Compositions comprising an endolysin and uses thereof

Abstract

The present invention relates to new pharmaceutical compositions comprising as active ingredient an effective amount of (a) an endolysin comprising the amino acid sequence provided in SEQ ID NO: 1; or (b) an endolysin comprising an amino acid sequence having 80% sequence identity to SEQ ID NO:1, wherein the endolysin has a killing activity against Gardnerella; and at least one pharmaceutically acceptable excipient. Furthermore, the present invention relates to therapeutic uses of said pharmaceutical composition.

Description

[0001] COMPOSITIONS COMPRISING AN ENDOLYSIN AND USES THEREOF

[0002] The present invention relates to new pharmaceutical compositions comprising as active ingredient an effective amount of (a) an endolysin comprising the amino acid sequence provided in SEQ ID NO: 1; or (b) an endolysin comprising an amino acid sequence having 80% sequence identity to SEQ ID NO: 1, wherein the endolysin has a killing activity against Gardnensl / a; and at least one pharmaceutically acceptable excipient. Furthermore, the present invention relates to therapeutic uses of said pharmaceutical composition.

[0003] BACKGROUND OF THE INVENTION

[0004] Bacterial vaginosis (BV), also been referred to in the literature as bacterial vaginitis, non-specific vaginosis and nonspecific vaginitis, is the most common vaginal infection worldwide and is associated with significant adverse consequences including preterm labor and delivery, post-partum endometritis and an increased risk of HIV acquisition. It is a dysbiosis of the vagina where the commensal Lactobacilli are displaced by a polymicrobial biofilm, the pH increases from the natural 3.5-4.5 up to 5.5, and a malodorous fluid forms. It is most commonly defined as a pathological state characterized by the loss of normal vagina flora, particularly of H2O2-producing species of Lactobacillus, and the simultaneous overgrowth of anaerobic bacteria including Gardnerella vaginalis G. vaginalis). This organism, first called Haemophilus vaginalis and repeatedly renamed as more information about its characteristics became available, is now classified as G. vaginalis which, until 2018, was considered to be the sole member of the genus Gardnerella. However, in early 2019 it was shown that the genus Gardnerella actually contains at least 13 species, and the most frequent ones were renamed G. vaginalis sensu stricto, G. leopoldii, G. piotii, and G. swidsinskii (Vaneechoutte et al., 2019 Int. J. Syst. Evol. Biol. 898661). Bacteria of the genus Gardnerella are special in that they are Gram-variable, i.e. they do not form the outer membrane defining the Gram-negative species. The cell wall is generally very thin and has only 10% or less content of peptidoglycan, which is why the crystal violet dye used for Gram staining does not always yield the deep purple color typical for Gram-positive species. Rather, Gardnerella cells can appear both Gram positive and negative in a Gram staining. Phylogenetic analysis based on 16S rRNA places Gardnerella in the gram-positive family Bifidobacteriales.

[0005] During BV, the epithelial surface is covered with a dense collection of Gardnerella bacteria forming an adherent biofilm on the vaginal epithelium, in which other species can proliferate, resulting in a polymicrobial biofilm that is frequently recalcitrant to treatment. Biofilms are adherent communities of microorganisms held together by a polymeric matrix composed of polysaccharides, proteins and / or nucleic acids. The distinct gene expression pattern, as well as the physical structure of biofilms increases bacterial resistance to many negative stimuli including chemical disinfectants, pH extremes, host immune defenses and antibiotics.

[0006] The recommended first-line therapy for BV is antibiotic treatment, predominately with nitroimidazole antibiotics such as metronidazole (MDZ), Tinidazole (TDZ) and secnidazole. and / or with clindamycin (CU). Metronidazole (MDZ) belongs to the group of nitroimidazoles, and only gains its full activity when it is metabolized into its hydroxy metabolite (MDZ-OH). Antibiotics are effective in quickly reducing BV symptoms, but are associated with a high recurrence rate of up to 60 % within six months of treatment. In a clinical trial where patients with recurrent BV were treated with 0.75 % MDZ vaginal gel over 16 weeks, the probability of lasting cure was 70 % after 16 weeks (i.e. 30% of patients had symptoms at the end of the 16 week treatment period) and declined to 34 % 12 weeks after the end of therapy, i.e. at week 28 (Sobel et al., 2006, Am. J. Obstet. Gynecol. 194, 1283-1289). Besides a possible re infection from sexual partners, the persistence of a residual infection has been postulated as a reason for recurrence, potentially due to the formation of a biofilm that protects BV-causing bacteria from antimicrobial therapy. Another reason may be antibiotic resistance of BV pathogens. Antibiotic resistance occurs naturally, but misuse of antibiotics in humans and animals is accelerating the process. Existing treatments thus fail to effectively penetrate biofilms. Accordingly, upon cessation of antibiotics treatment, biofilms re-grow, resulting in recurrent symptomatic presentations. Furthermore, treatment with antibiotics wipes the vaginal microbiome, despite leaving some rests of viable biofilm, which opens this ecological niche for other pathogens, e.g. fungi. A frequent effect of BV treatments is therefore candidiasis. Therefore, treatment failure and recurrent disease are common problems with antibiotic treatment.

[0007] More recently, endolysins that selectively act against Gardnerella have been described in the art. Endolysins are promising alternatives to the current antibiotics, due to their ability to eradicate biofilms, their low propensity to the development of resistance, and their specificity to individual genera or species of bacteria. Natural and genetically engineered Gardnerella-spedfic endolysins have been described (WO 2020 / 225335 Al, Landlinger et al. (2021, Pathogens 10, 1-19), or WO 2020 / 229802 Al). WO 2020 / 225335 describes specific recombinant Garr / nere / Za-specific endolysins (e.g., H2B10) for use in a method of treating a Gardnerella infection such as BV, wherein said bacterial vaginosis is caused by Gardnerella vaginalis sensu stricto, Gardnerella leopoldli, Gardnerella piotii and / or Gardnerella swidsinskii. Examples 6 and 7 of WO 2020 / 225335 demonstrate that the endolysin H2B10 (as a representative of recombinant Gardnerella-specific endolysins) is superior to the antibiotics Metronidazole and Clindamycin, particularly in terms of minimal inhibitory concentration (MIC) on the growth in suspension of the Gardnerella strains. The results of WO 2020 / 225335 therefore document that (recombinant) Gardnerella-spec fic endolysins are superior to antibiotics in the treatment of BV in a general manner. In other words, (recombinant) Gardnerella-spedfic endolysins have been shown to be generally more effective on the growth in suspension of the Gardnerella strains as can be derived from the generally lower MIC values of the endolysins across the different Gardnerella species in comparison to those of the antibiotics.

[0008] Despite these recent advances, there is still a need in the art for compositions and / or formulations that allow an even more effective treatment of infections caused by Gardnerella species.

[0009] Thus, the technical problem underlying the present invention is the provision of improved compositions comprising a Gardnerella-spedfic endolysin.

[0010] The technical problem is solved by the herein provided embodiments and claims.

[0011] SUMMARY OF THE INVENTION

[0012] That is, the present invention is characterized in the herein provided embodiments and claims. In particular, the present invention relates, inter alia, to the following embodiments:

[0013] 1. A pharmaceutical composition comprising as active ingredient an effective amount of a) an endolysin comprising the amino acid sequence provided in SEQ ID NO: 1; or b) an endolysin comprising an amino acid sequence having 80% sequence identity to SEQ ID NO: 1, wherein the endolysin has a killing activity against Gardnerella; and at least one pharmaceutically acceptable excipient.

[0014] 2. The pharmaceutical composition according to embodiment 1, wherein the composition is formulated as vaginal insert, preferably a vaginal tablet.

[0015] 3. The pharmaceutical composition according to embodiment 1 or 2, wherein the pharmaceutically acceptable excipient is or comprises an osmotic active agent.

[0016] 4. The pharmaceutical composition according to embodiment 3, wherein the osmotic active agent is a sugar alcohol, in particular wherein the sugar alcohol is sorbitol, mannitol or a mixture thereof.

[0017] 5. The pharmaceutical composition according to embodiment 4, wherein the sugar alcohol is present in about 10% to about 70% in weight based on the total weight of the composition.

[0018] 6. The pharmaceutical composition according to any one of embodiments 1 to 5, wherein the pharmaceutical composition has an osmolality of about 400 to 700 mOsmol / kg in simulated vaginal fluid (pH 6.0), preferably of about 590 mOsmol / kg.

[0019] 7. The pharmaceutical composition according to any one of embodiments 1 to 6, wherein the composition further comprises a gelling agent.

[0020] 8. The pharmaceutical composition according to embodiment 7, wherein the gelling agent is hydroxyethyl cellulose (HEC), in particular wherein HEC is present between 0 % and 5 % in weight based on the total weight of the composition, in particular wherein HEC is present in about 0.5 % in weight based on the total weight of the composition.

[0021] 9. The pharmaceutical composition according to embodiment 7, wherein the gelling agent is hydroxypropyl methyl cellulose (HPMC), in particular wherein HPMC is present between 0 % and 10 % in weight based on the total weight of the composition, in particular wherein HPMC is present in about 5 % in weight based on the total weight of the composition.

[0022] 10. The pharmaceutical composition according to any one of embodiments 1 to 9, wherein the composition further comprises a carbomer, in particular a carbomer homopolymer type B.

[0023] 11. The pharmaceutical composition according to embodiment 10, wherein the carbomer is Carbopol 974P NF or Carbopol 971P.

[0024] 12. The pharmaceutical composition according to embodiment 10 or 11, wherein the carbomer is present between 0 % and 5 % in weight based on the total weight of the composition, in particular wherein the carbomer is present in about 0.5 % in weight based on the total weight of the composition. 13. The pharmaceutical composition according to any one of embodiments 10 to 12, wherein the composition further comprises a salt, in particular wherein the salt reduces the interaction between the endolysin and the carbomer.

[0025] 14. The pharmaceutical composition according to embodiment 13, wherein the salt is sodium chloride (NaCI).

[0026] 15. The pharmaceutical composition according to embodiment 13 or 14, wherein the NaCI is present between 0 % to about 12.5 % in weight based on the total weight of the composition, in particular wherein the NaCI is present in about 5 % in weight based on the total weight of the composition.

[0027] 16. The pharmaceutical composition according to any one of embodiments 10 to 15, wherein the composition comprises a carbomer and NaCI, wherein the carbomer is present in about 0.5 % in weight based on the total weight of the composition and wherein the NaCI is present in about 5 % in weight based on the total weight of the composition.

[0028] 17. The pharmaceutical composition according to any one of embodiments 1 to 16, wherein the endolysin is present in about 0.1 % to about 5 % in weight based on the total weight of the composition.

[0029] 18. The pharmaceutical composition according to any one of embodiments 1 to 17, wherein the endolysin is present in the composition as a spray dried powder.

[0030] 19. The pharmaceutical composition according to embodiment 18, wherein the spray dried powder further comprises a stabilizing agent and / or a carrier.

[0031] 20. The pharmaceutical composition according to embodiment 19, wherein the stabilizing agent is a modified cyclodextrin and / or wherein the carrier is mannitol.

[0032] 21. The pharmaceutical composition according to embodiment 20, wherein the ratio between mannitol and the modified cyclodextrin in the spray dried powder is about 1: 1, 1.5:1, 2: 1, 3:1, 4: 1, 5:1, 6:1, 7:1, 8:1 or 9:1.

[0033] 22. The pharmaceutical composition according to embodiment 20 or 21, wherein the modified cyclodextrin is hydroxypropyl -cyclodextrin.

[0034] 23. The pharmaceutical composition according to any one of embodiments 18 to 22, wherein the spray dried powder further comprises one or more pH buffering agents, in particular wherein the pH buffering agent is sodium acetate trihydrate.

[0035] 24. The pharmaceutical composition according to any one of embodiments 1 to 23, wherein the composition further comprises a lubricant.

[0036] 25. The pharmaceutical composition according to embodiment 24, wherein the lubricant is magnesium stearate, in particular wherein the magnesium stearate is present between 0 % and 5 % in weight based on the total weight of the composition, in particular wherein the magnesium stearate is present in about 1 % in weight based on the total weight of the composition.

[0037] 26. The pharmaceutical composition according to any one of embodiments 1 to 25, wherein the composition further comprises a glidant.

[0038] 27. The pharmaceutical composition according to embodiment 26, wherein the glidant is selected from the list consisting of: silica, talc, stearic acid and stearyl alcohol.

[0039] 28. The pharmaceutical composition according to embodiment 26 or 27, wherein the glidant is present between 0 % and 5 % in weight based on the total weight of the composition, in particular wherein magnesium stearate is present in about 1 % to about 2% in weight based on the total weight of the composition.

[0040] 29. The pharmaceutical composition according to any one of embodiments 1 to 28, wherein the composition further comprises a filler, in particular wherein the filler is microcrystalline cellulose (MCC).

[0041] 30. The pharmaceutical composition according to any one of embodiments 1 to 23, wherein the composition further comprises a silicified microcrystalline cellulose (SMCC).

[0042] 31. The pharmaceutical composition according to any one of embodiments 1 to 30, wherein the composition comprises about:

[0043] - 0.2 to 2 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder;

[0044] - 30 to 55 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0045] - 0 to 5 % (w / w) of a carbomer, preferably a carbomer homopolymer type B;

[0046] - 0 to 12.5 % (w / w) of a salt, preferably sodium chloride;

[0047] - 0 to 5 % (w / w) of a gelling agent, preferably hydroxyethyl cellulose;

[0048] - 0 to 5 % (w / w) of a glidant, preferably talc;

[0049] - 0 to 5 % (w / w) of a lubricant, preferably magnesium stearate; and

[0050] - microcrystalline cellulose ad 100 % (w / w).

[0051] 32. The pharmaceutical composition according to any one of embodiments 1 to 30, wherein the composition comprises about:

[0052] - 0.2 to 2 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder;

[0053] - 30 to 55 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0054] - 0.5 % (w / w) of a carbomer, preferably a carbomer homopolymer type B;

[0055] - 5 % (w / w) of a salt, preferably sodium chloride;

[0056] - 0.5 % (w / w) of a gelling agent, preferably hydroxyethyl cellulose;

[0057] - 5 % (w / w) of a glidant, preferably talc; - 1 % (w / w) of a lubricant, preferably magnesium stearate; and

[0058] - microcrystalline cellulose ad 100 % (w / w).

[0059] 33. The pharmaceutical composition according to any one of embodiments 1 to 30, wherein the composition comprises about:

[0060] - 2 % (w / w) of a spray dried powder comprising the endolysin, wherein the spray dried powder comprises about 10% (w / w) of the endolysin;

[0061] - 51 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, in particular wherein the sugar alcohol is mannitol;

[0062] - 0.5 % (w / w) of a carbomer, in particular a carbomer homopolymer type B;

[0063] - 5 % (w / w) of sodium chloride;

[0064] - 0.5 % (w / w) of hydroxyethyl cellulose;

[0065] - 5 % (w / w) of talc;

[0066] - 1 % (w / w) of magnesium stearate; and

[0067] - microcrystalline cellulose ad 100 % (w / w).

[0068] 34. The pharmaceutical composition according to any one of embodiments 1 to 30, wherein the composition comprises about:

[0069] - 5 % (w / w) of a spray dried powder comprising the endolysin, wherein the spray dried powder comprises about 10% (w / w) of the endolysin;

[0070] - 48 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, in particular wherein the sugar alcohol is mannitol;

[0071] - 0.5 % (w / w) of a carbomer, in particular a carbomer homopolymer type B;

[0072] - 5 % (w / w) of sodium chloride;

[0073] - 0.5 % (w / w) of hydroxyethyl cellulose;

[0074] - 5 % (w / w) of talc;

[0075] - 1 % (w / w) of magnesium stearate; and

[0076] - microcrystalline cellulose ad 100 % (w / w).

[0077] 35. The pharmaceutical composition according to any one of embodiments 1 to 30, wherein the composition comprises about:

[0078] - 20 % (w / w) of a spray dried powder comprising the endolysin, wherein the spray dried powder comprises about 10% (w / w) of the endolysin;

[0079] - 33 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, in particular wherein the sugar alcohol is mannitol;

[0080] - 0.5 % (w / w) of a carbomer, in particular a carbomer homopolymer type B;

[0081] - 5 % (w / w) of sodium chloride;

[0082] - 0.5 % (w / w) of hydroxyethyl cellulose;

[0083] - 5 % (w / w) of talc;

[0084] - 1 % (w / w) of magnesium stearate; and

[0085] - microcrystalline cellulose ad 100 % (w / w). The pharmaceutical composition according to any one of embodiments 1 to 30, wherein the composition comprises about:

[0086] - 0.2 to 2 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder;

[0087] - 50 to 70 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0088] - 0.5 % (w / w) of a carbomer, preferably a carbomer homopolymer type B;

[0089] - 5 % (w / w) of a salt, preferably sodium chloride;

[0090] - 0 to 5 % (w / w) of a gelling agent, preferably hydroxyethyl cellulose;

[0091] - 0 to 2 % (w / w) of a g lidant;

[0092] - 1 to 2 % (w / w) of a lubricant, preferably magnesium stearate; and

[0093] - microcrystalline cellulose ad 100 % (w / w). The pharmaceutical composition according to any one of embodiments 1 to 30, wherein the composition comprises about:

[0094] - 0.2 to 2 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder;

[0095] - 30 to 55 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0096] - 0.5 % (w / w) of a carbomer, preferably a carbomer homopolymer type A and / or B;

[0097] - 5 % (w / w) of a salt, preferably sodium chloride;

[0098] - 0.5 % (w / w) of a gelling agent, preferably hydroxyethyl cellulose;

[0099] - 1 % (w / w) of a lubricant, preferably magnesium stearate; and

[0100] - microcrystalline cellulose ad 100 % (w / w). The pharmaceutical composition according to any one of embodiments 1 to 30, wherein the composition comprises about:

[0101] - 2.2 % (w / w) of a spray dried powder comprising the endolysin, wherein the spray dried powder comprises about 22.5% (w / w) of the endolysin;

[0102] - 53 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, in particular wherein the sugar alcohol is mannitol;

[0103] - 0.5 % (w / w) of a carbomer, in particular a carbomer homopolymer type A and / or B;

[0104] - 5 % (w / w) of sodium chloride;

[0105] - 0.5 % (w / w) of hydroxyethyl cellulose;

[0106] - 1 % (w / w) of magnesium stearate; and

[0107] - microcrystalline cellulose ad 100 % (w / w). The pharmaceutical composition according to any one of embodiments 1 to 30, wherein the composition comprises about:

[0108] - 8.9 % (w / w) of a spray dried powder comprising the endolysin, wherein the spray dried powder comprises about 22.5% (w / w) of the endolysin;

[0109] - 49 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, in particular wherein the sugar alcohol is mannitol; - 0.5 % (w / w) of a carbomer, in particular a carbomer homopolymer type A and / or B;

[0110] - 5 % (w / w) of sodium chloride;

[0111] - 0.5 % (w / w) of hydroxyethyl cellulose;

[0112] - 1 % (w / w) of magnesium stearate; and

[0113] - microcrystalline cellulose ad 100 % (w / w).

[0114] 40. The pharmaceutical composition according to any one of embodiments 31 to 39, wherein the spray dried powder comprises about 10 to 30 % (w / w) of the endolysin.

[0115] 41. The pharmaceutical composition according to embodiment 40, wherein the spray dried powder further comprises 10 to 20 % (w / w) or 20 to 50% (w / w) of a stabilizing agent, in particular wherein the stabilizing agent is hydroxypropyl p-cyclodextrin.

[0116] 42. The pharmaceutical composition according to embodiment 40 or 41, wherein the spray dried powder further comprises a carrier, in particular wherein the carrier is mannitol.

[0117] 43. The pharmaceutical composition according to any one of embodiments 1 to 42, wherein said composition is suitable for vaginal administration.

[0118] 44. The pharmaceutical composition according to any one of embodiments 1 to 43, for use in the treatment of bacterial infections in a subject.

[0119] 45. The pharmaceutical composition for use according to embodiment 44, wherein the bacterial infection is bacterial vaginosis.

[0120] 46. The pharmaceutical composition for use according to embodiment 44 or 45, wherein the bacterial infection or bacterial vaginosis is characterized by the presence of one or more Gardnerella species.

[0121] 47. The pharmaceutical composition for use according to embodiment 45 or 46, wherein the composition is to be administered locally into the vagina of a female subject.

[0122] 48. The pharmaceutical composition for use according to embodiment 47, wherein the female subject is not menstruating and / or is not expected to menstruate during the treatment.

[0123] 49. The pharmaceutical composition for use according to any one of embodiments 45 to 48, wherein a single dose of the pharmaceutical composition is administered to said subject.

[0124] 50. The pharmaceutical composition for use according to any one of embodiments 45 to 48, wherein at least one dose of the pharmaceutical composition is administered to said subject on n consecutive days, wherein n is an integer from 2 to 14, in particular wherein n is 5.

[0125] 51. The pharmaceutical composition for use according to any one of embodiments 45 to 50, wherein the efficacy of the bacterial vaginosis treatment is assessed during the treatment. 52. The pharmaceutical composition for use according to embodiment 51, wherein the efficacy of the bacterial vaginosis treatment is assessed based on one or more of: Nugent score, amount and / or appearance of vaginal discharge, outcome of whiff test, vaginal pH, and / or proportion of clue cells among total epithelial cells.

[0126] 53. A method of treating a bacterial infection in a subject in need, comprising administering to the subject the pharmaceutical composition according to any one of embodiments 1 to 43.

[0127] 54. The method according to embodiment 53, wherein the bacterial infection is bacterial vaginosis.

[0128] 55. The method according to embodiment 53 or 54, wherein the bacterial infection or bacterial vaginosis is characterized by the presence of one or more Gardnerella species.

[0129] 56. The method according to embodiment 54 or 55, wherein the pharmaceutical composition is administered locally into the vagina of a female subject.

[0130] 57. The method according to embodiment 56, wherein the female subject is not menstruating and / or is not expected to menstruate during the treatment.

[0131] 58. The method according to any one of embodiment 53 to 57, wherein a single dose of the pharmaceutical composition is administered to the subject.

[0132] 59. The method according to any one of embodiment 53 to 57, wherein at least one dose of the pharmaceutical composition is administered to the subject on n consecutive days, wherein n is an integer from 2 to 14, in particular wherein n is 5.

[0133] 60. The method according to any one of embodiment 54 to 59, wherein the efficacy of the bacterial vaginosis treatment is assessed during the treatment.

[0134] 61. The method according to embodiment 60, wherein the efficacy of the bacterial vaginosis treatment is assessed based on one or more of: Nugent score, amount and / or appearance of vaginal discharge, outcome of whiff test, vaginal pH, and / or proportion of clue cells among total epithelial cells.

[0135] 62. Use of the pharmaceutical composition according to any one of embodiments 1 to 43 in the manufacture of a medicament for the treatment of bacterial infections in a subject.

[0136] 63. The use according to embodiment 62, wherein the bacterial infection is bacterial vaginosis.

[0137] 64. The use according to embodiment 62 or 63, wherein the bacterial infection or bacterial vaginosis is characterized by the presence of one or more Gardnerella species.

[0138] 65. The use according to embodiment 63 or 64, wherein the medicament is to be administered locally into the vagina of a female subject.

[0139] 66. The use according to embodiment 65, wherein the female subject is not menstruating and / or is not expected to menstruate during the treatment.

[0140] 67. The use according to any one of embodiments 62 to 66, wherein a single dose of the medicament is administered to the subject.

[0141] 68. The use according to any one of embodiments 62 to 66, wherein at least one dose of the medicament is administered to the subject on n consecutive days, wherein n is an integer from 2 to 14, in particular wherein n is 5.

[0142] 69. The use according to any one of embodiments 63 to 68, wherein the efficacy of the bacterial vaginosis treatment is assessed during the treatment.

[0143] 70. The use according to embodiment 69, wherein the efficacy of the bacterial vaginosis treatment is assessed based on one or more of: Nugent score, amount and / or appearance of vaginal discharge, outcome of whiff test, vaginal pH, and / or proportion of clue cells among total epithelial cells.

[0144] 71. A method comprising:

[0145] (a) obtaining a biological sample from a subject suffering from bacterial vaginosis;

[0146] (b) analyzing one or more of the following in the biological sample from the subject:

[0147] (i) Nugent score;

[0148] (ii) amount and / or appearance of vaginal discharge;

[0149] (iii) outcome of whiff test;

[0150] (iv) vaginal pH; and / or

[0151] (v) proportion of clue cells among total epithelial cells;

[0152] (c) administering the pharmaceutical composition of any one of embodiments 1-43 to the subject;

[0153] (d) obtaining a second biological sample from the subject; and

[0154] (e) re-analyzing the one or more parameters obtained in step (b) to determine safety and / or effectiveness of the pharmaceutical composition in treating bacterial vaginosis in the subject.

[0155] Accordingly, in a particular embodiment, the invention relates to a pharmaceutical composition comprising as active ingredient an effective amount of (a) an endolysin comprising the amino acid sequence provided in SEQ ID NO: 1; or (b) an endolysin comprising an amino acid sequence having 80% sequence identity to SEQ ID NO:1, wherein the endolysin has a killing activity against Gardnerella; and at least one pharmaceutically acceptable excipient.

[0156] That is, in its broadest sense, the invention relates to a pharmaceutical composition comprising an endolysin comprising an amino acid sequence as set forth in SEQ ID NO:1, or an active variant thereof having killing activity against Gardnerella. The term "endolysin" as used herein refers to a polypeptide usually produced by bacteriophages to digest the host bacteria cell wall and release bacteriophage progeny. An endolysin is a cell-wall lytic enzyme encoded by bacteriophages which have the ability to hydrolyze the cell-wall of target bacteria when added exogenously (lysis- from- without). This novel class of antibacterials has important advantages over classical antibiotics, e.g. a novel mode of action; a narrow spectrum of susceptible bacteria; rapid killing of both stationary- and exponentially- growing bacteria; activity on mucous membranes and bacterial biofilms; low probability of developing resistances; and reduced impact on normal microbiota. These unique features have boosted the interest on the biotechnological and pharmacological exploitation of lysins and their recent inclusion among the top current alternatives to fight antibiotic resistances. Endolysins often consist of two or more domains: at least one catalytic domain, such as a hydrolase domain (typically located at the N-terminal of the polypeptide), which cleaves specific motifs in the peptidoglycan layer, and often one or more cell wall binding domains (classically located at the C-terminal of the polypeptide), which is involved in the specific binding and processing of the bacterial peptidoglycan. Although providing a general organization for endolysin structure, this typical architecture is not a defined characteristic of all endolysins. Endolysins from Gram-positive bacteria and their phages usually comprise 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 only contain the catalytic domain, though modular endolysins have also been reported. The catalytic units dictate the type of peptidoglycan (PG) bond to be cleaved, whereas the cell wall-binding domain(s) largely determines the lytic spectrum by specific recognition of cell wall elements distributed in genus-, or species / stra in -specific manner.

[0157] The endolysin comprised in the pharmaceutical composition of the present invention is preferably a recombinant endolysin which is Gardnerella- enus specific, i.e. it specifically targets bacteria that belong to the genus Gardnerella. The endolysin comprised in the pharmaceutical composition of the present invention further preferably has killing activity against species in the genus Gardnerella. For example, the endolysin comprised in the pharmaceutical composition of the present invention may have killing activity against Gardnerella vaginalis sensu sfricto, Gardnerella leopoldii, Gardnerella piotii and / or Gardnerella swidsinskii, preferably against all of them. The killing activity of the endolysin comprised in the pharmaceutical composition of the present invention against Gardnerella is more preferably a genus-selective killing activity against Gardnerella. Herein "genus-selective killing activity" or "genus-specific bacteriolytic effect" means that the endolysin comprised in the pharmaceutical composition of the present invention does not have killing activity or bacteriolytic effect against bacteria in general. In particular, the endolysin comprised in the pharmaceutical composition of the present invention preferably does not have killing activity against bacteria other than Gardnerella spp. Preferably, the endolysin comprised in the pharmaceutical composition of the present invention has genus-selective killing activity against Gardnerella, but not against Lactobacilli. In particular, it is preferred that said endolysin has no killing activity against Lactobacilli crispatus, Lactobacilli gasseri, and / or Lactobacilli Jensenii. More preferably, said endolysin has no killing activity against all of these Lactobacilli, i.e. Lactobacilli crispatus, Lactobacilli gasseri, and Lactobacilli jensenii.

[0158] As used herein, the "killing activity" of an endolysin against particular bacteria can be defined as a reduction in the number of viable bacteria cells caused by the lysing activity of said endolysin. The killing activity of the endolysin against said bacteria can be complete, meaning that 100% of the bacterial cells have been lysed 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 have been lysed. The killing activity of an endolysin on a particular microorganism may be determined by standard procedures in the field including those based on the determination of the Minimum Inhibitory Concentrations (MICs) of an antimicrobial agent defined as the lowest concentration of said antimicrobial agent that inhibits the visible growth of a microorganism after overnight incubation as described in Andrews, 2001, J Antimicrobial Chemotherapy, 48, Suppl. SI, 5-16 or in "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 Clinical and Laboratory Standards Institute. Another suitable method for determining the killing activity of an endolysin is described in the example section of WO 2020 / 225335 and consists in measuring a decrease in optical density at 610-620 nm of a bacterial cell suspension and / or a decrease in Colony Forming Units (CFU) per milliliter of a bacterial cell suspension after exposure to the endolysin to be tested. The decrease of the Optical Density measured at 610-620 nm of a suspension of bacteria, the susceptibility of which is to be tested, can be determined in an in vitro turbidity assay performed in presence of purified endolysin. According to another embodiment, in an in vitro turbidity test, an endolysin has killing activity against Gardnerella when said endolysin decreases the OD(610-620 nm) of a suspension of at least one strain of Gardnerella bacteria 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%.

[0159] The endolysin comprised in the pharmaceutical composition of the present invention is preferably a functional polypeptide, wherein the function comprises specifically targeting bacteria from the Gardnerella genus, more preferably specially killing bacteria from the Gardnerella genus. The endolysin comprised in the pharmaceutical composition of the present invention further preferably comprises a catalytic domain or a functional fragment thereof and / or a cell wall binding domain or a functional fragment thereof. The endolysin to be used in the therapeutic uses of the present invention might be a natural or a recombinant endolysin. The endolysin comprised in the pharmaceutical composition of the present invention most preferably is a recombinant endolysin.

[0160] The endolysin comprised in the pharmaceutical composition of the present invention is more preferably a recombinant endolysin comprising or consisting of

[0161] (i) a N-terminal catalytic domain, or a functional variant thereof;

[0162] (ii) a C-terminal cell-wall binding region, or a functional variant thereof, wherein the C-terminal cell-wall binding region comprises or consists of at least one cell-wall binding domain; and

[0163] (ill) optionally a linker region between the N-terminal catalytic domain and the C-terminal cell-wall binding region, and has preferably a killing activity, more preferably genus-selective killing activity, against GardnerellaceWs / strams.

[0164] In the context of the present disclosure, the term "recombinant endolysin" preferably refers to an endolysin which has been domain-swapped, as defined in WO 2020 / 225335. In line with this definition, the person skilled in the art readily understands that the "domain-swapped" or "recombinant" endolysins as described herein are non-naturally occurring endolysins. That is, the recombinant endolysin for use of the present invention has been modified by hand of man and excludes, by definition, natural endolysins, i.e. as it can be naturally found in nature. The appended examples as well as the teaching of WO 2020 / 225335 provide suitable method(s) how to generate the artificial endolysin of the invention.

[0165] The term "catalytic domain" or "enzymatic domain" refer to the part of the protein chain which contains the region where the catalyzed chemical reaction takes place. The "catalytic domain" as used herein refers to a functional polypeptide, wherein the function comprises the ability to lyse the cell wall of Gardnerella. In particular, the catalytic domain as a described herein can preferably modify and / or cleave a substrate in Gardnerella c&\\ walls, preferably peptidoglycan. Preferably, the catalytic domain cleaves peptidoglycan in in Gardnerella cell walls and can cause Gardnerella cell lysis. Preferably, the catalytic domain can modify and / or cleave bonds that are present in the cell wall and / or peptidoglycan of Gardnerella spp., such as Gardnerella vaginalis sensu stricto, Gardnerella leopoldii, Gardnerella piotii an / or Gardnerella swidsinskii, preferably all of them. Suitably, the catalytic domain does not modify and / or cleave a susbtrate, preferably peptidoglycan, present in cell wall of bacteria other than Gardnerella spp., preferably beneficial vaginal commensal bacteria, such as Lactobacillus spp. Including Lactobacilli crispatus, Lactobacilli gasseri, and / or Lactobacilli jensenii. The catalytic domain may be a N -acetylmuramidase, N- acetylmuramoyl-L-alanine amidases, L-alanoyl-D-glutamate endopeptidases, interpeptide bridge endopeptidases or N-acetyl-beta-D-glucosaminidases. Preferably, the N-terminal catalytic domain is a N-acetylmuramidase, most preferably a 1,4-beta-N-acetylmuramidase. The catalytic domain is preferably located N-terminally within the (recombinant) endolysin, thereby referred to as "N-terminal catalytic domain", even more preferably the N-terminal catalytic domain is located N-terminally from the C-terminal cell-wall binding region within the (recombinant) endolysin.

