Methods for targeting cancer cells

Abstract

Methods for targeting and causing osmotic lysis of cancer cells by administering a voltage-gated sodium channel enhancer, a Na+, K+-ATPase inhibitor, and a pulsed electric field.

Description

METHODS FOR TARGETING CANCER CELLSFIELD OF INVENTION

[0001] This disclosure relates generally to the targeting of cancer cells. More specifically, this disclosure concerns the associated delivery of pulsed electric fields (PEFs), and inhibitor of the sodium, potassium-adenosine triphosphatase (Na+, K+-ATPase or “sodium pump”), and an enhancer of voltage-gated sodium channels (VGSCs or “sodium channels”).BACKGROUND

[0002] Chemotherapy and radiotherapy of metastatic cancer, because of toxicity to both normal and abnormal tissues, present the clinician with the difficult challenge of trying to kill the neoplastic disease before killing the patient; a balance between treatment and rescue. All traditional cancer treatments are associated with toxicity, an increase in morbidity, and a reduction in quality of life that may extend far beyond the period of treatment. A major focus of current anti -neoplastic treatments is targeting treatment to the cancer cells, for example, targeting proteins expressed or over-expressed by cancer cells, but not by normal tissue.

[0003] Many invasive cancer cell types over-express VGSCs by more than 10-fold greater than normal cells. Cancer cells that over-express VGSCs are epithelial carcinomas that include, but are not limited to, highly invasive breast cancer, prostate cancer, small cell lung cancer, non-small cell lung carcinoma, neuroblastoma, and cervical cancer. Some sarcomas, notable rhabdomyosarcoma, mesothelioma, osteosarcoma, also overexpress VGSCs. Mesothelioma not classified as an epithelial cancer is also known to over-express VGSCs. When these sodium channels are activated, Na+is conducted into the cells. In these cancers, the degree of metastasis is directly related to an increased expression of VGSCs. Physiologically, these cancer cells share certain cellular properties with normal excitable cells such as neurons and cardiac myocytes (for example, the conduction of action potentials).

[0004] Of the 1.6 million people contracting epithelial cell cancer each year in the U.S., 40% are considered to be “highly invasive” and over-express VGSCs. These patients diagnosed with malignant / metastatic carcinomas are treated currently with major and often disfiguring surgical procedures, chemotherapy and / or radiation. More than 400,000 people die from epithelial cell carcinoma each year in the United States and an estimated 10 times thatworldwide. In addition, another 1.2 million U.S. patients diagnosed with invasive cancer are successfully treated with traditional surgery, chemotherapy and / or radiation. Breast cell carcinoma is an example of a highly invasive cancer. More than 40,000 people die from breast cell carcinoma each year in the United States and 465,000 worldwide. Greater than 90% of these deaths are due to metastasis of the primary tumor. In addition, another 170,000 U.S. women diagnosed with invasive breast cancer are successfully treated with traditional mastectomy, lumpectomy, chemotherapy and / or radiation. Of the 207,000 people contracting breast cancer each year, 40% of the cancers are considered to be “highly invasive”, and over-express VGSCs.

[0005] The family of sodium channels named “voltage-gated sodium channels” was so designated due to the sensitivity to small changes (about 30 mV; from a resting membrane potential of -70 mV to threshold of -40 mV) in the voltage across the cellular membrane. They have also been shown to be activated by many forms of stimulation — electric current, mechanical disturbances in the membrane, ultrasound, magnetic fields, and several drugs. There are nine members of the VGSC family, with nine different isoforms. They are designated Navl.X, where X represents 1-9. One different channel, NaX, does not respond to shifts in voltage, but to extracellular sodium concentrations.

[0006] Na+, K+-ATPase is a ubiquitous transmembrane protein in animal cells, and functions to maintain an ion imbalance across the cell membrane where more charged ions are located outside of the cell, largely sodium ions, than inside. This produces an electrochemical gradient that is in homeostatic balance. When ionic imbalance shifts in the presence of a change in voltage an action potential is generated causing a transient osmotic shift toward an intracellular hypertonic state. The restoration of the sodium imbalance is an essential function performed by Na+, K+-ATPase. When Na+, K -ATPase does not function properly, water follows sodium into the cell to restore osmotic balance thereby increasing cell volume. In normal cells this shift in cell volume is tolerated due to membrane compliance. Blocking Na+, K+-ATPase function can lead to a loss of cellular excitability and an increase in cellular volume. Many inhibitors are known, including the cardiac glycosides. The isozymes vary in their sensitivity to each of the cardiac glycoside drugs. More than 30 drugs have been shown to inhibit sodium pump activity. These include ouabain, digitalis, and its active ingredients digoxin and digitoxin.

