WO1999032620A1 - Insulin-like growth factor binding protein fragments and the utilization thereof - Google Patents

Insulin-like growth factor binding protein fragments and the utilization thereof Download PDF

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Publication number
WO1999032620A1
WO1999032620A1 PCT/EP1998/008405 EP9808405W WO9932620A1 WO 1999032620 A1 WO1999032620 A1 WO 1999032620A1 EP 9808405 W EP9808405 W EP 9808405W WO 9932620 A1 WO9932620 A1 WO 9932620A1
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Prior art keywords
peptides
acid sequence
amino acid
peptide
igfbp
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PCT/EP1998/008405
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German (de)
French (fr)
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WO1999032620A9 (en
Inventor
Wolf-Georg Forssmann
Ludger STÄNDKER
Maik Obendorf
Lothar Kling
Hans-Georg Opitz
Hossein Mostafavi
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Forssmann Wolf Georg
Staendker Ludger
Maik Obendorf
Lothar Kling
Opitz Hans Georg
Hossein Mostafavi
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Application filed by Forssmann Wolf Georg, Staendker Ludger, Maik Obendorf, Lothar Kling, Opitz Hans Georg, Hossein Mostafavi filed Critical Forssmann Wolf Georg
Priority to CA002315974A priority Critical patent/CA2315974A1/en
Priority to JP2000525539A priority patent/JP2002508931A/en
Priority to EP98965865A priority patent/EP1042476A1/en
Publication of WO1999032620A1 publication Critical patent/WO1999032620A1/en
Publication of WO1999032620A9 publication Critical patent/WO1999032620A9/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4743Insulin-like growth factor binding protein
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00Drugs for skeletal disorders
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00Drugs for skeletal disorders
    • A61P19/08Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00Drugs for disorders of the muscular or neuromuscular system
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00Drugs for disorders of the muscular or neuromuscular system
    • A61P21/02Muscle relaxants, e.g. for tetanus or cramps
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides

Definitions

  • Insulin-like growth factor binding protein fragments and their use
  • the present invention relates to peptides with cell proliferative and cell protective properties, complexes of the peptides with human insulin-like growth factor I and II (IGF) and the associated nucleic acids, antisense nucleotides, antibodies and inhibitors.
  • IGF insulin-like growth factor I and II
  • Insulin-like growth factor binding proteins are described, inter alia, by Shimasaki, S. and Ling, N. in Prog. Growth Factor Res. 3 (1991) 243-266 and Zapf, J. in Eur. J. Endocrinol. 132 (1995) 645-654.
  • the present invention relates to peptides whose amino acid sequence corresponds in part to the amino acid sequence of insulin-like growth factor binding proteins, and to cyclic, glycosylated, phosphorylated, acetylated, acidated and / or sulfated derivatives thereof. These peptides according to the invention are referred to as IGFBP or IBP.
  • Preferred peptides are those that occur naturally and can be isolated, for example, from hemofiltrate.
  • the peptides preferably have a length of 61 to 115 amino acids.
  • Peptides which have sequences which correspond to N- or C-terminal sequences of insulin-like growth factor binding proteins are particularly preferred.
  • Preferred peptides are peptides with the amino acid sequence of the formula
  • R NH 2 , an amino acid or a peptide with an amino acid sequence comprising up to 41 amino acids
  • X a peptide with an amino acid sequence comprising 24 to 31 amino acids
  • X represents a peptide with an amino acid sequence comprising 9 amino acids
  • X 3 represents a peptide with an amino acid sequence comprising 10 amino acids
  • X 4 represents a peptide with an amino acid sequence comprising 18 to 24 amino acids
  • the peptide has cell proliferative and cell protective properties.
  • the peptides according to the invention can have disulfide bridges, so that they have the general formula
  • the peptides have a glycine at one or more of the following positions in the amino acid sequence.
  • X ] at position 8 is L or V and / or X, at position 11 is L or I and / or X 2 at position 1 D or.
  • N is and / or X 2 at position 9 is K or R and / or X 3 at position 3 is S or A and / or at position 8 is R or A.
  • R is selected from
  • APSEEDHSILWDAISTYDGSKALHVTNIKKWKEP SEQ ID NO: 1
  • GGKHHLGLEEPKKLRPPPARTP SEQ ID NO: 2
  • GKGGKHHLGLEEPKKLRPPPARTP SEQ ID NO: 3
  • NKNGFYHSR SEQ ID NO 14
  • DHRGFYRKR (SEQ ID NO 19) and / or
  • RSSQGQRRGP (SEQ ID NO 25) and / or
  • YP NGKRIPGSPEIRGDPN (SEQ ID NO 26), NPNTGKLIQGAPTIRGDPE (SEQ ID NO 27), DKYGQPLPGYTTKGKEDVH (SEQ ID NO 28), DRKTGVKLPGGLEPKGELD (SEQ ID NO 29), DKYYGMGGGLGQD and or
  • R 2 selected from
  • Preferred peptides have the following sequences IGFBP-1
  • the peptides according to the invention can be obtained by purification from human blood filtrate or urine, by solid phase peptide synthesis or by expression in recombinant microorganisms.
  • the invention furthermore relates to the complexes of the peptides according to the invention with human insulin-like growth factor-I and / or human insulin-like growth factor-II and their physiologically active fragments and / or derivatives, in particular amidated, acetylated, sulfated, phosphorylated and / or glycosylated derivatives.
  • the invention furthermore relates to nucleic acids which code for the peptides according to the invention, antisense nucleotides which bind under stringent conditions to a nucleic acid which codes for the peptide according to the invention, antibodies which bind to the peptides according to the invention, inhibitors which inhibit the biological activity of the insulin -like growth factor binding proteins, inhibitors that inhibit the expression of insulin-like growth factor binding proteins.
  • the peptides, complexes, nucleic acids, antisense nucleotides, antibodies and inhibitors according to the invention are particularly suitable for the manufacture of a medicament for the treatment of over- or under-expression of insulin-like growth factor binding proteins, for the treatment of muscle loss, osteoporosis, diabetes, amyloidal lateral sclerosis, peripheral - Central and central neuropathies, inflammatory processes, disturbed inflammatory reactions, tumor disorders, inflammatory and neoplastic diseases, growth disorders, musculoskeletal disorders, diseases of the bone apparatus and / or for wound and bone healing.
  • IGFBP IGF-I or IGF-II are particularly suitable for the treatment of bone diseases.
  • the peptides according to the invention and the complexes of the peptides with insulin-like growth factor have a cell proliferative activity.
  • the peptides according to the invention regulate the release of IGF-I and IGF-II from the complexes at their site of action.
  • the co-administration of the peptides according to the invention with IGF-I or IGF-II extends the biological half-life and thus the availability of the latter.
  • the hypoglycemia induced by injection of free IGF-I or IGF-II is prevented by the co-administration of the peptides according to the invention.
  • the peptides according to the invention furthermore have a growth-promoting effect on bone cells and lead to an enhancement or modulation of the action of growth hormones.
  • the peptides, complexes, nucleic acids, antisense nucleotides, antibodies and inhibitors according to the invention are contained in a pharmaceutical in a pharmaceutical dosage form. They are suitable for oral, intravenous, intramuscular, intracutaneous, intrathecal use or as an aerosol for transpulmonary application.
  • the amount of peptide to be administered is 1 ⁇ g to 1 g per administration unit per day.
  • nucleic acids and / or antisense nucleotides according to the invention are also suitable for the manufacture of a medicament for the treatment of somatic or non-somatic genetic diseases.
  • the diagnostic agent according to the invention contains the peptides, complexes, nucleic acids, antisense nucleotides, antibodies and / or inhibitors according to the invention.
  • the diagnostic agent preferably contains poly- or monoclonal antibodies to the peptide according to the invention, it being possible for the antibodies to be labeled with fluorescence or radioactive in order to be able to be used in the known ELISA or RIA.
  • the diagnostic agent can also contain nucleic acids, which are used in modified or labeled form in tests known to those skilled in the art, such as PCR or finger printing.
  • the diagnostic agents according to the invention are particularly suitable for the diagnosis of functional disorders of the bones, the muscles, the nervous system, the lymph organs, the gastric Intestinal tract, immune system, diabetes, inflammatory and neoplastic processes as well as a marker for cancer.
  • the simultaneous appearance of several fragments of BP-4 or BP-5 in the plasma, in particular the N- and C-termmal domains, is suitable as a marker for diseases of the bone metabolism.
  • the corresponding peptides can either be detected by mass spectroscopy or, preferably by an immune reaction with the corresponding antibodies.
  • FIG. 1 shows an alignment of the consensus sequences of C-terminal fragments of the insulin-like growth factor proteins.
  • FIG. 2 shows the schematic structure of the insulin-like growth factor proteins with the cysteine-rich N- and C-terminal domains.
  • FIG. 3 shows the schematic structure of the insulin-like growth factor proteins and the sequence of the biologically active fragments isolated from hemofiltrate.
  • FIG. 4 shows the isolation of the osteoanabolic factor IGFBP-4-11 from human hemofiltrate (see example 3).
  • Figure 5 shows the sequence and sulfur bridge analysis of the osteoanabolic factor IGFBP-4- 11.
  • the cysteines 153-183, 194-205 and 207-228 are bridged.
  • Figure 6 shows the biological effect of the osteoanabolic factor IGFBP-4-11. After incubation of serum-free primary rat osteoblasts for (A) 24 hours, (B) 48 hours and (C) 72 hours with IGFBP-4-11, the proliferation-promoting effect of IGFBP-4-11 can be seen. An increase in the rate of DNA synthesis is found in a dose-dependent manner, measured as the incorporation of bromodeoxyuridine (BrdU).
  • Figure 7 shows the specific binding to osteoblasts and the possible receptor for the osteoanabolic factor IGFBP-4-11 (designated as IGFBP-4 136 "237 ).
  • A Radioactively labeled IGFBP-4-11 shows an unlabelled by increasing amounts IGFBP-4-11 displaceable specific binding to primary osteoblast cells
  • B Radioactively labeled IGFBP-4-11 could, after binding to osteoblasts, be chemically cross-linked with its putative receptor molecule and then by gel electrophoresis and subsequent authorization. diography can be demonstrated.
  • the ligand-receptor complex has a molecular weight of approximately 120 kDa. The formation of the complex is promoted by saponin, a membrane pore former. The complex formation is prevented by adding an excess of unlabelled IGFBP-4-11 to the incubation batch.
  • the peptides according to the invention can be obtained from a human hemofiltrate by a purification process.
  • This patented process (Forssmann, W.-G. (1988), publication DE 36 33 707 AI), which was developed for the extraction of proteins from hemofiltrate, was also used in a modified form to purify the peptide complex.
  • H. hemofiltrate accumulates in large quantities in the ultrafiltration of the blood of kidney patients. 800 to 1,000 1 hemofiltrate are adjusted to a pH of 2.1 with HCl and diluted with water to a conductivity of 5.5 mS / cm and applied to a strong cation exchanger at a flow rate of 3 1 / min.
  • Buffer A Hemofiltrate pH 2.1, conductivity 5.5 mS / cm
  • Buffer B 0.5 M ammonium acetate
  • ammonium acetate eluates of the batch extraction are combined in amounts of 5,000 to 10,000 1 hemofiltrate peptide.
  • the peptide extract is applied to the preparative cation exchanger with the addition of demineralized water with a conductivity of 5.5 mS / cm.
  • the column is rinsed with 0.01 M HCl until the conductivity is below 1 mS / cm.
  • the elution is carried out in several stages using the buffers specified below
  • Eluates 1-7 are referred to as pH pool I-VII. They are collected separately and then rinsed with demineralized water. The elution takes place until a new baseline is reached. never, with elution volumes of 10 to 25 1 being achieved for the individual pH pools I to VII.
  • the individual pH pools are separated for fractionation and simultaneous desalination using a reverse phase chromatography
  • Pillar FineLine 100 (Pharmacia, Freiburg)
  • Buffer B 80% acetonitrile in 10 mM HCl
  • the column is rinsed with buffer A. Fractions of 200 ml are collected during the elution. Aliquots of the fractions are tested in the bioassay. The fractions are freeze-dried and stored at -20EC.
  • the bioactive fractions 11 and 12 from pH pool V in the assay were separated using a semi-preparative reverse-phase column. Fractions 21 to 25 contained the substance according to the invention.
  • Buffer B 0.1% TFA, 80% acetonitrile
  • Buffer A 0.1% TFA, 20% methanol
  • Buffer B 0.1% TFA, 100% methanol
  • Buffer A 20 mM sodium phosphate, pH 3.0
  • Buffer B 20 mM sodium phosphate, pH 3.0, 1.5 M NaCl
  • Fraction 56 contained the substance according to the invention.
  • Buffer B 0.1% TFA, 80% acetonitrile
  • the bioactive fraction 56 from the previous separation step was further purified on an analytical reverse phase column.
  • Filling material YMC RP-C18, 5 ⁇ m, 300 ⁇
  • Buffer B 0.1% TFA, 80% acetonitrile
  • bioactive fraction 45 from the previous separation step was subjected directly to the mass and sequence analysis. Another portion was reduced and alkylated (as described in Example 2) and then further purified on an analytical reverse phase column.
  • Filling material Zorbax RP-C3, 5 ⁇ m, 300 ⁇ analytics is used, clearly.
  • IGF-II 7471 Da; IGFBP-2, 12,681 Da; IGFBP-2, 12 865 Da
  • Cysteines can be detected in peptide sequencing after prior chemical derivatization, for example after reduction with ß-mercaptoethanol and carboxamidomethylation with iodoacetamide. After derivatization, desalting is preferably followed by analytical reverse phase chromatography with a Vydac RP-C18 column (4.6 mm x 25 cm). Some of the peptides modified in this way are fed to the sequence analysis, with the other part the mass determinations give a corresponding molecular weight. From the mass difference to the native peptide, it is concluded that the peptides from hemofiltrate contain six cysteines, which are also connected to one another with three disulfide bridges.
  • IGFBP-2-13 MW 12681
  • IGFBP-2-13 MW 12865
  • the peptide sequences are 100% identical to the amino acids of human IGFBP-2 derived from the cDNA or to the amino acid sequence of IGF-II.
  • IGFBP-2 has so far been described as a 34 kD binding protein, the complete sequence analysis of which was carried out by analysis of the associated cDNA (Binkert, C. et al., EMBO Journal Vol. 8 (1989), pages 2497 to 2502).
  • IGF-II and IGF-I which also binds to IGFBP-2, have been extensively described in their structure at the protein and DNA sequence level (as a review: Rechler, MM, & Nissley, SP (1990) insulin-like growth factors In: Peptide growth factors and their receptors (Spori, MB, Roberts, AB eds.), pages 263 to 367, Springer-Verlag, Berlin).
  • the IGF / IGFBP-2-13 was isolated on the basis of its biological activity in a survival assay of the PC-12 (pheochromocytoma cells) cell line. For this purpose, aliquots of the individual chromatography steps described in Example 1 were freeze-dried and then fed to the biological assay. The fractions, which each gave a positive signal, were subjected to further purification.
  • the assay measures cell survival after being serum-free by determining mitochondrial enzyme activity 24 hours after serum deprivation.
  • Nerve growth factor (NGF) or fetal calf serum (FCS) is used as a positive control in this assay.
  • 10,000 PC-12 cells per hole are sown in 96-well plates in serum-free medium. Aliquots (approx. 100 ml equivalent of the starting material) are added to the wells. 20 hours later, the survival rate of the cells is measured using a Wst-1 substrate. This substrate is converted by mitochondrial enzymes. The resulting dye intensity is measured at 405 nm in an ELISA reader, the reference wavelength is over 600 nm.
  • the IGF / IGFBP-2-13 complex has a dose-dependent, survival-promoting effect on PC-12 cells. These cells correspond to neuronal progenitor cells, so that it can be assumed that IGF / IGFBP-2-13 is a neuroprotective factor.
  • the bioactive fraction 33 from pH pool IV in the assay was separated on an analytical reverse phase column. Fraction 34 contained the substance according to the invention.
  • Buffer B 0.1% TFA, 80% acetonitrile
  • Mass determinations The mass determinations were carried out on an electrospray mass spectrometer. The molecular weight of the peptide was as
  • the C-terminus was determined by comparing the measured molecular mass with the mass calculated from the sequence. The agreement of these masses lies in the measuring accuracy of the electrospray mass spectrometer of 0.1% of the total mass.
  • the peptide sequence is 100% identical to the amino acids of human IGFBP-4 derived from the cDNA.
  • the sulfur bridge linkage was analyzed by cleaving the native peptide IGFBP-4-11 in parallel in two different batches with the endoproteases chymotrypsin and Arg-C. The cleavage fragments obtained were then separated from one another by means of analytical reverse phase chromatography and subjected to the molecular mass and sequence analysis. The following fragments, each containing two cysteines and a sulfur bridge, were obtained: HPKQCHPALDGQRGKCW, MW 1960
  • the IGFBP-4-11 was isolated on the basis of its biological activity in a proliferation assay with primary bone cells (osteoblasts), which are initially isolated from the skullcap of rat embryos
  • TGF-beta Transforming growth factor-beta
  • FCS fetal calf serum
  • the proliferation rate (DNA synthesis rate) of the cells is measured by adding and incorporating radioactive thymidine.
  • the peptide IGFBP-4-11 has a proliferation-promoting effect on these primary osteoblasts. These cells correspond to typical bone cells, so it can be assumed that IGFBP-4-11 is an osteoanabolic factor.
  • the theoretical mass is 12.5 kD, so it can be assumed that the peptide is glycosylated on serine or theronine.

