EP1042364A2

EP1042364A2 - Antivirally active oligopeptide isolated from royal jelly

Info

Publication number: EP1042364A2
Application number: EP98965731A
Authority: EP
Inventors: Eberhard Bengsch; Antonius Kettrup; Jürgen POLSTER
Original assignee: Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
Current assignee: Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
Priority date: 1997-12-27
Filing date: 1998-12-02
Publication date: 2000-10-11
Also published as: WO1999033866A2; BR9814506A; DE19757932C2; DE19757932A1; WO1999033866A3

Abstract

The invention relates to a family of oligopeptides with a completely new structure which occurs in selectively obtained royal jellies of certain vegetable origin and has been correlated with marked antiviral properties. The pilot structure is a molecule with 52 amino acids. Numerous analogous, nearly identical molecules are present in a satellite-like manner. The substances have a broad spectrum of antiviral activity, including against retroviruses, and have no toxic effect on humans even at high doses. A further surprising characteristic is their extreme resistance to all controlled proteases and other enzymes. The molecules are highly convoluted and resistant to attack. Despite their convolution they are geometrically very flexible and can form several conformers. The native state is easily soluble in water. Chemical synthesis results in a water insoluble form which can, however, be easily transformed into a soluble bioactive conformer which is similar to the native form. These new oligopeptides are therefore the first to combine in an optimal manner all the properties required of a potentially antiviral substance category.

Description

Oligopeptides

All living organisms, from the lowest to the most organized, are constantly threatened by ventral infections. There are currently 7,000 viruses known. 500 of these are pathogenic for humans and cause direct infectious diseases, some of which are incurable.

Viral infections, including those that seem to be easily overcome by the immune system, sometimes leave profound changes in the genome and metabolism of the host.

In particular, 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 Alzheimer's as well as genetic illnesses and trisomy (Mongolism) also appear to involve viral activity.

Unexpectedly, viruses have recently been identified as the cause of other, completely different diseases:

- 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.

With the current state of knowledge in virology it can be assumed that the majority of the serious diseases which cannot be cured with the current state of the art (antibiotics) have viral or para-viral activity as a trigger for short-term or longer periods. The morphological, genetic and / or metabolic dysfunctions are then to be regarded as the late consequence and end result of a virus intrusion. The mistake was repeatedly made to regard the spectacular end results of such Dys function series as their causer (see prion protein in BSE, which, in reverse, is associated with autonomous replicative pathogen properties in the context of an event series which has not yet been understood). This leads to unsuccessful therapy approaches and neglect of prevention. In fact, the virus is at the center of diseases of all kinds in humans, animals and plants.

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.

Numerous viruses can be transmitted via the air we breathe: flu and rhinos, adeno and papilloma tumor viruses; at high concentrations even Lyssa viruses (rabies). The rapid or slow spread of such viruses can lead to worldwide epidemics. In 1919, 20 million people died from the A H1N1 influenza virus. Approved vaccines have so far only existed against 25 human pathogenic viruses.

Alimentary and latrogenic accidents can cause viral infections of an unmistakable extent. In an enormous vaccine accident in England, the cause of the scrapie disease of the sheep was spread worldwide and then transmitted through negligent species passage to cattle (BSE) and then spread across the entire mammal area, including humans.

In the 1960s, a large part of humanity was simultaneously moculated with the global vaccination campaign against the poliovirus, 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.

The concentration of humans and animals, the misery and the absence of hygiene in the third world, the modern means of transport and the unlimited world trade with its sacred priority over all other considerations of caution and reason ensure that the viral pathogens become more and more ever faster transmission vectors are available.

But not only viruses are transported, transmitted, mixed and recombined faster.

The mixture of terrain through alternating passages of the pathogens across different races can also contribute to virulence enhancement. As is well known, the Eppstein-Barr virus (EBV) 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.

On the other hand, environmental degradation, the toxicity of the food and its depletion of secondary plant tabolites, which ultimately serve to protect against parasites in plants and animals, increasingly weaken the immune systems of the host organisms concerned, in particular humans.

