WO2014124504A1

WO2014124504A1 - Compositions and methods for treating biofilms

Info

Publication number: WO2014124504A1
Application number: PCT/BE2014/000010
Authority: WO
Inventors: Bruno Cammue; Katrijn DE BRUCKER; Nicolas DELATTIN; Karin Thevissen
Original assignee: Katholieke Universiteit Leuven Ku Leuven Research & Development
Priority date: 2013-02-13
Filing date: 2014-02-13
Publication date: 2014-08-21

Abstract

The present invention relates to novel methods and compositions comprising a compound, preferably perhexiline (maleate), drospirenone or toremiphene (citrate), which increases the susceptibility and sensitivity of biofi!ms to antifungal drugs such as amphotericin B and caspofungin. Accordingly, the present invention relates to use of said composition for the treatment or prevention of biofilms, particularly fungal or yeast biofilms, such as Candida biofilms, in a subject or on a surface or other medium susceptible to biofilm formation. Coating of medical devices, like implants and plastics with said compounds, in combination with conventional antifungal therapy for the treatment or prevention of biofilms is also contemplated.

Description

COMPOSITIONS AND METHODS FOR TREATING BIOFILMS

FIELD OF THE INVENTION

The present invention relates to novel methods and compositions for the treatment or prevention of microbial biofilms, preferably a fungal and/or yeast biofilm, preferably when grown on a medical device, particularly an indwelling medical device, by increasing the susceptibility and sensitivity of said biofilm to antifungal drugs. More in particular, said methods and compositions comprise combining an antifungal agent, with a potentiating compound, preferably perhexiline (maleate), drospirenone or toremiphene (citrate), for increasing the antibiofilm activity of the antifungal agent, and for reducing, eradicating, inhibiting or preventing fungal biofilms or fungal biofilm formation in a subject and/or on a surface or other medium susceptible to biofilm formation.

BACKGROUND OF THE INVENTION

Biofilms of microbial pathogens consist of dense layers of microorganisms surrounded by an extracellular polymer matrix, adherent to a surface, thereby protecting the microbes from the action of antifungal agents, and are believed to be involved in at least 80% of human bacterial infections. Nowadays, it becomes more and more clear that biofilms are critical to the development of clinical infections in general. Due to the increasing number of immunocompromised patients, combined with the advances in medicai technology, fungi have emerged as a major cause of infectious disease, with Candida sp., particularly C. albicans, being the major pathogen.

Apart from their existence under free-living or planktonic form, Candida sp. are known to form biofilms upon contact with various surfaces. C. albicans cells are able to colonize and subsequently form biofilms on surfaces of indwelling medicai implants and devices, such as (dental) implants, intravascular and urinary catheters, voice prostheses and heart valves. C. albicans is an opportunistic human fungal pathogen, causing not only superficial infections, but aiso life-threatening systemic diseases. C. albicans is now recognized as the fourth most common cause of bloodstream infections in the United States, with a high attributable mortality rate, namely 20-40%. C. glabrata has become an increasingly prevalent pathogen worldwide. After C. albicans, it represents the second cause of fungal infections of the bloodstream, oropharynx and urinary tract. C, glabrata ceils are also able to colonize host tissues as well as abiotic surfaces, where they develop as multilayered biofilm structures. The formation of biofilms on medical devices, particularly fungal/Candida biofiims that are resistant to current antifungals, is the major factor responsible for biofilm associated infections, particularly implant infections. In many cases, the implant has to be removed to cure the infection. Implant failure leads to burdensome and costly revision surgery and sometimes severe suffering of the patient.

Today, there are 4 main classes of established antifungal drugs on the market: (i) the polyenes (e.g. Amphotericin B (AniB), nystatin, natamycin), (ii) the azoles (e.g. miconazole, fluconazole, itraconazole, voriconazole), (iii) ailylamines (e.g. terbinafine), and (iv) echinocandins (e.g. caspofungin). Of these classes, only the polyenes, azoles and echinocandins are used to treat systemic fungal infections, not the ailylamines. Among the current antifungals in clinical use, only the liposomal formula of AmB and echinocandins have shown unique and consistent in vitro and in vivo activity against C. albicans biofilms. Caspofungin disturbs the integrity of the fungal ceil wall by inhibiting the β- (1 ,3}-D-Glucan synthase and appears to lyse sessile cells within C. albicans biofilms. However, caspofungin doses that are effective against planktonic cells (i.e. 0.05 μΜ - 0.5 μΜ) cannot decrease the metabolic activity of C. albicans biofilm cells.

Fungal biofilms, especially those of the pathogen C. albicans, are a cause of infections associated with medical devices like indwelling intravascular catheters and implants. Such infections are particularly serious because biofilm-associated Candida cells are relatively resistant to a wide spectrum of antifungal drugs, including ROS-inducing antifungal compounds, such as Amphotericin B (AmB) and azoles.

All the currently marketed antifungal drugs have major drawbacks, including no broad- spectrum activity, no per oral absorption, side-effects, low antifungal activity, no fungicidal activity, drug-drug interactions and/or high costs. In the case of biofilm treatments or of treatments of sessile cells, these drawbacks become prohibiting. Also, antifungal agents that are active against microbial biofilms often result in only partial killing of the biofilm cells, even when applied at high doses, leaving a subpopulation of the biofilm cells alive, the so called persisters. Persisters are antifungal-tolerant cells that survive treatments with high antifungal concentrations. Because they start growing again when the antifungal pressure drops, persisters are considered as one of the most important reasons for the recurrence of biofilm- associated infections.

An antifungal activity of perhexiiine against planktonic cultures of Cryptococcus neoformans and C. albicans was recently reported (Butts et al, (2013) Eukaryotic Cell, 12:278-287). However, an antibiofilm activity of perhexiiine or a synergy with conventional antifungal agents has not been described before. Accordingly, there is a clear need for effective strategies to prevent and to eliminate deleterious biofiims, in particular fungal or yeast biofilms associated with the surface of medical devices, such as implants.

SUMMARY OF THE INVENTION

The present invention addresses the increasing problems of biofilms, in particular of fungal biofilms on different surfaces including medical devices, outside or within the human body and which escape conventional antifungal treatment. The present invention provides novel methods and compositions with improved anti-biofilm properties by enhancing the efficacy of antifungal drugs, such as by increasing the susceptibility and sensitivity of biofilms, particularly of fungal biofilms to said drugs and/or by a continued, highly localised treatment with said drugs.

The present invention is based on the surprising finding by the inventors that drospirenone, perhexiline (maleate) and toremiphene (citrate), even at subinhibitory concentrations, are potentiating compounds which can increase the antibiofi!m activity of known antifungal compounds, such as amphotericin B and caspofungin against C. albicans and the more resistant C. glabrata, both in vitro as in an in vivo worm C. albicans biofilm infection model, in contrast, perhexiline (maleate), drospirenone or toremiphene (citrate) alone were not very effective against these biofilms. In addition, because of the synergistic, sensitizing effect by perhexiline (maleate), drospirenone or toremiphene (citrate), combination therapy comprising perhexiline (maleate), drospirenone or toremiphene (citrate), and an antifungal compound, preferably a polyene such as amphotericin B or an echinocandin such as caspofungin, allows reducing the dose of said antifungal compound or even applying an antifungal concentration which would be sub-lethal for the antifungal biofilm in the absence of said potentiating compounds. Also, combination therapy comprising perhexiline (maleate), drospirenone or toremiphene (citrate) and said antifungal compound allows applying such sub-lethal dose of said potentiating compounds and such sub-lethal dose of said antifungal agent. Particularly, the present invention shows that the antibiofilm activity of polyenes, such as amphotericin B, or echinocandins, such as caspofungin, can be improved by combining said antifungal compound with perhexiline (maleate), drospirenone or toremiphene (citrate), and thus concomitantly resulting in a reduction of its minimal inhibitory concentration (MIC) against Candida biofilms.

Therefore, the first aspect of the present invention provides a composition for use in the treatment or prevention of a fungal biofilm associated condition in a human or animal subject, said composition comprising at least one of the compounds selected from the group consisting of drospirenone, toremiphene (citrate), perhexiline (maleate) and a derivative of any of the preceeding compounds, and at least one antifungal agent preferably selected from the group consisting of polyenes and echinocandins. Preferably, said at least one antifungal agent is amphotericin B or caspofungin. The composition of the present invention is particularly useful for inhibiting, reducing, eradicating and preventing fungal biofiims comprising or consisting of Candida species. More preferably, said composition further comprises one or more physiologically acceptable compounds, carriers and/or adjuvants. Typically, said subject is a human or an animal.

In one embodiment of this aspect of the present invention, said subject has been implanted with a medical device which is infected or at risk of being infected with a fungal biofilm. Preferably, said medical device is selected from the group including but not limited to catheters, stents, surgical plates, prostheses, valves or pins, artificial joints, pacemakers, contact lenses and bio-implants.

Another aspect of the present invention provides an implant comprising on at least part of its surface a composition comprising at least one of the compounds selected from the group consisting of drospirenone, toremiphene (citrate), perhexiline (maleate) and a derivative of any of the preceeding compounds, referred to as potentiating compounds, and at least one antifungal agent preferably selected from the group consisting of polyenes and echinocandins. More preferably, said at least one antifungal agent is amphotericin B or caspofungin. Preferably, at least part of the surface of said implant is coated with said composition.

In one embodiment of this aspect of the present invention, said at least one potentiating compound is applied to at least part of the surface of said implant before, after or concurrent with said at least one antifungal agent.

In another embodiment of this aspect of the present invention, said implant comprises an internal cavity or a reservoir comprising said composition, wherein said reservoir or internal cavity is connected to at least part of the surface of said implant in order to allow the transport of said composition towards the connected surface.

Preferably, said implant is a medical device selected from the group including but not limited to catheters, stents, surgical plates, prostheses, valves or pins, artificial joints, pacemakers, contact lenses and bio-implants.

Alternatively, the present invention provides a medical device which is coated with or treated with or comprising at least one compound selected from the group consisting of perhexiline (maleate), drospirenone, toremiphene (citrate) and a derivative of any of the preceeding compounds. A patient treated with such implant may be treated via oral or parenteral administration with an antifungal compound, preferably selected from the group consisting of polyenes (such as amphotericin B) or echinocandins (such as caspofungin), when clinical parameters indicate a fungal biofi!m associated infection or an increased risk of a fungal biofilm associated infection of said implant.

