EP1663246A2 - Utilisation de la famille genique sgk pour diagnostiquer et pour traiter la cataracte et le glaucome - Google Patents

Utilisation de la famille genique sgk pour diagnostiquer et pour traiter la cataracte et le glaucome

Info

Publication number
EP1663246A2
EP1663246A2 EP04708350A EP04708350A EP1663246A2 EP 1663246 A2 EP1663246 A2 EP 1663246A2 EP 04708350 A EP04708350 A EP 04708350A EP 04708350 A EP04708350 A EP 04708350A EP 1663246 A2 EP1663246 A2 EP 1663246A2
Authority
EP
European Patent Office
Prior art keywords
hsgkl
hsgk3
gene
glaucoma
diabetic neuropathy
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04708350A
Other languages
German (de)
English (en)
Inventor
Florian Lang
Andreas Busjahn
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of EP1663246A2 publication Critical patent/EP1663246A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4355—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having oxygen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/12—Ophthalmic agents for cataracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/16—Ophthalmology
    • G01N2800/166—Cataract
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/16—Ophthalmology
    • G01N2800/168—Glaucoma

Definitions

  • the invention relates to the use of a functional inhibitor of the hsgkl or hsgk3 protein or a negative transcription regulator of the hsgkl or hsgk3 gene for the manufacture of a medicament for the therapy and / or prophylaxis of a cataract, glaucoma or diabetic neuropathy.
  • the invention relates to the use of a single- or double-stranded nucleic acid comprising the hsgkl sequence according to Acc No. NM_005627 or one of its fragments or comprising the hsgk3 sequence according to Acc. No. AF169035 or one of its fragments for diagnosing a predisposition to form cataract, glaucoma and / or diabetic neuropathy, and a kit for diagnosing a predisposition to form cataract, glaucoma and / or diabetic neuropathy, which comprises the above-mentioned nucleic acid.
  • Another object of the invention are various screening methods for the identification and characterization of therapeutically active substances from a large number of test substances for the therapy and / or prophylaxis of at least one disease selected from cataract, glaucoma and diabetic neuropathy.
  • the serum and glucocorticoid-inducible kinase hsgkl was originally cloned as a glucocorticoid-sensitive gene [Webster et al, Characterization of sgk, a novel member of the serine / threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum. Mol Cell Biol 1993; 13: 2031-2040].
  • hsgkl is under the influence of a variety of stimuli [Lang F, Cohen P. Regulation and physiological roles of serum- and glucocorticoid-induced protein kinase isoforms. Science STKE. 2001 Nov 13; 2001 (108): RE17], such as the mineralocorticoids [Chen et al, Epithelial sodium Channel regulated by aldosterone-induced protein sgk. Proc Natl Acad Sei USA 1999; 96: 2514-2519; Näray-Fejes-T ⁇ th et al, sgk is an aldosterone-induced kinase in the renal collecting duet. Effects on epithelial Na + channels.
  • sgk serum- and glucocorticoid-regulated kinase
  • the hsgkl is stimulated by the "insulin-like growth factor IGF1", by insulin and by oxidative stress via a signal cascade by phosphoinositol-3-kinase (PI3 kinase) and phosphoinositol-dependent kinase PDK1
  • PI3 kinase phosphoinositol-3-kinase
  • PDK1 phosphoinositol-dependent kinase
  • the hsgkl is a potent stimulator of the renal epithelial Na + channel [De la Rosa et al, The serum and glucocorticoid kinase sgk increases the abundance of epithelial sodium Channels in the plasma membrane of Xenopus oocytes. J Biol Chem 1999; 274: 37834-37839; Böhmer et al, The Shrinkage-activated Na + Conductance of Rat Hepatocytes and its Possible Correlation to rENaC. Cell Phys Biochem. 2000; 10: 187-194; Lang et al., Deranged transcriptional regulation of cell volume sensitive kinase hSGK in diabetic nephropathy.
  • the hsgkl and its human homologues should have considerable potential for the diagnosis of numerous diseases.
