WO2009096502A1 - Marqueur pour la dépression et un état déprimé et détection et diagnostic l'utilisant - Google Patents

Marqueur pour la dépression et un état déprimé et détection et diagnostic l'utilisant Download PDF

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Publication number
WO2009096502A1
WO2009096502A1 PCT/JP2009/051527 JP2009051527W WO2009096502A1 WO 2009096502 A1 WO2009096502 A1 WO 2009096502A1 JP 2009051527 W JP2009051527 W JP 2009051527W WO 2009096502 A1 WO2009096502 A1 WO 2009096502A1
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Prior art keywords
peptide
chain
fibrinogen
marker
depression
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PCT/JP2009/051527
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English (en)
Japanese (ja)
Inventor
Noriyuki Kawamura
Kenji Tanaka
Lyang-Ja Lee
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Japan As Represented By President Of National Center Of Neurology And Psychiatry
Protosera Inc.
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Priority to JP2009551587A priority Critical patent/JP5410997B2/ja
Publication of WO2009096502A1 publication Critical patent/WO2009096502A1/fr

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    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/745—Assays involving non-enzymic blood coagulation factors
    • G01N2333/75—Fibrin; Fibrinogen
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/81—Protease inhibitors
    • G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • G01N2333/811—Serine protease (E.C. 3.4.21) inhibitors
    • G01N2333/8114—Kunitz type inhibitors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/30—Psychoses; Psychiatry
    • G01N2800/304—Mood disorders, e.g. bipolar, depression

Definitions

  • the present invention relates to a detection / diagnosis marker for a depressive disorder, a diagnosis method of the disease using the same, and the like.
  • Depression is a type of mood disorder and is a mental disorder characterized by depressed mood, anxiety / irritability, decreased mental activity, decreased appetite, insomnia and the like. Depression is now being diagnosed as a “major depressive disorder” of mood disorders [Mental Disease Diagnosis and Statistical Manual, 4th Edition, published by the American Psychiatric Association (APA) (See Diagnostic and Statistical, Manual, of Mental, Disorder, IV, DSM-IV). Furthermore, the name “Mild Depression” is used for depression with mild symptoms, and the name “Mood Dysfunction Disorder” (mood dysfunction) is used for long-lasting mild depression. It is called “depressive disorder” or depression.
  • Diagnosis of depression and other mental illnesses and assessment of individual psychological stress depend on the subjectivity of doctors and psychologists, or on the subjectivity of patients and individuals reporting symptoms and stress, and are said to be objective. It ’s hard. In fact, if you have some advantage of being sick, you can avoid the prejudice or inconvenience caused by the disease gain that overwhelms the symptoms, and conversely that others know that you are mentally ill. In many cases, accurate diagnosis and assessment are difficult.
  • Patent Document 2 proposes identification of a causative gene of depression and a method for screening a drug for treatment and / or prevention of depression Has been.
  • a method for objectively grasping depression by identifying substances that are found excessively in depression patients has also been studied.
  • depression is identified based on the amount of testosterone and cortisol present in the blood or saliva of men.
  • JP-T-2002-529706 polyglutamine-containing protein in neuropsychiatric disorders, Patent Document 4
  • neuropsychiatric disease is diagnosed using the polyglutamine-containing protein as a marker.
  • Fibrinogen is an abundant protein in the plasma (200-400mg / dl) with a molecular weight of about 340,000 and exists as a dimer in which three polypeptide chains of A ⁇ , B ⁇ , and ⁇ are linked by SS bonds. To do. By the action of thrombin, the N terminus (fibrinopeptide A (FPA) and fibrinopeptide B (FPB)) of the A ⁇ chain and the B ⁇ chain are sequentially cut out. In addition, various peptides that appear to be degradation products of fibrinogen ⁇ chain generated by excision of FPA have been found in plasma. The physiological functions of these fibrinogen degradation products are not well understood. Inter ⁇ -trypsin inhibitor heavy chain (ITIH4) exists in plasma in a complex with a peptide having weak protease inhibitor activity called bikunin. The physiological function of ITIH4 has not been fully elucidated.
  • ITIH4 Inter ⁇ -trypsin inhibitor heavy chain
  • Non-Patent Document 2 discloses proteolytic product patterns in the sera of several cancer patients, and FPA and its fragments, fibrinogen alpha chain fragments and ITIH4 fragments varied in prostate cancer, bladder cancer and breast cancer It has been reported (Non-Patent Document 2). However, there is no report on the relationship between these peptides and depressive disorders.
  • An object of the present invention is to provide a diagnostic marker for a depressive disorder and an effective diagnostic method for a depressive disorder using the same.
  • the present inventors examined the serum collected from a patient with a depressive disorder by mass spectrometry. As a result, the patient significantly increased or decreased in a patient with depressive disorder as compared with a healthy person. 7 types of peptides were found. As a result of analyzing the amino acid sequence, two of these peptides were peptides consisting of a partial amino acid sequence of fibrinogen A ⁇ chain, four were peptides consisting of a partial amino acid sequence of fibrinogen B ⁇ chain, and the remaining one was a portion of ITIH4 It was found to be a peptide consisting of an amino acid sequence. Based on the above findings, the present inventors have identified these peptides and proteins derived from them (polypeptides) as diagnostic markers for depressive disorders, and have completed the present invention.
  • the present invention [1] Measuring the amount of one or more peptides selected from the group consisting of fibrinogen A ⁇ chain, fibrinogen B ⁇ chain, inter- ⁇ -trypsin inhibitor heavy chain and their degradation products in a biological sample collected from a subject.
