WO2013135571A1

WO2013135571A1 - Compositions with enhanced therapeutic efficacy against infective agents of the eye comprising miltefosine and polyhexamethylene biguanide

Info

Publication number: WO2013135571A1
Application number: PCT/EP2013/054707
Authority: WO
Inventors: Andreas Obwaller
Original assignee: Orphanidis Pharma Research Gmbh
Priority date: 2012-03-14
Filing date: 2013-03-08
Publication date: 2013-09-19

Abstract

An antibiotic composition comprising miltefosine and polyhexamethylene biguanide (PHMB) and its use in the prevention or treatment of a condition of the eye or the skin is provided by the present invention.

Description

COMPOSITIONS WITH ENHANCED THERAPEUTIC EFFICACY AGAINST INFECTIVE AGENTS OF THE EYE COMPRISING MILTEFOSINE AND POLYHEXAMETHYLENE

BIGUANIDE

FIELD OF THE INVENTION

Hie present invention relales to antibiotic compositions such as topical administration forms, containing miltefosine and polyhexamethylene biguanide (PHMB). Furthermore the invention relates to the use of the compositions for the removal and eradication of microbial parasites.

BACKGROUND

Acanthamoeba keratitis is a corneal disease associated predominantly with contact lens wear. The occurrence of Acanthamoeba keratitis (AK) has been rising since 1990 in correlation to the enhanced number of contact lens wearers. Approximately 3000 cases of AK have been reported around the world. Meanwhile, Acanthamoebae are, besides the pseudomonads, the most common causative agents of contact lens-associated keratitis. It is estimated, that the annual incidence of AK in the USA is 1 :250.000 inhabitants. In Europe the most cases have been documented in the UK (-400).

Acanthamoeba keratitis has often been a medical challenge to most ophthalmologists. It is generally treated with a combination of polyhexamethylene biguanide (PHMB) or chlor- hexidinc and propamidine isethionate (Brolene). The treatment protocol used at Moorfields Eye Hospital, London, is empirical but based on both the clinical and laboratory data discussed previously and is outlined. Therapy is usually started with a biguanide (PHMB 0.02% or ehlorhexidine 0.02%) and a diamidine (propamidine 0.1% or hexamidine 0.1%), although there is no clinical evidence to suggest that this is more effective than monotherapy with PHMB alone. PHMB 0.02% and hexamidine drops are administered every hour, day, and night, for 48 hours initially, followed by hourly drops by day only for a further 72 hours. Intensive early treatment is given because organisms may be more susceptible before cysts have fully matured. Epithelial toxicity is common if the dosage is maintained at this intensity. For this reason the frequency of therapy is reduced after 5 days to 2-hourly by day for 3 to 4 weeks, and then tailored to the individual case. These anti-amoebics are tapered with the goal of maintaining topical therapy 4 times daily for several, weeks.

More recently Polat et al.( Parasitology Research 201 2, 1 10 515-520) have shown, that miltefosine exhibited highest efficacy against Acanthamoeba keratitis in a Syrian hamster model, whereas Vasseneix et al. (Cornea, 2006, 25, 597-602), using a rat model, found highest activ ity of a combination of PHMB + hexamidine diisethionate.

EP1850888B1 describes the use of miltefosine as antimicrobial agent in contact lens care composition. It discloses that PHMB does not have any effect on the viability of

Acanthamoeba ceils.

Compositions comprising PHMB as disinfectant are disclosed in US 1432345 and

EP0788797A1 and EP04501 17A1.

Walochnik J. et al. (Antimicrobial Agents and Chemotherapy, 2002, 695-701 ) describe activities of alkyiphosphochoiines against clinical isolates of Acanthamoeba spp.

Lark in et al. (Ophthalmology, 99, 2, 1992, 190- 191 ) describe the use of polyhexamethylcnc biguanide (PHMB) for the treatment of Acanthamoeba infections.

The use of selected amidoamines for the treatment of Acanthamoeba infections are described in WO02/49633A2

Combination therapies may show higher efficacy than single therapy strategies, however, toxicity may also arise and make a combination therapy impossible due to unacceptable side effects.

Thus, there is a need for prophylactic as well as therapeutic compositions for short-term and long-term treatment of eye infections due to bacterial, fungal or protozoan infections without unwanted negative side effects.

SHORT DESCRIPTION OF THE INVENTION:

The object of the inv ention is achiev ed by prov iding the embodiments described in the claims.

The inventors have surprisingly shown that an antibiotic preparation c PHMB and miltefosine shows synergistic therapeutic effects in the treatment of eye infections compared to single administration of the compounds and has minor toxic effects. The inventive composition is highly tolerable and was shown to have almost no side effects when tested in animal systems ev en after prolonged treatment. Therefore the present invention provides an antibiotic composition comprising miltefosine and PHMB.

Specifically, miltefosine and PHMB are contained in an effective amount to treat a microbial infection of the eye or the skin.

