WO2016072939A1

WO2016072939A1 - Formulations comprising antimicrobial agents with hydrophobic moieties and uses thereof

Info

Publication number: WO2016072939A1
Application number: PCT/SG2015/050435
Authority: WO
Inventors: Chi Lui Paul HO; Chun Hwee Desmond TEO; Woon Pei Jeanette TEO; Hai Ning PEE
Original assignee: National University Of Singapore; National University Hospital(S) Pte Ltd
Priority date: 2014-11-07
Filing date: 2015-11-05
Publication date: 2016-05-12

Abstract

The present disclosure discusses formulations comprising hydrophobic antimicrobial agents and uses thereof. More precisely, the present disclosure refers to a composition comprising a hydrophobic antimicrobial agent and a cyclodextrin.

Description

FORMULATIONS COMPRISING ANTIMICROBIAL AGENTS WITH

HYDROPHOBIC MOIETIES AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of priority of Singapore provisional application No. 10201407358Q, filed November 07, 2014, the contents of it being hereby incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

[002] The present invention relates to the field of microbiology. In particular, the present invention relates to the use of a composition comprising antimicrobial agents for the prevention or treatment of infections, or for decolonization.

BACKGROUND OF THE INVENTION

[003] Nosocomial, or hospital-acquired, infections (HAIs) have a significant impact on global healthcare costs. Surgical site infections (SSIs) are the second most prevalent hospital- acquired infections in the United States with approximately 300,000 occurrences yearly, preceded only by urinary tract infections. Surgical site infections complicate about 5% of surgeries, where the severity ranges from superficial skin infections to life-threatening conditions. Staphylococcus aureus (SA) is commonly isolated in surgical site infections, where most strains are methicillin -resistant (MRS A). The high costs and morbidities associated with surgical site infections have drawn great concern.

[004] Intranasal mupirocin and photodisinfection have shown to reduce surgical site infections risks by a significant degree. However, their effectiveness may be limited by the anatomical restrictions of the nasal cavity, as the current methods have a finite capacity to reach the tortuous areas of the nose, especially the sinuses. For the application of ointments, for example, the extent of application is dependent on the skill of the caregiver; while for photodisinfection, it varies based on the flexibility and length of the probe used.

[005] In light of the problems encountered in the art, there is a need for a composition that can be used for reducing the risk of such infections. SUMMARY OF THE INVENTION

[006] In one aspect, the present invention refers to a composition comprising one or more antimicrobial agent with hydrophobic moieties and a cyclodextrin.

[007] In another aspect, the present invention refers to a composition as defined herein for use in treating an infection.

[008] In yet another aspect, the present invention refers to a method of treating or preventing an infection in a patient, or for decolonizing an orifice of a patient, wherein the method comprised administering to the patient a therapeutically effective amount of the compositions as defined herein

[009] In a further aspect, the present invention refers to use of the composition as defined herein in the manufacture of a medicament for preventing or treating an infection, or for decolonizing an orifice of a patient.

[010] In one aspect, the present invention refers to a method of producing the compositions as described herein, the method comprising: (a) mixing a ground cyclodextrin, as defined herein, and one or more ground antimicrobial agent, as defined herein; and (b) constantly agitating and heating the mixture formed under (a) .

BRIEF DESCRIPTION OF THE DRAWINGS

[011] 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:

[012] Fig. 1 shows a chromatogram of mupirocin obtained with the mupirocin HPLC assay parameters as described in the experimental section and the example section below.

[013] Fig. 2 shows further chromatograms depicting mupirocin undergoing acid degradation at 40°C. Indicated in arrows are peaks from the degraded products of mupirocin.

[014] Fig. 3 shows further chromatograms depicting mupirocin undergoing base degradation at room temperature. Indicated in arrows are peaks from the degraded products of mupirocin.

[015] Fig. 4 shows further chromatograms showing mupirocin undergoing base degradation at 40°C. Indicated in arrows are peaks from the degraded products of mupirocin. [016] Fig. 5 shows a line chart depicting the stability of MPC-HPpCD-NaCl in aqueous solution, evaluated over 8 weeks stored at room temperature with light exposure. The composition is considered to be stable when exposed to this condition.

[017] Fig. 6 shows a line chart depicting the stability of MPC-HPpCD-NaCl in aqueous solution, evaluated over 8 weeks stored at room temperature without light exposure. The formulation is considered to be stable when exposed to this condition.

[018] Fig. 7 shows a scatterplot depicting the increase in concentration of 2-hyrdoxypropyl-P-cyclodextrin (HPpCD) increases the solubility of mupirocin in water when reconstituted. The composition tested was a mupirocin composition produced using the non-aqueous method as described herein.

[019] Fig. 8 shows a column chart showing the concentration of mupirocin at saturation in 2-hydroxypropyl-P-cyclodextrin (HPpCD) and varying concentrations of carboxymethyl cellulose (CMC) and sodium chloride (NaCl). The addition of CMC beyond 1.0% w/v reduces the saturation concentration of mupirocin (MPC) in HPpCD. The addition of NaCl does not impact the saturation concentration of MPC in HPpCD.

[020] Fig. 9 shows a photograph of a bacterial plate with a result of the time-dependent bactericidal activity for a mupirocin composition. MRSA 2 was added to mupirocin- 2-hydroxypropyl-P-cyclodextrin (MPC-HPpCD) composition and the bacteria was plated at 15 minutes. This plate was observed after 24 hours incubation. The bacteria had been plated at dilutions of undiluted (ND), lxlO¹ (1E1) dilution, lxlO³ (1E3) dilution and lxlO² (1E2) dilution (clockwise from left top). It is observed that there were no bacteria colonies in undiluted and lxlO¹ (1E1) dilution quadrants due to the residual drug inhibiting the growth of surviving bacteria. In the 1x10 (1E2) dilution quadrant, the uneven spread and differently sized bacteria colonies indicate possible partial inhibition of bacteria growth.

[021] Fig. 10 shows a line chart depicting the time-kill curves of mupirocin against MRSA 2 (A) and MRSA 7 (B). Bactericidal activity is defined as a "reduction of 99.9% (> 3 logio) of the total number of CFU/mL in the original inoculum". The bactericidal level is indicated by the dashed line. The MRSA 2 bacteria strain is MPC resistant.

[022] Fig. 11 shows a chromatogram of octenidine obtained with the octenidine HPLC assay parameters as described below. [023] Fig. 12 shows the Ql MS spectrum of OCT-HPpCD-NaCl dry powder formulation prepared two months prior to the formulation. Shown here is the total ion count (TIC) comparable to the freshly prepared formulation in Fig. 13 indicating stability of the sample.

[024] Fig. 13 shows the Ql MS Spectrum of OCT-HPpCD-NaCl dry powder formulation freshly formulated two months later than the composition shown in Fig. 12. Comparing the two spectrum and considering the ratio of the intensity of the parent ion (m/w = 551.7) and total ion count (TIC), there is no degradation observed in the OCT-HPpCD- NaCl dry powder formulation.

[025] Fig. 14 shows a scatterplot of data points depicting octenidine saturation in a composition comprising 2-hyrdoxypropyl-P-cyclodextrin (HPpCD) or a-cyclodextrin (a-CD). The increase in concentration of HPpCD and a-CD increases the solubility of octenidine in water in the presence of 2.8% NaCl.

[026] Fig. 15 shows a column chart showing that the increase in concentration of 2-hyrdoxypropyl-P-cyclodextrin (HPpCD) and a-cyclodextrin (a-CD) increases the solubility of octenidine in water in the presence of 2.8% sodium chloride (NaCl). It is observed that the concentration of octenidine dissolved in water in the presence of 2.8% NaCl, without cyclodextrin, is only 0.11 mg/ml.

[027] Fig. 16 shows a diagram of a 96-well, round-bottom, microtiter plate layout for Minimum Inhibitory Concentration (MIC) performed using the Broth Microdilution Method with 7 different strains of Staphylococcus aureus. The minimum inhibitory concentration was recorded as the lowest antimicrobial concentration where no bacteria growth is observed after overnight incubation.

[028] Fig. 17 shows a photograph of a bacterial plate with a result of the time-dependent bactericidal activity for an octenidine composition. MRSA 2 was added to OCT-HPpCD formulation and the bacteria were plated at 30 minutes. This plate was observed after 24 hours incubation. The bacteria were plated at dilutions of undiluted (ND), lxlO¹ (1E1) dilution, 1x10 3 (1E3) dilution and 1x102 (1E2) dilution (clockwise from left top). It is observed that there were no bacteria colonies in non-diluted and lxlO¹ (1E1) dilution quadrants due to the residual drug inhibiting the growth of surviving bacteria. In the 1x10 (1E2) dilution quadrant, the uneven spread of bacteria colonies indicates possible partial inhibition of bacteria growth. [029] Fig. 18 shows a line chart depicting the time-kill curves of Octenidine against MRSA 7. Bactericidal activity is defined as a "reduction of 99.9% (> 3 logio) of the total number of CFU/mL in the original inoculum". The bactericidal level is indicated by the dashed line.

[030] Fig. 19 shows a chromatogram of triclosan obtained with the triclosan HPLC assay parameters.

[031] Fig. 20 shows a scatterplot of data points depicting the solubility of triclosan in compositions produced using the non-aqueous (dry heat mixing) method as described herein. The non-aqueous dry physical mixing method produces a dry powder complex for reconstitution. Increasing the concentrations of 2-hyrdoxypropyl-P-cyclodextrin (HPpCD) and carboxymethyl cellulose (CMC) improved the aqueous solubility of triclosan (TCS) remarkably, with an optimal composition of 40mM HPpCD and 1% w/v CMC, for a concentration of 0.3% w/v TCS.

[032] Fig. 21 shows a scatterplot of data points depicting the solubility of triclosan in lyophilised compositions produced as described herein. Similar observations as shown in Fig. 18 above were seen for the aqueous method of slurry complexation. The slurry obtained from kneading is placed under a freeze-drying process to obtain a dry powder. The optimal composition is also at 40mM HPpCD and 1% w/v CMC, for a concentration of 0.3% w/v TCS.

[033] Fig. 22 shows a line chart depicting the viscosities of MilliQ water (negative control/baseline) and the formulations containing concentrations of carboxymethyl cellulose (CMC) between 0.25% to 5% w/v CMC were measured. Concentrations up to 1% w/v CMC had comparable viscosities to MilliQ water, allowing for use in a nebuliser suitably.

[034] Fig. 23 shows the Ql MS Spectrum of TCS-HPpCD-NaCl dry powder formulation prepared two months prior to the formulation shown in Fig. 24. Shown here is the total ion count (TIC) comparable to the freshly prepared formulation in Fig. 24 indicating stability of the sample.

[035] Fig. 24 shows the Ql MS Spectrum of TCS-HPpCD-NaCl dry powder formulation freshly formulated two months later than the formulation shown in Fig. 23 above. Comparing the two spectrum and considering the ratio of the intensity of the parent ion (m/w = 288.9) and total ion count (TIC), there is no degradation observed in the TCS-HPpCD-NaCl dry powder formulation. [036] Fig. 25 shows a line plot depicting data showing the stability of aqueous TCS- HPpCD-NaCl formulation evaluated over 72 hours stored at room temperature with light exposure. The formulation is considered to be stable when exposed to this condition.

[037] Fig. 26 shows a line plot depicting data showing the stability of aqueous TCS- HPpCD-NaCl formulation evaluated over 72 hours stored at room temperature without light exposure. The formulation is considered to be stable when exposed to this condition.

[038] Fig. 27 is a photograph showing the full experimental set-up of the nebulising procedure for swab samples obtained from twenty healthy volunteers.

[039] Fig. 28 is a photograph showing examples of experimental culture tubes before 72 hour incubation at 37°C. Negative control ("Control"), positive control ("CI 9") and test ("T19") are shown here.

[040] Fig. 29 is a photograph showing examples of experimental culture tubes after 72 hour incubation at 37°C. Negative control ("Control"), positive control ("CI 3") and test ("T13") are shown here. Turbidity can be observed for the tube containing the positive control.

[041] Fig. 30 is a photograph showing the side -by-side comparison of a positive control ("C13") and test ("T13") after 72 hour incubation at 37°C. Turbidity can be observed for the positive control, while a clear solution is seen for the test.

[042] Fig. 31 shows a line chart depicting the time-kill curves of triclosan against MRS A 2 (A) and MRSA 7 (B). Bactericidal activity is defined as a "reduction of 99.9% (> 3 logio) of the total number of CFU/mL in the original inoculum". The bactericidal level is indicated by the dashed line.

DETAILED DESCRIPTION

[043] Nosocomial, or hospital-acquired infections (HAIs) are an important category of hospital- acquired conditions and are commonly transmitted when health care providers become complacent and/or do not practice correct hygiene regularly. Also, increased use of outpatient treatment in recent decades means that a greater percentage of people who are hospitalized today are likely to be seriously ill with more weakened immune systems than in the past. Moreover, some medical procedures bypass the body's natural protective barriers. Since medical staff routinely moves from patient to patient, the staff themselves serve as a means for spreading pathogens, thus becoming possible vectors for the transmission of such infections. Microbes found in these environments can more often than not survive for a long time on surfaces in the hospital and enter the body through, for example, wounds, catheters, and ventilators. Orifices in the body of a patient can thus become susceptible to and can even facilitate various infections in a hospital setting, especially where the immune system is weak or when the patient becomes exposed to resistant microbes.

[044] The problem with compositions used for such purposes and which are known in the art is the inferior solubility of active substances, such as antimicrobial agents with hydrophobic moieties or hydrophobic antimicrobial agents, resulting in formulations which are, for example, not easily applicable.

[045] The inventors surprisingly found that mixing of such antimicrobial agents with hydrophobic moieties or hydrophobic antimicrobial agents with cyclodextrin enhances the solubility in aqueous solution and thus applicability of such compositions.

[046] The present disclosure therefore describes compositions comprising an antimicrobial agent and a cyclodextrin. The terms "composition" and "formulation" are used herein are interchangeable. In one example, the composition comprises an antimicrobial agent with hydrophobic moieties and a cyclodextrin. In another example, the antimicrobial agent is hydrophobic. As used herein, the term "hydrophobic" refers to a physical property of any molecule to seemingly repelled from a mass of water, that is to say, there is a lack of attractive forces between the molecule and the solvent, in this case water. As used herein, the term "hydrophobic moiety" refers to the physical property of a portion of a molecule to lack an affinity to water. This means that a molecule with hydrophobic moieties may also contain hydrophilic moieties and may also display the same characteristics as a hydrophobic molecule. A hydrophobic molecule, on the other hand, is understood to be hydrophobic in all moieties. This hydrophobicity of the antimicrobial agent also plays a role in its ability to be dissolved in water. It is understood in the art that hydrophobic compounds are or compounds with hydrophobic moieties may be difficult to dissolve, that is the solubility of hydrophobic compounds is low, whereas hydrophilic compounds are easily dissolved in water.

[047] As used herein, the term "antimicrobial agent" refers to a hydrophobic agent or agents that are able to complex with cyclodextrins or their derivatives used to kill or inhibit the growth of microbes and microorganisms. This agent may be chemical or biological in nature, or, synthetic or natural in origin. An antimicrobial agent may include, but is not limited to, antibacterial agents, antiseptic agents, antibiotics, fungicides, bacteriostats, sanitisers, disinfectants and sterilisers. Examples of an antimicrobial agent may be, but are not limited to ethyl alcohol, isopropyl alcohol, benzalkonium chloride, cetrimide, methylbenzethonium chloride, benzethonium chloride, cetalkonium chloride, cetylpyridinium chloride, dofanium chloride, domiphen bromide, chlorhexidine gluconate, chlorhexidine acetate, proflavine hemisulphate, triphenylmethane, brilliant green, crystal violet, gentian violet, hydrogen peroxide solution, potassium permanganate solution, benzoyl peroxide, chlorocresol, chloroxylenol, chlorophene, hexachlorophane/hexachlorphene, mupirocin calcium, octenidine dihydrochloride, triclosan, polyhexanide, chlorhexidine triclocarban ,hydroxyquinoline sulphate, potassium hydroxyquinoline sulphate, chlorquinaldol, dequalinium chloride, di-iodohydroxyquinoline, Burow's solution (aqueous solution of aluminium acetate), iodine, bleach, azelaic acid, derivatives and combinations thereof. Depending on the microbe in question, the antimicrobial agent may be triclosan, octenidine dihydrochloride, mupirocin calcium or derivatives thereof. In one example, the antimicrobial agent is mupirocin (also mupirocin calcium: MPC). In another example, the antimicrobial agent is octenidine (also octenidine dihydrochloride; OCT). In yet another example, the antimicrobial agent is triclosan (TCS). Combinations of various antimicrobial agents may also be used. These combinations may comprise at least one or more antimicrobial agents, for example two, three or more antimicrobial agents. For example, triclosan may alternatively be used as a preservative. In a low concentration, it may be formulated with, for example, mupirocin, to overcome resistance to some mupirocin-resistant MRSA. Therefore, in one example, the composition may comprise mupirocin and triclosan. In another example, the composition may comprise mupirocin and octenidine. In yet another example, the composition may comprise octenidine and triclosan.

[048] The bispyridinamine octenidine dihydrochloride is a cationic compound with two positively-charged active centers together with long hydrocarbon chains. It is a broad spectrum antimicrobial agent, effective against both Gram-negative and Gram-positive bacteria including MRSA, as well as certain fungi. This is due to its ability to adhere and interact with the cell wall and cell wall components. Aside from its wide spectrum of activity, octenidine dihydrochloride has generally superior antimicrobial efficacy in vitro and bactericidal activity as compared to chlorhexidine and alexidine. It is also proven to be more effective than chlorhexidine and alexidine in the inhibition of plaque-forming enzymes of Streptococcus mutans leading to better oral health. Hence, octenidine dihydrochloride can also be formulated as a mouthwash. Octenidine is not absorbed during oral or topical administration. Its ability to bind readily to negatively-charged surfaces allows it to exert a long residual effect. Furthermore, bacteria are unlikely to develop resistance to octenidine dihydrochloride due to its mechanism of action. There is also no difference in its effectiveness against MRSA and MSSA bacteria strains. Hence, in one example, the antimicrobial agent is octenidine or a derivative thereof. In another example, the antimicrobial agent is octenidine dihydrochloride.

