WO1997020049A1 - Peptide humain circulant dans le sang et possedant un effet insulinotrope - Google Patents

Peptide humain circulant dans le sang et possedant un effet insulinotrope Download PDF

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Publication number
WO1997020049A1
WO1997020049A1 PCT/EP1996/005183 EP9605183W WO9720049A1 WO 1997020049 A1 WO1997020049 A1 WO 1997020049A1 EP 9605183 W EP9605183 W EP 9605183W WO 9720049 A1 WO9720049 A1 WO 9720049A1
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Prior art keywords
gcap
leu
peptide
cys
diseases
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PCT/EP1996/005183
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German (de)
English (en)
Inventor
Wolf-Georg Forssmann
Andreas Kist
Mogens KRUHØFFER
Markus Meyer
Andreas Pardigol
Gabriele Heine
Original Assignee
Forssmann Wolf Georg
Andreas Kist
Kruhoeffer Mogens
Markus Meyer
Andreas Pardigol
Heine Gaby
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Application filed by Forssmann Wolf Georg, Andreas Kist, Kruhoeffer Mogens, Markus Meyer, Andreas Pardigol, Heine Gaby filed Critical Forssmann Wolf Georg
Priority to AU10313/97A priority Critical patent/AU1031397A/en
Publication of WO1997020049A1 publication Critical patent/WO1997020049A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a human peptide with insulinotropic activity and its GCAP analogs, in particular (GCAP-II- (89-112), guanylyl cyclase C activating peptide II) and GCAP-I- (99 115), its use as a pharmacological shear Active ingredient and use of its principle of action for providing new GC-C-dependent insulinotropic active ingredients.
  • peptides have become accessible which can activate guanylyl cyclase C and are able to influence insulin secretion from the ⁇ cell of the pancreas.
  • the msulmotropic peptides according to the invention open up a new class of active substances.
  • the peptide designated GCAP-II - (89-112) is preferably formed on the gastric mucosa and is found as a biologically active molecule in the plasma of healthy and sick people.
  • the hitherto unknown mechanism of action of the peptides according to the invention on the ⁇ cell has been elucidated. The therapeutic and economic use has been examined. It was z. B.
  • GCAP-11 - (89-112) surprisingly recognized as an important circulating peptide of human blood.
  • the peptides according to the invention can be used as a means to screen and further develop further ⁇ -cell activating substances.
  • the complete amino acid sequence of GCAP-I- (99-115) and the associated messenger RNA were sequenced.
  • the peptides according to the invention are obtainable by a process for obtaining proteins in pure or partially purified form from human body fluids which have the ability to regulate bodily functions related to metabolism, that is to say in the present case to control the secretion of insulin from the pancreas. These substances are characterized in that they can be obtained in particular from hemofiltrate or hemodialysate from human blood.
  • GCAPs guanylyl cyclase C activating peptides
  • the substances of the GCAP group known to date have so far been obtained from the tissue of various animal species (Currie, MG et al, Proc. Natl. Acad. Sei. USA, 89, 967-951, 1992; Hamra, et al. Proc. Natl.
  • the GCAP-II- (89-112) (Seq. ID No. 1) formed in the gastric mucosa, which is a peptide of 24 amino acids, is preferably used for the medicament of the peptides according to the invention.
  • GCAP-II - (89-112) was previously isolated and analyzed in the method disclosed in DE 36 33 707 AI (For ⁇ smann, 1988) from blood filtrate, which was originally developed for the extraction of proteins from hamofiltrate. With these methods, molecules with a molecular weight of up to about 20 kDalton can be isolated, which are filtered out of the blood in the case of a veno-venous or arterio-venous shunt connection.
  • the GCAP-I ⁇ - (pc, 112) can now be produced by chemical synthesis or by genetic engineering and can be used for numerous other purposes, for example for analysis in human blood as a pathognomonic diagnostic feature of metabolic diseases and the treatment of these diseases, in particular diabetes mellitus.
  • GCAP-II- 89-112
  • GCAP analogues in particular GCAP-I- (99-115) (Seq. ID No. 2) and this compares to GCAP-II - (89-112) can be used.
  • a new class of active substances is therefore made available as pharmaceuticals, in particular for the treatment of diabetes mellitus.
  • the mechanism described on known cell lines is used for the screening of further analogs, especially for the screening of synthetic ones! Peptides and also non-peptide active ingredients, possibly using modifications.
