ANTICOAGULANT FUSION PROTEINS AND USES THEREOF

Abstract

Disclosed is a fusion protein including an Ixodes ricinus salivary gland polypeptide. In particular, it relates to a fusion protein including at least one Ixodes ricinus salivary gland polypeptide, at least one serum albumin polypeptide and at least one linker peptide. Also disclosed is the use of such a fusion protein for preventing or treating thrombus formation and/or thrombus growth, as well as pharmaceutical compositions, medicaments and methods including such a fusion protein.

Claims

1.-19. (canceled)

20. A fusion protein comprising (i) at least one Ixodes ricinus salivary gland (IrCPI) polypeptide, having an amino sequence at least 75% identical to SEQ ID NO: 1, (ii) at least one serum albumin polypeptide and (iii) at least one peptide linker, wherein the fusion protein has an increased circulatory half-life compared to an unfused IrCPI polypeptide.

21. The fusion protein according to claim 20, wherein said at least one serum albumin polypeptide is a human serum albumin polypeptide.

22. The fusion protein according to claim 20, wherein said at least one peptide linker is selected from a group comprising Gly-rich linkers, Ser-rich linkers, Gly-rich and Ser-rich linkers, Pro-rich linkers, helical linkers, linkers non-cleavable by a protease, linkers cleavable by a protease and a combination thereof.

23. The fusion protein according to claim 20, wherein said at least one peptide linker is a Gly-rich and Ser-rich linker selected from a group comprising (G.sub.nS).sub.m linker wherein n is a number from 1 to 10 and wherein m is a number from 1 to 10; (G.sub.nS).sub.mA linker, wherein n is a number from 1 to 10 and wherein m is a number from 1 to 10; and a GS(GGS).sub.nGS linker wherein n is a number from 1 to 20.

24. The fusion protein according to claim 20, wherein said at least one peptide linker is a Pro-rich linker selected from a group comprising (AP).sub.n and P(AP).sub.n linker; wherein n is a number from 1 to 20.

25. The fusion protein according to claim 20, wherein said at least one peptide linker is a Pro-rich linker having the amino acid sequence (AP).sub.14 (SEQ ID NO: 26).

26. The fusion protein according to claim 20, wherein said at least one peptide linker is a helical linker selected from a group comprising (EAAAK).sub.n linker, wherein n is a number from 1 to 10 (SEQ ID NO: 14); [A(EAAAK).sub.nA].sub.m, wherein n is a number from 2 to 10 and m is a number from 1 to 5 (SEQ ID NO: 15); and A(EAAAK).sub.nALEA-(EAAAK).sub.mA wherein n is a number from 1 to 10 and m is a number from 1 to 10 (SEQ ID NO: 22).

27. The fusion protein according to claim 20, wherein the fusion protein is selected from a group of fusion proteins having an amino acid sequence comprising or consisting in the sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48.

28. A pharmaceutical composition or a medicament comprising a fusion protein comprising (i) at least one Ixodes ricinus salivary gland (IrCPI) polypeptide, having an amino sequence at least 75% identical to SEQ ID NO: 1, (ii) at least one serum albumin polypeptide and (iii) at least one peptide linker, wherein the fusion protein has an increased circulatory half-life compared to an unfused IrCPI polypeptide or a polynucleotide thereof, and at least one pharmaceutically acceptable excipient.

29. The pharmaceutical composition or medicament according to claim 28, wherein said at least one peptide linker is selected from a group comprising Gly-rich linkers, Ser-rich linkers, Gly-rich and Ser-rich linkers, Pro-rich linkers, helical linkers, linkers non-cleavable by a protease, linkers cleavable by a protease and a combination thereof.

30. The pharmaceutical composition or medicament according to claim 28, wherein said at least one peptide linker is a Gly-rich and Ser-rich linker selected from a group comprising (G.sub.nS).sub.m linker wherein n is a number from 1 to 10 and wherein m is a number from 1 to 10; (G.sub.nS).sub.mA linker, wherein n is a number from 1 to 10 and wherein m is a number from 1 to 10; and a GS(GGS).sub.nGS linker wherein n is a number from 1 to 20.

31. A medical device coated with a fusion protein comprising (i) at least one Ixodes ricinus salivary gland (IrCPI) polypeptide, having an amino sequence at least 75% identical to SEQ ID NO: 1, (ii) at least one serum albumin polypeptide and (iii) at least one peptide linker, wherein the fusion protein has an increased circulatory half-life compared to an unfused IrCPI polypeptide.

