslmked polymers include polyolefin, polyamide, polyurethane, polyacrylate, polyaerylamide, polycarbonate, polystyrene, polyvinyl acetates, polyvinyl alcohols, polyester, halogenated vinyl polymers such as PVC, natural and synthetic rubbers, alkyd resins, epoxy resins, unsaturated polyesters, unsaturated polyamides, polyimides, silicon containing and carbamate polymers, ftuorraated polymers, crosslinkable acrylic resins derived from substituted acrylic esters, e.g. from epoxy acrylates, urethane acrylates or polyester acrylates. The polymers may also be blends and copolymers of the preceding chemistries.
[0175] Biocompatible coating polymers, such as, poly[-alkoxyalkanoate-co-3- hydroxyalkenoate] (PHAE) polyesters, Geiger, et. al, Polymer Bulletin 52, 65-70 (2004), can also serve as binders in the present invention. Alkyd resins, polyesters, polyurethanes, epoxy resins, silicone containing polymers, polyacrylates, polyacrylamides, fluorinated polymers and polymers of vinyl acetate, vinyl alcohol and vinyl amine are non-limiting examples of common coating binders useful in the present invention. Other known coating binders are part of the present disclosure.
[0176] Coatings can be crosslinked with, for example, melamine resins, urea resins, isocyanates, isocyanurates, polyisocyanates, epoxy resins, anhydrides, poly acids and amines, with or without accelerators. The compositions described herein can be, for example, a coating applied to a surface which is exposed to conditions favorable for bi ©accumulation. The presence of one or more guanidines, or combinations of guanidines and D-amino acids, described herein in said coating can prevent the adherence of organisms to the surface.
[0177] The coating may be solvent borne or aqueous. Aqueous coatings are typically considered more environmentally friendly. In some examples, the coating can be an aqueous dispersion of one or more guanidines, or combinations of guanidines and D-amino acids, described herein and a binder or a water based coating or paint. For example, the coating can comprise an aqueous dispersion of one or more guanidines, or combinations of guanidines and D-amino acids, and an acrylic, methaeryiic or acrylamide polymers or co-polymers or a poly[-alkoxyalkanoate-co-3-hydroxyalkenoate] polyester.
[0178] In some instances, the coating composition can be applied to a surface by any conventional means including spin coating, dip coating, spray coating, draw down, or by brush, roller or other applicator. A drying or curing period can be performed.
[0179] Coating or film thickness can vary depending on the application and can readily be determined by one skilled in the art after limited testing. [0180] In some instances, a composition described herein can be in the form of a protective laminate film. Such a film can comprise thermoset, thermoplastic, elastomeric, or crosslinked polymers. Examples of such polymers include, but are not limited to, polyolefin, polyamide, polyurethane, polyacrylate, polyaerylamide, polycarbonate, polystyrene, polyvinyl acetates, polyvinyl alcohols, polyester, halogenated vinyl polymers such as PVC, natural and synthetic rubbers, alk d resins, epoxy resins, unsaturated polyesters, unsaturated polyamides, polyimides, fiuorinated polymers, silicon containing and carbamate polymers. The polymers can also be blends and copolymers of the preceding chemistries.
[0181] When a composition described herein is a preformed film, it can be applied to a surface by, for example, the use of an adhesive, or co-extruded onto the surface. It can also be mechanically affixed via fasteners which may require the use of a sealant or caulk wherein the esters of the instant invention may also be advantageously employed. A plastic film can also be applied with heat which includes calendaring, melt applications and shrink wrapping.
[0182] Given the wide array of applications for the guanidines, or combinations of guanidines and D-amino acids, described herein, a guanidine-containing composition, or a composition containing a combination of a guanidine and D-amino acid, can include other additives such as antioxidants, UV absorbers, hindered amines, phosphites or phosphonites, henzofuran-2-ones, thiosynergists, polyamide stabilizers, metal stearates, nucleating agents, fillers, reinforcing agents, lubricants, ernu!sifiers, dyes, pigments, dispersants, other optical brighteners, flame retardants, antistatic agents, blowing agents and the like, such as the materials listed below, or mixtures thereof.
[0183] Medical devices prepared from plastic can incorporate a guanidine, or combination of a guanidine and D-amino acid, during the forming, e.g., molding, process. Plastic-based medical devices that benefit from the present method include, but are not limited to, plastics articles used in the field of medicine, e.g. dressing materials, syringes, catheters etc., so-called "medical devices", gloves and mattresses. Exemplary of such plastics are polypropylene, polyethylene, PVC, POM, polysulfones, polyethersulfones, polystyrenics, polyamides, polyurethanes, polyesters, polycarbonate, polyaerylics and methaerylies, polybutadienes, thermoplastic polyolefins, ionomers, unsaturated polyesters and blends of polymer resins including ABS, SAN and PC/ABS.
[0184] The guanidines, or combinations of guanidines and D-amino acids, especially in low concentrations, can be safely used even in applications where ingestion is possible, such as reusable water bottles or drinking fountains where a biofilm may develop. The surfaces of such water transport devices can be rinsed with a formulation containing one or more guanidines, or combinations of guanidines and D-amino acids, described herein, or low levels of one or more guanidines, or combinations of guanidines and D-amino acids, can be introduced into the water that passes through the containers of conduits. For example, about 0.0001% or less or up to about 1%, typically less than about 0.1% by weight of one or more guanidines, or combinations of guanidines and D-amino acids, may be introduced into such water. Given the high activity of the instant guanidines, or combinations of guanidines and D-amino acids, very small amounts are effective in many circumstances and concentrations of about 0.000001% to about, 0.1%, for example, about 0.000001 % to about 0.01 %, or about 0.000001% to about 0.001 %, or 0.000001% to 0.1%, 0.000001% to 0.01 %, or 0.000001 % to 0.005 %, can be used in such applications.
[0185] When used in a coating or film, small amounts of one or more guanidines, or combinations of guanidines and D-amino acids, can be present for short term use, for example, one use, seasonal or disposable items, especially those applications which involve possible human contact, splints, catheters, tubing, dental equipment etc. In general, about 0.001% or less up to about 5%, for example up to about 3% or about 2%, or preferably 0.001% or less up to 5%, up to 3% or 2% by weight of one or more amino acids may be used in such coatings or films. Given the high activity of the instant guanidines, or combinations of guani dines and D-amino acids, very small amounts are effective in many circumstances and concentrations of about 0.0001% to about 1%, for example, about 0.0001 % to about 0.5%, or about 0.0001% to about 0.01 % can be used in coating applications, or preferably 0.0001% to 1%, 0.0001 % to 0.5%, or 0.0001% to 0.01 % by weight of one or more guanidines, or combinations of guanidines and D- amino acids.
[0186] For incorporation into a molded plastic article, about 0.00001 % to about 10% of one or more guanidines, or combinations of guanidines and D-amino acids, can be used, for example about 0.0001 % to about 3%, for example about 0.005 % up to about 1% one or more guanidines, or combinations of guanidines and D-amino acids, can be used, or preferably, 0.00001% to 10%, 0.0001% to 3 0.001% up to 1 % by weight one or more guanidines, or combinations of guanidines and D-amino acids, can be used, in situations in which the guanidines, or combinations of guanidines and D-amino acids, are impregnated into the surface of an already prepared molded article or fiber, the actual amount of a guanidine, or combination of a guanidine and D-amino-acid, present at, the surface can depend on the substrate material, the formulation of the impregnating composition, and the time and temperature used during the impregnation step. Given the high activity of the instant guanidines, or combinations of guanidines and D-amino acids, very small amounts are effective in many circumstances, and concentrations of about 0.0001 % to about 5 %, for example, about 0.0005 % to about 0.1%, or about 0.0001% to about 0.01 % can be used in plastics, or preferably 0.0001 % to 1 %, 0.0001% to 0.1%, or 0.0001% to 0.01 % by weight of one or more amino acids can by used..
[0187] Inhibition or reduction in a biofiim by treatment with a guanidine, or combination of a guanidine and D-amino acid, can be measured using techniques well established in the art. These techniques enable one to assess bacterial attachment by measuring the staining of the adherent biomass, to view microbes in vivo using microscopy methods; or to monitor cell death in the biofilm in response to toxic agents. Followi g treatment, the biofilm can be reduced with respect to the surface area covered by the biofilm, thickness, and consistency (for example, the integrity of the biofilm). Non-limiting examples of biofilm assays include microtiter plate biofilm. assays, fluorescence-based biofilm. assays, static biofilm assays according to Walker, et ah, Infect. Immun. 73:3693-3701 (2005), air-liquid interface assays, colony biofilm. assays, and Kadouri Drip-Fed Biofilm assays (Merritt, et at, (2005) Current Protocols in Microbiology l .B.1.1 -1 .B.l .17). Such assays can be used to measure the activity of a D-amino acid on the disruption or the inhibition of formation of a biofilm (Lew, et aL, (2000) Curr. Med. Chem. 7(6):663-72; Werner, et aL, (2006) Brief Funct. Genomic Froteomic 5(l):32-6).
[0188] In other instances, treatment can be assayed by measuring the growth of bacteria and/or can be quantified by measuring the density of a biofilm-forming bacteria in a biological sample, Non- limiting examples of biological samples include blood , serum, sputum, lacrimal secretions, semen, urine, vaginal secretions, and tissue samples. The reduction in the growth of bacteria can also be measured by chest X-rays or by a pulmonary function test (PFT) (for example, spirometry or forced expiratory volume (FEVi)).
[0189] In other situations, the presence or growth of biofilm-producing bacteria can be measured by detecting the presence of antigens of biofilm-producing bacteria in a biological sample, such as those described above. For example, an antibody to S. pneumoniae components can be used to assay colonization/infection in a subject afflicted with a biofilm-related condition or disorder, such as by assaying the presence of Streptococcus antigens in a biological sample. Such antibodies can be generated according to methods well established in the art or can be obtained commercially (for example, from Abeam, Cambridge, MA; Cell Sciences Canton, MA; Novus Biologicals, Littleton, CO; or GeneTex, San Antonio, TX).
[0190] Appropriate therapies for the treatment of biofilm-re fated disorders with a guanidine, or combination of a guanidine and D-amino acid, can be determined using techniques well established in the art. For example, animal models using mammals can be used to assess the efficacy of treatment with guanidines, or combinations of guanidines and D-amino acids. Non- limiting examples include implanting polymer beads, e.g., polymethylmethacrylate (PMMA) beads loaded with the guanidines, or combinations of guanidines and D-amino acids, in rats and assessing their ability to prevent biofilms.) polymethylmethacrylate (PM MA) beads in rats and catheters in rabbits have been used as animal models for biofilm formation for Staph aureus. See, e.g., Anguita-Alonzo, et ah, Antimicrobial Agents and Chemotherapy, July 2007, p. 2594- 2596, and Beenken, et al. J ournal o f B acterio 1 o y , July 2004, p. 4665-4684, which are hereby incorporated in their entirety by reference. .
Combination Therapy
[0191 Biofilms are understood, very generally, to be aggregations of living and dead microorganisms, especially bacteria, that adhere to living and non-living surfaces, together with their metabolites in the form of extracellular polymeric substances (EPS matrix), e.g. polysaccharides. The activity of antibiofiim substances that normally exhibit a pronounced growth-inhibiting or lethal action with respect to planktonic cells may be greatly reduced with respect to
microorganisms that are organized in biofilms, for example because of inadequate penetration of the active substance into the biological matrix.
[0192] In some instances, a guanidine, or combination of a guanidine and D-amino acid, can be administered alone or in combination with a second agent, e.g., a biocide, an antibiotic, or an antimicrobial agent, to treat a biofiim or to prevent the formation of a biofilm. An antibiotic can be co-administerecl with the guanidine, or combination of a guanidine and D-amino acid, either sequentially or simultaneously. For example, any of the compositions described herein can be formulated to include one or more guanidines, or combinations of giianidines and D-amino acids, and one or more second agents.
[0193] The antibiotic can be any compound known to one of ordinary skill in the art that can inhibit the growth of, or kill, bacteria. Useful, non-limiting examples of antibiotics include lineosamides (clindomycin); chloramphenicols; tetracyclines (such as Tetracycline,
Chlortetracycline, Demecioeycline, Mefhacycline, Doxycycline, Minocycline); aminoglycosides (such as Gentamicin, Tobramycin, Netilmicin, Amikacin, Kanamycin, Streptomycin,
Neomycin); beta-lactams (such as penicillins, cephalosporins, Imipenem, Aztreonam);
glycopeptide antibiotics (such as vancomycin); polypeptide antibiotics (such as bacitracin): macrolides (erythromycins), amphotericins; sulfonamides (such as Sulfanilamide,
Sulfamethoxazole, Sulfacetamide, Sulfadiazine, Sulfisoxazole, Sulfacytine, Sulfadoxine, Mafenide, p-Aminobenzoic Acid, Trimethoprim-Sulfamethoxazole); Methenamin;
Nitrofurantoin; Phenazopyridine; trimethoprim; rifampicins; metronidazoles; cefazolins;
Lincomycin; Spectinomycin; mupirocins; quinolones (such as Nalidixic Acid, Cinoxacin, Norfloxacin, Ciprofloxacin, Perfloxacin, Ofloxacin, Enoxacin, Fleroxacin, Levofloxacin);
novobiocins; polymixins; gramicidins; and antipseudomonals (such as Carbenicillin,
Carbenicillin Indanyl, Ticarcillin, Azlociilin, Mezlocillin, Piperacillin) or any salts or variants thereof. Such antibiotics are commercially available, e.g., from Daiichi Sankyo, Inc.