  • the present invention thus relates to a new peptide class of the GCAP type, e.g. B. GCAP-II- (89-112) (Guanylyl Cyclase C Activating Peptide II), GCAP-I- (99-115) and their analogues, their preparation, use as medicaments and preparations of the GCAP analogues and their use ⁇ Lich, the principle of action described according to the invention can be used via the newly discovered signal transduction in the control of insulin secretion in order to develop, isolate and screen analogous substances for this type of action and also to make them usable for economic purposes. It also relates to the known, resulting applications, namely the biotechnological production and construction of probes for gene diagnostic purposes and the use of gene therapy for GCAP gene-dependent diseases
  • the peptides according to the invention each of which has a different structure, have one principle in common, which is presented here for the first time, in each case when the islands of the pancreas are acted upon, they cause an increased insulin secretion.
  • FIG. 1 shows partial fragments of the GCAP-I precursor which were obtained by natural endoprotease fragmentation.
  • the endoproteases Arg-C, Lys C, Asp-N, Trysm, Chemotrypsm, Endoprotease for two basic amino acids, endoprotease Glu-C and Sureo U8 were used as endoproteases.
  • Figure 1 also relates to partial fragments of GCAP-II- (89-112)
  • FIG. 2 shows the nucleic acid sequence of the guanylate cyclase C (GC-C) receptor of the rat Dit used PCR liimei to detect the GC C Tianskrjpt ⁇ m ⁇ olieiten Langerhan ⁇ 'islands and msulm-producing cells smd indicated by arrows.
  • GC-C guanylate cyclase C
  • FIG. 3 shows an electrophoresis gel for the detection of guanylyl cyclase C in the cell-producing cells (INS-I) by means of the PCR method.
  • Lane No. 1 relates to a primer, GCC 77f GCC 492r (N-terminal)
  • Lane No. 2 relates to a primer GCC 3004f-Un ⁇ p3 (C-terminal)
  • Lane No. 3 relates to a primer GCC1786f-GCC1999r (intermediate)
  • Lane No. 4 relates to a primer GCC 77f (control)
  • lane No. 5 relates to a primer GCC 492r (control)
  • lane No. 6 relates to a primer GCC 3004f (control)
  • lane No. 7 relates to a primer Un ⁇ p3 (control)
  • lane No. 8 relates to a large Prime brand
  • FIG. 4 relates to the stimulation of insulin secretion on (A) isolated pancreatic arms of the mouse in vitro.
  • the administration of the GCAP and the glucose control are each indicated by arrows.
  • the abscissa relates to the diffusion time in minutes, the coordinate gives * - the insulin stimulation in customary units
  • FIG. 5 relates to the stimulation of the cGMP generation parallel to the insulin secretion in a msulm-producing cell myia (INS-I) in vitro.
  • the strong increase in the intra-cellular cGMP under the influence of the GCAP I (99 115) addition becomes clear.
  • the cGMP generation becomes clear at the same time the insulin delivery into the medium was observed
  • FIG. 6 relates to the detection of b-cell activation in the cytosensor, which measures the metabolic activation by protein release. Addition of GCAP-I- (99-115) produces an acidification of the pericellular milieu, which shows the activation of the cell metabolism in two b-cell minias (INS-I and RIN cells).
  • FIG. 7 relates to a schematic representation of the signal transduction path of the interaction of the GCAP-type peptides according to the invention with the b-cell.
  • Figure 8 shows primers for obtaining the cDNA from GCAP-II- (81-112).
  • the primers used are shown in 5 '-> 3' orientation. Underlined areas contain recognition sequences for restriction endonucleases which ensure efficient cloning of the PCR products These primers are named as sequences Ni 28 to 34 m in the sequence list
  • FIG. 9 relates to cDNA obtained from human gastric and colon extract.
  • the translated areas are shown in capital letters, the non-translated areas in g-letters.
  • the ATG start codon, the TGA stop codon and the AATAAA polyadenylation signal smd are underlined.
  • the positions of the primers used are marked with arrows.
  • the first amino acid of the isolated GCAP-II- (99-115) is marked with a triangle, the first amino acid of the uroganylm with a diamond.
  • the nucleic acid sequence in FIG. 9 corresponds to the sequence ID no. 36, whereas the correspondingly encoded amino acid sequence of the polypeptide as Seq ID No. 35 is filed.
  • FIG. 10 relates to the schematic representation of the elements of the GCAP I - (99-115) gene.
