Intravaginal formulations of gardnerella endolysin

Abstract

The present invention relates to a novel pharmaceutical composition comprising an effective amount of an active agent and a pharmaceutically acceptable excipient wherein the active agent is a polypeptide having killing activity against Gardnebacterium genus, and wherein the excipient is a polyacrylic acid polymer. The invention also relates to the use of the pharmaceutical composition in the treatment of Gardnebacterium infections, in particular bacterial vaginosis.

Description

Technical Field

[0001] This invention relates to a treatment for Gardnerella spp. ( Gardnerella Novel pharmaceutical compositions for bacterial infections. The pharmaceutical compositions of the present invention are characterized in that they comprise a Gardnerella spp.-specific polypeptide as an active agent and a polyacrylic acid polymer as an excipient. The present invention also relates to the pharmaceutical use of the pharmaceutical compositions of the present invention and to methods of treatment using the pharmaceutical compositions. Background Technology

[0002] Bacterial vaginosis (BV), also known in the literature as bacterial vaginosis, nonspecific vaginosis, or nonspecific vaginitis, is the most common vaginal infection worldwide and is associated with serious adverse consequences, including premature birth, postpartum endometritis, and an increased risk of HIV infection. It is a vaginal flora imbalance caused by the presence of commensal lactobacilli in the vagina (…). Lactobacilli The vaginal flora is replaced by a multimicrobial biofilm, raising the pH from the natural 3.5-4.5 to 5.5 and forming a foul-smelling liquid. The reported prevalence ranges from 10-40%, depending on the population studied. However, suboptimal diagnostic methods and a high proportion of asymptomatic patients make it difficult to determine the true prevalence of BV. Gardnerella vaginalis (… Gardnerella vaginalis ( G.vaginalis )) is a bacterial species associated with BV.

[0003] The pathogenesis of vaginal vaginitis (BV) remains poorly understood. It is most commonly defined as a pathological condition characterized by abnormal vaginal flora, particularly H2O2-producing lactobacilli. Lactobacillus The loss of ) and anaerobic bacteria (including Gardnerella vaginalis, Molybditis spp.) Mobiluncus ) species and human mycoplasma ( Mycoplasma hominis The overgrowth of Gardnerella vaginalis. However, recent data suggest that Gardnerella vaginalis plays a major role as a specific sexually transmitted pathogen in BV (Muzny et al., 2016, J. of Infect. Dis. 214 Suppl. 1., S1).

[0004] In the 1950s, numerous small, pleomorphic Gram-negative bacilli were observed in the reproductive tract of women with BV. These organisms were initially named *Haemophilus vaginalis*. Haemophilus vaginalis As more information about its characteristics became available, it was repeatedly renamed, and it is now classified as Gardnerella vaginalis. G. vaginalis Until 2018, it was considered to be from the genus Gardnerella (…). Gardnerella The only member of the genus Gardnerella. However, in early 2019, it was revealed that the genus Gardnerella actually comprises at least 13 species, with the most common species being renamed Gardnerella vaginalis (Gardnerella vaginalis).G. vaginalis sensu stricto Leopoldellagardneri, Piotella gardneri, Sweginnellagardneri (Vaneechoutte et al., 2019 Int. J. Syst. Evol. Biol. 898661). G. leopoldii G. piotii G. swidsinskii

[0005] Gardnerella bacteria are special in that they are Gram-variable, i.e. they do not form an outer membrane that defines Gram-negative bacteria. Their cell wall is usually very thin and has a peptidoglycan content of only 10% or less, so the crystal violet dye used for Gram staining does not always take on the deep purple color that is typical for Gram-positive bacteria. Instead, Gardnerella cells can appear Gram-positive and Gram-negative in Gram staining. Based on phylogenetic analysis of 16S rRNA, the genus Gardnerella was assigned to the Gram-positive family of the order of the Bifidobacteriales (Vaneechoutte et al., 2019 Int. J. Syst. Evol. Biol. 898661). Bifidobacteriales

[0006] During BV, the epithelial surface is covered with dense Gardnerella vaginalis, a usually refractory biofilm. Biofilms are adherent microbial communities comprising a polymeric matrix of polysaccharides, proteins and / or nucleic acids together. The unique gene expression pattern as well as the physical structure of biofilms enhances the resistance of bacteria against many negative stimuli, including chemical disinfectants, extreme pH, host immune defenses and antibiotics. The standard treatment for BV is the antibiotics Metronidazole and Clindamycin, but they usually fail to eradicate the biofilm, so the recurrence rate within 6 months is as high as 60%. Moreover, antibiotic treatment depletes the vaginal microbiota, although some live biofilm remains, this opens an ecological niche for other pathogens, e.g. fungi. Thus, a common effect of BV treatment is candidosis. Attempts have also been made to treat BV with probiotics, in particular with beneficial lactobacilli, which are said to repopulate the vagina. However, several clinical trials failed to prove their benefit.

[0007] Endolysins are promising alternatives to antibiotics at present, because of their ability to clear biofilms, their low propensity to develop resistance and their specificity for each bacterial genus or species. Naturally occurring and genetically engineered Gardnerella-specific endolysins have been described (WO 2020 / 225335 Al, Landlinger et al., (2021, Pathogens 10(1), 1-13). 10 ​​​​WO 2020 / 229802 Al). WO 2020 / 225335 describes specific recombinant Gardnerella- specific endolysins (e.g. H2B10) for the treatment of Gardnerella infections (e.g. BV), wherein the bacterial vaginosis is caused by Gardnerella vaginalis (in the narrow sense) Gardnerella vaginalis sensu stricto ), Leopoldella gardneri ( Gardnerella leopoldii ), Piottella gardneri ( Gardnerella piotii ) and / or Sweginnella gardneri ( Gardnerella swidsinskii ). Examples 6 and 7 of WO 2020 / 225335 show that, for example, endolysin H2B10 (as a representative of recombinant Gardnerella-specific endolysins) is superior to the antibiotics metronidazole and clindamycin, in particular with respect to the minimum inhibitory concentration (MIC) for Gardnerella strain suspensions grown. Thus, the results of WO 2020 / 225335 show that (recombinant) Gardnerella-specific endolysins are generally superior to antibiotics in the treatment of BV. In other words, (recombinant) Gardnerella-specific endolysins have shown to be generally more effective against Gardnerella strain suspensions, which can be derived from the fact that the MIC values for endolysins from different Gardnerella species are generally lower than the MIC values for antibiotics.

[0008] Despite these recent advances, there is still a need in the art for compositions and / or formulations that allow for a more effective treatment of Gardnerella infections. Thus, the technical problem can be formulated as providing an improved pharmaceutical composition for the treatment of infections caused by Gardnerella bacteria.

[0009] The embodiments and claims provided herein solve the technical problem. SUMMARY

[0010] The present invention relates in particular to the following embodiments: 1. A pharmaceutical composition comprising an effective amount of an active agent and a pharmaceutically acceptable excipient, wherein the active agent is a polypeptide having a killing activity against the genus Gardnerella, and wherein the excipient is a polyacrylic acid polymer.

[0011] 2. The pharmaceutical composition according to embodiment 1, wherein the polypeptide is an endolysin having a killing activity against the genus Gardnerella.

[0012] 3. The pharmaceutical composition according to embodiment 2, wherein the endolysin comprises or consists of

[0013] (i) an N-terminal catalytic domain or a functional variant thereof; (ii) a C-terminal cell wall binding region or a functional variant thereof, wherein the C-terminal cell wall binding region comprises or consists of at least one cell wall binding domain; and (iii) optionally, a linker between the N-terminal catalytic domain and the C-terminal cell wall binding region.

[0014] 4. The pharmaceutical composition according to any preceding embodiment, wherein the polypeptide is a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 37 and having a killing activity against Gardnerella.

[0015] 5. The pharmaceutical composition according to any preceding embodiment, wherein the polypeptide is a recombinant endolysin comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 37.

[0016] 6. The pharmaceutical composition according to any preceding embodiment, wherein the polypeptide has a killing activity against Gardnerella vagnalis, Gardnerella leopoldii, Gardnerella piotii and / or Gardnerella swegmarkii, or any other species in the genus Gardnerella.

[0017] 7. The pharmaceutical composition according to any preceding embodiment, wherein the polypeptide has a killing activity against Lactobacillus iners Lactobacillus iners ).

[0018] 8. The pharmaceutical composition according to any preceding embodiment, wherein the polypeptide has no killing activity against Lactobacillus crispatus Lactobacilli crispatus ), Lactobacillus gasseri Lactobacilli gasseri ) and / or Lactobacillus jensenii Lactobacilli jensenii ).

[0019] 9. The pharmaceutical composition according to any preceding embodiment, wherein the excipient is present at a concentration of about 0.01% to 10%, preferably about 0.1% to 1%, more preferably about 0.2% to 0.5%, most preferably about 0.25% or about 0.35%.

[0020] 10. The pharmaceutical composition according to any preceding embodiment, wherein the polyacrylic acid polymer is a cross-linked polyacrylic acid polymer or a carbomer.

[0021] 11. The pharmaceutical composition according to any preceding embodiment, wherein the combination of the active agent and the excipient increases the pharmaceutical activity of the active agent.

[0022] 12. The pharmaceutical composition according to any preceding embodiment, wherein the composition is suitable for intravaginal delivery of the active agent.

[0023] 13. The pharmaceutical composition according to any preceding embodiment, for use in the treatment of a bacterial infection in a subject, preferably wherein the bacterial infection is bacterial vaginosis.

[0024] 14. The pharmaceutical composition for use according to embodiment 13, wherein the subject has previously failed treatment with an antibiotic and / or wherein the infectious bacteria is resistant to antibiotic treatment.

[0025] 15. The pharmaceutical composition for use according to embodiment 14, wherein the antibiotic is a nitroimidazole and / or clindamycin.

[0026] 16. The pharmaceutical composition for use according to embodiment 14 or 15, wherein the antibiotic is metronidazole, Tinidazole, Secnidazole, clindamycin or any combination thereof.

[0027] 17. The pharmaceutical composition for use according to any one of embodiments 13 to 16, wherein the subject has recurrent bacterial vaginosis, preferably wherein the subject has two or more episodes of BV within 6 months or three or more episodes of BV within 12 months.

[0028] 18. The pharmaceutical composition for use according to any one of embodiments 13 to 17, to be administered topically into the vagina of a female subject and / or into or onto the glans penis, foreskin or urethral opening of a male subject.

[0029] 19. Use of the pharmaceutical composition according to any one of embodiments 1 to 12 for the manufacture of a medicament for the treatment or prevention of a bacterial infection, preferably bacterial vaginosis.

[0030] 20. A method of treating or preventing a bacterial infection, preferably bacterial vaginosis, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to any one of embodiments 1 to 12.

[0031] That is, the present application is based on the surprising and unexpected finding that polyacrylic acid polymers can increase the killing activity of bactericidal polypeptides against Gardnerella bacteria. In the appended Example 2, it is shown that the killing activity of the bactericidal polypeptides H2B10 (PM-477) and H2B10B11 in combination with polyacrylic acid polymers against Gardnerella biofilm is higher than the sum of the individual killing activities of the components, clearly indicating a synergistic mode of action.

[0032] While polyacrylic acid polymers show a certain degree of killing activity against Gardnerella biofilm when used alone (see Example 2), they show no killing activity against planktonic Gardnerella cells (Example 4, Figure 4 ), and also very limited killing activity against other BV-associated pathogens (Example 4, Figure 5). While polyacrylic acid polymers have limited or absent killing activity against planktonic bacteria, the attached Examples 3 and 5 (Figures 3 and 6) surprisingly demonstrate that polyacrylic acid polymers improve the killing activity of bactericidal polypeptides against planktonic Gardnerella bacterial species as well as other BV-associated pathogens such as Lactobacillus iners, Mobiluncus mulieris ), Atopobium vaginae Atopobium vaginae ), and Prevotella bivia Prevotella bivia ). Notably, bactericidal polypeptides alone have no killing activity against other BV-associated pathogens Figure IB ). Thus, it was unexpectedly discovered that polyacrylic acid polymers not only increase the killing activity of bactericidal polypeptides against Gardnerella bacterial species, but also increase the antimicrobial spectrum of bactericidal polypeptides to target other BV-associated pathogens. Notably, this surprising effect was observed in two different polyacrylic acid polymers, namely linear polyacrylic acid polymers and cross-linked polyacrylic acid polymers (Carbopol 974P).

[0033] Thus, compelling evidence is provided that bactericidal polypeptides, in particular Gardnerella-specific bactericidal polypeptides, in combination with polyacrylic acid polymer formulations can significantly increase the killing activity of bactericidal polypeptides against Gardnerella bacterial species and other BV-associated bacteria.

[0034] Thus, in one specific embodiment, the present application relates to a pharmaceutical composition comprising an effective amount of an active agent and a pharmaceutically acceptable excipient, wherein the active agent is a polypeptide having killing activity against Gardnerella, and wherein the excipient is a polyacrylic acid polymer.

[0035] The active agent comprised in the pharmaceutical composition of the present application is preferably a Gardnerella-specific bactericidal polypeptide, i.e. it specifically targets bacteria belonging to the genus Gardnerella. Thus, the polypeptide comprised in the pharmaceutical composition of the present application is preferably having killing activity against bacterial species within the genus Gardnerella. For example, the polypeptide comprised in the pharmaceutical composition of the present application can have killing activity against Gardnerella vaginalis, Gardnerella leopoldii, Gardnerella piottii, and / or Gardnerella swinkii, preferably against all of these bacterial species. More preferably, the polypeptide comprised in the pharmaceutical composition of the present application has killing activity against bacteria of the genus Gardnerella is genus-selective killing activity against bacteria of the genus Gardnerella.

