Compositions and methods for enhancing overheating therapy

Abstract

The name of the invention is compositions and methods for enhancing overheating therapy. A method for reducing cancer cell proliferation comprises the step of administering a sensitizing agent. The sensitizer for cancer cells may include selenium, fish oil, and a combination of selenium and fish oil. The methods contemplate administration of selenium and fish oil in amounts effective to increase sensitivity to cancer cells, respectively, and administration of selenium and fish oil in combination in an amount effective to synergistically increase sensitivity to cancer cells that is better than administration of selenium or fish oil alone. In addition, the method exposes cancer cells to temperatures exceeding 37 DEG C.

Description

[0001] This application is a divisional application of a patent application filed on June 13, 2018, with application number 2018800489052, entitled “Compositions and methods for enhancing hyperthermia therapy”.

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 519,090, filed June 13, 2017. These and all other referenced extrinsic materials are incorporated herein by reference in their entirety. To the extent a definition or use of a term in a reference incorporated by reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein shall be deemed controlling. Technical Field

[0003] The field of the invention is the use of hyperthermia to treat cancer. Background Art

[0004] The description of the background art includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication explicitly or implicitly referenced is prior art.

[0005] Hyperthermia (i.e., exposure to temperatures exceeding normal body temperature) is used to treat a variety of cancers. In some cases, hyperthermia is used to make cancer cells more sensitive to chemotherapy drugs or radiation and as an adjunct to these therapies. In other cases, hyperthermia can be used to directly kill or damage cancer cells, however, in these applications, the temperatures used carry the risk of damaging normal cells.

[0006] Hyperthermia can be applied locally, regionally, or to the entire body. Local hyperthermia is often used to generate very high temperatures confined to the tumor site, which results in thermal ablation. This is usually limited to local tumors that are exposed on the surface of the body or that can be touched by a fine needle or probe. The size of tumors that can be treated in this way is also limited (usually about two inches or less). Regional hyperthermia provides heat to a specific body part, such as a limb, organ, or body cavity. This can be accomplished by isolated perfusion (i.e., using an external device to heat the blood and guide it into the circulatory system that supplies the area) or by applying RF or microwave energy. The temperatures used for regional hyperthermia are too low to result in killing cancer cells alone, so the technology is generally used as an adjunct to chemotherapy and / or radiotherapy.

[0007] In whole-body hyperthermia, a patient's body temperature is raised to feverish levels by applying heat (e.g., using heated blankets or immersion in warm water). Temperatures as high as 107°F have been used. In theory, this simulates a fever and provides short-term activation of certain immune cells; however, whole-body hyperthermia is currently used as an adjunct to chemotherapy.

[0008] Attempts have been made to improve the performance of hyperthermia therapy by using a variety of sensitizers. For example, the U.S. Provisional Patent 60 / 290681 of Faulk describes conjugating sensitizing compounds (such as chemotherapeutics) to transferrins to produce a metastasis conjugate that tends to be located in cancer cells. However, it is unclear how specific this targeting is, how much sensitization is achieved, or what side effects are produced by protein-conjugated drugs. All publications herein are incorporated by reference to the extent that each individual publication or patent application is clearly and individually indicated to be incorporated by reference. When the definition of a term in the reference incorporated or the use thereof is inconsistent or contrary with the definition of the term provided herein, the definition of the term provided herein is applicable, and the definition of the term in the reference is not applicable.

[0009] Another approach is suggested in U.S. Patent Application Publication No. 2004 / 0072775 to Sobol and Gjerset. This patent application teaches genetically modifying cancer cells to restore the function of a mutant gene (specifically, p53), which confers sensitivity to a variety of cancer treatments, including hyperthermia. However, it is unclear how this selective genetic modification would be implemented in a clinical setting or how effective it would be in increasing cancer cell sensitivity to hyperthermia alone.

[0010] International Patent Application Publication No. WO2014 / 054884 by Cheon et al. proposes the use of magnetic nanoparticles in hyperthermia therapy. These magnetic nanoparticles are believed to provide both a sensitizing effect and a heat source by applying a high-frequency magnetic field. The need to localize these particles at the tumor site limits the application of this approach. Furthermore, the side effects of introducing these magnetic nanoparticles, especially on a repeated or long-term basis, are unclear.

[0011] Lamb et al., U.S. Patent Application Publication No. 12 / 833,207, propose the use of a conductive "button" placed proximate to the tumor. The conductive button can be made of metals such as gold, silver, aluminum, copper, or alloys and can be implemented in a variety of shapes and sizes. As with Cheon et al., the need to position the conductive button at the tumor site and to target heat application through the conductive button limits the application of this approach.

