Compositions, in particular pharmaceutical compositions comprising bacteriophages of the class caudoviricetes, and associated method and uses

Abstract

The present invention relates to compositions, and in particular to pharmaceutical compositions, comprising, or even consisting of, a suspension of bacteriophages of the class Caudoviricetes, the bacteriophages having lytic activity against at least one Gram-positive bacterial strain, at a titer of at least 108 PFU / mL, in a dispersing medium comprising, or even consisting of: - at least one non-ionic surfactant, and in particular a single non-ionic surfactant, selected from the polysorbate family, at a molar concentration greater than its critical micelle concentration, but not exceeding 2 mg / mL, and / or the poloxamer family, at a concentration ranging from 0.005% (w / v) to 30% (w / v); - one or more neutral salts formed from a pair of monovalent ions, the total molar concentration of which is in the range from 75 to 160 mM; - a buffer mixture imparting to the composition a pH from 6.0 to 7.9, and preferentially from 7.0 to 7.5, typically selected from hydrogen phosphate ion / dihydrogen phosphate, citric acid / citrate ion and citric acid / hydrogen phosphate ion ion pairs, wherein the molarity of the buffer mixture is in the range from 4 mM to 100 mM; - water, wherein the composition comprises less than 0.9 bacterial endotoxin units per mL and a total protein concentration of less than 0.09 mg / mL, and wherein the bacteriophage suspension in the dispersing medium has an osmolality of between 150 mOsm / kg and 600 mOsm / kg; the invention also relates to associated methods and uses.

Description

Description Title of the invention: Compositions, in particular pharmaceutical compositions of bacteriophages of the Caudoviricetes class, process and associated uses. Technical field

[0001] The present invention relates to the field of therapy. More specifically, it relates to novel pharmaceutical compositions containing an aqueous suspension of bacteriophages of the class Caudoviricetes having lytic activity against at least one strain of Gram-positive bacteria, and in particular, bacteriophages directed against at least one strain of Staphylococcus aureus; such pharmaceutical compositions for their use as a medicinal product and in various medical applications; as well as their method of preparation. Previous technique

[0002] In July 2022, the European Medicines Agency (EMA) published a "list of key therapeutic groups in crisis preparedness" in response to the increasing number of drug shortages exacerbated by the COVID-19 crisis (European Medicines Agency 2022). This list includes "systemic antibacterials." However, the growing threat of antibiotic resistance is compromising the effectiveness of this therapeutic group. Indeed, in April 2019, the World Health Organization predicted that, at the current rate, 10 million people could die each year from multidrug-resistant bacteria by 2050 (Interagency Coordination Group on Antimicrobial Resistance 2019). New approaches to treating bacterial infections are therefore being sought, and older ones, such as phage therapy, are being re-evaluated.

[0003] Bacteriophages are viruses that infect bacteria. They specifically recognize a bacterium, inject their genetic material into it, and hijack the host bacterium's metabolic pathway to produce new virions. This leads to lysis of the bacterial cell wall and the release of phage particles into the immediate environment of the lysed bacterium (Stone et al. 2019). When present at the site By targeting infectious bacteria, they can significantly reduce bacterial load and combat bacterial infections. In 1919, Félix d'Hérelle successfully treated bacillary dysentery with phages (d'Hérelle 1931), and phage-based products were marketed in France from 1930 to 1976 (Dublanchet 2014; Vidal 1943). However, conflicting clinical results, the advent of mass antibiotic production, and a bilateral geopolitical context led to the decline of this therapeutic strategy. Nevertheless, some countries (e.g., Georgia, Russia, Poland) have maintained this therapy for over a century (Miçdzybrodzki et al. 2018; Zaczek et al. 2022).

[0004] The European Pharmacopoeia, which sets standards for pharmaceutical products in Europe, includes a general chapter “5.31. MEDICINAL PRODUCTS USED IN PHAGOTHERAPY”. This chapter contains a set of requirements relating to active substances and medicinal products for human or veterinary use used in phage therapy, as well as their production and control. These standards are essential to ensure the quality and consistency of bacteriophage-based products throughout Europe. However, the diversity of production methods reported in the literature is subject to significant quantitative and qualitative fluctuations, making it difficult to apply standard Good Manufacturing Practices (GMP) (Ferry et al. 2021; Tanir et al. 2021). In 2018, at the initiative of the Belgian authorities, a generic monograph was developed on how to safely prepare and test phages intended for patients (Pirnay et al.2018; Verbeken and Pirnay 2022; Tanir et al. 2021). However, no mention of formulation allowing prolonged storage of phage suspensions, purified or not, with and without agitation, is specified in these texts.

[0005] Over the past fifty years, more than 5,100 bacteriophages have been identified and studied, over 90% of which possess a tail and belong to the myovirus, siphovirus, and podovirus morphotypes (Harada et al. 2018). Virus taxonomy was completely revised in 2021, and the order Caudovirales, as well as the families Myoviridae, Siphoviridae, and Podoviridae, were abolished. The taxonomy used in this patent application is defined in Walker et al. 2021 (Walker et al. 2021). Phages, although specific to bacterial types, exhibit similarities in protein structures and nucleic acids. Schematically, a prototypical bacteriophage particle is composed of one or two strands of nucleic acid protected by the protein icosahedral capsid, which is attached to the contractile sheath terminated at its base by a hexagonal plate coordinating the movement of the tail fibers.

[0006] The stability of bacteriophage preparations is affected by the external protein structure, which can undergo unfolding that promotes aggregation, reducing the phages' infectivity. These aggregation phenomena depend on a large number of factors, including the nature of the dispersing phase (water, water-co-solvent mixture) (Mendez et al. 2002), the concentrations of dissolved substances (monovalent and divalent cations) (Drab 2018), the pH (Meyer et al. 2017), mechanical agitation (Hoe et al. 2014; Francius et al. 2021), the production and storage temperature (Ergin 2022), and the packaging (glass, polypropylene) (Richter et al. 2021). The higher the bacteriophage titer, the more difficult it is to obtain stable bacteriophage suspensions over time. The bacteriophage titer is a key element of their stability and activity in suspension (Duyvejonck et al. 2021).

[0007] Furthermore, to be used in compositions administered to humans, bacteriophages must undergo a purification process, notably to reduce the presence of endotoxins and residual bacterial proteins. The purification steps disrupt the interactions between phages and the dispersing medium, as well as between phages and the packaging material, and are a source of instability for purified phage suspensions. The more purified the bacteriophage suspensions, the more the bacteriophages tend to aggregate, which can impair their lytic activity against target bacteria. In fact, to limit instability (including aggregation) in bacteriophage suspensions, they are often sold unpurified, with bacteriophages frozen inside their host cell.

[0008] The chemical structure (protein and nucleic) of bacteriophages greatly differentiates them from proteins and antibodies, making the preparation of bacteriophage formulations with good stability and preserved anti-infective properties very specific (Batinovic et al. 2019).

[0009] In this context, the objective of the invention is to provide compositions comprising a high bacteriophage titer and a low level of endotoxins and residual bacterial proteins, which are administrable to humans or animals, particularly in the form of an injectable composition, and therefore usable as a medicinal product or raw material for pharmaceutical, medical, or veterinary use, or as a laboratory reagent. These compositions must be stable over time, reduce bacteriophage aggregation, and ensure the maintenance of antibacterial lytic activity. Description of the invention

[0010] The present invention relates to compositions comprising a suspension of bacteriophages of the class Caudoviricetes having lytic activity against at least one strain of Gram-positive bacteria, at a titer of at least 10 8 PFU / mL, preferably at a concentration of 10 8 at 10 10 PFU / mL, and in particular at a concentration of 10 9 at 10 10 PFU / mL, in a dispersing medium. Said dispersing medium comprises: - at least one non-ionic surfactant, and in particular only one non-ionic surfactant, chosen from: - the polysorbate family, at a molar concentration higher than its critical micellar concentration, but not exceeding 2 mg / mL (which corresponds to 0.2% (w / v)), and / or - the poloxamer family, at a concentration ranging from 0.005% (w / v) to 30% (w / v), - one or more neutral salts formed from a pair of monovalent ions, whose total molar concentration is in the range of 75 to 160 mM, - a buffer mixture giving the composition a pH of 6.0 to 7.9, and preferably 7.0 to 7.5, typically chosen from the ion pairs hydrogen phosphate / dihydrogen phosphate ion, citric acid / citrate ion and citric acid / hydrogen phosphate ion, the molarity of the buffer mixture being in the range of 4 mM to 100 mM, - water.

[0011] The composition comprises less than 0.9 bacterial endotoxin units (EU) per mL, a total protein concentration of less than 0.09 mg / mL, and the bacteriophage suspension in the dispersing medium has an osmolality ranging from 150 mOsm / kg to 600 mOsm / kg. This is particularly advantageous for compositions intended for parenteral administration. In the context of the invention, the "bacteriophage suspension" or "aqueous suspension" of bacteriophages is the bacteriophage suspension included in the composition according to the invention, this composition being able to be in liquid or viscous form as detailed below.

[0012] Advantageously, in the compositions according to the invention, the dispersing medium in which the bacteriophages are suspended consists of: - of at least one non-ionic surfactant, and in particular only one non-ionic surfactant, chosen from: - the polysorbate family, at a molar concentration higher than its critical micellar concentration (CMC), but not exceeding 2 mg / mL (or 0.2% (w / v)), and / or - the poloxamer family, at a concentration ranging from 0.005% (w / v) to 30% (w / v), - of one or more neutral salts formed from a pair of monovalent ions, whose total molar concentration is in the range of 75 to 160 mM, - a buffer mixture giving the composition a pH of 6.0 to 7.9, and preferably of 7.0 to 7.5, typically chosen from the pairs hydrogen phosphate / dihydrogen phosphate ion, citric acid / citrate ion and citric acid / hydrogen phosphate ion, the molarity of the buffer mixture being in the range of 4 mM to 100 mM, - of water.

[0013] Advantageously, in the compositions according to the invention, the dispersing medium in which the bacteriophages are suspended comprises, preferably: - of at least one non-ionic surfactant, and in particular a single non-ionic surfactant, chosen from the polysorbate family, at a molar concentration higher than its critical micellar concentration (CMC), but not exceeding 2 mg / mL (or 0.2% (w / v)), and / or - of one or more neutral salts formed from a pair of monovalent ions, whose total molar concentration is in the range of 75 to 160 mM, - a buffer mixture giving the composition a pH of 6.0 to 7.9, and preferably of 7.0 to 7.5, typically chosen from the pairs hydrogen phosphate / dihydrogen phosphate ion, citric acid / citrate ion and citric acid / hydrogen phosphate ion, the molarity of the buffer mixture being in the range of 4 mM to 100 mM, - of water.

[0014] In particular, the present invention relates to compositions consisting of a suspension of bacteriophages of the class Caudoviricetes having lytic activity against at least one strain of Gram-positive bacteria, the titer of said bacteriophages being at least 10 8 PFU / mL, preferably 10 8 at 10 10 PFU / mL, and in particular 10 9 at 10 10 PFU / mL, in a dispersing medium consisting of: - of at least one non-ionic surfactant, and in particular only one non-ionic surfactant, chosen from: - the polysorbate family, at a molar concentration higher than its critical micellar concentration, but not exceeding 2 mg / mL (which corresponds to 0.2% (w / v)), and / or - the poloxamer family, at a concentration ranging from 0.005% (w / v) to 30% (w / v), - of one or more neutral salts formed from a pair of monovalent ions, whose total molar concentration is in the range of 75 to 160 mM, - a buffer mixture giving the composition a pH of 6.0 to 7.9, and preferably 7.0 to 7.5, typically chosen from the ion pairs hydrogen phosphate / dihydrogen phosphate ion, citric acid / citrate ion and citric acid / hydrogen phosphate ion, the molarity of the buffer mixture being in the range of 4 mM to 100 mM, - water, said aqueous suspension comprising less than 0.9 units of bacterial endotoxins (UE) per mL, a total protein concentration of less than 0.09 mg / mL and said bacteriophage suspension in said dispersing medium having an osmolality between 150 mOsm / kg and 600 mOsm / kg.

