Therapeutic composition and method combining an alternating electric field and a DNA-dependent protein kinase inhibitor

Abstract

This specification discloses the use of alternating electric fields and DNA-dependent PK inhibitors for cancer treatment. This specification discloses the use of alternating electric fields and DNA-dependent PK inhibitors for inducing cell death. This specification discloses the use of alternating electric fields and DNA-dependent PK inhibitors to inhibit DNA repair in cancer cells having DNA strand breaks (e.g., double-strand DNA breaks). This specification discloses the use of alternating electric fields and DNA-dependent PK inhibitors to enhance the effectiveness of radiotherapy.

Description

Background Art

[0001] DNA is damaged by various causes throughout the entire cell cycle, including the tumor cell cycle. In particular, radiotherapy and chemotherapy are used to induce DNA damage in tumor cells and cause tumor cell death. DNA damage is repaired through various DNA repair pathways depending on the type of damage. DNA repair pathways include direct repair (DR), base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), DNA strand break repair pathways, and the like. DNA-protein kinase (DNA-PK) regulates the major pathway (non-homologous end joining) responsible for the repair of DNA double-strand breaks induced by radiation.

[0002] When the normal repair process is impaired or altered, DNA damage is not repaired and causes cell death in tumor cells. The DNA repair status in tumor cells is associated with the therapeutic response to anticancer agents, and DNA repair pathways have been established as promising targets in cancer treatment.

[0003] A tumor treatment field (TT field, also referred to as an alternating current electric field) delays the DNA damage repair after radiotherapy for glioblastoma cells. The TT field affects the DNA repair ability of cells by changing the homologous recombination repair pathway.

[0004] Therefore, changing both the homologous recombination repair pathway and the non-homologous recombination repair pathway may result in effective cancer treatment.

Summary of the Invention

[0005] A method of treating a subject having cancer is disclosed, the method comprising applying an alternating current electric field to a target site of a subject containing one or more cancer cells for a certain period of time, and administering to the subject a therapeutically effective amount of a DNA-dependent PK inhibitor.

[0006] Also disclosed is a DNA-dependent protein kinase inhibitor for use in a method of treating a subject with cancer, the method comprising: a) applying an alternating electric field for a certain period of time to a target site of the subject containing one or more cancer cells; and b) administering a therapeutically effective amount of the inhibitor to the subject.

[0007] Also disclosed is a kit for treating cancer cells, the kit comprising one or more DNA-dependent protein kinase PK inhibitors and one or more materials for delivering an alternating electric field to a target site containing one or more cancer cells.

[0008] Furthermore, an in vitro method for treating a target site is disclosed, which comprises a) applying an alternating electric field to a target site containing one or more cancer cells for a certain period of time, and b) administering a therapeutically effective amount of a DNA-dependent protein kinase (PK) inhibitor to the target site.

[0009] A method for inducing cell death in cancer cells is disclosed, which includes exposing cancer cells to an alternating electric field for a certain period of time and exposing cancer cells to a DNA-dependent PK inhibitor.

[0010] Furthermore, a DNA-dependent protein kinase (PK) inhibitor used in a method for inducing cell death in cancer cells is disclosed, the cancer cells being present in a subject, and the method comprising exposing the cancer cells to an alternating electric field for a certain period of time and exposing the cancer cells to a DNA-dependent PK inhibitor.

[0011] A method for inhibiting DNA repair in cancer cells that have been exposed to or have been exposed to radiation is disclosed, the method comprising exposing cancer cells to an alternating electric field for a certain period of time and exposing cancer cells to a DNA-dependent PK inhibitor.

[0012] Furthermore, a DNA-dependent protein kinase (PK) inhibitor used in a method to inhibit DNA repair in cancer cells that have been or have been exposed to radiation is disclosed, wherein the cancer cells are present in a subject, and the method comprises exposing the cancer cells to an alternating electric field for a certain period of time and exposing the cancer cells to a DNA-dependent PK inhibitor.

[0013] A method for enhancing the effectiveness of radiotherapy in a subject is disclosed, which includes applying an alternating electric field for a certain period of time to a target site of the subject that is receiving or has received radiotherapy, and administering a therapeutically effective amount of a DNA-dependent PK inhibitor to the subject.

[0014] Also disclosed is a DNA-dependent protein kinase (PK) inhibitor used in a method to enhance the effectiveness of radiotherapy in a subject, the method comprising: a) applying an alternating electric field having frequency and electric field strength to a target site of the subject, including the site receiving or having received radiotherapy, for a certain period of time; and b) administering a therapeutically effective amount of the DNA-dependent protein kinase inhibitor to the subject.

[0015] Furthermore, DNA containing one or more double-strand breaks is disclosed, which is used in a method to enhance the effectiveness of radiotherapy in a subject, the method comprising applying an alternating electric field having a frequency and electric field intensity for a certain period of time to a target site of the subject including the site receiving or having received radiotherapy, the one or more double-strand breaks being formed by the application of the alternating electric field.

[0016] Furthermore, DNA containing one or more double-strand breaks is disclosed, and the DNA is used in a method for inducing cell death in cancer cells present in a subject, the method comprising exposing cancer cells to an alternating electric field for a certain period of time to form one or more double-strand breaks.

[0017] Further disclosed is DNA containing one or more double-strand breaks, which is used in a method for treating a subject having cancer, the method comprising forming one or more double-strand breaks at a target site in the subject containing one or more cancer cells by applying an alternating electric field for a certain period of time.

[0018] Furthermore, DNA containing one or more strand breaks is disclosed, which is used in a method to induce cell death in cancer cells present in a subject, the method comprising exposing cancer cells to an alternating electric field for a certain period of time to form one or more strand breaks. In such embodiments, the strand breaks may be one or more single-strand breaks and double-strand breaks.

[0019] Furthermore, DNA containing one or more strand breaks is disclosed, and the DNA is used in a method for treating a subject having cancer, the method comprising forming one or more strand breaks by applying an alternating electric field for a certain period of time to a target site in the subject containing one or more cancer cells. In such embodiments, the strand breaks may be one or more single-strand breaks and double-strand breaks.

[0020] Additional advantages of the disclosed methods and compositions may be some described in the following description, some understood from the description, or acquired through practice of the disclosed methods and compositions. The advantages of the present invention are realized and achieved by the elements and combinations specifically pointed out in the appended claims. It should be understood that the above general description and the following detailed description are for illustrative and explanatory purposes only and do not limit the claimed invention. [Brief explanation of the drawing]

[0021] The accompanying drawings incorporated herein and forming part thereof illustrate several embodiments of the disclosed methods and compositions and, together with the description, are useful in illustrating the principles of the disclosed methods and compositions.

[0022] [Figure 1] A schematic diagram of the DNA repair mechanism is shown. [Figure 2] A schematic diagram of the DNA repair mechanism is shown. [Figure 3] Examples of cell treatment with and without radiation and with and without DNA-dependent PK inhibitors are shown. Figure 3 shows the 3D colony formation viability of MGMT-negative CX18 central 1 and peripheral 2 primary glioblastoma stem cell-like cells (GSCs) treated with DNA-PK inhibitors (DNA-PKi) or radiation (IR) alone or in combination (DNA-PKi treatment 1 hour before radiation). The upper panel shows Western blots of specified proteins highlighting the effective inhibition of DNA-PK kinase activity (indicated by a decrease in radiation-induced Ser2056 autophosphorylation) at the indicated doses of DNA-PK inhibitors. Figure 3 also shows proposed studies using the TT field in combination with DNA-PK inhibitors and radiation. [Figure 4] Examples of cell treatment with and without radiation and with and without DNA-dependent PK inhibitors are shown. Figure 4 shows the 3D colony formation viability of MGMT-positive OX5 central and peripheral primary glioblastoma stem cell-like cells (GSCs) treated with DNA-PK inhibitors (DNA-PKi) or radiation (IR) alone or in combination (DNA-PKi treatment 1 hour before radiation). The upper panel shows Western blots of specified proteins highlighting the effective inhibition of DNA-PK kinase activity (indicated by a decrease in radiation-induced autophosphorylation at Ser2056) at specified DNA-PK inhibitor doses. Figure 4 also shows proposed studies using the TT field in combination with DNA-PK inhibitors and radiation. [Figure 5]The TT field in combination with VX984 (DNAPKi) was shown to enhance glioblastoma cell death by radiosensitization in the CX18 margin 2, CX18 center 1, OX5 margin, and OX5 center of primary-derived tumor resection specimens in 3D Alvetex culture. Western blot analysis verified DNAPK inhibition by VX984 (250 nM) through depletion of the pDNAPK (pS2056) signal against radiation (5 Gy) irradiation in the CX18 margin 2, CX18 center 1, OX5 margin, and OX5 center. Colony formation survival assay of cells pretreated with VX984 (250 nM, 1 hour) before TT field exposure (200 kHz, 72 hours) in CX18 margin 2, CX18 center 1, OX5 margin, and OX5 center cells. n = 3, statistical significance was shown by one-way analysis of variance (ANOVA). Representative images of cell death after TT field treatment in CX18 margin 2, CX18 center 2, OX5 margin, and OX5 center cells. [Figure 6] This is a table showing an overview of the survival data of the TT field in combination with VX984 and radiotherapy. The average intratumoral survival rate (INTRA); OX5 center vs. margin and CX18 center vs. margin, as well as the average intertumoral survival rate; OX5 INTRA vs. CX18 INTRA are shown. To determine whether cell death is synergistic or additive, the Bliss index was calculated using the product of the survival rates of DMSO+TTF and Combo-TTF as the ratio to Combo+TTF. A ratio < 1.1 is considered additive cell death, and > 1.1 is considered synergistic cell death. [Figure 11] Shows the titration of Nedisertib. [Figure 12] The TT field was shown to enhance the cytotoxic and overall effects of Nedisertib in A549 cells. 0.97 V / cm, 72 hours, 150 kHz, N = 6. [Figure 13] The TT field was shown to enhance the cytotoxic and overall effects of Nedisertib in H1299 cells. 0.97 V / cm, 72 hours, 150 kHz, N = 5. [Figure 14]Combined treatment with TT field and Nedisertib is shown to promote cell cycle arrest in A549 cells. 0.97 V / cm, 150 kHz, N = 3. [Figure 15] Combined treatment with TT field and Nedisertib is shown to promote cell cycle arrest in H1299 cells. 0.97 V / cm, 150 kHz, N = 3. [Figure 16] Shows the titration of CC-115. [Figure 17] TT field is shown to enhance the cytotoxic effect and overall effect of CC-115 in A549 cells. 0.97 V / cm, 72 hours, 150 kHz, N = 6. [Figure 18] TT field is shown to enhance the cytotoxic effect and overall effect of CC-115 in H1299 cells. 0.97 V / cm, 72 hours, 150 kHz, N = 4. [Figure 19] Combined treatment with TT field and CC-115 is shown to promote cell cycle arrest in A549 cells. 0.97 V / cm, 150 kHz, N = 6. [Figure 20] Combined treatment with TT field and CC-115 is shown to promote cell cycle arrest in H1299 cells. 0.97 V / cm, 150 kHz, N = 6.

Mode for Carrying Out the Invention

[0023] The disclosed methods and compositions may be more readily understood by reference to the following detailed description of specific embodiments and the examples contained therein, as well as the figures and the description before and after them.

[0024] It should be understood that the disclosed methods and compositions are not limited to a particular synthesis method, a particular analytical technique, or a particular reagent, and may vary, unless otherwise specified. It is also understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0025] Materials, compositions, and components that can be used in, in combination with, or in preparation of the disclosed methods and compositions, or that are products thereof, are disclosed herein. While specific reference to various individual and collective combinations and permutations of these materials may not be explicitly disclosed, it is understood that each is specifically considered and described herein. For example, if a peptide is disclosed and discussed, and several modifications that can be made to multiple molecules containing amino acids are discussed, then, unless otherwise explicitly stated, all combinations and permutations of the peptide and possible modifications are specifically assumed. Thus, if classes A, B, and C are disclosed, and further classes D, E, and F and examples of combined molecules AD are disclosed, then each is considered individually and collectively, even if not individually described. Therefore, in this example, each combination of AE, AF, BD, BE, BF, CD, CE, and CF is specifically considered and should be considered disclosed from the disclosure of A, B, and C, D, E, and F and example combination AD. Similarly, these subsets or combinations are also specifically considered and disclosed. Therefore, for example, the subgroups AE, BF, and CE are specifically envisioned and should be considered disclosed from the disclosures of A, B, and C, D, E, and F, as well as example combinations A through D. This concept applies to all embodiments of the Application, including but not limited to steps of methods for manufacturing and using the disclosed compositions. Therefore, where there are various additional steps that can be implemented, each of these additional steps can be implemented in any particular embodiment or combination of embodiments of the disclosed method, and each such combination is specifically considered and should be considered disclosed. A.Definition

[0026] The disclosed methods and compositions are not limited to the specific methods, protocols, and reagents described, and it is understood that these may vary. Furthermore, the terms used herein are for illustrative purposes only to describe specific embodiments and are not intended to limit the scope of the invention, and the scope of the invention is limited only by the appended claims.

