Pentaza macrocyclic complex and hormone therapy combination cancer treatment

Abstract

The present invention provides a method for detecting and characterizing a mouse model of stem cell lineage plasticity characterized by (i) levels of sirtuin (SIRT3) protein below a first predetermined threshold, (ii) levels of manganese superoxide dismutase acetylated at lysine 68 (AcK68) above a second predetermined threshold, and (iii) levels of hypoxia-inducible factor 2 (HIF2) above a third predetermined threshold, which are indicative of stemness and lineage plasticity. α (HIF2 α and optionally administering an additional anti-cancer therapeutic agent, said method comprising administering to said mammalian subject a therapeutically effective amount of a pentaaza macrocyclic ring complex corresponding to formula (I):

Description

[Technical Field]

[0001] This invention was made possible with government support from grants numbered 1R01CA214025-01, R01CA152601-06A1, and R01CA168292, awarded by the National Institutes of Health (NIH) / National Cancer Institute (NCI). The government has certain rights to this invention.

[0002] This disclosure relates to combination therapies for the treatment of cancer, generally including the administration of a pentaza macrocyclic complex in combination with an endocrine therapy agent. [Background technology]

[0003] Transition metal-containing pentaza macrocyclic complexes having a macrocyclic ring system corresponding to formula A have been shown to be effective in treating many animal and cell models of human diseases, as well as in the treatment of conditions in human patients suffering from these diseases. [ka] Formula A For example, in a rodent model of colitis, GC4403, one such compound, has been reported to significantly reduce damage to the colon of rats used in the colitis experimental model (see Cuzzocrea et al., Europ. J. Pharmacol., 432, 79-89 (2001)). [ka] Furthermore, GC4403 has been reported to attenuate radiation damage occurring in both clinically relevant hamster models of acute radiation-induced oral mucositis (Murphy et al., Clin. Can. Res., 14(13), 4292 (2008)) and lethal whole-body irradiation in adult mice (Thompson et al., Free Radical Res., 44(5), 529-40 (2010)). Similarly, another such compound, GC4419, has been shown to attenuate VEGFr inhibitor-induced lung disease in rat models (Tuder, et al., Am. J. Respir. Cell Mol. Biol., 29, 88-97 (2003)). Furthermore, another such compound, GC4401, has been shown to produce protective effects in animal models of septic shock (S. Cuzzocrea, et al., Crit. Care Med., 32(1), 157 (2004)) and pancreatitis (S. Cuzzocrea, et al., Shock, 22(3), 254-61 (2004)). [ka]

[0004] Some of these compounds have been shown to possess potent anti-inflammatory activity and preventative effects against oxidative damage in vivo. For example, GC4403 has been reported to suppress inflammation in an inflammatory rat model (Salvemini, et.al., Science, 286, 304 (1999)) and prevent joint disease in a rat model of collagen-induced arthritis (Salvemini et al., Arthritis & Rheumatism, 44(12), 2009-2021 (2001)). Furthermore, other of these compounds, MdPAM and MnBAM, have demonstrated in vivo activity in inhibiting colonic tissue injury and neutrophil accumulation in colonic tissue (Weiss et al., The Journal of Biological Chemistry, 271(42), 26149-26156 (1996)). Furthermore, these compounds have been reported to have analgesic effects and reduce inflammation and edema in a rat carrageenan-induced hyperalgesia model of the paw (see, for example, U.S. Patent No. 6,180,620).

[0005] Furthermore, compounds in this class have been shown to be safe and effective in the prevention and treatment of diseases in humans. For example, GC4419 has been shown to reduce oral mucositis in head and neck cancer patients undergoing chemoradiotherapy (Anderson, C., Phase 1 Trial of Superoxide Dismutase (SOD) Mimetic GC4419 to Reduce Chemoradiotherapy (CRT)-Induced Mucositis (OM) in Patients (pts) with Mouth or Oropharyngeal Carcinoma (OCC), Oral Mucositis Research Workshop, MASCC / ISOO Annual Meeting on Supportive Care in Cancer, Copenhagen, Denmark (June 25, 2015)).

[0006] Furthermore, pentaza macrocyclic ring complexes containing transition metals in this class have shown efficacy in treating various cancers. For example, certain compounds in this class have been provided in combination with drugs such as paclitaxel and gemcitabine to enhance the treatment of cancer, such as colorectal cancer and lung cancer (non-small cell lung cancer) (see, for example, U.S. Patent No. 9,198,893). The 4403 compounds described above have also been used in in vivo models of Meth A spindle cell squamous cell carcinoma and RENCA renal cell carcinoma (Samlowski et al., Nature Medicine, 9(6), 750-755 (2003)), and in in vivo models of spindle cell squamous cell carcinoma metastasis (Samlowski et al., Madame Curie Bioscience Database (Internet), 230-249 (2006)).

[0007] Endocrine therapies (hormone therapies) such as tamoxifen have proven effective in treating various types of cancer, including estrogen receptor-positive breast cancer, and are currently available as chemopreventive agents in women at high risk of breast cancer (Minsun Chang, Biomolecules and Therapeutics, 20(3):256-267 (2012)). However, a problem with certain endocrine therapies such as tamoxifen is that certain tumors may be inherently resistant (i.e., resistant to the measure even before such treatment begins), and / or tumors that initially responded may develop resistance to the endocrine therapy over time (Zhu et al, Nature Communications, 9 (1595): 1-11(2018); Wu et al., Cancer Research, 78(3): 671-684 (2017)). Therefore, the development of cancer cell resistance to treatment can lead to cancer recurrence in previously treated individuals, or make it impossible to fight cancer in individuals currently receiving treatment. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] Therefore, there is a need for an improved method of treating cancer that, while improving the effect of killing cancer cells, also reduces the resistance of cancer cells to cancer treatment.

Means for Solving the Problem

[0009] Thus, briefly stated, aspects of the present disclosure are (i) levels of sirtuin (SIRT3) protein below a first predetermined threshold, (ii) levels of manganese superoxide dismutase acetylated at lysine 68 residue (AcK68) above a second predetermined threshold, and (iii) hypoxia-inducible factor 2 α (HIF2 α ) having an expression level of one or more of a tumor signature characterized by treating cancer in a mammalian subject,

[0010] A method comprising administering to the mammalian subject a therapeutically effective amount of a pentaazamacrocyclic ring complex corresponding to the following formula (I):

Chemical

[0011] In another embodiment, a method for treating cancer in a mammalian subject is a step of selecting a mammalian subject suitable for treatment with a pentaza macrocyclic complex corresponding to formula (I), the selection being: (i) whether the level of sirtuin (SIRT3) protein in the tumor cells of the tissue sample is below a first predetermined threshold; (ii) whether the level of (AcK68) manganese superoxide dismutase acetylated at lysine 68 residues is above a second predetermined threshold; and (iii) hypoxia-inducible factor 2 α (HIF2 α The process includes evaluating the test tissue sample to determine a criterion that includes whether the expression level of ) exceeds a third predetermined threshold indicating stem cell lineage plasticity, determining that the subject is suitable for treatment if one or more of the criteria (i), (ii), and / or (iii) are met, and administering a therapeutically effective amount of a pentaza macrocyclic complex corresponding to formula (I) if the subject is selected as suitable for treatment.

[0012] In another embodiment, an assay for analyzing a tissue sample obtained from a subject and containing tumor cells, comprising: (i) whether the level of sirtuin (SIRT3) protein in tumor cells of the tissue sample is below a first predetermined threshold; (ii) whether the level of (AcK68) manganese superoxide dismutase acetylated at lysine 68 residue is above a second predetermined threshold; and (iii) hypoxia-inducible factor 2 α (HIF2 α An assay is provided that can determine whether the expression level of ) exceeds one or more of a third predetermined threshold indicating stem cell lineage plasticity, and a kit for treating cancers of mammalian subjects is provided, comprising a therapeutically effective amount of a pentaza macrocyclic ring complex corresponding to formula (I).

[0013] In a further embodiment, a method for treating a tumor in a mammal affected by a tumor resistant to a chemotherapy agent, wherein the tumor has (i) levels of sirtuin (SIRT3) protein below a first predetermined threshold, and (ii) levels of K68 acetylated manganese superoxide dismutase (MnSOD) above a second predetermined threshold. K68 (iii) the level of (iii) hypoxia-inducible factor 2 exceeding a third predetermined threshold indicating stem cell lineage plasticity. α (HIF2 α A method is provided for having a tumor signature characterized by one or more expression levels of ) of ). Aspects of the method include obtaining a test tissue sample containing tumor cells from a subject, (i) whether the level of sirtuin (SIRT3) protein activity in the tumor cells of the tissue sample is below a first predetermined threshold, (ii) whether the level of acetylated (AcK68) manganese superoxide dismutase at lysine 68 residue is above a second predetermined threshold, and (iii) hypoxia-inducible factor 2 α (HIF2 α The method includes evaluating a tissue sample to determine one or more criteria, including whether the expression level of ) exceeds a third predetermined threshold indicating stem cell-derived lineage plasticity; selecting a subject suitable for treatment by determining that the subject is suitable for treatment if one or more of criteria (i) to (iii) are met; and treating the subject by administering a therapeutically effective amount of the pentaza macrocyclic ring complex corresponding to formula (I) to the subject, if the subject has been selected as suitable for treatment.

[0014] In a further embodiment, a method for treating a tumor in a mammalian subject suffering from a tumor resistant to ionizing radiotherapy, wherein the tumor has (i) levels of sirtuin (SIRT3) protein below a first predetermined threshold, and (ii) levels of K68 acetylated manganese superoxide dismutase (MnSOD) above a second predetermined threshold. K68 (iii) the level of (iii) hypoxia-inducible factor 2 exceeding a third predetermined threshold indicating stem cell lineage plasticity. α (HIF2α The tumor signature is characterized by one or more expression levels of ), and the method is to (a) obtain a test tissue sample containing tumor cells from the subject, (b) (i) whether the level of sirtuin (SIRT3) protein activity in tumor cells of the tissue sample is below a first predetermined threshold, (ii) whether the level of acetylated (AcK68) manganese superoxide dismutase at lysine 68 residue is above a second predetermined threshold, and (iii) hypoxia-inducible factor 2 α (HIF2 α A method is provided for treating cancer in a mammalian subject, comprising: (c) evaluating a tissue sample to determine a criterion including whether the expression level of exceeds a third predetermined threshold indicating stem cell lineage plasticity; (c) selecting a subject suitable for treatment by determining that the subject is suitable for treatment if one or more of criteria (i) to (iii) are met; and, if the subject is selected as suitable for treatment, treating the subject by administering a therapeutically effective amount of a pentaza macrocyclic ring complex corresponding to formula (I) to the subject.

[0015] In a further embodiment, a method for treating cancer in a mammal affected by cancer is provided, comprising the steps of administering a therapeutically effective amount of a therapeutic agent that inhibits a hormone receptor pathway related to the growth or progression of cancer to the subject, and administering a therapeutically effective amount of a pentaza macrocyclic complex corresponding to formula (I) to the subject before, simultaneously with, or after administration of the therapeutic agent.

[0016] In another embodiment, a method is provided for treating cancer recurrence and / or reducing the likelihood of cancer recurrence in a mammalian subject at risk of cancer, comprising the step of administering to the subject a therapeutically effective amount of a pentaza macrocyclic complex corresponding to formula (I), which may be used in combination with further anticancer agents.

[0017] According to one embodiment, a method is provided for treating a tumor in a mammal that is resistant to a therapeutic agent that inhibits a hormone receptor pathway related to the growth or progression of cancer, the method comprising administering to the subject a therapeutically effective amount of a pentaza macrocyclic complex corresponding to formula (I), and further anticancer drugs may be used in combination.

[0018] In another embodiment, a method for treating cancer in a cancer-affected mammalian subject is provided, comprising the steps of administering a therapeutically effective amount of an endocrine therapy agent to the subject, and administering a therapeutically effective amount of a pentaza macrocyclic complex corresponding to formula (I) to the subject before, simultaneously with, or after administration of the endocrine therapy agent.

[0019] In yet another embodiment, a method for preventing and / or reducing the likelihood of cancer development and / or recurrence in a mammalian subject at risk of cancer includes the steps of administering a therapeutically effective amount of an endocrine therapy agent to the subject, and administering a therapeutically effective amount of a pentaza macrocyclic complex corresponding to formula (I) to the subject before, concurrently with, or after administration of the endocrine therapy agent.

[0020] Further embodiments provide a method for treating cancer in a mammalian subject that has cancer, or for preventing and / or reducing the likelihood of cancer development and / or recurrence in a mammalian subject at risk of cancer, comprising the steps of: determining whether the mammalian subject exhibits a biomarker indicating the expression of K68-acetylated manganese superoxide dismutase (MnSOD) above a predetermined level; and, if it is determined that the mammalian subject exhibits a biomarker indicating the expression of K68-acetylated MnSOD above a predetermined level, administering a therapeutically effective amount of a pentaza macrocyclic complex corresponding to formula (I) to the subject.

[0021] In yet another embodiment, a method for reducing resistance to endocrine therapy in mammalian subjects exhibiting resistance to endocrine therapy includes the steps of administering a therapeutically effective amount of the endocrine therapy agent to the subject, and administering a therapeutically effective amount of a pentaza macrocyclic complex corresponding to the following formula (I) to the subject before, simultaneously with, or after administration of the endocrine therapy agent. [Brief explanation of the drawing]

[0022] [Figure 1A] Figures 1a–1d are graphs and images illustrating that MnSODK68Q expression promotes transformation tolerance phenotypes in vitro; Figure 1a shows the immortalization of pMEFs infected with lenti-MnSODWT, lenti-MnSODK68R, and lenti-MnSODK68Q, as well as either lenti-Myc or lenti-Ras, i.e., growth beyond 15 passages. Experiments were performed in triplicates. Scale bar: 20 μm. [Figure 1B] Figures 1a–1d are graphs and images illustrating that MnSODK68Q expression promotes a transformation tolerance phenotype in vitro; Figure 1b shows the above cell lines tested for growth (top) and colony formation (bottom panel) on soft agar. Experiments were performed in triplicates. Scale bar: 20 μm. [Figure 1C] Figures 1a–1d are graphs and images illustrating that MnSODK68Q expression promotes a transformation tolerance phenotype in vitro; Figure 1c shows pMEF infected with RasG12V, tested for immortalization, doubling time, and growth on soft agar. Experiments were performed in triplicates. Scale bar: 20 μm. [Figure 1D] Figures 1a–1d are graphs and images illustrating that MnSODK68Q expression promotes a transformation tolerance phenotype in vitro; Figure 1d shows NIH 3T3 cells expressing MnSODWT, MnSODK68R, and MnSODK68Q being tested for proliferation (top) and colonization (bottom panel) on soft agar. The experiment was performed in triplicates. Scale bar: 20 μm. [Figure 2A]Figures 2a–2f are graphs and plots illustrating that MnSODK68Q expression increases xenograft tumor growth and in vitro growth; Figures 2a and 2b show that MCF7 cells expressing MnSODWT, MnSODK68R, and MnSODK68Q were transplanted into the hind limbs of nude mice (n=10 per group) and tested for xenograft tumor growth. All experiments were performed in triplicate. Error bars represent ±1 SEM. One-way ANOVA analysis with Tukey's post-test was used. **p<0.01 and ***p<0.001 [Figure 2B] Figures 2a–2f are graphs and plots illustrating that MnSODK68Q expression increases xenograft tumor growth and in vitro growth; Figures 2a and 2b show that MCF7 cells expressing MnSODWT, MnSODK68R, and MnSODK68Q were transplanted into the hind limbs of nude mice (n=10 per group) and tested for xenograft tumor growth. All experiments were performed in triplicate. Error bars represent ±1 SEM. One-way ANOVA analysis with Tukey's post-test was used. **p<0.01 and ***p<0.001 [Figure 2C] Figures 2a–2f are graphs and plots illustrating that MnSODK68Q expression increases xenograft tumor growth and in vitro growth; Figure 2c shows IF stained with Ki-67 and DAPI. All experiments were performed in triplicate. Error bars represent ±1 SEM. One-way ANOVA analysis with Tukey's post-test was used. **p<0.01 and ***p<0.001 [Figure 2D] Figures 2a–2f are graphs and plots illustrating that MnSODK68Q expression increases xenograft tumor growth and in vitro growth; Figure 2d shows the quantification by Ki-67 intensity measured by ImageJ analysis. All experiments were performed in triplicate. Error bars represent ±1 SEM. One-way ANOVA analysis with Tukey's post-test was used. **p<0.01 and ***p<0.001 [Figure 2E]Figures 2a–2f are graphs and plots illustrating that MnSODK68Q expression increases xenograft tumor growth and in vitro growth; Figures 2e and 2f show T47D cells expressing MnSODWT, MnSODK68R, and MnSODK68Q, with Figure 2e showing IF stained with Ki-67 and DAPI. All experiments were performed in triplicate. Error bars represent ±1 SEM. One-way ANOVA analysis with Tukey's post-test was used. **p<0.01 and ***p<0.001 [Figure 2F] Figures 2a–2f are graphs and plots illustrating that MnSODK68Q expression increases xenograft tumor growth and in vitro growth; Figures 2e and 2f show T47D cells expressing MnSODWT, MnSODK68R, and MnSODK68Q, and Figure 2f quantifies Ki-67 intensity. All experiments were performed in triplicates. Error bars represent ±1 SEM. One-way ANOVA analysis with Tukey's post-test was used. **p<0.01 and ***p<0.001 [Figure 3A] Figures 3a–3e are images and graphs illustrating how MnSOD-K68Q alters the three-dimensional structure of MnSOD and exhibits peroxidase activity. Figure 3a shows MCF7 (left panel) and T47D (right panel) cells expressing MnSODWT, MnSODK68R, or MnSODK68Q, analyzed by semi-native crosslinking and blotting with anti-MnSOD antibodies. All experiments were performed in triplicates. One-way ANOVA statistical analysis with Tukey's post-test was used. [Figure 3B] Figures 3a–3e are images and graphs illustrating how MnSOD-K68Q alters the three-dimensional structure of MnSOD and exhibits peroxidase activity, while Figure 3b shows MCF7 (left) and T47D (right) cells expressing shSIRT3, analyzed by cross-linking. All experiments were performed in triplicates. One-way ANOVA statistical analysis with Tukey's post-test was used. [Figure 3C]Figures 3a–3e are images and graphs illustrating how MnSOD-K68Q alters the three-dimensional structure of MnSOD and exhibits peroxidase activity, while Figure 3c shows Flag-MnSODWT, Flag-MnSODK68R, and Flag-MnSODK68Q expressed in MCF7 cells with measured peroxidase activity. Error bars represent ±1 SEM. **p<0.01; all experiments were performed in triplicates. One-way ANOVA statistical analysis with Tukey's post-test was used. [Figure 3D] Figures 3a–3e are images and graphs illustrating how MnSOD-K68Q alters the three-dimensional structure of MnSOD and exhibits peroxidase activity, while Figure 3d shows immortalized MnSOD- / -pMEFs expressing MnSODWT, MnSODK68R, or MnSODK68Q, analyzed by seminative crosslinking and immunoblotted with anti-MnSOD antibody. All experiments were performed in triplicate. One-way ANOVA statistical analysis with Tukey's post-test was used. [Figure 3E] Figures 3a–3e are images and graphs illustrating how MnSOD-K68Q alters the three-dimensional structure of MnSOD and exhibits peroxidase activity. Figure 3e shows measurements of transformations of MnSOD- / -pMEFs expressing MnSODWT, MnSODK68R, or MnSODK68Q, with or without Ad-Mito-Cat or Ad-Empty. All experiments were performed in triplicate. One-way ANOVA statistical analysis with Tukey's post-test was used. [Figure 4-1]Figures 4a–4i are images and plots illustrating that peroxidase activity arises from the physical acetylation of MnSOD-K68; Figures 4a–4d show immortalized MnSOD- / -MEFs expressing Flag-MnSODWT cultured in NAM+TSA or NAD+ and isolated using a 50kDa molecular cutoff membrane; Figure 4a shows the measurement of MnSOD-K68-Ac, MnSOD, and actin immunoreactive protein levels; Figure 4b shows peroxidase activity; Figure 4c shows MnSOD activity in fractions less than 50kDa; Figure 4d shows MnSOD activity in fractions greater than 50kDa; Figure 4e shows recombinant MnSOD-WT and MnSOD-K68-Ac proteins produced and purified by bacteria, characterized by size exclusion column chromatography. Standard values ​​are shown. All experiments were performed in triplicate. Errors represent ±1 SEM. ***p<0.01. A t-test was used to compare the means of the two groups. [Figure 4-2] Figures 4a–4i are images and plots illustrating the generation of peroxidase activity by physical acetylation of MnSOD-K68; Figures 4f and 4g show elution volumes 13 and 14 mL, corresponding to peaks 1 (4f) and 2 (4g) from Figure 4e, as well as elution volumes 16 and 17 mL, analyzed for MnSOD and MnSOD-K68-Ac immunoblotting (upper panel) or Coomassie brilliant blue staining (lower panel). All experiments were performed in triplicate. Errors represent ±1 SEM. ***p<0.01. A t-test was used to compare the mean values ​​of the two groups. [Figure 4-3] Figures 4a–4i are images and plots illustrating the generation of peroxidase activity by physical acetylation of MnSOD-K68; Figures 4h and 4i show the results of analyzing peak 1 (elution volumes 13 and 14 mL) and peak 2 (elution volumes 16 and 17 mL) for superoxide dismutase activity, as shown in Figure 4h, and for peroxidase activity, as shown in Figure 4i. All experiments were performed in triplicate. Errors represent ±1 SEM. ***p<0.01. A t-test was used to compare the mean values ​​of the two groups. [Figure 5] Figures 5a-5h are plots showing that MnSODK68Q expression leads to oxidative stress in human mammary cells, with Figures 5a and 5b showing MnSOD activity, Figure 5a showing MCF7-MnSODWT, MCF7-MnSODK68R, and MCF7-MnSODK68Q in whole-cell homogenates, and Figure 5b showing T47D-MnSODWT, T47D-MnSODK68R, and T47D-MnSODK68Q; Figures 5c and 5d show MCF7-Mn as shown in Figure 5c. Figures 5d and 5f show steady-state levels of O2·- measured in SODWT, MCF7-MnSODK68R, and MCF7-MnSODK68Q cells, as well as T47D-MnSODWT, T47D-MnSODK68R, and T47D-MnSODK68Q cells; Figures 5e and 5f show H2O2 levels measured in these cells by flow cytometry-mediated CDCFH2 oxidation; and Figures 5g and 5h show glutathione levels measured in whole-cell homogenates of these cells. All experiments were performed in triplicate. Error bars represent ±1 SEM. *p<0.05, **p<0.01, and ***p<0.001. One-way ANOVA statistical analysis with Tukey's post-test was used. [Figure 6-1]Figures 6a–6h are plots and images illustrating that hydroxy-Tam-resistant breast cancer cells exhibit the MnSOD-K68-Ac signature; Figures 6a–6c show clonal cell survival experiments for MCF7-MnSODWT cells, MCF7-MnSODK68R cells, and MCF7-MnSODK68Q cells (Figure 6a), MCF7-shCtrl cells, and MCF7-shSIRT3 cells (Figure 6b), and experiments measuring cytotoxicity of MCF and MCF7-HTR cells with and without 120 hours of exposure to 1 μM hydroxy-Tam (HT) (Figure 6c); Figures 6d and 6e show immunoblots of MCF7 and MCF7-HTR, as well as T47D and T47D-HTR cell lysates, for MnSOD-K68-Ac, MnSOD, SIRT3, and actin. All experiments were performed in triplicates. Error bars represent ±1 SEM. **p<0.01 and ***p<0.001. Three groups were analyzed by one-way ANOVA with Tukey's post-test, and two groups were analyzed by t-test. [Figure 6-2]Figures 6a–6h are plots and images illustrating that hydroxy-Tam-resistant breast cancer cells exhibit the MnSOD-K68-Ac signature; Figures 6d and 6e show immunoblots of MCF7 and MCF7-HTR, as well as T47D and T47D-HTR cell lysates, for MnSOD-K68-Ac, MnSOD, SIRT3, and actin; Figure 6e shows quantified immunoreaction protein levels; Figures 6f–6h show MC The following shows clonal cell survival experiments for F7-HTR cells. Figure 6f shows cells expressing MnSODWT, MnSODK68Q, or MnSODK68R after treatment with 1 μM hydroxy-Tam for 120 hours; Figure 6g shows cells expressing SIRT3WT or SIRT3DN (S3DN; deacetylated null SIRT3 gene) after treatment with 1 μM hydroxy-Tam; and Figure 6h shows cells treated with 5 μM GC4419 for 5 days. All experiments were performed in triplicate. Error bars represent ±1 SEM. **p<0.01 and ***p<0.001. Three groups were analyzed by one-way ANOVA statistical analysis with Tukey's post-test, and two groups were analyzed by t-test. [Figure 7-1]Figures 7a-7k are images and plots illustrating that hydroxy-Tam exposure increases oxidative stress; Figure 7a shows whole cell lysates of MCF7 and MCF7-HTR used for the analysis of total MnSOD activity; Figures 7b and 7c show whole cell homogenates of MCF7 and MCF7-HTR, as well as T47D and T47D-HTR used for the measurement, with Figure 7b showing the steady-state level of O2·- due to MitoSox oxidation and Figure 7c showing H2O2 due to CDCFH2 oxidation; Figure 7d shows, Figure 7e shows glutathione levels in MCF7 and MCF7-HTR whole cell homogenates; Figure 7e shows seminative gel analysis of MCF7 and MCF7-HTR, as well as T47D and T47D-HTR cell lysates; Figures 7f-7h show whole cell homogenates of MCF7-HTR cells expressing MnSODWT, MnSODK68Q, or MnSODK68R, analyzed for steady-state levels of O2·- for Figure 7f, H2O2 for Figure 7g, and glutathione levels for Figure 7h. All experiments were performed in triplicate. Error bars represent ±1 SEM. *p<0.05 and ***p<0.001. Three groups were analyzed by one-way ANOVA statistical analysis with Tukey's post-test, and two groups were analyzed by t-test. [Figure 7-2] Figures 7a–7k are images and plots illustrating that hydroxy-Tam exposure increases oxidative stress; Figure 7i shows MCF7 and MCF7-HTR cells stained with Ki-67 and DAPI; Figures 7j and 7k show clonal survival experiments of MCF7-MnSODK68Q cells expressing AdMitoCat. All experiments were performed in triplicate. Error bars represent ±1 SEM. *p<0.05 and ***p<0.001. The three groups were analyzed by one-way ANOVA statistical analysis with Tukey's post-test, and the two groups were analyzed by t-test. [Figure 7-3]Figures 7a–7k are images and plots illustrating that hydroxy-Tam exposure increases oxidative stress; Figures 7j and 7k show clonal survival experiments of MCF7-MnSODK68Q cells expressing AdMitoCat. Cells were treated with 1 μM hydroxy-Tam for 120 hours and quantified in Figure 7k. All experiments were performed in triplicates. Error bars represent ±1 SEM. *p<0.05 and ***p<0.001. Three groups were analyzed by one-way ANOVA statistical analysis with Tukey's post-test, and two groups were analyzed by t-test. [Figure 8-1] Figures 8a–8g are images and plots illustrating that luminal B human mammary gland tumors exhibit the SIRT3 / MnSOD-K68-Ac signature. Figures 8a and 8b show MCF7 and MCF7-HTR cells (5.0 × 10⁶) transplanted into both hind limbs of nude mice, as well as tumor volume measured over 6 weeks, with error bars in Figure 8a representing ±1 SEM. [Figure 8-2] Figures 8a–8g are images and plots illustrating that luminal B human mammary gland tumors exhibit the SIRT3 / MnSOD-K68-Ac signature. Figures 8a and 8b show MCF7 and MCF7-HTR cells (5.0 × 10⁶) transplanted into both hind limbs of nude mice, as well as tumor volume measured at 6 weeks. Figure 8b shows representative images of tumors from MCF7-HTR (left panel) and MCF7 cells (right panel) at 6 weeks. [Figure 8-3] Figures 8a–8g are images and plots illustrating that luminal B human mammary gland tumors exhibit the SIRT3 / MnSOD-K68-Ac signature. Figure 8c shows MCF7-HTR doxycycline-inducible MnSODK68R cells transplanted into the hind limbs of nude mice, with tumor volume monitored for 4 weeks, where error bars represent ±1 SEM. [Figure 8-4] Figures 8a–8g are images and plots illustrating that luminal B human mammary tumors exhibit a SIRT3 / MnSOD-K68-Ac signature. Figure 8d shows luminal mammary cancer sample TMA stained with anti-MnSOD-K68-Ac or anti-SIRT3 antibody. [Figure 8-5] Figures 8a–8g are images and plots illustrating that luminal B human mammary tumors exhibit the SIRT3 / MnSOD-K68-Ac signature. Figures 8e and 8f show quantified TMA from luminal A (n=37) and luminal B (n=38) samples immunostained for MnSOD-K68-Ac in Figure 8e and for SIRT3 in Figure 8f. Shading represents the interquartile range, and whiskers represent the 10–90th percentile range. Experiments were performed in triplicates. *p<0.05. A t-test was used to compare data between two groups. Figure 8g is a schematic diagram of the dichotomous roles of MnSOD in normal cells (i.e., protection) versus tumor promoter and / or Tam resistance. [Figure 9]Figures 9a-9b are graphs and plots showing that MnSODK68Q expression shortens the doubling time, enabling xenograft proliferation and leading to estrogen independence. For Figure 9a, pMEFs were infected with lenti-Myc (control) and either lenti-MnSODK68R or lenti-MnSODK68Q. Cells were selected in puromycin for 14 days, and then the culture medium was changed every 2 days for 28 days, and the cell proliferation rate was evaluated. The doubling times for pMEFs-control, pMEF-Myc-MnSODK68R, and pMEF-Myc-MnSODK68Q cells (middle row) were determined by Td = (t241) * log(2) / log(q2 / q1). MCF7 cells infected with lenti-Myc (control) and either lenti-MnSODK68R or lenti-MnSODK68Q were also used in xenograft growth experiments in which 1 million cells were transplanted into both hind limbs of nude mice. Tumor volume was measured every 3 days. Control and Myc-MnSODK68R cells did not form tumors, but Myc-MnSODK68Q cells formed xenograft tumors. For Figure 9b, selected MCF7 cells infected with lenti-MnSODK68Q and treated in puromycin for 14 days were then transplanted into both hind limbs of nude mice with or without estrogen supplementation (black squares) or with estrogen supplementation (red circles). Tumor volume was measured every 7 days (1.0 × 10⁶ cells). The three upward arrows indicate that all infections of the hind limbs of 10 nude mice resulted in xenograft growth. All experiments were performed in 3-series. Error bars represent ±1 SEM. [Figure 10-1]Figures 10a–10f show images of MCF7 cells and MCF7-MnSODWT cells stained for Ki-67 levels (Figures 10a–10b). Figure 10a shows MCF7 cells and MCF7-MnSOD'AFT cells, and Figure 10b shows asynchronous growth cultures of T47D cells and T47D-MnSOD'AFT cells constructed by infection with lenti-MnSODWT or empty control lentivirus. After growing on glass coverslips for 24 hours, the cells were fixed and stained with anti-Ki-67 antibody and anti-DAPI antibody. Figures 10c–10d show images of MCF7-MnSODK68Q cells exposed to either estrogen or Tam and stained for Ki-67. MCF7-MnSODWK68QT cells were exposed to estrogen (E2) for 5 days, as shown in Figure 10c, or to 1 μM 4-hydroxy-Tam (HT) for 5 days, as shown in Figure 10d. The cells were re-seeded on glass coverslips with the same concentration of E2 or HT for 24 hours. After fixing, the cells were stained with anti-Ki-67 antibody and anti-DAPI antibody. Figures 10e-10f show plots, represented as bar graphs, quantifying the mean Ki-67 intensity in the panels shown in Figures 10c and 10d. All experiments were performed in triplicate. Error bars represent ±1 SEM. Representative IHC images are shown. [Figure 10-2] Figures 10e-10f show plots quantifying the average Ki-67 intensity in the panels shown in Figures 10c and 10d, and these are presented as bar graphs. All experiments were performed in triplicate. Error bars represent ±1 SEM. Representative IHC images are shown. [Figure 11-1]Figures 11a–11d are plots and images showing that MnSODK68Q expression promotes a transformation-tolerant phenotype in vitro. Figure 11a shows selected MnSOD- / -MEFs obtained by infecting cells with lenti-MnSODWT, lenti-MnSODK68R, and lenti-MnSODK68Q and culturing them in puromycin for 14 days. MnSOD- / -MEFs expressing MnSODK68Q showed a more transformed phenotype compared to cells expressing MnSODK68R or MnSODWT, and uninfected cells (MnSOD- / -). Figure 11b shows the results of measuring growth at low density by plating 100 or 250 cells per 60 mm dish from all four cell lines and staining the cells with crystal violet after 14 days. All experiments were performed in triplicate. Error bars represent ±1 SEM. ***p<0.001. A representative image is shown. [Figure 11-2] Figures 11a–11d are plots and images showing that MnSODK68Q expression promotes a transformation-tolerant phenotype in vitro. Figure 11c shows the results of counting colonies after plating 10,000 cells from all four cell lines onto 0.3% agar medium on 0.6% agar medium for 21 days. All experiments were performed in triplicate. Error bars represent ±1 SEM. ***p<0.001. Representative images are shown. [Figure 11-3] Figures 11a–11d are plots and images showing that MnSODK68Q expression promotes a transformation-tolerant phenotype in vitro. Figure 11d shows results for 20,000 cells from all four of these cell lines, plated per 60 mm dish, measured daily, and with doubling time determined by Td = (t2-t1)*log(2) / log(q2 / q1). All experiments were performed in triplicate. Error bars represent ±1 SEM. ***p<0.001. Representative images are shown. [Figure 12]Figures 12a–12c are images and plots illustrating the generation of peroxidase activity by physical acetylation of MnSOD-K68. For Figure 12a, 293T cells were transfected with a plasmid expressing Flag-MnSODWT, treated with either 10 mM NAM and 1 μM TSA or 10 mM NAD+, collected at 40 hours, and subjected to IP with anti-Flag antibody. The IP-treated samples were separated using a 50 kDa centrifuge filter, and protein extracts above and below 50 kDa were isolated and subsequently subjected to immunoblotting with anti-MnSOD, MnSOD-K68-Ac, and actin antibodies. For Figure 12b, samples expressing Flag-MnSODWT and treated with 10 mM NAM and 1 μM TSA or 10 mM NAD+ were separated using a 50 kDa centrifuge filter. Subsequently, the samples were run on seminative gels and immunoblotted with anti-MnSOD antibody. For Figure 12c, MnSOD- / - immortalized MEF cells were transfected with a plasmid expressing Flag-MnSODWT, treated with either 10 mM NAM and 111 M TSA, or 10 mM NAD+, and cells were collected at 40 hours for IP with anti-Flag antibody. The IP-treated samples were separated using a 50 kDa centrifuge filter, and protein extracts greater than 50 kDa were isolated. The purified proteins were used for biochemical analysis of peroxidase activity. All experiments were performed in triplicate. Error bars represent ±1 SEM. Representative images are shown. [Figure 13-1]Figures 13a–13f are images and plots illustrating the peroxidase activity observed when MnSOD-K68 is acetylated. BL21(DE3) bacteria were transformed with pEVOL-AcKRS along with either pET21a-MnSODWT or pET21a-MnSODK68TAG. Cells were collected and lysed, and the eluted proteins were strung on a Superdex 20Increase 10 / 300 GL column, fractions 1'2'3, and these samples were subsequently used for further analysis. Figure 13a shows chromatograms from a size exclusion column of purified protein from bacteria with pET21a-MnSODwT (upper panel). Retention fractions 11–20 were further analyzed by either Coomassie staining (center panel) or immunoblotting with anti-MnSOD antibody (lower panel) to confirm MnSOD levels. Figure 13b shows chromatograms of purified proteins from bacteria possessing pEVOL-AcKRS and pET21a-MnSODK68TAG. All fractions were further analyzed by either Coomassie staining (center panel) or immunoblotting with anti-MnSOD-K68-Ac antibody (bottom panel). The raw data are displayed with the Y-axis in mAU (280 nm) to show that peak 2 is smaller than peak 1, likely due to slightly less protein flowing through the Superdex 200 Increase 10 / 300GL column (5.5 mg vs. 4.8 mg). All experiments were performed in triplicates. Representative images are shown. [Figure 13-2]Figures 13a–13f are images and plots illustrating the peroxidase activity observed when MnSOD-K68 is acetylated. BL21(DE3) bacteria were transformed with pEVOL-AcKRS along with pET21a-MnSODWT or pET21a-MnSODK68TAG. Cells were collected and lysed, and the eluted proteins were passed through a Superdex 20Increase 10 / 300 GL column, fractions 1'2'3, and these samples were subsequently used for further analysis. For Figure 13c, three separate MnSOD-K68-WT samples were analyzed via mass spectrometry, yielding 32 exclusive and unique peptides, 164 spectra, and 999 whole spectra, each with a coverage of 100%, which is the average of the run. For Figure 13d, three separate MnSOD-K68-Ac samples showed 24 exclusive and unique peptides identified, 99 unique spectra, 531 total spectra, and a 95% coverage average across each run. All experiments were performed in triplicates. Representative images are shown. [Figure 13-3] Figures 13a–13f are images and plots illustrating the peroxidase activity observed when MnSOD-K68 is acetylated. BL21(DE3) bacteria were transformed with pEVOL-AcKRS along with pET21a-MnSODWT or pET21a-MnSODK68TAG. Cells were collected and lysed, and the eluted proteins were passed through a Superdex 20Increase 10 / 300 GL column, with fractions 1, 2, and 3, and these samples were subsequently used for further analysis. Figure 13e is a table showing the average percentage of the total number of unique K68 acetylated peptides as a percentage of the total number of unique peptides. Data on the total number of unique peptides, unique spectra, and total spectra from bacteria expressing pET21a-MnSODWT or pET21a-MnSODK68TAG are also shown. For Figure 13f, peak 1 (volumes 13 and 14 ml) and peak 2 (volumes 16 and 17 ml) were separated by SDS-PAGE and immunoblotted with anti-MnSOD-K68-Ac antibody. All experiments were performed in triplicate sets. Representative images are shown. [Figure 14]Figures 14a-14g are images and plots illustrating that the absence of SIRT3-induced MnSOD-K68 deacetylation leads to hydroxy-Tam resistance in human breast cancer cells. For Figure 14a, cells with hydroxy-Tam resistance at 1 μM for 3 months were selected from the T47D-MnSODWT, T47D-MnSODK68R, and T47D-MnSODK68D permanent cell lines, and clonal cell survival experiments were completed. For Figure 14b, T47D-shCtrl and T47D-shSIRT3 permanent cell lines were exposed to 1 μM 4-hydroxy-Tam for 24 hours (HT), and clonal cell survival experiments were conducted. For Figure 14c, cytotoxicity was measured by clonal survival experiments for T47D and T47D-HTR cells, comparing exposure to 1 μM 4-hydroxy-Tam for 24 hours (HT) with and without exposure. Regarding Figure 14d, MCF7 cells (left) and T47D cells (right) were cultured for 3 months in standard DMEM containing 1 μM hydroxy-Tam (HT). Cell lysates were analyzed by immunoblotting using anti-MnSOD-K122-Ac (validated as a SIRT3 deacetylation target by Tao et al., 2010, Cancer Cell), anti-MnSOD, anti-OSCP-K139-Ac (validated as a SIRT3 deacetylation target by Tao et al., 2010, Cancer Cell), anti-OSCP, anti-IDH2K413-Ac (validated as a SIRT3 deacetylation target by Someya et al., 2010, Cancer Cell), anti-IDH2, and anti-actin. For Figure 14e, T47D-HTR cells were infected with lenti-MnSODWT, lenti-MnSODK68D, or lenti-MnSODK68R, treated with 1 μM 4-HT for 24 hours, and then subjected to a clonal cell survival assay. For Figure 14f, T47D-HTR cells were infected with lenti-SIRTWT(S3) or lenti-SIRTDN(S3DN; dominant-negative deacetylated-null gene), treated with 1 μM hydroxy-Tam for 24 hours, and then subjected to a clonal cell survival assay.For Figure 14g, T47D-HTR cells were incubated with 5 μM GC4419 for 5 days, followed by a clonal cell viability assay. All experiments were performed in triplicate. Error bars represent ±1 SEM. *p<0.05, **p<0.01, and ***p<0.001. [Figure 15] Figures 15a–15e are graphs showing that increased oxidative stress in hydroxy-tam resistant human breast cancer cells can be reversed by MnSODK68R expression. For Figures 15a and 15b, selected T47D cells were collected over 3 months in 1 μM hydroxy-tam, and whole cell homogenates were used as follows: Figure 15a for biochemical analysis of total MnSOD activity, and Figure 15b for biochemical analysis of glutathione levels. For Figures 15c–15e, T47D-HTR cells were collected after infection with lenti-MnSODWT, lenti-MnSODK68R, or lenti-MnSODK68Q. Whole cell homogenates were used: Figure 15c for biochemical analysis of MitoSox oxidation, Figure 15d for biochemical analysis of H2O2 detected by CDCFH2 oxidation, and Figure 15e for biochemical analysis of glutathione levels. All experiments were performed in triplicates. Error bars represent ±1 SEM. *p<0.05, **p<0.01, and ***p<0.001. [Figure 16A] Figures 16a–16g show images and plots demonstrating that the MnSOD mimetic GC4419 reduced Ki-67 levels in T47D-HTR cells. For Figure 16a, data from Figure 7i, in which MCF7-HTR cells were stained with Ki-67 similarly to DAPI, were counted in ImageJ and the mean Ki-67 intensity was quantified as shown in the bar graph. All experiments were performed in triplicate. Error bars represent ±1 SEM. ***p<0.001. Representative images are shown. [Figure 16B]Figures 16a–16g are images and plots showing that the MnSOD mimetic GC4419 reduced Ki-67 levels in T47D-HTR cells. For Figures 16b and 16c, T47D and T47D-HTR cells were stained for Ki-67 as well as DAPI, and intranuclear particles were counted in ImageJ and quantified for mean Ki-67 intensity, as shown in the bar graph. All experiments were performed in triplicate. Error bars represent ±1 SEM. ***p<0.001. Representative images are shown. [Figure 16C] Figures 16a–16g are images and plots showing that the MnSOD mimetic GC4419 reduced Ki-67 levels in T47D-HTR cells. For Figures 16b and 16c, T47D and T47D-HTR cells were stained for Ki-67 as well as DAPI, and intranuclear particles were counted in ImageJ and quantified for mean Ki-67 intensity, as shown in the bar graph. All experiments were performed in triplicate. Error bars represent ±1 SEM. ***p<0.001. Representative images are shown. [Figure 16D] Figures 16a–16g show images and plots demonstrating that the MnSOD mimetic GC4419 reduced Ki-67 levels in T47D-HTR cells. In Figure 16d, MCF7-HTR cells were treated with 5 μM GC4419 and / or 1 μM 4-hydroxy-Tam for 5 days and stained for Ki-67 as with DAPI. The quantification of mean Ki-67 intensity is shown in the bar graph. All experiments were performed in triplicate. Error bars represent ±1 SEM. ***p<0.001. Representative images are shown. [Figure 16E] Figures 16a–16g show images and plots demonstrating that the MnSOD mimetic GC4419 reduced Ki-67 levels in T47D-HTR cells. In Figure 16e, MCF7-HTR cells were treated with 5 μM GC4419 and / or 1 μM 4-hydroxy-Tam for 5 days and stained for Ki-67 as with DAPI. The quantification of mean Ki-67 intensity is shown in the bar graph. All experiments were performed in triplicate. Error bars represent ±1 SEM. ***p<0.001. Representative images are shown. [Figure 16F] Figures 16a–16g show images and plots demonstrating that the MnSOD mimetic GC4419 reduced Ki-67 levels in T47D-HTR cells. In Figure 16f, T47D-HTR cells were treated with 5 μM GC4419 and / or 1 μM 4-hydroxy-Tam for 5 days and stained for Ki-67 as with DAPI. The quantification of mean Ki-67 intensity is shown in the bar graph. All experiments were performed in triplicate. Error bars represent ±1 SEM. ***p<0.001. Representative images are shown. [Figure 16G] Figures 16a–16g show images and plots demonstrating that the MnSOD mimetic GC4419 reduced Ki-67 levels in T47D-HTR cells. In Figure 16g, T47D-HTR cells were treated with 5 μM GC4419 and / or 1 μM 4-hydroxy-Tam for 5 days and stained for Ki-67 as with DAPI. The quantification of mean Ki-67 intensity is shown in the bar graph. All experiments were performed in triplicate. Error bars represent ±1 SEM. ***p<0.001. Representative images are shown. [Figure 17-1] Figures 17a–17e are images and plots illustrating that the MnSOD mimetic GC4419 reduced Ki-67 levels in MCF7 and T47D cells expressing MnSODK68Q. MCF7-MnSODK68Q cells (Figures 17a and 17b) were treated with 5 μM GC4419 and / or 1 μM 4-hydroxy-Tam for 5 days and then stained for Ki-67 as well as DAPI. Mean Ki-67 intensity was quantified and shown in bar graphs. Representative images are shown. All experiments were performed in triplicate. Error bars represent ±1 SEM. ***p<0.001. [Figure 17-2]Figures 17a–17e are images and plots illustrating that the MnSOD mimetic GC4419 reduced Ki-67 levels in MCF7 and T47D cells expressing MnSODK68Q. MCF7-MnSODK68Q cells (Figures 17a and 17b) were treated with 5 μM GC4419 and / or 1 μM 4-hydroxy-Tam for 5 days and then stained for Ki-67 as well as DAPI. Mean Ki-67 intensity was quantified and shown in bar graphs. Representative images are shown. All experiments were performed in triplicate. Error bars represent ±1 SEM. ***p<0.001. [Figure 17-3] Figures 17a–17e are images and plots illustrating that the MnSOD mimetic GC4419 reduced Ki-67 levels in MCF7 and T47D cells expressing MnSODK68Q. T47D-MnSODK68Q cells (Figures 17c and 17d) were treated with 5 μM GC4419 and / or 1 μM 4-hydroxy-Tam for 5 days and then stained for Ki-67 as with DAPI. Mean Ki-67 intensity was quantified and shown in bar graphs. Representative images are shown. All experiments were performed in triplicate. Error bars represent ±1 SEM. ***p<0.001. [Figure 17-4] Figures 17a–17e are images and plots illustrating that the MnSOD mimetic GC4419 reduced Ki-67 levels in MCF7 and T47D cells expressing MnSODK68Q. T47D-MnSODK68Q cells (Figures 17c and 17d) were treated with 5 μM GC4419 and / or 1 μM 4-hydroxy-Tam for 5 days and then stained for Ki-67 as well as DAPI. Mean Ki-67 intensity was quantified and shown in a bar graph. In Figure 17e, MCF7 and MCF7-HTR cells were transplanted into both hind limbs of nude mice, and tumor volume was measured every 3 days for 6 weeks, showing the number of successfully transplanted tumors relative to the total number of mice infected with MCF7 and MCF7-HTR, as well as mean tumor weight and tumor size. Representative images are shown. All experiments were performed in triplicate. Error bars represent ±1 SEM. ***p<0.001. [Figure 18]Figures 18a–18d are images and plots illustrating that Tet-On-induced expression of MnSODK68R inhibits xenograft proliferation of MCF7-HTR cells. Figure 18a shows MCF7-HTR cells infected with pTet-DualOn (Clontech), selected with puromycin, then infected with pTre-Dual2-Flag-MnSODK68R, and selected with hygromycin. Cells not exposed to tetracycline and exposed cells (MCF7-HTR-Tet-On-MnSODK68Q cells) were examined by immunofluorescence imaging for both green (presence of pTet-DualOn) and red (presence of pTre-Dual2-Flag-MnSODK68R). Figure 18b also shows the above MCF7-HTR-Tet-ON-MnSODK68Q cells, which were also isolated, separated by SDS-PAGE, and immunoblotted with anti-MnSOD, Flag, and tubulin antibodies. A subgroup of human luminal B tumors showed high levels of MnSOD-K68-Ac. Figures 18c and 18d show human breast cancer TMAs consisting of defatted luminal A (n=37) and luminal B (n=38) samples immunostained with either anti-MnSOD-K68-Ac (Figure 18c) or anti-SIRT3 antibody (Figure 18d). MnSOD-K68-Ac and SIRT3 staining were grouped into low, intermediate, and high levels, and the number of samples in each of these groups is shown in the table below each TMA. Red circles indicate tumor samples containing high MnSOD-K68-Ac staining. All experiments were performed in triplicates. A representative image is shown. [Figure 19]Figures 19a–19c are images and plots showing that LNCaP-ENZR increased MnSOD-K68-Ac. In Figure 19a, LNCaP-ENZR cells were selected by growing them in ENZ (10 μM) for several months (more than 3 months). Extracts from these cells were immunoblotted with anti-MnSOD-K68-Ac antibody and MnSOD antibody. In Figure 19b, control and LNCaP-ENZR cells were glutaraldehyde crosslinked, collected, and the extracts were separated by SDS-PAGE and immunoblotted with anti-MnSOD antibody. In Figure 19c, the extracts were assayed for MnSOD activity. Error bars are ±1 SEM. Triple experiments were performed. **p<0.01. [Figure 20] Figures 20a-20b are plots showing that MnSODK68R inverts ENZR in LNCaP-ENZR cells, while MnSODK68Q induces ENZR in LNCaP cells. In Figure 20a, clonal cell survival experiments were performed in LNCaP-ENZR cells infected with lenti-MnSODK68R or lenti-MnSODK68Q in the presence of ENZ (10 μM). In Figure 20b, clonal survival assays were performed in LNCaP cells infected with lenti-MnSODWT, lenti-MnSODK68R, and lenti-MnSODK68Q, selected with neomycin, and placed in 10 μM ENZ for 72 hours. Error bars represent ±1 SEM. The experiment was performed in 3 series. **p<0.01. [Figure 21]Figures 21a and 21b are charts and graphs showing that GC4419 inverted ENZR in LNCaP-ENZR / LNCaP-MnSODK68Q cells. In Figure 21(a), clonal cell survival experiments were conducted for 5 days with and without GC4419 (20 μM) in LNCaP-ENZR (two bars on the left) and LNCaP-MnSODK68Q (two bars on the right) cells treated with ENZ. In Figure 21b, LNCaP-MnSODK68Q cells exhibiting ENZR were transplanted into the hind limbs of male nude mice and treated with GC4419 (10 mg / kg, once a week), ENZ (25 mg / kg / day), or GC4419 + ENZ. Tumor volume was measured three times a week for 46 days. n=10 in each group. Error bars ±1 SEM. All experiments were performed in triplicate. **p<0.01. [Figure 22] Figures 22a–22b are plots and images showing that MnSOD-K68-Ac staining correlates with an increase in Gliason grade. In Figure 22a, samples were stained for MnSOD-K68-Ac and quantified by relative IHC staining intensity. Shading represents the interquartile range, and whiskers represent the 10–90th percentile. Figure 22b provides images showing MnSOD-K68-Ac staining in PIN, G3, and G4 prostate tumor tissue samples. [Figure 23] Figures 23a–23b show images and plots demonstrating that LNCaP-MnSODK68Q cells do not exhibit AR-related changes. In Figure 23a, LNCaP-MnSODK68Q cells were treated with ENZ for 3 months and immunoblotted with anti-AR antibody and actin antibody. In Figure 23b, LNCaP cells containing an AR promoter upstream of mCherry were infected with lenti-MnSODK68Q, and mCherry levels were measured. Error bars represent ±1 SEM. [Figure 24]Figures 24a–24b are images and plots showing that dysregulation of the MnSOD-Ac-K68-ROS-HIF2α axis indicates the stem cell phenotype in LNCaP. In Figure 24a, LNCaP and LNCaP-MnSODK68Q cells were collected and immunoblotted with antibodies against HIF2α, SOX2, Oct4, and actin. In Figure 24b, LNCaP (striped lines) and LNCaP-MnSODK68Q cells (black dots) were measured by clonal cell viability assays with and without ENZ and by infection with scrambled (con) or HIF2α shRNA. Experiments were performed in triplicate. Error bars are ±1 SEM. *P<0.05. [Figure 25] Figures 25a–25c are graphs showing Fulv-R (resistance to fulvestrant) and Palb-R (resistance to palbociclib) in MCF7-MnSODK68Q cells. Figures 25a–25b show the survival tests of clonal MCF7-MnSODK68Q cells exposed to either 100 nM Fulv (Figure 25a) or 0.5 μM Palb (Figure 25b) using a standard method. Figure 25c shows the results of exposure of MCF7-MnSODK68Q cells to GC4419 (10 μM) or Palb (0.5 μM) alone or in combination. Error bars represent ±1 SEM. Triple experiments were performed. ***p<0.001. [Figure 26] Figures 26a–26b are images and graphs demonstrating that dysregulation of the MnSOD-Ac-K68 / HIF2α axis leads to a stem cell phenotype. In Figure 26a, MCF7 and MCF7-MnSODK68Q cells were collected and immunoblotted with antibodies against HIF2α, SOX2, OCT4, and actin. In Figure 26b, MCF7 and MCF7-MnSODK68Q cells were measured by clonal cell viability experiments with either scrambled (Con) or HIF2 shRNA, both unexposed to Tam and exposed to Tam. All experiments were performed in triplicate. Error bars are ±1 SEM. *p<0.05. [Figure 27]Figures 27a–27c are images and graphs demonstrating that dysregulation of MnSOD-Ac-K68 / HIF2α induces PanR (resistance to cancer drugs). Figure 27a shows MCF7-Cispl-R cells (cisplatin-resistant cells-R) immunoblotted with anti-MnSOD-K68-Ac, MnSOD, HIF2α, or actin antibodies compared to control (C) MCF7 cells. In Figure 27b, ROS was measured in MCF7-Cispl-R cells compared to MCF7 cells using the Amplex Red assay. In Figure 27c, MCF7 and MCF7-MnSODK68Q cells were measured by scrambled (C) or HIF2α shRNA-infected clonal cell viability assays, both unexposed to Cispl and exposed to Cispl. All experiments were performed in triplicate. Error bars are ±1 SEM. *p<0.05. [Figure 28] Figures 28a–28c are images and graphs showing that cisplatin and doxorubicin-resistant breast cancer cells exhibit increased MnSOD-Ac levels. In Figures 28a–28b, cell lysates were collected from cisplatin-resistant (250 nM, 500 nM, 1 μM) and doxorubicin-resistant (500 pM, 1 nM, 2 nM) MCF7 cells (cultured in drug-containing medium for 3 months) and immunoblotted for MnSOD-K68-Ac, MnSOD, actin, and tubulin. In Figure 28c, 10,000 MCF7 cells overexpressing empty vectors, MnSODWT, MnSODK68R, or MnSODK68Q were plated in 96-well plates and treated the following day with 1 mM CDDP or 2 nM DXR. After 48 hours, an MTT assay was performed to measure cell viability after chemotherapy. [Figure 29]Figures 29a and 29b are plots showing that exposure to the MnSOD mimetic GC4419 inhibits the growth of mammary gland allograft tumors. Figure 29a shows Sirt3- / --MT-SIRT3DN, and Figure 29b shows Sirt3- / --MT-SIRT3WT tumor cells (1.0 × 10⁶ cells) bilaterally injected into the hind limbs of nude mice (n=10) and treated with 2 mg / kg GC4401 without IP infusion and with IP infusion of GC4401, starting on day 4. Subsequently, the mice were injected with luciferin potassium (120 mg / kg) weekly, and signal intensity was quantified. Error bars represent 1 SD from the mean. [Figure 30] Figures 30a and 30b are plots showing that MnSODK68Q expression induces ionizing radiation resistance (IRR) in MCF7 cells. Figure 30a shows that MCF7-MnSODWT and MCF7-MnSODK68Q cells were plated and exposed to 5 Gy of ionizing radiation, and clonal cell viability was measured. Figure 30b shows that MCF7-MnSODK68Q cells were treated with 5 μM GC4419 for 5 days, or exposed to 5 Gy of ionizing radiation without treatment. All experiments were performed in triplicate. Error bars represent ±1 SEM. ***p<0.001. Data were analyzed by t-test. [Modes for carrying out the invention]

