Therapeutic use of atomic quantum clusters

Abstract

Provided are inventions relating to compositions and therapeutic uses of atomic quantum clusters (AQCs), in particular compositions consisting essentially of AQCs comprising five zero-valent transition metal atoms, for the treatment of cell proliferative disorders.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201980063142.3, with an application date of September 25, 2019, a priority date of September 26, 2018, and an invention title of "Therapeutic Use of Atomic Quantum Clusters". Technical Field

[0002] The present invention relates to the therapeutic use of atomic quantum clusters, particularly atomic quantum clusters composed of five zero-valent transition metal atoms. Background Art

[0003] Redox homeostasis is essential for cell survival. Thiols play an important role in maintaining the redox balance. The sulfur atom in the side chain of the amino acid cysteine can exist in a variety of different oxidation states. Under physiological conditions, the sulfur atom of cysteine reversibly transitions between the thiol and disulfide states (reduced and oxidized, respectively), but the transition to higher oxidation states (other than sulfonic acid) is irreversible, meaning that proteins can only be replaced by synthesizing new proteins. Cells in their different compartments (except for the endoplasmic reticulum) are constantly reducing proteins, which spontaneously oxidize in the presence of oxygen. Inside the cell, protein function depends on their sulfur oxidation state. There are two overlapping systems, the glutathione and thioredoxin systems, which have been well conserved throughout evolution and function to keep protein cysteines in a functional reduced state.

[0004] During normal metabolism of cells, reactive oxygen species (ROS) are produced, and the glutathione and thioredoxin systems protect cells from oxidative damage by maintaining the reduced state. If the ROS level increases and exceeds the buffering capacity of the glutathione and thioredoxin systems, activation of signaling pathways and gene expression can occur, inducing apoptosis. Actively proliferating tumor cells show increased respiration and thus higher ROS levels. Moreover, human tumors show insufficient angiogenesis, which contributes to glucose starvation and increased ROS (attributed to redox homeostasis imbalance).

[0005] WO2012 / 059572 describes a combination of at least one AQC and at least one anti-tumor drug for the prevention and / or treatment of cell proliferative disorders. This application describes AQCs composed of 2 to 25 zero-valent transition metal atoms, which have cytotoxic and anti-proliferative effects on cancer cell lines and can therefore be used in combination with anti-tumor agents to treat cell proliferative disorders.

[0006] An object of the present invention is to provide an improved therapeutic composition of AQCs. Summary of the Invention

[0007] Brief Description of the Invention

[0008] In a first aspect, the present invention provides a composition for treating a cell proliferative disorder, which comprises atomic quantum clusters (AQCs) composed of five zero-valent transition metal atoms. In another aspect, the present invention provides the use of the composition as defined herein in the preparation of a pharmaceutical composition for treating a cell proliferative disorder. In another aspect, the present invention provides the use of a composition comprising atomic quantum clusters (AQCs) composed of five zero-valent transition metal atoms as a radiosensitizer for proliferating cells in radiotherapy. In another aspect, the present invention provides a method for preventing or treating a cell proliferative disorder, which comprises administering to a patient in need thereof a therapeutically effective amount of the composition as defined herein. In another aspect, the present invention provides a method for preventing or treating a cell proliferative disorder, which comprises administering to a patient in need thereof a therapeutically effective amount of a composition comprising atomic quantum clusters (AQCs) composed of five zero-valent transition metal atoms, wherein the method does not include treating the patient with an additional anti-tumor drug.

[0009] In another aspect, the present invention provides a method for preventing or treating a cell proliferative disorder, which comprises administering to a patient in need thereof a therapeutically effective amount of a composition comprising atomic quantum clusters (AQCs) composed of five zero-valent transition metal atoms in combination with radiotherapy.

[0010] These and other aspects are described in more detail in the following specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 : Interaction of Ag5-AQC with Escherichia coli (E.Coli) thioredoxin. As can be seen, Ag5-AQC (large grey pentameric molecule) binds to cysteine (molecule highlighted by black arrow) residues, forming an active site. The binding energy is favorable (-167 kJ / mol).

[0012] Figure 2 : Normalized sulfur K-edge X-ray absorption near-edge structure (S-K XANES) spectra of cysteine (a) and glutathione (b) and those with added Ag5-AQCs (c & d respectively). The right panel shows an enlarged region of the left panel. The vertical solid and dashed lines refer to energy sites associated with different S-oxidation states.

[0013] Figure 3 : Normalized S-K XANES spectra of (a) cysteine and cysteine treated with different concentrations of Ag5-AQC, (b) diluted 1:106 relative to the reference stock concentration (RSC), (c) diluted 1:103 relative to the RSC, and (d) the RSC. The vertical lines represent the corresponding energies of different S-oxidation states.

[0014] Figure 4 : Normalized S-KXANES spectra of an aqueous solution of thioredoxin containing PBS before (a) and after (b) treatment with Ag5-AQC. The vertical lines indicate the corresponding energies of different S-oxidation states.

[0015] Figure 5 : Percentages of thioredoxin (TRX) oxidized by Ag5-AQCs, hydrogen peroxide (H 2 O 2 ), and hydroxyl radicals (HO·) alone or in various combinations, as shown on the x-axis.

[0016] Figure 6 : Number of sulfur oxidations of thioredoxin cysteine after various treatments. Sulfur oxidation is affected by Ag5-AQCs, hydrogen peroxide (H 2 O 2 ), and hydroxyl radicals (HO·). The combination with Ag5-AQC greatly enhances the effects of H 2 O 2 and HO·.

[0017] Figure 7 : Escherichia coli (E.Coli) survival was measured after adding different concentrations of dithiothreitol (DTT) alone (control) or in combination with Ag5-AQC. In the absence of DTT (0 mM), low concentrations of Ag5-AQC killed the bacteria. When increasing concentrations of DTT (0.1 mM) were co-administered with Ag5-AQC, bacterial viability was partially restored, indicating that DTT rescued E.Coli from the action of Ag5-AQC. Thus, 10 mM DTT was toxic to the bacteria, however, co-administration with Ag5-AQC reversed the effect of DTT.

[0018] Figure 8 : Dose-response (0.24 - 1.2 mg / L) graphs for each cell line when Ag5-AQCs were added.

[0019] Figure 9 : Results show the percentage of cell viability of the A549 cell line compared to the control when 5-atom clusters made of copper (Cu 5 -AQCs) were added.

[0020] Figure 10: Ag5-AQC oxidation of thiols in proteins. A549 cells were transduced with Premo Cellular Redox Sensor. After 48 hours, time-lapse imaging was performed using a Leica TCS SP5 confocal microscope. The samples were excited with 405 and 488 nm lasers, and the emission ratio (405 / 488 ratio) in the green channel (500 - 530 nm) was calculated. Images were taken every 10 seconds for 10 minutes after the addition of Ag5-AQC (IC50). The cell pseudocolor ratio maps at the specified time points highlighted the changes in the redox state. In each experiment, the ratios of two individual cells (arrows) were quantified and plotted against time.

[0021] Figure 11 : (a) The MTF1 location in response to Ag5-AQCs was detected by indirect immunofluorescence. A549 cells were treated with Ag5-AQCs for 1 hour and 2 hours, after which they were fixed and stained with anti-MTF1 antibody and DAPI to counterstain the nuclei. Ag5-AQCs showed that MTF-1 was significantly translocated into the nuclei compared to control cells (left panel) (right panel). (b) In HEK293 cells, Ag5-AQCs induced the translocation of Nrf2 from the cytoplasm to the nucleus. Immunofluorescence staining was performed using anti-Nrf2 antibody (red) and anti-Keap1 antibody (green). The nuclei were counterstained with Hoechst (blue). The overlay images showed the nuclear location of Nrf2 after treatment with Ag5-AQC (IC50) or N-ethylmaleimide (NEM) (100 μM, positive control) for 30 minutes.

[0022] Figure 12 : Treatment with Ag5-AQC reduced the size of A549 multicellular tumor spheroids (MCTS). (a) Images of MCTS controls and those treated with Ag5-AQCs showed the differences in MCTS size and cell density in the central region. White asterisks indicate the number of days of treatment, and the arrows point to the central region with lower cell density due to Ag5-AQC treatment. (b) Growth kinetics of MCTS controls or those treated with Ag5-AQCs. Data are represented as mean ± SD. Error bars indicate standard deviation; n = 8. Mann Whitney test ((* )p < 0.05). (c) Images using hypoxia reagent and Hoechst staining showed the hypoxia levels in tumouroids with or without (control) Ag5-AQC treatment.

[0023] Figure 13: Proliferating cells are more sensitive to the action of Ag5-AQCs than non-proliferating cells. (A) Proliferating and non-proliferating A549 cells, (B) proliferating and non-proliferating U251 cells, and (C) serum-starved A549 cells were exposed to different concentrations of Ag5-AQC for 1 hour, and cell viability was determined by MTT assay. Data are shown as the mean ± SD of three independent experiments. (D) Serum-starved or serum-fed A549 cells were treated with 1.2 mg / L Ag5-AQC alone or in combination with H 2 O 2 . When Ag5-AQC and H 2 O 2 were co-administered, a synergistic effect was clearly seen.

[0024] Figure 14 : In vivo action of Ag5-AQC. (a) Ag5-AQCs caused a reduction in tumor growth in mice with orthotopic brain cancer. Experimental groups: Ag5-AQCs (0.25 mg / kg) and control (untreated). (b-d) Ag5-AQC treatment caused a reduction in tumor growth in mice with orthotopic lung cancer. (b) Tumor growth was measured in vivo by luminescence ( spectroscopy). Black arrows indicate the time of treatment administration in the study. (c) Quantification of luciferase activity in excised lungs and mediastinal lymph nodes. (d) Immunohistochemical staining of lung tumors (arrows indicate tumor nodules). Experimental groups: CDDP (4 mg / kg), Ag5-AQCs (0.25 mg / kg), and control (untreated). (e) Body weight throughout the experiment. Experimental groups: CDDP (4 mg / kg), Ag5-AQCs (0.25 mg / kg), and control (untreated). Data are presented as the mean ± SD. Error bars represent the standard deviation; n = 5. Mann Whitney test ((*p < 0.05)).

[0025] Figure 15 : Ag5-AQC treatment led to a decrease in cell viability of B-CLL cells from patients. (a) Concentration-dependent decrease in viability of B-CLL primary cells after Ag5-AQC treatment. Cells were exposed to different concentrations of Ag5-AQC for 30 minutes, and cell viability was evaluated by MTT assay after 24 hours. (b) Ag5-AQC treatment increased the percentage of DHE-positive cells 2.5-fold compared to the control. B-CLL cells were treated with Ag5-AQC for 1 hour, and DHE-positive cells were quantified by flow cytometry 4 hours later. (c) TEM images of B-CLL cells treated with Ag5-AQC showed obvious signs of apoptosis, such as chromatin margination (black arrows) and mitochondrial disruption (black arrows).

[0026] Figure 16: Ag5-AQC treatment caused a reduction in tumor volume in a multiple myeloma xenograft model. Tumor volumes were measured over a 26-day period between three different treatment groups: control (intravenously administered saline solution, n = 4), Ag5-AQCs (intravenously administered at 0.0125 mg / kg, n = 4), and bortezomib (intraperitoneally administered at 0.25 mg / kg, n = 4).

[0027] Figure 17 : W3T3 cells carrying the doxycycline-inducible RasV12 allele were exposed to doxycycline (white bars) or vehicle (control - black bars) for 24 hours and then treated with different concentrations of Ag5-AQCs. (a) RasV12 expression was evaluated by Western blot when doxycycline was added, and (b) cell viability was determined by MTT assay 24 hours later. Data are shown as the mean ± SD of at least three independent experiments.

[0028] Figure 18 : Ag5-AQC treatment increased the radiosensitivity of A549 cells. Results of radiation in A549 cells treated with Ag5-AQCs as shown in A) colony-forming assay and B) DNA damage assay using anti-pH2AX staining.

[0029] Figure 19 : Ag5-AQC treatment increased the radiosensitivity of U251 cells. Results of radiation in U251 cells treated with Ag5-AQCs as shown in A) colony-forming assay and B) DNA damage assay using anti-pH2AX staining. Detailed Description of the Invention Detailed Description of the Invention

[0031] Definition

[0032] All technical and scientific terms used throughout the specification have the same meanings as those commonly understood by one of ordinary skill in the art.

[0033] Throughout the specification, the term "about" when used in reference to any quantity is considered to include that quantity.

[0034] Throughout the specification, unless the context requires otherwise, the word "comprising" will be understood to mean including the said whole, step, whole collection or collection of steps, but not excluding any other whole, step, whole collection or collection of steps.

[0035] Throughout the specification, unless the context requires otherwise, the term "consisting essentially of" will be understood to mean including the recited integer, step, group of integers or group of steps, excluding any other integer, step, group of integers or group of steps that materially affects the basic characteristics of the recited integer, step, group of integers or group of steps.

[0036] Throughout the specification, unless the context requires otherwise, the term "consisting of" will be understood to mean including the recited integer, step, group of integers or group of steps, excluding any other integer, step, group of integers or group of steps.

[0037] As used herein, the term "atomic quantum cluster" or "AQCs" refers to a group / cluster of 2 to 500 zero-valent transition metal atoms, such as 2 to 200, 2 to 100, 2 to 50 or 2 to 25 transition metal atoms, having a size less than 2 nm, such as less than 1 nm. AQCs can contain zero-valent transition metal atoms of the same (mononuclear cluster) or different (heteronuclear cluster) transition metals. It should be understood that the term does not include metal ions.

