WO1999032620A1

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

Info

Publication number: WO1999032620A1
Application number: PCT/EP1998/008405
Authority: WO
Inventors: Wolf-Georg Forssmann; Ludger STÄNDKER; Maik Obendorf; Lothar Kling; Hans-Georg Opitz; Hossein Mostafavi
Original assignee: Forssmann Wolf Georg; Staendker Ludger; Maik Obendorf; Lothar Kling; Opitz Hans Georg; Hossein Mostafavi
Priority date: 1997-12-22
Filing date: 1998-12-22
Publication date: 1999-07-01
Also published as: DE19757250A1; JP2002508931A; EP1042476A1; CA2315974A1; WO1999032620A9

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 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.

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 _r CX, -PNC-X ₂ -QC-X ₃ -CWCV-X ₄ -CR ₂

wherein

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.

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

R _r CX _r PNC-X ₂ -QC-X ₃ -CWCV-X ₄ -CR ₂

correspond.

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.

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 a particularly preferred embodiment, R is selected from

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) and / or

X selected from

RIELYRWESLAKAQETSGEEISKFYL (SEQ ID NO: 8), QQELDQVLERIST RLPDERGPLEHLYSLHI (SEQ ID NO: 9), RREMEDTLNHLKFLNVLSPRGVHI (SEQ ID NO: 10), QSELHRALERLAASQSRTHEDLYIIPI (SEQ ID NO: 11), RRHMEASLQELVYVVLQQQ NOVASVLQQQ

X ₂ selected from

NKNGFYHSR (SEQ ID NO 14)

DKHGLYNLK (SEQ ID NO 15)

DKKGFYKKK (SEQ ID NO 16)

DRNGNFHPK (SEQ ID NO 17)

DRKGFYKRK (SEQ ID NO 18)

DHRGFYRKR (SEQ ID NO 19) and / or

X ₃ selected from

ETS DGEAGL (SEQ ID NO 20),

KMSLNGQRGE (SEQ ID NO 21),

RPSKGRKRGF (SEQ ID NO 22),

HPALDGQRGK (SEQ ID NO 23),

KPSRGRKRGI (SEQ ID NO 24),

RSSQGQRRGP (SEQ ID NO 25) and / or

x ₄ selected from

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

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).

Preferred peptides have the following sequences IGFBP-1

APSEEDHSIL DAISTYDGSKALHVTNIKKWKEPCRIELYRWESLAKAQETSGEEISKFYLP NCNKNGFYHSRQCETSMDGEAGLCWCWPWNGKRIPGSPEIRGDPNCQIYFNVQN (SEQ ID NO: 39)

IGFBP-2

GKGGKHHLGLEEPKKLRPPPARTPCQQELDQVLERISTMRLPDERGPLEHLYSLHIPNCDKHG LYNLKQCKMSLNGQRGECWCVNPNTGKLIQGAPTIRGDPECHLFYNEQQEARGVHTQRMQ) SE

GGKHHLGLEEPKKLRPPPARTPCQQELDQVLERISTMRLPDERGPLEHLYSLHIPNCDKHG LYNLKQCKMSLNGQRGECWCVNPNTGKLIQGAPTIRGDPECHLFYNEQQEARGVHTQRMQ (SEQ ID

IGFBP-3

GHAKDSQRYKVDYESQSTDTQNFSSESKRETEYGPCRREMEDTLNHLKFLNVLSPRGVHIPNC DKKGFYKKKQCRPSKGRKRGFCWCVDKYGQPLPGYTTKGKEDVHCYSMQSK (SEQ ID NO: 41

KVDYESQSTDTQNFSSESKRETEYGPCRREMEDTLNHLKFLNVLSPRGVHIPNC DKKGFYKKKQCRPSKGRKRGFC CVDKYGQPLPGYTTKGKEDVHCYSMQSK (SEQ ID NO: 46)

HPLHSKIIIIKKGHAKDSQRY (SEQ ID NO: 47)

IGFBP-4

DEAIHCPPCSEEKLARCRPPVGCEELVREPGCGCCATCALGLGMPCGVYTPRCGSGLRCYPPR GVEKPLHTLMHGQGVCMELAEIEAIQESLQPSDKDEGDHPNNSFSPCSAHDRRCLQKHFAKM DRSTSGG NOK

KVNGAPREDARPVPQGSCQSELHRALERLAASQSRTHEDLYIIPIPNCDRNGNFHPKQCHPAL DGQRGKC CVDRKTGVKLPGGLEPKGELDCHQLADSFRE (SEQ ID NO: 42)

IGFBP-5

LTQSKFVGGAENTAHPRIISAPEMRQESEQGPCRRHMEASLQELKASPRMVPRAVYLPNCDRK GFYKRKQCKPSRGRKRGIC CVDKYGMKLPGMEYVDGDFQCHTFDSSNVE (SEQ ID NO: 43)

KFVGGAENTAHPRIISAPEMRQESEQGPCRRHMEASLQELKASPRMVPRAVYLPNCDRK GFYKRKQCKPSRGRKRGIC CVDKYGMKLPGMEYVDGDFQCHTFDSSNVE (SEQ ID NO: 49)

HTRISELKAEAVKKDRRKKLTQS (SEQ ID NO: 50)

IGFBP-6

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.

