WO2019104213A1 - Antibiofilm formulations and use thereof - Google Patents

Abstract

Provided herein are compositions comprising modified antibiotics and antibiotics compositions comprising excipient(s). Also provided herein are methods of treating biofilm infections by administering the modified antibiotics alone or in combination with excipient(s).

Description

ANTIBIOFILM FORMULATIONS AND USE THEREOF

DESCRIPTION

[0001] This application claims the benefit of United States Provisional Patent

Application No. 62/589,988, filed November 22, 2017, the entirety of which is incorporated herein by reference.

BACKGROUND

1. Field

[0002] The present invention relates generally to the field of medicine. More particularly, it concerns formulations for the treatment or prevention of biofilms.

2. Description of Related Art

[0003] Bacterial biofilm infections are particularly difficult to treat and eradicate using currently available drugs. Pseudomonas aeruginosa (PA) is an opportunistic pathogen that represents a major health care issue. It is responsible for up to 20% of nosocomial infections, such as pneumonia, and blood stream, surgical site, and urinary tract infections (Micek et al, 2015; Weinstein et al, 2005). Cystic fibrosis (CF) lung infection is a notable case of a chronic infection that has been the focus of extensive research during the last decades. The abnormal mucociliary clearance observed in these patients favors the development of recurrent infections (Fick, 1989). PA is the main pathogen isolated from the lungs of CF patients. This infection leads to a chronic inflammatory state that causes a progressive impairment in the lung function until respiratory failure (Bjamsholt et al, 2009). Long-term antibiotic courses are necessary to treat PA lung infections and subsequently prevent the exacerbations and decrease the rate of lung dysfunction (Murphy et al, 2004).

[0004] A primary challenge in the treatment of CF lung infection and other chronic infections is the eradication of developed biofilms (Hoiby et al, 2010). A biofilm is a form of life where bacteria live in aggregates, attached to each other, and encased in an extracellular polymeric substance (EPS). Biofilms can be hundreds of times more resistant to antimicrobials than individual bacteria in the planktonic state. Biofilms have been identified to cause or complicate many chronic infections like the mentioned CF pneumonia, as well as periodontitis and chronic wounds infections. There are several mechanisms described to explain the increased resistance to therapy of bacteria in biofilms compared to planktonic bacteria. These include the physical protection that the EPS provides to the community (Nichols et al, 1988) and the altered metabolic state of a small subpopulation of bacteria within the biofilms (Walters et al, 2003), known as persisters. Persister bacteria have a low metabolic state and therefore, can survive treatments that require a minimum cell activity level to be effective (Percival et al, 2011).

[0005] Different strategies have been tested to face these challenging infections and to improve the activity of current therapies. Some strategies are aimed to improve the penetration of antibiotics through the EPS by chemical modification of existing drugs (Du et al, 2015), by the use of enzymes to degrade the EPS components (Chiang et al, 2013), and by physical disruption with sound waves or magnetic fields (Bandara et al, 2015). However, there is an unmet need for improved formulations for the treatment of biofilms.

SUMMARY

[0006] In a first embodiment, the present disclosure provides pharmaceutical compositions comprising an antibiotic and at least one excipient selected from the group consisting of D-alanine, L-alanine, L-homoserine, L-proline, D-threonine, adenosine, 2-deoxy adenosine, inosine, citric acid, glycolic acid, glyoxylic acid, succinic acid, monomethyl succinate, quinic acid, d-amino valeric acid, chondroitin, D-cellobiose, D-fructose, D-fructose- 6-phosphate, D-galactose, b-methyl-D-galactoside, a-methyl-D-glucoside, D-glucose-l- phosphate, D-gluconic acid, b-methyl-D-glucuronic acid, N-acetyl-D-glucosaminitol, D- mannose, D-melezitose, D-melibiose, D-trehalose, inulin, L-arabitol, maltose, mannan, melibionic acid, m-inositol, palatinose, stachyose, sucrose, turanose, xylitol, phenylethylamine, propylene glycol, sec-butylamine, tween 20, tween 40, and tween 80. Excipients may be benzoic acid, potassium sorbate, alginic acid, carrageenan, citric acid, edetic acid, coconut oil, EDTA, and/or xylitol. In particular aspects, the at least one excipient is selected from the group consisting of N-acetyl-D-glucosaminitol, L-proline, L-alanine, 2- deoxy-adenosine, chondroitin, and D-gluconic acid. In specific aspects, the composition is further defined as an anti-biofilm composition. The biofilm may be an oral biofilm, such as an oral biofilm associated with dental caries, gingivitis, or periodontal disease.

[0007] The excipients may be L-alanine, L-homoserine, L-proline, D- threonine, adenosine, 2-deoxy adenosine, inosine, citric acid, glycolic acid, glyoxylic acid, succinic acid, monomethyl succinate, quinic acid, d-amino valeric acid, chondroitin, D- cellobiose, D-fructose, D-fructose-6-phosphate, D-galactose, b-methyl-D-galactoside, a- methyl-D-glucoside, D-glucose-l -phosphate, D-gluconic acid, b-methyl-D-glucuronic acid, N-acetyl-D-glucosaminitol, D-mannose, D-melezitose, D-melibiose, D-trehalose, inulin, L- arabitol, maltose, mannan, melibionic acid, m-inositol, palatinose, stachyose, sucrose, turanose, xylitol, phenylethylamine, propylene glycol, sec-butylamine, tween 20, tween 40, and/or tween 80. The at least one excipient may be selected from the group consisting of L- alanine, L-homoserine, L-proline, adenosine, 2-deoxy adenosine, inosine, citric acid, glycolic acid, glyoxylic acid, succinic acid, monomethyl succinate, quinic acid, d-amino valeric acid, chondroitin, D-cellobiose, D-fructose, D-fructose-6-phosphate, D-galactose, b-methyl-D- galactoside, a-methyl-D-glucoside, D-glucose-l -phosphate, D-gluconic acid, b-methyl-D- glucuronic acid, N-acetyl-D-glucosaminitol, D-mannose, D-melezitose, D-melibiose, D- trehalose, inulin, L-arabitol, maltose, mannan, melibionic acid, m-inositol, palatinose, stachyose, sucrose, turanose, xylitol, phenylethylamine, propylene glycol, sec-butylamine, tween 20, tween 40, and tween 80. The at least one excipient may be selected from the group consisting of adenosine, 2-deoxy adenosine, inosine, citric acid, glycolic acid, glyoxylic acid, succinic acid, monomethyl succinate, quinic acid, d-amino valeric acid, chondroitin, D- cellobiose, D-fructose, D-fructose-6-phosphate, D-galactose, b-methyl-D-galactoside, a- methyl-D-glucoside, D-glucose-l -phosphate, D-gluconic acid, b-methyl-D-glucuronic acid, N-acetyl-D-glucosaminitol, D-mannose, D-melezitose, D-melibiose, D-trehalose, inulin, L- arabitol, maltose, mannan, melibionic acid, m-inositol, palatinose, stachyose, sucrose, turanose, xylitol, phenylethylamine, propylene glycol, sec-butylamine, tween 20, tween 40, and tween 80. The at least one excipient can be selected from the group consisting of adenosine, 2-deoxy adenosine, inosine, glycolic acid, glyoxylic acid, monomethyl succinate, quinic acid, d-amino valeric acid, chondroitin, D-cellobiose, D-fructose, D-fructose-6-phosphate, D- galactose, b-methyl-D-galactoside, a-methyl-D-glucoside, D-glucose-l -phosphate, D- gluconic acid, b-methyl-D-glucuronic acid, N-acetyl-D-glucosaminitol, D-mannose, D- melezitose, D-melibiose, D-trehalose, inulin, L-arabitol, maltose, mannan, melibionic acid, m- inositol, palatinose, stachyose, sucrose, turanose, xylitol, phenylethylamine, propylene glycol, sec-butylamine, tween 20, tween 40, and tween 80. In some aspects, the at least one excipient is selected from the group consisting of N-acetyl-D-glucosaminitol, L-proline, L-alanine, 2- deoxy-adenosine, chondroitin, and D-gluconic acid. In particular aspects, at least one excipient is selected from the group consisting of N-acetyl-D-glucosaminitol, L-proline, L-alanine, and succinic acid. For example, the excipients are N-acetyl-D-glucosaminitol, L-proline, L-alanine, and succinic acid. In some aspects, the excipients are N-acetyl-D-glucosaminitol, L-proline, and L-alanine. The excipients may be L-alanine and N-acetyl-D-glucosaminitol. In particular aspects, the excipients are L-proline and N-acetyl-D-glucosaminitol. In some aspects, the excipients are succinic acid and N-acetyl-D-glucosaminitol. In certain aspects, the excipients are L-alanine and L-proline.

[0008] The excipient(s) may be present at a concentration of 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM. 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM. In some aspects, the composition comprises more than one excipient at the same concentration. In other aspects, the composition comprises more than one excipient at different concentrations.

[0009] In some aspects, the antibiotic is an aminoglycoside, a polymyxin, a monobactam, or a fluoroquinolone. In particular aspects, the aminoglycoside is tobramycin, streptomycin, kanamycin, gentamicin, or neomycin. In some aspects, the polymyxin is colistin sulfate. In certain aspects, the monobactam is aztreonam, nocardicin A, or tabtoxin. In some aspects, the fluoroquinolone is ciprofloxacin, levofloxacin, or trovafloxacin. The antibiotic may be a macrolide, such as erythromycin, clarithromycin, or azithromycin. The erythromycin may be present at a concentration of 0.7-800 pg/mL, such as 1-10, 10-20, 20-30, 30-40, 40-50, 50-100, 100-150, 150-200, 200-300, 400-500, 500-600, 600-700, or 700-800 pg/mL.

[0010] In certain aspects, the antibiotic comprises a modification. In particular aspects, the modification is PEGylation or conjugation to a polymer and/or peptide, such as a polymer and/or peptide hydrophilic and/or has a neutral charge. In some aspects, the antibiotic is PEGylated-tobramycin, PEGylated-colistin, or PEGylated-aztreonam.

[0011] In further aspects, the composition comprises 2, 3, 4, or 5 excipients. In some aspects, the excipients are selected from the group consisting of D-alanine, L-alanine, L- homoserine, L-proline, D-threonine, adenosine, 2-deoxy adenosine, inosine, citric acid, glycolic acid, glyoxylic acid, succinic acid, monomethyl succinate, quinic acid, d-amino valeric acid, chondroitin, D-cellobiose, D-fructose, D-fructose-6-phosphate, D-galactose, b-methyl-D- galactoside, a-methyl-D-glucoside, D-glucose-l -phosphate, D-gluconic acid, b-methyl-D- glucuronic acid, N-acetyl-D-glucosaminitol, D-mannose, D-melezitose, D-melibiose, D- trehalose, inulin, L-arabitol, maltose, mannan, melibionic acid, m-inositol, palatinose, stachyose, sucrose, turanose, xylitol, phenylethylamine, propylene glycol, sec-butylamine, tween 20, tween 40, tween 80, benzoic acid, potassium sorbate, alginic acid, carrageenan, citric acid, edetic acid, coconut oil, and EDTA. The excipients may be L-proline, succinic acid and EDTA. In some aspects, the excipients are L-proline, succinic acid, EDTA, and benzoic acid. The excipients may be L-proline, succinic acid, EDTA, benzoic acid, and alginic acid.

[0012] In some aspects, the antibiotic comprises 0.1-25%, such as 0.1-6%, 0.1-

0.3%, 1-3%, 2-5%, 5-20%, 10-20%, 20-25%, or 0.l-0.5%, of the composition. In certain aspects, the 1, 2, 3, or 4 excipients (individually or in combination) comprise 1-50%, such as 2-30%, 5-10%, 10-15%, 15-20%, 20-30%, 30-40%, or 40-50%, of the composition. In certain aspects, the composition comprises a formulation as described in Table 1.

[0013] In some aspects, the composition is free of or essentially free of a carboxylic acid or sugar, such as mannitol, fructose, and pyruvate. The composition may be free of or essentially free of D-methionine, D-tryptophan, D-leucine, D-tyrosine, D- phenylalanine, and D-proline. The composition may be free of or essentially free of D-proline.

[0014] In particular aspects, the biofilm is associated with Pseudomonas aeruginosa, Staphylococcus aureus, Helicobacter pylori, Burkholderia cepacia complex, Haemophilus influenzae, Escherichia coli, Streptococcus pneumoniae, Enterococcus faecalis, Pseudomonas fluorescens, Staphylococcus epidermidis or Acinetobacter baumannii. In one specific aspect, the biofilm is associated with Pseudomonas aeruginosa. In some aspects, the biofilm is associated with an ocular infection, cystic fibrosis infection, wound infection, otic infection, oral caries, or rhinosinusitis infection. In some aspects, the dental caries is associated with Streptococcus mutans, Streptococcus gordonii, Candida albicans, Actinomyces odontolyticus, Actinomyces naeslundii, Streptococcus sobrinus, Lactobacillus spp, or Veillonella spp. In some aspects, the oral biofilm is associated with gingivitis or periodontal disease. In certain aspects, the gingivitis or periodontal disease is associated with Streptococcus gordonii, Fusobacterium nulceatum, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Aggregatibacter actinomycetemcomitans, or Actinomyces naeslundii.

[0015] In some aspects, the composition is formulated to be administered orally, topically, rectally, percutaneously, by parenteral injection, intranasally or by inhalation. In particular aspects, the composition is a topical preparation, otic preparation, or ophthalmic preparation. In some aspects, the ophthalmic preparation is further defined as an ophthalmic solution, ophthalmic suspension. [0016] In another embodiment, there provided a method for treating a biofilm infection in a subject comprising administering an effective amount of the pharmaceutical composition comprising an antibiotic and at least one excipient selected from the group consisting of D-alanine, L-alanine, L-homoserine, L-proline, D-threonine, adenosine, 2-deoxy adenosine, inosine, citric acid, glycolic acid, glyoxylic acid, succinic acid, monomethyl succinate, quinic acid, d-amino valeric acid, chondroitin, D-cellobiose, D-fructose, D-fructose- 6-phosphate, D-galactose, b-methyl-D-galactoside, a-methyl-D-glucoside, D-glucose-l- phosphate, D-gluconic acid, b-methyl-D-glucuronic acid, N-acetyl-D-glucosaminitol, D- mannose, D-melezitose, D-melibiose, D-trehalose, inulin, L-arabitol, maltose, mannan, melibionic acid, m-inositol, palatinose, stachyose, sucrose, turanose, xylitol, phenylethylamine, propylene glycol, sec-butylamine, tween 20, tween 40, and tween 80. In particular aspects, the at least one excipient is selected from the group consisting of N-acetyl- D-glucosaminitol, L-proline, L-alanine, 2-deoxy-adenosine, chondroitin, and D-gluconic acid. Excipients may be benzoic acid, potassium sorbate, alginic acid, carrageenan, citric acid, edetic acid, coconut oil, and/or EDTA. In some aspects, the at least excipient is selected from the group consisting of N-acetyl-D-glucosaminitol, L-proline, L-alanine, 2-deoxy-adenosine, chondroitin, and D-gluconic acid. In additional aspects, the antibiotic is modified, such as PEGylation, polymer, and/or a peptide.

[0017] In particular aspects, the antibiotic is an aminoglycoside, a polymyxin, a monobactam, or a fluoroquinolone. In some aspects, the aminoglycoside is tobramycin, streptomycin, kanamycin, gentamicin, or neomycin. In particular aspects, the aminoglycoside is tobramycin sulfate. In some aspects, the polymyxin is colistin sulfate. In some aspects, the monobactam is aztreonam, nocardicin A, or tabtoxin. In certain aspects, the monobactam is aztreonam. In some aspects, the fluoroquinolone is ciprofloxacin, levofloxaxin, or trovafloxacin. In some aspects, the antibiotic is a macrolide, such as erythromycin.

[0018] In some aspects, the biofilm infection is bacterial and/or fungal. In some aspects, the biofilm infection is a nosocomial infection. In particular aspects, the infection is in a bloodstream or surgical site of a subject. In some aspects, the infection is a lung infection, wound, otitis media, urinary tract infection, or pneumonia. In certain aspects, the infection is chronic or recurrent. In particular aspects, the biofilm infection is a Staphylococcus infection or a Pseudomonas infection. In specific aspects, the biofilm infection is associated with Pseudomonas aeruginosa, Staphylococcus aureus, Helicobacter pylori, Burkholderia cepacia complex, Haemophilus influenzae, Escherichia coli, Streptococcus pneumoniae, Enterococcus faecalis, Pseudomonas fluorescens, Staphylococcus epidermidis or Acinetobacter baumannii.

[0019] In some aspects, the biofilm is an oral biofilm, such as dental caries which may be associated with Streptococcus mutans, Streptococcus gordonii, Candida albicans, Actinomyces odontolyticus, Actinomyces naeslundii, Streptococcus sobrinus, Lactobacillus spp, or Veillonella spp. In some aspects, the oral biofilm is associated with gingivitis or periodontal disease. In certain aspects, the gingivitis or periodontal disease is associated with Streptococcus gordonii, Fusobacterium nulceatum, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Aggregatibacter actinomycetemcomitans, or Actinomyces naeslundii.

[0020] In some aspects, the biofilm is associated with an artificial substance in vivo. In specific aspects, the artificial substance is an implant, catheter, tube, or prosthesis.

[0021] In some aspects, the composition comprises 2, 3, or 4 excipients. In certain aspects, the 2, 3, or 4 excipients are selected from the group consisting of D-alanine, L- alanine, L-homoserine, L-proline, D-threonine, adenosine, 2-deoxy adenosine, inosine, citric acid, glycolic acid, glyoxylic acid, succinic acid, monomethyl succinate, quinic acid, d-amino valeric acid, chondroitin, D-cellobiose, D-fructose, D-fructose-6-phosphate, D-galactose, b- methyl-D-galactoside, a-methyl-D-glucoside, D-glucose-l -phosphate, D-gluconic acid, b- methyl-D-glucuronic acid, N-acetyl-D-glucosaminitol, D-mannose, D-melezitose, D- melibiose, D-trehalose, inulin, L-arabitol, maltose, mannan, melibionic acid, m-inositol, palatinose, stachyose, sucrose, turanose, xylitol, phenylethylamine, propylene glycol, sec- butylamine, tween 20, tween 40, and tween 80.

[0022] In certain aspects, the at least one excipient enhances antimicrobial activity of the antibiotic against the biofilm infection. In some aspects, the enhanced activity is further defined as a greater percent decrease (e.g., 1.5 -fold to 3 -fold, such as 2-fold) in microbial proliferation.