[0007] U.S. Patent No. 8,921,320 (which is herein incorporated in its entirety for all purposes) discloses methods for treating cancer in a mammal involving co-administering totumor cells that over-express voltage-gated sodium channels a first agent and a second agent, wherein the first agent inhibits Na+, K+-ATPase, and wherein the second agent stimulates voltage-gated sodium channels, to cause the osmotic lysis of the tumor cells, resulting in a substantial reduction in tumor cell viability within about one hour after said co-administration. In view of the difference in VGSCs between cancerous and non-cancerous tissue, highly malignant cancer cells may be selectively killed by blocking sodium pumps and simultaneously stimulating the opening of sodium channels in these cells. The first agents may include ouabain, or digitoxin, and the second agents may include electrical currents, ultrasound, and magnetic fields.

[0008] U.S. Patent Publication No. US20230075948 (which is herein incorporated in its entirety for all purposes) discloses methods and devices for generating PEFs in large volumes with high uniformity using a plurality of coaxial, electrically conductive ring structures. The PEFs may be delivered to achieve a therapeutically effective dose at an 18 V / m field amplitude for 2 hours for two successive days.

[0009] A need remains for even more efficacious methods for targeting cancer cells that over-express VGSCs and causing osmotic lysis of these cancer cells by inhibiting Na+, K+- ATPase.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] It is believed that the disclosure will be more fully understood from the following description taken in conjunction with the accompanying drawings. Some of the figures may have been simplified by the omission of selected elements for the purpose of more clearly showing other elements. Such omissions of elements in some figures are not necessarily indicative of the presence or absence of particular elements in any of the exemplary embodiments, except as may be explicitly delineated in the corresponding written description. None of the drawings are necessarily to scale.

[0011] Figure 1 shows the in vivo survival of mice treated with riboci clib or ispinesib in association with TOL treatment.DETAILED DESCRIPTION OF THE EMBODIMENTS

[0012] It is an object of the present invention to overcome or ameliorate one or more of the disadvantages of prior art, or at least to provide a useful alternative.

[0013] This disclosure relates to the targeted osmotic lysis (TOL), which includes the delivery of pulsed electric fields (PEFs) and a Na , K+-ATPase inhibitor, with an enhancer of VGSC expression in cancer cells. While cancer cells over-express VGSCs as compared to noncancer cells, VGSC expression in cancer cells may be enhanced further by an immune response modifier that stimulates production and release of inflammatory mediators such as tumor necrosis factor alpha (TNF-a), prostaglandins (PGs) and interleukins (ILs), and / or a cancer cell cycle inhibitor such as ispinesib. The enhancement of VGSC expression in cancer cells can increase the efficacy of TOL treatment.

[0014] This disclosure describes the novel use of imiquimod and 5 -fluorouracil (5-FU), which are two topically applied drugs commonly prescribed by dermatologists to enhance the immune system’s attack of keratoses and some skin cancers. These drugs purportedly work by stimulating production and release of inflammatory mediators such as TNF-a, PGs and IL-6, which results in an increase in the immunological infiltrates in the affected area.

[0015] This disclosure introduces the previously unknown effect of inflammatory mediators in increasing the expression of VGSCs in treated cells. By enhancing the expression of VGSCs in cancer cells, inflammatory mediators (or drugs used to stimulate their production) may be combined with the associated delivery of PEFs and Na+, K+-ATPase inhibitors such as ouabain, digitalis, and its active ingredients digoxin and digitoxin, to enhance the killing of cancer cells through TOL.