[0027] In this specification and the attached claims, the singular forms "a," "an," and "the" also include plural references unless the context clearly indicates otherwise. Therefore, for example, a reference to "DNA-dependent protein kinase inhibitors" includes multiple such inhibitors, and a reference to "cells" refers to one or more cells and their equivalents known to those skilled in the art.

[0028] As used herein, the term "or" means any one member of a given list, and unless otherwise indicated, in other embodiments it may also include any combination of members of that list.

[0029] In this specification, “target site” refers to a specific site or location located inside or on the surface of a subject or patient. For example, “target site” may refer to, but is not limited to, cells (e.g., cancer cells), a population of cells, an organ, a tissue, or a tumor. Thus, the term “target cell” may be used to refer to a target site, and the target site is a cell. In some embodiments, “target cell” may be a cancer cell. In some embodiments, organs that may be a target site include, but are not limited to, the brain. In some embodiments, cells or populations that may be a target site or target cell include, but are not limited to, cancer cells (e.g., ovarian cancer cells). In some embodiments, “target site” may be a tumor target site.

[0030] A “tumor target site” is a site or location within or on the subject or patient that contains, is adjacent to, previously contained, or is suspected to contain one or more cancer cells. For example, a tumor target site may refer to a site or location within or on the body of a subject or patient that is prone to metastasis. In addition, a target site or tumor target site may refer to the site or location of resection of a primary tumor located within or on the surface of the subject or patient. In addition, a target site or tumor target site may refer to a site or location adjacent to the resection of a primary tumor located within or on the body of a subject or patient.

[0031] In this specification, “alternating current field” refers to a very low-intensity, directional, medium-frequency alternating current field delivered to a subject, a sample taken from a subject, or a specific location within a subject or patient (e.g., a target site such as a cell). In some embodiments, the alternating current field can be unidirectional or multidirectional. In some embodiments, the alternating current field can be delivered through two pairs of transducer arrays that generate a field perpendicular to the target site. For example, in the Optune® system (alternating current field delivery system), one pair of electrodes is positioned to the left and right (LR) of the target site, and the other pair of electrodes is positioned to the front and back (AP) of the target site. By circulating the electric field between these two directions (LR and AP), it is ensured that the maximum range of cell directions is targeted.

[0032] As used herein, an alternating electric field applied to a targeted site of a tumor may be called a tumor therapeutic field or TT field. TT fields are established as an anti-mitotic carcinoma therapy because they disrupt proper microtubule assembly during metaphase and ultimately destroy cells during telophase, cytokinesis, or subsequent interphase. TT fields target solid tumors and are described in U.S. Patent No. 7,565,205, which, as teachings of TT fields, is incorporated herein by reference in its entirety.

[0033] In vivo and inhydro studies have shown that the effectiveness of TT field therapy increases as the intensity of the electric field increases. Therefore, optimizing the array placement on the subject to increase the intensity of the subject site or subject cells is a standard technique of the Optune system. Optimization of array placement may be carried out by “rules of thumb” (e.g., positioning the array as close as possible to the subject’s target site or target cells), the patient’s body shape, the dimensions of the target site, and / or measurements representing the location of the target site or cells. Measurements used as input may be obtained from image data. Image data includes all kinds of visual data, such as single-photon emission computed tomography (SPECT) image data, X-ray computed tomography (X-ray CT) data, magnetic resonance imaging (MRI) data, positron emission tomography (PET) data, and data that can be acquired by optical instruments (e.g., photographic cameras, charge-coupled device (CCD) cameras, infrared cameras, etc.). In certain embodiments, image data may include 3D data acquired from or generated by a 3D scanner (e.g., point cloud data). Optimization may depend on understanding how the electric field is distributed within the target site or target cell as a function of the array's position, and in some embodiments, taking into account variations in the electrical property distribution within the heads of different patients.

[0034] The term "subject" refers to a subject of administration, such as an animal. Therefore, the subject of the disclosed method may be a vertebrate, such as a mammal. For example, the subject could be a human. This term does not indicate a specific age or sex. "Subject" may be used interchangeably with "individual" or "patient." For example, the subject being administered may mean a person receiving an alternating electric field. For example, the subject being administered may be a subject with ovarian cancer or lung cancer.

[0035] "To treat" means administering or applying therapeutic agents, such as alternating electric fields or vectors, to subjects, such as humans or other mammals (e.g., animal models), who have cancer or are at increased susceptibility to developing cancer, in order to prevent or slow the worsening of the effects of the disease or infection, or to partially or completely reverse the effects of cancer. For example, treating a subject with glioblastoma may involve delivering therapeutic agents to cells within the subject.

[0036] "Prevention" means minimizing or reducing the likelihood that a subject will develop cancer.

[0037] As used herein, the terms “administering” and “administration” refer to any method of delivering a DNA-dependent PK inhibitor directly or indirectly to a target site in a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral, transdermal, inhalation, nasal, topical, vaginal, intraocular, intraocular, intracerebral, rectal, sublingual, buccal, and parenteral administration, including injections such as intravenous, intra-arterial, intramuscular, and subcutaneous. Administration may be continuous or intermittent. In various embodiments, the formulation may be administered therapeutically, i.e., administered to treat cancer. In various further embodiments, the formulation may be administered prophylactically, i.e., administered to prevent cancer. In one embodiment, a skilled technician may determine an effective dose, effective schedule, or effective route of administration to treat a subject. In some embodiments, administration may include contact, exposure, or application. Therefore, in some embodiments, exposing a target site or subject to an alternating electric field, applying an alternating electric field to a target site or subject, or contacting a target site or subject with an alternating electric field means applying an alternating electric field to the target site or subject. In some embodiments, contact, exposure, and application can be used interchangeably.

[0038] In this specification, “subject” refers to the subject to which the administration is performed, for example, an animal. Therefore, the subject of the disclosed method may be a vertebrate such as a mammal. For example, the subject may be a human. This term does not indicate a specific age or sex. “Subject” can be used interchangeably with “individual” or “patient.”

[0039] In this specification, ranges may be expressed as "approximately" from a particular value and / or "approximately" to another particular value. Where such ranges are expressed, unless otherwise specified in the context, the range from a particular value and / or to another particular value is also specifically assumed and disclosed. Similarly, where the use of the antecedent "approximately" expresses a value as an approximation, it is understood that, unless otherwise specified in the context, that particular value forms another specifically considered embodiment that should be considered disclosed. Furthermore, unless otherwise specified in the context, it is understood that each endpoint of a range is important both in relation to and independently of other endpoints. Finally, it should be understood that all individual values ​​and subranges of values ​​included within an explicitly disclosed range are also specifically assumed and should be considered disclosed unless otherwise indicated in the context. The foregoing applies regardless of whether some or all of these embodiments are explicitly disclosed in a particular case.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in which the disclosed methods and compositions belong. Any methods and materials similar or equivalent to those described herein may be used in carrying out or testing the methods and compositions of the present invention, but the methods, apparatus and materials described herein are particularly useful. Publications cited herein and the materials from which they are cited are expressly incorporated herein by reference. Nothing in this specification should be construed as admitting that the present invention does not have prior rights to this disclosure by prior art. No reference is acknowledged to constitute prior art. The descriptions of references state the claims of their authors, and the applicant reserves the right to object to the accuracy and validity of the cited documents. Numerous publications are referenced herein, but it is clear that such references do not acknowledge that any of these documents constitute part of the common general knowledge in the art.

[0041] Throughout this specification and the claims, the word “comprise” and its variations “comprising” and “comprises” mean “not limited to” and are not intended to exclude, for example, other additives, components, integers, or steps. In particular, where a method is described as comprising one or more steps or operations, it is specifically assumed that each step includes what is listed (unless the step contains a limiting term such as “consisting of”). In other words, each step is not intended to exclude other additives, components, integers, or steps, etc., that are not listed in that step. B. AC electric field

[0042] The methods disclosed herein involve applying an alternating current (AC) electric field. In some embodiments, the AC electric field used in the methods disclosed herein is a tumor treatment electric field. In some embodiments, the AC electric field may vary depending on the type or state of the cells to which the AC electric field is applied. In some embodiments, the AC electric field may be applied via one or more electrodes placed on the body of a subject. In some embodiments, two or more electrode pairs may be present. For example, an array may be placed on the front / back and sides of the patient and may be used with the systems and methods disclosed herein. In some embodiments, when two pairs of electrodes are used, the AC electric field may be generated alternately between the electrode pairs. For example, a first electrode pair may be placed on the front and back of the subject, and a second electrode pair may be placed on either side of the subject, and then the AC electric field may be applied alternately between the front and back electrodes, and then between the left and right electrodes.

[0043] In some embodiments, the frequency of the AC electric field is 100 kHz to 500 kHz. In some embodiments, the frequency of the AC electric field is 50 kHz to 1 MHz. The frequency of the AC electric field may also be, but is not limited to, 50 to 500 kHz, 100 to 500 kHz, 100 to 300 kHz, 25 kHz to 1 MHz, 50 to 190 kHz, 25 to 190 kHz, 180 to 220 kHz, or 210 to 400 kHz. In some embodiments, the frequency of the AC electric field may be 50 kHz, 100 kHz, 150 kHz, 200 kHz, 250 kHz, 300 kHz, 350 kHz, 400 kHz, 450 kHz, 500 kHz, or any frequency in between. In some embodiments, the frequency of the AC electric field is approximately 200 kHz to approximately 400 kHz, approximately 250 kHz to approximately 350 kHz, and may also be approximately 300 kHz.

[0044] In some embodiments, the electric field strength of an AC electric field may be in the range of 0.5 to 4 V / cm RMS. In some embodiments, the electric field strength of an AC electric field may be in the range of 1 to 4 V / cm RMS. In some embodiments, different electric field strengths (e.g., 0.1 to 10 V / cm) may be used. In some embodiments, the electric field strength may be 1.75 V / cm RMS. In some embodiments, the electric field strength is at least 1 V / cm RMS. In some embodiments, the electric field strength may be 0.9 V / cm RMS. In other embodiments, combinations of electric field strengths are applied, for example, by combining two or more frequencies simultaneously or by applying two or more frequencies at different times.

[0045] In some cases, an electric field in at least a portion of the target site / subject / cancer cells is induced by an applied voltage of at least 50 V RMS, and optionally, the applied voltage is at least 100 V RMS. In some embodiments, an applied voltage of at least 50 V RMS induces an electric field having an electric field intensity of at least 1 V / cm (e.g., at least 5 V / cm) in at least a portion of the target site / subject / cancer cells.

[0046] In some embodiments, the alternating current field can be applied at various intervals ranging from 0.5 hours to 72 hours. In some embodiments, different periods can be used (e.g., 0.5 hours to 14 days). In some embodiments, the application of the alternating current field can be repeated periodically. For example, the alternating current field can be applied at 2-hour intervals daily.

[0047] In some embodiments, exposure may last for at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours. In some embodiments, exposure may be continuous or cumulative. In some embodiments, continuous exposure may last for at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours. In some embodiments, cumulative exposure may last for at least 42 hours, at least 84 hours, at least 168 hours, at least 250 hours, at least 400 hours, at least 500 hours, or at least 750 hours. In some embodiments, interruptions in treatment are possible, and the alternating electric field is applied for at least 50%, 60%, 70%, or 80% of the treatment time. For example, in some embodiments, cumulative exposure can be at least 12 hours out of 24 hours.

[0048] The disclosed method involves applying one or more alternating electric fields to a cell or a subject. In some embodiments, the alternating electric fields are applied to a target site or a tumor target site. When the alternating electric field is applied to a cell, this may mean applying the alternating electric field to the subject that constitutes the cell. Thus, when the alternating electric field is applied to a target site of the subject, the alternating electric field is applied to the cell. C. DNA-dependent protein kinase inhibitors

[0049] The methods and kits disclosed herein involve administering one or more DNA-dependent protein kinase inhibitors. In some embodiments, the DNA-dependent protein kinase inhibitors may be, but are not limited to, Nedisertib, VX-984, CC-115, CC-122, AZD7648, matinib, vemurafenib, gefitinib, Peposertib (M3814), MSC2490484A, LY294002, JU-57788, CC-115, BAY-8400, SF2523, LTURM34, Compound 401, AMA-37, IC 86621, DNA-PK-IN-1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9, NU7026, NU 7441, or combinations thereof. D. Composition

[0050] In some embodiments, in any of the disclosed methods, the DNA-dependent protein kinase inhibitor may be administered together with a pharmaceutically acceptable carrier and / or diluent.