[049] Formulations of octenidine dihydrochloride with and without hypertonic saline can have applications in nasal decolonisation, reducing the risk of surgery-related infections. In addition, octenidine dihydrochloride compositions with hypertonic saline can be used as an adjunctive treatment for many sino-nasal conditions such as sinusitis, rhinitis and respiratory diseases such as bronchiolitis. As a saline concentration of more than 3.0% could cause irritation to the mucous membrane, a concentration of 2.8% sodium chloride was adopted during formulation development. The nasal spray, irrigation or nebulised solution of octenidine dihydrochloride formulation with or without sodium chloride may also be administered before an operation for nasal decolonization. Hence, a formulation of the compositions as described herein comprising an antimicrobial agent is considered for nasal decolonisation.

[050] Mupirocin is an antibiotic produced by the bacterium Pseudomonas fluorescens which acts on isoleucyl tRNA synthetase and thereby inhibiting protein synthesis, eventually leading to the bacteria's death. It has activity against most gram-positive and gram-negative bacteria. Presently, mupirocin is used as an ointment at 2% concentration. Presently, the standard of case is application of mupirocin 2% ointment two to three times daily, for up to ten days. The nasal carriage is generally decolonized after five to seven days of treatment. Although mupirocin is established as the best topical antimicrobial agent against Gram- positive bacteria, there is emergence of mupirocin resistance characterized by the mutation of the mupA gene which is reported to range from 1% to 81%. Octenidine dihydrochloride thus can be formulated together with mupirocin to overcome mupirocin resistance. Thus, in one example, the present disclosure refers to a composition comprising an antimicrobial agent, wherein the antimicrobial agent is mupirocin or a derivative thereof. In another example, the antimicrobial agent is mupirocin calcium. [051] Triclosan (TCS) is an antibacterial and antifungal agent which is shown to have broad spectrum activity, and is particularly effective against Methicillin-resistant Staphylococcus aureus (MRSA). At high concentrations, triclosan acts as a biocide with multiple cytoplasmic and membrane targets. At the lower concentrations, such as those concentrations of triclosan seen in commercial products, triclosan appears bacteriostatic, and it targets bacteria primarily by inhibiting fatty acid synthesis. Use of triclosan in consumer products is prevalent. Such widespread use resulted in the EU Scientific Committee on Consumer Safety undertaking an extensive review of antimicrobial resistance related to triclosan (TCS) in 2010 and found no evidence of human pathogenic resistance, although they suggested its non-medical use to be more restricted. Thus, in one example, the compositions as described herein comprise the antimicrobial agent triclosan or a derivative thereof.

[052] Antimicrobial agents may be effective at different concentrations, depending on the microorganism in question and the desired effect, that is, if the intention is to eradicate or to impede growth and/or proliferation of the microorganism. Thus, in one example, the concentration of the antimicrobial agent in the composition may be between about 0.1% w/v to about 10% w/v, between about 0.05% w/v to about 5% w/v, between about 0.1% w/v to about 4% w/v, between about 0.3% w/v to about 2% w/v, between about 0.1% w/v to about 1.5% w/v, between about 0.15% w/v to about 2% w/v, between about 2% w/v to about 3% w/v, between about 0.1% w/v to about 2% w/v, about 0.05% w/v, about 0.1% w/v, about 0.15% w/v, about 0.2% w/v, about 0.25% w/v, about 0.3% w/v, about 0.35% w/v, about 0.4% w/v, about 0.5% w/v, about 0.75% w/v, about 1.0% w/v, about 1.25% w/v, about 1.5% w/v, about 2% w/v, about 2.5% w/v, about 3% w/v or about 3.5% w/v. In one example, the concentration of the antimicrobial agent in the composition is between about 0.1% w/v and about 2% w/v. In one example, the concentration of the antimicrobial agent in the composition is 0.1% w/v. In another example, the concentration of the antimicrobial agent in the composition is 0.3% w/v. In another example, the concentration of the antimicrobial agent in the composition is 2% w/v. As used herein, the term "w/v" refers to the ratio of weight per volume.

[053] As uses herein, the term "cyclodextrin (CD)" or "cyclodextrins" refers to cyclic oligosaccharides that contain 6 or more D-(+) glucopyranose units that are attached by α, β, or γ-(1 ,4) glucosidic bonds. It has been shown that cyclodextrins are able to form complexes with a variety of hydrophobic molecules due to their unique structure. Cyclodextrin derivatives are extensively used in research labs, for example to remove cholesterol from cultured cells and they are well known in the pharmaceutical industry for their ability to solubilise drugs. Cyclodextrins are able to form inclusion complexes with, for example, many drugs by taking up the whole drug, or, more commonly, the lipophilic moiety of the drug molecule, into a cyclodextrin cavity. Examples of cyclodextrins can be, but are not limited to, a-cyclodextrin (a-CD), β-cyclodextrin (β-CD) and γ-cyclodextrin (γ-CD), each containing six, seven, and eight glucopyranose units, respectively. Of these cyclodextrins, β-cyclodextrin is the most useful pharmaceutical complexing agent due to its cavity size, availability, low cost and other properties. Accordingly, cyclodextrin is used in the compositions to improve the aqueous solubility of the antimicrobial agent in aqueous solution. In one example, cyclodextrin is used to improve the aqueous solubility of triclosan (TCS), which is known as a poorly water-soluble phenolic antiseptic. The composition therefore comprises triclosan and cyclodextrin. In another example, cyclodextrin is used to improve the aqueous solubility of mupirocin, the composition thus comprising mupirocin and cyclodextrin. In another example, cyclodextrin is used to improve the aqueous solubility of octenidine dihydrochloride, the composition thus comprising octenidine dihydrochloride and cyclodextrin. Examples of a cyclodextrin or derivatives thereof may be, but are not limited to, hydroxypropyl-β- cyclodextrins and methylated cyclodextrins. Thus, in one example, the cyclodextrin is β- cyclodextrin. 2-hydroxypropyl^-cyclodextrin (ΗΡβΟϋ) is known to have a low toxicity as compared to other cyclodextrins. Therefore, in another example, the cyclodextrin is 2-hydroxypropyl^-cyclodextrin (ΗΡβΟϋ).

[054] Even though octenidine dihydrochloride has comparatively good solubility in water, it is unable to co-dissolve with 2.8% w/v sodium chloride (NaCl) and will precipitate in saline solution. The formulation of octenidine dihydrochloride together with 2-hydroxypropyl^-cyclodextrin (ΗΡβΟϋ) greatly improved the aqueous solubility of octenidine dihydrochlroide in water in the presence of sodium chloride (NaCl). This formulation, when prepared with the addition of 2.8% w/v sodium chloride, improves the antiseptic and irrigative properties of the formulation. In one example, the composition of the formulation comprises 0.1% w/v octenidine dihydrochloride (OCT) and 50 mM 2-hydroxypropyl^-cyclodextrin (ΗΡβΟϋ). 2.8% w/v sodium chloride can be added when required. The pH of the octenidine dihydrochloride compositions (OCT-HPpCD and OCT- HPpCD-NaCl) is between 6 and 7, making it compatible with the nasal cavity which has an average pH of approximately 6.3.

[055] The concentration of cyclodextrin required to improve the aqueous solubility of a antimicrobial agent may vary depending on the physic-chemical characteristics of the antimicrobial agent, such as chemical polarity, hydrophobicity, solubility, temperature, pressure, the solvent and the presence of other solutes in the solvent. Therefore, the cyclodextrin may be present in a range between about 20 mM to about 200 mM, between about 20 mM to 100 mM, between about 20 mM to about 30 mM, between about 25 mM to about 45 mM, between about 35 mM to about 50 mM, between about 55 mM to about 70 mM, between about 65 mM to about 85 mM, between about 80 mM to about 90 mM, between about 90 mM to about 100 mM, between about 85 mM to about 95 mM, or may be present in amount of about 20 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 49 mM, about 50 mM, about 55 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 95 mM, about 100 mM, about 110 mM, or about 200mM. In one example, cyclodextrin is present in a concentration of about 40 mM to about 100 mM. In another example, cyclodextrin is present in a concentration of about 40 mM. In yet another example, cyclodextrin is present in a concentration of about 50 mM. In a further example, cyclodextrin is present in a concentration of about 100 mM.

[056] In addition to cyclodextrin, polymers may be used to further enhance the aqueous solubility, at the same time allowing for lesser amounts of cyclodextrin required. The polymers may also enhance the viscosity of the formulation to confer better adherence to the applied surface. In one example, the composition may further comprise a polymer. In another example, the polymer is absent. As used herein, the term "polymer" refers to a large molecule, or macromolecule, composed of many repeated subunits. In the present instance, the polymer can be pharmaceutically or medically acceptable, especially for compositions intended for external, as well as internal use on a patient. Therefore, the polymer should not be toxic and be well soluble in aqueous solution, and should not compromise the solubility of the compound if it is used solely as a viscosity enhancer.

[057] The concentration of the polymer present in the composition also influences the viscosity of the composition. The higher the concentration of a polymer, the more viscose the composition becomes, therefore also effecting the efficacy of the active compound and other pharmaceutical properties of the resulting composition. For example, increasing the viscosity of a composition may increase the local bioavailability of the active compounds, for example in topical applications. Therefore, the polymer may be present in the composition in an amount between about 0.1% w/v to about 2% w/v, about 0.1% w/v to about 0.5% w/v, about 0.4% w/v to about 0.8% w/v, about 0.5% w/v to about 1% w/v, about 0.2% w/v to about 0.3% w/v, about 0.25% w/v to about 0.3% w/v or in an amount of about 0.1% w/v, about 0.18% w/v, about 0.20% w/v, about 0.23% w/v, about 0.24% w/v, about 0.25% w/v, about 0.3% w/v, about 0.8% w/v, about 0.9% w/v or about 0.12% w/v. In one example, the polymer is present in an amount of between about 0.1% w/v to 2% w/v. In another example, the polymer is present in an amount of about 0.25% w/v. In yet another example, the polymer is present in an amount of about 1% w/v.

[058] Polymers used for this purpose can include, but are not limited to cellulose derived polymers, such as salt derivatives of carboxymethyl cellulose (CMC), microcrystalline cellulose and derivatives thereof; soluble cellulose derivatives, such as hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl cellulose (HPC), methyl cellulose, and derivatives thereof, xanthan gum; insoluble cellulose derivatives, such as ethylcellulose, microcrystalline cellulose (MCC), and derivatives thereof; polyacrylates, such as carbomers, polycarbophil, and derivatives thereof, polyvinyl alcohol; starch, chitosan and derivatives thereof, alginates, acacia, and combinations thereof. In one example disclosed herein, it was seen that carboxymethyl cellulose sodium also increases the viscosity of the formulation, allowing for better adhesion, for example to the mucosal surfaces of the nasal passage, thus facilitating drug delivery. Therefore, in another example, the composition may further comprise a cellulose derivative, such as carboxymethyl cellulose or carboxymethyl cellulose sodium.

[059] The compositions as described herein may also further comprise a salt. The addition of a salt may be used to improve tonicity of the resulting composition and therefore enable an improved pharmaceutical, medical or non-medical application. As used herein, the term "tonicity" refers to is a measure of the effective osmotic pressure gradient (as defined by the water potential of two solutions) of two solutions separated by a semipermeable membrane. In other words, tonicity is the relative concentration of solutions that determine the direction and extent of diffusion. It is commonly used when describing the response of cells immersed in an external solution. Unlike osmotic pressure, tonicity is influenced only by solutes that cannot cross the membrane, as only these exert an effective osmotic pressure. Solutes able to freely cross the membrane do not affect tonicity because they will always be in equal concentrations on both sides of the membrane.

[060] The salt may be provided in the form of a crystalline compound, a partially crystalline compound or as a solution. For example, a salt, such as sodium chloride (NaCl), may be provided in the form of a saline solution. As used herein, the term "saline" refers to a sterile or non-sterile solution of sodium chloride (NaCl) in water. Sodium chloride is generally used, particularly if a buffer containing sodium ions is used in the composition, and is typically present in an amount that is physiologically equivalent to the tonicity of the nasal membranes. The saline solution may be a hypertonic, an isotonic or a hypotonic solution depending on the intended application. The term "saline" can be used interchangeably with the term "salt water solution". Clinical trials indicate that use of a hypertonic saline can enhance the ciliary beating frequency (CBF), which provides a further benefit to the patients.

[061] Randomised controlled trials (RCT) found that for patients with sinusitis, daily nasal irrigation with hypertonic saline was able to improve sinus-related quality of life and reduced the severity of symptoms. Another randomised controlled trial conducted in patients with symptomatic allergic rhinitis similarly reported improved nasal clearance time and reduced nasal symptoms, which was defined as nasal obstruction, nasal itching, nasal discharge and sneezing. Treatments using hypertonic saline are concluded to be more efficacious than normal saline (0.9% w/v sodium chloride) treatments. Hypertonic saline may be formulated with octenidine dihydrochloride-2-hydroxypropyl-P-cyclodextrin (OCT- HPPCD), mupirocin calcium-2-hydroxypropyl-P-cyclodextrin (MPC-HPpCD) or triclosan-2- hydroxypropyl-P-cyclodextrin-carboxymethyl cellulose (TCS-HPpCD-CMC), respectively in aqueous solution. Hypertonic saline nasal irrigation is widely regarded to be beneficial in supporting the mechanical clearance of mucus. Other than that, it is also proposed that hypertonic saline can facilitate reduction of mucosal edema, enhance ciliary beat activity, decrease inflammation and removal of antigen, leading to a protective effect on sino-nasal mucosa.

[062] Hypertonic saline is indicated as adjunctive treatment in many sino-nasal conditions. These include acute sinusitis, chronic sinusitis, allergic rhinitis, non-allergic rhinitis, atrophic rhinitis, sinonasal sarcoid, post-operative care and other scab-forming conditions. [063] There are side effects associated with the use of hypertonic saline for nasal irrigation, such as nasal irritation, nasal itching, burning sensation and nausea. These minor side effects are generally reported to disappear over time or with the discontinuation of treatment. Even though side effects may be common, the beneficial effect of saline outweighs its drawbacks for majority of the patients. The minimal and transient side effects, together with its high tolerability, make hypertonic saline an effective, safe and inexpensive treatment.

[064] Accordingly, the concentration of the salt present in the compositions described herein may vary based on the required application. Therefore, the salt present in the composition may be in an amount between about 2% w/v to about 4% w/v, between about 2% w/v to about 3% w/v, between about 2.5% w/v to about 3.5% w/v, between about 3.5% w/v to about 4% w/v, between about 2.75% w/v to about 3.15% w/v or in an amount of about 1.8% w/v, about 2% w/v, about 2.2% w/v, about 2.4% w/v, about 2.6 % w/v, about 2.8% w/v, about 3.0% w/v or about 3.2% w/v. In one example, the salt is present in the composition at an amount of about 2.8% w/v. In another example, the salt is present in the composition at an amount of about 3% w/v. In another example, the salt may be absent. In one example, the salt is sodium chloride.

[065] In compositions disclosed herein, both 2-hydroxypropyl-P-cyclodextrin (HPpCD) and carboxymethyl cellulose (CMC) serve to improve the aqueous solubility of the formulation, at the same time allowing for lesser amounts of HPpCD required, and modulate the release of triclosan in the nasal cavity when nebulised. CMC, when present, also increases the viscosity of the formulation, allowing for better adhesion to the mucosal surfaces of the nasal passage, facilitating drug delivery. Saline, when added, is added for its bacteriostatic and irrigative properties, and also enhances the aqueous solubility of the formulation.

[066] In one example, the present disclosure refers to a composition comprising triclosan (TCS), carboxymethyl cellulose (CMC) and 2-hydroxypropyl-P-cyclodextrin (HPpCD). In another example, the composition may further comprise a salt, whereby the composition then comprises triclosan (TCS), carboxymethyl cellulose (CMC), 2-hydroxypropyl-P-cyclodextrin (HPPCD) and sodium chloride (NaCl). In one example, the present disclosure refers to a composition comprising about 0.2% w/v to about 0.5% w/v triclosan (TCS), about 0.5% w/v to about 1.5% w/v carboxymethyl cellulose (CMC) and about 30 mM to about 50 mM 2 hydroxypropyl-P-cyclodextrin (HPpCD). In another example, the composition comprises about 0.2% w/v to about 0.5% w/v triclosan (TCS), about 0.5% w/v to about 1.5% w/v carboxymethyl cellulose (CMC), about 30 niM to about 50 niM 2-hydroxypropyl-P- cyclodextrin (HPpCD) and about 0.5% w/v to about 3% w/v sodium chloride (NaCl). More precisely, in one example, a composition comprises 0.3% w/v triclosan (TCS), 1% w/v carboxymethyl cellulose (CMC) and 40 mM 2-hydroxypropyl-P-cyclodextrin (HPpCD). In a further example, the present disclosure refers to a composition comprising 0.3% w/v triclosan (TCS), 1% w/v carboxymethyl cellulose (CMC), 40 mM 2-hydroxypropyl-P-cyclodextrin (HPPCD) and 2.8% w/v sodium chloride. HPpCD is used to improve the aqueous solubility of TCS, a poorly water-soluble phenolic antiseptic.

[067] In one example, the composition of the triclosan-2-hydroxypropyl-P-cyclodextrin- carboxymethyl cellulose complex (TCS-HPpCD-CMC) is 0.3% w/v triclosan, 40mM 2-hydroxypropyl-P-cyclodextrin (HPpCD), 1% w/v carboxymethyl cellulose (CMC). Concentrations above 1% carboxymethyl cellulose improved the aqueous solubility of the formulation further but also increased the viscosity of the solution formed, making it less suitable for nebulisation. 1% w/v saline can also be added to the formulation to improve the antiseptic and irrigative properties of the formulation. The formulation can be combined with a hypertonic saline solution for the nebulisation process, which may be used as an adjunctive treatment for many sino-nasal conditions such as sinusitis, rhinitis. This composition is for nasal decolonisation, that is, to reduce the risks of surgical site infections in patients undergoing surgery. The formulation may also have secondary uses such as for nasal irrigation, nasal cleaning, or as an antiseptic. Formulation of the antiseptic solution is also considered. The pH of the triclosan-based compositions ranges between about 6.43 to about 6.70. For formulations with 1% w/v saline added, the pH of the triclosan -based compositions ranges between about 6.72 to about 6.96. The osmolality of an antimicrobial solution as described herein containing triclosan, without saline, is between about 309 to about 320 mmol/kg and between about 387 to about 392 mmol/kg for the compositions with saline added.