[0166] The endolysin to be used in the therapeutic uses of the present invention preferably comprises a catalytic domain consisting of a polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID Nos: 2 to 10 or any functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the amino acid sequence of any one of SEQ ID Nos: 2 to 10. As shown in WO 2020 / 225335, the most active catalytic domain is "H2" (SEQ ID NO: 3). Accordingly, in a preferred aspect of the present invention the catalytic domain is consisting of a polypeptide which comprises or consists of the amino acid sequence of SEQ ID NO: 3, or any functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the amino acid sequence of SEQ ID NO: 3, whereby the endolysin is functional, wherein the function comprises the ability to lyse the cell wall of Gardnerella. The catalytic domain is preferably located N-terminally within the (recombinant) endolysin, thereby referred to as "N-terminal catalytic domain", even more preferably the (recombinant) endolysin further comprises a cell-wall binding region and the N-terminal catalytic domain is located N-terminally from the C- terminal cell-wall binding region within the (recombinant) endolysin.

[0167] The "cell-wall binding region" as used herein refers to a functional polypeptide, wherein the function comprises the ability to bind to the cell wall of Gardnerella. The cell-wall binding region may comprise or consist of one, two, three or more cell-wall binding domains. Cell wall binding domains are polypeptides that interact with and / or bind to bacteria cell walls and / or specific substrates within bacteria cell walls. In particular, the cell call binding domain described herein can preferably specifically bind to the cell wall (e.g., to the peptidoglycan) of Gardnerella spp., such as Gardnerella vaginalis sensu stricto, Gardnerella leopoldii, Gardnerella piotii and / or Gardnerella swidsinskii, preferably all of them. The cell-wall binding region is preferably located C-terminally within the (recombinant) endolysin, thereby referred to as "C-terminal cell-wall binding region", even more preferably the C-terminal cellwall binding region is located C-terminally from the N-terminal catalytic domain within the (recombinant) endolysin. The endolysin to be used in the therapeutic uses of the present invention preferably comprises a cell-wall binding region comprising or consisting of at least one cell-wall binding domain selected from the group consisting of polypeptides comprising or consisting of the amino acid sequence of any one of SEQ ID Nos: 11 to 28, and any functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the amino acid sequence of any one of SEQ ID Nos: 11 to 28. As shown in WO 2020 / 225335, the most active cell-wall binding region is "BIO" (comprising the cell-wall binding domains of SEQ ID Nos: 23 and 24), followed by "Bl 1" (comprising the cell-wall binding domains of SEQ ID Nos: 25 and 26). Thus, in a preferred aspect of the present invention the cell-wall binding domain(s) of is / are selected from the group consisting of polypeptides comprising or consisting of the amino acid sequence of any one of SEQ ID Nos: 23, 24, 25 and 26, and any functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the amino acid sequence of any one of SEQ ID Nos: 23, 24, 25 and 26, whereby the endolysin is functional, wherein the function comprises the ability to lyse the cell wall of Gardnerella. The cell-wall binding region is preferably located C-terminally within the (recombinant) endolysin, thereby referred to as "C-terminal cell-wall binding region", even more preferably the (recombinant) endolysin further comprises a catalytic domain and the catalytic domain is located N -terminally from the C-terminal cell-wall binding region within the (recombinant) endolysin.

[0168] The endolysin comprised in the pharmaceutical composition of the present invention comprises preferably two cellwall binding domains (within the cell-wall binding region). In one preferred aspect of the present invention the cellwall binding domains of the endolysin of the invention each consists of a polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID Nos: 23, 24, 25 and 26, and any functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the amino acid sequence of any one of SEQ ID Nos: 23, 24, 25 and 26, whereby the endolysin is functional, wherein the function comprises the ability to lyse the cell wall of Gardnerella. In one even more preferred aspect of the present invention, the endolysin comprises a first cell-wall binding domain and a second cell-wall binding domain, wherein said first cell-wall binding domain is selected from the group consisting of SEQ ID Nos: 23 and 25, and said second cell-wall binding domain is selected from the group consisting of SEQ ID Nos: 24 and 26. Preferably, said first cell-wall binding domain is located N- terminally of said second cell-wall binding domain.

[0169] In one more preferred embodiment, the endolysin to be used in the therapeutic uses of the present invention comprises

[0170] (i) a N-terminal catalytic domain consisting of a polypeptide which comprises or consists of the amino acid sequence of SEQ ID NO: 3, or any functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the amino acid sequence of SEQ ID NO: 3; and

[0171] (ii) a C-terminal cell-wall binding region comprising or consisting of a first cell-wall binding domain and a second cell-wall binding domain, wherein said first cell-wall binding domain is selected from the group consisting of SEQ ID Nos: 23 and 25, and any functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the amino acid sequence of any one of SEQ ID Nos: 23 and 25, and wherein said second cell-wall binding domain is selected from the group consisting of SEQ ID Nos: 24 and 26 and any functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the amino acid sequence of any one of SEQ ID Nos: 24 and 26; whereby the endolysin is functional, wherein the function comprises the ability to lyse the cell wall of Gardnerella. Preferably, said first cell-wall binding domain is located N-terminally of said second cell-wall binding domain.

[0172] In one particularly preferred embodiment, the endolysin to be used in the therapeutic uses of the present invention comprises

[0173] (i) a N-terminal catalytic domain consisting of a polypeptide which comprises or consists of the amino acid sequence of SEQ ID NO: 3; and

[0174] (ii) a C-terminal cell-wall binding region comprising or consisting of a first cell-wall binding domain and a second cell-wall binding domain, wherein said first cell-wall binding domain is selected from the group consisting of SEQ ID Nos: 23 and 25, and said second cell-wall binding domain is selected from the group consisting of SEQ ID Nos: 24 and 26. Preferably, said first cell-wall binding domain is located N-terminally of said second cell-wall binding domain.

[0175] The endolysin comprised in the pharmaceutical composition of the present invention further preferably comprises 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 a length of 9 to 15 amino acids, even more preferably a length of 12 amino acids. Preferably, the linker region may consist of a polypeptide comprising or consisting of the amino acid sequence (i) (XXX)n, wherein each X can be independently G, A or S, preferably wherein the amino acid sequence (XXX)n is (GGS)n, wherein n corresponds to the number of repetitions of the sequence XXX, preferably wherein n is 2, 3, 4, 5 or 6, or (ii) X1X2GLNGX3X4NGGS (SEQ ID NO: 36), wherein Xi 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. Another exemplary linker region consists of a polypeptide comprising or consisting of the amino acid sequence NVGLNGYKNGGS (SEQ ID NO:95). Illustrative examples of particularly preferred endolysins for use in the therapeutic uses of the present invention are "H2B10" (comprising from N-terminal to C-terminal : SEQ ID Nos: 3, 23, and 24), "H2B11" (comprising from N-terminal to C-terminal : SEQ ID Nos: 3, 25, and 26) as defined in WO 2020 / 225335, and "H2B10B11" (comprising from N-terminal to C-terminal : SEQ ID Nos: 3, 23, and 26).

[0176] In a particular preferred embodiment, the endolysin comprised in the pharmaceutical composition of the present invention is "H2B10B11", also referred to herein as "PM-477", the sequence of which is set forth in SEQ ID NO: 1 or is "H2B10", the sequence of which is set forth in SEQ ID NO: 37. Further endolysins that may be comprised in the pharmaceutical composition of the present invention are disclosed in WO 2020 / 229802, which is fully incorporated herein by reference. Specifically, any one of the endolysins disclosed in Table 4 of WO 2020 / 229802 may be comprised in the pharmaceutical composition of the present invention. The endolysins disclosed in Table 4 of WO 2020 / 229802 are listed herein as SEQ ID Nos: 38-94.

[0177] The terms "peptide", "polypeptide", "protein" and variations of these terms refer to peptide, oligopeptide, oligomer or protein including fusion protein, respectively, comprising at least two amino acids joined to each other by a normal or modified peptide bond, such as in the cases of the isosteric peptides, for example. These terms also include herewith "peptidomimetics" which are defined as peptide analogs containing non-peptidic structural elements, which peptides are capable of mimicking or antagonizing the biological action(s) of a natural parent peptide. A peptidomimetic lacks classical peptide characteristics such as enzymatically scissile peptide bonds. A peptide or polypeptide can be composed of amino acids other than the 20 amino acids defined by the genetic code. It can be composed of L-amino acids and / or D-amino acids. A peptide or polypeptide can equally be composed of amino acids modified by natural processes, such as post-translational maturation processes or by chemical processes, which are well known to a person skilled in the art. Such modifications are fully detailed in the literature. These modifications can appear anywhere in the polypeptide: in the peptide skeleton, in the amino acid chain or even at the carboxy- or amino-terminal ends. A peptide or polypeptide can be branched following an ubiquitination or be cyclic with or without branching. This type of modification can be the result of natural or synthetic post- translational processes that are well known to a person skilled in the art. For example, peptide or polypeptide modifications can include acetylation, acylation, ADP-ribosylation, amidation, covalent fixation of a nucleotide or of a nucleotide derivative, covalent fixation of a lipid or of a lipidic derivative, the covalent fixation of a phosphatidylinositol, covalent or non-covalent cross-linking, cyclization, disulfide bond formation, demethylation, glycosylation including pegylation, hydroxylation, iodization, methylation, myristoylation, oxidation, proteolytic processes, phosphorylation, prenylation, racemization, seneloylation, sulfatation, amino acid addition such as arginylation or ubiquitination. Such modifications are fully detailed in the literature and well-known by the killed person of the art.

[0178] The term "variant" refers to a polypeptide including insertions, deletions, and / or substitutions, either non- conservative or preferably conservative, relative to the native amino acid sequence. For example, the polypeptide may comprise an amino acid sequence with at least 80% identity to the native amino acid sequence, preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity to said amino acid sequence. Percent identity can be determined by methods well known in the art, using suitable computer programs for example MatGAT 2.0 {Myers and Miller, CABIOS (1989). Preferably, % identity is identified over the whole lengths of the sequences to be compared. It will be appreciated that percent identity is calculated in relation to polypeptides whose sequence has been aligned optimally. Fragment and variants of an amino acid sequence may be made using any of the methods of protein engineering, directed evolution and / or site-directed mutagenesis well known in the art (for example, see Molecular Cloning: a Laboratory Manual, 3rdedition, Sambrook & Russell, 2001, Cold Spring Harbor Laboratory Press'). It will be appreciated by skilled persons that a polypeptide according to the invention, or fragment, variant, or fusion thereof, may comprise or consist of a derivative of a native amino acid sequence, or a fragment or variant thereof. Chemical derivatives of one or more amino acids may be achieved by reaction with a functional side group. Such derivatized molecules include, for example, those molecules in which free amino acid groups have been derivatized to form amine hydrochlorides, p-toluene sulphonyl groups, carboxybenzoxy groups, f-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters and hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. Also included as chemical derivatives are those peptides which contain naturally occurring amino acid derivatives of the twenty standard amino acids. For example: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine and ornithine for lysine. Derivatives also include peptides containing one or more additions or deletions as long as the requisite activity is maintained. Other included modifications are amidation, amino terminal acylation (e.g., acetylation or thioglycolic acid amidation), terminal carboxylamidation (e.g, with ammonia or methylamine), and the like terminal modifications. It will be further appreciated by persons skilled in the art that peptidomimetic compounds may also be useful. Thus, by 'polypeptide' we include peptidomimetic compounds which exhibit endolysin activity. The term 'peptidomimetic' refers to a compound that mimics the conformation and desirable features of a particular polypeptide as a therapeutic agent.

[0179] Methods for the production of endolysins are well known in the art. Conveniently, the endolysin is or comprises a recombinant endolysin. The endolysin can be produced by standard techniques of genetic engineering comprising the use of a recombinant vector comprising a polynucleotide encoding an endolysin as described herewith. Numerous expression systems can be used including bacterial plasmids and derived vectors, transposons, yeast episomes, insertion elements, yeast chromosome elements, viruses such as baculovirus, papilloma viruses such as SV40, vaccinia viruses, adenoviruses, fox pox viruses, pseudorabies viruses, retroviruses, cosmid or phagemid derivatives. The nucleotide sequence can be inserted in the recombinant expression vector by methods well known to a person skilled in the art such as, for example, those that are described in MOLECULAR CLONING: A LABORATORY MANUAL, Sambrook et al., 4thEd., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001. The recombinant vector can include nucleotide sequences that control the regulation, the expression, the transcription, and / or the translation of the polynucleotide encoding the endolysin, these sequences being selected according to the host cells that are used. The recombinant vector can further include nucleotide sequences such as those encoding His tags for facilitating the purification step. Subsequently, such a recombinant vector is introduced in a host cell according to methods that are well known to a person skilled in the art, such as those described in BASIC METHODS IN MOLECULAR BIOLOGY, Davis et al., 2nded., McGraw-Hill Professional Publishing, 1995, and MOLECULAR CLONING: A LABORATORY MANUAL, supra, such as transfection by calcium phosphate, transfection by DEAE dextran, transfection, microinjection, transfection by cationic lipids, electroporation, transduction or infection. The host cell can be, for example, bacterial cells such as E. coli, cells of fungi such as yeast cells and cells of Aspergillus, Streptomyces, insect cells, Chinese Hamster Ovary cells (CHO), C127 mouse cell line, BHK cell line of Syrian hamster cells, Human Embryonic Kidney 293 (HEK 293) cells. Preferably, the host cell is E. coli. Said host cells are then cultivated in appropriate conditions so as to produce the endolysin described herewith, which can then further be purified from the culture medium or from the host cell lysate by any standard purification methods including, Immobilized-Metal Affinity Chromatography (IMAC) Block et al. 2008, Protein Expr. Purif. 27, 244-254).

[0180] The pharmaceutical composition according to the invention is preferably formulated for vaginal administration, even more preferably intravaginal administration. That is, in a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition is formulated as a vaginal insert, preferably a vaginal tablet.

[0181] A "vaginal insert" as used herein refers to a specific type of pharmaceutical composition designed for intravaginal use. The vaginal insert is intended for the localized administration of a therapeutic agent within the vaginal cavity. Vaginal inserts encompass, without limitation, tablets, filled capsules, and suppositories.

[0182] Preferably, the pharmaceutical composition according to the invention is formulated in solid form and is suitable for insertion into the vagina. Accordingly, in certain embodiments, the pharmaceutical composition according to the invention is a solid pharmaceutical composition. More preferably, said solid pharmaceutical composition is formulated such that it disintegrates when placed into the vagina.

[0183] In a particularly preferred embodiment, the pharmaceutical composition according to the invention is formulated as a tablet. Due to the limited amount of fluid in the vaginal cavity, tablets are inherently prone to poor disintegration. In the present invention, however, vaginal tablets have been identified that disintegrate rapidly in the vaginal cavity, resulting in an efficient release of the therapeutic agent, i.e., the endolysin, within the vaginal cavity.

[0184] The term "tablet" as used herein is intended to encompass compressed pharmaceutical dosage formulations of all shapes and sizes. A "vaginal tablet" is a tablet that is specifically designed to be inserted into the vagina. Preferably, a "vaginal tablet" is formulated to disintegrate rapidly in the vaginal cavity and is free or essentially free of ingredients and / or excipients that may be unfavorable or harmful to the vaginal environment. In certain embodiments, a "vaginal tablet" is a tablet that drains sufficient amounts of fluid from the vaginal tissue to facilitate disintegration of the tablet. At the same time, the "vaginal tablet" according to the invention is formulated such that it prevents leakage of the active ingredient from the vagina.

[0185] To achieve that, the pharmaceutical composition according to the invention comprises at least one pharmaceutically acceptable excipient. It is preferred herein that the pharmaceutical composition according to the invention comprises an active pharmaceutical ingredient, i.e., the endolysin, and one or more of the following excipients: an osmotic active agent, a gelling agent, an acidifier, a lubricant, a glidant, a flow regulator, a dispersant, a binder and / or a filler.

[0186] Preferred excipients comprised in the pharmaceutical composition according to the invention will be defined herein below:

[0187] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the pharmaceutically acceptable excipient is or comprises an osmotic active agent. The term "osmotic active agent" refers to a water-attracting agent, e.g., a hygroscopic, hydroscopic or other agent, which drives the osmotic flow in a hyperosmotic solution. Various osmotic active agents are known in the art and may be comprised in the pharmaceutical composition according to the invention, including, but not limited to colloidal osmotic active agents and a crystalloid osmotic active agents. Crystalloid osmotic agents that may be used in the compositions of the invention include, but are not limited to: sodium chloride (NaCI), dextrose, sucrose, glycerol, mannitol, sorbitol, polyethylene glycol 3350 NF, magnesium citrate, lactulose, and combinations thereof. Colloidal osmotic agents suitable for use in the compositions of the invention include, but are not limited to: hetastarch, pentastarch, gelatin polypeptides cross-linked with urea, dextran 70, dextran 40, albumin, icodextrin, bentonite USP, MgAI silicate NF type 2A, alginic acid / sodium alginate NF, microcrystalline cellulose and CMC NF, carbomer, gellan gum, and combinations thereof.

[0188] Optimizing the osmotic properties of the pharmaceutical composition according to the invention with an osmotic active agent is important for successful administration of the endolysin into the vagina. That is, the osmolality of the composition must be high enough to allow rapid disintegration of the composition, i.e. the tablet, in the vagina. On the other hand, too high osmolality would also be unfavorable, as it would cause the composition to leak out of the vagina.

[0189] Within the present invention, it was surprisingly found that adjusting the osmolality of the pharmaceutical composition in simulated vaginal fluid (pH 6.0) to about 590 mOsmol / kg resulted in optimal disintegration of a tablet and, at the same time, prevented leaking of the composition from the vagina. This effect was confirmed in human subjects in Example 15.

[0190] Accordingly, in a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the pharmaceutical composition has an osmolality in simulated vaginal fluid (pH 6.0) of about 400 to about 700 mOsmol / kg, preferably of about 450 to 650 mOsmol / kg, more preferably of about 500 to about 600 mOsmol / kg, most preferably of about 590 mOsmol / kg.

[0191] In certain embodiments, the pharmaceutical composition has an osmolality in simulated vaginal fluid (pH 6.0) of about 400 mOsmol / kg, of about 410 mOsmol / kg, of about 420 mOsmol / kg, of about 430 mOsmol / kg, of about 440 mOsmol / kg, of about 450 mOsmol / kg, of about 460 mOsmol / kg, of about 470 mOsmol / kg, of about 480 mOsmol / kg, of about 490 mOsmol / kg, of about 500 mOsmol / kg, of about 510 mOsmol / kg, of about 520 mOsmol / kg, of about 530 mOsmol / kg, of about 540 mOsmol / kg, of about 550 mOsmol / kg, of about 560 mOsmol / kg, of about 570 mOsmol / kg, of about 580 mOsmol / kg, of about 590 mOsmol / kg, of about 600 mOsmol / kg, of about 610 mOsmol / kg, of about 620 mOsmol / kg, of about 630 mOsmol / kg, of about 640 mOsmol / kg, of about 650 mOsmol / kg, of about 660 mOsmol / kg, of about 670 mOsmol / kg, of about 680 mOsmol / kg, of about 690 mOsmol / kg, or of about 700 mOsmol / kg.

[0192] Osmolality of a pharmaceutical composition may be determined as disclosed herein. That is, Osmolality may be determined using a cryoscopic osmometer (Osmomat 030, gonotec, Germany). First, the zero point of the instrument may be defined using highly purified water and the apparatus may be calibrated with 300 mOsmol / kg (9.463 mg / ml NaCI in water) and 700 mOsmol / kg (22.380 mg / ml NaCI in water) reference solutions. Thereafter, a commercially available physiological sodium chloride solution (sodium chloride 0.9 % Freeflex, Fresenius Kabi, Germany) may be used as control. Afterwards, samples (50 pl) may be investigated. Therefore, 500 pl of SVF (pH6.0) may be added to 40 mg of powder blends of the pharmaceutical composition according to the invention. After sonication for 30 min and subsequent centrifugation for 10 min at 12,500 RCF (Eppendorf minispin), the osmolality of supernatant may be determined.

[0193] Alternatively, an advanced Micro Osmometer (Model 3320 Advanced Instruments Inc, Norwood, MA, US), calibrated with 50 and 850 mOsm / kg standards may be used for determining osmolality. Osmolality may be measured using the freezing-point method (Ph. Eur.2.2.35). For that, 40 mg powder blend may be swirled in 500 pL SVF (pH6.0) and vortexed for 1 min at lowest speed. After centrifugation for 10 min at 12500 g, the osmolality of supernatant may be determined.

[0194] SVF may be prepared as described by Owen and Katz (Contraception, 1999, 59(2):91-5). In certain embodiments, SVF may comprise 3.51 mg / ml NaCI, 1.40 mg / ml KOH, 0.22 mg / ml Ca(OH)2, 2.00 mg / ml lactic acid, 1.00 mg / ml acetic acid, 0.40 mg / ml urea, 0.018 mg / ml bovine serum albumin and 5.00 mg / ml glucose.

[0195] The composition according to the present invention may comprise any osmotic active agent, or combination of osmotic active agents, that result in favorable osmotic properties of the composition, as defined herein above. That is, the osmotic agent may be used to tune the osmolality of the pharmaceutical composition according to the invention. Based on the teaching provided herein, the skilled person is capable of identifying suitable osmotic active agents, such as any one of the osmotic active agents disclosed herein, and suitable concentrations thereof, that will lead to a desired osmolality in SVF.

[0196] Within the present invention, it has been demonstrated that sugar alcohols are well suited to confer favorable osmotic properties to the pharmaceutical composition according to the invention. Thus, in a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the osmotic active agent is a sugar alcohol, in particular wherein the sugar alcohol is sorbitol, mannitol or a mixture thereof.

[0197] That is, in certain embodiments, the osmotic active agent comprised in the pharmaceutical composition according to the invention is a sugar alcohol, in particular an osmotic active sugar alcohol. The term "sugar alcohol," as used herein, refers to organic compounds, typically derived from a sugar, containing one hydroxyl group attached to each carbon atom. Although the term "sugar alcohol" as used in the present invention is not limited, xylitol, mannitol, sorbitol, maltitol, erythritol, pentitol, arabitol, ribitol, galactitol, lactitol and the like may be used. Preferably the sugar alcohol is sorbitol, mannitol or a mixture thereof. More preferably, the sugar alcohol is D- sorbitol, D-mannitol, or a mixture thereof. Most preferably, the sugar alcohol is D-mannitol.

[0198] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the sugar alcohol is present in about 10% to about 70% in weight based on the total weight of the composition.

[0199] That is, the concentration of the sugar alcohol in the pharmaceutical composition according to the invention may range from about 10% to about 70% (w / w), preferably from about 20% to about 70% (w / w), more preferably from about 30% to about 70% (w / w), even more preferably from about 40% to about 70% (w / w), most preferably from about 50% to about 70% (w / w). Preferably, the sugar alcohol is (D-)sorbitol, (D-)mannitol or a mixture thereof. That is, in certain embodiments, the total concentration of (D-)mannitol and / or (D-)sorbitol in the pharmaceutical composition according to the invention may range from about 10% to about 70% (w / w), preferably from about 20% to about 70% (w / w), more preferably from about 30% to about 70% (w / w), even more preferably from about 40% to about 70% (w / w), most preferably from about 50% to about 70% (w / w).

[0200] In certain embodiments, the concentration of sugar alcohols in the pharmaceutical composition according to the invention, in particular the total concentration of (D-)sorbitol and / or (D-)mannitol, may be about 10% (w / w), about 20% (w / w), about 35% (w / w), about 40% (w / w), about 45% (w / w), about 50% (w / w), about 55% (w / w), about 60% (w / w), about 65% (w / w), or about 70% (w / w).

[0201] The pharmaceutical composition may comprise (D-)sorbitol and (D-)mannitol in any ratio, i.e., in any ratio ranging from 0:100 to 100:0. In certain embodiments, the ratio of (D-)sorbitol and (D-)mannitol in the pharmaceutical composition according to the invention is about 0:100, about 10:90, about 20:80, about 30:70, about 40:60, about 50:50, about 60:40, about 70:30, about 80:20, about 90:10, or about 100:0. In certain embodiments, the sugar alcohol is Compressol®, a directly compressible pharmaceutical excipient consisting of mannitol and sorbitol.

[0202] In certain embodiments, the pharmaceutical composition according to the invention comprises only mannitol, preferably D-mannitol.

[0203] In certain embodiments, the pharmaceutical composition according to the invention further comprises a gelling agent. Thus, in a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition further comprises a gelling agent.

[0204] The pharmaceutical composition according to the invention preferably comprises a gelling agent to control release of the endolysin at the target site. The term "gelling agent," as used herein, refers to a pharmaceutically acceptable excipient known in the art to produce a gel upon mixing with a solvent (e.g., an aqueous solvent). Non-limiting examples of gelling agents include hyaluronan, a polyoxyethylene-polyoxypropylene block copolymer (e.g., a poloxamer), poly(lactic-co-glycolic) acid, polylactic acid, polycaprolactone, alginic acid or a salt thereof, polyethylene glycol, a cellulose, a cellulose ether, a carbomer (e.g., Carbopol®), agar-agar, gelatin, glucomannan, galactomannan (e.g., guar gum, locust bean gum, or tara gum), xanthan gum, chitosan, pectin, starch, tragacanth, carrageenan, polyvinylpyrrolidone, polyvinyl alcohol, paraffin, petrolatum, silicates, fibroin, and combinations thereof.

[0205] Preferably, the gelling agent is added to the pharmaceutical composition in an amount that allows for a sufficient degree of gelling without significantly inhibiting the distribution of the endolysin at the target site.

[0206] In certain embodiments, the gelling agent comprised in the pharmaceutical composition according to the invention is a cellulose, such as without limitation, hydroxyethyl cellulose (HEC) or hydroxypropyl methyl cellulose (HPMC). HEC and HPMC are particularly attractive for use in intravaginal administration, as they can enhance adherence of the drug product to mucosal surfaces. In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the gelling agent is hydroxyethyl cellulose (HEC).

[0207] Hydroxyethyl cellulose (HEC) is a partially substituted poly (hydroxyethyl) ether of cellulose. Within the present invention, HEC is preferably present in the pharmaceutical composition according to the invention between 0 % and about 5 % in weight based on the total weight of the composition, between 0 % and about 4 % in weight based on the total weight of the composition, between 0 % and about 3 % in weight based on the total weight of the composition, between 0 % and about 2 % in weight based on the total weight of the composition, or between 0 % and about 1 % in weight based on the total weight of the composition.

[0208] In certain embodiments, HEC is present in the pharmaceutical composition according to the invention at about 0.1% in weight based on the total weight of the composition, at about 0.2% in weight based on the total weight of the composition, at about 0.3% in weight based on the total weight of the composition, at about 0.4% in weight based on the total weight of the composition, at about 0.5% in weight based on the total weight of the composition, at about 0.6% in weight based on the total weight of the composition, at about 0.7% in weight based on the total weight of the composition, at about 0.8% in weight based on the total weight of the composition, at about 0.9% in weight based on the total weight of the composition, at about 1% in weight based on the total weight of the composition, at about 1.5% in weight based on the total weight of the composition, at about 2% in weight based on the total weight of the composition, at about 2.5% in weight based on the total weight of the composition, at about 3% in weight based on the total weight of the composition, at about 3.5% in weight based on the total weight of the composition, at about 4% in weight based on the total weight of the composition, at about 4.5% in weight based on the total weight of the composition, at about 5% in weight based on the total weight of the composition. In particularly preferred embodiment, HEC is present in the pharmaceutical composition according to the invention at about 0.5 % in weight based on the total weight of the composition.

[0209] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the gelling agent is hydroxypropyl methyl cellulose (HPMC).

[0210] HPMC is a water soluble nonionic cellulosic polymer in which some of the hydroxyl groups are substituted with methoxy and hydroxypropyl groups. Within the present invention, HPMC is preferably present in the pharmaceutical composition according to the invention between 0 % and about 10 % in weight based on the total weight of the composition, between about 1 % and about 9 % in weight based on the total weight of the composition, between about 2 % and about 8 % in weight based on the total weight of the composition, between about 3 % and about 7 % in weight based on the total weight of the composition, or between about 4 % and about 6 % in weight based on the total weight of the composition.

[0211] In certain embodiments, HPMC is present in the pharmaceutical composition according to the invention at about 0.5% in weight based on the total weight of the composition, at about 1% in weight based on the total weight of the composition, at about 1.5% in weight based on the total weight of the composition, at about 2% in weight based on the total weight of the composition, at about 2.5% in weight based on the total weight of the composition, at about 3% in weight based on the total weight of the composition, at about 3.5% in weight based on the total weight of the composition, at about 4% in weight based on the total weight of the composition, at about 4.5% in weight based on the total weight of the composition, at about 5% in weight based on the total weight of the composition, at about 5.5% in weight based on the total weight of the composition, at about 6% in weight based on the total weight of the composition, at about 6.5% in weight based on the total weight of the composition, at about 7% in weight based on the total weight of the composition, at about 7.5% in weight based on the total weight of the composition, at about 8% in weight based on the total weight of the composition, at about 8.5% in weight based on the total weight of the composition, at about 9% in weight based on the total weight of the composition, at about 9.5% in weight based on the total weight of the composition, at about 10% in weight based on the total weight of the composition. In particularly preferred embodiment, HPMC is present in the pharmaceutical composition according to the invention at about 5 % in weight based on the total weight of the composition.

[0212] The inventors of the present invention surprisingly found that balancing the amounts of the osmotic active agent, i.e., mannitol / sorbitol, and the gelling agent, i.e., HEC or HPMC, in the pharmaceutical composition has an impact on the disintegration rate of the pharmaceutical composition, as well as the release rate of the endolysin from the composition, thereby affecting the pharmacokinetic parameters of the composition.

[0213] Accordingly, in certain embodiments, the pharmaceutical composition according to the invention comprises about 10% to about 70% (w / w) of mannitol and / or sorbitol and between 0 % and about 5 % (w / w) of HEC, preferably about 20% to about 70% (w / w) of mannitol and / or sorbitol and between 0 % and about 4 % (w / w) of HEC, more preferably about 30% to about 70% (w / w) of mannitol and / or sorbitol and between 0 % and about 3 % (w / w) of HEC, even more preferably about 40% to about 70% (w / w) of mannitol and / or sorbitol and between 0 % and about 2 % (w / w) of HEC, most preferably about 50% to about 70% (w / w) of mannitol and / or sorbitol and between 0 % and about 1 % (w / w) of HEC. In certain embodiments, the pharmaceutical composition according to the invention comprises about 50% to about 70% (w / w) of mannitol and / or sorbitol and about 0.5 % (w / w) of HEC. In certain embodiments, the pharmaceutical composition according to the invention comprises about 40% to about 60% (w / w) of mannitol and / or sorbitol and about 0.5 % (w / w) of HEC.

[0214] In certain embodiments, the pharmaceutical composition according to the invention comprises about 10% to about 70% (w / w) of mannitol and / or sorbitol and between 0 % and about 10 % (w / w) of HPMC, preferably about 20% to about 70% (w / w) of mannitol and / or sorbitol and about 1 % and about 9 % (w / w) of HPMC, more preferably about 30% to about 70% (w / w) of mannitol and / or sorbitol and about 2 % and about 8 % (w / w) of HPMC, even more preferably about 40% to about 70% (w / w) of mannitol and / or sorbitol and about 3 % and about 7 % (w / w) of HPMC, most preferably about 50% to about 70% (w / w) of mannitol and / or sorbitol and about 4 % and about 6 % (w / w) of HPMC. In certain embodiments, the pharmaceutical composition according to the invention comprises about 50% to about 70% (w / w) of mannitol and / or sorbitol and about 5 % (w / w) of HPMC. In certain embodiments, the pharmaceutical composition according to the invention comprises about 40% to about 60% (w / w) of mannitol and / or sorbitol and about 5 % (w / w) of HPMC.

[0215] It was shown in Example 9 that formulations comprising at least 50% (w / w) of mannitol and / or sorbitol and about 5% (w / w) of HPMC resulted in the best pharmacokinetic (PK) properties. HPMC may be replaced with HEC without affecting the PK parameters. Preferably, about 5% (w / w) HPMC may be replaced with about 0.5% (w / w) HEC.

[0216] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition further comprises a carbomer. It was surprisingly found by the inventors that combining a Carafjere / Za-specific endolysin with a carbomer improves the killing activity of the endolysin against Gardnerella species (see Examples 4-8). Accordingly, it is preferred herein that the pharmaceutical composition of the invention comprises a carbomer.

[0217] The term "carbomer" refers to a series of polymers of acrylic acid that are commonly used in pharmaceutical compositions. Carbomers are high molecular weight homopolymers or copolymers of acrylic acid that are crosslinked with polyalkenyl ethers of sugars or polyalcohols. The carbomer comprised in the pharmaceutical composition according to the present invention is preferably a carbomer that follows the US and / or European Pharmacopeia requirements. Carbomers are marketed under the trade name Carbopol. Thus, in a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the excipient is a carbomer, preferably a Carbopol.

[0218] In certain embodiments, the pharmaceutical composition according to the invention comprises a specific type of carbomer. In certain embodiments, the pharmaceutical composition according to the invention comprises a mixture of two or more different types of carbomers.

[0219] The carbomer comprised in the pharmaceutical composition according to the invention may be defined based on one or more physicochemical properties.

[0220] Preferably, the carbomer comprised in the pharmaceutical composition according to the invention may be defined based on its viscosity. That is, the carbomer comprised in the pharmaceutical composition according to the invention may have a viscosity ranging from 300 - 115,000 mPa s when in the form of a 0.5% (w / v) gel (pH 7, 3-7, 8 at 25 °C).

[0221] In certain embodiments, the carbomer comprised in the pharmaceutical composition according may be a type A carbomer according to US Pharmacopeia having a viscosity of 4,000 - 11,000 mPa-s when in the form of a 0.5% (w / v) gel (pH 7, 3-7,8 at 25 °C). In certain embodiments, the type A carbomer is Carbopol 981, Carbopol 971 P or Carbopol71G.