[0012] In yet another approach, U.S. Patent Publication No. 5,810,888 to Fenn et al. suggests using a thermodynamic therapy system utilizing a radiation delivery system to heat the treatment area with focused radiation to activate liposomes containing thermosensitive drugs. However, this approach does not disclose specific drugs or adjuvants to be delivered via liposomes containing thermosensitive drugs.

[0013] Therefore, there remains a need for well-tolerated and / or non-toxic sensitizers that effectively induce cancer cell death by using hyperthermia without the use of adjuvant chemotherapy and / or radiotherapy. Summary of the Invention

[0014] The present subject matter provides compositions and methods wherein selenium, fish oil, and / or selenium in combination with fish oil enhance or potentiate the effect of hyperthermia in reducing tumor cell proliferation. In preferred embodiments, the selenium is in the form of selenium yeast, amino acids derived from selenium yeast, and / or peptides derived from selenium yeast.

[0015] The present subject matter contemplates administering to cancer cells a sensitizer selected from selenium, fish oil, and a combination of selenium and fish oil. Selenium, fish oil, or a combination of selenium and fish oil is contemplated to be administered at sufficiently high doses, depending on the application, to increase the sensitivity of target cancer cells to high temperatures. In a preferred embodiment, both selenium and fish oil are administered to advantageously increase the sensitivity of cancer cells to thermotherapy, which is superior to administering either fish oil or selenium alone.

[0016] One embodiment of the present inventive concept is a method of treating cancer cells comprising administering a fish oil formulated as listed in Table 1 and inducing hyperthermia in a patient, wherein the fish oil is provided in an amount that provides a synergistic effect in reducing cancer cell proliferation. In a preferred embodiment, the fish oil is provided to the patient prior to the initiation of hyperthermia. In other embodiments, the fish oil and hyperthermia may be administered simultaneously.

[0017] Another embodiment of the present inventive concept is a method of treating cancer cells comprising administering selenium in the form of selenium yeast formulated as listed in Table 1 and inducing hyperthermia in a patient, wherein the selenium is provided in an amount that provides a synergistic effect in reducing cancer cell proliferation. In a preferred embodiment, the selenium is provided to the patient prior to the initiation of the hyperthermia therapy. In other embodiments, the selenium and the hyperthermia therapy may be administered simultaneously.

[0018] A preferred embodiment of the present inventive concept is a method of treating cancer cells comprising administering both fish oil and selenium in the form of selenium yeast, as formulated as listed in Table 1, and inducing hyperthermia in a patient, wherein the fish oil and selenium are provided in amounts that provide a synergistic effect in reducing cancer cell proliferation. In a preferred embodiment, the fish oil and selenium are provided to the patient prior to the initiation of thermotherapy. In other embodiments, the fish oil and selenium may be administered simultaneously with the thermotherapy.

[0019] It is contemplated that thermotherapy may be administered at any temperature that elevates the temperature of body tissue above normal body temperature, which effectively reduces cancer cell proliferation by allowing for better perfusion of oxygen and drugs into the cancer cells. However, the present invention contemplates that thermotherapy may be administered at any temperature or combination of temperatures (e.g., a poikilothermic hyperthermia treatment) between 37°C and 44°C. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a bar graph depicting the effect of fish oil on sensitization of MDA-MB-231 breast cancer cells to hyperthermic conditions.

[0021] Figure 2is a bar graph depicting the effects of fish oil and selenium, alone or in combination, on the sensitization of HT-29 colon cancer cells to hyperthermia.

[0022] Figure 3 is a bar graph depicting the effects of fish oil and selenium, alone or in combination, on the sensitization of BFTC-905 bladder cancer cells to hyperthermia.

[0023] Figure 4 is a line graph depicting the effects of selenium, fish oil, and a selenium / fish oil combination on the proliferation of A549 lung cancer cells.

[0024] Figure 5 Regulation of pAMPKα and COX-2 concentrations by selenium yeast and fish oil is depicted. DETAILED DESCRIPTION

[0025] Hyperthermia is typically induced in cancer-affected tissues, such as tumors, in order to reduce, inhibit, or reverse the growth of cancer cells. Typically, hyperthermia therapy is accompanied by radiotherapy and chemotherapy in order to maximize the efficacy of the anti-cancer treatment. However, conventional hyperthermia therapy does not incorporate the use of adjuvants to enhance the efficacy of hyperthermia in reducing cancer cell proliferation. Disclosed herein are methods and compositions for using fish oil and selenium, alone or in combination, to enhance the anti-cancer effects of hyperthermia therapy. Such selenium can be in the form of selenium yeast, amino acids derived from selenium yeast, and / or peptides derived from selenium yeast. As used herein, approximately is defined as ±5% of a specified value.

