Field of the invention

The subject matter of the invention is a composition containing lipoic acid and selenite and a mixture of certain small molecules, applied for the prevention and treatment of neoplastic disorders. Background of the invention

Alpha-lipoic acid is an endogenous, disulfide bridge-containing short chain fatty acid. The basic biological function of alpha-lipoic acid is that it is the cofactor of certain mitochondrial multi-enzyme complexes such as the pyruvate-dehydrogenase enzyme complex through which it has an important role in the decarboxylation of alpha-ketoacids and thus in the mitochondrial energy production. The alpha-lipoic acid contains one chiral carbon atom, of its stereoisomers the R-enantiomer is the biologically active form as a cofactor of the above mentioned enzyme complex. In addition to its role in energy production the alpha-lipoic acid is a strong antioxidant [1] and utilizing this property it is applied as a pharmaceutical in the treatment of diabetic neuropathy. Both the alpha-lipoic acid and its reduced form the dihydrolipoic acid have been shown to be able to capture reactive oxygen species (e.g. hydroxyl radicals, superoxide radicals, hypochlorous acid) and to form complexes with the ions of various transition metals [2-4]. It has been shown that the alpha-lipoic acid is able to increase the level of reduced glutathione which is an important intracellular antioxidant molecule [5]. In addition to its above mentioned antioxidant properties it has been shown that the alpha-lipoic acid causes cell death in various cancer cell lines (Jurkat, H460, MCF-7 etc.) [6-10]. There is a lot of data about the mechanism of the anticancer effect of alpha-lipoic acid: treatment with alpha-lipoic acid increases the production of superoxide anions in the mithochondria, decreases the amount of anti-apoptotic proteins (bcl-2, bcl-xl), induces cytochrome c release from the mitochondria, activates caspase-9 and PARP-1, cumulatively induces the activation of the mitochondrial pathway of apoptosis in cancer cells [6, 7, 11]. In addition to the activation of the mitochondrial pathway of apoptosis, caspase-independent cell death and autophagy also may have a role in the anti-cancer effect of alpha-lipoic acid [12, 13]. Besides the activation of processes leading to cell death the treatment with alpha-lipoic acid is also capable to inhibit the PBK-Akt-mTOR cascade which is an important oncogenic signaling pathway. Treatment with alpha-lipoic acid reduces both the total amount of Akt protein and the level of phosphorylated Akt [14]. The alpha-lipoic acid has been shown to increase the anti-tumor effect of certain cytostatics (5-fluorouracil, etoposide) [12, 15]. The antitumor activity of alpha-lipoic acid and its synthetic derivatives has been demonstrated in numerous preclinical animal models (SkBr3, NCI-H69, S180, H460) [16-19]. A phase I study has been performed on cancer patients with a synthetic derivative of alpha-lipoic acid (CPI- 613) [20]. Another phase I study and a phase II study are currently underway (NCTO 1835041, NCT01832857).

Selenium is an essential trace element, with the main biological function of being incorporated into selenoproteins and through the oxidoreductase activity of such proteins it contributes to the maintenance of the proper redox state of the cell [21]. On the basis of this selenium can be considered as an indirect antioxidant. Selenium is present in foodstuffs in the form of different organic compounds: selenomethionine (SeMet), selenocysteine (SeCys), seleno-methyl-selenocysteine (SMSC); while in the soil it occurs mainly in the form of selenate and selenite. However, in addition to the indirect antioxidant effect, the above mentioned compounds can be considered as redox-active, since in the presence of reduced glutathione, on the one hand as substrates of oxidoreductase enzymes they can be transformed to hydrogen selenide, accompanied by the production of reactive oxygen species, on the other hand selenide anions can be produced through a redox-cycle, also accompanied by the production of reactive oxygen species [22-24]. Numerous redox-active organic and inorganic selenium compounds have been shown to possess some degree of antitumor activity [25].

Hereafter we review the background art regarding selenite (anion of selenous acid), which is the selenium compound used in the present invention. Selenite has been shown to influence numerous signal transduction pathways which have vital importance for cancer cells: in addition to the production of reactive oxygen species mentioned above, selenite is able to induce apoptosis through the inhibition of the Akt pathway, it is able to shift autophagy toward apoptosis through the inhibition of the Nfkappab pathway, additionally it can also induce caspase-independent cell death (necroptosis) [26-28]. Selenite has been shown to increase the in vivo effect of cisplatin in the A2780 tumor model [29]. Treatment with selenite inhibits the development of in vivo resistance against different cytostatic agents (melphalan, carboplatin, cisplatin) [29-32]. A phase I study has been performed on 34 cancer patients with selenite, in which the maximum tolerated dose, safety profile and pharmacokinetics of intravenously administered sodium-selenite have been determined [33]. During our research we have made the surprising discovery that upon the simultaneous application of lipoic acid (also known as alpha-lipoic acid) and a selenite, preferably sodium- selenite, the two compounds cause a significant cell death in various cancer cell lines by synergistically enhancing each other's effect. The extent of synergism was quantified by computing the so-called "combination index" based on cell culture experiments performed with various concentrations of the two compounds (see examples 1-2 and 15-17).

The synergistic antitumor activity of alpha-lipoic acid and sodium-selenite, which represents the preferred embodiment of the invention, has also been verified in vivo (see example 3). We have shown that both the R-, and the S-enantiomers of alpha-lipoic acid are able to partner with sodium-selenite to induce a synergistic antitumor effect (see examples 4- 5).

The surprising nature of the found effect is underscored by the fact that the simultaneous application of selenium-containing compounds other than selenite (sodium- selenate, selenomethionine, selenocysteine, seleno-methyl-selenocysteine, methylseleninic acid) and alpha-lipoic acid has not resulted in a synergistic increase of antitumor activity (see examples 6-10).

The combined application of alpha-lipoic acid and sodium-sulfite has not resulted in a synergistic increase of antitumor activity (see example 11), thus excluding the possibility that a redox reaction between alpha-lipoic acid and the selenite anion taking place in the cell culture media would account for the synergism of alpha-lipoic acid and sodium-selenite.

The combined application of (±)-dihydrolipoic acid (the reduced form of alpha-lipoic acid containing free SH-groups) and sodium-selenite also resulted a synergistic antitumor effect, while the combined application of reduced L-glutathione or L-cysteine (both compounds also contain free SH-groups) and sodium-selenite did not (see examples 12-14). On the basis of these results the synergistic antitumor activity seen upon the combined application of alpha-lipoic acid and sodium-selenite is not caused by reactive oxygen species formed in the cell culture media from sodium-selenite triggered by free SH-groups formed during the incidental reduction of alpha-lipoic acid.

We note here, that although dihydrolipoic acid is formed intracellularly in the human body from alpha-lipoic acid, it is not a stable compound (it is easily oxidized), therefore its application in a composition according to the invention is not expedient.

During our research we have made the further surprising discovery that the joint antitumor activity of alpha-lipoic acid and sodium-selenite can be further increased in a synergistic manner by the individual combination of the following active substances with a mixture of alpha-lipoic acid and sodium-selenite: L-tryptophan, D-tryptophan, L-methionine, D-phenylalanine, agmatine, phenylpyruvic acid, gentisic acid, pyrrole-2-carboxylic acid (see examples 18-25).

The aim to partially or completely substitute the common chemotherapeutic substances (e.g. docetaxel, doxorubicin, taxane-derivatives, cisplatin etc.), which in general have serious toxicity, during a chemotherapeutic treatment became achievable with our invention.

Some of the documents found during our search performed to get acquainted with the state of the art describe the application of lipoic acid and/or selenite as a supplementary substance in conjunction with an antitumor substance. Such a document is for example the US2011008418 (where the antitumor substance is e.g. carboplatin, cyclophosphamide, doxorubicin, 5-Fluorouracil, methotrexate and paclitaxel), and the US2002086894 (where the antitumor substance is rofecoxib), etc.

Another significant portion of the documents found during our search describe dietary supplements aimed to be applied during the treatment of cancer, such a document is for example the EP2296643. However, in these cases lipoic acid and/or selenite are only element(s) of lengthy lists in addition to certain other substances (vitamins, trace elements, minerals, hydrocarbons, etc.).

Thus during our search we have found no documents describing a composition which contains only the above two compounds as the active substances without other, typically large-molecular active substance generally applied in the treatment of cancer.

Brief description of the invention

On the basis of the above the invention relates to therapeutical compositions for the prevention and treatment of neoplastic disorders and uses thereof.

1. The subject matter of the invention, more specifically, is a synergistic composition containing exclusively lipoic acid or a therapeutically acceptable salt thereof and selenite as the primary active substances, optionally in conjunction with one or more secondary active substance(s) with a maximal molecular weight of 280 daltons, and optionally in conjunction with one or more usual therapeutically acceptable carrier(s), diluent(s) and/or other excipient(s).

In a typical embodiment the composition contains exclusively the above mentioned primary active substances. In another typical embodiment the composition, in addition to the two primary active substances, comprises exclusively one or more secondary active substance(s) mentioned in points 4 and 5 below. However, in these cases the composition may also comprise one or more usual pharmaceutical excipient(s) without a therapeutic effect (e.g. usual therapeutically acceptable carriers, diluents and/or other excipients).

The further subject matters of the invention are the following:

2. The synergistic composition according to point 1, wherein the lipoic acid is (±)- alpha-lipoic acid.

3. The synergistic composition according to point 1 or 2, wherein the selenite is sodium-selenite.

4. The synergistic composition according to any of points 1-3 comprising one or more compound(s) as secondary active substance(s) selected form the following group: L- tryptophan, D-tryptophan, L-methionine, D-phenylalanine, agmatine, phenylpyruvic acid, gentisic acid, pyrrole-2-carboxylic acid, L-arginine, D-arginine, L-phenylalanine, L-histidine, L-tyrosine, pyridoxine, D(+)-biotin, L(+)-ascorbic acid, L(-)-malic acid, D-(+)-glucosamine, ketoisocaproic acid (4-methyl-2-oxovaleric acid), cinnamic acid, adenosine, melatonin, p- coumaric acid and therapeutically acceptable salts thereof.

5. The synergistic composition according to point 4 comprising one or more compound(s) as secondary active substance(s) selected form the following group: L- tryptophan, D-tryptophan, L-methionine, D-phenylalanine, agmatine, phenylpyruvic acid, gentisic acid, pyrrole-2-carboxylic acid and therapeutically acceptable salts thereof.

6. The synergistic composition according to any of points 1-5 for use in the prevention or treatment of neoplastic disorders in mammals, particularly in humans.

7. Synergistic composition for use according to point 6, where the neoplastic disorder is a malignant (cancerous) disease.

8. A method for the prevention or treatment of neoplastic disorders characterized by that the patient in need of treatment is administered a synergistic composition according to any of points 1-5 in a therapeutically effective amount, preferably in an amount of 5-500 mg per kg body weight in 1-15 portion(s) per day.

Brief description of the drawings

Figure 1. part "A": dose-antitumor effect curves obtained on HELA cells with (±)- alpha-lipoic acid applied in 0.5 mM, 0.4 mM, 0.3 mM concentrations, with sodium-selenite applied in 1 μΜ, 0.75 μΜ, 0.5 μΜ concentrations, and with the combined application of the two compounds in the above concentrations. Figure 1. part "B": plot of the antitumor effect on a normalized isobologram [34] obtained on HELA cells with the combined application of (±)-alpha-lipoic (LA) acid and sodium-selenite (SEL) in the indicated concentrations.

