HAEMOSTATIC MATERIAL

Abstract

Haemostatic materials are described, particularly haemostatic materials comprising an oxidised cellulose substrate covalently immobilised to a plurality of fibrinogen-binding peptides. Methods are described for covalently attaching fibrinogen binding peptides to oxidised cellulose substrates ad other substrates that have carboxyl groups on their surface.

Claims

1. A haemostatic material comprising an oxidised cellulose substrate covalently immobilised to a plurality of fibrinogen-binding peptides.

2. The material according to claim 1, in which each peptide is immobilised to the substrate via a carbonyl group of the substrate.

3. The material according to claim 1, in which each peptide is immobilised to the substrate via a spacer.

4. The material according to claim 3, wherein the spacer is covalently linked to the peptide via an amide bond.

5. The material according to claim 1, wherein the spacer is covalently linked to the substrate via an amide bond.

6. The material according to claim 3, in which the spacer comprises a peptide spacer.

7. The material according to claim 3, in which the spacer comprises a non-peptide spacer.

8. The material according to claim 7, wherein the non-peptide spacer comprises a straight chain, preferably wherein the non-peptide spacer comprises the group (CH.sub.2).sub.a, wherein a is 1-20, preferably 1-15, 1-10, 1-5, or 2-4.

9. The material according to any preceding claim 1, in which each peptide is immobilised to the substrate via the C-terminus of the peptide.

10. The material according to claim 1, comprising the following structure:
CONHNHCONH.sub.2 where =fibrogen-binding peptide.

11. The material according to claim 1, in which each peptide is immobilised to the substrate via the N-terminus of the peptide.

12. The material according to claim 1, comprising the following structure:
CONHCONHCOOH where =fibrinogen-binding peptide.

13. The material according to claim 1, in which each fibrinogen-binding peptide comprises the sequence Gly-(Pro/His)-Arg-Xaa (SEQ ID NO: 1) where Xaa is any amino acid and Pro/His means that either proline or histidine is present at that position.

14. The material according to claim 1, in which each fibrinogen-binding peptide is 4-60 residues in length.

15. The material according to claim 1, in which the haemostatic material is in the form of a wound dressing.

16. A method of making a haemostatic material comprising covalently immobilising a plurality of fibrinogen binding peptides to an oxidised cellulose substrate.

17. The method according to claim 16, comprising: providing a plurality of moieties, each moiety comprising a fibrinogen-binding peptide and a first reactive group in the form of a carboxyl-reactive group; providing an oxidised cellulose substrate comprising a plurality of second reactive groups in the form of carboxyl groups; and reacting the first reactive groups with the second reactive groups to covalently immobilise each peptide to the substrate.

18. The method according to claim 17, in which the first reactive group is an amino group.

19. The method according to claim 17, in which each moiety comprises a non-peptide portion which provides the first reactive group.

20. The method according to claim 19, in which the non-peptide portion of each moiety is covalently linked to the -carbonyl group via the C-terminus of the peptide.

21. The method according to claim 19, in which the non-peptide portion is covalently linked to the peptide via an amide bond.

22. The method according to claim 19, in which the non-peptide portion of each moiety comprises a straight chain group of formula (CH.sub.2).sub.a, wherein a is 1-20, preferably 1-15, 1-10, 1-5, or 2-4.

23. The method according to claim 19, in which each moiety comprises the following structure:
H.sub.2N(CH.sub.2).sub.aNHCONH.sub.2 where a=1-20, preferably 1-15, 1-10, 1-5, or 2-4; and where =fibrinogen-binding peptide.

24. The method according to claim 17, in which each moiety is protected by one or more protecting groups, such that only the first reactive group is capable of reacting with the second reactive group.

25. The method according to claim 16, in which the substrate has been modified by reacting carboxyl groups on the substrate with modifying groups to form spacers on the substrate, in which the second reactive groups are positioned at the end of the spacers.

26. The method according to claim 16, comprising modifying the substrate by reacting carboxyl groups on the substrate with modifying groups to form spacers on the substrate, in which the second reactive groups are positioned at the end of the spacers.

27. The method according to claim 25, in which each modifying group has a first reactive group which is a carboxyl-reactive group, preferably an amino group, and a second reactive group which is a carboxyl group, and the first reactive group is capable of reacting with carboxyl groups on the substrate to form an amide bond.

28. The method according to claim 27, in which the modifying group comprises a peptide.

29. The method according to claim 16, comprising: providing a plurality of moieties, wherein each moiety comprises a fibrinogen-binding peptide and a first reactive group; providing a modified substrate comprising a plurality of second reactive groups, in which the second reactive groups are formed by modifying carboxyl groups of the oxidised cellulose substrate; and reacting the first reactive groups with the second reactive groups.

30. The method according to claim 29, in which the first reactive group is a carboxyl group, preferably the carboxyl group at the C-terminal end of the peptide.

31. The method according to claim 29, in which the second reactive group is a carboxyl-reactive group, preferably an amino group.

32. The method according to claim 29, in which the carboxyl groups on the substrate have been modified by reacting the carboxyl groups with modifying groups, preferably to form spacers on the substrate, in which the second reactive groups are positioned at the end of the spacers.

33. The method according to claim 29, comprising modifying the carboxyl groups on the substrate by reacting the carboxyl groups with modifying groups, preferably forming spacers on the substrate, in which the second reactive groups are positioned at the end of the spacers.

34. The method according to claim 32 in which each modifying group comprises a first carboxyl reactive group and a second carboxyl reactive group, wherein the first and second carboxyl reactive groups are preferably amino groups.

35. The method according to claim 34, in which each modifying group comprises the following structure:
H.sub.2N(CH.sub.2).sub.aNH.sub.2 where a is 1-20, preferably 1-15, 1-10 or 1-6.