  • a test method for diagnosis of a depressive disorder in the subject [2]
  • the degradation product of fibrinogen A ⁇ chain is a peptide group consisting of each amino acid sequence shown in SEQ ID NOs: 1 and 2, and the degradation product of fibrinogen B ⁇ chain consists of each amino acid sequence shown in SEQ ID NOs: 3 to 6
  • the method according to [1] or [2] above, wherein the biological sample is a body fluid; [4]
  • the body fluid is selected from the group consisting of blood, plasma, serum, saliva, urine, spinal fluid, bone marrow fluid, pleural effusion, ascites, joint fluid, tear fluid, aqueous humor, vitreous humor and lymph fluid [3] ]
  • the method according to [5] The method according to
  • a depressive disorder can be determined quickly and accurately, early detection and early treatment of the disease are possible.
  • the present invention provides a marker peptide for depressive disorder (hereinafter sometimes referred to as “depressive marker of the present invention”).
  • the “depressive disorder” in the present invention is used as a meaning defined as one kind of mood disorder in the revised version of DSM-IV (DSM-IV-TR). Depressive disorders are further divided into “major depressive disorders”, “dysthymic disorders” and “depression-related syndromes” according to symptoms.
  • the depression marker of the present invention is fibrinogen A ⁇ chain, fibrinogen B ⁇ chain, ITIH4, or a degradation product thereof.
  • the amino acid sequences of fibrinogen A ⁇ chain, fibrinogen B ⁇ chain and ITIH4 are shown in SEQ ID NOs: 8, 9 and 10, respectively.
  • the degradation product of fibrinogen A ⁇ chain is not particularly limited as long as it is a peptide consisting of an arbitrary partial amino acid sequence of the amino acid sequence shown in SEQ ID NO: 8, but preferably consists of each amino acid sequence shown in SEQ ID NOs: 1 and 2 And peptides (also referred to as “peptide ⁇ 1” and “peptide ⁇ 2”, respectively).
  • the amino acid sequence shown in SEQ ID NO: 1 corresponds to a fragment from the 407th Arg residue to the 424th Arg residue from the N-terminus of the fibrinogen A ⁇ chain.
  • the amino acid sequence shown in SEQ ID NO: 2 corresponds to a fragment from the 529th Gly residue to the 556th Lys residue from the N-terminus of the fibrinogen A ⁇ chain.
  • the degradation product of the fibrinogen B ⁇ chain is not particularly limited as long as it is a peptide consisting of an arbitrary partial amino acid sequence of the amino acid sequence shown in SEQ ID NO: 9, but preferably consists of each amino acid sequence shown in SEQ ID NOs: 3 to 6 And peptides (also referred to as “peptide ⁇ 1” to “peptide ⁇ 4”, respectively).
  • the amino acid sequence shown in SEQ ID NO: 3 corresponds to a fragment from the N-terminal Gln residue to the 21st Lys residue of the fibrinogen B ⁇ chain.
  • the amino acid sequence shown in SEQ ID NO: 4 corresponds to a fragment from the 15th Gly residue to the 41st Tyr residue from the N-terminus of the fibrinogen B ⁇ chain.
  • the amino acid sequence shown in SEQ ID NO: 5 corresponds to a fragment from the 22nd Lys residue to the 42nd Arg residue from the N-terminus of the fibrinogen B ⁇ chain.
  • the amino acid sequence shown in SEQ ID NO: 6 corresponds to a fragment from the 23rd Arg residue to the 42nd Arg residue from the N-terminus of the fibrinogen B ⁇ chain.
  • the degradation product of ITIH4 is not particularly limited as long as it is a peptide consisting of an arbitrary partial amino acid sequence of the amino acid sequence shown in SEQ ID NO: 10.
  • a peptide consisting of the amino acid sequence shown in SEQ ID NO: 7 (hereinafter referred to as “ Peptide H4 ").
  • the amino acid sequence shown in SEQ ID NO: 7 corresponds to a fragment from the 624th Gln residue to the 643rd Arg residue from the N-terminus of ITIH4.
  • peptides ⁇ 1 and ⁇ 2 are fragments (degradation products) of fibrinogen A ⁇ chain
  • peptides ⁇ 1 to ⁇ 4 are fragments of fibrinogen B ⁇ chain
  • peptide H4 is a fragment of ITIH4.
  • the patient to be tested includes a polymorphism comprising one or more amino acid substitutions, deletions, insertions or additions or combinations thereof within the partial amino acid sequence corresponding to these peptides of fibrinogen A ⁇ , B ⁇ or ITIH4
  • the amino acid sequence of the peptide to be detected should be understood as “the amino acid sequence having the polymorphism or allelic mutation in each amino acid sequence shown in SEQ ID NOs: 1 to 7”. It will be obvious to the contractor.
  • the depression marker of the present invention includes those in which the N-terminus, C-terminus or side chain is modified in vivo.
  • the marker when it has a Gln residue at the N-terminus, it includes a pyroglutamine-oxidized one.
  • the present invention also provides for the treatment of a depressive disorder in a patient by measuring the amount of one or more depressive markers of the present invention in a biological sample from the patient suspected of suffering from the depressive disorder.
  • test for diagnosis means measurement of the amount of the marker peptide and, if necessary, comparison with the measured value in a control sample.
