Peptide dendrimers comprising fibrinogen-binding peptides

Abstract

Peptide dendrimers and agents are described, which can be used for polymerising fibrinogen and as haemostatic agents. The peptide dendrimers comprise a branched core, and a plurality of fibrinogen-binding peptides separately covalently attached to the branched core. The branched core comprises: i) 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; il) 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; Hi) 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; iv) 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 y) 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, 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.

Claims

1. A peptide dendrimer that comprises a branched core, and a plurality of fibrinogen-binding peptides separately covalently attached to the branched core by a non-peptide linker, wherein the peptide dendrimer comprises the structure of Formula (I): ##STR00023## where: FBP is a fibrinogen-binding peptide; -(linker)- is a non-peptide linker comprising —NH(CH.sub.2).sub.nCO—, where n is 1-10; X is a tri-functional amino acid residue; Y is -FBP or —NH.sub.2; and Z is -[—X-(linker)-FBP].sub.a-(linker)-FBP, where a is 1-10.

2. The peptide dendrimer according to claim 1, wherein the tri-functional amino acid residues of Formula (I) comprise a lysine, ornithine, arginine, aspartic acid, glutamic acid, asparagine, glutamine, or cysteine residue.

3. The peptide dendrimer according to claim 1, wherein the fibrinogen-binding peptides bind preferentially to hole ‘a’ of fibrinogen over hole ‘b’ of fibrinogen.

4. The peptide dendrimer according to claim 1, wherein the fibrinogen-binding peptides bind preferentially to hole ‘b’ of fibrinogen over hole ‘a’ of fibrinogen.

5. The peptide dendrimer according to claim 1, which does not comprise the following: ##STR00024##

6. A composition comprising the peptide dendrimer according to claim 1, and a peptide conjugate comprising two or more fibrinogen-binding peptides.

7. The composition according to claim 6, wherein the peptide conjugate is a peptide dendrimer comprising a branched core and a plurality of fibrinogen-binding peptides separately covalently attached to the branched core by a non-peptide linker, wherein the branched core of said peptide dendrimer comprises: from two to ten multi-functional amino acid residues, wherein each fibrinogen-binding peptide is separately covalently attached, via a non-peptide linker, 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, via a non-peptide linker, 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, via a non-peptide linker, 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, via a non-peptide linker, 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.

8. A pharmaceutical composition, which comprises the peptide dendrimer according to claim 1, and a pharmaceutically acceptable carrier, excipient, or diluent.

9. The pharmaceutical composition according to claim 8, which is a ready-to-use haemostatic formulation in which the pharmaceutically acceptable carrier, excipient, or diluent comprises hydrated gelatin.

10. The peptide dendrimer according to claim 1, which is sterile.

11. A method of sterilising the peptide dendrimer according to claim 1, which comprises: exposing the peptide dendrimer to gamma irradiation.

12. A method of polymerising fibrinogen, which comprises: contacting fibrinogen with the peptide dendrimer according to claim 1.

13. A kit for formation of a hydrogel, which comprises the peptide dendrimer according to claim 1 and, separately, fibrinogen.

14. A hydrogel comprising a copolymer of the peptide dendrimer according to claim 1, and fibrinogen.

15. A method of treating bleeding, or of treating a wound, which comprises: administering the peptide dendrimer according to claim 1 to a site of bleeding or to a wound.

16. A method according to claim 15, which comprises administering fibrinogen and the peptide dendrimer to the site of bleeding or to the wound.

17. A pharmaceutical composition, which comprises the composition according to claim 6 and a pharmaceutically acceptable carrier, excipient, or diluent.

18. The composition of claim 6, which is sterile.

19. A method of sterilising the composition of claim 6 comprising: exposing the composition to gamma irradiation.

20. A method of polymerising fibrinogen, which comprises: contacting fibrinogen with the composition of claim 6.

21. A kit for formation of a hydrogel comprising: the composition of claim 6 and, separately, fibrinogen.

22. A hydrogel comprising a copolymer of the composition of claim 6 and fibrinogen.

23. A method of treating bleeding, or of treating a wound, which comprises: administering the composition of claim 6 to a site of bleeding or to a wound.