32. The medical device according to claim 31, wherein said at least one peptide linker is selected from a group comprising Gly-rich linkers, Ser-rich linkers, Gly-rich and Ser-rich linkers, Pro-rich linkers, helical linkers, linkers non-cleavable by a protease, linkers cleavable by a protease and a combination thereof.

33. The medical device according to claim 31, wherein said at least one peptide linker is a Gly-rich and Ser-rich linker selected from a group comprising (G.sub.nS).sub.m linker wherein n is a number from 1 to 10 and wherein m is a number from 1 to 10; (G.sub.nS).sub.mA linker, wherein n is a number from 1 to 10 and wherein m is a number from 1 to 10; and a GS(GGS).sub.nGS linker wherein n is a number from 1 to 20.

34. A method for treating or preventing thrombus formation and/or growth, or thromboinflammation in a subject in need thereof, said method comprising administering to said subject an effective therapeutic dose of a fusion protein comprising (i) at least one Ixodes ricinus salivary gland (IrCPI) polypeptide, having an amino sequence at least 75% identical to SEQ ID NO: 1, (ii) at least one serum albumin polypeptide and (iii) at least one peptide linker, wherein the fusion protein has an increased circulatory half-life compared to an unfused IrCPI polypeptide.

35. The method according to claim 34, wherein said at least one peptide linker is selected from a group comprising Gly-rich linkers, Ser-rich linkers, Gly-rich and Ser-rich linkers, Pro-rich linkers, helical linkers, linkers non-cleavable by a protease, linkers cleavable by a protease and a combination thereof.

36. The method according to claim 34, wherein said at least one peptide linker is a Gly-rich and Ser-rich linker selected from a group comprising (G.sub.nS).sub.m linker wherein n is a number from 1 to 10 and wherein m is a number from 1 to 10; (G.sub.nS).sub.mA linker, wherein n is a number from 1 to 10 and wherein m is a number from 1 to 10; and a GS(GGS).sub.nGS linker wherein n is a number from 1 to 20.

37. A method for inhibiting platelet recruitment, neutrophil recruitment, neutrophil activation and/or neutrophil extracellular trap formation (NETosis) in a subject in need thereof, said method comprising administering to said subject an effective therapeutic dose of a fusion protein comprising (i) at least one Ixodes ricinus salivary gland (IrCPI) polypeptide, having an amino sequence at least 75% identical to SEQ ID NO: 1, (ii) at least one serum albumin polypeptide and (iii) at least one peptide linker, wherein the fusion protein has an increased circulatory half-life compared to an unfused IrCPI polypeptide.

38. The method according to claim 37, wherein said at least one peptide linker is selected from a group comprising Gly-rich linkers, Ser-rich linkers, Gly-rich and Ser-rich linkers, Pro-rich linkers, helical linkers, linkers non-cleavable by a protease, linkers cleavable by a protease and a combination thereof.

39. The method according to claim 37, wherein said at least one peptide linker is a Gly-rich and Ser-rich linker selected from a group comprising (G.sub.nS).sub.m linker wherein n is a number from 1 to 10 and wherein m is a number from 1 to 10; (G.sub.nS).sub.mA linker, wherein n is a number from 1 to 10 and wherein m is a number from 1 to 10; and a GS(GGS).sub.nGS linker wherein n is a number from 1 to 20.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0218] FIG. 1 shows pharmacokinetic profile of V13 and IrCPI in rabbits. Test-items were administered at 65 nmol/kg (iv). V13 and IrCPI concentrations were measured by ELISA except for time point T=0 h which corresponds to the theoretical administered dose. Results are expressed as meanSEM. N=2 per time point.

[0219] FIGS. 2A and 2B show effect of purified yeast recombinant IrCPI-fusion proteins on aPTT ratio in human plasma: effect of IrCPI-fusion proteins V2, V9, V13 and native IrCPI (A); effect of IrCPI-fusion proteins V12, V13, V15, V18, V19, V20, V27 and native IrCPI (B). Percentage of purity obtained and purification method used are indicated in brackets.

[0220] FIGS. 3A, 3B and 3C show pharmacokinetic and pharmacodynamic profiles of V13 in rabbits. V13 was administered at 260 nmol/kg (i.v.). (A) The pharmacokinetic profile was determined by measuring V13 concentrations by ELISA. (B) Pharmacodynamics were measured by aPTT on samples from the same animals. aPTT ratios correspond to the clotting time measured post-administration divided by the clotting time measured before administration. (C) The pharmacokinetic-pharmacodynamics relationship of V13 in rabbits. Results are expressed as meanSEM. N=2 per time point.

