WO2011067451A2 - Methods for determining the propensity to suffer haemorrhagic transformation following a stroke - Google Patents

Abstract

The invention relates to methods for diagnosing a stroke and the risk of suffering a haemorrhagic disorder after suffering a stroke in response to treatment with an antithrombotic agent. Said methods are based on the various fluorescent properties of samples taken from patients having suffered a stroke or displaying a risk of having a stroke. The invention also provides personalised therapy methods for said patients.

Description

 ME T O D O S FOR D E T E RMI NAR P RO P E N S I O N A S UF RI R HEMORRAGICAL TRANSFORMATION AFTER AN ICTUS

TECHNICAL FIELD OF THE INVENTION

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. BACKGROUND OF THE INVENTION

 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.

To date, the only treatment for ischemic stroke during the arterial occlusion phase is the use of thrombolytic agents to recover cerebral perfusion. In particular, intravenous administration of tissue plasminogen activator (t-PA) is the treatment of choice for patients who have suffered ischemic stroke.

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.

Therefore, it is necessary to identify those patients in which there is a greater risk of hemorrhagic transformation in order to be able to select those that may benefit most from the treatment with t-PA. Surnii T. et al (Stroke, 2002, 33: 831-836) and Montaner et al. (Circulation, 2003, 107: 598-603) have described that patients who have a high risk of hemorrhagic transformation show elevated levels of matrix 9 metalloprotease (MMP-9) so that the determination of MMP- levels 9 can be used to predict the probability of hemorrhagic complications after thrombolytic stroke treatment.

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.

On the other hand, it has been shown that patients who suffered a hemorrhagic transformation had lower levels of Plasminogen Activator Inhibitor 1 (PAI-1) and higher levels of Thrombin Activable Fibrinolysis Inhibitor (TAFI), and that the combination of PAI-1 levels <21.4 ng / mL and TAFI> 180% had the best sensitivity and specificity to predict the occurrence of these hemorrhagic complications (Ribó et al, 2004, Stroke 35: 2123-2127).

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.

 However, the use of MMP-9, cellular fibronectin and / 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.

Additionally, it has been described that 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). However, 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.

Therefore, there is a need in the art for additional 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.

SUMMARY OF THE INVENTION In a first aspect, 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.

 In a second aspect, the invention 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.

In another aspect, 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. In another aspect, 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

to. the fluorescence intensity in a sample of said subject in the wavelength range of 400-500 nm in response to the excitation in of said sample in a wavelength range of 280-400 nm and b. the fluorescence intensity in a reference sample in the wavelength range of 400-500 nm in response to the excitation of said sample in a wavelength range of 280-400 nm, wherein the compound is considered to be It is effective in preventing the onset of bleeding disorder when said compound causes a decrease in the level of fluorescence compared to the level of said subject before administration of the candidate agent.

BRIEF DESCRIPTION OF THE FIGURES

The following figures form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention can be better understood by reference to one or more of these figures in combination with the detailed description of specific embodiments presented herein.

Figure 1. Normal distribution of fluorescence in plasma samples in the study population (n = 192 patients with stroke), shown by a histogram. 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.

Figure 4. Baseline fluorescence levels in relation to the subsequent occurrence of symptomatic TH (p <0.001).

Figure 5. ROC curves to identify fluorescence limits with the best sensitivity and specificity to predict the occurrence of symptomatic TH.

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 9. Baseline level of fluorescence with respect to the neurological outcome in the acute phase, according to the modification of the NIHSS scale, which allowed differentiating patients who improved, remained stable or worsened.

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 11. The combination of low plasma levels of fluorescence and SSAO / VAP-1 activity, measured at the arrival of the patient in the emergency room, which are below the indicated cut-off points, allows to select with great precision those patients who they will not present symptomatic bleeding complications 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.

DETAILED DESCRIPTION OF THE INVENTION

Method to diagnose a stroke in a patient

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. Thus, as seen in the example accompanying the present invention, 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).

Thus, in a first aspect, 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.

The term "diagnose," as used in the present invention, refers to assessing the probability that a subject suffers from a disease, in particular, a stroke. As those skilled in the art will understand, such evaluation, although preferred, may not be correct for 100% of the subjects to be diagnosed. The term, however, 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.

The term "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 anemia or hyperviscosity.