(Parsipanny, NJ), Merck (Whitehouse Station, NJ), Pfizer (New York, NY), Glaxo Smith Kline (Research 'Triangle Park, NC), Johnson & Johnson (New Brunswick, NJ), AstraZeneca
(Wilmington, DE), Novartis (East Hanover, NJ), and Sanofi-Aventis (Bridgewater, NJ). The antibiotic used will depend on the type of bacterial infection. [0194] Additional known biocides include biguanide, chiorliexidine, triclosan, chlorine dioxide, and the like.
J0195) Useful examples of antimicrobial agents include, but are not limited to, Pyrithiones, especially the zinc complex (ZPT); Octopirox®; Dimethyklimethyloi Hydantoin (Glydant®); Methyichloroisofhiazolinoiie/methylisothiazolinone ( athon CG®); Sodium Sulfite; Sodium Bisulfite; Imidazolidinyl Urea (Germail 115®, Diazolidinyl Urea (Germaill II®); Benzyl Alcohol; 2-Bronio-2-nitropropane~l ,3-diol (Bronopol®); Formalin (formaldehyde);
Iodo-pro-penyl Butylcarbamate (Polyphase P 00®); Chloroacetamide; Methanamine;
Methyldibromo"Tiitrile Glutaronitrile ( l,2-Dibromo-2,4-dicyanobutane or Tektamer®);
Glutaraldehyde; 5~hro"^no-5-nitro~l ,3-dioxane (Bronidox®); Phenethyl Alcohol; o- Phenylphenol/sodium o-phenyi""phenoi; Sodium Hydroxymethyiglycinate (Suttocide A®); Polymethoxy Bicyclic Oxazolidine (Nuosept C®); Dimethoxane; Thimersal; Dichlorobenzyl Alcohol; Captan; Chlorphenenesin; Dichiorophene; Chlorbutanol; Glyceryl Laurate;
Halogenated Diphenyl Ethers; 2,4,4'-triehloro-2'-hydroxy-diphenyl ether (Triclosan®. or TCS); 2,2,~dihydroxy-5,5'-dibromo-diphenyl ether; Phenolic Compounds; Phenol; 2 -Methyl Phenol; 3- Methyl Phenol; 4 -Methyl Phenol; 4-Ethyl Phenol; 2,4-Dimethyl Phenol; 2,5-Dimethyl Phenol; 3,4-Dimethyl Phenol; 2,6-Dimethyl Phe^nol; 4-n-Propyl Phenol; 4-n-Butyl Phenol; 4-n-Amyl Phenol; 4-tert-Amyl Phenol; 4-n-Hexyl Phenol; 4-n-Heptyl Phenol; Mono- and Poly-Alkyl and Aromatic Halophenols; p-Chloro'~phe~Tiol; Methyl p-Chlorophenol; Ethyl p-Chlorophenol; n- Propyl p-Chlorophenol; n- Butyl p-Chloro"~phenol; n-Amyl p-Chlorophenol; sec-Amyl p- Chlorophenol; Cyciohexyi p-Chloro-phe^nol; n-Heptyl p-Chlorophenol; n-Octyl p- Chlorophenol; o-Chlorophenoi; Methyl o-Chloro~phenol; Ethyl o-Chlorophenoi; n-Propyi o- Chlorophenol; n-Butyl o-Chiorophenol: n-Amyl o-Chioro~phenol; tert-Amyl o-Chlorophenol; n- Hexyl o-Chlorophenol; n-Heptyl o-Chlorophenol; o-Ben^zyl p-Chlorophenol; o-Benxyl-m- niethyl p-Chlorophenol; o-Benzyl-m; m-dimethyl p~Chloro~phenol; o-Phenylethyl p- Chlorophenol; o-Phenylethyl-m-methyl p-Chlorophenol; 3 -Methyl p-Chlorophenol; 3,5- Dimethyl p-Chlorophenol; 6-Ethyl-3-methyl p-Chlorophenol; 6-n-Propyl-3-methyl p- Chlorophenol; 6-iso-Propy 1-3 -methyl p-Chlorophenol; 2 -Emy 1-3 ,5-dimethyl p-Chloro-phenol: 6-sec-Butyl-3-methyl p-Chlorophenol; 2-iso-Propyl-3,5-dimethyi p-Chlorophenol; 6- Diethylmethyl-3-methyl p-Chlorophenol; 6-iso-Propyl-2-ethyl-3-methyl p-Chlorophenol; 2-sec- Amy 1-3 ,5-dimethyi p-Chlorophenol; 2-DiethylmethyI-3 ,5-dimethyl p-Chlorophenol; 6-sec- Octyl-3-methyl p-Chlorophenol; p-Ch oro-m-cresol: p-Bromopheno ; Methyl p-Bromophenol; Ethyl p-Bromophenol; n-Propy! p-Bromophenol; n-Butyl p-Bromophenol; n-Amyl p- Bromo "phenol; sec-Amyl ρ-Bromophenol; n-Hexyl ρ-Bromophenol; Cyclohexyl p- Bromophenol; o-Bromophenol; tert-Amyl o-Bromophenol; n-Hexyl o-Bromophenol; n-Propyl- m,m-Dimethyl o-Bromophenol; 2 -Phenyl Phenol; 4-Chloro-2-methyl phenol; 4-Chloro-3-methyl phenol; 4-Chioro-3,5-dimethyl phenol; 2,4-Dichloro-3,5-dimethyiphenol; 3,4,5,6-Terabromo-2- methyl-phenol; 5-Methyl-2-pentylphenol; 4-Isopropyl-3-methylphenol; Para-chloro-meta- xylenol (PCMX); Chloro thymol; Phenoxyethanol; Phenoxvisopropanol; 5-Chloro-2- hydroxydi-phenyl-methane; Resorcinoi and its Derivatives; Resorcinoi; Methyl Resorcinoi; Ethyl Resorcinoi; n-Propyl Resorcinoi; n-Butyl Resorcinoi; n-Amyl Resorcinoi; n-Hexyl Resorcinoi; n-Heptyl Re^sorcinol; n-Octyl Resorcmol; n-Nonyl Resorcinoi; Phenyl Resorcinoi; Benzyl Resorcinoi; Phe^nylethyl Resorcinoi; Phenyipropyl Resorcinoi; p-Chloro benzyl Resorcinoi; 5-Chloro 2,4-Dihy_,droxy_idiphenyl Methane; 4'-Chloro 2,4-Dihydroxydiphenyl Methane; 5-Bromo 2,4-Dihydroxy~idiphenyl Methane; 4'-Bromo 2,4-Dihydroxydiphenyl Methane; Bisphenolic Compounds; 2,2 -Methylene bis-(4-chlorophenol); 2, 2 '-Methylene bis- (3,4,6-trichlorophenol); 2,2 -Methylene bis-(4-chioro-6-bromophenol): bis(2-hydroxy-3,5- dichlorophenyl)sulfide; bis(2-hydroxy-5-ehlo-ro-'benzyl)sulfide; Benzoic Esters (Parabens); Methylparaben; Propylparaben; Butylpara-feen; Ethylparaben; Isopropylparaben;
Isobutylparaben; Benzylparaben; Sodium Methylpara- en; Sodium Propylparaben; Halogenated Carbanilides; 3,4,4'-Trichlorocarbani!ides (Triclo"xar"ban® or TCC); 3-Trifluoromethyl-4,4'- dichlorocarbanilide; 3,3',4-Trichlorocarbanilide; Chlorohexkline and its digluconate; diacetate and ^hydrochloride; Undecenoic acid; thiabendazole, Hexetidine;
poly(hexamefhyienebiguanide) hydrochloride (Cosmocil®); silver compounds such as organic silver salts ir anorganic silver salts, silver chloride including formulations thereof such as JM Acticare® and micronized silver particles.
Biofilm-Related Disorders
[0196] Methods and treatments using guanidines, or combinations of gua idines and D- amino acids, include inhibiting or preventing the formation of biofilm, even or especially without inhibiting organism growth, and alos the disruption of a biofilm once formed.
[0197] A guanidine, or combination of a guanidine and D-amino acid, can be used to treat, biofilm -related disorders in a subject by administering to the subject an effective amount of guanidine, or combination of a guanidine and D-amino acid, that reduces biofilm formation in the subject. A reduction in bacterial growth is indicative of the reduction in, or inhibition of, biofilm production in the subject.
[0198] In some instances, a guanidine, or combination of a guanidine and D-amino acid, can inhibit or reduce biofilm formation by diminishing adherence of biofilm- forming bacteria to a surface or by increasing bacterial death. This therapeutic approach can be useful for the treatment of biofilm-related disorders or conditions, or medical device-related infections associated with the formation of micro bial biofilms.
[0199] Non-limiting examples of biofilm-related disorders include otitis media, prostatitis, cystitis, bronchiectasis, bacterial endocarditis, osteomyelitis, dental caries, periodontal disease, infectious kidney stones, acne, Legionnaire's disease, chronic obstructive pulmonary disease (COPD), and cystic fibrosis. In one specific example, subjects with cystic fibrosis display an accumulation of biofilm in the lungs and digestive tract. Subjects afflicted with COPD, such as emphysema and chronic bronchitis, display a. characteristic inflammation of the airways wherein airflow through such airways, and subsequently out of the lungs, is chronically obstructed.
[0200] Bio film-related disorders can also e compass infections derived from
implanted/inserted devices, medical device-related infections, such as infections from biliary stents, orthopedic implant infections, and catheter-related infections (kidney, vascular, peritoneal). An infection can also originate from sites where the integrity of the skin and'Or soft tissue has been compromised. Non-limiting examples include dermatitis, ulcers from peripheral vascular disease, a burn injury, and trauma. For example, a Gram-positive bacterium, such as S. pneumoniae, can cause opportunistic infections in such tissues. The ability of S. pneumoniae to infect burn wound sites, e.g., is enhanced due to the breakdown of the skin, burn- related immune defects, and antibiotic selection.
[0201] In some instances, a subject is treated. A subject can be a mammal including, but not limited to, a. primate (e.g., a monkey, such as a cynomolgous monkey, a chimpanzee, and a human). A subject can be a non-human animal such as a bird (e.g., a. quail, chicken, or turkey), a farm animal (e.g., a cow, goat, horse, pig, or sheep), a pet (e.g., a cat, dog, or guinea pig, rat, or mouse), or laboratory animal (e.g. , an animal model for a disorder). Non -limiting representative subjects can be a human infant, a pre-adolescent child, an adolescent, an adult, or a senior/elderly adult.
[0202] In some instances, a subject in need of treatment can be one afflicted with one or more of the infections or disorders described herein. In some instances, the subject is at risk of developing a biofilm on or in a biologically relevant surface, or already has developed such a biofilm. Such a subject at risk can be a candidate for treatment with a guanidine, or combination of a guanidine and D-amino acid, in order to inhibit the development or onset of a biofilm- production-related disorder/condition or prevent the recurrence, onset, or development of one or more symptoms of a. biofilm-related disorder or condition. Such a subject can be harboring an immature biofilm that is clinically evident or detectable to the skilled artisan, but that has not yet fully formed. A subject at risk of developing a biofilm can also be one in which implantation of an indwelling device, such as a medical device, is scheduled. The risk of developing a biofilm can also be due to a propensity of developing a biofilm-related disease (such as the presence of a channel transporter mutation associated with cystic fibrosis). In such subjects, a biofilm-related disorder can be at an early stage, e.g., no bacterial infection and/or biofilm formation is yet detected.
[0203] In a specific example, the methods described herein can be used to prevent biofilm formation in the airways of a cystic fibrosis patient. Such a patient can be treated while free of bacterial infection of the airways or upon detection of a bacterial infection.
[0204] In other examples, the methods described herein can be used to prevent biofilm formation in superficial wounds of a patient. Such wounds can include burns. In some examples, the methods described herein can be used to prevent biofilm formation in patients with diabetic leg syndrome.
[0205] The invention is further described in the following examples, which does not limit the scope of the invention described in the claims. Room temperature denotes a temperature from the range of 20-25°C.
