Epsilon polylysine (EPL) as an antibiotic adjuvant for fluoroquinolone eye drop formulation

Abstract

The present invention relates to a formulation comprising epsilon polylysine and an antimicrobial compound, wherein the epsilon polylysine is present in the formulation at a concentration in the range of 0.0001 µg / mL to 70 µg / mL. The present invention also relates to a method of preparing such a formulation and a method of treating or preventing microbial infection using said formulation.

Description

[0001] EPSILON POLYLYSINE (ΕPL) AS AN ANTIBIOTIC ADJUVANT FOR FLUOROQUINOLONE EYE DROP FORMULATION

[0002] FIELD OF THE INVENTION

[0003] The present invention relates generally to the field of pharmaceuticals. In particular, the present invention relates to a formulation comprising epsilon polylysine and an antimicrobial compound, a method of preparing said formulation, and a method of treating microbial infection using said formulation.

[0004] BACKGROUND OF THE INVENTION

[0005] The growing incidents of antimicrobial resistance (AMR) are considered by clinicians as the clinical superchallenge of the 21stcentury. AMR has been classified as a leading public health threat and a priority’ of global significance. Ocular infections are the fifth leading cause of ocular morbidity and their severity ranges from self-limiting bacterial conjunctivitis to vision threatening keratitis and endophthalmitis. Infectious keratitis, the infection of cornea, is the major cause of comeal blindness in both developed and developing countries. The estimated incidence ranges from 2.5 to 799 per 100,000 population.

[0006] Ocular infection after any fonn of ocular surgery remains a significant risk globally, and surgical antibiotic prophylaxis regimes incorporating topical broad spectrum antibiotics (namely fluoroquinolones) are almost always used for all forms of ocular surgery, including cataract surgery and all forms of refractive laser surgery’. Endophthalmitis following cataract surgery’, is an uncommon but the most devastating complication, and many antibiotic prophylaxis regimes exist. The Asian study on Perioperative Antibiotic prophylaXis for infection prevention (APAX) consortium recently published an evaluation of various clinical regimes involving varying practice patterns with topical antibiotics, and concluded further evidence-based improvements remain important.

[0007] Fungal keratitis (FK), or keratomycosis, is an infection of the cornea caused by fungal pathogens and is the leading cause of comeal blindness in developing countries. It accounts for 6-20% of infectious keratitis in the US, with increasing prevalence reported in tropical countries, particularly in the agricultural communities of developing countries. It has been estimated that the annual incidence of FK exceeds 1 million, ranging from 736251-1367323 cases per year, with a higher ratio of fungi vs bacteria as the cause of microbial keratitis occurring in tropical and subtropical climates. Approximately three quarters of patients with FK will lose an eye or ey’e sight owing to late diagnosis and poor initial management of the disease.Asia Cornea Society Infectious Keratitis Study (ACSIKS), a pan-Asian study set up by the Singapore National Eye Centre identified Fusarium solani (18.3%) and Pseudomonas aeruginosa (10.7%) as the most common aetiological agents in the Asian population. ACSIKS reported that fungal pathogens accounted for 32.7% of FK cases, next to bacterial keratitis (38.0%). Members of the genera Fusarium, Candida, and Aspergillus are the most common causative fungal pathogens identified in FK patients. The common perception that ocular infections may not be life-threatening was invalidated by the recent outbreak of carbapenem-resistant Pseudomonas aeruginosa due to contaminated lubricant eye drops in the United States of America. The outbreak affected 81 patients and there were 4 deaths reported while 14 patients lost their vision and 4 patients required complete removal of the eye ball. P. aeruginosa exhibits multiple resistance mechanisms against contemporary antibiotics while treatment for fungal keratitis requires prolonged use of antifungal medications that are fungistatic, toxic, costly, and usually, not available commercially for topical utility and often requires invasive surgical intervention for infection control. Resistance to commonly used antifungals, such as echinocandins, coupled with poor corneal penetration of the effective antifungal agents further complicate treatment strategies for Fusarium keratitis. The increased prevalence of bacterial and fungal pathogens that are non-responsive to available medications pose unprecedented challenges for healthcare systems and community.

[0008] In view of the unprecedented challenges, there is a need for an alternative formulation that at least partially ameliorates the problem towards antimicrobial resistance.

[0009] SUMMARY

[0010] In one aspect, the present disclosure refers to a formulation comprising:

[0011] i) epsilon polylysine: and

[0012] li) an antimicrobial compound,

[0013] wherein the epsilon polyly sine is present in the formulation at a concentration in the range of 0.0001 pg / mL to 70 pg / mL.

[0014] Advantageously, exposure of epsilon polylysine in the range of 0.0001 pg / mL to 70 pg / mL, which is a sub-inhibitory concentration, may not provoke resistance induction of a microbial. More advantageously, elevated sub-inhibitory concentrations of epsilon polylysine may not induce any observable adverse events, which may be useful when the formulation is used as prophylaxis during ocular surgery, where intraocular tissues may be exposed to antimicrobials.

[0015] Further advantageously, epsilon polylysine may have a synergistic effect with the antimicrobial compound. Advantageously, the epsilon polylysine may be an effective adjuvant for antibiotics that target intracellularcomponents. In one example, epsilon poly lysine may sensitise drug-resistant pathogen to fluoroquinolines (FQ) antibiotics by increasing the permeability of bacterial outer membrane and fungal cell wall, resulting in an increased intracellular concentration of FQs for antimicrobial action towards drug -resistant pathogen. This may decrease the concentration of FQs required to inhibit microbial growth. In another example, epsilon polylysine may sensitise fungal pathogen to voriconazole antifungals. Also advantageously, the polymer - antimicrobial combination may have broad-spectrum activity covering bacterial, fungal, viral and parasitic pathogens.

[0016] In another aspect, the present disclosure refers to a method of preparing a formulation as defined above, comprising a step of mixing:

[0017] i) epsilon polylysine; and

[0018] ii) an antimicrobial compound,

[0019] wherein the epsilon polylysine is present in the formulation at a concentration of 0.0001 pg / mL to 70 pg / mL.

[0020] Advantageously, the method provides a facile, versatile and efficient way of preparing a formulation comprising epsilon polylysine and an antimicrobial compound.

[0021] In another aspect, the present disclosure refers to a formulation as defined above, comprising:

[0022] i) epsilon polylysine: and

[0023] ii) an antimicrobial compound,

[0024] wherein the epsilon polylysine is present in the formulation at a concentration in the range of 0.0001 pg / mL to 70 pg / mL, for use in treating and / or preventing a microbial infection.

[0025] In another aspect, the present disclosure refers to a method of treating and / or preventing a microbial infection, comprising a step of administering a formulation as defined above, comprising:

[0026] i) epsilon polylysine: and

[0027] ii) an antimicrobial compound,

[0028] wherein the epsilon polyly sine is present in the formulation at a concentration in the range of 0.0001 pg / mL to 70 pg / mL.

[0029] Tn another aspect, there is provided the use of a formulation as defined above, comprising:

[0030] i) epsilon polylysine: and

[0031] ii) an antimicrobial compound,

[0032] wherein the epsilon polylysine is present in the formulation at a concentration in the range of 0.0001 μg / mL to 70 μg / mL, in the manufacture of a medicament for treating and / or preventing a microbial infection.Advantageously, the formulation may be a therapeutically safe ophthalmic antimicrobial-el’L combination formulation for the treatment and / or prevention of drug resistant microbial infections More advantageously, the formulation may safely mitigate antimicrobial resistance and enhance antimicrobial efficacy against multi-drug resistant ophthalmic microorganisms. More advantageously, the use of the formulation as defined above may provide heightened safety of epsilon polylysine as surgical prophylaxis for ocular tissues for ophthalmic surgery to prevent postoperative infections.

[0033] BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0035] [Figure 1] refers to graphs showing serial passage resistance induction of εPL and polymyxin B over days against quality control and multi-drug resistant P. aeruginosa strains, where (A) is P. aeruginosa ATCC 27853, (B) is P. aeruginosa DX 783 and (C) is P. aeruginosa DX 744.

[0036] [Figure 2] refers to graphs showing bacterial viability of DX 744 and DX 783 strains in the presence of sub-inhibitory concentrations of εPL and fluoroquinolone antibiotics, where (A) is εPL and levofloxacin in P. aeruginosa DX 744, (B) is εPL and moxifloxacin in P. aeruginosa DX 744, (C) is εPL and levofloxacin in P. aeruginosa DX 783 and (D) εPL and moxifloxacin in P. aeruginosa DX 783. The details of various groups are presented in the table. Note that, no bacterial growth was observed in the presence of sub-inhibitory concentrations of εPL and FQs. The dotted line indicates initial inoculum.

[0037] [Figure 3] refers to graphs showing intracellular accumulation of FQs at various sub-inhibitory concentrations of εPL, where (A) is εPL and levofloxacin in P. aeruginosa DX 744, (B) is εPL and moxifloxacin in P. aeruginosa DX 744, (C) is εPL and levofloxacin in P. aeruginosa DX 783 and (D) εPL and moxifloxacin in P. aeruginosa DX 783. The amount of drug was quantitatively determined by RP-HPLC.

