Patent Application
20250152577
The present invention is in the field of anti-tumor drugs and relates to a pharmaceutical composition of ethidium bromide and, in particular, to the potential use of ethidium bromide in the preparation of a drug for the treatment of cancer.
Cancer is a disease caused by multiple factors. The current prevailing view of carcinogenesis is that it is a multi-step process controlled by multiple genes, which is similar to Darwin's theory of evolution. But based on them it is difficult to find effective methods and strategies to treat cancer. Likewise, another hypothesis regarding tumorigenesis, the abnormal glycolysis hypothesis of carcinogenesis, known as the “Warburg effect”, was awarded the Nobel Prize in 1923.
For many years, the most important and widespread method of tumor diagnosis has still been PET-CT, the most important theoretical basis for which is the “Warburg effect”. It is generally believed to be a tumor diagnostic technique developed using the characteristics of tumor tissue that have a high demand for energy to cope with its rapid proliferation, resulting in high glucose uptake. However, a contradiction arises in this process: why do tumor cells that require more energy instead choose an inefficient glycolytic pathway to consume glucose and gain energy? Undoubtedly, clarifying this paradoxical metabolic process in tumor cells will not only contribute to an in-depth understanding of tumor properties and provide better clinical diagnostics but will also expand anti-tumor strategies and potentially lead to the screening of more potent and broad-spectrum anti-tumor drugs.
Although only 5% of glucose in cancer cells is thought to produce ATP via the mitochondrial pathway, it accounts for more than half of the energy source. Given that cancer cells have a higher energy requirement and are more sensitive to changes in energy supply, it is possible that they are more sensitive to drugs that target mitochondrial function. Therefore, further inhibition of the already dysfunctional mitochondria is likely to be the “last straw that breaks the back of tumor cells”.
Ethidium bromide is an organic compound that can induce mitochondrial damage and has the following structural formula.
Ethidium bromide is commonly used as a nucleic acid dye for nucleic acid staining, and its tricyclic planar moiety can be embedded between DNA stacking bases in a non-base sequence-specific manner. Ethidium bromide is generally considered to be a mutagen with carcinogenicity. However, according to the Pharmacopeia of Compounds edited by the FDA, US, there is no evidence that ethidium bromide can induce DNA mutations and carcinogenesis in cells or organisms. (National Toxicology Program, 1239-45-8). In contrast, studies have shown that cells treated with long-term ethidium bromide do not create genetic mutations (Mutation Research, 174(1986) 175-178).
The present invention surprisingly found that ethidium bromide has excellent anti-tumor effects and thus has very high clinical application value.
In one aspect, provided by the present invention is a pharmaceutical composition comprising a therapeutically effective amount of ethidium bromide, or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, and a pharmaceutically acceptable carrier.
In another aspect, provided by the present invention is use of ethidium bromide, or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, in the preparation of a pharmaceutical composition.
In another aspect, provided by the present invention is a method for treating cancer, comprising administering to a subject in need thereof the aforementioned pharmaceutical composition.
In another aspect, provided by the present invention is a method for inhibiting tumor metastasis, comprising administering to a subject in need thereof the aforementioned pharmaceutical composition.
In another aspect, provided by the present invention is a method for activating an mTOR signaling pathway, comprising administering to a subject in need thereof the aforementioned pharmaceutical composition.
In another aspect, provided by the present invention is a method for selectively inducing apoptosis in cancer cells, comprising contacting said cancer cells with ethidium bromide, or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof.
The present invention surprisingly found that ethidium bromide has good targeting effects on cancer cells, can selectively induce apoptosis in cancer cells, and effectively inhibit tumor migration. It has been proved that ethidium bromide can effectively clear tumors in vivo, such as colorectal tumor tissue, and has a very good inhibitory effect on the proliferation and migration of cancer cells, such as the migration of pancreatic cancer cells in pancreas, liver and lung tissues.
It is even more difficult and surprising that ethidium bromide has little or almost no side effects, which is in contrast to the general knowledge of the prior art. Therefore, ethidium bromide has extremely high clinical application value and is expected to promote clinical trials and its application.
“Pharmaceutical composition” is a preparation containing a compound of the present invention in a form suitable for administration to a subject.
“Pharmaceutically acceptable” refers to those compounds, materials, compositions, carriers, and/or dosage forms which, within sound medical judgment, are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit/risk ratio.
