Patent Description:
Neoplasms or tumors originate from alteration of cells that multiply and spread throughout the body, escaping regulatory mechanisms. There are numerous types of neoplasms that can affect substantially any organ. They are usually classified into benign or malignant according to the ability of the neoplastic cells to invade surrounding structures and more distant organs. In addition to tumors of solid organs, the term neoplasm includes also tumors of blood cells, such as lymphomas and leukaemias.

Neoplasms continue to be among the leading causes of death and deterioration in quality of life despite the major successes in the clinical field achieved in recent decades thanks to targeted prevention measures, early diagnosis and therapy.

Tumor therapy is essentially based on surgery, especially in localised forms, radiotherapy and drugs.

Glioblastoma multiforme (GBM) represents one of the most aggressive tumors for humans due to the ability of GBM neoplastic cells to infiltrate the parenchyma adjacent to the tumor lesion (DeAngelis et al, <NUM>).

GBM patients usually undergo resection, possibly complete, of the lesion by surgery, followed by radiotherapy and chemotherapy with the drug temozolomide, an alkylating antitumor drug (Alifieris et al, <NUM>).

However, despite a multi-pronged therapeutic approach, GBM is characterized by a high recurrence rate, drug resistance and devastating neurological deterioration (Wilson et al, <NUM>).

GBM is a particularly infiltrative tumor, and the performance of a complete surgical resection of the lesion is therefore substantially impossible, so that almost all patients develop a relapse and have a survival time of only <NUM> months (Becker et al, <NUM>). Additionally, <NUM> years after surgery, less than <NUM>% of patients are still alive (Ostrom et al, <NUM>).

It is therefore clear how necessary it is to develop new therapeutic strategies to improve the efficacy of the drugs in use and to reduce the corresponding adverse effects, thus improving patients' quality of life.

In this sense, one of the most promising therapies comprises the use of nonionizing electromagnetic fields (EMFs).

This therapy is particularly promising as it appears to have an excellent degree of safety, low toxicity and the possibility of combination with conventional drug therapies (Mattson et al, <NUM>).

In recent years, several EMFs technologies have been tested and have shown to have a good level of efficacy against different types of tumors, both when used alone and in combination with chemotherapy (Pasi et al, <NUM>).

It has been found that different frequency ranges trigger different response mechanisms in the cells that can lead to the reduction of cell proliferation of neoplastic cells by acting on the formation and stability of the mitotic spindle of these cells (Giladi et al, <NUM>).

In <NUM>, the American Food and Drug Administration approved the use of Tumor Treating Fields (TTFields) for the treatment of patients with recurrent GBM.

Several prior art documents have been published to this effect, such as, by way of example, documents <CIT> and <CIT>.

The object of the present description is to realize an alternative device and method to those of a known type, adapted to block or inhibit the growth of neoplastic cells in a target region of a living organism.

In particular, it is an object of the present description that such a device and method may be used in the treatment of neoplasms, even of an aggressive type, such as glioblastoma multiforme.

Still, it is an object of the present description that such method and use of the device in a patient may also be of the non-invasive type.

Furthermore, it is an object of the present description to realize an antitumor drug which has greater therapeutic efficacy and a reduced toxicological profile, improving the patient's quality of life. According to the present invention, and to achieve the objects mentioned above, an antitumor drug as set out in claim <NUM> is provided.

The present invention also pertains to a device as set forth in claim <NUM>.

Furthermore, the objects are also achieved by a non-claimed method for treating neoplastic cells, according to what will be described below.

In particular, the present description relates to an antitumor drug for use in a method for blocking or inhibiting the growth of neoplastic cells in a target region of a living organism, wherein said method comprises treating said neoplastic cells with the antitumor drug prior to, simultaneously with or subsequent to the application of electric current waves to the target region for a predefined period of time, and wherein the electric current waves have a waveform with a fundamental frequency higher than or equal to <NUM>.

Preferably, such electric current waves have a waveform with a fundamental frequency ranging between <NUM> and <NUM>, more preferably ranging between <NUM> and <NUM>, even more preferably ranging between <NUM> and <NUM>.

Much more preferably, such electric current waves have a waveform with a fundamental frequency of about <NUM>.

It is specified that the application of such electric current waves will also be referred to in this document as Quantum Molecular Resonance or QMR.

Furthermore, according to an aspect of the present description, such electric current waves have a sinusoidal waveform.

According to one aspect of the description, this waveform is distorted by the presence of harmonics.

According to another aspect of the present description, the sinusoidal waveform is distorted by the presence of at least second and third order harmonics.

According to one aspect of the description, the treatment of neoplastic cells with the antitumor drug is carried out simultaneously with or subsequent to the application of the aforesaid electric current waves.

Preferably, according to one aspect of the description, the treatment of the neoplastic cells with the antitumor drug is carried out simultaneously with the application of the electric current waves to the target region, while the neoplastic cells are treated with this antitumor drug.

According to one aspect of the description, the aforesaid antitumor drug comprises temozolomide.

