EP1451590A2 - Peptides et procede permettant de detecter la maladie d'alzheimer et de differencier cette maladie par rapport aux autres maladies demencielles - Google Patents

Peptides et procede permettant de detecter la maladie d'alzheimer et de differencier cette maladie par rapport aux autres maladies demencielles

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Publication number
EP1451590A2
EP1451590A2 EP02798252A EP02798252A EP1451590A2 EP 1451590 A2 EP1451590 A2 EP 1451590A2 EP 02798252 A EP02798252 A EP 02798252A EP 02798252 A EP02798252 A EP 02798252A EP 1451590 A2 EP1451590 A2 EP 1451590A2
Authority
EP
European Patent Office
Prior art keywords
peptides
mac3
peptide
disease
alzheimer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02798252A
Other languages
German (de)
English (en)
Inventor
Norbert Lamping
Hans-Dieter Zucht
Hartmut Selle
Michael JÜRGENS
Gabriele Heine
Rüdiger HESS
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
DIGILAB Inc
Original Assignee
Biovision AG
Biovision GmbH and Co KG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Biovision AG, Biovision GmbH and Co KG filed Critical Biovision AG
Publication of EP1451590A2 publication Critical patent/EP1451590A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/472—Complement proteins, e.g. anaphylatoxin, C3a, C5a
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • the invention relates to a method for the detection of Alzheimer's disease, in particular a method which enables a distinction between Alzheimer's disease and other dementia diseases. For this purpose, the concentration of certain peptides in body fluids or other samples of the patient is determined.
  • the invention further relates to peptides, antibodies, nucleic acids etc. which have been found for determining the presence and / or the degree of the disease, and their uses for diagnosis and therapy.
  • dementia diseases are an increasing problem, as they appear with increasing age. Most dementia diseases are not curable and require long-term care of the sick.
  • Alzheimer's disease is the most common disease in the group of dementia diseases [1].
  • the diagnosis and therapy of Alzheimer's disease is therefore of particular importance.
  • Alzheimer's disease is a neurodegenerative disease that is characterized by the following symptoms: decrease in mental ability, short-term and long-term memory disorders, confusion and reduced self-preservation and self-care ability.
  • the deterioration in mental performance occurs in addition to the age-related deterioration in mental ability [1].
  • the diagnosis of Alzheimer's disease is difficult because, like other dementia diseases, it begins insidiously and is associated with slowly progressive destruction of nerve cells in the brain and the associated reduced mental performance.
  • Various other dementia diseases, such as vascular dementia have very similar symptoms, which makes it difficult to differentiate between Alzheimer's disease and other dementia diseases.
  • MMSE Score Minimum-Mental Status Examination
  • Alzheimer's disease cannot yet be distinguished with certainty from other dementia diseases [3].
  • the invention has for its object to avoid the disadvantages of Alzheimer's diagnosis in the prior art and to provide an early and reliable method for the detection and differentiation of Alzheimer's disease from other dementia diseases.
  • the peptides are fragments of the precursor molecule Complement C3, in particular a 17 amino acid long, defined peptide derived from Complement C3, which is referred to as C3f [4].
  • C3f a further C3f peptide, in which the C-terminal amino acid arginine has been split off (C3f-des-Arg), was detected in cerebrospinal fluid of cerebral Alzheimer's patients in concentrations which differ from the concentrations of the respective peptide in non- Alzheimer's dementia or healthy people deviates.
  • C3f occurs, but also the term C3F, whereby C3F is not identical to C3f. Rather, C3F stands for "C3-fast", a complement C3 polymorphism that causes the complete precursor molecule of this C3 variant to move faster in the electric field of gel electrophoresis.
  • MAC3 peptides The sequence of C3f is identical to the sequence for Seq. ID 1 from the sequence listing, as well as with the sequence of MAC3-1.
  • MAC3 peptides contain at least 8 and at most 17 amino acids, which are identical to the amino acids at the corresponding sequence positions in the sequence of C3f, according to Seq. ID 1, are.
  • MAC3 peptides can contain two point mutated, two deleted or two additional internally inserted amino acids, as well as N-terminal and / or C-terminal extensions according to Seq. Include ID 1.
  • C3f peptides and C3f peptide fragments are also referred to as MAC3 peptides, which are derived from naturally occurring complement C3 polymorphisms and from naturally occurring C3 mutants.
  • the invention in a method for the detection of Alzheimer's disease by determining the concentration of at least one marker peptide in a biological sample of a patient, that at least one MAC3 peptide is used as the marker peptide, a concentration increase or concentration reduction specific for the respective marker peptide Marker peptide is detected in the sample and a change in the concentration of the marker peptide is evaluated in the aforementioned manner as a positive detection result for Alzheimer's disease.
  • MAC3 peptides that can be understood as fragments of the complement C3f sequence or the C3f peptide itself are referred to as MAC3 peptides. They include homologous peptides and peptide fragments derived from the complement C3f fragment. They include descendants of naturally occurring alleles of these peptides and homologous mutants, in particular point-mutated mutants with preferably no more than two amino acids which differ from complement C3.
  • Preferred markers according to the invention are specified in the sequence listing and are numbered from MAC3-1 to MAC3-16, corresponding to Seq. ID 1 to 16, where MAC3-1 of Seq. ID 1, MAC3-2 of Seq. ID 2, MAC3-3 of Seq. Correspond to ID 3, etc.
  • homologous sequences are spoken of in the present application, this refers to sequences which have at least 70 percent homology.
  • computer programs such as the GCG program package (Genetics Computer Group, University of Wisconsin, Madison, Wl, USA), including GAP [5], BLASTP, BLASTN, FASTA [6] or the well-known Smith Waterman algorithm for determining homologies.
  • Preferred parameters for the amino acid sequence comparison include the algorithm by Needleman and Wunsch [7], the comparison matrix BLOSUM 62 [8], a gap value (gap penalty) of 12, a gap length value (gap length penalty) of 4 and a Homology threshold of 0.
  • the GAP program is also suitable for use with the above parameters.
  • the above parameters are the error parameters (default parameters) for amino acid sequence comparisons, with gaps at the ends not reducing the homology value. In the case of very short sequences compared to the reference sequence, it may also be necessary to increase the expectation value to up to 100,000 (expectation value) and, if necessary, to reduce the word size to up to 2. Further exemplary algorithms, gap opening penalties, gap extension penalties, comparison matrices including those mentioned in the program manual, Wisconsin package, version 9, September 1997, can be used. The selection will depend on the comparison to be performed and also on whether the comparison is carried out between pairs of sequences, with GAP or the best fit being preferred, or between a sequence and an extensive sequence database, with FASTA or BLAST being preferred.
