Patent Application
20200190172
The present invention relates in particular to polyclonal antibodies directed against peptides, particularly derived from the propeptide of sequence QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR (SEQ ID NO 1) hereinafter called PE.
The present invention also relates to polyclonal antibodies directed against peptides, particularly those derived from the propeptide of sequence QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR (SEQ ID NO 1) with the exception of fragment 12-18 of said propeptide, hereinafter called Mini-Spadin.
The present invention also relates to a method for detecting depression and a method for monitoring the effectiveness of an antidepressant therapy.
Advantageously, the present invention enables peptide assay to be used to diagnose the response to antidepressant therapy in major depression without being affected by treatment with mature mini-spadin or with its modified extremities. The present invention is applicable particularly in the pharmaceutical industry and particularly in the development of diagnostic tools used in the prevention and/or surveillance of psychiatric illnesses, in particular of patients suffering or having suffered from depression.
In the description below, the references in brackets ([ ]) refer to the list of references provided at the end of the text.
Psychiatric illnesses represent a real problem for Public Health. The most recent works have confirmed the high prevalence of depression: throughout their lives, 20% of women and 10% of men have experienced, are experiencing or will experience a depressive episode [1]. These figures are clearly significant; they are even more so when considering the major complication of depression, suicide, which accounts for 12,000 deaths per year in a country like France [2].
Depression is a very common and often disabling illness. It can affect up to 20% of the population in industrialized countries. Its origins are numerous and varied. This pathology affects both the psyche and the behavior and physiology of patients. Treatments for depression are also numerous 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 cause of disability in 2020. The personal and family suffering that depression represents is added to the huge social burden of this pathology. Depression already represents one of the main causes for time off work, with an economic burden amounting to over 30 billion euros per year. Despite the therapeutic arsenal available to medical professionals, in particular SSRI (Selective Serotonin Reuptake Inhibitors) and SNRI (Serotonin Norepinephrine Reuptake Inhibitors), no treatment exists for 30% of the depressed population. Furthermore, the time to onset of action of antidepressants is in the order of 3 to 6 weeks and the side effects are often severe.
Generally speaking, it is estimated that 15% of depressed patients commit suicide. For most sufferers, depression is due to the interaction between a genetic predisposition and environmental factors such as stress or emotional trauma [3]. The illness is common and the market for antidepressants (AD) is huge (at least 10 billion euros per year).
However, although these antidepressants improve the condition of patients in around 70% of cases, they result in a complete remission of the illness in only 30 to 40% of them. Moreover, almost a third of the subjects treated resist existing treatments. This therefore means that new treatments must be envisaged, capable of taking into account the mechanisms of depression [3].
In the therapeutic arsenal available to medical professionals, tricyclic antidepressants (TCAs) with amitriptyline and imipramine were the first to be discovered, followed by monoamine oxidase inhibitors (MAOIs), irreversible and non-selective, such as phenelzine and pargyline. Adverse effects, particularly the cardiotoxicity of TCAs (especially in the event of an overdose) and the hypertensive crises of MAOIs (interactions with alimentary tyramine, the famous ‘cheese effect’) have pushed research towards new molecules with identical therapeutic effectiveness but better acceptability.
The notion of selectivity then appeared with noradrenaline (NA) or serotonin (5-hydroxytryptamine or 5HT) reuptake selective inhibitors. For these new molecules, Phase III clinical trials have shown an effectiveness equivalent to first-generation antidepressants, as well as improved safety, particularly in the event of an overdose.
Selective serotonin reuptake inhibitors (SSRI) and selective noradrenalin reuptake inhibitors (SNRI) are currently the most widely used molecules [4-5]. ADs are thus most often associated with a facilitation of transmission in monoaminergic systems.
Although serotonin, noradrenalin and dopamine are certainly involved, it is now agreed that changes to the rates of monoamines produced by ADs and the adaptive processes resulting therefrom, particularly the alteration of the sensitivity of some of their receptors, cannot alone explain the mechanism of action of antidepressants.
Thus, it is difficult to correlate the period of 3 to 6 weeks necessary for ADs to become effective with the increase in the synaptic rates of monoamines, which occurs from the first administration of the product. For nearly half a century, the number of theories on the pathogenesis of depression and its treatment has continued to grow.
For example, high concentrations of glucocorticoids are generally associated with a negative effect on mood, as well as structural alterations of the hippocampus, through a reduction in the synthesis of BDNF (Brain-Derivated Neurotrophic Factor), an excessive secretion of glutamic acid and/or a reduction in glucose uptake [6]. According to these observations, inhibitors of the synthesis of glucocorticoids and antagonists of glucocorticoid receptors produce AD type effects [7].
Antagonists acting on substance P receptors, particularly NK1, or the CRF receptor (Corticotropin-Releasing Factor), as well as antagonists of NMDA receptors have been developed with a certain degree of effectiveness [8-10].