[0023] Abbreviations and definitions The following definitions and methods are provided to better define the present invention and to guide those skilled in the art in carrying it out. Unless otherwise specified, terms shall be understood in accordance with the conventional usage by those skilled in the art in the relevant field.

[0024] As used herein, the term "AcK68" refers to the acetylated form of manganese superoxide dismutase (MnSOD) having acetylation at the K68 residue of the MnSOD protein, and may also be referred to herein as MnSOD-K68-Ac.

[0025] "Acyl" means the -COR moiety where R is an alkyl, haloalkyl, optionally substituted aryl, or optionally substituted heteroaryl as defined herein, such as acetyl, trifluoroacetyl, benzoyl, etc.

[0026] "Acyloxy" means the -OCOR moiety where R is an alkyl, haloalkyl, optionally substituted aryl, or optionally substituted heteroaryl as defined herein, such as acetyl, trifluoroacetyl, benzoyl, etc.

[0027] "Alkoxy" refers to the -OR portion where R is an alkyl group as defined above, such as methoxy, ethoxy, propoxy or 2-propoxy, n-butoxy, isobutoxy or tert-butoxy.

[0028] "Alkyl" refers to a linear saturated monovalent hydrocarbon moiety consisting of 1 to 6 carbon atoms, or a branched saturated monovalent hydrocarbon moiety consisting of 3 to 6 carbon atoms, such as C1-C6 alkyl groups including methyl, ethyl, propyl, 2-propyl, butyl (including all isomers), and pentyl (including all isomers).

[0029] Furthermore, unless otherwise indicated, the term “alkyl” as used herein is intended to include both “unsubstituted alkyl” and “substituted alkyl,” the latter referring to an alkyl moiety having substituents that substitute hydrogens on one or more carbons of a hydrocarbon skeleton. In practice, unless otherwise indicated, all groups enumerated herein are intended to include both substituted and unsubstituted options.

[0030] "C x-y The term "C" when used with chemical parts, for example alkyl and aralkyl, includes a group containing x to y carbon atoms in the chain. For example, "C x-yThe term "alkyl" refers to a substituted or unsubstituted saturated hydrocarbon group, and includes straight-chain alkyl and branched-chain alkyl groups containing x to y carbon atoms in the chain.

[0031] Unless otherwise specified, "alkylene" refers to a straight-chain saturated divalent hydrocarbon moiety (e.g., 1 to 6 carbon atoms) or a branched-chain saturated divalent hydrocarbon moiety (e.g., 3 to 6 carbon atoms), such as methylene, ethylene, propylene, 1-methylpropylene, 2-methylpropylene, butylene, pentylene, etc.

[0032] "Alkenyl" refers to a straight-chain unsaturated monovalent hydrocarbon moiety (e.g., 2 to 6 carbon atoms) or a branched-chain saturated monovalent hydrocarbon moiety (e.g., 3 to 6 carbon atoms), such as ethenyl (vinyl), propenyl, 2-propenyl, butenyl (including all isomers), and pentenyl (including all isomers).

[0033] "Alkalyl" refers to a monovalent part derived from the aryl part by substituting one or more hydrogen atoms with an alkyl group.

[0034] "Alkenylcycloalkenyl" refers to a monovalent part derived from the alkenyl moiety by substituting one or more hydrogen atoms with a cycloalkenyl group.

[0035] "Alkenylcycloalkyl" refers to a monovalent part derived from a cycloalkyl moiety by substituting one or more hydrogen atoms with an alkenyl group.

[0036] "Alkylcycloalkenyl" refers to a monovalent part derived from a cycloalkenyl moiety by substituting one or more hydrogen atoms with an alkyl group.

[0037] "Alkylcycloalkyl" refers to a monovalent part derived from a cycloalkyl moiety by substituting one or more hydrogen atoms with an alkyl group.

[0038] "Alkynyl" refers to a straight-chain unsaturated monovalent hydrocarbon moiety (e.g., 2 to 6 carbon atoms) or a branched-chain saturated monovalent hydrocarbon moiety (e.g., 3 to 6 carbon atoms), such as ethynyl, propynyl, butynyl, isobutynyl, hexynyl, etc.

[0039] "Alkoxy" refers to a monovalent part derived from an alkyl part by substituting one or more hydrogen atoms with a hydroxyl group.

[0040] "Amino" is -NR a R b Group(R a and R b (Independently, this means hydrogen, alkyl, or aryl.)

[0041] As used herein, “antibodies” include Fab, F(ab')2, Fd, and antibodies of class IgG, IgM, IgA, IgD, or IgE, or fragments or derivatives thereof, including single-chain antibodies, diabodies, bispecific antibodies, and bifunctional antibodies. Antibodies may be monoclonal antibodies, polyclonal antibodies, affinity-purified antibodies, or mixtures thereof, exhibiting sufficient binding specificity to a desired epitope or sequence derived therefrom. Antibodies may also be chimeric antibodies. Antibodies may be derivatized by attaching one or more chemicals, peptides, or polypeptide moieties known in the art. Antibodies may be conjugated with chemical moieties. Antibodies may be human or humanized antibodies.

[0042] "Aralkyl" refers to a monovalent part derived from an alkyl part by substituting one or more hydrogen atoms with an aryl group.

[0043] "Aryl" refers to a monovalent monocyclic or bicyclic aromatic hydrocarbon moiety consisting of 6 to 10 ring atoms, such as phenyl or naphthyl.

[0044] The term "ring" refers to a carbon-cyclic saturated monovalent hydrocarbon portion consisting of 3 to 10 carbon atoms.

[0045] "Cycloalkyl" refers to a cyclic saturated monovalent hydrocarbon moiety consisting of 3 to 10 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0046] "Cycloalkylalkyl" refers to a monovalent moiety derived from an alkyl moiety by substituting one or more hydrogen atoms with a cycloalkyl group, such as cyclopropylmethyl, cyclobutylmethyl, cyclopentylethyl, or cyclohexylethyl.

[0047] "Cycloalkylcycloalkyl" refers to a monovalent part derived from a cycloalkyl moiety by substituting one or more hydrogen atoms with a cycloalkyl group.

[0048] "Cycloalkenyl" refers to a cyclic monounsaturated monovalent hydrocarbon moiety consisting of 3 to 10 carbon atoms, such as cyclopropenyl, cyclobutenyl, cyclopentenyl, or cyclohexenyl.

[0049] "Cycloalkenylalkyl" refers to a monovalent moiety derived from an alkyl moiety by substituting one or more hydrogen atoms with a cycloalkenyl group, such as cyclopropenylmethyl, cyclobutenylmethyl, cyclopentenylethyl, or cyclohexenylethyl.

[0050] "Ether" refers to the monovalent part derived from the alkyl part by substituting one or more hydrogen atoms with an alkoxy group.

[0051] "Halo" means fluoro, chloro, bromo, or iodine, preferably fluoro or chloro.

[0052] A "heterocycle" or "heterocyclyl" is a ring with one or two ring atoms that are N, O, or S(O). n A heteroatom selected from the above, where n is an integer from 0 to 2, and the remaining ring atoms are carbon atoms, meaning a saturated or unsaturated monovalent monocyclic group of 4 to 8 ring atoms. The heterocyclyl ring may be fused to a (single) aryl or heteroaryl ring as defined herein, provided that the aryl and heteroaryl rings are monocyclic. A heterocyclyl ring fused to a monocyclic aryl or heteroaryl ring is also referred to herein as a “bicyclic heterocyclyl” ring. Furthermore, one or two ring carbon atoms of the heterocyclyl ring may be substituted with a -CO- group. More specifically, the term heterocyclyl includes, but is not limited to, pyrrolidino, piperidino, homopiperidino, 2-oxopyrrolidinyl, 2-oxopiperidinyl, morpholino, piperazino, tetrahydropyranyl, and thiomorpholino. If the heterocyclyl ring is unsaturated, it may contain one or two cyclic double bonds, provided the ring is not aromatic. If the heterocyclyl group is a saturated ring and is not fused to an aryl or heteroaryl ring as described above, it is also referred to herein as a saturated monocyclic heterocyclyl.

[0053] "Heteroaryl" refers to a monovalent monocyclic or bicyclic aromatic moiety of 5 to 10 ring atoms, where one or more, preferably one, two, or three ring atoms are selected from N, O, or S, and the remaining ring atoms are carbon. Typical examples include, but are not limited to, pyrrolyl, pyrazolyl, thienyl, thiazolyl, imidazolyl, furanyl, indolyl, isoindolyl, oxazolyl, isoxazolyl, benzothiazolyl, benzoxazolyl, benzimidazolyl, quinolinyl, isoquinolinyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridadinyl, triazolyl, and tetrazolyl.

[0054] "Nitro" means -NO2.

[0055] "Organosulfur" refers to a monovalent partial-SR group (where R is hydrogen, alkyl, or aryl).

[0056] "Substituted alkyl," "substituted ring," "substituted phenyl," "substituted aryl," "substituted heterocycle," and "substituted nitrogen heterocycle" mean alkyl, ring, aryl, phenyl, heterocycle, or nitrogen-containing heterocycle, respectively, which may be appropriately substituted with one, two, or three substituents independently selected from alkyl, alkoxy, alkoxyalkyl, halo, hydroxy, hydroxyalkyl, or organosulfur. In general, the term "substituted" includes C 1-4 Alkyl, C 2-4 The group includes one or more alkenyl, halogen, alcohol, and / or amine groups.

[0057] "Thioether" refers to a monovalent part derived from an alkyl moiety by substituting one or more hydrogen atoms with an -SR group (where R is alkyl).

[0058] As used herein, (i) the compounds described herein and in the drawings as compound 401, 4401 or GC4401 are described as the same compound; (ii) the compounds described herein and in the drawings as compound 403, 4403 or GC4403 are described as the same compound; (iii) the compounds described herein and in the drawings as compound 419, 4419 or GC4419 are described as the same compound; and (iv) the compounds described herein and in the drawings as compound 444, 4444 or GC4444 are described as the same compound.

[0059] Furthermore, the use of the term "essentially consisting of" in reference to a treatment method means that the method substantially does not include a step of providing other treatments and / or other activators other than those specifically described in the claims, in an amount and / or condition that would be sufficient to provide treatment. Similarly, the use of the term "essentially consisting of" in reference to a treatment kit means that the kit substantially does not include other treatments and / or other activators other than those specifically described in the claims, provided in an amount and / or condition that would be sufficient to provide treatment. Detailed explanation

[0060] In one embodiment, aspects of the present disclosure relate to treating cancers having a specific tumor signature using a pentaza macrocyclic complex in combination with other therapeutic agents as appropriate. Specifically, it has been unexpectedly discovered that a particular pentaza macrocyclic complex may treat patients with cancers characterized by increased levels of acetylation of manganese superoxide dismutase (MnSOD) having acetylation at the K68 residue of the MnSOD protein, and / or decreased levels of SIRT3 protein (see Zhu et al., Lysine 68 Acetylation Directs MnSOD as a Tetrameric Detoxification Complex Versus a Monomeric Tumor Promoter, Nature Communications, 10: 2399 (2019)). In a particular further embodiment, a particular pentaza macrocyclic complex may be used to treat hypoxia-inducible factor 2 exhibiting stem cell-like lineage plasticity. α (HIF2 αIt was unexpectedly discovered that it is possible to treat patients with cancer characterized by having increased expression levels of ). For example, in certain embodiments, the pentaza macrocyclic complex may be able to treat cancer that has inherent resistance to a particular therapeutic agent, and / or may be able to reduce and halt the development of resistance to a particular therapeutic agent, such as endocrine therapy agents such as tamoxifen and / or enzalutamide, and increase its effectiveness. That is, in some embodiments, the pentaza macrocyclic complex may provide treatment to tumors that are resistant to treatment with a therapeutic agent (e.g., tamoxifen) before treatment is initiated, and / or provide treatment to tumors that acquire resistance during treatment with such therapeutic agents. According to other embodiments, it may reduce resistance to other therapeutic agents used to treat cancer, such as chemotherapeutic agents including cisplatin and doxilubicin, thereby increasing effectiveness, and similar effects are achieved with respect to either innate and / or acquired resistance of tumors to chemotherapeutic agents. According to certain other embodiments, the particular pentaza macrocyclic complex described herein has relatively high levels of AcK68 and / or HIF2 α For cancers having a tumor signature characterized by and / or relatively low levels of SIRT3, treatment may be offered as monotherapy (i.e., without the need for further therapeutic agents, such as endocrine or chemotherapeutic agents).

[0061] In certain embodiments, manganese superoxide dismutase (MnSOD) functions as a tumor suppressor; however, when tumorigenesis occurs, clinical data suggest that MnSOD levels correlate with more aggressive human tumors, implying a potential dual function of MnSOD in metabolic regulation. The unexpected finding was that the MnSOD-K68 acetylation (Ac) mimic mutant (MnSODK68Q) functions as a tumor promoter. Interestingly, in various breast cancer cell types and primary cell types, MnSODK68Q expression is accompanied by a change in the stoichiometry of MnSOD from the known homotetrameric complex to a monomeric form. Biochemical experiments using MnSOD-K68Q Ac-mimic, or physically K68-Ac (MnSOD-K68-Ac), suggest that these monomers function as peroxidases distinct from the established MnSOD superoxide dismutase activity. MnSODK68Q-expressing cells showed resistance to tamoxifen (Tam), and cells selected for Tam resistance showed increased levels of K68-Ac and monomeric MnSOD. These results suggest a MnSOD-K68-Ac metabolic pathway for Tam resistance, oncogenesis, and tumor progression (see Zhu et al., Lysine 68 Acetylation Directs MnSOD as a Tetrameric Detoxification Complex Versus a Monomeric Tumor Promoter, Nature Communications, 10: 2399 (2019)).

[0062] According to further embodiments, tumors with elevated AcK68 levels exhibit cellular metabolic disturbances, increased levels of reactive oxygen species (ROS), and HIF2 αIt also demonstrated stabilization at the level of [specific level], which was an unexpected finding as it resulted in a phenotype of lineage plasticity, and therefore tumor cells resistant to therapeutic agents such as prostate cancer cells resistant to endocrine agents like enzalutamide. In further embodiments, unexpectedly, it was found that AcK expression leads to dysregulation of mitochondrial morphology and ultrastructure and disrupts mitochondrial metabolism, and that dysregulation and / or disruption of the physiological MnSOD-K68-Ac axis results in a phenotype of chemotherapy resistance in certain cancers such as ER+ breast cancer.

[0063] According to a particular embodiment, a method for treating cancer in a mammalian subject, wherein the cancer is characterized by (i) a level of sirtuin (SIRT3) protein below a first predetermined threshold, (ii) a level of lysine 68-residue acetylated (AcK68) manganese superoxide dismutase above a second predetermined threshold, and (iii) a level of hypoxia-inducible factor 2 above a third predetermined threshold indicating stem cell-like lineage plasticity. α (HIF2 α A method is provided comprising administering a therapeutically effective amount of a pentaza macrocyclic complex corresponding to formula (I) described herein to a mammalian subject, wherein the tumor signature is characterized by one or more expression levels of ). Cancer having a tumor signature that satisfies any of criteria (i) to (iii) may be resistant to treatments including, for example, treatment with one or more of the following: certain chemotherapeutic agents, endocrine agents, ionizing radiation, and / or other anticancer agents. According to certain embodiments, administration of a pentaza macrocyclic complex may reduce the cancer's resistance to treatment and improve its efficacy.

[0064] In yet another embodiment, a method for treating cancer in a mammalian subject is provided, comprising the step of selecting a subject suitable for treatment with a pentaza macrocyclic complex corresponding to formula (I). For example, the subject may be selected on the basis of exhibiting biomarkers such as tumor signatures that indicate potential responsiveness to such treatment. In another example, the subject may have and / or have acquired inherent resistance to other therapeutic agents such as endocrine and / or chemotherapeutic agents. In one embodiment, the method may include the steps of selecting a subject by obtaining a test tissue sample from a subject containing tumor cells, and testing the tissue sample for the presence of specific biomarkers. The test tissue sample can be obtained, for example, by biopsy or other conventional methods. In a particular embodiment, the tissue sample may be, for example, one or more of breast cancer and prostate cancer, as further described herein, including AcK68, SIRT3 and / or HIF2 α Subjects are selected from those suffering from a type of cancer involving one or more of the following dysregulations. SIRT3 may be, for example, the level of mitochondrial SIRT3. SIRT3 is a protein encoded in humans by the SIRT3 gene [sirtuin(silent mating type information regulation 2 homolog)3 (S. cerevisiae)], and SIRT3 is sometimes referred to as NAD-dependent deacetylase sirtuin-3. According to one embodiment, a tissue sample is evaluated based on criteria that: i) the level of sirtuin (SIRT3) protein in tumor cells of the tissue sample is below a first predetermined threshold; (ii) the level of lysine 68-residue acetylated (AcK68) manganese superoxide dismutase is above a second predetermined threshold; and (iii) hypoxia-inducible factor 2 α (HIF2 αIt can be tested by evaluating a tissue sample to determine whether the expression level of exceeds one or more of a third predetermined threshold indicating stem cell lineage plasticity. In yet another embodiment, a diagnostic method can be provided separate from the treatment with the pentaazamacrocyclic ring complex, the diagnostic method including the step of analyzing a tissue sample to determine any of the criteria (i)-(iii) described herein.

