Compositions for use in the treatment of bacterial vaginosis

Abstract

The present invention provides a composition comprising an imidazole or a salt thereof, and a quaternary ammonium compound for use in the treatment and / or prevention of a bacterial vaginosis. The present invention also provides a vaginal composition and a kit comprising said vaginal composition and an applicator for topically applying the vaginal composition to the vagina of a subject.

Description

[0001] COMPOSITIONS FOR USE IN THE TREATMENT OF BACTERIAL VAGINOSIS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to vaginal compositions and their use in the treatment and / or prevention of bacterial vaginosis, in particular bacterial vaginosis caused by pathogens of the Phylum Actinomycetota, such as Atopobium vaginae and Gardnerella vaginalis.

[0004] BACKGROUND OF THE INVENTION

[0005] Mucosal biofilm-related bacterial infections are very common and the incidence of recurrent bacterial vaginosis is significant. Bacterial vaginosis (BV) is characterized by a change in the microbial composition of the vagina: the Lactobacillus spp., associated with a healthy vaginal microbiome, are outnumbered by microaerophilic and anaerobic organisms, including Gardnerella vaginalis and Atopobium vaginae. Various compounds have been described to combat Gardn ere 11 a / A topobium i n f ect i o n s .

[0006] Due to its strong adherence to vaginal epithelial cells and biofilm-forming capacities, it has been suggested that G. vaginalis initiates the colonisation of the vaginal epithelium and serves as a scaffolding to which other species like A. vaginae can then attach. Moreover, it has also been suggested that A. vaginae plays a major part in the establishment of a biofilm, together with G. vaginalis.

[0007] Current treatment options for bacterial vaginosis are limited and discontinuation of therapy often results in recurrence of the bacterial vaginosis. Moreover, some Gardnerella / Atopobium strains have been shown to be resistant to some of the current therapies.

[0008] In view thereof, there remains a need to develop further therapies for bacterial vaginosis.

[0009] SUMMARY OF THE INVENTION

[0010] Present inventors found that the specific combination of an imidazole or a salt thereof and a quaternary ammonium compound allows for the treatment and / or prevention of bacterial vaginosis. More particularly, present inventors have identified a synergy between imidazoles like miconazole or metronidazole, and quaternary ammonium compounds, like domiphen bromide or dequalinium chloride, on biofilm eradication of bacterial species belonging to the Phylum Actinomycetota, preferably G. vaginalis and / or A. vaginae, which are typically involved in bacterial vaginosis.

[0011] Accordingly, a first aspect provides a composition comprising an imidazole or a salt thereof, and a quaternary ammonium compound for use in the treatment (i.e. treatment and / or prevention) of a bacterial vaginosis in a subject, wherein the imidazole is miconazole or metronidazole, and wherein the quaternary ammonium compound is domiphen bromide or dequalinium chloride.

[0012] In particular embodiments, the bacterial vaginosis is caused by one or more bacteria of the Phylum Actinomycetota. In further particular embodiments, the one or more bacteria of the Phylum Actinomycetota comprise Atopobium vaginae (A. vaginae) and / or Gardnerella vaginalis (G. vaginalis).

[0013] In particular embodiments, the subject has been diagnosed with a vaginal biofilm predominantly formed by one or more bacteria of the Phylum Actinomycetota; preferably by Atopobium vaginae and / or Gardnerella vaginalis.

[0014] In particular embodiments of the embodiments described above, the composition comprises a molar excess of the imidazole or a salt thereof over the quaternary ammonium compound of 0.02 to 21. More particularly, the composition comprises from 0.5 to 5 % (w / w) of imidazole or salt thereof.

[0015] In particular embodiments of the invention, such as in the embodiments described above, the imidazole or a salt thereof and the quaternary ammonium compound are the only active ingredients in said composition.

[0016] Irrespective of the above, in particular embodiments, the composition may further comprise a mucoadhesive, such as a mucoadhesive selected from the group consisting of synthetic polycarbophil, chitosan, cellulose derivatives, pectin, hyaluronic acid derivatives, polyacrylates, tragacanth, carrageenan, sodium alginate, thiolated polymers, or combinations thereof.

[0017] Irrespective of the above-described features of the composition, in particular embodiments, the composition has a pH from 2 to 4, preferably from 2.75 to 3.5. In particular embodiments, the composition is a pharmaceutical composition, and optionally further comprises one or more pharmaceutically acceptable carriers. The composition as described with its different embodiments herein can be in a form suitable for topical application, preferably a gel or a cream.

[0018] In particular embodiments, the composition as described herein above is administered topically to the vagina. The application also provides, related thereto a vaginal composition comprising metronidazole or a salt thereof, and a quaternary ammonium compound, wherein the quaternary ammonium compound is domiphen bromide or dequalinium chloride, and wherein the vaginal composition has a pH from 2 to 4, preferably from 2.75 to 3.5.

[0019] The application also provides, related to the above, a kit comprising the vaginal composition as taught herein and an applicator for topically applying the vaginal composition to the vagina.

[0020] BRIEF DESCRIPTION OF DRAWINGS

[0021] Fig. 1. Dequalinium chloride shows synergistic activity with miconazole and metronidazole against Atopobium vaginae biofilms. Average FICI (Z FIC) for different imidazole - quaternary ammonium compound combinations against A topobium vaginae biofilms and more particularly for MET and DOM in a ratio of 9.4: 1, MET and DEQ in a ratio of 0.4: 1, MIC and DOM in a ratio of 3.8 to 1 and MIC and DEQ in a ratio of 10.5 to 1. The FICI was calculated by the formula FICI=[CBECA / BECA] + [C(BECB) / BECB], in which C(BECA) and C(BECB) are the BEC (biofilm eradication concentration) values of the antimicrobial drugs in combination, and BECA and BECB are the BEC values of antimicrobial drugs A and B alone. Synergy is defined as a FICI< 1. MIC- miconazole, MET-metronidazole, DOM-domiphen bromide, DEQ-dequalinium chloride.

