Incorporation by Cross-Reference

This application claims priority from Australian provisional patent application number 2020904800, filed on 22 December 2020, the entire contents of which are incorporated herein by cross-reference.

Technical Field

The present invention relates generally to the fields of biology and medicine, and more specifically to compositions and methods for the topical delivery of bacteriophage. In certain forms, the present invention provides compositions and methods for the topical delivery of therapeutic bacteriophage, along with methods for preparing these compositions.

Background

The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the invention.

Multidrug-resistant (MDR) bacteria are rapidly emerging as a major public health problem. The overuse and misuse of antibiotics has provoked the emergence of MDR bacteria, which are responsible for 16% of hospital-acquired infections. Wound infections are reported in one third of hospital-acquired infections in surgical patients and account for up to 80% of mortality. The rise in the number of MDR pathogens is adversely impacting the incidence and severity of burn wound infections with complications such as sepsis and death. The majority of nosocomial infections are caused by ESKAPE pathogens Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) and these infections bear the highest risk of mortality. P. aeruginosa is one of the most prevalent pathogens in acute and chronic burn wound infection. Burn wound infections caused by P. aeruginosa often result in rapid deterioration, followed by systemic spread and death within days or weeks. The high pathogenicity of P. aeruginosa is primarily due to intrinsic resistance to several antibiotics as well as acquired multi-drug resistance within the health care system. Broad spectrum antibiotic resistance in these superbugs significantly limits effective therapeutic options. Aminoglycosides and polymyxins are the last line antibiotics, but are associated with adverse effects such as ototoxicity, nephrotoxicity and neurotoxicity. Furthermore, there is growing evidence that superbugs are developing resistance to these last resort agents. Hence, it is imperative to develop alternative methods to combat MDR pathogens.

Bacteriophage (commonly known in the art as “phage”) have been recognized as potential new antimicrobial therapeutics to replace or supplement antibiotics. Phage are natural predators of bacteria that exploit bacterial cells for survival and replication. Lytic phage infect, replicate and then kill their bacterial host cells by lysing the cell wall. During bacteriolysis, hundreds of phage progenies are released for subsequent infection. The highly specific nature of phage avoids disturbance of non-targeted bacteria, providing low inherent toxicity. Furthermore, phage can kill bacteria irrespective of the antibiotic-resistance profile and can penetrate biofilms, a problematic state of bacteria in chronic wound infections.

The wound healing potential of phage has been demonstrated previously. However, dripped liquid topical formulations can easily run off the infection site, which complicates dose control and temporarily restricts the mobility of patients. Dripped liquid topical formulations often fail to keep wounds moist and offer little physical protection.

A need exists for compositions which can effectively deliver therapeutic bacteriophage to a specific site.

Summary of the Invention

The present invention addresses at least one of the problems associated with current compositions and/or methods for the delivery of phage.

The present inventors have found that, by combining phage with an agent comprising at least one non-ionic polymer selected from the group consisting of polyethylene oxide (PEO), polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP), compositions may be formed which do not easily run off the site of administration, for example, a wound. The inventors have also found that combining phage with an agent comprising at least one non-ionic polymer can provide compositions which may be administered via spraying. The compositions of the present invention may help to deliver and/or retain moisture at the site of administration and/or form a protective barrier over the site.

The present invention relates at least in part to the following aspects and embodiments. Embodiment 1. A composition for the topical delivery of bacteriophage, the composition comprising:

- an agent comprising at least one non-ionic polymer selected from the group consisting of polyethylene oxide (PEO), polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP); and

- the bacteriophage for topical delivery.

Embodiment 2. The composition according to embodiment 1, wherein:

- the concentration of the PEO in the agent is between 5% and 15% (w/w), 6% and 9% (w/w), 6.3% and 8.6% (w/w), or 6.6 and 8.4% (w/w); and/or

- the concentration of the PVA in the agent is between 12% and 40% (w/w), 14% and 26% (w/w), 16% and 24% (w/w), or 17% and 23% (w/w); and/or

- the concentration of the PVP in the agent is between 52.5% and 97.50% (w/w), 60% and 90% (w/w), 63.75% and 86.25% (w/w), or 66% and 84% (w/w).

Embodiment 3. The composition according to embodiment 1 or embodiment 2, wherein: the concentration of the PEO in the agent is about 7.5% (w/w); and/or the concentration of the PVA in the agent is about 20% (w/w); and/or the concentration of the PVP in the agent is about 75% (w/w).

Embodiment 4. The composition according to any one of embodiments 1 to 3, wherein the composition and/or the agent is a hydrogel.

Embodiment 5. The composition according to any one of embodiments 1 to 4, wherein the bacteriophage is a tailed bacteriophage.

Embodiment 6. The composition according to any one of embodiments 1 to 5, wherein the bacteriophage is a myovirus and/or a podovirus.

Embodiment 7. The composition according to any one of embodiments 1 to 6, wherein the bacteriophage is a Pseudomonas bacteriophage.

Embodiment 8. The composition according to any one of embodiments 1 to 7, wherein the bacteriophage is PEV 1 and/or PEV31.

Embodiment 9. The composition according to any one of embodiments 1 to 8, further comprising a preservative.

Embodiment 10. The composition according to embodiment 9, wherein the preservative is selected from the group consisting of: methylparaben, propylparaben, ethylparaben, butylparaben, sodium benzoate, potassium sorbate, chlorhexidine, chlorocresol, benzoic acid, polymyxin, phenoxyethanol and any combination thereof. Embodiment 11. A method of preparing a composition for the topical delivery of bacteriophage, the method comprising:

(i) providing an agent comprising at least one non-ionic polymer selected from the group consisting of polyethylene oxide (PEO), polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP); and

(ii) providing the bacteriophage for topical delivery, and

(iii) mixing (i) and (ii).

Embodiment 12. The method according to embodiment 11, wherein:

- the concentration of the PEO in the agent is between 5% and 15% (w/w), 6% and 9% (w/w), 6.3% and 8.6% (w/w), or 6.6 and 8.4% (w/w); and/or

- the concentration of the PVA in the agent is between 12% and 40% (w/w), 14% and 26% (w/w), 16% and 24% (w/w), or 17% and 23% (w/w); and/or

- the concentration of the PVP in the agent is between 52.5% and 97.50% (w/w), 60% and 90% (w/w), 63.75% and 86.25% (w/w), or 66% and 84% (w/w).

Embodiment 13. The method according to embodiment 11 or embodiment 12, wherein:

- the concentration of the PEO in the agent is about 7.5% (w/w); and/or

- the concentration of the PVA in the agent is about 20% (w/w); and/or

- the concentration of the PVP in the agent is about 75% (w/w).

Embodiment 14. The method according to any one of embodiments 11 to 13, wherein the composition and/or the agent is a hydrogel.

Embodiment 15. The method according to any one of embodiments 11 to 14, wherein the bacteriophage is a tailed bacteriophage.

Embodiment 16. The method according to any one of embodiments 11 to 15, wherein the bacteriophage is a myovirus and/or a podovirus.

Embodiment 17. The method according to any one of embodiments 11 to 16, wherein the bacteriophage is a Pseudomonas bacteriophage.

