Composition and method for combination therapy of alternating electric field and N-cadherin inhibitor

Abstract

A method for treating a target requiring treatment is disclosed, comprising applying an alternating electric field at a certain frequency for a certain period of time to a target site of the target requiring treatment, and administering a neuronal cadherin (N-cadherin) inhibitor to the target requiring treatment. A method for preventing metastasis is disclosed, comprising applying an alternating electric field at a certain frequency for a certain period of time to a cell population containing one or more cancer cells, and contacting the cell population with an N-cadherin inhibitor.

Description

Technical Field

[0001] The present invention relates to a composition comprising a neural cadherin (N-cadherin) inhibitor for use in a method of treating a subject in need thereof, said method comprising: applying an alternating electric field to a target site of the subject who needs it; administering a heat cadherin (N-cadherin) inhibitor to the subject who needs it.

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 493,389, filed Mar. 31, 2023, and U.S. Provisional Patent Application No. 63 / 593,160, filed Oct. 25, 2023, the entire contents of which are incorporated herein by reference.

Background Art

[0003] Neural (N)-cadherin is a calcium-dependent single-pass transmembrane glycoprotein that mediates homotypic and heterotypic cell-cell adhesion. As an important member of the cadherin family, N-cadherin plays an important role in the development and functional regulation of the nervous system, brain, heart, skeletal muscle, blood vessels, and hematopoietic microenvironment. However, abnormal expression of N-cadherin has been found in many cancers such as lung cancer, breast cancer, prostate cancer, and squamous cell carcinoma. Abnormal expression of N-cadherin has been increasingly recognized as being closely related to aspects of malignant tumor progression in humans, including the ability of N-cadherin to be a therapeutic target for tumor invasion and metastasis, transformation, adhesion, apoptosis, angiogenesis, invasion, and metastasis.

[0004] N-cadherin is closely involved in the formation of blood vessels (a process known as angiogenesis) and the maintenance of its integrity.

[0005] The N-cadherin antagonist LCRF-0006 inhibits neurite outgrowth and bone marrow endothelial cell (BMEC) adhesion in vitro. LCRF-0006 can also disrupt the BMEC monolayer, prevent endothelial duct formation in Matrigel, and disrupt mature endothelial ducts.

[0006] Inhibition of N-cadherin function destabilizes microvessels. For example, antibodies against N-cadherin disrupt periendothelial cell adhesion complexes, causing microvessels to bleed.

[0007] Furthermore, N-cadherins partially regulate tumor cell behavior through their interaction with and activation of fibroblast growth factor receptors. FGFR has also been shown to be upregulated after TT field expression.

[0008] Therefore, preventing or reducing the expression of N-cadherin is beneficial in the treatment of cancer. [Overview of the project] [Means for solving the problem]

[0009] The TT site is shown herein to induce N-cadherin expression. Due to the adverse effects that N-cadherins can have on cancer cells, the present invention relates to a combination therapy of the TT site and an N-cadherin inhibitor.

[0010] A method for treating a target requiring treatment is disclosed, comprising applying an alternating electric field at a certain frequency for a certain period of time to a target site of the target requiring treatment, and administering a neuronal cadherin (N-cadherin) inhibitor to the target requiring treatment.

[0011] A method for preventing metastasis is disclosed, comprising applying an alternating electric field at a certain frequency for a certain period of time to a cell population containing one or more cancer cells, and contacting the cell population with an N-cadherin inhibitor.

[0012] A method for reducing AKT phosphorylation in response to an alternating electric field is disclosed, comprising applying an alternating electric field at a certain frequency for a certain period of time to a cell population containing one or more fibroblasts, and contacting the cell population with a calcium chelating agent.

[0013] A method is disclosed for inhibiting the recruitment of the p85 subunit of PI3K to the N-cadherin complex in response to an alternating electric field, comprising applying an alternating electric field to a cell population at a certain frequency for a certain period of time, and contacting the cell population with a calcium chelating agent.

[0014] Additional advantages of the disclosed methods and compositions are partially described in the following description, partially 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]

[0015] 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.

[0016] [Figure 1] This image shows immunofluorescence imaging of non-small cell lung cancer (NSCLC) cells (H1299 cells) treated with TT field application for 72 hours. [Figure 2] A schematic diagram of an experimental design to test the dependence of N-cadherin accumulation in intercellular junctions on calcium and N-cadherin activity is shown. [Figure 3] Immunofluorescence imaging of NSCLC cells treated with TT field application 72 hours later is shown. [Figure 4] This study demonstrates that AKT s473 phosphorylation is mediated by extracellular Ca+ in H1299 cells. [Figure 5] This shows that AKT s473 phosphorylation is mediated by extracellular Ca+ in A2780. [Figure 6] This document presents an experimental example of immunosensory assessment of N-cadherin after 3 days of TT field treatment. (A) N-cadherin was immunoprecipitated from H1299 cell lysates using an anti-N-cadherin monoclonal antibody. The presence of the p85 subunit of PI3-kinase in anti-N-cadherin immunoprecipitation was detected by immunoblotting with PI3Kp85 antibody. Untreated cells were included as a control. (B) Western blot analysis of N-cadherin, PI3K phosphorylated on the p85 subunit, total PI3K, Akt phosphorylated with ser473, and total AKT levels in control and TT field treated cells. (C) Quantification of standardized relative levels of P-AKT compared to total AKT levels, standardized relative levels of P-PI3Kp85 compared to total PI3K levels, and standardized N-cadherin levels in control and TT field treated cells. A value of 1 was assigned to the expression level in untreated cells. *p<0.05 [Figure 7] This study demonstrates that AKT activation after TT entanglement correlates with the recruitment of the PI3Kp85 regulatory subunit to N-cadherin. (A) N-cadherin was immunoprecipitated from A2780 cell lysates using an anti-N-cadherin monoclonal antibody. The presence of the p85 subunit of PI3-kinase in anti-N-cadherin immunoprecipitation was detected by immunoblotting with PI3Kp85 antibody. Untreated cells were included as a control. (B) Western blot analysis of N-cadherin, PI3K phosphorylated on the p85 subunit, total PI3K, Akt phosphorylated with ser473, and total AKT levels in control and TT entanglement-treated cells. (C) Quantification of standardized relative levels of P-AKT compared to total AKT levels, standardized relative levels of P-PI3Kp85 compared to total PI3K levels, and standardized N-cadherin levels in control and TT entanglement-treated cells. A value of 1 was assigned to the expression levels in untreated cells. *p<0.05 [Figure 8] This study demonstrates that AKT phosphorylation on s473 after TT is mediated by N-cadherin in H1299 cells. [Figure 9] This study demonstrates that AKT phosphorylation on s473 after TT is mediated by N-cadherin in A2780 cells. [Figure 10][Figure 10A] Shows the upstream N-cadherin involvement in AKT activation during prolonged TT field application. H1299 cells were left untreated or treated with a TT field (150 kHz) for 72 hours. Left panel: Confocal fluorescence microscopy images of N-cadherin in control and TT field-treated cells. Blue, DAPI-stained DNA; Red, F-actin; Green, N-cadherin; Scale bar, 20 μm. Right panel: Quantification of N-cadherin expression shown as mean ± SEM. *p<0.05; Unpaired t-test; N≧2. [Figure 10B] Shows the upstream N-cadherin involvement in AKT activation during prolonged TT field application. A2780 and H1299 cells were treated in a TT field (200 kHz and 150 kHz, respectively) for 72 hours, then left untreated (without EGTA), then treated with 4 mM EGTA (EGTA), or treated with 4 mM EGTA, and subsequently replaced with serum-free calcium-containing medium (EGTA / Ca+2). This is a representative phase-contrast image. The scale bar is 100 μm. [Figure 10C] Shows the upstream N-cadherin involvement function of AKT activation during prolonged TT field application. A2780 and H1299 cells were treated in a TT field (200 kHz and 150 kHz, respectively) for 72 hours, then left untreated (without EGTA), then treated with 4 mM EGTA (EGTA), or treated with 4 mM EGTA, and subsequently replaced with serum-free calcium-containing medium (EGTA / Ca+2). Top panel: Samples were immunoblotted for AKT, pAKT (Ser473), and GAPDH. Bottom panel: Concentration measurement analysis (arbitrary units normalized to the expression of housekeeping protein GAPDH) is shown as mean ± SEM. *p<0.05 and ***p<0.001, followed by Tukey's post-hoc test after one-way ANOVA, N≧2. [Figure 10D] Shows the upstream N-cadherin involvement function of AKT activation during prolonged TT field application. A2780 and H1299 cells were left untreated or treated with a TT field (200 kHz and 150 kHz, respectively) for 72 hours, after which N-cadherin neutralization was performed using an N-cadherin neutralizing antibody (N-cad nAb) against the extracellular domain of the protein.Upper panel: Samples immunoblotted for AKT, pAKT (Ser473), and GAPDH. Lower panel: Concentration measurement analysis is shown as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001, and after one-way ANOVA, Tukey's post hoc test was performed, N ≥ 2. [Fig. 10E] Shows the function of N-cadherin involvement upstream of AKT activation during long-term TT field application. After treating A2780 and H1299 cells with TT fields (200 kHz and 150 kHz, respectively) for 72 h, immunoprecipitation was performed using an anti-N-cadherin antibody (α-N-cad). Samples were immunoblotted for N-cadherin and the p85 regulatory subunit of PI3K. Nonspecific IgG was used as a negative control.