[068] In another example, the present disclosure refers to a composition comprising octenidine dihydrochloride (OCT) and 2-hydroxypropyl-P-cyclodextrin (HPpCD). In yet another example, the composition comprises about 0.05% w/v to about 0.15% w/v octenidine dihydrochloride (OCT) and about 40 mM to about 60 mM 2-hydroxypropyl- β-cyclodextrin (HPpCD). More precisely, in one example, a composition comprises 0.1% w/v octenidine dihydrochloride (OCT) and 50 mM 2-hydroxypropyl-P-cyclodextrin (FfPpCD). Any of the compositions described herein may further comprise a salt. Therefore, in a further example, a composition comprises octenidine dihydrochloride (OCT), 2-hydroxypropyl-P- cyclodextrin (HPpCD) and sodium chloride (NaCl). In yet another example, the composition comprises about 0.05% w/v to about 0.15% w/v octenidine dihydrochloride (OCT), about 40 mM to about 60 mM 2-hydroxypropyl-P-cyclodextrin (HPpCD) and about 0.5% w/v to 3% w/v sodium chloride (NaCl). More precisely, in another example, the present disclosure refers to a composition comprising 0.1% w/v octenidine dihydrochloride (OCT), 50 mM 2-hydroxypropyl-P-cyclodextrin (HPpCD) and 2.8% w/v sodium chloride (NaCl).

[069] The formulation of mupirocin (MPC) together with 2-hydroxypropyl- β-cyclodextrin (HPpCD) greatly improved its solubility in water. This formulation can also be prepared with the addition of 2.8% w/v sodium chloride (NaCl) to improve the antiseptic and irrigative properties of the formulation. Also described herein is a composition comprising mupirocin comprises of 2% w/v mupirocin (MPC) and 100 mM 2-hydroxypropyl-P-cyclodextrin (HPpCD). 2.8% w/v sodium chloride (NaCl) may be added when required. The pH of the MPC compositions (MPC-HPpCD and MPC-HPpCD-NaCl) is between 6 and 7, making it compatible with the nasal cavity which has an average pH of approximately 6.3.

[070] In one example, the present disclosure refers to a composition comprising mupirocin calcium (MPC) and 2-hydroxypropyl-P-cyclodextrin (HPpCD). In one example, the composition comprises about 1% w/v to 3% w/v mupirocin calcium (MPC) and about 50 mM to about 150 mM 2-hydroxypropyl-P-cyclodextrin (HPpCD). More precisely, in another example, the composition comprises 2% w/v mupirocin calcium (MPC) and 100 mM 2-hydroxypropyl-P-cyclodextrin (HPpCD). In yet another example, the composition comprises mupirocin calcium (MPC), 2-hydroxypropyl-P-cyclodextrin (HPpCD) and sodium chloride (NaCl). In a further example, a composition comprises about 1% w/v to 3% w/v mupirocin calcium (MPC), about 50 mM to about 150 mM 2-hydroxypropyl-P-cyclodextrin (HPPCD) and about 0.5% w/v to 3% w/v sodium chloride (NaCl). More precisely, in one example, the composition comprises 2% w/v mupirocin calcium (MPC), 100 mM 2- hydroxypropyl-P-cyclodextrin (HPpCD) and 2.8% w/v sodium chloride (NaCl). In one example, the compositions disclosed herein comprise carboxymethyl cellulose (CMC). In one example, described herein is a composition which further comprises about 0.1% w/v to about 0.3% w/v carboxymethyl cellulose (CMC). [071] The present composition may also contain various pharmaceutically acceptable additives such as tolerance enhancers (sometimes more specifically referred to as humectants), absorption enhancers (sometimes also referred to as surfactants), preservatives, viscosity modifying agents (e.g., thickening agents), osmolality adjusters, complexing agents, stabilizers, solubilizers, or any combination thereof.

[072] A tolerance enhancer may be used in order to inhibit drying of the nasal membrane or mucosa. A tolerance enhancer may also serve the purpose of inhibiting or relieving irritation of the nasal membranes. Examples of suitable tolerance enhancers include, for example, humectants such as sorbitol, propylene glycol, glycerol, glycerin, hyaluronan, aloe, mineral oil, vegetable oil, soothing agents, membrane conditioners, sweeteners, and mixtures thereof. The selection and concentration of a tolerance enhancer may depend on a number of factors, including, for example, the type and concentration of antimicrobial agent being used in the composition. When used, the concentration of the tolerance enhancer in the composition will typically be in amounts from about 0.01% w/w to about 20% w/w.

[073] A preservative may also be employed to increase the shelf -life of the composition. A number of well-known and pharmaceutically acceptable preservatives may be used in the present composition, including, for example, parabens, thimerosal, phenylcarbinol, chlorobutanol, benzalkonium chloride, or benzyl alcohol and combinations thereof. Other ingredients which extend shelf life can be added such as for example, antioxidants. Examples of antioxidants include sodium metabisulfite, potassium metabisulfite, ascorbyl palmitate and other pharmaceutically acceptable antioxidants. Typically, the antioxidant will be present in the composition in a concentration of from about 0.01% w/w to about 5% w/w.

[074] A surfactant or absorption enhancer may also be used in the composition in order to enhance the absorption of the antimicrobial compound across the nasal membrane. However, in cases where the active agent is intended for topical use or dermal application, no such surfactant is added, as the active ingredient in not intended for internalization in a patient, that is, the active ingredient is not intended to pass the nasal mucosal membrane.

[075] In cases where the uptake and internalization of an antimicrobial composition is intended, suitable absorption enhancers include non-ionic, anionic and cationic surfactants. Any of a number of well-known surfactants may be used, including, for example, polyoxyethylene derivatives of fatty acids, partial esters of sorbitol anhydrides, sodium lauryl sulfate, sodium salicylate, oleic acid, lecithin, dehydrated alcohol, Tween (e.g., Tween 20, Tween 40, Tween 60, Tween 80 and the like), Span (e.g., Span 20, Span 40, Span 80 and the like), polyoxyl 40 stearate, polyoxy ethylene 50 stearate, edetate disodium, propylene glycol, glycerol monooleate, fusieates, bile salts, octoxynol and combinations thereof. When used, the concentration of the surfactant in the composition will typically be from about 0.1% w/w to about 50% w/w. By way of example, concentrations of sodium salicylate, sodium lauryl sulfate and edetate disodium may be from about 0.01% to about 5% w/w of the composition. Concentrations of polyoxyl 40 stearate, lecithin, dehydrated alcohol, can be from about 0.1% to about 10% w/w of the composition. Concentrations of oleic acid can be from about 0.01% to about 5% w/w of the composition. Concentrations of propylene glycol and Tween 20 can be from about 0.1% to about 25% w/w of the composition.

[076] A sweetening agent may also be added to the composition as described herein. In formulations where any one of the components may result in a bitter or otherwise displeasing taste, it would be possible to ameliorate this bitter taste by including an acceptable sweetening agent to the formulation. This may be of use in formulations intended for oral application, nasal irrigation or as nasal sprays. Examples of sweetening agents can be, but are not limited to, sugars such as monosaccharides, disaccharides and polysaccharides. Examples of suitable sugars include but are not limited to xylose, ribose, glucose, mannose, galactose, fructose, dextrose, sucrose, sucralose, maltose, partially hydrolyzed starch or corn syrup solids, stevia, and sugar alcohols such as sorbitol, xylitol, mannitol, glycerin and combination thereof. If the addition of such a sweetening agent is contemplated, the amount added would need to be sufficient in order to mask the bitter or otherwise displeasing taste of the composition, but without impeding the efficacy of the resulting composition in its intended purpose. The sweetening agent may, for example, be added together with a preservative as defined herein. The amount of the sweetening agent present in the formulation may be between about 1% to about 40%, between about 0.01% to about 0.1%, between about 0.05% to about 0.5%, between about 1% to about 5%, between about 4% to about 10%, between about 8% to about 15%, between about 12% to about 15%, between about 15% to about 20%, between about 20% to about 35%, between about 25% to about 40%, about 8%, about 10%, about 15%, about 20%, about 30% or about 40%. A sweetening agent in a formulation may, for example, result in the composition comprising 100 parts of water, 10 parts of xylitol/xylose, 0.65 parts of sodium chloride and effective amounts of benzalkonium chloride and phenylcarbinol as preservatives. [077] Also disclosed herein is the use of antimicrobial compositions for treating or preventing infections, whereby the infection may be, but is not limited to, an ear infection, a nose infection, a throat infection, a sino-nasal infection, a sinus infection, a respiratory infection, rhinitis and bronchiolitis. The composition of the invention may also be used as an adjunctive treatment for many sino-nasal conditions such as sinusitis, rhinitis. In one example, the composition described herein is administered to a patient before or after surgery. Administration may also take place both before and after surgery.

[078] The present disclosure also describes compositions for treating or preventing infection in a patient. As used herein, the term "infection" refers to the invasion of a living organism by disease-causing microorganisms. As used herein, the term "microorganism" may be used interchangeably with "microbe". The microorganisms referred to herein may be pathogenic and non-pathogenic, as well as prokaryotic and eukaryotic microorganisms, such as, but not limited to bacteria, fungi, mould, protozoan and yeast. In one example, the bacteria may be, but is not limited to, Salmonella spp., Streptococcus spp., Campylobacter spp., Mycobacterium spp., Helicobacter pylori, Staphylococcus spp., Staphylococcus aureus, Methicillin- sensitive Staphylococcus aureus (MSSA), Methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa and Escherichia coli, whereby the term "spp.", as used herein, refers to a species. In one example, the composition is used to treat or prevent an infection caused by, but is not limited to MRSA 1, MRSA 2, MRSA 3, MRSA 7, Mu50, WIS and MSSA 2590. In some examples, the described composition is used to treat a fungal infection. In this example, the fungus may be, but is not limited to Candida albicans, Aspergillus brasiliensis and Aspergillus niger.

[079] In one example, the present disclosure refers to a method of treating or preventing an infection in a patient, or for decolonizing an orifice of a patient, wherein the method comprised administering to the patient a therapeutically effective amount of the compositions as defined herein. In another example, a composition as defined herein is used for treating or preventing an infection. In another example, the use of a composition in the manufacture of a medicament for treating an infection is disclosed. In one example, a method is disclosed wherein the composition is administered nasally via nasal irrigation or nasal fuming. The present disclosure also refers to use of the composition as defined herein in the manufacture of a medicament for preventing or treating an infection, or for decolonizing an orifice of a patient. [080] As used herein, the term "decolonization" refers to the local transient or permanent reduction of the amount of microorganisms in a patient. This decolonization may take place in, but is not limited to, a single, particular location in or on the body of the patient. Decolonization may take place in various locations in or on the body of the patient simultaneously or sequentially. As used herein, the term "orifice" refers to the entrance or outlet of any body cavity. Example of an orifice may be, but is not limited to, nose, ears, mouth, nostrils and throat. In one example, the compositions, as described herein, are used to treat or prevent an infection in an orifice, whereby the orifice may be, but is not limited to ear, nose, nasal cavity, sinus, nasal passageway, Eustachian tube and throat.

[081] As such, the compositions described herein may be administered orally, topically, nasally, endosinually, intrasinally, transdermally, locally or combinations thereof. Examples of nasal administration are, but not limited to, nasal irrigation, nasal fuming, nasal sprays and the like. More specifically, the compositions of the invention can also be administered by the nasal route. When formulated for nasal administration the compositions may comprise a compound of the invention in a liquid carrier; such compositions may be administered for example in the form of a spray or as drops. The liquid carrier may be water, which may contain further components to provide the desired isotonicity and viscosity of the composition. The composition may also contain additional excipients such as preservatives, surface active agents and the like. The compositions may be contained in a nasal applicator that enables the composition to be administered as drugs or as a spray. For administration from an aerosol container the composition should also include a propellant. The composition may also be in unit dosage form, e.g. as tablets or capsules. In such form, the composition is sub-divided in unit dose containing appropriate quantities of the active ingredient; the unit dosage forms can be packaged composition, for example packeted powders, vials, ampoules, pre-filled syringes or sachets containing liquids. The unit dosage form can be, for example, a capsule or tablet itself, or it can be the appropriate number of any such compositions in package form. Thus, in one example, the present disclosure refers to the composition as disclosed herein, which may be provided in the form of, but not limited to dry powder, aqueous solution, non-aqueous solution, slurry, aerosols, nasal spray, nebulizer, inhalers, gels, creams, ointments, pastes, salves, suspensions, sterile solids, crystalline solids, amorphous solids, solids for reconstitution or combinations thereof. In one example, the present disclosure refers to the composition as a nasal spray. In another example, the composition is provided as a gel. In yet another example, the composition is provided as a cream.

[082] Nasal sprays are in liquid form such as an aqueous solution or suspension, an oil solution or suspension, or an emulsion, depending on the properties of the composition components. Optional ingredients ensure minimal irritation, proper spray composition, and adequate delivery. Buffers such as citrate, phosphate, and glycine adjust the pH of the nasal spray to prevent irritation to the nose. Moisturizing agents such as propylene glycol and glycerine are also useful in the nasal spray. Other optional ingredients such as polyphosphoesters, polyethylene glycol, high molecular weight polylactic acid, microsphere encapsulations such as polyvinylpyrrolidone, hydroxypropyl cellulose, chitosan, and polystyrene sulfonate enhance the retention time of the composition.

[083] With respect to the dose of the composition delivered to a patient per administration, it should be understood that each administration may comprise one or a plurality of applications or sprays of the claimed compositions, delivered to the nasal mucosa of the patient through one or both nostrils, the number of applications or sprays being dependent upon the concentration of the antimicrobial agent in the composition, the quantity of the composition delivered per spray, and the desired dose per administration as readily determined by one skilled in the art. The composition can be dispensed from a spray bottle including a pump (e.g., a manually actuated pump) capable of delivering a metered spray of the composition of predetermined volume (typically about 0.1 mL). Furthermore, and by way of example, a daily dose of 1% w/v of mupirocin in the composition may be administered in a single administration comprising one or more applications or metered sprays containing a total of 1% w/v of mupirocin (e.g., for 20μg mupirocin, a single administration comprising two applications or metered sprays, one in each nostril and each containing 10 μg of mupirocin) or in multiple administrations (e.g., four administrations at six hour intervals, each administration comprising one or more applications or metered sprays, in one or both nostrils, each administration containing a total of 5 μg of mupirocin).

[084] When intended for use as an aerosol, the antimicrobial composition will be stored in and dispensed from a sealed container equipped with a metering valve and pump capable of being actuated to deliver or emit an aerosol (e.g., mist or spray) of the composition of predetermined volume into the patient's nostril and having a suitable droplet size distribution as known to those skilled in the art. Generally, the size of the droplets are large enough to prevent them from passing directly through the nasal passages and into the lungs, but small enough that they do not coalesce into large drops which either run out of the nose or down into the throat.

[085] Suitable containers and metering valves for dispensing the antimicrobial composition according to the methods of the invention are available commercially and are known to those of skill in the art. The container and valve system used to deliver the antimicrobial composition may incorporate any of the conventional aerosol formation techniques. These include, for example, mechanical pumps; compressed air mechanisms in which delivery is made by hand pumping air into the container; compressed gas techniques in which delivery is made by the controlled release of a compressed gas (such as, for example, carbon dioxide, nitrogen, and dinitrogen oxide) into the antimicrobial containing composition; and liquid propellant techniques in which a low boiling liquid hydrocarbon (such as, for example, butane, isobutane, propane, and other low boiling hydrocarbons in either pure or mixed forms), halohydrocarbon, fluorocarbons (such as, for example, FC- 152A), chlorofluorocarbons (such as Freon or Freon like fluorocarbons, such as, for example, CFC-11, CFC-12 and CFC-114), and hydrofluorocarbons, also referred to as hydrofluoroalkanes (such as, for example, HFA-134a and HFA-227) are vaporized to exert a pressure and force the composition through the metering valve.

[086] The antimicrobial composition can be stored for administration in a container or bottle including a pump and metering valve adapted for delivery of a metered spray of the composition and designed to inhibit or prevent degradation or spoilage of and bacterial growth in the composition contained therein.

[087] To date, nasal nebulisers are operated by pushing fumes through both nostrils. This mechanism results in a substantial amount of fumes being forced down into the lungs or lower respiratory tract, rather than to the inner nasal cavity. Studies have revealed that humans mostly breathe through one nostril at a time. Therefore, this natural phenomenon was mimicked by administering the composition through only one nostril each time, allowing the fumes to escape via the other nostril. This may cause the fume pressure to be high enough to reach the upper nasal cavity and sinuses, but not the lower respiratory tract. The nasal irrigation has also been administered through one nostril at a time alternating between nostrils as a common clinical practice. The nasal spray, irrigation or nebulised solution of antimicrobial compositions with or without sodium chloride (NaCl) may also be administered before an operation for nasal decolonization. Use of a saline nasal spray and irrigation several times per day has been shown in the art to help prevent scab formation in the nose.

[088] The composition described herein may also be used in form of a gel or a cream. In situations where a topical application is desired, providing the composition as a gel or a cream allows for efficient, sterile and fuss-free application, for example, when the infection is present in the nose, the nostrils or within the nasal passageways of a patient. The same also applies for applications for nasal decolonization in a patient, which may also utilize the compositions described herein, formulated or provided as gels or creams. In one example, the composition comprises octenidine and cyclodextrin without a salt. In another example, the composition comprises octenidine and cyclodextrin without a salt, wherein the composition is provided as a gel. In another example, the composition comprises octenidine and cyclodextrin without a salt, wherein the composition is provided as a cream.

[089] The formulation of compositions of the antimicrobial agent with and without hypertonic saline increases the scope of application of the compositions. Also described herein are alternative approaches to the standard of care in, for example, pre- and post-surgical nasal treatments, by using, for example, spray or irrigation as a douche or a nebuliser to deliver fumes of antiseptic into the nasal cavity. Methods, such as a douche or a nebulizer, may be used to deliver fumes of antiseptic into the nasal cavity, for example during nose and sinus irrigations performed after functional sinus surgery. The composition described herein may also have further secondary uses, such as but not limited to surgical sutures and for inhalation. Other uses may be non-medical uses, such as, but not limited to, mouth wash, toothpaste, face wash, soaps, hand wash, surface disinfectants and cleaning products. For example, gel or cream formulations of octenidine dihydrochloride with or without sodium chloride can be used for antimicrobial applications.