[0015] In particular, the present invention relates to compositions consisting of a suspension of bacteriophages of the class Caudoviricetes having lytic activity against at least one strain of Gram-positive bacteria, the titer of said bacteriophages being at least 10 8 PFU / mL, preferably 10 8 at 10 10 PFU / mL, and in particular 10 9 at 10 10 PFU / mL, in a dispersing medium consisting of: - of at least one non-ionic surfactant, and in particular a single non-ionic surfactant, chosen from the polysorbate family, at a molar concentration higher than its critical micellar concentration, but not exceeding 2 mg / mL (which corresponds to 0.2% (w / v)), - of one or more neutral salts formed from a pair of monovalent ions, whose total molar concentration is in the range of 75 to 160 mM, - a buffer mixture giving the composition a pH of 6.0 to 7.9, and preferably of 7.0 to 7.5, typically chosen from the pairs hydrogen phosphate / dihydrogen phosphate ion, citric acid / citrate ion and citric acid / hydrogen phosphate ion, the molarity of the buffer mixture being in the range of 4 mM to 100 mM, - water, said aqueous suspension comprising less than 0.9 units of bacterial endotoxins (UE) per mL, a total protein concentration of less than 0.09 mg / mL and said bacteriophage suspension in said dispersing medium having an osmolality between 150 mOsm / kg and 600 mOsm / kg.

[0016] In the definition of the compositions according to the invention, the total molar concentration given in the range from 75 to 160 mM corresponds to the molar concentration of the neutral salt(s) formed from a pair of monovalent ions present in the composition.

[0017] The pH and osmolality are chosen to be compatible with administration in humans and animals, and in particular to allow for parenteral or enteral administration. The osmolality of the suspension is chosen to maintain blood and tissue isotonicity.

[0018] Preferably, the compositions according to the invention are pharmaceutical compositions. In particular, the present invention relates to pharmaceutical compositions comprising a bacteriophage suspension as described herein, and especially to pharmaceutical compositions consisting of such a bacteriophage suspension.

[0019] In the compositions according to the invention, the bacteriophages form a colloidal suspension in the dispersing medium. That is to say, the bacteriophages are dispersed in the dispersing medium in the form of particles whose size, at least in one direction, is between 1 nanometer and 1 micrometer.

[0020] The inventors have shown, in a non-obvious manner, that the compositions according to the invention make it possible to obtain a stable suspension of bacteriophages. Stability (shelf life) is, in particular, evaluated, after a period of storage, without agitation, under refrigerated conditions (temperature in the range of 2°C to 8°C), by maintaining the bacteriophage titer at a value of 10 8 PFU / mL or more. "PFU" stands for "plaque forming unit," a definition well-known to those skilled in the art. Bacteriophage titers can be determined, in particular, by spot titration. Bacteriophages with lytic activity lyse the host cell, creating a clearing zone (or plaque) on a bed of bacteria targeted by the lytic activity of the bacteriophages; these zones are counted to determine the titer.

[0021] In the examples given below, tests were carried out highlighting other characteristics of the compositions according to the invention that are related to the observed stability, including (i) a pH variation of less than 1, and / or (ii) maintenance of the suspension's osmolality (error less than 10%), and / or (iii) an aggregation index, determined by UV spectrophotometry, of less than 7. The compositions according to the invention exhibit long-term stability, in particular over a period exceeding one year, and preferably two years or more, under refrigerated conditions (temperature in the range of 2°C to 8°C) and one month at room temperature (temperature in the range of 15°C to 25°C). In particular, it was found that for the compositions in the examples illustrating the invention, stability at 5°C for a period of at least one year could be achieved.

[0022] The dispersing medium is a continuous aqueous phase (liquid or viscous, as will be detailed later) in which the bacteriophages are dispersed. The dispersing medium, present in the compositions according to the invention, was selected to optimize the stability of the resulting bacteriophage suspensions. To this end, stability studies were conducted (i) under stress conditions (L, temperature, pH, ionic strength, horizontal and rotary agitation promoting ventilation and cavitation) to induce aggregation and fragmentation of the bacteriophages, which inactivate them; (ii) in different packaging formats, for which the wettability of the compositions was studied; and (iii) under storage conditions associated with shelf life.Thus, the present invention reports the unexpected stabilizing effect of a specific combination of a neutral surfactant and a particular buffered saline solution on suspensions of bacteriophages of the class Caudoviricetes, and in particular bacteriophages of the class Caudoviricetes belonging to the genus Siiviavirus, having, notably, a myovirus morphotype and lytic activity against at least one strain of Staphylococcus aureus. Furthermore, the stabilizing effect against pH variations and mechanical stress, which are known to inactivate bacteriophages during storage, has been demonstrated.

[0023] In the compositions according to the invention, it has been demonstrated that aggregation between bacteriophages was reduced and that antibacterial activity was preserved over time.

[0024] In particular, in the compositions according to the invention, and especially in the pharmaceutical compositions according to the invention, the bacteriophages of the class Caudoviricetes belong to the genus SHviavirus, and have, in particular, a myovirus morphotype, said bacteriophages having lytic activity against at least one strain of Staphylococcus aureus.

[0025] In the context of the invention, for the sake of simplicity, the term bacteriophages will be used to designate bacteriophages of the class Caudoviricetes having lytic activity against at least one strain of Gram positive bacteria, and in particular bacteriophages of the class Caudoviricetes belonging to the genus SHviavirus, and having, in particular, a myovirus morphotype, and having lytic activity against at least one strain of Staphylococcus aureus.

[0026] According to advantageous embodiments, in the compositions according to the invention, and in particular in the pharmaceutical compositions according to the invention, the dispersing medium has an ionic strength of 150 to 400 mM. The ionic strength is chosen (i) to further limit the aggregation phenomena induced by electrostatic interactions (attraction / repulsion) related to the charges carried by the bacteriophages, (ii) to facilitate the destabilization of the bacterial cell wall of the target bacteria.

[0027] Advantageously, the compositions according to the invention, and in particular the pharmaceutical compositions according to the invention, are stable for at least one year at a temperature in the range of 2 to 8°C, that is to say, they exhibit a bacteriophage titer that remains greater than or equal to 10 8PFU / mL. Within the scope of the invention, storage at a temperature within the range of 2 to 8°C is carried out without agitation. In particular, such storage of the compositions according to the invention is carried out in a type I or type II glass container.

[0028] In particular, before the storage period of at least one year at a temperature in the range of 2 to 8°C, said compositions according to the invention, and in particular said pharmaceutical compositions according to the invention, have a bacteriophage titer in the range of 10 9 at 10 10PFU / mL. Also, preferably, the compositions according to the invention, and in particular the pharmaceutical compositions according to the invention, are stable for at least one year at a temperature in the range of 2 to 8°C, that is to say, they exhibit a bacteriophage titer of the class Caudoviricetes belonging to the genus SHviavirus, and which have, in particular, a myovirus mophotype, and which have lytic activity against at least one strain of Staphylococcus aureus that remains greater than or equal to 10 8PFU / mL. In particular, before a storage period of at least one year at a temperature in the range of 2 to 8°C, said compositions according to the invention, and in particular said pharmaceutical compositions according to the invention, have a bacteriophage titer of the class Caudoviricetes belonging to the genus SHviavirus, and which, in particular, have a myovirus mophotype, and which have lytic activity against at least one strain of Staphylococcus aureus, which belongs to the range of 10 9 at 10 10 PFU / mL. In particular, no decrease in bacteriophage titer of more than one log was observed over the period considered.

[0029] It can be noted that in the compositions according to the invention, and in particular in the pharmaceutical compositions according to the invention, the bacteriophages are neither in conjugated nor complexed form. Thus, the compositions according to the invention are simple to prepare.

[0030] According to advantageous embodiments, the compositions according to the invention, and in particular the pharmaceutical compositions according to the invention, comprise polysorbate 80, as a non-ionic surfactant, at a molar concentration greater than 0.013 mM (equivalent to 0.017 mg / mL, which corresponds to the CMC of polysorbate 80) and less than or equal to 1.6 mM (equivalent to 2.0 mg / mL), preferably in the range of 0.16 mM (equivalent to 0.2 mg / mL) to 0.38 mM (equivalent to 0.5 mg / mL), or of polysorbate 20, at a molar concentration greater than 0.05 mM (equivalent to 0.06 mg / mL, which corresponds to the CMC of polysorbate 20) and less than or equal to 1.77 mM (equivalent to 2.0 mg / mL), preferably in the range of 0.18 mM (equivalent to 0.2 mg / mL) to 0.44 mM (equivalent to 0.5 mg / mL). According to an advantageous embodiment, the compositions according to the invention, and in particular the pharmaceutical compositions according to the invention, comprise, as a non-ionic surfactant, polysorbate 80 and polysorbate 20, at a total concentration greater than the CMC of the mixture of polysorbate 80 and polysorbate 20 and less than 2.0 mg / mL, preferably in the range of 0.2 mg / mL to 0.5 mg / mL. The total concentration here is understood as the concentration of polysorbate 80 and polysorbate 20.According to this embodiment, the polysorbate 80 / polysorbate 20 ratio can vary according to the desired final characteristics of the composition and the container used, and can be adjusted by a person skilled in the art.

[0031] According to advantageous embodiments, the compositions according to the invention, and in particular the pharmaceutical compositions according to the invention, comprise, as a non-ionic surfactant, poloxamer 188 and / or poloxamer 124 and / or poloxamer 237 and / or poloxamer 338 and / or poloxamer 407, at a concentration greater than or equal to 0.005% (w / v) and less than or equal to 30% (w / v), preferably in the range of 0.01% (w / v) to 5% (w / v) for a liquid composition, and preferably in the range of 15% (w / v) to 25% (w / v) for a viscous composition. When several poloxamers are used, the total poloxamer concentration ranges from 0.005% (w / v) to 30% (w / v), and preferably from 0.01% (w / v) to 5% (w / v) for a liquid composition and from 15% (w / v) to 25% (w / v) for a viscous composition.According to this embodiment, the ratio between the different poloxamers can vary according to the final characteristics of the desired composition and the container used, and can be adjusted by a person skilled in the art.

[0032] In the context of the invention, a "viscous composition" means a composition having an apparent viscosity of at least 100 mPa·s at 20 °C. The liquid composition has an apparent viscosity of less than 100 mPa.s. Apparent viscosity can be measured with a rotary viscometer according to monograph 2.2.10. Viscosity - rotary viscometer method of the European Pharmacopoeia.