Modes for Carrying Out the Invention

[0017] The disclosed methods and compositions may be more readily understood by reference to the following detailed description of certain embodiments and the examples contained therein, as well as the figures and the description before and after them.

[0018] It is to be understood that the disclosed methods and compositions are not limited to specific synthetic methods, specific analytical techniques, or specific reagents, unless otherwise specified, and may vary. It is also to be understood that the terminology used herein is for the purpose of describing certain embodiments only and is not intended to be limiting.

[0019] Materials, compositions, and components that can be used in the disclosed methods and compositions, used in combination with them, used in their preparation, or are products thereof are disclosed. These and other materials are disclosed herein, and where combinations, subsets, interactions, groups, etc. of these materials are disclosed, it may not be explicitly disclosed to specifically reference each of the various individual and collective combinations and permutations of these compounds, but it is understood that each is specifically contemplated and described herein. Thus, if classes of molecules A, B, and C are disclosed and further an example of a combination molecule A-D with classes of molecules D, E, and F is disclosed, each is considered individually and collectively even if not described individually. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F is specifically contemplated and should be considered disclosed from the disclosure of A, B, and C, D, E, and F, and the example combination A-D. Similarly, these subsets or combinations are specifically contemplated and disclosed. Thus, for example, subgroups of A-E, B-F, and C-E are specifically envisioned and should be considered disclosed from the disclosure of A, B, and C, D, E, and F, and the example combination A-D. This concept applies to all aspects of this application, including but not limited to the steps of methods of making and using the disclosed compositions. Thus, if there are various additional steps that can be carried out, it is understood that each of these additional steps can be carried out in any particular embodiment or combination of embodiments of the disclosed method, and that each such combination is specifically contemplated and should be considered disclosed.

[0020] The headings are provided for convenience only and should not be construed as limiting the invention in any way. Embodiments shown under any heading or any part of this disclosure may be combined with embodiments shown under the same or other headings or other parts of this disclosure. A. Definitions

[0021] 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.

[0022] In this specification and the attached claims, the singular forms "a," "an," and "the" also include plural references unless the context clearly indicates otherwise. For example, a reference to "an N-cadherin inhibitor" includes multiple such N-cadherin inhibitors, and a reference to "the N-cadherin inhibitor" includes one or more N-cadherin inhibitors and their equivalents known to those skilled in the art.

[0023] 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 or cancer-associated fibroblasts), cell populations, organs, tissues, or tumors. Thus, the term “target cell” can be used to refer to a target site, where 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 ovaries or lungs. In some embodiments, cells or cell populations that may be a target site or target cell include, but are not limited to, cancer cells (e.g., lung cancer cells). In some embodiments, “target site” may be a tumor target site.

[0024] 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.

[0025] 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, for example, alternating directions across the target site. 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 cellular directions is targeted.

[0026] 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.

[0027] 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.

[0028] The term "subject" refers to the subject of administration, 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." For example, the subject to be administered may mean one that is subjected to an alternating electric field. For example, the subject to be administered may be a patient suffering from cancer, such as ovarian cancer or lung cancer.

[0029] "To treat" means administering or applying therapeutic agents, such as alternating electric fields and N-cadherin inhibitors, 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 lung cancer may include delivering therapeutic agents to cells within the subject.

[0030] "Preventing" means minimizing or reducing the likelihood that a subject will develop cancer.

[0031] As used herein, the terms “administering” and “administration” refer to any method of directly or indirectly delivering an N-cadherin inhibitor 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, parenteral administration, including oral, transdermal, inhalation, nasal, topical, vaginal, intraocular, intraocular, intracerebral, rectal, sublingual, buccal, and intravenous, intra-arterial, intramuscular, and subcutaneous injections. 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 exposure or application. Therefore, in some embodiments, exposing a target site or subject to an alternating electric field, or applying an alternating electric field to a target site or subject, means applying an alternating electric field to the target site or subject.

[0032] "Arbitrary" or "arbitrarily" means that it is uncertain whether the event, situation, or material described thereafter will occur or exist, and the description will include both cases where the event, situation, or material occurs or exists, and where it does not occur or does not exist.

[0033] 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. Such methods are well known to those skilled in the art and include, but are not limited to, parenteral administration, including oral administration, transdermal administration, inhalation administration, nasal administration, topical administration, vaginal administration, intraocular administration, intraocular administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration.

[0034] 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.