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Abstract

The invention relates to peptides which are characterized in that the amino acid sequence parts thereof correspond to the amino acid sequence of insulin-like growth factor binding protein. The invention also relates to cyclic, glycosylated, phosphorylated, acetylated, amidated and/or sulfatized derivatives.

Description

Insulin-like Growth Factor Binding Protein Fragmente und ihre Verwendung Insulin-like growth factor binding protein fragments and their use
Gegenstand der vorliegenden Erfindung sind Peptide mit zellproliferativen und zellprotektiven Eigenschaften, Komplexe der Peptide mit humanem Insulin-like growth factor I und II (IGF) sowie die damit in Verbindung stehenden Nucleinsäuren, Antisensenucleotide, Antikörper und Inhibitoren.The present invention relates to peptides with cell proliferative and cell protective properties, complexes of the peptides with human insulin-like growth factor I and II (IGF) and the associated nucleic acids, antisense nucleotides, antibodies and inhibitors.
Insulin-like Growth Factor Binding Proteine sind unter anderem von Shimasaki, S. und Ling, N. in Prog. Growth Factor Res. 3 (1991) 243-266 und Zapf, J. in Eur. J. Endocrinol. 132 (1995) 645-654 beschrieben worden.Insulin-like growth factor binding proteins are described, inter alia, by Shimasaki, S. and Ling, N. in Prog. Growth Factor Res. 3 (1991) 243-266 and Zapf, J. in Eur. J. Endocrinol. 132 (1995) 645-654.
Gegenstand der vorliegenden Erfindung sind Peptide, deren Aminosäuresequenz Teilen der Aminosäuresequenz von Insulin-like Growth Factor Binding Proteinen entspricht sowie cy- clische, glycosylierte, phosphorylierte, acetylierte, a idierte und/oder sulfatierte Derivate davon. Diese erfindungsgemäßen Peptide werden als IGFBP oder IBP bezeichnet.The present invention relates to peptides whose amino acid sequence corresponds in part to the amino acid sequence of insulin-like growth factor binding proteins, and to cyclic, glycosylated, phosphorylated, acetylated, acidated and / or sulfated derivatives thereof. These peptides according to the invention are referred to as IGFBP or IBP.
Bevorzugte Peptide sind solche, die natürlicherweise vorkommen und beispielsweise aus Hämo- filtrat isoliert werden können. Vorzugsweise weisen die Peptide eine Länge von 61 bis 115 Aminosäuren auf. Besonders bevorzugt sind Peptide, die Sequenzen aufweisen, die N- oder C- terminalen Sequenzen von Insulin-like Growth Factor Binding Proteinen entsprechen.Preferred peptides are those that occur naturally and can be isolated, for example, from hemofiltrate. The peptides preferably have a length of 61 to 115 amino acids. Peptides which have sequences which correspond to N- or C-terminal sequences of insulin-like growth factor binding proteins are particularly preferred.
Bevorzugte Peptide sind Peptide mit der Aminosäuresequenz der FormelPreferred peptides are peptides with the amino acid sequence of the formula
RrC-X,-PNC-X2-QC-X3-CWCV-X4-C-R2 R r CX, -PNC-X 2 -QC-X 3 -CWCV-X 4 -CR 2
worinwherein
R, NH2, eine Aminosäure oder ein Peptid mit einer Aminosäuresequenz umfassend bis zu 41 Aminosäuren, X, ein Peptid mit einer Aminosäuresequenz umfassend 24 bis 31 Aminosäuren, X, ein Peptid mit einer Aminosäuresequenz umfassend 9 Aminosäuren, X3 ein Peptid mit einer Aminosäuresequenz umfassend 10 Aminosäuren, X4 ein Peptid mit einer Aminosäuresequenz umfassend 18 bis 24 Aminosäuren, R2 COOH, CONH2 oder ein Peptid mit bis zu 12 Aminosäuren darstellt und cyclische, glycosylierte, phosphorylierte, acetylierte, amidierte, sulfatierte Derivate sowie Fragmente mit der physiologischen Fähigkeit des IGFBP.R, NH 2 , an amino acid or a peptide with an amino acid sequence comprising up to 41 amino acids, X, a peptide with an amino acid sequence comprising 24 to 31 amino acids, X represents a peptide with an amino acid sequence comprising 9 amino acids, X 3 represents a peptide with an amino acid sequence comprising 10 amino acids, X 4 represents a peptide with an amino acid sequence comprising 18 to 24 amino acids, R 2 COOH, CONH 2 or a peptide having up to 12 amino acids and cyclic, glycosylated, phosphorylated, acetylated, amidated, sulfated derivatives and fragments with the physiological ability of IGFBP.
Das Peptid weist zellproliferative und zellprotektive Eigenschaften auf.The peptide has cell proliferative and cell protective properties.
Die erfindungsgemäßen Peptide können Disulfidbrücken aufweisen, so daß sie der allgemeinen FormelThe peptides according to the invention can have disulfide bridges, so that they have the general formula
RrC-XrPNC-X2-QC-X3-CWCV-X4-C-R2 R r CX r PNC-X 2 -QC-X 3 -CWCV-X 4 -CR 2
entsprechen.correspond.
In einer bevorzugten Ausführungsform weisen die Peptide an einer oder mehrerer der folgenden Positionen der Aminosäuresequenz ein Glycin auf. X2 an Position 4, X3 an Position 9, X4 an Position 4 oder 5 und/oder X4 an Position 9 oder 10.In a preferred embodiment, the peptides have a glycine at one or more of the following positions in the amino acid sequence. X 2 at position 4, X 3 at position 9, X 4 at position 4 or 5 and / or X 4 at position 9 or 10.
Es ist weiter bevorzugt, daß X] an der Position 8 L oder V ist und/oder X, an der Position 11 L oder I ist und/oder X2 an der Position 1 D oder. N ist und/oder X2 .an der Position 9 K oder R ist und/oder X3 an der Position 3 S oder A ist und/oder an der Position 8 R oder A ist.It is further preferred that X ] at position 8 is L or V and / or X, at position 11 is L or I and / or X 2 at position 1 D or. N is and / or X 2 at position 9 is K or R and / or X 3 at position 3 is S or A and / or at position 8 is R or A.
In einer besonders bevorzugten Ausfuhrungsform wird R, ausgewählt ausIn a particularly preferred embodiment, R is selected from
APSEEDHSILWDAISTYDGSKALHVTNIKKWKEP (SEQ ID NO: 1), GGKHHLGLEEPKKLRPPPARTP (SEQ ID NO: 2), GKGGKHHLGLEEPKKLRPPPARTP (SEQ ID NO: 3) , GHAKDSQRYKVDYESQSTDTQNFSSESKRETEYGP (SEQ ID NO : 4), KVNGAPREDARPVPQGS (SEQ ID NO: 5) ,APSEEDHSILWDAISTYDGSKALHVTNIKKWKEP (SEQ ID NO: 1), GGKHHLGLEEPKKLRPPPARTP (SEQ ID NO: 2), GKGGKHHLGLEEPKKLRPPPARTP (SEQ ID NO: 3), GHAKDSQESKVTQNQSPGKPDQQ
LTQSKFVGGAENTAHPRIISAPEMRQESEQGP (SEQ ID NO : 6), PQAGTARPQDVNRRDQQRNPGTSTTPSQPNSAGVQDTEMGP (SEQ ID NO: 7) und/oderLTQSKFVGGAENTAHPRIISAPEMRQESEQGP (SEQ ID NO: 6), PQAGTARPQDVNRRDQQRNPGTSTTPSQPNSAGVQDTEMGP (SEQ ID NO: 7) and / or
X, ausgewählt ausX selected from
RIELYRWESLAKAQETSGEEISKFYL (SEQ ID NO: 8), QQELDQVLERIST RLPDERGPLEHLYSLHI (SEQ ID NO : 9), RREMEDTLNHLKFLNVLSPRGVHI (SEQ ID NO : 10) , QSELHRALERLAASQSRTHEDLYIIPI (SEQ ID NO: 11), RRHMEASLQELKASPRMVPRAVYL (SEQ ID NO: 12), RRHLDSVLQQLQTEVYRGAQTLYV (SEQ ID NO: 13) und/oderRIELYRWESLAKAQETSGEEISKFYL (SEQ ID NO: 8), QQELDQVLERIST RLPDERGPLEHLYSLHI (SEQ ID NO: 9), RREMEDTLNHLKFLNVLSPRGVHI (SEQ ID NO: 10), QSELHRALERLAASQSRTHEDLYIIPI (SEQ ID NO: 11), RRHMEASLQELVYVVLQQQ NOVASVLQQQ
X2 ausgewählt ausX 2 selected from
NKNGFYHSR (SEQ ID NO 14 )NKNGFYHSR (SEQ ID NO 14)
DKHGLYNLK (SEQ ID NO 15 )DKHGLYNLK (SEQ ID NO 15)
DKKGFYKKK (SEQ ID NO 16 )DKKGFYKKK (SEQ ID NO 16)
DRNGNFHPK (SEQ ID NO 17 )DRNGNFHPK (SEQ ID NO 17)
DRKGFYKRK (SEQ ID NO 18 )DRKGFYKRK (SEQ ID NO 18)
DHRGFYRKR (SEQ ID NO 19 ) und/oderDHRGFYRKR (SEQ ID NO 19) and / or
X3 ausgewählt ausX 3 selected from
ETS DGEAGL (SEQ ID NO 20) ,ETS DGEAGL (SEQ ID NO 20),
KMSLNGQRGE (SEQ ID NO 21) ,KMSLNGQRGE (SEQ ID NO 21),
RPSKGRKRGF (SEQ ID NO 22) ,RPSKGRKRGF (SEQ ID NO 22),
HPALDGQRGK (SEQ ID NO 23) ,HPALDGQRGK (SEQ ID NO 23),
KPSRGRKRGI (SEQ ID NO 24) ,KPSRGRKRGI (SEQ ID NO 24),
RSSQGQRRGP (SEQ ID NO 25) und/oderRSSQGQRRGP (SEQ ID NO 25) and / or
x4 ausgewählt ausx 4 selected from
YP NGKRIPGSPEIRGDPN (SEQ ID NO 26 ) , NPNTGKLIQGAPTIRGDPE (SEQ ID NO 27 ) , DKYGQPLPGYTTKGKEDVH (SEQ ID NO 28 ) , DRKTGVKLPGGLEPKGELD (SEQ ID NO 29) , DKYGMKLPGMEYVDGDFQ (SEQ ID NO: 30 ) , DRMGKSLPGSPDGNGSSS (SEQ ID NO: 31 ) und/oderYP NGKRIPGSPEIRGDPN (SEQ ID NO 26), NPNTGKLIQGAPTIRGDPE (SEQ ID NO 27), DKYGQPLPGYTTKGKEDVH (SEQ ID NO 28), DRKTGVKLPGGLEPKGELD (SEQ ID NO 29), DKYYGMGGGLGQD and or
R2 ausgewählt ausR 2 selected from
QIYFNVQN (SEQ ID NO: 32), HLFYNEQQEARGVHTQRMQ (SEQ ID NO: 33; HLFYNEQQE (SEQ ID NO: 34), YSMQSK (SEQ ID NO: 35), HQLADSFRE (SEQ ID NO: 36), HTFDSSNVE (SEQ ID NO: 37), PTGSSG (SEQ ID NO: 38) .QIYFNVQN (SEQ ID NO: 32), HLFYNEQQEARGVHTQRMQ (SEQ ID NO: 33; HLFYNEQQE (SEQ ID NO: 34), YSMQSK (SEQ ID NO: 35), HQLADSFRE (SEQ ID NO: 36), HTFDSS: NOVE 37), PTGSSG (SEQ ID NO: 38).
Bevorzugte Peptide weisen folgende Sequenzen auf IGFBP-1Preferred peptides have the following sequences IGFBP-1
APSEEDHSIL DAISTYDGSKALHVTNIKKWKEPCRIELYRWESLAKAQETSGEEISKFYLP NCNKNGFYHSRQCETSMDGEAGLCWCWPWNGKRIPGSPEIRGDPNCQIYFNVQN (SEQ ID NO: 39)APSEEDHSIL DAISTYDGSKALHVTNIKKWKEPCRIELYRWESLAKAQETSGEEISKFYLP NCNKNGFYHSRQCETSMDGEAGLCWCWPWNGKRIPGSPEIRGDPNCQIYFNVQN (SEQ ID NO: 39)
IGFBP-2IGFBP-2
GKGGKHHLGLEEPKKLRPPPARTPCQQELDQVLERISTMRLPDERGPLEHLYSLHIPNCDKHG LYNLKQCKMSLNGQRGECWCVNPNTGKLIQGAPTIRGDPECHLFYNEQQEARGVHTQRMQ (SEQ ID NO: 40)GKGGKHHLGLEEPKKLRPPPARTPCQQELDQVLERISTMRLPDERGPLEHLYSLHIPNCDKHG LYNLKQCKMSLNGQRGECWCVNPNTGKLIQGAPTIRGDPECHLFYNEQQEARGVHTQRMQ) SE
GGKHHLGLEEPKKLRPPPARTPCQQELDQVLERISTMRLPDERGPLEHLYSLHIPNCDKHG LYNLKQCKMSLNGQRGECWCVNPNTGKLIQGAPTIRGDPECHLFYNEQQEARGVHTQRMQ (SEQ ID NO: 45)GGKHHLGLEEPKKLRPPPARTPCQQELDQVLERISTMRLPDERGPLEHLYSLHIPNCDKHG LYNLKQCKMSLNGQRGECWCVNPNTGKLIQGAPTIRGDPECHLFYNEQQEARGVHTQRMQ (SEQ ID
IGFBP-3IGFBP-3
GHAKDSQRYKVDYESQSTDTQNFSSESKRETEYGPCRREMEDTLNHLKFLNVLSPRGVHIPNC DKKGFYKKKQCRPSKGRKRGFCWCVDKYGQPLPGYTTKGKEDVHCYSMQSK (SEQ ID NO: 41)GHAKDSQRYKVDYESQSTDTQNFSSESKRETEYGPCRREMEDTLNHLKFLNVLSPRGVHIPNC DKKGFYKKKQCRPSKGRKRGFCWCVDKYGQPLPGYTTKGKEDVHCYSMQSK (SEQ ID NO: 41
KVDYESQSTDTQNFSSESKRETEYGPCRREMEDTLNHLKFLNVLSPRGVHIPNC DKKGFYKKKQCRPSKGRKRGFC CVDKYGQPLPGYTTKGKEDVHCYSMQSK (SEQ ID NO: 46)KVDYESQSTDTQNFSSESKRETEYGPCRREMEDTLNHLKFLNVLSPRGVHIPNC DKKGFYKKKQCRPSKGRKRGFC CVDKYGQPLPGYTTKGKEDVHCYSMQSK (SEQ ID NO: 46)
HPLHSKIIIIKKGHAKDSQRY (SEQ ID NO: 47)HPLHSKIIIIKKGHAKDSQRY (SEQ ID NO: 47)
IGFBP-4IGFBP-4
DEAIHCPPCSEEKLARCRPPVGCEELVREPGCGCCATCALGLGMPCGVYTPRCGSGLRCYPPR GVEKPLHTLMHGQGVCMELAEIEAIQESLQPSDKDEGDHPNNSFSPCSAHDRRCLQKHFAKIR DRSTSGGKM (SEQ ID NO: 48)DEAIHCPPCSEEKLARCRPPVGCEELVREPGCGCCATCALGLGMPCGVYTPRCGSGLRCYPPR GVEKPLHTLMHGQGVCMELAEIEAIQESLQPSDKDEGDHPNNSFSPCSAHDRRCLQKHFAKM DRSTSGG NOK
KVNGAPREDARPVPQGSCQSELHRALERLAASQSRTHEDLYIIPIPNCDRNGNFHPKQCHPAL DGQRGKC CVDRKTGVKLPGGLEPKGELDCHQLADSFRE (SEQ ID NO: 42)KVNGAPREDARPVPQGSCQSELHRALERLAASQSRTHEDLYIIPIPNCDRNGNFHPKQCHPAL DGQRGKC CVDRKTGVKLPGGLEPKGELDCHQLADSFRE (SEQ ID NO: 42)
IGFBP-5IGFBP-5
LTQSKFVGGAENTAHPRIISAPEMRQESEQGPCRRHMEASLQELKASPRMVPRAVYLPNCDRK GFYKRKQCKPSRGRKRGIC CVDKYGMKLPGMEYVDGDFQCHTFDSSNVE (SEQ ID NO: 43)LTQSKFVGGAENTAHPRIISAPEMRQESEQGPCRRHMEASLQELKASPRMVPRAVYLPNCDRK GFYKRKQCKPSRGRKRGIC CVDKYGMKLPGMEYVDGDFQCHTFDSSNVE (SEQ ID NO: 43)
KFVGGAENTAHPRIISAPEMRQESEQGPCRRHMEASLQELKASPRMVPRAVYLPNCDRK GFYKRKQCKPSRGRKRGIC CVDKYGMKLPGMEYVDGDFQCHTFDSSNVE (SEQ ID NO: 49)KFVGGAENTAHPRIISAPEMRQESEQGPCRRHMEASLQELKASPRMVPRAVYLPNCDRK GFYKRKQCKPSRGRKRGIC CVDKYGMKLPGMEYVDGDFQCHTFDSSNVE (SEQ ID NO: 49)
HTRISELKAEAVKKDRRKKLTQS (SEQ ID NO: 50)HTRISELKAEAVKKDRRKKLTQS (SEQ ID NO: 50)
IGFBP-6IGFBP-6
