CA2315974A1

CA2315974A1 - Insulin-like growth factor binding protein fragments and the utilization thereof

Info

Publication number: CA2315974A1
Application number: CA002315974A
Authority: CA
Inventors: Ludger Standker; Maik Obendorf; Lothar Kling; Hans-Georg Opitz; Hossein Mostafavi
Original assignee: Individual
Current assignee: Individual
Priority date: 1997-12-22
Filing date: 1998-12-22
Publication date: 1999-07-01
Also published as: WO1999032620A1; JP2002508931A; DE19757250A1; EP1042476A1; 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

SMB
Insulin-like Growth Factor Bindin4 Protein Fragments and the Utilization Thereof The present invention relates to peptides having cell-proliferative and cell-protective properties, complexes of the peptides with human insulin-like growth factors (IGF) I and II, and the related nucleic acids, antisense nucleotides, antibodies and inhibitors.
Insulin-like growth factor binding proteins have been 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 the amino acid sequence of which corresponds to parts of the amino acid sequence of insulin-like growth factor binding proteins, and cyclic, glycosylated, phosphorylated, acetylated, amidated and/or sulfated derivatives thereof. These peptides according to the invention are designated as IGFBP or IBP.
Preferred peptides are those which are naturally occurring and can be isolated, for example, from hemofiltrate. Preferably, the peptides have lengths of from 61 to 115 amino acids. Particularly preferred are peptides having sequences which correspond to N- or C-terminal sequences of insulin-like growth factor binding proteins.
Preferred peptides are peptides having an amino acid sequence of formula Rl-C-X1-PNC-XZ-QC-X3-CWCV-X4-C-R2 wherein Rl is NH2, an amino acid or a peptide having an amino acid sequence comprising up to 41 amino acids, Xl is a peptide having an amino acid sequence comprising from 24 to 31 amino acids, X2 is a peptide having an amino acid sequence com-prising 9 amino acids, X3 is a peptide having an amino acid sequence comprising amino acids, X4 is a peptide having an amino acid sequence comprising from 18 to 24 amino acids, R2 is COOH, CONH2 or a peptide having up to 12 amino acids, and cyclic, glycosylated, phosphorylated, acetylated, amidated, sulfated derivatives and or fragments thereof having the physiological activity of IGFBP.
The peptide has cell-proliferative and cell-protective properties.
The peptides according to the invention can have disulfide bridges to correspond to the general formula:.
---.~ ~ r----z R,-C-X,-PNC-Xz-QC-X,-C~~hCV-Xa-C-R, In a preferred embodiment, the peptides have a glycine on one or more of the following positions of the amino acid sequence. X2 on position 4, X3 on position 9, X4 on position 4 or 5, and/or X4 on position 9 or 10.
It is further preferred that X1 is L or V on position 8, and/or X1 is L or I
on position 11, and/or XZ is D or N on position 1, and/or XZ is K or R on position 9, and/or X3 is S or A on position 3 and/or R or A on position 8.
In a particularly preferred embodiment, Rl 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), LTQSKFVGGAENTAHPRIISAPEMRQESEQGP (SEQ ID NO: 6), PQAGTARPQDVNRRDQQRNPGTSTTPSQPNSAGVQDTEMGP (SEQ ID NO: 7); and/or X1 is selected from RIELYRWESLAKAQETSGEEISKFYL (SEQ ID NO: 8), QQELDQVLERISTMRLPDERGPLEHLYSLHI (SEQ ID NO: 9), RREMEDTLNHLKFLNVLSPRGVHI (SEQ ID NO: 10), QSELHRALERLAASQSRTHEDLYIIPI (SEQ ID NO: 11), RRHMEASLQELKASPRMVPRAVYL (SEQ ID NO: 12), RRHLDSVLQQLQTEVYRGAQTLYV (SEQ ID NO: 13); and/or XZ is 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 X3 is selected from ETSMDGEAGL (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 X4 is selected from YPWNGKRIPGSPEIRGDPN (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); and/or R2 is 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).
Preferred peptides have the following sequences:

APSEEDHSILWDAISTYDGSKALHVTNIKKWKEPCRIELYRVVESLAKAQETSGEEISKFYL
PNCNKNGFYHSRQCETSMDGEAGLCWCVYPWNGKRIPGSPEIRGDPNCQIYFNVQN
(SEQ ID NO: 39) GKGGKHHLGLEEPKKLRPPPARTPCQQELDQVLERISTMRLPDERGPLEHLYSLHIPNCDK
HGLYNLKQCKMSLNGQRGECWCVNPNTGKLIQGAPTIRGDPECHLFYNEQQEARGVHTQ
RMQ (SEQ ID NO: 40) GGKHHLGLEEPKKLRPPPARTPCQQELDQVLERISTMRLPDERGPLEHLYSLHIPNCDKH
GLYNLKQCKMSLNGQRGECWCVNPNTGKLIQGAPTIRGDPECHLFYNEQQEARGVHTQR
MQ (SEQ ID NO: 45) GHAKDSQRYKVDYESQSTDTQN FSSESKRETEYGPCRREM EDTLN H LKFLNVLSPRGVHI
PNCDKKGFYKKKQCRPSKGRKRGFCWCVDKYGQPLPGYTTKGKEDVHCYSMQSK (SEQ
ID NO: 41) KVDYESQSTDTQNFSSESKRETEYGPCRREMEDTLNHLKFLNVLSPRGVHIPNCDK
KGFYKKKQCRPSKGRKRGFCWCVDKYGQPLPGYTTKGKEDVHCYSMQSK (SEQ ID NO:
46) HPLHSKIIIIKKGHAKDSQRY (SEQ ID NO: 47) DEAIHCPPCSEEKLARCRPPVGCEELVREPGCGCCATCALGLGMPCGVYTPRCGSGLRCYP
PRGVEKPLHTLMHGQGVCMELAEIEAIQESLQPSDKDEGDHPNNSFSPCSAHDRRCLQKH
FAKIRDRSTSGGKM (SEQ ID NO: 48) KVNGAPREDARPVPQGSCQSELHRALERLAASQSRTHEDLYIIPIPNCDRNGNFHPKQCHP
ALDGQRGKCWCVDRKTGVKLPGGLEPKGELDCHQLADSFRE (SEQ ID NO: 42) LTQSKFVGGAENTAHPRIISAPEMRQESEQGPCRRHMEASLQELKASPRMVPRAVYLPNC
DRKGFYKRKQCKPSRGRKRGICWCVDKYGMKLPGMEYVDGDFQCHTFDSSNVE (SEQ
ID NO: 43) KFVGGAENTAHPRIISAPEMRQESEQGPCRRHMEASLQELKASPRMVPRAVYLPNCDR
KGFYKRKQCKPSRGRKRGICWCVDKYGMKLPGMEYVDGDFQCHTFDSSNVE (SEQ ID
NO: 49) HTRISELKAEAVKKDRRKKLTQS (SEQ ID NO: 50) PQAGTARPQDVN RRDQQRN PGTSTTPSQPNSAGVQDTEMGPCRRH LDSVLQQLQTEVY
RGAQTLYVPNCDHRGFYRKRQCRSSQGQRRGPCWCVDRMGKSLPGSPDGNGSSSCPTG
SSG (SEQ ID NO: 44).
The peptides according to the invention can be obtained by purification from human hemofiltrate or urine, by solid-phase peptide synthesis, or by expression in recombinant microorganisms.