[0038] [Figure 4] refers to images of the cornea of New Zealand White Rabbits and a graph, showing comeal wound healing in New Zealand White rabbits as measured by fluorescent slit lamp (fSL) biomicroscopy. A) Fluorescent slit lamp (fSL) photographs showing fluorescein staining of the wounded area at various time points. B) Quantitative determination of wound area determined from fSL photographs. The wound area is expressed in terms of % in response to various treatment groups.

[0039] [Figure 5] refers to graphs showing the concentration-dependent changes in the fluorescence intensity at 405 nm of hydrophobic fluorescent probe N-phenyl naphthyl amine (NPN) upon addition of εPL to intactFusarium solani ATCC 46492 over time. The increasing fluorescence intensity of NPN upon addition of ePL suggests higher permeability of the probe in the presence of polymer.

[0040] [Figure 6] refers to graphs showing the change in maximum fluorescence intensity of NPN at 405 nm at various concentrations of ePL, where (A) is F. solani ATCC 46492, (B) is F. solani ATCC 3636, (C) is F. solani ATCC 62877, and (D) is F. solani ATCC 52628.

[0041] [Figure 7] refers to graphs showing viability of F. solani strains at various amount of εPL and / or 0.5% w / v moxifloxacin (in the form of Vigamox®), where (A) is F. solani ATCC 46492 at 24 hours post treatment, (B) is F. solani ATCC 46492 at 48 hours post treatment, (C) is F. solani ATCC 62877 at 24 hours post treatment, and (D) is_F. solani ATCC 62877 at 48 hours post treatment. The dotted horizontal lines in the graph indicate initial inoculum.

[0042] [Figure 8] refers to photographs showing the presence or absence of the fungal growth in PBS buffer (pH 7.0) upon treatment with buffer alone, 0.5% w / v moxifloxacin, and εPL and 0.5% w / v moxifloxacin (in the form of Vigamox®) combination.

[0043] [Figure 9] refers to slit lamp images of the mouse cornea, showing the effect of treatment of vehicle, ePL and / or 0.5% w / v moxifloxacin (in the form of Vigamox®).

[0044] [Figure 10] refers to graphs showing the effect of treatment of vehicle, ePL and / or 0.5% w / v moxifloxacin (in the form of Vigamox®), showing the fungal bioburden as determined by plating for colony forming unit (CFU) enumeration.

[0045] DEFINITIONS

[0046] As used herein, the term "epsilon polylysine" refers to a naturally occurring or synthetically produced homopolyamide in which the backbone amide group is formed between a-carboxyl and e-amino group of the amino acid lysine. The amino acid lysine may be -lysine, L-lysine, or any mixture thereof The term “epsilon-poly lysine” may also be referred to in the form of “ePL”, “PPL”, “epsilon-polylysine”, “e-polylysine” or “E-polylysine”.

[0047] As used herein, the term “antibacterial” and “antibiotics” are synonymous.

[0048] As used herein, the term “minimum inhibitory concentration (MIC)” refers to the minimum concentration of a compound required for the complete inhibition of microbial growth. The MIC may be in the form of “MICx%”, where it refers to the minimum concentration of a compound required to inhibit > X% ofmicrobial growth. For example, MlCso% refers to minimum concentration of a compound to inhibit > 90% of microbial growth. “MIC90%” may refer to “IC50”, which is the half inhibitory concentration required to inhibit 50 % of microbial growth.

[0049] As used herein, the term “sub-inhibitory concentration” or “sub-minimum inhibitor} concentration” (Sub-MIC) of a compound may be used interchangeably, and may refer to a concentration that is below minimum inhibitory concentration of said compound. For the avoidance of doubt, the term “sublethal concentration” is based on lethality where the cells have a complete loss of metabolic activity, whereas for the term “sub-inhibitory concentration”, the cells are still viable but do not lead to further growth.

[0050] As used herein, the terms "treat," "treatment," and grammatical variants thereof, refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological condition, disorder or disease or obtain beneficial or desired clinical results. Such beneficial or desired clinical results include, but arc not limited to, alleviation of symptoms; diminishment of extent of condition, disorder or disease: stabilized (i.e. not worsening) state of condition, disorder or disease; delay or slowing of condition, disorder or disease progression; prevention of infection; amelioration of the condition, disorder or disease state, remission (whether partial or total), whether detectable or undetectable; or enhancement or improvement of condition, disorder or disease. Treatment includes eliciting a cellular response that is clinically significant, without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.

[0051] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0052] The word “substantially” does not exclude “completely” e g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary', the word “substantially” may be omitted from the definition of the invention.

[0053] As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a genetic marker” includes a plurality of genetic markers, including mixtures and combinations thereof.As used herein, the term “about”, in the context of concentrations of components of the formulations, typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically, + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0054] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0055] Certain embodiments may also be described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the embodiments with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0056] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.DETAILED DESCRIPTION OF THE PRESENT DISCLOSURE

[0057] There is provided a formulation comprising:

[0058] i) epsilon polylysine: and

[0059] ii) an antimicrobial compound,

[0060] wherein the epsilon polylysine is present in the formulation at a concentration in the range of 0.0001 pg / m L to 70 pg / mL.

[0061] The epsilon polyly sine may be a naturally occurring or synthetically produced homopolyamide in which the backbone amide group is formed between a-carboxyl and e-amino group of the amino acid lysine. It is a polymer that may be cationic, proteolytically stable and may have broad-spectrum antimicrobial properties. The epsilon polylysine may contain about 10 to about 45, about 10 to about 12, about 10 to about 14, about 10 to about 16, about 10 to about 18, about 10 to about 20, about 10 to about 25, or about 10 to about 35 residues. The epsilon polylysine may contain either D-lysine residues or L-lysine residues, or any mixture thereof.

[0062] The formulation as defined above may be in a buffer.

[0063] The buffer may be selected from the group consisting of phosphate buffered saline (PBS), borate buffered saline (BBS), citrate buffer, tris(hydroxymethyl)aminomethane (TRIS), 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-[Tris(hydroxymethyl)methyl] -2 -aminoethanesulfonic acid (TES), 1,4-Piperazinediethanesulfonic acid (PIPES), 4-Morpholineethanesulfonic acid (MES) or any mixture thereof

[0064] The formulation as defined above may be in a buffer with a pH about 5.5 to about 8.

[0065] The pH may be in the range of about 5.5 to about 8, about 5.5 to about 6.0, about 6 to about 6.5, about 6 to about 7, about 6 to about 7.5, about 6.5 to about 7, about 6.5 to about 7.5, about 6.5 to about 8, about 7 to about 7.5, about 7 to about 8, or about 7.5 to about 8.

[0066] The formulation as defined above may be in the form of a solid, a semi-solid or a liquid.

[0067] The solid formulation may be in the form of a tablet, a powder, a capsule or a pill.

[0068] The semi-solid formulation may be in the form of a gel or a cream.

[0069] The formulation may be a liquid. The liquid may be in the form of a solution, an ointment, a lotion, a suspension, an emulsion or a syrup.The antimicrobial compound may be selected from the group consisting of an antibacterial compound, an antifungal compound, an antiviral compound, an antiparasitic compound or any mixture thereof.

[0070] The antimicrobial compound may be present in the formulation at a concentration in the range of about 0.1 pg / mL to about 300 pg / mL, about 0.1 pg / mL to about 0.15 pg / mL, about 0.1 pg / mL to about 0.25 pg / mL, about 0.1 pg / mL to about 0.5 pg / mL, about 0.1 pg / mL to about 1 pg / mL, about 0.1 pg / mL to about 2 pg / mL, about 0.1 pg / mL to about 4 pg / mL, about 0.1 pg / mL to about 8 pg / mL, about 0.1 pg / mL to about 10 pg / mL, about 0.1 pg / mL to about 16 pg / mL, about 0.1 pg / mL to about 32 pg / mL, about 0.1 pg / mL to about 64 pg / mL, about 0.1 pg / mL to about 100 pg / mL, or about 0.1 pg / mL to about 128 pg / mL, about 0.1 pg / mL to about 150 pg / mL, about 0.1 pg / mL to about 200 pg / mL, about 0.1 pg / mL to about 256 pg / mL.

[0071] The antimicrobial compound may be an antibacterial compound.

[0072] The antibacterial compound may be present in the formulation at a concentration in the range of about 0.1 pg / mL to about 6500 pg / mL, about 0.1 pg / mL to about 0.15 pg / mL, about 0.1 pg / mL to about 0.25 pg / mL, about 0.1 pg / mL to about 0.5 pg / mL, about 0.1 pg / mL to about 1 pg / mL, about 0.1 pg / mL to about 2 pg / mL, about 0.1 pg / mL to about 4 pg / mL, about 0.1 pg / mL to about 8 pg / mL, about 0.1 pg / mL to about 16 pg / mL, about 0.1 pg / mL to about 32 pg / mL, about 0.1 pg / mL to about 64 pg / mL, about 0.1 pg / mL to about 128 pg / mL, about 0.1 pg / mL to about 150 pg / mL, about 0.1 pg / mL to about 500 pg / mL, about 0.1 pg / mL to about 1000 pg / mL, about 0.1 pg / mL to about 2000 pg / mL, about 0.1 pg / mL to about 3500 pg / mL, about 0.1 pg / mL to about 5000 pg / mL, or about 0.1 pg / mL to about 6500 pg / mL.