  • a match of, for example, 70% determined with the above-mentioned algorithm is referred to as 70% homology.
  • homologous sequences are understood in principle to be all sequences which have at least a 70 percent match, the match being calculated in accordance with the above description. This relates to both amino acid and nucleic acid sequences.
  • the marker peptides can be present in post-translational modifications and / or in enzymatically and / or chemically modified form, preferably as Peptide oxides, and can be detected in this form.
  • a chemically or post-translationally modified complement C3 peptide or MAC3 peptide can consist of both D- and L-amino acids, as well as combinations of D- and L-amino acids, and can occur naturally, recombinantly or enzymatically, or be chemically synthesized , These peptides can also contain unusual amino acids, ie amino acids that do not belong to the 20 standard amino acids. Numerous examples of unusual amino acids and post-translational modifications such as phosphorus and sulfate groups, glycosylation, amidation, deamidation, pyroglutamic acid etc. are described in the literature and in databases [9].
  • one or more amino acids or the entire peptide can be replaced by structures consisting of peptidomimetics.
  • peptidomimetics is used in the present application in the form of the broadest possible definition.
  • a peptidomimetic is a substance that contains non-peptide structural elements and is able to mimic or antagonize the biological action of the natural mother molecule. Numerous works are known in the prior art which deal in detail with possibilities of using peptidomimetics as a replacement for conventional peptide structures.
  • nucleic acids DNA, RNA and DNA-RNA hybrid molecules of both natural origin and also synthetically, enzymatically or recombinantly produced are regarded as nucleic acids. Also included are nucleic acids that contain modified nucleotides with changed in vivo stability, such as phosphorothioates. Such stabilized nucleic acids are already used when using ribozyme, antisense RNAi ("RNA-mediated interference") and triplex nucleic acid techniques.
  • the method according to the invention is a method in which specific biomarkers are detected, the concentration of which has been changed in the case of neurodegenerative diseases, in particular in Alzheimer's disease, and which also have the disease at a very early stage and an increased probability of Show disease risk early. This is important in order to provide a reliable clinical marker for the diagnosis of these diseases.
  • the concentration of the MAC3 peptides in the sample but also the characteristic pattern of the occurrence of several specific MAC3-
  • Alzheimer's disease underlines Alzheimer's disease, since reliable detection of the disease is a prerequisite for the development of a therapy.
  • MAC3 peptides also makes it possible, in the context of clinical studies to develop new therapies for the treatment of Alzheimer's disease with high specificity, to select only those patients who are suffering from Alzheimer's disease and not from other similar diseases. This is important in order to obtain meaningful study results, since patients misdiagnosed as Alzheimer's disease patients negatively affect the quality of the results of an Alzheimer's therapy study. Complement C3 biology
  • the complement system plays a central role in the specific and non-specific immune defense and consists of more than 30 proteins, some of which are proteases and some of which are substrates of these proteases.
  • the functioning of the complement system is based on a kind of chain reaction, at the beginning of which a substance, e.g. a bacterial cell membrane, a component of the complement system activates, this complement factor in turn activates the next complement factor, etc.
  • the complement activation can also lead to the lysis of healthy, host-own cells which happen to be in the vicinity of the complement activation site. This process is also called the "bystander" effect, and it can result in extensive tissue destruction.
  • the anaphylatoxin C3a has, for example, cytotoxic, blood vessel-expanding and cell-stimulatory properties
  • a number of other complement C3 cleavage products such as C3b, C3c, C3d, C3g, C3e and C3f, are known (Fig. 1) Of these C3 peptides, some, but not all, are known to have biological functions.
  • C3d peptide is the C3 peptide in which the thiolester group is present in the intact C3 molecule and which therefore occurs covalently linked to complement-activating substances.
  • Scientific studies show that C3f sometimes mediates biological effects similar to C3a. C3f and C3a lead to contraction smooth muscles and increase vascular permeability [1 1].
  • C3a and C3f the C-terminal amino acid is an arginine, and the cleavage of this amino acid alters the biological activity of both C3a and C3f.
  • the resulting products are called C3a-des-Arg and C3f-des-Arg.
  • C3f and C3a presumably use, at least in part, the same receptors, which could explain their similar biological effects [1 1].
  • C3 is mainly synthesized by the liver, but also locally by monocytic cells and macrophages.
  • C3-concentrations in the cerebrospinal fluid are about 20 times higher.
  • the C3 concentration in the cerebrospinal fluid also increases, but only by a factor of two, while in other inflammatory reactions in the brain, e.g. in aseptic meningitis the C3 concentration in the cerebrospinal fluid remains unchanged [12].
  • Complement proteins and their receptors are also synthesized by microglia, astrocytes and neurons, among others.
  • the amount of mRNA and protein of the complement proteins C1 to C9 is increased in the brain of Alzheimer's disease patients relative to the brain of healthy people.
  • the concentration of C3 and C4 is the least changed with a 2-fold increase in the brain and unchanged concentrations in the liver, relative to healthy people.
  • Various other complement proteins are, compared to healthy individuals, significantly higher in the brain of Alzheimer's disease patients: C1 q mRNA is increased 23-fold, C1 r 5-fold, C7 6-fold and C9 is 22-fold increased [12 ]. It is therefore surprising and unexpected that fragments of the complement C3 protein and not fragments of other complement proteins are suitable as markers for Alzheimer's disease.
  • Deposits in the form of "plaques” and “tangles” in the brain of Alzheimer's disease patients consist primarily of beta-amyloid and tau protein.
  • these proteins include 1-anti-chymotrypsin, synaptophysin, cystatin C, heme oxigenase, various apolipoproteins such as Apo E4, Apo J and Apo A1, IL-6, the AMY antigen, prion protein, beta spectrin, alpha-2 macroglobulin, lactoferrin and various
  • Complement proteins such as C1 q, C3 and C4d.
  • these deposits also contain sugar structures, e.g. Heparan sulfate. These deposits thus represent complex structures made of proteins and other substances.
  • Eikelenboom et al. discovered that deposits in the brain of Alzheimer's disease patients contain, among other things, complement C3 and that beta-amyloid presumably activates the complement cascade, which was shown experimentally in subsequent work [13].
  • MAC3-3 to MAC3-16 represent C3f peptide fragments that have never been described in the literature and are therefore new as substances.
  • MAC3-1 and MAC3-2 are already known from the literature [4, 1 1].
  • none of the peptides MAC3-1 to MAC3-16 have been discussed in connection with neurodegenerative diseases, and consequently their use as diagnostic markers for the detection of Alzheimer's disease is novel.