[090] Also, the composition described herein may be used in conjunction with other forms of treatment, or as adjunctive treatment. The described composition may thus be administered before, after, during or together with a further treatment. As used herein, the term "adjunctive treatment" refers to another second treatment used together with the primary treatment, wherein the purpose of the secondary treatment is to assist the primary treatment.

[091] [Two methods are described herein for producing the described compositions. Thus, the compositions described herein can be obtained or are obtainable by the methods disclosed herein. [092] In one exemplary method each compound can be ground separately, after which they can be mixed together under constant agitation and heat to form the compositions as described herein. The method may comprise mixing a ground cyclodextrin, as defined herein, and a ground antimicrobial agent, as defined herein, and constantly agitating and heating the resulting mixture. Heating and agitating can be done using means known in the art, e.g. in a thermostatically controlled shaker oven or water bath.

[093] As used herein, the term "ground" or "grinding" refers to the mechanical process of reducing the particle size of compounds or compositions, thereby producing a powdered form of the compound or composition. This grinding may be performed using methods known in the art, but generally encompass methods requiring that the compound be rubbed against a rough surface, whereby the roughness of the surface dictates how fine or coarse the resulting powder will be.

[094] As used herein, the term "agitation" may be replaced with "stirring" and refers to the constant moving of a mixture or a compound. "Constant" agitation can be applied to ensure the homogeneity of the resulting mixture, or to prevent the mixture from adhering to the sides of the mixing vessel during reaction.

[095] As used herein, the term "heat" refers to subject the compositions to a temperature that is higher than the ambient room temperature. Without being bound by theory, the inventors found that heating the composition described herein whilst constantly agitating it resulted in the antimicrobial agent being taken up into the pores of cyclodextrin, thus resulting in the complexation of the antimicrobial agent within cyclodextrin. This is also shown to be the case for antimicrobial agents that are known to be difficult to dissolve in, for example aqueous solutions. In order to be able to use this process, individual components of the compositions are subjected to heat. In one example these components are cyclodextrin, the antimicrobial agent or both cyclodextrin and the antimicrobial agent. The temperature used in order to enable the complexation of the antimicrobial agent and cyclodextrin depends on the melting temperature of the antimicrobial agent. When heated to above this temperature, the antimicrobial agent becomes liquid and flows into the pores of cyclodextrin, thus enabling the complexation of the antimicrobial agent within cyclodextrin. However, it is to be noted that the heating temperature may not exceed the melting point of cyclodextrin, as this may inhibit the complexation of the antimicrobial agent with cyclodextrin. For example, the melting point of 2-hydroxypropyl-P-cyclodextrin is 278°C, and its degradation point is at around 300°C. Therefore, without being bound by theory, it would not be practical to melt cyclodextrin to form the complexation as there is a risk of degradation due to the proximity of its melting point to its degradation point. Economically, it is cheaper to use lower temperature to facilitate complexation of the compositions than higher temperature, as the lower temperature would require less energy. Thus, it will be more practical to melt the compound with low melting point to facilitate the complexation of the antimicrobial agent with cyclodextrin rather than to melt cyclodextrin for the complexation with the antimicrobial agent. Therefore, in one example, the heat applied in the method as described herein is the melting temperature of the antimicrobial agent. The heat of applied may be between about 40°C to about 100°C, between about 50°C to about 200°C, between about 55°C to about 90°C, between about 60°C to about 80°C, between about 80°C to about 120°C, between about 70°C to about 90°C, between about 75°C to about 110°C, between about 95°C to about 145°C, between about 150°C to about 180°C, about 40°C, about 50°C, about 55°C, about 59°C, about 60°C, about 61°C, about 65°C, about 70°C, about 75°C, about 79°C, about 80°C, about 81°C, about 84°C, about 85°C, about 90°C, about 100°C, about 120°C, about 130°C, about 150°C, about 165°C, about 170°C, about 180°C or about 200°C.

[096] Methods in the art describe agitation of the compounds for extended periods of time, for example in solution, which is considered to be time-consuming and tedious. The method described herein differs from those methods known in the art in that the time required for producing the described powdered compositions is markedly shortened. While the methods known in the art require an agitation time of at least 48 hours or more, the methods described herein allow agitation of the compounds and the composition to be completed in less than 48 hours. The agitation time may be between about 0.5 hours to about 48 hours, between about 1 hour to about 48 hours, between about 1 hour to about 5 hours, between about 1 hour to about 10 hours, between about 5 hour to about 15 hours, between about 15 hour to about 20 hours, between about 18 hour to about 24 hours, between about 24 hour to about 36 hours, between about 36 hour to about 48 hours, between about 1 hour to about 1.5 hours, between about 1.5 hours to about 2 hours, between about 1.25 hours to about 2.25 hours, between about 2.5 hours to about 3 hours, about 1 hour, about 1.25 hours, about 1.75 hours, about 2.25 hours, about 2.75 hours, about 3 hours, about 4 hours, about 5 hours, about 10 hours, about 12 hours, about 20 hours, about 24 hours, about 28 hours, about 36 hours, about 40 hours, or about 48 hours. In one example, the composition is agitated for between about 1 hour to about 24 hours. In another example, the composition is agitated for 1 hour. In yet another example, the composition is agitated for 2 hours.

[097] The state of the compositions or mixtures may not only be ground, as previously described. The state of the compositions may independently be dry or slurry, depending on the required downstream application. As used herein, the term "slurry" refers to a semi-liquid or fluid mixture, typically comprising fine particles of insoluble matter or matter with low solubility in a liquid solvent. The solvent required for the formation of a slurry would vary depending on the components to be used and the downstream application. For example, in this method, water may be used as a solvent. In the case of water as a solvent, the term "slurry" would refer to a watery mixture. Another factor that may influence the formation of a slurry is the amount of solvent required. This varies according to the hydrophobicity of the individual compounds, the viscosity of the solvent and the particle size of the individual compounds. In one example, the state of the mixture is dry or slurry. Therefore, the slurry formed by the method, as described herein, is a paste formed by adding a small amount of solvent to the mixed powder of the antimicrobial agent and cyclodextrin. This mixture is then agitated and/or triturated to form a slurry. As used herein, the term "triturate" refers to the process of grinding a compound or a mixture or a composition to a fine powder.

[098] The methods described herein utilise the low melting temperatures of the antimicrobial compositions in order to enable complexation with cyclodextrin and enable the solubilisation of antimicrobial agents that otherwise may not have been possible. For example, it is known in the art that octenidine will precipitate when mixed with sodium chloride in solution. Without being bound by theory, the methods described herein enable the mixing of compounds, for example, the antimicrobial agent and cyclodextrin, before the addition of sodium chloride resulted in a water-soluble composition without precipitation of any of the components. Therefore, in on example, the method disclosed herein may further comprise mixing the ground cyclodextrin with solvent to form a slurry; adding the ground antimicrobial agent to the slurry; subjecting the slurry to constant agitation as defined herein; grinding the resulting composition and mixing the resulting compositing with a salt as defined herein. In another example, the disclosure refers to the method as described herein, wherein the antimicrobial agent is octenidine.

[099] 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.

[0100] 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.

[0101] 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.

[0102] 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 recognised 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.

[0103] Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognise that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

EXPERIMENTAL SECTION

[0104] Octenidine dihydrochloride (OCT) is formulated with 2-hydroxypropyl-P- cyclodextrin (HPpCD) using a slurry approach as described herein. This formulation can also be supplemented with 2.8% sodium chloride (NaCl). Stored as a dry powder, it can be reconstituted into an aqueous solution to be administered as irrigation or through a nebulizer for nasal and sinus cleansing and decolonization. The composition can be stored as a ready- to-use solution. The details on the methodology of preparing the composition comprising antimicrobial octenidine dihydrochloride are discusses herein.

[0105] The compositions disclosed herein involve the use of cyclodextrins and its derivatives to enhance the solubility of mupirocin calcium (MPC) in water. 2.8% sodium chloride (NaCl) can also be added to the formulation. 2-hydroxypropyl-P-cyclodextrin (HPpCD) is chosen as it has low toxicity as compared to other cyclodextrins. The formulation of MPC with and without hypertonic saline increases the scope of potential application of the formulation.

[0106] A dry powder containing triclosan (TCS) and 2-hydroxypropyl-P-cyclodextrin (HPPCD) is formulated using the respective aqueous and non-aqueous approach. The dried powder is reconstituted into aqueous solution and administered to patients as nasal nebulization with any known nebulizer or simply as nasal douche by irrigation. The composition can also be supplied as solution in ampoules for reconstitution by dilution for the same nasal nebulization or nasal irrigation. A suitable method to administering the composition by nebulisation is provided. The composition can be stored as a dry powder, ready-to-use solution, or concentrated composition. The process for preparation of triclosan nebulising solution is also described herein.

Non-Aqueous Approach (Dry physical mixing)

[0107] In this method, 2-hyrdroxypropyl-P-cyclodextrin (HPpCD) and the antimicrobial agent were weighed out and ground in a glass mortar. Following that, the ground physical mixture was subject to constant agitation in glass vials at 275 rpm in a thermostatically controlled shaker oven at 85+l°C for 1 to 2 hours to facilitate complexation. The temperature of the controlled shaker oven was adjusted according to the melting point of the antimicrobial agent. This allowed the antimicrobial agent to melt into a liquid, which facilitated the incorporation into the cyclodextrin cavities. The heated and agitated mixture was then allowed to cool to room temperature (25°C). After cooling, the mixture was ground again for 10 minutes to ensure uniformity of mixture and facilitate rapid reconstitution later. In the case of formulation with hypertonic saline, sodium chloride (NaCl) was weighed and ground together with 2-hydroxypropyl-P-cyclodextrin (HPpCD) and the antimicrobial agent before being subjected to heating in the shaker oven. [0108] Aqueous Approach (slurry complexation-dry heat)

For the preparation of, for example, octenidine-cyclodextrin (OCT-CD) inclusion complexes, a 'slurry complexation-dry heat' method is used. In this method, 2-hydroxypropyl-P- cyclodextrin (HPpCD; Cavasol W7 HP Pharma, Wacker Chemicals) and the antimicrobial agent were weighed out and ground in a glass mortar. Following that, water and 0.1N sodium hydroxide (NaOH) were added per milligram of ground mixture (for example, 10ml of lmg/ml octenidine in water). This allows the dissolution of cyclodextrin and the formation of a viscous slurry with constant stirring. Saturating the aqueous phase with cyclodextrin first allows for complexation with antimicrobial agent to occur spontaneously, due to entropic forces from the release of bound water molecules present within the cyclodextrin cavities. The resulting complexes will then saturate out of the aqueous phase, allowing the formation of more complexes in solution.

[0109] After the formation of the slurry, the slurry was then dried in a thermostatically controlled shaker oven at 80 + 1°C for 1 hour, in order to remove any access water from the slurry and thereby forming a powder. The temperature of the controlled shaker oven was adjusted according to the melting point of the antimicrobial agent. The resulting powder was then ground to ensure uniformity. In the case of formulation with hypertonic saline, 2.8% w/v sodium chloride (NaCl) was weighed and ground together with the dried slurry powder.

High Performance Liquid Chromatography (HPLC) method

[0110] High Performance Liquid Chromatography (HPLC) was used to quantify the antimicrobial agent saturation concentration in the composition in order to identify a suitable 2-hyrdroxypropyl-P-cyclodextrin (HPpCD) concentration for the solubilisation of the antimicrobial agent in water. [0111] The system used consisted of a Shimadzu LC-10AT pump, SCL-IOA system controller, SIL-10AD auto-sampler and CTO-10AS column oven (Shimadzu Corporation, Kyoto, Japan). Chromatographic separation was achieved on Mightysil RP-18 GP 150 mm X 4.6 mm, particle size 5 μιη (Kanto Chemical Co.) analytical column with an isocratic mobile phase of 70% acetonitrile and 30% water, whereby the composition of the mobile phase is adjusted according to the sample in question. HPLC grade acetonitrile was used. All parameters for chromatographic separation were also adjusted according to the physic- chemical properties of the antimicrobial agent in question, according to methods known in the art. For example, a flow rate of 1.2 ml/min was used and the UV detection wavelength was set at 243 nm to monitor analyte peak elution. Flow rate and UV detection wave length are also adjusted according to the sample to be analysed. The oven temperature was maintained at 50°C.

Composition viscosity measurements

[0112] The viscosities of the compositions in solution were evaluated using the parallel plate method. The rheometer used was the ARES-G2 Rheometer, TA Instruments, USA. The plate temperature was set at 25 °C. A total of 100 data points was measured with a shear rate from 0.5 s^"1 to 60 s^"1, for a total time of 10 minutes per run. The viscosities of MilliQ water and the complexes containing 0.25% to 5% CMC were obtained and plotted in a graph of shear stress (Pa) against shear rate (s^_1).

Antimicrobial agent saturation in 2-hydroxypropyl-fi-cyclodextrin (ΗΡβΟΌ) and

a-cyclodextrin (a-CD)

[0113] The saturation concentration of the antimicrobial agent in the presence of 2.8% sodium chloride (NaCl) under varying concentration of 2-hydroxypropyl-P-cyclodextrin (FTPPCD) and a-cyclodextrin (a-CD) (Tokyo Chemical Industry) was determined. HPpCD and a-CD was first dissolved in water at varying concentrations from 50 mM to 300 mM and 50 mM to 100 mM respectively. 2.8% of sodium chloride (NaCl) was added to the dissolved cyclodextrins. Excess antimicrobial agent was added to the solution and shaken at room temperature for an hour. Undissolved antimicrobial agent was removed by filtration using 0.22 μηι Millex® GP Filter Units. The resulting solution was then diluted appropriately and quantified using the HPLC assay. The saturation of the antimicrobial agent in water without sodium chloride and antimicrobial agent in water with 2.8% sodium chloride (NaCl) was also evaluated as a control. After analysing the results of the saturation study, 2-hydroxypropyl-P- cyclodextrin (HPpCD) at a concentration of 50 mM was chosen to formulate compositions of an antimicrobial agent in the presence of 2.8% NaCl. This was because 2-hydroxypropyl-P- cyclodextrin had greater solubility than a-cyclodextrin in water, with the maximum solubility at 300 mM and 100 mM respectively. Furthermore, at 50 mM, it was sufficient to dissolve more than 10 times a target concentration of antimicrobial agent (set at 1 mg/ml) in the presence of hypertonic saline. At this 2-hydroxypropyl-P-cyclodextrin concentration, the antimicrobial composition could be solubilised quickly and the slurry could be manipulated more easily due to its lower viscosity.

Stability Assay validation

[0114] In order to establish the stability-indicating nature of the method, a sample was forcibly degraded by acid (IN hydrochloric acid) and base (IN sodium hydroxide) at room temperature and at 40°C for an hour. Both drugs were also placed under UV for 24 hours. Both drugs were dissolved in 50% acetonitrile and 50% water in screw cap glass tubes and for each degradation condition, triplicates were prepared. The samples were neutralised with an equal amount of IN sodium hydroxide and IN hydrochloric acid respectively. The samples were diluted in mobile phase before analysis.

Minimum Inhibitory Concentration (MIC) - Macrodilution (tube) broth assay

[0115] A series of 15 mL glass tubes were filled with double- strength (D/S) nutrient broth (Acumedia® Nutrient Broth 7146, Lot: 106490B), placed in a test-tube rack and labelled accordingly. The composition to be analysed is dissolved in MilliQ water and filtered through 0.22 μηι Millex® GP Filter Units. Varying volumes of the dissolved composition were added to each glass tube giving a range of concentrations for the determination of the minimum inhibitory concentration (MIC). Sterile water was then added to each glass tube. The tubes were then mixed thoroughly by rotating between the palms of the hands.

[0116] 0.2 ml of a diluted 24-hour broth culture of Staphylococcus aureus (MicroBiologics® S. aureus; Lot No.: 827963; RFF 0827S; ATCC 6538P) standardised at 0.5 McFarland Standard, was next added to each tube. A positive control containing only the cultured organism, sterile water and double-strength (D/S) nutrient broth was also prepared. The tubes were then mixed thoroughly again by rotating between the palms of the hands, and incubated at 37°C for 72 hours. The tubes were observed for turbidity, indicative of microbiological growth at 72 hours and quantified by comparing against McFarland Equivalence Standards (Batch No.: 1313947; TM4000T10). The minimum inhibitory concentration was recorded as the lowest antimicrobial concentration where no bacteria growth is observed after 72 hours incubation. All apparatus (i.e. glass tubes, measuring cylinders, pipette tips, stock bottles, and volumetric flasks), nutrient broth solution and sterile water were placed through a moist heat sterilisation process (autoclave) at 121°C for 15 minutes before they were used. Each set of tubes included a positive (bacteria without the addition of antimicrobial formulation) and negative control (broth only). The experiments were performed in duplicates.

Minimum Inhibitory Concentration (MIC) - Microdilution Broth assay

[0117] The minimum inhibitory concentration (MIC) of a composition is determined against 7 Staphylococcus aureus strains: MRSA 1, MRSA 2, MRSA 3, MRSA 7, Mu50, WIS and MSSA 2590 using the Minimum Inhibitory Concentration (MIC) Broth Microdilution Assay. The assay was conducted in accordance to the guidelines set out in 'Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically - Eighth Edition' (Clinical and Laboratory Standards Institute, 2012). All Staphylococcus aureus strains were kindly provided by Jeanette Teo from National University Hospital. MRSA 1, MRSA 2, MRSA 3 and MRSA 7 are clinically isolated MRSA strains. MRSA strains Mu50, WIS and MSSA strain 2590 were obtained from ATCC. Out of the clinically-isolated MRSA strains, MRSA 1 and MRSA 2 are resistant to mupirocin. They are characterised by the presence of the mupA gene.

[0118] The drug compositions were dissolved in MilliQ water and filtered through 0.22 μηι Millex® GP Filter Units. It was further diluted in Mueller Hinton (MH) broth (Ref: 275730, Lot: 0224378) to appropriate concentrations for the determination of the MIC. Sterile 96-well round-bottom microtiter plates (Nunc, Roskilde, Denmark) were used. 24- hour broth culture of S. aureus was diluted with MH broth (and standardised at between 1 X 105 - 1 X 106 CFU/ml. This was determined by the plating of the bacteria on LB agar at the time of inoculation. Each row of the microplate was loaded with a different S. aureus strain. 50 μΐ of test inoculum was loaded into each well. 50 μΐ of drug solution was then added into first column and serial two-fold dilution is performed across the columns. The final volume of the well was 50 μΐ. The 96-well microtiter plates were then covered with a plate cover and incubated overnight at 37°C. The minimum inhibitory concentration was recorded as the lowest antimicrobial concentration where no bacteria growth is observed after overnight incubation. The experiments were performed in duplicates.

