Classifications

• • 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
  • C07K14/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Cardionatrin; Cardiodilatin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P23/00—Anaesthetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/827—Proteins from mammals or birds
  • Y10S530/841—Muscles; heart

Definitions

• Cardiodilatin a new peptide hormone and process for its production.
• the invention relates to a new peptide hormone and a method for its production.
• the right atrium of the pig contains two different cell types, one of which has the morphology of endocrine cells (granular secretions). Through morphological and histochemical examinations of the right atrium, however, no hormone substance such as that of the already known type in myoendocrine cells of the auricle (auricle) could be determined. In contrast, many of the known neuropeptide hormones have been found in cardiac nerves.
• Atrium extracts cardiac antechamber
• atrium extracts cardiac antechamber
• they have effects on the ionotropy of the heart muscle itself or influences on the smooth vascular muscles, as well as influences on sweat secretion.
• these biological effects are caused by a new one.
• Peptide hormone are caused, which is of great clinical and therapeutic importance with regard to its effects, in particular with regard to the diagnosis and therapy of hypertension.
• the invention therefore relates to the new peptide hormone cardiodilatin with the N-terminal amino acid sequence:
• fragments in particular C-terminal fragments and the fragments of the peptide hormone cardiodilatin located between two Met residues of the peptide chain also have the biological activities of cardiodilatin, albeit in a weakened form in some cases.
• the invention therefore also relates to the fragments of the peptide hormone cardiodilatin located between two Met residues of the peptide chain, such as, for. B. the fragment with the amino acid sequence Asp-Phe-Lys-Asn-Leu-Leu-Asp-His-Leu-Glu-Asp-Lys-Hse (1) or the fragment with the amino acid sequence Pro-Leu-Glu-Asp-Glu - Ala-Hse (2), and the N-terminal fragment Asn-Pro-Val-Tyr-Gly-Ser-Val-Ser-Asn-Ala-Asp-Leu-Hse as well as the respective C-terminal remaining trunk sequences of the cardiodilatin 126, shortened by the homosin peptides listed.
• a number of cardiodilatin fragments were also produced and investigated either synthetically by Merrifield synthesis or by cleaving the cardiodilatin with specifically cleaving proteases.
• the Merrifield synthesis was carried out in such a way that starting at the C-terminal end, the amino acid chain was extended by two amino acids, split off, examined, and extended by a further two amino acids according to the same principle. This procedure was repeated as often as required for the fragments obtained and defined in more detail below.
• the fragments produced have an interesting biological activity and in fact they are suitable for the formation of antibodies which are able to recognize the entire cardiodilatin and can therefore also be used for its detection in an immunoassay. Both the numerous known embodiments of the RIA (radio-immunoassay) and the EIA (enzyme immunoassay) are suitable. It was also found that some of the fragments also have the vasodilatory activity of cardiodilatin, in particular the fragment comprising amino acids 39 to 126 and the corresponding fragments which are extended N-terminally to amino acid 7.
• the invention therefore further relates to the fragments listed below, which are characterized by the HPLC data: Fragments comprising amino acid positions 1 to 7:
• Fragments 108 to 114 with an additional Tyr (position 107), Tyr-Gly-Arg-
• cardiodilatin is obtained by using atrium extract as the starting material and the material extractable with aqueous solvents is fractionated according to conventional biochemical cleaning methods using a test in which the active fraction is determined by the relaxing effect on smooth muscles.
• the pure representation of the peptide hormone cardiodilatin from atrium extracts eg. B. from porcine atria (V. Mutt, Arkiv Kemi 15, 69-74 (1959).
• Cardiodilatin is preferably prepared by extracting cooked atrium material with dilute acid.
• the dilute acid is primarily carboxylic acids, but also mineral acids can be Acetic acid is preferably used, particularly preferably in a concentration of 0.1 to 0.3 M.
• Further purification according to the invention can be carried out by adsorbing the acid extract on alginic acid, eluting the peptides from the alginic acid again with dilute mineral acid, subjecting the eluate to salt precipitation, dissolving the precipitate formed and re-precipitating with ethanol. The ethanol precipitate is then dissolved and chromatographed on carboxymethyl cellulose.
• a material obtained in this way is generally sufficiently pure for pharmaceutical or analytical purposes.
• the precipitate obtained by salt precipitation described above can also be directly desalted and purified by gel chromatography, preferably on Sephadex G 25.
• Fine purification is possible by means of high pressure liquid chromatography (HPLC) on a reversed phase silica gel (gradient elution: 0.1% trifluoroacetic acid in water against acetonitrile, 0 to 60%) and separation of the active fractions.
• HPLC high pressure liquid chromatography
