WO2017152038A1 - Method of inhibiting bacterial biofilm formation - Google Patents

Method of inhibiting bacterial biofilm formation Download PDF

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Publication number
WO2017152038A1
WO2017152038A1 PCT/US2017/020641 US2017020641W WO2017152038A1 WO 2017152038 A1 WO2017152038 A1 WO 2017152038A1 US 2017020641 W US2017020641 W US 2017020641W WO 2017152038 A1 WO2017152038 A1 WO 2017152038A1
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adhesin
epidermidis
protein
biofilm
aureus
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PCT/US2017/020641
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French (fr)
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James M. RYNERSON
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Rynerson James M
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • 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
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/047—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates having two or more hydroxy groups, e.g. sorbitol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • 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
    • A61K31/63—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide
    • A61K31/635—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide having a heterocyclic ring, e.g. sulfadiazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1271—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Micrococcaceae (F), e.g. Staphylococcus

Definitions

  • the present invention relates to methods for treating a biofilm-related disorder, and more particularly, to methods and compositions of treating a biofilm-related disorder by inhibiting biofilm formation.
  • a biofilm is a group of adherent microbial cells embedded within a matrix of composed of extracellular DNA, proteins, and polysaccharides produced by the microbial cells. Microbial cells growing in a biofilm are physiologically distinct from planktonic cells of the same organism, which are single cells that can float or swim in a liquid medium.
  • Biofilms can form on living or non-living surfaces and are prevalent in natural, industrial and hospital settings.
  • the exopolysaccharide matrix that holds the biofilm together protects the cells within the biofilm, resulting in increased resistance to detergents and to anti-microbial reagents, such as antibiotics.
  • Biofilms are involved in a wide variety of microbial infections in the body, including infections in cystic fibrosis, endocarditis, urinary tract infections, middle-ear infections, chronic sinusitis, chronic tonsillitis, formation of dental plaque, gingivitis, periodontal disease, and infections of implanted devices, such as catheters, heart valves, intrauterine devices and joint prostheses. Studies have shown that sub-therapeutic levels of antibiotics can induce biofilm formation in some microbial infections.
  • Biofilms are complex structures produced by virtually all bacteria and are the prevailing microbial lifestyle seen in nature. Biofilms may occur anywhere in nature where there exists a combination of moisture, nutrients and a surface. The biofilm is initiated by the binding of the bacteria to a surface, such as the endocardium or the eyelid margin, and facilitated by a protein molecule called an adhesin.
  • the bacteria manufacture the protein and polysaccharide components of the biofilm, which is inherently sticky and also binds tightly to the surface.
  • This biofilm continues to accumulate on the lining of the heart throughout the years of an individual's life, eventually allowing the densities and populations of bacteria to increase dramatically.
  • the biofilm allows the bacteria to resist antibiotics and avoid the host's defense mechanisms, effectively sheltering in place.
  • quorum-sensing gene activation occurs activating dormant bacterial genes to begin producing highly inflammatory virulence factors such as cytolytic toxins, enzymes, more adhesins, and exotoxins. Without being bound to any particular theory, these virulence factors may be primarily responsible for the various inflammatory manifestations of biofilm-related disorders.
  • Embodiments of the present invention address shortcomings in the numerous disorder treatments currently available by providing an easy pharmacological solution that inhibits the adherence and accumulation of biofilm along a surface.
  • the method includes administering to a subject an effective amount of one or more adhesin activity blocking compounds to a subject that prevents at least one of the adherence, binding, or production of a biofilm along a surface in or on the subject's body to treat or prevent a biofilm-related disorder.
  • the biofilm-related disorders may include, without limitation, cystic fibrosis, bacterial vegetation, endocarditis, urinary tract infections, middle-ear infections, chronic sinusitis, chronic tonsillitis, prostate disease, formation of dental plaque, gingivitis, periodontal disease, eyelid disorders such as blepharitis and dry eye syndrome, and infections of implanted devices, such as catheters, heart valves, intrauterine devices and joint prostheses. Additionally, the treatment may also be used to inhibit non-infectious bacteria.
  • Bacteria live in one of two states: planktonic (i.e., individual free-floating bacteria) or within a biofilm. Bacteria in the planktonic state are more susceptible to attack from host defenses, antibiotics, or can be washed away more easily than bacteria living within biofilms. Without being bound to any particular theory, by preventing bacterial adherence to surfaces or to an existing early biofilm results in one or more of the following: preventing or reducing biofilm accumulation, delaying or avoiding increases in bacterial densities, delaying or reducing quorum-sensing, and avoiding pathogenesis of biofilm-related disorders since production of virulence factors is delayed or reduced.
  • Bacteria synthesize the protein/polysaccharide components of the biofilm.
  • adhesins Bacteria also produce a class of proteins collectively termed "adhesins.” These adhesins allow binding of bacterial cell surface components to either host cell surface components, or to an intermediary substrate, which then binds to the host cell surface, or to components of extracellular biofilm matrix. Adhesins also promote binding of the biofilm to the host surface.
  • researchers have identified several adhesion proteins. Without being bound to a particular theory, one option for treating or preventing biofilm-related disorders is blocking or inhibiting the activity of adhesin proteins. Accordingly, an aspect of the invention is directed to treating biofilm-related disorders or preventing biofilm formation by blocking or inhibiting the activity of adhesin proteins.
  • adhesin activity may be blocked or inhibited with monoclonal antibodies (Mab) directed against adhesin proteins or their binding sites, or through compounds, such as small molecules that block or inhibit the same.
  • Mob monoclonal antibodies
  • one or more compounds having adhesin activity blocking properties are applied to eyelid surfaces in an amount effective to block adhesin activity.
  • adhesin proteins that may be targeted by embodiments of the invention are identified below.
  • Embodiments of the invention may block the activity of one or more adhesin proteins. In an embodiment, the activity of a single adhesin protein is blocked. In another embodiment, the activity of a plurality of adhesin proteins is blocked.
  • An exemplary adhesin protein with activity that may be block in embodiments of the invention is the polysaccharide intercellular adhesin.
  • the polysaccharide intercellular adhesin (PIA) is significantly involved in biofilm accumulation and is produced by both S. aureus and S. epidermidis.
  • Another exemplary class of bacterial adhesin proteins is made up of proteins covalently anchored to bacterial cell peptidoglycans, which specifically attach to the plasma or extracellular matrix (ECM) of the subject in need of treatment. These proteins are collectively referred to as microbial-surface-component-recognizing-adhesive-matrix- molecules (MSCRAMMs). MSCRAMMs recognize and bind to one or more components of the ECM, plasma, including collagens, fibrinogen (fibrinogen-binding protein, clumping factors A and B), fibronectin, elastin, laminin, bone sialoprotein, and for vitronectin.
  • SasG Staph aureus surface protein G
  • Staphylococcal protein A is another exemplary adhesin protein.
  • Embodiments of the invention include an anti-SpA antibody, such as an anti-SpA monoclonal antibody (Mab) that would be beneficial in preventing adhesin activity.