Figure 2. part "A": dose-antitumor effect curves obtained on HELA cells with (±)- alpha-lipoic acid applied in 0.5 mM, 0.4 mM, 0.3 mM concentrations, with sodium-selenite applied in 4 μΜ, 3 μΜ, 2 μΜ concentrations, and with the combined application of the two compounds in the above concentrations. Figure 1. part "B": plot of the antitumor effect on a normalized isobologram obtained on HELA cells with the combined application of (±)-alpha- lipoic (LA) acid and sodium-selenite (SEL) in the indicated concentrations.

Figure 3. : in vivo antitumor effect obtained on the C26 tumor model with (±)-alpha- lipoic acid (LA) applied per se in a dose of 50mg/kg, with sodium-selenite applied per se in a dose of 1.5 mg/kg, and with the combined application of the two compounds in the above doses (LA+SEL). Part "A": tumor volumes measured during the treatment period (*p < 0,001 vs. CTRL, vs. LA, vs. SEL). Part "B": tumor weights measured on the 10 ^th day of the treatment (*p < 0,05 vs. CTRL, vs. LS; #p < 0,001 vs. SEL).

Detailed description of the invention

In the frame of the present description the term "lipoic acid" designates racemic lipoic acid [(±)-alpha-lipoic acid, which represents a preferred embodiment], and in addition enantiomers thereof [(+)-alpha-lipoic acid and (-)-alpha-lipoic acid], or any mixtures thereof.

The lipoic acid can be applied in the form of a salt, consequent from the intended use obviously in the form of a therapeutically acceptable salt. The cation present in the salt can be derived from an organic or inorganic acid. Preferred examples are salts formed with alkali metal and alkaline earth metal cations, or with a quaternary ammonium cation (or with other quaternary nitrogen-containing cation). Among these particularly preferred are the alkali metal and alkaline earth metal salts, more preferred is the sodium salt.

In the frame of the present description the term "selenite" used alone designates a salt derived from selenous acid (thus in this general name we refer to the salt, for the sake of simplicity, with the name of the anion present in it). In such salts the cation can be an alkali metal and alkaline earth metal cation, or a quaternary ammonium cation (or other quaternary nitrogen-containing cation). Among these salts particularly preferred are the following selenites: sodium-selenite, potassium-selenite, magnesium-selenite and calcium-selenite, particularly preferred is the sodium-selenite.

In the set of claims the ratio of the active substances is given as mass ratio. In the present description, unless otherwise specified, the mass ratios and mass percentage values in case of therapeutically acceptable salts of the listed active substances refer to the free bases/acids.

In the compositions according to the invention the mass ratio of the primary active substances can vary in a wide range, however it is typical, that the lipoic acid is present in a substantially higher amount than selenite.

In preferred embodiments the mass ratio of lipoic acid:selenite falls into the range of 10-10000: 1, preferably into the range of 50-5000: 1, more preferably into the range of 100- 3000: 1.

The group of secondary active substances does not include the conventionally applied high molecular weight chemotherapeutic compounds (such as e.g. cisplatin, doxorubicin, etoposide, paclitaxel, irinotecan HCL, vincristine). In case of the present invention the group of secondary active substances includes compounds with a maximal molecular weight of 280 daltons (the compounds are typically acids). Preferred cases of such compounds are the following: L-tryptophan, D-tryptophan, L-methionine, D-phenylalanine, agmatine, phenylpyruvic acid, gentisic acid, pyrrole-2-carboxylic acid, L-arginine, D-arginine, L- phenylalanine, L-histidine, L-tyrosine, pyridoxine, D(+)-biotin, L(+)-ascorbic acid, L(-)-malic acid, D-(+)-glucosamine, ketoisocaproic acid (4-methyl-2-oxovaleric acid), cinnamic acid, adenosine, melatonin, p-coumaric acid and therapeutically acceptable salts of said compounds. More preferred secondary active substances are the following: L-tryptophan, D- tryptophan, L-methionine, D-phenylalanine, agmatine, phenylpyruvic acid, gentisic acid, pyrrole-2-carboxylic acid and therapeutically acceptable salts of said compounds.

The secondary active substances are also typically applied in a high mass ratio compared to the selenite (if salts are applied then the calculation is also based on the free base/acid form of the substances). In preferred embodiments the mass ratio of the "secondary active substance": selenite falls into the range of 10-10000: 1, preferably into the range of 50- 5000: 1, more preferably into the range of 100-3000: 1.

The term "composition" used in the present description designates preferably a therapeutical composition, which may comprise usual therapeutically/pharmaceutically acceptable excipients (see in more detail below). However, the composition can also be formulated as a dietary supplement or foodstuff.

The active substances according to the invention and their therapeutically acceptable salts, and the therapeutical compositions made from them can be administered in any ordinary way. They can be used for example orally, parenterally (including subcutaneous, intramuscular and intravenous administration), buccally, sublingually, nasally, rectally or transdermally. The therapeutical compositions are prepared in unit dosage form using the ordinary pharmaceutical procedures, the usual therapeutically/pharmaceutically acceptable carriers, diluents and/or other excipients.

Hereafter, unless we specify otherwise, the active substances according to the invention define the active substances according to the invention and/or therapeutically acceptable salts thereof.

Compositions containing active substances according to the invention that are orally active can be prepared in a liquid or solid form. For example syrup, suspension, emulsion, tablets, capsules or lozenges can be prepared.

If the active substances according to the invention are prepared in liquid form, for example as a suspension solution, it will contain the active substances according to the invention in a suitable liquid carrier or carriers. Aqueous solvents (e.g. water, ethanol or glycerine) or non-aqueous solvents (e.g. polyethylene glycol or some sort of oil) can be used. The composition may also comprise suspending agents, preservatives, flavouring and colouring agents.

If the solid composition is a tablet, it can be formulated with any suitable carrier routinely used in the preparation of medicaments. Solid carriers can be for example lactose, suitable silicates, sucrose, talcum, gelatine, agar, pectin, gum arabic, magnesium stearate and stearic acid, etc. Optionally a standard aqueous or non-aqueous coating can be applied on the tablets.

From the compositions according to the invention tablets can be prepared by pressing or moulding, optionally using one or more absorption promoting agents or adjuvants. Tablets can be manufactured for example by means of a suitable tablet press machine; the active substance can be pressed in powder or granular form, optionally mixed with excipients, lubricants, inert diluents, surface active or dispersive agents.

If the solid composition is a capsule, it can be manufactured using any ordinary encapsulation method. For example pellets can be made from the active substances combined with a standard carrier, and then they can be filled in hard gelatine capsules. As an alternative solution, a dispersion or suspension can be prepared from the active substances combined with a suitable pharmaceutical carrier, and then it can be filled in soft gelatine capsules. Suitable pharmaceutical carriers can be for example water-dispersible gums, cellulose, silicates or oils.

The typical forms of parenteral compositions are solutions or suspensions which contain the active substances according to the invention in sterile aqueous carriers or parentally administrable non-aqueous carriers, e.g. in polyethylene glycol, polyvinylpyrrolidone, lecithin, peanut oil or sesame oil. As an alternative solution the solution can be lyophilized, and directly before administration it is transformed into a solution again with a suitable solvent.

The forms of the compositions according to the invention suitable for nasal administration contain the active substances according to the invention in aerosols, drops, gel or powder form. The aerosols according to the invention contain the active substances according to the invention typically in a solution or a finely dispersed suspension, in a physiologically acceptable aqueous or non-aqueous solvent. The sterile aerosol can be packaged in a closed container containing a single dose or several doses, which container enables administration or refilling and is generally equipped with a nebuliser. Aalternatively, the closed container may also be suitable for the administration of unit doses; for example it can be an inhaler ensuring single doses, or an aerosol dispenser with a dispensing valve, which can be disposed of when the container is empty. If administration is carried out by an aerosol dispenser, a propellant, e.g. a compressed gas (e.g. compressed air) or an organic propellant (e.g. chlorinated/fluorinated hydrocarbons) can be used. The administration of the aerosol can also be carried out by a nebuliser pump.

The compositions according to the invention containing the active substances according to the invention are also suitable for buccal and sublingual administration, for example in the form of tablets, lozenges or pastilles; they contain the active substances formulated with a carrier (e.g. sugar and gum arabic, tragacanth, or gelatine, glycerine, etc.).

The composition containing the active substances according to the invention is also suitable for rectal administration. Generally suppositories are prepared, which contain the active substances in a suppository base such as cocoa butter or some other known carrier. Suppositories are prepared by usual method, by mixing the components, then softening and melting the mixture and pouring the melt into a mould and cooling it.

The composition containing the active substances according to the invention is also suitable for transdermal administration, e.g. in the form of an ointment, gel or plaster.

The daily dose of the composition according to the invention is influenced by numerous factors such as the kind of the disease to be treated, the condition of the patient, the other ongoing treatments, the route of administration etc. Suitably a composition comprising 5-500 mg, preferably 10-300 mg active substances per kg adult body weight, should be administered daily. Considering the fast elimination of the components the daily dose should be administered in 1-15 portion(s) in the form of tablet, capsule, pill, drink powder, solution for infusion etc. The suitable daily dose has been determined for mammals, specifically for humans.

On the basis of the synergistic effect of the active substances described in the invention, the different types of cancer cells (e.g. with different histological origin and type), the metabolism of which differ from the normal cells at different extent, can show a various degree of sensitivity to combinations of the active substances according to the invention. On the basis of this the qualitative and quantitative composition of the mixtures described in the present invention can be optimized for the given type of cancer by using the synergism as selection criterion. Furthermore the sensitivity of cancer cells isolated from the tumor tissue of a given individual to a composition according to the invention can also be determined, and the composition of the mixtures can be optimized (individualized therapy).

The composition according to the invention can be preferably used for the prevention and treatment of the following neoplastic disorders: malignant (cancerous) tumors, such as malignant neoplasms of lip, oral cavity and pharynx, malignant neoplasms of digestive organs, malignant neoplasms of respiratory and intrathoracic organs, malignant neoplasms of bone and articular cartilage, melanoma and other malignant neoplasms of skin, malignant neoplasms of mesothelial and soft tissue, malignant neoplasm of breast, malignant neoplasms of female genital organs, malignant neoplasms of male genital organs, malignant neoplasms of urinary tract, malignant neoplasms of eye, brain and other parts of central nervous system, malignant neoplasms of thyroid and other endocrine glands, malignant neoplasms of ill- defined, secondary and unspecified sites, malignant neoplasms, stated or presumed to be primary, of lymphoid, haematopoietic and related tissue, malignant neoplasms of independent (primary) multiple sites, in situ neoplasms, neoplasms of uncertain or unknown behavior.

The invention is illustrated in more detail by the examples presented in the following tables and figures.

Example 1

Through example 1 presented in Table 1 and Figure 1 we show that 0.5 mM, 0.4 mM, 0.3 mM concentrations of (±)-alpha-lipoic acid (LA) applied in combination with 1 μΜ, 0.75 μΜ, 0.5 μΜ concentrations of sodium-selenite produce a synergistic antitumor effect on HELA cells.

For testing the active substances of the invention we used an in vitro experimental system based on TELA (American Type Culture Collection, product number: CCL-2) cancer cells. The cancer cells were incubated with the investigated substances for 48 hours, and then the number of cells that were still alive after the treatment was determined. HELA cells were seeded at a density of 4xl0 ³ cells/well on 96-well plates, and then treated for 48 hours in triplicates. Cells were cultured and the investigated active substances were dissolved in MEM media (Sigma-Aldrich, product number: M2279) containing 10% fetal bovine serum. At the end of the treatment cells were washed with phosphate buffered saline, and then 50 μΐ methylthiazolyldiphenyl-tetrazolium bromide (MTT, Sigma-Aldrich, product number: M2128) dissolved in phenol-red free RPMI media (Sigma-Aldrich, product number: R7509) was added to the cells. The cells were incubated for 4 h at 37 °C, then following the aspiration of the substrate solution 50 μΐ dimethyl-sulfoxide was added to the cells and the plate was shaken for 10 min. After this the optical density of each well was measured at 570 nm. The results are presented as averages of values obtained from three independent experiments. During statistical analysis normal distribution of the data was examined by Shapiro-Wilk test, equal variance was tested with Brown-Forsythe's test, and then one-way analysis of variance (ANOVA) followed by Bonferroni test was used for pairwise comparisons [35]. P values below 0.05 were considered statistically significant.