Embodiment 18. The method according to any one of embodiments 11 to 17, wherein the bacteriophage is PEV 1 and/or PEV31.

Embodiment 19. The method according to any one of embodiments 11 to 18, wherein the composition further comprises a preservative.

Embodiment 20. The method according to embodiment 19, wherein the preservative is selected from the group consisting of: methylparaben, propylparaben, ethylparaben, butylparaben, sodium benzoate, potassium sorbate, chlorhexidine, chlorocresol, benzoic acid, polymyxin, phenoxyethanol and any combination thereof.

Embodiment 21. A composition for the topical delivery of bacteriophage obtained or obtainable by the method according to any one of embodiments 11 to 20.

Embodiment 22. A method for the topical delivery of bacteriophage, the method comprising applying the composition according to any one of embodiments 1 to 10 or embodiment 21 to the site for topical application.

Embodiment 23. Use of the composition according to any one of embodiments 1 to 10 or embodiment 21 in the manufacture of a medicament for the topical delivery of bacteriophage.

Embodiment 24. A composition according to any one of embodiments 1 to 10 or embodiment 21 for use in the topical delivery of bacteriophage.

Embodiment 25. A composition for the topical delivery of bacteriophage, the composition comprising: an agent comprising at least one non-ionic polymer; and the bacteriophage for topical delivery, wherein the composition is formulated for administration by spraying.

Embodiment 26. The composition according to embodiment 25, wherein the at least one non-ionic polymer is selected from the group consisting of polyvinyl alcohol (PVA), hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), polyvinylpyrrolidone (PVP), polyethylene oxide (PEO), hydroxypropyl methylcellulose (HPMC), agarose and guar gum.

Embodiment 27. The composition according to embodiment 25 or embodiment 26, wherein:

- the concentration of the PVA in the agent is between 0.05% and 10% (w/v), 0.25% and 5% (w/v), 0.5% and 2.5% (w/v), or 0.75% and 1.25% (w/v); and/or

- the concentration of the HEC in the agent is between 0.05% and 2.5% (w/v), 0.1% and 2% (w/v), 0.15% and 1% (w/v), or 0.2% and 0.5% (w/v); and/or

- the concentration of the HPC in the agent is between 0.1% and 20% (w/v), 0.5% and 10% (w/v), 1% and 5% (w/v), or 1.5% and 2.5% (w/v); and/or

- the concentration of the PVP in the agent is between 0.1% and 20% (w/v), 0.5% and 10% (w/v), 1% and 5% (w/v), or 1.5% and 2.5% (w/v); and/or

- the concentration of the HPMC in the agent is between 0.05% and 2.5% (w/v), 0.1% and 2% (w/v), 0.15% and 1% (w/v), or 0.2% and 0.5% (w/v); and/or - the concentration of the PEO in the agent is between 0.1% and 5% (w/v), 0.2% and 4% (w/v), 0.3% and 3% (w/v), or 0.4% and 2% (w/v).

Embodiment 28. The composition according to any one of embodiments 25 to 27, wherein:

- the concentration of the PVA in the agent is about 1% (w/v); and/or

- the concentration of the HEC in the agent is about 0.25% (w/v); and/or

- the concentration of the HPC in the agent is about 2% (w/v); and/or

- the concentration of the PVP in the agent is about 2% (w/v); and/or

- the concentration of the HPMC in the agent is about 0.25% (w/v); and/or

- the concentration of the PEO in the agent is about 0.5% (w/v).

Embodiment 29. The composition according to any one of embodiments 25 to 28, wherein the bacteriophage is a tailed bacteriophage.

Embodiment 30. The composition according to any one of embodiments 25 to 29, wherein the bacteriophage is a myovirus and/or a podovirus.

Embodiment 31. The composition according to any one of embodiments 25 to 30, wherein the bacteriophage is a Pseudomonas bacteriophage.

Embodiment 32. The composition according to any one of embodiments 25 to 31, wherein the bacteriophage is PEV1 and/or PEV31.

Embodiment 33. The composition according to any one of embodiments 25 to 32, further comprising a solvent and/or a preservative.

Embodiment 34. The composition according to embodiment 33, wherein the solvent and/or preservative is selected from the group consisting of: ethanol, methylparaben, propylparaben, ethylparaben, butylparaben, sodium benzoate, potassium sorbate, chlorhexidine, chlorocresol, benzoic acid, polymyxin, phenoxyethanol, and any combination thereof.

Embodiment 35. A method of preparing a composition for the topical delivery of bacteriophage, the method comprising:

(i) providing an agent comprising at least one non-ionic polymer,

(ii) providing the bacteriophage for topical delivery, and

(iii) mixing (i) and (ii), wherein the composition is formulated for administration by spraying.

Embodiment 36. The method according to embodiment 35, wherein the non-ionic polymer is selected from the group consisting of polyvinyl alcohol (PVA), hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), polyvinylpyrrolidone (PVP), hydroxypropyl methylcellulose (HPMC), agarose and guar gum.

Embodiment 37. The method according to embodiment 35 or embodiment 36, wherein:

- the concentration of the PVA in the agent is between 0.05% and 10% (w/v), 0.25% and 5% (w/v), 0.5% and 2.5% (w/v), or 0.75% and 1.25% (w/v); and/or

- the concentration of the HEC in the agent is between 0.05% and 2.5% (w/v), 0.1% and 2% (w/v), 0.15% and 1% (w/v), or 0.2% and 0.5% (w/v); and/or

- the concentration of the HPC in the agent is between 0.1% and 20% (w/v), 0.5% and 10% (w/v), 1% and 5% (w/v), or 1.5% and 2.5% (w/v); and/or

- the concentration of the PVP in the agent is between 0.1% and 20% (w/v), 0.5% and 10% (w/v), 1% and 5% (w/v), or 1.5% and 2.5% (w/v); and/or

- the concentration of the HPMC in the agent is between 0.05% and 2.5% (w/v), 0.1% and 2% (w/v), 0.15% and 1% (w/v), or 0.2% and 0.5% (w/v); and/or

- the concentration of the PEO in the agent is between 0.1% and 5% (w/v), 0.2% and 4% (w/v), 0.3% and 3% (w/v), or 0.4% and 2% (w/v).

Embodiment 38. The method according to any one of embodiments 35 to 37, wherein,

-the concentration of the PVA in the agent is about 1% (w/v); and/or

-the concentration of the HEC in the agent is about 0.25% (w/v); and/or

-the concentration of the HPC in the agent is about 2% (w/v); and/or

-the concentration of the PVP in the agent is about 2% (w/v); and/or

-the concentration of the HPMC in the agent is about 0.25% (w/v); and/or

-the concentration of the PEO in the agent is about 0.5% (w/v).

Embodiment 39. The method according to any one of embodiments 35 to 38, wherein the bacteriophage is a tailed bacteriophage.

Embodiment 40. The method according to any one of embodiments 35 to 39, wherein the bacteriophage is a myovirus and/or a podovirus.

Embodiment 41. The method according to any one of embodiments 35 to 40, wherein the bacteriophage is a Pseudomonas bacteriophage.

Embodiment 42. The method according to any one of embodiments 35 to 41, wherein the bacteriophage is PEV 1 and/or PEV31.