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.

Complexes of IGFBP with 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.

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.

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.

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. 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.

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.

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.

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. 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 purification of the peptide according to the invention or its complex is illustrated by the following examples:

example 1

Purification and peptide chemical analysis of IGFBP-2-13

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.

Hemofiltrate batch extraction

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.

Chromatography conditions:

Column: Vantage VA 250 (Amicon, hosts)

Column material: Fractogel TSK SP 650 (M), 25 cm x 20 cm

Flow: 3 1 / min

Detection: 280 nm, pH, conductivity

Buffer A: Hemofiltrate pH 2.1, conductivity 5.5 mS / cm

Buffer B: 0.5 M ammonium acetate

Attachment: Autopilot Chromatography System, (PerSeptive Biosystems, Wiesbaden)

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.

First preparative separation

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.

Chromatography conditions:

Column: Vantage 250 VA

Column material: Fractogel TSK SP 650 (M), 25 cm x 20 cm

Flow: up to 3 1 / min during the order,

0.5 to 1 1 during elution

Detection: 280 nm, pH, conductivity

Sample: Hemofiltrate pH 2.7, conductivity 5.5 mS / cm

System: autopilot chromatography system,

(PerSeptive Biosystems, Wiesbaden)

After 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

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.

Second preparative separation:

The individual pH pools are separated for fractionation and simultaneous desalination using a reverse phase chromatography

Chromatography conditions:

Pillar: FineLine 100 (Pharmacia, Freiburg)

Column material: Source RPC, 15 μm 10 x 12.5 cm (FineLine 100)

Flow: 150 mL / min (FineLine 100)

Detection: 280 nm, conductivity, pH

Buffer A: 10 mM HCl

Buffer B: 80% acetonitrile in 10 mM HCl

Gradient: 0 to 60% buffer B in 5 column volumes

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.

Semi-preparative reverse phase C18 chromatography:

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.

Chromatography conditions:

Column: 4.7 cm x 30 cm steel column

Filling material: Vydac RP-C 18 15-20 μm, 300 Ä

Buffer A: 0.1% TFA

Buffer B: 0.1% TFA, 80% acetonitrile

Gradient: 5 to 50% B in 45 min, 50 to 100% B in 10 min

Flow: 42 ml / min

Detection: 214 nm and 280 nm

Chromatography system: BioCad

Fractions: 1.5 minutes from the start of the gradient

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.

Chromatography conditions:

Column: 4.7 cm x 30 cm steel column

Filling material: Vydac RP-C18 15-20 μm, 300 Ä

Buffer A: 0.1% TFA, 20% methanol

Buffer B: 0.1% TFA, 100% methanol

Gradient: 0 to 20% B in 6.5 min, 20 to 80% B in 55 min,

80 to 100% B in 13 min

Flow: 30 ml / min

Detection: 214 nm and 280 nm

Chromatography system: BioCad

Fractions: 1.5 minutes from the start of the gradient

Cation exchange chromatography:

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.

Chromatography conditions:

Column: 1 cm x 5 cm steel column

Filling material: Pepkat, Biotek 5 μm, 300 Ä

Buffer A: 20 mM sodium phosphate, pH 3.0

Buffer B: 20 mM sodium phosphate, pH 3.0, 1.5 M NaCl

Gradient: 0 to 50% B in 50 min, 50 to 100% B in 10 min

Flow: 3 ml / min

Detection: 280 nm

Chromatography system: BioCad Sprint

Fractions: a 1.5 min from the start of the gradient

Analytical reverse phase chromatography:

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.

Chromatography conditions:

Column: 1 cm x 25 cm steel column

Filling material: Vydac RP-C 18 5 μm, 300 Ä

Buffer A: 0.1% TFA

Buffer B: 0.1% TFA, 80% acetonitrile

Gradient: 5 to 50% B in 45 min, 50 to 100% B in 10 min

Flow: 2 ml / min

Detection: 220 nm

Chromatography system: Kontron

Fractions: ä 1 min from the start of the gradient Second analytical reverse phase C18 chromatography:

The bioactive fraction 56 from the previous separation step was further purified on an analytical reverse phase column.