[0035] 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, ingredients, 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, ingredients, integers, or steps, etc., that are not listed in that step. B. AC electric field

[0036] The methods disclosed herein involve applying an alternating current field. In some embodiments, the alternating current field used in the methods disclosed herein is a tumor treatment field. In some embodiments, the alternating current field may vary depending on the type or state of the cells to which the alternating current field is applied. In some embodiments, the alternating current 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 alternating current 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 alternating current field may be applied alternately between the front and back electrodes, and then between the left and right electrodes.

[0037] 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 can be, but is not limited to, 50 to 500 kHz, 100 to 500 kHz, 25 kHz to 1 MHz, 50 to 190 kHz, 25 to 190 kHz, 150 to 300 kHz, 180 to 220 kHz, or 210 to 400 kHz. In some embodiments, the frequency of the AC electric field can 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 be approximately 300 kHz.

[0038] In some embodiments, the electric field strength of the AC electric field can be in the range of 0.5 to 4 V / cm RMS. In some embodiments, the electric field strength of the AC electric field can 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 RMS) can be used. In some embodiments, the electric field strength can 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.

[0039] 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 durations 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 for 2 hours per day. For example, the alternating current field can be applied for at least 4 hours per day, at least 8 hours per day, at least 12 hours per day, at least 16 hours per day, or at least 20 hours per day. In some embodiments, the alternating current field can be applied for at least 2 days, for at least 4, 8, 12, 16, or 20 hours per day. In some embodiments, the alternating current field can be applied for at least 3 days, for at least 4, 8, 12, 16, or 20 hours per day. In some embodiments, the alternating current field can be applied for at least 7 days, for at least 4, 8, 12, 16, or 20 hours per day.

[0040] In some embodiments, continuous exposure may continue 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.

[0041] 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 or more.

[0042] The disclosed method includes the step of 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. Treatment method

[0043] A method for treating a target requiring treatment is disclosed, comprising applying an alternating electric field at a certain frequency for a certain period of time to a target site of the target requiring treatment, and administering a neuronal cadherin (N-cadherin) inhibitor to the target requiring treatment.

[0044] In some embodiments, the N-cadherin inhibitor is a calcium chelating agent. In some embodiments, the calcium chelating agent is ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), EDTA, citric acid, disodium anhydrous EDTA, or disodium calcium anhydrous EDTA.

[0045] In some embodiments, the N-cadherin inhibitor is an N-cadherin antagonist. In some embodiments, the N-cadherin antagonist is LCRF-0006, ADH-1 (CHAVC, SEQ ID NO: 1), HAV-containing peptide (e.g., LRAHAVDNG, SEQ ID NO: 2), Trp-containing peptide (e.g., SWTLYTPSGQSK, SEQ ID NO: 3), compound 15, HAV dimer (e.g., CHAVDINGHAVDIC, SEQ ID NO: 4), HAV biomaterial (a linear peptide conjugated to a hydrogel, the linear peptide may be any of those disclosed herein), or an N-cadherin antibody. In some embodiments, the N-cadherin antibody is GC4, 2A9, or 1H7.

[0046] In some aspects, the subjects requiring it have cancer. In some aspects, the cancer may be, but is not limited to, ovarian cancer, non-small cell lung cancer, or breast cancer. In some aspects, the target site includes cancer cells. In some aspects, the cancer cells may be, but are not limited to, ovarian cancer cells, non-small cell lung cancer cells, breast cancer cells, brain tumor cells, liver cancer cells, pancreatic cancer cells, or prostate cancer cells.

[0047] In some embodiments, N-cadherin inhibitors reduce AKT phosphorylation. In some embodiments, AKT phosphorylation is phosphorylation of Ser473.

[0048] In some embodiments, N-cadherin inhibitors reduce PI3K / p85 recruitment to N-cadherin.

[0049] In some embodiments, N-cadherin inhibitors reduce metastasis.

[0050] In some embodiments, an alternating electric field is applied before, after, or concurrently with the administration of the N-cadherin inhibitor. In some embodiments, the step of applying the alternating electric field begins at least one hour before the administration of the N-cadherin inhibitor. In some embodiments, the step of applying the alternating electric field begins at least 30 minutes before the administration of the N-cadherin inhibitor. In some embodiments, concurrent application of the alternating electric field can mean applying it before or after the administration of the N-cadherin inhibitor within 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. In some embodiments, the alternating electric field is applied and the N-cadherin inhibitors are administered at intervals of at least 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 from each other.

[0051] In some embodiments, N-cadherin inhibitors are administered via intratumor, intracranial, ventricular, intrathecal, epidural, intradural, intravascular, intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, oral, intranasal, topical, intratumor injection, or inhalation.

[0052] In some embodiments, the frequency and / or intensity of the alternating electric field may be any of those described herein and may be applied in any of the methods described herein.

[0053] In some embodiments, the frequency of the AC electric field is 50 kHz to 1 MHz. In some embodiments, the frequency of the AC electric field is approximately 150 kHz or 250 kHz. In some embodiments, the AC electric field has an electric field strength of 0.5 to 10 V / cm RMS. In some embodiments, the AC electric field has an electric field strength of approximately 0.9 V / cm RMS.

[0054] In some embodiments, the treatment method further includes administering an anticancer drug. In some embodiments, the anticancer drug may be any known anticancer drug, such as a chemotherapeutic agent or an anti-inflammatory agent, but is not limited to these.

[0055] In some embodiments, an increase in N-cadherin expression in a subject or cell is detected after applying an alternating electric field and before administering an N-cadherin inhibitor. Thus, in some embodiments, the method comprises only administering an N-cadherin inhibitor to those subjects whose N-cadherin levels increase after the application of an alternating electric field.

[0056] In some embodiments, the N-cadherin inhibitor is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the application of the alternating electric field. In some embodiments, the N-cadherin inhibitor is administered simultaneously with the application of the alternating electric field. In some embodiments, the N-cadherin inhibitor is administered within several hours, several days, or several weeks after the application of the alternating electric field.

[0057] In some embodiments, nanoparticles can be used in the disclosed methods. For example, in some embodiments, a chelating agent such as an N-cadherin inhibitor can be supported by nanoparticles (e.g., encapsulated by nanoparticles) to provide specific chelation at a target site, thus avoiding systemic effects of calcium chelation. In some embodiments, nanoparticles can be induced to release an N-cadherin inhibitor in the presence of an alternating electric field. For example, in some embodiments, an alternating electric field can rupture the nanoparticles, thus releasing the N-cadherin inhibitor. In some embodiments, the nanoparticles can be polymer nanoparticles, liposomes, micelles, or metal nanoparticles. In some embodiments, the nanoparticles include a site-specific targeting component. In some embodiments, the site-specific targeting component can be a cancer cell-specific targeting component. In some embodiments, the targeting moiety can guide or target the nanoparticles to a specific target site. The site-specific targeting moiety can be a chemical substance, compound, peptide, or nucleic acid. Examples of targeting components include, but are not limited to, molecules that recognize receptors on specific cell types. D. Methods to prevent metastasis

[0058] A method for preventing metastasis is disclosed, comprising applying an alternating electric field at a certain frequency for a certain period of time to a cell population containing one or more cancer cells, and contacting the cell population with an N-cadherin inhibitor.