Another aspect of the present invention provides a method for reducing, eradicating, inhibiting or preventing fungal biofilms or fungal biofilm formation, characterized in that a surface or medium, outside the body of a human or animal subject, carrying said fungal biofilm or susceptible said fungal biofilm formation, is treated with at least one compound selected from the group consisting of drospirenone, toremiphene (citrate), perhexiline (maleate) and a derivative of any of the preceeding compounds, and at least one antifungal agent preferably selected from the group consisting of polyenes and echinocandins. More preferably, said at least one antifungal agent is amphotericin B or caspofungin. Preferably, said fungal biofilm is a Candida biofilm.

More particularly, the present invention provides a method for coating at least part of the surface of medical devices, such as but not limited to implants, plastics or (subcutaneous) catheters, (voice) prostheses and (heart) valves with at least one compound selected from the group consisting of perhexiline (maleate), drospirenone, toremiphene (citrate) and a derivative of any of the preceeding compounds, for the treatment or prevention of fungal biofilms, preferably Candida biofilms, in combination with conventional antifungal compounds. Said antifungal compounds are preferably selected from the group consisting of polyenes and echinocandins. More preferably, said at least one antifungal agent is amphotericin B or caspofungin.

In another embodiment of this aspect of the present invention, said fungal biofilm is exposed to said at least one compound before, after or concurrent with exposing said fungal biofilm to said antifungal agent. Preferably, said compound is selected from the group consisting of drospirenone, toremiphene (citrate), perhexiline (maleate) and a derivative of any of the preceeding compounds. Said antifungal agent is preferably selected from the group consisting of polyenes and echinocandins. More preferably, said at least one antifungal agent is amphotericin B or caspofungin. Preferably, said fungal biofilm is a Candida biofilm. Another aspect of the present invention provides a method for the treatment of infections involving fungal biofilms in a human or animal subject, said method comprising administering a composition comprising at least one of the compounds selected from the group consisting of drospirenone, toremiphene (citrate), perhexiline (maleate) and a derivative of any of the preceeding compounds, and at least one antifungal agent preferably selected from the group consisting of polyenes and echinocandins. More preferably, said at least one antifungal agent is amphotericin B or caspofungin. Preferably, said fungal biofilm is a Candida biofilm.

Alternatively, the present invention provides a method for treatment and/or for the prevention of a fungal biofilm, preferably a Candida biofilm, upon a (solid support) surface comprising the steps of: treating (coating) the surface with an efficient amount of a potentiating compound, preferably selected from perhexiline (maleate), drospirenone or toremiphene (citrate); introducing the said coated surface in a mammal body, including a human patient body; exposing the said introduced coated surface and/or the said mamma!, including said human, with an efficient amount of an antifungal agent. Said antifungal agent is preferably selected from the group consisting of polyenes and echinocandins. Preferably, said antifungal agent is amphotericin B or caspofungin. More preferably, the amount of said antifungal agent is an amount which would be sublethal to said fungal biofilm in absence of said coating of the implant. Even more preferably, the efficient amount of said antifungal agent is the amount efficient against the fungal cells infection in a pianktonic form.

Preferably, said introduced coated surface is exposed to said antifungal agent by administrating said agent to the human or animal subject, for instance by oral or parenteral administration.

Another aspect of the present invention provides a composition for use in reducing, eradicating, inhibiting or preventing fungal biofilms or fungal biofilm formation, comprising at least one of the compounds selected from the group consisting of drospirenone, toremiphene (citrate), perhexiline (maleate) and a derivative of any of the preceeding compounds, and at least one antifungal agent preferably selected from the group consisting of polyenes and echinocandins. Preferably, said at least one antifungal agent is amphotericin B or caspofungin. Preferably, said fungal biofilm is a Candida biofilm. More preferably, said composition further comprises one or more physiologically acceptable compounds, carriers and/or adjuvants.

DETAILED DESCRIPTION OF THE INVENTION

Legends to the Figures

Figure 1. Toremiphene citrate acts synergisticaliy with caspofungin in a C. elegans infection assay. C. elegans worms (nematodes) were infected with C. albicans by feeding them on an YPD plate containing C. albicans for two hours and treated with 6.25 μΜ toremiphene citrate, 0.095 μΜ CAS and 6.25 μΜ toremiphene citrate + 0.095 μΜ CAS. Untreated worms and non-infected worms served as controls with a DMSO background of 0.6%. Worms were counted regularly for 10 days post infection. ^*, P < 0.05; ^**, P < 0.01 ; ^***, P < 0.001.

Figure 2. represents a schematic view showing a cross section of an implantable device with injection port in which (a) constitutes a fully dense or solid part serving as structural basis for the device, (b) is a port allowing attachment of a syringe, (c) a porous structure suitable for bone tissue attachment or ingrowth and (d) an optional cavity allowing a larger volume of fluid to be injected.

Figure 3. represents a schematic view of a cross section of a dental implant screw composed of a solid base with incorporated injection port and a porous screw of which the internal pore surface is coated with amorphous mesoporous silica (AMS) which acts as slow release medium and the external screw surface is (optionally) coated with a biofilm inhibiting coating, particularly a peptide coating.

Description

The present invention provides novel methods and compositions comprising combining an antifungal agent, preferably a polyene such as amphotericin B, or an echinocandin such as caspofungin, with a potentiating compound, preferably perhexiline (maleate), drospirenone or toremiphene (citrate), for reducing, eradicating, inhibiting or preventing (fungal) biofiims or (fungal) biofilm formation, particularly in a subject and/or on a solid support surface or other medium susceptible to biofilm formation.

Particularly, the inventors have developed means for increasing the susceptibility and/or sensitivity of biofiims, particularly of fungal biofiims, to antifungal drugs.

The present invention may also provide a medical device with the capacity to inhibit or prevent biofilm formation on the surface of such medical device, thus imparting a highly localised treatment with said drugs, and/or by combining said antifungal drugs, preferably polyenes such as amphotericin B, or echinocandins such as caspofungin, even at sublethal dosages, with a suitable biofilm sensitizing, or potentiating compound, particularly perhexiline (maleate), drospirenone or toremiphene (citrate) and/or by imparting a highly localized treatment with said biofilm sensitizing or potentiating compound, particularly perhexiline (maleate), drospirenone or toremiphene (citrate). Particularly, said medical device is adapted to allow administering one or more bio-active agents, including but not limited to potentiating compounds, antibiotics, antibacterial, antifungal, and/or other bio-active compounds (e.g. analgesic agents, anti-inflammatory agents), through the medical device porous structure to the surrounding tissue, resulting in a localized prevention or treatment of complications associated with implant surgery. When using the methods, means and compositions of the present invention, dosages of antifungal agents may be reduced but remain effective in eradicating, inhibiting, preventing or reducing biofilms. Particularly, such treatment may be continued during a defined period of treatment and at a constant (localised) dose without antifungal pressure drops so that development of resistance, persisters and recurrence of biofilm-associated infections can be prevented.

The scope of the applicability of the present invention will become apparent from the detailed description and drawings provided below. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

DEFINITIONS

As used herein the terms "reducing", "inhibiting", "eradicating" or the like in reference to a biofi!m or biofiim formation means complete or partial inhibition (more than 50%, preferably more than 90%, still more preferably more than 95% or even more than 99%) of biofiim formation (in the term of number of remaining cells) and/or development and also includes within its scope the reversal of biofiim development or processes associated with biofiim formation and/or development. Further, inhibition may be permanent or temporary. In terms of temporary inhibition, biofiim formation and/or development may be inhibited for a time sufficient to produce the desired effect (for instance at least 5 days, preferably at least 10 days). Preferably, the inhibition of biofiim is complete and/or permanent (no persisters) ("eradicating").

As used herein, "preventing" or the like in reference to a biofiim or biofiim formation means complete or partial prevention (more than 50%, preferably more than 90%, still more preferably more than 95% or even more than 99%) of biofiim formation (in the term of number of remaining cells) and also includes within its scope processes associated with biofiim formation. Further, prevention may be permanent or temporary. In terms of temporary prevention, biofiim formation may be inhibited for a time sufficient to produce the desired effect (for instance at least 5 days, preferably at least 10 days). Preferably, the prevention of biofiim is complete and/or permanent. As used herein the term "exposing" means administering to, or otherwise bringing into contact with. A microorganism or biofiim may be exposed to an active agent directly or indirectly. Typically direct exposure refers to administration of the agent to the microorganism or biofilm to be treated or otherwise bringing the microorganism or biofilm into contact with the agent itself. Typically indirect exposure refers to the administration of a precursor of the active agent or a compound or molecule capable of generating, either solely or in reaction with other compounds or molecules, the active agent to the microorganism or biofilm or otherwise bringing the microorganism or biofilm into contact therewith. Similarly, the terms "treat" and "treating" and variations thereof as used herein mean administering to, or otherwise bringing into contact with.

As used herein the term "biofiim" refers to a mode of microbial growth comprising sessile ceils, usually within a complex and highly heterogeneous matrix of extracellular polymers, and characterized by a reduced sensitivity to antifungal agents.

In the context of the present invention, "biofilms" can contain single species (e.g. a fungi/yeast such as C. albicans or C. glabrata) or multiple species microorganisms (such as C. albicans, C. glabrata and other microorganisms, preferably yeasts and/or fungi or even prokaryotes). Preferably, this biofilm is a fungal biofilm, more preferably a Candida biofilm, comprising C. albicans, C. glabrata and/or C. Krusei and/or consisting essentially of C. albicans or C. glabrata.

The term "consisting essentially of C, albicans or C. glabrata" refers to a percentage (number of C, albicans or C. glabrata celktotal cell) in the biofilm. Preferably the percentage is above 50%, more preferably above 75%, still more preferably above 90% and/or this term refers to the fact that C. albicans is present in an amount (concentration) sufficient to provoke the biofilm.

The composition of the present invention in first instance aims at biofilms which are associated with microbial infection (e.g., bums, wounds or skin ulcers) or a disease condition including, without limitation, dental caries, periodontal disease, prostatitis, osteomyelitis, septic arthritis, and cystic fibrosis.