[0026] As used herein, fish oil can comprise about 220mg of docosahexaenoic acid (DHA) and about 330mg of eicosapentaenoic acid (EPA), which are precursors of specific eicosanoids that can reduce inflammation in the body. In a preferred embodiment, the fish oil comprises between about 110mg and about 330mg of DHA and between about 160mg and about 500mg of EPA. It is contemplated that the preferred combination of DHA and EPA is administered in an amount no greater than 3 grams in total within 24 hours. However, it is also contemplated that when used in conjunction with thermotherapy, DHA and EPA can be present in any amount that effectively reduces cancer cell proliferation.

[0027] It is contemplated that a combination of DHA and EPA may include a fish oil content between about 8% and about 80%, depending on a variety of factors, such as the source of omega-3 fatty acids, the processing of the oil, and the amount of other ingredients in the oil. Sources of fish oil include "oily" fish. For example, herring, Spanish mackerel, salmon, halibut, tuna, anchovies, and sardines can be sources of concentrated omega-3 fatty acids. However, it is also contemplated that any marine source can serve as a source of fish oil comprising omega-3 fatty acids. In some embodiments, fish oil can be derived from fish whose tissues have low concentrations of omega-3 fatty acids, such as cod, flounder, and sea bream. In some embodiments, the fish oil equivalent comprising omega-3 fatty acids can be derived directly from algae rather than from oily fish. Alternatively, the fish oil equivalent comprising EPA and / or DHA and being suitable for use in the formulations of the present invention can be obtained from non-marine sources. For example, the non-marine sources of EPA and DHA can include flaxseed, chia seeds, walnuts, and soybeans.

[0028] As used herein, it is contemplated that selenium is administered at a concentration between about 500 ng / ml and about 1500 ng / ml. If administered directly, selenium salts may be toxic. The present invention contemplates obtaining selenium from selenium yeast by cultivating saccharomyces cerevisiae or other suitable yeasts in a selenium-rich culture medium to produce selenium. By cultivating yeast in a selenium-rich culture medium, selenium can replace sulfur in certain amino acids (e.g., methionine, cysteine), thereby providing a source of non-toxic selenium. It is contemplated that selenium from animal sources can be in the form of selenomethionine, selenocysteine, and / or methylselenocysteine, as well as proteins and peptides incorporating these amino acids.

[0029] In some embodiments, selenium can also be derived from plants. For example, bioconcentrated selenium can be derived from plants. In other embodiments, soluble selenium found in soil (e.g., selenate) can be a source of selenium. In still other embodiments, selenium can be derived from seawater.

[0030] Surprisingly, the present inventors have discovered that the use of fish oil and / or selenium yeast can complement the effects of hyperthermia on cancer cells, and can do so in a synergistic (eg, better than additive) manner. Figure 1 is a bar graph that depicts fish Oil for MDA-MB-231 Effects of sensitization of breast cancer ("BC") cells to hyperthermia. Breast cancer ("BC")Cells were subjected to either control (37°C) or hyperthermic temperatures (39°C, 41°C). At each temperature, each group of BC cells was (1) exposed to fish oil at concentrations of 0 μM (control at 37°C), 12.5 μM, 25 μM, and 50 μM, (2) maintained at 37°C, 39°C, or 41°C for 2 hours, (3) maintained in a CO2 incubator for 72 hours, and (4) examined for cell proliferation as a percentage of control BC cells (cells at control temperature and 0 μM fish oil concentration). The fish oil administered to the non-control concentration BC cell cultures contained 220 mg of docosahexaenoic acid (DHA) and 330 mg of eicosapentaenoic acid (EPA) per gram.

[0031] BC cells maintained at temperatures exceeding 37°C for 2 hours without fish oil administration showed a significant decrease in cell proliferation as a percentage of control BC cell cultures. At 39°C, a decrease of at least 25% in BC cell proliferation was observed compared to control BC cell cultures. At 41°C, a further decrease of approximately 50% in BC cell proliferation was observed compared to control BC cell cultures.

[0032] BC cells maintained for 2 hours at all tested temperatures and administered fish oil at a concentration of 12.5 μM showed an overall reduction in BC cell proliferation, with mixed results at different temperatures. At 37°C, an approximately 30% reduction was observed compared to the BC control. At 39°C, an approximately 60% reduction was observed compared to the BC control, indicating a synergistic effect. At 41°C, an approximately 50% reduction in BC cell proliferation was observed compared to the BC control.

[0033] BC cells maintained for 2 hours at all tested temperatures and administered fish oil at a concentration of 25 μM showed an overall reduction in BC cell proliferation, with mixed results at the different temperatures. At 37°C, an approximately 60% reduction was observed compared to control BC cell cultures. At 39°C, an approximately 65% ​​reduction was observed compared to BC cell cultures. At 41°C, an approximately 65% ​​reduction in BC cell proliferation was observed compared to control BC cell cultures.

