EP3526243A2

EP3526243A2 - Method for diagnosing/determining the effectiveness of treatment for depression

Info

Publication number: EP3526243A2
Application number: EP17794364.4A
Authority: EP
Inventors: Jean Mazella; Marc Borsotto; Catherine Heurteaux; Morgane ROULOT
Original assignee: Centre National de la Recherche Scientifique CNRS; Universite de Nice Sophia Antipolis UNSA
Current assignee: Centre National de la Recherche Scientifique CNRS; Universite de Nice Sophia Antipolis UNSA
Priority date: 2016-10-11
Filing date: 2017-10-11
Publication date: 2019-08-21
Also published as: EP3526244A1; JP2019534259A; FR3057268A1; US11220527B2; WO2018069640A2; HRP20231567T1; FI3526244T3; DK3526244T3; JP2020502995A; ES2965080T3; FR3068697A1; PT3526244T; WO2018069640A3; US20200190172A1; FR3057268B1; WO2018069641A1; CA3040054A1; US20190270771A1; EP3526244B1; FR3057267A1

Abstract

The present invention relates in particular to polyclonal antibodies directed against peptides, particularly derivatives of the propeptide of sequence QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR (SEQ ID NO 1). The present invention also relates to a method for the production of the aforementioned antibodies and to a method for dosing same. In addition, the present invention relates to a method for detecting/determining depression in an individual and a method for monitoring the effectiveness of a treatment for depression.

Description

DIAGNOSTIC METHOD / DETERMINATION OF TREATMENT EFFICIENCY OF DEPRESSION

Technical area

The present invention relates in particular to polyclonal antibodies directed against peptides, in particular derived from the sequence propeptide QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR

(SEQ ID NO: 1), hereinafter referred to as PE.

The present invention also relates to polyclonal antibodies directed against peptides, especially those derived from the sequence propeptide

QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR

(SEQ ID NO: 1) with the exception of the 12-18 fragment of said propeptide, hereinafter called Mini-Spadine.

The present invention also relates to a method of detecting depression and to a method of monitoring the effectiveness of an anti-depressant treatment.

The present invention advantageously makes it possible to use the peptide assay for diagnosing the response to antidepressant treatment in major depression without being affected by treatment with mature Mini-Spadine or with its modified ends. The present invention finds application particularly in the pharmaceutical industry and in particular in the development of diagnostic tools used in the prevention and / or monitoring of psychiatric diseases, in particular patients suffering or having suffered from depression.

In the description below, references in brackets ([]) refer to the list of references at the end of the text. State of the art

Psychiatric diseases represent a real problem of public health. The most recent work has confirmed the high prevalence of depression: over their lifetime, 20% of women and 10% of men have done, do or will have a depressive episode [1]. Such figures are obviously striking; they are even more so when we look at the major complication of depression, suicide, which amounts to 12000 deaths a year in countries like France [2].

Depression is a very common and often debilitating disease. It can affect up to 20% of the population in industrialized countries. Its origins are diverse and multiple. This pathology affects the psyche as well as the behavior and physiology of the patients. The treatments for depression are also multiple and the mechanisms of action of the drugs used are not clearly established.

The World Health Organization (WHO) predicts that unipolar depression will be the second leading cause of disability in 2020. To the personal and family suffering of depression is added the important social weight of this pathology. Depression is already one of the leading causes of work stoppage, with an economic burden of more than 30 billion euros per year. Despite the therapeutic arsenal available to the medical profession, particularly SSRI ("selective serotonin reuptake inhibitors") and SNRI ("serotonin norepinephrine reuptake inhibitors"), 30% of the depressed population has no treatment. In addition, the time of action of antidepressants is of the order of 3 to 6 weeks and side effects are often important.

In general, it is estimated that 15% of depressed patients die by suicide. In most patients, depression is due to the interaction between a genetic predisposition and environmental factors such as stress or trauma emotional [3]. The disease is common and the antidepressant (AD) market is huge (at least 10 billion euros a year).

Nevertheless, if these antidepressants improve the state of the patients in approximately 70% of the cases, they bring about a complete remission of the disease only in 30 to 40% of them. In addition, almost a third of the subjects treated are resistant to existing treatments. This state of affairs makes it necessary to consider new treatments that can take into account the mechanisms of depression [3].

In the therapeutic arsenal made available to the medical profession, tricyclic antidepressants (TCAs) with amitriptyline and imipramine were first discovered, followed by irreversible and non-selective monoamine oxidase inhibitors (MAOIs) such as phenelzine and pargyline. The adverse effects, in particular the cardiotoxicity of the TCA (especially in case of overdose) and the hypertensive crises of the MAOI (interactions with the food tyramine, the famous "cheese effect") pushed the research towards new molecules of identical therapeutic efficacy but better acceptability.

The notion of selectivity then appeared with the specific inhibitors of the reuptake of norepinephrine (NA) or serotonin (5-hydroxytryptamine or 5HT). Phase III clinical trials have demonstrated for these new molecules an efficacy equivalent to first-generation antidepressants and better safety, especially in case of overdose.

Specific serotonin reuptake inhibitors (SSRIs) and specific norepinephrine reuptake inhibitors

(SNRI) are currently the most used molecules [4-5]. DAs are thus most often associated with facilitation of the transmission of monoaminergic systems.

Although serotonin, norepinephrine and dopamine are definitely implicated, it is now accepted that changes in monoamine levels produced by ADs and the adaptive processes that result from them, in particular the alteration of the sensitivity of some of their receptors alone can not explain the mechanism of action of antidepressants.

Thus, it is difficult to correlate the time of 3 to 6 weeks necessary for the effectiveness of AD with the increase in synaptic levels of monoamines, which occurs as soon as the first administration of the product. In almost half a century, the number of hypotheses about the pathogenesis of depression and its treatment has continued to evolve.

For example, high concentrations of glucocorticoids are generally associated with a negative mood effect, as well as structural alterations of the hippocampus, through decreased brain-derived neurotrophic factor (BDNF) synthesis. ), Excessive secretion of glutamic acid and / or decreased glucose uptake [6]. According to these observations, glucocorticoid synthesis inhibitors and glucocorticoid receptor antagonists exert AD-like effects [7].

Antagonists acting on substance P receptors, particularly NK1, or CRF receptor ("corticotropin-releasing factor"), as well as NMDA receptor antagonists have been developed with some efficiency [8-10].

Various recent studies in stressful situations and models of depression have implicated neurogenesis in the etiology of major depressive disorders [11 -13]. It has been shown that all chronic antidepressant treatments, including electroshock, stimulate the proliferation of progenitor cells at the origin of the neurons of the granular layer of the hippocampus.

Antidepressants are also known to modulate the expression of various factors involved in cell survival and growth, such as CREB, Bcl2, or MAP kinases. However, the functional importance of these neoformed neurons in the pathophysiology of mood disorders remains controversial [14]. All these indications show that depression is a complex disease whose pathophysiology is multifactorial and, consequently, the treatment of such a pathology remains a challenge.