PQAGTARPQDVNRRDQQRNPGTSTTPSQPNSAGVQDTEMGPCRRHLDSVLQQLQTEVYRGAQT LYVPNCDHRGFYRKRQCRSSQGQRRGPCWCVDRMGKSLPGSPDGNGSSSCPTGSSG (SEQ ID NO: 44) Die erfindungsgemäßen Peptide lassen sich durch Aufreinigung aus humanem Blutfiltrat oder Urin, durch Festphasenpeptidsynthese oder durch Expression in rekombinanten Mikroorganismen erhalten.PQAGTARPQDVNRRDQQRNPGTSTTPSQPNSAGVQDTEMGPCRRHLDSVLQQLQTEVYRGAQT LYVPNCDHRGFYRKRQCRSSQGQRRGPCWCVDRMGKSLPGSPDGNGSSSCPTGSSG (SED The peptides according to the invention can be obtained by purification from human blood filtrate or urine, by solid phase peptide synthesis or by expression in recombinant microorganisms.
Gegenstand der Erfindung sind weiterhin die Komplexe der erfindungsgemäßen Peptide mit humanem Insulin-like growth factor-I und/oder humanem Insulin-like growth factor-II sowie dessen physiologisch aktiven Fragmenten und/oder Derivaten, insbesondere amidierte, acety- lierte, sulfatierte, phosphorylierte und/oder glykosylierte Derivate.The invention furthermore relates to the complexes of the peptides according to the invention with human insulin-like growth factor-I and / or human insulin-like growth factor-II and their physiologically active fragments and / or derivatives, in particular amidated, acetylated, sulfated, phosphorylated and / or glycosylated derivatives.
Gegenstand der Erfindung sind darüber hinaus Nucleinsäuren, die für die erfindungsgemäßen Peptide kodieren, Antisensenucleotide, die unter stringenten Bedingungen an eine Nucleinsäure binden, die für das erfindungsgemäße Peptid kodiert, Antikörper, die an die erfindungsgemäßen Peptide binden, Inhibitoren, die die biologische Aktivität der Insulin-like Growth Factor Binding Proteine hemmen, Inhibitoren, die die Expression von Insulin-like Growth Factor Binding Proteinen hemmen.The invention furthermore relates to nucleic acids which code for the peptides according to the invention, antisense nucleotides which bind under stringent conditions to a nucleic acid which codes for the peptide according to the invention, antibodies which bind to the peptides according to the invention, inhibitors which inhibit the biological activity of the insulin -like growth factor binding proteins, inhibitors that inhibit the expression of insulin-like growth factor binding proteins.
Die erfindungsgemäßen Peptide, Komplexe, Nucleinsäuren, Antisensenucleotide, Antikörper und Inhibitoren eignen sich insbesondere zur Herstellung eines Arzneimittels zur Behandlung der Über- oder Unterexpression von Insulin-like Growth Factor Binding Proteine, zur Behandlung von Muskelschwund, Osteoporose, Diabetes, amyloidaler lateraler Sklerose, periphe- ren und zentralen Neuropathien, entzündlichen Prozessen, gestörten Entzündungsre-aktionen, TumorerJ ankungen, entzündlichen und neoplastischen Erkrankungen, Wachstumsstörung, Erkrankung der Muskulatur, Erkrankung des Knochenapparates und/oder zur Wund- und Knochenheilung.The peptides, complexes, nucleic acids, antisense nucleotides, antibodies and inhibitors according to the invention are particularly suitable for the manufacture of a medicament for the treatment of over- or under-expression of insulin-like growth factor binding proteins, for the treatment of muscle loss, osteoporosis, diabetes, amyloidal lateral sclerosis, peripheral - Central and central neuropathies, inflammatory processes, disturbed inflammatory reactions, tumor disorders, inflammatory and neoplastic diseases, growth disorders, musculoskeletal disorders, diseases of the bone apparatus and / or for wound and bone healing.
Insbesondere Komplexe von IGFBP mit IGF-I oder IGF-II eignen sich zur Behandlung von Knochenerkrankungen.Complexes of IGFBP with IGF-I or IGF-II are particularly suitable for the treatment of bone diseases.
Die erfindungsgemäßen Peptide und die Komplexe der Peptide mit Insulin-like growth factor weisen eine zellproliferative Aktivität auf. Die erfindungsgemäßen Peptide regulieren die Freisetzung des IGF-I und IGF-Il aus den Komplexen an ihrem Wirkort. Die Coadministration der erfmdungsgemäßen Peptide mit IGF-I oder IGF-II verlängert die biologische Halbwertzeit und damit die Verfügbarkeit der letztgenannten. Die durch Injektion von freiem IGF-I oder IGF-II induzierte Hypoglykämie wird durch die Coadministration der erfindungsgemäßen Peptide verhindert.The peptides according to the invention and the complexes of the peptides with insulin-like growth factor have a cell proliferative activity. The peptides according to the invention regulate the release of IGF-I and IGF-II from the complexes at their site of action. The co-administration of the peptides according to the invention with IGF-I or IGF-II extends the biological half-life and thus the availability of the latter. The hypoglycemia induced by injection of free IGF-I or IGF-II is prevented by the co-administration of the peptides according to the invention.
Die erfindungsgemäßen Peptide haben desweiteren eine wachstumsfördernde Wirkung auf Knochenzellen und führen zu einer Verstärkung oder Modulation der Wirkung von Wachstumshormonen.The peptides according to the invention furthermore have a growth-promoting effect on bone cells and lead to an enhancement or modulation of the action of growth hormones.
In einer bevorzugten Ausführungsform sind die erfindungsgemäßen Peptide, Komplexe, Nucleinsäuren, Antisensenucleotide, Antikörper und Inhibitoren in einer pharmazeutisch üblichen Darreichungsform in einem Arzneimittel enthalten. Sie eignen sich zur oralen, intravenösen, intramuskulären, intracutanen, intrathekalen Anwendung oder als Aerosol zur transpulmo- nalen Applikation. Die Menge an zu verabreichenden Peptid beträgt 1 μg bis 1 g pro Darreichungseinheit pro Tag.In a preferred embodiment, the peptides, complexes, nucleic acids, antisense nucleotides, antibodies and inhibitors according to the invention are contained in a pharmaceutical in a pharmaceutical dosage form. They are suitable for oral, intravenous, intramuscular, intracutaneous, intrathecal use or as an aerosol for transpulmonary application. The amount of peptide to be administered is 1 μg to 1 g per administration unit per day.
Die erfindungsgem.äßen Nucleinsäuren und/oder Antisensenucleotide eignen sich auch zur Herstellung eines Medikamentes zur Behandlung somatischer oder nicht-somatischer Generkrankungen.The nucleic acids and / or antisense nucleotides according to the invention are also suitable for the manufacture of a medicament for the treatment of somatic or non-somatic genetic diseases.
Das erfindungsgemäße Diagnostikmittel enthält die erfindungsgemäßen Peptide, Komplexe, Nucleinsäuren, Antisensenucleotide, Antikörpern und/oder Inhibitoren.The diagnostic agent according to the invention contains the peptides, complexes, nucleic acids, antisense nucleotides, antibodies and / or inhibitors according to the invention.
Bevorzugterweise enthält das Diagnostikmittel poly- oder monoklonale Antiköφer gegen das erfindungsgem.äße Peptid, wobei die Antiköφer fluoreszenz- oder radioaktiv-markiert sein können, um in den bekannten ELISA oder RIA eingesetzt werden zu können. Jedoch kann das Diagnostikmittel auch Nucleinsäuren enthalten, die in modifizierter oder markierter Form in dem Fachmann bekannten Tests wie PCR oder Finger-Printing zum Einsatz kommen.The diagnostic agent preferably contains poly- or monoclonal antibodies to the peptide according to the invention, it being possible for the antibodies to be labeled with fluorescence or radioactive in order to be able to be used in the known ELISA or RIA. However, the diagnostic agent can also contain nucleic acids, which are used in modified or labeled form in tests known to those skilled in the art, such as PCR or finger printing.
Die erfindungsgemäßen Diagnostikmittel eignen sich insbesondere zur Diagnose von Funktionsstörungen des Knochens, der Muskeln, des Nervensystems, der Lymphorgane, des Magen- Darm-Traktes, des Immunsystems, von Diabetes, inflammatorischen und neoplastischen Prozessen sowie als Marker bei Krebs.The diagnostic agents according to the invention are particularly suitable for the diagnosis of functional disorders of the bones, the muscles, the nervous system, the lymph organs, the gastric Intestinal tract, immune system, diabetes, inflammatory and neoplastic processes as well as a marker for cancer.
Insbesondere das gleichzeitige Auftreten mehrerer Fragmente von BP-4 oder BP-5 im Plasma, insbesondere der N- und C-termmalen Domänen eignet sich als Marker für Erkrankungen des Knochenstoffwechsels. Die entsprechenden Peptide können entweder massenspektroskopisch nachgewiesen oder, bevorzugt durch Immunreaktion mit entsprechenden Antiköφer.In particular, the simultaneous appearance of several fragments of BP-4 or BP-5 in the plasma, in particular the N- and C-termmal domains, is suitable as a marker for diseases of the bone metabolism. The corresponding peptides can either be detected by mass spectroscopy or, preferably by an immune reaction with the corresponding antibodies.
Figur 1 zeigt ein Alignment der Konsensussequenzen von C-terminalen Fragmenten der Insulin- like Growth Factor Proteine.FIG. 1 shows an alignment of the consensus sequences of C-terminal fragments of the insulin-like growth factor proteins.
Figur 2 zeigt die schematische Struktur der Insulin-like Growth Factor Proteine mit den cystein- reichen N- und C-terminalen Domänen.FIG. 2 shows the schematic structure of the insulin-like growth factor proteins with the cysteine-rich N- and C-terminal domains.
Figur 3 zeigt die schematische Struktur der Insulin-like Growth Factor Proteine sowie die Sequenz der aus Hämofiltrat isolierten biologisch aktiven Fragmente.FIG. 3 shows the schematic structure of the insulin-like growth factor proteins and the sequence of the biologically active fragments isolated from hemofiltrate.
Figur 4 zeigt die Isolierung des osteoanabolen Faktors IGFBP-4-11 aus humanem Hämofiltrat (siehe Beispiel 3).FIG. 4 shows the isolation of the osteoanabolic factor IGFBP-4-11 from human hemofiltrate (see example 3).
Figur 5 zeigt die Sequenz- und Schwefelbrückenanalyse des osteoanabolen Faktors IGFBP-4- 11. Die Cysteine 153-183, 194-205 und 207-228 sind verbrückt.Figure 5 shows the sequence and sulfur bridge analysis of the osteoanabolic factor IGFBP-4- 11. The cysteines 153-183, 194-205 and 207-228 are bridged.
Figur 6 zeigt die biologische Wirkung des osteoanabolen Faktors IGFBP-4-11. Nach einer Inkubation von serumfrei gehaltenen primären Rattenosteoblasten für (A) 24 Stunden, (B) 48 Stunden und (C) 72 Stunden mit IGFBP-4-11 zeigt sich die proliferationsfordernde Wirkung des IGFBP-4-11. In dosisabhängiger Weise wird ein Anstieg der DNA-Syntheserate gefunden, gemessen als Einbau von Bromodesoxyuridin (BrdU).Figure 6 shows the biological effect of the osteoanabolic factor IGFBP-4-11. After incubation of serum-free primary rat osteoblasts for (A) 24 hours, (B) 48 hours and (C) 72 hours with IGFBP-4-11, the proliferation-promoting effect of IGFBP-4-11 can be seen. An increase in the rate of DNA synthesis is found in a dose-dependent manner, measured as the incorporation of bromodeoxyuridine (BrdU).
