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
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems
• • 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

Definitions

• the invention relates to a method for the detection of a chronic, dementia disease or a predisposition to a chronic dementia disease, in particular Alzheimer's disease or related neurological diseases, e.g. Lewy body dementia or vascular dementia.
• the invention further relates to peptides which have been found for the detection of the presence of these diseases, for monitoring the course of the diseases and the degree of the diseases.
• detection reagents such as antibodies and nucleic acids and the like, by means of which these peptides or the corresponding nucleic acids can be detected.
• the invention further relates to pharmaceutical applications, which include VGF, VGF peptides, VGF antibodies, VGF nucleic acids, VGF protein antagonists, VGF protein agonists, VGF peptide agonists or VGF peptide antagonists, for therapy or Prevention of neurological diseases, especially Alzheimer's disease. Furthermore, the invention relates to methods for determining patients with neurological diseases, in particular Alzheimer's disease, which are suitable for taking part in clinical studies for examining these diseases.
• the peptides are fragments of the protein VGF, which is also referred to as "neuroendocrine specific protein VGF".
• VGF is also used for the protein "vaccinia growth factor” or for “vaccinia virus growth factor” and used for "vascular permeability factor", these proteins not corresponding to the VGF protein which is the subject of this invention.
• Dementia is an increasing problem in industrialized countries due to the higher average life expectancy. Dementia is largely incurable and requires long-term care for the sick. About half of these patients are cared for in hospital. More than 60 dementia disorders are known, including those that cause dementia.
• Alzheimer's disease Alzheimer's disease
• AD Alzheimer's disease
• non-Alzheimer's dementias are known, among others: vascular dementia, Lewy body dementia, Binswanger dementia and dementia diseases which are an accompanying effect of other diseases, such as Parkinson's disease, Huntington's disease, Pick's Disease, Gerstmann-St Hurssler-Scheinger disease, Wienfeldt-Jakob disease etc. occur.
• Alzheimer's disease is a neurodegenerative disease characterized by the following symptoms: decrease in mental ability, confusion and reduced self-preservation and self-care.
• a strongly limited short-term memory in particular is characteristic of Alzheimer's disease, while long-standing memories of the patient, for example of one's own childhood, are far less affected by the disease.
• CERAD Consortium to Establish a Registry for Alzheimer's Disease
• the following criteria-based diagnostic systems are currently used to diagnose Alzheimer's disease: The "International classification of Diseases, lOth revision" (ICD-10), the
• Alzheimer's disease is also difficult because, like other dementia diseases, it begins insidiously and is associated with slowly progressive destruction of nerve cells in the brain.
• Alzheimer's disease No causal therapy for the treatment of Alzheimer's disease is currently available.
• the disease is only symptomatic e.g. treated by the administration of neurotransmitters such as acetylcholine.
• neurotransmitters such as acetylcholine.
• the administration of antioxidants, radical scavengers, calcium channel blockers, anti-inflammatory substances, secretase inhibitors, anti-amyloid antibodies etc. and immunization against amyloid peptides are currently being tested as further possible therapeutic strategies. So far, however, no causal therapy of this disease is possible.
• the invention has for its object to avoid the disadvantages in the diagnosis of Alzheimer's disease in the prior art and an early and reliable method to provide evidence of chronic dementia diseases, especially Alzheimer's disease. It is also the object of the invention to provide a new therapy for the treatment of Alzheimer's disease, since so far only unsatisfactory therapeutic approaches for the treatment of Alzheimer's disease have been available.
• VGF proteins or peptides according to Accession No. NM_003378 and Y12661:
• VGF proteins The peptides derived from the nucleic acid sequences NM_003378 and Y12661 are also referred to as VGF proteins and include all naturally occurring alleles, mutants and polymorphisms of VGF proteins as well as tissue-specific expressed VGF variants.
• VGF variants are also included which occur as a result of diseases or as a result of neurological diseases, in particular chronic dementia diseases, in particular Alzheimer's disease.
• VGF proteins with and without a signal sequence, pro-forms of VGF proteins that have not yet been processed, as well as VGF proteins that have already been processed, soluble VGF proteins and membrane-bound VGF proteins, the membrane-bound VGF proteins both can be connected to a cell or organelle membrane via transmembrane amino acid sequences, and also via a post-translational modification, for example a glycosyl-phosphatidyl-inositol (GPI) anchor.
• GPI glycosyl-phosphatidyl-inositol
• VGF peptides and VGF peptide variants are referred to as VGFARP ("VGF Alzheimer related peptide") peptides.
• VGFARP peptides can be derived from both the VGF sequences mentioned above (NM_003378 and Y1266) and from further VGF protein variants which may occur in nature.
• VGFARP peptides can contain two point-mutated, two deleted or two additionally inserted amino acids, as well as N-terminal and / or C-terminal extensions. However, they must retain at least 8 amino acids from the VGF protein sequence. Only those amino acids that occur in the VGF protein sequence at this sequence position in the VGF protein are suitable as N- or C-terminal extensions.
• VGFARP peptides derived from naturally occurring VGF polymorphisms and from naturally occurring VGF mutants are referred to as VGFARP peptides.
