Antineoplastic Preparation and the Use of Antineoplastic Preparation

Abstract

A method for manufacturing an antineoplastic preparation containing alpha-ketoglutarate (AKG) or/and glutamine or/and glutamate or/and alpha-ketoglutarate of ornithine or/and dipeptides of glutamine and other amino acids or/and tripeptides of glutamine and other amino acids or/and di- and tripeptides of glutamate with other amino acid, or/and mono- and divalent metal salts and other of alpha-keto-glutarate or/and glutamine or/and glutamate or/and ornithine of alpha-ketoglutarate.

Description

An object of the invention is antineoplastic preparation and the use of antineoplastic preparation.

In spite of the significant progress in the chemotherapy, radiotherapy and immunotherapy in last years the problem of the effective antineoplastic therapy is still the serious challenge in a present medicine. Epidemiological research indicates that in countries fully developed one of three people suffers from the different neoplastic diseases. In that group each fourth case is a lethal one. Cytostatics actually used in treatment are at the risk of side effects occurence limiting the effectiveness and decreasing the quality of patients life.

Therefore the development of new drugs acting specifically on the neoplastic cells with the simultaneous protective activity on the normal cells is especially urgent challenge.

The antineoplastic preparation distinguishes itself that contains alpha-ketoglutarate (AKG) or/and glutamine or/and glutamate or/and alpha-ketoglutarate of ornithine or/and dipeptides of glutamine and other amino acids or/and tripeptides of glutamine and other amino acids or/and di- and tripeptides of glutamate with other amino acid or/and mono- and divalent metal salts and other of alpha-keto-glutarate or/and glutamine or/and glutamate or/and ornithine of alpha-ketoglutarate.

The use of antineoplastic preparation is that the preparation containing alpha-ketoglutarate (AKG) or/and glutamine or/and glutamate or/and alpha-ketoglutarate of ornithine or and dipeptides of glutamine and other amino acids or/and tripeptides of glutamine and other amino acids or/and di- and tripeptides of glutamate with other amino acid or/and mono- and divalent metal salts and other of alpha-keto-glutarate or/and glutamine or/and glutamate or/and ornithine of alpha-ketoglutarate is used in the prophylaxis of neoplastic diseases.

Other use of antineoplastic preparation is that the preparation containing alpha-ketoglutarate (AKG) or/and glutamine or/and glutamate or/and alpha-ketoglutarate of ornithine or/and dipeptides of glutamine and other amino acids or/and tripeptides of glutamine and other amino acids or/and di- and tripeptides of glutamate with other amino acid or/and mono- and divalent metal salts and other of alpha-keto-glutarate or/and glutamine or/and glutamate or/and ornithine of alpha-keto-glutarate is used for metastases inhibition.

The preparation and the use of the preparation lets for the inhibition of the migration of neoplastic cells reflecting the potential role of AKG in the metastases inhibition. The preparation added to the diet plays a role as neo-adjuvant supporting existing methods used in the neoplasm treatment. It may improve the quality of the patients life through the synergic action with antineoplastic drugs and simultaneous protective activity to normal cells.

The example of the preparation action and its use is shown as a way of invention executing:

FIG. 1—the curve presenting the proliferation of A549 cells with the stimulation by AKG,

FIG. 2—the proliferation of C6 cells with AKG and

FIG. 3—the proliferation of HT-29 cells with AKG stimulation, whereas

FIG. 4 presents the proliferation of human neoplastic cells A549 in the presence of the cyclophosphamide with AKG, while

FIG. 5—the proliferation of human neoplastic cells A549 in the presence of iphosphamide with AKG,

FIG. 6—the proliferation of human neoplastic cells A549 in the presence of thiotepa with AKG,

FIG. 7—the inhibition of C6 cells migration due to AKG.

The cultures of neoplastic cells:

A549—human neoplastic cells of the lung cancer; the continuous line obtained from the Institute of the Immunology and the Experimental Therapy of Polish Academy of Science in Wroclaw

HT-29—human neoplastic cells of the large intestine cancer; the continuous line obtained from the Institute of the Immunology and the Experimental Therapy of Polish Academy of Science in Wroclaw

C6—the rat neoplastic cells of the brain cancer (glioma); the continuous line obtained from the Department of Neonatology, Humboldt University, Berlin, Germany

The bases of the culture.

