WO2003038437A2 - Procede de detection de principes actifs d'action pharmacologique agissant sur le fonctionnement des cellules nerveuses - Google Patents

Procede de detection de principes actifs d'action pharmacologique agissant sur le fonctionnement des cellules nerveuses Download PDF

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WO2003038437A2
WO2003038437A2 PCT/DE2002/004063 DE0204063W WO03038437A2 WO 2003038437 A2 WO2003038437 A2 WO 2003038437A2 DE 0204063 W DE0204063 W DE 0204063W WO 03038437 A2 WO03038437 A2 WO 03038437A2
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igfbp
igf
cell
activity
sample
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PCT/DE2002/004063
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German (de)
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WO2003038437A3 (fr
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Michael Anton Sendtner
Jennifer Gunnersen
Hans-Harald Sedlacek
Stefan Wiese
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MedInnova Gesellschaft für medizinische Innovationen aus akademischer Forschung mbH
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Priority to AU2002357426A priority Critical patent/AU2002357426A1/en
Priority to EP02802277A priority patent/EP1442304A2/fr
Publication of WO2003038437A2 publication Critical patent/WO2003038437A2/fr
Publication of WO2003038437A3 publication Critical patent/WO2003038437A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4745—Insulin-like growth factor binding protein
    • 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
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • 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

Definitions

  • the invention relates to a method for finding pharmacologically active substances which influence the function of nerve cells, a sample being brought into contact with a prospective active substance or a mixture of such active substances, with or simultaneously the activity of a biologically active molecule is determined, and a prospective active ingredient or a mixture of such active ingredients is selected if the activity of the biologically active molecule is reduced or increased when it is brought into contact.
  • the invention further relates to uses of active substances identified in this way.
  • Neuropathies are nerve disorders that are associated with motor and / or sensitive nerve conduction disorders.
  • the cause of neuropathies can be toxic, metabolic, infectious or inflammatory, with an inflammatory component involved in practically all cases of neuropathy.
  • the most common neuropathies are motor neuropathies that develop in the course of diabetes mellitus.
  • a special feature of these diabetogenic neuropathies is the degeneration of the motor end plates, the loss of the axons and the atrophy of the motor neurons.
  • the causes of neuropathy ie the toxic, metabolic, infectious or inflammatory causes, are often known, the molecular processes that are involved in the pathogenesis are largely unclear.
  • IGF-1 or -2 IGF-1 and IGF-2 are among those growth factors that support the survival of neurons and glial cells in vitro and in vivo. These factors promote the division of neural progenitor cells, the maturation and survival of motor neurons and Schwanschen cells (Arakawa et al. (1990) J. Neurosci. 10: 3507-3515; Syroid et al. (1999) J Neurosci. 19 : 2059-2068).
  • mice that either contained noisy IGF-1 or IGF-2 or overexpressed IGF-1 indicated that IGF stimulates the formation of myelin (Liu et al. (1993) Gell 75: 59-72 ; Beck et al. (1995) Neuron 14: 717-730; Carson et al. (1993) Neuron 10: 729-740).
  • IGFs In contrast to most growth factors, IGFs are present in the bloodstream in large quantities, but only a very small part of them are in the form of free IGF. The majority forms complexes with the so-called “Insulin-like Growth Factor Binding Proteins” (IGFBP) and another large protein called “Acid Labile Subunit” (ALS) (Blum et al. (1991) in: Modern Concepts in Insulin-Like Growth Factors, published by Spencer, Elsevier, New York, 381-893). So far, six different IGFBPs have been identified (Yamanaka et al. (1997) J Biol. Chem. 272: 30729-30734).
  • IGFBP Insulin-like Growth Factor Binding Proteins
  • ALS Acid Labile Subunit
  • IGFBP-5 is a glycosylated protein with a molecular weight of approximately 26 kDa, which binds to IGF-1 and IGF-2 with high affinity (Drop et al. (1992) Growth Regulation 2: 69-79 ; WO 97/03470).
  • IGFBP-5 can be detected in other organs to a considerable extent in the nervous system, for example in glial cells and in Purkinje cells and cerebellar granule cells (Bondy and Li (1993) J. Neurosci. 13: 5092-5104; Stenvers et al. (1994) J Comp. Neurol. 339: 91-105; Rochier et al. (2001) J Neurochem. 76: 11-20). Immunohistochemical studies show a localization of the protein on the cell membrane of Purkinje cells. IGFBP-5 can also be detected immunohistologically on and in myelinated axons.
  • IGFs promote the growth of nerve cells, complexes of IGF-1 and IGFBP-3 (WO 95/13823 and WO 99/62526), antisense oligonucleotides that are specific for IGFBP, antibodies that are specific for IGFBP, and peptides have been used
  • IGFBP Inhibition of the binding of IGFBP (WO 98/32022, WO 98/45427 and WO 99/32620) for the treatment of a number of diseases is described.