• VGFARP peptides can also be used with post-translational modifications such as Glycosylation and phosphorylation and / or in chemically modified form, preferably as
• VGFARP-12 has been identified as both an unoxidized and an oxidized peptide.
• a chemically or post-translationally modified peptide can consist of both D- and L-amino acids as well as combinations of D- and L-amino acids. These peptides can also contain unusual amino acids, ie amino acids that do not belong to the 20 standard amino acids. Examples of unusual amino acids include: alpha-aminobutyric acid, beta-aminobutyric acid, beta-alanine, beta-aminoisobutyric acid, norvaline, homoserine, norleucine, gamma-aminobutyric acid, thioproline, 4-hydroxyproline, alpha-aminoadipic acid, diaminobutyric acid, 4-
• aminobenzoic acid homocysteine, alpha-aminopenicillanic acid, histamine, ornithine, glycine-proline dipeptide, hydroxylysine, proline-hydroxyproline dipeptide, cystathionine, ethionine, seleno-cysteine.
• Post-translational or chemical modifications include modifications of the amino acid sequences by the following structures: Binding of free cysteine to one
• Biotinyl, stearoyl, palmityl, lipoyl, C-mannosyl, phosphorus and sulfate groups, glycosylations, amidations, deamidations, pyroglutamic acid, citrulline, etc. are possible.
• nucleic acids DNA, RNA and DNA-RNA hybrid molecules of natural origin as well as synthetically or recombinantly produced are regarded as nucleic acids. Also included are chemically modified nucleic acids that contain modified nucleotides with high in vivo stability, e.g. Phosphorothioates. Such stabilized nucleic acids are already used when using ribozyme, antisense and triplex nucleic acid techniques.
• Sensitivity defines the proportion of sick patients who receive a positive diagnosis result when diagnosed with the disease, ie the diagnosis correctly indicates the disease. Specificity:
• Specificity is defined as the proportion of healthy patients who receive a negative diagnosis result when diagnosed with the disease, i.e. the diagnosis correctly indicates that there is no disease.
• the invention comprises a method for the detection of a neurological, in particular a chronic dementia, in particular of Alzheimer's disease or a predisposition to such a disease by identifying one or more VGF peptides which are of the sequence with the gene bank accession No. NM_003378 or Accession No. Y12661 of the DNA Data Bank of Japan are derived in a biological sample from an individual. Since these VGF peptides are thought to be causal with the disease
• the present invention also includes the use of these peptides for the therapy of Alzheimer's disease or related neurological diseases.
• These peptides or peptide fragments are called “VGF derived Alzheimer related peptide "(VGFARP) and are numbered VGFARP-1 to VGFARP-38.
• the two VGF protein variants NM_003378 and Y12661 differ only in 13 positions of their amino acid sequence and VGF peptides were identified from both VGF proteins, one The VGFARP peptides VGFARP-11 and -32 come from the VGF variant with accession no.
• the VGFARP peptides VGFARP-25, -30, -31, -36 and - 37 come from the VGF variant with the accession no. NM_003378 All other VGFARP peptides can be derived from both VGF variants based on their amino acid sequence, since VGFARP peptides derived from two different VGF variants have been identified so far that there are other VGFARP peptides derived from these or other VGF variants, and these VGFARP peptides are also the subject of the invention.
• the invention provides a method for the detection of Alzheimer's disease by determining the relative concentration of at least one marker peptide in a biological sample of a patient, compared to the concentration of the marker peptide in a control sample, in which the following points must be fulfilled: 1. At least one VGFARP peptide or a peptide that is derived from the nucleic acids with the accession numbers NM_003378 or Y12661 or homologous sequences is used as the marker peptide. 2. A specific increase or decrease in the concentration of the marker peptide in the patient's sample relative to the concentration of the marker peptide in the control sample occurs for the respective marker peptide. 3.
• a significant change in the concentration of the marker peptide in the aforementioned manner is regarded as a positive detection result for a neurological disease, preferably Alzheimer's disease.
• a neurological disease preferably Alzheimer's disease.
• only a rise in the peptide concentration in Alzheimer's disease patients can occur for a certain VGFARP peptide, or in principle only a decrease in the peptide concentration in Alzheimer's disease patients can occur for this VGFARP peptide.
• an increased VGFARP petid concentration cannot occur simultaneously in an individual Alzheimer's patient and in another Alzheimer's patient a reduced concentration relative to the control group.
• VGFARP peptides which can be regarded as fragments of the VGF sequence are referred to as VGFARP peptides. They include homologous peptides derived from the VGF. 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 other than VGF.
• Preferred markers according to the invention are given in the sequence listing and are labeled VGFARP-1 to VGFARP-38, according to Seq. ID 1 to 35 named.
• the sequences of the VGFARP peptides are shown in Figure 1 and Table 1. The assignment of the VGFARP peptides to their respective Seq. ID No. is shown in Table 1.
• the method according to the invention is a method in which specific biomarkers are detected, in the case of neurodegenerative diseases, in particular in Alzheimer's disease, the concentration of which has changed and which also indicate the disease at a very early stage and an increased risk of disease. This is important in order to provide a reliable clinical marker for the diagnosis of these diseases.