[0059] In some embodiments, the N-cadherin inhibitor is a calcium chelating agent. In some embodiments, the calcium chelating agent is ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), EDTA, citric acid, disodium anhydrous EDTA, or disodium calcium anhydrous EDTA.

[0060] In some embodiments, the N-cadherin inhibitor is an N-cadherin antagonist. In some embodiments, the N-cadherin antagonist is LCRF-0006, ADH-1 (CHAVC, SEQ ID NO: 1), HAV-containing peptide (e.g., LRAHAVDNG, SEQ ID NO: 2), Trp-containing peptide (e.g., SWTLYTPSGQSK, SEQ ID NO: 3), compound 15, HAV dimer (e.g., CHAVDINGHAVDIC, SEQ ID NO: 4), HAV biomaterial (a linear peptide conjugated to a hydrogel, the linear peptide may be any of those disclosed herein), or an N-cadherin antibody. In some embodiments, the N-cadherin antibody is GC4, 2A9, or 1H7.

[0061] In some aspects, cancer may be, but is not limited to, ovarian cancer, non-small cell lung cancer, breast cancer, brain tumor, liver cancer, pancreatic cancer, or prostate cancer. In some aspects, the target site may include cancer cells. In some aspects, cancer cells may be, but are not limited to, ovarian cancer cells, non-small cell lung cancer cells, brain tumor cells, liver cancer cells, pancreatic cancer cells, or prostate cancer cells.

[0062] In some embodiments, the method is a method for preventing metastasis in a subject having cancer. For example, in some embodiments, the cell population is in vivo, and therefore, in some embodiments, the cell population is within the subject.

[0063] In some embodiments, the cell population includes cancer cells. In some embodiments, the target site includes cancer cells. In some embodiments, the cancer cells are ovarian cancer cells or non-small cell lung cancer cells.

[0064] In some embodiments, an alternating electric field is applied before, after, or concurrently with the administration of the N-cadherin inhibitor. In some embodiments, the step of applying the alternating electric field begins at least one hour before the administration of the N-cadherin inhibitor. In some embodiments, the step of applying the alternating electric field begins at least 30 minutes before the administration of the N-cadherin inhibitor. In some embodiments, concurrent application of the alternating electric field can mean applying it before or after the administration of the N-cadherin inhibitor within 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. In some embodiments, the alternating electric field is applied and the N-cadherin inhibitors are administered at intervals of at least 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 from each other.

[0065] In some embodiments, N-cadherin inhibitors are administered via intratumor, intracranial, ventricular, intrathecal, epidural, intradural, intravascular, intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, oral, intranasal, topical, intratumor injection, or inhalation.

[0066] In some embodiments, the frequency and / or field strength of the AC electric field may be any of those described herein and may be applied in any of the methods described herein. In some embodiments, the frequency of the AC electric field is 50 kHz to 1 MHz. In some embodiments, the frequency of the AC electric field is about 150 kHz or 250 kHz. In some embodiments, the AC electric field has a field strength of 0.5 to 10 V / cm RMS. In some embodiments, the AC electric field has a field strength of about 0.9 V / cm RMS.

[0067] In some embodiments, the treatment method further includes administering an anticancer drug. In some embodiments, the anticancer drug may be any known anticancer drug, such as a chemotherapeutic agent or an anti-inflammatory agent, but is not limited to these.

[0068] In some embodiments, an increase in N-cadherin expression in a subject or cell is detected after applying an alternating electric field and before administering an N-cadherin inhibitor. Thus, in some embodiments, the method comprises only administering an N-cadherin inhibitor to those subjects whose N-cadherin levels increase after the application of an alternating electric field.

[0069] In some embodiments, the N-cadherin inhibitor is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the application of the alternating electric field. In some embodiments, the N-cadherin inhibitor is administered simultaneously with the application of the alternating electric field. In some embodiments, the N-cadherin inhibitor is administered within several hours, several days, or several weeks after the application of the alternating electric field.

[0070] In some embodiments, nanoparticles can be used in the disclosed methods. For example, in some embodiments, a chelating agent such as an N-cadherin inhibitor can be supported by nanoparticles (e.g., encapsulated by nanoparticles) to provide specific chelation at a target site, thus avoiding systemic effects of calcium chelation. In some embodiments, nanoparticles can be induced to release an N-cadherin inhibitor in the presence of an alternating electric field. For example, in some embodiments, an alternating electric field can rupture the nanoparticles, thus releasing the N-cadherin inhibitor. In some embodiments, the nanoparticles can be polymer nanoparticles, liposomes, micelles, or metal nanoparticles. In some embodiments, the nanoparticles include a site-specific targeting component. In some embodiments, the site-specific targeting component can be a cancer cell-specific targeting component. In some embodiments, the targeting moiety can guide or target the nanoparticles to a specific target site. The site-specific targeting moiety can be a chemical substance, compound, peptide, or nucleic acid. Examples of targeting components include, but are not limited to, molecules that recognize receptors on specific cell types. Methods to reduce E.AKT phosphorylation

[0071] A method for reducing AKT phosphorylation in response to an alternating electric field is disclosed, comprising applying an alternating electric field at a certain frequency for a certain period of time to a cell population containing one or more fibroblasts, and contacting the cell population with a calcium chelating agent. In some embodiments, the calcium chelating agent is ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), EDTA, citric acid, disodium anhydrous EDTA, or disodium calcium anhydrous EDTA.

[0072] In some embodiments, since N-cadherins are activated by calcium binding, calcium can act as an inhibitor of N-cadherin activity. In some embodiments, other N-cadherin inhibitors can be used in addition to calcium chelators. Accordingly, a method for reducing AKT phosphorylation in response to an alternating electric field is disclosed, comprising applying an alternating electric field at a certain frequency for a certain period of time to a cell population containing one or more fibroblasts, and contacting the cell population with an N-cadherin inhibitor. In some embodiments, the N-cadherin inhibitor can affect neurotransmission and cardiac signaling. In some embodiments, the N-cadherin inhibitor is an N-cadherin antagonist. In some embodiments, the N-cadherin antagonist is LCRF-0006, ADH-1 (CHAVC, SEQ ID NO: 1), HAV-containing peptide (e.g., LRAHAVDNG, SEQ ID NO: 2), Trp-containing peptide (e.g., SWTLYTPSGQSK, SEQ ID NO: 3), compound 15, HAV dimer (e.g., CHAVDINGHAVDIC, SEQ ID NO: 4), HAV biomaterial (a linear peptide conjugated to a hydrogel, the linear peptide may be any of those disclosed herein), or an N-cadherin antibody. In some embodiments, the N-cadherin antibody is GC4, 2A9, or 1H7.

