WO2010041068A1 - Biomarqueurs - Google Patents

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Publication number
WO2010041068A1
WO2010041068A1 PCT/GB2009/051335 GB2009051335W WO2010041068A1 WO 2010041068 A1 WO2010041068 A1 WO 2010041068A1 GB 2009051335 W GB2009051335 W GB 2009051335W WO 2010041068 A1 WO2010041068 A1 WO 2010041068A1
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Prior art keywords
schizophrenia
quantifying
biomarker
peptide
detecting
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PCT/GB2009/051335
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English (en)
Inventor
Paul Guest
Lan Wang
Yishai Levin
Sabine Bahn
Agnes Ernst
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Cambridge Enterprise Limited
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Publication of WO2010041068A1 publication Critical patent/WO2010041068A1/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
    • G01N2333/575—Hormones
    • G01N2333/62—Insulins
    • 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
    • G01N2800/302—Schizophrenia

Definitions

  • Schizophrenia is a multifaceted neuropsychiatric disease with an onset and aetiology driven by a complex interplay of genetic, developmental, nutritional and environmental factors [1]. It affects at least 1 % of the world population and costs hundreds of billions of U.S. dollars in healthcare provision and lost earnings [2].
  • the present invention is based on the discovery that insulin secretory granule peptide biomarkers are useful in the diagnosis and/or monitoring of schizophrenia and other psychotic disorders.
  • the present invention is the use of one or more first peptides selected from proinsulin, des 31 ,32 proinsulin and C-peptide, as a biomarker for schizophrenia or other psychotic disorders, or predisposition thereto.
  • the present invention is the use of two or more second peptides selected from insulin, Chromogranin A, VGF, dopamine ⁇ hydroxylase and cathepsin L2, as a biomarker for schizophrenia or other psychotic disorders, or predisposition thereto.
  • the present invention is the use of a combination of proinsulin, des 31 ,32 proinsulin, insulin and C-peptide, as a biomarker for schizophrenia or other psychotic disorders, or predisposition thereto.
  • the present invention is a method of diagnosing or monitoring schizophrenia or other psychotic disorders, or predisposition thereto, comprising detecting and/or quantifying, in a sample from a test subject, one or more of the first peptide biomarkers as defined above.
  • the present invention is a method of diagnosing or monitoring schizophrenia or other psychotic disorders, or predisposition thereto, comprising detecting and/or quantifying, in a sample from a test subject, two or more of the second peptide biomarkers as defined above.
  • the present invention is a method of diagnosing or monitoring schizophrenia or other psychotic disorders, o r predisposition thereto, comprising detecting and/or quantifying, in a sample from a test subject, proinsulin, des 31 ,32 proinsulin, insulin and C-peptide.
  • the present invention is a method of monitoring efficacy of a therapy in a subject having, suspected of having, or of being predisposed to schizophrenia or other psychotic disorders, comprising detecting and/or quantifying, in a sample from said subject, one or more of the first peptide biomarkers as defined above.
  • the present invention is a method of monitoring efficacy of a therapy in a subject having, suspected of having, or of being predisposed to schizophrenia or other psychotic disorders, comprising detecting and/or quantifying, in a sample from said subject, two or more of the second peptide biomarkers as defined above.
  • the present invention is a method of monitoring efficacy of a therapy in a subject having, suspected of having, or of being predisposed to schizophrenia or other psychotic disorders, comprising detecting and/or quantifying, in a sample from said subject, proinsulin, des 31 ,32 proinsulin, insulin and C-peptide.
  • a further aspect of the invention provides ligands, such as naturally occurring or chemically synthesised compounds, capable of specific binding to the peptide biomarker.
  • a ligand according to the invention may comprise a peptide, an antibody or a fragment thereof, or an aptamer or oligonucleotide, capable of specific binding to the peptide biomarker.
  • the antibody can be a monoclonal antibody or a fragment thereof capable of specific binding to the peptide biomarker.
  • a ligand according to the invention may be labelled with a detectable marker, such as a luminescent, fluorescent or radioactive marker; alternatively or additionally a ligand according to the invention may be labelled with an affinity tag, e.g. a biotin, avidin, streptavidin or His (e.g. hexa-His) tag.
  • ligands as described herein, which may be naturally occurring or chemically synthesised, and is suitably a peptide, antibody or fragment thereof, aptamer or oligonucleotide, or the use of a biosensor of the invention, or an array of the invention, or a kit of the invention to detect and/or quantify the peptide.
  • the detection and/or quantification can be performed on a biological sample such as from the group consisting of CSF, whole blood, blood serum, plasma, urine, saliva, or other bodily fluid, breath, e.g. as condensed breath, or an extract or purification there from, or dilution thereof. Diagnostic or monitoring kits are provided for performing methods of the invention.
  • kits will suitably comprise a ligand according to the invention, for detection and/or quantification of the peptide biomarker, and/or a biosensor, and/or an array as described herein, optionally together with instructions for use of the kit.
  • a further aspect of the invention is a kit for monitoring or diagnosing schizophrenia or other psychotic disorders, comprising a biosensor capable of detecting and/or quantifying one or more of the first peptide biomarkers listed above.
