ES2913121B2 - METHOD TO DETERMINE THE EVOLUTION OF ACUTE BRAIN DAMAGE AND PHARMACEUTICAL COMPOSITION FOR ITS TREATMENT - Google Patents

Description

DESCRIPTION

METHOD TO DETERMINE THE EVOLUTION OF ACUTE BRAIN DAMAGE

AND PHARMACEUTICAL COMPOSITION FOR ITS TREATMENT

Object of the invention

The object of the present invention includes the use of combinations of SAA1 and S100P biomarkers to determine the long-term evolution of acute brain damage, as well as the use of a device to measure the expression product of said biomarkers.

Background of the invention

Acute brain damage is caused by very different causes, the most frequent being trauma that affects the nervous system, ischemic or hemorrhagic injury. Traumatic brain injury (TBI) is a type of acute brain damage and is one of the main causes of mortality and disability in people under 40 years of age in our surrounding countries. It entails a high economic, social and family cost due to severe and multiple disabilities, which are concentrated in surviving patients.

Globally, the incidence of traumatic brain injuries has not stopped growing. In Europe it is estimated that approximately one million patients a year will be hospitalized for this cause, and of these approximately 75,000 will die. Although the greatest disability has been related to those derived from traffic accidents in young people, the causes of TBI are very varied and its epidemiology has changed with the changes produced in advanced societies. Therefore, there is an urgent need for improved screening tools, both for early detection of TBI severity and for early knowledge of patients' prognosis.

Craniocerebral trauma is a complex nosological entity, difficult to classify given the different injury mechanisms, variety in severity, different response of the individual to the injury given the great variety of ages at which it occurs and the different injury pattern produced by the interaction. mechanismintensity-age of the subject receiving the injury. In recent decades, there has been great progress in the pathophysiology and molecular mechanisms of brain damage. However, these advances have not been reflected in an improvement in the final outcome of TBI seen in the many clinical trials carried out to date [1 and 2]. One of the possibilities why this happens may be due to inaccuracy in the classification of TBI due to the heterogeneity that this disease presents [1]. On the other hand, All the pathways and mechanisms by which damage occurs after trauma are not yet known exactly, both from the point of view of the acute injury, as well as the mechanisms that would condition a delayed or continued injury over time and whether those Mechanisms are similar in the different types of injuries that we face in patients who have suffered a TBI.

The initial injury will therefore produce a first wave of cell death and axonal dysfunction, determined by impact, deformation, cell membrane alteration, irreversible metabolic cell damage and/or excitotoxicity. Brain involvement due to primary brain injury develops extraordinarily quickly and it is unlikely that effective pharmacological treatments will be achieved for this first phase. The early application of treatments based on neuroprotection could have an application in improving the results in these patients, however, until now there is no pharmacological treatment.

acute cerebral

Systemic trauma also associates a situation of systemic dysfunction and the induction of a state of systemic inflammatory response that could increase the local inflammatory brain damage associated with head trauma. These alterations will be potentially modifiable main determinants of disability and poor functional evolution. Despite the frequency of secondary injury and its importance in the final prognosis of patients, it is frequently underdiagnosed. There are different means to diagnose the severity of the initial injury and the evolution of patients affected by acute brain damage, such as imaging tests, which are often very inaccurate, and the measurement of biomarkers.

After brain injury, biomarkers can be detected in the cerebrospinal fluid or bloodstream. Generally, peripheral blood collection is the most commonly used method, as it is a non-invasive and cost-effective practice. Changes in the expression profiles of certain proteins or genes can be determined in blood cells or serum. In the context of traumatic brain injury, biomarkers could be useful in diagnosing the severity of the injury, monitoring the response to treatment, or predicting the extent of the injury. The identification of markers of this type of injury can establish strategies to minimize the damage that has occurred, avoid its deleterious effect, as well as establish prognostic implications and subsequent management of the patient.

In recent years, several serum biomarkers have been evaluated as diagnostic and prognostic markers of traumatic brain injury (TBI). Among these, the The most studied are the S-1 proteins ÜÜp, GFAP, NSE and UCH-L1 [3, 4, 5, 6]. However, the present invention refers to a combination of biomarkers to determine the long-term evolution of acute brain injury or damage.

Furthermore, there is currently no approved pharmacological treatment for the treatment of patients with such lesions. The present invention also relates to a pharmaceutical composition for treating the sequelae caused by an acute brain injury and improving the long-term outcome of the individual. Experiments carried out on animal models with acute brain damage have shown that this compound improves the neurobehavioral abilities of these animals.

For all these reasons, the object of this invention is a method to determine the evolution of long-term acute brain damage in individuals who have suffered acute brain injuries, as well as the use of a device to detect the level of biomarkers and a pharmaceutical composition. for the treatment of acute brain damage.

Description of the invention

The following terms of the invention are defined below in detail:

“Brain injury or damage” refers to any involvement of the central nervous system that produces cellular or tissue destruction.

“Acute brain injury” or “acute brain damage”: damage to the Central Nervous System (CNS) that begins suddenly, presents with intense clinical manifestations and evolves relatively quickly and briefly. Acute brain injury is selected from: traumatic brain injury, ischemic stroke, spontaneous intracerebral hemorrhage, spontaneous subarachnoid hemorrhage, status epilepticus, and central nervous system infection.

“TBI” or “cranial trauma” refers to any acute brain injury or damage caused to the cranial, encephalic or meningeal components by a traumatic impact, causing neurological deficits of different severity depending on location and extent.

“Long term” refers to the presence of sequelae of at least 6 months after the moment in which the cause of the acute brain damage occurred.

“Progression” or “evolution” of the lesion are synonyms and are used interchangeably.

“Intracranial injury” refers to injury to the brain or its coverings.

“Severity” or “intensity” or “extent” of the injury are terms that are used interchangeably.

“Assay” refers to any method generally known in the art for detecting acute brain injury, such as an ELISA or any other standard method for detecting biomarkers.

The term "immunoassay", as used herein, refers to any analytical technique that is based on the conjugation reaction of an antibody with an antigen.

The term “antibody” refers to an immunoglobulin that specifically recognizes an epitope on a target determined by the binding characteristics of the immunoglobulin variable domains of the heavy and light chains (VHS and VLS), more specifically the regions that determine complementarity. (CDRs). Many forms of potential antibodies are known in the art, which may include, but are not limited to, a plurality of intact monoclonal antibodies or polyclonal mixtures comprising intact monoclonal antibodies, antibody fragments (e.g. Fab, Fab', and Fv fragments, linear antibodies (single chain antibodies and multispecific antibodies comprising antibody fragments), single chain variable fragments (scFvs), multispecific antibodies, chimeric antibodies, humanized antibodies and fusion proteins comprising the domains necessary for the recognition of a given epitope on a target.