More and more and more virulent viruses are confronted with increasingly weakened hosts.

The situation evolves towards a collective immune collapse of all higher organisms.

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.

Such substances have so far not been able to be supplied by the predominantly chemically-synthetically oriented research despite enormous search work.

The discrepancy between the enormous advances in knowledge of the detection, morphology and genetics of the viruses on the one hand and the clinical helplessness towards the virus-infected individual on the other has never been so great.

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

- only affects a part of the patients in whom an addressable virus serotype dominates

- sooner or later leads to the development of resistance due to the appearance of highly aggressive, non-treatable virus strains, which replace the strains eliminated by the therapy during the medicinal selection run in the host and ultimately control the terrain of the infected person.

- The transmission of such virus strains to third parties leads to particularly fulgurant disease courses in the newly infected

- is also accompanied by serious clinical side effects, the end effects of which are not yet foreseeable today

- In order to treat a sick person reasonably comfortably under the current conditions and with a maximum remaining lifespan until death, there is an enormous expenditure in terms of costs (medication for the virus itself and against opportunistic infections) and for highly qualified work performance (ongoing PCR dosing of the virus sequencing of the newly formed virus variants and their proteins) is required, a "luxury" that could hardly be up for debate for the majority of the approximately 100 million people infected with AIDS worldwide.

The problem with post-infection therapy, which is directed against the other 500 human pathogenic viruses known to date, is also unsolved.

The situation is even more obscure in epidemic neurodegenerative diseases such as BSE, the pathogens of which have not yet been found due to the lack of adequate detection methods. One step towards solving the problem: antiviral oligopeptides with a new structure and surprising properties

In the systematic search for antiviral natural substances, which have an effect at the plant / insect interface, particularly in the area of life and in the products of the social insect Apis mellifera, a previously unknown, simple substance class was found that is central to the antiviral defense strategy of this highly populated life system (50,000 individuals per 100 dm ³ ) is correlated.

In general, such substances are involved in epidemic protection throughout the bee state and are ubiquitous there, e.g. in the hemolymphs of the insects and their metamorphotic developmental stages (larva, pupa, nymph) and in the embryonic feeding juices for the 3 classes of workers, drones and queen.

Enriched and available in larger quantities, 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.

Indeed, 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. This occurs in the various secretion glands of 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:

- to eliminate viral pathogens without side effects,

- or at least to suppress their symptom expression to such an extent that the infected host can realize a normal life and reproduction cycle in the presence of the virus that has been rid of its virulence

- to ensure that the pathogens are not caused by resistance and

- recapture the terrain by reviving virulent tribes.

Royal jelly is the key substance to the functioning of such a system.

In order to find the antiviral active substances according to the invention, the first step was to look for optimal starting products.

Selection of raw materials

60 quasi-monofloral royal jellies were produced on the basis of 60 different bee-rich plant species.

Their effect was observed in the beehive itself, in which there are permanent map-parent viral infections with no or only weak symptom expression. The antiviral activity of the raw substances originating from monofloral sources was then tested in vitro, m-vivo and in the human field.

Among the monofloral jellies, two of them showed surprisingly selectively high antiviral activity: those based on Crataegus oxyacantha (hawthorn) and Genista tinctoπa (Farbergmster) as a pollen and thus as a protein source. A number of other plant species followed at a certain distance: Brassica nappus, Taraxacum officmale, in particular numerous Raphanus species, mustard waxes and other cruciferous plants, almost exclusively wild plants.

Test systems used

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:

- Coxsackie B 3 virus (Picorna virus) / Heia cells in the classic plaque essai: At a concentration of 200 ppm royal jelly in the usual nutrient solution, a total virus titer reduction was carried out (no infection site on the test plaque more)

- 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.

