Biomarker identification method for predicting and monitoring ophthalmological diseases, biomarker identification kit and uses thereof

Abstract

The present invention relates to a method for identifying and detecting the expression of biomarkers present in choroidal and retinal pigmented epithelium disorders or in ocular angiogenic, epithelial and neuronal disorders. The invention further encompasses a kit for biomarker identification and uses thereof. The present invention pertains to the field of chemical biology, more specifically in the healthcare sector, and describes a method for identifying and detecting the expression of a set of biomarkers present in severe ophthalmological diseases.

Description

BIOMARKER IDENTIFICATION METHOD FOR PREDICTING AND MONITORING OPHTHALMOLOGICAL DISEASES, BIOMARKER IDENTIFICATION KIT AND USES Field of invention

[0001] The present invention relates to a method for identifying and detecting the expression of biomarkers present in disorders of the choroid and retinal pigment epithelium, or ocular angiogenic, epithelial, and neuronal disorders. Additional objects include a kit for identification and uses.

[0002] The present invention falls within the field of chemical biology, more precisely in the health area, and describes a method for identifying and detecting the expression of a set of biomarkers present in serious ophthalmological diseases. Fundamentals of the invention

[0003] Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in individuals over 55 worldwide. It is characterized by the loss of central vision in the retina (macula), interfering with the ability to perform basic daily activities. The pathogenesis of the disease is associated with risk factors such as aging, smoking, and heredity. AMD can be classified as "dry" (dysfunction of the retinal pigment epithelium (RPE) and lipoprotein deposition (drusen)) or "wet" (exudative) (neovascularization of the choroid).

[0004] AMD is asymptomatic in its early stages and is diagnosed only through routine ophthalmological examinations (visual acuity, Amsler grid test, biomicroscopy of the posterior ocular segment) and complementary examinations based on visual acuity and fundus imaging (such as fluorescein angiography and optical coherence tomography), identifying pigmentary changes in the RPE, drusen, RPE detachment, geographic atrophy, hemorrhage, intra- or subretinal hard exudates, fibrotic lesions, and serous retinal detachment. The main symptoms are metamorphopsia and changes in central vision.

[0005] Thus, the disease often remains undetected until it is more advanced and results in vision loss. Its rapid progression is an important factor, and the earlier it is detected, the greater the chance of effectiveness and selection of appropriate treatment with better prediction. of the disease.

[0006] Thus, the use of new tools such as blood biomarkers that reflect pathobiological changes in AMD at different stages becomes necessary for rapid referral to specialized care, prevention of disease progression, better therapeutic choice and prognosis of cases.

[0007] The patent literature reveals biomarkers, methods and kits for identifying and monitoring the effectiveness of therapy adopted for ocular disorders such as age-related macular degeneration.

[0008] Patent KR10-2451938 B1 relates to a proteomic analysis of age-related macular degeneration (AMD), a composition for diagnosing AMD using biomarkers, a method for providing information for diagnosing AMD, and screening for a substance for the treatment of AMD. The described technology aims to measure the mRNA or protein expression levels of an AMD-related biomarker. However, neuroretina and retinal pigment epithelium (RPE) tissue are difficult to access and impractical for patient treatment. In contrast, the present invention, in addition to covering both "wet" and "dry" forms of the disease, identifies readily available blood biomarkers that are specific to each form of AMD at different stages of the disease's development.

[0009] Patent KR102414152 B9 relates to a marker composition, a kit, a gene panel, and a method that provide information for predicting the occurrence, diagnosis, or treatment of age-related macular degeneration. However, it does not specify the patient's form of AMD. However, the present invention provides information for predicting the occurrence, diagnosis, or treatment of age-related macular degeneration with excellent sensitivity and can be easily analyzed without the use of a biopsy, thus being effective for the early diagnosis of age-related macular degeneration. Furthermore, the present invention utilizes biomarkers specific to each form of AMD and the different stages of the pathologies' development.

[0010] Patent KR10-2316999 B1 relates to a blood marker for the diagnosis of eye diseases causing severe blindness and a A diagnostic method using a marker to diagnose at least one blinding eye disease selected from a group consisting of age-related macular degeneration (AMD), diabetic retinopathy, cataracts, and glaucoma. The technology described has excellent sensitivity and uses patient plasma to obtain an immunological diagnosis. This information, analyzed together with basic clinical information, would be sufficient for a diagnosis of blinding eye diseases. In contrast, the present invention uses other biomarkers, detects changes in protein expression by ELISA or any other method, and enables identification at different stages of the pathologies' development.