[0073] In some embodiments, the cell population is in vivo, and therefore, in some embodiments, the cell population is within the subject. In some embodiments, contacting the cell population with a calcium chelator or N-cadherin inhibitor may include administering the calcium chelator or N-cadherin inhibitor to a subject containing the cell population.

[0074] In some embodiments, the frequency and / or field strength of the AC electric field may be any of those described herein and may be applied in any of the methods described herein. In some embodiments, the frequency of the AC electric field is 50 kHz to 1 MHz. In some embodiments, the frequency of the AC electric field is about 150 kHz or 250 kHz. In some embodiments, the AC electric field has a field strength of 0.5 to 10 V / cm RMS. In some embodiments, the AC electric field has a field strength of about 0.9 V / cm RMS. Methods to hinder F.p.85 mobilization

[0075] A method is disclosed for inhibiting the recruitment of the p85 subunit of PI3K to the N-cadherin complex in response to an alternating electric field, comprising applying an alternating electric field to a cell population at a certain frequency for a certain period of time, and contacting the cell population with a calcium chelating agent. In some embodiments, the calcium chelating agent is ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), EDTA, citrate, disodium anhydrous EDTA, or disodium calcium anhydrous EDTA. In some embodiments, the cell population comprises one or more fibroblasts.

[0076] In some embodiments, other N-cadherin inhibitors can be used in addition to calcium chelators. Accordingly, a method for reducing AKT phosphorylation in response to an alternating electric field is disclosed, comprising applying an alternating electric field at a certain frequency for a certain period of time to a cell population containing one or more fibroblasts, and contacting the cell population with an N-cadherin inhibitor. In some embodiments, the N-cadherin inhibitor can affect neurotransmission and cardiac signaling. In some embodiments, the N-cadherin inhibitor is an N-cadherin antagonist. In some embodiments, the N-cadherin antagonist is LCRF-0006, ADH-1 (CHAVC, SEQ ID NO: 1), HAV-containing peptide (e.g., LRAHAVDNG, SEQ ID NO: 2), Trp-containing peptide (e.g., SWTLYTPSGQSK, SEQ ID NO: 3), compound 15, HAV dimer (e.g., CHAVDINGHAVDIC, SEQ ID NO: 4), HAV biomaterial (a linear peptide conjugated to a hydrogel, the linear peptide may be any of those disclosed herein), or an N-cadherin antibody. In some embodiments, the N-cadherin antibody is GC4, 2A9, or 1H7.

[0077] In some embodiments, the cell population is in vivo, and therefore, in some embodiments, the cell population is within the subject. In some embodiments, contacting the cell population with a calcium chelator or N-cadherin inhibitor may include administering the calcium chelator or N-cadherin inhibitor to a subject containing the cell population.

[0078] In some embodiments, the frequency and / or field strength of the AC electric field may be any of those described herein and may be applied in any of the methods described herein. In some embodiments, the frequency of the AC electric field is 50 kHz to 1 MHz. In some embodiments, the frequency of the AC electric field is about 150 kHz or 250 kHz. In some embodiments, the AC electric field has a field strength of 0.5 to 10 V / cm RMS. In some embodiments, the AC electric field has a field strength of about 0.9 V / cm RMS. G. Composition

[0079] Compositions and formulations comprising one or more N-cadherin inhibitors are disclosed. In some embodiments, the formulations further comprise a pharmaceutically acceptable carrier or diluent. For example, a pharmaceutical composition comprising an N-cadherin inhibitor and a pharmaceutically acceptable carrier is disclosed. For example, a pharmaceutical composition comprising LCRF-0006, ADH-1, HAV-containing peptide, Trp-containing peptide, compound 15, HAV dimer, HAV biomaterial, N-cadherin antibody, or H-SWTLYTPSGQSK-NH2 and a pharmaceutically acceptable carrier is disclosed. Pharmaceutical compositions comprising an N-cadherin inhibitor and a pharmaceutically acceptable diluent are also disclosed.

[0080] In some embodiments, N-cadherin inhibitors may be administered in any of the disclosed methods together with a pharmaceutically acceptable carrier and / or diluent.

[0081] 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 may 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. AR Gennaro, Mack Publishing Company, Easton, PA 1995. 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 about 5 to about 8, or about 7 to about 7.5. Further carriers include sustained-release formulations such as semipermeable matrices of solid hydrophobic polymers containing the composition, the matrices being in the form of molded articles such as films, stents (implanted in blood vessels during angioplasty), gels (including hydrogels), 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 containing solutions such as sterile water, saline solution, or a buffer at physiological pH.

[0082] 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 the site to be treated.

[0083] In some embodiments, the composition comprises nanoparticles carrying one or more N-cadherin inhibitors. In some embodiments, the nanoparticles may be polymer nanoparticles, liposomes, micelles, or metal nanoparticles. In some embodiments, the nanoparticles may include a site-specific targeting component. In some embodiments, the site-specific targeting component may be a cancer cell-specific targeting component. In some embodiments, the targeting moiety may guide or target the nanoparticles to a specific target site. The site-specific targeting moiety may be a chemical substance, compound, peptide, or nucleic acid. Examples of targeting components include, but are not limited to, molecules that recognize receptors on specific cell types. 1. Delivery of the composition

[0084] 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.

[0085] 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.

[0086] 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. H. Kit

[0087] 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 of several N-cadherin inhibitors and one or more materials for delivering an alternating electric field, such as an Optune® system. For example, a kit is disclosed comprising one N-cadherin inhibitor and one or more materials for delivering an alternating electric field, such as an Optune® system. In some embodiments, the kit may also include a cancer treatment agent. Embodiment

[0088] Embodiment 1: A method for treating a subject requiring treatment, A method comprising applying an alternating electric field at a certain frequency for a certain period of time to a target site of the subject requiring treatment, and administering a neuronal cadherin (N-cadherin) inhibitor to the subject requiring treatment.

[0089] Embodiment 2: The method according to Embodiment 1, wherein the N-cadherin inhibitor is a calcium chelating agent.

[0090] Embodiment 3: The method according to Embodiment 2, wherein the calcium chelating agent is EGTA, EDTA, citric acid, disodium anhydrous EDTA, or disodium calcium anhydrous EDTA.

[0091] Embodiment 4: The method according to Embodiment 1, wherein the N-cadherin inhibitor is an N-cadherin antagonist.

[0092] Embodiment 5: The method according to Embodiment 4, wherein the N-cadherin antagonist is LCRF-0006, ADH-1, HAV-containing peptide, Trp-containing peptide, compound 15, HAV dimer, HAV biomaterial, N-cadherin antibody, and H-SWTLYTPSGQSK-NH2.

[0093] Embodiment 6: The method according to Embodiment 5, wherein the N-cadherin antibody is GC4, 2A9, or 1H7.

[0094] Embodiment 7: The method according to any one of Embodiments 1 to 6, wherein the subject has cancer.

