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WO1999033866A2 - Oligopeptides - Google Patents

Oligopeptides Download PDF

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Publication number
WO1999033866A2
WO1999033866A2 PCT/EP1998/007811 EP9807811W WO9933866A2 WO 1999033866 A2 WO1999033866 A2 WO 1999033866A2 EP 9807811 W EP9807811 W EP 9807811W WO 9933866 A2 WO9933866 A2 WO 9933866A2
Authority
WO
WIPO (PCT)
Prior art keywords
virus
antiviral
royal jelly
oligopeptides
substances
Prior art date
Application number
PCT/EP1998/007811
Other languages
German (de)
English (en)
Other versions
WO1999033866A3 (fr
Inventor
Eberhard Bengsch
Antonius Kettrup
Jürgen POLSTER
Original Assignee
Gsf - Forschungszentrum Für Umwelt Und Gesundheit, Gmbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Gsf - Forschungszentrum Für Umwelt Und Gesundheit, Gmbh filed Critical Gsf - Forschungszentrum Für Umwelt Und Gesundheit, Gmbh
Priority to BR9814506-1A priority Critical patent/BR9814506A/pt
Priority to EP98965731A priority patent/EP1042364A2/fr
Publication of WO1999033866A2 publication Critical patent/WO1999033866A2/fr
Publication of WO1999033866A3 publication Critical patent/WO1999033866A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43572—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from bees
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • viruses can induce tumor formation and cause cell destruction by auto-immune reactions.
  • Viruses and virus-assimilated particles are also involved in the development of slowly evolving diseases, such as multiple sclerosis, Parkinson's, Creutzfeld Jacob and cardiovascular syndromes (cardiomyopathy), which break out after long latency periods.
  • diseases such as multiple sclerosis, Parkinson's, Creutzfeld Jacob and cardiovascular syndromes (cardiomyopathy), which break out after long latency periods.
  • Adenovirus 36 causes a particularly severe, non-therapeutic form of obesity
  • the equine Bornavirus can trigger behavioral disorders, depression and manic-depressive insanity in humans
  • a type C retrovirus is responsible for certain forms of uvemlen diabetes. Contrary to previous beliefs, the viral origin of such diseases also includes their transferable character. The transmission can take place horizontally and / or vertically. Clinical names in connection with such and other serious illnesses, terms such as “familial accumulation” and “genetic pre-disposition” should be re-examined from the point of view of an increased viral load that has spread in the life of the person concerned.
  • Viruses are transmitted horizontally by infection from individual to individual or vertically by genetic transfer of endogenous viruses from the genomes of the ancestors.
  • Endogenous viruses can be activated, for example by other viruses, by transposons or by all kinds of stress factors.
  • the highly active and non-eliminable tumor virus SV 40 which was subsequently introduced into the Simian cell cultures, and which subsequently became effective in multiple variations in the affected individual and in the subsequent generations becomes.
  • the SV 40 is involved in the explosive spread of tumor diseases.
  • Eppstein-Barr virus causes Pfeiffer's glandular fever (infectious mononucleosis) in adolescents of the white race, Burkitt's sarcoma in African children and Rhmo-Phar in Asian adults. ynx carcinoma. In mixed-race societies, Burkitt sarcoma also appears in whites after some time: the permissivity for the viral symptom expression has increased.
  • Finding substances with a selective antiviral effect without cytotoxicity and with high resistance to premature body degradation is a question of survival for humans and animals on the increasingly overpopulated globe.
  • the long-standing lack of success in efficient virus control without damaging the unaffected host cells is best characterized by the current situation in clinical AIDS treatment.
  • the currently available tritherapy for HIV 1 and 2 consisting of two different nucleoside analogues such as AZT and 3 TC as inhibitors of reverse virus transknptase, combined with an inhibitor of the viral protease (indinavir, ritonavir or saqui- navir), allows a temporary reduction of the virus load in more or less advanced disease stages
  • the discovered compounds can occur in the lifelong feed juice of the queen bee (royal jelly), from where the discovery and the first isolation according to the invention also took place.
  • a condition for the occurrence of these compounds in amounts as were already required for the characterization and function assignment from the raw substance (royal jelly) is that the jellies are preferably produced selectively by the bees on the basis of certain plant species.
  • the royal jelly products are mainly derived from plant pollen, which is selectively introduced by the working bees and processed by the allaitation bees into an emulsion of a highly complex composition.
  • Apis mellifera which is the most massive glandular system of all known organisms in terms of dimensions.
  • Apis mellifera is a flying enzyme factory, where the plant substances collected are immediately processed further. The highest mechanical, biochemical and reproductive performance as well as efficiency in the defense against parasites are combined in one organism.
  • Apis mellifera is probably the best, evolutionarily oldest and longest proven system for solving the task according to the invention:
  • Royal jelly is the key substance to the functioning of such a system.
  • the first step was to look for optimal starting products.
  • Virus / host systems were used both m-vitro and m-vivo as test systems with the help of which the selectively high antiviral activity of the royal jelly varieties obtained from hawthorn and gorse pollen were found:
  • Influenza virus A2 (orthomyxovirus, pandemic flu,) / mouse: With peritoneal application of 200 mg jelly / kg body weight, a survival rate of 100% was achieved for the infection which was fatal in mice in a blind test.
  • the survival rate could be significantly increased by pe ⁇ tonal, pre- and / or post-functional application of the described royal jellies in concentrations of 1-500 mg / kg body weight, with an intermediate amount of 50 mg / kg having a clear maximum effect in gorse jellies.
  • sy toms suppression up to hm to spontaneous remission were observed in the following virus infections:
  • Influenza B orthomyxo virus, non-pandemic flu
  • Herpes-2-V ⁇ rus herpes virus
  • Hepatitis B virus Hepadna virus
  • some retroviruses HTLV-2 virus hairy cell leukemia
  • the individual bee is permanently mapparent cerebrally infected with numerous viruses, in particular with the ubiquitously widespread paralysis virus APV (Picorna virus), without there being any symptom expressions under normal conditions.
  • APV paralysis virus
  • the beehive is pollen-maternity leave, d. that is, the starting product for obtaining the antiviral protein molecules is no longer available, or if these are removed from the hamolymph of the normally protected bee by parasites (the mite Varroa jacobsoni), the activated virus breaks through the brain-glandular barrier of the insect and infects the larvae via the protein-depleted juices. The entire beehive is affected by the epidemic.