Embodiment 43. The method according to any one of embodiments 35 to 42, wherein the composition further comprises a solvent and/or a preservative. Embodiment 44. The method according to embodiment 43, wherein the solvent and/or preservative is ethanol.

Embodiment 45. A composition for the topical delivery of bacteriophage obtained or obtainable by the method according to any one of embodiments 35 to 44.

Embodiment 46. A method for the topical delivery of bacteriophage, the method comprising applying the composition according to any one of embodiments 25 to 34 or embodiment 45 to the site for topical application.

Embodiment 47. Use of the composition according to any one of embodiments 25 to 34 or embodiment 45 in the manufacture of a medicament for the topical delivery of bacteriophage.

Embodiment 48. A composition according to any one of embodiments 25 to 34 or embodiment 45 for use in the topical delivery of bacteriophage.

Definitions

As used in this application, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.

As used herein, the term “comprising” means “including”, in a non-exhau stive sense. Variations of the word “comprising”, such as “comprise” and “comprises” have correspondingly varied meanings. Thus, for example, a composition “comprising” a given component A may consist exclusively of component A, or may include one or more additional components such as component B. Similarly, a composition “comprising” an agent “comprising” at least one non-ionic polymer selected from the group consisting of A, B and C; and a bacteriophage for topical delivery may consist exclusively of an agent comprising at least one non-ionic polymer selected from the group consisting of A, B and C; and a bacteriophage for topical delivery or may include one or more additional components, for example, water.

As used herein, the term “between” when used in reference to a range of numerical values encompasses the numerical values at each endpoint of the range.

As used herein, the term “about”, when used in reference to a recited numerical value, includes the recited numerical value and numerical values within plus or minus ten percent of the recited value.

As used herein, the term “bacteriophage”, also known in the art as “phage”, will be understood to mean a virus that infects bacteria. “Bacteriophage” and “phage” may be used interchangeably herein and include plural references unless the context clearly dictates otherwise. “Bacteriophage” and “phage”, when used in the plural sense, may refer to more than one phage of the same type or to more than one type of phage.

As used herein, the term “hydrogel” will be understood to mean a gel in which the liquid component comprises water. Hydrogels may contain a high proportion of water and may be hydrophilic.

As used herein, the terms “tailed bacteriophage” and “tailed phage” will be understood to mean phage which use a structure resembling a tail to initiate a connection to their bacterial host prior to phage genome injection.

Brief Description of the Figures

Preferred embodiments of the present invention will now be described by way of example only, with reference to the accompanying figures wherein:

Figure 1 provides graphs depicting in vitro release profiles of myovirus phage PEV1 from 75% PVP (la), 20% PVA (lb), 5% HPMC (1c), 30% HPC (Id), 5% HEC (le) and 7.5% PEO (If) gel formulations containing 0.1% methylparaben and 0.02% propylparaben over 8 hours (n=3). Franz diffusion cells were used with polyethersulfone membranes (0.45 pm). Phage titer in the collected samples was determined using plaque assay (n=3). Error bars represent standard deviation.

Figure 2 provides graphs depicting in vitro release profiles of podovirus phage PEV31 from 75% PVP (2a), 20% PVA (2b), 5% HPMC (2c), 30% HPC (2d), 5% HEC (2e) and 7.5% PEO (2f) gel formulations containing 0.1 %methylparaben and 0.02% propylparaben over 8 hours (n=3). Franz diffusion cells were used with polyethersulfone membranes (0.45 pm). Phage titer in the collected samples was determined using plaque assay (n=3). Error bars represent standard deviation.

Figure 3 provides graphs depicting in vitro release profiles of PEV1 (3a) and PEV31 (3b) from 30% HPC gel formulation containing 0.1% methylparaben and 0.02% propylparaben in the presence and absence of P. aeruginosa FADDI-PA001. Bacterial culture (10 ⁸ cfu) was placed on top of polyethersulfone membranes (0.45 pm). Phage release profile was assayed over 8 hours (n=3) and the phage titer in the collected samples was determined using plaque assay (n=3). Error bars represent standard deviation. Figure 4 provides graphs depicting titers of myovirus-type phage PEV 1 and podovirus- type phage PEV31 in 75% PVP (4a), 20% PVA (4b), 5% HPMC (4c), 30% HPC (4d), 5% HEC (4e) and 7.5% PEO (4f) gel formulations containing 0.1% methylparaben and 0.02% propylparaben after 0, 1, 2, 4, 6 and 8 weeks storage at 4 °C. Phage titer was determined using plaque assay (n=3). Error bars represent standard deviation.

Figure 5 provides graphs depicting in vitro release profiles of phage PEV 1 from PVP (5a), PVA (5b), HPMC (5c), HPC (5d), HEC (5e) and PEO (5f) spray formulations over 8 h (n=3). Franz diffusion cells were used with polyethersulfone membranes (0.45 pm). Phage titer in the collected samples was determined using a plaque assay (n=3). Error bars represent standard deviation. The titer of phage spray formulation loaded is presented as a dotted line.

Figure 6 provides graphs depicting in vitro release profiles of phage PEV31 from PVP (6a), PVA (6b), HPMC (6c), HPC (6d), HEC (6e) and PEO (6f) spray formulations over 8 h (n=3). Franz diffusion cells were used with polyethersulfone membranes (0.45 pm). Phage titer in the collected samples was determined using a plaque assay (n=3). Error bars represent standard deviation. The titer of phage spray formulation loaded is presented as a dotted line.Figure 7 provides graphs depicting storage stability of PEV 1 (7a) and PEV31 (7b) in the optimized spray phage formulations. Phage titer in the spray formulations in glass vials is shown after 0, 1, 2, 3, 4, 6 and 8-week storage at 4°C. The glass vials were sealed to minimize evaporation of the solvent.

Detailed Description

The following detailed description conveys exemplary embodiments of the present invention in sufficient detail to enable those of ordinary skill in the art to practice the present invention. Features or limitations of the various embodiments described do not necessarily limit other embodiments of the present invention, or the present invention as a whole. Hence, the following detailed description does not limit the scope of the present invention, which is defined only by the claims.

It will be appreciated by persons of ordinary skill in the art that numerous variations and/or modifications can be made to the present invention as disclosed in the specific embodiments without departing from the spirit or scope of the present invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. Compositions

The present inventors have developed compositions for the topical delivery of bacteriophage. The compositions may comprise an agent and the bacteriophage for topical delivery. The agent may comprise one or more non-ionic polymers. Without wishing to be bound by theory, the present inventors have observed that certain non-ionic polymers are able to form agents that enable the controlled release of bacteriophage. In the present invention, the agent may comprise at least one non-ionic polymer selected from the group consisting of polyethylene oxide (PEO), polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP). The composition may comprise an agent comprise at least one non-ionic polymer selected from the group consisting of PEO, PVA and PVP and the bacteriophage for topical delivery, and be in the form of a hydrogel. Additionally or alternatively, a composition of the present invention may comprise comprise an agent comprising at least one non-ionic polymer and the bacteriophage for topical delivery, and be formulated for administration by spraying. In certain embodiments, a spray formulation may comprise at least one non-ionic polymer selected from the group consisting of polyvinyl alcohol (PVA), hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), polyvinylpyrrolidone (PVP), polyethylene oxide (PEO), hydroxypropyl methylcellulose (HPMC), agarose and guar gum. In some embodiments of the invention, the composition and/or agent and/or non-ionic polymer is more viscous than water. The non-ionic polymers of the present invention may have linear alkane backbone. In some embodiments, the non-ionic polymers have a side chain containing oxygen. The non-ionic polymers of the invention may be alkenes.