36. The method according to claim 29, in which each spacer is covalently linked to the substrate by amide bonds.

37. The method according to claim 29, in which the modified substrate comprises the following structure:
CONHNH.sub.2

38. The method according to claim 16, in which each fibrinogen-binding peptide comprises the sequence Gly-(Pro/His)-Arg-Xaa (SEQ ID NO: 1) where Xaa is any amino acid and Pro/His means that either proline or histidine is present at that position.

39. A method of controlling bleeding comprising administering the haemostatic agent according to claim 1, to a wound.

40. A method of covalently immobilising a peptide to a substrate, comprising: providing a moiety comprising a peptide and a first reactive group in the form of a carboxyl-reactive group linked via the C-terminus of the peptide; providing a substrate comprising a second reactive group in the form of a carboxyl group; and reacting the first reactive group with the second reactive group to covalently immobilise each peptide to the substrate, such that the peptide is covalently immobilised to the substrate via its C-terminus.

41. The method according to claim 40, in which the first reactive group is an amino group.

42. The method according to claim 40, in which each moiety comprises a non-peptide portion which provides the first reactive group.

43. The method according to claim 40, in which the non-peptide portion is covalently linked to the peptide by an amide bond.

44. The method according to claim 40, in which the non-peptide portion of the moiety comprises a straight chain group of formula (CH.sub.2).sub.a, wherein a is 1-20, preferably 1-15, 1-10, 1-5, or 2-4.

45. The method according to claim 40, in which the moiety comprises the following structure:
H.sub.2N-(CH.sub.2).sub.aNHCONH.sub.2 where a=1-20, preferably 1-15, 1-10, 1-5, or 2-4.

46. The method according to claim 40, in which the substrate has been modified by reacting carboxyl groups on the substrate with modifying groups to form spacers on the substrate, in which the second reactive groups are positioned at the end of the spacers.

47. The method according to claim 46, in which each modifying group has a first reactive group which is a carboxyl-reactive group, preferably an amino group, and a second reactive group which is a carboxyl group, and the first reactive group is capable of reacting with carboxyl groups on the substrate to form an amide bond.

48. The method according to claim 47, in which the modifying group comprises a peptide.

49. A method of covalently immobilising a peptide to a substrate, comprising: providing a moiety comprising a peptide and a first reactive group, in which the first reactive group is the carboxyl group at the C-terminus of the peptide, or in which the first reactive group is linked via the C-terminus of the peptide; providing a modified substrate comprising a second reactive group formed by modifying a carboxyl group of the substrate; and reacting the first reactive group with the second reactive group to covalently immobilise the peptide to the substrate, such that the peptide is covalently attached to the substrate via its C-terminus.

50. The method according to any of claim 49, in which the second reactive group is a carboxyl-reactive group, preferably an amino group.

51. The method according to claim 49, in which the carboxyl group on the substrate has been modified by reacting the carboxyl group with a modifying group, preferably to form a spacer on the substrate, in which the second reactive group is positioned at the end of the spacer.

52. The method according to claim 49 in which the modifying group comprises a first carboxyl reactive group and a second carboxyl reactive group, wherein the first and second carboxyl reactive groups are preferably amino groups.

53. The method according to claim 52, in which the modifying group comprises the following structure:
H.sub.2N(CH.sub.2).sub.aNH.sub.2 where a is 1-20, preferably 1-15, 1-10 or 1-6.

54. The method according to claim 49, in which the spacer is covalently linked to the substrate by an amide bond.

55. The method according to claim 49, in which the modified substrate comprises the following structure:
CONHNH.sub.2

56. The method according to claim 40, in which the peptide is a fibrinogen-binding peptide.

57. The method according to claim 40, in which the substrate comprises oxidised cellulose.

58. The method according to claim 40, in which the substrate is a wound dressing.

59. The method according to claim 40, in which the moiety is protected by one or more protecting groups, such that only the first reactive group is capable of reacting with the second reactive group.

60. An oxidised cellulose substrate covalently immobilised to peptide, in which the peptide is covalently immobilised via its C-terminus.

61.-62. (canceled)

63. The method according to claim 49, in which the peptide is a fibrinogen-binding peptide.

64. The method according to claim 49, in which the substrate comprises oxidised cellulose.

65. The method according to claim 49, in which the substrate is a wound dressing.

66. The method according of claim 49, in which the moiety is protected by one or more protecting groups, such that only the first reactive group is capable of reacting with the second reactive group.

Description

[0217] Embodiments of the invention are now described by way of example only, with reference to the accompanying drawings, in which:

[0218] FIG. 1 shows a scheme for synthesising a haemostatic dressing of the invention;

[0219] FIG. 2 shows the results of the Kaiser test (Ninhydrin test) used to monitor presence of fully deprotected fibrinogen peptide remaining bound on a dressing of the invention;

[0220] FIG. 3a shows samples of a control dressing and a haemostatic dressing of the invention;

[0221] FIG. 3b shows improved clotting activity of a dressing of the invention compared to a control;

[0222] FIG. 4 shows a reaction scheme for modifying the surface of a dressing;

[0223] FIG. 5 shows a reaction scheme for synthesising, a haemostatic dressing of the invention;

[0224] FIG. 6 shows the results of the Kaiser Test (Ninhydrin test) used to monitor presence of fully deprotected fibrinogen peptide remaining bound on a dressing of the invention;

[0225] FIG. 7a shows samples of a control dressing and a haemostatic dressing of the invention;

[0226] FIG. 7b shows improved clotting activity of a dressing of the invention compared to a control;

[0227] FIG. 7c shows a haemostatic material of the invention and a control material placed into polypropylene tubes;

[0228] FIG. 7d shows polymerisation of human fibrinogen by a haemostatic material of the invention;

[0229] FIG. 7e shows a fibrinogen clot on a haemostatic material of the invention;

[0230] FIG. 8 shows a reaction scheme for modifying the surface of a dressing;

[0231] FIG. 9 shows a reaction scheme for synthesising a haemostatic dressing of the invention;

