Composition and use thereof in animals

Abstract

A composition, comprising at least two of: (a) a targeting agent for cancer cell mitochondria, (b) an ion chelating agent, and (c) a multivalent ion having pharmacological activity, wherein the weight ratio of (a) is 22.0%-98.4% on the basis of the total weight of the composition, the weight ratio of (b) is 37.9%-98.8% on the basis of the total weight of the composition, and the weight ratio of (c) is 0.69%-64.5% on the basis of the total weight of the composition; and the use thereof.

Description

Composition and use thereof for animals

Technical field

[0001] The present invention relates to a composition, a kit comprising the composition, and uses thereof. Specifically, the present invention is directed to a composition for treating malignant diseases caused by abnormal growth of animal cells, a kit comprising the composition, and uses thereof. [Background Technology]

[0002] Cancer is a major global health problem that affects not only humans but also other species. Malignant tumors develop from abnormal cells caused by genetic mutations. These cells divide and proliferate continuously, forming tumors that have the ability to invade and destroy nearby normal tissues and organs.

[0003] Furthermore, they can spread to distant organs through the bloodstream or lymphatic system or develop drug resistance, making treatment more challenging. Malignant tumor cells are highly active and destructive, with high recurrence rates and difficult to cure. The causes of cancer are highly complex, and a complete cure remains elusive. Therefore, developing drugs that effectively inhibit cancer cells is crucial for patients.

[0004] There are many similarities in the mechanisms of cancer between animals and humans, but there are also important differences. These differences are mainly reflected in genetic susceptibility, environmental influences, immune system response and viral infection. Understanding these differences is crucial for developing effective cancer treatment strategies for each species. Pet tumors are a serious threat to pet health and they may affect the life and quality of life of pets. Tumors can occur in various organs and tissues of pets, including skin, breasts, bones and internal organs. These tumors can be benign or malignant, and malignant tumors are more life-threatening to pets.

[0005] The efficacy and timeliness of cancer treatments in animals vary from those in humans, but are generally inferior. Common anti-cancer therapies include surgery, radiation therapy, chemotherapy, or the use of targeted drugs for human cancer therapy. Unfortunately, while inhibiting tumor growth, these conventional treatments often carry the risk of harming or toxicating normal tissue. Furthermore, alternative therapies designed to toxicate cancer cells without producing side effects are still under investigation and in preclinical testing.

[0006] As the incidence of animal cancer continues to increase, the need for cancer treatment and management is becoming increasingly urgent. The hope is to provide more effective and safer treatment options to help animals overcome cancer, extend their lifespan, and improve their quality of life. This represents a commitment to the health and well-being of pets. Innovation and improvement in therapeutic drugs will help improve the quality of life of pets and provide more options for pet owners. Currently, the cost of common pet treatments is very expensive for pet owners, so the cost of pet treatment is a heavy burden for pet owners. Therefore, it is very important to provide pet owners with drugs that are more affordable, have better therapeutic effects, and are affordable for pet owners.

[0007] [Summary of the invention]

[0008] In view of this, the present invention proposes a composition that is effective against malignant tumors, a kit containing this composition, and its use in treating malignant diseases caused by abnormal growth of animal cells. This drug combination can be applied in the field of oncology. The composition proposed by the present invention designs functional interactions between multiple existing drugs to synergistically interfere with the growth mechanism of cancer cells. The composition proposed by the present invention is simple to prepare, inexpensive, and can show statistically significant efficacy in inhibiting the growth of malignant tumors. In addition, the components of the composition proposed by the present invention can also be administered separately and show the efficacy of inhibiting cancer cells in individuals. The composition of the present invention can be used as a drug for treating animal cancer, a pharmaceutical composition for treating animal cancer, or a health food for promoting animal health.

[0009] In one aspect, the present invention first provides a composition for use in animals, comprising at least two of (a) a cancer cell mitochondrial targeting agent, (b) an ion chelator, and (c) a pharmacologically active multivalent ion. The cancer cell mitochondrial targeting agent is niclosamide. The ion chelator is disulfiram. The pharmacologically active multivalent ion is selected from the group consisting of magnesium ion, calcium ion, manganese ion, ferrous ion, copper ion, and zinc ion. The weight ratio of (a) is 22.0% to 98.4% of the total weight of the pharmaceutical composition, the weight ratio of (b) is 37.9% to 98.8% of the total weight of the pharmaceutical composition, and the weight ratio of (c) is 0.69% to 64.5% of the total weight of the pharmaceutical composition.

[0010] In one embodiment of the present invention, the dosage of (a) in the combination of (a) + (b) is 800 mg / Kg to 1.67 mg / Kg per administration, and the dosage of (b) per administration is 1,376 mg / Kg to 2.14 mg / Kg per administration.

[0011] In another embodiment of the present invention, the dosage of (a) in the combination of (a) + (c) is 800 mg / Kg to 1.67 mg / Kg, and the dosage of (c) is 1,456 mg / Kg to 2.65×10 -2 mg / Kg.

[0012] In another embodiment of the present invention, the dosage of (b) in the combination of (b) + (c) is 1,376 mg / Kg to 2.14 mg / Kg, and the dosage of (c) is 1,456 mg / Kg to 2.65×10 -2 mg / Kg.

[0013] In another embodiment of the present invention, the dosage of (a) in the combination of (a) + (b) + (c) is 800 mg / Kg to 1.67 mg / Kg, the dosage of (b) is 1,376 mg / Kg to 2.14 mg / Kg, and the dosage of (c) is 1,456 mg / Kg to 2.65×10 -2 mg / Kg.

[0014] In another aspect, the present invention further provides a use of the aforementioned composition for animals for preparing a medicament for treating or preventing malignant tumors in animals in need thereof.

[0015] In one embodiment of the present invention, the malignant tumor is selected from the group consisting of osteosarcoma, breast tumor, mast cell tumor, melanocytoma, breast cancer, and lung cancer.

[0016] In one embodiment of the present invention, the malignant tumor is selected from the group consisting of canine malignant osteosarcoma, feline mammary tumor, mouse mammary carcinoma, and lung cancer.

[0017] Different animals have different surface areas per unit volume, resulting in different metabolism and, therefore, different dosing ratios. In one embodiment of the present invention, the standard unit of dosage per kilogram is human. This unit can be converted according to the metabolic ratio of each animal. The kilogram range is 0.01 kg to 60 kg. (a) The dosage per kilogram is 800 mg to 1.67 mg per dose, (b) The dosage per kilogram is 1,376 mg to 2.14 mg per dose, and (c) The dosage per kilogram is 1,456 mg to 2.65 × 10 -2 mg.

[0018] In one embodiment of the present invention, the administration order of (b) is no later than the administration order of (c).

[0019] In one embodiment of the present invention, the drug is administered orally, by injection, transdermally, or by inhalation.

[0020] In another aspect, the present invention further provides a use of the aforementioned composition for animals for preparing food for promoting animal health, wherein the food may be in the form of a health food or a healthy food.

Brief Description of the Drawings

[0021] FIG1 shows the correlation between the inhibitory efficacy of the pharmaceutical composition of the present invention and a conventional targeted drug group on the tumor volume of canine osteosarcoma cancer cells in animals and the administration time.

[0022] FIG2 shows the correlation between the inhibitory efficacy of the pharmaceutical composition of the present invention and the conventional targeted drug group on the tumor weight of canine osteosarcoma cancer cells in animals and the administration time.

[0023] [Implementation Method]

[0024] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the present invention. Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0025] Animals: Any male or female of the orders Carnivora, Lagomorpha, Rodentia, or Primates (except Homo sapiens).

[0026] Malignant tumor (tumor, neoplasm): also known as cancer, is a disease of abnormal cell growth and division, characterized by the potential for unlimited cell proliferation, as well as a tendency to invade surrounding tissues and organs and metastasize to other sites. Malignant tumors are complex diseases that may involve multiple genetic mutations and molecular mechanisms. Early detection, accurate diagnosis and multidisciplinary treatment strategies are essential to controlling the development of malignant tumors. Treatment methods may include surgical resection, radiotherapy, chemotherapy, targeted therapy and immunotherapy, depending on the patient's specific situation and cancer type. Malignant tumors are different from benign tumors, which are usually localized in growth, rarely invade adjacent tissues, and rarely metastasize to other sites.