EXAMPLES
Synthesis of Guanidines
Materials
[0206] Chemicals and solvents were purchased from Sigma Aklrich or EMD chemicals. Solvents were anhydrous or HPLC grade and chemicals were of reagent grade or better and used without further purification. Temperatures were measured externally. !H NMR spectra and l3C NMR spectra were recorded on a Varian 400 MHz NMR-System and referenced to the residua] proton and carbon signals of the deuterated solvent, respectively. Spectra in D20 were referenced to 3-(trimethylsily3)propionic-2,2,3,3-i ,/ acid sodium salt (0.05 wt %).
[0207| Low resolution mass spectra were obtained by LC/MS (ESI) with an Agilent 6130 Quadrupole LC/MS coupled to an Agilent 1200 series HPLC system. High resolution mass spectrometry was carried out by the mass spectrometry laboratory of the University of Illinois by Q-TOF MS in positive mode or by the WM Keck Foundation Biotechnology Resource
Laboratory of Yale University using a 9.4T Bruker Qe FT-ICR MS in negative mode. Infrared (IR) spectra were measured using an ALPHA FT-IR Spectrometer (Bruker).
Example 1; Preparation of lJ.'-(ethane-l,2-diyl.)digManid e sulfate (1)
Figure imgf000083_0001
10208] Ethyl enedi amine (5.67 mL, 25 ramol) was added to a solution of S-metbylisothiourea ½. H2S04 (1 .39 g, 10 mmol) in 8 mL of 1 :3 H20/EtOH and stirred for 24 hours at room temperature. A white viscous precipitate had formed. The supernatant was decanted and the residue washed with ethanol. Crystallization from ethanol/H20 afforded transparent crystals which were sucked off and washed with 50% ethanol in water and ethanol (yield: 214 nig, 18%).
[0209] \H NMR (400 MHz, D20): δ 3.44 (s, 4H, (Ή.·(Ή>).
[0210] ¾3C NMR (400 MHz, D20): δ 159.95 (2x C Ml).42.95 (O f.-Cf S ·)·
[0211] The structure was confirmed via gCOSY, HSQCAD and gHMBCAD.
[0212] IR (solid): v(cm"]) 3319, 3160, 3111, 3034, 1686, 1627, 1153, 1078, 1029,970,596, 529, 472, 437.
J] LC-MS (LR) m/z: 145.2 [YMIj (caic: 145.1). [0214] HR ESl-MS, m/z: 145.1200 |M III (calc: 145.1202).
[0215] Anal, calcd for C H N SO (C H N · H SO ): C.19.83; H, 5.82: N, 34.69; S,
1 ' 4 14 6 4 4 12 ό 2 4' ' ' ' ' ' '
13.24. Found: C, 19.81; H, 5.74; N, 33.96; S, 13.23.
Example 2: Preparation of N,N-di(2-guamdiaylethyl)am e sulfate hydrate (2a)
Figure imgf000084_0001
[0216] Diethyienetriamine (540 μί^, 5 mmol) was added to a solution of iS-methylisothiourea ½H^SO (1392 mg, 10 mmol) in 5 mL of 1:1 H O/EtOH and stirred for 16 hours at room temperature. A white crystalline precipitate formed, which was sucked off and washed with efhanol. The raw crystals were dissolved in the minimum volume H^Q at 30°C and isopropanol was carefully layered on top of the solution. The pure product crystallized over night in at 4°C as fine white needles (910 nig, 60%).
[0217] iH NMR (400 MHz, 6.0
Figure imgf000085_0001
Hz, 4H, C 'U, --N i l-i ll, ).
[0218] NMR (400 MHz, D O): δ 1 59.92 (2x C=NH), 49.43 (Ci ί ~N I 1-C 1 1 43.47
(2x GuaNH- CHj.
[0219] The structure was confirmed via gCOSY, HSQCAD and gHMBCAD.
[0220] ]R (solid): v (cm-i) 3334, 3282, 3151 , 2852, 1626, 1459, 1345, 5046, 749, 601 , 508. HR ESI-M8, m/z: 188.1626 [M+H]+ (calc: 188.1 624).
[0221] Anal, calcd for C H N SO (C H N ■ H SO · H O): C, 23.76; H, 6.98; N. 32.32:
6 21 7 5 6 J7 7 2 4 2 1 ' ' ' ' '
S, 10.57.
[0222] Found: C, 23.75; H, 6.87; N, 32.12; S, 10.59.
[0223] Free base (2h): xhe suifate salt (200 mg, 0.659 mmol) was dissolved in 10 mL H20 and a solution of ^a(0¾, ^ nig, 0.659 mmol) in 6 mL H O was added. Precipitation was completed at 4°C, the suspension was filtered through celite and lyophilized The residue was extracted with hot methanol and filtered through celite giving the free base in quantitative yield. The free base was well soluble in hot methanol, the sulfate salt and barium salts were insoluble. Example 3: Preparation of l-l -A^^'V'-diisopropylguaiiidinyiethaae-l-yll-l '-fethane-l,!-
Figure imgf000086_0001
[0224] Diethylenetriamine (216 pL, 2,0 mniol) was dissolved in 4 mL acetonitril, Ν,Ν'- diisopropylcarbodiimide (1 174 p,L, 7,5 mniol) was added and the solution was stirred at 70°C for 24 hours. The solvent was removed, the oily residue was taken up in 6 mL ethanol and 4 mL of a 1 : 1 mixture of ethanol and 37% HC1 aq. were added. After evaporation of the solvent, the solid residue was taken up in a small volume of ethanol and acetone was added until a precipitate appeared. The liquid phase was decanted and the solid material taken up in ethanol. After filtration the solution was evaporated until a highly viscous oil was obtained. Isopropanoi was layered atop the oil and after standing for three days at room temperature, crystalline material had formed. The crystals were collected, washed with ice cold ethanol and isopropanoi and re- crystallized twice from isopropanol/ethanol and isopropanol/Ή,-,Ο yielding 58.6 mg (4.7 %) of the product as the trihydro chloride dihydrate.
[0225] iH NM (400 MHz, D O); δ 3,82-3.72 (m, 6H, 2x C!.l(( ! f ; } ;. 4x C!.l(( H : ) ). 3.62 (t ./ 4.8 Hz, 4H, ( ll N -C l! ,). 3.53 (t, ./ 4.7 Hz, 4H, 2x ( Ή -\ H ). 1.29 (d, 6,5 Hz, 12H, 2x CH(CH^)J, 1.25 (d, J = 6.4 Hz, 24H, 4x CH(CH,)?), [0226] i3C NMR (400 MHz, D O): δ 161.95 (C=NH), 155.60 (2x C=NH), 50.56 (2x CH(CH ) ), 49.71 (CH. N-CH2), 47.07 (4x n i{CH J ,}. 40.78 (2x CH -NH), 25.03 (2x CI IiT I l ;· ). 24.45 (4x CH(CH..). ).
[0227] The structure was confirmed via gCOSY, HSQCAD and gHMBCAD.
[0228] IR (solid): v (cm-i) 3199, 2973, 1615, 1430, 1388, 1368, 1335, 1268, 1 167, 1 147, 1 133, 1073, 734, 591.
[0229] LC-MS (LR) m/z: 161.6 [M+3H]3+ (calc: 161.5), 241.8 | M · IW y (caic: 241.7), 482.4 (calc: 482.5)
[0230] HR hS I - YI S . ηχ/ζ: 482.4653 ( .Vi H j (calc: 482.4659)
[0231] Anal, calcd for C H O N CI (C H 3HC1 2H O): C, 47.88; H, 9.96; N,
1 1 25 62 2 9 3 ' 25 55 9 2 ' '
20.10; CI, 16.96. Found: C, 48.01 ; H, 10.35; N, 19.73; CI, 17.44.
Example 4: Preparation of l,lHpropane-l,3-diyl)digiiaiiidiiie sislfate (4)
Figure imgf000087_0001
[0232] Compound 2 (740 mg, 3.19 mmol) and S-methylisothiourea ½ H2S04 (481 nig, 3.45 mmol) were dissolved in 5 mL H20 and the solution wras stirred under reflux. 5 M sodium hydroxide solution (690 μΐ,, 3.45 mmol) was added drop wise and stirred for 4 hours. Upon cooling to 4°C a mixture of 4 and 2 crystallized which was collected, dissolved in 5 mL H O, and again treated with 5-methylisothiourea ½ H^SO^ (481 mg, 3.45 mmol) and sodium hydroxide solution (690 fiL, 3.45 mmol) under reflux for 4 h. Large white crystals formed at 4°C which were sucked off and re-crystallized from H O, yielding 206.5 mg (25%) of the product as sulfate salt.
[0233] ; i l NMR (400 MHz, D O): δ 3.29 (t, J ------- 6.8 Hz, 41 1. 2x CH2-NHGua), ] .9 (p, J ------ 6.8
Hz, 2H, CH?).
[0234] !3C NMR (400 MHz, D.G): δ 159.71 (2x C=NH), 41.22 (2x CH,-NHGua), 29.85 (CH ).
[0235] The structure was confirmed via gCOSY, HSQCAD and gHMBCAD.
[0236] IR (solid): v (cm-i) 3304, 3131 , 3 , 1690, 1667, 1636, 1465, 1223, 1085, 1
729, 628, 603, 556, 452.
[0237] HR ESI-MS, m/z: 159.1362 j .Yi - i i i (calc: 159.1358).
[0238] Anal, calcd for C H N SO (C H N ■ H SO ): C, 23.43; H, 6.29; N
1 ! 5 ¾ 6 6 4 - 5 i 4 6 2 V
12.51. Found: C, 23.47; H, 6.58; N, 32.36; S, 12.53. example 5: rreparai V-di-i3-euanidinylpropyl)amine sesauisulfate hydrate (5a)
Figure imgf000088_0001
[0239] Norspermidine (700 uL, 5 mmol) was a""e" to a solution of S-Methylisothiourea ½ H-SO.< 2505 mg, 18 mmol) in 7 mL H„0 and stirred for 3 days at 105°C. A white precipitate formed after standing overnight at 4°C. The precipitate was sucked off and washed with ice cold H^O followed by ethanol. Reerystallization from H O afforded the sesquisulfate hydrate of the product (1275 mg, 67%) as white solid.
[0240] ; l ! NM R (400 MHz, D O): δ 3.33 (t, ,/ 6.8 Hz, 4H, 2x I IX ί '-ΧΠ-ί Ί Π. 3.15 (Ψί, J = 7.9 Hz, 4H, CH NH-CHJ, 2.02 ^q, J ------ 7.4 Hz, 4H, 2x CH^-CH^-CHJ.
[0241] i3C NMR (400 MHz, D O): δ 159.73 (2x C M l ). 47.85 (2x CH ,- H-CH ), 41.04
2 2
(2x H\ ( ··· NH-CHJ, 27.83 (2x ( "i 1 ,·-Π I -CI 1 J.
[0242] The structure was confirmed via gCOSY, HSQCAD and gHMBCAD.
[0243] IR (solid): v (cm ; } 1682, 1638, 1479, 1392, 1049, 969, 770, 6 7, 595, 555, 462. LC- MS (LR) m/z: 108.8 | Μ · 2ϋ Ι · (calc: 1 08.6), 216.2 | M ! ! ! (calc: 216.2). HR ESI-MS, m/z: 216.1930 [ M i l l (calc: 216.1937).
[0244] Anal, calcd for C H N S O (C H N · 1.5H SO · H O): C. 25.26; 1 1. 6.89: N.
1 S 26 7 1.5 ? 8 2i 7 2 4 2 ' ' '
25.77; S, 12.64. Found: C, 25.65; H, 7.06; N, 25.42; S, 12.72.
[0245] Free base The sulfate salt (200 nig, 0,526 mmol) was dissolved in 10 mL H,0 and a solution of Ba(OH),, (135 mg, 0,789 mmol) in 6 mL H O was added. Precipitation was completed at 4°C, the suspension was filtered through celite and lyopliilized. The residue was extracted with hot methanol giving the free base in quantitative yield. The free base was well soluble in hot methanol, the sulfate salt and barium salts were insoluble. Octanoic acid salt: Free base 5b (13.7 mg, 0.0635 mol) was mixed with 635 uL of 300 mM octanoic acid in H20/ethanol (2: 1 vol ) to give a lOOmM stock solution of the octanoic acid salt.
Example 6: Preparation of l-(3-guanidinylpropaiie-l-YlVl.l '-(propane-lJ-diyi -
Figure imgf000090_0001
[0247] Norspermidine (1399 ,u,L, 10 mmol) was dissolved in 7 mL ethanoi, 1.5 mL of concentrated hydrochloric acid were added whereupon a white precipitate formed, C anamide (1640 mg, 39 mmol) was added and the reaction mixture was refluxed for 36.5 hours and let to cool. The solvent was removed resulting in a highly viscous mass which crystallized after 8 days at room temperature. The crystalline material was washed with isopropanol and ice cold ethanoi.
[0248] It was recrystaliized from hot methanol/isopropanoi, yielding the trihydrochloride hydrate of the product (910 mg, 23.7 %) as white crystals.
