EA044080B1 - COMPOSITIONS AND METHODS FOR TREATING MALIGNANT CELLS BY INDUCING CYTOTOXIC OXIDATIVE STRESS - Google Patents

Description

Many metastatic malignancies are currently incurable, and 5-year survival rates are still very low, despite decades of research and ever-increasing funding. For example, KRAS mutations are common in pancreatic cancer, colorectal cancer, and lung cancer, and there is currently no effective intervention for the KRAS oncogene. One possible approach for treating malignant tumors, such as KRAS mutant malignancies, is to induce oxidative stress, which can selectively kill malignant cells relative to surrounding normal tissue. However, there remains a need today for methods and compositions that are capable of inducing sufficient oxidative stress that is selective for malignant cells to provide therapeutic efficacy.

The essence of the invention

In a first embodiment, there is provided a method of treating a cancer in an individual, comprising exposing the cancer cells to oxidative stress and administering to the individual an effective amount of D-VC or pharmaceutically acceptable salts thereof. In some aspects, exposing cancer cells to oxidative stress includes administering an effective amount of an oxidant to the individual. For example, in some aspects, the oxidizing agent is selected from the group consisting of, but not limited to, arsenic trioxide (ATO), 2-cyano-3,12-dioxooleane-1,9-dien-28-oic acid methyl ester (CDDO-Me), curcumin, betulinic acid, synthetic non-steroidal anti-inflammatory drug (NSAID), GT-094, celastrol, tolperisone and lanperisone (a tolperisone derivative).

In some embodiments, the present invention provides methods for treating cancer, comprising administering to an individual in need thereof a therapeutically effective dose of arsenic trioxide (ATO) and D-vitamin C (D-VC, also known as D-ascorbic acid) or pharmaceutically acceptable salts thereof . In some aspects, the cancer is a KRAS mutant cancer. In some aspects, the cancer is tested and determined to have a KRAS mutation. In some aspects, the cancer exhibits increased expression of GLUT1. In some aspects, the cancer is tested and determined to exhibit increased expression of GLUT1 or exhibit high glucose uptake. In some aspects, the cancer is a KRAS mutant cancer and exhibits increased expression of GLUT1.

In some aspects, oxidative stress is used separately from D-VC administration. In the following aspects, oxidative stress is applied within 24 hours of administration of D-VC. In further aspects, oxidative stress is applied within 8, 6, 4 or 2 hours of D-VC administration. In the following aspects, oxidative stress is applied within 1 hour of D-VC administration. In some aspects, oxidative stress is administered substantially simultaneously with the administration of D-VC. In other aspects, D-VC is administered following the application of oxidative stress. In further aspects, D-VC is administered approximately 2 hours after the application of oxidative stress.

In some aspects, the oxidizing agent, such as ATO, and D-VC are administered in separate compositions. In further aspects, the oxidizing agent, such as ATO, and D-VC are administered within 24 hours of each other. In further aspects, the oxidizing agent (eg, ATO) and D-VC are administered within 8, 6, 4, or 2 hours of each other. In further aspects, the oxidizing agent (eg, ATO) and D-VC are administered within 1 hour of each other. In some aspects, ATO and D-VC are administered together. In other aspects, D-VC is administered after an oxidizing agent (eg, ATO). In further aspects, D-VC is administered approximately 2 hours after administration of the oxidizing agent (eg, ATO).

In some aspects, the individual is hungry. In some aspects, the subject fasts for at least 1 hour before administration of D-VC. In other aspects, the individual fasts for at least 2, 4, 6, 8, 10, 12, 18, or 24 hours before administration of D-VC. In some aspects, the methods further include testing the individual's blood glucose level prior to administration of D-VC. In some aspects, the oxidizing agent (eg, ATO) and D-VC are formulated in the same composition. In some aspects, the oxidizing agent (eg, ATO) and D-VC are added at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 times. In some aspects, the oxidizing agent (eg, ATO) and D-VC are administered every day for one week, two weeks, one month, two months, or three months. In some aspects, the oxidizing agent is ATO and is administered at a dose of from about 10 μg/kg to about

- 1 044080 mg/kg. In further aspects, ATO is administered at a dose of from about 100 μg/kg to about 50 mg/kg. In further aspects, ATO is administered at a dose of from about 0.1 mg/kg to about 5 mg/kg.

For example, ATO can be administered at a dose of at least 0.1 mg/kg to about 0.5, 1, 2, 3 or 4 mg/kg. In further aspects, ATO is administered at a dose of approximately 0.2 mg/kg.