Figur 7 zeigt die spezifische Bindung an Osteoblasten und den möglichen Rezeptor für den osteoanabolen Faktor IGFBP-4-11 (als IGFBP-4136"237 bezeichnet). A: Radioaktiv markiertes IGFBP-4-11 zeigt eine durch steigende Mengen an nicht-markiertem IGFBP-4-11 verdrängbare spezifische Bindung an primäre Osteoblastenzellen. B: Radioaktiv markiertes IGFBP-4-11 konnte, nachdem es an Osteo-blasten gebunden hat, chemisch mit seinem putativen Rezeptormolekül vernetzt werden und anschließend durch Gelelektrophorese und nachfolgende Autora- diographie nachgewiesen werden. Der Ligand-Rezeptor-Komplex hat ein Molekulargewicht von etwa 120 kDa. Die Bildung des Komplexes wird begünstigt durch Saponin, einen Membranporenbildner. Die Komplexbildung wird verhindert durch Zusatz eines Überschusses an nicht- markiertem IGFBP-4-11 zum Inkubationsansatz.Figure 7 shows the specific binding to osteoblasts and the possible receptor for the osteoanabolic factor IGFBP-4-11 (designated as IGFBP-4 136 "237 ). A: Radioactively labeled IGFBP-4-11 shows an unlabelled by increasing amounts IGFBP-4-11 displaceable specific binding to primary osteoblast cells B. B: Radioactively labeled IGFBP-4-11 could, after binding to osteoblasts, be chemically cross-linked with its putative receptor molecule and then by gel electrophoresis and subsequent authorization. diography can be demonstrated. The ligand-receptor complex has a molecular weight of approximately 120 kDa. The formation of the complex is promoted by saponin, a membrane pore former. The complex formation is prevented by adding an excess of unlabelled IGFBP-4-11 to the incubation batch.
Die Aufreinigung des erfmdungsgemäßen Peptids bzw. seine Komplexes wird durch die nachfolgenden Beispiele erläutert:The purification of the peptide according to the invention or its complex is illustrated by the following examples:
Beispiel 1example 1
Aufreinigung und peptidchemische Analyse des IGFBP-2-13Purification and peptide chemical analysis of IGFBP-2-13
Die erfindungsgemäßen Peptide sind durch ein Reinigungsverfahren ausgehend vom humanem Hämofiltrat erhältlich. Dieses patentierte Verfahren (Forssmann, W.-G. (1988), Offenlegungs- schrift DE 36 33 707 AI), welches für die Gewinnung von Eiweißstoffen aus Hämofiltrat entwickelt wurde, wurde in modifizierter Form auch zur Aufreinigung des Peptidkomplexes eingesetzt.The peptides according to the invention can be obtained from a human hemofiltrate by a purification process. This patented process (Forssmann, W.-G. (1988), publication DE 36 33 707 AI), which was developed for the extraction of proteins from hemofiltrate, was also used in a modified form to purify the peptide complex.
Hämofiltrat-Batch-ExtraktionHemofiltrate batch extraction
H.ämofiltrat fällt bei der Ultrafiltration des Blutes von Nierenkranken in großen Mengen an. 800 bis 1.000 1 Hämofiltrat werden mit HCl auf einen pH- Wert von 2,1 eingestellt und mit Wasser auf eine Leitfähigkeit von 5,5 mS/cm verdünnt und mit einer Flußrate von 3 1/min auf einen starken Kationenaustauscher aufgetragen.H. hemofiltrate accumulates in large quantities in the ultrafiltration of the blood of kidney patients. 800 to 1,000 1 hemofiltrate are adjusted to a pH of 2.1 with HCl and diluted with water to a conductivity of 5.5 mS / cm and applied to a strong cation exchanger at a flow rate of 3 1 / min.
Chromatographiebedingungen:Chromatography conditions:
Säule: Vantage VA 250 (Amicon, Wirten)Column: Vantage VA 250 (Amicon, hosts)
Säulenmaterial: Fractogel TSK SP 650 (M), 25 cm x 20 cmColumn material: Fractogel TSK SP 650 (M), 25 cm x 20 cm
Fluß: 3 1/minFlow: 3 1 / min
Detektion: 280 nm, pH, LeitfähigkeitDetection: 280 nm, pH, conductivity
Puffer A: Hämofiltrat pH 2,1, Leitfähigkeit 5,5 mS/cmBuffer A: Hemofiltrate pH 2.1, conductivity 5.5 mS / cm
Puffer B: 0,5 M AmmoniumacetatBuffer B: 0.5 M ammonium acetate
Anlage: Autopilot Chromatographiesystem, (PerSeptive Biosystems, Wiesbaden)Attachment: Autopilot Chromatography System, (PerSeptive Biosystems, Wiesbaden)
Nach Auftragung der insgesamt 1.000 1 Flüssigkeit über Nacht wird mit mehreren Säulenvolumina 5 mM HCl gespült. Die Elution der gebundenen Peptide erfolgt als Batch-Elution mit 0,5 M Ammoniumacetat. Hierbei wird eine komplette Elution der Peptide über steigenden pH- Wert (6,8 bis 7,2) und steigende Leitfähigkeit (56 mS/cm) in etwa 5 1 Eluat erreicht.After applying the total of 1,000 l of liquid overnight, 5 mM HCl is rinsed with several column volumes. The bound peptides are eluted as a batch elution with 0.5 M ammonium acetate. Here, a complete elution of the peptides over increasing pH (6.8 to 7.2) and increasing conductivity (56 mS / cm) is achieved in about 5 1 eluate.
Erste präparative AuftrennungFirst preparative separation
Die Ammoniumacetat-Eluate der Batch-Extraktion werden in Mengen von 5.000 bis 10.000 1 Hämofiltrat-Peptid vereinigt. Nach pH-Einstellung auf 2,1 wird das Peptidextrakt unter Zumi- schung von VE- Wasser mit einer Leitfähigkeit von 5,5 mS/cm auf den präparativen Kationenaustauscher aufgetragen.The ammonium acetate eluates of the batch extraction are combined in amounts of 5,000 to 10,000 1 hemofiltrate peptide. After adjusting the pH to 2.1, the peptide extract is applied to the preparative cation exchanger with the addition of demineralized water with a conductivity of 5.5 mS / cm.
Chromatographiebedingungen:Chromatography conditions:
Säule: Vantage 250 VAColumn: Vantage 250 VA
Säulenmaterial: Fractogel TSK SP 650 (M), 25 cm x 20 cmColumn material: Fractogel TSK SP 650 (M), 25 cm x 20 cm
Fluß: bis zu 3 1/min während des Auftrages,Flow: up to 3 1 / min during the order,
0,5 bis 1 1 während der Elution0.5 to 1 1 during elution
Detektion: 280 nm, pH, LeitfähigkeitDetection: 280 nm, pH, conductivity
Probe: Hämofiltrat pH 2,7, Leitfähigkeit 5,5 mS/cmSample: Hemofiltrate pH 2.7, conductivity 5.5 mS / cm
Anlage: Autopilot Chromatographiesystem,System: autopilot chromatography system,
(PerSeptive Biosystems, Wiesbaden)(PerSeptive Biosystems, Wiesbaden)
Nach Auftrag des Rohextraktes über 240 min wird die Säule mit 0,01 M HCl gespült, bis die Leitfähigkeit unter 1 mS/cm ist. Die Elution erfolgt dabei in mehreren Stufen mit den im folgenden angegebenen PuffernAfter applying the crude extract over 240 min, the column is rinsed with 0.01 M HCl until the conductivity is below 1 mS / cm. The elution is carried out in several stages using the buffers specified below
Figure imgf000011_0001
Figure imgf000011_0001
Die Eluate 1-7 werden als pH-Pool I-VII bezeichnet. Sie werden separat gesammelt und abschließend mit VE- Wasser gespült. Die Elution erfolgt bis zum Erreichen einer neuen Basisli- nie, wobei für die einzelnen pH-Pools I bis VII Elutionsvolumina von 10 bis 25 1 erreicht werden.Eluates 1-7 are referred to as pH pool I-VII. They are collected separately and then rinsed with demineralized water. The elution takes place until a new baseline is reached. never, with elution volumes of 10 to 25 1 being achieved for the individual pH pools I to VII.
Zweite präparative Auftrennung:Second preparative separation:
Die einzelnen pH-Pools werden zur Fraktionierung und gleichzeitigen Entsalzung über eine Reverse Phase Chromatographie getrenntThe individual pH pools are separated for fractionation and simultaneous desalination using a reverse phase chromatography
Chromatographiebedingungen:Chromatography conditions:
Säule: FineLine 100 (Pharmacia, Freiburg)Pillar: FineLine 100 (Pharmacia, Freiburg)
Säulenmaterial: Source RPC, 15 μm 10 x 12,5 cm (FineLine 100)Column material: Source RPC, 15 μm 10 x 12.5 cm (FineLine 100)
Fluß : 150 mL/min (FineLine 100)Flow: 150 mL / min (FineLine 100)
Detektion: 280 nm, Leitfähigkeit, pHDetection: 280 nm, conductivity, pH
Puffer A: lO mM HClBuffer A: 10 mM HCl
Puffer B : 80% Acetonitril in 10 mM HClBuffer B: 80% acetonitrile in 10 mM HCl
Gradient: 0 bis 60% Puffer B in 5 SäulenvolumenGradient: 0 to 60% buffer B in 5 column volumes
Nach Auftrag der pH-Pools wird die Säule mit Puffer A gespült. Während der Elution werden Fraktionen zu 200 ml gesammelt. Aliquots der Fraktionen werden im Bioassay getestet. Die Fraktionen werden gefriergetrocknet und bei -20EC gelagert.After the pH pools have been applied, the column is rinsed with buffer A. Fractions of 200 ml are collected during the elution. Aliquots of the fractions are tested in the bioassay. The fractions are freeze-dried and stored at -20EC.
Semipräparative Reverse-Phase C18-Chromatographie:Semi-preparative reverse phase C18 chromatography:
Die im Assay bioaktiven Fraktionen 11 und 12 aus pH-Pool V wurden über eine semipräparative Reverse-Phase Säule aufgetrennt. Die Fraktionen 21 bis 25 enthielten die erfindungsgemäße Substanz.The bioactive fractions 11 and 12 from pH pool V in the assay were separated using a semi-preparative reverse-phase column. Fractions 21 to 25 contained the substance according to the invention.
Chromatographiebedingungen:Chromatography conditions:
Säule: 4,7 cm x 30 cm StahlsäuleColumn: 4.7 cm x 30 cm steel column
Füllmaterial: Vydac RP-C 18 15-20 μm, 300 ÄFilling material: Vydac RP-C 18 15-20 μm, 300 Ä
Puffer A: 0,1% TFABuffer A: 0.1% TFA
Puffer B : 0, 1 % TFA, 80% AcetonitrilBuffer B: 0.1% TFA, 80% acetonitrile
Gradient: 5 bis 50% B in 45 min, 50 bis 100% B in 10 minGradient: 5 to 50% B in 45 min, 50 to 100% B in 10 min
Fluß: 42 ml/minFlow: 42 ml / min
Detektion: 214 nm und 280 nmDetection: 214 nm and 280 nm
Chromatographieanlage: BioCadChromatography system: BioCad
Fraktionen: ä 1,5 min ab Start des GradientenFractions: 1.5 minutes from the start of the gradient
Semipräparative Reverse-Phase Cl 8-Chromatographie: Die bioaktiven Fraktionen 21 bis 25 aus der vorhergehenden Chromatographie wurden über die gleiche semipräparative Reverse Phase Säule aufgetrennt. Als Laufmittel wurde jedoch Methanol verwendet. Die Fraktion 24 enthielt die erfindungsgemäße Substanz.Semi-preparative reverse phase Cl 8 chromatography: The bioactive fractions 21 to 25 from the previous chromatography were separated on the same semi-preparative reverse phase column. However, methanol was used as the eluent. Fraction 24 contained the substance according to the invention.
Chromatographiebedingungen:Chromatography conditions:
Säule: 4,7 cm x 30 cm StahlsäuleColumn: 4.7 cm x 30 cm steel column
Füllmaterial: Vydac RP-C18 15-20 μm, 300 ÄFilling material: Vydac RP-C18 15-20 μm, 300 Ä
Puffer A: 0, 1 % TFA, 20% MethanolBuffer A: 0.1% TFA, 20% methanol
Puffer B : 0, 1 % TFA, 100% MethanolBuffer B: 0.1% TFA, 100% methanol
Gradient: 0 bis 20% B in 6,5 min, 20 bis 80% B in 55 min,Gradient: 0 to 20% B in 6.5 min, 20 to 80% B in 55 min,
80 bis 100% B in 13 min80 to 100% B in 13 min
Fluß: 30 ml/minFlow: 30 ml / min
Detektion: 214 nm und 280 nmDetection: 214 nm and 280 nm
Chromatographieanlage: BioCadChromatography system: BioCad
Fraktionen: ä 1,5 min ab Start des Gradienten Fractions: 1.5 minutes from the start of the gradient
Kationenaustauschchromatographie:Cation exchange chromatography:
Die bioaktiven Fraktionen 19 und 20 aus der vorhergehenden Chromatographie wurden über eine Kationenaustauscher-Säule aufgetrennt. Die Fraktionen 45 bis 47 enthielten die erfindungsgemäße Substanz.Bioactive fractions 19 and 20 from the previous chromatography were separated on a cation exchange column. Fractions 45 to 47 contained the substance according to the invention.
