DEVICE AND ANTITUMOR DRUGS FOR THE TREATMENT OF NEOPLASTIC CELLS - Patent application

Abstract

Provided is an anti-tumor drug for use in a method of preventing or inhibiting the proliferation of tumor cells in a target area of ​​a living body, the method comprising the steps of treating neoplastic cells with the anti-tumor drug while simultaneously applying an electrical current wave to the target area for a predetermined period of time during which the neoplastic cells are treated with the anti-tumor drug, the electrical current wave having a waveform with a fundamental frequency of 2 MHz or greater.

Description

[Technical field]

[0001] The present invention relates to devices configured to prevent or inhibit the proliferation of neoplastic cells in a target area of ​​a living body, and in particular to the use of the apparatus to prevent or inhibit the proliferation of neoplastic cells in combination with an anti-tumor drug. [Background technology]

[0002] Neoplasms or tumors result from cellular changes that escape regulatory mechanisms, grow, and spread throughout the body. There are many types of neoplasms that can affect virtually any organ. They are usually classified as benign or malignant, depending on the ability of the tumor cells to invade surrounding structures and more distant organs. In addition to tumors of solid organs, the term neoplasm also includes tumors of blood cells, such as lymphomas and leukemias.

[0003] Despite great advances in clinical practice over the last decades with targeted preventive measures, early diagnosis and treatment, neoplasms remain a leading cause of death and impaired quality of life.

[0004] Tumor treatment is essentially based on surgery, in particular local surgery, radiation therapy and drug therapy.

[0005] Glioblastoma multiforme (GBM) is one of the most aggressive tumors in humans due to the high ability of GBM tumor cells to invade parenchymal tissue adjacent to the tumor lesion (Non-Patent Document 1).

[0006] Patients with GBM usually undergo surgical resection of the lesion, preferably complete resection, followed by radiation therapy and chemotherapy with the alkylating antitumor agent temozolomide (Non-Patent Document 2).

[0007] However, despite multifaceted therapeutic approaches, GBM is characterized by high recurrence rates, drug resistance, and devastating neurological deterioration (Non-Patent Document 3).

[0008] GBM is a particularly invasive tumor, and therefore complete surgical resection of the lesion is virtually impossible to perform, so almost all patients experience recurrence and have a survival time of only 14 months (Non-Patent Document 4). Furthermore, the survival rate of patients 5 years after surgery is only less than 5% (Non-Patent Document 5).

[0009] There is therefore a clear urgent need to improve the quality of life of patients by developing new therapeutic strategies, improving the efficacy of the drugs used and reducing the associated side effects.

[0010] In this sense, one of the most promising therapeutic approaches is the use of non-ionizing electromagnetic fields (EMF).

[0011] This treatment is particularly promising due to its excellent safety profile, low toxicity, and the possibility of combining it with conventional drug therapies (Non-Patent Document 6).

[0012] In recent years, several EMF technologies have been tested and shown to have good levels of efficacy against various types of tumors, both when used alone and in combination with chemotherapy (Non-Patent Document 7).

[0013] It has been found that different frequency ranges trigger different response mechanisms in cells, which can result in the inhibition of cell proliferation in tumor cells by affecting the formation and stability of the mitotic spindle in these cells (Non-Patent Document 8).

[0014] In 2011, the U.S. Food and Drug Administration approved the use of Tumor Treating Fields (TTFields) to treat patients with recurrent glioblastoma multiforme.

[0015] Several prior art documents to this effect have been published, such as, for example, US Pat. No. 5,399,633 and US Pat. No. 5,499,623. [Prior art documents] [Patent documents]

[0016] [Patent Document 1] U.S. Patent Application Publication No. 2020 / 269041 [Patent Document 2] European Patent No. 3439733 [Patent Document 3] European Patent No. 1087691 [Non-Patent Document]

[0017] [Non-Patent Document 1] L. M. DeAngelis, “Brain tumors”, The New England Journal of Medicine, 2001, Vol. 344, pp. 114 - 123 [Non-Patent Document 2] C. Alifieris, D. T. Trafalis, “Glioblastoma multiforme: Pathogenesis and treatment”, Pharmacology & therapeutics, 2015, Vol. 152, pp. 63 - 82 [Non-Patent Document 3] T. A. Wilson, M. A. Karajannis, D. H. Harter, “Glioblastoma multiforme: State of the art and future therapeutics”, Surg Neurol Int, 2014, Vol. 5, pp. 64 - 64 [Non-Patent Document 4] K. P. Becker, J. Yu, “Status quo--standard-of-care medical and radiation therapy for glioblastoma”, Cancer journal (Sudbury, Massachusetts, United States of America), 2012, Vol. 18, pp. 12 - 19 [Non-Patent Document 5] Q. T. Ostrom et al., “The epidemiology of glioma in adults: a “state of the science” review”, Neuro-Oncology, 2014, Vol. 16, pp. 896-913 [Non-Patent Document 6] MO Mattsson, M. Simko “Emerging medical applications based on non-ionizing electromagnetic fields from 0 Hz to 10 THz” Medical devices (Auckland, New Zealand), 2019, Vol. 12, pp. 347-368 [Non-Patent Document 7] F. Pasi et al. “Pulsed Electromagnetic Field with Temozolomide Can Elicit an Epigenetic Pro-apoptotic Effect on Glioblastoma T98G Cells”, Anticancer Res. 2016, No. 36, pp. 5821-5826 [Non-Patent Document 8] M. Giladi et al., “Mitotic Spindle Disruption by Alternating Electric Fields Leads to Improper Chromosome Segregation and Mitotic Catastrophe in Cancer Cells”, Scientific Reports, 2015, Volume 5, p.18046 [Non-Patent Document 9] G. Pozzato and G. Vignato, “Teoria della risonanza quantica molecolare nella realizzazione del bisturi elettronico “Vesalius””, Quintessence International, 2003, 5 / 6:153-155 [Non-Patent Document 10] S. Sella et al., “In-vitro analysis of quantum molecular resonance effects on human mesenchymal stromal cells” PLoS One, 2018, Vol. 13, e0190082 Summary of the Invention [Problem to be solved by the invention]

[0018] SUMMARY OF THE PRESENT EMBODIMENT It is an object of the present invention to provide an alternative device and method to known types for preventing or inhibiting the proliferation of neoplastic cells in a target area of ​​the living body.

[0019] In particular, it is an object of the present invention that such devices and methods may also be used in the treatment of aggressive neoplasms such as glioblastoma multiforme.

[0020] It is a further object of the present invention that such devices and methods may be used on patients suffering from non-aggressive neoplasms.

[0021] Furthermore, it is an object of the present invention to achieve an antitumor drug with greater therapeutic efficacy and reduced toxicological profile, improving the quality of life of patients. [Means for solving the problem]

[0022] The above object is achieved by an antitumor drug as claimed in the main claim.

[0023] The above object is also achieved by a device as claimed in claim 7.

[0024] Moreover, these objectives are also achieved by the methods of treating neoplastic cells described below.

[0025] In particular, the present invention relates to an anti-tumor drug for use in a method of preventing or inhibiting the proliferation of neoplastic cells in a target area of ​​a living body, the method comprising treating the neoplastic cells with the anti-tumor drug before, simultaneously with or after application of an electric current wave to the target area for a predetermined period of time, the electric current wave being characterized by having a waveform having a fundamental frequency of 2 MHz or greater.

[0026] Preferably, such a current wave has a fundamental frequency in the range of 2 to 64 MHz, more preferably in the range of 2 to 20 MHz, and even more preferably in the range of 4 to 20 MHz.

[0027] Even more preferably, such a current wave has a waveform having a fundamental frequency of about 4 MHz.

[0028] Such application of a current wave is also referred to herein as quantum molecular resonance or QMR.

[0029] Further, in accordance with an aspect of the present invention, such current wave has a sinusoidal waveform.

[0030] According to one aspect of the invention, this waveform is modified by the presence of harmonics.