[0232] FIG. 10 shows the results of the Kaiser Test (Ninhydrin test) used to monitor presence of fully deprotected fibrinogen peptide remaining bound on a dressing of the invention;

[0233] FIG. 11 shows a reaction scheme for modifying the surface of a dressing;

[0234] FIG. 12 shows a reaction scheme for synthesising a haemostatic dressing of the invention;

[0235] FIG. 13 shows the results of the Kaiser Test (Ninhydrin test) used to monitor presence of fully deprotected fibrinogen peptide remaining bound on a dressing of the invention;

[0236] FIG. 14 shows the ability of a peptide dendrimer to polymerise fibrinogen at varying concentrations;

[0237] FIG. 15 shows the ability of several different peptide dendrimers to polymerise fibrinogen at varying concentrations. The numbering refers to the identity of the peptide dendrimer;

[0238] FIG. 16 shows the ability of several different peptide dendrimers to polymerise fibrinogen at varying concentrations. The numbering refers to the identity of the peptide dendrimer;

[0239] FIG. 17 shows the ability of several different peptide dendrimers to polymerise fibrinogen at varying concentrations. The numbering refers to the identity of the peptide dendrimer;

[0240] FIG. 18 shows a photograph of hydrogels formed by polymerisation of f different peptide dendrimers;

[0241] FIG. 19 shows the ability of different combinations of peptide dendrimers with peptide conjugates to polymerise fibrinogen at varying concentrations; and

[0242] FIG. 20 shows the ability of several different peptide dendrimers to polymerise fibrinogen in human plasma.

EXAMPLE 1

One Step Coupling of Boc-GPR (Pbf) PGNHCH.SUB.2.CH.SUB.2.NH.SUB.2 .(Boc-FBP-) on Oxidized Regenerated Cellulose Fabric

[0243] Boc-CPP (Pbf) PGNHCH.sub.2CH.sub.2NH.sub.2 (Boc-FBP-) moieties were assembled from the C to N terminus exclusively by Fmoc-chemistry. During the last synthetic point of the synthesis, the moieties were fully protected (including a Pbf protection group on Arg), except for a free amino group on the C-termini, and. Protected moieties were purchased from Almac Ltd.

[0244] Commercially available Surgicel* Absorbable Hemostat (oxidized regenerated cellulose (ORC)) made by Ethicon Inc. of Johnson & Johnson Medical Limited was used as the substrate. Carboxylic acid content in Surgical was adopted from the literature (See EP 0659440). 50 grams of Surgicel Nu-Knit* cloth has 20% carboxylic acid content (0.22 moles of carboxylic acid).

[0245] ORC fabric used in the synthesis was pre-washed with 21 ml dichloromethane (DCM) (1 min) and dried at 33 C. After drying, the ORC fabric -50 mg (0.2 mmolof carboxylic acid COOH) was immersed in a 1 ml dimethylformamide (DMF) solution and mixed with O-benzotriazole-N,N,N,N-tetramethyl-uronium-hexafluoro-phosphate (HBTU; 90 mg, 0.2 mmol). 1-hydroxy-1H-benzotriazole (HOBT; 30 mg, 0.2 mmol) then dressing was activated for 15 renins at room temperature. N,N-Diisopropylethylenediamine (0.4 mmol, 0.075 ml, d=0.798) (or N,N-Diisopropylethylamine DIPEA) was then added and resulting solution reacted for another 15 min. After this, 50 mg, 0.05 mmol of Boc-GPR (Pbf) PGNHCH.sub.2CH.sub.2NH.sub.2 dissolved in DMF was added to the reaction mixture2 ml in total. The coupling reaction was carried on at room temperature for 5 hours. The fabric was washed with DMF (31 ml), Methanol (MeOH) (31 ml) and with DMF (3m1). The Boc-GPR (Pbf) PGNHCH.sub.2CH.sub.2NH.sub.2 coupling step was repeated and incubated overnight at room temperature then washed with DMF (21 ml), MeOH (11 ml) and with DMF (21 ml). The ORC fabric was then washed with DMF (35 ml) and with DCM (35 ml). Removal of protecting groups with 95% TFA, 2.5% TIS, 2.5% water (3 ml) after the c produced GPRPGNHCH.sub.2CH.sub.2NHCOORC (GPRPG-ORC).

[0246] FIG. 1 summarises the reaction scheme and structures. FIG. 2 shows the results of a Kaiser Test (Ninhydrin test) tests to monitor the presence of fully deprotected peptide remaining bound on the fabric (ORC control (top); GPRPG-ORC (bottom)).

EXAMPLE 2

Functionality Test

[0247] Samples of GPRPG-FBP and ORC (control) were weighed out, treated with 100 l of human plasma (Alpha Labs-Plasma Lot# A1162 Exp 2015-03) and incubated for 1.5 or 3 min at 33C. Tested samples and controls were removed from the plasma and then were weighed to determine any difference. The test was repeated 3 times. The results in Table 1 show that the mass remaining on the GPRPG-ORC is significantly higher compared to control samples, indicating that fibrinogen binding peptides retain activity when conjugated to the fabric.

