Gardnerella endolysin vaginal preparation

Abstract

The present invention relates to a novel pharmaceutical composition comprising an effective amount of an activator and a pharmaceutically acceptable excipient, wherein the activator is a polypeptide having toxic activity against Gardnerella and the excipient is a polyacrylic acid polymer. The present invention further relates to the use of the pharmaceutical composition in the treatment of Gardnerella infections, particularly bacterial vaginosis.

Description

[Technical Field]

[0001] This invention relates to a novel pharmaceutical composition for the treatment of infections caused by bacteria of the genus Gardnerella. The pharmaceutical composition of the present invention is characterized by comprising a Gardnerella-specific polypeptide as an activator and a polyacrylic acid polymer as an excipient. This invention further relates to the medical use of the pharmaceutical composition of the present invention and to therapeutic methods using the same. [Background technology]

[0002] Bacterial vaginosis (BV), also known in the literature as bacterial vaginosis, nonspecific vaginitis, and nonspecific vaginosis, is the most common vaginal infection worldwide and is associated with significant adverse outcomes, including premature delivery, postpartum endometritis, and an increased risk of acquiring HIV. It is a dysbiosis of the vagina, in which commensal Lactobacillus are replaced by a multibacterial biofilm, increasing the pH from the natural 3.5–4.5 to 5.5 and forming a foul-smelling fluid. Reported prevalences vary from 10–40% depending on the population tested. However, determining the true prevalence of BV is difficult due to less-than-optimal diagnostic methods and a high proportion of asymptomatic cases. Gardnerella vaginalis (G. vaginalis) is a bacterial species associated with BV.

[0003] The etiology of BV is still not fully understood. It is most commonly defined as a pathological condition characterized by the loss of the normal vaginal microbiota, particularly H2O2-producing Lactobacillus species, and the simultaneous overgrowth of anaerobic bacteria, including G. vaginalis, Mobiluncus, and Mycoplasma hominis. However, recent data suggest that G. vaginalis plays a major role as a specific and sexually transmissible causative agent in BV (Muzny et al., 2016, J. of Infect. Dis. 214 Suppl. 1., S1).

[0004] In the 1950s, a large number of small, pleomorphic Gram-bacilli were observed in the reproductive tracts of women infected with BV. This organism was initially called Haemophilus vaginalis, but as more information about its characteristics became available, it was repeatedly renamed and is now classified as G. vaginalis. Until 2018, it was thought to be the sole member of the genus Gardnerella. However, in early 2019, it was discovered that the genus Gardnerella actually includes at least 13 species, the most frequent of which have been renamed G. vaginalis sensu stricto, G. leopoldii, G. piotii, and G. swidsinskii (Vaneechoutte et al., 2019 Int.J.Syst.Evol.Biol.898661).

[0005] Bacteria of the genus Gardnerella are unique in that they are Gram-indeterminate, meaning they do not form the outer membrane that defines Gram-negative species. Their cell walls are generally very thin, and their peptidoglycan content is less than 10%, which is why the crystal violet dye used in Gram staining does not always produce the deep purple color typical of Gram-positive species. Rather, Gardnerella cells can appear both Gram-positive and Gram-negative in Gram staining. Based on phylogenetic analysis using 16S rRNA, Gardnerella are classified as Gram-positive bacteria belonging to the family Bifidobacteriales.

[0006] In BV, the epithelial surface is covered with dense colonies of G. vaginalis within a biofilm, which is often refractory to treatment. The biofilm is an adhesive community of microorganisms held together by a polymer matrix composed of polysaccharides, proteins, and / or nucleic acids. Different gene expression patterns, as well as the physical structure of the biofilm, enhance bacterial resistance to 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 these often fail to eradicate the biofilm, resulting in recurrence rates of up to 60% within six months. Furthermore, antibiotic treatment often wipes out the vaginal microbiome, leaving some viable biofilm intact, thus opening this ecological niche to other pathogens (e.g., fungi). Therefore, a common side effect of BV treatment is candidiasis. Treatment for BV has also been attempted with probiotics, specifically beneficial Lactobacillus species thought to re-establish themselves in the vagina. However, some clinical trials failed to demonstrate any benefit.

[0007] Endolysins are promising alternatives to existing antibiotics due to their ability to eradicate biofilms, their low tendency to develop resistance, and their specificity to individual bacterial genera or species. Natural and genetically engineered Gardnerella-specific endolysins have been reported (International Publication 2020 / 225335(A1), Landlinger et al. (2021, Pathogens 10, 1-19), or International Publication 2020 / 229802(A1)). International Publication 2020 / 225335 describes a specific recombinant Gardnerella-specific endolysin (e.g., H2B10) for use in methods of treating Gardnerella infections such as BV, caused by Gardnerella vaginalis, Gardnerella leopoldii, Gardnerella piocii, and / or Gardnerella swidsynskii. Examples 6 and 7 of International Publication No. 2020 / 225335 demonstrate, for example, that endolysin H2B10 (as a representative example of recombinant Gardnerella-specific endolysin) is superior to the antibiotics metronidazole and clindamycin, particularly in terms of the minimum inhibitory concentration (MIC) for the growth of Gardnerella strains in suspension. Therefore, the results of International Publication No. 2020 / 225335 demonstrate that (recombinant) Gardnerella-specific endolysins are generally superior to antibiotics in the treatment of BV. In other words, (recombinant) Gardnerella-specific endolysins are generally shown to be more effective against the growth of Gardnerella strains in suspension. This may be due to the fact that endolysin generally exhibits lower MIC values ​​across various Gardnerella species compared to antibiotics.

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

[0009] This technical problem is solved by providing embodiments and claims available herein. [Overview of the project]

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

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

[0012] 3. The endolysin (i) 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 includes 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 domain, The pharmaceutical composition according to Embodiment 2, comprising or consisting of the following.

[0013] 4. The pharmaceutical composition according to any one of Embodiments 1 to 3, wherein the polypeptide has at least 80% sequence identity with the amino acid sequence provided in SEQ ID NO: 1 or SEQ ID NO: 37 and is a polypeptide having toxic activity against Gardnerella.

[0014] 5. The pharmaceutical composition according to any one of Embodiments 1 to 4, wherein the polypeptide is a recombinant endolysin comprising the amino acid sequence provided in SEQ ID NO: 1 or SEQ ID NO: 37.

[0015] 6. The pharmaceutical composition according to any one of embodiments 1 to 5, wherein the polypeptide has bactericidal activity against Gardnerella vaginalis in the narrow sense, Gardnerella leopoldii, Gardnerella piotii and / or Gardnerella swidsinskii, or any other species of the genus Gardnerella.

[0016] 7. The pharmaceutical composition according to any one of embodiments 1 to 6, wherein the polypeptide has bactericidal activity against Lactobacillus iners.

[0017] 8. The pharmaceutical composition according to any one of embodiments 1 to 7, wherein the polypeptide does not have bactericidal activity against Lactobacillus crispatus, Lactobacillus gasseri, and / or Lactobacillus jensenii.

[0018] 9. The pharmaceutical composition according to any one of embodiments 1 to 8, 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%, and most preferably about 0.25% or about 0.35%.

[0019] 10. The pharmaceutical composition according to any one of embodiments 1 to 9, wherein the polyacrylic acid polymer is a crosslinked polyacrylic acid polymer or carbomer.

[0020] 11. The pharmaceutical composition according to any one of embodiments 1 to 10, wherein the combination of the active agent and the excipient increases the pharmaceutical activity of the active agent.

[0021] 12. The pharmaceutical composition according to any one of embodiments 1 to 11, wherein the composition is suitable for intravaginal delivery of the active agent.

[0022] 13. The pharmaceutical composition according to any one of embodiments 1 to 12, which is for use in treating a bacterial infection in a subject, preferably wherein the bacterial infection is bacterial vaginitis.

[0023] 14. The pharmaceutical composition for use according to Embodiment 13, wherein the subject has previously failed to treat with antibiotics and / or the infectious bacteria are resistant to antibiotic treatment.

[0024] 15. A pharmaceutical composition for use according to Embodiment 14, wherein the antibiotic is nitroimidazole and / or clindamycin.

[0025] 16. A pharmaceutical composition for use according to Embodiment 14 or 15, wherein the antibiotic is metronidazole, tinidazole, secnidazole, clindamycin, or any combination thereof.

[0026] 17. A pharmaceutical composition for use according to any one of Embodiments 13 to 16, wherein the subject suffers from recurrent bacterial vaginosis, preferably having had two or more episodes of BV within the last six months, or three or more episodes of BV within the last twelve months.

[0027] 18. A 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 on the glans penis, foreskin, or urethral opening of a male subject.

[0028] 19. Use of any one of Embodiments 1 to 12 for the manufacture of a pharmaceutical for the treatment or prevention of a bacterial infection, preferably bacterial vaginosis.

[0029] 20. A method for treating or preventing a bacterial infection, preferably bacterial vaginosis, in a subject requiring treatment or prevention of a bacterial infection, preferably bacterial vaginosis, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition described in any one of Embodiments 1 to 12.

[0030] In other words, the present invention is based on the surprising and unexpected finding that polyacrylic acid polymers can increase the bactericidal activity of bactericidal polypeptides against Gardnerella bacteria. In Appendix Example 2, it is shown that formulations of bactericidal polypeptides H2B10 (PM-477) and H2B10B11 with polyacrylic acid polymers exhibited a bactericidal activity against Gardnerella biofilms that exceeded the combined bactericidal activity of each component alone, clearly demonstrating a synergistic mechanism of action.

[0031] While polyacrylic acid polymers alone exhibit some killing activity against Gardnerella biofilms (see Example 2), they have no killing activity against planktonic Gardnerella cells (Example 4, Figure 4) and only very limited killing activity against other BV-related pathogens (Example 4, Figure 5). Despite this limited or no killing activity of polyacrylic acid polymers against planktonic bacteria, surprisingly, in the appended Examples 3 and 5 (Figures 3 and 6), polyacrylic acid polymers were shown to enhance the killing activity of bactericidal polypeptides against planktonic Gardnerella species and other pathogens associated with bacterial vaginosis, such as Lactobacillus inersu, Mobilunchus murielis, Atopovium vaginae, and Prevotella bivia. Notably, the bactericidal polypeptides alone have no killing activity against other pathogens associated with bacterial vaginosis (Figure 1B). Therefore, surprisingly, it was found that polyacrylic acid polymers can not only enhance the bactericidal activity of bactericidal polypeptides against Gardnerella species, but also broaden the antimicrobial spectrum of bactericidal polypeptides to target other pathogens associated with bacterial vaginosis. It is noteworthy that this remarkable effect was observed for two different types of polyacrylic acid polymers, namely linear polyacrylic acid polymer and cross-linked polyacrylic acid polymer (Carbopol 974P).

[0032] Therefore, compelling evidence is provided that formulating bactericidal polypeptides, particularly Gardnerella-specific bactericidal polypeptides, together with polyacrylic acid polymers can significantly enhance the bactericidal activity of bactericidal polypeptides against Gardnerella species and other BV-related bacteria.

[0033] Accordingly, in certain embodiments, the present invention relates to a pharmaceutical composition comprising an effective amount of an activator and a pharmaceutically acceptable excipient, wherein the activator is a polypeptide having toxic activity against Gardnerella and the excipient is a polyacrylic acid polymer.

[0034] The active agent contained in the pharmaceutical composition of the present invention is preferably a bactericidal polypeptide specific to the genus Gardnerella, that is, it specifically targets bacteria belonging to the genus Gardnerella. Therefore, the polypeptide contained in the pharmaceutical composition of the present invention preferably has toxic activity against species of the genus Gardnerella. For example, the polypeptide contained in the pharmaceutical composition of the present invention may have toxic activity against Gardnerella vaginalis, Gardnerella leopoldii, Gardnerella piocii and / or Gardnerella swidsinskii, preferably all of them. The toxic activity of the polypeptide contained in the pharmaceutical composition of the present invention against Gardnerella is more preferably genus-selective toxic activity against Gardnerella.

[0035] In this specification, "genus-selective toxic activity" or "genus-specific lytic effect" means that the polypeptide contained in the pharmaceutical composition of the present invention does not have toxic activity or lytic effect against bacteria in general. Preferably, the polypeptide contained in the pharmaceutical composition of the present invention has genus-selective toxic activity against Gardnerella, but not against Lactobacillus crispatus, Lactobacillus gasseri, and / or Lactobacillus gensenii. More preferably, the polypeptide does not have toxic activity against any of those Lactobacillus species, namely Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus gensenii.

[0036] Those skilled in the art will understand that the term “genus-selective killing activity” should not be understood in its most strict sense, given the overwhelming diversity of bacteria and the blurred boundaries between different species and genera. For example, Lactobacillus inersu is classified as a species of the genus Lactobacillus, but it differs significantly from other species of the genus Lactobacillus, such as Lactobacillus crispatus, which encodes more than twice as much protein as Lactobacillus inersu (see France et al., Applied and Environmental Microbiology. 82(24):7063-7073). Therefore, it should be understood that endolysins possessing “genus-selective killing activity” may have lytic activity against bacterial species of other genera, but preferably do not have killing activity against the vast majority of bacteria.

