Composition and methods for inhibiting the growth of tumor cells and tumor using

 

(57) Abstract:

The invention can be used in medicine to prevent or reduce tumor growth and metastasis. The method of suppressing the growth of tumor cells include the stage at which the tumor cells are exposed to an effective dose of a composition containing tocotrienol and ionon, or statin and ionon, or tocotrienol, a statin and Inon. The method of suppressing tumor growth includes the stage at which the patient is exposed to an effective dose of a composition comprising at least one tocotrienol and at least one ionon, or at least one tocotrienol, at least one statin and at least one ionon, or at least one statin and at least one ionon. The claimed pharmaceutical composition comprising an effective dose of at least one tocotrienol and at least one ionone, or at least one tocotrienol, at least one statin and at least one ionone, or at least one statin and at least one ionone. The claimed combination of the active components of the composition when it is used to create a synergy effect. This will increase effektivnye derivative fruit, vegetables and grains derived mevalonata (isoprenoids), have the ability to suppress initiated by chemical carcinogenesis. This was explained by the ability of isoprenoid indirectly stimulate action, neutralizing toxic substances, and anti-oxidant actions of some of isoprenoids. Neither one of these properties is not due to the potential impact isoprenoids have on stage stimulation/development initiated by chemical carcinogenesis and growth formed by chemical and implanted tumors (for review see (Elson, 1995; Elson and Yu, 1994). The impact of isoprenoids on the growth of tumors varies considerably. Through posttranscriptional steps (see Correll and others , 1994; Parker and others , 1993; D. M. Peffley and A. K. Gayen, personal correspondence) isoprenoids suppress the activity of the reductase the coenzyme And 3-hydroxy-3-methylglutaryl (HGM-CoA), the activity is considered to be the factor limiting the rate of cholesterol synthesis. Statins are competitive inhibitors of HMG-CoA-reductase. There is an almost complete correlation between the ability of various isoprenoids suppress tumors late-stage development is aktivnosti reductase of the liver, that it is resistant to regulate the regeneration of the Sterol. However, the tumor is still highly susceptible to post-transcriptional regulation, caused by a variety of isoprenoids. As a result of suppression activity MG-CoA-reductase-mediated isoprenoids, group of intermediates of the pathway of metabolism mevalonata become limiters posttranslational development related growth proteins (for review see (Elson, 1995, Elson and Yn, 1994).

In one of the recent reviews were given a list of isoprenoids a variety of buildings with different abilities to inhibit the synthesis mevalonata (see Elson, 1995).

In one of the embodiments of the present invention, a method for inhibiting the growth of tumor cells. The method includes providing exposure to a tumor cell a combination of at least two products to share mevalonata selected from the group including statins, ionone and tocotrienols.

In a preferred embodiment, ionon chosen from the group comprising beta-ionon; 6-10-dimethyl-3,5-undecen-2,9-dione; 6,10-dimethyl-9,10-epoxy-3,5-undecene-2; 9,10-diacetoxy-6,10-dimethyl-3,5-undecene-2; 6-10-dimethyl-9,10-diol-3,5-undecene-2 and alpha-ionan. In the and d-tocotrienol. In a preferred statins include lovastatin, pravastatin, simvastatin and fluvastatin.

In another embodiment, the present invention proposed a pharmaceutical composition for the treatment or prevention of tumors, comprising an effective amount of at least two substances selected from the group including tocotrienols, statins and ionone.

One of the purposes of the present invention is the prevention or reduction of tumor growth and metastasis.

Another objective of the present invention is to increase the duration of survival of patients suffering from tumors of patients after the diagnosis of the tumor.

Another objective of the present invention is to prevent the formation of tumors.

Other objectives, advantages and features of the present invention will become apparent to the person skilled in the art after reading the description, patent claims and drawings.

In Fig. 1 shows a diagram proliferation of B16 melanoma cells from affecting dose Sokolov.

In Fig. 2A and illustrates the effect of combinations of gamma-tocotrienol, beta-ionone and Charvak the ETA-ionone.

In Fig. 2B illustrates the effect of carvacrol and beta ionone.

In Fig. 3 shows the survival curve of mice-media tumors treated with enriched isoprenoids food after detection of dense implanted B16 melanoma.

In one of the embodiments of the present invention, a method for inhibiting the growth of tumor cells by exposing them to a combination of isoprenoids. In one of the embodiments of the present invention the combination comprises at least one tocotrienol and at least one ionone. In another embodiment of the present invention the combination comprises at least one tocotrienol and at least one statin. In the third embodiment of the present invention, the combination consists of at measures of one statin and at least one ionone. We assume, in other acceptable embodiment of the present invention the combination will consist of all three products pathways malonate.

Under the "tocotrienol" refers to a substance that is included in the following group. The family of vitamin e consists of a mixture of vitomirov, mainly consisting of Tocopherols and koverola and tocotrienols.

