Dry biocompatible disintegratable films for delivering particulate egg shell membrane to a wound

Abstract

The invention provides a dry biocompatible film comprising at least one film forming material and particulate egg shell membrane (ESM), wherein said particulate ESM is distributed substantially uniformly in and/or on the film and wherein said film, or portion thereof, disintegrates upon contact with a wound or an exudate thereof. The invention further provides methods for preparing the films of the invention and the uses thereof in methods to promote the healing of wounds.

Claims

1. A dry biocompatible film comprising at least one film forming material and particulate egg shell membrane (ESM), wherein (i) said film does not have a knitted, woven or felted structure, said particulate ESM is distributed uniformly in the film and said film, or portion thereof, disintegrates upon contact with a wound or an exudate thereof, (ii) said at least one film forming material is a cellulose which is not in insoluble fibrous or filamentous form, and (iii) said particulate ESM is, or is formed from, at least one particle of ESM having a mean particle diameter of less than 100 m, and is free from egg shell.

2. The dry biocompatible film of claim 1, wherein the film forming material of the film is dissolvable in water and other aqueous liquids.

3. The dry biocompatible film of claim 1, wherein the film degrades and/or is degraded upon contact with a wound component.

4. The dry biocompatible film of claim 3, wherein said wound component is an enzyme, an inflammatory cell, a wound cell, a wound microorganism, a salt, or a reactive oxygen species.

5. The dry biocompatible film of claim 4, wherein: (i) the enzyme is selected from a matrix metalloproteinase, collagenase, elastase, and chymase, (ii) the inflammatory cell is selected from a macrophage, a neutrophil, and a monocyte.

6. The dry biocompatible film of claim 1, wherein said cellulose is selected from oxidised regenerated cellulose, hydroxyethylcellulose, hydroxypropylcellulose and carboxymethylcellulose.

7. The dry biocompatible film of claim 1, wherein upon contact with a wound or exudate thereof, said film disintegrates in about 3 to about 25 minutes.

8. The dry biocompatible film of claim 1, wherein the film comprises (i) about 5% to about 95% w/w particulate ESM, the remainder being made up of said at least one film forming material and optionally further excipients or active agents; or (ii) about 5% to about 95% w/w of said at least one film forming material, the remainder being made up of particulate ESM and optionally further excipients or active agents.

9. The dry biocompatible film of claim 1, wherein said particulate ESM and the at least one film forming material are present at a ratio of 1:19 to 19:1 ESM:film forming material.

10. The dry biocompatible film of claim 1, wherein said film consists essentially of particulate ESM, a cellulose derivative selected from hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, and mixtures thereof and, optionally, a plasticiser.

11. The dry biocompatible film of claim 10, wherein the ESM and cellulose derivative are present at ratios of 1:19 to 1:1.

12. The dry biocompatible film of claim 1, wherein said film is bioadhesive.

13. The dry biocompatible film of claim 1, wherein said particulate ESM is essentially spherical, prismatoidal, cylindrical, rod-shaped, needle-shaped or fibrous.

14. The dry biocompatible film of claim 13, wherein said particulate ESM has an aspect ratio between a first length dimension and a second length dimension arranged perpendicular thereto of at least 1.5 first length dimension:second length dimension.

15. The dry biocompatible film of claim 1, wherein said ESM is ESM of the eggs of Gallus gallus domesticus.

16. The dry biocompatible film of claim 1, wherein said film further comprises a clinically-useful anti-microbial agent, a growth factor, or an anti-inflammatory agent.

17. A method for preparing a dry biocompatible film as defined in claim 1, said method comprising (i) providing particulate ESM and said at least one film forming material in a liquid suspension, and (ii) drying the suspension, optionally in a mould or on a flat surface, thereby obtaining said film.

18. A method for preparing a dry biocompatible film as defined in claim 1, said method comprising (i) providing a dry biocompatible film formed from at least one film forming material, wherein said film, or portion thereof, disintegrates upon contact with a wound or an exudate thereof, and (ii) applying particulate ESM uniformly to at least one surface of the film.

19. A dry biocompatible film comprising at least one film forming material and particulate ESM obtained or obtainable by the method of claim 17.

20. A composite wound dressing comprising a dry biocompatible film as defined in claim 1 and a further wound dressing adapted such that the film is exposed to the wound or wound exudate when in use.

21. An implantable medical device whose susceptible surfaces, or a portion thereof, carry a dry biocompatible film as defined in claim 1.

22. A method for the delivery of particulate ESM to a wound, said method comprising applying a dry biocompatible film as defined in claim 1 to said wound.

23. A method to promote the healing of a wound, wherein a dry biocompatible film as defined in claim 1 is applied to said wound in an amount sufficient to promote the healing of the wound.

Description

BRIEF DESCRIPTION OF THE DRAWING

(1) FIG. 1 shows the effect of a disintegratable ESM-containing collagen film compared with recombinant growth factors on wound healing in a diabetic mouse model. Positive control (growth factors): triangles; negative control: squares; ESM film: diamonds. All groups: means.e.m.

EXAMPLES

Example 1Preparation of Collagen-ESM and Gelatin-ESM Dry Disintegratable Films

(2) Eggshell membrane was prepared in accordance with WO 2015058790 and then ground into fine particles using a rotational blade mill.

(3) Collagen suspensions were prepared by adding either 1 or 2 g of bovine dermal collagen to 200 mL of 0.5M acetic acid (0.5 to 1 wt % suspensions). These suspensions were mixed using an overhead blender (Ultra Turrax, IKA Works) for 15 minutes in a cooled (7 C.) reaction vessel. ESM powder (2 to 12 g, 1 to 6 wt %) was then added to the suspension and mixed for a further 15 minutes. By this method, a range of ratios of collagen:ESM mixtures were prepared.

