WO2024046332A1 - 医药组合物及其用途 - Google Patents

医药组合物及其用途 Download PDF

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WO2024046332A1
WO2024046332A1 PCT/CN2023/115614 CN2023115614W WO2024046332A1 WO 2024046332 A1 WO2024046332 A1 WO 2024046332A1 CN 2023115614 W CN2023115614 W CN 2023115614W WO 2024046332 A1 WO2024046332 A1 WO 2024046332A1
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pharmaceutical composition
cancer
present
drug
ions
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PCT/CN2023/115614
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English (en)
French (fr)
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石贵中
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石贵中
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/145—Amines having sulfur, e.g. thiurams (>N—C(S)—S—C(S)—N< and >N—C(S)—S—S—C(S)—N<), Sulfinylamines (—N=SO), Sulfonylamines (—N=SO2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/34—Copper; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention generally relates to a pharmaceutical composition, a kit containing the pharmaceutical composition, and uses thereof. Specifically, the present invention relates to a pharmaceutical composition that can treat malignant diseases caused by abnormal growth of animal cells, a kit containing the pharmaceutical composition, and the use of the pharmaceutical composition and the pharmaceutical kit.
  • 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 continue to divide and proliferate, forming tumors that have the ability to invade and destroy nearby normal tissues and organs.
  • Common anti-cancer treatments include surgery, radiation therapy and chemotherapy, such as the use of cancer treatment target drugs.
  • these well-known treatments often come with the risk of damaging or poisoning normal tissue.
  • alternative treatments designed to kill cancer cells without causing side effects are still in the research and preclinical stages.
  • the present invention proposes a pharmaceutical composition that is effective against malignant tumors, a kit containing the pharmaceutical composition, and its use in treating malignant diseases in which animal cells grow abnormally.
  • the pharmaceutical composition of the present invention can be applied in the treatment field of oncology.
  • the pharmaceutical composition proposed by the present invention is designed to synergistically interfere with the growth mechanism of cancer cells through functional interactions between multiple existing drugs.
  • the pharmaceutical composition proposed by the present invention is simple to prepare, cheap, and can exhibit statistically significant effects in inhibiting the growth of malignant tumors.
  • the components of the pharmaceutical composition proposed by the present invention can also be administered separately and exhibit the efficacy of inhibiting cancer cells in an individual.
  • the present invention first proposes a pharmaceutical composition, which includes (a) a targeting agent for cancer cell mitochondria, (b) an ion chelator, and (c) pharmacologically active multivalent ions.
  • the targeting agent for cancer cell mitochondria is Niclosamide.
  • the ion chelating agent is Disulfiram.
  • the pharmacologically active multivalent ions are selected from the group consisting of magnesium ions, calcium ions, manganese ions, ferrous ions, copper ions, and zinc ions.
  • the weight ratio of (a) is 64.5% to 43.7% based on the total weight of the pharmaceutical composition
  • the weight ratio of (b) is 56.2% to 16.1% based on the total weight of the pharmaceutical composition
  • the weight ratio of (c) The total weight of the pharmaceutical composition is 19.4% to 0.000116%.
  • the present invention also proposes the use of the aforementioned pharmaceutical composition for preparing a medicament for treating malignant tumors in individuals in need.
  • the malignant tumor is selected from the group consisting of solid tumors and hematological malignancies.
  • the malignant tumor is selected from the group consisting of lung cancer and brain cancer.
  • the amount of application per time is 2000 mg ⁇ 100 mg
  • the amount of application per time is 500 mg ⁇ 128.6 mg
  • the amount of application per time is 600 mg ⁇ 2.65 ⁇ 10 -4 mg.
  • the order of administration of (c) is no later than the order of administration of (b).
  • the drug is administered no more than 2 times a day.
  • the drug is administered orally, by injection, by transdermal, or by inhalation.
  • Figure 1 shows the correlation between the inhibitory efficacy of the pharmaceutical composition of the present invention and the well-known target drug group on the cancer cell tumor volume of human non-small cell lung cancer cell line (A549) in animals and the administration time.