In a preferred tocotrienols are d-gamma-tocotrienol and 2-desmethyltramadol. Other preferred tocotrienols are d-beta-tocotrienol, d-Delta-tocotrienol and d-tocotrienol.

Under the "mononom" refers to a substance that is included in the following group. Ionone are widespread in nature connections, related carotenoid. Alpha ionon and beta ionon, and some of them saturated with oxygen derivatives are widely present in plants in free and bound forms. Their biological activity is to act as phytoalexins. Ionone are also formed by means of thermal and photochemical oxidation of carotenes.

Among the preferred Ivanov includes beta ionon (4-2,6,6-trimethyl-1-cyclohexenyl-1)-3-butene-2; 6-10-dimethyl-3,5-undecen-2,9-dione; 6,10-dimethyl-9-10-epoxy-3,5-undecene-2; 9,10-diacetoxy-6,10-dimethyl-3,5-undecene-2 and 6-10-dimethyl-9,10-diol-3,5-undecene-2. Other preferred Ivanov consisting of alpha-Ionen (4-(2,6,6-trimethyl-2-cyclohexenyl-1))-3-butene-2.

Under the "statin" refers to a substance that is included in the following group. Statins are derived metabolites of fungi (ML-236B/compaction/monacolin) vydelennyi (HMG) are competitors of HMG-CoA at the connection point on the substrate HMG-CoA-reductase. In the United States statins dispensed on prescription. For example, lovastatin (Mevacor/Merck), simvastatin (Zocor/Merck), pravastatin (Pravachol/Bristol-Myers Squibb) and fluvastatin (Lescol/Sandoz). We know several clinical research, including research carried out in the company of Warner-Lambert in the tests at a later stage. A large part of the lipophilic statins are known to cause diseases of the musculoskeletal system (myositis, acute necrosis of skeletal muscles), but the majority of side effects observed in clinical trials were mild and easily portable (headache, abdominal pain, constipation, flatulence, and diarrhea). (See Pedersen, T. R. , N. Engl. , J Med. 333: 1350-1351, 1995; Kohashigawa, J. A. et al. , N. Engl. J. Med. 333: 621-627, 1995). In table B lists some preferred statins.

In table. In the above chemical formula preferred statins.

In a preferred statins include lovastatin, pravastatin, simvastatin and fluvastatin.

The following examples illustrate the synergy effect in case of B16 melanoma cells were exposed to combinations of tocotrienols, lovastatin and ionone.

In one of the following examples were fewer cells chalk the ptx2">

When tested in vivo, which were measured by the survival rate of mice carrier after implantation of melanoma, the three values R for isoprenoid, when comparing the mixed treatment, ranged from 0.66 to 0.16 while the three values R for isoprenoid obtained when compared with treatment isoprenoids exceeded 0,88. First, it should be noted that in all cases significantly increased the duration of survival (P < 0.03 in). Differences between effects in a single application was not significant. Indicators R nonparametric tests were in the range of 0.64 to 0.95. When it was tested the difference between the result of a single treatment and combined treatment, non-parametric indices of P was in the range of 0.16 - 0.64 in. The tendency to decrease R suggests a possible synergistic effect.

Under the "synergy" refers expressed in percentage terms, the decrease in the number of cells constituting at least an additional 5% above the total sum of individual figures, or increase survival media, amounting to 5% above the total sum of individual indicators.

For the purposes of the present invention was evaluated by the ability in vitro real. We assumed that the isoprenoids, with between a few similarities, except for the common predecessor isopentyl pyrophosphate - inhibit proliferation of melanoma cells, and showed that individual action isoprenoids tested in two-component mixtures, is additive. We also noted that the consumption of d-gamma-tocotrienol as part of the food ration is suppressed growth of implanted tumors.

The following examples show the calculation of the concentration of isoprenoids a variety of structures necessary to contain 50% increase in the population of cells murine melanoma b 16 (F10) under cultivation for 48 h (figure IC50). Figures IC50the d-limonene and parallelotope alcohol monoterpenes on the 1st stage of the trials, respectively 450 and 250 µmol/l; in related cyclic monoterpenes (the railing aldehyde, carvacrol, thymol), acyclic monoterpene (geraniol) and similar end rings of beta-carotene (beta-ionon) IC50was in the range of 120-150 mmol/l Indicator IC50farnesol, similar side chain of tocotrienols (50 µmol/l) was in the range between the rate of alpha-Toco is present novelty tocotrienol, has no methyl group on tatalovich rings, has demonstrated an extremely high chilling capacity (figure IC500.9 µmol/l). During the first of the two studies taken from the mother in breastfeeding females of mice 57L for 10 days before and 28 days after implantation, characterized by aggressive growth and high metastatic activity of murine melanoma B16 (F10), was given the experimental diet. As a result of replacement in the diet IN-76A d-gamma-tocotrienol, dl-alpha-tocopherol in terms of smolyary equivalent (116 µmol/kg diet) and the equivalent of vitamin E (928 µmol/kg diet) was marked slowing of tumor growth by 36% and 50%, respectively (P < 0,05). In the second study, melanoma were identified before the mice began to give the experimental diet, which consisted of 2 µmol/kg d-gamma-tocotrienol and beta-ionone separately and in combination. In the fourth part of the diet consisted of 4 µmol/kg d-gamma-tocotrienol (see Fig. 3). The result of each treatment period survival media increased (P < 0.03 in). The present invention is based on the fact that, as we have established, private dei is synergy. This phenomenon would suggest that some combination of isoprenoids can be extremely effective for use in chemotherapy.