[0016] This disclosure also introduces the previously unknown effect of cell cycle inhibitors in increasing the expression of VGSCs in treated cells. While such agents may inhibit the cell cycle for all cell types, cell cycle inhibitors may be more effective for cancer because cancer cells go through the cell cycle more often than non-cancer cells. By enhancing the overall expression of VGSCs in cancer cells, cell growth cycle inhibitors may be combined with the associated delivery of PEFs and Na+, K+-ATPase inhibitors such as ouabain, digitalis, and its active ingredients digoxin and digitoxin, to enhance the killing of cancer cells through TOL.

[0017] In addition to enhancing efficacy of the TOL treatment, these novel methods of incorporating an immune response modifier and / or a cancer cell cycle inhibitor can serve to identify previously undetected tumors, including tumors that are undetectable by other means.

[0018] This disclosure also provides pharmaceutical kits for TOL treatment of cancer. The kits may include a therapeutically effective amount of one or more Na+, K+-ATPaseinhibitor(s) and enhancer(s) of VGSC expression in cancer cell. The kits preferably also will contain instructions for simultaneous, separate or sequential administration of the Na+, K+- ATPase inhibitor(s) and enhancer(s) of VGSC expression for TOL treatment of cancer.

[0019] As used in the specification and appended claims, unless specified to the contrary, the following terms have the meaning indicated below.

[0020] “Tumor” as used herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.

[0021] “Cancer” and “cancerous” relate to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Benign and malignant as well as dormant tumors or microwound metastases are included in this definition.

[0022] “Subject” means a mammal, such as, but not limited to, a human or non-human mammal, such as a cow, equine, dog, sheep or cat.

[0023] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and / or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0024] A pharmacological agent for blocking the exit of the sodium from the cell, such as a Na1, K1-ATPase inhibitor, may be used together with PEF s to enhance the therapeutic efficacy. Non-limiting examples of pharmaceutical compounds that can be used to block Na+, K+ ATPase include ouabain (g Strophantin); dihydroouabain; ouabain octahydrate; ouabagenin; digoxin; digitoxin; digitalis; acetyldigitoxin; acetyldigoxin; lanatoside C; deslanoside; metildigoxin; gitoformate; oleanderin; oleandrigenin; bufotoxin; bufotalin; marinobufagenin (3,5 dihydroxy 14,15 epoxy bufodienolide); palytoxin; oligomycins A, B, C, E, F, and G; rutamycin (oligomycin D); rutamycin B; strophanthin (g strophanthin, Acocantherine); k P strophanthin; strophanthidin; k strophanthoside; cymarin; erysimoside (cardenolide); helveticoside; peruvoside; hypothalamic Na+, K+ ATPase inhibitory factor (HIF); the aglycone of HIF; arenobufagin; cinobufagin;marinobufagin; proscillaridin; scilliroside; daigremontianin; 3, 4, 5, 6, tetrahydroxyxanthone; and all other inhibitors of Na+, K+ ATPase, combinations and derivatives of each.

[0025] The Na+, K+ ATPase inhibitor may be delivered to a single tumor via direct or intravenous administration, to a single organ or area via intravenous or intraluminal administration, or the entire body via intravenous, subcutaneous, intramuscular or oral administration. Pulsed electric field stimulation of sodium channels can be delivered to a single tumor, a single organ, a section of the body, or the entire body. All types and subtypes of the VGSCs family should be equally susceptible to this technology. For example, cell lines that over-express Navl.l, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.5n, Nav1.6, Nav1.7, Nav1.8 and Nav1.9 are susceptible to mediated targeted lysis.

[0026] The delivery of PEFs may involve an electric field amplitude falling in the range of 0.1 V / m to 100 V / m in free space. The pulses may consist of a forward polarization of approximately 1-50 milliseconds, followed by a reverse polarization of similar duration and amplitude. The pulses may be separated by 5-50 milliseconds from finish to start. The precise details of timing, duration, and amplitude may vary widely in the application. For example, the PEF delivery during a treatment session may be increased from about 2 hours to longer. As another example, in the context of skin lesions, the time between intervals of treatment may be adjusted as the skin heals.

[0027] The voltage-gated sodium channel enhancer according to this disclosure may be administered as a pretreatment to or coincidentally with the TOL administration.