  • a further aspect of the invention is a kit for monitoring or diagnosing schizophrenia or other psychotic disorders, comprising a biosensor capable of detecting and/or quantifying two or more of the second peptide biomarkers listed above.
  • a further aspect of the invention is a kit for monitoring or diagnosing schizophrenia or other psychotic disorders, comprising a biosensor capable of detecting and/or quantifying proinsulin, des 31 , 32 proinsulin, insulin and C- peptide.
  • Biomarkers for schizophrenia or other psychotic disorders are essential targets for discovery of novel targets and drug molecules that retard or halt progression of the disorder. As the level of the peptide biomarker is indicative of disorder and of drug response, the biomarker is useful for identification of novel therapeutic compounds in in vitro and/or in vivo assays. Biomarkers of the invention can be employed in methods for screening for compounds that modulate the activity of the peptide.
  • a ligand as described, which can be a peptide, antibody or fragment thereof or aptamer or oligonucleotide according to the invention; or the use of a biosensor according to the invention, or an array according to the invention; or a kit according to the invention, to identify a substance capable of promoting and/or of suppressing the generation of the biomarker.
  • biomarker means a distinctive biological or biologically derived indicator of a process, event, or condition.
  • Peptide biomarkers can be used in methods of diagnosis, e.g. clinical screening, and prognosis assessment and in monitoring the results of therapy, identifying patients most likely to respond to a particular therapeutic treatment, drug screening and development. Biomarkers and uses thereof are valuable for identification of new drug treatments and for discovery of new targets for drug treatment.
  • Biosensors according to the invention may comprise a ligand or ligands, as described herein, capable of specific binding to the peptide biomarker. Such biosensors are useful in detecting and/or quantifying a peptide of the invention.
  • a biosensor according to the invention may comprise the peptide biomarker or a structural/shape mimic thereof capable of specific binding to an antibody against the peptide biomarker.
  • an array comprising a ligand or mimic as described herein.
  • one or more of the first peptide biomarkers as defined above may be used in combination with one or more second peptide selected from insulin, chromogranin A, VGF, dopamine ⁇ hydroxylase and cathepsin L2, as a biomarker for schizophrenia or other psychotic disorders, or predisposition thereto.
  • proinsulin, des 31 , 32 proinsulin, insulin and C-peptide are used together as a biomarker for schizophrenia or other psychotic disorders, or predisposition thereto. These may be the only biomarkers used to monitor and/or diagnose the psychotic disorder. Alternatively, these may be used in combination with one or more of the third peptide biomarkers, as defined above.
  • one or more of the biomarkers may be replaced by a molecule, or a measurable fragment of the molecule, found upstream or downstream of the biomarker in a biological pathway.
  • references herein to "other psychotic disorder” relate to any appropriate psychotic disorder according to DSM-IV Diagnostic and Statistical Manual of Mental Disorders, 4th edition, American Psychiatric Assoc, Washington, D. C, 2000.
  • the other psychotic disorder is a psychotic disorder related to schizophrenia.
  • Examples of psychotic disorders related to schizophrenia include brief psychotic disorder delusional disorder, psychotic disorder due to a general medical condition, schizoeffective disorder, schizophreniform disorder, and substance-induced psychotic disorder.
  • biosensor means anything capable of detecting the presence of the biomarker. Examples of biosensors are described herein.
  • kits for the diagnosis and monitoring of schizophrenia and other psychotic disorders are described herein.
  • they contain a biosensor capable of detecting and/or quantifying one or more biomarkers.
  • Monitoring methods of the invention can be used to monitor onset, progression, stabilisation, amelioration and/or remission.
  • detecting and/or quantifying the peptide biomarker in a biological sample from a test subject may be performed on two or more occasions. Comparisons may be made between the level of biomarker in samples taken on two or more occasions. Assessment of any change in the level of the peptide biomarker in samples taken on two or more occasions may be performed. Modulation of the peptide biomarker level is useful as an indicator of the state of the schizophrenia or other psychotic disorders or predisposition thereto. An increase in the level of the biomarker, over time is indicative of onset or progression, i.e. worsening of this disorder, whereas a decrease in the level of the peptide biomarker indicates amelioration or remission of the disorder, or vice versa.
  • a method of diagnosis of or monitoring according to the invention may comprise quantifying the peptide biomarker in a test biological sample from a test subject and comparing the level of the peptide present in said test sample with one or more controls.
  • the control used in a method of the invention can be one or more control(s) selected from the group consisting of: the level of biomarker peptide found in a normal control sample from a normal subject, a normal biomarker peptide level; a normal biomarker peptide range, the level in a sample from a subject with schizophrenia or another psychotic disorder, or a diagnosed predisposition thereto; schizophrenia or another psychotic disorder biomarker peptide level, or schizophrenia or another psychotic disorder biomarker peptide range.
  • a preferred method of diagnosing schizophrenia or other psychotic disorders, or predisposition thereto comprises:
  • a higher level of the peptide biomarker in the test sample relative to the level in the normal control is indicative of the presence of schizophrenia or other psychotic disorders, or predisposition thereto; an equivalent or lower level of the peptide in the test sample relative to the normal control is indicative of absence of schizophrenia or other psychotic disorders and/or absence of a predisposition thereto.