  • DE 197 08 173 AI shows in particular that the hsgkl for diagnosis in many diseases in which cell volume changes play a crucial pathophysiological role, such as hypernatremia, hyponatremia, diabetes mellitus, renal failure, hypercatabolism, hepatic encephalopathy and microbial or viral infections can be used.
  • WO 00/62781 has described that the hsgkl activates the endothelial Na + channel, which increases renal Na absorption. Since this increased renal Na absorption is associated with hypertension, it was assumed here that an increased expression of the hsgkl should lead to hypertension, a reduced expression of the hsgkl should ultimately lead to hypotension. -
  • DE 100 421 37 also describes a similar relationship between the overexpression or overactivity of the human homologues hsgk2 and hsgk3 with the overactivation of the ENaC, the resulting increased renal Na absorption and the hypertension that develops from this. Furthermore, the diagnostic potential of the kinases hsgk2 and hsgk3 regarding arterial hypertension has already been discussed.
  • WO02 / 074987 A2 the connection between the occurrence of two different polymorphisms (single nucleotide polymorphism (SNP)) of individual nucleotides in the hsgkl gene with a genetically determined predisposition to hypertension was disclosed. This is a polymorphism in intron 6 (T ⁇ C) and a polymorphism in exon 8 (C ⁇ T) in the hsgkl gene.
  • SNP single nucleotide polymorphism
  • nucleic acids containing polymorphic gene regions of the human homologues of the sgk family could be used to diagnose a predisposition for these further diseases . It was therefore an object of the invention to uncover further correlations between the function of the human homologs of the sgk family and new diseases and thus to provide new diagnostic uses for nucleic acids which contain polymorphic gene regions of the human homologues of the sgk family.
  • the glucose transporter Glutl provides, among other things, glucose uptake into different cells of the eye [Busik et al., Glucose-induced activation of glucose uptake in cells from the inner and outer blood-retinal barrier. luvest Ophthalmol Vis Be. 2002; 43: 2356-63; Takata K, Kasahara T, Kasahara M, Ezaki O, Hirano H. Ultracytochemical localization of the erythrocyte / HepG2- type glucose transporter (GLUT1) in the ciliary body and iris of the rat eye.
  • Glutl for the development of cataracts [Gong et al., Development of cataractous macrophthalmia in mice expressing an active MEK1 in the lens. Invest Ophthalmol Vis Sei. 2001; 42: 539-48].
  • Glutl overexpression requires the formation and deposition of connective tissue proteins £ Ayo et al., Increased extracellular matrix synthesis and mRNA in mesangial cells grown in high-glucose medium. At the J Physiol.
  • the above-mentioned disorders would occur in all conditions in which the hsgkl has increased activity, that is to say in excess of all the above-mentioned hormones.
  • Certain polymorphisms of the hsgkl gene that correlate with increased blood pressure [Busjahn et al., Serum- and glucocorticoid-regulated kinase (SGK1) gene and blood pressure. Hypertension 40 (3): 256-260, 2002], could at the same time increase Lead to occurrence of cataract and glaucoma.
  • the same modifications of the gene should also correlate with premature cataract and / or glaucoma.
  • the present findings reveal a completely new mechanism in the regulation of the glucose transporter Glutl.
  • An increased activity of the hsgkl should therefore lead to an increased uptake of glucose in the cells.
  • the transcription of the hsgkl is carried out by serum [Webster et al. 1993], by glucocorticoids [Brenan & Corder 2000, Webster et al. 1993], by mineralocorticoids [Chen et al. 1999, Naray-Fejes-Toth et al. 1999, Shigaev et al.
  • FSH Follicle-Stimulating hormone
  • PKA protein kinase B
  • Sgk serum and glucocorticoid-induced kinase
  • the hsgkl transcription is increased by cell shrinkage, as the Waldegger et al. from 1997 shows.
  • An increased glucose concentration as occurs in diabetes mellitus, stimulates the expression of hsgkl by cell shrinkage and / or by increased formation of TGF-ß [Lang et al. 2000].