  • r1 - either represents the amino acid serine or there is no amino acid at this sequence position
  • r2- represents a sequence which corresponds to the sequence or parts of the sequence of complement C3 from amino acid 1313 to 1320, where r2, starting from amino acid 1312 des C3 precursor, can be between 0 and 8 amino acids long.
  • r3- represents the C3 precursor sequence from amino acid 1304 to 131 1 or parts thereof, where r3, starting from the C3 amino acid 1312, between 0 and 8 amino acids can be long.
  • r4 corresponds to the amino acid arginine or, alternatively, there is no further amino acid at this position in the peptide sequence.
  • the MAC3 peptides according to the invention can exist in post-translational or chemical modification forms, which among other things. on their masses and thus the mass spectrometric identification and also on their elution behavior in chromatography, e.g. in reverse phase chromatography.
  • the peptides can be oxidized, phosphorylated, glycosylated, sulfated, amidated etc. in the sample to be examined.
  • Naturally occurring C3f peptides and C3f peptide fragments derived from complement C3 polymorphisms or C3 mutants are also referred to as MAC3 peptides.
  • C3f sequence Seq. ID 1
  • the same computer programs and algorithms are used that are also used to determine the homology of sequences [5-8].
  • the peptides are also considered to be MAC3 peptides if a maximum of 2 of their amino acids differ from the corresponding sequence of the C3 precursor molecule. Point mutations, deletions, insertions of amino acids and N-terminal and / or C-terminal extensions are permitted as long as the peptide sequence is between 8 and 17 amino acids long, at least 8 amino acids are conserved relative to the C3 precursor sequence, and a maximum of 2 amino acids from the C3f sequence deviate.
  • the concentration of the identified MAC3 peptide (s) is changed for each of these peptides in a defined concentration that is either always specifically higher or always specifically lower for the respective peptide.
  • concentrations of the respective peptides can be used to determine the severity of Alzheimer's disease. especially as a replacement or supplement to the implementation of a "mini-mental status examination" (MMSE).
  • Control samples that may be used can be a pool sample from various controls.
  • the Alzheimer's disease sample to be examined can also be a pool sample, with individual examinations being carried out if the result is positive.
  • a reference value for the respective individual MAC3 peptide can be determined from the results of the determination of individual MAC3 peptides in control samples. In future measurements to differentiate between Alzheimer's disease patients and non-Alzheimer's disease patients, the determined measurement value can then be compared with this reference value in order to arrive at a diagnosis.
  • the body fluid sample can preferably be (human) cerebrospinal fluid (cerebrospinal fluid, CSF) or a sample of another biological material such as serum, plasma, urine, stool, tear fluid, synovial fluid, lymph fluid, etc. This depends, among other things, on the sensitivity of the chosen detection method ( Mass spectrometry, ELISA etc.). Line or tissue samples can also be used if necessary. It is therefore provided in a further embodiment of this invention that cell or tissue homogenates are prepared for the preparation of the sample to be examined, for example from human tissue samples obtained in the context of biopsies or from blood cells.
  • These tissues can be comminuted, for example, with manual homogenizers, with ultrasonic homogenizers or with electrically operated homogenizers such as Ultraturrax, and then in a manner known to the person skilled in the art in acidic, aqueous solutions with, for example, 0.1 to 0.2 M acetic acid for Be cooked for 10 minutes.
  • the extracts are then subjected to the respective detection method, for example a mass spectrometric examination.
  • the samples can be in the prepared in the usual way, eg diluted or concentrated if necessary, and stored.
  • the invention further comprises the use of at least one of the MAC3 peptides according to the invention for the diagnosis of neurodegenerative diseases, in particular Alzheimer's disease, and the use of MAC3 peptides for the production of antibodies or other agents which, owing to their MAC3 Peptide-specific binding properties are suitable for the development of diagnostic reagents for the detection of this disease.
  • the invention also encompasses the use of MAC3 peptides to obtain phage particles which specifically bind these peptides or which, conversely, present MAC3 peptides on their surface and thus enable the identification of binding partners of MAC3 peptides.
  • MAC3 peptides can be used within the scope of the invention. All methods are suitable for this purpose which make it possible to specifically detect MAC3 peptides in a sample from a patient. Suitable methods include physical methods such as e.g. Mass spectrometry or liquid chromatography, molecular biological methods such as Reverse transcriptase polymerase chain reaction (RT-PCR) or immunological detection techniques, e.g. "Enzyme linked immunosorbent assays" (ELISA).
  • physical methods such as e.g. Mass spectrometry or liquid chromatography
  • molecular biological methods such as Reverse transcriptase polymerase chain reaction (RT-PCR) or immunological detection techniques, e.g. "Enzyme linked immunosorbent assays" (ELISA).
  • RT-PCR Reverse transcriptase polymerase chain reaction
  • ELISA Enzyme linked immunosorbent assays
  • One embodiment of the invention is the use of physical methods which can indicate the peptides according to the invention qualitatively or quantitatively. These methods include mass spectrometry, liquid chromatography,
  • the peptides in the sample are chromatographically separated before identification, preferably with reverse phase chromatography, and separation of the peptides in the sample with high-resolution reverse phase high-performance liquid chromatography (RP.) Is particularly preferred HPLC).
  • RP. reverse phase high-performance liquid chromatography
  • Another embodiment of this invention is to carry out precipitation reactions to fractionate the sample using precipitants such as e.g. Ammonium sulfate, polyethylene glycol, trichloroacetic acid, acetone, ethanol etc.
  • the fractions thus obtained are then subjected to the respective detection method, e.g. the mass spectrometric examination.
  • the liquid phase extraction is used.
  • the sample is e.g.
  • an organic solvent such as polyethylene glycol (PEG) and an aqueous saline solution. Due to their physical properties, certain constituents of the sample accumulate in the organic phase and others in the aqueous phase and can thus be separated from one another.
  • PEG polyethylene glycol
  • a particularly preferred embodiment of this invention comprises the use of reverse phase chromatography, in particular a C18 reverse phase chromatography column using eluents consisting of trifluoroacetic acid and acetonitrile, for the separation of peptides in human cerebrospinal fluid. For example, fractions are collected, each Include 1/100 of the volume of solvent used.
  • the fractions obtained in this way are analyzed using a mass spectrometer, preferably using a MALDI (“matrix-assisted laser desorption and ionization”) mass spectrometer using a matrix solution consisting of, for example, L (-) fucose and alpha Cyano-4-hydroxycinnamic acid, dissolved in a mixture of acetonitrile, water, trifluoroacetic acid and acetone, was analyzed to determine the presence of certain masses and to quantify the signal intensity, which correspond to the masses of the peptides MAC3-1 to MAC3-16 according to the invention.