  • diagnosis encompasses identification, confirmation, and/or characterisation of schizophrenia or other psychotic disorders, or predisposition thereto. By predisposition it is meant that a subject does not currently present with the disorder, but is liable to be affected by the disorder in time.
  • Methods of monitoring and of diagnosis according to the invention are useful to confirm the existence of a disorder, or predisposition thereto; to monitor development of the disorder by assessing onset and progression, or to assess amelioration or regression of the disorder.
  • Methods of monitoring and of diagnosis are also useful in methods for assessment of clinical screening, prognosis, choice of therapy, evaluation of therapeutic benefit, i.e. for drug screening and drug development.
  • Efficient diagnosis and monitoring methods provide very powerful "patient solutions” with the potential for improved prognosis, by establishing the correct diagnosis, allowing rapid identification of the most appropriate treatment (thus lessening unnecessary exposure to harmful drug side effects), reducing "down- time” and relapse rates.
  • test samples may be taken on two or more occasions.
  • the method may further comprise comparing the level of the biomarker(s) present in the test sample with one or more control(s) and/or with one or more previous test sample(s) taken earlier from the same test subject, e.g. prior to commencement of therapy, and/or from the same test subject at an earlier stage of therapy.
  • the method may comprise detecting a change in the level of the biomarker(s) in test samples taken on different occasions.
  • the invention provides a method for monitoring efficacy of therapy for schizophrenia or other psychotic disorders in a subject, comprising:
  • a decrease in the level of the peptide biomarker in the test sample relative to the level in a previous test sample taken earlier from the same test subject is indicative of a beneficial effect, e.g. stabilisation or improvement, of said therapy on the disorder, suspected disorder or predisposition thereto.
  • Methods for monitoring efficacy of a therapy can be used to monitor the therapeutic effectiveness of existing therapies and new therapies in human subjects and in non-human animals (e.g. in animal models). These monitoring methods can be incorporated into screens for new drug substances and combinations of substances.
  • the time elapsed between taking samples from a subject undergoing diagnosis or monitoring will be 3 days, 5 days, a week, two weeks, a month, 2 months, 3 months, 6 or 12 months. Samples may be taken prior to and/or during and/or following an anti-depressant therapy. Samples can be taken at intervals over the remaining life, or a part thereof, of a subject.
  • detecting means confirming the presence of the peptide biomarker present in the sample.
  • Quantifying the amount of the biomarker present in a sample may include determining the concentration of the peptide biomarker present in the sample. Detecting and/or quantifying may be performed directly on the sample, or indirectly on an extract therefrom, or on a dilution thereof.
  • the presence of the peptide biomarker is assessed by detecting and/or quantifying antibody or fragments thereof capable of specific binding to the biomarker that are generated by the subject's body in response to the peptide and thus are present in a biological sample from a subject having schizophrenia or other psychotic disorders or a predisposition thereto.
  • Detecting and/or quantifying can be performed by any method suitable to identify the presence and/or amount of a specific protein in a biological sample from a patient or a purification or extract of a biological sample or a dilution thereof.
  • quantifying may be performed by measuring the concentration of the peptide biomarker in the sample or samples.
  • Biological samples that may be tested in a method of the invention include cerebrospinal fluid (CSF), whole blood, blood serum, plasma, urine, saliva, or other bodily fluid (stool, tear fluid, synovial fluid, sputum), breath, e.g. as condensed breath, or an extract or purification therefrom, or dilution thereof.
  • Biological samples also include tissue homogenates, tissue sections and biopsy specimens from a live subject, or taken post-mortem. The samples can be prepared, for example where appropriate diluted or concentrated, and stored in the usual manner.
  • Detection and/or quantification of peptide biomarkers may be performed by detection of the peptide biomarker or of a fragment thereof, e.g. a fragment with C-terminal truncation, or with N-terminal truncation. Fragments are suitably greater than 4 amino acids in length, preferably 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.
  • the biomarker may be directly detected, e.g. by SELDI or MALDI-TOF.
  • the biomarker may be detected directly or indirectly via interaction with a ligand or ligands such as an antibody or a biomarker-binding fragment thereof, or other peptide, or ligand, e.g. aptamer, or oligonucleotide, capable of specifically binding the biomarker.
  • the ligand may possess a detectable label, such as a luminescent, fluorescent or radioactive label, and/or an affinity tag.
  • detecting and/or quantifying can be performed by one or more method(s) selected from the group consisting of: SELDI (-TOF), MALDI (-TOF), a 1-D gel-based analysis, a 2-D gel-based analysis, Mass spec (MS), reverse phase (RP) LC, size permeation (gel filtration), ion exchange, affinity, HPLC, UPLC and other LC or LC MS-based techniques.
  • SELDI SELDI
  • MALDI MALDI
  • MS mass spec
  • RP reverse phase
  • size permeation gel filtration
  • ion exchange affinity
  • HPLC HPLC
  • UPLC UPLC
  • LC MS-based techniques e.g.