  • the exon / intron structure of the gene and the 5 ′ - flanking one are shown Sequence area.
  • the AC-rich sequence region with potential Z-DNA conformation is highlighted.
  • FIG. 11 relates to the DNA sequence of the GCAP-I (99-115) gene and its flanking sequences with regulatory elements.
  • the corresponding sequence is stored as Seq ID No 39 in the sequence listing. Coding areas are capitalized, intron sequences are printed in small letters. Potential binding sites for transcription factors are capitalized and underlined. The potential binding of the factors on the sense or antisense strand is indicated by arrows The substance and stait codons are printed in italics. Direct and inverted repeats (direct and inverted repeats) are numbered with Roman numerals.
  • the oligo nucleotides Gua-Pl, Gua-P2 and Gua-P3 shown in the illustration were used to amplify the promoter fragments for the luciferase / reporter gene assay
  • FIG. 12 relates to promoter activities of the GCAP-I (99-115) promoter fragments.
  • the promoter / reporter gene constructs with the promoter sequences of different lengths are shown schematically.
  • the relative activities represented in the right part each represent a multiple of the definition standardized to 1 Basal activity of the promoterless clone PGL-2-Bas ⁇ c represents
  • the peptides according to the invention are surprisingly characterized in that when they act on msulm-producing cells, they react with increased insulin products. This effect was previously unknown. Only for GCAP-II- (89-112) had it been referred in the international application PCT / EP 96/03429 that this peptide can be used for the treatment of diabetes. However, it does not result for the person skilled in the art that this peptide itself is used as The present invention also contains the IT' ⁇ in the HmbLt 'au 4 96/03429 new information for the expert. Fragments claimed according to the invention are to be understood as fragments of guanylm which have the msulmotropic effect, in particular those which are mentioned here in the sequence listing.
  • the peptide GCAP-II- (89-112) according to the invention containing 24 amino acids has the following amino acid sequence
  • Seq. ID No 1 and 2 include their salts, natural and pharmacologically contractual derivatives, in particular amidated, acetylated, phosphorylated and glycosylated GCAP II (89-112) or GCAP-I (99-115) derivatives and further fragments of the peptides name, especially those according to Seq ID No. 3 - 25
  • Mass spectrometry revealed a molecular weight of 2,597.7 daltons for GCAP-II- (89-112) and 1,702 daltons for GCAP-I- (99-115) (Examples 1 and 2).
  • the amino acid sequence determined for GCAP-II - (89-112) corresponds to the amino acid sequence derived from the cDNA from position 89 to position 112 (COOH terminus)
  • the use of GCAP-II- (89-112), the peptides which are obtained from the cDNA sequence as cleavable partial fragments predominantly via natural endoproteases (FIG. 1) and its analogues as a pharmaceutical for the treatment of metabolic disorders, particularly diabetes mellitus is also the subject of the present invention.
  • a corresponding usability could be determined by functional analysis (Example 5).
  • the peptides according to the invention can be produced by various methods. These are characterized in that they are produced by prokaryotic or eme eukaryotic expression and purified chromatographically, by isolation from human blood using chromatographic methods in a manner known per se or by customary methods of solid-phase and liquid-phase synthesis from protected amino acids , which are contained in the specified sequence, couples, deblocks and cleans with common chromatography methods.
  • GCAP-II- (89-112) and GCAP-I- (99-115) were chemically synthesized (see Example 3) and prepared as a medicament.
  • Genetic engineering production using conventional vectors has also been developed:
  • the GCAP-II (89-112) peptide is produced by genetic engineering, both (1) in pro- and (2) in eukaryotic organisms.
  • Various organisms and vectors are available for eukaryotic expression, for example insect cells (Summers and Smith, Tex. Agric. Exp.
  • GCAP-I- 99-115) and GCAP-II- (89-112) are purified using methods of chromatography, preferably as indicated in Examples 1 and 2.
  • the medicament according to the invention containing the msulmotropic peptide according to the invention contains GCAP-1 - (99-115) or GCAP-II- (89-112) or their physiologically tolerable salts.
  • the form and composition of the medicinal product containing GCAP-I- (99-115) or GCAP-11 - (89-112) depends on the mode of administration.
  • Human GCAP-I - (99-115) or GCAP-II- (89-112) can be administered parenterally, intranasally, orally and by inhalation.
  • GCAP-II- (89-112) is preferably made up into an injection preparation, either as a solution or as a lyophilisate for dissolution immediately before use.