TABLE-US-00001 TABLE 1 Incubation time with Starting End 100 l of human Tested samples mass (mg) mass (mg) plasma (min) ORC (control) 6 42 3 GPRPG-ORC 6 66 3 ORC (control) 5 21 1.5 GPRPG-ORC 5 45 1.5 ORC (control) 3 27 1.5 GPRPG-ORC 3 42 1.5

[0248] Samples of ORC (control) and GPRPG-ORC (6 mg each) were placed on weighing boat (See FIG. 3acontrol (left); GPRPG-ORC (right)) and treated with 100 l of human plasma then incubated for 3 min at 33 C. The resulting clots were placed (tilted) to 90 angle, and any run-off from the clot was observed. ORC (control) released fluid, but GPRPG-ORC did not (See FIG. 3b control (left); GPRPG-ORC (right)),

EXAMPLE 3

Preparation of Surface-Modified Oxidised Cellulose fabric with a Gly-Gly Spacer

[0249] Introduction of a Gly-Gly spacer into the oxidised cellulose fabric was accor through base catalysed HBTU/HOBT amide bond formation. Fabric used in the synthesis was pre-washed with 25 ml dichloromethane (DCM) (1 min) and dried at 33 C. After drying, the fabric285 mg (1.25 mmolCOOH concentration) was immersed in a 5 ml dimethylformamide (DMF) solution containing and mixed with O-benzotriazole-N,N,N,N-tetramethyl-uronium-hexafluoro-phosphate (HBTU; 597 mg, 1.156 mmol), 1-hydroxy-1H-benzotriazole (HOBT; 217 mg, 1.56 mmol) then the fabric was activated for 15 min at room temperature, N,N-Diisopropylethylenediamine (3.14 mmol, 0.505 ml, d=0.798) (or N,N-Diisopropylethylamine DIPEA) was then added and resulting solution reacted for another 15 min. After this, 24 mg, 0.31 mmol of Gly-OH dissolved in Dimethylsulfoxide (DMSO) was added to the reaction mixture.

[0250] The coupling reaction was carried out at room temperature for 140 min. Oxidised cellulose fabric was washed with DMF (35 ml), Methanol (MeOH) (35 ml) and with DMF (35 ml). The Gly-OH coupling step was repeated and incubated for 30 min at room temperature then washed with DMF (25 mi), MeOH (15 ml) and with DMF (25 ml).

[0251] FIG. 4 summarises the reaction scheme and structures.

EXAMPLE 4

Coupling of Boc-FBP to Gly-Gly-Functionalised Oxidised Cellulose Fabric

[0252] The coupling of the Boc-FBP to the Gly-Gly-functionalised fabric was accomplished by a base-catalysed synthesis approach. First, Gly-Gly-functionalised fabric was immersed in a DMF (5 ml) and mixed with HBTU (475 mg, 1.25 mmol), HOBT (159 mg, 1.25 mmol). After stirring at room temperature for 2 min, N,N-Diisopropylethylenediamine (0.406 ml, 2.5 mmol) (or DIPEA) was added and mixed for 2 min. 275 mg (0.31 mmol) of Boc-FBP peptide was dissolved in DMF (200 l) and this was added to the reaction mixture. The coupling reaction was carried out overnight (17 hours) at room temperature. The dressing then was washed with DMF (35 ml) and with DCM (35 ml). Removal of protecting groups with 95% TFA, 2.5% TIS, 2.5% water (3 ml) after the coupling reaction produced GPRPGNHCH.sub.2CH.sub.2NHCO-G-G-ORC (GPRPG-G-G-ORC).

[0253] The Kaiser test (Ninhydrin test) was used to monitor presence of fully deprotected peptide remaining bound on the fabric (See FIG. 6ORC control (top); GPRPG-G-G-ORC (bottom)).

[0254] The Kaiser Test showed a strong positive result that was more intense comp Example 1 (without Gly-Gly spacer).

Example 5

Functionality Test

[0255] The tilt test (described in Example 2) was repeated for GPRPG-G-G-ORC. ORC (control) and GPRPG-G-G-ORC (8 mg each) were each placed in a weighing boat and treated with 100 l of human plasma than incubated for 1.5 min at 33 C. The resulting clots were placed (tilted) to 90 angle. The strength of the clot was observed. FIGS. 7a and 7b illustrate that GPRPG-G-G-ORC samples formed a stronger clot with plasma than ORC control (left); (GPRPG-G-G-ORC (right)).

[0256] Samples of GPRPG-G-G-FBP and ORC (control) were weighed out and treated with 150 l of human plasma (Alpha Labs-Plasma Lot# A1174 Exp 2016-03) and incubated for 1.5 min at 33 C. Tested samples and controls were removed from the plasma and then were weighed to determine if any difference was observable. The test was repeated four times. The results in table 2 showed that the mass remaining on GPRPG-G-G-ORC was significantly higher compared to control samples, suggesting that fibrinogen-binding peptides retain activity when conjugated to the regenerated oxidized cellulose fabric.

TABLE-US-00002 TABLE 2 Incubation time with Starting End 150 l of human Tested samples mass (mg) mass (mg) plasma (min) ORC (control) 8 65 1.5 GPRPG-G-G-ORC 8 89 1.5 ORC (control) 9 50 1.5 GPRPG-G-G-ORC 9 83 1.5 ORC (control) 6 43 1.5 GPRPG-G-G-ORC 7 50 1.5 ORC-control 10 78 1.5 GPRPG-G-G-ORC 10 85 1.5

[0257] FIG. 7c shows GPRPG-G-G-ORC (tube labelled SC+ (top)) and ORC (control) (tube labelled SC (bottom)) fibres placed into separate polypropylene tubes. 150 l of Human Plasma solution (Alpha Labs-Plasma Lot# A1162 Exp 2016-03) was added to each sample and the fibres were incubated at 37C for 1.5 minutes. There is a cl SC+ shown in FIG. 7d.

[0258] Visual examination of threads removed from the polyethylene tubes was also undertaken. FIG. 7e shows that GPRPG-G-G-ORC fibre formed a clot with human fibrinogen. The GPRPG-G-G-ORC fibre removed from the container was thicker than the control sample.

EXAMPLE 6

Preparation of Surface-Modified Oxidised Cellulose Fabric with -Ahx Spacer

[0259] Introduction of 6-amino hexanoic acid (-Ahx) spacer into the oxidsed cellulose fabric was accomplished through base catalysed HBTU/NHOBT amide bond formation. The synthetic method employed was substantially the same as described above in Example 3 for modification of ORC fabric with Gly-Gly spacers.