In some aspects, D-VC is administered at a dose of from about 5 mg/kg to about 30 g/kg. In further aspects, D-VC is administered at a dose of from about 15 mg/kg to about 20 g/kg. In further aspects, D-VC is administered at a dose of from about 150 mg/kg to about 15 g/kg. In further aspects, D-VC is administered at a dose of approximately 1.5 g/kg. In some aspects, the individual is fasted upon administration of an oxidizing agent (eg, ATO) and D-VC. In some aspects, the subject fasts for at least 2 hours before administration of the oxidizing agent (eg, ATO) and D-VC. In some aspects, the subject continues to fast for at least 2 hours after administration of the oxidizing agent (eg, ATO) and D-VC.

In some aspects, the cancer is leukemia, colorectal cancer, pancreatic cancer, or lung cancer. In the following aspects, the leukemia is acute promyelocytic leukemia (APL). In some aspects, the cancer is pancreatic cancer, colorectal cancer, or multiple myeloma. In some aspects, the cancer has a KRAS mutation and/or exhibits increased expression of GLUT1. In some aspects, ATO and/or D-VC are administered intravenously, intraperitoneally, intrathecally, intratumorally, intramuscularly, endoscopically, intralesional, subcutaneously, regionally, or by direct injection or perfusion. In other aspects, ATO and/or D-VC are administered by a non-parenteral route. In further aspects, ATO and/or D-VC are administered by the topical, epidermal or mucosal route. In some aspects, ATO and D-VC are administered intranasally, orally, vaginally, rectally, sublingually, or locally. In some aspects, ATO and D-VC are administered by the same route of administration. In some aspects, the methods further comprise treatment with at least one other anti-cancer therapy. In the following aspects, at least one other therapy against a malignant tumor is selected from tumor resection, chemotherapy, immunotherapy, and radiation therapy. In further aspects, the at least one other anti-cancer therapy includes an oxidizing agent. In specific aspects, the oxidizing agent is selected from the group consisting of 2-cyano-3,12dioxooleane-1,9-dien-28-oic acid methyl ester (CDDO-Me), curcumin, betulinic acid, a synthetic nonsteroidal anti-inflammatory drug (NSAID), GT-094, celastrol, tolperisone and lanperisone.

In further embodiments, the invention relates to pharmaceutical compositions containing an oxidizing agent (for example, arsenic trioxide (ATO), 2-cyano-3,12-dioxooleane-1,9-dien-28-oic acid methyl ester (CDDO-Me), curcumin , betulinic acid, a synthetic non-steroidal anti-inflammatory drug (NSAID), GT-094, celastrol, tolperisone and lanperisone (a tolperisone derivative)) and D-VC or pharmaceutically acceptable salts thereof formulated in a pharmaceutically acceptable excipient. In further embodiments, the invention relates to pharmaceutical compositions containing arsenic trioxide (ATO) and D-VC or pharmaceutically acceptable salts thereof formulated in a pharmaceutically acceptable excipient.

In other embodiments, the present invention provides methods for identifying an individual for treatment with an oxidant (eg, ATO) and D-VC, comprising testing a sample from the individual to determine whether the individual has a cancer that contains a KRAS mutation and/or exhibits increased expression of GLUT1 where if the cancer contains a KRAS mutation and/or exhibits increased GLUT1 expression or high glucose uptake, then the individual is a candidate for therapy through ATO and D-VC. In some aspects, the methods further include administering ATO and D-VC therapy to the identified individual.

In some embodiments, the present invention provides methods for treating a cancer in an individual, comprising exposing the cancer cells to oxidative stress by administering an effective amount of arsenic trioxide (ATO) or pharmaceutically acceptable salts thereof and an effective amount of a second oxidizing agent to the individual. In some aspects, the second oxidizing agent is selected from the group consisting of 2-cyano3,12-dioxooleane-1,9-dien-28-oic acid methyl ester (CDDO-Me), curcumin, betulinic acid, synthetic nonsteroidal anti-inflammatory drug (NSAID ), GT-094, celastrol, tolperisone and lanperisone. In some aspects, the cancer is a KRAS mutant cancer. In some aspects, the cancer is tested and determined to have a KRAS mutation. In some aspects, the cancer has increased GLUT1 expression or high glucose uptake. In some aspects, a cancer is tested and determined to have increased tumor expression of GLUT1. In some aspects, the cancer is a KRAS mutant cancer and exhibits increased expression of GLUT1. In some aspects, the second oxidizing agent is administered within 24 hours of the ATO administration. In some aspects, the second oxidizing agent is administered within 8, 6, 4, or 2 hours of ATO administration. In some aspects, the second oxidizing agent is administered within 1 hour of administration of the ATO. In some aspects, ATO is administered after the second oxidizing agent is introduced. In further aspects, ATO is administered approximately 2 hours after the second oxidant. In some aspects, the individual is hungry. In some aspects, the subject is fasted for at least 1 hour before administering ATO. In further aspects, the individual is fasted for at least 2, 4, 6, 8, 10, 12, 18, or 24 hours before ATO administration. In some aspects, the methods further include testing the individual's blood glucose level before administering ATO.

