FORMULATIONS

Abstract

The invention provides liquid formulations comprising dissolved oxygen (e.g. 10 mg/L up to 100 mg/L dissolved oxygen), and a substance which is capable of forming a gel upon contact with a body surface or body tissues, wherein said gel is capable of releasing a therapeutically effective amount of dissolved oxygen. The substance capable of forming a gel may be thermogelling and may, for example, comprise a blend of poloxamers. The liquid formulations can be applied to body tissues whereupon they form a gel suitable for the treatment of a compromised tissue, for example a wound (acute or chronic), a burn, a skin disorder (e.g. psoriasis, acne, rosacea, or other dermatological condition such as atopic dermatitis), skin sores, or tissue necrosis. The liquid formulations are also suitable for use in the prevention or treatment of a bacterial biofilm on a body surface, e.g. on the skin. The invention further provides delivery devices (e.g. wound dressings or bandages) having incorporated therein such a liquid formulation or a gel formed therefrom.

Claims

1. A liquid formulation comprising dissolved oxygen and a substance which is capable of forming a gel upon contact with a body surface or body tissues, and wherein said gel is capable of releasing a therapeutically effective amount of dissolved oxygen.

2. A formulation as claimed in claim 1 which comprises at least about 10 mg/L dissolved oxygen, and preferably up to about 100 mg/L dissolved oxygen, e.g. from about 15 to 70 mg/L, or from about 25 to 50 mg/L dissolved oxygen.

3. A formulation as claimed in claim 1 or claim 2, wherein said substance which is capable of forming a gel upon contact with a body surface or body tissues comprises at least one in-situ gelling agent.

4. A formulation as claimed in claim 3, wherein said in-situ gelling agent is thermogelling and capable of forming a gel at body temperature.

5. A formulation as claimed in claim 4, wherein said in-situ gelling agent is selected from the group consisting of: surface active block copolymers, polysaccharides (e.g. cellulose derivatives, xyloglycan, and chitosan) and N-isopropylacrylamide.

6. A formulation as claimed in claim 4, wherein said in-situ gelling agent comprises one or more poloxamers.

7. A formulation as claimed in claim 4, wherein said in-situ gelling agent comprises at least one poloxamer having a thermogelling temperature above about 25 C., e.g. in the range from about 25 to about 37 C.

8. A formulation as claimed in claim 6 or claim 7, wherein said in-situ gelling agent comprises one or more poloxamers selected from Poloxamer 407, Poloxamer 188, Poloxamer 338, Poloxamer 124, and Poloxamer 237.

9. A formulation as claimed in claim 8, wherein said in-situ gelling agent comprises Poloxamer 407.

10. A formulation as claimed in any one of claims 6 to 9 which comprises a blend of poloxamers.

11. A formulation as claimed in claim 10, wherein said blend comprises two poloxamers in a ratio of from 5:1 to 50:1, preferably from 10:1 to 25:1, e.g. from 10:1 to 15:1.

12. A formulation as claimed in claim 10 or claim 11, wherein said blend comprises Poloxamer 407 and Poloxamer 188, or Poloxamer 407 and Poloxamer 124.

13. A formulation as claimed in any one of claims 6 to 12, wherein said poloxamer or blend of poloxamers is present in an amount in the range of from 10 to 30 wt. %, preferably 15 to 25 wt. %. e.g. 16 to 20 wt. % (based on the weight of the formulation).

14. A formulation as claimed in any one of claims 4 to 13 which further comprises one or more thickening agents selected from the following: Carbopol, cellulose derivatives (e.g. hydroxypropyl cellulose or hydroxypropyl methylcellulose), carrageenans, gelatin, pectin, hydrocolloids, alginates, hydrogels, polyurethane, collagen, chitosan, and hyaluronic acid.

15. A formulation as claimed in any one of the preceding claims which further comprises one or more bioadhesives, e.g. one or more mucoahesive polymers.

16. A formulation as claimed in any one of the preceding claims, wherein the oxygen present in the formulation is dissolved in an aqueous medium which is physiologically tolerable, for example a physiological salt solution (e.g. saline) or water.

17. A formulation as claimed in any one of the preceding claims which comprises an oxygenated liquid (e.g. physiological saline or water) obtainable by (e.g. obtained by) a process comprising the following steps: introducing a pressurized liquid into a piping network to form a flow stream; injecting gaseous oxygen into the flow stream to produce a mixture of liquid and oxygen bubbles, providing a linear flow accelerator including a venturi; and passing the flowing mixture of liquid and gaseous oxygen bubbles through the linear flow accelerator to accelerate the flowing mixture and to subsequently decelerate the flowing mixture to subsonic speed to break up the gaseous oxygen bubbles.

18. A formulation as claimed in any one of the preceding claims which further comprises one or more active substances selected from the group consisting of: antibacterial agents, antifungal agents, antiviral agents, antibiotics, growth factors, cytokines, chemokines, nucleic acids, vitamins, minerals, anaesthetics, anti-inflammatory agents, moisturizers, extracellular matrix proteins, enzymes, stem cells from plants, extracts from eggs and eggshells, botanical extracts, fatty acids, and skin penetration enhancers.