The methods according to the present invention can be implemented in several different ways. In one embodiment, the implementation of the human or animal is introduced combination of isoprenoids in the form of a pharmaceutical or veterinary composition comprising a safe and effective dose. In another embodiment, the patient receives a diet enriched with a combination of isoprenoids. Because statins currently are sold only on prescription, it is preferable not to add them to food.

In the composition of the diet of humans and animals and pharmaceutical preparations used in the methods according to the present invention, includes a selected combination of isoprenoids, supplemented conventional animal feed, food supplements for humans or approved pharmaceutical diluents and carriers.

High activity of tocotrienols include d-gamma-tocotrienol, d-Delta-tocotrienol and 2-desmethyltramadol, and the last two are of natural origin, d-gamma-tocotrienol and the MoEHE oil using techniques known from methods. 2-Desmethyltramadol can be extracted from the tocotrienol-rich fractions of oil from rice bran is known from the technology of ways.

Beta Ionen can be obtained from a variety of industrial sources. For example, beta-ionon you can brush up on the directory Aldrich Flavors and Fragrances no. W25950-0; beta ionon also listed in the directory Aldrich Fine Chemical and Sigma. In the specified directories, and Bedoukian Research Distinctive Perfume & Flavor Ingredients precisley and other ionone.

Lovastatin is the most easily available industrial substance.

In addition to the diet of the patient, the combination of isoprenoids can be put into the form of pharmaceutical or veterinary compositions, such as tablets intersolubility coating, disintegrating in the gut, capsules, powders, solutions or emulsions. Be taking the exact dose depends on the type of isoprenoid, route of administration, the patient's weight and the nature of the state. Pay attention to that used later in the example, the line of melanoma cells is extremely resistant to chemotherapeutic effects. Thus, the actual dosage in the treatment of a patient can be lower than proposed in our experimental model.

Typical decaying in the gut tablet with intersolubility coating has the following composition, mg:

Active start:

d - gamma-Tocotrienol - 50

6,10-Dimethyl-9,0-epoxy-3,5 undecene-2 - 400

Statin - 30

Carriers/excipients: microcrystalline cellulose, glycolate, sodium, corn starch, hydrogenated paraffin, vegetable oil, magnesium stearate, talc.

Suppose the tap-hole of any desired shape, and then add a small amount of liquid containing active beginning, mg:

d-gamma Tocotrienol - 50

6,10-Dimethyl-9,0-epoxy-3,5 undecene-2 - 400

Preferably, in the tile or tablet attended appropriate flavorings to give an acceptable taste. It is essential that existing start was added after the implementation of all operations associated with baking or heating, and the completion of cooling, because tocotrienols do not have thermal stability. Such tiles or tablet can be Packed in a hermetically sealed package.

As tocotrienols and ionone are relatively non-toxic substances can be injected dose in excess of the above maximum value, in particular, it concerns decaying in the gut composition with intersolubility coating. However, as a result of taking higher doses can significantly affected the intestinal flora, leading to zhedudochno-intestinal disorders. In the case of compositions according to the present invention, it is recommended to limit the daily intake of vitamin E. the Most part lipophilic (absorbing fat) statins can be cennych in reports of clinical trials, side effects were mild and easily portable (headache, abdominal pain, constipation, flatulence, and diarrhea). Patients (see Thibault, et al. Clinical Cancer Research 2: 483-491, 1996) carried the receiving doses approaching 2 g/day (25 mg/kg body weight).

For specialists in this area it is obvious that the present invention can be made many modifications and changes, without going beyond its scope and nature. Thus, the invention is limited only by the following claims.

EXAMPLES

Used abbreviations: HGM COA-3-hydroxy-3-methylglutaryl-COA-reductase; IC50the concentration needed to reduce by 50% the population growth of melanoma cells; TRF - tocotrienol rich fraction of palm oil; TRF25 - containing oryzanol rich in tocotrienol (fraction of oil from rice bran.