[0221] FIG. 4 shows inhibitory effect of V13 and IrCPI on FXI and FXII coagulation activities in human plasma. FXI and FXII curves represent the fitting to a hyperbolic equation. Black and white circles represent individual data.

[0222] FIG. 5 shows effects of Ir-CPI and V13 on thrombus weight (mg) in the rat AV-shunt. UFH was used as positive control of the test. One-way ANOVA followed by a Dunnett's multiple comparison test was used for statistical analysis (*p<0.05; **p<0.01; ***p<0.001). All data were compared to the vehicle condition.

[0223] FIG. 6 shows effect of V13 administration on the aPTT ratio after 20 min of rat AV-shunt. V13 was administered as a bolus, 5 min before AV-shunt opening. Vehicle corresponds to a bolus of PBS. Results are expressed as meanSEM (n>5 per group). Statistical significance versus corresponding vehicle group as determined by a one-way ANOVA and a Dunnett's multiple comparison post-hoc test (***p<0.001).

[0224] FIGS. 7A, 7B, 7C and 7D show effect of V13 on thrombin generation. Human plasma was incubated with various concentrations of V13. Thrombin production was monitored after activation by Actin FS (A) or by tissue factor (TF, three concentrations: 1 pM, 5 pM and 20 pM, B-D), i.e. activators of the intrinsic and of the extrinsic coagulation pathway, respectively.

[0225] FIGS. 8A, 8B, 8C and 8D show effect of V13 on thrombin generation: Analysis of dose-response curves. Endogenous Thrombin Potential (ETP, A) i.e. area under the curve of thrombin generation; Maximal amplitude of thrombin generation (Peak, B); Delay up to start and up to maximal generation of thrombin (Lag time and time to Peak, C and D respectively).

[0226] FIGS. 9A and 9B show effect of V13 and synthetic IrCPI on thrombin generation in the presence of Actin FS or tissue factor (5 pM): peak (A) and lag time (B). Results are expressed as percentage compared to control (i.e. absence of V13 or IrCPI).

EXAMPLES

[0227] The present invention is further illustrated by the following examples.

Example 1: Construction of the Fusion Proteins

[0228] Twenty-three constructions of IrCPI in fusion with human serum albumin (HSA) were investigated (Table 1).

TABLE-US-00001 TABLE 1 Constructions of fusion proteins comprising Ir-CPI and human serum albumin Fusion protein name Constructions MW (kDa) V1 IrCPI-HSA 74.13 V2 HSA-IrCPI 74.13 V3 IrCPI-GGGGS-HSA 74.44 V4 HSA-GGGGS-IrCPI 74.44 V5 IrCPI-IrCPI-HSA 81.78 V10 HSA-IrCPI-IrCPI 81.78 V7 IrCPI-PAPAP-HSA 74.56 V6 HSA-PAPAP-IrCPI 74.56 V8 IrCPI-AEAAAKEAAAKA-HSA 75.21 V9 HSA-AEAAAKEAAAKA-IrCPI 75.21 V12 IrCPI-(AP).sub.7-HSA 75.3 V18 HSA-(AP).sub.7-IrCPI 75.3 V13 IrCPI-(AP).sub.14-HSA 76.5 V19 HSA-(AP).sub.14-IrCPI 76.5 V14 IrCPI-(G.sub.4S).sub.3A-HSA 75.1 V20 HSA-(G.sub.4S).sub.3A-IrCPI 75.1 V15 IrCPI-GS(GGS).sub.9GS-HSA 76.2 V21 HSA-GS(GGS).sub.9GS-IrCPI 76.2 V16 IrCPI-A(EAAAK).sub.4A-HSA 76.2 V22 HSA-A(EAAAK).sub.4A-IrCPI 76.2 V17 IrCPI-A(EAAAK).sub.4ALEA-(EAAAK).sub.4A- 78.4 HSA V23 HSA-A(EAAAK).sub.4ALEA-(EAAAK).sub.4A- 78.4 IrCPI V27 IrCPI-(AP).sub.7-IrCPI-(AP).sub.14-HSA 85.3

[0229] These differentiate by the presence or absence of peptide linker between the HSA and IrCPI molecule, the nature and the size of the linker used and the position of HSA relative to the IrCPI molecule (amino terminal or carboxy terminal of IrCPI). IrCPI-fusion proteins and IrCPI were produced in yeast Pichia pastoris with an expression vector under the control of AOX1 promoter. A point mutation in the oligonucleotide coding sequence of IrCPI was introduced in all constructions, leading to the replacement of Asparagine (Asp54) by Glutamine in order to prevent N-glycosylation. This modification in the amino acid sequence of IrCPI has been proven to have no consequence on the pharmacological activity of the molecule.