For more than forty years, research on depression and the development of effective drugs has been dominated by the monoaminergic hypothesis. Although monoaminergic neurotransmitters (serotonin, norepinephrine and dopamine) are indisputably involved, the number of hypotheses on the pathophysiology of depression and the mechanisms of action of antidepressants has continued to evolve.

The antidepressant drugs used today produce a range of side effects, including dry mouth, blurred vision and impaired bowel function (diarrhea or constipation). Although many side effects are transient (such as nausea), some appear to be consistent over time (such as sexual effects) and may affect long-term treatment adherence. This is why the search for new molecules acting on newly identified receptors or channels in depression is crucial.

Some proteins, receptors and channels, have been implicated in the molecular mechanisms of depression. This is particularly the case of the neurotensin receptor 3 (NTRS3), originally called sortiline, whose inactivation in mice produces a depression resistance phenotype, similar to that observed in mice invalidated for the channel. basal potassic TREK-1.

The recent STAR ^* D study [15] has pharmacogenically identified TREK-1 as a gene involved in the antidepressant response in humans. This study also suggests the utility of an animal model for the search for antidepressant treatments in the identification of candidate genes for a study in humans. Channel blockers TREK-1 represents a new concept in the field of antidepressant drug design.

Episodes of major depressive disorder (MDD) are treated with different classes of drugs. However, the current main treatments, after several weeks, allow about 35% remissions, and about 30% of patients with major depressive disorder are classified as suffering from drug-resistant depression (DRT)

[1, 16, 17]. Despite significant research efforts aimed at understanding the neurobiological basis of Major Depressive Disorder (MDD), treatments are still based solely on the relatively subjective assessment of symptoms. Because of the low remission rate, the identification of solid biomarkers predicting the clinical course of major depressive disorder and characterizing the extent of treatment outcome is therefore mandatory [3]. Clinical and preclinical studies have identified a number of factors that may serve as putative biomarkers for the diagnosis and treatment of major depressive disorder. However, the utility of any given marker for serving as a clinically useful CTM biomarker is limited by a lack of sensitivity and specificity [18-19].

Recent studies have identified Brain Derived Neurotrophic Factor (BDNF) as a potential biomarker of the clinical response in antidepressant pharmacotherapy and clinical outcome [20-21].

Sortilin is known to control the regulation of intracellular trafficking in the secretory pathway of BDNF neurotrophic factor [22].

In addition, increased serum levels of sortilin have been reported to be associated with CT and correlated with BDNF neurotrophic factor.

[23-24].

Proteins have also been identified in connection with depression. Spadin is a partial peptide (12-28) of the 44 amino acid (PE) propeptide generated from the maturation of [25], also known as the neurotensin-3 receptor [26]. When they are injected intravenously or intraperitoneally in mice, spadine and propeptide demonstrated potent antidepressant (AD) activity by inhibiting TREK-1 potassium channel activity [27] which is a target for the treatment of depression [28].

However, despite intense research on the underlying mechanisms of depressive pathophysiology, reliable biomarkers for assessing the response to antidepressant treatment are still lacking.

There is therefore a real need to find a way to overcome these defects, disadvantages and obstacles of the prior art, in particular a method for physiologically detecting a depressive state, in particular a major depressive disorder.

There is also a real need to find a way to overcome these defects, disadvantages and obstacles of the prior art, including a method for evaluating the effectiveness of a treatment of depression, in particular to reduce costs and improve the effectiveness of treatment of depression, especially a major depressive disorder.

Description of the invention

The inventors have interestingly demonstrated that spadine and propeptide are effective biological markers of depression, in particular major depressive disorder.

Thus, the present invention makes it possible precisely to solve and overcome the obstacles and disadvantages of the aforementioned prior art, in particular the absence of reliable markers of depression and its treatment by providing polyclonal AB1 antibodies binding in particular to spadin and to its propeptide but not to the minispadine.

The subject of the present invention is also a polyclonal anti-peptide antibody derived from PE with the exception of the 12-18 fragment and an antibody directed against the 12-18 sequence of the PE.

The inventors are the first to have developed antibodies for measuring the concentration of spadin and the propeptide and its fragments in a biological sample. In particular, the inventors demonstrated by the antibodies of the invention that two human peptides, namely Propeptide and / or Spadine and Mini-Spadine can be independently measured from samples of human sera. In particular, the subject of the present invention is polyclonal antibodies (AB1) directed against the sequence peptide APLPRWSGPIGVSWGLR (SEQ ID NO 2) (Spadine).

The subject of the present invention is also polyclonal antibodies (AB2) directed against peptides comprising the sequence GVSWGLR (Mini-Spadine).

The inventors have surprisingly demonstrated that the AB1 polyclonal antibody binds to the peptides selected from the group consisting of:

QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR

(SEQ ID NO: 1), APLPRWSGPIGVSWGLR (SEQ ID NO: 2), APLPRWSGPIGVSWGL (SEQ ID NO: 3), LPRWSGPIGVSW (SEQ ID NO: 4) and WSGPI (SEQ ID NO: 5) with the exception of peptide GVSWGLR (SEQ ID NO: 6). ) alone.

The present invention relates to polyclonal antibodies (AB1) directed against peptides in the group comprising: QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR

In particular, the polyclonal antibody (AB1) is directed against the peptides in the group consisting of:

QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR

(SEQ ID NO: 1), APLPRWSGPIGVSWGLR (SEQ ID NO: 2), APLPRWSGPIGVSWGL (SEQ ID NO: 3), LPRWSGPIGVSW (SEQ ID NO: 4), and WSGPI (SEQ ID NO: 5). The inventors have also surprisingly demonstrated that the polydonal AB2 antibody binds to the peptides selected from the group consisting of:

QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR

(SEQ ID NO: 1), APLPRWSGPIGVSWGLR (SEQ ID NO: 2) and GVSWGLR (SEQ ID NO: 6).

The present invention relates to polyclonal antibodies (AB2) directed against peptides in the group comprising QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR

(SEQ ID NO: 1), APLPRWSGPIGVSWGLR (SEQ ID NO: 2) and GVSWGLR (SEQ ID NO: 6).

In particular, the polydonal antibody (AB2) is directed against the peptides in the group consisting of

QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR

(SEQ ID NO: 1), APLPRWSGPIGVSWGLR (SEQ ID NO: 2) and GVSWGLR (SEQ ID NO: 6).

Also, the inventors are the first to have developed antibodies for measuring the concentration of spadin and the propeptide and its fragments in a biological sample. In particular, the inventors have demonstrated that two human peptides, namely Propeptide and / or Spadine and Mini-Spadine can be independently measured from human serum samples.

Hereinafter, propeptide or PE is understood to mean the peptide of sequence:

QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR

(SEQ ID NO1).

