Composition for treating 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 prophylaxis of bacterial vaginosis. The invention also provides a vaginal composition and a kit comprising the vaginal composition and an applicator for topically applying the vaginal composition to the vagina of a subject.

Description

Technical Field

[0001] This invention relates to vaginal compositions and their use in the treatment and / or prevention of bacterial vaginosis, particularly bacterial vaginosis caused by actinomycete pathogens such as Atobobacter vaginosis and Gardnerella vaginalis. Background Technology

[0002] Mucosal biofilm-associated bacterial infections are very common, and the incidence of recurrent bacterial vaginosis is significant. BV is characterized by a change in the vaginal microbiota composition: certain species of *Lactobacillus* spp., associated with a healthy vaginal microbiota, are outnumbered by microaerophilic and anaerobic organisms, including *Gardnerella vaginalis* and *Atopobacter vaginalis*. Several compounds have been described for use against *Gardnerella vaginalis* / *Atopobacter vaginalis* infections.

[0003] Due to its strong adhesion to vaginal epithelial cells and biofilm formation ability, *Gardnerella vaginalis* has been proposed to initiate vaginal epithelial colonization and act as a scaffold for the attachment of other bacterial species (such as *Atopobacterium vaginalis*). Furthermore, *Atopobacterium vaginalis*, along with *Gardnerella vaginalis*, has been proposed to play an important role in biofilm establishment.

[0004] Currently, treatment options for bacterial vaginosis are limited, and discontinuation of treatment often leads to recurrence. Furthermore, some Gardnerella vaginalis / Atopobia strains have been shown to be resistant to some current therapies.

[0005] Therefore, further treatments for bacterial vaginosis still need to be developed. Summary of the Invention

[0006] The inventors have discovered that specific combinations of imidazoles or their salts with quaternary ammonium compounds can treat and / or prevent bacterial vaginosis. More specifically, the inventors have confirmed that imidazoles (e.g., miconazole or metronidazole) and quaternary ammonium compounds (e.g., domiphen bromide or dequinoline chloride) have a synergistic effect on biofilm clearance by species belonging to the Actinobacteria phylum, preferably Gardnerella vaginalis and / or Atobacillus vaginalis, which are commonly involved in bacterial vaginosis.

[0007] Therefore, a first aspect provides a composition comprising imidazole or a salt thereof and a quaternary ammonium compound for use in treating (i.e. treating and / or preventing) bacterial vaginosis in a subject, wherein the imidazole is miconazole or metronidazole, and wherein the quaternary ammonium compound is domiphen bromide or desquinodilium chloride.

[0008] In a particular embodiment, bacterial vaginosis is caused by one or more bacteria of the phylum Actinobacteria. In a further particular embodiment, one or more bacteria of the phylum Actinobacteria include Atopobium vaginae (A. vaginae) and / or Gardnerella vaginalis (G. vaginalis).

[0009] In a particular embodiment, the subject has been diagnosed with a vaginal biofilm formed primarily of one or more bacteria of the Actinobacteria phylum, preferably Atobacillus vaginalis and / or Gardnerella vaginalis.

[0010] In a specific embodiment of the above-described embodiments, the composition comprises an imidazole or a salt thereof in a molar excess of 0.02 to 21 relative to the quaternary ammonium compound. More specifically, the composition comprises 0.5 to 5% (w / w) imidazole or a salt thereof.

[0011] In certain embodiments of the present invention, such as those described above, imidazole or its salts and quaternary ammonium compounds are the only active ingredients in the composition.

[0012] Regardless of the foregoing, in certain embodiments, the composition may further comprise a mucosal adhesive, such as a mucosal adhesive selected from the group consisting of synthetic polycarbofil, chitosan, cellulose derivatives, pectin, hyaluronic acid derivatives, polyacrylates, tragacanth gum, carrageenan, sodium alginate, thiolized polymers, or combinations thereof.

[0013] Regardless of the above-described characteristics of the composition, in a particular embodiment, the pH of the composition is 2 to 4, preferably 2.75 to 3.5.

[0014] In a particular embodiment, the composition is a pharmaceutical composition and optionally further comprises one or more pharmaceutically acceptable carriers.

[0015] The compositions described herein, with different embodiments, may be in a form suitable for topical application, preferably a gel or cream.

[0016] In a particular embodiment, the composition as described above is administered topically to the vagina.

[0017] This application also provides a vaginal composition comprising metronidazole or a salt thereof and a quaternary ammonium compound, wherein the quaternary ammonium compound is domiphen bromide or desquinodilium chloride, and wherein the pH of the vaginal composition is 2 to 4, preferably 2.75 to 3.5.

[0018] This application also provides a kit in connection with the foregoing, comprising a vaginal composition as taught herein and an applicator for topically applying the vaginal composition to the vagina. Attached Figure Description

[0019] Figure 1 Dequinoline chloride exhibited synergistic activity against *Atopodium vaginalis* biofilms with miconazole and metronidazole. The mean FICI (∑FIC) of different imidazole-quaternary ammonium compound combinations against *Atopodium vaginalis* biofilms was calculated, more specifically, for the ratios of MET and DOM (9.4:1), MET and DEQ (0.4:1), MIC and DOM (3.8:1), and MIC and DEQ (10.5:1). FICI was expressed by the formula FICI = [CBEC]. A / BEC A ]+[C(BEC B ) / BEC B ]Calculate, where C(BEC) A ) and C (BEC) B ) is the BEC (biofilm clearance concentration) value of the antimicrobial drug combination. A and BEC B These are the individual BEC values ​​of antimicrobial drugs A and B. Synergistic effects are defined as FICI < 1. MIC - miconazole, MET - metronidazole, DOM - domifene bromide, DEQ - desquinal chloride.