  • diseases also include diseases of the nervous system, such as amyotropic lateral sclerosis (WO 99/32620 and WO 99/62536), central and peripheral neuropathies (WO 99/32620), or Alzheimer's disease or Huntington's disease (WO 95 / 13823), a.
  • the invention is based on the technical problem of specifying a screening method by means of which further substances, in particular also non-natural substances, can be identified that are highly effective, in particular also more effective than IGF-1 or IGF-2, promote the survival of nerve cells.
  • the invention teaches a method for finding pharmacologically active substances which influence the function of nerve cells, which comprises the following steps: a) contacting a sample containing IGFBP-5 or an active part thereof with a potential active substance or a mixture of such active substances, b) determining the activity of IGFBP-5 in the sample and c) selecting an active substance or mixture of active substances which increases or decreases, in particular decreases or inhibits, the activity of IGFBP-5 in the sample.
  • the invention is based on the finding that the increased formation of IGFBP-5 in nerve cells leads to an impairment of the function of the nerve cells.
  • transgenic mice which overexpress in their motor neurons IGFBP-5 show a loss of their axons and a degeneration of their motor neurons and develop the clinical picture of neuropathy which is associated with the typical functional losses of the nerve cells.
  • Another surprising finding in addition to the findings from transgenic animal experiments is that increased amounts of the RNA coding for IGFBP-5 could be detected in nerve cells of patients with diabetes mellitus. This increase in IGFBP-5 is specific to diabetogenic forms of neuropathy because it was not found in the nerves of patients with non-diabetic forms of neuropathy.
  • the surprising finding is that one cause of diabetogenic neuropathy is an increase in the concentration of IGFBP-5 within or in the vicinity of the nerve cells.
  • This finding now enables the targeted search for active substances that allow the prophylaxis or therapy of neuropathies, preferably diabetic forms of neuropathy, with the help of Substances that specifically reduce the concentration and / or the activity of IGFBP-5 within the nerve cells or in the vicinity of the nerve cells.
  • “Functions” of nerve cells are understood to mean, for example, the stimulus conduction and all the biochemical and / or electrochemical processes involved.
  • the term function preferably includes the survival of nerve cells.
  • the survival of nerve cells for example in a cell culture dish, can take place by determining the number of cells that are dying in the cell culture.
  • the death of cells in the central nervous system can be observed, for example, by test methods that detect apoptosis. Such test methods are, for example, "tunnel assay” (Gavrielli et al. (1992) J. Cell Biol. 119: 493-501; Gold et al. (1994) Lab. Invest. 71: 219-225), chromatin fragmentation (Götz et al.
  • the contacting of a sample with at least one potential active ingredient comprises any form of mixing the sample with the potential active ingredient, wherein both the sample can be added to the potential active ingredient and the potential active ingredient to the sample.
  • the sample and / or the potential drug is (s) preferably as a fixed ⁇ material, solution, suspension, slurry or bound to a solid phase. If the sample with which at least one potential active substance is brought into contact is a cell, the step of contacting also includes processes known in the prior art which allow the introduction of substances into intact cells, such as, for example, DNA "guns", infection, transfection and / or transformation.
  • the potential active substance is naked DNA, viruses, viroids, virosomes and / or liposomes, the liposomes or virosomes also being suitable, in addition to a potentially effective nucleic acid olekül, further potential active substances contact with the sample.
  • a number of further methods are known to the person skilled in the art which are used to introduce potential active substances into cells and which are equally suitable for bringing the sample and potential active substance into contact.
  • a sample in the sense of this invention is, for example, at least one cell, at least one cell extract, at least one protein mixture and / or a mixture, the cell, the cell extract, the protein mixture or the mixture containing IGFBP-5 or an active part thereof.
  • the cells include, for example, pro- and eukaryotic cells, in particular, however, cells which express IGFBP-5 as wild-type cells. In cells which do not or only to a small extent express IGFBP-5 as wild-type cells, IGFBP-5 can be expressed or overexpressed by methods known to the person skilled in the art.
  • Such methods include, for example, infection, transfection or transformation of cells with vectors which contain nucleic acids which code for IGFBP-5 or an active part thereof and are under the control of a suitable promoter, in particular for constitutive expression.
  • a preferred sample which can be used in the method according to the invention is a cell which has an increased IGFBP-5 activity compared to wild-type cells of the same cell or tissue type.
  • Such a cell for example, from heterozygous or ho o- zygotic IGFBP-5 transgenic mice • be won. This cell h ⁇ len then the introduced genes included depending on the number, for example, between 1 to about 40 additional genes encoding for IGFBP-5 or an active portion thereof.
  • cells are made from heterozygotes or homozygous IGFBP-5 transgenic mice or mouse embryos are preferred, in particular neurons obtained from them or, more preferably, spinal motor neurons. These cells can be isolated, for example, using the panning technique (Metzger et al. (1998) J. Neurosci. 18: 1735-1742).