• the concentration of the VGFARP peptides in the sample can preferably be correlated with the severity of the disease.
• These new markers therefore make it possible to develop and monitor therapies for the treatment of Alzheimer's disease, since the course and a possible healing success due to a therapy or a reduced progression of the disease can be determined.
• An effective therapy for Alzheimer's disease is currently not possible, which underlines the urgency of providing a safe detection method for Alzheimer's disease, since reliable detection of the disease is a prerequisite for the development of a therapy.
• VGFARP peptides also makes it possible to select, within the scope of clinical studies for the development of new therapies for the treatment of Alzheimer's disease with high specificity, only those patients who are suffering from Alzheimer's disease and not from other diseases. This is important in order to obtain meaningful study results. Patients misdiagnosed as Alzheimer's disease negatively affect the quality of the results of an Alzheimer's therapy study.
• the detection of VGFARP peptides enables stratification of patients, whereby subgroups of Alzheimer's disease patients can be specifically selected for specific diseases Alzheimer's therapy strategies or clinical studies are particularly suitable.
• VGFARP peptides In Alzheimer's disease, the concentrations of VGFARP peptides have changed significantly, relative to healthy individuals. Another aspect of the invention is therefore to bring the VGFARP concentrations in Alzheimer's patients to normal concentrations. This procedure can be used to treat Alzheimer's disease or related neurological diseases. With increased VGF protein or VGFARP peptide concentrations, the concentrations of these substances can be increased by therapeutic administration of e.g. VGF protein or VGFARP peptide-specific antibodies or VGF-specific antisense nucleic acids, ribozymes or triplex nucleic acids or VGFARP peptide antagonists, VGF protein antagonists can be reduced.
• VGF protein or VGFARP peptide-specific antibodies or VGF-specific antisense nucleic acids, ribozymes or triplex nucleic acids or VGFARP peptide antagonists VGF protein antagonists can be reduced.
• substances can also be administered which suppress the body's own expression of VGF protein or the processing of VGF protein into VGFARP peptides. If there is a lack of VGF protein or VGFARP peptides as the cause of the disease, therapeutic administration of VGF protein, VGFARP peptides, VGFARP peptide agonists or VGF protein agonists can be carried out.
• the body's own production of VGF protein or VGFARP peptides can be achieved through the therapeutic administration of substances such as e.g. NGF, BNDF or NT-3 or other suitable substances can be increased as this
• the invention therefore also includes the use of VGF proteins, VGFARP peptides, VGFARP peptide agonists and antigens. gonists, VGF protein agonists and antagonists, anti-VGF
• antibody fragments, antibody fusion proteins, or other substances which bind selectively to VGF proteins, VGFARP peptides, NGF, BNDF or NT-3 can also be used.
• proteins and peptides mentioned fusion proteins of the proteins mentioned can also be used.
• the invention also includes the use of antisense nucleic acids, triplex nucleic acids and ribozymes which modulate the expression of the proteins and peptides mentioned.
• the invention also includes agonists and antagonists which modulate the activity of the proteins mentioned.
• VGF peptides, VGF proteins, nucleic acids, agonists or antagonists can be modified in such a way that they become, for example, lipophilic, which favors the passage into the subarachnoid space. This can be achieved by inserting hydrophobic molecular constituents or also by “packaging” the substances in hydrophobic agents, for example liposomes.
• peptide sequences can be added to these peptides, proteins, nucleic acids, agonists or antagonists, which convert in the subarachnoid space favor or, conversely, make it difficult to move out of the subarachnoid space.
• the invention also encompasses the administration of the said therapeutic agents in various ways, e.g. as an intravenous injection, as an orally administrable substance, as an inhalable gas or aerosol, or the administration in the form of direct injection into the subarachnoid space, or into tissues such as muscle, fat, brain, etc.
• This can increase the biological availability and effectiveness of these therapeutic agents ,
• Peptides or proteins that are administered orally can be protected against porteolytic degradation in the stomach by acid-resistant capsules. Strongly hydrophobic substances can be made more hydrophilic by suitable galenical preparations and therefore more suitable for e.g. intravenous injections, etc.
• Another embodiment of the invention is the use of VGFARP peptides or VGF proteins 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.
• positions in the VGF protein can be experimentally detected for the first time at which the VGF protein is processed in vivo.
• These processing sites are, based on the VGF protein sequence from NM_003378, the following sequence positions: 371/372, 418/419, 479/480, 480/481, 481/482, 482/483 and 483/484.
• the following processing sites are involved: 371/372, 419/420, 480/481, 483/484, 484/485 and 485/486. All experimental identified positions of dosing represent dibasic
• prohormone convertases convert prohormones to hormones, which creates new biologically active substances (peptide hormones).
• biologically active peptides generated from their pro forms in this way are proNGF / NGF, proBDNF / BNDF etc. [1].
• the VGFARP peptides according to the invention are peptide hormones which are suitable as targets for therapeutic agents in connection with neurological diseases, preferably Alzheimer's disease.
• the modulation of the VGFARP peptide concentrations can thus be used for the therapy of neurological diseases, preferably Alzheimer's disease.
• VGF proteins identified in the context of this invention are selectively synthesized as approximately 68 kDa proteins in neuroendocrine and neuronal cells, their expression decreasing with increasing age [2].