[0011] US patent application 2022 / 0146530 A1 relates to a composite marker for the diagnosis of age-related macular degeneration (AMD) and a use thereof, and more specifically, to a composite marker for the diagnosis of age-related macular degeneration, consisting of two or more blood markers specific for the diagnosis of age-related macular degeneration. The present invention uses different biomarkers to detect changes in protein expression that characterize AMD in its forms and identify the different stages of the development of the pathologies.

[0012] US Patent 11,099,185 B2 relates to a method for treating an individual with wet AMD, using biomarkers to determine the individual's responsiveness to treatment with a VEGF antagonist, and aiding in the diagnosis and distinction between dry and wet AMD and in determining the risk of conversion from dry AMD to wet AMD. In contrast, the present invention utilizes other biomarkers, detects changes in protein expression by ELISA or any other method, and enables identification at different stages of the development of pathologies, often not detected in imaging studies.

[0013] Patent application US2011 / 0117083A1 relates to methods and compositions for detecting the expression of one or more biomarkers for monitoring the efficacy of treatment with VEGF antagonists, identifying patients sensitive or responsive to treatment. Furthermore, it provides kits and articles of manufacture for use in the methods. In contrast, the present invention utilizes other biomarkers to identify diseases related to disorders of the choroid and retinal pigment epithelium or ocular angiogenic, epithelial, and neuronal disorders, such as for monitoring patients undergoing anti-VEGF treatments. Brief description of the invention

[0014] The present invention relates to a method for identifying and detecting the expression of biomarkers present in disorders of the choroid and retinal pigment epithelium, or ocular angiogenic, epithelial, and neuronal disorders. Additional objects include a kit for identification and uses.

[0015] The biomarker identification method detects changes in the retinal pigmented epithelium or choroidal vessels (epithelial or angiogenic disorders) and comprises the following steps: obtaining a biological sample from an individual; detecting the change in the expression of at least one of the biomarkers ZO-1, n-Cadherin, Vimentin or Zeb-1; and analyzing the change in expression of at least one of the biomarkers ZO-1, n-Cadherin, Vimentin or Zeb-1.

[0016] The kit for identifying and detecting the expression of biomarkers detects changes in the pigmented epithelium of the retina or choroidal vessels and comprises the following components: (1) a primary ZO-1, n-Cadherin, Vimentin and / or Zeb-1 antibody; (2) a secondary antibody conjugated to bind to the primary antibody; (3) a 96-well plate; (4) a buffer solution; (5) a blocking solution; (6) a washing solution; (7) a chromogenic substrate solution; (8) a reaction stop solution; and (9) a control sample.

[0017] Additional objectives include the use of the method and kit to identify and detect the expression of a set of biomarkers present in serious ophthalmological diseases and to monitor pharmacological therapies. Brief description of the figures

[0018] Figure 1 shows the preclinical model of dry AMD. Retinal pigment epithelial degeneration in C57BI6J mice was experimentally induced. A) Representative images of RPE / choroid from a control animal and 3, 7, and 14 days after induction, prepared flat and stained with Phalloidin (green). White arrows indicate loss of epithelial integrity. B) Animal weights before and after induction at the time points studied. Control and Day 14, n=10 animals; Day 3 and Day 7, n=8 animals. C) Tissue micrographs were used to quantify the total cytoskeleton. Image acquisition and quantification were performed blinded to the conditions. Control and Day 14, n=5 animals; Day 3 and Day 7, n=4 animals. In the graph, values ​​are expressed as mean ± SD. Data were analyzed by t-test. ***P < 0.0001 compared with control. The conditions studied feature males and females.

[0019] Figure 2 shows the identification of biomarkers of epithelial disorders in a preclinical model of dry AMD. RPE degeneration in C57BI6J mice was experimentally induced or not. Blood was collected 3, 7, and 14 days after induction, samples were prepared, and the biomarkers ZO-1 (A) and n-Cadherin (B) were analyzed. Control and Day 14 n=10 animals; Day 3 and Day 7 n=8 animals. In the graphs, values ​​are expressed as mean ± SD. Data were analyzed by t-test. *P < 0.05; ***P < 0.0001 compared with control. The studied conditions include males and females.