Example 2: Pharmacokinetic Profile of Fusion Proteins in Rabbits

[0230] Material and Methods

[0231] Male New-Zealand white rabbits (3-4 kg), received IrCPI-fusion protein (65 nmol/kg or 260 nmol/kg) or IrCPI (65 nmol/kg or 130 nmol/kg) under a volume of administration of 1 mL/kg (for 65 nmol/kg V2, V9, V13 and V20 variants) or 2 mL/kg (for 65 nmol/kg V13 and for 260 nmol/kg V12, V13, V18 and V19 variants) as a slow bolus, by intravenous route (marginal ear vein). The day before administration, a catheter was introduced into a carotid artery under isoflurane anesthesia for blood sampling. Blood samples were collected 5 min, 10 min, 30 min, 45 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 24 h, 32 h, 48 h and 56 h after intravenous administration of the test items. Blood was collected from the catheter at each time-point in 0.109 M 3.2% sodium citrate tubes (citrate/blood, v/v 1/9) and plasma prepared after two consecutive centrifugations at 2500 g for 15 min.

[0232] Concentrations of IrCPI and of IrCPI-fusion proteins in the rabbit plasma were determined by sandwich ELISA using an anti-IrCPI monoclonal antibody (as capture antibody) and an anti-IrCPI polyclonal antibody (as detection antibody). As shown in Table 2, IrCPI and some IrCPI-fusion proteins were measured by an ELISA technique in which the anti-IrCPI polyclonal antibody was coupled to acetylcholinesterase to allow spectrophotometric monitoring using acetylthiocholine as the substrate and DTNB as the chromophore. The other IrCPI-fusion proteins were measured by an ELISA technique in which the anti-IrCPI polyclonal antibody is biotinylated and used in association with streptavidin-HRP to allow a spectrophotometric monitoring using 3, 3, 5, 5-tetra-methylbenzidine (TMB Sureblue) as the substrate.

TABLE-US-00002 TABLE 2 Dose administered and ELISA method used for native IrCPI and IrCPI fusion protein tested IrCPI or IrCPI- Dose fusion protein (nmol/kg) ELISA method (detection antibody) IrCPI 65 Polyclonal coupled to acetylcholinesterase 130 Polyclonal coupled to acetylcholinesterase V2 65 Polyclonal coupled to acetylcholinesterase V9 65 Polyclonal coupled to acetylcholinesterase V12 260 Polyclonal biotinylated + streptavidin-HRP V13 65 Polyclonal biotinylated + streptavidin-HRP 260 Polyclonal biotinylated + streptavidin-HRP V15 65 Polyclonal biotinylated + streptavidin-HRP V18 260 Polyclonal coupled to acetylcholinesterase V19 260 Polyclonal coupled to acetylcholinesterase V20 65 Polyclonal biotinylated + streptavidin-HRP V27 260 Polyclonal biotinylated + streptavidin-HRP

[0233] Pharmacokinetic parameters were estimated using the PK Solutions 2.0 software. A compartmental analysis (intravenous bolus and two-compartments) was performed. The individual plasma concentration data were plotted against time after dosing for each dose. The pharmacokinetic parameters were derived from these data.

[0234] Results

[0235] Results are presented in Table 3.

TABLE-US-00003 TABLE 3 Pharmacokinetic parameters of IrCPI-fusion proteins and native IrCPI in rabbits Parameters IrCPI V2 V9 V12 V13 Dose 65-130 65 65 260 65-260 (nmol/kg, iv) Dose 0.5-1 5 5 20 5-20 (mg/kg, iv) AUC 12116- 618109 344942 398165* 264953- (min * 11430* 318651* nmol/kg) CL (mL/ 5.40-5.76 0.10 0.21 0.17 0.25-0.22 min/kg) T1/2 alpha 39-35 211 154 142 185-146 (min) T1/2 beta 415-474 2.792 1.072 1.115 1.579-987 (min) Vc 0.677-0.878 0.087 0.122 0.099 0.236-0.125 (L/kg) Vz 3.225-3.913 0.425 0.289 0.268 0.566-0.292 (L/kg) Parameters V15 V18 V19 V20 V27 Dose 65 260 260 65 260 (nmol/kg, iv) Dose 5 20 20 5 20 (mg/kg, iv) AUC 475274 267681* 177882* 544296 29059* (min * nmol/kg) CL (mL/ 0.10 0.20 0.40 0.12 2.20 min/kg) T1/2 alpha 78 218 190 141 56 (min) T1/2 beta 1.047 1.020 1.012 1.082 1.010 (min) Vc 0.106 0.191 0.253 0.092 0.268 (L/kg) Vz 0.209 0.366 0.552 0.190 3.233 (L/kg) *AUC: Area Under the Curve normalized for a same administered dose of 65 nmol/kg