  • the expressed hsgkl is activated by the "insulin like growth factor" IGF1, by insulin and by oxidative stress [Kobayashi & Cohen 1999, Park et al. 1999, Kobayashi et al. 1999].
  • the increased expression of the hsgkl increases the activity of the glucose transporter Glut-1. This means that more glucose is absorbed into the cells and the water that follows through osmosis causes cell swelling. In this way, there is increased water retention in the cornea and lens, which leads to cataracts by reducing transparency [Gong et al. 2001]. Glaucoma could also develop in a similar way and additionally through the incorporation of connective tissue [Fingert et al. 2001].
  • hsgkl plays a role in this mechanism and is therefore suitable as a target protein for the diagnosis and therapy of glaucoma.
  • One object of the invention thus relates to the use of a functional inhibitor of the hsgkl or hsgk3 protein or a negative transcription regulator of the hsgkl or hsgk3 gene to reduce cell swelling.
  • Another object of the invention relates to the use of a 'functional inhibitor of the hsgkl or hsgk3 protein or a negative transcription regulator of the hsgkl or hsgk3 gene for the production of a medicament for the therapy and / or prophylaxis of a cataract, a glaucoma or the diabetic neuropathy.
  • This functional inhibitor of the hsgkl protein or the hsgk3 protein can be a chemical substance of any kind which inhibits the normal physiological activity of the hsgkl protein or the hsgk3 protein.
  • the functional inhibitor of the hsgkl or hsgk3 protein is preferably a low-molecular chemical substance (a "small molecule") or a protein or peptide.
  • the functional inhibitor of the hsgkl protein or the hsgk3 protein can in particular be an antagonist of these enzymes, which blocks the substrate binding site of the hsgkl protein or the hsgk3 protein, but at the same time, no catalytic conversion is accessible through the hsgkl or hsgk3.
  • suitable antagonists are preferably those molecules which have structural similarity to the natural substrate of the hsgkl protein or the hsgk3 protein, that is to say in particular a structural similarity to the phosphorylatable amino acids serine and threonine.
  • Staurosporin and chele-rythrin are two known functional inhibitors of hsgkl.
  • either staurosporin or chelerythrine is therefore used as the functional inhibitor of the hsgkl or the hsgk3 for the therapy and / or prophylaxis of at least one of the diseases cataract, glaucoma or diabetic neuropathy.
  • a negative transcription regulator of the hsgkl gene or the hsgk3 gene is defined as a substance which activates the expression of the hsgkl gene or the hsgk3 gene at the transcription level.
  • the medicament according to the invention for the therapy and / or prophylaxis of a cataract, glaucoma or diabetic neuropathy can, in addition to the actual active substance, the functional inhibitor or the negative transcription regulator of the hsgkl or the hsgk3, additionally stabilizers and / or carrier substances, such as starch, lactose , Stearic acid, fats, waxes, alcohols or other additives such as preservatives, colors or flavors.
  • the drug can be administered in any way, in particular orally, in the form of tablets, granules, capsules or as a solution.
  • Other particularly suitable dosage forms relate to direct applications (e.g. on the skin or eye) in the form of ointments, tinctures, sprays or any type of injection (e.g. subcutaneous, intravenous) or infusion.
  • Another object of the invention is the use of a single or double-stranded nucleic acid comprising the hsgkl sequence according to Acc No. NM_005627 or one of its fragments to diagnose a predisposition to develop cataracts, glaucoma and / or diabetic neuropathy.
  • the fragment of hsgkl, which the single or double-stranded nucleic acid can comprise is at least 10 nucleotides / base pairs long, preferably at least 15 nucleotides / base pairs long and in particular at least 20 nucleotides / base pairs long.