In order to quantitatively measure the synergy, additive effect, or antagonism occurring upon the combined application of the investigated active substances, various concentrations of the active substances were applied individually and in combinations. The obtained results were analyzed with the Compusyn software which computes the so-called combination index (CI) [34, 36, 37]. The CI is the quantitative measure of interaction between the effect of the individual active substances: if the value of the CI<1 then synergism is present, if the value of the CI>1 then antagonism is present, if the value of the CI is nearly 1 then the effect of the investigated active substances is additive [34, 36, 37]. In more detail, if the value of the CI is between 0.85 and 0.90 then slight synergism; if the value of the CI is between 0.7 and 0.85 then moderate synergism; if the value of the CI is between 0.3 and 0.7 then synergism; if the value of the CI is between 0.1 and 0.3 then strong synergism; if the value of the CI<0.1 then very strong synergism is present.

The obtained results are the following Table 1.

0.5 mM, 0.4 mM, 0.3 mM concentrations of (±)-alpha-lipoic acid applied in combination with 1 μΜ, 0.75 μΜ, 0.5 μΜ concentrations of sodium-selenite produce a synergistic antitumor effect on HELA cells

Example 1

Number of living

(±)-alpha- cells expressed as

Sodium-selenite

lipoic acid % of untreated Combination concentration

concentration cells (average ± index (CI)

(μΜ)

(mM) standard

deviation)

2 - 33.87 ± 1.32 * -

1 - 64.81 ± 3.86 * -

0.5 - 82.40 ± 3.60 * -

0.3 - 93.67 ± 1.56 * -

- 1 92.86 -

- 0.75 99.74 -

- 0.5 99.97 -

0.5 1 33.44 ± 0.98 0.84

0.4 1 31.51 ± 1.35 0.81

0.3 1 34.44 ± 1.42 0.78

0.5 0.75 37.60 ± 3.65 *' ⁺ ' ^& 0.75

0.4 0.75 36.97 ± 3.75 *' ⁺ ' ^& 0.69

0.3 0.75 39.77 ± 1.99 *' ' ^& 0.66

0.5 0.5 45.55 ± 4.05 0.67

0.4 0.5 45.50 ± 1.40 0.60

0.3 0.5 54.16 ± 3.68 0.59

* pO.001 vs. untreated control

⁺ pO.001 vs. 0.5 mM (±)-alpha-lipoic acid

pO.001 vs. 0.3 mM (±)-alpha-lipoic acid

pO.001 vs. 1 μΜ sodium-selenite

&

pO.001 vs. 0.75 μΜ sodium-selenite

^§ p<0.001 vs. 0.5 μΜ sodium-selenite

P values were computed with one-way analysis of variance (ANOVA), followed by Bonferroni-test. On the basis of our results presented in Table 1, LA applied per se in 2 mM, 1 mM, 0.5 mM, 0.3 mM concentrations significantly reduced the number of living cells compared to the untreated control, while sodium-selenite applied per se in 1 μΜ, 0.75 μΜ, 0.5 μΜ concentrations did not cause a significant decrease in the number of cells. The combined application of various concentrations of the two compounds significantly enhanced the antitumor effect, resulting in a significantly lower number of living cells compared to the per se application of LA or sodium-selenite in the given concentration (see the symbols indicating significance in Table 1). On the basis of the CI values presented in Table 1 (0.59-0.84) there is synergism - moderate synergism in the antitumor effect between LA and sodium-selenite at the investigated concentrations. In Figure 1 part "A" we present the dose-effect curves obtained with the two compounds applied per se and in combination. In Figure 1 part "B" we present the normalized isobologram obtained with various concentrations of the two compounds applied in combination. The normalized isobologram is an equipotent graph with the normalized doses of the two investigated compounds (ΌΙίΌχΙ) and (02/Ε ⁾ χ2) on the x-, and y-axes, respectively. Dl and D2 are the doses of the two compounds in the D1+D2 combination which produce an inhibition of X%, while ϋχΐ and Όχ2 represents the doses of the two compounds which produce an inhibition of X% when the two compounds are applied per se. If the data points on the normalized isobologram are localized lower-left from the line connecting the ends of the two axes then synergism is present, if data points are localized along the connecting line then the effect of the two compounds is additive, and if data points are localized upper-right from the connecting line then antagonism is present. The normalized isobologram shown in Figure 1 part "B" confirms that there is synergism in the antitumor effect between LA and sodium-selenite at the investigated concentrations. Example 2

Through example 2 presented in Table 2 and Figure 2 we show that 0.5 mM, 0.4 mM, 0.3 mM concentrations of LA applied in combination with 4 μΜ, 3 μΜ, 2 μΜ concentrations of sodium-selenite produce a synergistic antitumor effect on HELA cells.

The measurements were performed with the in vitro experimental system described at example 1. The results are presented as averages of values obtained from three independent experiments. The statistical analysis and calculation of the CI were performed as described at example 1.

The obtained results are the following: Table 2.

0.5 mM, 0.4 mM, 0.3 mM concentrations of (±)-alpha-lipoic acid applied in combination with 4 μΜ, 3 μΜ, 2 μΜ concentrations of sodium-selenite produce a synergistic antitumor effect on HELA cells

Example 2

Number of living

(±)-alpha- cells expressed as

Sodium-selenite

lipoic acid % of untreated Combination concentration

concentration cells (average ± index (CI)

(μΜ)

(mM) standard

deviation)

2 - 33.26 ± 1.02 * -

1 - 63.63 ± 1.50 * -

0.5 - 83.94 ± 2.38 * -

0.3 - 96.85 ± 1.42 -

- 4 83.19 ± 2.07 * -

- 3 94.23 ± 1.55 -

- 2 99.37 ± 1.01 -

0.5 4 0.75 ± 0.13 0.32

0.4 4 0.68 ± 0.26 0.30

0.3 4 1.31 ± 0.29 0.34

0.5 3 2.46 ± 0.19 *' ⁺ ' ^& 0.33

0.4 3 5.67 ± 0.73 *' ⁺ ' ^& 0.40

0.3 3 15.44 ± 0.91 *' ^# ' ^& 0.50

0.5 2 19.47 ± 1.60 0.47

0.4 2 25.75 ± 3.55 0.48

0.3 2 27.82 ± 0.72 *' ' ^§ 0.45

* pO.001 vs. untreated control

⁺ pO.001 vs. 0.5 mM (±)-alpha-lipoic acid

pO.001 vs. 0.3 mM (±)-alpha-lipoic acid

pO.001 vs. 4 μΜ sodium-selenite

&

p<0.001 vs. 3 μΜ sodium-selenite

^§ p<0.001 vs. 2 μΜ sodium-selenite

P values were computed with one-way analysis of variance (ANOVA), followed by Bonferroni-test. On the basis of our results presented in Table 2, LA applied per se in 2 mM, 1 mM, 0.5 mM concentrations significantly reduced the number of living cells compared to the untreated control, while LA applied per se in a concentration of 0.3 mM produced no significant decrease in the number of cells. Sodium-selenite applied per se in a concentration of 4 μΜ significantly reduced the number of living cells compared to the untreated control, while sodium-selenite applied per se in 3 μΜ or 2 μΜ concentrations did not cause a significant decrease in the number of cells. The combined application of various concentrations of the two compounds significantly enhanced the antitumor effect, resulting in a significantly lower number of living cells compared to the per se application of LA or sodium-selenite in the given concentration (see the symbols indicating significance in Table 2). On the basis of the CI values presented in Table 2 (0.30-0.50) there is strong synergism - synergism in the antitumor effect between LA and sodium-selenite at the investigated concentrations. In Figure 2 part "A" we present the dose-effect curves obtained with the two compounds applied per se and in combination. In Figure 2 part "B" we present the normalized isobologram obtained with various concentrations of the two compounds applied in combination (the definition of the normalized isobologram is given at example 1). The normalized isobologram shown in Figure 2 part "B" confirms that there is synergism in the antitumor effect between LA and sodium-selenite at the investigated concentrations. Example 3

Through example 3 presented in Figure 3 we show that the combined application of LA in a dose of 50 mg/kg and sodium-selenite in a dose of 1.5 mg/kg produces a synergistic in vivo antitumor effect on the C26 tumor model.

The in vivo experiment was performed with the C26 colon adenocarcinoma tumor model in Balb/c mice. Tumor fragments 3-4 mm in diameter and with an app. weight of 20 mg were transplanted subcutaneously into 20 Balb/c mice per group. The treatment was initiated on the first day after tumor transplantation. Solutions were administered intraperitoneally once a day for 10 consecutive days in a volume of 1 ml. Control mice were injected with saline. After the measurement of the dimensions of the tumor with a digital caliper the tumor volume (V) was calculated with the following formula: V = a ² x b x π/6 where„a" and„b" stand for the shortest and the longest diameter of the tumor, respectively.

On the 10 ^th day of treatment the animals were terminated and the weight of the excised tumors has been measured. The inhibition of tumor growth (TGI) was determined by comparing the values measured in the treatment groups to the values measured in the control group (TGI% = 100 - [value of the treated group/value of the control group] x 100). For statistical analysis normal distribution of the data was examined by Shapiro-Wilk test, equal variance was tested with Brown-Forsythe's test, and then one-way analysis of variance (ANOVA) followed by Bonferroni test was used for pairwise comparisons. P values below 0.05 were considered statistically significant.

On the basis of the results of the experiment neither LA applied per se nor the sodium- selenite applied per se produced a significant tumor inhibition compared to the control. In contrast to this the combined treatment caused a significant inhibition both in the tumor volumes measured during the experiment (Figure 3 "A") and in the tumor weight measured at the end of the experiment (Figure 3 "B"). TGI values calculated on the basis of tumor volumes were the following: 25.8%, 34.4%, 23.3% on the 6 ^th , 8 ^th and 10 ^th days of the treatment, respectively. p<0.001 vs. control, vs. LA applied per se, and vs. sodium-selenite applied per se at all of the three time points. The TGI calculated on the basis of tumor weight was 35.2%), p<0.05 vs. control and vs. LA applied per se, p<0.001 vs. sodium-selenite applied per se.

The result of the experiment provides in vivo confirmation of the in vitro demonstrated synergistic antitumor effect of LA and sodium-selenite.

Examples 4-5

Through examples 4-5 presented in Table 3 we show that both the R- and the S- enantiomers of alpha-lipoic acid applied at 0.5 mM, 0.4 mM, 0.3 mM concentrations in combination with 1 μΜ, 0.75 μΜ, 0.5 μΜ concentrations of sodium-selenite produce a synergistic antitumor effect on HELA cells.

The measurements were performed with the in vitro experimental system described at example 1. The results are presented as averages of values obtained from three independent experiments. The statistical analysis and calculation of the CI were performed as described at example 1.

The obtained results are the following: Table 3.

Enantiomers of alpha-lipoic acid applied individually at 0.5 mM, 0.4 mM, 0.3 mM concentrations in combination with 1 μΜ, 0.75 μΜ, 0.5 μΜ concentrations of sodium-selenite produce a synergistic antitumor effect on HELA cells.