As used herein, “pharmaceutically acceptable salts” refers to the derivative of the compound of the present invention wherein the parent compound is modified by preparing an acid salt or base salt thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues (e.g., amines), base salt or organic salts of acidic residues (e.g., carboxylic acids), and the like. For the ethidium bromide of the present invention, pharmaceutically acceptable salts include ethidium bromide, such as conventional non-toxic salts that form from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include, but are not limited to, those obtained from inorganic and organic acids selected from the group consisting of: 2-acetoxybenzoic acid, 2-hydroxyethanesulfonic acid, acetic acid, ascorbic acid, benzenesulfonic acid, benzoic acid, acid carbonic acid, carbonic acid, citric acid, ethylenediaminetetraacetic acid, ethanedisulfonic acid, 1,2-ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hexylresorcinolic acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxymaleic acid, hydroxynaphthoic acid, hydroxyethanesulfonic acid, lactic acid, lactobionic acid, laurylsulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, naphthalenesulfonic acid, nitric acid, oxalic acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, polygalacturonic acid, propionic acid, salicylic acid, stearic acid, basic acetic acid, succinic acid, sulfamic acid, sulfanilic acid, sulfuric acid, tannic acid, tartaric acid, toluenesulfonic acid and the like.
The compound of the present invention can exist in a solvated or non-solvated form together with other solvent molecules. “Solvate” refers to a solvent addition form containing stoichiometric or non-stoichiometric amounts of solvent. Some compounds or salts have a tendency to trap a fixed molar ratio of solvent molecules in the crystalline solid state, resulting in the formation of the solvate. If the solvent is water, the solvates formed are hydrates; if the solvent is an alcohol, the solvates formed are alcoholates. Hydrates are formed from the combination of one or more molecules of water with one molecule of a substance in which the water retains its H2O molecular state.
The term “carrier” encompasses carriers and excipients and means a material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in carrying or transporting a pharmaceutical agent from one organ, or portion of the body, to another organ, or portion of the body of a subject. Carriers include solvents, dispersion media, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, salts, preservatives, drug stabilizers, buffers (e.g., maleic acid, tartaric acid, lactic acid, citric acid, acetic acid, sodium bicarbonate, sodium phosphate, and the like), and the like, and combinations thereof, which are generally recognized as safe. Unless any conventional carrier is incompatible with the active ingredient, its use in a therapeutic or pharmaceutical composition is contemplated. Excipients include any material that acts as a carrier for the release of the active ingredient into the body of the patient and any material that, for example, is added to the active ingredient to improve its handling or to allow the resulting composition to be formed into an orally deliverable unit dose having the desired shape and consistency. Excipients can include but are not limited to, for example, diluents, disintegrants, binders, adhesives, wetting agents, lubricants, glidants, substances that mask or counteract an undesirable taste or odor, flavoring agents, dyes, substances that improve the appearance of the dosage form, and any other substances besides the active ingredients commonly used in preparing oral dosage forms.
The pharmaceutical composition of the present invention is prepared to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenously, intra-arterially, intradermally, subcutaneously, orally (e.g., by inhalation), transdermally (topically), and transmucosally. Suitable pharmaceutical compositions for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for ad hoc preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include saline, bacteriostatic water, or phosphate buffer saline (PBS). In all cases, said compositions must be sterile and should be fluid to such an extent that easy injectability exists. Also included are antibacterial and antifungal agents, e.g., parabens, tert-butyl trichloride, phenol, ascorbic acid, thimerosal, etc.; antioxidants, e.g., ascorbic acid or sodium bisulfite; chelating agents, e.g., ethylenediaminetetraacetic acid; buffers, e.g., acetates, citrates, or phosphates, and isotonic agents, e.g., sugar, polyols (e.g., mannitol, sorbitol), sodium chloride. The pH can be adjusted with an acid or base (e.g., hydrochloric acid or sodium hydroxide).
Sterile injectable solutions may be prepared by combining the active compound in the desired amount in a suitable solvent with one of the above-listed components or a combination thereof, as desired, and subsequently sterilized by filtration. Typically, dispersions are prepared by incorporating active compounds into sterile carriers containing a base dispersion medium and other required components from those listed above. In the case of sterile powder used for preparing sterile injectable solutions, the preparation method includes vacuum drying and freeze-drying, which yield powder containing active ingredients plus any additional required ingredients from the previously sterile-filtered solution. They may be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.
Oral compositions typically include inert diluents or pharmaceutically acceptable carriers that are edible. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally, swished, and expectorated or swallowed. Pharmaceutically compatible binders, and/or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches, and the like may contain any of the following ingredients, or compounds of a similar nature: a binder, e.g., microcrystalline cellulose, gum tragacanth, or gelatin; a diluent, e.g., starch or lactose; a disintegrant, e.g., alginic acid or cornstarch; a lubricant, e.g., magnesium stearate; a glidant, e.g., colloidal silicon dioxide; a sweetening agent, e.g., sucrose or saccharin; or a flavoring agent, e.g., menthol, methyl salicylate, or an orange flavoring agent.
The pharmaceutical composition can be included in the container, packaging, or dispenser along with the instructions for administration.