[0222] In certain embodiments, the carbomer comprised in the pharmaceutical composition according may be a type B carbomer according to US Pharmacopeia having a viscosity of 25,000 - 45,000 mPa-s when in the form of a 0.5% (w / v) gel (pH 7, 3-7,8 at 25 °C). In certain embodiments, the type B carbomer is Carbopol 974P, Carbopol 984 or Carbopol 5984, preferably Carbopol 974P.

[0223] In certain embodiments, the carbomer comprised in the pharmaceutical composition according may be a type C carbomer according to US Pharmacopeia having a viscosity of 40,000 - 60,000 mPa-s when in the form of a 0.5% (w / v) gel (pH 7, 3-7, 8 at 25 °C). In certain embodiments, the type C carbomer is Carbopol 980.

[0224] In certain embodiments, the carbomer comprised in the pharmaceutical composition according to the invention has a viscosity of 300 - 115,000 mPa-s when in the form of a 0.5% (w / v) gel (pH 7, 3-7, 8 at 25 °C). In certain embodiments, the carbomer comprised in the pharmaceutical composition according to the invention has a viscosity of 300 - 60,000 mPa's when in the form of a 0.5% (w / v) gel (pH 7, 3-7, 8 at 25 °C). In certain embodiments, the carbomer comprised in the pharmaceutical composition according to the invention has a viscosity of 300 - 45,000 mPa-s when in the form of a 0.5% (w / v) gel (pH 7, 3-7, 8 at 25 °C). In certain embodiments, the carbomer comprised in the pharmaceutical composition according to the invention has a viscosity of 300 - 11,000 mPa s when in the form of a 0.5% (w / v) gel (pH 7, 3-7,8 at 25 °C). In certain embodiments, the carbomer comprised in the pharmaceutical composition according to the invention has a viscosity of 300 - 4,000 mPa s when in the form of a 0.5% (w / v) gel (pH 7, 3-7,8 at 25 °C). In certain embodiments, the carbomer comprised in the pharmaceutical composition according to the invention has a viscosity of 300 - 2000 mPa-s when in the form of a 0.5% (w / v) gel (pH 7, 3-7, 8 at 25 °C).

[0225] In a preferred embodiment, the polyacrylic acid polymer comprised in the pharmaceutical composition according to the invention is a type B carbomer, such as Carbopol 974P, having a viscosity of 25000 - 45000 mPa's when in the form of a 0.5% (w / v) gel (pH 7, 3-7,8 at 25 °C). Thus, in a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the carbomer is a carbomer homopolymer type B, in particular Carbopol 974P NF.

[0226] In another preferred embodiment, the polyacrylic acid polymer comprised in the pharmaceutical composition according to the invention is a type A carbomer, such as Carbopol 971P, having a viscosity of 4,000 - 11,000 mPa s when in the form of a 0.5% (w / v) gel (pH 7, 3-7, 8 at 25 °C). Thus, in a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the carbomer is a carbomer homopolymer type A, in particular Carbopol 971P.

[0227] The carbomer may be present in the pharmaceutical composition according to the invention at any suitable concentration. In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the carbomer is present between 0 % and 5 % in weight based on the total weight of the composition.

[0228] That is, the polyacrylic acid polymer may be present in the pharmaceutical composition of the invention at a concentration of about 0.1% (w / w), about 0.2% (w / w), about 0.25% (w / w), about 0.3% (w / w), about 0.35% (w / w), about 0.4% (w / w), about 0.5% (w / w), about 0.6% (w / w), about 0.7% (w / w), about 0.8% (w / w), about 0.9% (w / w), about 1% (w / w), about 1.1% (w / w), about 1.2% (w / w), about 1.3% (w / w), about 1.4% (w / w), about 1.5% (w / w), about 1.6% (w / w), about 1.7% (w / w), about 1.8% (w / w), about 1.9% (w / w), about 2% (w / w), about 2.1% (w / w), about 2.2% (w / w), about 2.3% (w / w), about 2.4% (w / w), about 2.5% (w / w), about 2.6% (w / w), about 2.7% (w / w), about 2.8% (w / w), about 2.9% (w / w), about 3% (w / w), about 3.1% (w / w), about 3.2% (w / w), about 3.3% (w / w), about 3.4% (w / w), about 3.5% (w / w), about 3.6% (w / w), about 3.7% (w / w), about 3.8% (w / w), about 3.9% (w / w), about 4% (w / w), about 4.1% (w / w), about 4.2% (w / w), about 4.3% (w / w), about 4.4% (w / w), about 4.5% (w / w), about 4.6% (w / w), about 4.7% (w / w), about 4.8% (w / w), about 4.9% (w / w), or about 5% (w / w). In a preferred embodiment, the carbomer is present in about 0.5 % in weight based on the total weight of the composition.

[0229] In certain embodiments, the invention relates to the pharmaceutical composition according to the invention, wherein the carbomer is present at a concentration of about 0.1% to 5%, more preferably about 0.1% to 2.5%, even more preferably about 0.1% to 1%, most preferably about 0.2% to 0.75%. While carbomers positively affect the activity of Gardnerella-s ea \c endolysins, carbomers were also found by the inventors to form complexes with the endolysin, thereby inhibiting the release of the endolysin at the target site when formulated as a tablet. The inventors surprisingly found that this disadvantage can be overcome by the addition of salts to the pharmaceutical composition. Accordingly, in a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition further comprises a salt, in particular wherein the salt reduces the interaction between the endolysin and the carbomer.

[0230] The salt may be any salt that is suitable for reducing the interaction between the endolysin and the carbomer. As used herein, the term "salt" is given its ordinary meaning in the art and refers to a neutral, ionic compound comprising a cation and an anion. The salt may be organic or inorganic, and may be a binary salt, complex salt, or the like. In some cases, two or more salts may be comprised in the pharmaceutical composition according to the invention.

[0231] In certain embodiment, the salt may be sodium chloride (NaCI). However, other salts having a similar effect as NaCI on the interaction of the endolysin and the carbomer are also encompassed.

[0232] The potential of a salt to reduce the interaction between the endolysin and the carbomer may be tested as follows: 40 mg tablets comprising at least the endolysin, the carbomer and different concentrations of salt (e.g., 0-12.5 %) may be incubated in 160 pL SVF (pH 6) at 37 °C and 300 rpm for 240 min. The endolysin in the soluble fraction may then subsequently be quantified via HPLC, as described herein in Example 1. A salt is determined to reduce the interactions between the endolysin and the carbomer, if more endolysin is present in the soluble fraction in the presence of the salt than in the absence of the salt. Alternatively, a salt may be defined to improve the release of the endolysin from a complex formed between the endolysin and the carbomer.

[0233] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the NaCI is present between 0 % to about 12.5 % in weight based on the total weight of the composition.

[0234] That is, it has been demonstrated by the inventors that salt concentrations up to 12.5% (w / w) result in increased release of the endolysin from the carbomer, whereas higher concentrations tend to have an inhibitory effect on the endolysin.

[0235] Thus, in certain embodiments, the pharmaceutical composition according to the invention comprises NaCI at a concentration ranging from about 0% to about 12.5% (w / w), preferably from about 1% to about 10% (w / w), more preferably from about 2.5% to about 7.5% (w / w). in certain embodiments, the pharmaceutical composition according to the invention comprises NaCI at a concentration ranging from about 0% to about 12.5% (w / w), preferably from about 1.5% to about 12.5% (w / w), more preferably from about 3% to about 12.5% (w / w).

[0236] In certain embodiments, the pharmaceutical composition according to the invention comprises NaCI at a concentration of about 0.5% (w / w), about 1% (w / w), about 1.5% (w / w), about 2% (w / w), about 2.5% (w / w), about 3% (w / w), about 3.5% (w / w), about 4% (w / w), about 4.5% (w / w), about 5% (w / w), about 5.5% ( j / vi), about 6% (w / w), about 6.5% (w / w), about 7% (w / w), about 7.5% (w / w), about 8% (w / w), about 8.5% (w / w), about 9% (w / w), about 9.5% (w / w), about 10% (w / w), about 10.5% (w / w), about 11% (w / w), about 11.5% (w / w), about 12% (w / w), or about 12.5% (w / w).

[0237] In a particularly preferred embodiment, the pharmaceutical composition according to the invention comprises NaCI at a concentration of about 5 % in weight based on the total weight of the composition.

[0238] At mentioned above, optimizing the ratio of the carbomer and the salt may improve the activity and / or pharmacokinetics of the endolysin. In certain embodiments, the pharmaceutical composition according to the invention comprises a carbomer and NaCI, wherein the carbomer is present at a concentration between 0 % and 5 % in weight based on the total weight of the composition and wherein the NaCI is present at a concentration between 0 % to about 12.5 % in weight based on the total weight of the composition.

[0239] More preferably, the pharmaceutical composition according to the invention comprises a carbomer and NaCI, wherein the carbomer is present at a concentration between 0.1 % and 4 % in weight based on the total weight of the composition and wherein the NaCI is present at a concentration between 1 % to about 10 % in weight based on the total weight of the composition.

[0240] Even more preferably, the pharmaceutical composition according to the invention comprises a carbomer and NaCI, wherein the carbomer is present at a concentration between 0.1 % and 2.5 % in weight based on the total weight of the composition and wherein the NaCI is present at a concentration between 2.5 % to about 7.5% in weight based on the total weight of the composition.

[0241] Even more preferably, the pharmaceutical composition according to the invention comprises a carbomer and NaCI, wherein the carbomer is present at a concentration between 0.1 % and 1 % in weight based on the total weight of the composition and wherein the NaCI is present at a concentration between 4 % to about 6% in weight based on the total weight of the composition.

[0242] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises a carbomer and NaCI, wherein the carbomer is present in about 0.5 % in weight based on the total weight of the composition and wherein the NaCI is present in about 5 % in weight based on the total weight of the composition.

[0243] As mentioned above, other salts than NaCI may be used in the pharmaceutical composition according to the invention. Based on the teaching provided herein, the skilled person is capable of identifying the optimal concentration of such salts to achieve sufficient release of the endolysin from a carbomer. In a preferred embodiment, grinded NaCI is used in the composition according to the invention.

[0244] The endolysin may be present in the pharmaceutical composition in any suitable amount. In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the endolysin is present in about 0.1 % to about 5 % in weight based on the total weight of the composition.

[0245] That is, in certain embodiments, the endolysin is present in the pharmaceutical composition at a concentration of about 0.1% (w / w), about 0.2% (w / w), about 0.3% (w / w), about 0.4% (w / w), about 0.5% (w / w), about 0.6% (w / w), about 0.7% (w / w), about 0.8% (w / w), about 0.9% (w / w), about 1% (w / w), about 1.1% (w / w), about 1.2% (w / w), about 1.3% (w / w), about 1.4% (w / w), about 1.5% (w / w), about 1.6% (w / w), about 1.7% (w / w), about 1.8% (w / w), about 1.9% (w / w), about 2.0% (w / w), about 2.1% (w / w), about 2.2% (w / w), about 2.3% (w / w), about 2.4% (w / w), about 2.5% (w / w), about 2.6% (w / w), about 2.7% (w / w), about 2.8% (w / w), about 2.9% (w / w), about 3.0% (w / w), about 3.1% (w / w), about 3.2% (w / w), about 3.3% (w / w), about 3.4% (w / w), about 3.5% (w / w), about 3.6% (w / w), about 3.7% (w / w), about 3.8% (w / w), about 3.9% (w / w), about 4.0% (w / w), about 4.1% (w / w), about 4.2% (w / w), about 4.3% (w / w), about 4.4% (w / w), about 4.5% (w / w), about 4.6% (w / w), about 4.7% (w / w), about 4.8% (w / w), about 4.9% (w / w), or about 5.0% (w / w).

[0246] In a preferred embodiment, the endolysin is present in the pharmaceutical composition at a concentration of about 0.2% (w / w). In another preferred embodiment, the endolysin is present in the pharmaceutical composition at a concentration of about 0.5% (w / w). In another preferred embodiment, the endolysin is present in the pharmaceutical composition at a concentration of about 1% (w / w). In another preferred embodiment, the endolysin is present in the pharmaceutical composition at a concentration of about 2% (w / w).

[0247] The endolysin may be present in the pharmaceutical composition of the invention in any form. Since the pharmaceutical composition is preferably formulated as a tablet, the endolysin is preferably present in solid form, such as a lyophilized powder or a spray-dried powder. In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the endolysin is present in the composition as a spray dried powder. Methods for lyophilizing and spray drying protein solutions are well known in the art.

[0248] Protein solutions are preferably mixed with stabilizing agents and / or carriers before spray drying to improve stability of the proteins in the spray dried powder. Thus, in a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the spray dried powder further comprises a stabilizing agent and / or a carrier.

[0249] In the context of spray drying proteins, a "stabilizing agent" refers to a substance or compound used to protect and maintain the structural integrity, functionality, and stability of proteins during the drying process and subsequent storage. These agents help prevent denaturation, aggregation, and other forms of protein damage. Stabilizing agents include, without limitation, cyclodextrins, maltodextrin, gelatin, whey protein, trehalose, polysaccharides and hydrocolloids. In a preferred embodiment, the stabilizing agent is a cyclodextrin, in particular a modified cyclodextrin. In certain embodiments, the modified cyclodextrin is 2-Hydroxypropyl-B-cyclodextrin (HPBCD). Thus, in a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the stabilizing agent is 2-hydroxypropyl p-cyclodextrin.

[0250] In the context of spray drying proteins, a "carrier" refers to a substance or material that is used to help stabilize and protect the protein during the drying process. The carrier can serve several purposes, including protecting the protein, enhancing flow properties and / or facilitating reconstitution. Carriers include, without limitation, mannitol, maltodextrin, starch, dextrose, or lactose. In a preferred embodiment, the carrier is mannitol, more preferably D- mannitol.

[0251] Accordingly, in a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the stabilizing agent is a modified cyclodextrin and / or wherein the carrier is mannitol. Mannitol and the modified cyclodextrin may be present in the spray dried powder at any suitable ratio. In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the ratio between mannitol and the modified cyclodextrin in the spray dried powder is about 1:1, 1.5: 1, 2:1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8:1 or 9:l.

[0252] In certain embodiments, the spray dried powder may comprise between about 5% and about 40% (w / w) of the endolysin, preferably between about 10% and about 30% (w / w) of the endolysin. In certain embodiments, the spray dried powder may comprise about 10% (w / w) of the endolysin. In certain embodiments, the spray dried powder may comprise between about 20% and about 30% (w / w) of the endolysin, preferably about 22.5% (w / w) of the endolysin.

[0253] In certain embodiments, the spray dried powder may comprise between about 5% and about 30% (w / w) of the endolysin, between about 60% and about 90% (w / w) of D-mannitol and between about 5% and about 25% (w / w) of the cyclodextrin, preferably 2-Hydroxypropyl-B-cyclodextrin (HPBCD).

[0254] In a preferred embodiment, the spray dried powder comprises about 10% (w / w) of the endolysin, about 75% (w / w) of D-mannitol and about 15% (w / w) of the cyclodextrin, preferably 2-Hydroxypropyl-B-cyclodextrin (HPBCD).

[0255] In certain embodiments, the spray dried powder may comprise between about 10% and about 30% (w / w) of the endolysin, between about 30% and about 60% (w / w) of D-mannitol and between about 20% and about 50% (w / w) of the cyclodextrin, preferably 2-Hydroxypropyl-B-cyclodextrin (HPBCD).

[0256] In certain embodiments, the spray dried powder may comprise between about 15% and about 25% (w / w) of the endolysin, between about 40% and about 60% (w / w) of D-mannitol and between about 20% and about 40% (w / w) of the cyclodextrin, preferably 2-Hydroxypropyl-B-cyclodextrin (HPBCD).

[0257] In another preferred embodiment, the spray dried powder comprises about 22.5% (w / w) of the endolysin, about 45% (w / w) of D-mannitol and about 32% (w / w) of the cyclodextrin, preferably 2-Hydroxypropyl-B-cyclodextrin (HPBCD).

[0258] Besides the endolysin, the stabilizing agent and the carrier, the spray dried powder may comprise additional agents. In particular, the spray dried powder may comprise one or more pH buffering agents. Thus, in a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the spray dried powder further comprises one or more pH buffering agents.

[0259] The skilled person is aware of various pH buffering agents that are suitable of obtaining a desired pH. Non limiting examples include sodium acetate trihydrate and acetic acid. Accordingly, in a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the pH buffering agent is sodium acetate trihydrate. In certain embodiments, the pH buffering agent, preferably the sodium acetate trihydrate are present at a concentration between about 0% and about 5% (w / w), preferably between about 0% and about 2% (w / w), more preferably between about 0.5% and about 1% (w / w). The pharmaceutical composition according to the invention may further comprise a lubricant. Thus, in a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition further comprises a lubricant.

[0260] The term "lubricant", as used herein, refers to an inactive ingredient used to prevent sticking of ingredients to one another when dry granulated, filled in capsules or compressed to tablets. A lubricant reduces powder sticking to the roll surface of roller compactors and sliding friction of the tableting material and punches in the die during the tableting operation and prevents sticking to the tablet punches. Suitable lubricants are alkaline-earth metal salts of fatty acids, such as magnesium stearate or calcium stearate, fatty acids, such as stearic acid, higher fatty alcohols such as cetyl alcohol or stearyl alcohol, fats such as glyceryl dipalm itostearate, glyceryl distearate, stearin or glyceryl dibehenate, alkaline-earth metal salts of C16-C18 alkyl substituted dicarbonic acids such as sodium stearyl fumarate, hydrated vegetable oils such as hydrated castor oil or hydrated cotton seed oil, or minerals such as talc. Preferred lubricants are sodium stearyl fumarate, esters of glycerol with fatty acids, stearic acid or pharmaceutically acceptable salts of stearic acid and divalent cations, preferably magnesium stearate. Lubricants can be present in the pharmaceutical composition according to the invention in a proportion of 0 to 5% (w / w), preferably 0.1 to 3% (w / w), particularly preferably 0.5 to 2.5 % (w / w), most preferably about 1% to about 2% (w / w), based on the total weight of the solid formulation.

[0261] Accordingly, in a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the lubricant is magnesium stearate, in particular wherein the magnesium stearate is present between 0 % and 5 % in weight based on the total weight of the composition, in particular wherein the magnesium stearate is present in about 1 % to about 2% in weight based on the total weight of the composition.

[0262] The pharmaceutical composition according to the invention may further comprise a glidant. Thus, in a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition further comprises a glidant.

[0263] The term "glidant", as used herein, refers to an inactive ingredient used as a flow aid that improves the flow characteristics of particulates such as powders or granules. In the present invention flow characteristics of the solid preparation or the mixtures containing the solid preparation during further processing such as encapsulation or tableting. Nonlimiting examples of glidants for use in the present invention include colloidal silicon dioxide (Aerosil 200, Cab-O- Sil), talc, magnesium carbonate, stearic acid, stearyl alcohol and combinations thereof. In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the glidant is selected from the list consisting of: silica (silicon dioxide), talc, stearic acid and stearyl alcohol.

[0264] Glidants are present in the pharmaceutical preparation according to the invention in a proportion of 0 to 5% (w / w), preferably 0 to 4% (w / w), more preferably 0 to 3% (w / w), even more preferably 1 to 2% (w / w), based on the total weight of the solid formulation. Accordingly, in a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the glidant is present between 0 % and 5 % in weight based on the total weight of the composition, in particular wherein the glidant is present in about 1 % to about 2% in weight based on the total weight of the composition. In a particular embodiment, talc is present in the pharmaceutical composition at a concentration of 0 to 5% (w / w), preferably 0 to 4% (w / w), more preferably 0 to 3% (w / w), even more preferably 1 to 2% (w / w), based on the total weight of the solid formulation.

[0265] In a particular embodiment, silica or a colloidal silicon dioxide is present in the pharmaceutical composition at a concentration of 0 to 5% (w / w), preferably 0 to 4% (w / w), more preferably 0 to 3% (w / w), even more preferably 1 to 2% (w / w), based on the total weight of the solid formulation.

[0266] In certain embodiments, the pharmaceutical composition is free, or essentially free, of glidants. That is, in certain embodiments, the pharmaceutical composition is free, or essentially free, of talc. Compositions without talc are disclosed herein.

[0267] The pharmaceutical composition according to the invention may further comprise a filler and / or binder. Thus, in a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition further comprises a filler and / or binder, in particular wherein the filler and / or binder is microcrystalline cellulose (MCC).

[0268] The term "filler" as used herein is an agent increasing the bulk of the pharmaceutical preparation by providing the quantity of material which is needed to form a solid preparation. A filler may also serve to create desired flow properties and compression characteristics in the preparation of the solid preparation as well as of solid pharmaceutical preparations such as tablets and capsule fillers. Fillers usable in the present invention may be a sugar alcohol such as sorbitol or mannitol, dulcitol, xylitol or ribitol, a sugar such as glucose, fructose, mannose, lactose, saccharose or maltose, a starch such as potato starch, rice starch, maize starch or pregelatinized starch.

[0269] The term "binder", as used herein, refers to an agent that provides cohesion and strength to a solid preparation. Binders which can be employed in the present invention are, for example, polyvinylpyrrolidone, polyvinyl acetate, a vinylpyrrolidone-vinyl acetate copolymer, polyethylene glycol, a starch paste, such as maize starch paste, a cellulose derivative, such as hydroxypropyl methylcellulose, hydroxypropyl cellulose or microcrystalline cellulose, preferably microcrystalline cellulose. Therefore, the present invention is as well directed to a solid pharmaceutical preparation, wherein the binder is polyvinylpyrrolidone, polyvinyl acetate, a vinylpyrrolidone-vinyl acetate copolymer, polyethylene glycol, a starch paste, such as maize starch paste, a cellulose derivative, such as hydroxypropyl methylcellulose, hydroxypropyl cellulose or microcrystalline cellulose, preferably microcrystalline cellulose.

[0270] In certain embodiments, the lubricant, the glidant and / or the filler / binder may be replaced with silicified microcrystalline cellulose (SMCC). Accordingly, in a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition further comprises a silicified microcrystalline cellulose (SMCC).

[0271] Silicified microcrystalline cellulose (SMCC) is a tableting excipient which can improve binding capability as a material and in tablet formulations. The material also shows improved resistance to the degrading effects of magnesium stearate compared with regular microcrystalline cellulose. In the following exemplary pharmaceutical compositions according to the invention will be disclosed:

[0272] In one embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0273] - 0.1 to 10 % (w / w), preferably 0.2% to 2% (w / w), of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder; and

[0274] - 30 to 70 % (w / w), preferably 40% to 60% (w / w), of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof.

[0275] Said composition may further comprise a gelling agent, such as HEC or HPMC at a concentration ranging from 0 to 10 % (w / w). As discussed in more detail above, optimizing the ratio between the osmotic active agent and the gelling agent may result in optimized pharmacokinetics.

[0276] Said composition may further comprise a carbomer, preferably a carbomer homopolymer type A and / or B, preferably at a concentration ranging from 0 to 5 % (w / w). It has been shown herein that carbomers act synergistically with the endolysin of the invention.

[0277] Said composition may further comprise salts, preferably sodium chloride, preferably at a concentration ranging from 0 to 12.5 % (w / w). It has been shown herein that optimizing the ratio between the carbomer and the salts can improve the release of the endolysin.

[0278] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0279] - 0.1 to 10 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder;

[0280] - 30 to 70 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0281] - 0.1 to 10 % (w / w) of a gelling agent, preferably HEC or HPMC;

[0282] - 0.1 to 5 % (w / w) of a carbomer, preferably a carbomer homopolymer type A and / or B; and

[0283] - 0.1 to 12.5 % (w / w) of a salt, preferably sodium chloride.

[0284] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0285] - 0.2 to 5 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder;

[0286] - 30 to 70 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0287] - 0.1 to 5 % (w / w) of a gelling agent, preferably HEC;

[0288] - 0.1 to 2 % (w / w) of a carbomer, preferably a carbomer homopolymer type A and / or B; and

[0289] - 1 to 12.5 % (w / w) of a salt, preferably sodium chloride.

[0290] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0291] - 0.2 to 5 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder; - 50 to 70 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0292] - 0.2 to 1 % (w / w) of a gelling agent, preferably HEC;

[0293] - 0.2 to 1 % (w / w) of a carbomer, preferably a carbomer homopolymer type A and / or B; and

[0294] - 2.5 to 7.5 % (w / w) of a salt, preferably sodium chloride.

[0295] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0296] - 0.2 to 5 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder;

[0297] - 30 to 70 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0298] - 1 to 10 % (w / w) of a gelling agent, preferably HPMC;

[0299] - 0.1 to 2 % (w / w) of a carbomer, preferably a carbomer homopolymer type A and / or B; and

[0300] - 1 to 12.5 % (w / w) of a salt, preferably sodium chloride.

[0301] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0302] - 0.2 to 5 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder;

[0303] - 50 to 70 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0304] - 2.5 to 7.5 % (w / w) of a gelling agent, preferably HPMC;

[0305] - 0.2 to 1 % (w / w) of a carbomer, preferably a carbomer homopolymer type A and / or B; and

[0306] - 2.5 to 7.5 % (w / w) of a salt, preferably sodium chloride.

[0307] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0308] - 0.1 to 10 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder;

[0309] - 30 to 70 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0310] 0 to 10 % (w / w) of a gelling agent, preferably HEC or HPMC;

[0311] - 0 to 5 % (w / w) of a carbomer, preferably a carbomer homopolymer type A and / or B;

[0312] - 0 to 12.5 % (w / w) of a salt, preferably sodium chloride;

[0313] - 0 to 5 % (w / w) of a lubricant, preferably magnesium stearate;

[0314] - 0 to 5 % (w / w) of a glidant, preferably talc or silica; and

[0315] - microcrystalline cellulose ad 100 % (w / w).

[0316] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0317] - 0.1 to 10 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder;

[0318] - 30 to 70 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0319] - 0.1 to 10 % (w / w) of a gelling agent, preferably HEC or HPMC;

[0320] - 0.1 to 5 % (w / w) of a carbomer, preferably a carbomer homopolymer type A and / or B; - 0.1 to 12.5 % (w / w) of a salt, preferably sodium chloride;

[0321] - 0 to 5 % (w / w) of a glidant, preferably talc or silica;

[0322] - 0.1 to 5 % (w / w) of a lubricant, preferably magnesium stearate; and

[0323] - microcrystalline cellulose ad 100 % (w / w).

[0324] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0325] - 0.2 to 5 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder;

[0326] - 30 to 70 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0327] - 0.1 to 5 % (w / w) of a gelling agent, preferably HEC;

[0328] - 0.1 to 2 % (w / w) of a carbomer, preferably a carbomer homopolymer type A and / or B;

[0329] - 1 to 12.5 % (w / w) of a salt, preferably sodium chloride;

[0330] - 0.5 to 5 % (w / w) of a glidant, preferably talc or silica;

[0331] - 0.1 to 2.5 % (w / w) of a lubricant, preferably magnesium stearate; and

[0332] - microcrystalline cellulose ad 100 % (w / w).

[0333] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0334] - 0.2 to 5 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder;

[0335] - 50 to 70 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0336] - 0.2 to 1 % (w / w) of a gelling agent, preferably HEC;

[0337] - 0.2 to 1 % (w / w) of a carbomer, preferably a carbomer homopolymer type A and / or B;

[0338] - 2.5 to 7.5 % (w / w) of a salt, preferably sodium chloride;

[0339] - 0.5 to 5 % (w / w) of a glidant, preferably talc or silica;

[0340] - 0.1 to 2.5 % (w / w) of a lubricant, preferably magnesium stearate; and

[0341] - microcrystalline cellulose ad 100 % (w / w).

[0342] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0343] - 0.2 to 5 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder;

[0344] - 30 to 70 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0345] - 1 to 10 % (w / w) of a gelling agent, preferably HPMC;

[0346] - 0.1 to 2 % (w / w) of a carbomer, preferably a carbomer homopolymer type A and / or B; and

[0347] - 1 to 12.5 % (w / w) of a salt, preferably sodium chloride;

[0348] - 0.5 to 5 % (w / w) of a glidant, preferably talc or silica;

[0349] - 0.1 to 2.5 % (w / w) of a lubricant, preferably magnesium stearate; and

[0350] - microcrystalline cellulose ad 100 % (w / w).

[0351] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about: - 0.2 to 5 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder;

[0352] - 50 to 70 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0353] - 2.5 to 7.5 % (w / w) of a gelling agent, preferably HPMC;

[0354] - 0.2 to 1 % (w / w) of a carbomer, preferably a carbomer homopolymer type A and / or B; and

[0355] - 2.5 to 7.5 % (w / w) of a salt, preferably sodium chloride;

[0356] - 0.5 to 5 % (w / w) of a glidant, preferably talc or silica;

[0357] - 0.1 to 2.5 % (w / w) of a lubricant, preferably magnesium stearate; and

[0358] - microcrystalline cellulose ad 100 % (w / w).

[0359] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0360] - about 0.2 to 2 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder;

[0361] - about 30 to 55 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0362] - about 0.5 % (w / w) of a carbomer, preferably a carbomer homopolymer type A and / or B;

[0363] - about 5 % (w / w) of a salt, preferably sodium chloride;

[0364] - about 0.5 % (w / w) of a gelling agent, preferably hydroxyethyl cellulose;

[0365] - about 5 % (w / w) of a glidant, preferably talc;

[0366] - about 1 % (w / w) of a lubricant, preferably magnesium stearate; and

[0367] - microcrystalline cellulose ad 100 % (w / w).

[0368] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0369] - about 2 % (w / w) of a spray dried powder comprising the endolysin, wherein the spray dried powder comprises about 10% (w / w) of the endolysin;

[0370] - about 51 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, in particular wherein the sugar alcohol is mannitol;

[0371] - about 0.5 % (w / w) of a carbomer, in particular a carbomer homopolymer type A and / or B;

[0372] - about 5 % (w / w) of sodium chloride;

[0373] - about 0.5 % (w / w) of hydroxyethyl cellulose;

[0374] - about 5 % (w / w) of talc;

[0375] - about 1 % (w / w) of magnesium stearate; and

[0376] - microcrystalline cellulose ad 100 % (w / w).

[0377] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0378] - about 5 % (w / w) of a spray dried powder comprising the endolysin, wherein the spray dried powder comprises about 10% (w / w) of the endolysin;

[0379] - about 48 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, in particular wherein the sugar alcohol is mannitol;

[0380] - about 0.5 % (w / w) of a carbomer, in particular a carbomer homopolymer type A and / or B; - about 5 % (w / w) of sodium chloride;

[0381] - about 0.5 % (w / w) of hydroxyethyl cellulose;

[0382] - about 5 % (w / w) of talc;

[0383] - about 1 % (w / w) of magnesium stearate; and

[0384] - microcrystalline cellulose ad 100 % (w / w).

[0385] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0386] - about 20 % (w / w) of a spray dried powder comprising the endolysin, wherein the spray dried powder comprises about 10% (w / w) of the endolysin;

[0387] - about 33 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, in particular wherein the sugar alcohol is mannitol;

[0388] - about 0.5 % (w / w) of a carbomer, in particular a carbomer homopolymer type A and / or B;

[0389] - about 5 % (w / w) of sodium chloride;

[0390] - about 0.5 % (w / w) of hydroxyethyl cellulose;

[0391] - about 5 % (w / w) of talc;

[0392] - about 1 % (w / w) of magnesium stearate; and

[0393] - microcrystalline cellulose ad 100 % (w / w).

[0394] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0395] - 0.2 to 2 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder;

[0396] - 50 to 70 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0397] - 0.2 to 1 % (w / w) of a carbomer, preferably a carbomer homopolymer type A and / or B;

[0398] - 1 to 12.5% (w / w) of a salt, preferably sodium chloride;

[0399] - 0 to 5 % (w / w) of a gelling agent, preferably hydroxyethyl cellulose;

[0400] - 0 to 2 % (w / w) of a glidant;

[0401] - 1 to 2 % (w / w) of a lubricant, preferably magnesium stearate; and

[0402] - microcrystalline cellulose ad 100 % (w / w).

[0403] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0404] - 0.2 to 2 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder;

[0405] - 30 to 55 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0406] - 0.2 to 1 % (w / w) of a carbomer, preferably a carbomer homopolymer type A and / or B;

[0407] - 1 to 12.5% (w / w) of a salt, preferably sodium chloride;

[0408] - 0 to 5 % (w / w) of a gelling agent, preferably hydroxyethyl cellulose;

[0409] - 0 to 2 % (w / w) of a glidant;

[0410] - 1 to 2 % (w / w) of a lubricant, preferably magnesium stearate; and

[0411] - microcrystalline cellulose ad 100 % (w / w). In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0412] - 0.2 to 2 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder;

[0413] - 50 to 70 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0414] - about 0.5 % (w / w) of a carbomer, preferably a carbomer homopolymer type A and / or B;

[0415] - about 5 % (w / w) of a salt, preferably sodium chloride;

[0416] - 0 to 5 % (w / w) of a gelling agent, preferably hydroxyethyl cellulose;

[0417] - 0 to 2 % (w / w) of a glidant;

[0418] - 1 to 2 % (w / w) of a lubricant, preferably magnesium stearate; and

[0419] - microcrystalline cellulose ad 100 % (w / w).

[0420] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0421] - 0.2 to 2 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder;

[0422] - 30 to 55 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0423] - about 0.5 % (w / w) of a carbomer, preferably a carbomer homopolymer type A and / or B;

[0424] - about 5 % (w / w) of a salt, preferably sodium chloride;

[0425] - 0 to 5 % (w / w) of a gelling agent, preferably hydroxyethyl cellulose;

[0426] - 0 to 2 % (w / w) of a glidant;

[0427] - 1 to 2 % (w / w) of a lubricant, preferably magnesium stearate; and

[0428] - microcrystalline cellulose ad 100 % (w / w).