Number of living cells

(±)-alpha- (R)-(+)-alpha- (S)-(-)-alpha- Sodium- expressed as % of

lipoic acid lipoic acid lipoic acid selenite Combination untreated cells

concentration concentration concentration concentration index (CI)

(average ± standard

(mM) (mM) (mM) (μΜ)

deviation)

2 - - - 30.30± 1.01 * -

1 - - - 63.62 ± 1.83 * -

0.5 - - - 83.01 ±2.03 * -

0.3 - - - 93.88 ±0.32 -

- 2 - - 29.45 ±3.93 * -

- 1 - - 49.19 ±2.23 * -

- 0.5 - - 58.51 ± 1.57 * -

- 0.3 - - 62.95 ±2.34 * -

- - 2 - 17.55 ±0.97 * -

- - 1 - 48.51 ±0.79* -

- - 0.5 - 58.34 ±2.91 * -

- - 0.3 - 64.84 ±2.36 * -

- - - 1 97.78 ±4.13 -

- - - 0.75 98.03 ± 1.99 -

- - - 0.5 99.38 ± 1.74 -

0.5 - - 1 26.45 ± 1.95 0.31

0.4 - - 1 26.13 ± 1.67 0.27

0.3 - - 1 29.11 ±3.52 0.24

0.5 - - 0.75 28.66 ± 1.32 *' ⁺ ' ^& 0.31

0.4 - - 0.75 29.32 ±2.30 *' ⁺ ' ^& 0.27

0.3 - - 0.75 33.11 ± 1.51 *' ^# ' ^& 0.24

0.5 - - 0.5 40.88 ±3.93 0.38

0.4 - - 0.5 37.93 ± 1.43 *' ⁺ ' ^§ 0.30

0.3 - - 0.5 42.13 ±3.18 0.26

Example 4

- 0.5 - 1 23.06 ± 1.80 0.21 - 0.4 - 1 23.62 ± 1.43 0.19

- 0.3 - 1 23.61 ± 1.76 0.17

- 0.5 - 0.75 30.56 ± 1.95 *' ⁺ ' ^& 0.30

- 0.4 - 0.75 29.54 ± 1.59 *' ⁺ ' ^& 0.24

- 0.3 - 0.75 30.20 ± 2.14 *' ^# ' ^& 0.20

- 0.5 - 0.5 39.43 ± 2.28 0.44

- 0.4 - 0.5 37.96 ± 4.69 0.34

- 0.3 - 0.5 36.14 ± 0.57 0.24

Example 5

- - 0.5 1 30.01 ± 0.41 0.46

- - 0.4 1 30.87 ± 0.66 0.40

- - 0.3 1 29.97 ± 0.28 0.32

- - 0.5 0.75 33.82 ± 0.54 *' ⁺ ' ^& 0.51

- - 0.4 0.75 34.81 ± 0.57 *' ⁺ ' ^& 0.44

- - 0.3 0.75 34.08 ± 1.04 *' ^# ' ^& 0.34

- - 0.5 0.5 42.44 ± 3.28 *' ⁺ ' ^§ 0.66

- - 0.4 0.5 44.62 ± 2.71 0.58

- - 0.3 0.5 42.03 ± 2.27 0.42

* pO.001 vs. untreated control

⁺ p<0.001 vs. 0.5 mM concentration of the alpha-lipoic acid enantiomer present in the given combination p<0.001 vs. 0.3 mM concentration of the alpha-lipoic acid enantiomer present in the given combination pO.001 vs. 1 μΜ sodium-selenite

&

pO.001 vs. 0.75 μΜ sodium-selenite

^§ pO.001 vs. 0.5 μΜ sodium-selenite

P values were computed with one-way analysis of variance (ANOVA), followed by Bonferroni-test.

On the basis of our results presented in Table 3, enantiomers of alpha-lipoic acid applied per se in 2 mM, 1 mM, 0.5 mM, 0.3 mM concentrations significantly reduced the number of living cells compared to the untreated control, while sodium-selenite applied per se in 1 μΜ, 0.75 μΜ, 0.5 μΜ concentrations did not cause a significant decrease in the number of cells. The combined application of various concentrations of alpha-lipoic acid enantiomers and sodium-selenite significantly enhanced the antitumor effect, resulting in a significantly lower number of living cells compared to the per se application of the given alpha-lipoic acid enantiomer or sodium-selenite in the given concentration (see the symbols indicating significance in Table 3). On the basis of the CI values presented in Table 3 (example 4, CI: 0.17-0.44 and example 5, CI: 0.32-0.66 for the R-, and S-enantiomers, respectively) there is strong synergism - synergism in the antitumor effect between either (R)-(+)-alpha-lipoic acid or (S)-(+)-alpha-lipoic acid and sodium-selenite.

Examples 6-10

Through examples 6-10 presented in Table 4 we show that 0.5 mM, 0.4 mM, 0.3 mM concentrations of LA applied in combination with selenium-containing active substances other than sodium-selenite do not produce a synergistic antitumor effect on HELA cells.

The measurements were performed with the in vitro experimental system described at example 1. The results are presented as averages of values obtained from three independent experiments. The statistical analysis and calculation of the CI were performed as described at example 1.

The obtained results are the following:

On the basis of our results presented in Table 4, LA applied per se in 2 mM, 1 mM, 0.5 mM concentrations significantly reduced the number of living cells compared to the untreated control, while LA applied per se in a concentration of 0.3 mM produced no significant decrease in the number of cells. The combined application of 0.5 mM, 0.4 mM, 0.3 mM concentrations of LA and various concentrations (0.5-4 μΜ) of selenium-compounds other than sodium-selenite did not produce a synergistic enhancement of the antitumor effect: sodium-selenate (example 6.), selenomethionine (example 7), selenocysteine (example 8), seleno-methyl-selenocysteine (example 9), methylseleninic acid (example 10). On the basis of the CI values presented in Table 4 selenium-compounds other than sodium-selenite antagonize the antitumor effect of LA. An exception to this is selenocysteine applied in concentrations of 2-4 μΜ, in case of which an additive antitumor effect was observed upon the combined application with LA.

Example 11

Through example 11 presented in Table 5 we show that 0.5 mM, 0.4 mM, 0.3 mM concentrations of LA applied in combination with 0.5-4 μΜ of sodium-sulfite do not produce a synergistic antitumor effect on HELA cells.

The measurements were performed with the in vitro experimental system described at example 1. The results are presented as averages of values obtained from three independent experiments. The statistical analysis and calculation of the CI were performed as described at example 1.

The obtained results are the following:

Table 5.

0.5 mM, 0.4 mM, 0.3 mM concentrations of (±)-alpha-lipoic acid applied in

combination with 0.5-4 μΜ sodium-sulfite do not produce a synergistic

antitumor effect on HELA cells

Example 11

Number of living

(±)-alpha-

Sodium-sulfite cells expressed as

lipoic acid Combination

concentration % of untreated cells

concentration index (CI)

(μΜ) (average ± standard

(mM)

deviation)

2 - 28.99 ± 0.60 ** -

1 - 63.37 ± 1.32 ** - 0.5 - 83.58 ± 0.85 ** -

0.3 - 94.16 ± 0.63 -

- 1 97.50 ± 2.04 -

- 0.75 97.33 ± 0.68 -

- 0.5 98.37 ± 1.27 -

0.5 1 89.42 ± 2.26 ** 1.34

0.4 1 94.19 ± 2.06 1.68

0.3 1 98.69 ± 0.11 5.41

0.5 0.75 85.10 ± 4.19 ** 1.04

0.4 0.75 90.84 ± 2.24 ** 1.18

0.3 0.75 98.03 ± 1.67 3.12

0.5 0.5 93.75 ± 2.07 * 1.79

0.4 0.5 95.66 ± 1.84 1.87

0.3 0.5 97.50 ± 1.82 2.22

- 4 96.29 ± 3.13 -

- 3 98.47 ± 1.53 -

- 2 98.51 ± 2.02 -

0.5 4 81.28 ± 1.06 ** 1.07

0.4 4 86.81 ± 0.35 ** 1.14

0.3 4 99.14 ± 0.48 5.69

0.5 3 82.17 ± 1.02 ** 1.05

0.4 3 86.65 ± 0.86 ** 1.06

0.3 3 97.77 ± 0.85 2.72

0.5 2 87.36 ± 1.88 ** 1.25

0.4 2 88.79 ± 1.97 ** 1.11

0.3 2 97.43 ± 1.56 2.20

* p<0.05 vs. untreated control

** pO.001 vs. untreated control

P values were computed with one-way analysis of variance (ANOVA),

followed by Bonferroni-test.

On the basis of our results presented in Table 5, LA applied per se in 2 mM, 1 mM, 0.5 mM, concentrations significantly reduced the number of living cells compared to the untreated control, while LA applied per se in a concentration of 0.3 mM produced no significant decrease in the number of cells. The combined application of 0.5 mM, 0.4 mM, 0.3 mM concentrations of LA and various concentrations (0.5-4 μΜ) of sodium-sulfite did not cause the synergistic enhancement of the antitumor effect. On the basis of the CI values presented in Table 5 (1.05-5.69) sodium-sulfite antagonizes the antitumor effect of LA. We have investigated the combination of LA and sodium-sulfite in order to exclude the possibility that a redox reaction between alpha-lipoic acid and the selenite anion taking place in the cell culture media would account for the synergism of alpha-lipoic acid and sodium-selenite. If this would be the case we should have observed the synergistic enhancement of the antitumor effect upon the combined application of LA and sodium-sulfite due to a redox reaction between the alpha-lipoic acid and the sulfite anion taking place in the cell culture media. On the basis of our results we have excluded the possibility that a redox reaction between alpha- lipoic acid and the selenite anion taking place in the cell culture media would account for the synergism of alpha-lipoic acid and sodium-selenite.

Examples 12-14

Through examples 12-14 presented in Table 6 we show that the combined application of free SH-group-containing compounds other than (±)-dihydrolipoic acid (the reduced form of alpha-lipoic acid), namely reduced L-glutathione and L-cysteine at concentrations of 0.5 mM, 0.4 mM, 0.3 mM and sodium-selenite do not produce a synergistic antitumor effect on HELA cells.

The measurements were performed with the in vitro experimental system described at example 1. The results are presented as averages of values obtained from three independent experiments. The statistical analysis and calculation of the CI were performed as described at example 1.

The obtained results are the following:

pO.OOl vs. the concentration of sodium-selenite applied in the given combination P values were computed with one-way analysis of variance (ANOVA), followed by Bonferroni-test.

On the basis of our results presented in Table 6 (±)-dihydrolipoic acid applied per se in 2 mM and 1 mM concentrations significantly reduced the number of living cells compared to the untreated control, while (±)-dihydrolipoic acid applied per se in concentrations of 0.3 mM and 0.5 mM and the per se applied reduced L-glutathione and L-cysteine produced no significant decrease in the number of cells. The combined application of various concentrations of (±)-dihydrolipoic acid and sodium-selenite significantly enhanced the antitumor effect, resulting in a significantly lower number of living cells compared to the per se application of (±)-dihydrolipoic acid or sodium-selenite in the given concentration (example 12). On the basis of the CI values presented in Table 6 at example 12 (0.33-0.55) there is synergism in the antitumor effect between (±)-dihydrolipoic acid and sodium-selenite at the investigated concentrations. In contrast to this the combined application of various concentrations of either reduced L-glutathione (example 13) or L-cysteine (example 14) and sodium-selenite produced no synergistic antitumor effect (CI: 1.00-3,41 and CI: 1.10-2,22 for examples 13 and 14, respectively). On the basis of these results the synergistic antitumor activity seen upon the combined application of alpha-lipoic acid and sodium-selenite is not caused by reactive oxygen species formed from sodium-selenite in the cell culture media triggered by free SH-groups formed during the incidental reduction of alpha-lipoic acid, since if this would be the case we should have obtained a synergistic antitumor effect upon the combination of free SH-group containing compounds other than LA (reduced L-glutathione and L-cysteine) with sodium-selenite.