[0065] For example, in one embodiment, since a low level of SIRT3 can indicate the likelihood of tumor responsiveness to treatment with the pentaazamacrocyclic ring complex of formula (I), a tissue sample can be tested to determine whether criterion (i) of showing a relatively low level of SIRT3, as indicated by being below a predetermined threshold, is met. As another example, since a high level of AcK68 can indicate a high likelihood of tumor responsiveness to treatment with the pentaazamacrocyclic ring complex of formula (I), a tissue sample can be tested to determine whether criterion (ii) of tumor cells showing a relatively high level of AcK68, as indicated by exceeding a predetermined threshold, is met. As yet another example, a high level of HIF2 α can indicate a high likelihood of tumor responsiveness to treatment with the pentaazamacrocyclic ring complex of formula (I), so a tissue sample can be tested to determine whether criterion (iii) of tumor cells showing a relatively high level of HIF2 α is met, as indicated by exceeding a predetermined threshold. Thus, in certain embodiments, the treatment method can include the step of determining that the subject is suitable for treatment if any one or more of criteria (i), (ii), and / or (iii) are met. If the subject is selected as suitable for treatment, the treatment method includes the step of administering a therapeutically effective amount of the pentaazamacrocyclic ring complex corresponding to formula (I) in combination with additional therapeutic agents such as, as appropriate, endocrine therapy agents and / or chemotherapeutic agents and / or other suitable agents such as those described herein.

[0066] According to a particular embodiment, AcK68, SIRT3 and / or HIF2 αOne or more of these levels can be measured by appropriate methods such as immunohistochemistry or other similar methods. Immunohistochemistry uses antibodies against the protein and / or portion (e.g., a specific region of a protein) of interest to detect a specific target (e.g., a protein) in a sample. For example, in one embodiment, the antibody used for immunohistochemistry may include an anti-AcK68 monoclonal antibody that specifically binds to a region (epitope) of AcK68 containing an acetylated lysine residue. The presence of an antibody bound to a protein in a sample (e.g., tissue or cell) can be determined by various methods, including tagging or labeling the antibody at a detectable portion, such as a fluorescent dye detectable by a fluorescence detector and / or a peroxidase chromogenically that gives a colored product detectable by methods such as optical microscopy. In a further embodiment, one or more secondary antibodies can be used that are labeled and / or tagged at a detectable portion (e.g., peroxidase or fluorescent dye) and bind to a primary antibody targeting the protein of interest in the sample. In a further embodiment, the primary antibody may be labeled with a small molecule that interacts with a high-affinity binding partner linked to an enzyme or fluorescent moiety, such as by using a biotin-streptavidin interaction. Examples of immunostaining methods include immunohistochemical (IHC) methods for staining tissue samples, or immunocytochemical staining methods for staining cells. Other techniques that can be used to perform and / or complement immunostaining techniques include flow cytometry, Western blotting, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy. In a particular embodiment, immunoprecipitation may also be used to separate the target protein from the sample for further analysis (e.g., by coupling it with beads). In a further embodiment, other indirect methods for measuring the level of the target protein of interest include methods by measuring the activity level of the protein, or by measuring other factors that indicate expression, protein activation, and / or deactivation.

[0067] According to one embodiment, a kit for treating cancer in a mammalian subject is provided. According to a particular embodiment, the kit includes an assay for analyzing a tissue sample obtained from a subject and containing tumor cells, the assay determining whether (i) the level of sirtuin (SIRT3) protein in tumor cells of the tissue sample is below a first predetermined threshold, (ii) the level of lysine 68-residue acetylated (AcK68) manganese superoxide dismutase is above a second predetermined threshold, and (iii) hypoxia-inducible factor 2 α (HIF2 αIt is possible to determine a criterion including one or more of whether the expression level of ) exceeds a third predetermined threshold indicating stem cell lineage plasticity. According to a further aspect, the kit can further include a pentaazamacrocyclic ring complex corresponding to formula (I) in a therapeutically effective amount for the treatment of cancer when any of the criteria (i)-(iii) determined by the assay is satisfied. In one embodiment, the assay includes an immunostaining assay, such as an immunohistochemical assay or an assay corresponding to any of the immunostaining techniques described herein, for measuring the level of a target protein in a tissue sample. In a further embodiment, the assay can include an anti-AcK68 antibody that can selectively bind to AcK68 for measuring the level of AcK68. The assay can alternatively or additionally include tests that utilize techniques other than immunostaining to directly or indirectly evaluate the level of the target protein. In a further embodiment, the kit can further include any one or more of the use of an assay for measuring the target protein level, instructions for evaluating whether any of the criteria (i)-(ii) is satisfied based on the results of the assay, and / or instructions regarding the administration of the pentaazamacrocyclic ring complex. In a further embodiment, the kit can include an instrument and / or reagent for obtaining a tissue sample from a subject. The kit can also include one or more instruments and / or reagents for preparing a tissue sample for analysis, such as an instrument and / or reagent for forming a formalin-fixed paraffin-embedded tissue section. The kit can also include one or more instruments and / or reagents for performing an analysis of a tissue, such as a primary antibody, a secondary antibody, a label, a blocking reagent, a buffer, a dye, a peroxidase, a developing reagent. In yet another embodiment, a diagnostic kit can be provided separately from the pentaazamacrocyclic ring complex, and this diagnostic kit includes, for example, an assay for analyzing a tissue sample for determining any of the criteria (i)-(iii) described herein when the diagnosis is performed separately from the treatment.

[0068] According to one embodiment, the level of one or more of AcK68, SIRT3, and / or HIF2α is compared to a threshold level to determine whether the subject has a type of tumor that would benefit from treatment with the pentaza macrocyclic complex of formula (I), either alone or in combination with further therapeutic agents. That is, the target proteins (AcK68, SIRT3, and / or HIF2α) measured in tumor cells obtained from the subject are compared to a threshold level. α The level of the detected protein can be compared to a predetermined threshold to determine whether any of criteria (i) to (iii) are met. In one embodiment, the comparison to the threshold may include evaluating the ratio of the detected level of one or more target proteins in tumor cells to the level in "normal" or non-cancerous tissue of the same tissue type. For example, the threshold may be met if the ratio of the detected value to the "normal" value is a predetermined value or exceeds it. In another embodiment, the comparison to the threshold level may include comparing the value of the detected level of one or more target proteins in tumor cells to the value of the level in "normal" or non-cancerous tissue of the same tissue type, for example, to a predetermined level that exceeds the standard deviation of the level for "normal" or non-cancerous tissue. Other comparisons between the detected level and the threshold level may also be provided. For example, other metrics of the threshold level may be provided based on the level at which tumor resistance to treatment is observed. In one embodiment, the threshold is the level at which the detection level deviates from the mean level of each target protein in non-cancerous tissue of the same tissue type, obtained from multiple different individuals. For example, the mean level may be the average for each target protein measured in non-cancerous (normal) tissue obtained from at least six different individuals having the same histological type as the cancerous tissue (e.g., comparing cancerous breast tissue with non-cancerous breast tissue). The mean level for each target protein may constitute the normal score for that target protein in each histological type tested.

[0069] According to certain embodiments, threshold levels for comparison with the detection level of a target protein in tumor cells can be set to levels indicating that treatment with the Pentaza macrocyclic ring complex is beneficial. In one embodiment, the threshold level for each target protein can be set according to the relationship between the detection level of the target protein in tumor cells and the standard deviation from the normal score of that protein in non-cancerous tissue. That is, the threshold can be set to levels that are at least half a standard deviation from the normal score, at least one standard deviation from the normal score, at least one and a half standard deviations from the normal score, at least two standard deviations from the normal score, at least two and a half standard deviations from the normal score, at least three standard deviations from the normal score, at least four standard deviations from the normal score, and / or at least five standard deviations from the normal score. Thus, the detected level of one or more target proteins outside a given threshold indicates tumor tissue that may respond to treatment with the Pentaza macrocyclic ring complex. According to yet another embodiment, the normal score may constitute an average obtained over a large population, such as the value for members of a large population, for a particular type of immunohistochemical assay, and may provide a reference value that can be used in subsequent determinations. According to further embodiments, the target protein (AcK68, SIRT3, and / or HIF2 α One or more of the threshold levels of ) may be set according to alternative diagnostic methods and / or diagnostic correlations that provide a correlation between target level and therapeutic suitability. For example, the threshold levels may be set according to a method that provides substantially equivalent results to the immunohistochemical staining methods described herein, which enable determination of whether tumor cells are resistant to anticancer drugs (e.g., SIRT3, AcK68 and / or HITF). α They may be equivalent in that they provide an assessment of the target protein level (based on the level of other diagnostic results).

[0070] According to certain embodiments, target proteins (AcK68, SIRT3 and / or HIF2) in tumor cells αThe level of one or more of the following is measured by immunohistochemistry. According to a particular embodiment, the level of the target protein can be compared to its respective threshold level measured according to the same immunohistochemistry technique, such as obtaining levels for non-cancerous tissue of the same tissue type as the tumor cells (e.g., breast cancer cells, prostate cancer cells) from at least six different individuals and measuring the normal score. According to one embodiment, a first predetermined threshold for sirtuin (SIRT3) protein activity in tumor tissue is a standard deviation level less than 1 from the normal score for non-cancerous tissue of the same tissue type as the tumor tissue, and the normal score is determined by taking the average of at least six non-cancerous tissue samples of the same tissue type from at least six different individuals measured by immunohistochemistry. According to another embodiment, a second predetermined threshold for acetylated (AcK68) manganese superoxide dismutase at lysine 68 residues is a standard deviation level greater than 1 from the normal score for non-cancerous tissue of the same tissue type as the tumor tissue, and the normal score is determined by taking the average of at least six non-cancerous tissue samples of the same type from at least six different individuals measured by immunohistochemistry. According to yet another embodiment, hypoxia-inducible factor 2 α (HIF2 α A third predetermined threshold for the expression level of ) is a standard deviation level greater than 1 from the normal score of non-cancerous tissue of the same type as the tumor tissue, where the normal score is determined by taking the mean of at least six non-cancerous tissue samples of the same type from at least six different individuals, determined by immunohistochemistry. In other embodiments, each threshold may be set as different multiples and / or fractions of the standard deviation from the normal score, or according to other correlations, as described above.

[0071] According to one embodiment, the therapeutic method herein may include administering a therapeutic anticancer agent comprising one or more chemotherapeutic agents and therapeutic agents that inhibit hormone receptor pathways associated with cancer growth or progression (e.g., hormone therapy agents such as endocrine agents) before, concurrently with, or after administration of the pentaza macrocyclic ring complex of formula (I). Such further therapeutic agents may also be included, for example, as part of any kit described herein to provide co-therapy with the pentaza macrocyclic ring complex of formula (I). In one embodiment, the therapeutic agent may comprise a chemotherapeutic agent comprising either a platinum-containing chemotherapeutic agent or an anthracycline-based chemotherapeutic agent, and / or combinations thereof. In further embodiments, the therapeutic agent comprises at least one platinum-containing chemotherapeutic agent and / or a pharmaceutically acceptable salt thereof selected from the group consisting of cisplatin, oxaliplatin, carboplatin, nedaplatin, lovaplatin, heptaplatin, dicycloplatin, lipoplatin, LA-12, phosphaplatin, phenance lipatin, prolindac, triplatin tetranitrate, picoplatin, and satraplatin, and / or an anthracycline chemotherapeutic agent and / or a pharmaceutically acceptable salt thereof selected from the group consisting of doxorubicin, daunorubicin, epirubicin, and idarubicin.

[0072] In yet another embodiment, the therapeutic anticancer agent may include a therapeutic agent that inhibits a hormone receptor pathway associated with the growth or progression of cancer (e.g., an endocrine therapy agent). In one embodiment, the therapeutic agent that inhibits a hormone receptor pathway associated with the growth or progression of cancer targets one or more of the estrogen receptor pathway, the progesterone receptor pathway, and the androgen receptor pathway (e.g., a hormone therapy agent and / or an endocrine therapy agent). For example, a therapeutic agent that targets one or more of the estrogen receptor pathway, the progesterone receptor pathway, and the androgen receptor pathway may be any of the group consisting of estrogen receptor inhibitors, estrogen receptor degraders / downregulators, selective estrogen receptor modulators (SERMs), aromatase inhibitors, GnRH agonists, and CDK4 / 6 inhibitors, as well as combinations thereof. According to one embodiment, a therapeutic agent targeting the estrogen receptor pathway includes at least one selected from the group consisting of tamoxifen, clomiphene, 4-hydroxytamoxifen, toremifene, raloxifene, napoxidine, rasofoxifen, bazedoxifen, ospemifene, fulvestrant, brilliantrant, elastrant, palbociclib, abemaciclib, ribociclib and its derivatives, salts, and / or prodrugs. According to another embodiment, a therapeutic agent targeting the androgen receptor pathway includes any one selected from the group consisting of androgen receptor antagonists, androgen synthesis inhibitors, and antigonadotropins.For example, therapeutic drugs that target the androgen receptor pathway include cyproterone acetate, megestrol acetate, chlormadinone acetate, spironolactone, oxendron, osaterone acetate, flutamide, bicalutamide, nilutamide, topirutamide, enzalutamide, apalutamide, dienogest, drospirenone, medgestone, nomegestrol acetate, promegestone, trimegestone, ketoconazole, abiraterone acetate, ceviteronel, aminoglutethimide, finasteride, dutasteride, episteride, alpha-tradial, cyproterone acetate, spironolactone, medrogen It may contain at least one selected from the group consisting of ston, flutamide, nilutamide, bifluranol, leuprorelin, cetrorelix, allylestrenol, chlormadinone acetate, cyproterone acetate, gestolone caproate, hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, osaterone acetate, oxendrone, estradiol, estradiol esters, ethinylestradiol, conjugated estrogens, diethylstilbesterol, and their derivatives, salts, and / or prodrugs. In another embodiment, a therapeutic agent targeting the progesterone receptor pathway may include a type I, type II, or type III selective modulator (SPRM) of progesterone, which is at least one selected from the group consisting of onapristone, mifepristone, lonaprisan, agrepristone, Org31710, Org31806, CDB-2914, and CDB-4124, as well as their derivatives, salts, and / or prodrugs (see also Antiprogestins in Breast Cancer Treatment: Are We Ready? by Lanari et al., Endocrine-Related Cancer (2012) 19 R35-R500).

[0073] In one embodiment, a method is provided for treating tumors resistant to therapeutic anticancer drugs, such as chemotherapeutic agents, in mammals suffering from cancer. For example, tumors resistant to chemotherapeutic agents are characterized by (i) levels of sirtuin (SIRT3) protein below a first predetermined threshold, and (ii) levels of K68-acetylated manganese superoxide dismutase (MnSOD) above a second predetermined threshold. K68 (iii) the level of (iii) hypoxia-inducible factor 2 exceeding a third predetermined threshold indicating stem cell lineage plasticity. α (HIF2 α The tumor may have a tumor signature characterized by one or more expression levels of (i) to (iii). That is, a tumor signature characterized by any of (i) to (iii) may indicate resistance of tumor cells to treatment with chemotherapeutic agents. In a particular embodiment, the method obtains a test tissue sample containing tumor cells from a patient and determines whether (i) the level of sirtuin (SIRT3) protein activity in the tumor cells of the tissue sample is below a first predetermined threshold, (ii) the level of acetylated (AcK68) manganese superoxide dismutase at lysine 68 residue is above a second predetermined threshold, and (iii) hypoxia-inducible factor 2 α (HIF2 αThe method may include a step of selecting a subject suitable for treatment by evaluating a tissue sample to determine whether the expression level of (i) exceeds a third predetermined threshold indicating stem cell lineage plasticity, or any one or more of the following criteria are met. The method further includes a step of determining that the subject is suitable for treatment if one or more of criteria (i) to (iii) are met. According to a particular embodiment, if the subject is selected as suitable for treatment, the method may include a step of administering to the subject a therapeutically effective amount of the pentaza macrocyclic ring complex corresponding to formula (I), or administering any further therapeutic agent such as any of the agents described herein. Alternatively, or additionally, a diagnostic method can be performed to determine whether a tumor is resistant to a chemotherapeutic agent by evaluating the tissue to determine whether any of criteria (i) to (iii) are met, without requiring administration of the pentaza macrocyclic ring complex by formula (I). The treatment and / or diagnosis may also be performed by a kit that includes an assay to evaluate any of criteria (i) to (iii), such as any kit described herein. In one embodiment, the method may further include the step of administering a chemotherapeutic agent before, concurrently with, or after administration of the pentaza macrocyclic complex of formula (I), the chemotherapeutic agent being any of those described herein. The chemotherapeutic agent may also be provided as part of a kit for carrying out the treatment method, and / or the kit may include instructions for administering the chemotherapeutic agent as part of the treatment.

[0074] In another embodiment, a method is provided for treating tumors resistant to ionizing radiotherapy in cancer-affected mammalian subjects. For example, a tumor resistant to radiotherapy is characterized by (i) a level of sirtuin (SIRT3) protein below a first predetermined threshold, or (ii) a level of K68-acetylated manganese superoxide dismutase (MnSOD) above a second predetermined threshold. K68 (iii) the level of (iii) hypoxia-inducible factor 2 exceeding a third predetermined threshold indicating stem cell lineage plasticity. α (HIF2 αThe tumor signature may be characterized by one or more expression levels of (i) to (iii). That is, a tumor signature characterized by any of (i) to (iii) may indicate resistance of tumor cells to ionizing radiation therapy. According to a particular embodiment, the therapeutic method obtains a test tissue sample containing tumor cells from a subject and determines whether (i) the level of sirtuin (SIRT3) protein activity in the tumor cells of the tissue sample is below a first predetermined threshold, (ii) the level of (AcK68) manganese superoxide dismutase acetylated at lysine 68 residue is above a second predetermined threshold, and (iii) hypoxia-inducible factor 2 α (HIF2 α The method may include a step of selecting a subject suitable for treatment by evaluating a tissue sample to determine one or more criteria, including whether the expression level of (i) exceeds a third predetermined threshold indicating stem cell lineage plasticity. In a particular embodiment, if one or more of criteria (i) to (iii) are met, the subject is determined to be suitable for treatment. In a further embodiment, if the subject is selected as suitable for treatment, the method may include a step of treating the subject by administering a therapeutically effective amount of the pentaaza macrocyclic complex corresponding to formula (I) to the subject. That is, in a particular embodiment, the pentaaza macrocyclic complex corresponding to formula (I) can be administered to reduce the cancer / tumor's resistance to radiotherapy, including ionizing radiation. Accordingly, in a particular embodiment, the method may further include a step of administering ionizing radiation to the subject in the course of radiotherapy, for example, before, concurrently with, or after administration of the pentaaza macrocyclic complex, in accordance with and / or in combination with any of the radiotherapy / radiotherapy described further herein. In certain further embodiments, additional anticancer agents, such as endocrine and / or chemotherapeutic agents comprising any of those described herein, may also be provided.

[0075] According to one embodiment, a method for treating cancer in a mammalian subject afflicted with cancer comprises the steps of administering a therapeutically effective amount of a therapeutic agent (e.g., an endocrine agent) that inhibits a hormone receptor pathway associated with the growth or progression of cancer to the subject, and administering a therapeutically effective amount of a pentaza macrocyclic complex corresponding to formula (I) before, concurrently with, or after administration of the therapeutic agent. For example, according to a particular embodiment, the pentaza macrocyclic complex can reduce the resistance of tumor cells to the therapeutic agent or otherwise enhance the effect of the therapeutic agent. According to yet another embodiment, a method for treating and / or reducing the likelihood of cancer recurrence in a mammalian subject at risk of cancer recurrence comprises the steps of administering a therapeutically effective amount of a pentaza macrocyclic complex corresponding to formula (I) to the subject, which may be administered to the subject in combination with further therapeutic agents (e.g., chemotherapeutic agents or endocrine agents). For example, according to a particular embodiment, administration of the pentaza macrocyclic complex can be effective in treating cancer recurrence in the subject, for example, recurrence of tumors resistant to other treatments, and / or can reduce the likelihood of tumor recurrence by reducing the likelihood of the development of resistance to treatments. In yet another embodiment, a method for treating a tumor in a mammalian subject that is resistant to a therapeutic agent that inhibits a hormone receptor pathway associated with the growth or progression of cancer endocrine therapy includes administering a therapeutically effective amount of the pentaza macrocyclic ring complex equivalent to formula (I) to the subject, which may be administered to the subject in combination with the therapeutic agent. In further embodiments, the tumor's resistance to the therapeutic agent can be determined according to the method herein, for example, by determining whether criteria (i) to (iii) described herein are met. Furthermore, the method herein may further include administering one or more of the therapeutic agents described herein before, concurrently with, or after administration of the pentaza macrocyclic ring complex. Kits including assays such as those described herein for determining criteria (i) to (iii) may also be provided as diagnostic and / or therapeutic kits to carry out any part of the whole of the method herein, with or without the pentaza macrocyclic ring complex and / or additional therapeutic agents.

[0076] Therefore, in certain embodiments, the pentaza macrocyclic ring complex described herein may favorably treat and / or reduce the likelihood of recurrence or relapse of certain cancers, as provided in combination with therapeutic and / or endocrine therapy agents and / or to reduce the resistance of cancer cells to therapeutic treatment.

[0077] According to one embodiment, a method for treating cancer in a cancer-affected mammalian subject includes the steps of administering a therapeutically effective amount of an endocrine therapy agent to the subject, and administering a therapeutically effective amount of a pentaza macrocyclic complex corresponding to the following formula (I) before, simultaneously with, or after the administration of the endocrine therapy agent. For example, the endocrine therapy agent and the pentaza macrocyclic complex may constitute a combination therapy administered to treat cancer in an affected individual.

[0078] In yet another embodiment, a method for reducing the likelihood of cancer recurrence in mammalian subjects at risk of cancer recurrence includes the steps of administering a therapeutically effective amount of an endocrine therapy agent to the subject, and administering a therapeutically effective amount of a pentaza macrocyclic complex corresponding to formula (I) before, concurrently with, or after administration of the endocrine therapy agent. For example, a method for reducing the likelihood of recurrence may include administering a combination therapy of an endocrine therapy agent and a pentaza macrocyclic complex to a subject at risk of cancer recurrence and / or who has experienced cancer recurrence. In yet another embodiment, the subject may be in cancer remission, and the combination therapy is administered to reduce the likelihood of cancer recurrence.

[0079] In yet another embodiment, the use of predetermined thresholds in determining criteria (i) to (ii) is such that the predetermined threshold is such that the target protein (e.g., SIRT3, AcK68 and / or HIF2) is therapeutically significant, such as the degree of K68-acetylation. αThe threshold for the target protein can be set relative to the mean or median of the target protein in the general population so as to correlate with a therapeutically significant amount of the target protein. In one embodiment, a predetermined threshold for AcK68 is the level at which significant peroxidase activity occurs, indicating the presence of K68-acetylated and / or monomeric MnSOD. In another embodiment, a predetermined threshold for the target protein may correlate with a level indicating enhanced resistance to anticancer drug treatments such as endocrine therapy and / or chemotherapy, and / or an increased risk of cancer recurrence and / or cancer growth or proliferation in the subject.

[0080] In yet another embodiment, a method for reducing resistance to anticancer drugs, such as endocrine therapy and / or chemotherapy, in a mammalian subject resistant to anticancer drugs comprises the steps of administering a therapeutically effective amount of the anticancer drug to the subject, and administering a therapeutically effective amount of a pentaaza macrocyclic complex corresponding to formula (I) before, concurrently with, or after administration of the anticancer drug. For example, the pentaaza macrocyclic complex can reduce and / or reverse the resistance inherent in and / or developed in cancer cells to specific anticancer drugs, such as specific endocrine therapy agents and / or chemotherapy agents, thereby increasing and / or restoring the effect of treatment with endocrine therapy agents and / or chemotherapy agents. In one embodiment, the combination therapy can be provided, for example, when a mammalian subject has and / or acquired inherent resistance to endocrine therapy as a result of receiving an endocrine therapy regimen to treat the cancer the subject has. In another embodiment, the combination therapy can be provided when a mammalian subject has acquired resistance to endocrine therapy as a result of receiving an endocrine therapy regimen to reduce the likelihood of cancer recurrence in a mammalian subject at risk. According to another embodiment, combination therapy can be provided when a mammalian subject has acquired resistance to chemotherapy as a result of receiving a chemotherapy treatment regimen to treat the cancer it is suffering from. According to a particular embodiment, the pentaza macrocyclic complex can unexpectedly and favorably restore the sensitivity of cancer cells to anticancer drugs (e.g., endocrine therapy agents and / or chemotherapeutic agents) so that treatment with anticancer drugs can be achieved.

[0081] In one embodiment, the cancer and / or tumor to be treated and / or have a reduced likelihood of recurrence according to any method herein may be one selected from the group consisting of breast cancer, prostate cancer, testicular cancer, glioma, glioblastoma, head and neck cancer, ovarian cancer, endometrial cancer, hepatocellular carcinoma, tendonoid, pancreatic cancer, melanoma, and renal cell carcinoma (see also the paper, SIRT3 is a Mitochondrial-Localized Tumor Suppressor Required for Maintenance of Mitochondrial Integrity and Metabolism during Stress, by Kim et al, Cancer Cell, Vol. 16, 41-52 (2010)). In certain embodiments, the cancer may be one that is known to be treatable and / or acceptable with one or more endocrine therapy agents and / or chemotherapeutic agents, but in other embodiments, other cancers may also be treated and / or prevented. In one embodiment, the cancer to be treated and / or prevented according to any method herein is hormone receptor-positive (HR+) breast cancer. According to yet another embodiment, the cancer is either luminal type A breast cancer and / or luminal type B breast cancer. For example, in one embodiment, the cancer is luminal type B breast cancer. According to yet another embodiment, the cancer is characterized by impaired dismutase function associated with resistance to endocrine therapy and / or chemotherapy, an increased level of the acetylated form of manganese superoxide dismutase (MnSOD) having acetylation at the K68 residue of the MnSOD protein, and / or a decreased level of SIRT3 protein, and / or HIF2 α This includes cancer cells showing an increased level of [unclear]. According to yet another embodiment, cancers and / or tumors that may be treated and / or whose likelihood of recurrence may be reduced may be hormone receptor-positive (HR+) cancers such as estrogen receptor-positive (ER+) cancers, progesterone receptor-positive (PR+) cancers and / or androgen receptor-positive (AR+) cancers.

[0082] In yet another embodiment, the method described herein may further include the steps of: performing an evaluation of a mammalian subject to determine whether the mammalian subject would benefit from treatment with the pentaza macrocyclic complex as part of a combination therapy; and administering the pentaza macrocyclic complex as part of a combination therapy in response to the results of the evaluation. For example, the evaluation may consist of determining whether the mammalian subject has any of the characteristics described herein, such as cancer treatable with endocrine therapy agents and / or chemotherapeutic agents, cancer with inherent and / or acquired resistance to treatment with endocrine therapy agents and / or chemotherapeutic agents, and / or cancer exhibiting a feature of disrupted dismutase function (e.g., any of the criteria (i) to (iii) described herein); and whether there is a risk of recurrence, in particular, whether there are other characteristics that may indicate that administration of the pentaza macrocyclic complex would be advantageous. If the subject is identified as one that would benefit from the treatment and belongs to a population that would accept the treatment, the pentaza macrocyclic complex may be administered to improve the effect of anti-cancer therapy (e.g., endocrine therapy and / or chemotherapeutic therapy). According to certain embodiments, the pentaza macrocyclic complex can be administered in therapeutically effective doses that result in an increased cancer response corresponding to any of the following, selected from the group consisting of a reduction in tumor volume, a decrease in tumor growth rate, an increase in survival rate of mammalian subjects, a decrease in the occurrence and / or extent of metastases, and a decrease in cancer cell proliferation and / or a decrease in cancer complications. Furthermore, the methods herein may include additional cancer treatments in combination with any of the treatments described herein, such as radiotherapy, immunotherapy, and / or the administration of further chemotherapeutic agents. Transition metal pentaza macrocyclic ring complexes

[0083] In one embodiment, the pentaza macrocyclic ring complex is given by formula (I): [ka] (I) (In the formula, M is Mn 2+ or Mn 3+ and; R1, R2, R'2, R3, R4, R5, R'5, R6, R'6, R7, R8, R9, R'9, and R 10 are each independently hydrogen, hydrocarbyl, substituted hydrocarbyl, heterocyclyl, an amino acid side chain moiety, or -OR 11 , -NR 11 R 12 , -COR 11 , -CO2R 11 , -CONR 11 R 12 , -SR 11 , -SOR 11 , -SO2R 11 , -SO2NR 11 R 12 , -N(OR 11 )(R 12 ), -P(O)(OR 11 )(OR 12 ), -P(O)(OR 11 )(R 12 ), and -OP(O)(OR 11 )(OR 12 ), and is a moiety selected from the group consisting of; R 11 and R 12 are each independently hydrogen or alkyl; U together with adjacent carbon atoms of the macrocyclic ring forms a fused substituted or unsubstituted saturated, partially saturated or unsaturated ring or heterocycle having from 3 to 20 ring carbon atoms; V together with adjacent carbon atoms of the macrocyclic ring forms a fused substituted or unsubstituted saturated, partially saturated or unsaturated ring or heterocycle having from 3 to 20 ring carbon atoms; W together with the nitrogen of the macrocyclic ring and the carbon atom of the macrocyclic ring to which it is attached forms an aromatic or alicyclic substituted or unsubstituted saturated, partially saturated or unsaturated nitrogen-containing fused heterocycle having from 2 to 20 ring carbon atoms, provided that when W is a fused aromatic heterocycle, the hydrogen attached to the nitrogen which is part of both the heterocycle and the macrocyclic ring, and R1 and R 10 which are attached to the carbon atoms which are part of both the heterocycle and the macrocyclic ring, are considered not to be present; X and Y represent suitable ligands derived from either monodentate or polydentate ligands or ligand systems, or anions corresponding thereto; Z is a counterion; n is an integer from 0 to 3; The dotted line represents a coordination bond between the nitrogen atom of the macrocyclic ring and the transition metal manganese) corresponding to the complex.

[0084] As described above for the pentaazamacrocyclic complex of formula (I), M is Mn 2+ or Mn 3+ is. In one specific embodiment where the pentaazamacrocyclic complex corresponds to formula (I), M is Mn 2+ is. In another specific embodiment where the pentaazamacrocyclic complex corresponds to formula (I), M is Mn 3+ is.

[0085] R1, R2, R'2, R3, R4, R5, R'5, R6, R'6, R7, R8, R9, R'9, and R 10 In embodiments where one or more of are hydrocarbyl, for example, suitable hydrocarbyl moieties include, but are not limited to, alkenyl, alkenylcycloalkenyl, alkenylcycloalkyl, alkyl, alkylcycloalkenyl, alkylcycloalkyl, alkynyl, aralkyl, aryl, cycloalkenyl, cycloalkyl, cycloalkylalkyl, cycloalkylcycloalkyl, cycloalkenylalkyl, and aralkyl. In one embodiment, R1, R2, R'2, R3, R4, R5, R'5, R6, R'6, R7, R8, R9, R'9, and R 10 are independently hydrogen, hydrocarbyl, substituted hydrocarbyl, or heterocyclyl. More preferably, in this embodiment, R1, R2, R'2, R3, R4, R5, R'5, R6, R'6, R7, R8, R9, R'9, and R 10R is independently either hydrogen or a lower alkyl (e.g., C1-C6 alkyl, more typically C1-C4 alkyl). Thus, for example, R1, R2, R'2, R3, R4, R5, R'5, R6, R'6, R7, R8, R9, R'9, and R 10 R1, R2, R'2, R3, R4, R5, R'5, R6, R'6, R7, R8, R9, R'9, and R 10 These are independently hydrogen or methyl.

[0086] In one preferred embodiment where the pentaza macrocyclic complex corresponds to formula (I), R1, R2, R'2, R3, R4, R5, R'5, R7, R8, R9, R'9, and R 10 Each of these is hydrogen, and one of R6 and R'6 is hydrogen, while the other of R6 and R'6 is methyl. In this embodiment, for example, R1, R2, R'2, R3, R4, R5, R'5, R6, R7, R8, R9, R'9, and R 10 Each of these may be hydrogen, and R'6 is methyl. Alternatively, for example, R1, R2, R'2, R3, R4, R5, R'5, R'6, R7, R8, R9, R'9, and R 10 Each of these may be hydrogen, but R6 is methyl. In another preferred embodiment where the pentaza macrocyclic complex corresponds to formula (I), R1, R3, R4, R5, R'5, R'6, R7, R8, and R 10 Each of R1 and R'2 is hydrogen, one of R2 and R'2 is hydrogen, the other of R2 and R'2 is methyl, and one of R9 and R'9 is hydrogen, the other of R9 and R'9 is methyl. In this embodiment, for example, R1, R'2, R3, R4, R5, R'5, R7, R8, R9, and R 10 Each of these may be hydrogen, but R2 and R'9 are methyl. Alternatively, for example, R1, R2, R3, R4, R5, R'5, R7, R8, R'9, and R 10R'2 and R'9 may each be hydrogen, but R'2 and R'9 are methyl. In another embodiment where the pentaza macrocyclic complex corresponds to formula (I), R1, R2, R'2, R3, R4, R5, R'5, R6, R'6, R7, R8, R9, R'9, and R 10 These are hydrogen atoms, respectively.

[0087] In certain embodiments, the U and V portions are independently substituted or unsubstituted condensed cycloalkyl portions having 3 to 20 ring carbon atoms, more preferably 4 to 10 ring carbon atoms. In certain embodiments, the U and V portions are each trans-cyclohexanyl condensed rings.

[0088] In certain embodiments, the W moiety is a substituted or unsubstituted condensed heteroaromatic moiety. In certain embodiments, the W moiety is a substituted or unsubstituted condensed pyridino moiety. When W is a substituted condensed pyridino moiety, for example, the W moiety is typically substituted with a hydrocarbyl or substituted hydrocarbyl moiety (e.g., alkyl, substituted alkyl) at the ring carbon atom located para relative to the nitrogen atom of the heterocycle. In one preferred embodiment, the W moiety is an unsubstituted condensed pyridino moiety.