[0027] The main characteristics of malignant tumors include:

[0028] 1. Unlimited proliferation: The cells of malignant tumors lose the restrictions on the growth of normal cells. They can continue to divide and proliferate indefinitely to form tumor tissue.

[0029] 2. Infiltration and invasion: Malignant tumor cells can invade surrounding normal tissues and organs, destroying their structure and function. This invasiveness can make surgical resection difficult and increase the challenge of treatment.

[0030] 3. Metastasis: Malignant tumor cells sometimes spread to other parts of the body through the blood or lymphatic system, forming metastatic tumors (also called uterine tumors). This can cause cancer to appear in multiple parts of the body at the same time, increasing the difficulty of treatment.

[0031] 4. Abnormal cell morphology: The cells of malignant tumors are often different in morphology from normal cells. They may be irregular, deformed, and of uneven size.

[0032] 5. Cellular heterogeneity: Within the same malignant tumor, the properties and characteristics of cells may vary, which is called cellular heterogeneity. This makes treatment more complicated because different cell subpopulations may respond differently to treatment.

[0033] 6. Angiogenesis: Malignant tumors usually induce the formation of new blood vessels to provide them with nutrients and oxygen, thereby maintaining their continued growth.

[0034] Drug synergy: Also known as drug synergy, it refers to the effect of two or more drugs when used together that exceeds the sum of the effects of each drug used alone. In drug treatment, drug synergy can lead to stronger, longer-lasting or wider therapeutic effects, while potentially reducing the side effects that may occur when using a single drug. Drug synergy can be observed in different types of drugs, including antibiotics, anticancer drugs, antiviral drugs, etc. However, drug synergy is not applicable to all drugs and all diseases, and the interactions between drugs can be complex and varied. The meaning of drug synergy can be described more specifically as follows:

[0035] 1. Enhanced efficacy: Drug synergy may lead to enhanced therapeutic effects. This means that when multiple drugs are given, their combined effect may be more powerful than that of a single drug, thereby more effectively controlling or treating the disease.

[0036] 2. Reduce the risk of drug resistance: Frequent use of a single drug can lead to the development of drug resistance in bacteria, viruses, or tumors. By using multiple drugs with different mechanisms, the risk of the disease developing resistance to any one drug can be reduced, thereby maintaining the effectiveness of treatment.

[0037] 3. Reduce side effects: In some cases, using low doses of multiple drugs can replace the use of high doses of a single drug, thereby reducing the possibility of adverse side effects.

[0038] 4. Multi-target intervention: Different drugs may exert their effects by affecting different targets or pathways of the disease, intervening in the disease through multiple pathways, increasing the diversity and comprehensiveness of treatment.

[0039] 5. Addressing Complex Diseases: Some complex diseases may involve multiple pathological mechanisms, making it difficult for a single drug to fully address them. Drug synergy can impact different aspects of the disease at multiple levels, leading to more effective responses to complex diseases.

[0040] Hematological malignancies refer to diseases that arise from the abnormal proliferation of white blood cells and can be divided into leukemia or lymphoma.

[0041] Cancer cell lethality rate: refers to the percentage of cancer cells in a cancer patient's body that are killed by treatment methods (such as chemotherapy, radiotherapy, targeted therapy, etc.) within a certain period of time. It is often used to evaluate the effectiveness of treatment methods to understand the impact of treatment on cancer conditions. The meaning of cancer cell lethality rate can be explained as follows:

[0042] Treatment efficacy: Cancer cell lethality is one of the important indicators for evaluating the impact of treatment on cancer. A higher lethality generally means that the treatment is more effective in killing cancer cells.

[0043] Disease control: By monitoring the cancer cell lethality rate, we can understand whether treatment is effectively controlling or reducing the spread and progression of cancer. If the lethality rate is low, it may be necessary to re-evaluate the treatment to achieve better control.

[0044] Predicting patient prognosis: A higher cancer cell lethality rate may be associated with improved patient prognosis. Effective cancer cell killing may reduce the risk of disease recurrence and progression, thereby improving patient survival.

[0045] Treatment selection: Cancer cell lethality can help medical professionals evaluate the pros and cons of different treatments. Treatment selection is often based on factors such as treatment lethality, possible side effects, and drug resistance.

[0046] Treatment Monitoring: Tracking cancer cell lethality can help healthcare professionals monitor the progress of treatment. If lethality does not improve significantly over time, adjustments to the treatment regimen may need to be considered. The term "treat" includes the partial or complete prevention, amelioration, alleviation, and / or management of cancer-related symptoms, secondary disorders, or conditions. As used herein, the term "treat" also refers to the application or administration of one or more agents described herein to a subject suffering from a cancer-related symptom, secondary disorder, or condition to partially or completely alleviate, slow down, cure, delay the onset of, inhibit the progression of, reduce the severity of, and / or reduce the occurrence of one or more cancer-related symptoms, signs, or conditions. Cancer-related symptoms, secondary disorders, and / or conditions include, but are not limited to, fever, weakness, fatigue, weight loss, pain, cough, bleeding, skin changes, diarrhea or constipation, nausea, vomiting, and loss of appetite. "Treatment" herein may also refer to administration to a subject with early signs or symptoms of a cancer to reduce the subject's risk of developing signs, secondary signs, and / or symptoms associated with the cancer. "Treatment" herein means effectively reducing one or more signs or clinical markers. In other words, treatment herein may also refer to reducing, slowing, or halting the progression of the disease, signs, or symptoms.

[0047] As used herein, an "effective amount" refers to an amount of a drug sufficient to produce a desired therapeutic response. An effective amount also refers to a compound or composition whose therapeutic benefit outweighs any toxic or deleterious effects. The specific effective amount depends on a variety of factors, such as the specific condition being treated, the patient's physiological condition (e.g., weight, age, or sex), the type of mammal or animal being treated, the duration of treatment, the nature of the current therapy (if applicable), the specific formulation used, and the structure of the compound or derivative thereof. For example, an effective amount can be expressed as the total weight of the drug (e.g., in grams, milligrams, or micrograms) or as the ratio of drug weight to body weight (in milligrams / kilogram (mg / kg)). Alternatively, an effective amount can be expressed as the concentration of the active ingredient (e.g., an agent of the present invention), such as molar concentration, weight concentration, volume concentration, weight molar concentration, molar fraction, weight fraction, and mixture ratio. Specifically, the term "therapeutically effective amount" refers to an amount of an agent administered that is sufficient to slow or alleviate symptoms associated with cancer in a subject. Those skilled in the art can use the body surface area method (BSA) to calculate the human equivalent dose (HED) for different animal species, such as the agents described herein. The BSA method is a common method for converting doses between species. This method assumes that drug dose is proportional to body surface area, not body weight.

[0048] As used herein, the term in vitro refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in cell culture, etc., rather than in an organism.

[0049] As used herein, the term "in vivo" or "in vivo" refers to events that occur within cells within an organism, such as a non-human animal. In cell-based descriptions, this may refer to events that occur within living cells (as opposed to, for example, in vitro systems).

[0050] As used herein, the term "subject" may refer to an organism to which a subject is administered, for example, for experimental, diagnostic, preventive, cosmetic, and / or therapeutic purposes. Typical subjects may be female or male animals of the orders Carnivora, Lagomorpha, Rodentia, and Primates (excluding Homo sapiens), e.g., mammals such as dogs, cats, mice, rats, rabbits, and non-human primates. In some embodiments, the subject may be a dog, a cat, a lagomorph, or a rodent.

[0051] As used herein, the term pharmaceutically acceptable or pharmacologically active may refer to a substance that is suitable for use in contact with the tissues of animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment.

[0052] As used herein, the term effective amount may refer to an amount sufficient to prevent and / or delay the onset of symptoms when administered to an individual. One of ordinary skill in the art will appreciate that an effective dose is typically administered via a dosing regimen comprising at least one unit dose.