Examples 15-17

Through examples 15-17 presented in Table 7 we show that 0.5 mM, 0.4 mM, 0.3 mM concentrations of LA applied in combination with 0.5-4 μΜ sodium-selenite produce a synergistic antitumor effect on MCF-7, PC-3 and CACO-2 cells.

The measurements were performed with the use of the in vitro experimental system described at example 1 on the MCF-7 human breast adenocarcinoma, on the PC-3 human prostate adenocarcinoma, and on the CACO-2 human colon adenocarcinoma cell lines. The results are presented as averages of values obtained from three independent experiments. The statistical analysis and calculation of the CI were performed as described at example 1.

The obtained results are the following:

0.4 0.5 41.81 ± 6.10 0.33 0.4 2 1.66 ± 0.72 0.03

0.3 0.5 38.46 ± 7.19 0.23 0.3 2 3.08 ± 0.52 0.04

* pO.001 vs. untreated control

⁺ pO.001 vs. 0.5 mM (±)-alpha-lipoic acid

^# p<0.001 vs. 0.3 mM (±)-alpha-lipoic acid

p<0.001 vs. 1 μΜ sodium-selenite

^& p<0.001 vs. 0.75 μΜ sodium-selenite

^ p<0.001 vs. 0.5 μΜ sodium-selenite

P values were computed with one-way analysis of variance (ANOVA). followed by Bonferroni-test.

On the basis of our results presented in Table 7, LA applied per se in 2 mM, 1 mM, 0.5 mM concentrations significantly reduced the number of living cells compared to the untreated control in all of the three cell lines. The combined application of various concentrations of LA and sodium-selenite significantly enhanced the antitumor effect in all of the three cell lines, resulting in a significantly lower number of living cells compared to the per se application of LA or sodium-selenite in the given concentration (see the symbols indicating significance in Table 7). On the basis of the CI values presented in Table 7 there is synergism - strong synergism in the antitumor effect between LA and sodium-selenite at the investigated concentrations on all of the three cell lines: MCF-7 (example 15, CI: 0.13-0.33), PC-3 (example 16, CI: 0.12-0.41), CACO-2 (example 17, CI: 0.01-0.46). On the basis of the above results the synergistic antitumor effect of LA and sodium-selenite has been verified on various cancer cell lines with different tissue origin, thus the synergistic antitumor effect can be considered general. Example 18

Through example 18 presented in Table 8 we show that L-tryptophan applied in 4.5 mM, 3 mM, 1.5 mM concentrations synergistically enhances the antitumor effect of the mixture of LA and sodium-selenite on HELA cells.

The measurements were performed with the in vitro experimental system described at example 1. The results are presented as averages of values obtained from three independent experiments. The statistical analysis and calculation of the CI were performed as described at example 1.

The obtained results are the following:

Table 8.

L-tryptophan synergistically enhances the antitumor effect of the mixture of (±)-alpha-lipoic acid and sodium- selenite on HELA cells

Example 18

Number of living cells

Sodium-

L-tryptophan expressed as % of

(±)-alpha-lipoic acid selenite Combination concentration untreated cells

concentration (mM) concentration index (CI)

(mM) (average ± standard

(μΜ)

deviation)

2 - - 33.71 ± 1.05 * -

1 - - 66.61 ± 2.28 * -

0.5 - - 84.34 ± 2.12 * - .3 - - 95.63 ± 1.30 -

- 6 - 61.04 ±2.24 * -

- 5 - 75.89 ± 1.14 * -

- 4 - 86.39 ±2.09 * -

- 3 - 98.59 ± 1.29 -

- - 4.5 13.88 ±0.31 * -

- - 3 21.91 ±0.98 * -

- - 1.5 62.40 ±0.23 * -.4 1 - 24.95 ± 1.85 * -.4 0.75 - 29.63 ± 1.10 * -.4 0.5 - 36.88 ±3.74 * -.4 0.25 - 58.78 ±2.74 * -.5 - 4.5 18.10 ±0.79 * 1.39.4 - 4.5 18.57 ±0.27 * 1.38.3 - 4.5 19.42 ± 1.27 * 1.38.5 - 3 28.04 ±0.69 * 1.28.4 - 3 29.37 ± 1.32 * 1.28.3 - 3 31.14 ± 0.35 * 1.28.5 - 1.5 46.82 ±0.87 * 1.11.4 - 1.5 47.42 ± 2.06 * 1.05.3 - 1.5 51.84 ±2.43 * 1.07

- 5 4.5 16.10 ±0.81 * 1.74

- 4 4.5 15.80 ±0.65 * 1.61

- 3 4.5 16.17 ±0.58 * 1.51

- 5 3 24.44 ± 0.27 * 1.62

- 4 3 25.21 ± 1.41 * 1.52

- 3 3 25.77 ±0.45 * 1.40

- 5 1.5 31.81 ± 1.21 * 1.27

- 4 1.5 34.78 ± 1.73 * 1.18

- 3 1.5 38.36 ±2.39 * 1.10.4 0.75 4.5 0.57 ±0.13 ^* · ⁺ · ^& 0.24.4 0.5 4.5 1.21 ±0.18 ^* · ⁺ · ^§ 0.35.4 0.25 4.5 3.39 ± 0.19 ^{* +} · ^$ 0.56.4 0.75 3 2.25 ±0.52 ^* · · ^& 0.35.4 0.5 3 4.61 ±1.30 ^* · ^# · ^§ 0.49.4 0.25 3 11.33 ±0.79 ^* · ^$ 0.74.4 0.75 1.5 11.98 ± 0.51 ^* ' ^& 0.67.4 0.5 1.5 16.67 ± 1.28 ^* " 0.72.4 0.25 1.5 27.78 ± 1.48 ^{* I>$} 0.83 * pO.001 vs. untreated control

⁺ pO.001 vs. 4.5 mM L-tryptophan

p<0.001 vs. 3 mM L-tryptophan

* p<0.001 vs. 1.5 mM L-tryptophan

&

pO.001 vs. the mixture of 0.4 mM (±)-alpha-lipoic acid and 0.75 μΜ sodium-selenite

p<0.001 vs. the mixture of 0.4 mM (±)-alpha-lipoic acid and 0.5 μΜ sodium-selenite

$

p<0.001 vs. the mixture of 0.4 mM (±)-alpha-lipoic acid and 0.25 μΜ sodium-selenite

P values were computed with one-way analysis of variance (ANOVA), followed by Bonferroni-test.

On the basis of our results presented in Table 8, L-tryptophan applied per se in 4.5 mM, 3 mM, 1.5 mM concentrations significantly reduced the number of living cells compared to the untreated control. The combined application of L-tryptophan with the mixture of LA and sodium-selenite significantly enhanced the antitumor effect, which resulted in that the number of living cells after the combined application of the three compounds was significantly lower than after the per se application of L-tryptophan or after the per se application of the mixture of LA and sodium-selenite in the given concentrations (see the symbols indicating significance in Table 8). On the basis of the CI values presented in Table 8 (0.24-0.83) there is moderate synergism - strong synergism in the antitumor effect between L- tryptophan and the LA-sodium-selenite mixture at the investigated concentrations. In contrast to this the combination of L-tryptophan with LA or sodium-selenite did not produce a synergistic enhancement of the antitumor effect, but on the contrary - on the basis of the CI values presented in Table 8 - slight antagonism could be observed (CI for the L-tryptophan- LA combination: 1.05-1.39; CI for the L-tryptophan-sodium-selenite combination: 1.10-1.74). In summary of these results L-tryptophan synergistically enhances the antitumor effect of the mixture of LA and sodium-selenite, and the simultaneous presence of both LA and sodium- selenite is necessary for the occurrence of this synergism. Example 19

Through example 19 presented in Table 9 we show that D-tryptophan applied in 4.5 mM, 3 mM, 1.5 mM concentrations synergistically enhances the antitumor effect of the mixture of LA and sodium-selenite on HELA cells.

The measurements were performed with the in vitro experimental system described at example 1. The results are presented as averages of values obtained from three independent experiments. The statistical analysis and calculation of the CI were performed as described at example 1.

The obtained results are the following:

Table 9.

D-tryptophan synergistically enhances the antitumor effect of the mixture of (±)-alpha-lipoic acid and sodium- selenite on HELA cells

Example 19

Number of living cells

Sodium-

D-tryptophan expressed as % of

(±)-alpha-lipoic acid selenite Combination concentration untreated cells

concentration (niM) concentration index (CI)

(niM) (average ± standard

(μΜ)

deviation)

2 - - 33.71 ± 1.05 * -

1 - - 66.61 ± 2.28 * -

0.5 - - 84.34 ± 2.12 * -

0.3 - - 95.63 ± 1.30 -

- 6 - 61.04 ± 2.24 * -

- 5 - 75.89 ± 1.14 * -

- 4 - 86.39 ± 2.09 * -

- 3 - 98.59 ± 1.29 -

- - 4.5 28.99 ± 0.74 * -

- - 3 48.90 ± 0.32 * -

- - 1.5 69.09 ± 3.31 * -

0.4 1 - 24.95 ± 1.85 * -

0.4 0.75 - 29.63 ± 1.10 * -

0.4 0.5 - 36.88 ± 3.74 * -

0.4 0.25 - 58.78 ± 2.74 * -

0.5 - 4.5 25.33 ± 1.38 * 1.04

0.4 - 4.5 31.81 ± 1.50 * 1.22

0.3 - 4.5 30.18 ± 1.97 * 1.12

0.5 - 3 38.33 ± 3.80 * 1.11

0.4 - 3 43.41 ± 3.52 * 1.20

0.3 - 3 38.92 ± 3.59 * 1.01

0.5 - 1.5 59.81 ± 4.92 * 1.18

0.4 - 1.5 70.21 ± 2.81 * 1.45

0.3 - 1.5 72.96 ± 3.91 * 1.16

- 5 4.5 28.47 ± 1.55 * 1.60

- 4 4.5 28.34 ± 2.03 * 1.46

- 3 4.5 28.34 ± 1.16 * 1.33 - 5 3 45.23 ± 1.02 * 1.77

- 4 3 48.62 ±0.20 * 1.73

- 3 3 49.96 ± 1.90 * 1.61

- 5 1.5 55.43 ± 2.95 * 1.49

- 4 1.5 66.25 ± 1.74 * 1.61

- 3 1.5 67.04 ± 1.61 * 1.46

0.4 0.75 4.5 0.57 ± 0.19 ^* · ⁺ · ^& 0.07

0.4 0.5 4.5 2.78 ± 0.35 ^* · ⁺ · ^§ 0.21

0.4 0.25 4.5 8.00 ± 0.84 ^{* +} · ^$ 0.41

0.4 0.75 3 7.06 ± 1.02 ^* · · ^& 0.40

0.4 0.5 3 14.24 ± 1.07 ^* · · ^§ 0.62

0.4 0.25 3 24.15 ± 0.99 ^* · ^$ 0.79

0.4 0.75 1.5 20.13 ± 1.36 0.84

0.4 0.5 1.5 24.99 ± 2.04 ^{* 1 §} 0.80

0.4 0.25 1.5 32.40 ± 1.64 ^{* I> $} 0.72

* ρθ.001 vs. untreated control

+ ρθ.001 vs. 4.5 mM D-tryptophan

pO.001 vs. 3 mM D-tryptophan

i pO.001 vs. 1.5 mM D-tryptophan

&

pO.001 vs. the mixture of 0.4 mM (±)-alpha-lipoic acid and 0.75 μΜ sodium-selenite

p<0.001 vs. the mixture of 0.4 mM (±)-alpha-lipoic acid and 0.5 μΜ sodium-selenite

$

p<0.001 vs. the mixture of 0.4 mM (±)-alpha-lipoic acid and 0.25 μΜ sodium-selenite

P values were computed with one-way analysis of variance (ANOVA), followed by Bonferroni-test.