[0023] In some aspects, the antibiotic and at least one excipient are administered topically, intralesionally, by inhalation, intranasally, opthalmically, parenterally or by the otic route. [0024] In yet another embodiment, there is provided a method of preventing biofilm formation and/or growth on a medical device, the method comprising coating surfaces of the medical device exposed to bodily fluids and/or tissues with an effective amount of a pharmaceutical composition comprising an antibiotic and at least one excipient selected from the group consisting of D-alanine, L-alanine, L-homoserine, L-proline, D-threonine, adenosine, 2-deoxy adenosine, inosine, citric acid, glycolic acid, glyoxylic acid, succinic acid, monomethyl succinate, quinic acid, d-amino valeric acid, chondroitin, D-cellobiose, D- fructose, D-fructose-6-phosphate, D-galactose, b-methyl-D-galactoside, a-methyl-D- glucoside, D-glucose-l -phosphate, D-gluconic acid, b-methyl-D-glucuronic acid, N-acetyl-D- glucosaminitol, D-mannose, D-melezitose, D-melibiose, D-trehalose, inulin, L-arabitol, maltose, mannan, melibionic acid, m-inositol, palatinose, stachyose, sucrose, turanose, xylitol, phenylethylamine, propylene glycol, sec-butylamine, tween 20, tween 40, and tween 80. In particular aspects, the at least one excipient is selected from the group consisting of N-acetyl- D-glucosaminitol, L-proline, L-alanine, 2-deoxy-adenosine, chondroitin, and D-gluconic acid. In some aspects, the device a prosthetic heart valve, venous catheter, urinary catheter, endoscope, contact lenses, intubation tube, or intrauterine device. In particular aspects, the antibiotic is tobramycin, colistin, aztreonam, or ciprofloxacin. In some aspects, the antibiotic is modified, such as PEGylated.

[0025] A further embodiment provides composition comprising an effective amount of an antibiotic and at least one excipient selected from the group consisting of D- alanine, L-alanine, L-homoserine, L-proline, D-threonine, adenosine, 2-deoxy adenosine, inosine, citric acid, glycolic acid, glyoxylic acid, succinic acid, monomethyl succinate, quinic acid, d-amino valeric acid, chondroitin, D-cellobiose, D-fructose, D-fructose-6-phosphate, D- galactose, b-methyl-D-galactoside, a-methyl-D-glucoside, D-glucose-l -phosphate, D- gluconic acid, b-methyl-D-glucuronic acid, N-acetyl-D-glucosaminitol, D-mannose, D- melezitose, D-melibiose, D-trehalose, inulin, L-arabitol, maltose, mannan, melibionic acid, m- inos itol, palatinose, stachyose, sucrose, turanose, xylitol, phenylethylamine, propylene glycol, sec-butylamine, tween 20, tween 40, tween 80, benzoic acid, potassium sorbate, alginic acid, carrageenan, citric acid, edetic acid, coconut oil, and EDTA for the treatment of a biofilm infection in a subject. In some aspects, the composition is formulated for oral, intravenous, intraarticular, parenteral, enteral, topical, subcutaneous, intramuscular, buccal, sublingual, rectal, intravaginal, intrapenile, intraocular, epidural, intracranial, or inhalational administration. [0026] In some aspects, the antibiotic is an aminoglycoside, a polymyxin, a monobactam, or a fluoroquinolone. In certain aspects, the antibiotic is tobramycin, colistin, aztreonam, ciprofloxacin, or erythromycin. In specific aspects, the antibiotic is modified, such as PEGylated.

[0027] In certain aspects, the biofilm infection is bacterial and/or fungal. In some aspects, the biofilm infection is a nosocomial infection. In some aspects, the infection is a lung infection, wound, otitis media, or urinary tract infection. In certain aspects, the infection is chronic or recurrent. In particular aspects, the biofilm infection is a Staphylococcus infection or a Pseudomonas infection. In certain aspects, the biofilm is associated with Pseudomonas aeruginosa, Staphylococcus aureus, Helicobacter pylori, Burkholderia cepacia complex, Haemophilus influenzae, Escherichia coli, Streptococcus pneumoniae, Enterococcus faecalis, Pseudomonas fluorescens, Staphylococcus epidermidis or Acinetobacter baumannii. In some aspects, the biofilm is associated with an ocular infection, cystic fibrosis infection, wound infection, otic infection, or rhinosinusitis infection. In certain aspects, the biofilm is associated with an otic infection and the composition is further defined as an otic composition. In some aspects, the biofilm is associated with an ocular infection and the composition is further defined as an ophthalmic solution, ophthalmic suspension, or ophthalmic ointment. In certain aspects, the biofilm is associated with a chronic wound or burn wound and the composition is further defined as a topical composition.

[0028] In some aspects, the topical composition is an emulsion, emulgel, lotion, or topical spray. In certain aspects, the biofilm infection is associated with a lung infection and the composition is further defined as a pulmonary composition. In some aspects, the pulmonary composition is a dry powder formulation or solution for inhalation. In certain aspects, the biofilm infection is associated with chronic rhinosinusitis infection and the composition is further defined as a nasal spray.

[0029] In another embodiment, there is provided a compound comprising a polymyxin covalently bound to a polyethylene glycol (PEG). In some aspects, the polymyxin is colistin or polymyxin B. In certain aspects, the PEG is linear. In certain aspects, the PEG comprises the formula CH₃-PEG-. In some aspects, the PEG comprises the formula CH₃— [O— CH₂— 0¾] -· In particular aspects, the PEG comprises the formula CH₃-[0-CH₂-CH₂]_n-0C(0)CH₂CH₂C(0)-, wherein n=2-3000, such as n=l00-2000. [0030] In some aspects, the compound is comprised in a pharmaceutical composition. In some aspects, the pharmaceutical preparation is formulated for topical, inhalational, parenteral, intravenous, or injection administration. In particular aspects, the compound comprises at least one excipient selected from the group consisting of D-alanine, L- alanine, L-homoserine, L-proline, D-threonine, adenosine, 2-deoxy adenosine, inosine, citric acid, glycolic acid, glyoxylic acid, succinic acid, monomethyl succinate, quinic acid, d-amino valeric acid, chondroitin, D-cellobiose, D-fructose, D-fructose-6-phosphate, D-galactose, b- methyl-D-galactoside, a-methyl-D-glucoside, D-glucose-l -phosphate, D-gluconic acid, b- methyl-D-glucuronic acid, N-acetyl-D-glucosaminitol, D-mannose, D-melezitose, D- melibiose, D-trehalose, inulin, L-arabitol, maltose, mannan, melibionic acid, m-inositol, palatinose, stachyose, sucrose, turanose, xylitol, phenylethylamine, propylene glycol, sec- butylamine, tween 20, tween 40, and tween 80. In specific aspects, the compound comprises at least one excipient is selected from the group consisting of N-acetyl-D-glucosaminitol, L- proline, L-alanine, 2-deoxy-adenosine, chondroitin, and D-gluconic acid. In further aspects, the compound comprises 2, 3, or 4 excipients.

[0031] In yet another embodiment, there is provided a method of treating a biofilm infection in a subject comprising administering to the subject a therapeutic amount of a PEGylated polymyxin. In some aspects, the pegylated polymyxin is a composition of the embodiments. In some aspects, the subject is human.

[0032] In certain aspects, the biofilm infection comprises gram-negative bacteria. In some aspects, the biofilm is associated with Pseudomonas aeruginosa, Staphylococcus aureus, Helicobacter pylori, Burkholderia cepacia complex, Haemophilus influenzae, Escherichia coli, Streptococcus pneumoniae, Enterococcus faecalis, Pseudomonas fluorescens, Staphylococcus epidermidis or Acinetobacter baumannii. In some aspects, the subject is immunocompromised or has an immune dysfunction. In certain aspects, the subject has a burn or a wound infection. In certain aspects, the biofilm infection is on or in the skin of the subject. In some aspects, the biofilm infection is on or adjacent to a medical device implanted in the subject. In certain aspects, the medical device is a catheter, sutures, a staple, or a pin.

[0033] Further provided herein is a medical device, wherein at least a portion of a surface of the device is coated with a compound of the embodiments. In some aspects, the medical device is a glove, a catheter, a stent, a staple, a pin, an electrical nerve stimulation device, a screw, a rod, a wire, a collar, a tube, or a surgical drain.

[0034] In another embodiment, there is provided a composition comprising chlorhexidine and at least one excipient selected from the group consisting of N-acetyl- glucosaminitol, succinic acid, L-alanine, L-proline, benzoic acid, potassium sorbate, alginic acid, carrageenan, citric acid, edetic acid, coconut oil, EDTA, and xylitol. In some aspects, the composition comprises 2, 3, 4, or 5 excipients. In certain aspects, the composition comprises L-proline and succinic acid. In certain aspects, the composition further comprises EDTA. In some aspects, the composition further comprises benzoic acid. In some aspects, the composition further comprises alginic acid. In certain aspects, the antibiotic further comprises carrageenan. In particular aspects, the composition comprises L-alanine and L-proline. In some aspects, the composition comprises L-proline and N-acetyl-D-glucosaminitol. In specific aspects, the composition comprises N-acetyl-D-glucosaminitol and succinic acid. In some aspects, the composition comprises L-proline and succinic acid. In certain aspects, the composition comprises L-proline and N-acetyl-D-glucosaminitol. In some aspects, the chlorhexidine is at a concentration of 1 to 100 pg/mL.

[0035] In some aspects the oral biofilm is associated with dental caries, gingivitis, or periodontal disease. In particular aspects, the dental caries is associated with Streptococcus mutans, Streptococcus gordonii, Candida albicans, Actinomyces odontolyticus, Actinomyces naeslundii, Streptococcus sobrinus, Lactobacillus spp, or Veillonella spp. In specific aspects, the oral biofilm is associated with gingivitis or periodontal disease. In some aspects, the gingivitis or periodontal disease is associated with Streptococcus gordonii, Fusobacterium nulceatum, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Aggregatibacter actinomycetemcomitans, or Actinomyces naeslundii.

[0036] In certain aspects, the composition is formulated for oral administration.

In some aspects, the composition is formulated as a solution, suspension, semisolid, varnish, paste, sublingual dissolvable tablet, lozenge, spray or mist for local oral cavity.

[0037] In additional aspects, the composition further comprises nanoparticles.

In some aspects, the nanoparticles are iron oxide nanoparticles, such as LeO, Fe^CL, Le₄0₅, Ee₂q₃ nanoparticles. In certain aspects, the iron oxide nanoparticles are coated. In some aspects, the iron oxide nanoparticles are citric acid coated or PEG/amine coated. In particular aspects, the nanoparticles are at a concentration of 50-200 pg/mL, such as 75, 100, 125, 150, or 175 pg/mL.

[0038] Further provided herein is a method of treating an oral biofilm comprising administering to the subject an effective amount of a composition of the embodiments. In some aspects, the at least one excipient enhances antimicrobial activity of the chlorhexidine against the oral biofilm infection. In certain aspects, the enhanced activity is further defined as a greater percent decrease in microbial proliferation.

[0039] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0041] FIG. 1: Proliferation of planktonic P. aeruginosa when treated with different excipients. This figure shows the results of the excipients that caused more than 20% or a statistically significant change in proliferation with respect to control (Mean ± S.D., n=5). *p<0.05 and **p<0.0l. The excipients in bold also reduced planktonic proliferation in combination with Tobramycin (TOB) (e.g., L-homoserine, see FIG. 2). Biofilm viability was also reduced with the excipients alone (L-proline, see FIG. 3) and in combination with TOB (e.g., L-proline and L-alanine in FIGS. 4 and 6).

[0042] FIG. 2: Proliferation of planktonic P. aeruginosa when treated with different excipients combined with tobramycin. This figure shows the treatments of TOB and excipient combinations that changed PA proliferation more than 20% with respect to control (Mean ± S.D., n=3). Black bars represent the excipients alone and hatch bars represent the combination with 0.5 pg/mL TOB. Symbols on top of the bars indicate a statistical difference with respect to control (*) and TOB alone (+). *p<0.05, **p<0.0l, and ***p<0.00l. The excipients in bold also reduced planktonic proliferation alone (L-homoserine, see FIG. 1) and biofilm viability in combination with Tob (N-acetyl-D-glucosaminitol in FIG. 4).

[0043] FIG. 3: Proliferation of P. aeruginosa biofilms when treated with different excipients. This figure shows excipients that caused a change of 20% or more in proliferation with respect to the control (Mean ± S.D, n=3; *p<0.05, **r<0.01, ***p<0.00l, and ****p<0.000l). L-proline (in bold) also reduced planktonic proliferation alone (see FIG. 1) and biofilm viability in combination with Tob (see FIGS. 4 and 6).

[0044] FIG. 4: Proliferation of P. aeruginosa biofilms when treated with different excipients combined with tobramycin. This figure only shows combinations that caused a change of 20% or more in proliferation with respect to control (Mean ± S.D., n=3). Black bars represent the excipients alone and hatch bars represent the combination with TOB. Symbols on top of the bars indicate a statistical difference with respect to control (*) and TOB alone (+) *p<0.05, **r<0.01, ***p<0.00l, and ****p<0.000l. The excipients in bold also reduced planktonic proliferation alone (L-alanine and L-proline, see FIG. 1) and in combination with Tob (N-acetyl-D-glucosaminitol in FIG. 2). Biofilm viability was also reduced by the excipients alone (L-proline in FIG. 3 and L-alanine and succinic acid in FIG. 6) and in combination with Tob (L-proline in FIG. 6).

[0045] FIGS. 5A-5D: Bacterial proliferation of PA14 biofilms treated with combinations of tobramycin and selected excipients. UCBPP-PA14 proliferation was measured by luminometry every 20 min during 20 h (Mean, n=3). Biofilms were treated with (A) excipients alone and in combination with (B) 0.5 pg/mL, (C) 2 pg/mL, and (D) 64 pg/mL of TOB. The luminescence emitted by the bacteria was normalized with respect to the values recorded at time zero of the treatment.

[0046] FIG. 6: Changes in surviving CFUs from treated PA14 biofilms. Log changes after treatment with excipients alone (0 pg/mL of TOB) and with tobramycin (2 and 64 pg/mL). Data was normalized to the survival of untreated biofilms (Mean + S.D, n=6). Asterisks on top of the bars indicate a statistical difference with respect the treatment with the same concentration of TOB but without excipient. *p<0.05, ***p<0.00l, and ****p<0.000l. [0047] FIG. 7 : PA proliferation when treated with tobramycin and PEGylated- tobramycin in combination with different excipients for 12 hours.

[0048] FIG. 8: Surviving PA biofilm colony forming units (CFU/well) after treatment with tobramycin and PEGylated-tobramycin in combination with different excipients for 12 hours.

[0049] FIG. 9 : PA proliferation when treated with tobramycin in combination with different excipients for 24 hours.

[0050] FIG. 10: Surviving PA biofilm colony forming units (CFU/well) after treatment with tobramycin in combination with different excipients for 24 hours.

[0051] FIG. 11: PA proliferation when treated with colistin and PEGylated- colistin for 24 hours.

[0052] FIG. 12: PA proliferation when treated with colistin and PEGylated- colistin combined with different excipients for 24 hours.

[0053] FIGS. 13A-13B: Effect of the combination of Tob with excipients on P. aeruginosa biofilms. Biofilms of a bioluminescent P. aeruginosa strain (PAOl:pl6Slux) were treated for 20 h with 64 mg/mL Tob alone and with binary combinations of the excipients L- alanine (L-ala), L-proline (L-pro), succinic acid (SA), and N-acetyl-D-glucosaminitol (NADG). The excipients were combined at 1:1 concentration equivalent to 10 or 20 mM each.

(A) Mean changes in bacterial proliferation by luminometry with respect the time zero (n ¼ 4). p >0.05 between Tob, FI, F2, and F3 groups at 20 h. (B) Logarithmic changes in the number of surviving biofilm CFU (per mL) with respect to the untreated control (n ¼ 4; mean ± SD). ****p <0.0001 with respect effect of Tob alone.

[0054] FIGS. 14A-14G: In vitro evaluation of dry powder formulations on P. aeruginosa biofilms. Biofilms were treated with Tob solution (64 mg/mL) and with the dry powder formulations 1, 2, and 3 (at equivalent Tob concentration). (A) The treatment was applied at 0 and 12 h (arrows) and diluted to approximately 2.5 mg/mL at 5 and 17 h (stars).

(B) Mean changes in bacterial proliferation with respect to time zero by luminometry (n ¼ 3). p values indicate significant statistical differences between Tob, Fl, F2, and F3 groups at 1.5, 12, and 24 h. (C) Logarithmic changes in surviving biofilm colonies (log reduction of CFU/mL) with respect to the number of colonies at time zero of the treatment (n ¼ 9; mean ± SD). *p <0.05, **p <0.01, and ****p <0.0001. (D) Alanine dose response curve. (E) Proline dose response curve. (F) Succinic acid response curve. (G) NADG dose response curve.

[0055] FIGS. 15A-15G: (A) Dose curve of erythromycin for oral biofilm. (B)

Oral biofilm viability after treatment with erythromycin alone or in combination with different excipients at 5 mM. (C) Oral biofilm viability after treatment with erythromycin alone or in combination with different excipients at 20 mM. (D) Dose response curve for L-proline for oral biofilm. (E) Dose response curve for succinic acid for oral biofilm. (F) Dose response curve for L-alanine for oral biofilm. (G) Dose response curve for N-acetyl-D-glucosaminitol for oral biofilm.

[0056] FIGS. 16A-16F: (A) Dose response of additional excipients for oral biofilm treatments including example preservatives, suspending agents, chelating agents, and others. (B) Dose curve of chlorhexidine for oral biofilm. (C) Oral biofilm viability after treatment with chlorhexidine alone or in combination with different excipients at 20 mM. (D) Oral biofilm viability after treatment with chlorhexidine alone or in combination with different excipients at 5 mM. (E) Oral biofilm viability after 30-minute treatment with excipients alone or in combination with chlorhexidine. (F) Oral biofilm viability after 30-minute treatment with excipients alone or in combination with chlorhexidine. Biofilms grown for 8 hours in fresh media post treatment before assessing viability. FIGS. 16A, C-F data was normalized to the survival of untreated biofilms (Mean + S.D, n=6). ^*p<0.1 , *p <0.05, **p <0.01, and ***p <0.001.