  • the “patient suspected of having a depressive disorder” may be subjectively suspected by the patient or based on some objective basis, but preferably, As a result of conventionally known clinical tests and / or examinations, it is a patient who has been judged by a doctor to have a reasonable possibility of suffering from these diseases, or a human having an equivalent medical condition.
  • the patient-derived biological sample to be the test sample is not particularly limited, but is preferably one that is less invasive to the patient, for example, blood, plasma, serum, saliva, urine, tears that can be easily collected from the living body And those collected relatively easily such as cerebrospinal fluid, bone marrow fluid, pleural effusion, ascites, joint fluid, aqueous humor, and vitreous humor.
  • serum or plasma it can be prepared by collecting blood from a patient according to a conventional method and separating the liquid component.
  • the depressive marker of the present invention to be measured is a peptide having a relatively low molecular weight (for example, 10000 or less, 5000 or less, etc.), if necessary, the peptide is concentrated using a spin column or the like, and a high molecular weight protein fraction is previously obtained. Etc. can be separated and removed.
  • the detection of the depressive marker of the present invention in a biological sample can be carried out, for example, by subjecting a biological sample to various molecular weight measurement methods such as gel electrophoresis and various separation and purification methods (eg, ion exchange chromatography, hydrophobic chromatography, Affinity chromatography, reverse phase chromatography, etc.), ionization (eg, electron impact ionization, field desorption, secondary ionization, fast atom bombardment, matrix-assisted laser desorption / ionization (MALDI), electrospray Ionization methods, etc.), mass spectrometers (eg, double-focusing mass spectrometer, quadrupole analyzer, time-of-flight mass spectrometer, Fourier transform mass spectrometer, ion cyclotron mass spectrometer, etc.) A band or spot that matches the molecular weight of the marker peptide, or peak It can be performed by detecting, without limitation.
  • various molecular weight measurement methods
  • the amino acid sequence of the depression marker of the present invention is known, a method of preparing an antibody that recognizes the amino acid sequence and detecting the peptide by Western blotting or various immunoassays can be used more preferably. Furthermore, the hybrid detection method of the above method is also effective.
  • Peptides ⁇ 1, ⁇ 2, ⁇ 1 to ⁇ 4 and H4 of the present invention are 2188.16, 2843.41, 2355.10 (when the N-terminal Gln residue is pyroglutamine oxidized), 2885.53, 2234.19, 2106.10 and 2166.06 (where the N-terminal Gln residue is Although it has a molecular weight (calculated value) of pyroglutamine oxidation), it goes without saying that the actual measurement value may slightly vary depending on the measurement method and measurement instrument used. For example, in the case of a method using a mass spectrometer, it is preferable to measure the peak intensity appearing at the position of the calculated value ⁇ 0.5% (preferably ⁇ 0.3%, more preferably ⁇ 0.1%).
  • One of the particularly preferable measurement methods in the inspection method of the present invention is that a test sample is brought into contact with the surface of a plate used for time-of-flight mass spectrometry, and the mass of a component captured on the plate surface is measured using a time-of-flight mass spectrometer.
  • the method of measuring by is mentioned.
  • the plate that can be adapted to the time-of-flight mass spectrometer may be any plate as long as it has a surface structure that can efficiently adsorb the peptide of the present invention to be detected.
  • Such surface structures include, for example, functionalized glass, Si, Ge, GaAs, GaP, SiO 2 , SiN 4 , modified silicon, a wide range of gels or polymers (eg (poly) tetrafluoroethylene, (poly And vinylidene difluoride, polystyrene, polycarbonate, or combinations thereof).
  • functionalized glass Si, Ge, GaAs, GaP, SiO 2 , SiN 4
  • modified silicon eg (poly) tetrafluoroethylene, (poly And vinylidene difluoride, polystyrene, polycarbonate, or combinations thereof).
  • Examples of surface structures having a plurality of monomer or polymer sequences include linear and cyclic polymers of nucleic acids, polysaccharides, lipids, peptides having ⁇ -, ⁇ - or ⁇ -amino acids, gel surfaces used in chromatography Carriers (anionic / cationic compounds, hydrophobic compounds composed of carbon chains 1-18, hydrophilic compounds (eg, carriers cross-linked with silica, nitrocellulose, cellulose acetate, agarose, etc.), artificial homopolymers) (For example, polyurethane, polyester, polycarbonate, polyurea, polyamide, polyethyleneimine, polyarylene sulfide, polysiloxane, polyimide, polyacetate, etc.) Any known drug or natural compound is bound to any of the above compounds (covalent and non-covalent bonds). ) Coated with heteropolymer etc. Packaging and the like.
  • the support used as a plate for mass spectrometry is a substrate coated with a thin layer of polyvinylidene difluoride (PVDF), nitrocellulose or silica gel, particularly preferably PVDF [usually a plate for mass spectrometry
  • PVDF polyvinylidene difluoride
  • insulators glass, ceramics, plastics, resins, etc.
  • metals aluminum, stainless steel, etc.
  • conductive polymers composites thereof, etc.
  • an aluminum plate is preferably used, refer to WO 2004/031759.
  • the shape of the support can be appropriately devised into a shape suitable for the sample analyzer, in particular, the sample introduction port, but is not limited thereto.
  • a blot chip registered trademark
  • Protocera is preferably used as such a plate for mass spectrometry coated with a thin layer with PVDF.
  • the coating refers to a thin layer formed by depositing on a support in a state where coating molecules are dispersed, instead of overlaying a pre-formed structure like a membrane on the support.
