PDGF-B /PDGF-BB binding variants of heparan sulfates

Abstract

Affinity purification of platelet-derived growth factor-binding heparan sulphate from porcine mucosa (HS6) is disclosed. Also disclosed is the use of HS6 in repair and regeneration of the skin for treating wounds, burns, ulcers and other skin injuries.

Claims

1. A method of treating a disease, condition or injury to skin in a patient, the method comprising administration of a therapeutically effective amount of heparan sulphate HS6 to the patient leading to repair and/or regeneration of the skin, wherein the heparan sulphate HS6 is capable of binding a peptide or polypeptide having, or consisting of, the amino acid sequence RAKTPQTRVTIRTVRVRRPPKGKHRKFKHTHDK (SEQ ID NO:1); wherein following digestion with heparin lyases I, II, and III and then subjecting the resulting disaccharide fragments to HPLC analysis, the heparan sulphate HS6 has a disaccharide composition comprising: TABLE-US-00005 Disaccharide Normalised weight percentage UA,2S-GlcNS,6S 12.9 3.0 UA,2S-GlcNS 6.8 2.0 UA-GlcNS,6S 11.7 3.0 UA-GlcNS 24.5 3.0 UA,2S-GlcNAc 0.5 0.5 UA-GlcNAc,6S 12.1 3.0 UA-GlcNAc 31.6 3.0.

2. The method of claim 1 wherein the method comprises administering the heparan sulphate HS6 to tissue at or surrounding a wound or location on the patient's body at which regeneration or repair of skin is required.

3. The method of claim 1, wherein the method further comprises administering PDGF-B, PDGF-BB or a heterodimer comprising PDGF-B to the patient.

4. A method of treating a disease, condition or injury to skin in a patient, the method comprising surgically implanting a biocompatible implant or prosthesis, which implant or prosthesis comprises a biomaterial and heparan sulphate HS6, into tissue of the patient at or surrounding the site of the disease, condition or injury leading to repair and/or regeneration of the skin, wherein the heparan sulphate HS6 is capable of binding a peptide or polypeptide having, or consisting of, the amino acid sequence RAKTPQTRVTIRTVRVRRPPKGKHRKFKHTHDK (SEQ ID NO:1); wherein following digestion with heparin lyases I, II, and III and then subjecting the resulting disaccharide fragments to HPLC analysis, the heparan sulphate HS6 has a disaccharide composition comprising: TABLE-US-00006 Disaccharide Normalised weight percentage UA,2S-GlcNS,6S 12.9 0.5 UA,2S-GlcNS 6.8 0.2 UA-GlcNS,6S 11.7 0.5 UA-GlcNS 24.6 1.5 UA,2S-GlcNAc 0.5 0.2 UA-GlcNAc,6S 12.1 0.5 UA-GlcNAc 31.6 0.8.

5. The method of claim 1, wherein following digestion with heparin lyases I, II, and III and then subjecting the resulting disaccharide fragments to HPLC analysis, the heparan sulphate HS6 has a disaccharide composition comprising: TABLE-US-00007 Disaccharide Normalised weight percentage UA,2S-GlcNS,6S 12.9 0.5 UA,2S-GlcNS 6.8 0.2 UA-GlcNS,6S 11.7 0.5 UA-GlcNS 24.6 1.5 UA,2S-GlcNAc 0.5 0.2 UA-GlcNAc,6S 12.1 0.5 UA-GlcNAc 31.6 0.8.

6. The method of claim 1, wherein following digestion with heparin lyases I, II, and III and then subjecting the resulting disaccharide fragments to HPLC analysis, the heparan sulphate HS6 has a disaccharide composition comprising: TABLE-US-00008 Disaccharide Normalised weight percentage UA,2S-GlcNS,6S 12.8 0.5 UA,2S-GlcNS 6.8 0.2 UA-GlcNS,6S 11.6 0.5 UA-GlcNS 24.6 0.5 UA,2S-GlcNAc 0.5 0.1 UA-GlcNAc,6S 12.0 0.4 UA-GlcNAc 31.6 0.5.

7. The method of claim 1, wherein the heparan sulphate HS6 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 having the amino acid sequence RAKTPQTRVTIRTVRVRRPPKGKHRKFKHTHDK (SEQ ID NO:1); (ii) contacting the solid support with a mixture comprising glycosaminoglycan 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.

8. The method of claim 7, wherein the mixture comprising glycosaminoglycans is a heparan sulphate preparation obtained from porcine mucosa (HSPM).

9. The method of claim 1, wherein the disease, condition or injury to tissue is a skin wound and the method comprises administration of a therapeutically effective amount of heparan sulphate HS6 to the subject leading to repair and/or regeneration of skin at the wound.

10. The method of claim 9, wherein the skin wound is a skin burn, ulcer, excisional wound, cut, stab or puncture wound.

11. The method of claim 9, wherein the method involves skin graft healing, skin reconstruction, or skin plastic surgery.

12. The method of claim 9, wherein the heparan sulphate HS6 is formulated for topical or transdermal administration.

13. The method of claim 9, wherein the method further comprises administration of a growth factor.

14. The method of claim 9, wherein the heparan sulphate HS6 is formulated as a combined preparation with a growth factor.

