Methods of administering neutralizing anti-protease nexin-1 antibodies to treat hemophilia A

Abstract

The present invention relates to a method and compositions for the treatment of haemorrhagic diseases.

Claims

1. A method for treating mild or moderate haemophilia A in a subject in need thereof, comprising the step of administering to said subject a protease nexin-1 activity inhibitor, wherein the protease nexin-1 activity inhibitor is an anti-protease nexin-1 neutralizing antibody.

2. The method of claim 1, wherein the protease nexin-1 activity inhibitor is administered in combination with one or more coagulation factors.

3. The method of claim 2, wherein the one or more coagulation factors is selected from the group consisting of factor VIII, factor IX, factor Vila, plasma-derived activated prothrombin complex and fibrinogen.

4. The method of claim 1, wherein the protease nexin 1 activity inhibitor is fused to a platelet targeting agent.

Description

FIGURES

(1) FIG. 1: Thrombin generation in haemophilic mice.

(2) The thrombogram of FVIII-KO mice (-.-. curve) displays a prolonged time to peak and a reduced peak thrombin. The addition of the neutralizing anti-PN-1 antibody enhances thrombin generation in PRP from haemophilia mice (black curve). This is illustrated by the acceleration of thrombin generation (shorter time to peak) and the increase of the peak thrombin. These changes give rise to an increase thrombin generation velocity.

(3) FIG. 2: Thrombin Generation in Haemophilica A Patient.

(4) The thrombogram of patient with haemophilia (--- curve) displays prolonged lag time and time to peak and a reduced peak thrombin and a reduced endogenous thrombin potential. As observed with the addition of recombinant FVIII (-.-. curve), the neutralizing anti-PN-1 antibody enhances thrombin generation in PRP from patients with haemophilia A (black curve). This is illustrated by the acceleration of thrombin generation (shorter time to peak) and the increase of the peak thrombin. These changes give rise to an increase thrombin generation velocity.

(5) FIG. 3: Thrombin generation in mild, moderate and severe haemophilica A patient.
velocity=Peak thrombin/(time to peak-lagtime)

(6) As observed with recombinant FVIIII, the neutralizing anti-PN-1 antibody improves velocity of thrombin generation in patients with mild and moderate haemophilia. In contrast it has no effect in PRP from patients with severe haemophilia.

EXAMPLE

(7) Material & Methods

(8) The inventors determined thrombin generation in platelet-rich plasma (PRP) samples using the calibrated automated thrombogram (CAT) system containing the Fluoroskan Ascent fluorescence plate reader (Thrombinoscope BV, Maastricht, Netherlands). This is a method for quantifying continuous and dynamic properties of thrombin generation after addition of tissue factor in the sample. According to the manufacturer's instructions, thrombin generation is conducted, in triplicate, with 80 l PRP in a total volume of 120 l. Samples spiked with 20 l thrombin calibrator (Diagnostica Stago, Asnieres, France) is run in parallel with each cycle of test sample.

(9) PRP is incubated with the neutralizing anti-PN-1 polyclonal antibody for 20 min at 37 C., before being transferred to a pre-warmed (37 C.) microtiter plate with a mixture of TF and phospholipids. Twenty microliters of FluCaKit (fluorogenic substrate for thrombin) with CaC12 is then be automatically injected into all of the wells, starting the reaction. The fluorescence signal is read every 20 sec for 60 min in a fluoroskan Ascent reader (Thermo Labsystems) equipped with a 390/460 nm filter set, and thrombin generation curves is calculated using thrombinoscope software.

(10) The thrombin generation assay (TGA) is a practical tool to determine global coagulation.

(11) The parameters determined from a thrombin generation curve (thrombogram) are: i) the endogenous thrombin potential (ETP), which corresponds to the area under the thrombin generation curve ii) the peak thrombin, which corresponds to the maximal level of thrombin iii) the lag time, which corresponds to the time taken to reach 2 nM thrombin iv) the time to peak, which corresponds to the time taken to reach the peak height.

(12) Thrombin Generation in Haemophilic Mice.

(13) Citrated blood is drawn from the inferior vena cava from WT mice or FVIII-KO mice to prepare PRP (150 000 platelets/l), which is preincubated or not with the anti-PN-1 antibody (150 g/ml) for 30 min at RT. TGA is conducted in triplicate with 20 l PRP plus 20 l mixture reagent containing TF (0.5 pM) and phospholipids and 20 l of fluorogenic substrate for thrombin with Cacl2 in a total volume of 60 l.