[0051] Accordingly, compositions and formulations comprising a DNA-dependent protein kinase inhibitor and a pharmaceutically acceptable carrier or diluent are disclosed. Suitable DNA-dependent protein kinase inhibitors include, but are not limited to, any of the DNA-dependent protein kinase inhibitors described herein. For example, a pharmaceutical composition comprising VX984 and a pharmaceutically acceptable carrier or diluent is disclosed.

[0052] For example, the compositions described herein may include pharmaceutically acceptable carriers. “pharmaceutically acceptable” means a material or carrier selected to minimize degradation of the active ingredient and minimize adverse side effects to subjects, as is well known to those skilled in the art. Examples of carriers include dimyristoyl phosphatidylcholine (DMPC), phosphate-buffered saline, or polyvesicular liposomes. For example, PG:PC:cholesterol:peptide or PC:peptide can be used as a carrier in the present invention. Other suitable pharmaceutically acceptable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. ARGennaro, Mack Publishing Company, Easton, PA 1995, and teachings regarding pharmaceutically acceptable carriers are incorporated herein by reference. Typically, an appropriate amount of pharmaceutically acceptable salt is used in the formulation to make it isotonic. Other examples of pharmaceutically acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution may be approximately 5 to 8, or approximately 7 to 7.5. Further carriers include sustained-release formulations such as a semipermeable matrix of a solid hydrophobic polymer containing the composition, which may be in the form of molded articles such as films, stents (implanted in blood vessels during angioplasty), liposomes, or microparticles. It will be apparent to those skilled in the art that certain carriers may be more preferred depending on the route of administration and the concentration of the composition being administered. These are standard carriers for drug administration to humans, typically including solutions such as sterile water, saline solution, or a buffer at physiological pH.

[0053] The pharmaceutical composition may also include carriers, thickeners, diluents, buffers, preservatives, etc., as long as the intended activity of the polypeptide, peptide, nucleic acid, or vector of the present invention is not impaired. In addition to the composition of the present invention, the pharmaceutical composition may also include one or more active ingredients such as antibacterial agents, anti-inflammatory agents, or anesthetics. In the methods described herein, delivery of the disclosed composition to cells may occur through various mechanisms. The pharmaceutical composition may be administered in various ways depending on whether topical or systemic treatment is preferred and on the site of treatment. 1. Delivery of the composition

[0054] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions, or suspensions (including physiological saline and buffer media). Carriers for parenteral administration include sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, lactated ringer's solution, and fixative oils. Bases for intravenous administration include water and nutritional supplements, electrolyte supplements (e.g., those based on ringer's dextrose), etc. Preservatives such as antibacterial agents, antioxidants, chelating agents, and inert gases, as well as other additives, may also be present.

[0055] Preparations for optical administration may include ointments, lotions, creams, gels, eye drops, suppositories, sprays, liquids, powders, etc. Conventional drug carriers, aqueous, powder, or oily bases, thickeners, etc. may be required or desirable.

[0056] Compositions for oral administration include powders or granules, suspensions or solutions in water or a non-aqueous medium, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersants, or binders may be desirable. Part of the composition may be administered as pharmaceutically acceptable acids or base addition salts formed by the reaction of inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid with organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by the reaction of inorganic bases such as sodium hydroxide, ammonium hydroxide, and potassium hydroxide with organic bases such as monoalkylamines, dialkylamines, trialkylamines, arylamines, and substituted ethanolamines. E. Method

[0057] Disclosed are methods for administering a DNA-dependent PK inhibitor to a subject by applying an alternating electric field, or by bringing cells into contact with a DNA-dependent PK inhibitor. In some embodiments, the alternating electric field can inhibit the homologous recombination pathway, while the DNA-dependent PK inhibitor can inhibit the non-homologous end-join repair pathway, resulting in increased cell death as DNA damage is not repaired. 1.Treatment method

[0058] This specification discloses the use of alternating electric fields and DNA-dependent PK inhibitors for cancer treatment.

[0059] A method for treating a subject with cancer is disclosed, which includes applying an alternating electric field to a target site of the subject containing one or more cancer cells for a certain period of time, and administering to the subject a therapeutically effective amount of a DNA-dependent PK inhibitor.

[0060] In some aspects, cancer may be, but is not limited to, ovarian cancer, hepatobiliary cancer, prostate cancer, pancreatic cancer, head and neck cancer, glioblastoma, gliosarcoma, leukemia, or non-small cell lung cancer. Thus, in some aspects, cancer cells originate from one or more of these cancers. In some aspects, the subject has ovarian cancer, hepatobiliary cancer, prostate cancer, pancreatic cancer, head and neck cancer, glioblastoma, gliosarcoma, leukemia, or non-small cell lung cancer.

[0061] In some embodiments, the application of the alternating electric field is performed on day 1, 2, 3, 4, 5, 6, or 7 prior to the administration of the DNA-dependent PK inhibitor. In some embodiments, the application of the alternating electric field is performed 1, 2, 3, 4, 5, 6, or 7 days after the administration of the DNA-dependent PK inhibitor. In some embodiments, the application of the alternating electric field is performed 1, 2, 3, or 4 weeks prior to the administration of the DNA-dependent PK inhibitor. In some embodiments, the application of the alternating electric field is performed 1, 2, 3, or 4 weeks after the administration of the DNA-dependent PK inhibitor. In some embodiments, the alternating electric field and the DNA-dependent PK inhibitor are administered in combination. In some embodiments, combination means within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours of each other. In some embodiments, subjects can be examined to confirm the presence of DNA-dependent PK inhibitors in the bloodstream before an alternating electric field is applied.

[0062] In some embodiments, subjects with cancer have received or are currently receiving radiotherapy. In some embodiments, the disclosed method includes the step of administering radiotherapy. In some embodiments, radiotherapy may be administered in combination with an alternating electric field. In some embodiments, radiotherapy may be administered in combination with a DNA-dependent PK inhibitor. In some embodiments, radiotherapy and an alternating electric field may be administered in combination with a DNA-dependent PK inhibitor. In some embodiments, combination means within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours of each other.

[0063] In some embodiments, radiotherapy may be administered 1, 2, 3, 4, 5, 6, or 7 days before or after the application of the alternating electric field. In some embodiments, radiotherapy may be administered 1, 2, 3, or 4 weeks before or after the application of the alternating electric field. In some embodiments, the alternating electric field may be applied several days, several weeks before, several days after, or several weeks after the application of radiotherapy and a DNA-dependent PK inhibitor. In some embodiments, the DNA-dependent PK inhibitor may be administered several days, several weeks before, several days after, or several weeks after the application of radiotherapy and the alternating electric field. In some embodiments, radiotherapy may be administered several days, several weeks before, several days after, or several weeks after the application of the DNA-dependent PK inhibitor and the alternating electric field. In some embodiments, radiotherapy, the DNA-dependent PK inhibitor, and the alternating electric field are applied sequentially (without regard to order) with an interval of at least one day. In some embodiments, radiotherapy is performed before the application of the alternating electric field. In some embodiments, radiotherapy is performed after the application of the alternating electric field.

[0064] In some embodiments, the disclosed method further includes the step of administering chemotherapy to a subject. In some embodiments, chemotherapy may be administered before, after, or concurrently with the application of an alternating electric field. In some embodiments, chemotherapy may be performed before, after, or concurrently with the administration of a DNA-dependent PK inhibitor.

[0065] In some embodiments, a therapeutically effective dose of a DNA-dependent PK inhibitor refers to a dose sufficient or effective to prevent or reduce (delay or prevent, inhibit, reduce or reverse) the effects of DNA-dependent PK, including assisting DNA repair. For example, the DNA-dependent PK inhibitor CC-115 can be administered at 5-10 mg twice daily. In some embodiments, a therapeutically effective dose of VX-984 is 50-720 mg per day in a 28-day cycle. In some embodiments, a therapeutically effective dose of VX-984 is 120, 240, 480, or 720 mg per day. In some embodiments, VX-984 can be administered from day 2 to day 4 for up to six 28-day cycles. In some embodiments, VX-984 can be administered at concentrations of 250 nM, 125 nM, or 63 nM.

[0066] In some embodiments, one or more cancer cells at a target site have one or more DNA strand breaks, e.g., one or more single-strand or double-strand DNA breaks. In some embodiments, at least some of the DNA strand breaks may be caused by an alternating electric field. In some embodiments, at least some of the DNA strand breaks may be caused by radiotherapy. In some embodiments, at least some of the DNA strand breaks may be caused by chemotherapy, including but not limited to DNA alkylating agents and DNA crosslinking agents. Thus, in some embodiments, subjects with cancer have received or are currently receiving chemotherapy. In some embodiments, at least some of the DNA strand breaks may be caused by other compounds known to induce DNA damage directly or indirectly. Thus, in some embodiments, subjects with cancer have received or are currently receiving cancer treatment that causes DNA strand breaks.

[0067] In some embodiments, one or more cancer cells at a target site have double-strand DNA breaks. In some embodiments, at least some double-strand DNA breaks may be caused by an alternating electric field. In some embodiments, at least some double-strand DNA breaks may be caused by radiotherapy. In some embodiments, at least some double-strand DNA breaks may be caused by chemotherapy, including but not limited to DNA alkylating agents and DNA crosslinking agents. Therefore, in some embodiments, subjects with cancer have received or are currently receiving chemotherapy. In some embodiments, at least some double-strand DNA breaks may be caused by other compounds known to induce DNA damage directly or indirectly. Therefore, in some embodiments, subjects with cancer have received or are currently receiving cancer treatment that causes double-strand DNA breaks.

[0068] In some embodiments, at least one DNA repair mechanism within one or more cancer cells at a target site is inhibited. In some embodiments, the inhibited DNA repair mechanism is homologous recombination repair, non-homologous end joining repair, or both. For example, an alternating electric field can inhibit the homologous recombination repair pathway, and DNA-dependent PK inhibitors can inhibit non-homologous end joining (recombination) repair, thereby making it possible to inhibit both DNA repair pathways.

[0069] In some embodiments, one or more cancer cells at the target site undergo cell death. In some embodiments, inhibition of DNA repair by an alternating electric field and a DNA-dependent PK inhibitor induces cell death.

[0070] In some embodiments, the DNA-dependent PK inhibitor may be Nedisertib, VX-984, CC-115, CC-122, AZD7648, matinib, vemurafenib, gefitinib, Peposertib (M3814), MSC2490484A, LY294002, JU-57788, CC-115, BAY-8400, SF2523, LTURM34, Compound 401, AMA-37, IC 86621, DNA-PK-IN-1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9, NU7026, NU 7441, or a combination thereof.

[0071] In some embodiments, these methods may further include administering a therapeutically effective amount of an ATR inhibitor, a PARP inhibitor, or a combination thereof to a subject. In some embodiments, the ATR inhibitor may be, but is not limited to, Schisandrin B, Nu6027, Dactolisib, EPT-46464, VE-821, AZ20, Berzosertib, Torin-2, Ceralasertib (AZD6738), Tetrahydropyrazolo[1,5-a]pyrazine, Azabenzimidazoles, Gartisertib (M4344 or VX-803), Bayl895344 (Elimsuretib), CGK 733, Camonsertib, ATR-IN-4, VE-821, AZ20, ETP-46464, or ATR inhibitor 1. In some embodiments, the PARP inhibitor may be, but is not limited to, olaparib, niraparib, talazoparib, rucaparib, or AZD9574.

[0072] In some embodiments, therapeutically effective amounts of DNA-dependent PK inhibitors, ATR inhibitors, or PARP inhibitors can be administered orally, subcutaneously, or intravenously. In some embodiments, DNA-dependent PK inhibitors, ATR inhibitors, or PARP inhibitors can be delivered as compositions by any of the delivery mechanisms described herein.

[0073] In some embodiments, the alternating current field has frequency and electric field strength. In some embodiments, the frequency of the alternating current field is 50 kHz to 1 MHz. In some embodiments, the frequency of the alternating current field is 100 kHz to 1 MHz. In some embodiments, the frequency of the alternating current field is 100 to 500 kHz. In some embodiments, the frequency of the alternating current field is 200 kHz. In some embodiments, the alternating current field can be any of the ranges described herein.

[0074] In some embodiments, the alternating electric field has an electric field strength of 0.1 to 10 V / cm RMS. In some embodiments, the alternating electric field has an electric field strength of 0.5 to 4 V / cm RMS. In some embodiments, the alternating electric field has an electric field strength of 1 V / cm RMS. In some embodiments, the alternating electric field has any of the electric field strengths described herein.