[0236] Circulating exposure to the compound (AUC, area under the curve normalized for a same administered dose of 65 nmol/kg) was between 2.5-fold (V27) and 54-fold (V2) higher with fusion proteins than that obtained with native IrCPI. Moreover, fusion proteins have a lower clearance (CL) than native IrCPI and, except for V27, a lower volume of distribution (Vz) which translates also to a longer terminal half-life (t.sub.1/2), up to 3.8-fold higher than native IrCPI. As t.sub.1/2 relates to the time for a molecule to be degraded or eliminated from the blood circulation, a longer t.sub.1/2 means a longer duration of action.

[0237] The volume of distribution of the IrCPI-fusion proteins is lower than that of native IrCPI meaning the IrCPI-fusion proteins remain advantageously in the vascular compartment where they may exert their activity.

[0238] The study conducted on different IrCPI-fusion proteins indicates that the fusion of IrCPI with human serum albumin improves consistently and whatever the variant, the PK profile of the native IrCPI molecule.

[0239] As example, FIG. 1 illustrates the larger and longer exposure obtained with V13 as competed to IrCPI administered at same molar dose.

Example 3: Anticoagulant Activity

[0240] Material and Methods

[0241] The anticoagulant activities of the IrCPI-fusion proteins were determined by a coagulation test on a human plasma pool using an activator of the contact phase to induce coagulation. IrCPI-fusion proteins were diluted in a buffer of 20 mM Hepes, 140 mM NaCl (pH 7.35). Native IrCPI was used as a comparator in this assay.

[0242] Activated Partial Thromboplastin Time (aPTT)

[0243] Plasma (platelet poor plasma, PPP) was mixed with different concentrations of an IrCPI fusion protein or synthetic IrCPI to reach final concentrations ranging from 0 to 2 M. Next, 50 L of each IrCPI fusion protein-containing plasma or IrCPI-treated plasma, was incubated for 240 seconds with 50 l of Actin FS (ellagic acid+phospholipids). The clotting reaction was started by adding 50 l of 25 mM CaCl.sub.2) and the clotting time (aPTT) was monitored. aPTT ratios were calculated by dividing the clotting time measured in the presence of test item (i.e. IrCPI-fusion protein or Ir-CPI) by the clotting time obtained without test substance.

[0244] Results

[0245] FIG. 2 shows that IrCPI fusion proteins prolonged the aPTT in a concentration-dependent manner with variable degree of efficacy. aPTT results were obtained from 2 independent laboratories and divided in 2 separates graphs. On FIG. 2A, by decreasing order, V13 was the most active variant, followed by V9 and V2. V13 increased the time to coagulate (aPTT) by 67% at 0.5 M, 84% at 1 M and 106% at 2 M, respectively. On FIG. 2B, V27 was the most active variant, followed by V13, next V12, V19, V18, V15 and then V20. V27 increased the time to coagulate (aPTT) by 58% at 0.5 M, 98% at 1 M and 152% at 2 M, respectively.

Example 4: Pharmacokinetic and Pharmacodynamics Relationship with V13

[0246] Material and Methods

[0247] Male New Zealand white rabbits (2-3.5 kg), received V13 (20 mg/kg corresponding to 260 nmol/kg) under a volume of administration of 2 mL/kg as a slow bolus, by intravenous route (marginal ear vein).

[0248] Blood was collected from the catheter at each time-point in 0.109 M 3.2% sodium citrate tubes (citrate/blood, v/v 1/9) and plasma prepared after two consecutive centrifugations at 2500 g for 15 min.

[0249] Concentrations of V13 in rabbit plasma was determined by sandwich ELISA using an anti-IrCPI monoclonal antibody (as capture antibody) and an anti-IrCPI polyclonal antibody (as detection antibody) coupled to acetylcholinesterase to allow spectrophotometric monitoring using acetylthiocholine as the substrate and DTNB as the chromophore.

[0250] Pharmacodynamics were measured by aPTT, according to the same protocol as Example 3, at the time points 45 min, 4 h, 10 h and 24 h post-administration.