The biofilm as used herein is preferably a fungal and/or yeast biofilm, more preferably a Candida species (e.g. C. albicans, C. glabrata, C. krusei) biofilm, an Aspergillus species (e.g. A. flavus, A. fumigatus, A. clavatus) biofilm or a Fusarium species (e.g. F. oxysporum, F. cuimorum) biofiim, even more preferably a C. albicans or C. glabrata biofilm, and can be associated with fungal infection on medical devices like indwelling intravascular catheters and in the oral cavity (e.g. on dental implants). The biofilms can be associated with a surface, e.g., a solid support surface. Such surface can be the surface of any industrial structure, e.g., pipeline or the surface of any structure in animals or humans. Preferably, such surface can be any epithelial surface, mucosal surface, or any host surface associated with microbial infection, e.g., persistent and chronic microbial infections. More preferably, the surface can also include any surface of a bio-device in an animal or human, including without limitation, bio-implants such as (dental) implants, (bone/voice) prostheses, heart valves, pacemakers and indwelling catheters. In a preferred aspect, the microbial or fungal biofilm is associated with the oral cavity, including the surface of dental implants or speech prostheses.

In addition to surfaces associated with biofilm formation in a biological environment, the surfaces can also be any surface associated with industrial biofilm formation. For example, the surfaces being treated can be any surface associated with biofouling of pipelines, heat exchangers, air filtering devices, or contamination of computer chips or water-lines in surgical units like those associated with dental hand-pieces.

The term "controlled release" as used herein refers to a relatively slow or delayed or prolonged release of a bio-active compound from a device in its environment. Particularly, an 80% release of the bio-active compound into an aqueous fluid at a pH between 1.0 and 8.0 is only obtained when a period of time of at least 30 minutes, preferably at least 60 min, at least 24 hours, or at least 48 hours has passed, even more preferably when a period of time lasting several hours, days, weeks or even months has passed (i.e. 20% (or more) of the bio- active compound remains in the device after at least 30 min, 60 min, 24 h, 48 h or even several days).

"Perhexiline" or "perhexiline maieate" is a prophylactic antianginal agent. Perhexiline is thought to act by inhibiting mitochondrial carnitine palmitoyltransferase-1. This shifts myocardial metabolism from fatty acid to glucose utilisation which results in increased ATP production for the same 02 consumption and consequently increases myocardial efficiency. "Drospirenone", also known as 1 ,2-dihydrospirorenone, is a synthetic hormone used in birth control pills and hormone replacement therapy. In combination with ethinyl estradiol it is used as contraception. It is also approved by the FDA to treat premenstrual dysphoric disorder and moderate acne vulgaris.

"Toremiphene" or "Toremiphene citrate" is an oral selective estrogen receptor modulator (SERM) which helps oppose the actions of estrogen in the body. Tore is used in the treatment of ER-positive breast cancer and is approved for treatment of this type of cancer in several countries. Furthermore Tore shows promising results in preventing prostate cancer.

One aspect of the present invention provides novel compositions comprising an antifungal agent, preferably selected from the group consisting of polyenes (such as amphotericin B) and echinocandins (such as caspofungin), and a potentiating compound, preferably perhexiline (maleate), drospirenone or toremiphene (citrate), for reducing, eradicating, inhibiting or preventing (fungal) biofilms or (fungal) biofilm formation, particularly on a solid support surface or other medium susceptible to biofilm formation. The term "potentiating compounds" as used herein in reference to biofilms or biofilm formation, refers to compounds that work synergistically with other compounds to increase the susceptibility and sensitivity of biofilms (and/or sessile cells) of microorganisms, in particular fungal species, to antifungal drugs, even in situations where the potentiating compounds alone, or the antifungal drugs alone, do not reduce, eradicate, inhibit or prevent biofilm formation and/or development.

Potentiating compounds such as perhexiline (maleate), drospirenone or toremiphene (citrate) have been shown by the inventors to work synergistically with antifungal agents and to increase the susceptibility and sensitivity of biofilms (and/or of sessile cells) of microorganisms, in particular fungal species, and/or yeast {Candida) to antifungal drugs such as amphotericin B and caspofungin, even in situations where the potentiating compounds alone do not reduce, eradicate, inhibit the biofilm structure, nor prevent biofilm formation.

In contrast to the potentiating compounds (perhexiline, drospirenone or toremiphene) or the antifungai(s) (amphotericin or caspofungin) alone, the combination of both synergizes against these microorganisms in the form of biofilms (and/or of sessile cells) in reducing the number of microbial cells, in particular fungal species and/or yeast (Candida) or of frequency of infections.

In addition, because of the sensitizing effect of the potentiating compound (perhexiline maleate, drospirenone or toremiphene (citrate)), combination therapy comprising one or more potentiating compounds and one or more antifungal compounds, preferably selected from the group consisting of polyenes (such as amphotericin B) and echinocandins (such as caspofungin), allows reducing the antifungal dose or even applying an antifungal concentration which would be sub-lethal (for a microorganism in the form of a btofilm and/or of sessile cells) in the absence of the potentiating compound (perhexiline maleate, drospirenone or toremiphene (citrate)).

Particularly, the present invention shows that the activity of antifungal compounds, particularly polyenes, such as amphothericin B, or echinocandins, such as caspofungin, against a microorganism, in particular a fungal species, and/or yeast such as Candida, in the form of a biofilm and/or sessile cells, can be improved by combining said antifungal compound(s) with a potentiating compound, preferably perhexiline (maleate), drospirenone or toremiphene (citrate), and thus concomitantly resulting in a reduction of its minimal inhibitory concentration (MIC) or against a microorganism infection, especially fungal infection and/or Candida (C. albicans or C. glabrata) in the form of a biofilm.

The inventors have further found that the potentiating compound (perhexiline (maleate), drospirenone or toremiphene (citrate)) allows for the use of one or more antifungal agent(s) against a fungal species, and/or yeast, such as Candida infection in the form of a biofilm at a reduced amount, which is the amount effective against this (fungal) infection in pianktonic form (non sessile, non forming a biofilm).

Preferably, the amount of said antifungal agent is the amount effective against the fungal cell infection in a pianktonic form (i.e. an about 5-fold to an about 10-fold lower amount than the amount effective against this fungal infection in biofilm form). More preferably, the amount of said antifungal agent is a sub-lethal amount against the fungal biofilm formation.

Accordingly, the present invention also provides the combined use of a potentiating compound (perhexiline maleate, drospirenone or toremiphene (citrate)) and of one or more antifungal agents, preferably selected from the group consisting of polyenes (such as amphotericin B) and echinocandins (such as caspofungin), for use in the treatment or prevention of (fungal infections in the form of) biofilms, particularly fungal and/or yeast biofi!ms, such as Candida biofilms.

Thus, the present invention also relates to a method for the treatment and/or prevention of a condition (fungal infection) associated with biofilm development, comprising administering to a subject an effective amount of a potentiating compound (perhexiline, drospirenone or toremiphene) and one or more antifungal agent(s), preferably selected from the group consisting of polyenes (such as amphotericin B) and echinocandins (such as caspofungin). Thus, a method of the invention for treating or preventing formation of biofilms (e.g. a fungal infection in the form of a biofilm) may comprise administering to a subject an effective amount of a potentiating compound (perhexiline, drospirenone or toremiphene) together with at least one antifungal agent, preferably selected from the group consisting of polyenes (such as amphotericin B) and echinocandins (such as caspofungin) for the treatment or prevention of a (fungal) biofilm-associated condition in this subject.

Suitable antifungal agents include the polyenes (e.g. amphotericin B, nystatin, natamycin), the azoles (e.g. miconazole, fluconazole, itraconazole, voriconazole), allylamines (e.g. terbinafine), the echinocandins (e.g. caspofungin) or the piperazine-1-carboxamidine derivatives described in WO2010068296. A preferred aspect relates to a combination therapy of a potentiating compound (perhexiline (maleate), drospirenone or toremiphene (citrate), or a derivative thereof) with one or more antifungal drug(s), preferably polyenes (such as amphotericin B) or echinocandins (such as caspofungin) (or other conventional antifungal agents) to treat or prevent (Candida) biofilm associated infections in a mammal subject, preferably a human patient.

The addition of a potentiating compound allows for the use of caspofungin doses (on a daily basis) of about 0.05 μΜ to about 0.5 uM and/or of doses of about between about 0.5 μΜ and about 15 μΜ, preferably between about 0.5 mg/kg and 15 mg/kg, more preferably between about 1 mg/kg and about 10 mg/kg, even more preferably between about 2 and about 5 or about 3 mg/kg body weight of a patient; or for the use of amphotericin B doses between about 1 μg/ml and about 10 g ml, preferably between about 2 pg/mi and about 5 pg/ml, more preferably of about 3 pg/ml of amphotericin, more preferably between about 0.05 mg/kg and about 1.5 mg/kg, preferably between about 0.1 mg/kg and about 1.0 mg/kg, more preferably between about 0.2 mg/kg and about 0.5 mg/kg, still more preferably of about 0.3 mg/kg body weight of amphotericin B in a patient.

According to another aspect of the invention, there is provided a composition for promoting dispersal of, or preventing formation of a microbial biofilm, the composition comprising a potentiating compound, more preferably perhexi!ine, drospirenone or toremiphene, and at least one antifungal agent, more preferably a polyene, such as amphotericin B, or an echinocandin, such as caspofungin.

It will be readily appreciated by those skilled in the art that according to the methods of the invention each component of the combination may be administered at the same time, or sequentially in any order, or at different times, so as to provide the desired effect.

Alternatively, the components may be formulated together in a single dosage unit as a combination product.

Another related aspect of the present invention is the combination of a medical device, particularly an implantable medical device, and a pharmaceutical composition comprising at least one potentiating compound, medicament or bio-active agents for preventing or suppressing biofilms in a patient, preferably a mammal, more preferably a human.

Another preferred aspect relates to the preparation (e.g by coating, incorporation) of a solid support surface, such as a medical device, like implants, plastics or (subcutaneous) catheters, with the addition of a sufficient amount (or dose) of a potentiating compound (perhexiline, drospirenone or toremiphene) upon this solid support surface (or inside the solid support), for the elimination (reducing, destroying, eradicating, inhibiting) or prevention of Candida biofilms in combination with conventional antifungal therapy (i.e. addition of one or more conventional antifungal compound(s) at the dose effective against the pianktonic form), preferably in combination with an antifungal therapy involving the administration of at least one antifungal agent, preferably selected from the group consisting of polyenes (such as amphotericin B) and echinocandins (such as caspofungin).

