HAEMOSTATIC COMPOSITIONS

Abstract

A sterile, ready-to-use, flowable haemostatic composition comprises a soluble haemostatic agent comprising a plurality of carriers and a plurality of fibrinogen binding peptides immobilised to the carrier; a biocompatible liquid; and particles of biocompatible cross-linked polysaccharide suitable for use in haemostasis and which are insoluble in the biocompatible liquid. Such compositions may be used for the control of bleeding, especially in surgical procedures.

Claims

1. A sterile, ready-to-use, flowable haemostatic composition comprising: a soluble haemostatic agent comprising a plurality of carriers and a plurality of fibrinogen-binding peptides immobilised to each carrier; a biocompatible liquid; and particles of a biocompatible cross-linked polysaccharide suitable for use in haemostasis and which are insoluble in the biocompatible liquid.

2. The composition according to claim 1, wherein the haemostatic composition has been sterilized by steam sterilization, or by dry-heat sterilization.

3. The composition according to claim 1, wherein the biocompatible liquid provides a continuous liquid phase, and the polymer particles are substantially homogenously dispersed throughout the liquid phase.

4. The composition according to claim 1, wherein each carrier of the haemostatic agent comprises a branched core, and the fibrinogen-binding peptides are separately covalently attached to the branched core.

5. The composition according to claim 4, wherein the branched core comprises: from two to ten multi-functional amino acid residues, wherein each fibrinogen-binding peptide is separately covalently attached to a multi-functional amino acid residue of the branched core; a plurality of multi-functional amino acid residues, wherein one or more fibrinogen-binding peptides are separately covalently attached to each of at least two adjacent multi-functional amino acid residues of the branched core; a plurality of multi-functional amino acid residues, wherein two or more fibrinogen-binding peptides are separately covalently attached to at least one of the multi-functional amino acid residues of the branched core; a plurality of multi-functional amino acid residues, wherein two or more multi-functional amino acid residues are covalently linked through a side chain of an adjacent multi-functional amino acid residue; or a single multi-functional amino acid residue, and a fibrinogen-binding peptide is separately covalently attached to each functional group of the multi-functional amino acid residue; wherein the multi-functional amino acid residues comprise tri- or tetra-functional amino acid residues, or tri- and tetra-functional amino acid residues, or the single multi-functional amino acid residue is a tri- or tetra-functional amino acid residue.

6. The composition according to claim 5, wherein the branched core comprises a plurality of consecutive multi-functional amino acid residues.

7. The composition according to claim 5, wherein the branched core comprises up to ten multi-functional amino acid residues.

8. The composition according to claim 5, wherein the multi-functional amino acid residue, or the plurality of multi-functional amino acid residues, comprise a lysine, ornithine, arginine, aspartic acid, glutamic acid, asparagine, glutamine, or cysteine residue.

9. The composition according to claim 5, wherein the haemostatic agent is of the following general formula (I): ##STR00026## where: FBP is a fibrinogen-binding peptide; -(linker)- is an optional linker, preferably a non-peptide linker; X is a tri-functional amino acid residue, preferably a lysine, ornithine, arginine, aspartic acid, glutamic acid, asparagine, glutamine, or cysteine residue; Y is FBP, or NH.sub.2; Z is -(linker)-FBP or [X.sub.n-(linker)-FBP].sub.a-(linker)-FBP when Y is FBP, or Z is [X.sub.n-(linker)-FBP].sub.a-(linker)-FBP when Y is NH.sub.2; where: X.sub.n is a tri-functional amino acid residue, preferably a lysine, ornithine, arginine, aspartic acid, glutamic acid, asparagine, glutamine, or cysteine residue; and a is 1-10, preferably 1-3.