  • This antibody may be used alone or in conjunction with other antibodies, such as antibodies directed against binding targets for the SpA, such as Mabs to clumping factor (Of) A and B proteins.
  • An additional exemplary MSCRAMM adhesin protein in S. epidermidis is the fibrinogen-binding protein SdrG.
  • embodiments of the invention include an antibody directed against SdrG, such as a monoclonal antibody, which would be beneficial in preventing adhesin activity.
  • An additional exemplary MSCRAMM adhesin protein in S. epidermidis is the fibrinogen-binding protein SdrF, which binds collagen.
  • SdrF fibrinogen-binding protein
  • embodiments of the invention include an antibody directed against SdrF, such as a monoclonal antibody, which would be beneficial in preventing adhesin activity.
  • An additional exemplary adhesin protein is the S. epidermidis Extracellular Matrix Binding Protein (Embp), which is a 1.1 -MDa protein that can bind fibronectin.
  • Embp S. epidermidis Extracellular Matrix Binding Protein
  • embodiments of the invention include an antibody directed against Embp, such as a monoclonal antibody, which would be beneficial in preventing adhesin activity.
  • Another exemplary adhesin protein is LPXTG motif-containing cell wall- anchored proteins termed S. epidermidis surface proteins, which have been implicated in biofilm production, specifically SesC.
  • Polyclonal anti-SesC reduced the in vitro biofilm- forming and fibrinogen-binding and, therefore, may be beneficial when added to an administered medication.
  • embodiments of the invention include an antibody directed against LPXTG motif-containing cell wall-anchored proteins such as SesC, such as a monoclonal antibody, which would be beneficial in preventing adhesin activity.
  • Staphylococcus aureus collagen-binding protein is another exemplary cell surface adhesin. It has been shown that the topical application of a collagen-binding peptide before bacterial challenge decreased S. aureus adherence to de-epithelialized corneas. The collagen- binding adhesin is therefore involved in the pathogenesis of S. aureus infection of the cornea epithelium and may well be also involved in binding to other cell or tissue surfaces, such as the endocardium, thus promoting biofilm formation. Endocardium adherence and invasion may be blocked by cytochalasin D and genistein, two potential adhesin blocking molecules. Thus, embodiments of the invention include at least one of cytochalasin D and genistein. Further embodiments of the invention include an antibody directed against Staphylococcus aureus collagen-binding protein, such as a monoclonal antibody, which would be beneficial in preventing adhesin activity.
  • S. aureus fibronectin-binding protein may play the major role in bacterial adhesion.
  • Fibronectin is a glycoprotein found in the extracellular matrix (ECM) and body fluids. It serves as a substrate for the adhesion of normal host cells, expressing the appropriate integrins, to one another.
  • Bacteria have evolved to take advantage of the existence of fibronectin by producing fibronectin-binding proteins A and B (FnBPA and FnBPB), which cause a tight binding of the bacteria with the extracellular matrix (ECM).
  • FnBPA and FnBPB fibronectin-binding proteins A and B
  • the current model for FnBP-mediated adhesion suggests a fibronectin-dependent bridging between S. aureus FnBPs and host cell ⁇ 5 ⁇ 1 integrin.
  • Fibronectin increases in quantity with capillary dilation due to inflammation. It is likely that, once bound using existing fibronectin as the bridge, the subsequent bacterial virulence factor-induced inflammation accelerates further exudation of fibronectin from the serum of dilated capillaries, providing more substrate for further binding and therefore biofilm accumulation, resulting in a vicious cycle.
  • Competitive inhibition using anti-FnBP antibodies or FnBP-derived peptides should be effective in blocking receptor-ligand interactions at the cell surface. In one study, S. aureus invasion was substantially but not completely eliminated by the absence of FnBP. Therefore, biofilm-related disorders should be also substantially decreased by competitive binding of bacterial FnBP.
  • FnBP also binds directly to a host cell surface protein, Heat Shock Protein 60, so providing a Mab or a compound similar in action, to competitively bind the FnBP may effectively block two bacterial separate binding mechanisms.
  • HSP 60 is a 57-kDa protein that was originally thought to be associated with the mitochondrial matrix, but is now known to be typical of eukaryotic cells and cytoplasmic membranes in general, so a HSP 60 Mab could be very effective along the cell or tissue surface.
  • the integrin protein has also been found to be a binding site for human lymphocytes invasion as well, when involved with host response inflammation, and is the basis for the mechanism of action for a potent LFA- 1/ICAM- 1 antagonist SAR 1118 (Shire, Lifitegrast), such as that described in U.S. Patent Nos. 8592450 and 8748391, which competitively binds the integrin protein, thus preventing binding and activation of lymphocytes.
  • U.S. Patent Nos. 8748391, 8592450, 9085553, 9051297, etc. are for inhibiting Lymphocyte Function-associated Antigen- 1 in an effort to reduce inflammation by shutting down white cell responses to inflammatory factors (virulence factors).
  • embodiments of the present invention do not shut down white cells by blocking integrin, rather, an embodiment of the present invention shuts down the formation of biofilms by blocking adhesins, such as by antagonism of LFA- 1/ICAM- 1 with SAR 1118, thus inhibiting the buildup of bacteria, thus inhibiting the quorum sensing gene activation of virulence factors.
  • Another exemplary adhesin is a staphylococcal 145 kDa cell wall adhesin, originally found in bovine mastitis, which allows Staph to bind to epithelial cells.
  • Antibodies against the 145 kDa adhesin protein were found to block bacterial adhesion to bovine epithelial cells as well as rat intestinal epithelial cells. This adhesin may also play a role in binding staph to human cells in moist environs.
  • bacterial proteins can be bifunctional adhesins/autolysins AtlE and Aae. These proteins have specific adherence functions (by binding noncovalently to vitronectin), and may also function to release eDNA, which is an important
  • Mabs to AtlE and Aae such as monoclonal antibodies, may be included in embodiments of the invention to reduce adherence and formation of biofilms.
  • Another effective mechanism of blocking adhesins could be the addition of actin microfilament inhibitor cytochalasin D (0.5 ⁇ g/ml; Sigma) or the tyrosine kinase inhibitor genistein (250 ⁇ g/ml; Calbiochem), which have been shown to reduce the binding and subsequent internalization of S. aureus by more than 99.9%.
  • embodiments of the present invention may include one or both of these compounds.
  • embodiments of the present invention may include iMab.
  • Hsp60 MAb a MAb specific for eukaryotic Hsp60
  • Hsp60 MAb should also significantly reduce the binding of S. aureus.
  • these 2 Mabs could block the multifunctional adhesin FnBP from binding directly to both host cell ligands, Hsp60 and ⁇ integrins.
  • Du-D4 is the functional fragment of FnBP, therefore, a Du-D4 antiserum should also block the association of Hsp60 with the bacterial cell surface and thus may be an effective adhesin inhibitor.
  • Fibronectin itself could serve as an adhesin blocker in high concentrations. This effect would be consistent with the function of fibronectin as a bifunctional molecule in which a blocking effect at high concentrations would be due to saturation of the integrin- FnBP system.