[0022] Fig. 2. Miconazole shows synergistic activity with domiphen bromide and dequalinium chloride against Gardnerella vaginalis biofilms. Average FICI (Z FIC) for different imidazole - quaternary ammonium compound combinations against Gardnerella vaginalis biofilms and more particularly for MET and DOM in a ratio of 2.3: 1, MET and DEQ in a ratio of 2.2: 1, MIC and DOM in a ratio of 3.9 to 1 and MIC and DEQ in a ratio of 0.1 to 1. The FICI was calculated by the formula FICI=[CBECA / BECA] + [C(BECB) / BECB], in which C(BECA) and C(BECB) are the BEC (biofilm eradication concentration) values of the antimicrobial drugs in combination, and BECA and BECB are the BEC values of antimicrobial drugs A and B alone. Synergy is defined as a FICI< 1. Abbreviations: MIC- miconazole, MET- metronidazole, DOM-domiphen bromide, DEQ-dequalinium chloride.

[0023] DETAILED DESCRIPTION OF THE INVENTION

[0024] The term "about", when used in relation to a numerical value, has the meaning generally understood in the relevant art. In certain embodiments the term "about" may be left out or may be interpreted to mean the numerical value +10%; or + 5%; or +2%; or +1%.

[0025] Whenever used herein in relation to a percentage, w / w means weight / weight and w / v means weight / volume.

[0026] As used herein, the singular forms "a", "an", and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0027] The terms "comprising", "comprises" and "comprised of" as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms "comprising", "comprises" and "comprised of" when referring to recited members, elements or method steps also include embodiments which "consist of" said recited members, elements or method steps.

[0028] Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements or steps and not necessarily for describing a sequential or chronological order, unless specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments described herein are capable of operation in other sequences than described or illustrated herein.

[0029] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment envisaged herein. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

[0030] Any references cited herein are hereby incorporated by reference. Bacterial vaginosis is a condition defined by a shift from Lactobacillus dominance to a polymicrobial, anaerobic bacterial community. Currently, metronidazole monotherapy may be used as a first line treatment of the condition. As a pro-drug, metronidazole is reduced to its active state under anaerobic conditions - a mechanism that spares beneficial Lactobacillus. However, the complete removal of bacterial vaginosis-associated bacteria is likely complicated by the presence of biofilms, which shield bacterial vaginosis-associated bacteria by preventing metronidazole penetration. As a result thereof, bacterial vaginosis recurrence rates are high after metronidazole monotherapy.

[0031] Present inventors have now found that the combination of an imidazole or a salt thereof and a quaternary ammonium compound allows to treat and / or prevent bacterial vaginosis very effectively. More particularly, present inventors have shown that an imidazole or salt thereof, like miconazole or metronidazole, in combination with a quaternary ammonium compound, like domiphen bromide or dequalinium chloride, displays synergistic antibiofilm eradication activity on biofilms of bacteria belonging to the Phylum Actinomycetota, preferably G. vaginalis and / or A. vaginae, based on determination of the fractional inhibitory concentration indices (FIC). FICs for bactericidal activity under anaerobiosis against mature biofilms may be determined according to the definition of Meletiadis (Meletiadis J, Pournaras S, Roilides E, Walsh TJ. Defining fractional inhibitory concentration index cutoffs for additive interactions based on self-drug additive combinations, Monte Carlo simulation analysis, and in vitro-in vivo correlation data for antifungal drug combinations against Aspergillus fumigatus. Antimicrob Agents Chemother. 2010 Feb;54(2):602-9. ) with FIC cut-off < 1, which is based on in vitro-in vivo correlation.

[0032] Accordingly, a first aspect provides a composition comprising, consisting essentially of or consisting of: an imidazole or a salt thereof, and a quaternary ammonium compound for use in the treatment (i.e. treatment and / or prevention, as described elsewhere herein) of bacterial vaginosis in a subject. In other words, provided herein is a method for treating (i.e. treating and / or preventing) bacterial vaginosis in a subject comprising administering a composition comprising, consisting essentially of or consisting of an imidazole or a salt thereof, and a quaternary ammonium compound to said subject, thereby treating said bacterial vaginosis. Also provided herein is the use of a composition comprising, consisting essentially of or consisting of an imidazole or a salt thereof, and a quaternary ammonium compound in the manufacture of a medicament for the treatment (i.e. treatment and / or prevention) of bacterial vaginosis.

[0033] The composition may comprise a synergistically, therapeutically and / or prophylactically effective amount, such as a synergistically and therapeutically effective amount, of the imidazole or a salt thereof and the quaternary ammonium compound.