[0031] According to another aspect of the invention, the sinusoidal waveform is modified by the presence of at least second and third harmonics.

[0032] According to one aspect of the invention, treatment of tumor cells with an antitumor drug is performed simultaneously with or subsequent to the application of said electrical current waves.

[0033] Preferably, according to one aspect of the present invention, the treatment of the neoplastic cells with an anti-tumor drug is performed simultaneously with the application of an electrical current wave to the target area, wherein the neoplastic cells are treated with the anti-tumor drug.

[0034] According to one aspect of the present invention, the antitumor drug comprises temozolomide.

[0035] According to one aspect of the invention, the tumor cells include glioblastoma cells.

[0036] In particular, the target region belongs to an organism, preferably a human.

[0037] Preferably, the target area includes the central nervous system.

[0038] Also part of the invention is an apparatus for preventing or inhibiting the proliferation of neoplastic cells in a target area of ​​a living body, the apparatus being configured to generate a current wave having a waveform with a fundamental frequency of 2 MHz or more, preferably in the range of 2-64 MHz, more preferably in the range of 2-20 MHz, even more preferably in the range of 4-20 MHz, and even more preferably about 4 MHz, the waveform being a sinusoidal waveform distorted by the presence of harmonics.

[0039] The device comprises one or more electrodes connected to at least one radio frequency circuit which supplies each of the electrodes with a current wave having a fundamental frequency of 2 MHz or greater.

[0040] Such one or more electrodes are configured to transmit waves of electrical current to a target area.

[0041] According to one aspect of the invention, the electrodes of the device are implantable electrodes, which are implanted at or near the aforementioned target area.

[0042] Methods for preventing or inhibiting the proliferation of tumor cells in a target area are also part of the invention.

[0043] The target region preferably belongs to an organism.

[0044] According to one embodiment of the method of the present invention, the method comprises treating tumor cells by applying an electric current wave to the target area for a predetermined period of time, the electric current wave having a waveform having a fundamental frequency of 2 MHz or more, preferably in the range of 2 to 64 MHz, more preferably in the range of 2 to 20 MHz, even more preferably in the range of 4 to 20 MHz, and even more preferably about 4 MHz.

[0045] Moreover, in accordance with one aspect of the method of the present invention, such waveform is a sinusoidal waveform.

[0046] Again, in accordance with one aspect of the present invention, this sinusoidal waveform is distorted by the presence of harmonics.

[0047] Again, according to another aspect of the method of the present invention, the sinusoidal waveform is modified by the presence of at least second and third harmonics.

[0048] Furthermore, according to one embodiment of the method of the present invention, the tumor cells comprise glioblastoma cells.

[0049] According to one aspect of the invention, the target area comprises the central nervous system.

[0050] According to a further aspect of the method of the present invention, the method comprises the steps of: Connecting to one or more electrodes a device configured to generate a current wave, the current wave having a fundamental frequency of 2 MHz or greater, preferably a sinusoidal waveform distorted by the presence of harmonics. - attaching at least one of these electrodes to a body surface of a living subject on which a target region containing tumor cells is located. Activating the device to transfer a current wave to the electrodes and leaving the device activated for a predetermined period of time.

[0051] Alternatively, according to another aspect of the method of the present invention, the method comprises the steps of: Connecting a device configured to generate a current wave, the current wave having a fundamental frequency of 2 MHz or greater, preferably a sinusoidal waveform modified by the presence of harmonics, to one or more implanted electrodes. Implanting at least one of these electrodes in a living body at or near a target area containing tumor cells. Activating the device to transfer a current wave to the electrodes and leaving the device activated for a predetermined period of time.

[0052] Preferably, the method of the present invention utilises the apparatus of the present invention.

[0053] Further features and advantages of the agents, devices and methods of the present invention will become apparent to those skilled in the art from the following description of preferred embodiments of the invention, given by way of illustration and not of limitation. [Brief description of the drawings]