[0034] BC cells maintained at all tested temperatures for 2 hours and administered 50 μM fish oil showed an overall reduction in BC cell proliferation, with mixed results at the different temperatures. At 37°C, a reduction of over 75% was observed compared to control BC cell cultures. At 39°C, a reduction of approximately 80% was observed compared to control BC cell cultures. At 41°C, a reduction of approximately 85% was observed compared to control BC cell cultures.

[0035] Exposing MDA-MB-231 breast cancer cells to hyperthermic conditions (e.g., 39°C or 41°C) alone resulted in a modest reduction in cell proliferation relative to 37°C controls. As shown, the effects of hyperthermia were significantly potentiated (in some cases synergistically) by simultaneous exposure to fish oil in a dose-dependent manner. It will be appreciated that the temperatures utilized are within the range of temperatures that can be safely produced in humans by conventional and relatively simple means, such as immersion in warm water and / or the use of heated blankets. It will be appreciated that these temperatures are significantly lower than the extremes that can be employed in hyperthermia therapy.

[0036] Without wishing to be bound by theory, applicants believe that the effect of fish oil in reducing cell proliferation in cancer cells is at least partially due to the effect that specific concentrations of fish oil have on the cell cycle of cancer cells when combined with elevated temperature.

[0037] Figure 2 is a bar graph depicting the effects of fish oil and selenium, alone and in combination, on the sensitization of HT-29 colon cancer cells ("HT cells") to hyperthermia. The sensitizing effects of selenium, fish oil, and the combination of selenium and fish oil are evident in HT-29 colon cancer cells exposed to hyperthermia.

[0038] HT cells were subjected to 37°C (control) and superheated temperatures (39°C and 41°C). At each temperature, each group of HT cells was (1) treated with 0.5 μg / ml of selenium, 25 μM concentration of fish oil, or both 0.5 μg / ml of selenium and 25 μM concentration of fish oil, (2) maintained at 37°C, 39°C, or 41°C for 2 hours, (3) maintained in a CO2 incubator for 72 hours, and (4) examined for cell proliferation as a percentage of the control HT cell culture. Selenium was administered in the form of selenium yeast. It is contemplated that the source of selenium is not limited to selenium yeast and can be administered using any method known in the art. In the absence of selenium and / or fish oil, incubation at 39°C reduced proliferation by approximately 20% compared to control HT cells, and incubation at 41°C reduced proliferation by approximately 45% compared to control HT cells.

[0039] HT cells maintained for 2 hours at all temperatures tested and treated with 0.5 μg / ml selenium, 25 μM fish oil concentration, or both 0.5 μg / ml selenium and 25 μM fish oil showed an overall reduction in HT cell proliferation compared to control HT cell cultures.

[0040] At 37°C, treatment of HT cells with 0.5 μg / ml of selenium resulted in approximately a 10% decrease in proliferation compared to control HT cells. At 39°C, treatment of HT cells with 0.5 μg / ml of selenium resulted in approximately a 20% decrease in proliferation compared to control HT cells. At 41°C, treatment of HT cells with 0.5 μg / ml of selenium resulted in approximately a 60% decrease compared to control HT cells.

[0041] At 37°C, treatment of HT cells with fish oil at a concentration of 25 μM resulted in approximately a 25% decrease in proliferation compared to control HT cells. At 39°C, treatment of HT cells with fish oil at a concentration of 25 μM resulted in approximately a 35% decrease in proliferation compared to control HT cells. At 41°C, treatment of HT cells with fish oil at a concentration of 25 μM resulted in approximately a 60% decrease in proliferation compared to control HT cells.

[0042] At 37°C, treatment of HT cells with a combination of 0.5 μg / ml selenium and 25 μM fish oil resulted in approximately a 30% decrease in proliferation compared to control HT cells. At 39°C, treatment of HT cells with 0.5 μg / ml selenium and 25 μM fish oil resulted in approximately a 35% decrease in proliferation compared to control HT cells, demonstrating a synergistic effect. At 41°C, exposure to 0.5 μg / ml selenium and 25 μM fish oil resulted in approximately a 60% decrease in proliferation compared to control HT cells.

[0043] Figure 2 The results depicted in Figure 2 show the surprising result of reduced cell proliferation in HT cells when cells were exposed to both fish oil and selenium. At each successively higher temperature, overall cell proliferation decreased. When 0.5 μg / ml of selenium or 25 μM concentration of fish oil were combined, cell proliferation was reduced relative to the control. Surprisingly, the combination of 0.5 μg / ml of selenium and 25 μM concentration of fish oil showed a synergistic effect in reducing cell proliferation that was better than either fish oil or selenium alone.