Accordingly, an aspect of the present invention relates to a method for the treatment or prevention of a microorganism infection in the form of a biofilm, preferably a fungal biofilm, wherein the microorganisms or fungi within this biofilm, or capable of forming a biofilm, are exposed to a (effective amount of a) potentiating compound (perhexiiine, drospirenone or toremiphene) and, before, after or concurrent with the potentiating compound, exposing the microorganisms within this biofilm or capable of forming a biofiim to at least one antifungal agent, preferably a polyene, such as amphotericin B, or an echinocandin, such as caspofungin.

The present invention also relates to a method for inhibiting biofilm formation and/or development, wherein a solid support surface or other medium susceptible to biofilm formation is treated or coated with a potentiating compound (perhexiiine (maieate), drospirenone or toremiphene (citrate)) or wherein this potentiating compound is incorporated in a solid support surface or medium susceptible to biofilm formation, such as incorporated in a controlled release medium or (porous) support, and wherein, concurrently or subsequently, this surface or other medium susceptible to biofilm formation is exposed to an antifungal agent.

The potentiating compound (perhexiiine (maieate), drospirenone or toremiphene (citrate)) and/or the antifungal agent, preferably selected from the group consisting of polyenes (such as amphotericin B) and echinocandins (such as caspofungin), may thus be coated onto at least part of the surface of, or be impregnated in or incorporated in, a suitable medical device such as a catheter, stent, prosthesis or other surgical or implantable device. Advantageously, said potentiating compound (perhexiiine (maieate), drospirenone or toremiphene (citrate)) and/or said antifungal agent, preferably selected from the group consisting of polyenes (such as amphotericin B) and echinocandins (such as caspofungin), is released locally in a slow, controlled and/or continuous manner by said medical device.

The present invention also relates to compositions for treating and/or preventing a (fungal) biofilm associated condition in a subject, typically a human or animal subject.

Preferred compositions with anti-biofilm properties comprise one or more of the following compounds: (i) antifungal agents, such as polyenes (e.g. amphotericin B, nystatin, natamycin); azoles (e.g. miconazole, fluconazole, itraconazole, voriconazole); aily!amines (e.g. terbinafine); echinocandins (e.g. caspofungin); gentamycin, ofloxacin, ciprofloxacin; piperazine-1-carboxamidine derivatives (WO2010068296); antifungal peptides with antibiofilm activity; other antimycotic substances such as cetyitrimethylammonium bromide and the like; (ii) agents that sensitize biofilms and/or persisters to existing antifungals, preferably a potentiating compound selected from perhexiiine (maieate), drospirenone or toremiphene (citrate). Preferably, said antifungal compounds are selected from the group consisting of polyenes and echinocandins. More preferably, said antifungal compounds are amphotericin B or caspofungin. Typically the compositions provide means for carrying out the methods of the invention.

Advantageously, the methods and compositions of the invention described above find application in a wide range of environments and circumstances. The composition may be an anti-fouling composition, incorporated in a medical device or component thereof, a coating for a medical device or a pharmaceutical composition. Preferably, the composition(s) of the invention or one or more components thereof, may (also) be used in coating medical devices, including implantable medical devices, including but not limited to venous catheters, urinary catheters, stents, prostheses such as artificial joints, hearts, heart valves or other organs, pacemakers, surgical plates and pins and contact lenses. Other medical equipment may also be coated, such as catheters and dialysis equipment. Alternatively, said implantable medical devices may have the intrinsic capacity to release the composition on the surface of such medical device, thus imparting a highly localised treatment with said composition.

If either the potentiating compound (perhexiline (maleate), drospirenone or toremiphene (citrate)) or the antifungal agent, preferably a polyene (such as amphotericin B) or an echinocandin (such as caspofungin), is not coated or released on said surface, the non- coated component (said potentiating compound or said antifungal agent) can then be administered in an alternative manner, for instance, by oral or parenteral administration.

Methods and compositions of the invention also find application in the management of infectious diseases. For example, a variety of infections associated with (fungal) biofilm formation may be treated with methods and compositions of the invention, such as urinary tract infections, pulmonary infections, dental plaque, dental caries and infections associated with surgical procedures or burns.

Accordingly, compositions of the invention may be formulated as pharmaceutical compositions or form components of, for example, surgical dressings, mouthwash, toothpaste or saline solutions.

Within the context of the present invention, this potentiating compound (perhexiline maleate, drospirenone or toremiphene (citrate)) and/or this antifungal agent, preferably a polyene (such as amphotericin B) or an echinocandin (such as caspofungin), may be applied or coated onto, or incorporated in the surface of an object/item of interest (such as to impart a slow or controlled release effect) well in advance of use of this object/item in, or exposure of this object/item to an environment which comprises biofilm-forming microorganisms, or said potentiating compound (perhexiline maleate, drospirenone or toremiphene (citrate)) and/or said antifungal agent may be applied or coated onto, or incorporated in the surface of an object/item of interest immediately before use of that object/item in, or exposure of this object/item to an environment which comprises (or is susceptible to comprise and/or to develop) biofilm-forming microorganisms.

Compositions according to the invention may be in any suitable form. For example a composition of the invention may be formulated as a paint, wax, other coating, emulsion, solution, gel, suspension, beads, powder, granules, pellets, flakes or spray.

As used herein the term "effective amount" includes within its meaning a non-toxic but sufficient amount or concentration of an agent to provide the desired effect. The exact amount/concentration required will vary depending on factors such as the species of microorganism(s) being treated, the extent, severity and/or age of a biofilm being treated, whether the biofilm is surface-associated, the particular agent(s) being administered and the mode of administration and so forth. Thus, it is not possible to specify an exact "effective amount". However, for any given case, an appropriate "effective amount" may be determined by one of ordinary skill in the art using only routine experimentation.

The antifungal agent(s) (compound(s) or drug(s)) of the present invention are preferably used (or present in composition, including in pharmaceutical composition) at a concentration effective against the planktonic form of the cells, which is a concentration too low to be effective against microorganisms in biofilms (in sessile form).

Medical device with the capacity to release a composition of the present invention

A preferred medical device, particularly a medical implant device in the context of the present invention has the capacity to release a composition of the present invention on at least part of the surface of such medical device, thus imparting a highly localised treatment with said composition. Particularly, said medical device comprises a means to increase in its direct environment the efficacy of an anti-biofilm treatment and/or the susceptibility of a biofilm, preferably a fungal and/or yeast biofilm, to said anti-biofilm treatment.

Preferably, said medical implant device is adapted to release in a controlled manner and/or provide locally a sustained dosage of one or more substances, particularly bioactive agents, including but not limited to an antifungal agent, such as amphotericin B or caspofungin, or a biofilm sensitizing agent, such as a potentiating compound (perhexiline (maleate), drospirenone or toremiphene (citrate)), in order to impart a highly localized treatment or prevention of a biofilm, particularly a fungal or yeast biofilm associated with the surface of said medical device.

Advantageously, the medical implant with improved anti-biofilm properties of the present invention is capable of applying a highly Iocalized, sustained treatment with a sensitizing compound, preferably perhexiline (maleate), drospirenone or toremiphene (citrate), preferably combined with an antifungal agent, which is sufficient to kill ail microbial cells in a biofilm, particularly a fungal biofilm, leaving no persisters and therefore preventing recurrence of biofilm-associated infections without using potentially toxic dosages of said antifungal agent, preferably selected from the group consisting of polyenes (such as amphotericin B) and echinocandins (such as caspofungin).

In the context of the present invention said medical device preferably relates to an implantable device with an incorporated injection port and internal cavity or reservoir connected to the environment of said implant through a porous network.

Figures 2 and 3 schematically show said preferable implantable device. Said porous network is made up of a first macroporous material that is in contact with a second porous material or "a controlled release medium", preferably comprising smaller pores (such as mesopores or micropores) adapted to form a diffusion barrier for a substance and/or to absorb and release said substance, such as a bioactive agent, such as a potentiating compound, from said internal cavity or reservoir in a controlled manner. Said injection port allows supplying various bioactive agents or compositions, including but not limited to (biofilm) sensitizing agents and antifungal agents, to the porous part of the implant and/or the surrounding tissue prior to, during and/or after implantation. Advantageously, prior to or upon the development of infections after full or partial implant fixation, the implant can be filled or injected with a suitable bioactive agent, such as a potentiating compound (including but not limited to perhexiline (maleate), drospirenone or toremiphene (citrate)) and/or a suitable antifungal agents, preferably selected from the group consisting of polyenes (such as amphotericin B) and echinocandins (such as caspofungin), providing localized treatment or prevention of the infection and thus preventing the need for implant extraction and replacement. Furthermore, the outer surface of the implantable device can be treated or coated with a bioactive agent using state-of-the-art or yet to be developed techniques, such as bioactive molecules which stimulate bone attachment or which inhibit, prevent or reduce microbial attachment or biofilm formation, or a combination thereof. Advantageously, the combination of a biocidal or sensitizing coating on the implant combined with a highly localised treatment after implantation maximizes prevention of (fungal) biofilm formation.

The term "microporous material" as used herein is in the meaning of solids, preferably solid silica, that contain pores with free diameters of molecular dimensions. The upper limit of the micropore diameter range according to lUPAC is 2 nm. The term "mesoporous material" as used herein is in the meaning of solids that contain pores with free diameters of 2 - 50 nm (preferably, between 3 nm and 30 nm, more preferably between 4 nm and 25 nm). A smaller diameter of the mesoporous (and of microporous) material was found beneficial for the (in vivo) controlled release of the drug. The term "macroporous material" as used herein is in the meaning of solids that contain pores with free diameters above 50 nm (for instance between 100 nm and 1000 pm, preferably between 200 nm and 20 pm, more preferably between 500 nm and 10 pm, still more preferably between (about) 1 pm and (about) 2 pm). A bigger diameter was found beneficial for allowing the surrounding tissue to invade parts of the implant.

Micropores are conveniently subdivided into ultramicropores narrower than 1.5 nm, and supermicropores with free diameters from 1.5 to 2 nm. Of the porous substances, those having uniform channels, such as zeolite, are defined as molecular sieves.