[0033] According to advantageous embodiments, the compositions according to the invention, and in particular the pharmaceutical compositions according to the invention, comprise, as a non-ionic surfactant, one or more of the following surfactants or a combination of several of the following surfactants: - polysorbate 80 at a molar concentration greater than 0.013 mM (equivalent to 0.017 mg / mL or 0.0017% (w / v), which corresponds to the CMC of polysorbate 80) and less than or equal to 1.6 mM (equivalent to 2.0 mg / mL or 0.2% (w / v)), preferably in the range of 0.16 mM (equivalent to 0.2 mg / mL or 0.02% (w / v)) to 0.38 mM (equivalent to 0.5 mg / mL or 0.05% (w / v)), - polysorbate 20, at a molar concentration greater than 0.05 mM (equivalent to 0.06 mg / mL, or 0.006% (w / v), which corresponds to the CMC of polysorbate 20) and less than or equal to 1.77 mM (equivalent to 2.0 mg / mL or 0.2% (w / v)), preferably in the range from 0.18 mM (equivalent to 0.2 mg / mL or 0.02% (w / v)) to 0.44 mM (equivalent to 0.5 mg / mL or 0.05% (w / v)), - poloxamer 188 at a concentration greater than or equal to 0.005% (w / v) and less than or equal to 30% (w / v), preferably in the range of 0.01% (w / v) to 5% (w / v) for liquid compositions, and preferably in the range of 15% (w / v) to 25% (w / v) for viscous compositions, - poloxamer 124 at a concentration greater than or equal to 0.005% (w / v) and less than or equal to 30% (w / v), preferably in the range of 0.01% (w / v) to 5% (w / v) for liquid compositions, and preferably in the range of 15% (w / v) to 25% (w / v) for viscous compositions, - poloxamer 237 at a concentration greater than or equal to 0.005% (w / v) and less than or equal to 30% (w / v), preferably in the range of 0.01% (w / v) to 5% (w / v) for liquid compositions, and preferably in the range of 15% (w / v) to 25% (w / v) for viscous compositions, - poloxamer 338 at a concentration greater than or equal to 0.005% (w / v) and less than or equal to 30% (w / v), preferably in the range of 0.01% (w / v) to 5% (w / v) for liquid compositions, and preferably in the range of 15% (w / v) to 25% (w / v) for viscous compositions, and - poloxamer 407 at a concentration greater than or equal to 0.005% (w / v) and less than or equal to 30% (w / v), preferably in the range of 15% (w / v) to 25% (w / v) for liquid compositions, and preferably in the range of 15% (w / v) to 25% (w / v) for viscous compositions.

[0034] When several nonionic surfactants are used, their total concentration preferably ranges from 0.005% (w / v) to 30% (w / v). According to this embodiment, the ratio between the different nonionic surfactants used can vary depending on the desired final characteristics of the composition and the container used, and can be adjusted by those skilled in the art.

[0035] According to advantageous embodiments which can be combined with the preceding ones, in the compositions according to the invention, and in particular in the pharmaceutical compositions according to the invention, the buffer mixture is a KH2PO4 / Na2HPO4 mixture, typically KH2PO4 at a molar concentration of 1 to 15 mM and Na2HPO4 at a molar concentration of 3 to 56 mM.

[0036] According to advantageous embodiments, the compositions according to the invention, and in particular the pharmaceutical compositions according to the invention, comprise only NaCl, as a neutral salt, which is present at a molar concentration of 75 to 160 mM, typically at a molar concentration of 150 to 160 mM.

[0037] According to advantageous embodiments, the compositions according to the invention, and in particular the pharmaceutical compositions according to the invention, do not contain any compounds of animal origin. Therefore, in this case, the non-ionic surfactants (polysorbate(s) and / or poloxamer(s)) and other excipients present in the composition will be of synthetic origin.

[0038] Advantageously, the compositions according to the invention, and in particular the pharmaceutical compositions according to the invention, are sterile, pyrogen-free and isotonic or hypertonic.

[0039] Furthermore, due to the presence of the non-ionic surfactant and the adjustment of its concentration, it is possible to modulate the contact angle of the composition with different types of surfaces. This is advantageous both for packaging and for the preparation of the composition according to the invention, particularly its sterilization, which requires a final filtration step, as it allows for a wider range of usable filters. In particular, the compositions according to the invention exhibit sufficiently low adsorption at the gas-liquid and liquid-material interfaces commonly used in the medical field, thus reducing surface adsorption phenomena.

[0040] Also, according to advantageous embodiments, the compositions according to the invention, and in particular the pharmaceutical compositions according to the invention, are packaged in a container such as a bottle, a bag or a syringe delimited by walls which are made of type I glass, type II glass, polystyrene, polypropylene, polyethylene, polyethylene terephthalate, treated or not with silicone oil, in particular closed with a stopper made of a rubber material (in particular, bromobutyl or chlorobutyl) perfluorinated or not.

[0041] In particular, the compositions according to the invention, and especially the pharmaceutical compositions according to the invention, exhibit a wettability of the walls of their container corresponding to a contact angle of 20 to 60°.

[0042] In particular, the container is closed by a cap and the compositions according to the invention, and in particular the pharmaceutical compositions according to the invention, exhibit a wettability of the cap closing their container corresponding to a contact angle of less than 90°.

[0043] According to particular embodiments which can be implemented regardless of the embodiment of the compositions described in this description, in the compositions according to the invention, and in particular in the pharmaceutical compositions according to the invention, said suspension of bacteriophages in said dispersing medium is supplemented with a viscous solution or a cellulose ether hydrogel - preferably, of hydroxypropyl methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, polyethylene glycol, polyvinyl alcohol, sodium alginate, PVA-sodium alginate, acrylate, methacrylate, polyacrylate, hyaluronic acid, acrylamide, polyacrylamide (e.g., poly(N-isopropylacrylamide), poly(N-isopropylacrylamide-co-allylamine)), polyethylene glycol maleimide, polyethylene oxide, guar gum, agarose, gelatin, collagen, chitosan, nanohydroxyapatite, silicate, or hydrated aluminum silicate. That is to say, the suspension of bacteriophages in the dispersing medium selected according to the invention is mixed with said viscous solution or hydrogel. It is then the resulting mixture that is administered as a medicinal product.

[0044] According to advantageous embodiments, the compositions according to the invention, and in particular the pharmaceutical compositions according to the invention, are adapted for enteral administration, or adapted for administration by injection, in particular by parenteral route, including intravenous, intraarterial, intramuscular, subcutaneous and / or intra-articular administrations.

[0045] The invention also relates to the compositions according to the invention, and in particular the pharmaceutical compositions according to the invention, for their use as a medicinal product, in particular for their use in the treatment of osteoarticular infections.

[0046] Furthermore, the compositions according to the invention are very simple to prepare, use widely available and inexpensive components, and are compatible with various applications, particularly for use as pharmaceutical compositions. The excipients used exhibit excellent safety and are compatible with different routes of administration, including enteral and parenteral administration, and are readily available as pharmaceutical raw materials (PRMs). Their molarity and osmolality are also suitable for administration in humans or animals, while ensuring the stability and activity of the bacteriophages present, which was a significant challenge in the current context.

[0047] The invention also relates to a method for preparing a composition, and in particular a pharmaceutical composition, according to the invention, comprising introducing it into a dispersing medium comprising, or even consisting of: - at least one non-ionic surfactant, and in particular only one non-ionic surfactant, chosen from: - the polysorbate family, at a molar concentration higher than its critical micellar concentration, but not exceeding 2 mg / mL (which corresponds to 0.2% (w / v)), and / or - the poloxamer family, at a concentration ranging from 0.005% (w / v) to 30% (w / v), - one or more neutral salts formed from a pair of monovalent ions, whose total molar concentration is in the range of 75 to 160 mM, - a buffer mixture giving the composition a pH of 6.0 to 7.9, and preferably of 7.0 to 7.5, typically chosen from the pairs hydrogen phosphate / dihydrogen phosphate ion, citric acid / citrate ion and citric acid / hydrogen phosphate ion, the molarity of the buffer mixture being in the range of 4 mM to 100 mM, - water, purified bacteriophages in the quantity necessary to obtain a final concentration of at least 10 in the composition 8 PFU / mL, and typically 10 8 at 10 10 PFU / mL in bacteriophages, and in particular 10 9 at 10 10 PFU / mL in bacteriophages.

[0048] Due to the purification of the bacteriophages, said bacteriophage suspension in the dispersing medium obtained, and therefore the composition which comprises it or which consists of said bacteriophage suspension in the dispersing medium, comprises less than 0.9 units of bacterial endotoxins (EU) per mL and a total protein concentration of less than 0.09 mg / mL.

[0049] Furthermore, the dispersing medium, and therefore the bacteriophage suspension within it, exhibits an osmolality ranging from 150 mOsm / kg to 600 mOsm / kg. Indeed, the presence of bacteriophages does not influence the resulting osmolality.

[0050] In particular, in the preparation process according to the invention, after the introduction of the bacteriophages into the dispersing medium, the resulting suspension is sterilized by implementing a terminal filtration operation, by passing through a filter made of polyethersulfone, polystyrene, poly(styrene-butadiene) or polyvinylidene fluoride.

[0051] According to another aspect, the invention relates to the use of a dispersing medium comprising, or even consisting of: - at least one non-ionic surfactant, and in particular only one non-ionic surfactant, chosen from: - the polysorbate family, at a molar concentration higher than its critical micellar concentration, but not exceeding 2 mg / mL (or 0.2% (w / v)), and / or - the poloxamer family, at a concentration ranging from 0.005% (w / v) to 30% (w / v), - one or more neutral salts formed from a pair of monovalent ions, whose total molar concentration is in the range of 75 to 160 mM, - a buffer mixture giving the composition a pH of 6.0 to 7.9, and preferably of 7.0 to 7.5, typically chosen from the pairs hydrogen phosphate ion / dihydrogen phosphate ion, citric acid / citrate ion and citric acid / hydrogen phosphate ion, the molarity of the buffer mixture being in the range of 4 mM to 100 mM, - water, to improve storage stability at a temperature within the range of 2 to 8°C, of ​​bacteriophages of the class Caudoviricetes having lytic activity against at least one strain of Gram-positive bacteria, said bacteriophages being suspended in said dispersing medium at a titer of at least 10 8 PFU / mL, and typically 10 8 at 10 10 PFU / mL in bacteriophages, and in particular 10 9 at 10 10PFU / mL and said composition comprising less than 0.9 units of bacterial endotoxins (UE) per mL, a total protein concentration of less than 0.09 mg / mL and said bacteriophage suspension in said dispersing medium having an osmolality between 150 mOsm / kg and 600 mOsm / kg.

[0052] In particular, according to this use, the stability obtained corresponds to a bacteriophage titer that remains greater than or equal to 10 8 PFU / mL, after storing the composition at a temperature within the range of 2 to 8°C, for at least one year.

[0053] In the processes and uses according to the invention, the selected dispersing medium is used as a dispersing medium for bacteriophages.

[0054] All the characteristics described in this description, in connection with the compositions according to the invention, apply to the preparation processes and uses according to the invention.

[0055] The more detailed description that follows, with reference to the attached figures and the examples given for illustrative purposes only, will provide a better understanding of the invention. The compositions according to the invention comprise a number of features and components which will be described in more detail.

[0056] In general, unless otherwise specified, the terms used in the present invention have the meanings conventionally recognized by those skilled in the art, and in particular are as defined by the European Pharmacopoeia. When reference is made to the European Pharmacopoeia (Ph. Eur.), it refers to the version applicable on January 1, 2024.

[0057] Osmolality is determined according to Ph. Eur. 2.2.35. Osmolality in mOsm / kg is measured by cryoscopic lowering with a cryoscopic osmometer.

[0058] The pH is determined according to Ph. Eur. 2.2.3.

[0059] Sterility is determined according to Ph. Eur. 2.6.1.

[0060] The CMC (Cellular Mass Concentration) is determined by the abrupt change (bend) in the surface tension curve (in mN / m) as a function of the logarithmic concentration of the surfactant in the water. Surface tension can be usefully measured using a tensiometer, for example, a ring tensiometer.

[0061] The concentration of bacterial endotoxins present in the composition, expressed in endotoxin units (EU) per mL, is determined according to Ph. Eur. 2.6.14.

[0062] The total protein concentration in the composition is determined according to the Bradford method (method 3 of Ph. Eur 01 / 2008: 20533 on the determination of total protein). A protein is defined as a macromolecule composed of one or more polypeptide chains, these polypeptide chains being a sequence of more than 20 amino acids linked together by peptide bonds.

