Chelating agents for cancer treatment

Abstract

The present invention provides a chelating agent for use in a method of treating a cancer in a subject, wherein the cancer is resistant to an anti-cancer therapeutic agent. Furthermore, the present invention provides a chelating agent for use in a method of sensitizing a subject to an anti-cancer therapy and / or counteracting resistance to an anti-cancer therapeutic agent wherein the method restores or reactivates tumor suppressor protein function that is preferably impaired due to aberrant tumor suppressor protein folding. The present invention also provides a pharmaceutical composition comprising 2, 3-dimercapto-1-propanesulfonic acid (DMPS) or DMSA, preferably miaDMSA, and a pharmaceutically acceptable excipient, and a pharmaceutical composition comprising a pharmaceutical composition comprising 2, 3-dimercapto-1-propanesulfonic acid (DMPS) or DMSA, preferably miaDMSA, wherein the DMPS is present in a dose of from 40 to 12000 mg.

Description

Technical Field

[0001] The present invention relates to the field of cancer treatment. More specifically, the present invention relates to the use of chelating agents to reverse the resistance of cancer to one or more anti-cancer therapeutic agents. Background Art

[0002] It is well-established that the tumor suppressor p53 is essential for both cancer prevention and tumor cell death following chemotherapy. However, the central gating function of p53 is known to be impaired by elevated levels of various metals (e.g., Cu, Pb, Cd, As, etc.) leading to loss of its protective function and increased invasiveness of aggressive tumors. Such metals are found elevated in a variety of cancer types.

[0003] Mutant p53 usually loses its tumor suppression and apoptosis induction functions, and it may acquire one or more of different function subsets (making the tumor more aggressive, more metastatic and resistant to chemotherapy), which is called function gain. The loss of function and function gain of p53 are mediated by alternative protein folding, such as misfolding or unfolding. The alternative folding can be caused by p53 gene mutation, but can also be attributed to environmental factors, such as the presence of elevated levels of certain metals, or a combination of p53 mutations and environmental factors (such as elevated levels of certain metals). In the literature, it is reported that zinc is required for normal p53 folding, while iron, copper, lead, mercury, cadmium, nickel, arsenic and vanadium are suspected to be drivers of abnormal p53 folding (Muller et al., Toxicology Research, 4:3, p.576-591 (2015)).

[0004] In cancer treatment, therapeutic agents, such as chemotherapeutic agents, are often used to stop tumor growth and / or reduce tumor size. However, tumors often have or develop resistance to the therapeutic agents, so that the patient no longer responds sufficiently to the treatment, resulting in an inability to effectively treat patients with cancer. As an example, small cell lung cancer (SCLC) and AML respond poorly to chemotherapy. AML is an example of a cancer associated with elevated levels of metals, including arsenic, copper, cadmium, nickel, and chromium, among others, as measured in serum.

[0005] There is a need in the art for methods of counteracting or mitigating resistance of cancer to anticancer therapeutic agents. In other words, there is a need for a therapeutic method that allows for the treatment of subjects with resistant cancer (i.e., cancer that has developed resistance to one or more anticancer therapeutic agents) and wherein the resistant cancer is made sensitive to treatment with the anticancer therapeutic agent by reducing or reversing its resistance to the anticancer therapeutic agent. Summary of the invention

[0006] Unexpectedly, the inventors have found that chelators can be successfully used to reverse the chemotherapeutic resistance (chemoresistance) of resistant cancers. Without wishing to be bound by theory, it is hypothesized that sensitization of this form of resistant cancer by chelation therapy (such as multimetal chelation therapy) results in functional restoration of tumor suppressors (such as p53), which subsequently allow or restore the induction of tumor cell death in response to treatment with chemotherapeutic agents (to which the cancer is resistant). The following examples and accompanying drawings herein particularly illustrate that (i) chelators can sensitize tumors with metal-induced chemotherapeutic resistance to treatment with chemotherapeutic agents to which the cancer exhibits resistance, (ii) wild-type tumor suppressor protein p53 is unfolded (unfolded) under conditions of elevated metal levels, and (iii) the restoration of chemosensitivity is more pronounced in cells expressing p53 that can refold into a wild-type p53 native conformation (e.g., wild-type p53 and mutants thereof that can refold back into a wild-type p53 native conformation).

[0007] Thus, the present invention provides, in a first aspect, a chelator for use in a method of treating cancer in a subject, wherein the cancer is resistant to an anticancer therapeutic agent. The present invention also provides a chelator for use in a method of treating cancer in a subject, wherein the chelator is used in a method of restoring the sensitivity of the cancer to an anticancer therapeutic agent. Preferably, the method provides restoration or reactivation (reactivation) of tumor suppressor protein function; more preferably, wherein, prior to administration of the chelator, the tumor suppressor protein function is impaired due to (or due to) abnormal tumor suppressor protein folding. In addition, the present invention provides a chelator for use in a method of sensitizing a subject to an anticancer treatment (e.g., sensitizing a subject to treatment with an anticancer therapeutic agent) and / or counteracting resistance to an anticancer therapeutic agent, wherein the method (effectively) restores tumor suppressor protein function that is preferably impaired due to (or due to) abnormal tumor suppressor protein folding. In other words, the present invention provides a chelator for use in a method of sensitizing a subject to an anti-cancer treatment (e.g., sensitizing a subject to treatment with an anti-cancer therapeutic agent) and / or counteracting resistance to an anti-cancer therapeutic agent, wherein the method provides for restoration or reactivation of tumor suppressor protein function; more preferably, wherein, prior to administration of the chelator, the tumor suppressor protein function is impaired due to abnormal tumor suppressor protein folding.

[0008] In a preferred embodiment of the medical (pharmaceutical) use of the present invention, the chelating agent is used in a method for counteracting the resistance of the cancer to the anticancer therapeutic agent.

[0009] In another preferred embodiment of the medical use of the present invention, the chelating agent is used in a method for counteracting the resistance of the cancer to the anticancer therapeutic agent, wherein the resistance is caused by cancer cells with impaired tumor suppressor protein function.

[0010] In another preferred embodiment of the medical use of the present invention, the chelating agent is used to restore or reactivate the function of tumor suppressor protein.

[0011] In another preferred embodiment of the medical use of the present invention, the chelating agent is administered before the anti-cancer therapeutic agent is administered.

[0012] In another preferred embodiment of the medical use of the present invention, the chelating agent is not administered simultaneously with the anti-cancer therapeutic agent (i.e., in the absence of the anti-cancer therapeutic agent). Instead, the anti-cancer therapeutic agent is preferably administered after the chelating agent has been administered (e.g., at least 10 minutes, at least 30 minutes, at least 1 hour, at least 12 hours, or at least 24 hours). This can also be referred to as "single" administration of the chelating agent, i.e., not administered simultaneously with the anti-cancer therapeutic agent, but administered in the absence of the anti-cancer therapeutic agent.

[0013] In another preferred embodiment of the medical use of the present invention, the chelator is administered prior to administration of the anti-cancer therapeutic agent, preferably wherein the chelator is administered prior to administration of the cancer therapeutic agent to (thereby) restore or reactivate tumor suppressor protein function, more preferably wherein the chelator is administered at least 10 minutes, at least 30 minutes, at least 1 hour or at least 3 hours before administration of the anti-cancer therapeutic agent to restore or reactivate tumor suppressor protein function, most preferably wherein the chelator is administered at least 10 minutes, at least 30 minutes or at least 1 hour before the first, second, third and / or each consecutive administration cycle of the cancer therapeutic agent to restore or reactivate tumor suppressor protein function.

[0014] In another preferred embodiment of the medical use of the present invention, the chelating agent is administered alone, preferably at least 10 minutes, at least 30 minutes, at least 1 hour, at least 12 hours or at least 24 hours before and / or after administration of the anticancer therapeutic agent or the combination of the anticancer therapeutic agent and the chelating agent.

[0015] In another preferred embodiment of the medical use of the present invention, the chelating agent is used for combined administration with the anti-cancer therapeutic agent.

[0016] In another preferred embodiment of the medical use of the present invention, the drug resistance is chemotherapy resistance (chemoresistance); and the anti-cancer therapeutic agent is a chemotherapeutic agent.

[0017] In another preferred embodiment of the medical use of the present invention, the cancer cells of the cancer have impaired tumor suppressor protein function.

[0018] In another preferred embodiment of the medical use of the present invention, the impaired tumor suppressor protein function is caused by abnormal tumor suppressor protein folding.

[0019] In another preferred embodiment of the medical use of the present invention, said cancer cells of said cancer comprise an abnormally folded tumor suppressor protein resulting in impaired tumor suppressor protein function.

[0020] In another preferred embodiment of the medical use of the present invention, the tumor suppressor protein is one or more tumor suppressor proteins selected from the group consisting of p53, p63 and p73.

[0021] In another preferred embodiment of the medical use of the present invention, the tumor suppressor protein is p53; and preferably the function of the tumor suppressor protein is one or more selected from the group consisting of: negative regulation of cell cycle and promotion of cell apoptosis.

[0022] In another preferred embodiment of the medical use of the present invention, the p53 is wild-type p53 or mutant p53.

[0023] In another preferred embodiment of the medical use of the present invention, the chelator is used in a method for counteracting the resistance of the cancer to the anti-cancer therapeutic agent, wherein the resistance is caused by cancer cells with impaired p53 protein function, preferably impaired wild-type p53 protein function and / or impaired mutant p53 protein function.

[0024] In another preferred embodiment of the medical use of the present invention, the chelator is used in a method for counteracting the resistance of the cancer to the anti-cancer therapeutic agent, wherein the resistance is caused by cancer cells with abnormal p53 protein folding, preferably abnormal wild-type p53 protein folding and / or abnormal mutant p53 protein folding.

[0025] In another preferred embodiment of the medical use of the present invention, the chelator is used in a method for counteracting the resistance of the cancer to the anti-cancer therapeutic agent, wherein the resistance is caused by cancer cells with abnormal p53 protein function, preferably abnormal wild-type p53 protein function and / or abnormal mutant p53 protein folding, wherein the chelator is administered alone, i.e., in the absence of the anti-cancer therapeutic agent, for example, at least 10 minutes, at least 30 minutes, at least 1 hour, at least 12 hours or at least 24 hours before and / or after the administration of the anti-cancer therapeutic agent or the combination of the anti-cancer therapeutic agent and the chelator; preferably, the chelator is administered alone, i.e., in the absence of the anti-cancer therapeutic agent, for example, at least 10 minutes, at least 30 minutes or at least 1 hour before and / or after the first, second, third or each consecutive administration cycle of the anti-cancer therapeutic agent and / or the combination of the anti-cancer therapeutic agent and the chelator. In another preferred embodiment of the medical use of the present invention, the cancer is characterized by the presence of at least two, more preferably at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or at least 23 metals, wherein the metal is selected from the group consisting of arsenic (As), aluminum (Al), antimony (Sb), barium (Ba), boron (B), cadmium (Cd), cerium (Ce), chromium (Cr), lead (Pb), mercury (Hg), neodymium (Nd), manganese (Mn), nickel (Ni), tin (Sn), titanium (Ti), uranium (U), vanadium (V), copper (Cu), iron (Fe), gold (Au), silver (Ag), palladium (Pd) and platinum (Pt).

[0026] In another preferred embodiment of the medical use of the present invention, the cancer is characterized by the presence of elevated levels (e.g., elevated levels in cancer cells) of at least two, more preferably at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or at least 23 metals selected from the group consisting of arsenic (As), aluminum (Al), antimony (Sb), barium (Ba), boron (B), cadmium (Cd), cerium (Ce), chromium (Cr), lead (Pb), mercury (Hg), neodymium (Nd), manganese (Mn), nickel (Ni), tin (Sn), titanium (Ti), uranium (U), vanadium (V), copper (Cu), iron (Fe), gold (Au), silver (Ag), palladium (Pd) and platinum (Pt). Elevated metal levels include levels that are at least 1.1 fold, more preferably at least 1.2 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold or at least 10 fold higher relative to the level of the metal in a suitable control, such as a level that may be measured in a cancer of the same cancer type that is not resistant to an anti-cancer therapeutic, and preferably wherein the tumor suppressor protein functions normally, e.g. wherein the tumor suppressor protein, such as p53, is in its native conformation (fold).

[0027] In another preferred embodiment of the medical use of the present invention, the cancer is characterized by the presence of elevated levels (e.g. elevated levels in cancer cells) of at least one, preferably at least two, more preferably at least 3, 4, 5, 6, 7, 8 or at least 9 metals selected from the group consisting of copper (Cu), iron (Fe), lead (Pb), mercury (Hg), cadmium (Cd), nickel (Ni), arsenic (As), vanadium (V) and chromium (Cr).

[0028] In another preferred embodiment of the medical use of the present invention, the cancer is characterized by the presence (e.g. elevated levels) of at least one, preferably at least two, more preferably at least 3, 4, 5 or at least 6 metals selected from the group consisting of chromium (Cr), manganese (Mn), copper (Cu), cadmium (Cd), mercury (Hg) and lead (Pb).

[0029] In another preferred embodiment of the medical use of the present invention, the aberrant tumor suppressor protein folding is induced by an elevated level of a metal as defined according to any one of the preceding embodiments.

[0030] In another preferred embodiment of the medical use of the present invention, the method for treating cancer is a method for chemosensitizing a subject's cancer chemotherapy.

[0031] In another preferred embodiment of the medical use of the present invention, the method for treating cancer is a method for enhancing the anti-cancer action (anti-cancer effect) of the anti-cancer therapeutic agent.

[0032] In another preferred embodiment of the medical use of the present invention, the enhanced anti-cancer effect is selected from the group consisting of cytotoxic effect, cytostatic effect, anti-invasiveness, anti-dissociation, anti-angiogenesis (anti-vascularization) and combinations thereof.

[0033] In another preferred embodiment of the medical use of the present invention, the resistance to the anticancer therapeutic agent is metal-induced resistance, preferably, wherein the metal-induced resistance is the result of the presence of at least two, more preferably at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or at least 23 metals, the metals being selected from arsenic (As), aluminum (Al), antimony (Sb), barium (Ba), boron (B), cadmium (Cd), cerium (Ce), chromium (Cr), lead (Pb), mercury (Hg), neodymium (Nd), manganese (Mn), nickel (Ni), tin (Sn), titanium (Ti), uranium (U), vanadium (V), copper (Cu), iron (Fe), gold (Au), silver (Ag), palladium (Pd) and platinum (Pt).

[0034] In another preferred embodiment of the medical use of the present invention, the resistance to the anti-cancer therapeutic agent is associated with, mediated by or a result of metal-induced abnormal folding of p53 protein in cancer cells.

[0035] In another preferred embodiment of the medical use of the present invention, the anticancer therapeutic agent is an anthracycline, more preferably doxorubicin; an antimetabolite, more preferably 5-fluorouracil (5-FU); and / or a taxane, more preferably Nab-paclitaxel (albumin-bound paclitaxel) and / or paclitaxel.

[0036] In another preferred embodiment of the medical use of the present invention, the chelating agent is 2,3-dimercaptosuccinic acid (DMSA), preferably monoisoamyl DMSA (miaDMSA), DMPS and / or EDTA.

[0037] In another preferred embodiment of the medical use of the present invention, the chelating agent is administered in combination with a second chelating agent.

[0038] In another preferred embodiment of the medical use of the present invention, (i) the chelating agent is 2,3-dimercapto-1-propanesulfonic acid (DMPS), and optionally wherein the second chelating agent (if present) is EDTA; or (ii) the chelating agent is 2,3-dimercaptosuccinic acid (DMSA), preferably monoisoamyl DMSA (miaDMSA), and optionally wherein the second chelating agent (if present) is EDTA.

[0039] In another preferred embodiment of the medical use of the present invention, the chelating agent and optionally the second chelating agent are provided in the form of a fixed-dose product (preferably a fixed-dose combination product), such as (i) a fixed-dose pharmaceutical composition comprising the chelating agent and optionally the second chelating agent, or (ii) a fixed-dose kit comprising a first container and a second container, wherein the first container comprises the chelating agent and the second container comprises the second chelating agent.