  • embodiments of the present invention may include fibronectin.
  • Delmopinol is a morpholino compound that has been demonstrated to have utility in the treatment of the oral cavity, especially tooth surface, and for the removal or inhibition of dental plaque, which is a biofilm.
  • the compound and its manufacture are disclosed in U.S. Patent No. 4894221, the disclosure of which is incorporated herein by reference in its entirety.
  • Delmopinol and its derivatives may be used to prevent biofilm formation along the surface of a cell or tissue.
  • delmopinol may work more effectively when formulated with a bacterial adhesin peptide (e.g., pl025) or an antibody that binds to a bacterial adhesin peptide.
  • delmopinol may act as an augmentation agent in any of proposed adhesion inhibitors described herein.
  • Extracellular polysaccharides can also act as adhesins.
  • Exemplary extracellular polysaccharides include PS/A, PNSG, PNAG, and the aforementioned PIA.
  • Embodiments of the invention may include blocking the activity of one or more of the identified adhesin proteins to thereby reducing the biofilm accumulation.
  • S. aureus Clf40 (Nl N2N3) - full length A domain of Clumping factor A (amino acids (AA) 40-559); S. aureus Clf41 (N2N3) - post protease site fragment of Clf 40 (AA 223-559); S. epidermidis SdrG (Nl N2N3) - full length A domain of SdrG (AA 50-597); S. epidermidis SdrG (N2N3) - post protease site fragment of SdrG (AA 273-597).
  • An additional adhesin activity blocking compound that may be included, individually or combination with each other or other adhesin blocking compounds, in embodiments of the invention are thymosin ⁇ 4 (or ⁇ 4), which is available from G-TreeBNT, lactoferrin, xylitol, mangainin I covalently linked to Il-mercapto undecanoic acid and 6- mercaptohexanol in 1 :3 ratio, and Fraction 7 from Terminalia chebula or its Gallic acid, cis- 2-decenoic acid, cytochalasin D, genistein (tyrosine kinase inhibitor), norspermidine, polyamines (alone or in synergistic combination with D-amino acids), alginate lyase, N- acetyl-hearosan lyase, hyaluronidase enzymes, sulphathiazole (suphonamide), iron chelating agents (alone or
  • compositions may be suitable for systemic administration.
  • the compositions may be systemically administered by other means known in the art, such as orally, parenterally (e.g., intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection), suppository, or even transdermally such as through a gel or patch formulation.
  • parenterally e.g., intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection
  • suppository e.g., transdermally
  • transdermally e.g., transdermally
  • Such formulations may be prepared as described above or as is known to those of ordinary skill in the art.
  • the adhesin activity blocking compounds are administered in a dose effective to inhibit adhesin activity and to prevent or decrease biofilm formation.
  • An effective dose is a dose at which biofilm formation is prevented or decreased.
  • Another aspect of the invention is directed to a method of treating the biofilm- related disorder in a subject that includes systemically administering via oral administration a therapeutically effective dose of adhesin activity blocking compound to inhibit biofilm formation.
  • This oral dose of adhesin activity blocking compound may be administered in a single dose or multiple doses in a 24 hour period and may generally be administered for a period of days, weeks, months, or even years.
  • adhesin activity blocking compound is orally administered to a subject over a period of at least two weeks, and in alternative embodiments, adhesin activity blocking compound is administered for a plurality of months or a year or more.
  • embodiments of the invention are directed to the long term administration of adhesin activity blocking compound to treat a biofilm-related disorder.
  • compositions of adhesin activity blocking compound can be administered in vivo in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable.
  • the compositions may be administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • the carrier would naturally be selected to minimize any degradation of the adhesin activity blocking compound and to minimize any adverse side effects in the subject, as would be known to one of skill in the art. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) A. R. Gennaro, Mack Publishing Company, Easton, Pa. 1995.
  • an appropriate amount of a pharmaceutic ally- acceptable salt is used in the formulation to render the formulation isotonic.
  • the pharmaceutically-acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution.
  • the pH of the solution is in a pharmaceutically acceptable range, preferably from about 5 to about 8.5, and more preferably from about 7.8 to about 8.2.
  • carriers include sustained release preparations such as semi-permeable matrices of solid hydrophobic polymers containing the pharmaceutical composition, which matrices are in the form of shaped articles (e.g., films, liposomes or microparticles). It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being
  • adhesin activity blocking compound may be formed into a tablet, capsulized, dissolved, or suspended in a liquid or gel as known to those of ordinary skill in the art for oral administration of a drug.
  • adhesin activity blocking compound is formulated for sustained release, such as with the use of one or more excipients that control the release of adhesin activity blocking compound over a specified period of time for absorption by the subject.
  • compositions of adhesin activity blocking compound may also include binders, thickeners, diluents, buffers, preservatives, surface active agents, and the like in addition to the adhesin activity blocking compound and carriers.
  • Another aspect of the invention is directed to a method of treating biofilm-related disorder in a subject comprising administering via transdermal administration, such as with a gel or patch, a therapeutically effective amount of adhesin activity blocking compound to inhibit biofilm formation.
  • the transdermal formulation maintains a therapeutic blood plasma concentration of adhesin activity blocking compound and its active metabolites for a duration ranging between about 18 hours/day and about 24 hours/day.
  • the transdermal formulation includes a total dose of adhesin activity blocking compound to inhibit biofilm formation clinically, resulting in an improvement in the biofilm- related disorder such as can be determined with a quantifiable clinical observations.
  • Adhesin activity blocking compound dosing may require titration to achieve and maintain the desired effect of preventing or reducing biofilm accumulation.
  • the dose required to adequately inhibit biofilm formation administered transdermally may vary widely for a given subject as treatment progresses as well as between subjects.
  • the injection of the therapeutically effective amount of adhesin activity blocking compound may be administered in a bolus injection, by continuous infusion, or using a combination of bolus injection and continuous infusion.
  • the term bolus injection is understood to be an injection wherein the dose is delivered over a relatively short period of time.
  • the term continuous infusion is understood to be an injection delivered, such as with an intravenous drip, wherein the dose is delivered in a metered manner over the period of time desired for adhesin activity blocking compound therapy.
  • the adhesin activity blocking compound is administered via continuous infusion over a period of time ranging between about 30 min/day to about 24 hours/day.
  • therapeutically effective amount of adhesin activity blocking compound is administered over a period of about 8 hours/day to about 24 hours/day.
  • adhesin activity blocking compound is administered via continuous infusion on at least one day and up to on seven days. In other circumstances, adhesin activity blocking compound may be
  • embodiments of the invention are directed to the long term administration of adhesin activity blocking compound to treat a biofilm-related disorder.
  • a combination of bolus injection with continuous infusion may be desired to a treat a subject.
  • a bolus injection may be utilized to deliver a loading dose, i.e., a dose of adhesin activity blocking compound to rapidly achieve a desired therapeutic level of adhesin activity blocking compound in the subject, and the continuous infusion may be utilized to maintain or even titrate the desired therapeutic levels over the desired duration of treatment.