15. The method of claim 1, wherein the heparan sulphate HS6 is provided in isolated or substantially purified form.

16. The method of claim 4, wherein following digestion with heparin lyases I, II and III and then subjecting the resulting disaccharide fragments to HPLC analysis the heparan sulphate HS6 has a disaccharide composition comprising: TABLE-US-00009 Disaccharide Normalised weight percentage UA,2S-GlcNS,6S 12.9 0.5 UA,2S-GlcNS 6.8 0.2 UA-GlcNS,6S 11.7 0.5 UA-GlcNS 24.6 1.5 UA,2S-GlcNAc 0.5 0.2 UA-GlcNAc,6S 12.1 0.5 UA-GlcNAc 31.6 0.8.

17. The method of claim 4, wherein the heparan sulphate HS6 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 having the amino acid sequence RAKTPQTRVTIRTVRVRRPPKGKHRKFKHTHDK (SEQ ID NO:1); (ii) contacting the solid support with a mixture comprising glycosaminoglycan 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.

18. The method of claim 1, wherein the heparan sulphate HS6 is capable of binding PDGF-B, PDGF-BB, or both PDGF-B and PDGF-BB.

19. The method of claim 4, wherein the heparan sulphate HS6 is capable of binding PDGF-B, PDGF-BB, or both PDGF-B and PDGF-BB.

20. The method of claim 13, wherein the growth factor is PDGF-B or PDGF-BB, or a heterodimer comprising PDGF-B.

21. The method of claim 1, wherein the heparan sulphate HS6 is capable of stimulating the proliferation of dermal fibroblasts and keratinocytes.

22. The method of claim 4, wherein the heparan sulphate HS6 is capable of stimulating the proliferation of dermal fibroblasts and keratinocytes.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

(2) FIG. 1. Chart showing PDGF-BB binding to heparin.

(3) FIG. 2. (A) Illustration of PDGF-B:HS binding assay; (B) Chart showing PSGF-B binds to heparin and HS.sup.PM.

(4) FIG. 3. Charts showing binding of heparin dp4-24 to PDGF-BB. (A) PDGF-BB+dp4, (B) PDGF-BB+dp8, (C) PDGF-BB+dp12, (D) PDGF-BB+dp16, (E) PDGF-BB+dp20, (F) PDGF-BB+dp24.

(5) FIG. 4. Chart showing that binding of PDGF-BB increases with chain length.

(6) FIG. 5. Charts showing that N-sulfation is crucial for PDGF-BB binding. Effect of (A) 2-O-desulfation; (B) 6-O-desulfation; (C) N-desulfation; (D) N-desulfated reN-Acetylated.

(7) FIG. 6. Chart showing that N-sulfation is crucial for PDGF-BB binding.

(8) FIG. 7. Amino acid sequence of PDGF-B showing the heparin binding domain of SEQ ID NO:1 in bold. PDGF-B peptide (SEQ ID NO:1) was selected based on the highly basic region of PDGF-BB sequence. Note the basic amino acids: Arginine (R), Lysine (K), Histidine (H).

(9) FIG. 8. Chromatogram from PDGF-B peptide coupled HiTrap Streptavidin column.

(10) FIG. 9. Illustration of HS6 purification by affinity chromatography.

(11) FIG. 10. Chromatogram showing HS6+ (bound fraction) and HS6 (unbound fraction).

(12) FIG. 11. Chart showing PDGF-BB binding to HS fractions. HS6+ binds to PDGF-BB.

(13) FIG. 12. Charts showing HS6+ interaction with growth factors. (A) Interaction with BMP-2, (B) Interaction with FGF2, (C) Interaction with VEGF-165.

(14) FIG. 13. Charts showing PDGF-BB and HS6+ dose dependently increase HDF proliferation. (A) PDGF-BB dosing, (B) HS6+ dosing, (C) PDGF-BB and HS6 dosing.

(15) FIG. 14. Chart showing HDF Proliferation with 5 ng/mL PDGF-BB+20 g/mL HS. PDGF-BB and HS6+ increase HDF proliferation.

(16) FIG. 15. Chart showing effect of PDGF-BB dosing in HDF migration studies in a Scratch Assay. PDGF-BB induces HDF migration.

(17) FIG. 16. Chart showing effect of HS6+ dosing in HDF migration studies in a Scratch Assay.

(18) FIG. 17. Chart showing PDGF-BB and HS6+ increase migration of HDFs in a Scratch Assay. 5 ng/mL PDGF-BB+20 g/mL HS fractions.

(19) FIG. 18. HS6+ increases phosphor-PDGFR in HDFs. (A) Photograph showing PDGFR phosphorylation in response to PDGF-BB, (B) Photograph showing PDGFR phosphorylation in response to HS6, (C) Chart showing PDGFR phosphorylation in HDF in response to PDGF-BB, PDGF-BB+HS6+, PDGF-BB+HS6.

(20) FIG. 19. Representation of nested wounds in pig skin model.

(21) FIG. 20. Photographs showing wound healing of 6 mm wounds at day 1, 3, 7 with CMC or HS6+.

(22) FIG. 21. Photographs showing wound healing of 10 mm wounds at day 9, 13, 15 with CMC or HS6+.

(23) FIG. 22. Differences in hematoxylin and eosin (H&E) staining at day 7. (A) Illustration of H&E staining at day 7 in CMC control and in HS6+ treatment group. (B) Graphical representation of differences in H&E staining, shown as area of granulation tissue at day 7. Each point represents a separate wound treated with either CMC carrier alone or CMC carrier containing HS6+. Line represents median score

(24) FIG. 23. Decreased cellular infiltrates with HS6+. (A) Illustration of cellular infiltration following HS6+ treatment. (B) Graphical representation of cellular infiltrates at day 7 in CMC control and HS6+ treatment group. Each point represents a separate wound treated with either CMC carrier alone or CMC carrier containing HS6+. Line represents median score.