(4) Gelatin suspensions were prepared by adding 20 g of gelatin to 200 mL of 0.5 M acetic acid (10 wt % suspension) which was heated to 40 C. on a hot plate and stirred using a magnetic stirrer for 20 min. ESM powder (6 g, 3 wt %) was then added to the suspension and mixed for a further 15 minutes.

(5) The ESM-[collagen/gelatin] suspensions were poured directly onto plastic acetate sheets with a raised edge to prevent run-off and to be able to modify the thickness of the final dried film. These were then dried at 37 C. in a drying oven. The films were then gamma irradiated (25 kGy) for sterilization.

Example 2Properties of Collagen-ESM and Gelatin-ESM Dry Disintegratable Films

(6) The films of Example 1 in the range of 1:1 to 1:6 ESM:collagen or ESM:gelatin were assessed for physical properties. 1:1 films were found to be more manageable with better handling characteristics than 1:6 films. 1:3 collagen films were found to be robust and to have an optimal solubilisation profile.

(7) The amount of ESM per cm.sup.2 was varied by modifying the depth of mixture in the acetate sheet mould. ESM-[collagen/gelatin] suspensions can be poured to depths of 1 mm to 5 mm. For a 1:6 ESM:collagen or ESM:gelatin film this corresponds to 3 mg ESM cm.sup.2 to 15 mg ESM cm.sup.2 and a final thickness of dry film of 0.2 mm to 1 mm. A film made with a 1:6 ESM:collagen ratio corresponding to 3 mg ESM cm.sup.2 in the final dried film (0.2 mm) was optimal. This provided a robust film with appropriate handling characteristics, an adequate solubility profile and an effective concentration of ESM particles for delivery and release to the wound surface.

Example 3Assessment of Dry Disintegratable ESM-Containing Film in Pre-Clinical Effectiveness Model

(8) The effectiveness of the 1:3 collagen ESM film of Example 1 and 2 was shown in the diabetic db/db mouse model of delayed wound healing. In this model, a 1 cm.sup.2 full skin thickness section was excised from the dorsal region. The wound was then covered with a simple protective transparent dressing and decrease in wound area was assessed over time relative to controls. The positive control was the active ingredient of Regranex, a recombinant human platelet derived growth factor based wound healing drug product. The model is challenging for a medical device as the control is a pharmacologically active material which represents maximum wound healing achievable within the model over 20 days.

(9) The healing response of wounds treated with this film formulation was compared to that of wounds exposed to (i) no treatment (negative control) and (ii) positive control treatment (platelet-derived growth factor-BB [rh-PDGF-BB]+Transforming Growth Factor-alpha [rh-TGF-]). In all cases, the wounds were covered post treatment with a transparent dressing, the Bioclusive Film Dressing (BFD), which provided a sterile barrier and protected the wound from infection and physical damage and irritation. The groups are shown in Table 1. The positive control was dosed daily for 6 days at the start of treatment and the ESM film was applied on days 0 and 4.

(10) TABLE-US-00001 TABLE 1 Details of treatment groups used in study Tx Treatment Group (BFD = Bioclusive Film Dressing) Group name 1 BFD only Negative control 2 ESM Film + BFD ESM-Film 3 rh-PDGF-BB [10 g] + rh-TGF- [1 g] + Positive control BFD

(11) The area of a given wound, at a given time point, was expressed as a percentage of the area of that wound immediately after injury (i.e. day 0). The mean percentage wound area remaining (& standard error of mean) was calculated for each group and was displayed graphically. Results are shown in FIG. 1.

(12) The following were shown: 1) Wound closure profiles were found to differ noticeably between the different treatment groups. Wounds in receipt of the growth factor combination (positive control) were found to display the fastest rate of closuredemonstrating close to full wound closure by day 16 post-wounding and significantly greater levels of wound closure relative to untreated wounds from day 8 onwards the study period (p=0.000, Mann-Whitney U test). 2) ESM film treatment resulted in significantly increased wound closure relative to no treatmentfrom day 8 onwards (p0.034, Mann-Whitney U test) 3) The effect of the ESM lags behind that of the PDGF/TGF positive control by approximately 4 days. 4) The effect shown with the ESM film is equivalent to that achieved with ESM powder at the same dosage and dosing frequency (data not shown), demonstrating that the collagen carrier is not having a negative effect on the potency of the ESM material in wound healing.

(13) In conclusion, ESM films of the invention have comparable activity to PDGF based products and the lag in wound healing response is acceptable in the light of reduced concerns of a cancer associated with use of growth factors. Together with the fact that ESM films are easy to apply and may be used with any secondary occlusive dressing currently available, such film represent a cost effective alternative to pharmacologically active wound healing products.

Example 4Carboxymethylcellulose Based Dry Disintegratable Film

(14) TABLE-US-00002 % dry w/w Carboxymethylcellulose 80-90 (representative 88%) Glycerol 2-4 (representative 2%) Particulate ESM 6-18 (representative 10%)

Example 5Hydroxypropylcellulose Based Dry Disintegratable Film

(15) TABLE-US-00003 % dry w/w Hydroxypropylcellulose e 80-90 (representative 84%) Glycerol 2-4 (representative 3%) Particulate ESM 6-18 (representative 13%)

Example 4Hydroxyethylcellulose Based Dry Disintegratable Film

(16) TABLE-US-00004 % dry w/w Hydroxyethylcellulose 80-90 (representative 82%) Glycerol 2-4 (representative 4%) Particulate ESM 6-18 (representative 14%)