[0040] In another preferred embodiment of the medical use of the present invention, the chelator and optionally the second chelator are administered parenterally (preferably intravenously or intratumorally) or enterally (preferably orally or rectally). In an embodiment, the (first) chelator and the second chelator are administered via the same administration route or via different administration routes.

[0041] In another preferred embodiment of the medical use of the present invention, the anti-cancer therapeutic agent is administered parenterally, such as intravenously or intratumorally.

[0042] In another preferred embodiment of the medical use of the present invention, the chelating agent and optionally the second chelating agent are administered daily at a dose of 1-100 mg / kg body weight / day, each cycle lasting 1-25 days, and provided in repeated cycles at intervals (e.g., intervals are usually 3-6 weeks).

[0043] In another preferred embodiment of the medical use of the present invention, the cancer is a solid tumor or a liquid tumor.

[0044] In another preferred embodiment of the medical use of the present invention, the cancer is mammary gland disease (breast cancer), lung cancer such as small cell lung cancer (SCLC), pancreatic cancer, or blood cancer such as acute myeloid leukemia (AML).

[0045] In another aspect, the present invention provides a pharmaceutical composition comprising 2,3-dimercapto-1-propanesulfonic acid (DMPS) and a pharmaceutically acceptable excipient; wherein the DMPS is present in a dose of 40-12000 mg, such as 40-6000 mg, 100-5000 mg, 200-4000 mg or 400-3600 mg; preferably wherein the composition is for daily administration.

[0046] In another aspect, the present invention provides a pharmaceutical composition comprising (i) 2,3-dimercapto-1-propanesulfonic acid (DMPS) or DMSA, preferably miaDMSA, (ii) EDTA, and (iii) a pharmaceutically acceptable excipient; preferably, wherein the DMPS or the DMSA is present in a dosage of, for example, 40-6000 mg, 100-5000 mg, 200-4000 mg or 400-3600 mg.

[0047] In a preferred embodiment of the pharmaceutical composition of the present invention, the composition further comprises an anticancer therapeutic agent, preferably a chemotherapeutic agent, more preferably an anthracycline drug, and most preferably doxorubicin.

[0048] In another aspect, the present invention provides a pharmaceutical combination comprising (i) a first container comprising a pharmaceutical composition comprising 2,3-dimercapto-1-propanesulfonic acid (DMPS) or DMSA, preferably miaDMSA and a pharmaceutically acceptable excipient; and (ii) a second container comprising a pharmaceutical composition comprising an anticancer therapeutic agent, preferably a chemotherapeutic agent, more preferably an anthracycline such as doxorubicin, an antimetabolite such as 5-fluorouracil (5-FU), and / or a taxane such as Nab-paclitaxel (albumin-bound paclitaxel) and / or paclitaxel, and a pharmaceutically acceptable excipient; and optionally wherein the combination comprises a third container comprising EDTA and a pharmaceutically acceptable excipient.

[0049] The present invention also provides a method of treating cancer in a subject, wherein the cancer is resistant to an anticancer therapeutic agent, the method comprising the step of administering to the subject a therapeutically effective amount of a chelating agent. The present invention also provides a method for restoring chemotherapy sensitivity in a subject having a cancer that is (at least partially) insensitive or resistant to chemotherapy, the method comprising the step of administering to the subject a therapeutically effective amount of a chelating agent.

[0050] In a preferred embodiment, the method provides for restoration or reactivation of tumor suppressor protein function; more preferably, wherein prior to administration of the chelator, the tumor suppressor protein function is impaired due to (or as a result of) abnormal tumor suppressor protein folding.

[0051] The present invention also provides a method of sensitizing a subject to an anti-cancer treatment (e.g., sensitizing a subject to treatment with an anti-cancer therapeutic agent) and / or counteracting resistance to an anti-cancer therapeutic agent, the method comprising the step of administering to the subject a therapeutically effective amount of a chelator. In a preferred embodiment, the method (effectively) restores tumor suppressor protein function, which is preferably impaired due to (or due to) abnormal tumor suppressor protein folding. In other words, the present invention provides a method of sensitizing a subject to an anti-cancer treatment (e.g., sensitizing a subject to treatment with an anti-cancer therapeutic agent) and / or counteracting resistance to an anti-cancer therapeutic agent, wherein the method provides restoration or reactivation of tumor suppressor protein function; more preferably, wherein prior to administration of the chelator, the tumor suppressor protein function is impaired due to abnormal tumor suppressor protein folding.

[0052] In a preferred embodiment of the treatment method of the invention or the sensitizing and / or counteracting method of the invention, the chelating agent is administered in combination with the anti-cancer therapeutic agent to which the cancer is resistant or insensitive.

[0053] All embodiments and / or aspects relating to the medical use of the invention also apply in relation to the treatment method of the invention or the sensitization and / or counteracting method, which is the medical use of the invention.

[0054] The invention also provides the use of a chelating agent in the preparation of a medicament for treating cancer in a subject; wherein the cancer is resistant to an anticancer therapeutic agent. The invention also provides the use of a chelating agent in the preparation of a medicament for restoring chemotherapy sensitivity in a subject with a cancer that is (at least partially) insensitive or resistant to chemotherapy.

[0055] In a preferred embodiment of the use of the chelating agent of the present invention, the chelating agent is used for combined administration with the anti-cancer therapeutic agent.

[0056] In another aspect, the present invention provides monoisoamyl DMSA (miaDMSA) for use in a method for treating cancer in a subject. Preferably, the miaDMSA is used for administration in combination with an anticancer therapeutic as described herein, preferably a chemotherapeutic as described herein. The cancer may be a solid tumor and / or a liquid tumor. Preferably, the miaDMSA is administered in the absence of the anticancer therapeutic, for example, at least 10 minutes, at least 30 minutes, or at least 1 hour, at least 12 hours, or at least 24 hours before and / or after the first, second, third, or each continuous administration cycle of the anticancer therapeutic and / or the combination of the anticancer therapeutic and the miaDMSA.

[0057] All embodiments and / or aspects described for the medical use of the invention or the method of treatment of the invention also apply in connection with the use of the chelating agent of the invention, which is the medical use of the invention.

[0058] In a preferred embodiment, the pharmaceutical composition or pharmaceutical combination disclosed herein is used in the medical method / use of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 Effect of metals on doxorubicin toxicity in Beas-2B cells. Beas-2B lung cells were subjected to different concentrations of a mixture of metals (a mixture consisting of chromium, manganese, zinc, copper, lead, mercury, and cadmium) (Table 1). A representative chemotherapeutic agent, doxorubicin, was added as indicated. In metal-treated cells, chemotherapy-induced cell death was reduced in a dose-dependent manner, demonstrating metal-induced resistance to chemotherapy.

[0060] Figure 2 .Effects of metals on p53 folding.

[0061] A. Exposure to Figure 1 Western blot of p53 folding status in cells at the metal concentrations described in A. Protein unfolding increases as the metal dose increases, as indicated by binding to Ab240, an antibody specific for unfolded p53. B) Quantification of Ab240 binding in Western blots (arbitrary units).

[0062] Figure 3 .The effect of metal chelation on chemosensitivity.

[0063] Addition of the representative chelator DMPS (150 mM) completely reversed chemoresistance and restored full chemoresistance to the three different cell types. Addition of chelators to cells not supplemented with metals had no effect, suggesting that sensitization of the cells is related to the removal of excess metals, without an effect on metals inherent in the cells.

[0064] A. Effect of chelation on the chemosensitivity of Beas-2B pneumocytes.

[0065] B. Effect of chelation on the chemosensitivity of MCF7 breast cancer cells.

[0066] C. Effect of chelation on the chemosensitivity of A549 lung cancer cells.

[0067] D. p53 dependence of chemoresistance and chelation restoration therapy

[0068] To demonstrate the p53 dependence of metal-induced chemoresistance and its reversal by chelators, the effects of metals and DMPS (150 μM) on sensitivity to doxorubicin were tested in p53KO clones of A549. It was shown that metal-induced chemoresistance and its reversal by DMPS were less pronounced compared with native A549 cells ( Figure 3 C).

[0069] Figure 4 Effect of different chelators on restoration of chemosensitivity. Addition of chelators reversed chemoresistance differently. In Beas-2B cells, DMPS completely reversed chemoresistance, whereas EDTA (given at a dose that provided equivalent binding capacity) only partially reversed resistance. Their combination also provided complete reversal. Chelators had little effect on cells not loaded with metals.

[0070] Figure 5 .Chelating agents at non-saturating doses.

[0071] In Beas-2B cells, potentiation was observed when the chelators were given at lower, non-saturating doses, where each chelator had only a small effect on its own but potentiated each other when combined. Without being bound by theory, the combined effect of chelators with different binding profiles may indicate that chemoresistance is related to the total metal load (multimetal toxicity) rather than to a single metal (monometal toxicity).

[0072] Figure 6 IC50 of doxorubicin in cells treated with metals and chelators

[0073] As shown in the figure, Beas-2B cells incubated with or without metals were treated with EDTA, DMPS or a combination thereof and exposed to different concentrations of doxorubicin. The survival rate was measured and the IC50 (the concentration of doxorubicin that kills 50% of the cells) was calculated. Metals have a significant effect on the sensitivity of cells to doxorubicin, inducing strong resistance. Chelators have no effect on the sensitivity of native cells to doxorubicin because it largely reverses the sensitivity of metal-loaded cells.

[0074] Figure 7 . Cellular uptake of doxorubicin after exposure to metals.

[0075] Beas-2B cells with and without a 1:128 metal mix (Table 1) were incubated with different concentrations of doxorubicin for 1 hour, and the intracellular fluorescence derived from doxorubicin was determined. No differences were observed between metal-loaded and metal-free cultures.

[0076] Figure 8 .Metal loading and chemoresistance in Beas-2B cells.

[0077] Beas-2B cells preloaded with metals ("metal followed by doxorubicin (dox)" group) have strong chemotherapy resistance, while simultaneous administration ("metal + doxorubicin" group) only provides weak chemotherapy resistance. This proves that the protective effect of metals on cancer cells is not the result of the interaction between metals and drugs, but the biological effect of metal uptake on cells.

[0078] Fig. 9 .Cellular metal uptake.

[0079] MCF7 breast cancer cells (A), Hec-1a endometrial adenocarcinoma cells (B) and Beas-2B bronchial epithelial cells (C) were incubated with metals for 24 hours, washed to remove external metal residues, and collected for metal analysis by ICP-MS. Observed that the metal uptake in all cell lines was dose-dependent. The results supplemented and supported the dose-responsiveness (dose-responsiveness) chemotherapy resistance and p53 unfolding observed with the increase of metal dosage by demonstrating that the metal added can lead to higher intracellular metal content.

[0080] Fig.10 .Metals and p53 in cell lysates.

[0081] Beas-2B cell lysates were exposed for 30 minutes in the absence of metal mixtures or in the presence of metal mixtures at 1:128, 1:256, or 1:512, and then immunoprecipitated with 240 antibody to detect unfolded p53. Unfolding was detected at all metal mixture concentrations, confirming that unfolding of p53 is a direct effect of protein exposure to the metal mixture.

[0082] Fig.11 .Metal loading and p53 unfolding in H2170 cells.

[0083] H2170 cells were exposed to increasing combinations of metal mixtures for 24 hours. p53 was immunoprecipitated using p53 folding specific antibodies (240 and 1620) and the amount of unfolded and folded p53 was detected on a Western blot using DO-1 as a p53 identification antibody. When exposed to 1:256 and 1:128 metal mixtures, p53 unfolding was observed.

[0084] Fig.12 .Metal loading and p53 mutation status in Hep3B cells.

[0085] WT p53, 175 mutant p53, 157 and 158 mutant p53 were transfected in Hep3B cells. The cells were then exposed to 1:512 or 1:64 metal for 8 hours, after which folded and unfolded p53 were immunoprecipitated using 240 or 1620 antibodies. In cells carrying WT p53 and mutants 157 and 158, exposure of cells to metal increased p53 unfolding in a dose-dependent manner. Surprisingly, p53 mutants were even more sensitive to low levels of metals compared to WT. The 175 mutant, which is common in many cancers, did not respond significantly to metals because it was already present in the cells in an unfolded state without the addition of metals.

[0086] Fig.13 .Metals and p53 knockdown in cell lysates.

[0087] Beas2B were transfected with siRNA targeting p53. This resulted in knockdown (kd) of p53 expression as demonstrated by Western blot (A). Control (Contr) and kd cells were plated for viability assays, exposed to metals, and then treated with doxorubicin for 72 hours as indicated. Cell viability was monitored and plotted (B), and IC50 was calculated (C). Based on the reduced level of p53 expression, chemoresistance was attenuated in p53 knockout compared to native cells. This confirms Figure 3 D presents the finding that metal-induced chemoresistance is mediated by p53.

[0088] Fig.14 .Metal exposure and chelation in Beas-2B cells.

[0089] Beas-2B cells were pretreated with metals 1:128 and then exposed to miaDMSA and doxorubicin for 72 hours, after which cell viability was determined (A). Panel B shows the IC50. The chelator miaDMSA completely reversed the chemoresistance induced in Beas-2B cells by treatment with the metal mixture.

[0090] Fig.15 .Metal exposure and chelation in Beas-2B cells.

[0091] Beas-2B cells were pretreated with metals 1:128 and then exposed to DMSA and doxorubicin for 72 hours, after which cell viability was determined (A). Panel B shows the IC50. The chelator DMSA only partially reversed the chemoresistance induced in Beas-2B cells treated with the metal mixture.

[0092] Fig.16 .Metal exposure and chelation in H2170 cells.

[0093] H2170 cells were pretreated with metals 1:128 and then exposed to DMPS and doxorubicin for 72 hours, after which cell viability was determined (A). Panel B shows the IC50. The chelator DMPS only partially reversed the chemoresistance induced in H2170 cells treated with the metal mixture.

[0094] Fig.17 .Metal exposure and chelation in BxPC-3 cells.

[0095] BxPC3 cells were pretreated with metals 1:128 and then exposed to 150uM DMSA or miaDMSA and 5-fluorouracil (5-FU) for 72 hours, after which cell viability was determined (A). The right figure shows the viability at 10ug / ml (B). Chelating agents DMSA and miaDMSA reversed the chemotherapeutic resistance to 5-FU induced in BxPC-3 cells treated with metal mixtures.

[0096] Fig.18 .Metal exposure and chelation in BxPC-3 cells.

[0097] BxPC3 cells were pretreated with metal 1:128 and then exposed to 150uM DMSA or miaDMSA and nab-paclitaxel for 72 hours, after which cell viability was determined (A). Panel B shows viability at 60ug / ml (B). At 60nM nab-paclitaxel, reduced resistance was observed in the case of miaDMSA.

[0098] Fig.19 .Metal exposure in SW1990 cells.

[0099] SW1990 cells were pretreated with metals 1:128 and then exposed to 150uM DMSA or miaDMSA and 5-FU for 72 hours, after which cell viability was determined (A). Panel B shows viability at 10ug / ml. The SW1990 pancreatic adenocarcinoma cell line, which does not contain any p53, did not show increased resistance to chemotherapy when exposed to metals. This finding included additional anticancer agents (not shown). This observation further confirms the dependence of metal-induced chemoresistance on p53.

[0100] Fig. 20 .p53 unfolding after metal exposure and its reversal by DMPS.

[0101] Beas-2B cells were incubated with metals in the presence or absence of the chelator DMPS as indicated. The folding state of p53 was monitored using a folding antibody (A). The ratios were determined and shown in Figure B. DMPS prevented unfolding in the presence of metals.

[0102] Fig.21 .p53 unfolding after metal exposure and its reversal by DMSA.

[0103] As indicated, Beas-2B cells were incubated in metals in the presence or absence of the chelating agent DMSA. The folding state of p53 was monitored using a folding antibody (A). The ratios were determined and shown in Figure (B). DMSA prevented unfolding in the presence of metals.

[0104] Fig. 22 .WT and mutant p53 unfolding upon metal exposure and chelation by miaDMSA.