The term "fluorescence", as used in the present invention, 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. In a preferred embodiment, 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. In a preferred embodiment, 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. 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. In a particular embodiment, 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. Hereinafter, the term 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.

The term "sample" as used in the present invention 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. In a particular embodiment, said sample is a sample of whole blood, serum, plasma, saliva or cells extracted from peripheral blood and tissue. In one embodiment, the sample is plasma.

Alternatively, the invention contemplates the determination of fluorescence in an extract of a certain tissue. By "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. The term "aqueous solvent", as used in the present invention, refers to a solution in which the solvent is water and contains an aqueous salt or a buffer solution.

The term "organic solvent", as used herein, 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. In a particular embodiment, the sample is extracted using ethaneheter (3: 1, v / v). In an even more preferred embodiment, the determinations are carried out from a plasma extract in ethanol: ether (3: 1, v / v).

Finally, 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. In accordance with the present invention, 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. Thus, by combining the fluorescence intensity values in a patient sample and one or more additional markers it is possible to make a more accurate diagnosis of the presence of stroke in a patient. Thus, in a preferred embodiment, the determination of the fluorescence intensity is carried out simultaneously with the determination of at least a second marker. In preferred embodiments, 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. By way of non-limiting example, 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. At the same time, the antibodies may be labeled or not. Illustrative, but not exclusive, examples of markers that can be used include radioactive isotopes, enzymes, fluorophores, chemiluminescent reagents, enzymatic substrates or cofactors, enzyme inhibitors, particles, dyes, etc. There is 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-blot or immunoblotting, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), EIA (enzyme immunoassay) competitive, DAS-ELISA (sandwich ELISA with double antibody), chemical immunochemical and immunohistochemical techniques, techniques based on the use of biochips or protein microarrays including specific antibodies or tests based on 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.

In a preferred embodiment, 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.

In another preferred embodiment, 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.

In another preferred embodiment, 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.

Additionally, 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.

Method to determine the probability of a patient who has suffered a stroke, of suffering a bleeding disorder in response to an antithrombotic agent The authors of the present invention have observed that fluorescence at 430 nm in response to excitation at 360 nm in a sample of the patient who has suffered a stroke and who has been treated with an antithrombotic agent allows to determine the probability that said patient undergo a hemorrhagic transformation. Thus, as observed in the examples of the present invention, the fluorescence intensity was increased among the patients who later developed a more severe HT and especially among those with symptomatic hemorrhagic transformation (HICS) (see Figures 3 and 4). This finding allows the use of fluorescence intensity for the identification of patients who have suffered HICS with high sensitivity and high specificity. Thus, as seen in Figure 6 to 8 of the present invention, a cut-off point of fluorescence intensity of 43.095 had a sensitivity of 100% and a specificity of 43.9% to detect the presence of HICS with a predictive value. positive = 5.6% and a negative predictive value = 100%. Another cut-off point of fluorescence intensity 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 = 14.8%) and a negative predictive value = 98.7%.

Without wishing to be bound by any theory, it is thought that the greater fluorescence observed in patients who are more likely to suffer hemorrhagic transformation after a stroke is due to the greater oxidative capacity that the samples of said patient present, which results in the first instance to the formation of different lipid peroxidation products that react in second instance with other biomolecules (proteins, carbohydrates, etc.) to give rise to molecules whose structure is unknown but which all have the ability to emit fluorescence at 430 nm when excited with a radiation of 360 nm.

Thus, in another aspect, 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. By "method to determine the probability", as used herein, it refers to methods to determine the probability that a patient suffers from a bleeding disorder. The person skilled in the art will appreciate that the prediction may not be correct for 100% of the patients under study. However, 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. Thus, "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 term "stroke" has been described in the first method of the invention. In a particular embodiment, the stroke is an ischemic stroke.

By "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. Thus, 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.

 In a preferred embodiment, 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.

In another preferred embodiment, 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. In the event that 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. or by a collagenopathy (systemic lupus erythematosus (SLE), polyarteritis nodosa (PAN), etc.), by 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. Alternatively, embolic thrombus diseases that can cause hemorrhagic transformation include deep vein thrombosis, pulmonary thromboembolism or acute myocardial infarction. The treatment of acute myocardial infarction involves the use of fibrinolytic agents thanks to their ability to achieve reperfusion and regenerate blood flow when administered within 12 hours after the onset of the infarction. Suitable fibrinolytic agents for the treatment of acute myocardial infarction include streptokinase, alteplase, reteplase, anistreplaseplase, anistreplase and the like.

