EP2943214A1 - Use of factor xa in the prevention or treatment of haemorrhagic events following the taking of anticoagulants - Google Patents
Use of factor xa in the prevention or treatment of haemorrhagic events following the taking of anticoagulantsInfo
- Publication number
- EP2943214A1 EP2943214A1 EP14703112.4A EP14703112A EP2943214A1 EP 2943214 A1 EP2943214 A1 EP 2943214A1 EP 14703112 A EP14703112 A EP 14703112A EP 2943214 A1 EP2943214 A1 EP 2943214A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- factor
- fxa
- anticoagulants
- rivaroxaban
- thrombin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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Classifications
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/482—Serine endopeptidases (3.4.21)
- A61K38/4846—Factor VII (3.4.21.21); Factor IX (3.4.21.22); Factor Xa (3.4.21.6); Factor XI (3.4.21.27); Factor XII (3.4.21.38)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
- A61K31/5375—1,4-Oxazines, e.g. morpholine
- A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/36—Blood coagulation or fibrinolysis factors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/56—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21006—Coagulation factor Xa (3.4.21.6)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
- G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
- G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
- G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
- G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
- G01N2333/96433—Serine endopeptidases (3.4.21)
- G01N2333/96441—Serine endopeptidases (3.4.21) with definite EC number
- G01N2333/96463—Blood coagulation factors not provided for in a preceding group or according to more than one of the proceeding groups
Definitions
- the present invention relates to the use of coagulation inhibitor antidotes indicated in the prevention or treatment of thromboembolic pathologies.
- Venous thrombosis is a widespread condition worldwide, affecting approximately 900,000 patients in the United States each year and 770,000 patients in Europe. Providing prophylactic venous thromboembolic events with effective and well-tolerated hemorrhagic treatments, especially in at-risk patients, is a public health need.
- Venous thrombosis is the formation of a clot in a vein. It can be “superficial” when it touches the small veins located between the skin and the muscles, or “deep” when it reaches a larger vein. The preferential location of deep thromboses is in the lower limbs (legs, thighs, groin folds). As a result of various factors, the clot can become detached from the vein that houses it, enter the bloodstream and cause pulmonary embolism (obstruction of the pulmonary artery) that may be life-threatening or have significant sequelae.
- venous thrombosis Although the prevalence of venous thrombosis increases significantly with age, other risk factors associated with venous thrombosis have been identified. These include inherited or acquired diseases of coagulation (antithrombin deficiency, protein C and S, factor V Leiden, prothrombin mutation), obesity, heart failure, bed rest after surgery surgical or pregnancy, taking oral contraceptives associated with smoking. More generally, any period causing a slowing of the blood circulation (venous stasis) promotes the appearance of small vascular lesions forming a fertile ground for the development of thromboses.
- the risk of venous thrombosis is particularly high in a patient who has undergone a major orthopedic surgery of the lower limbs, especially when placing a hip or knee prosthesis or the reduction fracture of the femoral neck.
- the clinician has systematically resorted to anticoagulants.
- Vitamin K antagonists including coumarins or 1,3-indandiones, target factors II, VII, IX and X of the coagulation cascade. These molecules, commonly used in situations where the risk of clot formation is major, require regular blood sampling to control the INR (International Normalized Ratio). Because of their ability to modify circulating plasma proteins, AVKs have a long half-life that makes their use inflexible.
- Standard heparin (unfractionated: heparin, calciparin) and low molecular weight heparins (LMWH) such as enoxaparin (Lovenox ® ), tinzaparin (Innohep ® ), bemiparin (Ivor ® ), nadroparin (Fraxiparine ® ), parnaparin (Flexum ® ), reviparin (Clivarine ® ) or deltaparin (Fragmine ® ) are glycosaminoglycans consisting of chains with alternating residues of D-Glucosamine and uronic acid.
- LMWH low molecular weight heparins
- LMWHs are capable of increasing the activity of antithrombin and thus of inhibiting activated X-factor (FXa) and activated II (Fila).
- FXa activated X-factor
- FXa activated X-factor
- Fila activated II
- LMWHs also directly inhibit FXa, thereby inhibiting the conversion of prothrombin to thrombin. Given their superior efficacy to unfractionated heparin and their lesser side effects in terms of thrombocytopenia, LMWHs are preferentially injected into patients.
- the compound PRT4445 currently under development, is the only therapeutic agent capable of binding and neutralizing all direct and indirect inhibitors of activated factor X, thereby restoring normal hemostatic function (WO 2011/008885 A1). This compound is in fact a protein derived from FX or FXa in which the active site and / or the Gla domain have been invalidated, thus reducing the intrinsic procoagulant activity of these factors (WO 2010/117729 A1). Phase II clinical trials of the use of the compound PRT4445 as an antidote for betrixaban (US 6,376, 515 and US 6,835,739) are imminent.
- Yet another object of the present invention is to provide an antidote for activated factor X inhibitors (FXa).
- the present invention relates to factor Xa (FXa) for its use in the prevention or treatment in a patient, in particular a human being or an animal, hemorrhagic events induced by the taking of anticoagulants.
- FXa factor Xa
- the invention is based on the unexpected finding that factor Xa is able to rapidly reverse the anticoagulant effect of anticoagulants, without inducing a risk of thrombosis, and to restore the "normal" haemostatic function at the occurrence of a consecutive haemorrhagic event. taking anticoagulants.
- factor Xa also called FXa or activated factor X
- FXa a serine protease of the coagulation cascade, obtained from the inactive factor X (Butenas & Mann, Biochemistry (Moscow), 2002, 67 (1) , 3-12).
- the proteolysis of FX in FXa is carried out either by the tenase complex (activated factor IX (FIXa) and its cofactor, activated factor VIII (FVIIIa) in the presence of calcium and phospholipids) or by the activated factor VII complex (FVIIa). Tissue factor in the presence of calcium.
- FXa forms, with activated factor V (FVa), the prothrombinase complex which catalyzes the conversion of prothrombin (factor II, FII) to thrombin (activated factor II, Fila).
- FII prothrombin
- Fila activated factor II
- Thrombin catalyzes the conversion of fibrinogen to fibrin responsible for blood clot formation.
- FXa is characterized by procoagulant activity. FXa occupies a central place in the intrinsic and extrinsic pathways of coagulation.
- Factor Xa serves as an antidote for anticoagulants.
- antidote is meant the molecules, including FXa, capable of neutralizing or reversing all or part of the anticoagulant activity of anticoagulants. This effect must be able to intervene in a more or less short time, in connection with the location and the magnitude of the haemorrhagic event to slow down, attenuate or completely interrupt this hemorrhagic event.
- the anticoagulant activity of anticoagulants can be measured by global coagulometric tests (Quick time (PT), activated partial thromboplastin time (aPTT)). In their presence, there is a decrease in the INR (International Normalized Ratio). Measurement of their effects can also be considered by coagulation tests that measure thrombin generation (TGT).
- Hemorrhagic events means visible or invisible bleeding resulting from rupture of blood vessels, including deep veins, superficial veins or venules. The extent of visible bleeding is assessed by simple observation. On the other hand, the following signs may reveal the existence of invisible bleeding: asthenia, dyspnoea, paleness, headaches resistant to usual treatments, discomfort. Hemorrhagic events within the meaning of the present invention are a deleterious effect of the misuse of anticoagulants. The severity of the bleeding depends on their location and intensity and is appreciated by the need for transfusion or the life-threatening condition of the patient.
- hemorrhagic events can lead to anemia.
- anticoagulants are meant compounds capable of inhibiting blood clot formation and lengthening coagulometric tests (aPTT and PT). The limitation of thrombin formation can also be evaluated in TGT.
- Anticoagulants can be listed according to their pharmacological target. There are thus antagonists of vitamin K, unfractionated or low molecular weight heparins, anti-tissue factor antibodies, specific inhibitors, direct or indirect factors IXa, Xa, Xla, Xlla or VIIa. Anticoagulants are administered alone or in combination in a patient at a dose and in a dosing regimen that can prevent or treat clot onset, thereby reducing the risk of thrombosis.
- the anticoagulants are heparins of low molecular weight.
- Low molecular weight heparins also known as LMWH, refers to heparins resulting from the cleavage of unfractionated heparin (Mousa SA, Methods Mol Biol, 2010, 663, 109-132). These anti-thrombotic compounds are administered intravenously, and their use concentrations are expressed in international anti-FXa units even if their effectiveness is not limited to this anti-FXa activity.
- LMWHs have a longer half-life than unfractionated heparins and have lower platelet interaction than unfractionated heparins.
- LMWHs have a molecular weight that ranges from 4000 to 6000 Da.
- heparins commonly used are enoxaparin (Lovenox ® ), tinzaparin (Innohep ® ), bemiparin (Ivor ® ), nadroparin (Fraxiparine ® ), parnaparin (Flexum ® ), reviparin (Clivarine ® ), certoparin (Sandoparine ® ) or deltaparin (Fragmine ® ).
- LMWHs are used preventively in postoperative lower limb surgery or when long-term immobilization is mandatory. By long-term immobilization is meant maintaining the patient in a bedded state for a period ranging from 3 days to several weeks.
- the low molecular weight heparins are chosen from bemiparin, certoparin, deltaparin, enoxaparin, nadroparin, parnaparin, reviparin or tinzaparin.
- anticoagulants are specific inhibitors of factor Xa.
- specific factor Xa inhibitors are meant compounds capable of directly or indirectly inhibiting the procoagulant activity of FXa which consists of the conversion of prothrombin to thrombin in vitro, and / or ex vivo, and / or in vivo.
- FXa inhibitors can be classified as peptide inhibitors, extracted and purified or genetically engineered, and non-peptide inhibitors obtained by chemical synthesis (Kher et al., 1998, The Pharmacologist's Letter, 12 (6), 222-226). Inhibitors that block the active site of FXa are called direct inhibitors while inhibitors that act by binding and catalyzing the effect of antithrombin on FXa are referred to as indirect inhibitors.
- FXa direct peptide inhibitors include, without restricting the scope of the invention, the TAP (tick anticoagulant peptide) extracted from tick saliva, antistasin extracted from the salivary gland of the leech Haementeria officinalis, ACAP (ancylostoma caninum anticoagulant peptide) isolated from the hookworm or its recombinant forms r Ac AP5, r Ac AP2 (also called NAP-5) or FXa I (factor Xa inhibitor) extracted from the saliva of the leech Hirudo medicinalis or the corresponding recombinant protein named Yagin.
- TAP tick anticoagulant peptide
- ACAP ancylostoma caninum anticoagulant peptide
- r Ac AP5 isolated from the hookworm or its recombinant forms r Ac AP5, r Ac AP2 (also called NAP-5)
- FXa I factor Xa inhibitor
- DX 9065a DX 9065a
- LY517717 xabans
- xabans eribaxaban, apixaban, betrixaban, edoxaban, otamixaban, rivaroxaban
- LMWH fondaparinux or idraparinous oligosaccharides and heparinoids (danaparoid, sulodexide, dermatan sulfate) are examples of indirect inhibitors of FXa.
- FXa is the antidote for specific FXa inhibitors.
- the catalytic structure of FXa used according to the present invention allows binding to specific FXa inhibitors.
- the use of factor Xa as an antidote for FXa inhibitors allows the titration of these inhibitors.
- the inhibitory activity of specific FXa inhibitors is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%. , 80%, 90% or 100%.
- the decrease in the anti-Xa inhibitory activity is measured by the return of a functional activity of FXa or an increase in procoagulant activity in the plasma.
- chromogenic measurement cleavage of FXa or thrombin specific peptide
- chronometric time aPTT, PT, INR increase
- global coagulation measurement tests measurement of the generation time of thrombin, TGT.
- the anticoagulants are chosen from the group consisting of low molecular weight heparins, fondaparinux or idraparinous oligosaccharides and factor Xa specific inhibitors.
- the anticoagulants are fondaparinux or idraparinous oligosaccharides.
- the oligosaccharide is fondaparinux.
- the factor Xa specific inhibitors are chosen from apixaban, betrixaban, edoxaban, otamixaban or rivaroxaban.
- factor Xa restores the procoagulant activity without the latter exceeding the basal level of the procoagulant activity measured in a group of healthy patients.
- procoagulant activity is meant the catalytic activity of FXa which consists in cleaving prothrombin to thrombin. This is called activation of prothrombin.
- the catalytic activity of FXa is comparable to the catalytic activity of prothrombinase, a protein complex that contains not only FXa but also FVa.
- basal level is meant the level of FXa of a patient or a group of healthy patients. Each patient has his own basal level of FXa that allows him to maintain a balance in terms of coagulation. This basal level, close to zero under normal physiological conditions, may vary from one individual to another, particularly depending on the basal level of FX present in the blood.
- patients any mammal, human or animal.
- the patients received at least one specific factor Xa inhibitor.
- healthy patients a group of patients who have no apparent pathology and who are not on Factor Xa-specific inhibitors, ie, who have not received any treatment with any of the specific inhibitors.
- FXa Factor Xa-specific inhibitors
- the advantage of measuring the level of FXa in several healthy patients is to be able to determine an average that serves as a reference value. The comparison of this average reference value of procoagulant FXa activity with that of a patient under FXa inhibitors and who receives FXa as an antidote makes it possible to follow the therapeutic effect of the antidote.
- the factor Xa for its use according to the invention is a native protein isolated from the plasma or a recombinant protein.
- native protein isolated from plasma is meant the FX found naturally in the plasma / serum of patients and which has undergone a purification step to isolate it from its natural matrix. The in vitro or ex vivo activation of the FX thus isolated results in FXa having the ability to activate prothrombin.
- recombinant protein is meant a protein prepared, expressed, isolated, purified by genetic engineering.
- the recombinant DNA technology makes it possible to insert a polynucleotide sequence coding for either FXa or FX in a host cell and thus to produce the polypeptide of interest, ie the FXa or the FX which can be activated after cell production. It is also possible to modify the polynucleotide sequence coding for the FX by inserting one or more intracellular cleavage sequences which will make it possible to directly obtain FXa.
- the recombinant FXa protein also has the ability to activate prothrombin.
- the factor Xa for its use according to the invention has improved properties compared to the native factor Xa isolated from the plasma.
- the factor Xa gene and / or protein is modified to enhance one or more of its biological properties relative to the native factor Xa isolated from the plasma.
- factor Xa may exhibit improved affinity for therapeutic FXa inhibitors, and / or improved ability to bind to its cofactor, activated Factor V coagulation, and / or enhanced activation properties. of prothrombin, relative to native factor Xa isolated from plasma.
- the factor Xa for its use according to the invention has an increased circulating half-life compared to the native factor Xa isolated from the plasma.
- half-life is meant the period of time during which a protein is capable of exerting its biological function. The shorter the half-life, the more labile the protein is. Conversely, the longer the half-life of a protein, the more stable the protein. Increasing the half-life of a protein is equivalent to increasing its stability in the bloodstream. Indeed, recent data suggest that the FXa used as a procoagulant in the haemophilic mouse does not have a sufficient half-life to allow a lasting restoration of the coagulation capacity (Invanciu et al., 2011, Nature Biotechnology, 29 (1 1), 1028-1035). Used in vitro in healthy plasma, FXa has the ability to normalize the rivaroxaban-induced thrombin generation failure. However, this ability could be impaired in vivo.
- FXa Factor Xa
- various chemical modification techniques such as, for example, and without limitation of method, pegylation, or by the generation of mutated molecules less sensitive to the action of plasma inhibitors such as antithrombin for example, or by the use of a chimeric molecule which would increase the half-life, such as, for example, and without being limited to their sole use, the fusion of factor Xa to the albumin or crystallizable fragment of antibody.
- the present invention also relates to a method of monitoring in vitro or ex vivo the occurrence of hemorrhagic events induced by the intake of factor Xa specific inhibitors comprising the following steps:
- analytical measurement is meant the determination of the concentration or the level of each of the markers of coagulation. The determination of these concentrations is carried out in vitro or ex vivo using diagnostic kits. The numerical values obtained are specific to each patient receiving a specific factor Xa inhibitor.
- coagulation markers coagulation time values are defined by measurement of aPTT, PT, or INR establishment.
- biological sample is meant a liquid fraction selected from the group consisting of blood, serum or plasma.
- haemorrhagic risk is meant the presumption of bleeding in a patient who has received at least one FXa inhibitor.
- the evaluation of the risk of haemorrhage can be carried out solely from the markers of coagulation or taken into consideration with medical imaging analyzes allowing the visualization of the haemorrhagic zones.
- Generation of thrombin less than 50% of normal generates a risk of bleeding (Van Veen et al., British J Haematol, 2008, 142, 889-903).
- the determination of the haemorrhagic risk is carried out in particular by comparing the numerical values determined for each of the markers in a patient receiving a specific inhibitor of FXa at reference numerical values.
- reference numerical values can correspond either to the average of the numerical values obtained in healthy subjects for each of the markers, or to be fixed or calculated in relation to international standards.
- the intended therapeutic purpose consists in the partial or total reduction of the haemorrhage.
- the evaluation of the therapeutic result may be based on the clinical picture of the patient or on any other biological index.
- the biological indices are in particular the neutralization of the FXa inhibitor administered to the patient, the reversal of the anticoagulant activity of the FXa inhibitor, the removal of the FXa inhibitor from the plasma of the patient, restoration of hemostasis and reduction or cessation of bleeding.
