Biomarkers of Drug-Induced Cytotoxicity and Depression

Abstract

The use of GFAP as a marker of drug-induced cytotoxicity and depression is described.

Description

Background Art

[0001] Post-traumatic stress disorder (PTSD) of the heart is a syndrome caused by exposure to actual or threatening serious injury, death, or sexual violence (1). PTSD affects civilians and especially active-duty military personnel. Risk factors include gender, previous exposure to mental trauma, existing mental illness, low socioeconomic status, mental retardation, and childhood adversity. Post-traumatic factors include the onset of acute stress disorder (ASD), other stresses such as business failure, subsequent adverse events in life, and lack of social support. Mental states such as PTSD are not well understood and vary widely in how the illness develops in an individual. After a mental trauma event, feelings of anxiety, sadness, stress, nightmares, intrusive memories of the event, and problematic sleep may exist. However, these symptoms do not necessarily mean that the individual has PTSD. The Diagnostic and Statistical Manual of Mental Disorders (DSM, the latest version is DSM-5) is used by clinicians and psychiatrists to diagnose mental illness. The DSM describes symptoms and provides statistical values including gender, age of onset, and effectiveness of treatment. The main issue with the DSM is its validity. In a statement by the National Institute of Mental Health (NIMH), it is stated that "DSM-5 presents the best information currently available for the clinical diagnosis of mental disorders." A diagnosis is valid when it accurately describes the patient's condition or disorder. However, the diagnoses described in DSM-5 are not objectively described physical medical conditions such as heart disease, diabetes, cancer, etc., but rather symptoms and traits reported by the patient and interpreted by the clinician. Possible risks of such an approach include misdiagnosis or overdiagnosis. The tendency of clinicians to anticipate, find, and interpret results can lead to confirmation bias. Therefore, patients may be easily labeled as having a disorder because these traits do not always conform to the current "ideal." So far, no clinically effective biomarker or combination of biomarkers (especially protein biomarkers) has been found to assist in the diagnosis, treatment, and management of patients with PTSD.Other mental and neurological disorders, such as symptoms and co - morbidities due to depression and anxiety, suggest that a single biomarker is unlikely to be diagnostic. Depression, a major co - morbidity of PTSD, represents a greater social problem than PTSD itself, and it is currently estimated that on average 10% of individuals will develop depression during their lifetime. Diagnosing depression is equally difficult and requires an in - depth personal history and clinical examination to identify important changes in mood, memory, physical illness, personality, etc. The subjective process can lead to misdiagnosis rates of up to 50%, ultimately affecting patient care. A more objective approach to diagnosing depression is a current medical challenge, and protein biomarkers for depression are an active area of research.

[0002] Research has been developed to investigate whether biomarkers or combinations of biomarkers with clinical risk factors can be used to help clinicians identify and / or stratify "at - risk" patients for PTSD. Unexpectedly, this research has identified biomarkers of drug - induced cell damage / toxicity.

Summary of the Invention

Problems to be Solved by the Invention

[0003] Surprisingly, during the investigation of potential blood biomarkers for post - traumatic stress disorder (PTSD), glial fibrillary acidic protein (GFAP) was found to be up - regulated in individuals from PTSD and non - PTSD cohorts who had ingested drugs.

Means for Solving the Problems

[0004] The present invention is an ex vivo method for evaluating the cellular effects of a drug to which an individual has been exposed or added to an in vitro cell line by measuring the amount of GFAP in an in vitro sample of the individual or in the in vitro cell line, wherein an increase in the measured amount of GFAP indicates cytotoxicity or damage.

[0005] From the same patient cohort, it was further identified that individuals in a depressive state had a greater amount of blood GFAP than individuals in a non-depressive state.

[0006] The use of GFAP as a biomarker for drug-induced cytotoxicity and depression may support its use in clinical medicine, clinical trials, and precision medicine as a diagnostic adjunct for identifying potentially toxic drugs. The use of GFAP as a biomarker for mental disorders may confer greater objectivity and accuracy to the clinical diagnosis of depression.

Brief Description of the Drawings

[0007]

Figure 1

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DETAILED DESCRIPTION OF THE INVENTION

[0008] A study including a PTSD cohort and a non-PTSD cohort, with the goal of detecting blood-based biomarkers in individuals to assist in PTSD diagnosis, unexpectedly identified biomarkers of drug-induced cell damage and depression. PTSD individuals are troubled by a group of co-existing conditions including depression and anxiety-related disorders, and are also defined in part by this group, and in many cases, several drugs are prescribed to counteract the condition. Several biomarker levels were evaluated in the PTSD study, but the variability in GFAP levels was significant, leading to further analysis.