[0249] iH NMR (400 MHz, D O): δ 3.46 (Ψί, J 7.6 Hz, 4H, ( I^- C H , ). 3.27 (t, J = 6.9 Hz, 4H, 2x CH. H), 1.98 i l q. J = 7.2 Hz, 4H, 2x C 'l l Π C 'l I J.
[0250] i3C NMR (400 MHz, D.G): δ 159.62 (2x C=NH), 158.91 (C M l ). 48.79 (CHINCH ), 41.18 (2x CH NH), 28.35 (2x Π \ i ,-('} I . ).
[0251] The structure was confirmed via gCOSY, HSQCAD and gHMBCAD. [0252] IR (solid): v (cm-i ) 3305, 3128, 1640, 1610, 1534, 1464, 1391 , 1 165, 1095, 1064, 464. LC-MS (LR) m/z: 129.7 j Y! 21 1 | " (calc: 129.6), 258.2 I M I I ] (calc.: 258.2).
[0253] HR ESI-MS, m/z: 258.2147 j .Yi - H i (calc: 258.2155).
[02541 Anal, calcd for C H N CI O (C H N -3HC1 H O): C, 28.10; H. 7.34; N, 32.77: CL
9 28 9 3 9 23 9 2 '
27.64.21. Found: C, 28.34; H, 7.54; N, 32.02; CI, 27.14.
[0255] Free base <£fe): xhe chloride salt (350 mg, 0.91 mmol) was dissolved in 10 raL H20 and a suspension of A 2SO « mI» 1 -36 mmol) was added i 6 mL H20. After sonication, precipitation was completed at 4°C, the suspension was filtered through celite and a solution of Ba.(OH)2 (234 mg, 1.36 mmol) in 5 mL H. Q was added. Precipitation was completed at 4CC and the suspension was filtered through celite and lyophilized. The residue was extracted with hot methanol and filtered through celite giving the free base in quantitative yield. The free base was well soluble in hot methanol, the sulfate salt, the silver and barium salts were insoluble.
[0256] Octanoic acid salt: Free base 6b (20.6 mg, 0.0802 mmol) was mixed with 802 \iL of 300 raM octanoic acid in H20/ethanol (2: 1 vol ) to give a 100 raM stoclv of the octanoic acid salt
Example 7: Preparation of N "-di-(3-gttanidiiiylpropyi)-l,3-propanediamine distrifate hydrate (7a) H NH
H H H H [0257] Norspermine (512 μί,, 2.5 mmol) was added to a solution of S-methylisothiourea ½ H-SO4 (1670 mg, 12 mmol) in 4 niL H O and stirred for 3 days at 105°C. The solvent was largely removed under reduced pressure and the residue dissolved by re fluxing in ethanol/H O.
A white precipitate formed upon cooling which was sucked off, washed with ethanol and re- crystallized from ethanol/H.0. The disulfate hydrate salt of the product ( 560 mg, 46%) was obtained as white solid.
[0258] : n NMR (400 MHz, D2°): δ 3.33 (t,•/ ;;; 6.8 Hz, 41 1. 2x HN-C-NH-CHJ, 3.21 -3.17 (m, 4H, 2x
Figure imgf000092_0001
2.22- 2.14 (m, 2H, 1W-CH,-CH^CH,-NH), 2.07-2.00 (m, 4Π. 2x I IN ί '~\ ! i~C 1 1 -O I J
[0259] i3C NMR (400 MHz, D20): δ 159.72 (2x C M l ). 47.88 (2x HN=C-NH-(CH2)2-CH2), 47 4 (2x i IN C~\I i-(i Π ^-ΧΠ-Π η. 41 .02 (2x I IN ί'-Π Π. 27.80 (2x HN-€-NH~CH
. C....H r }.. 25.52 ( vHN-CH 2 - .C....H 2 -CH 2 -NH) ).
[0260] The structure was confirmed via gCOSY, HSQCAD and gHMBCAD. [0261] ]R (solid): v (cm-i) 3118, 1685, 1638, 1478, 1060, 601, 557.
[0262] C -MS (LR) m/z: 137.2 | M 2! [ | ' (calc: 137.1), 273.3 i .Vi - l l j (calc: 273.3). [0263] HR ESl-MS, m/z: 273.2514 [M+H]+ (calc: 273.2515).
[0264] Anal, calcd for C H N S O (C H N 2H SO H O): C, 27.15: H, 7.04; N.
1 ii 34 8 2 9 i i 28 8 2 4 2 ' ' '
23.03; S, 13.18. Found: C, 27.27; H, 7.13; N, 23.23; S, 12.98. [0265] Free base Qfc): The su3fate sa[t 2oo mg, 0.41 1 mmol) was dissolved in 10 itiL H20 and a solution of *a(QH)., 041 mg, 0,822 mmol) in 6 mL H O was added. Precipitation was completed at 4°C, the suspension was filtered through celite and lyophilized. The residue was extracted with hot methanol and filtered through celite giving the free base in quantitative yield. The free base was well soluble in hot methanol, the sulfate salt and barium salts wrere insoluble.
[0266] Octanoie acid salt: Free base 7b (12.5 mg, 0.0458 mmol) was mixed with 458 iL of 400 mM octanoie acid in H2Q/ethanol (2: 1 vol) to give a 100 niM stock of the octanoie acid salt.
Figure imgf000093_0001
sesqiMSjsifate diiiydrate (8¾)
Figure imgf000093_0002
[0267] Spermidine (393 pL, 2.5 mmol) was added to a solution of S-Methyliso hiourea ½ H2S04 (1253 mg, 9.0 mmol) in 3.5 mL H O and stirred for 3 days at 105°C. After cooling to room temperature a white precipitate was sucked off and washed with ice cold ethanol followed by ethanol. Crystallization from ethanol/H O afforded the sesquisulfate dihydrate of the product
(557 mg, 54%) as white solid.
[0268] iH NMR (400 MHz, D O): 5 3.33 (t, J= 6.8 Hz, 2H, \ M-(. '_Η (( ί J - N 1 i-f Ί 1 3.25 (t, ./ 6.7 Hz, 2H, NH-CH i t 1 ! ) -NH-CH ), 3.15-3.12 (m. 2H, Μ ί- ί Π ! s -CH -NH-CH ), 3.12-3.08 (m, ' 2H, ' NH-( -CH 2 )"? -C—H 2 -NH-CH 2 ). 2.02 (tt, ,/ 7.9, 6.8 Hz, 2H, NH-CH 2 -C—H 2 -CH 2 - NH), 1.82-1.75 (m, 2H, NH-(CH2 CH CH NH-CH2), 1.724.65 (m, 2H, M S ·( !!-.· (Ή '·
(CHj. NH-CH J,
[0269] i3C NMR (400 MHz, D O : δ 159.72 (C=NH), 159.67 (C=NH), 50.05 (NH-(CH.) - 01..-Mi- CH ), 47.67 ( H-(CH ) -CH -NH-CH , 43.26 {\l l-CH -((Ί I .) I Ι·(Ή ).41.07 (NH-CH, (CH. ^-NH-CH ), 27.87 (\i i-C'H^ij ί -{Cil^-NH-Ci I }.27.81 ( M i~C 11 -CI I ~ < Ι,~ NH), 25.66 (^H-(CHJ7-CH2-CH, NH-CH2).
[0270] The structure was confirmed via gCOSY, HSQCAD and gHMBCAD. [0271] IR (solid): v (cm-i) 3118, 1629, 1553, 1478, 1044, 971, 748, 607.
[0272] LC-MS (LR) m/z: 115.7 [M+2H]2+ (calc: 115.6), 230.2 [M+H]÷ (eale.: 230.2). [0273] HR ESI-MS, m/z: 230.2094 j.Yi-iii (calc: 230.2093).
[0274] Anal, calcd for C H N S O (C H N 1.5H SO 2H O): C, 26.21; H, 7.33: N,
1 1 9 30 7 1.5 8 9 23 7 2 4 2 ' ' ' ' '
23.77; S, 11.66. Found: C, 26.22; H, 7.23; N, 23.45; S, 10.93.
[0275] Free base (8b): xhe sulfate salt (2(){) mg> 0.485 mmol) was dissolved in 10 itiL H20 and a solution of ^a(OH)„ (125 mg, 0.727 mmol) in 6 mL H?0 was added. Precipitation was completed at 4°C, the suspension was filtered through celite and lyophilized. The residue was extracted with hot methanol and filtered through celite giving the free base in quantitative yield. The free base was well soluble in hot methanol, the sulfate salt and barium salts were insoluble.
Example 9: Preparation of (3-Aminopropyl)guaiiidine sislfate hydrate (9)
Figure imgf000095_0001
[0276] 5-Methylisothiourea ½ H2S04 rag, 5 mmol) was added to a solution of 1 ,3- diaminopropane (1043 p.L, 12.5 mmol) in 5 mL of 1 :3 H„0/Et()H and stirred for a few minutes at room temperature until precipitation occurred. Another 5 mL of 1 :3 H O/EtOH were added and stirring continued for 30 minutes. The precipitate was sucked off and crystallized from hot H O (yield: 416 mg, 36%).
[0277] i H NMR 400 vi Hz. !.) ,()): g 3.32 {{. .! 6.9 Hz, 2H, GuaNH-CH2), 3.09 (Ψι . J = 7.8 Hz, 21 1. CH2~NH 2,03-1.96 (m, 2H, Gua H - CH ,-CH 2~ ί Ή , -N H
[0278] i3C NMR (400 MHz, D O): δ 159.74 (C=NH), 41.05 (GuaNH-CH ), 39.73 (CH -NH ),
2 ' ' 2 2 2'
28.95 (Π ί ).
[0279] The stmcture was confirmed via gCOSY, HSQCAD and gHMBCAD.
[0280] ]R (solid): v (cm"1) 2884, 2805, 1684, 1638, 1079, 863, 738, 618, 601 , 552, 469. [0281] LC-MS (LR) m/z: 1 17.2 [M+H]+ (calc: 1 17.1). [0282] HR ESI-MS, m/z: 1 17.1 140 [M+H]+ (calc: 1 17.1 149). [0283] Anal calcd for C H N SO (C H N ■ H SO · 1 1 O): ( ". 20.69; H. 6.94; N, 24.12;
1 * 4 16 4 5 4 12 4 2 4 2 ' '
S, 13.81 .
[0284] Found: C, 21.05; I I. 6.95; N, 23.87; S, 13.89.
Figure imgf000096_0001
diisopropylgMaaidinylprop>l)amme triformate (10)
Figure imgf000096_0002
[0285] AvV-Diisopropylcarbodiittiide (313 μΕ, 2.0 mrao!) was dissolved in 1 mL
dichloromethane and norspermidine (140 p.L, 1.0 mmol ) was added under vigorous stirring. After stirring overnight, the solvent was evaporated and the residue dissolved in a mixture of 1.1 mL water and 0.4 mL methanol. 50 uL of the solution were purified by HPLC with on a Hypercarb column (250 x 10 mm, 5 tun particle size, Thermo Scientific). Mobile phase A = water, B = acetonitril, gradient (T . ) T : B = 0%, T : B = 100%. The product eluted with a retention time of 8 min (yield 1.78 nig, 14%).
[0286] iH NMR (400 MHz, CD D): δ 3.82 (sept, J = 6.6 Hz, 2H, 2x CH(CHJ2), 3.36 (t, J === 7.0 Hz. 2 H, NHoua-CH ), 3.03 (t. J == 7.6, 2H, CH -NH k 2.99 OPt, J = 7.4 Hz, 2H, CH -
2 ' 2 2' ' " 2
(CI I ,) ,- NH ), 2.95 (Ψί, J ------ 7.4 Hz, 2H, N l 1<·^-ί ( Ί i j ^C H :-M I ). 2.01 ( H'q. J ------ 7.5 Hz, 2H, (Ή -
CH - NH }. 1.94 (sPq, J ------ 7 A Hz, 2H, CH, -CH -NH), 1 .27 (d, J ------ 6.4, 12H, 4x CH ).
2 2' 2 2 3' [0287] 13C NMR (400 MHz, CD OD): δ 154.50 (C=N), 46.61 (NHoua-(CH j -CH -NH), 46.50 (CH -(CH^-NH,), 45.52 ;Π S(C'S f .} .}.40.09 (NHoua-CHJ, 38.37 (ΠΙ,-ΝΠ,).27.86
(CH -Hi -NH), 26.53 (CH -CH -NH }.22.76 (CI I
2 2 ' ' ' 2 2 2' ' ' 3
[0288] The structure was confirmed via gCOSY, HSQCAD and gHMBCAD.
[0289] HR i.SI-MS. m /: 258.2662 (.Vi lli (ealc: 258.2658).
Example 11 s Pr eparation of i??-Piie8iyles¾edibigi8¾iiide dihydreriiloride (1 la)
Figure imgf000097_0001
[0290] 1 ,3-Diaminobenzene (1081 mg, 10 mmol) was dissolved in 7 mL H20, concentrated hydrochloric acid was added until pH :=: 1 and dicyandiamide (2186 mg, 26 mmol) was added. The reaction was refluxecl for 4 hours, let to cool and acetone was added until a white precipitate formed. After standing overnight the fine suspended material had settled and was washed with two times 20 mL acetone. After drvins, the material was crystallized from hot ethanol/H O.