“Sample” or “biological sample” refers to any fluid, tissue or cells of an organism, preferably blood, serum, plasma, CSF, saliva, tear fluid, urine or tissue, obtained by any method known to a person skilled in the art. , useful for performing a test or screening method to identify acute brain injuries. Preferably, the sample is a sample of blood plasma, tears, saliva, cerebrospinal fluid (CSF), or urine taken from an individual. The sample is treated according to generally known procedures to maintain or render feasible for biomarker analysis.

The term "biomarker" in the context of the present invention refers to a molecule present in a biological sample from a patient, the levels of which may be indicative of acute brain damage. Such molecules may include proteins or nucleic acids and derivatives thereof.

The term "expression product" refers to a biomarker and can be either a nucleic acid or a protein.

The term “SAA1” as used here refers to the serum amyloid protein A1 (UniProt number P0DJI8), or the gene encoding such protein (GenBank accession number: 6288).

The term “S100P” as used herein refers to the calcium-binding protein B S100 (UniProt number P04271), or the gene encoding such protein (GenBank accession number: 6285).

The terms “oligonucleotide” and “nucleic acid” are used interchangeably here, referring to polymeric forms of nucleotides of any length, both ribonucleotides (RNA) and deoxyribonucleotides (DNA).

The term "probe" refers to a molecule that is capable of specifically binding to a target molecule such that the target molecule can be detected as a consequence of said specific binding. Probes that can be used in the present invention include, for example, antibodies, aptamers, phages and oligonucleotides.

The “level” of a biomarker refers to the amount, expression level, or concentration of the biomarker within the sample. This level may be a relative level compared to another biomarker or a previous sample.

The term "protein" refers to a polymeric form of amino acids of any length, which may be coding or non-coding, chemically or biochemically modified.

The term "amount", as used in the description, refers to, but is not limited to, the absolute or relative amount of the proteins, as well as any other value or parameter related thereto or that may be derived from them. these. Said values or parameters include signal intensity values obtained from any of the physical or chemical properties of said proteins by direct or indirect measurement.

The term "comparison", as used in the description, refers to, but is not limited to, the comparison of the amount of the protein expression products or the biological sample to be analyzed, also called the biological sample. , with a desirable amount of proteins from one or more reference samples. The Reference sample can be analyzed, for example, simultaneously or consecutively, together with the biological test sample.

The reference value may be a range of values considered by the person skilled in the art as a normal level for the biomarker in a healthy individual. Those skilled in the art will appreciate that control values for a biomarker can be calculated by the user by analyzing the level of the biomarker in a sample from a healthy individual or by reference to typical values provided by the manufacturer of the assay used to determine the level of the biomarker. biomarker in the sample.

The term "individual", as used in the description, refers to animals, preferably mammals, and more preferably, humans. The term "individual" is not intended to be limiting in any aspect, and may be of any age, sex and physical condition.

The term "marker compound", as used in the present description, refers to a compound capable of giving rise to a chromogenic, fluorogenic, radioactive or chemiluminescent signal that allows the detection and quantification of the amount of antibodies against SAA1. and S100p.

The word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will emerge partly from the description and partly from the practice of the invention.

The terms “prognosis” and “outcome” as well as their grammatical variations are synonymous herein and can be used interchangeably.

Any other term used herein will have the usual meaning of the field of art to which the present invention relates.

The present invention relates to a method for determining the evolution of long-term acute brain damage in individuals, comprising:

to. determine the expression product of the biomarkers SAA1 and S100P in a biological sample taken from an individual and,

b. establish the statistical significance of the concentration of the expression product of the biomarkers of stage a,

where the SAA1 expression product is obtained from a biological sample taken between 24 hours and 1 week after the injury that produces acute brain damage.

Another additional embodiment of the method of the invention comprises, in step a) determining the expression product of the SAA1 and S100P biomarkers in a biological sample from the individual.

The expression product of a biomarker may be a nucleic acid and/or a protein. Nucleic acid, protein and combinations of both can be quantified.

Step b) of the method of the invention can be fully or partially automated without depending on the judgment of a specialist, such as a doctor or a laboratory technician.

The statistical significance that implies the determination of the evolution of long-term acute brain damage is obtained based on the result of the concentration of the expression product of each biomarker. To obtain a highly accurate determination, it is preferred that the determination is carried out after comparing the detected value obtained with the value detected using a reference concentration. The reference concentration can be obtained from a reference biological sample, obtained from a healthy individual, or from an individual who does not suffer an acute brain injury.

Reference concentrations can be determined by the method of the present invention from a reference biological sample that can be analyzed, for example, simultaneously or consecutively, together with the biological sample of the problem individual.

The method of the invention may also comprise carrying out a statistical methodology with each of the values of the expression product of each biomarker of step a) of the method and producing an output value with statistical significance that indicates whether the evolution of the damage individual's brain is favorable or unfavorable in the long term.

Statistical significance is obtained by comparing the level of the expression product of the individual's biological sample with the reference biological sample using a statistical methodology.

The statistical methodology may be a statistical methodology selected from a D'Agostino-Pearson test and a group classifier algorithm selected from a decision tree classifier, logistic regression classifier, classifier nearest neighbor, neural network classifier!, Gaussian mixture model (GMM), Support Vector Machine (SVM) classifier, nearest centroid classifier, linear regression classifier, linear discriminant analysis (LOA) classifier, classifier quadratic discriminant analysis (QDA) and random forest classifier.

The performance of the results of the applied statistical methods used in accordance with the present invention can be best described by their receiver operating characteristics (ROC). The ROC curve addresses both the sensitivity (the number of true positives) and the specificity (the number of true negatives) of the assay. Therefore, the sensitivity and specificity values for the biomarkers of the invention are an indication of the performance of the assay. For example, if a combination of biomarkers has a sensitivity and specificity value of 80%, out of every 100 individuals, 80 will be correctly identified from determining the presence of the particular combination of biomarkers as positive for the disease and a given prognosis, while for every 100 individuals who do not have the disease, 80 will give a test with negative accuracy for the disease and a certain prognosis.

It is preferred that the sensitivity of the method is greater than 80%, preferably 100%, and that the specificity is greater than 80%, preferably 100%.

An appropriate statistical classification model, such as logistic regression, can be derived for a combination of biomarkers. Additionally, the logistic regression equation can be extended to include other (clinical) variables such as the age and sex of the individual as well. As described in the previous paragraph, the ROC curve can be used to access the discrimination performance between individuals and controls using the logistic regression model. Therefore, the logistic regression equation can be used separately or combined with other clinical characteristics to assist in clinical decision making. Although a logistic regression equation is a common statistical procedure used in such cases and is preferred in the context of the present invention, other mathematical/statistical procedures, decision trees or machine learning may also be used.