According to one aspect of the present description, the neoplastic cells comprise glioblastoma cells.

In particular, the target region belongs to a living organism, preferably a human subject.

The aforesaid target region preferably comprises the central nervous system.

A device for blocking or inhibiting the growth of neoplastic cells in a target region of a living organism is also part of the present description, wherein said device is configured to generate electric current waves having a waveform having a fundamental frequency higher than or equal to <NUM>, preferably ranging between <NUM> and <NUM>, more preferably ranging between <NUM> and <NUM>, even more preferably ranging between <NUM> and <NUM>, much more preferably about <NUM>, and wherein said waveform is a sinusoidal waveform distorted by the presence of harmonics.

This device comprises one or more electrodes connected to at least one radiofrequency circuit adapted to supply each of these electrodes with a current wave having a fundamental frequency higher than or equal to <NUM>. Such one or more electrodes are configured so as to transmit said electric current waves to the aforesaid target region.

According to one aspect of the invention, the electrodes of the device are electrodes of the implantable type, adapted to be implanted at or near the aforesaid target region.

A non-claimed method for blocking or inhibiting the growth of neoplastic cells in a target region is also part of the present description.

This target region preferably belongs to a living organism.

According to one aspect of the method of the present description, it comprises treating the aforesaid neoplastic cells by applying electric current waves to the aforesaid target region for a predefined period of time, wherein said electric current waves have a waveform with a fundamental frequency higher than or equal to <NUM>, preferably ranging between <NUM> and <NUM>, more preferably ranging between <NUM> and <NUM>, even more preferably ranging between <NUM> and <NUM>, much more preferably about <NUM>.

Furthermore, according to one aspect of the method of the description, such a waveform is a sinusoidal waveform.

Again, according to one aspect of the description, this sinusoidal waveform is distorted by the presence of harmonics.

Again, according to another aspect of the method of the present description, the sinusoidal waveform is distorted by the presence of at least second and third order harmonics.

Furthermore, according to one aspect of the method of the description, the neoplastic cells comprise glioblastoma cells.

According to one aspect of the description, the target region comprises the central nervous system.

According to a further aspect of the method of the description, this method comprises the following steps:.

Alternatively, according to another aspect of the method of the description, this method comprises the following steps:.

Preferably, the method of the present description, makes use of the device of the present invention.

Further features and advantages of the drug, device and method of the description, will be apparent to one skilled in the art from the following description of preferred embodiments which is given by way of illustration, but not limitation.

As indicated above, the present description relates to a non-claimed method for blocking or inhibiting the growth of neoplastic cells in a target region of a living organism.

The term neoplastic cells is used in this document to refer to those cells that escape the proliferation control mechanisms and follow their own autonomous program of reproduction. They form a neoplasm, i.e. an abnormal mass of cells whose growth is excessive and uncoordinated compared to that of normal cells, and which progresses excessively even after the stimuli that evoked it have ceased.

The aforesaid neoplasm, and optionally the region of the body of the living organism where this tumor mass expands, also represents the target region mentioned above.

By way of example not to be considered limiting, this neoplasm may include lung, colorectal, mammary, prostate, pancreas, liver neoplasm, or haematological neoplasms of bone marrow cells, lymphatic system, immune system, etc..

In particular, the neoplastic cells of the present description comprise glioblastoma (GBM) cells, a malignant astrocytic tumor, and the target region comprises the central nervous system.

The method of the description provides for treating the aforesaid neoplastic cells by applying electric current waves to the aforesaid target region for a predefined period of time, wherein said electric current waves have a preferably sinusoidal waveform with a fundamental frequency higher than or equal to <NUM>.

The application of current waves having a sinusoidal waveform with a fundamental frequency higher than or equal to <NUM> transmits energy to the molecules to which these current waves are applied, which corresponds to the so-called "molecular resonance", defined by the term Quantum Molecular Resonance (QMR).

It is known from document <CIT> and from document Pozzato G et al, <NUM>, on behalf of the Applicant, that this QMR energy is just sufficient to break the bonds among the molecules hit by the passage of current, making it particularly useful when applied to a scalpel. This scalpel is in fact able to cut the regions of interest, without producing any effect of rupture, tear, necrosis, decrease or increase in the thickness of the tissues involved, alteration of the liquid content, coagulation or other degenerative effect around the cut.

However, it has now been surprisingly discovered that current waves having a waveform with a fundamental frequency higher than or equal to <NUM> perform an antitumor action when applied to cells of the neoplastic type.

The Applicants have in fact discovered that the application of electric current waves, such as to transmit the QMR resonance energy to the neoplastic cells, allows to obtain the reduction of the motility and aggressiveness of the aforesaid cells, decreasing their ability to migrate through the matrices, for example, to give rise to new tumor lesions.

Experiments on BM-MSC cells have also highlighted that the application of such current waves appears to be harmless towards normal, non-neoplastic type cells, thus ensuring that the method of the description is selective in blocking or inhibiting the growth of neoplastic cells only and not the growth of the supporting cells that are found around these neoplastic cells, as is the case, instead with standard type antitumor therapies.