[0009] GFAP is a structural protein mainly found in brain astrocytes and is a peripheral blood biomarker for stroke and traumatic brain injury (TBI) (WO2018096049, WO2018095872), which are conditions that can be risk factors for dementia. GFAP is present in one or more of three characterized full-length GFAP isoforms listed in the Uniprot database, GFAP isoform 1 of Uniprot number P14136-1, also known as GFAP alpha, GFAP isoform 2 of Uniprot number P14136-2, also known as GFAP delta, GFAP isoform 3 of Uniprot number P14136-3, also known as GFAP epsilon, with or without post-translational modification (PTM) respectively. They exist. "GFAP species" means all native GFAP isoforms with and without PTMs, as well as other GFAP isoforms such as GFAP beta, GFAP kappa, GFAP gamma, GFAPΔEx6, GFAPΔ135, GFAPΔ164, and GFAPΔEx7 (see Moeton et al 2016), GFAP multimers (dimers, tetramers, etc. based on any of the various isoforms), GFAP fragments and peptides that are structurally specific to GFAP, and any GFAP-related proteins. "GFAP fragments" (degradation products) are derived from native GFAP digested by enzymes; GFAP degradation products may also include PTMs. As used herein, the term "GFAP" corresponds to any GFAP species and any GFAP degradation product, unless otherwise described or implied by context (see WO2018096049 for a detailed definition of GFAP species and degradation products). The brain is composed of two main cell types, neurons and the more abundant glial cells, the latter of which are composed of oligodendrocytes and astrocytes. When an individual suffers from a stroke or TBI, brain cell death occurs, and the cytoplasmic contents of the cells are released into the extracellular environment and can enter the systemic circulation. As part of this release, GFAP can enter the systemic circulation, and a blood test to identify GFAP along with the patient's medical history can confirm the condition and whether a stroke or TBI has occurred, highlighting cell death and brain injury. GFAP is basically undetectable in the blood of healthy individuals (Mayer 2013).

[0010] Further analysis revealed the following categories: i. PTSD patients with depressive states during drug treatment versus patients during drug treatment versus PTSD patients not taking drugs but not in a depressive state, and ii. Depressive state patients during drug treatment versus patients during drug treatment and non-depressive state patients versus healthy patients (not taking drugs and disease-free) in the i. PTSD cohort and ii. non-PTSD cohort (control group). In both the PTSD cohort and the non-PTSD cohort, the blood GFAP levels in the drug treatment group and the non-drug treatment group were significantly different; in both cases, it was surprisingly found that the GFAP levels were higher in the drug treatment group.

[0011] A first aspect of the present invention is a method for evaluating the cytotoxicity of a drug, comprising measuring the amount of GFAP in an in vitro sample taken from an individual exposed to the drug, or in an in vitro cell line or cell model exposed to the drug, and comparing the measured amount of GFAP with a control measurement value; wherein a GFAP measurement value obtained from the in vitro sample or in vitro cell line or cell model that is greater than the control measurement value indicates cytotoxicity.

[0012] Further analysis emphasized that there was no difference in GFAP levels between untreated PTSD patients without co-existing diseases and healthy non-PTSD individuals (see Results and Figure 1). However, in depression in individuals in both the PTSD and non-PTSD cohorts, a small but significant increase in GFAP levels resulted (see Results and Figure 1).

[0013] A further aspect of the present invention is a method for assisting in the diagnosis of depression in an individual, comprising measuring the amount of GFAP in an in vitro sample of the individual and comparing the measured amount of GFAP with a control measurement value, wherein a GFAP measurement value obtained from the sample that is higher than the control measurement value suggests that the individual is in a depressive state.

[0014] In the context of the present invention, it is understood that a "control measurement value" or "control value" or "control level" is a GFAP level typically found in healthy individuals. A control biomarker level may be determined by analysis of samples isolated from healthy individuals, or may be a level of a biomarker that is understood by those skilled in the art to be typical of healthy individuals. A control value may be a range of values considered by those skilled in the art to be normal levels for a biomarker in healthy individuals. Those skilled in the art will recognize that a control value for a biomarker may be calculated by reference to typical values provided by a user analyzing the level of the biomarker in a sample from a healthy individual, or by the manufacturer of an assay used to determine the level of the biomarker in a sample. Alternatively, a control value may be a value taken from an in vitro sample of the same individual when classified as healthy. A control measurement value may be a threshold amount (also known as a cut-off value) or an absolute amount of a suitable GFAP species or GFAP degradation product. The term "individual" in the context of the present invention applies to any mammal, but is preferably a human. In a preferred embodiment, in relation to a patient's drug treatment or a patient's diagnosis of depression, the control measurement value is preferably the measured amount of GFAP in an in vitro sample taken from the individual before drug treatment or before the onset of depression. The phrase "before" implies at any point in time prior to the event described.