According to a specific embodiment of the invention, the composition may comprise about 5 to 200 fig ml, preferably about 20 to 100μg/ ml, more preferred about 50 to 75 pg/ml miltefosine. According to a further embodiment of the invention, the composition may comprise about 1 0 to 1000 ng ml, preferably about 4 to 500 jig/ml PHMB.

The composition o f the invention may be contained in any ratio of the components, for example, the ratio of miltefosine to PHMB may be in the range of about 1 :3.5.

Specifically, the composition may be free of endotoxins and/or proteases.

According to an embodiment of the invention, the composition may further comprise isotoni/ing agents, microbiozidal agents, stabilizing agents, one or more auxiliary agents, carriers or diluents. Pharmaceutically active agents may also be contained, for example, but not restricted to antifungals, analgesics, anesthetics and anti-inflammatory agents. The pH of said composition may be between 4 and 8, preferably between 6 and 8, more preferred at about 7.4.

The inventive composition may be for ophthalmic or dermal use, optionally in the form o f a gel, solution, cream, dispersion, spray, artificial tear solution.

The inventive composition may be an isotonic composition, specifically an isotonic solution.

According to a preferred embodiment, the composition can be used for topical application.

The inventive composition may be in single unit dose or multiple unit dose.

The present invention further prov ides the composition for use in the prevention or treatment of a condition of the eye or the skin of an indiv idual in need therefor, preferably for the eradication of protozoan parasites, for the prevention or treatment of bacterial and or protozoan and or fungal keratitis, preferably for the treatment of Acanthamoeba induced keratitis.

In a specific regimen, the composition may be administered at least every 1 0 hours, preferably at least every 5 hours, preferably at least every 2 hours, preferably hourly. A specific embodiment of the invention provides an antibiotic composition consisting essentially of miitefosine and PHMB as active agents.

The term "essentially" means that miitefosine and PHMB are the only active agents comprised, any additional isotonizing, stabilizing agents or buffers present in the composition are not considered as essential according to the embodiment of the invention.

A kit of single components, wherein a first component is miitefosine and a second component is PHMB is provided by the present invention as well.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : In vitro cytotoxic effect of 65.12 Lig/ml MLT, 0.02% CHX, 0.02% PHMB, 0.01% PI, 65. 1 2 μg/mi MLT plus 0.02% CHX, 65.12 jig ml MLT plus 0.02% PHMB, 65. 1 2 jig/ml

MLT plus 0.01% PI on L929 fibroblast ceils measured by XTT assay. *P<0.05 vs CHX, PHMB, PI, MLT plus CHX, MLT plus PHMB, and MLT plus PI.

DETAILED DESCRI PTION OF THE INVENTION

The present invention provides a pharmaceutical composition comprising miitefosine and polyhexamethylenc biguanide ( PHMB) wherein said composition has antibiotic activity.

M iitefosine is a hexaclecylphosphocholine, commercially av ailable, for example, under the trade names I mpav ido and Miltex. The term '"miitefosine" also covers any pharmaceutically acceptable salts or derivatives thereof.

The pharmaceutical composition of the present invention specifically has antibiotic activity toward microbes, i.e., said composition is antagonistic to the growth of microorganisms which more specifically means that said composition can destroy or inhibit the growth of microbes, thus hav ing a microbiocidal or antimicrobial or microbiostatic effect.

For the purpose of the present inv ention, the terms as used therein hav e the follow ing meanings:

The term "antimicrobial" is meant to include prev ention, inhibition, termination, or reduction of the function of a microbe. The term "microbiostatic" is meant to include inhibition or reduction or termination of the growth of a microbe. The term "microbiocidal" is meant to kill the microbe.

The terms "microbe" or "microbial agent" includes any organism of the phylogenetic domains bacteria and archaea, as well as unicellular and filamentous fungi (such as yeasts and molds), unicellular and filamentous algae, unicellular and multicellular parasites that causes ocular disease in a subject. Accordingly, such microbial agents include, but are not limited to, bacterial, fungal, or protozoan pathogens.

The term "bacterium" or "bacteria" includes all known aerobic bacteria, anaerobic bacteria and facultative anaerobic bacteria. These can be either gram-positive or gram-negativ e or atypical, do also comprise spore forming bacteria and bacterial spores and can for instance be members of Actinobacteria, Aquificae, Bacteroidetes, Chlamydiae, Chloroflexi,

Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus- Thermus, Dictyoglomi, Fibrobacteres Acidobacteria. Firmicutes. Fusobacteria, Gemmatimonadetes, N itrospirae, Omnibacteria, Planctomycetes, Proteobacteria, Spirochaetes, Thermodesulfo bacteria,

Thermomicrobia and/or Therm otogae.

The term "fungus" or " fungi" is intended to comprise all known single-cell unicellular and or multi-cellular members of the kingdom of fungi, such as Chytridiomycota, Zygomycota, Glomeromycota, Ascomycota and/or Basidiomycota, as well as in addition Myxomycota, Oomycota and/or tlypocln triomycota.