[0020] Figure 2 Miconazole exhibited synergistic activity against Gardnerella vaginalis biofilms with domiphen bromide and dequinoline chloride. The mean FICI (∑FIC) of different imidazole-quaternary ammonium compound combinations against Gardnerella vaginalis biofilms was calculated, more specifically, for the ratios of MET and DOM at 2.3:1, MET and DEQ at 2.2:1, MIC and DOM at 3.9:1, and MIC and DEQ at 0.1:1. FICI was expressed by the formula FICI = [CBEC]. A / BEC A ]+[C(BEC B ) / BEC B ]Calculate, where C(BEC) A ) and C (BEC) B ) is the BEC (biofilm clearance concentration) value of the antimicrobial drug combination. A and BEC B These are the individual BEC values ​​for antimicrobial drugs A and B. Synergistic effect is defined as FICI < 1. Abbreviations: MIC - miconazole, MET - metronidazole, DOM - domiphen bromide, DEQ - desquinal chloride. Detailed Implementation

[0021] When used in relation to numerical values, the term "about" has the meaning generally understood in the relevant art. In some implementations, the term "about" may be omitted or may be interpreted as a value +10%; or +5%; or +2%; or +1%.

[0022] Whenever used in relation to percentages in this article, w / w means weight / weight, and w / v means weight / volume.

[0023] As used herein, the singular forms “a,” “an,” and “the” include both singular and plural meanings, unless the context clearly indicates otherwise.

[0024] As used herein, the term "comprising / comprises / comprised of" is synonymous with "including / includes" or "containing / contains," and is inclusive or open-ended, not excluding additional undescribed members, elements, or method steps. When referring to described members, elements, or method steps, the term "comprising / comprises / comprised of" also includes embodiments that "consists of" the described members, elements, or method steps.

[0025] Furthermore, in the specification and claims, the terms first, second, and third, etc., are used to distinguish similar elements or steps and are not necessarily used to describe a sequence or chronological order unless specifically stated otherwise. It should be understood that the terms thus used are interchangeable where appropriate, and the embodiments described herein can operate in sequences other than those described or exemplified herein.

[0026] Throughout this specification, references to “one embodiment” or “implementation” mean that a particular feature, structure, or characteristic described in connection with said embodiment is included in at least one embodiment contemplated herein. Therefore, the phrases “in one embodiment” or “in an embodiment” appearing in various places throughout this specification do not necessarily all refer to the same embodiment, but may refer to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments, as will be apparent to those skilled in the art based on this disclosure. Moreover, while some embodiments described herein include some features included in other embodiments but not others, combinations of features from different embodiments are intended to be within the scope of the invention and form different embodiments, as will be understood by those skilled in the art. For example, in the following claims, any of the claimed embodiments may be used in any combination.

[0027] Any references cited in this article are incorporated herein by reference.

[0028] Bacterial vaginosis (BVV) is a condition defined by the transformation of a lactobacillus-dominant into an anaerobic community of various microorganisms. Currently, metronidazole monotherapy is used as a first-line treatment for this condition. As a prodrug, metronidazole is reduced to its active state under anaerobic conditions—a mechanism that does not damage beneficial lactobacilli. However, complete eradication of BVV-associated bacteria can be complicated by the presence of biofilms, which protect the bacteria by hindering metronidazole penetration. Therefore, the recurrence rate of BVV after metronidazole monotherapy is relatively high.

[0029] The inventors have now discovered that combinations of imidazole or its salts with quaternary ammonium compounds can very effectively treat and / or prevent bacterial vaginosis. More specifically, the inventors have demonstrated that, based on fractional inhibition concentration index (FIC) measurements, combinations of imidazole or its salts (e.g., miconazole or metronidazole) with quaternary ammonium compounds (e.g., domiphen bromide or dequinoline chloride) exhibit synergistic anti-biofilm clearance activity against biofilms of bacteria belonging to the phylum Actinomycetes, preferably Gardnerella vaginalis and / or Atobacillus vaginalis. The fractional inhibitory concentration index cutoffs (FICs) of bactericidal activity against mature biofilms under anaerobic conditions can 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 invitro-in vivo correlation data for antifungal drug combinations against Aspergillus fumigatus. Antimicrob Agents Chemother. 2010 Feb; 54(2):602-9.), with an FIC cutoff value <1. This definition is based on in vitro-in vivo correlation.

[0030] Therefore, a first aspect provides a composition comprising, substantially consisting of, or consisting of the following:

[0031] Imidazole or its salts, and

[0032] Quaternary ammonium compounds

[0033] This document provides a method for treating (i.e., treating and / or preventing, as described elsewhere herein) bacterial vaginosis in a subject. In other words, it provides a method for treating (i.e., treating and / or preventing) bacterial vaginosis in a subject, comprising administering to the subject a composition comprising imidazole or a salt thereof and a quaternary ammonium compound, substantially composed of imidazole or a salt thereof and a quaternary ammonium compound, or composed of imidazole or a salt thereof and a quaternary ammonium compound, thereby treating the bacterial vaginosis. This document also provides the use of a composition comprising imidazole or a salt thereof and a quaternary ammonium compound, substantially composed of imidazole or a salt thereof and a quaternary ammonium compound, or composed of imidazole or a salt thereof and a quaternary ammonium compound in the manufacture of a medicament for treating (i.e., treating and / or preventing) bacterial vaginosis.