[0015] Potential drug toxicity can be evaluated during the pharmaceutical development stage and prior to clinical trials by administering the drug to non-human mammals by administering / adding the drug to in vitro cell lines or in vitro cell models (including cells extracted from healthy or diseased animals / patients), and comparing the GFAP levels in ex vivo samples from the mammals or from the cellular environment before and after addition of the drug. The terms "cytotoxicity" and "cell damage" are used synonymously herein.

[0016]

[0017] ​Therefore, a preferred embodiment of the present invention is a method for evaluating the cytotoxicity of a drug, comprising measuring the amount of GFAP in an in vitro sample taken from an individual, or in an in vitro cell line or cell model, at a time point after the individual, or the in vitro cell line or cell model, has been exposed to the drug, and comparing the measured amount of GFAP with a control measurement value; the control measurement value used for comparing the GFAP measured in the in vitro sample taken from the individual, or in the in vitro cell line or cell model, is a GFAP measurement value obtained from an in vitro sample of the individual, or an in vitro cell line or cell model, before being exposed to the drug, and a higher GFAP measurement value than the control measurement value indicates cytotoxicity; the control measurement value, or the value used for comparing the amount of GFAP in the in vitro cell line or cell model after adding the drug to the in vitro cell line or cell model, may be a control measurement value in a stored database, or preferably, the amount of GFAP measured in the in vitro cell line or cell model before adding the drug, as described above.

[0018] Accordingly, in a further preferred embodiment of the method of the present invention, there is an evaluation of the cytotoxicity of a drug, comprising measuring the amount of GFAP in an in vitro cell line or cell model (control measurement value), then exposing the in vitro cell line or cell model to the drug, and further measuring the amount of GFAP in the in vitro sample or in the in vitro cell line or cell model, and a higher amount of GFAP than the control measurement value in the further measurement indicates cytotoxicity, as described above. The time point of exposure of the individual, cell line or cell model to the drug, ingestion, addition or application of the drug can be carried out several hours, days, weeks or months before implementing the method of the present invention for analyzing the GFAP concentration in the in vitro sample, cell line or cell model.

[0019] The term "cell model" is any cell population that is not a recognized cell line, including cells taken from an animal or individual. The cell population includes conventional cell cultures and cells structured at the organoid and tissue levels. The "amount" of a biomarker refers to the amount, expression level, or concentration of the biomarker in a sample. The amount of a biomarker may refer to a biomarker measurement expressed as a ratio or percentage of the amount of one or more other analytes. The amount of one or more other analytes may consistently remain in most samples or conditions. By way of example, the other analyte may be albumin, β-actin, or total matrix protein. The amount of a biomarker may refer to a biomarker measurement expressed as a ratio or percentage of the amount of one or more other analytes, where the amount of one or more other analytes is proposed to retain some biochemical significance in the clinical state of the subject.

[0020] In the majority of drugs (therapeutic drugs) used in research, the proposed mechanisms of action of the active ingredients include molecules that act on one or more cell receptor / neurotransmitter-affecting systems in the brain, such as serotonin transporter molecules, norepinephrine transporter molecules, dopamine transporter molecules, serotonin receptor agonists / antagonists (interact with the "5-HT" receptor family), dopamine receptor agonists / antagonists (interact with the "DT" receptor family), adrenergic receptor agonists / antagonists (interact with the "alpha" and "beta" receptor families, including beta-2-adrenergic receptor agonists), GABA receptor agonists / antagonists ("GABA A ", and "GABA B), acetylcholine receptor agonists / antagonists (interact with the "muscarinic" and "nicotinic" receptor families), glutamate receptor agonists / antagonists (e.g., NMDA receptor agonists / antagonists), opioid receptor agonists / antagonists (interact with the "delta", "kappa", "mu", "nociceptin" receptor families), and histamine receptor agonists / antagonists (the "H3" receptor). Other prescribed drugs include ACE inhibitors, antibacterials, and HMG-CoA reductase inhibitors (statins). References to "drugs" herein imply prescribed drugs, unless otherwise limited. The mechanisms of action and other pharmacodynamic properties of the various therapeutic drugs referred to herein can be found in standard pharmacopeias.