  • the manner in which the coating molecules are deposited is not particularly limited, but means exemplified in a method for preparing a plate for mass spectrometry described later is preferably used.
  • the thickness of the thin layer can be appropriately selected within a range that does not adversely affect the transfer efficiency of the molecules contained in the tissue or cells and the measurement sensitivity of mass spectrometry, etc., for example, about 0.001 to about 100 ⁇ m, Preferably, it is about 0.01 to about 30 ⁇ m.
  • the plate for mass spectrometry (support) can be prepared by a method known per se, for example, the above preferred mass spectrometry plate is prepared by thinly coating the surface of the support with a coating molecule such as PVDF.
  • the Preferred examples of the coating means include application, spraying, vapor deposition, dipping, printing (printing), and sputtering.
  • the coating molecule is dissolved in an appropriate solvent, for example, an organic solvent such as dimethyl formamide (DMF) at an appropriate concentration (for example, about 1 to about 100 mg / mL) ( The coating molecule-containing solution) can be applied to the substrate using a suitable tool such as a brush.
  • an appropriate solvent for example, an organic solvent such as dimethyl formamide (DMF) at an appropriate concentration (for example, about 1 to about 100 mg / mL)
  • DMF dimethyl formamide
  • the coating molecule-containing solution can be applied to the substrate using a suitable tool such as a brush.
  • the coating molecule-containing solution prepared in the same manner as described above may be put in a sprayer and sprayed so that PVDF is uniformly deposited on the substrate.
  • the coating molecule (which may be solid or solution) is heated and vaporized in a vacuum tank containing the base material using a normal vacuum deposition apparatus for producing an organic thin film. A thin layer of molecules can be formed.
  • immersion the substrate may be immersed in a coating molecule-containing solution prepared in the same manner as described above.
  • various printing techniques that can be normally used depending on the material of the substrate can be appropriately selected and used. For example, screen printing or the like is preferably used.
  • sputtering for example, a DC high voltage is applied between the substrate and the coating molecule while introducing an inert gas (eg, Ar gas) in a vacuum, and the ionized gas collides with the molecule.
  • an inert gas eg, Ar gas
  • the repelled coating molecules can be deposited on the substrate to form a thin layer.
  • the coating may be applied to the entire surface of the substrate, or may be applied only to the surface (fraction) subjected to mass spectrometry.
  • the coating molecule can be used in a suitable form depending on the coating means.
  • the coating molecule can be applied to the substrate in the form of a coating molecule-containing solution, a coating molecule-containing vapor, a solid coating molecule, etc. It is preferable to apply in the form. “Apply” refers to bringing a coating molecule into contact with the support so that the coating molecules remain and deposit on the support after contact.
  • the amount of application is not particularly limited, and examples of the coating molecular weight include about 10 to about 100,000 ⁇ g / cm 2 , preferably about 50 to about 5,000 ⁇ g / cm 2 . After the application, the solvent is removed by natural drying, vacuum drying or the like.
  • the surface of the substrate in the mass spectrometry plate may be modified (processed) in advance by an appropriate physical or chemical technique before coating with a coating molecule. Specifically, techniques such as polishing the plate surface, scratching, acid treatment, alkali treatment, glass treatment (tetramethoxysilane, etc.) are exemplified.
  • Transfer of the test sample to the mass spectrometric plate (support) can be performed by leaving the patient-derived biological sample as the test sample untreated or after removing and concentrating the high molecular weight protein using an antibody column or other method.
  • -It is performed by subjecting to polyacrylamide gel electrophoresis or isoelectric focusing, and transferring (blotting) the gel after contact with the plate.
  • a known transfer device can be used.
  • the transfer method itself is known.
  • electrotransfer is used.
  • the sample developed on the gel after the electrophoresis is transferred to the plate for mass spectrometry by various methods (diffusion, electric force, etc.).
  • As a buffer used for electrotransfer it is preferable to use a buffer having a pH of 7 to 9 and a low salt concentration.
  • Tris buffer Tris buffer, phosphate buffer, borate buffer, and acetate buffer.
  • the buffer include sodium borate-hydrochloric acid buffer, tris-borate / EDTA, borate / ACN, and the like.
  • the acetate buffer include tris-acetate / EDTA. Preferred are tris / glycine / methanol buffer and sodium borate-hydrochloric acid buffer.
  • compositions of the tris / glycine / methanol buffer include Tris 10-15 ⁇ mM, glycine 70-120 ⁇ mM, and methanol 7-13%.
  • An example of the composition of the sodium borate-hydrochloric acid buffer is about 5 to 20 mM sodium borate.
  • the molecules present in the test sample including the target molecules are efficiently captured on the surface of the support.
  • a reagent called matrix is added to absorb the laser light and promote ionization of analyte molecules through energy transfer, which is advantageous for later mass spectrometry (when using MALDI method) You can also.
  • the matrix those known in mass spectrometry can be used.
  • IAA indoleacrylic acid
  • DVB 2,5-dihydroxybenzoic acid
  • CHCA ⁇ -cyano-4-hydroxycinammic acid
  • it is DHB or CHCA.
  • the presence and amount of the depression marker of the present invention can be identified from the information on the molecular weight.
  • the mass spectrometer measures the molecular weight of a substance by ionizing a gaseous sample and then putting the molecule or molecular fragment into an electromagnetic field, separating it by mass number / charge number from its movement, and obtaining the spectrum of the substance.
  • -It is a device to detect.