On the basis of our results presented in Table 9 D-tryptophan applied per se in 4.5 mM, 3 mM, 1.5 mM concentrations significantly reduced the number of living cells compared to the untreated control. The combined application of D-tryptophan with the mixture of LA and sodium-selenite significantly enhanced the antitumor effect, which resulted in that the number of living cells after the combined application of the three compounds was significantly lower than after the per se application of D-tryptophan or after the per se application of the mixture of LA and sodium-selenite in the given concentrations (see the symbols indicating significance in Table 9). On the basis of the CI values presented in Table 9 (0.07-0.84) there is moderate synergism - strong synergism in the antitumor effect between D-tryptophan and the LA-sodium-selenite mixture at the investigated concentrations. In contrast to this the combination of D-tryptophan with LA or sodium-selenite did not produce a synergistic enhancement of the antitumor effect, but on the contrary - on the basis of the CI values presented in Table 9 - moderate antagonism could be observed (CI for the D- tryptophan-LA combination: 1.01-1.45; CI for the D-tryptophan-sodium-selenite combination: 1.33-1.77). In summary of these results D-tryptophan synergistically enhances the antitumor effect of the mixture of LA and sodium-selenite, and the simultaneous presence of both LA and sodium-selenite is necessary for the occurrence of this synergism.

Example 20

Through example 20 presented in Table 10 we show that L-methionine applied in 4.5 mM, 3 mM, 1.5 mM concentrations synergistically enhances the antitumor effect of the mixture of LA and sodium-selenite on HELA cells.

The measurements were performed with the in vitro experimental system described at example 1. The results are presented as averages of values obtained from three independent experiments. The statistical analysis and calculation of the CI were performed as described at example 1.

The obtained results are the following:

Table 10.

L-methionine synergistically enhances the antitumor effect of the mixture of (±)-alpha-lipoic acid and sodium- selenite on HELA cells

Example 20

Number of living cells

Sodium-

L-methionine expressed as % of

(±)-alpha-lipoic acid selenite Combination concentration untreated cells

concentration (mM) concentration index (CI)

(mM) (average ± standard

(μΜ)

deviation)

2 - - 33.71 ± 1.05 ** -

1 - - 66.61 ± 2.28 ** -

0.5 - - 84.34 ± 2.12 ** -

0.3 - - 95.63 ± 1.30 -

- 6 - 61.04 ± 2.24 ** -

- 5 - 75.89 ± 1.14 ** -

- 4 - 86.39 ± 2.09 * -

- 3 - 98.59 ± 1.29 -

- - 4.5 95.91 ± 3.05 -

- - 3 96.02 ± 5.27 -

- - 1.5 98.57 ± 1.38 - 0.4 1 - 24.95 ± 1.85 ** -

0.4 0.75 - 29.63 ± 1.10** -

0.4 0.5 - 36.88 ±3.74** -

0.4 0.25 - 58.78 ±2.74 ** -

0.5 - 4.5 90.65 ± 4.95 1.68

0.4 - 4.5 96.17 ±3.56 2.80

0.3 - 4.5 92.66 ± 4.42 1.46

0.5 - 3 89.95 ±3.35 1.44

0.4 - 3 97.52 ±2.88 3.25

0.3 - 3 90.53 ±4.68 1.03

0.5 - 1.5 87.38 ±3.18 * 1.11

0.4 - 1.5 89.98 ±2.67 1.07

0.3 - 1.5 87.87 ±2.45 * 1.05

- 5 4.5 75.58 ± 1.00 ** 1.16

- 4 4.5 88.88 ±3.81 * 1.36

- 3 4.5 92.78 ±5.52 1.42

- 5 3 68.96 ±0.97 ** 1.01

- 4 3 84.92 ±2.19 * 1.08

- 3 3 91.28 ±2.10 1.10

- 5 1.5 74.47 ±3.50** 1.03

- 4 1.5 91.43 ±7.15 1.18

- 3 1.5 93.94 ± 1.23 1.06

0.4 0.75 4.5 17.42 ± 1.21 ^™ ' ⁺ ' ^& 0.53

0.4 0.5 4.5 24.82 ±0.79 0.54

0.4 0.25 4.5 41.20 ± 5.19 ^{** +} ' ^$ 0.58

0.4 0.75 3 19.25 ± 1.18 " ' ^& 0.59

0.4 0.5 3 25.83 ±2.55 " 0.57

0.4 0.25 3 40.82 ±2.08 · '* 0.55

0.4 0.75 1.5 18.47 ±0.79 " ^l& 0.56

0.4 0.5 1.5 27.16 ±4.27 " 0.60

0.4 0.25 1.5 43.21 ±2.50 " ^l$ 0.59

* p<0.05 vs. untreated control

** pO.001 vs. untreated control

⁺ pO.001 vs.4.5 mM L-methionine

pO.001 vs.3 mM L-methionine

pO.001 vs.1.5 mM L-methionine

On the basis of our results presented in Table 10 L-methionine applied per se in 4.5 mM, 3 mM, 1.5 mM concentrations did not cause a significant decrease in the number of living cells. The combined application of L-methionine with the mixture of LA and sodium- selenite significantly enhanced the antitumor effect, which resulted in that the number of living cells after the combined application of the three compounds was significantly lower than after the per se application of L-methionine or after the per se application of the mixture of LA and sodium-selenite in the given concentrations (see the symbols indicating significance in Table 10). On the basis of the CI values presented in Table 10 (0.53-0.60) there is synergism in the antitumor effect between L-methionine and the LA-sodium-selenite mixture at the investigated concentrations. In contrast to this the combination of L-methionine with LA or sodium-selenite did not produce a synergistic enhancement of the antitumor effect, but on the contrary - on the basis of the CI values presented in Table 10 - antagonism could be observed (CI for the L-methionine-LA combination: 1.03-3.25; CI for the L- methionine-sodium-selenite combination: 1.01-1.42). In summary of these results L- methionine synergistically enhances the antitumor effect of the mixture of LA and sodium- selenite, and the simultaneous presence of both LA and sodium-selenite is necessary for the occurrence of this synergism. Example 21

Through example 21 presented in Table 11 we show that D-phenylalanine applied in 4.5 mM, 3 mM, 1.5 mM concentrations synergistically enhances the antitumor effect of the mixture of LA and sodium-selenite on HELA cells.

The measurements were performed with the in vitro experimental system described at example 1. The results are presented as averages of values obtained from three independent experiments. The statistical analysis and calculation of the CI were performed as described at example 1.

The obtained results are the following: Table 11.

D-phenylalanine synergistically enhances the antitumor effect of the mixture of (±)-alpha-lipoic acid and sodium-selenite on HELA cells

Example 21

Number of living cells

Sodium-

(±)-alpha-lipoic D-phenylalanine expressed as % of

selenite Combination acid concentration concentration untreated cells

concentration index (CI) (mM) (mM) (average ± standard

(μΜ)

deviation)

2 - - 33.71 ± 1.05 ** -

1 - - 66.61 ±2.28** -

0.5 - - 84.34 ±2.12 ** -

0.3 - - 95.63 ± 1.30 -

- 6 - 61.04 ±2.24 ** -

- 5 - 75.89 ± 1.14 ** -

- 4 - 86.39 ±2.09 ** -

- 3 - 98.59 ± 1.29 -

- - 4.5 88.88 ±3.64 * -

- - 3 97.45 ± 2.57 -

- - 1.5 98.45 ±0.71 -

0.4 1 - 24.95 ± 1.85 ** -

0.4 0.75 - 29.63 ± 1.10** -

0.4 0.5 - 36.88 ±3.74** -

0.4 0.25 - 58.78 ±2.74 ** -

0.5 - 4.5 85.10 ±5.36** 1.59

0.4 - 4.5 87.84 ±3.58 * 1.60

0.3 - 4.5 92.62 ±5.86 1.89

0.5 - 3 93.36 ±2.85 2.20

0.4 - 3 95.91 ±2.87 2.53

0.3 - 3 99.63 ± 1.12 8.13

0.5 - 1.5 97.49 ± 6.26 3.13

0.4 - 1.5 98.18 ±0.88 3.15

0.3 - 1.5 99.82 ± 1.69 8.99

- 5 4.5 70.32 ±4.84 ** 1.36

- 4 4.5 83.14 ± 1.41 ** 1.49

- 3 4.5 87.99 ±1.94** 1.49

- 5 3 83.36 ±3.62** 1.51

- 4 3 97.08 ±2.08 2.47

- 3 3 97.25 ± 1.73 2.22 - 5 1.5 86.88 ± 5.04 ** 1.39

- 4 1.5 96.72 ± 2.47 1.79

- 3 1.5 99.20 ± 1.90 2.49

0.4 0.75 4.5 17.28 ± 1.37 " ⁺ ' ^&& 0.62

0.4 0.5 4.5 23.06 ± 1.73 ^{** +} - ^ss 0.61

0.4 0.25 4.5 33.05 ± 2.28 ^** ' ⁺ ' * 0.56

0.4 0.75 3 18.45 ± 1.02 " · ^& 0.62

0.4 0.5 3 27.24 ± 2.01 " ^J 0.69

0.4 0.25 3 40.84 ± 3.30 ^** ' ^# ' 0.67

0.4 0.75 1.5 17.62 i l.l l ^{** 1} · ** 0.56

0.4 0.5 1.5 23.47 ± 1.97 " ^§ 0.53

0.4 0.25 1.5 42.84 ± 5.13 " *· * 0.64

* p<0.05 vs. untreated control

** pO.001 vs. untreated control

⁺ p<0.001 vs. 4.5 mM D-phenylalanine

pO.001 vs. 3 mM D-phenylalanine

^ ^■ pO.001 vs. 1.5 mM D-phenylalanine

&

p<0.05 vs. the mixture of 0.4 mM (±)-alpha-lipoic acid and 0.75 μΜ sodium-selenite

&&

p<0.001 vs. the mixture of 0.4 mM (±)-alpha-lipoic acid and 0.75 μΜ sodium-selenite

p<0.05 vs. the mixture of 0.4 mM (±)-alpha-lipoic acid and 0.5 μΜ sodium-selenite

^ pO.001 vs. the mixture of 0.4 mM (±)-alpha-lipoic acid and 0.5 μΜ sodium-selenite

$

p<0.001 vs. the mixture of 0.4 mM (±)-alpha-lipoic acid and 0.25 μΜ sodium-selenite

P values were computed with one-way analysis of variance (ANOVA), followed by Bonferroni-test.