[0260] After prewashing and drying steps. 114 mg of fabric was immersed in 2 ml of DMF solution and mixed with O-benzotriazole-N,N,N,N-tetramethyl-uronium-hexafluoro-phosphate (HBTU; 237 mg, 0.625 mmol), 1-hydroxy-1H-benzotriazole (HOST; 84 mg, 0.625 mmol) then the fabric was activated for 15 min at room temperature. N,N-Diisopropylethylenediamine (1.25 mmol, 0.200 ml, d=0.798) (or DIPEA) was then added and resulting solution reacted for another 15 min. After this, 16.4 mg, 0.125 mmol of -Ahx-OH dissolved in Dimethylsulfoxide (DMSO) was added to the reaction mixture, The coupling reaction was carried out at room temperature overnight.

[0261] The fabric was washed with DMF (33 ml), Methanol (MeOH) (3ml) and with DMF (33 ml).

[0262] FIG. 8 summarises the reaction scheme and the structures.

EXAMPLE 7

Coupling of Boc-FBP to the Ahx-Functionalised Dressing

[0263] The coupling of the Boc-FBP to the Ahx-functionalised fabric was accomplished by a base catalysed synthesis approach as described in Example 4.

[0264] A summary of the reaction scheme, and the structures, is shown in FIG. 9.

[0265] Firstly, Ahx-functionalised dressing was immersed in a DMF (2 ml) and mixed with HBTU (190 mg, 0.5 mmol), HOBT (67.4 mg, 0.5 mmol). After stirring at room temperature for 2 min N,N-Diisopropylethylenediamine (0.180 ml, 1.1 mmol) (or IPEA) was added and mixed for 2 min, 110 mg (0.125 mmol) of Boc-FOP peptide was dissolved in DMF (200 l) and this was added to the reaction mixture. The coupling reaction was carried out overnight (17 hours) at room temperature. The dressing then was washed with DMF (33 ml) and with DCM (33 ml). Removal of protecting groups with 95% TFA, 2.5% TIS, 2.5% water (3 ml) after the coupling reaction produced GPRPG-NHCH.sub.2CH.sub.2NHCO-Ahx-ORC (GPRPG-Ahx-ORC).

[0266] The Kaiser test (Ninhydrin test) was used to monitor presence of fully deprotected peptide remaining bound on the cellulose (See FIG. 10GPRPG-Ahx-ORC (left); GPRPG-G-G-ORC (right)).

EXAMPLE 8

Preparation of Surface-Modified Oxidised Cellulose Fabric with -Ala Spacer

[0267] Introduction of -alanine (-Ala) spacer into the oxidsed cellulose fabric was accomplished through base catalysed HBTU/HOBT amide bond formation. The synthetic method employed was substantially the same as described above in Example 3 for modification of ORC fabric with Gly-Gly spacers.

[0268] After prewashing and drying steps, 206 mg of fabric was immersed in 5 ml of DMF solution and mixed with O-benzotriazole-N,N,N,N-tetramethyl-uronium-hexafluoro-phosphate (HBTU; 442 mg, 1.165 mmol), 1-hydroxy-1H-benzotriazole (HOBT; 152 mg, 1.125 mmol) then the fabric was activated for 15 min at room temperature. N,N-Diisopropylethylenediamine (2.468 mmol, 0.319 ml) (or N,N-Diisopropylethylamine-DIEPA) was then added and resulting solution reacted for another 15 min. After this, 20 mg, 0.226 mmol of -Ata-OH dissolved in Dimethylsulfoxide (DMSO) was added to the reaction mixture. The coupling reaction was carried out at room temperature overnight.

[0269] The fabric was washed with DMF (35 ml), Methanol (MeOH) (35 ml) and with DMF (35 ml).

[0270] FIG. 11 summarises the reaction scheme and the structures.

EXAMPLE 9

Coupling of Boc-FBP to the -Ala-Functionalised Dressing

[0271] The coupling of the Boc-FBP to the -Ala-functionalised fabric was accomplished by a base catalysed synthesis approach as described in Example 4.

[0272] A summary of the reaction scheme, and the structures, is shown in FIG. 12.

[0273] Firstly, -Ala-functionalised dressing was immersed in a DMF (5 ml) and mixed with HBTU (350 mg, 0.923 mmol), HOBT (124 mg, 0.918 mmol). After stirring at room temperature for 2 min N,N-Diisopropylethylenediamine (0.247 ml, 1.911 mmol) (or N,N-Diisopropylethylamine DIEPA) was added and mixed for 2 min. 202 mg (0.231 mmol) of Boc-FBP peptide was dissolved in DMF (400 l) and this was added to the reaction mixture. The coupling reaction was carried out overnight (17 hours) at room temperature. The dressing then was washed with DMF (35 ml) and with DCM (35 ml). Removal of protecting groups with 95% TFA, 2.5% TIS, 2.5% water (3 ml) after the coupling reaction produced GPRPG-NHCH.sub.2CH.sub.2NHCO-Ahx-ORC (GPRPG-Ahx-ORC).

[0274] The Kaiser Test was used to monitor the presence of fully deprotected peptide remaining bound on the cellulose. See FIG. 13 from left Surgical control (labelled as ORC-Control), GPRPG--Ala-ORC and GPRPG-Ahx-ORC.

EXAMPLE 10

Functionality Test

[0275] Samples of GPRPG--Ala-FBP, GPRPG-Ahx-ORC and ORC (control) were weighed out and treated with 100 l of human plasma (Alpha Labs-Plasma Lot# A1174 Exp 2016-03) and incubated for 1.5 min at 33 C. Tested samples and controls were removed from the plasma and then were weighed to determine if any difference was observable. The test was repeated three times. The results in table 3 showed that the mass remaining on GPRPG--Ala-ORC, GPRPG-Ahx-ORC was significantly higher compared to control (Surgicel) samples, suggesting that fibrinogen-binding peptides retain activity when conjugated to the regenerated oxidised cellulose fabric.