[0429] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0430] - 0.2 to 2 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder;

[0431] - 50 to 70 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0432] - about 0.5 % (w / w) of a carbomer, preferably a carbomer homopolymer type A and / or B;

[0433] - about 5 % (w / w) of a salt, preferably sodium chloride;

[0434] - about 0.5 % (w / w) of a gelling agent, preferably hydroxyethyl cellulose;

[0435] - 0 to 2 % (w / w) of a glidant;

[0436] - 1 to 2 % (w / w) of a lubricant, preferably magnesium stearate; and

[0437] - microcrystalline cellulose ad 100 % (w / w).

[0438] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0439] - 0.2 to 2 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder;

[0440] - 30 to 55 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0441] - about 0.5 % (w / w) of a carbomer, preferably a carbomer homopolymer type A and / or B;

[0442] - about 5 % (w / w) of a salt, preferably sodium chloride; - about 0.5 % (w / w) of a gelling agent, preferably hydroxyethyl cellulose;

[0443] - 0 to 2 % (w / w) of a glidant;

[0444] - 1 to 2 % (w / w) of a lubricant, preferably magnesium stearate; and

[0445] - microcrystalline cellulose ad 100 % (w / w).

[0446] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0447] - 0.1 to 10 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder, preferably wherein the spray dried powder comprises between about 20 and about 30% (w / w) of the endolysin, more preferably about 22.5% (w / w) of the endolysin;

[0448] - 30 to 70 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0449] - 0 to 10 % (w / w) of a gelling agent, preferably HEC or HPMC;

[0450] - 0 to 5 % (w / w) of a carbomer, preferably a carbomer homopolymer type A and / or B;

[0451] - 0 to 12.5 % (w / w) of a salt, preferably sodium chloride;

[0452] - 0 to 5 % (w / w) of a lubricant, preferably magnesium stearate; and

[0453] - microcrystalline cellulose ad 100 % (w / w).

[0454] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0455] - 0.1 to 10 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder, preferably wherein the spray dried powder comprises between about 20 and about 30% (w / w) of the endolysin, more preferably about 22.5% (w / w) of the endolysin;

[0456] - 30 to 70 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0457] - 0.1 to 10 % (w / w) of a gelling agent, preferably HEC or HPMC;

[0458] - 0 to 5 % (w / w) of a carbomer, preferably a carbomer homopolymer type A and / or B;

[0459] - 0 to 12.5 % (w / w) of a salt, preferably sodium chloride;

[0460] - 0 to 5 % (w / w) of a lubricant, preferably magnesium stearate; and

[0461] - microcrystalline cellulose ad 100 % (w / w).

[0462] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0463] - 0.1 to 10 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder, preferably wherein the spray dried powder comprises between about 20 and about 30% (w / w) of the endolysin, more preferably about 22.5% (w / w) of the endolysin;

[0464] - 30 to 70 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0465] - 0.1 to 10 % (w / w) of a gelling agent, preferably HEC or HPMC;

[0466] - 0.1 to 5 % (w / w) of a carbomer, preferably a carbomer homopolymer type A and / or B;

[0467] - 0 to 12.5 % (w / w) of a salt, preferably sodium chloride;

[0468] - 0 to 5 % (w / w) of a lubricant, preferably magnesium stearate; and

[0469] - microcrystalline cellulose ad 100 % (w / w). In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0470] - 0.1 to 10 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder, preferably wherein the spray dried powder comprises between about 20 and about 30% (w / w) of the endolysin, more preferably about 22.5% (w / w) of the endolysin;

[0471] - 30 to 70 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0472] - 0.1 to 10 % (w / w) of a gelling agent, preferably HEC or HPMC;

[0473] - 0.1 to 5 % (w / w) of a carbomer, preferably a carbomer homopolymer type A and / or B;

[0474] - 0.1 to 12.5 % (w / w) of a salt, preferably sodium chloride;

[0475] - 0.1 to 5 % (w / w) of a lubricant, preferably magnesium stearate; and

[0476] - microcrystalline cellulose ad 100 % (w / w).

[0477] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0478] - 0.2 to 5 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder, preferably wherein the spray dried powder comprises between about 20 and about 30% (w / w) of the endolysin, more preferably about 22.5% (w / w) of the endolysin;

[0479] - 30 to 70 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0480] - 0.1 to 5 % (w / w) of a gelling agent, preferably HEC;

[0481] - 0.1 to 2 % (w / w) of a carbomer, preferably a carbomer homopolymer type A and / or B;

[0482] - 1 to 12.5 % (w / w) of a salt, preferably sodium chloride;

[0483] - 0.1 to 2.5 % (w / w) of a lubricant, preferably magnesium stearate; and

[0484] - microcrystalline cellulose ad 100 % (w / w).

[0485] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0486] - 0.2 to 5 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder, preferably wherein the spray dried powder comprises between about 20 and about 30% (w / w) of the endolysin, more preferably about 22.5% (w / w) of the endolysin;

[0487] - 40 to 60 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0488] - 0.2 to 1 % (w / w) of a gelling agent, preferably HEC;

[0489] - 0.2 to 1 % (w / w) of a carbomer, preferably a carbomer homopolymer type A and / or B;

[0490] - 2.5 to 7.5 % (w / w) of a salt, preferably sodium chloride;

[0491] - 0.1 to 2.5 % (w / w) of a lubricant, preferably magnesium stearate; and

[0492] - microcrystalline cellulose ad 100 % (w / w).

[0493] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about: - 0.2 to 5 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder, preferably wherein the spray dried powder comprises between about 20 and about 30% (w / w) of the endolysin, more preferably about 22.5% (w / w) of the endolysin;

[0494] - 30 to 70 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0495] - 1 to 10 % (w / w) of a gelling agent, preferably HPMC;

[0496] - 0.1 to 2 % (w / w) of a carbomer, preferably a carbomer homopolymer type A and / or B; and

[0497] - 1 to 12.5 % (w / w) of a salt, preferably sodium chloride;

[0498] - 0.1 to 2.5 % (w / w) of a lubricant, preferably magnesium stearate; and

[0499] - microcrystalline cellulose ad 100 % (w / w).

[0500] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0501] - 0.2 to 5 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder, preferably wherein the spray dried powder comprises between about 20 and about 30% (w / w) of the endolysin, more preferably about 22.5% (w / w) of the endolysin;

[0502] - 40 to 60 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0503] - 2.5 to 7.5 % (w / w) of a gelling agent, preferably HPMC;

[0504] - 0.2 to 1 % (w / w) of a carbomer, preferably a carbomer homopolymer type A and / or B; and

[0505] - 2.5 to 7.5 % (w / w) of a salt, preferably sodium chloride;

[0506] - 0.1 to 2.5 % (w / w) of a lubricant, preferably magnesium stearate; and

[0507] - microcrystalline cellulose ad 100 % (w / w).

[0508] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0509] - about 0.2 to 2 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder, preferably wherein the spray dried powder comprises between about 20 and about 30% (w / w) of the endolysin, more preferably about 22.5% (w / w) of the endolysin;

[0510] - about 40 to 60 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0511] - about 0.5 % (w / w) of a carbomer, preferably a carbomer homopolymer type A and / or B;

[0512] - about 5 % (w / w) of a salt, preferably sodium chloride;

[0513] - about 0.5 % (w / w) of a gelling agent, preferably hydroxyethyl cellulose;

[0514] - about 1 % (w / w) of a lubricant, preferably magnesium stearate; and

[0515] - microcrystalline cellulose ad 100 % (w / w).

[0516] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0517] - 1 to 10 % (w / w) of a spray dried powder comprising the endolysin, wherein the spray dried powder comprises between about 20 and about 30% (w / w) of the endolysin, preferably about 22.5% (w / w) of the endolysin;

[0518] - 40 to 60 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof; - 0.1 to 5 % (w / w) of a gelling agent, preferably HEC;

[0519] - 0.1 to 2 % (w / w) of a carbomer, preferably a carbomer homopolymer type A and / or B;

[0520] - 1 to 12.5 % (w / w) of a salt, preferably sodium chloride;

[0521] - 0.1 to 2.5 % (w / w) of a lubricant, preferably magnesium stearate; and

[0522] - microcrystalline cellulose ad 100 % (w / w).

[0523] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0524] - 1 to 10 % (w / w) of a spray dried powder comprising the endolysin, wherein the spray dried powder comprises about 20% to about 30% (w / w) of the endolysin, preferably about 22.5% (w / w) of the endolysin;

[0525] - 40 to 60 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;

[0526] - 0.2 to 1 % (w / w) of a gelling agent, preferably HEC;

[0527] - 0.2 to 1 % (w / w) of a carbomer, preferably a carbomer homopolymer type A and / or B;

[0528] - 2.5 to 7.5 % (w / w) of a salt, preferably sodium chloride;

[0529] - 0.1 to 2.5 % (w / w) of a lubricant, preferably magnesium stearate; and

[0530] - microcrystalline cellulose ad 100 % (w / w).

[0531] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0532] - about 2.22 % (w / w) of a spray dried powder comprising the endolysin, wherein the spray dried powder comprises about 22.5% (w / w) of the endolysin;

[0533] - about 53 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, in particular wherein the sugar alcohol is mannitol;

[0534] - about 0.5 % (w / w) of a carbomer, in particular a carbomer homopolymer type A and / or B;

[0535] - about 5 % (w / w) of sodium chloride;

[0536] - about 0.5 % (w / w) of hydroxyethyl cellulose;

[0537] - about 1 % (w / w) of magnesium stearate; and

[0538] - microcrystalline cellulose ad 100 % (w / w).

[0539] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the composition comprises about:

[0540] - about 8.9 % (w / w) of a spray dried powder comprising the endolysin, wherein the spray dried powder comprises about 22.5% (w / w) of the endolysin;

[0541] - about 49 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, in particular wherein the sugar alcohol is mannitol;

[0542] - about 0.5 % (w / w) of a carbomer, in particular a carbomer homopolymer type A and / or B;

[0543] - about 5 % (w / w) of sodium chloride;

[0544] - about 0.5 % (w / w) of hydroxyethyl cellulose;

[0545] - about 1 % (w / w) of magnesium stearate; and

[0546] - microcrystalline cellulose ad 100 % (w / w). The endolysin is preferably comprised in the pharmaceutical composition according to the invention in the form of a spray dried powder. In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the spray dried powder comprises about 5 to 30 % (w / w) of the endolysin.

[0547] In certain embodiments, the spray dried powder comprised in the pharmaceutical composition according to the invention comprises about 5 to about 30% (w / w) of the endolysin, preferably about 5% to about 20% (w / w) of the endolysin, more preferably about 10% (w / w) of the endolysins.

[0548] In certain embodiments, the spray dried powder comprised in the pharmaceutical composition according to the invention comprises about 5 to about 30% (w / w) of the endolysin, preferably about 10% to about 30% (w / w) of the endolysin, more preferably about 20% (w / w) to about 30% (w / w) of the endolysin, more preferably about 22.5% (w / w) of the endolysin.

[0549] In certain embodiments, the spray dried powder comprised in the pharmaceutical composition according to the invention comprises about 5% (w / w), about 6% (w / w), about 7% (w / w), about 8% (w / w), about 9% (w / w), about 10% (w / w), about 11% (w / w), about 12% (w / w), about 13% (w / w), about 14% (w / w), about 15% (w / w), about

[0550] 16% (w / w), about 17% (w / w), about 18% (w / w), about 19% (w / w), about 20% (w / w), about 21% (w / w), about

[0551] 22% (w / w), about 23% (w / w), about 24% (w / w), about 25% (w / w), about 26% (w / w), about 27% (w / w), about

[0552] 28% (w / w), about 29% (w / w), or about 30% (w / w) of the endolysin.

[0553] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the spray dried powder further comprises 5 to 20 % (w / w) of a stabilizing agent, in particular wherein the stabilizing agent is hydroxypropyl p-cyclodextrin.

[0554] In certain embodiments, the spray dried powder comprised in the pharmaceutical composition according to the invention comprises about 5% to about 20% (w / w) of the stabilizing agent, preferably about 10% to about 20% (w / w) of the stabilizing agent, more preferably about 10% to about 17.5% (w / w) of the stabilizing agent, even more preferably about 15% (w / w) of the stabilizing agent, preferably wherein the stabilizing agent is hydroxypropyl p-cyclodextrin.

[0555] In certain embodiments, the spray dried powder comprised in the pharmaceutical composition according to the invention comprises about 5% (w / w), about 6% (w / w), about 7% (w / w), about 8% (w / w), about 9% (w / w), about 10% (w / w), about 11% (w / w), about 12% (w / w), about 13% (w / w), about 14% (w / w), about 15% (w / w), about 16% (w / w), about 17% (w / w), about 18% (w / w), about 19% (w / w), or about 20% (w / w) of the stabilizing agent, preferably wherein the stabilizing agent is hydroxypropyl p-cyclodextrin.

[0556] In certain embodiments where the concentration of the endolysin in the spray-died powder is particularly high, i.e., above 10% (w / w) or between about 20% and 30% (w / w), higher concentrations of the stabilizing agent may be utilized. The skilled person is aware that the concentration of the endolysin in the spray-dried powder depends on the concentration of the endolysin in the protein solution before spray drying. For example, to obtain spray-dried powders comprising between about 20% and 30% (w / w) of the endolysin, a solution comprising about 6 mg / mL of the endolysin may be subjected to spray-drying. Increasing the concentration of the endolysin in the spray-dried powder will reduce the spray-dried powder volume needed for tableting. Without being bound to theory, this may improve powder flow and thus reduce the need for glidants, such as talc.

[0557] In such embodiments, the invention relates to the pharmaceutical composition according to the invention, wherein the spray dried powder further comprises 20 to 50 % (w / w) of a stabilizing agent, in particular wherein the stabilizing agent is hydroxypropyl 0-cyclodextrin.

[0558] In certain embodiments, the spray dried powder comprised in the pharmaceutical composition according to the invention comprises about 20% to about 50% (w / w) of the stabilizing agent, preferably about 20% to about 40% (w / w) of the stabilizing agent, more preferably about 25% to about 35% (w / w) of the stabilizing agent, even more preferably about 32% (w / w) of the stabilizing agent, preferably wherein the stabilizing agent is hydroxypropyl 0- cyclodextrin.

[0559] In certain embodiments, the spray dried powder comprised in the pharmaceutical composition according to the invention comprises about 20% (w / w), about 21% (w / w), about 22% (w / w), about 23% (w / w), about 24% (w / w), about 25% (w / w), about 26% (w / w), about 27% (w / w), about 28% (w / w), about 29% (w / w), about 30% (w / w), about 31% (w / w), about 32% (w / w), about 33% (w / w), about 34% (w / w), or about 35% (w / w) of the stabilizing agent, preferably wherein the stabilizing agent is hydroxypropyl 0-cyclodextrin.

[0560] In certain embodiments, the weight ratio between the stabilizing agent, in particular the hydroxypropyl 0- cyclodextrin, and the endolysin (HPBCD / endolysin ratio) in the spray-dried powder is between 0.5 and 2, in particular between 1 and 2, in particular between 1.2 and 1.6. In certain embodiments, the HPBCD / endolysin ratio in the spray-dried powder is about 1.4. In certain embodiments, the HPBCD / endolysin ratio in the spray-dried powder is about 0.7.

[0561] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein the spray dried powder further comprises a carrier, in particular wherein the carrier is mannitol.

[0562] In a preferred embodiment, the spray dried powder comprised in the pharmaceutical composition comprises the following ingredients:

[0563] - 5 to 30% (w / w) of the endolysin;

[0564] - 5 to 20% (w / w) of hydroxypropyl 0-cyclodextrin;

[0565] - 50 to 90% (w / w) of D-mannitol; and

[0566] - <1% (w / w) of Sodium Acetate trihydrate.

[0567] In a particularly preferred embodiment, the spray dried powder comprised in the pharmaceutical composition comprises the following ingredients:

[0568] - about 10% (w / w) of the endolysin;

[0569] - about 15% (w / w) of hydroxypropyl 0-cyclodextrin;

[0570] - about 75% (w / w) of D-mannitol; and

[0571] - <1% (w / w) of sodium acetate trihydrate. In another preferred embodiment, the spray dried powder comprised in the pharmaceutical composition comprises the following ingredients:

[0572] - 10 to 30% (w / w) of the endolysin;

[0573] - 20 to 50% (w / w) of hydroxypropyl 0-cyclodextrin;

[0574] - 30 to 60% (w / w) of D-mannitol; and

[0575] - <1% (w / w) of sodium acetate trihydrate.

[0576] In a more preferred embodiment, the spray dried powder comprised in the pharmaceutical composition comprises the following ingredients:

[0577] - 18 to 28% (w / w) of the endolysin;

[0578] - 27 to 37% (w / w) of hydroxypropyl 0-cyclodextrin;

[0579] - 40 to 50% (w / w) of D-mannitol; and

[0580] - <1% (w / w) of sodium acetate trihydrate.

[0581] In a particularly preferred embodiment, the spray dried powder comprised in the pharmaceutical composition comprises the following ingredients:

[0582] - about 22.5% (w / w) of the endolysin;

[0583] - about 32% (w / w) of hydroxypropyl 0-cyclodextrin;

[0584] - about 45% (w / w) of D-mannitol; and

[0585] - <1% (w / w) of sodium acetate trihydrate.

[0586] As mentioned herein above, the pharmaceutical composition according to the invention is preferably formulated as a solid, more specifically a tablet. The tablet preferably has a weight, size and / or shape that allows for vaginal insertion. Thus, in a particular embodiment, the invention relates to the pharmaceutical composition according to the invention, wherein said composition is suitable for vaginal administration.

[0587] In certain embodiments, the tablet has a weight ranging from about 10 mg to about 2 g, from about 20 mg to about 1.5 g, or from about 40 mg to about 1 g.

[0588] In certain embodiments, the tablet has a weight of about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 115 mg, about 150 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1500 mg, or about 2000 mg.

[0589] In certain embodiments, the tablet has a weight of 1000 mg and comprises between 2 and 20 mg of the endolysin. In a particular embodiment, the tablet has a weight of 1000 mg and comprises about 2 mg of the endolysin (0.2% (w / w)). In a particular embodiment, the tablet has a weight of 1000 mg and comprises about 5 mg of the endolysin (0.5% (w / w)). In a particular embodiment, the tablet has a weight of 1000 mg and comprises about 10 mg of the endolysin (1% (w / w)). In a particular embodiment, the tablet has a weight of 1000 mg and comprises about 20 mg of the endolysin (2% (w / w)).

[0590] In certain embodiments, the vaginal tablet may exhibit a tensile strength ranging from about 0.5 MPa to about 3 MPa, preferably from about 1 MPa to about 2 MPa. In certain embodiments, the tablet exhibits a tensile strength of about 1.7 MPa. In certain embodiments, the tablet exhibits a tensile strength of about 1.0 MPa. Without being bound by theory, softer tablets exhibiting a tensile strength of around 1.0 MPa may facilitate a faster release of the endolysin.

[0591] In certain embodiments, the hardness of the tablet may be in the range of about 100 to about 300 N, more preferably of about 100 to about 200 N, even more preferably of about 100 to about 150 N.

[0592] In certain embodiments, the tablet may have a tensile strength of about 1.0 MPa and / or a hardness of about 100 to about 150 N.

[0593] In certain embodiments, the tablet may have a tensile strength of about 1.7 MPa and / or a hardness of about 100 to about 300 N.

[0594] The skilled person is aware of methods for determining the hardness and / or tensile strength of a tablet. For example, hardness and tensile strength of a tablet may be analyzed using a Sotax ST50 hardness tester (Sotax, Allschwil, Switzerland). For that, a constant loading speed of 0.35 mm / sec may be applied. The tensile strength (TS) may be calculated based on tablet hardness, thickness, wall height and width, as shown in the following equation (Pitt et al., Powder Technology. Volume 238, April 2013, Pages 169-175): where otis the TS (MPa), P is the fracture load or hardness (N), D is the length of the short axis or width (mm), t is the overall thickness (mm), and W is the wall height of the tablet (mm).

[0595] In certain embodiments, the tablet may have a thickness of 5 to 10 mm, preferably of 5 to 7 mm.

[0596] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention for use in the treatment of bacterial infections in a subject.

[0597] That is, the pharmaceutical composition of the invention comprising the Gardnerella-specific endolysin can be used in the treatment of bacterial infections in a subject, in particular bacterial infections that are caused by bacteria of the genus Gardnerella. Accordingly, the pharmaceutical composition of the invention may be used for the treatment of infections caused, without limitation, by one or more of G. vaginalis sensu stricto, G. ieopo / dii, G. piotii, and / or G. swidsinskii.

[0598] In a particular embodiment, the invention relates to the pharmaceutical composition for use according to the invention, wherein the bacterial infection is bacterial vaginosis.

[0599] Gardnerella species, such as G. vaginalis is the most common cause of bacterial vaginosis. As used herein "Bacterial Vaginosis" (BV), also been referred to in the literature as bacterial vaginitis, non-specific vaginosis and non-specific vaginitis refers to the most common vaginal infection worldwide. In one embodiment, BV is defined as a pathological state characterized by the loss of normal vagina flora, particularly of ^Ch-producing species of Lactobacillus, and the simultaneous overgrowth of anaerobic bacteria, often including those from the genus Gardnerella. The genus Gardnerella contains at least 13 species, and the most frequent ones were renamed G. vaginalis sensu stricto, G. leopoldii, G. piotii, and G. sw / cfe / ns 7 / (Vaneechoutte et al., 2019 Int. J. Syst. Evol. Biol. 898661). In one preferred embodiment of the invention, the BV to be treated is a bacterial infection characterized by the presence of at least one strain of the Gardnerella genus selected from the group consisting of Gardnerella vaginalis sensu strict, Gardnerella leopoldii, Gardnerella piotii and Gardnerella swidsinskii, and any other Gardnerella species. Thus, in one embodiment, the pharmaceutical composition according to the invention comprises a Gardnerella-specific endolysin, preferably a recombinant Gardnerella-specific. endolysin, as described herein, for use in treating a bacterial vaginosis, wherein said bacterial vaginosis is characterized by the presence of infective bacteria of species Gardnerella vaginalis sensu stricto, Gardnerella leopoldii, Gardnerella piotii, Gardnerella swidsinskii, and / or any other species in the genus Gardnerella. It is understood herein that a bacterial vaginosis which "is characterized by the presence of" (or "is caused by" as used herein interchangeably) certain bacterial species in the genus Gardnerella refers to the overgrowth of said bacteria (also referred herein as "infective bacteria") in the vagina microflora of a patient, leading to vaginal dysbiosis and / or the loss of Lactobacillus dominance. Methods to determine whether a bacterial vaginosis is characterized by the presence of infective bacteria of bacterial species in the genus Gardnerella are known to the skilled person. As an example, a PCR test that checks for presence of Gardnerella strains to diagnose BV can be used.

[0600] Further tests may be performed to diagnose a female subject with bacterial vaginosis. For example, the Nugent score may be used to diagnose a female subject with bacterial vaginosis, as described in J Clin Microbiol. 1991 Feb;29(2):297-301, which is fully incorporated herein by reference. To determine the Nugent score, vaginal smears are Gram-stained and scored by the morphology of the observed bacteria (by quantifying gram-positive rods [Lactobacillus , gram-negative rods [such as Gardnerella, Prevotella and Bacteroides], and gram-labile rods [MobHuncus]'). K Nugent score of 7-10 is indicative of BV, while a score of 4 to 6 is considered intermediate vaginal microbiota and a score of 0 to 3 is considered normal vaginal microbiota.

[0601] Accordingly, in certain embodiments, a female subject may be diagnosed with bacterial vaginosis if the subject has a Nugent score >4, preferably of >7.

[0602] Alternatively or in addition, Amsel's criteria may be used to diagnose a female subject with bacterial vaginosis; see Am J Med, 1983 Jan;74(l):14-22, which is fully incorporated by reference herein. Per the Amsel's criteria, BV is defined as the presence of at least three of the following four criteria:

[0603] • Homogeneous, thin, grayish-white discharge that smoothly coats the vaginal walls.

[0604] • Vaginal pH >4.5.

[0605] • Positive Whiff-amine test, defined as the presence of a fishy odor when 10% KOH is added to a sample of vaginal discharge.

[0606] • Clue cells (i.e., vaginal epithelial cells covered with bacteria) on saline wet mount, comprising at least 20% of epithelial cells per high power field.

[0607] Accordingly, in certain embodiments, a female subject is diagnosed with bacterial vaginoses if at least three, preferably all, of the following criteria are met:

[0608] • homogeneous, thin, grayish-white discharge that smoothly coats the vaginal walls; • vaginal pH >4.5;

[0609] • positive Whiff-amine test, defined as the presence of a fishy odor when 10% KOH is added to a sample of vaginal discharge; and / or

[0610] • clue cells (i.e., vaginal epithelial cells covered with bacteria) on saline wet mount, comprising at least 20% of epithelial cells per high power field.

[0611] The skilled person, such as a practitioner in the field of gynecology is familiar with the above tests and capable of diagnosing a female patient with bacterial vaginosis.

[0612] Accordingly, in a particular embodiment, the invention relates to the pharmaceutical composition for use according to the invention, wherein the bacterial infection or bacterial vaginosis is characterized by the presence of one or more Gardnerella species.

[0613] In a particular embodiment, the invention relates to the pharmaceutical composition according to the invention for use in treating bacterial vaginosis, wherein the endolysin is to be administered to a patient who previously failed a treatment with antibiotics and / or who suffers from bacterial vaginosis wherein the infective bacteria are resistant to a treatment with antibiotics.

[0614] That is, in certain embodiments, the BV to be treated with the pharmaceutical composition described herein is characterized by the presence of a Gardnerella strain i.e., a strain from the genus Gardnerella) which is resistant to one or more antibiotics. In one preferred embodiment, said one or more antibiotic(s) is / are selected from the group consisting of the nitroimidazoles and Clindamycin. In one even more preferred embodiment, said Gardnerella strain is resistant to Metronidazole, Tinidazole, Secnidazole, Clindamycin or any combination thereof. In one even more preferred embodiment, said Gardnerella strain is resistant to Metronidazole and / or Clindamycin. In one even more preferred embodiment, the said Gardnerella strain is resistant to Metronidazole. In one most preferred embodiment, said Gardnerella strain is resistant to / yg / yresistant to Metronidazole.

[0615] As used herein, "a patient who previously failed a treatment with antibiotics" refers to a patient who has a history of bacterial vaginosis, i.e., who contracted (or already experienced) symptoms of bacterial vaginosis in the past, who has been treated for said bacterial vaginosis by antibiotics and who relapsed, i.e., for whom the symptoms reappeared. Such a patient can also be referred to as a patient suffering from recurrent BV. As used herein, the terms "a patient who has history with bacterial vaginosis", "a patient who already experienced symptoms of bacterial vaginosis" and "a patient suffering from recurrent BV" can be used interchangeably and include, but are not limited to, patients who had one or more episodes of BV, preferably two or more episodes of BV, more preferably two or more episodes of BV in 6 months, even more preferably two or more episodes of BV within the last 6 months, or preferably three or more episodes of BV, more preferably three or more episodes of BV in 12 months, even more preferably three or more episodes of BV within the last 12 months.

[0616] Methods to diagnose BV in a patient or to prove episodes of BV are known to the skilled person in the art, e.g., BV can be diagnosed clinically by using clinical criteria (such as, e.g., the Amsel's diagnostic criteria) or microscopically, by determining the Nugent score from a vaginal Gram stain. Without being bound by any theory, relapses (or recurrences) of BV in patients after a treatment with antibiotics might be caused by the persistence of a residual infection due to the resistance of the infective bacteria to the antibiotics used. Accordingly, in one preferred embodiment of the invention, the patient suffers from a bacterial vaginosis wherein the infective bacteria are resistant to antibiotics treatment. Thus, in one preferred aspect of the invention, the Garo ere / Zj-specific pharmaceutical composition described herein is for use in treating a bacterial vaginosis, wherein the pharmaceutical composition is to be administered to a patient who previously failed a treatment with antibiotics and who suffers from a bacterial vaginosis wherein the infective bacteria are resistant to antibiotics treatment. As defined herein below, the infective bacteria of the BV to be treated herein are more preferably highly resistant to antibiotics treatment. In one preferred embodiment of the therapeutic uses of the invention, the patient to be treated suffers from BV wherein the infective bacteria of said BV are resistant, preferably highly resistant, to a treatment with Metronidazole, Tinidazole, Secnidazole, Clindamycin or any combination thereof. In one more preferred embodiment of the therapeutic uses of the invention, the patient to be treated suffers from BV wherein the infective bacteria of said BV are resistant, preferably highly resistant, to a treatment with Metronidazole and / or Clindamycin. In one even more preferred embodiment of the therapeutic uses of the invention, the patient to be treated suffers from BV wherein the infective bacteria of said BV are resistant, preferably highly resistant, to a treatment with Metronidazole.

[0617] In the context of the present invention, "resistance" of bacterial strains (preferably Gardnerella strains) with regards to antibiotics refers to the ability of the strains to resist the activity of the antibiotic to which it was previously susceptible and permits them to survive the antibiotics treatment. Antibiotic resistance might occur naturally ("intrinsic resistance") or might be induced by the misuse of antibiotics in humans and animals ('acquired resistance"). Methods how to determine resistance or susceptibility of bacterial strains to antibiotics are known to the skilled person. As an example, EUCAST breakpoints for Gram-positive anaerobes (vll, 2021) can be used. An alternative definition is given in Petrina et al. (2017, Anaerobe 47, 115-119), where slightly higher resistance breakpoints are used, because the topical formulations of some nitroimidazoles and Clindamycin can establish concentrations in the mg / ml range in vaginal fluid, much higher than what is achievable with orally delivered antibiotics. According to this alternative, more stringent definition (also used in the appended examples), Resistance (R) can be defined as a MIC value superior or equal to 32 pg / ml for Metronidazole and as a MIC value superior 8 pg / ml for Clindamycin, whereas Sensitivity (S) can be defined as a MIC value inferior or equal to 8 g / ml for Metronidazole and inferior or equal to 2pg / ml for Clindamycin. The terms "Minimum Inhibitory Concentration" and "MIC" are used herein interchangeably and refer to the lowest concentration of a chemical, usually a drug, which prevents visible growth of bacterium. MIC can be defined as the minimal concentration of antibiotic at which no growth was detectable after 48h by OD measurement. Accordingly, in one preferred embodiment of the therapeutic uses of the invention, the patient to be treated suffers from BV wherein the infective bacteria are resistant to antibiotics treatment as defined by EUCAST breakpoints. Other generally accepted resistance criteria might be used instead of and / or besides the EUCAST definition or the alternative definition mentioned above (Petrina et al., 2017, Anaerobe 47, 115-119) to define the resistance of the infective bacteria to antibiotics treatment in the context of the present invention.

[0618] As will be known to the skilled person in the art, "resistance" and "susceptibility" might also be defined in terms of MBC and / or MBEC values. The terms "Minimum Bactericidal Concentration" or "MBC" refer to the lowest concentration of an antibacterial agent required to kill a particular bacterium. Usually, the MBC90 or MBC99.5 is measured, i.e., the antibiotic concentration killing 90% or 99.5%, respectively, of cells within a defined time. MBC can be defined as the minimal concentration fully eradicating a suspension of, e.g,, 2.5xl07CFU / ml. While MIC is the lowest concentration of an antibacterial agent necessary to inhibit visible growth, MBC is the minimum concentration of an antibacterial agent that results in bacterial death of all cells in suspension up to a defined limit of detection, resulting in killing of at least 90% (MBC90) or at least 99.5% (MBC99.5) of bacteria. The terms "Minimum Biofilm Eradicating Concentrations" or "MBEC" refer to the lowest concentration of an antibacterial agent required to reduce a population of bacteria growing as biofilm below the limit of detection.

[0619] Without being bound by any theory, the resistance of the infective bacteria to antibiotics might be involved in (or being partially or substantially responsible of) the relapses (or recurrences) of BV in patients suffering from recurrent BV. Therefore, a patient suffering from bacterial vaginosis wherein the infective bacteria are resistant to a treatment with antibiotics is likely to fail a treatment with antibiotics. Accordingly, in one preferred embodiment of the invention, the patient to be treated and who suffers from bacterial vaginosis wherein the infective bacteria are resistant to a treatment with antibiotics is a patient who is prone to fail a treatment with antibiotics. As used herein, a patient "who is prone to fail a treatment with antibiotics" refers to a patient who is at high risk of failing a treatment with antibiotics, i.e., for whom the likelihood of relapse ( .g., within 12 months) is very high if said patient were to be treated with antibiotics. Methods to determine whether a patient will be prone to fail a treatment with antibiotics are known to the skilled person. As an example, a clinical sample can be collected, and the resistance of the vaginal microflora strains assessed. Likewise, a patient suffering from bacterial vaginosis wherein the infective bacteria are resistant to a treatment with antibiotics is likely to have already (Ze., previously) failed a treatment with antibiotics (as defined above). Accordingly, in one further preferred embodiment of the invention, the patient to be treated and who suffers from bacterial vaginosis wherein the infective bacteria are resistant to a treatment with antibiotics is a patient who previously failed a treatment with antibiotics.