The invention further relates to complexes of the peptides according to the invention with human insulin-like growth factor I and/or human insulin-like growth factor II and its physiologically active fragments and/or derivatives, especially amidated, acetylated, sulfated, phosphorylated and/or glycosylated derivatives.
In addition, the invention relates to nucleic acids coding for the peptides according to the invention, antisense nucleotides which, under stringent conditions, bind to a nucleic acid coding for the peptide according to the invention, antibodies which bind to the peptides according to the invention, inhibitors which inhibit the biological activity of insulin-like growth factor binding proteins, inhibitors which 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 especially suitable for the preparation of a medicament for treating the over- or underexpression of insulin-like growth factor binding proteins, for treating muscular atrophy, osteoporosis, diabetes, amyloid lateral sclerosis, peripheral and central neuropathies, inflammatory processes, disordered inflammatory reactions, tumor diseases, inflammatory and neoplastic diseases, disturbance of growth, muscular affections, affections of the bone system, and/or for wound and bone healing.
Especially complexes of IGFBP with IGF-I or IGF-II are useful 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 control the release of IGF-I and IGF-II
from the complexes at their site of action. The coadministration 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. Hypoglycemia induced by the injection of free IGF-I or IGF-II is prevented by the coadministration of the peptides according to the invention.

_ 7 _ In addition, the peptides according to the invention have a growth-promoting effect on bone cells and lead to an enhancement or modulation of the activity 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 pharmaceutically acceptable dosage form in a medicament. They are suitable for oral, intravenous, intramuscular, intracutaneous, intrathecal administration, or as an aerosol for transpulmonary administration. The amount of peptide to be administered is from 1 Ng to 1 g per dosage unit per day.
The nucleic acids and/or antisense nucleotides according to the invention are also suitable for the preparation 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.
Preferably, the diagnostic agent contains poly- or monoclonal antibodies against the peptide according to the invention. Such antibodies may be fluorescence-labeled or radioactively labeled to be used in the known ELISA or RIA.
However, the diagnostic agent may also contain nucleic acids which, in a modified or labeled form, are employed in test known to those skilled in the art, such as PCR or fingerprinting.
The diagnostic agents according to the invention are especially useful for diagnos-ing functional disorders in bones, muscles, the nervous system, lymph organs, the gastrointestinal tract, the immune system, and of diabetes and inflammatory and neoplastic processes, and as a marker in cancer.
Especially the simultaneous occurrence of several fragments of BP-4 or BP-5 in the plasma, in particular, of the N- and C-terminal domains, is useful as a marker for 8 _ diseases of bone metabolism. The corresponding peptides can be detected by mass spectroscopy, preferably by an immune reaction with corresponding antibodies.
Figure 1 shows an alignment of the consensus sequences of C-terminal fragments of the insulin-like growth factor proteins.
Figure 2 shows the schematic structure of the insulin-like growth factor proteins with the cysteine-rich N- and C-terminal domains.
Figure 3 shows the schematic structure of the insulin-like growth factor proteins and the sequence of the biologically active fragments isolated from hemofiltrate.
Figure 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 primary rat osteoblasts, which had been kept in a serum-free medium, 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 of the DNA
synthetic rate in a dose-dependent way is found, measured as 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-413x-z3'). A:
Radioactively labeled IGFBP-4-11 exhibits specific binding to primary osteoblast cells which can be displaced by increasing amounts of non-labeled IGFBP-4-11. B: After having bound to osteoblasts, radioactively labeled IGFBP-4-11 could be chemically cross-linked with its putative receptor molecule and subsequently be detected by gel electrophoresis followed by autoradiography. The ligand-receptor complex has a molecular mass of about 120 kDa. The formation of the complex is favored by _g_ saponin, a membrane pore generator. Complex formation is prevented by adding an excess of unlabeled IGFBP-4-11 to the incubation mixture.
The purification of the peptide according to the invention or its complex is illus-trated by the following Examples:
Example 1 Purification and peptide-chemical analysis of IGFBP-2-13 The peptides according to the invention can be obtained by a purification method starting from human hemofiltrate. This patent method (Forssmann, W.-G. (1988), German Laid-Open DE 36 33 707 A1), which was developed for the recovery of proteins from hemofiltrate, was also employed, in a modified form, for the purification of the peptide complex.
Hemofiltrate batch extraction Hemofiltrate is obtained in large amounts in the ultrafiltration of the blood of kidney disease sufferers. 800 to 1,000 I of hemofiltrate are adjusted to pH
2.7 with HCI 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 I/min.
Conditions of chromatography column: Vantage VA 250 (Amicon, Witten) column material: Fractogel TSK SP 650 (M), 25 cm x 20 cm flow rate: 3 I/min detection: 280 nm, pH, conductivity buffer A: hemofiltrate, pH 2.7, conductivity 5.5 mS/cm buffer B: 0.5 M ammonium acetate plant: Autopilot Chromatographiesystem (PerSeptive Biosystems, Wiesbaden) After application of a total of 1,000 I of liquid over night, the column is washed with several column volumes of 5 mM HCI. The elution of the bound peptides is performed as a batch elution with 0.5 M ammonium acetate. Complete elution of the peptides is achieved with an increasing pH value (6.8 to 7.2) and increased conductivity (56 mS/cm) in about 5 I of eluate.
First preparative separation The ammonium acetate eluates from the batch extraction are combined in amounts of 5,000 to 10,000 I of hemofiltrate peptide. After adjusting the pH
to 2.7, the peptide extract is applied to the preparative cation-exchanger with admixing completely desalted water having a conductivity of 5.5 mS/cm.
Conditions of chromatography:
column: Vantage 250 VA
column material: Fractogel TSK SP 650 (M), 25 cm x 20 cm flow rate: up to 3 I/min during application 0.5 to 1 I during elution detection: 280 nm, pH, conductivity sample: hemofiltrate, pH 2.7, conductivity 5.5 mS/cm plant: Autopilot Chromatographiesystem (PerSeptive Biosystems, Wiesbaden) After application of the raw extract over a period of 240 min, the column is washed with 0.01 M HCI until the conductivity has fallen below 1 mS/cm. Elution is effected in several steps with the following buffers:

buffer pH value buffer substances conductivity (mS/cm) washing buffer2.0 0.01 M HCI 1 elution buffer3.6 0.1 M citric acid 1-hydrate2.9 elution buffer4.5 0.1 M acetic acid + 4.0 0.1 M sodium acetate elution buffer5.0 0.1 M malic acid 6.2 elution buffer5.6 0.1 M succinic acid 6.1 elution buffer6.6 0.1 M NaHZP04 4.9 elution buffer7.4 0.1 M NaHzP04 6.7 elution buffer9.0 0.1 M ammonium carbonate6.7 Eluates 1-7 are referred to as pH pool I-VII. They are separately collected and finally washed with completely desalted water. Elution is performed until a new baseline is reached. For the individual pH pools I to VII, elution volumes of 10 to 25 I are reached.
Second preparative separation The individual pH pools are separated by reversed-phase chromatography for fractionating and desalting at the same time.
Conditions of chromatography:
column: FineLine 100 (Pharmacia, Freiburg) column material: Source RPC, 15 Nm, 10 x 12.5 cm (FineLine 100) flow rate: 150 ml/min (FineLine 100) detection: 280 nm, conductivity, pH
buffer A: 10 mM HCI
buffer B: 80% acetonitrile in 10 mM HCI
gradient: 0-60% buffer B in 5 column volumes After application of the pH pools, the column is washed with buffer A. During the elution, fractions of 200 ml are collected. Aliquots of the fractions are tested in a bioassay. The fractions are freeze-dried and stored at -20 °C.
Semipreparative reversed-lahase C18 chromatogra~hy Fractions 11 and 12 from pH pool V, which had been biologically active in the assay, were separated through a semipreparative reversed-phase column.
Fractions 21 to 25 contained the substance according to the invention.
Conditions of chromatography column: 4.7 cm x 30 cm steel column column material:Vydac RP-C18 15-20 pm, 300 ~

buffer A: 0.1% TFA

buffer B: 0.1% TFA, 80% acetonitrile gradient: 5-50% B in 45 min, 50-100% B in 10 min flow rate: 42 ml/min detection: 214 nm and 280 nm chromatographic plant: BioCad fractions: every 1.5 min from the start of the gradient Seminreparative reversed-phase C18 chromatoara~hy The biologically active fractions 21 to 25 from the preceding chromatography were separated through the same semipreparative reversed-phase column. However, methanol was used as the eluent. Fraction 24 contained the substance according to the invention.
Conditions of chromatography column: 4.7 cm x 30 cm steel column column material: Vydac RP-C18 15-20 Nm, 300 r~
buffer A: 0.1% TFA, 20% methanol buffer B: 0.1% TFA, 100% methanol gradient: 0-20% B in 6.5 min, 20-80% B in 55 min, 80-100% B in 13 min flow rate: 30 ml/min detection: 214 nm and 280 nm chromatographic plant: BioCad fractions: every 1.5 min from the start of the gradient Cation exchange chromatographx The biologically active fractions 19 and 20 from the preceding chromatography were separated through a cation-exchange column. Fractions 45 to 47 contained the substance according to the invention.
Conditions of chromatoarap column: 1 cm x 5 cm steel column column material: Pepkat, Biotek 5 pm, 300 ~
buffer A: 20 mM sodium phosphate, pH 3.0 buffer B: 20 mM sodium phosphate, pH 3.0, 1.5 M NaCI
gradient: 0-50% B in 50 min, SO-100% B in 10 min flow rate: 3 ml/min detection: 280 nm chromatographic plant: BioCad Sprint fractions: every 1.5 min from the start of the gradient Analytical reversed-phase chromatographx The biologically active fractions 45 to 47 from the preceding chromatography were successively separated in several identical runs through a reversed-phase column.
Fraction 56 contained the substance according to the invention.