[0036] "genus-selective killing activity" or "genus-specific bacteriolysis" herein refers to the lack of killing activity or bacteriolysis of the polypeptide comprised in the pharmaceutical composition of the application against bacteria in general. Preferably, the polypeptide comprised in the pharmaceutical composition of the application has a genus-selective killing activity against Gardnerella, but no killing activity against Lactobacillus crispatus, Lactobacillus gasseri and / or Lactobacillus jensenii. More preferably, the polypeptide has no killing activity against all of these Lactobacilli, i.e. Lactobacillus crispatus, Lactobacillus gasseri and Lactobacillus jensenii.

[0037] The skilled person understands that the term "genus-selective killing activity" is not to be understood in the strictest sense, given the very diversity of bacteria and the unclear boundaries between different species and genera. For example, Lactobacillus iners is classified as a species of the genus Lactobacillus, although it is significantly different from other species of the genus Lactobacillus, such as Lactobacillus crispatus, which encodes more than twice the number of proteins than Lactobacillus iners (see France et al., Microbiology 158 (12): 3240-3251). It is thus understood that an endolysin having "genus-selective killing activity" can have lytic activity against other genera of bacterial species, but preferably has no killing activity against most bacteria. Applied and Environmental Microbiology . 82 (24): 7063-7073). It is thus understood that an endolysin having "genus-selective killing activity" can have lytic activity against other genera of bacterial species, but preferably has no killing activity against most bacteria.

[0038] In certain embodiments, the polypeptide comprised in the pharmaceutical composition of the application can have killing activity against other pathogens associated with BV. That is, in certain embodiments, the polypeptide comprised in the pharmaceutical composition of the application can have killing activity against one or more of Lactobacillus iners, Mobiluncus mulieris, Atopobium vaginae and / or Prevotella bivia, especially when formulated with a polyacrylic acid polymer. In certain embodiments, the polypeptide comprised in the pharmaceutical composition of the application can have killing activity against all of Lactobacillus iners, Mobiluncus mulieris, Atopobium vaginae and Prevotella bivia, especially when formulated with a polyacrylic acid polymer.

[0039] "bactericidal activity" of an active agent, such as a bacteriocidal polypeptide, against a particular bacterium can be defined as the reduction in the number of viable bacterial cells resulting from the activity of the active agent. The bactericidal activity of an active agent against a bacterium can be complete, i.e. 100% of the bacterial cells are killed, or it can be partial, i.e. at least about 80%, at least about 90%, at least about 95%, at least about 99% or at least about 99.9% of the bacterial cells are killed. The bactericidal activity of an active agent against a particular microorganism can be determined by standard operating procedures in the art, including those based on the determination of the minimum inhibitory concentration (MIC) of an antimicrobial agent, defined as the lowest concentration of the antimicrobial agent that inhibits visible growth of the microorganism after overnight incubation, as described in Andrews, 2001, J Antimicrobial Chemotherapy, 48, Suppl. SI, 5-16The article described in or published by the Clinical and Laboratory Standards Institute (CSIS) Document M7-A7, Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; Approved standards, As described in the 7th edition, January 2006, Volume 26, Issue 2. Another suitable method for determining the bactericidal activity of an active agent is described in the Examples section of WO 2020 / 225335 and consists of measuring the decrease in optical density at 610-620 nm and / or the decrease in colony-forming units (CFU) per milliliter of bacterial cell suspension after exposure to the test active agent. The susceptibility of the bacterial suspension to be tested can be determined by an in vitro turbidity assay in the presence of the active agent, measuring the decrease in optical density at 610-620 nm. According to another embodiment, in the in vitro turbidity test, the active agent has bactericidal activity against Gardnerella spp. when it reduces the OD (610-620 nm) of a suspension of at least one Gardnerella spp. strain by more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 95%.

[0040] The polypeptides included in the pharmaceutical compositions of the present invention preferably have bactericidal activity against antibiotic-resistant Gardnerella spp. strains, as described in more detail below. In a preferred embodiment, the polypeptides included in the pharmaceutical compositions of the present invention have bactericidal activity against Gardnerella spp. strains resistant to one or more antibiotics selected from nitroimidazoles and clindamycin. In a more preferred embodiment, the polypeptides included in the pharmaceutical compositions of the present invention have bactericidal activity against Gardnerella spp. strains resistant to metronidazole, tinidazole, secnidazole, clindamycin, or any combination thereof. In an even more preferred embodiment, the polypeptides included in the pharmaceutical compositions of the present invention have bactericidal activity against Gardnerella spp. strains resistant to metronidazole and / or clindamycin. In an even more preferred embodiment, the polypeptides included in the pharmaceutical compositions of the present invention have bactericidal activity against Gardnerella spp. strains highly resistant to the aforementioned antibiotics.

[0041] The bacteriocidal polypeptide comprised in the pharmaceutical composition of the present application can be any polypeptide having a killing activity against one or more bacterial species of the genus Gardnerella. Thus, in a particular embodiment, the present application relates to a pharmaceutical composition according to the present application, wherein the polypeptide has a killing activity against G. vagnalis, G. leopoldii, G. piotii and / or G. swegmarkii or any other species of the genus Gardnerella. Means and methods for analysing whether a given polypeptide has a killing activity against a bacterial species of the genus Gardnerella are provided herein and are known to the person skilled in the art.

[0042] The pharmaceutical composition of the present application is preferably for use in the treatment of bacterial vaginosis. In certain embodiments, the pharmaceutical composition can be effective in killing other bacteria associated with bacterial vaginosis, such as L. iners, A. vagnalis, P. bivia, M. modesti, M. curtisii Mubiluncus curtisii ) and S. agalactiae Streptococcus agalactiae ), in particular when the pharmaceutical composition comprises a polyacrylic acid polymer. Thus, in a particular embodiment, the present application relates to a pharmaceutical composition according to the present application, wherein the polypeptide has a killing activity against L. iners, A. vagnalis, P. bivia, M. modesti, M. curtisii and / or S. agalactiae, in particular when formulated with a polyacrylic acid polymer.

[0043] While it is preferred herein that the polypeptide comprised in the pharmaceutical composition of the present application has a killing activity against bacterial species of the genus Gardnerella and other pathogenic bacteria associated with BV, it is important that the polypeptide comprised in the pharmaceutical composition of the present application has no or essentially no killing activity against vaginal commensal bacteria. Vaginal commensal bacteria are essential for maintaining a normal vaginal flora and include, but are not limited to, L. crispatus, L. gasseri and L. jensenii. It has been shown in the accompanying Example 3 that the bacteriocidal polypeptide H2B10 (PM-477) and in the accompanying Example 5 that the bacteriocidal polypeptide H2B10B11 have no killing activity against the vaginal commensal bacterium L. crispatus, which is one of the most prevalent bacteria in a healthy vaginal flora. Thus, in a particular embodiment, the present application relates to a pharmaceutical composition according to the present application, wherein the polypeptide has no killing activity against L. crispatus, L. gasseri and / or L. jensenii. In certain embodiments, the present application relates to a pharmaceutical composition according to the present application, wherein the polypeptide has a killing activity against L. iners, but no killing activity against L. crispatus, L. gasseri and / or L. jensenii.

[0044] That is, in certain embodiments, the polypeptide comprised in the pharmaceutical composition of the application is an endolysin having selective killing activity against G. vaginalis and against at least one of G. leopoldii, G. piontii, G. swinkii, L. iners, A. vaginae, P. bicorporeum, M. modestus, M. curtisii and / or S. agalactiae, wherein the endolysin has no or essentially no killing activity against at least one of L. crispatus, L. gasseri and / or L. jensenii.

[0045] The terms "peptide", "polypeptide", "protein" and variations of these terms refer to a peptide, oligopeptide, oligomer or protein, including fusion proteins, comprising at least two amino acids, linked by normal or modified peptide bonds, e.g. in the case of isosteric peptides. These terms also include "peptide mimetics", which are defined as peptide analogs containing non-peptide moieties that are able to mimic or antagonize the biological activity of the parent peptide. Peptide mimetics lack the classical peptide characteristics, such as enzymatically cleavable peptide bonds. A peptide or polypeptide can comprise amino acids other than the 20 amino acids encoded by the genetic code. It can comprise L-amino acids and / or D-amino acids. A peptide or polypeptide can also comprise amino acids modified naturally, e.g. by post-translational maturation processes, or chemically, which are well known to the person skilled in the art. Such modifications are described in detail in the literature. These modifications can occur anywhere in the polypeptide: in the peptide backbone, in the amino acid chain, even at the carboxy- or amino terminus. A peptide or polypeptide can form branches after ubiquitination, or can form cyclic, with or without branches. This type of modification can be the result of natural or synthetic post-translational processes well known to the person skilled in the art. For example, peptide or polypeptide modifications can include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphatidylinositol, covalent or noncovalent cross-linking, cyclization, disulfide bond formation, demethylation, glycosylation (including pegylation), hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, seneloylation, sulfation, addition of amino acids (e.g. arginylation), or ubiquitination. Such modifications are described in detail in the literature and are well known to the person skilled in the art.

[0046] In a preferred embodiment of the application, the polypeptide comprised in the pharmaceutical composition of the application is an endolysin. Thus, in a particular embodiment, the present application relates to a pharmaceutical composition of the application, wherein the polypeptide is an endolysin having killing activity against the genus Gardnerella.

[0047] The term "endolysin" as used herein refers to a polypeptide, usually produced by a bacteriophage, to digest the host bacterial cell wall and release the phage progeny. Endolysins are cell wall lytic enzymes encoded by bacteriophages that, when added exogenously, have the ability to hydrolyze the cell wall of targeted bacteria (exogenous lysis). This new class of antibacterial agents has important advantages over traditional antibiotics, such as: new mode of action; narrow sensitive bacterial spectrum; rapid killing of stationary and exponentially growing bacteria; activity against mucous membranes and bacterial biofilms; low probability of developing resistance; and reduced impact on normal microbiota. These unique properties have generated interest in lysin biotechnology and pharmaceutical development, and they have recently been included as one of the top alternatives to combat antibiotic resistance.

[0048] Endolysins are usually composed of two or more domains: at least one catalytic domain, such as a hydrolase domain (usually located at the N-terminus of the polypeptide), which cleaves specific motifs in the peptidoglycan layer, and usually one or more cell wall binding domains (classically located at the C-terminus of the polypeptide), which are involved in the specific binding and processing of bacterial peptidoglycan. While providing a general organization for endolysin structures, this typical structure is not a defining feature of all endolysins. Endolysins from Gram-positive bacteria and their bacteriophages usually contain at least one catalytic domain and one or more cell wall binding domains. Conversely, many endolysins produced by Gram-negative bacteria or their bacteriophages contain only catalytic domains, although modular endolysins have also been reported. The catalytic unit determines the type of peptidoglycan (PG) bond to be cleaved, while the cell wall binding domain, by specific recognition of cell wall elements distributed in a genus- or species / strain-specific manner, largely determines the lysis spectrum.

[0049] The endolysin comprised in the pharmaceutical composition of the present application is preferably a functional polypeptide, wherein the functionality comprises specific targeting of Gardnerella bacteria, more preferably specific killing of Gardnerella bacteria. The endolysin comprised in the pharmaceutical composition of the present application is also preferably comprised of a catalytic domain or a functional fragment thereof and / or a cell wall binding domain or a functional fragment thereof. The endolysin comprised in the pharmaceutical composition of the present application can be a natural or a recombinant endolysin. The endolysin comprised in the pharmaceutical composition of the present application is most preferably a recombinant endolysin. The endolysin comprised in the pharmaceutical composition of the present application is more preferably a recombinant endolysin, which comprises or consists of: (i) an N-terminal catalytic domain or a functional variant thereof; (ii) a C-terminal cell wall binding region or a functional variant thereof, wherein the C-terminal cell wall binding region comprises or consists of at least one cell wall binding domain; and (iii) optionally, a linker region between the N-terminal catalytic domain and the C-terminal cell wall binding region, and preferably have a killing activity, more preferably a genus-selective killing activity, on Gardnerella cells / strains.

[0050] As defined herein, the terms "bind" and "binding" when referring to the ability of an endolysin to bind to a specific bacterial cell wall refer to the ability of said endolysin to specifically interact with and adhere to said bacterial cell wall. The binding ability of an endolysin to a bacterial cell wall can be determined by methods known in the art.

[0051] In the context of the present disclosure, the term "recombinant endolysin" preferably refers to an endolysin which has been subjected to a domain exchange, as defined in WO 2020 / 225335. In accordance with this definition, it is readily understood by the skilled person that the "domain-exchanged" or "recombinant" endolysin described herein is a non-naturally occurring endolysin. That is, the recombinant endolysin used by the present application has been artificially modified and, by definition, does not encompass a natural endolysin, i.e. it can not occur naturally in nature. The accompanying examples as well as the teachings of WO 2020 / 225335 provide suitable methods of how to generate the artificial endolysins of the present application.