Various recent studies carried out in situations of stress and some models of depression have involved neurogenesis in the etiology of major depressive disorders [11-13]. It has been proved that all antidepressant chronic treatments, including electroshock therapy, stimulate the proliferation of progenitor cells that generate the neurons in the granular layer of the hippocampus.
It is also known that antidepressants modulate the expression of different factors involved in the survival and growth of cells, such as CREB, Bcl2 or MAP-kinases. However, the functional importance of these neoformed neurons in the physiopathology of mood disorders remains controversial [14].
All of these indications show that depression is a complex illness with a multifactorial physiopathology and, consequently, the treatment of such a pathology remains a challenge.
For more than forty years, research into depression and the development of effective drugs have been dominated by the monoaminergic theory. Although monoaminergic neurotransmitters (serotonin, noradrenaline and dopamine) are undoubtedly involved, the number of theories on the physiopathology of depression and the mechanisms of action of antidepressants have continued to grow.
The antidepressant drugs used today produce a range of adverse effects, including dryness of the mouth, blurred vision and changes in intestinal function (diarrhea or constipation). Although many side effects are temporary (such as nausea), some appear to be constant over time (such as sexual effects) and risk affecting assiduity to treatment over the long term. This is the reason why research into new molecules acting on newly identified receptors or channels in depression is crucial.
Some proteins, receptors and channels have been involved in the molecular mechanisms of depression. This is particularly the case of the Neurotensin Receptor 3 (NTRS3), originally called sortilin, the inactivation of which in mice produces a depression-resistant phenotype, similar to that observed in invalided mice for the TREK-1 background potassium channel.
The recent STAR*D study (Sequenced Treatment Alternatives to Relieve Depression) [15] has pharmacogenetically identified TREK-1 as a gene involved in the antidepressive response in humans. This study also suggests the use of an animal model to research antidepressant treatments in the identification of candidate genes for a study on humans. TREK1 channel blockers therefore represent a new concept in the field of design of antidepressant drugs.
Episodes of Major Depressive Disorder (MDD) are treated with different classes of drugs. However, existing main treatments, after several weeks, enable around 35% of remissions, and around 30% of patients with major depressive disorder are classed as suffering from treatment-resistant depression (TRD) [1, 16, 17]. Despite intensive research aimed at understanding the neurobiological bases of major depressive disorder (MDD), treatments are still based soled on the relatively subjective evaluation of symptoms. Due to the low rate of remission, the identification of reliable biological markers predicting the clinical development of major depressive disorder and characterizing the extent of the result of treatment thus appears to be mandatory [3]. Clinical and preclinical trials have identified a certain number of factors that can serve as putative biomarkers for the diagnosis and treatment of major depressive disorder. However, the usefulness of any given marker to serve as clinically useful biomarker of MDD is limited by a lack of sensitivity and specificity [18-19].
Recent studies have identified the Brain Derived Neurotrophic Factor (BDNF) as a potential biomarker of clinical response in antidepressive pharmacotherapy and clinical outcome [20-21].
Sortilin is known to control the regulation of the intracellular traffic on the secretory pathway of BDNF [22]. Furthermore, increased serum levels of sortilin have been indicated as being associated with MDDs and correlated with BDNF [23-24].
Proteins have also been identified in connection with depression. Spadin is a partial peptide (12-28) of the propeptide of 44 amino acids (PE) generated from the maturation of sortilin [25], also called neurotensin receptor-3 [26]. When injected intravenously or intraperitoneally into mice, spadin and propeptide have shown powerful antidepressant (AD) activities by inhibiting the activity of the TREK-1 potassium channel [27], which is one of the goals in the treatment of depression [28].
However, despite intensive research into the underlying mechanisms of the pathophysiology of depression, reliable biomarkers to evaluate the response to antidepressant treatment are still lacking.
A real need therefore exists to find a way to address these shortcomings, drawbacks and obstacles of the prior art, in particular a method enabling a depressive state, particularly a major depressive disorder, to be detected physiologically.
There is also a real need to find a way to address these shortcomings, drawbacks and obstacles of the prior art, in particular a method enabling the effectiveness of a treatment for depression to be evaluated, in order particularly to reduce costs and improve the efficiency of the treatment for depression, particularly a major depressive disorder.
The inventors have interestingly shown that spadin and propeptide are effective biological markers of depression, particularly of major depressive disorder.
Thus, the present invention precisely resolves and overcomes the above-mentioned obstacles and drawbacks of the prior art, in particular the absence of reliable markers of depression and its treatment, by providing AB1 polyclonal antibodies bound particularly to spadin and its propeptide but not to mini-spadin.
The present invention also relates to an anti-peptide polyclonal antibody derived from PE with the exception of fragment 12-18 and an antibody directed against the 12-18 sequence of PE.