Conditions of chromatography column: 1 cm x 25 cm steel column column material:Vydac RP-C18 5 um, 300 ~

buffer A: 0.1% TFA

buffer B: 0.1% TFA, 80% acetonitrile gradient: 5-50% B in 45 min, 50-100% B in 10 min flow rate: 2 ml/min detection: 220 nm chromatographiclant: Kontron p fractions: every 1 min from the start of the gradient Second analytical reversed-phase C18 chromatography The biologically active fraction 56 from the preceding separation step was further purified on an analytical reversed-phase column.
Conditions of chromatoara~hv:
column: 0.46 cm x 25 cm steel column column material:YMC RP-C18, 5 Nm, 300 A

buffer A: 0.1% TFA

buffer B: 0.1% TFA, 80% acetonitrile gradient: 15-50% B in 75 min 75-100% B in 10 min , flow rate: 0.7 ml/min detection: 214 nm chromatographiclant: Kontron p Third analytical reversed-phase C3 chromatogr~~hX
Part of the biologically active fraction 45 from the preceding separation step was directly subjected to mass and sequence analyses. Another part was reduced and alkylated (as described under Example 2) and then further purified on an analytical reversed-phase column.

Conditions of chromatography column: 0.1 cm x 15 cm steel column , column material: Zorbax RP-C3, 5 Nm, 300 A
Mass determinations All mass determinations of the unmodified and modified peptides were performed on a MALDI TOF mass spectrometer. The molecular masses of the peptides were determined as:
IGF-II: 7,471 Da;
IGFBP-2: 12,681 Da;
IGFBP-2: 12,865 Da.
Determination of cysteines/modification ofa~eptides Cysteines can be detected after previous chemical derivatization, for example, after reduction with ~3-mercaptoethanol and carboxamidomethylation with io-doacetamide, in the peptide sequencing. Derivatization is followed by desalting, preferably through analytical reversed-phase chromatography on a Vydac RP-C18 column (4.6 mm x 25 cm). Part of the thus modified peptides are subjected to sequence analysis, and mass determinations performed on the rest yield a corresponding molecular mass. From the mass difference to the native peptide, it is concluded that the peptides from hemofiltrate contain six cysteines which are additionally interconnected by three disulfide bridges.
Sequence determination Both the purified native and the 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 of between 100 and 400 pmol. In accordance with the results of mass determinations, the following N-terminal sequences were found:

IGFBP-2-13, MW 12,681 (reduced molecule modified with iodoacetamide, MW 13,045) Amino acids GGKHHLGLEEPKKLRPPPARTPCQQELDQV...
IGFBP-2-13, MW 12,865 (reduced molecule modified with iodoacetamide, MW 13,223) Amino acids GKGGKHHLGLEEPKKLRPPPARTPCQQELDQV...
IGF-II, MW 7471 Amino acids AYRPSETLCGGEL....
The C terminus was determined by comparing the measured molecular mass with the mass calculated from the sequence. The identity of these masses is within the measuring accuracy of the MALDI TOF mass spectrometer, i.e., 0.1% of the total mass.
Data base comparison A data base comparison was performed on the SwissProt and EMBL nucleic acid data bases using the HUSAR program package. The peptide sequences have 100%
identity with the amino acids of human IGFBP-2 as derived from the cDNA or with the amino acid sequence of IGF-II.
To date, IGFBP-2 has been described as a 34 kD binding protein the sequence of which was completely analyzed by analyzing the related cDNA (Binkert, C., et al., EMBO Journal Vol. 8 (1989), pages 2497 to 2502). In contrast, IGF-II and also IGF-I, which also binds to IGFBP-2, have already been extensively described in terms of their structures on the protein and DNA sequence levels (as a review:
Rechler, M.M., & Nissley, S.P. (1990) "Insulin-like growth factors" in:
Peptide growth factors and their receptors (Spori, M.B., Roberts, A.B. eds.), pages 263 to 367, Springer-Verlag, Berlin).

Example 2 Determination of the biological activitx of IGF/IGFBP-2-13 The isolation of IGF/IGFBP-2-13 was based on its biological activity in a survival assay of the PC-12 (pheochromocytoma cells) cell line. Thus, aliquots of each of the individual chromatographic stages described under Example 1 were freeze-dried and then subjected to a biological assay. The fractions which gave a positive signal were respectively subjected to further purification.
The assay measures the survival of the cells after having been kept in serum-free medium by determining the activity of mitochondria) enzymes 24 hours after serum starvation. As a positive control, nerve growth factor (NGF) or fetal calf serum (FCS) are employed in this assay.
In 96-well plates, 10,000 PC-12 cells per well were seeded in a serum-free medium, following by the addition of aliquots (about 100 ml equivalent of starting material) to the wells. The survival rate of the cells is measured 20 hours later using a Wst-1 substrate. This substrate is converted by mitochondria) enzymes.
The color intensity obtained is measured at 405 nm in an ELISA reader; the reference wavelength was 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 precursor cells so that it can be considered 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 per-formed by complete analogy with the purification of IGFBP-2-13 described under Example 1 up to the step of the second preparative separation. The further purification was effected by:

Analytical reversed-phase C18 chromatogr~~hx Fraction 33 from pH pool IV, which was found biologically active in the assay, was separated through an analytical reversed-phase column. Fraction 34 contained the substance according to the invention.
Conditions of chromatograph rL:
column: 1 cm x 25 cm steel column column material:Vydac RP-C4 5 Nm, 300 A

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 rate: 2.5 ml/min detection: 230 nm chromatographic plant: Kontron fractions: every 1 min from the start of the gradient Mass determinations The mass determinations were performed on an electrospray mass spectrometer.
The molecular mass of the peptide was determined as:
IGFBP-4-11, 11,344 Da.
Sequence determination The amino acid sequence of the purified native biologically active peptide 11 was determined as described under Example 1.
The following N-terminal sequence was found:
IGFBP-4-11, MW 11,344 Da KVNGAPREDARPVPQGSXQSELIIRALERL...

