Therapeutic agents with improved fibrinogen binding

Abstract

Therapeutic agents with improved fibrinogen binding properties are described. The agents are suitable for use as artificial platelets, or for formation of biogels. Methods and intermediates for producing the agents, cross-linking agents for use in the methods, and biogels formed from, or comprising the agents, are also described.

Claims

1. An agent, comprising a carrier and a plurality of peptides that are selected from fibrinogen binding peptides and fibrinogen binding precursor peptides, wherein each peptide is covalently immobilised to the carrier by a non-peptide spacer that is covalently linked directly to a main chain α-carbonyl group at a carboxy-terminal end of the peptide.

2. The agent of claim 1, wherein the non-peptide spacers do not form intramolecular bonds.

3. The agent of claim 1, wherein each non-peptide spacer comprises a hydrophilic group.

4. The agent of claim 3, wherein at least one of: (i) the hydrophilic group is at least 17.6 Å in length, (ii) the hydrophilic group is a straight chain group, (iii) the hydrophilic group comprises an ethylene oxide group, (iv) the hydrophilic group comprises an ethylene oxide group that is a polyethylene glycol group of formula —(CH.sub.2CH.sub.2O).sub.x—, where x is 2-24, and (v) the hydrophilic group is covalently linked to the main chain α-carbonyl group of the peptide by a straight chain linker.

5. The agent of claim 3, wherein the hydrophilic group is covalently linked to the main chain α-carbonyl group of the peptide by a straight chain linker, and wherein the straight chain linker comprises at least one of (i) a group of formula —(CH.sub.2).sub.a—, wherein a is 1-4, and (ii) a group of formula —NH—(CH.sub.2).sub.a—NH—, wherein a is 1-4.

6. A pharmaceutical composition comprising the agent of claim 1; and a pharmaceutically acceptable carrier, excipient, or diluent.

7. A biogel formed from, or which comprises, the agent of claim 1.

Description

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

(2) FIG. 1 shows a reaction scheme for production of a peptide-spacer conjugate for reaction with a carrier according to a preferred embodiment of the invention: i) Ethlyelene diamine DIPEA, DCM, 30 min Loading Approx. 0.3-0.5 mmol/g; ii) Peptide Synthesis; iii) Cleavage from the resin, 3% TFA in DCM Neutralise immediately with pyridine/MeOH Evaporate solvent, purify on C18 column; iv) React the protected peptide with NHS-PEG-MAL and DIPEA in DMF:ratio 1:1:2. Reaction followed by ESMS when complete mixture acidified/neutralised with formic acid before purification by C18 HPLC; v) Freeze dried protected peptide side chain is deprotected in 96% TFA, 2% water, 2% triisopropyl silane for 60 min, solvent is removed by evaporation and purified by C18 HPLC;

(3) FIG. 2 shows a reaction scheme for immobilisation of a fibrinogen binding peptide to a carrier according to a preferred embodiment of the invention: i) Reaction 1 h at room temperature, unreacted 2-IT is removed by filtration; ii) Reaction 1 h at room temperature, unreacted peptide conjugate is removed by filtration; and

(4) FIG. 3 shows the results of stability testing of a preferred embodiment of the invention, comprising fibrinogen-binding peptides immobilised to HSA carrier, in solution stored at 37° C. for 6 months.

(5) A preferred embodiment of an agent of the invention comprises a fibrinogen binding peptide (in this embodiment, the fibrinogen binding peptide has the sequence H.sub.2N-Gly-Pro-Arg-Pro-Gly-(SEQ ID NO: 4)) covalently immobilised to a carrier by a non-peptide spacer that is covalently linked directly to a main chain α-carbonyl group at a carboxy-terminal end of the peptide. The agent of the preferred embodiment has the following formula:
[GPRPG-CONH—(CH.sub.2).sub.2—NHCO—(CH.sub.2CH.sub.2O).sub.12—(CH.sub.2).sub.2NHCO(CH.sub.2).sub.2-succinimide-S—(CH.sub.2).sub.3CNH.sub.2.sup.+-].sub.nCarrier
where n is >1.

(6) A preferred method for producing an agent of the preferred embodiment is shown in FIGS. 1 and 2, and described below.

(7) Ethylene diamine is reacted with a 2-chlorotrityl resin to produce a resin derivatised with a non-peptide linking group attached to a primary amine group: —NH(CH.sub.2).sub.2NH.sub.2 (step (i) of FIG. 1).