[0073] The antibacterial compound may be selected from the group consisting of an aminoglycoside, a tetracycline, an oxazolidinone, an amphenicol, a P-lactam, an antifolate, a fluoroquinolone, a rifamycin, a glycopeptide, a pleuromutilin, a nitrofuran or any mixture thereof.

[0074] The antibacterial compound may be an aminoglycoside. The aminoglycoside may be selected from the group consisting of streptomycin, neomycin, paromomycin, amikacin, spectinomycin, tobramycin, gentamicin, plazomicin or any mixture thereof.

[0075] The antibacterial compound may be a tetracycline. The tetracycline may be selected from the group consisting of doxycycline, chlortetracycline, clomocycline, demeclocycline, eravacycline, lymecycline, metacycline, minocycline, omadacycline, oxytetracycline, penimepicycline, rolitetracycline, sarecycline or any mixture thereof.The antibacterial compound may be an oxazolidinone. The oxazohdinone may be selected from the group consisting of eperezolid, linezolid, poiszolid, radezolid, radezoli, ranbezolid, sutezolid, tedizolid or any mixture thereof.

[0076] The antibacterial compound may be an amphenicol. The amphenicol may be selected from the group consisting of thiamphenicol, azidamfenicol, florfenicol, chloramphenicol or any mixture thereof.

[0077] The antibacterial compound may be a P-lactam. The P-lactam may be selected from the group consisting of benzylpenicillin, cloxacillin, amoxicillin, ampicillin, cefazolin, cefalexin, cefixime, cefiderocol or any mixture thereof.

[0078] The antibacterial compound may be an antifolate. The antifolate may be selected from the group consisting of pyrimethamine, trimethoprim, sulfadiazine, sulfamethoxazole or any mixture thereof.

[0079] The antibacterial compound may be a fluoroquinolone. The fluoroquinolone may be selected from the group consisting of levofloxacin, moxifloxacin, ciprofloxacin, gemifloxacin, ofloxacin, besifloxacin, finafloxacin, gatifloxacin, delafloxacin, danofloxacin, difloxacin, enrofloxacin, ibafloxacin, marbofloxacin, orbifloxacin, pradofloxacin or any mixture thereof.

[0080] The antibacterial compound may be a rifamycin. The rifamycin may be selected from the group consisting of rifampicin, rifabutin, rifapentine, rifaximin or any mixture thereof.

[0081] The antibacterial compound may be a glycopeptide. The glycopeptide may be selected from the group consisting of avoparcin, carbomycin, dalbavancin, oritavancin, ristocetin, teicoplanin, telavancin, vancomycin or any mixture thereof.

[0082] The antibacterial compound may be a pleuromutilin. The pleuromutilin may be selected from the group consisting of azamulin, Lefamulin, retapamulin, tiamulin, valnemulin or any mixture thereof.

[0083] The antibacterial compound may be a nitrofuran. The nitrofuran may be selected from the group consisting of furazolidone, nifuroxazide, nifurtoinol, nifurzide, nitrofurantoin, nitrofurazone or any mixture thereof. The antimicrobial compound may be an antifungal compound. The fungus may be filamentous The fungus may be yeast.

[0084] The antifungal compound may be present in the formulation at a concentration in the range of about 0.1 pg / mL to about 10 pg / mL, about 0.1 pg / mL to about 0.15 pg / mL, about 0.1 pg / mL to about 0.25 pg / mL, about 0.1 pg / mL to about 0.5 pg / mL, 0.1 pg / mL to about 1 pg / mL, about 0.1 pg / mL to about 2 pg / mL, about 0.1 pg / mL to about 4 pg / mL, or about 0.1 pg / mL to about 8 pg / mL.The antifungal compound may be selected from the group consisting of an azole, a polyene, an amine, an echinocandin or any mixture thereof.

[0085] The antifungal compound may be an azole. The azole may be selected from the group consisting of clotrimazole, miconazole, fluconazole, fosfluconazole, fosravuconazole, isavuconazole, itraconazole, luliconazole, osteseconazole, posaconazole, voriconazole or any mixture thereof.

[0086] The antifungal compound may be a polyene. The polyene may be selected from the group consisting of natamycin, nystatin, amphotericin B or any mixture thereof.

[0087] The antifungal compound may be an amine. The amine may be selected from the group consisting of naftifine, terbinafine, butenafine or any mixture thereof.

[0088] The antifungal compound may be an echinocandin. The echinocandin may be selected from the group consisting of anidulafungin, caspofungin, cilofungin, micafungin, rezafungin or any mixture thereof. The antimicrobial compound may be an antiparasitic compound. The antiparasitic compound may be selected from the group consisting of an antiprotozoal, an antihelminthic, an antinematodes, an anticestodes, an antiamoebics or any mixture thereof.

[0089] The antimicrobial compound may be an antiviral. The antiviral compound may be selected from the group consisting of a deoxyribonucleic acid (DNA) antiviral, a ribonucleic acid (RNA) antiviral, a capsid inhibitor, an entry inhibitor, an integrase inhibitor, a protease inhibitor, a reverse -transcriptase inhibitor or any mixture thereof.

[0090] The epsilon polylysine may be present in the formulation at a concentration in the range of about 0.0001 pg / mL to about 70 pg / mL, about 0.0001 pg / mL to about 1 pg / mL, about 0.0001 pg / mL to about 2 pg / mL, about 0.0001 pg / mL to about 4 pg / mL, about 0.0001 pg / mL to about 5 pg / mL, about 0.0001 pg / mL to about 8 pg / mL, about 0.0001 pg / mL to about 10 pg / mL, about 0.0001 pg / mL to about 16 pg / mL, about 0.0001 pg / mL to about 20 pg / mL, about 0.0001 pg / mL to about 32 pg / mL, or about 0.0001 pg / mL to about 64 pg / mL.

[0091] The epsilon polylysine may be present in the formulation at a concentration in the range of about 0.0001 pg / mL to about 20 pg / mL.

[0092] The epsilon polylysine may be present in the formulation at a concentration in the range of about 0.0001 pg / mL to about 10 pg / mL.

[0093] The epsilon polylysine may be present in the formulation at a concentration in the range of about 0.0001 pg / mL to about 5 pg / mL.The epsilon polylysine may have antiparasitic activity by itself at higher concentrations. Tire epsilon polylysine may have antiparasitic activity by itself at a concentration of about 17 pg / mL or higher. The epsilon polylysine may have antiparasitic activity by itself at a concentration of about 17 pg / mL to about 70 pg / mL, about 17 pg / mL to about 25 pg / mL, about 17 pg / mL to about 40 pg / mL, about 17 pg / mL to about 50 pg / mL. or about 17 pg / mL to about 60 pg / mL.

[0094] There is provided a method of preparing a formulation as defined above, comprising a step of mixing: i) epsilon polylysine: and

[0095] ii) an antimicrobial compound,

[0096] wherein the epsilon polylysine is present in the formulation at a concentration in the range of 0.0001 pg / mL to 70 pg / mL.

[0097] The method as defined above, may further comprise a step of dissolving the formulation in a buffer with a pH in the range of about 5.5 to about 8.

[0098] The buffer may be selected from the group consisting of phosphate buffered saline (PBS), borate buffered saline (BBS), citrate buffer, tris(hydroxymethyl)aminomethane (TRIS), 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-[Tris(hydroxymethyl)methyl ]-2-aminoethanesulfonic acid (TES), 1,4-Piperazinediethanesulfonic acid (PIPES), 4-Morpholineethanesulfonic acid (MES) or any mixture thereof.

[0099] The pH may be in the range of about 5.5 to about 8, about 5.5 to about 6, about 6 to about 6.5, about 6 to about 7, about 6 to about 7.5, about 6.5 to about 7, about 6.5 to about 7.5, about 6.5 to about 8, about 7 to about 7.5, about 7 to about 8, or about 7.5 to about 8.

[0100] The method as defined above, may further comprise a step of adding a polymer additive at a concentration in the range of about 0.001% to about 0.01% (w / v) to the formulation, to modulate viscosity and / or mucoadhesion of the formulation.

[0101] The polymer additive may be a natural polymer, synthetic polymer or any mixture thereof.