[0075] In some cases, an electric field in at least a portion of the target site / subject / cancer cells is induced by an applied voltage of at least 50 V RMS, and optionally, the applied voltage is at least 100 V RMS. In some embodiments, an applied voltage of at least 50 V RMS induces an electric field having an electric field intensity of at least 1 V / cm (e.g., at least 5 V / cm) in at least a portion of the target site / subject / cancer cells. 2.Cell death induction method

[0076] This specification discloses the use of alternating electric fields and DNA-dependent PK inhibitors to induce cell death.

[0077] A method for inducing cell death in cancer cells is disclosed, comprising exposing cancer cells to an alternating electric field for a certain period of time and exposing cancer cells to a DNA-dependent PK inhibitor. In some embodiments, these methods can be carried out in vitro. In some embodiments, these methods can be carried out in vivo.

[0078] In some embodiments, cancer cells may be simultaneously exposed to radiation or may have been exposed to radiation in the past.

[0079] In some embodiments, cancer cells are present within the subject. Therefore, in some embodiments, these methods are performed in vivo. In some embodiments, cancer cells may be in a cultured state. Therefore, in some embodiments, these methods are performed in vitro.

[0080] In some embodiments, exposing cancer cells to an alternating electric field involves applying the alternating electric field to a target site of a subject containing one or more cancer cells. In some embodiments, the cancer may be, but is not limited to, ovarian cancer, hepatobiliary cancer, prostate cancer, pancreatic cancer, head and neck cancer, glioblastoma, gliosarcoma, leukemia, or non-small cell lung cancer. Thus, in some embodiments, the cancer cells may originate from one or more of these cancers.

[0081] In some embodiments, exposing cancer cells to an alternating electric field is equivalent to applying an alternating electric field to cancer cells. In some embodiments, the application of the alternating electric field is performed on day 1, 2, 3, 4, 5, 6, or 7 before administration of a DNA-dependent PK inhibitor. In some embodiments, the application of the alternating electric field is performed 1, 2, 3, 4, 5, 6, or 7 days after administration of a DNA-dependent PK inhibitor. In some embodiments, the application of the alternating electric field is performed 1, 2, 3, or 4 weeks before administration of a DNA-dependent PK inhibitor. In some embodiments, the application of the alternating electric field is performed 1, 2, 3, or 4 weeks after administration of a DNA-dependent PK inhibitor. In some embodiments, the alternating electric field and the DNA-dependent PK inhibitor are administered in combination. In some embodiments, concomitant use means that the time between them is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some embodiments, subjects may be examined to confirm the presence of DNA-dependent PK inhibitors in the bloodstream before the application of an alternating electric field.

[0082] In some embodiments, cancer cells are simultaneously exposed to radiation or have been exposed to radiation in the past. In some embodiments, the radiation-exposed cancer cells are the same as those of a subject receiving radiation therapy. In some embodiments, radiation exposure can be carried out in combination with exposure to an alternating electric field. In some embodiments, radiation and exposure to an alternating electric field can be carried out in combination with a DNA-dependent PK inhibitor. In some embodiments, combination means that each exposure is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours of the other. In some embodiments, radiation exposure can be carried out 1, 2, 3, 4, 5, 6, or 7 days before or after the application of an alternating electric field. In some embodiments, radiation exposure can be carried out 1, 2, 3, or 4 weeks before or after the application of an alternating electric field. In some embodiments, radiation exposure and administration of DNA-dependent PK inhibitors can be carried out in combination, with the alternating electric field being applied several days, several weeks, several days, or several weeks prior. In some embodiments, radiation, DNA-dependent PK inhibitors, and the alternating electric field are applied sequentially (without regard to order), with an interval of at least one day. In some embodiments, radiation exposure is carried out after the application of the alternating electric field.

[0083] In some embodiments, exposure of cancer cells to a DNA-dependent PK inhibitor means exposure of cancer cells to a therapeutically effective amount of the DNA-dependent PK inhibitor. In some embodiments, a therapeutically effective amount of a DNA-dependent PK inhibitor means an amount sufficient or effective to prevent or reduce (delay or prevent, inhibit, reduce or reverse) the effects of DNA-dependent PK, including assisting DNA repair. For example, the DNA PK inhibitor CC-115 can be administered at 5-10 mg twice daily. In some embodiments, a therapeutically effective amount of VX-984 is 50-720 mg per day in a 28-day cycle. In some embodiments, a therapeutically effective amount of VX-984 is 120, 240, 480, or 720 mg per day. In some embodiments, VX-984 can be at concentrations of 250 nM, 125 nM, or 63 nM.

[0084] In some embodiments, one or more cancer cells at a target site have one or more DNA strand breaks, e.g., one or more single-strand or double-strand DNA breaks. In some embodiments, at least some of the DNA strand breaks may be caused by an alternating electric field. In some embodiments, at least some of the DNA strand breaks may be caused by radiotherapy. In some embodiments, at least some of the DNA strand breaks may be caused by chemotherapy, including but not limited to DNA alkylating agents and DNA crosslinking agents. Therefore, in some embodiments, subjects with cancer have received or are currently receiving chemotherapy. In some embodiments, at least some of the DNA strand breaks may be caused by other cancer treatments, such as poly(ADP-ribose) polymerase (PARP) inhibitors, but are not limited to these. Therefore, in some embodiments, subjects with cancer have received or are currently receiving cancer treatments that cause DNA strand breaks.

[0085] In some embodiments, one or more cancer cells at a target site have double-strand DNA breaks. In some embodiments, at least some double-strand DNA breaks may be caused by an alternating electric field. In some embodiments, at least some double-strand DNA breaks may be caused by radiotherapy. In some embodiments, at least some double-strand DNA breaks may be caused by chemotherapy, including but not limited to DNA alkylating agents and DNA crosslinking agents. Therefore, in some embodiments, subjects with cancer have received or are currently receiving chemotherapy. In some embodiments, at least some double-strand DNA breaks may be caused by other cancer treatments, such as poly(ADP-ribose) polymerase (PARP) inhibitors. Therefore, in some embodiments, subjects with cancer have received or are currently receiving cancer treatments that cause double-strand DNA breaks.

[0086] In some embodiments, at least one DNA repair mechanism within one or more cancer cells at a target site is inhibited. In some embodiments, the inhibited DNA repair mechanism is homologous recombination repair, non-homologous end joining repair, or both. For example, an alternating electric field can inhibit the homologous recombination repair pathway, and DNA-dependent PK inhibitors can inhibit non-homologous end joining (recombination) repair, thereby making it possible to inhibit both DNA repair pathways.

[0087] In some embodiments, one or more cancer cells undergo cell death. In some embodiments, inhibition of DNA repair by an alternating electric field and a DNA-dependent PK inhibitor enables cell death.

[0088] In some embodiments, the DNA-dependent PK inhibitor may be Nedisertib, VX-984, CC-115, CC-122, AZD7648, matinib, vemurafenib, gefitinib, Peposertib (M3814), MSC2490484A, LY294002, JU-57788, CC-115, BAY-8400, SF2523, LTURM34, Compound 401, AMA-37, IC 86621, DNA-PK-IN-1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9, NU7026, NU 7441, or a combination thereof.

[0089] In some embodiments, these methods may further involve exposing cancer cells to a therapeutically effective amount of an ATR inhibitor, a PARP inhibitor, or a combination thereof. In some embodiments, the ATR inhibitor may be, but is not limited to, Schisandrin B, Nu6027, Dactolisib, EPT-46464, VE-821, AZ20, Berzosertib, Torin-2, Ceralasertib (AZD6738), Tetrahydropyrazolo[1,5-a]pyrazine, Azabenzimidazoles, Gartisertib (M4344 or VX-803), Bayl895344 (Elimsuretib), CGK 733, Camonsertib, ATR-IN-4, VE-821, AZ20, ETP-46464, or ATR inhibitor 1. In some embodiments, the PARP inhibitor may be, but is not limited to, olaparib, niraparib, talazoparib, rucaparib, or AZD9574.

[0090] In some embodiments, if cancer cells are present in the subject, a therapeutically effective amount of a DNA-dependent PK inhibitor, ATR inhibitor, or PARP inhibitor can be administered orally, subcutaneously, or intravenously. In some embodiments, the DNA-dependent PK inhibitor, ATR inhibitor, or PARP inhibitor can be delivered as a composition by any of the delivery mechanisms described herein.

[0091] In some embodiments, the alternating current field has frequency and electric field strength. In some embodiments, the frequency of the alternating current field is 50 kHz to 1 MHz. In some embodiments, the frequency of the alternating current field is 100 kHz to 1 MHz. In some embodiments, the frequency of the alternating current field is 100 to 500 kHz. In some embodiments, the frequency of the alternating current field is 200 kHz. In some embodiments, the alternating current field can be any of the ranges described herein.

[0092] In some embodiments, the alternating electric field has an electric field strength of 0.1 to 10 V / cm RMS. In some embodiments, the alternating electric field has an electric field strength of 0.5 to 4 V / cm RMS. In some embodiments, the alternating electric field has an electric field strength of 1 V / cm RMS. In some embodiments, the alternating electric field has any of the electric field strengths described herein.

[0093] In some cases, an electric field in at least a portion of the target site / subject / cancer cells is induced by an applied voltage of at least 50 V RMS, and optionally, the applied voltage is at least 100 V RMS. In some embodiments, an applied voltage of at least 50 V RMS induces an electric field having an electric field intensity of at least 1 V / cm (e.g., at least 5 V / cm) in at least a portion of the target site / subject / cancer cells. 3. Methods for inhibiting DNA repair

[0094] This specification discloses the use of alternating electric fields and DNA-dependent PK inhibitors to inhibit DNA repair in cancer cells having one or more DNA strand breaks, particularly double-strand DNA breaks.

[0095] A method for inhibiting DNA repair in cancer cells that have been exposed to or have been exposed to radiation is disclosed, comprising exposing cancer cells to an alternating electric field for a certain period of time and exposing cancer cells to a DNA-dependent PK inhibitor. In some embodiments, these methods can be carried out in vitro. In some embodiments, these methods can be carried out in vivo.

[0096] In some embodiments, methods are disclosed for inhibiting DNA repair in cancer cells having at least one DNA strand break, these methods comprising exposing the cancer cells to an alternating electric field for a certain period of time and exposing the cancer cells to a DNA-dependent PK inhibitor. In some embodiments, the DNA strand break is a single-strand DNA break. In some embodiments, the DNA strand break is a double-strand DNA break.

[0097] In some embodiments, methods for inhibiting DNA repair in cancer cells having at least one double-strand DNA break are disclosed, these methods comprising exposing cancer cells to an alternating electric field for a certain period of time and exposing cancer cells to a DNA-dependent PK inhibitor.

[0098] In some embodiments, cancer cells may be simultaneously exposed to radiation or may have been exposed to radiation in the past.

[0099] In some embodiments, cancer cells are present within the subject. Therefore, in some embodiments, these methods are performed in vivo. In some embodiments, cancer cells may be in a cultured state. In some embodiments, these methods are performed in vitro.

[0100] In some embodiments, exposing cancer cells to an alternating electric field involves applying the alternating electric field to a target site of a subject containing one or more cancer cells. In some embodiments, the cancer may be, but is not limited to, ovarian cancer, hepatobiliary cancer, prostate cancer, pancreatic cancer, head and neck cancer, glioblastoma, gliosarcoma, leukemia, or non-small cell lung cancer. Thus, in some embodiments, the cancer cells may originate from one or more of these cancers.

[0101] In some embodiments, exposing cancer cells to an alternating electric field is equivalent to applying an alternating electric field to cancer cells. In some embodiments, the application of the alternating electric field is performed 1, 2, 3, 4, 5, 6, or 7 days before administering a DNA-dependent PK inhibitor. In some embodiments, the application of the alternating electric field is performed 1, 2, 3, 4, 5, 6, or 7 days after administering a DNA-dependent PK inhibitor. In some embodiments, the application of the alternating electric field is performed 1, 2, 3, or 4 weeks before administering a DNA-dependent PK inhibitor. In some embodiments, the application of the alternating electric field is performed 1, 2, 3, or 4 weeks after administering a DNA-dependent PK inhibitor. In some embodiments, the alternating electric field and the DNA-dependent PK inhibitor are administered in combination. In some embodiments, concomitant use means that the time between them is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some embodiments, subjects may be examined to confirm the presence of DNA-dependent PK inhibitors in the bloodstream before the application of an alternating electric field.