[0054] [Figure 1] Figure 1 shows representative images of glioblastoma A172 cell cultures taken under an inverted optical microscope, taken at 0, 24, and 48 hours after the end of QMR stimulation, as well as untreated control cells, and was taken at 10x magnification using an Axiovert40CFL inverted optical microscope. [Diagram 2] Figure 2 shows the cell viability of A172 cells 0, 24, and 48 hours after the end of QMR stimulation compared to control cells (mean ± standard error of at least 4–5 independent experiments; **=p<0.01; QMR vs. control (one-sample t-test)). [Figure 3a] Figure 3a shows the percentage of A172 cells in each phase of the cell cycle (G0-G1, S, G2-M). After the end of QMR stimulation, at 0 h (Figure 3a), cell cycle progression was monitored by flow cytometry (mean ± standard error of at least 4–6 independent experiments; *=p<0.05; QMR vs. control (unpaired t-test)). [Figure 3b] Figure 3b shows the percentage of A172 cells in each phase of the cell cycle (G0-G1, S, G2-M). After the end of QMR stimulation, 24 h later (Figure 3b), cell cycle progression was monitored by flow cytometry (mean ± standard error of at least 4–6 independent experiments; * = p < 0.05; QMR vs. control (unpaired t test)). [Figure 3c]Figure 3c shows the percentage of A172 cells in each phase of the cell cycle (G0-G1, S, G2-M). After the end of QMR stimulation, 48 h later (Figure 3c), cell cycle progression was monitored by flow cytometry (mean ± standard error of at least 4–6 independent experiments; * = p < 0.05; QMR vs. control (unpaired t test)). [Figure 4a] Figure 4a shows the induction of apoptosis in A172 cells at 0 h (Figure 4a) after the end of QMR stimulation. The histogram shows the percentage of live and dead cells, subdividing early apoptotic cells from late apoptotic and dead cells (mean ± SEM of at least 3–5 independent experiments; ** = p < 0.01; QMR vs. control (unpaired t test)). [Figure 4b] Figure 4b shows the induction of apoptosis in A172 cells 24 hours after the end of QMR stimulation (Figure 4b). The histogram shows the percentage of live and dead cells, subdividing early apoptotic cells from late apoptotic and dead cells (mean ± SEM of at least 3–5 independent experiments; ** = p < 0.01; QMR vs. control (unpaired t test)). [Figure 4c] Figure 4c shows the induction of apoptosis in A172 cells 48 h after the end of QMR stimulation (Figure 4c). The histogram shows the percentage of live and dead cells, subdividing early apoptotic cells from late apoptotic and dead cells (mean ± SEM of at least 3–5 independent experiments; ** = p < 0.01; QMR vs. control (unpaired t test)). [Figure 5a] Figure 5a shows the effect of QMR stimulation on mesenchymal stem cells (BM-MSCs). Figure 5a was acquired at 10x magnification using an Axiovert40CFL inverted optical microscope and shows representative images of BM-MSC cell cultures at 0 and 24 hours after 24 hours of QMR stimulation, as well as images of control cells. Results are presented as the mean ± standard error of at least 3-4 independent experiments; *=p<0.05; QMR vs. control. [Figure 5b]Figure 5b shows the effect of QMR stimulation on mesenchymal stem cells (BM-MSCs). Figure 5b was obtained by trypan blue test, in which the viability of BM-MSCs at 0 and 24 hours after 24 hours of QMR stimulation was compared with that of control cells. Results are shown as mean ± standard error of at least 3-4 independent experiments; *=p<0.05; QMR vs. control. [Figure 5c] Figure 5c shows the effect of QMR stimulation on mesenchymal stem cells (BM-MSCs). Figures 5c to 5f show the percentage of BM-MSCs in each phase of the cell cycle (G0-G1, S, G2-M) and the induction rate of apoptosis obtained by flow cytometry at 0 and 24 hours after the end of QMR stimulation (Figures 5c and 5e correspond to Figures 5d and 5f, respectively). Results are shown as the mean ± standard error of at least 3-4 independent experiments; *=p<0.05; QMR vs. control. [Figure 5d] Figure 5d shows the effect of QMR stimulation on mesenchymal stem cells (BM-MSCs). Figures 5c-5f show the percentage of BM-MSCs in each phase of the cell cycle (G0-G1, S, G2-M) and the induction rate of apoptosis obtained by flow cytometry at 0 and 24 hours after the end of QMR stimulation (Figures 5c and 5e correspond to Figures 5d and 5f, respectively). Results are shown as the mean ± standard error of at least 3-4 independent experiments; *=p<0.05; QMR vs. control. [Figure 5e] Figure 5e shows the effect of QMR stimulation on mesenchymal stem cells (BM-MSCs). Figures 5c to 5f show the percentage of BM-MSCs in each phase of the cell cycle (G0-G1, S, G2-M) and the induction rate of apoptosis obtained by flow cytometry at 0 and 24 hours after the end of QMR stimulation (Figures 5c and 5e correspond to Figures 5d and 5f, respectively). Results are shown as the mean ± standard error of at least 3-4 independent experiments; *=p<0.05; QMR vs. control. [Figure 5f]Figure 5f shows the effect of QMR stimulation on mesenchymal stem cells (BM-MSCs). Figures 5c-5f show the percentage of BM-MSCs in each phase of the cell cycle (G0-G1, S, G2-M) and the induction rate of apoptosis obtained by flow cytometry at 0 and 24 hours after the end of QMR stimulation (Figures 5c and 5e correspond to Figures 5d and 5f, respectively). Results are shown as the mean ± standard error of at least 3-4 independent experiments; *=p<0.05; QMR vs. control. [Figure 6] Figure 6 shows representative images of the karyotype of BM-MSC cells. These images compare the karyotype of BM-MSC cells under basal conditions (control) and after QMR stimulation. Chromosomes were in metaphase and detected by the GTG method. These images are representative of three independent experiments. [Figure 7a] Figure 7a shows the invasiveness and invasion of A172 cells after QMR stimulation. In Figure 7a, A172 cell colonies were labeled with calcein and counted using an AxiovertCFL inverted optical microscope. Using an inverted optical microscope, the percentage of scratch closure, an indicator of the rate of cell migration, was quantified using ImageJ software. Results are shown as the mean ± standard error of at least 3-5 independent experiments; *=p<0.05; ***=p<0.001; QMR vs. control. [Figure 7b] Figure 7b shows the invasiveness and invasion of A172 cells after QMR stimulation. In Figure 7b, A172 cells were cultured in silicone inserts directly in Petri dishes, and the cell migration rate was evaluated up to 48 h after QMR stimulation. Using an inverted optical microscope, the percentage of scratch closure, an index of cell migration rate, was quantified using ImageJ software. Results are shown as the mean ± standard error of at least 3-5 independent experiments; *=p<0.05; ***=p<0.001; QMR vs. control. [Figure 8a]Figure 8a shows the effect of QMR stimulation on T98G brain tumor cells. Figure 8a shows images of T98G cell cultures taken at 5x magnification using an Axiovert40CFL inverted optical microscope. Images are shown at 0 and 24 hours after 24 hours of QMR stimulation, and images of control cells are also shown. The results are presented as the mean ± standard error of at least 3-5 independent experiments; *=p<0.05; **=p<0.01; QMR vs. control. [Figure 8b] Figure 8b shows the effect of QMR stimulation on T98G brain tumor cells. Figure 8b shows the viability of T98G cells at 0, 24, and 48 hours after 24 hours of QMR stimulation, and the test was performed using trypan blue. The results are shown as the mean ± standard error of at least 3-5 independent experiments; *=p<0.05; **=p<0.01; QMR vs. control. [Figure 8c] Figure 8c shows the effect of QMR stimulation on T98G brain tumor cells. Figure 8c shows the results of counting the colonies of calcein-labeled T98G cells using an AxiovertCFL inverted optical microscope. These results are shown as the mean ± standard error of at least 3-5 independent experiments; *=p<0.05; **=p<0.01; QMR vs. control. [Figure 8d] Figure 8d shows the effect of QMR stimulation on T98G brain tumor cells. Figure 8d shows the percentage of T98G cells in each phase of the cell cycle (G0-G1, S, G2-M) and the induction rate of apoptosis obtained by flow cytometry at 0, 24, and 48 hours after the end of QMR stimulation. The results are shown as the mean ± standard error of at least 3-5 independent experiments; *=p<0.05; **=p<0.01; QMR vs. control. [Figure 8e]Figure 8e shows the effect of QMR stimulation on T98G brain tumor cells. Figure 8e shows the percentage of T98G cells in each phase of the cell cycle (G0-G1, S, G2-M) and the induction rate of apoptosis obtained by flow cytometry at 0, 24, and 48 hours after the end of QMR stimulation. The results are shown as the mean ± standard error of at least 3-5 independent experiments; *=p<0.05; **=p<0.01; QMR vs. control. [Figure 8f] Figure 8f shows the effect of QMR stimulation on T98G brain tumor cells. T98G cells were cultured in silicone inserts directly in Petri dishes, and the migration rate of the cells up to 72 hours after QMR stimulation was assessed using an inverted optical microscope. The percentage of scratch closure, an indicator of the migration rate of the cells, was quantified using ImageJ software. These results are shown as the mean ± standard error of at least 3-5 independent experiments; *=p<0.05; **=p<0.01; QMR vs. control. [Figure 9a] Figure 9a shows the effect of QMR stimulation on U87MG brain tumor cells. Figure 9a shows images of U87MG cell cultures taken at 5x magnification using an Axiovert40CFL inverted optical microscope. Images are shown at 0 and 24 hours after 24 hours of QMR stimulation, and images of control cells are also shown. The results are presented as the mean ± standard error of at least 3-5 independent experiments; *=p<0.05; **=p<0.01; QMR vs. control. [Figure 9b] Figure 9b shows the effect of QMR stimulation on U87MG brain tumor cells. Figure 9b shows the viability of U87MG cells at 0, 24, and 48 hours after 24 hours of QMR stimulation, and the test was performed using trypan blue. The results are shown as the mean ± standard error of at least 3-5 independent experiments; *=p<0.05; **=p<0.01; QMR vs. control. [Figure 9c]Figure 9c shows the effect of QMR stimulation on U87MG brain tumor cells. Figure 9c shows the results of counting colonies of calcein-labeled U87MG cells using an AxiovertCFL inverted optical microscope. These results are shown as the mean ± standard error of at least 3-5 independent experiments; *=p<0.05; **=p<0.01; QMR vs. control. [Figure 9d] Figure 9d shows the effect of QMR stimulation on U87MG brain tumor cells. Figure 9d shows the percentage of U87MG cells in each phase of the cell cycle (G0-G1, S, G2-M) and the induction rate of apoptosis obtained by flow cytometry at 0, 24, and 48 hours after the end of QMR stimulation. The results are shown as the mean ± standard error of at least 3-5 independent experiments; *=p<0.05; **=p<0.01; QMR vs. control. [Figure 9e] Figure 9e shows the effect of QMR stimulation on U87MG brain tumor cells. Figure 9e shows the percentage of U87MG cells in each phase of the cell cycle (G0-G1, S, G2-M) and the induction rate of apoptosis obtained by flow cytometry at 0, 24, and 48 hours after the end of QMR stimulation. The results are shown as the mean ± standard error of at least 3-5 independent experiments; *=p<0.05; **=p<0.01; QMR vs. control. [Figure 10a] Figure 10a shows the cell viability of A172, T98G, and U87MG cells after QMR stimulation. Figure 10a shows the cell viability of A172, T98G, and U87MG cells after 24 and 48 hours of QMR stimulation. The results are shown as the mean ± standard error of at least 1 to 4 independent experiments; *=p<0.05; **=p<0.01; ***=p<0.001; QMR vs. control. [Figure 10b]Figure 10b shows the cell viability of A172, T98G, and U87MG cells after QMR stimulation. In Figure 10b, T98G cells were stimulated with QMR at a 15% higher output than the other cell lines, and cell proliferation was evaluated 24 to 48 hours after QMR stimulation. These results are shown as the mean ± standard error of at least 1 to 4 independent experiments; *=p<0.05; **=p<0.01; ***=p<0.001; QMR vs. control. [Figure 11] Figure 11 shows the cell viability of A172 cells treated with the drug temozolomide (TMZ). The treatment time was 144 hours and the results were obtained by trypan blue test. The results are shown as the mean ± standard error of at least three independent experiments; *=p<0.05; **=p<0.01; ***=p<0.001; TMZ vs. control (one sample t-test)). [Figure 12a] Figure 12a shows the effect of combined treatment of TMZ drug and QMR stimulation on A172 cells. TMZ was administered for 144 h, and administration was performed after or simultaneously with 24 h of QMR stimulation. Cell viability was monitored by trypan blue test. Results are shown as mean ± SEM of at least 3–5 independent experiments; *=p<0.05; **=p<0.01; ***=p<0.001; treatment vs. control; +=p<0.05; ++=p<0.01; +++=p<0.001; combined treatment of TMZ and QMR vs. TMZ alone. [Figure 12b] Figure 12b shows the effect of combined treatment of TMZ drug and QMR stimulation on A172 cells. TMZ was administered for 144 h, either after or simultaneously with 24 h of QMR stimulation. Cell viability was monitored by trypan blue test. Results are shown as mean ± standard error of at least 3–5 independent experiments; *=p<0.05; **=p<0.01; ***=p<0.001; treatment vs. control; +=p<0.05; ++=p<0.01; +++=p<0.001; combined treatment of TMZ and QMR vs. TMZ alone. [Figure 13a]Figure 13a shows the values ​​of apoptosis induction in A172 cells treated with TMZ (10 μM and 25 μM). The values ​​of apoptosis induction were obtained by flow cytometry when TMZ was administered after (Figure 12a) or simultaneously (Figure 12b) with QMR stimulation. Results are shown as mean ± SEM of at least 4-6 independent experiments; *=p<0.05; **=p<0.01; ***=p<0.001; treatment vs. control; +=p<0.05; ++=p<0.01; +++=p<0.001; TMZ and QMR combined treatment vs. TMZ treatment alone. [Figure 13b] Figure 13b shows the values ​​of apoptosis induction in A172 cells treated with TMZ (10 μM and 25 μM). The values ​​of apoptosis induction were obtained by flow cytometry when TMZ was administered after (Figure 12a) or simultaneously (Figure 12b) with QMR stimulation. Results are shown as mean ± SEM of at least 4-6 independent experiments; *=p<0.05; **=p<0.01; ***=p<0.001; treatment vs. control; +=p<0.05; ++=p<0.01; +++=p<0.001; TMZ and QMR combined treatment vs. TMZ treatment alone. [Figure 14a] Figure 14a shows the percentage of A172 cells in each phase of the cell cycle (G0-G1, S, G2-M). Cell cycle progression was monitored by flow cytometry of TMZ-treated cells after QMR stimulation (Figure 14a). Results are shown as mean ± standard error of at least 3-5 independent experiments; *=p<0.05; **=p<0.01; ***=p<0.001; TMZ-treated and QMR vs. control; +=p<0.05; ++=p<0.01; +++=p<0.001; TMZ and QMR combined treatment vs. TMZ alone treatment. [Figure 14b]Figure 14b shows the percentage of A172 cells in each phase of the cell cycle (G0-G1, S, G2-M). Cell cycle progression was monitored by flow cytometry of TMZ-treated cells concomitantly with QMR stimulation (Figure 14b). Results are shown as mean ± standard error of at least 3-5 independent experiments; *=p<0.05; **=p<0.01; ***=p<0.001; TMZ-treated and QMR vs. control; +=p<0.05; ++=p<0.01; +++=p<0.001; TMZ and QMR combined treatment vs. TMZ alone treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0055] As indicated above, the present invention relates to a method for preventing or inhibiting the proliferation of neoplastic cells in a target area of ​​the body.

