COMPOSITIONS AND METHODS RELATING TO AMNION

Abstract

This invention relates to a composition comprising amnion homogenate in admixture with a biologically acceptable excipient such as a hydrogel, which may be useful as a therapeutic or cosmetic composition and in particular as a wound dressing, a scar dressing, a topical hydrogel or a topical ointment. This invention also relates to devices such as wound dressings and cosmetic patches comprising the composition and to methods of making and using the composition or device.

Claims

1. A composition comprising amnion homogenate in admixture with a biologically acceptable excipient.

2. The composition of claim 1, wherein the amnion homogenate is human amnion homogenate.

3. The composition of claim 1, wherein the amnion homogenate is free or substantially free of tissues other than amnion.

4. The composition of claim 1, wherein the amnion homogenate comprises homogenised amnion-containing donor tissue and a liquid medium.

5. The composition of claim 4, wherein the amnion homogenate comprises homogenised amnion-containing donor tissue at a concentration of at least 1% (w/v) and no more than 30% (w/v).

6. The composition of claim 1, wherein the composition is free or substantially free of fragments of donor tissue having a maximum dimension of greater than 50 μm.

7. The composition of claim 1, wherein the amnion homogenate is free or substantially free of cells and cell or extracellular matrix fragments.

8. The composition of claim 1, wherein the amnion homogenate comprises no more than 10% by weight of the cells and cell or extracellular matrix fragments present in the donor tissue.

9. The composition of claim 1, wherein the composition comprises at least one of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), keratinocyte growth factor (KGF), transforming growth factor α (TGF-α), transforming growth factor β (TGF-β), nerve growth factor (NGF) and hepatocyte growth factor (HGF), collagen, fibronectin, nidogen and proteoglycans.

10. The composition of claim 1, wherein the composition comprises amnion homogenate at a concentration of at least 1% (w/w), at least 3% (w/w), at least 5% (w/w) or at least 7% (w/w), and no more than 30% (w/w), no more than 25% (w/w), no more than 20% (w/w) or no more than 15% (w/w), and in particular about 10% (w/w).

11. The composition of claim 1, wherein the composition is a therapeutic composition or a cosmetic composition.

12. The composition of claim 11, wherein the composition is a wound dressing, a scar dressing, a topical hydrogel or a topical ointment.

13. The composition of claim 1, wherein the excipient is a hydrophilic polymeric material.

14. The composition of claim 13, wherein the excipient is a polysaccharide based or a polypeptide-based gel.

15. The composition of claim 13, wherein the excipient is a hydrogel.

16. The composition of claim 13, wherein the excipient comprises one or more water soluble polymeric materials and/or one or more water insoluble polymeric materials.

17. The composition of claim 1, wherein the composition has a viscosity at room temperature of at least about 1,000 centipoise.

18. The composition of claim 1, wherein the composition is in the form of a three-dimensional construct comprising a cross-linked gel.

19. The composition of claim 1, wherein the composition further comprises an effective amount of one of more of growth factors, cytokines, viscosity modifiers, surfactants, antioxidants, humectants, wetting agents, lubricants, thickeners, diluents, free-radical scavengers, plasticisers and stabilisers.

20. A device comprising the composition of claim 1 and a component scaffold to which the composition is applied or into which the composition is infused.

21. The device of claim 20, wherein the scaffold comprises an effective amount of one or more of growth factors, cytokines, haemostats, platelets, preservatives or antimicrobial agents.

22. A method of manufacturing a composition comprising the steps of: providing a quantity of placental donor tissue that comprises amnion; homogenising the donor tissue in order to produce an amnion homogenate; and, admixing the amnion homogenate with a biologically acceptable excipient.

23. (canceled)

24. The method of claim 22, wherein the amnion homogenate may comprise homogenised donor tissue at a concentration of at least 1% (w/v) and no more than 30% (w/v).

25. The method of claim 22, wherein the method further comprises centrifuging the amnion homogenate one or more times at a speed of at least 400 g for between 5 and 15 minutes.

26. The method of claim 22, wherein the method further comprises centrifuging the amnion homogenate one or more times at a speed of at least 6,000 g for between 5 and 15 minutes.

27. The method of claim 22, wherein the method further comprises a sterilisation step.

28. The method of claim 27, wherein the amnion homogenate is sterile filtered through a filter having a pore size of no more than 2.5 μm.

29. The method of claim 27, wherein the amnion homogenate is sterile filtered through a filter having a pore size of no more than 50 nm.

30. A kit for formation of a composition or a device, wherein the kit comprises an amnion homogenate and, separately, a biologically acceptable excipient.

31. (canceled)

32. An agent comprising amnion homogenate, wherein the agent is packaged with instructions to admix the agent with a biologically acceptable excipient in order to produce a composition.