[0038] The term "transition metal" will be understood to refer to the elements of the periodic table known as transition metals, but it does not refer to the electrical behavior of said elements. As reported in EP1914196, the confinement of electrons in AQCs causes quantum separation of energy levels, resulting in important changes in the properties of these materials. Thus, the metal atoms in the AQCs described herein can have behavior similar to that of semiconductors or even similar to that of insulators.

[0039] The term "substantially free of" can be used to refer to a composition that is substantially or completely free of the entity specifically recited thereafter, or at least does not contain an amount of the entity such that the entity affects the efficacy, storability, availability for necessary safety concerns, and / or stability of the composition.

[0040] The term "treatment" can include prevention, meaning improving symptoms, alleviating symptoms, temporarily or permanently eliminating the cause of symptoms or preventing or slowing the onset of symptoms of the specified disorder or disease. The compounds of the present invention can be used to treat humans and non-human animals.

[0041] The terms "effective amount", "therapeutically effective amount" or "effective dose" refer to an amount sufficient to cause the desired pharmacological or therapeutic effect, thereby resulting in effective prevention or treatment of a disease. Prevention of a disease is manifested by a medically significant delay in the onset of symptoms of the disease. Treatment of a disease is manifested by a reduction in symptoms associated with the disease or an improvement in the recurrence of symptoms of the disease.

[0042] Composition

[0043] The present inventors hereby provide the following evidence: Atomic quantum clusters (AQCs) composed of five zero-valent transition metal atoms affect both the glutathione and thioredoxin systems, thereby affecting cell viability. The fundamental feature of the action of AQCs with five atoms on biological systems is their specificity for two substrates - proteins and electron acceptors. Theoretical and experimental evidence is provided for the interaction of Ag5-AQC with cysteine, glutathione, and thioredoxin. And, importantly, the present inventors also provide evidence that the biological activity of Ag5-AQCs is dependent on the presence of an electron acceptor, as demonstrated by the fact that the activity of Ag5-AQC is greater in the presence of hydroxyl radical (HO·) >H 2 O 2 >O 2 (e.g. Figure 5 and 6 ). Without being limited by theory, the evidence provided herein indicates that AQCs with five atoms increase the action of ROS by acting as a catalytic bridge between ROS and sulfur atoms in proteins to increase the level of thiol oxidation. This mechanism of action is different from other chemotherapeutic agents currently known in the art. The mechanism of action of AQCs with five atoms has been demonstrated in in vitro 2D and 3D cell cultures, animal models, and primary cultures of tumor cells obtained from patients.

[0044] Accordingly, in a first aspect of the present invention, there is provided a composition for treating a cell proliferative disorder, which comprises an atomic quantum cluster (AQC) composed of five zero-valent transition metal atoms.

[0045] The potential therapeutic uses of compositions comprising AQCs composed of five zero-valent transition metal atoms will be described herein. It has been surprisingly found that the composition itself has a cytotoxic effect on eukaryotic cells without the need for the presence of additional anti-tumor agents. The theoretical and experimental evidence provided herein demonstrates that AQCs composed of five atoms selectively interact with cysteine residues present in proteins, leading to sulfur oxidation in the presence of reactive oxygen species (ROS). This mechanism is unique to clusters of this size. Thus, for the first time, the present application provides a motivation to use AQCs with five zero-valent transition metal atoms as a single therapy for treating cell proliferative diseases such as cancer.

[0046] The composition may consist essentially of atomic quantum clusters (AQCs) for treating a cell proliferative disorder, wherein the composition comprises AQCs composed of five zero-valent transition metal atoms. In one embodiment, the AQCs are the sole active ingredient of the composition, i.e., there are no other active ingredients in the composition.

[0047] In one embodiment, the composition does not contain an anti-tumor drug. In a further embodiment, the composition does not contain the anti-tumor drugs as described in WO2012 / 059572, such as alkylating agents (e.g., nitrogen mustard analogs, nitrosoureas, alkyl sulfonates, platinum-containing compounds, ethylenimines, and imidazotetrazines), cytotoxic antibiotics (e.g., anthracyclines, actinomycins), plant alkaloids and other natural products (e.g., camptothecin derivatives, epipodophyllotoxins, taxanes, and vinca alkaloids), antimetabolites (e.g., cytidine analogs, folic acid analogs, purine analogs, pyrimidine analogs, urea derivatives), and drugs for targeted therapy (e.g., kinase inhibitors and monoclonal antibodies).

[0048] In one embodiment, the composition is not used in combination with an anti-tumor drug. In one embodiment, the composition is not used in combination with the anti-tumor drugs as described in WO2012 / 059572, which are, for example, alkylating agents (e.g., nitrogen mustard analogs, nitrosoureas, alkyl sulfonates, platinum-containing compounds, ethylenimines, and imidazotetrazines), cytotoxic antibiotics (e.g., anthracyclines, actinomycins), plant alkaloids and other natural products (e.g., camptothecin derivatives, epipodophyllotoxins, taxanes, and vinca alkaloids), antimetabolites (e.g., cytidine analogs, folic acid analogs, purine analogs, pyrimidine analogs, urea derivatives), and drugs for targeted therapy (e.g., kinase inhibitors and monoclonal antibodies). It should be understood that the term "combination" as used herein refers to the effect of placing the composition (comprising AQCs) and the anti-tumor drug together. Thus, the term does not exclude the use of the anti-tumor drug at another time point during the course of cancer treatment, if such use is not for the purpose of using the anti-tumor drug in combination with the claimed composition.

[0049] In one embodiment, the composition is used as a single chemotherapy. The term "single chemotherapy" refers to the treatment of a cell proliferative disease such as cancer by using a single chemotherapeutic agent. As discussed herein, the composition of the present invention has its own chemotherapeutic effect and does not require combination with other drugs, and thus can be used as a single therapy, particularly a single chemotherapy, in cancer treatment.

[0050] The term "cell proliferative disorder" refers to a disorder that causes new abnormal growth of cells or growth of abnormal cells without physiological control. This can lead to an unstructured mass, i.e., a tumor. In one embodiment, the cell proliferative disorder is a tumor and / or cancer. The composition of the present invention can be used to treat cell proliferative diseases, including but not limited to primary tumors, metastases, and pre-cancerous conditions (pre-cancerous stages).

[0051] Cancers can include, but are not limited to: cancer of the spleen, colorectal and / or colon cancer, colon cancer, ovarian tumors, ovarian cancer, endometrial cancer, breast cancer, uterine cancer, lung cancer, gastric cancer, esophageal cancer, liver cancer, pancreatic cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, bone cancer, thyroid cancer, skin cancer such as melanoma, sarcoma, Kaposi's sarcoma, brain cancer such as glioma, medulloblastoma or neuroblastoma, blood cancers such as lymphoma and leukemia, fibroids and head and neck cancers. In one embodiment, the cancer is selected from lung cancer, breast cancer, colon cancer or brain cancer (particularly glioblastoma). In a further embodiment, the cancer is brain cancer, particularly brain cancer selected from gliomas (such as glioblastoma multiforme, oligodendroglioma, ependymoma, brainstem glioma), craniopharyngioma, hemangioblastoma, malignant meningioma, pineal region tumors and vestibular schwannoma. In still a further embodiment, the brain cancer is glioma, particularly glioblastoma.

[0052] The present invention can be particularly used for treating cancers / tumors having RAS mutations, such as KRAS, NRAS or HRAS mutations, particularly KRAS mutations. It has been demonstrated that such mutations cause oxidative stress in tumor cells, which leads to high levels of ROS, see for example Shaw et al., (2011) PNAS 108(21):8773 - 8778. As described herein, AQCs composed of 5 atoms are effective in cells containing high levels of ROS. Thus, in one embodiment, the cell proliferative disorder (such as cancer and / or tumor) comprises an RAS mutation. In a further embodiment, the RAS mutation is selected from KRAS, NRAS or HRAS mutations, particularly KRAS mutation. It should be understood that such cancers / tumors can also be referred to as RAS mutant cancers, such as KRAS, HRAS or NRAS mutant cancers or tumors. In still a further embodiment, the RAS mutation is an activating mutation, i.e., the mutation causes an increase in the activity or constitutive activity of the RAS protein. It should be noted that according to the mechanism of action of the AQCs of the present invention, the composition can be used to treat RAS mutant cancer cells, regardless of the mutation. This is contrary to current therapies, which are specific for particular mutations in the RAS gene (particularly the KRAS gene).

[0053] The RAS family of proteins are GTPases that hydrolyze GTP to GDP, thereby allowing activation of multiple downstream signaling pathways. For example, it has been demonstrated that KRAS is involved in the mitogen-activated kinase pathway. Common mutations in KRAS reduce its inherent GTPase function, preventing GTP hydrolysis to GDP, thereby locking KRAS in its active state. This results in constitutive activation of downstream signaling pathways that can drive tumorigenesis.

[0054] Multiple RAS mutations are known in the art, and KRAS mutations are the most common oncogenic mutations in human cancers. If one or more cells in a cancer contain a RAS mutation, then the cancer contains a RAS mutation. Individuals having a RAS mutation can be identified by methods known in the art, such as PCR, nucleic acid sequencing, allele-specific PCR methods, single-strand conformation polymorphism analysis, melting curve analysis, probe hybridization, pyrosequencing (i.e., nucleotide extension sequencing), genotyping, and other sequencing methods (see, e.g., Anderson (2011) Expert Rev Mol Diagn. 11(6):635-642 and Ogino et al., (2005) J. Mol. Diagn. 7:413-421). As shown herein, AQCs containing 5 atoms have a toxic effect on the A549 cell line, which has been shown to contain a KRAS mutation (e.g., KRAS G12S, where the glycine residue at position 12 is mutated). Moreover, cells containing an HRAS mutation (HRasV12, where the valine residue at position 12 is mutated) are more sensitive to the toxic effect of AQCs containing 5 atoms compared to control cells.

[0055] It is estimated that 30% of all human cancers carry a RAS mutation. For example, it is believed that 88% of pancreatic ductal adenocarcinomas, 52% of colorectal cancers, 43% of multiple myelomas, 32% of lung adenocarcinomas, 28% of melanomas, 25% of endometrial cancers, 13% of thyroid cancers, 12% of gastric cancers, 11% of acute myeloid leukemias, 11% of bladder cancers, 6% of head and neck squamous cell carcinomas, and 2% of breast cancers carry a RAS mutation (data compiled by the Cancer Cell Line Encyclopedia (CCLE); the International Cancer Genome Consortium (ICGC); and The Cancer Genome Atlas Data Portal (TCGA)). Thus, in one embodiment, the cell proliferative disorder (particularly a cell proliferative disorder having a RAS mutation) is selected from pancreatic cancer, colorectal cancer, blood cancer, lung cancer, skin cancer, endometrial cancer, thyroid cancer, gastric cancer, bladder cancer, head and neck cancer, or breast cancer. In a further embodiment, the cell proliferative disorder (particularly a cell proliferative disorder having a RAS mutation) is selected from pancreatic cancer, colorectal cancer, blood cancer, lung cancer, skin cancer, endometrial cancer, thyroid cancer, gastric cancer, bladder cancer, or head and neck cancer.

[0056] In one embodiment, the cell proliferative disorder is pancreatic cancer, such as pancreatic ductal adenocarcinoma, particularly RAS mutant pancreatic cancer, such as RAS mutant pancreatic ductal adenocarcinoma. In an alternative embodiment, the cell proliferative disorder is colorectal cancer, particularly RAS mutant colorectal cancer. In an alternative embodiment, the cell proliferative disorder is a blood cancer, such as multiple myeloma or acute myeloid leukemia, particularly RAS mutant blood cancer, such as RAS mutant multiple myeloma or RAS mutant acute myeloid leukemia. In an alternative embodiment, the cell proliferative disorder is lung cancer, such as non-small cell lung cancer, such as lung adenocarcinoma, particularly RAS mutant lung cancer, such as RAS mutant non-small cell lung cancer, such as RAS mutant lung adenocarcinoma. In an alternative embodiment, the cell proliferative disorder is skin cancer, such as melanoma, particularly RAS mutant skin cancer, such as RAS mutant melanoma. In an alternative embodiment, the cell proliferative disorder is endometrial cancer, particularly RAS mutant endometrial cancer. In an alternative embodiment, the cell proliferative disorder is thyroid cancer, particularly RAS mutant thyroid cancer. In an alternative embodiment, the cell proliferative disorder is gastric cancer, particularly RAS mutant gastric cancer. In an alternative embodiment, the cell proliferative disorder is bladder cancer, particularly RAS mutant bladder cancer. In an alternative embodiment, the cell proliferative disorder is head and neck cancer, such as head and neck squamous cell carcinoma, particularly RAS mutant head and neck cancer, such as RAS mutant head and neck squamous cell carcinoma.

[0057] The present invention can be particularly useful for treating cancers with low drug accessibility, such as large tumors with low levels of vascularization or brain tumors separated from the circulatory system by the blood-brain barrier. This is attributed to the neutral charge and small size of the therapeutic AQCs consisting of only 5 atoms, allowing them to reach regions of tumors or cancers that are not easily accessible to conventional anti-tumor drugs. The evidence provided herein shows that AQCs consisting of 5 atoms are able to penetrate into the central hypoxic region of multicellular tumor spheroids.