  • the climatic conditions are of importance: A high level of lumens and the absence of stress conditions such as excessive drought or pesticide demand the generation of all kinds of secondary metabolites, to which the antiviral oligopeptides according to the invention and their precursors also have to be paid.
  • the species of plants to be flown by the bees are large-scale wild crops of hedgerows and bush landscapes, and their roots reach through the bedrock through the bedrock.
  • the granite rock base is rich in gneiss (amphibiols, biotite, plagioclase), ferromagnesium, divalent cations of all kinds and sprinkling of pitchblende and thoanite mineral.
  • the area contains several open pit mines for the extraction of uranium and thorium ores. Natural radioactivity is high, but is largely discriminated by the plants and is not found in royal bee products such as royal jelly, pollen, honey, wax and propolis.
  • the optimum environmental conditions for the plant and insect for achieving the object according to the invention lead to a characteristic protein pattern which is significantly different from the average conditions in the royal jelly obtained.
  • the definitive assignment was finally carried out by a variant of MALDI mass spectroscopy specializing in low-molecular proteins, with the aid of which ionized molecules in the range from m / z 1000 to 10,000 Daltons are quantified with high selectivity and clear reproducibility.
  • ionized molecules in the range from m / z 1000 to 10,000 Daltons are quantified with high selectivity and clear reproducibility.
  • approx. 1 mg was dissolved in 500 ⁇ l of water and acidified with a little acetic acid solution for better solubility.
  • the corresponding amount of matrix (DHAP) was added to 2 ⁇ l of this clear solution. 0.5 ⁇ l of this was crystallized out on the sample holder. The sample prepared in this way could then be easily measured in the mass spectrometer.
  • each peak in the spectrum corresponds to one molecule.
  • royal jelly is like the composition of a living cell. This corresponds to its middle position as a post-cellular product of the plants and a pre-cellular mixture for the formation of the insect embryos.
  • the queen bee produces her 2 million offspring exclusively from royal jelly, with no condensed metabolic excretions.
  • the recently developed 2D electrophoresis proved to be the method of choice.
  • the first dimension is isoelectric focusing with immobilized pH gradients, followed by gel electrophoresis carried out perpendicular to it.
  • IPG strips immobilized pH gradient
  • the IPG strips (4mm wide, 18cm long) are made of polyacrylamide, in which a pH gradient of 4-9 is built up using real estate (from the Pharmacia Biotech company). Make sure that the IPG strips have swollen for about 16 hours beforehand. The following is used as the source solution
  • Deionized water is used to prepare the anode and cathode solution. Interfering C0 2 in the gas space of the electrophoresis chamber is bound by paper strips soaked with 3 M NaOH solution.
  • a horizontal SDS-PAGE sodium n-dodecyl sulphate-polyacrylamide gel electrophoresis based on a T-15% polyacrylamide separating gel and a T-6% collecting gel is used for the second electrophoresis dimension.
  • TRIS / HC1 serves as a buffer.
  • the pH of the bulk gel is adjusted to 6, 8 and that of the separating gel to 8.8.
  • Electrophoresis is stopped before the bromophenol blue front reaches the wick. Protein staining is done with Coomassie. In this way, spots with highly enriched and highly pure protein can be detected in the IPG strips. With the help of calibration proteins of different molecular weights, a low-molecular protein of molecular weight 5540 Daltons could be determined, localized and isolated by "blotting" or extraction using the running distance of a pronounced spot.
  • the protein isolated in this way was of unusual purity, no by-products could be detected.
  • the determined primary structure is just as unusual as the purity. It was determined by total sequencing: 52 amino acids are combined in a chain in the following way:
  • composition shows a protein chain without Cys, His, Phe and Trp.
  • hydrophilic and hydrophobic amino acid residues are present in approximately the same number, but are distributed unevenly.
  • a strongly hydrophobic central area (9 Val. 5 Leu, 4 Ile, 3 Ala) is flanked by two hydrophilic areas.
  • the high number of Ser residues (10) is striking, which explains the readily water-soluble character of the native oligopeptide.
  • the specified sequence leads to a molecular weight of 5414 D, in accordance with the values estimated by the MALDI measurements and the values found by the 2D electrophoresis separation using calibration proteins.
  • the peptide shows extreme resistance to all proteases and other enzymes tested: trypsin, chymotrypsin, pancreatic elastase, thermolysin, pepsin, collagenase, enzyme Glu C. and even pronase and proteinase K.
  • the molar ratio of the enzyme was able to / Substrate (normally 1/1000) can be increased by a thousand times (1/1) without the slightest proteolysis occurring:
  • the peptide according to the invention is exceptionally biologically stable, which, when used as a medicament, has good transport properties Serum and intestinal tract guaranteed.
  • the good solubility of the oligopeptide according to the invention in its native form is a good precondition for its bioavailability.
  • the oligopeptide according to the invention proved to be non-toxic to humans: in the form of royal jelly enriched thereon, test animals or test subjects were injected or administered mg amounts thereof or orally, without the slightest side effect being observed.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Insects & Arthropods (AREA)
  • Botany (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Toxicology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne une famille d'oligopeptides possédant une structure entièrement nouvelle qui apparaît dans des gelées royales d'origine végétale définie et obtenues par sélection, et qui a été corrélée avec des effets antiviraux marqués. La structure pilote est une molécule avec 52 aminoacides. De nombreuses molécules analogues pratiquement identiques sont présentes sous forme satellitaire. Les substances possèdent un large spectre d'activité antivirale, y compris contre les rétrovirus, et ne présentent aucun effet toxique chez l'homme, même à de fortes doses. Une autre caractéristique surprenante est leur extrême résistance à toutes les protéases régulées et les autres enzymes. Ces molécules sont extrêmement convolutées et résistantes à l'attaque. En dépit de leur convolution, elles sont géométriquement très souples et peuvent former plusieurs conformères. Il existe à l'état natif une forme aisément soluble dans l'eau. La synthèse chimique donne une forme insoluble dans l'eau pouvant toutefois être transformée aisément en un conformère bioactif soluble similaire à la forme native. Ces nouveaux oligopeptides sont donc les premiers à réunir de manière optimale toutes les propriétés exigées d'une classe de principes actifs potentiellement antiviraux.
PCT/EP1998/007811 1997-12-27 1998-12-02 Oligopeptides WO1999033866A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
BR9814506-1A BR9814506A (pt) 1997-12-27 1998-12-02 Oligopetìdios
EP98965731A EP1042364A2 (fr) 1997-12-27 1998-12-02 Oligopeptides