33. (canceled)

34. A method of treating a wound, wherein the method comprises a step of topically applying to the wound the composition of claim 1.

35. The method of claim 34, wherein the wound is a surgical incision, burn, diabetic ulcer or pressure sore.

36. The method of claim 34, wherein the method further comprises an initial step of surgically excising a pre-existing a keloid scar.

37-38. (canceled)

39. A method of reducing scar formation from a wound, the method comprising the step of topically applying amnion to the wound.

40. The method of claim 39, wherein the amnion is in the form of a composition comprising amnion homogenate in admixture with a biologically acceptable excipient.

41. The method of claim 39, wherein the method further comprises an initial step of surgically excising a pre-existing a keloid scar.

42. A method of improving the appearance of skin or treating a skin condition, the method comprising the step of topically applying amnion to the skin.

43. The method of claim 42, wherein the amnion is in the form of a composition comprising amnion homogenate in admixture with a biologically acceptable excipient.

44. The method of claim 42, which is a method of treating dry skin, treating erythema, treating acne, reducing acne-associated inflammation, treating sunburn, treating acute sun exposure, improving the smoothness of the skin, improving the appearance of skin blemishes, improving the appearance of skin aging, improving the appearance of striae gravidorum, improving the appearance of cellulite or improving the appearance of acute or chronic sun damage.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0094] FIGS. 1A-D depict the separation of amnion from chorion in donor placental tissue during term-elective Caesarean section surgery;

[0095] FIG. 2 depicts the separated amnion being cut to an appropriate size for application to a Caesarean incision;

[0096] FIG. 3 depicts a closed Caesarean incision to which amnion has been applied;

[0097] FIG. 4 depicts a composite wound dressing applied to a closed Caesarean incision comprising amnion in direct contact with the incision;

[0098] FIGS. 5-7 depict Caesarean scars in which FIGS. 5A, 6A and 7A depict scars from prior Caesarean surgery in three individuals, each of which exhibit keloid scarring, and FIGS. 5B, 6B and 7B depict Caesarean scars following the procedure set out in Example 1 in the same three individuals, none of which exhibit keloid scarring.

EXAMPLES

[0099] The invention is described further below by way of example only, with reference to the accompanying drawings.

Example 1—Topical Application of Amnion Reduces Cosmetic and Keloid Scarring at Caesarean Section

[0100] Amnion retrieved under sterile conditions at the time of term-elective Caesarean section was applied to the surgical incision post-surgery in order to assess its ability to reduce cosmetic and keloid scarring.

[0101] The routine procedure for closing the surgical incision was followed using a 2.0 interrupted vicryl suture to close the fat layer and a 2.0 subcuticular prolene suture to close the skin. In the case of a previous Caesarean section, the old scar was excised completely.

[0102] With reference to FIGS. 1A-D, at the time of Caesarean section, the donor placental tissue (10) was retained and placed on a sterile operating table and the amnion (20) was separated from the chorion (30) by hand.

[0103] With reference to FIGS. 2-4, the amnion (20) was cut to an appropriate size for application to the Caesarean incision (100) and washed in sterile water to remove blood and other contaminants. The amnion (20) was then applied directly to the Caesarean incision (100) and covered with a honeycomb dressing (40). The resulting composite dressing remained in place for 5 days and the subcuticular stitch was then removed at day 6.

[0104] This procedure was followed for more than 50 patients, three of which already had keloid scarring from previous Caesarean surgery (see FIGS. 5-7). At 6 weeks post-surgery, photographs were taken using a Panasonic camera with a ring flash in microscopic mode.

[0105] Visual analogue scales (VAS) were completed by the patients and two independent and blinded clinicians, the results of which demonstrate a significant improvement in scar appearance.

[0106] Referring now to FIGS. 5-7, the three patients with previous keloid scarring had their previous scar (200) excised (see FIGS. 5A, 6A and 7A), and in all three cases the scar that formed following the procedure outlined above (300) did not exhibit any recurrence of keloid scarring (see FIGS. 5B, 6B and 7B).

[0107] These results demonstrate the ability of amnion to reduce cosmetic scarring and prevent the formation or recurrence of keloid scarring.

Example 2—Preparation of Amnion Homogenate for Incorporation into a Hydrogel Wound Dressing

[0108] An amnion homogenate for incorporation into a hydrogel-based dressing was prepared from fresh placenta obtained under sterile conditions. The amnion was separated from the placenta and washed with phosphate buffered saline (PBS) to remove excess residue and blood. The amnion was then homogenised in PBS to provide an amnion homogenate at a final concentration of 10% (w/v).