TABLE-US-00003 TABLE 3 Incubation time with Starting End 100 l of human Tested samples mass (mg) mass (mg) plasma (min) ORC (control) 4 34 1.5 GPRPG--Ala-FBP 4 64 1.5 GPRPG-Ahx-ORC 4 54 1.5 ORC (control) 4 36 1.5 GPRPG--Ala-FBP 4 60 1.5 GPRPG-Ahx-ORC 4 50 1.5 ORC (control) 4 32 1.5 GPRPG--Ala-FBP 4 59 1.5 GPRPG-Ahx-ORC 4 57 1.5

EXAMPLE 11

Synthesis of Peptide Dendrimers and Peptide Conjugates

[0276] Peptides were synthesised on Rink amide MBHA low loaded resin (Novabiochem, 0.36 mmol/g), by standard Fmoc peptide synthesis, using Fmoc protected amino acids (Novabiochem).

[0277] In general, single-coupling cycles were used throughout the synthesis and HBTU activation chemistry was employed (HBTU and PyBOP (from AGTC Bioproducts) were used as the coupling agents). However, at some positions coupling was less efficient than expected and double couplings were required.

[0278] The peptides were assembled using an automated peptide synthesiser and HBTU up to the branch points and by manual peptide synthesis using PyBOP for the peptide branches.

[0279] For automated synthesis a threefold excess of amino acid and HBTU was used for each coupling and a ninefold excess of N,N-Diisopropylethylamine (DIPEA, Sigma) in dimethylformamide (DMF, Sigma).

[0280] For manual synthesis a threefold excess of amino acid and PyBOP was used for each coupling and a ninefold excess of DIPEA in N-methylpyrollidinone (NMP, Sigma).

[0281] Deprotection (Fmoc group removal) of the growing peptide chain using 20% piperidine (Sigma) in DMF likewise may not always be efficient and require double deprotection.

[0282] Branches were made using Fmoc-Lys(Fmoc)-OH, Fmoc-Lys(Boc)-OH, or F OH.

[0283] Final deprotection and cleavage of the peptide from the solid support was performed by treatment of the resin with 95% TFA (Sigma) containing triisopropylsilane (TIS, Sigma), water and anisole (Sigma) (1:1:1, 5%) for 2-3 hours.

[0284] The cleaved peptide was precipitated in cold diethyl ether (Sigma) pelleted by centrifugation and lyophilized. The pellet was re-dissolved in water (10-15 mL), filtered and purified via reverse phase HPLC using a C-18 column (Phenomenex at flow rate 20 ml/min) and an acetonitrile/water gradient containing 0.1% TFA. The purified product was lyophilized and analyzed by ESI-LC/MS and analytical HPLC and were demonstrated to be pure (>95%). Mass results all agreed with calculated values.

[0285] Peptide Dendrimers and Peptide Conjugates

[0286] The structures of peptide dendrimers and peptide conjugates synthesised using the methods described above are shown below.

[0287] The NH.sub.2 group at the end of a peptide sequence denotes an amino group at the amino-terminal end of the sequence. The -am group at the end of a peptide sequence denotes an amide group at the carboxy-terminal end of the sequence.

[0288] Peptide Conjugate No: 1:

##STR00020##

[0289] Peptide Conjugate No. 2:

##STR00021##

[0290] Peptide Dendrimer No. 3:

##STR00022##

[0291] Peptide Dendrimer No. 4:

##STR00023##

[0292] Peptide Dendrirner No. 5:

##STR00024##

[0293] Peptide Dendrimer No. 8:

##STR00025##

[0294] Peptide Dendrirner No. 9:

##STR00026##

[0295] Peptide Dendrimer No. 10:

##STR00027##

[0296] Peptide Dendrimer No. 11

##STR00028##

[0297] Peptide Dendrimer No. 12:

##STR00029##

[0298] Peptide Dendrimer No. 13:

##STR00030##

EXAMPLE 12

Co-Polymerisation of a Peptide Dendrimer With Fibrinogen

[0299] Dendrimer No. 12 comprises a branched core with five consecutive lysine residues. The lysine residues are covalently linked through a side chain of an adjacent lysine residue.

[0300] The ability of Peptide Dendrimer No. 12 to polymerise fibrinogen was assessed. 30 l of dendrimer in solution, at concentration ranging from 0.005-2 mg/ml, was added to 100 l purified human fibrinogen at 3 mg/ml (the level of fibrinogen found in the blood). Polymerisation of fibrinogen was analysed using a Sigma Amelung KC4 Delta coagulation analyser. FIG. 14 shows a plot of the polymerisation (clotting) times (in se increasing concentration of dendrimer.

[0301] The results show that the dendrimer was able to copolymerise with fibrinogen almost instantaneously, even at very low concentrations of dendrimer. The increase in clotting time with dendrimer concentrations above 0.5 mg/ml is thought to be explained by an excess of fibrinogen-binding peptides compared to the number of free binding pockets in fibrinogen. At higher concentrations, the fibrinogen-binding peptides of the dendrimer may saturate the fibrinogen binding pockets, resulting in a significant number of excess dendrimer molecules that are not able to copolymerise with fibrinogen.

EXAMPLE 7

Effect of Varying the Number of fibrinogen-Binding Peptides Per Dendrimer on the Speed of Copolymerisation With Fibrinogen

[0302] This example investigates the effect of varying the number of fibrinogen-binding peptides per peptide dendrimer on the speed of copolymerisation with fibrinogen.

[0303] The ability of Peptide Dendrimer Nos. 4, 5, 10, 11, and 12 to copolymerise with fibrinogen was assessed using the same method described in Example 11. The concentration of each dendrimer was varied from 0.005-0.5 mg/ml. FIG. 15 shows a plot of the clotting times (in seconds) with increasing concentration of each different dendrimer.