  • Figure 2 shows the correlation between the inhibitory efficacy of the pharmaceutical composition of the present invention and the well-known target drug group on the cancer cell tumor weight of human non-small cell lung cancer cell line (A549) in animals and the administration time.
  • Figure 3 shows the correlation between the inhibitory efficacy of the pharmaceutical composition of the present invention and the well-known target drug group on the cancer cell tumor volume of the human lung squamous epithelial cell line (H520) in animals and the administration time.
  • Figure 4 shows the correlation between the inhibitory efficacy of the pharmaceutical composition of the present invention and a well-known target drug group on the cancer cell tumor weight of human lung squamous epithelial cell carcinoma cell line (H520) in animals and the administration time.
  • Malignant tumor also known as cancer, is a disease of abnormal cell growth and division. It is characterized by the potential of cells to proliferate indefinitely, invade surrounding tissues and organs, and metastasize to other Part tendency. Malignant tumors are complex diseases that may involve multiple genetic variations and molecular mechanisms. Early detection, precise diagnosis, and multidisciplinary treatment strategies are crucial to control the progression of malignant tumors. Treatment options may include surgical resection, radiation therapy, chemotherapy, targeted therapy, and immunotherapy, depending on the patient's specific condition and type of cancer. Malignant tumors differ from benign tumors in that the latter usually grow locally, rarely invade adjacent tissues, and rarely metastasize to other sites.
  • Infinite proliferation Malignant tumor cells lose the restrictions on normal cell growth, and they can continue to divide and proliferate indefinitely to form tumor tissue.
  • Metastasis Malignant tumor cells sometimes metastasize to other parts of the body through the blood or lymphatic system, forming metastases (also called uterine tumors). This can cause cancer to appear in multiple parts of the body at the same time, making treatment more difficult.
  • Abnormal cell morphology Malignant tumor cells are often morphologically different from normal cells. They may be irregular, deformed, and unequal in size.
  • Cell heterogeneity In the same malignant tumor, the properties and characteristics of cells may be different, which is called cell heterogeneity. This makes treatment more complex, as different cell subpopulations may respond differently to treatment.
  • Angiogenesis Malignant tumors usually induce the formation of new blood vessels to provide them with nutrients and oxygen to maintain their continuous growth.
  • Drug synergism Also known as drug synergism, when two or more drugs are used together, their effects exceed the sum of the effects of each drug alone. In drug therapy, drug synergy can lead to a more powerful, longer-lasting, or broader therapeutic effect, while potentially reducing the side effects that may occur with a single drug. Drug synergy can be observed in different types of drugs, including antibiotics, anticancer drugs, antiviral drugs, etc. The meaning of drug synergy can be described more specifically as follows:
  • Enhanced therapeutic effect Drug synergy may lead to enhanced therapeutic effect. This means that when multiple drugs are given, their combined effect may be more powerful than a single drug, making it more effective at controlling or treating a disease.
  • Multi-target intervention Different drugs may work by affecting different targets or pathways of the disease, interfering with the disease through multiple pathways, increasing the diversity and comprehensiveness of treatment.
  • Coping with complex diseases Some complex diseases may involve multiple pathological mechanisms, and it is difficult for a single drug to comprehensively intervene. Drug synergies can affect different aspects of a disease at multiple levels, allowing for more effective responses to complex diseases.
  • Hematological malignant diseases refer to abnormal proliferation of white blood cells, which may include but are not limited to leukemia or lymphoma.
  • Cancer cell lethality rate refers to the percentage of cancer cells in cancer patients 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 treatments to understand the impact of treatments on cancer conditions. The meaning of cancer cell lethality can be explained as follows:
  • Cancer cell lethality is one of the important indicators to evaluate the impact of treatment methods on cancer. A higher lethality rate usually means the treatment is more effective at killing cancer cells.
  • Predicting patient prognosis Higher cancer cell lethality may be associated with improved patient prognosis. Effective killing of cancer cells may reduce the risk of disease recurrence and progression, thereby improving patient survival rates.