Cells of A549 line were cultured on the basis DMEM:F-12 HAM (2:1), HT-29 and C6 cells on the basis DMEM. To the culture basis 10% foetal beast serum (FBS), penicillin 100 i.u./ml and streptomycin 100 μg/ml were added. The bases DMEM:F-12 HAM, DMEM were produced by Sigma company (Sigma, St. Louis, Mo., U.S.A.). The foetal beast serum (FBS) was produced by Life Technologies company (Life Technologies, Karlsruhe, Germany). Remaining reagents were produced by Sigma company.

The preparation of cellular cultures.

Cells stored in a liquid nitrogen in a tissue bank were unfreezed in a temp. 37° C., then poured into the plastic bottles containing proper basis. They were cultured in a temperature 37° C. in the incubator with 5% CO2 flow. After cells reproduction liquid was poured out, cells were washed with PBS (without the calcium and magnesium ions) and processed with 0.25% trypsin solution+EDTA to receive the suspension of cells necessary in the experience.

Assessment of the antyproliferative activity of AKG in the cellular culture.

Prepared earlier, in the culture basis, suspension of cells with a density 1×10⁴ cells/ml (A549), 4×10⁴ cells/ml (HT-29) and 0.5×10⁴ cells/ml (C6) were poured into the 96-pits microplate with the flat bottom (NUNC company, Roskilde, Denmark) in the volume 100 μl/pit. After sticking of cells (24 hours) the liquid was carefully pulled down and then different concentrations of AKG and examined cytostatics (cyclophosphamide, iphosphamid, thiotepa) in liquid with 10% FBS (100 μl/pit) were added. The cultures on plates were sleft for 96 hours incubation in temp. 37° C., in the atmosphere 95% air and 5% CO2. The antyproliferative activity of examined substances was assessed with the method MTT.

The method MTT (according to kit “Cell proliferation kit III”, Boehringer Manheim).

This method was worked out to determine the proliferation and vitality of cells in studies on the cytotoxic and antyproliferative substances. In metabolically active cells tetrazolic yellow salt MTT is reduced to formazane blue with the mitochondrial dehydrases. Formazane crystals, insoluble in water, accumulate in cells and for their dissolutions the use of organic detergent, breaking the membrane and simultaneously solvent the dye, is necessary. For this purpose the buffer SDS-HCl with pH 7.4 is used. The concentration of released dye is evaluated quantitatively in the reader for 96-pits plates at the wavelength 570 nm. The colour intensity is directly proportional to the quantity of alive cells.

MTT solution in PBS condition in concentration 5 mg/ml was added into each pit on plastic plate in a dose 15 μl/pit. Plates were incubated for 3 hours in temp. 37° C. Thereafter buffer SDS-HCl in a dose 100 μl/pit was added and plates were left in temp. 37° C. all night. The results were evaluated next day with the use of E-max Reader (Molecular Devices Corporation, Menlo Park, Calif., U.S.A.).

Evaluation of the Cells Migration Degree with the Method “Wound Assay”

This method is for estimation the activity of substances affecting the modility of cells in vitro. It is used in research on wounds healing, angiogenesis and neoplastic metastases.

C6 cells (1×10⁶) suspended in the culture basis with the addition of 10% serum (FBS) were poured onto the culture plates (NUNC, Roskilde, Denmark) with 4 cm diameter. Next day in the equal layer of cells the flaw (wound) was done with the ending of automatic pipette and unsticked cells were removed by twice-rinsing of plates with PBS solution. Then AKG (10 and 20 mM) dissolved in the culture basis was added to the prepared culture. Plates were incubated for 24 hours in a temperature 37° C. and in a humid atmosphere 95% air and 5% CO₂. Thereafter the cultures were coloured with May-Grünwald-Giemza method. Then the microscopic analysis was performed with the microscope Olympus BX51 (Olympus Optical CO., LTD, Tokyo, Japan) with the use of the software analySIS® (Soft Imaging System GmbH, Münster, Germany). The degree of cells migration was assessed in cytometry as the number of cells which populated the wound done earlier in the layer of cells. At least 50 chosen fields on 8 photographs were assessed.