The inventors are the first to have developed antibodies enabling the measurement of the concentration of spadin and propeptide and its fragments in a biological sample. In particular, the inventors have shown, thanks to the antibodies of the invention, that two human peptides, namely Propeptide and/or Spadin and Mini-Spadin, can be independently measured on the basis of samples of human serums. In particular, the subject matter of the present invention relates to polyclonal antibodies (AB1) directed against the peptide of sequence APLPRWSGPIGVSWGLR (SEQ ID NO 2) (Spadin).
The present invention also relates to polyclonal antibodies (AB2) directed against peptides comprising the sequence GVSWGLR (Mini-Spadin).
Surprisingly, the inventors have shown that the AB1 polyclonal antibody binds to the peptides chosen from the group comprising: 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 sole exception of the peptide GVSWGLR (SEQ ID NO 6).
The present invention relates to polyclonal antibodies (AB1) directed against the peptides in the group comprising: 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 sole exception of the peptide GVSWGLR (SEQ ID NO 6).
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).
Surprisingly, the inventors have also shown that the AB2 polyclonal antibody is bound to the peptides chosen from the group comprising: 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 the peptides in the group comprising QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR (SEQ ID NO 1), APLPRWSGPIGVSWGLR (SEQ ID NO 2) and GVSWGLR (SEQ ID NO 6).
In particular, the polyclonal 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).
Furthermore, the inventors are the first to have developed antibodies that enable measurement of the concentration of spadin and propeptide and its fragments in a biological sample. In particular, the inventors have shown that two human peptides, namely propeptide and/or spadin and mini-spadin can be independently measured from samples of human serum.
In the present invention, propeptide or PE means the peptide of sequence:
In the present invention, 12-28 PE or spadin means the peptide fragment of the 12 to 28 propeptide amino acid sequence, namely APLPRWSGPIGVSWGLR (SEQ ID NO 2). In the present text, 12-27 PE means the peptide fragment of peptides 12 to 27 of the propeptide, namely:
In the present invention 14-25 PE means the peptide fragment of the 14 to 25 propeptide amino acid sequence, namely: LPRWSGPIGVSW (SEQ ID NO 4).
In the present invention 1-16 PE or pro-spadin means the peptide fragment of the 1 to 16 propeptide amino acid sequence, namely:
In the present invention 22-28 PE or spadin (12-18) or mini-spadin means the peptide fragment of the 22 to 28 propeptide amino acid sequence or the peptide fragment of the 12 to 18 spadin peptides, namely: GVSWGLR (SEQ ID NO 6).
The present invention relates in particular to a method for the production of the polyclonal antibodies of the invention.
The method for the production of the polyclonal antibodies of the invention can comprise the following steps:
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 can be an immunogenic composition. This may include, for example, any immunogenic composition known to a person skilled in the art. This may include, for example, a composition comprising at least one adjuvant.
In the present invention, an adjuvant means any immunogenic adjuvant known to a person skilled in the art. This may include, for example, a Freund or Alun adjuvant.
In the present invention, the animal can be any animal known to a person skilled in the art suitable for the production of antibodies; including, for example, a mouse, rat, rabbit, chicken, goat, sheep, donkey or horse.
In the present invention, administration can be achieved using any means and/or method known to a person skilled in the art. This may include, for example, an administration by injection, for example via a syringe. Administration can be achieved by any pathway known to a person skilled in the art; including, for example, an administration by a subcutaneous, intradermal, intramuscular and/or intravenous pathway.
In the present invention, the peptide can be administered at 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 invention, the recovery of the antibody can be achieved by any method known to a person skilled in the art. This may include, for example, a collection of serum from the animal, a bleeding and/or any means/method enabling serum to be recovered from the animal.
In the present invention, the isolation of the antibody can be achieved by any method known to a person skilled in the art. This may include, for example, a filtration, for example through a membrane. The filtration of the solution can be performed via a membrane comprising pores with a pore diameter ranging from 0.2 to 0.45 μm. This may include, for example, a sterile membrane. The membrane may be, for example, a cellulose, polyethersulfone, polycarbonate or nylon membrane. A method may also be included that comprises a step of coagulation of the blood, for example for 1 to 3 hours at a temperature ranging from 15 to 25° C., elimination of the clot formed, filtration of the solution, centrifugation and recovery of the supernatant containing the polyclonal antibodies. Centrifugation can be performed for a time ranging from 5 to 20 minutes, for example from 10 to 17 minutes, for example 15 minutes. Centrifugation can be performed, for example, at a speed of 5,000 to 25,000 rpm, for example from 10,000 to 20,000 rpm, for example equal to 15,000 rpm.
A person skilled in the art, from his general knowledge, will know how to adapt and/or chose the method for obtaining polyclonal antibodies.