[0052] The term "catalytic domain" or "enzymatic domain" refers to the part of a protein chain comprising a region where catalytic chemical reactions take place. The "catalytic domain" as used herein refers to a functional polypeptide, wherein the functionality comprises the ability to modify and / or cleave a substrate in a Gardnerella cell wall, preferably peptidoglycan. Specifically, the catalytic domain described herein preferably can modify and / or cleave a substrate, preferably peptidoglycan, in a Gardnerella cell wall. Preferably, the catalytic domain cleaves peptidoglycan in a Gardnerella cell wall and can lead to lysis of the Gardnerella cell. Preferably, the catalytic domain can modify and / or cleave a bond present in a cell wall and / or peptidoglycan of a Gardnerella bacterial species, such as Gardnerella vagnalis, Gardnerella leopoldii, Gardnerella piotii and / or Gardnerella swinkii, preferably all of them. Suitably, the catalytic domain does not modify and / or cleave a substrate, preferably peptidoglycan, present in a bacterial cell wall other than a Gardnerella bacterial species, preferably a healthy vaginal commensal bacterium, such as a Lactobacillus bacterial species, including Lactobacillus crispatus, Lactobacillus gasseri and / or Lactobacillus jensenii. The catalytic domain can be an N-acetylmuramidase, an N-acetylmuramoyl-L-alanine amidase, an L-alanyl-D-glutamate peptidase, an interpeptide bridge endopeptidase or an N-acetyl-beta-D-glucosaminidase. Preferably, the N-terminal catalytic domain is an N-acetylmuramidase, most preferably a 1,4-beta-N-acetylmuramidase. The catalytic domain is preferably located N-terminal within the (recombinant) endolysin, thereby being referred to as "N-terminal catalytic domain", even more preferably the N-terminal catalytic domain is located N-terminal to the C-terminal cell wall binding region within the (recombinant) endolysin.

[0053] The endolysin comprised in the pharmaceutical composition of the present application preferably comprises a catalytic domain consisting of a polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 2 to 10 or any functional variant thereof having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 2 to 10. As shown in WO 2020 / 225335, the catalytic domain with the highest activity is “H2” (SEQ ID NO: 3). Thus, in a preferred aspect of the present application, the catalytic domain consists of a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 3 or any functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) to the amino acid sequence of SEQ ID NO: 3, whereby the endolysin is functional, wherein the functionality comprises the ability to lyse cell walls of Gardnerella spp. The catalytic domain is preferably located N-terminal within the (recombinant) endolysin, whereby it is referred to as “N-terminal catalytic domain”, even more preferably, the (recombinant) endolysin further comprises a cell wall binding region, and the N-terminal catalytic domain is located N-terminal to the C-terminal cell wall binding region within the (recombinant) endolysin.

[0054] "Cell wall binding region" as used herein refers to a functional polypeptide, wherein the functionality comprises the ability to bind to the cell wall of Gardnerella. The cell wall binding region can comprise or consist of one, two, three or more cell wall binding domains. A cell wall binding domain is a polypeptide that interacts with and / or binds to the bacterial cell wall and / or a specific substrate within the bacterial cell wall. In particular, the cell wall binding domains described herein can bind specifically to the cell wall (e.g. peptidoglycan) of Gardnerella bacterial species, such as Gardnerella vaginalis, Gardnerella leopoldii, Gardnerella piotii and / or Gardnerella swinkii, preferably all of these species. The cell wall binding region is preferably located C-terminal within the (recombinant) endolysin, thereby referred to as "C-terminal cell wall binding region", even more preferably the C-terminal cell wall binding region is located C-terminal to the N-terminal catalytic domain within the (recombinant) endolysin.

[0055] The endolysin comprised in the pharmaceutical composition of the application preferably comprises a cell wall binding region comprising or consisting of at least one cell wall binding domain selected from the group of polypeptides comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 11 to 28 and any functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) to the amino acid sequence of any one of SEQ ID NOs: 11 to 28. As shown in WO 2020 / 225335, the most active cell wall binding region is “B10” (comprising the cell wall binding domains of SEQ ID NOs: 23 and 24), followed by “B11” (comprising the cell wall binding domains of SEQ ID NOs: 25 and 26).Thus, in a preferred aspect of the application, the cell wall binding domain is selected from the group of polypeptides comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 23, 24, 25 and 26 and any functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) to the amino acid sequence of any one of SEQ ID NOs: 23, 24, 25 and 26, whereby the endolysin is functional, wherein the function comprises the ability to lyse a cell wall of a Gardnerella species. The cell wall binding region is preferably located at the C-terminus within the (recombinant) endolysin, thus referred to as "C-terminal cell wall binding region", even more preferably, the (recombinant) endolysin further comprises a catalytic domain and the catalytic domain is located N-terminal to the C-terminal cell wall binding region within the (recombinant) endolysin.

[0056] The endolysin comprised in the pharmaceutical composition of the application preferably comprises two cell wall binding domains (within the cell wall binding region). In a preferred aspect of the application, the cell wall binding domains of the endolysin of the application each consist of a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 23, 24, 25 and 26 and any functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) to the amino acid sequence of any one of SEQ ID NOs: 23, 24, 25 and 26, or consists of a functional variant of the amino acid sequence of any one of SEQ ID NOs: 23, 24, 25 and 26 and any functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) to the amino acid sequence of any one of SEQ ID NOs: 23, 24, 25 and 26, whereby the endolysin is functional, wherein the functionality comprises the ability to lyse the cell wall of a Gardnerella species. In an even more preferred aspect of the application, the endolysin comprises a first cell wall binding domain and a second cell wall binding domain, wherein the first cell wall binding domain is selected from the group consisting of SEQ ID NOs: 23 and 25, and the second cell wall binding domain is selected from the group consisting of SEQ ID NOs: 24 and 26. Preferably, the first cell wall binding domain is located N-terminal of the second cell wall binding domain.

[0057] In a more preferred embodiment, the endolysin comprised in the pharmaceutical composition of the application comprises

[0058] (i) an N-terminal catalytic domain consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or any functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) to the amino acid sequence of SEQ ID NO: 3 or consisting of the amino acid sequence of SEQ ID NO: 3 or any functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) to the amino acid sequence of SEQ ID NO: 3; and

[0059] (ii) a C-terminal cell wall binding region comprising or consisting of a first cell wall binding domain and a second cell wall binding domain, wherein the first cell wall binding domain is selected from the group consisting of SEQ ID NOs: 23 and 25 and any functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) to the amino acid sequence of any of SEQ ID NOs: 23 and 25; and wherein the second cell wall binding domain is selected from the group consisting of SEQ ID NOs: 24 and 26 and any functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) to the amino acid sequence of any of SEQ ID NOs: 24 and 26; whereby the endolysin is functional, wherein the functionality comprises the ability to lyse a cell wall of a Gardnerella cell. Preferably, the first cell wall binding domain is located N-terminal of the second cell wall binding domain.

[0060] In a particularly preferred embodiment, the endolysin comprised in the pharmaceutical composition of the application comprises

[0061] (i) an N-terminal catalytic domain consisting of a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 3; and

[0062] (ii) a C-terminal cell wall binding region comprising or consisting of a first cell wall binding domain and a second cell wall binding domain, wherein the first cell wall binding domain is selected from the group consisting of SEQ ID NOs: 23 and 25, and the second cell wall binding domain is selected from the group consisting of SEQ ID NOs: 24 and 26.

[0063] Preferably, the first cell wall binding domain is located N-terminal to the second cell wall binding domain.

[0064] Exemplary examples of particularly preferred endolysins comprised in the pharmaceutical composition of the application are “H2B10” (comprising from N-terminal to C-terminal: SEQ ID NO: 3, 23 and 24), “H2B11” (comprising from N-terminal to C-terminal: SEQ ID NO: 3, 25 and 26) and “H2B10B11” (comprising from N-terminal to C-terminal: SEQ ID NO: 3, 23 and 26) as described in WO 2020 / 225335.

[0065] The endolysin comprised in the pharmaceutical composition of the application preferably further comprises a linker region between the N-terminal catalytic domain and the C-terminal cell wall binding region. The linker region can consist of a polypeptide having a length of 6 to 18 amino acids, preferably 9 to 15 amino acids, even more preferably 12 amino acids. Preferably, the linker region can consist of a polypeptide comprising or consisting of (i) (XXX)n, wherein each X can independently be G, A or S, preferably wherein the amino acid sequence (XXX)n is (GGS)n, wherein n corresponds to the number of repetitions of the sequence XXX, preferably wherein n is 2, 3, 4, 5 or 6, or (ii) X1X2GLNGX3X4NGGS (SEQ ID NO: 36), wherein X1 is N or K, X2 is A or V, X3 is Y or C, and X4 is K or Q. Non-limiting examples of such linker regions are provided in SEQ ID NOs: 29 to 35.

[0066] In a particularly preferred embodiment, the endolysin comprised in the pharmaceutical composition of the application is "H2B10", the sequence of which is shown in SEQ ID NO: 1, or "H2B10B11 ", the sequence of which is shown in SEQ ID NO: 37.

[0067] In a particular embodiment, the present application relates to a pharmaceutical composition according to the application, wherein the polypeptide is a polypeptide having at least 80% sequence identity to the amino acid sequence provided in SEQ ID NO: 1 and having a killing activity against the genus Gardnerella.

[0068] That is, in certain embodiments, the present application provides a pharmaceutical composition comprising an effective amount of a Gardnerella-specific endolysin, preferably a Gardnerella-specific recombinant endolysin, and a polyacrylic acid polymer, wherein the endolysin is a polypeptide having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 1 and having a killing activity against the genus Gardnerella. In a more preferred embodiment, the present application provides a pharmaceutical composition comprising an effective amount of a Gardnerella-specific endolysin, preferably a Gardnerella-specific recombinant endolysin, and a polyacrylic acid polymer, wherein the endolysin is a polypeptide having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 1 and having a killing activity against the genus Gardnerella. In an even more preferred embodiment, the present application provides a pharmaceutical composition comprising an effective amount of a Gardnerella-specific endolysin, preferably a Gardnerella-specific recombinant endolysin, and a polyacrylic acid polymer, wherein the endolysin is a polypeptide having at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 1 and having a killing activity against the genus Gardnerella. In an even more preferred embodiment, the present application provides a pharmaceutical composition comprising an effective amount of a Gardnerella-specific endolysin, preferably a Gardnerella-specific recombinant endolysin, and a polyacrylic acid polymer, wherein the endolysin is a polypeptide having at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 1 and having a killing activity against the genus Gardnerella. In a most preferred embodiment, the present application provides a pharmaceutical composition comprising an effective amount of a Gardnerella-specific endolysin, preferably a Gardnerella-specific recombinant endolysin, and a polyacrylic acid polymer, wherein the endolysin comprises or consists of the amino acid sequence shown in SEQ ID NO: 1.

[0069] Thus, in a particular embodiment, the present application relates to a pharmaceutical composition according to the application, wherein the polypeptide is a recombinant endolysin comprising the amino acid sequence shown in SEQ ID NO: 1.

[0070] An endolysin comprising or consisting of the amino acid sequence as shown in SEQ ID NO: 37 can also be used in the present application, i.e. in a pharmaceutical composition suitable for the treatment of bacterial vaginosis, the pharmaceutical composition comprising an effective amount of a Gardnerella-specific endolysin, preferably a Gardnerella-specific recombinant endolysin, and a polyacrylic acid polymer. Thus, an endolysin comprising or consisting of the amino acid sequence as shown in SEQ ID NO: 37 is an alternative to the Gardnerella-specific endolysin as shown in SEQ ID NO: 1.

[0071] Thus, in a specific embodiment, the present application relates to a pharmaceutical composition according to the present application, wherein the polypeptide is a polypeptide having at least 80% sequence identity to the amino acid sequence as shown in SEQ ID NO: 37 and having a killing activity against Gardnerella.

[0072] Thus, in certain embodiments, the present application provides a pharmaceutical composition comprising an effective amount of a Gardnerella-specific endolysin, preferably a Gardnerella-specific recombinant endolysin, and a polyacrylic acid polymer, wherein the endolysin is a polypeptide having at least 80% sequence identity to the amino acid sequence as shown in SEQ ID NO: 37 and having a killing activity against Gardnerella. In a more preferred embodiment, the present application provides a pharmaceutical composition comprising an effective amount of a Gardnerella-specific endolysin, preferably a Gardnerella-specific recombinant endolysin, and a polyacrylic acid polymer, wherein the endolysin is a polypeptide having at least 90% sequence identity to the amino acid sequence as shown in SEQ ID NO: 37 and having a killing activity against Gardnerella. In an even more preferred embodiment, the present application provides a pharmaceutical composition comprising an effective amount of a Gardnerella-specific endolysin, preferably a Gardnerella-specific recombinant endolysin, and a polyacrylic acid polymer, wherein the endolysin is a polypeptide having at least 95% sequence identity to the amino acid sequence as shown in SEQ ID NO: 37 and having a killing activity against Gardnerella. In an even more preferred embodiment, the present application provides a pharmaceutical composition comprising an effective amount of a Gardnerella-specific endolysin, preferably a Gardnerella-specific recombinant endolysin, and a polyacrylic acid polymer, wherein the endolysin is a polypeptide having at least 99% sequence identity to the amino acid sequence as shown in SEQ ID NO: 37 and having a killing activity against Gardnerella. In a most preferred embodiment, the present application provides a pharmaceutical composition comprising an effective amount of a Gardnerella-specific endolysin, preferably a Gardnerella-specific recombinant endolysin, and a polyacrylic acid polymer, wherein the endolysin comprises or consists of the amino acid sequence as shown in SEQ ID NO: 37.

[0073] Thus, in a particular embodiment, the present application relates to a pharmaceutical composition according to the present application, wherein the polypeptide is a recombinant endolysin comprising the amino acid sequence of SEQ ID NO: 37.

[0074] In one aspect, the present application relates to a pharmaceutical composition comprising a recombinant endolysin having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 37 and having a killing activity against Lactobacillus iners for use in the treatment of a bacterial infection involving and / or caused by Lactobacillus iners species.