10. The composition according to claim 5, wherein the haemostatic agent is of the following general formula (II): ##STR00027## where: FBP is a fibrinogen-binding peptide; -(linker)- is an optional linker, preferably comprising NH(CH.sub.2).sub.5CO; Y is FBP, or NH.sub.2; Z is: R-(linker)-FBP, when Y is FBP, or ##STR00028## when Y is NH.sub.2; or ##STR00029## when Y is NH.sub.2; or ##STR00030## when Y is NH.sub.2; or ##STR00031## when Y is FBP and a is 1-10, preferably 1-3 where R is (CH.sub.2).sub.4NH, (CH.sub.2).sub.3NH, or (CH.sub.2).sub.3NHCNHNH.

11. The composition according to claim 5, wherein the haemostatic agent is of the following general formula (III): ##STR00032## where: FBP is a fibrinogen-binding peptide; -(linker)- is an optional linker, preferably comprising NH(CH.sub.2).sub.5CO; Y is FBP, or NH.sub.2; Z is: (CH.sub.2).sub.4NH-(linker)-FBP, when Y is FBP; or ##STR00033## when Y is NH.sub.2; or ##STR00034## when Y is NH.sub.2; or ##STR00035## when Y is NH.sub.2; or ##STR00036## when Y is FBP and a is 1-10, preferably 1-3.

12. The composition according to claim 4, wherein each fibrinogen-binding peptide is attached to the branched core by a non-peptide linker.

13. The composition according to claim 12, wherein the linker comprises a straight-chain linker, preferably a straight-chain alkyl group.

14. The composition according to claim 13, wherein the linker comprises: NH(CH.sub.2).sub.nCO, where n is 1-10.

15. The composition according to claim 1, wherein the fibrinogen-binding peptides bind preferentially to hole a of fibrinogen over hole b of fibrinogen.

16. The composition according to claim 1, wherein the fibrinogen-binding peptides bind preferentially to hole b of fibrinogen over hole a of fibrinogen.

17. The composition according to claim 1, wherein the haemostatic agent comprises fibrinogen-binding peptides of different sequence.

18. The composition according to claim 17, wherein the plurality of carriers comprise a first plurality of carriers, and a second plurality of carriers, and wherein the fibrinogen-binding peptides attached to the first plurality of carriers are of different sequence to the fibrinogen-binding peptides attached to the second plurality of carriers.

19. The composition according to claim 17, wherein each carrier has fibrinogen-binding peptides of different sequence attached thereto.

20. The composition according to claim 17, wherein the fibrinogen-binding peptides of different sequence have different selectivity of binding to hole a over hole b of fibrinogen.

21. The composition according to claim 17, wherein the fibrinogen-binding peptides of different sequence comprise a first fibrinogen-binding peptide that binds preferentially to hole a of fibrinogen over hole b of fibrinogen, and a second fibrinogen-binding peptide that binds with higher selectivity of binding to hole a of fibrinogen over hole b of fibrinogen than the first fibrinogen-binding peptide.

22. The composition according to claim 21, wherein the first fibrinogen-binding peptide comprises an amino acid sequence GPRP (SEQ ID NO: 1) at its amino-terminal end.

23. The composition according to claim 21, wherein the second fibrinogen-binding peptide comprises an amino acid sequence -APFPRPG (SEQ ID NO: 14) at its carboxy-terminal end.

24. The composition according to claim 17, wherein the fibrinogen-binding peptides of different sequence comprise a first fibrinogen-binding peptide that binds preferentially to hole a of fibrinogen over hole b of fibrinogen, and a second fibrinogen-binding peptide that binds preferentially to hole b of fibrinogen over hole a of fibrinogen.

25. The composition according to claim 24, wherein the first fibrinogen-binding peptide comprises an amino acid sequence GPRP (SEQ ID NO: 1) at its amino-terminal end.

26. The composition according to claim 24, wherein the second fibrinogen-binding peptide comprises an amino acid sequence GHRP (SEQ ID NO: 10), preferably an amino acid sequence GHRPY (SEQ ID NO: 11), at its amino terminal end.