In some embodiments of the invention, the concentration of the PEO in the agent is between 5% and 15% (w/w), 6% and 9% (w/w), 6.3% and 8.6%, or 6.6 and 8.4%. Additionally or alternatively, the concentration of the PVA in the agent may be between 12% and 40% (w/w), 14% and 26% (w/w), 16% and 24% (w/w), or 17% and 23% (w/w). In further embodiments, the concentration of the PVP in the agent is between 52.5% and 97.50% (w/w), 60% and 90% (w/w), 63.75% and 86.25% (w/w), or 66% and 84% (w/w).

In yet further embodiments of the present invention, the concentration of the PEO in the agent is about 7.5% (w/w); and/or the concentration of the PVA in the agent is about 20% (w/w); and/or the concentration of the PVP in the agent is about 75% (w/w).

In certain formulations, the concentration of the PVA in the agent is between 0.05% and 10% (w/v), 0.25% and 5% (w/v), 0.5% and 2.5% (w/v), or 0.75% and 1.25% (w/v). Additionally or alternatively, the concentration of the HEC in the agent may be between 0.05% and 2.5% (w/v), 0.1% and 2% (w/v), 0.15% and 1% (w/v), or 0.2% and 0.5% (w/v). The concentration of the HPC in the agent may be between 0.1% and 20% (w/v), 0.5% and 10% (w/v), 1% and 5% (w/v), or 1.5% and 2.5% (w/v). The concentration of the PVP in the agent may be between 0.1% and 20% (w/v), 0.5% and 10% (w/v), 1% and 5% (w/v), or 1.5% and 2.5% (w/v). In further formulations of the invention, the concentration of the HPMC in the agent is between 0.05% and 2.5% (w/v), 0.1% and 2% (w/v), 0.15% and 1% (w/v), or 0.2% and 0.5% (w/v). In other formulations, the concentration of the PEO in the agent is between 0.1% and 5% (w/v), 0.2% and 4% (w/v), 0.3% and 3% (w/v), or 0.4% and 2% (w/v).

In some formulations, the concentration of the PVA in the agent is about 1 % (w/v), and/or the concentration of the HEC in the agent is about 0.25% (w/v), and/or the concentration of the HPC in the agent is about 2% (w/v); and/or the concentration of the PVP in the agent is about 2% (w/v); and/or the concentration of the HPMC in the agent is about 0.25% (w/v); and/or the concentration of the PEO in the agent is about 0.5% (w/v). In some embodiments, the compositions are formulated for administration by spraying.

The present inventors have identified optimal concentrations of non-ionic polymers in the agents of the present invention which are described above and in the Examples and claims of the present application. It will be understood that the concentrations of non-ionic polymers disclosed are exemplary only.

The composition and/or agent of the present invention may take the form of a hydrogel. In further embodiments, the composition and/or agent may be formulated for administration by spraying. Without limitation, the composition and/or agent may also be a cream, lotion, ointment, solution, gel, paste foam or any other form suitable for topical delivery. The composition and/or agent may comprise a preservative. Non-limiting examples of suitable preservatives include methylparaben, propylparaben, ethylparaben, butylparaben, sodium benzoate, potassium sorbate, chlorhexidine, chlorocresol, benzoic acid, polymyxin, phenoxyethanol and any combination thereof. In some embodiments of the invention, the composition and/or agent may comprise a solvent such as ethanol.

The compositions and/or agents of the present invention may include other suitable ingredients including water, saline, salt magnesium buffer, Tris magnesium buffer and/or phosphate buffer. Phage

The compositions described herein may be used for the delivery of any type of phage. The phage for delivery may be a component of the composition. In some embodiments of the invention, the bacteriophage for topical delivery is a tailed bacteriophage. In further embodiments, the bacteriophage for delivery is a myovirus, a podovirus and/or a siphovirus, and/or a mix of these bacteriophage types. The compositions of the present invention may be used for the delivery of any Pseudomonas phage. Non-limiting examples of suitable Pseudomonas phage include PEV 1 and PEV31. The compositions of the present invention may be used for the delivery of bacteriophage capable of infecting bacteria including, but not limited to Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermis, Streptococcus pyogenes, Escherichia coli, Proteus sp., Klebsiella sp., Propionibacterium acnes and Acinetobacter baumannii. In some embodiments of the invention, the bacteriophage are therapeutic bacteriophage. In further embodiments, the phage remain viable following storage.

Methods for preparing the compositions

The present invention also provides methods for preparing compositions for the topical delivery of bacteriophage. The methods may comprise providing an agent comprising at least one non-ionic polymer selected from the group consisting of polyethylene oxide (PEO), polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP). In further embodiments, the nonionic polymer selected from the group consisting of polyvinyl alcohol (PVA), hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), polyvinylpyrrolidone (PVP), hydroxypropyl methylcellulose (HPMC), agarose and guar gum. PEO, HEC, HPC, HPMC, PVA, PVP, agarose and guar gum are all readily available commercially.

In some embodiments, the agent is produced by dispersing particles of PEO, PVA and/or PVP in a solvent such as water, saline, salt magnesium buffer, Tris magnesium buffer and/or phosphate buffer. In further embodiments, particles of PVA, HEC, HPC, PVP, HPMC, agarose and/or guar gum are dissolved in a solvent. The water may be sterile water. In some embodiments, the water is distilled water. In further embodiments, hot water may be used. The sterile water used in the preparation of the agents and/or compositions may be any water substantially free from contaminants. Many types of sterile water are readily available commercially. Additionally or alternatively, the skilled person may prepare sterile water by any one of many well-known methods such as activated carbon, reverse osmosis, ion exchange, filtration and distillation. The solvent may be ethanol. In some embodiments, a final solvent concentration of between 20% and 50%, or between 25% and 45%, or between 30% and 40%, or about 35% ethanol in water is used.

In some embodiments of the invention, the pH of the agent is adjusted to about 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 7.9. The pH of the agent and/or composition may be about 7.4. The person skilled in the art will be familiar with ways in which the pH of a solution may be checked, for example, using a calibrated pH meter. One non-limiting example of a compound which may be used to adjust the pH is triethanolamine. The compositions and/or agents may include other suitable ingredients including water and/or phosphate buffer saline.

The agents of the present invention may be homogeneous. Without limitation, the agents may be transparent. The person skilled in the art would be familiar with ways in which to test the viscosity of an agent for suitability for topical application. The agents may be formulated for ease of spraying. The formulations may have minimal dripping upon spraying and/or a short drying time.

In the methods of preparation of the compositions, the concentration of the PEO in the agent may be between 5% and 15% (w/w), 6% and 9% (w/w), 6.3% and 8.6%, or 6.6 and 8.4%. Additionally or alternatively, the concentration of the PVA in the agent may be between 12% and 40% (w/w), 14% and 26% (w/w), 16% and 24% (w/w), or 17% and 23% (w/w). In further embodiments, the concentration of the PVP in the agent is between 52.5% and 97.50% (w/w), 60% and 90% (w/w), 63.75% and 86.25% (w/w), or 66% and 84% (w/w).