[0056] The term tumor cells is used herein to refer to cells that evade growth control mechanisms and follow their own autonomous reproductive program. These cells are abnormal cell populations that exhibit excessive and discordant proliferation compared to normal cells, and progress excessively even after the stimuli that triggered them have ceased.

[0057] The neoplasm, and optionally also the body area of ​​the living body into which this tumor mass extends, represent the target area mentioned above.

[0058] For example, but not limited to, such neoplasms may include lung, colorectal, breast, prostate, pancreatic, liver neoplasms, or hematological neoplasms of bone marrow cells, lymphatic system, immune system, and the like.

[0059] In particular, the neoplastic cells of the present invention include glioblastoma (GBM) cells, malignant astrocytic tumors, and the target areas include the central nervous system.

[0060] The method of the present invention provides for treating neoplastic cells by applying an electrical current wave to the aforementioned target area for a predetermined period of time, wherein the electrical current wave has a sinusoidal waveform, preferably with a fundamental frequency of 2 MHz or greater.

[0061] The application of electric current waves having a sinusoidal waveform with a fundamental frequency of 2 MHz or higher transfers energy to the molecules to which these electric current waves are applied, which corresponds to the so-called "molecular resonance", defined by the term quantum molecular resonance (QMR).

[0062] On behalf of the applicant, it is known from patent document 3 and non-patent document 9 that this QMR energy is sufficient to break the bonds between the molecules through which the current is passed, and is particularly effective when applied with a scalpel, which allows the cutting of the target area in practice, without the effects of rupture, tearing, necrosis, loss or increase in the thickness of the tissue involved, changes in the liquid content, coagulation or other degenerative effects around the cut.

[0063] However, it has been surprisingly discovered that when a current wave with a waveform having a fundamental frequency of 2 MHz or more is applied to neoplastic cells, an anti-tumor effect is exerted.

[0064] Applicants have discovered that it is possible to apply electrical current waves to deliver QMR resonant energy to neoplastic cells, thereby decreasing the motility and invasiveness of these cells, thereby reducing their ability to migrate through the matrix and give rise to new tumor lesions.

[0065] Experiments with BM-MSC cells also revealed that the application of such current waves was harmless to normal non-neoplastic cells, ensuring that the method of the invention is selective and only inhibits or suppresses the proliferation of neoplastic cells, but does not affect the proliferation of supporting cells surrounding these neoplastic cells, which is different from conventional standard anti-tumor therapies.

[0066] Moreover, it has been confirmed that application of such electrical current waves to glioblastoma cells induces global changes in their protein structure, ultimately leading to cell death.

[0067] An added benefit is that in healthy cells, this protein change is quickly addressed.

[0068] In the method of the invention, it has proven particularly advantageous to utilize fundamental frequency values ​​in the range of 2 to 64 MHz, more preferably in the range of 2 to 20 MHz, even more preferably in the range of 4 to 20 MHz.

[0069] Particularly preferred is the use of a current wave having a waveform with a fundamental frequency of about 4 MHz.

[0070] Preferably, such waveform is a sinusoidal type waveform. Moreover, according to one aspect of the method of the present invention, such sinusoidal waveform is modified by the presence of harmonics.

[0071] Again, according to another aspect of the method of the present invention, the sinusoidal waveform is modified by the presence of at least second and third harmonics.

[0072] Preferably, the sinusoidal waveform is modified by the presence of at least the first, second and third harmonics.

[0073] As the experimental results described below demonstrate, application of the aforementioned current waves to neoplastic cells can successfully prevent or inhibit the proliferation of neoplastic cells while being substantially harmless to healthy cells present in the target area.

[0074] The effect of combining the application of such electrical current waves with the treatment of neoplastic cells with known types of antitumor drugs was also examined.

[0075] The term anti-tumor agent refers to any molecule or combination of molecules, intended for medical use to inhibit or prevent the growth and spread of tumor formation.