[0058] Preventing and treating cancer metastasis is a key part of cancer treatment for preventing secondary cancers and recurrence. It has surprisingly been found that the compositions of the present invention have an additional beneficial effect of treating cancer metastasis as well as primary tumors ( Figure 14 ). Thus, according to one aspect of the present invention, there is provided a composition as described herein (particularly a composition comprising atomic quantum clusters (AQCs) consisting of 5 zero-valent transition metal atoms) for preventing and / or treating metastasis, such as lymph node metastasis, particularly for treating and / or preventing lung cancer metastasis. According to another aspect of the present invention, there is provided a composition as described herein for preventing and / or treating lymph node metastasis of cancer.

[0059] In a further embodiment, the lymph node is a mediastinal node. The mediastinal nodes are a group of lymph nodes located in the thoracic cavity of the body.

[0060] Combination therapy

[0061] The compositions described herein can be used in combination with AQCs composed of three zero-valent transition metal atoms. It has been found that the size of AQCs composed of three zero-valent transition metal atoms allows them to insert into DNA and cause chromatin decompression. Thus, this can be used to increase the radiosensitivity of treated cells and improve the effectiveness of radiotherapy.

[0062] In one embodiment, the composition (i.e., containing AQCs composed of five zero-valent transition metal atoms) and AQCs composed of three zero-valent transition metal atoms are administered simultaneously. In this embodiment, the two drugs are administered simultaneously or substantially simultaneously. They can also be administered by the same route and optionally in the same composition. Alternatively, they can be administered by different routes (i.e., separately), but simultaneously or substantially simultaneously.

[0063] In an alternative embodiment, the composition and AQCs composed of three zero-valent transition metal atoms are administered sequentially. In this embodiment, the two drugs are administered at different times such that one of the drugs is administered before the second drug. For example, the composition can be administered before or after AQCs composed of three zero-valent transition metal atoms. They can be administered by the same or different routes.

[0064] According to another aspect of the present invention, there is provided a composition for treating a cell proliferative disease, which comprises AQCs composed of three and five zero-valent transition metal atoms in combination with radiotherapy. In one embodiment, the composition consists of AQCs as follows: the AQCs are composed of two to five zero-valent transition metal atoms. In a further embodiment, the composition consists essentially of AQCs composed of three and five zero-valent transition metal atoms.

[0065] The inventors have surprisingly found that AQCs composed of five atoms have a catalytic effect on thiol oxidation, leading to cell death. Thus, these AQCs can be used as a cancer therapy by themselves, and thus in one embodiment, the compositions described herein do not include additional anti-tumor drugs.

[0066] In one embodiment, the compositions of the present invention may include additional therapeutic agents or may be used in combination with additional therapeutic agents. Such therapeutic agents may be active agents used in combination with cancer therapies, such as therapeutic agents used as palliative treatment to ameliorate undesirable side effects. Thus, in one embodiment, the additional therapeutic agent is a therapeutic agent used as palliative treatment. In a further embodiment, the palliative treatment is selected from: antiemetics, drugs intended to relieve pain such as opioids, drugs for reducing elevated blood uric acid levels such as allopurinol or rasburicase, antidepressants, sedatives, anticonvulsants, laxatives, antidiarrheals, and / or antacids.

[0067] In one embodiment, the additional therapeutic agent is not an anti-tumor agent. In an alternative embodiment, the additional therapeutic agent is an anti-tumor agent. In one embodiment, anti-tumor agents are selected from: alkylating agents (e.g., nitrogen mustard analogs, nitrosoureas, alkyl sulfonates, platinum-containing compounds, ethyleneimines, and imidazotetrazines), cytotoxic antibiotics (e.g., anthracyclines, actinomycins), plant alkaloids and other natural products (e.g., camptothecin derivatives, epipodophyllotoxins, taxanes, and vinca alkaloids), antimetabolites (e.g., cytidine analogs, folic acid analogs, purine analogs, pyrimidine analogs, urea derivatives), and drugs for targeted therapies (e.g., kinase inhibitors and monoclonal antibodies).

[0068] In one embodiment, the composition and the additional therapeutic agent are administered simultaneously. In this embodiment, the two drugs are administered simultaneously or substantially simultaneously. They may also be administered by the same route and optionally in the same composition. Alternatively, they may be administered by different routes (i.e., separately), but simultaneously or substantially simultaneously.

[0069] In an alternative embodiment, the composition and the additional therapeutic agent are administered sequentially. In this embodiment, the two drugs are administered at different times such that one of the drugs is administered before the second drug. They may be administered by the same or different routes.

[0070] In one embodiment, the composition is administered before the additional therapeutic agent. In an alternative embodiment, the composition is administered after the additional therapeutic agent.

[0071] Radiotherapy

[0072] Radiation therapy (also known as radiotherapy) uses high doses of radiation to damage cellular DNA and thus kill cancer cells and shrink tumors. This therapy may be in the form of external beam or as brachytherapy. The choice of radiation therapy may depend on the type of cancer, tumor size, tumor location, and other factors such as the patient's age, general health and medical history, and other types of cancer treatment being used.

[0073] Radiation therapy is administered worldwide to more than 50% of all cancers and is particularly important in developing and middle-income countries. However, the effectiveness of radiation therapy is limited by various factors, including damage to healthy surrounding tissues, proximity of adjacent organs, and tumors that develop radioresistance. Thus, there is a significant unmet need for therapeutic agents that improve the efficacy of radiation therapy.

[0074] Application of radiation therapy to cancer cells results in increased ROS production. As the evidence provided herein shows, the action of AQCs composed of 5 atoms is enhanced in the presence of ROS. Thus, the compositions of the present invention are particularly suitable for use as therapeutic agents that enhance the effectiveness of radiation therapy.

[0075] According to one aspect of the present invention, there is provided a composition as described herein for use in combination with radiation therapy for treating cell proliferative disorders such as cancer.

[0076] Radiation therapy (also known as radiotherapy) uses high doses of radiation to damage cellular DNA and thus kill cancer cells and shrink tumors. This therapy can be in the form of external beam or as brachytherapy. The choice of radiation therapy can depend on the cancer type, tumor size, tumor location, and other factors such as the patient's age, general health and medical history, and other types of cancer treatment being used.

[0077] According to one aspect of the present invention, there is provided the use of a composition as described herein as a radiosensitizer for radiation therapy. According to another aspect of the present invention, there is provided the use of a composition as described herein as a radiosensitizer for radiation therapy for proliferative cells. It should be understood that the term "radiosensitizer for radiation therapy", also known as "radiosensitizer", refers to a drug used to enhance / increase the cytotoxic effect of radiation therapy. A cancer or tumor affected by radiation therapy is called "radiosensitive".

[0078] According to another aspect, the present invention provides a composition comprising atomic quantum clusters (AQCs) composed of 5 zero-valent transition metal atoms for use as a radioprotectant for non-proliferative cells in radiation therapy.

[0079] Compositions comprising atomic quantum clusters (AQCs) composed of 5 zero-valent transition metal atoms can be used to protect non-proliferating (e.g., non-dividing) cells from radiation therapy. It should be understood that the term "radioprotectant for radiation therapy", also known as "radioprotectant", refers to a drug used to reduce / decrease the cytotoxic effect of radiation therapy.

[0080] Thus, the compositions of the present invention are particularly advantageous when used in combination with radiation therapy because they have the dual effect of enhancing the action of radiation therapy on proliferative cells (i.e., cancer cells) while also protecting non-proliferative cells (i.e., non-diseased cells) from harmful radiation.

[0081] The term "proliferating" is understood by those skilled in the art. As used herein, "proliferating cells" refers to cells that undergo cell proliferation such as cell growth and division. In particular, the present invention is used to target cancer cells having rapid, abnormal, and / or uncontrolled cell proliferation. In one embodiment, the proliferating cells are cancer cells, precancerous cells, or other abnormal rapidly dividing cells in an individual. Moreover, as used herein, "non-proliferating cells" refers to cells that do not undergo cell proliferation. These cells may also be described as "quiescent", "arrested", "resting", "non-dividing", "non-cycling", or "G 0 0 cells". In one embodiment, the non-proliferating cells are non-cancerous cells.

[0082] Radiation therapy can be in the form of external beam or as brachytherapy.

[0083] In one embodiment, radiation therapy includes external beam irradiation. External beam radiation therapy uses a radiation source outside the patient, typically a radioactive isotope such as cobalt-60 (60Co), cesium-137 (137Cs), or a high-energy X-ray source such as a linear accelerator (LINAC). The external source produces a collimated beam directed into the patient's body to reach the tumor site. By projecting the external radiation beam into the patient's body at various "gantry" angles such that the beam is focused on the tumor site, the adverse effects of healthy tissue irradiation can be reduced while maintaining a given dose of radiation in the tumor tissue.

[0084] Examples of external radiation therapy treatments include, but are not limited to, conformal radiation therapy, intensity-modulated radiation therapy (IMRT), image-guided radiation therapy (IGRT), 4-dimensional radiation therapy (4D-RT), stereotactic radiation therapy and radiosurgery, proton therapy, electron beam radiation therapy, and adaptive radiation therapy.

[0085] In an alternative embodiment, radiation therapy includes brachytherapy. In this embodiment, a radiopharmaceutical substance is administered to the patient and placed in the area to be treated. In one embodiment, the radiopharmaceutical substance includes a radioactive isotope that emits radiation. Radioactive isotopes are well known to those skilled in the art and may include metallic or non-metallic radioactive isotopes.

[0086] Suitable metallic radioactive isotopes include, but are not limited to: actinium-225, antimony-124, antimony-125, arsenic-74, barium-103, barium-140, beryllium-7, bismuth-206, bismuth-207, bismuth-212, bismuth-213, cadmium-109, cadmium-115m, calcium-45, cerium-139, cerium-141, cerium-144, cesium-137, chromium-51, cobalt-55, cobalt-56, cobalt-57, cobalt-58, cobalt-60, cobalt-64, copper-60, copper-62, copper-64, copper-67, erbium-169, europium-152, gallium-64, gallium-67, gallium-68, gadolinium-153, gadolinium-157, gold-195, gold-199, hafnium-175, hafnium-175-181, holmium-166, indium-110, indium-111, iridium-192, iron-55, iron-59, krypton-85, lead-203, lead-210, lutetium-177, manganese-54, mercury-197, mercury-203, molybdenum-99, neodymium-147, neptunium-237, nickel-63, niobium-95, osmium-185+191, palladium-103, palladium-109, platinum-195m, praseodymium-143, promethium-147, promethium-149, protactinium-233, radium-226, rhenium-186, rhenium-188, rubidium-86, ruthenium-97, ruthenium-103, ruthenium-105, ruthenium-106, samarium-153, scandium-44, scandium-46, scandium-47, selenium-75, silver-10m, silver-111, sodium-22, strontium-85, strontium-89, strontium-90, sulfur-35, tantalum-182, technetium-99m, tellurium-125, tellurium-132, thallium-204, thorium-228, thorium-232, thallium-170, tin-113, tin-114, tin-117m, titanium-44, tungsten-185, vanadium-48, vanadium-49, ytterbium-169, yttrium-86, yttrium-88, yttrium-90, yttrium-91, zinc-65, zirconium-89, and zirconium-95.

[0087] Suitable non-metallic radioactive isotopes include, but are not limited to: iodine-131, iodine-125, iodine-123, phosphorus-32, astatine-211, fluorine-18, carbon-11, oxygen-15, bromine-76, and nitrogen-13.

[0088] The type of radiation suitable for the present invention can vary. In one embodiment, radiotherapy includes electromagnetic radiation or particle radiation. Electromagnetic radiation includes, but is not limited to, X-rays and gamma rays. Particle radiation includes, but is not limited to, electron beams (beta particles), alpha particles, proton beams, neutron beams, and negative pions.

[0089] In one embodiment, radiotherapy includes brachytherapy. In brachytherapy, the radiation source is placed directly at the site of the cancer or tumor. This has the advantage that the irradiation affects only a very local area, thus minimizing the radiation exposure of healthy tissue. Moreover, this allows the tumor to be treated with a very high dose of local radiation while reducing the likelihood of unnecessary damage to the surrounding healthy tissue.

[0090] In one embodiment, brachytherapy includes intracavitary therapy or interstitial therapy. Intracavitary therapy involves placing a container holding a radiation source into a body cavity where a tumor is present or near where a tumor is present. Interstitial therapy involves placing a container holding a radiation source directly into a tumor or body tissue. These radiation sources can remain in the patient's body for a long time. Most commonly, the radiation sources are removed from the patient's body after a few days. The container can include needles, seeds, wires, or catheters.

[0091] In one embodiment, radiotherapy includes systemic radioisotope therapy. In systemic radioisotope therapy, a radiopharmaceutical substance containing a radioisotope is delivered by infusion or ingestion. The administered radioisotope can be targeted due to the chemical properties of the isotope, for example, radioactive iodine is preferentially taken up by the thyroid gland. Targeting can also be achieved by conjugating the radioisotope to a targeting moiety, such as a molecule or antibody that binds to the target tissue. In one embodiment, the radiopharmaceutical substance includes a radioactive conjugate. In a further embodiment, the radioactive conjugate is a radiolabeled antibody.

[0092] In one embodiment, the radiopharmaceutical substance is administered orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, buccally, intranasally, via inhalation, vaginally, intravitreally, topically, subcutaneously, intralipally, intraarticularly, or intrathecally. In one embodiment, the radiopharmaceutical substance is a sustained release formulation.

[0093] The choice of radiotherapy can depend on the type of cancer, tumor size, tumor location, and other factors such as the patient's age, general health, and medical history, as well as other types of cancer treatment being used.