27. The composition according to claim 1, wherein the biocompatible liquid is an aqueous solution.

28. The composition according to claim 27, wherein the aqueous solution is a saline solution.

29. The composition according to claim 1, wherein the polysaccharide comprises a glycosaminoglycan, oxidized cellulose, chitosan, chitin, alginate, oxidized alginate, or oxidized starch.

30. The composition according to claim 29, wherein the glycosaminoglycan comprises hyaluronic acid.

31. The composition according to claim 1, wherein the particles comprise cross-linked hyaluronic acid granules, wherein a majority of the granules have a diameter in the range 100-1500 ?m in partially or fully hydrated form.

32. The composition according to claim 1 having a solids content of 1-70%, or 5-20%, by weight of the composition.

33. The composition according to claim 1, wherein a weight ratio of the particles to the liquid is from 1:1 to 1:12, or from 1:3 to 1:8.

34. The composition according to claim 1, wherein the composition is transparent.

35.-37. (canceled)

38. A method of treating bleeding, which comprises administering an effective amount of the composition according to claim 1 to a bleeding wound.

39. A method of treating a wound, which comprises administering an effective amount of the composition according to claim 1 to a wound.

40. A method of making a sterile, ready-to-use, flowable haemostatic composition, which comprises: mixing together a biocompatible liquid, a soluble haemostatic agent, and particles of a biocompatible cross-linked polysaccharide suitable for use in haemostasis and which are insoluble in the biocompatible liquid, wherein the soluble haemostatic agent comprises a plurality of carriers and a plurality of fibrinogen-binding peptides immobilised to each carrier; and sterilizing the composition.

41. The method according to claim 40, wherein the composition is sterilized by steam sterilization, or by dry-heat sterilization.

42. The method according to claim 40, wherein the liquid, the agent, and the particles are mixed under conditions effective to form a continuous liquid phase comprising the particles substantially homogeneously dispersed throughout the liquid phase, thereby forming a substantially homogeneous haemostatic composition.

43. The method according to claim 40 claim 40, which further comprises centrifuging the mixture to remove air bubbles from the mixture.

44. The method according to claim 40, wherein the biocompatible liquid and the particles are mixed together before addition of the soluble haemostatic agent.

45. The method according to claim 44, which further comprises centrifuging the mixture of the biocompatible liquid and the particles to remove air bubbles from the mixture before addition of the soluble haemostatic agent.

Description

[0235] Embodiments of the invention are now described by way of example only, with reference to the accompanying drawings in which:

[0236] FIG. 1 shows the ability of a peptide dendrimer for use in a preferred embodiment to polymerise fibrinogen at varying concentrations;

[0237] FIG. 2 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. 3 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. 4 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. 5 shows a photograph of hydrogels formed by polymerisation of fibrinogen using different peptide dendrimers;

[0241] FIG. 6 shows the ability of different combinations of peptide dendrimers with peptide conjugates to polymerise fibrinogen at varying concentrations;

[0242] FIG. 7 shows the ability of several different peptide dendrimers to polymerise fibrinogen in human plasma;

[0243] FIG. 8A shows a schematic drawing of rabbit liver lobes indicating the approximate position of liver biopsy injuries. FIG. 8B illustrates how the degree of bleeding was assessed on a scale of 0 to 5;

[0244] FIG. 9 is a photograph of a biopsied rabbit liver. A biopsy site treated with a control is shown above the label Control. A biopsy site treated with a composition according to an embodiment of the invention is shown above the label HA paste+HXP12;

[0245] FIG. 10 shows a plot of the haemostatic effect (% haemostatic success) of different embodiments of a composition of the invention in treating bleeding of biopsied rabbit liver, compared with a control, over time; and

[0246] FIG. 11A shows a photograph of a transparent paste containing cross-linked hyaluronic acid gel particles. FIG. 11B shows a photograph of an embodiment of a composition of the invention formed by mixing the transparent paste with a haemostatic agent. FIG. 11C shows a photograph of a syringe containing an embodiment of a composition of the invention which has been sterilized in situ. FIG. 110 shows a photograph of an embodiment of a composition of the invention that has been extruded through a syringe. FIG. 11E shows a photograph of an embodiment of a transparent composition of the invention that has been deposited over surgical suture material.