  • Cancer cell lethality can help medical professionals evaluate the merits of different treatments.
  • the choice of treatment usually takes into account the lethality of the treatment as well as possible side effects, drug resistance and other factors.
  • Treatment monitoring Tracking cancer cell lethality can help medical professionals monitor the progress of treatment. If mortality does not improve significantly over time, treatment adjustments may need to be considered.
  • treatment includes the partial or complete prevention, amelioration, alleviation and/or treatment of symptoms, secondary disorders or conditions associated with cancer.
  • treatment in this specification also refers to the application or administration of one or more drugs of the present invention to an individual suffering from symptoms, secondary signs or symptoms associated with cancer to achieve partial or complete relief, Slow, cure, delay onset, inhibit progression, reduce disease severity, and/or reduce the occurrence of one or more cancer-related signs, symptoms, or minor symptoms.
  • Signs, secondary signs and/or symptoms associated with cancer include, but are not limited to, fever, weakness, tiredness, weight loss, pain, cough, bleeding, skin changes, diarrhea or constipation, nausea, vomiting and loss of appetite.
  • Treatment as used herein may also be administered to an individual suffering from early signs or symptoms to reduce the risk of the individual developing signs, secondary signs and/or symptoms associated with cancer.
  • Treatment as used herein may be effective in reducing one or more symptoms or clinical markers.
  • treatment here can also mean reducing, slowing down or stopping the course of a disease, the development of signs or symptoms.
  • Effective amount here refers to the amount of drug sufficient to produce the intended therapeutic response.
  • An effective amount is also that amount of a compound or composition whose therapeutically beneficial effects outweigh its toxic or harmful 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., patient weight, age, or sex), the type of mammal or animal being treated, the duration of treatment, current therapy (if where applicable) and the specific formulation used and the structure of the compound or its derivatives.
  • the effective amount may be expressed as the total weight of the drug (eg, in grams, milligrams, or micrograms) or as a ratio of drug weight to body weight (in milligrams per kilogram (mg/kg)).
  • the effective amount may be expressed as the concentration of the active ingredient (e.g., the agent of the present invention), such as molarity, weight concentration, volume concentration, weight molarity, mole fraction, weight fraction, and mixing ratio.
  • the term "therapeutically effective amount” refers to an amount of a drug administered that is sufficient to slow or alleviate an individual's cancer-related symptoms.
  • Technicians can calculate the human equivalent dose (HED) of a drug (such as the agent of the present invention) based on the dose in animal models.
  • HED human equivalent dose
  • in vitro refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in a cell culture, etc., rather than in an organism.
  • in vivo refers to events that occur within a living organism, such as cells in humans and cells in non-human animals. In the context of cell-based narratives, events occurring within living cells may be referred to (as opposed to, for example, in vitro systems).
  • the term "subject" may refer to any organism to which a provided medicament is administered, for example, for experimental, diagnostic, prophylactic, cosmetic and/or therapeutic purposes.
  • exemplary subjects may include animals, such as mammals, such as mice, rats, rabbits, non-human primates, and/or humans.
  • the individual may be a human.
  • the term "pharmaceutically acceptable” or “pharmacologically active” may mean a reasonable benefit/risk ratio commensurate with reasonable medical judgment, suitable for contact with human and animal tissue, and without undue toxicity , irritants, allergic reactions or other problems or complications.
  • prophylactically effective amount may refer to an amount that, when administered to an individual, is sufficient to prevent and/or delay the onset of symptoms based on the varying degrees of sensitivity of the individual or cancer cells. It will be understood by those of ordinary skill in the art that a prophylactically effective dose is generally administered by a dosage regimen containing at least one unit dose.
  • cycle cycle and “cycle of treatment” (cycle of treatment or treatment cycle) are interchangeable terms and refer to a period of time during which treatment is administered to a patient.
  • a cycle of treatment is followed by a period of rest without any treatment at all. After this rest period, one or more treatment cycles may be administered again, each treatment cycle being followed by a rest period.