[0028] The drugs and compounds that are suitable for use according to this disclosure include drugs that induce inflammation and cause the release of TNF, PGs and ILs, namely, immune response modifiers such as imiquimod (sometimes marketed as Aldara or Zyclara) and 5 -fluorouracil or 5-FU (sometimes marketed as Adrucil, Efudex, Fluoroplex, Tolak, or Carac), as well as compounds that induce VGSC RNA, namely, TNF-ot, PGE2, and IL-6. All salts, solvates, prodrugs, metabolites, derivatives, and analogs of these compounds are contemplated by this disclosure. This includes the 5-FU prodrug capecitabine (sometimes marketed as Xeloda).

[0029] The drugs and compounds that are suitable for use according to this disclosure include drugs that arrest, block, inhibit and / or synchronize the growth cycle in cancer cells at a stage where VGSC expression is heightened. One example drug is the kinesin spindle protein inhibitor ispinesib.

[0030] In addition, other drugs, compounds and / or agents, such as standard-of-care biologies for specific types of cancers, may facilitate additive or synergistic effects with TOL, and accordingly, are also contemplated by this disclosure. A non-exhaustive list includes Trastuzumab (sometimes marketed as Herceptin), Trastuzumab deruxtecan ((sometimes marketed as Enhertu), Olaparib (sometimes marketed as Lynparza), Lapatinib (sometimes marketed as Tykerb or Tyverb), Atezolizumab (sometimes marketed as Tecentriq), Bevacizumab (sometimes marketed as Avastin), Bevacizumab-bvzr (sometimes marketed as Zirabev), Bevacizumab-awwb (sometimes marketed as Mvasi), Erlotinib (sometimes marketed as Tarceva), Ramucirumab (sometimes marketed as Cyramza), Cetuximab (sometimes marketed as Erbitux), Nivolumab (sometimes marketed as Opdivo), and Everolimus (sometimes marketed as Zortress, Afinitor Disperz, or Afmitor).

[0031] The osmotic lysis of tumor cells according to this disclosure may occur at rates up to about 100% of treated cells.EXAMPLES

[0032] The following example(s), including any experiment s) conducted and result(s) achieved are provided for illustrative purposes only and are not to be construed as limiting of this disclosure.

[0033] Example 1. Metastatic squamous cell carcinoma. A patient presented with over 15 basal or squamous cell carcinomas on the face excised by Mohs surgery. Imiquimod (topical 5%) was prescribed to treat the lesions that had not been excised.

[0034] Pretreatment. 4 cancerous and 4 pre-cancerous lesions were treated with imiquimod for eight days prior to TOL treatment. The cancerous lesions had been previously biopsied and confirmed to be squamous cell carcinomas. They were located just below each temple, on the left forehead and on the chin. Precancerous actinic keratosis (AK) lesions were located below each eye, on the nose and medial to the right temple tumor. The imiquimod induced inflammation in the treated areas. As part of the TOL treatment protocol, the patient was pretreated with 0.25 mg digoxin for five days before PEF administration to achieve steady-state serum levels of this sodium pump blocker.

[0035] TOL treatment. The PEF delivery device was operated to treat the patient for 45 minutes on day one, followed by a two-hour treatment on day two. The PEF parameters were 18V / meter; 10 msec positive / 10 msec negative; 15 msec interstimulus interval. Three weeks later, the patient was again pretreated with imiquimod and digoxin for five days, then administered TOL for two hours on two consecutive days. This was repeated another three weeks later.

[0036] Results. On day one, after 15 minutes of PEF stimulation, the patient reported that cancerous tissues began to “tingle”, with a pins-and-needles feeling. At 20 minutes, serous fluid leaked from the right temple tumor and from the forehead tumor. Redness increased in all treated areas. On day two, digoxin was administered two hours before PEF administration. The patient again experienced tingling in both cancerous and AK lesions. Serous fluid leaked from all four tumors. The carcinoma on the chin became allodynic, which lasted for two days.

[0037] Three weeks later, during the second round of TOL treatment, the same procedure was followed. The patient was given three weeks between TOL treatments to allow for healing. During the two days of this second round, very little tingling was perceived. There was also much less serous exudate than during the first round. In the third round, there was no tingling and only a small amount of serous exudate from the left side tumor.

[0038] During the weeks between treatments, the patient’s lesion healing appeared to occur more rapidly (1 week now versus 2-3 weeks previously) with less scarring when compared to previous tumors excised using Mohs surgery.