  • sample also includes cell extracts that can be obtained, for example, from one of the cells listed above using standard methods known to the person skilled in the art. Suitable methods include, but are not limited to, freeze-thawing, sonification, or French pressing. If necessary, such a cell extract can be worked up or purified in a further step. Preferred steps include, for example, precipitation, filtration and chromatographic process steps. Suitable chromatographic methods are known to the person skilled in the art and include, for example, anion or cation exchange chromatography, affinity chromatography and / or size exclusion chromatography.
  • the sample can also be a mixture of purified or recombinant proteins containing IGFBP-5 or an active part thereof and / or a mixture containing IGFBP5 or an active part thereof, which additionally contains further components, such as, for example, components which are used for the determination of the Activity of IGFBP-5 can be used.
  • the activity of IGFBP-5 in the sample can be determined by a number of direct and indirect detection methods. The appropriate methods depend on the nature of the sample. In cells, the activity of 'is IGFBP-5 for example, determined by the amount of proteins expressed in the cell, IGFBP-5, or to bind by the ability of existing in the cell IGFBP-5 of IGF-1 or IGF-2.
  • the transcription of the gene coding for IGFBP-5 can be repressed, for example, by determining the amount of IGFBP-5 mRNA. Standard methods known in the prior art for determining the amount of IGFBP-5 mRNA include, for example
  • the IGFBP-5 activity which is based on the induction or repression of the transcription of the IGFBP-5 promoter, can also be determined by coupling the IGFBP-5 promoter to suitable reporter constructs.
  • suitable reporter genes are these Chloramphenicol transferase gene, the "green fluorescent protein” (GFP) and variants thereof, the luciferase gene and the Rennilla gene.
  • the expression of IGFBP-5 protein can also be detected at the protein level, in which case the amount of the protein is detected, for example, by antibodies directed against IGFBP-5 proteins, polyclonal or monoclonal.
  • the change in the binding ability of IGFBP-5 to IGF-1 and / or IGF-2 can be checked, for example, in a "two-hybrid" system, the two-hybrid system being carried out, for example, in yeast or in higher eukaryotic cells can be.
  • the interaction strength between IGFBP-5 and IGF-1 and / or IGFBP-5 and IGF-2 is then compared, with or without the addition of the potential active ingredient. If the sample is, for example, a cell extract, a protein mixture and / or a mixture containing IGFBP-5 or an active part thereof, the determination of the activity is preferably aimed at determining the binding ability of IGFBP-5 to IGF-1 and / or IGF-2.
  • IGF-1 and / or IGF-2 Either the amount of free, ie unbound IGFBP-5 and / or the amount of free IGF-1 or IGF-2 or the decrease or increase in free IGFBP, IGF-1 and / or IGF-2 can be determined .
  • Another possibility is the immunoprecipitation of IGFBP-5, the determination of the co-immunoprecipitated IGF-1 and / or IGF-2, the amount of the respectively precipitated IGF-1 or IGF-2 by immunological methods but also by radioactive labeling, fluorescent labeling or similar markings of the IGF can be determined. The amount of free IGFBP-5 or IGF in the treated and untreated sample is compared.
  • Active substances which increase or inhibit the activity of IGFBP-5 in the sample compared to the untreated sample (control) are, according to this invention, pharmacologically active substances which influence the function of nerve cells.
  • a pharmacologically active substance that influences the function of nerve cells changes the activity of IGFBP-5 compared to the control, each determined using the same method, more than approx. 10%, but preferably by at least approx. 50% or by at least 100% , more preferably by at least about 500%.
  • the potential active ingredient reduces the activity of IGFBP-5 compared to the untreated sample.
  • step a) can be followed by an incubation process step, which can vary in length depending on the sample.
  • the activity is increased after approximately one hour to 100 days, preferably after approximately 1 day to approximately 50 days, most preferably after approximately 3 days to approximately 10 days. determined especially after 3 days.
  • the sample is a cell extract, a protein mixture or a mixture
  • the activity can be determined, for example, after a period of about 0 seconds (measurement of the activity immediately when it comes into contact) to about 20 days.
  • the time period for the incubation after the sample has been brought into contact with the potential active ingredient is at least or is approximately 5, 10, 20, 30, 40, 50, 60, 90, 120, 150, 180 minutes (McDonald et al. (1999) Analyt. Biochem. 268: 318-329).
  • the present method also includes determining the activity of an active portion of IGFBP-5.
  • an active part of IGFBP-5 contains an N- and / or C-terminal deletion and / or optionally one or more internal deletion, but also alternatively or additionally mutations, the ability of the so deleted and / or mutated IGFBP-5 part, in transgenic mice Triggering loss of the axons and degeneration of the motor neurons is not lost.