A: Substances and methods

Isoprenoids: d-limonene (97%), parallelly alcohol (99%), the railing aldehyde (92%), carvacrol (98%), thymol (98%), beta-ionon (96%), geraniol (98%), farnesol (96) and dl-alpha-tocopherol (97%) were purchased from Aldrich Chemical company (Milwaukee, Wisconsin). Abbreviated list of concentrated natural sources and aromatic properties of these isoprenoids are shown below in table 1. In the preliminary studies it was found that n is Blarney distillation (see Dr. Laxman, Singh, Vitamins, Inc. , Chicago, IL), has a very powerful overwhelming tumor effect. Fraction consisted of 6% of d-alpha-tocopherol, 12.5% of d-alpha-tocotrienol, 21% d-gamma-tocotrienol, 10% d-Delta-tocotrienol, and 4.5% d-tocotrienol, 17% of d-2-desmethyltramadol, 18% not installed isomers of tocotrienol and 10% of sterols and triglycerides (see Qureshi, et al. , unpublished data). The main components of d - alpha-tocotrienol, d-gamma-tocotrienol, d-Delta-tocotrienol and d-2-desmethyltramadol were allocated by the company Advanced Medical Research (Madison, Wisconsin). For the separation of d-gamma-tocotrienol from tocotrienol rich fraction (TRF) of palm oil (containing 36% d-gamma tocotrienol, 18% d-alpha-tocotrienol, 12% d-Delta-tocotrienol and 22% d-alpha-tocopherol) was developed to chromatography provided as a gift by the Institute for the study of palm oil (Kuala Lumpur, Malaysia). Suspended in hexane silica gel (Merck, 60 μm, 150 g) was placed and a glass funnel with a volume of 350 ml with printovannymi disk. The gel was washed in 1 l of hexane, after which 20 ml of hexane was added 5 g of TRF. Tokoly were suirvey progressive introduction gecan 500 ml of diethyl ether mixtures(5-, 10, 12, 14-, 16-, 18, 20-, 22-, 25- and 30%). Re vacuum, resulting from the extraction of water. Eluate were dried under vacuum, the residues were subjected to repeated dissolution in hexane and identified on the basis of the delay time and the nature of the absorption with the use of system analysis HPLS. Allerona using 18% diethyl ether fraction predominantly (98%) consisted of d-gamma-tocotrienol.

Definition of the indicator IC50: obtained from Dr. William C. Ershler cells murine melanoma b 16 (F10), which is a line of tumor cells with high metastatic potential (see Tsukamoto, et al. , 1991), were grown in a single layer culture flasks with a size of 35 x 10 mm) in 3 ml of medium RMP1 (Sigma), which was added 10% newborn calf serum (GIBCOBRL, Grand Inland, new York) and 80 mg/l gentamicin (Sigma, St. Louis, Missouri). Culture is seeded with the use of 1-1,5 x 105cells were incubated for 24 h at 37oC in humidified air containing 5% CO2. After 24 h (time 0) were added isoprenoids, dissolved in absolutely pure ethanol; all cultures contained 5 ml of ethanol/l (85 µmol/l). Cultures were further incubated for 48 hours the Medium was removed, and ECENA 2 min, incubated in trypsin solution-add (Sigma) at 37oC. the Trypsin was deactivated by suspension of the cells in a medium containing 10% fetal cow serum (Sigma). Cells were granulated and subjected to re-suspendirovanie compensated in saline Hanks. Using hemocytometer they have counted the number of viable cells, i.e. cells not containing 0.4% of Trypanosoma blue (GIBCOBRL); based on the results of the final counting of the cells, brought the number of cells after 24 h and received estimate the net growth of cells. The calculation of the concentration of isoprenoid needed to reduce by 50% (IC50) net increase in cells for 48 h based on the experimental data obtained in the result of three or more evaluations.

Research using animals: Experience in assessing the ability of various isoprenoids suppress the tumor was extended by two studies of food rations. In the first of them was studied the effect of consumption of d-gamma-tocotrienol and beta-ionone as part of a diet on the survival of mice carrier after implantation they melanoma. First, there was prepared a base diet based on the recommendations of the AIN-76A (American inst is from vitamin E, corn oil were purchased from the company Teklad Test Diets (Madison, Wisconsin). The original solutions of dl-alpha-tocopherol (80 µmol/g) and d-gamma-tocotrienol (80 µmol/g) were obtained using purified from vitamin e, corn oil. Original solutions tokalov diluted purified from vitamin e, corn oil, mixed with a base diet, the result was obtained ready-made diet containing 5% corn oil and tocol at a given concentration. Where it is stipulated in oil was added to a beta ionon. The diet made weekly, kept chilled. Containers for food was cleaned and filled daily.