[0076] The use of the antitumor drug temozolomide (TMZ) is particularly preferred.

[0077] Surprisingly, it has been found that a current wave having a waveform with a fundamental frequency of 2 MHz or more can be applied to neoplastic cells in the aforementioned target area for a predetermined period of time before, during, or after treatment with an antitumor drug, and thus a higher antitumor effect of the antitumor drug can be obtained. In particular, a fundamental frequency in the range of 2 to 64 MHz, more preferably in the range of 2 to 20 MHz, even more preferably in the range of 4 to 20 MHz, and even more preferably about 4 MHz, is effective.

[0078] In particular, the use of an antitumor drug in combination with QMR can result in a greater reduction in the number of viable tumor cells than the use of the antitumor drug alone against tumor cells.

[0079] It is therefore emphasized that the combination of antitumor drug therapy with the application of the aforementioned current waves provides a more potent antitumor effect on the treated neoplastic cells than the use of antitumor drugs alone, which allows the administration of a reduced dose of the antitumor drug while still having the same antitumor effect.

[0080] As an added benefit, the reduced dose achieved by the use of QMR can provide a similar antitumor effect while significantly reducing the side effects caused by antitumor drug treatment, thereby reducing the burden of side effects associated with antitumor therapy.

[0081] These advantages are particularly evident when an anti-tumor drug is administered simultaneously with or after treatment with QMR.

[0082] This advantage is even more pronounced when the agent is administered concomitantly with QMR treatment.

[0083] As an added advantage, combined treatment with antitumor drugs and QMR can increase the number of cells undergoing early apoptosis.

[0084] It is clear that QMR can be used as an effective complement to conventional chemotherapy to enhance the efficacy of currently used antitumor drugs, while at the same time improving efficacy without increased toxicity.

[0085] In addition to these antitumor drugs, QMR can also be applied in combination with other types of antitumor treatments.

[0086] Examples of anti-tumor treatments include, but are not limited to, surgery, radiosurgery, ionizing radiation therapy, chemotherapy treatment with alkylating agents, metabolic inhibitors, anti-tumor antibiotics, topoisomerase inhibitors, anti-fungal agents, corticosteroids, biological agents, differentiation promoters, hormones, agents that stimulate the immune system, and monoclonal antibodies.

[0087] Therefore, the present invention also includes an antitumor drug for use in a method for inhibiting the proliferation of neoplastic cells present in a target area in a living body, which method comprises administering such an antitumor drug to the neoplastic cells after or simultaneously with the application of electrical current waves to said target area for a defined period of time. As mentioned above, these electrical current waves have a waveform with a fundamental frequency of 2 MHz or higher.

[0088] According to one aspect of the invention, it is not excluded that a treatment of the tumor cells with an antitumor drug precedes the application of said current waves.

[0089] Preferably, the waveform has a fundamental frequency in the range of 2 to 64 MHz, preferably in the range of 2 to 20 MHz, more preferably in the range of 4 to 20 MHz.

[0090] More preferably, the waveform has a fundamental frequency of about 4 MHz.

[0091] More preferably, said waveform is a sinusoidal waveform.

[0092] Furthermore, this sinusoidal waveform is distorted by the presence of harmonics.

[0093] Preferably, such a sinusoidal waveform is modified by the presence of at least second and third harmonics.

[0094] According to one aspect of the invention, the anti-tumor drug is used in a method of administering the anti-tumor drug to neoplastic cells simultaneously with the application of the aforementioned electrical current waves to a target area in vivo, in which the neoplastic cells are treated with the anti-tumor drug and the electrical current waves are applied to the target area at the same time.

[0095] Returning to the description of the method of the present invention, the method preferably comprises the following steps: Connecting to one or more electrodes a device configured to generate a current wave, the current wave having a fundamental frequency of 2 MHz or greater, preferably a sinusoidal waveform distorted by the presence of harmonics. - attaching at least one of these electrodes to a body surface of a living organism in which a target area containing tumor cells is located, or implanting at least one of these electrodes in the living organism at or near a target area containing tumor cells. · Activating the device to transfer a current wave to the one or more electrodes and leaving the device activated for a predetermined period of time.

[0096] Preferably, the living body is a human subject.

[0097] These electrodes have a suitable shape and configuration for transmitting the aforementioned electrical current waves to the target area.

[0098] By way of example, and not limitation, such electrodes may be non-implantable, e.g., in the form of a handpiece or in the form of a suction cup, or again essentially laminar in shape and applicable by adhesion to the skin of a subject, such electrodes may be in the form of a probe.

[0099] When the electrodes used in the methods of the present invention have a laminar geometry, they are essentially flexible so as to easily conform to the contours of the body surface, and also optionally comprise an adhesive material that facilitates maintaining contact with the body during application of the waveforms generated by the device.

[0100] It is not excluded that one or more of the aforementioned electrodes may be implanted.

[0101] Thus, a device for preventing or inhibiting the proliferation of neoplastic cells in a target area of ​​an organism also forms part of the present invention.

[0102] Such an apparatus can be used in the method of the present invention.

[0103] In particular, the device of the present invention is configured to generate a current wave having a waveform with a fundamental frequency of 2 MHz or greater.

[0104] The target region preferably belongs to an organism, as already indicated above, more preferably to a human.

[0105] According to a preferred embodiment of the invention, the device comprises a radio frequency circuit connected to one or more electrodes for supplying to each of said electrodes a current wave having a fundamental frequency of 2 MHz or higher.

[0106] Preferably, the device further comprises a rectifier circuit powered by the mains voltage, the rectifier circuit preferably providing a continuous, more preferably a stabilized voltage, to said radio frequency circuit.

[0107] Such a radio frequency circuit preferably comprises at least one electronic switch, powered by a voltage and driven by a drive circuit.

[0108] More specifically, the radio frequency circuit has a current wave with a fundamental frequency of 2 MHz or more on the output side and transmits the current wave to these electrodes. The fundamental frequency is preferably in the range of 2 to 64 MHz, more preferably in the range of 2 to 20 MHz, and even more preferably in the range of 4 to 20 MHz.

[0109] More preferably, the fundamental frequency corresponds to about 4 MHz.

[0110] This current wave also has a sinusoidal shape.

[0111] This current wave is also beneficially modified by the presence of harmonics.

[0112] These harmonics are preferably at least the second and third harmonics.

[0113] More preferably, the current wave has a sinusoidal shape modified by the presence of at least the first, second and third harmonics.

[0114] This current wave also circulates in a preferably wideband resonant circuit at the fundamental frequency of a modified sine wave shape.

[0115] According to a preferred embodiment of the present invention, the device generates a high frequency sinusoidal alternating current wave of 4 MHz with harmonics from 4 to 64 MHz.

[0116] According to one aspect of the device of the present invention, the electrodes of the device are configured to transmit the aforementioned electrical current waves to a target area.

[0117] It is specified that the electrodes of the device of the present invention may have shapes and configurations corresponding to those of the electrodes described above in relation to the method of the present invention.

[0118] According to one aspect of the device of the present invention, such electrodes are non-implantable and are applied to the surface of the organism's body at or near the location of the target area containing the neoplastic cells.

[0119] According to an alternative embodiment of the device, a helmet is provided, which is adapted to accommodate said electrodes and to allow both connection to said radio frequency circuitry and placement at or near the target area.

[0120] It should be noted that according to a particular variant of the device comprising the aforementioned helmet, the helmet is also adapted to accommodate a radio frequency circuit.

[0121] According to a further variant, the device comprises a wearable cap adapted to house the aforementioned electrodes and to enable them to be applied at or near the target area.

[0122] According to another variant, the device comprises at least one band provided with the aforementioned electrodes, optionally adapted to also accommodate the radio frequency circuitry of the device.

[0123] The band may also be configured to be positioned laterally and / or in front of and / or behind the subject's head.

[0124] It is not excluded that such a band may be arranged to be placed around a person's waist and worn by the person as a belt.

