DE19813802A1 - Anti-viral effects of propolis through inhibition of viral nucleic acid polymerases - Google Patents

Abstract

The invention relates to the anti-viral activity of propolis in relation to the inhibition of replication of polymerase-containing viruses in general. DOLLAR A In particular, the inhibition of the HIV virus and its key enzyme reverse transcriptase by propolis is described at the molecular level. The complete inhibition of the enzymatic polymerase activity of HIV reverse transcriptase is shown in the presence of crude propolis extract and compared with the action of the reference inhibitor azidothymidine triphosphate (AZT-TP). The inhibitory effect on reverse transcriptase can be observed up to a dilution of the crude extract of 1: 35000. DOLLAR A Furthermore, the inhibition of HIV replication by means of propolis is described at the cellular level or in cell culture. The complete inhibition of HIV replication in peripheral blood lymphocytes by means of propolis crude extract at non-toxic concentrations (dilution 1: 10000) is shown and compared with the effect of the reference inhibitor azidothymidine (AZT). DOLLAR A Due to its molecular and cellular anti-viral effect on polymerase-containing viruses, propolis can be used as a crude extract and / or fractions, pure substances or synthetic analogues for the therapy of virus-related diseases (AIDS, cancer, leukemia, hepatitis etc.) and for the inactivation of bio - / Gen-technological products are used in the pharmaceutical cosmetics and food industry.

Description

Nucleic acid polymerases are specific enzymes made up of monomers of nucleotide Synthesize triphosphates oligomeric or polymeric nucleic acids and during replication of genetic material (RNA, DNA) play an important role in all biological systems play. There is evidence that the enzymatic mechanism of nucleic acid polymerization (DNA, RNA) in nature has a more universal character (Steitz 1998) where the polymerases differ in their three-dimensional protein structure outside the active center according to the respective species (mammals, Bacteria differentiate more or less.

Viral polymerases are particularly important as target molecules for development of active ingredients that inhibit their enzymatic activity and as therapeutic agents used for viral diseases (e.g. AIDS, cancer, hepatitis, herpes, flu, etc.) can be.

AIDS is currently one of the biggest medical challenges facing scientists, Doctors, society and politics. The disease is infectious and, according to the current status science caused by a retrovirus, the HIV virus (Levy 1993, Pantaleo & Fauci 1996, De Vita et al. 1997). Retroviruses have the unpleasant quality of getting into it Injecting the genome of host cells irreversibly, with the "cell machinery" of the host multiply and thereby induce diseases.

The HIV virus primarily infects the cells of the immune system (T lymphocytes, macro phages), which gets out of control and becomes weaker and more vulnerable for other diseases that ultimately lead to human death (Pantaleo & Fauci 1996).

It is currently not possible to cure AIDS. The science and pharma Industry has so far been able to make great efforts over many years (approx. 15 years) Finding active substances and combination therapies that increase the multiplication of the HIV virus inhibit the virus load in the body temporarily below the detection limit (in the blood) lower and extend the life of an AIDS patient or improve their quality of life. The z. Active ingredients currently used are inhibitors of HIV reverse transcriptase and HIV protease, either as a substrate analogue (competitive) or as an allosteric inhibitor (non-competitive, incompetitive) act on the retroviral enzymes and their action in Inhibit virus replication cycle (De Clercq 1995). For the inhibition of the reverse Transcriptase there are the so-called nucleoside RT inhibitors (NRTI) and the non- Nucleoside RT inhibitors (NNRTI). As substrate analogs, the above Enzyme inhibitors cause no total and irreversible inhibition due to their competition with the natural enzyme substrates. This causes a complete inhibition of the virus in its Infection cycle also not possible.  

The following shows an overview of the inhibitors currently used as therapeutic agents Table.

According to the current state of science, it has been shown that the high mutation rate, due to the incorrect reactions of the reverse transcriptase and the inefficient HIV-Inhi bition, leads to the development and selection of drug-resistant virus strains, which are ineffective against the reverse transcriptase or protease inhibitors used (Richman 1993, Erickson & Burt 1996). As a result, the resistant HIV strains Me in the organism can replicate again, the viral load in the body increases and the AIDS patient's health deteriorates again.

Furthermore, the therapeutic agents developed so far show physiological side effects and burden the normal daily routine of the Patients (e.g. 18-22 tablets per day with a so-called triple therapy, a triple Combination of inhibitors).  

For these reasons, there is a need for another and better anti-viral Active ingredients and lead structures as well as a wider range of therapeutic agents different mechanisms of action to reduce the viral load again, resistance delaying or even avoiding education and physiological side effects and Minimize toxicity.

