WO2011067451A2 - Méthodes pour déterminer la propension à être atteint d'une transformation hémorragique après un ictus - Google Patents

Méthodes pour déterminer la propension à être atteint d'une transformation hémorragique après un ictus Download PDF

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WO2011067451A2
WO2011067451A2 PCT/ES2010/070796 ES2010070796W WO2011067451A2 WO 2011067451 A2 WO2011067451 A2 WO 2011067451A2 ES 2010070796 W ES2010070796 W ES 2010070796W WO 2011067451 A2 WO2011067451 A2 WO 2011067451A2
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Prior art keywords
fluorescence
stroke
patient
sample
levels
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PCT/ES2010/070796
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English (en)
Spanish (es)
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WO2011067451A3 (fr
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Joan Montaner Vilallonga
Carmen DOMÍNGUEZ LUENGO
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Fundació Institut De Recerca Hospital Universitari Vall D'hebron, Fundació Privada
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Publication of WO2011067451A3 publication Critical patent/WO2011067451A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2871—Cerebrovascular disorders, e.g. stroke, cerebral infarct, cerebral haemorrhage, transient ischemic event

Definitions

  • the invention falls within the field of diagnostic methods and personalized therapies and, more specifically, in the identification of biomarkers capable of distinguishing clinical conditions and allowing the individualized treatment of patients suffering from thromboembolic disorders.
  • Stroke stroke or stroke is the third leading cause of death and is the most common cause of permanent disability in the world. Stroke is a sudden interruption in blood supply to the brain. Approximately 80% of strokes are caused by a sudden blockage of the arteries that go to the brain (ischemic stroke). Other strokes are caused by a hemorrhage in the brain tissue caused by the rupture of a blood vessel (hemorrhagic stroke). Ischemic strokes can be divided into thrombotic and embolic strokes. Thrombotic strokes occur when a cerebral artery is blocked by a clot formed in the brain and accounts for approximately 50% of all strokes.
  • Embolic strokes are caused by a thrombus formed in a peripheral artery that travels to the brain where ischemia occurs.
  • Other causes of decreased cerebral blood flow are: occlusion of perforating arteries, intracranial artery stenosis with poor collateral circulation, arteritis, arterial dissection, venous occlusion, and significant anemia or hyperviscosity.
  • tissue plasminogen activator t-PA
  • t-PA Although treatment with t-PA is effective in increasing the proportion of patients free of disability and in decreasing the cost of medical care for individuals receiving it, t-PA is only administered at less than 5% from the patients with stroke. This is partly due to the risk of a 10-fold increase in intracranial hemorrhage (HIC) in patients treated with t-PA and a mortality rate of more than 50% for patients with this type of symptomatic hemorrhagic complications.
  • HIC intracranial hemorrhage
  • Millán et al (Stroke, 2007, 38: 90-95) have described that elevated levels of intracorporeal iron and ferritin are correlated with a worse prognosis in patients who have suffered stroke and with an increased risk of hemorrhagic transformation after thrombolytic therapy. .
  • WO2006036220 describes that elevated plasma levels of cellular fibronectin (c-Fn) correlate with a worse prognosis in patients who have suffered stroke and with a higher risk of hemorrhagic transformation after thrombolytic therapy.
  • PAI-1 Plasminogen Activator Inhibitor 1
  • TAFI Thrombin Activable Fibrinolysis Inhibitor
  • WO2010026272 describes a method to predict a bleeding disorder in a patient by determining the plasma levels of the Semicarbazide-sensitive amino oxidase (SSAO / VAP-1) in a plasma sample of said patient.
  • SSAO Semicarbazide-sensitive amino oxidase
  • MMP-9 cellular fibronectin
  • / or endogenous fibrinolysis inhibitors as risk markers of hemorrhagic transformation requires the determination of the expression levels of these proteins, which usually requires a longer analysis time. which is recommended to start thrombolytic treatment.
  • S 100B protein levels can be used to determine the risk of hemorrhagic complications after lytic thrombus therapy (Foerch, et al, 2007, Stroke, 38: 2491-2495).
  • the sensitivity of the diagnosis using S 100B levels as the only marker was too low for this marker to be used as a reliable marker in clinical practice.
  • biomarkers that allow predicting the propensity to undergo hemorrhagic transformation in patients who have suffered strokes and who have been treated with thrombolytic therapy and, in particular, biomarkers that can be determined more rapidly, since Thrombolytic treatment provides better results when administered shortly after the stroke episode.
  • the invention relates to a method for diagnosing a stroke in a patient, to determine the probability of a patient who has suffered a stroke from suffering a bleeding disorder in response to an antithrombotic agent or to determine the clinical and / or neurological evolution of a patient who has suffered a stroke and who has been treated with an antithrombotic agent that comprises determining in a sample of said patient the fluorescence intensity in the wavelength range of 400-500 nm in response to the excitation of said sample in a wavelength range of 280-400 nm where a fluorescence intensity greater than The intensity of fluorescence in a reference sample is indicative that the patient suffers a stroke, that he has a high probability of suffering from a bleeding disorder or a worse clinical course.