  • MALDI matrix-assisted laser desorption and ionization
  • the MAC3 peptides can be identified with the aid of a mass spectrometric determination, preferably a MALDI mass spectrometry.
  • the mass spectrometric determination further preferably comprises at least one of the following mass signals, each calculated on the basis of the theoretical, monoisotopic mass of the corresponding peptide. Slight deviations from the theoretical, monoisotopic mass can occur due to the experimental error and the natural isotope distribution.
  • a proton is added to the peptides in MALDI mass determinations due to the measurement method, which increases the mass by 1 dalton.
  • the following masses correspond to the theoretical, monoisotopic masses of the peptides we have identified; calculated with suitable software, here GPMAW 4.02.
  • the experimentally determined masses 2038 and / or 2054 occur.
  • These two experimentally determined masses are MAC3-1 as a single or double-oxidized peptide.
  • the symbol> (is larger or the same) is to be understood in such a way that not arbitrarily larger masses are possible for the MAC3 peptides concerned, but only the masses which can result from the amino acids which may also be located at the ends of these peptides.
  • the peptide can be a maximum of 17 amino acids in total. At the ends of these peptides it is not possible for any amino acids to be present in addition, but only those which, owing to the sequence of the complement C3 precursor molecule, can be located at this sequence position.
  • MAC3-1 to MAC3-16 C3f peptides and their descendants are referred to here as MAC3-1 to MAC3-16.
  • Their sequences are in the sequence listing under Seq. ID 1 to Seq. ID 16 specified.
  • the under Seq. MAC3 peptides (ID 15 and 16) (MAC3-15 and MAC3-16) may contain additional amino acids at the N and / or C terminus corresponding to the corresponding sequence of the complement C3f peptide (Seq. ID 1, MAC3-1).
  • the invention also includes the recombinantly or synthetically produced MAC3 peptides isolated from biological samples in unmodified, chemically modified or post-translationally modified form. Two point mutations as well as other deviations are possible as long as the MAC3 peptide has at least 8 amino acids which correspond in their identity and their position within the peptide sequence to the complement C3f peptide.
  • the invention also encompasses nucleic acids which correspond to complement C3 fragments, and in particular those which correspond to the MAC3 peptides according to the invention, and their use for the indirect determination and quantification of the associated protein molecules.
  • This also includes nucleic acids, e.g. non-coding sequences, such as 5'- or 3'-untranslated regions of the mRNA, or nucleic acids which have a sequence matching the nucleic acid sequence of Complement C3 which is sufficient for specific hybridization experiments and which therefore serve to indirectly detect the associated peptides, in particular the MAC3 Peptides are suitable.
  • RT-PCR Reverse transcriptase polymerease chain reaction
  • Northern blots can be used in a manner known to the person skilled in the art.
  • Results can be used to determine the likelihood of an Alzheimer's disease and / or its severity.
  • the identification of the MAC3 peptides can be carried out using an immunological detection system, preferably an ELISA (enzyme linked immunosorbent assay).
  • This immunological detection detects at least one MAC3 peptide.
  • a so-called “sandwich ELISA” should furthermore preferably be used, in which the detection of the MAC3 peptide depends on the specificity of two antibodies that recognize different epitopes within the same molecule.
  • MAC3 peptides can be used however, other immunological methods, for example direct or competitive immunological methods, can also be used, as well as other ELISA-like detection techniques, such as RIAs (radio immuno assays), ELI spot, luminescence, chemiluminescence, electrochemiluminescence, fluorescence or bioluminescence immunoassays - Methods etc. are suitable as immunological detection systems for MAC3 peptides, MAC3 peptides isolated, recombinantly produced or chemically synthesized from biological samples can be used as a standard for the quantification on the peptide or peptide fragment antibodies, i.e. an antibody specific for the peptide.
  • Other detection methods suitable for peptide detection include western blotting, immunoprecipitation, dot blots,
  • Plasmon resonance spectroscopy BIACORE TM
  • phage particles phage particles
  • PNAs Peptide Nucleic Acids
  • affinity matrices etc.
  • all substances / molecules are suitable as detection agents that allow a specific detection system to be set up.
  • Peptide-specific binding partners immobilized on a suitable carrier.
  • All known supports such as plastic tubes,
  • Microtiter plates, particles, microparticles, protein chips, etc. can be used.
  • the binding partner can be immobilized directly on this support or the solid phase in an adsorptive or covalently coupled manner.
  • Another possibility is to individually bind the MAC3 peptide via a pair of one
  • Immobilize binding pair such as biotin / avidin, biotin / streptavidin, antigen / antibody, etc.
  • Another object of the invention is a test kit for the detection of Alzheimer's disease or a predisposition to Alzheimer's disease, which contains at least one binding partner, preferably an antibody, which is directed against a MAC3 peptide.
  • This binding partner is present in an immobilized form bound to a suitable solid phase or a carrier or can be offered in a form which enables immobilization.
  • the binding partner can be biotinylated if the biotin / avidin interaction is to be used as indirect binding.
  • the binding partner can also be present in the test kit in a marked form or in a form which enables the marking.
  • the indirect labeling can be inserted via the interaction of a binding pair such as avidin / biotin, or streptavidin / biotin or digoxin / anti-digoxin antibodies. These methods are known to the person skilled in the art.
  • test kit can also contain a standard which consists of at least one MAC3 peptide.
  • MAC3 peptide concentrations are usually offered as standard.
  • a further embodiment of the invention is the extraction of MACSPeptides using recombinant expression systems, chromatography methods and chemical synthesis protocols which correspond to the Are known to those skilled in the art.
  • the MAC3 peptides obtained in this way can be used, inter alia, as standards for the quantification of the respective MAC3 peptides or as an antigen for producing MAC3 peptide-specific antibodies.
  • Recombinant expression of pepids is one of the methods known and suitable for the person skilled in the art for isolating and obtaining MAC3 peptides.
  • Cell systems such as bacteria such as Escherichia coli, yeast cells such as Saccharomyces cerevisiae, insect cells such as Spodoptera frugiperda (Sf-9) cells or mammalian cells such as "Chinese Hamster Ovary" (CHO) cells can be used to express the MAC3 peptides are available from the American Tissue Culture Collection (ATCC).
  • ATCC American Tissue Culture Collection
  • nucleic acid sequences which code for MAC3 peptides are inserted in combination with suitable regulatory nucleic acid sequences such as, for example, promoters, antibiotic selection markers, etc. into an expression vector using molecular biological methods.