[0057] FIGS. 17A-17F: (A) Oral biofilm viability after 30 minute treatment with 1 mg/mL chlorhexidine with combinations of iron oxide nanoparticles, negative (citric acid) and positive (PEG/ Amine) charges, alone or in combination with different excipients. (B) Oral biofilm viability after 30 minute treatment with 100 mg/mL chlorhexidine with combinations of iron oxide nanoparticles, negative (citric acid) and positive (PEG/ Amine) charges, alone or in combination with different excipients. (C) Oral biofilm viability after 30 minute treatment with combinations of iron oxide nanoparticles, negative (citric acid) and positive (PEG/ Amine) charges, alone or in combination with different excipients. (D) Oral biofilm viability after 30 minute treatment with 1 mg/mL chlorhexidine with combinations of iron oxide nanoparticles, negative (citric acid) and positive (PEG/ Amine) charges, alone or in combination with different excipients. Biofilms grown for 8 hours in fresh media post treatment before assessing viability. (E) Oral biofilm viability after 30 minute treatment with combinations of iron oxide nanoparticles, negative (citric acid) and positive (PEG/ Amine) charges, alone or in combination with different excipients. Biofilms grown for 8 hours in fresh media post treatment before assessing viability. (F) Oral biofilm viability after 30 minute treatment with 100 mg/mL chlorhexidine with combinations of iron oxide nanoparticles, negative (citric acid) and positive (PEG/ Amine) charges, alone or in combination with different excipients. Biofilms grown for 8 hours in fresh media post treatment before assessing viability. All data was normalized to the survival of untreated biofilms (Mean + S.D, n=6).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0058] Bacterial biofilm infections are particularly difficult to treat and eradicate using currently available drugs. For example, the current treatment of chronic Pseudomonas aeruginosa infections involves long-term administration of high doses of antibiotics, such as tobramycin. It was unknown if inactive pharmaceutical ingredients influence the activity of antibiotics they are co-formulated with. In an attempt to find excipient- drug synergism, the present studies comprised a screening study of potential excipients that could improve the antibiotic activity against PA biofilms. In this study, tobramycin (TOB) was selected as model antibiotic since it is the first line therapy against PA lung infections.

[0059] Specifically, 190 potential excipients alone, and in combination were screened with tobramycin sulfate against P. aeruginosa (strain PAOl) grown planktonic ally or as biofilms. After the excipient screening stage, the effect of 10 selected excipients were investigated against a more virulent strain (luminescent strain UCBPP-PA14). Temporal changes in luminescence, as an indicator of bacterial proliferation, and surviving colony forming units (CFUs) from the treated PA14 biofilms were quantified.48 materials tested caused a reduction of PAOl proliferation either alone or combined with tobramycin. L-alanine (p<0.05), D-alanine (p>0.05), and N-acetyl-D-glucosaminitol (p>0.05) improved the activity of tobramycin measured by PA14 luminometry. Additionally, L-alanine and succinic acid significantly reduced the survival of PA14 biofilms (p<0.05).

[0060] Accordingly, to solve the issue of ineffective and high dose treatments that can lead to significant side effects for the patient, certain embodiments of the present disclosure provide formulations comprising excipient combinations with antibiotics and/or antiseptics, such as chlorhexidine, which may be used as improved therapeutics for the treatment of biofilms, such as oral biofilms. Exemplary excipients include D-alanine, L- alanine, L-homoserine, L-proline, D-threonine, adenosine, 2-deoxy adenosine, inosine, citric acid, glycolic acid, glyoxylic acid, succinic acid, monomethyl succinate, quinic acid, d-amino valeric acid, chondroitin, D-cellobiose, D-fructose, D-fructose-6-phosphate, D-galactose, b- methyl-D-galactoside, a-methyl-D-glucoside, D-glucose-l -phosphate, D-gluconic acid, b- methyl-D-glucuronic acid, N-acetyl-D-glucosaminitol, D-mannose, D-melezitose, D- melibiose, D-trehalose, inulin, L-arabitol, maltose, mannan, melibionic acid, m-inositol, palatinose, stachyose, sucrose, turanose, xylitol, phenylethylamine, propylene glycol, sec- butylamine, tween 20, tween 40, tween 80, benzoic acid, potassium sorbate, alginic acid, carrageenan, citric acid, edetic acid, coconut oil, EDTA, and xylitol. Specifically, the formulations may comprise excipients including N-acetyl-D-glucosaminitol, L-alanine, L- proline, succinic acid, 2-deoxy-adenosine, chondroitin, and D-gluconic acid in order to improve antibiotic (e.g., tobramycin, colistin, or erythromycin) activity in several ranges of concentrations. These materials can improve the activity of the antibiotic even under inhibitory concentrations which can reduce the dose of the antibiotic and prevent the sides effects associated with long-term antibiotic treatments. The use of these materials that are excipients has the advantage of being classified as safe and inert. On the other side, these materials are organic molecules present in biologic systems. Therefore, they are predicted to have a low potential of development of resistance.

[0061] Further provided herein are methods for the treatment of biofilms, such as treating bacterial infections caused by Pseudomonas aeruginosa biofilms, oral biofilms, and/or fungal biofilms comprising administering the combination formulations provided herein.

[0062] Formulations containing either PEG-tobramycin alone or PEG- tobramycin and tobramycin combined with the present excipient(s) can be used to treat infections caused by bacterial biofilms, such as chronic respiratory, otic, wound, rhinosinusal, and device-related infections. These formulations can be developed as solutions, suspension, semisolids, and dry powders for local and pulmonary delivery.

[0063] Formulations containing either PEG-colistin alone, or PEG-colistin and colistin combined with the present excipient(s) can be used to treat infections caused by bacterial biofilms, such as chronic respiratory, otic, wound, rhinosinusal infections, and device- related infections. These formulations can be developed as solutions, suspension, semisolids, and dry powders for local and pulmonary delivery.

[0064] Formulations containing erythromycin combined with the present excipient(s) can be used to treat infections caused by bacterial biofilms, such as dental caries, gingivitis, and periodontal disease. These formulations can be developed as solutions, suspensions, semisolids, varnishes, paste, sublingual dissolvable tablets, lozenge, spray or mist for local oral cavity.

[0065] Formulations containing chlorhexidine combined with the present excipients and/or iron oxide nanoparticles can be used to treat infections caused by bacterial biofilms, such as dental caries, gingivitis, and periodontal disease. Iron oxide nanoparticles can also be used with selected excipients without chlorhexidine. These formulations can be developed as solutions, suspensions, semisolids, varnishes, paste, sublingual dissolvable tablets, lozenge, spray or mist for local oral cavity.

I. Definitions

[0066] As used herein,“essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 1%, such as below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

[0067] As used herein the specification,“a” or“an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word“comprising,” the words “a” or“an” may mean one or more than one.

[0068] The use of the term“or” in the claims is used to mean“and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and“and/or.” As used herein“another” may mean at least a second or more. [0069] Throughout this application, the term“about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0070] “Treatment” or“ treating” includes (1) inhibiting a disease in a subject or patient experiencing or displaying the pathology or symptomatology of the disease (e.g., arresting further development of the pathology and/or symptomatology), (2) ameliorating a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease (e.g., reversing the pathology and/or symptomatology), and/or (3) effecting any measurable decrease in a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease.

[0071] “Prevention” or“ preventing” includes: (1) inhibiting the onset of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease, and/or (2) slowing the onset of the pathology or symptomatology of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease.

[0072] As used herein, the term“patient” or“subject” refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human patients are adults, juveniles, infants and fetuses.

[0073] As generally used herein“pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.

[0074] "Administering" and/or "administer" as used herein refer to any route for delivering a pharmaceutical composition to a patient. In one embodiment, the compositions described herein are administered enterically to the small intestine. Routes of delivery may include non-invasive peroral (through the mouth), topical (skin, or wound), transmucosal (nasal, buccal/sublingual, vaginal, ocular and rectal) and inhalation routes, as well as parenteral routes, and other methods known in the art. Parenteral refers to a route of delivery that is generally associated with injection, including intra-articular, intraorbital, infusion, intraarterial, intracarotid, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrastemal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders.

[0075] The term“effective,” as that term is used in the specification and/or claims, means adequate to accomplish a desired, expected, or intended result.“Effective amount”, “therapeutically effective amount” or“pharmaceutically effective amount” when used in the context of treating a patient or subject with a compound means that amount of the compound which, when administered to a subject or patient for treating or preventing a disease, is an amount sufficient to effect such treatment or prevention of the disease.

[0076] As used herein, the term "antimicrobial" is meant to include prevention, inhibition or termination of a microbe. In some aspects, "prevention" can be considered to be the obstruction or hindrance of any potential microbial growth, and "inhibition" can be considered to be a reduction in microbial growth. This may occur via, but is not limited to, a microbiostatic mechanism such as interference in the synthesis of the cell wall or binding to ribosomal subunits to prevent production of microbial proteins. "Termination" can be considered to be actual killing of the microbes by the presence of the composition. This may occur via, but is not limited to, a microbiocidal mechanism such as a change in osmotic pressure leading to bursting of the cell or formation of leaky channels in the cell wall and membrane causing loss of cellular material.

[0077] As used herein, the term "microbe(s)" is meant to include any organism comprised of the phylogenetic domains bacteria and archaea, as well as unicellular and filamentous fungi (e.g., yeasts and molds), unicellular and filamentous algae, unicellular and multicellular parasites, and viruses.

[0078] An“excipient” is a pharmaceutically acceptable substance formulated along with the active ingredient(s) of a medication, pharmaceutical composition, formulation, or drug delivery system. Excipients may be used, for example, to stabilize the composition, to bulk up the composition (thus often referred to as“bulking agents,” “fillers,” or“diluents” when used for this purpose), or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption, reducing viscosity, or enhancing solubility. Excipients include pharmaceutically acceptable versions of antiadherents, binders, coatings, colors, disintegrants, flavors, glidants, lubricants, preservatives, sorbents, sweeteners, and vehicles. The main excipient that serves as a medium for conveying the active ingredient is usually called the vehicle. Excipients may also be used in the manufacturing process, for example, to aid in the handling of the active substance, such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation or aggregation over the expected shelf life. The suitability of an excipient will typically vary depending on the route of administration, the dosage form, the active ingredient, as well as other factors.

[0079] “Biofilm” refers to a structured consortium of bacteria embedded in a self-produced polymer matrix consisting of polysaccharides, protein and DNA.

II. Formulations

[0080] In some embodiments, the present disclosure provides formulations comprising an antibiotic and/or antiseptic in combination with one or more excipients. The formulation may comprise 2, 3, 4, 5, or more excipients. The antibiotic and/or antiseptic and excipient combination may be used to treat infections, such as biofilms, resulting from gram positive or gram-negative bacterial strains including, but not limited to, Pseudomonas aeruginosa, Staphylococcus aureus, Helicobacter pylori, Burkholderia cepacia complex, Haemophilus influenzae, Escherichia coli, Streptococcus pneumoniae, Enterococcus faecalis, Pseudomonas fluorescens, Staphylococcus epidermidis and Acinetobacter baumannii. The infection may be from a fungal strain, such as Candida albicans. In some aspects, the infection may be due to a bacteria and fungus. The biofilm may be an oral biofilm, such as an oral biofilm associated with dental caries, gingivitis, or periodontal disease.

[0081] The antibiotic may be an aminoglycoside (e.g., tobramycin, streptomycin, kanamycin, gentamicin, netilmicin, amikacin, or neomycin), a monobactam/b- lactam (e.g., aztreonam, nocardicin A, imipenem or tabtoxin), a polymyxin (e.g., colistin (also known as polymyxin E) or polymyxin B), or a fluoroquinolone (e.g., ciprofloxacin, levofloxacin, or trovafloxacin). Other antibiotics that may be used include, but are not limited to, quinolones/fluoroquinolones (e.g., ofloxacin, nalidixic acid, cinoxacin, norfloxacin, perfloxacin, enoxacin, and fleroxacin), ampicillin, amikacin, clindamycin, tetracyclines, rifampin, triclosan, macrolides (e.g., erythromycin, clarithromycin or azithromycin), penicillins, cephalosporins, amoxicillin/clavulanate, quinupristin/dalfopristin, amoxicillin/sulbactum, metronidazole, and ketolides.

[0082] Antipseudomonals that may be used include carbenicillin, carbenicillin indanyl, ticarcillin, azlocillin, mezlocillin, and piperacillin. Cephalosporins that may be used include first generation— cephalothin, cephaprin, cephalexin, cephradine, cefadroxil, cefazolin; second generation— cefamandole, cefoxitin, cefaclor, cefuroxime, cefotetan, ceforanide, cefuroxine axetil, cefonicid; and third generation— cefotaxime, moxalactam, ceftizoxime, ceftriaxone, cefoperazone, and ceftazidime. Other cephalosporins that may be used are cephaloridine and cefsulodin. b-Lactamase inhibitors that may be used include clavulanic acid, augmentin, and sulbactam. Sulfonamides that may be used include sulfanilamide, sulfamethoxazole, sulfacetamide, sulfadiazine, sulfisoxazole, sulfacytine, sulfadoxine, mafenide, p-aminobenzoic acid, and trimethoprim-sulfamethoxazole. Tetracyclines that may be used include tetracycline, chlortetracycline, demeclocycline, methacycline, doxycycline, and minocycline. Other Antibiotics that may be used include chloramphenicol (chlormycetin), erythromycin, lincomycin, clindamycin, spectinomycin, vancomycin, and bacitracin. Anti-fungal agents that may be used include amphotericin b, cyclosporine, and flucytosine.

[0083] Antibiotics that may be used in the present compositions and methods may include, but are not limited to, ampicillin, bacampicillin, carbenicillin indanyl, mezlocillin, piperacillin, ticarcillin, amoxicillin-clavulanic acid, ampicillin-sulbactam, benzylpenicillin, cloxacillin, dicloxacillin, methicillin, oxacillin, penicillin g, penicillin v, piperacillin tazobactam, ticarcillin clavulanic acid, nafcillin, cephalosporin, cefadroxil, cefazolin, cephalexin, cephalothin, cephapirin, cephradine cefaclor, cefamandol, cefonicid, cefotetan, cefoxitin, cefprozil, ceftmetazole, cefuroxime, loracarbef, cefdinir, ceftibuten, cefoperazone, cefixime, cefotaxime, cefpodoxime proxetil, ceftazidime, ceftizoxime, ceftriaxone, azithromycin, clarithromycin, clindamycin, dirithromycin, erythromycin, lincomycin, troleandomycin, cinoxacin, ciprofloxacin, enoxacin, gatifloxacin, grepafloxacin, levofloxacin, lomefloxacin, mozzxifloxacin, nalidixic acid, norfloxacin, ofloxacin, sparfloxacin, trovafloxacin, oxolinic acid, gemifloxacin, perfloxacin, imipenem-cilastatin, meropenem, aztreonam, amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, paromomycin, teicoplanin, vancomycin, demeclocycline, doxycycline, methacycline, minocycline, oxytetracycline, tetracycline, chlortetracycline, mafenide, silver sulfadiazine, sulfacetamide, sulfadiazine, sulfamethoxazole, sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole, sulfamethizole, rifabutin, rifampin, rifapentine, linezolid, streptogramins, quinopristin dalfopristin, bacitracin, chloramphenicol, fosfomycin, isoniazid, methenamine, metronidazol, mupirocin, nitrofurantoin, nitrofurazone, novobiocin, polymyxin, spectinomycin, trimethoprim, colistin, cycloserine, capreomycin, ethionamide, pyrazinamide, para-aminosalicyclic acid, erythromycin ethylsuccinate and combinations thereof.

[0084] The excipient(s) may include amino acids, such as D-alanine, L-alanine,

L-homoserine, L-proline, and D-threonine; nucleosides, such as Adenosine, 2-deoxy adenosine, and Inosine; organic acids and derivatives, such as Citric acid, Glycolic acid, Glyoxylic acid, Succinic acid, Monomethyl succinate, Quinic acid, and d-amino valeric acid; Saccharides, such as Chondroitin, D-cellobiose, D-fructose, D-fructose-6-phosphate, D- galactose, b-methyl-D-galactoside, a-methyl-D-glucoside, D-glucose-l -phosphate, D- gluconic acid, b-methyl-D-glucuronic acid, N-acetyl-D-glucosaminitol, D-mannose, D- melezitose, D-melibiose, D-trehalose, Inulin, L-arabitol, Maltose, Mannan, Melibionic acid, m-inositol, Palatinose, Stachyose, Sucrose, Turanose, and Xylitol; and/or others, such as Phenylethylamine, Propylene glycol, Sec-butylamine, Tween 20, Tween 40, Tween 80, benzoic acid, potassium sorbate, alginic acid, carrageenan, citric acid, edetic acid, coconut oil, EDTA, and xylitol..

[0085] Exemplary excipients in the present formulations may comprise, but are not limited to, N-acetyl-D-glucosaminitol, L-proline, L-alanine, succinic acid, 2-deoxy- adenosine, chondroitin, and/or D-gluconic acid.

[0086] Exemplary formulations may comprise a combination of tobramycin

(e.g., 10-50 pg/mL, particularly about 20, 30, or 40 pg/mL), N-acetyl-D-glucosaminitol (e.g., 5-30 mM, particularly about 10-20 mM or 1-5 mg/mL), L-alanine (e.g., 5-50 mM, particularly about 10, 20, or 30 mM or 1-5 mg/mL), and succinic acid (e.g., 5-50 mM, particularly about 10, 20, or 30 mM or 1-5 mg/mL, such as 1-2 mg/mL). In one specific composition, the composition comprises about 32 pg/mL tobramycin, 10-20 mM N-acetyl-D-glucosaminitol, 20 mM L-alanine, and 20 mM succinic acid.

[0087] Exemplary formulations may comprise a combination of tobramycin

(e.g., 0.1 to 82%, particularly about 0.3 to 6%), N-acetyl-D-glucosaminitol (e.g., 5 to 25%, particularly about 10 to 20%), L-alanine (e.g., 5 to 25%, particularly about 5 to 10%) and succinic acid (e.g., 5 to 25%, particularly about 5 to 10%). In one specific composition, the composition comprises about 0.3% tobramycin, 20% N-acetyl-D-glucosaminitol, 10% L- alanine, and 10% succinic acid.