Chromatography conditions:

Column: 0.46 cm x 25 cm steel column

Filling material: YMC RP-C18, 5 μm, 300 Ä

Buffer A: 0.1% TFA

Buffer B: 0.1% TFA, 80% acetonitrile

Gradient: 15 to 50% B in 75 min, 75 to 100% B in 10 min

Flow: 0J ml / min

Detection: 214 nm

Chromatography system: Kontron

Third analytical reverse phase C3 chromatography:

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.

Chromatography conditions:

Column: 0.1 cm x 15 cm steel column

Filling material: Zorbax RP-C3, 5 μm, 300 Ä analytics is used, clearly.

Mass determinations

All 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 Da

certainly.

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.

Sequence determination

Both the purified native and carboxamidomethylated peptides are analyzed by Edman degradation on an ABI 473 A sequencer using the standard program.

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 12681

(Reduced molecule modified with iodoacetamide, MW 13045)

amino acids

GGKHHLGLEEPKKLRPPPARTPCQQELDQV ...

IGFBP-2-13, MW 12865

(Reduced molecule modified with iodoacetamide, MW 13223)

amino acids

GKGGKHHLGLEEPKKLRPPPARTPCQQELDQV ...

IGF-π, MW 7471

amino acids

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.

Database comparison

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 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).

Example 2

Determination of the biological activity of the IGF / IGFBP-2-13

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.

Example 3

Purification of the peptide IGFBP-4-11 according to the invention

The 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

Analytical reverse phase C18 chromatography:

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.

Chromatography conditions:

Column: 1 cm x 25 cm steel column

Filling material: Vydac RP-C45 ym, 300 Ä

Buffer A: 0.1% TFA

Buffer B: 0.1% TFA, 80% acetonitrile

Gradient: 0 - 80% B in 80 min, 80 - 100% B in 10 min

Flow: 2.5 ml / min

Detection: 230 nm

Chromatography system: Kontron

Fractions: ä 1 min from the start of the gradient

Mass 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 Da

certainly.

Sequence determination

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.

The following N-terminal sequence resulted:

IGFBP-4- 11, MW 11344 Da

KVNGAPREDARPVPQGSXQSELIIRALERL ...

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.

Database comparison

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.

Determination of the sulfur bridge linkage of the IGFBP-4-11

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

CVDRKTGVKLPGGLEPKGELDCHQLADSF, MW 3112

PVPQGSCQSELHR

MW 3236

THEDLYIIPIPNCDR

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.

Example 4

Determination of the biological activity of IGFBP-4-1 1

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

For 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

Isolation of the C-terminal domain of IGFBP-3

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 DKKGFYKKKQCRPSKGRKRGFCWCVDKYGQPLPGYTTKGKEDVHCYSMQSK

Example 6

By a procedure similar to that in Examples 1 and 3, the N-terminal domain of IGFBP-4 with the sequence

DEAIHCPPCSEEKLARCRPPVGCEELVREPGCGCCATCALGLGMPCGVYTPRCGSGL RCYPPRGVEKPLHTLMHGQGVCMELAEIEAIQESLQPSDKDEGDHPNNSFSPCSAHD RRCLQKHFKMDRDRSGG

Example 7

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 RKGFYKRKQCKPSRGRKRGICWCVDKYGMKLPGMEYVDGDFQCHTFDSSNVE

The theoretical mass is 12.5 kD, so it can be assumed that the peptide is glycosylated on serine or theronine.

Claims

claims

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. Peptides according to claim 1, characterized in that the peptides can be isolated from hemofiltrate.

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. 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. Peptides according to at least one of claims 1 to 4 with an amino acid sequence of the formula

R ₁ -CX ₁ -PNC-X ₂ -QC-X ₃ -CWCV-X ₄ -CR ₂

wherein

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. Peptides according to at least one of claims 1 to 5 with disulfide bridges of the formula

R _r CX, -PNC-X ₂ -QC-X ₃ -CWCV-X ₄ -CR ₂

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. 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. Peptides according to at least one of claims 1 to 8, characterized in that R is selected from

APSEEDHSILWDAISTYDGSKALHVTNIKKWKEP,

GGKHHLGLEEPKKLRPPPARTP

GKGGKHHLGLEEPKKLRPPPARTP,

GHAKDSQRYKVDYESQSTDTQNFSSESKRETEYGP,

KVNGAPREDARPVPQGS,

LTQSKFVGGAENTAHPRIISAPEMRQESEQGP,

PQAGTARPQDVNRRDQQRNPGTSTTPSQPNSAGVQDTEMGP.

10. Peptides according to at least one of claims 1 to 9, characterized in that X is selected from

RIELYRVVESLAKAQETSGEEISKFYL,

QQELDQVLERISTMRLPDERGPLEHLYSLHI,

RREMEDTLNHLKFLNVLSPRGVHI,

QSELHRALERLAASQSRTHEDLYIIPI,

RRHMEASLQELKASPRMVPRAVYL,

RRHLDSVLQQLQTEVYRGAQTLYV.