In yet further embodiments of the present invention, the concentration of the PEO in the agent is about 7.5% (w/w); and/or the concentration of the PVA in the agent is about 20% (w/w); and/or the concentration of the PVP in the agent is about 75% (w/w).

In other embodiments of the methods, the concentration of the PVA in the agent is between 0.05% and 10% (w/v), 0.25% and 5% (w/v), 0.5% and 2.5% (w/v), or 0.75% and 1.25% (w/v). Additionally or alternatively, the concentration of the HEC in the agent may be between. 0.05% and 2.5% (w/v), 0.1% and 2% (w/v), 0.15% and 1% (w/v), or 0.2% and 0.5%. The concentration of the HPC in the agent may be between 0.1% and 20% (w/v), 0.5% and 10% (w/v), 1% and 5% (w/v), or 1.5% and 2.5% (w/v). The concentration of the PVP in the agent may be between 0.1% and 20% (w/v), 0.5% and 10% (w/v), 1% and 5% (w/v), or 1.5% and 2.5% (w/v). In further embodiments, the concentration of the HPMC in the agent is between 0.05% and 2.5% (w/v), 0.1% and 2% (w/v), 0.15% and 1% (w/v), or 0.2% and 0.5% (w/v). In other embodiments, the concentration of the PEO in the agent is between 0.1% and 5% (w/v), 0.2% and 4% (w/v), 0.3% and 3% (w/v), or 0.4% and 2% (w/v). In some methods of the invention, the concentration of the PVA in the agent is about 1% (w/v), and/or the concentration of the HEC in the agent is about 0.25% (w/v), and/or the concentration of the HPC in the agent is about 2% (w/v); and/or the concentration of the PVP in the agent is about 2% (w/v); and/or the concentration of the HPMC in the agent is about 0.25% (w/v); and/or the concentration of the PEO in the agent is about 0.5% (w/v). In some embodiments, the compositions are formulated for administration by spraying

The present inventors have identified optimal concentrations of non-ionic polymers in the agents of the present invention which are described above and in the Examples and claims of the present application. It will be understood by the person skilled in the art when practising the methods of preparing the compositions that the concentrations of non-ionic polymers disclosed are exemplary only.

In the methods of preparing the compositions of the present invention, the composition and/or agent may be prepared in the form of a hydrogel. In further embodiments, the compositions are formulated for administration by spraying. Without limitation, the composition and/or agent may also be a cream, lotion, ointment, solution, gel, paste foam or any other form suitable for topical delivery. The methods may comprise adding a preservative to the agent and/or composition. The person skilled in the art would be familiar with the wide range of preservatives commercially available for such use. Non-limiting examples of suitable preservatives include methylparaben, propylparaben, ethylparaben, butylparaben, sodium benzoate, potassium sorbate, chlorhexidine, chlorocresol, benzoic acid, polymyxin, phenoxyethanol and any combination thereof. Some methods of the invention involve adding a solvent such as ethanol.

The methods of preparing the compositions may also comprise providing the bacteriophage for topical delivery. The bacteriophage may be a tailed bacteriophage. In further embodiments, the bacteriophage for delivery is a myovirus, a podovirus and/or a siphovirus, and/or a mix of these phage types. The methods may involve the provision of any bacteriophage capable of infecting bacteria including, but not limited to, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermis, Streptococcus pyogenes, Escherichia coli, Proteus sp., Klebsiella sp., Propionibacterium acnes and Acinetobacter baumannii. Nonlimiting examples of suitable Pseudomonas phage include PE VI and PEV31.

The bacteriophage used in the methods of preparing the compositions may be a therapeutic bacteriophage. The person skilled in the art will be aware of the broad range of bacteriophage commercially available. The skilled person may also isolate phage for use in the compositions and methods of the invention. For a comprehensive review of methods of phage isolation and characterization, including ways in which procedures may be optimised in order to select phage with a broad host range, see Paul Hyman, “Phages for Phage Therapy: Isolation, Characterization, and Host Range Breadth.” Pharmaceuticals (Basel). 2019; 12(1): 35.

Several techniques for the concentration and purification of phage are commonly used in the art. These techniques involve, but are not limited to, centrifugation, filtration, ultrafiltration, precipitation with Polyethylene Glycol, ultracentrifugation in cesium chloride gradients and dialysis (see, for example, Yamamoto et al., “Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification” Virology. 1970; 40(3):734-744; Seeley and Primrose, “A Review: The isolation of bacteriophages from the environment” Journal of Applied Bacteriology. 1982; 53:1-17; Carlson, “Appendix: working with bacteriophages: common techniques and methodological approaches.” In: Kutter E, Sulakvelidze A, eds. Bacteriophages: Biology and Applications. Boca Raton: CRC Press. 2005; Vol. 1:439-490; Bourdin et al., “Amplification and Purification of T4-Like Escherichia coli Phages for Phage Therapy: from Laboratory to Pilot Scale” Applied and Environmental Microbiology, 2014; 80(4): 1469- 1476). As a non-limiting example, Bonilla et al., “Phage on tap-a quick and efficient protocol for the preparation of bacteriophage laboratory stocks.” PeerJ. 2016;4:e2261-e2261 provides a comprehensive description of a protocol for the quick and efficient preparation of homogenous phage stocks which could be easily applied by the skilled person.

The methods of preparing the compositions may also comprise mixing the agent comprising at least one non-ionic polymer selected from the group consisting of PEG, PVA and PVP with the phage for topical delivery. In further embodiments, the non-ionic polymer selected from the group consisting of PVA, HEC, HPC, PVP, HPMC, agarose and/or guar gum. The agent and the phage may be mixed by stirring, swirling, shaking, etc. Other suitable ingredients including water and/or phosphate buffer saline may be added to the mixture. The invention also provides compositions for the topical delivery of phage produced by the methods of the invention.

The present invention provides methods of delivering bacteriophage comprising applying the compositions of the invention to the site for topical application and use of the compositions of the invention in the manufacture of a medicament for the topical delivery of bacteriophage. No particular limitation exists as to in relation to the site for topical application. The invention provides compositions for use in the topical delivery of bacteriophage according to the requirements of the skilled person.

Examples

The present invention will now be described with reference to specific Examples, which should not be construed as in any way limiting.

Example One

Phage hydrogels were formulated using the non-ionic polymers hydroxypropyl methylcellulose (HPMC), hydroxyethyl cellulose (HEC), polyethylene oxide (PEO), polyvinyl alcohol (PVA), hydroxypropyl cellulose (HPC) and polyvinylpyrrolidone (PVP). Pseudomonas phage PEV1 (a myovirus) and PEV31 (a podovirus) were suspended in the hydrogels, followed by an assessment of in vitro infective phage release profile and storage stability.