  • the single- or double-stranded nucleic acid here preferably comprises at least one polymorphic nucleotide of the hsgkl gene, in particular a “single nucleotide polymorphism (SNP)” of the hsgkl gene.
  • the single- or double-stranded nucleic acid comprises at least one of the following SNPs of the hsgkl gene: a G insertion at position 732/733 in intron 2 of the hsgkl gene,
  • the above SNPs of the hsgkl gene in the patient's genomic DNA or cDNA can preferably be detected using the abovementioned single or double-stranded nucleic acids by the following methods: by direct sequencing of the genomic DNA or cDNA with the above nucleic acids, by specific hybridization the genomic DNA or cDNA with the above nucleic acids, by a PCR oligonucleotide elongation assay or by a ligation assay.
  • the patient's genomic DNA or cDNA is preferably isolated from a patient's body sample, in particular from saliva, blood, tissue or cells.
  • the activity of the expressed hsgkl gene depends on the version of this polymorphism in the patient's hsgkl gene and that nucleic acids which contain at least one of these polymorphisms are therefore particularly good for diagnosing a predisposition to the development of cataracts, glaucoma and / or diabetic neuropathy.
  • the invention further relates to the use of a single- or double-stranded nucleic acid comprising the hsgk3 sequence according to Acc No. AF169035 or one of its fragments to diagnose a predisposition to develop cataract, glaucoma and / or diabetic neuropathy.
  • the fragment of hsgk3, which the single or double-stranded nucleic acid can comprise, is at least 10 nucleotides / base pairs long, preferably at least 15 nucleotides / base pairs long and in particular at least 20 nucleotides / base pairs long.
  • the single- or double-stranded nucleic acid preferably comprises at least one polymorphic nucleotide of the hsgk3 gene, in particular a “single nucleotide polymorphism (SNP)” of the hsgk3 gene.
  • SNP single nucleotide polymorphism
  • certain antibodies which are directed against substrates of the human homologues of the sgk family, in particular against substrates of the hsgkl and hsgk3 are also suitable for diagnosing a predisposition to the formation of at least one of the diseases cataract, Glaucoma, diabetic neuropathy.
  • diagnostic antibodies are preferably directed against such an epitope of the human homologues of the sgk family (in particular the hsgkl and the hsgk3) which contains the phosphorylation site of the substrate either in phosphorylated form or in nonphosphorylated form.
  • an overexpression of the hsgkl protein that occurs due to the individual genetic makeup of the hsgkl gene could lead to an increased implementation of
  • Hsgk substrates i.e. to an increased enzymatic phosphorylation of the
  • Glutl glucose transporter
  • the ubiquitin protein ligase Nedd4-2 (Acc No. BAA23711) is used as the substrate of the human homologue of the sgk family.
  • This ubiquitin protein ligase is a protein which is specifically phosphorylated by the human homologues of the sgk family [Debonneville et al., Phosphorylation of Nedd4-2 by Sgkl regulates epithelial Na (+) channel cell surface expression. EMBO J., 2001; 20: 7052-7059; Snyder et al., Serum and glucocorticoid-regulated kinase modulates Nedd4-2-mediated inhibition of the epithelial Na (+) channel. J. Biol.
  • Phosphorylation sites for the hsgkl have the consensus sequence (R X R X X S / T), where R is arginine, S is serine, T is threonine and X is any amino acid.
  • R is arginine
  • S is serine
  • T is threonine
  • X is any amino acid.
  • Nedd4-2 2 Acc No. BAA23711
  • the abovementioned antibodies for diagnosing a predisposition to develop at least one of the diseases cataract, glaucoma, diabetic neuropathy are therefore preferably directed against the substrate Nedd4-2 and particularly preferably against directed a protein region of Nedd4-2 with the sequence of the potential phosphorylation site for the hsgkl, the consensus sequence (RXRXXS / T).