Hereinafter, PE 12-28 or spadin is understood to mean the peptide fragment of the amino acid sequence 12 to 28 of the propeptide namely APLPRWSGPIGVSWGLR (SEQ ID NO 2). Hereinafter, PE 12-27 is understood to mean the peptide fragment of peptides 12 to

27 of the propeptide namely: APLPRWSGPIGVSWGL (SEQ ID NO 3). Hereby PE 14-25 is understood to mean the peptide fragment of the amino acid sequence 14 to 25 of the propeptide namely: LPRWSGPIGVSW (SEQ ID NO 4).

In the present case, PE 1 -16 or pro-spadin is understood to mean the peptide fragment of the amino acid sequence 1 to 16 of the propeptide, namely: QDRLDAPPPPAAPLPR (SEQ ID No. 7).

Hereinafter, PE 22-28 or spadine (12-18) or minispadin means the peptide fragment of the amino acid sequence 22 to 28 of the propeptide or the peptide fragment of the peptides 12 to 18 of the spadine, namely: GVSWGLR (SEQ ID NO. 6).

The subject of the present invention is in particular a method for producing the polyclonal antibodies of the invention.

The process for producing the polyclonal antibodies of the invention may comprise the following steps:

at. administering a composition comprising a peptide of sequence APLPRWSGPIGVSWGLR (SEQ ID NO 2) or a peptide of sequence GVSWGLR (SEQ ID NO 6) to an animal b. recovering the antibody of said animal, and

vs. isolation of said antibody.

In the present invention, the composition comprising a peptide of sequence APLPRWSGPIGVSWGLR (SEQ ID NO 2) or a peptide of sequence GVSWGLR (SEQ ID No. 6) for administration to an animal may be an immunogenic composition. It may be for example any immunogenic composition known to those skilled in the art. It may be for example a composition comprising at least one adjuvant.

In the present, by adjuvant is meant any immunogenic adjuvant known to those skilled in the art. It may be for example a Freund's adjuvant, Alum. In the present the animal may be any animal known to those skilled in the art useful for the production of antibodies, it may be for example mouse, rat, rabbit, hen, goat, sheep, donkey, horse.

In the present administration may be carried out by any means and / or method known to those skilled in the art. This may be, for example, administration by injection, for example via a syringe. The administration may be carried out by any route known to those skilled in the art, it may be for example a subcutaneous, intradermal, intramuscular and / or intravenous administration.

In the present the peptide can be administered in step a) at a concentration of 1 to 4 mg per animal, for example from 1.5 mg to 3 mg per animal, for example at a concentration of 2 mg per animal.

In the present the recovery of the antibody can be carried out by any method known to those skilled in the art. This may be for example a sample of the serum of the animal, a bleeding and / or any means / method for recovering the serum of the animal.

In the present isolation of the antibody can be achieved by any method known to those skilled in the art. It may be for example a filtration, for example through a membrane. Filtration of the solution can be carried out via a membrane comprising pores with a pore diameter of 0.2 to 0.45 μm. It may be for example a sterile membrane. The membrane may be for example a cellulose membrane, polyethersulfone, polycaronate, nylon. It may also be a method comprising a step of coagulating the blood, for example for 1 to 3 hours at a temperature of

At 25 ° C, removal of the clot formed, filtration of the solution, centrifugation, and recovery of the supernatant comprising the polyclonal antibodies. The centrifugation can be carried out for a time of 5 to 20 minutes, for example 10 to 17 minutes, for example 15 minutes. The centrifugation can be carried out for example at a speed of 5,000 to 25,000 revolutions per minute, for example from 10,000 to 20,000 revolutions per minute, for example equal to 15,000 revolutions per minute. Those skilled in the art because of their general knowledge will adapt and / or choose the process for obtaining polyclonal antibodies.

In the present invention, the antibodies of the invention may be produced by any suitable method, for example the method described in Lee, BS, Huang, JS, Jayathilaka, LP, Lee, J., Gupta, S. 2016. Antibody production with synthetic peptides. High resolution imaging of cellular proteins, vol 1474 of the series Methods in Molecular Biology, 25-47. [29]

The inventors have also demonstrated that the antibodies according to the invention make it possible to measure the concentration of spadine and of the propeptide and its fragments in a biological sample. In particular, the inventors have demonstrated that two human peptides, namely Propeptide or Spadine and Mini-Spadine can be independently measured from human serum samples.

The present invention therefore also relates to the in-vitro use of the antibodies of the invention for the detection / assay of a peptide selected from propeptide, spadine and mini-spadin in a biological sample.

Antibodies useful for the detection and / or assay of the Propeptide, Spadine and Mini-Spadine are as defined above. It may be, for example, polyclonal antibodies AB1 or AB2.

The inventors have also demonstrated that the antibodies according to the invention make it possible to detect and / or measure the concentration of mini spadine in a biological sample. The inventors have also demonstrated that the antibodies according to the invention advantageously make it possible to detect and / or measure in a sample the concentration of mini spadine which has been injected into a mammal, for example an animal or a human being.

In particular, the inventors have demonstrated that two human peptides, namely Propeptide or Spadine and Mini-Spadine can be independently measured from human serum samples.

The inventors have also demonstrated that the antibodies according to the invention advantageously make it possible, with the AB1 antibody, to detect the measurement of the concentration of PE, spadine but not minispadine, and with the AB2 antibody the detection / measurement of the concentration of PE, spadine and mini-spadin, the absolute value of the difference in concentration between the measurement with the AB2 and AB1 antibody corresponding to the concentration of mini-spadine.

The present invention therefore also relates to the in-vitro use of the antibodies according to the invention for detecting and / or measuring the concentration of the mini-spadin in a biological sample.

As used herein, "biological sample" means any sample obtained from mammals, for example a mammal selected from the group consisting of the order Monotremata, Didelphimorphia, Paucituberculata, Microbiotheria, Notoryctemorphia, Dasyuromorphia, Peramelemorphia, Diprotodontia, Tubulidentata, Sirenia, Afrosoricida, Macroscelidea, Hyracoidea, Proboscidea, Cingulata, Pilosa, Scandentia, Dermoptera, Primates, Rodentia, Lagomorpha, Erinaceomorpha, Soricomorpha, Chiroptera, Pholidota, Carnivora, Perissodactyla, Artiodactyla and Cetacea. It can be for example a human or an animal.

In the present invention, the biological sample may be any biological fluid, for example, it may be a sample of blood, plasma, serum, cerebrospinal fluid (CSF), vaginal mucus, nasal mucus, saliva, urine and / or milk. Preferably the biological sample is a blood sample or a serum sample.

According to the invention, the antibodies of the invention can be used in any suitable assay / detection method known to those skilled in the art. It may be an immunoassay method, for example an ELISA or AlphaLISA assay. This may be for example a method of assay described in the document Immunoanalysis: From theory to the criteria of choice in clinical biology, Catherine Massart, EPD science 2009.