[0063] The ionic strength is calculated using the following formula: ∑ S(QXZ?) where q is the molar concentration of ionic species i, and Zj is the charge of the ion considered. The charges Zj of the ions considered are as follows:

[0064] In particular, the more detailed methods described in the examples can be used.

[0065] Bacteriophages

[0066] Bacteriophages of the class Caudoviricetes are phages with a head-tail structure (Zhu et al. 2022) that can exhibit lytic activity against bacteria. Lytic activity against a bacterial strain is defined as the bacteriophages causing the lysis of at least some of the bacteria of that strain. The lytic activity against target bacteria can be assessed using any technique known to those skilled in the art, particularly in vitro by counting plaques of lysis during bacteriophage titrations on a bed of at least one bacterial strain targeted by the phage. The titration can be performed, for example, by spot testing.

[0067] Within the scope of the invention, bacteriophages of the class Caudoviricetes that exhibit lytic activity against at least one strain of Gram-positive bacteria, and in particular against at least one strain of Staphylococcus aureus bacteria, are particularly advantageous and belong predominantly to the Herelleviridae family, Twortvirinae subfamily, and SHviavirus genus (ICTV 2023). In particular, phages exhibiting lytic activity against various strains of Gram-positive bacteria, and especially against at least one strain of Staphylococcus aureus, will be preferred. A tailed phage consists of a head containing its viral genome (in the context of this invention, this is often an icosahedral capsid containing double-stranded DNA), and a tail that plays a crucial role in the recognition and infection of target bacteria. The tail is composed of specific proteins that selectively interact with receptors on the surface of bacteria, enabling the phage to attach and efficiently inject its genetic material into bacterial cells, thereby initiating viral replication and ultimately leading to lysis of the host bacterium.

[0068] Bacteriophages, and in particular those of the class Caudoviricetes (Turner, Kropinski, and Adriaenssens 2021), and especially of the genus SHviavirus, exhibiting the following morphotypes can be used within the framework of the invention (Harada et al. 2018): - Myovirus: Myoviruses have a contractile tail. - Siphovirus: Siphoviruses have a long, non-contractile tail. - Podovirus: Podoviruses have a short, non-contractile tail.

[0069] Examples of bacteriophages that could be used in the context of the invention include: - Bacteriophages directed against Staphylococcus aureus, corresponding, for example, to PP1493 and PP1815 (https: / / www.hygienes.net / actualite / focus / lansm-autoriser-un-acces-compassionnel-pour-des-bacteriophages-dans-les-infections-osteo-articulaires), to one of the bacteriophages listed in the following documents: Plumet et al. 2022, Malik et al. 2017, WO 2018 / 162566, or one of those used in the examples that follow, namely vB_SauM-VlSA19 and vB_SauM-V1SA20 filed in GenBank under numbers ON814134.1 (https: / / www.ncbi.nlm.nih.gov / nuccore / ON814134) and ON814135.1 (https: / / www.ncbi.nlm.nih.gov / nuccore / ON814135). These last two Bacteriophages were also the subject of a publication presenting TEM images and comparisons of their genomes, notably (C. Kolenda, 2022). Any other bacteriophage having lytic activity against at least one strain of Staphylococcus aureus may be used. - Bacteriophages directed against Streptococcus pneumoniae, corresponding, for example, to phage Dp-1 (siphovirus, Taxonomy ID: 59241), Bacteriophage SOCP (podovirus, Taxonomy ID: 1498213) listed in the following document: (Qadir and Sajjad 2017). - Bacteriophages directed against Enterococcus faecalis, corresponding, for example, to Ben (myovirus), Bill (myovirus), Carl (myovirus), CCS1 (myovirus), CCS2 (siphovirus), SDS1 (siphovirus) and UMP (podovirus) listed in the following document: (Wandro et al. 2022). - Bacteriophages directed against Cutibacterium acnes, corresponding, for example, to phage Y3Z (siphovirus), deposited in GenBank under the number OQ411034 (https: / / www.ncbi.nlm.nih.gov / nuccore / OQ411034) (Xuan et al. 2023), or to the PA6 phage (siphovirus) deposited in GenBank under the number DQ431235 (https: / / www.ncbi.nlm.nih.gov / nuccore / DQ431235).

[0070] The compositions according to the invention may contain a single type of bacteriophage or a combination of different types of bacteriophages, and in particular a combination of different types of bacteriophages of the class Caudoviricetes, notably of the genus SHviavirus, and having, in particular, a myovirus morphotype, which exhibit lytic activity against at least one strain of Staphylococcus aureus bacteria. The use of a combination of different types of bacteriophages may be chosen to increase the spectrum of action, leading to the lysis of a greater number of target bacteria, of the compositions according to the invention.

[0071] In the compositions according to the invention, the bacteriophages in suspension were purified after in vitro culture and isolation from their natural environment. The in vitro culture can be performed using any conventional technique, particularly those described in the aforementioned publications, on host cells corresponding to one of the bacteria against which lytic activity is sought.

[0072] Purified bacteriophage suspensions can be obtained using methods described in the literature that employ filtration and / or centrifugation steps, notably those described in Kolenda et al. 2022. Specifically, bacteriophages are collected from the environment (e.g., upstream of wastewater treatment plants, from cattle effluents) or from clinical strains. So-called lytic bacteriophages are then isolated by successive spotting of lysis plaques on bacterial agar plates and subsequently genetically characterized; the bacteriophages are trained, if necessary, to increase their activity, spectrum, affinity, etc. (e.g., Appelmans protocol or other technique promoting recombination events) (Burrowes, Molineux, and Fralick 2019); produced on a bacterial strain of the species against which they are directed, free of prophages, resistance factors, major virulence; purified by ultracentrifugation with a cesium chloride gradient (densities of 1.6, 1.5 and 1.3) following which they are collected between the rings corresponding to the densities of 1.6 and 1.5 then they are dialyzed several times in a PBS type buffer (from the English "phosphate-buffered saline"), finally a polishing step allows them to be resuspended in the bacteriophage dispersing medium selected to form the composition according to the invention.

[0073] After such a purification step, the concentrations of bacterial endotoxins and residual bacterial proteins are considerably reduced. Thus, it is possible to obtain compositions comprising such purified bacteriophages as a therapeutic active ingredient with a bacterial endotoxin concentration of less than 0.9 EU / mL per composition. Advantageously, in the compositions according to the invention, the total protein concentration is less than 0.09 mg / mL, which corresponds almost exclusively to the constituent proteins of the bacteriophages.

[0074] The compositions according to the invention comprise or are composed of a suspension of bacteriophages of the class Caudoviricetes and a dispersing medium defined within the scope of the invention. This dispersing medium is composed of water, at least one non-ionic surfactant, at least one neutral salt consisting of monovalent ions and a buffer mixture, more details of which are given below.

[0075] In the context of this invention, the dispersing medium is an aqueous dispersing medium. This medium is used as a dispersing medium for bacteriophages. When the nonionic surfactant(s) are in liquid form, the dispersing medium is an aqueous solution. This is particularly true when the surfactant(s) are chosen from the polysorbate family. It is also true when the surfactant(s) are chosen from the poloxamer family and are present in a concentration such that they are not in a viscous form, as will be explained later.

[0076] Non-ionic surfactants

[0077] The compositions according to the invention comprise one or more nonionic surfactants selected from polysorbates and / or poloxamers, said nonionic surfactant being present at a concentration as defined in this description. Polysorbates are known esters of fatty acids and polyoxyethylene sorbitan. Poloxamers are block copolymers of polyethylene glycol and polypropylene glycol. More specifically, poloxamers comprise a central block of polypropylene glycol and two outer blocks of polyethylene glycol.

[0078] The presence of the nonionic surfactant helps limit the impact of cavitation, ventilation, and bacteriophage-bacteriophage aggregation. In addition to ensuring the stability of the composition and maintaining bacteriophage activity, the nonionic surfactant(s), in combination with the other components present, allow for adjustment of the composition's wettability and the contact angles between the composition and air, and between the composition and the walls of containers or caps. Furthermore, the nonionic surfactant(s) present allow for... It also helps to limit aggregation phenomena at gas-liquid and liquid-solid interfaces. The non-ionic surfactant of the polysorbate or poloxamer type, at the chosen concentration, is compatible with administration in humans or animals, and in particular, by injection, notably parenteral administration.

[0079] Advantageously, the quantity of polysorbate and / or poloxamer, and in particular the quantity of polysorbate 80 and / or polysorbate 20 and / or poloxamer 188 and / or poloxamer 124 and / or poloxamer 237 and / or poloxamer 338 and / or poloxamer 407, is chosen to ensure wettability of the packaging materials (container and cap when a cap is present) of the composition according to the invention corresponding to a material / composition contact angle of 20° to 60° for the walls of the containers and to a contact angle less than or equal to 90° for the caps.

[0080] Polysorbates are esters of fatty acids and polyoxyethylene sorbitan. In the context of this invention, polysorbate 80 (PS80 or Tween® 80) or polysorbate 20 (PS20 or Tween® 20) will preferably be used as the polysorbate, either alone or in combination with one or more nonionic surfactants defined in this invention. The numbers 20, 40, 60, and 80 following the term "polysorbate" correspond to the principal type of fatty acid associated with the polyoxyethylene sorbitan residue of the molecule; that is, monolaurate is indicated by 20, monopalmitate by 40, monostearate by 60, and monooleate by 80.

[0081] Within the framework of the invention, poloxamer 188, poloxamer 124, poloxamer 237, poloxamer 338, poloxamer 407 are preferably used as poloxamer, alone or in combination with one or more non-ionic surfactants defined within the framework of the present invention.

[0082] Neutral salts

[0083] The composition according to the invention comprises one or more neutral salts formed of monovalent ions. A typical example of a neutral salt is... NaCl and KG. The total molar concentration of the neutral salt is 75 to 160 mM. If only one neutral salt composed of monovalent ions, and in particular NaCl, is present in the composition according to the invention, its concentration in the composition is 75 to 160 mM. If several neutral salts composed of monovalent ions are present (typically NaCl and KG) in the composition according to the invention, their total concentration in the composition is 75 to 160 mM. Advantageously, the composition according to the invention comprises only NaCl as the neutral salt composed of monovalent ions.

[0084] Buffer mix

[0085] The composition according to the invention has a pH of 6.0 to 7.9, and preferably 7.0 to 7.5. This pH is adjusted and maintained stable by the presence of a buffer mixture. The buffer mixture may consist of a weak base and its conjugate acid, or even of an acid other than the conjugate acid, or alternatively, of a weak acid and its conjugate base, or even of a base other than its conjugate base. Examples of buffer mixtures that may be used in the compositions according to the invention include the hydrogen phosphate / dihydrogen phosphate, citric acid / citrate, and citric acid / hydrogen phosphate pairs. The molarity of the buffer mixture (i.e., the sum of the molarities of the base and the acid constituting the buffer mixture) is in the range of 4 mM to 100 mM.

[0086] The buffer mixture preferably has a buffer capacity in the range of 8 mM / ApH to 30 mM / ApH.

[0087] The buffer mixture is advantageously a KH2PO4 / Na2HPO4 mixture, typically KH2PO4 at a molar concentration of 1 to 15 mM and Na2HPO4 at a molar concentration of 3 to 56 mM. At such concentrations, the molarity of the buffer mixture is in the range of 4 to 71 mM.

[0088] It is possible to adjust the molarity of the buffer mixture according to the desired route of administration. More specifically, the molarity of the buffer mixture Tl may be chosen to allow intravenous and intramuscular administration: in this case, it will be less than or equal to 25 mM.

[0089] In the context of the invention, the water used is water suitable for pharmaceutical use, in particular water for injectable preparation, specifically as defined in Ph. Eur.