As used herein, substantially free of a particular component is used herein to indicate that no amount of said component is intentionally formulated and/or is present only as an impurity or in trace amounts. The total amount of said component resulting from unintentional contamination of the composition is preferably less than 0.01%. Most preferred is a composition in which no amount of said component can be detected using standard analytical methods.

As used herein in the specification and claims, the singular form means one or more. As used within the scope of the invention in the description and claims, when used with the word containing, the singular form can mean one or more than one. As used herein in the specification and claims, other or next may mean at least second or more.

As used within the scope of the invention in the description and claims, the term is approximately used to indicate that a value includes inherent variation therein as a result of the error of the device, the method used to determine the value, or the variation that exists among the individuals studied.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicative of certain embodiments of the invention, are provided by way of illustration only, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Brief description of drawings

The following drawings form part of the present description and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specific embodiments set forth herein.

Fig. 1 - Vitamin C/ATO and D-vitamin C/ATO combination treatments are effective in inducing cytotoxicity in KRAS mutant malignant cells. HCT116 colorectal cancer cells were treated with the indicated solutions for 72 hours: control (phosphate-buffered saline, PBS added to the culture medium at a dilution of 1:1000), 1 mM VC (vitamin C was used at a concentration of 1 mM), 1 mM D-VC (D-vitamin C was used at a concentration of 1 mM), 5 μM ATO (arsenic trioxide was used at a concentration of 5 μM), and a combination of 1 mM VC with 5 μM ATO or 1 mM D-VC with 5 μM ATO. Images of cells are presented with legends indicating cell processing;

fig. 2 - flow cytometric analysis of apoptosis of the HCT116 malignant tumor after drug treatment. HCT116 cells were treated with drugs as described in FIG. 1, and after treatment, cells were stained with Annexin-V FITC and sorted;

fig. 3 - graphical representation of apoptosis according to flow cytometry data. Flow cytometry data for atoptosis shown in FIG. 2, presented as a graph;

fig. 4 - detection of reactive oxygen species (ROS) by flow cytometry. HCT116 cells were treated with the indicated compounds as described in FIG. 1 and analyzed by flow cytometry to detect the presence of reactive oxygen species using Mito Sox Red reagent. Mito Sox Red is a reagent from Invitrogen™, part number #LSM360084;

fig. 5 is a graphical representation of the presence of reactive oxygen species (ROS) determined by flow cytometry. The ROS detection data shown in FIG. 4 are shown as a graph;

fig. 6 - xenografted HCT116 tumors after 9 injections. Xenografted tumors from each of 5 mice per injection condition are depicted. Mice were injected intraperitoneally with PBS (control), vitamin C (1.5 g/kg), D-vitamin C (1.5 g/kg), ATO 7 mg/kg), ATO (7 mg/kg)+vitamin C ( 1.5 g/kg) or ATO (7 mg/kg) + D-vitamin C (1.5 g/kg). The mice were injected every day for 6 days and after the 6th injection, the mice were left alone for one day. Scarring was observed in several tumors, including all tumors after D-VC+ATO administration;

fig. 7 - xenografted HCT116 tumors after 12 injections. Xenografted tumors from each of 5 mice are depicted under administration conditions as described in FIG. 6, after 12 injections - 3,044,080 injections (treatment for 2 weeks). Scarring was observed in several tumors, including all tumors after D-VC+ATO administration;

fig. 8 - xenografted HCT116 tumors after the 15th (final) injection after resection. Shown are excised tumors after 15 injections described in FIG. 6. Scarring was observed in several tumors, including all tumors after D-VC+ATO administration;

fig. 9 - mass of tumors after 15 injections. The p values for one-way ANOVA show a statistically significant difference in tumor burden between the mouse groups shown in Fig. 8, which were administered PBS against VC+ATO, PBS against D-VC+ATO, and VC+ATO against D-VC+ATO.