The term "amorphous" or "amorphous structure" as used herein means without an apparent long range order of the atom positions, therefore lacking crystallinity.

Said preferred implantable device in the context of the present invention thus typically comprises (i) a solid or dense structural element; (ii) an injection port incorporated in said solid or dense structural element; (iii) a first porous element, comprising or forming a macroporous network and acting as a macroporous backbone, which is attached to or bonded with said solid element and which can function as a reservoir of substances, molecules or agents, particularly bioactive molecules. Preferably, said implantable device further comprises (iv) a second porous material or controlled release medium in contact with and/or embedded in said (first) porous element, whereby said second porous material comprises smaller pores than said (macro)porous element and is adapted to act as absorbent and/or diffusion resistor or retarder of said substances, such as bioactive molecules that are incorporated in said implantable device.

In addition to the reservoir function of said (first) porous element, the implant may optionally further comprise (v) a cavity able to contain an injected fluid comprising said substances, molecules or agents, particularly bioactive agents, preferably said potentiating compound or composition. This cavity (d) may be located in the solid element of the implant, being connected with both said injection port and the porous element, but can extend into said porous element as well. The cavity, like the porous element, thus functions as a reservoir whereby a fluid with such agents, molecules or compounds, preferably bioactive agents, more preferably said potentiating compounds or composition comprising said potentiating compound, are stored in said reservoir, and are subsequently released via diffusion through the porous network (comprising macropores, mesopores and/or micropores) into the tissue environment of the implant. Typically, said cavity allows for a larger volume to be injected.

Said solid or dense element (a) functions as the structural basis of the implant device and typically contains the injection port (b). Said injection port may be adapted to expose part of the porous element that is normally covered by said solid element, it is understood that said solid element, more specifically the injection port, is to remain accessible after implantation. Said injection port may be adapted to easily introduce a fluid into said porous element. Particularly, said injection port may comprise a means to attach an injection device, particularly the proximate end of an injection device, for instance a syringe, for easy refilling of said implant.

Preferably, said incorporated injection port imparts the ability to administer a bioactive compound, particularly medication, post-surgery and allows easy modification of the treatment by changing or refilling the bioactive agents, such as an antifungal and/or a potentiating compound of the present invention, present in said cavity or reservoir, in the case of e.g. resistant microbial organisms or biofilms or if previously used medication remains ineffective.

Preferably, said porous element comprises a first porous material, particularly a macroporous material, wherein said macropores form a macroporous network that is in contact with the tissue environment of the implantable device and the central cavity or reservoir of the implant device. Typically, said macroporous network functions as the structural basis in which a second porous material is embedded or on which a second porous materia! is added.

Preferably, the porous element of the implantable device of the present invention is made of an inert, biocompatible material, more preferably said porous element comprises a macroporous titanium or a macroporous titanium alloy, forming a macroporous network or layer that acts as the delivery system of a bioactive agent from the implant to the surrounding tissue. Preferred pore size of said macroporous element is at least 100 nm, more preferably pore sizes range from 200 nm to 1000 nm, more preferably range from 500 nm to 100 nm, even more preferably range 1nm to 50 nm, such as from 1 nm to 20 nm or 1 to 10 nm or even 1 to 5 nm and 1 to 2 nm.

The porous element of the implantable device can be produced using any of the techniques available in the current state of the art. Said techniques include the use of sacrificial pore templates in powder metallurgical processes, the use of the sponge replication technique, emulsion templating of porous structures, rapid prototyping or additive manufacturing techniques such as selective laser sintering (SLS), selective lased melting (SLM) or electron beam deposition (EBD) or plasma spraying techniques, the partial sintering of metai powder or metal bead compacts or dehydrogenation of metal hydride powder compacts followed by partial sintering [e.g. WO2007000310].

Preferably, the macropores of said porous element are in contact with a second porous material. Said second porous material or said second porous layer has a smaller pore structure than the macroporous element and comprises small macropores, mesopores and micropores or combinations thereof. Said second porous material may be embedded in said macroporous element, thus partially or completely filling said macropores of the porous element. Alternatively, said second porous material may be coated onto or bonded with said macroporous element, either or both at the outside of said porous element or at the central cavity (if present). Such porous material embedded in or outside the macropores of a porous network have preferably a pore size in the range of 1 nm to 220 nm (or even from 1 to 300 nm), yet more preferably ranging from 2 to 100 nm, yet even more preferably ranging from 3 to 30 nm, such as ranging from 4 to 25 nm or even from 5 nm to 10 nm.

An optional additional feature of the implant of the present invention is that the porous network (layer) is foreseen with a zone of smaller pores in said network (layer) of pores adapted to shield against micro-organisms so that they cannot enter the implant reservoir, the implant central cavity or at least part of the network of pores. Alternatively, the implantable device is composed of a material with a functional gradient in porosity and or pore size. The resulting device can be considered to be fully dense at one side while a porosity suitable for implant purposes is achieved in another part of the device.

Preferably, said porous element (layer) allows implant fixation by bone attachment and/or bone ingrowth. More particularly, the implantable device comprises at least in part, preferably at the implant site, a porous surface, coating or layer that comprises a certain amount of interconnected porosity with adequate pore sizes to allow a sufficient tissue ingrowth, for instance bone ingrowth, so that a firm mechanical anchorage can be established.

Particularly, said implantable device may comprise a metal of the group consisting of Ti, Zr, Mg, Hf ,Ta, Nd, Nb, n, Mo, Al, Cr and Co, or alloys of these elements. More preferably, the implantable device of the present invention comprises Ti, even more preferably is made from ASTM GRADE 1 , 2, 3 or 4 Unalloyed Titanium. More preferably, the surface of said implantable device may be oxidized to titanium oxide or dioxide, which in its turn may be further cationic or anionic doped, as is known by the person skilled in the art.

The macroporous network of the porous element is, at least in part or in distinct zones or layers, in contact with a second porous material with smaller macropores, mesopores and/or micropores. Said second mesoporous and/or microporous material may be embedded in said macroporous network or may form a layer or zone surrounding or outside the macroporous network and may thus acts as a diffusion barrier (through which substances, compounds or molecules, particularly bioaciive molecules or therapeutic compounds elutes through diffusion) or as a barrier to shield the implant against invasion of microorganisms. More in particular, the rate at which said drugs or compositions are released is controlled through said second material with smaller pores located in the implant's macroporous network.

Preferably, said second porous material in contact with the macroporous layer of said implant is a non-bio-erodible (not disintegrating within a certain period of time by the action of body fluids and/or metabolic activity) porous material, more preferably is a porous oxide (which, in case the macroporous layer is made from an oxide, is typically different from the oxide of the macroporous layer), even more preferably is a silicate based nanoporous material. In the context of the present invention the term "silicate based nanoporous material" refers to porous material with a matrix based on silicon oxide with pore diameter less than 300 nm, preferably of less than 100 nm. The voids between the linked atoms have a free volume larger than a sphere with a 0.25 nm diameter. For pore shapes deviating from the cylinder, the above ranges of diameter of micropores and mesopores refer to equivalent cylindrical pores. Said porous oxides, preferably silicate based nano- or meso-porous material can be either amorphous, ordered or crystalline. Said porous oxides, preferably silicate based porous material can be mesoporous or microporous (or overlapping). Ordered microporous and mesoporous materials can be described in terms of a host structure, which defines a pore structure, which may contain guest species.

Also, current production processes allow to customize pore size and pore size distribution. This way, the release rate of a biologically active compound present in the porous network can be easily controlled. Such porous material embedded in or outside the macropores of a porous network have preferably a pore size in the range of 1 nm to 220 nm, yet more preferably ranging from 1 to 100 nm, yet even more preferably ranging from 1.5 to 30 nm, such as ranging from 2 to 20 nm, 3 to 15 nm or 4 to 10 nm. A narrow pore size distribution is preferred.

The open meso- and/or microporosity of such (amorphous, ordered or crystalline) materials makes them suitable as potential matrices for adsorption and subsequent delayed release of a variety of substances, molecules or compounds, particularly bioactive agents. Also, the diffusion of molecules inside a microporous solid is much slower than inside a mesoporous material, resulting in significantly smaller release rates for the former material. Preferably, said second porous material is an amorphous micro- or mesoporous silica material with pore size ranging from about 1 nm to about 20 nm.

Alternatively, amorphous microporous silica, (less preferably amorphous titania, amorphous zirconia and amorphous alumina) with a narrow monomodai pore-size distribution and a pore size maximum below 1 nm may be chosen as second porous material in contact with the macroporous element. Synthesis methods to prepare such microporous materials are known in the art, e.g. a sol-gel technique comprising polymerisation under acidic conditions.

The term "sol" as used in this application means a colloid that has a continuous liquid phase {e.g. an aqueous phase) in which a soiid with a particle size in the micrometer range or smaller is suspended. So! is synonymous to colloidal suspension.

The term "gel" as used herein refers to a material consisting of continuous solid and liquid phases of colloidal dimensions.

The term "sol-gel" as used herein means a gel derived from a sol, either by polymerising the so! into an interconnected solid matrix, or by destabilising the individual particles of a colloidal sol by means of an externa! agent. Sol-gel materials may be produced in a wide range of compositions (mostly oxides) in various forms, including powders, fibres, coatings, thin films, monoliths, composites, and porous membranes. In general, the sol-gel process involves the transition of a colloidal suspension system into a "gel" phase exhibiting a significantly higher viscosity.

Preferably, said implantable device is characterised whereby the implant site or implant zone is in the form of a screw adapted to be screwed into a tissue, preferably a bone tissue (Figure 3). This is particularly suitable for use as a dental implant. The solid or dense element of the dental implant device provides the structural basis for the attachment of a dental crown. The porous element allows fixation of the implant in the jaw bone by bone ingrowth. At any time after implantation the injection port can be accessed by removal of the dental crown, allowing administration of one or more biologically active agents into the implant. It is understood that the screw form is an example and that such implant can have different shapes. ] Another preferred aspect relates to an implantable device whereby said implantable device is a bone implant. Said implantable device may also be an abutment which provides the attachment for an external prosthesis, such as a prosthetic leg, finger or arm. As such an abutment permanently penetrates the skin, the tissue around the implant is sensitive to infection. With an injection port incorporated in the external part of the abutment localized administration of anti-inflammatory drugs or compounds and/or sensitizing agents with antibiofilm properties is rendered possible.