  • Drug repurposing refers to the use of existing drugs for new therapeutic applications. Based on the fact that these drugs are already approved 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.
  • Known drugs utilize new drug combination strategy models to generate new malignant tumor treatment applications, and drug combinations contribute to the interaction between different drugs.
  • Drug combinations can exert synergistic effects between drugs and interfere with the growth mechanism of cancer cells.
  • the cost of the drug of the present invention in treating cancer can be much lower than the out-of-pocket price of all current health care payment target drugs.
  • IRESSA Garfitinib
  • the currently lowest-priced cancer target drug even with the current national health care payment in Taiwan
  • patients still need to pay 573 yuan for a single pill of IRESSA and use it continuously every day.
  • the price of a single pill of the medicine of the present invention for treating cancer is only 184 yuan.
  • the inventors of the present invention provide a pharmaceutical composition effective against malignant tumors.
  • Each active ingredient in the pharmaceutical composition of the present invention is an existing known drug, and these known drugs have abundant clinical data and relatively high safety.
  • Approaches that utilize known drugs combined with drug combination strategies have significant multiple advantages over targeted drugs.
  • the present invention first proposes a pharmaceutical composition, which contains (a) a targeting agent for cancer cell mitochondria, (b) an ion chelating agent, and (c) pharmacologically active multivalent ions and other drugs.
  • a targeting agent for cancer cell mitochondria e.g., a targeting agent for cancer cell mitochondria
  • a targeting agent for cancer cell mitochondria e.g., a targeting agent for cancer cell mitochondria
  • a targeting agent for cancer cell mitochondria e.g
  • an ion chelating agent e.g
  • pharmacologically active multivalent ions and other drugs pharmacologically active multivalent ions and other drugs.
  • Three drugs form a drug combination, and the proportion of the three drugs is designed so that the dosage can be adjusted according to different therapeutic effects.
  • the therapeutic dosage unit of drugs (a), (b) (c), etc. is milligrams (mg).
  • 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 apoptosis.
  • GlobalData data no company has completed the development of niclosamide and entered the cancer treatment market for single use treatment of cancer.
  • the weight ratio of (a) is 64.5% to 43.7%.
  • the dosage range for niclosamide is daily use for seven days, no more than 2 times daily.
  • the effective dose of niclosamide is 857.2 mg.
  • the therapeutic dose range of niclosamide is 2000mg ⁇ 100mg.
  • the ion chelating agent may be Disulfiram.
  • Disulfiram can induce endoplasmic reticulum stress and autophagy to induce apoptosis, reduce angiogenesis, and can be used as a sensitizer for radiotherapy, or as an ion chelator.
  • the weight ratio of (b) is 56.2% to 16.1%.
  • the upper limit dose of Disulfiram per administration is 500 mg, and the lower limit dose is 128.6 mg.
  • the effective dose of Disulfiram is 257.2mg.
  • the therapeutic dose range of Disulfiram is 500mg ⁇ 128.6mg.
  • Pharmacologically active multivalent ions are pharmaceutically acceptable nutritional additive types of metal ions and their salts, such as chloride ions, oxygen ions, hydroxide ions, gluconate, sulfate, glycinate, succinate, stearate, and glycerin Phosphate, acetate, lactate, phosphate, borate, carbonate, fumarate, glutarate, fructose borate, cholate, malate, glycerate, oxalate, tartrate, citrate, HAP chelate ions, HVP chelate ions, etc., but the present invention is not limited thereto.
  • Multivalent active ions are typical element ions with two or more valences, or transition element ions.
  • Pharmacologically active multivalent ions are, for example, at least one of magnesium ions, calcium ions, chromium ions, manganese ions, ferrous ions, copper ions, and zinc ions. Based on the total weight of the pharmaceutical composition, the weight ratio of (c) is 19.4% to 0.000116%.
  • the dosage range of pharmacologically active multivalent ions is 1800mg ⁇ 2.65 ⁇ 10 -4 mg.