Chromatographiebedingungen:Chromatography conditions:
Säule: 1 cm x 5 cm StahlsäuleColumn: 1 cm x 5 cm steel column
Füllmaterial: Pepkat, Biotek 5 μm, 300 ÄFilling material: Pepkat, Biotek 5 μm, 300 Ä
Puffer A: 20 mM Natriumphosphat, pH 3,0Buffer A: 20 mM sodium phosphate, pH 3.0
Puffer B : 20 mM Natriumphosphat, pH 3,0, 1 ,5 M NaClBuffer B: 20 mM sodium phosphate, pH 3.0, 1.5 M NaCl
Gradient: 0 bis 50% B in 50 min, 50 bis 100% B in 10 minGradient: 0 to 50% B in 50 min, 50 to 100% B in 10 min
Fluß: 3 ml/minFlow: 3 ml / min
Detektion: 280 nmDetection: 280 nm
Chromatographieanlage: BioCad SprintChromatography system: BioCad Sprint
Fraktionen: a 1,5 min ab Start des GradientenFractions: a 1.5 min from the start of the gradient
Analytische Reverse-Phase Chromatographie:Analytical reverse phase chromatography:
Die bioaktiven Fraktionen 45 bis 47 aus der vorhergehenden Chromatographie wurden sukzessive in mehreren identischen Läufen über eine Reverse Phase - Säule aufgetrennt. Die Fraktion 56 enthielt die erfmdungsgem.äße Substanz.The bioactive fractions 45 to 47 from the previous chromatography were successively separated in several identical runs on a reverse phase column. Fraction 56 contained the substance according to the invention.
Chromato graphiebedingungen:Chromatography conditions:
Säule: 1 cm x 25 cm StahlsäuleColumn: 1 cm x 25 cm steel column
Füllmaterial: Vydac RP-C 18 5 μm, 300 ÄFilling material: Vydac RP-C 18 5 μm, 300 Ä
Puffer A: 0,1% TFABuffer A: 0.1% TFA
Puffer B: 0,1% TFA, 80% AcetonitrilBuffer B: 0.1% TFA, 80% acetonitrile
Gradient: 5 bis 50% B in 45 min, 50 bis 100% B in 10 minGradient: 5 to 50% B in 45 min, 50 to 100% B in 10 min
Fluß: 2 ml/minFlow: 2 ml / min
Detektion: 220 nmDetection: 220 nm
Chromatographieanlage: KontronChromatography system: Kontron
Fraktionen: ä 1 min ab Start des Gradienten Zweite Analytische Reverse-Phase C18-Chromatographie:Fractions: ä 1 min from the start of the gradient Second analytical reverse phase C18 chromatography:
Die bioaktive Fraktion 56 aus dem vorhergehenden Trennschritt wurde auf einer analytischen Reverse-Phase Säule weiter aufgereinigt.The bioactive fraction 56 from the previous separation step was further purified on an analytical reverse phase column.
Chromatographiebedingungen:Chromatography conditions:
Säule: 0,46 cm x 25 cm StahlsäuleColumn: 0.46 cm x 25 cm steel column
Füllmaterial: YMC RP-C18, 5 μm, 300 ÄFilling material: YMC RP-C18, 5 μm, 300 Ä
Puffer A: 0,1% TFABuffer A: 0.1% TFA
Puffer B : 0, 1 % TFA, 80% AcetonitrilBuffer B: 0.1% TFA, 80% acetonitrile
Gradient: 15 bis 50% B in 75 min, 75 bis 100% B in 10 minGradient: 15 to 50% B in 75 min, 75 to 100% B in 10 min
Fluß: 0J ml/minFlow: 0J ml / min
Detektion: 214 nmDetection: 214 nm
Chromatographieanlage: KontronChromatography system: Kontron
Dritte Analytische Reverse-Phase C3-Chromatographie:Third analytical reverse phase C3 chromatography:
Ein Teil der bioaktiven Fraktion 45 aus dem vorhergehenden Trennschritt wurde direkt der Massen- und Sequenzanalyse unterzogen. Ein anderer Teil wurde reduziert und alkyliert (wie unter Beispiel 2 beschrieben) und dann auf einer analytischen Reverse-Phase Säule weiter aufgereinigt.Part of the bioactive fraction 45 from the previous separation step was subjected directly to the mass and sequence analysis. Another portion was reduced and alkylated (as described in Example 2) and then further purified on an analytical reverse phase column.
Chromatographiebedingungen:Chromatography conditions:
Säule: 0,1 cm x 15 cm StahlsäuleColumn: 0.1 cm x 15 cm steel column
Füllmaterial: Zorbax RP-C3, 5 μm, 300 Ä Analytik verwandt wird, deutlich.Filling material: Zorbax RP-C3, 5 μm, 300 Ä analytics is used, clearly.
MassenbestimmungenMass determinations
Alle Massenbestimmungen der unmodifizierten und modifizierten Peptide wurden auf einem MALDI-TOF Massenspektrometer durchgeführt. Die Molekülmassen der Peptide wurden alsAll mass determinations of the unmodified and modified peptides were carried out on a MALDI-TOF mass spectrometer. The molecular weights of the peptides were as
IGF-II, 7471 Da; IGFBP-2, 12 681 Da; IGFBP-2, 12 865 DaIGF-II, 7471 Da; IGFBP-2, 12,681 Da; IGFBP-2, 12 865 Da
bestimmt.certainly.
Bestimmung von Cvsteinen/Modifizierung von Peptiden Cysteine lassen sich nach vorheriger chemischer Derivatisierung, zum Beispiel nach Reduktion mit ß-Mercaptoethanol und Carboxamidomethylierung mit Iodacetamid, in der Peptid- Sequenzierung nachweisen. Nach der Derivatisierung schließt sich eine Entsalzung vorzugsweise über eine analytische Reverse-Phase Chromatographie mit einer Vydac RP-C18 Säule (4,6 mm x 25 cm) an. Ein Teil der so modifizierten Peptide werden der Sequenzanalyse zugeführt, mit dem anderen Teil ergeben die Massenbestimmungen ein entsprechendes Molekulargewicht. Aus der Massendifferenz zum nativen Peptid wird geschlossen, daß die Peptide aus Hämofiltrat sechs Cysteine enthalten, welche zudem mit drei Disulfidbrücken untereinander verbunden sind.Determination of CV stones / modification of peptides Cysteines can be detected in peptide sequencing after prior chemical derivatization, for example after reduction with ß-mercaptoethanol and carboxamidomethylation with iodoacetamide. After derivatization, desalting is preferably followed by analytical reverse phase chromatography with a Vydac RP-C18 column (4.6 mm x 25 cm). Some of the peptides modified in this way are fed to the sequence analysis, with the other part the mass determinations give a corresponding molecular weight. From the mass difference to the native peptide, it is concluded that the peptides from hemofiltrate contain six cysteines, which are also connected to one another with three disulfide bridges.
SequenzbestimmungSequence determination
Sowohl die aufgereinigten nativen als auch die carboxamidomethylierte Peptide werden mittels Edman-Abbau auf einem ABI 473 A Sequenzer unter Verwendung des Standard-Programms analysiert.Both the purified native and carboxamidomethylated peptides are analyzed by Edman degradation on an ABI 473 A sequencer using the standard program.
Die Proben werden auf eine Polybrene-Membran in Mengen zwischen 100 und 400 pmol aufgetragen. In Übereinstimmung mit den Ergebnissen der Massenbestimmungen ergaben sich folgende N-terminale Sequenzen:The samples are applied to a Polybrene membrane in amounts between 100 and 400 pmol. In accordance with the results of the mass determinations, the following N-terminal sequences resulted:
IGFBP-2-13, MW 12681IGFBP-2-13, MW 12681
(reduziertes und mit Jodacet.amid modifiziertes Molekül, MW 13045)(Reduced molecule modified with iodoacetamide, MW 13045)
Aminosäurenamino acids
GGKHHLGLEEPKKLRPPPARTPCQQELDQV...GGKHHLGLEEPKKLRPPPARTPCQQELDQV ...
IGFBP-2-13, MW 12865IGFBP-2-13, MW 12865
(reduziertes und mit Jodacetamid modifiziertes Molekül, MW 13223)(Reduced molecule modified with iodoacetamide, MW 13223)
Aminosäurenamino acids
GKGGKHHLGLEEPKKLRPPPARTPCQQELDQV...GKGGKHHLGLEEPKKLRPPPARTPCQQELDQV ...
IGF-π, MW 7471IGF-π, MW 7471
Aminosäurenamino acids
AYRPSETLCGGEL.... Der C-Terminus wurde durch den Vergleich der gemessenen Molekularmasse mit der aus der Sequenz berechneten Masse bestimmt. Die Übereinstimmung dieser Massen liegt in der Meßgenauigkeit des MALDI-TOF-Massenspektrometers von 0,1% der Gesamtmasse.AYRPSETLCGGEL .... The C-terminus was determined by comparing the measured molecular mass with the mass calculated from the sequence. The agreement of these masses lies in the measuring accuracy of the MALDI-TOF mass spectrometer of 0.1% of the total mass.
DatenbankvergleichDatabase comparison
Ein Datenbankvergleich wurde mit Hilfe des HUSAR-Programmpakets an den SwissProl und EMBL-Nucleinsäure Datenbanken durchgeführt. Die Peptidsequenzen besitzt eine hundertprozentige Identität zu den aus der cDNA abgeleiteten Aminosäuren des humanen IGFBP-2 bzw. zur Aminosäuresequenz von IGF-II.A database comparison was carried out using the HUSAR program package on the SwissProl and EMBL nucleic acid databases. The peptide sequences are 100% identical to the amino acids of human IGFBP-2 derived from the cDNA or to the amino acid sequence of IGF-II.
IGFBP-2 wurde bisher als ein 34 kD großes Bindungsprotein beschrieben, dessen vollständige Sequenzanalyse durch Analyse der zugehörigen cDNA (Binkert, C. et al., EMBO Journal Vol. 8 (1989), Seiten 2497 bis 2502) erfolgte. IGF-II und auch IGF-I, welches ebenfalls an IGFBP-2 bindet, wurden dagegen in ihrer Struktur auf Protein- und DNA-Sequenzebene schon umfangreich beschrieben (als Review: Rechler, M.M., & Nissley, S.P. (1990) Insulin-like growth fac- tors In: Peptide growth factors and their receptors (Spori, M.B., Roberts, A.B. eds.), Seiten 263 bis 367, Springer- Verlag, Berlin).IGFBP-2 has so far been described as a 34 kD binding protein, the complete sequence analysis of which was carried out by analysis of the associated cDNA (Binkert, C. et al., EMBO Journal Vol. 8 (1989), pages 2497 to 2502). In contrast, IGF-II and IGF-I, which also binds to IGFBP-2, have been extensively described in their structure at the protein and DNA sequence level (as a review: Rechler, MM, & Nissley, SP (1990) insulin-like growth factors In: Peptide growth factors and their receptors (Spori, MB, Roberts, AB eds.), pages 263 to 367, Springer-Verlag, Berlin).
Beispiel 2Example 2
Bestimmung der biologischen Aktivität des IGF/IGFBP-2-13Determination of the biological activity of the IGF / IGFBP-2-13
Die Isolierung des IGF/IGFBP-2-13 erfolgte anhand seiner biologischen Aktivität in einem Überlebensassay der PC- 12 (Pheochromocytom-Zellen)-Zellinie. Dazu wurden jeweils Aliquots der unter Beispiel 1 beschriebenen einzelnen Chromatographiestufen gefriergetrocknet und anschließend dem biologischen Assay zugeführt. Die Fraktionen, die jeweils ein positives Signal ergaben, wurden der weiteren Aufreinigung unterzogen.The IGF / IGFBP-2-13 was isolated on the basis of its biological activity in a survival assay of the PC-12 (pheochromocytoma cells) cell line. For this purpose, aliquots of the individual chromatography steps described in Example 1 were freeze-dried and then fed to the biological assay. The fractions, which each gave a positive signal, were subjected to further purification.
Der Assay mißt das Überleben der Zellen, nachdem sie serumfrei gehalten wurden, indem 24 Stunden nach Serumentzug die Aktivität mitochondrialer Enzyme bestimmt wird. Als Positiv- Kontrolle wird in diesem Assay Nervenwachstumsfaktor (NGF) oder fötales Kälberserum (FCS) eingesetzt. In 96 Loch-Platten werden 10.000 PC- 12 Zellen pro Loch in serumfreien Medium ausgesät. Es erfolgt die Zugabe von Aliquots (ca. 100 ml Äquivalent des Ausgangsmaterials) in die wells. 20 Stunden später wird die Überlebensrate der Zellen mittels eines Wst-1 Substrats gemessen. Dieses Substrat wird von mitochondrialen Enzymen umgesetzt Die entstehende Farbstoffintensität wird bei 405 nm im ELISA- Reader gemessen, die Referenzwellenlänge liegt dabei bei über 600 nm.The assay measures cell survival after being serum-free by determining mitochondrial enzyme activity 24 hours after serum deprivation. Nerve growth factor (NGF) or fetal calf serum (FCS) is used as a positive control in this assay. 10,000 PC-12 cells per hole are sown in 96-well plates in serum-free medium. Aliquots (approx. 100 ml equivalent of the starting material) are added to the wells. 20 hours later, the survival rate of the cells is measured using a Wst-1 substrate. This substrate is converted by mitochondrial enzymes. The resulting dye intensity is measured at 405 nm in an ELISA reader, the reference wavelength is over 600 nm.