[0105] As indicated, Hep3B cells were transfected with 158 or WT p53 and incubated with metals followed by incubation with the chelator miaDMSA. The folding state of p53 was monitored using a folding antibody. In both WT and mutant 158 ​​p53, the chelator miaDMSA prevented or reduced p53 unfolding upon metal exposure.

[0106] Fig.23 . Refolding of p53 in cell lysates after exposure to chelation treatment.

[0107] Beas-2B lysates were incubated with metals for 30 minutes, followed by the addition of chelators for 1 hour to see if unfolded p53 could refold. These lysates were then immunoprecipitated with p53 refolding antibodies. EDTA and miaDMSA were clearly able to refold p53 in naive cell lysates ( Some p53 was refolded in both chelator-treated and loaded cell lysates, indicating that p53 is refolded by the chelator rather than replaced by new protein. DETAILED DESCRIPTION

[0108] definition

[0109] As used herein, the term "a" or "an" in relation to, for example, a chelator or an anti-cancer therapeutic includes reference to one or more than one chelator. Similarly, it refers to one or more than one anti-cancer therapeutic.

[0110] As used herein, the term "chelating agent" is used interchangeably with the term "chelator" and includes reference to agents that react with metal ions to form metal chelate complexes or chelates. This type of complexation generally involves the formation or presence of two or more separate coordination bonds between a multidentate ligand and a single central atom. Such ligands are also referred to as chelants, chelators, chelating agents, or sequestering agents. Those skilled in the art are familiar with a large number of available chelating agents, including but not limited to acetylacetone, alizarin, alizarin red S, amide oxime, amide oxime groups, and the like. group), aminoethylethanolamine, aminomethylphosphonic acid, aminopolycarboxylic acid, ATMP, BAPTA, Bathocuproine, BDTH2, benzotriazole, bipyridine, 2,2'-bipyridine, 2,2'-bipyrimidine, bis(dicyclohexylphosphino)ethane, 1,2-bis(dimethylarsino)benzene, 1,2-bis(dimethylphosphino)ethane, 1,4-bis(diphenylphosphino)butane, 1,2-bis(diphenylphosphino) )ethane, calixarene, molecular prison (Carcerand), catechol, cavitand (Cavitanad), citrate, citric acid, clathrochelate, corrole, 2.2.2-cryptand (2.2.2-cryptand, 2.2.2-cryptand), 1,4,8,11-tetraazacyclotetradecane (Cyclam), cyclocyclotene (Cyclen), cyclodextrin, deferasirox, deferrox Ketone, deferoxamine, Denticity, Dexrazoxane, diacetyl monoxime, trans-1,2-diaminocyclohexane, 1,2-diaminopropane, 1,5-diaza-3,7-diphosphacyclooctane, 1,4-diazacycloheptane, 1,5-diazacyclooctane, dibenzoylmethane, diethylenetriamine, diethylene glycol dimethyl ether (Diglyme), 2,3-dihydroxybenzoic acid, dimercaprol, 2,3-dimercapto- 1-Propanesulfonic acid, dimercaptosuccinic acid, 1,1-dimethylethylenediamine, 1,2-dimethylethylenediamine, dimethylglyoxime, DIOP, diphenylethylenediamine, 1,5-dithiacyclooctane, domoic acid, DOTA, DOTA-TATE, DTPMP, EDDHA, EDDS, EDTA, EDTMP, EGTA, 1,2-Ethanedithiol, Ethylenediamine, Ethylenediaminediacetic acid, Ethylenediaminetetraacetic acid, Etidronic acid, Fluo-4, Fura-2, Gallic acid, Gluconic acid, Glutamic acid, Glyoxal-bis(mesitylimine), Glyphosate, Hexafluoroacetylacetone, Homocitric acid, Iminodiacetic acid, Indole-1, Isosaccharinic acid, Kainic acid, Malic acid, Metal acetylacetonate, Metal disulfide complex, Metal crown ether, Nickel bis(stilbene dithiolate) (Bis(stilbene dithiol) nickel, Nickel bis (stilbenedithiolate), nitrilotriacetic acid, oxalic acid, oxime, pentotide, penicillamine, pentetic acid, phanehos, o-phenanthroline, o-phenylenediamine, phosphonate, phthalocyanine, phytochelatin, picolinic acid, pyridine-2-carboxylic acid, polyaspartic acid, porphine, porphyrin, 3-pyridylnicotinamide, 4-pyridylnicotinamide, pyrogallol, salicylic acid, sarcophagine, sodium citrate, sodium diethyldithiocarbamate, sodium polyaspartate, terpyridine, tetramethylethylenediamine, tetraphenylporphyrin, tetrasodium EDTA, thenoyltrifluoroacetone (Thenoyltrifluo The chelating agent may be lipophilic, such as dimercaprol (BAL), deferasirox (Exjade, TM 、Desirox TM 、Defrijet TM 、Desifer TM , Rasiroxpine TM and Jadenu TMN,N'-bis(2-mercaptoethyl)isophthalamide (also known as BDTH2, BDET and BDETH2; trade name B9 TM 、MetX TM and OSR#1 TM ), Prussian blue (Radiogardase TM ), α-lipoic acid, monoalkylated DMSA (e.g., monoisoamyl DMSA), oximes (e.g., dimethylglyoxime, salicylaldehyde oxime), diethyldithiocarbamate (DDC) and its derivatives (e.g., N-(methoxybenzyl)-D-glucosamine dithiocarbamate) and dextran; or hydrophilic, such as dimercaptosuccinic acid (DMSA), dimercaptopropionic acid (DMPA) or its derivatives, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), N-acetylcysteine ​​(NAC), malic acid, succinic acid, citric acid, tartaric acid, deferoxamine, penicillamine, nitrilotriacetate (NTA), histidine, 2,3-dimercapto-1-propanesulfonic acid (DMPS). When referring to a specific chelating agent, such as DMSA, its derivatives are included in the definition. As an example, monoisoamyl DMSA (miaDMSA) is a derivative of DMSA. The term "chelating agent" includes reference to metal complexes of the chelating agent, such as for EDTA, reference to Ca-EDTA, calcium sodium EDTA, and Zn-EDTA, and for DTPA, reference to, for example, Zn-DTPA, Ca-DTPA, and the like.

[0111] As used herein, the term "broad-spectrum chelator" includes reference to a chelator that is capable of chelating different metals, such as at least 2, 3, 4, 5, or at least 6 different metals.

[0112] Preferably, the chelating agent as disclosed herein is a broad spectrum chelating agent. Examples of broad spectrum chelating agents are EDTA, DMSA, DMPS, DTPA, BAL, etc. Preferably, the chelating agent as disclosed herein is miaDMSA.

[0113] As used herein, the term "tumor" includes reference to abnormal growth of tissue that may be benign, precancerous, malignant or metastatic. Tumors are preferably malignant, i.e. cancer. Examples of cancer include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, spleen cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia such as acute myeloid leukemia (AML), Hodgkin's lymphoma, non-Hodgkin's lymphoma, lymph node cancer, bone marrow cancer, lung cancer, gastric cancer, eye cancer, etc. As used herein, the term "liquid tumor" includes reference to a form of cancer, wherein cancer cells are mainly located in body fluids, such as blood, bone marrow and lymph. As used herein, the term "solid tumor" includes reference to a form of cancer, wherein cancer cells are mainly located in solid tissues, such as tissues of lungs, heart, brain, spleen, pancreas, liver, breast, prostate, intestines, stomach, bone, skin and cartilage.

[0114] As used herein, the terms "treatment" and "treating" include applying a form of therapy to a subject with the goal of, for example, curing the patient's disease, preventing or slowing the progression of a disease, prolonging the life of a subject, or relieving pain in a subject suffering from a disease or injury. For example, treatment may include chemotherapy, immunotherapy, radiotherapy, performing surgery, and any combination thereof. Prophylactic treatment, i.e., therapy intended to prevent the induction or onset of a disease, should also be understood to be part of the term "treatment."

[0115] As used herein, the term "subject" includes reference to a receptor for a chelator as described herein, i.e., a subject suffering from or suspected of suffering from cancer. Preferably, the subject is a mammal, more preferably a human. The terms "patient" and "subject" are used interchangeably herein. The subject is preferably a human, more preferably a human suffering from cancer. The subject may be older or younger than 50 years old, preferably older than 50 years old.

[0116] As used herein, the term "resistance" includes reference to the ability of cancer cells to at least partially tolerate one or more anticancer therapies, particularly one or more chemotherapies. In other words, resistance refers in particular to the reduced efficacy of the anticancer therapeutic agent used to treat a subject with cancer with elevated metal levels compared to the efficacy obtained with the anticancer therapeutic agent in the treatment of a subject with cancer without elevated metal levels. When the anticancer therapeutic agent is a chemotherapeutic agent, the resistance is referred to as chemotherapeutic resistance. When referring to the resistance of a cancer, it preferably refers to the resistance of the cancer cells of the cancer to the anticancer therapeutic agent. Preferably, the resistance is p53-dependent resistance or metal-induced resistance, such as metal-induced p53-dependent resistance.

[0117] As used herein, the term "metal-induced resistance" includes resistance induced by exposure to one or more metals, preferably a plurality of metals (i.e., multi-metal exposure), more preferably elevated levels of the plurality of metals, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or at least 23 metals selected from the list consisting of arsenic (As), aluminum (Al), antimony (Sb), barium (Ba), boron (B), cadmium (Cd), cerium (Ce), chromium (Cr), lead (Pb), mercury (Hg), neodymium (Nd), manganese (Mn), nickel (Ni), tin (Sn), titanium (Ti), uranium (U), vanadium (V), copper (Cu), iron (Fe), gold (Au), silver (Ag), palladium (Pd) and platinum (Pt). Ohanian et al., Am J Hematol. 2020; 95: 422-434 can be used Figure 1 The presence of elevated metal levels as disclosed herein is determined using a multi-metal scoring system disclosed in , which includes relevant metal level cutoffs. The contents of Ohanian et al., Am J Hematol. 2020; 95: 422-434 are incorporated herein by reference. Preferably, metal-induced resistance is caused by metal-induced abnormal folding of tumor suppressor proteins (such as p53), resulting in resistance to anticancer therapeutic agents (such as chemotherapeutic agents, e.g., doxorubicin). Abnormal folding may be misfolding and / or unfolding of tumor suppressor proteins (such as p53). Those skilled in the art can assess the p53 folding status in cancer cells, for example, by the p53 folding state assay described in the embodiments.

[0118] As an alternative to using the term "resistance" (such as "chemotherapeutic resistance") and counteracting resistance in the medical uses / methods of the present invention, it may instead refer to restoring sensitivity to treatment, such as restoring chemosensitivity. Thus, the phrase "counteracting (chemotherapeutic) resistance" may be used interchangeably with the phrase "restoring (chemotherapeutic) sensitivity".

[0119] As used herein, the term "restoration" relates to restoration of chemosensitivity, including at least partial restoration, such as complete restoration, of the chemosensitivity of a cancer to an anti-cancer therapeutic agent to which it was previously at least partially insensitive.

[0120] As used herein, the term "anticancer therapeutic agent" includes reference to a substance, drug, therapeutic agent and / or composition applied to a subject with cancer for the purpose of treating the subject. Anticancer therapeutic agents can be used as part of, for example, chemotherapy, immunotherapy, stem cell therapy, hormone therapy, radiotherapy and / or surgery. Most anticancer therapeutic agents have an anticancer effect on at least one type of cancer in at least one individual. Anticancer therapeutic agents as disclosed herein can be chemotherapeutics, targeted anticancer therapeutic agents (such as immunotherapeutics (e.g., immune checkpoint inhibitors), etc.).

[0121] As used herein, the term "anti-cancer effect" includes reference to the effect that an anti-cancer therapeutic has on cancer. The anti-cancer effect may, for example, be a cytotoxic effect and / or a cytostatic effect. As used herein, the term "cytotoxic effect" includes reference to an anti-cancer effect, wherein the treatment results in damage and subsequent death of cancer cells. In general, chemotherapeutic agents have a cytotoxic effect. As used herein, the term "cytostatic effect" includes reference to an anti-cancer effect, wherein the treatment results in inhibition of cell growth and / or proliferation.

[0122] As used herein, the term "sensitization" includes reference to the application of a chelator, preferably a broad-spectrum chelator, as part of an anticancer therapy to render a cancer more susceptible to the effects of an anticancer therapeutic agent, preferably an anticancer therapeutic agent to which the cancer has reversible resistance. This does not exclude the possibility that the chelator itself also has an anticancer effect. As used herein, the term "chemosensitization" includes reference to sensitization intended to render a cancer more susceptible to the effects of chemotherapy, wherein chemotherapy is used in conjunction with chelation therapy.

[0123] As used herein, the term "reversed" or "reversal" includes reference to partial or complete reversal of resistance of a cancer, preferably cancer cells of said cancer, to an anti-cancer therapeutic agent.

[0124] As used herein, the term "combination" or "combination therapy (combination therapy)" includes reference to the use of a chelator as disclosed herein and an anticancer therapeutic as disclosed herein in the same medical treatment. The chelator and anticancer therapeutic as disclosed herein can be administered together at the same time (such as in the form of a single pharmaceutical composition), administered separately from each other at the same time (for example in the form of separate pharmaceutical compositions) or administered separately from each other in time. It is explicitly contemplated that the chelator and anticancer therapeutic as disclosed herein are administered simultaneously, separately (separately) and sequentially in the same treatment regimen (treatment schedule). As an example, the time between the administration of the chelator and the anticancer therapeutic can be at least one minute, at least fifteen minutes, at least sixty minutes, at least four hours, at least one day, at least one week or at least one month or at least one year, or any time between, such as one minute and one year. Preferably, the chelator is administered before the anticancer therapeutic is administered. Alternatively, the anticancer therapeutic is administered together with the administration of the chelator, or after the administration of the chelator.

[0125] As used herein, the term "pharmaceutical combination" includes reference to, for example, a kit of parts comprising multiple containers containing different active ingredients.

[0126] As used herein, the term "therapeutically effective amount" means the amount of active ingredient administered that is sufficient to achieve the intended purpose, such as in this case, for chelators, to reverse chemotherapeutic resistance or restore chemosensitivity.

[0127] As used herein, the term "enhancing" includes reference to the use of chelators as part of an anti-cancer treatment that renders the cancer more susceptible to the anti-cancer therapeutic agent, thereby enhancing the anti-cancer effect of the anti-cancer therapeutic agent.

[0128] As used herein, the term "metal" includes reference to a chemical element that may be present in a subject's body. Preferably, the metal is present in the body in ionic form. In the context of the present invention, metalloids should also be understood to fall within the definition of "metal". Elements commonly referred to as metals or metalloids are lithium, beryllium, sodium, magnesium, aluminum, potassium, calcium, scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, gallium, rubidium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, indium, tin, cesium, barium, lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, hafnium, tantalum, tungsten, rhenium, Osmium, iridium, platinum, gold, mercury, thallium, lead, bismuth, polonium, francium, radium, actinium, thorium, protactinium, uranium, neptunium, plutonium, americium, curium, berkelium, californium, einsteinium, fermium, mendelevium, nobelium, lawrencium, rutherfordium, dubnium, seaborgium, bohrium, and hassium. Elements commonly referred to as metalloids, and therefore also referred to herein as the term "metals," include boron, silicon, germanium, arsenic, antimony, and tellurium.

[0129] As used herein, the term "tumor suppressor (protein) function" includes reference to the ability of a tumor suppressor protein to negatively regulate the cell cycle and / or promote apoptosis. Therefore, when referring to a tumor suppressor (protein) function, it preferably refers to negative regulation of the cell cycle and / or promotion of apoptosis. Preferably, the tumor suppressor protein function refers to the function of a wild-type tumor suppressor protein, rather than to an abnormal tumor suppressor protein caused by a gain of function or a loss of function mutation. Non-limiting examples of tumor suppressor proteins are retinoblastoma protein, p16, p53, FAS, NOTCH receptor, VHL, APC, MSH2, BRCA2, neurofibromin and PTCH1. Preferably, the tumor suppressor protein is p53.