[0620] According to the above definition of resistance, "High Resistance" (HR) can be defined as a MIC value superior or equal to 256 pg / ml for Metronidazole and a MIC value superior to 64 pg / ml for Clindamycin, i.e., 8-fold higher than the resistance breakpoints of the alternative definition, which are themselves already higher than the standard resistance breakpoints of the EUCAST definition. Accordingly, in one embodiment of the therapeutic uses of the invention, the patient to be treated suffers from BV wherein the infective bacteria of said BV are resistant, preferably highly resistant, to antibiotics treatment, preferably as defined by EUCAST breakpoints. In one preferred embodiment of the therapeutic uses of the invention, the patient to be treated suffers from BV wherein the infective bacteria of said BV are resistant, preferably highly resistant, to a treatment with Metronidazole, Tinidazole, Secnidazole, Clindamycin or any combination thereof. In one more preferred embodiment of the therapeutic uses of the invention, the patient to be treated suffers from BV wherein the infective bacteria of said BV are resistant, preferably highly resistant, to a treatment with Metronidazole and / or Clindamycin. In one even more preferred embodiment of the therapeutic uses of the invention, the patient to be treated suffers from BV wherein the infective bacteria of said BV are resistant, preferably highly resistant, to a treatment with Metronidazole.

[0621] In certain embodiments, the pharmaceutical composition according to the invention is used in treating bacterial vaginosis, wherein said patient previously failed a treatment with antibiotics and / or wherein the infective bacteria of said bacterial vaginosis are resistant to antibiotics treatment.

[0622] As used herein, the terms "treatment with antibiotics" and "antibiotics treatment" are used interchangeably and preferably refer to a treatment with the antibiotics recommended or approved for the treatment of BV. Antibiotics that are currently recommended or approved for the treatment of BV include the Nitroimidazoles, including but not limited to Metronidazole, Tinidazole, and Secnidazole, and Clindamycin. Thus, in one embodiment, said "treatment with antibiotics" or "antibiotics treatment" is a treatment with a nitroimidazole and / or Clindamycin. In a preferred embodiment, the treatment with antibiotics described herein is a treatment with Metronidazole, Tinidazole, Secnidazole, Clindamycin or any combination thereof. In a more preferred embodiment, the antibiotics treatment described herein is a treatment with Metronidazole, and / or Clindamycin. In an even more preferred embodiment, the antibiotics treatment described herein is a treatment with Metronidazole.

[0623] As used herein, "treatment" and "treating" and the like generally mean obtaining a desired pharmacological and physiological effect. The effect may be prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof and / or may be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease. The term "treating" as used herein covers any treatment of a bacterial infections of by bacteria of the genus Gardnerella, in particular vaginosis, in a mammal, particularly a human, and includes, without limitation: (a) preventing bacterial vaginosis from occurring in a patient which may be predisposed to bacterial vaginosis but has not yet been diagnosed as having it; (b) inhibiting bacterial vaginosis, i.e., arresting its development; or relieving the bacterial infection, i.e., causing regression of the bacterial infection and / or its symptoms or conditions such as improvement or remediation of damage. In particular, treatment of bacterial vaginosis comprises preventing, decreasing or even eradicating the infection, for instance by killing the infective bacteria and, thus, controlling, reducing or inhibiting bacterial proliferation as well as reducing the number of viable bacterial cells. Herein it is preferred that the disease, i.e., BV, is treated therapeutically in terms of a partial or complete cure of the disease or the symptoms.

[0624] The terms "patient" and "subjects" are used herein interchangeably and refer to mammals. For examples, mammals contemplated by the present invention include human, primates, domesticated animals such as cattle, sheep, pigs, horses, laboratory rodents and the like. It is preferred that the patient is a human being. It is even more preferred that the patient is a woman (also referred herein as a "female subject"). In certain embodiments, the female subject is pre-menopausal.

[0625] In a particular embodiment, the invention relates to the composition for use according to the invention, wherein the female subject is not menstruating and / or is not expected to menstruate during the treatment. Menstruation may interfere with local microbiome profile and local pharmacokinetics. In preferred embodiments, the composition is administered to the female subject either as a single dose or in multiple doses over a limited number of consecutive days. Consequently, the treatment can be scheduled such that the single or multiple doses are administered while the female subject is not menstruating. For example, treatment may be initiated after a menstrual cycle has ended. In certain embodiments, a female subject is not expected to menstruate within two to three weeks following her previous menstrual cycle. While it is preferred that the female subject is not menstruating during the treatment, treatment may be initiated at any time, including shortly before an expected menstruation or during menstruation, particularly in acute cases.

[0626] As discussed herein, the pharmaceutical composition according to the invention is preferably formulated as a vaginal insert, in particular a vaginal tablet, that is to be inserted into the vagina of a female subject in need, i.e., a female subject suffering from bacterial vaginosis. Accordingly, in a particular embodiment, the invention relates to the pharmaceutical composition for use according to the invention, wherein the composition is to be administered locally into the vagina of a female subject. Administering the pharmaceutical composition according to the invention into the vagina of a female subject may be achieved with an applicator. In a particular embodiment, the pharmaceutical composition according to the invention may be formulated as a tablet that can be inserted into the vagina of a female subject with an applicator. In certain embodiments, the invention relates to an applicator comprising the pharmaceutical composition according to the invention. In certain embodiments, the invention relates to a kit comprising the composition according to the invention and an applicator. The applicator may be any applicator suitable for intravaginal administration of vaginal inserts as known in the art.

[0627] To prevent and / or treat bacterial infections and, in particular bacterial vaginosis, in a subject in need, the pharmaceutical composition may be administered to said subject in a therapeutically effective amount.

[0628] The term "pharmaceutical composition" refers to a preparation which is in such a form as to permit biological activity of the active ingredient(s) to be unequivocally effective and which contains no additional component which would be toxic to patients to which the said composition would be administered. As used herein, "pharmaceutical composition" means a therapeutically effective formulation for use in the methods of the invention. A "therapeutically effective amount", or "effective amount", or "therapeutically effective", as used herein, refers to that amount which provides a therapeutic effect for a given condition and administration regimen. This is a predetermined quantity of active material calculated to produce a desired therapeutic effect in association with the required additive and diluent, i.e., a carrier or administration vehicle. Further, it is intended to mean an amount sufficient to reduce, and most preferably prevent, a clinically significant deficit in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in a host. As is appreciated by those skilled in the art, the amount of a compound or composition may vary depending on its specific activity. Suitable dosage amounts may contain a predetermined quantity of active composition calculated to produce the desired therapeutic effect in association with the required diluent. In the methods and use of compositions of the invention, a therapeutically effective amount of the active component is provided. A therapeutically effective amount can be determined by the ordinary skilled medical or veterinary worker based on patient characteristics, such as age, weight, sex, condition, complications, other diseases, etc., as is well known in the art.

[0629] In a particular embodiment, the invention relates to the pharmaceutical composition for use according to the invention, wherein a single dose of the pharmaceutical composition is administered to said subject.

[0630] That is, in certain embodiments, a single dose of the pharmaceutical composition according to the invention comprising the endolysin may be sufficient to treat a bacterial infection, in particular an infection caused by bacteria of the genus Gardnerella, in particular bacterial vaginosis. The single dose may comprise 2 to 100 mg of the Gardnerella-spe c endolysin, preferably 5 to 50 mg of the Carz / ereZZa-specific endolysin, more preferably 5 to 20 mg of the Garz / nereZZ -specific endolysin. In certain embodiments, a single dose comprising 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 mg may be administered to a patient for the treatment of an infection caused by bacteria of the genus Gardnerella.

[0631] In certain embodiments, multiple doses of the pharmaceutical composition according to the invention may be administered to a subject in need. In certain embodiments, one or more doses of the pharmaceutical composition according to the invention may be administered daily for n consecutive days. Thus, in a particular embodiment, the invention relates to the pharmaceutical composition for use according to the invention, wherein at least one dose of the pharmaceutical composition is administered to said subject on n consecutive days, wherein n is an integer from 2 to 14, in particular wherein n is 5.

[0632] In certain embodiments a dose of 2 to 100 mg, preferably 2 to 50 mg, more preferably 2 to 20 mg of the Ga / 'O’ne / 'e / Zs-specific endolysin may be administered to a subject in need for n consecutive days, wherein n preferably ranges from 2 to 14, preferably 2 to 10, more preferably 2 to 7.

[0633] In certain embodiments, a dose of 2 to 20 mg of the Garahere / Za-specific endolysin is administered to a subject in need for 5 consecutive days. In a particularly preferred embodiment, a dose of 2, 5, 10, or 20 mg of the Gardnerella- specific endolysin may be administered to a subject in need for 5 consecutive days.

[0634] The efficacy of the treatment for bacterial vaginosis may be evaluated at one or more points during or after the treatment. Various methods have been described in the art that can be used to assess the treatment efficacy of bacterial vaginosis. Thus, in a particular embodiment, the invention relates to the pharmaceutical composition for use according to the invention, wherein the efficacy of the bacterial vaginosis treatment is assessed during the treatment.

[0635] A well-established method for assessing the presence and / or severity of bacterial vaginosis is the Nugent score, as defined elsewhere herein. A Nugent score of 7 or higher is generally considered indicative of bacterial vaginosis. In certain embodiments, the Nugent score may be determined during the treatment, particularly when multiple doses are administered, or after the treatment. A decreased Nugent score compared to the start of the treatment is indicative of an effective treatment. A female subject may be considered cured of bacterial vaginosis if, in response to the treatment with the pharmaceutical composition according to the invention, a Nugent score below 4 is achieved.

[0636] Another method for assessing the treatment efficacy of bacterial vaginosis comprises assessing the amount and / or appearance of vaginal discharge. A homogeneous, thin, grayish-white discharge that smoothly coats the vaginal walls is generally considered indicative of bacterial vaginosis. In certain embodiments, the amount and / or appearance of vaginal discharge may be assessed during the treatment, particularly when multiple doses are administered, or after the treatment. Normalization of vaginal discharge compared to the start of the treatment is indicative of an effective treatment.

[0637] Another method for assessing the presence and / or severity of bacterial vaginosis is the "whiff test." This test involves the detection of a fishy odor when a sample of vaginal discharge is mixed with a potassium hydroxide (KOH) solution. The presence of a strong fishy odor is generally considered indicative of bacterial vaginosis. In certain embodiments, the whiff test may be performed during the treatment, particularly when multiple doses are administered, or after the treatment. The absence or reduction of the fishy odor compared to the start of the treatment is indicative of an effective treatment.

[0638] Another method for assessing the presence and / or severity of bacterial vaginosis involves determining vaginal pH. Vaginal pH is a measure of the acidity or alkalinity of the vaginal environment. In healthy women, the vaginal pH is typically between 3.8 and 4.5. However, in cases of bacterial vaginosis, the vaginal pH is often elevated above 4.5 due to the overgrowth of certain bacteria. In certain embodiments, the vaginal pH may be measured during the treatment, particularly when multiple doses are administered, or after the treatment. The reduction of vaginal pH to within the normal range compared to the start of the treatment is indicative of an effective treatment.

[0639] Another method for assessing the presence and / or severity of bacterial vaginosis comprises determining the proportion of clue cells among the total epithelial cells in a vaginal smear. Clue cells are vaginal epithelial cells that are covered with bacteria, giving them a distinctive stippled appearance. A high proportion of clue cells (>20%) is generally considered indicative of bacterial vaginosis. In certain embodiments, the proportion of clue cells may be evaluated during the treatment, particularly when multiple doses are administered, or after the treatment. A significant reduction in the proportion of clue cells compared to the start of the treatment is indicative of an effective treatment. In certain embodiments, a female subject may be considered cured of bacterial vaginosis if, in response to the treatment with the pharmaceutical composition according to the invention, the proportion of clue cells is below 20%, preferably below 15%, more preferably below 10%.

[0640] Accordingly, in a particular embodiment, the invention relates to the pharmaceutical composition for use according to the invention, wherein the efficacy of the bacterial vaginosis treatment is assessed based on one or more of: Nugent score, amount and / or appearance of vaginal discharge, outcome of whiff test, vaginal pH, and / or proportion of clue cells among total epithelial cells.

[0641] In a particular embodiment, the invention relates to a method comprising:

[0642] (a) obtaining a biological sample from a subject suffering from bacterial vaginosis;

[0643] (b) analyzing one or more of the following in the biological sample from the subject:

[0644] (i) Nugent score;

[0645] (ii) amount and / or appearance of vaginal discharge;

[0646] (iii) outcome of whiff test;

[0647] (iv) vaginal pH; and / or

[0648] (v) proportion of clue cells among total epithelial cells;

[0649] (c) administering the pharmaceutical composition according to the invention to the subject;

[0650] (d) obtaining a second biological sample from the subject; and

[0651] (e) re-analyzing the one or more parameters obtained in step (b) to determine safety and / or effectiveness of the pharmaceutical composition in treating bacterial vaginosis in the subject.

[0652] That is, the same parameters as described herein above may be used to determine the safety and / or effectiveness of the pharmaceutical composition in treating bacterial vaginosis in a subject. The skilled person, such as a practitioner in the field of gynecology, is familiar with these tests, including the Nugent score and Amsel's criteria, and is capable of determining, based on these parameters, whether a treatment with the composition according to the invention is effective and safe.

[0653] One or more of the parameters described hereinabove may be determined at one or more time points during or after the treatment. In certain embodiments, one or more of these parameters may be assessed prior to the treatment and at least once following the treatment. Specifically, one or more parameters may be evaluated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, and / or 31 days post-treatment. To determine the level of one or more parameter at the start of the treatment, a sample may be taken within 72 hours prior to the first dose. In particular embodiments, the Nugent score may be determined prior to the treatment and at least once after the treatment, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, l, 28, 29, 30, and / or 31 days post-treatment.

[0654] Embodiments and definitions described herein above for the therapeutic uses according to the invention apply in the context of the method of treating infections caused by bacteria of the genus Gardnerella, such as BV, mutatis mutandis.

[0655] The term "about", as used herein, refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term "about" generally refers to a range of numerical values e.g., + / - 1 -3% of the recited value) that one of ordinary skill in the art would consider equivalent to the recited value e.g., having the same function or result). In some instances, the term "about" may include numerical values that are rounded to the nearest significant figure.

[0656] The present invention is further described by reference to the following non-limiting figures and examples.

[0657] BRIEF DESCRIPTION OF THE DRAWINGS

[0658] FIG.l: Osmolality [Osmol / kg] of 40 mg of various powder blends incubated in 500 pl of SVF pH 6.0. Abbreviations: S: Sorbitol; M: Mannitol; MCC: Microcrystalline cellulose; REF: Reference; CP: Carbopol; SVF: Simulated vaginal fluid. Indicated values are means ± SD (n=4) with exception of * marked sample with n=l.

[0659] FIG.2: Swelling and gelation behavior of tablets after 1:2 (w / v) dilution with buffer. HPMC: Hydroxypropyl methylcellulose, Com: Compressol, M: Mannitol.

[0660] FIG.3: Drug release of tablet formulations containing Carbopol over 24 h. The nominal amount of H2B10B11 expected to be in the tablets was used as 100 % reference. Timepoint "1 h" of formulation F7 was not taken into account due to inconsistencies. Indicated values are means of n=4 ± SD.

[0661] FIG.4: Drug release of additional tablet formulations F7 and F8 over 24 h. The nominal amount of H2B10B11 expected to be in the tablets was used as 100 % reference. Timepoint "1 h" of formulation F7 was not taken into account due to inconsistencies. Indicated values are means of n=4 ± SD.

[0662] FIG.5: Interaction study of endolysin with single powder excipients. Recovery of endolysin after 17 h of incubation time. Indicated values are single values (n= 1).

[0663] FIG.6: Summary of interaction study of endolysin with different kinds of Carbopol (CP). Recovery of endolysin reference and acidic CP was determined after 17 h of incubation time whereas 50 % acidic / 50 % neutralized CP and pure neutralized CP were analyzed after 6 h of incubation time. Indicated values are single values (n=l).

[0664] FIG.7: Correlation between osmolality [mOsmol / kg] of powder blends incubated with SVF and the Compressol content [m / m %]. Green line designates osmolality of the previously developed formulation without NaCI for comparison. Indicated values are means of n=4 ± SD.

[0665] FIG.8: Recovered API [%] after incubation over 240 min of spray dried endolysin with Carbopol polymers. Indicated values are single values (n=l).

[0666] FIG.9: Free H2B10B11 [%] in the soluble fraction of selected tablet formulations over 240 min. The nominal amount of H2B10B11 expected to be in the tablets was used as 100% reference. Indicated values are means of n=4 ± SD. ** very significant difference (p<0.01) compared to F30% Naci control.

[0667] FIG.10: Activity [%] of H2B10B11 in the soluble as well as in the precipitate fractions of selected tablet formulations over 240 min. The nominal amount of H2B10B11 expected to be in the tablets was used as 100% reference. Indicated values are means of n=4 ± SD. * significant difference (p<0.05) *** highly significant difference (pcO.OOl) compared to F3 0% NaCI control.

[0668] FIG.ll: A, Kinetic study of H2B10 against G. vaginalis Gv9 (ATCC 14018T) shows a strong lytic effect which is time and dose dependent. Suspensions of Gardnerella cells were treated with recombinantly expressed H2B10 for 1 h, 5 h and 24 h at 2.5, 10, and 40 pg / ml and the colony forming units (CFU) per ml was determined in a logarithmic scale and compared to the buffer treated control. B, Suspensions of other vaginal BV pathogens (in grey) as well as probiotic Lactobacilli (in white boxes) were treated with H2B10 for 5 h at 10, and 100 pg / ml and the colony forming units (CFU) per ml was determined in a logarithmic scale and compared to the buffer treated control. LOD, indicates the limit of detection. Statistically significance (One-way ANOVA, comparison of control to 10 and 10 pg / mL H2B10 treatment for each strain) is indicated; ***, P< 0.001, **, P< 0.01, *, P< 0.05.

[0669] FIG.12: A, Gardnerella biofilms (using the type strain G. vaginalis ATCC 14018) were treated with 8 pg / ml H2B10 (and thus below the concentration that is typically used to eradicate Gardnerella biofilms), 0.35% (w / v) PAA and the combination thereof for 24 hours at pH 4.5 in sBHIG medium. B, Gardnerella biofilms (<7. vaginalis ATCC 14018) were treated with 32 pg / ml H2B10B11, 0.25% (w / v) PAA and the combination thereof for 24 hours at pH 6 in sNYC medium.

[0670] Cells were dislodged by pipetting and quantitative plating was used to determine the remaining viable cells (CFU / ml). The bactericidal effect is visualized by logic reductions in CFU / ml compared to the buffer treated control. The grey column indicates the theoretical additional effect of the mean log reduction of H2B10 / H2B10B11 and PAA. Mean values with standard deviation are shown.

[0671] FIG.13: Lytic activity of two H2B10 formulations on six BV-associated pathogens and one commensal Lactobacillus species. Sensitivity of the strains was tested by treating fresh liquid culture suspensions of the strains for 5 hours with a no-EL control, H2B10 formulated with PAA to pH 4.5 (panel A), or H2B10 formulated with PAA to a pH of 5.0 (Panel B). Viability after the indicated treatment was determined by quantitative spotting (Panel A & B) and viability reduction for both formulations (Panel C) was calculated by subtracting the loglO CFU / mL in replicates of the formulation treatments from the median of the respective unamended control. Shown are triplicates, range, and median. LOD: limit of detection. Points below the LOD indicate we reached the limit of detection. FIG.14: Influence of PAA concentration of the viability of planktonic Gv9 cells. Sensitivity to PAA was tested by treating fresh liquid culture suspensions of Gv9 with pH 5 sNYCB with varying concentrations of PAA for 90 minutes at 37°C. Viability after treatment was determined by quantitative spotting. Shown are triplicates and range with a horizontal line at the median. PAA: poly(acrylic acid). LOD: limit of detection.

[0672] FIG.15: Influence of PAA on the viability of five BV-associated pathogens and one commensal. Sensitivity of the strains to PAA was tested by treating fresh liquid culture suspensions with NYCB medium at pH 5 with concentrations of PAA varying from 0.01% to 1% for 90 minutes and comparing this to untreated controls. Viability after treatment was determined by quantitative spotting. Data are depicted in triplicate, with a line at the median. PAA: poly-acrylic acid. LOD: limit of detection. Points below the LOD indicate we reached the limit of detection.

[0673] FIG.16: Lytic activity of H2B10B11 formulated with Carbopol 974P on BV-associated pathogens and one commensal. Sensitivity of the strains was tested by treating fresh liquid culture suspensions of the strains for 20.5 h with a no-EL control or with a low concentration of H2B10B11 (2 pg / mL) formulated with 0.25 % (w / v) CP 974P in medium adjusted to pH 5. Viability after the indicated treatment was determined by quantitative spotting. Shown are triplicates and median. LOD: limit of detection. Points below the LOD indicate that the limit of detection was reached.

[0674] FIG.17: PK parameters determined for the H2B10B11 tablet formulation F3 (test item). The test item was administered to four sheep. 1, 2, 4, 8, and 24 h post-test item administration, two swab samples were taken from each animal at an inner (top panel) and outer (bottom panel) location in the vaginal canal. Swabs were analyzed in duplicates via sandwich ELISA. Each point represents the average of technical duplicates. Abbreviations: AUC = area under the curve; MBEC, minimum biofilm eradication concentration, 32 pg / mL; SD = standard deviation; MED, minimum effective dose.

[0675] FIG.18: ATP and ROS induction signal of cells treated with H2B10. The graphs show the ATP signal (A and B), and ROS signal (C and D) of Ectl and HeLa cells treated for 5 h (A and C) or 24 h (B and D), with 500 pg / mL H2B10 or controls. The ATP signal (A and B) was blanked with the luminescence signal of medium only. The three points in each group indicate experimental repetitions. Abbreviations: ATP = adenosine triphosphate; h = hour(s); HSA = human serum albumin (negative control); ROS =reactive oxygen species.

[0676] FIG.19: Cytokine release by PMNs upon H2B10B11 treatment. The graphs show the cytokines IL-ip, IL-6 and TNFo secreted by human PMNs upon treatment with H2B10B11. As positive control for the cytokine induction, Staphylococcus aureus,- TNFo = tumor necrosis factor-alpha.

[0677] FIG.20: H2B10B11 is not hemolytic. Human erythrocytes were treated for one hour with 500 pg / mL H2B10B11 or buffer control (MES), and then the hemolysis was assessed by measuring the hemoglobin release. Triton X-100 was used a lysis control (ctr.). Ordinary one-way ANOVA, (****p <0.0001). Abbreviations: ANOVA = analysis of variance; MES = 2-(N-morpholino)ethanesulfonic acid; ns = not significant. EXAMPLES

[0678] Example 1: Development and in vitro characterization of vaginal tablets for an endolysin

[0679] 1.1 Summary

[0680] Bacterial vaginosis (BV) is the most common vaginal infection in women of childbearing age causing not only physical and psychosocial discomfort but also increasing the risk of preterm birth, pelvic inflammatory disease, endometritis and transmission as well as acquisition of sexually transmitted infections. More recently, the inventors could successfully engineer an endolysin H2B10B11 (PM-477) providing a specific activity against Gardnerella bacteria, one of the primary organisms associated with BV. As next step, tablets comprising said endolysin were of high interest given their high storage stability. However, the vaginal milieu usually does not contain enough fluid to allow rapid disintegration of tablets, making release more difficult.

[0681] Within the present invention, this problem is addressed by using osmotic active agents in the formulation draining additional fluid from the vaginal tissue. It was therefore the aim of this study, to design osmotically active vaginal tablets for H2B10B11 (SEQ ID NO:1).

[0682] First, various osmotically active agents were investigated regarding their osmolality in simulated vaginal fluid (SVF). By using the cryoscopic measurement technique, the sugar alcohols sorbitol and mannitol were identified as the most potent osmotic candidates followed by the salts potassium chloride and sodium chloride. As these salts influence the endolysins activity, however, they were not further pursued, and the focus was set onto sugar alcohol- based formulations.

[0683] When preparing tablets by using an excenter tablet press, powder blends containing flow regulation agent, glidant, lubricant and dispersant resulted in tablets exhibiting the desired characteristics (adequate compressibility and sufficient disintegration). Accordingly, all prepared tablets consisted of optional microcrystalline cellulose (MCC) (filler, binder), sugar alcohol mannitol or Compressol comprised of mannitol and sorbitol (osmotic active agent, filler), Carbopol (CP), optional hydroxypropyl cellulose (HPMC) (gelling agent, dispersant, binder), magnesium stearate (glidant, lubricant) and highly dispersed silica (flow regulator). In order to ensure reproducibility of the manufacturing process, tablets were characterized according to European Pharmacopoeia (Ph. Eur.) regarding uniformity of mass, hardness and friability. All tablets met the requirements of Ph. Eur. with exception of formulation comprising only Compressol as filler which exhibited a friability of 1.8 %, whereas a maximum of 1 % is acceptable. Overall, increasing sugar alcohol component in the tablet formulation resulted in an increased mass and also hardness of the tablets. In disintegration studies, complete dissolution was observed for formulation based on only Compressol as filler and the formulation comprising no HPMC after an incubation time of 6 h. With HPMC serving on the one hand as dispersant but on the other hand acting as gelling agent, it is responsible for disintegration of tablets but also for immediate gel formation. Consequently, HPMC containing tablets might just have not appeared as disintegrated and only tablets without gelation behavior completely would be described as disintegrated in this experiment. Further to demonstrate swelling and gelation behavior, the size of tablets was assessed after SVF addition in a ratio of 1:2 (m / v) and most pronounced increase was observed for HPMC containing tablets comprising MCC and mannitol and hardly no increase for only sugar alcohol-based formulation. After distinguishing the most promising formulations, tablets with H2B10B11 were prepared by direct compression using a single punch press with a pressure of 10-11 kN for 30 sec. The loaded tablets were assessed regarding their drug release over 24 h. Hereby, it was particularly noticeable, that Fl - previously selected as fast disintegrating formulation - disintegrated slower in comparison to the other investigated formulations F2, F4 and F6. These formulations contained HPMC acting as disintegrant and leading to a rise of the tablets and therewith probably to a faster release. Even though F4 did almost completely disintegrate after 24 h of incubation, only a low but highest overall release of 12.3 % after 24 h was detected. Moreover, additional formulations - one with a reduced Compressol content of 70% (F7) and one without CP (F8) - were investigated. Thereby, F7 showed a faster increase in comparison to all previously tested formulations Fl, F2, F4 and F6. Nevertheless, the total release after 24 h incubation time was likewise limited to 13.4 % most likely associated to endolysin-CP interaction. This is also underlined by release of formulation F8 - same composition as F7 without CP - as a similar release pattern was observed but with remarkable higher concentrations such as 38 % of released endolysin after only 15 min and finally 63 % of released endolysin after 24 h of incubation time.

[0684] Additional interaction studies of H2B10B11 with single tablet excipients demonstrated a noteworthy interaction with the polymer CP. As a consequence, also neutralized and a mixture of neutralized and acidic CP were investigated and were found to interact even more with the endolysin. For this reason, tablets for in vivo studies in sheep were prepared with acidic CP and also without CP.

[0685] Overall, various tablet formulations were developed displaying all strong osmotically characteristics and offering different release behaviors.

[0686] 1.2 Methods

[0687] 1.2.1 HPLC quantification of H2B10B11

[0688] For quantification of H2B10B11, an HPLC method was applied. In brief, samples were separated on a C18 reversed phase (RP) column (250 x 4.6 mm, particle size 5 pm, pore size 130 A) in a water / acetonitrile mixture (gradient 60:40 to 10:90). Eluted endolysin was detected at a wavelength of 280 nm.

[0689] 1.2.2 Development of three different osmotic active vaginal tablets

[0690] In order to identify appropriate powder blends for tableting, they were prepared preliminary without the API. Therefore, tablet excipients such as filler, Carbopol 974P and osmotic active agents were mixed by geometric dilution for 5 min utilizing mortar and pestle. After identifying suitable powder blends based on their resulting osmolality, tablets were prepared by using an excenter tablet press. In order to ensure reproducibility of the manufacturing process, tablets were characterized regarding hardness, friability, disintegration and osmolality (as described below) and formulations were optimized. Based on the orientating evaluation of tablets without API, H2B10B11 was incorporated in the most promising powder blends and tablets were prepared by direct compression using a single punch press with a pressure of 10-11 kN for 30 sec. The loaded tablets were investigated regarding their drug release over 24 h. 1.2.2.1 Orientating evaluation of osmolality of powder blends in simulated vagina! fluid

[0691] Osmolarity was determined via a cryoscopic osmometer (Osmomat 030, gonotec, Germany). First, the zero point of the instrument was defined using highly purified water and the apparatus was calibrated with 300 mOsmol / kg (9.463 mg / ml NaCI in water) and 700 mOsmol / kg (22.380 mg / ml NaCI in water) reference solutions. Thereafter, a commercially available physiological sodium chloride solution (sodium chloride 0.9 % Freeflex, Fresenius Kabi, Germany) was used as control. Afterwards, samples (50 pl) were investigated. Therefore, 500 pl of simulated vaginal fluid (SVF) were added to 40 mg of powder blends. After sonication of 30 min and subsequent centrifugation for 10 min at 12,500 RCF (Eppendorf minispin), the osmolality of supernatant was determined. Further, SVF only served as reference.

[0692] 1.2.2.2 Improvement of powder blends for tablet pressing

[0693] To ensure a smooth pressing process and satisfying tablet performance such as disintegration, further excipient addition such as flow regulation agent, glidant, lubricant and dispersant was assessed.

[0694] 1.2.2.3 In vitro characterization of hardness and friability

[0695] The hardness of prepared vaginal tablets was determined using a Schleuniger 2-E / 205 tablet-hardness tester (Dr. K. Schleuniger and Co., Switzerland). Friability was calculated as the percentage weight loss after rotation of 4 min at 25 rpm (Erweka TAR-10 friabilator).

[0696] 1.2.2.4 Evaluation of the disintegration behavior

[0697] The stability of prepared tablets was analyzed in simulated vaginal fluid (SVF). In brief, tablets were added into Eppendorf tubes, SVF is added in an appropriate amount and samples are incubated at 37 °C while shaking (300 rpm) for 6 h.

[0698] 1.2.2.5 Investigation of swelling and gelation behavior

[0699] In order to identify the swelling and gelation behavior, tablets of approximately 200-300 mg were placed onto the bottom surface of a beaker and wetted with 500 pl of SVF buffer. After 5 min, the tablets were assessed regarding appearance and pictures were taken.

[0700] 1.2.2.6 Drug release studies in artificial vaginal fluid

[0701] The release behaviour of the endolysin from tablet formulations (Table 4) was evaluated by placing tablets of approx. 40 mg in glass vials. After addition of 400 pl of simulated vaginal fluid pH 6.0 preheated to 37 °C, samples were incubated at 37 °C while shaking for 24 h. After 0.25, 0.5, 1, 2, 6 and 24 h, samples of 100 pl were withdrawn, centrifuged (12,500 RCF, 10 min) and the amount of released H2B10B11 in the supernatant was analyzed via HPLC. The drawn volume was replaced with fresh pre-heated simulated vaginal fluid. As a reference, uncompressed powder blend was also mixed with the same amount of SVF and incubated for 6 h. 1.2.3 Excipient-H2B1OB11 interaction studies

[0702] 1.2.3.1 H2B10B11 interaction with tablet excipients

[0703] Since results from drug release studies indicated an interaction of endolysin with tablet excipients, endolysin powder was incubated with the single powder excipients in SVF for 17 h (overnight) and free concentration of EL was determined.

[0704] 1.2.3.2 Carbopol influence on pH of SVF buffer

[0705] Based on the thereafter found interaction between Carbopol (CP) and endolysin, various Carbopol mixtures compromising unmodified, acidic CP (R-COOH) and neutralized CP (R-COONa) were investigated regarding their influence on pH of simulated vaginal fluid (SVF) pH 6.0. Therefore, approximately 15 mg of CP mixture were placed in a plastic tube and 4 ml of SVF pH 6.0 were added. After sonication for 90 min, the pH of mixtures was determined.

[0706] 1.2.3.3 Carbopoi-H2B10B11 interaction

[0707] In the next step, endolysin powder was incubated with 100 % neutralized CP and a mixture of 50 % acidic and 50 % neutralized CP for 6 h and free concentration of EL was determined and compared to previously analyzed samples (reference and acidic CP).

[0708] 1.2.4 Statistical data analysis

[0709] All studies and tests were carried out with n>3 for each experimental setup unless otherwise stated.

[0710] 1.3 Results

[0711] 1.3.1 Development of three different osmotic active vaginal tablets

[0712] 1.31.1 Orientating evaluation of osmolality of powder blends in simulated vaginal fluid

[0713] In a first setup, powder blends with diverse osmotic active agents in different concentrations (0 %, 1 % and 5 %) were assessed regarding their osmolality when mixed with SVF (Figure 1). Thereby, highest osmolalities of approximately 675 mOsmol / kg were determined for sugar alcohol-based formulations. Salt addition of NaCI and KCI in concentrations of 1 % and 5 % to the powder blends resulted in an increase of about 30 and 100 mOsmol, respectively. In general, no impact on osmolality was observed by Carbopol addition.

[0714] By applying the cryoscopic measurement technique, no substantial influence on osmolality by using PEG as osmotic active agent could be determined.

[0715] 1.3.1.2 Improvement of powder blends for tablet pressing

[0716] Based on the obtained results of osmolality investigations of powder blends, tablets meeting all criteria regarding compressibility were developed (Table 1).

[0717] Table 1: Composition of investigated tablet formulations [%].:Compressol: directly compressible excipient consisting of mannitol and sorbitol;2Aerosil: highly-dispersed silicon dioxide.