  • MALDI Matrix-assisted laser deionization
  • a sample and a matrix that absorbs laser light are mixed, dried and crystallized, and ionized analytes are brought into vacuum by ionization by energy transfer from the matrix and instantaneous heating by laser irradiation.
  • MALDI-TOFMS method which uses time-of-flight mass spectrometry (TOFMS), which analyzes the mass number based on the time-of-flight difference of sample molecular ions by initial acceleration.
  • TOFMS time-of-flight mass spectrometry
  • the presence or absence and the amount of the target molecule in the test sample can be identified based on the molecular weight information of the target molecule.
  • information from the mass spectrometer as differential information by comparing it with mass spectrometry data in a biological sample derived from a healthy person using an arbitrary program. It will be appreciated that such programs are well known and those skilled in the art can easily construct or modify such programs using known information processing techniques.
  • each of the above steps is performed using a blot chip manufactured by Protocera as a plate for mass spectrometry, and the depression marker of the present invention is quantitatively compared (differential analysis) using a MALDI mass spectrometer. Further, if necessary, the marker peptide remaining on the same chip can be identified.
  • up to quantitative comparison (differential analysis) of the test sample is performed using a blot chip system manufactured by Protocera, and the marker peptide is identified by a combination device (LC-MS) of high performance liquid chromatography and an ion spray type mass spectrometer. / MS).
  • LC-MS combination device
  • the measurement of the depression marker of the present invention in the test method of the present invention can also be performed using an antibody against it.
  • an optimized immunoassay system is constructed and a kit is made, the peptide can be detected with high sensitivity and high accuracy without using a special device such as the mass spectrometer. It is particularly useful in that it can.
  • the antibody against the depression marker of the present invention can be prepared, for example, by isolating and purifying the depression marker of the present invention from a biological sample derived from a patient expressing the same and immunizing an animal using the marker peptide as an antigen. it can.
  • the marker peptide is partially digested with a peptidase or the like, the amino acid sequence of the obtained fragment is determined by the Edman method or the like, and a nucleic acid encoding the peptide based on the sequence
  • a cDNA encoding a protein containing the peptide is obtained by a hybridization method using a cDNA library derived from the patient as a template using this as a probe, or derived from the patient using the oligonucleotide as a primer.
  • RT-PCR is performed using the RNA of the above as a template to obtain a cDNA fragment encoding the peptide, the cDNA fragment is incorporated into an appropriate expression vector, introduced into an appropriate host cell, and the resulting transformant is obtained.
  • a large amount of the depression marker of the present invention is obtained by culturing the recombinant peptide. Can be prepared.
  • the depression marker of the present invention can also be obtained by using a cell-free transcription / translation system using the cDNA obtained as described above as a template. Further, it can be prepared in a large amount by an organic synthesis method.
  • the peptides ⁇ 1, ⁇ 2, ⁇ 1 to ⁇ 4 and H4 of the present invention are peptides consisting of amino acid sequences shown in SEQ ID NOs: 1 to 7, respectively. Therefore, the antibody against the peptides ⁇ 1, ⁇ 2, ⁇ 1 to ⁇ 4 or H4 of the present invention can be synthesized, for example, by synthesizing all or part of the amino acid sequence using a known peptide synthesis method based on the amino acid sequence information, Alternatively, a peptide fragment containing all or part of the sequence of the peptide ⁇ 1, ⁇ 2, ⁇ 1- ⁇ 4 or H4 of the present invention by cleaving a fibrinogen A ⁇ chain, B ⁇ chain or ITIH4 protein isolated by a conventional method with an appropriate peptidase or the like It is desirable to obtain and prepare as an immunogen.
  • the antibody against the depression marker of the present invention may be either a polyclonal antibody or a monoclonal antibody, and can be prepared by a known immunological technique.
  • the antibody includes not only a complete antibody molecule but also a fragment thereof, and examples thereof include Fab, F (ab ′) 2, ScFv, and minibody.
  • the polyclonal antibody may be prepared by a carrier protein such as bovine serum albumin or KLH (Keyhole Limpet Hemocyanin) prepared by any one of the above methods or other methods.
  • a carrier protein such as bovine serum albumin or KLH (Keyhole Limpet Hemocyanin) prepared by any one of the above methods or other methods.
  • KLH Keyhole Limpet Hemocyanin
  • the polyclonal antibody may be prepared by a carrier protein such as bovine serum albumin or KLH (Keyhole Limpet Hemocyanin) prepared by any one of the above methods or other methods.
  • a carrier protein such as bovine serum albumin or KLH (Keyhole Limpet Hemocyanin) prepared by any one of the above methods or other methods.
  • KLH Keyhole Limpet Hemocyanin
  • the polyclonal antibody may be prepared by a carrier protein such as bovine serum albumin or KLH (Keyhole Limpet Hemocyanin) prepared by any one of the above methods or other
  • Monoclonal antibodies can be obtained by cell fusion methods (for example, Takeshi Watanabe, principles of cell fusion methods and preparation of monoclonal antibodies, Akira Taniuchi, Toshitada Takahashi, “Monoclonal antibodies and cancer-basics and clinics”, 2-14. Page, Science Forum Publishing, 1985).
  • the depression marker of the present invention or a partial peptide thereof is administered to a mouse subcutaneously or intraperitoneally 2-4 times together with a commercially available adjuvant, and the spleen or lymph node is collected about 3 days after the final administration, and white blood cells are collected.