Aqueous Formulation Stability study

[0119] The compositions according to the invention were weighed and dissolved in 10 ml MilliQ water. Following that, the samples were filtered using 0.22 μιη Millex® GP Filter Units. The stability of the samples was evaluated for 8 weeks under two conditions:

Room temperature with light exposure and

Room temperature without light exposure.

[0120] At fixed time points, an aliquot of the sample would be obtained, diluted down with mobile phase, analysed and quantified according to the chromatographic conditions as stated earlier.

Time-dependent determination of bacterial activity

[0121] The time-dependent bactericidal kill profile of the compositions was determined at full-strength and half-strength against Staphylococcus aureus strains: MRS A 1, MRS A 2, MRSA7 and Mu50. The compositions were dissolved directly in MH broth in a 15ml falcon tube. Half-strength formulations were prepared by performing a two-fold dilution of the full- strength formulations.

[0122] At time zero, 2.5 μΐ of 24-hour broth culture of S. aureus was added to 5 ml of each formulation such that the bacteria concentration is between lxlO⁵ - lxlO⁶ CFU/ml. To obtain the initial bacteria count in the broth, 2.5 μΐ of 24-hour broth culture was added to 5 ml of MH broth. After which, an aliquot was obtained, diluted appropriately and plated on Luria Broth (LB) agar. At time point 15 minutes, 30 minutes, 2 hours, 6 hours and 24 hours, an aliquot was obtained from each tube, diluted appropriately and plated. A dilution of at least 1x10 is required to ensure that the drug in the solution has no bacteriostatic effect on the surviving bacteria during plating. The bacteria count at each time point was enumerated after overnight incubation of the plates at 37°C. A positive (bacteria without the addition of antimicrobial formulation) and negative control (broth only) were also included. The experiments were performed in duplicates.

Antimicrobial effectiveness testing of antiseptic solutions against bacteria and fungi

[0123] Preparation of the test inoculums were conducted using suitable mediums (Soybean-Casein Digest Broth and Sabouraud Dextrose Broth). The test microorganisms include: Escherichia coli (ATCC 8739), Pseudomonas aeruginosa (ATCC 9027), Staphylococcus aureus (ATCC 6538), Candida albicans (ATCC 10231) and Aspergillus brasiliensis (ATCC 16404). The test inoculums were incubated for microbial recovery. The incubated test inoculums were standardised such that the final concentration of the test preparations after inoculation is between lxlO⁵ and lxlO⁶ CFU/ml of the product. The initial concentration of viable test microorganisms in each test preparation is determined by the plate-count method.

[0124] 9ml of each formulation is transferred into 5 individual sterile containers for the investigation of the 5 test microorganisms. A duplicate set for each formulation was prepared as well. Each container was inoculated with one of the prepared and standardised inoculant and mixed well. The volume of the suspension inoculums used is between 0.5% and 1.0% of the volume of the product to minimise potential effects on the formulation. The inoculated containers were incubated at 22.5 + 2.5°C.

[0125] Each container was sampled and enumerated using the plate-count procedure at applicable test intervals: Day 1, Day 7, Day 14 and Day 28 of incubation. The logio values of the concentration of each microorganism at the applicable test intervals were calculated and reported to investigate the log reductions. The log reduction is defined as the difference between the loglO unit value of the starting concentration (in CFU/ml) in the suspension and the log 10 unit value of the surviving concentration (in CFU/ml) at that time point.

[0126] The requirements for antimicrobial effectiveness as indicated in USP 38-NF 33 Chapter 51 are met if the criteria specified below are met.

• Bacteria: Not less than 1.0 log reduction from the initial calculated count at 7 days, and not less than 3.0 log reduction from the initial count at 14 days, and no increase from the 14 days' count at 28 days.

• Yeasts and moulds: No increase from the initial calculated count at 7, 14 and 28 days. EXAMPLES

[0127] The invention will now be illustrated by way of examples. The examples are by way of illustration only and in no way restrict the scope of the invention. Various compositions according to the details of the invention were made according to the general methodology described above.

[0128] The main components of the formulation were 2-hydroxypropyl-P-cyclodextrin (HPPCD), sodium carboxymethyl cellulose (CMC), crystalline sodium chloride (NaCl), the poorly water-soluble phenolic antiseptic, triclosan (TCS), mupirocin calcium (MPC) and octenidine dihydrochloride (OCT). These methodologies can also likely be applied to other hydrophobic antiseptics as well. Other examples from well-established compositions are also shown for comparison.

Example 1: Non-Aqueous Approach (Dry physical mixing) to mupirocin compositions

[0129] Amorphous mupirocin characterised by a melting point of about 77°C to about 89° may be prepared by the same dry heat approach to melt mupirocin and triturate with 2-hydroxypropyl-P-cyclodextrin or other cyclodextrins and cyclodextrin derivative. The mixed dried powder may be reconstituted into solution for nasal nebulization or irrigation. The composition maybe modified by adding CMC to enhance the solubility and/or cohesive effects to the nasal membrane. Sodium chloride may also be added to the formulation for the additive/synergistic antimicrobial effects.

[0130] For the preparation of mupirocin-cyclodextrin inclusion complexes, a 'dry physical mixing' method is used. In this method, 100 mM of 2-hyrdroxypropyl-P-cyclodextrin (HPPCD) and 2% w/v mupirocin calcium (MPC) were weighed out and ground in a glass mortar. Following that, the ground physical mixture was subject to constant agitation in glass vials at 275 rpm in a thermostatically controlled shaker oven at 85 + 1°C for 2 hours. This allowed mupirocin calcium to melt into a liquid (melting point of 77 - 78°C), which facilitated the incorporation into the cyclodextrin cavities. The heated and agitated mixture was then allowed to cool to room temperature (25°C). After cooling, the mixture was ground again for 10 minutes to ensure uniformity of mixture and facilitate rapid reconstitution later. In the case of formulation with hypertonic saline, 2.8% w/v sodium chloride (NaCl) was weighed and ground together with 100 mM of 2-hydroxypropyl-P-cyclodextrin (HPpCD) and 2% w/v mupirocin calcium (MPC) before being subjected to heating in the shaker oven. [0131] To evaluate the efficacy of the formulations, four formulations were prepared from the dry powder. MPC-HPpCD was obtained by dissolving the dry formulation of MPC-HPpCD which consists of 20 mg/ml mupirocin calcium and 100 mM 2-hydroxypropyl- β-cyclodextrin. MPC-HPpCD-NaCl was obtained by dissolving the dry formulation of MPC-HPpCD-NaCl which consists of 20 mg/ml mupirocin calcium (MPC), 100 mM 2-hydroxypropyl-P-cyclodextrin (HPpCD) and 2.8% w/v sodium chloride (NaCl). Formulations with 0.25% w/v carboxymethyl cellulose sodium (CMC) were also evaluated. MPC-HPpCD-CMC was obtained by dissolving the dry formulation of MPC-HPpCD-CMC which consists of 20 mg/ml mupirocin calcium (MPC), 100 mM 2-hydroxypropyl-P- cyclodextrin (HPpCD) and 0.25% w/v carboy methyl cellulose (CMC). MPC-HPpCD- CMC-NaCl was obtained by dissolving the dry formulation of MPC-HPpCD-CMC-NaCl which consists of 20 mg/ml mupirocin calcium (MPC), 100 mM 2-hydroxypropyl-P- cyclodextrin (HPpCD), 2.8% w/v sodium chloride (NaCl) and 0.25% w/v carboxymethyl cellulose (CMC).

Example 2: Preparation of 2.0% w/v mupirocin calcium (MPC) in 100 mM 2 -hydroxy propy l- β -cyclodextrin (ΗΡβΟΌ)

[0132] For the preparation of mupirocin-cyclodextrin (MPC-CD) inclusion complexes, the 'dry physical mixing' method is used. In this method, 1400 mg of 2-hydroxypropyl-P- cyclodextrin (HPpCD) and 200 mg mupirocin (MPC) were weighed out and ground in a glass mortar. Following that, the ground physical mixture was subject to constant agitation in glass vials at 275 rpm in a thermostatically controlled shaker oven at 85 + 1°C for 2 hours. This allowed mupirocin to melt into a liquid (melting point of 77 - 78°C), which facilitated the incorporation into the cyclodextrin cavities. The heated and agitated mixture was then allowed to cool to room temperature (25°C). After cooling, the mixture was ground again for 10 minutes to ensure uniformity of mixture and facilitate rapid reconstitution later. When the powder is constituted in 10 ml of water, it will give a solution of 2.0% w/v MPC in 100 mM 2-hydroxypropyl-P-cyclodextrin. Example 3: Preparation of 2.0% w/v mupirocin chloride (MPC) in 100 mM 2-hydroxypropyl- β -cyclodextrin (ΗΡβΟΌ) with 2.8% w/v sodium chloride (NaCl) [0133] For the preparation of mupirocin-cyclodextrin (MPC-CD) inclusion complexes, the 'dry physical mixing' method is used. In this method, 1400 mg of 2-hydroxypropyl-P- cyclodextrin (HPpCD), 200 mg mupirocin chloride (MPC) and 280 mg sodium chloride (NaCl) were weighed out and ground in a glass mortar. Following that, the ground physical mixture was subject to constant agitation in glass vials at 275 rpm in a thermostatically controlled shaker oven at 85 + 1°C for 2 hours. This allowed mupirocin chloride to melt into a liquid (melting point of 77 - 78 °C), which facilitated the incorporation into the cyclodextrin cavities. The heated and agitated mixture was then allowed to cool to room temperature (25°C). After cooling, the mixture was ground again for 10 minutes to ensure uniformity of mixture and facilitate rapid reconstitution later. When the powder is constituted in 10 ml of water, it will give a solution of 2.0% w/v mupirocin chloride (MPC) in 100 mM 2-hydroxypropyl-P-cyclodextrin (HPpCD) with 2.8% w/v sodium chloride (NaCl).

Example 4: Preparation of 2.0% w/v mupirocin calcium (MPC) in 100 mM HPfCD with 2.8% w/v sodium chloride (NaCl) and 0.25% w/v carboxymethylcellulose sodium (CMC)

[0134] For the preparation of mupirocin-cyclodextrin (MPC-CD) inclusion complexes, the 'dry physical mixing' method is used. In this method, 1400 mg of 2-hydroxypropyl-P- cyclodextrin (HPpCD), 200 mg mupirocin calcium (MPC), 280 mg sodium chloride (NaCl) and 25 mg carboxymethyl cellulose (CMC) were weighed out and ground in a glass mortar. Following that, the ground physical mixture was subject to constant agitation in glass vials at 275 rpm in a thermostatically controlled shaker oven at 85 + 1°C for 2 hours. This allowed MPC to melt into a liquid (melting point of 77 - 78°C), which facilitated the incorporation into the CD cavities. The heated and agitated mixture was then allowed to cool to room temperature (25 °C). After cooling, the mixture was ground again for 10 minutes to ensure uniformity of mixture and facilitate rapid reconstitution later. When the powder is constituted in 10 ml of water, it will give a solution of 2.0% w/v MPC in 100 mM HPpCD with 2.8% w/v NaCl and 0.25% w/v CMC.

Example 5: High Performance Liquid Chromatography (HPLC) method (Mupirocin)

[0135] The method is performed as previously outlined. The retention time of mupirocin calcium (MPC) was 1.2 min with peak width of 0.4 min. Table 1. High Performance Liquid Chromatography (HPLC) Assay Parameters for Mupirocin.

Example 6: Stability Assay Validation (Mupirocin)

[0136] In order to establish the stability-indicating nature of the method, mupirocin was forcibly degraded by acid (IN hydrochloric acid) and base (IN sodium hydroxide) at room temperature and at 40°C for an hour. Both drugs were also placed under UV for 24 hours. Both drugs were dissolved in 50% acetonitrile and 50% water in screw cap glass tubes and for each degradation condition, triplicates were prepared. The samples were neutralised with an equal amount of IN sodium hydroxide and IN hydrochloric acid respectively. The samples were diluted in mobile phase before analysis. No degradation product peak was obtained for mupirocin under UV light for 24 hours. Degradation products were observed under acid and base degradation conditions at retention times of 1.2 min, 1.35 min and 5.0 min.

Example 7: Mupirocin Aqueous Formulation stability study

[0137] The MPC-HPpCD-NaCl composition was weighed and dissolved in 10 ml MilliQ water. Following that, it was filtered using 0.22 μιη Millex® GP Filter Units. The stability of the formulation was evaluated for 8 weeks under two conditions:

■ Room temperature with light exposure and

Room temperature without light exposure.

[0138] At fixed time points, an aliquot of formulation would be obtained, diluted down with mobile phase, analysed and quantified according to the chromatographic conditions as stated earlier. The reconstituted aqueous MPC formulation (MPC-HPpCD-NaCl) is observed to be stable for up to 8 weeks. Stability study of the composition is still ongoing to establish the long-term stability of the formulation.

Example 8: Mupirocin Saturation in 2-hydroxypropyl-fi-cyclodextrin (ΗΡβΟΌ) and a- cyclodextrin (a-CD)

[0139] The saturation concentration of mupirocin in water under varying concentrations of 2-hydroxypropyl-P-cyclodextrin (HPpCD) was determined. HPpCD was first weighed at varying concentrations from 20 mM to 150 mM. Excess mupirocin calcium was then added to the HPpCD powder and the mixture was ground in a glass mortar. It was then subjected to constant agitation in glass vials at 275 rpm in a thermostatically controlled shaker oven at 85 + 1°C for 2 hours before being reconstituted in MiUiQ water. Undissolved mupirocin calcium was removed by filtration using 0.22 μιη Millex® GP Filter Units. The resulting solution was then diluted appropriately and quantified using the HPLC assay.

[0140] The saturation concentration of mupirocin in water under in the presence of carboxymethyl cellulose (CMC) and sodium chloride (NaCl) and 100 mM 2-hydroxypropyl- β-cyclodextrin (HPpCD) was determined. Likewise, 100 mM HPpCD was first weighed. CMC of 0.25% w/v to 2.0% w/v and NaCl of 1.0% w/v were weighed added to the HPpCD powder depending on the formulation parameters. Excess mupirocin was then added to the mixture and ground in a glass mortar. It was then subjected to constant agitation in glass vials at 275 rpm in a thermostatically controlled shaker oven at 85 + 1°C for 2 hours before being reconstituted in MilliQ water. Undissolved mupirocin was removed by filtration using 0.22 μηι Millex® GP Filter Units. The resulting solution was then diluted appropriately and quantified using the HPLC assay. Example 9: Minimum Inhibitory concentration (MIC) for mupirocin - Macrodilution (TUBE) Broth Assay

[0141] A series of 15 mL glass tubes were filled with double- strength (D/S) nutrient broth (Acumedia® Nutrient Broth 7146, Lot: 106490B), placed in a test-tube rack and labelled accordingly. Four different formulations (MPC-HPpCD, MPC-HPpCD-NaCl, MPC-HPpCD- CMC, and MPC-HPpCD-CMC-NaCl, prepared by dry physical mixing) were dissolved in MilliQ water and filtered through 0.22 μιη Millex® GP Filter Units. Varying volumes of these formulations were added to each glass tube giving a range of concentrations for the determination of the minimum inhibitory concentration (MIC). Sterile water was then added to each glass tube. The tubes were then mixed thoroughly by rotating between the palms of the hands. Table 2. Preliminary Formulations for Minimum Inhibitory Concentration (MIC) assay using the Macrodilution (Tube) Broth Method with Staphylococcus aureus ATCC® 25923.

Name MPC Ml'fK^'D NaCI CMC

MPC-HPpCD 20mg/ml lOOmM

MPC-HPpCD-NaCl 20mg/ml lOOmM 3% w/v

IVl V_^-n ^pUlJ-^lVl^ umg/mi 1 l ΠuΠum.ηivi U. jyc w/v

MPC-HPpCD-CMC-NaC :\ 20mg/ml lOOmM 3% w/v 0.25% w/v

[0142] 0.2 ml of a diluted 24-hour broth culture of Staphylococcus aureus (MicroBiologics® S. aureus; Lot No.: 827963; RFF 0827S; ATCC 6538P) standardised at 0.5 McFarland Standard, was next added to each tube. A positive control containing only the cultured organism, sterile water and double-strength (D/S) nutrient broth was also prepared. The tubes were then mixed thoroughly again by rotating between the palms of the hands, and incubated at 37°C for 72 hours. The tubes were observed for turbidity, indicative of microbiological growth at 72 hours and quantified by comparing against McFarland Equivalence Standards (Batch No.: 1313947; TM4000T10). The minimum inhibitory concentration was recorded as the lowest antimicrobial concentration where no bacteria growth is observed after 72 hours incubation.

Table 3. Preliminary Minimum Inhibitory Concentration (MIC) results performed using the Macrodilution (Tube) Broth Method with Staphylococcus aureus ATCC® 25923.

Name of I ^'ormuhilioii M IC : Range i ng/rnh

MPC-HPpCD 800 - 1600

MPC-HPpCD-NaCl 80 - 160

MPC-HPpCD-CMC 800 - 1600

MPC-HPpCD-CMC-NaCl 800 - 1600

Example 10: Minimum Inhibitory Concentration (MIC) - Microdilution Broth assay

(Mupirocin) [0143] The minimum inhibitory concentration (MIC) of two formulations (MPC-HPpCD, MPC-HPpCD-NaCl) was determined against 7 Staphylococcus aureus strains: MRSA 1, MRSA 2, MRSA 3, MRSA 7, Mu50, WIS and MSSA 2590 using the Minimum Inhibitory Concentration (MIC) Broth Microdilution Assay. The assay was conducted in accordance to the guidelines set out in 'Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically - Eighth Edition' (Clinical and Laboratory Standards Institute, 2012). All Staphylococcus aureus strains were kindly provided by Jeanette Teo from National University Hospital. MRSA 1, MRSA 2, MRSA 3 and MRSA 7 are clinically isolated MRSA strains. MRSA strains Mu50, WIS and MSSA strain 2590 were obtained from ATCC. Out of the clinically-isolated MRSA strains, MRSA 1 and MRSA 2 are resistant to mupirocin. They are characterised by the presence of the mupA gene.