Preferably, said implantable device may comprise means to improve implant efficiency and to prevent implant failure by biofilm-associated infections by supplying a suitable bioactive agent, preferably a potentiating agent of the preent invention into the surrounding tissue through a slow or controlled release scheme. For instance, amorphous mesoporous silica can be synthesized by means of sol-gel processing inside the macroporous element of an implantable device with internal central cavity and/or porous reservoir and externally accessible injection port. The internal reservoir and/or central cavity itself can be filled with one or more slow release bioactive agents, yielding a long-lasting release (hours, days, weeks or even months) of the provided agent(s), particularly comprising one or more potentiating agent(s) of the present invention.

Accordingly, the present invention also relates to the combined use of such a potentiating compound (perhexiline (maieate), drospirenone or toremiphene (citrate)) and at least one antifungal agent, preferably selected from the group consisting of polyenes (such as amphotericin B) and echinocandins (such as caspofungin), for the treatment or prevention of fungal biofilm associated infections associated with an implantable device. Preferably, one or more sensitizing agents, particularly perhexiline (maieate), drospirenone or toremiphene (citrate), are released in a controlled way using the medical device and implant technology described above, and said antifungal agent is administered using a conventional route of administration, such as orally or intravenously. Alternatively, said potentiating compound may be coated on said implantable device. Advantageously, coating or incorporating only said potentiating compound in the implant will not induce multi-drug resistance of the microorganisms following sustained exposure to these agents because such agents are not or only little inhibitory themselves.

A suitable composition for use in the above described medical device typically comprise one or more potentiating agents, particularly perhexiline (maieate), drospirenone or toremiphene (citrate), and optionally one or more antifungal compounds, particularly a polyene (e.g. AmB) or an echinocandin (e.g. caspofungin), and optionally one or more biological active agents which are capable of providing direct or indirect therapeutic, physiologic and/or pharmacologic effect in a human or animal organism. The biological active agent may include a drug, pro-drug, a targeting group or a drug comprising a targeting group, including but not limited to, steroidal or non-steroidal antiinflammatory agents, anti-inflammatory peptides, antiviral compounds, analgesics, painkillers, local anaesthetics, anticoagulants, antihypertensive substances, vitamins, or contrast media. Suitable compounds are well known to and are routinely selected by the skilled person.

Another aspect of the present invention provides a method for inhibiting biofilm formation and/or development, wherein a surface or other medium susceptible to biofilm formation is treated with a potentiating compound (perhexiline (maieate), drospirenone or toremiphene (citrate)) or wherein this potentiating compound is incorporated in a surface or medium susceptible to biofilm formation, and wherein, concurrently or subsequently, said surface or other medium susceptible to biofilm formation is exposed to an antifungal agent, preferably selected from the group consisting of polyenes (such as amphotericin B) and echinocandins (such as caspofungin). Suitable antifungal agents include the polyenes (e.g. amphotericin B, nystatin, natamycin), the azoles (e.g. miconazole, fluconazole, itraconazole, voriconazole), allylamines (e.g. terbinafine), the newly introduced echinocandins (e.g. caspofungin) or the piperazine-1- carboxamidine derivatives. In a preferred embodiment said antifungal agent is a polyene or an echinocandin, more preferably said antifungal agent is amphotericin B or caspofungin.

The present invention also provides a method for the treatment and/or prevention of a condition associated with biofilm development, comprising administering to a subject an effective amount of perhexiline (maleate), drospirenone or toremiphene (citrate) and an antifungal agent, preferably selected from the group consisting of polyenes (such as amphotericin B) and echinocandins (such as caspofungin). Thus, a method of the invention for treating or preventing formation of biofilms may comprise administering to a subject an effective amount of perhexiline (maleate), drospirenone or toremiphene (citrate) together with said at feast one antifungal agent for the treatment or prevention of a biofilm-associated condition in said subject.

In this context, perhexiline (maleate), drospirenone or toremiphene (citrate) and/or the antifungal agent, preferably selected from the group consisting of polyenes (such as amphotericin B) and echinocandins (such as caspofungin), may be coated onto or be impregnated in or incorporated in the surface of a suitable medical device such as a catheter, stent, prosthesis or other surgical or implantable device.

According to another aspect of the invention, there is provided a composition for promoting dispersal of, or preventing formation of a microbial biofilm, the composition comprising perhexiline (maieate), drospirenone or toremiphene (citrate), and at least one antifungal agent, preferably selected from the group consisting of polyenes (such as amphotericin B) and echinocandins (such as caspofungin). Typically the compositions provide means for carrying out the methods of the invention.

In particular embodiments the composition may be an anti-fouling composition, a medical device or component thereof, a coating for a medical device or a pharmaceutical composition. The present invention also relates to compositions for treating and/or preventing a condition associated with biofilm development.

It will be readily appreciated by those skilled in the art that according to the methods of the invention each component of the combination may be administered at the same time, or sequentially in any order, or at different times, so as to provide the desired effect. Alternatively, the components may be formulated together in a single dosage unit as a combination product. The methods and compositions of the invention described above find application in a wide range of environments and circumstances.

In a preferred embodiment, compositions of the invention or one or more components thereof, may also be used in coating medical devices, including implantable medical devices, including but not limited to venous catheters, urinary catheters, stents, prostheses such as artificial joints, hearts, heart valves or other organs, pacemakers, surgical plates and pins and contact lenses. Other medical equipment may also be coated, such as catheters and dialysis equipment, if either perhexiline (maleate), drospirenone or toremiphene (citrate); or the antifungal agent, preferably selected from the group consisting of polyenes (such as amphotericin B) and echinocandins (such as caspofungin), is not coated on said surface, said non-coated component can then be administered in an alternative manner. Methods and compositions of the invention also find application in the management of infectious diseases. For example, a variety of infections associated with (fungal) biofilm formation may be treated with methods and compositions of the invention, such as urinary tract infections, pulmonary infections, dental plaque, dental caries and infections associated with surgical procedures or burns. Accordingly, compositions of the invention may be formulated as pharmaceutical compositions or form components of, for example, surgical dressings, mouthwash, toothpaste or saline solutions.

Within the context of the present invention, said perhexiline (maleate), drospirenone or toremiphene (citrate) and/or said antifungal agent, preferably selected from the group consisting of polyenes (such as amphotericin B) and echinocandins (such as caspofungin), may be applied or coated onto, or incorporated in the surface of an object/item of interest well in advance of use of said object/item in, or exposure of said object/item to an environment which comprises biofilm-forming microorganisms, or said perhexiline (maleate), drospirenone or toremiphene (citrate) and/or said antifungal agent may be applied or coated onto, or incorporated in the surface of an object/item of interest immediately before use of that object/item in, or exposure of said object/item to an environment which comprises biofilm-forming microorganisms.

Compositions according to the invention may be in any suitable form. For example a composition of the invention may be formulated as a paint, wax, other coating, emulsion, solution, gel, suspension, beads, powder, granules, pellets, flakes or spray. The skilled addressee will recognise that the appropriate formulation will depend on the particular application and the proposed route of delivery.

Compositions of the invention typically also include carriers, diluents or excipients. Suitable carriers, diluents and excipients are known to those skilled in the art. The diluents, adjuvants and excipients must be "acceptable" in terms of being compatible with the other ingredients of the composition, and in the case of pharmaceutical compositions, not deleterious to the recipient thereof.

Carriers may be liquid or solid. In the case of liquid carriers, the liquid may be an aqueous or non-aqueous solvent. Typically for anti-fouling and other industrial applications, the composition, for example in the form of a paint or other surface coating, employs a carrier enabling the controlled release of the active agent temporally and/or spatially.

Typically, the rate of release of the substance is determined by the properties of the polymer itself as well as environmental factors (such as pH, temperature etc). Controlled release systems are capable of delivering substances slowly and continuously for up to several years. Those skilled in the art will appreciate that a number of controlled release systems are applicable to the delivery of agents according to the present invention. By way of example only, release may be diffusion controlled, chemically controlled or solvent activated.

Examples of pharmaceutically acceptable diluents are demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils, including polysi!oxanes, such as methyl po!ysiloxane, phenyl polysiioxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcel!ulose or hydroxypropyimethylceliulose; lower alkanols, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1 ,3-butylene glycol or glycerin; fatty acid esters such as isopropyl paimitate, isopropyl myristate or ethyl oleate; polyvinylpyrrolidone; agar; carrageenan; gum tragacanth or gum acacia, and petroleum jelly. Typically, the carrier or carriers will form from 1% to 99.9% by weight of the compositions.

For pharmaceutical applications, compositions may be formulated for delivery by any route, for example oral, topical, intracavitary, intravesical, intramuscular, intraarterial, intravenous, intranasal, intrapulmonary or subcutaneous.

EXAMPLES: Identification of antibiofilm activity potentiating compounds & synergism with antifungal compounds

Study Setup Strains and chemicals. Two C. albicans strains and one C. glabrata were grown routinely on YPD (1% yeast extract, 2% peptone and 2% glucose) agar plates at 30°C for two days. Stock solutions of amphotericin B ("AmB" - Sigma, St Louis, MO) and caspofungin ("CAS" - Cancidas, Merck, Beeston Nottingham, United Kingdom) were prepared in DMSO. Rosweli Park Memorial Institute (RPMI) 1640 medium (pH 7.0) with L-g!utamine and without sodium bicarbonate was purchased from Sigma. RPMI medium was buffered with MOPS (Sigma). Drospirenone ("Dros" - 6β,7β,15β,16β -Dimethylene-3-oxo-17ct-pregn-4-ene-21 ,17- carbolactone), toremiphene ("Tore" - 2-{4-[(1Z)-4-chloro-1 ,2-dipheny!-but-1-en-1-yl]phenoxy}- N,N dimethylethanamine) and perhexiline (maleate) ("Perhex" 2-{2,2~ Dicyclohexy!ethyl)piperidine) were purchased from Sigma.