[0034] The term "bacterial vaginosis" or "BV" as used herein refers to a disease or condition resulting from an imbalance in the bacterial flora in the vagina, which may lead to one or more of the following symptoms: pain, itching, burning sensation, discomfort and abnormal vaginal discharge. A healthy or normal vaginal microbiome is typically dominated by Lactobacillus which produce various antimicrobial compounds. Bacterial vaginosis is characterized by the loss or sharp decline in the total number of Lactobacillus and a corresponding marked increase in the concentration of anaerobic bacteria. A number of potential microbial pathogens, singly and in combinations, have been implicated in the disease process., such as G. vaginalis, A. vaginae, Mobiluncus mulieris, Prevotella bivia, Fusobacterium nucleatum, and / or Peptoniphilus species. Species of the Phylum Actinomycetota, such as G. vaginalis and A. vaginae, are typically associated with the initiation of bacterial vaginosis. Due to its strong adherence to vaginal epithelial cells and biofilm-forming capacities, it has been suggested that G. vaginalis initiates the colonisation of the vaginal epithelium and serves as a scaffolding to which other species like A. vaginae can then attach, resulting in the formation of a biofilm. G. vaginalis is typically the predominant species of the biofilm. Other pathogens, such as Prevotella spp. and Mobiluncus spp., may act as secondary intruders. Bacterial vaginosis may be diagnosed by any means known in the art. For example, bacterial vaginosis may be diagnosed by microscopy. The presence of clue cells in vaginal discharge of the subject is diagnostic of bacterial vaginosis. Clue cells are epithelial cells that are so extensively coated by cocci bacteria that the cell membranes of the epithelial cells are obstructed from view.

[0035] In particular embodiments of the compositions as envisaged herein, the bacterial vaginosis is caused by one or more bacteria of the Phylum Actinomycetota. Within the meaning of present invention, bacterial vaginosis "caused by" one or more bacteria of the Phylum Actinomycetota refers to bacterial vaginosis caused by biofilm formation by one or more bacteria of the Phylum Actinomycetota. In particular embodiments, the one or more bacteria of the Phylum Actinomycetota comprise, consist essentially of or consist of A. vaginae and / or G. vaginalis. Accordingly, in particular embodiments, the bacterial vaginosis may be caused by A. vaginae and / or G. vaginalis.

[0036] The bacterial vaginosis as referred to herein may, in some embodiments, predominantly be caused by A. vaginae and / or G. vaginalis bacteria, or in other words, in particular embodiments, the biofilm formed upon bacterial vaginosis is predominantly formed by A. vaginae and / or G. vaginalis bacteria.

[0037] The terms "treat" or "treatment" encompass both the therapeutic treatment of an already developed disease or condition, such as the therapy of an already developed bacterial vaginosis, as well as prophylactic or preventive measures, wherein the aim is to prevent or lessen the chances of incidence of an undesired affliction, such as to prevent occurrence, development and progression of bacterial vaginosis. Beneficial or desired clinical results may include, without limitation, alleviation of one or more symptoms or one or more biological markers, diminishment of extent of disease, stabilised (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and the like.

[0038] The composition for vaginal administration as taught herein may be administered to the subject in a synergistically, therapeutically and / or prophylactically effective amount, such as a therapeutically and / or prophylactically effective. The term "therapeutically effective amount" as used herein, refers to an amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a subject that is being sought by a surgeon, researcher, veterinarian, medical doctor or other clinician, which may include inter alia alleviation of the symptoms of the disease or condition being treated. The term "prophylactically effective amount" refers to an amount of an active compound or pharmaceutical agent that inhibits or delays in a subject the onset of a disorder as being sought by a researcher, veterinarian, medical doctor or other clinician. Methods are known in the art for determining therapeutically and / or prophylactically effective doses of the composition for vaginal administration as taught herein.

[0039] The synergistic effect of the combination of an imidazole or a salt thereof, and a quaternary ammonium compound found by present inventors includes a synergistic bactericidal activity (e.g. biofilm eradication) against bacterial vaginosis-causing bacteria. A biofilm is a mode of microbial growth comprising sessile cells, usually within a complex and highly heterogeneous matrix of extracellular polymers, and is characterized by a reduced sensitivity to antibacterial agents. Biofilms can contain single species (e.g. a bacterium such as A. vaginae) or multiple species microorganisms (such as A. vaginae, G. vaginalis and other microorganisms, like Prevotella spp. and Mobiluncus spp. or even yeasts and / or fungi).

[0040] Accordingly, in particular embodiments, the treatment of bacterial vaginosis as taught herein encompasses a complete or partial reduction of the bacteria causing the bacterial vaginosis.

[0041] In further particular embodiments, the composition for vaginal administration as taught herein prevents and / or inhibits biofilm formation, and / or eradicates a biofilm. Preferably, the biofilm is a bacterial biofilm, more preferably a biofilm formed by species of the Phylum Actinomycetota like G. vaginalis and / or A. vaginae, even more preferably a biofilm formed by A. vaginae. In particular embodiments, the method of treatment (i.e. treatment and / or prevention) comprises a step of administering an effective amount of the composition for vaginal administration as taught herein sufficient to prevent and / or inhibit biofilm formation by species of the Phylum Actinomycetota like G. vaginalis and / or A. vaginae, and / or eradicating a biofilm formed by species of the Phylum Actinomycetota like G. vaginalis and / or A. vaginae.

[0042] Except when noted, the terms "subject" or "patient" can be used interchangeably and refer to animals, preferably warm-blooded animals, more preferably vertebrates, even more preferably mammals, still more preferably primates, and specifically includes human patients and non-human mammals and primates. Preferred subjects are human subjects. The terms "subject" or "patient" include subjects in need of treatment, more particularly subjects that would benefit from treatment of a given condition, particularly bacterial vaginosis. Such subjects may include, without limitation, those that have been diagnosed with said condition, those prone to develop said condition and / or those in who said condition is to be prevented. In particular embodiments, the subject has been diagnosed with bacterial vaginosis. In particular embodiments, the subject has been diagnosed with a biofilm, such as a vaginal biofilm, preferably a vaginal biofilm predominantly formed (or predominantly consisting of), such as by at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, such as at least 96%, at least 97%, at least 98%, at least 99%, or 100%, by one or more bacteria of the Phylum Actinomycetota. In particular embodiments, the subject has been diagnosed with a vaginal biofilm predominantly formed (or predominantly consisting of), such as by at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, such as at least 96%, at least 97%, at least 98%, at least 99%, by A. vaginae and / or G. vaginalis. In particular embodiments, the subject is a female subject, preferably a woman.