[0075] Lactobacillus iners is a bacterial species associated with an unbalanced vagina and is known to be potentially associated with BV situations. Patients receiving metronidazole (MDZ) treatment often shift to a Lactobacillus iners -dominated microbiota, followed by recurrence of BV. Thus, endolysins having (additional) killing activity against Lactobacillus iners are particularly relevant in the treatment of BV.

[0076] Preferably, the present application relates to a pharmaceutical composition comprising a recombinant endolysin comprising the amino acid sequence of SEQ ID NO: 1 or 37 for use in the treatment of a bacterial infection involving and / or caused by Lactobacillus iners. Figure IB , Figure 3 and Figure 6 Surprisingly, it was shown that endolysins PM-477 and H2B10B11 have a killing activity against Lactobacillus iners in the absence and in the presence of a polyacrylic acid polymer. In certain embodiments, the bacterial infection caused by Lactobacillus iners is a vaginal infection, in particular bacterial vaginosis. It is understood that any of the embodiments disclosed herein are equally applicable to the composition for use in the treatment of Lactobacillus iners. In particular, the composition can further comprise a polyacrylic acid polymer as disclosed elsewhere herein. In certain embodiments, the present application relates to a method for treating an infection involving and / or caused by Lactobacillus iners in a subject in need thereof, the method comprising the step of administering to said patient a recombinant endolysin having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 37 and having a killing activity against Lactobacillus iners. In certain embodiments, the present application relates to a method for reducing the number of Lactobacillus iners in the vagina of a subject in need thereof, the method comprising the step of administering to said patient a recombinant endolysin having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 37 and having a killing activity against Lactobacillus iners.

[0077] The term "variant" refers to a polypeptide comprising insertions, deletions and / or substitutions relative to the native amino acid sequence, which are non-conservative or are preferably conservative. For example, the polypeptide can comprise an amino acid sequence which is at least 80% identical to the native amino acid sequence, preferably at least 85% identical, more preferably at least 90% identical, even more preferably at least 95% identical, even more preferably at least 96% identical, even more preferably at least 97% identical, even more preferably at least 98% identical, even more preferably at least 99% identical, even more preferably at least 99.5% identical, most preferably at least 99.7% identical to said amino acid sequence. The percentage identity can be determined by methods known in the art using suitable computer programs, for example MatGAT 2.0 (Myers and Miller, CABIOS (1989)). Preferably, the % identity is determined over the entire length of the sequences to be compared. It will be appreciated that the percentage identity is calculated with respect to the polypeptides which have been optimally aligned. Fragments and variants of the amino acid sequence can be prepared using any of the protein engineering, directed evolution and / or site-directed mutagenesis methods known in the art (for example, see Molecular Cloning: a Laboratory Manual, 3rd edition , Sambrook & Russell, 2001, Cold Spring Harbor Laboratory Press). It will be appreciated by those skilled in the art that the polypeptides of the application, or fragments, variants or fusions thereof, can comprise or consist of derivatives of the native amino acid sequence, or fragments or variants thereof. Chemical derivatives of one or more amino acids can be obtained by reaction with functional side groups. Such derivatizing molecules include, for example, those in which free amino groups have been derivatized to form amine salts, p-toluenesulfonyl, carboxybenzyloxy, t-butyloxycarbonyl, chloroacetyl or formyl groups. Free carboxyl groups can be derivatized to form salts, methyl and ethyl esters or other types of esters and hydrazides. Free hydroxyl groups can be derivatized to form O-acyl or O-alkyl derivatives. Also included as chemical derivatives are peptides containing naturally occurring amino acid derivatives. For example: proline can be replaced with 4-hydroxyproline; lysine can be replaced with 5- hydroxylysine; histidine can be replaced with 3-methylhistidine; serine can be replaced with homoserine; and lysine can be replaced with ornithine. Derivatives also include peptides containing one or more additions or deletions, provided that the desired activity is retained. Other included modifications are amidation, amino-terminal acylation (e.g., acetylation or mercaptoacetic acid amidation), carboxy-terminal amidation (e.g., with ammonia or methylamine), and the like. It will further be appreciated by those skilled in the art that peptide mimetic compounds can also be used. Thus, "polypeptide" includes peptide mimetic compounds which exhibit endolysin activity. The term "peptide mimetic" refers to a compound which mimics the conformation and desired characteristics of a particular polypeptide as a therapeutic agent.

[0078] The compositions of the present application can comprise one or more endolysin polypeptides. In certain embodiments, the endolysin polypeptide can be present as an independent polypeptide, or can be present as a fusion protein comprising the endolysin polypeptide or a fragment thereof.

[0079] The term "pharmaceutical composition" refers to a preparation which is in such form as to permit biological activity and is in a form which is suitable for administration to a patient. As used herein, "pharmaceutical composition" refers to a therapeutically effective preparation for use in the methods of the present application. As used herein, "therapeutically effective amount" or "effective amount" or "therapeutically effective" refers to an amount that provides a therapeutic effect on the particular condition and dosage regimen being employed. This is a predetermined quantity of active material calculated to produce a desired therapeutic effect in conjunction with the required diluent, i.e., carrier or dosage vehicle. Further, it is intended to mean an amount adequate to alleviate and most preferably prevent, clinically significant deficiency in host activity, function and response. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in a host. Those skilled in the art understand that the amount of a compound can vary depending on its particular activity. Suitable dosages can comprise a predetermined quantity of active ingredient calculated to produce a desired therapeutic effect in conjunction with a required diluent. In the methods and uses for manufacture of the compositions of the present application, a therapeutically effective amount of active ingredient is provided. The therapeutically effective amount can be determined by an ordinarily skilled medical or veterinary worker depending on patient characteristics such as age, weight, sex, condition, complications, other diseases, etc., which are well known in the art.

[0080] In one embodiment of the present application, the pharmaceutical composition comprises a Gardnerella-specific endolysin as described herein and is used for the treatment of bacterial vaginosis as described herein. Thus, the pharmaceutical composition can comprise an amount of an endolysin or a fragment, variant, fusion or derivative thereof sufficient to inhibit the growth of at least some Gardnerella cells in a patient as described herein above, wherein the patient is infected or susceptible to infection with such cells. Preferably, the pharmaceutical composition comprises an amount of an endolysin or a fragment, variant, fusion or derivative thereof sufficient to kill Gardnerella cells in a patient to be treated as described herein above.

[0081] The pharmaceutical composition of the present application further comprises a pharmaceutically acceptable excipient. The term "pharmaceutically acceptable excipient" refers to a material that comprises no biologically or otherwise undesirable properties. The term "carrier" refers to any component present in the pharmaceutical formulation other than the active agent, and thus includes excipients, diluents, binders, lubricants, disintegrants, fillers, colorants, wetting or emulsifying agents, pH buffering agents, preservatives, etc.

[0082] The present inventors have surprisingly found that certain excipients, namely polyacrylic acid polymers, can increase the killing activity of bactericidal polypeptides, in particular Gardnerella-specific endolysins. Accordingly, in one specific embodiment, the present application relates to a pharmaceutical composition according to the present application, wherein the combination of the active agent and the excipient increases the pharmaceutical activity of the active agent.

[0083] In the present application, an excipient, in particular a polyacrylic acid polymer, "increases the pharmaceutical activity of an active agent, i.e. a Gardnerella-specific bactericidal polypeptide", if the excipient increases the killing activity of the active agent, i.e. a Gardnerella-specific bactericidal polypeptide, against bacteria of the bacterial species Gardnerella. The excipient can increase the pharmaceutical activity of the active agent by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or at least 80%. It can be seen from Table 1 that the excipient PAA increases the killing activity of the active agent PM-477 against Gardnerella biofilm by almost 100% and even more so for the active ingredient H2B10B11. Figure 2

[0084] The term "polyacrylic acid polymer" as used herein refers to polymers of acrylic acid and includes homopolymers, copolymers and cross-linked polymers of acrylic acid. That is, a polyacrylic acid polymer is a polymer comprising polymers of the formula (CH2-CHCO2H) n or consisting of polymers of the formula (CH2-CHCO2H) n .

[0085] In certain embodiments, the polyacrylic acid polymer is a linear polyacrylic acid polymer, in particular a linear polyacrylic acid homopolymer. Example 2 has shown that such polymers significantly increase the killing activity of endolysins against Gardnerella biofilm. Furthermore, Example 3 has shown that linear polyacrylic acid homopolymers can expand the antimicrobial spectrum of the endolysin PM-477 to other BV-associated bacteria.

[0086] In certain embodiments, the "polyacrylic acid polymer" is a cross-linked polyacrylic acid polymer, such as a carbomer or a carboxyvinyl polymer. Cross-linked polyacrylic acid polymers are homopolymers or copolymers of acrylic acid cross-linked with at least one other polymer. The term "carbomer" refers to a series of polymers of acrylic acid commonly used in pharmaceutical compositions. Carbomers are high molecular weight homopolymers or copolymers of acrylic acid cross-linked with a polyalkyi ether of a sugar or a polyol. The carbomer comprised in the pharmaceutical composition of the present application is preferably a carbomer complying with the requirements of the United States and / or European Pharmacopoeia. Carbomers are commercially available under the trade name Carbopol. Accordingly, in one specific embodiment, the present application relates to a pharmaceutical composition according to the present application, wherein the excipient is a carbomer, preferably a Carbopol. ​

[0087] In certain embodiments, the pharmaceutical composition of the present application comprises a specific type of carbomer. In certain embodiments, the pharmaceutical composition of the present application comprises a mixture of two or more different types of carbomer.

[0088] The polyacrylic acid polymer comprised in the pharmaceutical composition of the present application can be defined based on one or more physicochemical properties.

[0089] Preferably, the polyacrylic acid polymer comprised in the pharmaceutical composition of the present application is defined based on its viscosity. That is, the polyacrylic acid polymer comprised in the pharmaceutical composition of the present application can have a viscosity range of 300-115000 mPa-s in 0.5% (w / v) gel form (25°C pH 7.3-7.8).

[0090] In certain embodiments, the polyacrylic acid polymer comprised in the pharmaceutical composition is a Carbomer Type A according to the United States Pharmacopeia, which has a viscosity of 4000-11000 mPa-s in 0.5% (w / v) gel form (25°C pH 7.3-7.8). In certain embodiments, the Carbomer Type A is Carbopol 981, Carbopol 971 or Carbopol 71G.

[0091] In certain embodiments, the polyacrylic acid polymer comprised in the pharmaceutical composition is a Carbomer Type B according to the United States Pharmacopeia, which has a viscosity of 25000 - 45000 mPa-s in 0.5% (w / v) gel form (25°C pH 7.3-7.8). In certain embodiments, the Carbomer Type B is Carbopol 974P, Carbopol 984 or Carbopol 5984, preferably Carbopol 974P.

[0092] In certain embodiments, the polyacrylic acid polymer comprised in the pharmaceutical composition is a Carbomer Type C according to the United States Pharmacopeia, which has a viscosity of 40000 - 60000 mPa-s in 0.5% (w / v) gel form (25°C pH 7.3-7.8). In certain embodiments, the Carbomer Type C is Carbopol 980.

[0093] In certain embodiments, the polyacrylic acid polymer comprised in the pharmaceutical composition of the application has a viscosity of 300 - 115000 mPa-s in 0.5% (w / v) gel form (25°C pH 7.3 - 7.8). In certain embodiments, the polyacrylic acid polymer comprised in the pharmaceutical composition of the application has a viscosity of 300 - 60000 mPa-s in 0.5% (w / v) gel form (25°C pH 7.3 - 7.8). In certain embodiments, the polyacrylic acid polymer comprised in the pharmaceutical composition of the application has a viscosity of 300 - 45000 mPa-s in 0.5% (w / v) gel form (25°C pH 7.3 - 7.8). In certain embodiments, the polyacrylic acid polymer comprised in the pharmaceutical composition of the application has a viscosity of 300 - 11000 mPa-s in 0.5% (w / v) gel form (25°C pH 7.3 - 7.8). In certain embodiments, the polyacrylic acid polymer comprised in the pharmaceutical composition of the application has a viscosity of 300 - 4000 mPa-s in 0.5% (w / v) gel form (25°C pH 7.3 - 7.8). In certain embodiments, the polyacrylic acid polymer comprised in the pharmaceutical composition of the application has a viscosity of 300 - 2000 mPa-s in 0.5% (w / v) gel form (25°C pH 7.3 - 7.8).

[0094] In certain embodiments, the polyacrylic acid polymer comprised in the pharmaceutical composition of the application is a linear polyacrylic acid homopolymer having a viscosity of 300 - 2000 mPa-s in 0.5% (w / v) gel form (25°C pH 7.3 - 7.8).

[0095] In certain embodiments, the polyacrylic acid polymer comprised in the pharmaceutical composition of the application is a B-type carbomer, such as Carbomer 974P, having a viscosity of 25000 - 45000 mPa-s in 0.5% (w / v) gel form (25°C pH 7.3 - 7.8).

[0096] In one particular embodiment, the present application relates to a pharmaceutical composition according to the present application, wherein the excipient is present in a concentration of about 0.1% and 10%.