Antibiofilm screening assay. A repositioning library was screened in the presence of sub- BEC50 AmB against C. albicans biofiims, namely 0.156 μΜ. To this end, a C. albicans overnight culture, grown in YPD, was diluted to an optical density of 0.1 , which corresponds to 10⁶ cells/ml, in RPMI medium and 95 μΙ of this suspension was added to the wells of a round bottomed microtiterplate (TPP, Tradingen, Switzerland) in the presence of 200 μΜ of each compound (10 mM stock solution in DMSO), resulting in 2% DMSO background. Biofiims were allowed to form for 24h at 37°C. Afterwards, 0.156 μΜ of AmB was added (Final DMSO background 2.1%). Biofiims were incubated for an additional 24 hrs at 37°C. Finally, biofiims were washed and quantified with cell titre blue (CTB) by adding 100 μί CTB diluted 1/10 in PBS in each well. After 1 hr of incubation in the dark at 37°C, fluorescence was measured with a fluorescence spectrometer at an excitation wavelength of 535 nm and an emission wavelength of 590 nm. Fluorescence values of the samples were corrected by subtracting the average fluorescence value of CTB of uninoculated wells (blank). Percentage of surviving biofilm cells was calculated relative to the control treatment (2.1% DMSO).

Chequerboard antibiofilm assay. In order to determine possible synergistic interactions between AmB/CAS and Dros, Perhex and Tore against C. albicans/C. glabrata biofiims, chequerboard analysis was used and FICI coefficients were calculated. To this end, overnight cultures of C. albicans/C. glabrata were diluted to an optical density of 0.1 in RPMI 1640 medium. Dros (100-3.125 μΜ), Perhex (25-0.78 μΜ) and Tore (12.5-0.39 μΜ) were two-fold diluted across the columns of a round-bottomed 96-well plate in RPMI 1640 medium (TPP, Tradingen Switserland). To all wells, 5 μΙ of this compound solutions and 95 μΐ of the above cell suspension was added (DMSO background of 0.5%). After 1 hr of adhesion, the medium was aspirated and the biofiims were washed with 100 μΙ PBS to remove non- adherent cells, followed by the addition of 100 μΙ RPMi medium, containing the corresponding compound concentration. After 24 hrs of biofilm formation in the presence of the compounds, biofilms were washed with 100 μΙ PBS and 100 μΙ of a combination of AmB or CAS and the compounds, two-fold diluted in RPMI 1640 medium across the rows and columns of a microtiterplates, respectively, were added (final DMSO background 0.6%). The following range was used for AmB: 5-0.01 μΜ for C. albicans and 20 - 0.04 μΜ for C. glabrata. For CAS 1.25-0.002 μΜ was used for C. albicans and 20-0.04 was used for C. glabrata. After an additional 24 hrs of incubation at 37°C, biofilms of C, albicans were quantified with the CTB method as described above. Biofilms of C. glabrata were quantified with the XTT-assay (Teilier et al, 1992) as C. glabrata was not able to convert CTB within one hour. To this end, biofilms of C. glabrata were washed with 100 μΙ PBS and afterwards 00 μΙ XTT (0,25 mg/mi in PBS, 1 μΜ menadion; Sigma, St Louis, MO) was added to every well. After one hour of incubation at 37 °C absorbance was measured at 490 nm. Obtained values were corrected for the blank (XTT without cells). All assays were repeated at least 3 times. Interpretation of drug combination interactions against C. albicanslC. glabrata biofilms was determined on the basis of the fractional inhibitory concentration index (FICl). The FiCI was calculated by the formula FiCI = [C(BEC50A)/BEC50A] + [C(BEC50B)/BEC50B], in which C(BEC50A) and C(BEC50B) are the BEC50s of the antifungal drugs in combination and BEC50A and BEC50B are the BEC50s of antifungal drugs A and B alone. The interaction was defined as synergistic if FICI ≤0.5, indifferent if 0.5< FICI <4 and antagonistic if FICI >4.0 (Odds, J Antimicrob Chemother. 2003 52:1 ). The average F!CI-value of at least 3 independent experiments is shown.

Worm infection assay, In vivo experiments using the C. elegansJC. albicans model system were based on the procedure previously described (Breger et al. 2007. PLoS Pathog. 3: e18), with minor modifications. Briefly, larvae of glp-4 lsek-1A mutants of C. elegans were grown to the L4 stage on NGM agar plates containing a surface lawn of freshly inoculated OP50 Escherichia coli. Worms (nematodes) were collected, washed with M9 buffer, and incubated for 2 h on YPD agar plates containing freshly grown surface lawns of C. albicans SC5314. Next, worms were collected and washed with M9 buffer to remove C. albicans from their cuticles. Forty to 50 worms were then suspended in 0.25 ml M9 buffer (supplemented with 10 g ml cholesterol, 100 μg ml kanamycin and 75 g ml ampicilin) containing different drug combinations in separate wells of 24-well plates, and their survival was monitored regularly during 7 days. Worms were treated with 6.25 μΜ Tore; 0.095 μΜ CAS; 6.25 μΜ Tore + 0,095 μΜ CAS and 0.6% DMSO (negative control). As a control also the survival of non-infected worms was monitored. Worm survival was expressed as a percentage of their viability at day zero. The data shown below represent the mean and standard error of *** experiments with at least sextuple measurements. Results were analyzed for statistical significance by Student's f test. Values were considered to be statistically significant when the P value was <0.05, ***

Example 1. Drospirenone, perhexiline maleate and toremiphene citrate increase the activity of AmB against C. albicans and C. glabrata biofilms.

In a first instance, we assessed the effect of amphotericin B (AmB) on mature C. albicans biofilms using the fluorescent cell titre blue (CTB) method. We found the Biofilm eradicating concentration that results in a reduction of 50% of biofilm biomass (BEC50) of AmB against C. albicans biofilms to be 1 μ . Next, we screened 1600 off-patent drugs and other bioactive agents and looked for compounds that could increase the antibiofilm activity of AmB. To this end, C. albicans biofilms were preincubated with 200 μΜ of the repositioned compounds during adhesion and biofilm formation phase. The resulting biofilms were subsequently treated with a sub-inhibitory AmB concentration for 24h, namely 0.156 μΜ (highest concentration which results in 100% survival of biofilms), whereafter biofilm cells were quantified with CTB. We identified 50 compounds that resulted in less than 10% surviving C. albicans biofilm cells in the presence of AmB. Only 14 of these compounds were not characterized as antifungal compounds and were selected for further research. The potential inhibitory activity on biofilm formation (biofilm inhibiting concentration 50 (B1C50) of these 14 compounds in the absence of AmB was determined (Table 1).

Table 1. Compounds and corresponding BIC50 values

Compound BIC50 (μΜ)

Chlorprothixene hydrochloride 17

Clorgilini hydrochloride 24

Prochlorperazine edisylate 5.2

Danthron 12

Acamprosate calcium > 100

Dicyclomine hydrochloride 60

Toremiphene citrate 20

Perhexiline maleate 39

Drospirenone 400 Next, the effect of these 14 compounds on the antibiofilm activity of AmB was evaluated. To this end, C. albicans biofilms were incubated with a subinhibitory concentration of the compounds during adhesion and biofilm formation phase for 24h. The resulting biofilms were subsequently incubated with a concentration series of AmB, to determine the BEC50 of AmB in the presence of the compounds. From these 14 compounds, only Dros, Perhex and Tore were able to increase the AmB antibiofilm activity against C. albicans biofilms by at least 1.5- fold, in conclusion, Dros, Perhex and Tore enhance the antibiofilm activity of AmB.

Example 2. Assessment of synergism of drospirenone, perhexiline maleate and toremiphene citrate with AmB against C. albicans and C. glabrata biofilms.

To determine whether Dros, Perhex and Tore act synergistica!ly with AmB against C. albicans biofilms, we calculated the corresponding fractional inhibitory concentration index (FICI) for each combination by chequerboard analysis (Table 2). Compounds act synergistically when the FiCI index < 0.5. The BEC50 for Dros, Perhex and Tore alone is 400, 39 and 19.5 μ against C. albicans biofilms. We found that only Dros acts synergisticaily with AmB against C. albicans biofilms (FICi < 0.5 for AmB in combination with 50 μΜ Dros). Dros (100-25) can reduce the BEC50 of AmB by a 2 to 4 fold (Table 2A). Although the FICI for the other combinations is > 0,5, specific concentrations of Tore reduce the BEC50 of AmB significantly (P-value < 0.05; Table 2A). For example, the BEC50 of AmB is more than 3-fold reduced in combination with 6.25 μ Tore (Table 2A). Next, we assessed if the potentiation effect of these compounds is species specific. Therefore, we determined if these compounds could also potentiate AmB against C. glabrata biofilms. The BEC50 for Dros, Perhex and Tore alone, is >400, 60 and 28 μΜ against C. glabrata biofilms. Dros and Perhex act synergistically with AmB against C. glabrata biofilms (FiCI < 0.5; Table 2B). The BEC50 of AmB (3,89 μΜ) in combination with these compounds could be reduced up to 9 and 3.5-fold respectively (Table 2B). Whereas Tore does not act synergistically with AmB (FICI > 0,5) 6.25 μΜ Tore does significantly reduce the BEC50 of AmB (P-vaiue < 0,05).

Table 2. Activity of amphotericin B and combinations of drospirenone, perhexiline maleate and toremiphene citrate with amphotericin B against C. albicans and C. glabrata biofilms.

C. albicans SC5314 BEC50 (μΜ)

Compound Comp AmB Comp + AmB P-value Fold FICI

Cone. change

(μ )

Drospirenone 100 1.01 ± 0.09 0.27 ± 0.02 0.0010 3.8 0.514

50 0.32 ± 0.04 0.0017 3.2 0.438 25 0.46 ± 0.02 0.0067 2.2 0.517

Perhexifine 12.5 0.65 ±0.16 0.0747 1.5 0.967 maleate 6.25 0.91 ± 0.22 0.6228 1.1 1.060

3.125 0.96 ± 0.24 0.8106 1.1 1.030

Toremiphene 6.25 0.27 ± 0.02 0.0010 3.7 0.588 citrate 3.125 0.49 ±0.05 0.0097 2.0 0.648

1.56 0.60 ± 0.05 0.0307 1.7 0.677

C. glabrata BG2 BEC50 (μΜ)

Compound Comp AmB Comp + AmB p-value fold FICI

Cone. change

(MM)

Drospirenone 100 3.89 ± 0.41 0.43 ± 0.06 0.0007 9.1 0.360

50 1.17 ± 0.28 0.0040 3.3 0.425

25 1.33 ± 0.27 0.0056 2.9 0.405

Perhexiline 12.5 1.11 ± 0.35 0.0038 3.5 0.494 maleate 6.25 2.1 ± 0.21 0.0312 1.9 0.644

3.125 2.43 ± 0.27 0.0693 1.6 0.678

Toremiphene 6.25 1.23 ± 0.35 0.0049 3.2 0.540 citrate 3.125 2.67 ± 0.58 0.1443 1.46 0.798

1.56 2.33 ± 0.38 0.0594 1.7 0.656

Example 3. Drospirenone, perhexiline maleate and toremiphene citrate act synergistically with caspofungin against C. albicans and C. glabrata biofilms.