(8) The peptide is then synthesised starting at the carboxy-terminal end of the peptide. The carboxy-terminal amino acid is joined to the non-peptide linking group of the derivatised resin by reaction of the carboxy group of the amino acid with the primary amine reactive group attached to the linking group. Additional amino acids are then added using a conventional F-moc solid-phase peptide synthesis method (step (ii) of FIG. 1).

(9) The amino-terminal end, and the functional groups of the amino acid side chains of the peptide are protected by protecting groups. In this example, the amino-terminal end is protected with a t-Butoxycarbonyl (t-Boc) group, and the primary amine group of the arginine side chain is protected with a 2,2,4,6,7-pentamethyldihydrobenzofurane (Pbf) group. Other suitable protecting groups will be apparent to the skilled person depending on the particular amino acid residues present.

(10) Once the peptide has been synthesised, it is cleaved from the resin to provide a peptide-linker conjugate comprising a protected peptide with a primary amine group attached to the peptide by a non-peptide linking group (—NH(CH.sub.2).sub.2—) linked directly to the main chain α-carbonyl group at the carboxy-terminal end of the peptide (step (iii) of FIG. 1).

(11) This peptide-linker conjugate is then reacted with a heterobifunctional cross-linking agent comprising a thiol-reactive group (a maleimide group) joined to an amine-reactive group (an N-hydroxysuccinmide (NHS) group) by a non-peptide spacer group (a PEG.sub.n spacer). The NHS group of the cross-linker reacts with the primary amine group of the peptide-linker conjugate. The resulting peptide-spacer conjugate comprises a protected peptide attached to a non-peptide spacer group (PEG.sub.n) by a non-peptide linker (—NH(CH.sub.2).sub.2NHCO—), with a thiol-reactive group (maleimide group) attached to the non-peptide spacer group (step (iv) of FIG. 1). The protected peptide of the peptide-spacer conjugate is then deprotected (step (v) of FIG. 1).

(12) A carrier (a human serum albumin (HSA) carrier) is derivatised with thiol groups using the derivatising agent 2-iminothiolane (2-IT). 2-IT reacts with the primary amine group on the side chain of lysine residues of the carrier to provide the carrier with thiol groups linked to the carrier by a non-peptide linker (—(CH.sub.2).sub.3CNH.sub.2.sup.+—) (step (i) of FIG. 2). This derivatised carrier is then reacted with the peptide-spacer conjugate to produce the agent of the preferred embodiment (step (ii) of FIG. 2).

(13) A method of synthesis of the agent of the preferred embodiment is described in more detail in Example 4 below, with reference to FIGS. 1 and 2.

(14) Example 1 below describes synthesis of another preferred embodiment in which the peptide-spacer conjugate is reacted with thiol groups of an underivatised carrier. This embodiment has the following structure:
[GPRPG-CONH—(CH.sub.2).sub.2—NHCO—(CH.sub.2CH.sub.2O).sub.12—(CH.sub.2).sub.2NHCO(CH.sub.2).sub.2-succinimide-].sub.nCarrier
where n is >1.

EXAMPLE 1

Synthesis of an Agent Comprising a Fibrinogen Binding Peptide Immobilised to a Carrier by a Non-Peptide Spacer Linked Directly to a Main Chain α-Carbonyl Group at a Carboxy-Terminal End of the Peptide

(15) Synthesis of a Peptide-Linker Conjugate

(16) A 2 chlorotrityl chloride resin is firstly derivatised with ethylenediamine (CAS 107-15-3) (dry DCM, DIPEA), then Fmoc amino acids are coupled in series using standard Fmoc solid phase peptide synthesis techniques to generate the synthetic peptide Gly-Pro-Arg-Pro-Gly (SEQ ID NO: 4) attached to etheylenediamine. The N-terminal glycine amino acid is introduced as the Boc variant.

(17) The side chain, and N-terminally protected peptide is then cleaved from the resin. The peptide is first cleaved using 1% trifluoroacetic acid (TFA) in dichloromethane (DCM) 5×3 mins, then 1.5% TFA, 5×3 mins the fractions pooled and freeze dried. The protected peptide is HPLC purified and freeze dried.