The pharmaceutical compositions of the present invention may be in bulk or unit dosage form. The unit dosage form is in a variety of forms including, for example, any of capsules, injections, IV bags, tablets, single pumps on aerosol inhalers, or vials. The amount of active ingredient (e.g., the disclosed formulation of ethidium bromide or a pharmaceutically acceptable salt, solvate, or prodrug thereof) in a unit-dose composition is the effective amount and varies according to the particular therapy involved.
It is particularly advantageous to formulate oral or parenteral compositions in the form of dosage units in order to facilitate the administration and homogeneity of the dosage. As used herein, the dosage unit form refers to a physically dispersed unit suitable for use as a unit dose for a subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the desired drug carrier. The specification for the dosage unit forms of the present invention is dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved.
“Therapeutically effective amount” refers to an amount of an agent used to treat, alleviate, or prevent an identified disease or condition, or to exhibit a detectable therapeutic or inhibitory effect. Said effect can be detected by any assay method known in the art. The precise effective amount to be used for a subject will depend on the subject's weight, size, and health; the nature and extent of the disease condition; and a therapeutic agent or combination of therapeutic agents selected for administration. A therapeutically effective amount for a given situation can be determined by routine experimentation that is within the skill and judgment of the clinician.
For any compound, the therapeutically effective amount can be estimated initially either in cell culture assays (e.g., of neoplastic cells), or in animal models (typically rats, mice, rabbits, dogs, or pigs). Animal models can also be used to determine appropriate concentration ranges and routes of administration. Such information can subsequently be used to determine available dosages and routes of administration in humans. Therapeutic/prophylactic efficacy and toxicity can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. ED50 (the dosage that is therapeutically effective in 50% of the population) and LD50 (the dosage that is lethal in 50% of the population). The dosage ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio: LD50/ED50. Pharmaceutical compositions exhibiting a large therapeutic index are preferred.
The dosage may also vary within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration. Dosage and administration are adjusted to provide sufficient levels of the active agent or to maintain the desired effect. Factors that may be considered include the severity of the disease condition, the general health of the subject, age, weight, and sex of the subject, diet, time and frequency of administration, drug combinations, reaction sensitivities, and tolerance/response to therapy. Depending on the half-life and clearance of a particular formulation, a long-acting pharmaceutical composition may be administered every 3 to 4 days, weekly or biweekly.
The dosage will also depend on the route of administration. Various routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intra-arterial, intramuscular, intraperitoneal, inhalation, buccal, sublingual, intrapleural, intrathecal, intranasal, and on the like. In an embodiment, the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier, as well as with any preservative, buffer, or propellant desired.
In therapeutic applications, the dosage of the pharmaceutical compositions used in accordance with the present invention varies according to the agent, the age, weight, and clinical status of the recipient patient, and the experience and judgment of the clinician or practitioner administering the therapy, in addition to other factors affecting the selected dosage. Typically, said dosage should be sufficient to result in slowing down and preferably regressing the tumor growth and also preferably causing complete regression of the cancer. The dosage may range from about 0.01 mg/kg/day to about 5000 mg/kg/day. The effective amount of an agent is the amount that provides an objectively identifiable improvement as noted by a clinician or other qualified observer. For example, regression of a tumor in a subject can be measured with reference to tumor diameter. A decrease in tumor diameter indicates regression. Regression is also indicated by the inability of the tumor to recur after cessation of treatment.
The term “administration”, as used herein, refers to either directly administering a disclosed compound or a pharmaceutically acceptable salt or composition of the disclosed compound to a subject, or administering a prodrug, derivative, or analog of said compound or the pharmaceutically acceptable salt or composition of said compound to a subject, which can form an equivalent amount of the active compound in the body of the subject.
A “subject” is a cell, tissue, organ, or animal, e.g., a mammal, such as a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, or non-human primate, such as a monkey, chimpanzee, baboon, or rhesus monkey.
The term “treatment” with respect to a subject refers to ameliorating at least one symptom of the subject's condition. Treatment includes curing, ameliorating or at least partially alleviating the condition.
The methods provided herein are used to treat or alleviate symptoms of cancer. The term “cancer” includes solid tumors as well as hematologic and/or malignant tumors. A “pre-cancerous cell” or “precancerous cell” refers to a cell that exhibits a proliferative cellular disorder that is precancerous or a precursor to cancer. A “cancer cell” or “cancerous cell” is a cell that exhibits a proliferative disorder that is characterized by cancer. Any reproducible measurement can be used to identify cancer cells or precancerous cells. Cancer cells or precancerous cells can be identified by histological classification or grading of a tissue sample (e.g., a biopsy sample). Cancer cells or precancerous cells can be identified by using appropriate molecular markers.