[0037] In certain embodiments, polypeptides contained in the pharmaceutical composition of the present invention may have toxic activity against other pathogens associated with BV. That is, in certain embodiments, polypeptides contained in the pharmaceutical composition of the present invention may have toxic activity against one or more of Lactobacillus inersu, Mobilunchus murielis, Atopovium baginae, and / or Prevotella vivia, particularly when formulated with a polyacrylic acid polymer. In certain embodiments, polypeptides contained in the pharmaceutical composition of the present invention may have toxic activity against all of Lactobacillus inersu, Mobilunchus murielis, Atopovium baginae, and Prevotella vivia, particularly when formulated with a polyacrylic acid polymer.

[0038] As used herein, “killing activity” of an activator against a particular bacterium, such as a bactericidal polypeptide, can be defined as the reduction in the number of viable bacterial cells caused by the activity of the activator. The killing activity of the activator against the bacterium may be complete, meaning that 100% of the bacterial cells are killed, or partial, meaning that 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 killing activity of an activator against specific microorganisms can be measured by standard 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 an overnight incubation, as described in Andrews, 2001, J Antimicrobial Chemotherapy, 48, Suppl. SI, 5-16 or "Document M7-A7, Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; Approved standards, 7th Edition, January 2006, vol. 26, No. 2" published by the Clinical and Laboratory Standards Institute. Another preferred method for measuring the killing activity of an activator is described in the Examples section of International Publication No. 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 activator of test. The decrease in optical density measured at 610–620 nm in a suspension of bacteria whose susceptibility is being tested can be measured in an in vitro turbidity assay performed in the presence of an activator.According to another embodiment, if, in an in vitro turbidity test, the activator reduces the OD (610-620 nm) of a suspension of at least one Gardnerella bacterial 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%, the activator has killing activity against Gardnerella.

[0039] The polypeptides contained in the pharmaceutical composition of the present invention are more preferably active against antibiotic-resistant Gardnerella strains, as described in more detail below herein. In one preferred embodiment, the polypeptides contained in the pharmaceutical composition of the present invention are active against Gardnerella strains that are resistant to one or more antibiotics selected from the group consisting of nitroimidazole and clindamycin. In one more preferred embodiment, the polypeptides contained in the pharmaceutical composition of the present invention are active against Gardnerella strains that are resistant to metronidazole, tinidazole, secnidazole, clindamycin, or any combination thereof. In a further more preferred embodiment, the polypeptides contained in the pharmaceutical composition of the present invention are active against Gardnerella strains that are resistant to metronidazole and / or clindamycin. In a further more preferred embodiment, the polypeptides contained in the pharmaceutical composition of the present invention are active against Gardnerella strains that are resistant to metronidazole. In one further preferred embodiment, the polypeptides contained in the pharmaceutical composition of the present invention are active against Gardnerella strains that are highly resistant to the above antibiotics.

[0040] The bactericidal polypeptide contained in the pharmaceutical composition according to the present invention may be any polypeptide having toxic activity against one or more Gardnerella species. Accordingly, in certain embodiments, the present invention relates to a pharmaceutical composition according to the present invention in which the polypeptide has toxic activity against Gardnerella vaginalis in the narrow sense, Gardnerella leopoldii, Gardnerella piochii and / or Gardnerella swidsinskii, or any other species of the genus Gardnerella. Means and methods for analyzing whether a given polypeptide has toxic activity against Gardnerella species are provided herein and are known to those skilled in the art.

[0041] The pharmaceutical compositions according to the present invention are preferably used for the treatment of bacterial vaginosis. In certain embodiments, the pharmaceutical compositions may be effective in killing other bacteria associated with bacterial vaginosis, such as Lactobacillus inersu, Atopovium vaginae, Prevotella bivia, Mobiluncus murielis, Mobiluncus curtisii, and Streptococcus agalactie, particularly when the pharmaceutical compositions contain a polyacrylic acid polymer. Accordingly, in certain embodiments, the present invention relates to pharmaceutical compositions according to the present invention having toxic activity against Lactobacillus inersu, Atopovium vaginae, Prevotella bivia, Mobiluncus murielis, Mobiluncus curtisii, and / or Streptococcus agalactie, particularly when the polypeptide is formulated together with a polyacrylic acid polymer.

[0042] In this specification, it is preferable that the polypeptides contained in the pharmaceutical composition of the present invention have toxic activity against Gardnerella species and other pathogens associated with BV, but importantly, the polypeptides contained in the pharmaceutical composition of the present invention do not have toxic activity against vaginal commensal bacteria, or are essentially toxic. Vaginal commensal bacteria are important for maintaining a normal vaginal flora and include, but are not limited to, Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus gensenii. Appendix Example 3 shows that the bactericidal polypeptide H2B10 (PM-477), and Appendix Example 5 shows that the bactericidal polypeptide H2B10B11, do not have toxic activity against Lactobacillus crispatus, a vaginal commensal bacterium that is one of the most dominant bacteria in a healthy vaginal microbiome. Accordingly, in certain embodiments, the present invention relates to a pharmaceutical composition according to the present invention in which the polypeptide does not have toxic activity against Lactobacillus crispatus, Lactobacillus gasseri, and / or Lactobacillus gensenii. In certain embodiments, the present invention relates to a pharmaceutical composition according to the present invention in which the polypeptide has toxic activity against Lactobacillus inersu, but does not have toxic activity against Lactobacillus crispatus, Lactobacillus gasseri, and / or Lactobacillus gensenii.

[0043] In other words, in a particular embodiment, the polypeptide contained in the pharmaceutical composition of the present invention is an endolysin having selective toxic activity against Gardnerella vaginalis in the narrow sense, as well as at least one of Gardnerella leopoldii, Gardnerella piocii, Gardnerella swidosinskii, Lactobacillus inersu, Atopovium vaginae, Prevotella vivia, Mobiluncus murielis, Mobiluncus curtisii and / or Streptococcus agalactie, wherein the endolysin has no toxic activity, or is essentially inactive, against at least one of Lactobacillus crispatus, Lactobacillus gasseri and / or Lactobacillus gensenii.

[0044] The terms “peptide,” “polypeptide,” “protein,” and variations thereof refer to peptides, oligopeptides, oligomers, or proteins (including fusion proteins) that contain at least two amino acids linked to one another by normal or modified peptide bonds, such as in the case of isosteric peptides. These terms also include, as defined herein, “peptide mimes” as peptide analogs containing non-peptide structural elements, which can mimic or antagonize the biological action(s) of a native parent peptide. Peptide mimes lack classical peptide properties, such as enzymatically cleavable peptide bonds. A peptide or polypeptide may consist of amino acids other than the 20 amino acids defined by the genetic code. It may consist of L-amino acids and / or D-amino acids. A peptide or polypeptide may also consist of amino acids modified by natural processes, such as post-translational maturation processes, or by chemical processes, as are well known to those skilled in the art. Such modifications are fully described in the literature. These modifications may appear anywhere in the polypeptide, i.e., in the peptide backbone, in the amino acid chain, or even at the carboxyl or amino terminus. Peptides or polypeptides may be branched after ubiquitination, or they may be cyclic with or without branching. This type of modification may be the result of natural or synthetic post-translational processes well known to those skilled in the art. For example, peptide or polypeptide modifications may include acetylation, acylation, ADP-ribosylation, amidation, covalent fixation of nucleotides or nucleotide derivatives, covalent fixation of lipids or lipid derivatives, covalent fixation of phosphatidylinositol, covalent or non-covalent crosslinking, cyclization, disulfide bond formation, demethylation, glycation including pegylation, hydroxylation, iodization, methylation, myristoylation, oxidation, proteolytic processes, phosphorylation, prenylation, racemization, seneloylation, sulfation, amino acid addition, such as arginylation or ubiquitination. Such modifications are fully described in the literature and are well known to those skilled in the art.

[0045] In preferred embodiments of the present invention, the polypeptide contained in the pharmaceutical composition according to the present invention is endolysin. Therefore, in specific embodiments, the present invention relates to a pharmaceutical composition according to the present invention in which the polypeptide is endolysin having toxic activity against Gardnerella.

[0046] As used herein, the term “endolysin” refers to polypeptides typically produced by bacteriophages that digest host bacterial cell walls and release bacteriophage progeny. Endolysins are cell wall-lytic enzymes encoded by bacteriophages that, when added exogenously, have the ability to hydrolyze the cell walls of target bacteria (lysis-from-without). This novel class of antimicrobial agents offers significant advantages over classical antibiotics, such as novel modes of action, narrow-spectrum susceptibility to bacteria, rapid bacterial killing in both quiescent and logarithmic growth phases, activity against mucous membranes and bacterial biofilms, low likelihood of resistance development, and minimal impact on the normal microbiome. These unique characteristics have led to increased interest in the biotechnological and pharmacological uses of lysins, and they are now considered among the most promising alternatives to combat antibiotic resistance.

[0047] Endorticins often consist of two or more domains: at least one catalytic domain, such as a hydrolase domain (typically located at the N-terminus of the polypeptide) that cleaves specific motifs in the peptidoglycan layer, and often one or more cell wall-binding domains (classically located at the C-terminus of the polypeptide) that are involved in the specific binding and processing of bacterial peptidoglycans. This typical structure provides a general configuration of endolysin structures, but it is not a defined feature of all endolysins. Endorticins from Gram-positive bacteria and their phages usually contain at least one catalytic domain and one or more cell wall-binding domains. In contrast, many endolysins produced by Gram-negative species or their phages contain only a catalytic domain, 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 domains primarily determine the lysis spectrum by the specific recognition of cell wall elements that are distributed in a genus-specific or species / strain-specific manner.

[0048] The endolysin contained in the pharmaceutical composition of the present invention is preferably a functional polypeptide, whose function includes specifically targeting bacteria of the genus Gardnerella, and more preferably specifically killing bacteria of the genus Gardnerella. The endolysin contained in the pharmaceutical composition of the present invention more preferably includes a catalytic domain or a functional fragment thereof and / or a cell wall-binding domain or a functional fragment thereof. The endolysin contained in the pharmaceutical composition of the present invention may be natural or recombinant endolysin. The endolysin contained in the pharmaceutical composition of the present invention is most preferably recombinant endolysin. The endolysin contained in the pharmaceutical composition of the present invention is more preferably, (i) 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 includes 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 domain, Recombinant endolysins containing or consisting thereof It preferably has killing activity against Gardnerella cells / lines, and more preferably genus-selective killing activity.

[0049] As defined herein, the terms “to bind” and “to adhere” refer to the ability of endolysin to bind to the cell wall of a particular bacterium, and refer to the ability of endolysin to specifically interact with and adhere to the cell wall of said bacterium. The ability of endolysin to bind to the cell wall of a bacterium can be determined by methods known in the art.

[0050] In the context of this disclosure, the term “recombinant endolysin” preferably refers to domain-exchanged endolysin as defined in International Publication No. 2020 / 225335. In accordance with this definition, those skilled in the art will readily understand that the “domain-exchanged” or “recombinant” endolysins described herein are endolysins that do not exist in nature. That is, the recombinant endolysins for use in the present invention are artificially modified and, by definition, exclude natural endolysins, i.e., those that may exist naturally in nature. The appended examples and the teachings in International Publication No. 2020 / 225335 provide suitable methods(s) for producing the artificial endolysins of the present invention.

[0051] The terms “catalytic domain” or “enzyme domain” refer to a portion of a protein chain containing a region where a catalytic chemical reaction occurs. As used herein, “catalytic domain” refers to a functional polypeptide whose function includes the ability to dissolve the cell wall of Gardnerella. In particular, the catalytic domains described herein can preferably modify and / or cleave substrates, preferably peptidoglycans, in the Gardnerella cell wall. Preferably, the catalytic domain can cleave peptidoglycans in the Gardnerella cell wall, causing Gardnerella cell lysis. Preferably, the catalytic domain can modify and / or cleave bonds present in the cell walls and / or peptidoglycans of Gardnerella species, such as Gardnerella vaginalis, Gardnerella leopoldii, Gardnerella piochii, and / or Gardnerella swidsynskii, preferably all of their cell walls. Preferably, the catalytic domain does not modify and / or cleave substrates present in the cell walls of bacteria other than Gardnerella species, preferably healthy vaginal commensal bacteria, such as Lactobacillus crispatus, Lactobacillus gasseri, and / or Lactobacillus gensenii, preferably peptidoglycan. The catalytic domain may be N-acetylmuramidase, N-acetylmuramoyl-L-alanine amidase, L-alanoyl-D-glutamate endopeptidase, interpeptide cross-linking endopeptidase, or N-acetyl-β-D-glucosaminidase. Preferably, the N-terminal catalytic domain is N-acetylmuramidase, most preferably 1,4-β-N-acetylmuramidase. The catalytic domain is preferably located on the N-terminal side within (recombinant)endolysin and is therefore referred to as the "N-terminal catalytic domain," and more preferably, the N-terminal catalytic domain is located on the N-terminal side from the C-terminal cell wall binding region within (recombinant)endolysin.

[0052] The endolysin contained in the pharmaceutical composition of the present invention preferably comprises a polypeptide containing or consisting of any one amino acid sequence of SEQ ID NOs: 2 to 10, or a catalytic domain consisting of any functional variant thereof having at least 80% identity with any one amino acid sequence of SEQ ID NOs: 2 to 10. As shown in International Publication No. 2020 / 225335, the most active catalytic domain is "H2" (SEQ ID NO: 3). Accordingly, in a preferred embodiment of the present invention, the catalytic domain comprises or consists of a polypeptide comprising any functional variant thereof having at least 80% identity with the amino acid sequence of SEQ ID NO: 3, or at least 85% identity (preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99%, even more preferably at least 99.5%, and most preferably at least 99.7%), thereby the endolysin is functional, and its function includes the ability to dissolve the cell wall of Gardnerella. The catalytic domain is preferably located on the N-terminal side within (recombinant)endolysin and is thereby referred to as the "N-terminal catalytic domain," and more preferably, (recombinant)endolysin further comprises a cell wall binding region, and the N-terminal catalytic domain is located on the N-terminal side from the C-terminal cell wall binding region within (recombinant)endolysin.