  • these antibodies are directed against those Nedd4-2 protein regions which comprise at least one of the two potential phosphorylation sites serine at amino acid position 382 and / or serine at amino acid position 468.
  • kits for the diagnosis of one of the diseases cataract, glaucoma and diabetic neuropathy comprising at least one of the following components: " - Antibodies which are directed against hsgkl or hsgk3, single or double-stranded nucleic acids which with the hsgkl - Gene according to Acc No. NM_005627 or with the hsgk3 gene according to Acc No. AF169035 can hybridize under stringent conditions; in particular those single- or double-stranded nucleic acids which comprise polymorphic nucleotides, in particular “SNPs” of the hsgkl gene or the hsgk3 gene,
  • Antibodies directed against a substrate of a human homologue of the sgk family preferably antibodies directed against the phosphorylation site of this substrate in the phosphorylated or non-phosphorylated form; especially antibodies directed against the phosphorylation site of Nedd4 or Nedd4-2 in the phosphorylated or non-phosphorylated form.
  • the invention further relates to a screening method for identifying and characterizing therapeutically active substances from a large number of test substances for therapy and / or prophylaxis of at least one disease selected from the group consisting of cataract, glaucoma and diabetic neuropathy, comprising the following steps: a) heterologous coexpression of i) the glucose transporter Glutl and ii) the hsgkl and / or the hsgk3 in cells, b) cultivation of at least one cell portion Ai to A in the presence of at least one test substance, wherein the at least one test substance differs depending on the index 1 to X of the Zeil portion and Cultivation of a control cell part B in the absence of any test substance, c) determination of the activity of the glucose transporter Glutl in the Zeil fractions Ai to Ax compared to the activity of the glucose transporter Glutl in the control cell part B.
  • suitable cells preferably mammalian cells or cell lines, in particular human cells or cell lines with suitable expression vectors, contain suitable expression cassettes for the expression of Glutl and of hsgkl and / or hsgk3 according to standard methods, such as electroporation, CaPO4 precipitation, lipofection or the like, transfected.
  • the expression cassettes contain the genomic DNA or the cDNA of the target gene in question (Glutl, hsgkl, hsgk3) under the control of suitable promoters which are active in the cell type in question and can express the target gene in a suitable amount.
  • the expression vectors can also contain selection markers.
  • the transfected cells are then cultivated under conditions which allow expression of the target genes i) and ii).
  • step b) the transfected cells from a) are divided into different Zeil parts (aliquots) Ai to Ax and a control cell part B.
  • the Zeil parts Ai to Ax are each in the presence of at least one test Cultivated substance.
  • the test substance (s) added to the Zeil fractions Ai to Ax differ from one another (depending on the index 1 to X of the respective Zeil fractions A ⁇ to Ax).
  • the control cell portion B is cultivated in the absence of any test substance.
  • step c) the activity of the glucose transporter Glutl in the Zeil fractions Ai to Ax is quantitatively determined in comparison to the activity of the glucose transporter Glutl in the control cell fraction B.
  • a test substance that can functionally inhibit the hsgkl or the hsgk3 should have been added to the Zeil portions Ai to Ax, in which a significantly lower value of the Glutl activity is measured compared to the control Zeil portion B. or reduces their expression.
  • Such a substance could be suitable for the therapy of at least one of the diseases cataract, glaucoma or diabetic neuropathy.
  • Fig. 1 The invention is illustrated by the following Fig. 1.
  • the ordinate A in Fig. 1 shows the uptake of 2-deoxyglucose (in pmol 1/10 min / oocyte) (arithmetic mean ⁇ SEM).
  • Xenopus laevis oocytes were injected with cRNA from Glut-1 with or without cRNA from SGK1, SGK2, SGK3 or protein kinase B (PKB) (see Example 1).