The person skilled in the art by his general knowledge will know how to adapt the conditions / use the methods of assaying / detecting the antibodies according to the invention. The inventors are also the first to have demonstrated that the concentration of the peptide of sequence QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR

(SEQ ID NO: 1) is decreased in an individual with a depressive state.

The inventors have also demonstrated that the antibodies according to the invention make it possible to determine the concentration of the peptide of sequence

QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR (SEQ ID NO: 1) in a biological sample of a healthy control individual and also a depressed individual.

The present invention therefore also relates to the in-vitro use of the antibodies of the invention for the detection of a depressive state from a biological sample of an individual.

In the present "depressive state" or "depressive individual" is meant an individual with a mood disorder accompanied by sadness and mental suffering. It may be, for example, any depressive state known to a person skilled in the art, for example a major depressive disorder (MDD), neurotic depression / psychotic depression, endogenous psychogenic depression / depression and / or reaction depression / depression. autonomous.

In the present "healthy control individual" is meant an individual, for example a human being, not presenting a depressive state, for example a mood disorder accompanied by sadness and moral suffering, and / or having not presented with a depressive state and / or who does not have a major depressive disorder and / or has not presented major depressive disorder.

According to the invention, the term "individual" is intended to mean a mammal chosen from the group comprising the order Monotremata, Didelphimorphia, Paucituberculata, Microbiotheria, Notoryctemorphia, Dasyuromorphia,

Peramelemorphia, Diprotodontia, Tubulidentata, Sirenia, Afrosoricida, Macroscelidea, Hyracoidea, Proboscidea, Cingulata, Pilosa, Scandentia, Dermoptera, Primates, Rodentia, Lagomorpha, Erinaceomorpha, Soricomorpha, Chiroptera, Pholidota, Carnivora, Perissodactyla, Artiodactyla and Cetacea. It can be for example a human or an animal.

In the present invention, the in-vitro detection method can be any in-vitro method known to those skilled in the art adapted for the use of antibodies. It may be for example an immunochemical method, for example a western blot, ELISA, an immunocytochemical process, for example confocal microscopy, immunoelectronic microscopy, and / or an immunohistochemical method.

The present invention also relates to an in vitro method for detecting / determining depression in a patient comprising the following steps

at. Measuring the concentration (Cm) of the sequence peptide QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRG GRWRR (SEQ ID NO: 1) in a biological sample of an individual,

b. Comparison of the concentration (Cm) of the peptide measured in step a) with a reference concentration (Cref) of a healthy individual and calculation of a score (Si) according to the following formula:

S1 = Cm / Cref a value of S1 less than 1 indicating that the individual from which the sample was obtained is depressed.

According to the invention the measurement of the concentration of the sequence peptide QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR

(SEQ ID No. 1) can be carried out according to the method described in Mazella, J. et al. Spadine, a sortilin-derived peptide, targeting rodent TREK-1 channels: a new concept in the antidepressant drug design. PLoS Biol 8, e1000355 (2010) [27] incorporated herein by reference and / or with any suitable immunological method known to those skilled in the art using the polyclonal antibodies of the invention defined above. general knowledge can adapt the conditions for the implementation of known methods by using the polyclonal antibodies according to the invention.

As used herein, the term "healthy reference subject" is intended to mean a mammal, for example a human being, not presenting a depressive state and / or having not presented a depressive state and / or having no depressive disorder. major and / or not having major depressive disorder.

According to the invention, "group of reference subjects" is understood to mean a group making it possible to define a reliable reference value. It may be for example a group comprising at least 2 reference subjects as defined above, for example at least 40 reference subjects. It may be for example a group comprising from 40 to 200 reference subjects.

According to the invention, during the step of comparing the concentration (Cm) of the peptide measured with a reference concentration of the peptide (Cref), the reference concentration of the peptide (Cref) is preferably a concentration measured from d a biological sample of a subject or group of healthy subjects of reference with similar physiological characteristics for example chosen in the group including age, weight, sex, body mass, drug abuse, tobacco, alcohol.

According to the invention the "reference concentration of the peptide" (Cref) may be the concentration of said peptide in a healthy subject and / or group of healthy subjects of reference. For example, when the biological sample is a blood sample, the reference concentration of the peptide may be from 20 to 30 nM, for example from 22 to 28 nM.

The inventors have also demonstrated that the concentration of the peptide sequence QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR

(SEQ ID NO1) evolves during a pharmacological treatment of a depressive state in an individual. In particular, the inventors have demonstrated that an effective pharmacological treatment makes it possible to "restore" the concentration of the sequence peptide QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR

(SEQ ID NO1) and increase its concentration or even a return to a reference concentration.

The inventors have also demonstrated that the antibodies according to the invention make it possible to monitor the efficacy of a treatment on depression by monitoring the concentration of the peptide of sequence QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR

(SEQ ID NO1) in a biological sample of an individual.

The present invention therefore also relates to a method of monitoring the effectiveness of a treatment on depression comprising:

a) Measurement of the C1 concentration of the sequence peptide QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPR GGRWRR (SEQ ID NO1) in a biological sample of a depressed individual

b) Measurement of the concentration C2 of the sequence peptide

QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPR GGRWRR (SEQ ID NO1) in a biological sample of said individual after said treatment,

c) Comparison of the concentration values and calculation of a score (S2) according to the following formula:

S2 = C ₂ / Ci,

a value of S2 greater than 1, 2 indicating that the treatment is an effective antidepressant.

In the present "treatment" is meant for example a medical treatment, for example allopathic, involving the taking of molecules, for example chemical molecules, for example molecules obtained by organic synthesis, molecules of biological origin, for example proteins, molecules from living organisms, for example mammals, microorganisms, plants and / or synthesized by living organisms, for example proteins, nucleic acid molecules, or any other non-chemical treatment, for example example, re-education, or any other treatment based on cell therapy, for example injection of stem cells.

In the present the treatment may be any antidepressant treatment known to those skilled in the art. This may be, for example, treatment using SSRI ("selective serotonin reuptake inhibitors"), for example fluoxetine (Prozac®), paroxetine (Deroxat, Divarius, Paxil®), sertraline ( Zoloft (registered trademark)); citalopram (Seropram, Celexa®), escitalopram oxalate (Seroplex, Cipralex®), indalpine, zimelidine, dapoxetine (Priligy®), fluvoxamine maleate ( Floxyfral (registered trademark)); SNRI ("serotonin norepinephrine reuptake inhibitors"), for example venlafaxine (Effexor®), milnacipran (lxel, Savella®), duloxetine (Cymbalta®), tramadol (Topalgic®), registered trademark)), nefazodone, desvenlafaxine (Pristiq (registered trademark)), tricyclic antidepressants (TCA), for example amitriptyline (Laroxyl (registered trademark), Elavil (registered trademark)), amoxapine (Defanyl (trademark)), clomipramine (Anafranil (trademark), Clomipramine Merck (registered trademark)), dosulpin hydrochloride (Prothiaden, (trademark)), doxepin (Quitaxon, (registered trademark)), imipramine (Tofranil, (trade mark)), maprotiline (Ludiomil, (trade mark)), opipramol (Insidon, (trade mark)), quinupramine (Kinupril, (trade mark)), trimipramine (Surmontil, (trade mark) deposited)), irreversible and non-selective monoamine oxidase inhibitors (MAOIs), for example moclobemide, toloxatone, substance P receptor antagonists, NMDA receptor antagonists, noradrenergic and specific antidepressants Serotoninergic (ANSS).