11. 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.

12. Peptides according to at least one of claims 1 to 11, characterized in that X _{3 is} selected from

ETSMDGEAGL,

KMSLNGQRGE,

RPSKGRKRGF,

HPALDGQRGK,

KPSRGRKRGI,

RSSQGQRRGP.

13. Peptides according to at least one of claims 1 to 12, characterized in that X _{4 is} selected from

YPWNGKRIPGSPEIRGDPN,

NPNTGKLOQGAPTIRGDPE,

DKYGQPLPGYTTKGKEDVH,

DRKTGVKLPGGLEPKGELD,

DKYGMKLPGMEYVDGDFQ,

DRMGKSLPGSPDGNGSSS.

14. Peptides according to at least one of claims 1 to 13, characterized in that R _{2 is} selected from

QIYFNVQN,

HLFYNEQQEARGVHTQRMQ,

HLFYNEQQE,

YSMQSK,

HQLADSFRE,

HTFDSSNVE,

PTGSSG.

15. Peptides according to one of claims 1 to 14, characterized in that the peptides are selected from

IBP-1

APSEEDHSILWDAISTYDGSKALHVTNIKKWKEPCRIELYRVVESLAKAQETS

GEEISKFYLPNCNKNGFYHSRQCETSMDGEAGLCWCVYPWNGKRIPGSPEIRG

DPNCQIYFNVQN

IGFBP-2 GKGGKHHLGLEEPKKLRPPPARTPCQQELDQVLERISTMRLPDERGPLEHLYS LHIPNCDKHGLYNLKQCKMSLNGQRGECWCVNPNTGKLIQGAPTIRGDPECH LFYNEQQEARGVHTQRMQ

GGKHHLGLEEPKKLRPPPARTPCQQELDQVLERISTMRLPDERGPLEHLYSLH IPNCDKHGLYNLKQCKMSLNGQRGECWCVNPNTGKLIQGAPTIRGDPECHLF YNEQQEARGVHTQRMQ

IGFBP-3

GHAKDSQRYKVDYESQSTDTQNFSSESKRETEYGPCRREMEDTLNHLKFLNV LSPRGVHIPNCDKKGFYKKKQCRPSKGRKRGFCWCVDKYGQPLPGYTTKGK EDVHCYSMQSK

KVDYESQSTDTQNFSSESKRETEYGPCRREMEDTLNHLKFLNVLSPRGV HIPNCDKKGFYKKKQCRPSKGRKRGFCWCVDKYGQPLPGYTTKGKEDVHCY SMQSK

HPLHSKIIIIKKGHAKDSQRY

IGFBP-4

DEAIHCPPCSEEKLARCRPPVGCEELVREPGCGCCATCALGLGMPCGVYTPRC GSGLRCYPPRGVEKPLHTLMHGQGVCMELAEIEAIQESLQPSDKDEGDHPNNS FSPCSAHDRRCLQKHFKMDRDRSG

KVNGAPREDARPVPQGSCQSELHRALERLAASQSRTHEDLYIIPIPNCDRNGNF HPKQCHPALDGQRGKCWCVDRKTGVKLPGGLEPKGELDCHQLADSFRE

IGFBP-5

LTQSKFVGGAENTAHPRIISAPEMRQESEQGPCRRHMEASLQELKASPRMVPR AVYLPNCDRKGFYKRKQCKPSRGRKRGICWCVDKYGMKLPGMEYVDGDFQ

CHTFDSSNVE

KFVGGAENTAHPRIISAPEMRQESEQGPCRRHMEASLQELKASPRMVPRAV YLPNCDRKGFYKRKQCKPSRGRKRGICWCVDKYGMKLPGMEYVDGDFQCH TFDSSNVE

HTRISELKAEAVKKDRRKKLTQS

IGFBP-6

PQAGTARPQDVNRRDQQRNPGTSTTPSQPNSAGVQDTEMGPCRRHLDSVLQQ LQTEVYRGAQTLYVPNCDHRGFYRKRQCRSSQGQRRGPCWCVDRMGKSLPG SPDGNGSSSCPTGSSG

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. 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. Nucleic acid, characterized in that it codes for peptides according to at least one of claims 1 to 15.

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, characterized in that it binds to a peptide according to at least one of claims 1 to 15.

21. Inhibitor, characterized in that it inhibits the biological activity of peptides according to at least one of claims 1 to 15.

22. Inhibitor, characterized in that it inhibits the expression of peptides according to at least one of claims 1 to 15.

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. 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 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. 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. 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. 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. 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.