DEVELOPMENT OF BIOTECHNOLOGICALLY PRODUCED CORN EXTRACT (AVEMAR) HINDERING THE FORMATION OF TUMOROUS METASTASES

Contract number: OMFB-02663/2000 (BIO-072/2000)

Date of start: 11/15/2000.

Duration: 36 months

Total cost: 26,6 M HUF

OM contribution:16 M HUF

Acronym: Development of antimetastatic corn extract

BACKGROUND OF THE PROPOSAL

The literature published the first data about wheat germ fermented with yeast in 1940. It was found that the reduced glutation level of wheat germ gets eliminated during fermentation, but no detailed mechanism of the process was discovered then. In 1950, Vuataz managed to point out that the substances formed during fermentation were responsible for the reduction of the glutation level. In 1952, it was stated by Cosgrove et al that the forming molecules were metoxi-p-benzoquinone and 2.6-dimetoxi-p-benzoquinone.

The biochemical significance of the discoveries was pointed out by Albert Szent-Györgyi. Szent-Györgyi realized that wheat germ was a plant of „peroxidase type” and started to make experiments in the hope of finding the missing phenol components of the respiratory chain in that plant. Plants of the peroxidase type/model oxidize paraphenylene-diamine into a blue-coloured imine. These plant tissues display the colour reaction even after several thorough extractions, in other words, the process is bound to insoluble cell structures. In Szent-Györgyi’s views these compounds may play a primary role in the maintenance of immunity as well.

As a result of the development work based on the tumour-related research of Albert Szent-Györgyi, an antimetastatic plant extract has been developed which yields proven results in animal experiments and has produced factual results in the clinical tests presently in progress. Fermented wheat germ extract is a complex substance gained from a watery extract, containing biologically active molecules, standardized to its 2.6–dimethyl-benzoquinone content; it is water-soluble and administrable orally (per os), distributed by pharmacies under the trade name AVEMAR.

In the course of research on tumorous diseases, more and more attention should be paid to the discovery of the formation of metastases and the prevention of the process as metastases, rather than primary tumours, are the causes of death in the majority of cases with tumorous diseases.

A number of pre-clinical tests show that there are antimetastatic compounds with cytostatic effects. The majority of these compounds come from plants (Taxol, Iscador), but they also include synthetically produced molecules. Nevertheless, there is no agent at the moment that would only destroy cancerous cells selectively, without any detrimental effect to healthy cells. Most of them causes so serious side effects that make practical administration difficult.

The side effects of chemotherapeutic drugs also applied in clinical practice extremely overburden the organism of patients: their blood count changes pathologically; their immune functions are damaged; morover, they suffer from cahexia, which may even reduce the chances of treatment.

Therefore, the development and production of a non-toxic, antimetastatic preparation with no side effects can be considered as a significant result both from the scientific and the economic point of view.

The major observations concerning the effects of fermented wheat germ extract, obtained in the course of "in vitro" and "in vivo" experiments, can be summarized as follows:

• it inhibits metastasis formation in various experimental metastasis model systems.

• it significantly enhances the Concanavalin-A induced blastic transformation of the lymphocytes obtained from the spleen of mice.

• in co-isogenic skin-graft systems in thymectomized animals, it considerably shortened the survival time of the grafts, indicating that it exerted a restoring effect on the immune functions, nearly in identical proportions in two dose ranges different from each other by two orders of magnitude.

• Treatment of human leukemic leukocyte-cell lines (Jurkat-leukemic T-cells, BL-41-B-cells, and U937 myelomonocytes) resulted in 70-85% reduction / downregulation of the major histocompatibility complex (MHC) class-I proteins and induced pronounced apoptosis (20-40%) in 24 hours, preceded by the dose-dependently induced tyrosine phosphorylation of certain specific proteins, the influx of extracellular Calcium (Ca²⁺) ions, and the increase of intracellular Ca²⁺ concentration. However, it failed to induce a similar degree of apoptosis in healthy, resting peripheral blood mononuclear cells.