On the basis of our results presented in Table 11 D-phenylalanine applied per se in 4.5 mM concentration significantly reduced the number of living cells compared to the untreated control, while D-phenylalanine applied per se in 3 mM and 1.5 mM concentrations did not cause a significant decrease in the number of cells. The combined application of D- phenylalanine with the mixture of LA and sodium-selenite significantly enhanced the antitumor effect, which resulted in that the number of living cells after the combined application of the three compounds was significantly lower than after the per se application of D-phenylalanine or after the per se application of the mixture of LA and sodium-selenite in the given concentrations (see the symbols indicating significance in Table 11). On the basis of the CI values presented in Table 11 (0.53-0.69) there is synergism in the antitumor effect between D-phenylalanine and the LA-sodium-selenite mixture at the investigated concentrations. In contrast to this the combination of D-phenylalanine with LA or sodium- selenite did not produce a synergistic enhancement of the antitumor effect, but on the contrary - on the basis of the CI values presented in Table 11 - antagonism could be observed (CI for the D-phenylalanine-LA combination: 1.59-8.99; CI for the D-phenylalanine-sodium-selenite combination: 1.36-2.49). In summary of these results D-phenylalanine synergistically enhances the antitumor effect of the mixture of LA and sodium-selenite, and the simultaneous presence of both LA and sodium-selenite is necessary for the occurrence of this synergism.

Example 22

Through example 22 presented in Table 12 we show that agmatine-sulfate applied in 0.4 mM, 0.3 mM, 0.2 mM concentrations synergistically enhances the antitumor effect of the mixture of LA and sodium-selenite on HELA cells.

The measurements were performed with the in vitro experimental system described at example 1. The results are presented as averages of values obtained from three independent experiments. The statistical analysis and calculation of the CI were performed as described at example 1.

The obtained results are the following:

Table 12.

Agmatine-sulfate synergistically enhances the antitumor effect of the mixture of (±)-alpha-lipoic acid and sodium-selenite on HELA cells

Example 22

Number of living cells

Sodium-

(±)-alpha-lipoic Agmatine-sulfate expressed as % of

selenite Combination acid concentration concentration untreated cells

concentration index (CI) (mM) (mM) (average ± standard

(μΜ)

deviation)

2 - - 33.71 ± 1.05 ** -

1 - - 66.61 ± 2.28 ** -

0.5 - - 84.34 ± 2.12 ** -

0.3 - - 95.63 ± 1.30 -

- 6 - 61.04 ± 2.24 ** -

- 5 - 75.89 ± 1.14 ** -

- 4 - 86.39 ± 2.09 ** -

- 3 - 98.59 ± 1.29 - - - 0.4 66.96 ±0.73 ** -

- - 0.3 76.47 ± 1.06 ** -

- - 0.2 86.72 ±0.33 ** -

0.4 1 - 24.95 ± 1.85 ** -

0.4 0.75 - 29.63 ± 1.10** -

0.4 0.5 - 36.88 ±3.74** -

0.4 0.25 - 58.78 ±2.74 ** -

0.5 - 0.4 70.61 ± 1.18** 1.73

0.4 - 0.4 70.89 ±5.36** 1.63

0.3 - 0.4 75.42 ±3.71 ** 1.85

0.5 - 0.3 81.54 ±2.09 ** 2.16

0.4 - 0.3 78.79 ± 3.82 ** 1.74

0.3 - 0.3 82.56 ±4.15 ** 1.95

0.5 - 0.2 88.90 ± 1.78 * 2.28

0.4 - 0.2 88.58 ±6.38 * 2.01

0.3 - 0.2 90.66 ±2.99 2.15

- 5 0.4 53.67 ±3.75 ** 1.41

- 4 0.4 60.32 ±4.29 ** 1.44

- 3 0.4 54.66 ±8.67** 1.10

- 5 0.3 64.62 ± 1.02 ** 1.49

- 4 0.3 67.97 ±6.75 ** 1.42

- 3 0.3 58.26 ±2.94 ** 1.05

- 5 0.2 78.14 ±7.61 ** 1.58

- 4 0.2 87.40 ±6.51 * 1.97

- 3 0.2 81.20 ±4.62 ** 1.30

0.4 0.75 0.4 19.69 ±0.16 **. ⁺ · ^&& 0.73

0.4 0.5 0.4 24.95 ±0.60 **. ⁺ · ^§§ 0.71

0.4 0.25 0.4 38.08 ±2.27 **' ⁺ ' ^$ 0.79

0.4 0.75 0.3 22.79 ±0.61 **·*'* 0.82

0.4 0.5 0.3 30.28 ±0.23 **' ^# ' 0.83

0.4 0.25 0.3 49.66 ±0.92 **·*'* 0.82

0.4 0.75 0.2 22.30 ± 1.25 **' ^t& 0.74

0.4 0.5 0.2 30.97 ± 1.67 **· ^§ 0.78

0.4 0.25 0.2 50.51 ±0.86 **· ^t$ 0.78

* p<0.05 vs. untreated control

** pO.001 vs. untreated control

⁺ p<0.001 vs.0.4 mM agmatine-sulfate

On the basis of our results presented in Table 12, agmatine-sulfate applied per se in 0.4 mM. 0.3 mM, 0.2 mM concentrations significantly reduced the number of living cells compared to the untreated control. The combined application of agmatine-sulfate with the mixture of LA and sodium-selenite significantly enhanced the antitumor effect, which resulted in that the number of living cells after the combined application of the three compounds was significantly lower than after the per se application of agmatine-sulfate or after the per se application of the mixture of LA and sodium-selenite in the given concentrations (see the symbols indicating significance in Table 12). On the basis of the CI values presented in Table 12 (0.71-0.83) there is synergism in the antitumor effect between agmatine-sulfate and the LA-sodium-selenite mixture at the investigated concentrations. In contrast to this the combination of agmatine-sulfate with LA or sodium-selenite did not produce a synergistic enhancement of the antitumor effect, but on the contrary - on the basis of the CI values presented in Table 12 - antagonism could be observed (CI for the agmatine- sulfate -LA combination: 1.63-2.28; CI for the agmatine-sulfate-sodium-selenite combination: 1.05-1.97). In summary of these results agmatine-sulfate synergistically enhances the antitumor effect of the mixture of LA and sodium-selenite, and the simultaneous presence of both LA and sodium-selenite is necessary for the occurrence of this synergism. Example 23

Through example 23 presented in Table 13 we show that sodium -phenylpyruvate applied in 5 mM, 3.75 mM, 2.5 mM concentrations synergistically enhances the antitumor effect of the mixture of LA and sodium-selenite on HELA cells.

The measurements were performed with the in vitro experimental system described at example 1. The results are presented as averages of values obtained from three independent experiments. The statistical analysis and calculation of the CI were performed as described at example 1.

The obtained results are the following:

Table 13.

Sodium-phenylpyruvate synergistically enhances the antitumor effect of the mixture of (±)-alpha-lipoic acid and sodium-selenite on HELA cells

Example 23

Number of living cells

Sodium-

(±)-alpha-lipoic Sodium- expressed as % of

selenite Combination acid concentration phenylpyruvate untreated cells

concentration index (CI) (niM) concentration (mM) (average ± standard

(μΜ)

deviation)

2 - - 33.71 ± 1.05 ** -

1 - - 66.61 ± 2.28 ** -

0.5 - - 84.34 ± 2.12 * -

0.3 - - 95.63 ± 1.30 -

- 6 - 61.04 ± 2.24 ** -

- 5 - 75.89 ± 1.14 ** -

- 4 - 86.39 ± 2.09 * -

- 3 - 98.59 ± 1.29 -

- - 5 55.53 ± 6.54 ** -

- - 3.75 74.49 ± 3.06 ** -

- - 2.5 93.23 ±4.31 -

0.4 1 - 24.95 ± 1.85 ** -

0.4 0.75 - 29.63 ± 1.10 ** -

0.4 0.5 - 36.88 ± 3.74 ** -

0.4 0.25 - 58.78 ± 2.74 ** -

0.5 - 5 70.83 ± 8.26 ** 1.92

0.4 - 5 83.22 ± 4.37 * 2.56

0.3 - 5 84.49 ± 3.88 * 2.50

0.5 - 3.75 80.33 ± 6.93 ** 1.91

0.4 - 3.75 87.96 ± 1.33 2.32

0.3 - 3.75 86.64 ± 2.40 * 2.00

0.5 - 2.5 94.30 ± 4.31 2.66

0.4 - 2.5 96.48 ± 2.25 3.01

0.3 - 2.5 97.85 ± 1.34 3.33

- 5 5 27.15 ± 3.06 ** 1.25 - 4 5 39.00 ± 3.17 ** 1.33

- 3 5 43.31 ± 9.14 ** 1.26

- 5 3.75 30.42 ± 1.42 ** 1.10

- 4 3.75 33.68 ± 3.45 ** 1.01

- 3 3.75 41.62 ± 1.07 ** 0.97

- 5 2.5 38.19 ± 3.53 ** 0.98

- 4 2.5 61.04 ± 2.02 ** 1.07

- 3 2.5 70.51 ± 2.71 ** 1.02

0.4 0.75 5 12.31 ± 0.92 **. ⁺ . ^&& 0.72

0.4 0.5 5 18.87 ± 0.99 **. ⁺ · ^§§ 0.84

0.4 0.25 5 27.35 ± 2.47 **' ⁺ ' ^$ 0.88

0.4 0.75 3.75 17.78 ± 1.09 **· *' * 0.83

0.4 0.5 3.75 23.84 ± 1.13 **. ^# · ^§§ 0.86

0.4 0.25 3.75 33.96 ± 2.90 **· *' * 0.85

0.4 0.75 2.5 18.35 ± 1.04 **· ^& 0.70

0.4 0.5 2.5 26.48 ± 3.37 **· ^§ 0.76

0.4 0.25 2.5 36.24 ± 1.74 **· ^{t $} 0.67

* p<0.05 vs. untreated control

** pO.001 vs. untreated control

⁺ p<0.001 vs. 5 mM sodium-phenylpyruvate

pO.001 vs. 3.75 mM sodium-phenylpyruvate

p<0.001 vs. 2.5 mM sodium-phenylpyruvate

&

p<0.05 vs. the mixture of 0.4 mM (±)-alpha-lipoic acid and 0.75 μΜ sodium-selenite

&&

p<0.001 vs. the mixture of 0.4 mM (±)-alpha-lipoic acid and 0.75 μΜ sodium-selenite

p<0.05 vs. the mixture of 0.4 mM (±)-alpha-lipoic acid and 0.5 μΜ sodium-selenite

^ p<0.001 vs. the mixture of 0.4 mM (±)-alpha-lipoic acid and 0.5 μΜ sodium-selenite

$

p<0.001 vs. the mixture of 0.4 mM (±)-alpha-lipoic acid and 0.25 μΜ sodium-selenite

P values were computed with one-way analysis of variance (ANOVA), followed by Bonferroni-test.

On the basis of our results presented in Table 13 sodium-phenylpyruvate applied per se in 5 mM and 3.75 mM concentrations significantly reduced the number of living cells compared to the untreated control, while sodium-phenylpyruvate applied per se in 2.5 mM concentration did not cause a significant decrease in the number of cells. The combined application of sodium-phenylpyruvate with the mixture of LA and sodium-selenite significantly enhanced the antitumor effect, which resulted in that the number of living cells after the combined application of the three compounds was significantly lower than after the per se application of sodium-phenylpyruvate or after the per se application of the mixture of LA and sodium-selenite in the given concentrations (see the symbols indicating significance in Table 13). On the basis of the CI values presented in Table 13 (0.67-0.88) there is slight synergism - synergism in the antitumor effect between sodium-phenylpyruvate and the LA- sodium-selenite mixture at the investigated concentrations. In contrast to this the combination of sodium-phenylpyruvate with LA or sodium-selenite did not produce a synergistic enhancement of the antitumor effect, but on the contrary - on the basis of the CI values presented in Table 13 - antagonism and additive effect could be observed (CI for the sodium- phenylpyruvate-LA combination: 1.91-3.33; CI for the sodium-phenylpyruvate-sodium- selenite combination: 0.97-1.33). In summary of these results sodium-phenylpyruvate synergistically enhances the antitumor effect of the mixture of LA and sodium-selenite, and the simultaneous presence of both LA and sodium-selenite is necessary for the occurrence of this synergism.