19. A formulation as claimed in claim 18, wherein said antibacterial agent is selected from group consisting of: alcohols, chlorine, peroxides, aldehydes, triclosan, triclocarban, benzalkonium chloride, linezolid, quinupristin-dalfopristin, daptomycin, oritavancin and dalbavancin, quinolones, and moxifloxacin.

20. A formulation as claimed in any one of claims 1 to 17 which comprises at least one active agent selected from the group consisting of retinoids (e.g. vitamin A, acitretin, isotretinion, tretinion and tazarotene); peroxides (e.g. benzoyl peroxide); antibiotics (e.g. tetracycline, clindamycin, erythromycin, metronidazole, sulfacetamide, doxycycline, oxytetracycline, minocycline, and trimethoprim); hormones (e.g. co-cyprindiol); azelaic acid and derivatives thereof; adapalene; nicotinamide; salicylic acid; corticosteroids, vitamin D and derivatives thereof; antralin, and calcineurin inhibitors.

21. A formulation as claimed in any one of claims 1 to 17 which comprises at least one wound healing agent, e.g. collagen or chitosan.

22. A formulation as claimed in any one of the preceding claims which is a cosmetic formulation and which comprises one or more cosmetically active ingredients selected from the group consisting of peptides, amino acids, hyaluronic acid, hydroxy acids, vitamins or derivatives thereof, retinoids, ceramides, carbamide (urea) and coenzyme Q10.

23. A formulation as claimed in any one of the preceding claims which comprises from 50 to 99 wt. % water, preferably from 80 to 99 wt. % water.

24. A formulation as claimed in any one of the preceding claims which comprises one or more components which maintain a buffered pH, which maintain osmolality in a range suitable for application to a body surface or body tissues, or which maintain stability of the composition.

25. A formulation as claimed in any one of the preceding claims which comprises one or more components selected from the group consisting of: buffers, pH adjusting agents (e.g. sodium hydroxide or hydrochloric acid), osmolality adjusting agents, preservatives (e.g. anti-microbial agents), anti-oxidants, gel forming agents (e.g. Carbopols or cellulose derivatives), fragrances, and coloring agents, preferably a formulation which contains sufficient buffer to provide a pH in the range from 2 to 7, preferably 5 to 5.5, e.g. about 5.1 to about 5.5.

26. An oxygen-releasing gel obtainable by allowing the liquid formulation as claimed in any one of claims 1 to 25 to gel.

27. A formulation or oxygen-releasing gel as claimed in any one of claims 1 to 26 for use in medicine or for use as a medicament.

28. A formulation or oxygen-releasing gel for use as claimed in claim 27 in the treatment of a compromised tissue, for example a wound (acute or chronic), a burn, a skin disorder (e.g. psoriasis, acne, rosacea, or other dermatological condition such as atopic dermatitis), skin sores, or tissue necrosis.

29. A formulation or oxygen-releasing gel for use as claimed in claim 27 in the treatment of bacterial or fungal infections of the skin, for example cellulitis, infections in chronic wounds, gas gangrene, necrotizing fasciitis (infection by enterococcus, enterobacteriacea, clostridium, B. fragilis, streptococcus, pyogenis), or a fungal infection associated with mucorales or aspergilus.

30. A formulation or oxygen-releasing gel for use as claimed in claim 27 in the prevention or treatment of a bacterial biofilm on a body surface, preferably on an external body surface, e.g. on the skin.

31. A delivery device (e.g. a wound dressing or bandage) having incorporated therein a liquid formulation as claimed in any one of claims 1 to 25 or an oxygen-releasing gel as claimed in claim 26.

32. A method for the treatment of a compromised body surface or body tissue of a mammalian subject (e.g. a human), said method comprising: (i) applying to said surface or tissue an effective amount of a liquid formulation as claimed in any one of claims 1 to 25; and (ii) allowing said formulation to set to form a gel.

33. Use of a liquid formulation as claimed in any one of claims 1 to 25 in the manufacture of a medicament for use in a method for the treatment of a compromised body surface or body tissue of a mammalian subject (e.g. a human).

34. A kit for use in the treatment of a compromised body surface or body tissue of a mammalian subject (e.g. a human), the kit comprising: (a) a sealed container or package containing a liquid formulation as claimed in any one of claims 1 to 25; (b) a delivery device, e.g. a wound dressing or bandage; and optionally (c) instructions for use of components (a) and (b) in the topical treatment of a compromised tissue.

35. A method of cosmetic treatment performed on a mammalian subject (e.g. a human), said method comprising the step of administering to the surface of the skin of said subject a liquid formulation as claimed in any one of claims 1 to 25.

36. A method as claimed in claim 35 for improving or otherwise enhancing the appearance of the skin, for example in softening the skin, or in reducing any one of the following: hyper-pigmentation, roughness, dryness, fine lines and wrinkles of the skin.

Description

[0192] The invention will now be described further with reference to the following non-limiting Examples and the accompanying figures in which:

[0193] FIG. 1 shows the stability of a formulation in accordance with the invention (Oxy Dressing) when applied on a skin model. Dissolved oxygen levels in the Oxy Dressing with an original dissolved oxygen level of 32 mg/L were kept above 25 mg/L for more than 30 hours at 35 C. when kept in a closed system. The control dressing had an original dissolved oxygen concentration of 14 mg/L, and dropped to 2.5 mg/L after approximately 5 hours.