- the effective amount of FXa may vary depending on the type and dose of FXa inhibitor administered to the patient, the morphology and age of the patient, and the mode of administration of the FXa. The management of these various parameters is well known to those skilled in the art.
- the present invention also relates to a pharmaceutical composition
- a pharmaceutical composition comprising factor Xa and a factor Xa inhibitor as a combination product for separate or time-based use in prevention or treatment in a patient, including a human or an animal, events Venous thromboembolism, for example following an orthopedic intervention of the hip and / or knee.
- composition is meant the combination of an active compound, in particular FXa, with at least one pharmaceutically acceptable compound making the composition suitable for administration to a patient.
- combination product is meant a product containing at least two compounds A and B which may be administered together in one dose, or the product A is administered and then the product B is administered immediately thereafter, or the product A is administered and product B is administered several minutes, hours, days later.
- the combination product comprises the specific FXa inhibitor (product A) and FXa (product B).
- venous thromboembolic event is meant the formation of a clot that may clog the vessel in which it has formed or may leave the vessel in which it has formed to end up in the bloodstream.
- the formation of the blood clot is also called thrombosis.
- the frequency of occurrence of thromboembolic events increases when the patient undergoes prolonged immobilization. Orthopedic interventions of the lower limbs (hip, leg, knee) usually require prophylactic treatment of the patient with anticoagulants to prevent the occurrence of thrombosis.
- the concentration of factor Xa varies from 0.01 ng / ml to 2 ⁇ g / ml or from 10 ng / ml to 2 ⁇ g / ml.
- a determined volume of pharmaceutical composition according to the present invention makes it possible to define unit doses of FXa that can be administered to the patient receiving a specific inhibitor of FXa.
- unit dose is meant the specific amount of FXa administered to the patient in one dose.
- the unit dose is the appropriate presentation of a fixed amount of FXa in a single-dose container for administration to the patient.
- the present invention also relates to a method for preventing or treating hemorrhagic events induced by the administration of anticoagulants, in a mammal, in particular a human, comprising a step of administering factor Xa to said mammal, in particular man, by sufficient amount to restore a basal procoagulant activity.
- the goal is to stop haemorrhage occurring in a patient who has received at least one anticoagulant.
- anticoagulants are specific inhibitors of factor Xa.
- the specific inhibitors of factor Xa are chosen from apixaban, betrixaban, edoxaban, otamixaban or rivaroxaban.
- the factor Xa is administered to the mammal, in particular the man, during or after taking factor Xa specific inhibitors.
- Figure 1-A Thrombinogram obtained from the addition of 10 ⁇ g / ml of FX in a pool of normal plasma following activation by tissue factor:
- Figure 1-B Thrombinogram obtained from the addition of 10 ⁇ g / mL of FX in a plasma pool overloaded with 0.5 ⁇ g / mL rivaroxaban following activation by tissue factor:
- the normal plasma pool sample is represented by the black curve (1).
- the normal plasma pool sample overloaded with 0.5 ⁇ g / mL rivaroxaban is represented by curve (2).
- Factor X was added at 25 ⁇ g / mL in the normal plasma pool sample (curve (3)) and in the normal plasma pool sample and overloaded at 0.5 ⁇ g / mL rivaroxaban ( curve (4)).
- Figure 1-D Thrombinogram obtained from the addition of 100 ⁇ g / mL of FX in a pool of normal plasma overloaded with 0.5 ⁇ g / mL of rivaroxaban following activation by tissue factor:
- the normal plasma pool sample is represented by the thick black curve (1).
- the normal plasma pool sample overloaded with 0.5 ⁇ g / mL rivaroxaban is represented by the fine black curve (2).
- Factor X was added at 100 ⁇ g / mL in the normal plasma pool sample (gray curve (3)) and in the normal plasma pool sample and overloaded at 0.5 ⁇ g / mL rivaroxaban (light gray curve (4)).
- Figure 2 Thrombinogram obtained from the addition of 10 ⁇ g / mL of FX in a pool of normal plasma and overloaded with 0.35 ⁇ g / mL of rivaroxaban following an induction by the cephalin:
- the normal plasma pool sample is represented by the black curve (1).
- the normal plasma pool sample overloaded with 0.35 ⁇ g / mL rivaroxaban is represented by curve (3).
- Factor X was added at 10 ⁇ g / mL in the normal plasma pool sample and (curve (2)) in the normal plasma pool sample and overloaded at 0.35 ⁇ g / mL rivaroxaban ( curve (4)).
- Figure 3-A Thrombinogram obtained from the addition of increasing doses of FXa in a normal plasma pool:
- the normal plasma pool sample is represented by the black curve (1).
- Factor Xa was added at 0.5 ⁇ g / mL (curve (2)), 1.0 ⁇ g / mL (curve (3)), 1.5 ⁇ g / mL (curve (4)), 2, 0 ⁇ g / mL (curve (5)), 2.5 ⁇ g / mL (curve (6)), 5.0 ⁇ g / mL (curve (7)) in the normal plasma pool sample.
- Figure 3-B Thrombinogram obtained from the addition of increasing doses of FXa in a pool of normal plasma overloaded with 0.35 ⁇ g / mL rivaroxaban:
- the normal plasma pool sample is represented by the black curve (1).
- Factor Xa was added at 0.5 ⁇ g / mL (curve (3)), 1.0 ⁇ g / mL (curve (4)), 1.5 ⁇ / ⁇ ⁇ (curve (5)), 2.0 ⁇ / ⁇ ⁇ (curve (6)), 2.5 ⁇ g / mL (curve (7)), 5.0 ⁇ g / mL (curve (8)) in the overloaded normal plasma pool sample at 0.35 ⁇ s / ⁇ of rivaroxaban.
- Figure 4 Thrombograms obtained from the addition of 0.01 to 1 ⁇ / ⁇ ⁇ . activated FX in a normal plasma pool without activation:
- the normal plasma pool sample is represented by the ⁇ curve.
- the effect of the concentrations of FXa added to normal plasma is represented by the following curves: 0.01 ⁇ / ⁇ 1 (o); 0.05 ⁇ / ⁇ 1 ( ⁇ ); 0.1 ⁇ / ⁇ 1 ( ⁇ ); 0.5 ⁇ / ⁇ 1 (A) and 1 ⁇ / ⁇ 1 ( ⁇ ).
- Controls illustrating thrombin generation following induction of coagulation either by tissue factor / phospholipid (x) or by partial thromboplastin (0) are indicated.
- Figure 5 Thrombinograms obtained from the addition of 0.03 to 0.5 ⁇ g / mL of activated FX in a pool of normal plasma following activation by tissue factor:
- the normal plasma pool sample is represented by the ⁇ curve.
- the effect of the concentrations of FXa added to normal plasma is represented by the following curves: 0.0312 ⁇ g / ml (o); 0.0625 ⁇ g / ml ( ⁇ ); 0.125 ⁇ g / ml ( ⁇ ); 0.25 ⁇ g / ml (A) and 0.5 ⁇ g / ml ( ⁇ ).
- FIG. 6 Thrombograms obtained from the addition of 0.03 to 0.5 ⁇ / ⁇ , activated FX in a normal plasma pool in the presence of Rivaroxaban (0.35 ⁇ ⁇ ⁇ ⁇ ⁇ at activation by tissue factor:
- the normal plasma pool sample without Rivaroxaban (0.35 ⁇ / ⁇ 1) is represented by the ⁇ curve.
- the same plasma activated under the same conditions but in the presence of Rivaroxaban (0.35 ⁇ / ⁇ 1) is represented by the curve x.
- the effect of the FXa concentrations added to normal plasma in the presence of Rivaroxaban (0.35 ⁇ / ⁇ 1) is represented by the following curves: 0.0312 ⁇ / ⁇ 1 (o); 0.0625 ⁇ / ⁇ 1 ( ⁇ ); 0.125 ⁇ / ⁇ 1 ( ⁇ ); 0.25 ⁇ ⁇ ⁇ (A) and 0.5 ⁇ ⁇ ⁇ ( ⁇ ).
- FIG. 7 Thrombograms obtained from the addition of 0.03 to 0.5 ⁇ g / ml of activated FX in a pool of normal plasma following activation by the partial thromboplastin:
- the normal plasma pool sample is represented by the ⁇ curve.
- the effect of the concentrations of FXa added to normal plasma is represented by the following curves: 0.0312 ⁇ ⁇ ⁇ (o); 0.0625 ⁇ ⁇ ⁇ ( ⁇ ); 0.125 ⁇ ⁇ ⁇ ( ⁇ ); 0.25 ⁇ ⁇ ⁇ (A) and 0.5 ⁇ ⁇ . ( ⁇ ).
- FIG. 8 Thrombograms obtained from the addition of 0.03 to 0.5 ⁇ g / ml of activated FX in a pool of normal plasma following activation by the partial thromboplastin in the presence of Rivaroxaban (0.35 ⁇ ⁇ ⁇ ):
- the normal plasma pool sample without Rivaroxaban (0.35 ⁇ ⁇ ⁇ ) is represented by the ⁇ curve.
- the same plasma activated under the same conditions but in the presence of Rivaroxaban (0.35 ⁇ ⁇ ⁇ ) is represented by the curve x.
- the effect of concentrations in FXa added in normal plasma is represented by the following curves: 0.0312 ⁇ g / ml (o); 0.0625 ⁇ g / ml ( ⁇ ); 0.125 ⁇ g / ml ( ⁇ ); 0.25 ⁇ g / ml (A) and 0.5 ⁇ g / ml ( ⁇ ).
- the rivaroxaban stock solution at 436 ⁇ g / mL is diluted with 5% DMSO to obtain intermediate concentrations which, added to PNP (Normal Plasma Pool), give final concentrations of rivaroxaban in ⁇ g per mL of plasma.
- FX or FXa are introduced into the plasma without an incubation step.
- the thrombin generation test involves activating coagulation ex vivo either with a mixture of tissue factor and phospholipids, or with the aid of cephalin and then measuring the thrombin concentration generated over time.
- the thrombin generation tests are carried out on 80 ⁇ l of a plasma pool optionally containing FX and / or Rivaroxaban in the presence of either 20 ⁇ l of PPP reagent (Stago) containing finally 5 ⁇ M of Tissue Factor (FT) and 4 ⁇ l of phospholipids (PL), ie in the presence of 20 ⁇ l, cephalin (CK-Perst reconstituted with 5 ml of distilled H 2 O).
- the reaction is started by the addition of 20 ⁇ l of Fluca-kit (substrate + CaCl 2 ), which is the beginning of the measurement of the appearance of thrombin.
- the appearance of fluorescence is measured on a Fluoroskan Ascent fluorimeter (ThermoLabsystems) at an excitation wavelength of 390 nm and at an emission length of 460 nm.
- Thrombinograms curves representing the fluorescence intensity as a function of time
- Thrombinoscope TM software converts the fluorescence value into nM thrombin by comparative calculation.
- Table 1 Effect of factor X on normal or supplemented plasma in 0.5 ⁇ g / mL of Rivaroxaban.
- LT lag time
- ETP endogenous thrombin potential
- TTP time to peak
- ST start time
- mVRI mean velocity rate index
- PT prothrombin time.
- Rivaroxaban completely nullifies the ability to generate thrombin from normal plasma following activation by tissue factor and phospholipids.
- Supplementation with 10 ⁇ g / mL FX of normal plasma overloaded with Rivaroxaban at 0.5 ⁇ g / mL does not restore thrombin generation different from that obtained with Rivaroxaban overload ( Figure 1-B) .
- the commercial human FX (HTI) at 10 ⁇ g / mL final does not correct rivaroxaban overloaded plasma at 0.5 ⁇ g / mL extrinsically at 1 ⁇ M FT.
- Table 2 Factor X Factor Effect on Normal Plasma Containing 0.5 ⁇ l / ⁇ l of Rivaroxaban.
- LT lag time
- ETP endogenous thrombin potential
- TTP time to peak
- ST start time
- mVRI mean velocity rate index.
- FX 25 ⁇ / ⁇ 1 or 100 ⁇ / ⁇ 1) in a normal plasma increases the total amount of thrombin generated (+13 and +16% respectively), the height of the peak (+9.5 % o and 37% o respectively) and reduces the time between the onset of the reaction and the occurrence of thrombin but is unable to reverse the effects of Rivaroxaban.
- the thrombin generation tests are carried out on 80 ⁇ l of a normal plasma pool optionally containing FX and / or Rivaroxaban in the presence of 20 ⁇ l of cephalin (CK-Perst reconstituted with 5 ml of distilled H 2 0) and 20 ⁇ , fluca-kit (substrate + CaCl 2 ).
- Table 3 Effect of Factor X on a Normal Plasma Containing 0.35 ⁇ s / ml of Rivaroxaban.
- LT lag time
- ETP endogenous thrombin potential
- TTP time to peak
- ST start time
- mVRI mean velocity rate index.
- the addition of 10 ⁇ g / mL of FX in normal plasma increases the total amount of thrombin generated by 19%.
- the addition of 10 ⁇ g / mL of FX in normal plasma reduces the time between the initiation of the reaction and the onset of clot formation (LT) and the time between the initiation of the reaction and the onset of clot formation. peak concentration point of thrombin (TTP).
- Rivaroxaban completely nullifies the ability to generate thrombin from normal plasma following activation with Cephalin.
- Supplementation with 10 ⁇ g / mL FX of a normal plasma overloaded with Rivaroxaban at 0.35 ⁇ g / mL does not restore thrombin generation.
- FX does not correct a plasma overloaded with Rivaroxaban (at 0.35 ⁇ g / ml).
- the thrombin generation tests are carried out on 80 ⁇ l of a pool of normal plasma containing FXa and / or Rivaroxababn in the presence of 20 ⁇ l of PPP reagent low containing finally 1 ⁇ M of Tissue Factor (FT) and 4 ⁇ l of phospho lipids (PL), and 20 ⁇ , fiuca-kit (substrate + CaCl2).
- FT Tissue Factor
- PL phospho lipids
- Table 4 Effect of factor Xa on a normal plasma containing 0.35 ⁇ g / mL of Rivaroxaban.
- LT lag time
- ETP endogenous thrombin potential
- TTP time to peak
- ST start time
- mVRI mean velocity rate index
- PT prothrombin time
- aPTT activated partial thromboplastin time
- PTT-A prothromboplastin activated.
- the addition of FXa in normal plasma reduces the time between the triggering of the reaction in successive steps in a dose-dependent manner.
- the addition of 0.35 ⁇ g / mL of rivaroxaban in normal plasma increases this duration by a factor of between 2 and 3 and completely abolishes thrombin formation (Fig. 3- A and 3-B). .
- FXa in normal plasma reduces the time between the initiation of the reaction and the point of maximum concentration of thrombin (TTP) in successive steps in a dose-dependent manner.
- TTP point of maximum concentration of thrombin
- 0.35 ⁇ g / mL rivaroxaban in normal plasma increases this duration by a factor of between 3 and 4.
- Rivaroxaban 0.35 ⁇ g / mL restores both LT and ETP at the level observed for normal plasma alone.
- Factor Xa also enhances the thrombin generation of a normal plasma pool in a dose-dependent manner.
- FXa doses from 0.01 ⁇ g / ml to 0.05 ⁇ g / ml produce thrombin levels similar to that of a normal plasma activated by FT or the partial thrombin and follow the same kinetics of appearance. These results suggest that under these experimental conditions, FXa at a dose of 50 ng / ml should not be thrombogenic. The dose of 50 ng / ml would therefore correspond to the lowest dose of FXa that can be used to restore coagulation.
- Rivaroxaban significantly decreases thrombin generation (approximately 75% x-curve) compared to that obtained in untreated plasma ( ⁇ ).
- the supplementation with FXa of 0.03 ⁇ g / ml (o) does not allow to find a significant generation of thrombin.
- a correction of the thrombin generation is obtained from 0.06 ⁇ g / ml ( ⁇ ) up to a dose of 0.25 ⁇ g / ml (A).
- the amount of thrombin generated exceeds that of the control suggesting an FXa overdose and thus a possible thrombogenic effect despite Rivaroxaban.
- Table I Values of latency, thrombin potential, peak height and peak time of tissue-factor-induced thrombin generation in normal plasma with or without factor Xa and / or Rivaroxaban (data extracted from Figures 4 and 5).
- Table II Values of latency, thrombin potential, peak height and peak time of thrombin generation induced by the cephalin of normal plasma with or without factor Xa and / or rivaroxaban ( data extracted from Figures 7 and 8).
- FXa has the possibility of correcting the presence of Rivaroxaban in normal plasma following the induction of the intrinsic pathway by partial thromboplastin or extrinsic pathway by tissue factor.
- the data obtained in TGT do not identify a risk of major thrombogenicity.
- the thrombin generation time is very shortened and could reveal a thrombogenic risk.
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Abstract
The present invention concerns factor Xa for the use thereof in the prevention or treatment, in a patient, in particular a human or animal, of haemorrhagic events induced by the taking of anticoagulants.