Example 24

Through example 24 presented in Table 14 we show that sodium -genti sate applied in 4.5 mM, 3 mM, 1.5 mM concentrations synergistically enhances the antitumor effect of the mixture of LA and sodium-selenite on HELA cells.

The measurements were performed with the in vitro experimental system described at example 1. The results are presented as averages of values obtained from three independent experiments. The statistical analysis and calculation of the CI were performed as described at example 1.

The obtained results are the following:

Table 14.

Sodium-gentisate synergistically enhances the antitumor effect of the mixture of (±)-alpha-lipoic acid and sodium-selenite on HELA cells

Example 24

Number of living cells

Sodium-

(±)-alpha-lipoic Sodium-gentisate expressed as % of

selenite Combination acid concentration concentration untreated cells

concentration index (CI) (mM) (mM) (average ± standard

(μΜ)

deviation)

2 - - 33.71 ± 1.05 ** - 1 - - 66.61 ±2.28** -.5 - - 84.34 ±2.12 ** -.3 - - 95.63 ± 1.30 -

- 6 - 61.04 ±2.24 ** -

- 5 - 75.89 ± 1.14 ** -

- 4 - 86.39 ±2.09 ** -

- 3 - 98.59 ± 1.29 -

- - 4.5 81.47 ±2.81 ** -

- - 3 93.24 ± 1.99 -

- - 1.5 97.16 ± 1.08 -.4 1 - 24.95 ± 1.85 ** -.4 0.75 - 29.63 ± 1.10** -.4 0.5 - 36.88 ±3.74** -.4 0.25 - 58.78 ±2.74 ** -.5 - 4.5 84.01 ±4.20** 1.87.4 - 4.5 90.90 ±4.05 2.41.3 - 4.5 83.75 ± 1.32 ** 1.51.5 - 3 94.66 ±6.13 2.95.4 - 3 94.46 ± 0.49 2.57.3 - 3 87.45 ± 5.66 * 1.39.5 - 1.5 96.89 ± 1.65 3.09.4 - 1.5 98.39 ± 1.32 3.80.3 - 1.5 96.40 ± 1.20 2.04

- 5 4.5 84.02 ±5.76** 2.10

- 4 4.5 86.75 ± 6.72 ** 2.05

- 3 4.5 95.85 ±2.71 3.16

- 5 3 76.11 ± 1.18** 1.51

- 4 3 97.19 ±2.33 3.15

- 3 3 98.44 ±0.93 3.71

- 5 1.5 93.03 ±2.14 1.86

- 4 1.5 97.53 ±0.79 2.30

- 3 1.5 99.28 ± 1.20 3.26.4 0.75 4.5 19.45 ±2.28 **· ⁺ ' ^& 0.77.4 0.5 4.5 25.90 ±3.54 **. ⁺ · ^§§ 0.78.4 0.25 4.5 40.53 ±2.99 **· ⁺ ' ^$ 0.85.4 0.75 3 21.59 ± 1.42 **·*'* 0.80.4 0.5 3 26.28 ± 1.09 **·*' 0.72.4 0.25 3 39.18 ±4.04 **·*'* 0.71 0.4 0.75 1.5 21.46 ± 1.22 **· ^& 0.72

0.4 0.5 1.5 25.02 ± 0.72 **· ^t 0.60

0.4 0.25 1.5 43.78 ± 1.44 **· ^{t $} 0.71

* p<0.05 vs. untreated control

** pO.001 vs. untreated control

⁺ pO.001 vs. 4.5 mM sodium-gentisate

p<0.001 vs. 3 mM sodium-gentisate

* p<0.001 vs. 1.5 mM sodium-gentisate

&

p<0.05 vs. the mixture of 0.4 mM (±)-alpha-lipoic acid and 0.75 μΜ sodium-selenite

p<0.05 vs. the mixture of 0.4 mM (±)-alpha-lipoic acid and 0.5 μΜ sodium-selenite

^ p<0.001 vs. the mixture of 0.4 mM (±)-alpha-lipoic acid and 0.5 μΜ sodium-selenite

$

p<0.001 vs. the mixture of 0.4 mM (±)-alpha-lipoic acid and 0.25 μΜ sodium-selenite

P values were computed with one-way analysis of variance (ANOVA), followed by Bonferroni-test.

On the basis of our results presented in Table 14 sodium-gentisate applied per se in 4.5 mM concentration significantly reduced the number of living cells compared to the untreated control, while sodium-gentisate applied per se in 3 mM and 1.5 mM concentrations did not cause a significant decrease in the number of cells. The combined application of sodium- gentisate with the mixture of LA and sodium-selenite significantly enhanced the antitumor effect, which resulted in that the number of living cells after the combined application of the three compounds was significantly lower than after the per se application of sodium-gentisate or after the per se application of the mixture of LA and sodium-selenite in the given concentrations (see the symbols indicating significance in Table 14). On the basis of the CI values presented in Table 14 (0.60-0.85) there is moderate synergism - synergism in the antitumor effect between sodium-gentisate and the LA-sodium-selenite mixture at the investigated concentrations. In contrast to this the combination of sodium-gentisate with LA or sodium-selenite did not produce a synergistic enhancement of the antitumor effect, but on the contrary - on the basis of the CI values presented in Table 14 - antagonism could be observed (CI for the sodium-gentisate -LA combination: 1.51-3.80; CI for the sodium- gentisate-sodium-selenite combination: 1.51-3.71). In summary of these results sodium- gentisate synergistically enhances the antitumor effect of the mixture of LA and sodium- selenite, and the simultaneous presence of both LA and sodium-selenite is necessary for the occurrence of this synergism. Example 25

Through example 25 presented in Table 15 we show that pyrrole-2-carboxylic acid applied in 15 mM, 10 mM, 5 mM concentrations synergistically enhances the antitumor effect of the mixture of LA and sodium-selenite on HELA cells.

The measurements were performed with the in vitro experimental system described at example 1. The pyrrole-2-carboxylic acid solution was neutralized with a stoichiometric amount of sodium-hydrogencarbonate. The results are presented as averages of values obtained from three independent experiments. The statistical analysis and calculation of the CI were performed as described at example 1.

The obtained results are the following:

Table 15.

Pyrrole-2-carboxylic acid synergistically enhances the antitumor effect of the mixture of (±)-alpha-lipoic acid and sodium-selenite on HELA cells

Example 25

Number of living cells

Sodium-

(±)-alpha-lipoic Pyrrole-2- expressed as % of

selenite Combination acid concentration carboxylic acid untreated cells

concentration index (CI) (mM) concentration (mM) (average ± standard

(μΜ)

deviation)

2 - - 33.71 ± 1.05 * -

1 - - 66.61 ± 2.28 * -

0.5 - - 84.34 ± 2.12 * -

0.3 - - 95.63 ± 1.30 -

- 6 - 61.04 ± 2.24 * -

- 5 - 75.89 ± 1.14 * -

- 4 - 86.39 ± 2.09 * -

- 3 - 98.59 ± 1.29 -

- - 15 58.99 ± 3.78 * -

- - 10 69.89 ± 4.66 * -

- - 5 83.31 ± 3.78 * -

0.4 1 - 24.95 ± 1.85 * -

0.4 0.75 - 29.63 ± 1.10 * -

0.4 0.5 - 36.88 ± 3.74 * -

0.4 0.25 - 58.78 ± 2.74 * -

0.5 - 15 58.57 ± 1.44 * 1.41

0.4 - 15 63.04 ± 2.20 * 1.54 0.3 - 15 58.22 ± 0.32 * 1.22

0.5 - 10 68.72 ± 2.01 * 1.51

0.4 - 10 74.84 ± 1.23 * 1.77

0.3 - 10 72.09 ± 1.82 * 1.47

0.5 - 5 78.56 ± 0.91 * 1.47

0.4 - 5 84.27 ± 0.59 * 1.76

0.3 - 5 82.73 ± 3.21 * 1.45

- 5 15 63.49 ± 1.89 * 2.06

- 4 15 52.39 ± 5.31 * 1.44

- 3 15 54.81 ± 1.88 * 1.35

- 5 10 78.05 ± 2.14 * 2.49

- 4 10 70.66 ± 3.11 * 1.80

- 3 10 69.10 ± 3.21 * 1.54

- 5 5 85.09 ± 2.01 * 2.23

- 4 5 81.46 ± 2.58 * 1.74

- 3 5 81.82 ± 2.20 * 1.55

0.4 0.75 15 18.44 ± 2.07 *' ⁺ ' ^& 0.74

0.4 0.5 15 22.97 ± 2.26 *· ⁺ ' 0.72

0.4 0.25 15 26.93 ± 3.13 *· ⁺ ' ^$ 0.59

0.4 0.75 10 17.86 ± 2.00 *· *' ^& 0.65

0.4 0.5 10 23.81 ± 3.23 *· *' 0.67

0.4 0.25 10 27.34 ± 0.65 *· *' * 0.50

0.4 0.75 5 19.27 ± 0.97 *· ^& 0.65

0.4 0.5 5 23.47 ± 0.89 *· ^§ 0.57

0.4 0.25 5 40.24 ± 1.85 0.57

* pO.001 vs. untreated control

+ p<0.001 vs. 15 mM pyrrole-2-carboxylic acid

pO.001 vs. 10 mM pyrrole-2-carboxylic acid

^ pO.001 vs. 5 mM pyrrole-2-carboxylic acid

&

p<0.05 vs. the mixture of 0.4 mM (±)-alpha-lipoic acid and 0.75 μΜ sodium-selenite

p<0.001 vs. the mixture of 0.4 mM (±)-alpha-lipoic acid and 0.5 μΜ sodium-selenite

$

p<0.001 vs. the mixture of 0.4 mM (±)-alpha-lipoic acid and 0.25 μΜ sodium-selenite

P values were computed with one-way analysis of variance (ANOVA), followed by Bonferroni-test.

On the basis of our results presented in Table 15 pyrrole-2-carboxylic acid applied per se in 15 mM, 10 mM, 5 mM concentrations significantly reduced the number of living cells compared to the untreated control. The combined application of pyrrole-2-carboxylic acid with the mixture of LA and sodium-selenite significantly enhanced the antitumor effect, which resulted in that the number of living cells after the combined application of the three compounds was significantly lower than after the per se application of pyrrole-2-carboxylic acid or after the per se application of the mixture of LA and sodium-selenite in the given concentrations (see the symbols indicating significance in Table 15). On the basis of the CI values presented in Table 15 (0.50-0.74) there is synergism in the antitumor effect between pyrrole-2-carboxylic acid and the LA-sodium-selenite mixture at the investigated concentrations. In contrast to this the combination of pyrrole-2-carboxylic acid with LA or sodium-selenite did not produce a synergistic enhancement of the antitumor effect, but on the contrary - on the basis of the CI values presented in Table 15 - antagonism could be observed (CI for the pyrrole-2-carboxylic acid-LA combination: 1.22-1.77; CI for the pyrrole-2- carboxylic acid-sodium-selenite combination: 1.35-2.49). In summary of these results pyrrole- 2-carboxylic acid synergistically enhances the antitumor effect of the mixture of LA and sodium-selenite, and the simultaneous presence of both LA and sodium-selenite is necessary for the occurrence of this synergism.

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