The term protozoa" is intended to comprise all known single-cell unicellular and/or multicellular members of Sporozoa, Gregarinida. Coccida. Piroplasminda, Babesia, M icrosporidia, Giardiinae, Trichomonadida, Diplomonadida, Hypermastigida, Trypanosoma, Entamoebidae, Kinetoplasta, Tryposomatidea, Tryposomatidae, Apicomple.xa, Haemosporida, Plasmodiidae, Rhizopoda and or Amoebozoa like Acanthamoebidae.

According to an embodiment of the invention miltefosine and PHMB are contained in an effectiv e amount to prev ent or treat a microbial infection of the eye or the skin.

An "effectiv e amount" means the amount that, when administered to a subject for preventing or treating a disease, is sufficient to effect prev ention or treatment for that disease.

The composition specifically may comprise about 5-200pg/ml. preferably about 20 to 100 μ^ηιΐ, more preferred about 50 to 75 jig/ml of miltefosine.

The composition specifically may comprise about 10- ! 000 ng/ml, preferably about 4 to 500 tig/ml of PHMB.

The optimal ratio of the miltefosine to PHMB can be selected using comparative test systems or other systems known to the skilled person. Specifically it may be in the range of 1 :3.5 for miltefosine : PHMB.

According to a specific embodiment, the inventive composition may be free of endotoxins.

According to another embodiment, the composition of the invention may be free of proteases.

According to a specific embodiment of the present invention, the antibiotic composition consists essentially o f miltefosine and PHM B as active agents. More specifically it is a topical administration form consisting essentially of miltefosine and PHMB.

Alternatively, the inventive composition may further comprise microbiozidal agents.

The composition employed in the present invention may contain, in addition to miltefosine, PHMB and optionally further active agents as listed above, one or more other pharmaceutically acceptable components that are commonly present in ophthalmic solutions, for example, buffering agents: chelating agents; pH adjusting agents, viscosity modifying agents, demulcents and the like, which aid in making ophthalmic compositions more comfortable to the user and or more effective for their intended use.

According to a specific embodiment of the invention, the composition may comprise an isotonizing agent. The isotonizing agent may be chosen from known ones, such as sodium chloride or citric acid, glycerol, sorbitol, mannitol, ethylene glycol, propylene glycol, dextrose and is present within a concentration range of, for example, from 0 to 1% w/v, rendering the composition isotonic with lacrimal fluid (270-3 1 0 mOsm kg).

Alternatively also polyethylene glycol may be comprised in the inventive composition, specifically in the range of 0.0001 to 0. 1 wt%, specifically in the range of 0.0005 to

0.05 wt%, more specifically in the amount of about 0.001 wt%.

The terms "pharmaceutically acceptable", "physiologically tolerable" as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a subject without the production of undesirable physiological effects to a degree that would prohibit administration of the composition.

In exemplary embodiments of the present invention, pharmaceutically acceptable

compositions of the present invention comprising miitefosine and PHMB can be aqueous suspensions optionally containing a buffer, (e.g. carbonate salt, phosphate salt, acetate salt, glutamic acid, or citrate salt), an isotonizing agent (e.g. glycerol, mannitol, sorbitol, propylene glycol, sodium chloride, potassium chloride, boric acid), a stabilizer (e.g. sodium edetate, sodium citrate), a non-protein based surfactant (e.g.. polysorbate 80, polyoxyethylene, hydrogenated castor oil, tyloxapol, benzaikonium chloride, polyoxyethylene fatty acid esters, polyoxyethylene alkylphenyl ethers, and polyoxyethylene alky 1 ethers, or mixtures thereof), a preservative (e.g. p-hydroxybenzoate and its analogs, benzaikonium chloride, benzethonium chloride, chlorobutanol ), a pH control agent (e.g. hydrochloric acid, sodium hydroxide, phosphoric acid), a surfactant polyoxyethylene fatty acid esters, and or other additives.

The composition of the invention may be in a topical administration form, specifically in the form of a gel, solution, cream, dispersion, spray or artificial tear solution. Preferably, it is a solution.

The pharmaceutical composition may specifically be for ophthalmic or dermal use.

According to a specific embodiment of the invention the antibiotic composition may be in single unit dose or multiple unit dose.

The antibiotic composition may be provided for use in the prevention or treatment of a condition of the eye or the skin of an individual in need therefor.

Specifically it may be used for the removal of protozoan parasites from the skin or the eye or it may be used for the prevention or treatment of a microbial induced keratitis, for example from bacteria, protozoa or fungi, preferably it may be used for the treatment of Acanthamoeba induced keratitis.

Alternatively, the present composition may be used for the treatment to reduce the degrees of opacity of the cornea due to microbial infection. For the purpose of the present invention, the term "treatment" is also intended to include prophylactic treatment or alleviation.