  • the pharmaceutical preparation can also contain auxiliaries which are due to filling technology, which contribute to the solubility, stability or sterility of the pharmaceutical or which increase the efficiency of absorption into the body.
  • the daily dose of the insulmotropic peptide to be administered depends on the indication and the use of certain derivatives.
  • Injection is in the range of 100 to 1,200 units ( ⁇ g) / day, with daily subcutaneous injection, preferably 300-2400 units ( ⁇ g) / day.
  • the msulmotropic peptides according to the invention are particularly distinguished by the fact that they are also suitable for long-term therapy of diabetes mellitus. They have excellent biological properties Efficacy and, on the other hand, do not trigger an immune reaction even with Dauei treatment.
  • the biological effect shows that the preparation according to the invention can also be used as an agent for therapy for diarrheal diseases, kidney diseases, heart diseases, diseases of endocrine organs (as emphasized above, in particular diabetes mellitus), in intensive care and for lung diseases (example 4 and 5).
  • the msulmotropic peptide according to the invention can also be used as a means of therapy and diagnosis for metabolic disorders of the gastrointestinal tract (in particular the small intestine and pancreas), the respiratory tract, the cardiovascular system and the urogenital system, endocrine organs and the nervous system, since it is used for Production of human-compatible antibodies can be used, which are suitable for detecting or influencing changes in the metabolic position of these organs.
  • Hamofiltrate was used as the starting material, which accumulates in large quantities in the treatment of patients with kidney deficiency and contains all plasma constituents up to a molecular size of approximately 20,000 daltons.
  • the hamofiltrate was obtained by means of a Sartorius hamofiltration system using cellulose triacetate filters with an exclusion size of 20,000 daltons (type SM 40042, Sartorius, Göttingen, FRG).
  • the filtrate came from patients with kidney insufficiency who were in a stable metabolic state due to long-term hemoflltration.
  • 3,000 1 hamofiltrate were obtained and immediately protected against proteolytic degradation by acidification and cooling to 4 ° C.
  • a crude peptide fraction (570.5 g) was then obtained by four cation exchange extractions.
  • the Rohpeptidfr press from the ultrafiltration was mascara on a column packed with RP-C18 Materral (Vydac, Hespe ⁇ a, CA, USA) Kar ⁇ equilibrated with 0.01 N hydrochloric acid, chromato ⁇ graphically by means of HPLC separated •
  • the cGMP-generating effect of the fractions was grown on T84 cells (Currie, MG et al., Proc. Natl. Acad. Sei., 89, 947-951, 1992) in Fischer medium (Gibco) by measuring the Increase in intracellular cGMP was determined by means of a radioimmunoassay after 60 mm and was compared with controls in which 0.9% NaCl was added to the culture medium instead of the HF peptides.
  • Eluent B 80% acetonitrii, 20 o 0 water
  • Fractions 4-6 contained bioactivity for the cGMP generation test on T84 cells. Fraction 5 was used for the further separation. 1.2.3 Analytical cation exchange chromatography of the GCAP-II (89-112) -containing fraction from the preparative RP-Cl8 chromatography II (stage 3)
  • the bioactive GCAP-II- (89-112) -containing immunoreactive fraction (50 ml) was further purified by means of analytical cation exchange chromatography.
  • the biological activity appears in the range of a retention time of 8 and 9% of the eluent B.
  • Eluent A water with 0.1% trifluoroacetic acid
  • Eluent B 80% acetonitrii, 20% water
  • Eluent B 80% acetonitrii, 20 o water
  • the peptide activating T84 was detected in an area that eluted within one minute. The entire further material of the bioactive fractions was finally rechromatographed using the same column (see step 6)
  • Eluent B 80% acetonitrii, 20 W water
  • the sequence found is the NH 2 -terminally unknown Pepart, the C-termmus corresponds to human uroguanylms (Hamra, et al., Proc. Natl. Acad. Sei., USA, 90, 10464-10468, 1993) .
  • This surprisingly found peptide was designated as guanylyl cyclase C activating peptide II, GCAP-II- (89-112). It has structural homologies to those of human guanylm and the heat-stable enterotoxes from bacteria. The activating effect on guanylate cyclase-C is described for these peptides.
  • the analysis of the GCAP-II (89-112) sequence is confirmed by the cDNA (FIG. 7 and Example 6).