[0194] FIG. 2 shows the rheology of a formulation in accordance with the invention (Oxy Dressing). The influence of viscosity (G*: shear modulus complex component) on increased temperature for Lutrol F127 in MilliQ and in Oxy Water is shown. The presented data are representative measurements of one individual sample for each formulation.

[0195] FIG. 3 shows the shelf life of a formulation in accordance with the invention (Oxy Dressing). Dissolved oxygen was measured by Winkler titration in the Oxy Dressing after storage at room temperature (20 C.) or in the fridge (4 C.) for up to 7 weeks. The Oxy Dressing maintained stable dissolved oxygen levels above 30 mg/L when stored at 4 C., and above 20 mg/L when stored at room temperature in capped glass vials for 8 weeks. Data are presented as average SD.

[0196] FIG. 4 shows the pH stability of a formulation in accordance with the invention (Oxy Dressing). The pH value of Lutrol F127 formulated in 20 mM acetate buffer and MilliQ water, stored at 5 C. and 23 C. for 3 months, is shown. The Oxy Dressing kept a stable pH for 3 months when prepared in 20 mM acetate buffer. Results are presented as averages and standard deviations from individual measurements of 3-9 different samples.

[0197] FIG. 5 shows the in vitro effect of dissolved oxygen (DO) on human skin fibroblastsAdenosine triphosphate (ATP). ATP levels, presented as average nmol/10.sup.5 live cells SD, were measured in human skin fibroblasts incubated for 4 hours in control medium (DMEM w/10% FBS, 8 mg/L DO), DMEM w/10% FBS and 33.0 mg/L DO, or positive control (HeLa cells, DMEM w/10% FBS, 8 mg/L DO). The cells were re-stimulated with the respective conditions at 1, 2 and 3 hours. The cells treated with 33 mg/L DO (113.1 nm ATP/10.sup.5 cells) and positive control (191.6 nm ATP/10.sup.5 cells) had significantly higher ATP levels than the control (70.9 nm/10.sup.5 cells) after 4 h (n=3, *p<0.05).

[0198] FIG. 6 shows the in vitro effect of dissolved oxygen (DO) on human skin fibroblastsProliferation. Cells were grown in DMEM w/1% FBS with (23-50 mg/L) or without (11 mg/L) high levels of DO, or in DMEM w/10% FBS (11 mg/L, positive control). Medium was changed every day. At Day 2, 3 and 4 cells were harvested and manually counted using a hemocytometer. Treating cells with DO up to 50 mg/L did not significantly alter the proliferation rate of human skin fibroblasts when compared to control. Data are expressed as average SD (n=4).

[0199] FIG. 7 shows the in vitro effect of dissolved oxygen (DO) on human skin fibroblastsCell viability. Cells were grown in DMEM w/1% FBS with (23-50 mg/L) or without (11 mg/L) high levels of DO, or in DMEM w/10% FBS. 1% Triton X was used as positive control. Medium was changed every day. At Day 2, 3 and 4 cells were harvested, mixed with trypan blue and dead cells were counted using a TC20 Automated cell counter. Treating cells with DO up to 50 mg/L did not significantly alter the number of dead cells compared to control over the course of 4 days. Data are expressed as the percentage of dead cells relative to total cells and represent the average SD (n=4, *p<0.05).

[0200] FIG. 8 shows the in vitro effect of dissolved oxygen (DO) on human skin fibroblastsReactive Oxygen Species (ROS). ROS levels, presented as relative fluorescence (10.sup.3) SD, were measured in human skin fibroblasts incubated 30 min in control (DMEM w/5% FBS, 8.6 mg/L DO), 3 different concentrations of DO (DMEM w/5% FBS, 21.6, 26.5, or 34.9 mg/L DO) or H.sub.2O.sub.2, positive control (DMEM w/5% FBS, 8.6 mg/L DO and 500 M H.sub.2O.sub.2). The positive control (H.sub.2O.sub.2) had significantly higher levels of ROS compared to control. There were no significant differences between the ROS production in the cells treated with DO (21-35 mg/L) and control (n=3, *p<0.05).

EXAMPLES

Example 1Preparation of Oxygenated Water (Oxywater)

Method:

[0201] 1. Reverse osmosis (RO) water was chilled to 2-4 C. [0202] 2. Chilled water was fed into an oxygenation device as described in WO 2016/071691. [0203] 3. Oxygen gas was at the same time fed into the mixing chamber of the device. [0204] 4. Water and O.sub.2 gas were passed through the venturi and O.sub.2 gas dissolved in the water. [0205] 5. The gas input may vary depending on the flow rate of the gas and the process pressure and this may be used to adjust the O.sub.2 content of the water. For this production the process pressure employed was 42 psi. [0206] 6. The water was produced by continuous circulation through the device until a dissolved oxygen content of 100 mg/L was reached. [0207] 7. The oxygenated water was bottled in glass bottles and stored at ambient temperature.