• VGF gene deficient mice it was found that important functions in energy metabolism are affected [3].
• VGF gene deficient mice are small in size, hypermetabolic and hyperactive.
• VGF is also synthesized in the insulin-producing islet cells of the pancreas.
• VGF was discovered when examining a rat pheochromocytoma cell line (PC12 cell line) and the stimulation of this This cell line with "nerve growth factor” (NGF) causes a
• NGF is an important growth factor that regulates the differentiation of the peripheral and central nervous system.
• Other factors that regulate VGF expression are the "brain-derived neurotrophic factor” (BDNF) and neurotropin-3 (NT-3) [6].
• BDNF brain-derived neurotrophic factor
• NT-3 neurotropin-3
• VGF-mRNA is caused by neuronal activity, neuronal injuries and by biological Rhythm (circadian clock) regulates [2, 7-9].
• VGF is proteolytically processed with increasing differentiation of neuronal cells via neuronally expressed endoproteases, which presumably recognize basic amino acids.
• endoproteases As Trani et al. C-terminal VGF peptides with masses of 20, 18 and 10 kDa could be shown [10]. This VGF
• VGFARP peptides we identified, however, surprisingly represent fragments with a molecular weight significantly lower than 10 to 20 kDa and are therefore different from those of Trani et al. VGF peptides described. In addition, Trani et al.
• VGF peptides that recognize VGF epitopes that are different from the sequences of the VGFARP peptides.
• the peptides we have identified represent new VGF processing products that have not yet been described.
• the concentrations of the VGFARP peptides can be in one for each peptide specific way in the patient group relative to the control group either consistently increased or consistently decreased. So far, only other VGF peptides of unknown sequence, derived from the C-terminal region of the VGF protein and with a significantly higher molecular weight than the peptides newly identified by us and sequenced for the first time, were known [10].
• the chronic dementia disease detected by the method according to the invention is preferably Alzheimer's disease. So far, the change in the concentration of the peptides and peptide fragments according to the invention has been demonstrated in Alzheimer's patients. From this it can be concluded that the peptides according to the invention can be used for the detection and therapy of Alzheimer's disease and related neurological diseases.
• the identification preferably focuses on certain peptide fragments of the VGF proteins with the GeneBank Accession No. NM_003378, or the DDBJ Accession No. Y12661 i.e. for peptides that comprise partial sequences of these VGF proteins.
• VGF peptides VGF protein fragments
• VGFARP VGF derived Alzheimer related peptides
• Figure 1 The sequences of the peptides determined by us are given in the sequence listing.
• VGF peptides For the first time, we have detected various VGF peptides in biological samples, derived from two VGF protein variants. These peptides, designated VGFARP-1 to VGFARP-38, represent defined fragments of VGF proteins. These Fragments occur naturally in nature and have not previously been described in the literature. These fragments are different from peptides, which are often produced in the literature by in vitro proteolysis (by adding proteases such as trypsin). They thus represent new, previously unknown substances. These peptides were initially enriched and purified from biological samples using reverse phase chromatography and then separated from accompanying other peptides by mass spectrometry, so that these VGFARP peptides could then be sequenced.
• sequences of the peptides in the single letter amino acid code are as follows:
• * rl represents a sequence which corresponds to the sequence or parts of the sequence of the VGF protein from amino acid 49-23, where rl, starting from amino acid 50 of the VGF protein, can be between 0 and 27 amino acids long.
• r2 represents the VGF protein sequence from amino acid 58 to 64 or parts thereof, where r2, starting from VGF amino acid 57, can be between 0 and 7 amino acids long.
• the other peptide chains r3 to rl6 are composed according to the scheme exemplified above.
• ** VGFARP-12 was identified as an unoxidized and as a single oxidized peptide (molecular weight increased by approx. 16 Dal- ton) identified.
• the peptides can be in post-translational or chemical modification forms, which can be on their masses and thus the mass spectrometric identification and also on the elution behavior in chromatography, e.g. in reverse phase chromatography.
• the peptides can be glycosylated, phosphorylated, sulfated, amidated, oxidized, etc. in the sample to be examined.
• Modified peptides are preferably present as peptide oxide, e.g. the peptide VGFARP-12, which was identified both as an unmodified peptide and as a peptide oxide.
• the peptides are also regarded as VGFARP peptides in particular when individual amino acids differ from the corresponding sequence of the VGF protein, in particular when a maximum of 2 amino acids differ from the VGF protein sequence. Point mutations, deletions, internal insertions of amino acids, and N- and C-terminal extensions are permissible as long as the VGFARP peptide sequence contains at least 8 amino acids that are conserved relative to the amino acid sequence of the associated VGF protein, i.e. are unchanged.
• a further development of the invention provides that the concentration of the identified peptide (s) for each of these peptides is increased or decreased in a specific manner relative to the concentration of the respective peptide in a control sample.
• Concentration of the control sample can be used to determine the severity of the disease.
• the control sample can be a pool sample of different different controls.
• the sample to be examined can also be a pool sample, with individual examinations subsequently being carried out if the result is positive.