In the present invention, the antibodies of the invention can be produced by any suitable method, for example the method described in Lee, B. S., Huang, J. S., Jayathilaka, L. P., 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 shown that the antibodies according to the invention allow the concentration of spadin and the propeptide and its fragments to be measured in a biological sample. In particular, the inventors have shown that two human peptides, namely propetide or spadin and mini-spadin, can be measured independently from samples of human serum.
The present invention thus also relates to the in vitro use of the antibodies of the invention for the detection/assay of a peptide chosen from propeptide, spadin and mini-spadin in a biological sample.
The antibodies suitable for the detection and/or assay of propeptide, spadin and mini-spadin are those defined above. They may include, for example, the polyclonal antibodies AB1 or AB2.
The inventors have also shown that the antibodies according to the invention allow the concentration of mini-spadin to be detected and/or measured in a biological sample. The inventors have also shown that the antibodies according to the invention advantageously allow the concentration of mini-spadin that has been injected into a mammal, for example an animal or a human being, to be detected and/or measured in a sample.
In particular, the inventors have shown that two human peptides, namely propeptide or spadin and mini-spadin can be independently measured from samples of human serum.
The inventors have also shown that the antibodies according to the invention advantageously allow, with the antibody AB1, the detection/measurement of the PE concentration, spadin but not mini-spadin, and, with the antibody AB2, the detection/measurement of the PE concentration, spadin and mini-spadin, the absolute value of the difference in concentration between the measurement with antibodies AB2 and AB1 corresponding to the mini-spadin concentration.
The present invention also therefore relates to the in vitro use of the antibodies according to the invention for the detection and/or measurement of the concentration of mini-spadin in a biological sample.
In the present invention, a “biological sample” means any sample obtained from mammals, for example a mammal chosen from the group comprising the order of 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. These may include, for example, a human or an animal.
In the present invention, the biological sample may be any biological fluid, for example, this may include a sample of blood, plasma, serum, cerebrospinal fluid (CSF), vaginal mucus, nasal mucus, saliva, urine and/or milk. Preferably, the biological sample is a sample of blood or a sample of serum.
According to the invention, the antibodies of the invention can be used in any suitable assay/detection method known to a person skilled in the art. This may include an immunoassay method, for example an ELISA or AlphaLISA assay. This may include, for example, an assay method described in the document Immunoanalyse: De la théorie aux critères de choix en biologie clinique [Immunoanalysis: From theory to the selection criteria in clinical biology], Catherine Massart, EPD science 2009.
A person skilled in the art through his general knowledge will know how to adapt the conditions/use the antibody assay/detection methods according to the invention.
The inventors are also the first to have shown that the concentration of the peptide of sequence QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR (SEQ ID NO 1) is reduced in an individual in a depressive state.
The inventors have also shown that the antibodies according to the invention allow the concentration of the peptide of sequence QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR (SEQ ID NO 1) to be determined in a biological sample of a healthy control individual and also of a depressive individual.
The present invention thus 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 application, “depressive state” or “depressive individual” mean an individual suffering from a mood disorder accompanied by sadness and mental anguish. This may include, for example, any depressive state known to a person skilled in the art, for example a major depressive disorder (MDD), neurotic depression/psychotic depression, psychogenic depression/endogenic depression and/or reactive depression/autonomous depression.
In the present invention, “healthy control individual” means an individual, for example a human being, not presenting a depressive state, for example a mood disorder accompanied by sadness and mental anguish, and/or not having presented a depressive state and/or not presenting a major depressive disorder and/or not having presented a major depressive disorder.
According to the invention, “individual” means a mammal chosen from the group comprising the order of 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. This may include, for example, a human or an animal.
In the present invention, “biological sample” means any sample obtained from mammals, for example a mammal chosen from the group comprising the order of 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. This may include, for example, a human or an animal.
In the present invention, the in vitro detection method can be any in vitro method known to a person skilled in the art suitable for the use of antibodies. This may include, for example, an immunochemical method such as a western blot, ELISA, an immunocytochemical method such as confocal microscopy, immunoelectron 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:
S1=Cm/Cref
a value of S1 of less than 1 indicating that the individual from whom the sample was obtained is depressive.
According to the invention, the measurement of the concentration of the peptide of sequence QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR (SEQ ID NO 1) can be achieved according to the method described in Mazella, J. et al. Spadin, a sortilin-derived peptide, targeting rodent TREK-1 channels: a new concept in the antidepressant drug design. PLoS Biol 8, e1000355 (2010) [27] included here by reference and/or with any suitable immunological method known to a person skilled in the art using the polyclonal antibodies of the invention defined above. A person skilled in the art through his general knowledge would know how to adapt the conditions in order to implement known methods using the polyclonal antibodies according to the invention.
In the present application, “reference healthy subject” means a mammal, for example a human being, not presenting a depressive state and/or not having presented a depressive state and/or not presenting a major depressive disorder and/or not having presented a major depressive disorder.