A convenient goal for quantifying the diagnostic accuracy of a laboratory assay is to express its performance in a single number. The most common global measure is the area under the curve (AUC) of the ROC chart. The area under the ROC curve is a measure of the probability that the perceived measurement will allow correct identification of a condition. Values typically range between 1.0 (perfect separation of test values from the two groups) and 0.5 (no apparent distribution difference between the two groups of test values), any result below 0.5 will be considered worse than a random estimate. The area does not depend only on a particular part of the graph, such as the point closest to the diagonal or the sensitivity with 90% specificity, but on the entire graph. This is a quantitative and descriptive expression of how close the ROC graph is to perfect (area = 1.0). In the context of the present invention, the two different conditions may be whether a patient's acute brain injury progresses favorably or not.

The method of the invention may comprise placing the patient's biological sample in contact with a device comprising a substrate having an activated surface on which one or more antibodies specific for SAA1 and S100P are immobilized in discrete areas of said activated surface. in such a way that a device is used for each antibody, or a device for more than one.

In another preferred embodiment, the method of the invention comprises extracting nucleic acids from the patient's sample and performing an analysis thereof with specific oligonucleotides to detect one or more of the biomarkers SAA1, TLR4 and/or S100p.

In a further embodiment, the method of the invention comprises classifying an individual with the value of at least one marker selected from: age, temperature, a certain GCS (Glasgow Coma Scale), GOS ( Glasgow Outcome Scale), GOSE ( Glasgow Outcome Scale). extended), NIHSS ( National Institute of Health Stroke Scale), A2DS2 ( Age, Atrial fibrillation, Dysphagia, Sex, Stroke Severity) Rankin and modified Rankin, Barthel index, Fisher score or WFNS scale ( World Federation of Neurosurgical Societies).

The biological sample to carry out the method of the invention is selected from blood, serum, plasma, cerebrospinal fluid, saliva, tear fluid, tissues, cells, urine and combinations of these.

In a particular embodiment, the biological sample is selected from blood, plasma, serum and combinations of these.

Acute brain damage can be caused by head trauma (open or closed) and cerebral vascular pathology, of an ischemic or hemorrhagic type, preferably open or closed head trauma.

In a particular embodiment the acute brain damage is caused by open or closed head trauma.

Determining the evolution of long-term brain damage means determining the probability that the clinical sequelae of acute brain damage will decrease or disappear, with possible mechanisms of acute brain damage being:

1- Damage caused by intracranial processes such as the development of spontaneous or post-traumatic intracranial hematomas (epidural hematoma, subdural hematoma, cerebral contusion and spontaneous or post-traumatic intracerebral hematoma), subarachnoid hemorrhage (spontaneous or post-traumatic), diffuse axonal injury, intraventricular hemorrhage, cerebral edema, intracranial hypertension, cerebral herniation, cerebral hypoxia-ischemia, status epilepticus and acute infection of the central nervous system.

2- The damage that occurs in the central nervous system derived from extracranial processes such as systemic arterial hypotension, anemia, hypovolemia, hypoxemia, anoxia, cardiorespiratory arrest, hyponatremia, hypernatremia, hyperthermia, hyperglycemia and hypoglycemia.

Using the method of the invention we can determine the probability that an individual will have a favorable evolution. This means that at the end of follow-up, you obtain a score on the scores commonly used in clinical practice (including, but not only: Modified Ramkin Scale, Glasgow Outcome Scale and Glasgow Outcome Scale Extended) such that any expert in the field recognizes said score. result as “good prognosis”.

Determination of the expression product of biomarkers to determine the evolution of long-term acute brain injury can be performed at any time from the occurrence of the cause of acute brain injury to 6 months after the time of injury, preferably 1 hour after said cause, more preferably, 12h after, even more preferably 24h and even more preferably 72h after the cause of acute brain damage.

Regardless of the cause of acute brain damage, the physiological processes that give rise to post-traumatic sequelae and long-term brain damage are common to all of them. The present invention serves for the prognosis of long-term post-traumatic sequelae of patients who have suffered any type of acute brain damage.

The individual may be a human between 1 and 90 years old, both including, preferably, 5 and 80 years, more preferably 10 and 70 years, even more preferably 20 and 60 years, both limits being included in all cases.

Additionally, the method of the invention can be used to determine the effectiveness of a treatment with pharmaceutical compositions in the long-term evolution of acute brain damage. This use would require determining the concentration of the expression product of the biomarkers of the invention in a biological sample from an individual treated with a pharmaceutical composition, and comparing the biomarker levels with those of a healthy control or with levels from the same patient before treatment. with the drug, where a decrease in the level indicates the effectiveness of the pharmacological treatment. The comparison can be made using any of the statistical methods mentioned above.

The result obtained in the statistical method can be complemented by combining the result obtained in the method of the previous paragraph with a marker of the following: age, temperature, a certain GCS, GOS, GOSE, NIHSS, A2DS2, Rankin and modified Rankin, Barthel index, Fisher score or WFNS.

Another aspect of the present invention refers to the use of a device that comprises the elements necessary to detect and quantify the amount of expression product of one or more of the biomarkers selected from: SAA1, S100P and TLR4 to determine the evolution of brain damage. long-term acute in individuals from an in vitro biological sample taken from a patient, where the sample is placed in contact with the device on which specific probes for each of the biomarkers have been immobilized.

The device may comprise a substrate having an activated surface on which probes for SAA1 and S100B are immobilized in discrete areas of said activated surface. Use of the device may also comprise determining the efficacy of a treatment with pharmaceutical compositions in determining the progression of long-term acute brain damage in individuals.

The substrate can be any surface that can support one or more probes, but preferably is a chip. A chip is a flat substrate that may be, for example, based on a mineral or a polymer, but is preferably ceramic. When the various biomarkers/proteins of the invention are identified it will be evident to a person skilled in the art that in addition to identifying the full-length protein, the identification of a fragment or several fragments of a protein is possible, provided that this allows an accurate identification. of the protein.

Detection of the amount of SAA1 and S100P expression can be performed by any means known in the art, preferably an immunoassay.

The immunoassay includes: latex agglutination assay, fluoroimmunoassay (FIA method), enzyme immunoassay (EIA method), radioimmunoassay method (RIA method), linear immunoassay (LIA), chemiluminescent immunoassay (CLIA), x-map or chips protein and Western blot method. Examples of preferred measurement methods include the FIA method and the EIA method (including the ELISA method). These methods allow highly sensitive, rapid and simple detection. The FIA method uses a fluorescently labeled antibody and detects an antigen-antibody complex (immune complex) using fluorescence as a signal. On the other hand, the EIA method uses an enzyme-labeled antibody and detects an immune complex using color development or luminescence based on the enzymatic reaction as a signal.