[0034] The composition may contain synergistic, therapeutic and / or preventive effective amounts, such as synergistic and therapeutic effective amounts of imidazole or its salts and quaternary ammonium compounds.

[0035] As used herein, the term “bacterial vaginosis” or “BV” refers to a disease or condition caused by an imbalance in the vaginal bacterial flora, which can lead to one or more of the following symptoms: pain, itching, burning, discomfort, and abnormal vaginal discharge. A healthy or normal vaginal microbiota is typically dominated by lactobacilli that produce various antimicrobial compounds. Bacterial vaginosis is characterized by a decrease or sharp decline in the total number of lactobacilli and a corresponding significant increase in the concentration of anaerobic bacteria. Many potential microbial pathogens, either alone or in combination, are involved in the disease process, such as Gardnerella vaginalis, Atobacillus vaginalis, Mobiluncus mulieris, Prevotella bivia, Fusobacterium nucleatum, and / or certain species of Peptoniphilus. Actinobacteria, such as Gardnerella vaginalis and Atobacillus vaginalis, are often associated with the initiation of bacterial vaginosis. Due to its strong adhesion to vaginal epithelial cells and biofilm-forming ability, *Gardnerella vaginalis* has been proposed to initiate vaginal epithelial colonization and act as a scaffold for other bacterial species (such as *Atopobacterium vaginalis*) to attach, thereby forming a biofilm. *Gardnerella vaginalis* is usually the dominant species in biofilms. Other pathogens, such as certain species of *Prevotella* and *Mobiluncus*, may act as secondary invaders. Bacterial vaginosis can be diagnosed by any means known in the art. For example, bacterial vaginosis can be diagnosed by microscopy. The presence of clue cells in the vaginal discharge of a subject is sufficient for a diagnosis of bacterial vaginosis. Clue cells are epithelial cells that are so extensively covered by cocci that the cell membranes of the epithelial cells are not observable.

[0036] In specific embodiments of the compositions contemplated herein, bacterial vaginosis is caused by one or more bacteria of the phylum Actinomycetes. In the context of this invention, bacterial vaginosis “caused” by one or more bacteria of the phylum Actinomycetes refers to bacterial vaginosis caused by the formation of a biofilm by one or more bacteria of the phylum Actinomycetes. In specific embodiments, the one or more bacteria of the phylum Actinomycetes include, are substantially composed of, or consist of, Atobacillus vaginalis and / or Gardnerella vaginalis. Therefore, in specific embodiments, bacterial vaginosis may be caused by Atobacillus vaginalis and / or Gardnerella vaginalis.

[0037] In some embodiments, bacterial vaginosis as described herein may be caused primarily by Atobacillus vaginalis and / or Gardnerella vaginalis, or in other words, in certain embodiments, the biofilm formed in bacterial vaginosis is primarily formed by Atobacillus vaginalis and / or Gardnerella vaginalis.

[0038] The term "treat" or "treatment" encompasses both therapeutic management of an already developed disease or condition (e.g., treatment of an already developed bacterial vaginosis) and preventative or preventive measures aimed at preventing or reducing the likelihood of undesirable complications, such as preventing the occurrence, development, and progression of bacterial vaginosis. Beneficial or desired clinical outcomes may include, but are not limited to, relief of one or more symptoms or one or more biomarkers, reduction of the severity of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, and improvement or mitigation of the disease state.

[0039] Compositions for vaginal administration as taught herein may be administered to a subject in a synergistic, therapeutic, and / or preventatively effective amount, such as a therapeutically and / or preventatively effective amount. As used herein, the term "therapeuticly effective amount" refers to the amount of an active compound or agent sought by a surgeon, investigator, veterinarian, MD, or other clinician to elicit a biological or medical response in a subject, which may in particular include relief of symptoms of the treated disease or condition. The term "preventatively effective amount" refers to the amount of an active compound or agent sought by an investigator, veterinarian, MD, or other clinician to inhibit or delay the onset of a condition in a subject. Methods for determining the therapeutically and / or preventatively effective dose of compositions for vaginal administration as taught herein are known in the art.

[0040] The inventors have discovered that the synergistic effect of imidazole or its salts in combination with quaternary ammonium compounds includes synergistic bactericidal activity (e.g., biofilm clearance) against bacteria that cause bacterial vaginosis. A biofilm is a microbial growth pattern comprising sessile cells, typically within a complex and highly heterogeneous extracellular polymeric matrix, characterized by reduced sensitivity to antimicrobial agents. Biofilms may contain a single species (e.g., bacteria such as *Atopobacterium vaginalis*) or multiple species of microorganisms (e.g., *Atopobacter vaginalis*, *Gardnerella vaginalis*, and other microorganisms such as certain species of *Prevotella* and *Mobiluncus*, or even yeasts and / or fungi).

[0041] Therefore, in certain implementations, treatment of bacterial vaginosis, as taught herein, encompasses the complete or partial reduction of the bacteria that cause bacterial vaginosis.