  • a functionally active portion of IGFBP-5 that is expressed in transgenic mice has at least about 10%, preferably at least about 20%, more preferably at least 50% and most preferably at least about 100% of the activity of wild type IGFBP-5.
  • the sample contains at least one cell, at least one cell extract, at least one protein mixture and / or a mixture containing IGFBP-5 or an active part thereof.
  • the cell is a neuronal cell, in particular a sensory, empathic or motor-neuronal cell, a neuronal stem cell, a neuron, in particular a cholinergic neuron of the basal forebrain, a dopaminergic nerve cell of the midbrain, a granule cell Purkinje cell of the cerebellum or hypocampus, a retinal ganglion cell or a photoreceptor.
  • a cell extract or a protein mixture this is preferably produced or isolated from one of the aforementioned cells.
  • the cell has an increased IGFBP-5 activity compared to a wild-type cell, this increased activity, for example, on the stimulation of the expression of IGFBP-5 and / or the transfection by IGFBP-5 containing vectors and / or insertion of IGFBP-5 coding genes. The same applies in the case of active parts of the IGFBP-5.
  • the activity of IGFBP-5 in the cell is determined by the survival rate of the cells, optionally together with another IGFBP-5-specific method for determining the activity.
  • This determination of the activity is of particular interest in cells which, for example due to the introduction of additional IGFBP-5 genes, have increased IGFBP-5 activity, and which therefore have a comparison with the respective wild-type Cell have a reduced survival rate.
  • An extension of the survival rate of these cells after incubation with at least one potential active ingredient serves as an indirect means of determining the activity of IGFBP-5 in the sample.
  • a pharmacologically active ingredient extends the survival rate of the cells by at least about 20%, preferably by at least about 50%, more preferably by at least about 100%, and even more preferably by at least about 1000% compared to untreated cells. Most preferred are those active ingredients which, when added to the cells, lead to a permanent survival of the cells in the cell culture and / or prevent the loss of the axons and the degeneration of motor neurons in an IGFBP-5 transgenic mouse.
  • the activity of IGFBP-5 in the sample is determined by the amount of IGFBP-5 protein, the amount of nucleic acids coding therefor and / or the binding activity of IGFBP-5.
  • IGFBP-5 in the sample after contacting at least one potential active substance indicates that the potential active substance is a pharmacologically active substance which influences the function of nerve cells. If the potential active ingredient is a
  • Active ingredient mixture the pharmacologically active ingredient is isolated from this active ingredient mixture in a further step, for example by deconvolution.
  • the sample is compartmentalized, for example on a microtiter plate with a plurality of wells, for example 96, 384 or 1525 wells.
  • a microtiter plate with a plurality of wells, for example 96, 384 or 1525 wells.
  • Such microtiter plates are already routinely used in fully automatic, massively parallel test procedures (so-called "high throughput screens"), which allow hundreds of thousands of different active substances to be tested in a short time.
  • high throughput screens massively parallel test procedures
  • everyone is Compartmentalization suitable, which makes it possible to spatially limit the effect of the potential active ingredient brought into contact with the sample, so that the effects of the potential active ingredient used in each case on the activity of IGFBP-5 or an active part thereof in the sample are determined can.
  • the sample can be covalently or non-covalently linked to the surface of the sample carrier, such as a microtiter plate, or can be present as a solution, suspension or slurry.
  • the sample carrier such as a microtiter plate
  • planar sample carriers or, for example, structured by depressions or channels are also suitable.
  • sample areas can be rendered hydrophobic, drops being applied with the sample to such areas. Alternatively, the drops can be applied to delimited, non-hydrophobicized areas.
  • the sample carrier can be made of glass, silicon, metal or plastic, for example.
  • At least one potential active substance is linked covalently or non-covalently to a sample carrier, the surface of the sample carrier preferably being structured in the form of depressions, channels or also planar.
  • the sample is brought into contact with the immobilized potential active substances and the activity of IGFBP-5 in the sample is determined at the respective immobilization point of the potential active substance (s).
  • a protein chip can be produced which contains different peptide fragments on the surface, the influence of which on the activity of IGFBP -5 can be tested.
  • a large number of different chemical substances can be produced on a surface using combinatorial-chemical processes known in the prior art, the effect of which on Activity of IGFBP-5 can be examined by the method according to the invention.
  • the pharmacologically active ingredient or the isolated pharmacologically active ingredient is made up into a medicament or diagnostic agent in a further step, suitable auxiliaries and additives being added to the pharmacologically active ingredient, and a physiologically effective dosage is set.