"Prevention" can be considered, for example, to be the obstruction or hindrance of potential microbial growth. "Termination" or '"eradication" can be considered, for example, to be actual killing of the microbes by the presence of the composition. "Inhibition" can be considered, for example, to be a reduction in microbial growth or inhibiting v irulence factor expression or function of the microbe.

Preferably, the antibiotic composition of the present invention will inhibit the function of a microbe by more than 30%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, more preferably by more than 91%, 95% or 99%.

The inventive composition is speci fically effective in inhibiting the growth of protozoans including, but not limited to, Acanthamoebae, for example A. polyp haga, A.castellani, A. lenticulata, A. hatchetti, A. astronyxis, A. culbertsoni, and A. rhysodes. The inv ention may also effectiv e in inhibiting the growth of other protozoans.

Except when noted, the terms "subject" or "patient" or "individual" are used interchangeably and refer to mammals such as human patients and non-human primates, as well as

experimental animals such as rabbits, dogs, cats, rats, mice, and other animals. Accordingly, the term "subject" or "patient" as used herein means any mammalian patient or subject to the individual the composition of the inv ention can be administered.

In an exemplary embodiment of the present invention, to identify subjects for treatment with a pharmaceutical composition according the inv ention, accepted screening methods are employed to determine the status of a disease or condition in a subject or risk factors associated with a targeted or suspected disease or condition. These screening methods may include, for example, ocular examinations to determine whether a subject is suffering from an ocular disease. These and other routine methods allow the clinician to select subjects in need of therapy.

"Treating" or "treatment" of an ocular infection using the methods of the present inv ention includes prev ent ing the onset of symptoms in a subject that may be at increased risk of ocular infection but does not yet experience or exhibit symptoms of infection, inhibiting the symptoms of infection (slowing or arresting its development ), providing relief from the symptoms or side-effects of infection, and relieving the symptoms of infection (causing regression).

"Concomitant administration" o f a known drug with a pharmaceutical composition of the present invention means administration of the inventiv e composition together with said agent. Such concomitant administration may inv olv e concurrent (i.e., at the same time), prior, or subsequent administration of a further active agent with respect to the administration of the composition of the present invention.

The present invention provides methods of administering miitefosine and PHMB to the eye to treat ocular diseases, specifically to treat protozoan induced keratitis. Thus, compositions according to the present inv ention are suitable for direct administration to a subject's eye. By "direct administration" it is meant that the composition is applied topically or by injection or installation into the eye.

The composition of the present invention may be compounded with one or more agents to facilitate their use in a wide variety of contexts. Topical compositions for delivering miitefosine and PHMB to the eye according to the present invention will typically comprise miitefosine and PHMB present in a suitable pharmaceutically acceptable carrier.

Exemplary acceptable carriers include, but are not limited to, water, buffered aqueous solutions, isotonic mixtures of water and water-immiscible solvents, such as alkanois, aryl alkanols, vegetable oils, polyalkaiene glycols, petroleum-based jellies, ethylcellulose, ethyloleate, carboxymethylceliuioses, polyvinlprrolidones, and, isopropyl myristates. The compositions of the present inv ention can also include ophthalmically acceptable auxiliary components such as buffers, emulsifiers, preservatives, wetting agents, tonicity agents, thixotropic agents, e.g., polyethylene glycols, chelating agents, and additional antimicrobial agents.

The composition o f the present invention may be formulated as a sterile, substantially isotonic solution. Specifically the composition may be provided as sterile solution in single tubes or application forms like multi-dosage eye drop systems like ABAK® ( Laboratoires Thca), or

COMOD® (URSAPHARM) which allow liquid dosage forms to be kept sterile during storage and use. For treatment purposes, the pharmaceutical composition of the present invention may be, for example, administered to the subject in a single delivery, via continuous delivery over an extended time period, or as repeated administration (e.g., by an hourly, daily or weekly, repeated administration). The pharmaceutical composition of the present invention may be administered, for example, one or more times half-hourly, i.e., every half an hour for a 24 hour period, one or more times hourly, or one or more times daily. In certain embodiments, the pharmaceutical composition of the invention is administered two times daily, four times daily, six times daily, or twelve times daily. Typically, the formulations are self-administered.

Compared to other known substances or combinations of known substances the inventive composition was shown to be highly effective ev en after a short treatment time although there are no or only minor side effects even after long treatment. Administration time may be for several weeks, specifically it may long for 4 weeks, specifically for three weeks, specifically for two weeks, specifically for one week, more specifically it may last 6, 5, 4, 3 or 2 days.

Alternatively, also long term treatment using the inventive composition may be applicable. specifically in view of the lack of negative side effects of the active agents miitefosine and PHMB.

Specifically, the inv entiv e composition is administered at least every 10 hours, preferably at least ev ery 5 hours, preferably at least every 2 hours, preferably hourly.