  • a suitable vector is, for example, the vector pcDNA3.1 from Invitrogen.
  • the MAC3 peptide expression vectors obtained in this way can then be inserted into suitable cells, for example by electroporation.
  • the MAC3 peptides can be prepared by chemical synthesis, for example according to the Merrifield solid-phase synthesis protocol, using synthesizers which are available from various manufacturers.
  • Another embodiment of this invention is the isolation of MAC3 peptides from biological samples or from cell culture media or cell lysates from recombinant expression systems, for example with reverse phase chromatography, affinity chromatography, ion exchange chromatography, gel filtration, isoelectric focusing, etc.
  • Another embodiment of the invention is the production of monoclonal or polyclonal antibodies using MAC3 peptides.
  • the antibodies are obtained in a conventional manner known to the person skilled in the art.
  • a preferred embodiment is Production and production of MAC3 peptide-specific antibodies which recognize neo-epitopes which are only present on MAC3 peptides but which do not occur in the complete complement C3 precursor molecule.
  • Such anti-MAC3 peptide antibodies enable the specific immunological detection of MAC3 peptides in the presence of the complement C3 precursor molecule.
  • Another application example is the quantitative determination of the above-mentioned peptides to assess the effectiveness of a therapy in development against neurodegenerative diseases, in particular Alzheimer's disease.
  • the quantitative measurement results from a sample to be examined are compared with the measurement values obtained from a control group and a group of patients.
  • the effectiveness of a therapeutic agent can be derived from these results.
  • the efficacy test is of outstanding importance for the successful development of a therapeutic agent and so far there have been no clinically measurable parameters available for Alzheimer's disease that reliably enable this [3].
  • MAC3 peptides have changed significantly compared to healthy individuals.
  • Another aspect of the invention is therefore the MAC3 concentrations in Alzheimer's disease Bring patients to normal concentrations. This procedure can be used to treat Alzheimer's disease or related neurological diseases.
  • the concentrations of these substances can be given by therapeutic administration of, for example, anti-complement C3 or anti-MAC3 peptide antibodies or complement C3-specific antisense nucleic acids, ribozymes, RNAi nucleic acid molecules or triplex Nucleic acids or MAC3 peptide antagonists or complement C3 antagonists can be lowered.
  • substances can also be administered which suppress the body's own expression of complement C3 or the processing of complement C3 into MACS peptides. If there is a lack of complement C3 or MACS peptides as the cause of the disease, therapeutic additions of complement C3, MAC3 peptides, MAC3 peptide agonists or complement C3 agonists can be carried out. Substances that influence the processing of complement C3 into MAC3 peptides can also be used therapeutically. Of course, the combination of different therapy strategies may also be useful and possible.
  • the invention therefore also includes the use of complement C3 peptides, MAC3 peptides, MAC3 peptide agonists and MAC3 peptide
  • Antagonists anti-complement C3 antibodies and anti-MAC3 peptide
  • Complement C3 peptides and MAC3 peptides for the treatment of neurological diseases, especially Alzheimer's disease.
  • Antibodies can also be antibody fragments, antibody fusion proteins or other selectively binding to complement C3 peptides or MAC3 peptides
  • Peptides can also be used as fusion proteins of the proteins and peptides mentioned. Furthermore, the invention also includes the use of
  • the invention also includes agonists and antagonists which modulate the activity of the proteins mentioned.
  • Another embodiment of this invention is the galenical formulation or chemical modification of the peptides and nucleic acids described in a manner which enables them to cross the blood-brain barrier and / or the blood-liquor barrier more efficiently. This makes them particularly suitable for therapeutic use.
  • MAC3 peptides, complement C3 peptides, peptidomimetics, nucleic acids, agonists or antagonists are modified such that they e.g. lipophilic are what favors the passage into the subarachnoid space, the brain ventricles and the brain tissue. This can be done by inserting hydrophobic molecular components or by "packaging" the substances in hydrophobic agents, e.g. Liposomes can be achieved.
  • Peptide sequences are added to MAC3 or complement C3 peptides, nucleic acids, agonists or antagonists which favor entry into the subarachnoid space or, conversely, make it difficult to exit from the subarachnoid space.
  • the invention also encompasses the administration of the aforementioned therapeutic agents via various routes, such as, for example, as an intravenous injection, as an orally administrable substance, as an inhalable gas or aerosol, or as administration in the form of direct injection into the subarachnoid space, the brain ventricles, or into tissue such as muscle , Fat, brain, etc.
  • routes such as, for example, as an intravenous injection, as an orally administrable substance, as an inhalable gas or aerosol, or as administration in the form of direct injection into the subarachnoid space, the brain ventricles, or into tissue such as muscle , Fat, brain, etc.
  • peptides or proteins that are administered orally can be protected from proteolytic breakdown in the stomach by acid-resistant capsules. Strongly hydrophobic substances can be made more hydrophilic by suitable galenical preparations and thus more suitable for eg intravenous injections.
  • Dosage forms include the packaging of the active ingredients in polymers and gels (Atrix Labs, Fort
  • Another embodiment of the invention is the use of MACS peptides or of complement C3 peptides to identify receptors that selectively bind these molecules. These receptors can also be modulated by administration of agonists or antagonists, which is expedient for the therapy of neurological diseases, in particular of Alzheimer's disease.
  • Exemplary embodiments for checking the therapeutic effectiveness of complement C3 peptides, MAC3 peptides and of agents which modulate the expression and the biological availability of these substances include the cultivation of cell lines. These cell lines can be treated with complement C3 or MAC3 peptides or with peptidomimetics or with substances which promote the expression of complement C3 peptides or which promote the processing of complement C3 peptides into MAC3 peptides. In this way, biological properties of complement C3 and MAC3 peptides in connection with neurological diseases, in particular Alzheimer's disease, can be determined. Fusion proteins and fusion peptides can also be used to treat the cell lines, for example fusion proteins with peptide sequences which promote transport of the fusion protein into the interior of the cell.
  • cell lines can be transfected with expression vectors which directly or indirectly cause expression of complement C3 peptides or MAC3 peptides by the transfected cells.
  • expression vectors can encode, inter alia, MAC3 peptides or complement C3 peptides.
  • the simultaneous transfections with different MAC3 peptides and / or complement C3 peptides can also be carried out.
  • suitable cell lines can be used with anti-complement C3 peptide or anti-MAC3 peptide antibodies or with nucleic acids which suppress the expression of complement C3 peptides, such as, for example, complement C3 antisense nucleic acids, complement C3 triplex nucleic acids, complement C3 RNAi -Nucleic acids or ribozymes directed against complement C3 mRNA can be treated.