According to the invention, a “group of reference subjects” means a group allowing a reliable reference value to be defined. This may include, for example, a group comprising at least 2 reference subjects as defined above, for example at least 40 reference subjects. This may include, for example, a group comprising 40 to 200 reference subjects.
According to the invention, during the step of comparison of the concentration (Cm) of the measured peptide with a reference concentration of the peptide (Cref), the reference concentration of the peptide (Cref) preferably corresponds to a measured concentration from a biological sample of a reference healthy subject or group of healthy subjects with similar physiological characteristics, for example chosen from the group comprising the age, weight, sex, body mass and drug, tobacco and alcohol abuse.
According to the invention, the “peptide reference concentration” (Cref) can be the concentration of said peptide in a reference healthy subject and/or reference group of healthy subjects. For example, if the biological sample is a blood sample, the peptide reference concentration can range from 20 to 30 nM, for example from 22 to 28 nM.
The inventors have also shown that the concentration of the peptide of sequence QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR (SEQ ID NO 1) changes during a pharmacological treatment of a depressive state in an individual. In particular, the inventors have shown that an effective pharmacological treatment makes it possible to “restore” the concentration of the peptide of sequence QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR (SEQ ID NO 1) and to increase its concentration or even to return to a reference concentration.
The inventors have also shown that the antibodies according to the invention make it possible to monitor the effectiveness of a treatment for depression by monitoring the concentration of the peptide of sequence QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR (SEQ ID NO 1) in a biological sample of an individual.
The present invention thus also relates to a method for monitoring the effectiveness of a treatment for depression that involves:
S2=C2/C1,
a value of S2 of more than 1.2 indicating that the treatment is an effective antidepressant.
In the present application, “treatment” means, for example, a medical treatment, for example an allopathic treatment, involving taking molecules, for example chemical molecules, for example molecules obtained by organic synthesis, molecules of biological origin, for example proteins, molecules coming 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 re-education, or any other treatment based on cell therapy, for example the injection of stem cells.
In the present invention, the treatment can be any antidepressant treatment known to a person skilled in the art. This may, for example, include treatment using SSRIs (Selective Serotonin Reuptake Inhibitors), for example fluoxetine (Prozac (registered trademark)), paroxetine (Deroxat, Divarius, Paxil (registered trademark)), sertraline (Zoloft (registered trademark)); citalopram (Seropram, Celexa (registered trademark)), Escitalopram Oxalate (Seroplex, Cipralex (registered trademark)), indalpine, zimelidine, dapoxetine (Priligy (registered trademark)), the maleate of fluvoxamine maleate (Floxyfral (registered trademark)); SNRIs (Serotonin Norepinephrine Reuptake Inhibitors), for example venlafaxine (Effexor (registered trademark)), milnacipran (Ixel, Savella (registered trademark)), duloxetine (Cymbalta (registered trademark)), tramadol (Topalgic (registered trademark)), nefazodone, desvenlafaxine (Pristiq (registered trademark)), tricyclic antidepressants (TCA), for example amitriptyline (Laroxyl (registered trademark), Elavil (registered trademark)), amoxapine (Defanyl (registered trademark)), clomipramine (Anafranil (registered trademark), Clomipramine Merck (registered trademark)), dosulepin chlorhydrate (Prothiaden, (registered trademark)), doxepine (Quitaxon, (registered trademark)), imipramine (Tofranil, (registered trademark)), maprotiline (Ludiomil, (registered trademark)), opipramol (Insidon, (registered trademark)), quinupramine (Kinupril, (registered trademark)), trimipramine (Surmontil, (registered trademark)), monoamine oxidase inibitors (MAOIs), irreversible and non selective, for example moclobemide, toloxatone, antagonists acting on the receptors of substance P, antagonists of the receptors of NMDA and noradrenaline and specific serotoninergic antidepressants (NASSAs).
In the present invention, the biological sample is as defined above.
In the present invention, the individual is as defined above.
In the present invention, the measurement of the concentration can be performed using any method suitable and known to a person skilled in the art. This may include a method as defined above.
Advantageously, the present invention makes it possible to measure the quantity of sortilin-derived peptides in a biological sample, for example a blood sample from an individual, in particular a mammal, while differentiating it from the assay of mini-spadin that can correspond to the active molecule used in an antidepressant treatment.
Furthermore, the present invention advantageously makes it possible, if mini-spadin (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 sortilin-derived endogenous peptides from exogenous peptides, mini-spadin (12-18), present in the human serum.