• the HPLC on a reversed-phase silica gel is preceded by an HPLC on an ion exchange column and / or the HPLC on reversed-phase silica gel is repeated one or more times, preferably twice.
• the N-terminal fragments After conjugation with a carrier protein, the N-terminal fragments, which are obtained either synthetically or by cleavage at the N-terminal methionine residue or arginine residue, cause antibody formation when administered to other animal species such as rabbits or mice (Balb C), and therefore also have hapten properties .
• These antibodies can be used as a specific ligand in affinity chromatography for enrichment (isolation) and for the detection of the peptide hormone cardiodilatin. Affinity chromatography with a N-terminal fragment antibody as a specific ligand thus represents another suitable method for enriching (isolating) the peptide hormone, with which cardiodilatin can usually be obtained in a sufficiently pure form.
• Affinity chromatography can also be used with one of the above-mentioned chromatographic methods for isolating the peptide hormone, or else one of the chromatographic methods can be followed by further purification by one or more suitable methods; suitable procedures are e.g. B. electrophoresis, precipitation, adsorption chromatography, ion exchange chromatography, affinity chromatography, isoelectric focusing, or a combination of one or more of the same or different of these steps.
• suitable procedures are e.g. B. electrophoresis, precipitation, adsorption chromatography, ion exchange chromatography, affinity chromatography, isoelectric focusing, or a combination of one or more of the same or different of these steps.
• the fragments located between two Met residues of the peptide chain can be prepared from the cardiodilatin in a manner known per se by cleavage with suitable hydrolases.
• B. produce the above-mentioned fragments with the amino acid sequence (1) and (2) or N-terminal fragments, synthetically by known peptide synthesis, such as. B. by the Merrifield method or synthesis with mixed anhydrides in solution.
• the relaxing effect on the renal artery (rabbit), abdominal aorta (rabbit, rat) and inferior mesenteric artery (rabbit) is used as a differentiating bioassay for the action of the peptide Organ bath used, with the vascular muscle strips contracted after adrenaline pretreatment showing a marked relaxation after the addition of cardiodilatin.
• This relaxation of the muscle strip from the renal artery is detectable at low doses of approx. 10 ng to 100 ng / 10 ml organ bath.
• strips of the aorta as a test muscle there was evidence of relaxation at an approximately 10-fold higher dose, while hardly any activity was observed in the inferior mesenteric artery.
• the invention therefore also relates to medicaments for use in the above-mentioned diagnostic and therapeutic methods which contain the new peptide hormone cardiodilatin and / or active fragments thereof.
• the medicaments can be present in the usual forms of use for oral or parenteral administration, such as, for example, B. as tablets, suppositories, dragees, solutions, etc., optionally together with conventional, pharmacologically acceptable carriers and / or diluents.
• the amount of cardiodilatin is preferably 10 to 1000 ng / dose unit.
• This tissue of the right atrium described above was taken from 20,000 pig hearts and extracted using known methods - (cf. V. Mutt, Arkiv Kemi 15, 69-74 (1959), SI Said and V. Mutt, Eur. J. Biochem. 28, 199-204 (1972), V. Mutt Gut hormones, pp. 21-27, SR Bloom (editor) Edinburgh-London-New York: Churchill Livingstone, 1978).
• the column had previously been equilibrated with a buffer containing 0.03 M NaOH, 0.025 MH 3 PO 4 , 0.5% thiodiglycol, pH 6.4. After elution with the same buffer, another elution was carried out with a buffer containing 0.03 M NaOH, 0.025 M H..PO. and 0.2 M NaCl pH 6.4. The most active fractions were eluted.
• the fraction eluted with the salt-containing buffer is placed on an ion exchange column (TSK-carboxymethyl cellulose from LKB) and eluted with 0.03 M sodium phosphate buffer, pH 6.4 and a sodium chloride gradient from 0 to 0.5 M.
• the active fraction is collected over a molecular sieve column (Sephadex
• the peptide cardiodilatin emerges from the HPLC column at 45% acetonitrile and shows a retention time of 24.1 minutes.
• Fig. 1 shows a graphical representation of this HPLC.
• the active fraction obtained was then subjected to the same HPLC twice again.
• the isoelectric point I.P. was determined with 6 to 6.5.
• the peptide shows the following in amino acid analysis
• amino acid sequence was determined from the N-terminus by automated Edman degradation as follows: Asn-Pro-Val-Tyr-Gly-Ser-Val-Ser-Asn-Ala-Asp-Leu-Ket-Asp-Phe-Lys-Asn -Leu-Leu-Asp-His-Leu-Glu-Asp-Lys-Met-Pro-Leu-Glu-Asp-Glu-Ala-Met-Pro-Pro-Gln-Val-Leu-Ser- Glu-Gln-Asp -Glu-Val-Leu-Ser-Glu-Gln-Asn-Glu-Glu-Val-Gly-Ala-Pro-Leu-Pro-Leu-Leu-Glu-Glu-Val-Pro-Pro-Trp-Thr-Gly -Glu-Val-Asn-Pro:
• the molecular weight is calculated based on the amino acid composition with about 13,000 daltons.
• Bioassays were carried out for the various stages of the cleaning process, with an increase in specific activity as the degree of cleaning progressed.
• Bioassays were carried out on smooth vascular muscles, in which the relaxation in an organ bath was determined under the action of the fractions to be tested.