[0044] Figure 3 is a bar graph depicting the effects of fish oil and selenium alone and in combination on the sensitization of BFTC-905 bladder cancer cells (BFTC cells) to hyperthermia. The sensitizing effects of selenium, fish oil, and the combination of selenium and fish oil were evident in BFTC cells exposed to hyperthermia.

[0045] BFTC cells were subjected to 37°C (control) and superheated temperatures (39°C and 41°C). At each temperature, each group of BFTC cells was (1) treated with 0.5 μg / ml of selenium, 12.5 μM concentration of fish oil, or both 0.5 μg / ml of selenium and 12.5 μM concentration of fish oil, (2) maintained at 37°C, 39°C, or 41°C for 2 hours, (3) maintained in a CO2 incubator for 72 hours, and (4) examined for cell proliferation as a percentage of the BFTC control. Selenium was administered in the form of selenium yeast. Again, it is contemplated that the source of selenium is not limited to selenium yeast and can be administered using any method known in the art.

[0046] BFTC cells were kept for 2 hours at all tested temperatures and exposed to 0.5 μg / ml of selenium, 12.5 μM of fish oil concentration, or both 0.5 μg / ml of selenium and 12.5 μM of fish oil concentration, showing an overall reduction in BFTC cell proliferation compared to the BFTC control. In the absence of selenium and / or fish oil, incubation at 39°C reduced proliferation by approximately 13% compared to control BFTC cells, and incubation at 41°C reduced proliferation by approximately 28% compared to control BFTC cells.

[0047] At 37°C, treatment of BFTC cells with 0.5 μg / ml of selenium resulted in approximately a 10% decrease in proliferation compared to the BFTC control. At 39°C, treatment of BFTC cells with 0.5 μg / ml of selenium resulted in approximately a 15% decrease in proliferation compared to the BFTC control. At 41°C, treatment of BFTC cells with 0.5 μg / ml of selenium resulted in approximately a 30% decrease in proliferation compared to the BFTC control.

[0048] At 37°C, treatment of BFTC cells with fish oil at a concentration of 12.5 μM resulted in approximately a 30% decrease in proliferation compared to the BFTC control. At 39°C, treatment of BFTC cells with fish oil at a concentration of 12.5 μM resulted in approximately a 35% decrease in proliferation compared to the BFTC control. At 41°C, treatment of BFTC cells with fish oil at a concentration of 12.5 μM resulted in approximately a 45% decrease in proliferation compared to the BFTC control.

[0049] At 37°C, treatment of BFTC cells with 0.5 μg / ml of selenium and 12.5 μM of fish oil resulted in approximately a 15% decrease in proliferation compared to the BFTC control. At 39°C, treatment of BFTC cells with 0.5 μg / ml of selenium and 12.5 μM of fish oil resulted in approximately a 35% decrease in proliferation compared to the BFTC control. At 41°C, exposure to 0.5 μg / ml of selenium and 12.5 μM of fish oil resulted in approximately a 50% decrease in proliferation compared to the BFTC control.

[0050] Figure 3 The results depicted in Figure 2 show the surprising result of reduced cell proliferation in BFTC cells when the cells were exposed to both fish oil and selenium. Overall cell proliferation decreased at each successively higher temperature. When 0.5 μg / ml of selenium or 25 μM of fish oil were combined, cell proliferation was reduced relative to the control. With the exception of BFTC cell cultures treated at 37°C with 0.5 μg / ml of selenium and 12.5 μM of fish oil, the selenium and fish oil mixture demonstrated a synergistic effect in reducing cell proliferation that was superior to either fish oil or selenium alone.

[0051] Figure 4is a line graph depicting the effects of selenium, fish oil, and a selenium / fish oil combination on the proliferation of A549 lung cancer cells (A549 cells).

[0052] A549 cells were exposed to 0 μM (PBS), 25 μM, 50 μM, or 100 μM fish oil. At each concentration of fish oil, each group of A5 cells was (1) exposed to 0 μg / ml of selenium, 0.5 μg / ml of selenium, 1 μg / ml of selenium, 2 μg / ml of selenium, or 4 μg / ml of selenium, (2) maintained at 37°C, 39°C, or 41°C for 2 hours, (3) maintained in a CO2 incubator for 72 hours, and (4) examined for cell proliferation as a percentage of PBS. Selenium was administered in the form of selenium yeast. Again, it is contemplated that the source of selenium is not limited to selenium yeast and can be administered using any method known in the art.