[0089] As described above, X and Y represent appropriate ligands derived from monodentate or polydentate ligands or ligand systems, or their corresponding anions (e.g., benzoic acid or benzoate anion, phenol or phenoxide anion, alcohol or alkoxide anion). For example, X and Y are other possibilities, but include halo, oxo, aco, hydroxo, alcohol, phenol, dioxygen, peroxo, hydroperoxo, alkylperoxo, arylperoxo, ammonia, alkylamino, arylamino, heterocycloalkylamino, heterocycloarylamino, amine oxide, hydrazine, alkylhydrazine, arylhydrazine, nitrogen oxide, cyanide, cyanate, thiocyanate, isocyanate, isothiocyanate, alkylnitrile, arylnitrile, alkylisonitrile, arylisonitrile, nitrate, nitrite, azide, alkylsulfonic acid, arylsulfonic acid, alkylsulfoxide, arylsulfoxide, alkylarylsulfoxide, alkylsulfenic acid, arylsulfenic acid, alkylsulfinic acid, arylsulfinic acid, alkylthiol carboxylic acid, arylthiol carboxylic acid, alkylthiol thiocarboxylic acid, arylthiol thiocarboxylic acid, alkyl Carboxylic acid, arylcarboxylic acid, urea, alkylurea, arylurea, alkylarylurea, thiourea, alkylthiourea, arylthiourea, alkylarylthiourea, sulfuric acid, sulfite, bisulfite, bisulfite, thiosulfite, thiosulfite, hydrosulfite, alkylphosphine, arylphosphine, alkylphosphine oxide, arylphosphine oxide, alkylarylphosphine oxide, alkylphosphine sulfide, arylphosphine sulfide, alkylarylphosphine sulfide, alkylphosphonic acid, arylphosphonic acid, alkylphosphine acid, arylphosphine acid, alkylphosphinic acid, arylphosphorus acid, arylphosphorus acid, arylphosphorus acid, arylphosphorus acid, arylphosphorus acid, phosphoric acid, thiophosphate, phosphorous acid, pyrophosphorus acid, triphosphate, hydrogen phosphate, dihydrogen phosphate, alkylguanidino, arylguanidino, alkylarylguanidino, alkylcarbamate, arylcarbamate, alkylarylcarbamate, alkylthiocarbamate, arylthiocarbamate,The following may be selected from the group consisting of alkylarylthiocarbamates, alkyldithiocarbamates, aryldithiocarbamates, alkylaryldithiocarbamates, bicarbonate, carbonic acid, perchloric acid, chloric acid, chlorous acid, hypochlorous acid, perbromic acid, bromic acid, bromic acid, hypobromous acid, tetrahalomanganates, tetrafluoroboric acid, hexafluoroantimonic acid, hypophosphorous acid, iodic acid, periodic acid, metaboric acid, tetraarylboric acid, tetraalkylboric acid, tartaric acid, salicylic acid, succinic acid, citric acid, ascorbic acid, saccharic acid, amino acids, hydroxamic acid, thiotosylic acid, and anions of ion exchange resins, or corresponding anions therein. In one embodiment, if present, X and Y may be independently selected from the group consisting of halo, nitric acid, and bicarbonate ligands. For example, in this embodiment, if present, X and Y are halo ligands, e.g., chloro ligands.

[0090] Furthermore, in one embodiment, X and Y correspond to -OC(O)-X1, where each X1 is -C(X2)(X3)(X4), and each X1 is independently a substituted or unsubstituted phenyl or -C(-X2)(-X3)(-X4); Each X2 is independently substituted or unsubstituted phenyl, methyl, ethyl, or propyl; Each X3 independently contains hydrogen, hydroxyl, methyl, ethyl, propyl, amino, and -X5C(=O)R 13 (In the formula, X5 is NH or O, R 13 (is a C1-C18 alkyl, substituted or unsubstituted aryl, or a C1-C18 aralkyl, or -OR) 14 (In the formula, R 14 (is a C1-C18 alkyl, substituted or unsubstituted aryl, or a C1-C18 aralkyl, or together with X4 (=O); Each X4 is either hydrogen independently, or together with X3 (=O).

[0091] In yet another embodiment, X and Y are independently selected from the group consisting of charge-neutralizing anions derived from either monodentate or polydentate ligands, as well as ligand systems and their corresponding anions; or X and Y are independently selected from R1, R2, R'2, R3, R4, R5, R'5, R6, R'6, R7, R8, R9, R'9, and R 10 It is combined with one or more of the following.

[0092] In the pentaza macrocyclic complex corresponding to equation (I), Z is the counterion (e.g., a charge-neutralizing anion), and n is an integer between 0 and 3. In general, Z may be the counterion of the above part for X and Y.

[0093] In the combination, a particular preferred embodiment is a pentaaza macrocyclic ring complex corresponding to formula (I), M is Mn 2+ or Mn 3+ and; R1, R2, R'2, R3, R4, R5, R'5, R6, R'6, R7, R8, R9, R'9, and R 10 These are independently hydrogen or a lower alkyl group; U and V are trans-cyclohexanyl condensed rings, respectively; W is a substituted or unsubstituted condensed pyridino moiety; X and Y are ligands; Z is the composite, which, if present, is a charge-neutralizing anion.

[0094] More preferably, in these embodiments, M is Mn 2+ R1, R2, R'2, R3, R4, R5, R'5, R6, R'6, R7, R8, R9, R'9, and R 10is independently hydrogen or methyl; U and V are trans-cyclohexanyl condensed rings, respectively; W is an unsubstituted condensed pyridino moiety; X and Y are independently halo ligands (e.g., fluoro, chloro, bromo, iodine). Z may be a halide anion (e.g., fluoride ion, chloride ion, bromide ion, iodide ion) if present.

[0095] In yet another embodiment, the pentaza macrocyclic ring complex is given by the following formula (II): [ka] (In the formula, X and Y represent appropriate ligands or corresponding anions derived from either monodentate or polydentate ligands or ligand systems; R A , R B , R C , and R D These are independently hydrogen, hydrocarbyl, substituted hydrocarbyl, heterocyclyl, amino acid side chain moiety, or -OR 11 , -NR 11 R 12 , -COR 11 , -CO2R 11 ,-CONR 11 R 12 , -SR 11 -SOR 11 , -SO2R 11 -SO2NR 11 R 12 , -N(OR 11 )(R 12 ), -P(O)(OR 11 )(OR 12 ), -P(O)(OR 11 )(R 12 ), and -OP(O)(OR 11 )(OR 12 This is a part selected from the group consisting of R 11 and R 12 It is represented by (which is independently hydrogen or alkyl).

[0096] Furthermore, in one embodiment, the pentaaza macrocyclic ring complex is formula (III) or formula (IV): [ka] (In the formula, X and Y represent appropriate ligands or corresponding anions derived from either monodentate or polydentate ligands or ligand systems; R A , R B , R C , and R D These are independently hydrogen, hydrocarbyl, substituted hydrocarbyl, heterocyclyl, amino acid side chain moiety, or -OR 11 , -NR 11 R 12 , -COR 11 , -CO2R 11 ,-CONR 11 R 12 , -SR 11 -SOR 11 , -SO2R 11 -SO2NR 11 R 12 , -N(OR 11 )(R 12 ), -P(O)(OR 11 )(OR 12 ), -P(O)(OR 11 )(R 12 ), and -OP(O)(OR 11 )(OR 12 This is a part selected from the group consisting of R 11 and R 12 It is represented by (which is independently hydrogen or alkyl).

[0097] In yet another embodiment, the pentaza macrocyclic ring complex is given by formulas (V) to (XVI): [ka] [ka] [ka] [ka] It is a compound represented by a formula selected from the group consisting of the following.

[0098] In one embodiment, X and Y in any of the formulas herein are independently selected from the group consisting of fluoro, chloro, bromo, and iodo anions. In yet another embodiment, X and Y in any of the formulas herein are independently selected from the group consisting of alkyl carboxylates, aryl carboxylates, and arylalkyl carboxylates. In yet another embodiment, X and Y in any of the formulas herein are independently amino acids.

[0099] In one embodiment, the pentaaza macrocyclic ring complex is given by the following formula (IA): [ka] (IA) (In the formula, M is Mn 2+ or Mn 3+ and; R 1A , R 1B , R2, R3, R 4A , R 4B R5, R6, R 7A , R 7B R8, R9, R 10A , and R 10B These are independently hydrogen, hydrocarbyl, substituted hydrocarbyl, heterocyclyl, amino acid side chain moiety, or -OR 11 , -NR 11 R 12 , -COR 11 , -CO2R 11 -C(=O)NR 11 R 12 , -SR 11 -SOR 11 , -SO2R 11 -SO2NR 11 R 12 , -N(OR 11 )(R 12 ), -P(=O)(OR 11)(OR 12 ), -P(=O)(OR 11 )(R 12 ), and -OP(=O)(OR 11 )(OR 12 It is a part that is independently selected from the group consisting of ), R 11 and R 12 These are independently hydrogen or alkyl; U, together with adjacent carbon atoms in a macrocyclic ring, forms condensed substituted or unsubstituted saturated, partially saturated or unsaturated rings or heterocycles having 3 to 20 ring carbon atoms; V, together with adjacent carbon atoms of the macrocyclic ring, forms condensed substituted or unsubstituted saturated, partially saturated or unsaturated rings or heterocycles having 3 to 20 ring carbon atoms; W, together with the nitrogen of the macrocyclic ring and the carbon atoms of the macrocyclic ring to which it is bonded, forms an aromatic or alicyclic substituted or unsubstituted saturated, partially saturated or unsaturated nitrogen-containing condensed heterocycle having 2 to 20 ring carbon atoms, provided that if W is a condensed aromatic heterocycle, the hydrogen bonded to the nitrogen in both the heterocycle and the macrocyclic ring, as well as R5 and R6 in both the heterocycle and the macrocyclic ring, are not present; Each X1 is independently a substituted or unsubstituted phenyl, or -C(-X2)(-X3)(-X4); Each X2 is independently a substituted or unsubstituted phenyl or alkyl group; Each X3 independently consists of hydrogen, hydroxyl, alkyl, amino, and -X5C(=O)R 13 (In the formula, X5 is NH or O, R 13 C1-C 18 Alkyl, substituted or unsubstituted aryl, or C1-C 18 (It is an allatrical form), or -OR 14 (In the formula, R 14 C1-C 18 Alkyl, substituted or unsubstituted aryl, or C1-C 18(is aralkyl) or together with X4 (=O); Each X4 is either independently hydrogen or together with X3 (=O); The bond between the transition metal M and the nitrogen atom of the macrocyclic ring, as well as the bond between the transition metal M and the oxygen atom of the axial ligand -OC(=O)X1, is a coordinate covalent bond.

[0100] In one embodiment, within formula (IA) and the groups contained therein, in one group of the compound, X1 is -C(-X2)(-X3)(-X4), and each of X2, X3, and X4 in combination corresponds to one of the combinations specified in the table below: [Table 1]

[0101] Furthermore, in embodiment (IA) and within the groups contained therein, in one group of the compound, X1 is C(-X2)(-X3)(-X4) and X3 is -X5C(=O)R 13 The combinations of X2, X3, and X4 include one of the combinations identified in the table below: [Table 2] (In the formula, R 13 C1-C 18 Alkyl, substituted or unsubstituted aryl or C1-C 18 Aralquil, or -OR 14 (In the formula, R 14 C1-C 18 Alkyl, substituted or unsubstituted aryl, or C1-C 18 (It is Aralquil.)

[0102] In one embodiment, the pentaaza macrocyclic ring complex corresponding to formula (IA) is a complex formula (IE), for example, (IE R1 ), (IES1 ), (IE R2 ), (IE S2 ), (IE R3 ), or (IE S3 ): [ka] [ka] [ka] (In the formula, M is Mn +2 or Mn +3 and; Each X1 is independently a substituted or unsubstituted phenyl or -C(X2)(X3)(X4); Each X2 is independently substituted or unsubstituted phenyl, methyl, ethyl, or propyl; Each X3 is independently hydrogen, hydroxyl, methyl, ethyl, propyl, amino, or together with X4 is =O; Each X4 is either independently hydrogen or, together with X3, equals O; The bond between manganese and the nitrogen atom of the macrocyclic ring, and the bond between manganese and the oxygen atom of the axial ligand -OC(O)X1 are examples of coordinate covalent bonds.

[0103] In one embodiment, each X1 is -C(X2)(X3)(X4), and each -C(X2)(X3)(X4) corresponds to one of the combinations 1 to 9 shown in the table for the above formula (IA).

[0104] In yet another embodiment, X and Y in the pentaaza macrocyclic ring complex of formula (I) correspond to ligands in formula (IA) or (IE). For example, X and Y in the complex of formula (I) may correspond to -OC(O)-X1, where X1 is as defined for the complexes of formulas (IA) and (IE) above.

[0105] In one embodiment, a pentaza macrocyclic ring complex corresponding to formula (I) (for example, any subset of formula (I) corresponding to formula (I) or formulas (II)-(XIV), (IA), and (IE)) has the following structure: [ka] [ka] [ka] [ka] [ka] [ka] [ka] It may include any of the following:

[0106] In one embodiment, the pentaaza macrocyclic ring complex for use in the methods and compositions described herein includes formulas (2), (3), (4), (5), (6), and (7): [ka] This includes equivalents to (X and Y in each of formulas (2), (3), (4), (5), (6), and (7) are ligands independently). For example, according to one embodiment, a pentaza macrocyclic complex for use in the methods and compositions described herein includes equivalents to formulas (2), (3), (4), (5), (6), and (7) (where X and Y in each of these formulas are halos, e.g., chloro). Alternatively, X and Y may be ligands other than chloro, e.g., any of the ligands described above.

[0107] In another embodiment, the pentaza macrocyclic ring complex is given by formula (6) or formula (7): [ka] It corresponds to this.

[0108] The chemical structures of 6 (e.g., the dichloro complex form described herein, as in Riley, DP, Schall, OF, 2007, Advances in Inorganic Chemistry, 59: 233-263) and 7 (e.g., the dichloro complex form of 7) are identical except that they possess enantiomer chirality (i.e., their enantiomer structures cannot be superimposed).

[0109] For example, the pentaza macrocyclic complex is the following complex: [ka] It may correspond to at least one of the following.

[0110] In yet another embodiment, the pentaza macrocyclic ring complex is the following complex: [ka] [ka] and / or may correspond to at least one of these enantiomers.

[0111] In one embodiment, the enantiomer purity of the pentaza macrocyclic complex is 95% or higher, more preferably 98% or higher, more preferably 99% or higher, and most preferably 99.5% or higher. As used herein, the term "enantiomer purity" means the amount of compound having the indicated absolute stereochemistry, expressed as a percentage of the total amount of the indicated compound and its enantiomers. In one embodiment, the diastereomer purity of the pentaza macrocyclic complex is 98% or higher, more preferably 99% or higher, and most preferably 99.5% or higher. As used herein, the term "diastereomer purity" means the amount of compound having the indicated absolute stereochemistry, expressed as a percentage of the total amount of the indicated compound and its diastereomers. Methods for determining diastereomer and enantiomer purity are well known in the art. Diastereomer purity can be determined by any analytical method capable of quantitatively distinguishing a compound and its diastereomers, for example, high-performance liquid chromatography (HPLC). Similarly, enantiomer purity can be determined by any analytical method capable of quantitatively distinguishing a compound and its enantiomers. Suitable analytical methods for determining enantiomeric purity include, but are not limited to, polarimetric analysis using a polarimeter and HPLC using a chiral column packing material.

[0112] In one embodiment, the therapeutically effective dose of pentaza macrocyclic complex may be sufficient to provide a peak plasma concentration of at least 0.1 μM when administered to a patient. For example, in one embodiment, pentaza macrocyclic complex may be administered in an amount sufficient to provide a peak plasma concentration of at least 1 μM when administered to a patient. In yet another embodiment, pentaza macrocyclic complex may be administered in an amount sufficient to provide a peak plasma concentration of at least 10 μM when administered to a patient. Generally, pentaza macrocyclic complex is not administered in an amount that would provide a peak plasma concentration exceeding 40 μM when administered to a patient. For example, pentaza macrocyclic complex may be administered in an amount sufficient to provide a peak plasma concentration in the range of 0.1 μM to 40 μM in a patient. As another example, pentaza macrocyclic complex may be administered in an amount sufficient to provide a peak plasma concentration in the range of 0.5 μM to 20 μM in a patient. As another example, the pentaza macrocyclic complex may be administered in a sufficient amount to provide a peak plasma concentration in the range of 1 μM to 10 μM in the patient.

[0113] In yet another embodiment, the dose of pentaza macrocyclic ring complex administered per kg of patient body weight may be at least 0.1 mg / kg, for example, at least 0.2 mg / kg. For example, the dose of pentaza macrocyclic ring complex administered per kg of patient body weight may be at least 0.5 mg / kg. In another example, the dose of pentaza macrocyclic ring complex administered per kg of patient body weight may be at least 1 mg / kg. In yet another example, the dose of pentaza macrocyclic ring complex administered per kg of body weight may be at least 2 mg / kg, for example, at least 3 mg / kg, and even at least about 15 mg / kg, for example, at least 24 mg / kg, and even at least 40 mg / kg. In general, the dose of pentaza macrocyclic ring complex administered per kg of patient body weight will not exceed 1000 mg / kg. For example, the dose of pentaza macrocyclic ring complex administered per kg of body weight may range from 0.1 to 1000 mg / kg, for example, 0.2 mg / kg to 40 mg / kg, for example, 0.2 mg / kg to 24 mg / kg, and even from 0.2 mg / kg to 10 mg / kg. Another example is that the dose of pentaza macrocyclic ring complex administered per kg of body weight may range from 1 mg / kg to 1000 mg / kg, for example, 3 mg / kg to 1000 mg / kg, and even from 5 mg / kg to 1000 mg / kg, for example, 10 mg / kg to 1000 mg / kg. Yet another example is that the dose of pentaza macrocyclic ring complex administered per kg of body weight may range from 2 mg / kg to 15 mg / kg. And yet another example is that the dose of pentaza macrocyclic ring complex administered per kg of body weight may range from 3 mg / kg to 10 mg / kg. As another example, the dose of pentaza macrocyclic ring complex administered per kg of body weight of a patient may be in the range of 0.5 to 5 mg / kg. Furthermore, as yet another example, the dose of pentaza macrocyclic ring complex administered per kg of body weight of a patient may be in the range of 1 to 5 mg / kg.

[0114] In one embodiment, the above-described dosage and / or plasma concentration may be particularly suitable for a pentaza macrocyclic complex corresponding to GC4419, but may also be suitable for other pentaza macrocyclic complexes. Furthermore, those skilled in the art will recognize how to adjust the dosage and / or plasma concentration based on factors such as the molecular weight and / or activity of the specific compound used. For example, for a pentaza macrocyclic complex having twice the activity of GC4419, the dosage and / or plasma concentration may be halved, and for a pentaza macrocyclic complex having a higher molecular weight than GC4419, a correspondingly higher dosage may be used.

[0115] The administration schedule for the pentaza macrocyclic complex can be similarly selected depending on the treatment to be pursued. For example, in one embodiment, a preferred administration schedule may include administering the pentaza macrocyclic complex to the patient at least once a week during the course of treatment, for example, two, three, four, five, six, or seven days a week (e.g., daily). In another example, in one embodiment, administration may be at least once a day (qd) or at least twice a day (bid). In one embodiment, the course of treatment with the pentaza macrocyclic complex may continue for at least as long as the course of treatment with anticancer drugs such as endocrine agents (e.g., tamoxifen or 4-hydroxytamoxifen) and / or chemotherapeutic agents, and may even exceed the period during which the anticancer drugs are provided. The course of treatment with the pentaza macrocyclic complex may also begin on the same day as treatment with endocrine therapy agents, or it may begin some time after the first dose of anticancer drugs, as will be described in more detail below. For example, in one embodiment, an anticancer drug may be administered for a treatment period of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, while the pentaza macrocyclic complex may be administered for a treatment period of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months. Anti-cancer drugs

[0116] According to one embodiment, an anticancer drug is provided as part of a therapeutic method herein, in combination with a pentaza macrocyclic complex. The anticancer drug may be one or more of the following: a drug that inhibits hormone receptor pathways associated with the growth or progression of cancer (e.g., endocrine agents, sometimes referred to as hormone therapy agents), and / or chemotherapeutic agents. Secretory agents are compounds that can block or interfere with the action of hormones on cancer cells (Lumachi et al., Curr Med Chem, 18(4): 513-522 (2011); Awan et al., Curr Oncol, 25(4): 285-291 (2018)). Cancers and / or tumor cells that contain hormone receptors and / or depend on hormones for growth may respond particularly well to endocrine therapy, such as estrogen receptor-positive (ER-positive) cells that use estrogen for growth. According to one embodiment, therapeutic agents that inhibit hormone receptor pathways associated with cancer growth or progression target one or more of the estrogen receptor pathway, the progesterone receptor pathway, and the androgen receptor pathway. For example, therapeutic agents that target one or more of the estrogen receptor pathway, the progesterone receptor pathway, and the androgen receptor pathway may include one or more of estrogen receptor inhibitors, estrogen receptor degraders / downregulators, selective estrogen receptor modulators (SERMs), aromatase inhibitors, GnRH agonists, and CDK4 / 6 inhibitors. According to another embodiment, the endocrine therapeutic agent includes a SERM compound selected from the group consisting of tamoxifen, clomiphene, 4-hydroxytamoxifen, toremifene, raloxifene, napoxidine, rasofoxifen, bazedoxifen, ospemifene, and their derivatives, salts, and / or prodrugs. According to yet another embodiment, the endocrine therapeutic agent includes a SERM compound having a triphenylethylene structure and / or a benzothiophene structure.In yet another embodiment, the endocrine therapy agent comprises a SERM selected from the group consisting of tamoxifen, 4-hydroxytamoxifen, and their derivatives, prodrugs, and / or salts.

[0117] In yet another embodiment, the anticancer drug targets the androgen receptor pathway and comprises one or more of the following: androgen receptor antagonists, androgen synthesis inhibitors and antigonadotropins. For example, therapies that target the androgen receptor pathway include cyproterone acetate, megestrol acetate, chlormadinone acetate, spironolactone, oxendrone, osaterone acetate, flutamide, bicalutamide, nilutamide, topirutamide, enzalutamide, apalutamide, dienogest, drospirenone, medgestone, nomegestrol acetate, promegestone, trimegestone, ketoconazole, abiraterone acetate, ceviteronel, aminoglutethimide, finasteride, dutasteride, episteride, alphatrazial, cyproterone acetate, spironolactone, It may contain at least one selected from the group consisting of medrogestone, flutamide, nilutamide, bifluranol, leuprorelin, cetrorelix, allylestrenol, chlormadinone acetate, cyproterone acetate, gestolone caproate, hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, osaterone acetate, oxendrone, estradiol, estradiol esters, ethinylestradiol, conjugated estrogens, diethylstilbestrol, and their derivatives, salts, and / or prodrugs.

[0118] In yet another embodiment, the anticancer drug comprises one or more type I, type II, or type III selective modulators (SPRMs) of progesterone that target the progesterone receptor pathway and are selected from the group consisting of onapristone, mifepristone, ronaprisan, agrepristone, Org31710, Org31806, CDB-2914, and CDB-4124, as well as their derivatives, salts, and / or prodrugs.

[0119] In yet another embodiment, the anticancer drug comprises a chemotherapeutic agent such as either a platinum-containing chemotherapeutic agent or an anthracycline chemotherapeutic agent. For example, the chemotherapeutic agent may include any platinum-containing chemotherapeutic agent selected from the group consisting of cisplatin, oxaliplatin, carboplatin, nedaplatin, lovaplatin, heptaplatin, dicycloplatin, lipoplatin, LA-12, phosphaplatin, phenanceliplatin, prolindac, triplatin tetranitrate, picoplatin, and satraplatin, and / or an anthracycline chemotherapeutic agent selected from the group consisting of doxorubicin, daunorubicin, epirubicin, and idarubicin, and / or an anthracycline chemotherapeutic agent, and / or an anthracycline chemotherapeutic agent, and / or an anthracycline chemotherapeutic agent, as described elsewhere in this specification. Other chemotherapeutic agents described elsewhere in this specification may also be preferred.

[0120] The dosage of the anticancer drug can be selected according to the treatment provided and the specific anticancer drug used. Similarly, the administration schedule of the anticancer drug can be selected according to the intended treatment and the anticancer drug provided. For example, in one embodiment, a preferred administration schedule may include administering the drug to the patient once or twice per day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, or one month. Timing of administration

[0121] In one embodiment, the treatment process of the pentaaza macrocyclic complex, and the treatment process with an anticancer drug which may be administered as appropriate, may include one or more doses of the drug and / or complex, depending on the treatment to be provided. In one embodiment, the treatment process including one or more doses may include administering a dose of the pentaaza macrocyclic complex at a predetermined period prior to the administration of an anticancer drug. For example, the treatment process may include an initial dose of the anticancer drug and include one or more subsequent doses, with the initiation of administration with the pentaaza macrocyclic complex being performed at a predetermined period prior to the first anticancer drug. In another embodiment, the treatment process including one or more doses may include administering the pentaaza macrocyclic complex after a predetermined period has elapsed since the administration of the anticancer drug. That is, the treatment process may include administering an initial dose, with one or more subsequent doses of the anticancer drug being administered, with the initiation of administration with the pentaaza macrocyclic complex being delayed by a predetermined period from the first anticancer drug.

[0122] In one embodiment, at least one dose of the pentaza macrocyclic complex in the course of treatment is administered at least one week, at least five days, at least three days, at least two days, at least one day, at least 12 hours, at least eight hours, at least four hours, at least two hours, at least one hour, and / or at least 30 minutes before administration of the anticancer drug. In another embodiment, at least one dose of the pentaza macrocyclic complex in the course of treatment is administered at least one week, at least five days, at least three days, at least two days, at least one day, at least 12 hours, at least eight hours, at least four hours, at least two hours, at least one hour, and / or at least 30 minutes after administration of the anticancer drug. Furthermore, the timing of at least one dose of the pentaza macrocyclic complex can also be applied to multiple doses provided during the course of treatment, e.g., at least 25%, at least 50%, at least 75%, at least 90%, or even substantially all doses provided during the course of treatment. Other cancer therapies

[0123] In one embodiment, the treatment provided herein may further include treatment with other therapies other than those specifically described above, such as radiotherapy, immunotherapy, and / or one or more other chemotherapy therapies. For example, in one embodiment, radiotherapy may be administered to the target before, concurrently with, or after the administration of one or more pentaza macrocyclic complexes, together with an anticancer drug as appropriate. Further details regarding radiotherapy and other chemotherapy suitable for the treatment of cancer are described below.

[0124] In one embodiment, radiotherapy may be administered concurrently with the administration of one or more of the pentaza macrocyclic complex and any anticancer drugs. For example, the subject may receive one or more of the anticancer drugs and the pentaza macrocyclic complex during the course of radiotherapy, before, after, or on the same day as the administration of radiotherapy, so that the subject receives radiotherapy concurrently with the administration of one or more of the anticancer drugs and the pentaza macrocyclic complex.

[0125] In yet another embodiment, the pentaza macrocyclic complex and any anticancer drug may be administered in the absence of other cancer treatments. Unexpectedly, as further shown in the following examples, the pentaza macrocyclic complex has been found to enhance the response and / or efficacy to anticancer treatments such as endocrine therapy and chemotherapy, even when administered without radiotherapy. Thus, in one embodiment, the cancer treatment offered to a subject may consist essentially of the pentaza macrocyclic complex and include an anticancer drug, without radiation (i.e., without administering a radioactive dose or dose fraction). For example, the pentaza macrocyclic complex (and an anticancer drug, which may be used in combination as appropriate) may be administered to a subject that has not received radiotherapy, and / or has not received radiotherapy for at least one day, e.g., at least one week and / or at least one month. Method of administration

[0126] According to one embodiment, the anticancer drug is administered as co-therapy or combination therapy with the pentaza macrocyclic complex. The co-therapy or combination therapy according to the method herein is intended to encompass the administration of each compound sequentially in a regimen that provides the beneficial effects of drug combination, and is also intended to encompass the co-administration of these drugs substantially simultaneously, such as in a single capsule containing these active agents in immobilized proportions, or in multiple separate capsules for each agent, or in single or multiple parenteral administrations, or other routes of administration and dosage forms. Therefore, when administered in combination, the therapeutic agents (i.e., the pentaza macrocyclic complex and / or the anticancer drug) can be formulated as separate compositions administered simultaneously or sequentially at different times, or the therapeutic agents can be administered as a single composition. Pharmaceutical compositions and formulations are described elsewhere herein.

[0127] The pentaza macrocyclic complex and anticancer agents do not need to be administered simultaneously or essentially simultaneously; the drugs and compounds may be administered sequentially. The advantages of simultaneous, essentially simultaneous, or sequential administration are within the scope of a skilled clinician's judgment. For example, a pharmaceutical composition or formulation containing an anticancer agent may be advantageous to be administered first in a combination for a particular treatment before administration of the pentaza macrocyclic complex, while administering the pentaza macrocyclic complex beforehand may be advantageous in another treatment. It will also be understood that the combination of the pentaza macrocyclic complex and anticancer agents as described herein can be used in combination with other methods of treating cancer (typically malignant tumors), including but not limited to radiotherapy and surgery, or other chemotherapy. Furthermore, it will be understood that another active agent, such as a cell proliferation inhibitor or quiescent agent, or an antiemetic, may, in some cases, be administered sequentially or simultaneously with any or all of the other concurrent therapies.

[0128] Accordingly, embodiments of the treatment method include the step of administering the pentaza macrocyclic complex and an anticancer drug, or a combination thereof, simultaneously or sequentially. For example, aspects of the present disclosure encompass a method for treating cancer in which the pentaza macrocyclic complex and an anticancer drug are administered simultaneously or sequentially. Other active agents may also be administered simultaneously or sequentially with the pentaza macrocyclic complex and the anticancer drug.

[0129] As described above, if the pentaza macrocyclic ring complex and the anticancer drug are not administered simultaneously or essentially simultaneously, the initial administration order of the components may be changed. For example, the anticancer drug may be administered first, followed by the pentaza macrocyclic ring complex, or the pentaza macrocyclic ring complex may be administered first, followed by the anticancer drug. This alternating administration may be repeated during a single treatment protocol. Other administration orders for utilizing the effects described herein are contemplated, and other administration orders for other activators may also be provided.

[0130] In one embodiment, the subject is treated beforehand with an anticancer drug, and then the pentaza macrocyclic complex is administered, or vice versa. According to such embodiments, the pentaza macrocyclic complex may be administered at least one hour after administration of the anticancer drug, or even at least three days after, or vice versa. For example, in one embodiment, the pentaza macrocyclic complex is administered between one hour and three days after administration of the anticancer drug, or vice versa. In another embodiment, for example, the pentaza macrocyclic complex is administered between one hour and one day after administration of the anticancer drug, or vice versa. For example, the pentaza macrocyclic complex may be administered within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 9 weeks, 10 weeks, or 12 weeks after the administration of an anticancer drug, or conversely, within 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, or 12 weeks after the administration of an anticancer drug, or vice versa. In these and other embodiments, the anticancer drug may be administered in multiple doses before the administration of the pentaza macrocyclic complex, or vice versa.

[0131] Alternatively, the subject may be pre-treated with a pentaza macrocyclic complex, followed by the administration of an anticancer drug, or vice versa. In such embodiments, the pentaza macrocyclic complex may be administered within at least one plasma half-life of the anticancer drug, for example, within four plasma half-lives of the anticancer drug, or vice versa. For example, the pentaza macrocyclic complex may be administered within one, two, or three plasma half-lives of another anticancer drug, or vice versa.

[0132] In another embodiment, the subject may be pre-treated with an anticancer drug, followed by administration of the pentaza macrocyclic complex, and then one or more additional doses of the anticancer drug, or vice versa. For example, the subject may be pre-treated with a single dose of an anticancer drug, followed by administration of a single dose of the pentaza macrocyclic complex, then an additional (or partial) dose of the same or a different anticancer drug, and then another dose of the pentaza macrocyclic complex. Furthermore, the subject may be pre-treated with a partial or complete dose of the pentaza macrocyclic complex, followed by administration of an anticancer drug, and then an additional (or partial) dose of the pentaza macrocyclic complex.

[0133] As will be described in further detail below, the combinations disclosed herein may also be co-administered with other well-known therapeutic agents selected for their specific usefulness for the condition being treated. If multiple combination formulations are not appropriate, the combinations may instead be used sequentially with known pharmaceutically acceptable agents.

[0134] In one embodiment, the pentaza macrocyclic complex and / or anticancer drug may be administered according to a treatment protocol that may be known for these drugs. For example, the administration of various components may be varied depending on the disease being treated and the effects of the pentaza macrocyclic complex and the anticancer drug on that disease. Furthermore, according to the knowledge of a skilled clinician, the treatment protocol (e.g., dosage and timing of administration) may be varied in consideration of the observed effects of the drugs administered to the patient (i.e., the pentaza macrocyclic complex, the anticancer drug) and the disease's response to the administered drugs.

[0135] Furthermore, generally speaking, the pentaza macrocyclic complex and / or anticancer drugs should not be administered in the same pharmaceutical composition, as they have different physical and chemical properties and therefore need to be administered via different routes. For example, the pentaza macrocyclic complex may be administered orally to produce and maintain its good blood concentration, while anticancer drugs may be administered intravenously or via blood transfusion, or vice versa. The mode of administration may, if possible, be in the same pharmaceutical composition or in separate pharmaceutical compositions (e.g., two or three separate compositions). Moreover, once the initial dose has been administered, the dose, mode of administration, and timing of administration may be modified thereafter based on the observed effects.

[0136] The specific choice of pentaza macrocyclic complex, anticancer drugs, and other related therapies (such as radiotherapy, immunotherapy, or other chemotherapy) is at the discretion of the attending clinician, based on their diagnosis, the patient's condition, and the appropriate treatment protocol.

[0137] Therefore, clinicians can modify each protocol regarding the administration of the treatment components (pentaza macrocyclic complex and anticancer drugs) according to their experience and knowledge, as treatment progresses and according to the individual patient's needs.

[0138] When determining whether treatment is effective at the administered dose, the clinician should consider not only the patient's overall health but also more concrete signs such as relief of disease-related symptoms, suppression of tumor growth, actual tumor reduction, or suppression of metastasis. Tumor size can be measured using standard methods such as radiological examinations like CAT or MRI, and continuous measurements can be used to determine whether tumor growth has been suppressed or reversed. Relief of disease-related symptoms such as pain and improvement in overall condition can also be used to help determine the effectiveness of treatment.

[0139] Products comprising a combination may be administered simultaneously, separately, or at intervals over a period of time to obtain the maximum effect of the combination; the duration of each administration can vary from rapid administration of any of the components (in separate formulations or a single formulation) to relatively continuous perfusion. Consequently, for the purposes of this disclosure, the combination is not limited to those obtained by the physical association of its components, but may allow for separate administrations that can be administered simultaneously or at intervals over a period of time.

[0140] Therefore, the administration of the components described herein may be carried out as a single event of treatment or over a course of treatment. For example, the Pentaza macrocyclic complex and / or anticancer agents may be administered hourly (e.g., every hour, every two hours, every three hours, every four hours, every five hours, every six hours, etc.), daily, once a week, once every two weeks, or once a month (simultaneously or sequentially). For the treatment of acute symptoms, the course of treatment may be at least several hours or several days. In certain conditions, treatment may extend from several days to several weeks. For example, treatment may be extended over one, two, or three weeks. For more chronic conditions, treatment may be extended from several weeks to several months, more than a year, or over the lifetime of the patient requiring such treatment. Alternatively, compounds and agents may be administered hourly, once a day, once a week, once every two weeks, or once a month as a preventive measure for several weeks, several months, several years, or over the lifetime of the patient.

[0141] The dose or amount of a pharmaceutical composition containing the pentaza macrocyclic ring complex and / or anticancer agent administered to a patient should be an effective amount for the intended purpose, i.e., for the treatment or prevention of one or more of the diseases, pathological disorders and conditions described herein, particularly cancers. In general, the effective dose of the composition administered may vary depending on various factors such as age, weight, sex, diet, route of administration, and the condition of the patient requiring treatment. Particularly preferred doses are discussed more thoroughly herein. However, it is understood that the total daily dose of the compositions described herein should be determined by the attending physician or veterinarian within the bounds of sound medical judgment.

[0142] As described above, the combination can be co-administered (either through a jointly formulated dosage form or in separate dosage forms administered nearly simultaneously). The combination can also involve administering each drug separately in separate unit dosage forms at different times. Numerous approaches for administering the anticancer drug and the pentaza macrocyclic complex can be readily adapted for use in this disclosure. The pharmaceutical composition can be delivered orally, for example, in unit dosage forms of tablets or capsules, or parenterally, for example, in unit dosage forms of injection, or by any other route. For systemic administration, for example, the drug can be administered by intravenous infusion (sequential or bolus). The composition can be used in any therapeutic or prophylactic treatment in which the patient may benefit from the combined treatment.

[0143] The specific therapeutically effective dose level for any particular patient depends on a variety of factors, including the disorder being treated and its severity; the activity of the specific compound used; the patient's age, weight, overall health, sex, and diet; the time of administration; the route of administration; the rate of excretion of the specific compound used; the duration of treatment; drugs used concomitantly or concurrently with the specific compound used; and similar factors well known in the fields of medicine and / or veterinary medicine. If desired, the effective daily dose may be divided into multiple doses for administration purposes. Consequently, a single-dose composition may contain such an amount or multiples thereof to constitute the daily dose.