[0130] The term "impaired" as used herein in relation to tumor suppressor (protein) function includes reference to a biologically relevant reduction or inactivation of tumor suppressor (protein) function, which is preferably caused by abnormal tumor suppressor protein folding caused by elevated metal levels. Preferably, the abnormal tumor suppressor protein folding is metal-induced abnormal tumor suppressor protein folding.

[0131] As used herein, the term "inactivation" includes reference to at least a partial reduction or complete elimination of a tumor suppressor function (e.g., p53 tumor suppressor function). Within a cell, there is a population of the same protein. If for the population, the tumor suppressor activity is significantly reduced or completely eliminated, it can be considered to be inactivated.

[0132] As used herein with respect to tumor suppressor protein folding, the term "abnormal" includes reference to (referring to) incorrect (e.g., non-wild-type) folding of a tumor suppressor protein. Abnormal folding is preferably the extent to which the protein cannot function as a normal (preferably wild-type) protein because it is not in a normal (preferably wild-type) conformation. Abnormal folding can, for example, be abnormal protein misfolding, abnormal protein unfolding, or abnormal protein aggregation. Preferably, the abnormal folding is caused by elevated metal levels in cancer cells. Preferably, the abnormal tumor suppressor protein folding is (elevated) metal-induced abnormal tumor suppressor protein folding.

[0133] In a preferred embodiment, the cancer has wild-type p53 or mutant p53. For example, when subjected to elevated levels of metal, mutant p53 has abnormal protein folding. For example, mutant p53 easily refolds back to the native protein conformation of wild-type p53. When the cancer cells of the cancer express mutant p53, compared with wild-type p53, the p53 comprises one or more mutations, and the one or more mutations cause abnormal protein folding when subjected to elevated levels of one or more metals, but fold normally when not subjected to elevated levels of the one or more metals. For example, mutant p53 protein is as sensitive to metals as wild-type p53 protein. For example, mutant p53 protein is more sensitive to metals than wild-type p53 protein, so that mutant p53 protein will unfold at lower metal levels than wild-type p53 protein. Since mutant p53 proteins are more sensitive to metals than wild-type p53 proteins, administration of chelators in subjects and / or cancers with mutant p53 proteins will already aid in refolding and reactivation / restoration of tumor suppressor functions, thereby reversing chemotherapeutic resistance at moderate (i.e., normal or near-normal) metal levels. The skilled person can test mutations with these functions by expressing mutant p53 proteins in vitro, subjecting them to certain metal loads, and assessing the p53 folding state (the latter, for example, as described in the Examples section). The skilled person will appreciate that cells contain multiple copies of a protein, and that the folding state of a population of proteins in a cell is not absolute in the sense that all proteins are either abnormally folded or normally folded.

[0134] As used herein, the term "unfolding" includes reference to the partial or complete elimination of the tertiary and / or quaternary structure (also referred to as its conformation) of a protein. The term "unfolding" should be considered both as unfolding as part of the protein folding process and as unfolding (partial or complete unfolding) of the normal (wild-type) conformation of the protein. For example, unfolding may occur due to the presence of elevated metal levels. The unfolded protein has a different tertiary and / or quaternary structure than the same protein in its native (wild-type) conformation. Abnormal folding may result in loss of function and / or gain of function.

[0135] As used herein, the term "misfolding" includes reference to (referring to) protein folding that results in a non-native folded state, in which the tertiary and / or quaternary structure of the protein is different from that of the native (wild-type) folded protein. Misfolding can be the result of an intermediate but incomplete step of the native folding process, or a variation of normal folding due to environmental changes. For example, this may occur due to the presence of elevated levels of one or more metals and / or due to one or more mutations in tumor suppressor genes (such as p53). Misfolding may result in loss of function and / or gain of function.

[0136] As used herein, the term "aggregation" includes reference to the accumulation that occurs by the combination of two or more unfolded and / or misfolded proteins. The term aggregation refers, for example, to pathological protein aggregation. For example, aggregation may be caused by the presence of metal levels and / or by one or more mutations (such as mutations in tumor suppressor protein genes). Aggregation may result in loss of function and / or gain of function.

[0137] As used herein, the term "chemotherapeutic agent" includes reference to an anticancer drug used as part of chemotherapy, which is a type of cancer treatment. Chemotherapeutic agents can have cytotoxic and / or cytostatic effects, and can be used to cure a subject's cancer, alleviate subject symptoms, or extend the life of a subject. Non-limiting examples of chemotherapeutic agents include cyclophosphamide, mechlorethamine, chlorambucil, melphalan, dacarbazine, nitrosoureas, temozolomide, daunomycin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, cabazitaxel, lalotaxel, ortataxel, tesetaxel, paclitaxel, docetaxel, nab-paclitaxel (Abraxane), Taxotere, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone Azathioprine, vorinostat, romidepsin, irinotecan, topotecan, etoposide, teniposide, tafluposide, bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, vismodegib, azacitidine, azathioprine, capecitabine, cytarabine, doxifluridine, fluorouracil, gemcitabine, hydroxyurea, mercaptopurine, methotrexate, thioguanine, bleomycin, actinomycin, carboplatin, cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate Tetranitrate), Phenanthriplatin, Picoplatin, Satraplatin, Retinoic acid, Alitretinoin, Bexarotene, Vinblastine, Vincristine, Vindesine, Vinflunine, Vinorelbine, Aminopterin, Pemetrexed, Pralatrexate, Raltitrexed, Pentostatin, Cladribine, Clofarabine, Fludarabine, Nilarabine, Carmofur, Floxuridine, Tegafur, Cytarabine, Gemcitabine, Decitabine, Hydroxycarbamide, Belotecan, Camptothecin, Cositecan, Etirinotecanpegol), Exatecan, Gimatecan, Lurtotecan, Rubitecan, β-hydroxylactone silatecan (Silatecan), Aclarubicin, Amrubicin, Pirarubicin, Valrubicin, Zoorubicin, Mitoxantrone, Loxoxantrone, Pixantrone, Bendamustine, Nitrogen mustard, Ifosfamide, Trofosfamide, Prednimustine, Uramustine, Carmustine, Fotemustine, Lomustine Methylcyclohexyl Nitrosourea (Lomustine Semustine), Nimustine, Ranimustine, Streptozotocin, Mannosufan, Trioxosulfan, Carboquinone, Thiotepa, Triaziquone, Triethylenemelamine, Altretamine, Procarbazine, Mitobronitol, Pipobroman, Dacarbazine, Temozolomide, Dactinomycin, Bleomycin, Mitomycin, Plucamicin, Aminopyruvic Acid, Efaproxiral, Methylaminolevulinate, Paliporfin, Porfimer Sodium, Talaporfin, Temoporfin, Verteporfin, Tipirfa Abemaciclib, Alvocidib, Palbociclib, Ribociclib, Seliciclib, Bortezomib, Carfilzomib, Oprozomib, Ixazomib, Anagrelide, Tiazofurin, Masoprofen, Niraparib, Olaparib, Rucaparib, Belinostat, Entinostat, Panobinostat, Romidip, Vorinostat, Pi3K, Apellisib, Copanlisib, Duvelisib, Idelalisib, Umbralisib, Atrasentan, Bexarotene, Testolactone, Amsacrine, ArsenicThe combination of chemotherapeutic agents can be used to treat cancer. The treating doctor can establish or temporarily combine. The established combination is called a regimen, which includes dosage and administration interval in some cases.

[0138] The chemotherapeutic agent as disclosed herein can be (i) an alkylating agent (such as altretamine, bendamustine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, mechlorethamine, melphalan, oxaliplatin, temozolomide, thiotepa or trastuzumab), (ii) a nitrosourea (such as carmustine, lomustine or streptozotocin) , (iii) antimetabolites (azacitidine, 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine (Xeloda), cladribine, clofarabine, cytarabine (Ara-C), decitabine, floxuridine, fludarabine, gemcitabine (Gemzar), hydroxyurea, methotrexate, nilarabine, pemetrexed (Aninda), pentostatin, pralatrexate, thioguanine, or trifluridine / tipiracil (t (iv) anthracyclines (such as daunomycin, doxorubicin (adriamycin), liposomal doxorubicin, epirubicin, idarubicin, or valrubicin), (v) topoisomerase I or II inhibitors (such as irinotecan, liposomal irinotecan, topotecan, etoposide (VP-16), mitoxantrone, or teniposide), (vi) taxanes (cabazitaxel, docetaxel, Nab-purple or paclitaxel), (vii) a vinca alkaloid (such as vinblastine, vincristine, liposomal vincristine, or vinorelbine), (viii) a corticosteroid (such as prednisone, methylprednisone, or dexamethasone), or (ix) all-trans retinoic acid, arsenic (arsenic trioxide), asparaginase, eribulin, hydroxyurea, ixabepilone, mitotane, omacetin, pegaspargase, procarbazine, romidep, or vorinostat.

[0139] As used herein, the term "anthracycline" includes reference to members of the anthracycline class, which is a group of chemotherapeutic agents including doxorubicin, daunomycin, epirubicin and idarubicin or derivatives thereof. Those chemotherapeutic agents are naturally produced by the bacterium Streptomyces peucetius. As used herein, the term "doxorubicin" includes reference to chemotherapeutic agents belonging to the anthracycline class. It is sold under trade names including adriamycin, Doxil, Caelyx and Myocet. It is used to treat cancers including breast cancer, bladder cancer, Kaposi's sarcoma, lymphoma and acute lymphoblastic leukemia. Doxorubicin is part of a variety of chemotherapy regimens including AC, TAC, ABVD and FAC.

[0140] As used herein, the term "administer" includes reference to applying a substance or composition to a subject. The main route of administration is parenteral administration, enteral or enteral administration and topical administration. As used herein, the term "parenteral" includes administration without being applied to the skin or via any form of the gastrointestinal tract. The non-limiting example of parenteral administration includes administration via epidural, intracerebral, intraventricular, epidermal, sublingual, extra-amniotic, nasal, intraarterial, intraarticular, intracardiac, intracavernous, intradermal, intralesional, intramuscular, intraocular, intraosseous, intraperitoneal, intracapsular (intrathecal), intrauterine, intravaginal, intravenous, intravesical, intravitreal, subcutaneous, transdermal, perivascular, transmucosal, rectal or intratumoral administration. As used herein, the term "intravenous" includes reference to parenteral administration, wherein, for example, a hollow needle is used to inject a substance or composition into the vein of a subject. The substance or composition injected intravenously will directly reach the blood flow of the subject. As used herein, the term "intratumoral" includes reference to administering a substance or composition directly into a tumor, for example using a hollow needle. The tumor to which intratumoral administration is performed may be treated prior to administration, for example to improve visibility of the tumor. Intratumoral administration may, for example, be used for administration of anticancer therapeutic agents.

[0141] Chelating agents

[0142] The present invention relates to a chelating agent for use in a method of treating cancer in a subject, wherein the cancer is resistant to an anticancer therapeutic agent. Preferably, the chelating agent is used in combination with an anticancer therapeutic agent as disclosed herein. For example, the chelating agent is used in combination with an anticancer therapeutic agent to which the cancer is resistant and / or with a different anticancer therapeutic agent. It has been found that the use of a chelating agent in a subject that is resistant to one or more anticancer agents is beneficial from a therapeutic perspective.

[0143] Without being bound by theory, chelators bind metals in the body. Metals may induce inappropriate protein folding. Once the metal is chelated, the protein is less likely to fold improperly, thereby restoring the function of the protein in the body. One protein that typically misfolds in the presence of metals, thereby losing its wild-type tumor suppressor function and imparting chemotherapy resistance is p53. The inventors have demonstrated that chelation results in restoration of wild-type p53 function and leads to amelioration of chemotherapy resistance.

[0144] Preferably, the chelating agent binds metals such as lead, chromium, arsenic, mercury, cadmium, aluminum, antimony, barium, bismuth, copper, gold, iron, lithium, manganese, nickel, vanadium, platinum, silver, thallium, tin and / or titanium.

[0145] In some embodiments, the chelating agent is selected from the group consisting of acetylacetone, alizarin, alizarin red S, amide oxime, amide oxime group, aminoethylethanolamine, aminomethylphosphonic acid, aminopolycarboxylic acid, ATMP, BAPTA, bathocuproin, BDTH2, benzotriazole, bipyridine, 2,2'-bipyridine, 2,2'-bipyrimidine, bis(dicyclohexylphosphino)ethane, 1,2-bis(dimethylarsino)benzene, 1,2-bis(dimethylphosphino)ethane, 1,4-bis(diphenylphosphino)butane, 1,2-bis(diphenylphosphino)ethane, calixarene, molecular prison, catechol, hole ligand, citrate, citric acid, caged chelate, corrole, 2.2.2-cryptand, cyclolamin, cyclocyclotene, cyclodextrin, deferasirox, deferiprone, deferoxamine, dentate, dexrazoxane, diacetyl oxime, trans-1,2-diaminocyclohexane, 1,2-diaminopropane, 1,5-diaza-3,7-diphosphacyclooctane, 1,4-diazacycloheptane, 1,5-diazacyclooctane, dibenzoylmethane, diethylenetriamine, diglyme, 2,3-dihydroxybenzoic acid, dimercaptopropanol (BAL), 2,3-dimercapto-1-propanesulfonic acid (DMPS), dimercaptosuccinic acid (DMSA), miaDMSA, 1,1-dimethylethylenediamine, 1,2-dimethylethylenediamine, dimethylglyoxime, DIOP, di Phenylethylenediamine, 1,5-dithiooctane, domoic acid, DOTA, DOTA-TATE, DTPMP, EDDHA, EDDS, ethylenediaminetetraacetic acid (EDTA), EDTMP, EGTA, 1,2-ethanedithiol, ethylenediamine, ethylenediaminediacetic acid, ethylenediaminetetraacetic acid, etidronic acid, Fluo-4, Fura-2, gallic acid, gluconic acid, glutamic acid, glyoxal-bis(mesityl imide), glyphosate, hexafluoroacetylacetone, homocitric acid, iminodiacetic acid, indole-1 (Indo-1), isosaccharinic acid, kainite, malic acid, metal acetylacetonates, metal dithioene complexes, metal crown ethers, nickel bis(symmetrical di styryl dithiolate), nitrilotriacetic acid, oxalic acid, oxime, pentotide, penicillamine, pentetic acid, Phanehos, o-phenanthroline, o-phenylenediamine, phosphonates, phthalocyanine, phytochelatin, picolinic acid, polyaspartic acid, porphine, porphyrin, 3-pyridylnicotinamide, 4-pyridylnicotinamide, pyrogallol, salicylic acid, Sarcophagine, sodium citrate, sodium diethyldithiocarbamate, sodium polyaspartate, terpyridine, tetramethylethylenediamine, tetraphenylporphyrin, tetrasodium EDTA, thenoyltrifluoroacetone, thioglycolic acid, TPEN, 1,4,7-triazacyclononane, tributyl phosphate, tridentate, triethylenetetramine, 1,1,1-tri Fluoroacetylacetone, 1,4,7-trimethyl-1,4,7-triazacyclononane, bis(2-diphenylphosphinoethyl)phenylphosphine (Triphos), trisodium citrate, 1,4,7-trithiacyclononane, TTFA, N,N'-bis(2-mercaptoethyl)isophthalamide (also known as BDTH2, BDET and BDETH2), Prussian blue, alpha-lipoic acid, monoalkylated DMSA, diethyldithiocarbamate (DDC), dimercaptopropionic acid (DMPA) or its derivatives, diethylenetriaminepentaacetic acid (DTPA), N-acetylcysteine ​​(NAC), succinic acid, tartaric acid, nitrilotriacetate (NTA) and histidine, or a combination thereof.