By "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.

In the event that the bleeding disorder is a consequence of treatment with a thrombolytic agent after an episode of stroke, 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)

Also included are 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.

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, streptoptose, streptoptose, streptoptimose , tenecteplase, reteplase, microplasmin, ancrod, prourokinase).

Preferably, 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.

In a preferred embodiment, the antithrombotic agent is the tissue plasminogen activator (tPA). As one skilled in the art understands, the determination of fluorescence intensity can be performed before or after administration of the antithrombotic agent.

The terms "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. Thus, by combining the values of fluorescence intensity and one or more additional markers it is possible to make a more accurate prediction of the risk of a patient suffering from a bleeding disorder. Thus, in a preferred embodiment, the Fluorescence intensity determination is carried out simultaneously with the determination of at least a second marker. In preferred embodiments, 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. By way of non-limiting example, 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. At the same time, the antibodies may be labeled or not. Illustrative, but not exclusive, examples of markers that can be used include radioactive isotopes, enzymes, fluorophores, chemiluminescent reagents, enzymatic substrates or cofactors, enzyme inhibitors, particles, dyes, etc. 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.

In a particular embodiment, 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 .

 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 The authors of the present invention have shown that the determination of fluorescence intensity in samples of patients who have suffered a stroke is useful not only to determine the predisposition of such patients to undergo hemorrhagic transformation in response to a treatment with a thrombolytic agent, but also for the determination of the neurological evolution of the patient. Thus, Figure 9 accompanying the present invention shows how an increased fluorescence intensity determined after the stroke is associated with a greater degree of neuro logical worsening.

Therefore, in another aspect, 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.

The term "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). Specifically, 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). Alternatively, 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.

The terms "stroke", "antithrombotic agent", "fluorescence intensity", "sample" and "higher fluorescent intensity" have 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.

Method to treat a stroke patient

In another aspect, the invention 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 methods for measuring the intensity of fluorescence, as well as the terms "stroke", "sample", "reference sample" and "antithrombotic agent" have been described above and also apply to the method of treatment of the invention.

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. Thus, 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. In accordance with the present invention, 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 expression "similar to" or "equal to" the reference value refers to an expression level that is about 5%, 4%, 3%, 2%, 1% of an expression level of reference or control. In the same way as with the second method of the invention, 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. Thus, in a preferred embodiment, the method object of the second method of the invention comprises the determination of at least a second marker. In an even more preferred embodiment, 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

Method for designing personalized therapies

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. Thus, in another aspect, 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.

The term "personalized therapy", as used in the present invention, 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 expression "lower", when referring to the intensity of fluorescence with respect to the reference sample, indicates that the intensity determined in a sample of the patient is decreased 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%>, at least 45%, at least one 50%, at least 55% or, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%: at least 85%, 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 plus.

 The expression "similar", when referring to the fluorescence intensity with respect to the reference sample, indicates that the intensity determined in a patient sample is 80% to 120%) of the intensity determined in the fluorescence sample , more preferably between 85% and 1 15%, between 90% and 10%, between 95% and 105% and, even more preferably, 100%.

The terms "stroke", "fluorescence intensity", "sample", "reference sample and" antithrombotic agent "have been described in detail previously in other methods of the invention and are equally applied to the present method.

In the same way as in the other methods of the invention, 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. Preferably, 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.

Method for the identification of useful compounds to prevent the appearance of hemorrhagic disorders in patients who have suffered stroke

In another aspect, 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

to. the fluorescence intensity in a sample of said subject in the wavelength range of 400-500 nm in response to the excitation in of said sample in a wavelength range of 280-400 nm and b. the fluorescence intensity in a reference sample in the wavelength range of 400-500 nm in response to the excitation of said sample in a wavelength range of 280-400 nm, wherein the compound is considered to be It is effective for the appearance of hemorrhagic disorder when said compound causes a decrease in the level of fluorescence compared to the level of said subject before administration of the candidate agent. 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.

In accordance with the present 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%.