  • PKA protein kinase B
  • Fig. 1 shows the increased uptake of 2-deoxyglucose in those oocytes which, in addition to glut 1, express the hsgkl or the hsgk3 in comparison to those oocytes which express glut 1 alone. This shows that the function of the hsgkl and the hsgk3 efficiently stimulate the activity of the glucose transporter Glutl. A similar effect is not found for those oocytes which express the hsgk2 or PKBmut instead of hsgkl or hsgk3.
  • the invention is illustrated by the following example.
  • Example 1 Expression in Xenopus laevis oocytes and two-electrode voltage clamp cRNA of normal SGKl [Waldegger S, Barth P, Raber G, Lang F: Cloning and characterization of a putative human serine / threonine protein kinase transcriptionally modified during anisotonic and isotonic alterations of cell volume.
  • Xenopus laevis ovaries The dissection of Xenopus laevis ovaries and the collection and treatment of oocytes have already been described in detail [Wagner CA, Friedrich B, Setiawan I, Lang F, Bröer S: The use of Xenopus laevis ooeytes for the functional characterization of heterologously expressed membrane proteins. Cell Physiol Biochem 2000; 10: 1-12].
  • the oocytes were injected with 5 ng of human glutl, 7.5 ng of human SGKl and / or with 5 ng of Xenopus Nedd4-2. ControUocytes were injected with water. The uptake of radioactively labeled glucose was measured at room temperature 2 days after injection of the respective cRNAs.
  • the control bath solution contained 96 mM NaCl, 2 mM KC1, 1.8 mM CaCl 2 , 1 mM MgCl 2 and 5 mM HEPES, pH 7.4. All substances were used in the specified concentrations. The final solutions were nitrated to pH 7.4 with HC1 or NaOH.
  • n is the number of oocytes examined. All experiments were carried out in at least three different groups of oocytes. The results were tested for significant differences using the Student t-test. Only results with P ⁇ 0.05 were considered to be statistically significant.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Molecular Biology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Ophthalmology & Optometry (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Hematology (AREA)
  • General Physics & Mathematics (AREA)
  • Neurosurgery (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Pathology (AREA)
  • Neurology (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La présente invention concerne l'utilisation d'un inhibiteur fonctionnel de la protéine hsgk1 ou hsgk3 ou d'un régulateur de transcription négatif du gène hsgk1 ou hsgk3, pour la préparation d'un produit pharmaceutique qui sert à traiter et/ou à prévenir la cataracte, le glaucome ou la neuropathie diabétique. Un autre aspect de l'invention concerne l'utilisation d'un acide nucléique double ou simple brin comprenant la séquence hsgk1 selon Acc No. NM_005627 ou un fragment de celui-ci, ou comprenant la séquence hsgk3 selon Acc. No. AF169035 ou un fragment de celui-ci, pour diagnostiquer une prédisposition à l'apparition d'une cataracte, d'un glaucome et/ou d'une neuropathie diabétique, ainsi qu'un kit pour diagnostiquer une prédisposition à l'apparition d'une cataracte, d'un glaucome et/ou d'une neuropathie diabétique, ledit kit comprenant l'acide nucléique mentionné ci-dessus. L'invention a également pour objet différents procédés de criblage pour identifier et caractériser des substances à action thérapeutique, à partir d'une pluralité de substances d'essai, afin de traiter et/ou de prévenir au moins l'une des maladies que son la cataracte, le glaucome et la neuropathie diabétique.
EP04708350A 2003-02-07 2004-02-05 Utilisation de la famille genique sgk pour diagnostiquer et pour traiter la cataracte et le glaucome Withdrawn EP1663246A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10305212A DE10305212A1 (de) 2003-02-07 2003-02-07 Verwendung der sgk-Genfamilie zur Diagnose und zur Therapie von Katarakt und Glaukom
PCT/EP2004/001048 WO2004069258A2 (fr) 2003-02-07 2004-02-05 Utilisation de la famille genique sgk pour diagnostiquer et pour traiter la cataracte et le glaucome