Description of Illustrative Embodiments

Vitamin C in its natural form, also referred to herein as VC, L-VC or by its chemical name L-ascorbic acid, can induce oxidative stress in certain cancer cells, such as KRAS mutant cancers. The studies described herein demonstrate that the D-enantiomer of vitamin C (D-VC), alone or in combination with an oxidizing agent such as arsenic trioxide, can induce significant oxidative stress in malignant cells, such as KRAS mutant malignant cells. In particular, studies show that the optical isomer of vitamin C, D-vitamin C (also referred to herein as D-VC or D-ascorbic acid), is highly effective in inducing oxidative stress in KRAS mutant malignant cells. To enhance the oxidative effect of D-VC on KRAS mutant cells, D-VC was administered with another oxidizing compound, arsenic trioxide (ATO). The studies described herein demonstrate that the D-VC/ATO combination is highly cytotoxic to both human and mouse-derived malignant KRAS mutant cells. In addition, the data reported herein indicate that the combination of D-VC/ATO is significantly more effective in inhibiting the growth of KRAS mutant tumors in a xenograft mouse model than the combination of vitamin C and ATO. Since KRAS mutations are common in pancreatic, colorectal and lung cancers and there is currently no effective intervention for the KRAS oncogene, the present invention provides a potent cytotoxic combination that specifically targets malignant cells with mutant KRAS. In addition, this combination of D-VC and ATO can also be used for any type of malignancy associated with high GLUT1 expression.

Thus, in certain embodiments, the present invention provides compositions and methods for treating cancer with arsenic trioxide and D-VC.

I. Methods of treatment.

In one aspect, the present invention provides methods for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of arsenic trioxide and D-VC.

Tumors for which the present methods of treatment are suitable include any type of malignant cell, such as malignant cells found in a solid tumor or hematologic tumor. In particular, the cancer may be a cancer that overexpresses the GLUT1 transporter, such as a KRAS mutant cancer or cancers that exhibit high glucose uptake.

Malignancies that can be treated using the methods described herein include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and squamous cell lung carcinoma), peritoneal cancer, gastric cancer or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma , salivary gland carcinoma, kidney or renal cancer, prostate cancer, cancer of the female external genitalia, thyroid cancer, various types of head and neck cancer, and melanoma. Exemplary cancers that can be treated using the methods of the present invention may overexpress GLUT1, such as KRAS mutant cancers.

A malignant tumor may include, but is not limited to, the following histological types: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial coli-polyposis; solid carcinoma; carcinoid tumor, malignant; bronchiolo- 4 044080 alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophilic carcinoma; oxyphilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-encapsulating sclerosing carcinoma; adrenal cortex carcinoma; endometrioid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; tumor of fat cells, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; pheochromacytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; melanoma type lentigo maligna; acral lentiginous melanomas; nodular melanomas; malignant melanoma in a giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant tumor; müllerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; Brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; ovarian struma, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodenroglioma; oligodendroblastoma; primitive neuroectodermal tumor; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; fungoid mycosis; other specified non-Hodgkin's lymphomas; B cell lymphoma; low-grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate malignant/follicular NHL; intermediate malignant diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small cell NHL with non-split cells; NHL with a massive defeat; mantle cell lymphoma; AIDS-associated lymphoma; Waldenström's macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative disease of the small intestine; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; leukemia from lymphosarcoma cells; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; hairy cell leukemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML) and chronic myeloid leukemia.

In certain embodiments, a therapeutically effective amount of ATO and D-VC is administered to an individual intravenously, intratumorally, or intraperitoneally. The appropriate dosage of ATO and D-VC can be determined based on the type of malignancy being treated, the severity and course of the disease, the clinical condition of the individual, and the judgment of the treating physician.

A. Combined methods of therapy.

In certain embodiments, the methods described herein further include the step of administering to the individual at least one additional therapeutic agent. All additional therapeutic agents described herein are administered to an individual in accordance with good clinical practice for each particular composition or therapy, taking into account any potential toxicity, likely side effects and any other relevant factors.