Experiment 1: are Weaned from the mother during breastfeeding female C57BL (Harlan-Sprague Dawley, Madison, WIS.) were divided into four groups and placed in plastic cages with flooring from wood chips, where they were kept at 25oIn the alternate mode changing conditions of day and night, every 12 hours, All four groups of mice (20 mice in each) for 10 days before and 28 days after implantation of B16 melanoma cells were fed an experimental diet. According to the scheme the drugs were divided into four treatment consisting of two types of tokalov, dl-alpha-tocopherol and d-gamma-tocotrienol, and cost to make two comparisons of diets, contains equal doses of the Protocol, and a comparison of diets containing approximately equal to the dose in terms of the equivalent d-alpha-tocopherol. Without accurate estimates of biological activity of d-gamma-tocotrienol, we proceeded from the reports that 2,5,8 (d-beta) and 2,7,8 (d-gamma) trimethylthiazole have the same ability to absorb oxygen (to a maximum of 66% of the level of oxygen uptake 2,5,7,8 (d-alpha) tetramethylthiuram), but as a class of compounds, tocotrienols possess biological activity in the range of 5 to 30% of the biological activity of Tocopherols (see Karnal-Eldir and Appelqvist, 1996), the result of which was made a preliminary assessment, according to which the biological activity of d-alpha-tocopherol at least six times inferior to the biological activity of d-gamma-tocotrienol. Then we made a correction for the biological activity of dl-alpha-tocopherol (70% of the biological activity of d-alpha-tocopherol) and came to the conclusion that the correlation of biological activity of tocotrienol and tocopherol is 8: 1, which was used as the basis for formulation of the experimental diets. Melanoma cells, cultured and collected as described above (see Shoff et al. , 1991) were twice pomatiopsidae (98% viable cells) after dilution of 0.4% trypanosome blue dye. Then the cells were further diluted in the composition of RPMI 1640(1 x 108cells/l), and 0.1 ml of suspension (1 x 104cells/l) was subcutaneously injected in each mouse in the lateral region of the abdomen. The study was completed on the 28th day, when he killed the first mouse from the control group. Mice were euthanized excessive dose of CO2and tumors were eksterminowani and balanced. The Protocol was reviewed and approved by the Board of the Committee for the protection of farm animals and animals used in biological research.

Experiment 2: Weaned from the mother during breast-feeding female C57BL (60 individuals, the company Harlan-Sprague Dawley, Madison, WIS.) were acclimated to the conditions and diet AIN-76 A (116 mmol of dl-alpha-tocopherol per 1 kg of diet), as described above. Were implanted tumor, after which the mice continued to feed Razina AIN-76A. Starting from the 8th day after implantation produced daily palpation to identify the tumor. First tumors were detected on day 14. For each mouse in groups of five individuals by random selection was assigned a number corresponding to a particular type of diet. Experimental treatments PE on the survival of mice beta ionone (2 mmol/kg diet AIN-76A) and a mixture of two isoprenoids (2 mmol each/kg diet). Mice continued to feed the respective experimental diets; dying mouse detected by the observer, trained in the center for the study of animal resources were euthanized excessive dose of CO2and tumors were sclipiroare and balanced. The Protocol was reviewed and approved by the Board of the Committee for the protection of farm animals and animals used in biological research.

Statistical methods: To assess treatment-related effects were used software Staview and SuperNOVA (Abacus Concepts, Berkeley, California). Associated with treatment differences in body weight and tumor, the time until the appearance of the tumor and time to death were made using analysis techniques separate parameters variability and pairwise comparison lowest average quadratic value. Associated with treatment differences in terms of death were also evaluated using parametric (by pairwise comparison) and nonparametric (method relative rank Wilcoxon tests (see Haycock, et al. , 1992).

C. Results

In Fig. 1 shows a typical assessment based fast 5cells were incubated for 24 h prior to the introduction of Sokolov. Viable cells were counted after 48 h after injection of Sokolov. The dashed line shows the result of counting cells after 24 h (time 0). At the intersection of the solid horizontal line and the line is constructed for each of the Protocol shows the concentration at which total 50% suppressed the increase in the number of cells in the incubation period (figure IC50). Figures IC50(average, standard deviation, and n) for all tested isoprenoids in the table. In accordance with Fig. 1 dl-alpha-tocopherol had no impact on the number of cells. The ability to inhibit the cell growth of the individual tocotrienols were in inverse proportion to the number of methyl groups on the ring 6-chromanol: d-alpha-tocotrienol (methyl groups in positions of carbon 5, 7, 8, and 2) < < d-gamma-tocotrienol (methyl groups in positions of carbon 7, 8, and 2) <d-Delta-tocotrienol (methyl groups in positions of carbon 8 and 2) < <d-2-desmethyltramadol (methyl group is missing) (see table 1).

Figure IC50this sequence of tocotrienols was in inverse proportion to the number of methyl groups on the ring 6-chromanol. the x alcohols parallelotope alcohol ([R] 4-Isopropenyl-1-methanol-cyclohexen), perillaldehyde ([R] -4-Isopropenyl-1-carboxaldehyde-cyclohexen), thymol (5-methyl-2-isopropylphenol), carvacrol (5-isopropyl-2-METHYLPHENOL) and geraniol (3,7-transgenetic-2,6-octadienal-1) was also much lower than d-limonene ([R] 4-Isopropenyl-1-methyl-1-cyclohexen). Figure IC50beta ionone representing similar end rings of beta-carotene, was similar to the characteristics of most polar monoterpenes (see table 1). Figure IC50farnesol (3,7,11-trance, transtromer-2,6,10-dodecatrien-1), which sesquiterpenes and a structural analogue of the side chain of tocotrienols, was in the range between indicators d-alpha-tocotrienol and d-gamma and d-Delta tocotrienols.