[0053] Maintaining each group of A549 cells in a higher concentration of fish oil than PBS for 2 hours resulted in an overall reduction in A549 cell proliferation compared to PBS. Furthermore, treating cell cultures with increasing concentrations of selenium (i.e., from 0.5 to 4 μg / ml) showed the effects of increasing selenium concentration on cell proliferation relative to control (PBS only) A549 cells, as well as the effects of fish oil on A549 cells. The synergistic effect was particularly evident at high fish oil concentrations, with even low additions of selenium resulting in a substantial reduction in cell proliferation compared to the effects of either fish oil or selenium (alone or additively).

[0054] At 4 μg / ml of selenium, cell proliferation in each A549 cell culture was reduced to approximately 20% of that in PBS. However, exposure to increasing amounts of fish oil and selenium demonstrated an anti-proliferative effect at levels between 0 and 4 μg / ml of selenium, with high fish oil and selenium concentrations reducing cell proliferation more than low fish oil and selenium concentrations.

[0055] Table 1 shows the effects of fish oil and selenium on the cell cycle distribution of MDA-MB breast cancer cells (MDA-MB cells) at 39°C and 41°C.

[0056]

[0057] Table 1

[0058] The concentration of fish oil represents its DHA content. Each gram of fish oil contains 220mg DHA and 330mg EPA. The cells were warmed for 2 hours at superheated temperature and then placed in a CO2 incubator for 72 hours, and then cell cycle analysis was performed. Selenium was applied in the form of selenium yeast. As shown, using selenium, fish oil, and a combination of selenium and fish oil resulted in a significant redistribution of the cell cycle occupancy of these cells under superheated conditions. Specifically, the percentage of cells in the subG1 phase (which is associated with apoptosis) increased.

[0059] In addition to regulating cell cycle occupancy, selenium, fish oil, and the combination of selenium and fish oil can modulate the concentrations of certain proteins in cancer cells.

[0060] Figure 5 Described is the concentration regulation of pAMPKα and COX-2 by selenium yeast and fish oil. Increased AMPK signaling is believed to prevent the proliferation and metastasis of tumor cells. COX-2 is believed to regulate cell proliferation and apoptosis in solid tumors, and COX-2 inhibition is studied as a treatment model. Surprisingly, the inventors found that selenium, fish oil, and selenium / fish oil combinations lead to increased pAMPKα levels and reduced COX-2 levels in A549 lung cancer cells (including GAPDH as a control). When these are used in combination with superheat, these effects may contribute to visible enhancement and / or synergistic effects.

[0061] Suitable formulations incorporating fish oil and selenium yeast include the nutritional supplement formulations provided in Table 2, which incorporate fish oil and selenium yeast components along with other nutritional components. This nutritional supplement has been found to have a high level of acceptance and has unexpected beneficial anti-tumor activity in combination with conventional therapies. Therefore, applicants believe that the use of such a nutritional supplement can provide a beneficial enhancement to hyperthermia therapy and can be achieved at lower, relatively safe temperatures that are easily achieved using conventional methods and are safer for patients to use. Providing this safety margin with respect to body and local temperatures can lead to wider acceptance and application of this non-toxic treatment modality.

[0062]

[0063]

[0064]

[0065]

[0066] Table 2

[0067] The composition shown in Table 2 includes components with multiple physiological and biochemical effects, including anti-inflammatory activity, lowering blood sugar levels, reducing cholesterol and anti-tumor activity. Other components provide high-level essential vitamins, minerals and amino acid supplements. Other components (such as enzymes, lecithin) are used to help digest and absorb the components of the composition. This combination provides a synergistic effect, which exceeds the simple cumulative effect of a single component. It should be understood that the composition shown in Table 2 also includes some flavorings (such as brown sugar, honey, vanilla spices and masking agents), which are used to improve palatability and acceptance. Some components (such as honey, brown sugar, milk, rice protein, casein) can provide both fragrance and thermal energy. The inventors have found that the above-mentioned flavoring combination effectively provides the compliance of nutritional supplement consumption with an effective amount. In some embodiments, this type of flavoring can be excluded when the effect of the nutritional supplement is not adversely affected, thereby providing a functional nutritional supplement that only includes essential components.

[0068] It should be understood that the components of the nutritional supplement of the present invention's concept can be provided in powders, granules, liquids, suspensions and / or emulsions. In a preferred embodiment, the components of the nutritional supplement are provided in powders and / or granules. Similarly, in a preferred embodiment of the present invention's concept, the components of the nutritional supplement are provided in the relative amounts indicated in Table 2. In some embodiments, the components of the nutritional supplement are provided in single, mixed formulations. In other embodiments, the components of the nutritional supplement can be provided in kits or similar components that contain isolated or separately packaged different components of the formulation (e.g., to provide different storage conditions that are conducive to component stability).