[0125] Furthermore, it is not excluded that the band may be worn by the subject in different ways than described above.

[0126] According to a particular variant of the device of the invention, at least one of the electrodes of the device of the invention is an implanted electrode.

[0127] The term "implantable" refers to an electrode that is intended to be wholly or partially implanted in the human body by surgical or medical intervention.

[0128] According to another alternative embodiment of the invention, the device is adapted to be fully implantable.

[0129] By way of example, the implantable device may have a configuration substantially similar to that of an implantable pacemaker.

[0130] Other aspects and advantages of the present invention will become apparent from the following examples, which are illustrative and non-limiting. EXAMPLES

[0131] Example 1. Materials and Methods 1.1 Cell culture and cell growth Glioblastoma cell lines A172, T98G, and U87MG were used. A172 and T98G cells (Sigma-Aldrich, St. Louis, MO, USA) were cultured in Dulbecco's modified Eagle's medium / nutrient mixture F-12 (DMEM / F12 GlutaMAX, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (quality certified, Australian source, Gibco, Thermo Fisher Scientific) and 1% penicillin / streptomycin (Sigma-Aldrich).

[0132] U87MG cells (provided by Prof. Massimo Dominici, Institute of Cellular Therapy, Modena and Reggio Emilia University Hospital, Modena, Italy) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with GlutaMAX (Gibco, Thermo Fisher Scientific), 10% FBS (Gibco, Thermo Fisher Scientific) and 1% penicillin / streptomycin (Sigma-Aldrich).

[0133] Bone marrow-derived mesenchymal stromal cells (BM-MSCs) were also used as non-tumor-type control cells. BM-MSCs were produced at The Advanced Cellular Therapies Laboratory (LTCA) of the Hematology Multi-Operation Unit at ULSS8 Berica as previously described (Non-Patent Document 10). Briefly, MSCs were isolated from cells obtained by washing discarded bone marrow collection bags and filters from healthy donors (Ethics Committee Certification No. 107 / 18, December 2, 2019). After washing, the cells were centrifuged at 2,000 rpm for 10 min and aliquoted at 1x10 in DMEM containing GlutaMAX (Gibco, Thermo Fisher Scientific) supplemented with 10% FBS (Gibco, Thermo Fisher Scientific) and 1% penicillin / streptomycin (Sigma-Aldrich). 5 cells / cm 2 The BM-MSCs were seeded at a density of 2,000 cells / cm. The cell cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2. 72 h after seeding, non-adherent cells were removed and fresh medium was added. The medium was changed every 3–4 days. At 80% confluence, BM-MSCs were plated at 2,000 cells / cm. 2 The seeds were replated at a density of 1000 x 1000.

[0134] 1.2 QMR stimulation protocol The cell lines were subjected to QMR stimulation using a prototype QMR device from Telea (Telea Electronic Engineering, Sandrigo, Veneto, Italy), appropriately adapted for the present in vitro experiments, generating a high-frequency sinusoidal alternating current wave of 4 MHz with harmonics between 4 and 64 MHz. The specifications of the device are: power supply 100÷230V~50 / 60Hz, maximum output power 5W / 400Ω.

[0135] QMR was applied to the cells using a pair of electrodes placed at the inner edge of a 100 mm Petri dish and connected to a generator device. The transfer of the electric field to the medium of the cell culture generates heat (Joule effect). The average temperature increase of the medium of stimulated cells was 5 °C. This value was measured by three independent measurements with a data logger probe (iLog, Escorts Scunthorpe, UK) placed in the culture medium. To compensate for the temperature increase of the medium of stimulated cells and to achieve an average temperature of 37 °C in stimulated cells, the incubator used for stimulated cells was set to 32 °C with 5% CO2 and the laboratory temperature was maintained between 20 °C and 25 °C.

[0136] 1.3 Experimental setup Based on the cell line used, a defined number of cells were seeded in 100 mm plates (Greiner Bio-One, Frickenhausen, Germany) to be stimulated at 60% confluence. Cells were then exposed to QMR for 24 h and analyzed 0–24–48 h after the end of stimulation.

[0137] More specifically, cells were washed with D-PBS (Sigma-Aldrich), detached with 1X TrypLE Select (Gibco, Thermo Fisher Scientific), and the resulting aliquots were used to examine cell viability, apoptosis, cell cycle, karyotype, and proteomics. Cell aliquots were re-seeded for evaluation of cell proliferation potential in semi-solid medium (soft agar assay) and cell migration was directly monitored on QMR stimulation plates before and after stimulation.

[0138] The effect of combining QMR with the drug TMZ (Sigma-Aldrich) was also tested.

[0139] For these analyses, cells were treated for 144 h with 10–25 μM TMZ administered simultaneously with or after QMR stimulation. TMZ stock solutions were prepared in DMSO at a final concentration of 10 mM and kept at 4 °C. Subsequent dilutions were made in fresh culture medium and administered to cells. Cell viability, apoptosis, and cell cycle were assessed after 144 h.

[0140] 1.4 Cell viability: Trypan blue exclusion assay After stimulation, cells were harvested and suspended in culture medium with trypan blue solution (Gibco, Thermo Fisher Scientific) at a ratio of 1:1. Cells were counted using a Burker counter, and the viable cell number was calculated using the following formula: [(number of cells × 10,000 × 2 × V) / 9], where V represents the total volume of the cell solution obtained, and 2 represents the dilution factor of the solution with the dye (2-fold dilution).

[0141] 1.5 Apoptosis: Labeling with Annexin V / 7-aminoactinamycin D (7-AAD) After stimulation, 1.5×10 5 Cells were harvested and centrifuged at 400g for 6 min. After washing with binding buffer, cells were labeled with Annexin V / 7-AAD according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). After dilution with binding buffer, 2 × 10 cells were analyzed using an FC500 flow cytometer (Beckman Coulter, Brea, CA, USA). 4 The fluorescence of the cells / samples was detected and the cell population was divided into four groups: live cells with negative fluorescence for both Annexin V and 7-AAD, early apoptotic cells positive for Annexin V and negative for 7-AAD (Annexin V+ / 7-AAD-), late apoptotic cells double positive for Annexin V and 7-AAD (Annexin V+ / 7-AAD+), and dead cells negative for Annexin V and positive for 7-AAD (Annexin V- / 7-AAD+).

[0142] 1.6 Cell cycle After stimulation, 5×10 5 Cells were harvested and centrifuged at 400g for 6 min. Cells were then washed with D-PBS, fixed, and permeabilized with acetone (80% solution in water). After 1 h at 4°C, the acetone was removed by centrifugation, and cells were washed and labeled with 7-AAD (Invitrogen) at 1 μg / mL for 1 h at room temperature. 2.5 × 104 Fluorescence of cells / samples was analyzed using a FC500 flow cytometer (Beckman Coulter). The percentage of cells in different phases of the cell cycle was calculated using EXPO32 software (Beckman Coulter, Fullerton, CA, USA), taking into account the diploid cycle and correcting for cell clusters.

[0143] 1.7 Karyotype analysis To evaluate whether QMR stimulation is safe and therefore does not cause chromosomal alterations, BM-MSC mesenchymal stromal cells were stimulated with QMR for 24 h and examined by G-trypsin-Giemsa banding according to standard techniques at 350 / 400 band resolution. 20 metaphases were analyzed and 3 were karyotyped. Non-stimulated BM-MSC cells were used as control cells.

[0144] 1.8 Cell migration (scratch test) The scratch test represents a 2D cell migration approach to semi-quantitatively detect cell motility. The aim of the scratch is to create a cell-free area (or opening) to induce the surrounding cells to migrate and close this opening. Cells were seeded in 2-well silicone inserts (IBIDI, Gräfelfing, Germany) to obtain a cell-free opening of 500 μm above the cell monolayer.