[0146] In the medical use of the present invention, at least two, at least three, at least four or at least five chelating agents selected, for example, partially or completely from the chelating agents listed above can be used. Preferably, at least one chelating agent is selected from the group consisting of DMPS, EDTA, DTPA, dimercaprol, DMSA and derivatives thereof (such as miaDMSA), or a combination thereof. More preferably, the at least one chelating agent is a combination of DMPS and EDTA; a combination of DMPS and DTPA; a combination of DMPS and dimercaprol; a combination of DMPS and DMSA, preferably a combination of miaDMSA; a combination of EDTA and DTPA; a combination of EDTA and dimercaprol; a combination of EDTA and DMSA, preferably a combination of miaDMSA; a combination of DTPA and dimercaprol; a combination of DTPA and DMSA, preferably a combination of miaDMSA; a combination of dimercaprol and DMSA, preferably a combination of miaDMSA. Even more preferably, one or more chelating agents are selected from the group consisting of DMSA, DMPS, EDTA and derivatives thereof (such as miaDMSA). In a preferred embodiment, the chelating agent is DMPS; a combination of at least DMPS and EDTA; or a combination of at least DMSA, preferably miaDMSA, and EDTA.

[0147] In the medical use of the present invention, the chelating agent can be used, for example, in a dosage range of 1-100 mg / kg, preferably 2-50 mg / kg.

[0148] Preferably, the chelating agent as disclosed herein has binding properties allowing chelation of at least one metal selected from the group comprising vanadium, chromium, iron, copper, lead, arsenic, mercury and cadmium. More preferably, the chelating agent is capable of chelating at least one metal selected from the group comprising iron, copper, lead, mercury, cadmium and vanadium. Even more preferably, the chelating agent is capable of chelating a combination of metals selected from the group comprising iron, copper, lead, mercury and cadmium.

[0149] Chelators as disclosed herein can be used in methods for sensitizing a subject's cancer to an anticancer therapeutic agent. Preferably, the sensitization comprises sensitizing a subject's cancer to an anticancer therapeutic agent, wherein the cancer is resistant to the anticancer therapeutic agent. More preferably, the sensitization comprises chemosensitizing a subject's cancer to a chemotherapeutic agent (chemosensitization), wherein the cancer is resistant to the chemotherapeutic agent. Even more preferably, the chemosensitization comprises sensitizing a subject's cancer to a chemotherapeutic agent, wherein the cancer is p53-dependently resistant to the chemotherapeutic agent. More preferably, the p53-dependent resistance to the one or more chemotherapeutic agents is caused by abnormal p53 folding.

[0150] The chelating agents as disclosed herein can be used in a method of enhancing an anticancer therapeutic as described herein. Preferably, the anticancer effect of the anticancer therapeutic is enhanced. Thus, the anticancer effect of the anticancer therapeutic is enhanced. Preferably, the enhancement of the anticancer effect is greater than 10% compared to treatment with the anticancer therapeutic alone; more preferably greater than 25%; even more preferably greater than 50%; yet more preferably greater than 100%; most preferably greater than 200%.

[0151] In the medical use of the present invention, the chelating agent as disclosed herein can be co-administered to the subject with another agent (such as a reducing agent). Examples of reducing agents are antioxidants and vitamins. Alternatively, the chelating agent can be co-administered with essential minerals. The other agent is preferably co-administered with a chelating agent as disclosed herein and / or an anticancer therapeutic agent as disclosed herein. Suitable antioxidant vitamins include ascorbic acid (vitamin C) and alpha-tocopherol (vitamin E). Other antioxidant compounds include glutathione, lipoic acid, uric acid, carotenoids (e.g., beta-carotene, lycopene), flavonoids (e.g., quercetin), retinol, panthenol (coenzyme Q), taurine, N-acetylcysteine ​​(NAC) and amifostine. Essential minerals are those minerals necessary for the normal functioning of the body. They are sometimes classified as i) macrominerals (macrominerals), such as chloride, calcium, sodium, phosphorus, potassium, magnesium and sulfur; and trace minerals (trace minerals), such as zinc, selenium, magnesium, calcium and rubidium; a subset is zinc, selenium, magnesium and calcium. One or more of these antioxidants and / or one or more of these essential minerals can be administered in combination with the chelating agent.

[0152] In the medical use of the present invention, at least one, more preferably two, more preferably three, and even more preferably all of the following vitamins and minerals are administered to the subject: zinc, selenium, magnesium, and / or vitamin C; preferably, these compounds are administered to the subject in combination with the chelating agent and / or the anticancer therapeutic agent as disclosed herein. Examples of dosage ranges and forms of zinc, selenium, magnesium, and vitamin C that can be administered to a subject or patient may include, but are not limited to, the following: zinc (e.g., 5-75 mg of elemental zinc, zinc sulfate, zinc citrate, or zinc glycerate, such as 50 mg), vitamin C (e.g., 1000 mg to 50 g, such as orally or intravenously), magnesium citrate (e.g., 100 mg to 3 g, such as orally or intravenously, such as 3 g magnesium sulfate IV) and selenium (e.g., L-selenomethionine or equivalent, such as 100-200 mcg orally per day). In some embodiments, one or more or all of the following vitamins and minerals are administered to the subject: magnesium, selenium, rubidium, zinc, and / or vitamin C.

[0153] EDTA can be used as a chelating agent. The EDTA can also be provided in the form of calcium-EDTA (Ca-EDTA), zinc-EDTA (Zn-EDTA), sodium-EDTA (Na-EDTA), potassium EDTA or sodium calcium EDTA. In an embodiment, the EDTA is a dosage (e.g., a single unit dose) of 40-12000 mg. In some embodiments, DMPS and / or DMSA, preferably miaDMSA, is used as a chelating agent. DMSA can be, for example, in the form of Zn-DMSA. DMPS can be, for example, in the form of Zn-DMPS. As a non-limiting example, DMPS and / or DMSA, preferably miaDMSA, can be administered at a concentration of 10-30 mg / kg / day.

[0154] Preferably, the DMPS and / or DMSA, preferably miaDMSA, is in a dose (eg a single unit dose) of 40-12000 mg, such as 40-6000 mg, 100-5000 mg, 200-4000 mg or 400-3600 mg.

[0155] cancer

[0156] The chelators as disclosed herein are used to treat cancer. The cancer as described herein may be any cancer. Preferably, the cancer is a cancer in which the p53 tumor suppressor function is impaired, such as at least partially or completely inactivated. More preferably, the inactivation of the tumor suppressor function of p53 is not mediated solely by one or more mutations in the p53 gene. For example, the inactivation of the tumor suppressor function of p53 may be caused by one or more mutations in the p53 gene in combination with exposure to one or more metals, while in the absence of the one or more metals but in the presence of the one or more mutations, there is no impaired p53 function. More preferably, the inactivation of the tumor suppressor function of p53 is mediated by one or more mutations in the p53 gene at normal or near normal metal levels, wherein the tumor suppressor function can be restored by administering a chelator.

[0157] In the medical application of the present invention, the cancer may be selected from the group consisting of non-small cell lung cancer, renal cancer, renal cell carcinoma, clear cell renal cell carcinoma, lymphoma, blastoma, sarcoma, undifferentiated carcinoma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, bile duct cancer, pheochromocytoma, islet cell carcinoma, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma, neuroendocrine tumor, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, skin cancer, giant and spindle cell carcinoma carcinoma; small cell carcinoma; small cell lung cancer; papillary carcinoma; oral cancer; oropharyngeal cancer; nasopharyngeal cancer; respiratory tract cancer; genitourinary cancer; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrointestinal cancer; malignant gastrinoma; hepatobiliary carcinoma (cholangiocarcinoma); hepatocellular carcinoma; combined hepatocellular carcinoma and hepatobiliary carcinoma (combined hepatocellular carcinoma and cholangiocarcinoma); trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; malignant carcinoid tumor; branchioalveolar adenocarcinoma adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; oncocytic carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous gland carcinoma; ceraminous gland carcinoma;mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor (malignant); malignant thecoma (malignant); malignant granulosa cell tumor; malignant androblastoma (malignant testicular blastoma); Sertoli cell carcinoma; malignant Leydig cell tumor (malignant testicular stromal tumor) tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant nevus; lentigo maligna melanoma; acrrallentiginous melanoma; nodular melanoma; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromalsarcoma; mixed tumor, malignant; Mullerian mixed tumor mixed tumor); Wilms' tumor; hepatoblastoma; carcinosarcoma; malignant mesenchymoma (malignant); malignant Brenner tumor (malignant); malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant struma ovarii (malignant); choriocarcinoma; malignant mesonephroma; angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma;Juxtacortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; malignant odontogenic tumor; ameloblastic odontosarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; endocrine or neuroendocrine cancer or hematopoietic cancer); malignant pinealoma; chordoma; cancer of tissue of the central or peripheral nervous system; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant neurilemmoma (malignant); malignant granular cell tumor; B-cell lymphoma; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; low-grade / follicular non-Hodgkin's lymphoma; paragranuloma; small lymphocytic malignant lymphoma (malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; follicular malignant lymphoma; mycosis fungoides fungoides); mantle cell lymphoma; Waldenstrom's macroglobulinemia; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative intestinal disease; leukemia; preferably, the leukemia is a lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; acute myeloid leukemia (AML), chronic myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryocyte leukemia; myeloid sarcoma; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia;or acute lymphoblastic leukemia (ALL). In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a liquid tumor. In preferred embodiments, the cancer is acute myeloid leukemia (AML); pancreatic cancer; lung cancer, such as small cell lung cancer (SCLC); gastrointestinal cancer or breast cancer. More preferably, the cancer is AML, lung cancer (such as SCLC) or pancreatic cancer. ;

[0158] Anticancer therapeutic agents

[0159] The present invention relates to a chelating agent for use in a method of treating cancer in a subject, wherein the cancer is resistant to an anti-cancer therapeutic agent. The anti-cancer therapeutic agent may be any anti-cancer therapeutic agent.

[0160] The anti-cancer therapeutic agent as described herein can be an anti-cancer therapeutic agent used in a treatment type selected from chemotherapy, targeted therapy (such as immunotherapy), stem cell therapy, hormone therapy, radiotherapy and surgery, or a combination thereof; preferably, the treatment type is chemotherapy. The anticancer therapeutic agent can be selected from the group consisting of cyclophosphamide, dichloromethyl diethylamine, chlorambucil, melphalan, dacarbazine, nitrosourea, temozolomide, daunomycin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, cabazitaxel, lalotaxel, ortataxel, tesetaxel, paclitaxel, docetaxel, Abraxane, taxotere, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, vorinostat, romidep, irinotecan, topotecan, etoposide, teniposide, tafoside, bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, vismodegib, azacitidine, azathioprine, capecitabine, cytarabine, doxifluridine, Fluorouracil, gemcitabine, hydroxyurea, mercaptopurine, methotrexate, thioguanine, bleomycin, actinomycin, carboplatin, cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenoplatin, picoplatin, satraplatin, tretinoin, alitretinoin, bexarotene, vinblastine, vincristine, vindesine, vinflunine, vinorelbine, aminopterin, pemetrexed, pralatrexate, raltitrexed, pentostatin, cladribine, clofarabine, fludarabine, nilarabine, carmofur, floxuridine, cytarabine, gemcitabine, decitabine, hydroxyurea, belotecan, camptothecin, Cositecan, pegol-enotecan, exitecan, gimatecan, lortotecan, rubitecan, beta-hydroxylactone silanecamptothecin (slatitecan,Silatecan), aclarubicin, amrubicin, pirarubicin, valrubicin, daunorubicin, mitoxantrone, losoxantrone, pixenol, bendamustine, mechlorethamine, ifosfamide, trofosfamide, prednimustine, uracil mustard, carmustine, fotemustine, lomustine methyl cyclohexane nitrosourea, nimustine, ranoside mustard, streptozotocin, mannosulfamethoxazole, trooxsulfamethoxazole, carboquinone, thiotepa, trimethoprim-sulfamethoxazole, carboquinone, hexamethylmelamine, methylbenzalazine, dibromomannitol, guanidine, dacarbazine, temozolomide, dactinomycin, bleomycin, mitomycin, plicamycin, aminopyruvic acid, efaproxiral, methylaminolevulinate, padeliporfin, porfimer sodium sodium), Talaporfin, Temoporfin, Verteporfin, Tipifarnib, Abemaciclib, Alvocidib, Palbociclib, Ribociclib, Seliciclib, Bortezomib, Carfilzomib, Oprozomib, Ixazomib, Anagrelide, Thiazolam The group of drugs included tiazofurin, masoprocol, niraparib, olaparib, rucaparib, belinostat, entinostat, panobinostat, romidep, vorinostat, Pi3K, apellis, cupanisib, duvelice, idelalisib, erblisib, atrasentan, bexarotene, testolactone, amsacrine, arsenic, asparaginase, pegaspargase, beztifan, celecoxib, demeclocycline, ilisimol, elsamitrucin, eribulin, estramustine phosphate, ethidium, lonidamine, methionone, mitoguanazone, mitotane, oblimersen sodium, homoharringtonine, trastuzumab, alitretinoin, bexarotene, tretinoin, veliparib and venetoclax. In some embodiments, a combination of the above listed therapeutic agents is selected as an anti-cancer therapeutic agent as described herein. Preferably, the combination is a known chemotherapy regimen,Such as CMF (cyclophosphamide, methotrexate, 5-fluorouracil, vinorelbine), AC (doxorubicin, cyclophosphamide), DA (cytarabine, anthracycline antibiotics, daunomycin), IA (cytarabine, anthracycline antibiotics, idarubicin), DAT (daunomycin, cytarabine, thioguanine), FLAMSA (fludarabine, cytarabine, amsacrine), FLAMSA-BU (fludarabine, cytarabine, amsacrine) , busulfan), FLAMSA-MEL (fludarabine, adenosine, amsacrine, melphalan), TAD (thioguanine, cytarabine, daunomycin), CAF (cyclophosphamide, doxorubicin, fluorouracil), CLIA (cladribine, idarubicin and cytarabine), CLIA-M (milotibine, cladribine, idarubicin and cytarabine) and ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine). Preferably, anthracyclines (such as doxorubicin), antimetabolites (such as 5-fluorouracil (5-FU)) and / or taxanes (such as Nab-paclitaxel and / or paclitaxel) are selected as anticancer therapeutic agents to which cancers as described herein are resistant. ,

[0161] Anti-cancer therapeutics as disclosed herein can be administered by any acceptable mode of delivery, such as by liposome delivery.

[0162] Application

[0163] The present invention relates to a chelator for use in a method of treating cancer in a subject, wherein the cancer is resistant to an anticancer therapeutic agent. Preferably, the method of treating cancer in a subject further comprises co-administering an anticancer therapeutic agent as described herein. Preferably, the administration of the anticancer therapeutic agent is carried out parenterally. The parenteral administration may be epidural, intracerebral, intraventricular, epidermal, sublingual, extra-amniotic, nasal, intraarterial, intraarticular, intracardiac, intracavernous, intradermal, intralesional, intramuscular, intraocular, intraosseous, intraperitoneal, intracapsular, intrauterine, intravaginal, intravenous, intravesical, intravitreal, subcutaneous, transdermal, perivascular, transmucosal or intratumoral; more preferably, the administration is intravenous or intratumoral administration.

[0164] Administration of the chelating agents disclosed herein is carried out parenterally or enterally. The parenteral administration may be epidural, intracerebral, intraventricular, epidermal, sublingual, extra-amniotic, nasal, intraarterial, intraarticular, intracardiac, intracavernous, intradermal, intralesional, intramuscular, intraocular, intraosseous, intraperitoneal, intracapsular, intrauterine, intravaginal, intravenous, intravesical, intravitreal, subcutaneous, transdermal, perivascular, transmucosal, rectal or intratumoral; more preferably, the administration is intravenous or intratumoral, or the administration is oral or rectal.

[0165] A second and optionally additional chelating agent may be used in the medical use of the present invention. Preferably, the second chelating agent is administered together with the first chelating agent, for example, administered together at the same time (for example, in the form of a single pharmaceutical composition), administered separately at the same time as each other (for example, in the form of separate pharmaceutical compositions), or administered separately at the same time as each other. It is expressly contemplated that the chelating agents disclosed herein are administered simultaneously, separately, and sequentially in the same treatment regimen. The second chelating agent may be administered, for example, parenterally or enterally. More preferably, the parenteral administration is by epidural, intracerebral, intraventricular, epidermal, sublingual, extra-amniotic, nasal, intraarterial, intraarticular, intracardiac, intracavernous sinus, intradermal, intralesional, intramuscular, intraocular, intraosseous, intraperitoneal, intracapsular, intrauterine, intravaginal, intravenous, intravesical, intravitreal, subcutaneous, transdermal, perivascular, transmucosal, rectal, or intratumoral administration; yet more preferably, the administration is by intravenous or intratumoral administration, or by oral or rectal administration.