  • the effective dose of pharmacologically active multivalent ions is 0.139 mg ⁇ 5.3 ⁇ 10 -3 mg.
  • two Disulfirams can form a pharmacologically active chelate with a pharmacologically active polyvalent ion. Table 1 lists the dose ranges for different pharmacologically active multivalent ions.
  • the (a) component, (b) component and (c) component in the pharmaceutical composition of the present invention can have different dosage ratio ranges or combinations as necessary.
  • the dose ratio of items (a) and (b) can be Scale range.
  • the dosage ratio of items (a) and (c) can be Scale range.
  • the dose ratio of (a) to (b) + (c) can be Scale range.
  • the dose ratio of (a) + (b) and (c) can be Scale range.
  • the dose ratio of (a) + (c) and (b) can be Scale range.
  • the dose ratio of (b) to (a) can be Scale range.
  • the dosage ratio of items (b) and (c) can be Scale range.
  • the dose ratio of (b) to (a) + (c) can be Scale range.
  • the dose ratio of (b) + (c) to (a) can be Scale range.
  • the dose ratio of items (c) and (a) may be 13.35 ⁇ Scale range.
  • the dose ratio of items (c) and (b) may be 5.3 ⁇ Scale range.
  • the dose ratio of (c) to (a) + (b) can be Scale range.
  • the pharmaceutical composition of the present invention can produce corresponding cancer cell lethality effects within the dosage ratio ranges of (a), (b) and (c) above.
  • the first agent may be (c) pharmacologically active multivalent ions
  • the second agent may be (b) an ion chelator
  • the third agent may be (a) a targeting agent for cancer cell mitochondria.
  • the order of administration of the first agent (c) pharmacologically active multivalent ions is no later than the order of administration of the second agent (b) the ion chelator, which helps the pharmacologically active multivalent ions increase the ion concentration in the animal's body.
  • the order of administration of the third agent (a) cancer cell mitochondria-targeting agent is not particularly limited.
  • the order of administration of the first medicament (c), the second medicament (b), and the third medicament (a) can be as follows:
  • composition of the present invention may further contain one or more stabilizers, binding agents, fillers, disintegrants, excipients, and/or additives.
  • the pharmaceutical composition of the present invention can also be made into a pharmaceutical kit suitable for different administration sequences.
  • the pharmaceutical composition of the present invention is formulated into a pharmaceutical kit for treating an individual suffering from or suspected of suffering from cancer.
  • the pharmaceutical kit of the present invention includes a first container, an optional second container, an optional third container, and/or an optional fourth container.
  • the first container, the optional second container, and the optional third container respectively contain at least one of a first medicament, a second medicament, and a third medicament.
  • the first agent may be (c) pharmacologically active multivalent ions
  • the second agent may be (b) an ion chelator
  • the third agent may be (a) a targeting agent for cancer cell mitochondria.
  • Another aspect of the invention relates to a method of treating an individual suffering from or suspected of suffering from cancer using the pharmaceutical kit of the invention.
  • the method includes administering to the individual an agent of the invention in a first container; administering to the individual an agent of the invention in an optional second container; and administering to the individual an agent of the invention in an optional third container.
  • the first container contains at least one of a first medicament, a second medicament, and a third medicament.
  • the optional second container contains at least one of the first medicament, the second medicament and the third medicament.
  • the optional third container contains at least one of the first medicament, the second medicament and the third medicament.
  • the first agent may be (c) pharmacologically active multivalent ions
  • the second agent may be (b) an ion chelator
  • the third agent may be (a) a targeting agent for cancer cell mitochondria.
  • the first effective amount of the pharmacologically active multivalent ions of the first medicament (c) of the present invention may range from 1800 mg to 2.65 ⁇ 10 -4 mg.
  • the second agent is (b) an ion chelating agent, and the second effective amount thereof may range from 500 mg to 128.6 mg.
  • the third agent of the present invention includes (a) a targeting agent for cancer cell mitochondria, and the third effective amount thereof can range from 2000 mg to 100 mg.