[0021] In a preferred embodiment, the drug to which the individual is exposed preferably interacts with cell receptors (membrane and cytoplasmic) in the brain or with physiological pathways in the brain (acts on transporters), or is a drug that targets organs outside the brain that interact with cell receptors in the brain or affect / interfere with physiological pathways in the brain. Alternatively, the drug is for use in treating brain-related conditions; brain-related conditions can include depression, anxiety, stress, panic disorder, pain, epilepsy, dementia, suicidal ideation, Alzheimer's disease, and Parkinson's disease. Drugs administered to the individual are selective serotonin reuptake inhibitors (SSRI), selective norepinephrine reuptake inhibitors (SNRI), selective dopamine reuptake inhibitors (SDRI), serotonin receptor agonists / antagonists (interact with the "5-HT" receptor family), dopamine receptor agonists / antagonists (interact with the "DT" receptor family), adrenergic receptor agonists / antagonists (interact with the "alpha" and "beta" receptor families, including beta-2-adrenergic receptor agonists), GABA receptor agonists / antagonists (the "GABA A " and "GABA B), acetylcholine receptor agonists / antagonists (interact with the "muscarinic" and "nicotinic" receptor families), glutamate receptor agonists / antagonists (e.g., NMDA receptor agonists / antagonists), opioid receptor agonists / antagonists (interact with the "delta", "kappa", "mu", "nociceptin" receptor families), histamine receptor agonists / antagonists ("H3" receptor); or may include drugs targeting organs other than the brain that interact with cell receptors in the brain or indirectly disrupt physiological pathways in the brain. Preferably, the drug results in a change in homeostasis with respect to neurotransmitter-related physiological pathways. The agents (drugs) prescribed to the individuals involved in the study are listed in the Methods and Results sections. In a preferred embodiment of the present invention, the drug whose toxicity is being evaluated is a drug intended for use in treating a neuropsychiatric condition or is used in such treatment; the above condition is preferably depression, anxiety, panic disorder, suicidal thoughts or stress. The above condition is most preferably depression. The drug may be any drug tested for use in treating a neuropsychiatric condition or used in such treatment, but preferably a neurotransmitter reuptake inhibitor, e.g., an SSRI, SNRI, or SDRI or a neurotransmitter receptor agonist or antagonist (including partial agonists / antagonists), particularly serotonin or dopamine receptors; it has been found in studies that GFAP levels are particularly affected by drugs in the class of neurotransmitter reuptake inhibitors.

[0022] Preferably, the in vitro biological sample to be analyzed is a blood, plasma or serum sample, but may also be cerebrospinal fluid (CSF), urine or saliva. The determination of the biomarker level may be made in one or more samples from the patient. The sample can be obtained from the patient by methods routinely used in the art.

[0023] A further aspect of the invention is a method of treating an individual having a brain-related condition, wherein a drug is prescribed to the individual, and after ingestion of the drug, a sample is taken from the individual and the amount of GFAP in the sample is measured; when the amount of GFAP is greater than a control value, a determination is made as to whether to replace the drug with a different drug or supplement the drug with an agent that attenuates the side effects of the drug. The control value may be any GFAP value that is medically recognized as being a normal or healthy value for the individual, i.e., a value that is not considered to imply a brain-related disease or condition. Preferably, the control value is the level of GFAP measured in the individual at a time point prior to administration of the prescribed drug.

[0024] The invention also provides a method of treating a mammal or an in vitro cell line or cell model with a drug that binds to a receptor in the mammalian brain or interacts with a physiological pathway in the mammalian brain, wherein the drug is administered to the mammal or the in vitro cell line or cell model, and after administration of the drug, the level of GFAP is measured in an ex vivo sample taken from the mammal or in the cellular environment of the cell line or cell model, and the level of GFAP is compared to a control value. The control value may exhibit the values described above.