[0251] Results

[0252] Similar profiles were observed for V13 plasma concentrations and aPTT ratio in rabbit plasma ex vivo, i.e. both showed a progressive decrease over time after bolus administration (FIGS. 3A, B and C). The time to coagulate was increased by +408%, +220% and +45% at respectively 45 minutes, 4 h and 10 h post-administration and corresponded to mean plasmatic concentrations of 1.87 M, 0.97 M and 0.55 M. The results confirm that amount of the active principle (monitored by aPTT ratio) was consistent with the circulating concentration of V13 (monitored by ELISA method).

Example 5: Evaluation of the Inhibitory Effect of V13 on Factors XI and XII Activities

[0253] The effect of V13 on the inhibition of factors XI and XII activities was assessed in an aPTT-based coagulation assay using FXII or FXI deficient plasma complemented with diluted normal human plasma. Synthetic IrCPI was used as a comparator in this assay.

[0254] Material and Methods

[0255] Nine volumes of a human plasma pool were mixed with one volume of V13 or IrCPI and incubated during 30 minutes at 37 C. V13 or ICPI was added as a 10-fold solution and tested using a range of concentrations. Treatments were compared to controls (i.e. absence of V13 or IrCPI) consisting of nine volumes of the human plasma pool to which one volume of Tris (0.05 M), NaCl (0.15 M), BSA (1%) buffer (pH 7.50) was added.

[0256] After a 30 min incubation, samples were diluted 1:10 with imidazole buffer. Then, 50 L of each diluted sample was mixed with 50 L of Factor XI deficient human plasma or with 50 L Factor XII deficient human plasma (1 min at 37 C.), followed by an addition of 50 L of the aPTT reagent Cephen. The contact phase was activated by Cephen during an incubation of 10 min at 37 C. Clotting was initiated by the addition of 50 L of 0.025 M CaCl.sub.2) (preincubated at 37 C.) and clotting times were recorded.

[0257] FXI and FXII calibration curves were made for the calculation of plasma FXI and FXII activities after V13 treatment. Human plasma calibrator (duly titrated with FXI and FXII) was used at successive two-step dilutions with imidazole buffer (1:10 to 1:160 dilution). Each dilution was incubated with FXI or FXII deficient plasma, Cephen and with 0.025M CaCl.sub.2 as described above (ratio 1:1:1:1).

[0258] FXI and FXII calibration curves were obtained by plotting clotting times in function of the FXI/FXII activity of the different dilutions of the calibrator using a log-log plot. The 10-fold dilution of the calibrator is considered having 100% activity. There is an inverse linear relationship between the FXI or FXII activity and the corresponding clotting time, when plotted on a log-log-graph. The equations of the calibration curves were used to calculate the FXI and FXII activities in the human plasma pool treated with V13. The residual activity after V13 treatment was calculated as a percentage compared to control (untreated normal human plasma). Results are expressed as the percentage of inhibition of FXI and FXII activities after V13 treatment as compared to control, i.e. absence of V13, and calculated as follows:

% inhibition FXI=(1FXI.sub.%.sup.IrCPI/FXI.sub.%.sup.CTL)*100%

% inhibition FXII=(1FXII.sub.%.sup.IrCPI/FXII.sub.%.sup.CTL)*100.

[0259] Results

[0260] As shown in FIG. 4, patterns of inhibition of FXI and FXII followed similar hyperbolic curves as function of the concentration of V13. At the highest concentration (1.3 M), V13 inhibited the FXI and FXII activity by 44% and 45% respectively. Synthetic Ir-CPI inhibited the FXI/FXII activity by 77/85% at the same concentration.

[0261] The results indicate that the fusion of Ir-CPI molecule to human serum albumin allows (e.g. V13) a significant preservation of the activity of Ir-CPI keeping a selectivity and specificity of action on the contact phase of coagulation whilst improving the pharmacokinetic profile of the initial molecule.

[0262] The PK profiles of the IrCPI-HSA variants should allow administration of lower molar doses and within larger intervals to obtain a pharmacological effect on steady state.

Example 6: Antithrombotic Effect of V13 in the Rat AV-Shunt Model

[0263] The antithrombotic efficacy of V13 was evaluated in an extracorporeal arterio-venous (AV) shunt (silk thread) rat model.

[0264] Material and Methods

[0265] Rats were fasted overnight and were then anesthetized (ketamine/xylazine at 100/16 mg/kg i.m.). An arterio-venous shunt was prepared as follows: two 13 cm-long polyethylene tubes (0.85 mm i.d. and 1.27 mm o.d.) linked to a central part (6 cm-long; 1.14 mm i.d.) containing a 5-cm silk thread (USP 0, EP 3.5) and filled with physiological saline was placed between the left jugular vein and the right carotid artery (inserting the venous side first). After 20 minutes of extracorporeal blood circulation through the shunt, the blood flow was stopped at the arterial side. The central part of the shunt was then removed and the silk thread supporting the thrombus was extracted. The wet weight of the thrombus was immediately determined by subtracting the weight of the wet thread.