[0718] Excipient Function Fl F2 F3 F4 F5 F6 F1O

[0719] Avicel PH101 Filler, binder 54.4 78 63.7 51.8 - - 52.4

[0720] Mannitol Filler 43 14.3 28.7 40.9 - - 40.9

[0721] Compressol1Filler, binder - 97.4 92.4

[0722] Carbopol Gelling agent, acidifier 0.6 0.6 0.6 0.5 0.6 0.6

[0723] Gelling agent, binder, HPMC - 5 5 4.8 - 5 4.8 dispersant

[0724] Magnesium Lubricant, glidant 1 1 1 1 1 1 1 stearate

[0725] Aerosil2Flow regulation 1 1 1 1 1 1 1

[0726] 1.3.1.3 In vitro characterization of hardness and friability

[0727] All developed tablets were analyzed according to Ph. Eur. with respect to uniformity of mass, hardness and friability (Table 2). All tablets met the requirements of Ph. Eur. with only exception for F5 in friability by exhibiting a loss of 1.8 % whereas only 1 % is acceptable. By increasing the mannitol component in the MCC-mannitol based tablets, an increase of mass and therewith along in hardness was obtained deriving from the lower volume of the mannitol powder. Highest osmolality was determined for mainly sugar alcohol-based formulations F5 and F6 and increasing results for formulations with increasing mannitol component.

[0728] Table 2: Characterization of tablet formulations. HPMC: Hydroxypropyl methylcellulose, Com: Compressol, M: Mannitol.

[0729] Formulation Uniformity of mass Hardness Friability Osmolality

[0730] [mg] deviation [%] [N] [%] [Osmol / kg]

[0731] Fl - w / o HPMC 250 ± 3 1.1 58.8 ± 5.6 0.7 0.396 ± 0.006

[0732] F2 - 14 % M + HPMC 221 ± 2 0.9 45.5 ± 1.0 0.6 0.274 ± 0.006

[0733] F3 - 28 % M + HPMC 238 ± 1 0.6 59 ± 1.2 0.5 0.310 ± 0.010

[0734] F4 - 43 % M + HPMC 255 ± 2 0.9 63.5 ± 3.4 0.6 0.396 ± 0.014

[0735] F5 - Com w / o HPMC 296 ± 2 0.8 67 ± 10.4 1.8 0.644 ± 0.010

[0736] F6 - Com + HPMC 301 ± 3 1 79.5 ± 8.3 0.9 0.626 ± 0.013

[0737] F0 - Placebo 245 ± 3 1.1 56 ± 1.4 0.5 0.392 ± 0.002

[0738] 1.3.1.4 Evaluation of the disintegration behaviour

[0739] Disintegration evaluation of tablets in SVF is visualized in Table 3 by utilizing a number code representing the degree of disintegration. In general, complete disintegration could be observed for Fl and F5 exhibiting a high sugar alcohol concentration and no HPMC. With HPMC serving on the one side as dispersant but on the other side acting as gelling agent, it is responsible for disintegration of tablet but also for immediate gel formation. Consequently, HPMC containing tablets might just have not appeared as disintegrated and only tablets without gelation behavior could be described as completely disintegrated in this experiment.

[0740] Table 3: Disintegration evaluation of tablets after 1:12.5 (w / v) dilution with simulated vaginal fluid (SVF). HPMC: Hydroxypropyl methylcellulose, Com: Compressol, M: Mannitol.

[0741] Time after SVF 15 30 60 120 180 360 addition [min]

[0742] Fl - w / o HPMC 1 1 3 4 4 4

[0743] F2 - 14 % M + HPMC 1 1 2 2 2 2

[0744] F3 - 28 % M + HPMC 1 1 1 2 2 2

[0745] F4 - 43 % M + HPMC 1 1 1 2 3 3

[0746] F5 - Com w / o HPMC 1 2 4 4 4 4

[0747] F6 - Com + HPMC 1 1 2 2 3 3

[0748] FO - Placebo 1 1 2 3 3 3

[0749] 1: Tablet intact, sticking together, strong adhesion to tube wall

[0750] 2: Tablet mostly intact, slightly disintegrated, small pieces come off

[0751] 3: Tablet only partially intact / sticking together, mostly disintegrated

[0752] 4: No tablet shape visible, completely disintegrated

[0753] 1.3.1.5 Investigation of swelling and gelation behavior

[0754] To evaluate swelling and gelation properties of these tablets, SVF was added to the tablets in a ratio of 1:2 (w / v) and their size was visually assessed after 5 min incubation time (Figure 2). Overall, the highest swelling was observed for tablets containing gelling agent HPMC up to a diameter of 1.3 cm. But also, Formulation Fl without HPMC displayed a pronounced size increase to a diameter of approximately 1.2 cm, whereas the high sugar-based tablet formulations F5 and F6 remained with diameters of 0.9 and 1.0 cm, respectively.

[0755] 1.3.1.6 Drug release studies in artificial vagina! fluid

[0756] The release behaviour of endolysin from tablet formulations (Table 4) was evaluated over 24 h. After 6 h of incubation time, a maximum of 7.4 % of released endolysin could be detected for formulation F4, which increased within 24 h up to 12.3 % (Figure 3). It is especially noticeable, that Fl - originally selected as fast disintegrating formulation - did disintegrate slower in comparison to the other investigated formulations F2, F4 and F6. However, in comparison to previous disintegration studies, tablets were pressed with a single punch press (11 kN, 30 sec) and not an excenter press (no information of applied pressure available). These formulations all contained HPMC acting as disintegrant and leading to a rise of the tablets and therewith to a faster release. Even though F4 and F6 did almost completely disintegrate after 24 h of incubation, only a low but highest overall release was detected (Figure 3) indicating pronounced interaction of the endolysin with the excipients. This was furthermore underlined by 2.2- 2.8 % of free endolysin after incubating reference blends for 6 h.

[0757] Furthermore, additional formulations - one with a reduced Compressol content of 70% (F7) and one without CP (F8) - were investigated. Thereby, F7 with 70 % of Compressol and 0.5 % Carbopol (CP) showed a noticeable faster increase in comparison to previously tested CP containing formulations Fl, F2, F4 and F6 (Figure 3). However, the total release after 24 h incubation time was likewise limited to 13.4 % most likely associated to endolysin-CP interaction. This is also underlined by release of formulation F8 - same composition as F7 without CP - as a similar release pattern was observed but with pronounced higher concentrations such as 38 % of released endolysin after only 15 min and finally 63 % of released endolysin after 24 h of incubation time (Figure 4).

[0758] Table 4: Composition [%] of tablet formulations investigated in release studies.

[0759] Excipient Function Fl F2 F4 F6 F7 F8

[0760] H2B10B11 lyophilized API 1.3 1.3 1.3 1.3 1.3 1.3 powder

[0761] Avicel PH101 Filler, binder 53.2 77.2 48.2 - 20.1 20.1

[0762] Mannitol Filler 43 14 43 - - -

[0763] Compressol Filler, binder - - - 91.2 71.1 71.6

[0764] Gelling agent,

[0765] Carbopol 0.5 0.5 0.5 0.5 0.5 acidifier

[0766] HPMC Gelling agent, binder - 5 5 5 5 5

[0767] Magnesium Lubricant, glidant 1 1 1 1 1 1 stearate

[0768] Aerosil Flow regulation 1 1 1 1 1 1

[0769] 1.3.2 Excipient-H2B1OB11 interaction studies

[0770] 1.3.2.1 H2B10B11 interaction with tablet excipients

[0771] Since results from drug release studies indicated an interaction of endolysin with tablet excipients, endolysin powder was incubated with the single powder excipients in SVF for 17 h (overnight) and free concentration of EL was determined. As shown in Figure 5, after incubation of endolysin with Carbopol only 20 % free endolysin could be detected in solution, whereas minor interactions were observed with all other excipients.

[0772] 1.3.2.2 Carbopol influence on pH of SVF buffer

[0773] To investigate Carbopol influence on pH of SVF buffer, various acidic (R-COOH) and neutralized CP (R-COONa) mixtures were determined. Using 100 % of neutralized CP, resulted in the aimed pH of 5.0, whereas for all other investigated mixtures pH values between 4.7 and 4.8 were obtained (Table 5).

[0774] Table 5: Composition of CP mixtures (3.75 mg / ml) and resulting pH in SVF.

[0775] Acidic CP [%] Neutralized CP [%] pH

[0776] 100 - 4.6

[0777] 50 50 4.8

[0778] 67 33 4.7

[0779] 90 10 4.7

[0780] 80 20 4.7

[0781] 100 5.0

[0782] SVF buffer 6.1 1.3.3.3 Carbopo / - H2B10B11 interaction

[0783] In the next step, endolysin powder was incubated with 100 % neutralized CP and a mixture of 50 % acidic and 50 % neutralized CP for 6 h and free concentration of EL was quantified and compared to previously analyzed samples (reference and acidic CP).

[0784] As shown in Figure 6, endolysin interacts even more with neutralized CP or the CP mixture than with acidic CP underlined by 5-6 % of free endolysin. Further, the pH of EL interaction samples was analyzed in this setup as a higher amount of SVF was used than for the previous pH influence determination (Table 6).

[0785] Table 6: Outcome of pH evaluation of CP-endolysin interaction samples with a CP concentration of 0.625 mg / ml in SVF pH 6.0.

[0786] Acidic CP 50 % acidic - 50 % neutralized CP Neutralized CP

[0787] Incubation with EL n.d. 5.3 5.5

[0788] 1.4 Conclusion

[0789] The aim of this project was to develop osmotic active tablets for the vaginal administration of the endolysin H2B10B11. By focusing on sugar alcohol-based formulations, strong osmotically active tablets could be established. Hereby, powder blends containing flow regulation agent, glidant, lubricant and dispersant resulted in tablets exhibiting the desired characteristics as adequate compressibility and later sufficient disintegration. Especially, HPMC turned out to have an impact on disintegration when assessing the release behavior of tablet formulations. Moreover, a pronounced interaction of H2B10B11 with CP was observed in release studies and confirmed in follow up interaction studies.

[0790] Example 2: Development and in vitro characterization of different osmotically active vaginal tablets for an endolysin

[0791] 2.1 Summary

[0792] In Example 1, prototypes of osmotically active tablets exhibiting promising in vivo results in sheep were generated. As in one of the sheep an excess fluid secretion was observed probably due to high osmolality, adjustments of the formulation were tested. Further, the inventors identified a concentration of up to 400 mM NaCI as beneficial to improve the EL release from EL-Carbopol complexes. Based on these findings, it was the aim of this study to design different osmotically active tablets for an endolysin containing 5 % NaCI and different concentrations of sugar alcohol (Compressol) meeting required characteristics regarding disintegration.

[0793] First, powder blends containing different concentrations of Compressol and 5 % of NaCI were investigated regarding their osmolality in simulated vaginal fluid (SVF). Hereby, a linear correlation between Compressol content and resulting osmolality was observed. Compared to earlier developed formulations, addition of 5 % NaCI led to an increase of osmolality of about 100 mOsmol / kg. Based on these findings, three hyperosmolar formulations containing 5 % of hydroxypropyl methylcellulose (HPMC) exhibiting osmolalities of 588 mOsmol / kg (F3), 472 mOsmol / kg (F5) and 376 mOsmol / kg (F7) and two hyperosmolar formulations with 10 % HPMC exhibiting osmolalities of 588 mOsmol / kg (F8) and 472 mOsmol / kg (F9) were selected for further investigations. In order to ensure reproducibility of the manufacturing process, tablets were characterized according to European Pharmacopoeia (Ph. Eur.) regarding uniformity of mass, hardness and friability. Further, increasing the sugar alcohol component in the tablet formulation resulted in an increased mass. In disintegration studies, complete disintegration was observed for all tested formulations after 30 min of incubation time and no changes were observed over 24 h in total.

[0794] Within this timespan, complete disintegration was seen from formulations F3 and F5 already after 15 min, whereas F7 was still partially intact. Although 10 % HPMC containing formulations F8 and F9 were mostly disintegrated after 15 min, they displayed some gel-like lumps indicating a stronger gelation behavior than formulations with 5 % HPMC. Nevertheless, they were also fully disintegrated after 30 min incubation time.

[0795] Overall, by adding 5 % of NaCI to the tablet formulations with different osmolalities, a faster disintegration of 30 min compared to previously developed formulations could be achieved.

[0796] 2.2 Methods

[0797] 2.2.1 HPLC quantification of H2B10B11

[0798] See Example 1.

[0799] 2.2.2 Development of five different osmotic active vaginal tablets

[0800] In order to identify appropriate powder blends for tableting, powder blends were prepared preliminary without the API. Based on the previously developed tablet formulation (1.3 % API, 20.1 % microcrystalline cellulose (MCC), 71.1% Compressol, 0.5 % Carbopol 974P, 5 % HPMC, 1 % magnesium stearate and 1 % Aerosil), powder blends containing 5 % of NaCI and different amounts of sugar alcohol (Compressol) were prepared by mixing excipients by geometric dilution for 5 min utilizing mortar and pestle (Table 8). In the following, the mixtures were characterized regarding their osmolalities when mixed with SVF. After identifying powder blends meeting osmolalities of approximately 300, 450 and 600 mOsmol / kg, powder blends with different concentrations of HPMC (5 % and 10 %) were directly compressed into tablets of 1 g using an excenter press. In order to ensure reproducibility of the manufacturing process, powder blends as well as tablets were characterized regarding hardness, friability and disintegration (as described below). Based on the evaluation of tablets without H2B10B11, the API was incorporated in the most promising powder blends and resulting tablets were investigated regarding release behavior. Furthermore, selected formulations were prepared and transferred for in vivo studies in sheep. Table 8: Composition [%] of powder blends investigated regarding osmolality. pF: previously developed formulation, prepared for mouse tablets.

[0801] Excipient Function pF Fl F2 F3 F4 F5 F6 F7

[0802] H2B10B11 API 2.6 - - - - - - lyophilized

[0803] Osmotic active NaCI - 5 5 5 5 5 5 5 agent

[0804] Avicel Filler, binder 20 17.5 27.5 37.5 47.5 57.5 67.5 77.5

[0805] PH101

[0806] Compressol Filler, binder 70.1 70.0 60 50 40 30 20 10

[0807] Gelling agent, Carbopol 0.3 0.5 0.5 0.5 0.5 0.5 0.5 0.5 acidifier

[0808] Gelling agent, HPMC 5 5 5 5 5 5 5 5 binder, dispersant

[0809] Magnesium Lubricant, glidant 1 1 1 1 1 1 1 1 stearate

[0810] Aerosil Flow regulation 1 1 1 1 1 1 1 1

[0811] 2.2.2.1 Evaluation of osmolality of powder blends in simulated vagina! fluid

[0812] Osmolality was determined via a cryoscopic osmometer (Osmomat 030, gonotec, Germany). First, the zero point of the instrument was defined using highly purified water and the apparatus was calibrated with 300 mosmol / kg (9.463 mg / ml NaCI in water) and 700 mosmol / kg (22.380 mg / ml NaCI in water) reference solutions. Thereafter, a commercially available physiological sodium chloride solution (sodium chloride 0.9 % Freeflex, Fresenius Kabi, Germany) was used as control. Afterwards, samples (50 pl) were investigated. Therefore, 500 pl of SVF were added to 40 mg of powder blends. After sonication for 30 min and subsequent centrifugation for 10 min at 12,500 RCF (Eppendorf minispin), the osmolality of supernatant was determined. SVF only served as reference.

[0813] 2.2.2.2 In vitro characterization of hardness and friability

[0814] The hardness of selected tablet formulations was determined using a Schleuniger 2-E / 205 tablet-hardness tester (Dr. K. Schleuniger and Co., Switzerland). Friability was calculated as the percentage weight loss after rotation of 4 min at 25 rpm (Erweka TAR-10 friabilator).

[0815] 2.2.2.3 Evaluation of the disintegration behavior

[0816] The powder blends selected after osmolality determination were pressed into tablets of approximately 1 g by using an excenter press and tablet molds with a diameter of 1.5 cm. In the following, the disintegration of prepared tablets was analyzed in SVF. Therefore, tablets were placed in 50 ml Falcon tubes, 12 ml of SVF were added and samples were incubated at 37 °C while shaking (300 rpm) for 6 h. 2.2.3 Statistical data analysis

[0817] All studies and tests were carried out with n=4 for each experimental setup.

[0818] 2.3 Results

[0819] 2.3.1 Development of three different osmotic active vaginal tablets

[0820] 2.3.1.1 Evaluation of osmolality of powder blends in simulated vagina! fluid

[0821] Powder blends containing 5 % of NaCI and different amounts of sugar alcohol (Compressol) were assessed regarding their osmolality when mixed with SVF (Figure 7). Thereby, a linear correlation between Compressol concentration and osmolality was observed. Compared to previously developed salt free formulation (Example 1), addition of 5 % NaCI led to an increase of osmolality of about 100 mOsmol / kg. Based on these results, three hyperosmolar formulations containing 5 % of hydroxypropyl methylcellulose (HPMC) exhibiting different osmolalities (F3, F5, F7) and two hyperosmolar formulations with 10 % HPMC (F8 and F9) were selected for further investigations as summarized in Table 9.

[0822] Table 9: Composition [%] of tablets investigated regarding hardness, friability and disintegration.

[0823] Excipient Function F3 F5 F7 F8 F9

[0824] H2B10B11 API . . . . . lyophilized

[0825] Osmotic active

[0826] NaCI 5 5 5 5 5 agent

[0827] Avicel

[0828] Filler, binder 37.5 57.5 77.5 32.5 52.5

[0829] PH101

[0830] Compressol Filler, binder 50 30 10 50 30

[0831] Gelling agent,

[0832] Carbopol 0.5 0.5 0.5 0.5 0.5 acidifier

[0833] Gelling agent,

[0834] HPMC 5 5 5 10 10 binder, dispersant

[0835] Magnesium

[0836] Lubricant, glidant 1 1 1 1 1 stearate

[0837] Aerosil Flow regulation 1 1 1 1 1

[0838] 2.3.1.2 In vitro characterization of hardness and friability

[0839] The powder blends selected after osmolality determination (Table 9) were pressed into tablets of approximately 1 g by using an excenter press and automatic feeder. All developed tablets were analyzed according to Ph. Fur. with respect to uniformity of mass and hardness (Table 10). By decreasing the Compressol component (from F3 to F5 and F7), a decrease of mass was obtained deriving from the higher volume of the Avicel PH101 (MCC) powder. Table 10: Characterization of tablet formulations. Com: Compressol; HPMC: Hydroxypropyl methylcellulose.

[0840] Formulation Uniformity of mass Hardness

[0841] [g] deviation [%] [N]

[0842] F3 to 50 % Com, 5 % 1.01 ± 0.02 1.7 65.7 ± 1.5

[0843] HPMC

[0844] F5 to 30 % Com, 5 % 0.92 ± 0.02 2.3 70.8 ± 9.6

[0845] HPMC

[0846] F7 to 10 % Com, 5 % 0.84 ± 0.02 2.0 77.5 ± 4.4

[0847] HPMC

[0848] F8 to 50 % Com, 10 % 1.03 ± 0.02 2.0 100.0 ± 2.8

[0849] HPMC

[0850] F9 to 30 % Com, 10 % 0.92 ± 0.02 2.6 74 ± 11.7

[0851] HPMC

[0852] 2.3.1.3 Evaluation of the disintegration behavior

[0853] Disintegration of tablets in SVF is visualized by utilizing a number code representing the degree of disintegration (Table 11). In general, complete disintegration was observed for all tested formulations after 30 min incubation and no changes were observed over 24 h in total. However, a faster disintegration was observed with increasing Compressol concentration (10 % to 30 % to 50 %), whereas a higher HPMC concentration of 10 % led to gel-like lump formation and a slower disintegration

[0854] Table 11: Disintegration evaluation of tablets after 1:10 (w / v) dilution with simulated vaginal fluid (SVF). Com: Compressol; HPMC: Hydroxypropyl methylcellulose.

[0855] Time after SVF addition Over

[0856] 2 15 30 60 360

[0857] [min] night

[0858] F3 to 50 % Com, 5 % HPMC 3 5 5 5 5 5

[0859] F8 to 50 % Com, 10 % HPMC 3 4 5 5 5 5

[0860] F5 to 30 % Com, 5 % HPMC 3 5 5 5 5 5

[0861] F9 to 30 % Com, 10 % HPMC 2 4 5 5 5 5

[0862] F7 to 10 % Com, 5 % HPMC 2 3 5 5 5 5

[0863] 1: Tablet intact, sticking together, strong adhesion to tube wall

[0864] 2: Tablet mostly intact, slightly disintegrated, small pieces come off

[0865] 3: Tablet only partially intact / sticking together, mostly disintegrated

[0866] 4: Tablet only partially intact / sticking together, mostly disintegrated, gel-like lumps

[0867] 5: No tablet shape visible, completely disintegrated

[0868] 2.4 Conclusion

[0869] The aim of this project was to design different osmotically active tablets for an endolysin containing 5 % NaCI. By applying different concentrations of sugar alcohol in the tablet formulation, a linear correlation between the used Compressol content and the resulting osmolality was observed. In general, all selected formulations were comparable regarding their tablet characteristics such as hardness, friability as well as disintegration. In contrast to salt free formulations developed in the previous project, the new formulations containing 5 % NaCI displayed a faster disintegration within 30 min. All five selected formulations were chosen for in vivo studies in sheep.

[0870] Example 3: Investigation of tablet excipients on endolysin release from vaginal tablets via Dyerelease assay (DRA)

[0871] 3.1 Summary

[0872] Within recent projects, osmotically active tablets based on Carbopol achieving promising in vivo results were generated (see Example 1). As in one of the tested animals an excess fluid secretion was observed most likely as a result of high osmolality, adjustments of the formulation were needed. In the following project, the drug release from tablets containing 5% NaCI was identified as beneficial for disintegration of the tablet (Example 2).

[0873] Based on these latest findings, it was the aim of this study to investigate the influence of NaCI on the release and activity of endolysin from vaginal tablets quantified via HPLC and the dye-release assay (DRA), respectively. In comparison to previous studies, the payload was reduced by half (5 mg / g) and the spray-dried endolysin was used instead of a lyophilized API (Table 12).

[0874] Table 12: Composition [mg / ml] of feeding solution used to prepare spray-dried powder and the corresponding composition [%] of the powder.

[0875] Composition of feed solution Material Composition [%]

[0876] (mg / mL)

[0877] Sodium acetate 0.1365 0.9

[0878] Acetic acid 0.5006 3.2

[0879] Mannitol 13.5 86.3

[0880] H2B10B11 1.5 9.6

[0881] Firstly, the compressibility of non-loaded tablet formulations F3, based on a previously developed composition (5% NaCI, 37.5% Avicel PH101, 50% Compressol, 0.5% Carbopol 974 P, 5% hydroxypropyl methylcellulose, 1% magnesium stearate as well as 1% Aerosil) containing increasing amounts of NaCI and two Carbopol polymers was determined. On the one hand, tablets of approximately 115 mg were pressed by an excenter press exhibiting sufficient cohesive properties adjusting the pressure of the upper punch and utilizing grinded NaCI for the preparation of powder blends. On the other hand, 40 mg tablets comprising the highest and lowest amount of salt were successfully prepared utilizing a single punch press applying a pressure of 10-11.5 kN for 30 sec. All developed tablets met the requirements of Ph. Eur. regarding hardness values ranging from 40-60 N.

[0882] Secondly, an orientating recovery study of the spray dried powder (SDP, containing H2B10B11) was carried out as visual precipitation was observed in different investigated aqueous media. Therefore, areas under the curve (AUCs) of endolysin in the spray dried and lyophilized form were compared leading to a recovery of spray dried endolysin ofapproximately 20%. A similar recovery was detected after injection in the HPLC system of the standard H2B10B11 dissolved in MES buffer (provided by the sponsor) and further diluted in SVF. Despite the lower recovery, however, no visual precipitation was observed. Additionally, the AUC of the lyophilized batch utilized for the preparation of the SDP analysed within a previous project, was compared with the AUC obtained from the spray dried API and a recovery higher than 85% was calculated. The results suggested a significant difference of the protein content within the two lyophilized batches generating the variation of recovery for the SDP. Within this project, H2B10B11 in MES buffer was selected as internal reference standard for the quantification of released endolysin.

[0883] Thereafter, the release of endolysin from 40 mg tablets obtained utilizing a single punch press (F3 without NaCI and Carbopol, F3 comprising Carbopol 974 P and NaCI concentration ranging from 0% to 12.5% as well as F3 comprising Carbopol 971 P and 5% of NaCI) was quantified after 4 h of incubation with 160 pl of simulated vaginal fluid (SVF). Efficient release of endolysin (63.87% ± 7.42%) was detected for the control formulation. Tablets comprising Carbopol 974 P and NaCI in a concentration of 0% and 1.5% exhibited lower release of approximately 18% and 30% suggesting strong interactions between the drug and Carbopol, as subsequently confirmed by H2B10B11-Carbopol interactions studies. In contrast, the interactions of the drug with Carbopol were significantly reduced by increasing the salt concentration reaching endolysin release up to 70%. Additionally, the activity of PM- 477 in the soluble as well as in the precipitate fractions was quantified via DRA. The results of the activity studies confirmed the release of H2B10B11 quantified via HPLC leading to a constant increase of the activity of H2B10B11 while increasing NaCI content up to 5%. However, increasing the salt concentration up to 12.5% (corresponding to 535 mM NaCI in 160 pl of SVF) was not beneficial for drug release. Based on the activity of the samples, 5% of NaCI (corresponding to 215 mM NaCI in 160 pl of SVF) is most likely sufficient to disrupt the interactions of the spray dried H2B10B11 with Carbopol 974 P.

[0884] 3.2 Methods

[0885] 3.2.1 Quantification of H2B10B11 via HPLC

[0886] For quantification of endolysin, a gradient HPLC method was applied (see Example 1). H2B10B11 dissolved in MES buffer in a concentration of 1.348 mg / ml (determined via Nanodrop) was used as internal reference standard. Therefore, four calibration standards covering the range from 750 to 125 pg / ml H2B10B11, obtained after proper dilution of the standard stock solution with simulated vaginal fluid (SVF, 3.51 mg / ml NaCI, 1.40 mg / ml KOH, 0.22 mg / ml Ca(OH)2, 2.00 mg / ml lactic acid, 1.00 mg / ml acetic acid, 0.40 mg / ml urea, 0.018 mg / ml bovine serum albumin and 5.00 mg / ml glucose) were injected to calibrate the instrument. Based on the following equation where / stands for the response of the instrument (AUC), m stands for the gradient of the line and cstands for its intercept with the y-axis, the concentration of the API was calculated (x):

[0887] 3.2.2 Recovery studies of H2B10B11

[0888] In order to quantify the amount of solubilized protein in SVF, the spray dried H2B10B11 was suspended in a final endolysin concentration of 500 pg / ml (corresponding to 5200 pg / ml of SDP). Subsequently, samples were centrifuged at 12,500 RCF for 10 min and the supernatant was analysed via HPLC. For comparison reasons, H2B10B11 dissolved in MES buffer (drug loading of ^1.3 mg / ml, based on nanodrop quantification by the sponsor) was analysed after dilution with SVF reaching a final drug concentration of 162.5 pg / ml. Recovered API was calculated comparing the AUCs of the spray dried drug with values obtained from previous analysed lyophilized batches. 3.2.3 Quantification of H2B10B11 via dye release assay

[0889] The dye-release assay was utilized to determine the activity of the endolysin in a cell-free assay. Based on the endolysin activity, the Remazol brilliant blue R-labeled Gardnerella (Gv9) substrate (RBB-S) is hydrolyzed and released Remazol brilliant blue R (RBB) can be quantified optically. The protocol for the assay was based on the protocol provided in Farris et al. (J Vis Exp. 2016; (110): 53819). In brief, calibration standards of H2B10B11 (dissolved in MES buffer) were prepared ranging from 3.125 ng / ml to 50 ng / ml in SVF pH 6.0. To evaluate the activity of the endolysin, 10 pl of samples containing endolysin as well as 10 pl of RBB-S stock solution were combined in Eppendorf tubes. Thereafter, 80 pl of 22.5 mM Na-phosphate pH 7, 56.25 mM NaCI buffer were added and samples were incubated for 90 min at 37 °C while shaking at 180 rpm (Thermomixer compact, Eppendorf, Austria). As negative control, buffer without endolysin was used. To stop the reaction, 25 pl of EtOH were added to each sample. Resulting mixtures were vortexed, centrifuged for 5 min at 12,500 RCF and 90 pl of the supernatant were transferred into a 96 well plate without dipping into the pelleted substrate. The optical density (OD) was measured at 595 nm with a multimode plate reader (Infinite 200 Pro (M Nano+), Tecan, Austria). The activity was calculated according to the following equation: 100 where AU stands for activity units, XOD is the mean OD of the negative control samples at 595 nm and is the OD of samples containing endolysin.

[0890] 3.2.4 Investigation of compressibility

[0891] In order to evaluate compressibility of formulations containing different amounts of NaCI, powder blends were prepared preliminary without the API (Table 13). Based on the previously developed tablet formulation F3, increasing amounts of NaCI up to 12.5% (w / w) were incorporated and the percentages of Avicel PH101 exhibiting only minor influences on osmolality were decreased. In brief, powder blends were prepared mixing excipients by geometric dilution for 5 min utilizing mortar and pestle. Thereafter, tablets of approximately 115 mg were prepared by direct compression using an excenter press. In addition, 40 mg tablets containing the lowest and highest concentration of NaCI were prepared by direct compression utilizing a single punch press applying a pressure of 10-11.5 kN for 30 sec. In order to ensure reproducibility of the manufacturing process, tablets compressibility was assessed visually and characterized regarding hardness using a Schleuniger 2-E / 205 tablet-hardness tester (Dr. K. Schleuniger and Co., Switzerland).

[0892] Table 13: Composition [w / w %] of powder blends for compressibility evaluation. MCC: microcrystalline cellulose.

[0893] HPMC: Hydroxypropyl methylcellulose. *Carbopol 947P and Carbopol 971 P were evaluated.

[0894] 3.2.5 Carbopol-H2B10B11 interaction studies

[0895] The interaction of endolysin with the two utilized Carbopol polymers was investigated. In brief, Carbopol 974 P as well as Carbopol 971 P were suspended in SVF in a concentration of 1.25 mg / ml. Thereafter, 160 pl of each Carbopol suspension were added to the SDP in order to have the same ratio Carbopol: SDP applied in tablet formulations. Samples were incubated at 37 °C while shaking at 300 rpm over 240 min and treated as described in the drug release studies reported in Section 3.2.6.

[0896] 3.2.6 Drug release studies

[0897] The influence of different NaCI concentrations on the release of endolysin was investigated modifying the set-up of the orientating drug release studies. Thus, powder blends F30% formulations containing Carbopol 974 P and increasing concentrations of NaCI as well as F35% were prepared and pressed utilizing a single punch press into 40 mg tablets (Table 14). In the following, the release behaviour of H2B10B11 from tablet formulations was evaluated by placing the tablets in 1.5 ml glass vials without the cellulose filter. Thereafter, 160 pl of SVF pH 6.0 preheated to 37 °C were added to each 40 mg tablet retaining the same ratio tablet:SVF. Samples were incubated at 37 °C while shaking at 300 rpm. After 240 min of incubation, aliquots of 100 pl were withdrawn, centrifuged (12,500 RCF for 10 min) and the supernatant was separated from the precipitate. Released and active drug in the soluble fractions was quantified via HPLC as well as via dye-release, respectively (as described in Section 3.2.3). In addition, the amount of drug entrapped in the precipitate of F3 tablet formulations containing C974 P was quantified via DRA assay. Therefore, 100 pl of fresh SVF were added to each precipitate, vortexed for 30s, centrifuged for 10 min at 12,500 RCF and the activity of remaining endolysin in the supernatant was quantified. Drug concentrations were calculated based on calibration curve in SVF of the standard H2B10B11 in MES buffer. Table 14: Composition [%] of tablets for drug release studies. F30% F3i.s% F33% F35% F3S% and F3i2.5% NaCI correspond to 0 mM, 64.17 mM, 128.34 mM, 213.89 mM, 342.23 mM and 534.74 mM NaCI, respectively, in simulated vaginal fluid (40 mg tablet / 160 IJI SVF).

[0898] * Considering 9.6% of API load in the final spray dried powder.

[0899] ** NaCI was grinded with mortar and pestle prior incorporation.

[0900] *** As the spray dried powder consists of approximately 9.6% H2B10B11, 4.1% sodium acetate / acetic acid and 86.3% mannitol, the amount of Compressol (mannitol / sorbitol mixture) was reduced from 50% to 44.79% and 5.21% of the spray dried powder were incorporated.

[0901] **** Carbopol 974 P was utilized for F3 containing NaCI ranging from 0% to 12.5%, whereas Carbopol 971 P was investigated only with F3 containing 5% NaCI.

[0902] 3.2.7 Statistical data

[0903] All studies and tests were carried out with n=3 for each experimental setup unless otherwise stated. Statistical data analyses were performed using the Student t-test with p < 0.05 as the minimal level of significance.

[0904] 3.3 Results

[0905] 3.3.1 Quantification of H2B10B11 via HPLC

[0906] For quantification of H2B10B11, an HPLC method previously established was applied. The standard stock solution H2B10B11 in MES buffer was utilized to establish the calibration in SVF pH 6.0. The HPLC method provided sufficient accuracy within the investigated range underlined by the correlation coefficient of 1.00.

[0907] 3.3.2 Recovery studies of H2B10B11

[0908] In order to gain better knowledge on the solubility of SDP in SVF, orientating recovery studies were carried out. Precipitation was visually observed after dispersion of the SDP in different aqueous medium (water, SVF and MES buffer) even at concentration of 125 pg / ml suggesting insufficient solubility. Thereby, the amount of solubilized API in SVF was quantified via HPLC and resulting AUC compared to the value obtained by a previous established calibration curve of the lyophilized powder (assumed to contain 38.74% of protein content). A low recovery of 21.21% was detected for the SDP. Additionally, the standard H2B10B11 in MES buffer was injected in the HPLC system after dilution with SVF (no visible precipitation observed) in a final concentration of 162.5 pg / ml. Utilizing the lyophilized drug as reference, the MES buffer sample exhibited a similar recovery to the SDP of 27.95%.