The requirements of new active substances, lead structures and therapeutics against retroviral enzymes (reverse transcriptase, protease, RNAse, integrase) must meet the following characteristics and criteria based on current knowledge:

• a) total and irreversible inhibition of the HIV virus in minimal concentration ions (nM range)
• b) good bioavailability and pharmacokinetics
• c) Delaying or even avoiding resistance development of the HIV virus
• d) the active ingredients to be developed should be able to be administered orally,
• e) cause no toxic side effects and
• f) be of low molecular weight in order to be able to cross the blood-brain barrier.

One of the main strategies of pharmaceutical companies has been to have large collections available of biological or chemical substances to investigate their antiviral effect - a very Risky, time-consuming and costly project - because a pre-screening of this Fabrics without any clues and with an uncertain outcome was necessary.

To reduce the risks and costs, e.g. Currently pursuing other more rational strategies:

• a) molecular biological approaches to AIDS therapy with genome-based product development (e.g. antisense molecules, gene therapy approaches), their use and therapy effect is still in the future or is uncertain
• b) rational drug design at computer level based on known crystal structures of retroviral enzymes (e.g. protease inhibitors) and other active substances. Latter Development has shortcomings in that complex drug structures are based only known components can be modified.
• c) vaccine development based on recombinant retroviral proteins, cytokines, Nucleic acids etc., whose therapeutic successes are still a long way off. Chemokines and their cellular receptors, which play an essential role in the entry of the HIV virus into the Playing cells (Premack & Schall 1996) offer a completely new approach to AIDS therapy or the development of vaccines. However, since this therapeutic alternative in the In the initial phase, their chances of success cannot yet be realistically assessed.  
• d) Antiviral natural products: A crucial role in the development of new therapeutic agents plays the multitude of biological agents and structures from nature that through the development of natural defense mechanisms (principles of action) in animals and plants against pathogens (viruses, bacteria, parasites etc.) over millions of years have been selected and optimized and for the most part are still unexplored. Only about 5% of Approximately 2 million organisms that exist worldwide are currently biologically active Active substances examined. There is still enormous therapeutic potential and an important one The key to the drugs of the future.

The isolation and purification of natural active substances and lead substances herbal drugs (medicinal plants) is a great challenge. But there are many Medicines used every day still have a natural structural basis that due to slight chemical modification a better effect (tolerability, Effectiveness, applicability and also biodegradability and much more) than the pure one herbal active ingredient should be here in the almost inexhaustible variety of structure millions of years of evolution the basis of new drugs are sought. The previous Experience has shown that only because of the basic knowledge of the effect of natural substances Many synthetic drug analogs that could today be optimized as Medicines are on the market.

The effort to find natural products with anti-retroviral activity and in the AIDS Thera Pie is to be used using the example of hypericin and its derivatives from St. John's wort documented (Merueln et. al. 1992) as well as through reports on the international AIDS con in 1996 in Vancouver from Arkopharma using their own preparation SV30 Boxwood.

Propolis is a mixture of natural herbal substances that the bees make from buds Deciduous trees, conifers or shrubs collected and used to seal the honeycomb and their defense against infestation with bacteria, fungi, viruses and other pathogens is used.

The anti-bacterial effect of propolis has long been known in naturopathy as well the observation that so far no bacterial resistance to propolis have occurred (Hill 1995, Apimondia 1990). The antiviral effect in relation to herpes However, EBV or Varicella Zoster viruses have been described without target molecules or To investigate the mechanism of action (WO 91/13626, Apimondia 1990).

The anti-viral effect on enzymatic polymerase activity in retroviruses and other viruses containing polymerase have so far not been investigated or described. Furthermore, previous experience leaves no bacterial resistance to propolis have been observed, hope that viral resistance to propolis is also less true are likely or may not occur at all.  

Troubleshooting

It is an object of the present invention to provide a new medicament on a natural basis To provide treatment for viral diseases.

This object is achieved according to the invention in that propolis in a pharmaceutical effective amount to inhibit replication of polymerase-containing viruses provided.

example

Starting from the above Propolis crude extract became state of the art biochemically and cell biologically tested for the inhibition of HIV-1.

• a) Propolis extraction: dry substance from propolis was with equal volumes DMSO, ethanol or PBS added and shaken for 3 hours at room temperature. The dark brown solution was then filtered and the stock solution for the Investigations used.
• b) The molecular effect of the propolis extract on HIV reverse transcriptase was tested biochemically using the RETRO-KIT / RTA reverse transcriptase technology (see Appendix, Faff et al. in preparation and PCT / DE97 / 00391). Purified enzyme of HIV reverse transcriptase was preincubated with the propolis extract in various concentrations or dilutions and then tested in an enzymatic reaction. AZT-TP (azidothymidine triphosphate) ( FIGS. 2a-2b) and corresponding solvent controls, Blanc etc. were used as reference inhibitors.
  The results show a complete inhibition of the RT activity with up to approx. 700 µg / ml propolis extract (dilution 1: 3500) as well as a further significant inhibition of the RT activity up to a concentration of 7 µg / ml propolis extract or a dilution of 1: 35,000 ( Fig. 1a).
  Depending on the degree of inhibition, expressed in% inhibition, an IC ₅₀ concentration of approximately 150 μg / ml propolis extract can be determined ( FIG. 1b).
• c) The cell biological effect of the propolis extract on HIV was determined using the Infection of cells etc. PBL peripheral blood lymphocytes with HIV-1 (isolate IIIb, m.o.i. 0.001) in the presence of the crude RT extract in various concentrations / ver thinnings (1/100; 1/1000; 1/10 000) tested and their toxic effects (MTT test)  to the above Cells examined (triplicate). Replication of HIV in the cells was measured using p24 antigen in the cell culture supernatant after 4 days of incubation.