[0053] Although the numerical ranges and parameters used to define the broader scope of the present invention are approximate, the numerical values ​​in the specific embodiments have been presented as precisely as possible. Unless otherwise specified, the numerical ranges described herein include endpoints.

[0054] In this disclosure, the terms "cycle," "cycle of treatment," or "treatment cycle" are used interchangeably to refer to the period of time during which treatment is administered to a subject. Generally, in cancer treatment, a complete treatment cycle is followed by a rest period in which no treatment is administered. Following this rest period, one or more treatment cycles may be administered, each followed by another rest period.

[0055] Drug repurposing involves repurposing existing drugs for new therapeutic applications based on those drugs' approval for other treatments. This approach can reduce the cost and time required for development, as well as the risk of adverse drug reactions after marketing.

[0056] In cancer treatment, drug repurposing combined with drug combination strategies has significant advantages over traditional new targeted drugs for the following reasons:

[0057] 1. Old drugs utilize new drug combination strategy models to generate new applications for the treatment of malignant tumors. Drug combinations facilitate interactions between different drugs.

[0058] 2. Drug combinations can exert synergistic effects between drugs and interfere with the growth mechanism of cancer cells.

[0059] 3. Use old clinical drugs that have been used for a long time to find new therapeutic applications. These drugs have rich clinical data and relatively high safety.

[0060] 4. Inhibit multiple signaling pathway molecules related to cancer through different pharmacological mechanisms.

[0061] 5. Improve the therapeutic effect while reducing the dosage of individual drugs, making the treatment safer.

[0062] 6. The cost of treating malignant tumors with combination drugs is much lower than that with targeted drugs.

[0063] [Corrected 22 / 10 / 2024 per Regulation 26] Currently, Taiwan has no officially approved medications for the prevention and treatment of animal cancer. Consequently, animal owners must pay high treatment costs for their pets, ranging from NT$150,000 to NT$30,000, depending on the treatment method. This burden leads many owners to forgo treatment. Even if pets undergo cancer treatment, they are unable to express their true discomfort like humans do, which increases their risk of death and the time and cost of treatment and care for their owners. Therefore, there is a need for cancer treatment drugs specifically for animals that owners are willing to use and can easily afford.

[0064] Unexpectedly, according to various embodiments of the present invention, the inventors of this case have provided a composition effective against malignant tumors in animals. The active ingredients in the composition of this invention are existing, known drugs with extensive clinical data and relatively high safety profiles. Compared to targeted therapies, this approach, which combines existing drugs with a drug combination strategy, offers significant advantages.

[0065] The present invention first provides a composition comprising at least two of (a) a cancer cell mitochondrial targeting agent, (b) an ion chelator, and (c) a pharmacologically active multivalent ion. At least two of these three drugs can form a drug combination, and the ratio of at least two of these three drugs can be adjusted based on the animal's body weight and therapeutic effect. The therapeutic dosage of drugs (a), (b), and (c) is expressed in milligrams (mg).

[0066] In embodiments of the present invention, the standard unit of dosage per kilogram of human body weight is used. The dosage per kilogram of body weight can be converted based on the metabolic rate of each animal to obtain the dosage per kilogram of body weight for each animal. For example, the dosage conversion coefficients per kilogram of body weight for Carnivora, Rodentia, and Lagomorpha are as follows:

[0067] 1. The drug dosage is based on humans, and the body weight is calculated based on 60 kg.

[0068] 2.

[0069] 3.

[0070] 4.

[0071] 5.

[0072] 6.

[0073] The targeting agent for cancer cell mitochondria can be niclosamide. Niclosamide can target mitochondria in cancer cells to induce cell cycle arrest, inhibit cell growth and induce cell apoptosis. In GlobalData data, no company has developed niclosamide to enter the cancer treatment market for single use in the treatment of cancer. The amount of (a) is 22.0% to 98.4% by weight based on the total weight of the pharmaceutical composition. The dosage range of niclosamide is (calculated based on human drug dosage standards) daily use for seven days, not more than 2 times a day. The therapeutic dose range of niclosamide is from 800 mg / Kg to 1.67 mg / Kg. The upper limit dose is the known lethal dose value (LD 50 ), data comes from HAZARDOUS SUBSTANCESDATA BANK (HSDB).

[0074] The ion chelator can be disulfiram. Disulfiram can induce endoplasmic reticulum stress and autophagy to induce cell apoptosis, reduce angiogenesis, and can be used as a sensitizer for radiotherapy, or an ion chelate. In terms of single use for the treatment of cancer, no company has completed the development of disulfiram to enter the cancer treatment market. The amount of (b) is 37.9% to 98.8% by weight based on the total weight of the pharmaceutical composition. The therapeutic dose range of disulfiram (calculated based on human drug dosage standards) is from 1,376 mg / Kg to 2.14 mg / Kg. The upper limit dose is the known lethal dose (LD 50 ), data are from The Toxin and Toxin Target Database (T3DB).

[0075] Pharmacologically active multivalent ions are metal ions and their salts in the form of pharmaceutically acceptable nutritional additives, such as chloride, oxygen, hydroxide, gluconate, sulfate, glycine, succinate, stearate, glycerophosphate, acetate, lactate, phosphate, borate, carbonate, fumarate, glutarate, glycerophosphate, fructose borate, bile acid, malate, glycerate, lactate, oxalate, carbonate, tartrate, citrate, stearate, HAP chelate, HVP chelate, and yeast, etc., but the present invention is not limited thereto. Multivalent active ions are typical elemental ions with a valence of two or more, or transition element ions. Pharmacologically active multivalent ions are, for example, at least one of magnesium, calcium, chromium, manganese, ferrous, copper, and zinc. The amount of (c) is 0.69% to 64.5% by weight based on the total weight of the composition. The dosage range of pharmacologically active multivalent ions is 1,456 mg / Kg to 2.65×10 -2mg / Kg. The therapeutic dose range of pharmacologically active multivalent ions is 1,456 mg / Kg to 2.65×10 -2 mg / kg. For example, when used as an ion chelator, two disulfirams can form a pharmacologically active chelate with a pharmacologically active multivalent ion. Table 1 lists the dosage ranges for different pharmacologically active multivalent ions. The upper limit of ion absorption in animals differs from that in humans, so the upper limit of animal absorption is converted to the upper limit of human absorption per kilogram. Data are sourced from HSBD, T3DB, DrugBank, and other sources.

[0076] Table 1 Human dose ranges per kilogram for various pharmacologically active polyvalent gluconate ions (data source calculated using rodent body weight dose conversion factors)

[0077] The active ingredients in the composition of the present invention, such as component (a), component (b) and component (c), can be formulated in different weight dosage ratio ranges or combinations as needed.

[0078] In one embodiment of the present invention, the dosage ratio of (a) to (b) can be Proportional range.

[0079] In another embodiment of the present invention, the dosage ratio of (a) to (c) can be scale range.

[0080] In another embodiment of the present invention, the dosage ratio of (a) to (b) + (c) can be Proportional range.

[0081] In another embodiment of the present invention, the dosage ratio of (a) + (b) to (c) can be Proportional range.

[0082] In another embodiment of the present invention, the dosage ratio of (a) + (c) to (b) can be Proportional range.

[0083] In another embodiment of the present invention, the dosage ratio of (b) to (a) can be Proportional range.

[0084] In another embodiment of the present invention, the dosage ratio of (b) to (c) can be Proportional range.

[0085] In another embodiment of the present invention, the dosage ratio of (b) to (a) + (c) can be Proportional range.

[0086] In another embodiment of the present invention, the dosage ratio of (b) + (c) to (a) can be Proportional range.

[0087] In another embodiment of the present invention, the dosage ratio of (c) to (a) can be Proportional range.

[0088] In another embodiment of the present invention, the dosage ratio of (c) to (b) can be Proportional range.

[0089] In another embodiment of the present invention, the dosage ratio of (c) to (a) + (b) can be Proportional range.