[0304] The results show that dendrimer No. 5 (with only two fibrinogen-binding peptides/dendrimer) was not able to copolymerise with fibrinogen. As the number of fibrinogen-binding peptides was increased from three to five, at concentrations of dendrimer from0.125 to0.275 mg/ml, the speed of copolymerisation increased. At concentrations below0.125 mg/ml dendrimer, dendrimer No. 10 (with three fibrinogen-binding peptides/dendrimer) produced faster clotting times than dendrimer no. 4 (with four fibrinogen-binding peptides/dendrimer). In the range0.02-0.5 mg/ml, dendrimer no. 12 (with five fibrinogen-binding peptides/dendrimer) produced almost instantaneous clotting. In the range0.05-0.3 mg/ml, dendrimer no. 11 (with four fibrinogen-binding peptides/dendrimer) also produced almost instantaneous clotting.

[0305] It is concluded that the speed at which fibrinogen is polymerised by a dendrimer of the invention generally increases as the number of fibrinogen-binding peptides per dendrimer is increased.

EXAMPLE 13

Effect of Fibrinogen-Binding Peptide Orientation, and of Different Fibrinogen-Binding Peptide Sequences on Speed of Copolymerisation With Fibrinogen

[0306] To assess whether the orientation of a fibrinogen-binding peptide could affe a peptide dendrimer to copolymerise with fibrinogen, peptide dendrimers comprising three fibrinogen-binding peptides attached to a single tri-functional amino acid residue (lysine) were synthesised (referred to as three-branch dendrimers), but with one of the fibrinogen-binding peptides orientated with its amino-terminal end attached to the branched core, and amidated at its carboxy-terminal end. The ability of peptide dendrimers comprising different fibrinogen-binding peptide sequences to copolymerise with fibrinogen was also tested.

[0307] The fibrinogen-binding peptides of Peptide Dendrimer Nos. 3 and 10 are each of sequence GPRPG (SEQ ID NO: 18). Each fibrinogen-binding peptide of Peptide Dendrimer No. 10 is orientated with its carboxy-terminal end attached to the branched core. One of the fibrinogen-binding peptides of Peptide Dendrimer No. 3 is orientated with its amino-terminal end attached to the branched core. The carboxy-terminal end of that peptide comprises an amide group.

[0308] Two of the fibrinogen-binding peptides of Peptide Dendrimer No. 8 are of sequence GPRPG (SEQ ID NO: 18), and the third fibrinogen-binding peptide is of sequence APFPRPG (SEQ ID NO: 2) orientated with its amino-terminal end attached to the branched core. The carboxy-terminal end of that peptide comprises an amide group.

[0309] Two of the fibrinogen-binding peptides of Peptide Dendrimer No. 9 are of sequence GPRPFPA (SEQ ID NO: 7), and the third fibrinogen-binding peptide is of sequence APFPRPG (SEQ ID NO: 12) orientated with its amino-terminal end attached to the branched core. The carboxy-terminal end of that peptide comprises an amide group.

[0310] The sequence GPRPG (SEQ ID NO: 15) binds to hole a and hole b of fibrinogen, but with some preference for hole a. The sequence GPRPFPA (SEQ ID NO: 7) binds with high preference for hole a in fibrinogen. The sequence Pro-Phe-Pro stabilizes the backbone of the peptide chain and enhances the affinity of the knob-hole interaction (Stabenfeld et al., BLOOD, 2010, 116: 1352-1359).

[0311] The ability of the dendrimers to copolymerise with fibrinogen was assessed using the same method described in Example 11, for a concentration of each dendrimer ranging from 0.005-0.5mg/ml. FIG. 16 shows a plot of the clotting times (in seconds) obtained with increasing concentration of each different dendrimer.

[0312] The results show that changing the orientation of one of the fibrinogen-binding peptides of a three-branch dendrimer, so that the peptide is orientated with its amino-terminal end attached to the branched core (i.e. Dendrimer No. 3), reduced the ability of the dendrimer to copolymerise with fibrinogen (compare the dotting time of Dendrimer No. Dendrimer No. 10). However, at higher fibrinogen concentrations, Dendrimer No. 3 was able to copolymerise with fibrinogen (data not shown).

[0313] A three-branch dendrimer with a fibrinogen-binding peptide of different sequence orientated with its amino-terminal end attached to the branched core was able to copolymerise with fibrinogen (see the results for Dendrimer No. 8).

[0314] A three-branch dendrimer in which two of the fibrinogen-binding peptides comprise sequence that binds preferentially to hole b in fibrinogen (sequence GPRPFPA (SEQ ID NO: 7)), with these peptides orientated with their carboxy-terminal end attached to the branched core, and the other peptide comprising the reverse sequence (i.e. sequence APFPRPG (SEQ ID NO: 2)) orientated with its amino-terminal end attached to the branched core (Dendrimer No. 9) was also very active in copolymerising with fibrinogen.

[0315] EXAMPLE 14

Ability of Peptide Dendrimers With Different Fibrinogen-Binding Peptide Sequences to Copolymerise With Fibrinogen

[0316] The GPRPG (SEQ ID NO: 18) and GPRPFPA (SEQ ID NO: 8) motifs primarily bind to the a hole on fibrinogen. This example describes an assessment of the ability of a chimeric peptide dendrimer (i.e. a peptide dendrimer with different fibrinogen-binding peptide sequences attached to the same branched core) to copolymerise with fibrinogen.

[0317] Peptide dendrimer No. 13 is a chimeric four-branch peptide dendrimer comprising two fibrinogen-binding peptides with sequence GPRPG-(SEQ ID NO: 18) (which has a binding preference for the a hole), and two fibrinogen-binding peptides with sequence GHRPY-(SEQ ID NO: 15) (which binds preferentially to the b hole). Non-chimeric peptide dendrimers Nos. 11 and 12 are four- and five-arm peptide dendrimers, respectively. Each fibrinogen-binding peptide of these dendrimers has the sequence GPRPG-(SEQ ID NO: 18). Each fibrinogen-binding peptide of Dendrimers Nos. 11, 12, and 13 is attached at its carboxy-terminal end to the branched core.