• the biological sample can preferably be cerebrospinal fluid (cerebrospinal fluid, CSF) or a sample such as serum, plasma, urine, stool, tear fluid, synovial fluid, sputum etc. This depends, among other things. on the sensitivity of the chosen detection method (mass spectrometry, ELISA etc.). Homogenized tissue samples, tissue sections and biopsy specimens can also be used if necessary. It is therefore provided in a further embodiment of this invention that tissue homogenates are prepared for the preparation of the sample to be examined, e.g. from human tissue samples obtained from biopsies. These tissues can e.g.
• aqueous solutions with e.g. 0.1 to 0.2 M acetic acid can be boiled for 10 minutes.
• the extracts are then subjected to the respective detection method, e.g. subjected to a mass spectrometric examination.
• the samples can be prepared in the usual way, e.g. optionally diluted or concentrated, and stored.
• the invention comprises the use of at least one of the VGFARP peptides according to the invention or a VGF protein for the diagnosis of neurological diseases, in particular chronic dementia diseases, in particular Alzheimer's disease, and the use of VGFARP peptides for the production of antibodies or of other agents which are suitable for the development of diagnostic reagents for the detection of these diseases due to their VGFARP peptide-specific binding properties.
• the invention also encompasses the use of VGFARP peptides to obtain phage particles which specifically bind these peptides or which, conversely, present VGFARP peptides on their surface and thus enable the identification of binding partners such as, for example, receptors of VGF proteins or VGFARP peptides.
• VGFARP peptides can be used within the scope of the invention. All methods that make it possible to specifically detect VGFARP peptides in a patient's sample are suitable for this. 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, thin-layer chromatography, NMR (nuclear magnetic resonance) spectroscopy, etc.
• quantitative measurement results are obtained from a sample to be examined with the measurement values that are present in a group of neurological diseases, in particular chronic dementia Diseases, preferably Alzheimer's disease, suffering patients and a control group were compared. From these results, the presence of a neurological disease, in particular a chronic dementia disease, in particular Alzheimer's disease and / or the severity of this disease can be derived.
• 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-HPLC) is particularly preferred.
• RP-HPLC 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 obtained in this way are then individually subjected to the respective detection method, e.g. the mass spectrometric examination.
• Another embodiment of the invention is the use of liquid phase extraction. For this, 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 and then further analyzed.
• 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 containing 1/100 of the volume of eluent used.
• the fractions obtained in this way are analyzed using a mass spectrometer, preferably using a MALDI Mass spectrometers (matrix-assisted laser desorption ionization) 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, analyzed and so the presence of certain masses is determined and the signal intensity quantified.
• MALDI Mass spectrometers matrix-assisted laser desorption ionization
• the VGFARP peptides can be identified with the aid of a mass spectrometric determination, preferably a MALDI (matrix assisted laser desorption and ionization) mass spectrometry.
• the mass spectrometric determination preferably further 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, mono-isotopic 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 method according to the invention identified new, specific peptides of VGF proteins (VGFARP peptides) and recognized their importance. These VGFARP peptides and their descendants are referred to here as VGFARP-1 to VGFARP-38. Your Sequences are given in the sequence listing.
• the VGFARP peptides VGFARP-15, 16, -17, -27, -35, and VGFARP-38 can contain additional amino acids at the N- and / or C-terminus corresponding to the corresponding sequence of the associated VGF protein.
• the invention also encompasses the VGFARP peptides produced recombinantly or synthetically and 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 VGFARP peptide has at least 8 amino acids which correspond in their identity and their position within the peptide sequence to a VGF protein.
• the invention also encompasses nucleic acids which correspond to VGFARP peptides, and in particular those which correspond to the VGFARP peptides according to the invention, and their use for the indirect determination and quantification of the associated VGF proteins and peptides.
• This also includes nucleic acids that 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 VGF that is sufficient for specific hybridization experiments, and therefore for the indirect detection of the associated proteins, in particular the VGFARP Peptides are suitable.
• An embodiment of this includes obtaining tissue samples, e.g. of biopsy specimens, from patients and the subsequent determination of the concentration of an RNA
• Transcripts corresponding to the gene with the GeneBank Accession No. NM_03378 or Accession No. Y12661 of the "DNA Data Bank of Japan", DDBJ or corresponding to homologous VGF variants are quantitative measurement results (intensity ) from a sample to be examined with the measured values obtained from a group of patients suffering from Alzheimer's disease and a control group.
• Methods such as reverse transcriptase polymerease chain reaction (RT-PCR), quantitative real-time PCR (ABI PRISM® 7700 Sequence Detection System, Applied Biosystems, Foster City, CA, USA), in situ hybridizations or Northern blots in a person skilled in the art can be used for quantification known manner can be applied.
• RT-PCR reverse transcriptase polymerease chain reaction
• ABS quantitative real-time PCR
• in situ hybridizations or Northern blots in a person skilled in the art can be used for quantification known manner can be applied.
• the identification of the VGFARP peptides or the VGF proteins can be carried out using an immunological detection system, preferably an ELISA ("enzyme linked immunosorbed assay").
• an ELISA enzyme linked immunosorbed assay
• a so-called “sandwich ELISA” can furthermore preferably be used, in which the detection of the VGFARP peptides is based on the specificity of two antibodies that have different epitopes within the same mo - recognize leküls, is dependent.