[0095] Embodiment 8: The method according to Embodiment 7, wherein the cancer is ovarian cancer or non-small cell lung cancer.

[0096] Embodiment 9: The method according to any one of Embodiments 1 to 8, wherein the N-cadherin inhibitor reduces AKT phosphorylation.

[0097] Embodiment 10: The method according to any one of Embodiments 1 to 9, wherein the N-cadherin inhibitor reduces PI3K / p85 recruitment to N-cadherin.

[0098] Embodiment 11: The method according to any one of Embodiments 1 to 10, wherein the N-cadherin inhibitor reduces translocation.

[0099] Embodiment 12: A method for reducing AKT phosphorylation in response to an alternating electric field, comprising: a) applying an alternating electric field at a certain frequency for a certain period of time to a cell population comprising one or more fibroblasts; and b) contacting the cell population with a calcium chelating agent.

[0100] Embodiment 13: A method for inhibiting the recruitment of the p85 subunit of PI3K to the N-cadherin complex in response to an alternating electric field, comprising: a) applying an alternating electric field to a cell population at a certain frequency for a certain period of time; and contacting the cell population with a calcium chelating agent.

[0101] Embodiment 14: The method according to any one of Embodiments 12 to 13, wherein the calcium chelating agent is EGTA.

[0102] Embodiment 15: A method for preventing metastasis in a subject having cancer, comprising: a) applying an alternating electric field at a certain frequency for a certain period of time to a cell population containing one or more cancer cells; and b) contacting the cell population with an N-cadherin inhibitor.

[0103] Embodiment 16: The method according to Embodiment 15, wherein the N-cadherin inhibitor is a calcium chelating agent.

[0104] Embodiment 17: The method according to Embodiment 16, wherein the calcium chelating agent is EGTA, EDTA, citric acid, disodium anhydrous EDTA, or disodium calcium anhydrous EDTA.

[0105] Embodiment 18: The method according to Embodiment 15, wherein the N-cadherin inhibitor is an N-cadherin antagonist.

[0106] Embodiment 19: The method according to Embodiment 18, wherein the N-cadherin antagonist is LCRF-0006, ADH-1, HAV-containing peptide, Trp-containing peptide, compound 15, HAV dimer, HAV biomaterial, N-cadherin antibody, and H-SWTLYTPSGQSK-NH2.

[0107] Embodiment 20: The method according to any one of the prior embodiments, wherein the alternating current electric field is applied before, after, or simultaneously with the administration of the N-cadherin inhibitor.

[0108] Embodiment 21: The method according to any one of the prior embodiments, wherein the N-cadherin inhibitor is administered via intratumor, intracranial, intraventricular, intrathecal, epidural, intradural, intravascular, intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, oral, intranasal, topical, intratumor injection, or inhalation.

[0109] Embodiment 22: The method according to any one of Embodiments 12 to 21, wherein the cell population is in vivo.

[0110] Embodiment 23: The cell population is present in a subject, according to any one of Embodiments 12 to 21.

[0111] Embodiment 24: The method according to any one of the prior embodiments, wherein the frequency of the AC electric field is 50 kHz to 1 MHz.

[0112] Embodiment 25: The method according to any one of the prior embodiments, wherein the frequency of the AC electric field is approximately 150 or 250 kHz.

[0113] Embodiment 26: The method according to any one of the prior embodiments, wherein the AC electric field has an electric field strength of 0.5 to 10 V / cm RMS.

[0114] Embodiment 27: The method according to any one of the prior embodiments, wherein the alternating current electric field has an electric field strength of about 0.9 V / cm RMS.

[0115] Embodiment 28: The method according to any one of the prior embodiments, further comprising administering a cancer drug.

[0116] Embodiment 29: The method according to any one of Embodiments 1 to 28, wherein an increase in N-cadherin expression in the subject or cells is detected after step a) and before step b).

[0117] Embodiment 30: The method according to any one of Embodiments 1 to 29, wherein step b) is performed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after step a) has been performed.

[0118] Embodiment 31: The method according to any one of Embodiments 12 to 30, wherein the cell population includes cancer cells.

[0119] Embodiment 32: The method according to any one of Embodiments 1 to 30, wherein the target site includes cancer cells.

[0120] Embodiment 33: The method according to any one of Embodiments 31 or 32, wherein the cancer cells are ovarian cancer cells or non-small cell lung cancer cells.

[0121] Embodiment 34: A composition comprising a neuronal cadherin (N-cadherin) inhibitor for use in a method of treating a subject in need of treatment, the method comprising: applying an alternating electric field to a target site of the subject in need; and administering the thermal cadherin (N-cadherin) inhibitor to the subject in need.

[0122] Embodiment 35: The composition according to Embodiment 34, wherein the subject has cancer, and optionally the cancer is ovarian cancer or non-small cell lung cancer.

[0123] Embodiment 36: The composition according to any one of Embodiments 34 to 35, wherein the N-cadherin inhibitor reduces AKT phosphorylation, reduces the recruitment of PI3K / p85 to N-cadherin, or reduces its transfer in the subject.

[0124] Embodiment 37: A composition comprising a calcium chelating agent for use in a method for reducing AKT phosphorylation in response to an alternating electric field or inhibiting the recruitment of the p85 subunit of PI3K to an N-cadherin complex, the method comprising: a) applying an alternating electric field to a cell population comprising one or more fibroblasts; and b) bringing the calcium chelating agent into contact with the cell population.

[0125] Embodiment 38: A composition comprising a neuronal cadherin (N-cadherin) inhibitor for use in a method for preventing metastasis in a subject having cancer, the method comprising: a) applying an alternating electric field to a population of cells including one or more cancer cells; and b) bringing the N-cadherin inhibitor into contact with the population of cells.

[0126] Embodiment 39: The composition according to any one of Embodiments 34 to 38, wherein the N-cadherin inhibitor is a calcium chelating agent or an N-cadherin antagonist.

[0127] Embodiment 40: The composition according to Embodiment 39, wherein the calcium chelating agent is EGTA, EDTA, citric acid, disodium anhydrous EDTA, or disodium calcium anhydrous EDTA.

[0128] Embodiment 41: The composition according to Embodiment 39, wherein the N-cadherin antagonist is LCRF-0006, ADH-1, HAV-containing peptide, Trp-containing peptide, compound 15, HAV dimer, HAV biomaterial, N-cadherin antibody, or H-SWTLYTPSGQSK-NH2.

[0129] Embodiment 42: The composition according to any one of Embodiments 34 to 41, wherein the alternating electric field is applied before, after, or simultaneously with the administration of the N-cadherin inhibitor.

[0130] Embodiment 43: The composition according to any one of Embodiments 34 to 42, wherein the alternating electric field is applied at a frequency of 50 kHz to 1 MHz, optionally, about 150 or 250 kHz.