  • the derived precursor can be used to prepare the brologrsch available fiagments to the other biologically active peptides of the GCAP-II (89-112) group to win (Seq ID No 2)
  • the urine from healthy volunteers was used as the starting material - 11 l of urine were diluted with 44 l of deionized water immediately after collection and adjusted to pH 2.7 with 32% HCl after addition of 150 g Algmsaure was stirred for 2 hours at 4 ° C, sedimented for 10 minutes and stirred for a further 2 hours. The alginic acid was then sedimented over 1 hour and the supernatant checked for peptide / protein content hm. After stirring again, the suspension was transferred to a process filter and suction filtered through filter paper.
  • the alginic acid cake was sucked dry and washed twice with 5 mM HCl to remove the residual urine.
  • the alginic acid was then resuspended and washed four times with 600 ml of ethanol in order to remove lipids etc.
  • the alginic acid was suspended with 150 ml of 0.2 M HCl and sucked dry. This process was repeated four times, the filtrates were collected and immediately adjusted to pH 4.0 with 0.2 M sodium acetate.
  • the eluate was then dried by freeze-drying and the residues were taken up in 210 ml of demineralized water.
  • the dissolved peptides were desalted on a column filled with RP-C18 material.
  • the entire eluate of the preparative RP-C18 column was collected and then subjected to freeze drying.
  • the freeze-dried preparation was dissolved in 22 ml of 0.1 M acetic acid, 10% acetonitrile and separated into further constituents via cation exchangers.
  • stage IV The individual fractions of stage IV were subjected both to a direct coupling of the liquid chromatography and to a fractionation from a microbore column.
  • the mass could be determined with 1702 amu and thus corresponds to the mass of the human GCAP-I- (99-115) Example 3
  • the synthesis was carried out by m situ activation with the addition of 4.4 equivalents of TBTU [O- (1H-benzotriazol-1-yl) - N, N, N ', N' -tetramethyluronium tetrafluoroborate], 4.4 equivalents 1-Hydroxybenzotriazole (HOBt) and 8.8 equivalents of diisopropylethylamine in N, N-dimethylformamide.
  • Pre-occupied Fmoc-L-Leu-PEG-PS resin (0.20 mmol / g coverage, biosearch / per septive) was used as the carrier resin for the synthesis.
  • cysteine residues in positions 15 and 23 were used as otcotamidomethyl (Acm) -protected cysteine derivatives used while the system residues in positions 12 and 20 were used as trityl-protected derivatives.
  • Acm otcotamidomethyl
  • the resin was split off with trifluoroacetic acid / - ethanedithiol / water in a ratio of 94 3> (10 ml, v / v / v) within 90 minutes.
  • the solution is concentrated in vacuo and the product is precipitated by adding diethyl ether.
  • the crude peptide obtained was washed several times with diethyl ether, dried and then taken up in water and freeze-dried. 85 mg of crude peptide were obtained from 490 mg of peptide-resin.
  • the crude peptide was added at a concentration of 1 mg / ml m 0.1 M aqueous ammonium carbonate solution at pH 8.3. This mixture was stirred in air for 48 hours at room temperature and then lyophilized.
  • the product obtained was taken up in 80% methanol in water and 5 equivalents of solid iodine were added. This deep brown mixture was stirred vigorously for 3 hours under an atmosphere of plastic. The excess iodine is then added with the addition of 0.1 M sodium thiosulfate solution until it decolorises. The solution obtained is diluted with a volume of water. Then the peptide was purified.
  • the purification was carried out using a standard of GCAP-II- (89-112) prepared in independent synthesis, the correct sequence of which was confirmed by MS (mass spectrometry) and sequence analysis.
  • the yield was 3.6 mg.
  • the material obtained coelutes in an analytical RP-HPLC with C18 material with the independently produced standard.
  • the identity of the synthesized material with the given primary structure of GCAP-11 - (89-112) was determined by mass spectrometry (Sciex API III, Perkin-Elmer) and sequencing in a gas phase sequencer (model 470, Perkm-Elmer) confirmed.
  • the biological activity of the synthetic GCAP-II (89-112) was demonstrated in the functional test by the cGMP generation-stimulating effect on T84 colon carcinoma cells. This showed that the synthetic material has a biological activity
  • the cells were used after about 2-3 days.
  • the incubation was carried out with 0.5 ml of cell medium in the presence of 1 mM IBMX (Sigma) at 37 ° C.