  • the first container can carry an effective amount of the agent of the invention or a pharmaceutical dosage form thereof.
  • the second container can carry an effective amount of the agent of the invention or a pharmaceutical dosage form thereof.
  • the third container can carry an effective amount of the medicament of the present invention or its pharmaceutical dosage form.
  • the pharmaceutical kit of the present invention may further comprise instructions for use accompanying the first container and/or the second container and/or the third container.
  • the pharmaceutical kit of the present invention may optionally further include a fourth container containing a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution or glucose solution. It may also contain other commercial or user-required materials, including other buffers, diluents, filters, needles, and syringes.
  • Another aspect of the invention relates to a method of administering the pharmaceutical kit of the invention to treat an individual in need thereof (eg, an individual suffering from cancer, or an individual suspected of suffering from cancer).
  • the method includes: administering to the individual a first effective amount of a first container of the invention; and administering to the individual a second effective amount of a second container of the invention, as appropriate; or administering to the individual a third effective amount of a second container of the invention, as appropriate.
  • the invention requires a third container.
  • the first administration time point of the first container to the individual may be no later than the second administration time point of the second container to the individual, and the third administration time point of the third container to the individual may be no earlier than the second administration time point of the second container to the individual.
  • the second application time point of the second container and the third application time point may be no earlier than 10 minutes before the second application time point.
  • the medicines in the pharmaceutical kit of the present invention should be administered no more than twice a day.
  • a first container of the present invention is administered to the individual, in the form of a liposome, capsule or tablet.
  • the subject is administered a second container of the invention, in the form of liposomes, capsules, or tablets.
  • a third container of the invention in the form of liposomes, capsules, or tablets.
  • the subject is a human, for example a human weighing 60 kilograms, and the subject is administered the first agent at a dose of 10 mg to 4.42 ⁇ 10 -6 mg per kilogram of subject body weight per day, and the subject is administered the first agent at a dose of The second agent is administered at a dose of 8.33 mg to 2.14 mg per kilogram of the subject's body weight, and the subject is administered a third agent at a dose of 33.33 mg to 1.67 mg per kilogram of the subject's body weight per day.
  • administering the pharmaceutical composition of the present invention at a dose of 51.67 mg to 3.81 mg/kg/day is sufficient to reduce the number of cancer cells in this individual, thereby demonstrating the anti-cancer efficacy of the pharmaceutical composition of the present invention.
  • the first container contains a third medicament
  • the second container contains the first medicament
  • the third container contains a second medicament.
  • the subject is administered the agent of the present invention in the first container; at the second administration time point, the subject is administered the agent of the invention in the second container; and at the third administration time point, the subject is administered
  • the individual administers the medicament of the present invention in the third container such that the order of medicament administration in the method of using the pharmaceutical kit of the present invention to treat an individual suffering from or suspected of having cancer is first (a), then (c), then (b).
  • a first container contains a first medicament
  • a second container contains a second medicament
  • a third container contains a third medicament.
  • the subject is administered the agent of the present invention in the first container; at the second administration time point, the subject is administered the agent of the invention in the second container; and at the third administration time point, the subject is administered
  • the individual administers the medicament of the present invention in the third container such that the order of administration of the medicaments in the method of using the pharmaceutical kit of the present invention to treat an individual suffering from or suspected of having cancer is first (c), then (b), then (a).
  • the first container contains a first medicament and a third medicament
  • the second container contains a second medicament.
  • the individual is administered the pharmaceutical agent of the present invention in the first container; and at the second administration time point, the individual is administered the pharmaceutical agent of the present invention in the second container, so that the pharmaceutical kit of the present invention is used to A method of treating an individual with or suspected of having cancer in which the order of drug administration is simultaneous administration of (a) and (c) followed by administration of (b).
  • the first container contains a first medicament and a second medicament
  • the second container contains a third medicament.
  • the individual is administered the pharmaceutical agent of the present invention in the first container; and at the second administration time point, the individual is administered the pharmaceutical agent of the present invention in the second container, so that the pharmaceutical kit of the present invention is used to
  • the method of treating an individual with or suspected of having cancer is such that (c) is administered concurrently with (b) followed by (a).