[0094] In one embodiment, the composition and radiotherapy are applied simultaneously. In an alternative embodiment, the composition and radiotherapy are applied sequentially, preferably where the composition is applied before radiotherapy. If the drugs are administered separately, radiotherapy can be administered while the composition is still effective, i.e., the composition and radiotherapy are administered within a time frame in which they will exert a synergistic or at least a combined effect when administered to the patient. In one embodiment, the composition is administered no more than 6 hours before radiotherapy, such as between 1 and 6 hours before radiotherapy. In a further embodiment, the composition is administered approximately 6 hours, approximately 5 hours, approximately 4 hours, approximately 3 hours, approximately 2 hours, or approximately 1 hour before radiotherapy.

[0095] In one embodiment, the therapeutic effects of the composition and radiotherapy are synergistic. In one embodiment, the composition renders cancer cells in the patient sensitive to radiotherapy.

[0096] In one embodiment, compared to the efficacy of radiotherapy alone for treating a disorder, the composition of the present invention is capable of increasing the efficacy of radiotherapy by at least two-fold, such as three-fold, four-fold, five-fold, or more.

[0097] Pharmaceutical composition

[0098] According to one aspect of the present invention, there is provided a pharmaceutical composition comprising the composition as described herein.

[0099] The composition and combination (when appropriate) can be formulated into a pharmaceutical composition optionally comprising a pharmaceutically acceptable excipient, diluent, or carrier. The carrier, diluent, and / or excipient must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not harmful to its recipient.

[0100] Examples of pharmaceutically acceptable carriers can include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like and combinations thereof. Suitable pharmaceutical carriers, excipients, or diluents are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. The pharmaceutically acceptable carrier can further comprise small amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, or buffering agents, which increase the shelf life or effectiveness of the composition of the present invention. The pharmaceutical composition can also include anti-adhesives, binders, coatings, disintegrants, flavoring agents, coloring agents, lubricants, adsorbents, preservatives, sweetening agents, lyophilized excipients (including lyoprotectants), or compression aids.

[0101] The pharmaceutical composition of the present invention can be administered in a variety of pharmaceutical dosage forms, such as solids (e.g., tablets, pills, capsules, granules, etc.) or liquids (e.g., solutions, suspensions, syrups, ointments, creams, gels, or emulsions).

[0102] The pharmaceutical composition of the present invention may comprise a therapeutically effective amount. The therapeutically effective amount that can be administered to an individual (i.e., the amount that produces an effect that helps to alleviate or cure the condition to be treated) will depend on a variety of factors, such as the individual's disease state, age, sex, and weight, as well as the ability of the pharmaceutical composition to elicit the desired response in the individual. The therapeutically effective amount is also the amount in which the therapeutic beneficial effects exceed any toxic or adverse effects of the pharmaceutical composition of the present invention.

[0103] In one embodiment, the AQCs are present in an aqueous solution. In a further embodiment, the aqueous solution contains dissolved oxygen, for example, the concentration of AQCs present in the mixture (especially the concentration of AQCs containing 5 zero-valent transition metal atoms) is at least 2-fold or at least 3-fold.

[0104] In one embodiment, the composition is administered (or formulated for administration) by any suitable delivery means, such as intravenously, intra-arterially, intracardially, intradermally, subcutaneously, transdermally, intraperitoneally, intramuscularly, orally, sublingually, buccally, rectally, or by enema.

[0105] The composition of the present invention can be administered directly to the target site (i.e., the tumor site) or systemically (i.e., into the circulatory system). The advantage of targeted administration is to concentrate the therapeutic effect of the composition on the cancer or tumor to be treated. Such administration also minimizes side effects. However, the composition of the present invention is also suitable for systemic administration because the mode of action ensures that apoptosis occurs only in cells with high levels of ROS. The ROS level is high in proliferating cells such as cancerous cells. However, in normal non-proliferating cells, the level of ROS is relatively low, so the AQCs composed of 5 atoms have less effect on normal cells, which helps to minimize adverse side effects.

[0106] In one embodiment, the composition is administered orally, intravenously, or subcutaneously. In a further embodiment, the composition is administered orally. The advantages of the compositions of the present invention are that they can be depleted relatively quickly, and thus any side effects can be minimized because the AQCs do not remain in the body for an extended period of time.

[0107] Local application is also possible (e.g., for treating melanoma). The specific form of local application consists in introducing the composition into a carrier system, especially a drug delivery system, and implanting the carrier system into the cancerous tissue, where the carrier system then specifically releases the composition at the site of the cancerous tissue. In this way, side effects, such as those that may occur during systemic administration, can be avoided, i.e., reducing the overall burden on the body.

[0108] Use

[0109] According to one aspect of the present invention, there is provided the use of a composition as described herein for the treatment of a cell proliferative disorder.

[0110] According to one aspect of the present invention, there is provided the use of a composition as described herein for the treatment and / or prevention of cancer metastasis. In one embodiment, the composition is for the treatment and / or prevention of lymph node metastasis of cancer. In a further embodiment, the composition is for the treatment and / or prevention of lung cancer metastasis.

[0111] According to one aspect of the present invention, there is provided the use of a composition comprising atomic quantum clusters (AQCs) composed of 5 zero-valent transition metal atoms as a radiosensitizer for proliferative cells. The substance can be used for the treatment of cell proliferative disorders.

[0112] According to one aspect of the present invention, there is provided the use of a composition as described herein in combination with radiotherapy for the treatment of a cell proliferative disorder.

[0113] According to one aspect of the present invention, there is provided the use of a composition as described herein in the manufacture / preparation of a radiosensitizer for proliferative cells.

[0114] According to one aspect of the present invention, there is provided the use of a composition comprising atomic quantum clusters (AQCs) composed of 5 zero-valent transition metal atoms as a radio desensitizer for non-proliferative cells.

[0115] According to one aspect of the present invention, there is provided the use of a composition as described herein in the preparation of a pharmaceutical composition for the treatment of a cell proliferative disorder.

[0116] According to one aspect of the present invention, there is provided the use of a composition as described herein in the preparation of a medicament for the treatment of a cell proliferative disorder.

[0117] Atomic quantum clusters (AQCs)

[0118] The AQCs described herein are stable, i.e., they retain the number of atoms over time and thus retain their properties so that they can be isolated and manipulated like any other chemical compound. The AQCs can be stored for months or even years without the need for external stabilizers.

[0119] In one embodiment, the metal atoms are selected from silver (Ag), gold (Au), copper (Cu), platinum (Pt), iron (Fe), chromium (Cr), palladium (Pd), nickel (Ni), rhodium (Rh), lead (Pb), iridium (Ir), ruthenium (Ru), osmium (Os), cobalt (Co), titanium (Ti), vanadium (V), or any combination thereof. In a further embodiment, the metal atoms are selected from Ag, Au, Cu, Pt, or any combination thereof. In a further embodiment, the metal atoms are selected from Ag, Cu, or Pt. In yet a further embodiment, the metal atom is Ag.

[0120] Mixtures of AQCs can be synthesized by a variety of methods known in the art, such as those described in EP1914196, which is incorporated herein by reference.

[0121] The mixture can also be synthesized using the method described in Example 1 herein. More specifically, a method for synthesizing silver AQCs is provided, which includes carrying out the method in a three-electrode electrochemical cell that includes a hydrogen electrode as the reference electrode and two silver electrodes as the counter and working electrodes, wherein the silver electrodes have a surface area greater than 5 cm 2 , such as greater than 10 cm 2 , such as about 17 cm 2 . Clusters of 5 atoms can be obtained by applying a steadily increasing current for about 5 hours (300 minutes). For example, the increasing current can include: step (i) a current of about 200 - 300 μA (such as about 250 μA), step (ii) a current of about 430 - 530 μA (such as about 480 μA), step (iii) a current of about 800 - 1200 μA (such as about 1000 μA / 1 mA), step (iv) a current of about 2000 - 2400 μA (such as about 2200 μA / 2.2 mA), and / or step (v) a current of about 3800 - 4200 μA (such as about 4000 μA / 4 mA), or any combination of steps (i) - (v). In one embodiment, each step is carried out for at least 30 minutes, such as about 1 hour. The silver electrodes can be polished before and / or during the synthesis, for example, with sandpaper and / or alumina. The method can be carried out in purified degassed water, such as degassed MilliQ water. Optionally, any excess Ag+ ions can be removed by adding NaCl and subsequent precipitation and filtration.

[0122] The term AQCs as used herein includes those in the hydrate form, i.e., they have water molecules attached to the clusters via non-covalent bonds.

[0123] As described herein, the mechanism for increasing sulfur oxidation is unique to AQCs composed of 5 metal atoms because the size of these clusters allows for interaction between sulfur atoms and ROS. Thus, without being bound by theory, it should be understood that the compositions of the present invention do not need to be completely free of AQCs composed of clusters of other sizes (e.g., clusters containing fewer and / or more than 5 metal atoms). In one embodiment, the composition comprises greater than about 50%, such as greater than about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 99% of AQCs composed of 5 zero-valent transition metal atoms. In particular, greater than about 95% of the AQCs present in the composition are composed of 5 zero-valent transition metal atoms. In one embodiment, the composition consists essentially of AQCs as follows: AQCs composed of 5 zero-valent transition metal atoms. In a further embodiment, the composition consists of AQCs as follows: AQCs composed of 5 zero-valent transition metal atoms.

[0124] In one embodiment of the present invention, the composition is substantially free of AQCs composed of more than 5 zero-valent transition metal atoms. For example, the composition can contain less than about 10 mol% (mol percentage based on the total AQC content of the composition), such as less than about 7 mol%, less than about 5 mol%, less than about 2 mol%, less than about 1 mol% or less than about 0.5 mol% of AQCs composed of more than 5 zero-valent transition metal atoms.

[0125] In one embodiment of the present invention, the composition is substantially free of AQCs composed of less than 5 zero-valent transition metal atoms. For example, the composition can contain less than about 10 mol% (mol percentage based on the total AQC content of the composition), such as less than about 7 mol%, less than about 5 mol%, less than about 2 mol%, less than about 1 mol% or less than about 0.5 mol% of AQCs composed of less than 5 zero-valent transition metal atoms. AQCs composed of less than 5 zero-valent transition metal atoms include AQCs composed of 2, 3 or 4 zero-valent transition metal atoms.

[0126] In one embodiment, the composition is substantially free of metal ions. During the synthesis of AQCs, metal ions are often by-products. These can be removed using, for example, NaCl. It should be understood that the term metal ions refers to the ions of the transition metals contained in the AQCs.

[0127] In one embodiment, the composition contains less than about 20 mol%, such as less than about 15 mol%, 10 mol%, 5 mol%, 2 mol%, 1 mol% or 0.5 mol% of metal ions (i.e., free ions of the transition metals used to synthesize the AQCs).

[0128] According to a further aspect of the present invention, there is provided a composition comprising atomic quantum clusters (AQCs) composed of 2 to 5 zero-valent transition metal atoms, which is substantially free (i.e., less than 20%, 15%, 10%, 5%, 2%, 1%) of AQCs composed of more than 5 zero-valent transition metal atoms and / or metal ions. According to a further aspect of the present invention, there is provided a composition comprising atomic quantum clusters (AQCs) composed of 5 zero-valent transition metal atoms, which is substantially free (i.e., less than 20%, 15%, 10%, 5%, 2%, 1%) of AQCs composed of more than 5 zero-valent transition metal atoms and / or less than 5 zero-valent transition metal atoms and / or metal ions.

[0129] Methods for purifying a composition to remove AQCs composed of more or less than 5 zero-valent transition metal atoms are known in the art. For example, as described by Porto et al. (2018) Adv Mater. 30(33): e1801317. Such methods can include: (i) applying a solution of a mixture comprising AQCs to a separation medium, wherein the separation medium binds or does not bind AQCs composed of more than 5 zero-valent transition metal atoms; and (ii) isolating AQCs composed of 5 zero-valent transition metal atoms.

[0130] In one embodiment, the separation medium is used in a chromatographic method. Chromatography is a method for separating a mixture by passing a mobile phase comprising the mixture through a stationary phase (e.g., comprising the separation medium described herein). The mixture is separated based on how the components of the mobile phase interact with the stationary phase. It should be understood that whether a fraction is retained or discarded will depend on the content and the presence of 5 zero-valent transition metal atoms. For example, if the separation medium retains AQCs composed of more than 5 zero-valent transition metal atoms, the eluate (which contains AQCs composed of 5 or fewer zero-valent transition metal atoms) is collected. Alternatively, if the separation medium retains AQCs composed of 5 zero-valent transition metal atoms, the eluate (which contains AQCs composed of more than and / or less than 5 zero-valent transition metal atoms) is discarded. In one embodiment, the separation medium is present in a chromatographic column. Such chromatographic columns are commercially available.

[0131] The separation medium can comprise, for example, functional groups that bind to AQCs of a specific size, such as thiol groups that bind to AQCs composed of more than 3 zero-valent transition metal atoms. Alternatively, the functional groups can comprise aromatic groups, such as cyclic or polycyclic aromatic groups. The separation medium can also comprise, for example, deoxyribonucleic acid (DNA) that is substantially double-stranded. It has been found that clusters of three metal atoms interact with DNA by intercalation, which strictly depends on the number of atoms in the cluster and is independent of the type of base pairs (AT or GC) of the double helix. Thus, DNA can be used to separate AQCs composed of 3 zero-valent transition metal atoms.