[0144] The drug compositions were dissolved in MilliQ water and filtered through 0.22 μηι Millex® GP Filter Units. It was further diluted in Mueller Hinton (MH) broth (Ref: 275730, Lot: 0224378) to appropriate concentrations for the determination of the MIC. Sterile 96-well round-bottom microtiter plates (Nunc, Roskilde, Denmark) were used. 24-hour broth culture of S. aureus was diluted with MH broth (and standardised at between lxlO⁵ - lxlO⁶ CFU/ml. This was determined by the plating of the bacteria on LB agar at the time of inoculation. Each row of the microplate was loaded with a different S. aureus strain. 50 μΐ of test inoculum was loaded into each well. 50 μΐ of drug solution was then added into first column and serial two-fold dilution is performed across the columns. The final volume of the well was 50 μΐ. The 96-well microtiter plates were then covered with a plate cover and incubated overnight at 37°C. The minimum inhibitory concentration was recorded as the lowest antimicrobial concentration where no bacteria growth is observed after overnight incubation. The experiments were performed in duplicates.

Table 4. Minimum Inhibitory Concentration (MIC) performed using the Broth Microdilution Method with 7 different strains of Staphylococcus aureus. The MIC at 24 hours (MIC₂₄) for MRSA 1 and MRSA 2 is much higher than that of other strains due to the presence of mupA gene in those strains.

[0145] The minimum inhibitory concentration (MIC) of the MPC -based formulation was found to be between 0.1 to 0.2 μg/ml for Methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin-sensitive Staphylococcus aureus (MSSA) bacteria strains. MIC increases from 48 to 96 μg/ml for MPC-resistant MRSA bacteria strains.

Example 11: Antibacterial effectiveness of mupirocin compositions

[0146] Preparation of the test inoculums were conducted using suitable mediums and in accordance to the method as previously described. The test microorganisms include: Escherichia coli (ATCC 8739), Pseudomonas aeruginosa (ATCC 9027), Staphylococcus aureus (ATCC 6538), Candida albicans (ATCC 10231) and Aspergillus brasiliensis (ATCC 16404). The test inoculums were incubated for microbial recovery. The incubated test inoculums were standardised such that the final concentration of the test preparations after inoculation is between lxlO⁵ and lxlO⁶ CFU/ml of the product. The initial concentration of viable test microorganisms in each test preparation is determined by the plate-count method.

Table 5. Results of antimicrobial effectiveness test with 20mg/ml MPC in lOOmM HPpCD with 2.8% NaCl against 5 different microorganisms. This formulation meets the preservative effectiveness requirements of USP 38- F 33 Chapter 51 "Antimicrobial Effectiveness Testing".

Pseudomonas

Effective aeruginosa 5.5 3.3 2.1 <0

(As of Day 28) (ATCC 9027)

Staphylococcus

Effective aureus 5.2 3.5 2.1 <0

(As of Day 28) (ATCC 6538)

Candida albicans Effective

Yeasts 5.2 5.2 4.6 1.5

(ATCC 10231) (As of Day 28) and

Aspergillus niger Effective Molds 5.7 5.2 4.7 3.7

(ATCC 16404) (As of Day 28)

Example 12: Time-dependent bacterial activity of mupirocin compositions

[0148] The time-dependent bactericidal kill profile of two mupirocin calcium compositions (MPC-HPpCD, MPC-HPpCD-NaCl) at full-strength and half-strength were determined against Staphylococcus aureus strains: MRS A 1, MRS A 2, MRSA7 and Mu50. The compositions were dissolved directly in MH broth in a 15ml falcon tube. Half-strength formulations were prepared by performing a two-fold dilution of the full- strength formulations.

[0149] At time zero, 2.5 μΐ of 24-hour broth culture of S. aureus was added to 5 ml of each formulation such that the bacteria concentration is between lxlO⁵ - lxlO⁶ CFU/ml. To obtain the initial bacteria count in the broth, 2.5 μΐ of 24-hour broth culture was added to 5 ml of MH broth. After which, an aliquot was obtained, diluted appropriately and plated on Luria Broth (LB) agar. At time point 15 minutes, 30 minutes, 2 hours, 6 hours and 24 hours, an aliquot was obtained from each tube, diluted appropriately and plated. A dilution of at least 1x10 is required to ensure that the drug in the solution has no bacteriostatic effect on the surviving bacteria during plating. The bacteria count at each time point was enumerated after overnight incubation of the plates at 37°C. A positive (bacteria without the addition of antimicrobial formulation) and negative control (broth only) were also included. The experiments were performed in duplicates.

Table 6. Raw CFU/ml Data of Time-dependent Bactericidal Activity of Mupirocin formulations with MRSA 2.

Table 7. Logio CFU/ml Average Data o Time -dependent Bactericidal Activity or Mupirocin formulations with MRSA 2

Table 8. Raw CFU/ml Data or Time-dependent Bactericidal Activity or Mupirocin formulations with MRSA 7

3.50E

hour 0 0 0 0 0 0 0 0

+10

1.13E

hour 0 0 0 0 0 0 0 0

+14

Table 9. Logio CFU/ml Average Data of Time -dependent Bactericidal Activity of Mupirocin formulations with MRSA 7

Example 13: Slurry approach to octenidine compositions

[0150] For the preparation of octenidine-cyclodextrin (OCT-CD) inclusion complexes, a 'slurry complexation-dry heat' method is used. In this method, 50 mM of 2-hydroxypropyl-P- cyclodextrin (HPpCD; Cavasol W7 HP Pharma, Wacker Chemicals), 0.1% w/v octenidine dihydrochloride (Tokyo Chemical Industry) were weighed out and ground in a glass mortar. Following that, 200 μΐ of water and 3.5 μΐ of 0.1N sodium hydroxide (NaOH) were added per 710 mg of ground mixture (i.e. 10ml of lmg/ml octenidine in water). This allows the dissolution of cyclodextrin and viscous slurry is formed with constant stirring. Saturating the aqueous phase with cyclodextrin first allows for complexation with octenidine dihydrochloride to occur spontaneously, due to entropic forces from the release of bound water molecules present within the cyclodextrin cavities. The OCT-HPpCD complexes will then saturate out of the aqueous phase, allowing the formation of more complexes in solution.

[0151] After the formation of the slurry, the slurry was then dried in a thermostatically controlled shaker oven at 80 + 1°C for 1 hour. The resulting powder was then ground to ensure uniformity. In the case of formulation with hypertonic saline, 2.8% w/v sodium chloride (NaCl) was weighed and ground together with the dried slurry powder.

[0152] To evaluate the efficacy of the octenidine dihydrochloride formulations, three formulations were prepared from the dry powder. Octenidine dihydrochloride-HiO was obtained by dissolving 1 mg/ml of octenidine dihydrochloride in water. Octenidine dihydrochloride-HPpCD solution was obtained by dissolving the dry formulation of OCT- HPPCD which consists of 1 mg/ml octenidine dihydrochloride and 50 mM 2-hydroxypropyl- β-cyclodextrin (HPpCD). OCT-HPpCD-NaCl solution was obtained by dissolving the dry formulation of OCT-HPpCD-NaCl which consists of 1 mg/ml octenidine hydrochloride (OCT), 50 mM 2-hydroxypropyl-p-cyclodextrin (HPpCD) and 2.8% sodium chloride (NaCl).

Example 14: Preparation of 0.1% w/v octenidine in 50 mM 2-hydroxypropyl-fi-cyclodextnn 10 mg octenidine dihydrochloride (Tokyo Chemical Industry) and 700 mg 2-hydroxypropyl- β-cyclodextrin (HPpCD) (Cavasol W7 HP Pharma, Wacker Chemicals) was weighed out and ground in a glass mortar. Following that, 200μ1 of water and 3.5 μΐ of 0.1 N sodium hydroxide (NaOH) were added. This allowed the dissociation of cyclodextrin and viscous slurry was formed with constant stirring. The slurry was then dried in a thermostatically controlled shaker oven at 80 + 1°C for 1 hour. The resulting powder was then ground to ensure uniformity. The dried powder can be readily reconstituted with 10 ml water to give 0.1% w/v octenidine dihydrochloride in 50 mM 2-hydroxypropyl-P-cyclodextrin.

Example 15: Preparation of 0.1% w/v octenidine in 50 mMa -cyclodextrin

[0153] 10 mg octenidine dihydrochloride (Tokyo Chemical Industry) and 486.4 mg a- cyclodextrin (Cavamax W6, Wacker Chemicals) was weighed out and ground in a glass mortar. Following that, 200 μΐ of water and 3.5 μΐ of 0.1 N sodium hydroxide (NaOH) are added. This allowed the dissociation of cyclodextrin and viscous slurry was formed with constant stirring. The slurry was then dried in a thermostatically controlled shaker oven at 80+1 °C for 1 hour. The resulting powder was then ground to ensure uniformity. The dried powder can be readily reconstituted with 10 ml water to give 0.1% w/v octenidine dihydrochloride in 50 mM a-cyclodextrin.

Example 16: Preparation of 0.1% w/v octenidine in 50 mM 2-hydroxypropyl-fi-cyclodextrin (ΗΡβΟΌ) with 2.8% w/v sodium chloride (NaCl)

[0154] 10 mg octenidine dihydrochloride (Tokyo Chemical Industry) and 700 mg 2-hydroxypropyl-P-cyclodextrin (HPpCD. Cavasol W7 HP Pharma, Wacker Chemicals) were weighed out and ground in a glass mortar. Following that, 200 μΐ of water and 3.5 μΐ of 0.1 N sodium hydroxide (NaOH) was added. This allowed the dissociation of cyclodextrin and viscous slurry was formed with constant stirring. The slurry was then dried in a thermostatically controlled shaker oven at 80 + 1°C for 1 hour. The resulting powder was then ground with 280 mg sodium chloride (NaCl) to ensure uniformity. The dried powder can be readily reconstituted with 10 ml water to give 0.1% w/v octenidine dihydrochloride in 50 mM 2-hydroxypropyl-P-cyclodextrin (HPpCD) with 2.8% w/v sodium chloride (NaCl).

Example 17: Preparation of 0.1% w/v octenidine in 50 mM a-cyclodextrin with 2.8% w/v sodium chloride (NaCl)

[0155] 10 mg octenidine dihydrochloride (Tokyo Chemical Industry) and 486.4 mg a- cyclodextrin (Cavamax W6, Wacker Chemicals) were weighed out and ground in a glass mortar. Following that, 200μ1 of water and 3.5 μΐ of 0.1 N sodium hydroxide (NaOH) were added. This allowed the dissociation of cyclodextrin and viscous slurry was formed with constant stirring. The slurry was then dried in a thermostatically controlled shaker oven at 80 + 1°C for 1 hour. The resulting powder was then ground with 280 mg sodium chloride (NaCl) to ensure uniformity. The dried powder can be readily reconstituted with 10 ml water to give 0.1%) w/v octenidine dihydrochloride (OCT) in 50 mM α-cyclodextrin (a-CD) with 2.8% w/v sodium chloride (NaCl). Example 18: High Performance Liquid Chromatography Method for Octenidine

High Performance Liquid Chromatography (HPLC) was performed as disclosed above in order to quantify the octenidine dihydrochloride saturation concentration in the formulation to identify a suitable 2-hydroxypropyl-P-cyclodextrin (HPpCD) concentration for the co- dissolution of octenidine with hypertonic saline. The oven temperature was maintained at 50°C. The retention time of octenidine dihydrochloride was 1.2 min with peak width of 0.8 min.

Table 10. High Performance Liquid Chromatography (HPLC) Assay Parameters Octenidine.

Example 19: Stability Assay Validation of Octenidine compositions

[0156] In order to establish the stability-indicating nature of the method, octenidine dihydrochloride was forcibly degraded by acid (IN hydrochloric acid, HCl) and base (IN sodium hydroxide, NaOH) at room temperature and at 40°C for an hour. Both drugs were also placed under UV for 24 hours. Both drugs were dissolved in 50% acetonitrile and 50% water in screw cap glass tubes and for each degradation condition, triplicates were prepared. The samples were neutralised with an equal amount of IN sodium hydroxide (NaOH) and IN hydrochloric acid (HCl) respectively. The samples were diluted in mobile phase before analysis. No degradation product peak was obtained for octenidine under these conditions.

Example 20: Octenidine Powder composition stability study

[0157] The stability of powder octenidine dihydrochloride-2-hydroxypropyl-P- cyclodextrin-sodium chloride (OCT-HPpCD-NaCl) compositions was evaluated with the QTRAP 5500 LC-MS/MS system. The composition was reconstituted by dissolving in MilliQ water and diluted to a concentration of 1 μg/ml with 50% methanol and 50% water. The solution was directly injected into the LC-MS/MS system and the parent ion was scanned with in the positive scanning mode and a scan range of 500 to 700. The octenidine dihydrochloride parent ion peak was detected at m/z of 551.7. The parent ion or Ql scan, conducted at the first quadrupole mass filter was performed to identify degradation in octenidine dihydrochloride. Degradation can be concluded if there is a reduction in parent ion intensity or if there were other prominent peaks other than the parent ion peak. The stability of the formulation was evaluated after 8 weeks of storage. The dry powder OCT- HPpCD-NaCl formulation is observed to be stable for up to 8 weeks (the stability study is still on going to establish the long-term stability). Example 21: Octenidine saturation in 2-hydroxypropyl-fi-cyclodextrin (ΗΡβΟΌ) and a- cyclodextrin (a-CD)

[0158] The saturation concentration of octenidine dihydrochloride in the presence of 2.8% sodium chloride (NaCl) under varying concentration of 2-hydroxypropyl-P-cyclodextrin (HPpCD) and a-cyclodextrin (a-CD) (Tokyo Chemical Industry) was determined. HPpCD and a-CD was first dissolved in water at varying concentrations from 50 mM to 300 mM and 50 mM to 100 mM respectively. 2.8% of sodium chloride (NaCl) was added to the dissolved cyclodextrins. Excess octenidine dihydrochloride was added to the solution and shaken at room temperature for an hour. Undissolved octenidine dihydrochloride was removed by filtration using 0.22 μιη Millex® GP Filter Units. The resulting solution was then diluted appropriately and quantified using the HPLC assay. The saturation of octenidine dihydrochloride in water and octenidine in water with 2.8% sodium chloride (NaCl) was also evaluated as a control. After analysing the results of the saturation study, 2-hydroxypropyl-P- cyclodextrin (HPpCD) at a concentration of 50 mM was chosen to formulate octenidine dihydrochloride in the presence of 2.8% NaCl. This was because 2-hydroxypropyl-P- cyclodextrin had greater solubility than a-cyclodextrin in water, with the maximum solubility at 300 mM and 100 mM respectively. Furthermore, at 50 mM, it was sufficient to dissolve more than 10 times the target concentration of octenidine dihydrochloride (1 mg/ml) in the presence of hypertonic saline. At this 2-hydroxypropyl-P-cyclodextrin concentration, octenidine dihydrochloride formulation could be solubilised quickly and the slurry could be manipulated more easily due to its lower viscosity.

Example 22: Minimum Inhibitory Concentration (MIC) of octenidine compositions- Macrodilution ( tube ) broth assay

[0159] A series of 15 mL glass tubes were filled with double- strength (D/S) nutrient broth (Acumedia® Nutrient Broth 7146, Lot: 106490B), placed in a test-tube rack and labelled accordingly. Three different compositions (OCT-H₂0, OCT-HPpCD and OCT-HPpCD- NaCl, prepared by slurry complexation-dry heat method) were dissolved in MilliQ water and filtered through 0.22 μιη Millex® GP Filter Units. Varying volumes of these compositions were added to each glass tube giving a range of concentrations for the determination of the minimum inhibitory concentration. Sterile water was then added to each glass tube. The tubes were then mixed thoroughly by rotating between the palms of the hands. Table 11. Preliminary Formulations for Minimum Inhibitory Concentration (MIC) assay using the Macrodilution (Tube) Broth Method with Staphylococcus aureus ATCC® 25923.

0.2 ml of a diluted 24-hour broth culture of Staphylococcus aureus (MicroBiologics® S. aureus; Lot No.: 827963; RFF 0827S; ATCC 6538P) standardised at 0.5 McFarland Standard, was next added to each tube. A positive control containing only the cultured organism, sterile water and double-strength (D/S) nutrient broth was also prepared. The tubes were then mixed thoroughly again by rotating between the palms of the hands, and incubated at 37°C for 72 hours. The tubes were observed for turbidity, indicative of microbiological growth at 72 hours and quantified by comparing against McFarland Equivalence Standards (Batch No.: 1313947; TM4000T10). The minimum inhibitory concentration was recorded as the lowest antimicrobial concentration where no bacteria growth is observed after 72 hours incubation.

Table 12. Preliminary Minimum Inhibitory Concentration (MIC) results performed using the Macrodilution (Tube) Broth Method with Staphylococcus aureus ATCC® 25923.

Name of l -^'onmilai ioii M IC : R n e i ng/mh

OCT-H20 400 - 800

OCT-HPpCD 200 - 400

OCT-HPpCD-NaCl 200 - 400

Example 23: Minimum Inhibitory Concentration (MIC) of octenidine compositions- Microdilution ( tube ) broth assay

[0160] The minimum inhibitory concentration (MIC) of two octenidine dihydrochloride compositions (OCT-HPpCD, OCT-HPpCD-NaCl) were determined against 7 Staphylococcus aureus strains: MRSA 1, MRSA 2, MRSA 3, MRSA 7, Mu50, WIS and MSSA 2590 using the Minimum Inhibitory Concentration (MIC) Broth Microdilution Assay. The assay was conducted in accordance to the guidelines set out in 'Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically - Eighth Edition' (Clinical and Laboratory Standards Institute, 2012). The MIC of OCT-H₂0 was not determined due to its susceptibility to precipitate in the presence of Mueller Hinton (MH) broth (Ref: 275730, Lot: 0224378). All Staphylococcus aureus strains were kindly provided by Jeanette Teo from National University Hospital. MRSA 1, MRSA 2, MRSA 3 and MRSA 7 are clinically isolated MRSA strains. MRSA strains Mu50, WIS and MSSA strain 2590 were obtained from ATCC. Out of the clinically-isolated MRSA strains, MRSA 1 and MRSA 2 are resistant to mupirocin. They are characterised by the presence of the mupA gene. The drug formulations were dissolved in MiUiQ water and filtered through 0.22 μιη Millex® GP Filter Units. It was further diluted in MH broth to appropriate concentrations for the determination of the MIC. Sterile 96-well round-bottom microtiter plates (Nunc, Roskilde, Denmark) were used. 24-hour broth culture of S. aureus was diluted with MH broth (and standardised at between lxlO⁵ - lxlO⁶ CFU/ml. This was determined by the plating of the bacteria on Luria Broth (LB) Agar at the time of inoculation. Each row of the microplate was loaded with a different S. aureus strain. 50 μΐ of test inoculum was loaded into each well. 50 μΐ of drug solution was then added into first column and serial two-fold dilution is performed across the columns. The final volume of the well is 50 μΐ. The 96-well microtiter plates were then covered with a plate cover and incubated overnight at 37°C. The minimum inhibitory concentration was recorded as the lowest antimicrobial concentration where no bacteria growth is observed after overnight incubation. The experiments were performed in duplicates.