• VGFARP- enzyme linked immunosorbed assay
• peptides or VGF proteins can also use other ELISA systems, e.g. find direct or competitive ELISA.
• Other ELISA-like detection techniques such as RIA ("radio immuno assay"), EIA (enzyme immunoassay), ELI spot etc. are suitable as immunological detection systems
• VGFARP peptides or VGF proteins isolated, recombinantly produced or chemically synthesized can be used as standard for the quantification.
• the identification of the VGFARP peptide or peptides can, for example, commonly with the help of an antibody directed to the VGFARP peptide or VGF protein.
• Other methods suitable for such detection include western blotting, immunoprecipitation, dot blots, plasmon resonance spectrometry (BIACORE ® technology, Biacore International AB, Uppsala,
• phage particles eg ABICAP technology, ABION Society for Biosciences and Technology mbH, Irishlich, Germany
• affinity matrices eg ABICAP technology, ABION Society for Biosciences and Technology mbH, Irishlich, Germany
• all substances / molecules are suitable as detection agents that allow one to build a specific detection system because they specifically bind a VGFARP peptide or VGF protein.
• Another embodiment of the invention is the production of VGFARP peptides using recombinant expression systems, chromatography methods and chemical synthesis protocols, which are known to the person skilled in the art.
• the VGFARP peptides obtained in this way can be used, inter alia, as standards for the quantification of the respective VGFARP peptides or as an antigen for the production of VGFARP peptide antibodies.
• Recombinant expression of pepids is one of the methods known and suitable to the person skilled in the art for isolating and obtaining VGFARP peptides.
• VGFARP 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 for the expression of the VGFARP peptides Cells are available from the American Tissue Culture Collection (ATCC).
• ATCC American Tissue Culture Collection
• nucleic acid sequences which code for VGFARP peptides are used in combination with suitable regulatory nucleic acid sequences such as, for example, promoter sequences. Ren, antibiotic selection markers, etc. inserted into an expression vector using molecular biological methods.
• a suitable vector is, for example, the vector pcDNA3.1 from Invitrogen.
• the VGFARP peptide expression vectors obtained in this way can then be inserted into suitable cells, for example by electroporation.
• the VGFARP peptides produced in this way can be C- or N-terminal with heterologous sequences of peptides such as poly-histidine sequences, He agglutinin epitopes (HA-tag), or proteins such as maltose-binding proteins, glutathione-S-transferase (GST), or Protein domains such as the GAL-4 DNA binding domain or the GAL4 activation domain are fused.
• the VGFARP 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.
• a further embodiment of this invention is the isolation of VGFARP 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. or with other methods such as preparative immunoprecipitation, Ammonium sulfate precipitation, extraction with organic solvents, etc.
• a further embodiment of the invention is the production of monoclonal or polyclonal antibodies using VGFARP peptides. The antibodies are obtained in a customary manner familiar to the person skilled in the art.
• a preferred embodiment is the production and recovery of VGFARP peptide-specific antibodies
• a particularly preferred embodiment is the production of VGFARP peptide-specific antibodies which recognize neo-epitopes, that is to say epitopes which are only present on VGFARP peptides but which are not in a VGF protein.
• Such anti-VGFARP peptide antibodies enable provided specific immunological evidence of VGFARP-
• Polyclonal antibodies can be produced by immunizing experimental animals such as mice, rats, rabbits or goats. Monoclonal antibodies can be obtained, for example, by immunizing experimental animals and then using hybridoma techniques, or else using recombinant test approaches, such as, for example, using antibody banks such as the HuCAL ® antibody bank from MorphoSys, Martinsried, Germany, or other recombinant production processes known to the person skilled in the art be won. Antibodies can also be used in the form of antibody fragments, such as Fab fragments or Fab2 fragments, etc.
• Another application example is the quantitative or qualitative determination of the abovementioned VGFARP peptides or VGF proteins for assessing the effectiveness of a therapy in development against neurological diseases, in particular chronic dementia diseases, in particular Alzheimer's disease.
• the invention can also be used to identify suitable patients for clinical studies to develop therapies for these diseases, particularly 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 or the suitability of the patient for a clinical study can be derived from these results.
• the efficacy test and the selection of the right patients for therapies and for clinical studies is of outstanding importance for the successful application and development of a therapeutic agent and so far there is no clinically measurable parameter available for Alzheimer's disease that reliably enables [12].
• VGF proteins vascular endothelial growth factor proteins
• VGFARP peptides and agents that modulate the expression and bioavailability of these substances
• An embodiment of this includes the cultivation of cell lines and their treatment with VGF proteins, VGFARP peptides or with substances which promote the expression of VGF protein, such as e.g. Promote NGF, BNDF or NT-3, or the processing of VGF protein to VGFARP peptides, e.g. Prohormone.
• VGF protein and VGFARP 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, e.g.
• Fusion proteins consisting of prohormone convertases fused with peptide sequences that promote transport of the fusion protein into the interior of the cell.
• prohormone convertases are HIV-TAT sequences or antenna napedia sequences, etc.