Alternativ ely the composition may be administered for ten. or nine or eight or seven times per day during the first one or two or three weeks and two or three or four times per day for any further duration of the treatment.

The actual dosage of biologically activ e agents will of course vary according to factors such as the extent of exposure and particular status of the subject (e.g. the subject's age, size, fitness, extent of symptoms, susceptibility factors, etc.), time and route of administration, as well as other drugs or treatments being administered concurrently. Dosage regimens can be adjusted to provide an optimum prophylactic or therapeutic response. By "therapeutically effective dose" herein is meant a dose that produces effects for which it is administered. The exact dose will be ascertainable by one skilled in the art using known techniques. A therapeutically effective dose is also one in which any toxic or detrimental side effect of the biologically activ e agent is outweighed in clinical terms by therapeutically beneficial effects. The composition of the present invention is preferably administered topically or by intraocular injection (e.g. intrav itreal, subconjunctival, retrobulbar injection). Topical administration includes the application of the composition of the present invention to the cul- de-sac of the eye from a dropper controlled bottle or dispenser.

For each particular subject, specific dosage regimens should be evaluated and adjusted ov er time according to the individual need and pro lessional judgment of the person administering or supervising the administration of the pharmaceutical compositions.

Topical administration according to the present invention also includes the application of the composition containing miltefosine and PHMB to the eye. According to a specific

embodiment, the composition may include a base which permits diffusion of the drug into the ocular fluid.

The inventiv e antibiotic eye drop composition comprising miltefosine and PHMB used in the present invention can include any agents known to the skilled formulation chemist to be useful for eye drop preparations. For example, in certain embodiments of the present inv ention, the eye drops can contain an isotonic agent added to sterilized purified water, a preserv ativ e, a buffering agent, a stabilizer, a viscous v ehicle, and/or an additional antimicrobial agent.

A suitable pH range for use as an ophthalmic medicine is preferably within the range of about 5 to 9, more preferably in the range of about 6 to 8, and most preferably between about 7 to about 7.5, more preferably about 7.4.

Using the methods of the present invention, miltefosine and PHMB can be administered in combination with other known therapies to treat a subject suffering from keratitis. In some instances, drugs currently used to treat an eye disease will be effectiv e at lower dosages when administered in combination w ith the composition containing miltefosine and PHM B.

The precise dosage of a therapeutic agent to be delivered to a subject concomitantly w ith the miltefosine and PHMB formulation will depend on the age, weight and particular disease of the subject. The amount and precise regime will also depend on other factors including the severity of the ocular disease to be treated.

A topical solution in accordance w ith one embodiment of the present invention comprises incorporating a therapeutic dose of miltefosine and PHMB in an artificial tear formulation. The solution of the present invention can be adjusted with tonicity agents, to approximate the osmotic pressure of normal tear fluid. Tonicity adjusting agents are well known to those of skill in the art and include, but are not limited to, mannitol, sorbitol, dextrose, sucrose, urea, glycerin, propylene glycol and soluble salts, such as sodium, potassium, calcium and magnesium chlorides.

In some embodiments of the present invention, a preservative can be added for the purpose of preserving the compositions.

The composition o f the present invention may further include a demulcent, e.g. a water- soluble polymer that protects and lubricates mucous membrane surfaces and relieves dryness and irritation when applied to the eye.

Specifically, the pharmaceutical composition of the invention is an aqueous solution.

In another embodiment, the composition of this invention can optionally include viscosity adjusting agents.

In a preferred embodiment, the compounds o f the invention can be used for the manufacture of a medicament for the treatment or prophylaxis of diseases and/or pathophysiological conditions in mammals that are caused by microorganisms, where the microorganism is a bacterium.

In another preferred embodiment, the compounds of the invention can be used for the manufacture of a medicament used in the treatment or prophylaxis o f diseases and/or pathophysiological conditions in subjects that are caused by microorganisms, where the microorganism is a protozoon and selected from the group consisting of "Acanthamoeba spp., Amoeba spp., Babesia spp., Balantidium spp., Cryptosporidium spp., Cyclospora spp., Dientamoeba spp., Echinamoeba spp., EndoUmax spp., Entamoeba spp., Enterocytozoon spp., Giardia spp., Hartmanella spp., Isospora spp., Jodamoeba spp., Lamblia spp., Leishmania spp., Microsporidium spp., Naegleria spp., Nosema spp., Paramecium spp., Paramoeba spp., Penumocystis spp., Plasmodium spp., Sarcocystis spp., Tetrahymena spp., Toxoplasma spp., Trichomonas spp. and/or Trypanosoma spp.".

Alternatively, the antibiotic composition comprising miltefosine and PHMB is provided as a kit of single components, wherein a first component is miltefosine and a second component is PHMB.

As an illustration of the present invention, several examples are prov ided below. These examples serve only to further illustrate aspects of the inv ention and should not be construed as limiting the invention.