The C terminus was determined by comparing the measured molecular mass with the mass calculated from the sequence. The identity of these masses is within the measuring accuracy of the electrospray mass spectrometer, i.e., 0.1% of the total mass.
Data base comparison A data base comparison was performed on the SwissProt and EMBL nucleic acid data bases using the HUSAR program package. The peptide sequence has 100%
identity with the amino acids of human IGfBP-4 as derived from the cDNA.
Determination of the sulfur bridge cross-links in IGFBP-4-11 The analysis of the sulfur-bridge cross-linking was performed by cleaving the native peptide IGFBP-4-11 in two different parallel reactions with the endoprote-ases chymotrypsin and Arg-C. The cleaving fragments obtained were then sepa-rated by analytical reversed-phase chromatography and subjected to molecular mass and sequence analyses. The following fragments containing two cysteines and one sulfur bridge each were obtained:
HPKQCHPALDGQRGKCW, MW 1960 CVDRKTGVKLPGGLEPKGELDCHQLADSF, MW 3112 PVPQGSCQSELHR

THEDLYIIPIPNCDR
It can be seen therefrom that the sulfur bridges between cysteines 1 and 2, between cysteines 3 and 4 and between cysteines 5 and 6 are realized in the native IGFBP-4-11.

Example 4 Determination of the biological activity of IGFBP-4-11 The isolation of IGFBP-4-11 was based on its biological activity in a proliferation assay with primary bone cells (osteoblasts), which were first isolated from rat embryonal calvarias.
Thus, aliquots of each of the individual chromatographic stages described under Example 3 were freeze-dried and then subjected to a biological assay. The fractions which gave a positive signal were respectively subjected to further purification. The assay measures the proliferation of the cells by determining the incorporation of radioactive thymidine, i.e., the DNA synthesis rate, 48 or 72 hours after the addition of the fractions. As a positive control, transforming growth factor-beta (TGF-~) or fetal calf serum (FCS) are employed in this assay. In well plates, 5000 osteoblasts per well were seeded in a serum-containing medium, following by the addition of aliquots (about 100 ml equivalent of starting material) to the wells. The proliferation rate (DNA synthesis rate) of the cells is measured 48 or 72 hours later using the addition and incorporation of radioactive thymidine. The peptide IGFBP-4-11 has a dose-dependent proliferation-promoting effect on these primary osteoblasts. These cells correspond to typical bone cells so that it can be considered that IGFBP-4-11 is an osteoanabolic factor.
Example 5 Isolation of the C-terminal domain of IGFBP-3 By a similar method to that used in Examples 1 and 3, a peptide could be isolated from hemofiltrate, having a mass of 2,470 Dalton (MALDI: 2481 Dalton) and the following sequence:
HTRISELKAEAVKKDRRKKLTQS (?) from which the following sequence results as the C-terminal sequence of IGFBP-3:

KVDYESQSTDTQNFSSESKRETEYGPCRREMEDTLNHLKFLNVLSPRGVHIPNCDKK
GFYKKKQCRPSKGRKRGFCWCVDKYGQPLPGYTTKGKEDVHCYSMQSK
Example 6 By a similar method to that used in Examples 1 and 3, the N-terminal domain of IGFBP-4 could be isolated, having the following sequence:
DEAIHCPPCSEEKLARCRPPVGCEELVREPGCGCCATCALGLGMPCGVYTPRCGSGLRCYP
PRGVEKPLHTLMHGQGVCMELAEIEAIQESLQPSDKDEGDHPNNSFSPCSAHDRRCLQKH
FAKIRDRSTSGGKM
Example 7 Determination of the C-terminal sequence of 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 gave the following sequence:
KFVGGAENTAH PRIISAPEM RQESEQG PCRRH M EASLQELKASPRMVPRAVYLPNCDRKG
FYKRKQCKPSRGRKRGICWCVDKYGM KLPGMEYVDGDFQCHTFDSSNVE
The theoretical mass is 12.5 kD, and therefore, it has to be considered that the peptide is glycosylated at serine or threonine.

SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Prof. Dr. Wolf-Georg Forssmann (B) STREET: Feodor-Lynen-Str. 31 (C) CITY: Hannover (E) COUNTRY: Germany (F) POSTAL CODE: 30625 (ii) TITLE OF INVENTION: Insulin-like Growth Factor Binding Protein Fragments and the Utilization Thereof (iii) NUMBER OF SEQUENCES: 50 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER; IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Ala Pro Ser Glu Glu Asp His Ser Ile Leu Trp Asp Ala Ile Ser Thr Tyr Asp Gly Ser Lys Ala Leu His Val Thr Asn Ile Lys Lys Trp Lys Glu Pro (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Gly Gly Lys His His Leu Gly Leu Glu Glu Pro Lys Lys Leu Arg Pro Pro Pro Ala Arg Thr Pro (2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 Gly Lys Gly Gly Lys His His Leu Gly Leu Glu Glu Pro Lys Lys Leu Arg Pro Pro Pro Ala Arg Thr Pro (2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Gly His Ala Lys Asp Ser Gln Arg Tyr Lys Val Asp Tyr Glu Ser Gln Ser Thr Asp Thr Gln Asn Phe Ser Ser Glu Ser Lys Arg Glu Thr Glu Tyr Gly Pro (2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Lys Val Asn Gly Ala Pro Arg Glu Asp Ala Arg Pro Val Pro Gln Gly Ser Arg Pro Pro Pr (2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Leu Thr Gln Ser Lys Phe Val Gly Gly Ala Glu Asn Thr Ala His Pro Arg Ile Ile Ser Ala Pro Glu Met Arg Gln Glu Ser Glu Gln Gly Pro (2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE
DESCRIPTION:
SEQ ID
NO: 7:

Pro Gln Gly Thr Arg Gln Asp Val Asn Arg Arg Asp Ala Ala Pro Gln Gln Arg Pro Gly Ser Thr Pro Ser Gln Pro Asn Ser Asn Thr Thr Ala Gly Val Asp Thr Met Pro Gln Glu Gly (2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Arg Ile Glu Leu Tyr Arg Val Val Glu Ser Leu Ala Lys Ala Gln Glu Thr Ser Gly Glu Glu Ile Ser Lys Phe Tyr Leu (2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Gln Gln Glu Leu Asp Gln Val Leu Glu Arg Ile Ser Thr Met Arg Leu Pro Asp Glu Arg Gly Pro Leu Glu His Leu Tyr Ser Leu His Ile (2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Arg Arg Glu Met Glu Asp Thr Leu Asn His Leu Lys Phe Leu Asn Val Leu Ser Pro Arg Gly Val His Ile (2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
Gln Ser Glu Leu His Arg Ala Leu Glu Arg Leu Ala Ala Ser Gln Ser 1 5 10 . 15 Arg Thr His Glu Asp Leu Tyr Ile Ile Pro Ile _28_ (2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Arg Arg His Met Glu Ala Ser Leu Gln Glu Leu Lys Ala Ser Pro Arg Met Val Pro Arg Ala Val Tyr Leu (2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
Arg Arg His Leu Asp Ser Val Leu Gln Gln Leu Gln Thr Glu Val Tyr Arg Gly Ala Gln Thr Leu Tyr Val (2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Asn Lys Asn Gly Phe Tyr His Ser Arg (2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide -(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
Asp Lys His Gly Leu Tyr Asn Leu Lys (2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Asp Lys Lys Gly Phe Tyr Lys Lys Lys (2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
Asp Arg Asn Gly Asn Phe His Pro Lys (2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
Asp Arg Lys Gly Phe Tyr Lys Arg Lys (2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
Asp His Arg Gly Phe Tyr Arg Lys Arg (2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
Glu Thr Ser Met Asp Gly Glu Ala Gly Leu (2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
Lys Met Ser Leu Asn Gly Gln Arg Gly Glu (2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe (2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
His Pro Ala Leu Asp Gly Gln Arg Gly Lys (2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
Lys Pro Ser Arg Gly Arg Lys Arg Gly Ile (2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
Arg Ser Ser Gln Gly Gln Arg Arg Gly Pro (2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
Tyr Pro Trp Asn Gly Lys Arg Ile Pro Gly Ser Pro Glu Ile Arg Gly Asp Pro Asn (2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
Asn Pro Asn Thr Gly Lys Leu Ile Gln Gly Ala Pro Thr Ile Arg Gly Asp Pro Glu (2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:

Asp Lys Tyr Gly Gln Pro Leu Pro Gly Tyr Thr Thr Lys Gly Lys Glu Asp Val His (2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
Asp Arg Lys Thr Gly Val Lys Leu Pro Gly Gly Leu Glu Pro Lys Gly Glu Leu Asp (2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
Asp Lys Tyr Gly Met Lys Leu Pro Gly Met Glu Tyr Val Asp Gly Asp Phe Gln (2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
Asp Arg Met Gly Lys Ser Leu Pro Gly Ser Pro Asp Gly Asn Gly Ser Ser Ser (2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
Gln Ile Tyr Phe Asn Val Gln Asn (2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
His Leu Phe Tyr Asn Glu Gln Gln Glu Ala Arg Gly Val His Thr Gln Arg Met Gln (2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
His Leu Phe Tyr Asn Glu Gln Gln Glu (2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
Tyr Ser Met Gln Ser Lys (2) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
His Gln Leu A1a Asp Ser Phe Arg Glu (2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:

His Thr Phe Asp Ser Ser Asn Val Glu (2) INFORMATION FOR SEQ ID NO: 38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:
Pro Thr Gly Ser Ser Gly (2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 118 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE SEQ
DESCRIPTION: ID
NO:
39:

Ala Pro Glu Glu Asp Ser Ile Leu Asp Ala Ile Ser Ser His Trp Thr Tyr Asp Ser Lys Ala His Val Thr Ile Lys Lys Trp Gly Leu Asn Lys Glu Pro Arg Ile Glu Tyr Arg Val Glu Ser Leu Ala Cys Leu Val Lys Ala Gln Glu Thr Ser Gly Glu Glu Ile Ser Lys Phe Tyr Leu Pro Asn Cys Asn Lys Asn Gly Phe Tyr His Ser Arg Gln Cys Glu Thr Ser Met Asp Gly Glu Ala Gly Leu Cys Trp Cys Val Tyr Pro Trp Asn Gly Lys Arg Ile Pro Gly Ser Pro Glu Ile Arg Gly Asp Pro Asn Cys Gln Ile Tyr Phe Asn Val Gln Asn (2) INFORMATION FOR SEQ ID NO: 40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 123 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQ
SEQUENCE ID
DESCRIPTION: NO:
40:

GlyLys Gly GlyLys His HisLeu Gly Leu GluGlu Pro Lys LysLeu ArgPro Pro ProAla Arg ThrPro Cys Gln GlnGlu Leu Asp GlnVal LeuGlu Arg IleSer Thr MetArg Leu Pro AspGlu Arg Gly ProLeu GluHis Leu TyrSer Leu HisIle Pro Asn CysAsp Lys His GlyLeu TyrAsn Leu LysGln Cys LysMet Ser Leu AsnGly Gln Arg GlyGlu CysTrp Cys ValAsn Pro AsnThr Gly Lys LeuIle Gln Gly AlaPro ThrIle Arg GlyAsp Pro GluCys His Leu PheTyr Asn Glu GlnGln Glu Ala Arg Gly Val His Thr Gln Arg Met Gln (2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 114 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQ
SEQUENCE ID
DESCRIPTION: NO:
41:

Gly HisAla Lys AspSer GlnArg Tyr Lys Val AspTyr Glu Ser Gln Ser ThrAsp Thr GlnAsn PheSer Ser Glu Ser LysArg Glu Thr Glu Tyr GlyPro Cys ArgArg GluMet Glu Asp Thr LeuAsn His Leu Lys Phe LeuAsn Val LeuSer ProArg Gly Val His IlePro Asn Cys Asp Lys LysGly Phe TyrLys LysLys Gln Cys Arg ProSer Lys Gly Arg Lys ArgGly Phe CysTrp CysVal Asp Lys Tyr GlyGln Pro Leu Pro Gly TyrThr Thr LysGly LysGlu Asp Val His CysTyr Ser Met Gln Ser Lys (2) INFORMATION FOR SEQ ID NO: 42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 102 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQ
SEQUENCE ID
DESCRIPTION: NO:
42:

LysVal Asn Gly AlaPro ArgGlu Asp Ala ArgPro Val Pro GlnGly SerCys Gln Ser GluLeu HisArg Ala Leu GluArg Leu Ala AlaSer GlnSer Arg Thr HisGlu AspLeu Tyr Ile IlePro Ile Pro AsnCys AspArg Asn Gly AsnPhe HisPro Lys Gln CysHis Pro Ala LeuAsp GlyGln Arg Gly LysCys TrpCys Val Asp ArgLys Thr Gly ValLys LeuPro Gly Gly LeuGlu ProLys Gly Glu LeuAsp Cys His GlnLeu AlaAsp Ser Phe ArgGlu (2) INFORMATION FOR SEQ ID NO: 43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 113 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE
DESCRIPTION:
SEQ
ID
NO:
43:

Leu ThrGln Ser LysPhe Val GlyGly Ala Glu AsnThr Ala His Pro Arg IleIle Ser AlaPro Glu MetArg Gln Glu SerGlu Gln Gly Pro Cys ArgArg His MetGlu Ala SerLeu Gln Glu LeuLys Ala Ser Pro Arg MetVal Pro ArgAla Val TyrLeu Pro Asn CysAsp Arg Lys Gly Phe TyrLys Arg LysGln Cys LysPro Ser Arg GlyArg Lys Arg Gly Ile CysTrp Cys ValAsp Lys TyrGly Met Lys LeuPro Gly Met Glu Tyr ValAsp Gly AspPhe Gln CysHis Thr Phe AspSer Ser Asn Val Glu (2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 119 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE
DESCRIPTION:
SEQ
ID
NO:
44:

ProGln Ala Gly ThrAla Arg Gln Asp Asn Arg Arg Asp Gln Pro Val GlnArg Asn Pro GlyThr Ser Thr Pro Gln Pro Asn Ser Ala Thr Ser GlyVal Gln Asp ThrGlu Met Pro Cys Arg His Leu Asp Ser Gly Arg Val Leu Gln Gln Leu Gln Thr Glu Val Tyr Arg Gly Ala Gln Thr Leu Tyr Val Pro Asn Cys Asp His Arg Gly Phe Tyr Arg Lys Arg Gln Cys Arg Ser Ser Gln Gly Gln Arg Arg Gly Pro Cys Trp Cys Val Asp Arg Met Gly Lys Ser Leu Pro Gly Ser Pro Asp Gly Asn Gly Ser Ser Ser Cys Pro Thr Gly Ser Ser Gly (2) INFORMATION FOR SEQ ID NO: 45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 121 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQ
SEQUENCE ID
DESCRIPTION: NO:
45:

GlyGly Lys His HisLeu GlyLeu Glu Glu ProLys Lys Leu ArgPro ProPro Ala Arg ThrPro CysGln Gln Glu LeuAsp Gln Val LeuGlu ArgIle Ser Thr MetArg LeuPro Asp Glu ArgGly Pro Leu GluHis LeuTyr Ser Leu HisIle ProAsn Cys Asp LysHis Gly Leu TyrAsn LeuLys Gln Cys LysMet SerLeu Asn Gly GlnArg Gly Glu CysTrp CysVal Asn Pro AsnThr GlyLys Leu Ile GlnGly Ala Pro ThrIle ArgGly Asp Pro GluCys HisLeu Phe Tyr AsnGlu Gln Gln GluAla Arg Gly Val His Thr Gln Arg Met Gln (2) INFORMATION FOR SEQ ID NO: 46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 105 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:
Lys Val Asp Tyr Glu Ser Gln Ser Thr Asp Thr Gln Asn Phe Ser Ser Glu Ser Lys Arg Glu Thr Glu Tyr Gly Pro Cys Arg Arg Glu Met Glu Asp Thr Leu Asn His Leu Lys Phe Leu Asn Val Leu Ser Pro Arg Gly Val His Ile Pro Asn Cys Asp Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp Cys Val Asp Lys Tyr Gly Gln Pro Leu Pro Gly Tyr Thr Thr Lys Gly Lys Glu Asp Val His Cys Tyr Ser Met Gln Ser Lys (2) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
His Pro Leu His Ser Lys Ile Ile Ile Ile Lys Lys Gly His Ala Lys Asp Ser Gln Arg Tyr (2) INFORMATION FOR SEQ ID NO: 48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 135 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQ
SEQUENCE ID
DESCRIPTION: NO:
48:

AspGlu Ala Ile HisCys Pro ProCys Ser GluGlu Lys Leu AlaArg CysArg Pro Pro ValGly Cys GluGlu Leu ValArg Glu Pro GlyCys GlyCys Cys Ala ThrCys Ala LeuGly Leu GlyMet Pro Cys GlyVal TyrThr Pro Arg CysGly Ser GlyLeu Arg CysTyr Pro Pro ArgGly ValGlu Lys Pro LeuHis Thr LeuMet His GlyGln Gly Val CysMet GluLeu Ala Glu IleGlu Ala IleGln Glu SerLeu Gln Pro SerAsp Lys Asp Glu Gly Asp His Pro Asn Asn Ser Phe Ser Pro Cys Ser Ala His Asp Arg Arg Cys Leu Gln Lys His Phe Ala Lys Ile Arg Asp Arg Ser Thr Ser Gly Gly Lys Met (2) INFORMATION FOR SEQ ID NO: 49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 109 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQ
SEQUENCE ID
DESCRIPTION: NO:
49:

LysPhe Val Gly GlyAla GluAsn Thr Ala HisPro Arg Ile IleSer AlaPro Glu Met ArgGln GluSer Glu Gln GlyPro Cys Arg ArgHis MetGlu Ala Ser LeuGln GluLeu Lys Ala SerPro Arg Met ValPro ArgAla Val Tyr LeuPro AsnCys Asp Arg LysGly Phe Tyr LysArg LysGln Cys Lys ProSer ArgGly Arg Lys ArgGly Ile Cys TrpCys ValAsp Lys Tyr GlyMet LysLeu Pro Gly MetGlu Tyr Val AspGly AspPhe Gln Cys HisThr PheAsp Ser Ser AsnVal Glu (2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE type: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:
His Thr Arg Ile Ser Glu Leu Lys Ala Glu Ala Val Lys Lys Asp Arg Arg Lys Lys Leu Thr Gln Ser