[0043] The imidazole used in the composition for vaginal administration as taught herein may be any type of imidazole known in the art or salts thereof, including nitroimidazoles. Non-limiting examples of imidazoles include miconazole, clotrimazole, ketoconazole and metronidazole. In particular embodiments, the imidazole or salt thereof is miconazole or a salt thereof, or metronidazole or a salt thereof, or clotrimazole or a salt thereof, or ketoconazole or a salt thereof.

[0044] The quaternary ammonium compound used in the composition for vaginal administration as taught herein may be any quaternary ammonium compound known in the art, including mono-quaternary ammonium compounds, such as domiphen bromide, benzalkonium chloride and cetrimonium chloride, and bisquaternary ammonium compounds, such as dequalinium chloride and benzethonium chloride, but not being limited thereto.

[0045] In particular embodiments and irrespective of the other features of the composition, the imidazole or salt thereof is miconazole or a salt thereof, or metronidazole or a salt thereof.

[0046] The term "miconazole" as used herein refers to the compound (l-[2-(2,4- dichlorofenyl)-2-[(2,4-dichlorofenyl) methoxy] ethyl] imidazole) and is identified by CAS Registry Number 22916-47-8. Miconazole is an antifungal compound that interferes with the metabolism of ergosterol, a component of the yeast cell membrane. Miconazole is preferably formulated as its nitrate salt in the composition for vaginal administration as taught herein.

[0047] Accordingly, in particular embodiments, the imidazole or salt thereof is miconazole nitrate.

[0048] The term "metronidazole" as used herein refers to the compound 2-(2-methyl-5- nitroimidazol-l-yl)ethanol and is identified by CAS Registry Number 443-48-1. Metronidazole is a commonly used antibiotic, belonging to the nitroimidazole class of antibiotics. Metronidazole is a nitroimidazole, which is a prodrug that is reductively activated (i.e. the nitro-group is reduced) under low oxygen tension, leading to imidazole fragmentation and cytotoxicity. In particular embodiments, the quaternary ammonium compound is domiphen bromide or dequalinium chloride.

[0049] The term "domiphen bromide" as used herein refers to the compound [N,N- dimetyl-N-(2-phenoxyetyl)-, bromide] and is identified by CAS Registry Number 538-71-6. Domiphen bromide is a quaternary ammonium salt with antiseptic properties.

[0050] The term "dequalinium chloride" as used herein refers to the compound l-[10-(4- amino-2-methylqu inolin- 1-ium- 1-y l)decy I] -2-methylqu inolin -1-ium -4- amine;dichloride and is identified by CAS Registry Number 522-51-0. Dequalinium chloride is a quaternary ammonium cation antimicrobial agent used to treat common infections of the mouth and throat.

[0051] In further particular embodiments, the imidazole or salt thereof is miconazole or a salt thereof, or metronidazole or a salt thereof and the quaternary ammonium compound is domiphen bromide or dequalinium chloride.

[0052] In further particular embodiments of the invention as described above, the imidazole or salt thereof is metronidazole or salt thereof and the quaternary ammonium compound is domiphen bromide; the imidazole or salt thereof is metronidazole or salt thereof and the quaternary ammonium compound is dequalinium chloride; the imidazole or salt thereof is miconazole or salt thereof, preferably the nitrate salt thereof, and the quaternary ammonium compound is domiphen bromide; or the imidazole or salt thereof is miconazole or salt thereof, preferably the nitrate salt thereof, and the quaternary ammonium compound is dequalinium chloride.

[0053] Present inventors have found that the combination of miconazole or a salt thereof and dequalinium chloride has bacterididal activity against mature biofilms of both G. vaginalis and A. vaginae with a FIC of less than 0.5.

[0054] In particular embodiments, the molar excess of the imidazole or a salt thereof over the quaternary ammonium compound is from 0.02 to 21 or from 0.03 to 11, preferably from 0.4 to 10. In other words, in particular embodiments, the composition comprises a molar excess of the imidazole or a salt thereof over the quaternary ammonium compound of 0.02 to 21, or of 0.03 to 11, preferably of 0.4 to 10.

[0055] In particular embodiments, if the imidazole or salt thereof is metronidazole or salt thereof and the quaternary ammonium compound is domiphen bromide, metronidazole or salt thereof is present in the composition for vaginal administration as taught herein in a molar excess of from 3 to 11, preferably from 4 to 10, over the amount of domiphen bromide. In further particular embodiments, if the bacterial vaginosis is predominantly caused by Atopobium vaginae and if the imidazole or salt thereof is metronidazole or salt thereof and the quaternary ammonium compound is domiphen bromide, metronidazole or salt thereof is present in the composition for vaginal administration as taught herein in a molar excess of from 4 to 10, preferably from 4.4 to 9.4, more preferably from over the amount of domiphen bromide.