[0145] Once 60% cell confluence was reached, the inserts were removed and cells were stimulated with QMR for 24 h. Cell migration was monitored over time by image acquisition before and 0–24–48 h after stimulation. Image acquisition was performed with an Axiovert40CFL inverted optical microscope (Carl Zeiss, Oberkochen, Germany). The percentage of aperture closure was analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA), and migration ability was assessed by the migration rate curve, calculated as follows: % closure = [(area tx - area ty) / area tx] × 100, where tx represents the acquisition time and ty represents the next time point.

[0146] 1.9 Soft agar The colony formation assay in semi-solid medium (soft agar) is a method used to monitor cell growth independent of cell fixation to a substrate, measuring cell growth in semi-solid medium by optical colony counting. The rate of colony formation in soft agar varies depending on the cell line used. Therefore, the concentration of agarose, the number of cells seeded and the final day of the experiment were optimized for each cell line. Cell suspensions were prepared in a 0.4% agarose solution in cell medium and seeded on a solidified layer of 0.6% agarose in complete medium. After 1 h at room temperature, 160 μl of complete medium was added and another 100 μl was added every week until the end of the experiment. Plates were transferred to a 37 °C, 5% CO2 incubator for 21-24 days and then labeled with 100 μM calcein (Sigma-Aldrich) for 30 min. Colonies consisting of at least 50 cells were counted using an Axiovert40CFL inverted optical microscope (Carl Zeiss).

[0147] 1.10 Proteomics 1×10 6 Cells were harvested, centrifuged at 400g for 6 min, washed with D-PBS, and lysed on ice in lysis buffer (Pierce® RIPA buffer, Thermo Fisher Scientific) with protease inhibitor cocktail (Cell Signalling, Danvers, MA, USA). After standing on ice for 30 min, the cell lysates were centrifuged at 14,000g for 10 min at 4°C, and protein supernatants were quantified colorimetrically by BCA assay (Pierce® BCA Protein Assay Kit, Thermo Fisher Scientific). Bovine serum albumin (Sigma-Aldrich) was used as a standard.

[0148] 1.11 Liquid Chromatography-Mass Spectrometry Protein extraction Liquid chromatography-mass spectrometry (LC / MS-MS) is an analytical technique that combines the separation power of liquid chromatography with the high sensitivity and selectivity of triple quadrupole mass spectrometry. For sample preparation, 50 μg of protein lysate was precipitated with acetone and the resulting protein pellet was dissolved in a 6 M solution of urea and 100 mM ammonium bicarbonate (pH 8). Samples were reduced with 10 mM dithiothreitol for 1 h at room temperature and alkylated with 20 mM iodoacetamide in the dark for 30 min at room temperature. Subsequently, they were digested with the endopeptidase enzyme Lys-C (Promega) at an enzyme / protein ratio of 1:100 (w / w) for 3 h at room temperature. Proteins were then diluted 4-fold with 50 mM ammonium bicarbonate and digested overnight with trypsin (Promega) 1:100 (w / w) at room temperature. Proteolysis was interrupted by the addition of 1% trifluoroacetic acid. Samples were then desalted, vacuum dried, and resuspended in 0.1% formic acid for LC-MS / MS analysis.

[0149] Protein identification by specific databases and relative quantification Samples were analyzed using an Easy-nLC1200 system (Thermo Fisher) coupled in-line with an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher). A binary mobile phase of 0.1% formalin acid in water (buffer A) and 0.1% formalin acid in acetonitrile (buffer B) was used to separate the digested peptides using a reversed-phase column (Acclaim PepMap RSLC C18, 2 μm particle size, 100 Å pore size, 75 μm diameter, Thermo Fisher Scientific). Peptides were eluted with a gradient from 5% to 25% for 52 min, 25% to 40% for 8 min, and finally 40% to 98% for 10 min at a flow rate of 400 nL / min. The data-dependent acquisition (DDA) method was based on a full scan with a resolution power of 120,000 fwhm (full width at half max at 200 m / z) and a maximum injection time of 50 ms. The precursor range was examined from 350 to 1,100 m / z, and the first mass was set at 140 m / z for the fragments. A full scan was followed by a series of MS / MS (HCD) scans with a cycle time of 3 s, collision energy of 30%, and maximum injection time of 150 ms. The dynamic exclusion time was set at 50 s. The raw data were searched with ProteomeDiscoverer2.2 software (Thermo Fisher Scientific). Peptide searches were performed using human protein FASTA files (UniProt), and proteins were identified using the MASCOT search engine (Matrix Science, Boston, MA, USA). The mass tolerance for precursors was set at 10 ppm, and the mass tolerance for products was set at 0.6 Da.

[0150] 1.12 Bioinformatics Analysis The differential abundance of proteins expressed in the different experimental groups (t0 and t24, respectively) was used to perform a hierarchical cluster analysis using the Clustvis tool (Metsalu et al., 2015). Gene Ontology (GO) and pathway annotation of protein IDs was performed using the EnrichR gene set enrichment analysis server (http: / / amp.pharm.mssm.edu / Enrichr / ) by applying the biological process and reactome classification with a significance threshold set at p<0.05. Protein interaction networks were constructed using the STRING interaction database, version 11.0 (https: / / string-db.org / ) (Mering et al., 2003).

[0151] 1.13 Statistical analysis All data were analyzed using the GraphPad program (GraphPad Software, San Diego, CA, USA) and expressed as mean ± standard error. One-sample t-tests were used for analysis of results expressed as ratios / percentages of control (e.g., trypan blue exclusion assay). Unpaired Student's t-tests were used for all other analyses. Data with p < 0.05 were considered significant.

[0152] Example 2. Results 2.1 QMR treatment reduces glioblastoma cell proliferation by altering cell cycle progression The effect of QMR treatment on A172 glioblastoma cells was evaluated by analyzing the cell morphology, proliferation rate, apoptosis and cell cycle of the cells immediately after the end of treatment and 24 and 48 hours after the end of QMR stimulation. Adherence to these time frames allowed us to evaluate the durability of the therapeutic effects and the ability of treated cells to restore their cellular functions to pre-treatment levels.

[0153] Surprisingly, QMR treatment was found to alter the morphology of treated A172 cells compared to untreated control cells: indeed, QMR-treated cells appeared more swollen and granular than control cells, indicating that cell damage had occurred.

[0154] This damage was also found to persist over time, as shown in Figure 1.

[0155] The cell proliferation rate was also estimated by trypan blue assay. At 24 h after treatment, QMR-treated cells showed approximately 28% reduction in tumor proliferation. Moreover, the viable cell count tended to decrease even 24–48 h after treatment, suggesting an effect of QMR treatment on cell cycle progression, as shown in Figure 2.

[0156] To support this surprising finding, the cell cycle of A172 cells was also assessed using flow cytometry experiments. The results shown in Figure 3a and Figure 3c showed that QMR treatment significantly decreased the cell division rate (defined as the percentage of cells in S phase) and significantly increased the cells in G2 / M phase. These results were confirmed up to 48 h after the end of treatment, indicating that cell cycle arrest occurred in the G2 / M phase.

[0157] The efficacy of treating cells with QMR was also confirmed by the clear activation of apoptosis in treated cells. As shown by the results in Figures 4b and 4c, the number of live cells after QMR treatment was actually significantly lower than the number of control live cells not treated with QMR (p<0.01). This reduction also correlated with an approximately 10% increase in the number of cells in early apoptosis and a slight increase in the number of cells in late apoptosis.

[0158] 2.2 QMR treatment does not cause cytotoxic or genotoxic effects on BM-MSC cells To verify the safety of QMR treatment on non-neoplastic cells, we tested the effect of QMR treatment on healthy BM-MSC cells. Cell morphology, proliferation rate, apoptosis, cell cycle and karyotype were evaluated in treated BM-MSC cells and untreated control cells at the end of QMR stimulation and 24 h after the end of treatment. As shown in Figure 5b, a slight decrease in cell proliferation was noted at the end of QMR treatment, but this effect should not be considered comparable to that obtained with treated A172 glioblastoma cells shown in Figure 2.