The white crystalline material was sucked off and washed with cold ethanol (yield: 1.956 g, 56%).
[0291] Ή NMR (400 MHz, D20): δ 7.45 (t, J= 8.1 Hz, 1H, aromate: CH=CH-CH), 7.33 (t, J :::: 2 o Hz > f aromate: Ν-(ΧΉ-ΟΝ), 7.15 (dd, J ------ 8.5, 2.1 Hz, 2H, aromate: (11 (Ί !-('!.[. i3C NM (400 MHz, D.,0): δ 563.83 (2x £ \\).560.34 (2x £ Nil).540.16 (N-£= H-C- N), 133.12 (CH=CH-CH), 123.68 (CH=CH-CH), 121.68 (N-C=CH-C-N). 2] The structure was confirmed via gCOSY, HSQCAD and gHMBCAD.
[0293] IR (solid): v (cm-i) 3292, 3136, 1628, 1575, 1541 , 1525, 1448, 1394, 1283, 781 , 600, 468. i !R ESI-MS, m/z: 277.1632 [M+H]+ (calc: 277.1638).
[ 102941 ' Anal, calcd for C, 10H, 18 10 CI 2 (C 10 H 16 N 10 · 2HCD ' : C. 34.39; ' H,' 5.20: N,' 40.1 1 ; '
CL 20.30.
[0295] Found: C, 34.52; H, 5.05; N, 38.94; CI, 20.85.
[0296] Free base (lib): The chloride salt (350 mg, 1.00 mmol) was dissolved i 10 mL Η2<Τ3 and a suspension of Ag?SO (313 mg, 1.00 mmol) was added in 10 mL H.Q. After sonication, precipitation was completed at 4°C and a solution of Ba(OH) ;) (172 mg, 1.00 mmol) in 5 mL H„0 was added. Precipitation was completed at 4°C, 5 ml, ethanol were added and the suspension was filtered through celite and lyophilized. The residue was extracted with hot ethanol and filtered through celite giving the free base in 33% yield. The free base was well soluble in hot ethanol, the sulfate salt, the silver and barium salts were insoluble.
(0297] Octanoic acid salt: Free base 1 lb (12.8 mg, 0.0464 mmol) was mixed with 464 μΐ of 200 mM octanoic acid in H20/ethanol (2: 1 vol) to give a 100 mM stock of the octanoic acid salt.
Example J 2: Preparation of l-(2-(2-IniinoimidazoHdiii-l-vi)ethvnguaiiidine sulfate (12)
Figure imgf000098_0001
[0298] Diethylenetriamine (540 μL·, 5.0 mmol) was added to a solution of S- methylisothiourea (1392 mg, 10 mmol) in 2.5 niL, H.Q and refluxed for 3 days. After cooling to room temperature, 10 niL isopropanol were added followed by 1.3 niL of a 10% solution of sulfuric acid in isopropanol. The solvent was removed and the residue crystallized from hot efhanol/H.0. The crystals were sucked off, washed with ice cold ethanol and re-crystallized from hot ethanol/H O affording 794 mg (59 %) of white crystalline material. i l l NMR (400 MHz, D O): δ 3.81 -3.76 (m, 2H, cyclic X ! f-CI 1 -( H. -X ). 3.68-3.64 (m, 2H, cyclic H-CH -CH -1ST), 3.55-3.49 (m, 4H, N-CH -CH -NHGua and N-CH -CH -NHGua).
2 I I I i3C NMR (400 MHz, D ()): δ 161.93 (ring C=NH), 159.91 (C=NH), 50.70 (cyclic NH-CH - CH -N), 46.49 (N-CH -CH -NHGua), 43.59 (cvclic NH-CH -CH -N). 41.38 (N-CH -CH -
NHGua).
[0299] The structure was confirmed via gCOSY, HSQCAD and gHMBCAD.
[0300] IR (solid): v (cm-)) 3097, 2996, 2899, 1674, 1646, 1631 , 1587, 1462, 1312, 1074, 797, 647, 614, 593, 549.
[0301] HR ESI- S, m/z: 1 71.1367 [M+H]+ (calc: 171 .1358)
[0302] Anal, calcd for C H N SO (C H N · H SO ): C, 26.86; H, 6.01 ; N, 31 .32; S,
1 1 6 ¾ 6 6 4 6 i 4 6 2 ¥ ' ' ' ' ' ' '
1 1.95. Found: C, 26.85; H, 6.27; N, 31 .06; S, 1 1 .92.
Example 13: Preparation of Norspermidiae trihydrochloride
Figure imgf000100_0001
[0303] Norspermidine (2.80 mL, 20 mmol) was dissolved in 40 mL. ethanol and 12 mL of a 111 (vol.) mixture of 37% aqueous HC1 and ethanol was added. The precipitate was sucked off, washed with ethanol and the product crystallized from hot ethanol/ H20 (yield 3.73 g, 78%).
[0304] 1H NMR (400 MHz, D20): 5 3.20 (Ψΐ, J= 8.0 Hz, 4H, (11.··Λ ·( Ή -). 3.13 (Ψί, J = 7.9 Hz, 4H, 2x CH2NH2), 2.16-2.08 (m, 4H, 2x CH2-CH2-CH2).
! 3C NMR (400 MHz, !) >()): δ 47.55 (CH2-N-CH2), 39.40 (2x CH2NH2), 26.60 (2x CH2-CH2- CH2).
[0305] The structure was confirmed via gCOSY, HSQCAD and gHMBCAD.
[0306] IR (solid): v (cm"1) 2954, 2894, 2841 , 2751 , 2702, 2502, 2415, 2025, 1607, 1526, 1489, 1462, 1404, 1356, 1281 , 1 165, 1055, 1027, 994, 930, 779, 436.
[0307] HR ESI-MS, m/z: 132.1504 [M+H]+ (calc: 132.1501)
[0308] Anal, calcd for C0H20N CI3 (C6HJ7N3 3HC1): C, 29.95; H, 8.38: N, 17.46; CI, 44.21. Found: C, 30.12; H, 8.55: N, 17.18; CI, 44.16.
Figure imgf000100_0002
[0309] Norspermidine (700 iL, 5.0 mmol) was dissolved in 10 mL ethanol and 8 mL of 20% sulfuric acid in ethanol was added slowly under ice cooling. The precipitate was sucked off and dissolved in hot ethano !/¾(). Addition of isopropanol precipitated amorphous material which was crystallized from H20/acetone (yield 510 mg, 30%).
[0310] ¾H NMR (400 MHz, D20): δ 3.18 (Ψΐ, J = 7.8 Hz, 4H, CH2-N-CH2), 3.12 (Ψί, J = 7.9 Hz, 4H, 2x CH2NH2), 2.16-2.08 (m, 4H, 2x ( Ί I .··( '!_[>··( Ί I
! 3C NMR (400 MHz, !) -■();·. δ 47.63 (CH2-N-CH2), 39.50 (2x CH2 H2), 26.68 (2x CH2-CH2- CH2).
[0311] The structure was confirmed via gCOSY, HSQCAD and gHMBCAD.
[03121 IR (solid): v (cm"1) 3448, 2923, 1633, 1526, 1475, 1407, 1372, 1213, 1085, 996, 967, 895, 760, 595, 524, 420.
[0313] HR ESI-MS, m/z: 132.1505 [M+H]+ (calc: 132.1501).
[0314] Anal, calcd for (Vj I > · \ sS ·.( )·.> {(ν.Π ,-Ν , 2H2S04 I I -■( ) ;·. C, 20.86; H, 6.71 ; N, 12.17; S, 18.57. Found: C, 21.02; H, 6.96; N, 12.16; S, 18.43.
Example 15: Preparation of Norspermidine salts and micelle form¾Iatio¾s
Preparation of norspermidine hexanoic acid salt
[0315] Norspermidine (140 fiL, 1 .0 mrnol) was dissolved in 10 mL ethanol and hexanoic acid (375 ,uL, 3.0 rnmol) was added. The solvent was evaporated and the residue washed twice with 10 mL hexanes and with 30 mL ethyl acetate (yield 316 mg, 66%).
[0316] 1H NMR (400 MHz, CD30D): δ 3.02 (t, J = 7.3 Hz, 41 1. 2x CH2NH2), 2.92 (t, j 7.2 Hz, 4H, CH2-N-CH2), 2.18 (Ψί, J - 7.6 Hz, 6H, 3x CH2COO), 1.96 (Tp, J = 7.3 Hz, 4H, 2x CH2-CH2NH2), 1.60 (Ψρ, J = 7.5 Hz, 6H, 3x CH2-CH2COO), 1.40-1 .27 (m, 12H, 3x (CH2)2), 0.91 (t, J = 7.0 Hz, 9H, 3x C ! 13 ).
[0317] 13C MR (400 MHz, CD30D): δ 182.26 (COO), 46.72 (CH2-N-CH2), 38.57 (CH2NH2), 38.47 (CH2COO), 33.05 (CH2), 27.20 (CH2-CH2COO), 26.73 (CH2-CH2NH2), 23.63 (CH2--CH3), 14.44 (CH3).
Preparation of norspermidine trioctanoic acid salt
[0318] Norspermidine (140 ^iL, 1 .0 mmol) was dissolved in 10 mL ethanol and octanoic acid (475 uL, 3.0 mmol) was added. The solvent was evaporated and the residue washed twice with 10 mL liexanes, 30 mL diethyl ether, then sucked off and washed with 30 mL ethyl acetate (yield 467 mg, 83%).
[0319] 1H NMR (400 MHz, CD30D): δ 3.01 (Ψΐ, J = 7.3 Hz, 4H, 2x CH2NH2), 2.89 ( Ft, J = 7.2 Hz, 4H, CH2-N-CH2), 2.17 (Ψζ J = 7.6 Hz, 6H, 3x CH2COO), 1.94 (Ψρ, J = 7.2 Hz, 4H, 2x CH2-CH2NH2), 1.62-1.55 (m, 6H, 3x CH2-CH2COO), 1 .36- 1.27 (m, 24H, 3x (CH2)4), 0.90 (t, J = 6.9 Hz, 9H, 3x CH3).
[0320] 13C NMR (400 MHz, CD30D): 6 182.16 (COO), 46.88 (CH2-N-CH2), 38.70
(CH2NH2), 38.45 (CI 12C '()( )}. 33.05 (CH2), 30.77 (CH2), 30.36 (CI 12 ). 27.50 (( 1 12-C'1 12( ()( )). 26.93 (CH2-CH2NH2), 23.77 (CH2--CH3), 14.49 (CH3).
Preparation of norspermi dine tridodecanoic acid sal t
[0321] Dodecanoic acid (601 mg, 3.0 mmol) was dissolved in 10 mL ethanol and
norspermidme ( 140 μΐ,, 1.0 mmol) was added. The solvent was evaporated and the residue washed twice with 10 mL hexanes and 30 mL ethyl acetate (yield 663 mg, 91%). [0322] 1 H NMR (400 MHz, CD30D): δ 3.00 (t, J = 7.3 Hz, 4H, 2x CH2NH2), 2.82 (Ψί, J 7.1 Hz, 4H, CH2-N-CH2), 2.19 (Ψί, J = 7.6 Hz, 6H, 3x CH2COO), 1.89 (Ψρ, J = 7.2 Hz, 4H, 2x CH2-CH2NH2), 1.60 (p, J = 7.1 , 6H, 3x CH2-CH2COO), 1.39-1.23 (m, 48H, 3x (C '! 2)8 ). 0.90 (t J = 6.9 Hz, 9H, 3x C I 13).
[0323] 13C NMR (400 MHz, CD30D): δ 181.83 (COO), 47.29 (CH2-N-CH2), 39.06 (CH2NH2), 38.23 (CH2COO), 33.14 (CH2), 30.84 (CH2), 30.82 (CH2), 30.78 (2x CH2), 30.67 ( CM ?. ). 30.54 (CH2), 27.49 (CH2-CH2NH2), 27.42 (CH2-CH2COO), 23.80 (CH2--CH3), 14.50 (CH3).
Preparation of norspermidine tristearic acid salt
[0324] Stearic acid (341 mg, 1.2 mmoi) was dissolved in 15 mL etlianol and norspermidine (60 μΐ,, 0.4 mmol) was added. The solvent was evaporated a d the residue washed twice with 10 mL hexanes and 30 mL ethyl acetate (yield 353 mg, 90%).