Materials and methods

Phage

Two anti-Pseudomonas phage, PEV 1 (myovirus) and PEV31 (podovirus) were originally isolated from a sewage treatment plant in Olympia (WA, USA) using P. aeruginosa dog-ear strain PAV237. This strain was used as a reference bacterial strain for phage amplification and plaque assays. Phage were amplified and then purified according to the Phage On Tap protocol (see Bonilla et al., “Phage on tap-a quick and efficient protocol for the preparation of bacteriophage laboratory stocks.” PeerJ. 2016;4:e2261-e2261). Phage were stored in phosphate buffered saline (PBS) with pH adjusted to 7.4 until use.

Formulations

The compositions of the 18 hydrogel formulations are described in Table 1. Polymer particles including HEC, HPMC, PEO, HPC and PVP were weighed and thoroughly dispersed in 40°C sterile distilled water containing 0.1% methylparaben and 0.02% propylparaben by stirring. Hot water (>90°C) was used for PVA. The suspensions were continuously stirred for 2 h. The pH of each hydrogel formulation was measured and adjusted to 7.4 using triethanolamine. Briefly, one gram of the hydrogel formulation was weighed and mixed with 25 mL of purified water. The pH of the mixture was measured using a calibrated pH meter. A volume (100 pL) of phage lysate (5 x 10 ¹⁰ pfu/mL) was mixed with 1 g of optimized hydrogel formulation with geometric dilution.

Evaluation of hydrogel formulations

The hydrogel formulations were visually inspected after setting in the specimen jar overnight. The appearance, homogeneity and the presence of any polymer aggregates were assessed. The viscosity of the formulations was assessed and given a level - low: syrup-like, medium: gel-like and high: hard gel-like. For each polymer, transparent and homogeneous formulations with medium viscosity level were selected for further investigation. In vitro release of phage

The in vitro phage release profile of the phage hydrogel formulations was assessed using a Franz cell dissolution system. The dissolution cell temperature was set at 32°C using a digital immersion heater circulator and degassed PBS (pH 7.4) was used as dissolution medium. A membrane filter (polyethersulfone, 0.22 pm) was saturated with the dissolution media for 20 min, gently blotted, and then placed on top of the receptor chamber. A glass cap was placed on top of the membrane and then held together using a metal clamp. Phage hydrogel formulation (400 mg) was gently and evenly spread over the membrane filter. At predetermined time points (0, 1, 2, 3, 4, 6 and 8 h), 150 pL of the sample was withdrawn from the receptor chamber and replaced with the same amount of fresh PBS. Plaque assay method (see below) was used to assess the release profiles of infective phage. Each formulation was assessed in triplicate.

The HPC hydrogel formulation exhibited poor phage release (PEV 1 and PEV31) over an eight-hour period. As the phage titer would increase in the presence of a bacterial host, the release study was conducted using a clinical isolate of P. aeruginosa. The early-log of bacteria (ODeoo of 0.5) was prepared and 100 pL was placed on top of the membrane filter. Phage hydrogel formulations were loaded and assessed as above.

Plaque assay

Phage titer in the phage hydrogel formulations and in the Franz cell -collected samples was determined using a standard plaque assay. Briefly, 0.2 mL of reference bacterial strain (2 x 10 ⁸ colony forming units at stationary phase) was mixed with 5 mL of 0.4% nutrient broth soft agar. The mixture was poured on top of a nutrient agar plate. Then, serially diluted phage formulations were dropped (10 pL) on top of the overlay agar plate, air-dried and then incubated at 37 °C for 18 - 24 h. The assay was independently repeated three times.

Storage stability studies

Twelve optimized phage hydrogel formulations were assessed for biological stability during storage. The formulations were aliquoted in sterile polypropylene vials and stored at 4°C for up to eight weeks. The phage titer was assessed using a plaque assay at 0, 1, 2, 4, 6 and 8 weeks.

Results

All six non-ionic polymers formed transparent and homogenous hydrogels at the tested concentrations (Table 2). Table 2. Physical properties of gel formulations.

Formulation Polymer Appearance Viscosity Homogeneity

1 PVP 40 Transparent Low o

2 75 Transparent Medium o

3 80 Transparent Medium- o hard

4 PVA 10 Transparent Low o

5 20 Transparent Medium o

6 40 Transparent Hard o

7 HPMC 2 Transparent Low o

8 5 Transparent Medium o

9 10 Transparent Hard o with air bubbles

10 HPC 20 Transparent Low o

11 30 Transparent Medium o

12 40 Transparent Medium- o sticky

13 HEC 2 Transparent Low o

14 5 Transparent Medium o

15 10 Transparent Hard o

16 PEO 5 Transparent Low o

17 7.5 Transparent Medium o

18 10 Transparent Hard o

NOTE: PVP, polyvinylpyrrolidone; PVA, polyvinyl alcohol; HPMC, hydroxypropyl methylcellulose; HPC, hydroxypropyl cellulose; HEC, hydroxyethyl cellulose; PEO, polyethylene oxide. Polymers were dissolved in water containing 0.1% methylparaben and 0.02% propylparaben. Viscosity low: syrup-like, medium: gel-like, high: hard gel-like; symbol o: confirmed homogeneity.

HPMC, HEC and PEO formulations showed low viscosity (syrup-like) at concentrations <5%, medium viscosity (gel-like) at <7.5% and high viscosity (hard gel-like) at 10% (w/w). The formulations with medium viscosity were deemed suitable as hydrogels for topical application. PVA, HPC and PVP formulations exhibited medium viscosities at 20% (w/w), 30% (w/w) and 60% (w/w), respectively. The formulation containing 40% HPC also had medium viscosity, but the gel was too sticky, which is an undesirable trait for patient’ s cosmetic expectations.

Formulations including 75% PVP, 20% PVA, 5% HPMC, 30% HPC, 5% HEC and 7.5% PEO (all w/w) were selected to test for suitability as a potential delivery vehicle for myovirus (PEV1) and podovirus (PEV31) types of phage.

The optimized PVA, PVP and PEO hydrogel formulations demonstrated the feasibility of releasing a therapeutic dose of PEV 1 over eight hours. The highest cumulative PEV 1 release was achieved by PVA hydrogel, followed by PVP and PEO with 16% (3.2 x 10 ⁸ PFU), 7.5% (1.5 x 10 ⁸ PFU) and 7.4% (1.5 x 10 ⁸ PFU), respectively (Figure 1). Of these three polymers, PVA showed rapid release (1 x 10 ⁷ PFU released at 1 h), whereas PVP showed slow and extended release of PEV 1 (2.2 x 10 ³ PFU at 1 h).

PEV31 showed a similar trend with cumulative release of 36% (3.6 x 10 ⁸ PFU), 13% (1.3 x 10 ⁸ PFU) and 7.2% (7.2 x 10 ⁷ PFU) from PVA, PVP and PEO hydrogels respectively (Figure 2). The phage titer is expected to increase in the infection site during bacteriolysis.

In general, PEV31 (podovirus) exhibited slightly higher release in all hydrogel formulations as compared with PEV1 (myovirus).