  • Another aspect of the invention is directed to a method of treating biofilm-related disorder in a subject that includes administering an extended release formulation of adhesin activity blocking compound that maintains a therapeutic blood plasma concentration of adhesin activity blocking compound and its active metabolites for a duration ranging between about 18 hours/day and about 24 hours/day.
  • the extended release formulation may be an oral formulation, an injected formulation, or even a transdermal formulation.
  • the extended release formulation includes a total dose of adhesin activity blocking compound to inhibit biofilm formation, resulting in an improvement in the biofilm-related disorder such as can be determined with a quantifiable clinical observations.
  • Adhesin activity blocking compound dosing may require titration to achieve and maintain the desired effect of preventing or reducing biofilm accumulation.
  • the effect can be determined, for example, based on a comparison between observations made before and after the implementation of therapy.
  • the dose required to adequately reduce the biofilm formation may vary widely for a given subject as treatment progresses as well as between subjects.
  • the term day is understood to be a 24 hour cycle.
  • adhesin activity blocking compound is orally administered to a subject over a period of at least two weeks, and in alternative embodiments, adhesin activity blocking compound is administered for a plurality of months or a year or more.
  • embodiments of the invention are directed to the long term administration of adhesin activity blocking compound to treat a biofilm-related disorder.
  • the adhesin activity blocking compounds are administered for a duration sufficient to relieve the symptoms of biofilm formation for the subject being treated.
  • the duration is at least once daily treatment of the effective dose for a duration of one day to one month.
  • the duration is administered over longer periods of time to prevent the formation of new biofilms.
  • the dose is administered on a different schedule, such as multiple times a day or weekly.
  • the at least one adhesin activity blocking compound is an antibody or antibody fragment against a protein selected from the group consisting of Staphylococcal protein A (SpA), clumping factor A, clumping factor B, fibrinogen-binding protein SdrG, fibrinogen-binding protein SdrF, S. epidermidis
  • Extracellular Motif Binding Protein LPXTG motif-containing S. epidermidis surface protein SesC, S. aureus collagen-binding protein, S. aureus fibronectin-binding protein, S. aureus fibronectin-binding protein A, S. aureus fibronectin-binding protein B, Heat Shock Protein 60, staphylococcal 145 kDa cell wall adhesin, S. epidermidis AtlE, S. epidermidis Aae, S. epidermidis AtlE, S. epidermidis Aae, S. epidermidis polysaccharide intercellular adhesin, S. aureus polysaccharide intercellular adhesin, ⁇ integrin, and combinations thereof, and further wherein, the antibody is capable of blocking the activity of adhesin protein in biofilm formation.
  • the at least one adhesin activity blocking compound is selected from the group consisting of cytochalasin D, genistein, SAR 1118, fibronectin, delmopinol, S. aureus Clf40 (Nl N2N3), S. aureus Clf41 (N2N3), S. epidermidis SdrG (Nl N2N3), S. epidermidis SdrG (AA 50-597), S.
  • the adhesin activity blocking compounds may be combined with other therapeutic agents, such as antibacterial agents and anti-inflammatory agents.

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Abstract

The present invention relates to methods of treating a biofilm-related disorder by inhibiting the binding ability of bacteria, such as Staphylococcus aureus and Staphylococcus epidermidis, thus inhibiting biofilm formation and the increase in bacterial populations and densities that lead to quorum-sensing-gene activation and therefore, the production of inflammatory virulence factors.

Description

METHOD OF INHIBITING BACTERIAL BIOFILM FORMATION
Field
[0001] The present invention relates to methods for treating a biofilm-related disorder, and more particularly, to methods and compositions of treating a biofilm-related disorder by inhibiting biofilm formation.
Background
[0002] A biofilm is a group of adherent microbial cells embedded within a matrix of composed of extracellular DNA, proteins, and polysaccharides produced by the microbial cells. Microbial cells growing in a biofilm are physiologically distinct from planktonic cells of the same organism, which are single cells that can float or swim in a liquid medium.
Biofilms can form on living or non-living surfaces and are prevalent in natural, industrial and hospital settings. The exopolysaccharide matrix that holds the biofilm together protects the cells within the biofilm, resulting in increased resistance to detergents and to anti-microbial reagents, such as antibiotics.
[0003] Biofilms are involved in a wide variety of microbial infections in the body, including infections in cystic fibrosis, endocarditis, urinary tract infections, middle-ear infections, chronic sinusitis, chronic tonsillitis, formation of dental plaque, gingivitis, periodontal disease, and infections of implanted devices, such as catheters, heart valves, intrauterine devices and joint prostheses. Studies have shown that sub-therapeutic levels of antibiotics can induce biofilm formation in some microbial infections.
[0004] There is a need for improved treatments for biofilm-related disorders.
Detailed Description
[0005] Aspects of the present invention are directed to methods and compositions for treating biofilm-related disorders based a better understanding of the nature of flora bacteria, such as Staphylococcus aureus and Staphylococcus epidermidis. Biofilms are complex structures produced by virtually all bacteria and are the prevailing microbial lifestyle seen in nature. Biofilms may occur anywhere in nature where there exists a combination of moisture, nutrients and a surface. The biofilm is initiated by the binding of the bacteria to a surface, such as the endocardium or the eyelid margin, and facilitated by a protein molecule called an adhesin. Once adherent, the bacteria manufacture the protein and polysaccharide components of the biofilm, which is inherently sticky and also binds tightly to the surface. This biofilm continues to accumulate on the lining of the heart throughout the years of an individual's life, eventually allowing the densities and populations of bacteria to increase dramatically. The biofilm allows the bacteria to resist antibiotics and avoid the host's defense mechanisms, effectively sheltering in place. Once the population reaches a critical point, quorum-sensing gene activation occurs activating dormant bacterial genes to begin producing highly inflammatory virulence factors such as cytolytic toxins, enzymes, more adhesins, and exotoxins. Without being bound to any particular theory, these virulence factors may be primarily responsible for the various inflammatory manifestations of biofilm-related disorders.
[0006] Embodiments of the present invention address shortcomings in the numerous disorder treatments currently available by providing an easy pharmacological solution that inhibits the adherence and accumulation of biofilm along a surface. In particular, as discussed in greater detail below, in an embodiment, the method includes administering to a subject an effective amount of one or more adhesin activity blocking compounds to a subject that prevents at least one of the adherence, binding, or production of a biofilm along a surface in or on the subject's body to treat or prevent a biofilm-related disorder. The biofilm-related disorders may include, without limitation, cystic fibrosis, bacterial vegetation, endocarditis, urinary tract infections, middle-ear infections, chronic sinusitis, chronic tonsillitis, prostate disease, formation of dental plaque, gingivitis, periodontal disease, eyelid disorders such as blepharitis and dry eye syndrome, and infections of implanted devices, such as catheters, heart valves, intrauterine devices and joint prostheses. Additionally, the treatment may also be used to inhibit non-infectious bacteria.