[0132] In one embodiment, the separation medium is used in a dialysis method. Dialysis is a method of separating molecules based on the diffusion rate of molecules through a semipermeable membrane. For example, a solution of a mixture containing AQCs can be applied to the separation medium and then placed in a dialysis device (such as a dialysis cassette or dialysis tubing). Such dialysis cassettes, tubing, or devices are commercially available. The dialysis membrane can be selected, and the molecular weight cut-off can be selected according to the separation requirements (such as according to the molecular weight of the DNA used in the separation medium).

[0133] It should be understood that one or more of the purification methods disclosed herein can be carried out in combination and / or repeated one or more times. Carrying out the purification method multiple times can increase the purification of the sample and allow the desired purification to be achieved.

[0134] Therapeutic method

[0135] According to one aspect of the invention, there is provided a method for preventing and / or treating a cell proliferative disorder, the method comprising administering to a patient in need thereof a therapeutically effective amount of a composition comprising atomic quantum clusters (AQCs) composed of 5 zero-valent transition metal atoms. In one embodiment, the method does not include treating the patient with additional anti-tumor drugs.

[0136] According to one aspect of the invention, there is provided a method for preventing and / or treating a cell proliferative disorder, the method comprising administering to a patient in need thereof a therapeutically effective amount of the composition described herein.

[0137] According to one aspect of the invention, there is provided a method for treating a patient suffering from a cell proliferative disorder, the method comprising administering the composition as described herein. The embodiments of the composition described above can be applied to the treatment method (such as the time and manner of administration, formulation of the composition, etc.).

[0138] According to one aspect of the invention, there is provided a method for preventing and / or treating cancer metastasis, the method comprising administering the composition as described herein. In one embodiment, the method prevents and / or treats lymph node metastasis of cancer. In a further embodiment, the method prevents and / or treats lung cancer metastasis.

[0139] In one embodiment, the treatment methods described herein further include treating a patient with radiotherapy, such as treating the patient with radiotherapy after administering the composition. As described above, the compositions of the present invention can be particularly used as radiosensitizers.

[0140] In one embodiment, the composition is administered orally, intravenously, or subcutaneously.

[0141] In one embodiment, the composition is administered simultaneously with or before radiotherapy.

[0142] In one embodiment, the method further includes administering a therapeutically effective amount of a composition comprising AQCs consisting of three zero-valent transition metal atoms. In one embodiment, the composition comprising AQCs consisting of three zero-valent transition metal atoms is administered simultaneously or sequentially with a composition comprising AQCs consisting of five zero-valent transition metal atoms.

[0143] The patient can be any individual suffering from a disorder. In one embodiment, the patient is a mammal. In a further embodiment, the mammal is selected from a human or a mouse.

[0144] In one embodiment, the therapeutic effects of the composition and radiotherapy are synergistic. In one embodiment, the composition sensitizes the patient's cancer cells to radiotherapy.

[0145] The method includes administering a therapeutically effective amount of radiation. The amount of radiation used in radiotherapy is measured in Gray (Gy) units and varies according to the type and stage of the cancer being treated. In addition, to minimize adverse side effects, the total radiation dose can be divided into multiple smaller doses called "fractions" over a period of several days. A typical fractionation scheme for adults is 1.8 to 2 Gy per day, five days a week. A typical fractionation scheme for children is 1.5 to 1.8 Gy per day, five days a week.

[0146] In one embodiment, a total of at least about 10 Gy, such as 15 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, 50 Gy, 55 Gy, 60 Gy, 65 Gy, 70 Gy, 75 Gy, 80 G, 85 Gy, 90 Gy, 95 Gy, or 100 Gy is administered to a patient in need thereof. The patient can receive radiation three, four, or five times a week. Depending on the type of cancer and the purpose of treatment, the entire treatment process can last from one to seven weeks. In one embodiment, radiotherapy occurs over a period of at least 2, 3, or 4 weeks, such as 2 - 6 weeks, such as 2 - 4 weeks or 5 - 8 weeks, particularly a period of 5 - 7 weeks. For example, a patient can receive a dose of 2 Gy / day over approximately 30 days (i.e., 4 - 5 weeks).

[0147] In one embodiment, radiation is administered at least once per day for five consecutive days per week. For example, radiation is administered in fractions of at least about 2 Gy at least once per day. In one embodiment, radiation is administered every other day, three times per week. For example, radiation is administered in 10 Gy fractions every other day, three times per week.

[0148] In one embodiment, the radiation therapy is hypofractionated. Hypofractionation is a treatment regimen that delivers a higher radiation dose in fewer treatments. In an alternative embodiment, the radiation therapy is hyperfractionated. Hyperfractionation is a treatment regimen that divides the total dose into more fractions for delivery. It should be understood that multiple other factors are considered when selecting the dose, including whether the patient is receiving chemotherapy, the patient's co-morbidities, whether the radiation therapy is administered before or after surgery, and the degree of surgical success.

[0149] According to another aspect, the present invention provides a method for preventing non-proliferative cell damage in a patient receiving radiation therapy, the method comprising administering to the patient a therapeutically effective amount of a composition comprising AQCs consisting of five zero-valent transition metal atoms prior to the radiation therapy.

[0150] According to one aspect of the present invention, there is provided a method for treating metastases, such as lymph node metastases, the method comprising administering to a patient in need thereof a combination of a therapeutically effective amount of a composition comprising AQCs consisting of five zero-valent transition metal atoms and radiation therapy.

[0151] Kit

[0152] According to one aspect of the present invention, there is provided a kit comprising: a composition as described herein, optionally mixed with a pharmaceutically acceptable adjuvant, diluent or carrier. The kit of this aspect of the present invention can be used to treat cell proliferative disorders.

[0153] In one embodiment, the kit can be used in combination with radiation therapy for treating cell proliferative disorders.

[0154] Other aspects

[0155] According to one aspect of the present invention, there is provided an apoptosis agent comprising AQCs consisting of five zero-valent transition metal atoms. The apoptosis agent can comprise a composition as described herein.

[0156] According to another aspect of the present invention, there is provided a method for inducing thiol oxidation, the method comprising administering a composition as described herein, optionally in combination with reactive oxygen species (ROS). As described herein, AQCs composed of 5 atoms provide a catalytic bridge between ROS present in cells and sulfur atoms in cysteine residues of proteins. Thus, the compositions of the present invention can be used to enhance thiol oxidation. The addition of ROS will depend on whether the target already contains ROS.

[0157] The inventors also provided evidence that AQCs composed of 5 atoms have strong bactericidal effects. Thus, according to another aspect of the present invention, there is provided a composition as described herein for treating diseases caused by bacteria.

[0158] Herein, the present invention is illustrated in the following non-limiting examples.

[0159] Abbreviations

[0160] All units used herein should be understood with their standard definitions known in the art (unless otherwise specified).

[0161] A549 human lung adenocarcinoma cell line

[0162] Ag silver

[0163] Ag5 5 silver atoms

[0164] AQC atomic quantum cluster

[0165] ATCC American Type Culture Collection

[0166] BBB blood-brain barrier

[0167] B-CLL B-chronic lymphocytic leukemia

[0168] CDDP cisplatin

[0169] Cul3 Cullin 3

[0170] Cys cysteine

[0171] DAPI 4′,6-diamidino-2-phenylindole

[0172] DHE dihydroethidium

[0173] DMEM Dulbecco's Modified Eagle Medium

[0174] DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH DTT Dithiothreitol

[0175] FBS Fetal Bovine Serum

[0176] GBM Glioblastoma Multiforme

[0177] GPx Glutathione Peroxidase

[0178] GRX1 Glutaredoxin 1

[0179] GSH Glutathione

[0180] GSSG Glutathione Disulfide

[0181] H 2 O 2 Hydrogen Peroxide

[0182] HCT116 Human Colorectal Cancer Cell Line

[0183] HEK293 Human Embryonic Kidney 293 Cell Line

[0184] HMOX1 Heme Oxygenase-1

[0185] Keap1 Kelch-like ECH-associated Protein 1

[0186] Luc Firefly Luciferase Gene

[0187] MCF7 Human Breast Cancer Cell Line

[0188] MCTS Multicellular Tumor Spheroid

[0189] MEC-1 Human B-chronic Lymphocytic Leukemia Cell Line

[0190] MM.1S Human Multiple Myeloma Cell Line

[0191] MRE Metal Response Element

[0192] MT Metallothionein

[0193] MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide

[0194] MTF-1 Metal Response Transcription Factor 1 (MTF-1

[0195] NaCl Sodium Chloride

[0196] NAD(P)H Nicotinamide Adenine Dinucleotide Phosphate

[0197] Neh Nrf2-ECH Homologous Domain

[0198] NEM N-Ethylmaleimide

[0199] NQO-1 NAD(P)H Quinone Oxidoreductase

[0200] Nrf2 Nuclear Factor (Erythroid-Derived 2)-Like 2

[0201] O 2 Oxygen

[0202] PBS Phosphate Buffered Saline

[0203] PFA Paraformaldehyde

[0204] RL Human Non-Hodgkin Lymphoma Cell Line

[0205] roGFP Redox-Sensitive Green Fluorescent Protein

[0206] ROS Reactive Oxygen Species

[0207] TEM Transmission Electron Microscopy

[0208] U87 Human Glioblastoma Multiforme Cell Line

[0209] XANES X-Ray Absorption Near-Edge Structure

[0210] Materials and Methods

[0211] Reagents and materials

[0212] Unless otherwise stated, all reagents were purchased from Sigma Aldrich, Co., Spain. Silver foils (99%) were purchased from Goodfellow Cambridge Ltd., Huntingdon, UK. Alumina nanoparticles (average particle size ≈ 50 nm) and cloth pads were purchased from Buehler, Düsseldorf, Germany.

[0213] Sandpaper (1,000 grit) was provided by Wolfcraft S.L, Madrid, Spain. All aqueous solutions were prepared using MilliQ-grade water from a Direct-Q8UV system by Millipore (Millipore Ibérica S.A., Madrid, Spain). Mica sheets (V-1 grade muscovite) were purchased from SPI Supplies, West Chester, PA, USA.

[0214] X-ray absorption near-edge structure (XANES)

[0215] SK-edge (2470 eV) XANES experiments were carried out at the de SXS beamline of the Laboratório Nacional de Luz Síncrotron (LNLS, Campinas, Brazil) equipped with an InSb(111) double-crystal monochromator with a 1-mm slit to achieve a resolution of about 0.5 eV at the S K-edge. X-ray absorption spectra were recorded in fluorescence mode, collecting the X-rays emitted from the S Kα1,2 emission lines (at 2309.5 and 2308.4 eV, respectively) for each measurement edge. The absorption experiments were carried out at room temperature in a vacuum of 10-8 mbar and at room temperature and atmospheric pressure in a special liquid sample holder designed for experiments on reactive oxygen species. According to the criteria previously reported by Vairavamurthy (1998) Spectrochim.Acta.A.Mol.Biomol.Spectrosc.54:2009-2017, the photon energy was calibrated by assigning the 2481.5 eV value to the highest maximum of Na 2 S 2 O 3 corresponding to the so-called inner sphere. The final XANES spectra were obtained by the conventional method described elsewhere, after background subtraction and normalization to the intensity at the edge. XANES quantification was performed using Athena software and subsequently analyzed using Origin Lab software. For the characterization of glutathione, a portion of the solution was deposited on a carbon disk (Ted Pella, Inc) by drop casting to have a detectable Ag or S concentration. For the thioredoxin samples, they were carefully mounted in a special liquid sample holder designed for this experiment. PBS solution was used as the solvent in all reactions and mixed with thioredoxin with the aim of reproducing the same intracellular pH and ionic strength. The hydroxyl radical solution was prepared by the Fenton reaction using H 2 O 2 and FeCl 2 .

[0216] Cell line

[0217] Cell lines used for this work included: A549 (human lung adenocarcinoma, DSMZ No:ACC 107), A549 Luc-C8 MCF7 (human breast cancer, DSMZ No: ACC 115), HCT116 (human colorectal carcinoma, ATCC No: CCL-247), HEK293 (kidney, from human embryo), U87-luc (human glioblastoma multiforme, kindly provided by Joan Seoane), U251-Luc (human glioblastoma, kindly provided by Joan Seoane), MM.1S (human multiple myeloma, ATCC No: CRL-2974), RL (human non-Hodgkin lymphoma, ATCC No: CRL-2261) and MEC-1 (human B-chronic lymphocytic leukemia, DSMZ, No: ACC 497). A549, A549-Luc, MCF7, U87-Luc and HCT116 are all derived from solid tumors and grow as monolayer adherent, while MM.1S, RL and MEC-1 are all derived from malignant hematological diseases and grow in suspension. A549, A549-Luc, U251-Luc and HEK293 cell lines are maintained in low glucose DMEM (D6046, Sigma); MCF7 and U87-Luc cell lines are maintained in high glucose DMEM (D5671, Sigma); HCT116, MM.1S and RL are maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (R-5886, Sigma), and MEC-1 cell line is maintained in DMEM / Nutrient Mixture F-12 Ham mixture 1:1. The media are supplemented with 10% fetal bovine serum and 1% (v / v) L-glutamine, penicillin and streptomycin (Gibco). The modified cell lines (A549-Luc and U87-Luc) are supplemented with puromycin (1.3 μg / ml for A549-Luc and 5 μg / ml for U87-Luc) to select for stably transfected cells. All cell lines are cultured at 37 °C in a humidified atmosphere in the presence of 5% CO 2 and 95% air.