[0102] In some embodiments, cancer cells are simultaneously exposed to radiation or have been exposed to radiation in the past. In some embodiments, the radiation-exposed cancer cells are the same as those of a subject receiving radiation therapy. In some embodiments, radiation exposure can be carried out in combination with exposure to an alternating electric field. In some embodiments, radiation and exposure to an alternating electric field can be carried out in combination with a DNA-dependent PK inhibitor. In some embodiments, combination means that each exposure is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours of the other. In some embodiments, radiation exposure can be carried out 1, 2, 3, 4, 5, 6, or 7 days before or after the application of an alternating electric field. In some embodiments, radiation exposure can be carried out 1, 2, 3, or 4 weeks before or after the application of an alternating electric field. In some embodiments, radiation exposure and administration of DNA-dependent PK inhibitors can be carried out in combination, and an alternating electric field can be applied several days, several weeks, several days, or several weeks prior to or after the exposure. In some embodiments, radiation, DNA-dependent PK inhibitors, and alternating electric fields are applied sequentially (without regard to order), with an interval of at least one day between applications. In some embodiments, radiation exposure is performed after the application of the alternating electric field.

[0103] In some embodiments, exposing cancer cells to a DNA-dependent PK inhibitor means exposing cancer cells to a therapeutically effective amount of the DNA-dependent PK inhibitor. In some embodiments, a therapeutically effective amount of a DNA-dependent PK inhibitor means an amount sufficient or effective to prevent or reduce (delay or prevent, inhibit, reduce or reverse) the effects of DNA-dependent PK, including assisting DNA repair. For example, the DNA PK inhibitor CC-115 can be administered at 5-10 mg twice daily. In some embodiments, a therapeutically effective amount of VX-984 is 50-720 mg per day in a 28-day cycle. In some embodiments, a therapeutically effective amount of VX-984 is 120, 240, 480, or 720 mg per day. In some embodiments, VX-984 can be at concentrations of 250 nM, 125 nM, or 63 nM.

[0104] In some embodiments, one or more cancer cells at a target site have one or more DNA strand breaks, such as single-strand or double-strand DNA breaks. In some embodiments, at least some of the DNA strand breaks may be caused by an alternating electric field. In some embodiments, at least some of the DNA strand breaks may be caused by radiotherapy. In some embodiments, at least some of the DNA strand breaks may be caused by chemotherapy, including but not limited to DNA alkylating agents and DNA crosslinking agents. Therefore, in some embodiments, subjects with cancer have received or are currently receiving chemotherapy. In some embodiments, at least some of the DNA strand breaks may be caused by other cancer treatments, such as poly(ADP-ribose) polymerase (PARP) inhibitors, but are not limited to these. Therefore, in some embodiments, subjects with cancer have received or are currently receiving cancer treatments that cause DNA strand breaks.

[0105] In some embodiments, one or more cancer cells at a target site have double-strand DNA breaks. In some embodiments, at least some double-strand DNA breaks may be caused by an alternating electric field. In some embodiments, at least some double-strand DNA breaks may be caused by radiotherapy. In some embodiments, at least some double-strand DNA breaks may be caused by chemotherapy, including but not limited to DNA alkylating agents and DNA crosslinking agents. Therefore, in some embodiments, subjects with cancer have received or are currently receiving chemotherapy. In some embodiments, at least some double-strand DNA breaks may be caused by other cancer treatments, such as poly(ADP-ribose) polymerase (PARP) inhibitors. Therefore, in some embodiments, subjects with cancer have received or are currently receiving cancer treatments that cause double-strand DNA breaks.

[0106] In some embodiments, at least one DNA repair mechanism within one or more cancer cells at a target site is inhibited. In some embodiments, the inhibited DNA repair mechanism is homologous recombination repair, non-homologous end joining repair, or both. For example, an alternating electric field can inhibit the homologous recombination repair pathway, and DNA-dependent PK inhibitors can inhibit non-homologous end joining (recombination) repair, thereby making it possible to inhibit both DNA repair pathways.

[0107] In some embodiments, one or more cancer cells undergo cell death. In some embodiments, inhibition of DNA repair by an alternating electric field and a DNA-dependent PK inhibitor enables cell death.

[0108] In some embodiments, the DNA-dependent PK inhibitor may be Nedisertib, VX-984, CC-115, CC-122, AZD7648, matinib, vemurafenib, gefitinib, Peposertib (M3814), MSC2490484A, LY294002, JU-57788, CC-115, BAY-8400, SF2523, LTURM34, Compound 401, AMA-37, IC 86621, DNA-PK-IN-1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9, NU7026, NU 7441, or a combination thereof.

[0109] In some embodiments, these methods may further involve exposing cancer cells to a therapeutically effective amount of an ATR inhibitor, a PARP inhibitor, or a combination thereof. In some embodiments, the ATR inhibitor may be, but is not limited to, Schisandrin B, Nu6027, Dactolisib, EPT-46464, VE-821, AZ20, Berzosertib, Torin-2, Ceralasertib (AZD6738), Tetrahydropyrazolo[1,5-a]pyrazine, Azabenzimidazoles, Gartisertib (M4344 or VX-803), Bayl895344 (Elimsuretib), CGK 733, Camonsertib, ATR-IN-4, VE-821, AZ20, ETP-46464, or ATR inhibitor 1. In some embodiments, the PARP inhibitor may be, but is not limited to, olaparib, niraparib, talazoparib, rucaparib, or AZD9574.

[0110] In some embodiments, if cancer cells are present in the subject, a therapeutically effective amount of a DNA-dependent PK inhibitor, ATR inhibitor, or PARP inhibitor can be administered orally, subcutaneously, or intravenously. In some embodiments, the DNA-dependent PK inhibitor, ATR inhibitor, or PARP inhibitor can be delivered as a composition by any of the delivery mechanisms described herein.

[0111] In some embodiments, the alternating current field has frequency and electric field strength. In some embodiments, the frequency of the alternating current field is 50 kHz to 1 MHz. In some embodiments, the frequency of the alternating current field is 100 kHz to 1 MHz. In some embodiments, the frequency of the alternating current field is 100 to 500 kHz. In some embodiments, the frequency of the alternating current field is 200 kHz. In some embodiments, the alternating current field can be any of the ranges described herein.

[0112] In some embodiments, the alternating electric field has an electric field strength of 0.1 to 10 V / cm RMS. In some embodiments, the alternating electric field has an electric field strength of 0.5 to 4 V / cm RMS. In some embodiments, the alternating electric field has an electric field strength of 1 V / cm RMS. In some embodiments, the alternating electric field has any of the electric field strengths described herein.

[0113] In some cases, an electric field in at least a portion of the target site / subject / cancer cells is induced by an applied voltage of at least 50 V RMS, and optionally, the applied voltage is at least 100 V RMS. In some embodiments, an applied voltage of at least 50 V RMS induces an electric field having an electric field intensity of at least 1 V / cm (e.g., at least 5 V / cm) in at least a portion of the target site / subject / cancer cells. 4. Methods to improve the effectiveness of radiotherapy

[0114] This specification discloses the use of alternating electric fields and DNA-dependent PK inhibitors to enhance the effectiveness of radiotherapy.

[0115] A method for enhancing the effectiveness of radiotherapy in a subject is disclosed, which includes applying an alternating electric field for a certain period of time to a target site of the subject that is receiving or has received radiotherapy, and administering a therapeutically effective amount of a DNA-dependent PK inhibitor to the subject.

[0116] In some aspects, cancer may be, but is not limited to, ovarian cancer, hepatobiliary cancer, prostate cancer, pancreatic cancer, head and neck cancer, glioblastoma, gliosarcoma, leukemia, or non-small cell lung cancer. Thus, in some aspects, cancer cells originate from one or more of these cancers. In some aspects, the subject has ovarian cancer, hepatobiliary cancer, prostate cancer, pancreatic cancer, head and neck cancer, glioblastoma, gliosarcoma, leukemia, or non-small cell lung cancer.

[0117] In some embodiments, the application of the alternating electric field is performed on day 1, 2, 3, 4, 5, 6, or 7 prior to the administration of the DNA-dependent PK inhibitor. In some embodiments, the application of the alternating electric field is performed 1, 2, 3, 4, 5, 6, or 7 days after the administration of the DNA-dependent PK inhibitor. In some embodiments, the application of the alternating electric field is performed 1, 2, 3, or 4 weeks prior to the administration of the DNA-dependent PK inhibitor. In some embodiments, the application of the alternating electric field is performed 1, 2, 3, or 4 weeks after the administration of the DNA-dependent PK inhibitor. In some embodiments, the alternating electric field and the DNA-dependent PK inhibitor are administered in combination. In some embodiments, combination means within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours of each other. In some embodiments, subjects can be examined to confirm the presence of DNA-dependent PK inhibitors in the bloodstream before an alternating electric field is applied.

[0118] In some embodiments, subjects with cancer have received or are currently receiving radiotherapy. In some embodiments, radiotherapy can be administered in combination with an alternating electric field. In some embodiments, radiotherapy and alternating electric fields can be administered in combination with a DNA-dependent PK inhibitor. In some embodiments, combination means within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours of each other. In some embodiments, radiotherapy can be administered 1, 2, 3, 4, 5, 6, or 7 days before or after the application of the alternating electric field. In some embodiments, radiotherapy may be administered one, two, three, or four weeks before or after the application of an alternating electric field. In some embodiments, the alternating electric field may be administered several days, several weeks before, several days, or several weeks after radiotherapy and a DNA-dependent PK inhibitor in combination. In some embodiments, radiotherapy, the DNA-dependent PK inhibitor, and the alternating electric field are administered sequentially (without regard to order), with an interval of at least one day between them. In some embodiments, radiotherapy is administered after the application of the alternating electric field.

[0119] In some embodiments, a therapeutically effective dose of a DNA-dependent PK inhibitor refers to a dose sufficient or effective to prevent or reduce (delay or prevent, inhibit, reduce or reverse) the effects of DNA-dependent PK, including assisting DNA repair. For example, the DNA PK inhibitor CC-115 can be administered at 5-10 mg twice daily. In some embodiments, a therapeutically effective dose of VX-984 is 50-720 mg per day in a 28-day cycle. In some embodiments, a therapeutically effective dose of VX-984 is 120, 240, 480, or 720 mg per day. In some embodiments, VX-984 can be administered from day 2 to day 4 for up to six 28-day cycles. In some embodiments, VX-984 can be administered at concentrations of 250 nM, 125 nM, or 63 nM.

[0120] In some embodiments, one or more cancer cells at a target site have one or more DNA breaks, such as single-strand or double-strand DNA breaks. In some embodiments, at least some DNA breaks may be caused by an alternating electric field. In some embodiments, at least some DNA breaks may be caused by radiotherapy. In some embodiments, at least some DNA breaks may be caused by chemotherapy, including but not limited to DNA alkylating agents and DNA crosslinking agents. Therefore, in some embodiments, subjects with cancer have received or are currently receiving chemotherapy. In some embodiments, at least some DNA breaks may be caused by other cancer treatments, such as poly(ADP-ribose) polymerase (PARP) inhibitors, but are not limited to these. Therefore, in some embodiments, subjects with cancer have received or are currently receiving cancer treatments that cause DNA breaks.

[0121] In some embodiments, one or more cancer cells at a target site have double-strand DNA breaks. In some embodiments, at least some double-strand DNA breaks may be caused by an alternating electric field. In some embodiments, at least some double-strand DNA breaks may be caused by radiotherapy. In some embodiments, at least some double-strand DNA breaks may be caused by chemotherapy, including but not limited to DNA alkylating agents and DNA crosslinking agents. Therefore, in some embodiments, subjects with cancer have received or are currently receiving chemotherapy. In some embodiments, at least some double-strand DNA breaks may be caused by other cancer treatments, such as poly(ADP-ribose) polymerase (PARP) inhibitors. Therefore, in some embodiments, subjects with cancer have received or are currently receiving cancer treatments that cause double-strand DNA breaks.

[0122] In some embodiments, at least one DNA repair mechanism in one or more cancer cells at a target site is inhibited. In some embodiments, the inhibited DNA repair mechanism is homologous recombination repair, non-homologous end joining repair, or both. For example, an alternating electric field can inhibit the homologous recombination repair pathway, and a DNA-dependent PK inhibitor can inhibit non-homologous end joining (recombination) repair, thereby inhibiting both DNA repair pathways. In some embodiments, an alternating electric field can inhibit the homologous recombination repair pathway, and a DNA-dependent PK inhibitor can inhibit stress-induced DNA breaks.

[0123] In some embodiments, one or more cancer cells at the target site undergo cell death. In some embodiments, inhibition of DNA repair by an alternating electric field and a DNA-dependent PK inhibitor enables cell death.