[0007] Bacteria live in one of two states: planktonic (i.e., individual free-floating bacteria) or within a biofilm. Bacteria in the planktonic state are more susceptible to attack from host defenses, antibiotics, or can be washed away more easily than bacteria living within biofilms. Without being bound to any particular theory, by preventing bacterial adherence to surfaces or to an existing early biofilm results in one or more of the following: preventing or reducing biofilm accumulation, delaying or avoiding increases in bacterial densities, delaying or reducing quorum-sensing, and avoiding pathogenesis of biofilm-related disorders since production of virulence factors is delayed or reduced.
[0008] Bacteria synthesize the protein/polysaccharide components of the biofilm.
Bacteria also produce a class of proteins collectively termed "adhesins." These adhesins allow binding of bacterial cell surface components to either host cell surface components, or to an intermediary substrate, which then binds to the host cell surface, or to components of extracellular biofilm matrix. Adhesins also promote binding of the biofilm to the host surface. Researchers have identified several adhesion proteins. Without being bound to a particular theory, one option for treating or preventing biofilm-related disorders is blocking or inhibiting the activity of adhesin proteins. Accordingly, an aspect of the invention is directed to treating biofilm-related disorders or preventing biofilm formation by blocking or inhibiting the activity of adhesin proteins. For example, adhesin activity may be blocked or inhibited with monoclonal antibodies (Mab) directed against adhesin proteins or their binding sites, or through compounds, such as small molecules that block or inhibit the same. In an embodiment, one or more compounds having adhesin activity blocking properties are applied to eyelid surfaces in an amount effective to block adhesin activity.
[0009] Exemplary adhesin proteins that may be targeted by embodiments of the invention are identified below. Embodiments of the invention may block the activity of one or more adhesin proteins. In an embodiment, the activity of a single adhesin protein is blocked. In another embodiment, the activity of a plurality of adhesin proteins is blocked.
[0010] An exemplary adhesin protein with activity that may be block in embodiments of the invention is the polysaccharide intercellular adhesin. The polysaccharide intercellular adhesin (PIA) is significantly involved in biofilm accumulation and is produced by both S. aureus and S. epidermidis.
[0011] Another exemplary class of bacterial adhesin proteins is made up of proteins covalently anchored to bacterial cell peptidoglycans, which specifically attach to the plasma or extracellular matrix (ECM) of the subject in need of treatment. These proteins are collectively referred to as microbial-surface-component-recognizing-adhesive-matrix- molecules (MSCRAMMs). MSCRAMMs recognize and bind to one or more components of the ECM, plasma, including collagens, fibrinogen (fibrinogen-binding protein, clumping factors A and B), fibronectin, elastin, laminin, bone sialoprotein, and for vitronectin.
[0012] Staph aureus surface protein G (SasG) is an exemplary adhesin protein known for binding to mucosa or mucosal tissue. In particular, SasG variants with eight, six, and five repeats formed biofilm. Several other variants do not.
[0013] Staphylococcal protein A (SpA) is another exemplary adhesin protein.
Embodiments of the invention include an anti-SpA antibody, such as an anti-SpA monoclonal antibody (Mab) that would be beneficial in preventing adhesin activity. This antibody may be used alone or in conjunction with other antibodies, such as antibodies directed against binding targets for the SpA, such as Mabs to clumping factor (Of) A and B proteins. [0014] An additional exemplary MSCRAMM adhesin protein in S. epidermidis is the fibrinogen-binding protein SdrG. Thus, embodiments of the invention include an antibody directed against SdrG, such as a monoclonal antibody, which would be beneficial in preventing adhesin activity.
[0015] An additional exemplary MSCRAMM adhesin protein in S. epidermidis is the fibrinogen-binding protein SdrF, which binds collagen. Thus, embodiments of the invention include an antibody directed against SdrF, such as a monoclonal antibody, which would be beneficial in preventing adhesin activity.
[0016] An additional exemplary adhesin protein is the S. epidermidis Extracellular Matrix Binding Protein (Embp), which is a 1.1 -MDa protein that can bind fibronectin. Studies by Christner and colleagues found that a 460-kDa isoform of Embp is capable of binding fibronectin, which in turn can inhibit biofilm accumulation. Thus, embodiments of the invention include an antibody directed against Embp, such as a monoclonal antibody, which would be beneficial in preventing adhesin activity.
[0017] Another exemplary adhesin protein is LPXTG motif-containing cell wall- anchored proteins termed S. epidermidis surface proteins, which have been implicated in biofilm production, specifically SesC. Polyclonal anti-SesC reduced the in vitro biofilm- forming and fibrinogen-binding and, therefore, may be beneficial when added to an administered medication. Thus, embodiments of the invention include an antibody directed against LPXTG motif-containing cell wall-anchored proteins such as SesC, such as a monoclonal antibody, which would be beneficial in preventing adhesin activity.
[0018] Staphylococcus aureus collagen-binding protein is another exemplary cell surface adhesin. It has been shown that the topical application of a collagen-binding peptide before bacterial challenge decreased S. aureus adherence to de-epithelialized corneas. The collagen- binding adhesin is therefore involved in the pathogenesis of S. aureus infection of the cornea epithelium and may well be also involved in binding to other cell or tissue surfaces, such as the endocardium, thus promoting biofilm formation. Endocardium adherence and invasion may be blocked by cytochalasin D and genistein, two potential adhesin blocking molecules. Thus, embodiments of the invention include at least one of cytochalasin D and genistein. Further embodiments of the invention include an antibody directed against Staphylococcus aureus collagen-binding protein, such as a monoclonal antibody, which would be beneficial in preventing adhesin activity.
[0019] S. aureus fibronectin-binding protein (FnBP) may play the major role in bacterial adhesion. Fibronectin is a glycoprotein found in the extracellular matrix (ECM) and body fluids. It serves as a substrate for the adhesion of normal host cells, expressing the appropriate integrins, to one another. Bacteria have evolved to take advantage of the existence of fibronectin by producing fibronectin-binding proteins A and B (FnBPA and FnBPB), which cause a tight binding of the bacteria with the extracellular matrix (ECM). The current model for FnBP-mediated adhesion suggests a fibronectin-dependent bridging between S. aureus FnBPs and host cell α5β1 integrin. Fibronectin increases in quantity with capillary dilation due to inflammation. It is likely that, once bound using existing fibronectin as the bridge, the subsequent bacterial virulence factor-induced inflammation accelerates further exudation of fibronectin from the serum of dilated capillaries, providing more substrate for further binding and therefore biofilm accumulation, resulting in a vicious cycle. Competitive inhibition using anti-FnBP antibodies or FnBP-derived peptides should be effective in blocking receptor-ligand interactions at the cell surface. In one study, S. aureus invasion was substantially but not completely eliminated by the absence of FnBP. Therefore, biofilm-related disorders should be also substantially decreased by competitive binding of bacterial FnBP. FnBP also binds directly to a host cell surface protein, Heat Shock Protein 60, so providing a Mab or a compound similar in action, to competitively bind the FnBP may effectively block two bacterial separate binding mechanisms. HSP 60 is a 57-kDa protein that was originally thought to be associated with the mitochondrial matrix, but is now known to be typical of eukaryotic cells and cytoplasmic membranes in general, so a HSP 60 Mab could be very effective along the cell or tissue surface.