[0056] In particular embodiments, if the imidazole or salt thereof is metronidazole or salt thereof and the quaternary ammonium compound is dequalinium chloride, metronidazole or salt thereof is present in the composition for vaginal administration as taught herein in a molar excess of from 0.1 to 12.5, from 0.1 to 0.5, from 0.2 to 12.5 or from 0.2 to 0.5, preferably from 0.2 to 0.4, over the amount of dequalinium chloride. In further particular embodiments, if the bacterial vaginosis is predominantly caused by Atopobium vaginae and if the imidazole or salt thereof is metronidazole or salt thereof and the quaternary ammonium compound is dequalinium chloride, metronidazole or salt thereof is present in the composition for vaginal administration as taught herein in a molar excess of from 0.2 to 12.5, preferably from 0.2 to 0.4, over the amount of dequalinium chloride. In particular embodiments, if the imidazole or salt thereof is miconazole or salt thereof and the quaternary ammonium compound is domiphen bromide, miconazole or salt thereof is present in the composition for vaginal administration as taught herein in a molar excess of from 0.2 to 9, preferably from 0.3 to 8, over the amount of domiphen bromide. In further particular embodiments, if the bacterial vaginosis is predominantly caused by Atopobium vaginae and if the imidazole or salt thereof is miconazole or salt thereof and the quaternary ammonium compound is domiphen bromide, miconazole or salt thereof is present in the composition for vaginal administration as taught herein in a molar excess of from 1.5 to 4, preferably from 1.8 to 3.8, such as about 2, about 2.5, about 3, about 3.5 or about 4, over the amount of domiphen bromide. Thus, by means of example, a composition for vaginal administration as taught herein with a 3- fold molar excess comprising a 10 millimolar domiphen bromide comprises 30 millimolar miconazole nitrate. In further particular embodiments, if the bacterial vaginosis is predominantly caused by Gardnerella vaginalis and if the imidazole or salt thereof is miconazole or salt thereof and the quaternary ammonium compound is domiphen bromide, miconazole or salt thereof is present in the composition for vaginal administration as taught herein in a molar excess of from 0.2 to 9, preferably from 0.3 to 8, over the amount of domiphen bromide.

[0057] In particular embodiments, if the imidazole or salt thereof is miconazole or salt thereof and the quaternary ammonium compound is dequalinium chloride, miconazole or salt thereof is present in the composition for vaginal administration as taught herein in a molar excess of from 0.02 to 21, from 0.03 to 21, from 0.02 to 12, or from 0.03 to 12, preferably from 0.03 to 11, over the amount of dequalinium chloride. In further particular embodiments, if the bacterial vaginosis is predominantly caused by Atopobium vaginae and if the imidazole or salt thereof is miconazole or salt thereof and the quaternary ammonium compound is dequalinium chloride, miconazole or salt thereof is present in the composition for vaginal administration as taught herein in a molar excess of from 0.05 to 21, from 0.05 to 11, from 0.1 to 21, preferably from 0.1 to 10.5, over the amount of dequalinium chloride. In further particular embodiments, if the bacterial vaginosis is predominantly caused by Gardnerella vaginalis and if the imidazole or salt thereof is miconazole or salt thereof and the quaternary ammonium compound is dequalinium chloride, miconazole or salt thereof is present in the composition for vaginal administration as taught herein in a molar excess of from 0.02 to 5, preferably from 0.03 to 4, over the amount of dequalinium chloride.

[0058] In particular embodiments, the composition for vaginal administration as taught herein comprises from 0.5 % (w / w) to 5% (w / w), preferably from 1 % (w / w) to 5% (w / w), of imidazole or a salt thereof. For example, the composition for vaginal administration as taught herein may comprise about 1% (w / w), about 1.5% (w / w), about 2% (w / w), about 2.5% (w / w), about 3% (w / w), about 4%, or about 5% (w / w) imidazole or a salt thereof. In further particular embodiments, if the imidazole or salt thereof is metronidazole or salt thereof, the composition for vaginal administration as taught herein comprises from 0.5% (w / w) to 2% (w / w) or from 0.5% (w / w) to 1% (w / w), more preferably about 0.75% (w / w) of miconazole or a salt thereof. In further particular embodiments, if the imidazole or salt thereof is miconazole or a salt thereof, preferably miconazole nitrate, the composition for vaginal administration as taught herein comprises from 1% (w / w) to 3% (w / w), more preferably about 2% (w / w) of miconazole or a salt thereof.

[0059] In particular embodiments, the imidazole or a salt thereof and the quaternary ammonium compound are the only active ingredients, the only bactericidal ingredients, and / or the only antimicrobial ingredients in said composition for vaginal administration as taught herein. However, the composition for vaginal administration as taught herein may comprise one or more further antimicrobial agents, such as one or more antimicrobial agents which have a bactericidal effect on one or more bacteria of the Phylum Actinomycetota, preferably Atopobium vaginae and / or Gardnerella vaginalis.

[0060] In particular embodiments, the composition for vaginal administration as taught herein is a pharmaceutical composition, and optionally further comprises one or more pharmaceutically acceptable carriers.

[0061] The term "pharmaceutically acceptable" as used herein is consistent with the art and means compatible with the other ingredients of a pharmaceutical composition and not deleterious to the recipient thereof.

[0062] As used herein, "carrier" or "excipient" includes any and all solvents, diluents, buffers (such as, e.g., neutral buffered saline or phosphate buffered saline), solubilisers, colloids, dispersion media, vehicles, fillers, chelating agents (such as, e.g., EDTA or glutathione), amino acids (such as, e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavourings, aromatisers, thickeners, agents for achieving a depot effect, coatings, antifungal agents, preservatives, antioxidants, tonicity controlling agents, absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active substances, its use in the therapeutic compositions may be contemplated.

[0063] The compositions for vaginal administration as taught herein are preferably used topically and formulated to be compatible with the specific site of application (e.g. skin, mucosa). Accordingly, in particular embodiments, the composition for vaginal administration as taught herein is in a form suitable for topical application, preferably for topical application to the vagina, more particularly for topical application to the vaginal mucosa.

[0064] The compositions for vaginal administration as taught herein may include additives typically used for such forms including, but not limited to, binders, fillers, carriers, preservatives, stabilizing agents, emulsifiers, buffers and excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, or magnesium carbonate.