EXAMPLE 1

[0247] Synthesis of Peptide Dendrimers and Peptide Conjugates

[0248] Peptides were synthesised on Rink amide MBHA low loaded resin (Novabiochem, 0.36 mmol/g), by standard Fmoc peptide synthesis, using Fmoc or Boc protected amino acids (Novabiochem).

[0249] 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.

[0250] 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.

[0251] For automated synthesis a threefold excess of amino acid and HBTU was used for each coupling and a ninefold excess of diisopropylethylamine (DIPEA, Sigma) in dimethylformamide (DMF, Sigma).

[0252] 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).

[0253] 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.

[0254] Branches were made using Fmoc-Lys(Fmoc)-OH, Fmoc-Lys(Boc)-OH, or Fmoc-Lys(Mtt)-OH.

[0255] 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.

[0256] 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.

[0257] Peptide Dendrimers and Peptide Conjugates

[0258] The structures of peptide dendrimers and peptide conjugates synthesised using the methods described above are shown below.

[0259] 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.

##STR00020## ##STR00021## ##STR00022## ##STR00023## ##STR00024## ##STR00025##

EXAMPLE 2

[0260] Copolymerisation of a Peptide Dendrimer with Fibrinogen

[0261] 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.

[0262] 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. 1 shows a plot of the polymerisation (clotting) times (in seconds) with increasing concentration of dendrimer.

[0263] 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 3

[0264] Effect of Varying the Number of Fibrinogen-Binding Peptides Per Dendrimer on the Speed of Copolymerisation with Fibrinogen

[0265] This example investigates the effect of varying the number of fibrinogen-binding peptides per peptide dendrimer on the speed of copolymerisation with fibrinogen.

[0266] 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 2. The concentration of each dendrimer was varied from 0.005-0.5 mg/ml. FIG. 2 shows a plot of the clotting times (in seconds) with increasing concentration of each different dendrimer.

[0267] 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 from ?0.125 to ?0.275 mg/ml, the speed of copolymerisation increased. At concentrations below ?0.125 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 range ?0.02-0.5 mg/ml, dendrimer no. 12 (with five fibrinogen-binding peptides/dendrimer) produced almost instantaneous clotting. In the range ?0.05-0.3 mg/ml, dendrimer no. 11 (with four fibrinogen-binding peptides/dendrimer) also produced almost instantaneous clotting.

[0268] It is concluded that the speed at which fibrinogen is polymerised by a dendrimer generally increases as the number of fibrinogen-binding peptides per dendrimer is increased.

EXAMPLE 4

[0269] Effect of Fibrinogen-Binding Peptide Orientation, and of Different Fibrinogen-Binding Peptide Sequences on Speed of Copolymerisation with Fibrinogen

[0270] To assess whether the orientation of a fibrinogen-binding peptide could affect the ability of 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.

[0271] The fibrinogen-binding peptides of Peptide Dendrimer Nos. 3 and 10 are each of sequence GPRPG (SEQ ID NO: 17). 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.

[0272] Two of the fibrinogen-binding peptides of Peptide Dendrimer No. 8 are of sequence GPRPG (SEQ ID NO: 17), and the third fibrinogen-binding peptide is of sequence APFPRPG (SEQ ID NO: 14) orientated with its amino-terminal end attached to the branched core. The carboxy-terminal end of that peptide comprises an amide group.

[0273] Two of the fibrinogen-binding peptides of Peptide Dendrimer No. 9 are of sequence GPRPFPA (SEQ ID NO: 3), and the third fibrinogen-binding peptide is of sequence APFPRPG (SEQ ID NO: 14) orientated with its amino-terminal end attached to the branched core. The carboxy-terminal end of that peptide comprises an amide group.