  • the GCAP-II (89-112) peptide Preparations containing were taken up in the cell medium immediately before the start of the experiment.
  • FIG. 2 shows the GC-C cDNA the rat (Schulz DL, et al. Cell 63: 941-948, 1990), the 6 primers used being shown.
  • 3 shows as an example the result of the PCR for the detection of the GC-C transcript from INS-I cells.
  • the cGMP-specific protein kinases were detected in an analogous manner. The same amplifications can be found in isolated islets and on 3 other insulin-producing cells.
  • insulin-producing cell lines (INS-I, RIN, HIT) used in the studies and the isolated islets of the rat express the specific guanylin receptor
  • INS-I reads a slowly growing cell line which was established from an X-ray-induced insuloma of the rat.
  • the culture doubles time to 100 hours.
  • the Cells show the morphological characteristics of ß cells in the pancreas. They have an insulin content of about 20% of native ß cells. Glucose stimulates the insulin secretion of the INS-I cells.
  • the cells are cultivated in medium RPMI1640 + additives in an incubator at 5% CO, 37 ° C.
  • the cell chemistry is a suitable model for examining the influence of chemical substances on the insulin regulation in vitro .
  • RINm5F is also a cell measurement from an insuloma of the rat.
  • the stimulability of the cells for insulin secretion by glucose is about 1/50 that of INS-1 cells.
  • This cell myia which has comparable growth and culture restrictions, like INS-I, can also be used to study the regulatory mechanisms for the secretion of insulin.
  • the insulated-perifused pancreatic capsule reads an established tissue model for recording the effect of chemical substances on insulin secretion and the second messenger systems involved. Subject of the investigation was to demonstrate dec influence of CGAP-I] - (89 112) on the r In ul msekretrons antique natrver beta cells
  • the pancreatic tissue is removed from mice and, after digestion with collagenase, approximately 50 individual islands are transferred to a membrane and punctured with a physiological buffer. After an aquiliberation phase, the peptide to be tested is added to the buffer and the perfusionate is collected fractionally. The released insulin is then detected in the perfusate by the method already mentioned above.
  • Stimulation of the INS-I cells (FIG. 5) with GCAP-II- (89-112) resulted in an increase in the intracellular cGMP to 1016 pmol / ml at 10 M (negative control 362 pmol / ml)
  • the administration of 10 "7 M GCAP-II (89-112) stimulates INS-I cells (FIG. 5) to secrete insulin at a level of 6.08 ng / ml (negative control 1.15 ng / ml).
  • microphysiometer it is possible to record the cellular response to a chemical substance, in particular peptide factors as ligands for specific membrane receptors, via the acidification of the extracellular space.
  • concentration of released protons which is linear with the drop in the extracellular pH value, is measured using a light-addressable photometric silicon sensor (LAPS).
  • LAPS light-addressable photometric silicon sensor
  • GCAP-II- (89-112) binds to the membrane surface of the ß cell
  • cGMP-specific protein kinases eg cGK-II
  • the protein phosphorylation activates the mechanism of the Ca-dependent release of insulin via exocytosis (FIG. 7).
  • RNA was obtained from eight human tissues (duodenum, colon, stomach, liver, pancreas, kidney, adrenal gland and bladder) using standard methods.
  • MMLV reverse transcriptase and the oligo (dT) primer UNIP-5 (see FIGS. 8 and 9), which is partially complementary to the poly (A) tail of eukaryotic mRNA, a transcription was carried out in the first strand of cDNA.
  • PCR primer HUGU-5 (see FIGS. 8 and 9) was constructed, which contains the codons for the C-terminal fragment CVNVAC.
  • UNIP-6 the sequence of which is contained in UNIP-5, polymerase chain reactions (PCRs) (Saiki, RK et al ., Science, 239, 487-491, 1988).
  • PCRs polymerase chain reactions
  • the first potential translation start codon ATG appearing in the sequence is actually the beginning of an open reading frame of 336 base pairs, which codes for a 112 Ammosciui-n GCAP-11 (8 L UM Voi l.uf.i. As expected, the first 21 amino acids show the typical characteristics (high hydrophobicity) of a secretory signal peptide (Von Heij ne, G., Eur. J. Biochem., 133, 17-21, 1983). As already mentioned, the 3 'terminus of the cDNA sequence codes for the isolated peptide GCAP-II - (89 - 112)
  • luciferase reporter gene assay was used (Wood, KV, Biolummescence & Chemilumme ⁇ cence: Current Status, ed. P. Stanley and L. Kricka, John Wiley and Sons, Chicester , 1991).