  • Methods of diagnosing or monitoring according to the invention may comprise analysing a sample of cerebrospinal fluid (CSF) by SELDI TOF or MALDI TOF to detect the presence or level of the peptide biomarker. These methods are also suitable for clinical screening, prognosis, monitoring the results of therapy, identifying patients most likely to respond to a particular therapeutic treatment, for drug screening and development, and identification of new targets for drug treatment.
  • CSF cerebrospinal fluid
  • Detecting and/or quantifying the peptide biomarkers may be performed using an immunological method, involving an antibody, or a fragment thereof capable of specific binding to the peptide biomarker.
  • Suitable immunological methods include sandwich immunoassays, such as sandwich ELISA, in which the detection of the peptide biomarkers is performed using two antibodies which recognize different epitopes on a peptide biomarker; radioimmunoassays (RIA), direct, indirect or competitive enzyme linked immunosorbent assays (ELISA), enzyme immunoassays (EIA), Fluorescence immunoassays (FIA), western blotting, immunoprecipitation and any particle-based immunoassay (e.g. using gold, silver, or latex particles, magnetic particles, or Q-dots).
  • Immunological methods may be performed, for example, in microtitre plate or strip format.
  • Immunological methods in accordance with the invention may be based, for example, on any of the following methods.
  • Immunoprecipitation is the simplest immunoassay method; this measures the quantity of precipitate, which forms after the reagent antibody has incubated with the sample and reacted with the target antigen present therein to form an insoluble aggregate.
  • Immunoprecipitation reactions may be qualitative or quantitative.
  • particle immunoassays several antibodies are linked to the particle, and the particle is able to bind many antigen molecules simultaneously. This greatly accelerates the speed of the visible reaction. This allows rapid and sensitive detection of the biomarker.
  • biomarker In immunonephelometry, the interaction of an antibody and target antigen on the biomarker results in the formation of immune complexes that are too small to precipitate. However, these complexes will scatter incident light and this can be measured using a nephelometer.
  • the antigen, i.e. biomarker, concentration can be determined within minutes of the reaction.
  • Radioimmunoassay (RIA) methods employ radioactive isotopes such as I 125 to label either the antigen or antibody.
  • the isotope used emits gamma rays, which are usually measured following removal of unbound (free) radiolabel.
  • the major advantages of RIA compared with other immunoassays, are higher sensitivity, easy signal detection, and well-established, rapid assays.
  • the major disadvantages are the health and safety risks posed by the use of radiation and the time and expense associated with maintaining a licensed radiation safety and disposal program. For this reason, RIA has been largely replaced in routine clinical laboratory practice by enzyme immunoassays.
  • EIA Enzyme immunoassays were developed as an alternative to radioimmunoassays (RIA). These methods use an enzyme to label either the antibody or target antigen. The sensitivity of EIA approaches that for RIA, without the danger posed by radioactive isotopes.
  • One of the most widely used EIA methods for detection is the enzyme-linked immunosorbent assay (ELISA). ELISA methods may use two antibodies one of which is specific for the target antigen and the other of which is coupled to an enzyme, addition of the substrate for the enzyme results in production of a chemoluminescent or fluorescent signal.
  • Fluorescent immunoassay refers to immunoassays which utilize a fluorescent label or an enzyme label which acts on the substrate to form a fluorescent product. Fluorescent measurements are inherently more sensitive than colorimetric (spectrophotometric) measurements. Therefore, FIA methods have greater analytical sensitivity than EIA methods, which employ absorbance (optical density) measurement. Chemiluminescent immunoassays utilize a chemiluminescent label, which produces light when excited by chemical energy; the emissions are measured using a light detector.
  • Immunological methods according to the invention can thus be performed using well-known methods. Any direct (e.g., using a sensor chip) or indirect procedure may be used in the detection of peptide biomarkers of the invention.
  • Biotin-Avidin or Biotin-Streptavidin systems are generic labelling systems that can be adapted for use in immunological methods of the invention.
  • One binding partner hapten, antigen, ligand, aptamer, antibody, enzyme etc
  • biotin is labelled with avidin or streptavidin.
  • avidin or streptavidin is conventional technology for immunoassays, gene probe assays and (bio)sensors, but is an indirect immobilisation route rather than a direct one.
  • a biotinylated ligand e.g.
  • antibody or aptamer) specific for a peptide biomarker of the invention may be immobilised on an avidin or streptavidin surface, the immobilised ligand may then be exposed to a sample containing or suspected of containing the peptide biomarker in order to detect and/or quantify a peptide biomarker of the invention. Detection and/or quantification of the immobilised antigen may then be performed by an immunological method as described herein.
  • antibody as used herein includes, but is not limited to: polyclonal, monoclonal, bispecific, humanised or chimeric antibodies, single chain antibodies, Fab fragments and F(ab') 2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies and epitope-binding fragments of any of the above.
  • antibody as used herein also refers to immunoglobulin molecules and immunologically-a ct i ve p o rt i o n s of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen.
  • the immunoglobulin molecules of the invention can be of any class (e.
  • biosensors g., IgG, IgE, IgM, IgD and IgA
  • detecting and quantifying can be performed using a biosensor, microanalytical system, microengineered system, microseparation system, immunochromatography system or other suitable analytical devices.