Furthermore, it has been identified that the application of such current waves to glioblastoma cells causes a global alteration of their protein structure which ultimately leads to cell death.

Advantageously, instead, this protein alteration is quickly counteracted in healthy cells.

The use of fundamental frequency values ranging between <NUM> and <NUM>, more preferably ranging between <NUM> and <NUM>, even more preferably ranging between <NUM> and <NUM>, has proved particularly advantageous in the method of the description.

Particularly preferred is the use of electric current waves having a waveform with a fundamental frequency of about <NUM>.

Preferably, such a waveform is a waveform of the sinusoidal type.

Furthermore, according to one aspect of the method of the description, such a sinusoidal waveform is distorted by the presence of harmonics.

Preferably, the sinusoidal waveform is distorted by the presence of at least first, second and third order harmonics.

As shown by the results of the experiments described below, the application of the aforesaid electric current waves to neoplastic cells makes it possible to block or inhibit the growth of neoplastic cells with a high rate of success, leaving the healthy cells present in the target region substantially unharmed.

The effect of combining the application of such electric current waves with the treatment of neoplastic cells with antitumor drugs of known type was also tested.

The term antitumor drug refers to any molecule or combination of molecules, for pharmaceutical use, intended to inhibit or block the growth and spread of tumor formations.

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

It was surprisingly found that the combination of applying such electric current waves having a waveform with a fundamental frequency higher than or equal to <NUM>, preferably ranging between <NUM> and <NUM>, more preferably ranging between <NUM> and <NUM>, even more preferably ranging between <NUM> and <NUM>, much more preferably about <NUM>, to neoplastic cells of the aforesaid target region for a predefined period of time, either previously, simultaneously or subsequent to the treatment with an antitumor drug, allows obtaining a greater antitumor efficacy of the antitumor drug itself.

In particular, the use of the antitumor drug in combination with QMR allows to obtain a greater reduction in the number of live neoplastic cells than the use of the antitumor drug alone on neoplastic cells.

It is therefore highlighted that the combination of the treatment with an antitumor drug with the application of the aforesaid electric current waves exerts a greater antitumor effect in the treated neoplastic cells than the use of the antitumor drug alone on these neoplastic cells, thus allowing, with equal antitumor effect, to decrease the dosage of the antitumor drug.

Still advantageously, thanks to this reduction in dosage achieved through the use of QMR, it is possible to achieve a substantial reduction in the side effects caused by treatment with the antitumor drug, for the same antitumor effect achieved.

This advantage is particularly evident when the antitumor drug is administered simultaneously with or after the treatment with QMR.

This advantage is even more evident when the drug is administered simultaneously with QMR treatment.

Still advantageously, the combination of treatment with antitumor drug and QMR allows to obtain an increase in the number of cells in early apoptosis.

It is clear that QMR can be used as an effective adjunct to standard chemotherapy therapies to enhance the effects of currently used antitumor drugs, without a corresponding increase in toxicity.

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

Some examples, not to be considered limiting, of such antitumor treatments comprise surgical treatments, radiosurgery, ionizing radiation treatments, chemotherapy treatments with alkylating agents, antimetabolites, antitumor antibiotics, topoisomerase inhibitors, antifungal drugs, corticosteroids, biological drugs, differentiating agents, hormones, drugs that stimulate the immune system, monoclonal antibodies.

Therefore, an antitumor drug for use in a method for blocking or inhibiting the growth of neoplastic cells in a target region of a living organism also forms part of the present description, wherein the method comprises treating the neoplastic cells with such antitumor drug simultaneously or after the application of electric current waves to the aforesaid target region for a predefined period of time. As stated above, such electric current waves have a waveform with a fundamental frequency higher than or equal to <NUM>.

It is not excluded that, according to one aspect of the description, the treatment of the neoplastic cells with the antitumor drug takes place prior to the application of the aforesaid electric current waves.

Preferably, this waveform has a fundamental frequency ranging between <NUM> and <NUM>, preferably ranging between <NUM> and <NUM>, more preferably ranging between <NUM> and <NUM>.

More preferably, this waveform has a fundamental frequency of about <NUM>. Still preferably, the aforesaid waveform is a sinusoidal waveform.

Additionally, this sinusoidal waveform is distorted by the presence of harmonics.

Preferably, such a sinusoidal waveform is distorted by the presence of at least second and third order harmonics.

According to one aspect of the description, the antitumor drug is used for use in a method comprising treating neoplastic cells with the antitumor drug simultaneously with the application of the aforesaid electric current waves to the target region of a living organism while the neoplastic cells are treated with the antitumor drug.

Returning to the method of the present description, this method preferably comprises the following steps:.

Preferably, by a living organism it is intended a human subject.

These electrodes are of a suitable shape and configuration to transmit the aforesaid electric current waves to the target region.

By way of example not to be considered limiting, such electrodes may be of a non-implantable type and, for example, may be in the form of a handpiece or in the form of a suction cup, or again may be essentially laminar in shape and applicable by adhesion to the skin of the subject, or such electrodes may be in the form of a probe.