Hydrogels with cellulose esters, including HPC, HEC and HPMC (in this order) showed poor release of infective phage. PEV 1 release in HPC, HEC and HPMC hydrogels were 0.04% (7.4 x 10 ⁵ PFU), 1.5% (3.1 x 10 ⁷ PFU) and 1.8% (3.7 x 10 ⁷ PFU), respectively (Figure 1). A similar release pattern was observed for PEV31 with 0.08% (7.5 x 10 ⁵ PFU), 2.5% (2.5 x 10 ⁷ PFU) and 4.4% (4.4 x 10 ⁷ PFU) release respectively (Figure 2). The observed trend in cumulative release of phage aligns with the order of the mesh size, which is a critical factor for predicting the diffusion and release of the active ingredient. HPC was the most entangled polymer used with the smallest mesh size, followed by HEC and HPMC.

The phage titer is likely to increase at the infection site. Hence, P. aeruginosa was loaded on top of the membrane and then the HPC phage hydrogel was applied to assess the phage titer over time (Figure 3). The titer of PEV1 and PEV31 increased by 1.1 log and 1.5 log respectively in the presence of the bacterial host. However, the cumulative infective phage released at 8 h was still relatively low with 9.6 x 10 ⁶ and 2.4 x 10 ⁷ PFU for PEV1 and PEV31, respectively. Hence, polymers with a small mesh size such as HPC should not be considered for use in phage hydrogel formulations. In addition to mesh size, other phage-polymer interactions such as electrostatic and hydrophobic associations can impact phage release from hydrogels. As electrostatic interaction is non-specific, it may enable controlled and continuous delivery of different phage (i.e. a phage cocktail) from a single system. It is therefore essential to assess the influence of charge on phage stability as the presence of charged polymers may compromise the phage bioactivity. In the current Example, non-ionic polymers were used to eliminate any charge-induced interference with phage diffusion and subsequent release as well as phage instability.

In the current Example, the stability of PEV1 and PEV31 in the optimized hydrogel formulations was examined over eight weeks (Figure 4). PEV1 remained stable with no titer loss in PEO and PVA hydrogels, and minimal titer loss (0.4 log - 0.8 log) was observed in other formulations. PEV31 was relatively stable in PEO (0.3 log titer loss), HPMC (0.4 log) and PVA (0.8 log) hydrogels, while greater titer reduction was observed in all other formulations (1.2 - 1.4 log).

The presence of preservatives, including 0.1% methylparaben and 0.02% propylparaben, did not compromise the phage titer. Preservatives play an important role in maintaining an aseptic condition throughout shelf-life. Preservative systems, including 0.1% methylparaben and 0.02% propylparaben, 1% phenoxyethanol, and 0.2% propylparaben can potentially be used in phage hydrogel formulations.

Summary

This Example demonstrates controlled release of PE VI and PEV31 with high infectivity in PEO, PVA and PVP hydrogels. Cellulose esters exhibited poor phage release. PEV1 and PEV31 titers were well preserved (< 1 log titer loss) in PEO, PVA and HPMC hydrogels.

Example Two

The aim of this Example was to develop spray formulations containing anti- Pseudomonas phage Myoviridae PE VI or Podoviridae PEV31. Phage were formulated as spray formulations for topical application using non-ionic polymers in 35% ethanol in water. The phage-polymer spray formulations were assessed for various physical properties including appearance, viscosity, ease of spraying, dripping upon spraying, drying time, stickiness of the dried polymer film and in vitro release profile. The optimized phage-polymer spray formulations were then tested for antibacterial activity and storage stability. Materials and methods

Phage

Two anti-Pseudomonas phage, PEV1 (myovirus) and PEV31 (podovirus) were supplied at a titre of IO ¹⁰ plaque forming units (pfu)/mL. Phage were stored in phosphate buffered saline (PBS) with the pH adjusted to 7.4. PEV1 and PEV31 were originally isolated from the sewage treatment plant in Olympia (WA, USA) using P. aeruginosa dog-ear strain PAV237. This strain was used as a reference bacterial strain for plaque assay to assess the phage titer.

Polymers

The following polymers were tested: polyvinyl alcohol (PVA), hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), polyvinylpyrrolidone (PVP), polyethylene oxide (PEO) and hydroxypropyl methylcellulose (HPMC).

Formulations

The composition of the formulations is described in Table 3. Polymer particles were weighed and thoroughly dispersed in 40°C water by stirring. The suspensions were continuously stirred until room temperature was achieved. Then, ethanol was added to make up to a final solvent concentration of 35% ethanol in water. A volume (200 pL) of phage lysate (109 pfu/mL) was added to 2 mL of polymer in 35% ethanol in water.

Evaluation of polymer formulations

The liquid polymer formulations were visually inspected after setting in specimen jars overnight. Appearance, homogeneity and the presence of any polymer aggregates were assessed. The viscosity of the formulations was assessed and given a rating - low: liquid, medium: syrup-like and high: gel-like. Formulations with a viscosity level of 3 (Table 4) were excluded from further investigation. Evaluation of phage-polymer sprays

The phage-polymer spray formulations were sprayed using a spray bottle with an APF pump. The spray bottle was filled with the test formulation and then manually pumped three times to prime the bottle prior to evaluation.

Ease of spray: The sprayability of the formulations was assessed. Viscous formulations that could not be sprayed using the spray bottle were excluded from further evaluations.

Average weight per dose: The initial weight of the filled spray bottle was recorded. The formulation was sprayed once and then the bottle weighed again. The initial weight was subtracted from the final weight of the spray bottle to determine the weight per dose. The test was repeated four times to calculate the average weight per dose.

Dripping and drying time: The spray bottle was placed 10 cm away from a glass slide and then sprayed once. Dripping effect (the speed at which the droplets dripped down the glass slide) of the sprayed test formulation and drying time were recorded.

Appearance and stickiness of dried film: The cosmetic appearance of the polymeric films was assessed by visually inspecting the dried polymer films. The stickiness of the film was assessed by gently pressing a cotton bud and counting the number of retained cotton fibres.

For each polymer, the top formulation was selected based on the rating summarized in Table 4. These selected formulations were further investigated for in vitro phage release profiles, antibacterial activity and stability.

In vitro release of phage

The in vitro phage release profile of the phage-polymer formulations was assessed using a Franz cell dissolution system. Degassed PBS (pH 7.4) was used as dissolution medium and the dissolution cell temperature was maintained at 32°C using a digital immersion heater circulator. A polyethersulfone membrane filter (0.45 pm) was saturated with the dissolution media for 20 min, gently blotted with Kimwipes, and then placed on top of the receptor chamber. A glass cap was placed on top of the membrane and then held together by a metal clamp. Phage-polymer formulations (200 pF) were placed on top of the membrane filter. At predetermined timepoints (5, 10, 20 and 40 min, 1, 2, 3, 4, 6 and 8 h), an aliquot of the sample (approx. 150 pL) was withdrawn from the receptor chamber and replaced with the same amount of fresh PBS. Phage release was assessed using the plaque assay method described below. The phage release profile of each of the phage formulations was assessed in triplicate. Plaque assay

The phage titers in the phage-polymer formulations and in the Franz cell-collected samples were determined using a standard plaque assay. A volume (0.2 mL) of a reference bacterial strain (2 x 10 ⁸ colony forming units at stationary phase) was mixed with 5 mL of 0.4% nutrient broth soft agar. The mixture was poured on top of a nutrient agar plate. Then, 10 pL of serially diluted phage formulations was dropped on top of the top agar plate, air-dried and then incubated overnight at 37 °C. The assay was independently conducted three times.