[0069] The components shown in Table 2 can be provided in a single formulation (e.g., with a pill, tablet, capsule, powder, liquid, suspension, etc.) or can be isolated into different formulations (e.g., with a pill, tablet, capsule, powder, liquid, suspension, or a combination thereof). The amounts shown in Table 2 are exemplary and represent the typical daily doses provided to adults of normal stature and other normal health. These amounts can be adjusted to consider the differences in body weight, sex, medical conditions, etc. For example, a patient with a relatively small body weight of 40 kg or less can benefit from a dose provided at or below the lower limit of the provided range, while a patient with a relatively large body weight of 100 kg or more may need a dose provided at the upper limit (or higher) of the recorded range. In some embodiments, the daily dose can be distributed in multiple doses throughout the day. In some such embodiments, the individual compositions of the doses distributed in this manner can be identical. In other embodiments, the compositions of the doses distributed in this manner can be different, as long as the sum of these doses provides the required supplement.

[0070] It should be understood that the oils found in the formulations (e.g., linseed oil, rapeseed oil, borage oil, olive oil, fish oil, pure lemon oil, pure orange oil, mixed tocopherols) are at least consumer grade and preferably highly purified (>95% pure). It should also be understood that the mineral components (e.g., potassium, calcium, sodium, magnesium, iron, manganese) can be provided as any safe and absorbable salt (e.g., halide, phosphate, carbonate, sulfate), oxide, or organic complex (e.g., gluconate). It should also be understood that certain metals (e.g., chromium, molybdenum, selenium) can be provided in the form of yeast components, which can include yeast-containing powders or suspensions and / or complexes with peptides or amino acids resulting from yeast metabolism of such metals. Similarly, it should be understood that preparations of various non-yeast fungi (e.g., Cordyceps sinensis, Ganoderma lucidum, Shiitake mushrooms, Grifola frondosa, and Coriolus versicolor) can include powders or granular preparations derived from the dried / lyophilized fruiting bodies of these fungi.

[0071] The nutritional supplement of the present invention's concept can be provided as a unit dose with a range from about 1mg / kg body weight to about 100mg / kg body weight. This type of unit dose can be provided according to a schedule ranging from 4 times a day to once a week. The nutritional supplement can be provided with one or more pills or capsules. Alternatively, the nutritional supplement can be provided with a powder, granule and / or liquid formulation, which is added to food or beverage before consumption. In some embodiments, the nutritional supplement can be provided with foods, such as food or candy bars. In other embodiments, the nutritional supplement can be provided with a solution, suspension or beverage suitable for oral consumption and / or supplied by tube feeding.

[0072] It should be understood that packaging that excludes light, moisture, and / or oxygen can be used to extend the shelf life of the nutritional supplement. Similarly, the nutritional supplement of the present invention can be packaged with a hygroscopic agent (e.g., silica gel), a non-reactive gas (e.g., N2 or a rare gas), and / or under vacuum to extend the shelf life. For example, such packaging can provide a single unit dose of the nutritional supplement of the present invention and additionally provide instructions for preparation and / or dosing frequency.

[0073] The present invention contemplates the use of hyperthermia in conjunction with nutritional supplements having varying concentrations of fish oil and selenium. The hyperthermia regimen consists of heating body tissue to supranormal body temperatures to sensitize cancer cells to treatment methods or to directly kill cancer cells. For example, the hyperthermia regimen can be administered for 45-60 minutes over 4-12 treatment sessions. Hyperthermia can be applied locally, regionally, or to the entire body and can be derived from microwave energy, radiofrequency energy, ultrasound energy, or any other source of energy sufficient to heat tissue to supranormal temperatures. In some embodiments, heat can be applied directly to cancerous tissue, for example, by inserting a heated probe into a tumor.

[0074] In one embodiment, a nutritional supplement containing fish oil at a concentration of 25 μM is administered to the patient by oral swallowing. After waiting a sufficient amount of time to allow the fish oil to be absorbed into the bloodstream (e.g., within a 2-hour period), heat is applied to the cancerous tissue to raise the temperature of the tissue to supernormal levels for a period of 45 minutes. For example, a microwave heating element can be applied locally to the surface of the patient's skin above the tumor. In another example, a probe can be inserted into the tumor and heated. In still another example, the patient can be instructed to wear a full-body suit and the patient's body can be heated to 39°C.

[0075] In another embodiment, a nutritional supplement comprising 1000 ng / ml of selenium in the form of selenium yeast can be administered to the patient by oral swallowing. After waiting a sufficient amount of time to allow the selenium to be absorbed into the bloodstream (e.g., within a 2-hour period), heat is applied to the cancerous tissue to raise the temperature of the tissue to supernormal levels for a period of 60 minutes. As in the previous embodiment, a microwave heating element can be applied locally to the surface of the patient's skin above the tumor. In another example, a probe can be inserted into the tumor and heated. In still another example, the patient can be instructed to wear a full-body suit and the patient's body can be heated to 39°C.