  • the first container contains a first medicament, a second medicament, and a third medicament.
  • the agent of the present invention in the first container is administered to the individual, such that the order of drug administration of the method of using the pharmaceutical kit of the present invention to treat an individual suffering from or suspected of having cancer is (a) and ( b) administered simultaneously with (c).
  • the release form of the pharmaceutical composition of the present invention can be compressed into a double-layer or three-layer tablet.
  • the inner layer tablet is made by compressing a sustained-release excipient combined with the drug
  • the outer layer tablet is made by fast-disintegrating excipients.
  • the drug is combined with a preferentially released drug and compressed into tablets, and then the inner layer is wrapped with an outer layer to form the final tablet.
  • the drug release rate is the fastest in the outermost layer, and the release rate decreases across the inner layer.
  • pharmacologically active multivalent ions can enter the body first to increase the concentration in the body, and then chelate with the ion chelating agent released after disintegration to produce effects, etc.
  • Rapid disintegration release dosage forms of drugs are designed to release the drug rapidly for rapid drug action.
  • excipients may be used to prepare rapidly disintegrating release dosage forms, such as, but not limited to:
  • Lactose is a common auxiliary excipient and is often used in the preparation of oral tablets. It has excellent tableting performance and rapid disintegration properties, which promotes rapid drug release.
  • 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.
  • Microcrystalline cellulose is a fine crystalline excipient obtained by processing cellulose and is often used in the preparation of oral tablets. It increases the mechanical strength of tablets and promotes rapid drug release.
  • Water-soluble polymers Some water-soluble polymers, such as hydroxypropyl methylcellulose (HPMC), polyethylene glycol (PEG), etc., can be used as disintegrating agents when preparing fast-disintegrating dosage forms. Antidote use. They interact rapidly with water, causing tablets to rapidly disintegrate and release the drug.
  • HPMC hydroxypropyl methylcellulose
  • PEG polyethylene glycol
  • Protein excipients Certain proteins, such as gelatin and sodium starch glycolate, can rapidly expand and disintegrate under humid conditions, thereby achieving rapid release of drugs. These excipients can be used to prepare various rapidly disintegrating dosage forms, such as rapidly disintegrating tablets, disintegrating granules, and disintegrating capsules.
  • Sustained-release dosage forms of drugs often use special excipients.
  • this excipient can prolong the release time of the drug in the body, allowing the drug to enter the blood or target tissue at a stable rate, such as but not limited to:
  • Polymers are common excipients, such as polyethylene glycol (PEG), poly(lactic-co-glycolic acid), PLGA, etc. These polymers can form drug carriers that release drugs at a controlled rate.
  • PEG polyethylene glycol
  • PLA poly(lactic-co-glycolic acid)
  • PLGA poly(lactic-co-glycolic acid)
  • Silicate excipients such as the complex of silica dioxide and aluminum hydroxide, can delay the release rate of the drug by forming a drug coating layer or network structure.
  • 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 according to different needs to control the drug release rate.
  • Liposomes are tiny particles composed of lipid bilayers that can encapsulate drugs and form stable carriers. Liposomes can be taken orally, injected, or applied topically to achieve slow release of drugs.
  • Polymeric liposomes are an improved version of liposomes. By adding polymers or macromolecular substances, the release rate and stability of the drug can be further adjusted.
  • the release form of the pharmaceutical composition of the present invention is not limited to the aforementioned excipients and techniques that can be used to prepare drugs in sustained-release dosage forms. Appropriate excipients can be selected based on factors such as the characteristics of the drug, therapeutic needs, and route of administration.
  • the pharmaceutical composition of the present invention may have different effects on cancer or tumor cells under the influence of neutral, negative, or positive liposomes. Discuss the differences in the effects of liposomes with different charges on cells from two aspects:
  • Interaction mechanism There is electrostatic attraction between positively charged liposomes and negatively charged cell membranes, making it easier for positively charged liposomes to bind to the cell membrane, while it is relatively difficult for negatively charged liposomes to bind to the cell membrane. Electrically neutral liposomes have no significant electrostatic interactions. This interaction mechanism determines the affinity and uptake efficiency between different charged liposomes and cells.