[0109] The homogenate was then subjected to a low-speed centrifugation to remove larger debris, followed by further centrifugations at up to 8000 g for 10 min at 4° C., in order to remove cell debris from the homogenate, with the supernatant being recovered and the pellet discarded each time.

[0110] The supernatant was then sonicated twice for 5 min each time and the clarified and sonicated supernatant was then filtered through a Millex syringe (0.1 μm) PVDF filter (Millipore Corporation).

[0111] Western blot analysis for prion protein (PrP.sup.Sc) was carried out both pre- and post-filtration as described in the literature (Yunoki M, Tanaka H, Urayama T, Hattori S, Ohtani M, Ohkubo Y, Kawabata Y, Miyatake Y, Nanjo A, Iwao E, Morita M, Wilson E, MacLean C, Ikuta K. Prion removal by nanofiltration under different experimental conditions. Biologicals 2008; 36:27-36).

Example 3—Generation of a Hydrogel Wound Dressing Incorporating Amnion Homogenate

[0112] A novel hydrogel wound dressing was prepared incorporating the amnion homogenate prepared according to Example 2. Equal volumes of a 20% alginate dialdehyde solution in 0.1 M borax was reacted with a 15% solution of gelatin (Balakrishnan B, Mohanty M, Umashankar P R, Jayakrishnan A. Evaluation of an in situ forming hydrogel wound dressing based on oxidized alginate and gelatin. Biomaterials 2005; 26:6335-6342). Alginate dialdehyde, gelatin and borax were all used sterile. Gelation and cross-linking occurs rapidly at room temperature in order to produce a polysaccharide-based hydrogel.

[0113] Amnion homogenate was introduced into the hydrogel 10 minutes post-gelation at a ratio of between 1:1 (v/v) and 1:10 (v/v) and allowed to incorporate into the hydrogel at 37° C., in order to produce a hydrogel wound dressing incorporating amnion homogenate.

[0114] The laminin-derived SIKVAV peptide (Ser-Ile-Lys-Val-Ala-Val) chitosan hydrogel as previously reported (Chen X, Zhang M, Wang X, Chen Y, Yan Y, Zhang L, Zhang L. Peptide-modified chitosan hydrogels promote skin wound healing by enhancing wound angiogenesis and inhibiting inflammation. Am J Transl Res 2017; 9:2352-2362) may also be prepared according to the same method.

[0115] In addition, other peptides dissolved in water or PBS can be used, using standard protocols (Seow W-Y, Salgado G, Birgitte Lane E, Hauser C A E. Transparent crosslinked ultrashort peptide hydrogel dressing with high shape-fidelity accelerates healing of full-thickness excision wounds. Sci Rep 2016; 6:32670).

[0116] The hydrogel wound dressing can readily be applied topically to a wound in order to provide improved wound healing and reduced scarring by exposing the healing wound to the beneficial molecular constituents present in amnion. The hydrogel may also be combined with a scaffold or a second material or multiple materials, such as a membrane or three dimensional structure, in order to produce a composite wound dressing.

Example 4—Wound Dressings Incorporating Amnion Homogenate Reduce Scarring and Improve the Appearance of Skin

[0117] A number of patients (n=50) undergoing Caesarean section were randomly assigned to have their Caesarean incision treated for 5 days with either a standard dressing (control group) or a wound dressings incorporating amnion homogenate (treatment group).

[0118] The patients were reviewed after 6 months and photographs of their Caesarean incisions taken using a panasonic camera with ring flash and microspcopic view. The patients were asked to score the appearance of their Caesarean scars using a visual analogue scale with a score of 100 being the best result a score of 0 being the worst result. The two clinicians were also asked to independently score the the appearance of the Caesarean scars using the same scale.

[0119] Statistical analysis was performed on the resulting scores using the Mann-Whitney non-parametric test. The mean score for the treatment group was 87.68 (range 78-97) while the mean score for the control group was 71.36 (range 62-83). A highly significant improvement was seen in the treatment group using the Mann-Whitney test (p<0.0001). These results demonstrate the efficacy of the amnion dressing in improving the appearance of the Caesarean scar following Caesarean section.

[0120] Additionally, an improvement in the appearance of the skin surrounding the Caesarean scar that was covered by the amnion dressing in the treatment group was also observed. The improvements included improved skin tone and a reduction in visible stretch marks. This appears to indicate that the amnion dressing caused fibroblast proliferation and increased collagen production in the subdermal layers of the skin.

[0121] Accordingly, wound dressings comprising amnion homogenate not only significantly improve the appearance of surgical scars but also appear to have significant cosmetic benefits for the skin, apparently by increasing fibroblast activity and collagen production. A composition incorporating amnion homogenate therefore has the potential to be beneficial in a wide range of cosmetic and skin treatment applications.