Description
UTILISATION DU FACTEUR XA DANS LA PREVENTION OU LE TRAITEMENT USE OF XA FACTOR IN PREVENTION OR TREATMENT
D'EVENEMENTS HEMORRAGIQUES A LA SUITE DE LA PRISE D'ANTICOAGULANTS HAEMORRHAGIC EVENTS FOLLOWING THE TAKING OF ANTICOAGULANTS
La présente invention concerne l'utilisation d'antidotes d'inhibiteurs de la coagulation indiqués dans la prévention ou le traitement de pathologies thromboemboliques. The present invention relates to the use of coagulation inhibitor antidotes indicated in the prevention or treatment of thromboembolic pathologies.
La thrombose veineuse constitue une pathologie largement répandue dans le monde qui affecte chaque année environ 900 000 patients aux Etats-Unis et 770 000 patients en Europe. Disposer, en prophylaxie des événements thromboemboliques veineux, de traitements efficaces et bien tolérés sur le plan hémorragique, en particulier chez des patients à risque, constitue un besoin de santé publique. Venous thrombosis is a widespread condition worldwide, affecting approximately 900,000 patients in the United States each year and 770,000 patients in Europe. Providing prophylactic venous thromboembolic events with effective and well-tolerated hemorrhagic treatments, especially in at-risk patients, is a public health need.
La thrombose veineuse consiste en la formation d'un caillot dans une veine. Elle peut être "superficielle" lorsqu'elle touche les petites veines situées entre la peau et les muscles, ou "profonde" lorsqu'elle atteint une veine plus importante. La localisation préférentielle des thromboses profondes se situe dans les membres inférieurs (jambes, cuisses, plis de l'aine). Sous l'effet de différents facteurs, le caillot peut se détacher de la veine qui l'héberge, entrer dans la circulation sanguine et provoquer une embolie pulmonaire (obstruction de l'artère pulmonaire) pouvant engager le pronostic vital ou entraîner des séquelles importantes. Venous thrombosis is the formation of a clot in a vein. It can be "superficial" when it touches the small veins located between the skin and the muscles, or "deep" when it reaches a larger vein. The preferential location of deep thromboses is in the lower limbs (legs, thighs, groin folds). As a result of various factors, the clot can become detached from the vein that houses it, enter the bloodstream and cause pulmonary embolism (obstruction of the pulmonary artery) that may be life-threatening or have significant sequelae.
Bien que la prévalence de la thrombose veineuse augmente de manière significative avec l'âge, d'autres facteurs de risques associés à la thrombose veineuse ont été identifiés. Il s'agit notamment des maladies héréditaires ou acquises de la coagulation (déficits en antithrombine, protéines C et S ; facteur V Leiden, mutation de la prothrombine), l'obésité, l'insuffisance cardiaque, l'alitement consécutif d'une intervention chirurgicale ou d'une grossesse, la prise de contraceptifs oraux associée au tabagisme. De manière plus générale, toute période occasionnant un ralentissement de la circulation sanguine (stase veineuse) favorise l'apparition de petites lésions vasculaires formant un terrain propice au développement des thromboses. Although the prevalence of venous thrombosis increases significantly with age, other risk factors associated with venous thrombosis have been identified. These include inherited or acquired diseases of coagulation (antithrombin deficiency, protein C and S, factor V Leiden, prothrombin mutation), obesity, heart failure, bed rest after surgery surgical or pregnancy, taking oral contraceptives associated with smoking. More generally, any period causing a slowing of the blood circulation (venous stasis) promotes the appearance of small vascular lesions forming a fertile ground for the development of thromboses.
Le risque de thrombose veineuse est particulièrement élevé chez un patient ayant subi une intervention chirurgicale orthopédique lourde des membres inférieurs, notamment lors de la pose d'une prothèse de hanche ou de genou ou de la réduction
d'une fracture du col du fémur. Afin de prévenir la survenue de thrombose veineuse post-opératoire, le clinicien a systématiquement recourt aux anticoagulants. The risk of venous thrombosis is particularly high in a patient who has undergone a major orthopedic surgery of the lower limbs, especially when placing a hip or knee prosthesis or the reduction fracture of the femoral neck. In order to prevent the occurrence of postoperative venous thrombosis, the clinician has systematically resorted to anticoagulants.
Il existe plusieurs catégories d'anticoagulants réparties en fonction des protéines cibles de la cascade de coagulation. There are several categories of anticoagulants distributed according to the target proteins of the coagulation cascade.
Les antagonistes de la vitamine K (AVK), notamment les coumarines ou les 1,3- indandiones, ciblent les facteurs II, VII, IX et X de la cascade de coagulation. Ces molécules, couramment utilisés dans les situations ou le risque de formation de caillot est majeur, nécessitent des prélèvements sanguins réguliers pour contrôler l'INR (International Normalized Ratio). Compte tenu de leur capacité à modifier les protéines plasmatiques circulantes, les AVK possèdent un temps de demi- vie long qui rend leur utilisation peu flexible. Vitamin K antagonists (VKAs), including coumarins or 1,3-indandiones, target factors II, VII, IX and X of the coagulation cascade. These molecules, commonly used in situations where the risk of clot formation is major, require regular blood sampling to control the INR (International Normalized Ratio). Because of their ability to modify circulating plasma proteins, AVKs have a long half-life that makes their use inflexible.
L'héparine standard (non fractionnée : héparine, calciparine) et les héparines de bas poids moléculaire (HBPM) comme l'enoxaparine (Lovenox®), la tinzaparine (Innohep®), la bemiparine (Ivor®), la nadroparine (Fraxiparine®), la parnaparine (Flexum®), la reviparine (Clivarine®) ou la deltaparine (Fragmine®) sont des glycosaminoglycans constitués de chaînes comportant des résidus alternés de D- Glucosamine et d'acide uronique. Ces composés sont capables d'accroître l'activité de l'antithrombine et ainsi d'inhiber les facteurs X activé (FXa) et II activé (Fila). Les HBPM inhibent également directement le FXa, inhibant ainsi la conversion de la prothrombine en thrombine. Compte tenu de leur efficacité supérieure à celle de l'héparine non fractionnée et de leurs moindres effets secondaires en termes de thrombopénie, les HBPM sont préférentiellement injectées aux patients. Standard heparin (unfractionated: heparin, calciparin) and low molecular weight heparins (LMWH) such as enoxaparin (Lovenox ® ), tinzaparin (Innohep ® ), bemiparin (Ivor ® ), nadroparin (Fraxiparine ® ), parnaparin (Flexum ® ), reviparin (Clivarine ® ) or deltaparin (Fragmine ® ) are glycosaminoglycans consisting of chains with alternating residues of D-Glucosamine and uronic acid. These compounds are capable of increasing the activity of antithrombin and thus of inhibiting activated X-factor (FXa) and activated II (Fila). LMWHs also directly inhibit FXa, thereby inhibiting the conversion of prothrombin to thrombin. Given their superior efficacy to unfractionated heparin and their lesser side effects in terms of thrombocytopenia, LMWHs are preferentially injected into patients.
Plus récemment, de nouveaux anticoagulants oraux ont été développés. Il s'agit d'inhibiteurs spécifiques directs de la thrombine (dabigatran (Pradaxa®), hirudin) ou du facteur X activé (apixaban, betrixaban, edoxaban, otamixaban ou rivaroxaban (Xarelto®)). Ces composés sont rapidement métabolisés et atteignent une concentration plasmatique maximale dans les 2 à 4 heures suivant leur administration. Leur efficacité est comparable à celle des HBPM dans le traitement de la thrombose veineuse consécutive à une intervention chirurgicale de la hanche ou du genou. Toutefois, le recours à ces composés est contre-indiqué chez les patients souffrant d'un dysfonctionnement hépatique ou d'insuffisance rénale. More recently, new oral anticoagulants have been developed. These are specific direct inhibitors of thrombin (dabigatran (Pradaxa ® ), hirudin) or activated factor X (apixaban, betrixaban, edoxaban, otamixaban or rivaroxaban (Xarelto ® )). These compounds are rapidly metabolized and reach a peak plasma concentration within 2 to 4 hours of dosing. Their effectiveness is comparable to that of LMWHs in the treatment of venous thrombosis following surgery of the hip or knee. However, the use of these compounds is contraindicated in patients with hepatic dysfunction or renal failure.
La prise d'anticoagulants dans le cadre d'un traitement anti-thrombotique peut être à l'origine de la survenue de complications hémorragiques, rares mais parfois
fatales. La rapidité d'intervention est alors décisive et l'effet des anticoagulants doit être levé le plus rapidement possible. L'action des AVK peut être inversée par des moyens conventionnels comme la vitamine K ou la transfusion de plasma. Toutefois, les effets de la vitamine K perdurent pendant plusieurs jours, rendant son utilisation délicate. S'agissant des HBPM, la protamine permet d'inhiber l'activité anti-FIIa mais ne neutralise que partiellement l'activité anti-FXa ; seule une dialyse permet l'élimination complète de ces composés de la circulation sanguine, rétablissant ainsi la capacité coagulante naturelle du patient. L'hémodialyse est également envisageable dans le cas du dagitraban mais se révèle inefficace dans le cas du rivaroxaban. L'utilisation du complexe prothrombine concentré comme antidote des anticoagulants a été envisagée pour la première fois dans le milieu des années 90 (EP 0 700 684 A2). Plus récemment, les travaux d'Eerenberg et al. ont montré que ces composés permettent de renverser immédiatement et complètement l'effet anticoagulant du rivaroxaban chez des patients sains (Eerenberg et al., 2011, Circulation, 124, 1573-1579). Bien que le recours à des molécules procoagulantes comme le facteur VII activé (FVIIa) puissent être envisagé, sa faible activité procoagulante en l'absence de son cofacteur, le facteur tissulaire, en limite l'intérêt thérapeutique. Taking anticoagulants as part of an anti-thrombotic treatment may cause haemorrhagic complications, rare but sometimes fatal. The speed of intervention is then decisive and the effect of anticoagulants must be lifted as quickly as possible. The action of AVK can be reversed by conventional means such as vitamin K or plasma transfusion. However, the effects of vitamin K persist for several days, making its use delicate. With regard to LMWH, protamine inhibits anti-FIIa activity but only partially neutralizes anti-FXa activity; only dialysis allows the complete removal of these compounds from the bloodstream, thus restoring the natural coagulant capacity of the patient. Hemodialysis is also feasible in the case of dagitraban but is ineffective in the case of rivaroxaban. The use of the concentrated prothrombin complex as an antidote for anticoagulants was considered for the first time in the mid-1990s (EP 0 700 684 A2). More recently, the work of Eerenberg et al. have shown that these compounds can immediately and completely reverse the anticoagulant effect of rivaroxaban in healthy patients (Eerenberg et al., 2011, Circulation, 124, 1573-1579). Although the use of procoagulant molecules such as activated factor VII (FVIIa) may be considered, its low procoagulant activity in the absence of its cofactor, tissue factor, limits therapeutic interest.
Le composé PRT4445, actuellement en cours de développement, constitue l'unique agent thérapeutique capable de lier et de neutraliser tous les inhibiteurs directs et indirects du facteur X activé permettant ainsi de restaurer la fonction hémostatique normale (WO 2011/008885 Al). Ce composé est en fait une protéine dérivée du FX ou du FXa dans laquelle le site actif et/ou le domaine Gla ont été invalidés, réduisant ainsi l'activité procoagulante intrinsèque de ces facteurs (WO 2010/117729 Al). Des essais cliniques de phase la concernant l'utilisation du composé PRT4445 comme antidote du betrixaban (US 6,376 ,515 et US 6,835,739) sont imminents. The compound PRT4445, currently under development, is the only therapeutic agent capable of binding and neutralizing all direct and indirect inhibitors of activated factor X, thereby restoring normal hemostatic function (WO 2011/008885 A1). This compound is in fact a protein derived from FX or FXa in which the active site and / or the Gla domain have been invalidated, thus reducing the intrinsic procoagulant activity of these factors (WO 2010/117729 A1). Phase II clinical trials of the use of the compound PRT4445 as an antidote for betrixaban (US 6,376, 515 and US 6,835,739) are imminent.
Les cliniciens ne disposent à l'heure actuelle d'aucun antidote des inhibiteurs du FXa homologué par les autorités de santé publique. Il existe par conséquent un réel besoin d'identifier des agents thérapeutiques susceptibles de renverser rapidement l'effet anticoagulant des inhibiteurs du FXa tout en n'induisant pas de risque de thrombose. Clinicians currently have no antidote for FXa inhibitors approved by public health authorities. There is therefore a real need to identify therapeutic agents that can rapidly reverse the anticoagulant effect of FXa inhibitors while not inducing risk of thrombosis.
Le but de la présente invention est de proposer une méthode de prévention et de traitement d'un événement hémorragique consécutif de la prise d'anticoagulants.
Un autre but de la présente invention est de restaurer la fonction hémostatique normale lors de la survenue d'un événement hémorragique chez un patient placé sous anticoagulant. The aim of the present invention is to propose a method for preventing and treating a hemorrhagic event consecutive to taking anticoagulants. Another object of the present invention is to restore the normal haemostatic function during the occurrence of a haemorrhagic event in a patient placed on anticoagulant.
Encore un autre but de la présente invention est de fournir un antidote des inhibiteurs du facteur X activé (FXa). Yet another object of the present invention is to provide an antidote for activated factor X inhibitors (FXa).
La présente invention concerne le facteur Xa (FXa) pour son utilisation dans la prévention ou le traitement chez un patient, notamment un être humain ou un animal, des événements hémorragiques induits par la prise d'anticoagulants. The present invention relates to factor Xa (FXa) for its use in the prevention or treatment in a patient, in particular a human being or an animal, hemorrhagic events induced by the taking of anticoagulants.
L'invention repose sur la constatation inattendue que le facteur Xa est capable de renverser rapidement l'effet anticoagulant des anticoagulants, sans induire de risque de thrombose, et de restaurer la fonction hémostatique « normale » lors de la survenue d'un événement hémorragique consécutif à la prise d'anticoagulants. The invention is based on the unexpected finding that factor Xa is able to rapidly reverse the anticoagulant effect of anticoagulants, without inducing a risk of thrombosis, and to restore the "normal" haemostatic function at the occurrence of a consecutive haemorrhagic event. taking anticoagulants.
Par « facteur Xa», également nommé FXa ou encore facteur X activé, on désigne une protéase à sérine de la cascade de coagulation, obtenue à partir du facteur X inactif (Butenas & Mann, Biochemistry (Moscow), 2002, 67(1), 3-12). La protéolyse du FX en FXa est réalisée soit par le complexe ténase (facteur IX activé (FIXa) et son cofacteur, le facteur VIII activé (FVIIIa) en présence de calcium et de phospho lipides) soit par le complexe facteur VII activé (FVIIa) /Facteur tissulaire en présence de calcium. Le FXa forme avec le facteur V activé (FVa) le complexe prothrombinase qui catalyse la conversion de la prothrombine (facteur II, FII) en thrombine (facteur II activé, Fila). La thrombine catalyse la conversion du fibrinogène en fibrine responsable de la formation du caillot sanguin. Le FXa se caractérise par une activité procoagulante. Le FXa occupe une place centrale dans les voies intrinsèque et extrinsèque de la coagulation. By "factor Xa", also called FXa or activated factor X, is meant a serine protease of the coagulation cascade, obtained from the inactive factor X (Butenas & Mann, Biochemistry (Moscow), 2002, 67 (1) , 3-12). The proteolysis of FX in FXa is carried out either by the tenase complex (activated factor IX (FIXa) and its cofactor, activated factor VIII (FVIIIa) in the presence of calcium and phospholipids) or by the activated factor VII complex (FVIIa). Tissue factor in the presence of calcium. FXa forms, with activated factor V (FVa), the prothrombinase complex which catalyzes the conversion of prothrombin (factor II, FII) to thrombin (activated factor II, Fila). Thrombin catalyzes the conversion of fibrinogen to fibrin responsible for blood clot formation. FXa is characterized by procoagulant activity. FXa occupies a central place in the intrinsic and extrinsic pathways of coagulation.
Le facteur Xa sert d'antidote aux anticoagulants. Par « antidote », on désigne les molécules, et notamment le FXa, capables de neutraliser ou de renverser tout ou partie de l'activité anticoagulante des anticoagulants. Cet effet doit pouvoir intervenir dans un délai plus ou moins court, en relation avec la localisation et l'ampleur de l'événement hémorragique pour ralentir, atténuer ou interrompre totalement cet événement hémorragique. L'activité anticoagulante des anticoagulants peut être mesurée par des tests coagulométriques globaux (temps de Quick (PT), temps de céphaline activé (aPTT)). En leur présence, il y a diminution de l'INR (International Normalized Ratio).
La mesure de leurs effets peut aussi être envisagée par des tests de coagulation qui mesurent la génération de thrombine (TGT). Factor Xa serves as an antidote for anticoagulants. By "antidote" is meant the molecules, including FXa, capable of neutralizing or reversing all or part of the anticoagulant activity of anticoagulants. This effect must be able to intervene in a more or less short time, in connection with the location and the magnitude of the haemorrhagic event to slow down, attenuate or completely interrupt this hemorrhagic event. The anticoagulant activity of anticoagulants can be measured by global coagulometric tests (Quick time (PT), activated partial thromboplastin time (aPTT)). In their presence, there is a decrease in the INR (International Normalized Ratio). Measurement of their effects can also be considered by coagulation tests that measure thrombin generation (TGT).