- Vesicular stomatitis virus (rhabdo virus) / mouse:

The survival rate could be significantly increased by peπtonal, 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. In specific human applications, 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-Vιrus (herpes virus) Hepatitis B virus (Hepadna virus) some retroviruses HTLV-2 virus (hairy cell leukemia)

These experiments cover 6 main of the currently known 24 human pathogenic virus families.

Finally, the beehive itself was also used as a virological test and Expenmentalsysterα:

- In most cases, 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. If 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.

- A renewed pollen supply and / or the elimination of the hamolymph-sucking parasite leads to an immediate termination of the epidemic and a rapid regeneration of the bee colony. Certain types of pollen are more efficient than others. The effect of the pollen in the beehive correlates with the m-vitro and m-vivo effects of the corresponding royal jelly obtained from it. Obtaining the raw materials

In order to obtain the particularly antivirally active royal jelly described, a total of 50 beehives were converted in the usual way to the rearing of queens known in the professional state of the art of beekeepers (separating grid for queen, 20 to 60 larvae-filled rows of rows per stick). The sticks were placed in the vegetation to be flown at the appropriate time, taking care to ensure that there was no blossoming, attractive bee culture. 10 to 120 hours, preferably 72 hours, after the king larvae had been grown, the jellies contained in the cells were suctioned off.

In order to optimally preserve the antiviral properties, it has proven to be advantageous for all royal jelly, in any case suction from the white cells and storage (5 ° C) under protective gas, e.g. B. Argon (European Patent No. DE 42 32 732, EP 0 663 831, PCT 93 025 62, USA 5 580 297) and to do without the usual freezing or lyophilization.

The authenticity of the plant origin was checked and confirmed by pollen morphological analysis of the royal jelly varieties obtained in this way. Since gorse bleeding only has a limited attraction on the flying bees, they always collect only a limited but regular portion of it. This is reflected in the naturally low content of gorse pollen raw material in the rearing secretions (royal jelly). In order to obtain a royal jelly variety based almost exclusively on gorse costumes, a two-phase process was used:

- First, with the help of normal beehives provided with pollen wiping grids, several kg of gorse pollen were obtained, which mostly took place in remote wild regions - This material was offered in honey solution suspension of pollen-coated royal jelly bee colonies, which processed it without residue.

In this way, a royal jelly variety was obtained with a protein fraction derived almost exclusively from gorse pollen, which is naturally not attainable.

Simultaneously, however, there was an accumulation of gorse alkaloids, which set certain limits on the therapeutic use of the raw material obtained in this way.

Unused primary rock soils

Of particular importance for obtaining the active antiviral royal jelly varieties are the nature and nature of the soils, some of which are already defined by natural site conditions for the selected plant species.

It was found that acidic, unused, pesticide-free granite, possibly uraniciferous primary rock, and constantly renewing soils are necessary. For gorse bushes and hawthorn hedges, such soils are by themselves the preferred locations, the plants hardly grow on calcareous soils, the resulting royal jellies are of moderate activity.

Preliminary investigations indicated that certain trace elements such as cadmium and the elements of the uranium and thorium series, which had previously only been biologically negative, played an important role in the occurrence of the described antiviral properties or in the plant biosynthesis of the substances causing them (a Such metals have a highly specific effect, for example, in the recently discovered metalloproteases). A good supply of boron, which is inherently guaranteed in bedrock, also plays an important role (patent no .: DE 44 41 483, EP 95 93 82 85, PCT 95 044 94).

Furthermore, 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.

All of these conditions can be found in the test landscape that was ultimately selected: a wide hedge landscape without large-scale agricultural use in western France, between Cholet, Nantes and La Röche sur L'Yon. The area includes the Breton mainland plate and the bedrock of the Vendee. There is regular rainfall throughout the year. Nevertheless, the annual sunshine duration is high and comparable to that of the Mediterranean climate. The climate is mild and balanced: there is no extreme heat or extreme frost. The Atlantic near with the prevailing west winds ensures regular ventilation. The atmosphere is exposed to that of the Atlantic and is not exposed to any local and cumulative environmental pollution. The wild plant wide is fully intact. All in all, it is probably a uniquely favored large biotope that has apparently been forgotten by the global environmental threat.