Exemplary cancers include, but are not limited to, adrenocortical cancer, AIDS-related cancer, AIDS-related lymphoma, anal cancer, anorectal cancer, anal canal cancer, appendiceal cancer, childhood cerebellar astrocytoma, childhood cerebral astrocytoma, basal cell carcinoma, skin cancer (non-melanoma), gallbladder cancer, extrahepatic cholangiocarcinoma, intrahepatic cholangiocarcinoma, bladder cancer, bone and joint cancers, osteosarcoma and malignant fibrous histiocytoma, brain cancer, brain tumor, brain stem glioma, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway and hypothalamic gliomas, breast cancer, bronchial adenoma/carcinoma, carcinoid tumor, gastrointestinal, nervous system cancer, nervous system lymphoma, central nervous system cancer, central nervous system lymphoma, cervical cancer, cancer of childhood stage, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disease, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, lymphoid paraphyte, mycosis fungoides, endometrial cancer, esophageal cancer, extracranial blastoma, extragonadal blastoma, extrahepatic cholangiocarcinoma, ocular cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germinoma, germinal tumor of ovary, gestational trophoblastic tumor glioma, head and neck cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, ocular cancer, islet cell tumor (endocrine pancreas), Kaposi's sarcoma, renal cancer, laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, lip and oral cavity cancer, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, AIDS-related lymphoma, non-Hodgkin's lymphoma, primary central nervous system lymphoma, Waldenström's macroglobulinemia, medulloblastoma, melanoma, Merkel cell carcinoma, malignant mesothelioma, mesothelioma, metastatic squamous cervical carcinoma, oral cancer, tongue cancer, multiple endocrine neoplasia, mycosis fungoides, myelodysplastic syndrome, myelodysplastic/myeloproliferative disorder, chronic myeloid leukemia, acute myeloid leukemia, multiple myeloma, chronic myeloproliferative disease, nasopharyngeal cancer, neuroblastoma, oropharyngeal cancer, ovarian cancer, epithelial ovarian cancer, ovarian tumor of low malignant potential, pancreatic cancer, islet cell pancreatic cancer, paranasal sinus and nasal cavity cancer, parathyroid carcinoma, penile cancer, pharyngeal cancer, pheochromocytoma, pinealocytoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell paraphyte/multiple myeloma, pleuropulmonaryblastoma, prostatic cancer, rectal cancer, transitional-cell carcinoma of renal pelvic and ureteric, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcomatoid tumors of Ewing's family, Kaposi's sarcoma, soft-tissue sarcoma, uterine carcinoma, uterine sarcoma, skin cancer (non-melanoma), skin cancer (melanoma), Merkel cell skin cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, gastric (stomach) cancer, supratentorial primitive neuroectodermal tumor, testicular cancer, laryngeal cancer, thymoma, thymoma and thymic cancer, thyroid cancer, transitional cell carcinoma of the renal pelvis, ureter, and other urinary organs, gestational trophoblastic tumor, urethral cancer, endometrial cancer, uterine sarcoma, corpus uteri cancer, vaginal cancer, vulvar cancer and Wilms' tumor.
The compounds or pharmaceutical compositions of the present invention can be administered to a subject in a number of well-known methods currently used for chemotherapeutic treatment. For example, to treat cancer, the compounds of the present invention can be injected directly into a tumor, into the blood stream or into a body cavity, or administered orally or through the skin using a patch. The dosage selected should be sufficient to result in effective treatment, but not so high as to cause unacceptable side effects. It should be preferred that the status of the disease condition (e.g., cancer, pre-cancer, etc.) and the health of the patient be closely monitored during and for a reasonable period of time after treatment.
In one aspect, provided by the present invention is a pharmaceutical composition comprising a therapeutically effective amount of ethidium bromide, or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, and a pharmaceutically acceptable carrier.
In an embodiment, said pharmaceutical composition is for use in the treatment of cancer.
In another embodiment, said pharmaceutical composition selectively induces apoptosis in cancer cells or inhibits tumor metastasis.
Experiments have confirmed that ethidium bromide selectively induces apoptosis in cancer cells derived from a variety of different tissues. In contrast, ethidium bromide, although having an inhibitory effect on the growth of normal cells, cannot induce apoptosis in normal cells. This selectivity for specific killing of cancer cells is very valuable for potential clinical applications of ethidium bromide.
However, ethidium bromide plays a role in inhibiting tumor metastasis at different stages of tumor progression or even at advanced stages, which not only avoids further deterioration of cancer but also produces reversible or even eliminating effects on cancer progression, which will have a significant impact on survival and is of great clinical value.
Further experiments showed that ethidium bromide can selectively inhibit the mitochondrial function of cancer cells, indicating that ethidium bromide can selectively induce apoptosis in cancer cells by inhibiting mitochondrial function. This further demonstrates that defective mitochondrial function in cancerous cells plays a critical role in the selective induction of apoptosis in cancerous cells by ethidium bromide, and also suggests that mitochondria may play a unique and very important role both in the process of cancer development and in cancer treatment.