[0208] Oxygen content of the water was varied by adjusting the flow rate and pressure of O.sub.2 gas. In this way it was possible to prepare oxygenated water having a range of O.sub.2 contents according to need.

Example 2Preparation of Thermogelling Formulations and Determination of O.SUB.2 .Levels

Materials:

[0209] Oxygenated water (approx. 70 mg/L oxygen content)prepared by a method analogous to Example 1 [0210] PoloxamerLutrol F127 (BASF) [0211] TRIS buffer (Merck) [0212] 5M HCl

Method:

[0213] During preparation of the formulations the oxygenated water was handled with care in order to retain the high oxygen content. A fresh bottle of oxygenated water was opened for each formulation. Prior to use, the bottles were refrigerated at 2-4 C. in order to chill the water before preparation of the formulations.

[0214] A concentrated solution of TRIS buffer was prepared by adding 8-9 ml oxygenated water to 12.1 g TRIS base. The pH was adjusted to 7.5 using 5M HCl and the solution was made up to a volume of 10 ml by adding additional oxygenated water. In a mixing vial, 40 ml of a concentrated (40 wt. %) poloxamer solution was prepared by dissolving the poloxamer powder in a solution containing the 10 ml of TRIS buffer solution and 30 ml of oxygenated water.

[0215] The final formulation was prepared by dilution of the concentrated poloxamer solution (40 ml) to the final concentration (16 wt. % poloxamer) with fresh, ice-cooled oxygenated water (60 ml).

[0216] All steps were carried out without agitation. In order to minimise the tendency for bubble formation and loss of oxygen all work was carried out at 5 C. Head space in the mixing vial was kept to a minimum. In order to further minimise the loss of oxygen, mixing may be done under pressure (air or oxygen).

Analysis of Oxygen Content:

[0217] Prior to use, the oxygen content of the water was measured by Winkler titration and using an oxygen meter available from Orion, Thermo Scientific. For measurements using the oxygen meter, the oxygenated water was mixed 50:50 with MilliQ water and oxygenation levels were measured as 33-41 mg/L, i.e. 66-82 mg/L after compensation for the dilution.

[0218] Oxygen content of the formulations during gelling was monitored using an Oxygen meter developed by SP Technical Research Institute of Sweden. The oxygen meter (Oxymeter) supplied by Orion, Thermo Scientific was used to measure the oxygen content of the formulation before applying this to the skin model.

Gelling with Temperature:

[0219] Thermogelling was achieved either by lowering the vial containing the liquid formulation into a beaker of heated water at 35-37 C., or by direct application of the formulation onto the skin.

Results:

[0220] Thermogelling formulations having final oxygen contents in the range of from 25 to 30 mg/L were prepared according to the protocol described above. The resulting gels were capable of retaining the oxygen content at about 20 mg/L for at least 2 hours after application (i.e. following gel formation). When the gel was prevented from drying (i.e. stored in a closed system), oxygen levels above 25 mg/L were retained for at least 30 hours.

Conclusions:

[0221] The formulation protocol provided a thermogelling formulation of Oxywater with a final oxygen content of 25-30 mg/L with the potential to be used in wound healing and cosmetic applications.

[0222] The thermogelling formulation could oxygenate tissues for at least 30 hours under certain conditionsi.e. if the gel does not dry out and provided the oxygen is prevented from escaping to the surrounding air.

Example 3Preparation of Thermogelling Formulations and Determination of O.SUB.2 .Levels

Materials:

[0223] Oxygenated water (approx. 70 mg/L oxygen content)prepared by a method analogous to Example 1

PoloxamerLutrol F127 (BASF)

[0224] Acetic acid (Sigma-Aldrich)
Sodium acetate

10N NaOH

Method:

[0225] A concentrated solution of acetate buffer was prepared by adding oxygenated water to 54.43 g sodium acetate and 12 ml acetic acid to produce a total volume of 180 ml. The pH was adjusted to 7.5 using 10N NaOH and the solution was made up to a volume of 200 ml by adding additional oxygenated water.

[0226] In a mixing vial, 40 ml of a concentrated (40 wt. %) poloxamer solution was prepared by dissolving the poloxamer powder in a solution containing 10 ml of the acetate buffer solution and 30 ml of oxygenated water.

[0227] The final formulation was prepared by dilution of the concentrated poloxamer solution (40 ml) to the final concentration (16 wt. % poloxamer) with fresh, ice-cooled oxygenated water (60 ml).

Example 4Preparation of Thermogelling Formulations

[0228] Other poloxamer-based formulations were prepared with oxygenated water containing 58 mg/L dissolved O.sub.2. In a mixing vial, 40 ml of a concentrated (40 wt. %) poloxamer solution was prepared by dissolving the poloxamer powder in a solution containing 40 ml of the oxygenated water. The final formulations were prepared by dilution of the concentrated poloxamer solution (40 ml) to the final concentration with fresh, ice-cooled oxygenated water.

[0229] Thermogelling was assessed either by lowering a vial containing the formulation into a water bath at 35-37 C., by direct application of the formulation onto the skin, or with a kinexus Pro Rheometer with a plate-plate 20 nm diameter.

[0230] Table 1 provides details of the formulations and their thermogelling properties.