[0144] In one embodiment, preferred or desired doses of each component are used in or included in the composition described herein. A preferred dose of the pentaza macrocyclic complex may be, for example, in the range of 10 to 500 mg per patient per day. However, the dose may vary by an administration schedule that can be adjusted as needed to achieve the desired therapeutic effect. It should be noted that the range of effective doses provided herein is not intended to limit the disclosure and represents an exemplary range of doses. The most preferred dose will be adjusted to the individual subject, taking into account the specific combination used, and the patient's age, sex, weight, physical condition, diet, etc., so that it can be understood and determined by those skilled in the art without requiring excessive experimentation.

[0145] The cancer treatments or cancer therapies described herein include steps to achieve a therapeutic benefit, but therapies may also be administered to achieve a preventive benefit. A therapeutic benefit generally refers to at least partial eradication or improvement of the disorder that is being treated. For example, in a cancer patient, a therapeutic benefit includes the (partial or complete) eradication or improvement of the cancer that is causing the cancer. A therapeutic benefit is also achieved by at least partial or complete eradication or improvement of one or more physiological symptoms associated with the underlying disorder, such that improvement is observed in the patient despite the fact that the patient may still have the underlying disorder. With respect to a preventive benefit, the methods of this disclosure may be performed on patients at risk of developing cancer, or patients who are reporting one or more physiological symptoms of such a condition, even though a diagnosis of the condition may not have been made, or the compositions of the present invention may be administered to such patients. Cancer treatment methods

[0146] In general, any subject suffering from or suspected of suffering from cancer or other proliferative disorders can be treated with the compositions and methods of this disclosure. Subjects treated according to the methods described herein are mammalian subjects, typically human patients. Other mammals that can be treated according to this disclosure include companion animals such as dogs and cats, domesticated animals such as cattle, horses, and pigs, as well as birds and more rare animals (e.g., those found in zoos or nature reserves). One embodiment of this disclosure provides a method for the treatment of cancerous tumors, particularly solid tumors. Advantageously, the methods described herein can reduce tumor development, decrease tumor volume, or induce tumor regression in mammalian hosts. Cancer patients and individuals seeking cancer prevention can be treated with the combinations described herein.

[0147] Cancer and tumors generally refer to or describe physiological conditions in mammals typically characterized by uncontrolled cell proliferation. The drug combinations, co-formulations, and combination therapies of this disclosure can treat a variety of tumors, including tumors of the breast, heart, lung, small intestine, large intestine, spleen, kidney, bladder, head and neck, ovaries, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testes, cervix, and liver.

[0148] In one embodiment, the tumor or cancer is selected from adenoma, angiosarcoma, astrocytoma, epithelial carcinoma, germ cell tumor, glioblastoma, glioma, hamartoma, hemangioendothelioma, angiosarcoma, hematoma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma, and teratoma. Tumors include acral lentiginous melanoma, actinic keratosis, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, astrocytic tumor, Bartholin's gland carcinoma, basal cell carcinoma, bronchial gland carcinoma, capillary carcinoma, carcinoid, calcinoma, carcinosarcoma, cavernous carcinoma, cholangiocarcinoma, chondrosarcoma, choroid plexus papilloma / carcinoma, clear cell carcinoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymoma, epithelioid sarcoma, Ewing's sarcoma, lamellar type, focal nodular hyperplasia, gastrinoma, germ cell tumor, glioblastoma, glucagonoma, hemangioblastoma, hemangioendothelioma, hemangioma, hepatic adenoma, hepatic adenomatosis, hepatocellular carcinoma, insulinoma, intraepithelial neoplasia, squamous intraepithelial neoplasia, invasive squamous cell carcinoma, and large cell carcinoma. You can choose from cancer, leiomyosarcoma, lentigo malignant melanoma, malignant mesothelioma, medulloblastoma, medullary epithelioma, melanoma, meningioma, mesothelioma, metastatic carcinoma, mucoepidermal carcinoma, neuroblastic tumor, neuroepithelial adenocarcinoma, nodular melanoma, oat cell carcinoma, oligodendroglia, oligodendroglia, osteosarcoma, pancreatic, papillary serous adenocarcinoma, pineal cell carcinoma, pituitary tumor, plasmacytoma, pseudosarcoma, pulmonary blastoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, small cell carcinoma, soft tissue carcinoma, somatostatin-secreting tumor, squamous cell carcinoma, squamous cell carcinoma, solitary mesothelioma, superficial spreading melanoma, undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, vipomas, well-differentiated carcinoma, and Wilms' tumor.

[0149] Therefore, for example, this disclosure includes, but is not limited to, cancers of the bladder (including advanced and metastatic bladder cancer), breast, colorectal cancer (including colorectal cancer), kidney, liver, lung (including small cell and non-small cell lung cancer, and lung adenocarcinoma), ovary, prostate, testis, urinary tract, lymphatic system, rectum, larynx, pancreas (including exocrine pancreatic carcinoma), esophagus, stomach, gallbladder, cervix, thyroid and skin (including squamous cell carcinoma); leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkin lymphoma, hairy cell carcinoma. It provides methods for the treatment of various cancers, including lymphoid hematopoietic malignancies such as follicular lymphoma, histiocytic lymphoma, and Barketts lymphoma; myeloid hematopoietic malignancies such as acute and chronic myeloid leukemia, myelodysplastic syndrome, myeloid leukemia, and promyelocytic leukemia; central and peripheral nervous system tumors such as astrocytoma, neuroblastoma, glioma, and Schwann cell tumor; mesenchymal tumors such as fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors such as melanoma, xeroderma pigmentosum, keratoactantoma, seminoma, follicular thyroid carcinoma, and teratoma.

[0150] For example, certain leukemias that can be treated with the combinations and methods described herein include, but are not limited to, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, leukocyte leukemia, basal cell leukemia, blastocyte leukemia, bovine leukemia, chronic myeloid leukemia, cutaneous leukemia, fetal leukemia, eosinophilic leukemia, Gross leukemia, hairy cell leukemia, hemoblastic leukemia, hematocytic leukemia, histiocytic leukemia, and stem cell leukemia. Leukemia includes leukemia, acute monocytic leukemia, leukopenic leukemia, lymphocytic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphosarcomacytic leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myeloid leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Negeri's leukemia, plasma cell leukemia, plasmacytic leukemia, promyelocytic leukemia, leader cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemia, and anaplastic cell leukemia.

[0151] Lymphoma can also be treated in combinations and methods described herein. Lymphoma is generally a neoplastic transformation of cells, primarily present in lymphoid tissue. Lymphoma is a tumor of the immune system and generally exists as both T-cell-associated and B-cell-associated diseases. Among lymphomas, there are two major groups: non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma. In particular, the bone marrow, lymph nodes, spleen, and circulating cells may be involved. Treatment protocols involve removing bone marrow from the patient, removing the tumor cells, often using antibodies against antigens present in the type of tumor cells, and then preserving it. Subsequently, the patient is given a toxic dose of radiation or chemotherapy, and the washed bone marrow is reinfused to regrow the patient's hematopoietic system.

[0152] Other hematological malignancies that can be treated with the combinations and methods described herein include myelodysplastic syndromes (MDS), myeloproliferative syndromes (MPS), and myeloma (e.g., solitary myeloma and multiple myeloma). Multiple myeloma (also called plasma cell myeloma) is associated with the skeletal system and is characterized by multiple tumorous plasma cell nodules scattered throughout the body. It can also spread to other sites such as lymph nodes and skin. Solitary myeloma refers to a single lesion that tends to occur in the same location as multiple myeloma.

[0153] In one embodiment, the methods and pharmaceutical compositions described herein are used to treat any of the following cancers: breast cancer, melanoma, oral squamous cell carcinoma, lung cancer including non-small cell lung cancer, renal cell carcinoma, colorectal cancer, prostate cancer, brain cancer, spindle cell carcinoma, urothelial carcinoma, bladder cancer, colorectal cancer, squamous cell carcinoma, and pancreatic cancer. In yet another embodiment, the cancer to be treated is any one selected from the group consisting of breast cancer, prostate cancer, testicular cancer, glioma, glioblastoma, head and neck cancer, ovarian cancer, endometrial cancer, hepatocellular carcinoma, desmoid tumor, pancreatic cancer, melanoma, and renal cell carcinoma.

[0154] According to one embodiment, cancer treatment may involve administering one or both of a therapeutically effective amount of an anticancer agent and a pentaza macrocyclic complex that results in an increased cancer response corresponding to any of the following selected from the group: a reduction in tumor volume, a reduction in tumor growth rate, an increase in survival rate of the mammalian subject, a reduction in the occurrence and / or extent of metastasis, and / or a reduction in the proliferation of cancer cells, and / or a reduction in cancer complications. Pharmaceutical preparations

[0155] Another aspect of this disclosure relates to a pharmaceutical composition comprising the combinations described herein, together with pharmaceutically acceptable excipients. The pharmaceutical composition comprises a pentaza macrocyclic complex (e.g., corresponding to formula (I)), and at least an anticancer agent, which may optionally be included, and the above-described combination, and may typically be formulated as a pharmaceutical dosage form in combination with a pharmaceutically acceptable carrier, additive, or excipient. In one embodiment, for example, the pharmaceutical composition comprises a pentaza macrocyclic complex, an anticancer agent, and a pharmaceutically acceptable excipient. The pharmaceutical composition according to this disclosure may be used for the treatment of cancer.

[0156] The pharmaceutical compositions described herein are products resulting from mixtures or combinations of multiple active ingredients, and include both immobilized and unimmobilized combinations. Immobilized combinations involve the simultaneous administration of active ingredients, such as the pentaza macrocyclic complex and an anticancer drug, to a patient in the form of a single substance or single dose. Other active agents may also be administered as part of a single substance or single dose, or separately. Unimmobilized combinations involve the simultaneous, concurrent, or sequential administration of active ingredients, such as the pentaza macrocyclic complex and an anticancer drug, to a patient as separate components, without specific time constraints, thereby supplying effective levels of the compounds to the patient's body. The latter also applies to cocktail therapies, such as the administration of three or more active ingredients.

[0157] The pentaza macrocyclic complex and / or anticancer agents described above may be dispersed in a pharmaceutically acceptable carrier before administration to mammals; that is, the components described herein are preferably co-formulated. Carriers, also known in the art as excipients, vehicles, adjuvants, or diluents, are typically pharmaceutically inert substances that impart appropriate stiffness or form to a composition and do not diminish the efficacy of the compound. Carriers are generally considered "pharmaceutically or pharmacologically acceptable" if, when administered to mammals, particularly humans, they do not produce unacceptably harmful, allergic, or other unpleasant reactions.

[0158] The selection of a pharmaceutically acceptable carrier is also, in part, determined by the route of administration. Generally, the compositions described herein can be formulated according to conventional routes of administration for any route of administration, as long as the blood circulation system is available through that route. For example, preferred routes of administration include, but are not limited to, oral, parenteral (e.g., intravenous, intra-arterial, subcutaneous, rectal, intramuscular, intraorbital, intracapsular, intraspinal, intraperitoneal, or intrasternal), topical (transnasal, transdermal, intraocular), intravesical, subarachnoid, intraintestinal, intrapulmonary, intralymphatic, intraairway, intravaginal, transurethral, ​​intradermal, ear, intramammary, buccal, orthotopic, intratracheal, intrafocal, transdermal, endoscopic, transmucosal, sublingual, and intestinal administration.

[0159] pharmaceutically acceptable carriers for use in combination with the compositions of this disclosure are well known to those skilled in the art and are selected based on many factors: the specific compounds and agents used, and their / their concentrations, stability and bioavailability for the subject; the subject, its age, weight and overall condition; and the route of administration. Suitable non-aqueous, pharmaceutically acceptable polar solvents include alcohols (e.g., α-glycerol formal, 6-glycerol formal, 1,3-butylene glycol, aliphatic or aromatic alcohols having 2 to 30 carbon atoms, e.g., methanol, ethanol, propanol, isopropanol, butanol, t-butanol, hexanol, octanol, amylene hydrate, benzyl alcohol, glycerin (glycerol), glycol, hexylene glycol, tetrahydrofurfuryl alcohol, lauryl alcohol, cetyl alcohol, or stearyl alcohol, fatty acid esters of aliphatic alcohols, e.g., polyalkylene glycols (e.g., polypropylene glycol, polyethylene glycol), sorbitan, sucrose, and cholesterol); amides (e.g., dimethylacetamide (DMA), benzyl benzoate DMA, dimethylformamide, N-(6-hydroxyethyl)-lactamide, N,N-dimethylacetamideamide, 2-pyrrolidinone, 1-methyl-2-pyrrolidinone, or polyvinylpyrrolidone);Esters (e.g., 1-methyl-2-pyrrolidinone, 2-pyrrolidinone), acetates (e.g., monoacetin, diacetin, and triacetin), aliphatic or aromatic esters (e.g., ethyl caprylic or octanonate, alkyl oleate, benzyl benzoate, benzyl acetate, dimethyl sulfoxide (DMSO)), glycerin esters (e.g., mono, di, or triglyceryl citrate or tartarate), ethyl benzoate, ethyl acetate, ethyl carbonate, ethyl lactate, ethyl oleate, sorbitan fatty acid esters, fatty acid-derived PEG esters, glyceryl monostearate, glyceride esters (e.g., mono, di) (or triglycerides), fatty acid esters (e.g., isopropyl myristate), fatty acid-derived PEG esters (e.g., PEG-hydroxyoleic acid and PEG-hydroxystearic acid), N-methylpyrrolidinone, Pluronic 60, polyoxyethylene sorbitol oleate polyester, polyoxyethylene sorbitan esters, e.g., polyoxyethylene-monoleate sorbitan, polyoxyethylene-monopalmitate sorbitan, polyoxyethylene-monolaurate sorbitan, polyoxyethylene-monostearate sorbitan, and polysorbate® 20, 40, 60 or 80 (ICI Americas (derived from Wilmington, Delaware), polyvinylpyrrolidone, alkylene oxy-modified fatty acid esters, e.g., polyoxyl 40 hydrogenated castor oil and polyoxyethylated castor oil (e.g., Cremophor® EL solution or Cremophor® RH40 solution), sucrose fatty acid esters (i.e., condensation products of monosaccharides (e.g., pentoses, e.g., ribose, ribulose, arabinose, xylose, lyxose and xylulose, hexoses, e.g., glucose, fructose, galactose, mannose and sorbose, triose, tetrose, heptose and octose), disaccharides (e.g., sucrose, maltose, lactose and trehalose) or oligosaccharides, or C4-C; 22These mixtures with fatty acids (e.g., saturated fatty acids (e.g., caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, and stearic acid), and unsaturated fatty acids (e.g., palmitoleic acid, oleic acid, elaidic acid, erucic acid, and linoleic acid)), or steroidal esters); alkyl, aryl, or cyclic ethers having 2 to 30 carbon atoms (e.g., diethyl ether, tetrahydrofuran, dimethyl isosorbide, diethylene glycol monoethyl ether); glycoflores (tetrahydrofurfuryl alcohol polyethylene glycol ether); ketones having 3 to 30 carbon atoms (e.g., acetone, methyl ethyl ketone, methyl isobutyl ketone); aliphatic, cycloaliphatic, or aromatic hydrocarbons having 4 to 30 carbon atoms (e.g., benzene, cyclohexane, dichloromethane, dioxolane, hexane, n-decane, n-dodecane, n-hexane, sulfolane, tetramethylene sulfone, tetramethylene sulfoxide, toluene, dimethyl sulfoxide (DMSO)) , or tetramethylene sulfoxide); inorganic, plant, animal, natural or synthetic oils (e.g., mineral oil, e.g., aliphatic or wax-based hydrocarbons, aromatic hydrocarbons, mixed aliphatic and aromatic-based hydrocarbons, and refined paraffin oil, vegetable oil, e.g., linseed oil, tuna oil, safflower oil, soybean oil, castor oil, cottonseed oil, peanut oil, rapeseed oil, coconut oil, palm oil, olive oil, corn oil, corn germ oil, sesame oil, apricot kernel oil and peanut oil, and glycerides, e.g., mono-, Di- or triglycerides; animal oils, e.g., fish oil, aquatic animal oil, semen oil, cod liver oil, halibut oil, squalene, squalane, and shark liver oil, oleic acid oil, and polyoxyethylated castor oil); alkyl or aryl halides having 1 to 30 carbon atoms and optionally two or more halogen substituents; methylene chloride; monoethanolamine; petroleum benzine; trolamine; omega-3 polyunsaturated fatty acids (e.g., alpha-linolenic acid, eicosapentaenoic acid, docosapentaenoic acid, or docosahexaenoic acid);Examples include, but are not limited to, 12-hydroxystearic acid and polyethylene glycol polyglycol esters (Soltoll® HS-15 (derived from BASF, Ludwigshafen, Germany); polyoxyethylene glycerol; sodium laurate; sodium oleate; or sorbitan monooleate).

[0160] In some embodiments, oils or non-aqueous solvents may be used in the formulation to dissolve one or more compounds, for example, due to the presence of large lipophilic moieties. Alternatively, emulsions, suspensions, or other preparations, such as liposome preparations, may be used. With regard to liposome preparations, for example, several known methods for preparing liposomes may be used. See, for example, Bangham et al., J. Mol. Biol, 23: 238-252 (1965) and Szoka et al., Proc. Natl Acad. Sci 75: 4194-4198 (1978), which are incorporated herein by reference. Thus, in one embodiment, one or more compounds are administered in the form of liposome delivery systems, such as small monolayer vesicles, large monolayer vesicles, and multilayer vesicles. Liposomes can be formed from various phospholipids such as cholesterol, stearylamine, or phophatidylcholine. Ligands can also be attached to liposomes, for example, to guide these compositions to specific sites of action.

[0161] Other pharmaceutically acceptable solvents for use in the pharmaceutical compositions described herein are well known to those skilled in the art, as seen in The Chemotherapy Source Book (Williams & Wilkens Publishing), The Handbook of Pharmaceutical Excipients (American Pharmaceutical Association, Washington, DC, and The Pharmaceutical Society of Great Britain, London, England, 1968), Modern Pharmaceutics (G. Banker et al., eds., 3d ed.) (Marcel Dekker, Inc., New York, New York, 1995), The Pharmacological Basis of Therapeutics (Goodman & Gilman, McGraw Hill Publishing), Pharmaceutical Dosage Forms (H. Lieberman et al., eds.) (Marcel Dekker, Inc., New York, New York, 1980), Remington's Pharmaceutical Sciences (A. Gennaro, ed., 19th ed.) (Mack Publishing, Easton, PA, 1995), The United States This information is found in Pharmacopeia 24, The National Formulary 19 (National Publishing, Philadelphia, PA, 2000), and AJ Spiegel et al., Use of Nonaqueous Solvents in Parenteral Products, Journal of Pharmaceutical Sciences, Vol. 52, No. 10, pp. 917-927 (1963).

[0162] Formulations containing the pentaza macrocyclic ring complex and / or anticancer agents may be in solid, semi-solid, lyophilized powder, or liquid dosage forms, such as aerosols, capsules, creams, emulsions, foams, gels / jellys, lotions, ointments, pastes, powders, soaps, solutions, sprays, suppositories, suspensions, sustained-release formulations, tablets, tinctures, or transdermal patches, preferably in unit dosage forms suitable for easy administration of precise doses. When formulated as a fixed dose, such pharmaceutical compositions or formulation products use the pentaza macrocyclic ring complex and / or anticancer agents within an acceptable dose range.

[0163] In one embodiment, a formulation is provided that includes an anticancer drug as part of a liquid dosage form, such as a sterile liquid dosage form suitable for injection. For example, a liquid form containing the anticancer drug in combination with one or more further components, such as disodium edetate (EDTA). In one embodiment, the liquid form may contain an amount of EDTA suitable for acting as a preservative and / or metal chelating agent, for example, about 0.025%. The liquid form may further contain water and may also contain a pH adjuster, such as sodium bicarbonate, for pH adjustment in the range of pH 5.5 to 7.0. In one embodiment, the pentaza macrocyclic complex may also be provided as part of a sterile liquid dosage form suitable for injection, either in the same liquid dosage form as the anticancer drug or as a separate dosage form.

[0164] Formulations for specific pentaza macrocyclic complexes are also described, for example, in U.S. Patents 5,610,293, 5,637,578, 5,874,421, 5,976,498, 6,084,093, 6,180,620, 6,204,259, 6,214,817, 6,245,758, 6,395,725 and 6,525,041 (each of which is incorporated herein by reference in whole).

[0165] The co-formulation of the pentaza macrocyclic ring complex and anticancer drugs is intended to be carried out by either employing conventional formulation technologies for each component individually, or by using a different formulation route in combination, depending on the compatibility and efficacy of the various components.

[0166] The above-described pharmaceutical composition comprising the pentaza macrocyclic complex and / or an anticancer agent may further contain one or more additional pharmaceutically active ingredients. Suitable pharmaceutically active ingredients that may be included in compositions according to embodiments of the present invention include, for example, antiemetics, anesthetics, antihypertensives, anxiolytics, anticoagulants, anticonvulsants, hypoglycemic agents, decongestants, antihistamines, antitussives, anticancer agents, beta-blockers, anti-inflammatory agents, antipsychotics, cognitive enhancers, cholesterol-lowering agents, anti-obesity agents, autoimmune deficiency agents, anti-impotence agents, antibacterial and antifungal agents, hypnotics, anti-Parkinson's disease agents, anti-Alzheimer's disease agents, antibiotics, antidepressants, and antiviral agents. The individual components of such combinations may be administered sequentially or simultaneously in separate or combined pharmaceutical formulations.

[0167] In yet another embodiment, a kit comprising the pentaza macrocyclic complex and, as appropriate, an anticancer drug, may be provided for the treatment of a condition such as cancer and / or to reduce the likelihood of cancer recurrence. For example, the kit may include a first container or box containing a formulation comprising the pentaza macrocyclic complex (e.g., an oral or injectable formulation of the pentaza macrocyclic complex) and a second container or box containing a formulation comprising an anticancer drug, e.g., an injectable formulation of the anticancer drug. The kit may further include a label or other instructions relating to the administration of the active agent, recommended dosage, duration and administration regimen, warnings, a list of possible drug-drug interactions, and other relevant instructions, such as a label indicating a treatment regimen (e.g., dosage, number of doses, etc.) corresponding to any of those described herein. Combination therapy with cancer treatment

[0168] In one embodiment, the pentaza macrocyclic complex and / or anticancer drug can be administered in combination with another cancer drug to provide therapeutic treatment. For example, the pentaza macrocyclic complex and / or anticancer drug may be administered as part of a radiotherapy regimen.

[0169] In general, the timing of administration of the pentaza macrocyclic complex and / or anticancer drug may depend, for example, on the specific radiotherapy or type, nature, and / or duration of radiation exposure selected. Other considerations may include the disease or disorder being treated and its severity; the activity of the specific compound used; the specific composition used; the age, weight, relative health, sex, and diet of the subject; the timing, route of administration, and excretion rate of the specific compound used; the duration of treatment; drugs used in combination with or concurrently with the specific compound used; and similar factors. For example, the compound may be administered in various embodiments before, during, and / or after the administration of radiotherapy (e.g., before, during, or after a course of radiotherapy consisting of multiple exposures and / or doses). As another example, the compound may be administered in various embodiments before, during, and / or after exposure to radiation.

[0170] If necessary, the effective dose may be divided into multiple doses for the purpose of administration; as a result, a single-dose composition may contain such a dose or a small multiple thereof.

[0171] In one embodiment, for example, the pentaza macrocyclic complex and / or anticancer drug is administered to the patient before or concurrently with radiation therapy. In another embodiment, for example, the components are administered to the patient before radiation therapy but not after. In yet another embodiment, one or more of the pentaza macrocyclic complex and / or anticancer drugs are administered to the patient at least 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 180 minutes, 0.5 days, 1 day, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, or more before radiation therapy, such as the first radiation dose in the course of radiation therapy, or before another radiation dose that becomes one of the radiation doses or dose fractions in the course of treatment. In yet another embodiment, for example, the pentaza macrocyclic complex and / or anticancer drug is administered to the patient after radiation therapy; therefore, for example, the compound may be administered up to 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, or 180 minutes after radiation therapy, up to 0.5 days, 1 day, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, or more.

[0172] In one embodiment, the pentaza macrocyclic complex and / or anticancer drug is administered as part of a treatment process that includes radiotherapy. In radiotherapy, the patient receives a dose or dose fraction of ionizing radiation to kill or control the growth of cancer cells. The dose or dose fraction of radiation may be directed to a specific part of the body, and the radiation beam may also be embodied according to a predetermined treatment regimen to reduce adverse effects on parts of the body that are not affected by cancer. A typical radiotherapy process may involve one or more doses or dose fractions of radiation, which may be administered over several days, weeks, or even months. The total “dose” of radiation administered during a radiotherapy process typically refers to the amount of radiation the patient receives throughout the entire radiotherapy process, and this dose may be administered as dose “fractions” corresponding to multiple radiation exposures if the total dose is administered over several periods, with the sum of the administered fractions equaling the total dose.

[0173] In one embodiment, at least one of the pentaza macrocyclic complex and / or anticancer agents is administered within a predetermined time frame before or after radiation, for example, before or after a radiation dose or dose fraction. For example, the pentaza macrocyclic complex and / or anticancer agents may be administered within 1 week, 48 hours, 24 hours, 12 hours, 6 hours, 2 hours, 1 hour, or 30 minutes after the patient receives radiation, such as a dose or dose fraction (either before or after radiation corresponding to the radiation dose or dose fraction). Other periods between radiation and administration of compounds that enhance cancer cell death may also be appropriate. In one embodiment, one or more of the pentaza macrocyclic complex and / or anticancer agents may be administered before radiation, and the remaining one or more of the pentaza macrocyclic complex and / or anticancer agents may be administered after radiation. Alternatively, one or more of the pentaza macrocyclic complex and anticancer agents may be administered before or after radiation.

[0174] In one embodiment, the course of radiotherapy includes multiple radiation doses or dose fractions administered over a predetermined period, e.g., several hours, several weeks, several days, or even several months, wherein the multiple doses or dose fractions are either the same magnitude or vary. That is, the course of radiotherapy may include the administration of a series of multiple doses or dose fractions of radiation. In one embodiment, the pentaza macrocyclic complex and / or anticancer drug may be administered before one or more doses or dose fractions of a series of radiation, e.g., before each dose or dose fraction, or before several doses or dose fractions. Furthermore, the administration of the pentaza macrocyclic complex and / or anticancer drug during the course of radiotherapy can be selected to enhance the cancer-treating effect of radiotherapy, for example, by sensitizing cancer cells to radiotherapy. In one embodiment, the pentaza macrocyclic complex and / or anticancer drug is administered within a predetermined time before or after each dose or dose fraction, such as the predetermined time described above. In another embodiment, the pentaza macrocyclic complex and / or anticancer drug are administered within a predetermined period before or after only a selected dose or dose fraction. In yet another embodiment, at least one of the pentaza macrocyclic complex and / or anticancer drug is administered within a predetermined period before the dose, and another of the pentaza macrocyclic complex and / or anticancer drug is administered within a predetermined period after the dose or dose fraction. In yet another embodiment, at least one of the pentaza macrocyclic complex and / or anticancer drug is administered within a predetermined time before or after a selected dose or dose fraction, and another of the pentaza macrocyclic complex and / or anticancer drug is administered within a predetermined time before or after a dose or dose fraction other than the selected dose or dose fraction.

[0175] The appropriate total dose to be delivered during the course of treatment can be determined according to the type of treatment to be delivered, the patient's physical characteristics, and other factors, and the dose fraction to be delivered can be determined similarly. In one embodiment, the dose fraction of radiation delivered to the patient may be at least 1.8 Gy, e.g., at least 2 Gy, and even more at least 3 Gy, e.g., at least 5 Gy, and even more at least 6 Gy. In yet another embodiment, the dose fraction of radiation delivered to the patient may be at least 10 Gy, e.g., at least 12 Gy, and even more at least 15 Gy, e.g., at least 18 Gy, and even more at least 20 Gy, e.g., at least 24 Gy. Generally, the dose fraction of radiation delivered to the patient should not exceed 54 Gy. Furthermore, in one embodiment, the dose fraction delivered to the subject may refer to the amount delivered to a specific target area of ​​the subject, such as the target area of ​​a tumor, while other areas of the tumor or surrounding tissue may be exposed to more or less radiation than specified by the nominal dose fraction.

[0176] In yet another embodiment, the pentaza macrocyclic complex and / or anticancer drug is administered as part of a course of treatment that includes the administration of further chemotherapeutic agents. In chemotherapy, chemotherapeutic agents are administered to the patient to kill cancer cells or to control the growth of cancer cells. A typical course of chemotherapy may include the administration of one or more chemotherapeutic agents, which may be administered over a course of several days, weeks, or even months. Chemotherapeutic agents include: alkylating antineoplastic agents, e.g., nitrogen mustard (e.g., cyclophosphamide, chlorambucil), nitrosourea (e.g., n-nitroso-n-methylurea, carmustine, semustine), tetrazine (e.g., dacarbazine, mitozolimid), aziridine (e.g., thiotepa, mitomycin); antimetabolites, e.g., folate antimetabolites (e.g., methotrexate and pemetrexed), fluoropyrimidines (e.g., fluorouracil, capecitabine), anthracyclines These may include at least one of the following: phosphorus (e.g., doxorubicin, daunorubicin, epirubicin), deoxynucleoside analogs (e.g., cytarabine, gemcitabine, decitabine), and thiopurines (e.g., thioguanine, mercaptopurine); microtubule inhibitors (e.g., taxanes, paclitaxel, docetaxel); topoisomerase inhibitors (e.g., etoposide, doxorubicin, mitoxantrone, teniposide); and antitumor antibiotics (e.g., bleomycin, mitomycin). For example, the chemotherapeutic agent may be selected from the group consisting of all-trans retinoic acid, arsenic trioxide, azacitidine, azathioprine, bleomycin, carboplatin, capecitabine, cisplatin, chlorambutyl, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, eposilon, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, mechloretamine, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, teniposide, tiguanine, barrubicin, vinblastine, vincristine, vindesine, and vinorelbine.Many chemotherapy drug administrations are described in the "Physicians' Desk Reference" (PDR), for example, the 1996 edition (Medical Economics Company, Montvale, NJ 07645-1742, USA).

[0177] In one embodiment, the pentaza macrocyclic complex and / or anticancer drug is administered as part of a treatment process including a further chemotherapeutic agent selected from the group consisting of cisplatin, doxorubicin, bleomycin, and paclitaxel. Furthermore, in one embodiment, the further chemotherapeutic agent may be selected from the group consisting of taxanes, anticancer antibiotics, and anthracyclines. Other chemotherapeutic agents include arsenic trioxide and 5-FU, which can also be used in the methods and compositions described herein (Alexandre et al., Cancer Res. 67: (8), 3512-3517 (2007); Yen et al., J. Clin. Invest. 98 (5), 1253-1260 (1996); Masuda et al., Cancer Chemother. Pharmacol. 47(2), 155-160 (2001)).

[0178] In yet another embodiment, further chemotherapeutic agents may include at least one of antimetabolite anticancer agents and antimitotic anticancer agents, as well as combinations thereof, which may include some of the above-described agents, as well as other agents further described herein. Various antimetabolites and mitotic inhibitors can be used in the methods and compositions described herein.

[0179] Antimetabolites are generally structurally similar to natural metabolites and are involved in normal metabolic processes in cancer cells, such as the synthesis of nucleic acids and proteins. However, antimetabolites are sufficiently different from natural metabolites to inhibit the metabolic processes of cancer cells. Within cells, antimetabolites are mistaken for similar metabolites and processed by the cell in the same way as normal compounds. In the presence of "decoy" metabolites, cells become unable to perform essential functions and are unable to proliferate or survive. For example, antimetabolites may exert cytotoxic activity by substituting these incorrect nucleotides into cellular DNA, thereby interfering with cell division or inhibiting essential cellular enzymes and preventing DNA replication.

[0180] Therefore, in one embodiment, the antimetabolite is a nucleotide or a nucleotide analog. In a particular embodiment, for example, the antimetabolite includes a purine (e.g., guanine or adenosine) or an analog, or a pyrimidine (cytidine or thymidine) or an analog, having or not having a bound sugar moiety.

[0181] Suitable antimetabolites for use in this disclosure can generally be classified according to the metabolic processes they affect, and include, but are not limited to, analogs and derivatives of folate, pyrimidine, purine, and cytidine. Accordingly, in one embodiment, the antimetabolite is selected from the group consisting of cytidine analogs, folate analogs, purine analogs, pyrimidine analogs, and combinations thereof.

[0182] In certain embodiments, for example, the antimetabolite is a cytidine analog. According to this embodiment, for example, the cytidine analog may be selected from the group consisting of cytarabine (cytosine arabinoside), azacitidine (5-azacitidine), and their salts, analogs, and derivatives.

[0183] In another specific embodiment, for example, the antimetabolite is a folate analog. Folate analogs or folate antimetabolites generally function by inhibiting dihydrofolate reductase (DHFR), an enzyme involved in nucleotide formation; when this enzyme is inhibited, nucleotides are not formed, and DNA replication and cell division are inhibited. According to a particular embodiment, for example, the folate analog may be selected from the group consisting of denopterin, methotrexate (ametopterin), pemetrexate, pteropterin, larcitrexed, trimethrexate, and their salts, analogs, and derivatives.

[0184] In another specific embodiment, for example, the antimetabolite is a purine analog. Purine antimetabolites function by inhibiting DNA synthesis, for example by inhibiting the production of purine-containing nucleotides adenine and guanine, thereby stopping DNA synthesis and thus stopping cell division. Purine analogs may also be incorporated into the DNA molecule itself during DNA synthesis, thereby interfering with cell division. According to one embodiment, for example, the purine analog may be selected from the group consisting of acyclovir, allopurinol, 2-aminoadenosine, arabinosyladenine (ara-A), azacitidine, azathioprine, 8-aza-adenosine, 8-fluoro-adenosine, 8-methoxy-adenosine, 8-oxo-adenosine, cladribine, deoxycoformycin, fludarabine, ganciclovir, 8-aza-guanosine, 8-fluoro-guanosine, 8-methoxy-guanosine, 8-oxo-guanosine, guanosine diphosphate, guanosine diphosphate-beta-L-2-aminofucose, guanosine diphosphate-D-arabinose, guanosine diphosphate-2-fluorofucose, guanosine diphosphate fucose, mercaptopurine (6-MP), pentostatin, thiamiprine, thioguanine (6-TG), and salts, analogs, and derivatives thereof.

[0185] In yet another specific embodiment, for example, the antimetabolite is a pyrimidine analog. Similar to the purine analogs discussed above, pyrimidine antimetabolites inhibit the synthesis of pyrimidine-containing nucleotides (cytosine and thymine in DNA; cytosine and uracil in RNA). By acting as a "decoy," pyrimidine compounds can prevent the production of nucleotides and / or be incorporated into an elongating DNA strand, leading to its termination. In certain embodiments, for example, pyrimidine analogs include ancitabine, azacitidine, 6-azauridine, bromouracil (e.g., 5-bromouracil), capecitabine, carmofur, chlorouracil (e.g., 5-chlorouracil), cytarabine (cytosine arabinoside), cytosine, dideoxyuridine, 3'-azido-3'-deoxythymidine, 3'-dideoxycytidine-2'-ene, 3'-deoxy-3'-deoxythymidine-2'-ene, dihydrouracil, doxyfluridine, enocitabine, phloxyuridine, 5-fluorosto The pyrimidine analog may be selected from the group consisting of thymine, 2-fluorodeoxycytidine, 3-fluoro-3'-deoxythymidine, fluorouracil (e.g., 5-fluorouracil (also known as 5-FU)), gemcitabine, 5-methylcytosine, 5-propynylcytosine, 5-propynylthymine, 5-propynyluracil, thymine, uracil, uridine, and salts, analogs, and derivatives thereof. In one embodiment, the pyrimidine analog is anything other than 5-fluorouracil. In another embodiment, the pyrimidine analog is gemcitabine or a salt thereof.

[0186] In certain embodiments, the antimetabolite is selected from the group consisting of 5-fluorouracil, capecitabine, 6-mercaptopurine, methotrexate, gemcitabine, cytarabine, fludarabine, pemetrexed, and their salts, analogs, derivatives, and combinations. In other embodiments, the antimetabolite is selected from the group consisting of capecitabine, 6-mercaptopurine, methotrexate, gemcitabine, cytarabine, fludarabine, pemetrexed, and their salts, analogs, derivatives, and combinations. In one particular embodiment, the antimetabolite is other than 5-fluorouracil. In a particularly preferred embodiment, the antimetabolite is gemcitabine or a salt thereof (e.g., gemcitabine HCl (Gemzar®)).