[0020] Figure 3 shows the monitoring of the efficacy of KF6 treatment in the dry AMD model using the epithelial disorder biomarkers ZO-1 and n-Cadherin, currently under development by R&R NOVABIO PESQUISA E DESENVOLVIMENTO EM CIÊNCIAS NATURAIS LTDA. RPE degeneration was experimentally induced in C57BI6J mice. Representative images of RPE / choroid treated 7 days after induction with saline (AMD + vehicle) or KF6 (AMD + KF6) injection. After 7 days, the animals were euthanized, the eyes were enucleated, and the RPE / choroid was prepared flat and stained with Phalloidin (green). White arrows indicate loss of epithelial integrity. B) Tissue micrographs were used to quantify the total cytoskeleton. Image acquisition and quantification were performed blind to the conditions. of the treatments. Vehicle n=12; KF6 n=4 animals. C) After 7 days of induction, the animals were treated with intravitreal injection of saline (DM RI + vehicle) or KF6 (AMD + KF6). After 14 days of induction, blood was collected, samples prepared and the biomarkers ZO-1 and n-cadherin were analyzed. Control n=10; AMD+Vehicle and AMD+KF6 n=8 animals. In the graphs, the values ​​are expressed as mean ± SD of absorbance. Data were analyzed by t-test. **P < 0.01; ***P < 0.0001. The conditions studied present males and females.

[0021] Figure 4 shows the preclinical model of exudative AMD. Choroidal neovascularization (CNV) was experimentally induced in C57BI6J mice. A) Representative images of RPE / choroid from the control animal and 3, 7, and 14 days after induction injection, prepared flat and subjected to immunofluorescence assays for detection of the vascular marker IB4 (green) and nuclear marker DAPI (blue). B) Weight of animals before and after induction at the studied time points. Control and Day 14 n=14; Day 3 and Day 7 n=11 animals. C) Micrographs of IB4-labeled tissues were used to quantify the total CNV area per eye. Image acquisition and quantification were performed blinded to treatment conditions. Control and Day 14 n=5; Day 3 and Day 7 n=4 animals. In the graphs, values ​​are expressed as mean ± SD. Data were analyzed by t-test. **P < 0.01; ***P < 0.0001 compared with control. The studied conditions present males and females.

[0022] Figure 5 shows the identification of biomarkers of angiogenic disorders in a preclinical model of exudative AMD. Choroidal neovascularization (CNV) in C57BI6J mice was experimentally induced or not. 3, 7, and 14 days after induction after injection, blood was collected, samples were prepared, and the biomarkers vimentin (A) and Zeb-1 (B) were analyzed. Control and Day 14 n = 14; Day 3 and Day 7 n = 1 1 animals. In the graphs, values ​​are expressed as mean ± SD. Data were analyzed by t-test. *P < 0.05; ***P < 0.0001 compared with control. The studied conditions present males and females.

[0023] Figure 6 refers to the monitoring using biomarkers of angiogenic disorders, Vimentin and Zeb-1, in the efficacy of the treatment with KF7 under development by R&R NOVABIO PESQUISA E DESENVOLVIMENTO EM CIÊNCIAS NATURAIS LTDA in the exudative AMD model. A) Choroidal neovascularization (CNV) was experimentally induced in C57BI6J mice. Representative images of RPE / choroid treated 7 days after induction with intravitreal injection of PBS (AMD + vehicle) or KF7 (AMD + KF7). After 7 days, the animals were euthanized, the eyes were enucleated, the RPE / choroid was prepared flat and subjected to immunofluorescence assays for detection of the vascular marker IB4 (green) and the nuclear marker DAPI (blue). B) Micrographs of IB4-labeled tissues were used to quantify the total CNV area per eye. Image acquisition and quantification were performed blinded to treatment conditions. Vehicle n=7 animals; KF7 n=4 animals. C) After 7 days of induction, the animals were treated with an injection of PBS (AMD + vehicle) or KF7 (AMD + KF7).After 14 days of induction, blood was collected, samples prepared and the biomarkers Vimentin and Zeb-1 were analyzed. Control and AMD+Vehicle n=14 animals; AMD+KF7 n=1 1 animals. In the graphs, values ​​are expressed as mean ± SD of absorbances. Data were analyzed by t-test. *P < 0.05; **P < 0.01; ***P < 0.0001. The conditions studied included males and females. Detailed description of the invention

[0024] The present invention relates to a method for identifying and detecting the expression of biomarkers present in disorders of the mammalian retina, choroid, and pigmented epithelium, or ocular angiogenic, epithelial, and neuronal disorders. Additional objects include a kit for identification and uses.

[0025] The method for identifying and detecting the expression of biomarkers in changes in the pigmented epithelium of the retina or choroidal vessels (epithelial or angiogenic disorders) and comprises the following steps: Obtain a biological sample from an individual; Detect the change in the expression of at least one of the biomarkers ZO-1, n-Cadherin, Vimentin or Zeb-1; and Analyze changes in the expression of at least one of the biomarkers ZO-1, n-Cadherin, Vimentin or Zeb-1.