In another embodiment, said cancer is selected from one or more of gastric cancer, liver cancer, colorectal cancer, breast cancer, lung cancer, pancreatic cancer, leukemia, preferably colorectal cancer or pancreatic cancer.
Ethidium bromide has a good inhibitory effect against a variety of cancers or cancer cells, including but not limited to several verified by the present invention. Especially for pancreatic cancer, which is recognized to be highly malignant, rapidly progressive, poorly treated, and with a short survival period, ethidium bromide still has excellent inhibitory effects, including inhibition of cancer metastasis and induction of apoptosis in cancer cells.
In another embodiment, said pharmaceutical composition is in oral or injectable form.
The traditional way of administration by intraperitoneal injection (I.P.) not only has excessive side effects but also has a relatively poor anti-tumor effect, which may be one of the reasons why ethidium bromide has not been used for effective anti-tumor use in the prior art to date. In contrast, the present invention has for the first time found that ethidium bromide administered orally or by injection, in particular intravenous injection (I.V.), has a superior anti-tumor effect and is less toxic. Moreover, this anti-tumor effect targets in vivo tumor tissues, which is closer to the in vivo pathogenesis of cancer than studies targeting subcutaneous solid tumors, with better prospects for medical applications and greater value.
In another aspect, provided by the present invention is use of ethidium bromide, or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, in the preparation of the aforementioned pharmaceutical composition.
In another aspect, provided by the present invention is a method for treating cancer comprising administering to a subject in need thereof the aforementioned pharmaceutical composition.
In yet another aspect, provided by the present invention is a method for inhibiting tumor metastasis, comprising administering to a subject in need thereof the aforementioned pharmaceutical composition.
In yet another aspect, provided by the present invention is a method for activating the mTOR signaling pathway comprising administering to a subject in need thereof the aforementioned pharmaceutical composition.
Experiments have shown that ethidium bromide can rapidly activate mTOR, followed by rapid induction of downstream NFkB and Erk phosphorylation and Caspase3 cleavage, and the activation of these signaling molecules ultimately and selectively induced tumor cell apoptosis. This early event that induces apoptosis in tumor cells can occur both in vitro and be reproduced in vivo.
In one embodiment, said administration is performed orally, by injection, parenterally, subcutaneously, or by infusion, preferably orally or by injection, particularly preferably by injection, e.g., intravenously, intra-arterially, subcutaneously, or intramuscularly.
In another embodiment, the administration is performed at a dosage of from about 1 mg/Kg to 100 mg/Kg of body weight per day, e.g., about 1 mg/Kg, about 2 mg/Kg, about 5 mg/Kg, about 10 mg/Kg, about 15 mg/Kg, about 20 mg/Kg, about 25 mg/Kg, about 30 mg/Kg, about 35 mg/Kg, about 40 mg/Kg, about 50 mg/Kg, about 60 mg/Kg, about 70 mg/Kg, about 80 mg/Kg, about 90 mg/Kg, about 100 mg/Kg of body weight per day.
In general, said dosage should be sufficient to cause slowing and preferably regress the tumor growth and also preferably cause complete regression of the tumor. The dosage may for example range from about 0.01 mg/kg/day to about 5000 mg/kg/day.
When mice were treated in the present invention by conventional intraperitoneal administration, the dosage used was 10 mg/Kg, but most of the mice were very sensitive and died excessively. Gavage oral administration of 20 mg/Kg ethidium bromide per day was well tolerated and had better anti-tumor effects in mice. The intravenous dosage was 5 mg/Kg per day for 4 weeks. Intravenous administration has proved to have the lowest therapeutically effective dose, the least toxic side effects, and the best anti-tumor effect.
The specific ethidium bromide dosage will vary according to the dosage form employed, the sensitivity of the patient and the route of administration, and the experience and judgment of the clinician or practitioner administering the therapy.
In another embodiment, the administration is continuously performed for at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year.
After 3-4 weeks of continuous administration of ethidium bromide of the present invention, tumors regressed significantly or even completely. The specific period of administration will vary according to the dosage form employed, the sensitivity of the patient and the route of administration, and the experience and judgment of the clinician or practitioner administering the therapy. For example, for advanced tumors, the period of administration needs to be longer.
In another embodiment, the administration is performed for one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) courses of treatment, each course of treatment lasts at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks; and wherein there is an interval of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days, two weeks, three weeks, or four weeks between every two courses of treatment.
After one continuous course of continuous administration of ethidium bromide of the present invention, tumors regressed significantly or even completely. The specific course of administration will vary according to the dosage form employed, the sensitivity of the patient and the route of administration, and the experience and judgment of the clinician or practitioner administering the therapy.
In yet another aspect, provided by the present invention is a method for selectively inducing apoptosis in cancer cells comprising contacting said cancer cell with ethidium bromide, or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof.