[0266] The test substances were administered i.v. 5 minutes before the test (shunt), and compared with a respective vehicle control group. Sodium heparin (UFH), administered i.v. 5 minutes before the test (shunt), was used as positive control.

[0267] Concentrations used during the experiment were the following: V13: 1.21 or 2.08 mol/kg and UFH: 100 or 300 IU/kg.

[0268] Concentrations of V13 in the rat plasma were determined by sandwich ELISA using an anti-IrCPI monoclonal antibody (as capture antibody) and an anti-IrCPI polyclonal antibody (as detection antibody) biotinylated and used in association with streptavidin-HRP to allow a spectrophotometric monitoring using 3, 3, 5, 5-tetra-methylbenzidine (TMB Sureblue) as the substrate.

[0269] The effect on the activated partial thromboplastin time (aPTT) was measured using Actin FS (ellagic acid and phospholipids) as reagent. The analyzed plasmas were obtained immediately after shunt termination. Results are expressed as the ratio between the individual aPTT value in the presence of V13 divided by the mean aPTT value of the vehicle condition.

[0270] Results

[0271] As shown in FIG. 5, V13 (1.21 or 2.08 mol/kg), administered i.v., induced a significant reduction of thrombus weight after 20 min of the AV-shunt procedure.

[0272] At 2.08 mol/kg, V13 (mean thrombus weight: 12.17 mg) significantly reduced by 1.9-fold the thrombus weight when compared to the vehicle (control) condition (mean thrombus weight: 23.16 mg).

[0273] The positive control, UFH had a marked antithrombotic activity in this model. At 300 IU/kg, UFH (mean thrombus weight: 9.83 mg) significantly reduced by 2.4-fold the thrombus weight when compared to the vehicle (control) condition (mean thrombus weight: 23.16 mg).

[0274] Following a single bolus administration of 93 mg/kg (1.21 mol/kg) of V13, 5 min before the AV-shunt opening, the mean aPTT ratio (SEM) was 2.10.1 (n=6) (FIG. 6).

[0275] Following a single bolus administration of 160 mg/kg (2.08 mol/kg) of V13, 5 min before the AV-shunt opening, the mean aPTT ratio (SEM) was 4.40.9 (n=6). This aPTT ratio was significantly higher than the aPTT ratio of the vehicle group (p<0.001) (FIG. 6).

Example 7: Effect of V13 on Thrombin Generation

[0276] Material and Methods

[0277] The calibrated automated thrombin activity measurement of V13 was determined on human plasma pool. Synthetic IrCPI was used as a comparator in this assay. Plasma (platelet-poor plasma [PPP]) (450 L) was mixed with 50 L of solutions containing different concentrations of V13 or IrCPI (10-times concentrated). Eighty (80) L of spiked plasma and 20 L reagent (PPP LOW reagent (PPL1510/02, Thrombinoscope BV); PPP reagent (PPP1509/01, Thrombinoscope BV); PPP HIGH (PPPH1501/01, Thrombinoscope BV) or diluted Actin FS (batch 53851, Siemens, [1/20])) were mixed in a 96-well microtiter plate (Immulon 2HB; Thermo Fisher Scientific) and incubated for 5 min at 37 C. The coagulation process was triggered by the addition of 20 L of fluorogenic substrate in a calcium chloride buffer pre-warmed at 37 C. A calibration curve was also performed simultaneously using 80 L of normal pooled plasma, 10 L of PBS, 20 L of thrombin calibrator and 20 L of substrate/calcium chloride-buffered solution at 37 C. The substrate hydrolysis was monitored on a microplate fluorometer Fluoroskan Ascent FL (Thermo Labsystems) with a 390/460 nm filter set. Fluorescence was measured every 20 sec for 60 min. Eleven concentrations of V13 and IrCPI were tested which ranged from 0 to 2 M. A dedicated software program (Thrombinoscope 5.0, Synapse BV) calculated thrombin concentration generated in plasma depending on V13 or IrCPI concentrations added and displayed thrombin concentrations as function of time. Moreover, it enabled the calculation of the following TGT (Thrombin Generation Time) parameters: [0278] endogenous thrombin potential (ETP; [nM]): the sum amount of active thrombin which was present in the system over entire time of experiment (area under the thrombin generation curve), [0279] maximal thrombin concentration in the sample (Peak thrombin [nM)]), [0280] time to reach the maximal concentration of thrombin (ttPeak [0281] lag-time before beginning of accelerated thrombin production (LagTime