TABLE-US-00001 TABLE 1 G G Total (Pa) (Pa) Comment - Poloxamer F127 F68 O2 conc. Tgel Tgel at at application (wt. %) (wt. %) (wt. %) (mg/L).sup.1 (WB).sup.2 (Rheo).sup.3 35 C. 35 C. to skin.sup.4 16 16 0 23 32 31 805 9158 Low viscosity, slightly runny after application 20 18 2 20 36 Low viscosity, very runny after application 20 18.5 1.5 18 32 28 861 17140 Low viscosity, slightly runny after application 20 19 1 19 28 25 865 16930 Low viscosity, slightly runny after application 25 20 5 13 37 31 1167 13360 Low viscosity, runny after application .sup.1O.sub.2 levels for the formulations prior to gelling .sup.2Gelling temperature in water bath .sup.3Tgel measured by rheology (Tgel (Rheo)) and in the water bath (Tgel (WB)) differ due to differences in the Tgel definition .sup.4consistency immediately after application of the formulation to the skin

Conclusions:

[0231] In all cases, the low viscosity on application to the skin was acceptable. [0232] Increasing the poloxamer concentration from 16 wt. % to 20 wt. % leads to a higher gel strength and faster gelling onset. Further increasing the poloxamer concentration from 20 wt. % to 25 wt. % does not change the gel strength significantly. [0233] The oxygen content in the gels can be increased if the oxygenated water for use in the formulation has a higher oxygen content. [0234] The presence of oxygen does not significantly influence the gelling properties. [0235] When evaluating the gels on the skin, only very small differences were detected between the formulations.

Example 5Preparation of Thermogelling Formulation and Testing

Methods:

Production of Oxygenated Water (Oxy Water)

[0236] Water with elevated levels of dissolved oxygen was produced as described in WO 2016/07169. Reverse Osmosis (RO) water and oxygen was fed into a chamber of an oxygenation device. The water and oxygen were mixed and subsequently passed through a piping system including a venturi. The system ran continuously at 2.9 bar and was fed with 98% O.sub.2. At the outlet, oxygenated water containing up to 100 mg/L dissolved oxygen was collected, bottled in glass bottles and stored at room temperature until use.

Winkler Titration for Determination of Dissolved Oxygen Concentrations

[0237] Samples of the Oxy Water were mixed with manganese sulphate (2.2 M) and alkaline iodide azide (12 M NaOH, 0.86 M KI). The precipitate was allowed to settle halfway two times before 98% sulphuric acid was added, giving a dark amber colour. The solution was then titrated with sodium thiosulfate (0.0375 M). Starch indicator solution was added when the solution reached a light yellow colour. Each 1.0 ml of sodium thiosulfate used was equivalent to 3 mg/L dissolved oxygen.

Formulation of Oxygenated In-Situ Gelling Dressing (Oxy Dressing)

[0238] The Oxy Dressing was formulated with Oxy Water (65 mg/L) and Lutrol F127 (BASF). A fresh bottle of oxygenated water at 2-4 C. was opened for each formulation. In a mixing vial, concentrated (40-50 wt. %) Lutrol F127 solution was prepared by dissolving the poloxamer powder in the oxygenated water under gentle agitation. When the poloxamer was dissolved, the stock solution was diluted with Oxy Water (both 0 C.) to a final concentration of 15-16 wt %.

[0239] The Oxy Dressing was also formulated in 20 mM acetate buffer for pH control. 30 wt. % of Lutrol F127 was dissolved in a 38 mM acetate buffer by gentle mixing in an ice bath (0 C.) to minimise the viscosity of the blend. After dissolution of the Lutrol F127 in the stock solution, the 30 wt. % polymer solution was mixed with Oxy Water cooled to 0 C. to a final concentration of 15-16 wt % Lutrol F127.

Oxygen Stability after Application

[0240] An oxygen optode developed by RISE (Bors, Sweden) was used to evaluate the oxygen concentration and stability of the Oxy Dressing over time when applied at a temperature controlled surface of 35 C. (skin surface temperature). The optode was assembled on to a tip of a fibre optic probe (NeoFox, Ocean Optics) and mounted inverted (facing upwards) on a heating block controlling the surface temperature. The principle for the measurements was based on phase fluometry. Thus, the dye in the optode film was excited with a pulsed blue LED that caused a luminescence delay (phase shift) relative to the pulsed LED frequency, which is dependent on the oxygen concentration. A high oxygen level reduces, and low oxygen levels prolongs, the luminescent lifetimes as well as phase shifts. Oxy Water and calibrating solutions were measured using a brass pipe (15 ml) assembled onto a seal surrounding the fibre optic tip. Calibration of the heated (35 C.) optode was made by exposing the assembled sensor to argon and oxygen saturated ultra-purified water, respectively at 35 C. The oxygen contents of the calibrating solutions were also confirmed with Winkler titration. During measurements of the Oxy Dressing, a smaller black seal was used as a container during gel application facilitating an exact volume (1.5 ml) and surface area (3.5 cm.sup.2). The area of the gel measured corresponded to an uncured thickness of 4 mm. To avoid the gel from drying out, measurements were also performed when covering the gel with a glass lid. Temperature and oxygen levels were simultaneously and continuously measured during the experiments that lasted between 2-50 hours. Humidity and temperature in the room were determined before and after the measurements (29.7% relative humidity and 21.5 C.). Formulations prepared from MilliQ H.sub.2O served as a control.