(18) Synthesis of a Peptide-Spacer Conjugate

(19) The free C-terminal amine of the peptide-linker conjugate is reacted with the heterobifunctional cross-linker succinimidyl-[(N-maleimidopropionamido)-dodecaethyleneglycol]ester (NHS-PEG.sub.12-Maleimide) (Pierce Co. Product #22112) for 2-3 hours at room temperature in dimethylformamide (DMF) containing 100 ul N,N′-diisopropylethylamine (DIPEA), and the reaction monitored by mass spectrometry. The peptide is reacted in a 1.1 molar excess over the NHS-PEG.sub.12-Maleimide. Once the reaction is complete, the product is HPLC purified and freeze dried. Protecting groups are cleaved in 95% (v/v) TFA, 2.5% (v/v) triisopropylsilane, and 2.5% (v/v) water for 1 h at room temperature. The peptide-spacer conjugate is precipitated via the addition of ether, filtered, HPLC purified, and freeze dried to yield the final compound.

(20) Reaction of the Peptide-Spacer Conjugate with the Carrier

(21) The peptide-spacer conjugate is reacted with a carrier comprising spray-dried human serum albumin microparticles for 1 hour at room temperature. Sulfhydryl groups of the carrier react with the maleimide group of the peptide-spacer conjugate to form a stable thioether bond, and thereby immobilise the peptide to the carrier via a non-peptide spacer linked directly to a main chain α-carbonyl group at a carboxy-terminal end of the peptide. Unreacted peptide conjugate is removed by filtration.

(22) The non-peptide spacer group (PEG.sub.12) has the following advantages: increases the accessibility of the fibrinogen binding peptide to fibrinogen, helps maintain solubility, increases stability, high flexibility, reduced tendency toward aggregation, and reduced immunogenicity.

EXAMPLE 2

Synthesis of an Agent Comprising Fibrinogen Binding Peptide Immobilised to Carrier by a Non-Peptide Spacer Linked to a Lysine Residue at a Carboxy-Terminal End of the Peptide

(23) Synthesis of a Peptide with Carboxy-Terminal Lysine

(24) The synthetic peptide Gly-Pro-Arg-Pro-Lys (SEQ ID NO: 10) was prepared using standard Fmoc solid-phase peptide synthesis techniques. The N-terminal glycine amino acid is introduced as the Boc variant. The side chain, and N-terminally protected peptide is then cleaved from the resin. The peptide is first cleaved using 1% trifluoroacetic acid (TFA) in dichloromethane (DCM) 5×3 mins, then 1.5% TFA, 5×3 mins the fractions pooled and freeze dried. The protected peptide is HPLC purified and freeze dried.

(25) Synthesis of a Peptide-Spacer Conjugate

(26) The free amine of the C-terminal lysine of the protected peptide is reacted with the heterobifunctional cross-linker succinimidyl-[(N-maleimidopropionamido)-dodecaethyleneglycol]ester (NHS-PEG.sub.12-Maleimide) (Pierce Co. Product #22112) for 2-3 hours at room temperature in dimethylformamide (DMF) containing 100 ul N,N′-diisopropylethylamine (DIPEA), and the reaction monitored by mass spectrometry. The peptide is reacted in a 1.1 molar excess over the NHS-PEG.sub.12-Maleimide. Once the reaction is complete, the conjugate-protected peptide is HPLC purified and freeze dried. Protecting groups are cleaved in 95% (v/v) TFA, 2.5% (v/v) triisopropylsilane, and 2.5% (v/v) water for 1 h at room temperature. The peptide is precipitated via the addition of ether, filtered, HPLC purified, and freeze dried to yield the final compound.

(27) Reaction of the Peptide-Spacer Conjugate with the Carrier

(28) The peptide-spacer conjugate with the C-terminal lysine was immobilised to carrier in the same way as described in Example 1.

EXAMPLE 3

Fibrinogen Binding Efficiency of Agents with Differently Immobilised Fibrinogen-Binding Peptides

(29) The fibrinogen binding efficiency of the agents synthesised as described in Examples 1 and 2 was determined by measuring the amount of fluorescently labeled fibrinogen bound to the different agents by the following method:

(30) 5 ml of fibrinogen solution at 20 mg/ml was prepared in 20 mM sodium phosphate buffer pH 7.4 containing 0.15M NaCl to which 5.55 umoles of fluorescein isothiocyanate (FITC) is added in 0.54 ml of dimethyl sulfoxide (DMSO). This reaction was protected from light and incubated at room temperature for 1 h with gentle agitation. To separate the non-conjugated FITC from the FITC-fibrinogen the reaction (5 ml) was loaded on to Sephadex G25 column (XK16) equilibrated 20 mM sodium phosphate buffer, 0.15M NaCl, 1 mM EDTA (pH 7.2). The void fractions containing the fibrinogen were pooled and stored at −20° C. until needed. To determine the F/P ratio (mole/mole) the absorbance of a sample was read by a spectrophotometer at 280 and 495 nm. The calculation for determining the ratio is described by Xia et al, 1996 (Optimally functional fluorescein isothiocyanate-labelled fibrinogen for quantitative studies of binding to activated platelets and platelet aggregation. Br J. Haematol. 1996; 93:204-14). The F/P ratios for the human fibrinogen were typically between 1.5 and 4.