Der IGF/IGFBP-2-13 Komplex besitzt in dosisabhängigerweise eine Überlebensförderende Wirkung auf PC- 12 Zellen. Diese Zellen entsprechen neuronalen Vorläuferzellen, so daß man davon ausgehen kann, das IGF/IGFBP-2-13 ein neuroprotektiver Faktor ist.The IGF / IGFBP-2-13 complex has a dose-dependent, survival-promoting effect on PC-12 cells. These cells correspond to neuronal progenitor cells, so that it can be assumed that IGF / IGFBP-2-13 is a neuroprotective factor.
Beispiel 3Example 3
Aufreinigung des erfindungsgem.äßen Peptids IGFBP-4-11Purification of the peptide IGFBP-4-11 according to the invention
Die Aufreinigung des erfindungsgemäßen Peptid IGFBP-4-11 erfolgte bis zur Stufe der zweiten präparativen Auftrennung völlig analog zu der unter Beispiel 1 beschriebenen Aufreinigung des IGFBP-2-13. Die weitere Aufreinigung erfolgte durchThe purification of the peptide IGFBP-4-11 according to the invention was carried out up to the stage of the second preparative separation, completely analogously to the purification of IGFBP-2-13 described in Example 1. The further purification was carried out by
Analytische Reverse-Phase C18-Chromatographie:Analytical reverse phase C18 chromatography:
Die im Assay bioaktive Fraktion 33 aus pH-Pool IV wurden über eine analytische Reverse- Phase-Säule aufgetrennt. Die Fraktion 34 enthielt die erfindungsgemäße Substanz.The bioactive fraction 33 from pH pool IV in the assay was separated on an analytical reverse phase column. Fraction 34 contained the substance according to the invention.
Chromatographiebedingungen:Chromatography conditions:
Säule: 1 cm x 25 cm StahlsäuleColumn: 1 cm x 25 cm steel column
Füllmaterial: Vydac RP-C45 ym, 300 ÄFilling material: Vydac RP-C45 ym, 300 Ä
Puffer A: 0,1% TFABuffer A: 0.1% TFA
Puffer B : 0, 1 % TFA, 80% AcetonitrilBuffer B: 0.1% TFA, 80% acetonitrile
Gradient: 0 - 80% B in 80 min, 80 - 100% B in 10 minGradient: 0 - 80% B in 80 min, 80 - 100% B in 10 min
Fluß: 2,5 ml/minFlow: 2.5 ml / min
Detektion: 230 nmDetection: 230 nm
Chromatographieanlage: KontronChromatography system: Kontron
Fraktionen: ä 1 min ab Start des GradientenFractions: ä 1 min from the start of the gradient
Massenbestimmungen Die Massenbestimmungen wurden auf einem Elektrospray-Massenspektrometer durchgeführt. Die Molekülmasse des Peptids wurde alsMass determinations The mass determinations were carried out on an electrospray mass spectrometer. The molecular weight of the peptide was as
IGFBP-4-11, 11 344 DaIGFBP-4-11, 11 344 Da
bestimmt.certainly.
SequenzbestimmungSequence determination
Die Aminosäuresequenz des aufgereinigten, nativen, biologisch aktiven Peptids IGFBP-4-11 wurde wie unter Beispiel 1 auf der Seite 13 beschrieben durchgeführt.The amino acid sequence of the purified, native, biologically active peptide IGFBP-4-11 was carried out as described under Example 1 on page 13.
Es ergab sich die folgende N-terminale Sequenz:The following N-terminal sequence resulted:
IGFBP-4- 11 , MW 11344 DaIGFBP-4- 11, MW 11344 Da
KVNGAPREDARPVPQGSXQSELIIRALERL...KVNGAPREDARPVPQGSXQSELIIRALERL ...
Der C-Terminus wurde durch den Vergleich der gemessenen Molekul.arm.asse mit der aus der Sequenz berechneten Masse bestimmt. Die Übereinstimmung dieser Massen liegt in der Messgenauigkeit des Elektrospray-Massenspektrometers von 0,1% der Gesamtmasse.The C-terminus was determined by comparing the measured molecular mass with the mass calculated from the sequence. The agreement of these masses lies in the measuring accuracy of the electrospray mass spectrometer of 0.1% of the total mass.
DatenbankvergleichDatabase comparison
Ein Datenbankvergleich wurde mit Hilfe des HUSAR-Programmpakets an den SwissProt und EMBL-Nucleinsäuredatenbanken durchgeführt. Die Peptidsequenz besitzt eine hundertprozentige Identität zu den aus der cDNA abgeleiteten Aminosäuren des humanen IGFBP-4.A database comparison was carried out using the HUSAR program package at the SwissProt and EMBL nucleic acid databases. The peptide sequence is 100% identical to the amino acids of human IGFBP-4 derived from the cDNA.
Bestimmung der Schwefelbrückenverknüpfung des IGFBP-4-11Determination of the sulfur bridge linkage of the IGFBP-4-11
Die Analyse der Schwefelbrückenverknüpfung erfolgte, indem das native Peptid IGFBP-4-11 parallel in zwei verschiedenen Ansätzen mit den Endoproteasen Chymotrypsin und Arg-C gespalten wurde. Die erhaltenen Spaltfragmente wurden dann mittels analytischer Reverse Phase Chromatographie voneinander getrennt und der Molekularmassen- und Sequenzanalyse unterzogen. Folgende Fragmente, welche jeweils zwei Cysteine und eine Schwefelbrücke enthalten, wurden erhalten: HPKQCHPALDGQRGKCW, MW 1960The sulfur bridge linkage was analyzed by cleaving the native peptide IGFBP-4-11 in parallel in two different batches with the endoproteases chymotrypsin and Arg-C. The cleavage fragments obtained were then separated from one another by means of analytical reverse phase chromatography and subjected to the molecular mass and sequence analysis. The following fragments, each containing two cysteines and a sulfur bridge, were obtained: HPKQCHPALDGQRGKCW, MW 1960
CVDRKTGVKLPGGLEPKGELDCHQLADSF, MW 3112CVDRKTGVKLPGGLEPKGELDCHQLADSF, MW 3112
PVPQGSCQSELHRPVPQGSCQSELHR
MW 3236MW 3236
THEDLYIIPIPNCDRTHEDLYIIPIPNCDR
Daraus ist ersichtlich, daß im nativen IGFBP-4-11 die Schwefelbrücken zwischen Cystein 1 und 2, zwischen Cystein 3 und 4 sowie zwischen Cystein 5 und 6 ausgebildet sind.From this it can be seen that in the native IGFBP-4-11 the sulfur bridges are formed between cysteine 1 and 2, between cysteine 3 and 4 and between cysteine 5 and 6.
Beispiel 4Example 4
Bestimmung der biologischen Aktivität des IGFBP-4-1 1Determination of the biological activity of IGFBP-4-1 1
Die Isolierung des IGFBP-4-11 erfolgte anhand seiner biologischen Aktivität in einem Prolife- rationsassay mit primären Knochenzellen (Osteoblasten), die zunächst aus Schädeldecken von Rattenembryonen isoliert werdenThe IGFBP-4-11 was isolated on the basis of its biological activity in a proliferation assay with primary bone cells (osteoblasts), which are initially isolated from the skullcap of rat embryos
D.azu wurden jeweils Aliquots der unter Beispiel 3 beschriebenen einzelnen Chromatographiestufen gefriergetrocknet und .anschließend dem biologischen Assay zugeführt. Die Fraktionen, die jeweils ein positives Signal ergaben, wurden der weiteren Aufreinigung unterzogen. Der Assay mißt die Proliferation der Zellen, indem 48 oder 72 Stunden nach Zugabe der Fraktionen der Einbau von radioaktivem Thymidin, also die DNA-Syntheserate, bestimmt wird. Als Positiv-Kontrolle wird in diesem Assay Transforming Growth Factor-beta (TGF-beta) oder fötales Kälberserum (FCS) eingesetzt. In 96 Loch-Platten werden 5.000 Osteoblasten-Zellen pro Loch in serumhaltigem Medium ausgesät. Es erfolgt die Zugabe von Aliquots (ca. 100 ml Äquivalent des Ausgangsmaterials) in die wells. 48 oder 72 Stunden später wird die Proliferationsrate (DNA-Syntheserate) der Zellen mittels der Zugabe und des Einbaus von radioaktivem Thymi- dins gemessen. Das Peptid IGFBP-4-11 besitzt in dosisabhängigerweise eine proliferations- förderende Wirkung auf diese primären Osteoblasten. Diese Zellen entsprechen typischen Knochenzellen, so daß man davon ausgehen kann, das IGFBP-4-11 ein osteoanaboler Faktor ist. Beispiel 5For this purpose, aliquots of the individual chromatography stages described in Example 3 were freeze-dried and then fed to the biological assay. The fractions, which each gave a positive signal, were subjected to further purification. The assay measures the proliferation of the cells by determining the incorporation of radioactive thymidine, ie the rate of DNA synthesis, 48 or 72 hours after the addition of the fractions. Transforming growth factor-beta (TGF-beta) or fetal calf serum (FCS) is used as a positive control in this assay. 5,000 osteoblast cells per hole are sown in 96-well plates in serum-containing medium. Aliquots (approx. 100 ml equivalent of the starting material) are added to the wells. 48 or 72 hours later, the proliferation rate (DNA synthesis rate) of the cells is measured by adding and incorporating radioactive thymidine. Depending on the dose, the peptide IGFBP-4-11 has a proliferation-promoting effect on these primary osteoblasts. These cells correspond to typical bone cells, so it can be assumed that IGFBP-4-11 is an osteoanabolic factor. Example 5
Isolierung der C-terminalen Domäne des IGFBP-3Isolation of the C-terminal domain of IGFBP-3
Durch ein ähnliches Verfahren wie in den Beispielen 1 und 3 konnte aus Hämofiltrat ein Peptid isoliert werden mit einer Masse von 2.470 Dalton (MALDI:2481 Dalton) mit der Sequenz: HTRISELKAEAVKKDRRKKLTQS (?) wodurch sich als IGFBP-3 C-terminale Sequenz folgende Sequenz ergibt:Using a procedure similar to that in Examples 1 and 3, a peptide with a mass of 2,470 daltons (MALDI: 2481 daltons) with the sequence: HTRISELKAEAVKKDRRKKLTQS (?) Could be isolated from hemofiltrate, resulting in the following sequence as IGFBP-3 C-terminal sequence results in:
KVDYESQSTDTQNFSSESKRETEYGPCRREMEDTLNHLKFLNNLSPRGVHIPNC DKKGFYKKKQCRPSKGRKRGFCWCVDKYGQPLPGYTTKGKEDVHCYSMQSKKVDYESQSTDTQNFSSESKRETEYGPCRREMEDTLNHLKFLNNLSPRGVHIPNC DKKGFYKKKQCRPSKGRKRGFCWCVDKYGQPLPGYTTKGKEDVHCYSMQSK
Beispiel 6Example 6
Durch ein ähnliches Verfahren wie in den Beispielen 1 und 3 konnte die N-terminale Domäne des IGFBP-4 mit der SequenzBy a procedure similar to that in Examples 1 and 3, the N-terminal domain of IGFBP-4 with the sequence
DEAIHCPPCSEEKLARCRPPVGCEELVREPGCGCCATCALGLGMPCGVYTPRCGSGL RCYPPRGVEKPLHTLMHGQGVCMELAEIEAIQESLQPSDKDEGDHPNNSFSPCSAHD RRCLQKHFAKIRDRSTSGGKM DEAIHCPPCSEEKLARCRPPVGCEELVREPGCGCCATCALGLGMPCGVYTPRCGSGL RCYPPRGVEKPLHTLMHGQGVCMELAEIEAIQESLQPSDKDEGDHPNNSFSPCSAHD RRCLQKHFKMDRDRSGG
Beispiel 7Example 7
Bestimmung der C-terminalen Sequenz des IPB-5 durch ein Verfahren wie in den Beispielen 1 und 3 konnte ein Peptid mit einer Masse von 13,5 kD bestimmt werden. Die Sequenzbestimmung ergab folgende Sequenz:Determination of the C-terminal sequence of the IPB-5 by a method as in Examples 1 and 3, a peptide with a mass of 13.5 kD could be determined. The sequence determination resulted in the following sequence:
KFVGGAENTAHPRIISAPEMRQESEQGPCRRHMEASLQELKASPRMVPRAVYLPNCD RKGFYKRKQCKPSRGRKRGICWCVDKYGMKLPGMEYVDGDFQCHTFDSSNVEKFVGGAENTAHPRIISAPEMRQESEQGPCRRHMEASLQELKASPRMVPRAVYLPNCD RKGFYKRKQCKPSRGRKRGICWCVDKYGMKLPGMEYVDGDFQCHTFDSSNVE
Die theoretische Masse beträgt 12,5 kD, daher ist davon auszugehen, daß das Peptid an Serin oder Theronin glykosyliert ist. The theoretical mass is 12.5 kD, so it can be assumed that the peptide is glycosylated on serine or theronine.