[0090] Packaging

[0091] The composition according to the invention can be packaged in any suitable container, in particular in the form of a bottle, bag or syringe, delimited by one or more walls in contact with the composition which advantageously shall be made of type I glass (commonly used in the medical field and which generally has a contact angle with water of 40°), type II glass, polystyrene (which generally has a contact angle with water of 68°), polypropylene (which generally has a contact angle with water of 102°), polyethylene (which generally has a contact angle with water of 96°), polyethylene terephthalate (which generally has a contact angle with water of 64°), the walls being able or not to be coated with a silicone oil (which when present leads to a contact angle with water >> 90°).In particular, for packaging the compositions according to the invention, type I or II glass containers are preferred. Type I and II glasses are, in particular, defined in Ph. Eur. 3.2.1.

[0092] Preferably, the compositions, and in particular the liquid compositions, according to the invention exhibit a wettability of the packaging materials corresponding to a material / liquid medium contact angle of 20° to 60° with the container walls, and less than or equal to 90° with a cap capable of closing said container. Examples of materials that can constitute the cap include silicones, polypropylene, high-density polyethylene, rubbers, particularly chlorobutyl or perfluorinated bromobutyl, etc.

[0093] With such wettability, the compositions according to the invention can be filtered, particularly during sterilization operations, through a polyethersulfone filter (which generally has a water contact angle of 60°), a polystyrene filter (which generally has a water contact angle of 68°), or a poly(styrene-butadiene) filter (which generally has a water contact angle of 69°). Preferably, the composition ensures wettability of the filter materials such that the contact angles between the filter material and the liquid medium are less than 60°. This makes it possible to use polyvinylidene fluoride filters (water contact angle of 145°–89°).

[0094] Further details on the compositions, and in particular the pharmaceutical compositions according to the invention and their medical uses.

[0095] The composition according to the invention can directly be, as such, a pharmaceutical composition administrable to humans or animals. A pharmaceutical composition administrable to humans or animals can therefore consist of a suspension of bacteriophages of the class Caudoviricetes in the dispersing medium defined within the scope of the invention. It can be adapted for enteral and topical administration (e.g., cutaneous, mucosal, buccal, ophthalmic) or for administration by injection, via the parenteral route, including intravenous, intra-arterial, intramuscular, subcutaneous, and / or intra-articular administration.

[0096] Depending on the intended route of administration of a pharmaceutical composition according to the invention, in other embodiments, the composition may also include one or more components, and in particular one or more pharmaceutically acceptable excipients, making it suitable for the intended route of administration. In such a case, the pharmaceutical composition for administration to humans or animals comprises a composition according to the invention consisting of a suspension of Caudov / r / cetes class bacteriophages in the dispersing medium defined within the scope of the invention, mixed with one or more other pharmaceutically acceptable excipients or one or more other active ingredients.

[0097] The pharmaceutical compositions according to the invention are intended for the treatment of conditions related to the presence of Gram-positive bacteria, and in particular Staphylococcus aureus. They are administered to a subject, who may be a human or an animal, in an effective quantity. An "effective" quantity in the context of a treatment refers to a treatment that results in a decrease in the number of Gram-positive bacteria, and in particular Staphylococcus aureus, in a subject after said treatment compared to the number of Gram-positive bacteria, and in particular Staphylococcus aureus, before said treatment.

[0098] In the case of a pharmaceutical composition suitable for enteral and topical (e.g., cutaneous, mucosal, buccal, ophthalmic) administration, preferably, a pharmaceutical composition comprises a composition according to the invention consisting of a suspension of bacteriophages of the class Caudoviricetes in the dispersing medium defined within the scope of the invention, mixed with a viscous solution or hydrogel of cellulose ether—preferably, of hydroxypropyl methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, polyethylene glycol, polyvinyl alcohol (PVA), sodium alginate, PVA-sodium alginate, acrylate, methacrylate, polyacrylate, hyaluronic acid, acrylamide, polyacrylamide (e.g., poly(N-isopropylacrylamide), poly(N-isopropylacrylamide-co-allylamine)), polyethylene glycol-maleimide, polyethylene oxide, guar gum, agarose, gelatin, collagen, chitosan, nanohydroxyapatite, silicate, hydrated aluminum silicate.

[0099] A pharmaceutical composition administrable to humans or animals may comprise a composition according to the invention consisting of a suspension of bacteriophages of the class Caudoviricetes in the dispersing medium defined within the scope of the invention, mixed with an antibacterial agent exhibiting synergy and / or complementarity of action that enhances, increases, strengthens, and / or broadens the antibacterial activity of the composition. For example, in the case of bacteriophage suspensions directed against Staphylococcus aureus, the compounds may potentiate the effect Bacteriophage antibacterials can be chosen from the group of M penicillins (oxacillin, cloxacillin, flucloxacillin), A penicillins (amoxicillin / clavulanic acid), synergistins (pristinamycin), fusidic acid, mupirocin, cefazolin, gentamicin, macrolides, lincosamines, trimethoprim-sulfamethoxazole, rifampicin, glycopeptides (vancomycin), ceftaroline or linezolid.

[0100] Depending on the intended application, the composition according to the invention may have a bactericidal effect or a bacteriostatic effect and may be chosen according to the condition.

[0101] For example, a composition of bacteriophages of the class Caudoviricetes according to the invention is a pharmaceutical composition, in particular suitable for enteral administration or an injectable pharmaceutical composition for parenteral administration and, in particular, intended for the treatment of osteoarticular infection, septicemia or infection in a diabetic person.

[0102] The pharmaceutical compositions according to the invention are in particular intended and adapted for use in humans.

[0103] All preferred characteristics described in this description apply to the pharmaceutical compositions, medical uses, and treatment methods described in this description.

[0104] The compositions according to the invention can be used for the manufacture of a medicinal product, in particular for the manufacture of a medicinal product for the treatment of osteoarticular infection, septicemia, or infection in a diabetic person, or a medicinal product having a bactericidal or bacteriostatic effect against Staphylococcus aureus. Treatment methods comprising the administration to humans or animals of an effective quantity of a composition according to the invention, for the treatment of osteoarticular infection, septicemia, or infection in a diabetic person, or for achieving a bactericidal or bacteriostatic effect against Staphylococcus aureus, also form part of the present invention.

[0105] Examples

[0106] The examples below, with reference to the attached Figures, are given for illustrative purposes only.

[0107] Figure IA shows the UV-visible spectra of a bacteriophage suspension used in the examples during the purification steps. The UV-visible spectrum of a phage suspension obtained after the ultrafiltration step (“post-polishing”) formulated in medium F4 (KH2PO4: 1.06 mM; Na2HPO4: 2.97 mM; NaCl: 155.17 mM), shows two characteristic wavelengths (256 nm and 244 nm) corresponding to the absorbances of the phage protein-nucleic acid complex.

[0108] Figure IB shows the UV-visible spectra of a bacteriophage suspension obtained after successive dilutions in medium F4 (KH2PO4: 1.06 mM, Na2HPO4: 2.97 mM, NaCl: 154 mM, initial titer: 26.7 x 10 8 PFU / mL).

[0109] Figure IC shows the absorbance curve (256 nm) of a bacteriophage suspension formulated in medium F4 (KH2PO4: 1.06 mM; Na2HPO4: 2.97 mM; NaCl: 155.17 mM) as a function of bacteriophage titer (absorbance vs. PFU / mL). The limits of detection and quantification (LD, 3 x standard deviation / slope and 9 x standard deviation / slope) were 1.6 x 10-10, respectively. 8 PFU / mL and 4.7 x 10 8 PFU / mL. The impact of the filtration used for terminal sterilization on the bacteriophage titer is approximately 10% (non-significant variation).

[0110] Figure 2 shows the wettability characterization of (A) stoppers (1. BIIR: perfluorinated bromobutyl; 2. CIIR: chlorobutyl), (B) type I glass bottles, (C) polystyrene bottles, (D) polyethylene terephthalate bottles, and (E) poly(styrene-butadiene) filters as a function of PS80 concentration (%). The wettability of the materials is determined from the average of 3 (+) or 4 (4) solution / material contact angle measurements. The error bars show the standard deviation of these measurements. The composition of the buffer mixture and neutral salts in Fl is KH2PO4: 14.78 mM, Na2HPC>4: 56.38 mM, NaCl: 154 mM. The composition of the buffer mixture and neutral salts in formulation F4 is KH2PO4: 1.06 mM, Na2HPO4: 2.97 mM, NaCl: 154 mM. The variations in contact angle between suspensions supplemented and unsupplemented with PS80 are compared using a Student's t-test: NS: not significant. * p < 0.05; ** p < 0.01; *** p < 0.001.

[0111] Figure 3 shows the evolution of the aggregation index (AI) as a function of pH in the F4 bacteriophage suspension (KH2PO4: 1.06 mM, Na2HPO4: 2.97 mM, NaCl: 154 mM). Strong bacteriophage aggregation is observed at pH < 4. Each value corresponds to an experimental determination. AI > 30: strong aggregation; AI < 7: stability threshold.

[0112] Figure 4 shows (A) the evolution of the bacteriophage titer (initial titer: 10 9PFU / mL) of formulation F1-PS80-2 (KH2PO4: 14.78 mM; Na2HPO4: 56.38 mM; NaCl: 154 mM; PS80: 0.05% (corresponding to 0.38 mM)) packaged in type I glass bottles (5 mL filled to 3 mL; headspace: 40%) stored under shelf-life conditions (Le., 4°C and 25°C). (B) Detail of the evolution at 5°C and (C) detail of the evolution at 25°C. Each value is the mean ± standard deviation of three experimental determinations. The line represents the threshold below which the suspension no longer meets the stability criterion (pH <10 8 PFU / mL). Analysis of variance across all groups followed by multiple comparisons using Post Hoc tests (Tukey HSD): NS: not significant. * p<0.05; ** p<0.01; *** p<0.001. ND: not detectable.

[0113] Figure 5 shows (A) the evolution of the title (initial title: 10 9PFU / mL) of a myovirus morphotype bacteriophage suspension, formulated in F4 (KH2PO4: 1.06 mM, Na2HPO4: 2.97 mM, NaCl: 154 mM), after 21 days of horizontal shaking (25 Hz) in polystyrene bottles (15 mL filled to 10.6 mL; headspace: 30%) as a function of PS80 concentration. Each value is the mean ± standard deviation of two experimental determinations. (B) Analysis of variance over all groups followed by multiple comparisons using post-hoc tests (Tukey HSD or Bonferroni): NS: not significant. * p<0.05; ** p<0.01; *** p<0.001.

[0114] Figure 6 shows the evolution of the title (initial title >10 9PFU / mL) of a phage lysate (unpurified; hatched bars) and a purified suspension (white bars) in formulation F4 without PS80 (KH2PO4: 1.06 mM, Na2HPU4: 2.97 mM, NaCl: 154 mM), packaged in polystyrene bottles (15 mL filled to 5 mL; 67% headspace) and subjected to rotary shaking (0.7 Hz) for 28 days. Each value represents the mean ± standard deviation of 3 experimental determinations. Analysis of variance was performed across all groups followed by multiple comparisons using post-hoc tests (Tukey HSD). NS: not significant. * p < 0.05; ** p < 0.01; *** p < 0.001; ND: not detected.