• it enhances TNF induced cytotoxicity and the TNF production of macrophages. It decreases the phytohemaglutinine (PHA) induced proliferation of peripheral blood mononuclear cells in a dose dependent manner.

• treatment in an experimental mouse-model of the human systemic lupus erythematosus (SLE) results in a significant decrease of the humoral immune reaction to the antigene used for immunization, as well as a sustained decrease of auto-antibody (anti-dsDNA, mouse 16/6 Id and antihistone) production. Simultaneously with this, other parameters characteristic of the disease (erythrocyte sedimentation rate, count of leukocytes, proteinuria) also returned to normal.

• treatment also brought about the amelioration of the clinical manifestation of SLE by modulating - towards the restoration of balance - the immune reaction disorders in the Th1/Th2 cytokine network playing a decisive role in the pathogenesis of this disease.

• In the case of so called "rat adjuvant athritis" (AA) (induced by injection of Mycobacterium butyricum into the foot pad), a worldwide used experimental model of the human rheumatoid arthritis (RA), it inhibited both the primary and secondary (immune-mediated) response and exerted a significant anti-inflammatory effect similar to those elicited by treatment with Dexamethasone and Indomethacin.

• in macrophages and myelogenous tumor cell lines it alone induced a pronounced cytokine production; it enhanced synergistically the activation of these cells elicited by LPS (lipopolysaccharide) and PMA (phorbol-myristyl-acetate) and resulted in an over-expression of numerous cytokine-genes and in the release of several inflammation modulating cytokines by the activated cells. It increased TNF and IL-6 production the most. In higher dosages, however, it brought about cell-death due to the hyperactivation of the molecular mechanisms involved. In lymphoid cells, TNF production was not affected by the treatment.

• in other (HeLa) cell-lines it enhanced the activity of stress-kinases in a dose-dependent manner.

• it enhanced the inducing effect of TNF exerted upon the production of the adhesion molecule ICAM-1, while the VCAM expression of microvascular endothelial cells was not influenced by the compound.

It exerted a chemo - or rather "bio-preventive" effect in the Azoxymethane colon-carcinogenesis model in rats: colon tumors developed in a significantly smaller proportion of the rats and the number of tumors/animal developed was also considerably decreased.

The results of the above described studies strongly suggest that Avemar has a pronounced immune-modulating, immune-reconstitutive or even an immune-regulatory effect. Moreover – according to the investigation of hundreds of cancer patients  it does not elicit any toxic or other harmful side-effects. This is in full conformity with the negative results of the acute and subacute toxicological studies carried out in 2 species of mice and rats.

OBJECTIVE OF THE PROPOSAL

The objective of the proposal is to review the technology including the applied steps of fermentation, to increase and optimize its efficiency, at the same time to preserve the effects, efficiency, and quality features of the final product, and to recycle the large amount of by-products generated.

1. To decrease and optimize the duration of fermentation as well as the quantity of the raw materials used (other plant ingredients, microorganisms, enzymes, additives, water used), and to develop each step of the technology (condensation, filtering, drying).

This should also include selection of the following: germ fraction of the wheat species most suitable for the purpose; a ”food safe” microorganism (Saccharomyces phylum, lactic acid bacteria); optimal inoculum proliferation phase; conditions of fermentation (pH, time, temperature, etc.); implementation technology of fermentation; method for extracting agent (drying, solvent extraction, etc.).

Fermented wheat germ extract can be produced in larger quantities and more economically by establishing optimum parameters for fermentation, implementing the technology, and solving the problems of upsizing. Therefore the costs of the preparation produced for in vivo experiments and human tests will be decreased. As a result of the development of a more economical technology, the market price of the product may be decreased and the extract – distributed as a dietary supplement - will be available for more people.

2. The large amount of filter cakes and filter sludge produced in the technology and containing the water-soluble residues of wheat germ and baker’s yeast may be suitable for foddering. Preliminary tests show that the internal content data of filtering sludge are very good (high raw protein content). The project is intended to solve the problem of utilizing this valuable raw material rich in protein by optimization according to a biological value / price target function and to develop a corresponding technology.