[0282] Results

[0283] FIGS. 7 (thrombin generation time curves) and 8 (curves analysis) show the effects of V13 on thrombin generation induced by Actin FS or by tissue factor (TF, tested at 3 concentrations 1, 5 and 20 pM). When using Actin FS as inducer of the thrombin generation (FIG. 7A), V13 showed a concentration-dependent decrease of thrombin generation (Peak: maximum amplitude and ETP: AUC i.e. overall production) and a concentration-dependent delay in the thrombin generation (lag time and time to peak). At the highest concentration (2 M), V13 inhibited the endogenous thrombin potential (ETP) and peak by 46% and 79% respectively. At the same concentration, V13 increased the time to peak and the lag time by 3-fold and 3.2-fold respectively. When using tissue factor as inducer of the thrombin generation (FIG. 7B-D), V13 inhibited ETP maximally by 20% (2 M of V13 with TF concentration of 1 pM). V13 (2 M) decreased the peak thrombin generation by 11 to 41% and increased the lag time or time to peak by 1.3 to 1.5-fold depending on the concentration of tissue factor (relative effects more pronounced at lower than higher concentration of TF: 1>5>20 pM).

[0284] The results show that V13 inhibits more specifically the production of thrombin when induced by Actin FS than by TF. They are consistent with V13 inhibiting more specifically the activation of the contact phase (intrinsic coagulation pathway) than the activation of tissue factor pathway (extrinsic coagulation pathway), which was also observed for IrCPI (FIG. 9).

Example 8: Effect of V13 on Prothrombin Time

[0285] Material and Methods

[0286] Preparation of Human Platelet-Poor Plasma

[0287] Fifty-six individuals were included in the study. The exclusion criteria were thrombotic and/or hemorrhagic events, antiplatelet and/or anticoagulant medication, hormonal therapy, pregnancy and uptake of drugs potentially affecting the platelet and/or coagulation factor functions during the two weeks prior to the blood drawn. The study protocol is in accordance with the Declaration of Helsinki. Blood was taken by venipuncture in the antecubital vein and collected into 0.109M sodium citrate (9:1 v/v) tubes (Venosafe, Terumo, Belgium) using a 21-gauge needle (Terumo). The PPP was obtained from the supernatant fraction of blood tubes after a double centrifugation for 15 minutes at 1.500 g at room temperature. Immediately after centrifugation, PPP from the 40 donors were brought together to obtain the NPP which was frozen at 80 C. without any delay. Frozen NPP samples are thawed and heat to 37 C. for 5 minutes just before experiment. All tests were performed within 4 hours after thawing.

[0288] Preparation of Human Spiked Samples

[0289] Human plasma (PPP) (450 l) obtained was mixed with 50 l of solutions containing different concentrations of V13 (10-times concentrated) or synthetic IrCPI to reach final concentrations ranging from 0 to 2 M.

[0290] Measurement of Anticoagulant Activity of IrCPI in Spiked Samples

[0291] The prothrombin time consists of the use of calcium thromboplastin, prepared from human recombinant tissue factor and from phospholipids, to measure the clotting time of the plasma sample (as prepared above) and to compare it with that of a normal standard (pre-calibrated reagent). The test is performed according to the recommendations of the manufacturer (STA-Neoplastin R from Diagnostica Stagoapplication for a STA-R Evolution analyzer).

[0292] Briefly, fifty (50) L of plasma in presence of V13 or IrCPI was incubated for 240 seconds at 37 C. The clotting reaction was then started by adding 100 L of STA-Neoplastin-R (STAGO). The measurement was performed on a STA-R Evolution analyzer (Diagnostica Stago) using viscometric method for clot detection.

[0293] Results

[0294] Results are presented in Table 4.

TABLE-US-00004 TABLE 4 Effect of V13 and native IrCPI on prothrombin time Test item concentration V13 IrCPI (M) PT (sec) % PT (sec) % 2 14.6 105 14.3 103 1 14.7 105 14.4 103 0.5 14.3 103 14.0 101 0.25 14.1 101 14.1 101 0.125 14.3 102 14.1 101 0 14.0 100 13.9 100

[0295] The prothrombin time (PT) is an assay evaluating the activity of extrinsic coagulation factors (Factor II (FII), FV, FVII and FX).

[0296] The results of Table 4 show that no concentration-dependent effect was found of V13 or IrCPI on the prothrombin time.