(25) FIG. 24. Photographs showing presence of blood vessels following treatment with (A) CMC control or (B) HS6+.

(26) FIG. 25. HPLC-SEC-RI chromatograms of heparin lyase digests of HS6+, HS6 and Celsus HS.

(27) FIG. 26. Table showing proportion of disaccharides in heparin lyase digests of the HS samples as determined by HPLC-SEC-RI.

(28) FIG. 27. Table showing disaccharide composition of heparin lyase digested HS samples: Celsus HS, HS6+, HS6.

(29) FIG. 28. Chart showing disaccharide composition of HS-6 samples (SAX-HPLC of heparan lyase digests). UV SAX HPLC analysis of the disaccharide composition of heparin lyase digests of heparin sulphate preparations: Celsus HS, HS6+, HS6.

(30) FIG. 29. Charts showing proliferative effect of PDGF-BB and HS6 on human dermal fibroblasts: (A) PDGF-BB (1, 2.5, 5, 10, 25 ng/ml), (B) HS6 (0.25, 0.5, 1, 2.5, 5, 10, 25 g HS6), (C) 2.5 ng PDGF-BB+HS6 (0, 0.25, 0.5, 1, 2.5, 5, 10, 25 g).

(31) FIG. 30. Charts showing effect of PDGFR and FGFR inhibition. (A) Effect of PDGFR inhibitor JNJ-10198409; (B) Effect of FGFR1 inhibitor SU5402.

(32) FIG. 31. Charts showing results of scratch wound assay on keratinocytes (N-TERT/1): (A) PDGF-BB (0, 1, 5, 10, 25 ng); (B) HS6 (0, 0.25, 0.5, 1, 2.5, 5, 10, 25 g).

(33) FIG. 32. Chart showing result of scratch wound assay on human dermal fibroblasts.

(34) FIG. 33. Photographs showing results of co-immunoprecipitation studies investigating binding of PDGF-BB to Fc-PDGFR. HS6+ increases association of PDGF-BB with Fc-PDGFR.

EXAMPLES

Example 1: Surface Plasmon Resonance (SPR)

(35) 1. Heparin was biotinylated using N-hydroxysuccinimide-biotin (NHS-biotin) (Pierce). 2. Streptavidin (SA) sensor chip (GE Healthcare) was coated with biotinylated heparin using an in-built immobilisation protocol on Biacore T100 (GE Healthcare), with a targeted 40 response units (RUs). 3. Recombinant PDGF-BB (R&D) was prepared at 12.5 nM to 800 nM concentrations in HBS-EP-0.1 running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.1% Tween-20 (v/v) pH 7.4). 4. PDGF-BB samples were then injected over the heparin-coated surface at a flow rate of 30 L/min for 120 s, with HBS-EP-0.1 being subsequently passed over the surface for a further 600 s to monitor PDGF-BB dissociation. After dissociation, the sensor surface on the chip was regenerated by 2 washes of 2 M NaCl injected at 30 L/min for 60 s.
Results:

(36) PDGF-BB binds to the heparin coated chip in a dose dependent manner (FIG. 1).

(37) 50 nM PDGF-BB was selected for use in the competition experiments.

Example 2: PDGF-B Peptide Binds to Heparin and HS.SUP.PM

(38) GAG Protocol:

(39) 1. GAG binding plate (Iduron) was coated with 200 L/well of 5 g/ml Heparin or heparan sulphate from porcine mucosa (HS.sup.PM) prepared in Standard Assay Buffer (SAB, 100 mM NaCl, 50 mM Sodium Acetate, 0.2% (v/v) Tween-20, pH 7.2) overnight. Plates are protected from light at every incubation step. 2. Plates were washed thrice with SAB. 3. Add 250 L/well of 0.4% (w/v) gelatin blocking solution and incubate at 37 C. for 1 hour. 4. Plates were washed thrice with SAB. 5. Add 200 L/well of biotinylated PDGF-B peptide (SEQ ID NO:1) prepared at 100, 200 and 400 ng/mL in blocking solution and incubate at 37 C. for 1 hour. 6. Plates were washed thrice with SAB. 7. Add 200 L/well of 220 ng/mL ExtrAvidin prepared in blocking solution and incubate at 37 C. for 30 minutes. 8. Plates were washed thrice with SAB. 9. Add 200 L/well of Development Reagent SigmaFAST p-Nitrophenyl phosphate prepared in distilled water and incubate at room temperature for 40 minutes. 10. Read plate at 405 nm within one hour.
Results:

(40) Biotinylated PDGF-B peptide binds to both Heparin and HS.sup.PM (FIG. 2).

(41) PDGF-B peptide will bind to the highly sulfated Heparin more than HS.sup.PM.

(42) Results also shows that there are PDGF-B binding HS.sup.PM fractions.

(43) PDGF-B peptide was selected for use in isolating PDGF-BB binding HS fraction (HS6).