The ELISA ( Enzyme-Linked immunoSorbent Assay) is based on the premise that an immunoreagent (antigen or antibody) can be immobilized on a solid support, then putting that system in contact with a fluid phase that contains the complementary reagent that can bind to a marker compound. There are different types of ELISA: direct ELISA, indirect ELISA or sandwich ELISA.

The ELISA method has many advantages, such as high detection sensitivity, high specificity, marked quantitative performance, simple operation, and suitability for simultaneous treatment of many samples. Below is an example of the specific operation method for using the ELISA method. First, the anti-SAA1 antibody is immobilized on an insoluble support. Specifically, for example, the surface of a microplate is immunized (coated) with an anti-SAA1 antibody. The solid phase antibody is then brought into contact with the sample and the SAA1 protein. As a result of this operation, the solid phase antibody binds to the SAA1 protein to form an immune complex. After removing non-specific bindings by washing, the color is developed by the reaction of the enzyme with a substrate.

The device of the present invention may comprise at least one of the antibodies selected from anti-SAA1 and anti-S100P.

The device comprises secondary antibodies, positive and negative controls. The device may also include, without limitation, tampons, solutions of protein extraction, contamination prevention agents, and protein degradation inhibitors.

The device may be a DNA chip, to detect acute brain damage in individuals and its long-term evolution, comprising oligonucleotides or single-channel microarrays designed from a known sequence or a messenger RNA (mRNA) of at least one of the SAA1 and S100p genes.

Preferably, the device comprises pairs of oligonucleotides capable of detecting the mRNA of any of the SAA1 and S100p genes.

Thus, the oligonucleotide probes can be between 10 and 100 nucleotides, more preferably, between 20 and 70 nucleotides, and even more preferably, between 24 and 50 nucleotides. For the quantification of gene expression, approximately 40 oligonucleotides per gene are preferably used.

The probe is each of the samples on the chip. The target is the sample to be analyzed: messenger RNA, total RNA, or a fragment of an amplification by PCR (Polymerase Chain Reaction).

On the other hand, the device may include all the supports and containers necessary for its implementation and optimization in addition to the instructions to carry out the method of the invention.

Another additional aspect of the invention refers to a pharmaceutical composition that comprises at least one active ingredient that is an agent that modulates the expression levels of at least one of the biomarkers selected from SAA1, S100P and TLR4, for use in the treatment of acute brain damage achieving a favorable evolution of acute brain damage.

In a particular embodiment, the acute brain damage is caused by open or closed head trauma.

In a particular embodiment, the composition comprises one or more agents modulating all of the SAA1, S100P and TLR4 biomarkers, simultaneously.

In another particular embodiment, the composition comprises one or more agents that modulate the SAA1 and TLR4 biomarkers, simultaneously.

In another particular embodiment, the composition comprises one or more agents that modulate the SAA1 and S100P biomarkers, simultaneously.

In another particular embodiment, the composition comprises one or more agents that modulate the TLR4 and S100P biomarkers, simultaneously.

In another particular embodiment, the modulating agent is ethyl-(6R)-6-(N-(2-chloro-4-fluorophenyl)sulfamoyl)cyclohex-1-ene-1-carboxylate for use in the treatment of acute brain damage. , reducing the severity and/or number of long-term sequelae.

The pharmaceutical composition of the invention may additionally comprise at least one or more of the following:

- a pharmaceutically acceptable excipient,

- a pharmaceutically acceptable additive, and

- a second active ingredient.

The pharmaceutical composition of the invention can be used as a subsequent treatment or simultaneous with other therapies.

The pharmaceutical composition of the invention can be administered to an individual topically, intranasally, rectally, intraperitoneally, orally, subcutaneously or intramuscularly.

The pharmaceutical composition may have any formulation selected from the group consisting of a tablet, a lozenge, a powder, a granule, a capsule, a suspension, a solution, an emulsion, a syrup, a sterile aqueous solution, a non-aqueous solution, a suspension, an emulsion, a freeze dryer, a suppository and an injection.

These agents can be manufactured by a known process, for example, a general mixing, granulation, sugar manufacturing, dissolving or lyophilization process. For example, the pharmaceutical composition for oral administration can be prepared by mixing an active ingredient with a solid carrier, granulating the mixture, adding an appropriate additive if necessary, and then formulating the mixture or granule in the form of a tablet. or a dragee.

The pharmaceutically acceptable excipient may include, in particular, a filler, for example, sugar, such as lactose, sucrose, mannitol or sorbitol, a cellulose agent and/or calcium phosphate, such as tricalcium phosphate or calcium hydrogen phosphate, and a coupling agent, for example, starch pastes using corn, wheat, rice or potato starches, gelatin, tragacanth, methylcellulose and/or polyvinylpyrrolidone, and if necessary, a disintegrating agent, for example, the starches mentioned above, carboxymethyl starch , cross-linked polyvinylpyrrolidone, agar or alginic acid or a salt thereof, for example, sodium alginate.

Pharmaceutically acceptable additives include, in particular, a flow control agent and a lubricant, for example, silicic acid, talc, stearic acid or its salt, for example, magnesium, or calcium stearate and/or polyethylene glycol. An appropriate gastro-resistant coating is provided to a dragee core, and in particular, a concentrated sugar solution including gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide in an appropriate organic solvent or mixture of solvents, or a stirred solution is used, or, to form the gastro-resistant coating, a solution with appropriate cellulose agent, such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate, may be used.

Other pharmaceutically acceptable excipients for oral administration include encapsulating agents and dry-filled capsules made of gelatin, and soft sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The dry-filled capsule may include an active ingredient in particulate form, for example, a filler, such as lactose, a coupling agent, such as starches, and/or a modifier, such as talc or magnesium stearate, and a mixture with a stabilizer if appropriate. In the soft capsule, the active ingredient is dissolved or suspended in an appropriate liquid, for example, fixed oil, petroleum jelly or liquid polyethylene glycol, and a stabilizer may be added therein.

A pharmaceutical composition for parenteral administration may include a sterile aqueous solution, a non-aqueous solution, a suspension, an emulsion, a lyophilizing agent. The composition for parenteral administration can be an effective injection by various procedures, for example, intravenous, intraarterial, intramuscular, intraperitoneal, intranasal, intradermal and subcutaneous, preferably the intravenous procedure. The liquid necessary for parenteral administration is preferably an isotonic aqueous solution or a suspension that can be prepared from a lyophilizing agent that includes the active ingredient alone or in combination with a pharmaceutically acceptable carrier before use. The pharmaceutical composition may be sterilized and/or may include additives, such as preservatives, stabilizers, wetting and/or emulsifying agents, solubilizers, salts to control osmotic pressure and/or buffers.