Shelf Life Testing

[0241] For shelf life stability testing, the Oxy Dressing was gently filled in capped glass vials (Agilent HS, crimp, RB 20 ml with Hdspc Al crmp cap, PTFE/Si) with minimum head spacing, and stored at 20 C. or at 4 C. for up to 7 weeks. The dissolved oxygen concentrations were measured weekly by Winkler titration. Formulations prepared from MilliQ H.sub.2O served as a control.

Gelling Properties

[0242] Thermogelling was assessed either by visual inspection, by lowering a vial containing the formulation into a water bath at 35-37 C., by direct application of the formulation onto the skin, or with a kinexus Pro Rheometer with a cone-plate (4 degrees, 40 mm diameter). When using the kinexus Pro Rheometer, 1.9 ml of formulation was applied for each measurement and the temperature was increased 1 degree/min at a frequency of 0.3 Hz and a deformation of 1 Pa.

pH Stability Testing

[0243] The Oxy Dressing formulated in 20 mM acetate buffer was stored in capped glass vials at room temperature (23 C.) and at 5 C. for up to 3 months and pH was assessed regularly and compared to the pH of a 16 wt. % Lutrol F127 solution in MilliQ water.

Statistics

[0244] Winkler analysis was performed on 5 samples for each condition. Oxygen stability after application and rheology studies were performed on individual samples. The shelf life study was performed on two samples per group per condition and presented as average standard deviation. pH stability is presented as average and standard deviations from individual measurements of 3-9 different samples. Results were analysed statistically in SPSS using two-way analysis with unpaired t-test or ANOVA with Bonferroni post hoc test comparing the average values of each experiment. Values of p<0.05 were considered statistically significant.

Results:

[0245] The dissolved oxygen levels in the Oxy Dressing remained stable above 25 mg/L dissolved oxygen for more than 30 hours when applied on a skin model system with controlled temperature at 35 C. and covered with a glass lid (see FIG. 1). The covered Oxy Dressing did not dry out after 170 hours of operation, ending with an oxygen holding capacity exceeding 13 mg/L. The rheology and gelling properties of the dressing were not significantly influenced by the dissolved oxygen as shown in FIG. 2.

[0246] The Oxy Dressing maintained stable dissolved oxygen levels above 30 mg/L when stored at 4 C., and above 20 mg/L when stored at room temperature (20 C.) in capped glass vials for 7 weeks (see FIG. 3).

[0247] The results in FIG. 4 show that is possible to control the pH of the Oxy Dressing by formulating in a buffer and that the pH in the formulation is stable over time when stored at room temperature and under refrigerated conditions.

Conclusions:

[0248] In the present study a highly oxygenated and pH controlled, thermosensitive topical dressing (Oxy Dressing) was formulated and tested. The Oxy Dressing has high and stable levels of oxygen for more than 30 hours and can efficiently oxygenate tissue for the same period of time. The shelf life studies show that the thermosensitive in situ forming dressing can retain the high dissolved oxygen content when stored.

[0249] Preparation of the Oxy Dressing in a buffer enables pH control, with stable pH over time. While normal skin has a pH of approximately 5.5, chronic wounds have been shown to have an alkaline pH. An acidic environment in the wound has been shown to control wound infection, increase antimicrobial activity, altering protease activity, enhancing fibroblast growth in vitro, releasing oxygen, reducing toxicity of bacterial end products, and enhancing epithelization and angiogenesis (Percival et al., Wound Repair Regen. 22(2):174-86, 2014). In light of the importance of pH to wound healing, lowering the pH can itself be favourable for healing of wounds, and give an additional advantage to the wound healing properties of the Oxy Dressing.

Example 6Testing of Oxygenated Water

[0250] The effects of oxygen on wound healing are well established. Nevertheless, the understanding of the mechanisms is still limited and requires further evaluation, thus in vitro studies of the effect of dissolved oxygen on human skin cells was performed.

Methods:

Cultivation, Cells

[0251] Human skin fibroblasts (HSF) and HeLA cells (ATCC) were grown in complete cell medium (DMEM) with 10% Fetal bovine serum (FBS) and 1%

penicillin/streptomycin (PenStrep) (Sigma Aldrich). At confluence, cells were detached using 5% trypsin 1 mM ethylene diamine tetraacetic acid (EDTA) (Sigma Aldrich) and reseeded. Cells were cultured at 37 C. in an incubator with humidified atmosphere and 5% CO.sub.2.

Preparation of Oxygenated Cell Medium

[0252] Oxygenated medium was prepared using powder cell medium, diluted with Oxy Water prepared according to Example 5. The mixing ratio depended on the desired concentration of dissolved oxygen in the final cell medium. DMEM or DMEM medium without phenol red was used (Sigma Aldrich). The medium was supplied with additives and FBS. The phenol red free cell medium was used for the ROS experiment to avoid disturbance of the fluorescent readings.