[0909] Subsequently, AUCs obtained from a previous analysed sample of lyophilized powder and the spray dried API prepared utilizing the mentioned batch of lyophilized drug were compared and higher recovery of 86.29% was detected. The results suggested a significant difference of protein loaded in the different lyophilized batches influencing the recovery of the SDP.

[0910] 3.3.3 Quantification of H2B10B11 via dve release assay

[0911] Activity of H2B10B11 was evaluated based on the dye release assay. A calibration curve of H2B10B11 standard diluted in SVF pH 6.0 achieved sufficient linearity in the range of 3.125 ng / ml to 50 ng / ml indicated by an R2of 0.984.

[0912] 3.3.4 Investigation of compressibility

[0913] As summarized in Table 15, all developed tablets without the API pressed either by excenter press (115 mg tablets) or single punch press (40 mg tablets) were characterized regarding tablet weight as well as hardness. Firstly, a reduction of the cohesive properties of tablets pressed by excenter press was observed with increasing NaCI above 5%. This was furthermore underlined by a decrease of mass and therewith along in hardness reaching values of approximately 20 N. In order to optimize compressibility of tablets F38% Naci and F3 12.5% Naci containing both Carbopol polymers, NaCI was grinded prior the incorporation in the powder blends and the pressure applied on the upper punch was increased (no values of applied pressure available). Secondly, 40 mg tablets F3 0% Naci control and F3 12.5% Naci comprising C974 P and C971 P were prepared by direct compression utilizing a single punch press (applied pressure of 10-11 kN for 30 sec). All developed tablets met the requirements of Ph.Eur. in hardness by exhibiting values in the ranging of 40-60 N.

[0914] Table 15: Characterization of tablet formulations. Indicated values are means of n=3 ± SD. ♦Higher pressure applied.

[0915] Tablets weight Tablet press Carbopol Formulation Hardness [N]

[0916] [mg]

[0917] 3.3.5 CarbODOl-H2B10Bll interaction studies

[0918] To investigate the interactions between the spray dried drug with Carbopol 974 P as well as Carbopol 971 P, the recovery of the drug after incubation with Carbopol suspensions, applying the same ratio of H2B10Bll:Carbopol from drug release studies was investigated. As shown in Figure 8, only 14.53% and 13.18% of free endolysin was recovered after 240 min of incubation with Carbopol 974 P and Carbopol 971 P, respectively, suggesting strong interactions between endolysin and anionic polymers.

[0919] 3.3.6 Drug release studies

[0920] Release of the endolysin from 40 mg tablets in SVF was tested after 4 hours of incubation . The release behaviour of endolysin from all developed tablet formulations is illustrated in Figure 9. After 240 min of incubation, 63.87% ± 7.42% of free H2B10B11 was detected via HPLC in the soluble fraction from tablets F30% Naci control without NaCI and without Carbopol. Compared to the control, tablets F30% iw showed a limited release of 18.66% ± 1.48%, confirming the pronounced interactions of endolysin with the polymer (as mentioned above). In contrast, the interactions between endolysin and Carbopol 974 P were significantly reduced by increasing the salt concentration emphasized by an increase of drug release up to 32.77% ± 1.66%, 54.86% ± 5.44%, 69.71% ± 5.96%, 65.95% Comparably, 63.59% ± 2.77% was released from tablets containing 5% NaCI and Carbopol 971 P. The highest release of Carbopol 974 P Carbopol 971 P around 70% was observed with tablets comprising the 5% of NaCI corresponding to approximately 215 mM NaCI concentration in 160 pl of SVF. However, further increase of NaCI concentration up to 535 mM (corresponding to the NaCI concentration in tablets F3 in 160 pl of SVF) did not further improve the release of the drug implying a saturation of the provided effect by NaCI.

[0921] Additionally, the activity of H2B10B11 in the soluble fractions was explored (Figure 10). The outcome of the activity studies confirmed the HPLC results underlining improved activity while increasing the NaCI concentration (activity up to 70 % for F3 5% Naci C974 p). Therefore, released endolysin (measured via HPLC) was active (confirmed via DRA). Finally, residual endolysin entrapped in the precipitates obtained after centrifugation was analysed revealing an activity lower than 6% for all investigated tablets. Based on the above-mentioned results, NaCI concentration corresponding of 5% in tablets might be sufficient to suppress the interaction of endolysin with Carbopol.

[0922] 3.4 Conclusion

[0923] It was the aim of this project to investigate the influence of different NaCI concentrations on the release as well as activity of the spray dried endolysin from vaginal tablets comprising Carbopol polymers. As underlined by previous studies as well as Carbopol interaction studies, the anionic polymers Carbopol 974 P and Carbopol 971 P strongly interact with endolysin leading to a low amount of released and detectable API in the soluble fraction. By increasing the amount of NaCI incorporated in tablet formulations, enhanced endolysin release and activity was observed. Tablets comprising 5% of NaCI led to the highest release and activity of endolysin up to 70% suggesting an overall concentration of 215 mM of NaCI in the release medium as sufficient to suppress H2B10B11 interactions with Carbopol. This might be related to the increase of ionic strength attributed to NaCI reducing the interactions between endolysin and Carbopol. Nevertheless, further increase of NaCI content in tablet formulations up to 12.5% did not further improve drug release / activity.

[0924] Example 4: H2B10 endolysin is a highly active antibacterial enzyme that targets the genus Gardnerella and to a minor extent Lactobacillus iners

[0925] Bacterial vaginosis (BV) is characterized by an imbalance of the vaginal microbiome and a characteristic biofilm formed on the vaginal epithelium, which is initiated and dominated by Gardnerella bacteria. The inventors previously disclosed synthetic endolysins, originated from the type 1,4-beta-N-acetylmuramidase encoded on Gardnerella prophages (WO 2020 / 225335). These endolysins efficiently kill Gardnerella bacteria and to a minor extent also L. iners, another opportunistic pathogen in BV. Importantly, probiotic Lactobacilli such as L. crispatusan L. gasseri are not harmed by the treatment of H2B10. The inventors tested the killing activity of a representative endolysin H2B10 on Gardnerella vaginalis.

[0926] Gardnerella bacteria in suspension (OD=0.1) were treated with the endolysin H2B10 (stock solution 700 pg / ml H2B10 in 50 mM MES pH 5.5, 200 mM NaCI, 8 mM MgSCM) or with a buffer control and incubated anaerobically for 1, 5, and 24 hours. After the incubation period, the suspension was serially diluted, spotted on Chocolate agar plates, and the CFU / ml were counted. The efficacy was clearly time and dose dependent and already low concentrations of 10 and 40 pg / ml after 5 hours incubation reduced the cell number to the limit of detection (LOD). No regrowth was observed after 24 h. Interestingly, no further killing was recorded between 5 h and 24 h when treated with very low concentration of 2.5 / jg / m\, indicating that an equilibrium may have formed. The inventors also tested the activity of H2B10 on other vaginal opportunistic pathogens as well as probiotic vaginal Lactobacilli. H2B10 proved to be specific for Gardnerella bacteria without effecting beneficial Lactobacilli or other opportunistic BV pathogens. The only exception was L iners, where high concentrations of 100 pg / ml after 5 hours reduced the number of bacteria by 2.5 loglO units. L. iners clearly differs from other Lactobacilli regarding cell wall constitution and metabolism and is considered as a pathogen in BV.

[0927] In summary, a strong bactericidal effect against Gardnerella was demonstrated for the recombinantly expressed endolysin H2B10 without harming L. crispatus, L. gasserian^ L. jensenii, which are the most prevalent species in a healthy vaginal microbiome (see Figure 11).

[0928] Example 5: Poly acrylic acid (PAA) as an excipient enhances the killing effect of endolysins on Gardnerella bacteria growing as biofilm

[0929] The inventors tested PAA (Sigma Aldrich, catalog number 323667-100G) as a vehicle for the Gardnerella-s ea c endolysins for the use of intravaginal application. The viscosity and mucoadhesive property of PAA guarantee a slow release of the active pharmaceutical ingredient (the endolysin) on the mucus layer in the vagina and its acidifying effect helps to restore the healthy vaginal microbiome. Unexpectedly, the presence of PAA also enhanced the potency of endolysins and Gardnerella bacteria were killed more efficiently. The inventors tested the Carohere / Za-specific endolysins PM-477 (H2B10B11) and H2B10 formulated with PAA on in vitro mono-species Gardnerella biofilms. The biofilms (ff. vaginalis ATCC 14018) were generated in sBHIG (in g / L: Brain Heart Infusion: 37, gelatine: 20, yeast extract: 5, starch: 1, glucose: 2.5; water: up to 1000 mL) pH 7 for 48 hours on pre-coated tissue culture plates, then treated in sBHIG pH 4.5 for 24 hours with low concentrations of the endolysin H2B10 together with PAA, as well as H2B10 and PAA alone. Alternatively, the biofilms were generated in sNYC (in g / L: HEPES: 2.4, proteose peptone: 15, yeast extract: 3.8, NaCI: 5, glucose: 10; water: up to 1000 mL) pH 7 for 48 h, and were then treated with H2B10B11 or PAA or a combination of both in sNYC pH 6 for 24 h.

[0930] For the biofilm treatment with P and H2B10, a PM master stock (27% (w / v)) was used to prepare a 0.35 % PM (v / v) working stock, by dilution with sBHIG pH 5, containing 8 pg / ml H2B10 (H2B10 master stock of 700 pg / ml was used for dilutions). In addition, the final mixture of H2B10 with the excipient PM used for the biofilm treatment had pH 4.5 due to the buffering effect of 0.35% PM. For the biofilm treatment with PM and H2B10B11, a PM master stock (14.5 % (w / v), adjusted to pH 6) was used to prepare a treatment solution containing 0.25 % PM and 32 pg / mL H2B10B11 in sNYC pH 6. After incubation, the biofilm was washed and dislodged by vigorous pipetting. A serial dilution was done in PBS (lxPBS - DPBS, no calcium, no magnesium, Thermo Scientific, cat. no. 14190169) and spotted on Chocolate Agar plates (BD, cat. no. 254060) to count the number of viable cells (CFU / ml). For the tested strains, the Gardnerella biofilm eradication concentration of H2B10 was between 2 and 32 pg / ml. The concentration chosen herein was 8 pg / ml, and thus at the lower end of the range of the minimum biofilm eradication concentration (MBEC) for Gardnerella strains, to be able to see a potential synergistic effect of the excipient PM and H2B10. It is to be noted that the MBEC is influenced by the medium and pH used for the experiment. Since the biofilm experiment with H2B10B11 was carried out at pH 6, 32 pg / mL H2B10B11 were not sufficient to eradicate the biofilm in the absence of PM. H2B10 (8 pg / ml) alone reduced the CFU / ml by 1.6 logw units, PAA (0.35% w / v) alone by 0.6 logw units, while the combination of H2B10 and PAA resulted in a clearly synergistic effect with killing rates of 3 log io units. The difference to the potential additive effect (0.6 logw units of PAA and 1.6 logw units of H2B10 = 2.2 logw unit reduction) was 0.8 log units (3 log units in total) which translates into a 6-fold increased activity on a linear scale (Figure 12A).

[0931] No relevant effect was observed with H2B10B11 (32 g / mL) alone in medium at pH 6. PAA (0.25 % w / v) alone reduced the CFU / mL by 0.9 logw units, while the combination of H2B10B11 and PAA resulted in an obvious synergistic effect with a log reduction of 3.1 (Figure 12B).

[0932] Example 6: PAA as an excipient enhances the killing effect of endolysins on Gardnerella bacteria and other BV pathogens growing in the planktonic form

[0933] Next, the potency of two different formulations of H2B10 with 0.25% PAA (w / v; Sigma Aldrich, catalog number 323667-100G) as an excipient was tested against six BV associated pathogens and one commensal Lactobacillus species in a time-kill assay (Figure 13). Both formulations included 2 pg / mL H2B10 and 0.25% PAA and were adjusted to either pH 4.5 (Figure 13A) or pH 5 (Figure 13B) to simulate different PAA types and composition. Briefly, 4 g of PAA was added to 10 mL of dH2O and heated to 50 °C while mixing on a heated stir plate. The pH of PAA solutions was adjusted to either 4.5 or pH 5 using 5 M NaOH. Then, the volumes were adjusted to 27.5 mL (corresponding to 14.5 % PAA) and sterile filtered using a 0.22 pm syringe filter. These stocks were diluted to a final concentration of 0.25 % PAA in glucose-supplemented New York City Broth (sNYCB) adjusted to the respective pH. Supplemented NYCB consists 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 o-D-glucose (Sigma Aldrich)).

[0934] Bacteria were grown on Chocolate Agar plates anaerobically for 48h at 37°C, then scraped from the plates, and suspensions of 108CFU / ml were prepared for the experiment. For the time-kill experiments, the bacterial suspensions were mixed with the respective treatment stock solutions and incubated anaerobically for 5h at 37°C. Then, the surviving bacteria were quantified by spotting 2 pL of a 10-fold dilution series (lowest dilution 10s) on Chocolate Agar plates after anaerobic incubation at 37°C for 48h.

[0935] Both formulations were highly effective in reducing the bacterial load of all six pathogens but had no negative effect on the commensal L crispatus (Figure 13). The formulation set to a slightly more acidic pH (4.5) reduced viable Gv9 (6. vaginalis ATCC 14018), L. iners, M. mulieris, by > 3 logw and A. vaginae, and P. bivia by > 2 logw when compared to the controls (Figure 13C). When formulated to a less acidic pH (5.0), 2 pg / mL H2B10 reduced Gv24 (G. swidsinskii GS 9838-1), L. iners, and P. bivia by > 3 logw and Gv9, M. mulieris, and A. vaginae by > 2 logw when compared to the controls without having any negative affect on the commensal L. crispatus (Figure 13C).

[0936] In summary, formulation with PAA surprisingly extends the spectrum of activity of H2B10 to multiple BV-associated pathogens other than Gardnerella.

[0937] Example 7: PAA alone has no killing effect on Gardnerella and a weak killing effect on some other BV pathogens, but only at higher concentrations

[0938] Next, the effect of PAA (Sigma Aldrich, catalog number 323667-100G) alone on Gardnerella and other BV-associated pathogens was assessed. As described in the previous Examples, bacterial suspensions were adjusted to 108 CFU / ml, mixed with treatment stock solutions and incubated anaerobically for 90 minutes at 37°C, after which surviving bacteria were quantified by spotting of a 10-fold dilution series of the reactions. The spotting plates were incubated for 2-3 d prior to determination of CFU / mL. As shown in Figure 14, PAA concentrations up to 1% did not reduce the viability of Gardnerella after incubation for 90 min.

[0939] The sensitivity of five other BV associated pathogens and one commensal Lactobacillus species to PAA was also tested using the same assay. The four BV associated pathogens tested were L. iners, P. bivia, A. vaginae, and M. mulieris, while L. crispatus was used as the commensal (Figure 15). The inventors tested concentrations of PAA ranging from 0.01% to 1% set to pH 5 on all the five organisms using 90 minute potency assays. PAA concentrations up to 1% had minimal influence (< 0.6 logw decrease) on the viability of M. mulieris an L. crispatus. Viability of P. bivia, and A. vaginae decreased by 1.8 and 1.7 logw only in response to high concentrations of PAA (>0.5%), with lower concentrations having no effect (Figure 15). Viability of L. iners decreased by ~1.1 loglO in response to the lowest tested concentrations of PAA (0.01%) and did not decrease further with higher PAA concentrations (Figure 15).

[0940] Example 8: Carbopol 974P as an excipient enhances the killing effect of endolysins on planktonic BV-associated pathogens but not on the commensal Lactobacillus species

[0941] The potency of H2B10B11 formulated with 0.25 % (w / v) Carbopol 974P NF Polymer (CP 974; The Lubrizol Corporation) was tested against five BV-associated pathogens and one commensal Lactobacillus species in a planktonic setting for 5 h in sNYC medium at pH 5 (Figure 16). The formulation comprised 2 pg / mL H2B10B11 and 0.25 % CP 974P with a final pH of 5. To yield the formulated treatment solution, 50 mg CP 974P were added to 15 mL sNYC pH 5. After mixing, the pH was re-adjusted to pH 5 using 5 M NaOH, and the volume adjusted to 20 mL with sNYC pH 5. H2B10B11 was added to a final concentration of 2 pg / mL from a 1.343 mg / mL stock in MES pH 5.5 buffer.

[0942] Gardnerella was grown on a Chocolate Agar plate, the other bacterial strains on Schaedler Agar plates with vitamin KI and 5 % sheep blood (BD, catalog number 254042) anaerobically for 48 h at 37 °C, then scraped from the plates, and suspensions of 108CFU / ml were prepared for the experiment. The bacterial suspensions were mixed with either sNYC pH 5 or with sNYC containing formulated H2B10B11 pH 5 and incubated anaerobically for 20.5 h at 37 °C. Then, the surviving bacteria were quantified by spotting 2 pL of a 10-fold dilution series (lowest dilution IO-5) on Chocolate / Schaedler Agar plates and anaerobic incubation at 37 °C for 48 sh.

[0943] Formulated H2B10B11 eradicated the BV-associated pathogens G. vaginalis, L. iners, M. mulieris, A. vaginae, and P. bivia (Figure 16). This is an unexpected observation, since the endolysin alone is not effective on non- Gardnereiia strains except for to a smaller extend on L. iners see Figure 11B). Since the results obtained here with Carbopol 974P are comparable to the results presented in Example 3 (Figure 13), in which the formulation comprised PAA, it can be concluded that poly(acrylic acid) and its cross-linked pharmaceutical-grade form (carbomer) lead to a synergistic extension of lytic activity of the endolysin. Furthermore, likewise to H2B10 formulated with PAA, H2B10B11 formulated with CP 974P is not harming the beneficial L. crispatus (Figure 16).

[0944] Example 9: Single-dose PK study in sheep

[0945] Five osmotically different variants of vaginal tablet formulations (10 mg H2B10B11 active substance each) were compared to evaluate differences in dissolution and distribution of API within the vagina of sheep. As shown in Table 16, all five variants of vaginal tablets contained the same excipients. However, they varied in the concentration of mannitol / sorbitol mixture (10% to 50%), which drives the osmolality, and in the concentration of hydroxypropyl methylcellulose (HPMC, 5% or 10%), which is a gelling agent. Note, that in the final formulation, HPMC may be replaced with hydroxyethyl cellulose (HEC) to improve long-term storage properties.

[0946] Table 16: Composition (weight%) of tablets (1 g total weight) used in the single-dose sheep study

[0947] Component Function F3 F5 F7 F8 F9

[0948] H2B10B11 lyophilized Active substance 2.6 2.6 2.6 2.6 2.6

[0949] Sodium chloride Osmotic active agent 5 5 5 5 5

[0950] Microcrystalline cellulose Filler 34.9 54.9 74.9 29.9 49.9

[0951] Co-processed polyol with Filler 50 30 10 50 30 mannitol and sorbitol

[0952] Carbomer homopolymer

[0953] Mucoadhesive polymer 0.5 0.5 0.5 0.5 0.5 type B

[0954] HPMC Mucoadhesive polymer 5 5 5 10 10

[0955] Magnesium stearate Lubricant 1 1 1 1 1

[0956] Aerosil Flow regulation 1 1 1 1 1

[0957] Abbreviations: HPMC = hydroxypropylmethyl cellulose.

[0958] Individual sheep (n=10) were administered with single tablets intravaginally. The sheep were assigned to groups of n=2 individuals. Each group received two different formulations with a 3-week wash-out period between administrations, resulting in n=4 for each formulation variant.

[0959] Vaginal fluid was sampled by swabs using a custom-made swabbing device. Swab-sampling was done at 5 cm (outer location) and 10 cm (inner location) from the vulva (vaginal entrance) and at multiple time points (1 h, 2 h, 4 h, 8 h and 24 h post-dose), to measure the local concentration of H2B10B11 active substance. Formulation F3 had the best combination of PK properties with a median Cmaxinner=3400 pg / mL and median Qnax outer=3230 pg / mL indicating an even distribution along the vaginal canal. The median AUC0-24 for H2B10B11 F3 formulation at the inner location was 6929 pg*h / mL, and at the outer location 6478 pg*h / mL (Figure 17). The MED=768 pg*h / mL was defined as the theoretical minimum effective AUC for in vitro biofilm eradication (MBECg0=32 pg / mL) times 24 h. The median margin over the MED (ratio of the measured AUC0-24 and the MED) was 9.02 for the inner, and 8.43 for the outer location (Figure 17). The ti / 2of H2B10B11 active substance determined in sheep was 1.7-2.6 h.

[0960] Blood was drawn from sheep at the same time as the vaginal swabbing was performed, to assess a potential systemic exposure. None of the plasma samples of animals administered with vaginal tablets contained concentrations of H2B10B11 active substance above the LLOQ. The LLOQ of the sandwich ELISA was 1.6- 6.3 ng / mL. In summary, this study demonstrates that locally administered H2B10B11 does not lead to measurable systemic exposure in sheep, and that the LLOQ was sufficiently low to exclude pharmacologically relevant concentrations of active substance. Local tolerability was determined by visual inspection of the vaginal entrance at each timepoint of swabbing. After the 24 h sampling point, a speculum was inserted to check for local reactions in the vaginal tract and the vulva. There were no macroscopic signs of local intolerance noted. In summary, five different formulation variants of H2B10B11 were tested. Systemic exposure was not detected, and no adversities were observed. Local vaginal concentrations of H2B10B11 active substance in sheep were well above the MBEC for 6-7 h, and above MIC for 7-8 h. Cmax values in sheep vaginal fluid after application were between 1300 and 3400 pg / mL. The area under the plasma concentration -time curve over the 24 h dosing interval (AUC0-24) was estimated to be 6478 and 6929 pg*h / mL for H2B10B11 F3 formulation at the outer and inner location, respectively. The corresponding median margin over the MED values were 8.43- and 9.02-fold. The estimated halflife of H2B10B11 active substance in sheep vaginal fluid was 1.7-2.6 hours.

[0961] While this ti / 2is comparably short, it is known that the concentration of proteases in sheep vaginal fluid is up to ten times higher than in humans (Acarturk et al., J Pharm Pharmacol. 2001, 53(ll):1499-504). Spiking human vaginal fluid with purified H2B10B11 endolysin ex vivo indicated that the ti / 2 of H2B10B11 active substance in this matrix is in the range of 6.3 h to 23 h, while H2B10B11 active substance spiked into sheep vaginal fluid had a ti / 2of 1.5-2.6 h, consistent with the observation in vivo. The PK data determined for formulation F3 at the outer vagina location were used for fitting a one-compartment zero order PK / PD model to predict the efficacy of different doses of H2B10B11 active substance with an assumption of a prolonged ti / 2in human vaginal fluid (8-15 h) compared to sheep vaginal fluid. The model indicated that doses of 5 mg and 2 mg would be sufficient for efficacy in humans, as the yielded margins over the MED would be in the ranges 16-19- and 6-8-fold, respectively.

[0962] The excipient HPMC may be replaced with HEC to improve stability of the product during manufacturing. The performed in wfro HEC-HPMC comparison tests indicated no further effects on H2B10B11 other than stability, thus similar PK / tolerability data can be expected for H2B10B11 formulations comprising HEC instead of HPMC.

[0963] Example 10: Endolysins have no effect on mammalian cells in vitro

[0964] H2B10 (differs from H2B10B11 by one amino acid), at 500 pg / mL, was tested in HeLa and Ectl cells, and the survival, the reactive oxygen species (ROS) induction and the cell membrane leakage were assessed. H2B10 did not affect the survival of mammalian cells, nor induced any ROS or membrane leakage (Figure 18).

[0965] Human PMNs were stimulated for 24 h with 500 pg / mL of H2B10B11, and the concentrations of cytokines interleukins (IL)-ip, (IL)-6 and tumor necrosis factor-alpha in the supernatant were assessed with an enzyme- linked immunosorbent assay (ELISA). No significant induction of cytokines was observed for H2B10B11 (Figure 19).

[0966] H2B10B11 was also tested for hemolysis on human erythrocytes. Upon treatment of human erythrocytes with 500 pg / mL of H2B10B11 for one hour, no lysis was observed, compared to the buffer control (Figure 20).

[0967] To conclude, H2B10B11 was non-hemolytic in human erythrocytes and did not trigger cytokine release in human polymorphonuclear leukocytes in vitro.

[0968] A protein toxin database was established in-house, composed of all known to date toxins, including, but not limited to, snake and spider venom factors, plant toxins, and bacterial virulence factors or protein superantigens. A basic local alignment search tool (protein-protein BLAST [BLASTp]) search of H2B10 against this comprehensive database retrieved no significant hits, suggesting that H2B10 and any derived peptides can be predicted to be non-toxic. Furthermore, BLASTp search of H2B10B11 sequence against human proteome retrieved no significant hits as well, indicating lack of homology to human proteins.

[0969] Example 11: PK / safety study in sheep

[0970] In this non-GLP PK / safety study in sheep, H2B10B11 was administered as a vaginal tablet formulation (Table 17) to five sheep for seven consecutive days at a dose of 10 mg H2B10B11 per day. The aim of the study was to assess local and systemic tolerability of H2B10B11 upon repeated administration. Sheep tolerated the treatment well, and no clinical observations or signs of poor tolerability of the vaginal tablets were noted. No systemic exposure above 5 ng / mL (LLOQ in a GLP validated ELISA assay) and no test item-related histopathological changes in vaginal tissue were found.

[0971] Table 17. Composition (weight%) of tablets (1 g) used in the repeat-dose sheep study

[0972] Component Function Weight (° / o)

[0973] H2B10B11 drug substance API 10.00

[0974] Sodium chloride Osmotic active agent 5

[0975] Microcrystalline cellulose Filler 30.16

[0976] D-Mannitol Filler 43.25

[0977] Carbomer homopolymer type B Mucoadhesive polymer 0.5

[0978] HPMC E5 Mucoadhesive polymer 5

[0979] Talc G lidant and flow regulator 5

[0980] Magnesium stearate Lubricant 1

[0981] H2B10B11 SDP contains 97.64 mg / g H2B10B11, 741.46 mg / g D-mannitol, 8.33 mg / g sodium acetate and 152.56 mg / g HPBCD.

[0982] Abbreviations: API = active pharmaceutical ingredient; SDP = spray-dried powder; HPBCD = 2-Hydroxypropyl-B- cyclodextrin; HPMC = hydroxypropylmethylcellulose.

[0983] Example 12: Local tolerability study (GLP)

[0984] A GLP-compliant, 10-day vaginal irritation study was performed in female New Zealand White rabbits. The objective of the vaginal irritation study was to assess local tolerability of H2B10B11 vaginal tablets (19x10.41 mm, Table 18) in rabbits following daily intravaginal administrations. Rabbits (n=4 / group) were dosed for 10 days with either H2B10B11 vaginal tablets or with placebo tablets; a sham control group was also included (treated with an applicator only). The H2B10B11 content in the H2B10B11 drug product was 20 mg / tablet.

[0985] Table 18. H2B10B11 DP and placebo tablet (both 1 g) composition used in the study

[0986] H2B10B11 drug substance

[0987] (H2B10B11 API 20.48 0

[0988] / Mannitol / HPBCD / sodium acetate) Sodium chloride Osmotic active agent 5 5

[0989] Microcrystalline cellulose Filler 34.66 34.66 D-Mannitol Filler 32.86 53.54

[0990] Carbomer homopolymer type B Mucoadhesive polymer 0.5 0.5 Hydroxyethyl cellulose (HEC) Mucoadhesive polymer 0.5 0.5 Talc Glidant and flow regulator 5 5

[0991] Magnesium stearate Lubricant 1 1

[0992] Abbreviations: API = active pharmaceutical ingredients; DP = drug product; HPBCD = 2-Hydroxypropyl-B- cyclodextrin.

[0993] The following parameters and endpoints were evaluated in this study: mortality, clinical signs, body weight changes, food consumption, dermal irritation scoring, uterus / cervix weights, and macroscopic and microscopic examination of the vagina. All animals survived to the scheduled necropsy and tolerated the treatments. There were no apparent test article-related clinical observations evident during the course of the study. No test article-related body weight effects, as well as no effects on food consumption were observed during the study. Administration of H2B10B11 tablets resulted in non-adverse microscopic findings (minimal to mild) within the vagina, associated with a mild increase in vaginal irritation scores (Table 19). Administration of placebo tablets led to similar, but less pronounced changes in the vagina with a minimal increase in the vaginal irritation scores. Procedure (applicator insertion)- related effects (minimal to mild) were present in all groups. No H2B10Bll-related gross findings were noted at necropsy.

[0994] Table 19. Summary of vaginal irritation scoring

[0995] Vagina, Cranial 1.25 2.25 6

[0996] Vagina, Mid 1.75 2.75 7

[0997] Vagina, Caudal 0.50 1.25 1.25

[0998] An average for each group / vaginal section was calculated by taking the sum of all grades in that group and dividing by the number of animals observed (4). The maximum average grade per group cannot exceed 16. An irritation index is defined as: 0 - none, 1 to 4 - minimal, 5 to 8 - mild, 9 to 11 - moderate, 12 to 16 - severe.

[0999] Example 13: Repeated-dose toxicity study (GLP)

[1000] H2B10B11 is formulated as a tablet intended for intravaginal administration, thus the species -specific properties of vaginas were considered in toxicological species selection. Based on the performed PK / tolerability studies and considering similarities in gross anatomy, structural and biomechanical properties, and wound healing between a human and a rat vagina, a pivotal GLP-compliant repeated-dose toxicity study was conducted in rats.

[1001] In this GLP-compliant repeated dose toxicity study, rats were dosed daily, for 2 weeks, with vaginal H2B10B11 tablets (4 mm diameter, see Table 20). A 3-week recovery period was included to assess the reversibility of any observed effects. Local tolerance was also evaluated in this study by detailed clinical observations, colposcopy, macroscopic and microscopic examination of the dosing site (vagina). Plasma samples for toxicokinetics were collected and analyzed on days 1 and 14 (see Table 21 for a study design). Table 20. H2B10B11 drug products and placebo (50 mg tablets) composition used in the study Amount in placebo (%)

[1002] H2B10B11 drug substance

[1003] 2 (0.1 mg); 6 (0.3 mg);

[1004] (H2B10B11 / Mannitol / HPBCD / API 0

[1005] 20 (1 mg) sodium acetate)

[1006] Sodium chloride Osmotic active agent 5 5

[1007] Microcrystalline cellulose Filler 34.66 34.66

[1008] 51.29 (0.1 mg); 47.2

[1009] D-Mannitol Filler 53.34

[1010] (0.3 mg); 32.86 (1 mg)

[1011] Carbomer homopolymer

[1012] Mucoadhesive polymer 0.5 0.5 type B Hydroxyethyl cellulose (HEC) Mucoadhesive polymer 0.5 0.5

[1013] Talc Glidant and flow regulator 5 5

[1014] Magnesium stearate Lubricant 1 1

[1015] Abbreviations: API = active pharmaceutical ingredients; HPBCD = 2-Hydroxypropyl-B-cyclodextrin. Table 21. Design of the GLP-compliant repeated-dose toxicity study

[1016] Test item 4 mm H2B10B11 DP (0.1, 0.3, 1 mg) and H2B10B11 placebo

[1017] Test system Sprague-Dawley rat, females

[1018] Administration Daily for 14 days followed by a 3-week recovery period

[1019] Route Intravaginal

[1020] Dose groups 1. Control (Placebo) - 0 mg

[1021] 2. Low dose - 0.1 mg

[1022] 3. Intermediate dose - 0.3 mg

[1023] 4. High dose - 1 mg

[1024] Group size Group 1 to 4 (Toxicology): 10 / group + 5 recovery / group 1 and 4

[1025] Group 1 to 4 (Toxicokinetics): 3 / group

[1026] Necropsy Day 15- main phase animals

[1027] Day 36 - recovery phase animals

[1028] Abbreviations: DP = drug product; GLP = Good Laboratory Practice.

[1029] All rat plasma samples displayed levels of H2B10B11 below the limit of quantification (2.5 ng / mL), and therefore systemic exposure was not detected. H2B10B11 was tolerated up to the highest dose level tested (1 mg / animal). No mortality was observed during the treatment or recovery period. No test item-related clinical observations were recorded during the 14 days of treatment or delayed effects at completion of the 3 weeks of recovery period in female rats at all the doses tested. No test item-related changes in body weights and no test item-related changes in food consumption were recorded during the treatment nor at the recovery period in rats at all the doses tested. Histopathology findings noted in rat vagina (minimal to moderate multifocal inflammatory cell infiltrates within the vaginal wall) and in iliac lymph nodes (minimal to mild increase in lymphoid cellularity) at >0.1 mg / day displayed no dose-relationship and exhibited almost full reversibility. Based on the mild severity and transient nature of the test item-related findings the local No Observed Adverse Effect Level (NOAEL) of H2B10B11 was established as 1 mg / day in rats.

[1030] Overall, based on the absence of adverse effects and systemic exposure upon vaginal administration of H2B10B11 tablets in the pivotal GLP-compliant repeated-dose toxicity study in rats and considering similarity in physiology of rat vaginal mucosa to human vaginal mucosa, the risk for systemic toxicity in humans is low. Based on the mild irritation scores established for H2B10B11 DP in the GLP-compliant local tolerability study in rabbits (Example 12), as well as in the absence of macroscopic pathology in rat vaginas, the risk for local (vaginal) toxicity in humans is considered to be low. Data from non-clinical safety studies therefore indicate a favorable benefit / risk profile of the H2B10B11 formulation.