[0274] The sequence GPRPG (SEQ ID NO: 17) binds to hole a and hole b of fibrinogen, but with some preference for hole a. The sequence GPRPFPA (SEQ ID NO: 3) 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).

[0275] The ability of the dendrimers to copolymerise with fibrinogen was assessed using the same method described in Example 2, for a concentration of each dendrimer ranging from 0.005-0.5 mg/ml. FIG. 3 shows a plot of the clotting times (in seconds) obtained with increasing concentration of each different dendrimer.

[0276] 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 clotting time of Dendrimer No. 3 with that of Dendrimer No. 10). However, at higher fibrinogen concentrations, Dendrimer No. 3 was able to copolymerise with fibrinogen (data not shown).

[0277] 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).

[0278] 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: 3)), 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: 14)) orientated with its amino-terminal end attached to the branched core (Dendrimer No. 9) was also very active in copolymerising with fibrinogen.

EXAMPLE 5

[0279] Ability of Peptide Dendrimers with Different Fibrinogen-Binding Peptide Sequences to Copolymerise with Fibrinogen

[0280] The GPRPG (SEQ ID NO: 15) and GPRPFPA (SEQ ID NO: 3) 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.

[0281] Peptide dendrimer No. 13 is a chimeric four-branch peptide dendrimer comprising two fibrinogen-binding peptides with sequence GPRPG- (SEQ ID NO: 17) (which has a binding preference for the a hole), and two fibrinogen-binding peptides with sequence GHRPY (SEQ ID NO: 11) (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: 17). Each fibrinogen-binding peptide of Dendrimers Nos. 11, 12, and 13 is attached at its carboxy-terminal end to the branched core.

[0282] The ability of the dendrimers to copolymerise with fibrinogen was assessed using the same method described in Example 2, for a concentration of each dendrimer ranging from 0.005-0.5 mg/ml. FIG. 4 shows a plot of the clotting times (in seconds) obtained with increasing concentration of each different dendrimer.

[0283] 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. 5 shows a photograph of the hydrogels obtained using the different dendrimers. The gels are labelled 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 6

[0284] Ability of Mixtures of Peptide Dendrimers and Peptide Conjugates to Copolymerise with Fibrinogen

[0285] Fibrinogen-binding peptide of sequence GPRP (SEQ ID NO: 1) 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.

[0286] Fibrinogen-binding peptide of sequence GHRPY (SEQ ID NO: 11) binds strongly and preferentially to the b hole of fibrinogen (Doolittle and Pandi, Biochemistry 2006, 45, 2657-2667). Peptide Conjugate 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: 16)), 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.

[0287] The linker allows the peptides to extend away from each other.

[0288] 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 2, for a concentration of each dendrimer ranging from 0.025-0.5 mg/ml. FIG. 6 shows a plot of the clotting times (in seconds) obtained with increasing concentration of each different dendrimer.

[0289] 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-knob peptides) were not active when added to either Peptide Conjugate No. 2 or the peptide dendrimers.

EXAMPLE 7

[0290] Ability of Peptide Dendrimers to Polymerise Fibrinogen in Human Plasma

[0291] The ability of several different peptide dendrimers (Nos. 4, 5, 8, 9, 10, 11, 12, 13) to polymerise fibrinogen in human plasma was tested.

[0292] 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.

[0293] The clotting times for each dendrimer are shown in FIG. 7, 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 8

[0294] Effect of Steam Sterilisation on a Haemostatic Agent in Solution

[0295] This example describes the effect of steam sterilisation on the haemostatic activity of a haemostatic agent (Peptide Dendrimer No. 12 (see Example 1): HXP12) formulated in saline.