In particular, as the non-aqueous solution and suspension, propylene glycol, polyethylene glycol, vegetable fats, such as olive oil, injectable ester, such as ethyl oleate and the like can be used. If the composition is administered as a suppository, the excipients that can be used are witepsol, macrogol, tween 61, cocoa butter, laurin, glycerol gelatin and the like.

In one aspect of the present invention, the pharmaceutical composition can be administered with a variable administration interval, for example 1 to 3 times per week, in particular 1 to 2 times per week.

The dosage and administration interval of the pharmaceutical composition may vary depending on various factors, for example, individual conditions, such as availability and duration of the active ingredient, an administration procedure, sex, age and weight of the individuals, severity of the conditions. diseases and the like. The specific route of administration and dosage can be selected by one skilled in the art based on conditions such as age, weight, degree of disease activity and physical condition.

The individual is preferably a mammal, more preferably a human or a domestic animal, for example a dog or a cat, although alternative subjects may be treated in the course of disease research. The dose of the composition to be administered to a subject depends on the species and size of the subject, the nature of the condition being treated and can be easily determined by one skilled in the art.

In one aspect of the present invention, said modulating agent can be used for the claimed indication by administering to an individual a dose of 0.1 mg/kg/day to 1000 mg/kg/day, specifically a dose of 1 mg/kg/day. day at 500 mg/kg/day, preferably a dose of 3 mg/kg/day to 100 mg/kg/day. more preferably 3 mg/kg/day, of modulating agent.

A single administration of the pharmaceutical composition may be sufficient to achieve a decrease in the severity and/or number of sequelae, but in alternative embodiments, more than one dose of the pharmaceutical composition may be administered. For example, a first dose may be followed by a booster dose after one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, or longer intervals.

The invention is illustrated by the following examples that describe in detail the objects of the invention. These examples should not be considered as limiting the scope of the invention but as illustrative thereof.

Figure 1: Expression product levels of individual markers SAA1, S100P and TLR4 from patients who have suffered a TBI. Dot plots are individual samples with average serum levels based on prognosis.

Figure 2: ROC curves regarding the discrimination between favorable or unfavorable long-term prognosis (6 months) for SAA1 (72h), S100P (24h) and TLR4 (72h) in the performance of individual markers, (left, center and right , respectively). The x-axis is represented as 1- specificity value.

Figure 3: Combination of markers for long-term prognosis of patients who have suffered acute brain injury, TBI. Receiver operating characteristic (ROC) curve with respect to the discrimination between favorable or unfavorable prognosis for a predictive model based on the biomarkers SAA1 and S100P in combination. AUC = 0.931 (95% CI: 0.827 - 1.000). The x-axis is represented as 1 - specificity value.

Figure 4: Expression of TLR4 mRNA in leukocyte samples taken from blood in healthy subjects (controls) and in patients at 24 h, 72 h and 7 days after TBI.

Figure 5: Correlation between SAA1 protein and TLR4 mRNA in the same patients 72 hours after head trauma.

Figure 6: Serum expression of SAA1 in a murine model of traumatic brain injury. The upper panel (A) shows serum SAA1 levels from blood samples at 1 and 7 days after trauma. Brain SAA1 levels at 1 and 7 days in the ipsilateral and contralateral hemispheres are shown in the lower panel (B).

Figure 7: Serum and brain expression of SAA1 in a murine model of traumatic brain injury in the presence or absence of a TLR4 receptor blocker in serum and brain parenchyma 24 hours after injury, by ELISA (A and B) and Western Blot (C).

Figure 8: Effects of TLR4 receptor blockade with TAK ₂ 42 on the prognosis of animals subjected to TBI.

The following examples are intended to illustrate notable aspects of the invention and should not be construed as restrictive of the invention or any aspect thereof.

Example 1: Analysis of the SAA1, S100B proteins and the expression of TLR4 mRNA in samples from patients who have suffered a traumatic brain injury (TBI):

MATERIALS AND METHODS

Patients with TBI can serve as an exemplary pathology where the invention is applicable.

This prospective observational cohort study was carried out in 34 patients consecutively admitted to Hospital la Paz within 24 hours after TBI. It was performed in the Neurosurgery Service of the La Paz University Hospital between January 2017 and December 2018. Subjects aged between 15 and 85 years who showed clinical evidence of closed head trauma, loss of consciousness, score were included in the study. in post-resuscitation GCS less than 15 and brain lesions confirmed by the presence of abnormal findings on neuroimaging.

Mild injury was defined as a GCS of 13-15, moderate injury was defined as a GCS of 9 to 12, and severe injury was defined as a GCS score of 3 to 8 (regardless of CT findings).

The exclusion criteria were: presence of neurological disease or previous cognitive impairment, inability to perform a cranial computed tomography, collect biological samples or complete adequate patient follow-up.

Definition of clinical parameters upon admission

The clinical and demographic variables of the included patients were collected prospectively upon admission. This included age, sex, race, socioeconomic status and education, date, time and cause of injury, and medical history.

Evaluation of intracranial clinical severity: The degree of neurological dysfunction of the patient was evaluated by the Glasgow Coma Scale (GCS) score. Regarding the GCS score, the best total score and each of its subscales after non-surgical resuscitation in the best limb was determined. If sedative medication was administered, an attempt was made to perform a sedation window in the first 6 hours to determine the best score. To the components not evaluable, they were assigned a score of 1. A new score was assigned after the existence of clinical deterioration within 6 hours after hospital admission. Patients were classified according to the level of consciousness into mild (GCS score 13-15), moderate (GCS score 9-12), and severe (GCS score 3-8). For analysis, the worst evaluation at the end of the 6-hour period was used. Neurological deterioration was considered if there was a 2-point decrease in the GCS score or a control computed tomography (CT) scan worse than at admission.

Secondary systemic insults: Hypotension was defined as the presence or absence of any episode of systolic blood pressure <90 mmHg, including cardiorespiratory arrest within the first 6 hours after the traumatic event. Hypoxemia was established as pO2 ≤ 60 mmHg, cardiorespiratory arrest or oxygen saturation <90%.

Prognosis: the final outcome or functional deterioration was determined using the Extended Glasgow Outcome Scale (GOSE) at 6 months after the trauma through a quality of life questionnaire and/or follow-up visit in the neurosurgery service. For statistical purposes, the result obtained in GOSE was dichotomized according to the score obtained as favorable (8/7/6) vs. unfavorable (5/4/3/2/1).