• cell lines can be transfected with expression vectors which directly or indirectly cause expression of VGF protein or VGFARP peptides by the transfected cells.
• expression vectors can encode, inter alia, for VGFARP peptides, VGF proteins, NGF, BNDF, NT-3 or for prohormone convertases. The transfection of combinations of the proteins mentioned can also be carried out.
• suitable cell lines with anti-VGF protein or anti-VGFARP peptide antibodies or with nucleic acids which suppress the expression of VGF such as, for example, VGF antisense nucleic acids, VGF triplex nucleic acids or ribozymes directed against VGF mRNA .
• VGF antisense nucleic acids such as, for example, VGF antisense nucleic acids, VGF triplex nucleic acids or ribozymes directed against VGF mRNA
• VGF antisense nucleic acids such as, for example, VGF antisense nucleic acids, VGF triplex nucleic acids or ribozymes directed against VGF mRNA
• VGF anti-NGF, anti-BNDF or anti-NT-3 antibodies could also be used to suppress VGF protein expression.
• cell lines that are considered neurological Model systems in connection with VGF appear suitable can be used for such investigations.
• Tests that can be used as read-out systems for these examinations include the rate of proliferation of the treated cells, their metabolic activity, the rate of cell apoptosis, changes in cell morphology, the expression of cell-specific proteins or reporter genes or the release of cytosolic cell components as a marker for cell death.
• Suitable strains of experimental animals for example mice or rats, which are considered as a model for neurological diseases, in particular as a model for Alzheimer's disease, can be used as further test systems. These experimental animals can be used to investigate the effectiveness of therapy strategies which aim to modulate the concentration of VGFARP peptides or VGF proteins.
• proteins and peptides such as, for example, VGF protein, VGFARP peptides, NGF, BNDF, NT-3, prohormone convertases etc. can also be investigated in experimental animals, these peptides and proteins possibly being galenically processed in such a way that they Brain barrier and / or the blood-liquor barrier can pass better.
• Liposome-packed proteins and peptides, proteins and peptides fused with transport sequences, such as, for example, an 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 more lipophilic properties and can therefore penetrate cells more easily.
• 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 in the stomach that are to be administered orally. Read-out parameters in experiments with animal models can
• a memory test that is suitable for experimental animals is the "Morris water maze test”.
• body function such as blood tests, the measurement of brain waves, metabolism test, the expression rate of VGF protein and VGFARP- can be used as further parameters.
• FIG. 1 Alignment of the VGFARP peptides with the two known VGF proteins according to the database Accession No. NM_003378 and Y12661
• Figure 2 Reverse phase chromatography for the separation and enrichment of the VGFARP peptides from liquor or cerebrospinalis
• Figure 5 MS / MS fragment spectrum using the example of the peptide VGFARP-13
• Figure 6a-C "Box whisker plots" for the quantitative comparison of the concentrations of VGFARP-1, VGFARP-2, VGFARP-18, VGFARP-3, VGFARP-4, VGFARP-5, VGFARP-6, VGFARP-7, VGFARP-19, VGFARP-20, VGFARP-21, VGFARP-10, VGFARP-22, VGFARP-28, VGFARP-29, VGFARP-30/32, VGFARP-31, VGFARP-12, VGFARP-13, VGFARP-36 VGFARP-37 in Alzheimer's disease patients compared with control patients.
• FIG. 1 shows an alignment of the peptides according to the invention with two known variants of the VGF protein, which are shown in the figure with their database Accession No. NM_003378 and Y12661. Sequence positions that are identical in both variants of the VGF proteins are shown with an asterisk in the sequence of NM_003378. Differences in the sequences are shown by the amino acid code in white letters on a black background. The arrow at the end or at the beginning of partial sequences of VGFARP-12, -13 and 34 indicates that the respective sequence extends over two lines in the alignment.
• Figure 2 shows a chromatogram, recorded with reverse phase chromatography according to Example 2, for the separation and enrichment of the VGF peptides from cerebrospinal fluid.
• FIG 3 shows a spectrum that was obtained by MALDI mass spectrometric measurement according to Example 3 of VGFARP-7 with a theoretical, monoisotopic mass of 3686 Daltons, after reverse phase chromatography of human cerebrospinal fluid according to Example 2.
• VGFARP- 7 corresponds to the VGF sequence (Accession No. Y12661) of amino acid 26-62.
• Figure 4 shows by MALDI as relatively quantifying
• Figure 5 shows an MS / MS fragment spectrum according to Example 4 of the peptide VGFARP-13 according to the invention.
• VGFARP-13 corresponds to the VGF sequence (Accession No. Y12661) of amino acid 421-479.
• Figures 6A to 6C show in the form of "box whisker plots" a comparison of the integrated MALDI mass spectrometric signal intensities of different
• VGFARP peptides in controls compared to signal intensities in samples from Alzheimer's disease patients.
• Example 1 Obtaining cerebrospinal fluid to determine VGFARP peptides
• Cerebrospinal fluid or cerebrospinal fluid is that in the four brain ventricles and in the sub- fluid contained in the rachnoid space, mainly in the
• 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 80 ° C.
• Reverse phase chromatography is used as the separation process.
• Various RP chromatography resins and eluents are equally suitable.