[0325] 1H NMR (600 MHz, CD30D): δ 3.00 (t, J = 7.1 Hz, 4H, 2x CH2NH2), 2.80 (t, J = 6.8 Hz, 4H, CH2-N-CH2), 2.19 (t, J = 7.5 Hz, 6H, 3x CH2COO), 1 .86 (p, J = 6.8 Hz, 4H, 2x CH2-CH2NH2), 1 .63-5.56 (m, 6H, 3x CH2-CH2COO), 1.40-1.20 (m, 841 1. 3x (C! 12 ) 14). 0.90 (t, J = 6.7 Hz, 9H, 3x t Ί 13).
[0326] 13C NMR (400 MHz, CD30D): 6 181.77 (COO), 47.72 (CH2-N-CH2), 39.46 (CH2NH2), 38.34 (CH2COO), 33.13 (CH2), 30.84 (lOx ( 1 12 ). 30.69 (CH2), 30.54 (CH2), 28.24 (CH2-CH2NH2), 27.50 (CH2-CH2COO), 23.80 (CH2--CH3), 14.49 (C I 13).
Preparation of Norspermidine tripalmitic acid salt
[0327] Palmitic acid (769 rng, 3.0 nimol) was dissolved in 10 mL ethanol and norspermidine (140 μί,, 1.0 mmol) was added. The solvent was evaporated and the residue washed twice with 10 mL hexanes and 30 mL ethyl acetate (yield 827 mg, 92%).
[0328] 1 1 1 NMR (400 MHz, CD30D): δ 3.00 (t, J = 7.3 Hz, 4H, 2x CH2NH2), 2.80 (t, J
= 7.0 Hz, 4H, CH2-N-CH2), 2.19 (t, J - 7.6 Hz, 6H, 3x CH2COO), 1.88 (p, j = 7.1 Hz, 4H, 2x CH2-CH2 H2), 1.60 (p, J = 7.0 Hz, 6H, 3x CH2-CH2COO), 1.36-1.24 (m, 72H, 3x (CH2)12), 0.90 (t, J = 6.9 Hz, 9H, 3x CH3).
[0329] 13C NMR (400 MHz, CD30D): δ 181.63 (COO), 47.43 (CH2-N-CH2), 39.19 (CH2NH2), 38.08 (CH2COO), 33.14 (CH2), 30.85 (4x CH2), 30.83 (CH2), 30.82 (CI 12). 30.78 (CH2), 30.76 (CH2), 30.67 (CH2), 30.54 (CH2), 27.71 (CH2-CH2NH2), 27.37 (CH2- CI 12C ()()}. 23.80 (CH2--CH3), 14.50 (CH3).
Preparation of norspermidine trimyristic acid salt
[0330] Myristic acid (411 mg, 1.8 mmol) was dissolved in 20 mL ethanol and norspermidine (84 μΐ., 0.6 mmol) was added. The solvent was evaporated and the residue washed twice with 10 mL hex arte then sucked off and washed with 30 mL ethyl acetate (yield 431 mg, 88%).
[0331] 1H NMR (400 MHz, CD30D): δ 3.00 (Ψί:, J = 7.3 Hz, 4H, 2x CH2NH2), 2.82 (Ψί, J = 7.1 Hz, 4H, CH2-N-CH2), 2.19 (Ψζ J = 7.6 Hz, 6H, 3x CH2COO), 1.89 (Ψρ, J = 7.1 , 4H, 2x CH2-CH2NH2), 1 .60 (Ψρ, J = 7.2, 6H, 3x CH2-CH2COO), 1.33-1.29 (m, 60H, 3x (CH2)10), 0.90 (t, J = 6.9 Hz, 91 1. 3x CI 13) [0332] 13C MR (400 MHz, CD30D): 5 181.78 (COO), 47.30 (CH2-N-CH2), 39.07
(CH2 H2), 38.19 (CH2COO), 33.14 (CH2), 30.86 (CI 12 ;·. 30.85 (CH2), 30.84 (CH2), 30.83 (CH2), 30.78 (2x CH2), 30.67 (CH2), 30.54 (CH2), 27.40 (CH2-CH2COO), 27.51 (CH2- CH2NH2), 23.80 (CH2--CH3), 14.50 (CHS).
Preparation of micelle formulation 9: 1 Tetradecanoic acid : Norspermidine tetradecanoic acid salt (formulation C )
[0333] 90 iL of a 100 mM stock of tetradecanoic a cid in ethanol and 10 ,uL of a 100 mM stock of norspermidine tridodecanoate in ethanol were mixed and the solvent was evaporated. The compounds were emulsified by addition of 100 μL of 100 mM sodium taurocholate, 90 iL of 10% (w/v) saline and 810 p,L deionized water. The micelles were formulated at 50°C and homogenized by passing through a 0.2 μηι sterile filter to give a 1 mM micelle formulation with 10 mM sodium taurocholate in 0.9% saline.
Preparation of micelle formulation 1 : 1 Tetradecanoic acid : Norspermidine tetradecanoic acid salt
[0334] 50 p.L of a 100 mM stock of tetradecanoic acid in ethanol and 50 uL of a 100 mM stock of norspermidine tridodecanoate in ethanol were mixed and the sol vent was evaporated. The compounds were emulsified by addition of 100 ,uL of 500 mM sodium taurocholate, 90 p.L of 10% (w/v) saline and 810 ,u.L deionized water. The micelles were formulated at 50°C and homogenized by passing through a 0.2 tun sterile filter to give a 1 mM micelle formulation with 10 mM sodium taurocholate in 0.9% saline.
Preparation of micelle formulation with orspermidine dodecanoic acid salt (formulation A)
[0335] The sol vent of 100 μΐ., of a 100 mM stock of norspermidine tridodecanoic acid salt in ethanol was evaporated. The compound was emulsified by addition of 100 μΐ. of 100 mM sodium taurocholate, 90 μΐ. of 10% (w/v) saline and 810 μ deionized water. The micelles were formulated at 50°C and homogenized by passing through a. 0.2 μηι sterile filter to give a 1 mM micelle formulation with 10 mM sodium taurocholate in 0.9% saline.
Preparation of micelle formulation Norspermidine tetradecanoate (formulation B)
[0336] The solvent of 100 uL of a 100 mM stock of norspermidine tritetraecanoic acid salt in ethanol was evaporated. The compound was emulsified by addition of 100 μΡ. of 100 mM sodium taurocholate, 90 μΕ of 10% (w/v) saline and 810 μί deionized water. The micelles were formulated at 50°C and homogenized by passing through a. 0.2 μηι sterile filter to give a 1 mM micelle formulation with 10 niM sodium taurocholate in 0.9% saline.
Example 16: Inhibition Assays Bacterial strains
[0337] B. subtilis strain NCBI 3610: a wild strain of B. subtilis, which is capable of forming robust biofilms.5
[0338] Staphylococcus aureus SCOl was obtained from the Kolter lab collection.2
[0339] B. subtilis strain NCBI 3610 and S. aureus SCOl were isolated as a single colonies growing overnight at 37°C on solid LB medium.
Biofilm inhibition assay [0340] B. subtilis strain NCBI 3610 was grown for 2 hours with shaking at 37°C, then diluted 1 : 1000 in either 3 mi/well (12 well plates, polystyrene, VWR) or 1.5 mi/well (24 wells plates, polystyrene, VWR) of defined biofilm medium MSgg (pH 7.0). Cells were grown without shaking for 3 days at 25°C. For pH experiments bacteria, were additionally grown in MSgg at pH 6.0 and 8.0. Floating biofilms (pellicles) οΐΒ, subtilis were examined optically.
[0341] S. aureus strain SCOl was grown overnight with shaking at 37°C, then diluted 1 : 100 in 1 ml/well of TSB medium, 0.5% glucose, 3% NaCl (pH 7.0). Cells were grown without shaking for 24 hours at, 37°C. For pH experiments bacteria were additionally grown in MSgg at pH 6.0 and 8.0. Submerged biofilms of S. aureus were observed and quantified by crystal violet staining as below.
[0342] Inhibition of biofilm formation is shown in FIG . 1A and MBICs are shown in Table 3, supra. FIG. I B shows the results of contacting a preexisting biofilm with compound 7a,
[0343] Stock solutions of the fatty acid salts were diluted and added to the bac teria to test for biofilm inhibition. MBICs for fatty acid salts of guanidines and polyamines are shown in Table 5, supra.
[0344] Inhibition of biofilm formation by different norspermidine micelle formulations is shown in FIGS. 7A-B.
Example 17: Crystal violet staining
[0345] Crystal Violet (CV) staining was done as described previously except that the cells were grown in 24-well plates.3 Weils were stained with 300 μΐ of 1 .0 % Crystal-violet dye, rinsed twice with 2 ml deionised water and thoroughly dried. For quantification, 0.5 ml of 95 % ethanol were added to each well. Plates were incubated for one hour at room temperature with shaking. CV solution was diluted and the OD at 595 nm was measured using an IJltraspec 2000 (Pharmacia Biotech). The results of compound 7 as shown in FIG. I B demonstrate that the guanidines disclosed herein are also capable of disrupting formed biofilms. Likewise, the results of the different norspermidine micelle formulations shown in FIGS. 7A-B demonstrate that micelle formulations and other lipid-bound compositions disclosed herein are also capable of disrupting formed biofilms.
Example 18: Solubility data ( s values)
[0346] 20-30 mg of each compound was incubated with a volume of H?0 that was insufficient to fully dissolve the entire amount (typically 100-300 uL) and incubated for at, least 24 hours at, 20°C. The undissolved material was centrifuged off and a defined volume of the saturated solution was removed, lyophilized, and the weight of the product was determined. The K, values are shown in Table 6.
Table 6. Solubility data as Ks values for the compounds.
Figure imgf000108_0001
Figure imgf000109_0001
Example 19: pKa determination and ion speciation
[0347] In addition to structural and spatial properties, the charge of the compounds is also an important property for binding with the exopolysaecharide. To investigate this effect and the correlation with activity of the compounds, the pKa values of selected compounds were determined at 25°C with concentrations of 25 mM. Cumulative association constants were calculated by HypNMR13' u and values for pKa (D20) were finally converted to pKa. (H2O), The pKa values for the compounds are given in Table 5. For comparison, similar pKa values have been reported before for spermidine with pKl = 10.90, pK2 = 9.71, and pK3 = 8.25.16. (See, e.g., Kimberly, M. Anal. Ch em., 1981 , 53, (6), 789-93). As discussed above, the results shown in Table 7 demonstrate that the more effective guanidines have higher pKa values.
Table 7
Figure imgf000110_0001
[0348] Speciation data derived from. pKa values were generated using the program HySS and average charge of the molecules was plotted against pH (FIG. 2), For con venience protonation states will be given in the following by a digit string with (1 ) for a protonated site and (0) for a nonprotonated site. A fully protonated norspermidme is in this notation (1 1 1). Diethylenetriamine (DET) has one extremely low protonation constant of 3.9 that results in one uncharged amino group in a wide range of pH 5-8 (FIG. 2A). Although guanidine groups in the related structure 2 significantly increase the third pKa value to 6.3, the central amino group remains non-protonated (101) at physiological pH values, which was confirmed by X-ray structure analysis (FIG. 4). It could remain inactive in bioassays at pH 7 due to either its charge or structure. Similarly, guanidine groups on the scaffold of norsperrnidine or norsperniine increased all individual pKa values compared to the corresponding polyamines (FIG. 2A) causing the average degree of protonation to rise which is in line with the increased activity of 6 over norsperrnidine and 7 over norsperniine. However, 5 was active but did not display increased activity over norsperrnidine, despite higher degree of protonation.
[0349] The low availability of compound 5 may be a critical factor, which was confirmed by a novel method for the delivery of compounds into the biofiim. Norsperniine carries on average 3.3 positive charges at pH 7 corresponding to about 30% fully charged (1111) molecules, while the majority is only triple charged (1110 or 1101) and will likely populate predominantly the protonation micro-state (1101) with one non-charged secondary amine, 14, 16, 17. The protonation mico-state (1101) would not comply with the triple charged motif (111) and may contribute to the higher activity of norsperrnidine over norsperniine,
[0350] 'The active compound 11 exists at maximum in a double protonated form which is due to high pKa values virtually the only relevant species until pH 8.5. The structure and protonation ofbiguanides is in the literature frequently misrepresented as reported by Bharatam et al., who showed by theoretical studies that the central nitrogen ofbiguanides usually carries hydrogen neither in neutral nor in charged states (FIG. 2B and C). This N is rather partially negatively charged while the positi ve charge is delocahzed between the terminal nitrogens of any biguanide. The crystal stracture of protonated 11 confirmed these results (FIG. 2D and FIG. 6), making 11 analogous to the (1111) motif of fully charged compound 7, which explains its activity. [0351 ] Finally, confirming that charge is a critical factor for activity, the inhibition of biofilm formation in pH dependence, which should directly affect the average degree of protonation of the correspo ding compounds, was determined. MBIC values of B. subtilis and S. aureus were plotted against the degree of protonation inferred from speciation data for different pH values (FIG, 3). Although biofilm morphology and physiology as well as bioavailability of the compounds are expected to change with pH, the potency of active compounds correlated well with the degree of protonation as predicted by the model. For all active compounds, activity generally increased (lower MBICs) in both species at higher protonation states while the inactive compound 2 did not respond to changes in protonation. Absolute activities of different compounds, however, did not coincide with the degree of protonation, suggesting that not charge alone but the combination of structure and charge determine activity.