[0115] LIST OF RAW MATERIALS, MATERIALS AND EQUIPMENT USED IN THE EXAMPLES

[0116] The excipients used are as follows: sodium chloride (NaCl) FRESENIUS 0.9% (Fresenius Kabi, Sevres, France); water for injections Lavoisier (Laboratoires Chaix et du Marais, Gailletrous, France); PBS (phosphate-buffered saline) IX at pH 7.4 (GibcoTM, Life Technologies Corporation, Grand Island, NY, USA and Life TechnologiesTM, Paisley, England); monobasic potassium phosphate KH2PO4, CAS: 7778-77-0 (ReagentPIus®, Sigma-Aldrich®, Saint Louis, MO, USA); dibasic anhydrous sodium phosphate Na2HPO4, CAS: 7558-79-4 (CARLO ERBA Reagents SAS, Val de Reuil, France); Tween® 80 (polysorbate 80 or PS80) (EMPROVE® ESSENTIAL, Merck KGaA, Darmstadt, Germany); Tween® 20 (polysorbate 20 or PS20) (BioXtra, Merck KGaA, Darmstadt, Germany); poloxamer 188 or P188 (Kolliphor® P188, BASF Corporation, Geismar, LA, US).

[0117] A 1 M hydrochloric acid (HCl) solution was prepared extemporaneously to force bacteriophage aggregation in an acidic pH environment (see Figure 3): 10.8 g HCl R (CARLO ERBA Reagents SAS, Val de Reuil, France) diluted in demineralized water, sufficient to make 100 mL. This solution was used to modify the pH, in accordance with the results shown in Figure 3.

[0118] The packaging used is listed below: 5 mL type I clear glass vials (SGD Pharma, Puteaux, France); chlorobutyl, Teflon-coated bromobutyl and silicone stoppers (Gravis Trelazé, Trélazé, France); caps bromobutyl coated with Raydylyo® fluorinated polymer (ARaymond® Life SASU, St Egreve, France); 15 mL conical polystyrene centrifuge tubes with polypropylene stopper (Corning™ Falcon™, Fischer Scientific SAS, Illkirch, France).

[0119] GENETIC CHARACTERISTICS OF BACTERIOPHAGES USED WITH MYOVIRUS MORPHOTYPE

[0120] vB_SauM-VlSA19 - of myovirus morphotype (Kolenda et al. 2022) used in all the examples that follow: • Lineage: Viruses; Duplodnaviria; Heunggongvirae; Uroviricota; Caudoviricete, Herelleviridae; Twortvirinae; SUviavirus,' unclassified SHviavirus • Taxonomy ID: 2972385 • GenBank partial genome: ON814134.1 • 245 proteins; genome size I 138,507 bp.

[0121] MATERIALS AND METHODS:

[0122] OBTAINING PURIFIED BACTERIOPHAGE SUSPENSIONS:

[0123] Bacteriophages were isolated as described in (Kolenda et al. 2022). The S. aureus P2SA225 strain (CC6 clonal complex; methicillin-susceptible; isolated from a nasal swab obtained during clinical trial ERS1242607) was incubated for 2 hours at 37°C with shaking at 180 rpm in 1 L of Superior Broth™ culture medium (Athena Enzyme Systems™, Baltimore, MD, USA) in a 2.5 L Frenbach culture flask (Avantor™ VWR™, Radnor, PA, USA). Bacteriophage vB_SauM-VlSA19 was added to achieve a MOI (multiplicity of infection) of 10⁻⁶. 2 The mixture was then incubated for approximately 3 hours (Kolenda et al. 2022). The amount of double-stranded DNA in the Fernbach was then quantified using a Quantus™ Fluorometer (Promega, Madison, WI, USA) with the Quantifluor® dsDNA System kit (Promega, Madison, WI, USA). For each µg of DNA, 1 unit of Benzonase® Nuclease enzyme (Sigma-Aldrich Chimie SarL, Saint-Quentin de Fallavier, France) and (ii) 2 mL of 1 M MgCl (Invitrogen™, ThermoFischer Scientific Baltic UAB, Vilnius, Lithuania), an amount sufficient to reach a concentration of 2 mM in the Fernbach. The mixture was incubated for 1 hour at 37°C, with shaking at 180 rpm.

[0124] The product was collected and filtered using a Polycap TC 150 progressive filtration cartridge (0.8 µm - 0.22 µm) (Cytiva, Buckinghamshire, England) and packaged in 1 L uLDPE FlexFilm® bags (Avantor™, VWR™, Radnor, PA, USA). The resulting product is called phage lysate. The bacterial endotoxin concentration of this phage lysate was typically between 1 and 50 EU / mL, depending on the production lot. The total protein concentration of this phage lysate was typically greater than 0.2 mg / mL.

[0125] The product was then purified by ultracentrifuge (Boulanger 2009). Into an ultraclear, thin-walled tube (Beckman Coulter France SAS, Villepinte, France), 9 mL of (i) cesium chloride (CICs) solution (Sigma-Aldrich Chimie SarL, Saint-Quentin de Fallavier, France) at 1.6 g / L in 0.9% Fresenius NaCl (Fresenius Kabi, Sevres, France); (ii) CICs solution at 1.5 g / L; (iii) CICs solution at 1.3 g / L; and (iv) bacteriophage suspension to be purified were added. The tube was ultracentrifuged at 120,000 g (SW32Ti rotor, Beckman, Brea, CA, USA) for 2 hours at 4°C. The purified bacteriophages were then harvested between the density rings of 1.5 and 1.6 g / L before being dialyzed (10K MWCO cassettes, Serva Electrophoresis GmbH, Heidelberg, Germany) twice in 3 L of PBS IX buffer at pH 7.4 (Gibco™, Life Technologies Corporation, Grand Island, NY, USA and Life Technologies™, Paisley, England).

[0126] After purification, the quantities of endotoxins and total protein were evaluated. The concentration of bacterial endotoxins in the formulations was between 0.05 and 0.5 EU / mL, depending on the production batch, and their concentration of total protein was <0.01-0.08 mg / mL, depending on the production batch.

[0127] PREPARATION OF BACTERIOPHAGE COMPOSITIONS:

[0128] A 15 mL Amicon® Ultra ultrafiltration unit with an Ultracel-100 membrane made of regenerated cellulose (100 kD, Merck-Millipore, Sigma-Aldrich Chimie Sarl, Saint-Quentin de Fallavier, France) was filled with the purified bacteriophage suspension obtained and centrifuged (Centrifuge 5810 with rotor A-4-81 Eppendorf SE, Hamburg, Germany) for 15 minutes at 3100 g. This step is commonly referred to as polishing. The retentate was then resuspended in the selected formulation of bacteriophage dispersing medium (detailed below), and the resulting composition was stored in 125 mL polyethylene terephthalate (PET) bottles (PharmaTainer™, SaniSure®, Camarillo, CA, USA).

[0129] Then the formulation was sterilized by terminal filtration. More specifically, this filtration consists of filtering and aliquoting the suspension in the packaging of interest using a 3-piece 10 mL BD Plastipak™ syringe (Becton Dickson France SAS, Grenoble, France) connected to a 0.22 µm polyethersulfone filtration unit (Sterivex-GP, Millipore Merck KGaA, Damstadt, Germany).

[0130] Figure IA shows the evolution of the UV-visible spectrum of the bacteriophage suspension as a function of the purification steps. The ultrafiltration step during the preparation of the bacteriophage compositions, called polishing, is characterized by a UV-visible spectrum with two absorbances at 244 nm and 256 nm corresponding to the protein-nucleic acid complex (Morag et al. 2018). Determining the absorbance at 350 nm allows for the assessment of the aggregation index (AI) of the suspended bacteriophages (Pignataro, Herrera, and Dodero 2020). A linear relationship between the maximum absorbance and the bacteriophage titer was demonstrated. Figure IB shows the evolution of the UV spectrum as a function of dilution, and Figure IC illustrates the non-significant reduction in the bacteriophage titer following the final sterilizing filtration.

[0131] STABILITY TEST UNDER STIRRING

[0132] The prepared compositions were placed either in (i) vertical rotary stirring at 40 rpm (Cole-Parmer® TR-200D Series Stuart, Saint Neots, England); or in (ii) horizontal vibrating motion stirring between 150 and 2000 rpm (3 mm orbit, Multi Reax, Heidolph Instruments, Schwabach, Germany).

[0133] MEASURES TAKEN

[0134] Absorbance measurements were performed using a V-730 UV-Visible spectrophotometer (Jasco, Lisses, France). The aggregation index (AI) was calculated based on the 100 (Pignataro, Herrera, and Dodero 2020) with À max measured at 255 nm. With AI > 30: high aggregation; AI < 7: stability threshold. pH and osmolality measurements were performed respectively with the InLab Ultra-Micro ISM probe connected to the SevenCompact™ Duo S213 pH meter (Mettler-Toledo SAS, Viroflay, France) and the OsmoPRO (Radiometer SAS, Neuilly-Plaisance, France).

[0135] Bacteriophage titer measurements (PFU / mL) were performed by spot titration on a reference bacterial strain (in this case, P2SA225 for bacteriophage vB_SauM-VlSA19). P2SA225 was cultured the previous day at 180 rpm and 37°C in 9 mL of Trypto Casein Soy culture medium (TSB, bioMérieux SA, Marcy-L'Etoile, France). After approximately 20 hours of incubation, 500 µL of bacterial broth was transferred to a 120 x 120 mm square polystyrene Petri dish (Greiner Bio-one, Courtaboeuf, France). Approximately 30 mL of liquefied TSB-soft (30 g / L of solid TSB BD-Difco™ - 7.56 g / L of BD-Difco™ granulated agar (FisherScientific SAS, Illkirch, France)) was then poured over the mixture and homogenized before drying to form an agar plate. The suspension to be titrated was serially diluted 10⁻¹ emein TSB (bioMérieux SA, Marcy-L'Etoile, France) in a 96-well plate, then 5 µL from each well was loaded onto the agar. Each measurement was performed as a triplicate of broth bacterial. Bacteriophage titers were read after an incubation of 20 h at 37°C.

[0136] Contact angle measurements were performed by depositing 10 pL of liquid on the surface of interest, then a photo was taken and the angle at the interface was measured with the ImageJ software (Nih.gov).

[0137] Statistical analyses were performed, as indicated in the figure legends, by ANOVA followed by multiple comparisons by Post Hoc (Tukey HSD or Bonferroni) or Student's t-test with o=0.05, using KaleidaGraph software (version 3.6, Synergy Software).