[0088] The formulation may comprise tobramycin and L-proline; tobramycin,

L-proline, and N-acetyl-D-glucosaminitol; tobramycin, L-proline, and L-alanine; tobramycin, L-alanine, and N-acetyl-D-glucosaminitol; or tobramycin, L-alanine, L-proline, and N-acetyl- D-glucosaminitol. The tobramycin may be at a concentration of about 10-100 pg/mL, such as about 20-30, 30-40, 40-50, 50-60, or 60-70 pg/mL, particularly about 32 pg/mL or about 64 pg/mL. The L-proline may be present at a concentration of about 5-50 mM, such as 10-20 mM or 1.15-2.5 mg/mL. The N-acetyl-D-glucosaminitol may be present at a concentration of about 5-50 mM, such as about 10-20 mM or 2.23-4.47 mg/mL. The L-proline may be present at a concentration of about 5-50 mM, such as 10-20 mM or about 1.15-2.4 mg/mL. The L-alanine may be present at a concentration of about 5-50 mM, such as about 10-20 mM or 0.89-1.78 mg/mL.

[0089] The tobramycin may be at a concentration of about 0.3 to 21%, such as about 0.1 to 0.3%, 6 to 10%, or 70 to 80%, particularly about 1% or about 50%. The L-proline may be present at a concentration of about 5 to 20%, such as 5 to 10% or 50 to 100 mg/mL. The N-acetyl-D-glucosaminitol may be present at a concentration of about 15 to 25%, such as about 20% or 80 to 100 mg/mL. The L-proline may be present at a concentration of about 5 to 20%, such as 8 to 10% or about 80 to 120 mg/mL. The L-alanine may be present at a concentration of about5 to 20%, such as about 8 to 12% or 50 to 120 mg/mL.

[0090] The formulation may be provided as an ophthalmic solution, ophthalmic suspension, ophthalmic ointment, dry powder for inhalation, solution for inhalation, topical cream, otic solution, otic suspension or nasal spray. The ophthalmic solution may comprise about 0.1-1% of the antibiotic, such as about 0.2, 0.3, or 0.4% of the antibiotic, such as tobramycin, gentamicin or ciprofloxacin hydrochloride. The ophthalmic suspension may comprise 0.05-1% of the antibiotic, such as 0.1 -0.3% of the antibiotic, such as tobramycin. The ophthalmic ointment may comprise 0.1-1% of the antibiotic, such as 0.2, 0.3, or 0.4% of the antibiotic, such as tobramycin, gentamicin or ciprofloxacin hydrochloride. The dry powder for inhalation may comprise about 5-100 mg/dose, such as about 10-50 mg/dose, particularly about 20-40 mg/dose, specifically about 28 mg/dose. The solution for inhalation may comprise about 50-500 mg/l-lO mL, such as about 200-400 mg/4-6 mL of the antibiotic, such as tobramycin, and/or 10-150 mg/dose, such as about 50-100 mg/dose, particularly about 75 mg/dose, of the antibiotic, such as aztreonam. The topical cream may comprise about 0.05-1%, such as about 0.1, 0.2, or 0.3%, of the antibiotic, such as gentamicin sulfate. The otic solution may comprise 0.05-1%, such as 0.1, 0.2, or 0.3%, antibiotic, such as ciprofloxacin. The otic solution may comprise 1-5 mg/dose, such as 3-4 mg/dose, of the antibiotic, such as tobramycin, gentamicin, or ciprofloxacin hydrochloride. The nasal spray may comprise about 0.1 to 10%, such about 0.3, 1, or 6%, antibiotic such as tobramycin, colistin, ciprofloxacin or gentamicin.

[0091] Table 1: Exemplary formulations.

[0092] In some embodiments, the formulation is a powder for reconstitution and parenteral administration. Sterile tobramycin sulfate may be supplied as a sterile dry powder with L-alanine, L-proline and be intended for reconstitution with 30 mL of Sterile Water for Injection, USP. Sulfuric acid and/or sodium hydroxide may have been added during manufacture to adjust the pH. Each vial may contain tobramycin sulfate equivalent to 1200 mg of tobramycin. After reconstitution, the solution may contain 40 mg of tobramycin per mL, lg/mL of L-alanine, and 1 g/mL. The product preferably contains no preservatives or sodium bisulfite.

[0093] The formulations may comprise an antiseptic, such as chlorhexidine and one or more excipients and/or iron oxide nanoparticles. The nanoparticles may be coated with citric acid or PEG/amine. The iron oxide nanoparticle containing compositions may be administered in combination with exposing the subject to a magnetic field. The chlorhexidine composition may be used to treat biofilms, such as oral biofilms. The chlorhexidine composition may be formulated as a solution, suspension, semisolid, varnish, paste, sublingual dissolvable tablet, lozenge, spray or mist for local oral cavity.

[0094] The nanoparticle can be any suitable nanoparticle. The nanoparticle can be a magnetic nanoparticle. The iron in the nanoparticle can be at least one of iron oxide and zero-valent iron. The iron in the nanoparticle can be part of an iron compound that is at least one of LeO, Fe₃0₄, Le₄05, Fe₂0₃.

[0095] The concentration of the nanoparticles can be about 0.0001 pg/mL to about 1 g/mL, or about 0.0001 pg/mL or less, or less than, equal to, or greater than about 0.001 pg/mL, 0.01, 0.1, 1 pg/mL, 0.01 mg/mL, 0.1, 1 mg/mL, 0.01 g/mL, 0.1 g/mL, or about 1 g/mL or more.

[0096] The nanoparticle can have any suitable size, such as having a largest dimension of about 1 nm to about 999 nm, about 10 nm to about 400 nm, about 1 nm to about 100 nm, or about 1 nm or less, or less than, equal to, or greater than about 2 nm, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, or about 999 nm or more.

A. PEGylation

[0097] The antibiotic, such as tobramycin or colistin, may be modified. The modification may comprise PEGylation. In one exemplary formulation, the PEGylated tobramycin (e.g., about 0.1 to 85%, particularly about 0.3 to 6%, such as 5%) is combined with N-acetyl-D-glucosaminitol (e.g., about 5 to 20%, such as 8 to 12%).

[0098]“PEG”, “polyethylene glycol”, and “poly(ethylene glycol)” are used interchangeably herein to refer to a compound comprising the repeating unit — [O— CH₂— CH₂]n— . For example, the PEG may comprise the structure CH3-[0-CH2-CH2]_n- (mPEG) or H-[0-CH₂-CH₂]_n-. Polyethylene glycol is an example of a PEG and refers to a compound with the structure H-[0-CH₂-CH₂]_n-0H. As would be recognized by one of skill in the art, a wide variety of sizes of PEG may be used to pegylate an antibody, such as colistin. For example, in some embodiments, n = 2-4000, 2-3000, 2-2000, 50-4000, 50-3000, 50-2000, 100-2000, 100-750, or 250-1000. In some embodiments, the PEG has a molecular weight of 100-10000, 2000-20000, or 2000-10000 daltons. In some embodiments, the PEG has a molecular weight of 1500-7500, 4000-6000, or about 5000 daltons. In some embodiments, the PEG is PEG 5k. In some embodiments, the PEG has the formula CH₃-PEG-OC(0)CH₂CH₂C(0)OH or CH₃-[0-CH₂-CH₂]_n-OC(0)CH₂CH₂C(0)OH.

[0099] Modified PEG moieties are known and may comprise the formula H— [O— CH₂— CtEJ_n— The n in the formula of the modified PEG have a range as defined above. The modified PEG may have the structure H-[0-CH₂-CH₂]_n-(leaving group), wherein the leaving group is defined below. For example, the leaving group may be, e.g., -OH (e.g., as present in -0C(0)CH₂CH₂C(0)0H or other esters), -OMs, -OTf, -OMe, or -OTs. After pegylation, the PEG moiety may be covalently bound to the aminoglycoside moiety via an amide, etc. bond. Methods for PEGylating an antibody are described in US20170143842; incorporated herein by reference in its entirety.

[00100] A“leaving group” when used as described above is a functional group which converts the hydroxyl group into a better leaving group. This functional group makes the hydroxyl group a better leaving group by stabilizing the charge on the oxygen when the atom bears a negative charge. This functional group makes the hydroxyl group more susceptible to a nucleophilic attack and displacement by nucleophilic groups.

[00101] In certain aspects, methods and compositions of the embodiments relate to PEGylation of disclosed antibiotics, such as tobramycin or colistin. PEGylation is the process of covalent attachment of poly(ethylene glycol) polymer chains to another molecule, normally a drug or therapeutic protein. PEGylation is routinely achieved by incubation of a reactive derivative of PEG with the target macromolecule. The covalent attachment of PEG to a drug or therapeutic protein can“mask” the agent from the host's immune system (reduced immunogenicity and antigenicity) or increase the hydrodynamic size (size in solution) of the agent, which prolongs its circulatory time by reducing renal clearance. PEGylation can also enhance biofilm penetration, mucus penetration, and provide water solubility to hydrophobic drugs and proteins.

[00102] The first step of the PEGylation is the suitable functionalization of the PEG polymer at one or both terminals. PEGs that are activated at each terminus with the same reactive moiety are known as“homobifunctional,” whereas if the functional groups present are different, then the PEG derivative is referred as“heterobifunctional” or“heterofunctional.” The chemically active or activated derivatives of the PEG polymer are prepared to attach the PEG to the desired molecule.

[00103] The choice of the suitable functional group for the PEG derivative is based on the type of available reactive group on the molecule that will be coupled to the PEG. For proteins, typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, and tyrosine. The /V-terminal amino group and the C- terminal carboxylic acid can also be used.

[00104] The techniques used to form first generation PEG derivatives are generally reacting the PEG polymer with a group that is reactive with hydroxyl groups, typically anhydrides, acid chlorides, chloroformates, and carbonates. In the second generation PEGylation chemistry more efficient functional groups, such as aldehyde, esters, amides, etc., are made available for conjugation.

[00105] As applications of PEGylation have become more and more advanced and sophisticated, there has been an increase in need for heterobifunctional PEGs for conjugation. These heterobifunctional PEGs are very useful in linking two entities, where a hydrophilic, flexible, and biocompatible spacer is needed. Preferred end groups for heterobifunctional PEGs are maleimide, vinyl sulfones, pyridyl disulfide, amine, carboxylic acids, and NHS esters.

[00106] The most common modification agents, or linkers, are based on methoxy PEG (mPEG) molecules. Their activity depends on adding a protein-modifying group to the alcohol end. In some instances polyethylene glycol (PEG diol) is used as the precursor molecule. The diol is subsequently modified at both ends in order to make a hetero- or homo- dimeric PEG-linked molecule.

[00107] Proteins are generally PEGylated at nucleophilic sites, such as unprotonated thiols (cysteinyl residues) or amino groups. Examples of cysteinyl-specific modification reagents include PEG maleimide, PEG iodoacetate, PEG thiols, and PEG vinylsulfone. All four are strongly cysteinyl-specific under mild conditions and neutral to slightly alkaline pH but each has some drawbacks. The thioether formed with the maleimides can be somewhat unstable under alkaline conditions so there may be some limitation to formulation options with this linker. The carbamothioate linkage formed with iodo PEGs is more stable, but free iodine can modify tyrosine residues under some conditions. PEG thiols form disulfide bonds with protein thiols, but this linkage can also be unstable under alkaline conditions. PEG-vinylsulfone reactivity is relatively slow compared to maleimide and iodo PEG; however, the thioether linkage formed is quite stable. Its slower reaction rate also can make the PEG-vinylsulfone reaction easier to control.

[00108] Site-specific PEGylation at native cysteinyl residues is seldom carried out, since these residues are usually in the form of disulfide bonds or are required for biological activity. On the other hand, site-directed mutagenesis can be used to incorporate cysteinyl PEGylation sites for thiol- specific linkers. The cysteine mutation must be designed such that it is accessible to the PEGylation reagent and is still biologically active after PEGylation.

[00109] Amine- specific modification agents include PEG NHS ester, PEG tresylate, PEG aldehyde, PEG isothiocyanate, and several others. All react under mild conditions and are very specific for amino groups. The PEG NHS ester is probably one of the more reactive agents; however, its high reactivity can make the PEGylation reaction difficult to control on a large scale. PEG aldehyde forms an imine with the amino group, which is then reduced to a secondary amine with sodium cyanoborohydride. Unlike sodium borohydride, sodium cyanoborohydride will not reduce disulfide bonds. However, this chemical is highly toxic and must be handled cautiously, particularly at lower pH where it becomes volatile.

[00110] Due to the multiple lysine residues on most proteins, site-specific PEGylation can be a challenge. Fortunately, because these reagents react with unprotonated amino groups, it is possible to direct the PEGylation to lower-pK amino groups by performing the reaction at a lower pH. Generally the pK of the alpha-amino group is 1-2 pH units lower than the epsilon-amino group of lysine residues. By PEGylating the molecule at pH 7 or below, high selectivity for the 77-terminus frequently can be attained. However, this is only feasible if the 77-terminal portion of the protein is not required for biological activity. Still, the pharmacokinetic benefits from PEGylation frequently outweigh a significant loss of in vitro bioactivity, resulting in a product with much greater in vivo bioactivity regardless of PEGylation chemistry.

[00111] There are several parameters to consider when developing a PEGylation procedure. Fortunately, there are usually no more than four or five key parameters. The “design of experiments” approach to optimization of PEGylation conditions can be very useful. For thiol- specific PEGylation reactions, parameters to consider include: drug concentration, PEG-to-drug ratio (on a molar basis), temperature, pH, reaction time, and in some instances, the exclusion of oxygen. (Oxygen can contribute to intermolecular disulfide formation by the drug, which will reduce the yield of the PEGylated product.) The same factors should be considered (with the exception of oxygen) for amine-specific modification except that pH may be even more critical, particularly when targeting the N-terminal amino group.

[00112] In addition, the reactivity of the PEG linker should be known before starting the PEGylation reaction. For example, if the PEGylation agent is only 70 percent active, the amount of PEG used should ensure that only active PEG molecules are counted in the drug-to-PEG reaction stoichiometry.

B. Pharmaceutical Compositions

[00113] Certain of the methods set forth herein pertain to methods involving the administration of a pharmaceutically effective amount of a composition comprising an antibiotic and/or antiseptic, and at least one excipient and/or iron oxide nanoparticles of the present disclosure. [00114] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (Remington’s, 1990). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated. The compositions used in the present disclosure may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection.

[00115] The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions, and these are discussed in greater detail below. For human administration, preparations preferably meet sterility, pyrogenicity, general safety and purity standards as required by FDA.

[00116] The present disclosure contemplates methods using compositions that are sterile solutions for intravascular injection or for application by any other route as discussed in greater detail below. A person of ordinary skill in the art would be familiar with techniques for generating sterile solutions for injection or application by any other route. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients familiar to a person of skill in the art.

[00117] The present formulations may be combined with different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. A formulation of the present disclosure may be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, locally, via inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, spray, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington: The Science and Practice of Pharmacy, 2l^st Ed. Lippincott Williams and Wilkins, 2005).

[00118] Formulations, such as oral formulations, may comprise one or more additives, buffering agents, preservatives, flavorings, chelating agents, anti-oxidants, humectants, stabilizers (including antioxidants), colorants, and other additives used in preparations administered into the oral cavity.

[00119] The formulations of the present disclosure may be formulated into a composition in a free base, neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts, e.g., those formed with the free amino groups, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations may be easily administered in a variety of dosage forms such as formulated for parenteral administrations such as injectable solutions, or aerosols for delivery to the lungs, or formulated for alimentary administrations such as drug release capsules and the like.

[00120] The formulation of the composition may vary depending upon the route of administration· For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered and the liquid diluent first rendered isotonic with sufficient saline or glucose. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.

[00121] In addition to the compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, formulations for administration via an implantable drug delivery device, and any other form. One may also use nasal solutions or sprays, aerosols or inhalants in the present disclosure.

[00122] Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders. A person of ordinary skill in the art would be familiar with well-known techniques for preparation of oral formulations.

[00123] In certain embodiments, pharmaceutical composition includes at least about 0.1 % by weight of the active agent. The composition may include, for example, about 0.01%. In other embodiments, the pharmaceutical composition includes about 2% to about 85% of the weight of the composition, or between about 25% to about 60% by weight of the composition, for example, and any range derivable therein.

[00124] The pharmaceutical composition may comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g. , methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof. The composition may be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that exotoxin contamination should be kept minimally at a safe level, for example, less than 0.5 ng/mg protein.

[00125] In embodiments where the composition is in a liquid form, a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. In many cases, it will be preferable to include isotonic agents, such as, for example, sugars, sodium chloride or combinations thereof.

[00126] In other embodiments, one may use nasal solutions or sprays, aerosols or inhalants in the present disclosure. Nasal solutions may be aqueous solutions designed to be administered to the nasal passages in drops or sprays.

[00127] Sterile injectable solutions are prepared by incorporating the nanoparticles in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by sterilization.

[00128] The composition can be administered to the subject using any method known to those of ordinary skill in the art. For example, a pharmaceutically effective amount of a composition may be administered intravenously, intracerebrally, intracranially, intrathecally, intradermally, intraarterially, intraperitoneally, intralesionally, intratracheally, intranasally, topically, intramuscularly, intraperitoneally, subcutaneously, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (Remington’s, 1990). In particular embodiments, the composition is administered to a subject using a drug delivery device.

[00129] In other embodiments, the compositions are formulated for administration by routes including, but not limited to, oral, intranasal, enteral, topical, sublingual, intra-arterial, intramedullary, intrathecal, inhalation, ocular, transdermal, vaginal or rectal routes, and will include appropriate carriers in each case. For example, compositions for topical application may be prepared including appropriate carriers. Creams, lotions and ointments may be prepared for topical application using an appropriate base such as a triglyceride base. Such creams, lotions and ointments may also contain a surface active agent. Aerosol formulations may also be prepared in which suitable propellant adjuvants are used. Other adjuvants may also be added to the composition regardless of how it is to be administered, for example, anti-microbial agents, anti-oxidants and other preservatives may be added to the composition to prevent microbial growth and/or degradation over prolonged storage periods.

[00130] A pharmaceutically effective amount of the composition is determined based on the intended goal, for example inhibition of bacterial growth. The quantity to be administered, both according to number of treatments and dose, depends on the subject to be treated, the state of the subject, the protection desired, and the route of administration· Precise amounts of the therapeutic agent also depend on the judgment of the practitioner and are peculiar to each individual.

[00131] For example, a dose of the therapeutic agent may be about 0.0001 milligrams to about 1.0 milligrams, or about 0.001 milligrams to about 0.1 milligrams, or about 0.1 milligrams to about 1.0 milligrams, or even about 10 milligrams per dose or so. Multiple doses can also be administered. In some embodiments, a dose is at least about 0.0001 milligrams. In further embodiments, a dose is at least about 0.001 milligrams. In still further embodiments, a dose is at least 0.01 milligrams. In still further embodiments, a dose is at least about 0.1 milligrams. In more particular embodiments, a dose may be at least 1.0 milligrams. In even more particular embodiments, a dose may be at least 10 milligrams. In further embodiments, a dose is at least 100 milligrams or higher.