  • Two differently long, potential promoter fragments were first identified by PCR (Saiki, RK et al. Science 239, 487 491, 1988) using a proofreading heat-stable combi-polymerase (InViTec, Berlin, Germany) from a lysate of phage pHGU 35 ( Hill, O., et al., Proc. Natl. Acad. Sci.
  • the two plasmids GLuc25 and GLuc30 were cut with the residual endonucleases SstI and PstI (Life Technologies, Eggenstein, Germany) and the linear ones After electrophoresis in the agarose gel, plasmid DNA of the five plasmid constructs was religated (GLuc25, GLuc30, 25 ⁇ SstI, 25 ⁇ PstI and 30 ⁇ PstI) were prepared using TIP 100 columns (Qiagen, Hilden, Germany).
  • T84 cells were used using the calcium phosphate DNA coprecipitation method (Sambrook, J., et al. 1989, Molecular Cloning: A Laboratory Manual. Cold Spring Habor Laboratory, Cold Spring Habor, New York) with the promoter / reporter gene constructs for 7 hours.
  • 5 ⁇ g of promoter / report gene DNA together with 2 ⁇ g of pSVßGal DNA (Sigma-Aldrich, Deisenhofen, Germany) were propagated as the internal standard for later normalization of the luciferase values in the cells.
  • the relative promoter activities represented in FIG. 12 represent the mean values from two independent transformation experiments. All constructs show significant relative promoter activity. Due to a 3-fold drop in the activity of the clone GLuc25, binding motifs for potentially negative transcription factors could exist in the range between -1309 and -532.
  • MOLECULE TYPE Peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • MOLECULE TYPE Peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • MOLECULE TYPE Peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • MOLECULE TYPE Peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO: 6.
  • MOLECULE TYPE Peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • MOLECULE TYPE Peptide
  • HYPOTHETICAL NO Uv
  • ANTI ⁇ ENSE NO
  • MOLECULE TYPE Peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • MOLECULE TYPE Peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • CACGATTTCC TACCCTGATG GCACGTTCAT GGATTGGGAG TTTAAGATCT CTGTCTTAAA 1800
  • GGAAAATTCC TATGGGACGA AACCCTTCCG CCCAGATCTC TTCCTGGAAA CCGCAGATGA 2160
  • GCCCCCAAGT CATGTGGGTG TTCTGGGTTG GGTTGGGTTG GGTTTGGTTG GTTGGTTTTG 3420
  • ANTISENSE NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: CCTCAGCTGC AGCTCGAGTT TTTTTTTTTTTTTTTTTTTT TT 42
  • ANTISENSE NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: CTGCAGCTCG AGTAGAATCT TTATTCAGGA GCTG 34
  • ACCATCGCTA ACGACGACTG TGAGCTGTGT GTGAACGTTG CGTGTACCGG CTGCCTCTGA 360
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • CTGCAGAGCA CACAGTCAGT CTACATCCAG TACCAAGGCT TCCGGGTCCA GCTGGAATCC 120
  • GGTATTATTT TTGGTTCCCA CAGGGGACAC AAGGGACAAC GAGACTCAGA GAGGGGAAAG 1740
  • ACACAGCAGC ACAGGGCAGT CGTGAGTGAA TGTGCCTTGT TGGTGAATGA ATGCAGGACC 2160
  • CTCTTCGCAC TGTGCCTCCT TGGGGCCTGG GCCGCCTTGG CAGGAGGGGT CACCGTGCAG 2460
  • CTGATCTCTA TCAAAGCCAG ATTCCGGGAG GCCTTTTATT GCTTCTCAAA CCAAAACCAG 3060

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Toxicology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Endocrinology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un peptide insulinotrope issu du sang humain, notamment le GCAP-II-(89-112) (peptide II activateur de guanylatecyclase C), dont la structure a été étudiée en vue de son utilisation comme médicament à des fins diagnostiques, médicales et industrielles. L'invention concerne un GCAP-I-(99-115) analogue isolé à partir de l'urine. La forme moléculaire du GCAP-II-(89-112) a été déterminée par séquençage et spectrométrie de masse. Une activité biologique diversifiée et des différences dans la structure spatiale du peptide du GCAP-II-(89-112) par rapport à d'autres fragments GCAP-II-(89-112) prouvent que ce fragment GCAP-II représente un peptide naturel de la famille du groupe de peptides de l'invention, qui peuvent être utilisés dans le traitement de nombreuses maladies s'accompagnant de perturbations du transport du glucose et des électrolytes et de la sécrétion d'insuline dans les cellules, et plus particulièrement dans le traitement de maladies affectant les glandes endocrines. Le principe d'action par exemple du GCAP-II-(89-112) dans le cadre de la sécrétion d'insuline par le pancréas endocrine et donc de substances analogues a été décrit et peut donc être utilisé pour développer des antidiabétiques puissants. En outre, le GCAP-I-(99-115) et le GCAP-II-(89-112) (peptides I et II activateurs de guanylatecyclase C) peuvent être utilisés sous leur forme pure ou sous forme d'extrait brut naturel à des fins industrielles dans la recherche de nouvelles fonctions cellulaires. Les connaissances acquises sur les structures de l'ADNc et des gènes du GCAP-II-(89-112) et du GCAP-I-(99-115) peuvent avoir une importance diagnostique et thérapeutique.