24. The peptide dendrimer of claim 1, wherein said dendrimer comprises: ##STR00025##

25. The peptide dendrimer according to claim 1, wherein the tri-functional amino acid residues of Formula (I) are lysine residues.

Description

(1) Embodiments of the invention are now described by way of example only, with reference to the accompanying drawings in which:

(2) FIG. 1 shows the ability of a peptide dendrimer of a preferred embodiment to polymerise fibrinogen at varying concentrations;

(3) 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;

(4) 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;

(5) 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;

(6) FIG. 5 shows a photograph of hydrogels formed by polymerisation of fibrinogen using different peptide dendrimers of the invention;

(7) FIG. 6 shows the ability of different combinations of peptide dendrimers of the invention with peptide conjugates to polymerise fibrinogen at varying concentrations; and

(8) FIG. 7 shows the ability of several different peptide dendrimers of the invention to polymerise fibrinogen in human plasma.

EXAMPLE 1

Synthesis of Peptide Dendrimers and Peptide Conjugates

(9) 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).

(10) 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.

(11) 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.

(12) 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).

(13) 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).

(14) 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.

(15) Branches were made using Fmoc-Lys(Fmoc)-OH, Fmoc-Lys(Boc)-OH, or Fmoc-Lys(Mtt)-OH.

(16) 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.

(17) 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.

(18) Peptide Dendrimers and Peptide Conjugates

(19) The structures of peptide dendrimers and peptide conjugates synthesised using the methods described above are shown below.

(20) 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.

(21) ##STR00017## ##STR00018## ##STR00019## ##STR00020## ##STR00021## ##STR00022##

EXAMPLE 2

Copolymerisation of a Peptide Dendrimer with Fibrinogen

(22) 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.

(23) The ability of Peptide Dendrimer No. 12 to polymerise fibrinogen was assessed. 30 μl of dendrimer in solution, at concentration ranging from 0.005-2 mg/ml, was added to 100 μl 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.

(24) 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

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

(25) This example investigates the effect of varying the number of fibrinogen-binding peptides per peptide dendrimer on the speed of copolymerisation with fibrinogen.

(26) 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.

(27) 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.

(28) 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 4

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

(29) 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.

(30) The fibrinogen-binding peptides of Peptide Dendrimer Nos. 3 and 10 are each of sequence GPRPG (SEQ ID NO: 15). 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.

(31) Two of the fibrinogen-binding peptides of Peptide Dendrimer No. 8 are of sequence GPRPG (SEQ ID NO: 15), 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.

(32) 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.

(33) 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: 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).

(34) 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.

(35) 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).

(36) 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).

(37) 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

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

(38) 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.

(39) Peptide dendrimer No. 13 is a chimeric four-branch peptide dendrimer comprising two fibrinogen-binding peptides with sequence GPRPG- (SEQ ID NO: 15) (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: 15). Each fibrinogen-binding peptide of Dendrimers Nos. 11, 12, and 13 is attached at its carboxy-terminal end to the branched core.

(40) 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.

(41) 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

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

(42) 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.

(43) 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.

(44) The linker allows the peptides to extend away from each other.

(45) 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.

(46) 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

Ability of Peptide Dendrimers to Polymerise Fibrinogen in Human Plasma

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

(48) 30 μL of each dendrimer (at a concentration of 0.25 mg/ml) was added to 100 μL human plasma at 37° C., and polymerisation of fibrinogen was determined using a Sigma Amelung KC4 Delta coagulation analyzer.

(49) 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

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

(50) 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.

(51) 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.

(52) After storage, peptide dendrimers were extracted from each paste using 10 mM HEPES buffer. 30 μL of each extract (with a peptide concentration of about 0.25 mg/ml) was added to 100 μL 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.

(53) TABLE-US-00001 Clotting activity (seconds) Peptide Non- Storage for 2 Storage for 4 dendrimer irradiated weeks post weeks post no. control irradiation irradiation 12 1 1 1 13 4.3 9.4 10

(54) 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.