[0065] In particular embodiments, the composition for vaginal administration as taught herein is in the form of a solution, suspension (e.g. for use as a vaginal douche or sprays), gel (e.g. vaginal suppository gel), a capsule (e.g. vaginal capsule), a cream (e.g. vaginal cream), salve, tincture, lotion, pessary, transdermal patch, ointment, lubricant, or a tablet (e.g. a vaginal tablet). The composition for vaginal administration as taught herein can also be impregnated in a towelette or sponge. The composition for vaginal administration as taught herein may also be in the form of a sustained-release preparation.

[0066] Formulation of the composition for vaginal administration as taught herein as a gel or cream allows at the one hand the easy removal of the composition from a tube or syringe and on the other hand ensures that the composition remains attached at the site of application. Accordingly, in a preferred embodiment, the composition for vaginal administration as taught herein is in the form of a cream or a gel. A cream is typically an oil in water (o / w) or water in oil (w / o) emulsions, and may further comprise an emulsifier and / or a thickener. Gels are typically transparent preparations and may comprise cellulose ethers or carbomer in water or a water-alcohol mixture.

[0067] Since quaternary ammonium compounds are highly soluble in water and imidazoles and its salts are insoluble in water, the composition for vaginal administration as taught herein is preferably in the form of a cream further comprising one or more emulsifying agents. In particular embodiments, the composition for vaginal administration as taught herein may be in the form of an emulsion. Various oil / in water emulsions are known in the art. In the composition for vaginal administration as taught herein, liquid paraffin and / or lauroyl macrogol-6 glycerides are preferably used as oily phase. To stabilise the emulsion emulsifiers, such as for example a mixture of polyethylene glycol-6, palmitostearate, ethylene glycol stereate and PEG-32, stereate can be used.

[0068] To increase the viscosity of the composition for vaginal administration as taught herein, such as when the composition for vaginal administration as taught herein is in the form of an emulsion, one or more thickening agents such as cetostearyl may be used. Viscosity in the context of the present invention refers to resistance to gradual deformation by shear stress. Viscosity can be measured by any means known in the art, such as using a Brookfield HA viscometer.

[0069] For optimal attachment to the site of application (e.g. the vagina) the composition for vaginal administration as taught herein may further comprise one or more mucoadhesives. Suitable mucoadhesives for vulvovaginal application include polymers that are capable of forming hydrogels such as synthetic polycarbophil, chitosan, cellulose derivatives (hydroxyethycellulose, hydroxypropylcellulose and hydroxypropylmethylcellulose), pectin, hyaluronic acid derivatives, polyacrylates, tragacanth, carrageenan and sodium alginate, thiolated polymers. Accordingly, in particular embodiments, the composition for vaginal administration as taught herein further comprises a mucoadhesive, such as a mucoadhesive selected from the group consisting of synthetic polycarbophil, chitosan, cellulose derivatives, pectin, hyaluronic acid derivatives, polyacrylates, tragacanth, carrageenan and sodium alginate, thiolated polymers.

[0070] The pH of a healthy, normal vagina is typically acidic. However, as bacterial vaginosis reduces the amount of lactobacillus bacteria in the vagina pH levels increase. Preferably, the composition for vaginal administration as taught herein is acidic, so that it matches the pH of healthy, normal vagina. Accordingly, in particular embodiments, the composition for vaginal administration as taught herein has a pH from 2 to 4, preferably from 2.75 to 3.5. The composition for vaginal administration as taught herein may be made acidic by any means known in the art, such as by inclusion of benzoic acid, HCI or lactic acid.

[0071] In particular embodiments, the composition as taught herein is administered topically to the vagina. In other words, in particular embodiments, the method of treating bacterial vaginosis may comprise a step of administering the composition as taught herein topically to the vagina. The composition for vaginal administration as taught herein may be administered by any suitable means, for example by use of a vaginal cream applicator, such as the Gentle Dose® cavity applicator of Biosrx (SKU : GD-40). In particular embodiments, the composition as taught herein is administered to the infected region in the vagina.

[0072] The pharmaceutical applications as taught herein can be used as a prophylactic, but are typically used upon signs or symptoms of a bacterial vaginosis.

[0073] The duration of administration of the composition for vaginal administration as taught herein to the subject can extend over several days or longer, depending on the condition. For example, the administration may be continued until the symptoms of bacterial vaginosis are sufficiently reduced or eliminated. The progress of this therapy may be easily monitored by conventional techniques and assays. In particular embodiments, the composition for vaginal administration as taught herein may be administered over a period of at least 7, at least 14 or at least 21 consecutive days. In particular embodiments, the composition for vaginal administration as taught herein is administered every two days, daily, twice a day or three times a day. .

[0074] In particular embodiments, the composition for vaginal administration as taught herein is used for the treatment of recurrent bacterial vaginosis. Recurrent bacterial vaginosis within the meaning of present invention refers to the occurrence of at least four specific episodes of bacterial vaginosis in one year or the occurrence of at least three episodes of bacterial vaginosis unrelated to antibiotic therapy within one year. The compositions for vaginal administration as taught herein may be used for the treatment of a recurrent infection with pathogens of the Phylum Actinomycetota and specifically for recurrent infections with A. vaginae and / or G. vaginalis, which are less sensitive to monotherapies. The invention also provides a vaginal composition comprising an imidazole or a salt thereof, and a quaternary ammonium compound, wherein the vaginal composition has a pH from 2 to 4, preferably from 2.75 to 3.5. In particular embodiments, the imidazole is metronidazole, and the quaternary ammonium compound is domiphen bromide or dequalinium chloride. In particular embodiments, the vaginal composition comprises a synergistically, therapeutically and / or prophylactically effective amount, such as a synergistically and therapeutically effective amount, of the imidazole or a salt thereof, and the quaternary ammonium compound. The composition for vaginal administration as taught herein may be made acidic by any means known in the art, as described elsewhere herein. The features of the vaginal composition of the invention correspond to those of the compositions for vaginal administration as detailed herein above.