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE1997157932 DE19757932C2 (de) 1997-12-27 1997-12-27 Oligopeptide
DE19757932.9 1997-12-27

Publications (2)

Publication Number Publication Date
WO1999033866A2 true WO1999033866A2 (fr) 1999-07-08
WO1999033866A3 WO1999033866A3 (fr) 1999-09-10

Family

ID=7853425

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1998/007811 WO1999033866A2 (fr) 1997-12-27 1998-12-02 Oligopeptides

Country Status (4)

Country Link
EP (1) EP1042364A2 (fr)
BR (1) BR9814506A (fr)
DE (1) DE19757932C2 (fr)
WO (1) WO1999033866A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008208088A (ja) * 2007-02-27 2008-09-11 Ehime Univ 口内炎処置用組成物
WO2018188714A3 (fr) * 2018-09-06 2020-07-23 El Fiky Salem Ismaeil Salem Activités anti-leucémiques, anti-vih et sialidase de protéines de gelée royale

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10344561B4 (de) * 2003-09-25 2011-12-22 Eberhard Bengsch Verwendung eines Tetra-, Penta- und Octapeptids aus Gelée Royale

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1048323A (zh) * 1989-06-28 1991-01-09 北京朝阳三益日用化学厂 防治艾滋病、性病洗液的制备方法
GB8917295D0 (en) * 1989-07-28 1989-09-13 Ruffles Graham K Anti-viral compositions
DE4232732C1 (de) * 1992-09-30 1994-02-03 Gsf Forschungszentrum Umwelt Standardisiertes Gelee Royale, Verfahren zu seiner Gewinnung und seine Verwendung

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008208088A (ja) * 2007-02-27 2008-09-11 Ehime Univ 口内炎処置用組成物
WO2018188714A3 (fr) * 2018-09-06 2020-07-23 El Fiky Salem Ismaeil Salem Activités anti-leucémiques, anti-vih et sialidase de protéines de gelée royale

Also Published As

Publication number Publication date
WO1999033866A3 (fr) 1999-09-10
BR9814506A (pt) 2000-10-10
DE19757932A1 (de) 1999-07-08
EP1042364A2 (fr) 2000-10-11
DE19757932C2 (de) 2000-01-05

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