In the first study assessed the effect of d-gamma-tocotrienol and dl-alpha-tocopherol on the timing of detection of solid tumors and the growth of implanted B16 melanoma. As mentioned above, there were two comparison treatments using equal concentrations of the Protocol (116 and 928 µmol/kg diet) and a comparison of courses of treatment with approximately 80 μmol equivalents of d-alpha-tocopherol (35 mg)/kg diet. At the time of implantation of the tumor weight of mice fed a diet with you the La (see table 2). On the 28th day after implantation of melanoma accounted for 15% of the weight of mice fed the diet AIN-76A. Were tested two types of tokalov, each at two levels. Analysis of separate parameters variability showed a significant effect of Sokolov (P<0.001) and levels (P<0,04) on the 28th day after implantation of the tumor; there was no evidence of interaction between these two factors (P<0,77). The analysis comparing the lowest average quadratic value confirmed the overwhelming growth of the tumor action of gamma-tocotrienol (see table 2). Measurement of another parameter of tumor growth, namely the period from implantation to detect tumors, showed the value of the Protocol (P<0.03 in), level (P<0.01) and interaction (P<0,02). Analysis of sraania lowest average quadratic values showed that the effect of treatment using 928 µmol d-gamma-tocotrienol/kg of diet on the parameter of tumor growth is significantly different from the effects of other treatments.

Then it was determined that the impact of isoprenoids on the growth of B16 melanoma cells in culture is additive, and in some cases synergistic. In Fig. 2A illustrates the additive effect of gamma-tocotrienol and beta-ionone on cell population metre electron microscopy = 36 (X104). Counting the number of cells showing a decrease in their population relative to the control group, shown in the attached table.

In Fig. 2B illustrates the additive effect of carvacrol and beta ionone on populations of B16 melanoma cells. The values of the indicators averages standard deviation, n = 28; grouped data scanning electron microscopy = 62 (X104). Counting the number of cells showing a decrease in their population relative to the control group, shown in the attached table.

Average values a-h not containing Superscript sign (a>b>c>d>e>f>g>h) are different (P<0,001). In accordance with Fig. 2 during a single trial of B16 melanoma cells were incubated with beta-ionones and d-gamma-tocotrienol (see Fig. 2A). d-gamma-tocotrienol (7.5 µmol/l) and beta-ionon (50 µmol/l) reduced the number of cells after 48 h at 19 and 10%, respectively (see Fig. 2A). The decrease in the number of cells by 34%, achieved by using paired isoprenoids, exceeded the decrease of 29%, predicted on the basis of the summation of the individual effects (see Fig. 2A). At higher concentrations of d-gamma-tocotrienol (15 µmol/l) and beta ionone (100 µmol/l) number of cells che is also greater sum of individual impacts (59%) (see Fig. 2A).

While the above results indicate a synergistic effect of d-gamma-tocotrienol and beta-ionone, a study in which carvacrol was used in tandem with beta-ionones, showed only an additive effect (see Fig. 2B). Carvacrol (50 µmol/l) and beta-ionon (75 µmol/l) reduced the number of cells after 48 h at 25 and 31%, respectively; additive effect, namely the reduction in the number of cells at 48% was less than 56% of predicted on the basis of the summation of the individual effects. Carvacrol (100 µmol/l) and beta-ionon (150 µmol/l) reduced the number of cells after 48 h at 35 and 65%, respectively; additive effect, namely the reduction in the number of cells by 84% again proved to be less than 100% of predicted on the basis of the summation of the individual effects (see Fig. 2B). The decrease in the number of cells predicted on the basis of the summation of the individual effects of isoprenoids significantly correlated with achieved using paired isoprenoids (r = 0,91, n = 8, P <0,01).1the concentration was 2 mmol of isoprenoid per kg of diet; when using a mixture of 2 mmol of each isoprenoid per kg of diet; and when using a gamma-tocotrienol2- 4 mmol of gamma-tocotrienol on 1 kg of the diet.

According to the scheme of the experiment each animal in the group of 5 individuals could be combined with another member of the subgroup for testing. In the following table 4 shows the performance of R pairwise comparisons given node the average performance of the control and treatment groups; the differences between the averages in the treatment groups were not significant. The trend is illustrated in table 4, gives additional credibility to the results of in vitro assays, demonstrating the presence of at least additive action of both isoprenoids. While the performance P of the three comparisons isoprenoid with isoprenoid therapy exceed 0,88, indicators R three comparisons isoprenoid mixed therapy are in the range from 0.66 to 0.16.