[0318] The ability of the dendrimers to copolymerise with fibrinogen was assessed using the same method described in Example 11, for a concentration of each dendrimer ranging from 0.005-0.5 mg/ml. FIG. 17 shows a plot of the clotting times (in seconds) obtained with increasing concentration of each different dendrimer.

[0319] The results show that the clotting speed using the chimeric dendrimer was slower than the non-chimeric dendrimers at concentrations below 0.3 mg/ml. However, FIG. 18 shows a photograph of the hydrogels obtained using the different dendrimers, The g with the number of the peptide dendrimer used (11, 12, and 13), and P labels a hydrogel formed using a product in which several fibrinogen-binding peptides are attached to soluble human serum albumin. The hydrogel formed by the chimeric dendrimer was more dense and contained less fluid compared to the hydrogels formed using dendrimers Nos. 11 and 12 (at 3 mg/ml fibrinogen, or at higher concentrations of fibrinogen). Thus, although the clotting time was slower using the chimeric dendrimer, the hydrogel formed using this dendrimer was more dense.

EXAMPLE 15

Ability of Mixtures of Peptide Dendrimers and Peptide Conjugates to Copolymerise with Fibrinogen

[0320] Fibrinogen-binding peptide of sequence GPRP-(SEQ ID NO: 5) binds strongly and preferentially to the a hole of fibrinogen (Laudano et al., 1978 PNAS 7S). Peptide Conjugate No. 1 comprises two fibrinogen-binding peptides with this sequence, each attached to a lysine residue. The first peptide is attached its carboxy-terminal end by a linker to the lysine residue, and the second peptide is attached at its amino-terminal end by a linker to the lysine residue. The carboxy-terminal end of the second peptide comprises an amide group.

[0321] Fibrinogen-binding peptide of sequence GHRPY-(SEQ ID NO: 16) binds strongly and preferentially to the b hole of fibrinogen (Doolittle and Pandi, Biochemistry 2006, 46, 2657-2667). Peptide Conjuoate No. 2 comprises a first fibrinogen-binding peptide with this sequence, attached at its carboxy-terminal end by a linker to a lysine residue. A second fibrinogen-binding peptide, which has the reverse sequence (YPRHG (SEQ ID NO: 19)), is attached at its amino terminal end by a linker to the lysine residue. The carboxy-terminal end of the second peptide comprises an amide group.

[0322] The linker allows the peptides to extend away from each other.

[0323] Peptide Conjugate No. 1 or 2 (2 mg/ml) was mixed with Peptide Dendrimer No. 3 or 4, and fibrinogen, and the ability of the mixtures to copolymerise with fibrinogen was assessed using the same method described in Example 11, for a concentration of each dendrimer ranging from 0.025-0.5 mg/ml. FIG. 15 shows a plot of the clotting times (in seconds) obtained with increasing concentration of each different dendrimer.

[0324] The results show that, surprisingly, only mixtures containing Peptide Conjugate No. 2 (i.e. with the B-knob peptides) and the dendrimer peptides were synergistic and increased activity, whereas mixtures containing the Peptide Conjugate No. 1 (the A-kn were not active when added to either Peptide Conjugate No. 2 or the peptide dendrimers.

EXAMPLE 16

Ability of Peptide Dendrimers to Polymerise Fibrinogen in Human Plasma

[0325] The ability of several different peptide dendrimers (Nos. 4, 5, 8, 9, 10, 11, 12, 13) to polymerise fibrinogen in human plasma was tested.

[0326] 30 L of each dendrimer (at a concentration of 0.25 mg/ml) was added to 100 L human plasma at 37 C., and polymerisation of fibrinogen was determined using a Sigma Amelung KC4 Delta coagulation analyzer.

[0327] The clotting times for each dendrimer are shown in FIG. 20, and show that peptide dendrimers Nos. 10, 11, 4, 12 and 13 were able to polymerise fibrinogen in human plasma, with dendrimer No. 12 being particularly effective (with a clotting time of less than one second). However, peptide dendrimers Nos. 5, 8, and 9 were not able to polymerise fibrinogen in human plasma.

EXAMPLE 12

Effect of Sterilisation on Ready-to-Use Peptide Dendrimer Formulations

[0328] This example describes the effect of Gamma irradiation on the haemostatic activity of peptide dendrimers formulated as a ready-to-use paste with hydrated gelatin.

[0329] 2 ml of solution of Peptide Dendrimer No. 12 or 13 was mixed with SURGIFLO Haemostatic Matrix (a hydrated flowable gelatin matrix) to form a paste of each peptide. Each paste was sterilised by irradiation with .sup.60Co gamma rays at a dose of 30 kGy, and then stored at room temperature. Samples of the sterilised pastes were used for testing after storage for two and four weeks.

[0330] After storage, peptide dendrimers were extracted from each paste using 10 mM HEPES buffer. 30 L of each extract (with a peptide concentration of about 0.25 mg/ml) was added to 100 L of human fibrinogen at 3 mg/ml, and the ability of each dendrimer to polymerise fibrinogen (the clotting activity) at 37 C. was determined using a Sigma Amelung KC4 Delta coagulation analyser. The polymerisation activity of non-irradiated control samples was also determined. The results are summarized in the Table below.

TABLE-US-00004 Clotting activity (seconds) Peptide Storage for 2 Storage for 4 dendrimer Non-irradiated weeks post weeks post no. control irradiation irradiation 12 1 1 1 13 4.3 9.4 10

[0331] The results show that peptide dendrimers of the invention, formulated as a ready-to-use paste with hydrated gelatin, retain ability to polymerise fibrinogen after sterilization by irradiation.