[0053] As used herein, “cell wall binding region” refers to a functional polypeptide whose function includes the ability to bind to the cell wall of Gardnerella. A cell wall binding region may contain 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 a specific substrate in the bacterial cell wall and / or within the bacterial cell wall. In particular, the cell wall binding domains described herein are preferably capable of specifically binding to the cell walls (e.g., peptidoglycan) of Gardnerella species, such as Gardnerella vaginalis, Gardnerella leopoldii, Gardnerella piochii, and / or Gardnerella swidsinskii, preferably all of their cell walls. The cell wall binding region is preferably located on the C-terminal side within (recombinant) endolysin and is thereby referred to as the “C-terminal cell wall binding region,” and more preferably the C-terminal cell wall binding region is located on the C-terminal side of the N-terminal catalytic domain within (recombinant) endolysin.

[0054] The endolysin contained in the pharmaceutical composition of the present invention preferably contains or comprises a cell wall-binding region comprising at least one cell wall-binding domain selected from the group consisting of polypeptides comprising or comprising any one amino acid sequence of SEQ ID NOs. 11 to 28, and any functional variant thereof having at least 80% identity with any one amino acid sequence of SEQ ID NOs. 11 to 28 (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, most preferably at least 99.7% identity). As shown in International Publication No. 2020 / 225335, the most active cell wall-binding region is "B10" (containing the cell wall-binding domains of SEQ ID NOs. 23 and 24), followed by "B11" (containing the cell wall-binding domains of SEQ ID NOs. 25 and 26). Therefore, in a preferred embodiment of the present invention, the cell wall-binding domain(s) is selected from the group consisting of polypeptides comprising or comprising any one of the amino acid sequences 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, most preferably at least 99.7% identity), thereby the endolysin is functional, and its function includes the ability to lyse the cell wall of Gardnerella.The cell wall binding region is preferably located on the C-terminal side within (recombinant)endolysin and is therefore referred to as the "C-terminal cell wall binding region," and more preferably, the (recombinant)endolysin further comprises a catalytic domain, the catalytic domain being located on the N-terminal side of the C-terminal cell wall binding region within (recombinant)endolysin.

[0055] The endolysin contained in the pharmaceutical composition of the present invention preferably comprises two cell wall-binding domains (within the cell wall-binding region). In a preferred embodiment of the present invention, each of the multiple cell wall-binding domains of the endolysin of the present invention comprises a polypeptide containing or consisting of any one of the amino acid sequences 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, most preferably at least 99.7% identity), thereby the endolysin is functional, and its function includes the ability to dissolve the cell wall of Gardnerella. In a more preferred embodiment of the present invention, 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 at the N-terminus of the second cell wall binding domain.

[0056] In one more preferred embodiment, the endolysin contained in the pharmaceutical composition of the present invention is (i) an N-terminal catalytic domain comprising a polypeptide containing or consisting of the amino acid sequence of SEQ ID NO: 3, or any functional variant thereof having at least 80% identity with the amino acid sequence of SEQ ID NO: 3 (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, most preferably at least 99.7% identity), and (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, Includes, 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, most preferably at least 99.7% identity), 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, most preferably at least 99.7% identity), Thus, endolysin is functional, and its function includes the ability to lyse the cell wall of Gardnerella. Preferably, the first cell wall binding domain is located N-terminal to the second cell wall binding domain.

[0057] In one particularly preferred embodiment, the endolysin contained in the pharmaceutical composition of the present invention is (i) An N-terminal catalytic domain comprising a polypeptide containing or consisting of the amino acid sequence of SEQ ID NO: 3, and (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, Includes, The first cell wall-binding domain is selected from the group consisting of Sequence IDs 23 and 25, and the second cell wall-binding domain is selected from the group consisting of Sequence IDs 24 and 26.

[0058] Preferably, the first cell wall binding domain is located on the N-terminal side of the second cell wall binding domain.

[0059] Particularly preferred examples of endolysins included in the pharmaceutical composition of the present invention are "H2B10" (including the N-terminus to the C-terminus: SEQ ID NOs. 3, 23, and 24), "H2B11" (including the N-terminus to the C-terminus: SEQ ID NOs. 3, 25, and 26), and "H2B10B11" (including the N-terminus to the C-terminus: SEQ ID NOs. 3, 23, and 26), as defined in International Publication No. 2020 / 225335.

[0060] The endolysin contained in the pharmaceutical composition of the present invention more preferably includes a linker region between the N-terminal catalytic domain and the C-terminal cell wall binding region. The linker region may consist of a polypeptide having a length of 6 to 18 amino acids, preferably 9 to 15 amino acids, and more preferably 12 amino acids. Preferably, the linker region may consist of a polypeptide containing or comprising the amino acid sequence (i)(XXX)n [wherein each X can independently be G, A, or S], preferably the amino acid sequence (GGS)n [wherein n corresponds to the number of repeats of sequence XXX, preferably 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.

[0061] In a particularly preferred embodiment, the endolysin contained in the pharmaceutical composition of the present invention has a sequence of "H2B10" as shown in Sequence ID No. 1, or a sequence of "H2B10B11" as shown in Sequence ID No. 37.

[0062] In certain embodiments, the present invention relates to a pharmaceutical composition according to the present invention, wherein the polypeptide has at least 80% sequence identity with the amino acid sequence provided in SEQ ID NO: 1 and is a polypeptide having toxic activity against Gardnerella.

[0063] That is, in a particular embodiment, the present invention provides a pharmaceutical composition comprising an effective amount of Gardnerella-specific endolysin, preferably recombinant Gardnerella-specific endolysin, and a polyacrylic acid polymer, wherein the endolysin is a polypeptide having at least 80% sequence identity with the amino acid sequence provided in SEQ ID NO: 1 and having toxic activity against Gardnerella. In one more preferred embodiment, the present invention provides a pharmaceutical composition comprising an effective amount of Gardnerella-specific endolysin, preferably recombinant Gardnerella-specific endolysin, and a polyacrylic acid polymer, wherein the endolysin is a polypeptide having at least 90% sequence identity with the amino acid sequence provided in SEQ ID NO: 1 and having toxic activity against Gardnerella. In a further more preferred embodiment, the present invention provides a pharmaceutical composition comprising an effective amount of Gardnerella-specific endolysin, preferably recombinant Gardnerella-specific endolysin, and a polyacrylic acid polymer, wherein the endolysin is a polypeptide having at least 95% sequence identity with the amino acid sequence provided in SEQ ID NO: 1 and having toxic activity against Gardnerella. In a more preferred embodiment, the present invention provides a pharmaceutical composition comprising an effective amount of Gardnerella-specific endolysin, preferably recombinant Gardnerella-specific endolysin, and a polyacrylic acid polymer, wherein the endolysin is a polypeptide having at least 99% sequence identity with the amino acid sequence provided in SEQ ID NO: 1 and having toxic activity against Gardnerella. In a most preferred embodiment, the present invention provides a pharmaceutical composition comprising an effective amount of Gardnerella-specific endolysin, preferably recombinant Gardnerella-specific endolysin, and a polyacrylic acid polymer, wherein the endolysin comprises or consists of the amino acid sequence provided in SEQ ID NO: 1.

[0064] Accordingly, in certain embodiments, the present invention relates to a pharmaceutical composition according to the present invention, wherein the polypeptide is a recombinant endolysin comprising the amino acid sequence provided in SEQ ID NO: 1.

[0065] The endolysin comprising or consisting of the amino acid sequence provided in SEQ ID NO: 37 may also be used in the context of the present invention, namely in the context of a pharmaceutical composition suitable for treating bacterial vaginosis comprising an effective amount of Gardnerella-specific endolysin, preferably recombinant Gardnerella-specific endolysin, and a polyacrylic acid polymer. Therefore, the endolysin comprising or consisting of the amino acid sequence provided in SEQ ID NO: 37 is a substitute for the Gardnerella-specific endolysin provided in SEQ ID NO: 1.

[0066] Accordingly, in certain embodiments, the present invention relates to a pharmaceutical composition according to the present invention, wherein the polypeptide has at least 80% sequence identity with the amino acid sequence provided in SEQ ID NO: 37 and is a polypeptide having toxic activity against Gardnerella.

[0067] That is, in a particular embodiment, the present invention provides a pharmaceutical composition comprising an effective amount of Gardnerella-specific endolysin, preferably recombinant Gardnerella-specific endolysin, and a polyacrylic acid polymer, wherein the endolysin is a polypeptide having at least 80% sequence identity with the amino acid sequence provided in SEQ ID NO: 37 and having toxic activity against Gardnerella. In one more preferred embodiment, the present invention provides a pharmaceutical composition comprising an effective amount of Gardnerella-specific endolysin, preferably recombinant Gardnerella-specific endolysin, and a polyacrylic acid polymer, wherein the endolysin is a polypeptide having at least 90% sequence identity with the amino acid sequence provided in SEQ ID NO: 37 and having toxic activity against Gardnerella. In a further more preferred embodiment, the present invention provides a pharmaceutical composition comprising an effective amount of Gardnerella-specific endolysin, preferably recombinant Gardnerella-specific endolysin, and a polyacrylic acid polymer, wherein the endolysin is a polypeptide having at least 95% sequence identity with the amino acid sequence provided in SEQ ID NO: 37 and having toxic activity against Gardnerella. In a more preferred embodiment, the present invention provides a pharmaceutical composition comprising an effective amount of Gardnerella-specific endolysin, preferably recombinant Gardnerella-specific endolysin, and a polyacrylic acid polymer, wherein the endolysin is a polypeptide having at least 99% sequence identity with the amino acid sequence provided in SEQ ID NO: 37 and having toxic activity against Gardnerella. In a most preferred embodiment, the present invention provides a pharmaceutical composition comprising an effective amount of Gardnerella-specific endolysin, preferably recombinant Gardnerella-specific endolysin, and a polyacrylic acid polymer, wherein the endolysin comprises or consists of the amino acid sequence provided in SEQ ID NO: 37.

[0068] Accordingly, in certain embodiments, the present invention relates to a pharmaceutical composition according to the present invention, wherein the polypeptide is a recombinant endolysin comprising the amino acid sequence provided in SEQ ID NO: 37.

[0069] In one embodiment, the present invention relates to a pharmaceutical composition comprising recombinant endolysin having at least 80% sequence identity with the amino acid sequence provided in SEQ ID NO: 1 or 37 and having killing activity against Lactobacillus iners, for use in the treatment of bacterial infections involving and / or caused by Lactobacillus iners.

[0070] L. iners is a bacterial species associated with an imbalanced vaginal microbiome and is known to potentially be involved in pubescent venereal vein thromboembolism (BV). After metronidazole (MDZ) treatment, patients often transition to an L. iners-dominant microbiome, followed by a relapse of BV. Therefore, endolysin, which has (additional) killing activity against L. iners, is particularly relevant in the treatment of BV.

[0071] Preferably, the present invention relates to a pharmaceutical composition comprising recombinant endolysin comprising the amino acid sequence provided in SEQ ID NO: 1 or 37 for use in the treatment of bacterial infections involving and / or caused by the Lactobacillus iners species. Surprisingly, in Figures 1B, 3, and 6, endolysin PM-477 and H2B10B11 are shown to have toxic activity against L. iners, both in the presence and absence of polyacrylic acid polymers. In certain embodiments, the bacterial infection caused by L. iners is a vaginal infection, particularly bacterial vaginosis. It is understood that any embodiment disclosed herein is similarly applicable to compositions for use in the treatment of L. iners infections. In particular, the composition may further comprise polyacrylic acid polymers disclosed elsewhere herein. In certain embodiments, the present invention relates to a method for treating an infection involving and / or caused by L. iners in a subject requiring treatment, comprising the step of administering to a patient recombinant endolysin having at least 80% sequence identity with the amino acid sequence provided in SEQ ID NO: 1 or 37 and having killing activity against L. iners. In certain embodiments, the present invention relates to a method for reducing the number of L. iners in the vaginal microbiota of a subject requiring a reduction in the number of L. iners in the vaginal microbiota, comprising the step of administering to a patient recombinant endolysin having at least 80% sequence identity with the amino acid sequence provided in SEQ ID NO: 1 or 37 and having killing activity against L. iners.