When the electrodes used in the method of the description have a laminar shape, they are essentially flexible so as to follow the shape of the surface of the body without difficulty and, moreover, they are optionally also equipped with an adhesive substance which eases keeping them in contact with the body during the application of the waveform generated by the device.

It is not excluded that one or more of the aforesaid electrodes may be of the implantable type.

A device for blocking or inhibiting the growth of neoplastic cells in a target region of a living organism therefore also forms part of the present description.

Such a device is usable in the method of the description.

In particular, the device of the description is configured to generate electric current waves having a waveform with a fundamental frequency higher than or equal to <NUM>.

The target region preferably belongs to a living organism, more preferably a human subject, as already indicated above.

According to a preferred embodiment of the device of the description, it comprises a radiofrequency circuit connected to one or more electrodes and adapted to supply each of these electrodes with a current wave having a fundamental frequency higher than or equal to <NUM>.

Preferably, the device further comprises a rectifier circuit powered by the mains voltage which supplies preferably continuous voltage, more preferably stabilised, to the aforesaid radiofrequency circuit.

Such a radiofrequency circuit preferably comprises at least one electronic switch which is powered by the voltage and driven by a driving circuit.

More in detail, the radiofrequency circuit presents at its output a current wave with a fundamental frequency higher than or equal to <NUM> and is adapted to transmit this current wave to these electrodes. Preferably, this fundamental frequency is ranging between <NUM> and <NUM>, more preferably ranging between <NUM> and <NUM>, even more preferably ranging between <NUM> and <NUM>.

More preferably, the fundamental frequency corresponds to about <NUM>. This current wave also has a sinusoidal shape.

This current wave is also advantageously distorted by the presence of harmonics.

These harmonics are preferably harmonics of at least the second and third order.

More preferably, the current wave has a sinusoidal shape distorted by the presence of at least first, second and third order harmonics.

This current wave also circulates in a preferably broadband resonant circuit on the frequency of the fundamental wave of the distorted sinusoidal shape.

According to the preferred embodiment of the description, the device generates high frequency sinusoidal alternating electric current waves of <NUM> with harmonics from <NUM> to <NUM>.

According to one aspect of the device of the description, the electrodes of said device are configured in such a way that they can transmit the aforesaid current wave to the target region.

It is specified that the electrodes of the device of the description may have a shape and configuration corresponding to the shape and configuration of the electrodes described above for the method of the description.

According to one aspect of the device of the description, such electrodes are of the non-implantable type and are adapted to be applied to the surface of the body of the organism at or near which the target region comprising the neoplastic cells is located.

According to a variant embodiment of the device, it comprises a helmet configured to allow the housing of the aforesaid electrodes and to allow both their connection with the aforesaid radiofrequency circuit and their arrangement at or near the target region.

It should be noted that, according to a particular variant embodiment of the device comprising the aforesaid helmet, the latter is configured to also house the radiofrequency circuit.

According to a further variant of the device, it comprises a cap of the wearable type configured to house the aforesaid electrodes and allow them to be applied at or near the target region.

According to another variant embodiment of the device, it comprises at least one band provided with the aforesaid electrodes. Optionally, this band is configured to also house the device's radiofrequency circuit.

This band is also configured to be placed laterally and/or frontally and/or posteriorly to the head of the subject.

It is not excluded that such a band is configured to be placed at the person's waist and to be worn by the person as a belt.

Furthermore, it is not excluded that this band may be worn by the subject in a different way than indicated above.

According to a particular variant embodiment of the device of the description, at least one or more of the electrodes of the device of the description are electrodes of the implantable type.

The term "implantable" means an electrode intended to be implanted wholly or partially, by surgical or medical intervention, into the human body.

According to another variant embodiment of the device of the description, it is configured so as to be completely implantable.

By way of example, the present implantable device has a configuration substantially similar to the configuration of an implantable pacemaker.

Other aspects and advantages of the present description will appear when reading the following examples, which are to be considered as illustrative and non-limiting.

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-<NUM> (DMEM/F12 GlutaMAX, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) added with <NUM>% foetal bovine serum (FBS) (Qualified Australian, Gibco, Thermo Fisher Scientific) and <NUM>% penicillin/streptomycin (Sigma-Aldrich).

U87MG cells (courtesy of Prof. Massimo Dominici, Laboratory of Cellular Therapy, University Hospital of Modena and Reggio Emilia Modena, Italy) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) with GlutaMAX (Gibco, Thermo Fisher Scientific) added with <NUM>% FBS (Gibco, Thermo Fisher Scientific) and <NUM>% penicillin/streptomycin (Sigma-Aldrich).