(31) Spray-dried human serum albumin microparticles with immobilized peptide (Example 1) at 0.2 mg/ml protein equivalents were incubated with FITC-fibrinogen (1 mg/ml) for 30 min at room temperature. The reaction was fixed with 0.2% formyl saline and then analyzed by flow cytometry. Microparticles were identified on the basis of their forward and side scatter and their mean fluorescence measured in arbitrary units. The number of fibrinogen molecules bound per particle was calculated from a standard curve of fluorescence reference standard microbeads (Bangs Laboratories, Inc.).

(32) The results of the flow cytometry analysis are shown in Table 1 below:

(33) TABLE-US-00001 TABLE I Amount of Number of fibrinogen molecules peptide reacted bound per microparticle to microparticles (mg) Lysine Ethylenediamine 1:1 5607 8317 2:1 6501 10103

(34) These results show that an agent comprising fibrinogen binding peptide immobilised to carrier by a non-peptide spacer linked directly to the main chain α-carbonyl group at a carboxy-terminal end of the peptide was able to bind more fibrinogen molecules than an agent in which the fibrinogen binding peptide was immobilised by a non-peptide spacer linked to a side chain of a carboxy-terminal lysine residue of the peptide.

EXAMPLE 4

Synthesis of an Agent Comprising Fibrinogen Binding Peptide Immobilised to Derivatised Carrier by a Non-Peptide Spacer Linked Directly to a Main Chain α-Carbonyl Group at a Carboxy-Terminal End of the Peptide

(35) Synthesis of a Peptide-Spacer Conjugate

(36) A peptide spacer conjugate is synthesised as described in Example 1.

(37) Derivatisation of the Carrier with 2-Iminothiolane (2-IT)

(38) 2-IT is used to derivatise the carrier to provide thiol groups for reaction with the maleimide group of the peptide-spacer conjugate. The carrier (formed from HSA in this example), is incubated with 2-IT, which reacts with primary amines on the carrier to introduce sulfhydryl groups. Unreacted 2-IT is removed by filtration.

(39) Reaction of the Peptide-Spacer Conjugate with the Derivatised Carrier

(40) The peptide-spacer conjugate is reacted with the derivatised carrier for 1 hour at room temperature. Sulfhydryl groups of the derivatised carrier react with the maleimide group of the peptide-spacer conjugate to form a stable thioether bond, and thereby immobilised the peptide to the carrier via a non-peptide spacer linked directly to a main chain α-carbonyl group at a carboxy-terminal end of the peptide. Unreacted peptide conjugate is removed by filtration.

(41) The linker between the carrier and the non-peptide spacer group (—(CH.sub.2).sub.3CNH.sub.2.sup.4″) is a straight-chain aliphatic group. This has reduced immunogenicity compared with conventional linkages that include a cyclic group. This linker also preserves the original positive charge of the primary amine thereby helping to maintain the solubility of the protein.

EXAMPLE 5

Conjugation of Fibrinogen-Binding Peptide to an Albumin Carrier

(42) This example describes conjugation of a peptide-spacer conjugate of the invention, comprising a fibrinogen-binding peptide of sequence GPRPG (SEQ ID NO: 4) linked to a maleimide group (Mal) by a polyethylene glycol (PEG) linker, to an albumin carrier. The resulting product is referred to as “PeproStat”, and is a preferred embodiment of an agent of the invention.

(43) Human serum albumin is diluted to 50 mg/mL in reaction buffer (50 mM sodium phosphate, 150 mM sodium chloride, 100 mM Ethylenediaminetetraacetic acid [EDTA], pH 8.0±0.2) and thiolated by the addition of a sixty-fold molar excess of 2-iminothiolane hydrochloride. After incubation for one hour at room temperature, thiolated albumin is separated from unreacted 2-iminothiolane hydrochloride by tangential flow diafiltration using 20 mM sodium phosphate, 150 mM sodium chloride, 1 mM EDTA pH 7.2±0.2 (filtration buffer).