[0072] The term “mutant” refers to a polypeptide containing non-conservative or preferably conservative insertions, deletions, and / or substitutions to a native amino acid sequence. For example, a polypeptide may contain an amino acid sequence having at least 80% identity to a native amino acid sequence, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99%, even more preferably at least 99.5%, and most preferably at least 99.7% identity. The identity percentage can be determined by methods well known in the art using a suitable computer program, e.g., MatGAT 2.0 (Myers and Miller, CABIOS (1989)). Preferably, the identity percentage is identified over the entire length of the sequence being compared. It is understood that the identity percentage is calculated with respect to a polypeptide whose sequence is optimally aligned. Fragments and variants of amino acid sequences can be prepared using any of the protein engineering, directed evolution, and / or site-directed mutagenesis methods known in the art (see, for example, Molecular Cloning: A Laboratory Manual, 3rd edition, Sambrook & Russell, 2001, Cold Spring Harbor Laboratory Press). Those skilled in the art will understand that the polypeptides, or fragments, variants, or fusions thereof according to the present invention may include, or consist of, derivatives of, natural amino acid sequences, or fragments or variants thereof. Chemical derivatives of one or more amino acids can be achieved by reaction with functional side chain groups. Examples of such derivatized molecules include molecules in which a free amino acid group is derivatized to form an amine hydrochloride, a p-toluenesulfonyl group, a carboxybenzoxy group, a t-butyloxycarbonyl group, a chloroacetyl group, or a formyl group.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. Peptides containing naturally occurring amino acid derivatives of 20 standard amino acids are also included as chemical derivatives. For example, 4-hydroxyproline may be used instead of proline. 5-hydroxylysine may be used instead of lysine. 3-methylhistidine may be used instead of histidine. Homoserine may be used instead of serine, and ornithine instead of lysine. Derivatives also include peptides with one or more additions or deletions, as long as the desired activity is maintained. Other included modifications are terminal modifications such as amidation, amino-terminal acylation (e.g., acetylation or thioglycolic acid amidation), and terminal carboxylamilation (e.g., by ammonia or methylamine). It will be further understood by those skilled in the art that peptide mimetic compounds may also be useful. Therefore, "polypeptides" include peptide mimetic compounds exhibiting endolysine activity. The term "peptide mimetic" refers to a compound that mimics the three-dimensional structure and desirable characteristics of a particular polypeptide as a therapeutic agent.

[0073] The composition according to the present invention may contain one or more endolysin polypeptides. In certain embodiments, the endolysin polypeptide may exist as an independent polypeptide or as a fusion protein comprising the endolysin polypeptide or a fragment thereof.

[0074] The term “pharmaceutical composition” refers to a preparation in which the biological activity of the active ingredient is clearly effective and which does not contain additional ingredients that would be toxic to the patient to whom the composition is administered. As used herein, “pharmaceutical composition” means a therapeutically effective formulation for use in the methods of the present invention. As used herein, “therapeutic effective dose” or “effective dose” or “therapeutically effective” refers to an amount that provides a therapeutic effect for a given condition and administration regimen. This is a predetermined amount of the active substance calculated to produce the desired therapeutic effect in relation to the necessary additives and diluents, i.e., carriers or administration media. Furthermore, it is intended to mean an amount sufficient to reduce, and most preferably prevent, a clinically significant deficit in the host’s activity, function, and response. Alternatively, a therapeutic effective dose is sufficient to cause a clinically significant improvement in the host’s condition. As will be understood by those skilled in the art, the amount of a compound may vary depending on its specific activity. An appropriate dose may include a predetermined amount of the active composition calculated to produce the desired therapeutic effect in combination with the necessary diluents. In the methods for producing and using the compositions of the present invention, a therapeutically effective dose of the active ingredient is provided. The therapeutically effective dose can be determined by a conventionally skilled medical or veterinary professional based on patient characteristics such as age, weight, sex, condition, comorbidities, and other diseases, as is well known in the art.

[0075] In one embodiment of the present invention, the pharmaceutical composition comprises the Gardnerella-specific endolysin described herein and is intended for use in the treatment of bacterial vaginosis as described herein. Accordingly, the pharmaceutical composition may contain an amount of endolysin, or a fragment, variant, fusion, or derivative thereof, sufficient to at least partially inhibit the proliferation of Gardnerella cells in the patients described herein who are infected with or susceptible to infection by Gardnerella cells. Preferably, the pharmaceutical composition contains an amount of endolysin, or a fragment, variant, fusion, or derivative thereof, sufficient to kill Gardnerella cells in the patients targeted for treatment as defined herein above.

[0076] The pharmaceutical composition according to the present invention further comprises pharmaceutically acceptable excipients. The term “pharmaceutically acceptable excipient” means an excipient composed of a material that is not biologically or otherwise undesirable. The term “carrier” means any component other than the activator present in the pharmaceutical formulation, and therefore includes excipients, diluents, binders, lubricants, disintegrants, fillers, colorants, wetting agents or emulsifiers, pH buffers, preservatives, and the like.

[0077] Surprisingly, the inventors have found that certain excipients, namely polyacrylic acid polymers, can increase the toxic activity of bactericidal polypeptides, particularly Gardnerella-specific endolysins. Therefore, in certain embodiments, the present invention relates to pharmaceutical compositions according to the present invention, in which a combination of the activator and the excipient increases the pharmaceutically active of the activator.

[0078] Within the scope of the present invention, excipients, particularly polyacrylic acid polymers, "increase the pharmaceutically active" of an activator, i.e., a Gardnerella-specific bactericidal polypeptide, if they increase the activating activity of the activator against Gardnerella species bacteria. Excipients can increase the pharmaceutically active of an activator 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%. From Figure 2, it can be seen that the excipient PAA increases the activating activity of the activator PM-477 against Gardnerella biofilm by nearly 100%, and increases the activating activity of the active component H2B10B11 against Gardnerella biofilm by an even greater extent.

[0079] As used herein, the term "polyacrylic acid polymer" refers to polymers of acrylic acid, and includes homopolymers, copolymers, and crosslinked polymers of acrylic acid. That is, polyacrylic acid polymers are defined by the formula (CH2-CHCO2H) n It is a polymer containing or consisting of.

[0080] In certain embodiments, the polyacrylic acid polymer is a linear polyacrylic acid polymer, particularly a linear polyacrylic acid homopolymer. Example 2 demonstrates that such a polymer significantly increases the endolysin-causing activity against Gardnerella biofilms. Furthermore, Example 3 demonstrates that the linear polyacrylic acid homopolymer can extend the antimicrobial spectrum of endolysin PM-477 to other BV-related bacteria.

[0081] In certain embodiments, the “polyacrylic acid polymer” is a crosslinked polyacrylic acid polymer, such as a carbomer or carboxyvinyl polymer. A crosslinked polyacrylic acid polymer is a homopolymer or copolymer of acrylic acid crosslinked with at least one other polymer. The term “carbomer” refers to a range of polymers of acrylic acid commonly used in pharmaceutical compositions. A carbomer is a high molecular weight homopolymer or copolymer of acrylic acid crosslinked with a polyalkenyl ether of a sugar or polyalcohol. The carbomer contained in the pharmaceutical composition according to the present invention is preferably a carbomer that conforms to the requirements of the United States and / or European Pharmacopoeia. Carbomer is commercially available under the trade name Carbopol. Therefore, in certain embodiments, the present invention relates to a pharmaceutical composition according to the present invention, wherein the excipient is a carbomer, preferably Carbopol.

[0082] In certain embodiments, the pharmaceutical composition according to the present invention comprises a specific type of carbomer. In certain embodiments, the pharmaceutical composition according to the present invention comprises a mixture of two or more different types of carbomers.

[0083] The polyacrylic acid polymer contained in the pharmaceutical composition according to the present invention may be defined based on one or more physicochemical properties.

[0084] Preferably, the polyacrylic acid polymer contained in the pharmaceutical composition according to the present invention is defined based on its viscosity. That is, the polyacrylic acid polymer contained in the pharmaceutical composition according to the present invention may have a viscosity in the range of 300 to 115,000 mPa·s when in the form of a 0.5% (w / v) gel (pH 7.3 to 7.8 at 25°C).

[0085] In certain embodiments, the polyacrylic acid polymer contained in the pharmaceutical composition according to the present invention is a United States Pharmacopeia type A carbomer having a viscosity of 4,000 to 11,000 mPa·s when in the form of a 0.5% (w / v) gel (pH 7.3 to 7.8 at 25°C). In certain embodiments, the type A carbomer is Carbopol 981, Carbopol 971, or Carbopol 71G.

[0086] In certain embodiments, the polyacrylic acid polymer contained in the pharmaceutical composition according to the present invention is a United States Pharmacopeia type B carbomer having a viscosity of 25,000 to 45,000 mPa·s when in the form of a 0.5% (w / v) gel (pH 7.3 to 7.8 at 25°C). In certain embodiments, the type B carbomer is Carbopol 974P, Carbopol 984, or Carbopol 5984, preferably Carbopol 974P.

[0087] In certain embodiments, the polyacrylic acid polymer contained in the pharmaceutical composition according to the present invention is a US Pharmacopeia type C carbomer having a viscosity of 40,000 to 60,000 mPa·s when in the form of a 0.5% (w / v) gel (pH 7.3 to 7.8 at 25°C). In certain embodiments, the type C carbomer is Carbopol 980.

[0088] In certain embodiments, the polyacrylic acid polymer contained in the pharmaceutical composition according to the present invention has a viscosity of 300 to 115,000 mPa·s when in the form of a 0.5% (w / v) gel (pH 7.3 to 7.8 at 25°C). In certain embodiments, the polyacrylic acid polymer contained in the pharmaceutical composition according to the present invention has a viscosity of 300 to 60,000 mPa·s when in the form of a 0.5% (w / v) gel (pH 7.3 to 7.8 at 25°C). In certain embodiments, the polyacrylic acid polymer contained in the pharmaceutical composition according to the present invention has a viscosity of 300 to 45,000 mPa·s when in the form of a 0.5% (w / v) gel (pH 7.3 to 7.8 at 25°C). In certain embodiments, the polyacrylic acid polymer contained in the pharmaceutical composition according to the present invention has a viscosity of 300 to 11,000 mPa·s when in the form of a 0.5% (w / v) gel (pH 7.3 to 7.8 at 25°C). In certain embodiments, the polyacrylic acid polymer contained in the pharmaceutical composition according to the present invention has a viscosity of 300 to 4,000 mPa·s when in the form of a 0.5% (w / v) gel (pH 7.3 to 7.8 at 25°C). In certain embodiments, the polyacrylic acid polymer contained in the pharmaceutical composition according to the present invention has a viscosity of 300 to 2,000 mPa·s when in the form of a 0.5% (w / v) gel (pH 7.3 to 7.8 at 25°C).

[0089] In a particular embodiment, the polyacrylic acid polymer contained in the pharmaceutical composition according to the present invention is a linear polyacrylic acid homopolymer having a viscosity of 300 to 2000 mPa·s when in the form of a 0.5% (w / v) gel (pH 7.3 to 7.8 at 25°C).

[0090] In a particular embodiment, the polyacrylic acid polymer contained in the pharmaceutical composition according to the present invention is a type B carbomer, such as Carbomer 974P, having a viscosity of 25,000 to 45,000 mPa·s when in the form of a 0.5% (w / v) gel (pH 7.3 to 7.8 at 25°C).

[0091] In certain embodiments, the present invention relates to a pharmaceutical composition according to the present invention, wherein the excipient is present in a concentration of about 0.1% to 10%.

[0092] In other words, the polyacrylic acid polymer is present in the pharmaceutical composition of the present invention in amounts of approximately 0.1%, approximately 0.2%, approximately 0.25%, approximately 0.3%, approximately 0.35%, approximately 0.4%, approximately 0.5%, approximately 0.6%, approximately 0.7%, approximately 0.8%, approximately 0.9%, approximately 1%, approximately 1.1%, approximately 1.2%, approximately 1.3%, approximately 1.4%, approximately 1.5%, approximately 1.6%, approximately 1.7%, approximately 1.8%, approximately 1.9%, and approximately 2%. Approximately 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7% Approximately 4.8%, 4.9%, 5%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7%, 7.1%, 7.2%, 7.3%, 7.4%, 7. It may be present at concentrations of 5%, approximately 7.6%, approximately 7.7%, approximately 7.8%, approximately 7.9%, approximately 8%, approximately 8.1%, approximately 8.2%, approximately 8.3%, approximately 8.4%, approximately 8.5%, approximately 8.6%, approximately 8.7%, approximately 8.8%, approximately 8.9%, approximately 9%, approximately 9.1%, approximately 9.2%, approximately 9.3%, approximately 9.4%, approximately 9.5%, approximately 9.6%, approximately 9.7%, approximately 9.8%, approximately 9.9%, or approximately 10%.

[0093] In a particularly preferred embodiment, the present invention relates to a pharmaceutical composition according to the present invention, wherein the excipient is present in a concentration of about 0.1% to 5%, more preferably about 0.1% to 2.5%, even more preferably about 0.1% to 1%, and most preferably about 0.2% to 0.5%. In a particularly preferred embodiment, the excipient, in particular the polyacrylic acid polymer, is present in a concentration of about 0.25% or 0.35%.

[0094] In certain embodiments, the pharmaceutical composition according to the present invention further comprises a salt. Surprisingly, Figure 7 shows that increasing the salt concentration in the culture medium increases the activity of endolysin when formulated with a polyacrylic acid polymer. As used herein, the term “salt” generally refers to an ionic compound composed of both cations (positively charged ions) and anions (negatively charged ions), thereby resulting in an electrically neutral (no net charge) product. These constituent ions may be inorganic or organic ions, monatomic or polyatomic, monovalent or polyvalent. Preferably, the salt contained in the pharmaceutical composition according to the present invention is a pharmaceutically acceptable salt. In preferred embodiments, the salt contained in the pharmaceutical composition according to the present invention is sodium chloride (NaCl).