Claims

CLAIMS:
1. Peptides, characterized in that said peptides are selected from APSEEDHSILWDAISTYDGSKALHVTNIKKWKEPCRIELYRVVESLAKAQETSGEEI
SKFYLPNCNKNGFYHSRQCETSMDGEAGLCWCVYPWNGKRIPGSPEIRGDPNCQI
YFNVQN

GKGGKHHLGLEEPKKLRPPPARTPCQQELDQVLERISTMRLPDERGPLEHLYSLHIP
NCDKHGLYNLKQCKMSLNGQRGECWCVNPNTGKLIQGAPTIRGDPECHLFYNEQ
QEARGVHTQRMQ

GGKHHLGLEEPKKLRPPPARTPCQQELDQVLERISTMRLPDERGPLEHLYSLHIPN
CDKHGLYNLKQCKMSLNGQRGECWCVNPNTGKLIQGAPTIRGDPECHLFYNEQQ
EARGVHTQRMQ

GHAKDSQRYKVDYESQSTDTQNFSSESKRETEYGPCRREMEDTLNHLKFLNVLSP
RGVHIPNCDKKGFYKKKQCRPSKGRKRGFCWCVDKYGQPLPGYTTKGKEDVHCY
SMQSK

KVDYESQSTDTQNFSSESKRETEYGPCRREMEDTLNHLKFLNVLSPRGVHI
PNCDKKGFYKKKQCRPSKGRKRGFCWCVDKYGQPLPGYTTKGKEDVHCYSMQSK

HPLHSKIIIIKKGHAKDSQRY

DEAIHCPPCSEEKLARCRPPVGCEELVREPGCGCCATCALGLGMPCGVYTPRCGSG
LRCYPPRGVEKPLHTLMHGQGVCMELAEIEAIQESLQPSDKDEGDHPNNSFSPCSA
HDRRCLQKHFAKIRDRSTSGGKM

KVNGAPREDARPVPQGSCQSELHRALERLAASQSRTHEDLYIIPIPNCDRNGNFHP
KQCHPALDGQRGKCWCVDRKTGVKLPGGLEPKGELDCHQLADSFRE

LTQSKFVGGAENTAHPRIISAPEMRQESEQGPCRRHMEASLQELKASPRMVPRAV
YLPNCDRKGFYKRKQCKPSRGRKRGICWCVDKYGMKLPGMEYVDGDFQCHTFDS
SNVE

KFVGGAENTAHPRIISAPEMRQESEQGPCRRHMEASLQELKASPRMVPRAVYLP
NCDRKGFYKRKQCKPSRGRKRGICWCVDKYGMKLPGMEYVDGDFQCHTFDSSN
VE

HTRISELKAEAVKKDRRKKLTQS

PQAGTARPQDVNRRDQQRNPGTSTTPSQPNSAGVQDTEMGPCRRHLDSVLQQLQ
TEVYRGAQTLYVPNCDHRGFYRKRQCRSSQGQRRGPCWCVDRMGKSLPGSPDG
NGSSSCPTGSSG, and cyclic, glycosylated, phosphorylated, acetylated, amidated and/or sulfated derivatives thereof.
2. A method for the preparation of the peptides according to claim 1 by purification from human hemofiltrate or urine, by solid-phase peptide synthesis, or by expression in recombinant microorganisms.
3. Complexes of peptides according to claim 1 with hIGF-I (human insulin-like growth factor I, MW 7649) or hIGF-II (human insulin-like growth factor II, MW 7491) and its biologically active fragments and/or derivatives, especially amidated, acetylated, sulfated, phosphorylated and/or glycosylated derivatives.
4. A nucleic acid, characterized by coding for peptides according to claim 1.
6. Antibody, characterized by binding to a peptide according to claim 1.
7. Inhibitor, characterized by inhibiting the biological activity of peptides according to claim 1.

8. Inhibitor, characterized by inhibiting the expression of peptides according to claim 1.
9. Use of peptides according to claim 1, complexes according to claim 32, nucleic acids according to claim 4, for the preparation of a medicament for treating the underexpression of insulin-like growth factor binding proteins.
10. Use of antisense nucleotides according to claim 5, antibodies according to claim 6, inhibitors according to claim 7 and/or inhibitors according to claim 8 for the preparation of a medicament for treating the overexpression of insulin-like growth factor binding proteins.
11. A medicament containing the peptides according to claim 1, complexes according to claim 3, nucleic acids according to claim 4, antisense nucleotides according to claim 5, antibodies according to claim 20, inhibitors according to claim 7 and/or inhibitors according to claim 8 in a pharmaceutically acceptable dosage form for oral, intravenous, intramuscular, intracutaneous, intrathecal administration, or as an aerosol for transpulmonary administration.
12. Use of a medicament according to claim 11 for treating muscular atrophy, osteoporosis, diabetes, amyloid lateral sclerosis, peripheral and central neuropathies, inflammatory processes, disordered inflammatory reactions, tumor diseases, inflammatory and neoplastic diseases, disturbance of growth, muscular affections, affections of the bone system, and/or for wound and bone healing.
13. Use of the nucleic acid according to claim 4 and/or the antisense nucleotides according to claim 5 for the preparation of a medicament for treating somatic or non-somatic genetic diseases.
14. Diagnostic agents containing the peptides according to claim 1, complexes according to claim 3, nucleic acids according to claim 4, antisense nucleotides according to claim 5, antibodies according to claim 6, inhibitors according to claim 7 and/or inhibitors according to claim 8, and further auxiliaries.
15. Use of the diagnostic agents according to claim 14 for diagnosing functional disorders in bones, muscles, the nervous system, lymph organs, the gastrointestinal tract, the immune system, and of diabetes and inflammatory and neoplastic processes, and as a marker in cancer.