Example 3: Binding of Heparin Dp4-24 to PDGF-BB

(44) Competition Protocol:

(45) 1. Heparin and heparin derivatives (Iduron), including dp fragments and desulfated heparins, were prepared at 5 g/ml and 10 g/ml in HBS-EP-0.1 running buffer. 2. 50 nM PDGF-BB was prepared in HBS-EP-0.1 running buffer. 3. PDGF-BB and heparin/heparin derivatives were mixed. 4. PDGF-BB samples were then injected over the heparin-coated surface at a flow rate of 30 L/min for 120 s, with HBS-EP-0.1 being subsequently passed over the surface for a further 600 s to monitor PDGF-BB dissociation. After dissociation, the sensor surface on the chip was regenerated by 2 washes of 2 M NaCl injected at 30 L/min for 60 s.
Results:

(46) Binding of PDGF-BB increases with longer dp fragments (FIGS. 3 and 4).

(47) The increased affinity that PDGF-BB has to the dp fragments results in decreased PDGF-BB binding to the heparin coated chip.

Example 4: N-Sulfation is Crucial for PDGF-BB Binding

(48) The competition protocol from Example 3 was used, using the following heparin and heparin derivatives: Desulfated heparin (Iduron): heparin that has been digested with enzymes to remove specific sulfations 2-O-deS: 2-O-desulfated heparin 6-O-deS: 6-O-desulfated heparin N-deS: N-desulfated heparin N-deS/R: N-desulfated Re-acetylated heparin
Results:

(49) N-sulfation is crucial for PDGF-BB binding (FIGS. 5 and 6).

Example 5: HS6 Purification

(50) We investigated the purification of a new PDGF-BB binding heparan sulphate (HS) from commercially available Porcine Celsus Heparan sulphate sources (also called HS.sup.PM) suitable for scale up of heparan sulphate (HS) preparations that can be readily used in the clinic.

(51) The Heparin binding domain (HBD) peptide sequence RAKTPQTRVTIRTVRVRRPPKGKHRKFKHTHDK (SEQ ID NO: 1). from PDGF-B was selected and used to purify specific HS species capable of binding to PDGF-B and PDGF-BB.

(52) Protocol:

(53) 1. 4 mg of biotinylated PDGF-BB peptide ((Biotin)K-AHX-RAKTPQTRVTIRTVRVRRPPKGKHRKFKHTHDK [SEQ ID NO:2; AHX=6 aminohexanoic acid]) was coupled to HiTrap streptavidin HP column (GE Healthcare) at 1 mg/mL in low salt buffer (20 mM phosphate, 150 mM NaCl, pH 7.2) (FIG. 8). 2. HS.sup.PM (Celsus Laboratories Inc.) was dissolved at 1 mg/mL in low salt buffer. 3. For each run, 3 mL of 1 mg/mL HS.sup.PM was injected at 0.2 mL/min. 4. Column was washed in low salt buffer to elute the unbound material, until the baseline absorbance at 232 nm reached zero. 5. Bound HS was eluted with high salt buffer (20 mM phosphate, 1.5 M NaCl, pH 7.2) (FIG. 9). 6. Peak fractions of unbound and bound material were collected separately and freeze-dried (FIG. 10). 7. Samples were desalted on a HiPrepTM 26/10 desalting column (GE Healthcare) at a flow rate of 10 mL/min, freeze dried again and stored in a desiccator cabinet.
Results:

(54) HS6=HS fraction that does not bind to PDGF-B peptide in low salt buffer.

(55) HS6+ (also called HS6)=HS fraction that binds to PDGF-B peptide in low salt buffer and eluted with high salt buffer.

Example 6: Analysis of HS6

(56) Samples Used

(57) Dry weight of HS preparations:

(58) TABLE-US-00003 HS6 +ve 2.13 mg HS6 ve 2.1 mg

(59) Included in this study as a control:

(60) TABLE-US-00004 Celsus HS 10697 2.0 mg
Digestion of HS Samples with Heparin Lyase Enzymes

(61) Heparan sulfate (HS) was from Celsus Laboratories Inc. (HO-03103, Lot # HO-10697). Heparin lyase I (Heparitinase, EC 4.2.2.8, also known as hepartinase I), heparin lyase II (heparitinase II, no EC number assigned) and heparin lyase III (heparinase, EC 4.2.2.7, also known as heparitinase III) were obtained from Ibex Technologies, Quebec, Canada. The enzymes, supplied as solutions (0.5 IU/50 L) stabilised in 5% sucrose, were diluted with BSA (0.1% w/v solution in 50 mM sodium phosphate buffer; pH 7.1 containing 100 mM NaCl for heparin lyase I, pH 7.1 for heparin lyase II and pH 7.6 for heparin lyase III) and aliquots (5 mIU/5 L) were stored frozen (80 C.) until needed.

(62) HS samples were solubilised in water (1 mg/mL) and aliquots (21 mL) of each were freeze-dried for analysis. The HS samples were digested to di- and oligosaccharides by the sequential addition of heparin lyase enzymes as described below (modified from the method of Brickman et al., 1998, Structural modification of fibroblast growth factor-binding heparan sulfate at a determinative stage of neural development. The Journal of Biological Chemistry, 273, 4350-4359). The dry HS samples (1 mg) were re-solubilised in 100 mM sodium acetate containing 2 mM calcium acetate (pH 7.0, 470 L) and heparin lyase I (5 L; 5 mIU) was added to each sample. The samples were incubated (37 C., 2 h) with gentle mixing on a rotating wheel (11 rpm). Heparin lyase III (5 L; 5 mIU) was added and the samples were incubated for a further 1 h (as above). Heparin lyase II (5 L; 5 mIU) was then added and the samples were incubated as above, for a further 18 h. Finally, aliquots (5 L; 5 mIU) of all three heparin lyases were added simultaneously and the samples were incubated for a further 24 h. The enzyme digestion was terminated by heating (100 C., 5 min).