[0095] Salts, particularly NaCl, may be present in the composition at any suitable concentration. In certain embodiments, the pharmaceutical composition according to the present invention contains NaCl in concentrations ranging from about 0% to about 12.5%, preferably about 1% to about 10%, and more preferably about 2.5% to about 7.5%. In certain embodiments, the pharmaceutical composition according to the present invention contains NaCl in concentrations 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 according to the present invention contains NaCl in concentrations 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%.

[0096] It should be understood that the concentrations provided herein should be understood as w / v% (weight / volume percentage) when the composition is formulated as a liquid, and w / w% (weight / weight percentage) when the composition is formulated as a solid.

[0097] The pharmaceutical compositions provided herein can be used in the form of solids such as tablets or filled capsules, or liquids such as solutions, suspensions, emulsions, elixirs, or capsules filled therewith, all for oral use or in the form of suppositories for topical use (including vaginal use). Furthermore, other means of administration are also contemplated in the context of the present invention. Such means may, in particular, include parenteral administration.

[0098] Furthermore, pharmaceutical compositions for topical and / or local administration, such as pharmaceutical compositions in the form of (topical) gels, lotions, or creams, or pharmaceutical compositions via pessaries, i.e., vaginal suppositories, are also envisioned. Such pessaries / vaginal suppositories may be coated with the pharmaceutical compositions described herein. The pharmaceutical compositions and their unit dosage forms may contain components in conventional proportions, with or without additional active compounds or components, and such unit dosage forms may contain any appropriate effective amount of active component corresponding to the intended daily dose range for use. The compositions for use of the present invention may be liquid formulations, including but not limited to aqueous or oily suspensions, solutions, emulsions, syrups, and elixirs. The compositions may be formulated as dry products for reconstitution with water or other suitable media before use. Such liquid preparations may contain additives, including but not limited to suspending agents, emulsifiers, non-aqueous media, and preservatives. Examples of suspending agents include, but are not limited to, sorbitol syrup, methylcellulose, glucose / sugar syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, and hydrogenated edible oils and fats. Examples of emulsifiers include, but are not limited to, lecithin, sorbitan monooleate, and acacia. Examples of non-aqueous media include, but are not limited to, edible oils, almond oil, fractionated coconut oil, oily esters, propylene glycol, and ethyl alcohol. Examples of preservatives include, but are not limited to, p-hydroxybenzoate methyl or propyl and sorbic acid. Further materials and processing techniques are described in Part 5 of Remington's "The Science and Practice of Pharmacy," 22nd Edition, 2012, University of the Sciences in Philadelphia, Lippincott Williams & Wilkins.

[0099] The solid compositions of the present invention may be in the form of tablets or lozenges formulated in a conventional manner. The tablets may be coated according to methods well known in the art. The injectable compositions are typically based on injectable sterile saline or phosphate-buffered saline, or other injectable carriers known in the art.

[0100] The pharmaceutical compositions of the present invention may also be formulated as suppositories, which may comprise a suppository base (including, but not limited to, cocoa butter or glycerides). The compositions of the present invention may also be formulated as transdermal formulations comprising aqueous or non-aqueous media, including, but not limited to, creams, ointments, lotions, pastes, medicinal plasters, patches, or films. The compositions of the present invention may also be formulated for parenteral administration, including, but not limited to, injection or continuous infusion. Injectable formulations may be in the form of suspensions, solutions, or emulsions in oily or aqueous media, and may contain formulation agents, including, but not limited to, suspending agents, stabilizers, and dispersants. The compositions may also be provided in powder form for reconstitution in a suitable medium (including, but not limited to, sterile water free of pyrogens).

[0101] The pharmaceutical compositions of the present invention may also be formulated as depot preparations, which can be administered by implantation or intramuscular injection. The compositions may be formulated using suitable polymer or hydrophobic materials (e.g., as emulsions in acceptable oils), ion exchange resins, or as sparingly soluble derivatives (e.g., as sparingly soluble salts).

[0102] The pharmaceutical compositions of the present invention may also be administered in a sustained-release form or via a sustained-release drug delivery system. A description of typical sustained-release materials can also be found in Remington's "The Science and Practice of Pharmacy."

[0103] The pharmaceutical composition according to the present invention may further contain one or more pharmaceutically acceptable components (e.g., alum, stabilizers, antibacterial agents, buffers, colorants, flavoring agents, adjuvants, etc.). The pharmaceutical composition for use according to the present invention is preferably imidazole-free.

[0104] The pharmaceutical compositions according to the present invention are preferably used for the treatment of bacterial vaginosis. Therefore, it is preferable herein that the pharmaceutical compositions according to the present invention be formulated for intravaginal delivery. Accordingly, in certain embodiments, the present invention relates to pharmaceutical compositions according to the present invention that are suitable for intravaginal delivery of an active agent. Those skilled in the art can formulate pharmaceutical compositions comprising the active agent and excipients disclosed herein that are suitable for intravaginal delivery.

[0105] However, the pharmaceutical compositions of the present invention can also be used to treat bacterial infections in men, particularly Gardnerella infections. In such embodiments, the pharmaceutical compositions according to the present invention are preferably formulated for administration in or over the glans penis, foreskin, or urethral opening. Those skilled in the art can formulate pharmaceutical compositions comprising the activators and excipients disclosed herein that are suitable for delivery in or over the glans penis, foreskin, or urethral opening.

[0106] In certain embodiments, the present invention relates to a pharmaceutical composition according to the present invention for use in the treatment of bacterial infections, preferably bacterial vaginosis.

[0107] In other words, the pharmaceutical composition of the present invention, comprising a Gardnerella-specific polypeptide and a polyacrylic acid polymer, can be used to treat bacterial infections, particularly those caused by bacteria of the genus Gardnerella. Accordingly, the pharmaceutical composition of the present invention can be used to treat infections caused by one or more of the narrowly defined G. vaginalis, G. leopoldii, G. piochii, and / or G. swidosinskii.

[0108] Gardnerella species, such as G. vaginalis, are the most common cause of bacterial vaginosis. As used herein, “bacterial vaginosis” (BV), also referred to in the literature as bacterial vaginosis, and nonspecific vaginitis and nonspecific vaginosis refer to the most common vaginal infections worldwide. In one embodiment, BV is defined as a pathological condition characterized by the loss of the normal vaginal microbiota, particularly H2O2-producing Lactobacillus species, and the simultaneous overgrowth of anaerobic bacteria, often derived from the genus Gardnerella. The genus Gardnerella includes at least 13 species, the most frequent of which have been renamed G. vaginalis, G. leopoldii, G. piochii, and G. swidsinskii in the narrow sense (Vaneechoutte et al., 2019 Int.J.Syst.Evol.Biol.898661). In one preferred therapeutic embodiment of the present invention, BV to be treated is a bacterial infection characterized by the presence of at least one strain of the genus Gardnerella selected from the group consisting of Gardnerella vaginalis in the narrow sense, Gardnerella leopoldii, Gardnerella piocii and Gardnerella swidsynskii, and any other Gardnerella species. Accordingly, in one embodiment, the pharmaceutical composition according to the present invention comprises, for use in the treatment of bacterial vaginosis, Gardnerella-specific endolysin described herein, preferably recombinant Gardnerella-specific endolysin, said bacterial vaginosis characterized by the presence of infectious bacteria of Gardnerella vaginalis in the narrow sense, Gardnerella leopoldii, Gardnerella piocii, Gardnerella swidsynskii, and / or any other species of the genus Gardnerella. In this specification, bacterial vaginosis "characterized by the presence of" (or "caused by" as used interchangeably herein) certain bacterial species of the genus Gardnerella refers to an overgrowth of said bacteria (also referred to herein as "infectious bacteria") in the patient's vaginal microbiota, resulting in vaginal dysbiosis and / or loss of Lactobacillus dominance. Methods for determining whether bacterial vaginosis is characterized by the presence of infectious bacteria of the genus Gardnerella are known to those skilled in the art. As an example, a PCR test can be used to check for the presence of Gardnerella strains to diagnose BV.

[0109] It is important to understand that Gardnerella infection can also affect men, causing severe, foul-smelling inflammation and swelling of the inner foreskin and glans penis. Therefore, the pharmaceutical composition of the present invention may be used in the treatment of Gardnerella infection in men, particularly when the infection affects the glans penis, foreskin, or urethral opening.

[0110] Accordingly, in certain embodiments, the present invention relates to pharmaceutical compositions for use according to the present invention, which are administered topically into the vagina of female subjects and / or into or on the glans penis, foreskin, or urethral opening of male subjects.

[0111] In certain embodiments, the present invention relates to a pharmaceutical composition for use in the treatment of bacterial vaginosis, wherein a polypeptide, particularly endolysin, is administered to a patient suffering from bacterial vaginosis in which treatment with antibiotics has previously failed and / or the infectious bacteria are resistant to antibiotic treatment.

[0112] In other words, in certain embodiments, BV treated with the pharmaceutical compositions described herein is characterized by the presence of a Gardnerella strain (i.e., a strain of the genus Gardnerella) that is resistant to one or more antibiotics. In a preferred embodiment, the one or more antibiotics are selected from the group consisting of nitroimidazole and clindamycin. In a more preferred embodiment, the Gardnerella strain is resistant to metronidazole, tinidazole, secnidazole, clindamycin, or any combination thereof. In a still more preferred embodiment, the Gardnerella strain is resistant to metronidazole and / or clindamycin. In a still more preferred embodiment, the Gardnerella strain is resistant to metronidazole. In the most preferred embodiment, the Gardnerella strain is resistant to to highly resistant to metronidazole.

[0113] As used herein, “patients who have previously failed treatment with antibiotics” means patients with a history of bacterial vaginosis, i.e., patients who have previously suffered from (or have already experienced) symptoms of bacterial vaginosis and have been treated for bacterial vaginosis with antibiotics, but have experienced a relapse, i.e., symptoms have reappeared. Such patients may also be referred to as patients with recurrent BV. As used herein, the terms “patients with a history of bacterial vaginosis,” “patients who have already experienced symptoms of bacterial vaginosis,” and “patients with recurrent BV” are interchangeable and not limited to patients who have had one or more episodes of BV, preferably two or more episodes of BV, more preferably two or more episodes of BV within the last six months, even more preferably two or more episodes of BV within the last six months, or preferably three or more episodes of BV, more preferably three or more episodes of BV within the last twelve months, even more preferably three or more episodes of BV within the last twelve months.

[0114] Methods for diagnosing BV in patients, or demonstrating an episode of BV, are known to those skilled in the art. For example, BV can be diagnosed clinically by using clinical criteria (e.g., Amsel's diagnostic criteria) or microscopically by determining the Nugent score from vaginal Gram staining. While not bound by any theory, a relapse (or recurrence) of BV in patients after antibiotic treatment may be caused by persistent residual infection due to the infectious bacteria's resistance to the antibiotic used. Thus, in one preferred embodiment of the present invention, the patient suffers from bacterial vaginosis in which the infectious bacteria are resistant to antibiotic treatment. Thus, in one preferred aspect of the present invention, the Gardnerella-specific pharmaceutical composition described herein is for use in the treatment of bacterial vaginosis, which is administered to a patient who has previously failed to treat with antibiotics and suffers from bacterial vaginosis in which the infectious bacteria are resistant to antibiotic treatment. The infectious bacteria of BV treated herein, as defined below herein, are more preferably highly resistant to antibiotic treatment. In one preferred embodiment of the therapeutic use of the present invention, the patient being treated is infected with BV, and the infectious bacteria of BV are resistant, preferably highly resistant, to treatment with metronidazole, tinidazole, secnidazole, clindamycin, or any combination thereof. In one more preferred embodiment of the therapeutic use of the present invention, the patient being treated is infected with BV, and the infectious bacteria of BV are resistant, preferably highly resistant, to treatment with metronidazole and / or clindamycin. In an even more preferred embodiment of the therapeutic use of the present invention, the patient being treated is infected with BV, and the infectious bacteria of BV are resistant, preferably highly resistant, to treatment with metronidazole.

[0115] In relation to the present invention, “resistance” of a bacterial strain (preferably a Gardnerella strain) to an antibiotic refers to the ability of a strain to resist the activity of an antibiotic to which it was previously susceptible, enabling it to survive antibiotic treatment. Antibiotic resistance can occur naturally (“endogenous resistance”) or be induced by the misuse of antibiotics in humans and animals (“acquired resistance”). Methods for determining the resistance or susceptibility of bacterial strains to antibiotics are known to those skilled in the art. As an example, EUCAST breakpoints (v11, 2021) for Gram-positive anaerobic bacteria can be used. An alternative definition is presented by Petrina et al. (2017, Anaerobe 47, 115-119), where a somewhat higher resistance breakpoint is used. This is because some topical formulations of nitroimidazole and clindamycin can establish concentrations in vaginal fluid in the mg / ml range, which is much higher than the concentrations achievable with orally delivered antibiotics. According to this alternative, in a more precise definition (also used in the attached examples), resistance (R) can be defined as an MIC value of 32 μg / ml or greater for metronidazole and an MIC value greater than 8 μg / ml for clindamycin, while susceptibility (S) can be defined as an MIC value of 8 μg / ml or less for metronidazole and an MIC value of 2 μg / ml or less for clindamycin. The terms "minimum inhibitory concentration" and "MIC" are used interchangeably herein and refer to the lowest concentration of a chemical substance, usually a drug, that prevents visible growth of bacteria. MIC can be defined as the lowest concentration of an antibiotic in which no growth was detected after 48 hours by OD measurement. Thus, in one preferred embodiment of the therapeutic use of the present invention, the patient being treated is infected with BV, in which the infectious bacteria are resistant to antibiotic treatment as defined by the EUCAST breakpoint. Other generally accepted resistance criteria may be used instead of and / or in addition to the EUCAST definition or the alternative definition described above (Petrina et al., 2017, Anaerobe 47, 115-119) to define the resistance of infectious bacteria to antibiotic treatment in the context of the present invention.