[0042] In a further specific embodiment, the composition for vaginal administration, as taught herein, prevents and / or inhibits biofilm formation, and / or removes biofilms. Preferably, the biofilm is a bacterial biofilm, more preferably a biofilm formed by species of the Actinobacteria phylum such as Gardnerella vaginalis and / or Atobacillus vaginalis, and even more preferably a biofilm formed by Atobacillus vaginalis. In a specific embodiment, the method of treatment (i.e., treatment and / or prevention) comprises the steps of administering an effective amount of the composition for vaginal administration, as taught herein, sufficient to prevent and / or inhibit the formation of biofilms by species of the Actinobacteria phylum such as Gardnerella vaginalis and / or Atobacillus vaginalis, and / or remove biofilms formed by species of the Actinobacteria phylum such as Gardnerella vaginalis and / or Atobacillus vaginalis.

[0043] Unless otherwise stated, the terms "subject" or "patient" are used interchangeably and refer to animals, preferably warm-blooded animals, more preferably vertebrates, even more preferably mammals, and even more preferably primates, and particularly include human patients and non-human mammals and primates. Preferred subjects are human subjects. The terms "subject" or "patient" include subjects who require treatment, and more particularly subjects who will benefit from treatment for a given condition (especially bacterial vaginosis). Such subjects may include, but are not limited to, those who have been diagnosed with said condition, those who are prone to developing said condition, and / or those in whom said condition is to be prevented. In a particular embodiment, the subject has been diagnosed with bacterial vaginosis. In a particular embodiment, the subject has been diagnosed with a biofilm, such as a vaginal biofilm, preferably a vaginal biofilm formed (or composed primarily of) one or more bacteria of the phylum Actinobacteria, for example 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%, for example at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In a particular embodiment, the subject has been diagnosed with a vaginal biofilm formed (or composed primarily of) *Atopobia vaginalis* and / or *Gardnerella vaginalis*, for example 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%, for example at least 96%, at least 97%, at least 98%, at least 99%. In a particular embodiment, the subject is a female subject, preferably a woman.

[0044] The imidazole used in the compositions for vaginal administration as taught herein can be any type of imidazole or salt thereof known in the art, including nitroimidazoles. Non-limiting examples of imidazoles include miconazole, clotrimazole, ketoconazole, and metronidazole. In certain 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.

[0045] The quaternary ammonium compound used in the compositions for vaginal administration as taught herein can be any quaternary ammonium compound known in the art, including, but not limited to, monoquaternary ammonium compounds such as domiphen bromide, benzalkonium chloride, and cetrimonium chloride, and bisquaternary ammonium compounds such as dequalinium chloride and benzethonium chloride.

[0046] In a particular embodiment and regardless of other characteristics of the composition, imidazole or a salt thereof is miconazole or a salt thereof, or metronidazole or a salt thereof.

[0047] As used herein, the term "miconazole" refers to the compound (1-[2-(2,4-dichlorophenyl)-2-[(2,4-dichlorophenyl)methoxy]ethyl]imidazole) and is identified by CAS Registry No. 22916-47-8. Miconazole is an antifungal compound that interferes with the metabolism of ergosterol (a component of yeast cell membranes). Miconazole is preferably formulated as its nitrate in compositions for vaginal administration as taught herein.

[0048] Therefore, in a particular embodiment, imidazole or a salt thereof is miconazole nitrate.

[0049] As used herein, the term "metronidazole" refers to the compound 2-(2-methyl-5-nitroimidazole-1-yl)ethanol and is identified by CAS registration number 443-48-1. Metronidazole is a commonly used antibiotic belonging to the nitroimidazole class of antibiotics. Metronidazole is a nitroimidazole, a prodrug that is reductively activated under low oxygen stress (i.e., the nitro group is reduced), leading to imidazole cleavage and cytotoxicity.

[0050] In a particular embodiment, the quaternary ammonium compound is domiphen bromide or dequinoline chloride.

[0051] As used herein, the term "domiphen bromide" refers to the compound [N,N-dimetyl-N-(2-phenoxyethyl)-, bromide] and is identified by CAS registration number 538-71-6. Domiphen bromide is a quaternary ammonium salt with antibacterial properties.

[0052] As used herein, the term "dequinoline chloride" refers to the compound 1-[10-(4-amino-2-methylquinolin-1-onthium-1-yl)decyl]-2-methylquinolin-1-onthium-4-amine; dichloride (1-[10-(4-amino-2-methylquinolin-1-ium-1-yl)decyl]-2-methylquinolin-1-ium-4-amine; dichloride) and is identified by CAS registry number 522-51-0. Dequinoline chloride is a quaternary ammonium cationic antimicrobial agent used to treat common infections of the oral cavity and pharynx.

[0053] In a further specific embodiment, the imidazole or its salt is miconazole or its salt, or metronidazole or its salt, and the quaternary ammonium compound is domiphen bromide or desquinodilium chloride.

[0054] In a further specific embodiment of the invention as described above,

[0055] The imidazole or its salt is metronidazole or its salt, and the quaternary ammonium compound is domiphen bromide;

[0056] The imidazole or its salt is metronidazole or its salt, and the quaternary ammonium compound is diquinoline chloride;

[0057] Imidazole or its salt is miconazole or its salt, preferably its nitrate, and the quaternary ammonium compound is domiphen bromide; or

[0058] The imidazole or its salt is miconazole or its salt, preferably its nitrate, and the quaternary ammonium compound is dequinoline chloride.

[0059] The inventors have discovered that miconazole or a combination of its salts and dequinoline chloride has bactericidal activity against the mature biofilms of both Gardnerella vaginalis and Atobacillus vaginalis, with an FIC of less than 0.5.