  • the invention in a second aspect, relates to a method for treating a patient who has suffered a stroke which comprises administering an effective amount of an antithrombotic agent to said patient, wherein a sample of said patient exhibits a fluorescence intensity in the wavelength range of 400-500 nm in response to the excitation of said sample in a wavelength range of 280-400 nm that is similar to or lower the fluorescence intensity in a reference sample.
  • the invention relates to a method for designing a personalized therapy in a patient who has suffered a stroke comprising the determination in a sample of said patient of the fluorescence intensity in the wavelength range of 400-500 nm in response to the excitation of said sample in a wavelength range of 280-400 nm where if the level of fluorescence in the patient sample is similar or less than the level of fluorescence in a reference sample, it is selected a therapy based on an antithrombotic agent.
  • the invention relates to a method for the identification of compounds useful for preventing the occurrence of a bleeding disorder in response to treatment with an antithrombotic agent in a subject who has suffered a stroke which comprises comparing
  • Figure 2. Temporal profile of the plasma fluorescence level during the study period, showing a gradual decrease in the fluorescence level during the first days (p ⁇ 0.001). The dashed line indicates the reference range of the fluorescence level for healthy controls.
  • Figure 3. Baseline levels of fluorescence in plasma samples according to the subsequent presence or appearance of the main subtypes of hemorrhagic transformation (HT) based on the results of CT Computed Tomography.
  • HT hemorrhagic transformation
  • Figure 6 Number of patients with symptomatic HT (circles) and without symptomatic HT (stripes) in relation to the fluorescence cut-off points selected in the ROC curves.
  • Figure 7. Percentage of patients with symptomatic HT using a fluorescence cut-off point that offers excellent positive predictive value.
  • Figure 8. Percentage of patients with symptomatic HT using a fluorescence cut-off point that offers excellent negative predictive value.
  • Figure 10 The combination of high plasma levels of fluorescence and SSAO / VAP-1 activity, measured at the arrival of the patient in the emergency room, which are located above the indicated cut-off points, allows to select with great precision those patients who will present bleeding complications symptomatic after treatment with t-PA.
  • Figure 12 The combination of levels of fluorescence and cellular fibronectin, measured at the arrival of the patient in the emergency room, which are located above or below the indicated cut-off points, allows to select with great precision those patients who will present or not, symptomatic bleeding complications after treatment with t-PA.
  • Figure 13 The combination of fluorescence levels, SSAO / VAP-1 activity and cellular fibronectin, measured at the arrival of the patient in the emergency room, which are located above or below the indicated cut-off points, allows selection with large precision those patients who will present or not, symptomatic hemorrhagic complications after treatment with t-PA.
  • the authors of the present invention have observed that, surprisingly, the fluorescence emitted by a plasma sample isolated from a patient at 430 nm in response to the excitation of said sample in a wavelength range of 360 nm correlates with positive form with the presence in said patient of a situation of acute ischemic stroke.
  • patients who had suffered stroke had a mean baseline fluorescence level of 45.69 (36.65-60.84) that was significantly different from the fluorescence level of the healthy controls (34.56 (30.3-45.55) (see figure 2).
  • the invention relates to a method for diagnosing a stroke in a patient comprising determining in a sample of said patient the fluorescence intensity in the wavelength range of 400-500 nm in response to the Excitation of said sample in a wavelength range of 280-400 nm where a fluorescence intensity greater than the fluorescence intensity in a reference sample is indicative that the patient suffers a stroke.
  • diagnosis refers to assessing the probability that a subject suffers from a disease, in particular, a stroke.
  • evaluation although preferred, may not be correct for 100% of the subjects to be diagnosed.
  • the term requires that a statistically significant part of the subjects can be identified as having the disease or having a predisposition to it.
  • the person skilled in the art can determine if a part is Statistically significant without further delay using several well-known statistical evaluation tools, for example, determination of confidence intervals, determination of the p-value, Student's t-test, Mann-Whitney test, etc. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983.
  • Preferred confidence intervals are at least 50%, at least 60%, at least 70%>, at least 80%>, at least 90%>, at least 95%>.
  • P values are preferably 0.2, 0.1, 0.05.
  • stroke is used in the present invention interchangeably with “stroke (CVA)", or “stroke” and refers to a sudden interruption in blood supply to the brain.
  • a part of the strokes are caused by a sudden blockage of the arteries that go to the brain (ischemic stroke).
  • Other strokes are caused by a hemorrhage in the brain tissue caused by the rupture of a blood vessel (hemorrhagic stroke).
  • Ischemic strokes can be divided into thrombotic strokes and emboli.
  • Thrombotic strokes occur when a cerebral artery is blocked by a clot formed in the brain and accounts for approximately 50%) of all strokes.
  • Embolic strokes are caused by a thrombus formed in a peripheral artery that travels to the brain where ischemia occurs.