Claims

Patentansprüche claims
1. Peptide, dadurch gekennzeichnet, daß ihre Aminosäuresequenz Teilen der Aminosäuresequenz von Insulin-like growth factor binding protein entspricht sowie cyclische, gly- cosylierte, phosphorylierten, acetylierten, amidierten und/oder sulfatierten Derivate.1. Peptides, characterized in that their amino acid sequence corresponds to parts of the amino acid sequence of insulin-like growth factor binding protein and cyclic, glycosylated, phosphorylated, acetylated, amidated and / or sulfated derivatives.
2. Peptide nach Anspruch 1, dadurch gekemizeichnet, daß die Peptide aus Hämofiltrat isoliert werden können.2. Peptides according to claim 1, characterized in that the peptides can be isolated from hemofiltrate.
3. Peptide nach einem der Ansprüche 1 und/oder 2, dadurch gekennzeichnet, daß die Peptide eine Länge von 61 bis 115 Aminosäuren aufweisen.3. Peptides according to one of claims 1 and / or 2, characterized in that the peptides have a length of 61 to 115 amino acids.
4. Peptide nach einem der Ansprüche 1 bis 3, dadurch gekennzeichnet, daß die Peptide Sequenzen aufweisen, die N- oder C-terminalen Sequenzen von Insulin-like growth factor binding protein entsprechen.4. Peptides according to one of claims 1 to 3, characterized in that the peptides have sequences which correspond to the N- or C-terminal sequences of insulin-like growth factor binding protein.
5. Peptide gemäß mindestens einem der Ansprüche 1 bis 4 mit einer Aminosäuresequenz der Formel5. Peptides according to at least one of claims 1 to 4 with an amino acid sequence of the formula
R1-C-X1-PNC-X2-QC-X3-CWCV-X4-C-R2 R 1 -CX 1 -PNC-X 2 -QC-X 3 -CWCV-X 4 -CR 2
worinwherein
R, NH2, eine Aminosäure oder ein Peptid mit einer Aminosäuresequenz umfassend bis zu 41 Aminosäuren, Xj ein Peptid mit einer Aminosauresequenz umfassend 24 bis 31 Aminosäuren, X2 ein Peptid mit einer Aminosäuresequenz umfassend 9 Aminosäuren, X3 ein Peptid mit einer Aminosäuresequenz umfassend 10 Aminosäuren, X4 ein Peptid mit einer Aminosäuresequenz umfassend 18 bis 24 Aminosäuren, R2 COOH, CONH2 oder ein Peptid mit bis zu 12 Aminosäuren darstellt und cyclische, glycosylierte, phos- phorylierte, acetylierte, amidierte, sulfatierte Derivate sowie Fragmente mit der physiologischen Fähigkeit des IGFBP.R, NH 2 , an amino acid or a peptide with an amino acid sequence comprising up to 41 amino acids, Xj a peptide with an amino acid sequence comprising 24 to 31 amino acids, X 2 a peptide with an amino acid sequence comprising 9 amino acids, X 3 a peptide with an amino acid sequence 10 amino acids, X 4 is a peptide with an amino acid sequence comprising 18 to 24 amino acids, R 2 COOH, CONH 2 or a peptide with up to 12 amino acids and cyclic, glycosylated, phosphorylated, acetylated, amidated, sulfated derivatives and fragments with the physiological ability of the IGFBP.
6. Peptide gemäß mindestens einem der Anspruch 1 bis 5 mit Disulfidbrücken der Formel6. Peptides according to at least one of claims 1 to 5 with disulfide bridges of the formula
RrC-X,-PNC-X2-QC-X3-CWCV-X4-C-R2 R r CX, -PNC-X 2 -QC-X 3 -CWCV-X 4 -CR 2
7. Peptide gemäß mindestens einem der Ansprüche 1 bis 6, dadurch gekennzeichnet, daß X2 an Position 4 seiner Aminosäuresequenz ein Glycin aufweist und/oder X3 an der Position 9 seiner Aminosäuresequenz ein Glycin aufweist und/oder X4 an der Position 4 oder 5 seiner Aminosäuresequenz ein Glycin aufweist und/oder X4 an der Position 9 oder 10 seiner Aminosäuresequenz ein Glycin aufweist.7. Peptides according to at least one of claims 1 to 6, characterized in that X 2 has a glycine at position 4 of its amino acid sequence and / or X 3 has a glycine at position 9 of its amino acid sequence and / or X 4 at position 4 or 5 of its amino acid sequence has a glycine and / or X 4 has a glycine at position 9 or 10 of its amino acid sequence.
8. Peptide gemäß mindestens einem der Ansprüche 1 bis 7, dadurch gekennzeichnet, daß X, an der Position 8 oder V ist und/oder X, an der Position 1 1 oder I ist und/oder X2 an der Position 1 D oder N ist und/oder X2 an der Position 9 K oder R ist und/oder X3 an der Position 3 S oder A ist und/oder an der Position 8 R oder A ist.8. Peptides according to at least one of claims 1 to 7, characterized in that X, at position 8 or V and / or X, at position 1 is 1 or I and / or X 2 at position 1 is D or N. and / or X 2 at position 9 is K or R and / or X 3 at position 3 is S or A and / or at position 8 is R or A.
9. Peptide gemäß mindestens einem der Ansprüche 1 bis 8, dadurch gekennzeichnet, daß R, ausgewählt wird aus9. Peptides according to at least one of claims 1 to 8, characterized in that R is selected from
APSEEDHSILWDAISTYDGSKALHVTNIKKWKEP,APSEEDHSILWDAISTYDGSKALHVTNIKKWKEP,
GGKHHLGLEEPKKLRPPPARTPGGKHHLGLEEPKKLRPPPARTP
GKGGKHHLGLEEPKKLRPPPARTP,GKGGKHHLGLEEPKKLRPPPARTP,
GHAKDSQRYKVDYESQSTDTQNFSSESKRETEYGP,GHAKDSQRYKVDYESQSTDTQNFSSESKRETEYGP,
KVNGAPREDARPVPQGS,KVNGAPREDARPVPQGS,
LTQSKFVGGAENTAHPRIISAPEMRQESEQGP,LTQSKFVGGAENTAHPRIISAPEMRQESEQGP,
PQAGTARPQDVNRRDQQRNPGTSTTPSQPNSAGVQDTEMGP.PQAGTARPQDVNRRDQQRNPGTSTTPSQPNSAGVQDTEMGP.
10. Peptide gemäß mindestens einem der Ansprüche 1 bis 9, dadurch gekennzeichnet, daß X, ausgewählt wird aus10. Peptides according to at least one of claims 1 to 9, characterized in that X is selected from
RIELYRVVESLAKAQETSGEEISKFYL,RIELYRVVESLAKAQETSGEEISKFYL,
QQELDQVLERISTMRLPDERGPLEHLYSLHI,QQELDQVLERISTMRLPDERGPLEHLYSLHI,
RREMEDTLNHLKFLNVLSPRGVHI,RREMEDTLNHLKFLNVLSPRGVHI,
QSELHRALERLAASQSRTHEDLYIIPI,QSELHRALERLAASQSRTHEDLYIIPI,
RRHMEASLQELKASPRMVPRAVYL,RRHMEASLQELKASPRMVPRAVYL,
RRHLDSVLQQLQTEVYRGAQTLYV.RRHLDSVLQQLQTEVYRGAQTLYV.
11. Peptide gemäß mindestens einem der Ansprüche 1 bis 10, dadurch gekennzeichnet, daß X2 ausgewählt wird aus11. Peptides according to at least one of claims 1 to 10, characterized in that X 2 is selected from
NKNGFYHSR, DKHGLYNLK, DKKGFYKKK, DRNGNFHPK, DRKGFYKRK, DHRGFYRKR.NKNGFYHSR, DKHGLYNLK, DKKGFYKKK, DRNGNFHPK, DRKGFYKRK, DHRGFYRKR.
12. Peptide gemäß mindestens einem der Ansprüche 1 bis 11, dadurch gekennzeichnet, daß X3 ausgewählt wird aus12. Peptides according to at least one of claims 1 to 11, characterized in that X 3 is selected from
ETSMDGEAGL,ETSMDGEAGL,
KMSLNGQRGE,KMSLNGQRGE,
RPSKGRKRGF,RPSKGRKRGF,
HPALDGQRGK,HPALDGQRGK,
KPSRGRKRGI,KPSRGRKRGI,
RSSQGQRRGP.RSSQGQRRGP.
13. Peptide gemäß mindestens einem der Ansprüche 1 bis 12, dadurch gekeimzeichnet, daß X4 ausgewählt wird aus13. Peptides according to at least one of claims 1 to 12, characterized in that X 4 is selected from
YPWNGKRIPGSPEIRGDPN,YPWNGKRIPGSPEIRGDPN,
NPNTGKLOQGAPTIRGDPE,NPNTGKLOQGAPTIRGDPE,
DKYGQPLPGYTTKGKEDVH,DKYGQPLPGYTTKGKEDVH,
DRKTGVKLPGGLEPKGELD,DRKTGVKLPGGLEPKGELD,
DKYGMKLPGMEYVDGDFQ,DKYGMKLPGMEYVDGDFQ,
DRMGKSLPGSPDGNGSSS.DRMGKSLPGSPDGNGSSS.
14. Peptide gemäß mindestens einem der Ansprüche 1 bis 13, dadurch gekennzeichnet, daß R2 ausgewählt wird aus14. Peptides according to at least one of claims 1 to 13, characterized in that R 2 is selected from
QIYFNVQN,QIYFNVQN,
HLFYNEQQEARGVHTQRMQ,HLFYNEQQEARGVHTQRMQ,
HLFYNEQQE,HLFYNEQQE,
YSMQSK,YSMQSK,
HQLADSFRE,HQLADSFRE,
HTFDSSNVE,HTFDSSNVE,
PTGSSG.PTGSSG.
15. Peptide gemäß einem der Ansprüche 1 bis 14, dadurch gekennzeichnet, daß die Peptide ausgewählt werden aus15. Peptides according to one of claims 1 to 14, characterized in that the peptides are selected from
IBP-1IBP-1
APSEEDHSILWDAISTYDGSKALHVTNIKKWKEPCRIELYRVVESLAKAQETSAPSEEDHSILWDAISTYDGSKALHVTNIKKWKEPCRIELYRVVESLAKAQETS
GEEISKFYLPNCNKNGFYHSRQCETSMDGEAGLCWCVYPWNGKRIPGSPEIRGGEEISKFYLPNCNKNGFYHSRQCETSMDGEAGLCWCVYPWNGKRIPGSPEIRG
DPNCQIYFNVQNDPNCQIYFNVQN
IGFBP-2 GKGGKHHLGLEEPKKLRPPPARTPCQQELDQVLERISTMRLPDERGPLEHLYS LHIPNCDKHGLYNLKQCKMSLNGQRGECWCVNPNTGKLIQGAPTIRGDPECH LFYNEQQEARGVHTQRMQIGFBP-2 GKGGKHHLGLEEPKKLRPPPARTPCQQELDQVLERISTMRLPDERGPLEHLYS LHIPNCDKHGLYNLKQCKMSLNGQRGECWCVNPNTGKLIQGAPTIRGDPECH LFYNEQQEARGVHTQRMQ
GGKHHLGLEEPKKLRPPPARTPCQQELDQVLERISTMRLPDERGPLEHLYSLH IPNCDKHGLYNLKQCKMSLNGQRGECWCVNPNTGKLIQGAPTIRGDPECHLF YNEQQEARGVHTQRMQGGKHHLGLEEPKKLRPPPARTPCQQELDQVLERISTMRLPDERGPLEHLYSLH IPNCDKHGLYNLKQCKMSLNGQRGECWCVNPNTGKLIQGAPTIRGDPECHLF YNEQQEARGVHTQRMQ
IGFBP-3IGFBP-3
GHAKDSQRYKVDYESQSTDTQNFSSESKRETEYGPCRREMEDTLNHLKFLNV LSPRGVHIPNCDKKGFYKKKQCRPSKGRKRGFCWCVDKYGQPLPGYTTKGK EDVHCYSMQSKGHAKDSQRYKVDYESQSTDTQNFSSESKRETEYGPCRREMEDTLNHLKFLNV LSPRGVHIPNCDKKGFYKKKQCRPSKGRKRGFCWCVDKYGQPLPGYTTKGK EDVHCYSMQSK
KVDYESQSTDTQNFSSESKRETEYGPCRREMEDTLNHLKFLNVLSPRGV HIPNCDKKGFYKKKQCRPSKGRKRGFCWCVDKYGQPLPGYTTKGKEDVHCY SMQSKKVDYESQSTDTQNFSSESKRETEYGPCRREMEDTLNHLKFLNVLSPRGV HIPNCDKKGFYKKKQCRPSKGRKRGFCWCVDKYGQPLPGYTTKGKEDVHCY SMQSK
HPLHSKIIIIKKGHAKDSQRYHPLHSKIIIIKKGHAKDSQRY
IGFBP-4IGFBP-4
DEAIHCPPCSEEKLARCRPPVGCEELVREPGCGCCATCALGLGMPCGVYTPRC GSGLRCYPPRGVEKPLHTLMHGQGVCMELAEIEAIQESLQPSDKDEGDHPNNS FSPCSAHDRRCLQKHFAKIRDRSTSGGKMDEAIHCPPCSEEKLARCRPPVGCEELVREPGCGCCATCALGLGMPCGVYTPRC GSGLRCYPPRGVEKPLHTLMHGQGVCMELAEIEAIQESLQPSDKDEGDHPNNS FSPCSAHDRRCLQKHFKMDRDRSG
KVNGAPREDARPVPQGSCQSELHRALERLAASQSRTHEDLYIIPIPNCDRNGNF HPKQCHPALDGQRGKCWCVDRKTGVKLPGGLEPKGELDCHQLADSFREKVNGAPREDARPVPQGSCQSELHRALERLAASQSRTHEDLYIIPIPNCDRNGNF HPKQCHPALDGQRGKCWCVDRKTGVKLPGGLEPKGELDCHQLADSFRE
IGFBP-5IGFBP-5
LTQSKFVGGAENTAHPRIISAPEMRQESEQGPCRRHMEASLQELKASPRMVPR AVYLPNCDRKGFYKRKQCKPSRGRKRGICWCVDKYGMKLPGMEYVDGDFQLTQSKFVGGAENTAHPRIISAPEMRQESEQGPCRRHMEASLQELKASPRMVPR AVYLPNCDRKGFYKRKQCKPSRGRKRGICWCVDKYGMKLPGMEYVDGDFQ
CHTFDSSNVECHTFDSSNVE
KFVGGAENTAHPRIISAPEMRQESEQGPCRRHMEASLQELKASPRMVPRAV YLPNCDRKGFYKRKQCKPSRGRKRGICWCVDKYGMKLPGMEYVDGDFQCH TFDSSNVEKFVGGAENTAHPRIISAPEMRQESEQGPCRRHMEASLQELKASPRMVPRAV YLPNCDRKGFYKRKQCKPSRGRKRGICWCVDKYGMKLPGMEYVDGDFQCH TFDSSNVE
HTRISELKAEAVKKDRRKKLTQSHTRISELKAEAVKKDRRKKLTQS
IGFBP-6IGFBP-6
PQAGTARPQDVNRRDQQRNPGTSTTPSQPNSAGVQDTEMGPCRRHLDSVLQQ LQTEVYRGAQTLYVPNCDHRGFYRKRQCRSSQGQRRGPCWCVDRMGKSLPG SPDGNGSSSCPTGSSGPQAGTARPQDVNRRDQQRNPGTSTTPSQPNSAGVQDTEMGPCRRHLDSVLQQ LQTEVYRGAQTLYVPNCDHRGFYRKRQCRSSQGQRRGPCWCVDRMGKSLPG SPDGNGSSSCPTGSSG
16. Verfahren zur Herstellung der Peptide gemäß mindestens einem der Ansprüche 1 bis 15 durch Aufreinigung aus humanem Blutfiltrat oder Urin, durch Festphasenpeptid- synthese oder durch Expression in rekombinanten Mikroorganismen. 16. A process for the preparation of the peptides according to at least one of claims 1 to 15 by purification from human blood filtrate or urine, by solid phase peptide synthesis or by expression in recombinant microorganisms.
17. Komplexe von Peptiden gemäß mindestens einem der Ansprüche 1 bis 15 mit hIGF-I (humaner Insulin-like growth factor-I, MW 7649) oder hIGF-II (humaner Insulin-like growth factor-II, MW 7491) sowie dessen biologisch aktive Fragmenten und/oder Derivaten, insbesondere amidierten, acetyherten, sulfatierten, phosphoryherten und/oder glykosylierten Derivaten.17. Complexes of peptides according to at least one of claims 1 to 15 with hIGF-I (human insulin-like growth factor-I, MW 7649) or hIGF-II (human insulin-like growth factor-II, MW 7491) and its biological active fragments and / or derivatives, especially amidated, acetylated, sulfated, phosphorylated and / or glycosylated derivatives.
18. Nucleinsäure, dadurch gekennzeichnet, daß sie für Peptide gemäß mindestens einem der Ansprüche 1 bis 15 kodiert.18. Nucleic acid, characterized in that it codes for peptides according to at least one of claims 1 to 15.
19. Antisensenucleotid, dadurch gekennzeichnet, daß es unter stringenten Bedingungen eine Nucleinsäuresequenz bindet, die für ein Peptid gemäß mindestens einem der Ansprüche 1 bis 15 kodiert.19. Antisense nucleotide, characterized in that, under stringent conditions, it binds a nucleic acid sequence which codes for a peptide according to at least one of Claims 1 to 15.
20. Antiköφer, dadurch gekennzeichnet, daß er an ein Peptid gemäß mindestens einem der Ansprüche 1 bis 15 bindet.20. Antiköφer, characterized in that it binds to a peptide according to at least one of claims 1 to 15.
21. Inhibitor, dadurch gekennzeichnet, daß er die biologische Aktivität von Peptiden gemäß mindestens einem der Ansprüche 1 bis 15 hemmt.21. Inhibitor, characterized in that it inhibits the biological activity of peptides according to at least one of claims 1 to 15.
22. Inhibitor, dadurch gekennzeichnet, daß er die Expression von Peptiden gemäß mindestens einem der Ansprüche 1 bis 15 hemmt.22. Inhibitor, characterized in that it inhibits the expression of peptides according to at least one of claims 1 to 15.
23. Verwendung von Peptiden gemäß mindestens einem der Ansprüche 1 bis 15, Komplexen gemäß Anspruch 17, Nucleinsäuren gemäß Anspruch 18, zur Herstellung eines Arzneimittels zur Behandlung der Unterexpression von Insulin-like Growth Factor Binding Proteinen.23. Use of peptides according to at least one of claims 1 to 15, complexes according to claim 17, nucleic acids according to claim 18, for the manufacture of a medicament for the treatment of under-expression of insulin-like growth factor binding proteins.
24. Verwendung von Antisensenucleotiden gemäß Anspruch 19, Antiköφern gemäß Anspruch 20, Inhibitoren gemäß Anspruch 21 und/oder Inhibitoren gemäß Anspruch 22 zur Herstellung eines Arzneimittels zur Behandlung der Überexpression Insulin-like Growth Factor Bindmg Proteinen.24. Use of antisense nucleotides according to claim 19, antibodies according to claim 20, inhibitors according to claim 21 and / or inhibitors according to claim 22 for the manufacture of a medicament for the treatment of overexpression insulin-like growth factor binding proteins.
25 Arzneimittel enthaltend Peptide gemäß mindestens einem der Ansprüche 1 bis 15, Komplexe gemäß Anspruch 17, Nucleinsäuren gemäß Anspruch 18, Antisen- senucleotiden gemäß Anspruch 19, Antiköφern gemäß Anspruch 20, Inhibitoren gemäß Anspruch 21 und/oder Inhibitoren gemäß Anspruch 22 in einer pharmazeutisch üblichen Darreichungsform zur oralen, intravenösen, intramuskulären, intracutanen, intrathekalen Anwendung oder als Aerosol zur transpulmonalen Applikation.25 medicament containing peptides according to at least one of claims 1 to 15, complexes according to claim 17, nucleic acids according to claim 18, antise senucleotides according to claim 19, antibodies according to claim 20, inhibitors according to claim 21 and / or inhibitors according to claim 22 in a pharmaceutically customary dosage form for oral, intravenous, intramuscular, intracutaneous, intrathecal use or as an aerosol for transpulmonary application.
26. Verwendung eines Arzneimittels gemäß Anspruch 25 zur Behandlung von Muskelschwund, Osteroporose, Diabetes, amyloidaler lateraler Sklerose, peripheren und zentralen Neuropathien, entzündlichen Prozessen, gestörten Entzündungsreaktionen, Tumorerkrankungen, entzündlichen und neoplastischen Erkrankungen, Wachstumsstörung, Erkrankung der Muskulatur, Erkrankung des Knochenapparates und/oder zur Wund- und Rnochenheilung.26. Use of a medicament according to claim 25 for the treatment of muscle loss, osteoporosis, diabetes, amyloidal lateral sclerosis, peripheral and central neuropathies, inflammatory processes, disturbed inflammatory reactions, tumor diseases, inflammatory and neoplastic diseases, growth disorders, muscle diseases, diseases of the bone apparatus and / or for healing wounds and bones.
27. Verwendung der Nucleinsäure gemäß Anspruch 18 und/oder der Antisensenucleotide gemäß Anspruch 19 zur Herstellung eines Medikamentes zur Behandlung somatischer oder nicht-somatischer Generkrankungen.27. Use of the nucleic acid according to claim 18 and / or the antisense nucleotides according to claim 19 for the manufacture of a medicament for the treatment of somatic or non-somatic genetic diseases.
28. Diagnostikmittel enthaltend Peptide gemäß mindestens einem der Ansprüche 1 bis 15, Komplexe gemäß Anspruch 17, Nucleinsäuren gemäß Anspruch 18, Antisensenucleotide gemäß Anspruch 19, Antiköφern gemäß Anspruch 20, Inhibitoren gemäß Anspruch 21 und/oder Inhibitoren gemäß Anspruch 22 sowie weitere Hilfsmittel.28. Diagnostic agent containing peptides according to at least one of claims 1 to 15, complexes according to claim 17, nucleic acids according to claim 18, antisense nucleotides according to claim 19, antibodies according to claim 20, inhibitors according to claim 21 and / or inhibitors according to claim 22 and further auxiliaries.
29. Verwendung der Diagnostikmittel gemäß Anspruch 28 zur Diagnose von Funktionsstörungen des Knochens, der Muskeln, des Nervensystems, der Lymphorgane, des Magen-Darm-Traktes, des Immunsystems, von Diabetes, inflammatorischen und neoplastischen Prozessen sowie als Marker bei Krebs. 29. Use of the diagnostic agent according to claim 28 for the diagnosis of functional disorders of the bones, the muscles, the nervous system, the lymphatic organs, the gastrointestinal tract, the immune system, diabetes, inflammatory and neoplastic processes and as a marker in cancer.
PCT/EP1998/008405 1997-12-22 1998-12-22 Insulin-like growth factor binding protein fragments and the utilization thereof WO1999032620A1 (en)