  • the leukocytes and myeloma cells are cell-fused to obtain a hybridoma that produces a monoclonal antibody against the marker peptide.
  • the cell fusion may be PEG method [J. Immunol. Methods, 81 (2): 223-228 (1985)] or voltage pulse method [Hybridoma, 7 (6): 627-633 (1988)].
  • a hybridoma producing a desired monoclonal antibody can be selected by detecting an antibody that specifically binds to an antigen from the culture supernatant using a known EIA or RIA method or the like.
  • the hybridoma producing the monoclonal antibody can be cultured in vitro, or in vivo, such as mouse or rat, preferably mouse ascites, and the antibody can be obtained from the culture supernatant of the hybridoma and the ascites of the animal, respectively.
  • fibrinogen A ⁇ chain, B ⁇ chain, and ITIH4 fragment there may be a peptide other than the peptide that is a depression marker of the present invention.
  • These peptide groups do not show significant variation in depressive disorders, but any of these may be significantly higher or lower in other diseases.
  • an antibody against peptide ⁇ 1 of the present invention has cross-reactivity with one or more other fragments of fibrinogen A ⁇ chain, the possibility of misdiagnosing other diseases as a depressive disorder (false positive) increases.
  • the antibody against the depression marker of the present invention used in the test method of the present invention may be a highly specific antibody that does not cross-react with fibrinogen A ⁇ chain, B ⁇ chain or ITIH4 fragment other than the marker peptide. desirable.
  • Such an antibody can be obtained by reacting a plurality of monoclonal antibodies obtained as described above with the other fragments and selecting an antibody that does not cross-react with them.
  • the test method of the present invention using the antibody of the present invention is not particularly limited, and the amount of antibody, antigen or antibody-antigen complex corresponding to the amount of antigen in the test sample is determined by chemical or physical means. Any measurement method may be used as long as it is a measurement method that is detected and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competition method, immunometric method and sandwich method are preferably used.
  • a labeling agent used in a measurement method using a labeling substance for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like is used.
  • the radioisotope for example, [ 125 I], [ 131 I], [ 3 H], [ 14 C] and the like are used.
  • the enzyme is preferably stable and has a large specific activity.
  • ⁇ -galactosidase, ⁇ -glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used.
  • the fluorescent substance for example, fluorescamine, fluorescein isothiocyanate and the like are used.
  • luminescent substance for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used.
  • a biotin-avidin system can also be used for binding of an antibody or antigen and a labeling agent.
  • the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass.
  • a test sample is reacted with an insolubilized antibody of the present invention (primary reaction), and another labeled antibody of the present invention is reacted (secondary reaction), followed by a labeling agent on an insolubilized carrier.
  • primary reaction and secondary reaction may be performed in the reverse order, or may be performed simultaneously or at different times.
  • the monoclonal antibody against the depression marker of the present invention can also be used in measurement systems other than the sandwich method, such as a competitive method, an immunometric method, or nephrometry.
  • a competitive method the antigen in the test sample and the labeled antigen are reacted competitively with the antibody, and then the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated ( B / F separation), measure the amount of labeled B or F, and quantify the amount of antigen in the test sample.
  • a soluble antibody is used as an antibody
  • a B / F separation is performed using polyethylene glycol
  • a liquid phase method using a second antibody against the antibody and a solid-phased antibody is used as the first antibody, or
  • the first antibody is soluble
  • the second antibody is a solid phase method using a solid phase antibody.
  • the antigen of the test sample and the immobilized antigen are competitively reacted with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated, or the antigen and excess in the test sample are separated.
  • the solid phase antigen is added, and then the solid phase antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated.
  • the amount of label in any phase is measured to quantify the amount of antigen in the test sample.
  • the amount of insoluble precipitate produced as a result of antigen-antibody reaction in a gel or solution is measured. Laser nephrometry using laser scattering is preferably used even when the amount of antigen in the test sample is small and only a small amount of precipitate is obtained.
  • the antibody is immobilized on the surface of a probe that can be adapted to a mass spectrometer as described above, and a test sample is applied to the antibody on the probe.
  • a test sample is applied to the antibody on the probe. Examples include a method of detecting a peak corresponding to the molecular weight of a marker peptide recognized by the antibody by subjecting the biological sample component captured by the antibody to mass spectrometry.
  • the subject When the level of the depression marker of the present invention in a sample derived from a subject measured by any of the above methods is significantly different from the level of the marker peptide in a control sample derived from a healthy subject, the subject Can be diagnosed as likely to have a depressive disorder.
  • the depression marker of the present invention is fibrinogen A ⁇ chain, fibrinogen B ⁇ chain, or ITIH4
  • the level of the marker in the sample is significant compared to the marker level in a control sample derived from a healthy person. If so, the subject can be diagnosed with a high probability of suffering from a depressive disorder.
  • the depression marker of the present invention is peptide ⁇ 1, ⁇ 2, ⁇ 1, ⁇ 3, ⁇ 4 or peptide H4, the level of the marker in the sample is significantly increased compared to the marker level in the control sample derived from a healthy person. If so, the subject can be diagnosed as likely to have a depressive disorder.
  • the subject has a depressive disorder if the level of the marker in the sample is significantly reduced compared to the marker level in a control sample from a healthy subject. It can be diagnosed that there is a high possibility of being affected.
  • the test method of the present invention is preferably carried out by collecting biological samples from patients in time series and examining the changes over time in the expression of the depression marker of the present invention in each sample.