  • Other causes of decreased cerebral blood flow are: occlusion of perforating arteries, intracranial artery stenosis with poor collateral circulation, arteritis, arterial dissection, venous occlusion, and significant an
  • fluorescence refers to a type of luminescence in which the molecular absorption of a photon by one molecule causes the emission of another photon of a greater wavelength. Fluorescence intensity detection can be carried out using any known method, although it is preferred that it be carried out by spectrofluorometric measurement by exciting the sample in a wavelength range of 280-400 nm and detecting the intensity of radiation in response to the excitation emitted in the range of 400-500 nm.
  • the determination of the fluorescence intensity is carried out by excitation in a wavelength range of 300-380 nm, more preferably 320 to 360 nm and even more preferably, in the range from 340 to 350 nm, even more preferably between 355 to 365 nm or between 350 and 370 n ,.
  • Fluorescence detection is carried out in a wavelength range from 400 to 500 nm, more preferably from 410 to 490, even more preferably from 420 to 480 nm and preferably between 430 and 440 nm.
  • the measurement is performed using wavelengths of 360 (excitation) and 430 nm (emission).
  • Fluorescence can be determined as relative fluorescence units (UFRFI) per sample volume.
  • URFI relative fluorescence units
  • the use of standard curves may be necessary for the measurement of fluorescence. Methods for generating standards have been described extensively in the state of the art.
  • the standard used for the measurement of the fluorescence intensity and the calibration of the wavelengths is 1 ng of quinine sulfate / ml in H 2 SO 4 0, 1 N.
  • fluorescence is use to refer to the level of fluorescence measured at 430 nm in response to excitation at 360 nm and is indicated in arbitrary units of relative fluorescence.
  • sample refers to any sample that can be obtained from a patient and in which there is sufficient fluorescence intensity to be detected using the usual methods.
  • Samples suitable for use in the present invention include any biofluid and, in particular, serum, saliva, semen, sputum, cerebrospinal fluid (CSF), tears, mucus, sweat, milk, brain extracts and the like.
  • said sample is a sample of whole blood, serum, plasma, saliva or cells extracted from peripheral blood and tissue.
  • the sample is plasma.
  • extract as used in the present invention, is meant an acellular preparation obtained from a certain tissue that can be raw or partially purified or fractionated.
  • the extracts may be carried out using an aqueous solvent, in which case an aqueous extract will be obtained, preferably, using an organic solvent.
  • aqueous solvent refers to a solution in which the solvent is water and contains an aqueous salt or a buffer solution.
  • organic solvent refers to any organic substance that is liquid at room temperature and that is immiscible in water, usually being non-polar or apolar aprotic.
  • Organic solvents suitable for use in the present invention include, without limitation, ethanol, diethyl ether, chloroform, 1- propanol, isopropanol, benzene, toluene, dichloromethane and combinations thereof.
  • the sample is extracted using ethaneheter (3: 1, v / v).
  • the determinations are carried out from a plasma extract in ethanol: ether (3: 1, v / v).
  • the first method of the invention includes the step of comparing the fluorescence intensity in the patient sample with the fluorescence intensity in a reference sample, such that a high fluorescence intensity with respect to the intensity in the sample of reference is indicative that the patient shows a high probability of suffering a stroke.
  • the fluorescence intensity is considered to be high when it is increased with respect to a reference value of at least 5%, at least 10%, at least 15%, at least 20%> , at least 25%, at least 30%>, at least 35%, at least 40% or, at least 45%, at least 50%, at least 55%, at least 60% , at least 65%, at least 70%, at least 75%, at least 80%: at least 85% or, at least 90%, at least 95%, at least 100%) , at least 10%), at least 120%, at least 130%), at least 140%), at least 150%) or more.
  • a “reference sample”, as used herein, means a sample obtained from subjects, who are well documented from the clinical point of view and who do not present any disease or, alternatively, from the general population. Suitable reference expression levels of fluorescence intensity can be determined by measuring the fluorescence intensity in several suitable subjects, and such reference levels can be adjusted to specific subject populations. In determining the reference fluorescent intensity, It may be necessary to take into account some characteristics of the type of sample, such as the age, sex, physical condition and the like of the patient since oxidative stress may increase with age. For example, the reference sample can be obtained from identical quantities of a group of at least 2, at least 10, at least 100 to more than 1000 individuals, so that the population is statistically significant.
  • the authors of the present invention have observed that the ability of fluorescent intensity to diagnose the presence of stroke significantly improves if said determination is combined with one or more diagnostic markers of stroke.
  • the determination of the fluorescence intensity is carried out simultaneously with the determination of at least a second marker.
  • the additional marker or markers that can be determined are selected from the group of one or more of cellular fibronectin, SSAO / VAP-1, ferritin, MMP9, laminin, PAI-1, TAFI and S 100B and wherein high levels of plasma cellular fibronectin, SSAO / VAP-1, ferritin, MMP9, TAFI or SI 00b or low levels of PAI-1 or laminin, with respect to a reference value are indicative that the patient shows a high probability of suffering a bleeding disorder.