Publications (1)

Publication Number Publication Date
EP1663246A2 true EP1663246A2 (fr) 2006-06-07

Family

ID=32730900

Family Applications (1)

Application Number Title Priority Date Filing Date
EP04708350A Withdrawn EP1663246A2 (fr) 2003-02-07 2004-02-05 Utilisation de la famille genique sgk pour diagnostiquer et pour traiter la cataracte et le glaucome

Country Status (13)

Country Link
EP (1) EP1663246A2 (fr)
JP (1) JP2006519189A (fr)
KR (1) KR20050114214A (fr)
CN (1) CN1771039A (fr)
AU (1) AU2004210416A1 (fr)
BR (1) BRPI0407300A (fr)
CA (1) CA2514703A1 (fr)
DE (1) DE10305212A1 (fr)
MX (1) MXPA05008394A (fr)
PL (1) PL378399A1 (fr)
RU (1) RU2005127808A (fr)
WO (1) WO2004069258A2 (fr)
ZA (1) ZA200506280B (fr)

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1964705A (zh) * 2004-03-11 2007-05-16 默克专利有限公司 干扰纤维化的方法
RU2006135654A (ru) * 2004-03-11 2008-09-10 Мерк Патент ГмбХ (DE) Способы модуляции глутаматных рецепторов для лечения нейропсихиатрических расстройств, включающие применение модуляторов сывороточных и индуцируемых глюкокортикоидами киназ
DE102004059781A1 (de) * 2004-12-10 2006-06-22 Sanofi-Aventis Deutschland Gmbh Verwendung der Serum-/Glucocorticoid regulierten Kinase
WO2007002670A2 (fr) * 2005-06-28 2007-01-04 Bausch & Lomb Incorporated Procede pour faire baisser la pression intraoculaire
JPWO2007037560A1 (ja) * 2005-09-30 2009-04-16 リンク・ジェノミクス株式会社 Ｓｇｋ２遺伝子の治療的又は診断的用途
JP5249774B2 (ja) * 2005-11-22 2013-07-31 マギル ユニバーシティ 眼内圧調節初期遺伝子およびその使用
WO2008079980A1 (fr) * 2006-12-22 2008-07-03 Alcon Research, Ltd. Inhibiteurs de la protéine kinase c-delta pour le traitement du glaucome
DE102008029072A1 (de) * 2008-06-10 2009-12-17 Lang, Florian, Prof. Dr.med. Sgk3 als therapeutisches und diagnostisches Target für Alterserkrankungen
BR112013018374A2 (pt) * 2011-01-25 2016-10-11 Monell Chemical Senses Centre composições e métodos para fornecer ou modular o sabor doce e métodos para rastrear os mesmos
KR102357260B1 (ko) * 2020-12-10 2022-02-08 주식회사 레피겐엠디 마이크로rna를 이용한 당뇨병성 신경병증의 예측 및 진단 방법 및 이를 위한 키트
CN119303113A (zh) * 2024-09-03 2025-01-14 河北大学附属医院 Nedd4l基因在制备治疗血管内皮细胞增生性相关疾病药物中的应用

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996006863A1 (fr) * 1994-08-30 1996-03-07 University Of Dundee Agents utilises pour induire une apoptose et leurs applications therapeutiques
AU6263996A (en) * 1995-06-07 1996-12-30 Ligand Pharmaceuticals Incorporated Method for screening for receptor agonists and antagonists
US5925523A (en) * 1996-08-23 1999-07-20 President & Fellows Of Harvard College Intraction trap assay, reagents and uses thereof
DE19708173A1 (de) * 1997-02-28 1998-09-03 Dade Behring Marburg Gmbh Zellvolumenregulierte humane Kinase h-sgk
WO1999059559A1 (fr) * 1998-05-15 1999-11-25 Joslin Diabetes Center Regulation independante de transport de glucose basal et stimule par insuline
DE69937159T2 (de) * 1998-12-14 2008-06-26 University Of Dundee, Dundee Verfahren zur Aktivierung von SGK durch Phosphorylierung.
US6399655B1 (en) * 1998-12-22 2002-06-04 Johns Hopkins University, School Of Medicine Method for the prophylactic treatment of cataracts
DE19917990A1 (de) * 1999-04-20 2000-11-02 Florian Lang Arzneimittel enthaltend Hemmstoffe der zellvolumenregulierten humanen Kinase h-sgk
WO2001004145A2 (fr) * 1999-07-14 2001-01-18 University Of Lausanne Famille de polypeptides glutx et acides nucleiques les codant
US6416759B1 (en) * 1999-09-30 2002-07-09 The Regents Of The University Of California Antiproliferative Sgk reagents and methods
US20030236246A1 (en) * 2002-04-30 2003-12-25 Brazzell Romulus Kimbro Method for decreasing capillary permeability in the retina
DE10225844A1 (de) * 2002-06-04 2003-12-18 Lang Florian sgk und nedd als diagnostische und therapeutische targets

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2004069258A2 *

Also Published As

Publication number Publication date
CN1771039A (zh) 2006-05-10
BRPI0407300A (pt) 2006-02-07
AU2004210416A1 (en) 2004-08-19
PL378399A1 (pl) 2006-04-03
RU2005127808A (ru) 2006-05-27
CA2514703A1 (fr) 2004-08-19
JP2006519189A (ja) 2006-08-24
DE10305212A1 (de) 2004-08-19
WO2004069258A2 (fr) 2004-08-19
KR20050114214A (ko) 2005-12-05
MXPA05008394A (es) 2005-10-05
WO2004069258A3 (fr) 2005-02-24
ZA200506280B (en) 2006-05-31

