Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
  • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/08—Peptides having 5 to 11 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/10—Peptides having 12 to 20 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/08—Antiepileptics; Anticonvulsants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/24—Antidepressants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54306—Solid-phase reaction mechanisms
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates in particular to polyclonal antibodies directed against peptides, in particular derived from the sequence propeptide QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR
• PE amino acid sequence
• the present invention also relates to polyclonal antibodies directed against peptides, especially those derived from the sequence propeptide
• 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.
• 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.
• TCAs tricyclic antidepressants
• MAOIs monoamine oxidase inhibitors
• 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].
• BDNF brain-derived neurotrophic factor
• 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].
• Antidepressants are also known to modulate the expression of various factors involved in cell survival and growth, such as CREB, Bcl2, or MAP kinases.
• CREB CREB
• Bcl2 MAP kinases
• 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.
• NTRS3 neurotensin receptor 3
• sortiline whose inactivation in mice produces a depression resistance phenotype, similar to that observed in mice invalidated for the channel.
• basal potassic TREK-1 basal potassic TREK-1.
• DTD drug-resistant depression
• BDNF Brain Derived Neurotrophic Factor
• Sortilin is known to control the regulation of intracellular trafficking in the secretory pathway of BDNF neurotrophic factor [22].
• 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].
• PE 44 amino acid
• 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].
• spadine and propeptide are effective biological markers of depression, in particular major depressive disorder.
• 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.
• 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.
• 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 AB1 polyclonal antibody binds to the peptides selected from the group consisting of:
• the present invention relates to polyclonal antibodies (AB1) directed against peptides in the group comprising: QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR
• polyclonal antibody (AB1) is directed against the peptides in the group consisting of:
• polydonal AB2 antibody binds to the peptides selected from the group consisting of:
• the present invention relates to polyclonal antibodies (AB2) directed against peptides in the group comprising QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR
• polydonal antibody is directed against the peptides in the group consisting of
• 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.
• the inventors have demonstrated that two human peptides, namely Propeptide and / or Spadine and Mini-Spadine can be independently measured from human serum samples.
• propeptide or PE is understood to mean the peptide of sequence:
• 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).
• PE 12-27 is understood to mean the peptide fragment of peptides 12 to
• 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).
• 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).
• 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:
• 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
• 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.
• adjuvant any immunogenic adjuvant known to those skilled in the art. It may be for example a Freund's adjuvant, Alum.
• 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.
• 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.
• 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.
• 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.
• 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 ⁇ m. 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
• 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.
• 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.
• 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 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.
• the inventors have demonstrated that two human peptides, namely Propeptide or Spadine and Mini-Spadine can be independently measured from human serum samples.
• 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.
• 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.
• 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.
• the biological sample is a blood sample or a serum sample.
• 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.
• 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.
• depression 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.
• MDD major depressive disorder
• neurotic depression / psychotic depression neurotic depression / psychotic depression
• endogenous psychogenic depression / depression and / or reaction depression / depression. autonomous.
• health control individual 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.
• a depressive state for example a mood disorder accompanied by sadness and moral suffering
• 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.
• 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.
• 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.
• 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
• S1 Cm / Cref a value of S1 less than 1 indicating that the individual from which the sample was obtained is depressed.
• 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.
• 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.
• 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.
• 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.
• the "reference concentration of the peptide” may be the concentration of said peptide in a healthy subject and / or group of healthy subjects of reference.
• 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.
• the inventors have demonstrated that an effective pharmacological treatment makes it possible to "restore" the concentration of the sequence peptide QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR
• 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
• treatment 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.
• 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.
• 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)),
• the biological sample is as defined above.
• the individual is as defined above.
• 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.
• 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.
• 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.
• 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.
• 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.
• the present invention advantageously makes it possible to monitor the effectiveness of a treatment on depression whatever the treatment used.
• 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.
• 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.
• 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.
• FIG. 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.
• 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.
• MADRS Montgomery- ⁇ sberg depression rating scale
• ambient temperature means a temperature of 19 to 25 ° C, for example equal to 22 ° C.
• Peptides were synthesized by GeneCust (Dudelange, Germany). 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 ⁇ l 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.
• mice 10-12 per group
• mice 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).
• 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.
• MMSE Mental State Examination Mini
• 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).
• MDD major depressive disorder
• DSM-IV Diagnostic and Statistical Manual IV for Mental Disorders
• SCID-I DSM-IV Axis I Disorders
• 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.
• Axis I Generalized Anxiety Disorder (GAD)
• GAD Generalized Anxiety Disorder
• NOS anxiety disorder not otherwise specified
• 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).
• SSRIs serotonin reuptake inhibitors
• SNRI selective serotonin-noradrenaline inhibitors
• TCA tricyclic antidepressants
• ANSS noradrenergic and serotonin-specific antidepressants
• MADRS Montgomery and Asberg Depression Scale
• Table 1 Demographic and Clinical Characteristics of Control Groups and CT Patients
• 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 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.
• a polyclonal composition for specifically measuring the concentration of propeptide and spadin has thus been obtained.
• FIG. 1D shows the test results of Porsolt forced swimming (FST).
• FST Porsolt forced swimming
• the concentration of PE for each incubation period was determined using an AlphaLISA assay method.
• 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.
• PE Propeptide 44AA PE1 -44 2.18 1, 5 - 400 Yes (SEQ ID NO 1) or PE (1, 53- 3.09)
• 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
• 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.
• 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.
• MDD major depressive disorder
• 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.
• 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].
• 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.
• the change in the expression of sorbitin in the blood has already been observed in patients with CT.
• 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]. ].
• 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 ⁇ of blood and reproducible.
• Figure 1 shows the binding specificities of antibody functions.
• 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
• 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.
• MMSE Mental State Examination Mini
• 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).
• MDD major depressive disorder
• DSM-IV Diagnostic and Statistical Manual IV for Mental Disorders
• SCID-I Diagnostic and Statistical Manual IV for Mental Disorders
• 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.
• QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRWRR is carried out according to the method described in Example 1 prior to the administration of any anti-depressant treatment.
• 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.
• SSRIs serotonin reuptake inhibitors
• SNRIs selective serotonin-noradrenaline inhibitors
• TCAs tricyclic antidepressants
• ANSS serotonin-specific and serotonergic antidepressants
• 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.
• S2 C2 / C1 (C1, concentration before treatment, C2, concentration after treatment).
• S2 value was greater than 1, 2 the administered treatment was an effective antidepressant.
• Serum BDNF as a peripheral biomarker of treatment-resistant depression and the rapid antidepressant response A comparison of ketamine and ECT. J Affect Disord 186, 306-31 1.
• Sortilin controls intracellular sorting of brain-derived neurotrophic factor to the regulated secretory pathway. J Neurosci 25, 6156-6166.
• the 100-kDa neurotensin receptor is gp95 / sortilin, a non-G-protein-coupled receptor.

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