Bone marrow-derived mesenchymal stromal cells (BM-MSC) were also used as non-tumor type control cells. BM-MSC cells were produced in the Advanced Cellular Therapies Laboratory of the Complex Operative Unit of Haematology of ULSS8 Berica as described previously (Sella et al, <NUM>). Briefly, MSC were isolated from cells obtained by washing discarded bone marrow collection bags and filters from healthy donors (Ethics Committee auth. No. <NUM>/<NUM> of <NUM>. After washing, cells were centrifuged at <NUM> rpm for <NUM> and seeded at a density of 1x10<NUM> cells/cm<NUM> in DMEM with GlutaMAX (Gibco, Thermo Fisher Scientific,) added with <NUM>% FBS (Gibco, Thermo Fisher Scientific) and <NUM>% penicillin/streptomycin (Sigma-Aldrich). Cell cultures were incubated at <NUM> in a humid atmosphere with <NUM>% CO<NUM>. Cells that did not adhere were removed <NUM> hours after seeding and new medium was added. The medium was changed every <NUM>-<NUM> days. At <NUM>% confluence, BM-MSC cells were re-seeded at a density of <NUM> cells/cm<NUM>.

The cell lines were stimulated with QMR using a prototype QMR device from the company Telea (Telea Electronic Engineering, Sandrigo, VI, Italy). The device, suitably adapted for the present in vitro experiments, generated high-frequency sinusoidal alternating electric current waves of <NUM> with harmonics from <NUM> to <NUM> and had the following data: power supply <NUM>÷<NUM> V ~ <NUM>/<NUM>; maximum output power, <NUM> W/<NUM>Ω.

QMR was applied to the cells using a pair of electrodes that were placed inside a <NUM> Petri dish, at the edge, and connected to the generator device. The transmission of electric fields to the medium of the cell cultures generated heat (Joule effect). The average temperature increase in the medium of the stimulated cells was <NUM>. This value was measured by means of three independent measurements with a data-logger probe (iLog, Escort Scunthorpe, UK) placed in the culture medium. In order to compensate for the increased temperature of the medium of the stimulated cells and also to ensure an average temperature of <NUM> for the stimulated cells, the incubator used for the stimulated cells was set at <NUM> at <NUM>% CO<NUM> and the temperature of the laboratory was maintained between <NUM> and <NUM>.

Based on the cell line used, a defined number of cells were seeded in <NUM> plates (Greiner Bio-One, Frickenhausen, DE) in order to be stimulated at <NUM>% confluence. The cells were then exposed to QMR for <NUM> hours and analysed <NUM>-<NUM>-<NUM> hours after the end of the stimulation.

More specifically, the cells were washed with D-PBS (Sigma-Aldrich), detached with 1X TrypLE Select (Gibco, Thermo Fisher Scientific) and the aliquots obtained were used to investigate cell viability, apoptosis, cell cycle, karyotype and proteomics. A cell aliquot was re-seeded for evaluation of cell growth capacity in semi-solid medium (soft agar assay), while cell migration was monitored directly on QMR-stimulated plates before and after stimulation. The effect of the combined use of QMR and the drug TMZ (Sigma-Aldrich) was also tested.

For these analyses, the cells were treated for <NUM> hours with <NUM>-<NUM> of TMZ administered at the same time as or after QMR stimulation. The TMZ stock solution was prepared in DMSO at a final concentration of <NUM> and maintained at <NUM>. Subsequent dilutions were made in fresh culture medium and administered to the cells. Cell viability, apoptosis and the cell cycle were assessed after <NUM> hours.

After stimulation, the cells were harvested and suspended in a <NUM>:<NUM> ratio with a Trypan blue solution (Gibco, Thermo Fisher Scientific) in culture medium. The cells were counted using a Burker's chamber and the number of live cells was obtained by applying the following formula: [(number of cells x <NUM>,<NUM> x <NUM> x V)/<NUM>], where V is the total volume of cell solution obtained and <NUM> is the dilution factor of the solution with the dye (2X).

After stimulation, <NUM> x <NUM><NUM> cells were harvested and centrifuged at <NUM> for six minutes. After washing with binding buffer, the cells were labelled with Annexin V/<NUM>-AAD according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). After dilution with binding buffer, the fluorescence of <NUM> x <NUM><NUM> cells/sample was detected using an FC500 flow cytometer (Beckman Coulter, Brea, CA, USA). The cell population was separated into four groups: live cells with negative fluorescence for both Annexin V and <NUM>-AAD; cells in early apoptosis positive for Annexin V and negative for <NUM>-AAD (Annexin V+/<NUM>-AAD-); cells in late apoptosis with double positivity for Annexin V and <NUM>-AAD (Annexin V+/<NUM>-AAD+) and dead cells negative for Annexin V and positive for <NUM>-AAD (Annexin V-/<NUM>-AAD+).

After stimulation, <NUM> x <NUM><NUM> cells were harvested and centrifuged at <NUM> for six minutes. The cells were then washed with D-PBS and fixed and permeabilised with acetone (<NUM>% solution in water). After one hour at <NUM>, the acetone was removed by centrifugation, the cells were washed and labelled with <NUM>µg/mL with <NUM>-AAD (Invitrogen) for <NUM> hour at room temperature. The fluorescence of <NUM> x <NUM><NUM> cells/sample was analysed using a FC500 flow cytometer (Beckman Coulter). EXPO <NUM> software (Coulter Systems, Fullerton, CA, USA) was used to calculate the percentage of cells in the different phases of the cell cycle. Diplode cycles were considered and corrected for cell clusters.