Par « événements hémorragiques », on désigne les saignements visibles ou invisibles résultant de la rupture des vaisseaux sanguins, notamment des veines profondes, superficielles ou des veinules. L'ampleur des saignements visibles est évaluée par observation simple. En revanche les signes suivants peuvent révéler l'existence de saignements invisibles : asthénie, dyspnée, pâleur, maux de tête résistants aux traitements habituels, malaises. Les événements hémorragiques au sens de la présente invention sont un effet délétère du mésusage des anticoagulants. La gravité des saignements dépend de leur localisation et de leur intensité et est appréciée par la nécessité de transfusion ou la mise en jeu du pronostic vital du patient. En dehors des accidents hémorragiques intracrâniens, les formes cliniques sont très variées et peuvent se présenter comme des hémorragies extériorisées ou non (hémorragie digestive, hématome rétropéritonéal, saignements urologique, gynécologique, hématome des parties molles, épistaxis, hémoptysie, gingivorragies...). Pour les formes les moins graves, les événements hémorragiques peuvent conduire à une anémie. "Hemorrhagic events" means visible or invisible bleeding resulting from rupture of blood vessels, including deep veins, superficial veins or venules. The extent of visible bleeding is assessed by simple observation. On the other hand, the following signs may reveal the existence of invisible bleeding: asthenia, dyspnoea, paleness, headaches resistant to usual treatments, discomfort. Hemorrhagic events within the meaning of the present invention are a deleterious effect of the misuse of anticoagulants. The severity of the bleeding depends on their location and intensity and is appreciated by the need for transfusion or the life-threatening condition of the patient. Apart from intracranial haemorrhagic accidents, the clinical forms are very varied and can be presented as externalized hemorrhages or not (digestive haemorrhage, retroperitoneal hematoma, urological bleeding, gynecological, hematoma of the soft parts, epistaxis, haemoptysis, gingivorragies ...). For the less severe forms, hemorrhagic events can lead to anemia.
Par « anticoagulants », on désigne les composés capables d'inhiber la formation du caillot sanguin et d'allonger les tests coagulométriques (aPTT et PT). La limitation de la formation de thrombine peut aussi être évaluée en TGT. Les anticoagulants peuvent être répertoriés selon leur cible pharmaco logique. On distingue ainsi les antagonistes de la vitamine K, les héparines non fractionnées ou de bas poids moléculaire, les anticorps anti-facteur tissulaire, les inhibiteurs spécifiques, directs ou indirects des facteurs IXa, Xa, Xla, Xlla ou Vlla. Les anticoagulants sont administrés seuls ou en combinaison chez un patient à une dose et selon un schéma posologique qui permettent de prévenir ou de traiter l'apparition de caillot, réduisant ainsi le risque de thrombose. By "anticoagulants" are meant compounds capable of inhibiting blood clot formation and lengthening coagulometric tests (aPTT and PT). The limitation of thrombin formation can also be evaluated in TGT. Anticoagulants can be listed according to their pharmacological target. There are thus antagonists of vitamin K, unfractionated or low molecular weight heparins, anti-tissue factor antibodies, specific inhibitors, direct or indirect factors IXa, Xa, Xla, Xlla or VIIa. Anticoagulants are administered alone or in combination in a patient at a dose and in a dosing regimen that can prevent or treat clot onset, thereby reducing the risk of thrombosis.
Selon un mode de réalisation particulier, dans l'utilisation du facteur Xa selon la présente invention, les anticoagulants sont des héparines de bas poids moléculaire. According to a particular embodiment, in the use of the factor Xa according to the present invention, the anticoagulants are heparins of low molecular weight.
Par « héparines de bas poids moléculaires », également nommées HBPM, on désigne les héparines résultant du clivage de l'héparine non fractionnée (Mousa SA, Methods Mol. Biol, 2010, 663, 109-132). Ces composés anti-thrombotiques sont
administrés par voie intraveineuse, et leurs concentrations d'utilisation sont exprimées en unité internationales anti-FXa même si leur efficacité ne se limite pas à cette activité anti-FXa. Les HBPM ont une durée de demi-vie plus longue que les héparines non fractionnées et possèdent une interaction avec les plaquettes inférieure à celle des héparines non fractionnées. Les HBPM ont une masse moléculaire qui varie de 4000 à 6000 Da. Les héparines de bas poids moléculaire couramment utilisées sont l'enoxaparine (Lovenox®), la tinzaparine (Innohep®), la bemiparine (Ivor®), la nadroparine (Fraxiparine®), la parnaparine (Flexum®), la reviparine (Clivarine®), la certoparine (Sandoparine®) ou la deltaparine (Fragmine®). Les HBPM sont employées préventivement en chirurgie post-opératoire d'un membre inférieur ou lorsqu'une immobilisation de longue durée est obligatoire. On entend par immobilisation longue durée le maintien du patient à l'état alité pendant une période qui varie de 3 jours à plusieurs semaines. Selon un mode de réalisation plus particulier, dans l'utilisation du facteur Xa selon la présente invention les héparines de bas poids moléculaire sont choisies parmi la bemiparine, la certoparine, la deltaparine, l'enoxaparine, la nadroparine, la parnaparine, la reviparine ou la tinzaparine. Selon un autre mode de réalisation, dans l'utilisation du facteur Xa selon la présente invention, les anticoagulants sont des inhibiteurs spécifiques du facteur Xa. "Low molecular weight heparins", also known as LMWH, refers to heparins resulting from the cleavage of unfractionated heparin (Mousa SA, Methods Mol Biol, 2010, 663, 109-132). These anti-thrombotic compounds are administered intravenously, and their use concentrations are expressed in international anti-FXa units even if their effectiveness is not limited to this anti-FXa activity. LMWHs have a longer half-life than unfractionated heparins and have lower platelet interaction than unfractionated heparins. LMWHs have a molecular weight that ranges from 4000 to 6000 Da. The low molecular weight heparins commonly used are enoxaparin (Lovenox ® ), tinzaparin (Innohep ® ), bemiparin (Ivor ® ), nadroparin (Fraxiparine ® ), parnaparin (Flexum ® ), reviparin (Clivarine ® ), certoparin (Sandoparine ® ) or deltaparin (Fragmine ® ). LMWHs are used preventively in postoperative lower limb surgery or when long-term immobilization is mandatory. By long-term immobilization is meant maintaining the patient in a bedded state for a period ranging from 3 days to several weeks. According to a more particular embodiment, in the use of the factor Xa according to the present invention the low molecular weight heparins are chosen from bemiparin, certoparin, deltaparin, enoxaparin, nadroparin, parnaparin, reviparin or tinzaparin. According to another embodiment, in the use of factor Xa according to the present invention, anticoagulants are specific inhibitors of factor Xa.
Par « inhibiteurs spécifiques du facteur Xa », on désigne les composés capables d'inhiber, directement ou indirectement, l'activité procoagulante du FXa qui consiste en la conversion de la prothrombine en thrombine in vitro, et/ou ex vivo, et/ou in vivo. Les inhibiteurs du FXa peuvent être classés en inhibiteurs de nature peptidique, extraits et purifiés ou obtenus par génie génétique et en inhibiteurs de nature non peptidique obtenus par synthèse chimique (Kher et al., 1998, La Lettre du pharmacologue, 12(6), 222-226). Les inhibiteurs qui bloquent le site actif du FXa sont appelés inhibiteurs directs alors que les inhibiteurs qui agissent en se liant et en catalysant l'effet de l'antithrombine vis-à-vis du FXa sont appelés inhibiteurs indirects. Des exemples d'inhibiteurs peptidiques directs du FXa incluent, sans que cela ne restreigne la portée de l'invention, le TAP (tick anticoagulant peptide) extrait de la salive de la tique,
l'antistasine extraite des glandes salivaires de la sangsue Haementeria officinalis, l'ACAP (ancylostoma caninum anticoagulant peptide) isolé à partir de l'ankylostome ou ses formes recombinantes r Ac AP5, r Ac AP2 (également appelé NAP-5) ou encore FXa I (factor Xa inhibitor) extraite de la salive de la sangsue Hirudo medicinalis ou la protéine recombinante lui correspondant nommée Yagin. Parmi les inhibiteurs non peptidiques directs du FXa, on trouve le DX 9065a, le LY517717 et les xabans (eribaxaban, apixaban, betrixaban, edoxaban, otamixaban, rivaroxaban). Les HBPM, les oligosaccharides fondaparinux ou idraparinux et les héparinoïdes (danaparoïde, sulodexide, sulfate de dermatan) constituent des exemples d'inhibiteurs indirects du FXa. By "specific factor Xa inhibitors" are meant compounds capable of directly or indirectly inhibiting the procoagulant activity of FXa which consists of the conversion of prothrombin to thrombin in vitro, and / or ex vivo, and / or in vivo. FXa inhibitors can be classified as peptide inhibitors, extracted and purified or genetically engineered, and non-peptide inhibitors obtained by chemical synthesis (Kher et al., 1998, The Pharmacologist's Letter, 12 (6), 222-226). Inhibitors that block the active site of FXa are called direct inhibitors while inhibitors that act by binding and catalyzing the effect of antithrombin on FXa are referred to as indirect inhibitors. Examples of FXa direct peptide inhibitors include, without restricting the scope of the invention, the TAP (tick anticoagulant peptide) extracted from tick saliva, antistasin extracted from the salivary gland of the leech Haementeria officinalis, ACAP (ancylostoma caninum anticoagulant peptide) isolated from the hookworm or its recombinant forms r Ac AP5, r Ac AP2 (also called NAP-5) or FXa I (factor Xa inhibitor) extracted from the saliva of the leech Hirudo medicinalis or the corresponding recombinant protein named Yagin. Among the direct non-peptide inhibitors of FXa are DX 9065a, LY517717 and xabans (eribaxaban, apixaban, betrixaban, edoxaban, otamixaban, rivaroxaban). LMWH, fondaparinux or idraparinous oligosaccharides and heparinoids (danaparoid, sulodexide, dermatan sulfate) are examples of indirect inhibitors of FXa.
Dans ce mode de réalisation particulier, le FXa constitue l'antidote des inhibiteurs spécifiques du FXa. La structure catalytique du FXa utilisé selon la présente invention permet la liaison aux inhibiteurs spécifiques du FXa. L'utilisation du facteur Xa comme antidote des inhibiteurs du FXa permet la titration de ces inhibiteurs. Dans l'utilisation du FXa comme antidote selon la présente invention, l'activité inhibitrice des inhibiteurs spécifiques de FXa est réduite d'au moins environ 10%, 20%, 30%>, 40%, 50%, 60%, 70%, 80%, 90% ou 100%. La diminution de l'activité inhibitrice des anti-Xa est mesurée par le retour d'une activité fonctionnelle du FXa ou d'une augmentation de l'activité procoagulante dans le plasma. Ces activités peuvent être mesurées par mesure chromogénique (clivage de peptide spécifique du FXa ou de la thrombine), chronométrique (aPTT, PT, augmentation de l'INR) ou par des tests de mesure globale de la coagulation (mesure de temps de génération de thrombine, TGT). In this particular embodiment, FXa is the antidote for specific FXa inhibitors. The catalytic structure of FXa used according to the present invention allows binding to specific FXa inhibitors. The use of factor Xa as an antidote for FXa inhibitors allows the titration of these inhibitors. In the use of FXa as an antidote according to the present invention, the inhibitory activity of specific FXa inhibitors is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%. , 80%, 90% or 100%. The decrease in the anti-Xa inhibitory activity is measured by the return of a functional activity of FXa or an increase in procoagulant activity in the plasma. These activities can be measured by chromogenic measurement (cleavage of FXa or thrombin specific peptide), chronometric time (aPTT, PT, INR increase) or by global coagulation measurement tests (measurement of the generation time of thrombin, TGT).
Selon un autre mode de réalisation particulier, dans l'utilisation du facteur Xa selon la présente invention, les anticoagulants sont choisis dans le groupe constitué par les héparines de bas poids moléculaire, les oligosaccharides fondaparinux ou idraparinux et les inhibiteurs spécifiques du facteur Xa. According to another particular embodiment, in the use of the factor Xa according to the present invention, the anticoagulants are chosen from the group consisting of low molecular weight heparins, fondaparinux or idraparinous oligosaccharides and factor Xa specific inhibitors.
Selon encore un autre mode de réalisation particulier, dans l'utilisation du facteur Xa selon la présente invention, les anticoagulants sont les oligosaccharides fondaparinux ou idraparinux. According to yet another particular embodiment, in the use of factor Xa according to the present invention, the anticoagulants are fondaparinux or idraparinous oligosaccharides.
Selon encore un autre mode de réalisation particulier, dans l'utilisation du facteur Xa selon la présente invention, l'oligosaccharide est le fondaparinux.
Selon un autre mode de réalisation plus particulier, dans l'utilisation du facteur Xa selon la présente invention, les inhibiteurs spécifiques du facteur Xa sont choisis parmi l'apixaban, le betrixaban, l'édoxaban, l'otamixaban ou le rivaroxaban. Selon un autre mode de réalisation, dans l'utilisation du facteur Xa selon l'invention, le facteur Xa restaure l'activité procoagulante sans que cette dernière ne dépasse le niveau basai de l'activité procoagulante mesurée dans un groupe de patients sains. Par « activité procoagulante », on désigne l'activité catalytique du FXa qui consiste à cliver la prothrombine en thrombine. On parle alors d'activation de la prothrombine. L'activité catalytique du FXa est assimilable à l'activité catalytique de la prothrombinase, complexe protéique qui contient non seulement le FXa mais également le FVa. According to yet another particular embodiment, in the use of factor Xa according to the present invention, the oligosaccharide is fondaparinux. According to another more particular embodiment, in the use of the factor Xa according to the present invention, the factor Xa specific inhibitors are chosen from apixaban, betrixaban, edoxaban, otamixaban or rivaroxaban. According to another embodiment, in the use of factor Xa according to the invention, factor Xa restores the procoagulant activity without the latter exceeding the basal level of the procoagulant activity measured in a group of healthy patients. By "procoagulant activity" is meant the catalytic activity of FXa which consists in cleaving prothrombin to thrombin. This is called activation of prothrombin. The catalytic activity of FXa is comparable to the catalytic activity of prothrombinase, a protein complex that contains not only FXa but also FVa.
Par « niveau basai », on désigne le taux de FXa d'un patient ou d'un groupe de patients sains. Chaque patient possède son propre niveau basai de FXa qui lui permet de maintenir un équilibre en termes de coagulation. Ce niveau basai, proche de zéro en conditions physiologiques normales, peut varier d'un individu à l'autre, notamment en fonction du niveau basai de FX présent dans le sang. By "basal level" is meant the level of FXa of a patient or a group of healthy patients. Each patient has his own basal level of FXa that allows him to maintain a balance in terms of coagulation. This basal level, close to zero under normal physiological conditions, may vary from one individual to another, particularly depending on the basal level of FX present in the blood.
Par « patients », on désigne tout mammifère, humain ou animal. Dans le cadre de la présente invention, les patients ont reçu au moins un inhibiteur spécifique du facteur Xa. By "patients" is meant any mammal, human or animal. In the context of the present invention, the patients received at least one specific factor Xa inhibitor.
Par « patients sains», on désigne un groupe de patients qui ne présentent pas de pathologie apparente et non placés sous inhibiteurs spécifiques du facteur Xa, c'est-à- dire n'ayant reçu aucun traitement par l'un quelconque des inhibiteurs spécifiques du FXa. L'intérêt de réaliser la mesure du taux de FXa chez plusieurs patients sains est de pouvoir en déterminer une moyenne qui sert de valeur de référence. La comparaison de cette valeur moyenne de référence d'activité procoagulante du FXa avec celle d'un patient sous inhibiteurs de FXa et qui reçoit le FXa comme antidote permet de suivre l'effet thérapeutique de l'antidote. By "healthy patients" is meant a group of patients who have no apparent pathology and who are not on Factor Xa-specific inhibitors, ie, who have not received any treatment with any of the specific inhibitors. FXa. The advantage of measuring the level of FXa in several healthy patients is to be able to determine an average that serves as a reference value. The comparison of this average reference value of procoagulant FXa activity with that of a patient under FXa inhibitors and who receives FXa as an antidote makes it possible to follow the therapeutic effect of the antidote.
Selon un mode de réalisation particulier, le facteur Xa pour son utilisation selon l'invention est une protéine native isolée du plasma ou une protéine recombinante.
Par « protéine native isolée du plasma », on désigne le FX retrouvé à l'état naturel dans le plasma/sérum des patients et qui a subi une étape de purification permettant de l'isoler de sa matrice naturelle. L'activation in vitro ou ex vivo du FX ainsi isolé aboutit à l'obtention du FXa qui possède la capacité d'activer la prothrombine. According to a particular embodiment, the factor Xa for its use according to the invention is a native protein isolated from the plasma or a recombinant protein. By "native protein isolated from plasma" is meant the FX found naturally in the plasma / serum of patients and which has undergone a purification step to isolate it from its natural matrix. The in vitro or ex vivo activation of the FX thus isolated results in FXa having the ability to activate prothrombin.