  • the biosensor may incorporate an immunological method for detection of the biomarker(s), electrical, thermal, magnetic, optical (e.g. hologram) or acoustic technologies. Using such biosensors, it is possible to detect the target biomarker(s) at the anticipated concentrations found in biological samples.
  • an apparatus for diagnosing or monitoring schizophrenia or other psychotic disorders which comprises a biosensor, microanalytical, microengineered, microseparation and/or immunochromatography system configured to detect and/or quantify any of the biomarkers defined herein.
  • biomarker(s) of the invention can be detected using a biosensor incorporating technologies based on "smart" holograms, or high frequency acoustic systems, such systems are particularly amenable to "bar code” or array configurations.
  • a holographic image is stored in a thin polymer film that is sensitised to react specifically with the biomarker.
  • the biomarker reacts with the polymer leading to an alteration in the image displayed by the hologram.
  • the test result read-out can be a change in the optical brightness, image, colour and/or position of the image.
  • a sensor hologram can be read by eye, thus removing the need for detection equipment.
  • a simple colour sensor can be used to read the signal when quantitative measurements are required. Opacity or colour of the sample does not interfere with operation of the sensor.
  • the format of the sensor allows multiplexing for simultaneous detection of several substances. Reversible and irreversible sensors can be designed to meet different requirements, and continuous monitoring of a particular biomarker of interest is feasible.
  • biosensors for detection of one or more biomarkers of the invention combine biomolecular recognition with appropriate means to convert detection of the presence, or quantitation, of the biomarker in the sample into a signal.
  • Biosensors can be adapted for "alternate site” diagnostic testing, e.g. in the ward, outpatients' department, surgery, home, field and workplace.
  • Biosensors to detect one or more biomarkers of the invention include acoustic, plasmon resonance, holographic and microengineered sensors.
  • Imprinted recognition elements may be employed in biosensors for detection of the one or more biomarkers of the invention.
  • Methods involving detection and/or quantification of one or more peptide biomarkers of the invention can be performed on bench-top instruments, or can be incorporated onto disposable, diagnostic or monitoring platforms that can be used in a non-laboratory environment, e.g. in the physician's office or at the patient's bedside.
  • Suitable biosensors for performing methods of the invention include "credit" cards with optical or acoustic readers. Biosensors can be configured to allow the data collected to be electronically transmitted to the physician for interpretation and thus can form the basis for e-neuromedicine.
  • Any suitable animal may be used as a subject non-human animal, for example a non-human primate, horse, cow, pig, goat, sheep, dog, cat, fish, rodent, e.g. guinea pig, rat or mouse; insect (e.g. Drosophila), amphibian (e.g. Xenopus) or C. elegans.
  • a non-human primate horse, cow, pig, goat, sheep, dog, cat, fish
  • rodent e.g. guinea pig, rat or mouse
  • insect e.g. Drosophila
  • amphibian e.g. Xenopus
  • C. elegans e.g. Xenopus
  • the test substance can be a known chemical or pharmaceutical substance, such as, but not limited to, an anti-depressive disorder therapeutic; or the test substance can be a novel synthetic or natural chemical entity, or a combination of two or more of the aforesaid substances.
  • a method of identifying a substance capable of promoting or suppressing the generation of the peptide biomarker in a subject comprising exposing a test cell to a test substance and monitoring the level of the peptide biomarker within said test cell, or secreted by said test cell.
  • the test cell could be prokaryotic, however it is preferred that a eukaryotic cell be employed in cell-based testing methods.
  • the eukaryotic cell is a yeast cell, insect cell, Drosophila cell, amphibian cell (e.g. from Xenopus), C. elegans cell or is a cell of human, non-human primate, equine, bovine, porcine, caprine, ovine, canine, feline, piscine, rodent or murine origin.
  • non- human animals or cells can be used that are capable of expressing the peptide.
  • Screening methods also encompass a method of identifying a ligand capable of binding to the peptide biomarker according to the invention, comprising incubating a test substance in the presence of the peptide biomarker in conditions appropriate for binding, and detecting and/or quantifying binding of the peptide to said test substance.
  • High-throughput screening technologies based on the biomarker, uses and methods of the invention, e.g. configured in an array format, are suitable to monitor biomarker signatures for the identification of potentially useful therapeutic compounds, e.g. ligands such as natural compounds, synthetic chemical compounds (e.g. from combinatorial libraries), peptides, monoclonal or polyclonal antibodies or fragments thereof, which may be capable of binding the biomarker.
  • Methods of the invention can be performed in array format, e.g. on a chip, or as a multiwell array. Methods can be adapted into platforms for single tests, or multiple identical or multiple non-identical tests, and can be performed in high throughput format. Methods of the invention may comprise performing one or more additional, different tests to confirm or exclude diagnosis, and/or to further characterise a condition.
  • the invention further provides a substance, e.g. a ligand, identified or identifiable by an identification or screening method or use of the invention.
  • a substance e.g. a ligand, identified or identifiable by an identification or screening method or use of the invention.
  • Such substances may be capable of inhibiting, directly or indirectly, the activity of the peptide biomarker, or of suppressing generation of the peptide biomarker.