(44) Peptide conjugation is performed by dissolving peptide (GPRPG-PEG12-maleimide) (SEQ ID NO: 11) at a concentration of 50 mg/mL in filtration buffer and adding to thiolated albumin at a ratio of 0.95 mg peptide per 1 mg albumin. Following incubation for one hour at room temperature, excess peptide is removed by dialysing against a 60-fold excess of Tris-buffered saline (TBS; 20 mM Tris, 150 mM sodium chloride pH 7.2±0.2) using dialysis membrane with a molecular weight cut-off of 10-14 KD for at least 16 hours at 4° C., with one change of buffer. Recovered PeproStat is diluted to 5 mg/mL in TBS, sterile filtered through a 0.2 μm filter, dispensed and stored at 4° C.

(45) The protein content of the final product is estimated by measuring absorbance at 280 nm where E(280, 1%)=5.3.

(46) The activity of the final product is investigated using a Sigma Amelung KC4 coagulometer. Briefly, 30 μL test sample at 0.5 mg/ml is added to 100 μL purified human fibrinogen at 3 mg/ml, the KC4 registers clot formation (i.e. formation of a co-polymer of PeproStat and fibrinogen which has characteristics of a fibrin clot) in less than 6 seconds.

(47) The molecular weight of the final product is estimated by SDS-PAGE of reduced samples using 4-15% Tris-glycine precast gels stained with Coomassie by comparison with the band profile of unstained protein ladder.

(48) Product Profile

(49) TABLE-US-00002 Test Parameter Profile Adsorption at E280 Total protein 5 mg/ml Clotting activity Activity 0.5 mg/ml clots in <6 seconds SDS PAGE Molecular weight 85-120 KD

EXAMPLE 6

Haemostatic Properties of Gelatin Carriers with Immobilised Fibrinogen-Binding Peptides

(50) This example describes preparation of two different agents of the invention, and their haemostatic properties. Both agents comprise a gelatin carrier, with a plurality of fibrinogen-binding peptides covalently immobilised to the carrier via a PEG linker. To prepare the first agent, 2-iminothiolane was used to covalently attach a plurality of peptide-spacer conjugates of the invention (comprising fibrinogen-binding peptide covalently attached to PEG) to the gelatin carrier. To prepare the second agent, cystamine moieties were conjugated to the carboxyl groups of gelatin in the presence of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), followed by reductive cleavage of the introduced disulphide bond to generate a free thiol for attachment of the fibrinogen-binding peptide.

(51) Preparation of Gelatin Granules Conjugated with GPRPG-PEG-12-Mal (SEQ ID NO: 11) Using 2-Iminothiolane

(52) Gelatin granules were thiolated using 2-iminothiolane which modifies amine residues. The method used was that of Kommareddy S, Amiji M, 2005, Bioconjugate Chem 16: 1423-1432.

(53) 1 g of gelatin granules were weighed and hydrated in 40 ml of a buffer containing 50 mM sodium phosphate, 0.15M NaCl, 0.1M EDTA pH 8.0±0.2, by mixing on a roller mixer for 10 minutes at room temperature. 102 g of 2-iminothiolane are added to the hydrated gelatin and mixed on a roller mixer for 1 hour. The granules were then spun at 500 rpm-RCF 28 for 2 minutes, the supernatant removed, and volume replaced with 20 mM sodium phosphate, 0.15M NaCl, 0.1 M EDTA pH 7.2±0.2. This was repeated four times to remove the 2-iminothiolane.

(54) An Ellman's assay was performed to measure the number of —SH groups introduced onto the gelatin. Ellman's reagent 5,5′dithiobis(2-nitrobenzoic acid) reacts with sulphydryls under slightly alkaline conditions to release the highly chromogenic compound, 5-thio-2 nitrobenzoic acid (TNB) Ellman GL. (1959) Arch Bichem. Biophys. 82 70-77.

(55) Following quantitation of the —SH groups, 2.5 ml GPRPG-PEG-12-Mal (SEQ ID NO: 11) at 50 mg/ml was added to the granules and roller mixed for 1 hour. The granules were then washed four times with distilled water to remove excess peptide. A slurry of the granules was placed in a plastic container and dried by incubating at 37° C. for 15 hours.