[0026] In a preferred embodiment, the method comprises the following steps: Obtaining a biological sample from an individual: 1) Isolate plasma or blood; 2) dilute the plasma or blood obtained in step (1) in a 1:1 ratio with phosphate-saline buffer solution; 3) Apply the sample prepared in step (2) in triplicate to a 96-well plate and incubate at 4°C for 20 hours; 4) Add the blocking buffer for 1 hour at 25 e W; 5) Remove the locking plug; Detect the change in the expression of at least one of the biomarkers ZO-1, n-Cadherin, Vimentin or Zeb-1: 6) Incubate the primary antibodies or individual conjugates of ZO-1, n-Cadherin, Vimentin and Zeb-1 for 20 hours at 4°C; 7) Remove primary or conjugated antibodies; 8) Add wash buffer for 5 minutes at 25 e C, if primary antibodies: 8.1) Incubate conjugated secondary antibody for 1 hour at 25 e W; 8.2) remove the secondary antibody; 8.3) add wash buffer for 5 minutes at room temperature; 8.4) remove the wash buffer; 9) Add the chromogenic substrate for 30 minutes; 9.1) If secondary antibody is HRP conjugate, add a chromogenic substrate for peroxidases (3,3',5,5';-tetramethylbenzidine) and keep for 30 minutes; 10) Add the stop solution to the reaction; 11) Perform the absorbance reading at 450 nm / 650 nm; 12) Detect the expression of biomarkers by reading absorbance at 450 nm / 650 nm; and Analysis of the change in expression of at least one of the biomarkers ZO-1, n-Cadherin, Vimentin and Zeb-1 compared to the control: 13) Analysis of the change in expression of at least one of the biomarkers ZO-1, n-Cadherin, Vimentin and Zeb-1 compared to the control: low expression of the biomarker ZO-1 and high expression of the biomarker n-Cadherin represents an individual with changes in the RPE / retina (e.g. similar to age-related macular degeneration in their dry form), acquired or hereditary, compared to the expression of these biomarkers in individuals without epithelial alterations (control); The high expression of the biomarker ZO-1 and the low expression of the biomarker n-Cadherin represents the reversal of the levels of these biomarkers, that is, the increase in the expression of ZO-1 or the decrease in the expression of n-Cadherin, reflects the competence of the adopted therapy; The high expression of the biomarker Vimentin or Zeb-1 represents an individual with vascular alterations in the choroid (e.g. similar to the choroid in age-related macular degeneration in its exudative form) compared to the expression of these biomarkers in individuals without vascular alterations (control); The low expression of the biomarker Vimentin or Zeb-1 represents the restitution of Vimentin or Zeb-1, that is, its decrease represents the effectiveness of the therapy; and Failure to restore normal levels of biomarker expression compared to the expression of these proteins in individuals without alterations indicates resistance to treatment with anti-angiogenic drugs.

[0027] Whereas: Low expression refers to the decrease in biomarker levels that present values ​​with a statistically significant difference of p < 0.05 compared to the control sample; and High expression refers to increased levels of biomarkers that present values ​​with a statistically significant difference of p < 0.05 higher compared to the control sample.

[0028] The kit for identifying and detecting biomarker expression comprises the following components: (1) ZO-1, n-Cadherin, Vimentin and / or primary Zeb-1 antibodies; (2) a secondary antibody that binds to the primary antibody and is conjugated to a marker; (3) a 96-well plate; (4) a buffer solution; (5) a blocking solution; (6) a washing solution; (7) a chromogenic substrate solution; (8) a reaction stop solution; and (9) a control sample. Wherein: the buffer solution comprises phosphate saline; the blocking solution comprises bovine serum albumin and a detergent in phosphate saline; the wash solution comprises a detergent in phosphate saline; the chromogenic substrate solution comprises a chromogenic substrate for peroxidases and HRP-conjugated secondary antibody, 3, 3', 5, 5'-tetramethylbenzidine; the reaction stop solution comprises an acidic solution.

[0030] The control sample may be blood from an individual without epithelial or angiogenic changes and / or a biological sample from the same individual before and after therapy.

[0031] The present invention defines specific biological markers for changes mimicking age-related macular degeneration in its dry and exudative forms from an early stage, often undetected by imaging tests. The present invention also aids in monitoring disease progression and the chosen therapy, and presents diagnostic and therapy monitoring kits for manufacturing.