In the present, the biological sample is as defined above.

In the present, the individual is as defined above. In the present, the measurement of the concentration can be carried out by any method known and adapted to those skilled in the art. It can be a process as defined above.

Advantageously, the present invention makes it possible to measure the amount of peptides derived from Sortilin in a biological sample, for example a blood sample derived from an individual, in particular a mammal, while distinguishing it from the dosage of the mini-spadine which can correspond to the active molecule used in antidepressant treatment.

In addition, the present invention advantageously allows, when mini-spadine (12-18) is used as an antidepressant drug, to monitor and control the effective doses in each individual / patient. In other words, the antibodies of the invention advantageously make it possible to differentiate the endogenous peptides derived from Sortiline from exogenous peptides, mini-spadine (12-18), present in human serum.

The use of antibodies in methods for measuring concentrations according to the invention and / or monitoring the efficacy of a treatment antidepressant advantageously allows with AB1 to measure serum levels of Sepilin-derived peptides from healthy controls / individuals and depressive individuals / patients even if they are treated with mini-Spadine (12-18), and to measure the efficacy of the mini-Spadine bioavailability (12-18) with the difference obtained between the peptides measured with AB2 and the peptides measured with AB1 as described in FIG. 1A.

Advantageously, the present invention makes it possible to detect a depressive state in an individual, for example a patient, with sensitivity and specificity superior to known methods. In addition, the present invention advantageously makes it possible to monitor the effectiveness of a treatment on depression whatever the treatment used. In particular, the method of the invention, in particular by the advantageous use of the antibody AB1, makes it possible to monitor the effectiveness of a treatment on depression, even if the individual and / or the patient is treated with a peptide, for example exogenous, for example a peptide of sequence SEQ ID NO 6.

Advantageously, the present invention makes it possible, by virtue of the use of the AB1 and AB2 antibody, for example to monitor the evolution of the concentration, for example of the peptide of sequence SEQ ID No. 6.

Brief description of the figures:

Figure 1: Figure 1A is a diagram showing the structure-recognition relationships of AB1 and AB2 antibodies as a function of different fragments. In this figure + means that the antibody recognizes the fragment and - means that it is not recognized, n.t. Figure 1B and 1C show the relative affinities for Spadine, PE and analogs assayed by the detection method.

Figure 1D shows the results of the Porsolt Forced Swimming Test (FST), commonly used in the screening of antidepressants. Mice treated with Spadine or partial peptides had a shorter immobility time than those obtained with saline. (In a regular way ANOVA, F _5, ₅ 4 = 20.21, ^**** p <0.0001 for Spadine, the PE fragment 12-27 and 22-28 PE compared to saline treated mice with saline, ^** p = 0.0026 for the PE 14-25 fragment versus saline mice treated with saline, error bars, mean ± SEM.

Figure 2: Figure 2 shows the concentrations of PE in healthy patient sera, untreated CT (T0) and treated CT (T1) patients for 12 weeks. Statistical analysis was performed using the Mann-Whitney test between patients (CT T0, n = 37) and controls (n = 49), and using the Wilcoxon Rank test between untreated (T0 ) and treated (T1). ^* P <0.05; ns not significant. Figure 2 also represents the mean ± SEM of the Montgomery-Âsberg Scale of Depression Assessment (MADRS: Montgomery-Âsberg depression rating scale) for CT patients before (T0) and after (T1) a 12-week treatment (bars). ^*** P <0.001.

Figure 3: Figure 3 is a histogram showing the stability of PE in human serum. Sera with or without EDTA (n = 7 for each matrix) from healthy blood donors were tested for stability at room temperature for up to 24 hours. The concentration of PE for each incubation period was determined using an AlphaLISA assay method. Mean concentrations at different time points are expressed as mean ± SEM. ANOVA indicated no difference between serum group without EDTA or serum with EDTA. The unpaired t-test for the PE concentration between serum and serum-EDTA indicated no significant difference for each time.

EXAMPLES Example 1 Spadine Detection Method and Detection of Major Depressive Disorder (MDD)

In the example below the experiments were carried out with the products and processes as mentioned below. In the present example, ambient temperature means a temperature of 19 to 25 ° C, for example equal to 22 ° C.

Animals

The experiments were carried out on males of 20-25g C57BL / 6J 8-10 weeks old (January France Breeding) in accordance with the policies on the care and use of laboratory animals of the European Community legislation 2010/63 / EU . The local ethics committee (CIEPAL) approved the protocols used in this study (protocol number 00.893.02).

Antibody and preparation of the biotinylated peptide.

Peptides were synthesized by GeneCust (Dudelange, Luxembourg). Rabbit polyclonal antibodies against spadin (YAPLPRWSGPIGVSWGLR (SEQ ID No. 8)) were prepared by Eurogentec (Seraing, Belgium). The antibody used was the AB1 antibody as mentioned above. Spadine (5.4 mg, 2.7 mmol) was solubilized in 1.5 ml of 25 mM phosphate buffer, pH 6.7. Biotin N-hydroxysuccinimide (13.5 mmol) resuspended in 700 μl of 70% acetonitrile / 30% dimethylformamide was added to the spadin solution and incubated overnight at room temperature. Biotinylated spadin was purified by high performance liquid chromatography (HPLC) using a Waters apparatus equipped with a semi-preparative Lichrosorb RP18 column. The biotinylated spadin (elution at 27 min), identified by mass spectrometry, was collected, quantified by its absorption at 280 nm and lyophilized in aliquots.

Alpha-Lisa Test (registered trademark) According to the principles of the AlphaScreen technology (trademark (Perkin Elmer)), streptavidin-donating microbeads were recognized by biotinylated Spadine, whereas the accepting microspheres with rabbit anti-IgG were bound by the anti-human antibodies. Spadine. When both microbeads (acceptors and donors) were nearby, the signal was produced by a molecular interaction occurring between the binding partners on the beads. The propeptide present in the serum sample was able to interfere with this interaction leading to competition. Calibration curves were obtained by incubating, in 96-well plates, 10 nM of biotinylated Spadine with the anti-Spadine antibody (1: 1000) in AlphaLisa buffer (registered trademark) in the absence or presence of increasing concentrations of Spadine (10 ^{~ from 11} to 10 ^"6 M) for 1 h at room temperature. After the addition of donor and acceptor beads and further incubation for 2 hours at room temperature, the plates were read using the Enspire device (Perkin) For serum measurements, the same volume of serum was added instead of unlabeled spadin The amount of propeptide was determined from its percent signal inhibition and calculated using the standard curve.