Table 13. Minimum Inhibitory Concentration (MIC) performed using the Broth Microdilution Method with 7 different strains of Staphylococcus aureus. The MIC at 24 hours (MIC₂₄) for MRSA and MSSA bacteria strains are comparable.

1 mg/ml OCT

in 50mM 1.6 1.6 1.6 1.6 1.6 1.6 1.2

HPpCD with g ml g ml Mg/ml Mg/ml Mg/ml Mg/ml Mg/ml

2.8% NaCl

[0161] The minimum inhibitory concentration (MIC) of the octenidine dihydrochloride- based formulation was found to be about 1.6 Mg/ml for all Methicillin -resistant Staphylococcus aureus (MRSA) and Methicillin-sensitive Staphylococcus aureus (MSSA) bacteria strains tested. It was also found to have equal activity against mupirocin-resistant and mupirocin-responsive MRSA.

Example 24: Time-dependent determination of bacterial activity of octenidine compositions [0162] The time-dependent bactericidal kill profile of two compositions (OCT-HPpCD, OCT-HPpCD-NaCl) at full-strength and half-strength were determined against Staphylococcus aureus strains: MRSA 1, MRSA 2, MRSA7 and Mu50. The formulations were dissolved directly in MH broth in a 15ml falcon tube. Half-strength formulations were prepared by performing a two-fold dilution of the full-strength formulations. At time zero, 2.5 μΐ of 24-hour broth culture of S. aureus was added to 5 ml of each formulation such that the bacteria concentration is between 1x105 - 1x106 CFU/ml (colony forming units per ml). To obtain the initial bacteria count in the broth, 2.5 μΐ of 24-hour broth culture was added to 5 ml of Mueller-Hinton (MH) broth. After which, an aliquot was obtained, diluted appropriately and plated on Luria broth (LB) agar.

[0163] At time point 15 minutes, 30 minutes, 2 hours, 6 hours and 24 hours, an aliquot was obtained from each tube, diluted appropriately and plated. A dilution of at least 1x10 (1E3) is required to ensure that the drug in the solution has no bacteriostatic effect on the surviving bacteria during plating. The bacteria count at each time point was enumerated after overnight incubation of the plates at 37°C. A positive (bacteria without the addition of antimicrobial formulation) and negative control (broth only) are also included. The experiments were performed in duplicates.

Table 14. Raw CFU/ml Data of Time-dependent Bactericidal Activity of Octenidine formulations with MRSA 7

Table 15. Log 10 CFU/ml Average Data of Time-dependent Bactericidal Activity of

Octenidine formulations with MRSA 7

Example 25: Antimicrobial effectiveness testing of octenidine compositions against bacteria and fungi

[0164] The results of the effectiveness of octenidine antiseptic solutions against bacteria and fungi. This experiment was performed as outlined in the Experimental section above.

Table 16. Results of antimicrobial effectiveness test with lmg/ml OCT against 5 different microorganisms. This formulation meets the preservative effectiveness requirements of USP 38- F 33 Chapter 51 "Antimicrobial Effectiveness Testing".

Tesi M icroorgan isms ttesu li ( log i„ Cl ^'l ^'/nil ) Conclusion

Table 17. Results of antimicrobial effectiveness test with lmg/ml OCT in 50mM HPpCD with 2.8% NaCl against 5 different microorganisms. This formulation meets the preservative effectiveness requirements of USP 38- F 33 Chapter 51 "Antimicrobial Effectiveness Testing".

Example 26: Aqueous (slurry complexation) and non-aqueous (dry heat mixing) approach to triclosan compositions

[0165] Two methods were adopted in the preparation of the TCS-HPpCD-CMC-NaCl complexes. ' Slurry complexation' (aqueous method) was one method used to prepare the complexes. 40mM of 2-hydroxypropyl-P-cyclodextrin (HPpCD) were weighed out and ground in a glass mortar. A small quantity of water (less than 500 μΐ) was added to dissolve the cyclodextrin and stirred with constant agitation for about 15 minutes. Triclosan (TCS, 30 mg) was then added to the aqueous cyclodextrin and ground for another 30 minutes. Saturating the aqueous phase with cyclodextrin first allows for complexation with triclosan to occur spontaneously, due to entropic forces from the release of bound water molecules present within the cyclodextrin cavities. The TCS-HPpCD complexes will then saturate out of the aqueous phase, allowing the formation of more complexes in solution. 1% w/v carboxymethyl cellulose (CMC) was next added to the complex mixtures and the samples were ground for another 30 minutes. In the case of formulations with hypertonic saline, 1% w/v sodium chloride NaCl crystals were ground into the mixture before addition of the carboxymethyl cellulose. The completed slurries were collected in 1.5 ml Eppendorf® tubes and placed in a freeze-dryer (0.05 mBar, -47°C) for 2 hours. The dried powders were obtained and each was dissolved in 10 mL of MilliQ water, which were filtered immediately through 0.22 μιη Millex® GP Filter Units.

[0166] 'Dry physical mixing' (non-aqueous method) was another method proposed to prepare the complexes. 40 mM of 2-hydroxypropyl-P-cyclodextrin (HPpCD), 1% w/v carboxymethyl cellulose (CMC), 1% w/v sodium chloride (NaCl) and 0.3% w/v of triclosan (TCS, 30 mg) were weighed out and ground in a glass mortar for about 15 minutes. The ground physical mixtures were subject to constant agitation in glass vials at 275 rpm in a thermostatically controlled shaker oven at 60 + 1°C for 24 hours. This temperature was sufficient to melt triclosan into a liquid (melting point of 54 - 57°C), which facilitated the incorporation into the cyclodextrin cavities. The heated and agitated mixtures were then allowed to cool to room temperature (20 - 25°C) before dissolving in 10 ml of MilliQ water. The dissolved solutions were filtered immediately through 0.22 μπι Millex® GP Filter Units. To further enhance the solubility of TCS-FIPpCD, 80 mM of 2-hydroxypropyl-P-cyclodextrin (FIPpCD) can be used instead and formulated in the same manner.

Example 27: Dry physical mixture of HPfCD-CMC-TCS for nasal nebulisation

[0167] The dry powders of 2-hydroxypropyl-P-cyclodextrin (FIPpCD), carboxymethyl cellulose (CMC), and triclosan (TCS) were weighed out separately and ground in a glass mortar until a fine powder was obtained. The powdered mixture was subjected to constant agitation in glass vials at 275 rpm in a thermostatically controlled shaker oven at 60 + 1°C for 2 hours. The heated and agitated mixtures were then allowed to cool to room temperature (20 - 25°C) before dissolving in MilliQ water. The dissolved solutions were then immediately filtered through 0.22 μπι Millex® GP Filter Units. This triclosan-based solution composition can be used in a nebuliser to deliver fumes of antiseptic to the nasal cavities, providing decolonising effect. 2-hydroxypropyl-P-cyclodextrin and carboxymethyl cellulose serve to improve the aqueous solubility of the formulation and modulate the release of triclosan in the nasal cavity when nebulised. Example 28: Dry physical mixture of HP fCD-CMC-TCS-NaCl for nasal nebulisation

[0168] The dry powders of 2-hydroxypropyl-P-cyclodextrin (FIPpCD), carboxymethyl cellulose (CMC), sodium chloride (NaCl) and triclosan (TCS) were weighed out separately and ground in a glass mortar until a fine powder was obtained. The powdered composition was subjected to constant agitation in glass vials at 275 rpm in a thermostatically controlled shaker oven at 60 + 1°C for 2 hours. The heated and agitated compositions were then allowed to cool to room temperature (20 - 25°C) before dissolving in MilliQ water. The dissolved compositions were then immediately filtered through 0.22 μπι Millex® GP Filter Units. This triclosan-based solution composition can be used in a nebuliser to deliver fumes of antiseptic to the nasal cavities, providing decolonising effect. 2-hydroxypropyl-P-cyclodextrin (FIPpCD) and carboxymethyl cellulose (CMC) serve to improve the aqueous solubility of the formulation and modulate the release of triclosan in the nasal cavity when nebulised. Saline is added for its bacteriostatic and irrigative properties, and also enhances the aqueous solubility of the formulation. Example 29: Lyophilised mixture of HPfCD-CMC-TCS for nasal nebulisation

[0169] The two proposed methods of 'dry physical mixing' and 'slurry complexation' as previously described were used respectively to produce aqueous solutions of triclosan, which can be used intranasally with other administration techniques besides nebulisation. Examples of such administration methods can be in the form of nasal sprays, douches, and inhalations. FIPpCD and CMC serve to improve the aqueous solubility of the formulation and modulate the release of triclosan in the nasal cavity using these various administration techniques. [0170] 2-hydroxypropyl-P-cyclodextrin (HPpCD), triclosan (TCS) and carboxymethyl cellulose (CMC ) were added in sequence in order to produce slurry for lyophilisation. A small quantity of water (less than 500 pL) was added to dissolve 2-hydroxypropyl-P- cyclodextrin ( and stirred with constant agitation for about 15 minutes, until a clear solution was obtained. Triclosan was then added to the aqueous cyclodextrin and ground for another 30 minutes, producing a cloudy paste. The addition of carboxymethyl cellulose produced a viscous and sticky paste, which was then collected in Eppendorf® tubes and placed in a freeze-dryer (0.05 mBar, -47°C) for 24 hours. The dried powders obtained were then dissolved in MilliQ water, which were filtered immediately through 0.22 μπι Millex® GP Filter Units. This triclosan-based solution composition can be used in a nebuliser to deliver fumes of antiseptic to the nasal cavities, providing decolonising effect. 2-hydroxypropyl-P- cyclodextrin (FfPpCD) and carboxymethyl cellulose serve to improve the aqueous solubility of the formulation and modulate the release of triclosan in the nasal cavity when nebulised. Example 30: Lyophilised mixture ofHPfiCD-CMC-TCS-NaClfor nasal nebulisation.

[0171] The two methods of 'dry physical mixing' and 'slurry complexation', as previously described, were respectively used to produce aqueous solutions of triclosan, which can be used intranasally with other administration techniques besides nebulisation. Examples of such administration methods can be in the form of nasal sprays, douches, and inhalations.

[0172] HPPCD, TCS, NaCl and CMC were added in sequence in order to produce slurry for lyophilisation. A small quantity of water (less than 500 μΐ,) was added to dissolve FfPpCD and stirred with constant agitation for about 15 minutes, until a clear solution was obtained. TCS was then added to the aqueous CD and ground for another 30 minutes, producing a cloudy paste. NaCl was added to the paste next, and further grinding incorporated the crystals into the slurry. The addition of CMC produced a viscous and sticky paste, which was then collected in Eppendorf® tubes and placed in a freeze-dryer (0.05 mBar, -47°C) for 24 hours. The dried powders obtained were then dissolved in MilliQ water, which were filtered immediately through 0.22 μπι Millex® GP Filter Units. This triclosan-based solution formulated can be used in a nebuliser to deliver fumes of antiseptic to the nasal cavities, providing decolonising effect.

Example 31: High Performance Liquid Chromatography (HPLC) of triclosan compositions [0173] High Performance Liquid Chromatography (HPLC) was used to quantify the triclosan saturation concentration in the composition to identify a suitable 2-hydroxypropyl- β-cyclodextrin (HPpCD) concentration for the solubilisation of triclosan in water. The system used consisted of a Shimadzu LC-10AT pump, SCL-IOA system controller, SIL- 10AD auto-sampler and CTO-10AS column oven (Shimadzu Corporation, Kyoto, Japan). Chromatographic separation was achieved on Mightysil RP-18 GP 150 mm X 4.6 mm, particle size 5 μηι (Kanto Chemical Co.) analytical column with an isocratic mobile phase of 80% acetonitrile and 20% water supplemented with 0.1% formic acid. HPLC grade acetonitrile was used. A flow rate of 1.5 ml/min was used and the UV detection wavelength was set at 283 nm to monitor analyte peak elution. The oven temperature was maintained at 50°C. The retention time of TCS was 2.8 min with peak width of 0.4 min.

Table 18. High Performance Liquid Chromatography (HPLC) Assay Parameters for

Example 32: Stability Assay validation of triclosan compositions

[0174] In order to establish the stability-indicating nature of the method, triclosan was forcibly degraded by acid (IN hydrochloric acid, HC1) and base (IN sodium hydroxide, NaOH) at room temperature and at 40°C for an hour. Both acidic and basic samples were also placed under UV light for 24 hours. Both drugs were dissolved in 50% acetonitrile and 50% water in screw cap glass tubes and for each degradation condition, triplicates were prepared. The samples were neutralised with an equal amount of IN sodium hydroxide (NaOH) and IN hydrochloric acid (HC1) respectively. The samples were diluted in mobile phase before analysis. No degradation product peak was obtained for triclosan under these conditions. Example 33: Triclosan powder composition stability assay

[0175] The stability of powder TCS-HPpCD-NaCl composition was evaluated with the QTRAP 5500 LC-MS/MS system. The formulation was reconstituted by dissolving in MilliQ water and diluted to a concentration of 1 μg/ml with 50% methanol and 50% water. The solution was directly injected into the LC-MS/MS system and the parent ion was scanned with in the negative scanning mode and a scan range of 150 to 300. The triclosan parent ion peak was detected at m/z of 288.9. The parent ion or Ql scan, conducted at the first quadrupole mass filter was performed to identify degradation in triclosan. Degradation can be concluded if there is a reduction in parent ion intensity or if there were other prominent peaks other than the parent ion peak. The stability of the formulation was evaluated after 8 weeks of storage.

Example 34: Triclosan aqueous composition stability study

[0176] TCS-HPpCD-NaCl composition produced using the slurry method as described herein was weighed and dissolved in 10 ml MilliQ water. Following that, it was filtered using 0.22 μηι Millex® GP Filter Units. The stability of the composition was evaluated for 8 weeks under two conditions:

Room temperature with light exposure and

Room temperature without light exposure.

[0177] At fixed time points, an aliquot of formulation was obtained, diluted down with mobile phase, analysed and quantified according to the chromatographic conditions as stated in Example 36. The dry powder TCS-HPpCD-NaCl formulation was observed to be stable for up to 8 weeks. The aqueous formulation of TCS-HPpCD-NaCl was observed to be stable during the study period of 72 hours.

Example 35: Triclosan saturation in 2-hydroxypropyl-fi-cyclodextrin (ΗΡβΟΌ) with carboxymethyl cellulose (CMC)

[0178] The saturation concentration of triclosan in water under varying concentrations of 2-hydroxypropyl-P-cyclodextrin (HPpCD) and carboxymethyl cellulose (CMC) was determined. HPpCD was first weighed at varying concentrations from 10 mM to 60 mM. CMC was also weighed at concentrations from 0.25% w/v to 5.0% w/v. For the dry physical mixing, excess triclosan was then added to the HPpCD and CMC powder and the mixture was ground in a glass mortar. It was then subjected to constant agitation in glass vials at 275 rpm in a thermostatically controlled shaker oven at 60 + 1°C for 2 hours before being reconstituted in MilliQ water. Undissolved triclosan was removed by filtration using 0.22 μιη Millex® GP Filter Units. The resulting solution was then diluted appropriately and quantified using the HPLC assay.

[0179] For the slurry-complexation method, a small quantity of water (less than 500 μΐ) was added to dissolve the cyclodextrin and stirred with constant agitation for about 15 minutes. Excess TCS was then added to the aqueous cyclodextrin and ground for another 30 minutes. CMC was next added to the complex mixtures and the samples were ground for another 30 minutes. The completed slurries were collected in 1.5 ml Eppendorf® tubes and placed in a freeze-dryer (0.05 mBar, -47°C) for 24 hours. The dried powders were obtained and reconstituted in MilliQ water. Undissolved triclosan was removed by filtration using 0.22 μηι Millex® GP Filter Units. The resulting solution was then diluted appropriately and quantified using the HPLC assay.

Example 36: Minimum Inhibitory Concentration (MIC) of triclosan compositions- Macrodilution ( tube ) broth assay

[0180] The Minimum Inhibitory Concentration (MIC) of triclosan compositions was performed according to the Macrodilution (tube) broth assay as previously described. The minimum inhibitory concentration was recorded as the lowest antimicrobial concentration where no bacteria growth is observed after 72 hours incubation.

Tube N umber 1 2 3 4 5 6

TCS Cc mcentration

0 2.4 6 12 24 30 (ng mL 12.0 12.0 10.5 7.50 3.00

72-hour Cell Density* + + + + + 0

(1 x 10⁸ CFU/mL)

0.00 0.00 2.12 2.12 0.00

1 111K^> Number 7 s 9 10 1 1 12

TCS Concentration

150 300 1,500 3,000 30,000 75,000 (ng mL-¹)

72-hour Cell Density*

0 0 0 0 0 0

(1 x 10⁸ CFU/mL)

Table 19. The minimum inhibitory concentration (MIC) of the TCS-based formulation was found to be between 24 ng mL^"1 to 30 ng mL^"1, from a 72-hour incubation period at 37°C. The test samples were deliberately inoculated with a 24-hour broth culture of Staphylococcus aureus.

Table 20. The minimum inhibitory concentration (MIC) of the TCS-based formulation with 1% w/v saline was found to be between 12 ng mL^"1 to 24 ng mL^"1, after a 72-hour incubation period at 37°C. The test samples were deliberately inoculated with a 24-hour broth culture of Staphylococcus aureus.