[0032] The present invention also relates to the use of the method and kit for monitoring therapies adopted for angiogenic (figure 6) and epithelial (figure 3) disorders, in detecting sensitivity to anti-VEGF treatment among others in angiogenic and epithelial disorders, enabling the use of a co-treatment (figure 3 and 6). Definitions

[0033] The term “biological specimen” as used herein refers to any biological sample obtained from an individual, including body fluids, body tissues, cells, or other sources. Bodily fluids include, for example, lymph, serum, fresh whole blood, peripheral blood mononuclear cells, frozen whole blood, plasma (including fresh or frozen), urine, saliva, semen, synovial fluid, and spinal fluid. Specimens also include tissue. breast tissue, kidney tissue, colon tissue, brain tissue, muscle tissue, synovial tissue, skin, hair follicle, bone marrow and tumor tissue.

[0034] The term “subject” refers to a cell, tissue and animal, preferably in mammals, more specifically in humans, intended to diagnose, prevent or treat a particular disease or condition using the present invention.

[0035] The term “biomarker” used here refers to a DNA, RNA, protein, whose expression in a sample from an individual can be detected by standard methods (or methods disclosed here) to identify epithelial and angiogenic disorders and monitor the responsiveness or sensitivity of a subject to an adopted treatment.

[0036] The term "angiogenic disorders" used here refers to abnormal angiogenesis, that is, excessive, insufficient, or inappropriate growth of new blood vessels, accompanied or not by dedifferentiation (e.g., the location, timing, or onset of angiogenesis is medically undesirable) in a disease state or such that it causes a disease state, i.e., an angiogenic disorder. Excessive, inappropriate, or uncontrolled angiogenesis occurs when there is growth of new, dedifferentiated or undifferentiated blood vessels that contribute to the worsening of the disease state or cause a disease state. The new blood vessels can nourish diseased tissues, destroy normal tissues, and, in the case of cancer, the new vessels can allow tumor cells to escape into the circulation and lodge in other organs (tumor metastases).Disease states involving abnormal angiogenesis (i.e., angiogenic disorders) include both nonneoplastic and neoplastic conditions.

[0037] The term "epithelial disorders" used here refers to dysfunction and dedifferentiation resulting in degeneration of the epithelium, usually caused by persistent or recurrent injuries. Among the functions of an epithelium, nutrient transport, immunoregulation, barrier integrity, autophagy, phagocytosis, among others, are compromised in epithelial disorders. Examples of implementation

[0038] The present invention allows to identify or monitor the effectiveness of the Treatment applied to patients with disorders of the tissues of the posterior segment of the eye (changes in the retinal pigment epithelium / retina - Figures 1 and 2; or choroidal vessels - Figures 4 and 5), which is often not detected in fundus images, due to variation in the expression levels of at least one of the specific biomarkers (ZO-1; n-Cadherin; Vimentin and / or Zeb-1). Low expression refers to decreased levels of biomarkers that present values ​​with a statistically significant difference of p < 0.05 compared to the control sample. High expression refers to increased levels of biomarkers that present values ​​with a statistically significant difference of p < 0.05 compared to the control sample.

[0039] The expression of ZO-1, a peripheral membrane phosphoprotein belonging to the intercellular tight junction proteins, can be determined as lower, and the expression of the biomarker called n-Cadherin, a protein belonging to a family of transmembrane molecules that mediate calcium-dependent intercellular adhesion, can be determined as higher in a sample obtained from an individual with RPE / retina alterations (e.g., similar to age-related macular degeneration in its dry form), whether acquired or hereditary, compared to individuals without alterations in the posterior segment of the eye. The reversal of the levels of these biomarkers, that is, the increase in ZO-1 expression or decrease in n-Cadherin expression, reflects the effectiveness of the adopted therapy (Figure 3).

[0040] The expression of Vimentin, a protein of the intermediate filament family, or Zeb-1, a transcription factor, can be determined to be higher in a sample obtained from an individual with vascular alterations in the choroid (e.g., similar to the choroid in age-related macular degeneration in its exudative form) compared to individuals without vascular alterations. The restoration of Vimentin or Zeb-1 levels, that is, their decrease, represents the efficacy of the therapy (Figure 6). Failure to restore them indicates resistance to treatment with anti-angiogenic drugs.

[0041] Thus, the efficacy of a therapy or a patient's effective response (responsiveness or sensitivity) to treatment applied with VEGF antagonists, which inhibit or reduce the biological activity of the molecule to which they bind as an anti-VEGF antibody, includes cellular or biological responses, a complete response, a partial response, a stable disease (without progression or relapse) or a response with a subsequent relapse of the patient from or as a result of treatment with the VEGF antagonist.