The results show that the propolis extract in high concentrations (1/100 or 1/1000) has a toxic effect and only about 4% of the cells survive (Tab. 1). In the 10 × lower Concentration (dilution 1/10 000) survive approx. 65% of the cells.

If the supernatant of these cells is examined for p24 antigen, an almost total one can be obtained Decrease of the p24 amount to approx. 0.2 pg / ml compared to the control (39.3 ng / ml) measure up. This corresponds to a degree of inhibition of approx. 99.5% compared to the effect of the reference inhibitor azidothymidine (Tab. 2).

Table 1

Toxicity study of HIV-infected lymphocytes (PBL's) in the presence of the propolis extract

(MTT test according to T. Mossmann, J. Immunol. Methds. 65, 55-63, 1983)

Table 2

Inhibition of HIV infection & replication of lymphocytes (PBL's) in the presence of the propolis extract

(p24 test according to Müller et al .: Fresenius Z. Anal. Chem. 330, 352-353, 1988)

The experimental data show that the propolis extract has a complete inhibition of HIV-1 on both the molecular and the cellular level ( Fig. 1-2, Table 2). The anti-HIV effect can be measured in both test systems down to low concentrations (high dilutions 1/35 000, 1/10 000) where solvent or toxic effects can be excluded ( Fig. 1-2, Tab. 1 ).

The high anti-retroviral effect was measured in a crude extract that a mixture is made up of many (thousands) components and is neither optimized nor extracted is partially cleaned.

This very strong HIV inhibition with a very poor degree of purity of the propolis Extract leaves on a high quantitative proportion of the active components and / or very good dose / effect ratio.

Inhibition of HIV reverse transcriptase, and knowledge of its effects Mechanism through kinetic studies open up the possibility of prognostic sub conduct research on resistance development and also the effect of the Propolis extract to investigate other species of retro or other viruses that are Have enzyme reverse transcriptase or other nucleic acid polymerases. Consequently the extract active ingredients could possibly also be used in other retrovirus-related Diseases e.g. B. leukemia, autoimmune diseases, cancer, hepatitis etc. used become.

The inhibition of HIV replication in cell cultures of lymphocytes shows that the Active ingredient components of the propolis extract the HIV virus on one or more Inhibit stations of the replication cycle. That this is in reverse transcription of the retroviral RNA occurs, is due to the molecular investigations on the reverse Transcriptase enzyme close, but does not exclude, the inhibition of others viral target molecules (RNAseH, protease, integrase etc.).

Furthermore, the cellular HIV inhibition by propolis shows that its components are the Plasma membrane can pass through the cells, indicating good cellular bioavailability indicates availability and a chemical derivatization of the active ingredients, at least for the Makes the beginning superfluous.

Thus, the strong cellular HIV inhibition (bioavailability) as well as the Effect at low concentrations (dose / effect ratio) on good ones Requirements for the successful development of a potent anti-viral Therapeutic.  

bibliography

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Claims (10)

1. Use of propolis in a pharmaceutically effective amount to inhibit the Replication of viruses exhibiting polymerase activity. 2. Use according to claim 1, characterized in that propolis derivatives are used become. 3. Use according to claim 2, characterized in that the derivatives propolis Raw extracts, fractions of propolis, propolis pure substances or synthetic Active ingredient analogues of propolis, each with the effect of inhibiting polymerase Viruses exhibiting activity. 4. Use according to one or more of the preceding claims for inhibiting the enzymatic activity of viral nucleic acid polymerases. 5. Use according to claim 4, characterized in that the enzymatic activity of Reverse transcriptases, DNA polymerases and / or RNA polymerases is inhibited. 6. Use according to claim 5, characterized in that the reverse transcriptase is inhibited. 7. Use according to one or more of the preceding claims, characterized characterized in that the replication of the viruses is inhibited in vitro or in vivo. 8. Use according to claim 7, characterized in that the inhibition of replication the viruses occur in cell cultures. 9. Use according to one or more of the preceding claims for the therapy of Diseases caused by viruses that have polymerase activity. 10. Use according to one or more of the preceding claims, characterized characterized in that propolis in a pharmaceutically effective amount in drugs, Cosmetics or foods exist to replicate polymerase activity inhibiting viruses.