Par « protéine recombinante », on désigne une protéine préparée, exprimée, isolée, purifiée par génie génétique. La technologie des ADN recombinants permet d'insérer une séquence polynucléotidique codant soit pour le FXa, soit pour le FX, dans une cellule hôte et ainsi de faire produire le polypeptide d'intérêt, i.e. le FXa ou bien le FX qui pourra être activé après production cellulaire. Il est également possible de modifier la séquence polynucléotidique codant pour le FX en insérant une ou plusieurs séquences de clivage intracellulaire qui permettront d'obtenir directement du FXa. La protéine recombinante FXa possède également la capacité d'activer la prothrombine. By "recombinant protein" is meant a protein prepared, expressed, isolated, purified by genetic engineering. The recombinant DNA technology makes it possible to insert a polynucleotide sequence coding for either FXa or FX in a host cell and thus to produce the polypeptide of interest, ie the FXa or the FX which can be activated after cell production. It is also possible to modify the polynucleotide sequence coding for the FX by inserting one or more intracellular cleavage sequences which will make it possible to directly obtain FXa. The recombinant FXa protein also has the ability to activate prothrombin.
Selon un mode de réalisation particulier, le facteur Xa pour son utilisation selon l'invention possède des propriétés améliorées par rapport au facteur Xa natif isolé du plasma. Cela signifie que le gène et/ou la protéine du facteur Xa est modifïé(e) pour améliorer une ou plusieurs de ses propriétés biologiques par rapport au facteur Xa natif isolé du plasma. Notamment, le facteur Xa peut présenter une affinité améliorée vis-à- vis des inhibiteurs thérapeutiques du FXa, et/ou une capacité améliorée de liaison à son cofacteur, le Facteur V activé de la coagulation, et/ou des propriétés améliorées d'activation de la prothrombine, par rapport au facteur Xa natif isolé du plasma. Selon un mode de réalisation particulier, le facteur Xa pour son utilisation selon l'invention a une demi-vie circulante augmentée par rapport au facteur Xa natif isolé du plasma. Cela signifie que le gène et/ou la protéine du facteur Xa est modifïé(e) pour augmenter sa demi-vie circulante. Par « demi-vie », on désigne la période de temps pendant laquelle une protéine est capable d'exercer sa fonction biologique. Plus la demi-vie est courte et plus la protéine est labile. Inversement, plus la demi-vie d'une protéine est longue et plus la protéine est stable. Accroître la demi-vie d'une protéine équivaut à accroître sa stabilité dans la circulation sanguine.
En effet, des données récentes suggèrent que le FXa utilisé comme agent procoagulant chez la souris hémophile ne possède pas une demi-vie suffisante pour permettre une restauration durable de la capacité de coagulation (Invanciu et al., 2011, Nature Biotechnology, 29(1 1), 1028-1035). Utilisé in vitro dans du plasma sain, le FXa a la capacité de normaliser la défaillance de génération de thrombine induite par le rivaroxaban. Toutefois, cette capacité pourrait être altérée in vivo. According to a particular embodiment, the factor Xa for its use according to the invention has improved properties compared to the native factor Xa isolated from the plasma. This means that the factor Xa gene and / or protein is modified to enhance one or more of its biological properties relative to the native factor Xa isolated from the plasma. In particular, factor Xa may exhibit improved affinity for therapeutic FXa inhibitors, and / or improved ability to bind to its cofactor, activated Factor V coagulation, and / or enhanced activation properties. of prothrombin, relative to native factor Xa isolated from plasma. According to a particular embodiment, the factor Xa for its use according to the invention has an increased circulating half-life compared to the native factor Xa isolated from the plasma. This means that the gene and / or factor Xa protein is modified to increase its circulating half-life. By "half-life" is meant the period of time during which a protein is capable of exerting its biological function. The shorter the half-life, the more labile the protein is. Conversely, the longer the half-life of a protein, the more stable the protein. Increasing the half-life of a protein is equivalent to increasing its stability in the bloodstream. Indeed, recent data suggest that the FXa used as a procoagulant in the haemophilic mouse does not have a sufficient half-life to allow a lasting restoration of the coagulation capacity (Invanciu et al., 2011, Nature Biotechnology, 29 (1 1), 1028-1035). Used in vitro in healthy plasma, FXa has the ability to normalize the rivaroxaban-induced thrombin generation failure. However, this ability could be impaired in vivo.
L'activité du FXa pourrait être soutenue par différentes techniques de modification chimique telle que, par exemple, et sans limitation de méthode, la pegylation, ou bien par la génération de molécule mutée moins sensibles à l'action des inhibiteurs plasmatiques telle que l'antithrombine par exemple, ou par l'utilisation de molécule chimérique qui permettraient d'augmenter la demi-vie, telle que, par exemple et sans se limiter à leur seule utilisation, la fusion du facteur Xa à l'albumine ou au fragment cristallisable des anticorps. La présente invention concerne également un procédé de suivi in vitro ou ex- vivo de la survenue d'événements hémorragiques induits par la prise d'inhibiteurs spécifiques du facteur Xa comprenant les étapes suivantes : The activity of FXa could be supported by various chemical modification techniques such as, for example, and without limitation of method, pegylation, or by the generation of mutated molecules less sensitive to the action of plasma inhibitors such as antithrombin for example, or by the use of a chimeric molecule which would increase the half-life, such as, for example, and without being limited to their sole use, the fusion of factor Xa to the albumin or crystallizable fragment of antibody. The present invention also relates to a method of monitoring in vitro or ex vivo the occurrence of hemorrhagic events induced by the intake of factor Xa specific inhibitors comprising the following steps:
a) mesure analytique des marqueurs de la coagulation sanguine dans un échantillon biologique issu d'un patient recevant un inhibiteur spécifique du facteur Xa pour obtenir des valeurs numériques attribuables à chacun des susdits marqueurs, a) analytical measurement of the markers of blood coagulation in a biological sample from a patient receiving a specific factor Xa inhibitor to obtain numerical values attributable to each of the above markers,
b) détermination du risque hémorragique à partir des valeurs numériques établies à l'étape a), b) determining the haemorrhagic risk from the numerical values established in step a),
c) détermination d'une quantité adéquate de facteur Xa à administrer audit patient recevant un inhibiteur spécifique du facteur Xa. c) determining an adequate amount of factor Xa to be administered to said patient receiving a specific factor Xa inhibitor.
Par « mesure analytique », on désigne la détermination de la concentration ou du taux de chacun des marqueurs de la coagulation. La détermination de ces concentrations est réalisée in vitro ou ex-vivo à l'aide de kits de diagnostic. Les valeurs numériques obtenues sont propres à chacun des patients recevant un inhibiteur spécifique du facteur Xa. By "analytical measurement" is meant the determination of the concentration or the level of each of the markers of coagulation. The determination of these concentrations is carried out in vitro or ex vivo using diagnostic kits. The numerical values obtained are specific to each patient receiving a specific factor Xa inhibitor.
Par « marqueurs de la coagulation », on désigne des valeurs de temps de coagulation par mesure de l'aPTT, du PT ou de l'établissement de l'INR.
Par « échantillon biologique », on désigne une fraction liquide choisie parmi le groupe constitué par le sang, le sérum ou le plasma. By coagulation markers, coagulation time values are defined by measurement of aPTT, PT, or INR establishment. By "biological sample" is meant a liquid fraction selected from the group consisting of blood, serum or plasma.
Par « risque hémorragique », on désigne la présomption de saignement chez un patient ayant reçu au moins un inhibiteur du FXa. L'évaluation du risque hémorragique peut être effectuée à partir des seuls marqueurs de la coagulation ou pris en considération avec des analyses d'imagerie médicale permettant la visualisation des zones hémorragiques. Une génération de thrombine inférieure à 50% de la normale génère un risque hémorragique (Van Veen et al. British J Haematol, 2008, 142, 889- 903). By "haemorrhagic risk" is meant the presumption of bleeding in a patient who has received at least one FXa inhibitor. The evaluation of the risk of haemorrhage can be carried out solely from the markers of coagulation or taken into consideration with medical imaging analyzes allowing the visualization of the haemorrhagic zones. Generation of thrombin less than 50% of normal generates a risk of bleeding (Van Veen et al., British J Haematol, 2008, 142, 889-903).
La détermination du risque hémorragique s'effectue notamment par comparaison des valeurs numériques déterminées pour chacun des marqueurs chez un patient recevant un inhibiteur spécifique du FXa à des valeurs numériques de références. Ces valeurs numériques de référence peuvent correspondre soit à la moyenne des valeurs numériques obtenues chez des sujets sains pour chacun des marqueurs, soit être fixées ou calculées par rapport à des standards internationaux. The determination of the haemorrhagic risk is carried out in particular by comparing the numerical values determined for each of the markers in a patient receiving a specific inhibitor of FXa at reference numerical values. These reference numerical values can correspond either to the average of the numerical values obtained in healthy subjects for each of the markers, or to be fixed or calculated in relation to international standards.
Par « quantité adéquate », on désigne la quantité de FXa efficace pour atteindre le résultat thérapeutique visé. Dans le cadre de la présente invention, le but thérapeutique visé consiste en la réduction partielle ou totale de l'hémorragie. L'évaluation du résultat thérapeutique peut reposer sur le tableau clinique du patient ou sur toute autre indice de nature biologique. Dans le cadre de la présente invention, les indices biologiques sont notamment la neutralisation de l'inhibiteur du FXa administré au patient, le renversement de l'activité anticoagulante de l'inhibiteur du FXa, le retrait de l'inhibiteur du FXa du plasma du patient, la restauration de l'hémostase et la réduction ou la cessation du saignement. La quantité efficace de FXa peut varier en fonction du type et de la dose d'inhibiteur du FXa administré au patient, de la morphologie et de l'âge du patient ainsi que du mode d'administration du FXa. La gestion de ces différents paramètres est bien connue de l'homme du métier. By "adequate amount" is meant the amount of FXa effective to achieve the desired therapeutic outcome. In the context of the present invention, the intended therapeutic purpose consists in the partial or total reduction of the haemorrhage. The evaluation of the therapeutic result may be based on the clinical picture of the patient or on any other biological index. In the context of the present invention, the biological indices are in particular the neutralization of the FXa inhibitor administered to the patient, the reversal of the anticoagulant activity of the FXa inhibitor, the removal of the FXa inhibitor from the plasma of the patient, restoration of hemostasis and reduction or cessation of bleeding. The effective amount of FXa may vary depending on the type and dose of FXa inhibitor administered to the patient, the morphology and age of the patient, and the mode of administration of the FXa. The management of these various parameters is well known to those skilled in the art.
La présente invention concerne également une composition pharmaceutique comprenant le facteur Xa et un inhibiteur du facteur Xa comme produit de combinaison pour une utilisation séparée ou étalée dans le temps dans la prévention ou le traitement chez un patient, notamment un être humain ou un animal, des événements
thromboemboliques veineux, par exemple consécutifs à une intervention orthopédique de la hanche et/ou du genou. The present invention also relates to a pharmaceutical composition comprising factor Xa and a factor Xa inhibitor as a combination product for separate or time-based use in prevention or treatment in a patient, including a human or an animal, events Venous thromboembolism, for example following an orthopedic intervention of the hip and / or knee.
Par « composition pharmaceutique », on désigne la combinaison d'un composé actif, notamment le FXa, avec au moins un composé pharmaceutiquement acceptable rendant la composition apte à être administrée à un patient. By "pharmaceutical composition" is meant the combination of an active compound, in particular FXa, with at least one pharmaceutically acceptable compound making the composition suitable for administration to a patient.
Par « produit de combinaison », on désigne un produit contenant au moins deux composés A et B qui peuvent être administrés conjointement en une prise, ou le produit A est administré puis le produit B est administré immédiatement après, ou le produit A est administré puis le produit B est administré plusieurs minutes, heures, jours plus tard. Dans le cadre de la présente invention, le produit de combinaison comprend l'inhibiteur spécifique du FXa (produit A) et le FXa (produit B). By "combination product" is meant a product containing at least two compounds A and B which may be administered together in one dose, or the product A is administered and then the product B is administered immediately thereafter, or the product A is administered and product B is administered several minutes, hours, days later. In the context of the present invention, the combination product comprises the specific FXa inhibitor (product A) and FXa (product B).
Par « événement thromboembolique veineux », on désigne la formation d'un caillot susceptible d'obstruer le vaisseau dans lequel il s'est formé ou susceptible de quitter le vaisseau dans lequel il s'est formé pour se retrouver dans la circulation sanguine. La formation du caillot sanguin est également appelée thrombose. La fréquence de survenue d'événements thromboemboliques augmente lorsque le patient subit une immobilisation prolongée. Les interventions orthopédiques des membres inférieurs (hanche, jambe, genou) nécessitent généralement un traitement prophylactique du patient avec des anticoagulants afin d'éviter la survenue de thrombose. By "venous thromboembolic event" is meant the formation of a clot that may clog the vessel in which it has formed or may leave the vessel in which it has formed to end up in the bloodstream. The formation of the blood clot is also called thrombosis. The frequency of occurrence of thromboembolic events increases when the patient undergoes prolonged immobilization. Orthopedic interventions of the lower limbs (hip, leg, knee) usually require prophylactic treatment of the patient with anticoagulants to prevent the occurrence of thrombosis.
Selon un mode de réalisation particulier, dans la composition pharmaceutique selon la présente invention, la concentration de facteur Xa varie de 0,01 ng/ml à 2 μg/ml ou de 10 ng/ml à 2 μg/ml. According to a particular embodiment, in the pharmaceutical composition according to the present invention, the concentration of factor Xa varies from 0.01 ng / ml to 2 μg / ml or from 10 ng / ml to 2 μg / ml.
Un volume déterminé de composition pharmaceutique selon la présente invention permet de définir des doses unitaires de FXa administrables au patient recevant un inhibiteur spécifique du FXa. A determined volume of pharmaceutical composition according to the present invention makes it possible to define unit doses of FXa that can be administered to the patient receiving a specific inhibitor of FXa.
Par « dose unitaire », on désigne la quantité spécifique de FXa administrable au patient en une prise. La dose unitaire est la présentation appropriée d'une quantité déterminée de FXa dans un récipient unidose, destinée à l'administration au patient.
La présente invention concerne également une méthode pour prévenir ou traiter des événements hémorragiques induits par la prise d'anticoagulants, chez un mammifère, notamment l'homme, comprenant une étape d'administration de facteur Xa au dit mammifère, notamment l'homme, en quantité suffisante permettant de restaurer une activité procoagulante basale. By "unit dose" is meant the specific amount of FXa administered to the patient in one dose. The unit dose is the appropriate presentation of a fixed amount of FXa in a single-dose container for administration to the patient. The present invention also relates to a method for preventing or treating hemorrhagic events induced by the administration of anticoagulants, in a mammal, in particular a human, comprising a step of administering factor Xa to said mammal, in particular man, by sufficient amount to restore a basal procoagulant activity.
Il s'agit de permettre au patient ayant reçu l'antidote de retrouver une coagulation normale sans toutefois risquer de provoquer une thrombose. Le but est d'arrêter une hémorragie survenue chez un patient ayant reçu au moins un anticoagulant. This is to allow the patient who received the antidote to resume normal coagulation without risking thrombosis. The goal is to stop haemorrhage occurring in a patient who has received at least one anticoagulant.
Selon un mode de réalisation particulier, dans la méthode selon la présente invention, les anticoagulants sont des inhibiteurs spécifiques du facteur Xa. Selon un autre mode de réalisation plus particulier, dans la méthode selon la présente invention, les inhibiteurs spécifiques du facteur Xa sont choisis parmi l'apixaban, le betrixaban, l'édoxaban, l'otamixaban ou le rivaroxaban. According to a particular embodiment, in the method according to the present invention, anticoagulants are specific inhibitors of factor Xa. According to another more particular embodiment, in the method according to the present invention, the specific inhibitors of factor Xa are chosen from apixaban, betrixaban, edoxaban, otamixaban or rivaroxaban.
Selon un autre mode de réalisation encore plus particulier, dans la méthode selon la présente invention, le facteur Xa est administré au mammifère, notamment l'homme, pendant ou après la prise d'inhibiteurs spécifiques du facteur Xa. According to another even more particular embodiment, in the method according to the present invention, the factor Xa is administered to the mammal, in particular the man, during or after taking factor Xa specific inhibitors.
Les figures et les exemples suivants servent à illustrer la présente invention mais ne doivent en aucun cas servir à en restreindre la portée. The following figures and examples serve to illustrate the present invention but should in no way serve to restrict its scope.
Légende des figures Legend of figures
Figure 1-A : Thrombinogramme obtenu à partir de l'ajout de 10 μg/mL de FX dans un pool de plasma normal suite à l'activation par du facteur tissulaire: Figure 1-A: Thrombinogram obtained from the addition of 10 μg / ml of FX in a pool of normal plasma following activation by tissue factor:
en abscisses : le temps (en minutes) on the abscissa: the time (in minutes)
- en ordonnées : la concentration maximale de thrombine observée (en nM) - ordinate: the maximum concentration of thrombin observed (in nM)
L'échantillon de pool de plasma normal est représenté par la courbe noire (PT=11,9 secondes).
Le facteur X a été ajouté à raison de 10 μ§/ητΙ, (courbe grise pointillée) dans l'échantillon de pool de plasma normal (PT=11,1 secondes). The normal plasma pool sample is represented by the black curve (PT = 11.9 seconds). Factor X was added at 10μ§ / ητΙ, (dashed gray curve) in the normal plasma pool sample (PT = 11.1 seconds).