[0042] For example, an effective response may involve RPE cytoskeletal remodeling if there is an increase in the biomarker ZO-1 and / or a decrease in n-Cadherin expression compared to individuals with RPE degeneration (Figure 3), or a reduction in vascular lesion size if there is a decrease in Vimentin and Zeb-1 expression compared to individuals with choroidal neovascularization (Figure 6). Therefore, the lack of a decrease in Vimentin and Zeb-1 biomarkers in patients with wet AMD treated with anti-VEGF therapy indicates sensitivity or low responsiveness to treatment. Protein Expression Detection

[0043] The presence of the protein biomarker can be performed in several ways, such as Western blotting (with or without immunoprecipitation), two-dimensional SDS-PAGE, immunoprecipitation, fluorescence-activated cell sorting (FACS), flow cytometry, mass spectrometry, and enzyme-linked immunosorbent assay (ELISA) procedures to analyze a wide variety of tissues and samples, including blood.

[0044] In an enzyme immunoassay, an enzyme is conjugated to the secondary antibody. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to use fluorogenic substrates, which produce a fluorescent product instead of the chromogenic substrates mentioned above. In all cases, the secondary antibody is added to the primary antibody-molecular marker complex, allowing binding. A solution containing the appropriate substrate is then added to the antibody-antigen-antibody complex. The substrate will react with the enzyme bound to the secondary antibody, producing a qualitative visual signal, which can be further quantified, usually through spectrophotometry, to indicate the level of biomarker expression present in the sample.

[0045] Alternatively, fluorophores such as fluorescein and rhodamine can be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a specific wavelength, the fluorochrome-labeled antibody absorbs the light energy, inducing a state of excitability in the molecule, followed by the emission of light in a characteristic color detectable visually with a light microscope.

[0046] Enzyme-linked immunosorbent assay and immunofluorescence techniques are well-established in the state of the art. However, other reporter molecules, such as radioisotopes, chemiluminescent molecules, or bioluminescent molecules, can also be used. Gene Expression Detection

[0047] The biomarkers described herein can also be detected by gene expression. For example, mammalian tissue or cell samples can be conveniently analyzed for, for example, mRNAs or DNAs of a biomarker of interest using Northern blot, dot-blot, or polymerase chain reaction (PCR) analysis, array hybridization, RNase protection assay, or using commercially available DNA SNP chip microarrays, including DNA microarray snapshots.

[0048] For example, real-time PCR (RT-PCR) assays, such as quantitative PCR assays, are well known in the art. In an illustrative embodiment of the invention, a method for detecting mRNA of a genetic biomarker of interest in a biological sample comprises producing cDNA from the sample by reverse transcription using at least one primer, amplifying the cDNA thus produced; and detecting the presence of the amplified cDNA.

[0049] Additionally, such methods may include one or more steps that allow the determination of mRNA levels in a biological sample (e.g., by simultaneously examining the levels of a comparative control mRNA sequence of a housekeeping gene, such as a member of the actin family). Optionally, the sequence of the amplified cDNA may be determined. Statistic

[0050] The general form of a prediction rule consists of specifying a function of one or several biomarkers potentially including clinical covariates to predict angiogenic disorders similar to age-related macular degeneration in its exudative form or the process of epithelial degeneration mimetic to age-related macular degeneration in its dry form and to monitor the treatment applied or detect sensitivity to anti-VEGF therapy to predict benefit or lack of benefit in terms of adequately defined clinical endpoints.

[0051] The prediction rule consists of a univariate model without covariates, in which the prediction is determined by a cutoff or threshold. This can be expressed in terms of the Heaviside function for a specific cutoff ce and a biomarker measurement X, where the binary prediction A or B should be made, then: If H(xc)=0, then predict A. If H(xc)=1 , then predict B.

[0052] This is the simplest way to use univariate biomarker measurements in prediction rules. If such a simple rule is sufficient, it allows for easy identification of the direction of the effect—that is, whether high or low levels of expression are beneficial to the patient. Identification of biomarkers to detect changes present in the degeneration process of ocular epithelia similar to age-related macular degeneration in its dry form from its early stage

[0053] After 3, 7 and 14 days of experimental induction of retinal pigment epithelium degeneration in mice (dry AMD model), blood was collected, processed and the expression of the biomarkers ZO-1 and n-Cadherin were detected by enzyme-linked immunosorbent assay.