EXAMPLES

Treatment of Acanthamoeba infection

The activ ity of various known or already used substances for treatment of Acanthamoeba keratitis were tested against the trophozoites and cysts of Acanthamoeba polyphaga, A.

castellani and A. lenticulata. Trophozoits and cysts were isolated, cultured and treated as described by Polat et al (2012).

Animals

Sixty three male Wistar rats weighing approximately 1 25 g were used for the present study. The animals were fed with a standard laboratory diet, kept at 22 ± 2 °C, 50%-70% humidity, and 12-hour day-night cycle; drinking water was available ad libitum during the experiment. All corneas were examined before inoculation to exclude any abnormality. Experimental procedures involved in the study were approved by the Institutional Review and Animal Use Committee of the Cumhuriyet University, and conducted by following accepted guidelines for the care and use of laboratory animals for research.

Amoeba Culture

Corneas were infected with Acanthamoeba hatchetti strain 1 1 DS, a human corneal isolate ( Walochnick et al.. 2000). Vegetative forms were prepared in axenic cultures in 25-cm² flasks, containing 10-ml protease peptone, yeast extract, and glucose medium (Schuster 2002 ) at 37°C. Trophozoites in the stage of exponential growth (72 to 96 h) were concentrated by centrifugation at 500 g for 1 0 min. The amoebae were washed twice in a sterile Neff s saline solution ( 1 .2 g NaCl, 0.4 g MgS0₄.H₂0, 0.4 g CaCl₂.2H₂0, 1 .42 g Na₂HP0₄, 1.36 g KHP0₄ in 100 ml distilled water), counted in a hemacytometer, adjusted to a final concentration in Neff s saline solution at a density of 1 ^χ 10⁶ amoebae/ml (95.0% trophozoites), and used immediately for infection. Chemicals

Miltefosine (MLT) was provided by Orphanidis Pharma Research GmbH (Vienna, Austria) and prepared as a 2 niM stock solution in 5% ethanol. Chlorhexidine gluconate (CHX) was provided by Sigma- Aidrich (C93949, po ly h e x a methylene biguanide ( PI 1MB) ( Lavasept concentrate 20%) was purchased from Braun (Germany), and propamidine isethionate ( PI ) (Brolene®) was purchased from Sanofi-Aventis (GB). Chlorhexidine gluconate and

Polyhcxanid were diluted (to 0.02% final concentration) in artificial tear solution ( Dacrolux, Alcon, Spain).

Anesthesia

Ketamine HQ (Ketamine®, 10% 100 mg kg body wt intraperitoneally; cp-pharma, Burgdorf, Germany) and xylazine (Rompun®, 10 mg/kg body wt intraperitoneally; Bayer, Leverkusen, Germany) were administered by an intramuscular injection, and one drop of preservative- free benoxinate was applied to each of the eyes.

Inoculation

The inoculation was performed under an operating microscope (Leica-M841 ; Leica Microsystems GmbH, Wetzlar, Germany). Initially, a half-thickness linear blade incision was made approximately 2 mm from the center of the cornea. Using a 30-G needle attached to

1 id-syringe, the needle was advanced from the point of incision, through the lamella o f the stroma, to the center of the cornea. One microliter infection solution, including

1 x 10⁶ amoeba tnl, was injected into the stroma. After three days, the infection was repeated. Control animals received a mock inoculum of 1 μΐ Neff s saline.

Grading of Acanthamoeba keratitis

Under general anesthesia, the animals were examined under a slit-lamp microscope on days 1 , 3, and 5 after inoculation. Infected corneas and iris were examined by retro-illumination, and by transillumination. The oblique slit beam was used to illuminate lesser degrees of opacity. The following grading scheme was used ( Lark in and Easty 1 990): grade 0, normal; grade 1 , corneal opacity visible only by oblique slit beam; grade 2, corneal opacity v isible by retro- illumination, but not sufficient to obscure iris vessels, and grade 3, corneal opacity, visible by retro-illumination, and obscure iris vessel details. Therapy

On the fifth day after the incubation, all the corneas were examined to grade infections as grade 0, grade 1 , grade 2, or grade 3. Each grade included the same numbers of eyes to be able to use similar numerical parameters to better assess the effectiveness of the therapy. The therapy lasted for one month. During the first week therapeutic agents were applied 8 times per day, and for the last three weeks they were applied only 3 times per clay.

Acanthamoeba growth after the treatment

Non-nutrient agar was set in each well of 6- well Petri dishes. A layer of Escherichia coli was added to the agar surface in each well. Corneas were excised and then homogenized in 1 ml. Page's amoeba saline in a glass grinder. Hundred microliter of each of the homogenates were added onto E. coli containing wells, and the dishes were incubated for 14 days at 30°C. Amoebic growth was inspected daily under the phase contrast microscopy ( Eclipse TS 1 00; Nikon, Tokyo. Japan). Cultures containing no growth after 14 days of incubation were discarded.