[0116] As is known to those skilled in the art, "resistance" and "susceptibility" can be defined in relation to 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 particular bacterium. Typically, MBC90 or MBC99.5, i.e., the antibiotic concentration that kills 90% or 99.5% of cells, respectively, within a specified time. MBC is, for example, 2.5 × 10⁻⁶ 7 The minimum concentration (MIC) can be defined as the minimum concentration required to completely eliminate a suspension of CFU / ml. While the MIC is the minimum concentration of antimicrobial agent needed to inhibit visible growth, the MBC is the minimum concentration of antimicrobial agent needed to kill all cells in a suspension down to a defined detection limit, resulting in the death of at least 90% (MBC90) or at least 99.5% (MBC99.5) of bacteria. The term "minimum biofilm elimination concentration" or "MBEC" refers to the minimum concentration of antimicrobial agent needed to reduce a bacterial population growing as a biofilm to below the detection limit.

[0117] While not bound by any theory, antibiotic resistance of infectious bacteria may be involved in (or partially or substantially cause) the relapse (or recurrence) of BV in patients with recurrent BV. Therefore, patients with bacterial vaginosis in which the infectious bacteria are resistant to antibiotic treatment are likely to fail antibiotic treatment. Thus, in one preferred embodiment of the present invention, patients who are candidates for treatment and have bacterial vaginosis in which the infectious bacteria are resistant to antibiotic treatment are patients who are likely to fail antibiotic treatment. As used herein, patients who are “likely to fail antibiotic treatment” refer to patients at high risk of failure to antibiotic treatment, i.e., patients who are very likely to relapse (e.g., within 12 months) if treated with antibiotics. Methods for determining whether a patient is likely to fail antibiotic treatment are known to those skilled in the art. For example, a clinical sample can be taken and the resistance of vaginal microbiota strains can be evaluated. Similarly, patients suffering from bacterial vaginosis in which the infectious bacteria are resistant to antibiotic treatment are likely to have already failed (i.e., previously) with antibiotic treatment (as defined above). Therefore, in a more preferred embodiment of the present invention, the patient to be treated, suffering from bacterial vaginosis in which the infectious bacteria are resistant to antibiotic treatment, is a patient who has previously failed with antibiotic treatment.

[0118] According to the above definition of resistance, "high resistance" (HR) can be defined as an MIC value of 256 μg / ml or higher for metronidazole and an MIC value greater than 64 μg / ml for clindamycin. That is, it is 8 times higher than the resistance breakpoint under the alternative definition, which is already higher than the standard resistance breakpoint under the EUCAST definition. Therefore, in one embodiment of the therapeutic use of the present invention, the patient being treated is infected with BV, and the infectious bacteria of BV are resistant, preferably highly resistant, to antibiotic treatment, preferably as defined by the EUCAST breakpoint. In one preferred embodiment of the therapeutic use of the present invention, the patient being treated is infected with BV, and the infectious bacteria of BV are resistant, preferably highly resistant, to treatment with metronidazole, tinidazole, secnidazole, clindamycin, or any combination thereof. In one more preferred embodiment of the therapeutic use of the present invention, the patient being treated is infected with BV, and the infectious bacteria of BV are resistant, preferably highly resistant, to treatment with metronidazole and / or clindamycin. In a more preferred embodiment of the therapeutic use of the present invention, the patient being treated is infected with BV, and the infectious bacteria of BV are resistant, preferably highly resistant, to treatment with metronidazole.

[0119] In a particular embodiment, the pharmaceutical composition according to the present invention is used to treat bacterial vaginosis in a patient who has previously failed to treat with antibiotics and / or the infectious bacteria of the bacterial vaginosis are resistant to antibiotic treatment.

[0120] As used herein, the terms “antibiotic treatment” and “antibiotic therapy” are interchangeable and preferably refer to antibiotic treatment recommended or approved for the treatment of BV. Antibiotics currently recommended or approved for the treatment of BV include nitroimidazoles (metronidazole, tinidazole, and secnidazole, but not limited to these) and clindamycin. Thus, in one embodiment, “antibiotic treatment” or “antibiotic therapy” is treatment with nitroimidazole and / or clindamycin. In a preferred embodiment, the antibiotic treatment described herein is treatment with metronidazole, tinidazole, secnidazole, clindamycin, or any combination thereof. In a more preferred embodiment, the antibiotic therapy described herein is treatment with metronidazole and / or clindamycin. In an even more preferred embodiment, the antibiotic therapy described herein is treatment with metronidazole.

[0121] As used herein, “treatment” and “to treat” generally mean obtaining a desired pharmacological and physiological effect. The effect may be prophylactic in relation to preventing or partially preventing a disease, its symptoms, or condition, and / or therapeutic in relation to the partial or complete cure of a disease, condition, symptoms, or adverse effects resulting from the disease. As used herein, the term “to treat” encompasses any treatment of bacterial vaginosis in mammals, particularly humans. (a) preventing bacterial vaginosis from developing in patients who are susceptible to bacterial vaginosis but have not yet been diagnosed with it; (b) inhibiting bacterial vaginosis, i.e., preventing its development, or alleviating a bacterial infection, i.e., causing a regression of the bacterial infection and / or its symptoms or condition, such as improvement or repair of damage. In particular, treatment of bacterial vaginosis includes, for example, preventing, suppressing or even eradicating the infection by killing infectious bacteria, and thus controlling, reducing or inhibiting bacterial growth, and reducing the number of viable bacterial cells. In this specification, the disease, i.e., BV, is preferably treated therapeutically in relation to partial or complete cure of the disease or symptoms.

[0122] It will be understood by those skilled in the art that the activators used in accordance with the present invention are generally administered in mixture with appropriate pharmaceutically acceptable excipients, diluents, or carriers selected in relation to the intended route of administration and standard pharmacopoeia (see, for example, Remington: The Science and Practice of Pharmacy, 19th edition, 1995, Ed. Alfonso Gennaro, Mack Publishing Company, Pennsylvania, USA). For example, the activator may be administered topically, i.e., topically into the vagina of female subjects, and / or in or on the glans penis, foreskin, or urethral opening of male subjects.

[0123] In this specification, the term “intra-glans or on the glans penis” also includes “intra-glans and on the glans penis.” Accordingly, the term “intra-glans, foreskin or urethral opening of a male subject” also includes “intra-glans and on the glans penis, on the foreskin and on the urethral opening of a male subject.” In another embodiment, the activator may be co-administered with a compound or composition that adjusts the pH of the vagina. In some embodiments, the compound or composition adjusts the pH of the vagina to pH 4.0 to 6.0, preferably pH 5.0.

[0124] The terms “patient” and “subject” are used interchangeably herein and refer to mammals. For example, mammals targeted by the present invention include humans, primates, and domestic animals (e.g., cattle, sheep, pigs, horses, laboratory rodents, etc.). The patient is preferably human. It is even more preferable that the patient is female (also referred to herein as “female subject”).

[0125] It will be understood that the pharmaceutical compositions for use described herein may be administered to a subject in combination with one or more additional therapeutic agents. For example, endolysin and the pharmaceutical compositions described herein may be administered to a subject in combination with the following: (a) Treatment with one or more conventional antibiotics: such antibiotics may include clindamycin, metronidazole, or any other suitable antibiotic known to those skilled in the art; (b) One or more additional endolysins, or nucleic acid molecules, vectors, host cells, or bacteriophages capable of expressing them; (c) Compounds or compositions for adjusting the pH of the vagina to preferably pH 4.0 to 6.0, more preferably about pH 5.0: Such pH-adjusting compounds may include phosphates, lactic acid (for example, a natural acidifying substance secreted by Lactobacillus to establish an acidic environment), or other organic acids, such as carboxysubstituted polymers; (d) Therapies to neutralize toxins released during bacterial lysis of G. vaginalis cells in the vagina: Appropriate neutralization therapies may include antibodies (see Babcock et al., 2006, Infect.Immun. 74:6339-6347) and toxin absorbers such as trevamer (Barker et al., 2006, Aliment.Pharmacol.Ther. 24:1525-1534); (e) Probiotics.

[0126] In one embodiment of the use of the present invention, a host cell or a pharmacological composition containing a host cell is used to deliver endolysin (preferably a host cell).

[0127] In certain embodiments, the present invention relates to the use of a pharmaceutical composition according to the present invention for the manufacture of a medicament for the treatment or prevention of a bacterial infection, preferably bacterial vaginosis.

[0128] In certain embodiments, the present invention relates to a method for treating or preventing a bacterial infection, preferably bacterial vaginosis, in a subject requiring treatment or prevention of a bacterial infection, preferably bacterial vaginosis, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to the present invention.

[0129] The present invention also provides a method for treating a bacterial infection, particularly bacterial vaginosis, as described herein, comprising administering to a patient a therapeutically effective amount of a pharmaceutical composition comprising a Gardnerella-specific polypeptide, preferably a Gardnerella-specific endolysin, and a polyacrylic acid polymer.

[0130] With respect to the therapeutic use of the present invention, the embodiments and definitions described herein above shall apply with appropriate modifications in the context of methods for treating BV.

[0131] One aspect of the present invention relates to a composition comprising a polyacrylic acid polymer for use in the treatment and / or prevention of bacterial infections, wherein the polyacrylic acid polymer reduces biofilms.

[0132] In another aspect, the present invention relates to the non-therapeutic use of polyacrylic acid polymers in reducing bacterial biofilms.

[0133] In other words, the inventors have surprisingly found that polyacrylic acid polymers can reduce Gardnerella biofilms even in the absence of Gardnerella-specific polypeptides; see Figures 2A and 2B. In contrast, polyacrylic acid polymers do not have killing activity against suspended Gardnerella; see Figure 5. Thus, it has been shown that polyacrylic acid polymers act directly and specifically against bacterial biofilms. Based on these findings, polyacrylic acid polymers can be used as components of pharmaceutical compositions for the treatment and / or prevention of bacterial infections, particularly when bacterial infections involve biofilm formation.

[0134] A polyacrylic acid polymer is defined as reducing or dissolving a biofilm if, upon contact with the biofilm, the number of viable bacteria (CFU / mL) in the biofilm decreases by at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 times.

[0135] To this end, a biofilm (e.g., of G. vaginalis ATCC 14018) can be formed on a pre-coated tissue culture plate in sBHIG (in g / L: Brain Heart Infusion: 37, Gelatin: 20, Yeast Extract: 5, Starch: 1, Glucose: 2.5; Water: up to 1000 mL) at pH 7 for 48 hours. The resulting biofilm can then be treated with PAA in sBHIG pH 4.5 for 24 hours. Alternatively, the biofilm can be formed in sNYC (in g / L: HEPES: 2.4, Proteospeptone: 15, Yeast Extract: 3.8, NaCl: 5, Glucose: 10; Water: up to 1000 mL) at pH 7 for 48 hours, and then treated with PAA in sNYC at pH 6 for 24 hours. For biofilm treatment with PAA, a 0.35% PAA (v / v) working stock can be prepared using a PAA master stock (27% (w / v)). After incubation, the biofilm can be washed and detached by vigorous pipetting. Serial dilutions can be performed in PBS (1×PBS-DPBS, calcium-free, magnesium-free, Thermo Scientific, catalog no. 14190169) and spotted onto chocolate agar plates (BD, catalog no. 254060) to count the number of viable cells (CFU / ml).

[0136] In certain embodiments, the bacterial biofilm is a mucosal biofilm. In certain embodiments, the (mucosal) biofilm is formed by bacteria of the genus Gardnerella.

[0137] In certain embodiments, the bacterial infection is bacterial vaginosis as defined herein above.

[0138] Preferably, the pharmaceutical composition further comprises a polypeptide having toxic activity against Gardnerella, as described herein. Specifically, polyacrylic acid acts synergistically with endolysin PM-477 against Gardnerella biofilms, as shown in Figure 2.

[0139] The polyacrylic acid polymer contained in the pharmaceutical composition according to the present invention may be a crosslinked polyacrylic acid polymer or a carbomer. The above definition of polyacrylic acid polymer herein applies with appropriate modifications.

[0140] As used herein, the term “approximately” refers to numerical values, including, for example, integers, fractions, and percentages, whether expressly indicated or not. Generally, the term “approximately” refers to a range of numerical values ​​that a person skilled in the art would consider equivalent to (for example, having the same function or result as) the listed values ​​(e.g., ±1 to 3% of the listed values). In some examples, the term “approximately” may include numerical values ​​that are rounded to significant figures.