  • complement C3 antisense nucleic acids such as, for example, complement C3 antisense nucleic acids, complement C3 triplex nucleic acids, complement C3 RNAi -Nucleic acids or ribozymes directed against complement C3 mRNA
  • complement C3 peptides such as, for example, complement C3 antisense nucleic acids, complement C3 triplex nucleic acids, complement C3 RNAi -Nucleic acids or ribozymes directed against complement C3 mRNA.
  • cell lines that appear to be suitable as neurological model systems in connection with Complement C3 can be used for such
  • tests can be used, among other things, to determine the proliferation rate of the treated cells, their metabolic activity, the apoptosis rate of the cells, changes in cell morphology, the expression of cell-specific proteins or reporter genes or the release of cytosolic Identify cell components as markers for cell death.
  • Suitable strains of experimental animals for example mice, rats, or other species can be used as further test systems, which are considered to be a model for neurological diseases, in particular a model for Alzheimer's disease.
  • These experimental animals can be used to investigate the effectiveness of therapy strategies aimed at modulating the concentration of MAC3 peptides or of complement C3 peptides, and these peptides and proteins can possibly be galenically prepared in such a way that they Brain barrier and / or the blood-CSF barrier can pass better.
  • Liposome-packed proteins and peptides, proteins and peptides, covalently fused or non-covalently associated with transport peptides, such as the HIV-TAT sequence, etc. can be used as a galenical preparation method.
  • peptides and proteins can be chemically modified in such a way that they acquire lipophilic properties and are therefore easier to penetrate into cells.
  • Peptides that are only sparingly soluble in aqueous solutions can be chemically modified in such a way that they become more hydrophilic and can then be used, for example, as an intravenously injectable therapeutic agent.
  • Acid-resistant capsules can be used to protect sensitive substances to be administered orally in the stomach from being destroyed.
  • Read-out parameters in experiments with animal models can be the survival time of the animals, their behavior, their short-term memory performance, their ability to learn, etc.
  • An example of a memory test used for experimental animals such as Suitable for rats is the "Morris water maze test”.
  • experimental animals such as, for example, gene-deficient animals, or transgenic animals can be obtained using molecular biological methods, in whose organism complement C3 peptides or MAC3 peptides are not produced, or produced in reduced or increased quantities.
  • the expression can be changed both locally, for example in the brain, and in the entire organism of the test animal. Caenorhabditis elegans, Drosophila, zebra fish, mice, rats etc. are suitable as experimental animals.
  • Figure 1 Schematic representation of the complement C3 protein with the position of the identified MAC3 peptides
  • Figure 2 Reverse-phase chromatography for the separation and enrichment of the MAC3 peptides from cerebrospinal fluid
  • Figure 6 Box whisker plot for the quantitative comparison of the concentrations of MAC3-1 in healthy controls, in Alzheimer's disease patients and in patients with non-Alzheimer's dementia, and of chemically modified MAC3-1 (e.g. the single or double-oxidized peptide , as well as the double-charged MAC3-1 peptide ion)
  • chemically modified MAC3-1 e.g. the single or double-oxidized peptide , as well as the double-charged MAC3-1 peptide ion
  • Figure 7 Box whisker plot for quantitative comparison of the concentrations of MAC3-2 in healthy controls, in Alzheimer's disease patients and in patients with non-Alzheimer's dementias
  • Figure 8 Box-whisker plot for the quantitative comparison of the concentrations of MAC3-7 in healthy controls, in Alzheimer's disease patients and in patients with non-Alzheimer's dementias
  • Figure 9 Box whisker plot for the quantitative comparison of the concentrations of MAC3-8 in healthy controls, in Alzheimer's disease patients and in patients with non-Alzheimer's dementias
  • Figure 10 Box-whisker plot for the quantitative comparison of the concentrations of MAC3-9 in healthy controls, in Alzheimer's disease patients and in patients with non-Alzheimer's dementias
  • Figure 1 Box whisker plot for quantitative comparison of the concentrations of MAC3-13 in healthy controls, in Alzheimer's disease patients and in patients with non-Alzheimer's dementias
  • Figure 1 shows a schematic representation of the complement C3 protein with the position of the identified MAC3 peptides, which are shown with each other in the form of an alignment.
  • the Masses actually identified in the mass spectrometer deviate from these theoretical, monoisotopic masses due to the natural isotope distribution and a low measurement inaccuracy.
  • the measurement inaccuracy is 500 ppm for peptides in the mass range from 1000 to 4000 Daltons, the error of the measurement gradually increasing for peptides with a mass which is greater than 4000 Daltons.
  • peptide MAC3-1 could be identified in the form of peptide variants with one and with two additional, covalently linked oxygen atoms, with about 16 daltons corresponding to the mass of the peptide for each oxygen atom.
  • the experimentally determined masses for MAC3-1 are: 2038 and 2054 daltons, the mass of MAC3-1 being increased sequentially by the mass of one oxygen atom.
  • Figure 2 shows the elution profile of a sample that was subjected to reverse phase chromatography according to Example 2, for the separation and enrichment of the MAC3 peptides from cerebrospinal fluid.
  • the position at which MAC3-1 peptides elute is marked by an arrow.
  • Figure 3 shows a spectrum, which was created by MALDI mass spectrometric measurement of MAC3-1 according to Example 3, after reverse phase chromatography of human cerebrospinal fluid according to Example 2.
  • MAC3-1 corresponds to the sequence of the C3f peptide, derived of Complement C3, and is marked by an arrow.
  • Figure 4 shows an MS / MS fragment spectrum according to Example 4 of the peptide MAC3-1 according to the invention.
  • Figure 4A shows the raw data of
  • Figure 4B shows the converted, deconvoluted mass spectrum of MAC3-1.
  • the peak pattern in Figure 4B is characteristic of MAC3-1.
  • MAC3-1 corresponds to the C3f peptide of the complement C3 protein (Seq. ID 1).
  • Figure 5 shows data generated by MALDI as a relatively quantifying MS method.
  • Each peptide shows an individual, typical ratio of signal strength to concentration, which can be read in this diagram using the slope of the curve.
  • Figure 6 shows box whisker plots for the peptide MAC3-1 in its 2-fold oxidized form (Fig. 6A and B), in its simply oxidized form (Fig. 6C and D), in its non-oxidized form (Fig. 6E and F) and as a double-charged ion (Fig. 6G and H).
  • the single-charged ion is analyzed in Figs. 6A to F.