[0166] In some embodiments, a third, fourth, fifth or additional chelating agent is used in the medical use of the present invention. Preferably, the third, fourth, fifth or additional chelating agent is administered together with the first and second chelating agents as described above.

[0167] In some embodiments, the chelator and the optional second, third, fourth, fifth and / or additional chelators are administered together with one or more anticancer therapeutic agents as described herein. In some embodiments, one or more chelators are administered alone, i.e., administered in the absence of an anticancer therapeutic agent. For example, one or more chelators are administered alone, and then an anticancer therapeutic agent is administered, or an anticancer therapeutic agent and one or more chelators are administered in combination. For example, one or more chelators are administered alone, i.e., administered at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 8 hours, at least 12 hours, at least 24 hours, at least 48 hours, or at least 72 hours before and / or after administration of an anticancer therapeutic agent and / or a combination of an anticancer therapeutic agent and the chelator in the absence of an anticancer therapeutic agent. For example, one or more chelating agents are administered alone 10 minutes to 30 minutes, 10 minutes to 72 hours, 30 minutes to 48 hours, 1 hour to 24 hours, 2 hours to 12 hours, or 1 hour to 3 hours before and / or after the administration of the anticancer therapeutic agent and / or the combination of the anticancer therapeutic agent and the chelating agent. For example, the chelating agent is administered at least 10 minutes, 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 8 hours, at least 12 hours, or at least 24 hours before and / or after the first, second, third, and / or each continuous administration cycle of the anticancer therapeutic agent or the combination of the anticancer therapeutic agent and the one or more chelating agents. For example, one or more chelating agents are administered at least 10 minutes, 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 8 hours, at least 12 hours, or at least 24 hours before and / or after each administration cycle of the anticancer therapeutic agent or the combination of the anticancer therapeutic agent and the one or more chelating agents.

[0168] For example, administration of one or more chelating agents in the absence of an anti-cancer therapeutic (administered alone) is repeated after administration of an anti-cancer therapeutic and / or after administration of one or more chelating agents in combination with an anti-cancer therapeutic.

[0169] Preferably, the same route of administration is selected for the chelating agent and the optional second, third, fourth, fifth and / or additional chelating agents, and one or more anticancer therapeutic agents as described herein. However, the chelating agent and the anticancer therapeutic agent may also be administered by different routes of administration. As an example, the anticancer therapeutic agent may be administered parenterally and the chelating agent may be administered orally. In addition, as an example, the (first) chelating agent may be administered orally and the second or additional chelating agent may be administered parenterally.

[0170] As disclosed herein, chelating agents are preferably in compositions that also include pharmaceutically acceptable excipients (or carriers). As used herein, as those of ordinary skill in the art should know, pharmaceutically acceptable excipients or carriers include materials and combinations thereof such as any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delay agents, salts, preservatives, medicines, drug stabilizers, gels, adhesives, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, etc. Except that any conventional carrier is incompatible with active ingredient to a certain extent, otherwise its use in pharmaceutical compositions is predictable. Acceptable excipients, carriers or diluents for therapeutic use are well known in the pharmaceutical field, for example, described in standard textbook references such as Remington's Pharmaceutical Sciences (such as Mack Publishing Co., A. R. Gennaro edit., 1985).

[0171] In the medical use of the present invention, the chelating agent can be administered to the subject at least once, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, at least twelve times, at least fourteen times, at least sixteen times, at least eighteen times, at least twenty times, at least twenty-five times, at least thirty times, at least thirty-five times, at least forty times, at least fifty times, at least sixty times, at least seventy times, at least eighty times, at least ninety times, or at least one hundred times.

[0172] Preferably, the chelating agents as disclosed herein are employed in a treatment regimen involving daily, weekly or monthly administration of said chelating agents. Preferably, the treatment is maintained for at least 3 days, at least a week, at least a month, more preferably at least 6 months or at least 1 year, such as 2-5 years.

[0173] Preferably, the chelating agents and / or anticancer therapeutic agents as disclosed herein are used in a treatment regimen that involves administration in cycles, wherein each cycle includes repeated administration, such as administration of the chelating agent every day for multiple days, such as at least one day, at least three days, at least one week, at least two weeks, or at least three weeks. Preferably, the chelating agents and / or anticancer therapeutic agents as disclosed herein are administered in cycles every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks. Preferably, the chelating agents and / or anticancer therapeutic agents as disclosed herein are administered in cycles after 3-5 weeks, such as after three weeks or after five weeks. Preferably, the administration of the chelating agents and / or anticancer therapeutic agents as disclosed herein can be repeated at irregular intervals, but according to the needs of the patient. Preferably, the chelating agents and / or anticancer therapeutic agents as disclosed herein are administered in cycles repeated at least once, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, or at least 10 times. Preferably, the treatment is maintained for at least 3 days, at least 1 week, at least 1 month, more preferably at least 6 months or at least 1 year, such as 2-5 years.

[0174] Administration of a chelator as disclosed herein or an anti-cancer therapeutic as disclosed herein to a subject can follow general protocols for administration of such compounds (taking into account the toxicity, if any, of the formulation). Thus, in some embodiments, there is a step of monitoring toxicity due to the combination therapy.

[0175] The chelating agents as disclosed herein can be administered in any acceptable pharmaceutical dosage form (eg, as an aqueous medium, such as a solution, suspension, emulsion). The chelating agents can also be administered orally as pills, tablets, capsules, and the like.

[0176] In the medical use of the present invention, if a subject has cancer and the cancer cells show drug resistance as disclosed herein (such as chemotherapy resistance), they can be identified as eligible for treatment. Such patients can benefit from the medical use of the present invention.

[0177] Numbering implementation method

[0178] Embodiment 1. A method of treating a subject having cancer, wherein the cancer is resistant to one or more anti-cancer therapeutic agents, comprising the steps of:

[0179] -Administering a therapeutically effective amount of a chelating agent to a subject having cancer, wherein the cancer is resistant to one or more anti-cancer therapeutics.

[0180] Embodiment 2. The method according to embodiment 1, wherein the method further comprises the step of administering a therapeutically effective amount of the one or more anti-cancer therapeutic agents.

[0181] Embodiment 3. The method of embodiment 1 or embodiment 2, wherein the treatment method is a method of sensitizing the subject's cancer to one or more anti-cancer therapeutic agents.

[0182] Embodiment 4. The method according to any of the preceding embodiments, wherein the treatment method is a method of chemosensitizing (chemosensitizing) cancer in a subject.

[0183] Embodiment 5. A method according to any of the preceding embodiments, wherein the treatment method is a method of enhancing the anti-cancer effect of the one or more anti-cancer therapeutic agents.

[0184] Embodiment 6. The method according to embodiment 5, wherein the enhanced anti-cancer effect is selected from the group consisting of cytotoxic effect, cytostatic effect, anti-invasiveness; anti-dissociation; anti-vascularization and a combination thereof.

[0185] Embodiment 7. The method according to any of the preceding embodiments, wherein the resistance to the one or more anti-cancer therapeutic agents is metal-induced resistance, preferably wherein the metal is selected from the group consisting of iron, copper, lead, cadmium, mercury, chromium, vanadium (e.g., in the form of VO4 3- forms) and their combinations.

[0186] Embodiment 8. The method according to any one of the preceding embodiments, wherein the method further comprises the following steps:

[0187] - providing a sample from a subject suffering from cancer, preferably a sample comprising cancer cells;

[0188] - identifying said subject as having cancer resistant to one or more anti-cancer therapeutic agents by measuring the presence of a p53-inactivated cancer cell metallome in said sample, preferably wherein said p53-inactivated cancer cell metallome is characterized by the presence (elevated levels) of one or more metals selected from the group consisting of iron, copper, lead, cadmium, manganese, mercury, chromium, vanadium (e.g. as VO4 3- The invention further comprises a group consisting of the following: the cancer is resistant to one or more anti-cancer therapeutic agents and a combination thereof.

[0189] Embodiment 9. The method according to any of the preceding embodiments, wherein the one or more anti-cancer therapeutic agents are one or more chemotherapeutic agents.

[0190] Embodiment 10. The method according to any of the preceding embodiments, wherein the one or more anti-cancer therapeutic agents is an anthracycline drug, such as doxorubicin.

[0191] Embodiment 11. The method according to any of the preceding embodiments, wherein the chelating agent is administered in combination with a second chelating agent.

[0192] Embodiment 12. A method according to any one of the preceding embodiments, wherein the chelating agent is 2,3-dimercapto-1-propanesulfonic acid (DMPS), and optionally wherein if a second chelating agent is present, the second chelating agent is EDTA; or

[0193] wherein the chelating agent is 2,3-dimercaptosuccinic acid (DMSA), and optionally wherein if a second chelating agent is present, the second chelating agent is EDTA.

[0194] Embodiment 13. A method according to embodiment 11 or embodiment 12, wherein the chelating agent and optionally the second chelating agent are provided in the form of a fixed-dose product (preferably a fixed-dose combination product), such as (i) a fixed-dose pharmaceutical composition comprising the chelating agent and optionally the second chelating agent, or (ii) a fixed-dose kit comprising a first container and a second container, wherein the first container comprises the chelating agent and the second container comprises the second chelating agent.

[0195] Embodiment 14. The method according to any of the preceding embodiments, wherein the chelator and optionally the second chelator are administered parenterally, such as intravenously or intratumorally, or enterally, such as orally or rectally.

[0196] Embodiment 15. The method according to any of the preceding embodiments, wherein the one or more anti-cancer therapeutics are administered parenterally, such as intravenously or intratumorally.

[0197] Embodiment 16. A method according to any of the preceding embodiments, wherein the chelator and optionally the second chelator are administered daily at a dose of 1-100 mg / kg / day, with each cycle lasting 1-25 days, and provided in repeated cycles at intervals (e.g., typically 3-6 weeks apart).

[0198] Embodiment 17. A method according to any of the preceding embodiments, wherein the cancer is a solid tumor or a liquid tumor.

[0199] Embodiment 18. A method according to any of the preceding embodiments, wherein the cancer is breast cancer, lung cancer such as SCLC, pancreatic cancer, or a blood cancer such as AML.

[0200] Embodiment 19. A chelator for use in a method of treating cancer in a subject, wherein the cancer is resistant to an anti-cancer therapeutic.

[0201] Embodiment 20. The chelator for use according to embodiment 19, wherein the chelator is used in a method of counteracting resistance of the cancer to the anti-cancer therapeutic agent.

[0202] Embodiment 21. The chelator for use according to embodiment 19 or embodiment 20, wherein the chelator is for administration in combination with the anti-cancer therapeutic agent.

[0203] Embodiment 22. A chelator for use according to any one of embodiments 19-21, wherein the drug resistance is chemotherapy resistance; and wherein the anti-cancer therapeutic agent is a chemotherapeutic agent.

[0204] Embodiment 23. A chelator for use according to any one of embodiments 19-22, wherein the cancer cells of the cancer have impaired tumor suppressor protein function.

[0205] Embodiment 24. The chelator for use according to embodiment 23, wherein the impaired tumor suppressor protein function is the result of abnormal tumor suppressor protein folding.

[0206] Embodiment 25. A chelator for use according to any one of embodiments 19 to 24, wherein cancer cells of the cancer comprise abnormally folded tumor suppressor proteins resulting in impaired tumor suppressor protein function.

[0207] Embodiment 26. The chelator for use according to any one of embodiments 23-25, wherein the tumor suppressor protein is one or more tumor suppressor proteins selected from the group consisting of p53, p63 and p73.

[0208] Embodiment 27. A chelator for use according to any one of embodiments 23-26, wherein the tumor suppressor protein is p53; and preferably wherein the function of the tumor suppressor protein is one or more selected from the group consisting of negative regulation of the cell cycle and promotion of cell apoptosis formation.

[0209] Embodiment 28. The chelator for use according to embodiment 27, wherein the p53 is wild-type p53 or mutant p53.

[0210] Embodiment 29. A chelating agent for use according to any one of embodiments 19-28, wherein the cancer is characterized by the presence of at least two, more preferably at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or at least 23 metals selected from the group consisting of arsenic (As), aluminum (Al), antimony (Sb), barium (Ba), boron (B), cadmium (Cd), cerium (Ce), chromium (Cr), lead (Pb), mercury (Hg), neodymium (Nd), manganese (Mn), nickel (Ni), tin (Sn), titanium (Ti), uranium (U), vanadium (V), copper (Cu), iron (Fe), gold (Au), silver (Ag), palladium (Pd) and platinum (Pt).

[0211] Embodiment 30. A chelating agent for use according to any one of embodiments 19-29, wherein the cancer is characterized by the presence of elevated levels of at least two, more preferably at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or at least 23 metals selected from the group consisting of arsenic (As), aluminum (Al), antimony (Sb), barium (Ba), boron (B), cadmium (Cd), cerium (Ce), chromium (Cr), lead (Pb), mercury (Hg), neodymium (Nd), manganese (Mn), nickel (Ni), tin (Sn), titanium (Ti), uranium (U), vanadium (V), copper (Cu), iron (Fe), gold (Au), silver (Ag), palladium (Pd) and platinum (Pt).

[0212] Embodiment 31. A chelator for use according to any one of embodiments 19-30, wherein the cancer is characterized by the presence of elevated levels of at least one, preferably at least two, more preferably at least 3, 4, 5, 6, 7, 8 or at least 9 metals selected from the group consisting of copper (Cu), iron (Fe), lead (Pb), mercury (Hg), cadmium (Cd), nickel (Ni), arsenic (As), vanadium (V) and chromium (Cr).

[0213] Embodiment 32. A chelator for use according to any one of embodiments 25-31, wherein the aberrant tumor suppressor protein folding is induced by elevated metal levels as defined in any one of embodiments 23-31.

[0214] Embodiment 33. A chelator for use according to any one of embodiments 19-32, wherein the anti-cancer therapeutic is an anthracycline, such as doxorubicin.

[0215] Embodiment 34. A chelating agent for use according to any one of embodiments 19 to 33, wherein the chelating agent is administered in combination with a second chelating agent.

[0216] Embodiment 35. A chelating agent for use according to any one of embodiments 19-34,

[0217] (i) wherein the chelating agent is 2,3-dimercapto-1-propanesulfonic acid (DMPS), and optionally wherein the second chelating agent, if present, is EDTA; or

[0218] (ii) wherein the chelating agent is 2,3-dimercaptosuccinic acid (DMSA) and optionally wherein the second chelating agent, if present, is EDTA.

[0219] Embodiment 36. A chelating agent for use according to embodiment 34 or embodiment 35, wherein the chelating agent and optionally the second chelating agent are provided in the form of a fixed-dose product (preferably a fixed-dose combination product), such as (i) a fixed-dose pharmaceutical composition comprising the chelating agent and optionally the second chelating agent, or (ii) a fixed-dose kit comprising a first container and a second container, wherein the first container comprises the chelating agent and the second container comprises the second chelating agent.

[0220] Embodiment 37. A chelating agent for use according to any one of embodiments 19-36, wherein the chelating agent and optionally the second chelating agent are administered parenterally (such as intravenously or intratumorally), or enterally (such as orally or rectally).

[0221] Embodiment 38. A chelator for use according to any one of embodiments 19-37, wherein the anti-cancer therapeutic is administered parenterally, such as intravenously or intratumorally.

[0222] Embodiment 39. A chelating agent for use according to any one of embodiments 19-38, wherein the chelating agent and optionally the second chelating agent are administered daily at a dose of 1-100 mg / kg / day, each cycle lasting 1-25 days, and provided in repeated cycles at intervals (e.g., intervals are typically 3-6 weeks).