Putative synergies between Dros, Perhex and Tore with other commonly used antifungals like CAS were investigated against C. albicans and C. glabrata biofilms. However, in contrast with AmB, Dros, Perhex and Tore all act synergistically with CAS (FICI < 0.5) against biofilms of C. albicans and C. glabrata (Table 3 A, B). In C. albicans, the strongest potentiation of CAS is observed with Tore. The BEC50 of CAS against C. albicans biofilms (0.29 μ ) was reduced by a 21 -fold in the presence of 6.25 μΜ Tore. In addition, Dros and Perhex reduce the BEC50 of CAS by 4 and 6.5-fold respectively (Table 3A). In contrast with C. albicans, Perhex was the strongest potentiator for CAS in C. glabrata. The BEC50 (9.25 μΜ) of CAS was reduced by a 21 -fold (Table 3B). Dros and Tore were able to reduce the BEC50 of CAS against C. glabrata biofilms by a 4.7 and 18-fold respectively (Table 3B). Table 3. Activity of caspofungin and combinations of drospirenone, perhexiline maleate and toremiphene citrate with caspofungin against C. albicans and C. giabrata biofilms.

Example 4. Toremiphene citrate acts synergistically with caspofungin in a C. albicans worm infection assay.

To translate these in vitro findings to an in vivo infection model, we used the Caenorhabditis elegans infection assay, which is regarded as a good infection model for biofiim-associated infections. We selected the most potent combination against C. albicans biofilms based on the in vitro data, namely the Tore/CAS combination (Table 2&3). To this end, we used a concentration of CAS (0.095 μΜ) that only had a very small effect on the survival of the infected C. elegans worms compared to the control treatment (0.6 % DMSO). Subsequently, C. elegans worms were infected with C. albicans by feeding them on an YPD plate containing C. albicans and treated with 6.25 μΜ Tore or 0.095 μ CAS alone, or with 6.25 μΜ Tore and 0.095 μ CAS. Untreated worms and non-infected worms served as controls (Fig 2). Treatment of the infected worms with a combination of 6,25 μ Tore and 0.095 μΜ CAS significantly increased the survival of the worms compared to treatment with 6.25 μΜ Tore or 0.095 μΜ CAS alone or mock (0.6% DMSO) after 3, 5, and 7days of incubation. Ten days post infection, still 46.3 ± 4.7% of the worms survived after treatment with the combination of CAS and Tore. In contrast, treatment with Tore or CAS alone resulted in 16.6 ± 4.3% or 14.9 ± 1.7% surviving worms, respectively, whereas worms treated with mock (0.6% DMSO control) only 9.4 ± 5.3% of the worms survived. As there is no significant difference between treatment with Tore alone and mock (0.6% DMSO), it seems that there are no toxic side-effects apparent on the nematodes resulting from this specific Tore concentration. The non-toxicity of Tore on worms corroborates our other findings regarding the growth potential of the osteoblasts in the presence of Tore.

In conclusion, pre-incubation of C. albicans biofilms with subinhibitory concentrations of drospirenone, perhexiline maleate or toremiphene citrate significantly increased the activity of antifungal compounds against C. albicans or C. glabrata biofilms. Moreover, the compounds act synergistica!ly with said antifungal compounds (particularly CAS) against C. albicans and C. glabrata biofilms (FiCI < 0.5), while they did not affect growth potential of osteoblasts. These in vitro findings were translated to a worm C. albicans biofilm infection model and the synergy between toremiphene citrate and CAS in curing the infection was confirmed.

The above examples show that potentiation of antifungals against biofilms by combining them with compounds which act synergistica!ly with said antifungals, will allow to lower their effective dose and thus, reduce potential toxic side-effects and economic costs.

Three compounds, i.e. potentiators, that could enhance the activity of AmB and CAS against biofilms of C. albicans and C. glabrata showed synergistic activity for all three potentiators with CAS against biofilms of C. albicans and C. glabarata (Chequerboard analysis - (FICI)≤ 0.5). in several combinations, up to a 20-fold reduction of the CAS concentration necessary to eradicate 50% of the biofilm cells (BEC₅₀) of C. albicans or C. glabrata was achieved. Furthermore, these in vitro results could be translated to an C. elegans biofilm infection model for C. albicans. Treatment of infected worms with a combination of Tore and CAS resulted in a significant better survival of the C. elegans worms compared to single treatment with Tore, CAS or DMSO (control treatment).

The three identified compounds in this study are well characterized for their application in other medical domains. Dros is a synthetic hormone used in several birth control pil!s in combination with ethinylestradiol. Perhex has been used clinically as an anti-anginal agent for over 25 years. Finally, Tore is a selective estrogen receptor modulator (SER ), which binds to estrogen receptors (ERs). Interestingly, other selective estrogen receptor modulators (SERMs) have been identified as potentiators of fluconazole against pianktonic cultures of the yeast Saccharomyces cerevisiae (Spitzer et al., 2012), namely tamoxifen and clomiphene, while they act only in an additive way with fluconazole against C. albicans pianktonic cultures. The SERMs tamoxifen, toremiphene and clomiphene are triphenylethy!enes (Goldestein et al., 2000). Tamoxifen and toremiphene differ by a single chloride ion (Hirsimaki et al., 2002). Antifungal activity of tamoxifen has been reported for more than 20 years (Wiseman et ai., 1989; Beggs et al., 1993&1994; Dolan et al., 2009). The antifungal activity of tamoxifen is based on its membrane perturbing effects (Wiseman et al., 1993; Parsons et a!., 2006; Dolan et al., 2009). We have tested putative membrane perturbing activity of toremiphene against C. albicans biofilms, using the probe propidium iodide, and could not detect membrane perturbation induced by toremiphene in this setup. These data indicate that the toremiphene-induced potentiation of CAS against C. albicans biofilms is not attributed to its antifungal mode of action based on membrane perturbation. This is in line with our experimental setup in which we use subinhibitory concentrations for assessing potentiation. Taken together, all these data indicate that potentiation can be species-specific, and biofilm-specific.

Claims

1. A composition for use in the treatment or prevention of a fungal biofilm associated condition in a human or animal subject, said composition comprising at feast one of the compounds selected from the group consisting of drospirenone, toremiphene (citrate), perhexiline (maleate) and a derivative of any of the preceeding compounds, and at least one antifungal agent selected from the group consisting of polyenes and echinocandins.

2. The composition of claim 1 , wherein said at least one antifungal agent is amphotericin B or caspofungin,

3. The composition of claims 1 or 2, wherein said fungal biofilm is a Candida biofilm,

4. The composition of any one of the claims 1 to 3, further comprising one or more physiologically acceptable compounds, carriers and/or adjuvants,

5. The composition of any one of the claims 1 to 4, wherein said subject has been implanted with a medical device, which is infected or at risk of being infected with a funga! biofilm.

6. The composition of claim 5 wherein said medical device is selected from the group consisting of catheters, stents, surgical plates, prostheses, valves or pins, artificial joints, pacemakers, contact lenses and bio-implants.

7. An implant comprising on at least part of its surface a composition comprising at least one of the compounds selected from the group consisting of drospirenone, toremiphene (citrate), perhexiline (maleate) and a derivative of any of the preceeding compounds, and at least one antifungal agent selected from the group consisting of polyenes and echinocandins.

8. The implant of claim 7, wherein said at least one antifungal agent is amphotericin B or caspofungin,

9. The implant of claims 7 or 8, wherein at least part of the surface of said implant is coated with said composition.

10. The implant of any one of the claims 7 to 9, wherein said implant comprises an internal cavity or a reservoir comprising said composition, wherein said reservoir or internal cavity is connected to at least part of the surface of said implant in order to allow the transport of said composition towards the connected surface.

11. The implant of any one of the claims 7 to 10, wherein said implant is a medical device selected from the group consisting of catheters, stents, surgical plates, prostheses, valves or pins, artificial joints, pacemakers, contact lenses and bio-implants.

12. A method for reducing, eradicating, inhibiting or preventing fungal biofilms or fungal biofilm formation, characterized in that a surface or medium outside the body of a human or animal subject carrying said fungal biofilm or susceptible to said fungal biofilm formation, is treated with at least one compound selected from the group consisting of drospirenone, toremiphene (citrate), perhexiline (maieate) and a derivative of any of the preceeding compounds, and at least one antifungal agent selected from the group consisting of polyenes and echinocandins.

13. The method of claim 12 wherein said at least one antifungal agent is amphotericin B or caspofungin,

14. The method of any one of claims 12 or 13 wherein said biofilm is exposed to said at least one compound before, after or concurrent with exposing said biofilm to said antifungal agent.

15. The method of any one of claims 12 to 14 wherein said fungal biofilm is a Candida biofilm.

16. A method for the treatment of infections involving fungal biofilms in a human or animal subject, said method comprising administering a composition comprising at least one of the compounds selected from the group consisting of drospirenone, toremiphene (citrate), perhexiline (maieate) and a derivative of any of the preceeding compounds, and at least one antifungal agent selected from the group consisting of polyenes and echinocandins.

17. The method of claim 16 wherein said at least one antifungal agent is amphotericin B or caspofungin.

18. The method of any one of claims 16 or 17 wherein said fungal biofilm is a Candida biofilm.

19. A composition for use in reducing, eradicating, inhibiting or preventing fungai biofilms or fungai biofilm formation, comprising at least one of the compounds selected from the group consisting of drospirenone, toremiphene (citrate), perhexiline (maieate) and a derivative of any of the preceeding compounds, and at least one antifungal agent selected from the group consisting of polyenes and echinocandins.

20. The composition of claim 19, wherein said at least one antifungal agent is amphotericin B or caspofungin.