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.

From the active raw product to the active molecule

From the raw products optimized according to the invention (= selectively obtained royal jelly), the task now was to identify and define the substances which are mainly responsible for the antiviral effects. For this purpose, the usual analysis methods for the investigation of complex natural product mixtures were used:

classic column chromatography

HPLC one-dimensional electrophoresis

Capillary electrophoresis

From this it was only possible to hint at the affiliation of the antiviral activity to a protein fraction of low molecular weight.

A detection peak in MALDI spectroscopy

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. For this purpose, approx. 1 mg was dissolved in 500 μl of water and acidified with a little acetic acid solution for better solubility. The corresponding amount of matrix (DHAP) was added to 2 μl of this clear solution. 0.5 µl of this was crystallized out on the sample holder. The sample prepared in this way could then be easily measured in the mass spectrometer.

Since no fragmentation is observed with the MALDI-MS method, each peak in the spectrum corresponds to one molecule.

In contrast to the high molecular proteins of royal jelly, which are present in large numbers (some 100) and are grouped in species-related variations around a main storage protein of 55,000 D, there are only a dozen main oligopeptides in the low molecular weight range. Their relative amounts are approximately constant with the main peptides. Numerous analog, almost identical molecules exist like satellites, but their concentration fluctuates depending on the vegetable raw material and the age of the royal jelly emulsion. This somewhat constant analysis scheme of the lower proteins was confirmed by 2-D gel electrophoresis.

There is one exception to this fixed rule in the peptide scheme: At 5450 D there is a well-characterized MALDI peak, the intensity of which varies extremely from one royal jelly variety to another. This selection peak is most pronounced in spectra that arise from native royal jellies with a high proportion of gorse, hawthorn and certain wild plant cruciferous pollen proteins (e.g. Raphanus species, mustard waxes). These are the same raw substances that develop the most intense anti-viral activity in the test. The substance to be assigned to the peak is therefore directly or indirectly correlated with the antiviral effect sought.

An unusual oligopeptide of unusual purity The selected raw jelly products could be presented in large quantities, some of which could be used for direct therapy trials.

On the other hand, isolating the target molecules as in the previous analytical search procedure was very difficult. This is due to the highly complex composition of the raw product: some 1000 proven or detectable compounds; in total probably a few million z. T. ephemeral, mutually convertible trace components.

Indeed, 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.

Classical methods such as side exclusion chromatography (Sephades G 50), ion exchange chromatography and one-dimensional electrophoresis led to mixtures of substances with changing compositions.

What was achieved in the analysis with MALDI is ruled out as a separation method on a preparative scale.

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.

In this way, the peptide was isolated in the highest purity, but this had to be bought with the disadvantages inherent in the method:

minimal amounts of substance reduced biological activity The procedure was as follows:

First, royal jelly is mixed with water (1: 2 ratio of royal jelly to water). The insoluble components are then centrifuged off (for 15 minutes at 15,000 revolutions / minute). The supernatant is mixed with Pharmalyte pH 3-10 (for IEF, Pharmacia Biotech) and DTT (dithioerythritol). The amounts of these additives are each 1%.

For isoelectric focusing (IEF), 30 μl of this solution are applied to IPG strips (immobilized pH gradient) on the anodic side. 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

Mixture prepared: 0.5% CHAPS (= 3- [(3-cholamidopropyl) dimethylammonio] propane sulfate), 0.2% Pharmalyte (pH 3-10), 20 mM DTT and 8M urea.

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.