The use of antibodies in the methods for measuring concentrations according to the invention and/or for monitoring the effectiveness of an antidepressant treatment advantageously make it possible with AB1 to measure the serum levels of sortilin-derived peptides of healthy controls/individuals and depressive individuals/patients even if they are treated with mini-spadin (12-18), and to measure the effectiveness of mini-spadin (12-18) bioavailability with the difference obtained between the peptides measured with AB2 and the peptides measured with AB1 as described in
Advantageously, the present invention makes it possible to detect a depressive state in an individual, for example a patient, with a greater sensitivity and specificity than known methods. Furthermore, the present invention advantageously makes it possible to monitor the effectiveness of a treatment for depression whatever the treatment used. In particular, the method of the invention, particularly by the advantageous use of the antibody AB1, makes it possible to monitor the effectiveness of a treatment for depression even if the individual and/or the patient is treated with a peptide, for example an exogenous peptide, for example a peptide of sequence SEQ ID NO 6.
Advantageously, the present invention makes it possible, by using the antibodies AB1 and AB2, for example to monitor the change in concentration, for example of the peptide of sequence SEQ ID NO 6.
In the example below, the experiments were performed using the products and methods mentioned below. In the present example, room temperature means a temperature of between 19 and 25° C., for example equal to 22° C.
Animals
The experiments were performed on males of 20-25 g C57BL/6J of 8-10 weeks old (January France Elevage) in accordance with the policies on the welfare and use of laboratory animals set out in European Community Directive 2010/63/EU. The local ethics committee (CIEPAL) has approved the protocols used in this study (Protocol Number 00.893.02).
Antibodies and Preparation of Biotinylated Peptide
The peptides were synthesized by GeneCust (Dudelange, Luxembourg). The 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. The spadin (5.4 mg; 2.7 mmol) was dissolved in 1.5 ml of 25 nM pH 6.7 phosphate buffer. Some biotin N-hydroxysuccinimide (13.5 mmol) resuspended in 700 μl 70% acetonitrile/30% of dimethylformamide was added to the spadin solution and incubated for one night at room temperature. The biotinylated spadin was purified by High Performance Liquid Chromatography (HPLC) using a Waters system equipped with a semi-preparative Lichrosorb RP18 column. The biotinylated spadin (elution at 27 min), identified by mass spectroscopy, was collected, quantified by its absorption at 280 nm and lyophilized in aliquots.
Alpha-Lisa (Registered Trademark) Test
According to the principles of AlphaScreen technology (registered trademark (Perkin Elmer)), streptavidin donor microbeads were recognized by biotinylated spadin, while acceptor microbeads with rabbit anti-IgG were bound by anti-spadin antibodies. When two microbeads (acceptors and donors) were in close proximity, the signal was produced by a molecular interaction 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 incubation, in 96-well plates, of 10 nM of biotinylated spadin with the anti-spadin antibody (1:1000) in the AlphaLisa (registered trademark) buffer in the absence or presence of increasing concentrations of spadin (10−11 to 10−6 M) for 1 hour at room temperature. After the addition of donor and acceptor beads and an additional incubation for 2 hours at room temperature, the plates were read using an Enspire reader (Perkin). For the serum measurements, the same volume of serum was added instead of the unlabeled spadin. The quantity of propeptide was determined from its signal percent inhibition and calculated using the standard curve.
Porsolt Forced Swim Test (FST)
After intravenous injection of saline solution or various tested peptides, the mice (n=10-12 per group) were placed individually 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 immobility time was recorded during the final 4 min (Porsolt et al., 1977).
Human Blood Samples
The cohort of control individuals was made up of 49 healthy unrelated volunteers who were selected for diagnosis of DSM-IV Axis I disorders by expert psychologists using the Mini-International Neuropsychiatric Interview (MINI) [30]. Only healthy volunteers with no history of drug or alcohol abuse or dependency and no personal or first-degree relative family history of psychiatric disorders were enrolled in the trial. Furthermore, the absence of pertinent neurological illnesses, namely epilepsy and Parkinson's syndrome, was compulsory for enrolment in the trial. Lastly, subjects who obtained a score of less than 27/30 in the Mini Mental State Examination (MMSE) were excluded from the trial.
The cohort of patients was made up of 37 patients, having a Major Depressive Disorder (MDD) with moderate to severe depression, who met the criteria of the classification system in the Diagnostic and Statistical Manual IV (DSM-IV) of mental disorders. The diagnosis of unipolar depression was confirmed by a structured clinical interview for DSM-IV Axis I disorders (SCID-I). The exclusion criteria were as follows: a) a mental retardation or cognitive disorder; b) life history of schizophrenia, schizoaffective disorder or a bipolar disorder; c) personality disorder, addiction, alcohol abuse or dependency, obsessive-compulsive disorder or post-traumatic stress as a primary diagnosis; and d) comorbidity with an eating disorder.