(56) Preparation of Gelatin Granules Conjugated with GPRPG-PEG-12-Mal (SEQ ID NO: 11) Using EDC/Cystamine Chemistry

(57) 2.2 g of gelatin granules were weighed out and hydrated in 80 mL of 50 mM MES buffer, pH 6.0, for 15 minutes on a roller mixer. 625 mg of Cystamine, 350 mg of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and 130 mg of N-hydroxysuccinimide (NHS) were weighed out and added to hydrated granules. The reaction mixture was left for 2 hours on a roller mixer at ambient temperature, and then split into two 50 mL tubes. The granules were then washed and spun at 500 rpm with 4×40 mL volumes of MES buffer. 200 μL of 1M tris(2-carboxyethyl)phosphine (TCEP) stock was added to each tube and left for 10 minutes on the roller mixer at ambient temperature. A repeat washing with 4 volumes of MES was followed by an Ellman's Assay to determine free SH.

(58) 7.2 mL of GPRPG-PEG-12-Mal (SEQ ID NO: 11) at 50 mg/ml was mixed with 10.45 mL of N-ethyl-maleimide and then 8.8 mL of the mixture was added to each tube. The reaction was left for 1 hour on a roller mixer at ambient temperature.

(59) Reactions were washed and spun at 800 rpm with 4 volumes of Milli-Q water to remove excess peptide and N-ethyl-maleimide. Granules were then poured into a plastic box, covered with Nescofilm, the Nescofilm pierced and placed in a bench-top Freeze-Dryer for drying as follows:

(60) Drying Process for Conjugated Granules

(61) The shelves of the freeze-dryer were equilibrated at −36° C., and the gelatin granules placed on the pre-frozen shelf and subjected to thermal treatment steps bringing the temperature up to −20° C. over a period of 270 minutes. Primary drying was accomplished over 800 minutes decreasing the vacuum from 800 mTorr with a concomitant increase in temperature to 20° C. The freeze-dried granules were stored in a desiccator prior to testing.

(62) Testing of Dry Gelatin Granules in a Plug Disintegration Test

(63) 100 mg of dry conjugated gelatin granules were packed into a 3 ml syringe, and then 0.5 ml tris-buffered saline (TBS) (0.02M Tris, 0.15M NaCl, pH 7.2±0.2) was added, using a syringe connector, to suspend the granules. 0.5 ml thrombin at 500 u/ml or 0.5 ml TBS was added to 100 mg “blank” (non-conjugated gelatin granules). Using a syringe connector, TBS was passed from another 3 ml syringe into each formulation to suspend it. Each suspension was then mixed by passing it between the syringes approximately 40 times.

(64) The gelatin slurry was added to a third 3 ml syringe and the syringe plunger used to form a plug. 0.2 ml of plasma was then injected into the plug, and left to stand for 3 minutes. The bottom of the syringe was cut off and the plug pushed out into a 50 ml tube containing 0.9% saline. The tube was then mixed on a vortex mixer for up to 10 minutes. The plug was then scored over a period of 10 minutes as follows:

(65) 0=plug disintegrated entirely; 2=small lumps present (2-5 mm in size); 5=larger lumps present (5-8 mm); 8=large plug intact, signs of erosion; 10=plug completely intact.

(66) Results

(67) TABLE-US-00003 Sample Time point (minutes) Score Conjugated granules 1 10 3 10 7 10 10 10 Thrombin/granules 1 10 3 10 7 10 10 10 TBS/granules 1 0 3 0 7 0 10 0

(68) The results show that the mechanical durability of the plug formed using the conjugated gelatin granules of the invention is equivalent to that of the non-conjugated granules mixed with thrombin.

EXAMPLE 7

Stability of Fibrinogen-Binding Peptides Immobilised to Carrier in Solution

(69) This example describes the results of stability testing of fibrinogen-binding peptides immobilised to carrier in solution at 37° C.

(70) PeproStat (comprising fibrinogen-binding peptides, each of sequence GPRPG (SEQ ID NO: 4) immobilised to HSA carrier) was stored in solution for 6 months at 37° C. At time zero, and various times during the storage period, samples of the stored solution were assayed for ability to form a co-polymer with fibrinogen, as follows:

(71) Fibrinogen was diluted in 10 mM HEPES, 0.15M NaCl, pH 7.3+/1 0.2 to 6 mg/ml. 25 μl PeproStat at 5 mg/ml was added to 4000 of the diluted fibrinogen, and the time taken for formation of a visual clot comprising a co-polymer of PeproStat and fibrinogen was recorded.

(72) The results are shown in FIG. 3, and demonstrate that PeproStat is stable in solution at 37° C. for at least 6 months.