[0075] The invention also provides a kit comprising the vaginal composition as taught herein and an applicator for topically applying the vaginal composition to the vagina. In particular embodiments, the kit comprises a vaginal composition comprising metronidazole, and a quaternary ammonium compound wherein the quaternary ammonium compound is domiphen bromide or dequalinium chloride. The applicator may be any applicator known in the art to be suitable for topically applying a composition to the vagina, as described elsewhere herein. The features of the kit of the invention correspond to those of the vaginal composition for use in the methods as detailed herein above.

[0076] The following examples are meant to illustrate the present invention and should not be construed as a limitation of its scope. EXAMPLES

[0077] Example 1 : Quaternary ammonium compounds act synergistically with Imidazoles against biofilms of Atopobium vaginae.

[0078] Materials and methods

[0079] Strains and chemicals. A. vaginae strain BAA-55 was grown routinely on Columbia Blood Medium (2.3% peptone (International Medical Products NV); 0.1% starch (Merck Millipore), 0.5% sodium chloride (TCI Europe NV) supplemented with 5% defibrinated sheep blood) agar plates at 37° C, anaerobically. Brain Heart Infusion broth, purchased from Bio-Rad laboratories was used as overnight culture medium. New York City III broth (1.5% peptone (International Medical Products NV; 0.5% glucose (Sigma - Aldrich NV); 0.24% HEPES (Life Technologies Europe BV); 0.5% sodium chloride (TCI Europe NV) and 0.38% Yeast extract (VWR International)) was used as medium for biofilm formation. Stock solutions of miconazole (MIC) (Sigma-Aldrich) and metronidazole (MET) (Sigma-Aldrich) were prepared in DMSO (VWR International, Belgium). Domiphen bromide was purchased from Selleck Chemicals and dequalinium chloride from Sigma - Aldrich NV.

[0080] Antibiofilm screening assay. An Atopobium vaginae overnight culture, grown in BHI medium was diluted to an optical density of 0.1 (approximately 108cells) and further diluted 1 / 100 in New York City III medium. 200 pL of this suspension was added to the wells of a round bottomed microplate (TPP Techno Plastic Products AG, Switzerland). Biofilms were allowed to grow for 72h, anaerobically. Afterwards, the growth medium in all wells was discarded and combinations of imidazoles (Miconazole, metronidazole) and quaternary ammonium compounds (domiphen bromide, dequalinium chloride), two-fold diluted across rows and columns of a microplate respectively in New York City broth was added to the biofilms (DMSO background 1 %). After 24 h of anaerobic incubation at 37°C, biofilms were quantified by spot plating. To this end, biofilms were washed with PBS and thoroughly scraped off the bottom of the plate. Serial dilutions were spot plated (5 pL) on Columbia Blood agar plates and anaerobically incubated for 48 h at 37° C before determining the absence / presence of colony formation.

[0081] The fractional inhibitory concentration index (FICI) was calculated by the formula FICI=[CBECA / BECA] + [C(BECB) / BECB], in which C(BECA) and C(BECB) are the BEC values of the antimicrobial drugs in combination, and BECA and BECB are the BEC values of antimicrobial drugs A and B alone. BEC stands for biofilm eradication concentration which is the minimal concentration of a compound for which no colony growth is observed after spot plating. The interaction was defined as synergistic for a value of FICI < 1, according to Meletiadis J, et al. Defining fractional inhibitory concentration index cutoffs for additive interactions based on self-drug additive combinations, Monte Carlo simulation analysis, and in vitro-in vivo correlation data for antifungal drug combinations against Aspergillus fumigatus. Antimicrob Agents Chemother. 2010 Feb;54(2):602-9.

[0082] Results.

[0083] To determine whether quaternary ammonium compounds (domiphen bromide, dequalinium chloride) act synergistically with imidazoles (metronidazole and miconazole) against A. vaginae biofilms, checkerboard experiments and FICI calculations were performed (Fig. 1). The checkerboard data show that, when combined, lower concentrations of imidazole and quaternary ammonium compound can be used to eradicate biofilms of A. vaginae.

[0084] Table 1. Synergistic imidazole - domiphen bromide combinations against A. vaginae biofilms

[0085] *FICI calculations are based on the formula FICI = [C(BECA) / BECA] + [C(BECB) / BECB], in which C(BECA) and C(BECB) are the BEC values of the imidazoles and domiphen bromide in combination and BECA and BECB are the BEC values of metronidazole (250 pM) or Miconazole (100 pM) and domiphen bromide (50 pM) alone. ## synergistic values

[0086] For concentrations of 25 pM and 12.5 pM domiphen bromide, synergy was observed (FICI < 1) in combination with both imidazoles (i.e. miconazole or metronidazole) against A. vaginae biofilms (Table 1). Synergistic combinations were found for combinations of domiphen bromide and metronidazole in a ratio of 1 / 4.4 to 1 / 9.4 and for domiphen bromide and miconazole in a ratio of 1 / 1.8 to 1 / 3.8. Table 2. Synergistic imidazole - dequalinium chloride combinations against A. vaginae biofilms

[0087] *FICI calculations were performed as described above. BEC values of monotherapies: DEQ (37.5 pM), MET (250 pM) and MIC (250 pM). ## synergistic values

[0088] For concentrations of 18.8 pM and 9.4 pM dequalinium chloride, synergy was observed (FICI < 1) in combination with metronidazole against A. vaginae biofilms in a metronidazole - dequalinium chloride ratio of 1 / 2.6 to 1 / 5.5 (Table 2). For concentrations of 18.8 pM to 2.3 pM dequalinium chloride, synergy was observed (FICI < 1) in combination with miconazole against A. vaginae biofilms in a dequalinium chloride - miconazole ratio of 1 / 0.1 to 1 / 10.5.