CONTENT DESCRIPTION OF THE WORK

As stated in the objectives, the primary aims of the project are to optimize the production technology of AVEMAR as well as the raw materials used and the processes implemented; on the basis thereof, to modify and further develop the present technology. In order to achieve them, the following partial tasks should be completed:

1. To set up a model system by which the basic rules of the transformation of hydroquinone-glucoside into quinone – produced in plants in the course of fermentation – can be studied. By examining the model system, information can be obtained for the optimization of fermentation, preliminary data can be collected for selecting the proper microorganism and adjusting the conditions of the reaction.

   1. The first prerequisite for this is to produce – enzymatically and synthetically – the glucosides – present in wheat germ – of the quinone derivatives studied.

   2. To check for the impact of enzymes and enzyme systems in the model. On the basis thereof, select food safe microorganisms (Saccharomyces, lactic acid bacteria) with suitable enzymatic activity.

2. As a next step towards selecting a suitable raw material, to compare the germ fraction of various species of wheat from the point of view of the quantity of benzoquinones produced in them. In each research phase, the benzoquinone content of products is monitored by a highly efficient fluid chromatographic method developed by us.

3. In the knowledge of the suitable raw material, a microorganism can be selected for carrying out and optimizing fermentation.

   1. This should include specification of a suitable proliferation phase of the inoculum where enzime activity important from the viewpoint of the process is at a maximum.

4. In the knowledge of the raw material (wheat germ from wheat true to variety) and microorganism selected, the fermentation process can be optimized under laboratory conditions. As a first step, optimization will be performed in flasks in volumes varying between 100 and 200 ml. The efficiency of fermentation will be checked by monitoring the benzoquinone derivatives produced and enriched in the fermentation liquid. As regards the factors affecting fermentation, the impact of the following – as a first step - is intended to be investigated in the course of optimization: temperature, pH, duration of fermentation, nutrients, airing, mixing, execution of fermentation (mixed / filled column).

Other parameters arising in the course of optimization will also be included in the schedule for optimization.

Optimization will be performed by implementing experiment planning methods (factor plans, simplex, modified simplex methods). Results of the experiments will be evaluated by mathematical statistical methods.

5. Based on the results of laboratory optimization, fermentation will also be performed in greater volumes, in laboratory fermentors of 3 to 5 l and in pilot plant conditions as well. Tasks and problems coupled with upsizing may be solved / eliminated by further optimization steps adjusted to the particular conditions concerned. Increase of proportions in pilot plant fermentation will be performed in sizes of 300 to 400 l on the basis of summarization and evaluation of the preliminary partial results of optimization.

At the pilot plant level, the optimization of fermentation will also be supplemented by the optimization of the related operations of separation.

6. Using the results of the preceding steps, the technological improvement directed to the main product will intend to provide answers for the following questions:

• What are the fermentation parameters for yielding a product with the highest biological effect most economically?

• What is the series of separation operations that yields a fraction (filtrate) with the most favourable internal content value?

• How is the biological activity and other parameters (e.g. consistency, driability, particle distribution / volumetric mass of the resulting powder, etc.) of the preparation affected by the heat load applied in the course of supplementary operations (condensation, drying)?

• What quality assurance points should be built into the system and with what discrepancy intervals?

7. To examine the by-product (filter sludge) resulting from the new technology from the point of view of internal contents and foddering, coupled with feeding experiments.

8. To elaborate a filter sludge preservation process, including the inspection of drying methods and the dosage of preservatives from the technological and economical points of view.

9. In the course of processing the filter sludge produced as a by-product of the production process, technological variants will be sought for where – from the mass containing valuable components – a fodder additive or fodder component can be obtained which is sufficiently stable, its quality can be reproduced, and at the same time, it can be produced economically, including:

• Possibilities of on-site concentration and preservation; formation of transportable, manageable consistency.

• Drying variables: on a carrier or in itself, with a view to achieving the targeted nutrient component proportions based on tests of inside content.

PRESENT SITUATION, RESULTS

Several interim reports have been produced with extremely positive results. The obligations undertaken according to the objectives are expected to be performed in November, 2003.