[0131] Embodiment 44: The composition according to any one of Embodiments 34 to 43, wherein the alternating electric field has an electric field strength of 0.5 to 10 V / cm RMS, and optionally about 0.9 V / cm RMS.

[0132] Embodiment 45: The composition according to any one of Embodiments 34 to 44, further comprising administering a cancer drug.

[0133] Embodiment 46: The composition according to any one of Embodiments 34 to 45, wherein an increase in N-cadherin expression in the subject or cells is detected after step a) and before step b).

[0134] Embodiment 47: The composition according to Embodiments 34-446, wherein step b) is performed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after step a) has been performed.

[0135] Embodiment 48: The composition according to any one of Embodiments 34 to 47, wherein the target site comprises cancer cells, optionally ovarian cancer cells, or non-small cell lung cancer cells. [Examples]

[0136] A. Example 1 Preclinical data from the Novocure team showed that TT field therapy in NSCLC cells increased N-cadherin expression in a calcium-dependent manner compared to untreated cells in vitro.

[0137] The most studied cyclic peptide is N-Ac-CHAVC-NH2 (designated ADH-1), which can disrupt a wide variety of N-cadherin-mediated processes. Importantly, ADH-1 has been shown to inhibit angiogenesis and thus improve TT field therapeutic outcomes. Systemic ADH-1 has been reported to play a dual role in both (1) influencing vascular permeability in the tumor microenvironment and (2) regulating tumor growth through activation of the AKT pathway. 1.Results

[0138] Figure 1 shows that N-cadherin expression is enhanced after the application of the TT field. The results show an increase in N-cadherin after the TT field compared to untreated cells.

[0139] Figure 2 shows a schematic diagram of an experimental design to test the dependence of N-cadherin accumulation in intercellular junctions on calcium and N-cadherin activity. Exemplary Ca 2+ In the switch experiment, NSCLC cells were treated with a TT field for 72 hours. After 72 hours, the cells were either left untreated or treated with 4 mM EGTA (calcium chelator) for 30 minutes. Next, the EGTA-containing medium was replaced with calcium-containing medium for 30 minutes. The cells could be H1299 or A2780 cells.

[0140] Figure 3 shows that N-cadherin expression is enhanced after the TT field and is Ca+-dependent. N-cadherin accumulation in intercellular junctions is dependent on calcium and N-cadherin activity, as shown in Figure 3. The results indicate that N-cadherin activation after the application of the TT field is calcium-dependent, and that N-cadherin becomes reactive after calcium recovery.

[0141] Figures 4 and 5 show that phosphorylation of AKT s473 leads to extracellular Ca 2+This demonstrates that it is mediated by Ca after application of the TT field. NSCLC cells (Figure 4, H1299 cells) or ovarian cancer cells (Figure 5, A2780 cells) treated in a TT field for 72 hours were incubated with 4 mM EGTA to disrupt intercellular contact. Immunoblot analysis of phosphorylated Akt against the Ser-473 amino acid. Cell lysates were separated on 15% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with an antibody against phosphate-specific Akt on Ser-473. The results show that Ca after application of the TT field... 2+ We demonstrated that the activation of AKT phosphorylation by repair is calcium-dependent, and that AKT phosphorylation (Ser 473) is reactive after calcium repair. The relative protein density of Akt relative to the Ser-473 ratio was determined. Each relative protein value was further normalized to the value observed with TT field application for 72 hours. The data represent the mean of two independent experiments in Figure 4 and the mean of three experiments in Figure 5.

[0142] Figure 6 shows that AKT activation after TT entanglement correlates with the recruitment of the PI3Kp85 regulatory subunit to N-cadherin. (Figure 6A) N-cadherin was immunoprecipitated from H1299 cell lysates using an anti-N-cadherin monoclonal antibody. The presence of the p85 subunit of PI3-kinase in anti-N-cadherin immunoprecipitation was detected by immunoblotting with PI3Kp85 antibody. Untreated cells were included as a control. (Figure 6B) Western blot analysis of N-cadherin, PI3K phosphorylated on the p85 subunit, total PI3K, Akt phosphorylated with ser473, and total AKT levels in control and TT entanglement-treated cells. (Figure 6C) Quantification of standardized relative levels of P-AKT compared to total AKT levels, standardized relative levels of P-PI3Kp85 compared to total PI3K levels, and standardized N-cadherin levels in control and TT entanglement-treated cells. The expression level in untreated cells was assigned a value of 1. *p<0.05 Figure 7 shows that AKT activation after TT entanglement correlates with the recruitment of the PI3Kp85 regulatory subunit to N-cadherin. (Figure 7A) N-cadherin was immunoprecipitated from A2780 cell lysates using an anti-N-cadherin monoclonal antibody. The presence of the p85 subunit of PI3-kinase in anti-N-cadherin immunoprecipitation was detected by immunoblotting with PI3Kp85 antibody. Untreated cells were included as a control. (Figure 7B) Western blot analysis of N-cadherin, PI3K phosphorylated on the p85 subunit, total PI3K, Akt phosphorylated with ser473, and total AKT levels in control and TT entanglement-treated cells. (Figure 7C) Quantification of standardized relative levels of P-AKT compared to total AKT levels, standardized relative levels of P-PI3Kp85 compared to total PI3K levels, and standardized N-cadherin levels in control and TT entanglement-treated cells. The expression level in untreated cells was assigned a value of 1. *p<0.05

[0143] B. Example 2 To verify that AKT activation was specifically attributable to N-cadherin homoaffinity involvement, rather than to several other calcium-dependent pathways, we used an N-cadherin-targeted neutralization assay. The results demonstrate that applying an anti-N-cadherin monoclonal antibody, which prevents N-cadherin from forming intercellular contacts, to TT-field treated cells (for 3 days) resulted in a significant reduction in AKT phosphorylation in both cell lines (H1299 and A2780) (Figures 8 and 9).

[0144] N-cadherin neutralization assay: A2780 and H1299 cells (2 × 10⁶ cells) 4 Cells (coverslips) were treated in a TT field for 3 days, with serum depletion on the final treatment day. At the end of treatment, cells were incubated for 1 hour in the absence or presence of monoclonal anti-N-cadherin antibody (Sigma, C3865, clone GC4, 1:200). Then, as described above, the cells were lysed and subjected to Western blot analysis to determine AKT phosphorylation.

[0145] C. Example 3 Intercellular contact is involved in the rapid activation of the PI3K / AKT pathway via cadherin. The observation that AKT signal amplitude increased over time during TT field application facilitated the exploration of whether cadherin can influence the intracellular cascade that modulates PI3K / AKT activation during treatment. Confocal microscopy of H1299 cells untreated or treated with TT field for 72 hours revealed co-localization of N-cadherin and F-actin at the intercellular interface, with significantly increased levels of N-cadherin on the membrane of TT field-treated cells compared to control cells (Figure 10A).