C. Discussion

Melanoma B16 (F10) allows you to create a rigid model evaluation in vitro and in vivo actions of pharmacological substances (see Gruber et al. , 1992; Kuwasbima, et. al. , 1990; Mac Neil, et al. , 1992; Tsukamoto, et al. , 1991; Shoff, et al. , 1991). We found that consumption of a diet containing a daily dose of 0.4 µmol d-gamma-tocotrienol, was the suppression of growth of B16 melanoma implanted in the lateral region of the abdomen mouse-media. In the course we have the most stringent test diet containing a daily dose of 7 µmol d-gamma-tocotrienol and beta-ionone, was the suppression of growth identified B16 melanoma. In the in vitro tests, it was proved additive effect of the two isoprenoids (see Fig 2A). In remote increased by 35%. While doubling consumed daily dose of d-gamma-tocotrienol (14 µmol/day) did not lead to an additional increase of the period of survival (-0,21 day, P= 0,95), resulting in increased daily vines consumption of d-gamma-tocotrienol (7 (µmol/day) for 7 µmol/day due to beta ionone was observed trend of increasing period survival (+0,83 d, R= 0,65, see tables 3, 4). In this study was estimated reaction revealed melanoma B16 on the beta ionon and d-gamma-tocotrienol, representing two isoprenoid, two structurally related anti-oxidant nutrients, more recently regarded as having the effect of depressing the growth of tumors (see Greenberg and Sporn, 1996). Both isoprenoid provide a significant increase in the period of survival (see table 4). We noticed that it was established that significant consumption of ascorbic acid (733 µmol/day) inhibited the growth of implanted B16 melanoma (see Meadows et al. , 1991). Our research has shown that consumption of a diet containing eight-fold increased dose in terms of the equivalent d-alpha-tocopherol, have minimal impact on tumor growth, whereas the diet on the basis of d-gamma is Noah suppression of tumor growth.

D. Synergistic effect of lovastatin, tocotrienols and ionone

i. Prerequisites

Lovastatin, which is a fungal antibiotic, competitive suppresses the activity of the reductase the coenzyme And 3-hydroxy-3-methylglutaryl (MG SOA) (see J. L. Goldstein and M. S. Brown, Nature 343: 425-430, 1990). Beta ionon (see S. G. Yu, et al. , J. Agric. Food Chem. 42: 1493-1496, 1994), which is the pure isoprenoids, and related compounds inhibit the activity of HMG-COA reductase inhibitor by suppressing its synthesis (review N. Mo, et al. , Nutritional Oncology, publishing house of D. Huber & G. Blackburn, Academic press, NY, in press; C. E. Elson, et al. , Amer. Assoc. Cancer Res. , in press). Tocotrienols, which is a group of mixed isoprenoids suppress the activity of the reductase by initiating a proteolytic cleavage enzyme (see above C. E. Elson, in press). By suppressing the activity of the reductase lovastatin and subsequent derivatives (see above J. L. Goldstein and M. S. Brown. 1990; R. A. Parker, et al. , J. Biol. Chem. 268: l1230-11238, 1993), beta ionon and related net isoprenoids (see above S. G. Yu, et al. , 1994; N. Mo, et al. , in press) and tocotrienols (see above H. Mo, et al. , in press; R. A. Parker, et al. , 1993; A. A. Qureshi, et. al. , J. Biol. Chem. 261: 10544-10550, 1986; A. A. Qureshi, et. al. , Proceedings of the International Conference on palm oil, 1996, Kuala Lumpur, Malaysia, PP 168-18 the tsya content HMG-CoA-reductase, what happens as a result of increasing the activity of the reductase after removal of the inhibitor (see above J. L. Goldstein and M. S. Brown, 1990). Cellular activity underlying the increase in mass reductase, leads to increased transcription and efficiency of processing of the messenger RNA HMG-CoA-reductase and reduce proteolytic cleavage of the enzyme. Beta ionon (see above S. G. Yu, et al. 1994) and tocotrienols (see above R. A. Parker, et al. , 1993) weaken the degree of increased activity (see S. G. Yu, et al. 1994) and weight gain (see above R. A. Parker, et al. , 1993) reductase caused by exposure to lovastatin.

Intermediate products pathways mevalonata are essential for the posttranslational modification of proteins that play an important role in the rapid multiplication of cells (see above N. Mo, et al. , in press). Due to competitive inhibition of synthesis mevalonata, force caused by stanine, is curbing proliferation of cultured cells (see above J. L. Goldstein and M. S. Brown, 1990) and the growth of implanted tumors (see W. Maltese, et al. , J. Clin. Invest. , 76: 1748-1754, 1985; J. P. Jani, et. al. , Invasion Metastasia 13: 314-324, 1993). In the result of five years of control observations 745 patients with high contents of cholesterol is revealed cases 21 predicted) (see J. A. Tolbert, Arch. Intern. Med. , 153-1079-1087, 1993). At 1 stage trials assessed the tolerability of lovastatin be taken in gradually increasing doses (from 3 to 43 cycles held monthly 7-day course of treatment), 88 patients with thick tumors (39 of them with brain tumors, 24 - gormonalnozavisimyh prostate cancer. Take doses ranged from 2 to 45 mg/kg (maximum dose with increased content of cholesterol in the blood is 80 mg/day). At sixty patients were noted in a total of 128 cases of intoxication; the frequency and severity of cases of intoxication significantly increased when the dose level reached 25 mg/day. To prevent intoxication and improve tolerability of lovastatin group of patients taking more than 30 mg/kg lovastatin, was prescribed ubiquinone. Ubiquinone has not led to reduction in the frequency of cases of intoxication, however, substantially mitigate their severity. The authors concluded that gliomas at a late stage of development presumably serve as a suitable target for phase 2 trials, and is recommended to conduct a study of alternative treatment regimens designed to achieve long-term suppression of the synthesis of the mew is lovastatina in suppressing the growth of tumor cells