Cytotoxic potential of drugs

Cultivation of L929 mouse fibroblast cells

A mouse connective tissue fibroblast cell line, 1.929 (ATCC cell line, NCTC clone 929) was cultured in Dulbecco's minimum Eagle medium (DMEM) (Sigma, St. Louis, MO.USA ) supplemented with 10% fetal calf serum (Sigma, St. Louis, MO.USA ) and 2 mM/mi L-glutamine. No antibiotics were added to the cell culture medium. The cultures were cultivated in an incubator at 37°C and 5% C0₂, until the cell monolayer attained confluence, alter approximately 7 days. Assays were always performed in the exponential growth phase of the cells.

Fibroblast cells were selected because they are the predominant tissue type in the body, and are easy to cultiv ate and because of their favorable doubling time of 24 h. Moreover, these cells are recommended by many standard institutions.

Cell proliferation assay

The proliferation assay analyzed the number of viable cells by the cleav age of tetrazolium salts added to the culture medium, using the XTT labeling reagent ( Roche, Mannheim, Germany). During the assay, tetrazolium salts were cleaved to formazan by cellular enzymes. An expansion in the number of viable cells resulted in an increase in the overall activ ity o f mitochondrial dehydrogenases in the sample. This augmentation in enzyme activ ity lead to an increase in the amount of formazan dye formed, which correlates directly with the number of metabolically active cells in the culture. The formazan dye produced by the metabolically active cells was quantified by a scanning multiwell spectrophotometer by measuring the absorbance of the dye solution at 450 nm. Cells were seeded in 96- well microliter plates at a concentration of 1 x 10⁵ cells/ml in a final v olume of 100 μΐ per well. For the cytotoxicity test, 65. 1 2 Lig/ml MLT, 0.02% CHX, 0.02% PHMB, 0.01% PI, 65. 1 2 ug/ml MLT plus 0.02% CHX, 65. 1 2 ug/ml MLT plus 0.02% PHMB. 65. 1 2 Lig/ml MLT plus 0.01% PI concentrations were used. Cells were then incubated for 24 h in a humidified atmosphere (37°C, 5%> C(> ). After this, 10 μΐ o f the XT T labeling reagent were added to 10 μΐ of culture medium in each well, and the absorbance of the samples was measured at 450 nm against the control (the same cells w ithout any treatment ) using a microtiter plate reader (Thermo Scientific

M icroplate Photometer, Multiskan FC USA). The same volume of culture medium and XTT labeling reagent (10 μΐ of XTT labeling reagent /100 μΐ of culture medium) was added to one well to use as a background control (absorbance of culture medium plus XT T in the absence of cells) as a blank position for the EL ISA. reader. The absorbance was measured after 2 h from the start of the tetrazolium reaction. The experiments were conducted in quadruple. The optical density (OD) of the samples was compared with that of the negativ e control to obtain the percentage v iability, as follows: cell v iability (%) = [(OD450 ( sample )/OD450 (negativ e control)) x 100].

The results, expressed as mean ± SD from four replicates, were analyzed statistically by using one-way analysis o f v ariance ( A NOV A) at 95% confidence levels for multiple comparisons and student's t-test for two-group comparisons.

Five days after the incubation w ith Acanthamoeba trophozoite, all o f the eyes (100%) developed keratitis and they were grouped w ith respect to the degree of opacity observed as follow s: 35 of the eyes (31.25%), Gl ; 28 (25%), G2; and 49 (43.75%), G3. After this, eight different groups of eyes were formed, and into each of the groups, six G3, four G2, four G l eyes were included.

The best treatment results were obtained from PHMB plus MLT group: on day zero there were six G3, four G2, and four G 1 opacity eyes; on day 28 these number reduced to one G2 and nine G 1 eyes. The ratio of fully recov ered eyes was 28.4% (Table 1). In the group treated with PHMB there were two G3, five G2, six Gl , and one GO eyes on the 28^th day (Table 1). And in the MLT group the distribution of opacity was as follows: five G2, seven Gl , and two GO (Table 1). In the infected control group infection appeared to have progressed until the 28^!h day and six G3, five G2, and 3 G l eyes were obtained (Table 1 ). It was also observed on the 28^th day that opacity areas reduced, vascularisation intensified, and veins appeared to become thinner and shortened in the MLT, CHX, PHMB, MLT plus CHX, and MLT plus PHMB groups. In addition development of ghost veins were detected in MLT, CHX, PHMB, MLT plus CHX, , MLT plus PHMB groups at ratios 50%, 28.4%, 42.8%, 35.5%, 71.4%, respectively.

No changes were observed in the uninfected control group eyes during the 28 day of the experiment time.

The results are shown in Table 1 . Degrees of opacity after 7, 14, 2 1 and 28 days of treatment with PI ( Brolene), chlorhexidine, PHMB, miltefosine, miltefosine + PI, miltefosine + CHX, miltefosine + PHMB and infected control.