Serum biomarker measurements

Venous blood samples were taken at 24 hours, 72 hours and 7 days after admission. The blood was allowed to clot and after centrifugation at 2500 rpm for 10 minutes, the serum was separated from the blood cells. The serum was stored at −80°C until further analysis. To establish our own reference intervals, a control group consisting of 8 healthy volunteers was included. Serum S100P levels were analyzed using a chemiluminescent immunoassay (CLIA) (Liaison® S100, Diasorin, Saluggia, Italy). An ELISA kit was used to determine SAA1 concentrations (Human SAA Elisa kit second generation, EL10015L, Anogen, Mississauga, Canada). In all cases the manufacturer's instructions were followed.

The investigator was blinded to the clinical and neuroradiological findings during the measurement of biomarker levels.

Measurement of TLR4 mRNA in leukocyte samples

Venous blood samples were taken 24 hours, 72 hours and 7 days after admission. To establish our own reference intervals, a control group consisting of 8 healthy volunteers was included. The total RNA of the blood cells obtained Following the previously described protocol, it was extracted using a specific RNA extraction kit (QIAamp® RNA Blood Mini Kit, 52304, Qiagen, Germany). Subsequently, complementary DNA (cDNA) was synthesized with a cDNA synthesis kit (iScript cDNA synthesis kit, 52304, Biorad, USA) and real-time PCR was performed with the QuantStudio 5 PCR system device (Applied Biosystems, USA). TLR4 gene expression was analyzed using Taqman® probes (FAM probe, Hs00152939_m1). TLR4 mRNA expression was normalized against eukaryotic 18S rRNA expression (VIC probe, 4310893E) in the same samples and calculated by the N A Ct method.

Statistic analysis

The normality of the data distribution was evaluated using the D'Agostino-Pearson test. Categorical variables are presented as percentages and continuous variables are shown as mean ± standard deviation if normally distributed or as median (interquartile range) if not normally distributed. Significance for intergroup differences was assessed using the Mann-Whitney U test for continuous variables. Logistic regression analyzes were used to create explanatory models on the role of biomarkers in certain clinical parameters. The prognostic value of biomarker levels at different times after hospital admission was evaluated using Receiver Operating Characteristics (ROC) curves.

Data were analyzed with the statistical package GraphPad version 8.0 and SPSS Statistics version 21. A p value <0.05 was considered significant.

Study approval

The study was conducted in accordance with the ethical standards of the institutional review board (Clinical Research Ethics Committee of Hospital Universitario la Paz) and according to the 1964 Helsinki Declaration and its later amendments. Written informed consent was obtained from all participants prior to inclusion in the study.

RESULTS

The combination of SAA1 and S00B determines the long-term evolution of acute brain damage in individuals.

Table 1: Results of the expression of biomarkers and their division in patients according to the level of severity determined by the GCS scale

Table 1 shows the values detected at each of the analyzed times (24 and 72 hours) for the biomarkers SAA1, S100P and TLR4. The biomarkers S100p and SAA1 were measured at the protein level and TLR4 at the mRNA level; the reference values included the measurement of these biomarkers in healthy patients, that is, without TBI. The values obtained at the mRNA level are shown in proportion to the reference value, while the protein values are shown in μg/mL of blood sample both in the individuals who have suffered TBI and in the reference values of the patients. healthy.

Common clinical practice involves measuring markers within 24 hours after injury. However, in the present invention, the results obtained with the measurement of the SAA1 marker at 72 hours provide more effective results for separating patients into mild and moderate-severe patients than having measured it at 24 hours (Figure 1 upper panel).

Furthermore, a significant increase in serum SAA1 levels was observed at 24 hours (median = 280; interquartile range, (IQR) = 73.41 - 404.4), 72 hours (median = 381.2; IQR = 161.9 - 642.9) and 7 days later. of trauma (median = 313.2; IQR = 117.8 -500.4). A peak was reached at 72 h, which remained significantly elevated 1 week after injury.

To determine the relationship between the level of expression of the biomarkers and the level of consciousness of the patients and their classification into mild and moderate-severe, the Glasgow Coma Scale (GCS) was used. This scale is the most widely used for the evaluation of intracranial clinical severity in patients with TBI (Teasdale and Jennet, 1974). However, few mentions have been made regarding different measurement times of post-TBI biomarkers.

First, we classified patients according to the Glasgow Coma Scale (GCS) into two groups: on the one hand, severe TBIs (GCS 3-8) and moderate TBIs (GCS 9-13) and, on the other hand, mild TBIs ( GCS 14-15).

Figure 1 shows the biomarker expression levels of individual patients at 24, 48, and 72 hours divided by their GCS classification into mild and moderate-severe. Significant differences were found 72 hours after TBI between the mild and moderate-severe groups. In the other 2 times (24 hours and 7 days) a trend can be observed that those patients classified as moderate-severe have a greater amount of SAA1. In the case of S100P, the trend is also observed that patients classified as moderate-severe at 3 times have greater expression.

To evaluate whether the levels of the 3 biomarkers after a TBI could discriminate between patients with favorable or unfavorable long-term evolution, the score obtained on the GOSE scale was taken into account, the ROC curve of each of these biomarkers was calculated individually ( Figure 2).

The area under the curve (AUC) demonstrated that SAA1 levels 72 h after TBI (Figure 2, left) can discriminate between favorable and unfavorable prognosis of patients (AUC = 0.864, 95% CI: 0.726 - 1.000). With specificity set to 100%, SAA1 showed a sensitivity of 46.2%. We also evaluated the sensitivity of SAA1 in a specificity range of 90-95%. In this case, SAA1 reached 69.2% and 53.8% sensitivity respectively (Table 2).

Regarding S100P, the area under the curve (AUC) demonstrated that the levels of said biomarker at 24 hours after a TBI (Figure 2, center) can discriminate between the favorable and unfavorable prognosis of patients (AUC = 0.753, CI 95 %: 0.568 -0.937). With the specificity set to 100%, S100P showed a sensitivity of 21.1%. We also evaluated the sensitivity of S100P in a specificity range of 90-95%. In this case, S100P reached 42.9% and 35.7% sensitivity respectively (Table 2).

In conclusion, the level of SAA1 or S100P in blood discriminates patients according to the severity of the TBI and its measurement after the injury is capable of determining the prognosis of patients 6 months after the TBI. In the case of TLR4, Measuring this marker alone is not capable of determining the long-term prognosis of patients (Table 2 and Figure 2).

Table 2 shows the AUC values in the long-term prognosis of patients through the individual measurement of the biomarkers at 24 and 72 hours, as well as the combination of S100p and SAA1.

Table 2 AUC values for the determination of acute brain damage in individuals using different markers at different times.