  • the term "substances" includes substances that do not directly bind the peptide biomarker and directly modulate a function, but instead indirectly modulate a function of the peptide biomarker.
  • Ligands are also included in the term substances; ligands of the invention (e.g. a natural or synthetic chemical compound, peptide, aptamer, oligonucleotide, antibody or antibody fragment) are capable of binding, preferably specific binding, to the peptide.
  • the invention further provides a substance according to the invention for use in the treatment of schizophrenia or other psychotic disorders, or pre
  • a substance according to the invention in the treatment of schizophrenia or other psychotic disorder, or predisposition thereto.
  • kits for diagnosing or monitoring schizophrenia or other psychotic disorders, or predisposition thereto may contain one or more components selected from the group: a ligand specific for the peptide biomarker or a structural/shape mimic of the peptide biomarker, one or more controls, one or more reagents and one or more consumables; optionally together with instructions for use of the kit.
  • biomarkers for schizophrenia or other psychotic disorders permits integration of diagnostic procedures and therapeutic regimes.
  • many anti-depressant therapies have required treatment trials lasting weeks to months for a given therapeutic approach.
  • Detection of a peptide biomarker of the invention can be used to screen subjects prior to their participation in clinical trials.
  • the biomarkers provide the means to indicate therapeutic response, failure to respond, unfavourable side-effect profile, degree of medication compliance and achievement of adequate serum drug levels.
  • the biomarkers may be used to provide warning of adverse drug response.
  • Biomarkers are useful in development of personalized brain therapies, as assessment of response can be used to fine-tune dosage, minimise the number of prescribed medications, reduce the delay in attaining effective therapy and avoid adverse drug reactions.
  • patient care can be tailored precisely to match the needs determined by the disorder and the pharmacogenomic profile of the patient, the biomarker can thus be used to titrate the optimal dose, predict a positive therapeutic response and identify those patients at high risk of severe side effects.
  • Biomarker-based tests provide a first line assessment of 'new' patients, and provide objective measures for accurate and rapid diagnosis, in a time frame and with precision, not achievable using the current subjective measures.
  • diagnostic biomarker tests are useful to identify family members or patients at high risk of developing schizophrenia or other psychotic disorders. This permits initiation of appropriate therapy, or preventive measures, e.g. managing risk factors. These approaches are recognised to improve outcome and may prevent overt onset of the disorder.
  • Biomarker monitoring methods, biosensors and kits are also vital as patient monitoring tools, to enable the physician to determine whether relapse is due to worsening of the disorder, poor patient compliance or substance abuse. If pharmacological treatment is assessed to be inadequate, then therapy can be reinstated or increased; a change in therapy can be given if appropriate. As the biomarkers are sensitive to the state of the disorder, they provide an indication of the impact of drug therapy or of substance abuse. The following study illustrates the invention. Study
  • This study was designed to establish the utility of certain insulin secretory granule protein biomarkers for: 1 ) the early diagnosis of schizophrenia; 2) the prediction of drug responses with a focus on metabolic side effects; 3) the selection of appropriate drug treatments at first onset and; 4) translational medicine focussing on the validation of preclinical models for novel drug discovery and development.
  • these biomarkers were tested for patient selection and for monitoring their responses to novel therapeutic treatments which improve peripheral or central insulin action such as the insulin- sensitizing agents, thiazolidinediones.
  • biomarkers are associated with impaired insulin signalling.
  • the biomarkers that were elevated in schizophrenic patients were insulin, proinsulin, des 31 ,32 proinsulin conversion intermediate, proinsulin connecting (C)-peptide, and the secretory granule accessory proteins chromogranin A, VGF, dopamine ⁇ hydroxylase and cathepsin L2.
  • C proinsulin connecting
  • Many of these markers are metabolic in nature, indicative of perturbations in insulin signalling and glucoregulatory pathways.
  • Glucose was determined in 25 ⁇ l_ plasma/serum spectrophotometrically by measuring the extent of glucose phosphorylation and subsequent NAD to NADH conversion using a Dimension RXL Clinical Chemistry System (Dade Behring; Milton Keynes, UK). All reagents and calibrants were supplied by the manufacturer.
  • Insulin was determined in 25 ⁇ L of serum/plasma using a two-step time resolved fluorometric assay and reagents and calibrants recommended by the manufacturer (DAKO Ltd; Ely, UK). Cross-reactivity with intact proinsulin, des 31/32 proinsulin and C-peptide was less than 0.5, 1 and 0.1 %, respectively [12].
  • Intact proinsulin and des 31 ,32 proinsulin were determined in 250 ⁇ L of plasma samples using a two-site time-resolved fluorescence assay employing combinations of monoclonal antibodies 3B1 , A6 and CPT-3F11 which recognize epitopes in the insulin B-chain, the B-chain/C-peptide junction and the C-peptide, respectively [13].
  • the assay for proinsulin used the 3B1 antibody for capture and the A6 antibody for detection and, therefore, only recognizes the intact proinsulin molecule.
  • the assay for des 31 ,32 proinsulin used the 3B1 antibody for capture and the CPT-3F11 antibody for detection and recognized total proinsulin (does not distinguish between des 31 ,32 proinsulin and intact proinsulin) [13].