• the following is an example of the separation of VGF peptides using a C18 reverse phase chromatography column with the size 4 mm ⁇ 250 mm from Vydac.
• the following compositions were used: solvent A: 0.06% (v / v) trifluoroacetic acid, solvent B: 0.05% (v / v) trifluoroacetic acid, 80% (v / v) acetonitrile.
• Chromatography was carried out at 33 ° C. using an HP ChemStation 1100 from Agilent Technologies with a flow cell Micro from Agilent Technologies.
• Human cerebrospinal fluid was used as the sample. 440 ⁇ l CSF were diluted to 1650 ⁇ l with water, the pH to 2-3 adjusted, the sample was centrifuged for 10 minutes at 18000x 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 the solvent B concentration to 100% and then to at the time 53 min 100% buffer B constant. 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 spectrometers are manufactured by PerSeptive Biosystems Framingham (Voyager-DE, Voyager-DE PRO or Voyager-DE STR) or by Bruker Daltonik GmbH (BIFLEX).
• a matrix substance 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 lyophilized equivalent obtained according to reverse phase chromatography, corresponding to 500 ⁇ l human cerebrospinal fluid, is used.
• the chromatographed sample is dissolved in 15 ⁇ l of a matrix solution.
• This matrix solution contains, for example, 10 g / 1 ⁇ -cyano-4-hydroxycinnamic acid and 10 g / 1 L (-) fucose dissolved in a solution.
• mixed solvent consisting of acetonitrile, water, trifluoroacetic acid and acetone in a volume ratio of 49: 49: 1: 1.
• 0.3 ⁇ l 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 an example of a measurement of one of the VGFARP peptides according to the invention.
• MALDI-TOF mass spectrometry can be used to quantify peptides such as of the VGFARP peptides according to the invention are used when these peptides are present in a concentration which is in the dynamic measuring range of the mass spectrometer, thereby avoiding detector saturation. This is the case for the measurement of the VGFARP peptides according to the invention in cerebrospinal fluid with a liquor equivalent concentration of 33.3 ⁇ l per ⁇ l matrix solution. There is a specific relationship between the measurement signal and the concentration for each peptide, which means that MALDI mass spectrometry can preferably be used for the relative quantification of peptides. This is shown in Figure 4.
• a VGFARP 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 extracted by applying collision energy with a collision gas, e.g. Helium or nitrogen, fragmented and the resulting VGFARP peptide fragments detected in the mass spectrometer in an integrated analysis unit and corresponding m / z values determined (principle of tandem mass spectrometry) [13].
• a collision gas e.g. Helium or nitrogen
• the fragmentation behavior of peptides enables a mass accuracy of e.g. 50ppm an unambiguous identification of the VGFARP peptides according to the invention using computer-assisted search methods [14] in sequence databases in which the sequence of a VGF protein was entered.
• 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 are shown in Figure 5.
• Example 5 Mass spectrometric quantification of VGFARP peptides to compare their relative concentration in control samples compared to patient samples
• a subsequent MALDI measurement of the VGFARP peptides according to the invention according to Example 3 was carried out.
• Exemplary MALDI signal intensities are visualized in the form of "box whisker plots" in Figures 6A to 6C.
• the "box whisker plots" shown in Figure 6 are based on measured values, each based on 29 to 45 samples of Alzheimer's disease patients, or 13 to 44 control samples per experiment were carried out. A total of 4 experiments were carried out.
• the "box whisker plots” shown enable comparison of the integrated MALDI mass spectrometric signal intensities of various VGFARP peptides in controls with the MALDI signal intensities in samples from Alzheimer's disease patients.
• the “box” includes the columns in the diagrams in FIGS. 6A to 6C, the area of the MALDI signal intensities, in which 50% of the respective MALDI signal intensities are located, the lines pointing upwards and downwards from the “box” (“whiskers”) indicate the area, which contains the 25% of the measured values with the highest signal intensities (upper quartile) or the 25% of the measured values with the lowest signal intensities (lower quartile).
• the solid line in the columns indicates the median and the dashed line in the columns indicates the mean.
• VGF a novel role for this neuronal and neuroendocrine polypeptide in the regulation of energy balance. Front neuroendocrinol. 21: 199-219.
• VGF vascular endothelial growth factor

Applications Claiming Priority (3)

Publications (1)

Family

ID=7680795

Family Applications (1)

Country Status (7)

Cited By (1)

Families Citing this family (29)

Family Cites Families (4)

• 2002
  • 2002-04-08 WO PCT/DE2002/001376 patent/WO2002082075A2/de active Application Filing
  • 2002-04-08 DE DE10291524T patent/DE10291524D2/de not_active Expired - Fee Related
  • 2002-04-08 JP JP2002579794A patent/JP2004531250A/ja active Pending
  • 2002-04-08 AU AU2002338348A patent/AU2002338348A1/en not_active Abandoned
  • 2002-04-08 CA CA002446886A patent/CA2446886A1/en not_active Abandoned
  • 2002-04-08 EP EP02742678A patent/EP1373905A2/de not_active Withdrawn
• 2003
  • 2003-10-06 US US10/680,087 patent/US20040142388A1/en not_active Abandoned

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events