[0090] The composition of the present invention can produce a corresponding cancer cell lethality effect within the above dosage ratio range of (a), (b) and (c).

[0091] In one embodiment of the present invention, when The cancer cell lethality rate in mouse experiments was 42.93%.

[0092] In one embodiment of the present invention, when The cancer cell lethality rate in mouse experiments was 53.39%.

[0093] In one embodiment of the present invention, when The cancer cell lethality rate in mouse experiments was 42.90%.

[0094] In one embodiment of the present invention, when The cancer cell lethality rate in the cell experiment was 62.04%.

[0095] In one embodiment of the present invention, when The cancer cell lethality rate in the cell experiment was 66.02%.

[0096] In one embodiment of the present invention, when The cancer cell lethality rate in the cell experiment was 78.19%.

[0097] In one embodiment of the present invention, when The cancer cell lethality rate in the cell experiment was 79.50%.

[0098] In one embodiment of the present invention, when The cancer cell lethality rate in the cell experiment was 75.85%.

[0099] In one embodiment of the present invention, when The cancer cell lethality rate in cell experiments was 88.47%.

[0100] In one embodiment of the present invention, when The cancer cell lethality rate in cell experiments was 88.72%.

[0101] In one embodiment of the present invention, when The cancer cell lethality rate in cell experiments was 86.97%.

[0102] In one embodiment of the present invention, when The cancer cell lethality rate in cell experiments was 30.75%.

[0103] In one embodiment of the present invention, when The cancer cell lethality rate in the cell experiment was 34.17%.

[0104] In one embodiment of the present invention, when The cancer cell lethality rate in the cell experiment was 35.92%.

[0105] In one embodiment of the present invention, when The cancer cell lethality rate in the cell experiment was 37.05%.

[0106] In one embodiment of the present invention, when The cancer cell lethality rate in cell experiments was 69.87%.

[0107] In one embodiment of the present invention, when The cancer cell lethality rate in the cell experiment was 81.17%.

[0108] In one embodiment of the present invention, when The cancer cell lethality rate in cell experiments was 86.97%.

[0109] In one embodiment of the present invention, when The cancer cell lethality rate in cell experiments was 89.45%.

[0110] In one embodiment of the present invention, when The cancer cell lethality rate in cell experiments was 88.42%.

[0111] In one embodiment of the present invention, when The cancer cell lethality rate in cell experiments was 82.93%.

[0112] In one embodiment of the present invention, when The cancer cell lethality rate in the cell experiment was 77.90%.

[0113] The various pharmaceutical components in the composition of the present invention can be administered in a variety of different sequences, depending on the circumstances. According to embodiments of the present invention, the first agent can be (c) a pharmacologically active multivalent ion, the second agent can be (b) an ion chelator, and the third agent can be (a) a cancer cell mitochondrial targeting agent. The first agent (c) pharmacologically active multivalent ion should be administered no later than the second agent (b) ion chelator, to help the multivalent pharmacologically active ion increase ion concentrations in the animal. The third agent (a) cancer cell mitochondrial targeting agent can be administered in any order without particular limitation.

[0114] In some embodiments of the present invention, the order of administration of the first agent (c), the second agent (b), and the third agent (a) may be as follows:

[0115] 1. First (a), then (c), and then (b).

[0116] 2. First (a) + (c) together, then (b).

[0117] 3. (a) + (c) together, but without (b).

[0118] 4. First (c), then (b), and then (a).

[0119] 5. First (c) + (b) together, then (a).

[0120] 6. (c) + (b) together, but without (a).

[0121] 7. (a), (c), and (b) are administered simultaneously.

[0122] 8.(a) + (b) together, but without (c).

[0123] 9. (a) + (b) together, then (c).

[0124] 10. First (c), then (a), then (b).

[0125] If necessary, the pharmaceutical composition of the present invention may further comprise one or more stabilizers, binders, fillers, disintegrants, excipients, and additives.

[0126] The composition of the present invention can also be made into a pharmaceutical composition or pharmaceutical kit suitable for different dosing orders, or used to prepare food that promotes animal health, and the food form can be a health food or a health food. For example, the pharmaceutical composition of the present invention is made into a pharmaceutical kit for treating an individual suffering from or suspected of suffering from cancer. The pharmaceutical kit of the present invention comprises a first container, a second container as needed, a third container as needed, or a fourth container as needed. The first container, the second container as needed, and the third container as needed respectively contain at least one of the first agent, the second agent, and the third agent. According to the embodiment of the present invention, the first agent may be (c) a pharmacologically active multivalent ion, the second agent may be (b) an ion chelator, and the third agent may be (a) a targeting agent for cancer cell mitochondria.

[0127] Another aspect of the present invention relates to a method for treating a subject suffering from or suspected of suffering from cancer using the pharmaceutical kit of the present invention. The method comprises administering to a subject a first container of a pharmaceutical agent of the present invention; optionally administering to the subject a second container of a pharmaceutical agent of the present invention; and optionally administering to the subject a third container of a pharmaceutical agent of the present invention. The first container comprises at least one of a first agent, a second agent, and a third agent. The second container optionally comprises at least one of the first agent, the second agent, and the third agent. The third container optionally comprises at least one of the first agent, the second agent, and the third agent. According to embodiments of the present invention, the first agent may be (c) a pharmacologically active multivalent ion, the second agent may be (b) an ion chelator, and the third agent may be (a) a cancer cell mitochondrial targeting agent.

[0128] The first effective amount of the pharmacologically active multivalent ion of the first agent (c) of the present invention may be in the range of 1,456 mg / Kg to 2.65×10 -2 The second agent is (b) an ion chelator, and the second effective amount can range from 1,376 mg / Kg to 2.14 mg / Kg. The third effective amount of the third agent of the present invention comprises (a) a cancer cell mitochondrial targeting agent, and the third effective amount can range from 800 mg / Kg to 1.67 mg / Kg.

[0129] A first container, optionally a second container, and optionally a third container suitable for holding the agent of the present invention can be made of various materials, such as glass, liposomes, or plastic. The first container can hold an effective amount of the agent of the present invention or a pharmaceutical dosage form thereof. The second container can hold an effective amount of the agent of the present invention or a pharmaceutical dosage form thereof. The third container can hold an effective amount of the agent of the present invention or a pharmaceutical dosage form thereof. The pharmaceutical kit of the present invention may further include instructions for use accompanying the first container and / or the second container and / or the third container. Furthermore, the pharmaceutical kit of the present invention may optionally include a fourth container containing a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It may also include other materials required by the commercial or user, including other buffers, diluents, filters, needles, and syringes.

[0130] Another aspect of the present disclosure relates to a method for treating a subject in need thereof (e.g., a subject suffering from cancer or suspected of suffering from cancer) using the pharmaceutical kit of the present invention. This method comprises administering to the subject a first effective amount of a first container of the present invention; and optionally administering to the subject a second effective amount of a second container of the present invention; or optionally administering to the subject a third effective amount of a third container of the present invention. The first administration of the first container to the subject may be no later than the second administration of the second container to the subject, and the third administration of the third container to the subject may be no earlier than the second administration of the second container to the subject. The medication in the pharmaceutical kit of the present invention may be administered no more than twice daily.

[0131] At the first administration time point, the first container of the present invention is administered to the subject in the form of liposomes, capsules or tablets. At the second administration time point, the second container of the present invention is administered to the subject in the form of liposomes, capsules or tablets. At the third administration time point, the third container of the present invention is administered to the subject in the form of liposomes, capsules or tablets. According to some embodiments, the subject is an animal excluding humans. The different surface areas per unit volume of various animals result in different metabolism, and thus different dosing ratios. In one embodiment of the present invention, the standard unit of dosage is human per kilogram, which can be converted into the dosage of the subject according to the metabolic ratio of each animal. The subject is administered 1,456 mg to 2.65×10 -2In one embodiment, a first agent is administered to a subject at a dose of 1,376 mg to 2.14 mg of a second agent per kg of subject's body weight per day, and a third agent is administered to the subject at a dose of 800 mg to 1.67 mg per kg of subject's body weight per day. According to one working example of the present invention, administration of 21.98 mg to 10.98 mg / kg / day of the pharmaceutical composition of the present invention is sufficient to reduce the number of cancer cells in the subject, thereby demonstrating the anti-cancer efficacy of the pharmaceutical composition of the present invention.