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• Urology & Nephrology (AREA)
• Cardiology (AREA)
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• Public Health (AREA)
• Toxicology (AREA)
• Zoology (AREA)
• Gastroenterology & Hepatology (AREA)
• Animal Behavior & Ethology (AREA)
• Pharmacology & Pharmacy (AREA)
• Genetics & Genomics (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• General Chemical & Material Sciences (AREA)
• Biotechnology (AREA)
• Cell Biology (AREA)
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• General Physics & Mathematics (AREA)
• Food Science & Technology (AREA)
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• Bioinformatics & Cheminformatics (AREA)
• Heart & Thoracic Surgery (AREA)
• Anesthesiology (AREA)
• Peptides Or Proteins (AREA)

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• 1983
  • 1983-12-24 DE DE19833346953 patent/DE3346953A1/de active Granted
• 1984
  • 1984-12-21 US US06/769,627 patent/US4751284A/en not_active Expired - Lifetime
  • 1984-12-21 JP JP60500395A patent/JPH0822876B2/ja not_active Expired - Lifetime
  • 1984-12-21 AT AT85900447T patent/ATE89829T1/de not_active IP Right Cessation
  • 1984-12-21 DE DE8585900447T patent/DE3486154D1/de not_active Expired - Lifetime
  • 1984-12-21 EP EP85900447A patent/EP0167575B2/de not_active Expired - Lifetime
  • 1984-12-21 WO PCT/DE1984/000279 patent/WO1985002850A1/de not_active Ceased
• 1988
  • 1988-01-04 US US07/140,736 patent/US4782044A/en not_active Expired - Lifetime
  • 1988-08-08 US US07/229,706 patent/US4895932A/en not_active Expired - Lifetime
• 1994
  • 1994-01-13 JP JP6002150A patent/JPH0772146A/ja active Pending
• 1995
  • 1995-10-06 JP JP7260453A patent/JPH08224094A/ja active Pending

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