[0187] Other antimetabolites include acanthifolic acid, aminothiadiazole, brequinal sodium, Ciba-Geigy CGP-30694, cyclopentylcytosine, cytarabine phosphate stearate, cytarabine conjugate, Lilly DATHF, Merrel Dow DDFC, desaguanine, dideoxycytidine, dideoxyguanosine, Zidox, Yoshitomi DMDC, Wellcome EHNA, Merck&Co. EX-015, fazarabine, fludarabine phosphate, N-(2'-flanidyl)-5-fluorouracil, Daiichi Seiyaku FO-152, 5-FU-fibrinogen, isopropylpyrrolidine, Lilly LY-188011; Lilly LY-264618, metobenzaprim, Wellcome MZPES, norspermidine, and NCI. The following may be selected, but are not limited to, the group consisting of NSC-127716, NCI NSC-264880, NCI NSC-39661, NCI NSC-612567, Warner-Lambert PALA, pentostatin, pyritrexime, plicamycin, Asahi Chemical PL-AC, Takeda TAC-788, thiazophrine, Erbamont TIF, tyrosine kinase inhibitors, Taiho UFT, and uricin.

[0188] In one embodiment, the chemotherapeutic agent includes a mitotic inhibitor, which is a microtubule inhibitor or microtubule stabilizer. Generally, microtubule stabilizers, such as taxanes (any of those also described above) and eposilons, bind to the inner surface of beta microtubule chains, promoting the nucleation and elongation phases of polymerization and increasing microtubule association by lowering the concentration of the critical tubulin subunits required to construct microtubules. Unlike microtubule inhibitors such as vinca alkaloids that hinder microtubule association, microtubule stabilizers such as taxanes reduce the delay time and dramatically shift the dynamic equilibrium between tubulin dimers and microtubule polymers toward polymerization. Therefore, in one embodiment, the microtubule stabilizer is a taxane or eposilon. In another embodiment, the microtubule inhibitor is a vinca alkaloid.

[0189] One element of the therapies described herein may involve the use of taxanes or their derivatives or analogues, some of which have been described above. In one embodiment, the taxane may be a naturally occurring compound or a related form thereof having antitumor properties, or a chemically synthesized compound or a derivative thereof. Taxanes are terpenes, including but not limited to paclitaxel (Taxol®) and docetaxel (Taxotere®), mainly obtained from the Pacific yew tree, Taxus brevifolia, and have activity against certain tumors, particularly breast cancer and ovarian tumors. In one embodiment, the taxane is docetaxel or paclitaxel. Paclitaxel is a preferred taxane and is considered a mitotic inhibitor that stabilizes microtubules by promoting the association of microtubules from tubulin dimers and inhibiting depolymerization. This stability suppresses the normal dynamic rearrangement of the microtubule network, which is essential for important interphase and mitotic cellular functions.

[0190] Furthermore, various known taxane derivatives, including hydrophilic and hydrophobic derivatives, are also included. Examples of taxane derivatives include, but are not limited to, galactose and mannose derivatives described in WO99 / 18113; piperazino and other derivatives described in WO99 / 14209; taxane derivatives described in WO99 / 09021, WO98 / 22451, and U.S. Patent No. 5,869,680; 6-thio derivatives described in WO98 / 28288; sulfenamide derivatives described in U.S. Patent No. 5,821,263; deoxygenated paclitaxel compounds, e.g., those described in U.S. Patent No. 5,440,056; and taxol derivatives described in U.S. Patent No. 5,415,869. As described above, further examples of paclitaxel prodrugs include those described in WO98 / 58927; WO98 / 13059; and U.S. Patent No. 5,824,701. Taxanes may also be taxane conjugates, such as paclitaxel-PEG, paclitaxel-dextran, paclitaxel-xylose, docetaxel-PEG, docetaxel-dextran, and docetaxel-xylose. Other derivatives are mentioned in particular in "Synthesis and Anticancer Activity of Taxol Derivatives," DGI Kingston et al., Studies in Organic Chemistry, vol. 26, entitled "New Trends in Natural Products Chemistry" (1986), Atta-ur-Rabman, PW le Quesne, Eds. (Elsevier, Amsterdam 1986). Each of these references is incorporated herein by reference in its entirety.

[0191] Various taxanes may be readily prepared using techniques known to those skilled in the art (see also WO94 / 07882, WO94 / 07881, WO94 / 07880, WO94 / 07876, WO93 / 23555, WO93 / 10076; U.S. Patent Nos. 5,294,637; 5,283,253; 5,279,949; 5,274,137; 5,202,448; 5,200,534; 5,229,529; and EP590,267) (each of which is incorporated herein in whole by reference), or may be obtained from various commercial sources (including, for example, Sigma-Aldrich Co., St. Louis, Missouri).

[0192] Alternatively, a mitotic inhibitor may be a microtubule inhibitor; in one preferred embodiment, the microtubule inhibitor is a vinca alkaloid. Generally, vinca alkaloids are mitotic spindle toxins. Vinca alkaloids act during mitosis, when chromosomes begin to move along the tube of the mitotic spindle toward one of its poles before the chromosomes divide and the cell separates. Under the action of these spindle toxins, the spindle is disrupted by the dispersion of chromosomes during mitosis, affecting cell replication. According to a particular embodiment, for example, the vinca alkaloid is selected from the group consisting of vinblastine, vincristine, vindesine, vinorelbine, and their salts, analogs, and derivatives.

[0193] Mitotic inhibitors can also be eposilones. Generally, members of the eposilone family of compounds stabilize microtubule function through a mechanism similar to that of taxanes. Eposilones can also induce cell cycle arrest during the G2-M transition, leading to cytotoxicity and ultimately apoptosis. Suitable episiolons include eposilone A, eposilone B, eposilone C, eposilone D, eposilone E, and eposilone F, as well as their salts, analogs, and derivatives. One particular eposilone analog is ixabepyrone (Ixempra®), an eposilone B analog.

[0194] In certain embodiments, the anti-mitotic anticancer agent is selected from the group consisting of taxanes, eposilones, vinca alkaloids, and their salts and combinations. For example, in one embodiment, the mitotic inhibitor is a taxane. More preferably, in this embodiment, the mitotic inhibitor is paclitaxel or docetaxel, and even more preferably, paclitaxel. In another embodiment, the mitotic inhibitor is eposilone (e.g., an eposilone B analog). In yet another embodiment, the mitotic inhibitor is a vinca alkaloid.

[0195] In one embodiment, the pentaza macrocyclic complex and / or at least one anticancer drug is administered before or within a predetermined period after the administration of further chemotherapeutic agents. For example, the pentaza macrocyclic complex and / or anticancer drug may be administered within one week, 48 hours, 24 hours, 12 hours, 6 hours, 2 hours, 1 hour, or 30 minutes of the administration of further chemotherapeutic agents (either before or after the administration of the chemotherapeutic agents). Other periods between the administration of further chemotherapeutic agents and the administration of components that enhance cancer cell death may also be preferred. In one embodiment, one or more of the pentaza macrocyclic complex and / or anticancer drugs may be administered before the administration of further chemotherapeutic agents, and the remaining one or more of the pentaza macrocyclic complex and / or anticancer drugs may be administered after the administration of further chemotherapeutic agents. Alternatively, one or more of the pentaza macrocyclic complex and / or anticancer drugs may be administered both before and after the administration of further chemotherapeutic agents.

[0196] In one embodiment, the course of chemotherapy includes a single dose of an additional chemotherapeutic agent. In another embodiment, the course of chemotherapy includes multiple doses of an additional chemotherapeutic agent administered over a predetermined period of time, such as several hours, several weeks, several days, or even several months. The multiple doses may be the same or different doses and may include the administration of the same or different chemotherapeutic agents and / or combinations of chemotherapeutic agents. The administration of the pentaza macrocyclic complex and / or anticancer drug during the course of chemotherapy may be selected to enhance the cancer-treating effect of the chemotherapy, for example, by increasing intracellular levels of hydrogen peroxide and promoting oxidative stress in cancer cells. In one embodiment, the pentaza macrocyclic complex and / or anticancer drug is administered before or after each dose within a predetermined duration, such as the predetermined duration described above. In another embodiment, the pentaza macrocyclic complex and / or anticancer drug is administered only before or after a selected dose within a predetermined period. In yet another embodiment, at least one of the pentaza macrocyclic complex and / or anticancer drug is administered within a predetermined time before administration, and another of the pentaza macrocyclic complex and / or anticancer drug is administered within a predetermined time after administration. In yet another embodiment, at least one of the pentaza macrocyclic complex and / or anticancer drug is administered only within a predetermined period before or after a selected dose, and another of the pentaza macrocyclic complex and / or anticancer drug is administered only within a predetermined period before or after a dose other than the selected dose.

[0197] In yet another embodiment, the pentaza macrocyclic complex and / or at least one anticancer drug is administered in combination with both radiotherapy and chemotherapy, which includes the administration of additional chemotherapeutic agents. [Examples]

[0198] The following non-limiting embodiments are provided to further illustrate aspects of the invention. It should be understood by those skilled in the art that the techniques disclosed in the embodiments below demonstrate that the methods discovered by the inventors are well-suited for use in carrying out the invention and can therefore be considered to constitute examples of its embodiments. However, those skilled in the art should recognize that, taking this disclosure into consideration, many modifications can be made to the specific embodiments disclosed, and similar or equivalent results can be obtained without departing from the spirit and scope of the invention.

[0199] Example 1 SIRT3, a mitochondrial sirtuin, acts as a tumor suppressor (TS) protein, and manganese superoxide dismutase (MnSOD) 1,2,3,4 Several metabolite proteins, including those mentioned above, are targeted for deacetylation, leading to metabolic disorders. 5 It protects against this. Studies have shown that MnSOD acetylation inhibits normal cellular and mitochondrial metabolism, leading to tumor-tolerant phenotypes. 1,6 This has been shown, suggesting that MnSOD is an adaptive enzyme that responds to cellular oxidative stress. 7,8,9 MnSOD acetylation is associated with aging, energy state, metabolic stressors such as reactive oxygen species (ROS), carcinogenesis, and resistance to anticancer drugs. 2,3,4,8 While it has been proposed that this mechanism demonstrates physiological connections between cells and living organisms, the mechanism by which MnSOD acetylation directs these processes remains unclear.

[0200] Mammalian MnSOD is a homotetrameric antioxidant enzyme localized in the mitochondrial matrix, possessing four identical subunits, each containing a Mn2+ atom. 10 The primary function of MnSOD is to eliminate superoxide generated from various metabolic processes. While multiple MnSOD acetylation sites have been identified, recent studies suggest that K68 is central to the regulation of MnSOD superoxide dismutase activity. 1,6,8,9,11,12,13However, the specific cellular biological, biochemical, and / or physiological significance of MnSOD acetylation, as well as the underlying molecular mechanisms that regulate MnSOD detoxification activity and mitochondrial metabolism, are still not fully understood. Therefore, it has been proposed that MnSOD, in addition to directing mitochondrial metabolism, is a mitochondrial signaling hub that controls how cells adapt to ROS-induced metabolic stress. 14 This is thought to play an important role in late-onset diseases. 2,5 .

[0201] Mice lacking Sirt3, and therefore containing acetylated MnSOD (MnSOD-Ac), develop tumors. 7 This suggests that SIRT3 may function as a tumor suppressor (TS). Sirt3-deficient female mice spontaneously develop estrogen-positive (ER+), poorly differentiated, high-Ki-67 mammary tumors. This appears to be similar to human luminal type B mammary tumors, which are often diagnosed in elderly women. 2,5,7,15 Compared to luminal A-type ER+ breast cancer, luminal B-type subtypes tend to have increased growth markers and, most importantly, can exhibit an endocrine resistance phenotype. 5 Monoallelic knockout mice for MnSOD (MnSOD+ / -) exhibit decreased MnSOD activity, increased oxidative stress, and reduced lifespan, as well as aging-related phenotypes, particularly carcinogenesis. 16 This in vitro and in vivo evidence supports the possibility of a link between SIRT3-directed mitochondrial acetylome and reactive oxygen species detoxification, mitochondrial metabolism, and carcinogenesis. Furthermore, the examples herein suggest that under certain physiological conditions in which K68 is acetylated, MnSOD functions as a tumor promoter, consistent with data showing a positive correlation between MnSOD levels and invasive tumor phenotypes. In this regard, several publications in recent years have suggested disruption of the MnSOD-Ac axis, aging... 34 , neurodegeneration 35 , cardiovascular disease 36 , and insulin resistance37 Human diseases such as 4 This indicates a connection to [the above].

[0202] The examples herein present data showing that the acetylation state of MnSOD, particularly K68, directs ROS detoxification activity and links metabolic stress and mitochondrial repair pathways that maintain metabolic balance. The results revealed that MnSOD exists in both homotetrameric and monomeric forms, functioning as superoxide dismutase and peroxidase, respectively. Furthermore, while the homotetrameric form is TS, MnSOD K68Q By forcing the expression of [the specified molecule], it was shown that the modeled monomer functions as a tumor-promoting factor.

[0203] MnSOD K68Q The expression of promotes the transformation phenotype. MnSOD is a TS protein in vitro and in vivo. 17,18 The same is true for human tumor samples. 19 However, correlational findings in human tumor samples suggest that while MnSOD may function as a TS in the early stages of tumorigenesis, as tumorigenesis progresses, MnSOD levels are positively correlated with more invasive human tumors. 20 This suggests that certain isoforms of MnSOD, including acetylated forms, may function as tumor promoters. Furthermore, under certain conditions, MnSOD dysregulation and abnormal ROS levels in cells are observed. 21,22,23 There also appears to be a correlation between these findings and resistance to tamoxifen (Tam)-induced cytotoxicity. 24 This suggests a mechanistic relationship between the mitochondrial redox / ROS balance and the ecology of ER+ breast cancer.

[0204] To test this hypothesis, an acetylation mimetic of MnSODK68 (MnSOD) was developed, which mimics the acetylated amino acid state by substituting lysine with glutamine (Q) and deacetylation by substituting arginine (R).K68Q ) and deacetylation mimetic (MnSOD K68R ) Mutants were created. 8 MnSOD is a site-directed mutant that genetically mimics K68-Ac. K68Q To determine if it functions as a tumor promoter, we injected lenti-MnSOD into wild-type (WT) primary mouse embryonic fibroblasts (pMEFs) expressing either c-Myc or Ras with lentivirus. K68R Or wrench-MnSOD K68Q Co-infection was performed. In these experiments, immortalization and / or transformation of primary cells required at least two oncogenes, namely c-Myc and Ras (WT Ras gene). 25 A wrench MnSOD is required. K68Q pMEFs infected with either c-Myc or Ras were immortalized (i.e., divided beyond the 15-cell stage) similar to cells infected with both genes (Figure 1a, bottom panel). On the other hand, lenti-MnSOD K68R When infected with c-Myc or Ras, WT pMEFs infected with MnSOD do not become immortalized, and interestingly, MnSOD K68R This prevented the immortalization of cells infected with both genes (Figure 1a, middle column). In control, pMEF was immortalized when infected with c-Myc and Ras together, but not with c-Myc or Ras alone (Figure 1a, top panel). Furthermore, lenti-MnSOD K68Q pMEFs infected with [the virus] exhibited a more transformed in vitro phenotype, determined by growth on soft agar (Figure 1b, top panel), i.e., an indicator of immobilization-independent growth; increased colony formation when plated at low density, an indicator of proliferative capacity (bottom panel); decreased doubling time, an indicator of growth rate (Figure 9a, middle column); and xenograft tumor formation, an indicator of tumorigenicity phenotype in vivo (Figure 9a, right column).

[0205] To further characterize MnSOD-Ac and its function, and the relationship between TS and tumor promoters, we investigated the tumorigenicity of pMEF and Kras G12V (i.e., tumorigenic Kras gene) and lentiMnSODWT , wrench MnSOD K68R Or wrench MnSOD K68Q It co-infected with MnSOD. K68Q pMEFs expressing this gene were immortalized (Figure 1c, bottom panel, second column), and when measured by doubling time in culture (22 hours vs. 35 hours, third column) and growth on soft agar (bottom panel, right column), they showed a more transformed in vitro phenotype. Interestingly, the deacetylation-mimicking MnSOD mutant, lenti-MnSOD, was also found to exhibit this trait. K68R Infection caused by lenti-Kras G12V Co-infection with [another pathogen] inhibited immortalization (middle panel, second column). Finally, to confirm transformation in vitro, these experiments were repeated with immortalized NIH 3T3 cells, an in vitro established model. NIH 3T3 cells expressing MnSODK68Q proliferated on soft agar (Figure 1d, top panel), and colony formation increased when plated at low density (bottom panel).

[0206] MnSODK68Q increases the growth of both in vitro and xenografts. MnSOD K68Q To confirm the role of lenti-MnSOD expression in tumor growth characteristics, WT , wrench-MnSOD K68R , or wrench-MnSOD K68Q Human mammary gland ER+MCF7 tumor cells infected with lenti-MnSOD were engrafted into nude mice. Normally, MCF7 cells do not proliferate in nude mice without estrogen supplementation, but lenti-MnSOD was used. K68Q MCF7 cells infected with (MCF7-MnSOD) K68QMCF7 cells expressed with lenti-MnSODWT (MCF7-MnSODWT) or lenti-MnSODK68R (MCF7-MnSODK68R) did not form tumors (Figure 2a, b). These results suggest that xenograft tumors expressing MnSODK68Q exhibit increased growth characteristics; however, this may also reflect estrogen-independent growth characteristics. To address this, MCF7-MnSODK68Q cells were injected into the hind limbs of nude mice, and these xenograft experiments showed that estrogen supplementation did not alter the tumor growth curve (Figure 9b).

[0207] Luminal type B ER+ breast cancer cells are more invasive and exhibit increased proliferation compared to luminal type A cancer cells, as measured by Ki-67 staining. 5 Tumors in Sirt3-deficient mice containing MnSOD-Ac are Ki-67 5 It exhibits a luminal B-like tumor signature, including an increase in [specific component]. Consistent with these observations, MCF7-MnSOD stained with anti-Ki-67 antibody is observed. K68Q The cells produce MCF7-MnSOD K68R Or MCF7-MnSOD WT Compared to non-infected cells (Figure 2c), and quantified using ImageJ analysis, a significant increase in Ki-67 immunofluorescence (IF) staining was demonstrated (Figure 2d). MCF7-MnSODWT cells showed the same Ki-67 staining as uninfected control MCF7 cells (Figure 10a). Furthermore, experiments using a second ER+ human breast cancer cell line, T47D, also showed T47D-MnSOD K68R Cells and T47D-MnSOD WT Compared to cells, T47D-MnSOD K68Q Ki-67 staining increased (Figure 2e, Figure 2f). T47D-MnSOD WT The cells showed the same Ki-67 staining as the uninfected T47D cells used as a control (Figure 10b). Finally, MCF7-MnSOD K68QThe cells showed similar Ki-67 staining when exposed to either estrogen (Figure 10c, e) or Tam (Figure 10d, f).

[0208] MnSOD K68Q It is a monomer that exhibits peroxidase activity. MnSOD is composed of four subunits, each of which is bound to a manganese ion to form a homotetramer (approximately 88 kDa). 3,10 To investigate whether the acetylation state of K68 alters the three-dimensional structure and activity of MnSOD, we used MCF7-MnSOD. WT MCF7-MnSOD K68R , and MCF7-MnSOD K68Q Cells were collected, and the cell lysates were crosslinked with glutaraldehyde. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with anti-MnSOD antibody were then performed. These experiments revealed that MnSOD K68Q In cells expressing MnSOD, the tetrameric form was significantly reduced (Figure 3a, left panel), while both the dimer and monomer forms were slightly increased. Similar results were obtained with T47D-MnSOD. K68Q This was also observed in cells (right panel). Furthermore, when these experiments were repeated in MCF7 and T47D cells infected with lenti-shSIRT3, a significant decrease in the tetrameric form of MnSOD was observed in these cells, in contrast to the control cells where the oligomeric state of MnSOD remained largely unchanged (Figure 3b).

[0209] It has already been shown that MnSOD can exhibit peroxidase activity under specific conditions and when significantly overexpressed. 26 However, the mechanism explaining this observation is unknown. Therefore, when MnSODK68Q mutants immunoprecipitated (IP) from MCF7 cells were subjected to a peroxidase assay, it was found to function as a peroxidase (Figure 3c). On the other hand, IP-treated MnSOD K68RThis showed significantly lower peroxidase activity (i.e., a difference of approximately 50 times), suggesting that this peroxidase activity may require acetylation of K68 (Figure 3c). Ultimately, MnSOD- / -MEF expressing MnSODK68Q was found to be MnSOD WT Or MnSOD K68R Experiments confirmed that tetrameric MnSOD levels were significantly reduced compared to cells expressing the gene (Figure 3d).

[0210] In the above experiment, the gene variant MnSOD functions as a mimetic of K68-Ac. K68Q However, it has been shown to result in a tumor-promoting phenotype (Figure 1a, b). To more rigorously investigate this idea, untransformed, immortalized MnSOD- / -MEFs were used to infect the above-mentioned MnSOD site-specific mutants. These experiments showed that lenti-MnSOD K68Q MnSOD- / -MEF infected only with a single gene (i.e., a lenti-empty vector, lenti-MnSOD) was used so that growth, contact inhibition, and doubling time in soft agar could be measured (Figures 11b-d). WT Or wrench-MnSOD K68R Compared to cells infected with (Figure 11a), it was revealed that they exhibited a more transformed phenotype (Figure 3e, middle column). These results were obtained in cells lacking MnSOD, therefore, MnSOD K68Q It seems reasonable that this would function as an in vitro tumor promoter.

[0211] Ultimately, MnSOD K68Q If it acts as a peroxidase, then removing intracellular hydrogen peroxide, which is a necessary and essential substrate for peroxidase activity, may prevent it from functioning not only as a tumor promoter but also as a peroxidase. In this regard, lente-MnSOD K68R When co-infection was performed with AdMitoCat, which expresses catalase and reduces intracellular hydrogen peroxide, transformation was inhibited (Figure 3e, right column). Immortalized MnSOD- / -MEF is MnSOD K68QSince it has been shown that infection causes transformation, these results suggest that MnSOD enriches monomeric MnSOD. K68Q This suggests that it may be an in vitro tumor promoter that requires hydrogen peroxide.

[0212] MnSOD-K68-Ac exhibits peroxidase activity. The data shown above is from MnSOD mimicking K68-Ac. K68Q The expression of K68 is shown to enrich monomeric MnSOD (Figure 3a) and peroxidase activity (Figure 3c). However, it is also essential to show how physical acetylation of K68 affects enzyme activity. To address this, we first used an established tissue culture system containing large amounts of acetylated K68 compared to deacetylated K68. In this system, FLAG-MnSOD was added to MnSOD- / -MEF. WT After transfection, cells were either (i) exposed to 10 mM nicotinamide (NAM) and 1 μM trichostatin A (TSA) to inhibit SIRT3 deacetylase activity and enrich K68-Ac, or (ii) exposed to 10 mM NAD+ to activate SIRT3 activity and enrich deacetylated K68. As expected, whole cell extracts collected 40 hours after transfection were subjected to IP with anti-FLAG antibody, showing that NAM / TSA exposure increased MnSOD-K68-Ac (Figure 4a, top panel, left two lanes), while NAD+ exposure minimized MnSOD-K68-Ac (right two lanes). Similar results were observed in 293T cells (Figure 12a). The specificity of the MnSOD-K68-Ac antibody (Abcam, Inc, ab137037) was verified by two different methods. 12 .

[0213] These samples were then separated into fractions greater than or less than 50 kDa using a spin column. Immunoblotting with an anti-MnSOD antibody showed that samples from cells cultured in NAM / TSA were enriched with MnSOD in the fraction less than 50 kDa, suggesting that most of the MnSOD was in monomeric form and that there was little MnSOD in the fraction greater than 50 kDa (Figure 4a, left two lanes in the second and third columns). After processing the fraction less than 50 kDa with a seminative gel, immunoblotting for MnSOD confirmed enrichment of monomeric MnSOD (Figure 12b, left two lanes), and the fraction greater than 50 kDa contained the smallest tetrameric MnSOD (right panel, left two lanes). On the other hand, in cells cultured with NAD+, the level of MnSOD increased in fractions exceeding 50 kDa (Figure 4a, columns 2 and 3, right two lanes), and tetrameric MnSOD was concentrated in fractions exceeding 50 kDa (Figure 12b, right panel, right two lanes). These experiments confirmed that samples concentrated with MnSOD-K68-Ac mainly contained monomeric MnSOD, while samples with deacetylated MnSOD-K68 mainly contained tetrameric MnSOD.

[0214] Biochemical analysis of fractions of less than 50 kDa from cells exposed to NAM / TSA (i.e., enriched with MnSOD-K68-Ac and monomeric MnSOD) showed higher peroxidase activity compared to fractions of less than 50 kDa from cells treated with NAD+ (Figure 4b). On the other hand, MnSOD from both fractions of less than 50 kDa showed minimal MnSOD detoxification activity (Figure 4c). Analysis of fractions of more than 50 kDa from cells treated with NAD+ (i.e., enriched with tetrameric MnSOD) showed higher MnSOD detoxification activity compared to cells exposed to NAM / TSA (Figure 4d). Fractions of more than 50 kDa from cells treated with either NAD+ or NAM / TSA showed little to no MnSOD peroxidase activity (Figure 12c).

[0215] The second method was also used to clarify how physical acetylation of MnSOD-K68 alters its enzyme activity. Acetyl-lysyl tRNA synthetase / tRNA from M. barkeri CUA pET21a-MnSOD is a MnSOD bacterial expression vector that can site-specifically incorporate the paired pEVOL-AcKRS and N-(ε)-acetyl-L-lysine into K68. K68TAG It was transformed into E. coli to produce recombinant MnSOD-K68-Ac. Control (pET21a-MnSOD WT (containing) and acetylated form (pET21a-MnSOD) K68TAG Bacterial expression proteins from (containing) were purified by nickel affinity column chromatography followed by size exclusion chromatography (SEC). 13,27,28 Wild-type MnSOD purified from bacteria is found in other organisms (Knyphausenetal., 2016). 13 As shown, it eluted at a volume approximately corresponding to 92 kDa (Figure 4e, peak 1) on SEC, consistent with the known size of the homotetramer complex (Figure 13a, full chromatogram). pEVOL-AcKRS and pET21a-MnSOD K68TAG MnSOD-K68-Ac purified from bacteria possessing this compound eluted in a volume consistent with the monomeric form of MnSOD, which corresponds to approximately 25 kDa (Figure 4e, peak 2) (Figure 13b, full chromatograph).

[0216] Prior to further analysis, two eluted fractions corresponding to peak 1 (elution volumes 13 and 14 mL) and peak 2 (elution volumes 16 and 17 mL) were analyzed and confirmed to be MnSOD. The presence of MnSOD was confirmed by immunoblotting (Figure 4f, top panel) and Coomassie staining (bottom panel) of purified wild-type bacterial-expressed protein. Similar experiments confirmed the presence of pET21a-MnSOD. K68TAGThe presence of MnSOD was also confirmed in bacteria containing [the specified gene] (Figure 4g, upper and lower panels). These samples were also analyzed by mass spectrometry (Figure 13c-e) and staining with anti-MnSOD-K68-Ac antibody (Figure 13f), and it was confirmed that peak 2 was concentrated with MnSOD-K68-Ac protein.

[0217] pET21a-MnSOD WT Purified protein samples from bacterial cells expressing pET21a-MnSOD (elution volumes of 13 and 14 mL) exhibited minimal peroxidase activity (Figure 4i, left bar) and considerable superoxide dismutase activity (Figure 4h, left bar). On the other hand, pET21a-MnSOD K68TAG Recombinant MnSOD-K68-Ac protein (eluted at 16 and 17 mL) from bacterial cells expressing K68 showed minimal superoxide activity (Figure 4h, right bar) and considerable peroxidase activity (Figure 4i, right bar). These biochemical results demonstrate two methods for peroxidase enzyme function: isolation of MnSOD with physically acetylated K68 (bacterial expression system) or MnSOD rich in K68 acetylation (transfection expression system) to confirm conversion to monomeric MnSOD.

[0218] MnSOD-K68-Ac increases oxidative stress in mammary cells. MnSOD K68Q MCF7-MnSOD constitutively expresses K68Q (Figure 5a) and T47D-MnSODK68Q (Figure 5b) cells are also from other individuals. 6,12 This showed a significant decrease in MnSOD superoxide detoxification activity, consistent with what had been observed. Since the main function of MnSOD is to detoxify mitochondrial superoxide (O2·-), the mitochondrial oxidation / reduction state was measured in MCF7 and T47D cells expressing various MnSOD acetylation mutants. Among these cell lines, MCF7-MnSOD K68Q and T47D-MnSOD K68Q The cells produce MCF7-MnSOD K68R T47D-MnSODK68R Compared to control cell lines, the following were significantly increased: (1) MitoSox oxidation, an indicator of mitochondrial O2·- (Figure 5c, d); (2) CDCFH2 oxidation, an indicator of intracellular hydroperoxide levels (Figure 5e, f); and (3) the GSSG / GSH ratio, an indicator of intracellular oxidative stress (Figure 5g, h).

[0219] Tumor cells expressing MnSODK68Q exhibit Tam resistance. MnSOD dysregulation 29,30,31 And abnormalities in intracellular ROS levels and / or oxidative stress due to several different mechanisms. 23,32 The association between these and resistance to endocrine therapy has already been shown. These previous studies, and MnSOD K68Q Based on the above results, which identified MnSOD as an in vitro tumor-promoting factor, we have identified it as a similar factor to other oncogenes. K68Q It is reasonable to consider that enhanced expression of this gene could indirectly or directly lead to resistance to Tam.

[0220] To test this idea, we used MCF7 and MCF7-MnSOD. K68R MCF-MnSOD K68Q Cells were treated with 1 μM hydroxy-Tam for 5 days, and a clonal survival assay was performed. The results of these experiments showed that MnSOD K68Q MCF7 (Figure 6a) and T47D (Figure 14a) cells that constitutively express MnSOD K68WT Or MnSOD K68R Compared to cells expressing MnSOD, these cells showed significant resistance to hydroxy-Tam cytotoxicity. Furthermore, MCF7 (Figure 6b) and T47D (Figure 14b) cells expressing shSIRT3, which increases intracellular MnSOD-K68-Ac, also showed resistance to hydroxy-Tam cytotoxicity. In these experiments, MnSOD K68Q The expression of [substance] and its association with hydroxy-Tam-resistant tumor cells have been shown. Furthermore, these results suggest a link to the MnSOD pathway. 29,30,31 , and ROS level23,32 This has been added to the literature suggesting that it is involved in Tam resistance.

[0221] Tam-resistant mammary cells exhibit a MnSOD-K68-Ac signature. MnSOD K68Q Since breast cancer cells expressing hydroxy-Tam showed resistance to Tam-induced cytotoxicity in vitro, it is thought that MCF7 cells selected for resistance to hydroxy-Tam may also exhibit a MnSOD-K68-Ac signature. To address this idea, MCF7 (Figure 6c) cells and T47D (Figure 14c) cells were cultured for 3 months in the presence of 1 μM hydroxy-Tam to generate hydroxy-Tam-resistant (HTR) cells. Both MCF7-HTR cells and T47D-HTR cells showed an increase in MnSOD-K68-Ac (Figures 6d, e). Furthermore, staining with antibodies against several other SIRT3 deacetylation targets (MnSOD-K122-Ac, IDH2-K413-Ac, and OSCP-K139-Ac), which are surrogates for SIRT3 activity, also showed increased acetylation (Figure 14d), suggesting decreased SIRT3 activity. These experimental results demonstrate that selected ER+ breast cancer cell lines exhibit a MnSOD-K68-Ac signature in relation to resistance to Tam, suggesting its potential as a molecular biomarker.

[0222] Tam resistance is reversed by the expression of MnSOD K68R. To further demonstrate that MnSOD-K68-Ac is a potential marker of Tam resistance, we administered lenti-MnSOD to HTR cells. WT , wrench-MnSOD K68Q , wrench-MnSOD K68R The cells were infected, and hydroxy-Tam resistance was measured by a clonal cell survival assay. As a result, lenti-MnSOD WT Or wrench-MnSOD K68Q Instead, wrench-MnSOD K68RInfection with hydroxy-Tam reversed hydroxy-Tam resistance (Figure 6f and Figure 14e). Furthermore, lenti-SIRT3, which causes MnSOD deacetylation, was introduced into MCF7-HTR cells and T47D-HTR cells. WT When MnSOD-K68-Ac (Figure 6g and Figure 14f) was infected or treated with 5 μM of the pentaza macrocyclic ring complex GC4419 (Figure 6h and Figure 14g), which chemically removes superoxide from the catalytic mechanism used by homotetrameric MnSOD, it was shown that the cells became sensitive to hydroxy-Tam. These results suggest that MnSOD-K68-Ac is a promising molecular biomarker and / or tumor signature for resistance to Tam. These results also indicate that pentaza macrocyclic ring complexes such as GC4419 can treat cells with hydroxy-Tam resistance and / or reverse hydroxy-Tam resistance.

[0223] Exposure to Tam increases oxidative stress and monomeric MnSOD. MnSOD activity is closely related to mitochondrial metabolism, and HTR cells exhibit a MnSOD-K68-Ac signature (Figure 6d, e); therefore, we determined the mitochondrial metabolic profile in HTR ER+ cells. In this regard, MCF7-HTR cells (Figure 7a) and T47D-HTR cells (Figure 15a) showed decreased MnSOD activity, increased mitochondrial O2·- levels (Figure 7b), increased intracellular hydroperoxides as measured by CDCFH2 oxidation (Figure 7c), and increased GSSG / GSH ratio (Figures 7d and 15b). Finally, monomeric MnSOD was enriched in MCF7-HTR cells and T47D-HTR cells, consistent with decreased MnSOD activity (Figure 7a) and increased MnSOD-K68-Ac (Figure 6d) compared to control MCF7 and T47D cells (Figure 7e).

[0224] Furthermore, to investigate whether this increase in oxidative stress in HTR cells is due to the acetylation status of MnSOD-K68, we administered lenti-MnSOD to MCF7-HTR cells and T47D-HTR cells.WT , wrench-MnSOD K68R , wrench-MnSOD K68Q They infected it. From these experiments, MnSOD K68R When MnSOD is forcibly expressed, K68Q Or MnSOD WT Compared to cells expressing HTR, it was shown to reverse increases in mitochondrial O2·- (Figure 7f and Figure 15c), intracellular hydroperoxides (Figure 7g and Figure 15d), and GSSG / GSH ratio (Figure 7h and Figure 15e). These data indicate that HTR increases MnSOD-K68-Ac and suggest the possibility of a Tam-resistant tumor signature, including changes in intracellular ROS profiles as shown by others. 23,30 .

[0225] Tam-resistant MCF7 and T47D cells show increased Ki-67 levels. MCF7-HTR (Figures 7i and 16a) and T47D-HTR cells (Figures 16b, c) exhibiting the MnSOD-K68-Ac signature (Figure 6d) are similar to those of luminal type B breast malignancies, and MCF7-MnSOD K68Q and T47D-MnSOD K68Q The cells (Figure 2c, d) showed elevated Ki-67 levels. Furthermore, MCF7-HTR cells (Figure 16d, e) and T47D-HTR cells (Figure 16f, g) treated with the pentaza macrocyclic ring complex GC4419, or hydroxy-Tam and GC4419, showed reduced Ki-67 IHC staining. Additionally, GC4419, or hydroxy-Tam and GC4419, showed elevated Ki-67 levels in MCF7-MnSOD K68Q Reversing the increase in Ki-67 IHC staining in the T47D-MnSODK68Q cell line (Figures 17a, b) and T47D-MnSODK68Q (Figures 17c, d) suggested that chemically substituting the SOD activity of MnSOD reversed the increase in Ki-67.

[0226] MCF7-MnSOD K68QTo determine whether hydrogen peroxide is necessary for HTR observed in cells, the inventors infected these cells with AdMitoCat, removing and / or significantly reducing mitochondrial hydrogen peroxide levels, which are important and necessary substrates for peroxidase enzyme activity. Results from cloning cell survival experiments showed that when mitochondrial hydrogen peroxide levels decreased, MnSOD K68Q It was shown that HTR observed in MCF7 cells constitutively expressing was reversed (Figure 7j, k). These results suggest that cells expressing MnSOD acetylation mutants require hydrogen peroxide indirectly or directly to maintain resistance to Tam.