In the same way as in the other methods of the invention, it is possible to identify useful compounds to prevent the appearance of a bleeding disorder based on the intensity of fluorescence and one or more additional markers. Preferably, 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. EXAMPLES

EXAMPLE 1

Methods

Study population

 Patients with acute stroke admitted to the emergency department of a University Hospital were prospectively studied. The study group consisted of patients who had had an acute ischemic stroke admitted within the first 3 hours after the onset of symptoms. A total of 192 consecutive patients with a non-lacunar stroke affecting the vascular area of the middle cerebral artery (ACM) or the basilar artery were evaluated. All patients underwent transcranial Doppler (DTC) and urgent carotid ultrasound examinations. Patients received t-PA at a conventional dose of 0.9 mg / kg (10% in bolus, 90% in continuous infusion for 1 hour). Patients with cancer or a known inflammatory disease were excluded.

In addition, a group of healthy controls (n = 30) was evaluated to study normal fluorescence values at 430 nm among our population.

Clinical protocol

A detailed history of vascular risk factors was obtained for each patient. To identify the possible mechanism of cerebral infarction, a set of diagnostic tests were performed that included electrocardiogram, thoracic radiography, carotid ultrasonography, complete blood count and leukocyte formula and biochemical analysis of blood in all patients; when indicated, some patients also received special coagulation tests, transthoracic echocardiography and monitoring Holter With this information and with neuroimaging data, the previously defined etiological subgroups were determined (Adams HP et al. Stroke. 1993; 24: 35-41). The clinical examination was carried out at admission and at 12, 24 and 48 hours after the onset of symptoms. The severity of the stroke was assessed by using the Stroke Scale of the National Institutes of Health (NIHSS). We define neurological improvement as a decrease in the stroke scale score at 4 or more points and neurological impairment either as death or as an increase in the score by 4 or more points at 48 hours (Brott TG et al. Stroke. 1992; 23: 632-640).

No intravenous heparin was administered during the study period. This study was approved by the Hospital Ethics Committee and all patients or family members gave their written informed consent to participate in it. I take computerized spelling (CT)

 Upon arrival at the emergency room, all patients received a CT scan within the first 3 hours of the onset of the stroke, which was repeated after 24-48 hours (or earlier, in the event of rapid neurological deterioration) to evaluate the presence of TH.

CT scans were reviewed by a neuroradiologist with extensive experience in neurovascular pathology who was blind to clinical details and the results of fluorescence determination. The presence and type of TH were defined according to the criteria previously published by Hacke W et al. {JAMA. 1995; 274: 1017-1025) and Pessin M. et al. {Clin Neuropharmacol. 1990; 13: 271-289). Hemorrhagic infarction (HI) was defined as a heart attack with petechiae with no space-occupying effect and parenchymal hematoma (PH) was defined as hemorrhage with mass effect. For the statistical analysis, both subtypes of IH and HP (IH-1, small petechiae along the margins of the infarction were considered together. IH-2, more confluent petechiae within the infarcted area. HP-1, when the hematoma affects ≤ 30% of the infarcted area with some slight effect of space occupation and HP-2, when the hematoma affects> 30% of the infarcted area with the effect of substantial mass, or HP-R when the bruise was removed and affected a different area from the infarcted area). Symptomatic intracranial hemorrhage (HICS) was defined as bleeding at any site in the brain in the CT scan and documentation of neurological impairment probably related to such bleeding. No patient with a hypodensity that affected> 33% of the MCA area received t-PA in this study.

Determination of fluorescence

 Peripheral blood samples were taken from all patients (n = 192) upon entry into the study (before administration of t-PA). In a subset of patients (n = 101), serial samples were obtained to study the temporal fluorescence profile at the beginning, 1 and 2 hours after t-PA, and 12 and 24 h after the onset of symptoms.

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). In summary, 200 μΐ of plasma samples were mixed vigorously with 600 μΐ 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 χ 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. To calculate the sensitivity and specificity for fluorescence values to predict (symptomatic intracerebral hemorrhage) HICS, a characteristic receiver-operator curve (ROC) was configured. A logistic regression analysis was performed to determine factors that could be considered independent predictors of HICS. A value of p <0.05 was considered statistically significant.