[00132] In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non- limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.

[00133] The dose can be repeated as determined by those of ordinary skill in the art. Thus, in some embodiments of the methods set forth herein, a single dose is contemplated. In other embodiments, two or more doses are contemplated. Where more than one dose is administered to a subject, the time interval between doses can be any time interval as determined by those of ordinary skill in the art. For example, the time interval between doses may be about 1 hour to about 2 hours, about 2 hours to about 6 hours, about 6 hours to about 10 hours, about 10 hours to about 24 hours, about 1 day to about 2 days, about 1 week to about 2 weeks, or longer, or any time interval derivable within any of these recited ranges.

[00134] In certain embodiments, the method may provide a continuous supply of a pharmaceutical composition to the patient. This could be accomplished by catheterization, followed by continuous administration of the therapeutic agent. The administration could be intra-operative or post-operative.

C. Methods of Use

[00135] The disclosure provides compositions and methods for preventing, ameliorating or treating biofilm infections. The compositions and methods disclosed herein are suitable for preventing, ameliorating or treating biofilm infections comprising any bacterium, a member of the Fungi Kingdom (e.g., yeast, fungus), or single-celled protozoan, and are suitable for biofilms found in any vertebrate (e.g., mammal, including human), regardless of whether the biofilm is found associated with a natural structure (e.g., a joint) or an artificial structure (e.g., a catheter, a prosthesis, a replacement joint or bone, or an implant).

[00136] The formulations comprising an antibiotic (or antiseptic) and at least one excipient may be administered topically, intralesionally, by inhalation, intranasally, ophthalmically, orally, and by parenteral routes. More specifically, the present disclosure refers to a pharmaceutical composition that may be administrated in dosage form such as mouth rinses, nasal sprays, solutions, oral solutions, inhalation solution, oral liquids suspensions, and capsules, among others. The compositions may be administrated and manufactured by any suitable means known in the art, for example, topically (including via direct application to skin or to any epithelial tissue surface, including such surfaces as may be present in glandular tissues or in the respiratory and/or gastrointestinal tracts), vaginally, intraperitoneally, orally, parenterally, intravenously, intraarterially, transdermally, sublingually, subcutaneously, intramuscularly, transbuccally, intranasally, via inhalation, intraoccularly, subcutaneously, intraadiposally, and intraarticularly or intrathecally among others.

[00137] In topical administration the carrier may include a solution, emulsion, ointment or gel base. The base, for example, may include one or more components such as petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers, among others.

[00138] This formulation can be useful in the treatment of infections caused by P. aeruginosa biofilms, such as in chronic lung infections, chronic wounds, chronic rhinosinusitis, recurrent otitis media, and recurrent urinary tract infections. Also, the formulation can be used to rinse materials used as catheters and intubation tubes, which are known to cause catheter-associated urinary tract infections and ventilator-associated pneumonia.

[00139] The present formulations may be used to treat chronic infections involving biofilms, including urinary tract infection, gastritis, lung infection, ear infection, cystitis, pyelonephritis, arterial damage, leprosy, tuberculosis, benign prostatic hyperplasia, prostatitis, osteomyelitis, bloodstream infection, cirrhosis, skin infection, acne, rosacea, open wound infection, chronic wound infection, and sinus infection. [00140] For example, chronic lung infections are hard to eradicate due to the increased resistance of the bacteria growing as biofilms. The treatment involves a long-term administration of high doses of antibiotics. However, the infection usually re-occurs, and the treatment must be repeated by cycles of 28 days-in and -off antibiotics. On the other hand, it is reported that subinhibitory doses of aminoglycosides can stimulate the formation of biofilms. Within the lung surface, subinhibitory doses of antibiotics could be present in different microregions. Therefore, the co-administration with an excipient that can improve the activity of antibiotics, such as tobramycin, under these conditions would be beneficial for the current therapy.

[00141] The biofilm infection can be located on any part of the body and can be caused by any one or combination of microorganisms. Some examples of body parts that may contain a biofilm include the heart, stomach, intestines, lungs, a vein or artery, sinus, gums, bone, joint, kidney, jaw, liver, and bladder. The biofilm infection may also be located on medical devices, including, but not limited to catheters, orthopedic devices, implants, prosthetic heart valves, prosthetic joints, orthopedic implants, shunts, pacemaker and defibrillator, endotracheal intubation, hemodialysis/peritoneal dialysis devices, dental implants, intravascular catheters, intrauterine devices (IUDs), and any inert and chemically modified plastic used for implant or medical device purposes. The microorganism can be any type, including procaryote or eucaryote, e.g., bacteria, archaea, protozoa, fungi (e.g., yeast (such as Candida albicans ) or mold), and algae.

[00142] In one embodiment, the compositions of the present disclosure provide antimicrobial effect to a target microbial organism and can be used to treat a disease or infection associated with the target microbial organism. An antimicrobial effect includes inhibiting the growth or killing of the target microbial organisms, or interfering with any biological functions of the target microbial organisms. In general, the compositions of the present disclosure can be used to treat a disease or infection at any place in a host, e.g., at any tissue including surfaces of any tissue or implant. In one embodiment, the compositions are used to specifically kill or inhibit bacterial target microbial organisms in body fluid (e.g., blood, sputum).

[00143] The method, such as for formulations comprising iron oxide nanoparticles, may include exposing the biofilm infection to a magnetic field. The exposing of the microbe to the magnetic field can occur at any suitable temperature, such as about -100° C. to about 100° C., or about -100° C. or less, or less than, equal to, or greater than about -90° C., -80, -70, -60, -50, -40, -30, -20, -10, 0, 10, 20, 30, 40, 50, 60, 70, 80, 90° C., or about 100° C. or more.

[00144] The magnetic field can have any suitable strength, such as a strength of 0.001 kGs to about 10,000,000 kGs, about 0.01 kGs to about 10 kGs, about 0.001 kGs or less, or less than, equal to, or greater than about 0.01 kGs, 0.1, 1, 2, 3, 4, 5, 10, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1,000, 1,500, 2,000, 2,500, 5,000, 10,000, 20,000, 50,000, 100,000, 250,000, 500,000, 1,000,000, 5,000,000, or about 10,000,000 kGs or more.

[00145] In some embodiments, compositions of the present disclosure are effective against bacteria including Gram-positive and Gram-negative cocci, Gram-positive and Gram negative straight, curved and helical/vibroid and branched rods, sheathed bacteria, sulfur- oxidizing bacteria, sulfur or sulfate-reducing bacteria, spirochetes, actinomycetes and related genera, myxobacteria, mycoplasmas, rickettsias and chlamydias, cyanobacteria, archea, fungi, parasites, viruses and algae. For example, the target microbial organisms of the present disclosure include, without limitation, Escherichia coli, Candida, Salmonella, Staphylococcus, and Pseudomonas, especially Campylobacter jejuni, Candida albicans, Candida krusei, Chlamydia trachomatis, Clostridium difficile, Cryptococcus neoformans, Haempohilus influenzae, Helicobacter pylori, Moraxella catarrhalis, Neisseria gonorrhoeae, Pseudomonas aeroginosa, Salmonella typhimurium, Shigella disenteriae, Staphylococcus aureus, and Streptococcus pneumoniae. In addition, the microbial peptide composition may be used to treat chronic skin ulcers, infected acute wounds or bum wounds, infected skin eczema, impetigo, atopic dermatitis, acne, external otitis, vaginal infections, seborrhoic dermatitis, oral infections, paradontitis, conjunctivitis or pneumonia.

[00146] In particular embodiments, the compositions of the present disclosure are effective against gram-negative bacteria. Gram-positive and Gram-negative cocci include, but are not limited to, Aerococcus, Enterococcus, Halococcus, Leuconostoc, Micrococcus, Mobiluncus, Moraxella catarrhalis, Neisseria (including N gonorrheae and N meningitidis ), Pediococcus, Peptostreptococcus, Staphylococcus species (including S. aureus, methicillin- resistant S. aureus, coagulase-negative S. aureus, and S. saprophyticus), Streptococcus species (including S. pyogenes, S. agalactiae, S. bovis, S. pneumoniae, S. mutans, S. sanguis, S. equi, S. equinus, S. thermophilus, S. morbillorum, S. hansenii, S. pleomorphus, and S. parvulus ), and Veillonella. [00147] The Gram-positive and Gram-negative straight, curved, helical/vibrioid and branched rods include, but are not limited to, Acetobacter, Acinetobacter, Actinobacillus equuli, Aeromonas, Agrobacterium, Alcaligenes, Aquaspirillum, Arcanobacterium haemolyticum, Bacillus species (including B. cereus and B. anthracis), Bacteroides species (including B. fragilis), Bartonella, Bordetella species (including B. pertussis), Brochothrix, Brucella, Burkholderia cepacia, Calymmatobacterium granulomatis, Campylobacter species (including C. jejuni ), Capnocytophaga, Caulobacter, Chromobacterium violaceum, Citrobacter, Clostridium species (including C. perfringens, C. tetani and C. difficile ), Comamonas, Curtobacterium, Edwardsiella, Eikenella, Enterobacter, Erwinia, Erysipelothrix, Escherichia species (including E. coli), Flavobacterium species (including E. meninosepticum), Francisella species (including E. tularensis), Fusobacterium (including E. nucleatum), Gardnerella species (including G. vaginalis), Gluconobacter, Haemophilus species (including H. influenzae and H. ducreyi ), Hafnia, Helicobacter (including H. pylori ), Herpetosiphon, Klebsiella species (including K. pneumoniae ), Kluyvera, Lactobacillus, Legionella species (including E. pneumophila ), Leptotrichia, Listeria species (including E. monocytogenes ), Microbacterium, Morganella, Nitrobacter, Nitrosomonas, Pasteurella species (including P. multocida ), Pectinatus, Porphyromonas gingivalis, Proteus species (including E. mirabilis ), Providencia, Pseudomonas species (including P. aeruginosa, P. mallei, P. pseudomallei and R. solanacearum), Rahnella, Renibacterium salmoninarum, Salmonella, Serratia, Shigella, Spirillum, Streptobacillus species (including S. moniliformis), Vibrio species (including V. cholerae and V. vulnificus ), Wolinella, Xanthobacter, Xenorhabdus, Yersinia species (including Y. pestis and Y. enter ocoliticd), Zanthomonas and Zymomonas.

[00148] The clinical diseases or infections caused by Gram-positive and/or Gram negative bacteria, treatable with the present disclosure include biofilms, abscesses, bacteremia, contamination of peritoneal dialysis fluid, endocarditis, pneumonia, meningitis, osteomyelitis, cellulitis, pharyngitis, otitis media, sinusitis, scarlet fever, arthritis, urinary tract infection, laryngo tracheitis, erysipeloid, gas gangrene, tetanus, typhoid fever, acute gastroenteritis, bronchitis, epiglottitis, plague, sepsis, chancroid, wound and bum infection, cholera, glanders, periodontitis, genital infections, empyema, granuloma inguinale, Legionnaire's disease, paratyphoid, bacillary dysentery, brucellosis, diphtheria, pertussis, botulism, toxic shock syndrome, mastitis, rheumatic fever, cystic fibrosis, eye infections, plaque, and dental caries. Other uses include swine erysipelas, peritonitis, abortion, encephalitis, anthrax, nocardiosis, pericarditis, mycetoma, peptic ulcer, melioidosis, HaverhiU fever, tularemia, Moko disease, galls (e.g., crown, cane and leaf), hairy root, bacterial rot, bacterial blight, bacterial brown spot, bacterial wilt, bacterial fin rot, dropsy, columnaris disease, pasteurellosis, furunculosis, enteric redmouth disease, vibriosis offish, and fouling of medical devices.

[00149] More typically, the microorganism is one or more types of bacteria. The bacteria can be gram positive or gram negative. Some examples of genera of biofilm-causing bacteria include Staphylococcus, Cob forms {e.g., Citrobacter, Enterobacter, Escherichia, Hafnia, Klebsiella, Serratia and Yersinia), Lactic Acid Bacteria {e.g., Enterococcus, Streptococcus), Pseudomonas, and Aspergillus.

[00150] In some embodiments, the present disclosure is particularly directed to methods of detecting and/or treating biofilm infections caused by Staphylococcus or Pseudomonas bacteria. Some examples of particularly relevant species of Staphylococcus include Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus saprophyticus, Staphylococcus hominis, Staphylococcus warneri, Staphylococcus cohnii, Staphylococcus capitis, Staphylococcus camosis, Staphylococcus lugdunesis, Staphylococcus schleiferi, and Staphylococcus caprae.

[00151] The present disclosure also considers any antibiotic-resistant microorganism, particularly antibiotic -resistant bacteria. Of particular importance is the class of antibiotic-resistant Staphylococcus bacteria. Most notable of the antibiotic-resistant bacteria are the methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible Staphylococcus aureus (MSSA) types of bacteria.

[00152] Other species of bacteria that can be involved in biofilm infections include, for example, Escherichia coli, Yersinia pestis, Pseudomonas aeruginosa, Streptococcus mutans, Streptococcus sanguinis, Enterococcus faecalis, Streptococcus viridans, Klebsiella pneumoniae, Proteus mirabilis and Streptococcus veridans.

[00153] The pharmaceutical composition provided herein may be used for disrupting biofilms formed in chronic infections such as bacterial vaginosis, chronic nasal and sinus infections (sinusitis, chronic rhinosinusitis), oral and dental infections (periodontal diseases such as chronic periodontitis), cystic fibrosis, and chronic gangrene (by infection or ischemia). [00154] The present invention may be utilized to inhibit biofilms in or on urinary catheters and, further, to reduce or prevent bacterial colonization thereon. The compounds and compositions of the present disclosure also may be used to inhibit biofilms formed by E. coli that reside intracellularly in bladder cells, which resist conventional antibiotics and evade host immune systems. Not wishing to be bound by a particular theory, it is believed that by preventing or disrupting the attachment of E. coli to uroplakin or the proteins of the tight junctions of umbrella cells of the bladder, the compounds and compositions of the present invention may prevent, reduce, or control the re-occurrence of such urinary tract infections.

[00155] The compounds and compositions of the present disclosure also may be used to treat, i.e., prevent and/or reduce the risk of atherosclerosis and kidney stones. Again, not wishing to be bound by a particular theory, it is believed that bacterial colonization may cause atherosclerosis and the formation of kidney stones. For example, bacterial colonization has been identified in calcified human aneurysms, carotid plaques, femoral arterial plaques, and cardiac valves. Arterial calcification appears to resemble infectious lesion formation in models of atherosclerosis. Moreover, it is believed that a toxin produced by Cag-A positive Helicobacter pylori colonization of the stomach leads to tissue inflammation and lesions in arterial walls resulting in atherosclerosis. Accordingly, administering to a patient in need thereof one or more compounds of the present disclosure (or a composition containing one or more compounds of the present disclosure) may reduce the risk of, or treat atherosclerosis and kidney stones.

[00156] The compounds and compositions of the present disclosure may be used to treat cystic fibrosis. The principal organism found in the lungs of cystic fibrosis patients is Pseudomonas aeruginosa, existing within a biofilm. Thus, the compounds and compositions of the present disclosure may be used to prevent, inhibit or reduce the formation of biofilms in the lungs of such cystic fibrosis patients.

[00157] Diseased tissue, including certain tumors, are more susceptible to bacterial colonization. Based on this observation, Clostridia spores and attenuated Salmonella typhimurium have been used to deliver therapeutic proteins to tumors. These bacteria selectively colonize tumors versus normal tissue. Accordingly, further embodiments of the present disclosure include administering the compounds and compositions of the present disclosure to diseased tissues to reduce, treat or eradicate the biofilms within the diseased tissue, including tumors. Again, not wishing to be bound by a particular theory, it is believed that the eradication of biofilms and bacteria from such diseased tissue would enable the mammalian immune system, and/or other pharmaceutical compositions, to further treat the diseased tissue after bacterial colonization has been removed or reduced.

D. Disinfectant Compositions

[00158] The compositions of the present disclosure are useful in a variety of environments including industrial, clinical, the household, and personal care. The compositions of the present disclosure for industrial, pharmaceutical, household and personal care use may comprise at least one active ingredient, of which the antibiotic of the is an active ingredient acting synergistically with the at least one excipient against the target microbe.

[00159] Accordingly, the compositions of the present disclosure may be used to form contact-killing coatings or layers on a variety of substrates including personal care products (e.g., toothbrushes, contact lens cases and dental equipment), healthcare products, household products, food preparation surfaces and packaging, and laboratory and scientific equipment. Further, other substrates include medical devices such as catheters, urological devices, blood collection and transfer devices, tracheotomy devices, intraocular lenses, wound dressings, sutures, surgical staples, membranes, shunts, gloves, tissue patches, prosthetic devices (e.g., heart valves) and wound drainage tubes.

[00160] Bacterial and fungal biofilms develop on the various types of medical equipment which may be treated or prevented by the present compositions. This includes medical diagnostic devices, such as: stethoscopes, colposcopes, nasopharyngoscopes, angiography catheters, endoscopes, angioplasty balloon catheters; and various permanent, semi-permanent, and temporary indwelling devices, such as: contact lenses, intrauterine devices, dental implants, urinary tract prostheses and catheters, peritoneal dialysis catheters, indwelling catheters for hemodialysis and for chronic administration of chemotherapeutic agents (Hickman catheters), cardiac implants (pacemakers, prosthetic heart valves, ventricular assisting devices— VAD), synthetic vascular grafts and stents, prostheses, internal fixation devices, percutaneous sutures, tracheal and ventilator tubing, dispensing devices such as nebulizers, and cleaning devices such as sterilizers. The biofilms may form on various medical devices, including prosthetic heart valves, central venous catheters, urinary (Foley) catheters, contact lenses, intrauterine devices, and dental unit water lines. E. Dosage

[00161] The specific therapeutically effective dose level for any particular patient may depend upon a variety of factors, including the specific biofilm (and, preferably, taking into account the source of such biofilm) being treated or inhibited; the amount of existing biofilm to be treated, if any, within a given patient; the activity of the specific compound employed; the specific pharmacologic formulation employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or contemporaneously with the specific compound employed; and like factors well known in the medical arts. Furthermore, it may be appropriate to administer the required dose more than once in a twenty-four hour period, such as for example in two, three, four or more sub-doses at appropriate intervals throughout the day.