No patient showed psychotic symptoms; 11 (29.7%) showed existing comorbidity in Axis I (Generalized Anxiety Disorder (GAD)), panic attacks, panic disorders or anxiety disorders Not Otherwise Specified (NOS), 2 (5.4%) showed symptoms of Axis II disorders (dependent personality disorder) and no alcohol abuse, as a secondary diagnosis (the total number exceeded the number of subjects due to the presence of comorbidities).
All of the patients were either “drug-naïve”, and had never received prior treatment with an antidepressant drug, or “drug-free”, in other words they had been previously treated with one or two antidepressants but had had a period of withdrawal for at least 2 weeks before starting the new antidepressant treatment. All of the patients were treated with a monotherapy: thirty-five patients were treated with serotonin reuptake inhibitors (SSRI), namely escitalopram, and the other patients were treated with serotonin-noradrenalin reuptake inhibitors (SNRI) (venlafaxine, duloxetine, tricyclic antidepressants (TCA) (nortriptyline) or noradrenergic and specific serotonergic antidepressants (NSSA) (mirtazapine). The severity of the illness was assessed by the Montgomery and Asberg Depression Rating Scale (MADRS) before the start of the new antidepressant treatment (T0) and after 12 weeks of treatment (T1). All of the socio-demographic, clinical and pharmacological characteristics of patient treatment are presented in Table 1 below.
a the total number could exceed the number of subjects due to the presence of multiple administrations of drugs.
For the patients and controls, venous blood samples were taken in the morning between 8:00 a.m. and 9:00 a.m. in tubes without anticoagulant. The serum was separated by centrifugation (1620 g for 15 minutes). Blood samples for the PE measurements were collected at each time point.
The trials were approved by local ethics committees (CEIOC, IRCCS, Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia No: 50/2008 and Ethics Committee of the Province of Verona No: 4997/09.11.01), and written informed consent was obtained.
Statistics
The results are expressed as an average ±Standard Error of the Mean (SEM). Statistical analyses were conducted using GraphPad (version 6.0). The demographic and clinical characteristics in the samples from patients were described both in quantitative terms of average ±standard deviation (SD) or as proportions. After having checked normality, Student t tests were used if necessary to evaluate the differences in the quantitative variables whereas Analysis Of Variance (ANOVA) was used to calculate the possible differences between groups for the continuous variables (
Results
The specificity of the antibodies used was studied. In particular, the specificity of the antibodies used in the assay method was studied. A series of partial sequences of human PE was prepared in order to characterize the specificity of the antibodies used (
As shown in
Thus, from the analysis of the recognition structure of these peptides (
A polyclonal composition making it possible specifically to measure the propeptide and spadin concentration was obtained.
The antidepressant activity of the analogs was tested using the FST.
In order to understand better the variations in PE levels in the pathology of depression, all of the human serums coming from a clinical center in Italy were collected and a measurement of the PE concentration was made.
In the cohort, it was determined that the PE concentration was significantly lower in patients suffering from major depressive disorder (MDD) (18.9±1.3 nM) than in the healthy controls (23.7±1.5 nM) (z=−2.11, p=0.035) (
Discussion
As shown in this example, the antibody used recognized only one series of peptides derived from human PE, as well as PE itself, and not unrelated peptides like neurotensin or somatostatin.
As shown, the peptides recognized by the antibody have antidepressant activities as shown by the mouse forced swim test (
This example thus clearly shows that the PE concentration in the serum is downregulated. i.e. it is less in patients with a major depressive disorder (MDD) compared to healthy subjects. This example also clearly shows that the PE concentration varies with an antidepressant treatment and, particularly in patients presenting a propeptide concentration below normal, enables a return to the normal in correlation with the clinical development of the treatment for depression.
This example thus clearly shows 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 an antidepressant treatment. The serum PE concentration is an additional marker to the known serum level marker, for example BDNF [31].
As shown, the serum PE concentration is significant lower in patients with MDD and, taking into account the fact that the peptides measured in the present example have been generated from sortilin, a study of the possible correlation with the level and/or expression of sortilin has been carried out. Interestingly, the variation of the expression of sortilin in the blood has previously been observed in patients suffering from MDD. Firstly, in the mononuclear cells of the blood, the gene expression of sortilin decreases with clinical improvement [23], whereas sortilin is overexpressed in patients suffering from MDD, particularly in non-responders [32]. Moreover, in human serum, the increase in the level of sortilin is associated with the state of depression [24].
The results obtained show that the serum PE concentration varies depending on the mood symptoms in humans, and is an additional biomarker of depression, in addition to or in replacement of BDNF and/or VEGF [24]. Moreover, the possible future use of spadin as an effective therapy against the pathology, a peptide for which the serum level could also be controlled, will certainly greatly improve the outlook for new strategies to effectively manage depression worldwide.
This example thus clearly shows that the present invention makes it possible in particular to detect/determine the depressive state, monitor the effectiveness of a treatment for depression, measure the concentration and/or detect the propeptide or PE of sequence QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR (SEQ ID NO 1) in a biological sample, particularly by using the antibody AB1 in Example 1 described above.