[0097] That is, the polyacrylic acid polymer can be present in the pharmaceutical composition of the present application at a concentration of about 0.1%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5%, about 5.1%, about 5.2%, about 5.3%, about 5.4%, about 5.5%, about 5.6%, about 5.7%, about 5.8%, about 5.9%, about 6%, about 6.1%, about 6.2%, about 6.3%, about 6.4%, about 6.5%, about 6.6%, about 6.7%, about 6.8%, about 6.9%, about 7%, about 7.1%, about 7.2%, about 7.3%, about 7.4%, about 7.5%, about 7.6%, about 7.7%, about 7.8%, about 7.9%, about 8%, about 8.1%, about 8.2%, about 8.3%, about 8.4%, about 8.5%, about 8.6%, about 8.7%, about 8.8%, about 8.9%, about 9%, about 9.1%, about 9.2%, about 9.3%, about 9.4%, about 9.5%, about 9.6%, about 9.7%, about 9.8%, about 9.9%, or about 10%.

[0098] In a particularly preferred embodiment, the present application relates to the pharmaceutical composition of the present application, wherein the excipient is present at a concentration of about 0.1% to 5%, more preferably about 0.1% to 2.5%, even more preferably about 0.1% to 1%, most preferably about 0.2% to 0.5%. In a particularly preferred embodiment, the excipient, in particular the polyacrylic acid polymer, is present at a concentration of about 0.25% or 0.35%.

[0099] In certain embodiments, the pharmaceutical composition of the present application further comprises a salt. Figure 7Surprisingly, it has been shown that increasing the salt concentration in the culture medium enhances the activity of the endolysin when formulated together with the polyacrylic acid polymer. The term "salt" as used herein generally refers to ionic compounds that comprise both a cation (positively charged ion) and an anion (negatively charged ion), thus the product is electrically neutral (does not carry a net charge). These component ions can be inorganic or organic ions, monoatomic or polyatomic ions, monovalent or polyvalent ions. Preferably, the salt comprised in the pharmaceutical composition of the present application is a pharmaceutically acceptable salt. In a preferred embodiment, the salt comprised in the pharmaceutical composition of the present application is sodium chloride (NaCI).

[0100] The salt, in particular NaCI, can be present in the composition in any suitable concentration. In certain embodiments, the pharmaceutical composition of the present application comprises NaCI in a concentration ranging from about 0% to about 12.5%, preferably from about 1% to about 10%, more preferably from about 2.5% to about 7.5%. In certain embodiments, the pharmaceutical composition of the present application comprises NaCI in a concentration of about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 10.5%, about 11%, about 11.5%, about 12%, or about 12.5%. In certain embodiments, the pharmaceutical composition of the present application comprises NaCI in a concentration of about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 10.5%, about 11%, about 11.5%, about 12%, or about 12.5%.

[0101] It is to be understood that the concentrations provided herein are to be understood as weight / volume percent concentrations (w / v%) when the composition is formulated as a liquid, and as weight / weight percent concentrations (w / w%) when the composition is formulated as a solid.

[0102] The pharmaceutical composition provided herein can be used in a solid (e.g. tablet or filled capsule) or liquid (e.g. solution, suspension, emulsion, elixir) or a capsule filled with the pharmaceutical composition, all of which can be used for oral administration, or in the form of a suppository for local (including intravaginal) administration. Furthermore, other modes of administration are also contemplated by the present application. Such modes can include, inter alia, parenteral administration.

[0103] Also contemplated are pharmaceutical compositions for topical body surface and / or topical administration, such as pharmaceutical compositions in the form of (topical body surface) gels, lotions, or creams, or pharmaceutical compositions for administration via a vaginal suppository (i.e., a pessary). Such vaginal suppositories / pessaries can be coated with a pharmaceutical composition described herein. The pharmaceutical compositions and unit dosage forms thereof can comprise conventional ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms can contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage Remington of "The Science and Practice of Pharmacy” Fifth Part ， The University of Pennsylvania, Philadelphia, PA, 22ndEdition, 2012, Lippincott Williams & Wilkins are shown in

[0104] The solid compositions of the present application can be in unit dosage form, in conventional methods of preparation. Tablets can be coated according to methods well known in the art. Compositions for injection are typically based on injectable sterile saline or phosphate-buffered saline or other injectable carriers known in the art.

[0105] The pharmaceutical compositions of the present application can also be formulated as suppositories, which can contain the suppository base, including but not limited to cocoa butter or glycerides. The compositions of the present application can also be formulated for transdermal administration, including but not limited to creams, ointments, lotions, pastes, medicated patches, or films. The compositions of the present application can also be formulated for parenteral administration, including but not limited to injection or continuous infusion. Formulations for injection can be suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents including but not limited to suspending, stabilizing, and dispersing agents. The compositions can also be provided in powder form for reconstitution with a suitable vehicle, including but not limited to sterile pyrogen-free water.

[0106] The pharmaceutical compositions of the present application can also be formulated in a depot preparation, which can be administered by implantation or by intramuscular injection. The composition can be formulated with suitable polymers or hydrophobic materials (e.g., an emulsion in an acceptable oil), ion exchange resins, or sparingly soluble derivatives (e.g., sparingly soluble salts).

[0107] The pharmaceutical compositions of the present application can also be administered in sustained release forms or from sustained release drug delivery systems. Descriptions of representative sustained release materials can also be found in Remington "The Science and Practice of Pharmacy”

[0108] The pharmaceutical compositions of the present application can further comprise one or more pharmaceutically acceptable additional ingredients, such as alum, stabilizers, antimicrobial agents, buffers, colorants, flavoring agents, adjuvants, and the like. The pharmaceutical compositions of the present application preferably do not comprise imidazole.

[0109] The pharmaceutical compositions of the present application are preferably used for the treatment of bacterial vaginosis. Thus, the pharmaceutical compositions of the present application are preferably formulated for intravaginal delivery. Accordingly, in a particular embodiment, the present application relates to a pharmaceutical composition of the present application, wherein said composition is suitable for intravaginal delivery of said active agent. A person skilled in the art is able to formulate a pharmaceutical composition comprising the active agents disclosed herein and excipients suitable for intravaginal delivery.

[0110] However, the pharmaceutical compositions of the present application can also be used for the treatment of bacterial infections, in particular Gardnerella infections in male subjects. In such embodiments, the pharmaceutical compositions of the present application are preferably formulated for administration into or onto the glans, foreskin, or urethral opening. A person skilled in the art is able to formulate a pharmaceutical composition comprising the active agents disclosed herein and excipients suitable for delivery into or onto the glans, foreskin, or urethral opening.

[0111] In a particular embodiment, the present application relates to the use of a pharmaceutical composition of the present application for the treatment of a bacterial infection, preferably bacterial vaginosis.

[0112] That is, the pharmaceutical compositions of the present application comprising a Gardnerella-specific polypeptide and a polyacrylic acid polymer can be used for the treatment of bacterial infections, in particular bacterial infections caused by Gardnerella bacteria. Thus, the pharmaceutical compositions of the present application can be used for the treatment of infections caused by one or more G. vaginalis, G. leopoldii, G. piotii, and / or G. swegmarkii, but are not limited thereto.

[0113] ​​Gardnerella bacterial species, such as Gardnerella vaginalis, are the most common cause of bacterial vaginosis. As used herein, “bacterial vaginosis” (BV), also referred to in the literature as bacterial vaginitis, nonspecific vaginosis, and nonspecific vaginitis, refers to the most common vaginal infection in the world. In one embodiment, BV is defined as a pathological condition characterized by the loss of H2O2-producing species of the normal vaginal flora, in particular Lactobacillus species, and the simultaneous overgrowth of anaerobes, typically including anaerobes of the genus Gardnerella. The genus Gardnerella comprises at least 13 species, and the most common species have been renamed as Gardnerella vagnalis s.s., Gardnerella leopoldii, Gardnerella piotii, and Gardnerella swinkii (Vaneechoutte et al. 2019 Int. J. Syst. Evol. Biol. 898661). In a preferred embodiment of the therapeutic use of the present application, the BV to be treated is a bacterial infection characterized by the presence of at least one strain of the genus Gardnerella selected from Gardnerella vagnalis s.s., Gardnerella leopoldii, Gardnerella piotii, and Gardnerella swinkii and any other Gardnerella species. Thus, in one embodiment, the pharmaceutical composition of the present application comprises a Gardnerella-specific endolysin as described herein, preferably a recombinant Gardnerella-specific endolysin, for use in the treatment of bacterial vaginosis, wherein said bacterial vaginosis is characterized by the presence of an infectious bacterium of the species Gardnerella vagnalis s.s., Gardnerella leopoldii, Gardnerella piotii, Gardnerella swinkii and / or an infectious bacterium of any other Gardnerella species. It is understood that bacterial vaginosis “characterized by the presence of” certain species of the genus Gardnerella (or “caused by certain species of the genus Gardnerella” used interchangeably herein) refers to the overgrowth of said bacteria (also referred to herein as “infectious bacteria”) in the vaginal flora of a patient, leading to a vaginal flora dysbiosis and / or loss of lactobacilli dominance. Methods to determine whether bacterial vaginosis is characterized by the presence of infectious bacteria of a species of the genus Gardnerella are known to the person skilled in the art. For example, BV can be diagnosed using a PCR test that checks for the presence of Gardnerella strains.

[0114] It is important to understand that Gardnerella infections can also affect men, where they can cause severe and malodorous inflammation and swelling of the inner foreskin and glans penis. Thus, the pharmaceutical composition of the present application can also be used for the treatment of Gardnerella infections in male subjects, in particular when the infection affects the glans penis, foreskin, or urethral opening.

[0115] Thus, in a particular embodiment, the present application relates to a pharmaceutical composition for use according to the present application, to be locally administered into the vagina of a female subject and / or into or onto the glans penis, foreskin, or urethral opening of a male subject.

[0116] In one particular embodiment, the present application relates to the use of a pharmaceutical composition of the present application for the treatment of bacterial vaginosis, wherein the polypeptide, in particular endolysin, is to be administered to a patient who has failed a previous treatment with an antibiotic and / or who has bacterial vaginosis with an infectious bacterium that is resistant to antibiotic treatment.

[0117] That is, in certain embodiments, the BV to be treated with the pharmaceutical composition described herein is characterized by the presence of a Gardnerella strain (i.e., a strain from the genus Gardnerella) that is resistant to one or more antibiotics. In a preferred embodiment, the one or more antibiotics is selected from the group consisting of nitroimidazoles and clindamycin. In an even more preferred embodiment, the Gardnerella strain is resistant to metronidazole, tinidazole, secnidazole, clindamycin, or any combination thereof. In an even more preferred embodiment, the Gardnerella strain is resistant to metronidazole and / or clindamycin. In an even more preferred embodiment, the Gardnerella strain is resistant to metronidazole. In a most preferred embodiment, the Gardnerella strain is resistant to metronidazole to highly resistant.

[0118] As used herein, a "patient who has failed a previous treatment with an antibiotic" refers to a patient who has a history of bacterial vaginosis, i.e., who has previously been infected (or has experienced) symptoms of bacterial vaginosis, who has received antibiotic treatment for said bacterial vaginosis and whose condition has relapsed, i.e., who has symptoms again. This patient can also be referred to as a patient with recurrent BV. As used herein, the terms "patient who has a history of bacterial vaginosis", "patient who has experienced symptoms of bacterial vaginosis", and "patient with recurrent BV" can be used interchangeably and include, but are not limited to, a patient who has had one or more episodes of BV, preferably two or more episodes of BV, more preferably two or more episodes of BV within 6 months, even more preferably two or more episodes of BV within the past 6 months, or preferably three or more episodes of BV, more preferably three or more episodes of BV within 12 months, even more preferably three or more episodes of BV within the past 12 months.

[0119] Methods for diagnosing BV or confirming an episode of BV in a patient are well known to the skilled person, for example, BV can be diagnosed clinically by using clinical criteria (e.g. Amsel diagnostic criteria) or by microscopy, by determining the Nugent score from a vaginal Gram stain. Without being bound by any theory, recurrence (or reoccurrence) of BV in a patient after antibiotic treatment can be due to the persistence of a residual infection caused by an infective bacteria that is resistant to the antibiotic used. Thus, in a preferred embodiment of the application, the patient has bacterial vaginosis wherein the infective bacteria is resistant to antibiotic treatment. Thus, in a preferred aspect of the application, the Gardnerella-specific pharmaceutical composition described herein is used for the treatment of bacterial vaginosis, wherein the pharmaceutical composition is to be administered to a patient who has failed previous antibiotic treatment and has bacterial vaginosis, wherein the infective bacteria is resistant to antibiotic treatment. As defined below, the infective bacteria of the BV to be treated herein is more preferably highly resistant to antibiotic treatment. In a preferred embodiment of the therapeutic use of the application, the patient to be treated has BV wherein the infective bacteria of the BV is resistant, preferably highly resistant, to treatment with metronidazole, tinidazole, secnidazole, clindamycin or any combination thereof. In a more preferred embodiment of the therapeutic use of the application, the patient to be treated has BV wherein the infective bacteria of the BV is resistant, preferably highly resistant, to treatment with metronidazole and / or clindamycin. In an even more preferred embodiment of the therapeutic use of the application, the patient to be treated has BV wherein the infective bacteria of the BV is resistant, preferably highly resistant, to treatment with metronidazole.