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Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000054806A1 (en) * 1999-03-15 2000-09-21 Mayo Foundation For Medical Education And Research Insulin-like growth factor binding protein-4 protease
WO2001081394A1 (en) * 2000-04-27 2001-11-01 Biowindow Gene Development Inc. Shanghai A novel polypeptide, human insulin-like growth factor-binding protein 9 and the polynucleotide encoding thereof
WO2002012301A1 (en) * 2000-06-14 2002-02-14 Biowindow Gene Development Inc. Shanghai A novel polypeptide-insulin-like growth factor binding protein 16.17 and the polynucleotide encoding said polypeptide
WO2002012493A1 (en) * 2000-06-14 2002-02-14 Biowindow Gene Development Inc. Shanghai Novel polypeptide--- an insulin like growth factor binding protein 11.88 and polynucleotide encoding it
US6403764B1 (en) 1999-01-06 2002-06-11 Genentech, Inc. Insulin-like growth factor-1 protein variants
US6500630B2 (en) 2001-01-12 2002-12-31 Mayo Foundation For Medical Education And Research Marker for inflammatory conditions
US6506874B1 (en) 1999-01-06 2003-01-14 Genentech, Inc. IGF-I variants
EP1435986A2 (en) * 2001-09-18 2004-07-14 Bioexpertise, Llc Igf-binding protein-derived peptide or small molecule
JP2005508295A (en) * 2001-06-07 2005-03-31 エフ.ホフマン−ラ ロシュ アーゲー Variants of IGF binding proteins and methods for producing agonists thereof
EP1560933A2 (en) * 2002-11-14 2005-08-10 Wyeth Methods and compositions for treating neurological disorders
WO2006069029A1 (en) * 2004-12-24 2006-06-29 Insmed, Inc. Purified rhigf-i/rhigfbp-3 complexes and their method of manufacture
US7115382B1 (en) 1999-03-15 2006-10-03 Mayo Foundation For Medical Education And Research Method for detecting IGFBP-4 protease without detecting IGFBP-4 protease/proMBP Complex
US7192738B2 (en) 2003-10-03 2007-03-20 Genentech, Inc. IGF binding proteins
EP1806358A3 (en) * 2005-09-05 2007-10-03 Immatics Biotechnologies GmbH Tumor-associated peptides binding promiscuously to human leukocyte antigen (HLA) class II molecules
US7423017B2 (en) 1997-04-04 2008-09-09 Genentech, Inc. Method for treating cartilage disorders
WO2010099139A2 (en) 2009-02-25 2010-09-02 Osi Pharmaceuticals, Inc. Combination anti-cancer therapy
WO2013152252A1 (en) 2012-04-06 2013-10-10 OSI Pharmaceuticals, LLC Combination anti-cancer therapy
US20140100160A1 (en) * 2012-10-09 2014-04-10 Research & Business Foundation Sungkyunkwan University Novel use of c-terminal domain of igfbp-5 comprising heparin-binding domain as an angiogenesis inhibitor
WO2014165137A1 (en) * 2013-03-12 2014-10-09 The University Of North Carolina At Chapel Hill Compounds and methods for treating obesity and controlling weight
CN112566653A (en) * 2018-05-24 2021-03-26 阿莫利特制药公司 Role of heparin-binding domain of IGFBP-2 in treatment of metabolic disorders

Families Citing this family (5)

* Cited by examiner, † Cited by third party
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GB0521276D0 (en) * 2005-10-19 2005-11-30 Lewitt Moira S Medical uses and therapies based upon the action of azurocidin on IGFBP-1
BRPI0619179A2 (en) * 2005-11-30 2011-09-13 Nestec Sa Methods For Treating Muscle Loss
WO2008086813A2 (en) * 2007-01-19 2008-07-24 Kobenhavns Universitet Peptides derived from proteins of the insulin super-family
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US20220259265A1 (en) * 2019-07-30 2022-08-18 The University Of Sydney Inhibitors and Use Thereof in Cancer Treatment

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996001636A1 (en) * 1994-07-08 1996-01-25 Royal Children's Hospital Research Foundation A method for the prophylaxis and/or treatment of proliferative and/or inflammatory skin disorders
WO1996002565A1 (en) * 1994-07-20 1996-02-01 Celtrix Pharmaceuticals, Inc. Igf/igfbp complex for promoting bone formation and for regulating bone remodeling
EP0725080A1 (en) * 1994-06-27 1996-08-07 Snow Brand Milk Products Co., Ltd. Novel protein and process for producing the same
WO1997030084A1 (en) * 1996-02-15 1997-08-21 Genetics Institute, Inc. Tnf receptor death domain ligand proteins and inhibitors of ligand binding

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE247164T1 (en) * 1991-01-08 2003-08-15 Chiron Corp INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN
DE69333063T2 (en) * 1992-11-04 2004-05-13 Chiron Corp. (N.D.Ges.D. Staates Delaware), Emeryville SHORTENED INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN WITH MITOGENIC ACTIVITY

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0725080A1 (en) * 1994-06-27 1996-08-07 Snow Brand Milk Products Co., Ltd. Novel protein and process for producing the same
WO1996001636A1 (en) * 1994-07-08 1996-01-25 Royal Children's Hospital Research Foundation A method for the prophylaxis and/or treatment of proliferative and/or inflammatory skin disorders
WO1996002565A1 (en) * 1994-07-20 1996-02-01 Celtrix Pharmaceuticals, Inc. Igf/igfbp complex for promoting bone formation and for regulating bone remodeling
WO1997030084A1 (en) * 1996-02-15 1997-08-21 Genetics Institute, Inc. Tnf receptor death domain ligand proteins and inhibitors of ligand binding

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KALUS, WENZEL ET AL: "Structure of the IGF-binding domain of the insulin-like growth factor-binding protein-5 (IGFBP-5): implications for IGF and IGF-I receptor interactions", EMBO J. (1998), 17(22), 6558-6572 CODEN: EMJODG;ISSN: 0261-4189, 16 November 1998 (1998-11-16), XP002103022 *

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WO1999032620A9 (en) 1999-09-23

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