  • the pharmacologically active ingredient is modified in further process steps using methods known to the person skilled in the art, which include, for example, modifications with halogen, in particular with fluorine or chlorine and / or combinatorial chemical compositions, and is applied again in the process according to the invention investigated the influence on the activity of IGFBP-5, the activity of IGFBP-5 in the sample treated with the modified pharmacologically active ingredient being compared with the activity of IGFBP-5 in the sample using the starting active ingredient and a
  • Improving the effectiveness of the pharmacologically active ingredient in particular a greater reduction in the activity of IGFBP-5 in the sample compared to the starting active ingredient is used as a criterion for selection.
  • a reduction is at least about 5%, preferably at least about 10%, more preferably at least about 50%, compared to the starting active ingredient.
  • standard chemical and / or physical methods such as mass chromatography or GC-MS, 1 H-NMR chromatography, or IR can be used Spectroscopy to determine the structure of the modified drug.
  • Another object of the present invention is a test system for finding pharmacologically active substances, which function the cells of the nervous system influence, comprising: a) at least one sample containing IGFBP-5 or an active part thereof and b) at least one agent for determining the activity of IGFBP-5 in the sample.
  • the present invention furthermore relates to the use of at least one active ingredient for the prophylaxis and / or therapy of diseases which are associated with cell growth disorders in nerve cells, in particular diabetic neuropathy, the active ingredient used being found in the method according to the invention described above.
  • the present invention shows for the first time the importance of IGFBP-5 for the pathogenesis of diabetogenic neuropathy.
  • Another object of the present invention is therefore the use of at least one active substance for the prophylaxis and / or therapy of diabetogenic neuropathy, characterized in that the active substance inhibits the activity of IGFBP-5.
  • one of the following active substances is used: IGF-1, IGF-2, a mutant of IGF-1 and / or IGF-2 with increased binding affinity for IGFBP-5 (Luthi et al., Eur. J Biochem., 205, 483-480 (1992); Lowman et al., Bioche istry, 37, 8870-8878, (1998)), an active fragment of IGF-1 and / or IGF-2, a substance which binds to the heparin-binding domain of IGFBP-5 (Song et al., J. Mol.
  • a substance which inhibits the expression and / or release of IGF-1 and / or IGF-2 a nucleotide sequence coding for IGF-1 and / or IGF-2, a nucleotide sequence for a mutant of IGF-1 and / or a mutant of IGF-2 with increased binding affinity to IGFBP- 5 encodes a nucleotide sequence which encodes ment of IGF-2 for an active fragment of IGF-1 and / or an active Frag ⁇ , and / or a nucleotide sequence for a binding IGFBP-5 antibody or antibody fragment kodi ert.
  • an active substance which expresses and / or Release of IGF-1 and / or IGF-2 stimulated.
  • Preferred active substances which stimulate the expression and / or release of IGF-1 and / or IGF-2 are: antiprogestins, for example tamoxifen (Quin et al., Endocinology, 1,40, 2501-2508 (1999)), antioestrogens, for example Mifepristone (Bitar, et al, Surgery, 127, 687-695 (2000)), the osteogenic protein-1 (OP-1), the "bone morphogenic protein-2" (BMP-2), the "basic fibroblast growth factor "(bFGF), the" transforming growth factor ⁇ -1 "(TGFß-1), the" platelet derived growth factor BB “(PDGFBB), interleukin-1 receptor antagonists, for example IL-RA (Benbassat et al., Horm.
  • Mus musculus NM_010518, XM_129718, AF179369, X81583, AAC37636 and L12447.
  • Rattus norvegicus AF179370, AF139830, NM_012817, AAA53533 and M62781.
  • Active parts of this are in particular the C-terminal region or the heparin-binding domain or a partial sequence of the heparin-binding domain with a minimum length of 4, preferably 8, most preferably 10, amino acids.
  • the heparin-binding domain is in the case of the translation product according to accession number NM000599 aa216-266.
  • the heparin-binding domain is in the case of the translation product according to accession number M62781 aa215-265.
  • the heparin-binding domain is in the case of the translation product according to accession number L12447 aa215-265.
  • homologous sequences under the terms of the IGFBP-5, in particular Amino Text ⁇ rensequenzen to the above-mentioned sequences and partial sequences thereof.
  • the term homology is defined as follows. The expect value is used as the homology criterion, as it is when using the BLASTP program 2.2.4 [Aug-26-2002] results. Homology to a query amino acid sequence longer than 20 aa is when the expect value is less than 0.1, in particular less than 0.01 or 0.001. Homology to a query amino acid sequence of a length of less than or equal to 20 aa exists if the expect value is greater than or equal to 0.1, in particular 0.01 or 0.001, and less than 10, in particular 1.
  • a sample within the meaning of the invention can consequently contain, for example, a polypeptide or an oligopeptide which contains a partial sequence of IGFBP-5, in particular the heparin-binding domain of IGFBP-5, a minimum length of 4, 10, 20 or 40 amino acids, or a homolog For this. Minimum lengths of 20 to 40 amino acids are preferred. It is not critical whether and which amino acid sequences are connected to the partial sequence N- and / or C-terminal.