(63) HPLC-SEC-RI of Digested HS Samples

(64) The HPLC-SEC chromatograms were obtained using two Superdex Peptide 10/300 GL columns (30010 mm, GE Healthcare, Buckinghamshire, UK) in series, on a Waters 2690 Alliance system with a Waters 2410 refractive index detector (range 64). The do/dc for quantification from the RI was set at 0.129 (Knobloch and Shaklee, 1997). Samples (2 mg/mL) were injected (50 L; 100 g) and eluted with 50 mM ammonium acetate (0.5 mL/min) at room temperature. Heparin oligosaccharide standards (Iduron Ltd, Manchester, UK and Dextra Laboratories Ltd, Reading, UK), previously run under the same conditions, were used for identification purposes. Run times for these columns were 120 min. Data was collected and analysed using DAWN Astra software (Version 4.73.04, Wyatt Technology Corp., Santa Barbara, Calif., USA).

(65) The four late eluting signals correspond to the trisulfated disaccharides (28.8 mL), disulfated disaccharides (30.7 mL), mono- and un-sulfated disaccharides (32.8 mL) and sucrose (34.3 mL; from the enzyme solution), respectively (see FIG. 25). The large signal at approximately 36.3 mL is from buffer salts. Larger oligosaccharides and material elute before approximately 28 mL. The peak eluting at approximately 42-43 mL is NaCl.

(66) For Celsus HS 10697 (starting material) the disaccharides and >dp2 material accounted for approximately 75 g of the 100 g injected onto the columns. For the HS-6 samples this value was lower (67 g for HS-6 +ve and between 48-58 g for HS-6 ve). This reflects the increased NaCl contents of the HS-6 samples compared to Celsus HS 697. There was also a reasonably large unidentified peak (37 mL) present in the HS-6 ve samples.

(67) Disaccharide Compositional Analysis by HPLC

(68) Twelve disaccharide standards, derived from the digestion of high-grade porcine heparin by bacterial heparinases, were purchased from Iduron Ltd, Manchester, UK. A stock solution of each disaccharide standard was prepared by dissolving the disaccharide in water (1 mg/mL).

(69) To determine the calibration curves for the disaccharide standards, a standard mix containing 20 g/mL of each of the disaccharides was prepared from the stock solutions. From this twelve disaccharide standard mix a dilution series containing 20, 10, 5, 2.5, 1.25, 0.625 and 0.3125 g/mL of each disaccharide was prepared.

(70) The HS digests (2 mg/mL) were diluted with water to give 100 g/mL solutions and then filtered (hydrophilic PTFE disposable syringe filter units, 0.2 m, 13 mm, Advantec). The HPLC separation conditions were based on those of Skidmore et al. (2010). The analyses were performed on an Agilent 1260 Infinity liquid chromatography system (Agilent Technologies) with an Agilent 1260 MWD VL detector monitored at 232 nm. HS-derived disaccharides were separated on a ProPac PA1 column (Thermo Scientific, 4 mm250 mm) with a guard column. Gradient elution was performed using a binary solvent system. Eluent A was water at pH 3.5 (adjusted using HCl), and eluent B was 2 M NaCl at pH 3.5 (adjusted with HCl). The gradient program was as follows: 100% A from 0-1 min, then 0-35% B from 1-32 min, then 35-65% B from 32-47 min, then 100% B from 47-57 min, then 100% A from 57-60 min. The injection volume was 50 L. The column was eluted at a flow rate of 1.0 mL/min and maintained at 40 C.

(71) Disaccharides present in the HS digests were identified from their elution times by comparison with the elution times of the disaccharides in the twelve disaccharide standard mixes. A dilution series of the twelve disaccharide standard mix was run with the digested HS samples. The detector response was linear for all of the disaccharide standards at all of the concentrations tested. These linear calibration curves were used to calculate the proportions of the various disaccharides present in the digests.

(72) The HS samples (Celsus HS 10697, HS6 +ve and HS6 ve) were all digested in duplicate. The duplicate digests of all the samples were injected twice in the HPLC (total of four analyses for each sample). The disaccharide compositions (normalised % disaccharides) determined from each HPLC run are shown in FIG. 27, together with the mean averages. FIG. 28 shows the mean average normalised percentage of each disaccharide in each of the digests. The error intervals were determined using student's t-distribution with confidence limits set at 95.

(73) The proportions of the seven disaccharides in the Celsus HS digests were similar to those found in previous analyses of Celsus HS 10697. The predominant disaccharides were UA-GlcNAc and UA-GlcNS, with smaller proportions of UA-GlcNAc,6S, UA-GlcNS,6S and UA,2S-GlcNS,6S, a small proportion of UA,2S-GlcNS and trace amounts of UA,2S-GlcNAc.

(74) HS-6 has slightly less UA-GlcNAc and UA-GlcNS than Celsus HS 10697 and is slightly enriched in UA-GlcNS,6S and UA,2S-GlcNS,6S, though the differences are only 1-2%. The non-retained HS-6 contains more UA-GlcNAc and UA-GlcNS than HS-6 and Celsus HS 10697 and less UA-GlcNS,6S and UA,2S-GlcNS,6S.