[0159] Furthermore, BM-MSC cells treated with QMR showed no differences in cell morphology, cell cycle regulation and apoptosis, as shown in Figure 5a, Figure 5c / Figure 5d and Figure 5e / Figure 5f, respectively.

[0160] Furthermore, time did not influence the final effect, as indicated by substantial overlap between results at 0 and 24 hours post-treatment.

[0161] Karyotype analysis of QMR-treated BM-MSC cells also advantageously demonstrated that QMR treatment did not cause any changes in chromosome number or their structure, as seen in FIG.

[0162] 2.3 QMR treatment reduces the aggressiveness and invasiveness of A172 glioblastoma cells It is well known that neoplastic cells are characterized by uncontrolled proliferation and migration. Thus, the ability of cells to grow in semi-solid medium and their migration rate are considered markers of tumor aggressiveness and invasiveness. Therefore, cell colony formation and cell migration in soft agar were analyzed under basal conditions and after QMR treatment. The results shown in Figure 7a surprisingly showed that QMR treatment significantly reduced the proliferation of A172 cells in soft agar. Indeed, the number of cell colonies after QMR treatment was about 85% lower than that of untreated control cells. Surprisingly, moreover, the cell migration rate of QMR-treated A172 cells was also time-dependently lower than that of control cells, as shown in Figure 7b.

[0163] 2.4 QMR treatment modulates the proteomic fingerprint of A172 glioblastoma cell line The proteomic profiles of QMR-treated A172 and BM-MSC cells were also investigated. Analyses were performed at the end of QMR stimulation (time 0) and 24 h after the end of treatment. Untreated A172 and BM-MSC cells were used as controls.

[0164] A total of 5104 proteins were identified in the A172 cell line, of which 311 and 308 were found to be significantly altered after QMR treatment at 0 and 24 h after the end of treatment, respectively. Cluster analysis, not shown in the figure, highlighted two separate groups of proteins in both tested time frames, demonstrating that QMR treatment interferes with the activity of neoplastic cells.

[0165] Proteins were found to be differentiated between treated and untreated cells. Specifically, at time zero, 111 proteins were found to be significantly upregulated and 200 proteins were found to be downregulated. Instead, 24 hours after the end of treatment, 103 upregulated proteins and 205 downregulated proteins were found.

[0166] More specific analysis of gene ontology and pathway enrichment showed that the upregulated proteins were strongly associated with stress response mechanisms, protein folding and extracellular matrix modeling, suggesting that QMR treatment also acts as a toxic protein stimulus.

[0167] More specifically, QMR-treated cells underwent significant downregulation of key factors involved in pathways involved in protein translation, RNA processing, and cell cycle compared to untreated control cells, leading to cell cycle arrest and death of treated cells.

[0168] Advantageously, the effect of QMR stimulation on BM-MSC cells yielded completely different results compared to A172 glioblastoma cells. At the end of QMR treatment, activation of heat shock proteins was associated with extracellular matrix remodeling. However, this change was found to be completely normalized 24 hours after treatment. Thus, QMR treatment did not hinder the proliferation rate of BM-MSC cells, aiding the selectivity of QMR treatment against neoplastic cells.

[0169] 2.5 QMR treatment in T98G and U87MG cell lines The above experiments were also performed on glioblastoma cell lines that were more aggressive than A172 cell line. These more aggressive cell lines include T98G and U87MG cells. The proliferation rate after QMR treatment was found to be decreased in both cell lines, but there was no significant difference in apoptosis activation, cell cycle progression, clonogenic potential and cell motility, as shown in Figures 8a-f.

[0170] Cell viability was also examined 48 and 72 hours after QMR treatment. Surprisingly, it was found that the cell proliferation rates of both A172 and U87MG cells decreased significantly over time.

[0171] Even a 15% increase in treatment power has been shown to affect cell proliferation rates, and as shown in the figure, cell viability of QMR-treated cells at high power was found to decrease by up to 35%.

[0172] 2.6 Application of QMR enhances the effect of the drug temozolomide (TMZ) in A172 glioblastoma cells Until now, standard treatment for patients with glioblastoma has included total resection of the lesion, followed by radiation therapy and chemotherapy with the drug TMZ.

[0173] Unfortunately, the effectiveness of the drug is limited by the severity of side effects caused by the drug itself and the development of drug resistance mechanisms. Therefore, it was essential to find new therapeutic strategies to be used in combination with TMZ to reduce the dose of TMZ and improve the quality of life of patients.

[0174] Therefore, the efficacy of combined TMZ and QMR treatment on A172 glioblastoma cells was tested. These cells were treated with 10-25 μM TMZ either simultaneously with or after QMR treatment. Cell viability, apoptosis, and cell cycle values ​​were analyzed. TMZ drug concentrations were selected based on their IC50 (Figure 14).

[0175] The obtained results surprisingly show that when TMZ drug at a concentration of 10 μM was used in combination with QMR treatment, it caused a decrease in cell viability of about 12% more than the decrease in cell viability obtained in cells treated with TMZ drug alone, as shown in Figure 12 b.

[0176] Moreover, these data were found to be surprisingly correlated with those regarding cell apoptosis: indeed, combined administration of TMZ and QMR demonstrated that a decrease in the number of live cells was associated with an increase in the number of cells in early apoptosis, as shown in the graphs in Figures 13a and 13b.

[0177] Concurrent exposure of cells to 25 μM of the drugs TMZ and QMR activated greater changes in cell cycle progression compared to cells treated with the TMZ drug alone.

[0178] These data surprisingly demonstrate that the combined TMZ and QMR treatment strategy induced significant cell cycle arrest at the G2-M phase and reduced the percentage of cells in the G0-G1 phase, as shown in Fig. 14b.

Claims

1. 1. An anti-tumor agent for use in a method for preventing or inhibiting the proliferation of tumor cells in a target area of ​​a living body, the method comprising the step of treating neoplastic cells with the anti-tumor agent while simultaneously applying an electric current wave to the target area during a predetermined period during which the neoplastic cells are treated with the anti-tumor agent, the electric current wave having a waveform having a fundamental frequency of about 4 MHz modified by the presence of at least second and third harmonics.

2. The antitumor agent according to claim 1, wherein the waveform is a sine wave waveform.

3. The antitumor agent according to claim 1 or 2, characterized in that the antitumor agent comprises temozolomide.

4. The antitumor agent according to any one of claims 1 to 3, characterized in that the tumor cells comprise glioblastoma cells and the target area comprises the central nervous system.

5. 1. An anti-tumor agent for use in a method for preventing or inhibiting the proliferation of tumor cells in a target area of ​​a living body, the method comprising the step of treating said neoplastic cells with said anti-tumor agent for a predetermined period of time while simultaneously applying an electric current wave to said target area, said electric current wave having a waveform having a fundamental frequency of about 4 MHz modified by the presence of at least second and third harmonics.

6. 1. An anti-tumor agent for use in a method for preventing or inhibiting the proliferation of tumor cells in a target area of ​​a living body, the method comprising the step of treating the neoplastic cells with the anti-tumor agent prior to application of an electric current wave to the target area for a predetermined period of time, the electric current wave having a waveform having a fundamental frequency of about 4 MHz modified by the presence of at least second and third harmonics.

7. 1. An apparatus configured to inhibit or inhibit the proliferation of neoplastic cells in a target region of a living body, the apparatus configured to generate a current wave having a waveform having a fundamental frequency of about 4 MHz modified by the presence of at least second and third harmonics, one or more electrodes connected to at least one radio frequency circuit; the at least one radio frequency circuit supplies to each of the electrodes a current wave having a fundamental frequency of about 4 MHz modified by the presence of at least second and third harmonics; A device, wherein the one or more electrodes are implantable electrodes, the one or more electrodes being implanted at or near the target area.

8. 8. The apparatus of claim 7, wherein the waveform is a sinusoidal waveform.

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