[0223] Embodiment 40. The chelator for use according to any one of embodiments 19-39, wherein the cancer is a solid tumor or a liquid tumor.

[0224] Embodiment 41. A chelator for use according to any one of embodiments 19-40, wherein the cancer is breast cancer, lung cancer such as small cell lung cancer (SCLC), pancreatic cancer, or a blood cancer such as acute myeloid leukemia (AML).

[0225] Embodiment 42. A pharmaceutical composition comprising 2,3-dimercapto-1-propanesulfonic acid (DMPS), and a pharmaceutically acceptable excipient; wherein the DMPS is present in a dose of 40-12000 mg, preferably 400-3600 mg; preferably wherein the composition is for daily administration.

[0226] Embodiment 43. A pharmaceutical composition comprising (i) 2,3-dimercapto-1-propanesulfonic acid (DMPS) or DMSA, (ii) EDTA, and (iii) a pharmaceutically acceptable excipient; preferably wherein the DMPS or the DMSA is present in a dose of 40-12000 mg, preferably 400-3600 mg.

[0227] Embodiment 44. The pharmaceutical composition according to embodiment 42 or embodiment 43 further comprises a chemotherapeutic agent, preferably an anthracycline drug such as doxorubicin.

[0228] Embodiment 45. A pharmaceutical combination comprising (i) a first container comprising a pharmaceutical composition comprising 2,3-dimercapto-1-propanesulfonic acid (DMPS) or DMSA and a pharmaceutically acceptable excipient; and (ii) a second container comprising a pharmaceutical composition comprising a chemotherapeutic agent, preferably an anthracycline such as doxorubicin, and a pharmaceutically acceptable excipient; and optionally wherein the combination comprises a third container comprising EDTA and a pharmaceutically acceptable excipient.

[0229] Example

[0230] Example 1

[0231] Materials and methods

[0232] Cell lines

[0233] A549, MCF7 and Beas-2B cells were obtained from ATCC ( https: / / www.atcc.org ). Cells were maintained in DMEM high glucose (Invitrogen) and 10% FCS (Gibco) at 37°C and 5% CO2. A549 p53 KO cells were generated using a CRISPR p53 KO construct (pLV-U6g-EPCG with a target sequence of TCCATTGCTTGG GACGGCAAGG, Sigma). GFP-expressing cell lines were selected using FACS sorting, followed by clonal selection in neomycin (600 ng / ml, Sigma).

[0234] Metal / chelating agents and chemotherapy solutions

[0235] The metals were each dissolved in 10 mM HNO3 and combined in the desired ratios in a filter-sterilized parent mixture. Small amounts of the parent mixture were added to the incubation medium (incubation medium) to provide the cells with different amounts of the metals, as detailed in Table 1. An equal amount of NaOH was added to prevent pH fluctuations.

[0236] Table 1: Metal concentrations used in the experimental work

[0237]

[0238]

[0239] DMPS stock solution was 1 mM (in water) / filtered and stored in aliquots at -20°C, EDTA was dissolved as a 0.5 M solution in water with pH adjusted to 8 with 2M NaOH / filtered and stored at RT.

[0240] Doxorubicin was made into a 32 mM solution in water / filtered and stored in aliquots at -20 °C.

[0241] Cell viability assay

[0242] Resazurin cell viability assay was used to determine cell viability after chemotherapy and chelator challenge. 2 × 10 3 Cells were inoculated with 100 μl. After 24 hours, the medium was replaced with 90 μl of metal mixture or control medium for another 24 hours. Next, the combination of chelators and chemotherapy as shown was prepared and added to the cells as an additional 10 μl. The cells were then incubated for 72 hours (Beas-2B) or 96 hours (A549 and MCF7). A stock solution of resazurin (Sigma) was prepared in PBS as 880 μM (adjusted to pH 7.8) / filtered (0.2um) and kept at 4°C for 6 months. The medium was replaced with 75 μl of resazurin solution (final concentration 44 μM). The cells were incubated at 37°C for 3-4 hours and the fluorescence emission was measured at 583 nm using a spectrophotometer (Beckman), with excitation at 555 nm. In order to directly test the effect of metals on doxorubicin, doxorubicin was incubated with the metals in solution (as a 10X solution) for 24 hours before being added to the cells, while the control cells were incubated in the metals as previously described.

[0243] P53 folding / Western blot

[0244] Cells were seeded in 6-well plates at a cell density of 30%. After 24 hours, the culture medium was replaced with the metal mixture as indicated. The cells were incubated for 24 hours and lysed on ice for 15 minutes with 100 μl NP-40 lysis buffer (100 mM NaCl 100 mM Tris pH 8, 1% NP-40). The cell pellet was discarded by centrifugation (up to 10 minutes), 10% of the input was taken for protein blotting and mixed with 4X SB. The remaining lysate was immunoprecipitated with p53 Ab240 Ab (1 ul for each condition) using goat anti-mouse magnetic immunomagnetic beads (Dynabeads) (Thermo Fisher) washed with 30 μl NP-40 buffer. The lysate and beads were tumbled for 2 hours at 4°C and washed 3X with NO-40 buffer. The beads were placed in 2X SB and placed in a heat block together with the input denatured at 95°C. Western blots were run for all samples using 8% SDS page gels. The gels were transferred to nitrocellulose membranes, blocked with milk (TBS-tween) for 1 hour (rolling), and incubated at 4°C in p53 DO-1 (Santa Cruz Ab, 1:2000) ON. The membranes were washed 3X with TBS-tween, and the blots were incubated for 1 hour at room temperature using the second Ab (anti-mouse 700, Li-Cor) at 1:10.000. The blots were washed 3X with TBS-tween, and the signals were measured in Li-Cor Odyssey. Quantification was measured in Li-Cor's software, and the input levels for each sample were corrected and drawn.

[0245] Doxorubicin uptake assay

[0246] 1×10 4 Cells were seeded in 96 wells for 24 hours. The cells were then incubated with a metal mixture 1:128 for 24 hours. 10% volume of indicated concentration of doxorubicin was subsequently added, and after 2 hours, the accumulation of doxorubicin was measured under 564nm excitation using the Opera Phenix high-content imaging screening system. Representative images of each concentration are shown.

[0247] result

[0248] To test whether metals contribute to chemoresistance, immortalized Beas-2B lung cell lines were exposed to increasing doses of a mixture of metals for 24 hours. Cells were then subjected to a range of doxorubicin concentrations, and viability was measured after 72 hours using the resazurin viability assay ( Figure 1). It is clear that increasing metal doses increased resistance to doxorubicin in a dose-dependent manner. In companion assays using similar concentrations of metal, we determined p53 expression and folding state. These assays showed that p53 expression levels did not change after metal exposure, but a higher proportion of p53 was in an unfolded state after elevated metal levels ( Figure 2 A and B).

[0249] We next tested whether metal-induced chemoresistance could be reversed by adding high doses (150 μM) of the chelator DMPS to Beas2B cells. In cells primed with 1:128 and 1:256 dilutions of the metals, sensitivity to doxorubicin was fully restored when DMPS was added concomitantly with the metals ( Figure 3 A). In breast cancer cell line MCF7 ( Figure 3 B) and in the adenocarcinoma lung cancer cell line A549 ( Figure 3 C), suggesting that this effect can be observed in a variety of different cancer cells. Most interestingly, A549 p53 KO cells exposed to metals developed only a weak chemoresistance and chelators were unable to restore the same degree of sensitivity as in A549 control cells, although it is important to note that these cells showed an overall lack of growth compared to control A549 cells ( Figure 3 D).

[0250] EDTA was used with the same chelating capacity as DPMS. EDTA is a multidentate ligand that forms a 1:1 complex with the added metal, whereas DMPS is a bidentate ligand that will form complexes in varying stoichiometric ratios of 1:1, 1:2, or 1:3. The most common is 1:2, which has been used as a guideline for calculating equivalent chelating capacity. Therefore, to provide the same chelating capacity, the molar concentration of EDTA (e.g. 75 μM) is half the molar concentration of DMPS (e.g. 150 μM). EDTA at 75 μM was also able to restore chemosensitivity, but not to the extent seen with DMPS used at 150 μM ( Figure 4 ). We could not observe an additive effect of EDTA when used in combination with DMPS. However, given that DMPS alone almost restored the doxorubicin sensitivity of the cells to the levels seen in the absence of the metal, it was necessary to test the combination effect at lower doses of the chelators. Therefore, we reduced DMPS to 40 μM and EDTA to 20 μM. At these levels, each chelator alone failed to significantly restore chemosensitivity. However, in combination, they clearly enhanced each other in reversing resistance to doxorubicin ( Figure 5 ).

[0251] To quantify metal-induced chemoresistance, Beas-2B cells were incubated with or without metals and with EDTA, DMPS, and their combination and exposed to doxorubicin. IC 50 - The concentration of doxorubicin that will kill 50% of the cells ( Figure 6 ). It was observed that, in the absence of added metals, the IC 50 low and was not affected by chelators, while in cells loaded with metals (1:128), IC 50 More than 10 times higher. Chelating agent treatment largely reversed this resistance.

[0252] Previous studies have established that doxorubicin can complex with certain metals, which enhances its activity in breast cancer ( et al., Molecules, 22(7):1106 (2017)). This may contradict what we observed, but it does make us wonder whether our metal cocktail complexed with doxorubicin, thereby preventing its uptake. Since doxorubicin is fluorescent, we performed a fluorescent uptake assay in which Beas-2B cells were loaded with a 1:128 dilution of the metal cocktail and then exposed to increasing doses of doxorubicin for 2 hours. No difference was observed in doxorubicin uptake between metal-exposed cells and control cells ( Figure 7 We also tested whether pre-incubating doxorubicin with metals before adding it to the cells would render it ineffective. Premixing the metals with doxorubicin did not prevent doxorubicin from causing cell death. In this case, sensitivity to doxorubicin was better than in cells primed with metals, but worse than in cells that had not seen any metals ( Figure 8 ). Furthermore, it is apparent that preincubation of cells with metals prior to doxorubicin exposure, which promotes metal uptake, is effective in inducing chemoresistance, whereas co-administration of metals and doxorubicin is much less effective, suggesting that metal uptake is required for the induction of chemoresistance.

[0253] In summary, these data indicate that metals induce resistance to doxorubicin in multiple cancer cell lines. Metal-induced chemoresistance appears to be largely p53-dependent and is consistent with a dose-dependent unfolding of p53. Finally, chelators were able to fully restore doxorubicin sensitivity. Our data suggest that metals drive an as-yet unknown intracellular chemoresistance phenotype that is dependent on p53 signaling.

[0254] Example 2

[0255] Materials and methods

[0256] In general, the method disclosed in the first embodiment is also used in the second embodiment. In addition, the following method is also used:

[0257] Additional cells:

[0258] Obtain Hep3B and H2177 cells from ATCC, and maintain them in DMEM high glucose (DMEM high Glucose) (Invitrogen) and 10% FCS (Gibco) at 37 ℃ and 5% CO2.Beas2B cell is plated in 6-well plates, and then use 4.5 μ l liposomes (lipofectamine) and 500 μ l serum-free medium (Thermo Fisher Scientific) to transfect with 1.5 μ l 20 μ M p53 siRNA (from 4 pools of Dharmacon) or control siRNA (from 4 pools of Dharmacon) on the second day.Cells are counted subsequently, and added to 96 well plates, and survival rate is measured as previously described.Hep3B cells are transfected with p53 plasmid (described in Muller et al, Cell 2009) using liposomes (lipofectamine) 3000 (Thermo Fisher Scientific).

[0259] In vitro folding assay:

[0260] To evaluate the direct effects of metals and chelators on p53 folding, Beas-2B cells were lysed and the lysates were treated with metals in NP40 buffer on ice for 30 minutes. If a chelator was used, it was then added on ice for 1 hour. The lysates were then subjected to IP conditions as previously described.

[0261] Data analysis

[0262] IC50 values ​​were calculated using Graphpad Prism. Error bars represent the standard deviation of triplicate measurements.

[0263] Metal / chelating agents and chemotherapy solutions

[0264] Solutions were prepared as previously described. As detailed in Tables 2 and 3, different amounts of metals were provided to the cells.

[0265] Table 2: Metal concentrations used for MCF7 and Hec-1a cells

[0266]

[0267]

[0268] Table 3: Metal concentrations used for Beas-2B cells

[0269]

[0270] Cell preparation for metal uptake assessment

[0271] Cell cultures were grown according to the supplier's (ATCC) instructions and incubated with the metal mixtures for 24 hours as described.

[0272] At the end of the experiment, cells were harvested by incubation with EDTA-free trypsin for 8 minutes, collected and washed 3 times in phosphate buffered saline (PBS). The cells were centrifuged to produce a precipitate and transferred for metal evaluation. In a high-grade clean room, 200uL of pure HNO3 (15N) and 100uL of 30% H2O2 were added to the cell precipitate, mixed thoroughly, and incubated at room temperature for 2 hours, followed by the addition of 1180uL of double distilled water. The final sample was transferred for metal measurement.

[0273] ICP-MS measurements

[0274] After dilution, metal concentrations were determined using a quadrupole inductively coupled plasma mass spectrometer (Agilent Technologies, 8900 ICP-MS Triple Quad). The ICP-MS was calibrated with a series of multi-element standard solutions (Merck; ME VI) and blanks. Shifts were corrected with internal standards (750 μg / L Sc, 100 μg / L Re, and 50 μg / L Rh).

[0275] result

[0276] The results of Example 1 indicate that metal uptake is necessary for the full development of chemoresistance ( Figure 8 To determine the metal uptake into cells and the different effects of different metal mix concentrations added to the culture medium, cells were incubated with different concentrations of extended metal mix in culture medium for 24 hours, washed and collected for ICP-MS evaluation. Metals were taken up in a dose-dependent manner in the three different cell lines ( Fig. 9 , representative metals are shown). This finding is relevant to the dependence of chemoresistance on metal uptake, as increasing levels of added metals lead to higher intracellular metal levels, which in turn lead to increased chemoresistance.

[0277] In addition to the unfolding observed in Beas2B cells ( Figure 2 ), in addition to cell lysates exposed to low levels of metal mixtures in vitro ( Fig.10) and in other lung cancer cells H2170 (which has a 157 mutation in p53, which is particularly common in small cell cancers and exists in a folded state under normal conditions) also showed p53 unfolding ( Fig.11 Hep3B cells (p53 null) were used as a p53 null cell line sensitive to metal treatment to determine whether certain mutants were more sensitive than WT p53 to metal-induced unfolding ( Fig.12 ). The 175H mutant is an unfolded mutant that is the most prevalent in all cancers. Since the 175H p53 is already unfolded, no difference in folding can be detected after metal addition. In contrast, mutants 157 and 158 are more folded; however, it is clear that the ratio of unfolded to folded p53 in response to lower metal concentrations is higher in the 157 and 158 mutations than in WT p53. In other words, these mutants are more prone to unfolding than WT p53, making them interesting targets for chelation strategies.

[0278] Further extending these findings, it was demonstrated that metals act in a p53-dependent manner to induce chemoresistance. Example 1 shows this in A549 cells using a CRISPR KO line ( Figure 3 In Example 2, a similar effect was demonstrated in Beas2B cells (in which p53 was knocked out), which resulted in a significantly lower IC50 in the p53 knockout cells compared to the control ( Fig.13 ).

[0279] In Example 1, it was shown that EDTA and DMPS can reverse metal-induced resistance to cancer therapy in doxorubicin-treated Beas-2B cells ( Figure 3-5 In the current example, miaDMSA ( Fig.14 ) and DMSA( Fig.15 ) also reversed metal-induced chemoresistance, thus demonstrating the efficacy of different types of chelators.

[0280] Importantly, the chelator also restored chemosensitivity to doxorubicin in a different, more aggressive lung cancer cell line, H2170 ( Fig.16 Other chemotherapy regimens have also been investigated. Fig.17 Examples of this are shown, where chemosensitivity of BxPC-3 to 5-FU was reduced by metals and restored with DMSA and miaDMSA, whereas resistance of BxPC-3 to nab-paclitaxel in response to metals was more modest but still reversed by chelators ( Fig.18 In contrast, SW1990 cells did not show metal-induced resistance to any chemotherapeutic agents ( Fig.19). It is important to note that BxPC-3 cells have a mutant form of p53 - Y220 C - which has been shown to be refoldable, whereas SW1990 cells do not have any p53. Taken together, these findings support the notion that metals can induce resistance to various chemotherapies, whereas chelators are able to restore sensitivity.