The two antibodies were prepared from rabbit and the volumes obtained were approximately 200 ml of each. In the experimental protocols, these antibodies were used at dilutions of 1/1000 and 1/5000. The techniques used to measure the level of propeptide in the blood were multiple, immunological or fluorescence. The peptide assay can be achieved with 100 μI of blood and is reproducible.
In particular, antibodies AB1 and AB2 were obtained by injecting into two rabbits, namely Oryctolagus cuniculus, spadin coupled to KLH (Keyhole limpet hemocyanin protein) (AB1) or mini-spadin (22-28 PE) coupled to KLH (AB2) on day 0, DO (1 mg), day 7, D7 (1 mg), day 14, D14 (1 mg) and day 29, D29 (1 mg). Blood samples, namely 20 ml, were taken on day 27 (D27) and day 42 (D42). The polyclonal serum was obtained by centrifuging at 1200×g for 10 min. The polyclonal serum corresponding to the supernatant was recovered. The immune response was checked by ELISA for each antibody. The antibodies contained in the serum were kept at −20° C.
In the example below, the experiments were performed with the polyclonal antibodies AB1 described in Examples 1 and 2 above.
A measurement of the serum concentration of the peptide of sequence QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR was taken according to the method described in Example 1.
The cohort of control individuals was made up of 49 healthy unrelated volunteers who were selected for diagnosis of DSM-IV Axis I disorders by expert psychologists using the Mini-International Neuropsychiatric Interview (MINI) [30]. Only healthy volunteers with no history of drug or alcohol abuse or dependency and no personal or first-degree relative family history of psychiatric disorders were enrolled in the trial. Furthermore, the absence of pertinent neurological illnesses, namely epilepsy and Parkinson's syndrome, was compulsory for enrolment in the trial. Lastly, subjects who obtained a score of less than 27/30 in the Mini Mental State Examination (MMSE) were excluded from the trial.
An average of the concentration measured in the samples of the control subjects was determined and the value obtained corresponded to the reference concentration (Cref) of a healthy individual.
A measurement of the serum concentration (Cm) of the peptide of sequence QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR in a sample from an individual was made.
A comparison of the concentration (Cm) of the peptide measured in the depressive subject with the reference concentration (Cref) of the healthy subject was made via the calculation of a score (S1) according to the following formula: S1=Cm/Cref, a value of S1 of less than 1 indicating that the individual from whom the sample was obtained is depressive.
In this example, the effectiveness of a treatment for depression is monitored.
The cohort of patients was made up of patients having a Major Depressive Disorder (MDD) with moderate to severe depression who met the criteria of the classification system of Diagnostic and Statistical Manual IV (DSM-IV) of mental disorders. The diagnosis of unipolar depression was confirmed by a structured clinical interview for DSM-IV Axis I disorders (SCID-I). The exclusion criteria were as follows: a) a mental retardation or cognitive disorder; b) life history of schizophrenia, schizoaffective disorder or a bipolar disorder; c) personality disorder, addiction, alcohol abuse or dependency, obsessive-compulsive disorder or post-traumatic stress as a primary diagnosis; and d) comorbidity with an eating disorder.
A measurement of the serum concentration of the peptide of sequence QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR was made according to the method described in Example 1 prior to the administration of any antidepressant treatment. In particular, the measurement of the concentration was made from a blood sample. The concentration was measured independently for each patient prior to any treatment. The concentration measured was 19 nM.
The patients were then treated by monotherapy with selective serotonin reuptake inhibitors (SSRI), selective serotonin-noradrenalin inhibitors (SNRI), tricyclic antidepressants (TCA) or noradrenergic and specific serotonergic antidepressants (NSSA).
The mode of administration, frequency, assay and dose corresponded to that stated in the directions of use or instructions mentioned in the Vidal dictionary.
A measurement of the serum concentration of the peptide of sequence QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR according to the method described in Example 1 after 12 weeks of treatment was made independently for each patient. The concentration measured was 21 nM.
A comparison of the concentration (Cm) of the peptide measured in the depressive patient with the reference concentration (Cref) was made via the calculation of a score (S1) according to the following formula: S1=CM/Cref, a value of S1 of less than 1 indicating that the individual from whom the sample was obtained is depressive.
A comparison of the concentration values measured was measured via the calculation of a score (S2) according to the following formula: S2=C2/C1 (C1, concentration before treatment, C2, concentration after treatment). If the value of S2 was more than 1.2, the treatment administered 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 in the Diagnostic and Statistical Manual IV (DSM-IV) of mental disorders.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1659781 | Oct 2016 | FR | national |
| 1752162 | Mar 2017 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/FR2017/052787 | 10/11/2017 | WO | 00 |