[0296] HXP12 at a concentration of 50 mg/ml was diluted with 150 mM sodium chloride to a concentration of 0.5 mg/ml. The formulation was prepared as a 6 ml bulk solution (using 60 ?l of HXP12 stock). 400 ?l of this bulk solution was used for each 2 ml glass vial, with a screw-fit air-tight lid. Each vial was autoclaved (200 kPa) for 25 minutes at 121? C. After sterilisation, the vials were placed at 40? C. and stored for up to 27 weeks.

[0297] To test the ability of the stored samples to polymerise fibrinogen, each sample was diluted with 20 mM phosphate buffer, pH 7.6, to a concentration of 0.05 mg/ml. 30 ?l of each diluted sample was added to 100 ?l of human fibrinogen, at a concentration of 3 mg/ml, formulated in 20 mM phosphate buffer, pH 7.6. The ability of HXP12 in each diluted sample to polymerise fibrinogen (the clotting activity) at 37? C. was determined using a Sigma Amelung KC4 Delta coagulation analyser. The polymerisation activity of non-sterilised, control samples was also determined. The results are summarized in Table 1 below.

TABLE-US-00002 TABLE 1 Clotting activity (seconds) in human fibrinogen Non Autoclaved After sterilization storage @ 40? C. HXP12 autoclaved @ 121? C. 4 wks 7 wks 13 wks 27 wks c = 0.05 1.1 1.0 0.9 1.2 1.1 1.1 mg/ml

[0298] The results in Table 1 show that the haemostatic agent formulated in saline retains its ability to polymerise fibrinogen after sterilization by steam in an autoclave (200 kPa) for 25 minutes at 121? C., and that this activity is retained even after storage at 40? C. for at least 27 weeks.

EXAMPLE 9

[0299] Effect of Steam Sterilisation on a Ready-to-Use, Flowable, Haemostatic Composition

[0300] This example describes the effect of steam sterilisation on the haemostatic activity of a haemostatic agent (HXP12) formulated as a ready-to-use, flowable paste comprising Hyaluronic Acid (HA) cross-linked particles.

[0301] 0.6 ml of a solution of HXP12 dissolved in water was mixed with 1.4 g of HA hydrogel particles hydrated in 10 mM phosphate buffer (HA concentration 2.7%; cross-linking 5:1 [HA/divinyl sulfone DVS], fully hydrated particle size 400 ?m) to form a paste in which the concentration of HXP12 was 1 mg/ml. 200 mg of the paste was aliquoted into to glass vials, and each vial was closed with a lid. The vials were autoclaved (200 kPa) for 25 min at 121? C. After sterilisation, vials were placed at 80? C. for an extra 16 hours to simulate an accelerated aging process. The samples were assessed at 4 and 16 hours.

[0302] HXP12 was extracted from the stored samples, and diluted with 20 mM phosphate buffer, pH 7.2, to a concentration of 0.1 mg/ml. 30 ?l of each extracted sample was added to 100 ?l of human plasma (Alpha Labs), and the ability of HXP12 in each diluted sample to polymerise fibrinogen (the clotting activity) at 37? C. was determined using a Sigma Amelung KC4 Delta coagulation analyser. The polymerisation activity of non-sterilised, control samples was also determined. The results are summarized in Table 2 below.

TABLE-US-00003 TABLE 2 Clotting activity (seconds) in human plasma After sterilization, accelerated aging Non Autoclaved study @ 80? C. Extracted HXP12 autoclaved @ 121? C. 4 hours 16 hours c = 0.1 mg/ml 2.0 2.6 2.8 4.8

[0303] The results in Table 2 show that HXP12 peptide, formulated as a ready-to-use, flowable paste with HA hydrogel particles, retains ability to polymerise fibrinogen from human plasma after sterilization by steam in an autoclave (200 kPa) for 25 minutes at 121? C., and that this activity is retained even after storage at 80? C. for at least 4 hours.