In certain embodiments, the adjunctive therapy may be immunotherapy, radiation therapy, surgery (eg, surgical resection of a tumor), chemotherapy, bone marrow transplant, or a combination of the foregoing. Additional

- 5 044080 therapy may be targeted therapy. In certain embodiments, the additional therapy is administered prior to the primary treatment (ie, as adjuvant therapy). In certain embodiments, additional therapy is administered after primary treatment (ie, as neoadjuvant therapy).

In certain embodiments, the additional therapeutic agent includes treatment through radiation therapy. In certain embodiments, radiation therapy is selected from the group consisting of gamma rays (γ-rays), x-rays, microwaves, proton beam irradiation, ultraviolet radiation, and targeted delivery of radioisotopes to the tumor. In certain embodiments, radiation therapy includes treatment with x-rays. In certain embodiments, X-rays are administered in daily doses of 50 to 200 roentgens over a period of three to four weeks. In certain embodiments, X-rays are applied in a single dose of 2000 to 6000 roentgens. In certain embodiments, radiation therapy includes targeted delivery of radioisotopes to a tumor. Dosage ranges for radioisotopes vary widely depending on the half-life of the isotope, the strength and type of radiation emitted, and the extent of uptake by tumor cells, but one skilled in the art will be able to determine the appropriate therapeutically effective dose.

In certain embodiments, the additional therapeutic agent includes the administration of agents to treat side effects associated with the primary treatment (eg, nausea, cachexia, etc.). In certain embodiments, the adjunctive therapy includes immunotherapy. In certain embodiments, the adjunctive therapy includes radiation therapy. In some embodiments, the radiation therapy includes gamma radiation. In certain embodiments, the additional therapy includes surgery. In certain embodiments, the adjunctive therapy includes a combination of radiation therapy and surgery. In certain embodiments, the adjunctive therapy includes treatment with a class of chemotherapeutic agents selected from the group consisting of alkylating agents, anthracyclines, cytoskeleton disrupting agents, epothilones, histone deacetylase inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, kinase inhibitors, nucleotide analogs, and nucleotide precursor analogs , peptide antibiotics, platinum-based compounds, retinoids, vinca alkaloids and their derivatives.

Additional therapies contemplated by the present invention may be administered before, after, or concurrently with the administration of the compositions described herein. In certain embodiments, additional therapy is administered prior to administration of the compositions described herein. In certain embodiments, additional therapy is administered following administration of the compositions described herein. In certain embodiments, additional therapy is administered at one or more intervals before or after administration of the compositions described herein. Determining the appropriate interval for administering additional therapy so that the individual being treated will benefit from combination therapy is within the scope of one of ordinary skill in the art.

In certain embodiments, the adjunctive therapy is immune checkpoint inhibitor therapy. The immune checkpoint inhibitor inhibits an immune checkpoint protein selected from the group consisting of programmed cell death cascade protein 1 (PD-1/CD279) and its ligands (PD-L1/CD274 and PD-L2/CD273) associated with cytotoxic T -lymphocyte antigen 4 (CTLA-4/CD152), lymphocyte activation gene 3 (LAG3/CD223), B- and T-lymphocyte attenuator (BTLA), domains (TIGIT) of T-cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif (ITIM), T cell immunoglobulin and mucin domain protein 3 (TIM-3/HAVcr2), killer cell immunoglobulin-like receptor (KIR/CD158), immunoglobulin V domain suppressor of T cell activation (VISTA), and adenosine receptor A2a (A2aR).

Immune checkpoint inhibitors may be drugs, such as small molecules, recombinant forms of ligands or receptors, or, in particular, antibodies, such as human antibodies. Known inhibitors of immune checkpoint proteins or analogs thereof can be used, in particular chimerized, humanized or human forms of antibodies. As will be known to one skilled in the art, alternative and/or equivalent names may be used for certain antibodies mentioned herein. Such alternative and/or equivalent names are interchangeable in the context of the present invention. For example, it is known that lambrolizumab is also known by the alternative and equivalent names MK-3475 and pembrolizumab.

In certain embodiments, the immune checkpoint inhibitor is a PD-1 binding antagonist. In certain embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In certain embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In certain embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g.

- 6 044080 immunoadhesin containing an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to an immunoglobulin constant region (eg, the Fc region of an immunoglobulin sequence)).

In certain embodiments, the immune checkpoint inhibitor is a CTLA-4 binding antagonist. In certain embodiments, the CTLA-4 binding antagonist is an anti-CTLA-4 antibody. In certain embodiments, the anti-CTLA-4 antibody is selected from the group consisting of ipilimumab and tremelimumab.