Example 7: HS6+ Binds to PDGF-BB

(75) GAG Protocol:

(76) 1. GAG binding plate (Iduron) was coated with 200 L/well of 5 g/ml HS6+ and HS6 fractions prepared in Standard Assay Buffer (SAB, 100 mM NaCl, 50 mM Sodium Acetate, 0.2% (v/v) Tween-20, pH 7.2) overnight. Plates are protected from light at every incubation step. 2. Plates were washed thrice with SAB. 3. Add 250 L/well of 0.4% (w/v) gelatin blocking solution and incubate at 37 C. for 1 hour. 4. Plates were washed thrice with SAB. 5. Add 200 L/well of 125, 250 and 500 ng/mL PDGF-BB (R&D) prepared in blocking solution and incubate at 37 C. for 2 hour. 6. Plates were washed thrice with SAB. 7. Add 200 L/well of 250 ng/mL biotinylated anti-PDGF-BB antibody (R&D) prepared in blocking solution and incubate at 37 C. for 1 hour. 8. Plates were washed thrice with SAB. 9. Add 200 L/well of 220 ng/mL ExtrAvidin prepared in blocking solution and incubate at 37 C. for 30 minutes. 10. Plates were washed thrice with SAB. 11. Add 200 L/well of Development Reagent SigmaFAST p-Nitrophenyl phosphate prepared in distilled water and incubate at room temperature for 40 minutes. 12. Read plate at 405 nm within one hour.
Results:

(77) HS6+ selectively binds to PDGF-BB compared to HS6 (FIG. 11).

Example 8: HS6+ Also Interacts with Other Growth Factors

(78) Whilst HS6+ is isolated based on affinity for a peptide sequence contained within the amino acid sequence of PDGF-BB, HS6+ is also able to interact with other pro-healing factors present in wound sites such as BMP-2 and FGF-2, and displays some association with VEGF165 (FIG. 12). This binding may simply be based on the increased charge density of HS6+ and not related to sequence information. What is interesting to note is that HS6+ binds with high affinity to PDGF-BB whilst the flow through fraction HS6 does not bind much PDGF-BB, yet the HS6 fraction still contains chains with affinity for BMP-2, FGF-2 but not VEGF165 (FIG. 12). Thus HS6+ may generate its affect through a range of factors making it particularly useful for wound healing.

Example 9: PDGF-BB and HS6+ Dose Dependent Increase in HDF Proliferation

(79) Proliferation Protocol: 1. Thaw human dermal fibroblast (HDF, Cascade Biologics) into a T75 in maintenance medium at 510.sup.3 cells/cm.sup.2 (-MEM, 10% FCS, P/S) at 37 C. 5% CO.sup.2 humidified incubator. 2. At 70-80% confluence, wash cells in PBS twice, add 1 ml of 0.125% trypsin and incubate for 3 mins. Tap the dish and stop the trypsin by adding 5 ml of medium. Centrifuge cells down at 180 g for 7 mins. 3. Count total number of cells. 4. Seed cells at 500 l of 3000 cells/cm.sup.2 cell suspension per well and place the plates into the incubator for 24 hrs. 5. Change media to -MEM, 1% FCS, P/S media for 24 hrs. 6. Treat cells with PDGF-BB and HS fractions. 7. After 72 hrs, wash cells with PBS and add 100 ul of trypsin, incubate for 3 mins. 8. Add 100 ul of Guava Viacount dye mastermix into each well of trypsinize cells. 9. Transfer total volume of 200 ul of cells per well into a 96 well Guava plate.
Results:

(80) There is a PDGF-BB dose dependent increase in proliferation of HDF (FIG. 13). 10 ng/mL was the optimal concentration for proliferation.

(81) Higher concentration of PDGF-BB results in a decreased viability of HDF.

(82) A sub-optimal PDGF-BB concentration at 5 ng/mL was used for future proliferation experiments.

(83) Similarly, an increase in HS6+ concentration results in a gradual increase in proliferation. Proliferation plateaus with the addition of 20 g/mL HS6+ and 5 ng/mL.

(84) 20 g/mL was selected as the HS6+ concentration to be used to compare between the different fractions.

Example 10: PDGF-BB and HS6+ Increases HDF Proliferation

(85) The proliferation protocol described in Example 9 was used.

(86) Results are for 72 h proliferation:

(87) 20 g/mL HS6+ and HS6 alone is able to induce proliferation better than 5 ng/mL PDGF-BB (FIG. 14).

(88) In combination, 20 g/mL HS6+ with 5 ng/mL PDGF-BB increases HDF proliferation compared to HS6 with PDGF-BB (FIG. 14).

(89) Not shown in FIG. 14 is the different morphology of HDF treated with HS6+ and PDGF-BB. Cells look spindle shaped and may be stained for myofibroblast marker such as alpha smooth muscle actin.

(90) The HS6 fraction is able to also enhance the growth of HDFs presumably because it can bind and activate FGF-2 (FIG. 12). The fact that HS6 when combined with PDGF does not produce an additive affect, suggests there is little interaction between these two factors, consistent with FIG. 11.