[0141] The present invention will be further described by reference to the following non-limiting drawings and embodiments. [Brief explanation of the drawing]

[0142] [Figure 1A] In study A, the pharmacokinetic study of PM-477 against G. vaginalis Gv9 (ATCC 14018T) showed a strong time-dependent and dose-dependent lytic effect. Gardnerella cell suspensions were treated with recombinant PM-477 at 2.5 μg / ml, 10 μg / ml, and 40 μg / ml for 1 hour, 5 hours, and 24 hours. Colony-forming units (CFUs) per ml were measured on a logarithmic scale and compared with buffer-treated controls. In study B, suspensions of other vaginal BV pathogens (gray) and the probiotic Lactobacillus (white box) were treated with PM-477 at 10 μg / ml and 100 μg / ml for 5 hours. Colony-forming units (CFUs) per ml were measured on a logarithmic scale and compared with buffer-treated controls. LOD indicates the limit of detection. Statistical significance (one-way ANOVA, comparing each strain with the control and PM-477 treatment at 10 μg / mL and 10 μg / mL) is indicated as follows: ***: P<0.001, **: P<0.01, *: P<0.05. [Figure 1B]In study A, the pharmacokinetic study of PM-477 against G. vaginalis Gv9 (ATCC 14018T) showed a strong time-dependent and dose-dependent lytic effect. Gardnerella cell suspensions were treated with recombinant PM-477 at 2.5 μg / ml, 10 μg / ml, and 40 μg / ml for 1 hour, 5 hours, and 24 hours. Colony-forming units (CFUs) per ml were measured on a logarithmic scale and compared with buffer-treated controls. In study B, suspensions of other vaginal BV pathogens (gray) and the probiotic Lactobacillus (white box) were treated with PM-477 at 10 μg / ml and 100 μg / ml for 5 hours. Colony-forming units (CFUs) per ml were measured on a logarithmic scale and compared with buffer-treated controls. LOD indicates the limit of detection. Statistical significance (one-way ANOVA, comparing each strain with the control and PM-477 treatment at 10 μg / mL and 10 μg / mL) is indicated as follows: ***: P<0.001, **: P<0.01, *: P<0.05.

[0143] [Figure 2] In A, Gardnerella biofilms (using the reference strain G. vaginalis ATCC 14018) were treated with 8 μg / ml PM-477 (i.e., below the concentration typically used to eradicate Gardnerella biofilms), 0.35% (w / v) PAA, or combinations thereof, in sBHIG medium at pH 4.5 for 24 hours. In B, Gardnerella biofilms (G. vaginalis ATCC 14018) were treated with 32 μg / ml H2B10B11, 0.25% (w / v) PAA, or combinations thereof, in sNYC medium at pH 6 for 24 hours.

[0144] Cells were detached by pipetting, and the remaining viable cells (CFU / ml) were measured using quantitative plating. The bactericidal effect was measured in log CFU / ml compared to the buffer-treated control. 10 The decrease is visualized. The gray column shows the theoretical additive effect of the mean logarithmic decrease of PM-477 / H2B10B11 and PAA. The figure shows the mean values ​​with standard deviations.

[0145] [Figure 3A] The lytic activity of two PM-477 formulations against six BV-related pathogens and one commensal Lactobacillus species. Strain susceptibility was tested by treating fresh liquid culture suspensions of the strains for 5 hours with either an EL-free control, PM-477 formulated with PAA at pH 4.5 (Panel A), or PM-477 formulated with PAA at pH 5.0 (Panel B). The viability after treatment was determined by quantitative spotting (Panels A and B), and the decrease in viability for both formulations (Panel C) was calculated by subtracting the log10 CFU / mL in the replicated formulation from the median of the respective uncorrected control. The figure shows the triplicate, range, and median values. LOD: Limit of Detection. Points below the LOD indicate that the limit of detection was reached. [Figure 3B] The lytic activity of two PM-477 formulations against six BV-related pathogens and one commensal Lactobacillus species. Strain susceptibility was tested by treating fresh liquid culture suspensions of the strains for 5 hours with either an EL-free control, PM-477 formulated with PAA at pH 4.5 (Panel A), or PM-477 formulated with PAA at pH 5.0 (Panel B). The viability after treatment was determined by quantitative spotting (Panels A and B), and the decrease in viability for both formulations (Panel C) was calculated by subtracting the log10 CFU / mL in the replicated formulation from the median of the respective uncorrected control. The figure shows the triplicate, range, and median values. LOD: Limit of Detection. Points below the LOD indicate that the limit of detection was reached. [Figure 3C]The lytic activity of two PM-477 formulations against six BV-related pathogens and one commensal Lactobacillus species. Strain susceptibility was tested by treating fresh liquid culture suspensions of the strains for 5 hours with either an EL-free control, PM-477 formulated with PAA at pH 4.5 (Panel A), or PM-477 formulated with PAA at pH 5.0 (Panel B). The viability after treatment was determined by quantitative spotting (Panels A and B), and the decrease in viability for both formulations (Panel C) was calculated by subtracting the log10 CFU / mL in the replicated formulation from the median of the respective uncorrected control. The figure shows the triplicate, range, and median values. LOD: Limit of Detection. Points below the LOD indicate that the limit of detection was reached.

[0146] [Figure 4] Effect of PAA concentration on the viability of suspension Gv9 cells. Susceptibility to PAA was tested by treating fresh liquid culture suspensions of Gv9 cells with sNYCB at pH 5 containing various concentrations of PAA at 37°C for 90 minutes. Post-treatment viability was determined by quantitative spotting. The figure shows triple values ​​and ranges, with a horizontal line at the median. PAA: poly(acrylic acid). LOD: Limit of Detection.

[0147] [Figure 5] The effect of PAA on the viability of five BV-related pathogens and one commensal bacterium. The susceptibility of bacterial strains to PAA was tested by treating fresh liquid culture suspensions with NYCB medium at pH 5 with PAA concentrations ranging from 0.01% to 1% for 90 minutes, and comparing this to an untreated control. Post-treatment viability was determined by quantitative spotting. Data are shown in triplicate, with a line drawn at the median. PAA: polyacrylic acid. LOD: Limit of Detection. Points below the LOD indicate that the limit of detection was reached.

[0148] [Figure 6]The lytic activity of H2B10B11 formulated with Carbopol 974P against BV-related pathogens and one commensal bacterium. Strain susceptibility was tested by treating fresh liquid culture suspensions of the strains in pH 5 medium with either an EL-free control or a low concentration of H2B10B11 (2 μg / mL) formulated with 0.25% (w / v) CP 974P for 20.5 hours. Post-treatment viability was determined by quantitative spotting. The figure shows triple values ​​and median values. LOD: Limit of Detection. Points below the LOD indicate that the detection limit was reached.

[0149] [Figure 7] The activity of H2B10B11 formulated with Carbopol can be adjusted by controlling the salt content. A 90-minute solubility assay against Gardnerella vaginalis was performed using 10 μg / mL of H2B10B11 and / or 0.1% (w / v) Carbopol in pH 5.5 culture media with normal salt concentration (86 mM, round) and high salt concentration (345 mM, diamond). The potency of H2B10B11 was evaluated by quantitative spotting. The figure shows triple measurements along with the median and range.

[0150] [Figure 8] Decrease in L. inels in swab samples from BV-positive women after 19 hours of treatment. Thirty vaginal swab samples were treated overnight with 50 μg / mL H2B10B11 (black) or 50 μg / mL H2B10B11 (gray) formulated with 0.1% (w / v) CP 974P and 1% NaCl. Viable L. inels loading was measured by viability qPCR using L. inels-specific primer pairs. The DNA copy number / μL of the treated samples, minus the loading of the corresponding untreated baseline samples, was plotted against the loading of the baseline samples. Linear regression was performed across samples, and the slope was plotted on the graph. [Examples]

[0151] Example 1 - PM-477 endolysin is a highly active antimicrobial enzyme that targets Gardnerella species and also targets Lactobacillus inersu to a limited extent. Bacterial vaginitis (BV) is characterized by an imbalance in the vaginal microbiome and the formation of a distinctive biofilm on the vaginal epithelium, which is initiated and dominated by Gardnerella bacteria. We previously disclosed synthetic endolysins derived from the 1,4-β-N-acetylmuramidase type encoded by Gardnerella prophage (International Publication No. 2020 / 225335). These endolysins effectively kill Gardnerella bacteria and also slightly kill L. iners, another opportunistic pathogen in BV. Importantly, probiotic Lactobacillus such as L. crispatus and L. gasseri are not impaired by treatment with PM-477. We tested the killing activity of a representative endolysin (PM-477(H2B10)) against Gardnerella vaginalis.

[0152] Gardnerella bacteria (OD=0.1) in suspension were treated with endolysin PM-477 (700 μg / ml stock solution in 50 mM MES pH 5.5, 200 mM NaCl, 8 mM MgSO4) or a buffer control, and incubated anaerobically for 1 hour, 5 hours, and 24 hours. After incubation, the suspension was serially diluted, spotted onto chocolate agar plates, and the CFU / ml was counted. The efficacy was clearly time-dependent and dose-dependent, with cell counts already reduced to the limit of detection (LOD) after 5 hours of incubation even at low concentrations of 10 μg / ml and 40 μg / ml. No regrowth was observed after 24 hours. Interestingly, when treated with a very low concentration of 2.5 μg / ml, no further death was recorded between 5 and 24 hours, suggesting that equilibrium may have been established. The inventors also tested the activity of PM-477 against other vaginal opportunistic pathogens and probiotics against vaginal Lactobacillus. PM-477 proved to be specific to Gardnerella bacteria without affecting beneficial Lactobacillus or other opportunistic BV pathogens. The only exception was L. inersus, which showed a 2.5 log10 unit reduction in bacterial count after 5 hours at a high concentration of 100 μg / ml. L. inersus is distinctly different from other Lactobacillus in terms of cell wall composition and metabolism and is considered a pathogen in BV.

[0153] In summary, recombinant endolysin PM-477(H2B10) demonstrated a potent bactericidal effect against Gardnerella without harming L. crispatus, L. gasseri, and L. gensenii, the most dominant species in a healthy vaginal microbiome (see Figure 1).

[0154] Example 2 - PAA as an excipient enhances the endolysin-killing effect against Gardnerella bacteria growing as biofilms. The inventors tested PAA (Sigma Aldrich, catalog no. 323667-100G) as a medium for Gardnerella-specific endolysin for vaginal application. The viscosity and mucosal adhesion properties of PAA ensure the sustained release of the active pharmaceutical ingredient (endolysin) in the vaginal mucus layer, and its acidifying effect helps restore a healthy vaginal microbiome. Unexpectedly, the presence of PAA also enhanced the potency of endolysin, killing Gardnerella bacteria more effectively. The inventors tested Gardnerella-specific endolysins PM-477 and H2B10B11 formulated with PAA on a single Gardnerella species biofilm in vitro. Biofilms (G. vaginalis ATCC 14018) were generated in sBHIG (g / L: Brain Heart Infusion: 37, Gelatin: 20, Yeast Extract: 5, Starch: 1, Glucose: 2.5; Water: up to 1000 mL) at pH 7 for 48 hours on pre-coated tissue culture plates, and then treated with a low concentration of endolysin PM-477 and PAA in combination, as well as PM-477 alone and PAA alone, in sBHIG pH 4.5 for 24 hours. Alternatively, biofilms were generated in sNYC (g / L: HEPES: 2.4, Proteospeptone: 15, Yeast Extract: 3.8, NaCl: 5, Glucose: 10; Water: up to 1000 mL) at pH 7 for 48 hours, and then treated with H2B10B11 or PAA, or a combination of both, in sNYC pH 6 for 24 hours.

[0155] For biofilm treatment with PAA and PM-477, a working stock of 0.35% PAA (v / v) containing 8 μg / ml PM-477 was prepared by diluting a PAA master stock (27% (w / v)) with sBHIG pH 5 (700 μg / ml PM-477 master stock was used for dilution). Furthermore, the final mixture of PM-477 and the excipient PAA used for biofilm treatment had a pH of 4.5 due to the buffering effect of 0.35% PAA. For biofilm treatment with PAA and H2B10B11, a treatment solution containing 0.25% PAA and 32 μg / mL H2B10B11 in sNYC pH 6 was prepared using a PAA master stock (14.5% (w / v), adjusted to pH 6). After incubation, the biofilm was washed and removed by vigorous pipetting. Serial dilutions were performed in PBS (1×PBS-DPBS, calcium-free, magnesium-free, Thermo Scientific, catalog number 14190169), spotted onto chocolate agar plates (BD, catalog number 254060), and the number of viable cells (CFU / ml) was counted. For the strains tested, the Gardnerella biofilm eradication concentrations of PM-477 ranged from 2 to 32 μg / ml. The concentration selected here was 8 μg / ml, which is the lower limit of the minimum biofilm eradication concentration (MBEC) range for Gardnerella strains, allowing observation of the potential synergistic effect of the excipient PAA and PM-477. Note that MBEC is affected by the culture medium and pH used in the experiment. Since the biofilm experiment using H2B10B11 was performed at pH 6, 32 μg / mL of H2B10B11 was not sufficient to eradicate the biofilm in the absence of PAA.

[0156] PM-477 (8 μg / ml) alone produced a CFU / ml of 1.6 log 10 When the unit is reduced, PAA (0.35% w / v) alone is 0.6 log 10 Although the unit was reduced, the combination of PM-477 and PAA is 3log 10 This resulted in a clear synergistic effect in the unit mortality rate. Potential additive effect (0.6 log10 units of PAA and 1.6 log 10 units of PM-477 = 2.2 log 10 The difference from the unit decrease) is 0.8 log units (3 log units in total) 10 units), which corresponds to a 6-fold increase in activity on a linear scale (Figure 2A).