[0218] Animal

[0219] Female athymic nude mice, 8 - 12 weeks old and weighing approximately 20 - 25 g, were used in in vivo studies and were provided by Janvier Laboratories. Before the experiment, the animals were acclimatized for at least 1 week; they were housed in ventilated polypropylene cages at an average temperature of 22 °C and exposed to 12 hours of light and 12 hours of darkness per day. All mice had free access to a standard laboratory food diet and water. The experiment was conducted in accordance with the regulations of the Rules of the Santiago de Compostela University Bioethics Committee and adhered to the Principles of Laboratory Animal Care according to Spanish national law (RD 53 / 2013).

[0220] In vitro cytotoxicity assay

[0221] Toxicity was evaluated by MTT assay. Proliferating cells: A549 (4×10 3 cells / well) and U251 (5×10 3 cells / well) were seeded in 96 - well plates. After 24 hours, the medium was discarded, and the cells were replaced with serum - free medium containing different concentrations of Ag5 - AQCs (1.2 - 0.24 mg / L) for 1 hour and then allowed to grow for another 24 hours in complete medium. Primary B - CLL cultures (4×10 4 cells / well) were seeded in serum - free medium, and different concentrations of Ag5 - AQCs (1.2 - 0.24 mg / L) were immediately added to the wells and maintained for 30 minutes. Then, complete medium was added, and the cells were allowed to grow for 24 hours. Non - proliferating cells (confluent or serum - deprived): A549 cells (4×10 3 cells / well) and U251 (5×10 3 cells / well) were seeded in 96 - well plates. Confluent cells were grown in medium - 10% FBS for 96 hours to reach confluence. For serum - deprived cells, the medium was replaced with medium - 0.05% FBS 24 hours after seeding for 72 hours. In both conditions, the non - proliferating state of the cells was confirmed by flow cytometry. Then, the cells were treated with Ag5 - AQCs (1.2 - 0.24 mg / L) in serum - free medium for 1 hour and then cultured in complete medium for another 24 hours.

[0222] Then, for all test conditions, 10 μl of MTT solution (5 mg / ml) was added to each well and incubated at 37 °C in the dark. After 4 hours, 100 μl of solubilization solution (SDS / 0.1 N HCl) was added, and the samples were incubated at 37 °C for 18 hours. Using The microplate reader measures absorbance at 595 nm. A similar protocol was performed using the MTT assay to measure cell viability of other cell types.

[0223] Radioactive measurement of GSH oxidation in living cells

[0224] The PREMO Cell Redox Sensor Grx-1-roGFP (Molecular Probes, P36242) is a genetically encoded sensor used to detect changes in the glutathione redox state in live cells. This sensor is based on the introduction of two cysteines into the β-barrel structure of the GFP protein. Under oxidizing conditions, the formation of disulfide bonds alters the fluorescence properties of the biosensor, resulting in changes in the emission intensity after excitation at two different wavelengths (400 and 488 nm). The ratio of the emission intensities is related to the change in the redox state of roGFP.

[0225] To analyze the change in the oxidized state of GSH in the presence of Ag5-AQCs, 2.5×10 4 A549 cells were seeded in a 35 mm plate dish (Mattek, P35GC-0-10-C) and transduced with the PREMO Cell Redox Sensor Grx-1-roGFP immediately after cell seeding. The volume of the sensor was calculated according to the following equation:

[0226] Volume of sensor (ml) = (Number of cells) x (MOI) / (1x10 8 )

[0227] where "Number of cells" is the number of cells seeded per dish; MOI is the number of viral particles per cell, and 1×10 8 is the number of viral particles per ml of reagent.

[0228] According to the results obtained from this equation, 60 μl of the PREMO Cell Redox Sensor was added per ml of culture medium. The samples were cultured for 48 hours to obtain optimal expression of the sensor, and the redox changes in live cells were motorized using a Leica TCS SP5X confocal microscope. Ag5-AQCs (IC50) were added to the dish, and images were taken every 10 seconds for 10 minutes. The PREMO Cell Redox Sensor was excited at 400 and 488 nm, and the emission was collected at 500 - 530 nm. The fluorescence intensity emitted by each cell was measured under both excitations, and the 400ex / 488ex ratio was calculated. The images were processed using ImageJ software.

[0229] Immunofluorescence

[0230] MTF-1: Make A549 (2.5×10 4) Cells were grown on glass coverslips in 24-well plates, treated with Ag5-AQCs (IC50) in serum-free medium for 1 hour, and then the medium was replaced with complete medium for 2 hours. HEK293 (8×10 4 ) Cells were grown on glass coverslips in 24-well plates, treated with Ag5-AQCs (IC50), DTT (0.5 mM), or a combination of both in serum-free medium for 10 or 30 minutes, and then treated in medium. Then, the cells were washed twice with PBS Ca2+ / Mg2+, fixed with methanol / acetone (1:1 dilution) at -20 °C for 10 minutes. Then, they were blocked with PBS containing 10% FBS for 1 hour, washed twice with PBS, and incubated overnight at 4 °C with a primary antibody against MTF-1 (dilution 1:200) (sc-48775, Santa Cruz Biotechnology). Thereafter, the cells were washed and incubated for 45 minutes with a secondary antibody, Alexa Fluor-594 goat anti-rabbit IgG (dilution 1:500) (A11037, Life Technologies) at 0.25 μg / ml and Hoechst (dilution 1:1000) (Molecular Probes). The coverslips with stained cells were mounted on slides with Fluoroshield Mounting Medium (F6182, Sigma). Images were taken using a Leica TCS SP8 confocal microscope and analyzed using LasX software.

[0231] Nrf2: HEK293 cells (3×10 4)Cells were seeded on glass coverslips in 24-well plates overnight and treated with Ag5-AQCs (IC50), DTT (0.5 mM), or a combination of both in serum-free medium for 10 or 30 minutes. After treatment, the cells were fixed in formalin solution (10%) for 30 minutes, washed twice with PBS Ca2+ / Mg2+, and permeabilized with 0.5% Triton X-100 for 5 - 10 minutes. Then, the cells were washed again and blocked with PBS containing 1% BSA. Subsequently, the cells were incubated with a primary antibody against Nrf2 (1:100 dilution) (sc-722, Santa Cruz Biotechnology) for 2 hours at room temperature, washed twice with PBS, and incubated with an Alexa Fluor-594 goat anti-rabbit IgG secondary antibody (1:250 dilution) (A11037, Life Technologies) and Hoechst (1:1000 dilution) (Molecular Probes) for 45 minutes. A negative control without the primary antibody was included in the analysis (data not shown). The coverslips with stained cells were mounted on slides with Fluoroshield Mounting Medium (F6182, Sigma). Images were taken using a Leica TCS SP8 confocal microscope and analyzed using LasX software.

[0232] ROS measurement

[0233] Flow cytometry measurements were performed using the superoxide indicator dihydroethidium (DHE) (Molecular Probes, D11347). Cells were seeded in 12-well plate dishes and treated with Ag5-AQCs (IC50) in serum-free medium. Cells were harvested at 30 minutes, 1, 2, and 3 hours after treatment, washed twice with cold PBS, and incubated with DHE (3.17 mM) for 20 minutes at room temperature in the dark. Stained cells were analyzed using a Guava EasyCyte flow cytometer with InCyte software.

[0234] Multicellular tumor spheroids

[0235] A549 and U251 multicellular tumor spheroids (MCTSs) were generated by the hanging drop method. A 20 μl cell suspension containing 500 cells was dispensed into 60-well microplates (Nunc). Subsequently, the plates were inverted and cultured for 5 days under standard conditions. On the 5th day, the plates were righted, and the spheroids were transferred to a 96-well plate coated with 50 μl of 1% agarose. Then, the spheroids were treated with Ag5-AQCs four times every other day, and images of the spheroids were taken daily until the end of the treatment using an Olympus IX51 microscope equipped with an Olympus DP72 camera and CellSens imaging software. The images were processed using ImageJ to measure the spheroid area and the difference in gray values as an indirect indication of cell density.

[0236] At the end of the experiment, the spheroids were stained with Image-iT Green hypoxia reagent 5 μM (Molecular Probes, I14834) and Hoechst (1 μg / μl) for 1 hour. Images of the control and treated spheroids were taken using a Leica AOBS-SP5 confocal microscope and analyzed using ImageJ software.

[0237] In vivo efficacy of Ag5-AQCs

[0238] An A549luc orthotopic lung cancer model was developed according to the protocol described by Borrajo et al. (2016) J. Control Release 238: 263 - 271. 1 × 10 6 A549Luc cells suspended in 50 μl of PBS were injected into the left lung of athymic nude mice through the intercostal space. After tumor development, luciferin was injected into the intraperitoneal cavity at a dose of 150 mg / kg body weight approximately 5 minutes before imaging. Luciferase bioluminescence was imaged using an IVIS LIVING IMAGE system (Caliper Life Sciences) under vaporized isoflurane anesthesia.

[0239] For Ag5-AQCs treatment, the mice were divided into three groups (5 animals per group): The first group (control) was not treated, the second group was treated with cisplatin (CDDP) (four single doses, 4 mg / kg), and the third group was treated with Ag5-AQCs (four single doses, 0.25 mg / kg). The drugs were administered intravenously via the tail vein on days 20, 22, 24, and 26 after tumor inoculation. The mice were sacrificed on day 37. The lungs and mediastinal lymph nodes were removed, and the luminescence per microgram of protein in vivo was quantified as described by Borrajo et al. (2016).

[0240] Histological analysis

[0241] The lungs were fixed in 10% neutral buffered formalin for 24 hours and embedded in paraffin. Sections 4 mm thick were mounted on FLEX IHC microscope slides (Dako-Agilent, Glostrup, Denmark) and heated at 60 °C for 1 hour. Immunohistochemistry was performed automatically using an AutostainerLink 48 (Dako-Agilent). After dewaxing at 97 °C and epitope retrieval in EnVision FLEX Target Retrieval Solution (high pH) for 20 minutes, the slides were cooled to 65 °C in a PT Link and then cooled in Dako Wash Buffer for 5 minutes at room temperature (RT). The immunostaining protocol included incubation at RT as follows: (1) EnVision FLEX Peroxidase-Blocking Reagent (Dako-Agilent) for 5 minutes; (2) ready-to-use FLEX primary antibody (Dako-Agilent) anti-CK7 (clone OV-TL12 / 30) antibody for 20 minutes; (3) EnVision FLEX / HRP (dextran polymer conjugated to horseradish peroxidase and affinity-isolated goat anti-mouse and anti-rabbit immunoglobulins) for 20 minutes; (4) substrate working solution (mixture) (3,3'-diaminobenzidine tetrahydrochloride chromogen solution) (Dako-Agilent) for 10 minutes; and (5) EnVision FLEX hematoxylin (Dako-Agilent) for 9 minutes. The sections were examined and photographed using an Olympus PROVIS AX70 microscope equipped with an Olympus DP70 camera.

[0242] Radiation treatment

[0243] A549 (3×10 4 cells / well) and U251 (3.5×10 4 cells / well) cells were seeded on 24-well plates and cultured for 96 hours to reach confluence. Then, the medium was replaced with medium without FBS containing different dilutions of Ag5-AQCs (1:50, 1:75, and 1:100 for A549 and 1:150, 1:175, and 1:200 for U251). After pretreatment with Ag5-AQCs for 10 minutes, the cells were irradiated with a dose of 0 - 10 Gy using a linear accelerator from the Radiophysics Laboratory of the Universidade de Santiago.

[0244] Statistical analysis

[0245] All statistical analyses were performed using GraphPad Prism version 5.0 software (GraphPad Software, Inc., La Jolla, USA). For *P < 0.05, the difference was considered significant; for *P < 0.01, it was considered highly significant.

[0246] Example 1: Synthesis method of Ag5-AQCs

[0247] The synthesis of Ag5-AQC clusters was carried out at 25 °C using a Biologic VMP3 potentiostat (Seyssinet-Parisetm France). A Methrom adiabatic three-electrode electrochemical cell with a hydrogen electrode as the reference electrode and two Ag foils (17.5 cm 2 surface area) as the counter and working electrodes was used. These electrodes were opposite to each other and separated by a distance of 3 cm. The first step was 250 μA for 1 hour, the second step was 480 μA for 1 hour, the third step was 1 mA for 1 hour, the fourth step was 2.2 mA for 1 hour, and the last two steps were 4 mA for 30 minutes each at 25 °C. Before the synthesis and 4 hours and 4.5 hours after the synthesis, the two silver electrodes were polished with sandpaper and then polished with alumina (about 50 nm), thoroughly rinsed with MilliQ water, and sonicated (2 steps of 5 minutes each, changing the water each time). After sonication and before the synthesis, an electrochemical cleaning was carried out, which included a step of 250 mA for 5 minutes in water.

[0248] Purification: The amount of unreacted ions in the solution was estimated using an Ag ion-selective electrode (Hanna). Ag ions were precipitated using NaCl in an amount 1.5 times the Ag ion concentration. The system was left standing at 25 °C overnight to complete the precipitation.

[0249] Concentration: Sixteen portions of the synthesized product were collected together (8 L in total), filtered through a 0.1 μm membrane, and concentrated to a volume of 10 mL at 35 °C (vacuum ≈ 30 mbar) in a rotary evaporator (Heidolphlaborota 20). Finally, the solution was filtered through a 0.22 μm membrane and further concentrated to 2 mL in a vial. The concentration of Ag5-AQCs at the end of the purification and concentration processes was approximately 30 mg / L as evaluated by flame atomic absorption spectroscopy (performed with a Perkin-Elmer 3110 with a silver hollow cathode lamp Lumia (current 10 mA) from Perkin-Elmer (Madrid, Spain)). Mass spectrometry analysis showed the main presence of Ag5-AQCs species (about > 50%).