[00162] By way of example only, the total daily dose of one or more of the biofilm inhibitors disclosed herein may be provided to a patient in single or in divided doses, which may be in amounts from 0.01 to 50 mg/kg body weight or, more typically, from 0.1 to 25 mg/kg body weight. Single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. More preferably, treatment regimens according to the present invention may comprise administering to a patient about 10 mg to about 1000 mg of the biofilm inhibitor(s) disclosed herein, per day in single or multiple doses.

III. Examples

[00163] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Example 1 - Combination of Antibiotic and Excipient(s)

[00164] Screening of excipients in planktonic PA: Initially, the effect of 190 excipients on planktonic PA proliferation was evaluated. Those excipients that caused a statistically significant change or a change in more than 20% in PA proliferation compared to control are shown in FIG. 1. L-homoserine (-21%), D-alanine (-13%) and L-alanine (-10%) showed the highest reductions while salicylic acid (+19%) and sorbic acid (+18%) caused the largest increase in proliferation. In general, organic acids and amines caused an increase in planktonic PA proliferation while saccharides and amino acids caused a decrease.

[00165] To define the concentration of tobramycin that was used in the studies that included tobramycin in combination with the excipients, serially diluted tobramycin was tested between 0.5 and 256 pg/mL. When 1 pg/mL was used, the bacterial proliferation was strongly reduced (97%) compared to untreated biofilms (p<0.000l), therefore a lower concentration (0.5 pg/mL) was selected to facilitate the identification of any interaction between tobramycin and the excipients. This concentration did not significantly affect bacterial proliferation (p>0.05) (FIG. 2).

[00166] FIG. 2 shows the results of 7 combinations of excipients with tobramycin that caused a change in PA proliferation of 20% or more compared to the untreated control. L-homoserine (-37%) was the only excipient that maintained the same significant effect observed previously in the excipient alone studies. This reduction was also highly significant compared to the effect of tobramycin alone (p<0.00l).

[00167] It was observed that some of these excipients might act synergistically or antagonistically with tobramycin. In this context, synergism or antagonism is described when the effect of the combination of tobramycin and excipient on PA proliferation was significantly different from the effect observed with tobramycin alone, the excipient alone, and the control. From these excipients, N-acetyl-D-glucosaminitol (-20%) and sec-butylamine (- 18%) showed a potential synergistic effect with tobramycin, while lactulose (+17%) presented a potential antagonistic effect.

[00168] Screening of excipients on PA biofilms: The same excipients tested in planktonic bacteria were also tested on 24 h pre-formed PA biofilms. FIG. 3 shows the results of 60 excipients that caused a change of 20% or more in PA biofilms proliferation compared to control. In 18 of these excipients, the effect was statistically significant (p<0.05). In PA biofilms, unlike with planktonic growth, a trend was not observed between categories of excipients and the effect in bacterial proliferation. Overall, the excipients that reduced proliferation showed an average proliferation decrease of around -27%, whereas those that caused significant increase showed values up to +134%, as observed with acetoin. 2-deoxy adenosine (-32%), phenylethylamine (-27%), and chondroitin (-27%) caused the highest reductions.

[00169] L-proline (-22%) and turanose (-21%) were the only excipients that maintained the same proliferation trends in biofilms that were also observed in planktonic PA. Phenylethylamine also reduced PA proliferation in planktonic state, but this effect was observed only when it was combined with tobramycin. D-threonine (-25%), but not L- threonine (+23%), caused a significant reduction in proliferation compared to control. Tween 20 (-27%), tween 40 (-24%) and tween 80 (-27%) also reduced bacterial proliferation.

[00170] To evaluate the effect of the combination of excipients with tobramycin in biofilms, a subinhibitory concentration of tobramycin (2 mg/mL) was used which caused a statistically significant reduction in proliferation with respect to control after 24 h (-16%; p<0.05) (FIG. 4). This concentration allowed the quantification of the inhibitory effects of the combination of excipient with tobramycin compared to tobramycin alone. When 2 pg/mL tobramycin was added with the excipients in the screening studies, 5 excipient-tobramycin combinations showed significant changes compared to the effect of tobramycin alone. These five combinations caused significant increases in proliferation. In contrast, nucleosides, and most of the saccharides, combined with tobramycin caused reductions in the proliferation of biofilms compared to control after 24 h of treatment.

[00171] b-methyl-D-glucuronic acid (-20%), D,L-carnitine (+35%), 2-deoxy adenosine (-20%), L-proline (-32%) and most of the amino acids, also preserved the trends observed in planktonic bacteria when they were tested alone in biofilms. Hydroxy-L-proline (+121%), L-isoleucine (+116%), and L-malic acid (+105%) combined with TOB caused a significant increase in proliferation, which was also in agreement with the definition of the antagonistic effect defined previously.

[00172] Table 2 summarizes the“hits” obtained from the screening stage of this study. It includes those excipients that alone and in combination with tobramycin caused a reduction of more than 20% in PA proliferation or a statistically significant decrease (p<0.05). [00173] Table 2: Summary of excipients that resulted in either a statistically significant reduction in bacterial proliferation or a numeric reduction of more than 20% with respect to the untreated biofilm in screening studies (48 out of 190 tested). Table reports data as % change in proliferation with respect to untreated control.

The symbols (*) or (^#) indicate the selection criteria of excipients; which was either by statistical difference (*p<0.05) or numeric difference (^#more than 20% in change).

[00174] Effect of selected excipients on tobramycin activity against PA14 biofilms: From these screening studies described above, a total of 10 excipients (i.e.“hits”) were selected for further investigation: L-alanine, D-alanine, L-proline, L-homoserine (amino acids), D-melibiose, D-trehalose, N-acetyl-D-glucosaminitol (saccharides), glyoxylic acid, succinic acid, and monomethyl succinate (organic acids). These hit excipients were further tested in combination with tobramycin against a more virulent and bioluminescent strain of P. aeruginosa (PA14). The luminescence emitted from mutant PA14 bacteria treated with combinations of these excipients and TOB was monitored for 20 h as a measurement of bacterial proliferation with respect the proliferation at time zero.

[00175] D-serine and D-aspartic acid did not show significant reduction in proliferation of P. aeruginosa biofilms (strain PAOl) (Table 3).

[00176] Table 3: Summary of additional excipients tested.

The symbols (*) or (^#) indicate the selection criteria of excipients; which was either by statistical difference (*p<0.05) or numeric difference (^#more than 20% in change).

[00177] FIG. 5 shows bacterial proliferation measured by luminometry under different treatments of excipients with tobramycin. The controls without excipients at each tobramycin concentration are shown as filled squares. Untreated biofilms (No excipient and 0 pg/mL tobramycin) showed a short initial inhibition of proliferation, followed by a proliferative phase at around 10 h, to finally diminish in a second inhibitory phase to 38.1%. Similarly, when treated with 0.5 pg/mL of tobramycin alone (FIG. 5B), an initial decrease was followed by a proliferative phase. However, at 20 h proliferation was three-times higher than untreated biofilms with value of 122.5% (comparing FIG. 5A and B). In contrast, the incubation with 2 mg/mL tobramycin caused a continuous inhibition in proliferation to 16.4% (FIG. 5C) and down to 6.7% of proliferation with 64 mg/mL (FIG. 5D).

[00178] The effects of individual excipients are shown in FIG. 5A. Monomethyl succinate caused an extended proliferative phase with a significantly higher proliferation value after 20 h compared to untreated biofilms (175.5% vs. 38.1%; p<0.0l). The combination of this excipient with 2 mg/mL tobramycin (FIG. 5C) and 64 mg/mL tobramycin (FIG. 5D) also caused significantly higher proliferations compared to the effects of those concentrations of TOB alone (250.3% vs 16.4% p<0.000l, and 44.7% vs 6.7% p<0.000l, respectively)

[00179] L-alanine showed lower proliferation after 20 h in all combination with tobramycin compared to tobramycin alone (p>0.05) (FIG. 5B, C and D). The combination of D-alanine with 2 mg/mL of tobramycin (FIG. 5C) also resulted in lower proliferation after 20 h compared to tobramycin without excipients (p>0.05). N-acetyl-D-glucosaminitol also showed bacterial inhibition when it was combined with tobramycin. The combination with 0.5 mg/mL of tobramycin (FIG. 5B) resulted in the largest inhibition observed after 20 h compared to tobramycin (9.0% vs. 122.5%; p>0.05).

[00180] L-proline showed an effect that was dependent on tobramycin concentration. L-proline alone (191.8% vs. 38.1%; p<0.0l) (FIG. 5A) or combined with 0.5 mg/mL tobramycin (695.2% vs. 122.5%; p<0.000l) (FIG. 5B) resulted in significantly higher proliferation values after 20 h, compared to the untreated biofilms and 0.5 mg/mL tobramycin respectively. However, in combination with 64 mg/mL tobramycin (Fig. 5D), it showed the highest inhibition observed after 20h in this luminometric study (2.2% vs. 6.7%, p>0.05).

[00181] Glyoxylic acid alone caused a sustained inhibition of bacterial proliferation (8.9% vs. 38.1%; p>0.05) (FIG. 5A). L-homoserine, D-melibiose, D-trehalose, and succinic acid did not affect tobramycin activity significantly. L-alanine, L-proline, and N- acetyl-D-glucosaminitol with 64 mg/mL tobramycin (FIG. 5D) showed sustained reduction from time zero of the treatment and the lowest proliferation values after 20 h.

[00182] Survival ofPAM biofilms: To evaluate the effect of the combinations of tobramycin with excipients on PA 14 biofilms viability, the colony forming units (CFU’s) were quantified after the kinetic study. PA 14 biofilms were rinsed to remove the planktonic bacteria and then the biofilms were sonicated to disrupt the bacteria within the biofilms. FIG. 6 shows the results of PA14 viability expressed as log changes in the number of surviving CFU/mL with respect to the treatment with 2 pg/mL and 64 pg/mL of tobramycin alone. Untreated biofilms viability represents a zero-log change in survival. The treatment with 2 pg/mL tobramycin caused a minimal reduction in survival (0.21 log; p>0.05) while 64 pg/mL tobramycin reduced significantly the survival with respect to untreated biofilms (1.49 log; p<0.000l).

[00183] L-alanine and succinic acid reduced the number of surviving bacteria in all conditions. The reductions caused by L-alanine were highly significant when used alone (- 0.95 log) and in combination with 2 pg/mL of tobramycin (-0.96 log). Similarly, succinic acid alone (-0.59 log) and with 2 pg/mL tobramycin alone (-1.70 log) caused statistically significant reductions. On the other hand, D-trehalose and glyoxylic acid did not improve the activity of tobramycin against PA14 biofilms. In fact, these treatments resulted in significantly higher survival. Conflicting results were obtained with L-proline. L-proline alone caused a significant increase (+0.34 log), while when it was combined with 64 pg/mL of tobramycin it caused the highest reduction observed compared to tobramycin alone (-2.27 log). The effects of L- homoserine (+0.20 log) and monomethyl succinate (-0.69 log) were opposite and significant only when used alone. D-melibiose combined with 2 pg/mL of tobramycin (+0.70 log) resulted is more survival CFU compare to tobramycin alone.

[00184] L-alanine and succinic acid reduced the number of surviving bacteria in all conditions. The reductions caused by L-alanine were highly significant when used alone (- 0.95 log) and in combination with 2 pg/mL of tobramycin (-0.96 log). Similarly, succinic acid alone (-0.59 log) and with 2 pg/mL tobramycin alone (-1.70 log) caused statistically significant reductions. On the other hand, D-trehalose and glyoxylic acid did not improve the activity of tobramycin against PA14 biofilms. In fact, these treatments resulted in significantly higher survival. Conflicting results were obtained with L-proline. L-proline alone caused a significant increase (+0.34 log), while when it was combined with 64 pg/mL of tobramycin it caused the highest reduction observed compared to tobramycin alone (-2.27 log). The effects of L- homoserine (+0.20 log) and monomethyl succinate (-0.69 log) were opposite and significant only when used alone. D-melibiose combined with 2 pg/mL of tobramycin (+0.70 log) resulted is more survival CFU compare to tobramycin alone. [00185] Treatment of PA infections in CF patients involves a long-term administration of high doses of antibiotics (Mogayzel et al, 2014). An excipient that can prevent these exacerbations of PA proliferation in periods with transient subinhibitory concentrations of tobramycin can be useful to improve the therapy by broadening tobramycin therapeutic effect. Therefore, adjuvants like N-acetyl-D-glucosaminitol or L-alanine could improve tobramycin activity in both ranges of concentrations, improving current therapy.

[00186] Separately, lactulose, L-isoleucine, hydroxy-L-proline, and L-malic acid did not affect the proliferation of PA and could be described as inactive materials in this screening (FIG. 4). But each combination of these potential excipients with tobramycin, showed a significantly higher proliferation compared to the untreated controls. Although the mechanisms behind these antagonistic results are unknown, these outcomes highlight the importance of the evaluation of novel treatments with tobramycin as the final effect in biofilms could not be predicted by the individual effects of tobramycin and excipients alone. The aim of the study was to identify potential excipients with activity against biofilms of the opportunistic pathogen Pseudomonas aeruginosa (PA). 48 excipients were identified that presented activity against PA proliferation alone or when combined with tobramycin. L- proline, L-alanine, N-acetyl-D-glucosaminitol, and succinic acid, among other selected excipients showed promising results during the screening phase of the study PA. From these 10“hit” excipients tested on PA14, L-alanine and succinic acid significantly reduced the number of surviving bacterial from PA14 biofilms and represent the main candidates for further studies.

Example 2 - Materials and Methods

[00187] Microorganisms: Pseudomonas aeruginosa PAOl (ATCC^® 15692™) and a bioluminescent PA 14 (UCBPP-PA14 was kindly donated by Dr. Marvin Whiteley from the University of Texas at Austin, US) were maintained in frozen stocks at -80°C. PAOl was subcultured overnight in blood agar (Sigma Aldrich, St. Louis MO, USA) before being grown planktonic ally in brain heart infusion (BHI) medium (Sigma Aldrich, St. Louis MO, USA) in an orbital shaker at 180 rpm and 37°C for 18 h (MaxQ Mini 4450 Shaker, Thermo Fisher Scientific, Marietta, OH, US). PA14 was inoculated from frozen stocks into BHI containing 100 pg/mL of carbenicillin (BioVision, Inc. Milpitas, CA, US) to maintain the luminescent plasmid. [00188] Screening of excipients in planktonic PAOl: A bacterial suspension from an overnight culture was rinsed twice with PBS (Sigma Aldrich, St. Louis MO, USA) and harvested by centrifugation. Then, the obtained pellet was dispersed in M9 minimal medium supplemented with 10 mg/mL peptone (Sigma Aldrich, St. Louis MO, USA) at a final concentration of 5 x 10⁵ CFU/mL measured by spectrophotometry (Infinite M200 microplate reader, TECAN, Morrisville NC, US). Then, 100 pL of the adjusted bacterial suspension were inoculated into each well of two phenotypic microarray plates (PM1 and PM2A; Biolog INC. Hayward CA, US) and incubated in an orbital shaker at 75 rpm and 37°C.

[00189] After 24 hours, the absorbance of the bacterial suspension was measured at 600 nm. We expressed the results as a change in proliferation with respect to the untreated control. The same procedure was repeated with the addition of 0.5 pg/mL of tobramycin sulfate (TOB; Letco Medical. Decatur AL, US).

[00190] Screening of excipients in PAOl biofilms: For biofilm formation, an overnight bacterial suspension was rinsed twice with PBS and harvested by centrifugation as described above. The pellet was dispersed in M9 supplemented medium to a final bacterial concentration of 3 x 10⁷ CFU/mL. This suspension was added to the wells of MB EC™ biofilm inoculators (Innovotech Inc. AB Canada). The MB EC™ plates were incubated at 75 rpm and 37 °C for 24 h. After incubation, the MB EC™ lids containing biofilms were transferred to a 96-well plate prefilled with PBS for 30 seconds to wash the unadhered bacteria. PM1 and PM2A plates were filled with 100 pL of fresh supplemented M9 media with and without 2 pg/mL of tobramycin. The MBEC™ lids were then transferred from the rinsing plate to the PM1 and PM2 plates and incubated for 24 h.

[00191] An XTT cell proliferation assay was performed to evaluate the biofilm viability after the treatment using a solution in PBS of XTT sodium salt and menadione (Sigma Aldrich, St. Louis MO, USA). Briefly, a solution of menadione (7 mg/mL in acetone) was freshly prepared and then diluted in PBS. Before the experiment, we prepared a XTT working solution by combining PBS, XTT stock solution (1 mg/mL in PBS) and menadione solution (0.07 mg/mL in PBS) in the proportion 79:20:1. This solution was used to fill the wells of a new 96- well plate. The MBEC™ lid with biofilms was rinsed in a plate with PBS and transferred to the plate with the XTT working solution. The absorbance of the XTT reagent was measured at 492 nm after 5 h of incubation in the dark at 37°C. [00192] Effect of excipients on tobramycin activity against PA14 biofilms: For the screening of the excipients, P. aeruginosa PAOl, a standard and low-virulence strain, was used. The strain PA14 was selected for post-screening studies due to its higher virulence (Mikkelsen et al, 2011) and different EPS composition (Yang et al, 2011) which provides enhanced resistant to aminoglycoside antibiotics (Colvin et al, 2011).

[00193] PA14 biofilms were formed in the base of black 96-well plates with clear and flat bottoms (Corning Inc. Durham NC, US). An adjusted bacterial suspension (3 x 10⁷ CFU/mL) was used to fill the black plates. Then, the plates were incubated at 37 °C for biofilm formation. After 24 h, the media with planktonic bacteria were discarded, and the biofilms were rinsed with PBS before the addition of the treatment solutions. The plates were covered with a sterile breathable sealing film (Sigma Aldrich, St. Louis MO, USA) and immediately incubated in a plate reader at 37 °C for 20 h. Bacterial luminescence was recorded every 20 min as a measurement of bacterial proliferation (FlexStation 3 Reader, Molecular Devices LLC. Sunnyvale, CA, US).

[00194] Log reduction ofPA14 biofilms: After incubation and luminometry, the supernatant medium was aspirated and discarded, and the treated biofilms were rinsed twice with PBS to remove unattached bacteria. Then, the wells were filled with PBS and sonicated for 30 min to disperse the attached bacteria. Dispersed biofilms were serially diluted and spotted on blood agar plates using the drop plate method (Herigstad et al, 2001). Colony forming units (CFU) were quantified after incubation, and the results were reported as changes in log (CFU/mL) with respect to the untreated control.