Results

 192 patients (45.8% were women) with an acute ischemic stroke were included in the study (Table 1). Most of them included the area of the MCA (n = 176) and the rest were occlusions of the basilar artery (n = 13) or occlusions of the posterior cerebral artery (n = 2). The mean age was 71, 46 ± 11.8 years (interquartile range 65 to 79 years). A total of 56.1% of the patients were hypertensive, 32.3% or were dyslipidemic and 18.6% had a history of diabetes mellitus. The NIHSS score at admission was 17 (from 12 to 20) (Table 1). The initial DTC detected a proximal ACM occlusion in 72.0% and a distal occlusion in 28.0%> of the patients (Table 1). Regarding the etiology of stroke, we identified 49.5% of patients who were cardioembolic, 25.8% of them being atherothrombotic.

Stroke patients had a mean baseline fluorescence level of 45.69 (36.65 - 60.84) that was different from the fluorescence level among healthy controls, 34.56 (30.3 - 45.55) (p <0.001, figure 2). The temporal profile of fluorescence after the stroke is shown in Figure 2. The highest level of fluorescence was found at baseline: Start (before administration of t-PA) = 45.69 (36.65 - 60.84 ), at one hour = 41.41 (33.4 - 56.65), at two hours = 39.4 (30.99 - 53.58), at twelve hours = 31.53 (26.66 - 37.15), at twenty four hours = 29.07 ( 24.82 - 37.35), p value <0.001.

With respect to demographic factors related to the level of fluorescence, we only identified differences for fluorescence with respect to age (Table 1). In fact, age was correlated with fluorescence at several time points in the study [start (p = 0.02), first hour (p = 0.07) and two hours (p = 0.008)], with higher levels of fluorescence among older patients. In addition, baseline fluorescence levels tended to be higher among patients with a previous stroke (p = 0.092). Taking into account other cardiovascular risk factors, none of them were related to the baseline level of fluorescence; Except the blood pressure figures of the patient upon arrival at the emergency room.

Frequency Basal level of p

 (%) fluorescence

 according to clinical variables

 If not

 Qualitative data

 Women 88 (45.8%) 49.65 44,095 0.122

Hypertension 105 (56.1%) 5 44.17 1731

Dyslipidemia 60 (32.3%) 47.9 46.62 0.957

Diabetes mellitus 35 (18.6%) 45.79 44.82 0.136

Previous stroke 27 (14.4%) 49.7 44,925 0.092

Atrial fibrillation 75 (40.1) 52.2 44.67 0.106

Smoking 35 (19.7%) 47.7 44.91 0.328

Proximal occlusion 134 (72%) 49.61 46.92 0.84

 44.92

5 Quantitative data

 Age 71.46 ± 11.84 R = 0.17 0.02

Start NIHSS score 17 R = 0.06 0.425

TAS (systolic blood pressure)

 mmHg 154 ± 26 R = 0.02 0.017

ADT (diastolic blood pressure),

 mmHg 83 ± 15 R = 0.03 0.033

Glucose, mg / dl 131.68 ± 43.72 R = -0.03 0.648

Platelets, 241305.01 ± 85669.41 R = 0.08 0.282

Leucitos xlOE9 / l 9946.01 ± 9530.9 R = 0.04 0.607

TTP (thromboplastin time

 partial) 0.94 ± 0.1 R = -0.04 0.66

Fibrinogen, g / 1 3.49 ± 0.72 R = -0.14 0.094

Table 1. Demographic factors and other variables of stroke at the arrival of the patient in the emergency room and their influence on the level of fluorescence.

When the hemorrhagic transformation (TH) was explored, it was observed that it was present in 56 patients (30.4%) and that by subtypes of TH they corresponded to: 19 (10.3%) IH-1, 13 (7 , 1%) IH-2, 14 (7.6%) HP-1, 8 (4.3%) HP-2 and 2 (1.1%) HP-R]. Among those hemorrhagic complications, our target, HICS were found in 6 cases (3, 12% or). Baseline fluorescence levels were increased among patients who later developed a more serious HT and especially among those with HICS as shown in Figures 3 and 4.

The 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 43.095 had a sensitivity of 100% and a specificity of 43.9%> to detect the presence of HICS with a positive predictive value = 5.6%> and a negative predictive value = 100%.

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.