[0250] The cluster samples were characterized by UV-Vis and fluorescence spectroscopy, AFM (Atomic Force Microscopy), HRTEM (High-Resolution Transmission Electron Microscopy), XANES and ESI-TOF (Electrospray Ionization Time-of-Flight) mass spectrometry, showing that the composition mainly contains clusters with N = 5 atoms (Ag5-AQCs).

[0251] Example 2: Model of interaction with Ag5

[0252] Theoretical models of the interaction of Ag5-AQCs with glutathione and thioredoxin show that the reaction is thermodynamically possible. In addition, Ag5-AQCs selectively interact with the basal domain of thioredoxin, which is called the "thioredoxin fold" and is found in both prokaryotic and eukaryotic proteins. Although the sequences are variable in many regions of the fold, thioredoxins share a common active site sequence with two reactive cysteine residues: Cys-X-Y-Cys, where X and Y are usually but not necessarily hydrophobic amino acids. Without being bound by theory, Ag5-AQCs show interaction with these two cysteine residues, as Figure 1 shown.

[0253] Example 3: Ag5-AQCs promote sulfur oxidation

[0254] Ag5-AQCs promote the sulfur oxidation in cysteine and glutathione, as seen using X-ray absorption near-edge structure (XANES) ( Figure 2 ). In addition, the reaction is dose-dependent ( Figure 3 ). It was also shown that Escherichia coli (E.Coli) thioredoxin is oxidized in the presence of Ag5-AQCs ( Figure 4 ). As expected, pure thioredoxin molecules show only a peak at 2474.3 eV, which is consistent with the S(-2) oxidation state and corresponds to their two cysteine groups. Importantly, it was noted that the reduced form of the molecule could be confirmed before catalytic treatment with Ag5-AQCs, because no signal of disulfide with a characteristic splitting of about 1.5 eV in the range of 2473 to 2475 eV was observed. After treatment with AQCs, a strong peak associated with S+6 was clearly seen.

[0255] XANES analysis also showed the role of different electron acceptors in Ag5-AQC-mediated thioredoxin oxidation ( Figure 5 ). From a biological point of view, it is very important to find that Ag5-AQCs enhance oxygen, H 2 O 2The role of silver ions and hydroxyl radicals (HO·) in sulfur oxidation leads to an irreversible oxidation state in biological systems. This links the action of Ag5-AQC to cell metabolism and tumor angiogenesis ( Figure 6 ).

[0256] Example 4: Ag5-AQC has bactericidal activity

[0257] Ag5-AQCs have antibacterial and bactericidal effects on Escherichia coli (E. Coli). The responsible mechanism is thought to be thiol oxidation. In fact, dithiothreitol (DTT), a thiol reducing agent, rescues E. coli from the action of Ag5-AQC. The reverse is also true, i.e., Ag5-AQCs rescue E. coli from the action of DTT, as expected from redox agents with opposite actions. Clusters made of copper and platinum also have bactericidal activity.

[0258] In the absence of DTT (0 mM), low concentrations (1.2 mg / L) of Ag5-AQCs kill bacteria. When DTT is increased to 0.1 mM, bacterial viability is partially restored. 10 mM of DTT is toxic to bacteria, however co-administration of Ag5-AQCs reverses the action of DTT ( Figure 7 ).

[0259] Example 5: The effect of Ag5-AQCs on human cell lines

[0260] A panel of nine cell lines was used: A549 (human lung adenocarcinoma, DSMZ No.: ACC 107), A549 Luc-C8 MCF7 (human breast cancer, DSMZ No.: ACC 115), HCT116 (human colorectal cancer, ATCC No.: CCL-247), HEK293: (kidney, from human embryo), U87-luc (human glioblastoma multiforme, kindly provided by Joan Seoane), MM.1S (human multiple myeloma, ATCC No.: CRL-2974), RL (human non-Hodgkin lymphoma, ATCC No.: CRL-2261) and MEC-1 (human B-chronic lymphocytic leukemia, DSMZ No.: ACC 497).

[0261] All cell lines are sensitive to Ag5-AQCs. In Figure 8 , dose-response (0.24 - 1.2 mg / L) plots of various cell lines are presented. Importantly, when DTT is co-administered with Ag5-AQCs, the toxic effect is reduced, indicating that the action of Ag5-AQC is mediated by thiol oxidation.

[0262] Using the A549 cell line, it was also found that clusters made of copper show cytotoxic effects, seeFigure 9 .

[0263] The development of redox-sensitive GFP molecules allows the monitoring of the redox state within living cells by fluorescence microscopy. The roGFP-Grx1 chimera is a genetically encoded sensor used to measure changes in thiol oxidation of two cysteines introduced into the β-barrel structure of the GFP protein. The formation of a disulfide bond between the cysteines results in protonation of GFP and an increase in the 400 nm excitation spectrum at the expense of the 488 nm excitation spectrum. A549 cells were transduced with the sensor for 48 hours, and changes in fluorescence intensity were monitored for 10 minutes by confocal fluorescence microscopy after treatment with Ag5-AQC (IC50 - approximately 0.3 mg / L). While control cells did not change their redox state over time, the addition of Ag5-AQC to the samples resulted in a rapid oxidation response to a maximum signal 6 minutes after treatment. A total of 34 randomly selected cells (from a total of three experiments, with at least 10 cells per experiment) were analyzed, and 20 of these cells showed a clear change in their redox state after exposure to Ag5-AQC. Additionally, 13 of the remaining cells changed their oxidation state, although the effect was not as significant as in the above 20 cells. roGFP responds to GSH / GSSG levels through an electron exchange with glutaredoxin (GRX1), thus demonstrating the effect of Ag5-AQC on GSH( Figure 10 ).

[0264] Example 6: Ag5-AQCs act on key thiols present in proteins

[0265] Metallothioneins (MTs) are a group of low molecular weight, cysteine-rich intracellular metal-binding proteins that play a key role in protection against oxidants. The expression of MTs is controlled by metal response transcription factor 1 (MTF-1). Under normal conditions, MTF-1 shuttles between the cytoplasm and the nucleus, but upon various stresses, it accumulates in the nucleus and binds to metal response elements (MREs), inducing the expression of MTs in other genes. Under physiological conditions, MTs bind zinc through the sulfhydryl groups of their cysteine residues, forming two zinc / thiolate clusters, but under oxidative stress conditions, zinc is released via oxidation of the zinc / thiolate clusters, leading to the formation of MT-disulfides. This MT-disulfide state can be restored in a reducing environment, resulting in the formation of MT-thiols, which can associate with zinc ions to form MTs. This process constitutes the MT redox cycle, which plays a key role in the biological function of MTs.

[0266] It is inferred that Ag5-AQCs can catalyze the conversion of MT-thiol to MT-disulfide, thereby releasing zinc, activating MTF-1 and translocating it into the nucleus. To confirm this, the location of MTF-1 after treatment with Ag5-AQCs was analyzed. A549 cells were treated with Ag5-AQCs (IC50 - approximately 0.3 mg / L), fixed 2 hours later, and stained with an antibody against MTF-1. Immunofluorescence images showed clear nuclear accumulation of MTF-1 in the treated cells relative to control cells ( Figure 11 a). A total of 300 cells were counted for each condition, of which 242 cells were positive for nuclear location of MTF-1 in Ag5-AQC-treated cells and 9 cells in the control. In addition, microarray data obtained using the cell line MM.1S showed that MT genes were upregulated in response to Ag5-AQC treatment 4 hours after treatment, as expected from MTF-1 activation.

[0267] The Nrf2-Keap1 pathway is generally considered to be the major cellular defense pathway, which controls the expression of genes with antioxidant functions within cells. Under basal conditions, Nrf2 is transcriptionally repressed by Keap1 in the cytoplasm, which in turn promotes Cul3-mediated polyubiquitination of Nrf2, leading to its proteasomal degradation. Keap1 contains 27 cysteines, some of which have been reported to be targets of electrophiles and oxidants, and modification of them promotes the de-repression of Nrf2. Upon exposure to stress, Keap1 is inactivated by direct modification of cysteine thiol residues, and subsequently Nrf2 is stabilized, avoiding proteasomal degradation and translocating into the nucleus to mediate the activation of multiple genes involved in the antioxidant response, such as glutathione peroxidase (GPx), NAD(P)H quinone oxidoreductase (NQO-1), and heme oxygenase-1 (HMOX1). There are other mechanisms that regulate Nfr2 independent of Keap1, including the modification of cysteines in the Neh domain of Nrf2, which results in nuclear accumulation of Nrf2. It is hypothesized that Ag5-AQCs may be involved in the oxidation of sulfhydryls in Keap1 or Nrf2, leading to the release and translocation of Nrf2 into the nucleus. The localization of Nrf2 protein in response to N-ethylmaleimide (NEM, positive control) and Ag5-AQCs was evaluated using indirect immunofluorescence. NEM is an alkene reactive towards thiols and is commonly used to modify cysteine residues in proteins and peptides. The A549 cell line has a mutation in the Keap1 gene, resulting in altered Keap1 activity, which ceases to exert its inhibitory function on Nrf2, leading to the predominant localization of Nrf2 in the nucleus under normal conditions. Therefore, this cell line is not suitable for studying the cellular location of Nrf2. Instead, human embryonic kidney 293 (HEK293) cells were exposed to NEM (100 μM) and Ag5-AQC (IC50 - approximately 0.3 mg / L) for 30 minutes and then stained with specific antibodies against Keap1 and Nrf2. Compared to control cells in which Nrf2 was predominantly localized in the cytoplasm, Ag5-AQC treatment resulted in an increase in Nrf2 protein staining (red staining) after 30 minutes, indicating protein stabilization and nuclear accumulation (colocalized with blue Hoechst staining)( Figure 11 b). As expected, NEM treatment increased the nuclear accumulation of Nrf2. Clearly, Ag5-AQCs and NEM shared a similar staining pattern (increased Nfr2 expression due to reduced degradation and increased nuclear localization), thus supporting the thiol-reactive action of Ag5-AQC.

[0268] Example 7: Ag5-AQCs Reduce Multicellular Spheroid Tumor Growth

[0269] Multicellular tumor spheroids (MCTSs) are similar to multiple aspects of the pathophysiological conditions within human tumor tissues and are widely used in drug testing. Therefore, MCTSs of A549 cells were developed as an ex vivo tumor model to evaluate the activity of Ag5-AQC. The physiological state of cells in MCTS depends on its size; single MCTSs with a diameter of approximately 400 - 500 μm after 4 days of culture are often selected for drug testing. Thus, MCTSs were selected according to these criteria and treated four times with Ag5-AQC (2.4 mg / L) (on days 0, 2, 4, and 6, considering the first day of treatment as 0). From day 0 to day 7, images of control and treated MCTSs were taken daily. The images showed that Ag5-AQC treatment reduced the growth of MCTSs ( Figure 12 a), as evaluated by measuring the area of MCTSs using ImageJ. After the first dose of treatment, the size of MCTS decreased significantly and remained decreased over time, being significantly different from day 3 ( Figure 12 b). In addition, it is worth noting that there are translucent areas in the central part of MCTSs treated with Ag5-AQC, indicating the result of lower cellularity ( Figure 12 a, arrow). As mentioned before, large MCTSs (i.e., those with a size greater than 600 μm) are characterized by the presence of heterogeneous cell subpopulations, with actively proliferating cells in the periphery and quiescent, hypoxic, and necrotic cells in the inner region. The presence of these translucent areas in MCTSs after Ag5-AQC treatment is thought to be related to the ability of Ag5-AQC to penetrate into MCTSs due to their small size and neutral charge to reach these central hypoxic regions.

[0270] To verify this hypothesis, we evaluated the hypoxic levels in tumoroids using a fluorescent probe. As Figure 12 shown in c, the hypoxic levels increased inside tumoroids of 1,000 cells. Interestingly, exposure of tumoroids to increasing concentrations of Ag5-AQCs led to a dose-dependent decrease in hypoxic cells.

[0271] Example 8: H 2 O 2 Enhancing the effect of Ag5-AQC

[0272] The above examples provide evidence of the importance of the interaction of Ag5-AQC with O 2 、H 2 O 2 and hydroxyl radicals. The evidence provided here indicates that the same is true in cell cultures. The cytotoxic activity of Ag5-AQC was shown to be affected when cell respiration decreased and thus the ROS level decreased, thereby altering cell metabolism. In fact, A549 cells and U251 cells with weakened respiration (after allowing them to reach confluence) were less sensitive to the action of Ag5-AQC (Figure 13 A and 13B). As expected, serum-starved A549 cells were also less sensitive to Ag5-AQCs than proliferating cells ( Figure 13 C). If low doses of H 2 O 2 were co-administered with Ag5-AQCs, sensitivity was restored ( Figure 13 D).

[0273] Example 9: In Vivo Effects of Ag5-AQC

[0274] The in vivo effects of Ag5-AQC were tested in U87luc orthotopic glioma cancer models and A549luc orthotopic lung cancer models (metastasis to mediastinal lymph nodes).