  • Each MAC3-1 variant is compared between Alzheimer's disease patients and healthy controls (Fig. 6A, C, E and G) and between Alzheimer's disease patients and non-Alzheimer's patients (Fig. 6B, D, F and H) shown.
  • Figures 7 to 1 1 show in the form of box whisker plots a comparison of the integrated MALDI mass spectral signal intensities of MAC3- 2, MAC3-7, MAC3-8, MAC3-9 and MAC3-13 in healthy controls compared to disease -Alzheimer's patients (Fig. A) and non-Alzheimer's dementias, compared to Alzheimer's disease patients (Fig. B).
  • Example 1 Obtaining cerebrospinal fluid to determine MACS peptides.
  • CSF or cerebrospinal fluid cerebrospinal fluid
  • cerebrospinal fluid cerebrospinal fluid
  • Liquor cerebrospinalis is usually removed by lumbar puncture, more rarely by suboccipital puncture or ventricular puncture.
  • lumbar puncture spinal puncture
  • the spinal subarachnoid space between the 3rd and 4th or 4th and 5th lumbar spinous process is punctured with a long hollow needle and puncture is obtained.
  • the sample is then centrifuged for 10 minutes at 2000x g and the supernatant is stored at minus 70 ° C.
  • Example 2 Separation of peptides in cerebrospinal fluid (CSF) for mass spectrometric measurement of MAC3 peptides
  • MAC3 peptides For the mass spectrometric detection of MAC3 peptides in CSF, a separation of the peptide ingredients is necessary in this example. This sample pretreatment serves to enrich the peptides according to the invention and to separate components which can interfere with the measurement. Reverse phase chromatography is used as the separation process. Various RP chromatography resins and eluents are equally suitable. The separation of MAC3 peptides using a C18 reverse-phase chromatography column with the size 4 mm ⁇ 250 mm from Vydac is shown by way of example.
  • compositions were used: solvent A: 0.06% (v / v) trifluoroacetic acid, solvent B: 0.05% (v / v) trifluoroacetic acid, 80% (v / v) acetonitrile.
  • the chromatography was carried out at 33 ° C. using an HP ChemStation 1 100 from Agilent Technologies with a flow cell Micro from Agilent Technologies. Human cerebrospinal fluid was used as the sample. 440 ⁇ l of CSF were diluted to 1650 ⁇ l with water, the pH was adjusted to 2-3, the sample was centrifuged for 10 minutes at 18000 ⁇ g and finally 1500 ⁇ l of the sample prepared in this way were applied to the chromatography column.
  • the chromatography conditions were as follows: 5% solvent B at the time 0 min, from time 1 to 45 min continuous increase in the solvent B concentration to 50%, from Time 45 to 49 min continuous increase in mobile phase B concentration to 100% and then constant until time 53 min 100% buffer B. 10 minutes after the beginning of the chromatography, 96 fractions of 0.5 ml each are started.
  • the chromatogram of a cerebrospinal fluid sample, produced under the test conditions described here, is shown in Figure 2.
  • Example 3 Determination of the Masses of Peptides Using MALDI Mass Spectrometry
  • MALDI-TOF mass spectrometer matrix-assisted laser desorption ionization
  • Suitable MALDI-TOF mass spectrometers are manufactured by PerSeptive Biosystems Framingham (Voyager-DE, Voyager-DE PRO or Voyager-DE STR) or by Bruker Daltonik GmbH (BIFLEX). To prepare the samples, they are mixed with a matrix substance, which typically consists of an organic acid.
  • Typical matrix substances which are suitable for peptides are 3,5-dimethoxy-4-hydroxycinnamic acid, ⁇ -cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid.
  • a dried equivalent obtained according to reverse phase chromatography corresponding to 400 ⁇ ⁇ human cerebrospinal fluid, is used.
  • the chromatographed sample is dissolved in 15 ⁇ ⁇ of a matrix solution.
  • This matrix solution contains, for example, 10 g / l of ⁇ -cyano-4-hydroxycinnamic acid and 10 g / l of L (-) fucose dissolved in a solvent mixture consisting of acetonitrile, water, trifluoroacetic acid and acetone in a volume ratio of 49:49: 1: 1.
  • 0.3 ⁇ ⁇ of this solution are transferred to a MALDI carrier plate and the dried sample is analyzed in the MALDI mass spectrometer Voyager-DE STR from PerSeptive Biosystems. The measurement is carried out in "Linear Mode" with "Delayed Extraction TM".
  • Figure 3 shows the spectrum of MAC3-1 as an example of a measurement of one of the MAC3 peptides according to the invention.
  • the peak corresponding to the MAC3-1 peptide is marked by an arrow.
  • MALDI-TOF mass spectrometry can be used to quantify peptides such as, for example, the MAC3 peptides according to the invention, if these peptides are present in a concentration that is in the dynamic measuring range of the mass spectrometer, thereby avoiding detector saturation. This is the case for the measurement of the MAC3 peptides according to the invention in cerebrospinal fluid with a liquor equivalent concentration of 33.3 ⁇ per ⁇ matrix solution.
  • MAC3 peptides according to the invention To quantify the MAC3 peptides according to the invention, it must be ensured that the mass signals to be analyzed of peptides in the fractions, obtained by reverse phase chromatography of cerebrospinal fluid, according to Example 2, are actually the MAC3 peptides according to the invention.
  • the peptides according to the invention are identified in these fractions, for example using nanoSpray-MS / MS [14].
  • a MAC3 peptide ion is selected in the mass spectrometer on the basis of its specific m / z (mass / charge) value in a manner known to the person skilled in the art in the mass spectrometer.
  • This selected ion is then fragmented by supplying collision energy with a collision gas, for example argon or nitrogen, and the resulting MAC3 peptide fragments in the Mass spectrometer detected in an integrated analysis unit and corresponding m / z values determined (principle of tandem mass spectrometry) [15].
  • a collision gas for example argon or nitrogen
  • the fragmentation behavior of peptides given the mass accuracy of the devices used of 50 ppm, enables the MAC3 peptides according to the invention to be clearly identified using computer-assisted search methods [16] in sequence databases in which the sequence of the precursor molecule Complement C3 has been entered. Taking into account the positive shift of the molecular mass by approx.
  • oxidized peptides can also be identified with this method.
  • Other chemically or post-translationally modified peptides can also be identified accordingly.
  • the mass spectrometric analysis was carried out using a quadrupole TOF instrument, model "QStar-Pulsar” from Applied Biosystems-Sciex, USA. Exemplary MS / MS fragment spectra of MAC3-1 are shown in Figure 4.