Table 2 shows the main baseline characteristics of patients with and without HICS. Following the results of this univariate study, logistic regression showed that the level of fluorescence (> 79.4) was the main baseline predictor of the appearance of HICS [OR 13.65 (2.36-78.79), p = 0.003]; also the Systolic Arterial Tension (TAS) [OR 1,034 (1,002 - 1, 067), p = 0.04] and the previous stroke history [OR 6,696 (1,274 - 35,18), p = 0,025] were independent predictors of HICS

No HICS HICS

 n = 180 n = 6 P

 Women 81 (45) 5 (83.3) 0.097

 Age 71.4 76.3 0.316

 Hypertension * 95 (53.7) 6 (100) 0.034

 Dyslipidemia 58 (32.8) 2 (33.3) 0.977

 Diabetes Mellitus 34 (19.1) 1 (16.7) 0.881

 Stroke Previous * 23 (13) 3 (50) 0.038

 NIHSS Score 16 18 0.333

 Proximal occlusion 127 (72.2) 4 (66.7) 0.673

 TAS, mmHg * 1543.6 176.3 0.036

 TAD, mmHg 82.9 93.5 0.084

 Glucose, mg / dL 130.4 150.2 0.318

 Platelets, 237946 295000 0.139

 Leukocytes, x 10E9 / L 9906 9600 0.884

 TTPa 0.93 1.02 0.153

 Fibrinogen, g / L 3.47 3.46 0.986

 fluorescence> 43 * 56.1% 100% 0.039

 fluorescence> 79.4 * 12.8% 66.7% 0.004

 Table 2. Main characteristics of the onset of patients in

 according to the presence or absence of hemorrhagic transformation

 symptomatic (HICS). Values are means or n (%>), depending on

 applicable. * Factors with a value of p <0.05.

Regarding the neuro logical state, the clinical evaluation revealed that 22 (11.7%) patients worsened, 112 (59.6%>) improved and 54 (28.7%) remained stable during the first 48 hours after admission. A trend was observed at a higher baseline fluorescence level among patients with neurological worsening (p = 0.135, as shown in Figure 9). The NIHSS score evaluated at twenty-four hours was correlated with baseline fluorescence levels (p = 0.026), one hour fluorescence (p = 0.04), two hour fluorescence (p = 0.003), twelve hour fluorescence ( p = 0.037) and fluorescence at twenty-four hours (p = 0.055). Similarly, the NIHSS score evaluated at 48 hours was correlated with the start fluorescence (p = 0.027), the fluorescence at one hour (p = 0.043), the fluorescence at two hours (p = 0.005), the fluorescence at twelve hours (p = 0.014) and fluorescence at 24 hours (p = 0.03).

EXAMPLE 2

The combination of fluorescence levels with other biomarkers such as SSAO / VAP-1 and / or cellular fibronectin improves the predictive value of all of them taken individually as a marker of hemorrhagic transformation risk.

Methods

In a subgroup of patients from the previous study (example 1), in addition to fluorescence, the activity levels of SSAO / VAP-1 and the levels of cellular fibronectin were determined. All of them measured at baseline, before the patient receives thrombolytic treatment. 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 μΜ. Samples were pre-incubated for 30 minutes at 37 ° C with 1 μΜ L-deprenyl to inhibit the possible contamination of platelet MAO B. The reaction was carried out at 37 ° C in a final volume of 225 μΐ of 50 mM Tris-HCl buffer, pH 9, and stopped by the addition of 100 μΐ of 2 M citric acid. The radiolabeled products were extracted in toluene. / ethyl acetate (1: 1, volume / volume) containing 2.5- 0.6% diphenyloxazole (PPO) (weight / volume) before scintillation fluid. The protein concentration was determined by the Bradford method, and the resulting specific VAP-1 / SSAO activity is expressed as pmol / min-mg of protein.

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

 The combination of high levels of fluorescence and SSAO / VAP-1, measured at the arrival of the patient in the emergency room, which are above the indicated cut-off points (fluorescence = 96.02 and SSAO / VAP-1 = 3, 87), allows to select with great precision those patients who will present symptomatic hemorrhagic complications after treatment with t-PA. Figure 10 shows that these cut-off points have 66.7% sensitivity, 99% specificity, 66.7% positive predictive value and 99% negative predictive value. The use of the test would help not to treat those with a positive test and therefore would reduce the rates of HICS to <1%.