[0020] The integrin protein has also been found to be a binding site for human lymphocytes invasion as well, when involved with host response inflammation, and is the basis for the mechanism of action for a potent LFA- 1/ICAM- 1 antagonist SAR 1118 (Shire, Lifitegrast), such as that described in U.S. Patent Nos. 8592450 and 8748391, which competitively binds the integrin protein, thus preventing binding and activation of lymphocytes. U.S. Patent Nos. 8748391, 8592450, 9085553, 9051297, etc. are for inhibiting Lymphocyte Function-associated Antigen- 1 in an effort to reduce inflammation by shutting down white cell responses to inflammatory factors (virulence factors). However, embodiments of the present invention do not shut down white cells by blocking integrin, rather, an embodiment of the present invention shuts down the formation of biofilms by blocking adhesins, such as by antagonism of LFA- 1/ICAM- 1 with SAR 1118, thus inhibiting the buildup of bacteria, thus inhibiting the quorum sensing gene activation of virulence factors. [0021] Another exemplary adhesin is a staphylococcal 145 kDa cell wall adhesin, originally found in bovine mastitis, which allows Staph to bind to epithelial cells. Antibodies against the 145 kDa adhesin protein were found to block bacterial adhesion to bovine epithelial cells as well as rat intestinal epithelial cells. This adhesin may also play a role in binding staph to human cells in moist environs.
[0022] In S. epidermidis, bacterial proteins can be bifunctional adhesins/autolysins AtlE and Aae. These proteins have specific adherence functions (by binding noncovalently to vitronectin), and may also function to release eDNA, which is an important
adherence/aggregation factor in both S. aureus and S. epidermidis biofilm formation. Mabs to AtlE and Aae, such as monoclonal antibodies, may be included in embodiments of the invention to reduce adherence and formation of biofilms.
[0023] For pathology and virulence to occur, factors must exist that not only allow adherence of bacteria to ECM or host cell surfaces, but also allow for accumulation of the biofilm mass, within which the bacteria can increase their densities to quorum-sensing levels. Known adhesins allowing for biofilm accumulation (biofilm sticking to biofilm) are produced by S. epidermidis (and to some extent S. aureus) are polysaccharide intercellular adhesin (PIA) and accumulation associated protein (Aap). Antibodies, such as monoclonal antibodies, or compounds blocking these adhesin should prevent accumulation of biofilm mass, thereby significantly decreasing the incidence of biofilm-related disorders.
[0024] Another effective mechanism of blocking adhesins could be the addition of actin microfilament inhibitor cytochalasin D (0.5 μg/ml; Sigma) or the tyrosine kinase inhibitor genistein (250 μg/ml; Calbiochem), which have been shown to reduce the binding and subsequent internalization of S. aureus by more than 99.9%. Thus, embodiments of the present invention may include one or both of these compounds.
[0025] In another aspect of the present invention, since a monoclonal antibody (MAb) specific for βΐ integrins dramatically reduced S. aureus adherence and invasion, addition of a iMab should prevent the formation of a Fn bridge linking the host cell βΐ integrin and the FnBP adhesin. Thus, embodiments of the present invention may include iMab.
[0026] In another embodiment of the present invention, addition of a MAb specific for eukaryotic Hsp60, Hsp60 MAb, should also significantly reduce the binding of S. aureus. Combined, these 2 Mabs could block the multifunctional adhesin FnBP from binding directly to both host cell ligands, Hsp60 and βΐ integrins. Du-D4 is the functional fragment of FnBP, therefore, a Du-D4 antiserum should also block the association of Hsp60 with the bacterial cell surface and thus may be an effective adhesin inhibitor. [0027] Fibronectin itself could serve as an adhesin blocker in high concentrations. This effect would be consistent with the function of fibronectin as a bifunctional molecule in which a blocking effect at high concentrations would be due to saturation of the integrin- FnBP system. Thus, embodiments of the present invention may include fibronectin.
[0028] Delmopinol is a morpholino compound that has been demonstrated to have utility in the treatment of the oral cavity, especially tooth surface, and for the removal or inhibition of dental plaque, which is a biofilm. The compound and its manufacture are disclosed in U.S. Patent No. 4894221, the disclosure of which is incorporated herein by reference in its entirety. Delmopinol and its derivatives may be used to prevent biofilm formation along the surface of a cell or tissue. In an embodiment, delmopinol may work more effectively when formulated with a bacterial adhesin peptide (e.g., pl025) or an antibody that binds to a bacterial adhesin peptide. Thus, delmopinol may act as an augmentation agent in any of proposed adhesion inhibitors described herein.
[0029] Extracellular polysaccharides can also act as adhesins. Exemplary extracellular polysaccharides include PS/A, PNSG, PNAG, and the aforementioned PIA. Embodiments of the invention may include blocking the activity of one or more of the identified adhesin proteins to thereby reducing the biofilm accumulation.
[0030] The following surface adhesin activity blocking proteins, which may be used individually or in combination, may be obtained from Inhibitex, Inc., Alpharetta, GA., USA: S. aureus Clf40 (Nl N2N3) - full length A domain of Clumping factor A (amino acids (AA) 40-559); S. aureus Clf41 (N2N3) - post protease site fragment of Clf 40 (AA 223-559); S. epidermidis SdrG (Nl N2N3) - full length A domain of SdrG (AA 50-597); S. epidermidis SdrG (N2N3) - post protease site fragment of SdrG (AA 273-597).
[0031] An additional adhesin activity blocking compound that may be included, individually or combination with each other or other adhesin blocking compounds, in embodiments of the invention are thymosin β4 (or Τβ4), which is available from G-TreeBNT, lactoferrin, xylitol, mangainin I covalently linked to Il-mercapto undecanoic acid and 6- mercaptohexanol in 1 :3 ratio, and Fraction 7 from Terminalia chebula or its Gallic acid, cis- 2-decenoic acid, cytochalasin D, genistein (tyrosine kinase inhibitor), norspermidine, polyamines (alone or in synergistic combination with D-amino acids), alginate lyase, N- acetyl-hearosan lyase, hyaluronidase enzymes, sulphathiazole (suphonamide), iron chelating agents (alone or in combination with aminoglycosides), N-acetyl cysteine, D-amino acids, disperin B, DNase I, furanone, S-phenyl-l-cysteine sulphoxide and the derivative compound diphenyl disulfide. [0032] In embodiments of the invention, adhesin blocking compounds are administered systemically to block adhesin activity to inhibit biofilm formation. The disclosed
compositions may be suitable for systemic administration. For example, the compositions may be systemically administered by other means known in the art, such as orally, parenterally (e.g., intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection), suppository, or even transdermally such as through a gel or patch formulation. Such formulations may be prepared as described above or as is known to those of ordinary skill in the art.
[0033] The adhesin activity blocking compounds are administered in a dose effective to inhibit adhesin activity and to prevent or decrease biofilm formation. An effective dose is a dose at which biofilm formation is prevented or decreased.