[0025] There are many analytical techniques that can be used to measure GFAP, but the preferred method of the present invention involves passing through an immune system or antibody system test, which is common to most clinical laboratories and clinical trials. Such tests can use substrates, such as slides, chips, beads, microtiter plates, etc., that can contain hydrophobic or hydrophilic coatings and can be chemically activated so that binding or capture agents can be attached, and can include competitive assay formats, immunoturbidimetric assay formats, and sandwich assay formats. The term "antibody" refers to an immunoglobulin that specifically recognizes an epitope against a target, determined by the binding characteristics of the immunoglobulin variable domains of the heavy and light chains (VHS and VLS), and more specifically, the complementarity-determining regions (CDRs). Many potential antibody forms are known in the art, and in the context of the present invention, include, but are not limited to, multiple intact monoclonal antibodies or polyclonal mixtures containing intact monoclonal antibodies, antibody fragments (e.g., Fab, Fab’, and Fv fragments, linear antibodies containing antibody fragments, single-chain antibodies, and multispecific antibodies), single-chain variable fragments (scFvS), multispecific antibodies, chimeric antibodies, humanized antibodies, and fusion proteins containing the domains necessary for the recognition of a given epitope against a target. Antibodies can also be conjugated to various detectable labels, including, but not limited to, radionuclides, fluorophores, dyes, or enzymes, such as horseradish peroxidase, biotin, and alkaline phosphatase, to enable detection. The term "specifically binds" in the context of an antibody-epitope interaction means that the antibody and epitope are related by an interaction that is more frequent or rapid, or of greater persistence or affinity, or any combination thereof, than when either the antibody or the epitope is replaced by a substitute substance, such as an unrelated protein. Generally, although not necessarily, a reference to binding means specific recognition. As techniques known in the art for determining specific binding of a target by an antibody or its deletion, but not limited to, FACS analysis, immunocytochemical staining, immunohistochemistry, Western blotting / dot blotting, ELISA, affinity chromatography can be mentioned. By way of example, and not limitation, specific binding, or its deletion, can be determined by comparative analysis with a control that includes the use of an antibody known in the art to specifically recognize the above target and / or a control in which there is no or minimal specific recognition of the above target (e.g., the control includes the use of a non-specific antibody). The comparative analysis can be qualitative or quantitative. However, it is understood that an antibody or binding site that demonstrates exclusive specific recognition of a given target is said to be more specific for that target, for example, when compared to an antibody that specifically recognizes both the target and a homologous protein.

[0026] The accuracy of a diagnostic method is best described by the Receiver Operating Characteristic (ROC) (Zweig, M.H., and Campbell, G., Clin.Chem. 39 (1993) 561-577). An ROC graph is a plot of all sensitivity / specificity pairs obtained by continuously varying the discrimination threshold over the entire range of observed data. The ROC plot shows the overlap between two distributions by plotting sensitivity against 1-specificity for the complete range of the discrimination threshold. The y-axis is sensitivity, or the true positive fraction defined as [(number of true positive test results) / (number of true positive + false negative test results)]. This is also referred to as the positivity in the presence of the disease or condition. This is calculated from the diseased subgroup only. The x-axis is the false positive fraction, or 1-specificity [defined as (number of false positive results) / (number of true negative + false positive results)]. This is an indicator of specificity and is calculated overall from the non-diseased subgroup. Since the true and false positive fractions are calculated separately overall by using test results from two different subgroups, the ROC plot is independent of the prevalence of the disease in the sample. Each point on the ROC plot represents a sensitivity / specificity pair corresponding to a discrimination threshold. A test with perfect discrimination (no overlap in the distribution of the two results) has an ROC plot passing through the upper left corner, where the true positive fraction is 1.0 or 100% (perfect sensitivity) and the false positive fraction is 0 (perfect specificity). The theoretical plot for a test with no discrimination (identical distribution of results for the two groups) is a 45° diagonal line from the lower left corner to the upper right corner. Most plots fall between these two extremes. Qualitatively, the closer the plot is to the upper left corner, the higher the overall accuracy of the test. One convenient way to quantify the diagnostic accuracy of a laboratory test is to represent its performance by a single number. The most common comprehensive measure is the area under the ROC plot (AUC). The area under the ROC curve is a measure of the probability that a measured value captured enables an accurate identification of the state. By convention, this area is always ≥ 0.5. The value ranges between 1.0 (perfect separation of the test values for the two groups) and 0.5 (no obvious difference in distribution between the test values for the two groups). The area depends on the entire plot, not just on parts of the plot, for example, the point closest to the diagonal or the sensitivity at 90% specificity.This is an expression that quantitatively describes how close the ROC plot is to being perfect (area = 1.0).