  • Des 31 , 32 proinsulin levels were calculated by subtracting intact proinsulin from total proinsulin values.
  • C-peptide concentrations were determined in 25 ⁇ l_ serum by comparison against a standard curve using the LincoPlex Human Endocrine kit (Linco Diagnostic Services; St Charles, IL, USA). The software provided automated data analysis and generation of summary reports.
  • LC-MS E Liquid chromatography tandem mass spectrometry
  • LC-MS E Waters Protein Expression System comprised of a IOkpsi nanoAcquity Ultra Performance Liquid Chromatography System coupled to a Quadrapole-Time-of-Flight (Q-TOF) Mass Spectrometer Premier.
  • Samples (2 ⁇ L each) were injected in triplicate into a 0.18mm x 20mm Symmetry BEH nanoAcquity trapping column at a flow rate of 15 ⁇ L/min for 1 minute in solution A (99.9% H 2 O, 0.1 % formic acid).
  • samples were loaded into a nanoAcquity 0.075mm x 200mm BEH analytical column at 0.3 ⁇ L/min using a gradient comprised of the indicated percentages of solution A (above) and solution B (0.99% acetonitrile, 0.1 % formic acid): 0-1 min, 97/3; 60 min, 70/30; 80-90 min, 10/90; 91-110 min, 97/3.
  • Data were acquired in expression positive V mode at 10000 FWHM resolution.
  • a reference was acquired every 30 scans with a scan time of 0.6 sec (mass range, 50 - 1990 m/z; low collision energy, 4ev; high collision energy, 20-43ev).
  • Insulin is produced with the C-peptide within pancreatic ⁇ cell granules by complete endoproteolytic cleavage of the proinsulin molecule at paired basic ami n o acids ma rki ng the B-chain/C-peptide and the C-peptide/A-chain boundaries [15].
  • Insulin is released into the circulation along with unprocessed proinsulin, the proinsulin conversion intermediates and C-peptide in response to elevated glucose levels.
  • Plasma samples from 26 schizophrenia patients and 21 controls (Cohort 3) were analyzed using two-site time-resolved fluorescence assays as detailed in the methods section [13].
  • the assay for proinsulin is capable of detecting intact proinsulin and the des 64,65 proinsulin conversion intermediate.
  • plasma levels of des 64,65 proinsulin are virtually undetectable in normal humans after an overnight fast, it is not considered to contribute significantly to the intact proinsulin signal [13].
  • the assay for the des 31 ,32 proinsulin intermediate measures total proinsulin specifically and the actual levels of the intermediate were calculated after subtraction of the intact proinsulin values.
  • Total proinsulin is comprised of the intact proinsulin molecule and the des 31 ,32 proinsulin and des 64,65 proinsulin conversion intermediates.
  • the C- peptide is normally excised from proinsulin as described above.
  • Serum samples from 20 first-onset, drug-na ⁇ ve schizophrenia patients and 20 control subjects (Cohort 2) were analysed to determine the abundance of total proinsulin and C- peptide using a two-site enzyme-linked immunosorbent assay and the LincoPlex assay kit, respectively, as described in the Methods section. Quality controls and standards were also included in the assay and the observed concentrations in the clinical samples were within the normal ranges given by the suppliers.
  • both of these proteins are constituents of neuroendocrine secretory granules, including those of pancreatic ⁇ cells [18,19] thus lending further support to our hypothesis of increased insulin secretory granule turnover in first onset schizophrenia.
  • VGF is a secreted neuroendocrine polypeptide precursor that has been shown previously to be involved in regulation of energy balance and hippocampal synaptic plasticity [20,21]. Previous studies have shown that distinct VGF-derived peptides are produced in insulin-containing pancreatic ⁇ cells and are co- released with insulin in response to elevated glucose 22,23].
  • hyperinsulinaemia may play a role in the onset of schizophrenia and suggest that the insulin-related peptides and other secretory granule constituents may have utility as part of a molecular fingerprint for early and accurate diagnosis of the disorder.
  • markers may also be used for clinical screening, assessment of prognosis, evaluation of therapeutics and for drug screening and development.
  • Residual proinsulin and the conversion intermediates des 31 , 32 proinsulin and des 64, 65 proinsulin are co-secreted with mature insulin and C-peptide by exocytosis from pancreatic ⁇ cells in response to elevations in blood glucose and comprise approximately 15% of the total circulating insulin-related immunoreactivity in healthy individuals [13]. These precursor molecules accumulate in the circulation due to their longer half- lives relative to insulin although they have only weak hypoglycaemic activity [26].
  • Elevation of circulating proinsulin and the proinsulin conversion intermediates has been observed in a number of pathophysiological conditions including the prodrome of type 1 diabetes, metabolic syndrome, mild type 2 diabetes and insulin resistance [27], and has been variously attributed to either a deficiency of secretory granule prohormone convertase activity [28], asynchrony between stimulation of secretion and biosynthesis [29] or increased secretory granule turnover as a consequence of increased demands imposed by peripheral insulin resistance or decreased functional ⁇ cell mass [30-32].
  • insulin secretory granules may be co-secreted with the insulin-related peptides at elevated levels in the schizophrenia patients.