  • the collection interval of the biological sample is not particularly limited, but it is desirable to sample as frequently as possible within a range that does not impair the patient's QOL.
  • plasma or serum is used as a sample, blood is collected at intervals of about 1 minute to about 12 hours It is preferable to carry out.
  • fibrinogen A ⁇ chain, fibrinogen B ⁇ chain and ITIH4, or peptide ⁇ 2 tend to decrease in serum and plasma levels as the disease state of the depressive disorder progresses.
  • peptides ⁇ 1, ⁇ 2, ⁇ 1, ⁇ 3, ⁇ 4 and peptide H4 tend to increase in serum and plasma levels as the pathological state of depressive disorder progresses. Therefore, when the level of these markers decreases with time, it can be determined that there is a high possibility that the pathological condition of the depressive disorder in the patient is improved.
  • the method for examining depression disorder by the above time-series sampling is such that when a treatment for the disease is taken for the patient as a subject between the previous sampling and the current sampling, the measure is taken. It can be used to evaluate the therapeutic effect of. That is, for a sample sampled before and after treatment, when it is determined that the condition after treatment is improved compared to the condition before treatment, it can be evaluated that the treatment is effective. On the other hand, when it is determined that the condition after treatment is not improved or further deteriorated as compared with the condition before treatment, it can be evaluated that the treatment has no effect.
  • the depression marker of the present invention can also provide a drug discovery target for aggressive depression other than diagnosis. That is, when the marker peptide itself has a physiological function in the direction of treatment (remission) of the disease (referred to as “therapeutic peptide”), by administering to the patient a substance that increases the amount or activity of the peptide, When the marker peptide itself has a physiological function in the direction of exacerbation of the disease (referred to as “exacerbation peptide”), the disease can be treated by administering a substance that reduces the amount or activity of the peptide, respectively. .
  • the invention also increases the amount or activity of the peptide when the depressive marker of the invention acts as a therapeutic peptide and / or the peptide of the peptide when the depressive marker of the invention acts as an exacerbating peptide.
  • Methods of treating depressive disorders by reducing the amount or activity are provided.
  • the therapeutic method specifically includes an effective amount of a substance that increases the amount or activity of the depression marker of the present invention as a therapeutic peptide and / or a substance that decreases the amount or activity of the depression marker of the present invention as an exacerbation peptide. In a patient with a depressive disorder.
  • the present invention also comprises a substance that increases the amount or activity of the depressive marker of the present invention as a therapeutic peptide and / or a substance that decreases the amount or activity of the depressive marker of the present invention as an exacerbation peptide.
  • a therapeutic agent for depression is provided.
  • examples of the substance that increases the activity of the depression marker of the present invention as a therapeutic peptide include the peptide itself or a molecule having an agonistic action similar thereto.
  • examples of the substance that increases the activity of the depression marker of the present invention as a therapeutic peptide include non-neutralizing antibodies, preferably agonist antibodies, and the like of the peptide.
  • examples of the substance that reduces the activity of the depression marker of the present invention as an exacerbation peptide include molecules having an antagonistic action of the peptide, neutralizing antibodies against the peptide, and the like.
  • a degrading enzyme that liberates the peptide from a parent protein (fibrinogen A ⁇ chain, fibrinogen B ⁇ chain, ITIH4) existing in the living body, A substrate or substrate analog molecule of the decomposing enzyme, a molecule (including similar compounds) that promotes the production of the decomposing enzyme, further comprising an amino acid sequence that is recognized and cleaved by the decomposing enzyme on the N-terminal side and / or C-terminal side , Molecules that promote the activity of the decomposing enzyme, molecules that suppress the production of inhibitors of the decomposing enzyme, and the like.
  • a substrate or substrate analog molecule of a decomposing enzyme thus identified that is, a peptide molecule further comprising an amino acid sequence recognized and cleaved by the decomposing enzyme on the N-terminal side and / or C-terminal side of the peptide is a depressive disorder Since it is cleaved by the degrading enzyme in the body of a patient to release the depression marker of the present invention or its analog molecule as a therapeutic peptide, the same therapeutic effect can be obtained.
  • a substance that promotes the production and / or activity of the identified degrading enzyme can indirectly increase the production of the depression marker of the present invention as a therapeutic peptide.
  • the target degrading enzyme If the target degrading enzyme is identified, these substances can be screened or molecularly designed by a method known per se.
  • a substance that reduces the production of the depression marker of the present invention as an exacerbation peptide a molecule that suppresses the production of a degrading enzyme that liberates the peptide from a protein present in the living body, an inhibitor of the decomposing enzyme, Examples include molecules that promote production.
  • the degrading enzyme that liberates the depression marker of the present invention as an exacerbation peptide can be searched and identified by the same technique as the depression marker of the present invention as the therapeutic peptide.
  • a substance that suppresses (inhibits) the production or activity of the degrading enzyme directly or indirectly can be screened or molecularly designed by a method known per se.
  • compositions for oral administration include solid or liquid dosage forms, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules). Syrup, emulsion, suspension and the like.
  • Such a composition is produced by a method known per se, and contains a carrier, diluent or excipient usually used in the pharmaceutical field.
  • lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
  • a composition for parenteral administration for example, injections, suppositories and the like are used, and injections are intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, intravenous injections, intraarticular injections. Includes dosage forms such as agents.
  • Such an injection is prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the above compound or a salt thereof in a sterile aqueous or oily liquid usually used for injection.
  • aqueous solution for injection for example, isotonic solutions containing physiological saline, glucose and other adjuvants are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)) and the like may be used in combination.