[0102] The polymer additive may be selected from the group consisting of alginate, pectin, gelatin, gellan, chitosan, polyvinyl alcohol, polyamides, polycarbonates, polyalkylene glycols, polyvinyl ethers, methylcellulose, ethylcellulose, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose, hydroxypropyl methylcellulose (HPMC), carboxymethyl cellulose (CMC), polymethacrylate, hyaluronic acid, polycarbophil, or any mixture thereof. For example, the polymethacrylate may be a Eudragit®L100 polymer.The polymer additive may be present at a concentration in the range of about 0.001% to about 0.01% (w / v), about 0.001% to about 0.002% (w / v), about 0.001% to about 0.004% (w / v), about 0.001% to about 0.006% (w / v), or about 0.001% to about 0.008% (w / v).

[0103] There is provided a formulation as defined above, comprising:

[0104] i) epsilon polylysine: and

[0105] li) an antimicrobial compound,

[0106] wherein the epsilon polylysine is present in the formulation at a concentration in the range of 0.0001 pg / mL to 70 pg / mL, for use in treating and / or preventing a microbial infection.

[0107] There is provided a method of treating and / or preventing a microbial infection, comprising a step of administering a formulation as defined above, comprising:

[0108] i) epsilon polylysine: and

[0109] li) an antimicrobial compound,

[0110] wherein the epsilon polylysine is present in the formulation at a concentration in the range of 0.0001 pg / mL to 70 pg / mL

[0111] There is provided a formulation as defined above, comprising:

[0112] i) epsilon polylysine: and

[0113] ii) an antimicrobial compound,

[0114] wherein the epsilon polylysine is present in the formulation at a concentration in the range of 0.0001 μg / mL to 70 μg / mL, for use as a medicament.

[0115] There is provided the use of a formulation as defined above, comprising:

[0116] i) epsilon polylysine: and

[0117] ii) an antimicrobial compound,

[0118] wherein the epsilon polylysine is present in the formulation at a concentration in the range of 0.0001 μg / mL to 70 μg / mL, in the manufacture of a medicament for treating and / or preventing a microbial infection.

[0119] The prevention of the microbial infection may comprise prophylaxis, including surgical prophylaxis for ophthalmic surgery to prevent postoperative infection.

[0120] The microbial infection may be selected from the group consisting of Blepharitis, Hordeolum, Preseptal Cellulitis, Dacryocystitis, Orbital Cellulitis, Erysipelas, Keratomycosis, Keratoconjunctivitis, Conjunctivitis, Conjunctival Laceration, Superior Limbic Keratoconjunctivitis, Keratitis, CornealUlceration, Phlyctenulosis, Anterior Uveitis, Endophthalmitis, Abscess, Papillitis, Scleritis or Orbital Cellulitis.

[0121] The microbial infection may be a bacterial infection. The bacterium may be a Gram negative bacterium or a Gram positive bacterium.

[0122] The bacterium may be selected from the genus consisting of Acetobacter spp., Acinetobacter spp., Actinomyces spp., Agrobacterium spp., Azorhizobium spp., Azotobacter spp., Anaplasma spp., Bacillus spp., Bacteoides spp., Bartonella spp., Bordetella spp., Borrelia spp., Brucella spp., Burkholderia spp., Calymmatobacterium spp., Campylobacter spp., Chlamydia spp., Chlamydophila spp., Clostridium spp., Corynebacterium spp., Coxiella spp., Ehrlichia spp., Enterobacter spp.. Enterococcus spp., Escherichia spp., Francisella spp., Fusobacterium spp., Gardnerella, Haemophilus spp., Helicobacter spp., Klebsiella spp.. Lactobacillus spp.. Lactococcus spp., Legionella spp.. Listeria spp., Methanobacterium extroquens, Microbacterium multiforme, Micrococcus spp., Micrococcus luteus, Moraxella catarrhalis, Mycobacterium spp., Mycoplasma spp.. Neisseria spp., Pasteurella spp., Peptostreptococcus spp., Porphyromonas spp., Pseudomonas spp., Rhizobium spp., Rickettsia spp., Rochalimaea spp., Rothia spp., Salmonella spp., Serratia spp., Shigella spp., Staphylococcus spp., Stenotrophomonas spp., Streptococcus spp., Treponema spp., Vibrio spp., Wolbachia spp., or Yersinia spp.

[0123] The bacterium may be selected from the group consisting of Acetobacter aurantius. Acinetobacter baumannii, Actinomyces Israelii, Agrobacterium radiobacter, Agrobacterium tumefaciens, Azorhizobium caulinodans, Azotobacter vinelandii, Anaplasma phagocylophilum. Anaplasma marginale. Bacillus anthracis, Bacillus brevis, Bacillus cereus, Bacillus fusiformis, Bacillus licheniformis, Bacillus megaterium, Bacillus mycoides, Bacillus stearothermophilus, Bacillus subtilis, Bacteroides fragilis, Bacteroid.es gingivalis, Bacteroides melam.inogen.icus (Prevotella melaminogenica), Bartonella henselae, Bartonella quintana, Bordetella bronchiseptica, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella melitensis, Brucella suis, Burkholderia mallei, Burkholderia pseudomallei. Burkholderia cepacia complex, Burkholderia cenocepacia, Calymmatobacterium granulomatis, Campylobacter coli, Campylobacter fetus. Campylobacter jejuni, Campylobacter pylori. Chlamydia trachomatis, Chlamydophila. (such as C. pneumoniae, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani), Corynebacterium diphtheriae, Corynebacterium fusiforme, Coxiella burnetii, Ehrlichia chajfeensis, Enterobacter cloacae, Enterococcus avium, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus galllinarum, Enterococcus maloratus, Escherichia coli, Francisella tularensis, Fusobacterium. nucleatum, Gardnerella vaginalis, Haemophilus ducreyi, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus pertussis, Haemophilus vaginalis, Helicobacter pylori, Klebsiella pneumoniae, Lactobacillus acidophilus, Lactobacillus casei, Lactococcus lactis, Legionella pneumophila, Listeria monocytogenes,Methanobacterium extroquens, Microbacterium multiforme, Micrococcus luteus, Moraxella catarrhalis, Mycobacterium avium, Mycobacterium bovis, Mycobacterium diphtheriae, Mycobacterium intracellular, Mycobacterium leprae, Mycobacterium lepraemurium, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium tuberculosis. Mycoplasma fermentans, Mycoplasma genitalium, Mycoplasma hominis. Mycoplasma penetrans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Pasteurella tularensis Peptostreptococcus, Porphyromonas gingivalis, Pseudomonas aeruginosa, Rhizobium Radiobacter, Rickettsia prowazekii, Rickettsia psittaci, Rickettsia quintana, Rickettsia rickettsii, Rickettsia trachomae, Rochalimaea henselae, Rochalimaea quintana, Rothia dentocariosa, Salmonella enteritidis, Salmonella typhi, Salmonella typhimurium., Serratia marcescens, Shigella dysenteriae, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus, avium, Streptococcus bovis, Streptococcus cricetus, Streptococcus faceium, Streptococcus faecalis, Streptococcus ferus, Streptococcus gallinarum, Streptococcus laclis. Streptococcus mitior. Streptococcus mitis, Streptococcus mutans. Streptococcus oralis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus rattus, Streptococcus salivarius, Streptococcus sanguis, Streptococcus sobrinus. Treponema pallidum, Treponema denticola, Vibrio cholerae, Vibrio comma, Vibrio parahaemolyticus, Vibrio vulnificus, Wolbachia, Yersinia enterocolitica, Yersinia pestis or Yersinia pseudotuberculosis.

[0124] The bacterium may be selected from the group consisting of Acinetobacter baumannii, Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae. Pseudomonas spp., Staphylococcus spp., Streptococcus spp., Enterococcus faecalis or Enterococcus faecium. The microbial infection may be a drug resistant bacterial infection. The drug resistant bacterium may be selected from the group consisting of carbapenam-resistant Enterobacter strains (CRE), Methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin-resistant Enterococci (VRE), polymixin B -resistant Enterobacter cloacae, drug resistant Acinetobacter baumannii, prolific biofilm forming P. aeruginosa or S. epidermidis strains, multi-drug resistant Pseudomonas spp. strains, multi -drug resistant Staphylococcus spp., or multi -drug resistant Streptococcus spp..