[0034] Another aspect of the invention is directed to a method of treating the biofilm- related disorder in a subject that includes systemically administering via oral administration a therapeutically effective dose of adhesin activity blocking compound to inhibit biofilm formation. This oral dose of adhesin activity blocking compound may be administered in a single dose or multiple doses in a 24 hour period and may generally be administered for a period of days, weeks, months, or even years. In an embodiment, adhesin activity blocking compound is orally administered to a subject over a period of at least two weeks, and in alternative embodiments, adhesin activity blocking compound is administered for a plurality of months or a year or more. Thus, embodiments of the invention are directed to the long term administration of adhesin activity blocking compound to treat a biofilm-related disorder.
[0035] The compositions of adhesin activity blocking compound can be administered in vivo in a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable. Thus, the compositions may be administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the adhesin activity blocking compound and to minimize any adverse side effects in the subject, as would be known to one of skill in the art. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) A. R. Gennaro, Mack Publishing Company, Easton, Pa. 1995.
[0036] For intravenous administration, an appropriate amount of a pharmaceutic ally- acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution is in a pharmaceutically acceptable range, preferably from about 5 to about 8.5, and more preferably from about 7.8 to about 8.2.
Further, carriers include sustained release preparations such as semi-permeable matrices of solid hydrophobic polymers containing the pharmaceutical composition, which matrices are in the form of shaped articles (e.g., films, liposomes or microparticles). It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being
administered. For example, persons skilled in the art may choose a particular carrier suitable for introduction to the body by injection, as described above, or ingestion.
[0037] For ingestion, adhesin activity blocking compound may be formed into a tablet, capsulized, dissolved, or suspended in a liquid or gel as known to those of ordinary skill in the art for oral administration of a drug. In some embodiments, adhesin activity blocking compound is formulated for sustained release, such as with the use of one or more excipients that control the release of adhesin activity blocking compound over a specified period of time for absorption by the subject.
[0038] The pharmaceutical compositions of adhesin activity blocking compound may also include binders, thickeners, diluents, buffers, preservatives, surface active agents, and the like in addition to the adhesin activity blocking compound and carriers.
[0039] Another aspect of the invention is directed to a method of treating biofilm-related disorder in a subject comprising administering via transdermal administration, such as with a gel or patch, a therapeutically effective amount of adhesin activity blocking compound to inhibit biofilm formation. In an embodiment, the transdermal formulation maintains a therapeutic blood plasma concentration of adhesin activity blocking compound and its active metabolites for a duration ranging between about 18 hours/day and about 24 hours/day. In an embodiment, the transdermal formulation includes a total dose of adhesin activity blocking compound to inhibit biofilm formation clinically, resulting in an improvement in the biofilm- related disorder such as can be determined with a quantifiable clinical observations. The improvement can be determined, for example, based on a comparison between observations made before and after the implementation of therapy. Adhesin activity blocking compound dosing may require titration to achieve and maintain the desired effect of preventing or reducing biofilm accumulation. Thus, the dose required to adequately inhibit biofilm formation administered transdermally may vary widely for a given subject as treatment progresses as well as between subjects. [0040] The injection of the therapeutically effective amount of adhesin activity blocking compound may be administered in a bolus injection, by continuous infusion, or using a combination of bolus injection and continuous infusion. The term bolus injection is understood to be an injection wherein the dose is delivered over a relatively short period of time. The term continuous infusion is understood to be an injection delivered, such as with an intravenous drip, wherein the dose is delivered in a metered manner over the period of time desired for adhesin activity blocking compound therapy. In an embodiment, the adhesin activity blocking compound is administered via continuous infusion over a period of time ranging between about 30 min/day to about 24 hours/day. In another embodiment, therapeutically effective amount of adhesin activity blocking compound is administered over a period of about 8 hours/day to about 24 hours/day. In some circumstances, adhesin activity blocking compound is administered via continuous infusion on at least one day and up to on seven days. In other circumstances, adhesin activity blocking compound may be
administered for even longer periods of times, such as on days over multiple weeks, months, or even years, as necessary to treat the subject. Thus, embodiments of the invention are directed to the long term administration of adhesin activity blocking compound to treat a biofilm-related disorder.
[0041] In some instances a combination of bolus injection with continuous infusion may be desired to a treat a subject. For example, a bolus injection may be utilized to deliver a loading dose, i.e., a dose of adhesin activity blocking compound to rapidly achieve a desired therapeutic level of adhesin activity blocking compound in the subject, and the continuous infusion may be utilized to maintain or even titrate the desired therapeutic levels over the desired duration of treatment.
[0042] Another aspect of the invention is directed to a method of treating biofilm-related disorder in a subject that includes administering an extended release formulation of adhesin activity blocking compound that maintains a therapeutic blood plasma concentration of adhesin activity blocking compound and its active metabolites for a duration ranging between about 18 hours/day and about 24 hours/day. The extended release formulation may be an oral formulation, an injected formulation, or even a transdermal formulation. In an embodiment, the extended release formulation includes a total dose of adhesin activity blocking compound to inhibit biofilm formation, resulting in an improvement in the biofilm-related disorder such as can be determined with a quantifiable clinical observations. Adhesin activity blocking compound dosing may require titration to achieve and maintain the desired effect of preventing or reducing biofilm accumulation. The effect can be determined, for example, based on a comparison between observations made before and after the implementation of therapy. Thus, the dose required to adequately reduce the biofilm formation may vary widely for a given subject as treatment progresses as well as between subjects. The term day is understood to be a 24 hour cycle. In an embodiment, adhesin activity blocking compound is orally administered to a subject over a period of at least two weeks, and in alternative embodiments, adhesin activity blocking compound is administered for a plurality of months or a year or more. Thus, embodiments of the invention are directed to the long term administration of adhesin activity blocking compound to treat a biofilm-related disorder.
[0043] The adhesin activity blocking compounds are administered for a duration sufficient to relieve the symptoms of biofilm formation for the subject being treated. In an embodiment, the duration is at least once daily treatment of the effective dose for a duration of one day to one month. In another embodiment, the duration is administered over longer periods of time to prevent the formation of new biofilms. In embodiments, the dose is administered on a different schedule, such as multiple times a day or weekly.
[0044] In embodiments of the invention, the at least one adhesin activity blocking compound is an antibody or antibody fragment against a protein selected from the group consisting of Staphylococcal protein A (SpA), clumping factor A, clumping factor B, fibrinogen-binding protein SdrG, fibrinogen-binding protein SdrF, S. epidermidis
Extracellular Motif Binding Protein (Embp), LPXTG motif-containing S. epidermidis surface protein SesC, S. aureus collagen-binding protein, S. aureus fibronectin-binding protein, S. aureus fibronectin-binding protein A, S. aureus fibronectin-binding protein B, Heat Shock Protein 60, staphylococcal 145 kDa cell wall adhesin, S. epidermidis AtlE, S. epidermidis Aae, S. epidermidis AtlE, S. epidermidis Aae, S. epidermidis polysaccharide intercellular adhesin, S. aureus polysaccharide intercellular adhesin, βΐ integrin, and combinations thereof, and further wherein, the antibody is capable of blocking the activity of adhesin protein in biofilm formation.