[0132] In some embodiments of the present invention, the first container contains the third agent, the second container contains the first agent, and the third container contains the second agent. At a first administration time point, the subject is first administered the agent of the present invention in the first container; at a second administration time point, the subject is administered the agent of the present invention in the second container; and at a third administration time point, the subject is administered the agent of the present invention in the third container, such that the order of drug administration in a method of treating a subject suffering from or suspected of suffering from cancer using the pharmaceutical kit of the present invention is (a) first, then (c), and then (b).

[0133] In some embodiments of the present invention, the first container contains the first agent, the second container contains the second agent, and the third container contains the third agent. At a first administration time point, the subject is first administered the agent of the present invention in the first container; at a second administration time point, the subject is administered the agent of the present invention in the second container; and at a third administration time point, the subject is administered the agent of the present invention in the third container, such that the order of drug administration in a method of treating a subject suffering from or suspected of suffering from cancer using the pharmaceutical kit of the present invention is first (c), then (b), and then (a).

[0134] In some embodiments of the present invention, the first container contains the first agent and the third agent, and the second container contains the second agent. At a first administration time point, the subject is administered the agent of the present invention in the first container first; and at a second administration time point, the subject is administered the agent of the present invention in the second container, such that the order of drug administration in a method of treating a subject suffering from or suspected of suffering from cancer using the pharmaceutical kit of the present invention is to administer (a) and (c) simultaneously before administering (b).

[0135] In some embodiments of the present invention, the first container contains the first agent and the second agent, and the second container contains the third agent. At a first administration time point, the subject is administered the agent of the present invention in the first container first; and at a second administration time point, the subject is administered the agent of the present invention in the second container, such that the order of drug administration in a method of treating a subject suffering from or suspected of suffering from cancer using the pharmaceutical kit of the present invention is to administer (c) and (b) simultaneously before administering (a).

[0136] In some embodiments of the present invention, the first container contains a first agent, a second agent, and a third agent. At a first administration time point, the first container of the present invention is administered to the subject, such that the sequence of drug administration in the method of treating a subject suffering from or suspected of suffering from cancer using the pharmaceutical kit of the present invention is simultaneous administration of (a), (b), and (c).

[0137] The release form of the composition of the present invention can be formed into a double-layer or triple-layer tablet. The inner layer tablet is formed by combining a sustained-release excipient with the drug, with the drug release rate being fastest in the outermost layer and decreasing toward the inner layers. The outer layer tablet is formed by combining a fast-disintegrating excipient with the preferentially released drug and the inner layer tablet. The different disintegration rates can be utilized to allow pharmacologically active multivalent ions to enter the body, increase their concentration, and then chelate with the ion chelator released after disintegration to produce a chelating effect.

[0138] Excipients for rapidly disintegrating release dosage forms

[0139] Rapid disintegration release dosage forms of drugs are intended to release the drug quickly to achieve rapid drug effect. In some embodiments of the present invention, excipients can be used to prepare rapid disintegration release dosage forms, such as but not limited to:

[0140] 1. Lactose: Lactose is a common auxiliary excipient commonly used in the preparation of oral tablets. It has excellent tableting properties and rapid disintegration characteristics, which can promote the rapid release of drugs.

[0141] 2. Corn starch: Corn starch is a common natural polysaccharide excipient with good compressibility and disintegration properties. It can be used to prepare rapidly disintegrating tablets and granules.

[0142] 3. Microcrystalline cellulose: Microcrystalline cellulose is a finely crystalline excipient obtained by processing cellulose and is commonly used in the preparation of oral tablets. It can increase the mechanical strength of tablets and promote the rapid release of drugs.

[0143] 4. Water-soluble polymers: Some water-soluble polymers, such as hydroxypropyl methylcellulose (HPMC) and polyethylene glycol (PEG), can be used as disintegrants in the preparation of rapidly disintegrating dosage forms. They interact rapidly with water, promoting rapid tablet disintegration and drug release.

[0144] 5. Protein excipients: Certain proteins, such as gelatin and sodium starch glycolate, can rapidly swell and disintegrate under wet conditions, thereby achieving rapid drug release. These excipients can be used to prepare various rapidly disintegrating dosage forms, such as rapidly disintegrating tablets, disintegrating granules, and disintegrating capsules.

[0145] Excipients for sustained-release dosage forms

[0146] Sustained-release formulations of drugs often use special excipients. In some embodiments of the present invention, such excipients can prolong the release time of the drug in vivo, allowing the drug to enter the blood or target tissue at a steady rate, for example but not limited to:

[0147] 1. Polymers: Polymers are common excipients, such as polyvinyl glycol (PEG) and poly(lactic-co-glycolic acid) (PLGA). These polymers can form drug carriers, allowing the drug to be released at a controlled rate.

[0148] 2. Silicates: Silicate excipients, such as silica dioxide and aluminum hydroxide, can delay the release rate of drugs by forming a drug coating layer or network structure.

[0149] 3. Polylactic acid (PLA) and polylactic-co-glycolic acid (PLGA) microspheres: These microspheres can be injected or taken orally as carriers for sustained-release dosage forms. The size and structure of the microspheres can be adjusted to meet specific needs, thereby controlling the release rate of the drug.

[0150] 4. Liposomes: Liposomes are tiny particles composed of a lipid bilayer that can encapsulate drugs and form a stable carrier. Liposomes can be used for oral administration, injection, or topical application to achieve slow release of drugs.

[0151] 5. Polymerized liposomes: Polymerized liposomes are an improved version of liposomes. By adding polymers or high molecular substances, the release rate and stability of drugs can be further adjusted.

[0152] The release form of the composition of the present invention is not limited to the aforementioned excipients and techniques for preparing sustained-release dosage forms of the drug. Suitable excipients can be selected based on factors such as the characteristics of the drug, therapeutic needs, and route of administration.

[0153] In some embodiments of the present invention, the drugs of the present invention may have different effects on cancer or tumor cells under the influence of neutral, negatively charged, or positively charged liposomes. The differences in the effects of liposomes with different charges on cells are discussed in two aspects:

[0154] 1. Liposomes interact with cells.

[0155] 2. The effect of liposomes on cell (drug) delivery.

[0156] These differences and their impacts are described in the following aspects.

[0157] Interaction Mechanism: Positively charged liposomes experience an electrostatic attraction with the negatively charged cell membrane, making it easier for positively charged liposomes to bind to the cell membrane, while negatively charged liposomes are less likely to do so. Neutral liposomes exhibit no significant electrostatic interaction. This interaction mechanism determines the affinity and uptake efficiency of liposomes of varying charge levels.

[0158] Cellular Uptake Efficiency: Positively charged liposomes enhance cellular uptake due to their interaction with the negatively charged cell membrane. Cellular uptake primarily occurs through endocytosis, where positively charged liposomes bind to the cell membrane and can be actively or passively taken up into the cell. In contrast, negatively charged liposomes have a relatively low uptake efficiency due to electrostatic repulsion from the cell membrane.

[0159] Intracellular Delivery Efficiency: Positively charged liposomes facilitate the release and delivery of cargo, such as drugs or genes, into cells because they interact with negatively charged molecules within the cell, such as nucleic acids and proteins. Negatively charged liposomes can also deliver drugs or cargo to a certain extent, but compared to positively charged liposomes, negatively charged liposomes are better suited for stabilizing and protecting drugs and less suitable for direct intracellular delivery. Neutral liposomes lack charge interactions with cell membranes, thus, under certain parameters, reducing nonspecific interactions with cells and the likelihood of adsorption and phagocytosis. This contributes to improved in vivo stability and bioavailability of liposomes. They also typically possess a higher drug loading capacity, enabling more efficient encapsulation and stabilization of various drug types (both water-soluble and lipid-soluble).