To assess whether QMR stimulation was safe and therefore did not cause chromosome alterations, BM-MSC mesenchymal stromal cells were stimulated for <NUM> hours with QMR and tested by G-Trypsin-Giemsa banding following standard techniques with <NUM>/<NUM> band resolution. <NUM> metaphases were analysed and <NUM> were karyotyped. Unstimulated BM-MSC cells were used as control cells.

The scratch test represents a 2D cell migration approach to semi-quantitatively detect cell motility. The purpose of scratching (opening) is to create a cell-free area (or opening) in order to induce the surrounding cells to migrate and close this opening. The cells were seeded in two-well silicone inserts (IBIDI GmbH, Gräfelfing, DE) that achieve a <NUM> cell-free opening on the cell monolayer. When <NUM>% cell confluence was reached, the insert was removed and the cells were stimulated for <NUM> hours with QMR. Cell migration was monitored over time by image acquisition before stimulation and <NUM>-<NUM>-<NUM> hours after stimulation. Image acquisition was performed with an Axiovert <NUM> CFL inverted light microscope (Carl Zeiss, Oberkochen, DE). The percentage of closure of the opening was analysed with ImageJ software (National Institutes of Health, Bethesda, MD, USA) and the migratory capacity was assessed by means of a migration rate curve, which was calculated as shown below: Closure % = [(Area tx - Area ty)/ Area tx] x <NUM>, where tx represents the acquisition time and ty the next time point.

The colony formation assay in semi-solid medium (soft agar) is a method used to monitor cell growth independently of cell anchoring to the substrate and which measures cell proliferation in semi-solid medium by optical colony counting. The rate of colony formation in soft agar changes depending on the cell line used. Therefore, the concentration of agarose, the number of cells seeded and the final day of the experiment were optimised for each cell line. The cell suspension was prepared in a <NUM>% agarose solution in cell medium and seeded on a solidified layer of <NUM>% agarose in complete medium. After <NUM> hour at room temperature, <NUM>µl of complete medium was added and a further <NUM>µl were added every week until the end of the experiment. The plates were transferred to an incubator at <NUM> at <NUM>% CO<NUM> for <NUM>-<NUM> days before being labelled with <NUM> calcein (Sigma-Aldrich) for <NUM> minutes. The colonies consisting of at least <NUM> cells were counted using an Axiovert <NUM> CFL inverted light microscope (Carl Zeiss).

<NUM> x <NUM><NUM> cells were harvested and centrifuged at <NUM> for <NUM> minutes, washed with D-PBS and lysed with lysis buffer on ice (Pierce™ RIPA buffer, Thermo Fisher Scientific) added with protease inhibitor cocktail (Cell Signalling, Danvers, MA, USA). After <NUM> minutes on ice, the cell lysates were centrifuged at <NUM> for <NUM> minutes at <NUM> and the protein supernatant was determined colorimetrically by BCA assay (Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific). Bovine serum albumin was used as the standard (Sigma Aldrich).

Liquid chromatography - mass spectrometry (LC/MS-MS) is an analytical technique that combines the separating capacity of liquid chromatography with the high sensitivity and selectivity of the mass analysis of the triple quadrupole mass spectrometry. For sample preparation, <NUM>µg of protein lysate were precipitated with acetone and the obtained protein pellet was dissolved in a <NUM> solution of urea and <NUM> ammonium bicarbonate (pH <NUM>). Samples were reduced using <NUM> dithiothreitol for <NUM> hour at room temperature and alkylated with <NUM> iodoacetamide in the dark for <NUM> minutes at room temperature. Subsequently, the proteins were subjected to digestion with the endopeptidase enzyme Lys-C (Promega) at an enzyme/protein ratio of <NUM>:<NUM> (w/w) for <NUM> hours at room temperature. The proteins were then diluted <NUM> times in <NUM> ammonium bicarbonate and digested overnight with trypsin (Promega) <NUM>:<NUM> (w/w) at room temperature. Proteolysis was interrupted by the addition of <NUM>% trifluoroacetic acid. Samples were then desalinated, vacuum dried and resuspended in <NUM>% formic acid for LC-MS/MS analysis.