[0076] In a preferred embodiment, a nutritional supplement comprising 25 μM concentration of fish oil and 1000 ng / ml selenium in the form of selenium yeast can be administered to the patient by oral swallowing. After waiting for a sufficient amount of time to allow the fish oil and selenium to be absorbed into the bloodstream (e.g., within a 2-hour period), heat is applied to the cancerous tissue to raise the temperature of the tissue to a supernormal level for a period of 55 minutes. As in the previous embodiment, a microwave heating element can be applied locally to the surface of the patient's skin above the tumor. In another example, a probe can be inserted into the tumor and heated. In still another example, the patient can be instructed to wear a full-body suit, and the patient's body can be heated to 39°C.

[0077] It is contemplated that any method effective for exposing cancer cells to fish oil and / or selenium may be used, alone or in combination, and at any respective concentration. For example, the fish oil and selenium may be injected directly into the cancerous tissue. In another example, the fish oil and selenium may be applied topically to the skin or outermost tissue of an organ for absorption into the cancerous tissue.

[0078] It is also contemplated that heat may be applied to the cancerous tissue using any method known in the art. For example, a heating element may be applied directly to the cancerous tissue or tissue surrounding the cancerous tissue to transfer heat by conduction. In another example, heat may be transferred using a heating element that delivers electromagnetic energy waves, including, for example, an infrared heating element.

[0079] It will be appreciated that the technology of the present disclosure provides a number of advantageous technical effects, including sensitizing tumor cells to the effects of hyperthermia using well-tolerated substances and without the use of radiation and / or chemotherapeutic drugs.

[0080] The description of the inventive subject matter herein includes information that may be useful in understanding the invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any specific publication explicitly or implicitly referenced is prior art.

[0081] The grouping of optional elements or embodiments of the invention disclosed herein is not to be construed as limiting. Each group member may be cited and claimed individually or in any combination with other members of the group or other elements found herein. For reasons of convenience and / or patentability, one or more group members may be included or deleted in a group. When any such inclusion or deletion occurs, this specification is deemed to include the group of modifications, thereby achieving the written description of all Markush groups used in the appended claims.

[0082] The detailed description provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus, if one embodiment includes elements A, B, and C, and a second embodiment includes elements B and D, then the inventive subject matter is considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.

[0083] It should be apparent to one of ordinary skill in the art that many more modifications are possible in addition to those already described without departing from the inventive concept herein. Therefore, the subject matter of the present invention is not limited except in the spirit of the appended claims. Moreover, when interpreting the specification and claims, all terms should be interpreted in the broadest possible manner consistent with the context. Specifically, the terms "comprise" and "include" should be interpreted as referring to elements, parts or steps in a non-exclusive manner, indicating that the mentioned elements, parts or steps may exist or be utilized, or be combined with other elements, parts or steps not explicitly mentioned. When the specification claims refer to at least one selected from A, B, C... and N, the text should be interpreted as requiring only one element in the group, rather than A plus N, or B plus N, etc.

Claims

1. A method for reducing cancer cell proliferation, comprising: administering to the cancer cells a sensitizer selected from selenium, fish oil, and a combination of selenium and fish oil, wherein the selenium and fish oil are administered in amounts effective to respectively increase the sensitivity of the cancer cells, and wherein the administration of the combination of selenium and fish oil is in an amount effective to synergistically increase the sensitivity of the cancer cells over the administration of either selenium or fish oil alone; and The cancer cells were exposed to temperatures exceeding 37°C.

2. The method of claim 1, wherein the temperature is about 39°C.

3. The method of claim 1, wherein the temperature is about 41°C.

4. The method according to one of claims 1 to 3, wherein the cancer cells are breast cancer cells and the selenium is in the form of selenium yeast or a peptide derived from selenium yeast.

5. The method according to one of claims 1 to 3, wherein the cancer cells are bladder cancer cells and the selenium is in the form of selenium yeast or a peptide derived from selenium yeast.

6. The method according to one of claims 1 to 3, wherein the cancer cells are lung cancer cells and the selenium is in the form of selenium yeast or a peptide derived from selenium yeast.

7. The method according to claim 1, wherein the sensitizer comprises a plurality of components as shown in Table 2.

8. A method for modulating the pAMPKα level in cancer cells, comprising administering a sensitizer selected from the group consisting of selenium, fish oil, and a combination of selenium and fish oil. The method of claim 8 , wherein the cancer cells have increased levels of pAMPKα.

10. The method according to claim 8 or 9, wherein the selenium is in the form of selenium yeast or a peptide derived from selenium yeast.

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