Antibacterial activity studies

The antibacterial activity of the optimized phage-polymer formulations was determined against a Pseudomonas aeruginosa clinical isolate. Double agar overlay plates containing P. aeruginosa were prepared as described above. The optimized phage formulations were sprayed 10 cm away from the overlay plate, air-dried and then incubated overnight at 37 °C. The zones of inhibition were assessed.

Storage stability studies

Ten optimized phage-polymer formulations were assessed for biological stability during storage. The formulations were aliquoted in glass vials and stored at 4°C for up to eight weeks. The glass vials were tightly sealed to avoid any evaporation during storage. The phage titer was assayed using the plaque assay at 0, 1, 2, 3, 4, 6 and 8 weeks.

Results and discussion

A total number of 22 spray formulations were prepared according to the compositions shown in Table 3. Six non-ionic polymers (PVP, PVA, HPMC, HPC, HEC and PEO) were utilized as phage tend to become unstable in the presence of charged polymers. The capsid exhibits an overall net negative charge and the tail fibers possess an overall net positive charge at physiological pH. Hence, the use of non-ionic polymers would minimize any charge-induced phage destabilization. In the current study, 35% ethanol in water was used as the solvent system because our preliminary data showed biostability of both myovirus PEV1 and podovirus PEV31 over 8 weeks. All six polymers formed transparent and homogenous liquid suspensions at the tested concentrations in 35% ethanol in water (Table 5). PVP, PVA and HPC formulations showed low viscosity (liquid-like) at concentrations ≤5% (w/v) and medium viscosity (syrup-like) at 10% (w/v). HPMC, HEC and PEO formulations exhibited low viscosity at concentrations <0.5% (w/v) and medium viscosity between 1-2% (w/v). Table 5. Physical properties of spray formulations.

Concentration Viscosity

Formulation Polymer Appearance Homogeneity

(%, w/v) level

1 PVP 2 Transparent low o

2 5 Transparent low o

3 10 Transparent medium o

4 PVA 1 Transparent low o

5 2 Transparent low o

6 5 Transparent medium o

7 10 Transparent medium o

8 HPMC 0.25 Transparent low o

9 0.5 Transparent low o

10 1 Transparent medium o

11 2 Transparent medium o

12 HPC 2 Transparent low o

13 5 Milky low o

14 10 Milky medium o

15 HEC 0.25 Transparent low o

16 0.5 Transparent low o

17 1 Transparent medium o

18 PEO 0.5 Transparent low o

19 1 Transparent Medium o

20 2 Transparent medium o

PVP, polyvinylpyrrolidone; PVA, polyvinyl alcohol; HPLC, hydroxypropyl methylcellulose; HPC, hydroxypropyl cellulose; HEC, hydroxyethyl cellulose; PEO, polyethylene oxide. Polymers were dissolved in 35% ethanol in water. Viscosity low: liquid, medium: syrup-like, symbol o: confirmed homogeneity.

The amount of phage formulation delivered after each actuation was consistent for each formulation (Table 6). This implies that the use of metered-dose sprays would allow precise control of the dose delivered to the application site. In general, formulations with lower polymer concentrations exhibited high spray-ability with faster polymeric film drying time. The organic solvents in these formulations vaporized rapidly leaving behind a thin polymeric film. HPMC (0.25%, w/v) and PVP (2%, w/v) spray formulations showed the shortest drying times of 10 and 12 min, respectively. Formulations containing other polymers (PVA, HPC, HEC and PEO) exhibited a longer drying time of 20-35 min even at low polymer concentrations. When sprayed on a glass slide, minimal droplet dripping (0-1.2 cm/30 s) was observed for PVP, PVA, HPMC, HPC and PEO formulations at low polymer concentrations. Greater than 5 cm/30 s droplet dripping was observed for the HEC spray formulation at a low concentration of 0.25% (w/v). All the dried polymeric films showed no stickiness regardless of the polymer types or concentrations. The cosmetic appearance of the films was generally shiny and clear, but some bubbles were present in HPMC and HPC films at high polymer concentrations. The top performing spray formulation was selected for each polymer based on the rating criteria specified in Table 4 above. Selected formulations included 2% PVP, 1% PVA, 0.25% HPMC, 2% HPC, 0.25% HEC and 0.5% PEO (all w/v) in 35% ethanol in water.

In vitro release profile is one of the critical factors that determine the efficacy of therapeutically active ingredients. In the present Example, myovirus phage PEV1 was rapidly released within 20-40 min from all the selected phage-polymer formulations and the phage titer remained high for eight hours (Figure 5). Approximately 3 x 10 ⁸ PFU/mL of PEV1 was released (loaded dose: 10 ⁹ PFU) within the first 10 min from 1% PVA, 0.25% HPMC and 0.5% PEO (w/v) spray formulations, followed by complete release at 20 min. Other phage-polymer formulations, including 0.25% HEC, 2% HPC and 2% PVP (w/v) also exhibited rapid phage release with 10 ⁶ pfu at 10 min and total release in 40 min. Similar results were observed for the podovirus phage PEV31 (Figure 6). PEV31 was rapidly released from all the phage- polymer formulations with 100% release within 40 min. In the present Example, both myovirus- and podovirus -type phage were rapidly released from the selected phage-polymer formulations. Furthermore, sprayed phage-polymer formulations inhibited the growth of P. aeruginosa bacteria on overlay plates.

Summary

This Example provides spray formulations containing non-ionic polymers in 35% ethanol in water that are compatible with Myoviridae and Podoviridae PEV phages. The spray phage formulations allowed easy and dose-controlled topical application using a metered-dose spray. These formulations can potentially be applied to many other types of phage.

20 2 Difficult

PVP, polyvinylpyrrolidone K30; PVA, polyvinyl alcohol; HPLC, hydroxypropyl methylcellulose; HPC, hydroxypropyl cellulose; HEC, hydroxyethyl cellulose; PEO, polyethylene oxide. S.D., standard deviation. Dripping and drying times were assessed after spraying at a vertical distance of 10 cm from a glass slide. Stickiness of the dried film was assessed using cotton balls. ^a n = 4.

Liquid phage formulations are typically stored under refrigerated conditions to prolong phage stability. Commercial liquid phage products generally have recommended storage temperatures of 2-8 °C. The data of the present Example showed titer reduction of PEV1 and PEV31 formulations when stored at 25 °C. In the current Example, the phage-polymer formulations were subjected to storage stability study at 4 °C. Phage PEV1 remained biologically stable in all the spray formulations over eight-week storage at 4°C (Figure 7a). PEV31 in PVP formulation gradually lost titer with 1 -log 10 reduction by week 8 (Figure 7b). PEV31 remained relatively stable in PVA, HPC, HPMC, HEC and PEO formulations with less than 0.5-logio titer loss over the tested period. Both myovirus PEV1 and podovirus PEV31 were biologically stable with minimal titer reduction in the selected phage-polymer spray formulations. Alcohol is known to cause significant drop in the titer of some phage. However, this study has shown that both myovirus and podovirus PEV phages are biologically stable in 35% ethanol in water. The phage-polymer formulations in the present Example utilized an ethanol/water solvent system which speeds up the evaporation time and minimizes droplet dripping while maintaining the biological activity of the phage.