[0138] METHOD FOR PREPARING DISPERSING MEDIA FOR BACTERIOPHAGES IN THE COMPOSITIONS:

[0139] The formulations were prepared as detailed below, at the latest the day before the bacteriophages were suspended: • Fl: KH2PO4: 14.78 mM; Na2HPO4: 56.38 mM; NaCI: 154 mM In two sterile 180 mL polypropylene powder containers (CORNING® GOSSELIN™, Borre, France), weigh out 800 mg of Na₂HPO₄ and 200 mg of KH₂PO₄, respectively. Dissolve both powders in less than 100 mL of 0.9% NaCl added using a 30 mL BD Plastipak™ three-piece syringe (Becton Dickson France SAS, Grenoble, France). Then transfer the liquid into the powder container holding the Na₂HPO₄, which was tare-balanced before adding the powder. Finally, add 0.9% NaCl to 100 g. If dissolution is not complete, place the powder container in an ultrasonic bath for 10 minutes at 40 °C (FB15051, Fischerbrand®, FisherScientific SAS, Illkirch, France). • F2: water for injection preparation. • F4: KH2PO4: 1.06 mM; Na2HPO4: 2.97 mM; NaCl: 155.17 mM This composition corresponds to PBS IX at pH 7.4 (GibcoTM, Life Technologies Corporation, Grand Island, NY, USA and Life TechnologiesTM, Paisley, England) in a sterile 180 mL powder container (CORNING® GOSSELIN™, Borre, France). • F1-PS80-1 (composition according to the invention): KH2PO4: 14.78 mM; Na2HPÛ4: 56.38 mM; NaCl: 154 mM; PS80: 0.001% (corresponding to 0.008 mM) Same formulation as Fl. Then add 9.4 pL of PS80 to 1000 mL of Fl. • F1-PS80-2 (composition according to the invention): KH2PO4: 14.78 mM; N32HPO4: 56.38 mM; NaCl: 154 mM; PS80: 0.05% (corresponding to 0.38 mM) Same formulation as Fl. Then add 47 pL of PS80 to 100 mL of Fl. • F1-PS20 (composition according to the invention): KH2PO4: 14.78 mM; Na2HPO4: 56.38 mM; NaCl: 154 mM; PS20: 0.05% (corresponding to 0.44 mM) Same formulation as Fl. Then add 46 pL of PS20 to 100 mL of Fl. • F2-PS80: water for injection (WFI); PS80: 0.05% (corresponding to 0.38 mM) Weigh 100 mg of EPPI into a sterile 180 mL powder container (CORNING® GOSSELIN™, Borre, France). Add 47 pL of PS80. • F3-PS80: NaCl 0.9%; PS80: 0.05% (corresponding to 0.38 mM) Weigh 100 mg of NaCl 0.9% (corresponding to 154 mM) into a sterile 180 mL powder container (CORNING® GOSSELIN™, Borre, France). Add 47 pL of PS80. • F4-PS80-1 (composition outside the invention): KH2PO4: 1.06 mM; Na2HPÛ4: 2.97 mM; NaCl: 155.17 mM; PS80: 0.001% (corresponding to 0.008 mM) Same formulation as F4 then add 9.4 pL of PS80 to 1000 mL of F4. • F4-PS80-2 (composition according to the invention): KH2PO4: 1.06 mM; Na2HPC>4: 2.97 mM; NaCl: 155.17 mM; PS80: 0.005% (corresponding to 0.038 mM) Same formulation as F4 then add 24 pL of PS80 to 510 mL of F4. • F4-PS80-3 (composition according to the invention): KH2PO4: 1.06 mM; Na2HPC>4: 2.97 mM; NaCl: 155.17 mM; PS80: 0.05% (corresponding to 0.38 mM). Weigh 100 mg of PBS IX at pH 7.4 (GibcoTM, Life Technologies Corporation, Grand Island, NY, USA and Life TechnologiesTM, Paisley, England) into a sterile 180 mL powder container (CORNING® GOSSELIN™, Borre, France). Add 47 µL of PS80. • F1-P188-1 (composition according to the invention): KH2PO4: 14.78 mM; Na2HPÛ4: 56.38 mM; NaCl: 154 mM; P188: 0.041% (corresponding to 0.048 mM) Same formulation as Fl. Then add 0.0041 g of P188 to 10 mL of Fl. • F1-P188-2 (composition according to the invention): KH2PO4: 14.78 mM; Na2HPÛ4: 56.38 mM; NaCl: 154 mM; P188: 0.413% (corresponding to 0.48 mM) Same formulation as Fl. Then add 0.0413 g of P188 to 10 mL of Fl. • F1-P188-3 (composition according to the invention): KH2PO4: 14.78 mM; N32HPO4: 56.38 mM; NaCl: 154 mM; P188: 4.13% (corresponding to 4.8 mM) Same formulation as Fl. Then add 0.4126 g of P188 to 10 mL of Fl. • F1-P188-4 (composition according to the invention): KH2PO4: 14.78 mM; Na2HPO4: 56.38 mM; NaCl: 154 mM; P188: 12.38% (corresponding to 14.4 mM) Same formulation as Fl. Then add 1.2377 g of P188 to 10 mL of Fl.

[0140] The percentages are mass percentages relative to the volume of dispersing medium. The resulting compositions were then immediately packaged in the selected container.

[0141] The concentration of bacterial endotoxins in all formulations was between 0.05 and 0.5 EU / mL, and their total protein concentration was <0.01 to 0.08 mg / mL.

[0142] RESULTS OBTAINED

[0143] In the examples according to the invention, highly concentrated suspension compositions of phages of the class Caudoviricetes (>10 8PFU / mL) were obtained and proved resistant to pH variations, adsorption onto packaging materials, and mechanical stress known to reduce bacteriophage activity during (i) the production process, (ii) storage, and (iii) transport. The suspension composition was chosen based on intrinsic biochemical and biophysical properties evaluated during forced stress tests (mechanical agitation, pH, ionic strength, wettability of packaging materials) (Nowak et al. 2017; Dumas, Huille, and Prades 2019) to reduce (i) the exposure of hydrophobic regions of bacteriophage proteins, and (ii) the rate of non-covalent (hydrophobic and / or reversible electrostatic interactions) and covalent (disulfide bond formation) bacteriophage aggregates (potentially immunogenic).

[0144] To assess the stability of bacteriophage suspension compositions, and in particular to evaluate resistance to aggregation and pH variations, an aggregation test induced (i) by rotary agitation (40 rpm, 0.7 Hz), (ii) by horizontal agitation (1500 rpm, 25 Hz) of the final packaging was carried out for 14–21 days at room temperature (15–25°C) (Kopp et al. 2020). Measurement of the UV-visible spectrum recorded during the test allowed for the determination of the bacteriophage aggregation index (AI). A spot titration of bacteriophages was performed during and at the end of the test.

[0145] In order to further limit the aggregation phenomena (i) bacteriophage-bacteriophage, (ii) at gas-liquid and liquid-solid interfaces, a primary packaging (bottle and cap) was chosen taking into account: the filling volume in relation to the total volume offered by the packaging; the wettability of the primary packaging.

[0146] The selected packaging met the following criteria: - Polystyrene bottle. The wettability of the polystyrene bottle was significantly improved, achieving a value below 70°, by the presence and quantity of selected non-ionic surfactant, as shown in Figure 2(C), compared to a solution without surfactant. The hydrophilic surface area of ​​the bottle (assessed by a contact angle with water of less than 90°) helps to limit aggregation between the hydrophobic parts of the bacteriophage proteins and the bottle walls. - Type I pharmaceutical glass bottle. The wettability of the type I glass bottle has been significantly improved, achieving a value below 32°, by the presence and quantity of selected non-ionic surfactant, as shown in Figure 2 (B). The hydrophilic surface area of ​​such bottles (assessed by a contact angle with water of less than 90°) helps to limit aggregation between the hydrophobic parts of bacteriophage proteins and the bottle walls. - The cap material. The wettability of bromobutyl caps coated with a perfluorinated polymer (BIIR) and chlorobutyl (CIIR) has could be usefully improved, with obtaining a value below 90°, by the presence and quantity of selected non-ionic surfactant, as shown in Figure 2, parts Al and A-2.

[0147] For comparison, after 14 days of rotary agitation at 0.7 Hz, as detailed in Table 1, the bacteriophage, placed in surfactant-free suspensions (F1: buffered saline and F2: water for injection), exhibits a drop in its bacteriophage titer of at least 2 log, associated with a high aggregation index measured by UV spectrophotometry (25 and a negative value, respectively), indicating that the aggregates formed significantly disrupt the incident light. Thus, the presence of nonionic surfactant at the recommended concentration is necessary to maintain the titer and prevent aggregation. However, again according to Table 1, simply adding surfactant is not sufficient to reduce aggregation to an acceptable level (AI of 12 at day 14 for formulation F2-PS80; AI of 7 at day 14 for formulation F3-PS80).

[0148] As an illustration, Figure 3 shows the evolution of the aggregation rate of the F4-PS80-3 bacteriophage suspension as a function of pH (the pH is modified by the addition of HCl). The presence of phosphate saline buffer in the compositions used in the examples allows for maintaining a near-neutral pH. Unexpectedly and unpredictably, the addition of neutral salts composed of a pair of monovalent ions such as NaCl (which are in the first elements of the Hofmeister series and therefore likely to induce aggregation (Okur et al. 2017)) enhanced the antiaggregatory activity and the stability of the bacteriophage suspensions upon shaking. Indeed, according to Table 1: - With comparable packaging (5 mL type I glass filled to 3 mL), the reduction in bacteriophage titer is greater in formulation Fl (bacteriophage in a 338 mM phosphate saline buffer) than in formulation F2 (bacteriophages in water for injections).Thus, while bacteriophages exhibit relative stability in water under agitation, bacteriophage-water interactions are destabilized by the addition of kosmotropic anions (present in Fl). The Fl formulation without surfactant is not. sufficient to preserve the title under pressure. -The compositions F1-PS80-2 and F4-PS80-3 according to the invention, containing the electrolytes described above, preserve the bacteriophage titer and have an aggregation index close to 3, while the formulations F2-PS80 and F3-PS80 free of phosphate buffer have a preserved titer but an aggregation index greater than or equal to 7, which is predictive of instability.

[0149] The results in Table 1 illustrate that one without the other, Le., (i) saline solution containing a buffer mixture, of the phosphate type in the illustrated example and (ii) PS80, are insufficient to maintain the antibacterial activity of bacteriophages as well as acceptable aggregation rates throughout the stages through which a finished product passes, e.g., shelf-life storage; transport.

[0150] In Table 1, the particle size, electrokinetic, and bacteriophage titer analyses after 14 days of rotary shaking (0.7 Hz) at room temperature (15°C–25°C) are presented for the compositions outside the invention and according to the invention, corresponding to myovirus morphotype bacteriophage suspensions comprising a buffer mixture and a neutral surfactant, selected from the polysorbate (PS) or poloxamer (P) series, packaged (i) in polystyrene bottles (20 mm diameter, 2 or 5 mL fill volume in a 15 mL bottle closed with an HDPE cap), (ii) in type I bottles (20 mm diameter, 3 mL fill volume in a 5 mL bottle closed with a perfluorinated bromobutyl cap). The initial bacteriophage titer was 10 9 PFU / mL. Each value is the mean ± standard deviation of 3 (+) or 4 (4) experimental determinations. ND: not determined.

[0151] Aggregation indices of 7 or more are predictive of a decline in stability over time.

[0152] Table 1 Table 1 (continued 1) Table 1 (continued 2)

[0153] A measurement of the bacteriophage titer (PFU / mL) after rotary shaking (0.7 Hz) confirms the link between aggregation and antibacterial activity (Table 2). In an initial early phase (< 5 days), in the absence of PS80, significant aggregation is observed, followed, between 5 and 14 days, by probable irreversible aggregation and then fragmentation of the bacteriophages, resulting in a lack of antibacterial activity (Table 2).

[0154] Table 2 presents the bacteriophage titer of bacteriophage suspensions formulated in F4 (KH2PO4: 1.06 mM, Na2HPO4: 2.97 mM, NaCl: 154 mM), packaged in a polystyrene bottle (15 mL filled to 6 mL; headspace: 60%) with a high-density polyethylene (HDPE) cap, as a function of the PS80 concentration (%) after 14 days of rotary shaking (initial titer: 10 9 PFU / mL, 40 rotations per minute, 0.7 Hz). Each value is the mean ± standard deviation of 3 or 4 experimental determinations. Analysis of variance on all groups followed by multiple comparisons using post-hoc tests (Tukey HSD or Bonferroni): NS: not significant. * p < 0.05; *** p < 0.0001.

[0155] Table 2

[0156] For comparison, the aggregation rates and titers of Caudoviricetes bacteriophage suspensions subjected (i) to rotary shaking (14 days, 40 rotations per minute, 0.7 Hz), (ii) maintained in two buffered aqueous solutions of different ionic strengths (F1-PS80-2 and F4-PS80-3), (iii) containing 0.05% PS80, (iv) packaged in type I glass bottles (5 mL filled to 3 mL, headspace: 40%), and (v) closed with a perfluorinated BIIR stopper are shown in Table 1. The compositions F1-PS80-2, F4-PS80-3, F1-P188-1, F1-P188-2, F1-P188-3, and F1-P188-4 have sodium chloride (155 mM) - phosphate buffer (approximately 4 and 71 mM, respectively) - 0.05% PS80 or 0.041%, 0.413%, 4.13% and 12.38% P188 respectively - type I borosilicate glass closed with a perfluorinated BIIR stopper or a 15 mL polystyrene bottle preserve the bacteriophage titer under conditions of intense mechanical stress.