[0160] In some embodiments of the present invention, positively charged liposomes, such as but not limited to DOTAP (docosatetraenoic acid trimethylammonium bromide), DDAB (docosatetraenoic acid docosatetraenoic acid ammonium salt), DPTAP (docosatetraenoic acid triphenylphosphonate), DMTAP (docosapentaenoic acid dimethylphosphonate), DMTMA (docosapentaenoic acid dimethyltrimethylammonium bromide), DC-Chol (docosacholine carbonate), DOTMA (docosatetraenoic acid trimethylammonium salt), DPTMA (docosatetraenoic acid triphenylammonium salt), DPTTA (docosatetraenoic acid triphenyltriethylammonium salt), and DMRIE (dimethyldeoxycholine docosapentaenoate).

[0161] In some embodiments of the present invention, negatively charged liposomes, such as but not limited to DOPC (docosahexaenoic acid phosphatidylcholine), DOPS (phosphatidylserine), DOPG (docosahexaenoic acid phosphatidylglycerol), DOPA (docosahexaenoic acid phosphatidylserine), DOPS-Na (phosphatidylserine sodium salt), POPG (docosahexaenoic acid phosphatidylserine) SOPC (phosphatidylserine alcoholamine), SOPG (phosphatidylserine alcoholglycerol), POPE (phosphatidylserine alcohol phosphatidylcholine), POPA (phosphatidylserine alcohol phosphatidylserine).

[0162] In some embodiments of the present invention, the electrically neutral liposomes include, but are not limited to, DSPC (docosylphosphatidylcholine), DMPC (docosylphosphatidylglycerol), DPPC (docosylphosphatidylcholine), DOPC (docosahexaenoic acid phosphatidylcholine), DPPG (docosylphosphatidylglycerol), DOPG (docosahexaenoic acid phosphatidylglycerol), DPPA (docosylphosphatidylserine), DOPA (docosahexaenoic acid phosphatidylserine), DMPE (docosylphosphatidylglycerolamine), and DMPS (docosylphosphatidylglycerol thiol).

[0163] In some embodiments of the present invention, other ingredients used to prepare liposomes include, but are not limited to, cholesterol, polyethylene glycol (PEG), surfactants (such as Tween, Span), adjuvants (such as egg phosphatidylserine, egg phosphatidylcholine), liposome coating agents (such as egg phosphatidylcholine), targeting ligands (such as antibodies, ligands), dyes (such as fluorescent dyes, nucleic acid dyes), and liposome stabilizers (such as liposome structure regulators, preservatives).

[0164] In some embodiments of the present invention, drugs can be encapsulated using small and large spheres. For example, small sphere liposomes can first encapsulate two drugs: an ion chelator and a cancer cell mitochondrial targeting agent. Then, large sphere liposomes can encapsulate the small sphere liposomes and the pharmacologically active multivalent ions, forming a two-layer liposome. The large spheres can disintegrate first, releasing the pharmacologically active multivalent ions to increase their concentration. The small spheres then disintegrate to release the ion chelator and cancer cell mitochondrial targeting agent. The ion chelator can then react with the previously released pharmacologically active multivalent ions to produce a therapeutic effect.

[0165] In some embodiments of the present invention, bi- or tri-layer liposomes refer to processes that utilize bi- or tri-layer liposomes to simultaneously encapsulate and deliver three different drugs, employing a multi-layer lipid structure. For example, this can enhance the stability of individual drugs: each drug is encapsulated within its own liposome layer, protecting it from degradation, chemical interactions, and enzymatic degradation; or control drug release: the bi-layer liposome structure allows for the controlled release of each drug. By adjusting the composition and structure of the liposomes, the independent release of each drug can be achieved, allowing precise control of the release rate and timing. This helps maintain drug stability and integrity during storage and transportation. This is applicable to drug delivery strategies that enhance drug stability, reduce the likelihood of drug degradation or inactivation, and improve drug bioavailability. Therefore, by adjusting the lipid composition of the inner and outer layers, bi- or tri-layer liposomes can facilitate the control and regulation of drug delivery processes. For example, by enabling the drug to disintegrate before allowing pharmacologically active multivalent ions to enter the body and increase their concentration, which can then interact with chelators released later to produce chelating effects. Therefore, using liposomes of varying sizes for encapsulation is a strategy that can achieve gradual drug release. By first encapsulating one drug with small liposomes, and then encapsulating the pharmacologically active multivalent ions and small liposomes with larger liposomes, the larger liposomes disintegrate first during the release process, allowing the pharmacologically active multivalent ions within them to increase their concentration in the body. These ions then react with the ion chelators released by the smaller liposomes, which then disintegrate later. This delays drug release, optimizing therapeutic efficacy and mitigating side effects.

[0166] Exemplary cancers suitable for the pharmaceutical kits and / or compositions of the present invention include malignant tumors, such as solid tumors or hematological malignancies. Examples of suitable cancers include, but are not limited to, lymphoma, mast cell tumor, osteosarcoma, breast tumor, melanoma, angiosarcoma, adenocarcinoma, thyroid tumor, bladder cancer, liver cancer, brain tumor, squamous cell carcinoma, kidney tumor, breast tumor, intestinal tumor, bladder cancer, and leukemia. According to certain embodiments of the present invention, the cancer is resistant to chemotherapy, radiation therapy, or immunotherapy.

[0167] Exemplary administration routes of the pharmaceutical kits and / or pharmaceutical compositions of the present invention include, but are not limited to, enteral, oral, nasal, parenteral, topical, or transmucosal administration, wherein parenteral administration may be intratumoral, intramuscular, intravenous, intraarterial, subcutaneous, intraperitoneal, intracranial, intraventricular, or intrathecal injection.

[0168] Experimental procedures

[0169] Animal experimental materials and methods

[0170] experimental animals

[0171] [Corrected 22 October 2024 according to Regulation 26] Five-week-old female Balb / c CAnN.Cg-Foxnlnu / CrlNarl nude mice were purchased from the Animal Experimentation Center in Taiwan, China. They were given water and chow (LabDiet 5058, PMI Nutrition International Inc, MO, USA; 21.56% of energy from fat, metabolizable energy 3.46 kcal / gm) daily ad libitum. The temperature was maintained at 22 ± 2°C, the humidity at 55 ± 15%, and the light / dark cycle was 12 / 12 h (lights on at 1:00 AM, lights off at 1:00 PM). Experiments were initiated after the mice were raised for 6 weeks of age.

[0172] Induced cancer tumor animal models

[0173] Six-week-old nude mice were used to establish a malignant osteosarcoma animal model. This experiment used canine osteosarcoma cancer cells provided by the American Type Culture Collection (ATCC). The induction method was to add 1×10 7 100 μL of osteosarcoma cell suspension was subcutaneously injected into the dorsal part of the right hind limb. Two weeks after the administration of osteosarcoma cells, when the tumor diameter was 5 mm to 6 mm, the mice were grouped and drug administration was started, thus successfully establishing an induced osteosarcoma cell cancer tumor mouse model.

[0174] Administration of the test product

[0175] The drug dosage in the experiment is recorded in the form of human dosage per kilogram, and then the actual drug dosage is obtained through corresponding conversion.

[0176] After establishing osteosarcoma tumors in canine osteosarcoma cancer cells, mice were divided into five groups within each tumor group. These included: S1: Control (no drug treatment), S2: First-dose combination of the present invention (Niclosamide (7.10 mg) + Disulfiram (2.22 mg) + pharmacologically active multivalent ion (1.66 mg)), S3: Second-dose combination of the present invention (Niclosamide (14.21 mg) + Disulfiram (4.44 mg) + pharmacologically active multivalent ion (3.33 mg)), S4: Third-dose combination of the present invention (Niclosamide (14.21 mg) + Disulfiram (4.44 mg)), and S5: Fourth-dose combination of the present invention (Niclosamide (21.64 mg)). Each group received oral administration once daily for 29 consecutive days. In this experiment, the pharmacologically active multivalent ion was copper ion, in the form of copper gluconate.