[0089] Example 2: Quaternary ammonium compounds act synergistically with miconazole against biofilms of Gardnerella vaginalis.

[0090] Materials and methods

[0091] Strains and chemicals. G. vaginalis strain ATCC 14018 was grown routinely on Columbia Blood Medium (2.3% peptone (International Medical Products NV); 0.1% starch (Merck Millipore), 0.5% sodium chloride (TCI Europe NV) supplemented with 5% defibrinated sheep blood) agar plates at 37° C, anaerobically. Brain Heart Infusion broth, purchased from Bio-Rad laboratories was used as overnight culture medium. New York City III broth (1.5% peptone (International Medical Products NV; 0.5% glucose (Sigma - Aldrich NV); 0.24% HEPES (Life Technologies Europe BV); 0.5% sodium chloride (TCI Europe NV) and 0.38% Yeast extract (VWR International)) was used as medium for biofilm formation. Stock solutions of miconazole (MIC) (Sigma-Aldrich) and metronidazole (MET) (Sigma-Aldrich) were prepared in DMSO (VWR International, Belgium). Domiphen bromide was purchased from Selleck Chemicals and dequalinium chloride from Sigma - Aldrich NV.

[0092] Antibiofilm screening assay were performed as described in Example 1.

[0093] Results.

[0094] To determine whether quaternary ammonium compounds (domiphen bromide, dequalinium chloride) act synergistically with imidazoles (metronidazole and miconazole) against G. vaginalis biofilms, checkerboard experiments and FICI calculations were performed (Fig. 2). The checkerboard data show that, when combined, lower concentrations of miconazole and quaternary ammonium compound, more particularly domiphen bromide or dequalinium chloride, can be used to eradicate biofilms of G. vaginalis.

[0095] Table 3. Synergistic miconazole - quaternary ammonium compound combinations against G. vaginalis biofilms monotherapies against G. vaginalis biofilms: MIC (50 pM), DOM (50 pM) and DEQ (100 pM). ## synergistic values

[0096] For concentrations of 25 pM to 6.25 pM miconazole, synergy was observed (FICI < 1) in combination with domiphen bromide against G. vaginalis biofilms in a domiphen bromide - miconazole ratio of 1 / 0.3 to 1 / 8 (Table 3). For concentrations of 25 pM to 1.56 pM miconazole, synergy was observed (FICI < 1) in combination with dequalinium chloride against G. vaginalis biofilms in a dequalinium chloride - miconazole ratio of 1 / 0.03 to 1 / 4.

Claims

CLAIMS1. A composition comprising an imidazole or a salt thereof, and a quaternary ammonium compound for use in the treatment and / or prevention of a bacterial vaginosis in a subject, wherein the imidazole is miconazole or metronidazole, and wherein the quaternary ammonium compound is domiphen bromide or dequalinium chloride.

2. The composition for use according to claim 1, wherein the bacterial vaginosis is caused by one or more bacteria of the Phylum Actinomycetota.

3. The composition for use according to claim 2, wherein the one or more bacteria of the Phylum Actinomycetota comprise Atopobium vaginae and / or Gardnerella vaginalis.

4. The composition for use according to any one of claims 1 to 3, wherein the subject has been diagnosed with a vaginal biofilm predominantly formed by one or more bacteria of the Phylum Actinomycetota; preferably by Atopobium vaginae and / or Gardnerella vaginalis.

5. The composition for use according to any one of claims 1 to 4, wherein the composition comprises a molar excess of the imidazole or a salt thereof over the quaternary ammonium compound of 0.02 to 21.

6. The composition for use according to any one of claims 1 to 5, wherein the composition comprises from 0.5 to 5 % (w / w) of imidazole or salt thereof.

7. The composition for use according to any one of claims 1 to 6, wherein the imidazole or a salt thereof and the quaternary ammonium compound are the only active ingredients in said composition.

8. The composition for use according to any one of claims 1 to 7, wherein the composition further comprises a mucoadhesive.

9. The composition for use according to claim 8, wherein the mucoadhesive is a mucoadhesive selected from the group consisting of synthetic polycarbophil, chitosan, cellulose derivatives, pectin, hyaluronic acid derivatives, polyacrylates, tragacanth, carrageenan, sodium alginate, thiolated polymers, or combinations thereof.

10. The composition for use according to any one of claims 1 to 9, wherein the composition has a pH from 2 to 4, preferably from 2.75 to 3.5.

11. The composition for use according to any one of claims 1 to 10, wherein the composition is a pharmaceutical composition, and optionally further comprises one or more pharmaceutically acceptable carriers.

12. The composition for use according to any one of claims 1 to 11, wherein the composition is in a form suitable for topical application, preferably a gel or a cream.

13. The composition for use according to any one of claims 1 to 12, wherein the composition is administered topically to the vagina.

14. A vaginal composition comprising metronidazole or a salt thereof, and a quaternary ammonium compound, wherein the quaternary ammonium compound is domiphen bromide or dequalinium chloride, wherein the vaginal composition has a pH from 2 to 4, preferably from 2.75 to 3.

5.

15. A kit comprising the vaginal composition according to claim 14 and an applicator for topically applying the vaginal composition to the vagina of a subject.

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