[1031] Example 14: A First-in-human Safety Trial of a H2B10B11 DP Administered as Single Ascending Doses in Healthy Women and as Multiple Ascending Doses in Women Diagnosed With Bacterial Vaginosis

[1032] This is a two-part, randomized, double-blind, placebo-controlled study to assess the safety, tolerability, pharmacokinetics, pharmacodynamics, and efficacy (for Part B) of a H2B10B11 drug product (DP) in healthy women (Part A) and in women diagnosed with bacterial vaginosis (BV) (Part B).

[1033] Part A will include single ascending dose levels and will assess the safety of the H2B10B11 DP and describe the incidence of adverse events (AEs) for participants randomized at a ratio of 3: 1 to the H2B10B11 DP or placebo. Participants will receive one single dose of study treatment.

[1034] Part B will include multiple ascending dose levels. Participants will be randomized at a ratio of 2:1 to the H2B10B11 DP or placebo. Participants with BV will receive study treatment for five consecutive days.

[1035] The vaginal inserts will be self-administered by the participant. The participants will receive detailed instructions from the investigator on how to self-administer the vaginal inserts at home.

[1036] Table 22. Arms and Interventions

[1037] Outcome Measures

[1038] Primary Outcome Measure: 1. Part A - Percentage of participants with adverse events (AEs) with onset after first treatment dose and until 7 days post-dose

[1039] In participants who have received at least one dose of H2B10B11 DP or placebo. For each dose level cohort of H2B10B11 DP and for the combined placebo group. [Time Frame: from first dose of study treatment up to 7 days post-dose]

[1040] 2. Part B - Percentage of participants with adverse events (AEs) with onset after first treatment dose and until 120 days after the first dose

[1041] In participants who have received at least one dose of H2B10B11 DP or placebo. For each dose level cohort of H2B10B11 DP and for the combined placebo group. [Time Frame: from first dose of study treatment up to 120 days post-first dose]

[1042] 3. Part A - Percentage of participants with serious adverse events (SAEs) with onset after first treatment dose and until 7 days post-dose

[1043] In participants who have received at least one dose of H2B10B11 DP or placebo. For each dose level cohort of H2B10B11 DP and for the combined placebo group. [Time Frame: from first dose of study treatment up to 7 days post-dose]

[1044] 4. Part B - Percentage of participants with SAEs with onset after first treatment dose and until 120 days after the first dose

[1045] In participants who have received at least one dose of H2B10B11 DP or placebo. For each dose level cohort of H2B10B11 DP and for the combined placebo group. [Time Frame: from first dose of study treatment up to 120 days post-first dose]

[1046] Secondary Outcome Measure:

[1047] 5. Part A - Serum concentrations of H2B10B11 active substance at pre-specified timepoints

[1048] For each cohort. In participants who received one single administration. [Time Frame: from pre-dose up to 12 days post-dose]

[1049] 6. Part B - Serum concentrations of H2B10B11 active substance at pre-specified timepoints

[1050] For each cohort. In participants who received all scheduled administrations. [Time Frame: from pre-dose up to 30 days post-first dose]

[1051] 7. Part A - Anti-drug antibody (ADA) prevalence and change of binding titers against H2B10B11 active substance in blood before study treatment and at 7 days post-dose For each cohort. [Time Frame: from pre-dose up to 7 days post-dose]

[1052] 8. Part B - ADA prevalence or change of binding titers against H2B10B11 active substance in blood before study treatment and at 6 days after the first dose, 21 to 30 days after the first dose, and 120 days after the first dose

[1053] For each cohort. [Time Frame: from pre-dose up to 120 days post-first dose]

[1054] 9. Part B - Number of participants with clinical cure

[1055] For each cohort of H2B10B11 DP group and for the combined placebo group. Normalization of the vaginal discharge, a negative potassium hydroxide (KOH) "Whiff" test, and clue cells <20% of the total epithelial cells / high power field on microscopic examination of the vaginal fluid. |Time Frame: At 6 days post-first dose and 21 to 30 days after the first dose]

[1056] 10. Part B - Number of participants with Nugent score cure / Microbiological cure

[1057] For each cohort of H2B10B11 DP group and for the combined placebo group. Nugent score of <4. [Time Frame: At 6 days post-first dose and 21 to 30 days after the first dose]

[1058] 11. Part B - Responder outcome - Number of participants with clinical cure and normal Nugent score of <4

[1059] For each cohort of H2B10B11 DP group and for the combined placebo group. (Time Frame: At 6 days post-first dose and 21 to 30 days after the first dose]

[1060] Minimum Age: 18 Years

[1061] Sex: Female

[1062] Gender Based: No

[1063] Accepts Healthy Volunteers: Yes

[1064] Inclusion Criteria:

[1065] • Have given written informed consent by signing and dating the informed consent form (ICF) before initiation of any study-specific procedures.

[1066] • Participant reported assigned female sex at birth, at least 18 years of age and pre -menopausal, as determined by the investigator.

[1067] • Not menstruating or having vaginal bleeding:

[1068] • Part A and Part B: At Visit 0 and not expecting to menstruate during Visit 1 and until Visit 3.

[1069] • Part B only: At Visit 0 and Visit 1 and do not expect to menstruate within the next 6 days after Visit 1, until the Early Response Visit (Visit 2).

[1070] • Part A only: Are healthy according to screening procedures. Part B only: Participants suffering from BV but who are otherwise healthy in the clinical judgement of the investigator. • Note: Participants with pre-existing stable disease (e.g., obesity, hypertension, etc.), defined as disease not requiring significant change in therapy or hospitalization for worsening disease during the 90 days before Visit 0, can be included.

[1071] • Part A only: Should not have any clinical signs of BV as assessed by the absence of all Amsel's criteria and a normal Nugent score at screening, or other vaginal symptoms, including symptomatic vulvo-vaginal candidiasis (WC) or infection with sexually transmitted infection (STI) pathogens including Chlamydia trachomatis, Trichomonas vaginalis, or Neisseria gonorrhoeae.

[1072] • Able to participate in the study as an outpatient, to attend all required visits, and to comply with all study requirements.

[1073] • Women of childbearing potential must have a negative highly sensitive urine pregnancy test result prior to study treatment initiation.

[1074] • The participant must have been on the same form of highly effective contraception for at least 3 months prior to dosing (Visit 1) and must agree to keep this method until:

[1075] • Part A: 60 days after Follow-up Visit (Visit 3)

[1076] • Part B: at least 60 days after Test of Cure (ToC) Visit (Visit 3).

[1077] • Women of childbearing potential who agree not to donate or cryopreserve eggs (ova, oocytes) for the purposes of assisted reproduction during study:

[1078] • Part A: Within 3 months prior to dosing (Visit 1) and continuously until 60 days after Follow-up Visit (Visit 3)

[1079] • Part B: Starting at Visit 0 and continuously until the Late Follow-up Visit (Visit 5).

[1080] • Agree to abstain from vaginal intercourse:

[1081] • Part A: From 72 hours prior to dosing until the Follow-up Visit (Visit 3)

[1082] • Part B: For the duration of treatment (~5 days) and until ToC Visit (Visit 3).

[1083] • Agree to not use any vaginal products, e.g., creams, gels, foams, sponges, douches, and tampons (except during menstruation):

[1084] • Part A: From 72 hours prior to dosing until the Follow-up Visit (Visit 3)

[1085] • Part B: Until ToC Visit (Visit 3).

[1086] • Part B only: Have a clinical diagnosis of BV, defined as having all the following Amsel's criteria (4 / 4): a. Off-white (milky or gray), thin, homogeneous vaginal discharge. b. Vaginal pH >4.5. c. Presence of clue cells >20% of the total epithelial cells / high power field on microscopic examination of the vaginal saline wet mount. d. A positive 10% KOH Whiff test.

[1087] • Part B only: Have a sample collected within 72 h prior to first dose for a Gram stain slide to assess Nugent score by the central laboratory.

[1088] Exclusion Criteria:

[1089] • Pregnant, lactating, or planning to become pregnant during their study participation and for at least:

[1090] • Part A: 60 days after Follow-up Visit (Visit 3)

[1091] • Part B: 60 days after ToC Visit (Visit 3).

[1092] • Have genital lesions, including active herpes simplex virus or syphilitic lesions, or other vaginal or vulvar conditions.

[1093] • Part A only: Have active STI. • Had received antifungal or antimicrobial therapy (in Part A, systemic or topical; in Part B, systemic or vaginal) within 14 days prior to the Visit 1.

[1094] • Are using a Copper intrauterine device, or any vaginal hormonal products (including NuvaRing®) as a form of contraception.

[1095] • Had a history of drug or alcohol abuse within the past 12 months, as determined by the investigator.

[1096] • Had participated in any investigational study within 30 days before the Visit 1 or is currently participating or plans to participate in any investigational, or observational study.

[1097] • Has any history of allergies, hypersensitivities, or intolerance to the study treatments including any excipients thereof.

[1098] • Has any history of an abnormal Pap smear which required cervical biopsy and / or cervical cauterization within 6 months of Visit 1

[1099] • Malignancy within 5 years of screening, including but not limited to cervical carcinoma and carcinomas of the vagina and vulva.

[1100] • Has any condition including psychiatric illnesses that could interfere with their ability to understand or comply with the requirements of the study as determined by the investigator.

[1101] • Vulnerable individuals, i.e., are individuals whose willingness to participate in a clinical study may be unduly influenced by the expectation, whether justified or not, of benefits associated with participation, or of a retaliatory response from senior members of a hierarchy in case of refusal to participate. This includes all sponsor, study site, or third party (e.g., CRO, vendor) personnel directly involved in the conduct of the study and their family members or dependents, as well as all study site personnel otherwise supervised by the investigator.

[1102] • Part B only: Currently suspected clinically (or confirmed diagnostically) of having alternative causes of vaginal disease symptoms including symptomatic WC or infection with STI including Chlamydia trachomatis, Trichomonas vaginalis, or Neisseria gonorrhoeae.

[1103] Example 15: Disintegration and dissolution of vaginal inserts in humans

[1104] Methods

[1105] A Phase I first-in-human safety trial with H2B10B11 formulated as vaginal insert was carried out as outlined in Example 14. Part A followed a single ascending dose design, where single vaginal inserts were administered to three dose cohorts (cohorts I-III) of 8 healthy subjects each in a 3: 1 randomization for verurmplacebo. Part B followed a multiple ascending dose design, where 5 vaginal inserts were administered to two further cohorts (cohorts IV and V) of 36 BV patients each, once daily for 5 consecutive days, in a 2:1 randomization verurmplacebo. Pelvic examinations by a qualified clinician were conducted to assess local safety / tolerability, and where also the disintegration / dissolution of the vaginal insert was assessed by visual inspection. The composition of the vaginal inserts is shown in Table 23.

[1106] Table 23: Composition of vaginal inserts used in Phase I.

[1107] SDP: spray-dried powder; HPBCD: 2-Hydroxypropyl-beta-cyclodextrin.

[1108] Results

[1109] Table 24 summarizes preliminary results on the disintegration / dissolution by visual inspection during the pelvic examinations at 4 h and 24 h post administration of the single vaginal insert in healthy subjects in Part A. 4 h post administration, large fragments of vaginal insert were visible in 4, 8 and 6 subjects for the 2 mg, 5 mg, and 20 mg dose cohort, respectively (each cohort had 8 subjects, of which 6 were administered verum and 2 placebo). This indicates that 4 h are insufficient for full disintegration / dissolution of the vaginal insert in vivo. 24 h post administration, large fragments were no longer observed in any of the subjects in any cohort. At this timepoint, no residue or smear film were detected in 8 / 8 subjects of the 2 mg cohort and 7 / 8 subjects of the 20 mg cohort. Therefore, the formulation with an osmolality of 590 ±100 mOsmol / kg (for all doses) draws sufficient vaginal fluid in healthy volunteers (who in general have less vaginal fluid than BV patients) to allow the vaginal insert to disintegrate almost completely within 24 h. While the formulation used here also contained 5% Talc as a glidant, the excipients that influence the osmolality are mannitol and NaCI.

[1110] Table 24: Observations of pelvic examination after single administration of vaginal insert in healthy women in Part A.

[1111] Smear: only smear film visible; small fragments: vaginal insert was disintegrated into smaller fragments; large fragments: vaginal insert may have been partially disintegrated, but oval shape still visible. The 24 h data points for the 5 mg cohort are not yet available. Table 25 summarizes preliminary results on the disintegration / dissolution by visual inspection during the pelvic examinations 10-20 h after the last of 5 administrations of vaginal inserts in subjects diagnosed with BV (Part B of the study). The data were collected for the first 15 subjects (of 36 planned to be enrolled). No residues of the vaginal inserts or a smear was visible in 9 / 15 subjects, whereas small fragments were visible in 5 / 15 subjects and large fragments in only one subject. This means that no accumulation of residues from multiple vaginal inserts was observed after insertion for five consecutive days in any of the subjects. This further indicates that also for BV patients, with supposedly more vaginal discharge than healthy subjects, the disintegration / dissolution of the tablet was tuned appropriately to avoid accumulation of debris. This also allows for a homogeneous distribution of the active ingredient H2B10B11 along the vaginal canal in principle.

[1112] Table 25: Observations of pelvic examination after multiple doses once per day for 5 consecutive days in women diagnosed with BV (Part B).

[1113] Smear: only smear film visible; small fragments: vaginal insert was disintegrated into smaller fragments; large fragments: vaginal insert may have been partially disintegrated, but oval shape still visible. 15 participants completed the visit on day 6 when the data were collected.

[1114] Example 16: Stability of vaginal inserts (Phase I GMP clinical trial material)

[1115] A GMP stability study of blistered 2 mg and 20 mg vaginal inserts (Phase I, composition see Table 23) was conducted according to ICH guideline Q1A (R2). Table 26 summarizes the assay percentage (assay %w / w) at the time of release (TO) and time points up to 12 months at long-term storage condition (-20°C), up to 6 months at accelerated condition (5°C), and up to 1 month at stressed conditions (25°C / 60° / oRH).

[1116] Table 26: Assay by RP-UHPLC for 2 mg and 20 mg dose vaginal inserts.

[1117] RP-UHPLC: Reversed-phase ultra high-performance liquid chromatography.

[1118] All results of vaginal inserts stored at -20°C for up to 12 months and at 5°C up to 6 months were within the specification for the respective dose (2 mg, 20 mg) and scatter within the expected method variability. No trend was observed for both insert strengths stored for up to 12 months at -20°C. At 5 °C, a decreasing trend in assay %w / w from 80.4% at TO to 77.0% at T6m was observed for the 2 mg inserts. A similar decreasing trend in assay %w / w (94.5% at TO to 91.3% at T6m) was also observed for 20 mg inserts stored at 5°C. The 2 mg inserts (due to the low load of H2B10B11 as worst-case scenario) were additionally stored under stressed conditions (25°C / 60%RH) for up to 1 month. A decrease in assay %w / w was observed from 80.4% at TO to 75.0% at Tim.

[1119] At each time point, the potency of the vaginal inserts was measured via the dye-release assay (DRA). After storage of the 2 mg inserts for 12 months at -20°C, a potency of 73% was measured compared to 92% at TO. However, a similar value (72%) was observed after 1 month, therefore, the results are scattered within the expected method variability, no trend of decreasing potency was observed. Similar observations were made for the 20 mg inserts after 12 months storage at -20°C, where a potency of 88% was determined compared to 105% at TO. However, a potency of 88% was also measured at Tim and therefore is not considered as decreasing potency, but rather within the experimental variability.

[1120] Table 27: Potency by DRA 2 mg and 20 mg dose vaginal inserts.

[1121] The Phase I blistered vaginal inserts were considered stable for at least 12 months at long-term conditions (-20°C), 6 months at accelerated conditions (2-8°C) and for 1 month at stressed conditions (25°C / 60%RH).

[1122] Example 17: Stability of vaginal inserts (pre Phase II development-grade material)

[1123] A non-GMP stability study was performed on 5 mg blistered vaginal inserts using two different batches of drug substance spray-dried powder (SDP, Phase II development material). Batch 1 SDP is similar to Phase I material, but the vaginal inserts were compressed with a reduced compression force to target a tensile strength of Ts=1.0 MPa vs. Ts=1.7 MPa for the Phase I GMP material. The intention behind reducing the tensile strength was to ease the release of H2B10B11 from the vaginal insert, thereby increasing assay %w / w. H2B10B11 used for batch 2 SDP was expressed from a codon-optimized strain and purified with less purification steps compared to the material used in Phase I. Tablets were also compressed targeting a tensile strength of Ts=1.0 MPa. The composition of the vaginal inserts is shown in Table 28.

[1124] Table 28: Composition of vaginal inserts used in the technical stability study (Phase II development material).

[1125] SDP: spray-dried powder; HPBCD: 2-Hydroxypropyl-beta-cyclodextrin.

[1126] T: Assay %w / w of H2B10B11 in the SDP for batch 1: 220.11 mg / g, in batch 2 199.59 mg / g. The theoretical composition of the SDP is 5 mg / tablet H2B10B11, 10.13 mg / tablet mannitol, 0.11 mg / tablet sodium acetate, and 7 mg / tablet HPBCD.

[1127] Tensile strength Ts=1.0 MPa was used to compress the vaginal inserts.

[1128] Measurements of the assay %w / w at different time points after storage at -20°C, 25°C / 60%RH and 40°C / 75%RH are shown in Table 29. Both vaginal insert batches were stable for 1 week stored at -20°C or after ten freeze / thaw cycles. For batch 1, a decrease of 7% in the RP-UHPLC assay was observed compared to TO after storage for 1 month at 25°C / 60%RH, and a decrease of 11% after storage for 1 month at 40°C / 75%RH. For batch 2, a decrease of 4% was observed compared to TO after storage for 1 month at 25°C / 60%RH, and a decrease of 15% after storage for 1 month at 40°C / 75%RH. These preliminary data indicate that when stored at -20°C., the formulation is stable, with no degradation trend observable, for at least 1 week.

[1129] Table 29: Assay by RP-UHPLC for 2 batches of 5 mg dose vaginal inserts.

Claims

CLAIMS1. A pharmaceutical composition comprising as active ingredient an effective amount of a) an endolysin comprising the amino acid sequence provided in SEQ ID NO: 1; or b) an endolysin comprising an amino acid sequence having 80% sequence identity to SEQ ID NO: 1, wherein the endolysin has a killing activity against Gardnerella; and at least one pharmaceutically acceptable excipient.

2. The pharmaceutical composition according to claim 1, wherein the composition is formulated as vaginal insert, preferably a vaginal tablet.

3. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutically acceptable excipient is or comprises an osmotic active agent.

4. The pharmaceutical composition according to claim 3, wherein the osmotic active agent is a sugar alcohol, in particular wherein the sugar alcohol is sorbitol, mannitol or a mixture thereof.

5. The pharmaceutical composition according to claim 4, wherein the sugar alcohol is present in about 10% to about 70% in weight based on the total weight of the composition.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition has an osmolality of about 400 to 700 mOsmol / kg in simulated vaginal fluid (pH 6.0), preferably of about 590 mOsmol / kg.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the composition further comprises a gelling agent.

8. The pharmaceutical composition according to claim 7, wherein the gelling agent is hydroxyethyl cellulose (HEC), in particular wherein HEC is present between 0 % and 5 % in weight based on the total weight of the composition, in particular wherein HEC is present in about 0.5 % in weight based on the total weight of the composition.

9. The pharmaceutical composition according to claim 7, wherein the gelling agent is hydroxypropyl methyl cellulose (HPMC), in particular wherein HPMC is present between 0 % and 10 % in weight based on the total weight of the composition, in particular wherein HPMC is present in about 5 % in weight based on the total weight of the composition.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the composition further comprises a carbomer, in particular a carbomer homopolymer type B.

11. The pharmaceutical composition according to claim 10, wherein the carbomer is Carbopol 974P NF or Carbopol 971P.

12. The pharmaceutical composition according to claim 10 or 11, wherein the carbomer is present between 0 % and 5 % in weight based on the total weight of the composition, in particular wherein the carbomer is present in about 0.5 % in weight based on the total weight of the composition.

13. The pharmaceutical composition according to any one of claims 10 to 12, wherein the composition further comprises a salt, in particular wherein the salt reduces the interaction between the endolysin and the carbomer.

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

15. The pharmaceutical composition according to claim 13 or 14, wherein the NaCI is present between 0 % to about 12.5 % in weight based on the total weight of the composition, in particular wherein the NaCI is present in about 5 % in weight based on the total weight of the composition.

16. The pharmaceutical composition according to any one of claims 10 to 15, wherein the composition comprises a carbomer and NaCI, wherein the carbomer is present in about 0.5 % in weight based on the total weight of the composition and wherein the NaCI is present in about 5 % in weight based on the total weight of the composition.

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein the endolysin is present in about 0.1 % to about 5 % in weight based on the total weight of the composition.

18. The pharmaceutical composition according to any one of claims 1 to 17, wherein the endolysin is present in the composition as a spray dried powder.

19. The pharmaceutical composition according to claim 18, wherein the spray dried powder further comprises a stabilizing agent and / or a carrier.

20. The pharmaceutical composition according to claim 19, wherein the stabilizing agent is a modified cyclodextrin and / or wherein the carrier is mannitol.

21. The pharmaceutical composition according to claim 20, wherein the ratio between mannitol and the modified cyclodextrin in the spray dried powder is about 1:1, 1.5: 1, 2: 1, 3: 1, 4: 1, 5:1, 6:1, 7: 1, 8: 1 or 9:1.

22. The pharmaceutical composition according to claim 20 or 21, wherein the modified cyclodextrin is hydroxypropyl p-cyclodextrin.

23. The pharmaceutical composition according to any one of claims 18 to 22, wherein the spray dried powder further comprises one or more pH buffering agents, in particular wherein the pH buffering agent is sodium acetate trihydrate.

24. The pharmaceutical composition according to any one of claims 1 to 23, wherein the composition further comprises a lubricant.

25. The pharmaceutical composition according to claim 24, wherein the lubricant is magnesium stearate, in particular wherein the magnesium stearate is present between 0 % and 5 % in weight based on the total weight of the composition, in particular wherein the magnesium stearate is present in about 1 % in weight based on the total weight of the composition.

26. The pharmaceutical composition according to any one of claims 1 to 25, wherein the composition further comprises a glidant.

27. The pharmaceutical composition according to claim 26, wherein the glidant is selected from the list consisting of: silica, talc, stearic acid and stearyl alcohol.

28. The pharmaceutical composition according to claim 26 or 27, wherein the glidant is present between 0 % and 5 % in weight based on the total weight of the composition, in particular wherein magnesium stearate is present in about 1 % to about 2% in weight based on the total weight of the composition.

29. The pharmaceutical composition according to any one of claims 1 to 28, wherein the composition further comprises a filler, in particular wherein the filler is microcrystalline cellulose (MCC).

30. The pharmaceutical composition according to any one of claims 1 to 23, wherein the composition further comprises a silicified microcrystalline cellulose (SMCC).

31. The pharmaceutical composition according to any one of claims 1 to 30, wherein the composition comprises about:- 0.2 to 2 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder;- 30 to 55 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;- 0 to 5 % (w / w) of a carbomer, preferably a carbomer homopolymer type B;- 0 to 12.5 % (w / w) of a salt, preferably sodium chloride;- 0 to 5 % (w / w) of a gelling agent, preferably hydroxyethyl cellulose;- 0 to 5 % (w / w) of a glidant, preferably talc;- 0 to 5 % (w / w) of a lubricant, preferably magnesium stearate; and- microcrystalline cellulose ad 100 % (w / w).

32. The pharmaceutical composition according to any one of claims 1 to 30, wherein the composition comprises about:- 0.2 to 2 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder;- 30 to 55 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;- 0.5 % (w / w) of a carbomer, preferably a carbomer homopolymer type B;- 5 % (w / w) of a salt, preferably sodium chloride;- 0.5 % (w / w) of a gelling agent, preferably hydroxyethyl cellulose;- 5 % (w / w) of a glidant, preferably talc;- 1 % (w / w) of a lubricant, preferably magnesium stearate; and- microcrystalline cellulose ad 100 % (w / w).

33. The pharmaceutical composition according to any one of claims 1 to 30, wherein the composition comprises about:- 2 % (w / w) of a spray dried powder comprising the endolysin, wherein the spray dried powder comprises about 10% (w / w) of the endolysin;- 51 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, in particular wherein the sugar alcohol is mannitol;- 0.5 % (w / w) of a carbomer, in particular a carbomer homopolymer type B;- 5 % (w / w) of sodium chloride;- 0.5 % (w / w) of hydroxyethyl cellulose;- 5 % (w / w) of talc;- 1 % (w / w) of magnesium stearate; and- microcrystalline cellulose ad 100 % (w / w).

34. The pharmaceutical composition according to any one of claims 1 to 30, wherein the composition comprises about:- 5 % (w / w) of a spray dried powder comprising the endolysin, wherein the spray dried powder comprises about 10% (w / w) of the endolysin;- 48 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, in particular wherein the sugar alcohol is mannitol;- 0.5 % (w / w) of a carbomer, in particular a carbomer homopolymer type B;- 5 % (w / w) of sodium chloride;- 0.5 % (w / w) of hydroxyethyl cellulose;- 5 % (w / w) of talc;- 1 % (w / w) of magnesium stearate; and- microcrystalline cellulose ad 100 % (w / w).

35. The pharmaceutical composition according to any one of claims 1 to 30, wherein the composition comprises about:- 20 % (w / w) of a spray dried powder comprising the endolysin, wherein the spray dried powder comprises about 10% (w / w) of the endolysin;- 33 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, in particular wherein the sugar alcohol is mannitol;- 0.5 % (w / w) of a carbomer, in particular a carbomer homopolymer type B;- 5 % (w / w) of sodium chloride;- 0.5 % (w / w) of hydroxyethyl cellulose;- 5 % (w / w) of talc;- 1 % (w / w) of magnesium stearate; and- microcrystalline cellulose ad 100 % (w / w).

36. The pharmaceutical composition according to any one of claims 1 to 30, wherein the composition comprises about:- 0.2 to 2 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder;- 50 to 70 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;- 0.5 % (w / w) of a carbomer, preferably a carbomer homopolymer type B;- 5 % (w / w) of a salt, preferably sodium chloride;- 0 to 5 % (w / w) of a gelling agent, preferably hydroxyethyl cellulose;- 0 to 2 % (w / w) of a g lidant;- 1 to 2 % (w / w) of a lubricant, preferably magnesium stearate; and- microcrystalline cellulose ad 100 % (w / w).

37. The pharmaceutical composition according to any one of claims 1 to 30, wherein the composition comprises about:- 0.2 to 2 % (w / w) of the endolysin, in particular wherein the endolysin is comprised in a spray dried powder;- 30 to 55 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, preferably mannitol, sorbitol or a mixture thereof;- 0.5 % (w / w) of a carbomer, preferably a carbomer homopolymer type A and / or B;- 5 % (w / w) of a salt, preferably sodium chloride;- 0.5 % (w / w) of a gelling agent, preferably hydroxyethyl cellulose;- 1 % (w / w) of a lubricant, preferably magnesium stearate; and- microcrystalline cellulose ad 100 % (w / w).

38. The pharmaceutical composition according to any one of claims 1 to 30, wherein the composition comprises about:- 2.2 % (w / w) of a spray dried powder comprising the endolysin, wherein the spray dried powder comprises about 22.5% (w / w) of the endolysin;- 53 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, in particular wherein the sugar alcohol is mannitol;- 0.5 % (w / w) of a carbomer, in particular a cartoomer homopolymer type A and / or B;- 5 % (w / w) of sodium chloride;- 0.5 % (w / w) of hydroxyethyl cellulose;- 1 % (w / w) of magnesium stearate; and- microcrystalline cellulose ad 100 % (w / w).

39. The pharmaceutical composition according to any one of claims 1 to 30, wherein the composition comprises about:- 8.9 % (w / w) of a spray dried powder comprising the endolysin, wherein the spray dried powder comprises about 22.5% (w / w) of the endolysin;- 49 % (w / w) of an osmotic active agent, in particular wherein the osmotic active agent is a sugar alcohol, in particular wherein the sugar alcohol is mannitol;- 0.5 % (w / w) of a carbomer, in particular a carbomer homopolymer type A and / or B;- 5 % (w / w) of sodium chloride;- 0.5 % (w / w) of hydroxyethyl cellulose;- 1 % (w / w) of magnesium stearate; and- microcrystalline cellulose ad 100 % (w / w).

40. The pharmaceutical composition according to any one of claims 31 to 39, wherein the spray dried powder comprises about 10 to 30 % (w / w) of the endolysin.

41. The pharmaceutical composition according to claim 40, wherein the spray dried powder further comprises 10 to 20 % (w / w) or 20 to 50% (w / w) of a stabilizing agent, in particular wherein the stabilizing agent is hydroxypropyl 0-cyclodextrin.

42. The pharmaceutical composition according to claim 40 or 41, wherein the spray dried powder further comprises a carrier, in particular wherein the carrier is mannitol.

43. The pharmaceutical composition according to any one of claims 1 to 42, wherein said composition is suitable for vaginal administration.

44. The pharmaceutical composition according to any one of claims 1 to 43, for use in the treatment of bacterial infections in a subject.

45. The pharmaceutical composition for use according to claim 44, wherein the bacterial infection is bacterial vaginosis.

46. The pharmaceutical composition for use according to claim 44 or 45, wherein the bacterial infection or bacterial vaginosis is characterized by the presence of one or more Gardnerella species.

47. The pharmaceutical composition for use according to claim 45 or 46, wherein the composition is to be administered locally into the vagina of a female subject.

48. The pharmaceutical composition for use according to claim 47, wherein the female subject is not menstruating and / or is not expected to menstruate during the treatment.

49. The pharmaceutical composition for use according to any one of claims 44 to 48, wherein a single dose of the pharmaceutical composition is administered to said subject.

50. The pharmaceutical composition for use according to any one of claims 44 to 48, wherein at least one dose of the pharmaceutical composition is administered to said subject on n consecutive days, wherein n is an integer from 2 to 14, in particular wherein n is 5.

51. The pharmaceutical composition for use according to any one of claims 45 to 50, wherein the efficacy of the bacterial vaginosis treatment is assessed during the treatment.

52. The pharmaceutical composition for use according to claim 51, wherein the efficacy of the bacterial vaginosis treatment is assessed based on one or more of: Nugent score, amount and / or appearance of vaginal discharge, outcome of whiff test, vaginal pH, and / or proportion of clue cells among total epithelial cells.

53. A method of treating a bacterial infection in a subject, comprising administering to the subject the pharmaceutical composition according to any one of claims 1 to 43.

54. The method according to claim 53, wherein the bacterial infection is bacterial vaginosis.

55. The method according to claim 53 or 54, wherein the bacterial infection or bacterial vaginosis is characterized by the presence of one or more Gardnerella species.

56. The method according to claim 54 or 55, wherein the pharmaceutical composition is administered locally into the vagina of a female subject.

57. The method according to claim 56, wherein the female subject is not menstruating and / or is not expected to menstruate during the treatment.

58. The method according to any one of claim 53 to 57, wherein a single dose of the pharmaceutical composition is administered to the subject.

59. The method according to any one of claim 53 to 57, wherein at least one dose of the pharmaceutical composition is administered to the subject on n consecutive days, wherein n is an integer from 2 to 14, in particular wherein n is 5.

60. The method according to any one of claim 54 to 59, wherein the efficacy of the bacterial vaginosis treatment is assessed during the treatment.

61. The method according to claim 60, wherein the efficacy of the bacterial vaginosis treatment is assessed based on one or more of: Nugent score, amount and / or appearance of vaginal discharge, outcome of whiff test, vaginal pH, and / or proportion of clue cells among total epithelial cells.

62. Use of the pharmaceutical composition according to any one of claims 1 to 43 in the manufacture of a medicament for the treatment of bacterial infections in a subject.

63. The use according to claim 62, wherein the bacterial infection is bacterial vaginosis.

64. The use according to claim 62 or 63, wherein the bacterial infection or bacterial vaginosis is characterized by the presence of one or more Gardnerella species.

65. The use according to claim 63 or 64, wherein the medicament is to be administered locally into the vagina of a female subject.

66. The use according to claim 65, wherein the female subject is not menstruating and / or is not expected to menstruate during the treatment.

67. The use according to any one of claims 62 to 66, wherein a single dose of the medicament is administered to the subject.

68. The use according to any one of claims 62 to 66, wherein at least one dose of the medicament is administered to the subject on n consecutive days, wherein n is an integer from 2 to 14, in particular wherein n is 5.

69. The use according to any one of claims 63 to 68, wherein the efficacy of the bacterial vaginosis treatment is assessed during the treatment.

70. The use according to claim 69, wherein the efficacy of the bacterial vaginosis treatment is assessed based on one or more of: Nugent score, amount and / or appearance of vaginal discharge, outcome of whiff test, vaginal pH, and / or proportion of clue cells among total epithelial cells.

71. A method comprising:(a) obtaining a biological sample from a subject suffering from bacterial vaginosis;(b) analyzing one or more of the following in the biological sample from the subject:(I) Nugent score;(ii) amount and / or appearance of vaginal discharge;(iii) outcome of whiff test;(iv) vaginal pH; and / or(v) proportion of clue cells among total epithelial cells;(c) administering the pharmaceutical composition of any one of claims 1-43 to the subject;(d) obtaining a second biological sample from the subject; and(e) re-analyzing the one or more parameters obtained in step (b) to determine safety and / or effectiveness of the pharmaceutical composition in treating bacterial vaginosis in the subject.

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