[0060] In a particular embodiment, the imidazole or its salt is in a molar excess of 0.02 to 21, or 0.03 to 11, preferably 0.4 to 10, relative to the quaternary ammonium compound. In other words, in a particular embodiment, the composition comprises an imidazole or its salt in a molar excess of 0.02 to 21, or 0.03 to 11, preferably 0.4 to 10, relative to the quaternary ammonium compound.

[0061] In a particular embodiment, if imidazole or a salt thereof is metronidazole or a salt thereof and the quaternary ammonium compound is domiphen bromide, then metronidazole or a salt thereof is present in a molar excess of 3 to 11, preferably 4 to 10, relative to domiphen bromide in the composition for vaginal administration as taught herein. In a further particular embodiment, if bacterial vaginosis is primarily caused by Atobacillus vaginalis, and if imidazole or a salt thereof is metronidazole or a salt thereof and the quaternary ammonium compound is domiphen bromide, then metronidazole or a salt thereof is present in a molar excess of 4 to 10, preferably 4.4 to 9.4, relative to domiphen bromide in the composition for vaginal administration as taught herein.

[0062] In a particular embodiment, if imidazole or a salt thereof is metronidazole or a salt thereof and the quaternary ammonium compound is desquinal chloride, then metronidazole or a salt thereof is present in a molar excess of 0.1 to 12.5, 0.1 to 0.5, 0.2 to 12.5, or 0.2 to 0.5, preferably 0.2 to 0.4, relative to the amount of desquinal chloride. In a further particular embodiment, if bacterial vaginosis is primarily caused by Atobacillus vaginalis, and if imidazole or a salt thereof is metronidazole or a salt thereof and the quaternary ammonium compound is desquinal chloride, then metronidazole or a salt thereof is present in a molar excess of 0.2 to 12.5, preferably 0.2 to 0.4, relative to the amount of desquinal chloride.

[0063] In a particular embodiment, if imidazole or a salt thereof is miconazole or a salt thereof and the quaternary ammonium compound is domiphen bromide, then miconazole or a salt thereof is present in a molar excess of 0.2 to 9, preferably 0.3 to 8, relative to the amount of domiphen bromide in the composition for vaginal administration as taught herein. In a further particular embodiment, if bacterial vaginosis is primarily caused by *Atopobacterium vaginalis*, and if imidazole or a salt thereof is miconazole or a salt thereof and the quaternary ammonium compound is domiphen bromide, then miconazole or a salt thereof is present in a molar excess of 1.5 to 4, preferably 1.8 to 3.8, for example about 2, about 2.5, about 3, about 3.5, or about 4, relative to the amount of domiphen bromide in the composition for vaginal administration as taught herein. Thus, as an example, a composition for vaginal administration as taught herein containing 10 mmol of domiphen bromide with a 3-fold molar excess contains 30 mmol of miconazole nitrate. In a further specific embodiment, if the bacterial vaginosis is primarily caused by Gardnerella vaginalis, and if the imidazole or its salt is miconazole or its salt and the quaternary ammonium compound is domiphen bromide, then miconazole or its salt is present in a molar excess of 0.2 to 9, preferably 0.3 to 8, relative to the amount of domiphen bromide in the composition for vaginal administration as taught herein.

[0064] In a particular embodiment, if imidazole or a salt thereof is miconazole or a salt thereof and the quaternary ammonium compound is desquinal chloride, then miconazole or a salt thereof is present in a molar excess of 0.02 to 21, 0.03 to 21, 0.02 to 12, or 0.03 to 12, preferably 0.03 to 11, relative to the amount of desquinal chloride. In a further particular embodiment, if bacterial vaginosis is primarily caused by Atobacillus vaginalis, and if imidazole or a salt thereof is miconazole or a salt thereof and the quaternary ammonium compound is desquinal chloride, then miconazole or a salt thereof is present in a molar excess of 0.05 to 21, 0.05 to 11, 0.1 to 21, preferably 0.1 to 10.5, relative to the amount of desquinal chloride. In a further specific embodiment, if the bacterial vaginosis is primarily caused by Gardnerella vaginalis, and if the imidazole or its salt is miconazole or its salt and the quaternary ammonium compound is dequinoline chloride, then miconazole or its salt is present in a molar excess of 0.02 to 5, preferably 0.03 to 4, relative to the amount of dequinoline chloride in the composition for vaginal administration as taught herein.

[0065] In certain embodiments, the vaginal composition as taught herein comprises 0.5% (w / w) to 5% (w / w), preferably 1% (w / w) to 5% (w / w) of imidazole or a salt thereof. For example, the vaginal composition 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) of imidazole or a salt thereof. In further specific embodiments, if the imidazole or a salt thereof is metronidazole or a salt thereof, the vaginal composition as taught herein comprises 0.5% (w / w) to 2% (w / w) or 0.5% (w / w) to 1% (w / w), more preferably about 0.75% (w / w) of miconazole or a salt thereof. In a further specific embodiment, if the imidazole or its salt is miconazole or its salt, preferably miconazole nitrate, then the composition for vaginal administration as taught herein comprises 1% (w / w) to 3% (w / w), more preferably about 2% (w / w) of miconazole or its salt.

[0066] In certain embodiments, imidazole or its salts, as well as quaternary ammonium compounds, are the only active ingredients, the only bactericidal ingredients, and / or the only antimicrobial ingredients in the compositions for vaginal administration as taught herein.