Example 11: PDGF-BB Induces HDF Migration

(91) Migration Protocol: 1. Seed 10,000 HDF per well in maintenance media, into a 96 well ImageLock plate. 2. After 24 hrs, change media to 1% -MEM 3. Once cells are confluent, scratch the surface using the wound maker and wash once with PBS. 4. Add 100 l of treatment media into each well and place plate into IncuCyte and run for 40 hours. 5. Program is set up to take photo every 2 hours.
Results:

(92) Rate of wound closure increases up to 10 ng/mL PDGF-BB (FIG. 15).

(93) The addition of more than 10 ng/mL PDGF-BB does not speed up the rate of closure (FIG. 15).

(94) Sub optimal concentration of 5 ng/mL PDGF-BB was selected for use in future migration studies.

Example 12: HS6+ Dosing for Migration Studies

(95) The migration protocol from Example 11 was followed using a range of HS6+ doses (20 g/mL, 40 g/mL, 60 g/mL) with 5 ng/ml PDGF-BB.

(96) Results:

(97) Addition of 20 ug/ml of HS6+ significantly increases the rate of wound closure compared to 5 ng/ml PDGF-BB alone. Addition of more than 20 ug/ml HS6+ does not further speed up rate of wound closure (FIG. 16).

(98) HS6 was better than PDGF-BB alone, but has a lower rate of wound closure compared to HS6+(FIG. 17).

Example 13: HS6+ Increases Phosphor-PDGFR in HDFs

(99) Protocol:

(100) 1. Thaw human dermal fibroblast (HDF, Cascade Biologics) into a T75 in maintenance medium at 510.sup.3 cells/cm.sup.2 (-MEM, 10% FCS, P/S) at 37 C. 5% CO.sup.2 humidified incubator. 2. At 70-80% confluence, wash cells in PBS twice, add 1 ml of 0.125% trypsin and incubate for 3 mins. Tap the dish and stop the trypsin by adding 5 ml of medium. Centrifuge cells down at 180 g for 7 mins. 3. Count total number of cells. 4. Seed cells at 10,000 cells/cm.sup.2 cell suspension per well and place the plates into the incubator for 24 hrs. 5. Change media to -MEM, 1% FCS, P/S media for 24 hrs. 6. Treat cells with PDGF-BB and HS fractions. 7. Remove old media and wash cells twice with PBS. 8. Add Laemmli (2) Sigma into each well and scrap cells. 9. Collect samples into labeled eppendorf tubes and denature at 95 C. for 5 mins. 10. Store all samples at 20 C. 11. SDS Page.fwdarw.Transfer.fwdarw.antibody incubation
Results:

(101) HS6+ increases phosphor-PDGFR in HDFs (FIG. 18).

Example 14: Pick Skin Wound Healing Model

(102) Full thickness excisional wounds were created on the back of five adult micropigs. The back of the anaesthetized pigs was shaved and cleaned with povidone iodine solution. To serve the experimental timepoints at day 1, 3, 7, 9, 13 and 15, nested wounds were created using a 6 and 10 mm biopsy punch. A set of three wounds is required for the entire experimental timepoints. The initial three wounds were made with a 6 mm biopsy punch. At day 1, 3 and 7, each wound is cored out with a 10 mm biopsy punch. By day 16, the 10 mm wounds serve as day 9, 13 and 15 timepoints.

(103) Treatments were initiated by the topical application of 250 g/cm.sup.3 HS compound via 10 mg/ml carboxymethyl cellulose (CMC) (Sigma) gel (n=6), or CMC gel alone (untreated wound) (n=6). 56.5 L and 157 L of HS compound was applied respectively into the 6 and 10 mm wounds at day 0 and 1. The wounds were covered with Tegaderm dressing (3M Singapore). All of the wounds were examined and photographed following surgery.

(104) The pigs were euthanized at day 16 and full thickness skin samples of the wounds with surrounding unwounded skin were excised and cut into half. One half was fixed in 10% neutral buffered formalin for histological analysis. Another half was placed into TRIzol (Life Technologies) and frozen at 80 C. for subsequent molecular analysis.

(105) Treatment and dosage groups may be summarised as follows: Endogenous PDGF-BB Treatment groups Empty Carrier: 10 mg/mL carboxymethylcellulose (CMC) gel 250 g/cm.sup.3 HS6+ in CMC 250 g/cm.sup.3 HS6 in CMC HS dosage 6 mm.fwdarw.14.13 g in 56.5 L of CMC gel 10 mm.fwdarw.39.25 g in 157 L of CMC gel Treat at day 0 and 1

(106) Degree of wound healing is shown in FIGS. 20 and 21. HS6+ treatment group shows accelerated wound healing compared to CMC group.

(107) Differences in Hematoxylin and Eosin (H&E) Staining at Day 7:

(108) HS6+ treatment group shows (FIG. 22): Advanced epidermis structure Contraction of granulation tissue Increased matrix deposition Formation of blood vessels Decreased cellular infiltrates (inflammatory cells)
Decreased Cellular Infiltrates with HS6+

(109) Numbers of infiltrating leucocytes were counted in four 1 mm zones per wound bed (FIG. 23).

(110) Presence of Blood Vessels with HS6+

(111) FIG. 24 shows inflammation with carrier alone, and more matrix and blood vessel formation following treatment with HS6+

(112) Summary

(113) Treatment with HS6+ led to complete re-epithelization and formation of granulation tissue at Day 7, contraction of granulation tissue, increased matrix deposition, formation of blood vessels, decreased cellular infiltrates (inflammatory cells).