[0281] Given the ability of chelators to prevent metal-induced chemoresistance or restore chemosensitivity, we investigated whether chelators could prevent p53 unfolding or even restore folding once unfolding has occurred. Fig. 20 ) or DMSA( Fig.21 ) were used to incubate Beas-2B cells with metals. DMPS and DMSA significantly prevented metal-induced unfolding of p53. In the mutant context of Hep3B cells, miaDMSA was able to prevent 158 ​​mutant p53 and WT p53 from unfolding when exposed to metals ( Fig. 22 To determine whether any of these chelators were able to refold p53, we first incubated lysates from Beas-2B cells with the metals and thereafter incubated them with the chelators. miaDMSA and EDTA were able to refold p53 ( Fig.23 ).

[0282] discuss

[0283] A variety of different cell lines, different chemotherapies, and different chelators were added to show that the chelation strategy combined with chemotherapy works in cells with either WT p53 or mutant forms of p53 that are prone to refolding.

[0284] In many cell lines, including Beas-2B bronchial epithelial cells ( Figure 1 ; Figure 2 A), MCF7 breast cancer cells ( Figure 2 B) A549 lung cancer cells ( Figure 2 C) H2170 Squamous cell lung cancer ( Fig.16 ), BxPC3 epithelial pancreatic adenocarcinoma cells ( Figure 17-18 ) and to some extent in SW1990 pancreatic cells ( Fig.19 ), it has been demonstrated that mixtures of metals induce resistance to cancer therapies. Therefore, it can be concluded that metal-induced resistance to therapy is a general feature of cancer.

[0285] In addition to the p53 unfolding observed in Beas-2B cells by metal addition ( Figure 2 ), it was also demonstrated that the addition of metals could lead to the unfolding of p53 in H2170 lung cancer cells and Hep3B liver cancer cells ( Figure 11-12In vitro, p53 unfolding was also confirmed in cell lysates exposed to low amounts of metal mixtures ( Fig.10 and Fig.23 ), thus confirming that the unfolding of p53 is a direct effect of the protein's exposure to metal mixtures. In addition, it has been shown that certain variants of mutant p53 that are folded under normal conditions may be more easily unfolded by the addition of metals than WT p53. Therefore, the folding and unfolding patterns were explored in lung cancer cells H2170 and liver cancer Hep3B cells. H2170 has the R158G mutation in p53, which is a very frequent mutation in small cell lung cancer, and its p53 exists in a folded state under normal conditions ( Fig.11 Hep3B cells (p53 null) were used as a p53 null cell line to determine whether certain mutants were more sensitive to metal-induced unfolding than WT p53 ( Fig.12 ). The R175H mutant is an unfolded mutant that is the most prevalent in all cancers. In contrast, mutants R157L and R158G are more folded and enriched in SCLC. Since R175H is already unfolded, no metal-induced differences can be detected in the case of this mutant. However, it is clear that the ratio between unfolded and folded p53 is higher in mutants R157L and R158G than in WT p53, even in response to much lower metal concentrations. In other words, these mutants are more likely to unfold than wild-type p53, making these mutants interesting targets for chelation strategies in cancer.

[0286] Importantly, metals have been shown to act in cells to induce chemoresistance in a p53-dependent manner. Thus, in a CRISPR knockout (KO) line of A549 cells, addition of metals had only a minor effect on cell viability when exposed to doxorubicin ( Figure 2 D). Furthermore, exposure of p53 knockout Beas-2B cells to metals resulted in a much smaller increase in IC50 compared to native Beas-2B cells ( Fig.13 These observations were further supported by the SW1990 pancreatic adenocarcinoma cell line, which does not possess any p53 and does not display metal-induced resistance to any chemotherapy ( Fig.19 ).

[0287] When metal-loaded cells were exposed to doxorubicin, all tested chelators had a significant effect on cell viability ( Figure 3-6 ; 14-15), indicating that this strategy works with different types of chelators. It has been demonstrated that, in particular, miaDMSA ( Fig.14 ) is a highly effective chelating agent.

[0288] The ability of the chelators to restore sensitivity to doxorubicin was observed in different cell lines. Thus, in Beas-2B, A549, MCF7 ( Figure 3 ) and in a different, more aggressive lung cancer cell line, H2170 ( Fig.16 ), chelators can also restore chemosensitivity to doxorubicin. While doxorubicin has been widely used to demonstrate the effects of metals and chelators on p53 folding and chemoresistance, other chemotherapies have shown similar patterns. As an example, DMSA and miaDMSA restored chemosensitivity of BxPC-3 to 5-fluorouracil (5-FU) ( Fig.17 Interestingly, exposure of BxPC-3 cells to nab-paclitaxel did not result in metal-induced resistance to this chemotherapeutic agent, but addition of miaDMSA or DMSA did reduce viability. These data could suggest that endogenous metal levels in these cells are sufficient to promote resistance to nab-paclitaxel ( Fig.18 ). Significantly, BxPC-3 cells harbor a mutant form of p53 Y220C, which has been shown to be refoldable. Together, these findings support the notion that various chelators can restore sensitivity to various chemotherapies in a p53-dependent manner.

[0289] DMPS( Fig. 20 ) and DMSA( Fig.21 ) significantly prevented metal-induced p53 unfolding in Beas-2B cells. In the mutant context of Hep3B cells, miaDMSA was able to prevent the unfolding of R158G mutant p53 and WT p53 when exposed to metals ( Fig. 22 These results provide further evidence for the dependence of p53 unfolding on metals in a form that can be taken up by cells.

[0290] A consistent model of metal-induced chemoresistance by means of a p53 unfolding mechanism requires that restoration of chemosensitivity occurs either by refolding p53 or by the cell producing new, properly folded p53. To determine whether any of these chelators could refold p53, lysates of Beas-2B cells were exposed to metals and then incubated with the chelators. It was shown that miaDMSA and EDTA were able to refold p53 ( Fig.23 ), without the need for de novo synthesis of properly folded p53.

[0291] As evidence of intracellular metal accumulation, Beas-2B cells were exposed to metals for 24 h, washed to eliminate all traces of added metals, and collected for metal assessment by ICP-MS. Dose-dependent metal uptake into the cells was observed ( Fig. 9Similar results were obtained in other cell lines such as MCF7 and A549.

[0292] In conclusion, the data presented herein confirm the mechanism of action of the interaction between p53 and metals and further suggest the preferred use of miaDMSA as a chelator to restore sensitivity to chemotherapy and to restore folding of both WT p53 and mutant p53.

Claims

1. A chelating agent for use in a method of treating cancer in a subject, wherein the cancer is resistant to an anti-cancer therapeutic agent.

2. The chelating agent for use according to claim 1, wherein the chelating agent is for use in a method of counteracting resistance of the cancer to the anti-cancer therapeutic agent.

3. The chelating agent for use according to claim 1 or claim 2, wherein the chelating agent is for administration in combination with the anti-cancer therapeutic agent.

4. A chelator for use according to any one of the preceding claims, wherein the drug resistance is chemotherapy resistance; and wherein the anti-cancer therapeutic is a chemotherapeutic agent.

5. The chelating agent for use according to any one of the preceding claims, wherein cancer cells of the cancer have impaired tumor suppressor protein function.

6. The chelator for use according to claim 5, wherein the impaired tumor suppressor protein function is the result of abnormal tumor suppressor protein folding.

7. The chelator for use according to any one of the preceding claims, wherein cancer cells of the cancer comprise abnormally folded tumor suppressor proteins which result in impaired tumor suppressor protein function and / or result in resistance of the cancer to the anti-cancer therapeutic agent.

8. The chelator for use according to any one of claims 5 to 7, wherein the tumor suppressor protein is one or more tumor suppressor proteins selected from the group consisting of p53, p63 and p73.

9. The chelator for use according to any one of claims 5 to 8, wherein the tumor suppressor protein is p53; and preferably wherein the tumor suppressor protein function is one or more selected from the group consisting of: negative regulation of cell cycle and promotion of apoptosis.

10. The chelator for use according to claim 9, wherein p53 is wild-type p53 or mutant p53.

11. The chelator for use according to any one of claims 1 to 10, wherein the chelator is used for use in restoring or reactivating the function of a tumor suppressor protein.

12. The chelating agent for use according to any one of claims 3 to 11, wherein the chelating agent is administered before the anti-cancer therapeutic agent is administered, Preferably, wherein the chelating agent is administered prior to administration of the anti-cancer therapeutic agent to restore or reactivate tumor suppressor protein function, More preferably wherein the chelator is administered at least 10 minutes, at least 30 minutes, at least 1 hour, at least 12 hours or at least 48 hours prior to administration of the anti-cancer therapeutic agent to restore or reactivate tumor suppressor protein function, More preferably wherein the chelator is administered at least 10 minutes, at least 30 minutes, at least 1 hour, at least 12 hours or at least 24 hours prior to the first, second, third and / or each consecutive administration cycle of the anti-cancer therapeutic agent to restore or reactivate tumor suppressor protein function.

13. A chelating agent for use according to any one of claims 1 to 12, wherein the cancer is characterized by the presence of at least two, more preferably at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or at least 23 metals selected from the group consisting of arsenic (As), aluminum (Al), antimony (Sb), barium (Ba), boron (B), cadmium (Cd), cerium (Ce), chromium (Cr), lead (Pb), mercury (Hg), neodymium (Nd), manganese (Mn), nickel (Ni), tin (Sn), titanium (Ti), uranium (U), vanadium (V), copper (Cu), iron (Fe), gold (Au), silver (Ag), palladium (Pd) and platinum (Pt).

14. A chelating agent for use according to any one of claims 1 to 13, wherein the cancer is characterized by the presence of elevated levels of at least two, more preferably at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or at least 23 metals selected from the group consisting of arsenic (As), aluminum (Al), antimony (Sb), barium (Ba), boron (B), cadmium (Cd), cerium (Ce), chromium (Cr), lead (Pb), mercury (Hg), neodymium (Nd), manganese (Mn), nickel (Ni), tin (Sn), titanium (Ti), uranium (U), vanadium (V), copper (Cu), iron (Fe), gold (Au), silver (Ag), palladium (Pd) and platinum (Pt).

15. A chelating agent for use according to any one of claims 1 to 14, wherein the cancer is characterized by the presence of elevated levels of at least one, preferably at least two, more preferably at least 3, 4, 5, 6, 7, 8 or at least 9 metals selected from the group consisting of copper (Cu), iron (Fe), lead (Pb), mercury (Hg), cadmium (Cd), nickel (Ni), arsenic (As), vanadium (V) and chromium (Cr).

16. A chelator for use in a method of sensitizing a subject to an anti-cancer treatment and / or counteracting resistance to an anti-cancer therapeutic agent, wherein the method restores or reactivates a tumor suppressor protein function that is impaired, preferably due to aberrant tumor suppressor protein folding.

17. The chelating agent for use according to claim 16, wherein A tumor suppressor protein with impaired function caused by abnormal folding is p53.

18. A chelating agent for use according to claim 16 or claim 17, wherein the chelating agent is administered in combination with an anti-cancer therapeutic agent.

19. The chelating agent for use according to claim 18, wherein the chelating agent is administered prior to administering the anti-cancer therapeutic agent, Preferably, wherein the chelating agent is administered prior to administration of the anti-cancer therapeutic agent to restore or reactivate tumor suppressor protein function, More preferably wherein the chelator is administered at least 10 minutes, at least 30 minutes, at least 1 hour, at least 12 hours or at least 48 hours prior to administration of the anti-cancer therapeutic agent to restore or reactivate tumor suppressor protein function, More preferably, the chelator is administered at least 10 minutes, at least 30 minutes, at least 1 hour, at least 12 hours, or at least 48 hours prior to the first, second, third and / or each consecutive administration cycle of the anti-cancer therapeutic agent to restore or reactivate tumor suppressor protein function.

20. A chelator for use according to any one of claims 6-19, wherein the abnormal tumor suppressor protein folding is caused by an elevated level of a metal as defined in any one of claims 13-15.

21. A chelating agent for use according to any one of the preceding claims, wherein the anticancer therapeutic agent is an anthracycline, such as doxorubicin; an antimetabolite, such as 5-fluorouracil (5-FU) and / or a taxane, such as Nab-paclitaxel and / or paclitaxel.

22. A chelating agent for use according to any one of the preceding claims, wherein the chelating agent is administered in combination with a second chelating agent.

23. A chelating agent for use according to any one of the preceding claims, wherein the chelating agent is monoisoamyl DMSA (miaDMSA).

24. A chelating agent for use according to any one of claims 1 to 22, (i) wherein the chelating agent is 2,3-dimercapto-1-propanesulfonic acid (DMPS), and optionally wherein the second chelating agent, if present, is EDTA; or (ii) wherein the chelating agent is 2,3-dimercaptosuccinic acid (DMSA), preferably monoisoamyl DMSA (miaDMSA) and optionally wherein the second chelating agent, if present, is EDTA.

25. A chelating agent for use according to any one of claims 22 to 24, wherein the chelating agent and optionally the second chelating agent are provided in the form of a fixed dose product (preferably a fixed dose combination product), such as (i) a fixed dose pharmaceutical composition comprising the chelating agent and optionally the second chelating agent, or (ii) a fixed dose kit comprising a first container and a second container, wherein the first container comprises the chelating agent and the second container comprises the second chelating agent.

26. The chelator for use according to any one of the preceding claims, wherein the chelator and optionally the second chelator are administered parenterally (eg intravenously or intratumorally) or enterally (eg orally or rectally).

27. A chelating agent for use according to any one of the preceding claims, wherein the anti-cancer therapeutic is administered parenterally, such as intravenously or intratumorally.

28. A chelating agent for use according to any one of the preceding claims, wherein the chelating agent and optionally the second chelating agent are administered daily at a dose of 1-100 mg / kg / day, each cycle lasting 1-25 days, and provided in repeated cycles at intervals (e.g., typically 3-6 weeks apart).

29. A chelator for use according to any one of the preceding claims, wherein the cancer is a solid or liquid tumor.

30. A chelating agent for use according to any one of the preceding claims, wherein the cancer is breast cancer, lung cancer such as small cell lung cancer (SCLC), pancreatic cancer, or a blood cancer such as acute myeloid leukemia (AML).

31. A pharmaceutical composition comprising 2,3-dimercapto-1-propanesulfonic acid (DMPS) and / or DMSA, preferably miaDMSA, and a pharmaceutically acceptable excipient; wherein the DMPS and / or DMSA, preferably miaDMSA, is present in a dosage of 40-12000 mg, preferably 400-3600 mg; preferably wherein the composition is for daily administration.

32. A pharmaceutical composition comprising (i) 2,3-dimercapto-1-propanesulfonic acid (DMPS) or DMSA, preferably miaDMSA, (ii) EDTA, and (iii) a pharmaceutically acceptable excipient; preferably wherein the DMPS or the DMSA, preferably miaDMSA, is present in a dosage of 40-12000 mg, preferably 400-3600 mg.

33. The pharmaceutical composition according to claim 31 or claim 32, further comprising a chemotherapeutic agent, preferably an anthracycline such as doxorubicin, an antimetabolite such as 5-fluorouracil (5-FU), and / or a taxane such as Nab-paclitaxel and / or paclitaxel.

34. A pharmaceutical combination comprising (i) a first container comprising a pharmaceutical composition comprising 2,3-dimercapto-1-propanesulfonic acid (DMPS) or DMSA, preferably miaDMSA, and a pharmaceutically acceptable excipient; and (ii) a second container comprising a pharmaceutical composition comprising a chemotherapeutic agent, preferably an anthracycline such as doxorubicin, and a pharmaceutically acceptable excipient; and optionally wherein the combination comprises a third container comprising EDTA and a pharmaceutically acceptable excipient.

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