Porsolt Forced Swimming Test (FST).

After intravenous injection of saline solution or various peptides tested, the mice (n = 10-12 per group) were individually placed in a cylinder (height: 30 cm, diameter: 15 cm) filled with water to a depth of 12 cm (temperature: 22 ± 1 ° C) for 6 min. The total duration of immobility was recorded during the last 4 minutes (Porsolt et al., 1977). Human blood samples

The cohort of control individuals consisted of 49 unrelated healthy volunteers who were selected for Diagnosis of DSM-IV Axis I disorders by expert psychologists using the Mini-International Neuropsychiatry Interview (MINI) [30]. Only healthy volunteers with no history of abuse or addiction to drugs or alcohol and no personal or family history of first-degree psychiatric disorders were recruited into the study. In addition, the absence of relevant neurological diseases namely epilepsy, Parkinson's syndrome, was mandatory for inclusion in the study. Finally, subjects who scored less than 27/30 in the Mental State Examination Mini (MMSE) were excluded from the study.

The cohort of patients consisted of 37 patients with major depressive disorder (MDD) with moderate to severe depression who met the criteria for the classification system of the Diagnostic and Statistical Manual IV for Mental Disorders (DSM-IV). The diagnosis of unipolar depression was confirmed by a structured clinical interview for DSM-IV Axis I Disorders (SCID-I). Exclusion criteria were: a) mental retardation or cognitive impairment; b) a life history of schizophrenic, schizo affective, or bipolar disorder; c) personality disorder, substance abuse, alcohol abuse or addiction, obsessive-compulsive disorder, or post-traumatic stress disorder as the primary diagnosis; and d) comorbidity with an eating disorder.

No patient showed psychotic symptoms; 1 1 (29.7%) showed current comorbidity in Axis I (Generalized Anxiety Disorder (GAD)), panic attacks, panic disorder or anxiety disorder not otherwise specified (NOS ), 2 (5.4%) had symptoms of Axis II disorder (dependent personality disorder) and no alcohol abuse as a secondary diagnosis (the total number exceeded the number of subjects in because of the presence of comorbidities).

All patients were either "naïve drugs", and had never received prior treatment with antidepressant medication, or "no drugs" ie they were previously treated with one or two antidepressants, but had a weaning period for at least 2 weeks before starting the new antidepressant treatment. All patients were treated as monotherapy: thirty-five patients were treated with serotonin reuptake inhibitors (SSRIs) ie escitalopram, and the remaining patients were treated with selective serotonin-noradrenaline inhibitors ( SNRI) (venlafaxine, duloxetine, tricyclic antidepressants (TCA) (nortriptyline) or noradrenergic and serotonin-specific antidepressants (ANSS) (mirtazapine) .The severity of the disease was assessed by the Montgomery and Asberg Depression Scale ( MADRS) before the start of the new antidepressant treatment (T0) and after 12 weeks of treatment (T1) All the socio-demographic, clinical and pharmacological characteristics of patient treatment are presented in Table 1 below.

Table 1: Demographic and Clinical Characteristics of Control Groups and CT Patients

MDD checks P-

Characteristics

(N = 49) (N = 37) value

Age (years), average (SD) 44.9

45.1 (12.1) 0.95

(13,1)

Gender (% F) 65.3 75.7 0.30

Education (years), average (SD) 13.8 (5.1) 1 1, 9 (2.8) 0.04 ^*

BMI (Body Mass Index) 23.6 (3.1) 24.4 (2.8) 0.26

% smoker 16.3 37.8 0.02 ^*

Beginning age (years), average 40.9

(SD) (1 1, 1)

% MADRS at T0, average not 25.5 (5.2)

(SD)

% of AMADRS at T1, mean (SD) -60.1

(33,1)

% recurrence of CT 27.0

% of severe vs, moderate MDD 8.1

% comorbidity with disorders 5,4

personality

% comorbidity with disorders 29,7

anxiety

% comorbidity with alcohol abuse 0.0

% administration of SSRIs ^* 75.7

(Escitalopram)

% administration of SNRIs ^* 5.4

(Venlafaxine)

% administration of SNRIs ^* 2,7

(Duloxetine)

% administration of TCAs ^* 10.8

(Nortriptyline)

% administration of NaSSAs ^* 5.4

(Mirtazapine) ^has the total number could exceed the number of subjects due to the presence of multiple drug administrations

* Indicates significant p values (<0.05)

For patients and controls, venous blood samples were collected in the morning between 8:00 and 09:00 in tubes without anticoagulant. The serum was separated by centrifugation (1620 g for 15 minutes). Blood samples for PE measurements were collected at each time point.

The studies have been approved by the local ethics committees (CEIOC IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia N: 50/2008 and Verona Province Ethics Committee N: 4997 / 09.1 1 .01), and written informed consent was obtained.

Statistics

Results are expressed as mean ± SEM

(SEM). Statistical analyzes were performed using GraphPad (version 6.0). Demographic and clinical characteristics in patient samples have been described either in quantitative terms of mean ± standard deviation (SD) or as proportions. After verifying normality, t Student tests were used where appropriate to evaluate differences in quantitative variables while ANOVA was used to calculate possible group differences for continuous variables. (Fig. 1 D). The clinical and biological changes that occur during drug treatments were analyzed using a general linear model in a design of repeated measures over time (T0, T1) as an intra-subject factor, and the correction of Greenhouse-Geisser has been applied. Non-parametric tests of Mann-Whitney U and Wilcoxon Ranks were used to evaluate differences between groups.

Results

The specificity of the antibodies used has been studied. In particular, the specificity of the antibodies used in the assay method has been studied. A series of partial sequences of human PE was developed to characterize the specificity of the antibodies used (Figure 1A). As shown in Figure 1B, Spadine and all PE were recognized by anti-Spadine antibodies with an IC50 of 2.50 nM, respectively (95% CI: 1.85 to 3.37; = 6) and 2.18 nM (95% CI: 1.53 to 3.09, n = 3). Neurotensin (NT, black squares) and somatostatin-14 (SS14, white squares) were not recognized by the antibodies.

As shown in Figure 1C the 14-25 fragment of PE is bound to the antibodies with an IC50 of 1.73 nM, whereas NC50 of the 12-27 fragment of the PE was 3.44 nM. The PE fragment 22-28 (mini-spadin, white squares) and PE fragment 1 -16 (black squares) are not recognized by the antibodies. Each point corresponds to the mean ± SEM of 3 to 6 independent experiments.

Thus, from the analysis of the recognition structure of these peptides (FIG. 1B and 1C), it has been observed that PE, spadine, PE 12-27 and PE 14-25 have been recognized by the antibodies having identical affinities, whereas PE 1 -16 and PE 22-28 (mini-spadine) were not recognized. Unrelated peptides such as neurotensin or somatostatin were unable to interfere with the assay procedure (Figure 1B and 1C). These results made it possible to identify the epitope of the antibody used, namely the WSGPI sequence.