[0045] In embodiments of the invention, the at least one adhesin activity blocking compound is selected from the group consisting of cytochalasin D, genistein, SAR 1118, fibronectin, delmopinol, S. aureus Clf40 (Nl N2N3), S. aureus Clf41 (N2N3), S. epidermidis SdrG (Nl N2N3), S. epidermidis SdrG (AA 50-597), S. epidermidis SdrG (N2N3), thymosin β4, lactoferrin, xylitol, mangainin I covalently linked to Il-mercapto undecanoic acid and 6- mercaptohexanol in 1 :3 ratio, Fraction 7 from Terminalia chebula or its Gallic acid, cis-2- decenoic acid, norspermidine, polyamines, alginate lyase, N-acetyl-hearosan lyase, hyaluronidase enzymes, sulphathiazole, iron chelating agents, N-acetyl cysteine, D-amino acids, disperin B, DNase I, furanone, S-phenyl-l-cysteine, sulphoxide, diphenyl disulfide, FnBP-derived peptides, and combinations thereof.
[0046] In embodiments of the invention, the adhesin activity blocking compounds may be combined with other therapeutic agents, such as antibacterial agents and anti-inflammatory agents.
[0047] While the present invention has been illustrated by the description of
embodiments thereof, and while the embodiments have been described in considerable detail, it is not intended to restrict or in any way limit the scope of the appended claims to such detail. Additional advantages and modifications will readily appear to those skilled in the art. The invention in its broader aspects is, therefore, not limited to the specific details, representative apparatus and method, and illustrative examples shown and described.
Accordingly, departures may be made from such details without departing from the scope of the general inventive concept.
[0048] What is claimed is:

Claims

1. A method of treating a biofilm-related disorder in a subject comprising administering to the subject a dose of at least one adhesin activity blocking compound in a dose sufficient to prevent the formation of a biofilm or to disrupt a biofilm already formed.
2. A method of preventing the formation of a biofilm in a subject comprising administering to the subject a dose of at least one adhesin activity blocking compound in a dose sufficient to prevent the formation of a biofilm.
3. A method of disrupting a biofilm in a subject comprising administering to the subject a dose of at least one adhesin activity blocking compound in a dose sufficient to disrupt a biofilm.
4. The method of one of claims 1 to 3, wherein the at least one adhesin activity blocking compound is selected from the group consisting of a protein, protein fragment, an antibody, and a small molecule.
5. The method of one of claims 1 to 4, wherein the at least one adhesin activity blocking compound binds to an adhesin protein.
6. The method of one of claims 1 to 4, wherein the at least one adhesin activity blocking compound binds to a receptor for an adhesin protein.
7. The method of one of claims 1 to 5, wherein the at least one adhesin activity blocking compound is an antibody or antibody fragment against a protein selected from the group consisting of Staphylococcal protein A (SpA), clumping factor A, clumping factor B, fibrinogen-binding protein SdrG, fibrinogen-binding protein SdrF, S. epidermidis
Extracellular Motif Binding Protein (Embp), LPXTG motif-containing S. epidermidis surface protein SesC, S. aureus collagen-binding protein, S. aureus fibronectin-binding protein, S. aureus fibronectin-binding protein A, S. aureus fibronectin-binding protein B, Heat Shock Protein 60, staphylococcal 145 kDa cell wall adhesin, S. epidermidis AtlE, S. epidermidis Aae, S. epidermidis AtlE, S. epidermidis Aae, S. epidermidis polysaccharide intercellular adhesin, S. aureus polysaccharide intercellular adhesin, βΐ integrin, and combinations thereof, and further wherein, the antibody is capable of blocking the activity of adhesin protein in biofilm formation.
8. The method of one of claims 1 to 5, wherein the at least one adhesin activity blocking compound is selected from the group consisting of cytochalasin D, genistein, SAR 1118, fibronectin, delmopinol, S. aureus Clf40 (Nl N2N3), S. aureus Clf41 (N2N3), S. epidermidis SdrG (Nl N2N3), S. epidermidis SdrG (AA 50-597), S. epidermidis SdrG (N2N3), thymosin β4, lactoferrin, xylitol, mangainin I covalently linked to Il-mercapto undecanoic acid and 6- mercaptohexanol in 1:3 ratio, Fraction 7 from Terminalia chebula or its Gallic acid, cis-2- decenoic acid, norspermidine, polyamines, alginate lyase, N-acetyl-hearosan lyase, hyaluronidase enzymes, sulphathiazole, iron chelating agents, N-acetyl cysteine, D-amino acids, disperin B, DNase I, furanone, S-phenyl-l-cysteine, sulphoxide, diphenyl disulfide, FnBP-derived peptides, and combinations thereof.
9. A pharmaceutical composition comprising at least one adhesin activity blocking compound provided at a dose sufficient to block the activity of adhesin in biofilm formation.
10. The composition of claim 9, wherein the at least one adhesin activity blocking compound is selected from the group consisting of a protein, protein fragment, an antibody, and a small molecule.
11. The composition of claim 9, wherein the at least one adhesin activity blocking compound is an antibody or antibody fragment against a protein selected from the group consisting of Staphylococcal protein A (SpA), clumping factor A, clumping factor B, fibrinogen-binding protein SdrG, fibrinogen-binding protein SdrF, S. epidermidis
Extracellular Motif Binding Protein (Embp), LPXTG motif-containing S. epidermidis surface protein SesC, S. aureus collagen-binding protein, S. aureus fibronectin-binding protein, S. aureus fibronectin-binding protein A, S. aureus fibronectin-binding protein B, Heat Shock Protein 60, staphylococcal 145 kDa cell wall adhesin, S. epidermidis AtlE, S. epidermidis Aae, S. epidermidis AtlE, S. epidermidis Aae, S. epidermidis polysaccharide intercellular adhesin, S. aureus polysaccharide intercellular adhesin, βΐ integrin, and combinations thereof, and further wherein, the antibody is capable of blocking the activity of adhesin protein in biofilm formation.
12. The composition of claim 9,wherein the at least one adhesin activity blocking compound is selected from the group consisting of cytochalasin D, genistein, SAR 1118, fibronectin, delmopinol, S. aureus Clf40 (Nl N2N3), S. aureus Clf41 (N2N3), S. epidermidis SdrG (Nl N2N3), S. epidermidis SdrG (AA 50-597), S. epidermidis SdrG (N2N3), thymosin β4, lactoferrin, xylitol, mangainin I covalently linked to Il-mercapto undecanoic acid and 6- mercaptohexanol in 1 :3 ratio, Fraction 7 from Terminalia chebula or its Gallic acid, cis-2- decenoic acid, norspermidine, polyamines, alginate lyase, N-acetyl-hearosan lyase, hyaluronidase enzymes, sulphathiazole, iron chelating agents, N-acetyl cysteine, D-amino acids, disperin B, DNase I, furanone, S-phenyl-l-cysteine, sulphoxide, diphenyl disulfide, FnBP-derived peptides, and combinations thereof.
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