[00195] The statistical analysis was performed with JMP® 10.0.0 with ANOVA using Tukey-Kramer HSD test to compare differences between groups. A p- value of <0.05 was considered statistically significant.

Example 3 - Validation in Pseudomonas aeruginosa Biofilm Model

[00196] The combination of tobramycin with different excipients was tested in the Pseudomonas aeruginosa (PA) biofilm model. The proliferation of the PA strain PAOl was measured in response to treatment with tobramycin or PEGylated-tobramycin 137 mM in combination with different excipients for 12 hours (FIG. 7). The bacterial proliferation was found to be lowest when treated with tobramycin, or PEGylated tobramycin, in combination with L-proline and succinic acid. [00197] Next, a quantification of surviving colony forming units was performed by treating the PA biofilms with tobramycin, or PEGylated tobramycin, in combination with different excipients for 12 hours (FIG. 8) and 24 hours (FIG. 10). It was found that there was reduction in surviving PA biofilm colonies when treated with tobramycin, or PEGylated tobramycin, in combination with the different excipients including succinic acid, L-proline, N- acetyl-D-glucosaminitol (NADG), and L-alanine.

[00198] Further testing was performed using the PA biofilm model treated with tobramycin in combination with different excipients for 24 hours (FIG. 9). It was observed that all of the excipient combinations tested resulted in a decrease in bacterial proliferation with the combination of tobramycin, L-alanine, and NADG having the highest decrease.

[00199] In addition, bacterial proliferation was measured during the treatment with colistin or PEGylated colistin alone or combined with different excipients for 24 hours (FIGS. 11, 12). It was found that colistin in combination with NADG and succinic acid resulted in a high reduction in PA biofilm proliferation.

[00200] A further assay tested on PA01 biofilms showed that the combination of L-alanine, L-proline, N-acetyl-D-glucosaminitol and succinic acid with tobramycin showed a significantly higher anti-biofilm activity as compared to tobramycin alone (FIG. 13).

[00201] Thus, combinations of an antibiotic and excipients such as NADG, succinic acid, and L-alanine can be used for the treatment of biofilms, such as PA biofilms.

Example 4 - Oral Biofilms

[00202] To test the activity of the present compositions against oral biofilms, Streptococcus gordonii DL1.1, Streptococcus mutans UA159, and Candida albicans SC5314 were grown overnight in suspension and adjusted to the concentrations of lxlO⁷ CFU/mL, lxlO⁷ CFU/mL, and lxlO⁶ CFU/mL, respectively. S. gordonii and S. mutans were incubated statically at 37°C and 5% CO₂. C. albicans was incubated at 160 rpm and 32°C in an ambient atmosphere. For biofilm formation, 33.3 pL of each adjusted suspension was added in the ordered listed into the wells of a 96 well microtiter plate and incubated statically at 37°C and 5 % CO₂ for 24 hours.

[00203] After incubation, spent media was removed and 100 pL of the experimental treatment was added for 24 hours. The susceptibility of the biofilms to the treatments was evaluated using more than one viability assay: A metabolic assay by PrestoBlue, determination of viable counts (colony forming units -CFU-), and final biomass by crystal violet staining. Biofilm metabolism was primarily estimated by adding 10 pL of PrestoBlue^® Cell Viability Reagent (Invitrogen™, Carlsbad, CA, US.) and 90 pL of fresh growth media to each well. Plates were incubated in the dark at 37°C for 20 minutes and fluorescence intensity (Ex/Em: 560/590 nm) was measured using a microplate reader (Infinite M200 microplate reader, Tecan, Tecan Sp, Inc., Mannedorf, Switzerland).

[00204] In further studies, Streptococcus gordonii DL1.1, Streptococcus mutans UA159, and Candida albicans SC5314 were grown overnight in suspension and adjusted to the concentrations of lxlO⁷ CFU/mL, lxlO⁷ CFU/mL, and lxlO⁶ CFU/mL, respectively. S. gordonii and S. mutans were incubated statically at 37°C and 5% C0₂. C. albicans was incubated at 160 rpm and 32°C in an ambient atmosphere.

[00205] For biofilm formation, 33.3 pL of each adjusted suspension was added in the ordered listed into the wells of a 96 well microtiter plate and incubated statically at 37°C and 5% CO₂ for 24 hours. After incubation, spent media was removed and 100 pL of the experimental treatment was added for 30 minutes. The susceptibility of the biofilms to the treatment was assessed after the 30-minute treatment time using the PrestoBlue metabolic activity assay (Immediate Post Dose). Following this assay, the supernatant was removed and fresh media was added to the wells. The plate was incubated at the previously defined conditions for 8 more hours. The susceptibility of the biofilms to the treatments was evaluated after the 8 hour-post treatment incubation (8 hours Post-Dose) using more than one viability assay: A metabolic assay by PrestoBlue and final biomass by crystal violet staining.

[00206] To test the combinations of chlorhexidine, excipients, and iron oxide nanoparticles, an 8-hour dose treatment was performed on preformed biofilms. For biofilm formation, 33.3 pL of each adjusted suspension was added in the ordered listed into the wells of a 96 well microtiter plate and incubated statically at 37°C and 5% CO₂ for 24 hours. After incubation, spent media was removed and 100 pL of the experimental treatment was added for 8 hours. The susceptibility of the biofilms to the treatments was evaluated using more than one viability assay: A metabolic assay by PrestoBlue and final biomass by crystal violet staining.

[00207] To test the combinations of chlorhexidine, excipients, and iron oxide nanoparticles on biofilm prevention, an 8-hour dose was performed on planktonic cells. To test biofilm prevention, 33.3 pL of each adjusted suspension was added in the ordered listed into the wells of a 96 well microtiter plate and 100 pL of the experimental treatment was added for 8 hours. The plates were incubated statically at 37°C and 5% C0₂ for 8 hours. After incubation, the optical density of the wells was measured before the spent media and planktonic cells were removed and the formed biofilms were evaluated using more than one viability assay: A metabolic assay by PrestoBlue and final biomass by crystal violet staining.

[00208] As shown in Table 4, the combination of chlorhexidine with some of the different excipients enhances the antimicrobial activity.

[00209] Table 4: Summary of data for chlorhexidine formulations.

[00210] The studies of chlorhexidine combined with excipients L-proline, L- alanine N-actyl-D-glucosaminitol, succinic acid, benzoic acid, citric acid, edetic acid, alginic acid, carrageenan, and additional suspending agents are shown in FIGS. 16C-F. Iron oxide nanoparticles were also combined with excipients and excipients + chlorhexidine as shown in FIGS. 17A-F. Combinations of 1 m^hiΐ. chlorhexidine and 20 mM of (1) L-proline (p<0.l), (2) L-alaninine + L-proline + N-acetyl-D-glucosaminitol (NADG) (p<0.05), (3) L-proline + NADG (p<0.05), and (4) L-proline + Succinic acid (p<0.l) reduced oral biofilm viability when treated for 24 hours (FIG. 16C).

[00211] Further tests of excipients and others alone and in combination with chlorhexidine demonstrated limited effect immediately after a 30-minute dose (FIG. 16E). However, when biofilms were provided with fresh media after the 30 minute dose time and allowed to grow an additional 8 hours, reductions in biofilm viability were seen for combinations of 1 mg/mL chlorhexidine and (1) L-proline + Succinic acid, (2) L-proline + Succinic acid + EDTA, (3) L-proline + Succinic acid + EDTA + Benzoic acid, (4) L-proline + Succinic acid + EDTA + Benzoic acid + Alginic acid, (5) L-proline + Succinic acid + EDTA + Benzoic acid + Carrageenan compared to the untreated control and 1 mg/mL chlorhexidine only control (FIG. 16F).

[00212] The addition of iron oxide nanoparticles with chlorhexidine and these combinations of excipients were studied both immediately after a 30-minute dose (FIG. 17 A, B) and after 8 hours of subsequent growth in fresh media (FIG. 17C, D). Negatively charged (citric acid coated) iron oxide nanoparticles in combination with 1 mg/mL chlorhexidine and excipients showed better reduction in the oral biofilm immediately after the 30-minute dose compared with positively charged iron oxide nanoparticles. While positively charged (PEG/amine coated) iron oxide nanoparticles, had better reduction in the oral biofilm immediately after the 30-minute dose when in combination with 100 mg/mL chlorhexidine and excipients.

[00213] For biofilms allowed to grow an additional 8 hours in fresh media after treatment, combinations of 1 mg/mL chlorhexidine and excipients with both positively and negatively charged nanoparticles were capable of reducing oral biofilm viability. Table 4 summarizes additional studies that have been conducted for combinations of \\xglmL chlorhexidine, excipients, and iron oxide nanoparticles against oral biofilms when dosed for 2.5 minutes, 2.5 minutes + 8hrs growth in fresh media, and 8 hours, as well as, against planktonic cultures of oral bacteria for 8 hours. Numerous combinations demonstrated effectiveness for at least two of the biofilm forms tested compared to an untreated control.

* * * [00214] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

Bjamsholt et al, Pediatr Pulmonol. 2009;44(6):547-558.

Chiang et al, Antimicrob Agents Chemother. 20l3;57(5):2352-236l.

Colvin et al, PLoS pathogens. 20ll;7(l):el00l264.

Du et al., Mol Pharm. 20l5;l2(5): 1544-1553.

Fick et al, Chest. 1989;96(1): 158-164.

Herigstad et al, Journal of Microbiological Methods. 2001;44(2):121-129.

Hoiby et al, Int J Antimicrob Agents. 20l0;35(4):322-332.

Micek et al, Infection Control & Hospital Epidemiology. 20l5;36(l0): 1190-1197.

Mikkelsen et al, Plos One. 20l l;6(l2):e29l l3.

Mogayzel et al, Ann Am Thorac Soc. 2014;11(10):1640-1650.

Murphy et al, Pediatr Pulmonol. 2004;38(4):3l4-320.

Nichols et al, Antimicrob Agents Ch. 1988;32(4):518-523.

Percival et al, Wound repair and regeneration. 2011; 19(1): 1-9.

Remington: The Science and Practice of Pharmacy, 2l^st Ed. Lippincott Williams and

Wilkins, 2005.

U.S. Patent Publication No. 20170143842

Weinstein et al, Clinical Infectious Diseases. 2005;4l(6):848-854.

Walters et al, Antimicrob Agents Ch. 2003;47(l):3l7-323.

Yang et al, Environmental microbiology. 2011;13(7): 1705-1717.

Claims

CLAIMS What is Claimed is:

1. A pharmaceutical composition comprising an antibiotic and at least one excipient selected from the group consisting of D-alanine, L-alanine, L-homoserine, L-proline, D-threonine, adenosine, 2-deoxy adenosine, inosine, citric acid, glycolic acid, glyoxylic acid, succinic acid, monomethyl succinate, quinic acid, d-amino valeric acid, chondroitin, D-cellobiose, D-fructose, D-fructose-6-phosphate, D-galactose, b-methyl- D-galactoside, a-methyl-D-glucoside, D-glucose-l -phosphate, D-gluconic acid, b- methyl-D-glucuronic acid, N-acetyl-D-glucosaminitol, D-mannose, D-melezitose, D- melibiose, D-trehalose, inulin, L-arabitol, maltose, mannan, melibionic acid, m- inositol, palatinose, stachyose, sucrose, turanose, xylitol, phenylethylamine, propylene glycol, sec-butylamine, tween 20, tween 40, and tween 80.

2. The composition of claim 1, wherein the composition comprises 2, 3, 4, or 5 excipients.

3. The composition of claim 1 or 2, wherein the at least one excipient is selected from the group consisting of L-alanine, L-homoserine, L-proline, D-threonine, adenosine, 2- deoxy adenosine, inosine, citric acid, glycolic acid, glyoxylic acid, succinic acid, monomethyl succinate, quinic acid, d-amino valeric acid, chondroitin, D-cellobiose, D- fructose, D-fructose-6-phosphate, D-galactose, b-methyl-D-galactoside, a-methyl-D- glucoside, D-glucose-l -phosphate, D-gluconic acid, b-methyl-D-glucuronic acid, N- acetyl-D-glucosaminitol, D-mannose, D-melezitose, D-melibiose, D-trehalose, inulin, L-arabitol, maltose, mannan, melibionic acid, m-inositol, palatinose, stachyose, sucrose, turanose, xylitol, phenylethylamine, propylene glycol, sec-butylamine, tween 20, tween 40, and tween 80.

4. The composition of claim 1 or 2, wherein the at least one excipient is selected from the group consisting of L-alanine, L-homoserine, L-proline, adenosine, 2-deoxy adenosine, inosine, citric acid, glycolic acid, glyoxylic acid, succinic acid, monomethyl succinate, quinic acid, d-amino valeric acid, chondroitin, D-cellobiose, D-fructose, D-fructose-6- phosphate, D-galactose, b-methyl-D-galactoside, a-methyl-D-glucoside, D-glucose-l- phosphate, D-gluconic acid, b-methyl-D-glucuronic acid, N-acetyl-D-glucosaminitol, D -mannose, D-melezitose, D-melibiose, D-trehalose, inulin, L-arabitol, maltose, mannan, melibionic acid, m-inositol, palatinose, stachyose, sucrose, turanose, xylitol, phenylethylamine, propylene glycol, sec-butylamine, tween 20, tween 40, and tween 80.

5. The composition of claim 1 or 2, wherein the at least one excipient is selected from the group consisting of adenosine, 2-deoxy adenosine, inosine, citric acid, glycolic acid, glyoxylic acid, succinic acid, monomethyl succinate, quinic acid, d-amino valeric acid, chondroitin, D-cellobiose, D-fructose, D-fructose-6-phosphate, D-galactose, b-methyl- D-galactoside, a-methyl-D-glucoside, D-glucose-l -phosphate, D-gluconic acid, b- methyl-D-glucuronic acid, N-acetyl-D-glucosaminitol, D-mannose, D-melezitose, D- melibiose, D-trehalose, inulin, L-arabitol, maltose, mannan, melibionic acid, m- inositol, palatinose, stachyose, sucrose, turanose, xylitol, phenylethylamine, propylene glycol, sec-butylamine, tween 20, tween 40, and tween 80.

6. The composition of claim 1 or 2, wherein the at least one excipient is selected from the group consisting of adenosine, 2-deoxy adenosine, inosine, glycolic acid, glyoxylic acid, monomethyl succinate, quinic acid, d-amino valeric acid, chondroitin, D- cellobiose, D-fructose, D-fructose-6-phosphate, D-galactose, b-methyl-D-galactoside, a-methyl-D-glucoside, D-glucose-l -phosphate, D-gluconic acid, b-methyl-D- glucuronic acid, N-acetyl-D-glucosaminitol, D-mannose, D-melezitose, D-melibiose,

D-trehalose, inulin, L-arabitol, maltose, mannan, melibionic acid, m-inositol, palatinose, stachyose, sucrose, turanose, xylitol, phenylethylamine, propylene glycol, sec-butylamine, tween 20, tween 40, and tween 80.

7. The composition of claim 1 or 2, wherein the at least one excipient is selected from the group consisting of N-acetyl-D-glucosaminitol, L-proline, L-alanine, 2-deoxy- adenosine, chondroitin, and D-gluconic acid.

8. The composition of claim 1 or 2, wherein the at least one excipient is selected from the group consisting of N-acetyl-D-glucosaminitol, L-proline, L-alanine, and succinic acid.

9. The composition of claim 2, wherein the excipients are N-acetyl-D-glucosaminitol, L- proline, L-alanine, and succinic acid.

10. The composition of claim 2, wherein the excipients are N-acetyl-D-glucosaminitol, L- proline, and L-alanine.

11. The composition of claim 2, wherein the excipients are L-alanine and N-acetyl-D- glucosaminitol.

12. The composition of claim 2, wherein the excipients are L-proline and N-acetyl-D- glucosaminitol.

13. The composition of claim 2, wherein the excipients are succinic acid and N-acetyl-D- glucosaminitol.

The composition of claim 2, wherein the excipients are L-alanine and L-proline.

14. The composition of any of claims 1-13, wherein the excipient(s) is at a concentration of 5 mM.

15. The composition of any of claims 1-13, wherein the excipient(s) is at a concentration of 10 mM.

16. The composition of any of claims 1-17, wherein the excipient(s) is at a concentration of 20 mM.

17. The composition of any of claims 1-13, wherein the composition is further defined as an anti-biofilm composition.

18. The composition of claim 17, wherein the biofilm is an oral biofilm.

19. The composition of claim 18, wherein the oral biofilm is an oral caries biofilm.

20. The composition of any of claims 1-19, wherein the antibiotic is an aminoglycoside, a polymyxin, a monobactam, or a fluoroquinolone.

21. The composition of claim 20, wherein the aminoglycoside is tobramycin, streptomycin, kanamycin, gentamicin, or neomycin.

22. The composition of claim 20, wherein the aminoglycoside is tobramycin sulfate.

23. The composition of claim 20, wherein the polymyxin is colistin sulfate.

24. The composition of claim 20, wherein the monobactam is aztreonam, nocardicin A, or tab toxin.

25. The composition of claim 20, wherein the monobactam is aztreonam.

26. The composition of claim 20, wherein the fluoroquinolone is ciprofloxacin, levofloxacin, or trovafloxacin.

27. The composition of claim 20, wherein the fluoroquinolone is ciprofloxacin.

28. The composition of any of claims 1-19, wherein the antibiotic is a macrolide.

29. The composition of claim 28, wherein the macrolide is erythromycin, clarithromycin or azithromycin.

30. The composition of claim 28, wherein the macrolide is erythromycin.

31. The composition of claim 30, wherein the erythromycin is at a concentration of 0.7 to

800 pg/mL.

32. The composition of any of claims 1-31, wherein the composition further comprises one or more components selected from the group consisting of benzoic acid, potassium sorbate, alginic acid, carrageenan, citric acid, edetic acid, coconut oil, EDTA, and xylitol.

33. The composition of claim 32, wherein the composition comprises L-proline, succinic acid and EDTA.

34. The composition of claim 33, wherein the composition further comprises benzoic acid.

35. The composition of claim 34, wherein the composition further comprises alginic acid.

36. The composition of claim 34, wherein the composition further comprises carrageenan.

37. The composition of any of claims 1-36, wherein the antibiotic comprises a modification.

38. The composition of claim 35, wherein the modification is PEGylation.

39. The composition of claim 35, wherein the modification is conjugation to a polymer or peptide.

40. The composition of claim 39, wherein the polymer or peptide is hydrophilic and/or has a neutral charge.

41. The composition of claim 35, wherein the antibiotic is PEGylated- tobramycin, PEGylated-colistin, or PEGylated-aztreonam.