[0125] The microbial infection may be a fungal infection. The fungus may be selected from the genus consisting of Absidia spp., Ajellomyces spp., Arthroderma spp., Aspergillus spp., Blastomyces spp., Candida spp., Cladophialophora spp., Coccidioides spp., Cryptococcus spp., Cunninghamella spp., Epidermophyton spp., Exophiala spp., Filobasidiella spp., Fonsecaea spp., Fusarium spp., Geotrichum spp., Histoplasma spp., Horiaea spp., Issatschenkia spp., Madurella spp., Malassezia spp., Microsporum spp., Microsporidia spp., Mucor spp., Nectria spp., Paecilomyces spp., Paracoccidioides spp., Penicillium spp., Pichia spp., Pneumocystis spp., Pseudallescheria spp., Rhizopus spp., Rhodotorula spp., Scedosporium spp., Schizophyllum spp., Sporothrix spp., Trichophyton spp., or Trichosporon spp..The fungus may be selected from the group consisting of Absidia corymbifera, Ajellomyces capsulatus, Ajellomyces dermatilidis, Arthroderma benhamiae, Arthroderma fulvum, Arthroderma gypseum, Arthroderma incurvatum, Arthroderma otae, Arthroderma vanbreuseghemii, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Blastomyces dermatitidis, Candida albicans, Candida glabrata, Candida guilliermondii, Candida krusei, Candida parapsilosis, Candida tropicalis, Candida pelliculosa, Cladophialophora carrionii, Coccidioides immitis, Coccidioides posadasii, Cryptococcus neoformans, Cunninghamella Sp, Epidermophyton floccosum, Exophiala dermatitidis, Filobasidiella neoformans, Fonsecaea pedrosoi, Fusarium solani, Fusarium oxysporum, Geotrichum candidum, Histoplasma capsulatum, Hortaea sverneckii, Issatschenkia orientalis, Madurella grisae, Malassezia furfur, Malassezia globosa, Malassezia obtusa, Malassezia pachydermatis, Malassezia restricta, Malassezia slooffiae, Malassezia sympodialis, Microsporum canis, Microsporum fulvum, Microsporum gypseum, Microsporidia, Mucor circinelloides, Nectria haematococca, Faecilomyces variotii, Paracoccidioides brasiliensis, Penicillium marneffei, Pichia anomala, Pichia guilliermondii, Pneumocystis proved, Pneumocystis carinii, Pseudallescheria boydii, Rhizopus oryzae, Rhodolorula rubra, Scedosporium apiospermum, Schizophyllum commune, Sporothnx schenckii, Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton verrucosum, Trichophyton violaceum, Trichosporon asahii, Trichosporon cutaneum, Trichosporon inkin, or Trichosporon mucoides.

[0126] The fungus may be selected from the group consisting of Candida spp., Fusarium spp., Saccharomyces cerevisiae, or Aspergillus spp..

[0127] The microbial infection may be a drug resistant fungal infection. The drug resistant fungus may be selected from the group consisting of Absidia corymbifera, Ajellomyces capsulatus, Ajellomyces dermatitidis, Arthroderma benhamiae, Arthroderma fulvum, Arthroderma gypseum, Arthroderma incurvatum, Arthroderma otae, Arthroderma vanbreuseghemii, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Blastomyces dermatitidis, Candida albicans, Candida glabrata, Candida guilliermondii, Candida krusei, Candida parapsilosis, Candida tropicalis, Candida pelliculosa, Cladophialophora carrionii, Coccidioides immitis, Coccidioides posadasii, Cryptococcus neoformans, Cunninghamella Sp, Epidermophyton floccosum, Exophiala dermatitidis, Filobasidiella neoformans, Fonsecaea pedrosoi. Fusarium solani, Fusarium oxysporum, Geotrichum candidum, Histoplasma capsulatum, Hortaea ■werneckii, Issatschenkia orientalis, Madurella grisae, Malassezia furfur, Malassezia globosa, Malassezia obtusa, Malassezia pachydermatis, Malassezia restricta, Malassezia slooffae, Malassezia sympodialis, Microsporum canis, Microsporum fulvum, Microsporum gypseum, Microsporidia, Mucor circinelloides, Nectria haematococca, Faecilomyces variotii, Paracoccidioides brasiliensis, Penicillium marneffei, Pichia anomala, Pichia guilliermondii, Pneumocystis proved, Pneumocystis carinii, Pseudallescheria boydii, Rhizopus oryzae, Rhodotorula rubra, Scedosporium apiospermum, Schizophyllum commune, Sporothnxschenckii, Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton verrucosum, Trichophyton violaceum, Trichosporon asahii, Trichosporon cutaneum. Trichosporon inkin, or Trichosporon mucoides.

[0128] The drug resistant fungus may be selected from the group consisting of Candida spp., Fusarium spp., Saccharomyces cerevisiae, or Aspergillus spp..

[0129] The formulation as defined above, may be administered at a concentration in the range of about 0.5 % w / v to about 2.5 % w / v, about 0.5 % w / v to about 1.0 % w / v, about 0.5 % w / v to about 1.5 % w / v, about 0.5 % w / v to about 2.0 % w / v, about 1.0 % w / v to about 1.5 % w / v, about 1.0 % w / v to about 2.0 % w / v, about 1.0 % w / v to about 2.5 % w / v, about 1.5 % w / v to about 2 % w / v, about 1.5 % w / v to about 2.5 % w / v, or about 2.0 % w / v to about 2.5 % w / v.

[0130] The formulation as defined above, may be administered about once per day, about twice per day, about thrice per day, about four times per day, about five times per day, or about six times per day, over a period of about 5 days to about 24 days, about 5 days to about 7 days, about 5 days to about 10 days, about 5 days to about 15 days, about 5 days to about 18 days, about 5 days to about 21 days, about 7 days to about 10 days, about 7 days to about 15 days, about 7 days to about 18 days, about 7 days to about 21 days, about 7 days to about 24 days, about 10 days to about 15 days, about 10 days to about 18 days, about 10 days to about 21 days, about 10 days to about 24 days, about 15 days to about 18 days, about 15 days to about 21 days, about 15 days to about 24 days, about 18 days to about 21 days, about 18 days to about 24 days, or about 21 days to about 24 days.

[0131] The formulation as defined above, may be administered orally, intravenously, intramuscularly, intrathecally, subcutaneously, sublingually, buccally, rectally, vaginally, ocularly, oticly, nasally, intrastromally, transdermally, topically, by injection, by inhalation, or by nebulization.

[0132] The formulation as defined above, may be administered before, during, or after infection.

[0133] The formulation as defined above, may be administered to the eye of a subject.EXAMPLES

[0134] Non-limiting examples of the invention will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.

[0135] Materials and Methods

[0136] Pseudomonas aeruginosa ATCC 27853 and ATCC 9027 were obtained from ATCC, Manassas, Virginia, USA. Pseudomonas aeruginosa DX 744 and DX 783 were obtained from Asia Cornea Society for Infectious Keratitis Study (ACSIKS) repository (Singapore). Methicillin-resistant Staphylococcus aureus ATCC 700699 and ATCC 43300 were obtained from ATCC, Manassas, Virginia, USA. Methicillin-resistant Staphylococcus aureus DX 575, DX 578, DX 579, DX 590, DX 616 and DX 625 were obtained from ACSIKS repository (Singapore).

[0137] Example 1: Absence of resistance at Sub-MIC exposure of ePL.

[0138] The polymer (ePL) was challenged over a period of 21 days serial passage broth microdilution assay to determine the potential resistance induction. One quality control (. aeruginosa ATCC 27853) and two multi-drug resistant strains (DX744 and DX 783) strains were treated with either ePL or polymyxin B over a period of 21 days (Figure 1).

[0139] Sub-inhibitory exposure of ePL did not induce detectable resistance by P. aeruginosa 27853 and DX 783 strains whereas >4* increase in the MIC was observed after 12th passage by DX 744 strains. However, polymyxin B exposure resulted in >4* increase in the MIC as early as day 3 against all the strains. These results suggested sPL induced the least induction of resistance by both susceptible and drug -resistant P. aeruginosa strains. Based on these observations we hypothesized that sub -inhibitory concentrations of EPL in ophthalmic formulations did not provoke resistance induction.

[0140] Example 2: Sub-inhibitory concentration of EPL sensitizes P. aeruginosa to fluoroquinolone (FQ) antibiotics

[0141] To confirm if ePL sensitizes, the minimum inhibitory concentration of levofloxacin and moxifloxacin was determined in the presence of ePL. For a comparison, both quality control (P. aeruginosa ATCC 9027) as well as two FQ-resistant P. aeruginosa isolates collected from ACSIKS had been used for the study. Tables 1-3 show the fold reduction in the MIC of ciprofloxacin and moxifloxacin in the presence of sub-inhibitory concentrations of EPL.Table 1. Antimicrobial properties of ePL and fluoroquinolone combination against P. aeruginosa ATCC 9027

[0142] Sub-inhibitory concentration of εPL (μg / mL) MIC (fold reduction) in the presence of εPLaLevofloxacin Moxifloxacin 8 0.25 (4x)b0.25 (8x) 4 0.5 (2x) 0.5 (4x) 2 0.5 (2x) -

[0143]

[0144] aMIC of ePL, levofloxacin and moxifloxacin are 16, 1 and 2 ug / m I,. respectively.bNumbers in parenthesis indicates fold reduction in MIC.

[0145] Table 2. Antimicrobial properties of ePL and fluoroquinolone combination against clinical isolates of P. aeruginosa (DX 744), which are fluoroquinolone-resistant bacteria

[0146] Sub-inhibitory concentration of εPL (μg / mL) MIC (fold reduction) in the presence of εPLaLevofloxacin Moxifloxacin 8 64 (8x) 64 (4x) 4 - 128 (2x)

[0147]

[0148] aMIC of ePL, levofloxacin and moxifloxacin are 16, 512 and 256 ug / mL. respectively.bNumbers in parenthesis indicates fold reduction in MIC. indicates no change in MIC.