[0067] However, compositions for vaginal administration as taught herein may contain one or more additional antimicrobial agents, such as one or more antimicrobial agents that have bactericidal activity against one or more bacteria of the Actinobacteria phylum, preferably Atobacillus vaginalis and / or Gardnerella vaginalis.

[0068] In certain embodiments, the compositions for vaginal administration, as taught herein, are pharmaceutical compositions and optionally also comprise one or more pharmaceutically acceptable carriers.

[0069] As used herein, the term “pharmaceutical acceptable” is consistent with the art and means compatible with other components of a pharmaceutical composition and harmless to its recipient.

[0070] As used herein, "carrier" or "excipient" includes any and all solvents, diluents, buffers (such as, for example, neutral buffered saline or phosphate buffered saline), solubilizers, colloids, dispersion media, vehicles, fillers, chelating agents (such as, for example, EDTA or glutathione), amino acids (such as, for example, glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, aroma agents, thickeners, reagents for achieving depot function, coatings, antifungal agents, preservatives, antioxidants, tension control agents, and absorption delay agents, etc. The use of such media and reagents in pharmaceutically active substances is well known in the art. Unless any conventional media or reagent is incompatible with the active substance, its use in therapeutic compositions may be considered.

[0071] Compositions for vaginal administration as taught herein are preferably for topical use and formulated to be compatible with the specific application site (e.g., skin, mucous membrane). Thus, in certain embodiments, compositions for vaginal administration as taught herein are in a form suitable for topical application, preferably for topical application to the vagina, and more particularly for topical application to the vaginal mucosa.

[0072] Compositions for vaginal administration as taught herein may include additives commonly used in such forms, including but not limited to binders, fillers, carriers, preservatives, stabilizers, emulsifiers, buffers, and excipients such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, or magnesium carbonate.

[0073] In certain embodiments, the compositions for vaginal administration as taught herein are in the form of solutions, suspensions (e.g., used as vaginal douches or sprays), gels (e.g., vaginal suppository gels), capsules (e.g., vaginal capsules), creams (e.g., vaginal creams), salves, tinctures, lotions, vaginal suppositories, transdermal patches, ointments, lubricants, or tablets (e.g., vaginal tablets). The compositions for vaginal administration as taught herein may also be impregnated in wipes or sponges. The compositions for vaginal administration as taught herein may also be in the form of sustained-release formulations.

[0074] Formulating the compositions for vaginal administration as taught herein into gels or creams allows for easy removal from a tube or syringe while ensuring the composition remains adhered to the application site. Therefore, in preferred embodiments, the compositions for vaginal administration as taught herein are in the form of creams or gels. Creams are typically oil-in-water (o / w) or water-in-oil (w / o) emulsions and may further contain emulsifiers and / or thickeners. Gels are typically transparent formulations and may contain cellulose ethers or carbomers in water or a water-alcohol mixture.

[0075] Because quaternary ammonium compounds are highly soluble in water, while imidazoles and their salts are insoluble in water, compositions for vaginal administration as taught herein are preferably in cream form, further comprising one or more emulsifiers. In certain embodiments, compositions for vaginal administration as taught herein may be in emulsion form. Various oil-in-water emulsions are known in the art. In compositions for vaginal administration as taught herein, liquid paraffin and / or lauroyl polyoxyethylene-6 glyceryl ester are preferably used as the oil phase. To stabilize the emulsion, emulsifiers such as, for example, mixtures of polyethylene glycol-6 palmitate stearate, ethylene glycol stearate, and PEG-32 stearate can be used.

[0076] To increase the viscosity of compositions for vaginal administration as taught herein, such as when the compositions for vaginal administration as taught herein are in emulsion form, one or more thickeners, such as cetearyl, may be used. In the context of this invention, viscosity refers to resistance to gradual deformation caused by shear stress. Viscosity can be measured by any means known in the art, such as using a Brookfield HA viscometer.

[0077] For optimal adhesion to the application site (e.g., the vagina), compositions for vaginal administration as taught herein may further comprise one or more mucosal adhesives. Suitable mucosal adhesives for vulvovaginal administration include polymers capable of forming hydrogels, such as synthetic polycarbofil, chitosan, cellulose derivatives (hydroxyethyl cellulose, hydroxypropyl cellulose, and hydroxypropyl methyl cellulose), pectin, hyaluronic acid derivatives, polyacrylates, tragacanth gum, carrageenan, and sodium alginate, and thiolated polymers. Therefore, in certain embodiments, compositions for vaginal administration as taught herein further comprise mucosal adhesives, such as those selected from the group consisting of synthetic polycarbofil, chitosan, cellulose derivatives, pectin, hyaluronic acid derivatives, polyacrylates, tragacanth gum, carrageenan, and sodium alginate, and thiolated polymers.

[0078] The pH of a healthy, normal vagina is typically acidic. However, when bacterial vaginosis reduces the amount of lactobacilli in the vagina, the pH level rises. Preferably, the compositions for vaginal administration as taught herein are acidic to match the pH of a healthy, normal vagina. Thus, in certain embodiments, the pH of the compositions for vaginal administration as taught herein is 2 to 4, preferably 2.75 to 3.5. The compositions for vaginal administration as taught herein can be made acidic by any means known in the art, for example by containing benzoic acid, HCl, or lactic acid.

[0079] In certain embodiments, the compositions as taught herein are administered topically to the vagina. In other words, in certain embodiments, a method of treating bacterial vaginosis may include the step of topically administering a composition as taught herein to the vagina. Compositions as taught herein for vaginal administration may be administered by any suitable manner, such as by using a vaginal cream applicator, such as Biosrx's Gentle Dose® cavity applicator (SKU: GD-40). In certain embodiments, the compositions as taught herein are administered to the infected area in the vagina.