42. The composition of claim 1, wherein the composition comprises 2, 3, or 4 excipients.

43. The composition of claim 42, wherein the 2, 3, or 4 excipients are selected from the group consisting of D-alanine, L-alanine, L-homoserine, L-proline, D-threonine, adenosine, 2-deoxy adenosine, inosine, citric acid, glycolic acid, glyoxylic acid, succinic acid, monomethyl succinate, quinic acid, d-amino valeric acid, chondroitin, D- cellobiose, D-fructose, D-fructose-6-phosphate, D-galactose, b-methyl-D-galactoside, a-methyl-D-glucoside, D-glucose-l -phosphate, D-gluconic acid, b-methyl-D- glucuronic acid, N-acetyl-D-glucosaminitol, D-mannose, D-melezitose, D-melibiose,

D-trehalose, inulin, L-arabitol, maltose, mannan, melibionic acid, m-inositol, palatinose, stachyose, sucrose, turanose, xylitol, phenylethylamine, propylene glycol, sec-butylamine, tween 20, tween 40, and tween 80.

44. The composition of any of claims 1-43, wherein the antibiotic comprises 0.1-25% of the composition.

45. The composition of any of claims 1-43, wherein the antibiotic comprises 0.1-6% of the composition.

46. The composition of any of claims 1-43, wherein the antibiotic comprises 0.1-0.5% of the composition.

47. The composition of claim 42, wherein the 1, 2, 3, or 4 excipients comprise 1-50% of the composition.

48. The composition of claim 42, wherein the 1, 2, 3, or 4 excipients comprise 2-30% of the composition.

49. The composition of claim 42, wherein the 1, 2, 3, or 4 excipients comprise 5-10% of the composition.

50. The composition of any of claims 1-49, wherein the composition comprises a formulation of Table 1.

51. The composition of any of claims 1-50, wherein the composition does not comprise a carboxylic acid or sugar.

52. The composition of any of claims 1-51, wherein the composition is free of or essentially free of mannitol, fructose, and pyruvate.

53. The composition of any of claims 1-52, wherein the composition is free of or essentially free of D-methionine, D-tryptophan, D-leucine, D-tyrosine, D-phenylalanine, and D- proline.

54. The composition of any of claims 1-53, wherein the composition is free of or essentially free of D-proline.

55. The composition of any of claims 1-54, wherein the biofilm is associated with Pseudomonas aeruginosa, Staphylococcus aureus, Helicobacter pylori, Burkholderia cepacia complex, Haemophilus influenzae, Escherichia coli, Streptococcus pneumoniae, Enterococcus faecalis, Pseudomonas fluorescens, Staphylococcus epidermidis or Acinetobacter baumannii.

56. The composition of any of claims 1-54, wherein the biofilm is associated with Pseudomonas aeruginosa.

57. The composition of any of claims 18-54, wherein the oral biofilm is associated with dental caries.

58. The composition of claim 57, wherein the dental caries is associated with Streptococcus mutans, Streptococcus gordonii, Candida albicans, Actinomyces odontolyticus, Actinomyces naeslundii, Streptococcus sobrinus, Lactobacillus spp, or Veillonella spp.

59. The composition of any of claims 18-54, wherein the oral biofilm is associated with gingivitis or periodontal disease.

60. The composition of claim 59, wherein the gingivitis or periodontal disease is associated with Streptococcus gordonii, Fusobacterium nulceatum, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Aggregatibacter actinomycetemcomitans, or Actinomyces naeslundii.

61. The composition of any of claims 1-56, wherein the biofilm is associated with an ocular infection, cystic fibrosis infection, wound infection, otic infection, oral caries, or rhinosinusitis infection.

62. The composition of any of claims 1-61, wherein the composition is formulated to be administered orally, topically, rectally, percutaneously, by parenteral injection, intranasally or by inhalation.

63. The composition of any of claims 1-62, wherein the composition is a topical preparation, otic preparation, or ophthalmic preparation.

64. The composition of claim 63, wherein the ophthalmic preparation is further defined as an ophthalmic solution or ophthalmic suspension.

65. A method for treating a biofilm infection in a subject comprising administering an effective amount of the pharmaceutical composition of any of claims 1-64.

66. The method of claim 65, wherein the at least excipient is selected from the group consisting of N-acetyl-D-glucosaminitol, L-proline, L-alanine, 2-deoxy-adenosine, chondroitin, and D-gluconic acid.

67. The method of claim 65, wherein the antibiotic is an aminoglycoside, a polymyxin, a monobactam, or a fluoroquinolone.

68. The method of claim 67, wherein the aminoglycoside is tobramycin, streptomycin, kanamycin, gentamicin, or neomycin.

69. The method of claim 67, wherein the aminoglycoside is tobramycin sulfate.

70. The method of claim 67, wherein the polymyxin is colistin sulfate.

71. The method of claim 67, wherein the monobactam is aztreonam, nocardicin A, or tab toxin.

72. The method of claim 67, wherein the monobactam is aztreonam.

73. The method of claim 67, wherein the fluoroquinolone is ciprofloxacin, levofloxaxin, or trovafloxacin.

74. The method of claim 67, wherein the fluoroquinolone is ciprofloxacin.

75. The method of claim 65, wherein the biofilm infection is further defined as an oral biofilm infection.

76. The method of claim 75, wherein the antibiotic is erythromycin.

77. The method of claim 65, wherein the antibiotic is PEGylated.

78. The method of claim 65, wherein the biofilm infection is bacterial and/or fungal.

79. The method of claim 65, wherein the biofilm infection is bacterial.

80. The method of claim 65, wherein the biofilm infection is a nosocomial infection.

81. The method of claim 65, wherein the infection is in a bloodstream or surgical site of a subject.

82. The method of claim 65, wherein the infection is a lung infection, wound, otitis media, urinary tract infection, or pneumonia.

83. The method of claim 65, wherein the infection is chronic or recurrent.

84. The method of claim 65, wherein the biofilm infection is a Staphylococcus infection or a Pseudomonas infection.

85. The method of claim 65, wherein the biofilm infection is associated with Pseudomonas aeruginosa, Staphylococcus aureus, Helicobacter pylori, Burkholderia cepacia complex, Haemophilus influenzae, Escherichia coli, Streptococcus pneumoniae, Enterococcus faecalis, Pseudomonas fluorescens, Staphylococcus epidermidis or Acinetobacter baumannii.

86. The method of claim 65, wherein the biofilm infection is associated with Pseudomonas aeruginosa.

87. The method of claim 65, wherein the biofilm is associated with an artificial substance in vivo.

88. The method of claim 87, wherein the artificial substance is an implant, catheter, tube, or prosthesis.

89. The method of claim 65, wherein the composition comprises 2, 3, or 4 excipients.

90. The method of claim 89, wherein the 2, 3, or 4 excipients are selected from the group consisting of D-alanine, L-alanine, L-homoserine, L-proline, D-threonine, adenosine, 2-deoxy adenosine, inosine, citric acid, glycolic acid, glyoxylic acid, succinic acid, monomethyl succinate, quinic acid, d-amino valeric acid, chondroitin, D-cellobiose, D- fructose, D-fructose-6-phosphate, D-galactose, b-methyl-D-galactoside, a-methyl-D- glucoside, D-glucose-l -phosphate, D-gluconic acid, b-methyl-D-glucuronic acid, N- acetyl-D-glucosaminitol, D-mannose, D-melezitose, D-melibiose, D-trehalose, inulin, L-arabitol, maltose, mannan, melibionic acid, m-inositol, palatinose, stachyose, sucrose, turanose, xylitol, phenylethylamine, propylene glycol, sec-butylamine, tween 20, tween 40, and tween 80.

91. The method of claim 65, wherein the at least one excipient enhances antimicrobial activity of the antibiotic against the biofilm infection.

92. The method of claim 91, wherein the enhanced activity is further defined as a greater percent decrease in microbial proliferation.

93. The method of claim 65, wherein the antibiotic and at least one excipient are administered topically, intralesionally, by inhalation, intranasally, opthalmically, or parenterally.

94. A method of preventing biofilm formation and/or growth on a medical device, the method comprising coating surfaces of the medical device exposed to bodily fluids and/or tissues with an effective amount of the pharmaceutical composition of any of claims 1-64.

95. The method of claim 94, wherein the device a prosthetic heart valve, venous catheter, urinary catheter, endoscope, contact lenses, intubation tube, or intrauterine device.

96. The method of claim 94, wherein the antibiotic is tobramycin, colistin, aztreonam, or ciprofloxacin, or erythromycin.

97. The method of claim 94, wherein the antibiotic is PEGylated.

98. A composition comprising an effective amount of an antibiotic and at least one excipient selected from the group consisting of N-acetyl-D-glucosaminitol, L-proline, L-alanine, succinic acid, 2-deoxy-adenosine, chondroitin, and D-gluconic acid for the treatment of a biofilm infection in a subject.

99. The composition of claim 98, wherein the composition is formulated for oral, intravenous, intraarticular, parenteral, enteral, topical, subcutaneous, intramuscular, buccal, sublingual, rectal, intravaginal, intrapenile, intraocular, epidural, intracranial, or inhalational administration.

100. The composition of claim 98, wherein the antibiotic is an aminoglycoside, a polymyxin, a monobactam, or a fluoroquinolone.

101. The composition of claim 98, wherein the antibiotic is tobramycin, colistin, aztreonam, ciprofloxacin, or erythromycin.

102. The composition of claim 98, wherein the antibiotic is PEGylated.

103. The composition of claim 98, wherein the biofilm infection is bacterial and/or fungal.

104. The composition of claim 98, wherein the biofilm infection is bacterial.

105. The composition of claim 98, wherein the biofilm infection is a nosocomial infection.

106. The composition of claim 98, wherein the infection is a lung infection, wound, otitis media, urinary tract infection, or oral infection.

107. The composition of claim 98, wherein the infection is chronic or recurrent.

108. The composition of claim 98, wherein the biofilm infection is a Staphylococcus infection or a Pseudomonas infection.

109. The composition of claim 98, wherein the biofilm is associated with Pseudomonas aeruginosa, Staphylococcus aureus, Helicobacter pylori, Burkholderia cepacia complex, Haemophilus influenzae, Escherichia coli, Streptococcus pneumoniae, Enterococcus faecalis, Pseudomonas fluorescens, Staphylococcus epidermidis or

Acinetobacter baumannii.

110. The composition of claim 98, wherein the biofilm infection is Pseudomonas aeruginosa.

111. The composition of claim 98, wherein the biofilm is associated with an ocular

infection, cystic fibrosis infection, wound infection, otic infection, or rhinosinusitis infection.

112. The composition of claim 98, wherein the biofilm is associated with an otic infection and the composition is further defined as an otic composition.

113. The composition of claim 98, wherein the biofilm is associated with an ocular

infection and the composition is further defined as an ophthalmic solution, ophthalmic suspension, or ophthalmic ointment.

114. The composition of claim 98, wherein the biofilm is associated with a chronic wound or burn wound and the composition is further defined as a topical composition.

115. The composition of claim 114, wherein the topical composition is an emulsion,

emulgel, lotion, or topical spray.

116. The composition of claim 98, wherein the biofilm infection is associated with a lung infection and the composition is further defined as a pulmonary composition.

117. The composition of claim 116, wherein the pulmonary composition is a dry powder formulation or solution for inhalation.

118. The composition of claim 98, wherein the biofilm infection is associated with chronic rhinosinusitis infection and the composition is further defined as a nasal spray.

119. A compound comprising a polymyxin covalently bound to a polyethylene glycol (PEG).

120. The compound of claim 119, wherein the polymyxin is colistin or polymyxin B.

121. The compound of claim 119, wherein the PEG is linear.

122. The compound of claim 119, wherein the PEG comprises the formula CH₃-PEG-.

123. The compound of claim 119, wherein the PEG comprises the formula CH₃— [O— CH₂— CH₂]„— .

124. The compound of claim 119, wherein the poly(ethylene glycol) comprises the formula CH₃-[0-CH₂-CH₂]_n-0C(0)CH₂CH₂C(0)-, wherein n=2-3000.

125. The compound of claim 124, wherein n=l00-2000.

126. The compound of claim 119, wherein the compound is comprised in a pharmaceutical composition.

127. The compound of claim 126, wherein the pharmaceutical preparation is formulated for topical, inhalational, parenteral, intravenous, or injection administration.

128. The compound of claim 119, wherein the compound comprises at least one excipient selected from the group consisting of D-alanine, L-alanine, L-homoserine, L-proline, D-threonine, adenosine, 2-deoxy adenosine, inosine, citric acid, glycolic acid, glyoxylic acid, succinic acid, monomethyl succinate, quinic acid, d-amino valeric acid, chondroitin, D-cellobiose, D-fructose, D-fructose-6-phosphate, D-galactose, b-methyl- D-galactoside, a-methyl-D-glucoside, D-glucose-l -phosphate, D-gluconic acid, b- methyl-D-glucuronic acid, N-acetyl-D-glucosaminitol, D-mannose, D-melezitose, D- melibiose, D-trehalose, inulin, L-arabitol, maltose, mannan, melibionic acid, m- inositol, palatinose, stachyose, sucrose, turanose, xylitol, phenylethylamine, propylene glycol, sec-butylamine, tween 20, tween 40, and tween 80.

129. The compound of claim 119, wherein the compound comprises the pharmaceutical composition of claim 1.

130. The compound of claim 119, wherein the compound comprises at least one excipient is selected from the group consisting of N-acetyl-D-glucosaminitol, L-proline, L-alanine, 2-deoxy-adenosine, chondroitin, and D-gluconic acid.

131. The compound of claim 128, wherein the compound comprises 2, 3, or 4 excipients.

132. A method of treating a biofilm infection in a subject comprising administering to the subject a therapeutic amount of a PEGylated polymyxin.

133. The method of claim 132, wherein the pegylated polymyxin is further defined as the compound of any one of claims 119-131.

134. The method of claim 132, wherein the subject is human.

135. The method of claim 132, wherein the biofilm infection comprises gram-negative bacteria.

136. The method of claim 132, wherein the biofilm is associated with Pseudomonas aeruginosa, Staphylococcus aureus, Helicobacter pylori, Burkholderia cepacia complex, Haemophilus influenzae, Escherichia coli, Streptococcus pneumoniae, Enterococcus faecalis, Pseudomonas fluorescens, Staphylococcus epidermidis or Acinetobacter baumannii.

137. The method of claim 132, wherein the subject is immunocompromised or has an

immune dysfunction.

138. The method of claim 132, wherein the subject has a burn or a wound infection.

139. The method of claim 132, wherein the biofilm infection is on or in the skin of the subject.

140. The method of claim 132, wherein the biofilm infection is on or adjacent to a medical device implanted in the subject.

141. The method of claim 132, wherein the medical device is a catheter, sutures, a staple, or a pin.

142. A medical device, wherein at least a portion of a surface of the device is coated with a compound of any one of claims 119-131.

143. The device of claim 142, wherein the medical device is a glove, a catheter, a stent, a staple, a pin, an electrical nerve stimulation device, a screw, a rod, a wire, a collar, a tube, or a surgical drain.

144. A composition comprising chlorhexidine and at least one excipient selected from the group consisting of N-acetyl-glucosaminitol, succinic acid, L-alanine, L-proline, benzoic acid, potassium sorbate, alginic acid, carrageenan, citric acid, edetic acid, coconut oil, EDTA, and xylitol.

145. The composition of claim 144, wherein the composition comprises 2, 3, 4, or 5

excipients.

146. The composition of claim 144, wherein the composition comprises L-proline and succinic acid.

147. The composition of claim 146, wherein the composition further comprises EDTA.

148. The composition of claim 146 or 147, wherein the composition further comprises benzoic acid.

149. The composition of any of claims 146-148, wherein the composition further comprises alginic acid.

150. The composition of any of claims 146-149, wherein the antibiotic further comprises carrageenan.

151. The composition of claim 146, wherein the composition comprises L-alanine and L- proline.

152. The composition of claim 146, wherein the composition comprises L-proline and N- acetyl-D-glucosaminitol.

153. The composition of claim 146, wherein the composition comprises N-acetyl-D- glucosaminitol and succinic acid.

154. The composition of claim 146, wherein the composition comprises L-proline and succinic acid.

155. The composition of claim 146, wherein the composition comprises L-proline and N- acetyl-D-glucosaminitol.

156. The composition of any of claims 144-155, wherein the chlorhexidine is at a concentration of 1 to 100 pg/mL.

157. The composition of any of claims 144-156, wherein the oral biofilm is associated with dental caries, gingivitis, or periodontal disease.

158. The composition of claim 157, wherein the oral biofilm is associated with dental caries.

159. The composition of claim 158, wherein the dental caries is associated with Streptococcus mutans, Streptococcus gordonii, Candida albicans, Actinomyces odontolyticus , Actinomyces naeslundii, Streptococcus sobrinus, Lactobacillus spp, or Veillonella spp.

160. The composition of claim 157, wherein the oral biofilm is associated with gingivitis or periodontal disease.

161. The composition of claim 160, wherein the gingivitis or periodontal disease is associated with Streptococcus gordonii, Fusobacterium nulceatum, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Aggregatibacter actinomycetemcomitans, or Actinomyces naeslundii.

162. The composition of any of claims 144-157, wherein the composition is formulated for oral administration.

163. The composition of any of claims 144-162, wherein the composition is formulated as a solution, suspension, semisolid, varnish, paste, sublingual dissolvable tablet, lozenge, spray or mist for local oral cavity.

164. The composition of any of claims 144-163, wherein the composition further comprises nanoparticles.

165. The composition of claim 164, wherein the nanoparticles are iron oxide nanoparticles.

166. The composition of claim 165, wherein the iron oxide nanoparticles are FeO, Fe₃0₄, Fe₄0₅, Fe₂0₃ nanoparticles.

167. The composition of claim 165 or 166, wherein the iron oxide nanoparticles are coated.

168. The composition of claim 167, wherein the iron oxide nanoparticles are citric acid coated or PEG/amine coated.

169. The composition of any of claims 164-168, wherein the nanoparticles are at a concentration of 50-200 pg/mL.

170. A method of treating an oral biofilm comprising administering to the subject an effective amount of a composition of any of claims 144-169.

171. The method of claim 170, wherein the at least one excipient enhances antimicrobial activity of the chlorhexidine against the oral biofilm infection.

172. The method of claim 171, wherein the enhanced activity is further defined as a greater percent decrease in microbial proliferation.