Figure 1-B : Thrombinogramme obtenu à partir de l'ajout de 10 μg/mL de FX dans un pool de plasma surchargé à 0,5 μg/mL de rivaroxaban suite à l'activation par du facteur tissulaire: Figure 1-B: Thrombinogram obtained from the addition of 10 μg / mL of FX in a plasma pool overloaded with 0.5 μg / mL rivaroxaban following activation by tissue factor:
en abscisses : le temps (en minutes) on the abscissa: the time (in minutes)
en ordonnées : la concentration maximale de thrombine observée (en nM) on the ordinate: the maximum concentration of thrombin observed (in nM)
L'échantillon de pool de plasma surchargé à 0,5 μg/mL de rivaroxaban est représenté par la courbe grise (PT=32,5 secondes). The plasma pool sample overloaded with 0.5 μg / mL rivaroxaban is represented by the gray curve (PT = 32.5 seconds).
Le facteur X a été ajouté à raison de 10 μg/mL (courbe noire) dans l'échantillon surchargé à 0,5 μg/mL de rivaroxaban (PT=29,3 secondes). Figure 1-C : Thrombinogramme obtenu à partir de l'ajout de 25 μg/mL de FX dans un pool de plasma normal surchargé à 0,5 μg/mL de rivaroxaban suite à l'activation par du facteur tissulaire: Factor X was added at a rate of 10 μg / mL (black curve) in the sample overloaded at 0.5 μg / mL rivaroxaban (PT = 29.3 seconds). Figure 1-C: Thrombinogram obtained from the addition of 25 μg / mL of FX in a pool of normal plasma overloaded with 0.5 μg / mL rivaroxaban following activation by tissue factor:
en abscisses : le temps (en minutes) on the abscissa: the time (in minutes)
en ordonnées : la concentration maximale de thrombine observée (en nM) on the ordinate: the maximum concentration of thrombin observed (in nM)
L'échantillon de pool de plasma normal est représenté par la courbe noire (1). The normal plasma pool sample is represented by the black curve (1).
L'échantillon de pool de plasma normal et surchargé à 0,5 μg/mL de rivaroxaban est représenté par la courbe (2). The normal plasma pool sample overloaded with 0.5 μg / mL rivaroxaban is represented by curve (2).
Le facteur X a été ajouté à raison de 25 μg/mL dans l'échantillon de pool de plasma normal (courbe (3)) et dans l'échantillon de pool de plasma normal et surchargé à 0,5 μg/mL de rivaroxaban (courbe (4)). Factor X was added at 25 μg / mL in the normal plasma pool sample (curve (3)) and in the normal plasma pool sample and overloaded at 0.5 μg / mL rivaroxaban ( curve (4)).
Figure 1-D: Thrombinogramme obtenu à partir de l'ajout de 100 μg/mL de FX dans un pool de plasma normal surchargé à 0,5 μg/mL de rivaroxaban suite à l'activation par du facteur tissulaire: Figure 1-D: Thrombinogram obtained from the addition of 100 μg / mL of FX in a pool of normal plasma overloaded with 0.5 μg / mL of rivaroxaban following activation by tissue factor:
en abscisses : le temps (en minutes) on the abscissa: the time (in minutes)
en ordonnées : la concentration maximale de thrombine observée (en nM)
L'échantillon de pool de plasma normal est représenté par la courbe noire épaisse (1). L'échantillon de pool de plasma normal et surchargé à 0,5 μg/mL de rivaroxaban est représenté par la courbe noire fine (2). on the ordinate: the maximum concentration of thrombin observed (in nM) The normal plasma pool sample is represented by the thick black curve (1). The normal plasma pool sample overloaded with 0.5 μg / mL rivaroxaban is represented by the fine black curve (2).
Le facteur X a été ajouté à raison de 100 μg/mL dans l'échantillon de pool de plasma normal (courbe grise (3)) et dans l'échantillon de pool de plasma normal et surchargé à 0,5 μg/mL de rivaroxaban (courbe grise claire (4)). Factor X was added at 100 μg / mL in the normal plasma pool sample (gray curve (3)) and in the normal plasma pool sample and overloaded at 0.5 μg / mL rivaroxaban (light gray curve (4)).
Figure 2 : Thrombinogramme obtenu à partir de l'ajout de 10 μg/mL de FX dans un pool de plasma normal et surchargé à 0,35 μg/mL de rivaroxaban suite à une induction par la céphaline: Figure 2: Thrombinogram obtained from the addition of 10 μg / mL of FX in a pool of normal plasma and overloaded with 0.35 μg / mL of rivaroxaban following an induction by the cephalin:
en abscisses : le temps (en minutes) on the abscissa: the time (in minutes)
en ordonnées : la concentration maximale de thrombine observée (en nM) on the ordinate: the maximum concentration of thrombin observed (in nM)
L'échantillon de pool de plasma normal est représenté par la courbe noire (1). The normal plasma pool sample is represented by the black curve (1).
L'échantillon de pool de plasma normal et surchargé à 0,35 μg/mL de rivaroxaban est représenté par la courbe (3). The normal plasma pool sample overloaded with 0.35 μg / mL rivaroxaban is represented by curve (3).
Le facteur X a été ajouté à raison de 10 μg/mL dans l'échantillon de pool de plasma normal et (courbe (2)) dans l'échantillon de pool de plasma normal et surchargé à 0,35 μg/mL de rivaroxaban (courbe (4)). Factor X was added at 10 μg / mL in the normal plasma pool sample and (curve (2)) in the normal plasma pool sample and overloaded at 0.35 μg / mL rivaroxaban ( curve (4)).
Figure 3-A : Thrombinogramme obtenu à partir de l'ajout de doses croissantes de FXa dans un pool de plasma normal : Figure 3-A: Thrombinogram obtained from the addition of increasing doses of FXa in a normal plasma pool:
en abscisses : le temps (en minutes) on the abscissa: the time (in minutes)
en ordonnées : la concentration maximale de thrombine observée (en nM) on the ordinate: the maximum concentration of thrombin observed (in nM)
L'échantillon de pool de plasma normal est représenté par la courbe noire (1). The normal plasma pool sample is represented by the black curve (1).
Le facteur Xa a été ajouté à raison de 0,5 μg/mL (courbe (2)), 1,0 μg/mL (courbe (3)), 1,5 μg/mL (courbe (4)), 2,0 μg/mL (courbe (5)), 2,5 μg/mL (courbe (6)), 5,0 μg/mL (courbe (7)) dans l'échantillon de pool de plasma normal. Factor Xa was added at 0.5 μg / mL (curve (2)), 1.0 μg / mL (curve (3)), 1.5 μg / mL (curve (4)), 2, 0 μg / mL (curve (5)), 2.5 μg / mL (curve (6)), 5.0 μg / mL (curve (7)) in the normal plasma pool sample.
Figure 3-B : Thrombinogramme obtenu à partir de l'ajout de doses croissantes de FXa dans un pool de plasma normal surchargé à 0,35 μg/mL de rivaroxaban : Figure 3-B: Thrombinogram obtained from the addition of increasing doses of FXa in a pool of normal plasma overloaded with 0.35 μg / mL rivaroxaban:
en abscisses : le temps (en minutes)
en ordonnées : la concentration maximale de thrombine observée (en nM) on the abscissa: the time (in minutes) on the ordinate: the maximum concentration of thrombin observed (in nM)
L'échantillon de pool de plasma normal est représenté par la courbe noire (1). The normal plasma pool sample is represented by the black curve (1).
0,35 μ§/ι ί de rivaroxaban ont été ajouté à l'échantillon de pool de plasma normal (courbe (2)). 0.35 μ / sec of rivaroxaban was added to the normal plasma pool sample (curve (2)).
Le facteur Xa a été ajouté à raison de 0,5 μg/mL (courbe (3)), 1,0 μg/mL (courbe (4)), 1,5 μ§/ι ί (courbe (5)), 2,0 μ§/ι ί (courbe (6)), 2,5 μg/mL (courbe (7)), 5,0 μg/mL (courbe (8)) dans l'échantillon de pool de plasma normal surchargé à 0,35 μ§/ηιί de rivaroxaban. Factor Xa was added at 0.5 μg / mL (curve (3)), 1.0 μg / mL (curve (4)), 1.5 μ§ / ι ί (curve (5)), 2.0 μ§ / ι ί (curve (6)), 2.5 μg / mL (curve (7)), 5.0 μg / mL (curve (8)) in the overloaded normal plasma pool sample at 0.35 μs / ηιί of rivaroxaban.
Figure 4 : Thrombinogrammes obtenus à partir de l'ajout de 0,01 à 1 μ§/ι Ι. de FX activé dans un pool de plasma normal sans activation: Figure 4: Thrombograms obtained from the addition of 0.01 to 1 μ§ / ι Ι. activated FX in a normal plasma pool without activation:
en abscisses : le temps (en minutes) on the abscissa: the time (in minutes)
en ordonnées : la concentration maximale de thrombine observée (en nM) on the ordinate: the maximum concentration of thrombin observed (in nM)
L'échantillon de pool de plasma normal est représenté par la courbe ·. L'effet des concentrations en FXa ajouté en plasma normal est représenté par les courbes suivantes : 0,01 μ§/ι 1 (o) ; 0,05 μ§/ι 1 (■) ; 0,1 μ§/ι 1 (□) ; 0,5 μ§/ι 1 (A) et 1 μ§/ι 1 (Δ). Les témoins illustrant la génération de thrombine suite à l'induction de la coagulation soit par le facteur tissulaire/phospho lipides (x) soit par la céphaline (0) sont indiqués. The normal plasma pool sample is represented by the · curve. The effect of the concentrations of FXa added to normal plasma is represented by the following curves: 0.01 μ§ / ι 1 (o); 0.05 μ§ / ι 1 (■); 0.1 μ§ / ι 1 (□); 0.5 μ§ / ι 1 (A) and 1 μ§ / ι 1 (Δ). Controls illustrating thrombin generation following induction of coagulation either by tissue factor / phospholipid (x) or by partial thromboplastin (0) are indicated.
Figure 5 : Thrombinogrammes obtenus à partir de l'ajout de 0,03 à 0,5 μg/mL de FX activé dans un pool de plasma normal suite à l'activation par du facteur tissulaire: Figure 5: Thrombinograms obtained from the addition of 0.03 to 0.5 μg / mL of activated FX in a pool of normal plasma following activation by tissue factor:
- en abscisses : le temps (en minutes) - on the abscissa: the time (in minutes)
en ordonnées : la concentration maximale de thrombine observée (en nM) on the ordinate: the maximum concentration of thrombin observed (in nM)
L'échantillon de pool de plasma normal est représenté par la courbe ·. L'effet des concentrations en FXa ajouté en plasma normal est représenté par les courbes suivantes : 0,0312 μg/ml (o) ; 0,0625 μg/ml (■) ; 0,125 μg/ml (□) ; 0,25 μg/ml (À) et 0,5 μg/ml (Δ).
Figure 6 : Thrombinogrammes obtenus à partir de l'ajout de 0,03 à 0,5 μ§/ηιΙ, de FX activé dans un pool de plasma normal en présence de Rivaroxaban (0,35 μ^πιΓ^υΐίε à l'activation par du facteur tissulaire: The normal plasma pool sample is represented by the · curve. The effect of the concentrations of FXa added to normal plasma is represented by the following curves: 0.0312 μg / ml (o); 0.0625 μg / ml (■); 0.125 μg / ml (□); 0.25 μg / ml (A) and 0.5 μg / ml (Δ). FIG. 6: Thrombograms obtained from the addition of 0.03 to 0.5 μ§ / ηιΙ, activated FX in a normal plasma pool in the presence of Rivaroxaban (0.35 μ ^ πιΓ ^ υΐίε at activation by tissue factor:
en abscisses : le temps (en minutes) on the abscissa: the time (in minutes)
- en ordonnées : la concentration maximale de thrombine observée (en nM) - ordinate: the maximum concentration of thrombin observed (in nM)
L'échantillon de pool de plasma normal sans Rivaroxaban (0,35 μ§/ι 1) est représenté par la courbe ·. Le même plasma activé dans les même conditions mais en présence de Rivaroxaban (0,35 μ§/ι 1) est représenté par la courbe x. L'effet des concentrations en FXa ajouté en plasma normal en présence de Rivaroxaban (0,35 μ§/ι 1) est représenté par les courbes suivantes : 0,0312 μ§/ι 1 (o) ; 0,0625 μ§/ι 1 (■) ; 0,125 μ§/ι 1 (□) ; 0,25 μ^πιΐ (A) et 0,5 μ^πιΐ (Δ). The normal plasma pool sample without Rivaroxaban (0.35 μ§ / ι 1) is represented by the · curve. The same plasma activated under the same conditions but in the presence of Rivaroxaban (0.35 μ§ / ι 1) is represented by the curve x. The effect of the FXa concentrations added to normal plasma in the presence of Rivaroxaban (0.35 μ§ / ι 1) is represented by the following curves: 0.0312 μ§ / ι 1 (o); 0.0625 μ§ / ι 1 (■); 0.125 μ§ / ι 1 (□); 0.25 μ ^ πιΐ (A) and 0.5 μ ^ πιΐ (Δ).
Figure 7 : Thrombinogrammes obtenus à partir de l'ajout de 0,03 à 0,5 μg/mL de FX activé dans un pool de plasma normal suite à l'activation par la céphaline: FIG. 7: Thrombograms obtained from the addition of 0.03 to 0.5 μg / ml of activated FX in a pool of normal plasma following activation by the partial thromboplastin:
en abscisses : le temps (en minutes) on the abscissa: the time (in minutes)
en ordonnées : la concentration maximale de thrombine observée (en nM) on the ordinate: the maximum concentration of thrombin observed (in nM)
L'échantillon de pool de plasma normal est représenté par la courbe ·. L'effet des concentrations en FXa ajouté en plasma normal est représenté par les courbes suivantes : 0,0312 μ^πιΐ (o) ; 0,0625 μ^πιΐ (■) ; 0,125 μ^πιΐ (□) ; 0,25 μ^πιΐ (À) et 0,5 μ πύ. (Δ). The normal plasma pool sample is represented by the · curve. The effect of the concentrations of FXa added to normal plasma is represented by the following curves: 0.0312 μ ^ πιΐ (o); 0.0625 μ ^ πιΐ (■); 0.125 μ ^ πιΐ (□); 0.25 μ ^ πιΐ (A) and 0.5 μ πύ. (Δ).
Figure 8: Thrombinogrammes obtenus à partir de l'ajout de 0,03 à 0,5 μg/mL de FX activé dans un pool de plasma normal suite à l'activation par la céphaline en présence de Rivaroxaban (0,35 μ^ιηΐ): FIG. 8: Thrombograms obtained from the addition of 0.03 to 0.5 μg / ml of activated FX in a pool of normal plasma following activation by the partial thromboplastin in the presence of Rivaroxaban (0.35 μ ^ ιηΐ ):
en abscisses : le temps (en minutes) on the abscissa: the time (in minutes)
en ordonnées : la concentration maximale de thrombine observée (en nM) on the ordinate: the maximum concentration of thrombin observed (in nM)
L'échantillon de pool de plasma normal sans Rivaroxaban (0,35 μ^ιηΐ) est représenté par la courbe ·. Le même plasma activé dans les même conditions mais en présence de Rivaroxaban (0,35 μ^ιηΐ) est représenté par la courbe x. L'effet des concentrations en
FXa ajouté en plasma normal estreprésenté par les courbes suivantes : 0,0312 μg/ml (o) ; 0.0625 μg/ml (■) ; 0,125 μg/ml (□) ; 0,25 μg/ml ( À) et 0,5 μg/ml (Δ). The normal plasma pool sample without Rivaroxaban (0.35 μ ^ ιηΐ) is represented by the · curve. The same plasma activated under the same conditions but in the presence of Rivaroxaban (0.35 μ ^ ιηΐ) is represented by the curve x. The effect of concentrations in FXa added in normal plasma is represented by the following curves: 0.0312 μg / ml (o); 0.0625 μg / ml (■); 0.125 μg / ml (□); 0.25 μg / ml (A) and 0.5 μg / ml (Δ).
Exemples Examples
Exemple 1 : voie extrinsèque de la coagulation (FT lpM/PL 4μΜ) EXAMPLE 1 Extrinsic Coagulation Pathway (FT lpM / PL 4 μΜ)
1 - Protocole 1 - Protocol
1.1 - Réactifs 1.1 - Reagents
Thrombin calibrator (Stago) Thrombin calibrator (Stago)
TC1103 TC1103
PPP reagent 5 pM (Stago) PPP reagent 5 pM (Stago)
PPP1009 PPP1009
PPP reagent low (Stago) PPP reagent low (Stago)
PPL1101 PPL1101
CK-Prest (Stago) CK-Perst (Stago)
106025 106025
Fluo-buffer (Stago) Fluo-buffer (Stago)
FLB1012 FLB1012
Fluo-substrat (Stago) Fluo-substrate (Stago)
FLS1012 FLS1012
Rivaroxaban à 436 μg/mL Rivaroxaban at 436 μg / mL
PNP (Cryopep) A1112 PNP (Cryopep) A1112
FX humain à 6,8 mg/mL (HTI) Human FX at 6.8 mg / mL (HTI)
AA1108 AA1108
FXa bovin à 2,15 mg/mL (Hyphen Biomed) Bovine FXa at 2.15 mg / mL (Hyphen Biomed)
110728C 110728C
1.2 - Protocole expérimental 1.2 - Experimental Protocol
La solution stock de rivaroxaban à 436 μg/mL est diluée en DMSO 5% pour obtenir des concentrations intermédiaires lesquelles, ajoutées au PNP (Pool de Plasma Normal), donnent des concentrations finales de rivaroxaban en μg par mL de plasma. The rivaroxaban stock solution at 436 μg / mL is diluted with 5% DMSO to obtain intermediate concentrations which, added to PNP (Normal Plasma Pool), give final concentrations of rivaroxaban in μg per mL of plasma.