  • a MALDI measurement of the MAC3 peptides according to the invention according to Example 3 was carried out after sample preparation according to Examples 1 and 2.
  • Exemplary MALDI signal intensities are visualized in the form of "box whisker plots" in Figures 6 to 1 1.
  • the "box whisker plots" shown enable comparison of the integrated MALDI mass spectrometric signal intensities of various MAC3 peptides in controls (healthy subjects, patients with non-Alzheimer's dementias) with the MALDI signal intensities in samples from Alzheimer's disease patients.
  • the solid line in the columns indicates the median and the dashed line in the columns indicates the mean.

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Abstract

L'invention concerne des peptides définis et leur détermination quantitative dans des liquides corporels de patients souffrant de la maladie d'Alzheimer, quant à leur concentration dans un groupe témoin de patients sains ou souffrant d'autres maladies démentielles. Les peptides selon l'invention proviennent du fragment C3f du précurseur protéiniques complément C3 avec le gène correspondant et sont traités de manière spécifique et, le cas échéant, modifiés par post-translation ou modifiés chimiquement. Ils sont modifiés d'une manière spécifique pour chaque peptide, en ce qui concerne leurs concentrations chez des patients, par rapport au groupe témoin. Une modification spécifique et significative des concentrations de ces peptides par rapport à leurs concentrations chez des personnes saines présente une différenciation par rapport à des affections provoquées par la maladie d'Alzheimer. L'invention concerne en outre des utilisations de ces peptides en vue du contrôle de l'évolution, ainsi que pour le développement des diagnostics et des moyens thérapeutiques.
EP02798252A 2001-11-28 2002-11-27 Peptides et procede permettant de detecter la maladie d'alzheimer et de differencier cette maladie par rapport aux autres maladies demencielles Withdrawn EP1451590A2 (fr)

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DE10158180A DE10158180A1 (de) 2001-11-28 2001-11-28 Verfahren zum Nachweis von Morbus Alzheimer und zur Unterscheidung von Morbus Alzheimer gegenüber anderen demenziellen Erkrankungen, zugehörige Peptide und deren Verwendung
PCT/DE2002/004360 WO2003048775A2 (fr) 2001-11-28 2002-11-27 Procede permettant de detecter la maladie d'alzheimer et de differencier cette maladie par rapport aux autres maladies demencielles, peptides correspondants et leurs utilisations

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11592452B2 (en) 2017-07-14 2023-02-28 Mcbi Inc. Disease detection method

Families Citing this family (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7049397B2 (en) * 2001-04-30 2006-05-23 Syn X Pharma, Inc. Biopolymer marker indicative of disease state having a molecular weight of 1211 daltons
DE10303974A1 (de) 2003-01-31 2004-08-05 Abbott Gmbh & Co. Kg Amyloid-β(1-42)-Oligomere, Verfahren zu deren Herstellung und deren Verwendung
WO2005098446A2 (fr) 2004-03-31 2005-10-20 The Johns Hopkins University Biomarqueurs du cancer des ovaires
GB0421639D0 (en) * 2004-09-29 2004-10-27 Proteome Sciences Plc Methods and compositions relating to alzheimer's disease
US8871715B2 (en) 2005-10-14 2014-10-28 Alltech, Inc. Use of selenium compounds, especially selenium yeasts for altering cognitive function
CN101073584B (zh) * 2005-10-14 2012-10-10 全面技术公司 改变细胞功能的方法和组合物
US8865763B2 (en) 2005-10-14 2014-10-21 Alltech, Inc. Methods and compositions for altering cell function
PL2289909T3 (pl) 2005-11-30 2015-04-30 Abbvie Inc Sposób przeszukiwania, proces oczyszczania niedyfundujących oligomerów Abeta, selektywne przeciwciała przeciw niedyfundującym oligomerom Abeta i sposób wytwarzania tych przeciwciał
US8497072B2 (en) 2005-11-30 2013-07-30 Abbott Laboratories Amyloid-beta globulomer antibodies
US8455626B2 (en) 2006-11-30 2013-06-04 Abbott Laboratories Aβ conformer selective anti-aβ globulomer monoclonal antibodies
WO2008104386A2 (fr) * 2007-02-27 2008-09-04 Abbott Gmbh & Co. Kg Méthode de traitement d'amyloïdoses
US8987419B2 (en) 2010-04-15 2015-03-24 AbbVie Deutschland GmbH & Co. KG Amyloid-beta binding proteins
CA2808187A1 (fr) 2010-08-14 2012-02-23 Abbvie Inc. Proteines de liaison beta-amyloides
EP2651972B1 (fr) * 2010-12-16 2016-04-06 Autism Biotech Limited Nouveau biomarqueur et ses utilisations dans le diagnostic, le traitement de l'autisme
JP6012923B2 (ja) 2010-12-22 2016-10-25 株式会社Ｍｃｂｉ 認知機能障害疾患のバイオマーカーおよび該バイオマーカーを用いる認知機能障害疾患の検出方法
GB201310203D0 (en) * 2013-06-07 2013-07-24 Electrophoretics Ltd Materials and methods relating to Alzheimer's disease
GB2515334A (en) * 2013-06-20 2014-12-24 Neuro Bio Ltd Biomarkers for alzheimer's Disease
US20160018798A1 (en) * 2014-07-17 2016-01-21 Toyota Motor Engineering & Manufacturing North America, Inc. Home control system from a vehicle
JP7457300B2 (ja) * 2018-08-29 2024-03-28 国立大学法人 岡山大学 神経変性疾患の診断用ペプチドマーカー
CN114150057B (zh) * 2021-12-21 2024-04-26 贾龙飞 一种诊断阿尔茨海默病的外泌体蛋白及其用途

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6617308B2 (en) * 2001-04-30 2003-09-09 Syn X Pharma, Inc. Biopolymer marker indicative of disease state having a molecular weight of 1865 daltons
US20020160420A1 (en) * 2001-04-30 2002-10-31 George Jackowski Process for diagnosis of physiological conditions by characterization of proteomic materials
US6756476B2 (en) * 2001-04-30 2004-06-29 Syn X Pharma, Inc. Biopolymer marker indicative of disease state having a molecular weight of 2021 daltons
US7314717B2 (en) * 2001-04-30 2008-01-01 Nanogen Inc. Biopolymer marker indicative of disease state having a molecular weight of 1562 daltons
US20030100016A1 (en) * 2001-11-23 2003-05-29 George Jackowski Complement C3 precursor biopolymer markers predictive of Alzheimers disease

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO03048775A2 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11592452B2 (en) 2017-07-14 2023-02-28 Mcbi Inc. Disease detection method

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