The next step was to determine whether by combining the individual markers S100P and SAA1 we could obtain a better determination of the evolution of long-term acute brain damage than with both biomarkers separately (Figure 3 and Table 2). The model comprised two biomarkers: SAA1 at 72 h and S100p at 24 h. The area under the curve (AUC) demonstrated that the combination of the levels of S100P at 24h and SAA1 at 72h after TBI (Figure 3) can discriminate between the favorable and unfavorable evolution of patients better than each of these. biomarkers separately (AUC = 0.931, 95% CI: 0.827 - 1.000). With specificity set to 100%, the combination showed a sensitivity of 50.0%. We also evaluated the sensitivity of the combination in a specificity range of 90-95%. In this case, 75.0% and 58.3% sensitivity was achieved respectively (Table 2).

Furthermore, we developed a corresponding formula for clinical application of the predictive model. The calculation of the probability that a patient has a good prognosis (P ^bp ) was performed using a binary logistic regression that includes regression factors for all the two markers that are part of the predictive model: P ^bp = 1/(1 exp (-5.846 0.008 * [SAA1] 3.618 * [S1000]), where [SAA1] is the concentration of SAA1 obtained in the patient's serum at 72 h after trauma and [S100P] is the concentration of S100P obtained in the patient's serum 24 h after the trauma causing acute brain damage. For example, we observed a patient (#11) with the following serum levels for the two markers included in the predictive model: [SAA1] = 250.49 μg/mL and [S100P] = 0.67 μg/mL. Thus, the probability of having a good prognosis could be calculated with P _BP = 1/(1 exp (-5.846 0.008 * 250.49 3.618 * 0.67) = 0.81 and, therefore, 81% of having a good prognosis. That is, at the end of the follow-up, there will be an 81% probability of obtaining a score on the scores usually used in clinical practice for “good prognosis.” In this specific case, the patient has an 81% probability chances of obtaining a score of 8/7/6 on the GOSE scale at 6 months after traumatic brain injury.

Correlation between SAA1 and TLR4 expression levels

SAA1 is capable of signaling through Toll-like receptors 2/4 (TLR2/4) in different cells. To examine whether SAA1 is relevant to inflammation after TBI, we studied TLR4 expression in leukocytes from patients 24 h, 72 h, and 7 days after TBI. As in the case of SAA1, we observed an increase in TLR4 mRNA at 24 hours (median = 10.51; interquartile range (IQR) = 2.26 - 28.15), 72 hours (median = 11.24; IQR = 2.42 - 15.34), and 7 days post-TBI (median = 9.74; IQR = 2.42 -15.34) (Figure 4). Significant differences were obtained both at 24 hours and at 72 hours. Next, we examined the possible relationship between TLR4 expression and serum SAA1 levels. A positive correlation was found at 72 h post-TBI (Pearson coefficient = 0.6395, p < 0.05) (Figure 5). Therefore, serum SAA1 levels, TLR4 expression, and the severity and prognosis of patients are mechanistically linked.

Example 2: Analysis of the SAA1 protein in serum samples from murine experimental models of craniocerebral trauma.

MATERIALS AND METHODS

1. TCE experimental model.

The serum and brain expression of SAA1 has been explored in a preclinical model of brain damage called “Closed head injury” or closed TBI.

2. Validation of the models: the models have been validated through functional and histological analysis and markers of traumatic brain injury.

3. Analysis of SAA1 expression: Serum and brain expression of SAA1 was analyzed at the protein level by using ELISA.

4. Verification of the potential beneficial effect of treatment with the TLR4 blocker (TAK-242).

Closed TCE model

The closed TBI model consists of dropping a weight on the mouse's skull. The procedures and methodology were adapted from existing literature [7]. The injury occurred in the right hemisphere, between the sagittal and lambdoid sutures, by a 50 g weight that fell from a height of 34 cm to produce severe TBI. The mouse kept its head on a hard surface to minimize energy dissipation and cause a focal injury. One hour before subjecting the animals to trauma, they were treated with 0.05 mg/kg of buprenorphine subcutaneously for analgesia. Mice were anesthetized with inhaled isoflurane before impact and were administered oxygen after TBI until breathing returned to normal. The evaluation of the severity of the injury was done using the NSS test ( Neurological Severity Score), adapted and modified from the literature [7]. This test consists of the evaluation of neurological and behavioral parameters, in which a value of 10 means total deterioration. The animals were subjected to the test 1 hour and 24 hours after suffering the trauma. Mice that obtained a score of 6-8 on the NSS were considered severe TBI. Those who obtained a score of 9 or 10 in the 1-hour test were immediately euthanized to prevent their suffering, as dictated by the Guidelines for the Care and Use of Laboratory Animals. The mice were treated just after the 1-hour test with the compound TAK ₂ 42 at a concentration of 3 mg/kg intraperitoneally.

Detection of SAA1 by ELISA

Mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) for sacrifice, and blood was obtained by intracardiac puncture. The blood was deposited into EDTA-treated tubes and centrifuged at 2500 rpm for 10 minutes to obtain serum. In the case of brains, the cortex of the ipsilateral and contralateral hemispheres was disaggregated in RIPA lysis buffer. protein levels SAA1 were determined by a specific ELISA kit (ref. no. DY2948, R&D Systems) and following the manufacturer's instructions.

Western Blot

The cerebral cortex of mouse brains was disaggregated with RIPA lysis buffer. 30 μg of protein was run on SDS-PAGE gels and transferred to Immobilon-P membranes. The membranes were incubated with anti-SAA1/A2 antibody (concentration 1:1000; ref. no. AF2948, R&D Systems) and anti-p-actin (concentration 1:50,000; ref. no. A3854, Sigma-Aldrich) following the protocol marked by the manufacturer. Peroxidase-conjugated secondary antibodies (concentration 1:5,000; ref. no. sc-2354, Santa Cruz Biotechnology) were used to detect proteins by chemiluminescence. The intensity of the bands was quantified with the Scion Image program (RRID: SCR_008673).

Statistic analysis

Data were represented with the mean ± standard deviation. The analysis of normality in the distribution of the data was done using the D'Agostino-Pearson normality test. Differences between more than two groups with an independent factor were analyzed by 1-way ANOVA followed by Tukey's post-hoc test; Experiments with two independent variables were subjected to two-way ANOVA followed by Sidak's post-hoc test. The animals were randomly assigned to each group before undergoing TCE. All statistical analyzes and graph representation were performed with GraphPad Prism 6.0 software (GraphPad Software Inc., RRID:SCR_002798).

RESULTS

Serum and brain expression of SAA1 is increased 24 hours after trauma in mice.