ATP Concentration

[0253] HSF and HeLa cells (positive control) were seeded at a concentration of 2.510.sup.5 cells/9.5 cm.sup.2 and grown to confluence for 48 hours. The experiment included wells for ATP quantification and corresponding wells for cell counting. The cells were treated with complete cell medium (10% FBS) with dissolved oxygen: 8 mg/L or 33 mg/L. After stimulation, the cells were left at room temperature for 30 min, followed by incubation at 37 C. The cells were re-stimulated with the respective treatments after 1, 2 and 3 hours. After 4 hours, the cells were rinsed with PBS and lysed on ice with lysis buffer (200 mM Tris, pH 7.5, 2 M NaCl, 20 mM EDTA, 0.2% Triton X-100). The lysate was centrifuged at 10 000g for 5 min. The luminescence was read at a plate reader (FLUOStar Omega, BMG Labtech) and compared to an ATP standard curve (ATP Determination Kit, ThermoFisher, USA). Cells in corresponding wells were harvested, trypan blue (Sigma Aldrich) added and subsequently the number of cells was determined by manually counting live and dead cells using a hemocytometer. The data were corrected for blank readings and presented as nm ATP/10.sup.5 live cells.

[0254] Proliferation and Cell Viability

[0255] HSFs were seeded at a density of 510.sup.4 cells/25 cm.sup.2 and left overnight. The cells were treated with control cell medium (1% FBS, dissolved oxygen: 11 mg/L), oxygenated cell medium (1% FBS, dissolved oxygen: 23, 31, 41, 50 mg/L), or 10% FBS, dissolved oxygen: 10 mg/L. After stimulation, the cells were left at room temperature for 30 min, followed by incubation at 37 C. The treatment was repeated every 24 hours up to 4 days. On day 2, 3 and 4 the cells were harvested; the cell suspension was mixed with trypan blue and live cells were manually counted using a hemocytometer and dead cells were counted using a TC20 Automated cell counter (BioRad, USA). 1% Triton X was used as a positive control for cell death.

Reactive Oxygen Species (ROS) Production

[0256] HSFs were seeded at a density of 2.510.sup.4 cells/0.32 cm.sup.2 and were grown to confluence overnight. The cells were stained with 20 uM DCFDA solution (2,7-dichlorofluorescin diacetate) (DCFDA Cellular ROS Detection Assay Kit, Abcam, Cambridge, UK) and incubated for 45 min at 37 C. protected from light exposure. Control cell medium (5% FBS, dissolved oxygen: 8.6 mg/L), oxygenated cell medium (5% FBS, dissolved oxygen: 21.6, 26.5, 34.9 mg/L) and positive control (500 M H.sub.2O.sub.2, 5% FBS, dissolved oxygen: 8.6 mg/L) were added. The cells were incubated at room temperature. The fluorescence was read with an FLx800 plate reader (BioTek, Winooski, USA) with excitation wavelength at 485 nm and emission wavelength 520 nm, 30 min after adding the treatments. The values were corrected for blank readings and presented as relative fluorescence (10.sup.3). The experiments were repeated using 10% FBS.

Statistics

[0257] All in vitro experiments were performed in triplicates and repeated at least 3 times. Results were analysed statistically in SPSS using two-way analysis with unpaired t-test or ANOVA with Bonferroni post hoc test comparing the average values of each experiment. Values of p<0.05 were considered statistically significant.

Results:

[0258] In vitro studies showed that ATP levels were significantly higher in human skin fibroblasts after four hourly stimulations of 33 mg/L dissolved oxygen compared to control (8 mg/L dissolved oxygen). The levels of ATP were 70.920.5, 113.110.1, and 191.634.2 nM ATP/10.sup.5 live cells for control, 33 mg/L dissolved oxygen, and positive control, respectively (see FIG. 5). Increasing the concentration of dissolved oxygen up to 50 mg/L did not change cell proliferation of HSF (see FIG. 6), nor did it change the level of cell viability when compared to the control (11 mg/L dissolved oxygen) (see FIG. 7). Increasing the concentration of dissolved oxygen to 34.9 mg/L did not significantly change the ROS production after 30 min in HSF when compared to control (8.6 mg/L dissolved oxygen). The relative fluorescence (10.sup.3) was 13.53.9, 9.33.4, 15.24.5, 8.92.9, and 187.618.0 for control, 21.6 mg/L dissolved oxygen, 26.5 mg/L dissolved oxygen, 34.9 mg/L dissolved oxygen and positive control, respectively (see FIG. 8).

Conclusions:

[0259] The present studies show that treatment of cells with elevated dissolved oxygen levels significantly increased ATP production without affecting proliferation, cell viability, or oxidative stress in vitro.

[0260] 4 hourly stimulations of 33 mg/L dissolved oxygen resulted in increased ATP levels in HSFs when compared to the control. This indicates an advantageous effect of dissolved oxygen on the energy levels of human skin cells, thus facilitating increased wound healing. The results presented herein do not show any significant change in proliferation or viablility after treating the HSF with dissolved oxygen levels up to 50 mg/L for four days, indicating that topical treatment with dissolved oxygen up to 50 mg/L is safe and does not induce cellular toxicity.