  • the expression levels of the markers indicated above can be determined either by measuring the levels of AR m or protein of each of the markers indicated above.
  • the mRNA levels of each of the markers indicated above are determined by any technique known in the state of the art such as RT-PCR, Northern blot, etc.
  • the determination of the levels of the markers indicated above in a sample can be carried out using any conventional method.
  • the levels of the markers indicated above can be quantified using antibodies capable of specifically binding to the proteins of each of the markers indicated above (or fragments thereof containing the antigenic determinants) and subsequent quantification of the resulting antigen-antibody complexes.
  • the antibodies to be used in this type of assays can be, for example, polyclonal antibodies, hybridoma supernatants or monoclonal antibodies, antibody fragments, Fv, Fab, Fab 'and F (ab') 2, scFv, diabody, triabodies, humanized tetrabodies and antibodies.
  • the antibodies may be labeled or not.
  • markers include radioactive isotopes, enzymes, fluorophores, chemiluminescent reagents, enzymatic substrates or cofactors, enzyme inhibitors, particles, dyes, etc.
  • the diagnostic method of the invention contemplates the determination of fluorescent intensity and VAP-1 / SSAO levels where high fluorescence levels with respect to the reference sample and VAP levels. High 1 / SSAO with respect to the reference sample are indicative that the patient shows a high probability of having suffered a stroke.
  • the diagnostic method of the invention contemplates the determination of fluorescent intensity and cellular fibronectin levels where high fluorescence levels with respect to the reference sample and high cellular fibronectin levels with respect to to the reference sample are indicative that the patient shows a high probability of having suffered a stroke.
  • the diagnostic method of the invention contemplates the determination of fluorescent intensity, VAP-1 / SSAO levels and cellular fibronectin levels where high fluorescence levels with respect to the sample of reference, high levels of VAP-1 / SSAO with respect to the reference sample and high levels of cellular fibronectin with respect to the reference sample are indicative that the patient shows a high probability of having suffered a stroke.
  • Suitable methods for determining VAP-1 / SSAO levels include methods based on the determination of SSAO enzymatic activity, methods based on the determination of protein levels and methods based on the determination of AR m levels. These methods have been described in detail in WO2010026272.
  • Suitable methods for determining cellular fibronectin levels include methods based on the determination of protein levels and methods based on the determination of mRNA levels. These methods have been described in detail in the international patent application with publication number WO2006036220.
  • the diagnostic method of the invention may be accompanied by the determination of one or more clinical variables which, combined with the determination of fluorescence and, optionally, one or more additional biomarkers, allows to improve the reliability of the diagnosis of stroke.
  • Clinical variables suitable for use in the diagnostic method of the invention include, without limitation, smoking status, initial hypodensity in CT, hypertension, a history of diabetes, hyperglycemia, platelet disease, blood clotting disorder and age.
  • the invention relates to a method for determining the probability in a patient who has suffered a stroke that this patient suffers a bleeding disorder in response to an antithrombotic agent comprising determining in a sample of said patient the intensity of fluorescence in the wavelength range of 400-500 nm in response to the excitation of said sample in a wavelength range of 280-400 nm where an intensity of Fluorescence greater than fluorescence intensity in a reference sample is indicative that the patient has a high probability of suffering from a bleeding disorder or a worse clinical course.
  • method to determine the probability refers to methods to determine the probability that a patient suffers from a bleeding disorder.
  • the prediction may not be correct for 100% of the patients under study.
  • the expression requires that the prediction method provide correct results for a statistically significant portion of patients.
  • the determination of whether the method of the invention provides statistically significant predictions can be carried out using standard statistical techniques such as determination of confidence intervals, determination of p-value, Student's t-test, Mann-Whitney test such and as explained in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983.
  • Adequate confidence intervals are at least 50%>, at least 60%>, at least 70%), at least 80%>, at least 90%>, at least 95%>.
  • P values are preferably 0.2, 0.1, 0.05.
  • high probability of suffering a bleeding disorder means the situation where the subject has at least 5%, at least 10% or, at least 20%>, at least 30%>, at least 40%>, at least 50%>, at least 60%>, at least 70%>, at least 80%>, at least 90%>, at least 100% likely to develop or suffer from a bleeding disorder over time.
  • the stroke has been described in the first method of the invention.
  • the stroke is an ischemic stroke.
  • hemorrhagic disorder is meant, in the context of the present invention, a disorder in which inadequate blood clotting occurs so that excessive bleeding occurs and includes congenital, acquired bleeding disorders caused by trauma, spontaneous or caused by treatment with a thrombolytic agent.
  • the invention contemplates methods for predicting hemorrhagic conditions such as hemophilia A, hemophilia B, von Willebrand disease, idiopathic thrombocytopenia, deficiencies in one or more coagulation factors such as factor XI, factor XII, prekalikrein and high molecular weight kinogen, deficiencies associated with clinically significant bleeding such as deficiencies in factor V, factor VII, factor VIII, factor IX, factor X, factor XIII, factor II (hypoprothrombinemia) and von Willebrand factor, a deficiency in vitamin K, alterations in fibrinogen such as afibrinogenemia, hypofibrinogenemia and dysibrinogenemia, a deficiency in alpha 2-antiplasmin and excessive bleeding due to kidney disease, liver disease, thrombocytopenia, a platelet disorder, bruising, internal bleeding, hemarthros, surgery, trauma, hypothermia, menstruation and pregnancy.