Results: The Estimation of Antyproliferative Activity of AKG

Antyproliferative activity of AKG in the cultures was estimated in different kind of neoplastic cells: the lung cancer cells (A549), the large intestine cancer cells (HT-29) and glioma cells (C6). Cells were processed with AKG in concentrations 0.5, 1, 2.5, 5, 10 and 20 mM, for 96 hours.

The examined substance has had antyproliferative activity with relation to all neoplastic cells types (the diagram—FIG. 1, FIG. 2, FIG. 3). Statistically significant (4.5%) inhibition of cells growth was observed at AKG concentration 2.5 mM in A549 cell line in the comparison to the control group. That effect was correlated with a dose of AKG and was 7.8%, 12.4%, 17.5% with doses 5 mM, 10 mM and 20 mM respectively. (the diagram on FIG. 1). The growth inhibition in glioma cells (C6) was 12.6% with AKG dose 2.5 mM, 7.9%—5 mM, 16%—10 nmM and 19.8%—20 mM respectively. The growth inhibition of large intestine cancer cells (HT-29) hadn't had the lineal character with AKG concentrations between 1 and 10 mM. The dose 1 mM inhidited the cells growth by 11.8%. The similar effect was obtained with a dose 5 mM (11.8%) and 10 mM (11.5%). Only a dose 20 mM caused the significant (25%) inhibition of these cells growth (the diagram—FIG. 3).

The estimation of the interaction between AKG and antineoplastic drugs.

The research on the interaction between AKG and popular cytostatics used in cancer chemotherapy was performed in the culture of lung cancer cells (A549). For that purpose cells were treated with the following cytostatics: cyclophosphamide (1.5 mM), iphosphamid (1.5 mM) and thiotepa (5 μM), alone and in combination with AKG (5, 10 and 20 mM). The additive effect of AKG on cytostatic activity of used chemotherapeutics was observed (results are presented on FIG. 4, FIG. 5 and FIG. 6). Cyclophosphamide in a concentration 1.5 mM inhibited the growth of A549 cells by 21.4%. The addition of AKG increased its cytostatic activity by 6.4%, 9.8% and 14.4% respectively (the diagram—FIG. 4). Iphosphamid (1.5 mM) inhidited the cells growth by 7.3%. After AKG addition (5, 10 and 20 mM) that effect increased by 5%, 5.3% and 8.8% respectively (the diagram—FIG. 5). AKG intensified also cytostatic activity of thiotepa (5 μM—25.9%) by 4.2%(5 mM), 8% (10 mM) and 11.2% (20 mM) (results are presented on FIG. 6).

The Influence of AKG on the Migration of Neoplastic Cells

The research on the mobility of neoplastic cells was passed in the “wound assay” model. At the photograph 1 the wound in the layer of C6 cells (A), the wound population with cells after 24 hours of incubation without AKG (B) and significant inhibition of cells migration in the presence of AKG 20 mM (C) are presented. On the diagram—FIG. 7, the average number of cells migrating to one field of flaw done in the uniform layer of cells is presented. Statistically significant inhibition of cells migration in the presence of 10 mM and 20 mM AKG was shown.

The statistical analysis

The statistical analysis was performed with the use of t-student test. * p<0.05, ** p<0.01, ***p<0.001.

Claims

1. A method for the manufacture of a pharmaceutical preparation for the treatment or prophylaxis of neoplastic diseases, comprising the step of preparing an antineoplastic preparation using ketoglutarate (AKG), mono- or divalent metal salts of alpha-ketoglutarate and/or ornithine of alpha-ketoglutarate as the only active ingredients.

2. A method for the manufacture of a pharmaceutical preparation for inhibition of cancer metastases, comprising the step of preparing an antineoplastic preparation using alpha-ketoglutarate (AKG), mono- or divalent metal salts of alpha-ketoglutarate and/or ornithine of alpha-ketoglutarate as the only active ingredients.