[0149] Table 3 Antimicrobial properties of ePL and fluoroquinolone combination against clinical isolates of P. aeruginosa (DX 783), which are fluoroquinolone-resistant bacteria

[0150] Sub-inhibitory concentration of EPL (pg / mL) MIC (fold reduction) in the presence of ePLaLevofloxacin Moxifloxacin 8 64 (4x) 64 (4x) 4 - 128 (2x)

[0151]

[0152] aMlC of ePL, levofloxacin and moxifloxacin are 16, 256 and 256 ug / mL. respectively.bNumbers in parenthesis indicates fold reduction in MIC.‘-‘ indicates no change in MIC.

[0153] Example 3: Sub-inhibitory concentrations of EPL attenuated the bacterial growth in combination with fluoroquinolones

[0154] To confirm ePL-FQ combinations prevented the bacterial growth, the viability of dru -resistant DX 744 and DX 783 strains after sub-inhibitory exposure to EPL and the antibiotics was determined (Figure 2).

[0155] Both sPL-lcvofloxacin and sPL-moxifloxacin combinations at sub-inhibitory concentrations abrogated the bacterial growth, indicating potent synergism between the polymcr-dnig combinations.Example 4: EPL enhances the intracellular accumulation of FQs

[0156] To infer the mechanism of enhanced antimicrobial activity of FQs in the presence of ePL, the intracellular accumulation of the antibiotics was determined against the two drug-resistant strains. The amount of drug in the bacterial cells was determined by analytical reversed-phase high performance liquid chromatography. An increased intracellular accumulation of FQs was observed at 8 pg / mL of ePL, indicating that the polymer enhanced the permeability of the antibiotics against the drug resistant strains (Figure 3).

[0157] Fluoroquinolone resistant bacterial isolates from ACS1KS (PA 744 and PA 783) were cultured in Muller-Hinton broth (Becton-Dickinson, Franklin Lakes, NJ USA) overnight and adjusted to OD600 0.45 in phosphate buffered saline (PBS), pH 7.4. To about 1.5 mL of this suspension, levofloxacin or moxifloxacin (0.4 pg / ml, Sigma-Aldrich, Inc. St. Louis, MO, USA) followed by sub-inhibitory concentrations of EPL (2 - 8 pg / mL, TNJ Chemical, China) were added. The mixtures were then incubated at 37 °C with shaking at 100 rpm for 2-6 hours. The tubes were removed and centrifuged at 13200 rpm for 1 minute to remove the supernatants and washed with PBS, pH 7.4. The pellets were resuspended in 200 µl of water for injection (B. Braun Melsungen SE, Melsungen, Germany) and heated at 95 °C for 30 min. The tubes were then cooled overnight, centrifuged and the supernatants were transferred to a sampling vial. The amount of quinolones was determined by high performance liquid chromatography (Shimadzu LC-2050C, Shimadzu Pte Ltd., Kyoto, Japan), using a linear calibration method.

[0158] Example 5: Concentrated EPL solutions have no observable adverse events level (NOAEL) injured rabbit cornea

[0159] To be utilized as a topical agent, it is important to determine effect of polymer on corneal wound healing since safety and tolerability are key determinants that affect the successful outcome of the topical drugs. The biocompatibility of a high concentration of EPL (1.0% or 2.0%, w / v in saline) in a rabbit model of corneal wound healing was examined. Deep injury was created by de-epithelialization of the rabbit cornea. The rate of wound healing was assessed in terms of wound closure at day 7 post injury’ and time required to reach 50% wound closure.

[0160] EPL-treated animals had higher wound closure (93.5±11.0% for 2% EPL and 92.6±9.9% for 1% EPL) compared to saline treated (89.2±10.9%). The mean day for 50% wound closure for vehicle group was 2.5±1.0 days compared 2.3±0.9 days and2.0±0.5 days for 1% and 2% EPL, respectively. Statistical analysis indicated no significant difference in the rate of wound closure and time to 50% heal between saline or EPL instillation (Figure 4A and B). In addition, no corneal opacity or iritis, eye lid swelling, and conjunctival congestion were observed in eyes instilled with 1% or 2% PL solution. These observations establish excellent ocular tolerance of a concentrated EPL solution.Example 6: Synergism between EPL and other antibiotics against multi-drug MRSA strains From a list of 40 MRSA isolates collected from ACSIKS studies, 6 isolates that are multi -drug resistant to various classes of antibiotics and two ATCC strains were identified (Table 4). The results suggested that MIC of ePL against these strains ranged from 8-32 μg / mL. Next, whether sub-inhibitory concentrations of ePL sensitized the antibiotics was studied by determining the MIC of antibiotics in the presence of ePL.

[0161] Table 5 shows the fold decrease in MIC of various classes of antibiotics against three different MRSA isolates. In general, 2-8 fold decrease in MIC was observed for different classes of antibiotics. Sensitization was effective in combination w ith chloramphenicol, gatifloxacin and dclafloxacin.Table 4. MIC of antibiotics against multidrug resistant and quality control MRSA strains

[0162] MRSA strains Amikacin Tobra Vanco Levo Moxi Cipro Gati \ Olio Besi Chi Dela gPL

[0163] DX 575 4 16 0.5 8 2 64 4 \ 8 0.5 8 0.5 8

[0164] DX 578 4 16 0.5 4264 4 \ 16 0.5 8 0.5 8 DX579 4 16 0.5 8 2 64 2 \ 8 0.5 8 0.5 8

[0165] DX 590 8 32 0.5 8 2 64 4 \ 16 0.5 8 0.5 8 DX616 4 32 0.5 4 2 16 4 \ 16 0.5 8 0.5 8

[0166] DX 625 4 32 0.5 4 2 16 2 \ 8 0.5 8 0.5 8 ATCC 700699 32 | >256 2 32 4 64 4 \ 16 1 8 0.5 32 ATCC 43300 32 >256 0.5 <0.125 0.25 0.25 <0.125 \ <0.125 ND 8 <0.125 8

[0167]

[0168]

[0169] Table 5. MIC of antibiotics in the presence of sub-inhibitory concentrations of EPL and the fold decrease in MIC is shown in parenthesis. indicates no change in the MIC value. '*’ indicates fluoroquinolone-resistant bacteria. _

[0170] MRSA DX 578*

[0171] Sub-inhibitory

[0172] concentration of EPL Amik Tobra Van co Chi Oflo Cipro Levo Moxi Gati Besi Dela (jig / mL)

[0173] 4 2.0 (2x) 8.0 (2x) 4.0 (2.x) 16.0 (2x) 1.0 (2x) 1.0 (2.x) 0.125 (4x). 2. | -. - 4.0 (2x) - - l.0 (2\).. T.0 (2X) -. '>.125 (4 -). j14.0 (2x) 1.0 (2x) 0.125 (4x) |

[0174] M < SA DX 61 5*.

[0175] — —

[0176] 075

[0177] 4 2.0 (2x) 16.0 (2.x) 1.0 (8x) 8.0 (2x) 8.0 (2x) 1.0 (2x) 1.0 (4x) 0.0625 (8x) (2x)

[0178] 0.25 \ to 2 16.0 (2x) - 4.0 (2x) 8.0 (2x) 8.0 (2x) - 1.0 (2x) 2.0 (2x) 0.125 (4x) (2x)

[0179] 0.25

[0180] 116.0 (2x) - 4.0 (2x) 8.0 (2x) 8.0 (2x) - 1.0 (2x) 2.0 (2x) 0.125 ( L<) (2x)

[0181] MRSA ATCC 700699*

[0182] 0.25

[0183] 16 8.0 (4x) 64(8 x) 1 (2x) 1 (8x) 8.0 (2x) 8.0 (2x) - 2.0 (2x) 1.0 (4x) 0.0625 (8x) (4x)

[0184] 0.25

[0185] 8 16.0 (2x) 256 (2x) 4 (2x) 8.0 (2x) 2.0 (2x) 0.125 ( lx) (2x)

[0186] — — —

[0187] 075

[0188] 4 4 (2x) 8.0 (2x) - 2.0 (2x) 0.125 (4x)

[0189] (2x)

[0190]

[0191] Example 7: Synergism between EPL and voriconazole

[0192] Due to the broad-spectrum antimicrobial properties of EPL, we explored the synergism between EPL and voriconazole against Fusarium solani strains. Table 6 compares the MIC90% of ePL and voriconazole against a panel of F. solani strains. Table 7 shows the fold decrease in the MIC values of voriconazole in presence of sub-inhibitory concentrations of ePL. Overall, the results suggested appreciable synergism between ePL and voriconazole.