  • coagulation factors such as factor X
  • the hemorrhagic condition that can be predicted with the method of the invention is a parenchymal hemorrhage, that is, a release of blood to the cerebral parenchyma (white or gray substance) that can occasionally present openness to the ventricular system ( intraventicular hemorrhage), to the subarachnoid space (subarachnoid hemorrhage) or to the subdural space (subdural hemorrhage) or any other extravasation of blood in the brain.
  • a parenchymal hemorrhage that is, a release of blood to the cerebral parenchyma (white or gray substance) that can occasionally present openness to the ventricular system ( intraventicular hemorrhage), to the subarachnoid space (subarachnoid hemorrhage) or to the subdural space (subdural hemorrhage) or any other extravasation of blood in the brain.
  • the hemorrhagic condition that can be predicted with the method of the invention is a hemorrhagic transformation following a thromboembolic disease and a treatment with an antithrombotic agent.
  • thromboembolic diseases that can result in a haemorrhagic transformation in the brain include a stroke (stroke), acute myocardial infarction, massive pulmonary embolism and the like.
  • stroke a stroke
  • the thromboembolic disease that precedes the hemorrhagic episode is a stroke
  • it may be of an ischemic type caused by an atherosclerotic vascular disease, by a hypertensive vascular disease, by a hypertensive atherosclerotic vascular disease, by amyloid angiopathy, by a disease Vascular associated with an inflammation caused, among others, by infections such as bacterial meningitis, tuberculosis (TB), syphilis, etc.
  • a collagenopathy systemic lupus erythematosus (SLE), polyarteritis nodosa (PAN), etc.
  • a coagulation disorder by an embolism, by an acute myocardial infarction, by a cardiac aneurysm, by a vasospasm, due to systemic hypotension, extrinsic vascular compression, arterial dissection, venous thrombosis, which may be caused, in turn, by vascular rupture, coagulopathy and the like.
  • embolic thrombus diseases that can cause hemorrhagic transformation include deep vein thrombosis, pulmonary thromboembolism or acute myocardial infarction.
  • fibrinolytic agents for the treatment of acute myocardial infarction include streptokinase, alteplase, reteplase, anistreplaseplase, anistreplase and the like.
  • thrombolytic agent or “antithrombotic agent”, as used in the present invention, it is meant any compound that administered to the patient in a clinically effective amount will cause a rupture or lysis of the thrombus or clot to restore circulation.
  • said thrombolytic agent includes antiplatelet drugs such as thromboxane inhibitors (aspirin, ridogrel, S I 8886); PAR antagonists (E5555 / SCH530348); ADP antagonist-receptor (AZD6140, cangrelor, clopidogrel, prasugrel, ticlopidine); Iib / IIIa glycoprotein inhibitors (abciximab, eptifibatide, tirofiban); platelet adhesion antagonists (ClqTNF, DZ-697b); Phosphodiesterase (dipyridamole) inhibition, Heparin, Unfractionated or low molecular weight heparins (bemiparin, certroparin, dalteparin, enoxaparin, nadroparin, parnaparin, reviparin, tinzaparin)
  • antiplatelet drugs such as thromboxane inhibitors (aspirin, ridogrel, S
  • fibrinolytic agents such as tenecteplase, reteplase, plasmin, microplasmin, demoteplase, V10153, combinations of thrombolytics and antithrombotic agents such as t-PA and thyrofibran, t-PA and abciximab, repalase and abciximab, t-PA and eptifibatide and t-PA in combination with eptifibatide, aspirin and tinzaparin, combinations of thrombolytics and neuroprotectors.
  • fibrinolytic agents such as tenecteplase, reteplase, plasmin, microplasmin, demoteplase, V10153, combinations of thrombolytics and antithrombotic agents such as t-PA and thyrofibran, t-PA and abciximab, repalase and abciximab, t-PA and eptifibatide and t-PA
  • Oral anticoagulants (warfarin, acenocoumarol).
  • Other drugs that are included are terutroban, fetroban, variprost, ridogrel, picotamide and new antithrombotic drugs (rivaroxaban, epixaban, fondaparinux, idraparinux, desirudin, lepidurine, bivalirudin, argatroban, ximelagatran, dabigabatran, streptoptose, streptoptoapase, streptoptoapase, streptoptosepa, streptoptimat, streptoptimat, streptoptimat, streptophotash, streptophotash, streptoptimat, streptophotash, streptoptmat, streptophotash, streptoptose, streptoptose, streptoptimose , tenecteplase, reteplase, microplasmin, ancrod, prourokina
  • the thrombolytic agent is a plasminogen activator.