The increase of GO eyes upon treatment with miltefosine and PHMB already after 4 days treatment compared to the other treatments show that the combination is highly effective.

Acanthamoeba growth after the treatment

After completion of the treatment. Acanthamoeba w ere tried to be recovered from the excised eyes and eye tissue w ere inoculated in NNA solid media. After 14 days of incubation at 30° C four eyes in each of the MLT, CHX, and PI groups (28.5%); 3 eyes in each of the PHMB, MLT plus CHX, and MLT plus PI groups (21.4%), and two eyes in MLT plus PHMB group (14.2%). Amoeba growth was obtained from 10 eyes (71.42%) of the infected control inoculations; no growth was observed for uninfected control eyes.

Table 1 :

Chlorhexidine Chlorhexidine and Miltefosine

PHMB PHMB and Miltefosine

Miltefosine infected control

Cytotoxic potential of drags

Cytotoxicity of MLT, CHX, PHMB, PI, MLT plus CHX, MLT plus PHM B. and MLT plus PI was tested for 24 h by quantitative analysis using the XTT test ( Figure 1). The cytotoxicity value of MLT and control were compared with that of CHX, PHMB, PI, MLT plus CHX, MLT plus PHMB, and MLT plus PI, and found to be statistically di fferent ( Figure 1 , p< 0.05 ). As can be seen MLT 65.12 ^ig nil had no negativ e effect on cell v i ability.

In vivo Toxicity tests

To determine the local tolerance, the ocular tissue distribution and the systemic toxicity o f the test item in the rabbit following a 4-week treatment period with eight daily administrations by the ocular route (50μ1), 65 pg ml miltefosine or 65 pg ml miltefosine + 200 jig ml PHMB respectiv ely, to ev aluate the regression of any toxic signs during a 2-week treatment-free period and to assess systemic exposure under the defined experimental conditions. The determination of ocular irritation has been performed by v isual examination and based on the calculation of indices of ocular irritation according to the scale published in the OECD Guideline 405 and evaluation of pupillary reflex for both eyes.

The Index of Ocular Irritation ( 10! ) and the evaluation of papillary reflex for both eyes were not affected by the treatment.

All findings, were transient and lead to 101 generally equal or less than 6 (maximum possible score: 1 10) in treated animals consisting of conjunctival redness, chemosis or corneal opacity, except for two animals. However, in one animal corneal opacities on the right eye along the study were not considered to be of toxico logical relevance since they were already observed during pretest, 101 ( R) was 10. In the second animal the finding can be related to the slight corneal opacity noted during pretest detailed examination, so a toxico logical relev ance can be excluded.

Iris examination was found normal through the course of the study in all animals.

Thus, the ophthalmoiogical findings noted during the study (such as lenticular or corneal opacity) were considered to be incidental since they were already noted pretest or and they were also noted in control animals. In addition, these findings are known to can occur spontaneously in the rabbit.

Claims

1 . An antibiotic pharmaceutical composition comprising miltefosine and

polyhexamethylene biguanide (PHMB).

2. The composition according to claim 1 wherein miltefosine and PHMB are contained in an etTective amount to treat a microbial infection of the eye or the skin.

3. The composition according to claims 1 or 2 which is an isotonic composition.

4. The composition according to any one of claims 1 to 3, wherein the composition comprises about 5-200 μ g /ml, preferably about 20- 100 μ g/ml, more preferred about

50-75 jig inl o f miltefosine.

5. The composition according to any one of claims 1 to 4, wherein the composition comprises about 1 0- 1000 μg/ml, preferably about 4-500 μg/ml of PHMB.

6. The composition according to any one of claims 1 to 5, wherein the ratio of the miltefosine to PHMB is in the range of about 1 :3.5.

7. The composition according to any one of claims 1 to 6 which is free of endotoxins or proteases.

8. An antibiotic composition consisting essentially of miltefosine and PHMB as active agents.

9. The composition according to any one of claims 1 to 8, suitable for ophthalmic or dermal use.

10. The composition according to any one of claim 1 to 9, wherein the composition is in the form of a gel, solution, cream, dispersion, spray, artificial tear solution.

1 1. The composition according to any one of claims 1 to 10 for use in the prevention or treatment of a microbial infection of the eye or the skin of an individual in need thereof.

12. The composition according to any one of claims 1 to 1 1 for use in the eradication of protozoan parasites from the eye.

1 3. The composition according to any one of claims 1 to 1 1 for use in the prevention or treatment of a bacterial and/or protozoan and or fungal keratitis, specifically for the treatment of Acanthamocba induced keratitis.

14. The composition according to any one of claims 1 1 to 1 3 wherein said composition is administered at least every 10 hours, preferably at least every 5 hours, preferably at least every 2 hours, preferably hourly.

15. The composition according to any one of claims 1 to 1 1 provided as a k it of single components, wherein a first component is miltefosine and a second component is PHMB.