[0120] In the context of the present application, "resistance" of a bacterial strain, preferably a Gardnerella strain, to an antibiotic refers to the ability of the strain to resist the activity of an antibiotic to which it was previously susceptible and to allow the strain to survive after antibiotic treatment. Antibiotic resistance can occur naturally ("intrinsic resistance") or can be caused by the misuse of antibiotics by humans and animals ("acquired resistance"). Methods for determining the resistance or susceptibility of a bacterial strain to an antibiotic are well known to the person skilled in the art. For example, the EUCAST breakpoints for Gram-positive anaerobes (vll, 2021) can be used. An alternative definition is given by Petrina et al. (2017, Anaerobe 47, 115-119), wherein slightly higher resistance breakpoints are used, because topical formulations of some nitroimidazoles and clindamycin can establish concentrations in the vaginal fluid in the mg / ml range, much higher than what can be achieved with oral antibiotics. According to this alternative, more stringent definition (also used in the attached examples), resistance (R) can be defined as MIC value for metronidazole higher than or equal to 32 pg / ml and MIC value for clindamycin higher than 8 pg / ml, while susceptibility (S) can be defined as MIC value for metronidazole lower than or equal to 8 pg / ml and MIC value for clindamycin lower than or equal to 2 pg / ml. The terms "minimum inhibitory concentration" and "MIC" are used interchangeably herein to refer to the minimum concentration of a chemical substance, typically a drug, that prevents visible growth of bacteria. The MIC can be defined as the minimum antibiotic concentration at which no bacterial growth is detectable by OD measurement after 48 hours. Thus, in a preferred embodiment of the therapeutic use of the present application, the patient to be treated suffers from BV, wherein the infecting bacteria are resistant to antibiotic treatment, as defined by the EUCAST breakpoints. In the present application, other generally accepted resistance criteria can be used instead of or in addition to the EUCAST definition or the above-mentioned alternative definition (Petrina et al. 2017, Anaerobe 47, 115-119) to define the resistance of the infecting bacteria to antibiotic treatment.

[0121] As is well known to the person skilled in the art, "resistance" and "susceptibility" can also be defined in terms of MBC and / or MBEC values. The term "minimum bactericidal concentration" or "MBC" refers to the lowest concentration of an antimicrobial agent required to kill a specific bacterium. Typically, the MBC90or MBC99.5, i.e. the antibiotic concentration that kills 90% or 99.5% of the cells, respectively, within a specified time, is measured. The MBC can be defined as the complete elimination of, for example, 2.5xl07CFU / ml of bacteria. 7The lowest concentration of a suspension of CFU / ml. The MIC is the lowest antimicrobial agent concentration required to inhibit visible growth, while the MBC is the lowest antimicrobial agent concentration that results in death of all bacterial cells in suspension and to the defined detection limit, resulting in killing at least 90% (MBC90) or at least 99.5% (MBC99.5) of bacteria. The term "minimum biofilm eradication concentration" or "MBEC" refers to the lowest antimicrobial agent concentration required to reduce a population of bacteria growing in a biofilm below the detection limit.

[0122] Without being bound by any theory, the resistance of the infective bacteria to the antibiotic can be associated with (or partially or substantially responsible for) the recurrence of BV in the patient suffering from recurrent BV. Thus, a patient suffering from bacterial vaginosis with infective bacteria resistant to antibiotic treatment can fail treatment with an antibiotic. Therefore, in a preferred embodiment of the application, the patient to be treated and suffering from bacterial vaginosis with infective bacteria resistant to antibiotic treatment is a patient prone to fail antibiotic treatment. As used herein, "patient prone to fail antibiotic treatment" means a patient at high risk of failing treatment with an antibiotic, i.e. the likelihood of the patient to relapse (e.g. within 12 months) is very high if the patient is treated with an antibiotic. Methods to determine whether a patient is prone to fail antibiotic treatment are well known to the person skilled in the art. For example, a clinical sample can be collected and the resistance of the vaginal flora strains can be assessed. Likewise, a patient suffering from bacterial vaginosis and with infective bacteria resistant to antibiotic treatment is likely to have failed (i.e. previously) antibiotic treatment (as defined above). Therefore, in a further preferred embodiment of the application, the patient to be treated and suffering from bacterial vaginosis with infective bacteria resistant to antibiotic treatment is a patient who has previously failed antibiotic treatment.

[0123] According to the above definition of resistance, "High Resistance" (HR) can be defined as MIC values higher or equal to 256 pg / ml for metronidazole and higher or equal to 64 pg / ml for clindamycin, i.e. 8-fold higher than the alternative definition of resistance breakpoints, which are already higher than the standard resistance breakpoints defined by EUCAST. Thus, in one embodiment of the therapeutic use of the application, the patient to be treated suffers from BV, wherein the infecting bacteria of said BV are resistant, preferably highly resistant, to antibiotic treatment, preferably as defined by the EUCAST breakpoints. In a preferred embodiment of the therapeutic use of the application, the patient to be treated suffers from BV, wherein the infecting bacteria of said BV are resistant, preferably highly resistant, to metronidazole, tinidazole, secnidazole, clindamycin or any combination thereof. In a more preferred embodiment of the therapeutic use of the application, the patient to be treated suffers from BV, wherein the infecting bacteria of said BV are resistant, preferably highly resistant, to metronidazole and / or clindamycin. In an even more preferred embodiment of the therapeutic use of the application, the patient to be treated suffers from BV, wherein the infecting bacteria of said BV are resistant, preferably highly resistant, to metronidazole.

[0124] In certain embodiments, the pharmaceutical composition of the application is used for the treatment of bacterial vaginosis, wherein said patient has failed previous antibiotic treatment and / or wherein the infecting bacteria of said bacterial vaginosis are resistant to antibiotic treatment.

[0125] As used herein, the terms "treatment with antibiotics" and "antibiotic treatment" are used interchangeably and preferably refer to the use of an antibiotic recommended or approved for the treatment of BV. Antibiotics currently recommended or approved for the treatment of BV include nitroimidazoles, including but not limited to metronidazole, tinidazole and secnidazole, and clindamycin. Thus, in one embodiment, said "treatment with antibiotics" or "antibiotic treatment" is a treatment with a nitroimidazole and / or clindamycin. In a preferred embodiment, the treatment with antibiotics described herein is a treatment with metronidazole, tinidazole, secnidazole, clindamycin or any combination thereof. In a more preferred embodiment, the antibiotic treatment described herein is a treatment with metronidazole and / or clindamycin. In an even more preferred embodiment, the antibiotic treatment described herein is a treatment with metronidazole.

[0126] As used herein, "treatment" and "treating" and the like generally mean obtaining a desired pharmacologic and physiologic effect. The effect can be prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof and / or can be therapeutic in terms of a partial or complete cure for a disease, condition, symptom or adverse effect attributed to the disease. The term "treatment" as used herein covers any treatment of bacterial vaginosis in a mammal, particularly in a human, and includes: (a) preventing the bacterial vaginosis in a subject that can be predisposed to the bacterial vaginosis but has not yet been diagnosed as having it; (b) inhibiting the bacterial vaginosis, i.e., arresting its development; or relieving the bacterial infection, i.e., causing the bacterial infection and / or its symptoms or conditions to regress, e.g., improving or repairing damage. In particular, treatment of bacterial vaginosis includes preventing, reducing or even eradicating the infection, e.g., by killing the infective bacteria, thereby controlling, reducing or inhibiting bacterial proliferation and reducing the number of viable bacterial cells. It is preferred herein that the disease, i.e., BV, is treated therapeutically in terms of a partial or complete cure of the disease or symptoms.

[0127] Those of skill in the art understand that the active agents used in the present application are typically administered in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy, 19th Edition, 1995, Alfonso R. Gennaro, editor, Mack Publishing Company, Easton, PA). For example, the active agents can be administered topically, i.e., topically within the vagina of a female subject and / or topically within or on the glans penis, foreskin or urethral opening of a male subject. Remington: The Science and Practice of Pharmacy , 19th Edition, 1995, Alfonso R. Gennaro, editor, Mack Publishing Company, Easton, PA). For example, the active agents can be administered topically, i.e., topically within the vagina of a female subject and / or topically within or on the glans penis, foreskin or urethral opening of a male subject.

[0128] The term "(administered) within or on the glans penis" herein also includes "(administered) within and on the glans penis". Consistently with this, the term "(administered) within or on the glans penis, foreskin or urethral opening of a male subject" also includes "(administered) within and on the glans penis and on the foreskin and on the urethral opening of a male subject". In another embodiment, the active agents can be co-administered with a compound or composition that modulates the pH of the vagina. In some embodiments, the compound or composition modulates the pH of the vagina to a pH of 4.0 to 6.0, preferably to a pH of 5.0.

[0129] The terms "patient" and "subject" are used interchangeably herein and refer to a mammal. For example, mammals contemplated by the present application include humans, primates, domesticated animals such as cows, sheep, pigs, horses, laboratory test animals such as rodents and the like. Preferably, the patient is a human. Even more preferably, the patient is a female (also referred to herein as "female subject").

[0130] It will be appreciated that the pharmaceutical compositions for use described herein can be administered to a subject in combination with one or more additional therapeutic agents. For example, the endolysins and pharmaceutical compositions described herein can be administered to a subject in combination with: (a) one or more conventional antibiotic therapies. Such antibiotics can include clindamycin, metronidazole or any other suitable antibiotic known to the skilled person; (b) one or more additional endolysins, or nucleic acid molecules, vectors, host cells or bacteriophages capable of expressing the same; (c) a compound or composition that modulates the pH of the vagina, preferably to a pH of 4.0 to 6.0, more preferably to a pH of about 5.0. Such pH modulating compounds can include phosphate, lactic acid (e.g. the natural acidifying substance secreted by Lactobacillus species to establish an acidic environment) or other organic acids, such as carboxyl-substituted polymers; (d) a therapy that neutralises the toxins released following bacterial lysis of Gardnerella vaginalis cells within the vagina. Suitable neutralising therapies can include antibodies (see Babcock et al., 2006, Infect. Immun. 74:6339-6347 ) and toxin adsorbers such as tolevamer (see Barker et al., 2006, Aliment. Pharmacol. Ther.24: 1525-1534 ; (e) probiotic bacteria.

[0131] In one embodiment of the uses of the application, a host cell or a pharmaceutical composition comprising a host cell is used to deliver the endolysin (preferably the host cell).

[0132] In a particular embodiment, the application relates to the use of a pharmaceutical composition according to the application in the manufacture of a medicament for the treatment or prevention of a bacterial infection, preferably bacterial vaginosis.

[0133] In a particular embodiment, the application relates to a method of treating or preventing a bacterial infection, preferably bacterial vaginosis, in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a pharmaceutical composition according to the application.

[0134] The application also provides a method of treating a bacterial infection, in particular bacterial vaginosis, as described above, wherein the method comprises administering to said patient a therapeutically effective amount of a pharmaceutical composition comprising a Gardnerella-specific polypeptide, preferably a Gardnerella-specific endolysin, and a polyacrylic acid polymer.

[0135] The embodiments and definitions described above in relation to the therapeutic uses according to the application apply mutatis mutandis in the context of the method of treating BV.

[0136] One aspect of the present application relates to a composition comprising a polyacrylic acid polymer for use in the treatment and / or prevention of a bacterial infection, wherein the polyacrylic acid polymer reduces a biofilm.

[0137] In another aspect, the present application relates to a non-therapeutic use of a polyacrylic acid polymer in reducing a bacterial biofilm.

[0138] That is, the inventors surprisingly found that polyacrylic acid polymers are able to reduce a Gardnerella biofilm even in the absence of Gardnerella-specific polypeptides; see Figure 2 A and B. In contrast, polyacrylic acid polymers have no killing activity on planktonic Gardnerella; see Figure 5 Thus, it was shown that polyacrylic acid polymers specifically act directly on bacterial biofilms. Based on these findings, polyacrylic acid polymers can be used as a component of a pharmaceutical composition for the treatment and / or prevention of a bacterial infection, in particular when the bacterial infection involves biofilm formation.

[0139] A polyacrylic acid polymer is defined to reduce or dissolve a biofilm if the number of viable bacteria (CFU / mL) in the biofilm is reduced by at least a factor of 2, at least a factor of 3, at least a factor of 4, at least a factor of 5, at least a factor of 6, at least a factor of 7, at least a factor of 8, at least a factor of 9, or at least a factor of 10 after contacting the biofilm with the polyacrylic acid polymer.

[0140] To this end, a biofilm (e.g., of Gardnerella vaginalis ATCC 14018) can be generated on pre-coated tissue culture plates in sBHIG (in g / L: brain heart infusion: 37, gelatin: 20, yeast extract: 5, starch: 1, glucose: 2.5; water: q.s. to 1000 mL) pH 7 for 48 hours. The resulting biofilm can then be treated with PAA in sBHIG pH 4.5 for 24 hours. Alternatively, a biofilm can also be generated in sNYC (in g / L: HEPES: 2.4, proteose peptone: 15, yeast extract: 3.8, NaCl: 5, glucose: 10; water: q.s. to 1000 mL) pH 7 for 48 hours, and the biofilm can then be treated with PAA in sNYC pH 6 for 24 hours. For biofilm treatment with PAA, a PAA stock (27% (w / v)) can be used to prepare a 0.35% PAA (v / v) working solution. After incubation, the biofilm can be rinsed and removed by vigorous pipetting. Gradient dilutions can be made with PBS (1 x PBS-DPBS, calcium- and magnesium-free, Thermo Scientific, Cat. No. 14190169) and spotted on chocolate agar plates (BD, Cat. No. 254060) to count the number of viable cells (CFU / ml).

[0141] In certain embodiments, the bacterial biofilm is a mucosal biofilm. In certain embodiments, the (mucosal) biofilm is formed by bacteria of the genus Gardnerella.

[0142] In certain embodiments, the bacterial infection is bacterial vaginosis, as defined above.

[0143] Preferably, the pharmaceutical composition further comprises a polypeptide having killing activity against Gardnerella as described herein. That is, Figure 2 It has been shown that polyacrylic acid synergistically works with endolysin PM-477 against Gardnerella biofilm.