B. Pharmaceutical compositions.

In another aspect, the present invention provides pharmaceutical compositions and compositions containing arsenic trioxide (ATO), D-VC and a pharmaceutically acceptable carrier.

Pharmaceutical compositions and formulations as described herein can be prepared by mixing the active ingredients (ATO and D-VC) having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences ^22nd edition, 2012), in form of aqueous solutions such as normal saline (eg, 0.9%) and human serum albumin (eg, 10%). Pharmaceutically acceptable carriers are generally non-toxic in the dosages and concentrations used and include, but are not limited to: buffers such as phosphate, citrate and other organic acids; antioxidants; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethetonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg zinc-protein complexes) and/or non-ionic surfactants such as polyethylene glycol (PEG).

II. Kits and diagnostic tools.

In various aspects of the invention, a kit is provided containing diagnostic agents, therapeutic agents, and/or other therapeutic agents or delivery vehicles. In some embodiments, a kit is provided for obtaining and/or administering a therapeutic agent according to embodiments. The kit may contain reagents that can be used to administer the active or effective agent(s). For example, the kit reagents may include at least one D-VC pharmaceutical formulation and at least one ATO pharmaceutical formulation, as well as reagents for preparing, formulating and/or administering the components according to embodiments. In further aspects, the kit may contain diagnostic reagents, such as reagents for determining GLUT1 expression and/or KRAS status in a sample.

In some embodiments, the kit may also contain a suitable container, which is a container that does not react with the components of the kit, such as an Eppendorf tube, syringe, bottle or tube. The container can be made of sterilizable materials such as plastic or glass.

In addition, the kit may include instructions that describe the procedural steps of the methods and follow substantially the same procedures as those described herein or known to one of ordinary skill in the art.

III. Examples.

The following examples are included to demonstrate preferred embodiments of the invention. It will be understood by those skilled in the art that the methods described in the examples described below are methods designed by the inventor to function properly in the practice of the present invention and, thus, may be considered to constitute the preferred methods of practicing the same. However, those skilled in the art will, in view of the present invention, understand that many changes can be made to the specific embodiments that are described and the same or similar result can still be obtained without departing from the spirit and scope of the invention.

Example 1. In vitro test of D-VC treatment of malignant cells with mutant KRAS.

HCT116 colorectal cancer cells were treated for 72 h with PBS, 1 mM vitamin C (VC), 1 mM D-vitamin CVC (D-VC, also referred to as D-ascorbic acid), 5 μM arsenic trioxide (ATO) , vitamin C and ATO (VC/ATO), or D-VC and ATO (D-VC/ATO) (Fig. 1). As expected, ATO had a mild cytotoxic effect compared with buffer alone, while treatment with vitamin C and ATO induced significantly stronger cytotoxicity than treatment with buffer or vitamin C alone (Fig. 1). Interestingly, D-VC and ATO treatment induced equally robust cytotoxicity (Fig. 1). Similar synergistic cytotoxic effects of VC/ATO and D-VC/ATO were also observed in other KRAS mutant malignant cells, including HPAC and MiaPaCA2 (pancreas) and A549 (lung) malignant cells. However, neither combination induced cytotoxicity in PC3 (PTEN-null prostate cancer cells) or primary lung fibroblasts, indicating that the induction of cytotoxicity may be limited to KRAS mutant malignant cells.

To confirm these results, apoptosis was measured in a flow cytometric assay detecting FITC-conjugated annexin V to measure apoptosis (Fig. 2). Treatment with vitamin C or its D-enantiomer demonstrated levels of apoptosis similar to the buffer control (Figs. 2 and 3, comparing VC and D-VC versus PBS). As expected, treatment with 5 μM ATO induced a low increase in apoptosis relative to the buffer control, but treatment with vitamin C or the D-vitamin C analog together with ATO induced significantly more apoptosis than ATO alone (Figs. 2 and 3).

The cause of cytotoxic stress and apoptosis was expected to be accumulation of reactive oxygen species (ROS), so HCT116 cells were treated as described above and analyzed for the presence of ROS by flow cytometry (Fig. 4). Similar to the cytotoxicity and apoptosis assays, treatment with vitamin C or its D-enantiomer did not induce an increase in ROS production above baseline levels, and approximately 5% of cells were found to accumulate ROS (Figs. 4 and 5). Treatment with 5 μM ATO increased the number of cells demonstrating ROS accumulation to 26% (Figs. 4 and 5). Treatment with vitamin C or D-vitamin C in addition to ATO increased ROS production such that approximately 95% of the cells were found to accumulate ROS (Figs. 4 and 5).