  • Example 1 Induction of neuropathy in mice by overexpression of IGFBP-5
  • a lambda-ZAPII cDNA library from the brain of a mouse was tested with an 873 bp IGFBP-5 cDNA sample. This sample was analyzed using PCR (primer: forward reaction 5'-GCC CCG AGG TAA AGC CAG ACT-3 '(SEQ ID NO. 1), reverse reaction 5'-GGA TAG GGG GAG GAA GGG AGG-3' (SEQ ID NO . 2)) prepared and contained the entire coding sequence and 48 bp of the 5'-untranslated region.
  • the cDNA was inserted into the EcoRI site of the polylinker of a pßS II SK vector using the Exassist / SOLR system (from Stratagene, La Jolla Ca.) according to the manufacturer's instructions inserted.
  • the identity of the clones was confirmed by DNA sequencing.
  • This NFL-IGFBP-5 DNA construct was used to generate an 8 kB fragment using the restriction enzyme S a I, which is the human neurofilament light chain (NFL) promoter, the cDNA for IGFBP-5 of the mouse, the poly A signal from pMC-Cre and exons 2-4 of the NFL gene (in the downstream region).
  • S a I which is the human neurofilament light chain (NFL) promoter
  • the cDNA for IGFBP-5 of the mouse the poly A signal from pMC-Cre and exons 2-4 of the NFL gene (in the downstream region).
  • the fragment was purified twice over gel and then over Nucleotrap resin (Machery and Nagel, Düren, D), extracted with butanol, then desalted and finally purified with the aid of Elutip (Schleicher and Schüll, Dassel, D).
  • the eluted DNA was extracted with phenol / chloroform and chloroform and then precipitated with ethanol.
  • the DNA was suspended in sterile buffer solution (5 mM Tris-HCl, pH 7.4; 0.1 mM EDTA) (concentration approx. 500 copies / pl) and this suspension was infected in fertilized mouse oocytes.
  • Southern blot analysis can be identified.
  • the following primers were used for the PCR reaction to determine the genotype: Forward reaction: NFL-SEQ 5'-TCG CAG GCT GCG TCA GGA G-3 1 (SEQ ID NO. 3); Reverse reaction: BP5 PCR 5'- CTT GCA GGT AGA GCA GGT GCT CTC-3 '(SEQ ID NO. 4). 45 cycles of 45 s at 94 ° C, 45 s at 53 ° C and 30 s at 72 ° C were carried out.
  • the DNA sample was digested with Xho I and tested with a radiolabelled IGFBP-5 PCR sample.
  • RNA was presented for each RT-PCR reaction.
  • the first strand of DNA was synthesized with oligo (dT) primer (from Life Technologies).
  • the primers for amplifying IGFBP-5 were: 5 'primer: 5' CTT TCG TGC ACT GTG AAC C-3 '(SEQ ID NO. 5); 3 'primer: 5' -CTC AAC GTT ACT GCT GTC G-3 * (SEQ ID NO.6).
  • the separated proteins were transferred in an electroblotting apparatus to a nitrocellulose membrane (electroblotting apparatus from Biometra GmbH, Goettingen, D).
  • the membranes were labeled with iodine-labeled IGF-2 using the method described by Hossenlopp et al. (1986) Anal Biochem 154: 138-143. Radioactive iodine bound to IGFBP-5 was autoradiographed.
  • the results showed an increased expression of IGFBP-5 in the spinal cord and in the sciatic nerve of transgenic animals.
  • the muscle strength of the forelimbs was determined on the transgenic as well as on the wild-type control mice (method as described by Masu et al. (1993) Nature 365: 27-32). Although there was no difference in body weight between the two animals, a significant reduction in the gripping strength of the forelimbs was found in the transgenic mice. Histomorphological examinations were carried out to analyze these clinical symptoms. They consisted of a morphometric examination of the sciatic nerve, the peritoneum nerve and the ganglia of the anterior horn of the spinal cord. For these investigations, samples of the corresponding tissue after fixation in osmium tetroxide were embedded in epoxy resin and semi-thin sections were produced.
  • Tissue sections were analyzed under the light microscope and evaluated morphometrically using the Quantimed 500 software (Leica GmbH, Bensheim, Germany). The number of intact myelinated nerve fibers in the cross-cut nerves was counted. The number of degenerated fibers was determined using Waller's degeneration.
  • cholinesterase activity on the neuromuscular end plates in muscle samples was histochemically determined using the combined acetylcholinesterase-silver staining (Namba (1971) Exp. Neurol. 33: 322-328; Gurney et al. (1992) J. Neurosci. 12: 3241-3247).
  • acetylcholinesterase activity was carried out in samples of the Gastrocnemius muscle.
  • the muscle samples were homogenized with the addition of 0.5% Triton X-100 for the determination of the total (soluble as well as membrane-bound) enzyme activity and without the addition of Triton X100 for the determination of the soluble acetylcholine esterase activity.