The samples were analysed using the Easy-nLC <NUM> system (Thermo Fisher), coupled in-line with an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher). A reversed-phase column (Acclaim PepMap RSLC C18, <NUM> particle size, 100Å pore size, id <NUM>, Thermo Fisher Scientific) with a two-component mobile phase was used to separate the digested peptides: <NUM>% formic acid in water (buffer A) and <NUM>% formic acid in acetonitrile (buffer B). Peptides were eluted using a gradient from <NUM>% to <NUM>% for <NUM> minutes, followed by <NUM>% to <NUM>% for <NUM> minutes and finally by <NUM>% to <NUM>% for <NUM> minutes at a flow rate of <NUM> nL/min. The data-dependent acquisition (DDA) method is based on full scans performed at a resolution power of <NUM> fwhm (full width at half maximum) (at <NUM>/z), and a maximum injection time of <NUM>. The mass range of <NUM>-<NUM>/z was examined for the precursors, with the first mass set at <NUM>/z for the fragments. Full scans were followed by a series of MS/MS (HCD) scans for a cycle time of <NUM> seconds, at a collision energy of <NUM>% and detected in the ion trap with a maximum injection time of <NUM>. The dynamic exclusion time was set at <NUM> sec. Unprocessed data were searched in Proteome Discoverer <NUM> software (Thermo Fisher Scientific). Peptide searches were carried out using the Human protein FASTA file (uniprot). The proteins were identified with the MASCOT search engine (Matrix Science inc, Boston, MA, USA) using a precursor mass tolerance of <NUM> ppm and a product mass tolerance of <NUM> Da.

The different abundances of proteins expressed in the different experimental groups (at t0 and t24, respectively) were used to perform a hierarchical cluster analysis using the Clustvis tool (Metsalu T et al, <NUM>). Gene Ontology (GO) and Pathway annotation of protein IDs was performed using the EnrichR gene set enrichment analysis server (http://amp. edu/Enrichr/), by applying biological processes and Reactome categorisation with the significance threshold set at p <<NUM>. The network of protein interactions was constructed using the STRING interaction database, version <NUM> (https://string-db. org/) (Mering C et al, <NUM>).

All data were analysed using GraphPad program (GraphPad Software, San Diego, CA, USA) and were expressed as mean ± standard error. The One-sample t-test was used for the analysis of the results expressed as a ratio/percentage of the control (e.g. trypan blue exclusion assay). The unpaired Student's t-test was used for all the other analyses. Data with p < <NUM> were considered as significant.

The effects of QMR treatment on A172 glioblastoma cells were assessed by analysing cell morphology, proliferation rate, apoptosis and cell cycle of the cells immediately at the end of the treatment and <NUM> and <NUM> hours after the end of QMR stimulation. The compliance with these timeframes made it possible to obtain an assessment of the permanence of the effects obtained with the treatment and the ability of the treated cells to restore their cellular functions to pre-treatment levels.

It was surprisingly found that QMR treatment changes the morphology of treated A172 cells compared to untreated control cells. QMR-treated cells appeared in fact more swollen and more granular than control cells, indicating that cell damage had occurred.

This damage was also found to be persistent over time, as shown in <FIG>.

The cell proliferation rate was also estimated by testing with Trypan Blue. <NUM> hours after treatment, QMR-treated cells showed a reduction in tumor growth by about <NUM>%. Furthermore, the number of live cells tended to decrease even <NUM>-<NUM> hours after the treatment, suggesting an effect of QMR treatment on the cell cycle progression, as visible in <FIG>.

In support of this surprising finding, the cell cycle of A172 cells was also evaluated using flow cytometry experiments. The results, shown in <FIG>, showed that QMR treatment significantly reduces the rate of dividing cells (defined as the percentage of cells in S phase) while the cells in G<NUM>/M phase significantly increase. These results were also confirmed up to <NUM> hours after the end of the treatment, showing that cell cycle arrest had occurred in the G<NUM>/M phase.

Further, the efficacy of treating cells with QMR was confirmed by the clear apoptotic activation in the treated cells. The number of live cells after QMR treatment was in fact significantly lower (p<<NUM>) than the number of control live cells not treated with QMR, as shown by the results in <FIG>. This reduction was also correlated with an increase of about <NUM>% in the number of cells in early apoptosis and a slight increase in the number of cells in late apoptosis.

The effects of QMR treatment on healthy BM-MSC cells were tested in order to verify the safety of QMR treatment on non-neoplastic type cells. At the end of QMR stimulation and <NUM> hours after the end of the treatment, cell morphology, proliferation rate, apoptosis, cell cycle and karyotype were evaluated in treated BM-MSC cells and in untreated control cells. Although a slight decrease in cell growth was displayed at the end of the QMR treatment, as shown in <FIG>, this effect is not to be considered comparable to that obtained in the treated A172 glioblastoma cells, shown in <FIG>.

Furthermore, BM-MSC cells treated with QMR showed no differences in cell morphology, cell cycle modulation and apoptosis, as visible in <FIG>, respectively.

Furthermore, time did not influence the final effect obtained, as demonstrated by the results obtained which are substantially superimposable at <NUM> and <NUM> hours after treatment.

The analysis of the karyotype of BM-MSC cells treated with QMR also showed that the QMR treatment advantageously causes no changes in the number of chromosomes or in their structure, as can be seen in <FIG>.