PCT/EP1996/005183 1995-11-24 1996-11-23 Peptide humain circulant dans le sang et possedant un effet insulinotrope WO1997020049A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU10313/97A AU1031397A (en) 1995-11-24 1996-11-23 Human peptide circulating in the blood and possessing insulinotropic properties

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE1995143628 DE19543628A1 (de) 1995-11-24 1995-11-24 Humanes, im Blut zirkulierendes Peptid mit insulinotroper Wirkung (GCAP-II-(89-112), (Guanylyl Cyclase C Aktivierendes Peptid II) und seine GCAP-Analoga, insbesondere das GCAP-I-(99-115), seine Anwendung als pharmakologischer Wirkstoff und Benutzung seines Wirkungsprinzipes zur Bereitstellung neuer GC-C-abhängiger insulinotroper Wirkstoffe
DE19543628.8 1995-11-24

Publications (1)

Publication Number Publication Date
WO1997020049A1 true WO1997020049A1 (fr) 1997-06-05

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PCT/EP1996/005183 WO1997020049A1 (fr) 1995-11-24 1996-11-23 Peptide humain circulant dans le sang et possedant un effet insulinotrope

Country Status (3)

Country Link
AU (1) AU1031397A (fr)
DE (1) DE19543628A1 (fr)
WO (1) WO1997020049A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8173596B2 (en) 2004-05-14 2012-05-08 The University Of North Carolina At Chapel Hill Prouroguanylin, and synthetic analogs or proteolytic cleavage products derived from it, as therapeutic and diagnostic agents for diseases involving salt and/or fluid homeostasis

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AU2001256315A1 (en) * 2000-04-22 2001-11-07 Ipf Pharmaceuticals Gmbh Use of guanylate cyclase-c (gc-c) agonists as insulinotropic factors for treating diabetes type 2
US20050032684A1 (en) * 2001-06-05 2005-02-10 Yalcin Cetin Use of a peptide which activates guanylate-cyclase c for the treatment of respiratory airway problems via the airways, medicament, inhaltion devices and method of diagnosis
US7494979B2 (en) * 2003-06-13 2009-02-24 Ironwood Pharmaceuticals, Inc. Method for treating congestive heart failure and other disorders
JP2007501866A (ja) 2003-06-13 2007-02-01 マイクロバイア インコーポレイテッド 胃腸疾患の治療のための方法および組成物
HUE033371T2 (en) 2013-03-21 2017-11-28 Sanofi Aventis Deutschland Process for the preparation of peptide products containing a ring imide
AU2014234400B2 (en) 2013-03-21 2017-11-16 Sanofi-Aventis Deutschland Gmbh Synthesis of hydantoin containing peptide products

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WO1995003410A2 (fr) * 1993-07-20 1995-02-02 Forssmann Wolf Georg Gene codant les peptides i activant la guanylatcyclase (gcap-i)
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WO1995003410A2 (fr) * 1993-07-20 1995-02-02 Forssmann Wolf Georg Gene codant les peptides i activant la guanylatcyclase (gcap-i)
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8173596B2 (en) 2004-05-14 2012-05-08 The University Of North Carolina At Chapel Hill Prouroguanylin, and synthetic analogs or proteolytic cleavage products derived from it, as therapeutic and diagnostic agents for diseases involving salt and/or fluid homeostasis

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