  • Plasminogen activators that can be used in the treatment of thromboembolic diseases and that can lead to hemorrhagic transformation include the tissue plasminogen activator (tPA), the urokinase type plasminogen activator (uPA) and streptokinase.
  • the antithrombotic agent is the tissue plasminogen activator (tPA).
  • tPA tissue plasminogen activator
  • sample and “reference sample” have been described in the section of the first method of the invention.
  • the second method of the invention contemplates the determination of fluorescence intensity in a sample of said patient either in isolation or in conjunction with one or more additional markers whose predictive value of the risk of suffering a bleeding disorder is already known.
  • the Fluorescence intensity determination is carried out simultaneously with the determination of at least a second marker.
  • the additional marker or markers that can be determined are selected from the group of one or more of cellular fibronectin, SSAO / VAP-1, ferritin, MMP9, laminin, PAI-1, TAFI and S100B and where levels high plasma cellular fibronectin, SSAO / VAP-1, ferritin, MMP9, TAFI or SI 00b or low levels of PAI-1 or laminin, with respect to a reference value are indicative that the patient shows a high probability of suffering a bleeding disorder.
  • the expression levels of the markers indicated above can be determined either by measuring the levels of AR m or the protein of each of the markers indicated above.
  • the mRNA levels of each of the markers indicated above are determined by any technique known in the state of the art such as RT-PCR, Northern blot, etc.
  • the determination of the levels of the markers indicated above in a sample can be carried out using any conventional method.
  • the levels of the markers indicated above can be quantified using antibodies capable of specifically binding the proteins of each of the markers indicated above (or fragments thereof containing the antigenic determinants) and subsequent quantification. of the resulting antigen-antibody complexes.
  • the antibodies to be used in this type of assays can be, for example, polyclonal antibodies, hybridoma supernatants or monoclonal antibodies, antibody fragments, Fv, Fab, Fab 'and F (ab') 2, scFv, diabody, triabody , humanized tetrabodies and antibodies.
  • the antibodies may be labeled or not.
  • markers include radioactive isotopes, enzymes, fluorophores, chemiluminescent reagents, enzymatic substrates or cofactors, enzyme inhibitors, particles, dyes, etc.
  • assays there are a wide variety of well known assays that can be used in the present invention, which use unlabeled antibodies (primary antibody) and labeled antibodies (secondary antibodies); These techniques include Western blotting or immunoblotting, ELISA (assay enzyme-linked immunoabsorbent), RIA (radioimmunoassay), EIA (enzyme immunoassay) competitive, DAS-ELISA (sandwich ELISA with double antibody), immunocytochemical and immunohistochemical techniques, techniques based on the use of biochips or protein microarrays including specific antibodies or assays based in colloidal precipitation in formats such as test strips. Other ways of detecting and quantifying the protein of the markers indicated above include affinity chromatography techniques, ligand binding assays, etc.
  • the second method of the invention further comprises determining at least one clinical variable selected from the group: smoking status, initial hypodensity in CT, hypertension, history of diabetes, hyperglycemia, platelet disease, blood clotting disorder and age .
  • the invention relates to a method for determining the clinical and / or neurological evolution of a patient who has suffered a stroke and who has been treated with an antithrombotic agent which comprises determining in a sample of said patient the fluorescence intensity in the wavelength range of 400-500 nm in response to the excitation of said sample in a wavelength range of 280-400 nm where a fluorescence intensity exceeds the fluorescence intensity in a sample Reference is indicative of a worse clinical evolution.
  • the term "clinical evolution”, as used in the present invention refers to a significant decrease in the various symptoms and signs associated with a certain disorder. Since stroke occurs primarily with neurological symptoms, in the case of the present invention, the terms “clinical evolution” and “neurological evolution” can be used interchangeably.
  • Neurological evolution is understood, in the context of the present invention, as a change in the neurological characteristics of the patient that can be classified according to the NIHS S scale as described by Brott T and Bogousslavsky J (N. Engl. J. Med. 2000, 343: 710-722).
  • a neurological worsening is understood in the context of the present invention as an increase of 4 or more points on the neurological scale of the NIHSS. By passing this scale periodically we identify these changes and can define worsening or stability or improvement (decrease of 4 or more points on the scale).
  • the neurological worsening can be determined by the Rankin scale in which the value 0 indicates absence of symptoms, the value 1 indicates the absence of disabling symptoms, the value 2 indicates slight disability, the value 3 indicates moderate disability, the value 3 indicates severe / moderate disability, value 5 indicates severe disability and value 6 indicates that death has occurred.
  • stroke has been described in detail above and also apply to the method of the invention to determine clinical and / or neurological evolution of a patient who has suffered a stroke and who has been treated with an antithrombotic agent.
  • the invention in another aspect, relates to a method for treating a patient who has suffered a stroke which comprises administering an effective amount of an antithrombotic agent to said patient, wherein a sample of said patient has a fluorescence intensity in the range. of wavelengths of 400-500 nm in response to the excitation of said sample in a wavelength range of 280-400 nm that is similar or less than the fluorescence intensity in a reference sample.