[0054] Briefly, the plasma sample was applied to a 96-well plate and incubated overnight (20 hours) at 4°C. The next day, blocking buffer was added for 1 hour at room temperature. Subsequently, the blocking buffer was removed, and the primary antibody ZO-1 or n-Cadherin was incubated overnight (20 hours) at 4°C. The following day, the primary antibody was removed, and the wash buffer was used for 5 minutes at room temperature. Then, the HRP-conjugated secondary antibody was incubated for 1 hour at room temperature. The secondary antibody was removed, and the wash buffer was applied once for 5 minutes. After washing, the chromogenic substrate was added for 30 minutes or until color appeared. The reaction stop solution was added. The reading was performed at absorbance of 450 nm / 650 nm (Figure 2).

[0055] To identify changes in the expression of the biomarkers ZO-1 and n-Cadherin in retinal pigment epithelial degeneration, we compared them with a group of control animals without induction of epithelial degeneration. The RPE degeneration process is detected in the blood by the initial decrease in the expression of the biomarker ZO-1 and the increase in the expression of the biomarker n-Cadherin since the beginning of these changes, compared to the control group. This result also demonstrates the sensitivity of the technique used to capture these changes in the blood (Figure 2 A and B). Identification of useful biomarkers in mammals to monitor the effectiveness of applied therapy or to monitor changes present in the degeneration process of ocular epithelia similar to age-related macular degeneration in its dry form

[0056] After 7 days of experimental induction of retinal pigment epithelial degeneration in mice (dry AMD model), the animals were treated with a single injection of saline (AMD + vehicle) or KF6 (AMD + KF6). After 14 days of induction, blood was collected, samples prepared, and the biomarkers ZO-1 and n-Cadherin were analyzed.

[0057] Briefly, the plasma sample was applied to a 96-well plate and incubated overnight (20 hours) at 4°C. The next day, blocking buffer was added for 1 hour at room temperature. Subsequently, the blocking buffer was removed, and the primary antibody ZO-1 or n-Cadherin was incubated overnight (20 hours) at 4°C. The following day, the primary antibody was removed, and the wash buffer was used for 5 minutes at room temperature. Then, the HRP-conjugated secondary antibody was incubated for 1 hour at room temperature. The secondary antibody was removed, and the wash buffer was applied once for 5 minutes. After washing, the chromogenic substrate was added for 30 minutes or until coloration appeared. The reaction stop solution was added. The reading was performed at absorbance of 450 nm / 650 nm (Figure 3).

[0058] The RPE degeneration process is detected in the blood from the beginning of the transformations by the decrease in the expression of the biomarker ZO-1 and the increase from the initial time of the expression of the biomarker n-Cadherin in relation to the control group. The effectiveness of the adopted therapy is dependent on the reversal of this situation, that is, the increase in the expression of the biomarker ZO-1 and the reduction in the expression of the biomarker n-Cadherin observed through the use of KF6 therapy under development by R&R NOVABIO Pesquisa e Desenvolvimento em Ciências Naturais LTDA (figure 3 C). Identification of biomarkers to detect changes present in the process of ocular angiogenic disorders similar to age-related macular degeneration in its exudative form from its early phase

[0059] After 3, 7 and 14 days of experimental induction of choroidal neovascularization in mice (exudative AMD model), blood was collected, processed and the expression of the biomarkers Vimentin and Zeb-1 were detected by enzyme-linked immunosorbent assay.

[0060] Briefly, the plasma sample was applied to a 96-well plate and incubated overnight (20 hours) at 4°C. The next day, blocking buffer was added for 1 hour at room temperature. Subsequently, the blocking buffer was removed, and the primary antibody, vimentin or Zeb-1, was incubated overnight (20 hours) at 4°C. The following day, the primary antibody was removed, and the wash buffer was used for 5 minutes at room temperature. Then, the HRP-conjugated secondary antibody was incubated for 1 hour at room temperature. The secondary antibody was removed, and the wash buffer was applied once for 5 minutes. After washing, the chromogenic substrate was added for 30 minutes or until color appeared. The reaction stop solution was added. The reading was performed at absorbance of 450 nm / 650 nm (Figure 5).

[0061] The formation of choroidal neovessels is identified in the blood by the increase in the expression of the biomarkers vimentin and Zeb-1 from the initial stage and sustained throughout the progression of the disease compared to the control group. This finding also demonstrates the technique's ability to reveal these changes in blood samples (Figure 5). Identification of biomarkers to monitor the efficacy or sensitivity to anti-VEGF treatment in the process of ocular angiogenic disorders similar to age-related macular degeneration in its exudative form

[0062] After 7 days of experimental induction of neovascularization of the choroid in mice (exudative AMD model), the animals were treated with a single injection of saline (AMD + vehicle) or KF7 (AMD + KF7). After 14 days of induction, blood was collected, samples prepared and the biomarkers Vimentin and Zeb-1 were analyzed.