This event of selective induction of apoptosis in cancer cells can occur both in vitro and be reproduced in vivo.
In an embodiment, said cancer cells are derived from one or more of gastric cancer, liver cancer, colorectal cancer, breast cancer, lung cancer, pancreatic cancer, leukemia, preferably colorectal cancer or pancreatic cancer.
The present invention is further illustrated by the following specific examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. Furthermore, it will be appreciated that those skilled in the art, upon reading the teachings of the present invention, may make various changes and modifications to the present invention and that such equivalents are intended to fall within the scope of the appended claims of the present invention.
To verify the important role of mitochondrial function in tumor treatment and occurrence, cancerous and non-cancerous cells were treated with the mitochondrial damage-inducing reagent, ethidium bromide, in this Example.
Ethidium bromide was purchased from Sigma (E7637) and treated as follows. Cancer and normal cells of various tissue origins with approximately 40% growth abundance were treated with 0.1, 0.4, and 1 μg/ml for 48 h, followed by PI staining and cycle detection by flow cytometry, statistical analysis of genomic DNA break (Sub-G1) cells, or cell viability by MTT method.
The results showed that ethidium bromide can selectively induce the death of a wide range of cancer cells, including hepatocellular carcinoma cells (SK-Hep1, BEL-7404), and melanoma cells (B16), whereas for normal cells, such as hepatocellular cell lines (Chang, LO2), and fibroblast cell lines (3T3-L1), it inhibited growth at most and cannot induce apoptosis (
In support of this, cancer cells and normal cells from various tissues with growth abundance of about 40% were treated with 1, 2, and 5 μM for 48 h, respectively, and then observed the growth of the cells by light microscopy, followed by PI staining, detection and statistical analysis of genomic DNA break (Sub-G1) cells by flow cytometry (
The results showed that ethidium bromide can selectively induce the death of cancer cells in many different tissues, including hepatocellular carcinoma cells (SK-Hep1, 7404), gastric cancer cells (AGS), colorectal cancer cells (RKO), whereas for normal cells such as normal hepatocellular cell line (LO2, chang), gastric epithelial cell line (GES1), it only inhibited growth and cannot induce apoptosis (
In support of this, Table 1 summarizes the median lethal dosage of ethidium bromide on tumor cells and normal cells from various sources, and the data indicate that the vast majority of median lethal dosages of ethidium bromide against these tumor cells are below 10 μM, whereas those against normal cells are mostly above 50 μM.
In conclusion, ethidium bromide can specifically induce cancerous cell death, and the mode of death is mainly apoptosis. However, the effect of ethidium bromide on non-cancerous cells (normal cells) is only growth inhibition and does not result in cell death. This may be related to the mechanism by which ethidium bromide induces mitochondrial damage and alters energy metabolism in tumor tissues.
To further verify the effect of ethidium bromide on mitochondrial function, various cell lines were treated with ethidium bromide and mitochondrial function was tested by using Seahorse after 6 hours. The results showed that the parameters reflecting mitochondrial function, such as oxygen consumption, glycolysis level, and glycolysis rate, were significantly reduced in pancreatic cancer cells Panel and colorectal cancer cells RKO after being treated with ethidium bromide, and manifested as a certain concentration-dependent effect (the numbers are the concentration values in μM). In contrast, the normal cell line GES1 decreased only at higher concentrations such as 5 μM, and the effective concentration of ethidium bromide toxicity was significantly higher than that of cancer cells (
This indicates that ethidium bromide can selectively inhibit the mitochondrial function of cancer cells, and suggests that ethidium bromide can selectively induce apoptosis in cancer cells by inhibiting mitochondrial function. This further demonstrates that defective mitochondrial function in cancerous cells plays a critical role in the selective induction of death in cancerous cells by ethidium bromide, and also suggests that mitochondria may play a unique and very important role both in the process of cancer occurrence and in cancer treatment.
1) Administration after 1 Week
The classical intraperitoneal injection of AOM and the spontaneous colorectal cancer formation by three rounds of DSS were utilized to construct a tumor mouse model. Mice were treated with ethidium bromide (500 mg/L dissolved in drinking water) one week after completion of the construction. After 2 months of administration, control mice showed significant prolapse due to oversized colorectal tumors, whereas the dosing group did not (
To further verify the above results, we then expanded the sample size to about 10-16 mice per group and still used the above AOM+DSS protocol to induce spontaneous colorectal cancer formation in mice. Because it is very difficult to administer each mouse in drinking water, and because a significant proportion of mice do not drink drinking water containing ethidium bromide, we switched to gavage administration (20 mg/Kg once daily). Meanwhile, since it has been reported that zalcitabine (ddC) can effectively impair the function of mitochondria (mainly the replication of mtDNA, similar to ethidium bromide), we added the experimental group of zalcitabine (33 mg/Kg) as a control.