The combination of low levels of fluorescence and SSAO / VAP-1, measured at the arrival of the patient in the emergency room, which are below the indicated cut-off points (fluorescence = 48.2 and SSAO / VAP-1 = 2, 13), allows to select with great precision those patients who will not present symptomatic hemorrhagic complications after treatment with t-PA. Figure 11 shows that these cut-off points show 100%> sensitivity, 73.5% specificity, 10% positive predictive value and 100%) negative predictive value. That is, before a negative test we did not find patients with HICS and therefore we could safely extend the treatment to populations at risk of HT.

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 points of indicated cut-off (fluorescence = 74.85 and cellular fibronectin = 23), allows to select with great precision those patients who will present or not, symptomatic hemorrhagic complications after treatment with t-PA, as shown in Figure 12.

The combination of fluorescence levels, SSAO / VAP-1 and cellular fibronectin, measured at the arrival of the patient in the emergency room, which are above or below the indicated cut-off points (fluorescence = 74.85; SSAO / VAP -1 = 2.03 and cellular fibronectin = 23) allows to select even more accurately those patients who will present or not, symptomatic bleeding complications after treatment with t-PA, as shown in Figure 13, reaching a sensitivity of 100 % and a specificity of 96%>.

Claims

1. Method to diagnose 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 that has been treated with 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 range of lengths of 280-400 nm wave where a fluorescence intensity greater than the fluorescence intensity 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 evolution . 2. Method for treating a patient who has suffered a stroke that comprises administering an effective amount of an antithrombotic agent to said patient, wherein a sample of said patient has 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 or less than the fluorescence intensity in a reference sample. 3. Method for designing a personalized therapy in a patient who has suffered a stroke 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 shows in a range of wavelengths of 280-400 nm where if 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. 4. Method for the identification of useful compounds to prevent the occurrence of a bleeding disorder in response to treatment with an antithrombotic agent in a subject who has suffered a stroke that includes comparing to. the fluorescence intensity in a sample of said subject in the wavelength range of 400-500 nm in response to the excitation in of said sample in a wavelength range of 280-400 nm and b. the fluorescence intensity in a reference sample in the wavelength range of 400-500 nm in response to the excitation of said sample in a wavelength range of 280-400 nm, wherein the compound is considered to be It is effective in preventing the onset of hemorrhagic disorder when said compound causes a decrease in the level of fluorescence compared to the level of said subject before administration of the candidate agent. 5. Method according to any one of claims 1 to 4 wherein the antithrombotic agent is chosen from among antiplatelet drugs, fibrinolytic agents, oral anticoagulants, thrombolytic agent. 6. Method according to claim 5 wherein the antithrombotic agent is tPA. 7. Method according to any of the preceding claims wherein the fluorescence detection is carried out in the 420-440 nm range and / or the excitation is carried out in the 350-370 nm range. 8. Method according to any of the preceding claims wherein the sample is blood, serum, plasma, saliva or a tissue extract. 9. Method according to claim 8 wherein the extract of a tissue is an aqueous extract or an organic extract. 10. Method according to any of claims 1 to 9 wherein the level of fluorescence is determined by fluorescence spectrometry. 11. A method according to any one of claims 1 to 10 further comprising determining at least one second marker. 12. A method according to claim 11 wherein the second marker is selected from the group of VAP-1, cellular fibronectin, ferritin, MMP9, PAI-1, laminin, TAFI and S 100B and wherein high levels of VAP-1, of cellular fibronectin, ferritin, MMP9, TAFI and / or S 100B and / or low levels of PAI-1 and / or laminin, with respect to a reference value are indicative that the patient shows a high probability of suffering a bleeding disorder. 13. A method according to claim 12 wherein the levels of VAP-1 and cellular fibronectin are determined, wherein elevated levels of VAP-1 and fibronectin with respect to a reference value are indicative that the patient shows a high probability of suffering a bleeding disorder. 14. A method according to any of claims 1 to 13 wherein the bleeding disorder is a hemorrhagic transformation, preferably symptomatic. 15. A method according to claim 14 wherein the hemorrhagic transformation is a parenchymal hemorrhage. 16. Method according to any one of claims 1 to 15 wherein the stroke is an ischemic stroke. 17. Method according to any of the preceding claims, further comprising determining at least one clinical variable selected from the group of smoking status, initial hypodensity in CT, hypertension, history of diabetes, hyperglycemia, platelet disease, blood clotting disorder and age.