[0177] Observation of body weight and food intake

[0178] During the experiment, nude mice were given free access to feed and water, and their body weight and food intake were recorded weekly using an electronic scale.

[0179] Tumor volume, weight and inhibition rate measurement and evaluation

[0180] Tumor volume was measured weekly using a tumor measuring instrument (TM900, Peira, Turnhout, Belgium). Five weeks after administration, the nude mice were sacrificed and the tumors on the right hind limbs were removed. The tumor volume was measured by measuring the longest and shortest diameters. The tumor volume was calculated using the formula: volume = 0.5 × longest diameter × shortest diameter. 2 The actual tumor volume was calculated, and the tumor mass was weighed and recorded. The tumor inhibition rate of each group was calculated based on the control group.

[0181] Tumor volume inhibition rate % = 1-(tumor volume of drug group) / (tumor volume of control group) × 100%

[0182] Tumor mass weight inhibition rate % = 1-(tumor weight of drug group) / (tumor weight of control group) × 100%

[0183] Cancer cell test materials and methods

[0184] Cell culture

[0185] Canine osteosarcoma cancer cells, canine melanocytoma cells, feline melanocytoma cells, canine mammary tumor cells, feline mammary tumor cells, mouse mammary cancer cells, or mouse lung cancer cells were cultured in Minimum Essential Medium (MEM) containing glutamine and NEAA, supplemented with 10% (v / v) fetal bovine serum (Fetal Bovine Serum, Penicillin-Streptomycin Solution) (100X) (ACE Biolabs, CC1009). The cells were then placed in a 5% CO2, 37°C incubator (Astec-SCA-165DS).

[0186] Cell Viability

[0187] Different cancer cells were seeded into 96-well plates and allowed to adhere to the plate for 24 hours. The cells were then treated with a combination of three drugs. A control group, which did not receive any drug treatment, was treated 48 hours later. 20 μL of MTS reagent (Progema, USA) was added and the cells were incubated at 37°C for 1 hour. An ELISA reader (BioTeck EPOCH2) was used to measure absorbance at a wavelength of 490 nm to assess cell viability and proliferation. Cell viability was calculated using the following formula:

[0188] Cell viability % = (cell absorbance after adding drug) / (cell absorbance without drug) × 100%

[0189] In some embodiments of the present invention, the experimental dosage concentration of the drug administered to cancer cells is nM, wherein the effective dosage range is 0.25 nM to 1024 nM.

[0190] Example 1: Efficacy of the pharmaceutical composition of the present invention on canine osteosarcoma cancer cells

[0191] To evaluate whether different dosages of the pharmaceutical composition of the present invention could inhibit the survival of canine osteosarcoma cancer cells, we first analyzed the survival rates of canine osteosarcoma cancer cells. The results are listed in Tables 1-3. As shown in Tables 1-3, the survival rates of canine osteosarcoma cancer cells treated with the pharmaceutical composition of the present invention correlated with the dosage. The pharmacologically active copper ion used in this experiment was copper gluconate. Nis: Niclosamide, DSF: Disulfiram, Cu: pharmacologically active multivalent copper ion.

[0192] Table 1. Effect of the present invention's pharmaceutical composition on the survival rate of canine osteosarcoma cancer cells (%)

[0193] Table 2. Effect of the present invention's pharmaceutical composition on the survival rate of canine osteosarcoma cancer cells (%)

[0194] Table 3. Effect of the present invention's pharmaceutical composition on the survival rate of canine osteosarcoma cancer cells (%)

[0195] The experimental results in Tables 1-3 show that when niclosamide, disulfiram, and the pharmacologically active multivalent ion Cu were tested separately on cancer cells, niclosamide and disulfiram alone exhibited an inhibitory effect on cancer cells, while the pharmacologically active multivalent ion Cu exhibited no effective inhibitory effect. The pharmaceutical composition of the present invention exhibited inhibitory effects at various dose combinations. When niclosamide was administered at 3 nM and Cu at 32 nM, the cancer cell survival rate dropped to 39.13%. When niclosamide was administered at 4 nM, disulfiram at 32 nM, and Cu at 32 nM, the cancer cell survival rate dropped to 21.81%. When disulfiram at 32 nM and Cu at 16 nM were administered, the cancer cell survival rate dropped to 76.05%. When the pharmaceutical composition of the present invention was administered to cancer cells at higher drug concentrations, it exhibited significantly higher inhibitory effects on cancer cell survival, with the lowest osteosarcoma cancer cell survival rate reduced to 10.19%. This experiment demonstrates that the pharmaceutical combination of the present invention can produce a higher degree of synergistic efficacy among the three drugs.

[0196] The experimental results show that the pharmaceutical composition of the present invention has the effect of inhibiting the survival rate of animal cancer cells. The inhibition of cancer cell survival rate can be achieved by the different combinations and dosage modes of the pharmaceutical composition of the present invention.

[0197] Example 2: The efficacy of the pharmaceutical composition of the present invention in single use or multiple combination mode in inhibiting tumor growth of canine osteosarcoma cancer cells in animals

[0198] To evaluate whether the pharmaceutical composition of the present invention has an inhibitory effect on canine osteosarcoma cancer cells, whether the drug can effectively inhibit lung cancer tumor growth after absorption through the gastrointestinal tract, and whether the drug will cause discomfort, an experiment was conducted. A tumor model was first established by subcutaneous injection in nude mice. The tumor size was measured after 14 days of observation. The mice were then divided into 5 groups and drug administration began. The drug-treated group was then fed the test drug orally. The changes in tumor growth in the tumor model nude mice were evaluated up to 29 days after administration, and the inhibitory effect of the test drug on the tumor was observed. The results are listed in Tables 4 and 5. Figure 1 shows the correlation between the inhibitory effect of the pharmaceutical composition of the present invention on the tumor volume of canine osteosarcoma cancer cells in animals and the time of administration. Figure 2 shows the correlation between the inhibitory effect of the pharmaceutical composition of the present invention on the tumor weight of canine osteosarcoma cancer cells in animals and the time of administration.

[0199] Table 4. Correlation between tumor volume and dosage of the pharmaceutical composition of the present invention and conventional targeted drug combination on canine osteosarcoma cancer cells. Tumor volume is in mm 3 .

[0200] In Table 4, the days represent the date of drug administration, and day 29 represents the final tumor volume at the time of animal sacrifice.

[0201] Table 4 shows the tumor volume inhibition rates of mice bearing canine osteosarcoma cancer cells. Comparison of tumor volume in each group on day 29 after the animals were grown in the S1 group revealed that the tumor inhibition rates in the S2 group were 42.93%, 53.39%, and 42.90% in the S3 group. The tumor inhibition rates in the S5 group, which received the effective dose of the present invention's drug combination, were 26.40%, and those in the S3 group, which received the low dose of the present invention's drug combination, were 53.38%. The experimental results indicate that the S4 drug combination produced a more potent inhibitory effect on cancer cells than the most potent, and that S5 also produced effective inhibitory effects under different drug modes of use. This demonstrates that the pharmaceutical composition of the present invention exhibits superior tumor volume inhibition efficacy in both combined and individual doses.

[0202] Table 5. Correlation between the weight of cancer cell tumor masses and the dosage of the pharmaceutical composition of the present invention for canine osteosarcoma cancer cells. The unit of weight of cancer cell tumor masses is milligrams (mg).

[0203] Table 5 shows the average tumor mass weight inhibition rates of mice bearing canine osteosarcoma cancer cells after sacrifice. Compared to the experimental results of group S1, group S2 had a tumor inhibition rate of 44.6%, group S3 had a tumor inhibition rate of 59.3%, and group S4 had a tumor inhibition rate of 45.3%. Group S5, the effective dose group of the present invention's pharmaceutical combination, had a tumor inhibition rate of 32.2%. These experimental results demonstrate that S3 produces a more effective cancer-inhibiting effect on cancer cells, and that the pharmaceutical composition of the present invention exhibits superior tumor volume inhibition efficacy both in combination and when used alone.