[0193] Table 6. MIC of ePL and voriconazole against / *', solani strains

[0194] F. solani isolates

[0195] Antifungals

[0196] 46492 62877 3636 52628 EPL 64 64 64 256 Voriconazole 4 8 16 16

[0197]

[0198] Table 7. Synergism between EPL and voriconazole against four different F solani strains

[0199] Sub-inhibitory MIC of voriconazole against F.solani isolates concentration of EPL

[0200] 46492 62877 3636 52628 (pg / rnL)

[0201] 64 - - - 4 (4*) 32 2 (2*) 0.25 (32*) 4 (4*) 8 (2*) 16 2 (2*) 1.0 (8*) 4 (4*) 8 (2*) 8 2 (2*) 2.0 (4*) 4 (4*) 8 (2*) 4 - 2.0 (4*) 8 (2*) 8 (2*) 2 - - - -

[0202]

[0203] This synergism between ePL and voriconazole was further studied by determining the fractional inhibitory concentration index (FICI).

[0204] Freshly grown cells of Fusarium solani ATCC 46492, ATCC 3636, ATCC 62877, and ATCC 52628 were suspended in 5 mM HEPES buffer supplemented with 5 mM glucose (pH 7.4). The cell density was adjusted to an optical density (at a wavelength of 600 nm, OD600) of 0.4, followed by incubation with 10 μM N-phenyl-1-naphthylamine (NPN). A 600 pL aliquot of the dye-loaded cell suspension was transferred to a quartz cuvette, and fluorescence measurements were performed using a Quanta Master spectrofluorometer (Photon Technology International, NJ, USA) at an excitation wavelength of 355 nm and an emission wavelength of 405 nm. After a stable baseline was established, e-poly-L-lysine (ePL) was added, and changes in fluorescence intensity were recorded. The final concentrations of EPL tested ranged from 0.125× to 2× the minimum inhibitory concentration (MIC). For instance, Figure 5 shows the concentrationdependent changes in the fluorescence intensity at 405 nm of hydrophobic fluorescent probe N-phenylnaphthyl amine (NPN) upon addition of εPL to intact Fusarium solani ATCC 46492 over time. The increasing intensity of NPN upon addition of ePL suggests higher permeability in the presence of polymer.

[0205] Table 8 summarises the results of the FICI between ePL and voriconazole. Results from the N-phenyl-1-naphthylamine (NPN) assay indicated that at sub-minimum inhibitory concentration of ePL, the permeability of the hydrophobic fluorescent probe NPN towards intact F. solani cells increased. This led to a significant increase in the uptake of the hydrophobic fluroresecnt probe NPN at sub-MIC concentration of EPL (Figures 6A to 6D).

[0206] Table 8. Fractional Inhibitory Concentration Index

[0207] F.solani strains Fractional inhibitory concentration index with voriconazole ATCC 3636 0.26

[0208] ATCC 46492 0.56

[0209] ATCC 52628 0.37

[0210] ATCC 62877 0.25

[0211]

[0212] Example 8: ePL- Moxiflacin (in the form of Vigamox®) as a potential combination against Fusarium Solani strains

[0213] Fusarium solani ATCC 46492 and 62877 and Aspergillus flavus ATCC 204304 were cultured on potato-dextrose agar (PDA) and incubated at 29°C for 5 days. Upon reaching confluence, conidial suspensions were harvested using phosphate buffered saline (PBS; pH 7.0) and the cell concentration was adjusted to 1 x 104CFU / mL. 1 mL of the suspensions were added to 24 well plates and divided into various groups shown below in Tables 9 and 10. 70 pg of ePL was added to 0.5% w / v moxifloxacin (in the form of Vigamox®) preparations and the solution remained optically clear. 1 to 4 drops (40 pL per drop) of 0.5% w / v moxifloxacin (in the form of Vigamox®). ePL and the combination of moxifloxacin (in the form of Vigamox*) and ePL (in triplicates) were added to the fungal suspension and incubated at a temperature of either 29 °C (for growing fungal pathogen)_or 35 °C (close to body temperature). Viable fungal cells were quantified by plating on PDA at 24 and 48 hours post-treatment. Colony-forming unit (CFU) counts were enumerated, and plate images were acquired using a Scanl200 colony counter (Interscience, France).

[0214] Table 9. Monotherapy of ePL or 0.5% w / v moxifloxacin (in the form of Vigamox®) on F. solani strains Groups Details

[0215] I No treatment

[0216] II 0.5% w / v moxifloxacin (in the form of Vigamox®) - 1 drop (40 pL per drop) III 0.5% w / v moxifloxacin (in the form of Vigamox®) - 2 drops (40 pl, per drop) IV 0.5% w / v moxifloxacin (in the form of Vigamox®) - 3 drops (40 pL per drop)

[0217]

[0218] V 0.5% w / v moxifloxacin (in the form of Vigamox®) - 4 drops (40 pL per drop) VI ePL - 1 drop (40 pL per drop)

[0219] VII EPL - 2 drops (40 pL per drop)

[0220] VIII ePL - 3 drops (40 pL per drop)

[0221] IX ePL - 4 drops (40 pL per drop)

[0222]

[0223] Table 10. Combination therapy of ePL with 0.5% w / v moxifloxacin (in the form of Vigamox®) on F. solani strains

[0224] Groups Details

[0225] X No treatment

[0226] XI 0.5% w / v moxifloxacin (in the form of Vigamox®) + 70 pg / mL ePL - 1 drop (40 pL per drop) XII 0.5% w / v moxifloxacin (in the form of Vigamox®) + 70 pg / mL ePL - 2 drops (40 pL per drop)

[0227] XIII 0.5% w / v moxifloxacin (in the form of Vigamox®) + 70 pg / mL ePL - 3 drops (40 pL per drop)

[0228] XIV 0.5% w / v moxifloxacin (in the form of Vigamox®) + 70 pg / mL ePL - 4 drops (40 pL per drop)

[0229]

[0230] The results are shown in Figures 7A to 7D. At 24 or 48 hours post treatment, exposure of 0.5% w / v moxifloxacin (in the form of Vigamox®) alone resulted in a moderate decrease in viable fungal cells in a concentration-dependent manner. At 24 or 48 hours post treatment, no growth was observed in ePL monotherapy against both F. solani strains.

[0231] Against F. solani ATCC 46492 strain, no growth was observed at 24 or 48 hours post treatment when a combination of ePL with 0.5% w / v moxifloxacin (in the form of Vigamox®) was used. For 1 drop of the combination of ePL with 0.5% w / v moxifloxacin (in the form of Vigamox®), less than 10 cells were observed at 24 hours post treatment (Figure 7A) and no growth was observed at 48 h post treatment (Figure 7B). Against the F solani ATCC 62877 strain, a significant decrease in viable cell was observed at 24 hours post treatment (Figure 7C) and no growth was observed after 48 hours post treatment (Figure 7D).

[0232] These observations suggested that the combination of ePL with 0.5% w / v moxifloxacin (in the form of Vigamox®) decreased the fungal viability substantially compared to ePL or 0.5% w / v moxifloxacin (in the form of Vigamox®) alone.

[0233] Figure 8 is the representative photograph of the agar plate, showing that EPL (70 pg / mL) was dissolved in 0.5% moxifloxacin (in the form of Vigamox®) which confirmed the lack of fungal growth upon exposure to the combination therapy.Example 9: Synergism between EPL and 0.5% w / v moxifloxacin against Aspergillus flavus

[0234] No synergism between ePL and 0.5% w / v moxifloxacin (in the form of Vigamox®) against Aspergillus flavus was observed in vitro.

[0235] In a mice model randomly divided into four groups:

[0236] Group 1: Untreated control;

[0237] Group 2: ePL alone (70 pg / mL in PBS. pH 7.0);

[0238] Group 3: 0.5% w / v moxifloxacin (in the form of Vigamox®); and

[0239] Group 4: 0.5% w / v moxifloxacin (in the form of Vigamox®) with 70 pg / mL ePL

[0240] Each group consisted of 4 to 6 mice. All animals received systemic immunosuppression via intraperitoneal injection of methylprednisolone (100 mg / kg) one day prior to fungal inoculation. On the day of inoculation, mice were administered general anesthesia by intraperitoneal injection of xylazine hydrochloride (10 mg / kg) and ketamine hydrochloride (80 mg / kg) (Ilium Troy, Australia), along with topical anesthesia using proxymetacaine hydrochloride (0.5%) (Bausch & Lomb, UK) or one drop of 1 to 5% lignocaine hydrochloride. The central 2 mm corneal epithelium was gently debrided using sterile mini-blades (#64, Beaver, MA, USA). The wound site was denuded of epithelium, leaving the basal lamina intact and the cornea was irrigated with sterile saline to remove debris and residual topical anaesthetic agent. Discomfort was prevented by application of Buprenorphine, 0.05 to 0.1 mg / kg (subcutaneous administration for two times / day) for 1 day, immediately after surgery. After which, 10 pL of a suspension containing 7×105colony-forming units (CFU) Aspergillus flavus ATCC 204304 was applied to the wounded cornea. Topical treatments were initiated 24 hours post-inoculation (p.i.). Infected corneas received 10 pL of the assigned treatment eight times on day 1 post-infection and twice on day 2 post-infection (day 1 post treatment (p.t.)) At the study endpoint, cornea was collected from untreated and treated mice for estimating the CFU / mL. Disease progression was monitored daily using slit -lamp biomicroscopic photography and anterior segment optical coherence tomography (AS-OCT) (Figure 9). Two hours after the final treatment on day 2 postinfection, mice were humanely euthanized, corneas were excised, and viable fungal burden was determined by plating for CFU enumeration (Figure 10).