Example 15: Effects of HS6 on HDF and Keratinocyte Proliferation In Vitro

(114) The effects of HS6 on keratinocytes within the skin epidermis, and human dermal fibroblasts (HDFs) was investigated, because both cell types are important in wound healing.

(115) FIG. 29B shows that HS6 drives in vitro proliferation of HDFs by itself, and FIG. 29C shows that HS6 also increases proliferation-promoting effects of PDGF-BB (FIG. 29A) when HS6 and PDGF-BB are used in combination.

(116) FIGS. 30A and 30B show that stimulation of proliferation is very strongly inhibited in the presence of the PDGF-R inhibitor JNJ-10198409 (FIG. 30A), and is also inhibitedbut not as stronglyin the presence of FGFR1 inhibitor SU5402 (FIG. 30B).

(117) FIG. 31 shows that when human N-TERT/1 keratinocytes are grown to confluency in vitro, and then scratched using the IncuCyte system, the presence of increasing amounts of HS6, like the PDGF positive control, is able to improve the closure of the wound in vitro. The same effect, although less pronounced, is seen for HDFs (FIG. 32).

(118) We thus conclude that HS6 exerts effects both within the epidermis, on keratinocytes, and the dermis, on the dermal fibroblasts.

Example 16: Effects of HS6 on Association of PDGF-BB with PDGFR

(119) The influence of HS6 on association between PDGF-BB and PDGFR (platelet derived growth factor receptor beta) was investigated by co-immunoprecipitation analysis.

(120) 20 l of protein A/G agarose beads (Santa Cruz) were added to eppendorfs, and 1 g/mL PDGFR-Fc was added to the eppendorfs and immobilised on the beads by rotation at 4 C. for 1 hour. A negative control sample was prepared to which PDGFR-Fc was not added. After immobilisation, the beads were washed three times with PBS. Each time, beads were pelleted by centrifugation at 14,000 rpm for 1 min and the supernatant was discarded. 3 g/mL aliquots of PDGFBB were preincubated with 0, 125, 250 or 500 g/mL HS6+ on ice, for 10 min. The PDGF-BB/HS mixtures were then applied to the beads. The beads were then washed in PBS three times as above, and finally re-suspended in 40 l of Laemmli buffer. Samples were boiled for 5 minutes to denature proteins, and then loaded into a 10 well 4-12% Bis-Tris gel, and blotted onto a nitrocellulose membrane. Membranes were blocked, and then incubated with anti-PDGFR or anti-PDGF-BB antibodies.

(121) FIG. 33 shows the results of the co-immunoprecipitation studies. In the presence of HS6, there is much more association PDGF-BB and PDGFR, as can be seen by the increased intensity of the PDGF-BB bands of lanes 4 to 6 (i.e. HS6+ 125, 250 and 500 g/mL, respectively) relative to the PDGF-BB band of lane 3 (i.e. HS6+0 g/mL).

(122) The results suggest that HS6+ enhances binding between PDGF-BB and PDGFR.

REFERENCES

(123) 1. Brickman, Y. G., Ford, M. D., Gallagher, J. T., Nurcombe, V., Bartlett, P. F. & Turnbull, J. E. (1998) Structural modification of fibroblast growth factor-binding heparan sulfate at a determinative stage of neural development. The Journal of Biological Chemistry, 273, 4350-4359. 2. Knobloch, J. E., & Shaklee, P. N. (1997) Absolute molecular weight distribution of low-molecular-weight heparin and heparan sulfate by size-exclusion chromatography with multiangle laser light scattering detection. Analytical Biochemistry, 245, 231-241. 3. Skidmore, M. A., Guimond, S. E., Dumax-Vorzet, A. F., Yates, E. A. & Turnbull, J. E. (2010) Disaccharide compositional analysis of heparan sulfate and heparin polysaccharides using UV or high-sensitivity fluorescence (BODIPY) detection. Nature Protocols, 5 (12), 1983-1992. 4. The effect of controlled release of PDGF-BB from heparin-conjugated electrospun PCL/gelatin scaffolds on cellular bioactivity and infiltration. Lee J, Yoo J J, Atala A, Lee S J. Biomaterials. 2012 33(28):6709-20. 5. Heparan sulfate side chains have a critical role in the inhibitory effects of perlecan on vascular smooth muscle cell response to arterial injury. Gotha L, Lim S Y, Osherov A B, Wolff R, Qiang B, Erlich I, Nili N, Pillarisetti S, Chang Y T, Tran P K, Tryggvason K, Hedin U, Tran-Lundmark K, Advani S L, Gilbert R E, Strauss B H. Am J Physiol Heart Circ Physiol. 2014 Aug. 1; 307(3):H337-45. 6. Priming with proangiogenic growth factors and endothelial progenitor cells improves revascularization in linear diabetic wounds. Ackermann M, Pabst A M, Houdek J P, Ziebart T, Konerding M A. Int J Mol Med. 2014 April; 33(4):833-9. 7. Defective N-sulfation of heparan sulfate proteoglycans limits PDGF-BB binding and pericyte recruitment in vascular development. Abramsson A1, Kurup S, Busse M, Yamada S, Lindblom P, Schallmeiner E, Stenzel D, Sauvaget D, Ledin J, Ringvall M, Landegren U, Kjelln L, Bondjers G, Li J P, Lindahl U, Spillmann D, Betsholtz C, Gerhardt H. Genes Dev. 2007 Feb. 1; 21(3):316-31.