Example

[0027] Methods and Results Patients 78-year-old gender-matched participants were recruited in the US by Discovery Life Sciences (DLS), 1236 Los Osos Valley Rd, Suite T, Los Osos, CA 93402 USA and PrecisionMed, 132 N. Acacia Ave, Solana Beach, CA 92075, USA. The participants were composed of a PTSD cohort (N = 39) and a non-PTSD cohort (N = 39). Venous blood samples and detailed medical histories were collected from each study participant. The study was conducted in accordance with all data use agreements (DUA). Socio-demographic and clinical factors were collected from each participant and these included; age, gender, prescribed medications, and co-existing diseases such as depression, anxiety, panic disorder, diabetes, and hypertension. The medications prescribed to the patients at the time of blood collection were: Evrilify, Adderall, Atarax, Ativan, Baclofen, Benzarpil, Buspirone Hydrochloride, Celexa, Clozapine, Crestor, Cyclobenzaprine, Cymbalta, Depakote, Doxycycline, Effexor, Elavil, Fetzima, Gabapentin, Hydrochlorothiazide, Imitrex, Inderal, Irbesartan, Janumet, Klonopin, Lamictal, Lasix, Latuda, Lexapro, Lipitor, Maxalt, Metformin, Minocycline, Mobic, Nexium, Norco, Paroxetine, Plaquenil, Prozac, Prazosin, Saphris, Seroquel, Trazodone, Viberzi, Zanax, Zoloft and Zyrtec. The medications prescribed to the patients at the time of blood collection were: Evrilify, Adderall, Atarax, Ativan, Baclofen, Benzarpil, Buspirone Hydrochloride, Celexa, Clozapine, Crestor, Cyclobenzaprine, Cymbalta, Depakote, Doxycycline, Effexor, Elavil, Fetzima, Gabapentin, Hydrochlorothiazide, Imitrex, Inderal, Irbesartan, Janumet, Klonopin, Lamictal, Lasix, Latuda, Lexapro, Lipitor, Maxalt, Metformin, Minocycline, Mobic, Nexium, Norco, Paroxetine, Plaquenil, Prozac, Prazosin, Saphris, Seroquel, Trazodone, Viberzi, Zanax, Zoloft and Zyrtec.

[0028] In a follow-up study (N = 84), GFAP levels were measured in individuals without PTSD. The medications prescribed to the patients at the time of blood sampling were: atorvastatin, azathioprine, cetirizine, chlorpheniramine, cortisone, desmopressin, diazepam, dicloflex, ifexer, femoston, frixonase, fosamax, hydrocortisone, lansoprazole, levothyroxine, lisinopril, loperamide, metformin, methotrexate, midazolam, mycophenolate, omeprazole, perindopril, prednisone, premarin, propranolol, propecia, prozac, ramipril, rosuvastatin, sertraline, simvastatin, spironolactone, cymbort, thyroxine, warfarin.

[0029] Sampling and Laboratory Methods Scientists who could not see the participant data completed biomarker analysis at Randox Clinical Laboratory Services (RCLS) (Antrim, UK) using the cytokine assay (Randox Laboratories Ltd, Crumlin, UK) for the following proteins: Cytokine Array I: interleukin-1α, -1β, -2, -4, -6, -8, -10, VEGF, EGF, TNFα, IFNγ and MCP-1; Metabolic Assay I: ferritin, insulin, leptin, plasminogen activator inhibitor-1 (PAI-1), and resistin; Metabolic Assay II: C-reactive protein (CRP), adiponectin and cystatin C; Brain Assay I: brain-derived neurotrophic factor (BDNF), glial fibrillary acidic protein (GFAP), and heart-type fatty acid-binding protein (H-FABP); Brain Assay II: D-dimer, neuron-specific enolase (NSE), neutrophil gelatinase-associated lipocalin (NGAL), and soluble tumor necrosis factor receptor I (sTNFR1). Arrays were run on an Evidence Investigator© analyzer according to the manufacturer's instructions (Randox Laboratories Ltd, Crumlin, UK). Total cholesterol, HDL and LDL cholesterol were analyzed on a Randox RX Series analyzer (RCLS, Antrim, UK). Human tissue-type plasminogen activator (tPA) and human type 1 plasminogen activator inhibitor PAI-1 / tPA complex ELISA were obtained from AssayPro, 3400 Harry S. Truman Blvd, St. Charles, MO 63301. Assays were completed according to the manufacturer's instructions.The limits of detection (LOD) of the biomarkers under investigation were as follows: cytokine I - IL-2 2.97 pg / ml; IL-4 2.12 pg / ml; IL-6 0.12 pg / ml, IL-8 0.36 pg / ml; VEGF 3.24 pg / ml; IFNγ 0.44 pg / ml; TNFα 0.59 pg / ml; IL1α 0.19 pg / ml; MCP1 3.53 pg / ml; EGF 1.04 pg / ml; IL-10 0.37 pg / ml; IL-1β 0.26 pg / ml; metabolic assay I - ferritin 3.27 ng / ml, insulin 2.32 μIU / ml, leptin 1.10 ng / ml, PAI-1 2.34 ng / ml, and resistin 1.06 ng / ml; metabolic assay II - CRP 0.69 mg / l, adiponectin. 164 ng / ml, and cystatin C 60 ng / ml; brain assay I - BDNF 0 .59 pg / ml, and GFAP 0.18 ng / ml; brain assay II - D-dimer 2.1 ng / ml, NSE 0.26 ng / ml, NGAL 17.8 ng / ml, and STNFRI 0.24 ng / ml. Direct method HDL-cholesterol (HDL) 0.189 mmol / l (7.30 mg / dl), direct method LDL-cholesterol (LDL) 0.189 mmol / l (7.30 mg / dl), cholesterol: 0.865 mmol / l (33.4 mg / dl). AssayPro ELISA - human tPA ELISA - 0.013 ng / ml and human PAI-1tPA complex ELISA - 0.05 ng / ml. Thirty-two biomarkers in serum were investigated. Biomarker values below the LOD were assigned 90% of the LOD value.