  • Effective amount or “therapeutically effective amount” as applied to the biologically active compound means that amount of the compound that is generally sufficient to effect a desired change in the subject. For example, where the desired effect is the dissolution of thrombi, an effective amount of the compound is that dose that produces at least the desired dissolution of thrombi or clots without causing bleeding, and without producing a significant systemic toxicity reaction.
  • the third method of the invention includes the step of comparing the fluorescence intensity in a given range of wavelengths in a patient sample with a sample or reference value.
  • a patient who has suffered a stroke will be given an effective amount of an antithrombotic agent, if said patient has a fluorescence intensity in a sample obtained from the patient that is similar or less than the value in a reference sample.
  • the fluorescence intensity is considered to be decreased (is lower) with respect to a reference value when the levels in the patient sample are decreased by at least 5%, at least 10%, at least 15%, at least 20%>, at least 25%, at least 30%>, at least 35%, at least 40% or, at least 45%, at least 50% , at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%: at least 85% or at least 90%, at least 95%, at least 100%), at least 1 10%), at least 120%, at least 130%), at least 140%), at least 150%) or more.
  • the third method of the invention contemplates the simultaneous determination of the levels of one or more additional markers so as to increase the reliability of the determination of personalized therapy.
  • the method object of the second method of the invention comprises the determination of at least a second marker.
  • the second marker is selected from the group of cellular fibronectin, ferritin and MMP9 where the second marker is selected from the group of cellular fibronectin, SSAO / VAP-1, ferritin, MMP9, laminin, PAI- 1, TAFI and S100B and where plasma cell fibronectin levels, ferritin, SSAO / VAP-1, MMP9, TAFI or SI 00b lower or similar and / or low levels of PAI-1 and / or laminin, with respect to a reference value are indicative that the patient will be selected for treatment with an antithrombotic agent
  • the correlation between fluorescence in a sample of a patient and probability of suffering a hemorrhagic transformation in response to treatment with an antithrombotic agent as a result of a stroke can also be applied to the development of personalized therapies in patients who have suffered a stroke, so that patients with fluorescence levels similar or lower than the intensity of a reference sample are candidates for treatment with a therapy based on an antithrombotic agent.
  • the invention relates to a method for designing a personalized therapy in a patient who has suffered a stroke which comprises determining in a sample of said patient the fluorescence intensity in the wavelength range of 400 -500 nm in response to the excitation of said sample in a wavelength range of 280-400 nm where the level of Fluorescence in the patient sample is similar or lower than the level of fluorescence in a reference sample, a therapy based on an antithrombotic agent is selected.
  • personalized therapy refers to the adequacy of pharmaceutical compositions and medicine for a particular individual based on and taking into consideration the knowledge of the phenotype and genotype of the individual.
  • the determination of the most suitable therapy for a patient who has suffered from stroke can be carried out by simultaneously determining the fluorescence intensity in a sample of the patient and the level of expression of one or more additional markers whose involvement as biomarkers of risk of hemorrhagic transformation is known.
  • said one or more additional markers are selected from the group of cellular fibronectin, SSAO / VAP-1, ferritin, MMP9, laminin, PAI-1, TAFI and S100B and wherein levels of plasma cellular fibronectin, SSAO / VAP -1, of ferritin, MMP9, TAFI or SI 00b lower or similar and / or higher levels of PAI-o of laminin with respect to a reference value are indicative that the compound is considered to be effective for appearance of bleeding disorder.
  • Preferred combinations of biomarkers in the context of the method of the present invention include, without limitation, fluorescence intensity in combination with SSAO / VAP-1 levels, fluorescence intensity in combination with cellular fibronectin levels and fluorescence intensity in combination. with the levels of SSAO / VAP-1 and with the levels of cellular fibronectin.
  • the invention relates to a method for the identification of compounds useful for preventing the occurrence of a bleeding disorder in response to treatment with an antithrombotic agent in a subject who has suffered a stroke which comprises comparing
  • the terms "hemorrhagic disorder", “antithrombotic agent”, “stroke”, “fluorescent intensity”, “sample”, as well as the methods for measuring fluorescence intensity have been previously defined in relation to the first and second method of the invention.
  • the fluorescence intensity in the patient sample is considered to be decreased with respect to the fluorescence intensity of said subject before administration of the candidate agent when levels in the patient sample after administration. are decreased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%), at least 35%, at least 40% >, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or, at least 80% : at least 85%, at least 90%, at least 95%, at least 100%.
  • said one or more additional markers are selected from the group of cellular fibronectin, SSAO / VAP-1, ferritin, MMP9, laminin, PAI-1, TAFI and S 100B and wherein plasma cellular fibronectin levels of SSAO / VAP-1, ferritin, MMP9, TAFI or SI 00b lower or similar and / or higher levels of PAI-o laminin with respect to a reference value are indicative that the compound is considered to be effective for the appearance of bleeding disorder.