As an experimental model of TBI in mice, we used the model called "closed head injury” (CHI). As observed in humans, we detected a significant increase in SAA1 in plasma 24 hours after trauma, which was almost reduced to control levels 1 week after injury in mice (Figure 6A).

In the brain, SAA1 was also elevated in the hemisphere ipsilateral to the injury 1 day after injury, the accumulation of which disappeared after 7 days. We detected no changes in protein levels on the contralateral side (Figure 6B), indicating that the SAA1 protein contributes to the evolution of damage in the hemisphere affected by trauma.

Treatment with a TLR4 receptor blocker reduces SAA1 levels and improves the prognosis of the mice.

Mice subjected to the TBI model were treated 1 hour after trauma with the TLR4 receptor blocker TAK-242 (3 mg/kg) intraperitoneally. First, we evaluated possible changes in SAA1 in serum obtained from intracardiac puncture blood and in the brain parenchyma 24 hours after injury. We observed in both cases that TLR4 antagonism produced a significant reduction in protein levels compared to untreated animals; almost reached control levels in brain tissue (Figure 7A and B).

To confirm these findings, we performed Western blot analysis of the SAA1 protein. Western blot of brain tissue revealed that SAA1 is not present in the brains of uninjured animals (Figure 7C left). SAA1 levels increased on the ipsilateral side of mice subjected to TBI, while they were undetectable in the contralateral hemisphere. Furthermore, treatment of animals with TAK-242 decreased the accumulation of SAA1 in the brain parenchyma of the injured hemisphere (Figure 7C, right).

Finally, we explored the possible effects of TLR4 receptor blockade on the prognosis of animals subjected to TBI. We used the Neurological Severity Score (NSS) test, which assesses neurological, motor, and behavioral abilities in mice after TBI (24). A score of 10 points represents complete neurological impairment, while 0 points means normal function. One hour after TBI, we detected that the animals obtained an NSS between 6 and 8. After the 1-hour test, the animals were treated with TAK-242 (3 mg/kg) and the NSS was evaluated again 24 hours after the injury. Under these conditions, we found that animals treated with TAK-242 significantly improved neurobehavioral abilities compared to saline animals (Figure 8).

References

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2. Menon DK, Maas AI (2015). Traumatic brain injury in 2014. Progress, failures and new approaches for TBI research. Nature reviews. Neurology 11:71-72.

3. Welch, Robert D et al. “Ability of Serum Glial Fibrillary Acidic Protein, Ubiquitin C-Terminal Hydrolase-L1, and S100B To Differentiate Normal and Abnormal Head Computed Tomography Findings in Patients with Suspected Mild or Moderate Traumatic Brain Injury.” Journal of neurotrauma vol. 33.2 (2016): 203-14. doi:10.1089/neu.2015.4149

4. Lewis, Lawrence M et al. “Utility of Serum Biomarkers in the Diagnosis and Stratification of Mild Traumatic Brain Injury.” Academic emergency medicine: official journal of the Society for Academic Emergency Medicine vol. 24.6 (2017): 710-720. doi:10.1111/acem.13174.

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Claims (18)

1. Method to determine the evolution of long-term acute brain damage in individuals, comprising: to. determine the expression product of the biomarkers SAA1 and S100p in a biological sample taken from the individual and, b. establish the statistical significance of the concentration of the expression product of the biomarkers of stage a, where the SAA1 expression product is obtained from a biological sample taken between 24 hours and 1 week after the injury that produces acute brain damage. 2. Method according to any one of the preceding claims, comprising in step a) determining the expression product of the SAA1 and S100P biomarkers in a biological sample from the individual. 3. Method according to any one of the preceding claims, wherein the expression product of the biomarker is a nucleic acid and/or a protein. 4. Method according to any one of the preceding claims, which further comprises carrying out a statistical methodology with each of the values of the expression product of the biomarker and producing an output value with statistical significance that indicates whether the evolution of the brain damage of the individual is favorable or unfavorable in the long term. 5. Method according to the preceding claim, wherein the statistical methodology is selected from the D'Agostino-Pearson test and a group classifier algorithm selected from a decision tree classifier, logistic regression classifier, nearest neighbor classifier. , neural network classifier, Gaussian mixture model (GMM), Support Vector Machine (SVM) classifier, nearest centroid classifier, linear regression classifier, linear discriminant analysis (LOA) classifier, quadratic discriminant analysis classifier ( QDA) and random forest classifier. 6. Method according to any one of the preceding claims, comprising placing the patient's biological sample in contact with a device comprising a substrate having an activated surface on which one or more is immobilized, in discrete areas of said activated surface. antibodies specific for SAA1, and S100P, in such a way that one device is used for each antibody, or one device for more than one. 7. Method according to any one of the preceding claims, comprising extracting nucleic acids from the patient's sample and performing an analysis thereof with specific oligonucleotides to detect one or more of the biomarkers SAA1, and S100p. 8. Method according to any one of the preceding claims, comprising classifying an individual with the value of at least one marker selected from: age, temperature, a certain GCS, GOS, NIHSS, A2DS2, Rankin and modified Rankin, Barthel index , Fisher score or WFNS. 9. Method according to any one of the preceding claims, wherein the biological sample is selected from blood, serum, plasma, cerebrospinal fluid, saliva, tear fluid, tissues, cells, urine and combinations of these. 10. Method according to any one of the preceding claims, wherein the cause of the acute brain damage is selected from open head trauma, closed head trauma and cerebral vascular pathology, of an ischemic or hemorrhagic type. 11. Method according to the preceding claim, wherein the acute brain damage is caused by open or closed head trauma. 12. Method according to one of the preceding claims, wherein the individual is a human between 1 and 90 years old, both included. 13. Use of a device to carry out the method defined in any of claims 1 to 12, which comprises the elements necessary to detect and quantify the amount of expression product of one or more of the biomarkers selected from: SAA1 and S100p. 14. Use according to the preceding claim, wherein the device is an immunoassay selected from latex agglutination assay, FIA, EIA, RIA, LIA, CLIA, X-map, Western Blot and ELISA, preferably an ELISA, FIA and EIA . 15. Use according to the preceding claim, wherein the ELISA is selected from direct ELISA, indirect ELISA and sandwich ELISA. 16. Use according to one of claims 13 to 15, wherein the device comprises secondary antibodies, positive and negative controls, buffers, protein extraction solutions, and contamination prevention agents and protein degradation inhibitors. 17. Use according to claim 13, wherein the device comprises a DNA chip, comprising oligonucleotides or single channel microarrays designed from a known sequence or an mRNA of at least one of the biomarkers SAA1, S100p and TLR4. 18. Use according to any one of claims 13 to 17, wherein the device comprises all the supports and containers necessary for its implementation and optimization, as well as the instructions for carrying out the method of the invention.