  • the respective enzyme activity was determined after centrifugation of the homogenates (15 min, 15,000 g, 4 ° C.) in the respective supernatants according to the method of Ellmann (Schegg et al. (1990) Neurosci. Lett. 118: 197-200).
  • transgenic mice In order to clarify whether the loss of axons in the transgenic mice is the result or the cause of the death of motor neurons, the number of motor neurons in the facial nucleus as well as in the lumbar section of the spinal cord was counted in transgenic and control mice of different ages ,
  • mice transgenic for IGFBP-5 After cutting the facial nerve in newborn and 3-week-old transgenic mice, no reduction in the motor neurons was found 1 week later. This suggests that motor neuron degeneration in mice transgenic for IGFBP-5 is the cause of axon loss.
  • Example 2 Characterization of nerves from patients with diabetes mellitus.
  • the proteins from nerve biopsy samples were isolated from healthy individuals and from patients with diabetic neuropathy using standard methods (Life Technologies, Düsseldorf, D) and these on a 12% SDS-PAGE -Gel applied.
  • the separated protein samples were transferred to a nitrocellulose membrane, then these membranes were blocked for one hour by incubation in a buffer solution containing horse serum and fat-free dry milk powder.
  • IGFBP-5 was subsequently detected using an IGFBP-5-specific rabbit antibody (Santa Cruz, Göttingen, D, whose specific binding was detected by a peroxidase-coupled anti-rabbit antibody.
  • the peroxidase was determined using the ECL reagent (Messrs.
  • Example 3 Isolation and cultivation of nerve cells, which overexpress IGFBP-5, for testing active substances
  • Embryos 12.5 days old that were either normal or transgenic for IGFBP-5 were spinal motoneurons using the panning technique (Metzger et al. (1998) J Neurosci. 18: 1735-1742) using a monoclonal anti-p75 antibody isolated from the rat (Chemicon, Hofheim, Germany).
  • the ventrolateral parts of the lumbar spinal cord were mechanically comminuted, transferred to hepespuffer solution (containing 10 ⁇ M 2-mercaptoethanol) and incubated with trypsin (0.05%, 10 min).
  • the single cell suspension in the supernatant was transferred to a culture dish coated with the anti p75 antibody and at room temperature for 30 min. incubated.
  • the cells obtained in this way were sown at a density of 20.00 cells / cm 2 in culture plates (Greiner, Nuertingen, Germany) which were precoated with polyornithine and laminin.
  • the cells were kept at 37 ° C. in neurobasal medium (Life Technologies, supplemented with B27 supplement, 10% horse serum, 500 ⁇ M glutamate and 50 ⁇ g / ml apotransferrin) and in a 5% CO 2 atmosphere. 50% of the cell culture medium was replaced on the 1st day and every 2nd day following.
  • Sensory neurons were also isolated from embryos 12.5 days old that were normal or transgenic for the IGFBP-5 gene. For this, dorsal Root ganglia isolated, taken up in PBS and then with trypsin (0.05% in hepespuffer) for 30 min. incubated. The trypsin digestion was stopped by adding L15 medium which contained 10% horse serum and subsequently the cells were plated out in culture plates and incubated for 3-4 hours. Cells in the supernatant were centrifuged (10 min at 400 g) and the cell sediment was kept in the neurobasal medium in the same way as described for spinal motor neurons. Neural stem cells were derived from the brain of normal mouse embryos, or from mouse embryos that are transgenic
  • IGFBP-5 are isolated.
  • the area of the forebrain was removed under a dissecting microscope, and the area of the hippocampus and the periventricular zone in further developed embryos. These brain areas were then transferred to 200 ⁇ l HBSS (Hanks balanced salt solution (HBSS, Life Technologies (Karlsruhe), incubated with 0.1% trypsin (final concentration in HBSS) for 10 min at 37 ° C., the reaction with 0.1% trypsin inhibitor (trypsin inhibitor from egg yolk sack (Sigma, Deisenhofen), stock solution: 1% in HBSS / 25 mM HE-PES) final concentration in HBSS was stopped, then the cells were triturated ten times with a 200 ⁇ l pipette and in Medium [(Neurobasal Medium (Life Technologies), B27 Supplement (Life Technologies Stock 50x, EK lx) Glutamax II (Life Technologies Stock lOOx, EK lx), basicFGF (20ng / ml
  • the neural stem cells were frozen according to the standard protocol, i.e. after centrifugation, the cells were taken up in medium with 10% DMSO and first cooled to -86 ° C at 1 ° C / min (in MrFrosti), before being stored in liquid N2 at - 186 ° C.
  • Example 4 Method for Finding Nerve Cell Protective Substances Nerve cells obtained, for example, with the above-mentioned method can be used to search for substances which inhibit the expression or the function of IGFBP-5 in nerve cells.