The tumors in the pre-administration mice are shown in
We tested the mtDNA copy number of colorectal tumors in the dosing group and, surprisingly, ethidium bromide did not alter the mitochondrial mtDNA copy number (
In addition, in order to investigate in what way ethidium bromide clears tumors in mice, we also tested cell proliferation (
2) Administration after 4 Weeks
To further verify whether ethidium bromide is still effective against already existing larger tumors (advanced colorectal cancer), we set aside a group of control mice (started administration 5 weeks after the end of induction, equivalent to the control group of mice described above) to administer them by gavage (20 mg/Kg per day).
The results showed that ethidium bromide was similarly very effective for these larger tumors, with more than 40% of the mice achieving complete tumor clearance after 1 month of administration, and the remaining tumors that were not completely cleared were also very small (
Ectopic metastasis of tumors is the biggest challenge in tumor treatment. In order to further investigate whether ethidium bromide can effectively inhibit the migration of tumors, we firstly tested the effect of ethidium bromide on the migration of tumor cells in vitro. The scratch assay of cultured cells showed that ethidium bromide can indeed significantly inhibit the migration of pancreatic tumor cells Panc1 and colorectal cancer cells RKO (
To further verify whether ethidium bromide can inhibit tumor migration in vivo, we injected 2×106 pancreatic cancer cells Panc1 intraperitoneally into nude mice, which were then divided into 3 groups: control group, gavage, and intravenous injection administration. Mice in the gavage-administration group were started with gavage oral administration of 20 mg/Kg ethidium bromide per day by gavage 3 days after intraperitoneal injection of pancreatic cancer cells. Mice in the intravenous administration group were administered with 5 mg/Kg ethidium bromide per day intravenously (Tail-IV) 3 days after intraperitoneal injection of pancreatic cancer cells.
The results showed that ethidium bromide can significantly inhibit the proliferation of pancreatic cancer in vivo, especially in the intravenous injection group, no tumor was found in the pancreas of nude mice, indicating its very excellent anti-tumor effect. However, the pancreas of nude mice in the gavage group still had a small amount of tumor, but was significantly smaller than that in the control group (
The body weight data of nude mice showed that compared with gavage administration, intravenous injection administration did not cause a significant decrease in body weight of nude mice, indicating that intravenous injection administration is even slightly less toxic to nude mice than the other modes of administration, where the control nude mice weighed slightly more than the dosing group, partly due to the fact that ethidium bromide caused a slight decrease in the animals' body weight, and partly due to the fact that the body weights of the control mice increased with the increase of the tumor (
In order to further investigate the anti-tumor effect of ethidium bromide on nude mice with advanced tumorigenicity and to obtain its effect on the viability of nude mice, we intraperitoneally injected the nude mice with 2×106 pancreatic cancer cells Panc1, which were then divided into 3 groups, the control group (no-treatment group), the early-injection group (administered intravenously after 3 days) and the late-injection group (administered intravenously after 14 days). Mice in the early injection group were administered with 5 mg/Kg ethidium bromide intravenously per day for 3 weeks 3 days after intraperitoneal injection of pancreatic cancer cells (starting intravenous administration after 3 days for about 3 weeks). Mice in the late injection group were administered with 5 mg/Kg ethidium bromide intravenously per day for 3 weeks starting 2 weeks days after intraperitoneal injection of pancreatic cancer cells (starting intravenous administration after 14 days for about 3 weeks).
As the control nude mice had severe ascites due to oversized tumors during this process, the administration was stopped after 3 weeks of continuous administration. At the same time, the abdominal cavity of the control nude mice was filled with ascites; while the abdominal cavity of the dosing group was very normal (results not shown).
Meanwhile, the results showed that the pancreatic cancer tumor of nude mice in the dosing group was significantly smaller than that in the control group (
In addition, we utilize the most advanced nuclear magnetic spectrometer uMR 9.4T with a 30 cm aperture magnet jointly developed by our work unit and Shanghai United Imaging Healthcare Co., LTD, as well as our self-developed GC, GPA, and VTC42 coils specially designed for abdominal scanning, and an optional PET insert system to achieve the PET-MR imaging results of the temporal and spatial integrality. The development of pancreatic cancer was followed dynamically in the control and dosing groups following intraperitoneal injection of pancreatic cancer cells. Mice were injected intraperitoneally with 2×106 pancreatic cancer cells, Panc1, and left for 2 weeks after which a visible tumor mass can be detected in the abdominal cavity to begin intravenous administration of 5 mg/Kg ethidium bromide for 4 weeks. Similar to the above correlation results: the growth of pancreatic cancer in the abdominal cavity of the mice was significantly inhibited, or almost completely inhibited, after administration. Moreover, the abdominal cavity of the control mice was filled with ascites by the late stage (where the arrows are shown), whereas there was none in the dosing group (
In order to further investigate whether ethidium bromide also has a better inhibitory effect on other types of tumors, we administered nude mice 2 weeks after the intraperitoneal injection of 2×106 colorectal cancer cells RKO, and then tested the anti-tumor effect of ethidium bromide after 4 weeks of intravenous administration, as described above.