Example 37: Minimum Inhibitory Concentration (MIC) of triclosan compositions - Microdilution assay [0181] The minimum inhibitory concentration (MIC) of two compositions (TCS-HPpCD, TCS-HPpCD-NaCl) was determined against 7 Staphylococcus aureus strains: MRSA 1, MRSA 2, MRSA 3, MRSA 7, Mu50, WIS and MSSA 2590 using the Minimum Inhibitory Concentration (MIC) Broth Microdilution Assay. The assay was conducted as described previously. Out of the clinically-isolated MRSA strains, MRSA 1 and MRSA 2 are resistant to mupirocin. They are characterised by the presence of the mupA gene.

[0182] The minimum inhibitory concentration (MIC) of the triclosan-based formulation was found to be between 24 ng/ml to 30 ng/ml; and between 12 ng/ml to 24 ng/ml for the formulation with 1% w/v saline added. This suggests a possible additive/synergistic effect between saline and the TCS-HPpCD-CMC formulation. When the formulations were evaluated against a wider range of bacteria strains, the MIC of the TCS -based formulation was found to be between 1 ng/ml to 0.2 μg/ml for Methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin-sensitive Staphylococcus aureus (MSSA) bacteria strains.

Table 21. Minimum Inhibitory Concentration (MIC) performed using the Broth Microdilution Method with 7 different strains of Staphylococcus aureus. The MIC at 24 hours (MIC24) for MRSA 2 and MRSA 3 for 3mg/ml TCS in 80mM HPpCD with 2.8% NaCl was unable to be conclusively determined. MIC24 of MRSA and MSSA strains are comparable.

Table 22. Results of antimicrobial effectiveness test with 3mg/ml TCS in 80mM HPpCD with 2.8% NaCl against 5 different microorganisms. This formulation meets the preservative effectiveness requirements of USP 38- F 33 Chapter 51 "Antimicrobial Effectiveness Testing"

Example 38: Time-dependent bacterial activity of triclosan compositions

[0183] The time-dependent bactericidal kill profile of two compositions (TCS-HPpCD, TCS-HPpCD-NaCl) at full-strength and half-strength were determined against Staphylococcus aureus strains: MRS A 1, MRS A 2, MRSA7 and Mu50. The compositions were dissolved directly in Mueller Hinton (MH) broth in a 15 ml falcon tube. Half-strength concentrations were prepared by performing a two-fold dilution of the full-strength concentrations.

[0184] At time zero, 2.5 μΐ of 24-hour broth culture of S. aureus was added to 5 ml of each formulation such that the bacteria concentration is between lxl 0⁵ - lxl 0⁶ colony- forming units per ml (CFU/ml). To obtain the initial bacteria count in the broth, 2.5 μΐ of 24-hour broth culture was added to 5 ml of Mueller Hinton (MH) broth. After which, an aliquot was obtained, diluted appropriately and plated on Luria Broth (LB) agar.

[0185] At time point 15 minutes, 30 minutes, 2 hours, 6 hours and 24 hours, an aliquot was obtained from each tube, diluted appropriately and plated. A dilution of at least 1x10 (1E3) is required to ensure that the composition in the solution has no bacteriostatic effect on the surviving bacteria during plating. The bacteria count at each time point was enumerated after overnight incubation of the plates at 37°C. A positive (bacteria without the addition of antimicrobial formulation) and negative control (broth only) were also included. The experiments were performed in duplicates. Table 23. Raw CFU/ml Data of Time-dependent Bactericidal Activity of Triclosan formulations with MRSA 2

Table 24. LoglO CFU/ml Average Data of Time-dependent Bactericidal Activity of Triclosan formulations with MRSA 2

Table 25. Raw CFU/ml Data of Time-dependent Bactericidal Activity of Triclosan formulations with MRSA 7

Table 26. LoglO CFU/ml Average Data of Time-dependent Bactericidal Activity of Triclosan formulations with MRSA 7

Example 39: Ex-vivo swab culture test of triclosan compositions

[0186] The TCS-HPpCD-CMC-NaCl composition (0.3% w/v TCS) prepared by dry mixing was evaluated for its efficacy as a nebulising solution against the nasal swab samples of 'healthy' volunteers. A total of twenty volunteers were recruited for the study. These volunteers were screened preliminarily through an online sign-up process (Google Forms) and particulars were collated in accordance with the Personal Data Protection Act 2012. The exclusion criteria for the study was: (1) A history of any nose operations or procedures (2) History of allergic rhinitis, sinusitis or nasal mucositis (3) Chronic use of any nasal products (4) Chronic use of any antibiotics through any administration route. Volunteers fulfilling these criteria were deemed to be 'healthy'. Copan Innovation Sterile Cotton Swabs (150C Cotton; Lot: B25 GCGL00) were purchased for the study. [0187] Volunteers were requested to perform hand-washing steps with 4% w/v chlorhexidine gluconate solution, and lather on their entire hand surface 4% w/v chlorhexidine gluconate gel. The hands were allowed to either air-dry or dried using an automated hand-dryer. The volunteers then placed on Biomedia Powder-free Latex Examination Gloves (Lot: 110420011300) and then disinfected the gloves with 70% ethanol. Under close supervision of the investigator, the volunteers were asked to carefully remove the sterile cotton swabs from the protective casings, taking care not to allow the swab to come into contact with any surfaces. The swab was then inserted about two centimetres into one nostril, and rested against the nasal septum. Light pressure was applied on the outside of the nose against the swab, such that the swab was held steadily in place between the septum and mucosa membrane. The swab was rotated against the mucosa for a total of 5 revolutions lasting 1 minute. The swab was then placed back into the protective casing, and another swab was similarly performed for the other nostril.

[0188] The two swab samples collected from each volunteer were subjected to different procedures. One swab was placed directly into 10 ml of a normal-strength nutrient broth solution; the other was subject to 2 ml of the nebulised solution for a total of 5 minutes. The swab was clamped using a retort stand and aligned directly against the nebuliser (set-up as shown in Figure 25). During the nebulisation process, the swab was rotated 90° clockwise every 1 minute and 15 seconds to ensure maximal coverage of the swab by the nebulised solution. The treated swab was then placed in 10 ml of normal-strength nutrient broth solution. Both tubes were incubated at 37°C for 24 hours up to 72 hours. The tubes were observed for turbidity, indicative of microbiological growth, in intervals of 24 hours up to 72 hours and were measured against McFarland Equivalence Standards (Batch No.: 1313947; TM4000T10). All apparatus (i.e. glass tubes, measuring cylinders, pipette tips, stock bottles, and volumetric flasks), nutrient broth solution and sterile water were placed through a moist heat sterilisation process (autoclave) at 121°C for 15 minutes before they were used.

Table 27. Swab culture results from twenty 'healthy' volunteers. Two swabs were obtained from each subject; and one swab was treated with the TCS formulation using a nebuliser, the other swab was used as a control. There is a statistically significant difference (p-value < 0.001) between the 72 hour cell densities of the positive controls and tests. * Based on McFarland's Standards; readings are obtained after incubation at 37°C for 72

^Λ Sterile swab sample from subject, placed in nutrient broth.

# Sterile swab sample from subject, treated with antiseptic formulation, placed in nutrient broth.

Claims

1. A composition comprising one or more antimicrobial agent with hydrophobic moieties and a cyclodextrin.

2. The composition of claim 1, wherein the one or more antimicrobial agent is hydrophobic.

3. The composition of any one of claims 1 to 2, wherein the one or more antimicrobial agent in the composition is present in an amount of about 0.1% w/v to 2% w/v.

4. The composition of any one of the preceding claims, wherein the one or more antimicrobial agent is selected from the group consisting of triclosan (TCS), octenidine dihydrochloride (OCT), mupirocin calcium (MPC), polyhexanide, chlorhexidine, combinations and derivatives thereof.

5. The composition of any one of the preceding claims, wherein the cyclodextrin in the composition is present at an amount of about 40 mM to about 100 mM.

6. The composition of any one of the preceding claims, wherein the cyclodextrin is selected from the group consisting of a-cyclodextrin, β-cyclodextrin, γ-cyclodextrin and derivatives thereof.

7. The composition of claim 6, wherein the cyclodextrin is a β-cyclodextrin.

8. The composition of claim 7, wherein the β -cyclodextrin is 2-hydroxypropyl-P- cyclodextrin (HPpCD).

9. The composition of claim 1, wherein the composition further compromises a salt, a polymer or a combination thereof.

10. The composition of claim 9, wherein the polymer in the composition is present at an amount of about 0.25% w/v to about 1% w/v.

11. The composition of claim 9, wherein the polymer is selected from the group consisting of carboxymethyl cellulose (CMC), soluble cellulose derivatives, insoluble cellulose derivatives, polyacrylates, starch, chitosan and derivatives thereof.

12. The composition of claim 11, wherein the soluble cellulose derivative is selected from the group consisting of hydroxypropyl methylcellulose, hydroxypropyl cellulose (HPC), methylcellulose, and derivatives thereof.

13. The composition of claim 11, wherein the insoluble cellulose derivative is selected from the group consisting of ethylcellulose, microcrystalline cellulose (MCC), and derivatives thereof.

14. The composition of claim 9, wherein the polyacrylate is selected from the group consisting of carbomers, polycarbophil, and derivatives thereof.

15. The composition of claim 11, wherein the carboxymethyl cellulose (CMC) is selected from the group consisting of carboxymethyl cellulose sodium, salt derivatives of carboxymethyl cellulose sodium, microcrystalline cellulose and derivatives thereof.

16. The composition of claim 9, wherein the salt is present in the composition at an amount of about 2.5% w/v to about 3% w/v.

17. The composition of claim 9, wherein the salt is present in the composition at an amount of about 2.8% w/v.

18. The composition of any one of claims 9, 16 and 17, wherein the salt is sodium chloride.

19. The composition of claim 18, wherein the sodium chloride is present in the form of a saline solution.

20. The composition of claim 19, wherein the saline solution is a hypertonic or an isotonic saline solution.

21. The composition of any of claims 1 to 20, wherein the composition comprises triclosan (TCS), carboxymethyl cellulose (CMC) and 2-hydroxypropyl-P-cyclodextrin (HPpCD).

22. The composition of claim 21, wherein the composition comprises about 0.2% w/v to about 0.5% w/v triclosan (TCS), about 0.5% w/v to about 1.5% w/v carboxymethyl cellulose (CMC) and about 30 mM to about 50 mM 2-hydroxypropyl-P-cyclodextrin (HPpCD).

23. The composition of claim 21, wherein the composition comprises 0.3% w/v triclosan (TCS), 1% w/v carboxymethyl cellulose (CMC) and 40 mM 2-hydroxypropyl-P- cyclodextrin (HPpCD).

24. The composition of any of claims 1 to 20, wherein the composition comprises triclosan (TCS), carboxymethyl cellulose (CMC), 2-hydroxypropyl-P-cyclodextrin (HPpCD) and sodium chloride (NaCl).

25. The composition of claim 22, wherein the composition comprises about 0.2% w/v to about 0.5% w/v triclosan (TCS), about 0.5% w/v to about 1.5% w/v carboxymethyl cellulose (CMC), about 30 mM to about 50 mM 2-hydroxypropyl-P-cyclodextrin (HPpCD) and about 0.5% w/v to about 3% w/v sodium chloride (NaCl).

26. The composition of claim 25, wherein the composition comprises 0.3% w/v triclosan (TCS), 1% w/v carboxymethyl cellulose (CMC), 40 mM 2-hydroxypropyl-P- cyclodextrin (HPpCD) and 2.8% w/v sodium chloride.

27. The composition of any of claims 1 to 20, wherein the composition comprises octenidine dihydrochloride (OCT) and 2-hydroxypropyl-P-cyclodextrin (HPpCD).

28. The composition of claim 27, wherein the composition comprises about 0.05% w/v to about 0.15% w/v octenidine dihydrochloride (OCT) and about 40 mM to about 60 mM 2-hydroxypropyl-P-cyclodextrin (HPpCD).

29. The composition of claim 28, wherein the composition comprises 0.1% w/v octenidine dihydrochloride (OCT) and 50 mM 2-hydroxypropyl-P-cyclodextrin (HPpCD).

30. The composition of any of claims 1 to 20, wherein the composition comprises octenidine dihydrochloride (OCT), 2-hydroxypropyl-P-cyclodextrin (HPpCD) and sodium chloride (NaCl).

31. The composition of claim 30, wherein the composition comprises about 0.05% w/v to about 0.15% w/v octenidine dihydrochloride (OCT), about 40 mM to about 60 mM 2- hydroxypropyl-P-cyclodextrin (HPpCD) and about 0.5% w/v to 3% w/v sodium chloride (NaCl).

32. The composition of claim 31, wherein the composition comprises 0.1% w/v octenidine dihydrochloride (OCT), 50 mM 2-hydroxypropyl-P-cyclodextrin (HPpCD) and 2.8% w/v sodium chloride (NaCl).

33. The composition of any one of claims 1 to 20, wherein the composition comprises mupirocin calcium (MPC) and 2-hydroxypropyl-P-cyclodextrin (HPpCD).

34. The composition of claim 33, wherein the composition comprises about 1% w/v to

3% w/v mupirocin calcium (MPC) and about 50 mM to about 150 mM 2- hydroxypropyl-P-cyclodextrin (HPpCD).

35. The composition of claim 34, wherein the composition comprises 2% w/v mupirocin calcium (MPC) and 100 mM 2-hydroxypropyl-(3-cyclodextrin (HPpCD).

36. The composition of any one of claims 1 to 20, wherein the composition comprises mupirocin calcium (MPC), 2-hydroxypropyl-P-cyclodextrin (HPpCD) and sodium chloride (NaCl).

37. The composition of claim 36, wherein the composition comprises about 1% w/v to 3% w/v mupirocin calcium (MPC), about 50 mM to about 150 mM 2-hydroxypropyl- β-cyclodextrin (HPpCD) and about 0.5% w/v to 3% w/v sodium chloride (NaCl).

38. The composition of claim 37, wherein the composition comprises 2% w/v mupirocin calcium (MPC), 100 mM 2-hydroxypropyl-p-cyclodextrin (HPpCD) and 2.8% w/v sodium chloride (NaCl).

39. The composition of any one of claims 21 to 38, wherein the composition further comprises carboxymethyl cellulose (CMC).

40. The composition of claim 39, wherein the composition further comprises about 0.1% w/v to about 0.3% w/v carboxymethyl cellulose (CMC).

41. The composition of claim 41, the composition comprises octenidine dihydrochloride (OCT) and 2-hydroxypropyl-P-cyclodextrin (HPpCD) and wherein the composition is provided as a gel or a cream.

42. The composition of any one of the preceding claims, wherein the composition further comprises a sweetening agent.

43. The composition of any of the preceding claims, wherein the composition is provided as dry powder, aqueous solution, non-aqueous solution, slurry, aerosols, nasal sprays, nebulizers, inhalers, gels, creams, ointments, pastes, suspensions, sterile solids, crystalline solids, amorphous solids, solids for reconstitution or combinations thereof.

44. A composition of any one of claims 1 to 43 for use in treating an infection or for decolonizing an orifice of a patient.

45. A method of treating or preventing an infection in a patient, or for decolonizing an orifice of a patient, wherein the method comprised administering to the patient a therapeutically effective amount of the compositions according to any one of the preceding claims.

46. The method of claim 45 or the composition of claim 44, wherein the infection is a bacterial infection or a fungal infection

47. The method or the composition of claim 46, wherein the bacterial infection is caused by one or more agents selected from the group consisting of Staphylococcus aureus, Methicillin- sensitive Staphylococcus aureus (MSSA), Methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa and Escherichia coli.

48. The method or the composition of claim 47, wherein the Staphylococcus aureus is a strain selected from the group consisting of MRSA 1, MRSA 2, MRSA 3, MRSA 7, Mu50, WIS and MSSA 2590.

49. The method or the composition of claim 47, wherein the fungal infection is caused by one or more agents selected from the group consisting of Candida albicans, Aspergillus brasiliensis and Aspergillus niger.

50. The method of claim 45 or the composition of claim 44, wherein the infection is selected from a group consisting of ear infection, nose infection, throat infection, sino-nasal infections, sinus infection, respiratory infection, rhinitis, bronchiolitis and combinations thereof.

51. The method of claims 45 to 49 or the composition of claims 44 to 49, wherein the composition is administered prior to or after surgery.

52. The method of claims 45 to 49 or the composition of claims 44 to 49, wherein the composition is administered orally, or nasally, or topically.

53. The method or the composition of claim 52, wherein the composition is administered nasally.

54. The method of claim 53, wherein the composition is administered nasally, wherein the composition comprises octenidine dihydrochloride (OCT) and 2-hydroxypropyl-P- cyclodextrin (HPpCD).

55. The method of claim 54, wherein the method is for decolonizing an orifice of a patient.

56. The method or the composition of claim 53, wherein the composition is administered nasally via nasal irrigation or nasal fuming.

57. The method of claim 45 or the composition of claim 44, wherein the orifice is selected from the group consisting of ear, nose, nasal cavity, sinus, nasal passageway, Eustachian tube and throat.

58. Use of the composition as defined in claims 44 to 57 in the manufacture of a medicament for preventing or treating an infection, or for decolonizing an orifice of a patient.

59. A method of producing the compositions according to any one of claims 1 to 42, the method comprising:

(a) mixing a ground cyclodextrin, as defined in any one of claims 1 to 43, and one or more ground antimicrobial agent, as defined in any one of claims 1 to 43; and

(b) constantly agitating and heating the mixture formed under (a)..

60. The method of claim 59, wherein the heat is set to the melting temperature of the one or more antimicrobial agent.

61. The method of claim 60, wherein the melting temperature is between about 55°C to about 90°C.

62. The method of claim 61, wherein the melting temperature is about 60°C, or about 80°C, or about 85°C.

63. The method of any one of claims 59 to 62, wherein the mixture is agitated for about 1 to about 2 hours.

64. The method of claim 58, wherein the mixture was agitate for about 1 hour, or about 2 hours.

65. The method of any one of claims 59 to 64, wherein the state of the mixture is dry or slurry.

66. The method of any one of claims 59 to 65, the method comprising:

(c) mixing the ground cyclodextrin with solvent to form a slurry; (d) adding the one or more ground antimicrobial agent to the slurry formed in under (c);

(e) subjecting the slurry of (d) to constant agitation and heat as defined in any one of claims 60 to 64;

(f) grinding the composition resulting from (e); and

(g) mixing the compositing resulting from step (f) with a salt as defined in any one of claims 8 to 19.

67. The method of claim 66, wherein the one or more antimicrobial agent is octenidine.