[0146] To determine whether PI3K recruitment at the site of N-cadherin homoaffinity ligation is necessary for PI3K / AKT activation in response to the TT field, a calcium switch assay was performed in A2780 and H1299 cells. Epithelial cells require Ca+2 to establish cadherin homoaffinity ligation; therefore, Ca+2 ions were removed from the medium by the addition of a chelating agent (EGTA), and then Ca+2 ions were reintroduced to restore intercellular contact. Under both control and TT field (72h) conditions, EGTA-treated cells exhibited a round morphology, and this phenotype was slightly reversed upon calcium restoration (Figure 10B). Furthermore, calcium removal resulted in decreased AKT phosphorylation compared to TT field-treated cells under normal calcium levels. Involvement of intercellular adhesion by calcium repair re-induced AKT phosphorylation (Figure 10C).

[0147] Next, we used an N-cadherin neutralizing antibody to confirm that AKT activation is a result of N-cadherin homoaffinity ligation and not a disturbance of calcium homeostasis. The results demonstrated that applying a neutralizing antibody that inhibits N-cadherin-mediated intercellular contact significantly reduces AKT phosphorylation under normal conditions compared to cells treated with TT field therapy (Figure 10D).

[0148] Next, the recruitment of the p85 regulatory subunit of I3K to the PN-cadherin complex was tested by a pull-down assay using an antibody against N-cadherin. These experiments did not distinguish between membrane and cytoplasmic N-cadherin. This resulted in the discovery of comparable levels of N-cadherin in control and TT-field treated cell lysates (Figure 10E). In TT-field treated cell lysates, rather than control lysates, the PI3K-p85 subunit was found to be associated with N-cadherin, indicating increased recruitment of PI3K to the N-cadherin complex after TT-field application. Negative controls using nonspecific antibodies did not show precipitation of N-cadherin or PI3K-p85. These findings suggest that N-cadherin-mediated intercellular contact initiates PI3K-dependent signaling, leading to FAK-independent activation of AKT during longer TT-field exposure.

[0149] Those skilled in the art will recognize, or can confirm 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. References

[0150] 1.Shirayoshi et al, “Cadherin cell adhesion molecules with distinct binding specificities share a common structure,” The EMBO Journal, vol 5, issue 10, Oct 1986 2.Cao et al. “Aberrant N-cadherin expression in cancer,” Biomedicine & Pharmacotherapy,vol 118,109320,Oct 2019 3.Blaschuk “N-cadherin antagonists as oncology therapeutics”Phil Trans R Soc Lond B Biol Sci.2015 Feb 5;370(1661):20140039 4.Mrozik et al.“LCRF-0006,a small molecule mimetic of the N-cadherin antagonist peptide ADH-1,synergistically increases multiple myeloma response to bortezomib,”FASEB Bioadv.2020 Jun 15;2(6):339-353 5.Gerhardt et al.,“N-cadherin mediates pericytic-endothelial interaction during brain angiogenesis in the chicken,”Dev Dyn.2000 Jul;218(3):472-9 6.Tanaka et al.“Monoclonal antibody targeting of N-cadherin inhibits prostate cancer growth,metastasis and castration resistance,”Nature Medicine volume 16,pages 1414-1420(2010) 7.El-Kenawi et al.“Angiogenesis inhibitors in cancer therapy:mechanistic perspective on classification and treatment rationales,”Br J Pharmacol.2013 Oct;170(4):712-729 8.Hamidi et al.“Every step of the way:integrins in cancer progression and metastasis,”Nature Reviews Cancer,volume 18,pages 533-548(2018) 9.Tran et al.“Signal transduction from N-cadherin increases Bcl-2.Regulation of the phosphatidylinositol 3-kinase / Akt pathway by homophilic adhesion and actin cytoskeletal organization,”J Biol Chem.2002 Sep 6;277(36):32905-14 10.De Santis et al,2009;Oncogene 28,1206-1217 11.Li et al,2001;Cancer Research 61,3819-3825 12.Pece et al,1999;J Biol Chem 274:19347-19351. 13.Yamauchi et al,2011 Cancer Research 71(14);2989-97. 14.Volberg et al,1986,The J of Cell Biology,vol 102,1832-1842

Claims

1. A composition comprising a neuronal cadherin (N-cadherin) inhibitor for use in a method of treating a subject requiring treatment, wherein the method is Applying an alternating electric field to the target area of ​​the object that requires it, A composition comprising administering a thermal cadherin (N-cadherin) inhibitor to a subject in need thereof.

2. The composition according to claim 1, wherein the subject has cancer, and optionally the cancer is ovarian cancer or non-small cell lung cancer.

3. The composition according to any one of claims 1 to 2, wherein the N-cadherin inhibitor reduces AKT phosphorylation, reduces the recruitment of PI3K / p85 to N-cadherin, or reduces its transfer in the subject.

4. A composition comprising a calcium chelating agent for use in a method for reducing AKT phosphorylation in response to an alternating electric field or inhibiting the recruitment of the p85 subunit of PI3K to an N-cadherin complex, wherein the method is a) Applying an alternating electric field to a cell population containing one or more fibroblasts, b) A composition comprising bringing the calcium chelating agent into contact with the cell population.

5. A composition comprising a neuronal cadherin (N-cadherin) inhibitor for use in a method for preventing metastasis in a subject with cancer, wherein the method is a) Applying an alternating electric field to a cell population containing one or more cancer cells, b) A composition comprising contacting the cell population with the N-cadherin inhibitor.

6. The composition according to any one of claims 1 to 5, wherein the N-cadherin inhibitor is a calcium chelating agent or an N-cadherin antagonist.

7. The composition according to claim 6, wherein the calcium chelating agent is EGTA, EDTA, citric acid, disodium anhydrous EDTA, or disodium calcium anhydrous EDTA.

8. The composition according to claim 6, wherein the N-cadherin antagonist is LCRF-0006, ADH-1, HAV-containing peptide, Trp-containing peptide, compound 15, HAV dimer, HAV biomaterial, N-cadherin antibody, or H-SWTLYTPSGQSK-NH2.

9. The composition according to any one of claims 1 to 8, wherein the alternating electric field is applied before, after, or simultaneously with the administration of the N-cadherin inhibitor.

10. The composition according to any one of claims 1 to 9, wherein the alternating electric field is applied at a frequency of approximately 150 or 250 kHz, which is optionally between 50 kHz and 1 MHz.

11. The composition according to any one of claims 1 to 10, wherein the alternating electric field has an electric field strength of 0.5 to 10 V / cm RMS, and optionally about 0.9 V / cm RMS.

12. The composition according to any one of claims 1 to 11, further comprising the step of administering a cancer drug.

13. The composition according to any one of claims 1 to 12, wherein an increase in N-cadherin expression in the subject or cells is detected after step a) and before step b).

14. Step b) is performed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after step a) has been performed, the composition according to any one of claims 1 to 13.

15. The composition according to any one of claims 1 to 14, wherein the target site comprises cancer cells, optionally ovarian cancer cells, or non-small cell lung cancer cells.

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