[0275] High-grade gliomas - glioblastoma multiforme (GBM) - are the most aggressive and lethal form of brain tumors, with a survival rate of less than 5% after 5 years. One of the major limiting factors in the treatment of GBMs is the delivery of therapeutic agents across the blood-brain barrier (BBB) to the brain. This highly restrictive physiological barrier prevents 98% of small molecule drugs and virtually 100% of macromolecule drugs from reaching the central nervous system from the blood circulation. The small size of AQCs conjugated with their neutral charge at physiological pH favors (at least in theory) their diffusion in biological tissues. Consider whether these properties would allow Ag5-AQC to freely diffuse across the BBB to the tumor. For this purpose, a U87luc orthotopic glioma model was developed to test the potential of Ag5-AQC to cross the BBB and reduce tumors.

[0276] After orthotopic transplantation of U87luc cells, Ag5-AQC (0.5 mg / kg) was administered intravenously four times. Tumor growth was monitored by bioluminescence of tumor cells in the brain over a 14-day period using a spectral system. The results showed that tumor size increased exponentially throughout the experiment in control animals, while in animals treated with Ag5-AQC, tumor growth was reduced ( Figure 14 a). Thus, these results indicate that Ag5-AQC is able to cross the BBB, reach the tumor, and reduce its size.

[0277] Another limiting factor in cancer treatment is the occurrence of metastasis. Due to its systemic nature and the resistance of disseminated tumor cells to existing therapeutic agents, metastatic disease is mostly incurable. This explains why more than 90% of cancer mortalities are due to metastasis rather than the primary tumors that give rise to these malignant lesions. The ability of Ag5-AQC to reduce or eliminate both primary tumors and metastases was evaluated using the previously described A549luc orthotopic lung cancer model that metastasizes to mediastinal lymph nodes (Porto et al. (2018) Adv. Mater. e1801317). The A549 cell line is known to be a KRAS-mutated cancer cell line. In this model, the occurrence of metastasis to lymph nodes has been well established and can be detected 13 days after injection of tumor cells. Therefore, the effect of Ag5-AQC was evaluated starting on the 20th day after implantation of A549luc cells, when lymph node metastasis was already evident. Three groups were established: untreated control mice, mice treated with CDDP (4 mg / kg) as a positive control, and mice treated with Ag5-AQC (0.25 mg / kg). The treatments were administered intravenously four times (on days 20, 22, 24, and 26), and tumor progression was monitored in vivo by measuring the bioluminescence of tumor cells in the lungs over a 37-day period using a spectral system. The results showed that the tumor size was significantly reduced in mice treated with CDDP and Ag5-AQC compared to control animals in which the tumors grew exponentially throughout the experiment ( Figure 14 b). On day 37, the animals were sacrificed because the control mice showed obvious signs of morbidity, and the luciferase activity was quantified to measure the cancer cell burden. Mice treated with Ag5-AQC and CDDP showed a significantly lower luciferase activity in the primary tumors and mediastinal lymph nodes compared to control mice ( Figure 14 c). Then, the luciferase activities of mice treated with Ag5-AQC were compared to those of mice treated with CDDP. In mice treated with Ag5-AQC, the signals in the primary tumors and mediastinal lymph nodes were significantly lower than those in the other mice ( Figure 14 b, c). Immunohistochemical staining of lung sections with a monoclonal antibody against human cytokeratin that specifically stains tumor cells also confirmed these results ( Figure 14 d). Moreover, treatment with Ag5-AQC did not affect the body weight of the animals, ruling out severe toxicity of the compound ( Figure 14 e).

[0278] In summary, these results demonstrate that Ag5-AQC is able to reach both primary tumors and metastases and is able to significantly reduce their size without causing additional toxic effects. Therefore, due to their ability to cross the BBB and reach and reduce metastases, Ag5-AQC offers a new approach that could improve the treatment of human tumors.

[0279] Example 10: Effect of Ag5-AQC on primary cultures of human tumors

[0280] After diagnosis was established, Ag5-AQC sensitivity was evaluated ex vivo in cells from patients with B-chronic lymphocytic leukemia (B-CLL) obtained from routine bone marrow cultures. Evaluation of cytotoxic effects and ultrastructural morphological changes associated with Ag5-AQC treatment was performed in cells from 3 patients. B-CLL cells were cultured with different doses of Ag5-AQCs, and cytotoxicity was evaluated by MTT assay. Consistent with results previously obtained from established cell lines, a concentration-dependent decrease in cell viability was observed after 24 h ( Figure 15 a). Since Ag5-AQC increases O 2 ·- in cancer cell lines, we quantified changes in O 2 ·- levels in B-CLL cells exposed to Ag5-AQC by measuring the quantification of DHE-positive cells by flow cytometry. A significant increase, more than 2-fold, in DHE-positive cells was observed 4 h after treatment ( Figure 15 b). Moreover, TEM images showed distinct morphological changes of apoptosis and the presence of damaged mitochondria after Ag5-AQC treatment ( Figure 15 c). Thus, these results confirmed the results obtained with cell lines and spheroids discussed in the above examples.

[0281] Example 11: Effect of Ag5-AQC on a multiple myeloma tumor model

[0282] The effect of Ag5-AQC was tested in a multiple myeloma xenograft MM.1S mouse tumor model. Mice (n = 4 per treatment group) were treated with saline solution (control), 0.125 mg / kg Ag5-AQCs, or 0.25 mg / kg bortezomib, a proteasome inhibitor approved for the treatment of multiple myeloma in the United States and Europe. Tumor volume was monitored over several days, and the results are shown in Figure 16 . The results showed that Ag5-AQC treatment had an efficacy equivalent to that of bortezomib, even without an optimized dose.

[0283] Example 12: Ag5-AQCs preferentially cause cell death in Ras-transformed cells

[0284] It has been widely reported that activated oncogenes cause the accumulation of reactive oxygen species (Irani et al. (1997) Science). It was reasoned that transformed cells could be preferentially killed by treatment with Ag5-AQCs. To test this hypothesis, a doxycycline-inducible H-ras activating allele (RasV12) was introduced into non-transformed immortalized mouse fibroblasts (W3T3), and the effect of Ag5-AQCs on oncogene induction was analyzed. AsFigure 17 As shown in B, Ag5-AQCs caused a decrease in the survival of W3T3 in a dose-responsive manner. Interestingly, at all tested doses of AQCs, cells expressing RasV12 were more sensitive to the toxic effects of Ag5-AQCs compared to non-induced cells (control), and the viability was significantly reduced.

[0285] DTT treatment reversed this selective killing, suggesting that Ras-induced ROS accumulation causes different effects. This preferential effect of Ag5-AQCs on oncogene-transformed cells provides evidence for the use of Ag5-AQCs as anti-tumor agents, especially in RAS mutant cancers.

[0286] Example 13: Effect of AQCs in combination with radiotherapy

[0287] The effect of Ag5-AQC treatment on cells treated with radiation was tested. A549 cells and U251 cells were treated with different dilutions of Ag5-AQCs and immediately irradiated with different doses of radiation (0 - 10 Gy).

[0288] Cell survival was measured using clonogenic assays as described by Franken et al. (2006) Nat. Protocol. 1(5): 2315 - 2319. Briefly, after irradiation, cells were trypsinized and counted using a TC20 automated cell counter (Biorad). 100 cells from each sample were seeded in triplicate in 6-well plates and cultured at 37 °C in a humid atmosphere for 1 week to allow the formation of macroscopically visible colonies. Then, the cells were fixed and stained with crystal violet. Colonies with more than 50 cells were counted. After correcting for the plating efficiency of control cells, the survival fraction (SF) was calculated. The results are shown in Figure 18 Figures 19A. The results showed that the administration of Ag5-AQCs increased the cell killing effect of radiotherapy.

[0289] DNA damage in irradiated cells was also measured. After irradiation, cells were trypsinized, fixed with PFA (0.04%), and stained with an anti-pH2AX antibody (Millipore, product number: 16 - 202A) as described by Muslimovic et al. (2008) Nat. Protocol. 3: 1187 - 1193. Phosphorylated histone H2AX (pH2AX) expression is a marker of DNA damage (Sharma et al., DNA Repair Protocols, (edited by L. ), Springer Science, New York, 2012, Chapter 40). Stained cells were analyzed on a Guava EasyCyte flow cytometer using the InCyte program (Millipore). The results are shown in Figure 18 B and 19B.

[0290] The role of AQCs composed of five zero-valent transition metal atoms can be further tested in two different models to determine their effect in combination with radiotherapy. A) Ex vivo studies using multicellular tumor spheroids (MCTSs): MCTSs of U251-luc cells can be obtained by the hanging drop method. B) In vivo model: An orthotopic glioblastoma cancer model can be developed by injecting U251-luc cells (a human glioblastoma cell line carrying the luciferase gene to allow in vivo imaging of growing tumors) into the brains of athymic mice.

[0291] Conclusion

[0292] This paper describes the role of Ag5-AQCs in the oxidation of proteins with high cysteine content and accessible thiol groups such as metallothionein or glutaredoxin. It has been proposed that due to the small size of Ag5-AQCs, they should exhibit excellent permeability to enter tumor tissues. Experiments in MTCSs showed how Ag5-AQCs penetrate into the inner regions and kill hypoxic cells in these regions. The orthotopic lung cancer model with metastasis to mediastinal lymph nodes revealed the ability of Ag5-AQCs to reach tumors in vivo. A reduction in tumor size was observed in both the primary lung tumor and mediastinal lymph node metastases. These results highlight the ability of Ag5-AQCs to reach and reduce metastatic foci, constituting an innovative tool to address two major problems in cancer treatment.

[0293] The entire contents of all patents and patent applications cited herein are hereby incorporated by reference in their entirety. Moreover, all embodiments described herein can be used in all aspects of the present invention.

Claims

1. A composition for treating a cell proliferative disorder, comprising atomic quantum clusters (AQCs) composed of five zero-valent transition metal atoms.

2. The composition for such use according to claim 1, in combination with radiotherapy.

3. The composition for such use according to claim 2, for simultaneous or sequential administration with radiotherapy.

4. The composition for such use according to claim 2 or claim 3, wherein the radiotherapy is external beam radiotherapy.

5. The composition for such use according to any one of claims 1 to 4, wherein the composition is not used in combination with an anti-tumor drug.

6. The composition for such use according to any one of claims 1 to 5, wherein the AQCs are the sole active ingredient of the composition.

7. The composition for such use according to any one of claims 1 to 6, used as a single chemotherapy.

8. The composition according to any one of claims 1 to 4, for use in combination with an additional therapeutic agent, such as an anti-tumor agent, selected from: alkylating agents (such as nitrogen mustard analogs, nitrosoureas, alkyl sulfonates, platinum-containing compounds, ethyleneimines and imidazotetrazines), cytotoxic antibiotics (such as anthracyclines or actinomycins), plant alkaloids and other natural products (such as camptothecin derivatives, epipodophyllotoxins, taxanes and vinca alkaloids), antimetabolites (such as cytidine analogs, folic acid analogs, purine analogs, pyrimidine analogs, urea derivatives) and drugs for targeted therapy (such as kinase inhibitors and monoclonal antibodies).

9. The composition for such use according to any one of claims 1 to 8, wherein the metal atoms are selected from Ag, Au, Cu, Pt, Fe, Cr, Pd, Ni, Rh, Pb, Ir, Ru, Os, Co, Ti, V or any combination thereof.

10. The composition for such use according to claim 9, wherein the metal atoms are selected from Ag, Au, Cu, Pt or any combination thereof.

11. The composition for such use according to claim 9 or claim 10, wherein the metal atom is Ag.

12. The composition for such use according to any one of claims 1 to 11, wherein the composition comprises more than 50% of AQCs composed of five zero-valent transition metal atoms.

13. The composition for such use according to any one of claims 1 to 12, wherein the cell proliferative disorder is a tumor and / or cancer.

14. The composition for such use according to claim 13, wherein the cancer is selected from brain cancer, lung cancer, breast cancer or colon cancer.

15. The composition for such use according to claim 14, wherein the cancer is selected from brain cancer.

16. The composition for such use according to any one of claims 1 to 15, wherein the cell proliferative disorder comprises a RAS mutation.

17. The composition for such use according to claim 16, wherein the RAS mutation is a KRAS mutation.

18. A composition for the use according to any one of claims 1 to 17, wherein the composition comprises a pharmaceutically acceptable excipient, diluent or carrier.

19. A composition for the use according to any one of claims 1 to 18, which is combined with AQCs composed of 3 zero-valent transition metal atoms.

20. A composition for the use according to any one of claims 1 to 19, which: (i) is substantially free of AQCs composed of more than 5 zero-valent transition metal atoms, (ii) is substantially free of AQCs composed of less than 5 zero-valent transition metal atoms, and / or (iii) is substantially free of metal ions.

21. A composition for the use according to claim 20, wherein more than about 95% of the AQCs present in the composition are composed of 5 zero-valent transition metal atoms.

22. A composition according to any one of claims 1 to 21, for the prevention and / or treatment of cancer metastasis.

23. A composition according to claim 22, for the prevention and / or treatment of lymph node metastasis of cancer.

24. A composition according to claim 22 or claim 23, for the prevention and / or treatment of lung cancer metastasis.

25. A composition for the use according to any one of claims 1 to 24, which is administered orally, intravenously or subcutaneously.

26. Use of a composition according to any one of claims 1 to 25 in the preparation of a pharmaceutical composition for the treatment of a cell proliferative disorder.

27. Use of a composition comprising atom quantum clusters (AQCs) composed of 5 zero-valent transition metal atoms as a radiosensitizer for proliferating cells.

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