[0030] Statistical analysis and results No influence of gender was observed in any of the studies.

[0031] Main study. There were no significant differences in BMI, heart rate, or systolic blood pressure between the control (non-PTSD) group and the PTSD group. The diastolic blood pressure was higher in the PTSD group (p = 0.004). PTSD individuals presented significantly more comorbidities, such as panic disorder, depression, and anxiety, than the control group. Significantly more medications were prescribed to PTSD individuals than to the control group (2.8 ± 2.4 vs. 0.7 ± 1.2, respectively, p < 0.001). GFAP levels within PTSD patients (N = 39) with and without depression and medications and within non-PTSD patients (N = 39) were analyzed using one-way analysis of variance with Tukey's comparison; all other analyses were by t-tests. Significance levels of P < 0.05 and P < 0.10 were used. All data were analyzed using GraphPad Prism according to log 10 transformation if applicable.

[0032] Analyzed, two age-matched cohorts (gender did not affect GFAP levels) were 1. PTSD patients / individuals: with depression during medication treatment (D+M), without depression during medication treatment (D−+M), and without depression and not on medication (D−+M−) 2. Non-PTSD patients / individuals (control group): with depression during medication treatment (D+M), without depression during medication treatment (D−+M), and healthy patients (H) were.

[0033] The results are summarized in Table 1.

[0034]

Table 1

[0035] In the non-PTSD cohort, GFAP levels in the sera of individuals in both the depressed and non-depressed states during pharmacotherapy were significantly increased compared to healthy individuals (P < 0.001). In the PTSD cohort, it has also been shown that the drug significantly increased GFAP levels in the sera of individuals in both the depressed and non-depressed states during pharmacotherapy compared to healthy individuals (P < 0.05). ROC curve analysis supported these findings (Table 2). PTSD individuals who were neither on pharmacotherapy nor in a depressed state, and healthy individuals, had similar GFAP levels (0.26 vs. 0.28 ng / ml; Figure 1). In both the PTSD and non-PTSD cohorts , individuals in the depressed state during pharmacotherapy had a higher absolute amount of blood GFAP than individuals in the non-depressed state during pharmacotherapy.

[0036]

Table 2

[0037] The effect of the drug was further analyzed by drug type using the PTSD cohort. Individuals on pharmacotherapy taking selective serotonin reuptake inhibitors (SSRI) had significantly higher GFAP levels than individuals on pharmacotherapy not taking SSRI (P < 0.05; Figure 2). This analysis was further supported by comparing GFAP levels in depressed individuals taking SSRI drugs versus depressed individuals on non-SSRI pharmacotherapy (P = 0.05, upper panel of Figure 3). In non-depressed PTSD individuals, the absolute GFAP levels were higher in SSRI drug-treated individuals than in non-pharmacotherapy individuals (P > 0.05, lower panel of Figure 3). GFAP levels in depressed individuals increased compared to non-depressed individuals in both SSRI (P < 0.010) and non-SSRI pharmacotherapy groups (Figure 4).

[0038] Within the non-PTSD cohort, individuals taking over-the-counter, non-steroidal anti-inflammatory drugs (NSAIDs) had no detectable levels of GFAP. Individuals taking SSRI medications had higher levels of GFAP than those taking non-SSRI medications (1.01 ng / ml vs. 0.85 ng / ml). To determine whether there was a possible effect of the number of drugs taken per individual on GFAP levels, a correlation analysis of GFAP levels vs. number of drugs taken was performed; there was no significant correlation (r = 0.212, P>0.05). The results demonstrate that GFAP levels increase in the blood of both PTSD and healthy individuals taking prescribed medications, individuals taking SSRI medications, and individuals in a depressive state.