It is known that beta ionon, gamma-tocotrienol and lovastatin individually inhibit the proliferation of cells of the murine melanoma B16/a10. Figures IC50beta ionone and gamma-tocotrienol be 140 µmol (see L. No, et al. , J. Nutr. 127: 668-674, 1997) and 20 µm (see Not above L., et al. ). Using the research Protocol mentioned above is Not performed, et al. we found that B16 melanoma cells, incubated using (1,9 0,3) µmol/l of lovastatin, is growing at a speed component (61,4 4,6)% growth rate in the control groups. This figure confirms the data obtained in the quantitative analysis of the formation of colonies (see above J. P. Jani, et. al. , 1993). The results of in vivo studies show that lovastatin (50 mg/kg) taken every other day (see above J. L. Goldstein and M. S. Brown, 1990), beta ionon (2 mmol/kg diet) (see Not above L., et. al. 1997) and gamma-tocotrienol (116-2000 µmol/kg diet) individually inhibit the growth of implanted tumor murine melanoma 16F10.

As a result of our in vitro quantitative analyses was recorded synergistic relationship between the overwhelming growth of the tumor action of all three substances. The vast tumor d activities (see table 5).

Table 5 summarizes the results of our quantitative in vitro. It provides various concentrations of lovastatin, beta ionone and gamma-tocotrienols, as well as the ability to suppress tumor growth of each substance. The increase in the number of cells subjected to their influence, was recorded in the percentage growth in the number of cells in the control groups. Table 5 shows that 2 Microm lovastatin and 150 µmol beta ionone respectively 50% and 32% lower growth in the number of cells (50% and 68% increase in the control groups), and the result predicted by the combined action of the two compounds has been the growth of 82% (50% + 32%) or reduced cell growth to 18% relative to the control groups. The cell growth was reduced by 90% or to the level of 10% relative to the control groups. In a series of tests in which lovastatin was used in combination with isoprene (tocotrienol or inanam), we predicted a decrease in the number of cells by 66% (33 5% from the level in the control group), however, recorded a decrease of 87% (134% of control group). The combination of isoprenoids was provided for a reduction of cell growth by 50%, while the predicted reduction was 57% (50% predicted decrease in the control group, 43% of the floor is and tocotrienols helped reduce cell growth by an amount inferior to the predicted additive index.

In accordance with table 5 we predicted complete inhibition of cell growth using a mixture containing 2 mmol of lovastatin, 5 mmol tocotrienol and 50 µmol of ionone.

1. The method of suppressing the growth of tumor cells, including the stage at which the tumor cells are exposed to an effective dose of a composition containing tocotrienol and ionon, or statin and ionon, or tocotrienol, a statin and Inon.

2. The method according to p. 1, in which use tocotrienol selected from the group comprising d-gamma-tocotrienol, 2-desmethyltramadol, d-Delta-tocotrienol and d-tocotrienol.

3. The method according to p. 1 wherein the tocotrienol is a d-gamma-tocotrienol.

4. The method according to p. 1, in which ionon chosen from the group comprising beta-ionon and alpha ionon.

5. The method according to p. 1, in which ionon is a beta ionon.

6. The method according to p. 1, in which the statin is a lovastatin.

7. The method according to p. 1, in which stage the influence of the effective dose of the composition is a composition method as diet.

8. The method according to p. 1, in which stage of rendering damage to the tumor, including the stage at which the patient is exposed to an effective dose of a composition comprising at least one tocotrienol and at least one ionon, or at least one tocotrienol, at least one statin and at least one ionon, or at least one statin and at least one ionon.

10. The method according to p. 9, in which the daily consumption ionone is 2000 - 4000 mg.

11. The method according to p. 9, in which the daily intake of statin is 150 to 300 mg.

12. The method according to p. 9, in which the daily intake of tocotrienols is 250 - 500 mg, ionone - 2000 - 4000 mg statin - 150 - 130 mg.

13. Pharmaceutical composition containing an effective dose of at least one tocotrienol and at least one ionone, or at least one tocotrienol, at least one statin and at least one ionone, or at least one statin and at least one ionone.

14. The pharmaceutical composition according to p. 13 containing beta-ionon and d-gamma-tocotrienol.

15. The pharmaceutical composition according to p. 13, which represents a part of the diet.

 

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