[0241] Based on the results obtained in Figures 9 and 10, the untreated groups contained significant presence of fungal burden in 4 out of 5 groups, while a combination of EPL (70 pg / mL) and 0.5% w / v moxifloxacin (in the form of Vigamox®)(5 mg / mL) sterilized the cornea for all the treatment groups while corneas treated with monotherapies cleared fungal burden in only 4 out of 5 groups.INDUSTRIALLY APPLICABILITY

[0242] The disclosed method of preparation may be useful in facile, versatile and efficient preparation of a formulation as defined above. The formulation may be useful in treating a microbial infection. The formulation may be useful as a medicament, or for use in treatment of a microbial infection. The disclosed method of treatment may also be useful in treating a microbial infection. The formulation as defined above may be applied in antimicrobial ophthalmic eye drops formulation containing sub-inhibitory concentration of ePL. The formulation as defined above may be safe to use in the treatment of a microbial infection.

[0243] It will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.

Claims

CLAIMS1. A formulation comprising:i) epsilon polylysine; andii) an antimicrobial compound,wherein the epsilon polylysine is present in the formulation at a concentration in the range of 0.0001 μg / mL to 70 μg / mL.

2. The formulation according to claim 1, wherein the formulation is in a buffer with a pH in the range of 5.5 to 8.

3. The formulation according to claim 2, wherein the buffer is selected from the group consisting of phosphate buffered saline (PBS), borate buffered saline (BBS), citrate buffer, tris(hydroxymethyl)aminomethane (TRIS), 4-(2-hydroxyethy1)-l -piperazineethanesulfonic acid (HEPES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES), 1,4-Piperazinediethanesulfonic acid (PIPES) or 4- Morpholineethanesulfonic acid (MES).

4. The formulation according to any of claims 1 to 3, wherein the antimicrobial compound is an antibacterial compound.

5. The formulation according to claim 4, wherein the antibacterial compound is present in the formulation at a concentration in the range of 0.1 μg / mL to 6500 μg / mL.

6. The formulation according to claims 4 or 5, wherein the antibacterial compound is an amphenicol.

7. The formulation according to claim 6, wherein the amphenicol is selected from the group consisting of thiamphcnicol, azidamfcnicol, florfcnicol, chloramphenicol or any mixture thereof.

8. The formulation according to claims 4 or 5, wherein the antibacterial compound is a fluoroquinolone.

9. The formulation according to claim 8, wherein the fluoroquinolone is selected from the group consisting of levofloxacin, moxifloxacin, ciprofloxacin, gemifloxacin, ofloxacin, besifloxacin, finafloxacin, gatifloxacin, delafloxacin, danofloxacin, difloxacin, enrofloxacin, ibafloxacin, marbofloxacin, orbifloxacin, pradofloxacin or any mixture thereof.

10. The formulation according to any of claims 1 to 3, wherein the antimicrobial compound is an antifungal compound.

11. The formulation according to claim 10, wherein the antifungal compound is present in the formulation at a concentration in the range of 0.1 μg / mL to 10 μg / mL.

12. The formulation according to claims 10 or 11, wherein the antifungal compound is selected from the group consisting of fluconazole, fosfluconazole, fosravuconazole, hexaconazole, isavuconazole, itraconazole, luliconazole, osteseconazole, prosaconazole, voriconazole or any mixture thereof.

13. The formulation according to any of the preceding claims, wherein the epsilon polylysine is present in the formulation at a concentration in the range of 0.0001 pg / mL to 20 pg / mL.

14. The formulation according to any of the preceding claims, wherein the epsilon polylysine is present in the formulation at a concentration in the range of 0.0001 pg / mL to 10 pg / mL.

1. The formulation according to any of the preceding claims, wherein the epsilon polylysine is present in the formulation at a concentration in the range of 0.0001 pg / mL to 5 pg / mL.

16. A method of preparing a formulation according to any of the preceding claims, comprising a step of mixing:i) epsilon polylysine; andii) an antimicrobial compound,wherein the epsilon polylysine is present in the formulation at a concentration in the range of 0.0001 pg / mL to 70 pg / mL.

17. The method according to claim 16, further comprising a step of dissolving the formulation in a buffer with a pH in the range of 5.5 to 8.

1. The method according to claim 17, wherein the buffer is selected from the group consisting of phosphate buffered saline (PBS), borate buffered saline (BBS), citrate buffer, tris(hydroxymethyl)aminomethane (TRIS), 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES), 1,4-Piperazinediethanesulfonic acid (PIPES) or 4- Morpholineethanesulfonic acid (MES).

19. The method according to claim 16 or 17, further comprising a step of adding a polymer additive at a concentration in the range of about 0.001% to about 0.01% (w / v) to the formulation.

20. The method according to claim 19, wherein the polymer additive is be selected from the group consisting of alginate, pectin, gelatin, gellan, chitosan, polyvinyl alcohol, polyamides, polycarbonates, polyalkylene glycols, polyvinyl ethers, methylcellulose, ethylcellulose, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose, hydroxypropyl methylcellulose (HPMC), carboxymethyl cellulose (CMC), polymethacrylates, hyaluronic acid, polycarbophil, or any mixture thereof.

21. A formulation according to any of claims 1 to 15, comprising:i) epsilon polylysine; andii) an antimicrobial compound,wherein the epsilon polylysine is present in the formulation at a concentration in the range of 0.0001 μg / mL to 70 μg / mL. for use in treating and / or preventing a microbial infection.

22. A method of treating and / or preventing a microbial infection, comprising a step of administering a formulation according to any of claims 1 to 15, comprising:i) epsilon polylysine; andii) an antimicrobial compound,wherein the epsilon polylysine is present in the formulation at a concentration in the range of 0.0001 μg / mL to 70 μg / mL.

23. A formulation according to any of claims 1 to 15, comprising:i) epsilon polylysine; andii) an antimicrobial compound,wherein the epsilon polylysine is present in the formulation at a concentration in the range of 0.0001 μg / mL to 70 μg / mL, for use as a medicament.

24. Use of a formulation according to any of claims 1 to 15, comprising:i) epsilon polylysine; andii) an antimicrobial compound,wherein the epsilon polylysine is present in the formulation at a concentration in the range of 0.0001 μg / mL to 70 μg / mL, in the manufacture of a medicament for treating and / or preventing a microbial infection.

25. The use according to claim 24, wherein the microbial infection is selected from the group consisting of Blepharitis, Hordeolum, Preseptal Cellulitis, Dacryocystitis, Orbital Cellulitis, Erysipelas,Keratomycosis, Keratoconjunctivitis, Conjunctivitis, Conjunctival Laceration, Superior Limbic Keratoconjunctivitis, Keratitis, Comeal Ulceration, Phlyctenulosis, Anterior Uveitis, Endophthalmitis, Abscess, Papillitis, Scleritis or Orbital Cellulitis.

26. The use according to claim 24 or 25, wherein the microbial infection is a bacterial infection.

27. The use according to claim 26, wherein the bacterium is selected from the group consisting of Acinetobacter baumannii, Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Pseudomonas spp., Staphylococcus spp., Streptococcus spp., Enterococcus faecalis or Enterococcus faecium.

28. The use according to claim 26 or 27, wherein the microbial infection is a drug resistant bacterial infection.

29. The use according to claim 28, wherein the drug resistant bacterium is selected from the group consisting of carbapenam-resistant Enterobacter strains (CRE), Methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin-resistant Enterococci (VRE), polymixin B -resistant Enterobacter cloacae, drug resistant Acinetobacter baumannii, prolific biofilm forming P. aeruginosa or S. epidermidis strains, multi -drug resistant Pseudomonas spp., multi-drug resistant Staphylococcus spp., or multi-drug resistant Streptococcus spp..

30. The use according to claim 24 or 25, wherein the microbial infection is a fungal infection.

31. The use according to claim 30, wherein the fungus is selected from the group consisting of Candida spp., Fusarium spp., Saccharomyces cerevisiae, or Aspergillus spp..

32. The use according to claim 30 or 31, wherein the microbial infection is a drug resistant fungal infection.

33. The use according to claim 32, wherein the drug resistant fungus is selected from the group consisting of Candida spp.. Fusarium spp., Saccharomyces cerevisiae, or Aspergillus spp..

34. The use according to any one of claims 24 to 33, wherein the formulation is to be administered at a concentration in the range of 1.0 % w / v to 2.0% w / v.

35. The use according to any one of claims 24 to 34, wherein the formulation is to be administered over a period of 7 days to 21 days.

36. The use according to any one of claims 24 to 35, wherein the formulation is to be administered before, during, or after infection.

37. The use according to any one of claims 24 to 36, wherein the formulation is to be administered to the eye of a subject.

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