  • alcohol eg, ethanol
  • polyalcohol eg, Propylene glycol, polyethylene glycol
  • nonionic surfactants eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)
  • oily liquid for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent.
  • the prepared injection solution is usually filled in a suitable amp
  • compositions are conveniently prepared in dosage unit form to suit the dosage of the active ingredient.
  • dosage form of such a dosage unit include tablets, pills, capsules, injections (ampoules), suppositories, etc., and usually 5 to 500 mg, particularly 5 to 100 mg for injections, Other dosage forms preferably contain 10 to 250 mg of the above compound.
  • Each of the above-mentioned compositions is preferably combined with a substance that increases the amount or activity of the depression marker of the present invention as the therapeutic peptide or a substance that decreases the amount or activity of the depression marker of the present invention as an exacerbation peptide.
  • Other active ingredients may be included as long as no interaction occurs.
  • the preparation thus obtained is safe and has low toxicity, it can be administered, for example, orally or parenterally to humans.
  • the substance that increases the amount or activity of the depressive marker of the present invention as a therapeutic peptide and the dose of a substance that decreases the amount or activity of the depressive marker of the present invention as an exacerbating peptide depend on its action, administration route, and patient severity. Although there are differences depending on the degree, age, body weight, drug acceptability, etc., for example, the amount of active ingredient per day for an adult is about 0.0008 to about 25 mg / kg, preferably about 0.008 to about 2 mg / kg. Yes, this can be administered in one or several divided doses.
  • Example 1 Profiling Analysis Using BlotChip Serum / plasma of various patients with depressive disorder and 1.5 ⁇ L of healthy subject serum / plasma with electrophoresis sample treatment solution (NuPAGE (registered trademark) LDS Sample Buffer 4x; Invitrogen) 4.5 ⁇ L After mixing and heating at 70 ° C. for 10 minutes, the mixture was applied to a 4-12% gradient polyacrylamide gel (Invitrogen) and electrophoresed. After the completion of electrophoresis, the gel was cut out, layered on BLOTCHIP (registered trademark) (Protosera, Inc.), and transferred in an electric transfer buffer (BLOTBuffer TM ; Protosera, Inc.) at 90 mA for 120 minutes.
  • BLOTCHIP registered trademark
  • BLOTBuffer TM Electric transfer buffer
  • Example 2 Identification of peptides by de novo MS / MS analysis on BlotChip
  • matrix-assisted laser desorption ionization Using time-of-flight (MALDI-TOF) mass spectrometer (Ultra-FlexII manufactured by Bruker Daltnics), Bradykinin, Angiotensin II, Angiotensin I, Substance P, Bombesin, Renin Substrate, ACTH Clip ⁇ 1-17 ⁇ , ACTH Mass calibration was performed using Clip ⁇ 18-39 ⁇ , Somatostatin.
  • peptides 1 to 7 were identified as peptides having the respective amino acid sequences shown in Table 2 (the amino acids at both ends represent adjacent amino acid residues in the parent protein).
  • peptides 1, 4, 5, and 6 were fibrinogen B ⁇ chain fragments
  • peptide 2 was an ITIH4 fragment
  • peptides 3 and 7 were fibrinogen A ⁇ chain fragments.
  • the clinical examination method using the depression marker of the present invention is useful in that a depressive disorder can be determined promptly and accurately, so that early detection and early treatment of the disease are possible.
  • the depression marker of the present invention which is a measurement target in the present invention, can itself be a drug discovery target in a depressive disorder, it is used for screening a novel therapeutic drug for the disease and treating the disease using them. This is extremely useful.
  • the present invention is based on Japanese Patent Application No. 2008-021719 filed in Japan (filing date: January 31, 2008), the contents of which are incorporated in full herein.

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Abstract

L'invention concerne un marqueur de diagnostic pour un trouble dépressif. L'invention concerne un procédé de test pour diagnostiquer un trouble dépressif chez un sujet caractérisé en ce qu'il consiste à mesurer la quantité d'un ou plusieurs peptides choisis dans le groupe constitué par la chaîne Aα du fibrinogène, la chaîne Bβ du fibrinogène, la chaîne lourde de l'inhibiteur inter-α de la trypsine et des produits de décomposition de ceux-ci dans un échantillon biologique prélevé chez le sujet.
PCT/JP2009/051527 2008-01-31 2009-01-30 Marqueur pour la dépression et un état déprimé et détection et diagnostic l'utilisant WO2009096502A1 (fr)

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WO2018117064A1 (fr) * 2016-12-21 2018-06-28 国立大学法人徳島大学 Procédé de test de maladie impliquant un état dépressif
WO2020202923A1 (fr) 2019-03-29 2020-10-08 日本たばこ産業株式会社 Procédé d'évaluation de dépression et procédé permettant d'évaluer un procédé de traitement de dépression, et procédé d'acquisition de données permettant d'évaluer une dépression ou un résultat d'un procédé de traitement de dépression

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WO2018117064A1 (fr) * 2016-12-21 2018-06-28 国立大学法人徳島大学 Procédé de test de maladie impliquant un état dépressif
WO2020202923A1 (fr) 2019-03-29 2020-10-08 日本たばこ産業株式会社 Procédé d'évaluation de dépression et procédé permettant d'évaluer un procédé de traitement de dépression, et procédé d'acquisition de données permettant d'évaluer une dépression ou un résultat d'un procédé de traitement de dépression

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