[0352] For pH and pD measurements, a FHR-146B Micro combination pH electrode (Cole Farmer) was used. The electrode as calibrated for pH using commercial standard solutions of pH 4.0, 7.0 and 10.0 and for pD applying the relation4 pD = pH + 0.40 to convert pD to pH which was additionally verified by two pD calibration standards of 2DPO4/ D2PO4 (0.025 M K2DP04 and 0.025 M KD2P04 in D20) and K2C03/KDC03 (0.025 M 2C03 and 0.025 M
Figure imgf000112_0001
[0353] To measure p a values in the range of 2<pH<12 the strategy of Blagbrough et, al. was applied. Titrations were conducted in D20 using Ή or 13C NMR as described before without correction for ionic strength/1 Solutions of 25 mM of the compounds in D20 were prepared (~1 mL to compensate for losses during titration steps). Small amounts of DC! (35 wt.% in D20, 99 atom % D, Sigma Aklrich) and KOD (40 wt.% in D20, 98 atom% D, Sigma Aldrich) were added successively and pD values were measured followed by !H or L,C NMR spectroscopy at 25°C.
I l l [0354] Higher pKa values were determined by the method recommended by Popov et al. for 12<pH<14 in H20 by i C MR at fixed ionic strength of I = 1 M with OH/KC1 using calculated pH values.4 Compounds were dissolved in the pre-adjusted OH KC1 solutions to give about 500 uL of 25 mM concentration. Corrections for the deprotonation of compound were applied as suggested. Close below pH 12.5, the calculated values were checked and if necessary corrected by pH measurements. In some cases, pH values below 12 were added to expand the pH range and directly measured by the microelectrode after compound addition. Chemical shifts were plotted as function of pH or pD and cumulative association constants were calculated by HypN R2008 (Protonic Software)6' ' with the setting pKw ::: 13.80s and using the relation pD = pH + 0.40 to finally convert p a (D20) to p a (H20).4
(0355] Ion speciations were calculated from p a values using the program HySS2009 - Hyperquad Simulation and Speciation (Protonic Software).
Example 20: Crystal structures
(0356] Compounds 2a, 5a and 11a were re-crystallized from deionized water and structures were determined by X-ray diffraction at the Harvard University Center for Crystallographic Studies. Data were collected at 100 from a crystal mounted on a Bruker APEX II CCD diffractometer (Μθκα radiation, λ=0.71073 A) equipped with an Oxford Cryosystems nitrogen flow apparatus. The collection method involved 0.5° scans in ω at 28° in 2Θ. Data integration down to 0.78 A resolution was carried out using SAINT V7.46 A (Broker diffractometer, 2009) with reflection spot size optimization. Absorption corrections were made with the program SADABS (Bruker diffractometer, 2009). The structure was solved by the direct methods procedure and refined by least-squares methods again F2 using SHELXS-97 and SHELXL-979 with OLEX 2 interface.30 Non-hydrogen atoms were refined anisotropically, and hydrogen atoms were allowed to ride on the respective atoms. Crystal data as well as details of data collection and refinement are summarized in Table 8 and FIGS. 4-6.
Table 8. Crystal data, and data on collection and refinement.
Figure imgf000114_0001
EQUIVALENTS
[0357] It is to be understood that 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. A method of treating, reducing, or inhi biting biofiim formation by bacteria, the method comprising: contacting an article with a composition comprising an effective amount of a guanidine of Formula ( I) or Formula (II),
Figure imgf000116_0001
or a base, salt, hydrate, or tautomer thereof, wherein
Y is C( R;:R(,}. O, S, NFL or NR10;
M is C(RiR2)-Y-C(R$R4), meta-plienylene, or 1 -C5-C7 cycioalkane, where the meta- phenylene or cycioalkane may be substituted by R10 or have one or more N heteroatoms in its ring; each j, R2, R3, R4, R5, and § is H, C1-Q2 alkyl, C-.-Ci?. alkenyl, Ci-Ci2 alkynyl, Ci- Cj2 alkoxy, alkaryl, aryl, or heteroaryl, so that each Rj , R2, R3, R4, R5, and Re may be the same or different; each R7, R¾>, and jo is H, C]-C]6 alkyl, C C , alkenyl, C3-C16 alkynyl, aryl, heteroaryl, or C7-C22 aralkyl, so that each R7, R9, and R10 may be the same or different; each R8 is H or -C(=NR7)-NR7H, so that each R8 may be the same or different; and each x an integer from 1 to 6, inclusive, thereby treating, reducing or inhibiting formation of the biofilm.
2. The method of claim 1, wherein Y is QRsRe).
3. The method of claim 1 or 2, wherein x is 2 or 3,
4. The method of any one of the preceding claims, wherein each R¾, R2, R3, R4, R5, and Re is H.
5. 'The method of claim 1 wherein the guanidine is a guanidine of Formula (II), wherein M is meta-phenylene.
6. The method of claim 1, wherein the guanidine is at least one compound selected from the group consisting of:
Figure imgf000117_0001
Figure imgf000118_0001
7. The method of any one of the preceding claims, wherein the composition further comprises an effective amount of a D-amino acid, thereby treating, reducing or inhibiting formation of the biofilm,
8. The method of any one of the preceding claims, wherein the composition comprises a fatty acid salt of the guanidine, wherein the fatty acid has a chain length between C4 and C20.
9. The method of claim 1 , wherein the composition comprises a carrier structure.
10. A coated article resistant to biofilm formation, comprising: an article comprising a coating on at least one exposed surface, the coating comprising an effective amount of a guanidine of Formula (I) or Formula (II),
Figure imgf000119_0001
or a base, salt, hydrate, or tautomer thereof, wherein Y is Ci'RsRe), O. S, NH, or NR] 0;
M is C(R]R2)-Y-C(R3R4), meta-phenylene, or 1 ,3-Cs-C cycloalkane, where the meta- phenyiene or cycloalkane may be substituted by Rio or have one or more N heteroatoms in its ring; each Ri, R2, R3, R4, R5, and R$ is H, C1-C12 alkyl, (' ·-( * r- alkenyl, C1-C12 alkynyl, Ci- C12 alkoxy, alkaryl, aryi, or heteroaryl, so that each Ri, R.2, R3, R4, R5, and Re may be the same or different; each Ry, R9, and io is H, Ci-Cie alkyl, C3-C16 alkenyl, C3-C16 alkynyl, aryi, heteroaryl, or C7-C22 aralkyl, so that each Ry, R9, and io may be the same or different; each R is H or -C(=NR7)- R7H, so that each R8 may be the same or different; and each x an integer from. 1 to 6, inclusive, thereby reducing or inhibiting formation of the biofilm.
1 1. The coated article of claim 10, wherein the guanidine is a. compound selected from the group consisting of:
Figure imgf000120_0001
Figure imgf000121_0001
12. The coated article of any one of claims 10-1 1 , wherein the coating further comprises an effective amount of a D-amino acid, thereby treating, reducing or inhibiting formation of the biofilm.
13. The coated article of any of claims 10-12, wherein the article is one or more selected from the group consisting of comprises an industrial equipment, plumbing systems, bodies of water, household surfaces, textiles and paper.
14. The coated article of any of claims 10-12, wherein the article is one or more components involved in water condensate collection, water recirculation, sewerage transport, paper pulping and manufacture, and water processing and transport.
15. The coated article of any of claims 10-12, wherein the article is a drain, tub, kitchen
appliance, countertop, shower curtain, grout, toilet, industrial food or beverage production facility, floor, boat, pier, oil platform, water intake port, sieve, water pipe, cooling system, or powerplant.
16. The coated article of any of claims 10- 12, wherein the article is made from a material selected from the group consisting of metal, metal alloy, synthetic polymer, natural polymer, ceramic, wood, glass, leather, paper, fabric, nom-metallie inorganics, composite materials and combinations thereof.
17. The coated article of any of claims 10-16, wherein the coating further comprises a binder. 58. The coated article or composition of any of claims 10-17, further comprising a biocide.
19. The coated article or composition of claim 18, wherein the biocide comprises
polyhexamethylerie biguanide, chlorhexidirie, xylitol, triclosan, or chlorine dioxide.
20. A composition resistant to biofilm formation, comprising: a carrier; and an effective amount of a guanidine of Formula (Γ) or Formula (II),
Figure imgf000123_0001
or a base, salt, hydrate, or tautomer thereof, wherein
Y is C(R5R<s), O, S, NFL or NR10;
M is C(R1R2)-Y-C(R3R4), meta-phenylene, or 1 J-CyC- cycloalkane, where the meta- phenylene or cycloalkane may he substituted by R10 or have one or more N heteroatoms in its ring; each R3 , R2, R3, R4, R5, and Re is H, CrC12 alkyl, Ci-C12 alkenyl, C3-C12 alkynyl, Q- C12 aikoxy, alkaryl, aryl, or heteroarvl, so that each Ri, R2, R3, R4, R5, and Re may be the same or different; each R7, R9, and Rj0 is H, C1-C16 alkyl, C3-C16 alkenyl, C3-Ci6 alkynyl, aryl, heteroarvl, or C7-C22 aralkyl, so that each R7, R9, and RJQ may be the same or different; each Rg is H or -C(= R7)-1S1 R?H, so that each Rg may be the same or different; each x an integer from 1 to 6, inclusive, thereby reducing or inhibiting formation of the biofi!m.
The composition of claim 20, wherein the guanidme is a compound selected from group consisting of:
Figure imgf000124_0001
Figure imgf000125_0001
22. The composition of any one of claims 20-21 , further comprising an effective amount of a D-amino acid distribiited in the base, thereby treating, reducing or inhibiting formation of the biofilm.
23. The composition of any one of claims 20-22, wherein the carrier is a polymeric binder or a fluid base.
24. The composition of any one of claims 20-22, wherein carrier is a fluid base selected from a liquid, gel, paste,
25. The composition of any one of claims 20-24, wherein the composition is selected from the group consisting of water, washing formulations, disinfecting formulations, paints and coating formulations. A method of treating a biofilni-related disorder in a subject in need thereof, the method comprising administering to the subject a composition comprising an effective amount of a guanidine of Formula (I) or Formula (Π),
Figure imgf000126_0001
or a base, salt, hydrate, or tautomer thereof, wherein
Y is C(R5R6), O, S, NH, or NR10;
M is C(RiR.2)-Y-C(R3R4), meta-phenylene, or 1 ,3-Cs-C6 cycloalkane, where the meta- phenylene or cycloalkane may be substituted by Rio or have one or more N heteroatoms in its ring; each Rj, R2, R3, R4, R5, and Re is H, C3-C12 alkyl, C1-C32 alkenyl, C1-C12 alkynyl, Cj- C12 alkoxy, alkaryl, aryl, or heteroaryl, so that each Rj , R2, R3, R4, R5, and may be the same or different; each 7, R¾ and Rio is H, C\-C\e alkyl, C3-C16 alkenyi, ( " --·( ' ·;. alkynyl, aryl, heteroaryl, or C7-C22 aralkyl, so that each R?, R¾ and R10 may be the same or different; each Rg is H or -C^NR?)-!^ R?H, so that each Rg may be the same or different; and each x an integer from 1 to 6, inclusive, thereby treating the biofilm -related disorder.
The method of claim 26, wherein the guamdine is a compound selected from the group consisting of:
Figure imgf000127_0001
Figure imgf000128_0001
28. The method of any one of claims 1-27, wherein the composition is administered to a surface of the subject selected from the group of dermal and mucosal surfaces and combinations thereof.
29. The method of claim 28, wherein the surface is an oral surface, a skin surface, a urinary tract, surface, a vaginal tract surface, or a lung surface.
30. The method of claims 1-28, wherei the composition is administered to the subject via subcutaneous, intra-museular, intra-peritoneal, intravenous, oral, nasal, or topical administration, and a combination thereof.
31. The method any of claims 1-28, wherein the subject is a human.
32. The method of any of claim 1-28, wherein the biofilm-related disorder is selected from the group consisting of pneumonia, cystic fibrosis, otitis media, chronic obstructive pulmonary disease, and a urinary tract infection and combinations thereof.