[0124] In some embodiments, the DNA-dependent PK inhibitor may be Nedisertib, VX-984, CC-115, CC-122, AZD7648, matinib, vemurafenib, gefitinib, Peposertib (M3814), MSC2490484A, LY294002, JU-57788, CC-115, BAY-8400, SF2523, LTURM34, Compound 401, AMA-37, IC 86621, DNA-PK-IN-1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9, NU7026, NU 7441, or a combination thereof.

[0125] In some embodiments, these methods may further include administering a therapeutically effective amount of an ATR inhibitor, a PARP inhibitor, or a combination thereof to a subject. In some embodiments, the ATR inhibitor may be, but is not limited to, Schisandrin B, Nu6027, Dactolisib, EPT-46464, VE-821, AZ20, Berzosertib, Torin-2, Ceralasertib (AZD6738), Tetrahydropyrazolo[1,5-a]pyrazine, Azabenzimidazoles, Gartisertib (M4344 or VX-803), Bayl895344 (Elimsuretib), CGK 733, Camonsertib, ATR-IN-4, VE-821, AZ20, ETP-46464, or ATR inhibitor 1. In some embodiments, the PARP inhibitor may be, but is not limited to, olaparib, niraparib, talazoparib, rucaparib, or AZD9574.

[0126] In some embodiments, therapeutically effective amounts of DNA-dependent PK inhibitors, ATR inhibitors, or PARP inhibitors can be administered orally, subcutaneously, or intravenously. In some embodiments, DNA-dependent PK inhibitors, ATR inhibitors, or PARP inhibitors can be delivered as compositions by any of the delivery mechanisms described herein.

[0127] In some embodiments, the alternating current field has frequency and electric field strength. In some embodiments, the frequency of the alternating current field is 50 kHz to 1 MHz. In some embodiments, the frequency of the alternating current field is 100 kHz to 1 MHz. In some embodiments, the frequency of the alternating current field is 100 to 500 kHz. In some embodiments, the frequency of the alternating current field is 200 kHz. In some embodiments, the alternating current field can be any of the ranges described herein.

[0128] In some embodiments, the alternating electric field has an electric field strength of 0.1 to 10 V / cm RMS. In some embodiments, the alternating electric field has an electric field strength of 0.5 to 4 V / cm RMS. In some embodiments, the alternating electric field has an electric field strength of 1 V / cm RMS. In some embodiments, the alternating electric field has any of the electric field strengths described herein.

[0129] In some cases, an electric field in at least a portion of the target site / subject / cancer cells is induced by an applied voltage of at least 50 V RMS, and optionally, the applied voltage is at least 100 V RMS. In some embodiments, an applied voltage of at least 50 V RMS induces an electric field having an electric field intensity of at least 1 V / cm (e.g., at least 5 V / cm) in at least a portion of the target site / subject / cancer cells. F. Kit

[0130] The materials described above and other materials may be packaged together in any suitable combination as a kit useful for carrying out or assisting in the carrying out of the disclosed method. A kit is useful if the kit components within a given kit are designed and adapted to be used together in the disclosed method. For example, a kit is disclosed comprising one or more DNA-dependent PK inhibitors and one or more materials for delivering an alternating electric field. The materials may include a transducer array that generates an electric field within a target site. The materials may also include one or more electrodes configured to be attached (possibly via adhesive) to a subject or target site, and these electrodes are connected to a generator that produces a voltage signal that induces an alternating electric field for the disclosed method when in use. In one example, the material for delivering the alternating electric field is an Optune system. For example, a kit is disclosed comprising one or more of Nedisertib, VX-984, CC-115, CC-122, AZD7648, matinib, vemurafenib, and gefitinib and one or more materials for delivering an alternating electric field, such as an Optune system. Examples A. Example 1

[0131] DNA-PK regulates the major pathway (non-homologous end joining) responsible for repairing radiation-induced DNA double-strand breaks. Mutations or inhibition of DNA-PK result in a marked increase in radiosensitivity of cells, tumors, and tissues. This has been demonstrated by a 2-3-fold increase in radiosensitivity in cells and tissues of severe combined immunodeficiency mice with DNA-PK mutations [Biedermann et al. PNAS 88(4):1394-1397, Feb 1991].

[0132] DNA-PK inhibition inhibits non-homologous end joining (NHEJ) and increases DNA double-strand breaks (DSBs).

[0133] The TT field has already been reported to delay DNA damage repair and to show synergistic effects with PARP inhibitors and ATR / ATM inhibitors [Biedermann, 1991; Giladi et al. Radiat Oncol. 2017 Dec 29;12:206; Mumblat et al. Lung Cancer. 2021 Oct;160:99-110]. The TT field induces the formation of DNA double-strand breaks by inhibiting the DNA damage repair mechanism. The TT field inhibits DNA damage repair in MPM cells, particularly the FA-BRCA pathway.

[0134] Experiments will be conducted using TT field and the DNA-dependent protein kinase (PK) inhibitor VX-984 in combination. The doses will be 250 nM, 125 nM, and 63 nM, which correspond to 1x, 0.5x, and 0.25x of the doses validated by Western blotting, respectively. Western blotting data using patient-derived glioblastoma cells will also be obtained.

[0135] Experiments using CC-115 and Nedisertib were conducted, and cytotoxicity was evaluated by cell count measurement using flow cytometry, apoptosis assay, and colony formation assay.

[0136] TTFields demonstrate efficacy when used in combination with ATR / ATM / PARP inhibitors (Karanam et al. Transl Res. 2020 Mar;217:33-46;Karanam et al. Cell Death Dis. 2017 Mar 30;8(3):e2711;Karanam et al. International J. of Radiation Oncology Biology Physics 2454, volume 111, issue 3, supplement E230-E231). TTFields plus PARP inhibitor data show synergy (Martinez-Conde et al. International J Radiation Biology Physics, vol 114, issue 3, supplement, Nov. 2022, page e276; US. Patent Number 10, 953, 209).

[0137] DNA-PK phosphorylation (pS2056) was clearly increased 1 and 4 hours after radiotherapy (RT), and a similar increase was observed when the TT field was applied after RT. (No increase in DNA-PK phosphorylation was observed with the TT field alone) (Giladi et al. Radiat Oncol. 2017 Dec 29;12:206).

[0138] Nedisertib (M3814, pepocertib, MSC2490484A) is currently recruiting patients for a Phase I clinical trial targeting leukemia, solid tumors (ovarian cancer, hepatobiliary tract cancer, prostate cancer, pancreatic cancer, head and neck cancer), gliosal coma, and glioblastoma. VX-984 (M9831) is being tested for advanced solid tumors (Phase I, 2017 Trial 1). The small molecule mTOR and DNA-PK inhibitor CC-115 is currently undergoing a Phase I clinical trial involving 44 patients with advanced solid tumors or hematological malignancies, with CC-115 monotherapy being administered. All of these DNA-PK inhibitors can be used in combination with TT. B. Example 2

[0139] Cellular DNA is exposed to various types of DNA damage inducers. When DNA damage is recognized, a complex signaling cascade is activated, and the cell cycle is temporarily suspended. This gives the cell enough time to repair the damage. However, if the damage is extensive, it can ultimately lead to cell death. In fact, in clinical practice, some anticancer drugs are used to induce DNA damage within cells with the aim of causing cell death.

[0140] As a defense mechanism, cells possess various DNA damage response systems, each specific to the type of damage. Key proteins in the repair pathway are being targeted as promising anti-cancer therapies (Figure 7). This project focuses on the repair of double-strand breaks through two major systems: homologous recombination (HR) and non-homologous end joining (NHEJ). It is noteworthy that double-strand breaks are also formed during the repair of interstrand crosslinks by Fanconi anemia-related proteins and are subsequently repaired by these two systems.

[0141] The primary mechanism for repairing double-strand breaks is the non-homologous end joining (NHEJ) system. This system ligates broken ends of DNA without requiring a homologous template. For this reason, NHEJ can function throughout the cell cycle, but it is most important in the G1 phase. NHEJ repair is mediated via Ku70 / Ku80 heterodimers that recognize and bind double-strand breaks and recruit DNA-PK proteins in the early stages.

[0142] On the other hand, because HR functions under the induction of template DNA with complete homology, it is most active in the S / G2 phase and almost absent in the G1 phase. This highly regulated pathway is known as the error-free repair pathway, and the ATM, RAD51, and BRCA1 / 2 genes play major roles in it.

[0143] The TT site has been shown to downregulate intracellular FANC / BRCA protein levels, thereby inhibiting the HR-mediated repair process and leading to replication stress and DNA damage.

[0144] Previous studies have established that the TT field influences the cellular DNA repair capacity by altering homologous recombination repair pathways, and have shown that the TT field does not inhibit the phosphorylation and activation of DNA-PK in glioma cells. Furthermore, bioinformatics analysis (Figure 8) showed that in almost all cell lines examined, there was no significant increase or decrease in NHEJ pathway-related RNA expression levels after administration of the TT field, while other DNA damage repair pathways were significantly downregulated.

[0145] The theoretical basis of this project is to induce cell death by using a combination of a TT site that inhibits the HR pathway and a DNA-PK inhibitor that inhibits the NHEJ pathway.

[0146] The inhibitors used in these studies were Nedisertib and CC-115 (Figure 9). Nedisertib is a potent inhibitor of the catalytic unit of DNA-PK. Several Phase I / II clinical trials testing this inhibitor for leukemia and various solid tumors are currently recruiting patients.

[0147] CC-115 is a dual inhibitor of DNA-PK and mTOR. A Phase I clinical trial evaluating the safety and preliminary efficacy of CC-115 has been completed.

[0148] This inhibitor was tested because it has dual inhibitory activity against mTOR, which regulates proliferation and survival, and is associated with the AKT signaling pathway, which is known to be affected by the TT field.

[0149] The purpose of this study is to evaluate the cytotoxicity, apoptosis induction effect, and colony formation effect of combination therapy with TT field and Nedisertib or CC-115 in non-small cell lung cancer (A549 and H1299 cells). Furthermore, this study will also investigate the mechanism of action of the combination therapy.

[0150] In this project, A549 and H1299 cell lines were used and maintained in F12K medium supplemented with 10% FCS or RPMI medium, respectively (Figure 10). Gene mutations in the homologous recombination (HR) and non-homologous end repair (NHEJ) pathways in the cells are shown in the table. Both cell lines had mutations that inhibited the HR pathway, and in H1299 cells, amplified mutations were observed in PRKDC, the catalytic unit of DNA-PK.

[0151] This study began with Nedisertib titrations. In these experiments, 15,000 A549 or H1299 cells per dish were seeded into 12-well plates and incubated overnight to promote cell adhesion. These cells were then treated with various concentrations of Nedisertib for 72 hours. The number of viable cells was counted using a flow cytometer (Guava easyCyte HT) (Figure 11). The IC50 values ​​for both cell lines were similar to previously reported results, with H1299 having a higher IC50 value than A549. This is likely due to amplification mutations in the catalytic unit of DNA-PK.

[0152] The effects of combination therapy with Nedisertib and TT-en on cytotoxicity, apoptosis induction, and colony formation ability were investigated, and the overall effect was calculated (Figure 12).

[0153] In these experiments, 15,000 A549 cells per dish were seeded into in vitro culture dishes and incubated overnight to allow cell adhesion. The cells were then treated with a TT field (0.97 V / cm RMS, 150 kHz, 72 hours) alone or with different concentrations of Nedisertib. Efficacy was measured by cell count analysis using a flow cytometer (Guava easyCyte HT), colony formation, and apoptosis induction by Annexin V / 7AAD staining. The overall effect was calculated by multiplying cell count by colony formation. The results showed that the TT field synergistically enhanced the cytotoxicity and overall effect of Nedisertib.

[0154] The apoptotic effect of combination therapy was significant only with high doses of Nedisertib, suggesting that this treatment primarily affects cell proliferation.

[0155] For H1299 cells, an additive effect was observed with combination therapy of TT and Nedisertib (Figure 13). Similarly, in A549 cells, the apoptotic effect of combination therapy was significant only in the high-dose Nedisertib group.

[0156] In these experiments, 15,000 H1299 cells per dish were seeded into in vitro culture dishes and incubated overnight to allow cell adhesion. The cells were then treated with a TT field (0.97 V / cm RMS, 150 kHz, 72 hours) alone or with different concentrations of Nedisertib. Efficacy was measured by cell count analysis using a flow cytometer (Guava easyCyte HT), colony formation, and apoptosis induction by Annexin V / 7AAD staining. The overall effect was calculated by multiplying the cell count by colony formation.