  • IGFBP-5 overexpressing and normal motor neurons and sensory neurons are obtained as described above, sown in cell cultures and mixed with the test substance or a mixture of test substances. Substances or mixtures that inhibit the function or expression of IGFBP-5 are able to prevent the death of IGFBP5 overexpressing neurons without affecting the survival of normal neurons.

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Abstract

L'invention concerne un procédé de détection de principes actifs d'action pharmacologique agissant sur le fonctionnement de cellules nerveuses. Ce procédé comporte les étapes suivantes : a) mise en contact d'un échantillon contenant la protéine IGFBP-5 (protéine de liaison du facteur 5 de croissance analogue à l'insuline) ou une fraction active de cette protéine contenant au moins un principe actif protentiel et b) détermination de l'activité de la protéine IGFBP-5 dans l'échantillon.
PCT/DE2002/004063 2001-10-29 2002-10-29 Procede de detection de principes actifs d'action pharmacologique agissant sur le fonctionnement des cellules nerveuses WO2003038437A2 (fr)

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DE2001153335 DE10153335A1 (de) 2001-10-29 2001-10-29 Verfahren zum Auffinden von pharmakologisch aktiven Wirkstoffen, die die Funktion von Nervenzellen beeinflussen

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Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
ANDERSON RICHARD A ET AL: "The insulin-like growth factor system in the GT1-7 GnRH neuronal cell line" NEUROENDOCRINOLOGY, Bd. 70, Nr. 5, November 1999 (1999-11), Seiten 353-359, XP009021284 ISSN: 0028-3835 *
BEILHARZ ERICA J ET AL: "Differential expression of insulin-like growth factor binding proteins (IGFBP) 4 and 5 mRNA in the rat brain after transient hypoxic-ischemic injury" MOLECULAR BRAIN RESEARCH, Bd. 18, Nr. 3, 1993, Seiten 209-215, XP009021270 ISSN: 0169-328X *
BONDY CAROLYN ET AL: "Correlation between insulin-like growth factor (IGF)-binding protein 5 and IGF-I gene expression during brain development" JOURNAL OF NEUROSCIENCE, Bd. 13, Nr. 12, 1993, Seiten 5092-5104, XP009021215 ISSN: 0270-6474 *
CHENG HSIN-LIN ET AL: "Immunohistochemical localization of insulin like growth factor binding protein-5 in the developing rat nervous system" DEVELOPMENTAL BRAIN RESEARCH, Bd. 92, Nr. 2, 1996, Seiten 211-218, XP001153291 ISSN: 0165-3806 *
GREGORY CHRISTOPHER W ET AL: "Androgen receptor up-regulates insulin-like growth factor binding protein-5 (IGFBP-5) expression in a human prostate cancer xenograft" ENDOCRINOLOGY, Bd. 140, Nr. 5, Mai 1999 (1999-05), Seiten 2372-2381, XP002261944 ISSN: 0013-7227 *
LEE WEI-HUA ET AL: "Altered IGFBP5 gene expression in the cerebellar external germinal layer of weaver mutant mice" MOLECULAR BRAIN RESEARCH, Bd. 30, Nr. 2, 1995, Seiten 259-268, XP002261945 ISSN: 0169-328X *
PETERSSON SUSANNA ET AL: "The megencephaly mouse has disturbances in the insulin-like growth factor (IGF) system" MOLECULAR BRAIN RESEARCH, Bd. 72, Nr. 1, 8. September 1999 (1999-09-08), Seiten 80-88, XP001155888 ISSN: 0169-328X *
ROSCHIER MAARIA ET AL: "Insulin-like growth factor binding protein 5 and type-1 insulin-like growth factor receptor are differentially regulated during apoptosis in cerebellar granule cells" JOURNAL OF NEUROCHEMISTRY, Bd. 76, Nr. 1, Januar 2001 (2001-01), Seiten 11-20, XP001155887 ISSN: 0022-3042 *
STENVERS KAYE L ET AL: "Expression of insulin-like growth factor binding protein-4 and -5 mRNAs in adult rat forebrain" JOURNAL OF COMPARATIVE NEUROLOGY, Bd. 339, Nr. 1, 1994, Seiten 91-105, XP009021210 ISSN: 0021-9967 *
TANNO BARBARA ET AL: "Expression of insulin-like growth factor-binding protein 5 in neuroblastoma cells is regulated at the transcriptional level by c-Myb and B-Myb via direct and indirect mechanisms" JOURNAL OF BIOLOGICAL CHEMISTRY, Bd. 277, Nr. 26, 28. Juni 2002 (2002-06-28), Seiten 23172-23180, XP002261943 ISSN: 0021-9258 *

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AU2002357426A1 (en) 2003-05-12
DE10153335A1 (de) 2003-05-15

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