[0144] The polyacrylic acid polymer comprised in the pharmaceutical composition of the present application can be a cross-linked polyacrylic acid polymer or a carbomer. The definition of polyacrylic acid polymer above applies to the present application.

[0145] The term "about" as used herein in reference to a numerical value includes, for example, integers, fractions, and percentages, whether explicitly stated or not. The term "about" generally refers to a range of values that a person of skill in the art would consider equivalent to the stated value (e.g., + / - 1-3% of the stated value). In some cases, the term "about" can include values rounded to the nearest significant figure.

[0146] The present application will be further described by the following non-limiting figures and embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0147] Figure 1 : A, Kinetic studies of PM-477 against Gardnerella vaginalis Gv9 (ATCC 14018 T ) show strong lytic effects in a time- and dose-dependent manner. Cell suspensions of Gardnerella were treated with recombinantly expressed PM-477 at 2.5, 10 and 40 pg / ml for 1 h, 5 h and 24 h and colony forming units (CFU) per ml were determined on a logarithmic scale and compared to buffer-treated controls. B, Other vaginal BV pathogens (grey) as well as probiotic Lactobacillus suspensions (white boxes) were treated with PM-477 at 10 and 100 pg / ml for 5 h and colony forming units (CFU) per ml were determined on a logarithmic scale and compared to buffer-treated controls. LOD indicates limit of detection. Statistical significance (one-way ANOVA, comparing control to 10 and 10 pg / mL PM-477 treatment for each strain); P < 0.001, P < 0.01, P < 0.05.

[0148] Figure 2A. Gardnerella biofilm (using the typical strain Gardnerella vaginalis ATCC 14018) was treated with 8 pg / ml PM-477 (thus below the concentration usually used to eradicate Gardnerella biofilm), 0.35% (w / v) PAA and combinations thereof in sBHIG medium at pH 4.5 for 24 hours. B. Gardnerella biofilm (Gardnerella vaginalis ATCC 14018) was treated with 32 pg / mL H2B10B11, 0.25% (w / v) PAA and combinations thereof in sNYC medium at pH 6 for 24 hours.

[0149] Cells were removed by pipette aspiration and quantitative plating was used to determine the remaining viable cells (CFU / ml). The log reduction of CFU / ml compared to the buffer treated control 10 The reduction is visually reflected in the bactericidal effect. The grey column represents the theoretical additive effect of the average log reduction of PM-477 / H2B10B11 and PAA. Mean and standard deviation are shown.

[0150] Figure 3: Lytic activity of two PM-477 formulations against six BV-associated pathogens and one commensal Lactobacillus species. Strains were tested for susceptibility by treating fresh liquid culture suspensions of the strains with no EL control, PM-477 formulated with PAA to pH 4.5 (panel A) or PM-477 formulated with PAA to pH 5.0 (panel B) for 5 hours. Post-treatment viability was determined by quantitative spotting (panels A and B) and reduction in viability of the two formulations was calculated by subtracting the logio CFU / mL in the formulation-treated replicates from the median of the corresponding untreated controls (panel C). Three replicates, range and median are shown. LOD: limit of detection. Points below the LOD indicate we have reached the limit of detection.

[0151] Figure 4 : Effect of PAA concentration on viability of planktonic Gv9 cells. Gv9 fresh liquid culture suspensions were treated with pH 5 sNYCB with different concentrations of PAA for 90 minutes at 37 °C to test for susceptibility to PAA. Post-treatment viability was determined by quantitative spotting. Three replicate values are shown, with range and median horizontal line. PAA: polyacrylic acid. LOD: limit of detection.

[0152] Figure 5: Effect of PAA on the viability of five BV-associated pathogens and one commensal. Strains were tested for sensitivity to PAA by treating fresh liquid culture suspensions with pH 5 NYCB medium with PAA concentrations ranging from 0.01% to 1% for 90 minutes and comparing them to untreated controls. Viability after treatment was determined by quantitative spotting. Data are presented in triplicate with median line. PAA: polyacrylic acid. LOD: limit of detection. Points below the LOD indicate we have reached the limit of detection.

[0153] Figure 6 : Lytic activity of H2B10B11 against BV-associated pathogens and one commensal with Carbopol 974P formulation. Strains were tested for sensitivity by treating fresh liquid culture suspensions of the strains with no EL control or with low concentrations of H2B10B11 (2 pg / mL) with 0.25% (w / v) CP 974P formulation in medium adjusted to pH 5 for 20.5 hours. Viability after treatment was determined by quantitative spotting. Three replicates and median are shown. LOD: limit of detection. Points below the LOD indicate the limit of detection has been reached.

[0154] Figure 7 : The activity of H2B10B11 with Carbopol formulation can be modulated by adjusting the salt content. A 90-minute lysis efficacy assay was performed on G. vaginalis with 10 pg / mL H2B10B11 and / or 0.1% (w / v) Carbopol in medium adjusted to pH 5.5 with normal salt concentration (86 mM, circles) and high salt concentration (345 mM, diamonds). The efficacy of H2B10B11 was assessed by quantitative spotting. Median and range of three replicates are shown.

[0155] Figure 8 : Reduction of L. iners in BV-positive female swab samples after 19 hours of treatment. Thirty vaginal swab samples were treated overnight with 50 pg / mL H2B10B11 (black) or with 50 pg / mL H2B10B11 with 0.1% (w / v) CP 974P and 1% NaCl formulation (gray). Live L. iners load was determined by viability qPCR using a primer pair specific for L. iners. DNA copies per pL in treated samples were subtracted from the load in the corresponding untreated baseline samples and plotted against the load in the baseline samples. DETAILED DESCRIPTION

[0156] Example 1 - PM-477 endolysin is a highly active antibacterial enzyme that targets Gardnerella and to a lesser extent Lactobacillus iners

[0157] Bacterial vaginosis (BV) is characterized by an imbalance in the vaginal microbiota and the formation of a characteristic biofilm on the vaginal epithelium, initiated and dominated by bacteria of the genus Gardnerella. The inventors previously disclosed synthetic endolysins derived from 1,4-beta-N-acetylmuramidases (muramidases) encoded by phages of the genus Gardnerella (WO 2020 / 225335). These endolysins efficiently kill Gardnerella bacteria and to a lesser extent kill the inert Lactobacillus iners (another opportunistic pathogen in BV). Importantly, probiotic Lactobacillus species such as Lactobacillus crispatus and Lactobacillus gasseri are not harmed by PM-477. The inventors tested the killing activity of a representative endolysin (PM-477 (H2B10)) against vaginal Gardnerella bacteria.

[0158] Gardnerella bacteria in suspension (OD = 0.1) were treated with endolysin PM-477 (stock solution: 700 pg / ml PM-477 in 50 mM MES pH 5.5, 200 mM NaCl, 8 mM MgS04) or with buffer control and incubated anaerobically for 1, 5 and 24 hours. After the incubation period, the suspensions were serially diluted, spotted on chocolate agar plates and CFU / ml were counted. The effect is clearly time- and dose-dependent and already after 5 hours of incubation with low concentrations of 10 and 40 pg / ml the cell number was reduced to the limit of detection (LOD). No regrowth was observed after 24 hours. Interestingly, when treated with an extremely low concentration of 2.5 pg / ml no further killing was recorded between 5 hours and 24 hours, suggesting that a kind of equilibrium might have been established. The inventors also tested the activity of PM-477 against other vaginal opportunistic pathogens as well as probiotic Lactobacillus species of the vaginal microbiota. It was confirmed that PM-477 is specific for Gardnerella bacteria and has no effect on beneficial Lactobacillus species or other opportunistic BV pathogens. The only exception is Lactobacillus iners, which at high concentrations of 100 pg / ml the bacterial number was reduced by 2.5 logio units after 5 hours. Lactobacillus iners is clearly different from other Lactobacillus species in cell wall composition and metabolism and is considered a pathogen of BV.

[0159] In summary, the recombinantly expressed endolysin PM-477 (H2B10) has a strong bactericidal effect on Gardnerella bacteria and does not harm Lactobacillus crispatus, Lactobacillus gasseri and Lactobacillus jensenii, which are the most prevalent bacterial species in the healthy vaginal microbiota (see Figure 1).

[0160] Example 2 - PAA as excipient enhances the killing effect of endolysins on Gardnerella bacteria growing as biofilm

[0161] The inventors tested PAA (Sigma Aldrich, Catalog # 323667-100G) as a vehicle for Gardnerella-specific endolysins for intravaginal administration. The viscosity and mucoadhesive properties of PAA ensure slow release of the active pharmaceutical ingredient (endolysin) at the vaginal mucosal layer and its acidifying effect helps restore a healthy vaginal microbiota. Unexpectedly, the presence of PAA also enhanced the efficacy of the endolysins and more effectively killed Gardnerella bacteria. The inventors tested the use of Gardnerella-specific endolysins PM-477 and H2B10B11 with PAA formulation on mono-species Gardnerella biofilms in vitro. Biofilms (vaginal Gardnerella ATCC 14018) were generated on pre-coated tissue culture plates in sBHIG (in g / L: brain heart infusion: 37, gelatin: 20, yeast extract: 5, starch: 1, glucose: 2.5; water: to 1000 mL) pH 7 for 48 hours, then treated with low concentrations of endolysin PM-477 and PAA, as well as PM-477 and PAA alone in sBHIG pH 4.5 for 24 hours. Alternatively, biofilms were generated in sNYC (in g / L: HEPES: 2.4, peptone: 15, yeast extract: 3.8, NaCl: 5, glucose: 10; water: to 1000 mL) pH 7 for 48 hours, then treated with H2B10B11 or PAA or a combination of both in sNYC pH 6 for 24 hours.

[0162] For biofilm treatment with PAA and PM-477, a 0.35% PAA (v / v) working stock was prepared using PAA stock (27% (w / v)) diluted in sBHIG pH 5 containing 8 pg / ml PM-477 (dilution from 700 pg / ml PM-477 stock). In addition, due to the buffering effect of 0.35% PAA, the final mixture of PM-477 with excipient PAA for biofilm treatment had a pH 4.5. For biofilm treatment with PAA and H2B10B11, a treatment solution containing 0.25% PAA and 32 pg / mL H2B10B11 was prepared in sNYC pH 6 using PAA stock (14.5% (w / v), adjusted to pH 6). After incubation, biofilms were washed with forceful pipetting and removed. Serial dilutions were performed in PBS (1 x PBS-DPBS, calcium-free, magnesium-free, Thermo Scientific, Cat# 14190169) and spotted on chocolate agar plates (BD, Cat# 254060) to count the number of viable cells (CFU / ml). The Gardnerella biofilm eradication concentration for PM-477 was between 2 and 32 pg / ml for this tested strain. The chosen concentration herein was 8 pg / ml, thus at the lower limit of the Gardnerella strain minimum biofilm eradication concentration (MBEC) range in order to be able to observe potential synergy of excipient PAA and PM-477. It is important to note that the MBEC is affected by the medium and pH used for the experiment. Since the biofilm experiment with H2B10B11 was performed at pH 6, 32 pg / mL H2B10B11 was not sufficient to eradicate biofilms in the absence of PAA.

[0163] A reduction of 1.6 log CFU / ml using PM-477 (8 pg / ml) only 10 units, a reduction of 0.6 log CFU / ml using PAA (0.35% w / v) only 10 units, while the combination of PM-477 and PAA resulted in a clear synergy with a kill rate of 3 log 10 units. Compared to the potential additive effect (PAA 0.6 log 10 units + PM-477 1.6 log 10 units = 2.2 log 10 units reduction), the difference between both is 0.8 log 10 units (total 3 log 10 units), which means a 6-fold increase in activity in the linear range Figure 2 A).

[0164] No relevant effect was observed with H2B10B11 (32 pg / mL) alone in medium at pH 6. The use of PAA (0.25% w / v) alone reduced CFU / mL by 0.9 log 10 units, while the combination of H2B10B11 and PAA resulted in a clear synergistic effect, with a log 10 reduction of 3.1 Figure 2 B).

[0165] Example 3 - PAA as excipient enhances the killing effect of endolysin on planktonically grown Gardnerella bacteria and other BV pathogens

[0166] Next, the efficacy of PM-477 with two different formulations of 0.25% PAA (w / v; Sigma Aldrich, Cat. No. 323667-100G) as excipient was tested in a time-kill assay against six BV-associated pathogens and one commensal Lactobacillus species (Figure 3). Both formulations contained 2 pg / mL PM-477 and 0.25% PAA and were adjusted to pH 4.5 Figure 3A ) or pH 5 Figure 3B , respectively, to mimic different PAA types and compositions. Briefly, 4 g PAA was added to 10 mL dH20 and heated to 50°C while stirring on a heated stir plate. The pH of the PAA solution was adjusted to 4.5 or pH 5 with 5 M NaOH. Then, the volume was adjusted to 27.5 mL (corresponding to 14.5% PAA) and sterile filtered using a 0.22 pm syringe filter. These stock solutions were diluted to a final concentration of 0.25% PAA in glucose-supplemented New York City Broth (sNYCB) adjusted to the respective pH values. The supplemented NYCB consisted of 10 mM HEPES (Sigma Aldrich), 15 g / L peptone from B. subtilis (Sigma Aldrich), 3.8 g / L yeast extract (Thermo Fisher Scientific), 86 mM sodium chloride (Carl Roth), and 10 g / L a-D-glucose (Sigma Aldrich).