[0039] Example 2. Ispinesib pretreatment. Mice were assigned to 12 groups shown in Figure 1 where A group were pretreated with Ribociclib; B group were pretreated with Ispinesib; and C group were not pretreated. Respective letter group Is (n=16) were treated with vehicle; Respective letter group 2s (n=12) were treated with stim; Respective letter group 3s (n=12) were treated with digoxin; and Respective letter group 4s (n=12) were treated with TOL. In other words, for example, in Figure 1, Al represents data from mice (n=16) pretreated with Ribociclib and treated with vehicle.

[0040] BALB / c mice were injected with 500,0004T1 cells subcutaneously on their backs on day 0. On day 6, mice were given Ribociclib at 150 mg / kg, Ispinesib at 0.8 mg / ml, or the respective vehicle (10% DMSO, 5% ETOH and 85% PBS - ispinesib vehicle and 0.5% methylcellulose - ribociclib vehicle). Eighteen hours later, digoxin doses were administered hourly for 8 hours. After the fourth digoxin dose, hourly PEFs were delivered at 18V / m, 10 msec positive / 10 msec negative, 15 msec interstimulus interval (i.e., delivered for a 30 minute period,administered 30 minutes after each digoxin dose). Animals were monitored and tumors were measured daily until they reached ethical endpoint criteria, when they were sacrificed.

[0041] Average survival time was measured in each group. Average survival time in the TOL treatment alone group was 7 days. The group combining ribociclib and TOL had an average survival time of 6 days. In the group of mice pretreated with ispinesib before TOL treatment the average survival was 10 days.

[0042] It will be appreciated that the foregoing description provides examples of the disclosed methods and techniques. However, it is contemplated that other implementations of the disclosure may differ in detail from the foregoing examples. All references to the disclosure or examples thereof are intended to reference the particular example being discussed at that point and are not intended to imply any limitation as to the scope of the disclosure more generally. All language of distinction and disparagement with respect to certain features is intended to indicate a lack of preference for those features, but not to exclude such from the scope of the disclosure entirely unless otherwise indicated.

[0043] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0044] Although the preceding text sets forth a detailed description of different embodiments of the invention, it should be understood that the legal scope of the invention is defined by the words of the claims set forth at the end of this patent. The detailed description is to be construed as exemplary only and does not describe every possible embodiment of the invention because describing every possible embodiment would be impractical, if not impossible. Numerous alternative embodiments could be implemented, using either current technology or technology developed after the filing date of this patent, that would still fall within the scope of the claims defining the invention.

[0045] It should also be understood that, unless a term is expressly defined in this patent using the sentence “As used herein, the term ‘x’ is hereby defined to mean ‘y’” or a similar sentence, there is no intent to limit the meaning of that term, either expressly or by implication, beyond its plain or ordinary meaning, and such term should not be interpreted to be limited inscope based on any statement made in any section of this patent (other than the language of the claims). To the extent that any term recited in the claims at the end of this patent is referred to in this patent in a manner consistent with a single meaning, that is done for sake of clarity only so as to not confuse the reader, and it is not intended that such claim term be limited, by implication or otherwise, to that single meaning. Finally, unless a claim element is defined by reciting the word “means” and a function without the recital of any structure, it is not intended that the scope of any claim element be interpreted based on the application of 35 U.S.C. § 112, sixth paragraph.

Claims

CLAIMSI / We claim1. A method for targeting cancer cells comprising administering to tumor cells that overexpress voltage-gated sodium channels: a voltage-gated sodium channel enhancer selected from an immune response modifier and a cancer cell growth cycle inhibitor; a Na+, K+-ATPase inhibitor selected from ouabain, digitalis, digoxin, and digitoxin; and a pulsed electric field generated from a plurality of ring structures made of an electrically conductive material that are charged to different voltage levels, coaxially arranged and spatially separated, wherein at least about 80% of the tumor cells exhibit osmotic lysis.

2. The method of claim 1 wherein the immune response modifier is imiquimod or 5- fluorouracil.

3. The method of claim 1 wherein the immune response modifier is capecitabine.

4. The method of claim 1 wherein the cancer cell growth cycle inhibitor is ispinesib.

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