[0261] In the studies described herein, no significant increase in ROS was found after 30 min in HSFs stimulated with increased levels of dissolved oxygen when compared to the control, either when using 5% or 10% FBS. Thus, increasing the dissolved oxygen levels up to 34.9 mg/L does not induce damaging levels of ROS. This is further reflected in results from quantification of cell death after daily stimulations with highly oxygenated cell medium (up to 50 mg/L), where no difference in toxicity was observed after 4 days when compared to the control.

[0262] The oxygenated water can be used in the production of dressings with various dissolved oxygen concentrations and pH. With proper monitoring of a wound, it is expected that the dressings can optimize chronic wound treatment.

Example 7Preparation of Thermogelling Formulation

[0263] An oxygenated thermogelling formulation was prepared by oxygenation of a poloxamer-containing composition.

Materials:

[0264] Poloxamers: Kolliphor P407 (Sigma Aldrich) [0265] Kolliphor P188 (Sigma Aldrich) [0266] Buffer: Tri-sodium citrate (anhydrous) [0267] NaCl [0268] Citric acid [0269] MilliQ H.sub.2O [0270] pH 5.1

Methods:

Preparation of 10 mM Citrate Buffer:

[0271] Base (10 mM tri-sodium citrate/140 mM NaCl): 11.76 g of tri-sodium citrate (anhydrous) and 32.73 g NaCl were dissolved in MilliQ H.sub.2O to a final volume of 4000 ml.

[0272] Acid (10 mM citric acid/140 mM NaCl): 1.92 g citric acid and 8.18 g NaCl were dissolved in MilliQ H.sub.2O to a final volume of 1000 ml. 3000 ml of base was mixed with 750 ml of acid. pH was adjusted to 5.1 using the acid or base and the buffer was placed in a fridge or cold room.

Preparation of Formulation:

[0273] Cold buffer equal to 80 wt. % of the final volume of the formulation was placed in a wide flask and stirred with a magnetic stir bar. Slowly the poloxamers were added to buffer whilst stirring to produce a solution containing 18.5 wt. % Kolliphor P407 and 1.5 wt. % Kolliphor P188. The solution was mixed on ice in a cold room overnight or until the solution was completely clear and lump free.

Oxygenation of Formulation:

[0274] The poloxamer-containing formulation was oxygenated by passing it through the device described in WO 2016/071691. The O.sub.2 pressure was set to 4.1 bar (60 psi). The formulation was passed through the device once at a flow rate of 100 ml/min O.sub.2.

Testing:

[0275] Samples of the oxygenated formulation were taken for Winkler titration according to the protocol described in Example 5, and for gelling and pH measurements. Gelling ability included gelling of both 100 l and 1 ml samples to compare gelling of large and small volumes of the formulation on a 35 C. surface.

Results:

[0276] Passing the formulation through the oxygenation device once with 100 ml/min O.sub.2 did not lead to a significant foam build-up, even without the addition of foam reducing agents. The average dissolved oxygen content measured with Winkler titration was 8.21.1 (n=4), 10.45.9 (n=4), and 36.13.8 mg/L (n=4) for the baseline, no oxygen (single pass), and oxygenated (single pass), respectively. The oxygenated sample had dissolved oxygen values significantly higher than baseline.

[0277] The pH did not change when circulating the formulation through the device without/with oxygen, with an average pH of 5.470.04 and 5.470.05, respectively. The gelling of 100 l formulation and 1 ml formulation applied to a 35 C. surface took approximately 3 min and 5 min, respectively, independent of the addition of oxygen.

Conclusions:

[0278] Oxygenation of the poloxamer-containing formulation up to 36.13.8 mg/L was achieved after a single pass through the oxygenation device and without changing the pH or the thermogelling properties. A single pass did not result in foam build-up even without the addition of foam reducing agents.

Example 8Preparation of Thermogelling Formulation

Method:

[0279] The method of Example 7 was repeated subject to the following changes: EX CELL foam reducer (simethicone emulsion, Sigma Aldrich) was added to the buffer or to the formulation immediately prior to oxygenation.

[0280] The formulation was passed through the oxygenation device either once or twice at a flow rate of 200 ml/min O.sub.2.

Results:

[0281] The average dissolved oxygen content measured with Winkler titration was 11.01.7 (n=3), 12.42.8 (n=3), 46.85.7 mg/L (n=3), and 42.73.2 mg/L (n=3) for the baseline, no oxygen, oxygenated (single pass) and oxygenated (multiple passes), respectively. The oxygenated samples had dissolved oxygen values significantly higher than baseline.

[0282] With a starting pH of 5.1 in the buffer, the pH at baseline in the final formulation was 5.540.04. This did not change after circulating the formulation through the device without/with oxygen. The gelling of 100 l formulation and 1 ml formulation applied to a 35 C. surface took approximately 3 min and 5 min, respectively, independent of the addition of oxygen.

Conclusions:

[0283] 18.5 wt. %/1.5 wt. % Kolliphor 407/188 in a 10 mM citrate buffer has thermogelling properties and can be regulated to a pH of 5.5. Oxygenation of the formulation up to 46.85.7 mg/L was achieved without changing the pH or the thermogelling properties. Circulating the formulation several times through the device did not increase the dissolved oxygen levels, but increased the foam-build up.