[0039] Follow-up study. A follow-up study comparing individuals without drug treatment, during brain non-affecting drug treatment, and during brain-affecting drug treatment was analyzed using one-way analysis of variance with Tukey's comparison. As described above, brain-affecting drugs include drugs that interact with cell receptors in the brain (both on-target and off-target drugs), or interfere with the physiological pathways of the brain, including neurotransmission processes. In the follow-up study results (Table 3), it was confirmed that brain-affecting drugs increase GFAP levels (one-way analysis of variance P = 0.0113). Individuals taking drugs active against receptors in the brain or the physiological pathways of the brain had significantly higher serum GFAP levels than those not taking drugs (P<0.01). Also, individuals receiving other drug treatments (the middle bar graph in Figure 5) had higher mean absolute GFAP levels than patients not taking drugs. Reanalysis of the data excluding five patients with depression did not affect the results.

[0040]

Table 3

[0041] Among the possible interpretations of these findings, astrocytes (or other GFAP-containing cells) are driven by the drug and depression to cease functioning and leak into the peripheral circulation. As with most drugs, the systemic response to a drug can vary on an individual basis; some individuals may experience more severe brain cell effects and damage than other individuals, while others are unaffected and the levels of GFAP in the blood vary accordingly. The advantage of the present invention is that by identifying individuals in whom GFAP blood levels increase after drug treatment, particularly drug treatment used to treat mental disorders, such as depression, which is said to interfere with the brain's neurotransmission systems, clinicians can perform a cost-benefit assessment based on more information on an individual basis and potentially prescribe different drugs to manage the patient's condition. Point-of-care testing can easily enable clinicians (hospital doctors or general practitioners) to collect blood samples from patients before and after drug treatment and appropriately measure the amount of GFAP and the corresponding response. Alternatively, patients can easily and rapidly self-monitor their GFAP blood levels at home during drug treatment (e.g., using blood from a finger prick sample) using a suitable medical device. The effect of the drug on cell damage evaluated by GFAP concentration can also be easily applied using in vitro cell models and methods that can be very useful during the drug development process. A further advantage derived from the current findings is that it is possible to assist clinicians in diagnosing depression using an objective measure of depression, namely, the GFAP concentration. Considering the difficult nature of diagnosing mental health-related illnesses and the social stigma often associated with these conditions, this discovery can significantly benefit patients.

[0042] References 1. American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders [Internet]. American Psychiatric Association; 2013 [cited 2019 Jul 4]. Available from https: / / psychiatryonline.org / doi / book / 10.1176 / appi.books.9780890425596 2. Moeton M. et al. (2016), Cellular and Molecular Life Sciences, 73: 4101 - 4120. 3. Mayer C.A. et al. (2013), PLoS ONE, 8(4): e62101.

Claims

1. A method for evaluating the cytotoxicity of a drug, comprising measuring the amount of GFAP in an in vitro sample obtained from an individual exposed to the drug, or in an in vitro cell line or cell model exposed to the drug, and comparing the measured amount of GFAP with a control measurement value, wherein a GFAP measurement value obtained from the in vitro sample or in vitro cell line or cell model that is greater than the control measurement value indicates cytotoxicity, and the drug is a selective serotonin reuptake inhibitor.

2. The method according to claim 1, wherein the control measurement value is a GFAP level measurement value obtained from the in vitro sample or in vitro cell line or cell model before the individual, cell line or cell model is exposed to the drug.

3. The method according to claim 1 or 2, wherein the drug is for use in brain-related medical conditions.

4. The method according to claim 3, wherein the brain-related medical condition is depression, anxiety, stress, panic disorder, pain, epilepsy, dementia, suicidal ideation, Alzheimer's disease or Parkinson's disease.

5. The method according to claim 3, wherein the brain-related medical condition is depression.

6. A method for assisting in the diagnosis of depression, comprising measuring the amount of GFAP in an in vitro sample obtained from an individual who has been exposed to a drug in the past, and comparing the measured amount of GFAP with a control measurement value, wherein a GFAP measurement value obtained from the in vitro sample that is higher than the control measurement value supports that the individual is in a depressive state, and the drug is a selective serotonin reuptake inhibitor.

7. The method according to claim 6, wherein the control measurement value is a GFAP level measurement value obtained from an in vitro sample of the individual before exposure to the drug.

8. The method according to claim 7, wherein the control measurement value is a GFAP level measurement value obtained from an in vitro sample of the individual at a time when the individual did not suffer from depression before exposure to the drug. described method.

9. The method according to any one of claims 1 to 8, wherein the sample is blood, serum or plasma.

10. Use of GFAP as a marker for drug-induced cytotoxicity, wherein the drug is a selective serotonin reuptake inhibitor.

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