[0157] The mechanisms of action of combination therapies were investigated, and cell cycle analysis was performed. In these experiments, 15,000 A549 cells per dish were seeded into in vitro culture dishes and incubated overnight to allow cell adhesion. Subsequently, the cells were treated for different durations in a TT field (0.97 V / cm RMS, 150 kHz) under conditions of single treatment or with the addition of 6 μM Nedisertib (Figure 14). For each single and combination treatment, the cell cycle distribution was analyzed using a Cytek® Northern Lights® flow cytometer.

[0158] Results from the A549 cell line showed that both the TT field and Nedisertib induced G1 phase arrest at most measurement points, resulting in a decrease in S phase cell counts, with the highest level of arrest observed among combination therapies. No significant changes were observed in the subG1 phase, given the low levels of apoptosis measured at this Nedisertib concentration.

[0159] Similar results were obtained with the H1299 cell line as with the A549 cell line, and combination therapy induced G1 phase arrest (Figure 15). In these experiments, 15,000 H1299 cells per dish were seeded into in vitro culture dishes and incubated overnight to allow cell adhesion. Subsequently, the cells were treated in a TT field (0.97 V / cm RMS, 150 kHz) for different durations. Treatment was performed either as a single treatment or as a combination treatment with 6.25 μM Nedisertib. For each single and combination treatment, the cell cycle distribution was analyzed using a Cytek® Northern Lights® flow cytometer.

[0160] In this experiment, titration of CC-115 was also performed (Figure 16). In these experiments, 15,000 A549 or H1299 cells per dish were seeded into 12-well plates and incubated overnight to promote cell adhesion. Subsequently, the cells were treated with various concentrations of CC-115 for 72 hours. The number of viable cells was counted using a flow cytometer (Guava easyCyte HT). The IC50 values ​​of CC-115 in both cell lines were significantly lower than those of Nedisertib, similar to previously reported results. Here again, the IC50 value of H1299 was higher than that of A549.

[0161] Similar to Nedisertib, the TT field synergistically enhanced the cytotoxicity and overall effect of CC-115 in the A549 cell line (Figure 17). Due to the dual inhibitory effect of CC-115, the synergistic effect in cytotoxicity was more pronounced compared to Nedisertib. In these experiments, 15,000 A549 cells per dish were seeded into in vitro culture dishes and incubated overnight to allow cell adhesion. Subsequently, cells were treated with the TT field (0.97 V / cm RMS, 150 kHz, 72 hours) alone or with the addition of different concentrations of CC-115. Efficacy was measured by cell number analysis using a flow cytometer (Guava easyCyte HT), colony formation, and apoptosis induction by Annexin V / 7AAD staining. The overall effect was calculated by multiplying the cell number by colony formation.

[0162] For H1299, a synergistic and additive overall effect on cytotoxicity was observed in combination therapy with TT field and CC-115 (Figure 18). In these experiments, 15,000 H1299 cells per dish were seeded into in vitro culture dishes and incubated overnight to allow cell adhesion. Subsequently, the cells were treated with TT field (0.97 V / cm RMS, 150 kHz, 72 hours) alone or with the addition of different concentrations of CC-115. Efficacy was measured by cell count analysis using a flow cytometer (Guava easyCyte HT), colony formation, and apoptosis induction by Annexin V / 7AAD staining. The overall effect was calculated by multiplying the cell count by colony formation.

[0163] Combination therapy with CC-115 and TT fields, which affect the cell cycle, has been investigated (Figure 19). In these experiments, 15,000 A549 cells per dish were seeded into in vitro culture dishes and incubated overnight to allow cell adhesion. Subsequently, the cells were treated in a TT field (0.97 V / cm RMS, 150 kHz) for different durations. Treatment was performed either alone or with the addition of 0.11 μM CC-115. For each monotherapy and combination therapy, the cell cycle distribution was analyzed using a Cytek® Northern Lights® flow cytometer. The results for the A549 cell line showed that combination therapy induced G1 phase arrest and reduced the number of S phase cells. Furthermore, no significant changes were observed in the subG1 phase, as indicated by the low apoptosis levels measured at this CC-115 concentration.

[0164] Similar results were obtained with the H1299 cell line, and combination therapy resulted in G1 phase arrest (Figure 20). In these experiments, 15,000 H1299 cells per dish were seeded into in vitro culture dishes and incubated overnight to allow cell adhesion. Subsequently, the cells were treated in a TT field (0.97 V / cm RMS, 150 kHz) for different durations. Treatment was performed either alone or with the addition of 0.3125 μM CC-115. For each monotherapy and combination therapy, the cell cycle distribution was analyzed using a Cytek® Northern Lights® flow cytometer.

[0165] The combination of TT-field and Nedisertib or CC-115 showed a synergistic interaction in A549 cells. In H1299 cells, combination therapy with TT-field and Nedisertib showed an additive interaction in cytotoxicity, while combination therapy with TT-field and CC-115 showed both a synergistic interaction and an additive overall effect. Exemplary Embodiments

[0166] One example of the many embodiments described herein is a method for treating a subject having cancer, the method comprising applying an alternating electric field for a certain period of time to a target site of the subject containing one or more cancer cells, and administering to the subject a therapeutically effective amount of a DNA-dependent PK inhibitor.

[0167] One example of the many embodiments described herein is a DNA-dependent protein kinase inhibitor used in a method for treating a subject with cancer, the method comprising applying an alternating electric field for a certain period of time to a target site in the subject containing one or more cancer cells, and administering a therapeutically effective amount of the inhibitor to the subject.

[0168] One example of the many embodiments described herein is an in vitro method for treating a target site, which comprises applying an alternating electric field to a target site in a subject containing one or more cancer cells for a set period of time, and administering a therapeutically effective amount of a DNA-dependent protein kinase (PK) inhibitor to the target site.

[0169] In many embodiments described herein, the cancer is ovarian cancer, hepatobiliary cancer, prostate cancer, pancreatic cancer, head and neck cancer, glioblastoma, gliosarcoma, leukemia, or non-small cell lung cancer.

[0170] In one example of the many embodiments described herein, a subject with cancer has received or is currently receiving radiotherapy or chemotherapy.

[0171] In one example of many embodiments described herein, one or more cancer cells at a target site have DNA breaks, such as single-strand DNA breaks or double-strand DNA breaks.

[0172] In one example of the many embodiments described herein, one or more cancer cells at the target site have double-strand DNA breaks.

[0173] In one example of the many embodiments described herein, at least one DNA repair mechanism within one or more cancer cells at a target site is inhibited.

[0174] In many embodiments described herein, the inhibited DNA repair mechanism is homologous recombination repair, non-homologous end joining repair, or both.

[0175] In one of the many embodiments described herein, one or more cancer cells at the target site undergo cell death.

[0176] In many of the embodiments described herein, a therapeutically effective dose of a DNA-dependent protein kinase (PK) inhibitor is administered orally, subcutaneously, or intravenously.

[0177] One example of the many embodiments described herein is a method for inducing cell death in cancer cells, comprising exposing cancer cells to an alternating electric field for a certain period of time and exposing cancer cells to a DNA-dependent PK inhibitor.

[0178] One example of the many embodiments described herein is a DNA-dependent protein kinase (PK) inhibitor used in a method for inducing cell death in cancer cells, wherein the cancer cells are present in a subject, and the method comprises exposing the cancer cells to an alternating electric field for a certain period of time, and exposing the cancer cells to a DNA-dependent PK inhibitor.

[0179] In one example of many embodiments described herein, cancer cells are simultaneously exposed to radiation or have been exposed to radiation in the past.

[0180] One example of the many embodiments described herein is a method for inhibiting DNA repair in cancer cells that have been exposed to or have been exposed to radiation, and includes exposing cancer cells to an alternating electric field for a certain period of time and exposing cancer cells to a DNA-dependent PK inhibitor.

[0181] One example of the many embodiments described herein is a DNA-dependent protein kinase (PK) inhibitor used in a method to inhibit DNA repair in cancer cells that have been exposed to or have been exposed to radiation, wherein the cancer cells are present in a subject, and the method comprises exposing the cancer cells to an alternating electric field for a certain period of time and exposing the cancer cells to a DNA-dependent PK inhibitor.

[0182] In one example of the many embodiments described herein, cancer cells are present within the subject.

[0183] In one example of the many embodiments described herein, exposing cancer cells to an alternating electric field includes supplying an alternating electric field to a target site of a subject containing one or more cancer cells.

[0184] In one example of the many embodiments described herein, the cancer cells originate from ovarian cancer, hepatobiliary cancer, prostate cancer, pancreatic cancer, head and neck cancer, glioblastoma, gliosarcoma, leukemia, or non-small cell lung cancer.

[0185] In many embodiments described herein, the DNA-dependent PK inhibitor is Nedisertib, VX-984, CC-115, CC-122, AZD7648, matinib, vemurafenib, gefitinib, Peposertib (M3814), MSC2490484A, LY294002, JU-57788, CC-115, BAY-8400, SF2523, LTURM34, Compound 401, AMA-37, IC 86621, DNA-PK-IN-1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9, NU7026, or NU 7441, or a combination thereof.

[0186] In one example of the many embodiments described herein, the method further comprises administering to a subject a therapeutically effective amount of an ATR inhibitor, ATM inhibitor, or PARP inhibitor.

[0187] In many embodiments described herein, the ATR inhibitor is Schisandrin B, Nu6027, Dactolisib, EPT-46464, VE-821, AZ20, Berzosertib, Torin-2, Ceralasertib (AZD6738), Tetrahydropyrazolo[1,5-a]pyrazine, Azabenzimidazole, Gartisertib (M4344 or VX-803), Bayl895344 (Elimsuretib), CGK 733, Camonsertib, ATR-IN-4, VE-821, AZ20, ETP-46464, or ATR Inhibitor 1.

[0188] In many embodiments described herein, the ATM inhibitor is M4076, KU-55933, KU-60019, CP-466722, Wortmannin, Torin 2, AZD0156, or AZ31.

[0189] In many embodiments described herein, the PARP inhibitor is olaparib, niraparib, talazoparib, lucaparib, or AZD9574.

[0190] In one example of the many embodiments described herein, the therapeutically effective dose of VX-984 is 50 to 720 mg / day.

[0191] In many embodiments described herein, cancer cells have one or more DNA strand breaks, such as single-strand DNA breaks and / or double-strand DNA breaks.

[0192] In one example of the many embodiments described herein, cancer cells have double-strand DNA breaks.

[0193] In one example of the many embodiments described herein, at least one DNA repair mechanism in cancer cells is inhibited.

[0194] In many embodiments described herein, the inhibited DNA repair mechanism is homologous recombination repair, non-homologous end joining repair, or both.

[0195] In many embodiments described herein, one or more cancer cells undergo cell death.

[0196] In one example of the many embodiments described herein, the alternating electric field has frequency and electric field strength.

[0197] In one example of the many embodiments described herein, the frequency of the AC electric field is in the range of 100 kHz to 1 MHz.

[0198] In many embodiments described herein, the frequency range is 100 to 500 kHz.

[0199] One example of the many embodiments described herein is a method for enhancing the effectiveness of radiotherapy in a subject, which includes applying an alternating electric field having a frequency and electric field strength to a target site of the subject, including the site receiving or having received radiotherapy, for a certain period of time, and administering to the subject a therapeutically effective amount of a DNA-dependent protein kinase (PK) inhibitor.

[0200] One example of the many embodiments described herein is a DNA-dependent protein kinase (PK) inhibitor used in a method to enhance the effectiveness of radiotherapy in a subject, the method comprising applying an alternating electric field having a frequency and electric field intensity for a certain period of time to a target site of the subject, including the site receiving or having received radiotherapy, and administering a therapeutically effective amount of the DNA-dependent protein kinase inhibitor to the subject.

[0201] One example of the many embodiments described herein is an in vitro method for increasing the effectiveness of radiotherapy in a subject, the method comprising applying an alternating electric field having frequency and electric field intensity for a certain period of time to a target site of the subject including the site receiving or having received radiotherapy, and administering to the subject a therapeutically effective amount of a DNA-dependent protein kinase (PK) inhibitor.

[0202] In one example of the many embodiments described herein, the target site includes one or more cancer cells.

[0203] In one example of the many embodiments described herein, the subject has cancer.

[0204] In many embodiments described herein, the cancer is ovarian cancer, hepatobiliary cancer, prostate cancer, pancreatic cancer, head and neck cancer, glioblastoma, gliosarcoma, leukemia, or non-small cell lung cancer.

[0205] In one example of many embodiments described herein, one or more cancer cells at a target site have one or more DNA strand breaks, such as single-strand DNA breaks and / or double-strand DNA breaks.

[0206] In one example of the many embodiments described herein, one or more cancer cells at the target site have double-strand DNA breaks.