A polyclonal composition for specifically measuring the concentration of propeptide and spadin has thus been obtained.

The anti-depressant activity of the analogs was tested using the FST. Figure 1D shows the test results of Porsolt forced swimming (FST). As shown, mice treated with Spadine or partial peptides had a shorter immobility time than those obtained with mice receiving saline. (Ordinarily ANOVA, F5.54 = 20.21, ^**** p <0.0001 for Spadine, PE fragment 12-27 and PE 22-28 compared to saline mice treated with saline solution, ^** p = 0.0026 for PE fragment 14-25 versus saline mice treated with saline, error bars, mean ± SEM.) Thus, as shown in FIG.

1D, all peptides bearing the epitope sequence (PE, Spadine, PE 14-25 and PE 12-27) demonstrated antidepressant activity in the Porsolt swim test similar to the antidepressant activity of the spadin itself. The relative affinities and range of detection values of the peptides tested were summarized in Table 2 below. These results indicated that both PE, Spadine and the major partial sequences of PE corresponded to active peptides that can be quantified (Spadine-like immunoreactivity, SLI). A study of PE stability in human serum was performed. Figure 3 is a histogram showing the stability of PE in human serum. Sera with or without EDTA (n = 7 for each matrix) from healthy blood donors were tested for stability at room temperature (22 ° C) up to 24 h. The concentration of PE for each incubation period was determined using an AlphaLISA assay method. In FIG. 3, the average concentrations at the different time points are expressed as an average ± SEM. The determination of ANOVA was performed and indicated no difference between serum group without EDTA or serum with EDTA. The unpaired t-test for the PE concentration between serum and serum-EDTA indicated no significant difference for each time. Therefore, the stability of the SLI measurement up to 24 hours at room temperature (Figure 3) validated subsequent measurements in mice and humans.

Table 2: Relative affinities of PE and partial peptide analogues recovered in the AlphaLISA assay.

Peptide sequence Name IC50 Quantity Activity

(nM) detectable antidepressant

(Ng / mL)

Propeptide (PE) 44AA PE1 -44 2.18 1, 5 - 400 Yes (SEQ ID NO 1) or PE (1, 53- 3.09)

APLPRWSGPIGVSWGLR spadine 2.50 1, 5 - 400 Yes (SEQ ID NO 2) PE12-28 (1.85- 3.37)

QDRLDAPPPPAAPLPR PE1 -16> no No (SEQ ID NO 7) (pro 1000 detectable

spadine)

LPRWSGPIGVSW (SEQ PE14-25 3.44 0.8 - 300 Yes ID NO 4) (2.44-4.55) APLPRWSGPIGVSWGL PE12-27 1, 73 1, 5 - 400 Yes (SEQ ID NO: 3) (1, 34-2.24)

GVSWGLR (SEQ ID NO PE22-28> No Yes 6) 1000 detectable

To better understand changes in PE levels in the pathology of depression, all human sera from a clinical center in Italy were collected and a measure of the concentration of PE performed.

In the cohort, it was determined that PE concentration was significantly lower in patients with major depressive disorder (MDD) (18.9 ± 1, 3 nM) than in healthy controls (23.7 ± 1, 5nM) (z = -2.1 1, p = 0.035) (Figure 2). Patients with major depressive disorder treated for 12 weeks with antidepressants showed a significantly higher PE level than before treatment (21.0 ± 1.5 nM) (z = -1.98, p = 0.047) (Figure 2). The effectiveness of the treatment was confirmed by the significant decrease in the Montgomery-Besberg Depression Rating Scale (MADRS: Montgomery-Asberg depression rating scale) MADRS (Figure 2). In particular, there was a 2.5-fold decrease in the score after 12 weeks of treatment (T1).

Discussion

As demonstrated in this example, the antibody used recognizes that a series of peptides derived from human PE, as well as the PE itself and not unrelated peptides such as neurotensin or somatostatin.

As demonstrated, the peptides recognized by the antibody have antidepressant activities as demonstrated by the forced swimming test in mice (FIG. 1D). This example clearly demonstrates that the concentration of PE in serum is downregulated, i.e., is lower in patients with major depressive disorder (MDD) compared to healthy subjects. This example also clearly demonstrates that the concentration of PE varies with antidepressant treatment and in particular allows patients with a concentration of propeptide lower than normal to return to normal in correlation with the clinical course of treatment of depression.

This example clearly demonstrates that the propeptide concentration is a marker of the depressive state and can be used to detect / determine a depressive state, for example a major depressive disorder, and also to determine / monitor the effectiveness of a treatment. antidepressant. The serum PE concentration is an additional marker to the known serum level marker, for example BDNF [31].

As demonstrated, the serum PE concentration is significantly lower in patients with CT, and taking into account that the peptides measured in this example were generated from sortilin, a study of the possible correlation with the level and / or the expression sortiline was performed. Interestingly, the change in the expression of sorbitin in the blood has already been observed in patients with CT. First, in the mononuclear cells of the blood, the gene expression of sortilin decreases with clinical improvement [23], while the sorbitin is overexpressed in patients suffering from CT, especially in non-responders [32]. ].

On the other hand, in the human serum, the increase of the level of the spell is associated with the state of depression [24].

The results show that the serum concentration of PE varies according to mood symptoms in humans, and is an additional biomarker of depression, in addition to or in replacement of BDNF and / or VEGF [24]. In addition, the possible subsequent use of Spadine as an effective therapy against pathology, a Peptide for which serum level could also be controlled, will certainly greatly improve the prospects for new strategies to effectively manage depression worldwide.

This example clearly demonstrates that the present invention makes it possible in particular to detect / determine the depressive state, monitor the effectiveness of a treatment on depression, measure the concentration and / or detect the propeptide or PE of sequence: QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGR WRR (SEQ ID NO1) in a biological sample, in particular using the AB1 antibody in Example 1 described above.

Example 2 Method for Obtaining AB1 and AB2 Antibodies and Determining Their Specificities

Both antibodies were prepared from rabbit and the volumes obtained are about 200 ml each. In the experimental protocols, these antibodies are used at dilutions of 1/1000 and 1/5000. The techniques used to measure the level of the pro-peptide in the blood are multiple, immunological or fluorescent. The peptide assay can be obtained with 100 μΙ of blood and reproducible.

Figure 1 shows the binding specificities of antibody functions.