Le FX ou le FXa sont introduits dans le plasma sans étape d'incubation. FX or FXa are introduced into the plasma without an incubation step.
On considère que 1 unité de FX = 10 μg/mL dans le plasma, correspondant à 100% de taux de FX dans le plasma. It is considered that 1 unit of FX = 10 μg / mL in plasma, corresponding to 100% of FX level in the plasma.
Le test de génération de thrombine consiste à activer la coagulation ex vivo soit à l'aide d'un mélange de facteur tissulaire et de phospholipides, soit à l'aide de céphaline et à mesurer ensuite la concentration de thrombine générée au cours du temps.
Les tests de génération de thrombine sont réalisés sur 80 μΐ d'un pool de plasma contenant éventuellement le FX et/ou le Rivaroxaban en présence soit de 20 μΐ de PPP reagent (Stago) contenant au final 5 pM de Facteur Tissulaire (FT) et 4 μΜ de phospho lipides (PL), soit en présence de 20 μΐ, de céphaline (CK-Prest reconstituée avec 5 mL d'H20 distillée). The thrombin generation test involves activating coagulation ex vivo either with a mixture of tissue factor and phospholipids, or with the aid of cephalin and then measuring the thrombin concentration generated over time. The thrombin generation tests are carried out on 80 μl of a plasma pool optionally containing FX and / or Rivaroxaban in the presence of either 20 μl of PPP reagent (Stago) containing finally 5 μM of Tissue Factor (FT) and 4 μl of phospholipids (PL), ie in the presence of 20 μl, cephalin (CK-Perst reconstituted with 5 ml of distilled H 2 O).
La réaction est démarrée par l'ajout de 20 μΐ, de Fluca-kit (substrat + CaCl2) qui constitue le début de la mesure de l'apparition de thrombine. L'apparition de fluorescence est mesurée sur un fluorimètre de type Fluoroskan Ascent (ThermoLabsystems) à une longueur d'onde d'excitation de 390 nm et à une longueur d'émission de 460 nm. Les thrombinogrammes (courbes représentant l'intensité de fluorescence en fonction du temps) sont ensuite analysés grâce au logiciel Thrombinoscope™ qui transforme la valeur de fluorescence en nM de thrombine par calcul comparatif. The reaction is started by the addition of 20 μl of Fluca-kit (substrate + CaCl 2 ), which is the beginning of the measurement of the appearance of thrombin. The appearance of fluorescence is measured on a Fluoroskan Ascent fluorimeter (ThermoLabsystems) at an excitation wavelength of 390 nm and at an emission length of 460 nm. Thrombinograms (curves representing the fluorescence intensity as a function of time) are then analyzed using the Thrombinoscope ™ software which converts the fluorescence value into nM thrombin by comparative calculation.
2 - Résultats 2 - Results
2.1 - Effets du FX (10 μ /ml) sur un plasma surchargé en 2.1 - Effects of FX (10 μ / ml) on a plasma overloaded with
Rivaroxaban rivaroxaban
L'ensemble des résultats figurent dans le tableau 1 ci-dessous et sont illustrés par les thrombinogrammes des figures 1-A et 1-B : The overall results are shown in Table 1 below and are illustrated by the thrombograms of FIGS. 1-A and 1-B:
Tableau 1 : Effet du facteur X sur un plasma normal ou supplémenté en 0,5 μg/mL de Rivaroxaban. LT=lag time, ETP=endogenous thrombin potential, TTP=time to peak, ST=start time, mVRI= mean velocity rate index, PT=prothrombine time. Table 1: Effect of factor X on normal or supplemented plasma in 0.5 μg / mL of Rivaroxaban. LT = lag time, ETP = endogenous thrombin potential, TTP = time to peak, ST = start time, mVRI = mean velocity rate index, PT = prothrombin time.
L'ajout de 10 μg/mL de FX dans un plasma normal accroît légèrement (+13%) la quantité totale de thrombine générée (ETP), la hauteur du pic (+7,3%) et diminue la durée comprise entre le déclenchement de la réaction et le point de concentration maximale de la thrombine (TTP) par rapport à des valeurs observables pour un plasma normal (Figure 1-A). The addition of 10 μg / mL of FX in a normal plasma slightly increases (+ 13%) the total amount of thrombin generated (ETP), the height of the peak (+ 7.3%) and decreases the duration between the triggering of the reaction and the point of maximum concentration of thrombin (TTP) compared to observable values for a normal plasma (Figure 1-A).
L'ajout de 0,5 μg/mL de Rivaroxaban annihile intégralement la capacité de générer de la thrombine à partir du plasma normal, suite à une activation par du facteur tissulaire et des phospho lipides. La supplémentation par 10 μg/mL de FX d'un plasma normal surchargé en Rivaroxaban à 0,5 μg/mL ne permet pas de restaurer une génération de thrombine différente de celle obtenue lors d'une surcharge en Rivaroxaban (Figure 1-B). The addition of 0.5 μg / mL Rivaroxaban completely nullifies the ability to generate thrombin from normal plasma following activation by tissue factor and phospholipids. Supplementation with 10 μg / mL FX of normal plasma overloaded with Rivaroxaban at 0.5 μg / mL does not restore thrombin generation different from that obtained with Rivaroxaban overload (Figure 1-B) .
En conclusion, Le FX humain commercial (HTI) à 10 μg/mL final ne corrige pas un plasma surchargé en rivaroxaban à 0,5 μg/mL en voie extrinsèque à 1 pM de FT. In conclusion, the commercial human FX (HTI) at 10 μg / mL final does not correct rivaroxaban overloaded plasma at 0.5 μg / mL extrinsically at 1 μM FT.
2.2- Effets du FX (25 μ^ιηΐ ou 100 μ /ml) sur un plasma surchargé en Rivaroxaban (0,5 μ /ml) 2.2- FX effects (25 μ ^ ιηΐ or 100 μ / ml) on a plasma overloaded with Rivaroxaban (0.5 μ / ml)
L'ensemble des résultats figurent dans le tableau 2 ci-dessous et sont illustrés par les thrombinogrammes des figures 1-C et 1-D: The overall results are shown in Table 2 below and are illustrated by the thrombograms of Figures 1-C and 1-D:
Tableau 2 : Effet-dose du facteur X sur un plasma normal contenant 0,5 μ§/ι Ε de Rivaroxaban. LT=lag time, ETP=endogenous thrombin potential, TTP=time to peak, ST=start time, mVRI= mean velocity rate index. L'ajout de FX (25 μ§/ι 1 ou 100 μ§/ι 1) dans un plasma normal augmente la quantité totale de thrombine générée (+13 et +16% respectivement), la hauteur du pic (+9,5%o et 37%o respectivement) et réduit la durée comprise entre le déclenchement de la réaction et l'apparition de thrombine mais est incapable de renverser les effets du Rivaroxaban. Table 2: Factor X Factor Effect on Normal Plasma Containing 0.5 μl / μl of Rivaroxaban. LT = lag time, ETP = endogenous thrombin potential, TTP = time to peak, ST = start time, mVRI = mean velocity rate index. The addition of FX (25 μ§ / ι 1 or 100 μ§ / ι 1) in a normal plasma increases the total amount of thrombin generated (+13 and +16% respectively), the height of the peak (+9.5 % o and 37% o respectively) and reduces the time between the onset of the reaction and the occurrence of thrombin but is unable to reverse the effects of Rivaroxaban.
En conclusion, le FX humain commercial (HTI) augmente de façon dose- dépendante la génération de thrombine d'un pool de plasma normal en voie extrinsèque à 1 pM de FT final. Par contre il ne corrige pas la génération de thrombine d'un plasma surchargé en Rivaroxaban à 0,5 μg/mL jusqu'à des concentrations de 100 μg/ml soit 10 U/ml. In conclusion, commercial human FX (HTI) increases dose-dependently the generation of thrombin from a pool of normal extrinsic plasma to 1 μM of final FT. On the other hand, it does not correct the thrombin generation of a plasma overloaded with Rivaroxaban at 0.5 μg / mL up to concentrations of 100 μg / ml or 10 U / ml.
Exemple 2 : Voie intrinsèque de la coagulation (céphaline seule) Example 2 Intrinsic Route of Coagulation (Partial Cephalin)
1 - Protocole 1 - Protocol
Les réactifs, l'automate et le protocole expérimental sont identiques à ceux décrits dans l'exemple 1. The reagents, the automaton and the experimental protocol are identical to those described in Example 1.
Les tests de génération de thrombine sont réalisés sur 80 μΐ d'un pool de plasma normal contenant éventuellement le FX et/ou le Rivaroxaban en présence de 20 μΐ de céphaline (CK-Prest reconstitué avec 5 mL d'H20 distillée) et de 20μΙ, fluca-kit (substrat + CaCl2). The thrombin generation tests are carried out on 80 μl of a normal plasma pool optionally containing FX and / or Rivaroxaban in the presence of 20 μl of cephalin (CK-Perst reconstituted with 5 ml of distilled H 2 0) and 20μΙ, fluca-kit (substrate + CaCl 2 ).
2 - Résultats 2 - Results
2.1 - Effet du FX sur un plasma normal surchargé en Rivaroxaban 2.1 - Effect of FX on normal plasma overloaded with Rivaroxaban
L'ensemble des résultats figurent dans le tableau 3 ci-dessous et sont illustrés les thrombinogrammes de la figure 2 :
The overall results are shown in Table 3 below and are illustrated by the thrombograms of Figure 2:
Tableau 3 : Effet du facteur X sur un plasma normal contenant 0,35 μ§/ι Ε de Rivaroxaban. LT=lag time, ETP=endogenous thrombin potential, TTP=time to peak, ST=start time, mVRI= mean velocity rate index. Table 3: Effect of Factor X on a Normal Plasma Containing 0.35 μs / ml of Rivaroxaban. LT = lag time, ETP = endogenous thrombin potential, TTP = time to peak, ST = start time, mVRI = mean velocity rate index.
L'ajout de 10 μg/mL de FX dans un plasma normal augmente la quantité totale de thrombine générée de 19%. Par ailleurs, l'ajout de 10 μg/mL de FX dans un plasma normal réduit la durée comprise entre le déclenchement de la réaction et le début de la formation du caillot (LT) et la durée comprise entre le déclenchement de la réaction et le point de concentration maximale de la thrombine (TTP). The addition of 10 μg / mL of FX in normal plasma increases the total amount of thrombin generated by 19%. In addition, the addition of 10 μg / mL of FX in normal plasma reduces the time between the initiation of the reaction and the onset of clot formation (LT) and the time between the initiation of the reaction and the onset of clot formation. peak concentration point of thrombin (TTP).
L'ajout de 0,35 μg/mL de Rivaroxaban annihile intégralement la capacité de générer de la thrombine du plasma normal suite à une activation par de la Céphaline. La supplémentation par 10 μg/mL de FX d'un plasma normal surchargé en Rivaroxaban à 0, 35 μg/mL ne permet pas de restaurer la génération de thrombine. The addition of 0.35 μg / mL Rivaroxaban completely nullifies the ability to generate thrombin from normal plasma following activation with Cephalin. Supplementation with 10 μg / mL FX of a normal plasma overloaded with Rivaroxaban at 0.35 μg / mL does not restore thrombin generation.
En conclusion, le FX humain commercial (HTI) à 10 μg/mL final ne corrige pas un plasma surchargé en Rivaroxaban à 0,35 μg/mL en voie intrinsèque. In conclusion, commercial human FX (HTI) at 10 μg / mL final does not correct a plasma overloaded with Rivaroxaban at 0.35 μg / mL intrinsically.
En voie intrinsèque, comme en voie extrinsèque, le FX (HTI) ne corrige pas un plasma surchargé en Rivaroxaban (à 0,35 μg/ml). Intrinsically, as well as extrinsically, FX (HTI) does not correct a plasma overloaded with Rivaroxaban (at 0.35 μg / ml).
Exemple 3 : Génération de thrombine par activation de la voie extrinsèque de la coagulation (FT 5pM/PL 4μΜ) Example 3 Generation of Thrombin by Activation of the Extrinsic Coagulation Pathway (FT 5pM / PL 4μΜ)
1 - Protocole
Les réactifs, l'automate et le protocole expérimental sont identiques à ceux décrits dans l'exemple 1. 1 - Protocol The reagents, the automaton and the experimental protocol are identical to those described in Example 1.
Les tests de génération de thrombine sont réalisés sur 80 μΐ d'un pool de plasma normal contenant du FXa et/ou du Rivaroxababn en présence de 20 μΐ de PPP reagent low contenant au final 1 pM de Facteur Tissulaire (FT) et 4 μΜ de phospho lipides (PL), et de 20μΙ, fiuca-kit (substrat + CaC12). The thrombin generation tests are carried out on 80 μl of a pool of normal plasma containing FXa and / or Rivaroxababn in the presence of 20 μl of PPP reagent low containing finally 1 μM of Tissue Factor (FT) and 4 μl of phospho lipids (PL), and 20μΙ, fiuca-kit (substrate + CaCl2).
2 - Résultats 2 - Results
L'ensemble des résultats figurent dans le tableau 4 ci-dessous et sont illustrés par les thrombinogrammes des figures 3-A et 3-B : The overall results are shown in Table 4 below and are illustrated by the thrombograms of Figures 3-A and 3-B:
Tableau 4 : effet du facteur Xa sur un plasma normal contenant 0,35 μg/mL de Rivaroxaban. LT=lag time, ETP=endogenous thrombin potential, TTP=time to peak, ST=start time, mVRI= mean velocity rate index, PT=prothrombine time, aPTT=activated partial thromboplastin time, PTT-A=prothromboplastin activated.
L'ajout de FXa dans un plasma normal réduit la durée comprise entre le déclenchement de la réaction par paliers successifs de manière dose dépendante. A l'inverse, l'ajout de 0,35 μg/mL de rivaroxaban dans un plasma normal augmente cette durée d'un facteur compris entre 2 et 3 et abolit complètement la formation de thrombine (Fig 3- A et 3-B). Table 4: Effect of factor Xa on a normal plasma containing 0.35 μg / mL of Rivaroxaban. LT = lag time, ETP = endogenous thrombin potential, TTP = time to peak, ST = start time, mVRI = mean velocity rate index, PT = prothrombin time, aPTT = activated partial thromboplastin time, PTT-A = prothromboplastin activated. The addition of FXa in normal plasma reduces the time between the triggering of the reaction in successive steps in a dose-dependent manner. Conversely, the addition of 0.35 μg / mL of rivaroxaban in normal plasma increases this duration by a factor of between 2 and 3 and completely abolishes thrombin formation (Fig. 3- A and 3-B). .
L'ajout de FXa dans le plasma normal réduit la durée comprise entre le déclenchement de la réaction et le point de concentration maximale de la thrombine (TTP) par paliers successifs de manière dose dépendante. A l'inverse, l'ajout de 0,35 μg/mL de rivaroxaban dans un plasma normal augmente cette durée d'un facteur compris entre 3 et 4. The addition of FXa in normal plasma reduces the time between the initiation of the reaction and the point of maximum concentration of thrombin (TTP) in successive steps in a dose-dependent manner. In contrast, the addition of 0.35 μg / mL rivaroxaban in normal plasma increases this duration by a factor of between 3 and 4.
L'ajout de FXa dans le plasma normal augmente d'environ 14% la quantité totale de thrombine générée (ETP) pour la dose de FXa la plus élevée (5μg/mL). A l'inverse, l'ajout de 0,35 μg/mL de rivaroxaban dans un plasma normal diminue la quantité totale de thrombine générée. The addition of FXa in normal plasma increases the total thrombin generated (FTE) by about 14% for the highest FXa dose (5 μg / mL). In contrast, the addition of 0.35 μg / mL rivaroxaban in normal plasma decreases the total amount of thrombin generated.
L'ajout de quantités croissantes de FXa dans un plasma normal surchargé en The addition of increasing amounts of FXa in a normal plasma overloaded with
Rivaroxaban à 0,35 μg/mL restaure les deux paramètres LT et ETP au niveau observé pour le plasma normal seul. Rivaroxaban 0.35 μg / mL restores both LT and ETP at the level observed for normal plasma alone.
Le FXa de 0,5 à 5 μg/mL final corrige partiellement un plasma surchargé en Rivaroxaban à 0,35 μg/mL de façon dose-dépendante jusqu'à 2,5 μg/mL en voie extrinsèque à 5 pM de FT. Le facteur Xa améliore aussi la génération de thrombine d'un pool de plasma normal de façon dose-dépendante. FXa 0.5 to 5 μg / mL final partially corrects Rivaroxaban overloaded plasma to 0.35 μg / mL dose-dependently up to 2.5 μg / mL extrinsically at 5 μM FT. Factor Xa also enhances the thrombin generation of a normal plasma pool in a dose-dependent manner.