EXAMPLE 10

[0304] Effect of Steam Sterilisation on a Ready-to-Use, Flowable, Haemostatic Composition

[0305] This example describes the effect of steam sterilisation on the haemostatic activity of a haemostatic agent (HXP12) formulated as a ready-to-use, flowable paste made of Hyaluronic Acid (HA) cross-linked particles.

[0306] 0.6 ml of a solution of HXP12 formulated in 10 mM phosphate buffer, 160 mM Arg.HCl, pH 6.8, was mixed with 1.4 g of HA hydrogel particles (HA concentration 2.7%; cross-linking 5:1 [HA/divinyl sulfone DVS], fully hydrated particle size 400 ?m) to form a paste in which the concentration of HXP12 was 1 mg/ml. 200 mg of the paste was aliquoted into glass vials, and each vial was closed with a lid. The vials were autoclaved (200 kPa) for 25 min at 121? C. After sterilisation, vials were placed at 40? C. The samples were assessed at 0, 2 and 4 weeks.

[0307] HXP12 was extracted from the stored samples, and diluted with 20 mM phosphate buffer, pH 7.2, to a concentration of 0.06 mg/ml. 30 ?l of each extracted sample was added to 100 ?l of human fibrinogen at a concentration of 3 mg/ml (the level of fibrinogen found in the blood) formulated in 20 mM phosphate buffer, pH 7.2. The ability of HXP12 in each diluted sample to polymerise fibrinogen (the clotting activity) at 37? C. was determined using a Sigma Amelung KC4 Delta coagulation analyser. The polymerisation activity of non-sterilised, control samples was also determined. The results are summarized in Table 3 below.

TABLE-US-00004 TABLE 3 Clotting activity (seconds) in human fibrinogen @ c = 3 mg/ml After sterilization, accelerated aging Non Autoclaved study @ 40? C. Extracted HXP12 autoclaved @ 121? C. 2 weeks 4 weeks c = 0.06 mg/ml 1.0 3.3 3.6 5.4

[0308] The results in Table 3 show that HXP12 peptide, formulated as a ready-to-use, flowable paste with HA hydrogel particles, retains ability to polymerise fibrinogen from human fibrinogen after sterilization in an autoclave for 25 minutes at 121? C. (200 kPa), and that this activity is retained even after storage at 40? C. for at least 2 weeks.

EXAMPLE 11

[0309] Assessment of the Haemostatic Activity of a Haemostatic Composition of the Invention in a Rabbit Liver Biopsy Injury Model

[0310] This example describes testing of the haemostatic activity of three different compositions of the invention, each with a different concentration of a haemostatic agent (HXP12 peptide dendrimer).

[0311] Methods

[0312] 7 g of HA paste (HA concentration 2.7%; 5:1; HA/DVS, fully hydrated particle size 400 ?m) was prefilled into a syringe and mixed with 3 ml of HXP12 peptide dendrimer at one of three different concentrations, resulting in 10 ml of the final product. The final HXP12 concentration for each 10 ml product was: sample B, 1 mg/ml; B2, 0.5 mg/ml; B3, 1.4 mg/ml. As a control (C), 7 g of HA paste was mixed with 3 ml of saline.

[0313] Heparinised rabbits (Breed: New Zealand White; Sex: Males) were anaesthetized. All three lobes of the liver were withdrawn from the abdominal cavity and laid on saline-wet gauze swabs. Samples were tested on biopsy injuries which were created sequentially on the three liver lobes as set out below in Table 3. FIG. 8A shows the approximate orientation and order of liver injury on the three lobes.

TABLE-US-00005 TABLE 4 Order and location of injury to liver lobes Cut No: 1 2 3 4 5 6 7 Lobe: Left Left Central Central Right Right Right Cut name: LL1 LL2 CL1 CL2 RL1 RL2 RL3

[0314] Biopsies were created on the lobes of the liver using a 6 mm biopsy punch to approximately 5 mm depth. A pre-weighed dry swab was used to collect blood exiting the wound for 15 seconds. The swab was then weighed as a measure of bleeding severity. After removal of the swab, the wound was dried with another swab and then the test samples applied.