[0167] Bacteria were grown on chocolate agar plates at 37°C anaerobically for 48 h, then bacteria were scraped from the plates and a suspension with a concentration of 10 8 CFU / ml was prepared for the experiments. In the time-kill experiments, the bacterial suspension was mixed with the respective treatment stock and incubated at 37°C anaerobically for 5 h. Then, 2 pL of a 10-fold dilution series (with the lowest dilution being 10 -5Bacterial spotting of the bacteria of the genus Gardnerella onto chocolate agar plates, quantification of surviving bacteria after anaerobic incubation at 37°C for 48 hours.

[0168] Both formulations efficiently reduced the bacterial load of all six pathogens, but had no negative effect on the commensal Lactobacillus crispatus (Figure 3). Compared to the control group, the formulation set to a slightly more acidic pH (4.5) reduced the viable bacteria of Gv9 (Gardnerella vaginalis ATCC 14018), Lactobacillus iners, Mobiluncus mulieris by > 3 log 10 and the viable bacteria of Atopobium vaginae and Prevotella bivia by > 2 log 10 Figure 3C When the formulation was set to a less acidic pH (5.0), 2 pg / mL PM-477 reduced the viable bacteria of Gv24 (Gardnerella swegini GS 9838-1), Lactobacillus iners and Prevotella bivia by > 3 log 10 and the viable bacteria of Gv9, Mobiluncus mulieris and Atopobium vaginae by > 2 log 10 without any negative effect on the commensal bacteria Lactobacillus crispatus Figure 3C

[0169] In summary, the formulation with PAA surprisingly extended the activity spectrum of PM-477 to multiple BV-associated pathogens other than Gardnerella bacteria.

[0170] Example 4 - PAA alone has no killing effect on Gardnerella and weak killing effect on some other BV pathogens, but only at higher concentrations

[0171] Next, the effect of PAA alone (Sigma Aldrich, Cat. No. 323667-100G) on the bacteria of the genus Gardnerella and other BV-associated pathogens was evaluated. Bacterial suspensions were adjusted to 10 8 CFU / ml, mixed with the treatment stock solution and incubated anaerobically at 37°C for 90 minutes, after which surviving bacteria were quantified by spotting a 10-fold dilution series of the reaction. The spotting plates were incubated for 2-3 days, after which CFU / mL were determined. As shown in Figure 4 PAA concentrations up to 1% did not reduce the viability of Gardnerella bacteria after 90 minutes of incubation.

[0172] The susceptibility of five other BV-associated pathogens and one commensal Lactobacillus species to PAA was also tested using the same assay. The four BV-associated pathogens tested were Lactobacillus iners, Prevotella bivia, Atopobium vaginae and Mobiluncus mulieris, while Lactobacillus crispatus was used as a commensal bacteria Figure 5 ​​). The inventors tested the efficacy of 0.01% to 1% PAA concentrations set to pH 5 against all five organisms using a 90-minute efficacy assay. PAA concentrations up to 1% had minimal impact on the viability of C. curvatus and L. crispatus (reductions < 0.6 log 10 ). Only in response to high concentrations of PAA (> 0.5%) did the viability of P. bivia and A. vaginae decrease by 1.8 and 1.7 log 10 , respectively, with no impact at lower concentrations Figure 5 ). In response to the lowest tested PAA concentration (0.01%), the viability of L. iners decreased by approximately 1.1 log10, and at higher PAA concentrations, its viability did not further decrease Figure 5

[0173] Example 5 - Carbopol 974P as an excipient enhances the killing effect of endolysin on planktonic BV-associated pathogens, but not on commensal lactobacillus species

[0174] Five-hour tests in planktonic environment were performed on five BV-associated pathogens and one commensal lactobacillus species in sNYC medium at pH 5 Figure 6 ), testing the efficacy of H2B10B11 formulated with 0.25% (w / v) Carbopol 974P NF polymer (CP 974; The Lubrizol Corporation). The formulation contained 2 pg / mL H2B10B11 and 0.25% CP 974P, with a final pH of 5. To obtain the formulated treatment, 50 mg CP 974P was added to 15 mL sNYC pH 5. After mixing, the pH was re-adjusted to pH 5 using 5 M NaOH, and the volume was adjusted to 20 mL with sNYC pH 5. H2B10B11 was added from a 1.343 mg / mL stock in MES pH 5.5 buffer to a final concentration of 2 pg / mL.

[0175] Gardnerella was grown on chocolate agar plates, and the other strains were grown on Schaedler agar plates with vitamin K1 and 5% sheep blood (BD, Cat# 254042) anaerobically at 37°C for 48 hours, then scraped from the plates and prepared as 10 8 CFU / mL bacterial suspensions for the experiments. The bacterial suspensions were mixed with sNYC pH 5 or with sNYC containing H2B10B11 pH 5 formulation and incubated anaerobically at 37°C for 20.5 hours. Then, 2 pL of a 10-fold dilution series (with the lowest dilution being 10 -5 ​Bacterial suspensions of Gardnerella vaginalis ATCC 14025 were spotted on chocolate / Schaedler agar plates and incubated anaerobically at 37°C for 48 hours. Surviving bacteria were quantified.

[0176] H2B10B11 formulation of the preparation eliminates BV-associated pathogenic bacteria Gardnerella vaginalis, Lactobacillus iners, Mobiluncus mulieris, Atopobium vaginae and Dialister pigrus Figure 6 This is an unexpected finding, as endolysin alone was not effective against non-Gardnerella strains and only had a small effect on Lactobacillus iners (see Example 1). Since the results obtained here with Carbopol 974P are comparable to those in Example 3 (Figure 3), which contains polyacrylic acid in the formulation, it can be concluded that polyacrylic acid and its cross-linked, pharmaceutical grade form (carbomer) leads to a synergistic enhancement of the lytic activity of endolysins. Furthermore, H2B10B11 formulated with CP 974P does not impair the beneficial Lactobacillus crispatus Figure 1B Figure 6

[0177] Example 6 - Activity of H2B10B11 formulated with Carbopol can be modulated by adjusting the salt content

[0178] H2B10B11 alone or formulated with Carbopol 974P NF polymer or Carbopol 971P NF polymer (The Lubrizol Corporation) was tested for efficacy against Gardnerella vaginalis in a planktonic setting incubated for 90 minutes in pH 5.5 NYC medium with normal (86 mM) or elevated (345 mM) NaCl concentration Figure 7 The formulation contained 10 pg / mL H2B10B11 and 0.1 % Carbopol with a final pH of 5.5. To obtain the formulated treatment, 40 mg CP 974P or CP 971P were added to 40 mL NYC medium with normal or elevated NaCl concentration. After mixing, the pH was adjusted to 5.5 with 5 M NaOH. H2B10B11 was added from a 1.345 mg / mL stock in MES pH 5.5 buffer to a final concentration of 10 pg / mL.

[0179] Gardnerella bacteria were grown on chocolate agar plates anaerobically at 37°C for 48 hours, after which the bacterial bodies were scraped from the plates using PBS. Bacterial suspensions were mixed with the respective medium containing H2B10B11, Carbopol or a combination of both to yield a final bacterial OD of 0.1. The reaction plates were incubated anaerobically at 37°C for 90 minutes. Then, 2 pL of a 10-fold dilution series (lowest dilution 10 -5 ​​Bacterial suspensions of L. iners (ATCC 49248) were spotted on chocolate agar plates and incubated anaerobically at 37°C for 48 hours and quantified for surviving bacteria.

[0180] Example 7 - Reduction of L. iners in BV-positive female vaginal swab samples after 19 hours of treatment

[0181] Vaginal swab samples from 30 BV-positive females were treated for 19 hours with 50 pg / mL H2B10B11 or with 50 pg / mL H2B10B11 formulated with 0.1 % (w / v) CP974P and 1 % NaCl. For this, two vaginal swab samples in Amies medium (Copan 480 CE) were combined, 400 pL NYC pH 5.5 + 10 % horse serum were added, mixed thoroughly and frozen. Treatment reactions were performed in 96-well plates using 114 pL sample and treatment in MES buffer pH 5.5. The reaction plates were incubated for 19 hours at 37°C under anaerobic conditions. Both baseline (untreated samples) and treated samples were treated with PMAxx (Biotium) which intercalates DNA of dead cells, thus minimizing any qPCR signal from dead cells. Briefly, 45 pL 50 pM PMAxx premix I were added to the cell pellet, incubated on ice for 15 minutes and then exposed to light in a PMA-Lite LED photolysis device for 15 minutes. The PMAxx treatment was repeated twice with 2.25 pL 1 mM PMAxx premix II. The tubes were centrifuged at 8000 rpm for 8 minutes and the supernatant was discarded. The cell pellet was washed with physiological saline and centrifuged at 8000 rpm for 10 minutes. Genomic DNA was extracted using the DNeasy 96 Blood and Tissue Kit.

[0182] Viable L. iners load was determined by viability qPCR using a L. iners specific primer pair. EvaGreen® qPCR Supermix (Solis Biodyne) was used in a LightCycler 480 (Roche) with 2 pK extracted DNA per reaction in a total volume of 10 pL. PCR conditions were as follows: initial denaturation at 95°C for 12 minutes followed by 40 cycles of 95°C for 15 seconds, 57°C for 40 seconds and 72°C for 30 seconds, and finally a melting analysis between 55 and 95°C with a ramping rate of 2.5°C / s.

[0183] DNA copy numbers / pL in treated samples were subtracted from the load in the corresponding untreated baseline samples and plotted against the load in the baseline samples. Linear regression analysis was performed on the samples and the slope is shown in the graph. The slope reflects the overall reduction of L. iners in the patient samples, i.e. 99% reduction of L. iners using H2B10B11 formulation.

Claims

1. A pharmaceutical composition comprising an effective amount of an active agent and a pharmaceutically acceptable excipient, wherein the active agent is a Gardnerella spp. Gardnerella The polypeptide has killing activity, wherein the excipient is a polyacrylic acid polymer.

2. The pharmaceutical composition of claim 1, wherein the polypeptide is an endolysin active against Gardnerella.

3. The pharmaceutical composition of claim 2, wherein the endolysin comprises or consists of (i) an N-terminal catalytic domain or a functional variant thereof; (ii) a C-terminal cell wall binding region or a functional variant thereof, wherein the C-terminal cell wall binding region comprises or consists of at least one cell wall binding domain; and (iii) optionally, a linker region between the N-terminal catalytic domain and the C-terminal cell wall binding region.

4. The pharmaceutical composition of any preceding claim, wherein the polypeptide is a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 37 and is active against Gardnerella.

5. The pharmaceutical composition of any preceding claim, wherein the polypeptide is a recombinant endolysin comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO:

37.

6. The pharmaceutical composition of any preceding claim, wherein the polypeptide has a killing activity against Gardnerella vagnalis ( Gardnerella vaginalis sensu stricto ), Gardnerella leopoldii ( Gardnerella leopoldii ), Gardnerella piotii ( Gardnerella piotii ) and / or Gardnerella swinkii ( Gardnerella swidsinskii ), or any other bacterial species in the genus Gardnerella.

7. The pharmaceutical composition according to any of the preceding claims, wherein the polypeptide has a target for inert lactobacilli ( Lactobacillus iners ( ) kills activity.

8. The pharmaceutical composition of any preceding claim, wherein the polypeptide has no killing activity against Lactobacillus crispatus (L. crispatus) Lactobacilli crispatus ), Lactobacillus gasseri (L. gasseri) Lactobacilli gasseri ), and / or Lactobacillus jensenii (L. jensenii) Lactobacilli jensenii ).

9. The pharmaceutical composition of any preceding claim, wherein the excipient is present at a concentration of about 0.01% to 10%, preferably about 0.1% to 1%, more preferably about 0.2% to 0.5%, most preferably about 0.25% or about 0.35%.

10. The pharmaceutical composition of any preceding claim, wherein the polyacrylic acid polymer is a cross-linked polyacrylic acid polymer or a carbomer.

11. The pharmaceutical composition of any preceding claim, wherein the combination of the active agent and the excipient increases the pharmaceutical activity of the active agent.

12. The pharmaceutical composition of any preceding claim, wherein the composition further comprises a salt.

13. The pharmaceutical composition of claim 12, wherein the salt is sodium chloride (NaCI).

14. The pharmaceutical composition of any preceding claim, wherein the composition is suitable for intravaginal delivery of the active agent.

15. The pharmaceutical composition of any preceding claim, for use in treating a bacterial infection in a subject, preferably wherein the bacterial infection is bacterial vaginosis.

16. The pharmaceutical composition for use of claim 15, wherein the subject has previously failed treatment with an antibiotic and / or wherein the infective bacteria are resistant to antibiotic treatment.

17. The pharmaceutical composition for use of claim 16, wherein the antibiotic is a nitroimidazole and / or clindamycin.

18. The pharmaceutical composition for use according to claim 16 or 17, wherein the antibiotic is metronidazole, tinidazole, secnidazole, clindamycin or any combination thereof.

19. The pharmaceutical composition for use according to any one of claims 15 to 18, wherein the subject has recurrent bacterial vaginosis, preferably wherein the subject has two or more episodes of BV within 6 months or three or more episodes of BV within 12 months.

20. The pharmaceutical composition for use according to any one of claims 15 to 19, to be administered topically into the vagina of a female subject and / or into or onto the glans penis, foreskin or urethral opening of a male subject.

21. Use of the pharmaceutical composition according to any one of claims 1 to 14 for the manufacture of a medicament for the treatment or prevention of a bacterial infection, preferably bacterial vaginosis.

22. A method of treating or preventing a bacterial infection, preferably bacterial vaginosis, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to any one of claims 1 to 14.

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