Example 2 In vivo xenograft study of D-VC treatment of KRAS mutant malignancy.

VC has been shown to act through competition with glucose. To avoid fluctuations in blood glucose levels, food was removed from mice 2 h before drug injections and food was returned to mice 2 h after VC or D-VC injection.

Initially, VC or D-VC (2 g/kg) was injected with ATO (8 mg/kg) simultaneously. Injections for 2 weeks were not toxic to mice: body weight did not decrease). Tumor size showed no measurable change when determined using a thickness gauge.

To optimize drug injections, a 2-hour break (time interval) was used between ATO and VC or D-VC injections. After the second injection, all mice (group of 5 mice) in the ATO+VC injection group died, and only 2 mice died in group 5, which received ATO+D-vitamin C injections. This showed that using a 2-hour break between injections caused significant toxic effects, indicating that the drug combination acted more effectively by promoting oxidative stress in mouse tissue. This also indicates that D-VC is less toxic than VC when combined with ATO. Drug concentrations were reduced to avoid toxicity and optimized for injection in mice (VC or Dvitamin C 1.5 g/kg and ATO 7 mg/kg). The optimized drug concentrations were effective in inhibiting the growth of KRAS mutant tumors.

1.5 million human malignant tumor cells HCT116 (KRAS mutant) were injected into nude mice, 5 mice per experimental group. After 10 days and after the tumor reached a size of at least 0.8 cm, mice were injected every day with PBS, PBS+1.5 g/kg vitamin C, PBS+7 mg/kg ATO, PBS+7 mg/kg ATO+1 .5 g/kg vitamin C, PBS+1.5 g/kg D-vitamin C, or PBS+7 mg/kg ATO+1.5 g/kg D-vitamin C. Mice were injected every day, 6 times a day week, and were left alone for one day a week, with a total of 15 drug injections. Two days before the first injections, lower doses of drugs were injected at these concentrations. Initially, ATO was injected at a dose of 3 mg/kg, while vitamin C or D-VC was injected at a dose of 1 g/kg. The next day, ATO was used at a dose of 7 mg/kg, while vitamin C and D-VC were used at a dose of 1 g/kg.

After 9 injections, tumor xenografts were photographed (Fig. 6). Scarring was observed in D-VC (2/5 mice) and ATO+D-VC (5/5 mice) tumors in mice treated during the first week of injections (Fig. 6). After 12 injections, the tumors were again visualized (Fig. 7). Scarring was present in 2/5 tumors treated with ATO, 2/5 mice treated with D-VC, 1/5 mice treated with ATO plus vitamin C, and all mice treated with ATO plus D-VC (Fig. 7).

Tumors were then removed from the mice after the 15th injection. The scarring observed with ATO plus D-VC administration resulted in these tumors having a flat appearance (Figure 8). Initial scarring was observed after 6 injections. The scarring observed in the other injection groups tended to appear much later and was less frequent than with ATO plus D-VC. Tumors from each administration group were then weighed (Fig. 9). The average weight of tumors treated with ATO, vitamin C, and D-VC was lower compared to tumors treated with PBS, but these differences were not statistically significant (Fig. 9). Both tumors treated with ATO plus vitamin C and tumors treated with ATO plus D-VC had significantly less weight.

- 8 044080 than tumors treated with PBS, and tumors treated with D-VC plus ATO had significantly less weight than tumors treated with vitamin C plus ATO (Fig. 9).

Compared with PBS injection (control), all components alone (ATO, vitamin C or D-VC) showed an inhibitory effect on tumor growth, and D-VC alone was more effective, but its effect was not sustained (2 mice from group that included 5). The combination treatment was found to be more effective than the injection of these drugs alone, as indicated by a decrease in tumor weight. Importantly, the combination of D-VC plus ATO was significantly more effective than the combination of vitamin C plus ATO. Interestingly, it was also observed that administration of a combination of D-VC plus ATO resulted in scarring or burning of tumors over the course of 6 injections. The tumors in these mice became flat after 15 injections due to a scarring effect. Tumors with scarring were observed in all 5 mice injected with the combination of ATO and D-VC, while the scarring effect induced by the combination of ATO plus vitamin C was observed only after 15 injections and was present in only 3 of 5 samples.