33. The method of claim 1-28, wherein the biofilm-related disorder is a medical device- related infection.
34. The method of any of claims 1 -28, wherein the biofilm-related disorder is caused by bacteria.
35. A method of treating, reducing, or inhibiting biofilm formation by bacteria on a
biologically-related surface, the method comprising: contacting a biological surface with a composition comprising an effective amount of a guanidine of Formula (I) or Formula (II),
Figure imgf000129_0001
or a base, salt, hydrate, or tautomer thereof, wherein Y is Ci'RsRe), O. S, NH, or NR] 0;
M is C(R]R2)-Y-C(R3R4), meta-phenyiene, or 1 ,3-Cs-C cycloalkane, where the meta- phenyiene or cycloalkane may be substituted by Rio or have one or more N heteroatoms in its ring; each Ri, R2, R3, R4, R5, and R$ is H, C1-C12 alkyl, (' ·-( * r- alkenyl, C1-C12 alkyny], Ci- C12 alkoxy, alkaryl, aryi, or heteroaryl, so that each Ri , R.2, R3, R4, R5, and Re may be the same or different; each Ry, R9, and io is H, Ci-Cie alkyl, C3-C16 alkenyl, C3-C16 alkynyl, aryi, heteroaryl, or C7-C22 aralkyl, so that each Ry, R9, and io may be the same or different; each R is H or -C(=NR7)- R7H, so that each R8 may be the same or different; and each x an integer from. 1 to 6, inclusive, thereby treating, reducing, or inhibiting formation of the biofilm.
The method of claim 35, wherein the guanidine is a compound selected from the group consisting of:
Figure imgf000130_0001
Figure imgf000131_0001
37. The method of any one of claims 35-36, wherein the surface comprises a medical device, a wound dressing, a contact lens, or an oral device.
38. The method of claim 37, wherein the medical device is selected from the group
consisting of a clamp, forcep, scissors, skin hook, tubing, needle, retractor, scaler, drill, chisel, rasp, saw, catheter, orthopedic device, artificial heart valve, prosthetic joint, voice prosthetic, stent, shunt, pacemaker, surgical pin, respirator, ventilator, and an endoscope and combinations thereof.
39. The method of any one of claims 1-38, wherein the composition further comprises a D-amino acid, or pharmaceutically acceptable salts, esters, or derivatives thereof.
40. The method of any one of claims 1-39, further comprising administering a biocide. 45. The method of claim 40, wherein the biocide is an antibiotic.
42. A composition comprising: a pharmaceutically acceptable carrier; and a guanidine of Formula (I) or Formula (II),
Figure imgf000132_0001
Figure imgf000133_0001
or a base, salt, hydrate, or tautomer thereof, wherein
Y is C(R5R6), O, S, M S . or NR!0:
M is C( jR2)-Y-C(RjR4), meta-phenylene, or 1 -C5-C7 cyeioalkane, where the meta- phenylene or cyeioalkane may be substituted by Ri0 or have one or more N heteroatoms in its ring; each Rj, R2, R3, R4, R5, and Re is H, C 1-C12 alkyl, C1-C12 alkenyl, C¾-C]? alkynyL C¾ - C12 alkoxy, alkaryl, aryi, or heteroaryl, so that each R\, R2, R3, R4, R5, and 5 may be the same or different; each R7, R9, and R¾o is H, Cj-Cie alkyl, C3-Cj6 alkenyl, C3-C16 alkynyl, aryl, heteroaryl, or C7-C22 aralkyl, so that each R7, R9, and Rjo may be the same or different; each Rs is H or -C(= R7)-1S1 R7H, so that each Rg may be the same or different; and each x an integer from 1 to 6, inclusive, in an amount effective to treat, reduce, or inhibit biofilm formation.
43. The composition of claim 42, wherein the guanidine is a compound selected from the group consisting of:
Figure imgf000134_0001
, and
Figure imgf000135_0001
44. The composition of any one of claims 42-43, further comprising a D-amino acid in an amount effective to treat, reduce, or inhibit biofiim formation.
45. The composition of any one of claims 42-44, wherein the effective amount is an amount effective to treat or prevent a biofilm-related disorder.
46. The composition of claim 45, wherein the biofilm-related disorder is pneumonia, cystic fibrosis, otitis media, chronic obstructive pulmonary disease, or a urinary tract infection.
47. The composition of claim 45, wherein the biofilm-related disorder is a medical device- related infection.
48. The composition of any of claims 42-47, wherein the composition is formulated as a wash solution, a dressing, a wound gel, a synthetic tissue, tablets, pills, troches, capsules, aerosol spray, solutions, suspensions, gels, pastes, creams, or foams.
49. The composition of any of claims 42-47, wherein the composition is formulated for parenteral, intravenous, intradermal, subcutaneous, oral, inhalation, transdermal, topical, transmucosal, vaginal or rectal administration.
50. A composition resistant to biofiim formation, comprising: a pharmaceutically or cosmetically suitable base; and an effective amount of a guanidine of Formula (Γ) or Formula (II),
Figure imgf000136_0001
Figure imgf000136_0002
or a base, salt, hydrate, or tautomer thereof, wherein
Y is C(R5R6), O, S, NH, or NR10;
M is C(RiR2)-Y-C(R3R4), meta-phenylene, or 1 ,3-Cs-C7 cycloalkane, where the meta- phenylene or cycloalkane may be substituted by io or have one or more N heteroatoms in its ring; each Rj, R2, R3, R4, R5, and e is H, C3-C12 alkyl, C1-C32 alkenyl, Ci~Ci2 alkynyl, Cj- C12 alkoxy, alkaryl, aryl, or heteroaryl, so that each i, R2, R3, R4, R5, and Re may be the same or different; each R7, R9, and Rio is H, Cj-Qe alkyl, C3-C16 alkenyl, C3-C16 alkynyl, aryl, heteroaryl, or C7-C22 aralkyl, so that each R7, R9, and Rio may be the same or different; each -8 is H or -C(= R7)-NR7H, so that each Rg may be the same or different; and each x an integer from 1 to 6, inclusive, said guanidine being distributed in the base, thereby treating, reducing or inhibiting formation of the biofilm.
The composition of claim 50, wherein the guanidine is a compound selected from group consisting of:
Figure imgf000137_0001
Figure imgf000138_0001
52. The composition of any one of claims 50-5 , further comprising a D-araino acid.
53. The composition of any one of claims 50-52, wherein the base is selected from a liquid, gel, paste, or powder.
54. The composition of claim 53, wherein the composition is selected from the group
consisting of shampoos, bath additives, hair care preparations, soaps, lotions, creams, deodorants, skin-care preparations, cosmetic personal care preparations, intimate hygiene preparations, foot care preparations, light protective preparations, skin tanning preparations, insect, repellants, antiperspirants, sharing preparations, hair removal preparations, fragrance preparations, dental care, denture care and mouth care preparations and combinations thereof.
55. An oral composition comprising: an orally acceptable carrier; and an effective amount of a guanidine of Formula (1) or Formula (IS),
Figure imgf000139_0001
or a base, salt, hydrate, or tautomer thereof, wherein
Y is C(R5R6), O, S, NH, or NR10;
M is C(RiR.2)-Y-C(R3R4), meta-phenylene, or 1 ,3-Cs-C6 cycloalkane, where the meta- phenylene or cycloalkane may be substituted by Rio or have one or more N heteroatoms in its ring; each Rj, R2, R3, R4, R5, and Rg is H, C¾-C]? alkyl, Ci-C12 alkenyl, Ci-Cj2 alkynyl, Cj- C12 alkoxy, alkaryl, aryl, or heteroaryl, so that each Rj , R2, R3, R4, R5, and may be the same or different; each 7, R¾ and Rio is H, C\-C\e alkyl, C3-C16 alkenyi, ( " --·( ' ·;. alkynyl, aryl, heteroary or C7-C22 aralkyl, so that each R7, R¾ and R10 may be the same or different; each Rg is H or -C(= R7)-1S1 R7H, so that each Rg may be the same or different; and each x an integer from 1 to 6, inclusive, thereby treating, reducing or inhibiting formation of the biofilm.
The oral corapositior! of claim 55, wherein the guanidine is a compound selected from the group consisting of:
Figure imgf000140_0001
Figure imgf000141_0001
The oral composition of any one of claims 55-56, further comprising an effective amount of a D-amino acid, thereby treating, reducing or inhibiting formation of the biofilm.
The oral composition of any one of claims 55-57, wherein the oral composition is in the form of a toothpaste, tooth gel, tooth powder, a mouthwash, mouth rinse, mouth spray, a dental solution, or an irrigation fluid.
The method, article, composition, device, or liquid of any one of the preceding claims, wherein the bacteria are Gram-negative, Gram-positive bacteria, or mycobacteria.
The method, article, composition, device, or liquid of claim 59, wherein the bacteria are of the genus ActinobacUlus, Acinetobacter, Aeromonas, Bordetella, Brevibacillns, Brucella, Bacteroides, Biirkholderia, Boreiia, Bacillus, Campylobacter,
Capnocytophaga, Cardio bacterium, Citrobacter, Clostridium-, Chlamydia, Eikenella, Enierobacter, Escherichia, Entembacter, Francis ella, Fusobacterium, Flavobacterium, Haemophilus, Helicobacter, Kingella, Klebsiella, Legionella, Listeria, Leptospirae, Moraxella, Morganella, Mycoplasma, Mycobacterium, Neisseria, PasteureUa, Proteus, Prevotella, Plesiomonas, Pseudomonas, Providencia, Rickettsia, Stenotrophomonas, Staphylococcus, Streptococcus, Streptomyces, Salmonella, Serratia, Shigella, Spirillum, Treponema, Veillonella, Vibrio, Yersinia, or Xanthomonas.
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US9439433B2 (en) 2013-05-22 2016-09-13 Curza Global, Llc Compositions and methods comprising a biocidal polyamine
US9550345B2 (en) 2013-05-16 2017-01-24 Universiteit Twente Process for the preparation of an object supporting a lipid bilayer
RU2646488C2 (en) * 2016-04-25 2018-03-05 Государственное бюджетное образовательное учреждение высшего профессионального образования "Оренбургский государственный медицинский университет" Министерства здравоохранения Российской Федерации (ГБОУ ВПО ОрГМУ Минздрава России) Means for selective effect on biofilm formation by microorganisms
US10786448B2 (en) 2018-01-18 2020-09-29 Christian Arnold Chewing gum composition comprising polyhexanide
WO2021222393A1 (en) * 2020-04-28 2021-11-04 Zelos Energy Ltd. Polymer coating process for electrode assemblies incorporating ion exchange materials
US11352315B2 (en) 2017-04-05 2022-06-07 Curza Global, Llc Compositions and methods comprising a triaryl polyamine

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012151554A1 (en) * 2011-05-04 2012-11-08 President And Fellows Of Harvard College Polyamines for treating biofilms

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012151554A1 (en) * 2011-05-04 2012-11-08 President And Fellows Of Harvard College Polyamines for treating biofilms

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BONCHER* T ET AL: "Polyamine-based analogues as biochemical probes and potential therapeutics", BIOCHEMICAL SOCIETY TRANSACTIONS, PORTLAND PRESS LTD, GB, vol. 35, no. 2, 1 April 2007 (2007-04-01), pages 356 - 363, XP009161409, ISSN: 0300-5127 *
KARIN THEVISSEN ET AL: "Novel fungicidal benzylsulfanyl-phenylguanidines", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, PERGAMON, AMSTERDAM, NL, vol. 21, no. 12, 19 April 2011 (2011-04-19), pages 3686 - 3692, XP028387827, ISSN: 0960-894X, [retrieved on 20110424], DOI: 10.1016/J.BMCL.2011.04.075 *
THOMAS BÖTTCHER ET AL: "Synthesis and Activity of Biomimetic Biofilm Disruptors", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 135, no. 8, 27 February 2013 (2013-02-27), pages 2927 - 2930, XP055101437, ISSN: 0002-7863, DOI: 10.1021/ja3120955 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9550345B2 (en) 2013-05-16 2017-01-24 Universiteit Twente Process for the preparation of an object supporting a lipid bilayer
US9034927B2 (en) 2013-05-22 2015-05-19 Curza Global, Llc Methods of use for compositions comprising a biocidal polyamine
US9220267B2 (en) 2013-05-22 2015-12-29 Curza Global, Llc Methods of use comprising a biocidal polyamine
US9439433B2 (en) 2013-05-22 2016-09-13 Curza Global, Llc Compositions and methods comprising a biocidal polyamine
US9839219B2 (en) 2013-05-22 2017-12-12 Curza Global, Llc Compositions comprising a biocidal polyamine
US10440955B2 (en) 2013-05-22 2019-10-15 Curza Global, Llc Methods comprising a biocidal polyamine
RU2646488C2 (en) * 2016-04-25 2018-03-05 Государственное бюджетное образовательное учреждение высшего профессионального образования "Оренбургский государственный медицинский университет" Министерства здравоохранения Российской Федерации (ГБОУ ВПО ОрГМУ Минздрава России) Means for selective effect on biofilm formation by microorganisms
US11352315B2 (en) 2017-04-05 2022-06-07 Curza Global, Llc Compositions and methods comprising a triaryl polyamine
US10786448B2 (en) 2018-01-18 2020-09-29 Christian Arnold Chewing gum composition comprising polyhexanide
WO2021222393A1 (en) * 2020-04-28 2021-11-04 Zelos Energy Ltd. Polymer coating process for electrode assemblies incorporating ion exchange materials

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