[0227] Tam-resistant xenografts exhibit a more invasive phenotype. To investigate whether MCF7-HTR cells exhibiting the MnSOD-K68-Ac signature (Figure 6d) form more invasive xenograft tumors in vivo, MCF7 cells and MCF7-HTR cells were injected into immunodeficient mice and tumor growth was monitored. As expected, control MCF7 cells failed to form tumors in vivo without estrogen supplementation. In contrast, MCF7-HTR cells, even without estrogen supplementation, grew an average of 859 mm² over 6 weeks. 3The cells formed tumors (Figure 8a, b), and the engraftment rate of the xenografts was 100% (Figure 17e), indicating that these cells exhibited a high tumorigenic phenotype. Finally, a Tet-On expression system was constructed using these MCF7-HTR cells to inductively express a deacetylation mimic mutant (MnSODK68R). Thus, MCF7-HTR cells were first infected with pTet-DualOn (Clontech), selected with puromycin, then infected with pTre-Dual2-MnSODK68R, selected with hygromycin, and finally, these cells were examined for MnSODK68R Tet-induction (Figure 18a, b). MCF7-HTR-Dual2-MnSODK68R xenografts were grown to 100 mm, and mice were exposed to doxycycline to induce MnSODK68R expression. These experiments demonstrated that forced expression of MnSODK68R inhibited the proliferation of MCF7-HTR xenograft tumor cells in vivo (Figure 8c).

[0228] Human luminal type B tumors show high levels of MnSOD-K68-Ac. Sirt3-deficient mice develop mammary tumors with a luminal B-like phenotype, exhibiting ER+, poor differentiation, and high levels of Ki-67. 5,7,33To investigate whether a subgroup of human ER+ tumors exhibiting SIRT3 / MnSOD-Ac signature deficiency exists, tissue microarray (TMA) slides from breast cancer patients, including all four subtypes of breast malignancy, were analyzed. TMAs were stained with anti-MnSOD-K68-Ac (see Figures 12a, b for antibody specificity) and anti-SIRT3 antibodies to show representative IHC images of luminal A and B tumor samples (Figures 8d and 18c, d). Subsequently, quantification of staining intensity using automated HistoQuest software revealed significantly higher MnSOD-K68-Ac levels (Figure 8e) and significantly lower SIRT3 protein levels (Figure 8f) in luminal B tumor samples compared to luminal A tumor samples. Furthermore, stratification of luminal A type TMA staining intensity against luminal B type TMA into low, medium, and high staining levels suggested the possibility of a subgroup of luminal B type tumors showing significant MnSOD-K68-Ac staining (Figure 18c, d). These results suggest that the SIRT3 / MnSOD-Ac signature is a useful marker for identifying specific subgroups of women with luminal B type breast cancer.

[0229] method cell line ER+MCF7 and T47D human mammary cells were both obtained from ATCC, authenticated using STR profiling with CellCheck 9 Plus by IDEXX Bioresearch, and tested for mycoplasma in April 2016 using the Mycoplasma Detection Kit from InvivoGen, Inc. Cells were cultured in Dulbecco's modified Eagle medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS; Sigma) and antifungal solution (Sigma). Cells were maintained in a humidified environment at 5% CO2 and 37°C. pMEFs were isolated from E14.5 isogenic SIRT3+ / + mice (following a protocol approved by the Institutional Animal Care and Use Committee (IACUC) and adhering to relevant animal research ethics guidelines) and maintained in a 37°C incubator at 5% CO2 and 6% oxygen unless otherwise noted. MCF7 and T47D cells were cultured in 1 μM hydroxy-Tam for 3 months to create MCF7-HTR and T47D-HTR permanent cell lines, and several different subclones were frozen. MCF7-HTR and T47D-HTR were not used beyond 5 passages; new cell lines were used instead. All experiments were performed using cell cultures that grew exponentially at 50% confluence.

[0230] Viral plasmids, short hairpin RNA (shRNA) constructs and mutagenicity To package lentiviruses, 293T cells (obtained from ATCC) were transfected with 5 μg of DNA, 5 μg of psPAX2 packaging plasmid, and 500 ng of VSV.G envelope plasmid. After 72 hours, the viral supernatant was collected and filtered through a 0.45 μm filter (Corning). Lentivirus-SIRT3 WT and deacetylated null mutant (lenticular-SIRT3 DNThe pLKO.1 human SIRT3 shRNA was donated by Dr. Toren Finkel (NIDDK). The pLKO.1 human SIRT3 shRNA was purchased from OpenBiosystem. The lenti-MnSOD plasmid (human) is MnSOD WT It was used as a plasmid and for site-directed mutagenesis, i.e., mutagenesis from K68 to arginine (R: deacetyl mimic) or glutamine (Q: acetyl mimic) (Bioinnovatise). MCF7, T47D, and NIH3T3 cells were infected with 5 MOI lentivirus and selected for 14 days in DMEM containing 2 μg / mL puromycin (Invitrogen) or 100 μg / mL G418 sulfate (Invitrogen). After a 2-week selection period, the cells were cultured in DMEM containing 10% FBS.

[0231] Transduction of antioxidant enzymes Non-replicating adenovirus vectors, AdCMV Bgl II (AdBglII) and AdCMV Mito-Catalase (AdMitoCat), were received as donations from Dr. Douglas Spitz (University of Iowa) and Dr. Marcelo Bonini (Medical College of Wisconsin, WI). Cells were plated the day before virus administration. After adding the desired number of virus particles for 24 hours, the culture medium was replaced with fresh medium before each experiment and left for a further 48 hours.

[0232] In vitro cell transformation assay In this study, spontaneous immortalization of pMEFs is defined as the ability to continue dividing even after 15 passages. For in vitro immortalization experiments, MnSOD, or one of its site-specific mutants (KR or KQ), was co-infected with c-Myc and / or Kras and introduced into third-stage pMEFs. To prevent confluence, cells were cultured and divided every two days, resulting in 3.0 × 10⁶ cells. 5 Individual cells were plated onto new 100 mm dishes. After 15 further passages (18 in total), the cells were considered immortalized.

[0233] Analysis of the survival rate of clonal cells In the clonal survival analysis, exponentially growing cells were reseeded at appropriate dilution ratios, and clonal survival was evaluated after 14 days in standard growth medium. Cells were stained with crystal violet, and more than 50 colonies were counted and used to calculate clonal survival. 46 .

[0234] Soft agar colony formation assay analysis 10,000 cells were plated onto a growth medium consisting of a 0.6% agar base layer topped with 0.3% agar. 7,8 21 days later, the colonies were visualized using a 20x microscope (Zeiss) and images were acquired.

[0235] Analysis of tumor formation in in vivo xenografting MnSOD K68WT MnSOD K68R , or MnSOD K68Q Five million MCF7, MCF7-HTR, or MCF7 cells (obtained from ATCC) expressing [specific gene] were injected into 6-week-old Foxn1nu atomic nude mice (Jackson Laboratory) (conducted according to a protocol approved by the Institutional Animal Care and Use Committee (IACUC), in compliance with relevant animal research ethics regulations). Tumor size was measured every 2-3 days using calipers, and volume was calculated using V = 1 / 2 × W² × L. The average tumor size was 1000 mm². 3 At this point, the mice were sacrificed, and the tumors were recovered and analyzed for weight and size.

[0236] MCF-HTR MnSOD K68R A TetOn induction system for xenografts. MCF7-HTR cells were infected with the pLenti-CMV-IE-Tet-On Advanced-IRES2-ZsGreen1-P2A-Puro plasmid (modified by Clontech, Mountain View, California and Bioinnovatise, Inc., Baltimore, Maryland), and selected for their green color under puromycin (1 μg / mL) (MCF7-HTR TetOn). Subsequently, MCF7-HTR TetOn cells were treated with pLenti-SV40 promotor-HygroR-SV40 poly(A) / pTreDual2MnSOD K68R -mCherry(MCF7-HTR TetOn MnSOD K68R Cells were infected with (Clontech, Inc.) and selected using mCherry and hygromycin (50 μg / ml). To confirm activation of the TetOn system, 1 μg / mL of doxycycline (Acros Organics, New Jersey) was added, and after 24 hours, expression was confirmed by the presence of mCherry, and fluorescence images were taken. MnSOD was selected using Western blotting. K68R We verified that the expression of MCF7-HTR TetOn MnSOD was activated during the implantation of tamoxifen pellets (5 mg pellets, Innovative Research of America, Sarasota, Florida). K68R Cells were injected into the right posterior flank of 8-week-old nude mice (Jackson Labs). The experimental group was fed a diet containing doxycycline (625 ppm, Envigo Teklad Diets, Madison, Wisconsin), while the control group continued to receive standard diet provided by Northwestern Animal Facility. The diet was changed every three days during the experiment. Tumors were measured every other day, and at the end of the experiment, the tumors were excised for analysis according to a protocol approved by the Facility Animal Care and Use Committee (IACUC) and in accordance with relevant animal research ethics regulations.

[0237] Immunohistochemical staining and analysis. Breast cancer tissue array slides (Biomax) were immersed twice: once in 100% xylene (Sigma) for 5 minutes, and again in 100% ethanol (Sigma) for 5 minutes. The slides were then sequentially immersed in 95%, 80%, and 50% ethanol for 5 minutes each, followed by immersion in water and fixation in 95 mL of 95% ethanol and 5 mL of 37% formaldehyde for 2 minutes. Subsequently, the slides were treated with 1% Triton X-100 (Corning) in 1×PBS for 20 minutes, washed three times in 1×PBS for 5 minutes each, and the reaction was stopped in 0.3% H2O2 in 1×PBS for 20 minutes. Slides were blocked for 2 hours with 10% donkey serum (Sigma), 1% bovine serum albumin (BSA; Sigma), and 0.3% Triton X-100 (Sigma) in 1×PBS, and then treated with anti-MnSOD-K68-Ac antibody (1:250 dilution, Abcam, #ab137037) at 4°C for 48 hours. These slides were then incubated at room temperature for 1 hour and washed three times with 1×PBS for 5 minutes each. Rabbit secondary antibody (1:200 dilution, A0545, Sigma) was diluted in antibody solution and applied to the slides for 1 hour, followed by three washes with 1×PBS for 5 minutes each. The slides were treated with the VECTASTAIN ABC kit (Vector Laboratories) for 45 minutes according to the manufacturer's protocol to detect the avidin / biotinylation enzyme complex. Slides were treated with a DAB peroxidase substrate kit (Vector Laboratories) according to the manufacturer's protocol and stained with hematoxylin (Sigma) for 10 minutes. The slides were then destained with 100 mL of 70% ethanol and 1 mL of 37% hydrochloric acid, followed by dehydration. Intensity was quantified using HistoQuest software (Tissuegnostics).

[0238] Peroxidase activity assay MnSOD with Flag tag WT MnSOD K68R , and MnSOD K68QOne million cells expressing [protein name] were lysed for 30 minutes in 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% NP-40, 5% glycerol, a protease inhibitor (BioTool), and TSA (Trichostatin A, Sigma). The lysates were quantified using the Bradford assay (BioRad), and IP was performed using an anti-Flag antibody (Sigma). The peroxidase enzyme activity of these IP-treated proteins was measured using pyrogallol as a substrate. The final concentrations in the reaction mixture were 14 mM potassium phosphate (Sigma), 0.027% (v / v) hydrogen peroxide (Sigma), and 0.5% (w / v) pyrogallol (Sigma). The plate was tapped to mix the sample and reaction reagents, incubated at room temperature for 10 minutes, and then read at OD420 nm. The increase in A420 was recorded every 3 minutes. For all test samples and blanks, ΔA420 / 20s was determined using the maximum linear velocity or 0.5-minute intervals. Peroxidase activity was calculated using the following formula: Units / mL = [(ΔA420 / 20s Test Sample - ΔA420 / 20s Blank) (Reaction Volume) (Dilution Factor)] / [(12)(0.1)].

[0239] Glutathione analysis One million cells at 70-80% confluence were lysed with 1.34 mM diethylenetriaminepentaacetic acid (DETAPAC, Sigma) and then lysed with 143 mM sodium phosphate (Sigma). Subsequently, 6.3 mM EDTA (Sigma) and 5% 5-sulfosalicylic acid (Sigma) were added to the lysate. 50 μL of the lysate was mixed with 700 μL of 0.298 mM NADPH (Sigma) dissolved in sodium phosphate buffer, 100 μL of 6 mM 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB, Sigma) dissolved in sodium phosphate buffer, 100 μL of water, and 50 μL of 0.023 U / μL glutathione reductase (GR) dissolved in water (Sigma). Using an xMark® microplate spectrophotometer (BioRad), velocity absorbance was read every 15 seconds at 412 nm for 2.5 minutes, and the velocity was compared to a standard curve. Tumors were lysed with DETAPAC buffer before the assay, and protein concentrations were measured to standardize GSH levels, which were standardized by the BCA method.

[0240] MnSOD / SOD enzyme activity Total SOD activity and MnSOD activity were evaluated using an indirect competitive inhibition assay. 47 The following measurements were taken: Superoxide is produced from xanthine by xanthine oxidase and detected by recording the reduction rate of nitroblue tetrazolium (NBT). SOD removes superoxide and competitively inhibits the reduction of NBT. One unit of SOD activity is defined as the amount of protein required to inhibit 50% of the maximum reduction of NBT. To obtain the amount of MnSOD activity, 5 mM sodium cyanide was added to inhibit CuZnSOD enzyme activity. Protein levels in each sample were measured using the BCA protein assay. 48,49,50,51 The measurement was performed using [a specific method / tool].

[0241] Western blot analysis Cells and tissues were washed three times with chilled 1×PBS, collected, and then treated with a protease inhibitor (BioTool) and TSA (Sigma) in 25mM Tris-HCl pH 7.4, 150mM NaCl, and 1mM After dissolving in EDTA, 0.1% NP-40, and 5% glycerol for 30 minutes, the results were quantified by the Bradford assay. Protease inhibitors (BioTool) and TSA (Sigma) were added to 1% NP-40 and 5% glycerol, and the results were quantified by the Bradford assay. Anti-MnSOD (1:1000 dilution, Millipore, #06-984), anti-MnSOD-K68-Ac (1:1000 dilution, Abcam, #Ab137037), anti-MnSOD-K122-Ac (1:500, Abcam, #Ab214675), anti-SIRT3 (1:1000 dilution, Cell Signaling, #D22A3), anti-IDH2 (1:1000 dilution, Cell Immunoblotation was performed with Cell Signaling (#56439), anti-IDH2-K413-Ac (1:1000 dilution, Epitomics, Inc., Burlingame, California (this company was acquired by Abcam, Inc.)), anti-OSCP (1:1000 dilution, Santa Cruz Biotechnology, #sc-365162), anti-OSCP-K139-Ac (1:1000 dilution, Epitomics, Inc., Burlingame, California), and anti-actin (1:10,000 dilution, Cell Signaling, #4970). Secondary antibodies included anti-rabbit and anti-mouse (1:10,000 dilution, Cell Signaling, #7074, #7076). In the MnSOD4 merization assay, lysed cells were treated with 0.1% glutaraldehyde at room temperature for 10 minutes, and then the samples were immunoblotted with anti-MnSOD antibody.

[0242] Measurement of intracellular superoxide levels using MitoSox. The steady-state level of mitochondrial O2·- was estimated using the oxidation of the fluorescent dye dihydroethidium (DHE) (Life Technologies). Cells were trypsinized, washed, and then labeled with MitoSox Red (2 μM in 0.1% DMSO) in 5 mM pyruvate containing 1 × PBS at 37°C for 20 minutes. After labeling, cells were stored on ice. Samples were analyzed using a Fortessa flow cytometer (Becton Dickinson Immunocytometry System, Inc., Mountain View, California; excitation 488 nm, emission 585 nm, 25 nm bandpass filter). The mean fluorescence intensity (MFI) of 10,000 cells in each sample was analyzed and corrected for autofluorescence from unlabeled cells. MFI data were normalized to control levels.

[0243] Evaluation of intracellular H2O2 levels using CDCFH2 oxidation. The steady-state level of H2O2 was assessed using oxidation-sensitive 5-(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (CDCFH2) (Life Technologies). Cells were trypsinized, washed once with 1×PBS, and then labeled with CDCFH2 or CDCF (10 μg / mL in 0.1% DMSO, 15 min) at 37°C. After labeling, cells were stored on ice. Samples were analyzed using a Fortessa flow cytometer (Becton Dickinson Immunocytometry System, Inc., Mountain View, California; excitation 488 nm, emission 530 nm, 25 nm bandpass filter). MFI was analyzed from 10,000 cells in the sample, corrected for autofluorescence from unlabeled cells. MFI data were normalized to control levels.

[0244] Survival experiment using the MnSOD mimetic GC4419. To test parameters indicating oxidative stress, clonalization assays were performed using hydroxy-Tam and antioxidants. Cells were plated at a density of 50,000 cells per 60 mm dish and treated with 1 μM hydroxy-Tam (Sigma) and 5 μM GC4419 (Galera Therapeutics) for a total of 120 hours. This protocol was repeated for 5 days, with fresh medium being changed every 24 hours. On day 6, cells were trypsinized, counted, re-seeded in control medium using appropriate dilutions, and clonal viability was assessed.

[0245] Incorporation of N-(ε)-acetyllysine into K68. BL21(DE3) pMAGIC chemically competent E-Coli cells, a kind donation from Andrzej Joachimiak of Argonne National Laboratory, express MnSOD-K68-Ac and MnSOD-WT proteins using pEVOL-AcKRS and pET21a-MnSOD. K68TAG Co-transformed with plasmid or pET21a-wtMnSOD. pEVOL-AcKRS and pET21a-MnSOD K68TAG Cells containing the plasmid were incubated at 37°C for 3 hours in 100 mL of LB containing 300 μg / mL ampicillin, 50 μg / mL kanamycin, and 50 μg / mL chloramphenicol (37°C, 220 rpm). 50 mM nicotinamide (Sigma) was added to the culture. Once the OD600 reached 1.1, 2 mM Nε-acetyllysine (Sigma) was added to the culture, followed by 0.4 mM IPTG and 0.2% arabinose, and the cells were further induced for 20 hours (25°C, 180 rpm). (The bacterial MnSOD expression and lysine acetylated tRNA mutant plasmids used to produce physically acetylated MnSOD-K68-Ac were kindly donated by Dr. Jiangyun Wang of the Institute of Biophysics, Chinese Academy of Sciences (Beijing, China).)

[0246] BL21(DE3) cells containing the pET21a-wtMnSOD plasmid were cultured in 5 mL of LB medium containing 300 μg / mL ampicillin and 50 μg / mL kanamycin. 1 mL of this culture was incubated overnight in 100 mL of LB medium containing 300 μg / mL ampicillin and 50 μg / mL kanamycin (37°C, 200 rpm). The following day, 1 L of LB containing 300 μg / mL ampicillin and 50 μg / mL kanamycin was inoculated into 10 mL of overnight culture, and cells were induced overnight in 0.4 mM IPTG for approximately 2.5 hours until OD600 = 0.6 (25°C, 180 rpm). All purification steps were performed on ice. E. coli cells in 1 L of LB were collected by centrifugation (6000 rpm, 10 min, 4°C) and conditioned in 50 mL of Buffer containing 20 mM imidazole, 50 mM Tris salt HCl, 200 mM NaCl, 5 mM MgCl2, 50 mM nicotinamide, pH = 8.0. The pellet was washed with Buffer I. Next, the centrifugated pellet was suspended in 50 mL of Buffer I supplemented with 1 mM PMSF and approximately 1 mg / mL of lysozyme, and the lysate was incubated at 4°C for 10 minutes. Then, the protein was extracted by sonication cycling (5 seconds on, 6 seconds off, 25 minutes). The extract was clarified by centrifugation (13,000 × g, 30 minutes, 4°C), and the pellet was discarded. A total of 0.2 mL of Ni2+-NTA beads were added to the supernatant, and the mixture was incubated at 4°C for 1 hour with stirring.

[0247] The beads were transferred to a column and washed three times with Buffer I containing increasing imidazole gradients (50, 75, 100 mM). The protein was then eluted with 1 mL of Buffer I supplemented with 200 mM imidazole. The protein was analyzed by SDS-PAGE and then concentrated using an Ultra-15 Centrifugal Filter Unit (10 kDa, Millipore Amicon®, USA UFC800324). Subsequently, the eluted protein was rebuffered in Buffer II (50mM Tris-HCl, 200mM NaCl, 5mM MgCl2, 50mM nicotinamide, pH 8.0) and loaded into an equilibrated Ni2+-NTA AKTA FPLC Purifier system equipped with a GE HisTrap HP column (product number GE17524701). Further purification was performed using a Superdex 200 Increase 10 / 300GL column (GE Healthcare, product number GE28-9909-44) in a buffer containing 50mM potassium phosphate (pH=7.8). Peak fractions were collected using an automated fraction collector. A280 as a function of elution volume / time was also recorded. 13,27,28 The peak protein fraction concentrations were measured and immunoblotted with anti-MnSOD and anti-MnSOD-K68-Ac. Peroxidase activity and MnSOD activity were measured for the remaining purified protein. The eluted fractions were then further analyzed. Calibration curves were created using gel filtration low and high molecular weight kits (GE Healthcare) according to the manufacturer's instructions and used to determine the relative sizes of peak 1 and peak 2, as shown in Figure 4e.

[0248] Sample preparation and image acquisition for immunofluorescence imaging. Cells seeded on glass coverslips were fixed with 4% paraformaldehyde and blocked with 1% BSA and 10% normal goat serum in 1× PBS. Cells were incubated in 1× PBS with anti-Ki-67 antibody (c-bioscience), and then incubated in 1× PBS containing 5% goat serum with goat anti-rabbit IgG conjugated with AlexaFluor647 (Invitrogen). Cells were washed in 1× PBS, mounted, and imaged with a fluorescence microscope. Fluorescence images were acquired using a laser scanning confocal microscope (Nikon A1R). Paired images for all figures were acquired with the same gain and offset settings. Post-acquisition processing was applied uniformly to all paired images. Images were displayed as a single optical layer after acquisition by z-series stack scans from individual regions, or as a maximum intensity projection to represent the confocal stack.

[0249] statistical analysis Statistical analysis was performed using GraphPad Prism for Windows (GraphPad Software, San Diego, California). Error bars represent the mean ± SEM. One-way ANOVA with Tukey's post-test was used to examine differences between three or more groups. For two-column bar graphs (i.e., whether there was a significant difference between the means of two groups), a t-test was used. All experiments were repeated at least three times. Statistical significance was defined as p < 0.05.

[0250] Example 2 Most men diagnosed with metastatic castration-resistant prostate cancer (mCRPC) initially respond well to androgen deprivation therapy (ADT), including ENZs that target the androgen receptor (AR) signaling pathway. 81 However, over time, most men begin to experience disease progression. Thus, the mortality rate in men with ADTR is at least largely due to the development of resistance to ADT drugs and, importantly, the lack of effective systemic treatments. 82Therefore, identifying the specific processes leading to ENZ resistance (ENZR), their underlying mechanisms, novel therapeutic interventions, and predictive signatures is crucial. In this regard, several ADTR mechanisms contributing to ADTR / ENZR have been identified, primarily focusing on AR, with AR amplification and hypersensitivity, AR mutations leading to disorder, mutations in activating cofactors / cosuppressors, and AR-independent intratumoral androgen production being used as escape pathways that provide alternative growth and survival stimuli. 81,83,84 While most men develop ADTR through mechanisms involving altered AR signaling, new resistance mechanisms are centered on the expression of lineage plasticity properties. 85-87 .

[0251] NCI White Paper (Beltran, 2019) and Overview of the Workshop (Yuan, 2019, Cancer Discovery) 88 It has been proposed that microenvironmental cues, probabilistic genetic and / or epigenetic changes, or selective pressures imposed by treatment contribute to the development of tumor heterogeneity and resistant tumor cell phenotypes. The development of phenotypes related to lineage plasticity has recently emerged as an important mechanism of treatment resistance in prostate cancer. 81,84 It occurs in approximately 20% of patients with advanced prostate cancer and has clinical and therapeutic significance. 86,87 In this regard, long-term exposure to ADT is associated with a subset of tumor cells exhibiting loss of AR signaling-dependent and luminal prostate markers, as well as induction of stem cell-like developmental programs. 85,86 Disruptions in mitochondrial metabolism, including abnormal ROS levels, are one of the potential mechanisms that lead to drug resistance phenotypes rooted in the development of lineage plasticity-like characteristics. 87 Therefore, lineage plasticity is defined as CRPC, in which plasticity is driven by the disruption of the normal metabolic physiology of cells, resulting in loss of AR-regulating lineage characteristics, including AR independence, sustained tumor cell proliferation, and the acquisition of new phenotypes, including ENZR.

[0252] In tumors, the detoxification activity of antioxidant enzymes is dysregulated. 89,90 The subsequent loss of metabolic homeostasis corresponds to tumors that show resistance to anticancer drugs, including ADT / ENZ. 89-91 MnSOD is a mitochondrial detoxification enzyme (i.e., superoxide dismutase) that, when deficient or dysregulated, plays a role in metabolic, carcinogenic, and treatment-resistant phenotypes. MnSOD-Ac may act as a link between metabolic and bioenergetic balance and tumor cell proliferation and / or survival, and under certain cellular conditions, it may function as an in vivo driver of the ENZR tumor phenotype. A mitochondrial signaling axis centered on MnSOD-Ac has been identified, and dysregulation of this axis leads to disrupted cellular metabolism and abnormal ROS (Zhu, 2019, Nature Commun). 52 Also, abnormally stabilized HIF2 α This activates the dedifferentiation program (He, 2019; Proc. Natl. Acad. Sci.). 55 In certain cases, MnSOD-K68-Ac is MnSOD K68Q When present outside of its normal physiological context modeled by the expression of HIF2, it disrupts cellular metabolism, increases ROS levels, and α This stabilizes the phenotype of phylogenetic formation and ENZR tumor cells. 89,90 Therefore, targeting the MnSOD-K68-Ac axis with appropriate therapeutic agents (i.e., pentaza macrocyclic complexes such as GC4419) may offer an effective therapeutic option.

[0253] ENZR prostate tumor cells show increased MnSOD-K68-Ac and decreased MnSOD activity. To investigate whether there is a correlation between MnSOD-K68-Ac and ENZR, we used two ENZ-sensitive prostate tumor cell lines, LNCaP or 22RV1, and established a method for selecting ENZR cells. 91Cells were cultured in 5 μM ENZ for 3 weeks, followed by continuous culture at 10 μM. Immunoblotting of LNCaP-ENZR cells (Figure 19a) or 22RV1-ENZR cells (data not shown) showed an increase in MnSOD-K68-Ac, suggesting that biological changes in MnSOD may be one possible mechanism for inducing ENZR. Since K68-Ac alters the surface charge of the MnSOD tetramerization interface, we evaluated the oligomerization state of MnSOD in LNCaP-ENZR cells. Crosslinking experiments showed a decrease in the tetramer complex (Figure 19b). In this gel, trimer and dimer bands not observed by the seminative separation method were also observed, suggesting that these are artifact bands likely due to the harsh glutaraldehyde crosslinking separation method.

[0254] LNCaP-ENZR cells (Figure 19c) and 22RV1-ENZR (data not shown) cells showed decreased MnSOD detoxification activity and elevated intracellular ROS levels (data not shown). These results suggest that chronic exposure to ENZ or the MnSODK68 acetylation mimic mutant gene (MnSOD K68Q The forced expression of MnSOD-K68-Ac, when present outside of normal physiological conditions, disrupts cellular metabolism, including abnormal ROS levels, leading to the ENZR phenotype in prostate tumor tissue culture cells. Ultimately, since these are pooled cells, it is likely that a subset of cells has different ENZR mechanisms, but it also appears that dysregulated MnSOD is at least one mechanism.

[0255] MnSOD K68R When it is expressed, ENZR is reversed, and conversely, MnSOD K68Q This induced ENZR in PCa cells. To investigate whether the MnSOD-K68-Ac-ROS axis is involved in the ENZR phenotype, we enriched tetrameric MnSOD in our ENZR tissue culture model cells and introduced a MnSOD deacetylation mimic mutant (MnSOD) that increases MnSOD activity. K68RCells were infected with lenti-MnSOD. Cloning cell survival experiments performed 72 hours after infection on cells cultured in 10 μm ENZ showed that lenti-MnSOD K68R LNCaP-ENZR (Figure 20a, bar 1:2) and 22RV1-ENZR (data not shown) cells infected with (deacetylation mimic mutants) were shown to be converted from ENZR to a susceptible phenotype. MnSOD-K68R levels were confirmed by immunoblotting using anti-Flag antibodies. K68R The expression of MnSOD-K68-Ac also reduces intracellular ROS levels (data not shown), suggesting that the presence of MnSOD-K68-Ac outside of its normal physiological state disrupts cellular metabolism, leading to the ENZR phenotype. Therefore, the expression of acetylation mimetic substances, which would also disrupt the biology of MnSOD, would also induce the ENZR phenotype. In fact, the inventors previously showed that an acetylation mimetic mutant gene (MnSOD) mimics MnSOD-K68-Ac. K68Q LNCaP cells infected with a lentivirus expressing ) (Zhu, 2019, Nature Common.) also showed the ENZR phenotype.

[0256] LNCaP-ENZR and LNCaP-MnSOD K68Q ENZR observed in cells was inverted by GC4419 treatment. The data from Figures 20a-20b suggested that the disruption of MnSOD's ecosystem due to abnormal MnSOD-K68-Ac and ROS levels may be at least partially involved in the development of the ENZR phenotype. Therefore, it was hypothesized that restoring SOD activity using the pentaza macrocyclic complex GC4419, a chemical detoxifier of superoxide, could reverse / convert ENZR into a susceptible phenotype. Indeed, LNCaP-ENZR cells treated with GC4419 showed a significant decrease in tumor cell viability in the presence of ENZ, as measured in clonal survival experiments (Figure 21a, leftmost two bars). Furthermore, LNCaP-MnSOD K68QThe ENZR observed in cells (Figure 21a, the two bars on the left) was also converted to a susceptible phenotype upon exposure to GC4419 (the two bars on the right). LNCaP-MnSOD showed ENZR. K68Q The cells (Figure 20b, bar 3) were also used in in vivo mouse amphibole xenograft experiments with exposure to GC4419. The dose and pharmacokinetics of GC4419 are based on data from the mouse model. 92-93 LNCaP-MnSOD grown in mice using ENZ K68Q The cells (plots above the boxes in the graph in Figure 21b) showed similar proliferation characteristics to control LNCaP cells (triangles), and LNCaP-MnSOD K68Q Xenografts were identified as ENZR. In contrast, LNCaP-MnSOD treated with GC4419 (second to last plot from the bottom of the box in the graph) and large amounts of GC4419+ENZ (plot below the box in the graph) were identified. K68Q Mouse xenografts significantly inhibited xenograft proliferation.

[0257] These results may explain the surprising clinical data showing a positive correlation between MnSOD levels and more invasive breast cancer under certain conditions, suggesting that MnSOD can act as a tumor promoter, rather than its more established function as a tumor suppressor (TS). 95-97 These studies and previous data 52,55This suggests a dual role for MnSOD, with the tetramer theoretically functioning as a TS during the early, proliferative phase of tumorigenesis. However, as carcinogenesis progresses, monomeric MnSOD-K68-Ac may establish a more invasive tumor phenotype. Therefore, MnSOD may be converted from tetrameric SOD to monomeric peroxidase under certain conditions such as nutritional status, genetic damage, or cellular stress. In this model, it is hypothesized that cells and / or mitochondria are stressed from normal metabolic demands necessary for energy production, but continuous exposure to ENZ disrupts the MnSOD ecosystem via MnSOD-K68-Ac, shifting the balance towards higher levels of monomeric MnSOD. Thus, this process may play a role in resulting in ENZR in tumors. Finally, substituting MnSOD detoxification activity with specific pentaza macrocyclic complexes such as GC4419 may functionally increase intracellular SOD activity, potentially restoring cellular metabolism and, importantly, reversing the phenotype of ENZR tumor cells.

[0258] The malignancy of prostate tumors correlates with increased MnSOD-K68-Ac levels. Genomics has shown a correlation between MnSOD-Ac and prostate cancer. 98 To expand on this data, tissue microarrays (TMAs) containing 21 PINs, 28 grade 3 prostate tumors, and 25 grade 4 prostate tumors were stained with our anti-MnSOD-K68-Ac antibody, and the samples were scored according to staining intensity. Quantification using Tissue-Gnostics software revealed a significant increase in MnSOD-K68-Ac staining correlated with increasing tumor grade (Figure 22a, b). These results indicate the existence of human prostate tumors exhibiting a MnSOD-K68-Ac signature.

[0259] MnSOD-K68-Ac / ROS / HIF2 α Axial dysregulation indicates the phenotype of ENZR tumors with systemic plasticity. The mechanisms of ENZR include a wide range of gene mutations, AR splice variants, AR dysregulation, and AR-related signaling pathways. However, some metastatic prostate tumors exhibit ENZR through stem cell-like mechanisms that are independent of AR signaling. 87 MnSOD K68Q To investigate whether the mechanism by which expression triggers ENZR is due to dysregulation of AR signaling, we used LNCaP-3xAR-LNCaP cells containing the mCherry reporter gene downstream of the 3xAR binding site and a minimal promoter as a surrogate for measuring AR signaling. These cells were then treated with lenti-MnSOD. K68Q When the cells were infected with (LNCaP-3xAR-Cherry-MnSOD), the subsequent cells (LNCaP-3xAR-Cherry-MnSOD) K68Q ) is LNCaP-MnSOD K68Q The cells exhibited ENZR similar to that of the control cells (see Figure 20b, bar 3). Surprisingly, these cells showed no change in AR protein levels (Figure 23a) or AR transcriptional activity (Figure 23b) compared to the control cells. Rather, these cells showed HIF2, one of two downstream biomarkers related to lineage plasticity. α (Figure 24b) and elevated SOX2 / Oct4 levels were observed. HIF2 α This is important because stabilization can induce a phenotype of systemic plasticity and ENZR. Ultimately, HIF2 α Knockdown of MnSOD reduced both SOX2 / Oct4 levels (data not shown) and returned ENZR tumor cells to a sensitive phenotype (Figure 24b, bar 5 vs 6). Therefore, MnSOD K68Q Disruption of cellular metabolism due to the expression of HIF2 α It is hypothesized that this stabilizes and brings about the characteristics of systemic plasticity, which is a potentially novel mechanism for the ENZR phenotype in a subset of prostate tumors. Therefore, these results suggest that MnSOD-K68-Ac / ROS / HIF2 α This suggests that it could be a therapeutic target for new interventions.

[0260] Example 3 Estrogen receptor-positive (ER+) invasive ductal carcinoma in situ (IDC), the most common type of breast cancer, is generally treated with selective estrogen receptor modulators (SERMs), and several studies have shown improved clinical outcomes. 99,100 ER+ IDCs are classified into luminal A and luminal B (LuBCa) types. LuBCa accounts for the majority of breast cancer deaths in the United States and exhibits invasive tumor characteristics such as elevated growth index (high Ki-67), poor differentiation (high grade), and an increased risk of recurrence and metastasis. 101,102 The mortality rate in women with LuBCa is at least partially attributable to the development of resistance to selective estrogen receptor modulators (SERMs) and the failure to receive alternative systemic therapy. 103 Therefore, identifying the mechanisms of SERM resistance is clinically very important.

[0261] The ER pathway plays a central role in breast cancer, and endocrine therapy that blocks ER signaling is highly effective. However, over time, a small percentage of ER-positive women experience recurrence due to the development of endocrine resistance. 99,100 Multiple mechanisms of endocrine resistance have been identified, including deregulation of various components of the ER signaling pathway, alterations in cell cycle and cell survival processes, and activation of escape pathways that provide alternative growth and survival stimuli to tumors. 99,104 Most resistance to SERMs is related to one of these processes, but an increasingly accepted mechanism is that it is related to the plasticity of the generative lineage. In this regard, see the recent NCI White Paper (Beltran, 2019) 86 and an overview of the workshop (Yuan, 2019, Cancer Discovery) 88It is stated that lineage plasticity, driven by microenvironmental cues, stochastic genetic / epigenetic processes, metabolic changes, or selective pressures from other therapies, is important in contributing to tumor heterogeneity and the expression of resistance phenotypes. Lineage plasticity is understood as a reversible or irreversible reprogramming process in which mature cells exhibit plasticity through changes in cellular "identity," dedifferentiation into progenitor-like states, or transformation into other differentiated cell types, resulting in the emergence of new phenotypes. 105-109 Disruption of mitochondrial physiological function may be a novel mechanism that leads to phylogenetic plasticity and could be a way for tumor cells to establish a phenotype resistant to therapeutic intervention. 110,111 Therefore, lineage plasticity may lead to treatment-resistant phenotypes in the ER+LuBCa IDC subgroup.