En conclusion, on note que l'ajout de FXa dans un plasma normal semble apporter un effet thrombogénique modéré en permettant la génération de quantités de thrombine plus importantes et de manière plus précoce que celles obtenues à partir d'un plasma non supplémenté. En revanche, l'effet inhibiteur du Rivaroxaban à 0,35μg/ml est abolit en grande partie par l'adjonction de FXa avec un effet plateau à 2^g/ml qui ne permet toutefois pas d'atteindre les valeurs de thrombine générées dans du plasma normal. In conclusion, it is noted that the addition of FXa in a normal plasma seems to provide a moderate thrombogenic effect by allowing the generation of larger amounts of thrombin and earlier than those obtained from an unsupplemented plasma. On the other hand, the inhibitory effect of Rivaroxaban at 0.35 μg / ml is largely abolished by the addition of FXa with a plateau effect at 2 μg / ml, which does not, however, make it possible to reach the thrombin values generated in normal plasma.
Exemple 4 : Evaluation des doses utiles de facteur Xa (FXa) induisant la coagulation ou requises pour contrebalancer l'effet du Rivaroxaban Example 4 Evaluation of Useful Doses of Factor Xa (FXa) Inducing Coagulation or Required to Counterbalance the Effect of Rivaroxaban
1 - Induction de la génération de thrombine par le seul facteur Xa
L'effet d'un surdosage en FXa d'un plasma normal a été évalué en mesurant la génération de thrombine (TGT) sans induction additionnelle de la coagulation (figure 4). Des doses de 0.01 μg/ml à 1 μg/ml de FXa ont été comparées. La quantité de thrombine générée dépend de la dose de FXa ajoutée. L'effet du FXa a été comparé à du plasma normal sans induction de coagulation (·), activé par du facteur tissulaire (X) ou de la céphaline (O).Les doses de FXa de 0.01 μg/ml à 0.05 μg/ml produisent des quantités de thrombine similaires à celle d'un plasma normal activé par le FT ou la céphaline et suivent la même cinétique d'apparition. Ces résultats suggèrent que dans ces conditions expérimentales, le FXa à la dose de 50 ng/ml ne devrait pas être thrombogène. La dose de 50 ng/ml correspondrait donc à la dose la plus faible de FXa utilisable permettant de restaurer la coagulation. 1 - induction of thrombin generation by factor Xa alone The effect of an FXa overdose of normal plasma was evaluated by measuring thrombin generation (TGT) without additional coagulation induction (Figure 4). Doses of 0.01 μg / ml to 1 μg / ml of FXa were compared. The amount of thrombin generated depends on the dose of FXa added. The effect of FXa was compared to normal plasma without coagulation induction (·), activated by tissue factor (X) or partial thromboplastin (O). FXa doses from 0.01 μg / ml to 0.05 μg / ml produce thrombin levels similar to that of a normal plasma activated by FT or the partial thrombin and follow the same kinetics of appearance. These results suggest that under these experimental conditions, FXa at a dose of 50 ng / ml should not be thrombogenic. The dose of 50 ng / ml would therefore correspond to the lowest dose of FXa that can be used to restore coagulation.
2 - Induction de la génération de thrombine par le facteur tissulaire 2 - Induction of Thrombin Generation by Tissue Factor
L'effet d'un surdosage de FXa en plasma normal a été évalué en mesurant la génération de thrombine (TGT) suite à une induction par du facteur tissulaire (0.5 pM) (figure 5, table I). Des doses de 0,03 μg/ml à 0,5 μg/ml de FXa ont été comparées. La quantité de thrombine générée dépend de la dose de FXa ajoutée. Des doses de FXa de 0,03 μg/ml à 0,125 μg/ml produisent des quantités de thrombine similaires à celle d'un plasma normal activé (·) et suivent la même cinétique d'apparition. Ces résultats suggèrent que dans ces conditions expérimentales, le FXa ne semble pas induire de thrombogènicité supplémentaire à celle apportée par l'inducteur pour des doses de 0,03 μg/ml à 0,125 μg/ml. The effect of an overdose of FXa in normal plasma was assessed by measuring thrombin generation (TGT) following induction by tissue factor (0.5 μM) (Figure 5, Table I). Doses of 0.03 μg / ml to 0.5 μg / ml of FXa were compared. The amount of thrombin generated depends on the dose of FXa added. FXa doses of 0.03 μg / ml to 0.125 μg / ml produce thrombin levels similar to that of activated normal plasma (·) and follow the same kinetics of appearance. These results suggest that under these experimental conditions, FXa does not appear to induce thrombogenicity additional to that provided by the inducer for doses of 0.03 μg / ml to 0.125 μg / ml.
Une expérience similaire a été répétée en présence d'une dose thérapeutique de 0,35 μg/ml de Rivaroxaban (figure 6, table I). La dose utilisée de Rivaroxaban diminue la génération de thrombine de manière très importante (environ 75% ; courbe x) par rapport à celle obtenue dans un plasma non traité (·). La supplémentation en FXa de 0,03 μg/ml (o) ne permet pas de retrouver une génération de thrombine importante. En revanche, une correction de la génération de thrombine est obtenue dès 0,06 μg/ml (■) et ce jusqu'à la dose de 0,25 μg/ml (A). Pour la dose supérieure, la quantité de thrombine générée dépasse celle du contrôle suggérant un surdosage en FXa et donc un effet thrombogène possible malgré le Rivaroxaban.
A similar experiment was repeated in the presence of a therapeutic dose of 0.35 μg / ml Rivaroxaban (Figure 6, Table I). The dose of Rivaroxaban significantly decreases thrombin generation (approximately 75% x-curve) compared to that obtained in untreated plasma (·). The supplementation with FXa of 0.03 μg / ml (o) does not allow to find a significant generation of thrombin. On the other hand, a correction of the thrombin generation is obtained from 0.06 μg / ml (■) up to a dose of 0.25 μg / ml (A). For the higher dose, the amount of thrombin generated exceeds that of the control suggesting an FXa overdose and thus a possible thrombogenic effect despite Rivaroxaban.
Table I : Valeurs du temps de latence, du potentiel de thrombine, de la hauteur du pic et du temps au pic de la génération de thrombine induite par le facteur tissulaire d'un plasma normal en présence ou non de facteur Xa et/ou Rivaroxaban (données extraites des figures 4 et 5). Table I: Values of latency, thrombin potential, peak height and peak time of tissue-factor-induced thrombin generation in normal plasma with or without factor Xa and / or Rivaroxaban (data extracted from Figures 4 and 5).
3 - Induction de la génération de thrombine par la céphaline 3 - Induction of thrombin generation by the thromboplastin
Une série d'expérience similaire est réalisée en induisant l'initiation de la coagulation par de la céphaline. La série d'expérience préliminaire mime l'activation extrinsèque de la coagulation. Cette seconde série d'expérience mime l'activation intrinsèque de la coagulation. La présence de FXa en quantité croissante dans du plasma normal (·) diminue la vitesse d'apparition de la thrombine sans changer la quantité totale de thrombine générée (figure 7, table II). La vitesse d'apparition de la thrombine est corrélée avec la dose de FXa ajoutée. Des temps au pic plus courts sont obtenus en présence de FXa. Cette différence est significative et suggère un risque thrombotique à partir de 0,25 μg/ml de FXa. A similar series of experiments is carried out by inducing the initiation of coagulation by cephalin. The preliminary experiment series mimics the extrinsic activation of coagulation. This second series of experiments mimics the intrinsic activation of coagulation. The presence of increasing amounts of FXa in normal plasma (·) decreases the rate of occurrence of thrombin without changing the total amount of thrombin generated (Figure 7, Table II). The rate of occurrence of thrombin is correlated with the dose of FXa added. Shorter peak times are obtained in the presence of FXa. This difference is significant and suggests a thrombotic risk from 0.25 μg / ml of FXa.
L'effet du FXa suite à l'induction de la génération de thrombine par la céphaline est ensuite mesurée en présence de Rivaroxaban à 0,35 μg/ml (figure 8, table II). Le Rivaroxaban inhibe complètement la génération de thrombine induite par la céphaline (courbe x). La présence de FXa en concentration croissante permet d'augmenter de
manière dose-dépendante la quantité de thrombine générée ainsi que de diminuer le temps au pic. Une quantité maximale de thrombine est obtenue pour une concentration comprise entre 0,125 et 0,25 μ^ιηΐ. The effect of FXa following the induction of thrombin generation by the cephalin is then measured in the presence of Rivaroxaban at 0.35 μg / ml (FIG. 8, Table II). Rivaroxaban completely inhibits the thrombin generation induced by the cephalin (x-curve). The presence of FXa in increasing concentration makes it possible to increase dose-dependent manner the amount of thrombin generated as well as decrease the time at peak. A maximum amount of thrombin is obtained for a concentration between 0.125 and 0.25 μ ^ ιηΐ.
Table II : Valeurs du temps de latence, du potentiel de thrombine, de la hauteur du pic et du temps au pic de la génération de thrombine induite par la céphaline d'un plasma normal en présence ou non de facteur Xa et/ou rivaroxaban (données extraites des figures 7 et 8). Table II: Values of latency, thrombin potential, peak height and peak time of thrombin generation induced by the cephalin of normal plasma with or without factor Xa and / or rivaroxaban ( data extracted from Figures 7 and 8).
En conclusion, les données présentées montrent que le FXa a la possibilité de corriger la présence de Rivaroxaban dans du plasma normal suite à l'induction de la voie intrinsèque par la céphaline ou de la voie extrinsèque par le facteur tissulaire. Pour des doses inférieures ou égales à 0,25 μg/ml de facteur Xa et pour une dose de Rivaroxaban à 0.35 μg/ml, les données obtenues en TGT n'identifient pas un risque de thrombogénicité majeur. Pour des doses supérieures à 0,25 μg/ml, le temps de génération de thrombine est très raccourci et pourrait révéler un risque thrombogène.
In conclusion, the data presented show that FXa has the possibility of correcting the presence of Rivaroxaban in normal plasma following the induction of the intrinsic pathway by partial thromboplastin or extrinsic pathway by tissue factor. For doses less than or equal to 0.25 μg / ml of factor Xa and for a dose of Rivaroxaban at 0.35 μg / ml, the data obtained in TGT do not identify a risk of major thrombogenicity. For doses greater than 0.25 μg / ml, the thrombin generation time is very shortened and could reveal a thrombogenic risk.
Claims
1. Facteur Xa pour son utilisation dans la prévention ou le traitement chez un patient, notamment un être humain ou un animal, des événements hémorragiques induits par la prise d'anticoagulants. 1. Factor Xa for its use in the prevention or treatment in a patient, particularly a human being or an animal, of hemorrhagic events induced by taking anticoagulants.
2. Facteur Xa pour son utilisation selon la revendication 1, dans laquelle les anticoagulants sont des héparines de bas poids moléculaire. 2. Factor Xa for its use according to claim 1, in which the anticoagulants are low molecular weight heparins.
Facteur Xa pour son utilisation selon la revendication 2, dans laquelle les héparines de bas poids moléculaire sont choisies parmi la bemiparine, la certoparine, la deltaparine, l'enoxaparine, la nadroparine, la parnaparine, la reviparine ou la tinzaparine. Factor
Facteur Xa pour son utilisation selon la revendication 1, dans laquelle les anticoagulants sont des inhibiteurs spécifiques du facteur Xa. Factor Xa for its use according to claim 1, in which the anticoagulants are specific inhibitors of factor Xa.
Facteur Xa pour son utilisation selon la revendication 4, dans laquelle les inhibiteurs spécifiques du facteur Xa sont choisis parmi l'apixaban, le betrixaban, l'édoxaban, l'otamixaban ou le rivaroxaban. Factor Xa for its use according to claim 4, in which the specific inhibitors of factor Xa are chosen from apixaban, betrixaban, edoxaban, otamixaban or rivaroxaban.
6. Facteur Xa pour son utilisation selon la revendication 1, dans laquelle les anticoagulants sont les oligosaccharides fondaparinux ou idraparinux, en particulier l'oligosaccharide fondaparinux. 6. Factor Xa for its use according to claim 1, in which the anticoagulants are the oligosaccharides fondaparinux or idraparinux, in particular the oligosaccharide fondaparinux.
7. Facteur Xa pour son utilisation selon l'une quelconque des revendications 1 à 6, dans laquelle le facteur Xa restaure l'activité procoagulante sans que cette dernière ne dépasse le niveau basai de l'activité procoagulante mesurée dans un groupe de patients sains. 7. Factor Xa for its use according to any one of claims 1 to 6, in which factor
8. Facteur Xa pour son utilisation selon l'une quelconque des revendications 1 à 7, dans laquelle le facteur Xa est une protéine native isolée du plasma ou une protéine recombinante.
8. Factor Xa for its use according to any one of claims 1 to 7, in which factor Xa is a native protein isolated from plasma or a recombinant protein.
9. Facteur Xa pour son utilisation selon l'une quelconque des revendications 1 à 8, dans laquelle le facteur Xa a une demi- vie circulante augmentée par rapport au facteur Xa natif isolé du plasma. 9. Factor Xa for its use according to any one of claims 1 to 8, in which factor Xa has an increased circulating half-life compared to native factor Xa isolated from plasma.
10. Procédé de suivi in vitro ou ex-vivo de la survenue d'événements hémorragiques induits par la prise d'inhibiteurs spécifiques du facteur Xa comprenant les étapes suivantes : 10. Method for monitoring in vitro or ex-vivo the occurrence of hemorrhagic events induced by taking specific factor Xa inhibitors comprising the following steps:
a) mesure analytique des marqueurs de la coagulation sanguine dans un échantillon biologique issu d'un patient recevant un inhibiteur spécifique du facteur Xa pour obtenir des valeurs numériques attribuables à chacun des susdits marqueurs, a) analytical measurement of blood coagulation markers in a biological sample from a patient receiving a specific factor Xa inhibitor to obtain numerical values attributable to each of the above markers,
b) détermination du risque hémorragique à partir des valeurs numériques établies à l'étape a), b) determination of the hemorrhagic risk from the numerical values established in step a),
c) détermination d'une quantité adéquate de facteur Xa à administrer audit patient recevant un inhibiteur spécifique du facteur Xa. c) determining an adequate quantity of factor Xa to administer to said patient receiving a specific factor Xa inhibitor.
11. Composition pharmaceutique comprenant le facteur Xa et un inhibiteur du facteur Xa comme produit de combinaison pour son utilisation dans la prévention ou le traitement chez un patient, notamment un être humain ou un animal, des événements thromboemboliques veineux, par exemple consécutifs à une intervention orthopédique de la hanche et/ou du genou. 11. Pharmaceutical composition comprising factor Xa and a factor orthopedic treatment of the hip and/or knee.
12. Composition pharmaceutique selon la revendication 11, dans laquelle la dose unitaire de facteur Xa varie de 0,01 ng/ml à 2 μg/ml, notamment de 10 ng/ml à 2 μg/ml. 12. Pharmaceutical composition according to claim 11, in which the unit dose of factor Xa varies from 0.01 ng/ml to 2 μg/ml, in particular from 10 ng/ml to 2 μg/ml.
13. Méthode pour prévenir ou traiter des événements hémorragiques induits par la prise d'anticoagulants, chez un mammifère, notamment l'homme, comprenant une étape d'administration de facteur Xa audit mammifère, notamment l'homme, en quantité suffisante permettant de restaurer une activité procoagulante basale.
13. Method for preventing or treating hemorrhagic events induced by taking anticoagulants, in a mammal, in particular man, comprising a step of administering factor Xa to said mammal, in particular man, in sufficient quantity to restore basal procoagulant activity.
14. Méthode selon la revendication 13, dans laquelle les anticoagulants sont des inhibiteurs spécifiques du facteur Xa. 14. Method according to claim 13, in which the anticoagulants are specific inhibitors of factor Xa.
15. Méthode selon la revendication 14, dans laquelle les inhibiteurs spécifiques du facteur Xa sont choisis parmi l'apixaban, le betrixaban, l'édoxaban, l'otamixaban ou le rivaroxaban. 15. Method according to claim 14, in which the specific factor Xa inhibitors are chosen from apixaban, betrixaban, edoxaban, otamixaban or rivaroxaban.
16. Méthode selon la revendication 15, dans laquelle le facteur Xa est administré au mammifère, notamment l'homme, pendant ou après la prise d'inhibiteurs spécifiques du facteur Xa.
16. Method according to claim 15, in which factor Xa is administered to the mammal, in particular humans, during or after taking specific factor Xa inhibitors.
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FR1350278A FR3000895B1 (en) | 2013-01-11 | 2013-01-11 | USE OF ANTIDOTES OF COAGULATION INHIBITORS INDICATED IN THE PREVENTION OR TREATMENT OF THROMBOEMBOLIC PATHOLOGIES |
PCT/FR2014/050020 WO2014108632A1 (en) | 2013-01-11 | 2014-01-08 | Use of factor xa in the prevention or treatment of haemorrhagic events following the taking of anticoagulants |
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US10676731B2 (en) | 2014-08-19 | 2020-06-09 | The Children's Hospital Of Philadelphia | Compositions and methods for modulating factor IX function |
CN114681597A (en) * | 2022-03-11 | 2022-07-01 | 兆科药业(合肥)有限公司 | Application of viper venom hemocoagulase in preparation of drugs for reversing anticoagulation of coagulation factor Xa inhibitor |
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