Similar Documents

Publication Publication Date Title
DE60217152T2 (de) Knochen-morphogene proteine (bmp), bmp-rezeptoren und bmp-bindungsproteine und ihre verwendung bei der diagnose und behandlung des glaukoms
DE19917990A1 (de) Arzneimittel enthaltend Hemmstoffe der zellvolumenregulierten humanen Kinase h-sgk
EP1663246A2 (fr) Utilisation de la famille genique sgk pour diagnostiquer et pour traiter la cataracte et le glaucome
EP1523571B1 (fr) Sgk et nedd utilises comme cibles diagnostiques et therapeutiques
EP1313476B1 (fr) Sgk3 en tant que cible diagnostique et therapeutique
Etheredge et al. Functional compensation by other voltage-gated Ca2+ channels in mouse basal forebrain neurons with CaV2. 1 mutations
DE10305213A1 (de) Verwendung eines neuen Polymorphismus im hsgk1-Gen zur Diagnose der Hypertonie und Verwendung der sgk-Genfamilie zur Diagnose und Therapie des Long-Q/T-Syndroms
Tao et al. Expression of urocortin 2 and its inhibitory effects on intracellular ca2+ via L-type voltage-gated calcium channels in rat pheochromocytoma (PC12) cells
WO2004005540A2 (fr) Utilisations de substances qui se lient a ngal pour le diagnostic et le traitement de maladies cancereuses
EP1386615B1 (fr) Antagonistes du récepteur EG-VEGF/prokineticin 2
DE102004059781A1 (de) Verwendung der Serum-/Glucocorticoid regulierten Kinase
Kroumpouzou et al. Common pathways for primary hypertrophic and dilated cardiomyopathy
DE10234901A1 (de) Verwendung von am Mrp4 bindenden Substanzen zur Diagnose und Behandlung von Krebserkrankungen
EP0816848A1 (fr) Méthode pour le criblage comparatif des substances avec l'activité pharmacologique
DE69813677T2 (de) Humäne Serumglucocorticoidregulierte Kinase, ein Ziel für chronischer Nierenerkrankung und diabetische Nephropathie
DE10120834A1 (de) Neue Schmerzmittel, die Inhibitoren von TRP-Kanälen sind
DE19856301C1 (de) Regulatorisches Protein pKe#83 aus humanen Keratinozyten
DE19811326C1 (de) Verfahren zur Herstellung von Mitteln zur Behandlung von Tumorerkrankungen und zur Immunsuppression
Lee et al. Changes in inward rectifier K+ channels in hepatic stellate cells during primary culture
Deng et al. Delayed development of dendritic spines in Fxr2 knockout mouse
DE10360956A1 (de) System und Verfahren zum Nachweis von diabetischer Nephropatie oder einer Prädisposition dafür
EP1877803A2 (fr) Procede pour caracteriser une activite de transactivation et de transrepression de ligands du recepteur glucocorticoide dans des cellules immunitaires primaires
EP1517146A2 (fr) Diagnostic et traitement de cancer par l'intermédiaire des (ant)agonistes de Flamingo.
EP1287030A1 (fr) Substance regulee par l'insuline (irs-2) induite par la pioglitazone, dosage et utilisation de cette derniere
DE102006021267A1 (de) Verfahren zur Charakterisierung der Transaktivierungs- und Transrespressionsaktivität von Glukokortikoid-rezeptor-Liganden in primären Immunzellen

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20050829

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: LT LV

17Q First examination report despatched

Effective date: 20061130

17Q First examination report despatched

Effective date: 20061130

17Q First examination report despatched

Effective date: 20061130

GRAJ Information related to disapproval of communication of intention to grant by the applicant or resumption of examination proceedings by the epo deleted

Free format text: ORIGINAL CODE: EPIDOSDIGR1

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

RIC1 Information provided on ipc code assigned before grant

Ipc: G01N 33/50 20060101AFI20090407BHEP

RTI1 Title (correction)

Free format text: SREENING METHOD BASED ON THE SGK GENE FAMILY AND GLUT1

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

RTI1 Title (correction)

Free format text: SCREENING METHOD BASED ON THE SGK GENE FAMILY AND GLUT1

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20090901