[0157] In the absence of agitation and surfactant, the composition of sodium chloride (155 mM) - phosphate buffer (4-71 mM) - type I borosilicate glass sealed with a perfluorinated BIIR stopper maintains the bacteriophage titer as a function of storage temperature (Figure 4-A and Figure 4-B). At 5°C, the bacteriophage titer shows no significant decrease at one year compared to day 0 (Figure 4-B), whereas a decrease of approximately 4 log from the initial titer is observed at a storage temperature of 25°C (Figure 4-C). These differences are partly explained by the reduction of Brownian motion at low temperatures, thus reducing material-bacteriophage and bacteriophage- Bacteriophages (i.e., bacteriophages are non-motile colloidal particles (Kasman and Porter 2022)) cause adsorption onto materials, aggregation / agglomeration between bacteriophages, and then fragmentation of viral particles. It should be noted that after one year, the titer of a bacteriophage suspension stored at 5°C is still above the stability threshold of 10 8 PFU / mL (8 logio).

[0158] To distinguish the effects of cavitation and / or ventilation (produced by the rotation and settling of the suspension) from purely mechanical effects, a bacteriophage aggregation test induced by horizontal agitation (1500 rotations per minute, 25 Hz) was performed for 21 days at room temperature (15°C–25°C) in a polystyrene bottle (15 mL filled to 10.6 mL, headspace: 30%). The results of the reduction in bacteriophage titer over time for different concentrations of PS80 are shown in Figure 5(A) and Figure 5(B). It should be noted that the tests at 0.02% and 0.05% are above the CMC of polysorbate 80.

[0159] The instability under shaking of a purified suspension formulated without PS80, compared to a phage lysate (unpurified), is shown in Figure 6. Indeed, after only two days of shaking, the titer of the purified suspension is less than 10 8PFU / mL while the titer of the unpurified suspension is always greater than 10 8 PFU / mL at one month of agitation. References

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Claims

Demands

1. A composition, and in particular a pharmaceutical composition, comprising or even consisting of a suspension of bacteriophages of the class Caudoviricetes having lytic activity against at least one strain of Gram-positive bacteria, at a titer of at least 10 8 PFU / mL, typically at a concentration of 10 8 at 10 10 PFU / mL, and in particular 10 9 at 10 10 PFU / mL, in a dispersing medium comprising, or even consisting of: - at least one non-ionic surfactant, and in particular only one non-ionic surfactant, chosen from: - the polysorbate family, at a molar concentration higher than its critical micellar concentration, but not exceeding 2 mg / mL, and / or - the poloxamer family, at a concentration ranging from 0.005% (w / v) to 30% (w / v), - one or more neutral salts formed from a pair of monovalent ions, whose total molar concentration is in the range of 75 to 160 mM, - a buffer mixture giving the composition a pH of 6.0 to 7.9, and preferably of 7.0 to 7.5, typically chosen from the pairs hydrogen phosphate / dihydrogen phosphate ion, citric acid / citrate ion and citric acid / hydrogen phosphate ion, the molarity of the buffer mixture being in the range of 4 mM to 100 mM, - water, said composition comprising less than 0.9 units of bacterial endotoxins (EU) per mL, a total protein concentration of less than 0.09 mg / mL and said bacteriophage suspension in said dispersing medium having an osmolality between 150 mOsm / kg and 600 mOsm / kg.

2. Composition according to claim 1, characterized in that the bacteriophages of the class Caudoviricetes belong to the genus Siiviavirus, having lytic activity against at least one strain of Staphylococcus aureus.

3. Composition according to claim 1 or 2, characterized in that said bacteriophages have a myovirus morphotype.

4. Composition according to any one of the preceding claims, characterized in that the dispersing medium has an ionic strength of 150 to 400 mM.

5. A composition according to any one of the preceding claims, characterized in that it is stable for at least one year at a temperature in the range of 2 to 8°C, i.e., it has a bacteriophage titer that remains greater than or equal to 10 8 PFU / mL.

6. Composition according to any one of the preceding claims, characterized in that the bacteriophages are neither in conjugated form nor in complexed form.

7. Composition according to any one of the preceding claims, characterized in that it comprises, as a non-ionic surfactant, one or more of the following surfactants: - polysorbate 80, at a molar concentration greater than 0.013 mM and less than or equal to 1.6 mM, preferably in the range of 0.16 mM to 0.38 mM, - polysorbate 20, at a molar concentration greater than 0.05 mM and less than or equal to 1.77 mM, preferably in the range of 0.18 mM to 0.44 mM, - poloxamer 188 at a concentration greater than or equal to 0.005% (w / v) and less than or equal to 30% (w / v), preferably in the range of 0.01% (w / v) to 5% (w / v) for liquid compositions, and preferably in the range of 15% (w / v) to 25% (w / v) for viscous compositions, - poloxamer 124 at a concentration greater than or equal to 0.005% (w / v) and less than or equal to 30% (w / v), preferably in the range of 0.01% (w / v) to 5% (w / v) for liquid compositions, and preferably in the range of 15% (w / v) to 25% (w / v) for viscous compositions, - poloxamer 237 at a concentration greater than or equal to 0.005% (w / v) and less than or equal to 30% (w / v), preferably in the range of 0.01% (w / v) to 5% (w / v) for liquid compositions, and preferably in the range of 15% (w / v) to 25% (w / v) for viscous compositions, - poloxamer 338 at a concentration greater than or equal to 0.005% (w / v) and less than or equal to 30% (w / v), preferably in the range of 0.01% (w / v) to 5% (w / v) for liquid compositions, and preferably in the range of 15% (w / v) to 25% (w / v) for viscous compositions, and - poloxamer 407 at a concentration greater than or equal to 0.005% (w / v) and less than or equal to 30% (w / v), preferably in the range of 15% (w / v) to 25% (w / v) for liquid compositions, and preferably in the range of 15% (w / v) to 25% (w / v) for viscous compositions, the viscous compositions having an apparent viscosity of at least 100 mPa.s as measured according to monograph 2.2.

10. Viscosity - European Pharmacopoeia rotary viscometer method, it being understood that the total concentration of non-ionic surfactants is from 0.005% (w / v) to 30% (w / v).

8. Composition according to any one of the preceding claims, characterized in that the buffer mixture is a KH2PO4 / Na2HPO4 mixture, typically KH2PO4 at a molar concentration of 1 to 15 mM and Na2HPO4 at a molar concentration of 3 to 56 mM.

9. Composition according to any one of the preceding claims, characterized in that it comprises only NaCl, as a neutral salt, which is present at a molar concentration of 75 to 160 mM, typically at a molar concentration of 150 to 160 mM.

10. Pharmaceutical composition according to any one of the preceding claims, characterized in that it contains no compound of animal origin.

11. Composition according to any one of the preceding claims, characterized in that it is sterile, pyrogen-free and isotonic or hypertonic.

12. Composition according to any one of the preceding claims, characterized in that it is packaged in a container such as a bottle, a bag or a syringe delimited by walls which are of type I glass, type II glass, polystyrene, polypropylene, polyethylene, polyethylene terephthalate, treated or not with silicone oil, in particular closed with a stopper of a perfluorinated or non-perfluorinated rubber material.

13. Composition according to claim 12, characterized in that it exhibits a wettability of the container walls corresponding to a contact angle of 20 to 60°.

14. Composition according to claim 12 or 13, characterized in that the container is closed by a stopper and the composition has a stopper wettability corresponding to a contact angle of less than 90°.

15. Composition according to any one of the preceding claims, characterized in that said bacteriophage suspension in said dispersing medium is supplemented with a viscous solution or hydrogel of cellulose ether, polyethylene glycol, polyvinyl alcohol, sodium alginate, PVA-sodium alginate, acrylate, methacrylate, polyacrylate, hyaluronic acid, acrylamide, polyacrylamide, polyethylene glycol-maleimide, polyethylene oxide, guar gum, agarose, gelatin, collagen, chitosan, nanohydroxyapatite, silicate, or hydrated aluminum silicate.

16. Composition according to any one of the preceding claims, characterized in that it is suitable for enteral administration, or suitable for administration by injection, in particular by parenteral route, including intravenous, intraarterial, intramuscular, subcutaneous and / or intra-articular administrations.

17. Composition according to any one of the preceding claims, for its use as a medicinal product, in particular for its use in the treatment of osteoarticular infections.

18. A method for preparing a composition, and in particular a pharmaceutical composition, according to any one of claims 1 to 16, comprising introducing into a dispersing medium comprising, or even consisting of: - at least one non-ionic surfactant, and in particular only one non-ionic surfactant, chosen from: - the polysorbate family, at a molar concentration higher than its critical micellar concentration, but not exceeding 2 mg / mL, and / or - the poloxamer family, at a concentration ranging from 0.005% (w / v) to 30% (w / v), - one or more neutral salts formed from a pair of monovalent ions, whose total molar concentration is in the range of 75 to 160 mM, - a buffer mixture giving the composition a pH of 6.0 to 7.9, and preferably of 7.0 to 7.5, typically chosen from the pairs hydrogen phosphate ion / dihydrogen phosphate ion, citric acid / citrate ion and citric acid / hydrogen phosphate ion, the molarity of the buffer mixture being in the range of 4 mM to 100 mM, - water, purified bacteriophages in the quantity necessary to obtain a final concentration of at least 10 in the composition 8 PFU / mL, and typically 10 9 at 10 10 PFU / mL in bacteriophages.

19. A preparation method according to claim 18 characterized in that after the introduction of the bacteriophages into the dispersing medium, the resulting suspension is sterilized by carrying out a filtration operation, by passing through a filter made of polyethersulfone, polystyrene, poly(styrene-butadiene) or polyvinylidene fluoride.

20. Use of a dispersing medium comprising, or even consisting of: - at least one non-ionic surfactant, and in particular only one non-ionic surfactant, chosen from: - the polysorbate family, at a molar concentration higher than its critical micellar concentration, but not exceeding 2 mg / mL, and / or - the poloxamer family, at a concentration ranging from 0.005% (w / v) to 30% (w / v), - one or more neutral salts formed from a pair of monovalent ions, whose total molar concentration is in the range of 75 to 160 mM, - a buffer mixture giving the composition a pH of 6.0 to 7.9, and preferably of 7.0 to 7.5, typically chosen from the pairs hydrogen phosphate / dihydrogen phosphate ion, citric acid / citrate ion and citric acid / hydrogen phosphate ion, the molarity of the buffer mixture being in the range of 4 mM to 100 mM, - water, to improve storage stability at a temperature within the range of 2 to 8°C, of ​​bacteriophages of the class Caudoviricetes having lytic activity against at least one strain of Gram-positive bacteria, said bacteriophages being suspended in said dispersing medium at a titer of at least 10 8 PFU / mL, and typically 10 8 at 10 10 PFU / mL in bacteriophages, and in particular 10 9 at 10 10PFU / mL and said composition comprising less than 0.9 units of bacterial endotoxins per mL, less than 0.09 mg / mL or 90 nm / mL of total protein and the osmolality of said bacteriophage suspension in said dispersing medium being in the range of 150 mOsm / kg to 600 mOsm / kg.

21. Use according to claim 20, characterized in that the stability obtained corresponds to a bacteriophage titer that remains greater than or equal to 10 8 PFU / mL, after storing the composition at a temperature within the range of 2 to 8°C for at least one year.

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