In particular, antibodies AB1 and AB2 were obtained by injection into two rabbits, namely Oryctolagus cuniculus, spadin coupled to KLH (Keyhole limpet hemocyanin) protein (AB1) or mini-spadine (PE 22 -28) coupled to KLH (AB2) at day 0, J0 (1 mg), day

7, J7 (1 mg), day 14, 14 (1 mg) and day 29, J29 (1 mg). Blood samples, ie 20 ml were taken at day 27 (day 27) and day 42 (day 42). The polyclonal serum was obtained by centrifugation at 1200xg for 10 min. The polyclonal serum corresponding to the supernatant was recovered. The immune response was monitored by ELISA for each antibody. The antibodies contained in the serum were stored at -20 ° C. Example 3: Method for Detecting / Monitoring the Efficacy of a Treatment on Depression

In the example below the experiments are carried out with the polyclonal AB1 antibodies described in Examples 1 and 2 above.

A measurement of the serum concentration of the sequence peptide

QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR is carried out according to the method described in Example 1.

The cohort of control individuals was composed of 49 unrelated healthy volunteers who were selected for diagnosis of DSM-IV Axis I disorders by expert psychologists using the Mini-International Neuropsychiatry Interview (MINI) [30]. Only healthy volunteers with no history of abuse or addiction to drugs or alcohol and no personal or family history of first-degree psychiatric disorders were recruited into the study. In addition, the absence of relevant neurological diseases namely epilepsy, Parkinson's syndrome, was mandatory for inclusion in the study. Finally, subjects who score less than 27/30 in the Mental State Examination Mini (MMSE) were excluded from the study.

An average of the concentration measured in the samples of control subjects was carried out and the value obtained corresponded to the reference concentration (Cref) of a healthy individual.

A measurement of the serum concentration (Cm) of the sequence peptide

QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR was performed in a sample of an individual.

A comparison of the concentration (Cm) of the peptide measured in the depressed subject with the reference concentration (Cref) of the healthy subject is performed by calculating a score (S1) according to the following formula:

S1 = Cm / Cref, a value of S1 less than 1 indicating that the individual from which the sample was obtained is depressed. In this example a follow-up of the effectiveness of a treatment on the depression is carried out.

The patient cohort consisted of patients with major depressive disorder (MDD) with moderate to severe depression who met the criteria of the classification system of the Diagnostic and Statistical Manual IV for Mental Disorders (DSM-IV). The diagnosis of unipolar depression was confirmed by a structured clinical interview for DSM-IV Axis I Disorders (SCID-I). Exclusion criteria were: a) mental retardation or cognitive impairment; b) a life history of schizophrenic, schizo affective, or bipolar disorder; c) personality disorder, substance abuse, alcohol abuse or addiction, obsessive-compulsive disorder, or post-traumatic stress disorder as the primary diagnosis; and d) comorbidity with an eating disorder.

A measurement of the serum concentration of the sequence peptide

QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR is carried out according to the method described in Example 1 prior to the administration of any anti-depressant treatment. In particular the measurement of the concentration was carried out from a blood sample. The concentration was measured independently for each patient prior to any treatment. The measured concentration was 19 nM.

Patients were then treated as monotherapy with serotonin reuptake inhibitors (SSRIs), selective serotonin-noradrenaline inhibitors (SNRIs), tricyclic antidepressants (TCAs) or serotonin-specific and serotonergic antidepressants (ANSS). .

The mode of administration, frequency, dosage and dosage corresponded to that of the instructions for use or the indications mentioned in the Vidal dictionary.

A measurement of the serum concentration of the sequence peptide QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR is performed according to the method described in Example 1 after 12 weeks of treatment was performed independently for each patient. The measured concentration was 21 nM.

A comparison of the concentration (Cm) of the peptide measured in the depressed patient with the reference concentration (Cref) was performed by calculating a score (S1) according to the following formula: S1 = Cm / Cref, a value of S1 less than 1 indicating that the individual from which the sample was obtained is depressed.

A comparison of the measured concentration values was measured by calculating a score (S2) according to the following formula: S2 = C2 / C1 (C1, concentration before treatment, C2, concentration after treatment). When the S2 value was greater than 1, 2 the administered treatment was an effective antidepressant.

This result was confirmed by an evaluation of the psychological state of the patient via an evaluation of the criteria of the classification system of the Diagnostic and Statistical Manual IV of Mental Disorders (DSM-IV).

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Claims

1. Polyclonal antibodies (AB1) directed against the APLPRWSGPIGVSWGLR sequence peptide (SEQ ID NO 2) (Spadine), said antibody binds to the peptides selected from the group consisting of: QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR

(SEQ ID NO: 1), APLPRWSGPIGVSWGLR (SEQ ID NO: 2), APLPRWSGPIGVSWGL (SEQ ID NO: 3), LPRWSGPIGVSW (SEQ ID NO: 4), and WSGPI (SEQ ID NO: 5) with the exception of the peptide GVSWGLR (SEQ ID NO. 6) alone.

2. Polyclonal antibodies (AB2) directed against peptides comprising the sequence GVSWGLR (SEQ ID No. 6) (Mini-Spadine).

The polyclonal antibody of claim 2, said antibody binds to the peptides selected from the group consisting of: QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR

(SEQ ID NO: 1), APLPRWSGPIGVSWGLR (SEQ ID NO: 2) and GVSWGLR (SEQ ID NO: 6).

The method of making the antibodies of claim 1 comprising:

at. administering a composition comprising a peptide of sequence APLPRWSGPIGVSWGLR (SEQ ID NO 2) to an animal b. recovering the antibody of said animal, and

vs. isolation of said antibody.

A method of making the antibodies according to any one of claims 2 or 3 comprising:

at. administering a composition comprising a peptide of sequence GVSWGLR (SEQ ID No. 6) to an animal

b. recovering the antibody of said animal, and

vs. isolation of said antibody

6. In vitro use of antibodies according to any one of claims 1 to 3 for the detection / assay of spadin in a biological sample.

7. In vitro use of antibodies according to any one of claims 2 to 3 for the detection of mini-spadin in a biological sample.

8. In vitro use of the antibodies according to any one of claims 1 to 3 for the detection of peptide sequence SEQ ID NO 1 from a biological sample of an individual for the detection of a depressive state.

9. In-vitro method for detecting / determining depression in a patient comprising the following steps

at. Measuring the concentration (Cm) of the QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGR WRR sequence peptide (SEQ ID NO: 1) with antibodies according to any one of Claims 1 to 3 in a biological sample of a depressed individual,

b. Comparison of the concentration of the peptide measured in step a) with a reference concentration (Cref) of a healthy individual and calculation of a score (Si) according to the following formula:

S1 = Cm / Cref

a value of S1 less than 1 indicating that the individual from which the sample was obtained is depressed.

10. A method of monitoring the efficacy of a treatment on depression comprising:

a) Measurement of the concentration C1 of the sequence peptide QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPR GGRWRR (SEQ ID NO. 1) with antibodies according to one any of claims 1 to 3 in a biological sample of an individual,

b) measuring the concentration C2 of the sequence peptide QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPR GGRWRR (SEQ ID No. 1) with antibodies according to any one of claims 1 to 4 in a biological sample of said individual after said treatment,

S2 = C ₂ / Ci,