[0207] In one example of the many embodiments described herein, at least one DNA repair mechanism within one or more cancer cells at a target site is inhibited.

[0208] In many embodiments described herein, the inhibited DNA repair mechanism is homologous recombination repair, non-homologous end joining repair, or both.

[0209] In one of the many embodiments described herein, one or more cancer cells at the target site undergo cell death.

[0210] In many of the embodiments described herein, a therapeutically effective dose of a DNA-dependent protein kinase (PK) inhibitor is administered orally, subcutaneously, or intravenously.

[0211] In many embodiments described herein, the DNA-dependent PK inhibitor is Nedisertib, VX-984, CC-115, CC-122, AZD7648, matinib, vemurafenib, gefitinib, Peposertib (M3814), MSC2490484A, LY294002, JU-57788, CC-115, BAY-8400, SF2523, LTURM34, Compound 401, AMA-37, IC 86621, DNA-PK-IN-1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9, NU7026 or NU 7441, or a combination thereof.

[0212] In one example of the many embodiments described herein, the therapeutically effective dose of VX-984 is 50 to 720 mg / day.

[0213] In many embodiments described herein, cancer cells have one or more DNA strand breaks, such as single-strand DNA breaks and / or double-strand DNA breaks.

[0214] In one example of the many embodiments described herein, cancer cells have double-strand DNA breaks.

[0215] In one example of the many embodiments described herein, at least one DNA repair mechanism in cancer cells is inhibited.

[0216] In many embodiments described herein, the inhibited DNA repair mechanism is homologous recombination repair, non-homologous end joining repair, or both.

[0217] In many embodiments described herein, one or more cancer cells undergo cell death.

[0218] In one example of the many embodiments described herein, the alternating electric field has frequency and electric field strength.

[0219] In one example of the many embodiments described herein, the frequency of the AC electric field is in the range of 100 kHz to 1 MHz.

[0220] In many embodiments described herein, the frequency range is 100 to 500 kHz.

[0221] One example of the many embodiments described herein is a DNA-dependent PK inhibitor used in a method for treating a subject in need, the method comprising applying an alternating electric field to a target site of a subject in need of the DNA-dependent PK inhibitor, and administering the DNA-dependent PK inhibitor to the subject in need.

[0222] One example of the many embodiments described herein is a combination of alternating electric field and DNA-dependent PK inhibitors for use in the treatment of subjects who require it.

[0223] One example of the many embodiments described herein is a DNA-dependent PK inhibitor used in a method to induce cell death in a subject who requires it, the method comprising applying an alternating electric field to a target site in a subject who requires the DNA-dependent PK inhibitor, and administering the DNA-dependent PK inhibitor to the subject who requires it.

[0224] One example of the many embodiments described herein is a combination of an alternating electric field and a DNA-dependent PK inhibitor used in a method for inducing cell death in a subject requiring it.

[0225] One example of the many embodiments described herein is a DNA-dependent PK inhibitor used in a method to inhibit DNA repair in a subject requiring it, the method comprising applying an alternating electric field to a target site in a subject requiring the DNA-dependent PK inhibitor, and administering the DNA-dependent PK inhibitor to the subject requiring it.

[0226] One example of the many embodiments described herein is a combination of an alternating electric field and a DNA-dependent PK inhibitor for use in a method of inhibiting DNA repair in a subject requiring it.

[0227] One example of the many embodiments described herein is a DNA-dependent PK inhibitor used in a method to enhance the effectiveness of radiotherapy in a subject in need thereof, the method comprising applying an alternating electric field to a target site of a subject in need of the DNA-dependent PK inhibitor, and administering the DNA-dependent PK inhibitor to the subject in need thereof.

[0228] One example of the many embodiments described herein is a combination of an alternating electric field and a DNA-dependent PK inhibitor for use in a method to enhance the effectiveness of radiotherapy in subjects requiring it.

[0229] In one example of the many embodiments described herein, an alternating electric field is applied to a target area of ​​a subject requiring it at a predetermined frequency for a predetermined time.

[0230] In one example of the many embodiments described herein, the subject has cancer. In some embodiments, the cancer may be ovarian cancer, hepatobiliary cancer, prostate cancer, pancreatic cancer, head and neck cancer, glioblastoma, gliosarcoma, leukemia, or non-small cell lung cancer.

[0231] In one example of the many embodiments described herein, the target site includes one or more cancer cells.

[0232] In many embodiments described herein, an alternating electric field is applied before, after, or concurrently with the administration of one or more DNA-dependent PK inhibitors.

[0233] In many embodiments described herein, the cancer is ovarian cancer, hepatobiliary cancer, prostate cancer, pancreatic cancer, head and neck cancer, glioblastoma, gliosarcoma, leukemia, or non-small cell lung cancer.

[0234] In one example of the many embodiments described herein, a subject with cancer has received or is currently receiving radiotherapy or chemotherapy.

[0235] In one example of many embodiments described herein, one or more cancer cells at a target site have one or more DNA strand breaks, such as single-strand DNA breaks and / or double-strand DNA breaks.

[0236] In one example of the many embodiments described herein, one or more cancer cells at the target site have double-strand DNA breaks.

[0237] In one example of the many embodiments described herein, at least one DNA repair mechanism within one or more cancer cells at a target site is inhibited.

[0238] In many embodiments described herein, the inhibited DNA repair mechanism is homologous recombination repair, non-homologous end joining repair, or both.

[0239] In one of the many embodiments described herein, one or more cancer cells at the target site undergo cell death.

[0240] In many of the embodiments described herein, a therapeutically effective dose of a DNA-dependent protein kinase (PK) inhibitor is administered orally, subcutaneously, or intravenously.

[0241] In one example of the many embodiments described herein, the alternating electric field has frequency and electric field strength.

[0242] In one example of the many embodiments described herein, the frequency of the AC electric field is in the range of 100 kHz to 1 MHz.

[0243] In many embodiments described herein, the frequency range is 100 to 500 kHz.

[0244] One example of the many embodiments described herein is a kit for enhancing the effectiveness of radiotherapy in a subject, the kit comprising one or more DNA-dependent protein kinase PK inhibitors and one or more materials for delivering an alternating electric field having frequency and electric field intensity for a period of time to a target site of the subject, including the site receiving or having received radiotherapy.

[0245] A further example of the many embodiments described herein is a kit for inducing cell death of cancer cells, wherein the cancer cells are located at a target site in a subject, and the kit comprises one or more DNA-dependent protein kinase PK inhibitors and one or more materials for delivering an alternating electric field to expose the cancer cells at the target site in the subject to the alternating electric field for a set period of time.

[0246] A further example of the many embodiments described herein is a kit for inhibiting DNA repair in cancer cells that have been exposed to or have been previously exposed to radiation, wherein the cancer cells are located at a target site in the subject, and the kit comprises one or more DNA-dependent protein kinase PK inhibitors and one or more materials for delivering an alternating electric field to expose the cancer cells at the target site in the subject to the alternating electric field for a set period of time.

[0247] Furthermore, DNA containing one or more strand breaks is disclosed, which is used in a method to enhance the effectiveness of radiotherapy in a subject, the method comprising applying an alternating electric field having a frequency and electric field strength for a certain period of time to a target site of the subject including the site receiving or having received radiotherapy, the one or more strand breaks being formed by the application of the alternating electric field.

[0248] A further example involves DNA containing one or more strand breaks, which is used in a method to induce cell death in cancer cells present in a subject, the method comprising exposing the cancer cells to an alternating electric field for a certain period of time to form one or more strand breaks.

[0249] Furthermore, the DNA includes one or more strand breaks, and the DNA is used in a method for treating a subject with cancer, the method including forming one or more strand breaks by applying an alternating electric field for a certain period of time to a target site in the subject containing one or more cancer cells.

[0250] In the three examples above, the method may further include administering a therapeutically effective dose of a DNA-dependent protein kinase (PK) inhibitor to the subject.

[0251] Also disclosed is DNA containing one or more double-strand breaks, which are used in a method to enhance the effectiveness of radiotherapy in a subject, the method comprising applying an alternating electric field having a frequency and electric field strength to a target site of the subject, including the site receiving or having received radiotherapy, for a certain period of time, wherein one or more strand breaks are formed by the application of the alternating electric field.

[0252] Further examples include DNA containing one or more double-strand breaks, which are used in a method to induce cell death in cancer cells present in a subject, the method comprising exposing cancer cells to an alternating electric field for a certain period of time to form one or more double-strand breaks.

[0253] Furthermore, the DNA includes one or more double-strand breaks, and the DNA is used in a method for treating a subject with cancer, the method including forming one or more double-strand breaks by applying an alternating electric field for a certain period of time to a target site in the subject containing one or more cancer cells.

[0254] In the three examples above, the method may further include administering a therapeutically effective dose of a DNA-dependent protein kinase (PK) inhibitor to the subject.

[0255] Those skilled in the art will recognize, or can verify by routine experimentation, numerous equivalents to the specific embodiments of the methods and compositions described herein. Such equivalents are intended to be covered by the following claims.

Claims

1. A DNA-dependent protein kinase inhibitor used in a method for treating a subject with cancer, wherein the method is: a) Applying an alternating electric field to a target site of the subject containing one or more cancer cells for a certain period of time, b) A DNA-dependent protein kinase inhibitor comprising administering a therapeutically effective amount of the inhibitor to the subject.

2. It is a kit for treating cancer cells, One or more DNA-dependent protein kinase PK inhibitors, A kit comprising one or more materials for delivering an alternating electric field to a target site containing one or more cancer cells.

3. An in vitro method for treating a target site, a) Applying an alternating electric field to a target site containing one or more cancer cells for a certain period of time, b) A method comprising administering a therapeutically effective amount of a DNA-dependent protein kinase (PK) inhibitor to the target site.

4. A method for treating a subject with cancer, a) Applying an alternating electric field to a target site of the subject containing one or more cancer cells for a certain period of time, b) A method comprising administering to the subject a therapeutically effective amount of a DNA-dependent protein kinase (PK) inhibitor.

5. The DNA-dependent protein kinase (PK) inhibitor according to claim 1, or the method according to claim 4, wherein the cancer is ovarian cancer, hepatobiliary tract cancer, prostate cancer, pancreatic cancer, head and neck cancer, glioblastoma, gliosarcoma, leukemia, or non-small cell lung cancer.

6. The subject having cancer has received or is currently receiving radiotherapy or chemotherapy, the DNA-dependent protein kinase (PK) inhibitor according to claim 1 or claim 5, or the method according to any one of claims 4 to 5.

7. The method according to any one of the claims 1 or 5 to 6, wherein the one or more cancer cells in the target site have double-strand DNA breaks.

8. A DNA-dependent protein kinase (PK) inhibitor according to claim 1 or any one of claims 5 to 7, wherein at least one DNA repair mechanism is inhibited in one or more cancer cells at the target site, or the method according to any one of claims 3 to 7.

9. The DNA-dependent protein kinase (PK) inhibitor according to claim 7, or the method according to claim 8, wherein the inhibited DNA repair mechanism is homologous recombination repair, non-homologous end joining repair, or both.

10. The DNA-dependent protein kinase (PK) inhibitor according to claim 1 or any one of claims 5 to 9, or the method according to any one of claims 3 to 9, wherein one or more cancer cells in the target site undergo cell death.

11. A DNA-dependent protein kinase (PK) inhibitor according to claim 1 or any one of claims 5 to 10, or the method according to any one of claims 4 to 10, administered orally, subcutaneously, or intravenously in a therapeutically effective amount of the DNA-dependent protein kinase (PK) inhibitor.

12. A DNA-dependent protein kinase (PK) inhibitor used in a method for inducing cell death in cancer cells, wherein the cancer cells are present in a subject, and the method is a) Exposing the cancer cells to an alternating electric field for a certain period of time, b) A DNA-dependent protein kinase (PK) inhibitor comprising exposing the cancer cells to the DNA-dependent PK inhibitor.

13. A DNA-dependent protein kinase (PK) inhibitor used in a method to inhibit DNA repair in cancer cells that have been exposed to or have been exposed to radiation, wherein the cancer cells are present in the subject, and the method is a) Exposing the cancer cells to an alternating electric field for a certain period of time, b) A DNA-dependent protein kinase (PK) inhibitor comprising exposing the cancer cells to the DNA-dependent PK inhibitor.

14. A method for inducing cell death in cancer cells, a) Exposing the cancer cells to an alternating electric field for a certain period of time, b) A method comprising exposing the cancer cells to the DNA-dependent PK inhibitor.

15. The DNA-dependent protein kinase (PK) inhibitor according to claim 12, or the method according to claim 14, wherein the cancer cells are simultaneously exposed to radiation or have been exposed to radiation in the past.

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