  • additional proteins include the proinsulin-converting proteases PC1 , PC2 and CPH , enzymes involved in neurotransmitter biosynthesis such as glutamic acid decarboxylase, dopamine ⁇ hydrozylase and cathepsin L, and other proproteins such as neuropeptide Y, substance P, galanin, dynorphin, 7B2, VGF, and chromogranin A, B and C and their proteolytic products [33-39].
  • proinsulin-converting proteases PC1 , PC2 and CPH
  • enzymes involved in neurotransmitter biosynthesis such as glutamic acid decarboxylase, dopamine ⁇ hydrozylase and cathepsin L
  • proproteins such as neuropeptide Y, substance P, galanin, dynorphin, 7B2, VGF, and chromogranin A, B and C and
  • peripheral hyperinsulinemia has been implicated in the pathogenesis of Alzheimer's disease and associated with such phenomena as aberrant phosphorylation of filamentous proteins, translocation of signalling molecules, increased central nervous system inflammation and ⁇ - amyloid plaque deposition [41 ,42].
  • Hyperinsulinaema is also known to perturb the function of various neurotransmitter systems such as that of norepinephrine [43]. Recent studies have also shown that hyperinsulinaemia increases dopamine levels in the rat nucleus accumbens [44]. This may be important since most researchers subscribe to the hypothesis that the clinical symptoms of schizophrenia are caused by a disturbed and hyperactive dopaminergic signal transduction [45]. Taken together, these findings show, for the first time, that hyperinsulinamia may play a role in the onset of schizophrenia and suggests that drugs which improve insulin signalling may represent a novel treatment strategy for this debilitating disorder. In this case, the above insulin secretory granule markers would have utility as biomarkers not only for patient selection but also for monitoring responses to novel therapeutic treatments.
  • BMI (kg/m 2 ) 24.3 ⁇ 5.8 23.2 ⁇ 3.4 Ages are shown as mean ⁇ standard deviation. BMI: body mass index. ND: not determined.
  • Table II List of secretory granule biomarkers identified from profiling serum/plasma of drug na ⁇ ve first onset schizophrenics using targeted immunoassays, LC-MS E profiling and SELDI-TOF mass spectrometry.
  • P01308 total proinsulin 2.59 0.0045
  • Insulin, intact proinsulin and des 31,32 proinsulin levels were determined across multiple cohorts and the mean ⁇ SD values were calculated after averaging the means.

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Abstract

La présente invention porte sur un procédé de diagnostic ou de surveillance de la schizophrénie ou d’autres troubles psychotiques.
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CN103630692A (zh) * 2013-06-02 2014-03-12 刘辉宇 快速检测尿液c肽的胶体金免疫层析试剂盒及其检测方法
WO2015095930A1 (fr) 2013-12-23 2015-07-02 Stephanie Fryar-Williams Modèle de maladie mentale et test d'évaluation du risque de développer une maladie mentale pour la psychose schizophrénique
EP2939024A4 (fr) * 2012-12-26 2016-08-24 Quest Diagnostics Invest Inc Détection du peptide c par spectrométrie de masse
US11513130B2 (en) 2010-09-15 2022-11-29 Quest Diagnostics Investment Incoporated Detection of vitamins A and E by tandem mass spectrometry

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11513130B2 (en) 2010-09-15 2022-11-29 Quest Diagnostics Investment Incoporated Detection of vitamins A and E by tandem mass spectrometry
EP2939024A4 (fr) * 2012-12-26 2016-08-24 Quest Diagnostics Invest Inc Détection du peptide c par spectrométrie de masse
US9885724B2 (en) 2012-12-26 2018-02-06 Quest Diagnostics Investments Incorporated C peptide detection by mass spectrometry
US10191065B2 (en) 2012-12-26 2019-01-29 Quest Diagnostics Investments Incorporated C peptide detection by mass spectrometry
US10309972B1 (en) 2012-12-26 2019-06-04 Quest Diagnostics Investments Incorporated C peptide detection by mass spectrometry
US10782305B2 (en) 2012-12-26 2020-09-22 Quest Diagnostics Investments Incorporated C peptide detection by mass spectrometry
EP3875959A1 (fr) * 2012-12-26 2021-09-08 Quest Diagnostics Investments Incorporated Détection de peptides c par spectrométrie de masse
US11346845B2 (en) 2012-12-26 2022-05-31 Quest Diagnostics Investments Incorporated C peptide detection by mass spectrometry
US11789026B2 (en) 2012-12-26 2023-10-17 Quest Diagnostics Investments Incorporated C peptide detection by mass spectrometry
CN103630692A (zh) * 2013-06-02 2014-03-12 刘辉宇 快速检测尿液c肽的胶体金免疫层析试剂盒及其检测方法
WO2015095930A1 (fr) 2013-12-23 2015-07-02 Stephanie Fryar-Williams Modèle de maladie mentale et test d'évaluation du risque de développer une maladie mentale pour la psychose schizophrénique
EP3087392A4 (fr) * 2013-12-23 2017-07-26 Stephanie Sue Williams Modèle de maladie mentale et test d'évaluation du risque de développer une maladie mentale pour la psychose schizophrénique

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