Heparan sulphates for use in repair and/or regeneration of skin

Patent metadata

Field	Value
Publication number	US-10471091-B2
Application number	US-201515527972-A
Country	US
Kind code	B2
Filing date	Nov 19, 2015
Priority date	Nov 19, 2014
Publication date	Nov 12, 2019
Grant date	Nov 12, 2019

Abstract

Affinity purification of fibroblast growth factor 2-binding heparan sulphate from porcine mucosa (HS8) is disclosed. Also disclosed is the use of HS8 in repair and regeneration of the skin for treating wounds, burns, ulcers and other skin injuries.

First claim

The invention claimed is:

1. A method of healing a skin wound comprising contacting the skin wound with an effective amount of a pharmaceutical composition comprising heparan sulphate HS8, wherein the heparan sulphate HS8 is capable of specific binding to a peptide or polypeptide consisting of the amino acid sequence YCKNGGF (SEQ ID NO:2) and optionally 1 to 20 additional amino acids at one or both ends of the amino acid sequence SEQ ID NO:2, thereby promoting growth of skin cells.

2. A method of treating a subject having a skin wound, the method comprising administration of a therapeutically effective amount of heparan sulphate HS8 to the subject leading to repair and/or regeneration of skin at the wound, wherein the heparan sulphate HS8 is capable of binding a peptide or polypeptide consisting of the amino acid sequence YCKNGGF (SEQ ID NO:2) and optionally 1 to 20 additional amino acids at one or both ends of the amino acid sequence SEQ ID NO:2.

3. The method of claim 1 wherein the skin wound is a skin burn, ulcer, excisional wound, cut, stab or puncture wound.

4. A method of skin grafting, skin reconstruction, or skin plastic surgery comprising contacting skin at or adjacent the region of grafting, reconstruction or plastic surgery with an effective amount of a pharmaceutical composition comprising heparan sulphate HS8, wherein the heparan sulphate HS8 is capable of specific binding to a peptide or polypeptide consisting of the amino acid sequence YCKNGGF (SEQ ID NO:2) and optionally 1 to 20 additional amino acids at one or both ends of the amino acid sequence SEQ ID NO:2, thereby promoting growth of skin cells.

5. The method of claim 1 wherein the heparan sulphate HS8 or pharmaceutical composition is formulated for topical or transdermal administration.

6. The method of claim 1 wherein the heparan sulphate HS8 or pharmaceutical composition is formulated as a gel, spray, paste, ointment, cream, lotion, salve, oil, aqueous solution, suspension, dispersion, patch, adhesive plaster, bandage, dressing, depot, or reservoir.

7. The method of claim 1 wherein the method further comprises administration of a growth factor and/or mesenchymal stem cells.

8. The method of claim 1 wherein the heparan sulphate HS8 or pharmaceutical composition is formulated as a combined preparation with a growth factor and/or with mesenchymal stem cells.

9. The method of claim 1 wherein the heparan sulphate HS8 is provided in isolated or substantially purified form.

10. The method of claim 1 wherein the heparan sulphate HS8 is capable of binding a peptide or polypeptide comprising the amino acid sequence GHFKDPKRLYCKNGGF (SEQ ID NO: 1).

11. The method of claim 1 wherein following digestion with heparin lyases I, II and III and then subjecting the resulting disaccharide fragments to capillary electrophoresis analysis the heparan sulphate HS8 has a disaccharide composition comprising:

Disaccharide
Normalised weight percentage

ΔUA,2S-GlcNS,6S
12.7 ± 3.0 

ΔUA,2S-GlcNS
7.2 ± 2.0

ΔUA-GlcNS,6S
15.5 ± 3.0 

ΔUA,2S-GlcNAc,6S
6.5 ± 2.0

ΔUA-GlcNS
15.7 ± 3.0 

ΔUA,2S-GlcNAc
1.0 ± 0.5

ΔUA-GlcNAc,6S
8.9 ± 3.0

ΔUA-GlcNAc
32.5 ± 3.0. 

12. The method of claim 1 wherein the heparan sulphate HS8 is obtained by a method comprising:
(i) providing a solid support having polypeptide molecules adhered to the support, wherein the polypeptide comprises a heparin-binding domain comprising the amino acid sequence YCKNGGF;
(ii) contacting the polypeptide molecules with a mixture comprising glycosaminoglycans such that polypeptide-glycosaminoglycan complexes are allowed to form;
(iii)partitioning polypeptide-glycosaminoglycan complexes from the remainder of the mixture;
(iv)dissociating glycosaminoglycans from the polypeptide-glycosaminoglycan complexes; and
(v) collecting the dissociated glycosaminoglycans.

13. The method of claim 2 wherein the heparan sulphate HS8 is capable of binding a peptide or polypeptide comprising the amino acid sequence GHFKDPKRLYCKNGGF (SEQ ID NO:1).

14. The method of claim 2 wherein following digestion with heparin lyases I, II and III and then subjecting the resulting disaccharide fragments to capillary electrophoresis analysis the heparan sulphate HS8 has a disaccharide composition comprising:

Disaccharide
Normalised weight percentage

ΔUA,2S-GlcNS,6S
12.7 ± 3.0 

ΔUA,2S-GlcNS
7.2 ± 2.0

ΔUA-GlcNS,6S
15.5 ± 3.0 

ΔUA,2S-GlcNAc,6S
6.5 ± 2.0

ΔUA-GlcNS
15.7 ± 3.0 

ΔUA,2S-GlcNAc
1.0 ± 0.5

ΔUA-GlcNAc,6S
8.9 ± 3.0

ΔUA-GlcNAc
32.5 ± 3.0. 

15. The method of claim 2 wherein the heparan sulphate HS8 or pharmaceutical composition is formulated for topical or transdermal administration.

16. The method of claim 2 wherein the skin wound is a skin burn, ulcer, excisional wound, cut, stab or puncture wound.

17. The method of claim 4 wherei

Assignees

• Agency Science Tech & Res

Inventors

• Cool Simon
• Nurcombe Victor

Classifications

• A61L26/0023

  Polysaccharides · CPC title

• A61K31/727 Primary

  Heparin; Heparan · CPC title

• A61K38/1825

  Fibroblast growth factor [FGF] · CPC title

• C08B37/0075

  Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof · CPC title

• A61K9/0014

  Skin, i.e. galenical aspects of topical compositions (non-active ingredients are additionally classified in A61K47/00; A61K9/0009, A61K9/0021, A61K9/7015, A61K9/7023 take precedence; cosmetic preparations A61K8/00, A61Q; preparations for wound dressings or bandages A61L26/00) · CPC title