[0080] The medications taught in this article can be used as preventative agents, but are generally used when there are signs or symptoms of bacterial vaginosis.

[0081] The duration of administration of the vaginal composition as taught herein to a subject may be extended for several days or longer, depending on the condition. For example, administration may continue until the symptoms of bacterial vaginosis are adequately relieved or eliminated. Progression of this treatment can be easily monitored using routine techniques and assays. In certain embodiments, the vaginal composition as taught herein may be administered for a continuous period of at least 7 days, at least 14 days, or at least 21 days. In certain embodiments, the vaginal composition as taught herein may be administered every two days, daily, twice daily, or three times daily.

[0082] In certain embodiments, the vaginal compositions as taught herein are used to treat recurrent bacterial vaginosis. Recurrent bacterial vaginosis, in the context of this invention, refers to at least four specific episodes of bacterial vaginosis within one year or at least three episodes of bacterial vaginosis within one year that are unrelated to antibiotic treatment. The vaginal compositions as taught herein can be used to treat recurrent infections of Actinobacteria pathogens, particularly recurrent infections of *Atopobacterium vaginalis* and / or *Gardnerella vaginalis* that are not very responsive to monotherapy.

[0083] The present invention also provides a vaginal composition comprising imidazole or a salt thereof and a quaternary ammonium compound, wherein the pH of the vaginal composition is 2 to 4, preferably 2.75 to 3.5. In a particular embodiment, the imidazole is metronidazole, and the quaternary ammonium compound is domiphen bromide or desquinal chloride. In a particular embodiment, the vaginal composition comprises a synergistic, therapeutic, and / or preventative effective amount, such as a synergistic and therapeutic effective amount of imidazole or a salt thereof and a quaternary ammonium compound. Compositions for vaginal administration as taught herein can be made acidic by any means known in the art, as described in other parts of this document. The characteristics of the vaginal compositions of the present invention correspond to the characteristics of the compositions for vaginal administration as detailed above.

[0084] The present invention also provides a kit comprising a vaginal composition as taught herein and an applicator for topically applying the vaginal composition to the vagina. In a particular embodiment, the kit comprises a vaginal composition comprising metronidazole and a quaternary ammonium compound, wherein the quaternary ammonium compound is domiphen bromide or desquinal chloride. The applicator may be any applicator known in the art suitable for topically applying the composition to the vagina, as described in other sections herein. The features of the kit of the present invention correspond to the features of the vaginal composition used in the methods detailed above.

[0085] The following examples are intended to illustrate the invention and should not be construed as limiting its scope.

[0086] Example

[0087] Example 1: Quaternary ammonium compounds and imidazoles showed a synergistic effect on the biofilm of Atobacillus vaginalis.

[0088] Materials and methods

[0089] Strains and chemicals. *Atopobacterium vaginalis* strain BAA-55 was typically grown anaerobically at 37°C on Columbia blood agar plates (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). Brain-heart infusion broth purchased from Bio-Rad laboratories was used as the 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 the biofilm formation medium. 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 desquinal chloride was purchased from Sigma-Aldrich NV.

[0090] Anti-biofilm screening assay. Overnight cultures of *Atopobacterium vaginalis* grown in BHI medium were diluted to an optical density of 0.1 (approximately 10⁻⁶). 8 (1 cell), and further diluted 1 / 100 in New York City III medium. 200 µL of this suspension was added to the wells of a round-bottom microplate (TPP Techno Plastic Products AG, Switzerland). The biofilm was allowed to grow anaerobically for 72 hours. Afterward, the growth medium in all wells was discarded, and a combination of imidazoles (miconazole, metronidazole) and quaternary ammonium compounds (domiphen bromide, desquinal chloride), diluted 2-fold in New York City broth, was added to the biofilm along the rows and columns of the microplate (1% DMSO background). After anaerobic incubation at 37°C for 24 hours, the biofilm was quantified by spot plating. For this, the biofilm was washed with PBS and thoroughly scraped from the bottom of the plate. Serial dilutions were spotted (5 µL) onto Columbia blood agar plates and incubated anaerobically at 37°C for 48 hours to determine if colony formation had occurred.

[0091] Fractional Inhibition Concentration Index (FICI) is calculated using the formula FICI = [CBEC]. A / BEC A ]+[C(BEC B ) / BEC B ]Calculate, where C(BEC) A ) and C (BEC) B (BEC) is the BEC value of the antimicrobial drug combination. A and BEC B These are the individual BEC values ​​for antimicrobial drugs A and B. BEC represents the biofilm clearance concentration, which is the minimum concentration of a compound at which no colony growth is observed after spotting. According to Meletiadis J et al., "Defining fractional inhibitory concentration indexcutoffs 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", when the FICI value < 1, the interaction is defined as synergistic.

[0092] result.

[0093] To determine whether quaternary ammonium compounds (domiphen bromide and dequinoline chloride) exhibit synergistic effects with imidazoles (metronidazole and miconazole) on vaginal atorbozyme biofilms, a checkerboard experiment and FICI calculations were performed. Figure 1 Chessboard data shows that, when combined, lower concentrations of imidazole and quaternary ammonium compounds can be used to clear biofilms of vaginal atobacillus.