[0315] For the application of tested samples, a saline-moistened sterile gauze swab was applied against the bleeding surface, and the syringe was used to dispense up to 2 ml of Sample B, B2, or B3, or 2 ml of the control (C), between the gauze and the bleeding surface into the biopsy wound. Gentle pressure was applied to the gauze swab for one minute after application. Upon removal of the moist gauze, the wound was evaluated for haemostasis at 1, 3, 6, 9 and 12 min after the application of the test sample (i.e. including one minute application of pressure).

[0316] Bleeding scores of 0, 1, 2, 3, 4 and 5 were assigned for no bleeding, oozing, very mild, mild, moderate, and severe bleeding, respectively (FIG. 8B). The scores for the degree of bleeding were adapted from Adams et al (J Thromb Thrombolysis DOI 10.1007/s 11239-008-0249-3). On successful haemostasis, the lobe was covered with a saline soaked swab and the procedure repeated until each lobe had received treatment as described above.

[0317] Results

[0318] FIG. 9 is a photograph of one of the biopsied livers. Blood can be seen flowing from a biopsy site treated with the control (shown above the label Control), whereas the haemostatic effect at a biopsy site treated with a composition of the invention comprising HA paste and HXP12 (shown above the label HA paste+HXP12) is clearly visible.

[0319] FIG. 10 shows a plot of the haemostatic effect (% haemostatic success) of samples B, B2, and B3, compared with the control, over the time (in minutes) of the evaluation. In contrast to the control, each of the different compositions comprising HA paste and HXP12 peptide dendrimer demonstrated strong coagulant activity. This activity was dose-dependent, with the compositions having higher concentrations of HXP12 (samples B and B3) demonstrating approximately 80%-100% haemostatic activity throughout the 12-minute evaluation. The composition with the lowest concentration of HXP12 (sample B2) demonstrated 100% haemostatic activity for the first three minutes of the evaluation, but this then reduced to ?75% over the remaining 9 minutes.

[0320] This example shows that an embodiment of a composition of the invention comprising HA particles that are essentially not haemostatic, and a peptide dendrimer that has coagulant properties, is surprisingly effective in controlling bleeding.

EXAMPLE 12

[0321] A Sterile, Ready-to-Use, Flowable Haemostatic Composition Comprising Cross-Linked HA Gel Particles, and a Haemostatic Agent

[0322] A flowable paste made from cross-linked hyaluronic acid (HA) gel particles (HA concentration 2.7%; 5:1; HA/DVS, fully hydrated particle size 400 ?m) was made transparent by centrifuging the paste at 600 rpm for 5 minutes. The transparent paste is shown in FIG. 11A.

[0323] 7 g of the transparent HA paste was mixed with 3 ml of HXP12 peptide dendrimer (formulated in 10 mM phosphate buffer, 160 mM Arg.HCl, pH 6.8) resulting in 10 ml of the final product. The final concentration of HXP12 was 1.05 mg/mI. FIG. 11B shows a photograph of some of the resulting composition. The photograph shows that the composition is sufficiently cohesive to form a continuous layer over a wound, and thus can be used to seal a wound.

[0324] The composition was placed in a glass vial, and sterilised by steam sterilisation in an autoclave (200 kPa) for 25 minutes at 121? C.

[0325] FIG. 11C shows a photograph of a syringe containing the composition. FIG. 11D shows a syringe containing the composition, in which some of the composition has been extruded though the opening at the tip of the syringe barrel using its plunger.

[0326] FIG. 11E shows a photograph of an embodiment of a transparent composition of the invention that has been deposited over surgical suture material of size code 0, diameter 0.3-0.39 mm. The suture material is clearly visible through the transparent composition.

[0327] A surgeon can see through a transparent composition of the invention when administering it. This makes it much easier to administer the composition correctly, and determine whether it has been effective in controlling or stopping bleeding.