[0262] Antioxidant enzymes are dysregulated in tumors. 108,109 This corresponds to the fact that tumor cells become resistant to treatment when metabolic homeostasis is subsequently lost. 108-110 Manganese superoxide dismutase (MnSOD) is a crucial mitochondrial detoxification enzyme, and its deficiency or dysregulation can lead to involvement in metabolism, carcinogenesis, and, importantly, treatment resistance. While a mechanistic link between mitochondrial ROS dysregulation, MnSOD detoxification activity, and tumor cell resistance has long been suggested, rigorous models supporting this idea have limitations. Non-physiological levels of MnSOD acetylation (Ac) may, at least partially, be associated with metabolic and bioenergetic balance, as well as tumor cell proliferation and / or survival, and under certain cellular conditions, could function as an in vivo driver of tumor resistance by inducing phylogenetic plasticity. For example, if a mitochondrial signaling axis centered on MnSOD-Ac exists and is dysregulated, cellular metabolism is disrupted, leading to abnormal ROS levels (Zhu, 2019, Nature Commun). 52 HIF2 α This abnormally stabilizes the cell and activates the dedifferentiation program (He, 2019; Proc. Natl. Acad. Sci.).55 Therefore, MnSOD appears to exhibit a dual nature based on the acetylation state of lysine 68 (K68), with the deacetylated homotetramer form functioning as a protective detoxification enzyme against persistent / abnormal ROS. On the other hand, K68-Ac inhibits homotetramer formation, shifting the equilibrium of MnSOD to primarily monomeric MnSOD, which functions as a peroxidase and / or oncoprotein. Thus, MnSOD-K68-Ac is associated with MnSOD. K68Q When present in conditions other than the normal physiological state modeled by its expression, cellular metabolism is disrupted, ROS levels increase, and HIF2 α This stabilizes the system and promotes systemic plasticity and the PanR (treatment resistance) phenotype. 112,113 Therefore, targeting the MnSOD-K68-Ac axis with an appropriate pentaza macrocyclic complex (e.g., GC4419) may provide an effective new therapeutic option.

[0263] MCF7-MnSOD K68Q The cells showed resistance to fulvestrant (Fulv) and palbociclib (Palb). PALOMA-3 114 The trial showed that combination therapy with Fulv and Palb improved progression-free survival (PFS) compared to Fulv monotherapy, but no improvement in overall survival was observed in women with endocrine resistance. MnSOD K68Q To evaluate whether cells expressing MCF7-MnSOD would show resistance to these drugs, a clonal tumor cell survival test was performed, and MCF7-MnSOD K68Q Cells produce MnSOD WT Compared to MCF7 cells expressing , these cells showed resistance to Fulv (Fulv-R, Figure 25a) and Palb (Palb-R, Figure 25b) (bar 2 vs 4). Furthermore, the combined use of the pentaza macrocyclic ring complex GC4419 and Palb increased tumor cell death (Figure 25c), confirming that the macrocyclic ring complex is effective in reducing resistance and improving tumor cell death efficacy in co-therapy.

[0264] MnSOD-Ac-K68 / HIF2 α Disruption of the axis leads to phylogenetic plasticity in breast cancer cells. By disrupting mitochondrial metabolism, tumors, including breast cancer, exhibit phylogenetic plasticity, a cellular developmental process that results in alternative cell "fates" and tumor-resistant phenotypes due to changes in the cellular environment, such as genetic / epigenetic damage or exposure to therapeutic drugs. 115 It will be reprogrammed to do so. 104,116 MCF7-MnSOD K68Q and T47D-MnSOD K68Q Since the cells did not show any changes in ER signaling or ER-related pathways, it was suggested that TamR emerges via an ER-independent mechanism. 54 However, MCF7-MnSOD K68Q Cells are known to promote lineage plasticity through HIF2 α It has also been shown that levels of were increased (Figure 26a). Furthermore, levels of two established stem cell markers, OCT4 and SOX2, were also increased (Figure 26a). Importantly, HIF2 α Knockdown of HIF2 also reduced SOX2 and OCT4 levels (data not shown), and the TamR cell phenotype was converted to that of susceptible tumor cells (Figure 26b, bar 5 vs 6). These results suggest that HIF2 α This is consistent with previously published data showing that it correlates with an increased risk of distant recurrence and poor outcomes. This suggests that the induction of systemic plasticity is linked to MnSOD K68Q This suggests that it may be a mechanism that leads to more invasive tumors and the TamR phenotype in mammary tumor cells expressing it.

[0265] Abnormal HIF2 α The level promotes systemic plasticity, leading to chemotherapy resistance and metastasis. MnSOD K68QBreast tumor cells expressing may exhibit the PanR tumor cell phenotype to drugs commonly used in the treatment of women with LuBCa. Therefore, cisplatin-resistant (Cispl-R) MCF7 cells were selected by exposing them to 5 μM Cispl for 3 months. These MCF7-Cispl-R cells were selected for MnSOD-K68-Ac and HIF2 α The levels show an increase (Figure 27a), and the ROS levels also show an increase (Figure 27b), which indicates that abnormal MnSOD-K68-Ac is causing an increase in ROS levels in HIF2 α This means that it leads to stabilization. To test this idea, it has been shown that exposure to GC4419 or knockdown of HIF2 reduces ROS levels in cells (data not shown). Finally, GC4419 or shHIF2 α MCF7-Cispl-R cells treated with [method / method] showed reduced SOX2 and OCT4 levels (data not shown) and converted Cispl-R cells to a susceptible phenotype (Figure 27c). This data is in contrast to HIF2 due to abnormal MnSOD-K68-Ac and ROS levels. α This suggests that the stabilization of leads to systemic plasticity and the Cispl-R phenotype. Based on this data, we propose that MnSOD-K68-Ac leads to abnormal ROS and HIF2 α We propose promoting the development of the PanR phenotype in mammary gland tumor cells and enriching them with stem cell-like properties.

[0266] Example 4 Acetylation (Ac) of manganese superoxide dismutase (MnSOD) is an important post-translational modification with significant regulatory detoxification activity in various disease models. Acetylation of lysine-68 (K68) of MnSOD (K68-Ac) alters its function from a homotetramer that removes superoxide to a peroxidase-directed monomer. Estrogen receptor-positive (ER+) breast cancer cell lines (MCF7 and T47D) selected for serial proliferation with cisplatin (CDDP) and doxorubicin (DXR) have been shown to increase MnSOD-K68-Ac. Furthermore, effective acetylation-mimicking mutant genes (MnSOD) K68Q MnSOD-K68-Ac, modeled by the expression of (), also causes therapeutic resistance to CDDP and DXR, loss of tetrameric MnSOD, changes in mitochondrial structure and morphology, and cellular metabolic abnormalities. MnSOD in mouse embryonic fibroblasts (MEF) K68Q Expression of [the gene] induced a transformation-tolerant phenotype in vitro.

[0267] Cisplatin and doxorubicin-resistant breast cancer cells show increased levels of MnSOD-K68-Ac. MnSOD-K68-Ac is concentrated in women with luminal type B breast malignancy that typically recurs on endocrine therapy. 52 This describes the mitochondrial-based signaling network for tamoxifen resistance (TamR) expression determined using breast cancer tissue culture cells. In this example, we investigated whether this resistance phenotype is broadly applicable to other standard treatments in women with luminal B breast cancer, such as cisplatin (CDDP) and doxorubicin (DXR). To address this question, standard methods for selecting resistance to anticancer drugs in tissue culture were used in both MCF7 and T47D, two established ER+ breast cancer cell lines. 53MCF7 cells were selected for resistance for 3 months to three doses of CDDP (250 nM, 500 nM, 1 μM) and DXR (500 pM, 1 nM, and 2 nM). Both MCF7 CDDP-resistant (MCF7-CDDP-R) and MCF7 DXR-resistant (MCF7-DXR-R) cells showed no change in total MnSOD protein levels, while MnSOD-K68-Ac increased in a dose-dependent manner (Figures 28a, 28b). K68 acetylation mimics (MnSOD) mimic the constitutive acetylation state by substituting lysine (K) with glutamine (Q) and the constitutive deacetylation state by substituting arginine (R). K68Q ) and deacetylation mimetic (MnSOD K68R We created a sample and specifically investigated the effect of K68 acetylation on drug resistance. 54,52,55 MnSOD K68Q MCF7 cells overexpressing MnSOD-Ac showed high resistance to short-term treatment (48 hours) with the highest doses of CDDP (1 μM) and DXR (2 nM). These results clearly suggest the role of dysregulated MnSOD-Ac in the disruption of MnSOD in vivo and the pan-resistance (PanR) tumor cell phenotype in MCF7 and T47D breast cancer cells.

[0268] cell line Wild-type ER+MCF7 human breast cancer cells and immortalized MnSOD - / - Mouse embryonic fibroblasts (MEFs) were cultured in Dulbecco's modified Eagle medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS; Sigma) and 1% antibiotic antifungal solution (Sigma) at 5% CO2 and 37°C using standard growth medium. Primary MEFs were isolated from mouse embryos of the same strain (E13.5) and cultured at 37°C, 5% CO2, and 6% oxygen. Lentivirus-infected MCF7 and MEFs were cultured in medium supplemented with 1 μg / ml puromycin. Cisplatin and doxorubicin-resistant MCF7 cells were treated for more than 3 months to establish permanent cell lines. All experiments were performed using exponentially growing cells at 50%–70% confluence.

[0269] Lentivirus infection Human lentivirus-MnSOD plasmid was used for site-directed mutagenesis, inducing mutations at position 68 from lysine to either arginine (deacetylation mimicry) or glutamine (acetylation mimicry) (Bioinnovation). 293T cells were transfected with 5 μg of the target DNA, 5 μg of psPAX2 packaging plasmid, and 300 ng of VSV.G envelope plasmid. After overnight incubation, fresh medium was added, and the viral supernatant was collected after a further 48 hours and filtered using a 0.45 μm filter (Corning). MCF7 and MEF cells were infected with lentivirus at 40% confluence with 10 μg / ml polyblen for 72 hours. Cells were then collected in standard medium for 24 hours and selected with 1 μg / ml puromycin.

[0270] Cloning cell survival assay To evaluate the viability of clonal cells by testing cell proliferation at low densities, 500 exponentially growing cells were serially diluted and triple-plated in 6-well plates. Cell proliferation was observed over 14 days in standard growth medium. After fixing the cells with 70% ethanol for 5 minutes, they were stained with 0.5% crystal violet (in 25% methanol) for 20 minutes. Photographs were taken of the stained plates, and the number of cell colonies with more than 50 cells was counted to calculate the clonal viability.

[0271] Soft agar colony formation assay 10,000 cells were plated in three separate batches onto 0.3% agar in 1X growth medium on a 0.6% basal agar base layer in 1X growth medium (2X growth medium was DMEM supplemented with 20% FBS, 2% penicillin-streptomycin, 1% 2.5M glucose, and 2% GlutaMax 100X). Colony size was monitored for three weeks, and colony growth was visualized and images acquired under a microscope by the end of the three-week period.

[0272] MTT cell proliferation assay Cell proliferation was measured using the MTT proliferation assay kit (ab211091). 10,000 exponentially growing cells were plated in triples in a 96-well plate using standard growth medium. After overnight, the cells were treated with the specified reagent and incubated for 48 hours. The treatment solution was then discarded, and a mixture of 50 μl of MTT reagent and 50 μl of serum-free medium was added to each well. The mixture was incubated at 37°C for 3 hours. Subsequently, 150 μl of MTT solvent was added, and the plate was shaken for 15 minutes, away from light. Absorbance was read at OD=590 nm and used to evaluate cell proliferation.

[0273] Incorporation and isolation of N-(ε)-acetyllysine into MnSOD-K68 Acetyl-lysyl-tRNA synthetase / tRNA derived from M. Barkeli CUA pEVOL-AcKRS expresses a pair of proteins, and pET21a-MnSOD expresses a site-directed mutation that incorporates N-(ε)-acetyl-l-lysine (AcK) into K68. K68TAG These were co-transformed into BL21(DE3)pMAGIC bacteria. BL21(DE3)pMAGIC cells transformed with pEVOL-AcKRS and pET21a-MnSODK68TAG were cultured in 3 ml of sterile LB medium supplemented with 300 μg / ml ampicillin, 50 μg / ml kanamycin, and 50 μg / ml chloramphenicol. 1 ml of this culture was cultured overnight in 100 ml of LB medium containing 300 μg / ml ampicillin, 50 μg / ml kanamycin, and 50 μg / ml chloramphenicol (200 rpm, 37°C). 1 ml of the culture cultured overnight was inoculated into 100 ml of LB medium with the same antibiotic concentration and shaken at 220 rpm, 37°C until OD=600 nm was 0.6. Bacterial cultures were induced with 0.4 mM IPTG, nicotinamide, arabinose, and N-acetyllysine, and then shaken overnight at 180 rpm at room temperature.

[0274] BL21(DE3)pMAGIC cells transformed with pEVOL-AcKRS and pET21a-MnSODK68TAG were cultured in 3 ml of sterile LB medium supplemented with 300 μg / ml ampicillin, 50 μg / ml kanamycin, and 50 μg / ml chloramphenicol. 1 ml of the culture was then cultured overnight in 100 ml of LB medium containing 300 μg / ml ampicillin, 50 μg / ml kanamycin, and 50 μg / ml chloramphenicol (200 rpm, 37°C). 1 ml of the culture cultured overnight was inoculated into 100 ml of LB medium with the same antibiotic concentrations and shaken at 220 rpm, 37°C until the OD600 reached 0.6. Bacterial cultures were induced with 0.4 mM IPTG, nicotinamide, arabinose, and N-acetyllysine and shaken overnight at 180 rpm, room temperature.

[0275] The protein was dissolved in a buffer containing 1.5 mg / ml PMSF and 1 mg / ml lysozyme (20 mM imidazole, 50 mM Tris-HCl, 200 mM NaCl, pH=8). The lysate was incubated on ice for 10 minutes, sonicated for 20 minutes (10 seconds on, 5 seconds off, 50% amplitude), and the supernatant was centrifuged (13,000 g, 30 minutes). 0.2 mL of Ni2+ NTA beads were added to the recovered supernatant and rotated at 4°C for 1 hour. The supernatant was filtered through a Probond Purification System filter column to separate the protein. The column was washed three times with lysis buffer. The protein was then eluted with elution buffer (250 mM imidazole, 50 mM Tris-HCl, 200 mM NaCl, pH=8) and quantified for further experiments.

[0276] Peroxidase activity assay MnSOD with Flag tag WT MnSOD K68R , and MnSOD K68QOne million cells expressing [protein name] were lysed for 30 minutes in 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% NP-40, 5% glycerol, a protease inhibitor (BioTool), and TSA (Tri chostatin A, Sigma). The lysates were quantified by the Bradford assay (BioRad), and IP was performed using an anti-Flag antibody (company). The peroxidase enzyme activity of these IP-treated proteins was measured using pyrogallol as a substrate. The final concentrations of the reaction mixture were 14 mM potassium phosphate (Sigma), 0.027% (v / v) hydrogen peroxide (Sigma), and 0.5% (w / v) pyrogallol (Sigma). The plate was tapped to mix the sample and reaction reagents, incubated at room temperature for 10 minutes, and then read at OD=420 nm. The increase in A420 was recorded every 3 minutes. For all test samples and blanks, ΔA420 / 20s was determined using the maximum linear velocity or 0.5-minute intervals. Peroxidase activity was calculated using the following formula: Units / mL = [(ΔA420 / 20s Test Sample - ΔA420 / 20s Blank) (Reaction Volume) (Dilution Factor)] / [(12)(0.1)].

[0277] statistical analysis Statistical analysis was performed using GraphPad Prism for Windows (GraphPad Software, San Diego, California). Unless otherwise specified, data were presented as mean SEM. One-way ANOVA with Tukey's post-test was used to examine differences between three or more means. Significance was determined by p<0.05 and 95% confidence intervals.

[0278] Example 5 Allogeneic transplant mammary gland tumors isolated from Sirt3-deficient mice exhibit marked cytotoxic sensitivity to GC4419. We evaluated whether superoxide-removing agents could reverse cytotoxicity and / or Tam resistance in cells exhibiting the SIRT3-MnSOD-Ac signature. IP injection of 2 mg / kg of GC4419 (a specific Mn-based MnSOD mimetic that removes superoxide) 30 minutes prior to IR attenuated liver injury in Sirt3-deficient mice via a mechanism shown to prevent ROS accumulation (Colemanet al., 2014, Antioxid. Redox Signal). To address whether this Tam resistance is restored, we used tumor cell lines derived from mammary gland tumors that spontaneously occurred in Sirt3 knockout mice (Sirt3 - / - - Breast tumor cells (Sirt3 - / - We used a cell line called -MT). Subsequently, these cell lines were classified as SIRT3 wild-type (Sirt3 - / - -MT-SIRT3 WT ) or deacetylated null(Sirt3 - / - -MT-SIRT3 DN The mice were infected with the ) gene and lenticularase and expressed, and selected to evaluate tumor growth by bioluminescence. Allogeneic tumor mice were (1) control and untreated Sirt3 - / - -MT-SIRT3 DN (2) GC4419 processed Sirt3 - / - -MT-SIRT3 DN (3) Control, untreated Sirt3 - / - -MT-SIRT3 WT ; and (4) GC4419 processed Sirt3 - / - -The experiment was conducted by dividing the subjects into four groups: MT-SIRT3WT. These experiments revealed that Sirt3 - / - -MT-SIRT3 DN When the graft was injected via IP at 5-week intervals, it demonstrated an antiproliferative effect, showing a significant reduction in tumor growth compared to control mice (Figure 29a, upper panel: control vs. lower panel: GC4419). In contrast, Sirt3 mice exposed to GC4419... - / - -MT-SIRT3 WTIn the grafts, no changes were observed in tumor growth (Figure 29b), although these tumors grew slightly slower (Figures 29a-b; in Figure 7b, the line ending highest on the y-axis at 4 weeks represents the control, and the line ending low on the y-axis at 6 weeks represents the presence of GC4419). In vitro data using various Tam-resistant and Sirt3-deficient tumor cell lines also indicate that GC4419 reverses Tam resistance (data not shown).

[0279] Example 6 MnSOD K68Q Expression of (i.e., the K68-Ac mimic mutant) induces ionizing radiation resistance (IRR) in MCF7 cells. To investigate whether the MnSOD-K68-Ac-ROS axis plays a role in IRR, we hypothesized that MnSOD acetylation may be involved in how tumor cells are reprogrammed to produce the IRR phenotype. To address this idea, we lenticular MnSOD into the established tumor cell line MCF-7. K68Q Infect with MnSOD K68Q We induced and validated the expression of MCF-7-MnSOD. WT and MCF-7-MnSOD K68Q Cells were treated with or without the addition of 5 Gy of IR. Cloning cell survival experiments showed that MnSOD was present in MCF7 cells. K68Q Forced expression of MCF-7-MnSOD was shown to reduce IR-induced cell death (Figure 30a, black bars vs. checked bars). Furthermore, exposure to GC4419, which has been shown to chemically remove superoxide, substituted MnSOD activity, leading to MCF-7-MnSOD. K68Q It was hypothesized that the IRR phenotype of the cells would be reversed. Indeed, clonal cell viability tests showed that exposure to GC4419 reversed the IRR phenotype (Figure 30b, 2 checked bars vs 4 bars). These experiments were compared with MCF-7-MnSOD. K68Q The IRR observed in cells is converted to a sensitive phenotype upon exposure to GC4419, suggesting that substitution of SOD activity is the mechanism in this process.

[0280] Example of a protocol The following protocol example allows for the detection of AcK68, SIRT3, and HIF2 in tissue samples containing tumor cells. α Methods for measuri...

Claims

1. (i) a level of sirtuin (SIRT3) protein below a first predetermined threshold, (ii) a level of lysine 68-residue acetylated (AcK68) manganese superoxide dismutase above a second predetermined threshold, and (iii) a third predetermined threshold of hypoxia-inducible factor 2 exceeding a stem cell-like lineage plasticity. α (HIF2 α A drug for treating cancer in mammalian subjects having a tumor signature characterized by one or more of the expression levels of ), comprising a pentaza macrocyclic complex corresponding to the following formula, A drug characterized by being used in the process of administering a pentaza macrocyclic ring complex corresponding to the following formula to the mammalian subject. formula: 【Chemistry 1】

2. A step of selecting a mammalian subject suitable for treatment with a pentaza macrocyclic complex corresponding to the following formula, The aforementioned selection is, (a) Obtain a test tissue sample containing tumor cells from the subject, (b) (i) whether the level of sirtuin (SIRT3) protein in tumor cells of the tissue sample is below a first predetermined threshold, (ii) whether the level of (AcK68) manganese superoxide dismutase acetylated at lysine 68 residue is above a second predetermined threshold, and (iii) hypoxia-inducible factor 2 α (HIF2 α Evaluating the test tissue sample to determine whether the expression level of ) exceeds a third predetermined threshold indicating stem cell lineage plasticity, and then (c) Determining that the subject is suitable for treatment if one or more of the above criteria (i), (ii), and / or (iii) are met. A process that involves, If the subject is selected as suitable for treatment, the process involves administering a pentaza macrocyclic complex corresponding to the following formula. A drug for treating mammalian cancer, characterized by being used in the above-mentioned context, and containing a pentaza macrocyclic ring complex corresponding to the above formula. formula: 【Chemistry 2】

3. The agent according to claim 2, wherein the step of selecting the target includes measuring the level of AcK68 by an immunohistochemical method that involves contacting the tissue with an anti-AcK68 monoclonal antibody and measuring the level of the anti-AcK68 monoclonal antibody that binds to AcK68 in the tissue sample.

4. The agent according to claim 3, wherein the anti-AcK68 monoclonal antibody includes a binding region that specifically binds to a region of MnSOD containing acetylated lysine 68 residues.

5. A step of selecting a mammalian subject suitable for treatment with a pentaza macrocyclic complex corresponding to the following formula, The aforementioned selection is, (a) Obtain a test tissue sample containing tumor cells from the subject, (b) Evaluate the test tissue sample to determine one or more of the following criteria: (i) whether the level of sirtuin (SIRT3) protein in tumor cells of the tissue sample is below a first predetermined threshold; (ii) whether the level of (AcK68) manganese superoxide dismutase acetylated at lysine 68 residues is above a second predetermined threshold; and (iii) whether the expression level of hypoxia-inducible factor 2α (HIF2α) is above a third predetermined threshold indicating stem cell lineage plasticity; then (c) Determining that the subject is suitable for treatment if one or more of the above criteria (i), (ii), and / or (iii) are met. A process that involves, If the subject is selected as suitable for treatment, the process involves administering a pentaza macrocyclic ring complex corresponding to the following formula and further anticancer drugs. A drug for treating mammalian cancer, characterized by being used in the above-mentioned context, and containing a pentaza macrocyclic ring complex corresponding to the above formula. formula: 【Transformation 3】

6. (a) A test kit for performing an assay to analyze a tissue sample obtained from a subject and containing tumor cells, comprising: (i) whether the level of sirtuin (SIRT3) protein in tumor cells of the tissue sample is below a first predetermined threshold; (ii) whether the level of (AcK68) manganese superoxide dismutase acetylated at lysine 68 residue is above a second predetermined threshold; and (iii) hypoxia-inducible factor 2 α (HIF2 α A test kit for performing an assay that can determine whether the expression level of ) exceeds a third predetermined threshold indicating stem cell lineage plasticity, including one or more of these criteria; and (b) A pentaza macrocyclic complex corresponding to the following formula, characterized in that it is used in the step of administering to the subject as a drug for treating cancer when any one or more of the above criteria (i) to (iii) are met. A kit for treating cancer in mammals, including [mention specific product / technology]. formula: 【Chemistry 4】

7. The kit according to claim 6, wherein the assay comprises an immunohistochemical assay comprising an anti-AcK68 antibody selectively capable of binding to AcK68 for measuring the level of AcK68 in a tissue sample.

8. The drug and / or kit according to any one of claims 1 to 7, wherein a first predetermined threshold for sirtuin (SIRT3) protein activity in tumor tissue is a standard deviation level less than 1 from a normal score of non-cancerous tissue of the same type as the tumor tissue, the normal score being measured by taking the average of at least six non-cancerous tissue samples of the same tissue type from at least six different individuals, measured by immunohistochemistry.

9. The agent and / or kit according to any one of claims 1 to 8, wherein a second predetermined threshold for (AcK68) manganese superoxide dismutase acetylated at lysine 68 residues is a standard deviation level higher than 1 from the normal score of non-cancerous tissue of the same type as tumor tissue, the normal score being measured by taking the average of at least six non-cancerous tissue samples of the same type from at least six different individuals, measured by immunohistochemistry.

10. Hypoxia-inducible factor 2 α (HIF2 α The drug and / or kit according to any one of claims 1 to 9, wherein a third predetermined threshold for the expression level of ) is a standard deviation level higher than 1 from the normal score of non-cancerous tissue of the same type as the tumor tissue, the normal score being measured by taking the average of at least six non-cancerous tissue samples of the same type from at least six different individuals, measured by immunohistochemistry.

11. The agent and / or kit according to any one of claims 1 to 10, further comprising the step of administering ionizing radiation to the subject as part of a radiotherapy process that provides treatment for cancer.

12. The drug and / or kit according to any one of claims 1 to 11, further characterized in that the drug is used in a method comprising the step of administering a pentaza macrocyclic ring complex of the formula, and further administering an anticancer agent comprising one or more chemotherapeutic agents and therapeutic agents that inhibit hormone receptor pathways related to the growth or progression of cancer, before, simultaneously with, or after the administration of the pentaza macrocyclic ring complex of the formula, and / or the kit further comprises an anticancer agent used in the step of administering the drug as a drug for treating cancer.

13. The agent and / or kit according to claim 12, wherein the chemotherapeutic agent comprises either a platinum-containing chemotherapeutic agent or an anthracycline-based chemotherapeutic agent.

14. The agent and / or kit according to claim 13, wherein the chemotherapeutic agent comprises at least one platinum-containing chemotherapeutic agent selected from the group consisting of cisplatin, oxaliplatin, carboplatin, nedaplatin, lovaplatin, heptaplatin, dicycloplatin, lipoplatin, LA-12, phosphaplatin, phenantriplatin, prolyndac, triplatin tetranitrate, picoplatin, satraplatin, and / or pharmaceutically acceptable salts thereof, and / or an anthracycline chemotherapeutic agent selected from the group consisting of doxorubicin, daunorubicin, epirubicin, idarubicin, and / or pharmaceutically acceptable salts thereof.

15. The agent and / or kit according to claim 12, wherein the therapeutic agent inhibits a hormone receptor pathway associated with the growth or progression of cancer.

16. The agent and / or kit according to claim 15, wherein the therapeutic agent inhibiting the hormone receptor pathway associated with the growth or progression of the cancer targets one or more of the estrogen receptor pathway, the progesterone receptor pathway, and the androgen receptor pathway.

17. The agent and / or kit according to claim 16, wherein the therapeutic agent targeting one or more of the estrogen receptor pathway, the progesterone receptor pathway, and the androgen receptor pathway is selected from the group consisting of estrogen receptor inhibitors, estrogen receptor degraders / downregulators, selective estrogen receptor modulators (SERMs), aromatase inhibitors, GnRH agonists, and CDK4 / 6 inhibitors.

18. The agent and / or kit according to claim 17, wherein the therapeutic agent targeting the estrogen receptor pathway comprises at least one selected from the group consisting of tamoxifen, clomiphene, 4-hydroxytamoxifen, toremifene, raloxifene, napoxidine, rasofoxifen, bazedoxifen, ospemifene, fulvestrant, brillanestrant, elastrant, palbociclib, abemaciclib, ribociclib, and derivatives, salts, and / or prodrugs thereof.

19. The agent and / or kit according to claim 16, wherein the therapeutic agent targeting the androgen receptor pathway comprises any one selected from the group consisting of androgen receptor antagonists, androgen synthesis inhibitors, and antigonadotropins.

20. The therapeutic agents that target the androgen receptor pathway include cyproterone acetate, megestrol acetate, chlormadinone acetate, spironolacon, oxendron, osaterone acetate, flutamide, bicalutamide, nilutamide, topirutamide, enzalutamide, apalutamide, dienogest, drospirenone, medgestone, nomegestrol acetate, promegestone, trimegestone, ketoconazole, abiraterone acetate, ceviteronel, aminoglutethimide, finasteride, dutasteride, episteride, alpha-tradial, cyproterone acetate, spironolactone, medrogestone, and flutamide. The agent and / or kit according to claim 19, comprising at least one selected from the group consisting of , nilutamide, biflulanol, leuprorelin, cetrorelix, allylestrenol, chlormadinone acetate, cyproterone acetate, gestolone caproate, hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, osaterone acetate, oxendrone, estradiol, estradiol esters, ethinylestradiol, conjugated estrogens, diethylstilbestrol, and their derivatives, salts, and / or prodrugs.

21. The agent and / or kit according to claim 17, wherein the therapeutic agent targeting the progesterone receptor pathway comprises a progesterone type I, type II, or type III selective receptor modulator (SPRM), which is at least one selected from the group consisting of onapristone, mifepristone, ronaprisan, agrepristone, Org31710, Org31806, CDB-2914, and CDB-4124, and their derivatives, salts, and / or prodrugs.

22. A medicament for treating a tumor in a mammalian subject suffering from a tumor resistant to a chemotherapeutic agent, the medicament comprising a pentaaza macrocyclic ring complex corresponding to the following formula, wherein the tumor has (i) a level of sirtuin (SIRT3) protein below a first predetermined threshold, (ii) a level of manganese superoxide dismutase acetylated at lysine 68 residue (AcK68) above a second predetermined threshold, and (iii) hypoxia-inducible factor 2 above a third predetermined threshold indicating stem cell lineage plasticity α (HIF2 α ) having a tumor signature characterized by any one or more of the expression levels, and the medicament is (a) Obtain a test tissue sample containing tumor cells from the subject; (b) (i) whether the level of sirtuin (SIRT3) protein activity in tumor cells of the tissue sample is below a first predetermined threshold, (ii) whether the level of (AcK68) manganese superoxide dismutase acetylated at lysine 68 residue is above a second predetermined threshold, and (iii) hypoxia-inducible factor 2 α (HIF2 α Evaluating the tissue sample to determine a criterion including whether the expression level of ) exceeds a third predetermined threshold indicating stem cell lineage plasticity, or any one or more of these criteria; then (c) If one or more of the above criteria (i) to (iii) are met, the subject is deemed suitable for treatment. This is a process of selecting a target suitable for treatment; If the subject is selected as suitable for treatment, the process involves administering a pentaza macrocyclic complex corresponding to the following formula to the subject for treatment. A drug for treating tumors in mammals, characterized by its use in the following way. formula: 【Transformation 5】

23. The agent according to claim 22, further characterized in that it is used in a method comprising the step of administering a pentaza macrocyclic ring complex of the above formula, and further the step of administering a chemotherapeutic agent to a subject suffering from a resistant tumor before, simultaneously with, or after administration of the pentaza macrocyclic ring complex of the above formula.

24. A drug for treating a tumor resistant to ionizing radiotherapy in a mammalian subject suffering from a tumor containing a pentaza macrocyclic ring complex corresponding to the following formula, wherein the tumor has (i) a level of sirtuin (SIRT3) protein below a first predetermined threshold, (ii) a level of lysine 68-residue acetylated (AcK68) manganese superoxide dismutase above a second predetermined threshold, and (iii) a third predetermined threshold of hypoxia-inducible factor 2 exhibiting stem cell-like lineage plasticity above a third predetermined threshold. α (HIF2 α The drug has a tumor signature characterized by one or more expression levels of ) and the drug is (a) Obtain a test tissue sample containing tumor cells from the subject; (b) (i) whether the level of sirtuin (SIRT3) protein activity in tumor cells of the tissue sample is below a first predetermined threshold, (ii) whether the level of (AcK68) manganese superoxide dismutase acetylated at lysine 68 residue is above a second predetermined threshold, and (iii) hypoxia-inducible factor 2 α (HIF2 α A step of evaluating the tissue sample to determine a criterion including whether the expression level of ) exceeds a third predetermined threshold indicating stem cell lineage plasticity, or any one or more of these criteria; then (c) If one or more of the above criteria (i) to (iii) are met, the subject is deemed suitable for treatment. This is a process of selecting a target suitable for treatment; A drug for treating tumors in mammals, characterized in that, when the subject is selected as suitable for treatment, it is used in a step of treating the subject by administering a pentaza macrocyclic ring complex corresponding to the following formula to the subject. formula: 【Transformation 6】

25. The agent according to any one of claims 22 to 24, further characterized in that it is used in a method comprising the step of administering a pentaza macrocyclic complex of the above formula, and further the step of administering ionizing radiation to a target as part of a radiotherapy process that provides treatment for cancer.

26. The agent according to claim 24, further characterized in that, in addition to the step of administering the pentaza macrocyclic ring complex of the above formula, it is used in a step of administering a chemotherapeutic agent to a target before, simultaneously with, or after the administration of the pentaza macrocyclic ring complex of the above formula.

27. The agent according to claim 23 or 26, wherein the chemotherapeutic agent comprises either a platinum-containing chemotherapeutic agent or an anthracycline-based chemotherapeutic agent.

28. The agent according to claim 27, wherein the chemotherapeutic agent comprises one or more platinum-containing chemotherapeutic agents comprising at least one of cisplatin, oxaliplatin, carboplatin, nedaplatin, lovaplatin, heptaplatin, dicycloplatin, lipoplatin, LA-12, phosphaplatin, phenanthriplatin, prolyndac, triplatin tetranitrate, picoplatin, satraplatin, and / or pharmaceutically acceptable salts thereof, and / or anthracycline chemotherapeutic agents comprising at least one of doxorubicin, daunorubicin, epirubicin, idarubicin, and / or pharmaceutically acceptable salts thereof.

29. The agent according to any one of claims 22 to 28, wherein a first predetermined threshold for sirtuin (SIRT3) protein activity in tumor tissue is a standard deviation level less than 1 from a normal score of non-cancerous tissue of the same type as the tumor tissue, the normal score being measured by taking the average of at least six non-cancerous tissue samples of the same tissue type from at least six different individuals, measured by immunohistochemistry.

30. The agent according to any one of claims 22 to 28, wherein a second predetermined threshold for (AcK68) manganese superoxide dismutase acetylated at lysine 68 residues is a standard deviation level higher than 1 from the normal score of non-cancerous tissue of the same type as tumor tissue, the normal score being measured by taking the average of at least six non-cancerous tissue samples of the same type from at least six different individuals, measured by immunohistochemistry.

31. Hypoxia-inducible factor 2 α (HIF2 α The agent according to any one of claims 22 to 28, wherein a third predetermined threshold for the expression level of ) is a standard deviation level higher than 1 from the normal score of non-cancerous tissue of the same type as tumor tissue, the normal score being measured by taking the average of at least six non-cancerous tissue samples of the same type from at least six different individuals, measured by immunohistochemistry.

32. The agent and / or kit according to any one of claims 1 to 31, wherein the pentaza macrocyclic ring complex is administered to the subject in a dose ranging from 0.2 mg / kg to 40 mg / kg.

33. The agent and / or kit according to any one of claims 1 to 31, wherein the pentaza macrocyclic ring complex is administered to the subject in a dose ranging from 0.2 mg / kg to 24 mg / kg.

34. The agent and / or kit according to any one of claims 1 to 31, wherein the pentaza macrocyclic ring complex is administered to the subject in a dose ranging from 0.2 mg / kg to 10 mg / kg.

35. The agent and / or kit according to any one of claims 1 to 34, wherein the pentaza macrocyclic ring complex is administered via at least one of a parenteral route and an oral route.

36. The agent and / or kit according to any one of claims 1 to 35, wherein the pentaza macrocyclic ring complex is administered intraperitoneally or intravenously.

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