[0094] Table 1. Synergistic effect of imidazole-domiphen bromide combination on vaginal atobacillus biofilm

[0095]

[0096] *FICI calculation is based on the formula FICI = [C(BEC)] A ) / BEC A ] + [C(BEC B ) / BEC B ], where C(BEC)A ) and C (BEC) B () is the BEC value of the combination of imidazole and domiphen bromide. A and BEC B These are the individual BEC values ​​for metronidazole (250 µM) or miconazole (100 µM) and domomiphen (50 µM). ## Synergistic effect value

[0097] For domomiphen bromide at concentrations of 25 µM and 12.5 µM, synergistic effects (FICI < 1) were observed in combination with two imidazoles (i.e., miconazole or metronidazole) on vaginal attobacter biofilms (Table 1). The synergistic combination ratios of domomiphen bromide and metronidazole were found to be 1 / 4.4 to 1 / 9.4, and the synergistic combination ratios of domomiphen bromide and miconazole were 1 / 1.8 to 1 / 3.8.

[0098] Table 2. Synergistic effect of imidazole-dequinoline chloride combination on vaginal atobacillus biofilm

[0099]

[0100] *FICI calculations were performed as described above. BEC values ​​for monotherapy: DEQ (37.5 µM), MET (250 µM), and MIC (250 µM). ## Synergistic effect values

[0101] For dequinoline chloride at concentrations of 18.8 µM and 9.4 µM, a synergistic effect (FICI < 1) was observed with metronidazole in combination with dequinoline chloride at a metronidazole-dequinoline chloride ratio of 1 / 2.6 to 1 / 5.5 on the biofilm of *Atopobacterium vaginalis* (Table 2). For dequinoline chloride at concentrations of 18.8 µM to 2.3 µM, a synergistic effect (FICI < 1) was observed with miconazole in combination with dequinoline chloride at a dequinoline chloride-miconazole ratio of 1 / 0.1 to 1 / 10.5 on the biofilm of *Atopobacterium vaginalis* (Table 2).

[0102] Example 2: Quaternary ammonium compounds and miconazole showed a synergistic effect on Gardnerella vaginalis biofilm.

[0103] Materials and methods

[0104] Strains and chemicals. Gardnerella vaginalis strain ATCC 14018 was typically grown anaerobically at 37°C on Columbia blood agar plates (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). Brain-heart infusion broth purchased from Bio-Rad laboratories was used as the 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 the biofilm formation medium. 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 desquinal chloride was purchased from Sigma-Aldrich NV.

[0105] Anti-biofilm screening and detection were performed as described in Example 1.

[0106] result.

[0107] To determine whether quaternary ammonium compounds (domiphen bromide and dequinoline chloride) exhibit synergistic effects with imidazoles (metronidazole and miconazole) on Gardnerella vaginalis biofilms, a checkerboard experiment and FICI calculations were performed. Figure 2 Chessboard data shows that, when combined, lower concentrations of miconazole and quaternary ammonium compounds, particularly domiphen bromide or dequinoline chloride, can be used to clear biofilms of Gardnerella vaginalis.

[0108] Table 3. Synergistic effect of miconazole-quaternary ammonium compound combination on Gardnerella vaginalis biofilm

[0109]

[0110] *FICI calculations were performed as described above. BEC values ​​of monotherapy against Gardnerella vaginalis biofilms: MIC (50 µM), DOM (50 µM), and DEQ (100 µM). ## Synergistic effect values

[0111] For miconazole concentrations ranging from 25 µM to 6.25 µM, a synergistic effect (FICI < 1) was observed when combined with domomiphen bromide at a ratio of 1 / 0.3 to 1 / 8 on Gardnerella vaginalis biofilm (Table 3). For miconazole concentrations ranging from 25 µM to 1.56 µM, a synergistic effect (FICI < 1) was observed when combined with dequinoline chloride at a ratio of 1 / 0.03 to 1 / 4 on Gardnerella vaginalis biofilm (Dequinoline chloride).

Claims

1. A composition for treating and / or preventing bacterial vaginosis in a subject, comprising: Imidazole or its salts, and Quaternary ammonium compounds, in, The imidazole is miconazole or metronidazole, and The quaternary ammonium compound is domiphen bromide or dequinoline chloride.

2. The composition according to claim 1, wherein, Bacterial vaginosis is caused by one or more bacteria of the phylum Actinobacteria.

3. The composition according to claim 2, wherein, One or more bacteria in the Actinobacteria phylum include 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 mainly composed of one or more bacteria of the phylum Actinobacteria, preferably Atobacillus vaginalis and / or Gardnerella vaginalis.

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

6. The composition for use according to any one of claims 1 to 5, wherein, The composition contains 0.5 to 5% by weight of imidazole or its salt.

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

8. The composition for use according to any one of claims 1 to 7, wherein, The composition also includes a mucosal adhesive.

9. The composition for application according to claim 8, wherein, The mucosal adhesive is selected from the group consisting of synthetic polycarbofil, chitosan, cellulose derivatives, pectin, hyaluronic acid derivatives, polyacrylate, tragacanth gum, carrageenan, sodium alginate, thiolized polymers, or combinations thereof.

10. The composition for use according to any one of claims 1 to 9, wherein, The pH of the composition is 2 to 4, preferably 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 may optionally further comprise 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 cream.

13. The composition for the application 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 its salts, and Quaternary ammonium compounds, among which, The quaternary ammonium compound is domiphen bromide or desquinodim chloride, wherein the pH of the vaginal composition is 2 to 4, preferably 2.75 to 3.

5.

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

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