It is well known that neoplastic type cells are characterized by uncontrolled proliferation and migration. The ability of cells to grow in semi-solid medium and their mobility rate are therefore considered markers of tumor aggressiveness and invasiveness. Cell colony formation in soft agar and cell migration were therefore analysed under basal conditions and after QMR treatment. The results, shown in <FIG>, surprisingly showed that the QMR treatment significantly reduces the growth of A172 cells in soft agar. In fact, the number of cell colonies after the QMR treatment was about <NUM>% lower than the number of colonies of untreated control cells. Surprisingly, moreover, the cell migration rate of the A172 cells treated with QMR was also lower in a timedependent manner than that of control cells, as shown in <FIG>.

The proteomic profile of A172 and BM-MSC cells treated with QMR was also investigated. The analysis was conducted at the end of the QMR stimulation (time <NUM>) and <NUM> hours after the end of the treatment. Untreated A172 and BM-MSC cells were used as control groups.

A total of <NUM> proteins were identified in the A172 cell line, of which <NUM> and <NUM> were found to be significantly altered after QMR treatment at time zero and <NUM> hours after the end of the treatment, respectively. Cluster analysis, not shown in the figures, highlighted two separate groups of proteins at both testing timeframes, demonstrating that QMR treatment interferes with neoplastic cell activity.

The proteins were found to be differentiated between treated and untreated cells. Specifically, at time zero, <NUM> proteins were found to be significantly up-regulated, while <NUM> proteins were found to be down-regulated. Instead, <NUM> hours after the end of the treatment, <NUM> up-regulated proteins and <NUM> down-regulated proteins were found.

More specific analyses of gene ontology and pathway enrichment showed that up-regulated proteins were strongly associated with stress response mechanisms, protein folding and extracellular matrix modelling, suggesting that QMR treatment also acts as a toxic-protein stimulus.

More specifically, QMR-treated cells, compared to untreated control cells, had undergone a significant down-regulation of key factors involved in protein translation, RNA processing and cell cycle involved pathways, leading to cell cycle arrest and death of treated cells.

Advantageously, the effects of the QMR stimulation on BM-MSC cells provided completely different results compared to A172 glioblastoma cells. At the end of QMR treatment, the activation of heat shock proteins was associated with a remodelling of the extracellular matrix. However, this alteration was found to be totally normalised <NUM> hours after treatment. Therefore, QMR treatment does not interfere with the proliferation rate of BM-MSC cells, supporting the selectivity of QMR treatment towards neoplastic type cells.

The experiments described above were also performed on glioblastoma cell lines that were more aggressive than the A172 cell line cells. These more aggressive cell lines comprise T98G and U87MG cells. Although the proliferation rate was found to be reduced in both cell lines following QMR treatment, there were no significant differences in apoptosis activation, cell cycle progression, clonogenic capacity and cell motility, as shown in <FIG>.

Cell viability was also tested after <NUM> and <NUM> hours of QMR treatment. Surprisingly, the cell proliferation rate for both A172 and U87MG cells was found to be significantly reduced over time.

Even a <NUM>% increase in treatment power has been shown to affect the rate of cell proliferation. In fact, the cell viability of the QMR-treated cells at higher power was found to be decreased by up to <NUM>%, as shown in the figures.

Standard therapies for patients with glioblastoma include, to date, total resection of the lesion, followed by radiotherapy and chemotherapy with the drug TMZ.

Disadvantageously, the efficacy of the drug is limited by the severity of the side effects caused by the drug itself and by the establishment of drug resistance mechanisms. It is therefore essential to find new therapeutic strategies to be used in combination with TMZ in order to lower the dosage thereof and improve the patient's quality of life.

The efficacy of the combined therapy of TMZ and QMR treatment on A172 glioblastoma cells was therefore tested. These cells were treated with <NUM>-<NUM> of TMZ at the same time as or after the QMR treatment. Cell viability, apoptosis and cell cycle values were analysed. The TMZ drug concentration was selected on the basis of its IC50 (<FIG>).

The results obtained have surprisingly shown that, when the TMZ drug at a concentration of <NUM> was used in combination with QMR treatment, cell viability was reduced by about <NUM>% more than the reduction in cell viability obtained in cells treated with TMZ drug alone, as shown in <FIG>.

Furthermore, these data were found to be surprisingly correlated with the data on cell apoptosis. Indeed, the combined administration of TMZ and QMR showed that the reduction in the number of live cells is associated with an increase in the number of cells in early apoptosis, as shown in the graphs in <FIG>.

The simultaneous exposure of the cells to the drug TMZ <NUM> and to QMR activates a greater alteration in cell cycle progression, compared to cells treated with the TMZ drug alone.

These data surprisingly demonstrate that the combined therapeutic strategy of TMZ with QMR induces significant cell cycle arrest in the G2-M phase and reduces the percentage of cells in the G0-G1 phase, as shown in <FIG>.

Claim 1:
An antitumor drug for use in a method for blocking or inhibiting the growth of neoplastic cells in a target region of a living organism, said method comprising treating said neoplastic cells with said antitumor drug simultaneously with the application of electric current waves to said target region for a predefined period of time while said neoplastic cells are treated with said antitumor drug, said electric current waves having a waveform with a fundamental frequency of about <NUM> distorted by the presence of at least second and third order harmonics.