[0204] Based on the experimental results, it can be clearly understood that the pharmaceutical composition of the present invention can effectively inhibit the weight and volume of tumor masses in animals. After the pharmaceutical composition of the present invention is effectively absorbed through the gastrointestinal tract, the drug clearly targets cancer cells and tumor tissues, and the animals do not experience any discomfort during the drug administration process.

[0205] Example 3 The composition of the present invention is used in a single or multiple combination mode to prepare a canine dry food formula to promote the health of canine animals

[0206] Dogs can range from small to large breeds, and the dry feed range for dogs is 15g / kg to 30g / kg, depending on their weight. The main feed ingredients are chicken meal, beef meal, fish meal, brown rice, oats, barley, etc., and vitamins, minerals, fiber, and other nutritional supplements are also added.

[0207] During the preparation process, the meat powder and other additives are mixed uniformly according to the appropriate dosage ratio. The mixture is then extruded into pellets. The pellets are then dried at low temperature to reduce the moisture content of the dry feed to 10-12%. Table 6 shows the acceptable dosage of the composition of the present invention as an additive per gram of dry feed.

[0208] Table 6. Dosage (mg) of the composition of the present invention as an additive per gram of dog dry feed, expressed as human dosage per kilogram.

[0209] Example 4 The composition of the present invention is used in a single or multiple combination mode to prepare a canine wet dog food formula to promote the health of canine animals

[0210] Wet feed for dogs ranging from small to large breeds ranges from 60g / kg to 100g / kg, depending on the dog's weight. Main ingredients include fresh chicken, beef, fish, rice, potatoes, oats, and other sources, along with added vitamins, minerals, fiber, and other nutritional supplements.

[0211] During the preparation process, fresh meat is cut into small pieces and mixed evenly with other additives according to the appropriate dosage. The mixture is then placed in a pressure cooker and cooked until thoroughly cooked. Finally, the cooked wet feed is filled into containers and sterilized at high temperature. Table 7 lists the acceptable dosages of the composition of the present invention as an additive per gram of wet feed.

[0212] Table 7. Dosage (mg) of the composition of the present invention as an additive per gram of canine wet feed, reported as human dosage per kilogram.

[0213] Example 5 The composition of the present invention is used in a single or multiple combination mode to prepare a cat dry cat food formula to promote the health of cats

[0214] Cat dry feed ranges from 20g / kg to 50g / kg, depending on the cat's weight. Main ingredients include chicken meal, turkey meal, fish meal, brown rice, oats, barley, etc., and vitamins, minerals, fiber, and other nutritional supplements are also added.

[0215] During the preparation process, the meat powder and other additives are mixed uniformly according to the dosage ratio. The mixture is then extruded into pellets. The pellets are then dried at low temperature to reduce the moisture content of the dry feed to 10-12%. Table 8 shows the acceptable dosage of the composition of the present invention as an additive per gram of dry feed.

[0216] Table 8. Dosage (mg) of the composition of the present invention per gram of dry feed additive for cats, recorded in the form of human dosage per kilogram.

[0217] Example 6 The composition of the present invention is used in a single or multiple combination mode to prepare a cat wet cat food formula to promote the health of cats

[0218] Cat wet feed ranges from 70g / kg to 150g / kg, depending on the cat's weight. Mainly made from fresh chicken, fresh turkey, fresh fish, etc., with added vitamins, minerals, fiber, and other nutritional supplements.

[0219] During the preparation process, fresh meat is cut into small pieces and mixed evenly with other additives according to the appropriate dosage. The mixture is then placed in a pressure cooker and cooked until thoroughly cooked. Finally, the cooked wet feed is filled into containers and sterilized at high temperature. Table 9 lists the acceptable dosages of the composition of the present invention as an additive per gram of wet feed.

[0220] Table 9. Dosage (mg) of the composition of the present invention as an additive per gram of cat wet feed, reported as human dosage per kilogram.

[0221] Example 7 The composition of the present invention is used alone or in combination to prepare a lagomorph food formula to promote the health of lagomorphs.

[0222] The range for lagomorph feed is 8g / kg to 15g / kg, depending on the lagomorph's body weight. The main feed ingredients are timothy hay meal, alfalfa hay meal, soy meal, pea protein, and flaxseed, with vitamins, minerals, fiber, and other nutritional supplements added.

[0223] During the preparation process, hay is ground into a powder and mixed evenly with other additives according to a proportional dosage. The mixture is then placed in an extruder to form pellets. The pellets are then dried at low temperature to ensure that the moisture content of the dry feed is reduced to 10-12%. Table 10 shows the acceptable dosage of the composition of the present invention as an additive per gram of dry feed.

[0224] Table 10. Dosage (mg) of the composition of the present invention as an additive per gram of dry lagomorph feed, reported as human dosage per kilogram.

[0225] Example 8 The composition of the present invention is used alone or in combination to prepare a rodent food formula to promote the health of rodents.

[0226] Rodent feed ranges from 5g to 25g depending on the rodent's body weight. Main ingredients include timothy hay meal, alfalfa hay meal, oats, barley, wheat, corn, soybean meal, pea protein, dried fava bean meal, flaxseed, dried carrots, and dried apples. Vitamins, minerals, fiber, and other nutritional supplements are also added.

[0227] During the preparation process, grains, hay, and seeds are ground into appropriate sizes and mixed with other additives in a proportional dosage. The mixture is then placed in an extruder to form pellets. The pellets are then dried at low temperatures to reduce the moisture content of the dry feed to 10-12%. Table 11 lists the acceptable dosages of the compositions of the present invention as additives per gram of dry feed.

[0228] Table 11. Dosage (mg) of the composition of the present invention as an additive per gram of dry rodent feed, reported as human dosage per kilogram.

[0229] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A composition for animals, comprising: (a) Agents targeting mitochondria of cancer cells; (b) ion chelating agents; and (c) pharmacologically active multivalent ions; at least two of them; in, The cancer cell mitochondrial targeting agent is niclosamide, the ion chelator is disulfiram, and the pharmacologically active multivalent ion is selected from a group consisting of magnesium ion, calcium ion, manganese ion, ferrous ion, copper ion, and zinc ion. The weight ratio of (a) is 22.0% to 98.4% based on the total weight of the composition, the weight ratio of (b) is 37.9% to 98.8% based on the total weight of the composition, and the weight ratio of (c) is 0.69% to 64.5% based on the total weight of the composition.

2. The composition for animals according to claim 1, wherein the dosage of (a) in the combination of (a)(b) is 800 mg / Kg to 1.67 mg / Kg, and the dosage of (b) in each administration is 1,376 mg / Kg to 2.14 mg / Kg.

3. The composition for animals according to claim 1, wherein the dosage of (a) in the combination of (a) and (c) is 800 mg / Kg to 1.67 mg / Kg, and the dosage of (c) in the combination of (a) and (c) is 1,456 mg / Kg to 2.65×10 -2 mg / Kg.

4. The composition for animals according to claim 1, wherein the dosage of (b) in the combination of (b) and (c) is 1,376 mg / Kg to 2.14 mg / Kg, and the dosage of (c) in the combination of (b) and (c) is 1,456 mg / Kg to 2.65×10 -2 mg / Kg.

5. The composition for animals according to claim 1, wherein the dosage of (a) in the combination of (a), (b) and (c) is 800 mg / Kg to 1.67 mg / Kg, the dosage of (b) in the combination is 1,376 mg / Kg to 2.14 mg / Kg, and the dosage of (c) in the combination is 1,456 mg / Kg to 2.65×10 -2 mg / Kg.

6. Use of the composition according to claim 1 for animals, for preparing a medicament for treating or preventing a malignant tumor in the animal.

7. The use of the composition for animals according to claim 6, wherein the malignant tumor is selected from the group consisting of osteosarcoma, breast tumor, mastocytoma, melanoma, breast cancer, and lung cancer.

8. The use of the composition for animals according to claim 6, wherein the administration order of (b) is no later than the administration order of (c).

9. The use of the composition for animals according to claim 6, wherein the drug is administered orally, by injection, percutaneously, or by inhalation.

10. Use of the composition according to claim 1 for animals for preparing food for improving the health of the animals.

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