  • Preferred combinations of biomarkers in the context of the method of the present invention include, without limitation, fluorescence intensity in combination with SSAO / VAP-1 levels, fluorescence intensity in combination with cellular fibronectin levels and fluorescence intensity in combination. with the levels of SSAO / VAP-1 and with the levels of cellular fibronectin.
  • the invention is described below by means of the following examples that are merely illustrative and in no way limiting the scope of the invention.
  • TH Hemorrhagic infarction
  • HI Hemorrhagic infarction
  • PH parenchymal hematoma
  • HICS intracranial hemorrhage
  • EDTA tubes were used to collect blood and to measure fluorescence. Plasma was immediately separated by centrifugation at 3000 rpm for 15 minutes and stored in aliquots at -80 ° C until analysis.
  • Fluorescence determination was based on spectrofluorimetric detection according to Shimasaki (Methods Enzymol; 1994, 233: 338-46).
  • 200 ⁇ of plasma samples were mixed vigorously with 600 ⁇ of ethanol: ether (3: 1, v / v) in Pyrex borosilicate glass tubes (99445-12, Corning, New York) followed by centrifugation 10 min. at 1000 g.
  • a 0.5 ml aliquot of the supernatant was pipetted into a quartz cuvette for measurement of fluorescence at 430 nm emission and 360 nm excitation on a Hitachi F-2500 spectrofluorimeter and expressed as ng per mi (ng / ml ).
  • Duplicate fluorescence levels were measured and the coefficient of variation was less than 5% in all cases.
  • Quinine sulfate in dilute SO 4 H 2 0.1 N was used for the standard calibration curve and to calculate the relative fluorescence intensities of the samples.
  • Samples were retested with plasma fluorescence levels> 100 ng / ml after a previous dilution of 5 or 10 times with distilled water. The inter-test mean variation coefficients were ⁇ 10%.
  • Statistic analysis Frequency and descriptive statistical analyzes were obtained and comparisons were made using the SPSS statistical package, version 15.0. Statistical significance for the differences between the groups was assessed by Fisher's exact test and Pearson's ⁇ 2 (chi-square) for categorical variables and the Mann-Witney and Kruskal-Wallis test for continuous variables. Fluorescence values were not normally distributed. To evaluate the variations in the temporal fluorescence profile, a test for repeated measurements was performed (Friedman test). The baseline fluorescence level (pre-tPA) was used for any other analysis. When indicated, the Mann-Whitney and Pearson U tests were used.
  • ROC receiver-operator curve
  • the initial DTC detected a proximal ACM occlusion in 72.0% and a distal occlusion in 28.0%> of the patients (Table 1).
  • Atrial fibrillation 75 (40.1) 52.2 44.67 0.106
  • ADT diastolic blood pressure
  • ROC curves used to better identify the cut-off points for the association of fluorescence with HICS are shown in Figure 5.
  • a fluorescence cut-off point of 79.4 had a sensitivity of 66.7%> and a specificity of 87.2%> to detect the presence of HICS with a positive predictive value of 14.8%) and a negative predictive value of 98.7%>. These two cut points are they represent in Figure 6 and the percentage of patients with and without HICS using those cut-off points is shown in Figure 7 and 8.
  • HICS symptomatic
  • VAP-1 / SSAO activity was determined radiochemically at 37 ° C as previously described (Fowler, CJ and Tipton, KF, 1981, Biochem. Pharmacol. 30: 3329-3332); using as substrate [ 14 C] -benzylamine (3 mCi / mmol, Amersham, RU) 100 ⁇ .
  • Cellular fibronectin was determined by ELISA techniques, using a commercial kit (quantitative sandwich ELISA kits from Biohit Pie Finland), and working in duplicate, with coefficients of variation ⁇ 10%>. Results

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Abstract

L'invention concerne des méthodes pour le diagnostic d'ictus et du risque d'être atteint d'un trouble hémorragique après un ictus en réponse au traitement à l'aide d'un agent antithrombotique. Lesdites méthodes reposent sur les différentes propriétés fluorescentes que présentent les échantillons provenant de patients qui ont été atteints d'un ictus ou qui présentent ledit risque. L'invention concerne également des méthodes de thérapie personnalisée pour lesdits patients.
PCT/ES2010/070796 2009-12-03 2010-12-02 Méthodes pour déterminer la propension à être atteint d'une transformation hémorragique après un ictus WO2011067451A2 (fr)

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WO2004103313A2 (fr) * 2003-05-20 2004-12-02 The American National Red Cross Methodes de reduction de la permeabilite vasculaire d'un tissu par inhibition de l'activateur tissulaire du plasminogene (tpa) et inhibiteurs tpa utilises dans ces methodes
EP1733683A1 (fr) * 2004-04-06 2006-12-20 National University Corporation Okayama University Appareil de contrôle de l'ischémie cérébrale
WO2006036220A2 (fr) * 2004-09-22 2006-04-06 Prediction Sciences Llc Fibronectine cellulaire en tant que marqueur diagnostique dans l'accident vasculaire cerebral et ses procedes d'utilisation

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