The results showed that ethidium bromide still remained very effective in inhibiting colorectal cancer of human origin and its metastasis. The abdominal tumors of nude mice in the dosing group were significantly smaller than those in the control group (
In addition, to further investigate the effect of ethidium bromide on ectopic tissue metastatic tumors, we injected 2×106 pancreatic cancer cells Panel intravenously into nude mice. After ectopic tissue metastasis, tumors formed primarily in the lungs. After approximately 3 weeks, intravenous administration of 5 mg/Kg ethidium bromide per day was started after the tumor had already grown in the lung tissue (
Finally, we also conducted a preliminary study on the toxicity of ethidium bromide by collecting whole blood and serum from the control and the dosing groups (administered intravenously for about 1 month) and tested their routine blood, and liver and kidney functions, respectively.
The data demonstrated that after 4 weeks of intravenous administration, ethidium bromide showed no significant difference in liver or kidney function in nude mice compared with the control group (
We investigated the mechanism of ethidium bromide-induced apoptosis in tumor cells. First, tumor tissues treated with ethidium bromide (mice shown in
The results showed that ethidium bromide treatment resulted in a significant increase in cleaved Caspase3 in tumor tissues, as well as phosphorylation of Erk and the level of LC3, an important protein for autophagy (
In order to further determine how ethidium bromide activates phosphorylation of Erk and Caspase3 cleavage and induces apoptosis, we also tested with different cell lines treated with ethidium bromide for 10 min, 30 min, 1, 2, and 6 h. The results showed that mTOR was already activated by ethidium bromide very quickly at 10 min, while p-NFkB and p-Erk were also activated at 30 min and 1 h, respectively, suggesting that the activation of these signaling molecules is an early event in ethidium bromide-induced apoptosis in tumor cells.
In order to further test whether ethidium bromide still has strong anti-tumor activity against solid tumors in vivo, C57 mice were first injected subcutaneously with 2×106 mouse melanoma cells B16, and the administration was initiated after about 10 days when the subcutaneous tumor volume of the mice reached about 30 mm3. We first treated the mice by traditional intraperitoneal administration (10 mg/Kg). The results showed that the mice were very sensitive to intraperitoneal injection of ethidium bromide, and after 3 days of continuous injection, mice started to die one after another and began to lose weight significantly. So after 3 days, we switched to injecting every other day. By the end of the experiment, only 1 of the 19 mice survived (
In addition, we also tested for nude mice utilizing intraperitoneal administration, where 2×106 colorectal cancer cells, RKO, were first injected intraperitoneally in nude mice, and about 3 days later, we treated the nude mice using intraperitoneal administration (10 mg/Kg). Similar to C57 mice, once daily until day 5, administration was stopped due to excessive deaths in nude mice (
We also tested the anti-tumor activity of ethidium bromide against subcutaneous solid tumors. 2×106 colorectal cancer cells RKO were injected subcutaneously. About 1 week later, when solid tumors grew subcutaneously in nude mice, gavage administration (20 mg/Kg) was started. The size of the tumors was measured twice a week. It was found that although ethidium bromide exhibited some tumor proliferation inhibitory activity and also contributed to improve the viability of nude mice, the inhibitory effect of ethidium bromide on subcutaneous solid tumors was very unsatisfactory compared to its very strong inhibitory effect on tumor cell proliferation and migration in vivo (
In addition, we also grafted isolated tumor tissue blocks (PDX) from patients with colorectal cancer subcutaneously in nude mice and similarly found that the anti-tumor effect of ethidium bromide was very limited and much less than its tumor inhibitory effect on various tissues in vivo (
However, when we injected ethidium bromide directly into the subcutaneous tumor mass (20 mg/Kg ethidium bromide dissolved in PBS, injected once a day for three consecutive injections), the subcutaneous tumor mass was rapidly necrotic and eventually died and fell off (
Taken together, we hypothesize that the limitations of ethidium bromide for subcutaneous solid tumors may be primarily due to the difficulty of drug molecules administered in vivo to reach the subcutis. The value of subcutaneous neoplasia is to facilitate scientific research, but the test results of subcutaneous neoplasia may not be repeated in vivo, and there is great difference between subcutaneous neoplasia and in vivo, so it is not practical. In contrast, the present invention has a very good inhibitory effect on the proliferation and metastasis of tumor tissues in vivo by infusion or intravenous injection of ethidium bromide with little or almost no side effect, showing great medical application value.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202210475952.3 | Apr 2022 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2023/090596 | 4/25/2023 | WO |