[0157] No relevant effect was observed with H2B10B11 (32 μg / mL) alone in the medium at pH 6. Alone with PAA (0.25% w / v), the CFU / mL decreased by 0.9 log 10 units, but the combination of H2B10B11 and PAA brought about a clear synergistic effect with a 3.1 log 10 decrease (Figure 2B).

[0158] Example 3 - PAA as an excipient enhances the killing effect of endolysin on Gardnerella bacteria and other BV pathogens growing in a floating form Next, the potency of two different formulations of PM-477 containing 0.25% PAA (w / v; Sigma Aldrich, catalog no. 323667-100G) as an excipient was tested against six BV-related pathogens and one commensal Lactobacillus species using a time-kill assay (Figure 3). Both formulations contained 2 μg / mL of PM-477 and 0.25% PAA, and were adjusted to either pH 4.5 (Figure 3A) or pH 5 (Figure 3B) to simulate different PAA types and compositions. Briefly, 4 g of PAA was added to 10 mL of dH2O and heated to 50°C while mixing on a heated stirring plate. The pH of the PAA solution was adjusted to either 4.5 or pH 5 using 5 M NaOH. The volume was then adjusted to 27.5 mL (equivalent to 14.5% PAA) and sterile filtered using a 0.22 μm syringe filter. These stocks were diluted to a final concentration of 0.25% PAA in glucose-supplemented New York City Broth (sNYCB) adjusted to their respective pH levels. Supplemented NYCB consisted of 10 mM HEPES (Sigma Aldrich), 15 g / L proteose peptone (Sigma Aldrich), 3.8 g / L yeast extract (Thermo Fisher Scientific), 86 mM sodium chloride (Carl Roth), and 10 g / L α-D-glucose (Sigma Aldrich).

[0159] The bacteria were grown anaerobically on a chocolate agar plate at 37°C for 48 hours, then scraped off the plate and prepared for the experiment. 8 CFU / ml suspensions were prepared. For the time-kill experiment, the bacterial suspensions were mixed with their respective treatment stock solutions and incubated anaerobically at 37°C for 5 hours. Subsequently, after 48 hours of anaerobic incubation at 37°C, 2 μL of a 10-fold dilution series (minimum dilution 10) was placed on a chocolate agar plate. -5 The number of viable bacteria was quantified by spotting the bacteria.

[0160] Both formulations were highly effective in reducing the bacterial load of all six pathogens, but did not adversely affect commensal L. crispatus (Figure 3). The formulation set to a slightly acidic pH (4.5) reduced the survival rate of Gv9 (G. vaginalis ATCC 14018), L. inels, and M. murielis by 3 log compared to the control. 10 Ultra, A. baginae and P. vivia 2log 10 The levels were greatly reduced (Figure 3C). When formulated at a lower acidic pH (5.0), 2 μg / mL PM-477 had no adverse effect on commensal L. crispatus, and compared to the control, reduced Gv24 (G. swidsinskii GS 9838-1), L. iners, and P. vivia by 3 log. 10 Super, Gv9, M. murielis, and A. baginae in 2 logs 10 It was drastically reduced (Figure 3C).

[0161] In summary, formulations containing PAA surprisingly extend the activity spectrum of PM-477 to multiple BV-related pathogens other than Gardnerella.

[0162] Example 4 - PAA alone has no lethal effect against Gardnerella, and only at high concentrations does it have a weak lethal effect against some other BV pathogens. Next, the effect of PAA (Sigma Aldrich, catalog number 323667-100G) alone against Gardnerella and other BV-related pathogens was evaluated. As described in the previous example, the bacterial suspension was 10 8 The solution was adjusted to CFU / ml, mixed with the treated stock solution, and incubated anaerobically at 37°C for 90 minutes. Viable bacteria were then quantified by spotting a 10-fold dilution series of the reaction mixture. After incubating the spotting plates for 2-3 days, the CFU / mL was measured. As shown in Figure 4, PAA concentrations up to 1% did not reduce the viability of Gardnerella after 90 minutes of incubation.

[0163] The susceptibility of five other BV-related pathogens and one commensal Lactobacillus species to PAA was also tested using the same assay. The four BV-related pathogens tested were L. inersu, P. vivia, A. baginae, and M. murieris, with L. crispatus used as a commensal bacterium (Figure 5). The inventors tested PAA concentrations ranging from 0.01% to 1% at pH 5 for all five organisms using a 90-minute potency assay. PAA concentrations up to 1% had minimal effect on the viability of M. murieris and L. crispatus (<0.6 log). 10 (Decrease). The viability of P. vivia and A. vaginae responded only to high concentrations of PAA (>0.5%) to 1.8 and 1.7 log, respectively. 10 The effect decreased, and was negligible at lower concentrations (Figure 5). The viability of L. inellus decreased by approximately 1.1 log10 in response to the lowest test concentration of PAA (0.01%), and did not decrease further at higher PAA concentrations (Figure 5).

[0164] Example 5 - Carbopol 974P as an excipient enhances the killing effect of endolysin against airborne BV-related pathogens, but does not enhance the killing effect against commensal Lactobacillus species. The efficacy of H2B10B11 formulated with 0.25% (w / v) Carbopol 974P NF polymer (CP 974; The Lubrizol Corporation) was tested against five BV-related pathogens and one commensal Lactobacillus species in a suspension environment for 5 hours at pH 5 in sNYC medium (Figure 6). The formulation contained 2 μg / mL of H2B10B11 and 0.25% CP 974P, with a final pH of 5. To obtain the formulated treatment solution, 50 mg of CP 974P was added to 15 mL of sNYC (pH 5). After mixing, the pH was readjusted to pH 5 using 5 M NaOH, and the volume was adjusted to 20 mL with sNYC pH 5. H2B10B11 was added from a stock of 1.343 mg / mL in MES pH 5.5 buffer to a final concentration of 2 μg / mL.

[0165] Gardnerella was grown anaerobically for 48 hours at 37°C on chocolate agar plates, and the other bacterial strains were grown anaerobically on Sheldler agar plates containing vitamin K1 and 5% sheep blood (BD, catalog number 254042), then scraped off the plates and 10 8 CFU / ml suspensions were prepared for the experiment. The bacterial suspensions were mixed with either sNYC (pH 5) or sNYC (pH 5) containing formulated H2B10B11, and incubated anaerobically at 37°C for 20.5 hours. The viable bacteria were then subjected to a 10-fold dilution series of 2 μL (minimum dilution 10 -5 The substance was quantified by spotting it onto a chocolate / Schödler agar plate and incubating it anaerobically at 37°C for 48 hours.

[0166] The formulated H2B10B11 eradicated BV-related pathogens G. vaginalis, L. iners, M. murielis, A. vaginae, and P. vivia (Figure 6). This was an unexpected observation, because endolysin alone was only slightly effective against L. iners and ineffective against non-Gardnerella strains (see Figure 1B). The results obtained here using Carbopol 974P are comparable to those shown in Example 3 (Figure 3) where the formulation contained PAA, so we can conclude that poly(acrylic acid) and its cross-linked pharmaceutical-grade form (carbomer) result in a synergistic enhancement of endolysin's solubility. Furthermore, H2B10B11 formulated with CP 974P, like PM-477 formulated with PAA, does not harm beneficial L. crispatus (Figure 6).

[0167] Example 6 - The activity of H2B10B11 formulated with Carbopol can be adjusted by controlling the salt content. The efficacy of H2B10B11 alone, or formulated with Carbopol 974P NF polymer or Carbopol 971P NF polymer (The Lubrizol Corporation), was tested against Gardnerella vaginalis in a suspension environment for 90 minutes in NYC medium at pH 5.5 with normal NaCl concentration (86 mM) or high NaCl concentration (345 mM) (Figure 7). The formulations contained 10 μg / mL of H2B10B11 and 0.1% Carbopol, with a final pH of 5.5. To obtain the formulated treatment solution, 40 mg of CP 974P or CP 971P was added to 40 mL of NYC with normal or high NaCl concentration. After mixing, the pH was adjusted to pH 5.5 using 5 M NaOH. H2B10B11 was added from a stock concentration of 1.345 mg / mL in MES pH5.5 buffer to a final concentration of 10 μg / mL.

[0168] Gardnerella was grown anaerobically on chocolate agar plates at 37°C for 48 hours, then scraped off the plates using PBS. The bacterial suspension was mixed with respective media containing H2B10B11, Carbopol, or a combination of both, to achieve a final bacterial OD of 0.1. The reaction plates were incubated anaerobically at 37°C for 90 minutes. The viable bacteria were then refractory to 2 μL of a 10-fold dilution series (minimum dilution 10). -5 The substance was quantified by spotting it onto a chocolate agar plate and incubating it anaerobically at 37°C for 48 hours.

[0169] Example 7 - Reduction in L. inellus in vaginal swab samples from BV-positive women after 19 hours of treatment. Thirty vaginal swab samples from BV-positive women were treated with 50 μg / mL H2B10B11, or with 50 μg / mL H2B10B11 formulated with 0.1% (w / v) CP 974P and 1% NaCl, for 19 hours. For this purpose, two vaginal swabs were pooled in Amies medium (Copan 480CE), 400 μL of NYC pH 5.5 + 10% horse serum was added, mixed well, and frozen. The treatment reaction was carried out in a 96-well plate using 114 μL of sample and treatment solution in MES buffer pH 5.5. The reaction plate was statically incubated at 37°C for 19 hours under anaerobic conditions. Baseline (untreated) and treated samples were treated with PMAxx (Biotium). PMAxx intercalates into the DNA of dead cells, thereby minimizing the qPCR signal from dead cells. In short, 45 μL of 50 μM PMAxx premix I was added to the cell pellet, incubated on ice for 15 minutes, and exposed to light in a PMA-Lite LED photodegradation device for 15 minutes. The PMAxx treatment was repeated twice with 2.25 μL of 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 saline and centrifuged at 8000 rpm for 10 minutes. Genomic DNA was extracted using the DNeasy 96 Blood & Tissue Kit.

[0170] The viability of L. iners was measured by viability qPCR using L. iners-specific primer pairs. qPCR was performed on a LightCycler 480 (Roche) using EvaGreen® qPCR Supermix (Solis Biodyne) with 2 μK of extracted DNA per reaction in a total volume of 10 μL. The PCR conditions were as follows: initial denaturation at 95°C for 12 minutes, followed by 40 cycles of 15 seconds at 95°C, 40 seconds at 57°C, and 30 seconds at 72°C, followed by thawing analysis at a ramp rate of 2.5°C / second between 55 and 95°C.

[0171] The DNA copy number / μL of the treated sample, minus the loading of the corresponding untreated baseline sample, is plotted against the loading of the baseline sample. Linear regression is performed across the samples, and the slope is plotted on the graph. This slope reflects the overall decrease in L. inellus across patient samples, specifically a 99% decrease in formulated H2B10B11.

Claims

1. A pharmaceutical composition comprising an effective amount of an activator and a pharmaceutically acceptable excipient, wherein the activator is a polypeptide having toxic activity against Gardnerella, and the excipient is a polyacrylic acid polymer.

2. The pharmaceutical composition according to claim 1, wherein the polypeptide is endolysin having killing activity against Gardnerella.

3. The endolysin (i) 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 includes 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, The pharmaceutical composition according to claim 2, comprising or consisting of the following.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the polypeptide has at least 80% sequence identity with the amino acid sequence provided in SEQ ID NO: 1 or SEQ ID NO: 37 and is a polypeptide having toxic activity against Gardnerella.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the polypeptide is a recombinant endolysin comprising the amino acid sequence provided by SEQ ID NO: 1 or SEQ ID NO:

37.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the polypeptide has toxic activity against Gardnerella vaginalis sensu stricto, Gardnerella leopoldii, Gardnerella piotii, and / or Gardnerella swidsinskii, or any other species of the genus Gardnerella.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the polypeptide has toxic activity against Lactobacillus iners.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the polypeptide does not have toxic activity against Lactobacillus crispatus, Lactobacillus gasseri, and / or Lactobacillus gensenii.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the excipient is present in a concentration of about 0.01% to 10%, preferably about 0.1% to 1%, more preferably about 0.2% to 0.5%, and most preferably about 0.25% or about 0.35%.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the polyacrylic acid polymer is a crosslinked polyacrylic acid polymer or a carbomer.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the combination of the activator and the excipient increases the pharmacokinetic activity of the activator.

12. The pharmaceutical composition according to any one of claims 1 to 11, further comprising a salt.

13. The pharmaceutical composition according to claim 12, wherein the salt is sodium chloride (NaCl).

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the composition is suitable for intravaginal delivery of the activator.

15. A pharmaceutical composition according to any one of claims 1 to 14, for use in treating a bacterial infection in a subject, preferably the bacterial infection being bacterial vaginosis.

16. The pharmaceutical composition for use according to claim 15, wherein the subject has previously failed to treat with antibiotics and / or the infectious bacteria are resistant to antibiotic treatment.

17. The pharmaceutical composition for use according to claim 16, wherein the antibiotic is 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 suffers from recurrent bacterial vaginosis, and preferably the subject has had two or more episodes of BV within the last six months, or three or more episodes of BV within the last twelve months.

20. A pharmaceutical composition for use according to any one of claims 15 to 19, to be administered topically into the vagina of a woman and / or into or on the glans penis, foreskin, or urethral opening of a man.

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

22. A method for treating or preventing a bacterial infection, preferably bacterial vaginosis, in a subject requiring treatment or prevention of a bacterial infection, preferably bacterial vaginosis, 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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