Initial injections of drug combinations showed only a minor effect on tumor growth, so the dose was increased as shown above. Secondly, the effect was enhanced by applying a 2-hour break (time interval) between ATO and D-VC injections. The injection schedule was: ATO injection/2-hour break/vitamin C or D-VC injection. It is likely that vitamin C or D-VC inactivates the oxidizing compound ATO in vivo, since both vitamin C and D-VC are known antioxidants. However, this inactivation was observed only in tumor xenografts and not under cell culture conditions. In addition, it is also known that the effect of vitamin C administration on malignant cells with mutant KRAS is glucose dependent. To avoid fluctuations in blood glucose levels and enhance the effect of vitamin C and D-VC, drug injections were administered after food was withdrawn from mice 2 h before ATO injections, and food was reintroduced only 2 h after vitamin C or D-VC injections.

Thus, D-configuration VC (D-vitamin C or D-VC) was found to be effective in inducing oxidative stress in KRAS mutant malignant cells. To enhance the oxidative effect of D-VC on mutant KRAS cells, D-VC was administered in combination with another oxidant, arsenic trioxide (ATO). The D-VC/ATO combination was determined to be highly cytotoxic to KRAS mutant malignant cells of human or mouse origin. This study also indicates that the use of the DVC/ATO combination is more effective in inhibiting KRAS mutant tumor growth in a mouse xenograft model than the combination of L-form vitamin C (VC) and ATO. Thus, the combination of D-VC and ATO can be used to treat KRAS mutant malignancies. KRAS mutations are common in pancreatic, colorectal, and lung cancers, and there is a lack of effective KRAS oncogene intervention. The invention described herein provides a potent cytotoxic combination for the treatment of mutant malignant cells and, in particular, malignant cells with mutant KRAS. The D-VC and ATO combination approach can also be applied to any cancer type associated with high GLUT1 expression or to cancers with high glucose uptake.

All methods described and claimed herein can be carried out and carried out without undue experimentation in view of the present invention. While the compositions and methods of the present invention are described by way of preferred embodiments, those skilled in the art will appreciate that variations in the methods and steps or sequence of steps of the method described herein can be used without departing from the spirit, spirit and scope of the invention. More specifically, it will be understood that certain agents that are both chemically and physiologically related can be substituted for the agents described herein while achieving the same or similar results. All such similar substitutions and modifications apparent to those skilled in the art are intended to be within the spirit, scope and spirit of the invention as defined in the appended claims.

Links

The following references, to the extent that they provide illustrative methodological or other details additional to those described herein, are expressly incorporated herein by reference.

Dinnen et al., Mol Cancer Ther., 12(12):2792-2803, 2013.

Grad et al., Blood, 96(3):805-813, 2001.

Yedjou et al., Arch Drug Info., 2:59-65, 2009.

Yun et al., Science, 350(6266): 1391-1396, 2015.

Claims (11)

CLAIM 1. A method of treating a cancer, comprising administering to an individual in need thereof a therapeutically effective dose of arsenic trioxide (ATO) and D-vitamin C (D-VC) or pharmaceutically acceptable salts thereof, wherein the cancer contains a KRAS mutation. 2. The method of claim 1, wherein the malignant tumor exhibits increased expression of GLUT1 or high glucose uptake. 3. The method according to claim 1, where ATO and D-VC are administered in separate compositions. 4. The method according to claim 1, where ATO and D-VC are composed in one composition. 5. The method according to claim 1, wherein the malignant tumor is leukemia, colorectal cancer, pancreatic cancer or lung cancer. 6. The method of claim 5, wherein the leukemia is acute promyelocytic leukemia (APL). 7. The method according to claim 1, wherein the malignant tumor is pancreatic cancer, colorectal cancer or multiple myeloma. 8. The method according to claim 1, wherein the malignant tumor is colorectal cancer. 9. A pharmaceutical composition containing arsenic trioxide (ATO) and D-vitamin C (D-VC) or pharmaceutically acceptable salts thereof, formulated in a pharmaceutically acceptable excipient. 10. A method of identifying an individual for treatment with arsenic trioxide (ATO) and D-vitamin C (D-VC), comprising testing a sample from the individual to determine whether the individual has a cancer that has a KRAS mutation, where if the cancer has a KRAS mutation , then the individual is a candidate for treatment with ATO and DVC. 11. The method of claim 10, further comprising administering therapy via ATO and D-VC to the identified individual.