(14) Thrombin Generation in Haemophilica A Patient.

(15) Samples from patients with severe (FVIII<1%), moderate (1%<FVIII<5%) and mild (5%<FVIII<40%) haemophilia are used. Citrated whole blood is drawn from patients to prepare PRP (150 000 platelets/l), which is preincubated or not with the anti-PN-1 antibody (150 g/ml), or recombinant FVIII (1 U/ml) for 30 min at RT. TGA is conducted in triplicate with 80 l PRP plus 20 l mixture reagent containing TF (0.5 pM) and phospholipids and 20 l of fluorogenic substrate for thrombin with Cac12 in a total volume of 120 l.

(16) Thrombin Generation in Mild, Moderate and Severe Haemophilica A Patient.
velocity=Peak thrombin/(time to peak-lagtime)

(17) Velocity of thrombin generation was measured in PRP from patients with mild, moderate and severe haemophilia with recombinant FVIIII and the neutralizing anti-PN-1 antibody.

(18) Results

(19) PN-1 Inhibition in Mice

(20) The thrombogram (thrombin generation) in haemophilic mice (FVIII-KO mice) (-.-. curve) displays a prolonged time to peak and a reduced peak thrombin. The addition of the neutralizing anti-PN-1 antibody enhances thrombin generation in PRP from haemophilia mice (black curve). This is illustrated by the acceleration of thrombin generation (shorter time to peak) and the increase of the peak thrombin. These changes give rise to an increase thrombin generation velocity (FIG. 1).

(21) PN-1 Inhibition in Human

(22) Thrombin Generation in Haemophilica A Patient.

(23) As previously described, the classic thrombogram of patient with haemophilia (--- curve) displays prolonged lag time and time to peak and a reduced peak thrombin and a reduced endogenous thrombin potential. As observed with the addition of recombinant FVIII (-.-. curve), the neutralizing anti-PN-1 antibody can enhance thrombin generation in PRP from patients with haemophilia A. (- curve) This is illustrated by the acceleration of thrombin generation (shorter time to peak) and the increase of the peak thrombin. These changes give rise to an increase thrombin generation velocity (FIG. 2).

(24) Thrombin Generation in Mild, Moderate and Severe Haemophilica A Patient.

(25) Severe, Moderate and Mild phenotypes are defined by plasma FVIII of less than 1%, 1-5% and 5%-40%, respectively. Moderate and mild patients account for about 60% of the haemophilia A population.

(26) As observed with recombinant FVIIII, the neutralizing anti-PN-1 antibody improves velocity (Peak thrombin/(time to peak-lagtime)) of thrombin generation in patients with mild and moderate haemophilia. In contrast it has no effect in PRP from patients with severe haemophilia (FIG. 3).

(27) Anti-PN-1 antibody improves thrombin generation in mild and moderate haemophilia patients but not in severe haemophilia patients. These findings establish a requirement for PN-1 inhibition as a specific anticoagulant in platelets and demonstrated that blocking PN-1 have a role in haemorrhagic disease treatment.

REFERENCES

(28) Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. Boulaftali Y, Adam F, Venisse L, Ollivier V, Richard B, Taieb S, Monard D, Favier R, Alessi M C, Bryckaert M, Arocas V, Jandrot-Perrus M, Bouton M C. Anticoagulant and antithrombotic properties of platelet protease nexin-1. Blood. 2010; 115: 97-106. Bouton M C, Boulaftali Y, Richard B, Arocas V, Michel J B, Jandrot-Perrus M. Emerging role of serpinE2/protease nexin-1 in hemostasis and vascular biology. Blood. 2012 Mar. 15; 119(11):2452-7. Evans D L, McGrogan M, Scott R W, Carrell R W. Protease specificity and heparin binding and activation of recombinant protease nexin I. The Journal of biological chemistry. 1991; 266: 22307-12. Knauer D J, Majumdar D, Fong P C, Knauer M F. SERPIN regulation of factor XIa. The novel observation that protease nexin 1 in the presence of heparin is a more potent inhibitor of factor Xia than Cl inhibitor. The Journal of biological chemistry. 2000; 275: 37340-6. McGrogan M, Kennedy J, Li M P, et al. Molecular cloning and expression of two forms of human protease nexin I. Biotechnology. 1988; 6:172-177.