After isoelectric focusing (IEF), several IPG strips are equilibrated with two buffer solutions for 15 minutes each. The buffer solutions (50mM TRIS / HCl; pH = 8.8) contain 6M urea, 30% glycerol and 2% SDS. DTT (final concentration 90mM) and the second iodoacetamide (260mM) are added separately to the first buffer solution. A buffer solution with 1.4% glycine, 0.3% TRIS and 0.1% SDS is used for the anode and cathode 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:

Lys (l) - Thr - Ser - -Ile - -Ser (5)

- Val - Lys - Gly - Glu - Ser (10) -

- Asn - Val - Asp - Val - Val (15) -

- Ser - Gin - Ile - Asn -Ser (20) -

- Leu - Val - Ser - Ser - Ile (25;

- Val - Ser - Gly - Ala - Asn (30!

Val - Ser - Ala - Val - Leu (35) - Leu - Ala - Gin - Thr - Leu (40)

.- Val - Asn - Ile - Leu - Tyr (45)

- Thr - Asp - Ser - Asp - Pro (50)

Met - Arg (52)

The composition shows a protein chain without Cys, His, Phe and Trp.

Since with increasing sequencing the signal levels of the amino acid split off and derivatized from the protein chain decrease continuously due to the sequencing technique, a safe sequencing generally cannot be carried out beyond 40 amino acids. Nevertheless, the oligopeptide according to the invention, which is composed of 52 amino acids, was completely sequenced. However, the last approximately 3 amino acids could not be identified with absolute certainty, so that any hormologists may also be biologically active.

The search for homologies to the specified sequence in the databases did not match any known sequences: the substance therefore has a completely new structure.

The structural analysis shows that 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. There is only one aromatic amino acid (type 45). 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. Despite the absence of stabilizing disulfide bridges, 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: Despite its good solubility in the physiological environment, 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.

In addition to its biological stability, the good solubility of the oligopeptide according to the invention in its native form is a good precondition for its bioavailability.

Even in high doses, 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.

In order to be able to dispose of larger amounts of the peptide, its total synthesis was carried out according to a modified Merrifield method. The synthetic product was practically insoluble in water and showed no biological activity in this form. In order to approximate the native form, as it is excreted from the secretion glands of Apis mellifera, the synthesis product was taken up in trifluoroethanol and then carefully transferred into an aqueous medium (5 mg peptide in 5 ml water), with no further precipitation taking place. Freeze-drying gave a lyophilisate which dissolved excellently in the presence of sodium bicarbonate (pH = 8) and approached the native product in its biological and physicochemical properties. The 2D gel electrophoresis of the low molecular weight proteins in the royal jellies examined showed that the described main peptide is flanked by quasi-identical satellites in small amounts in most cases. No differences can be found in the molecular masses, but the isoelectric points differ by up to a few tenths of pH units. It appears that the antiviral activity of these accompanying substances is assigned a function in such a way that a kind of “oligo therapy” makes it more difficult for the viruses to avoid the formation of resistant strains.

It is proposed to use this action scheme, which is drawn by nature, in accordance with the invention by specifically modifying the pilot structure of the main peptide and optimizing it for the biological action against one or the other virus.

As a possible point of attack of the oligopeptide according to the invention and possibly its satellites on the viral cycle, a reduction in the effectiveness of the virus proteases is the most obvious at the current state of knowledge.

Claims

claims

1. oligopeptide with antiviral activity, characterized in that it consists of at least 52 amino acid residues with the sequence specified in the description.

2. Oligopeptides according to claim 1, characterized in that amino acid residues can be replaced by others in particular from position 48 of the sequence.

3. Oligopeptides according to claims 1 and 2, characterized in that their antiviral effect is optimized by targeted exchange of any amino acid residue.

4. A process for the preparation of oligopeptides according to claims 1 and 2, characterized in that royal jellies are selectively obtained in the manner described under the specified conditions, which contain these substances to a particular extent.

5. A method for activating a synthetic peptide according to claims 1 to 4, characterized in that it by gradually changing the solvent, followed by lyophilization and subsequent inclusion in sodium hydrogen sulfate solution from an insoluble inactive in a soluble active, the biological form related conformation is converted.