[0063] Briefly, the plasma sample was applied to a 96-well plate and incubated overnight (20 hours) at 4°C. The next day, blocking buffer was added for 1 hour at room temperature. Subsequently, the blocking buffer was removed, and the primary antibody, vimentin or Zeb-1, was incubated overnight (20 hours) at 4°C. The following day, the primary antibody was removed, and the wash buffer was used for 5 minutes at room temperature. Then, the HRP-conjugated secondary antibody was incubated for 1 hour at room temperature. The secondary antibody was removed, and the wash buffer was applied once for 5 minutes. After washing, the chromogenic substrate was added for 30 minutes or until color appeared. The reaction stop solution was added. The reading was performed at absorbance of 450 nm / 650 nm (Figure 6).

[0064] Vascular changes and lesions are detected in the blood by increased expression of the biomarkers vimentin or Zeb-1 compared to the control group from the onset of the process and maintained throughout disease progression. The success of a therapy is the reduction of this scenario, that is, the reduction of the expression of the biomarkers vimentin or Zeb-1, as observed with the KF7 treatment under development by R&R NOVABIO Pesquisa e Desenvolvimento em Ciências Naturais LTDA to control levels (Figure 6). An increase in these biomarkers also indicates non-responsiveness to anti-VEGF treatment and the possibility of co-treatment. The expression levels of the biomarkers vimentin and Zeb-1 must be reduced for antiangiogenic therapy to be effective in this case.

Claims

CLAIMS 1. Biomarker identification method characterized by detecting changes in the pigmented epithelium of the retina or choroidal vessels (epithelial or angiogenic disorders) and comprising the following steps: - Obtain a biological sample from an individual; - Detect the change in the expression of at least one of the ZO- biomarkers 1, n-Cadherin, Vimentin or Zeb-1; and - Analyze changes in the expression of at least one of the biomarkers ZO-1, n-Cadherin, Vimentin or Zeb-1.

2. Method according to claim 1, characterized by comprising the following steps: - Obtain a biological sample from an individual: 1) Isolate plasma or blood; 2) dilute the plasma or blood obtained in step (1) in a 1:1 ratio with phosphate-saline buffer solution; 3) Apply the sample prepared in step (2) in triplicate to a 96-well plate and incubate at 4°C for 20 hours; 4) Add the blocking buffer for 1 hour at 25 e W; 5) Remove the locking plug; - Detect the change in the expression of at least one of the biomarkers ZO-1, n-Cadherin, Vimentin or Zeb-1: 6) Incubate the primary antibodies or individual conjugates of ZO-1, n-Cadherin, Vimentin and Zeb-1 for 20 hours at 4°C; 7) Remove primary or conjugated antibodies; 8) Add wash buffer for 5 minutes at 25 e C, if primary antibodies: 8.1) Incubate conjugated secondary antibody for 1 hour at 25 e W; 8.2) remove the secondary antibody; 8.3) add wash buffer for 5 minutes at room temperature; 8.4) remove the wash buffer; 9) Add the chromogenic substrate for 30 minutes; 9.1) If secondary antibody is HRP conjugate, add a chromogenic substrate for peroxidases (3,3',5,5';-tetramethylbenzidine) and keep for 30 minutes; 10) Add the stop solution to the reaction; 1 1) Perform the absorbance reading at 450 nm / 650 nm; 12) Detect the expression of biomarkers by reading absorbance at 450 nm / 650 nm; and 13) Analysis of the change in expression of at least one of the biomarkers ZO-1, n-Cadherin, Vimentin or Zeb-1 compared to the control.

3. Kit to identify and detect the expression of biomarkers characterized by detecting changes in the pigmented epithelium of the retina or choroidal vessels and comprising the following components: -(1) ZO-1, n-Cadherin, Vimentin and / or primary Zeb-1 antibodies; -(2) a secondary antibody conjugated to bind to the primary antibody; -(3) a 96-well plate; -(4) a buffer solution; -(5) a blocking solution; -(6) a washing solution; -(7) a chromogenic substrate solution; -(8) a reaction stopping solution; and -(9) a control sample.

4. Use of the method as described in claims 1 and 2, characterized by identifying and detecting the expression of a set of biomarkers present in serious ophthalmological diseases and monitoring pharmacological therapies.

5. Use of the kit as described in claim 3, characterized by identifying and detecting the expression of a set of biomarkers present in serious ophthalmological diseases and monitoring pharmacological therapies.

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