  • Positively charged liposomes can promote intracellular uptake of liposomes due to their negative charge interaction with the cell membrane. Cellular uptake is mainly carried out through endocytosis. Positively charged liposomes can be actively or passively taken into cells after binding to the cell membrane. In contrast, negatively charged liposomes have relatively low uptake efficiency due to electrostatic repulsion with cell membranes.
  • Positively charged liposomes can promote the release and delivery of payload materials such as drugs or genes into cells because they can interact with negatively charged molecules in cells, such as nucleic acids, proteins, etc. Negatively charged liposomes can also deliver drugs or payload substances to a certain extent, but compared to positively charged liposomes, negatively charged liposomes are more suitable for stabilizing and protecting drugs, and are less suitable for direct delivery to in the cell. Electroneutral liposomes have no charge interaction with cell membranes, so under certain parameters, they can reduce non-specific interactions with cells and reduce the possibility of adsorption and phagocytosis. This helps improve the stability and bioavailability of liposomes in the body and generally has a higher drug loading capacity, which can more effectively coat and stabilize various types of drugs (water-soluble and lipid-soluble).
  • materials for preparing positively charged liposomes include, but are not limited to, DOTAP (Dioleoyl-3-trimethylammonium propane).
  • materials for preparing negatively charged liposomes include, but are not limited to, DOPG (dioleoylphosphatidylglycerol).
  • materials for preparing electrically neutral liposomes include, but are not limited to, DSPC (Distearoylphosphatidylcholine).
  • liposomes such as but not limited to cholesterol, polyethylene glycol (PEG), surfactants (such as Tween, Span), auxiliaries (such as lecithin) Serine, lecithin), liposome coating agents (such as lecithin choline), targeting ligands (such as antibodies, ligands), dyes (such as fluorescent dyes, nucleic acid dyes), liposome stabilization Agents (such as liposome structure regulators, preservatives).
  • PEG polyethylene glycol
  • surfactants such as Tween, Span
  • auxiliaries such as lecithin Serine, lecithin
  • liposome coating agents such as lecithin choline
  • targeting ligands such as antibodies, ligands
  • dyes such as fluorescent dyes, nucleic acid dyes
  • liposome stabilization Agents such as liposome structure regulators, preservatives.
  • the coating of pharmaceutical agents can be carried out in ball-in-ball.
  • two drugs are first coated with small spherical liposomes: an ion chelator + a targeting agent for cancer cell mitochondria, and then the small spherical liposomes and pharmacologically active multivalent ions are coated with large spherical liposomes. Formation of two-layered liposomes.
  • the big ball can be disintegrated first to release pharmacologically active multivalent ions to increase the concentration.
  • the small ball can be disintegrated to release an ion chelator + a targeting agent for cancer cell mitochondria.
  • the ion chelator can be combined with the pharmacologically active multivalent ions released earlier. Active multivalent ions chelate and thereby produce therapeutic effects.
  • the big ball disintegrates first for 10 minutes, and then the small ball disintegrates again.
  • bilayer or trilamellar liposomes refer to a process that uses bilayer or trilamellar liposomes to simultaneously coat and deliver three different drugs, with a structure of multiple lipid layers.
  • the stability of individual drugs can be enhanced: each drug is encapsulated within its own liposome layer, protecting it from degradation, chemical interactions and enzymatic degradation; or drug release can be controlled: lipid bilayers
  • the body structure allows for individual release and control of each drug. By adjusting the composition and structure of liposomes, independent release of each drug can be achieved, thereby precisely controlling the release rate and time of the drug. This will help maintain the stability and integrity of the drug during storage and transportation.
  • bilayer or trilayer liposomes made by adjusting the preparation process of the lipid components of the inner and outer layers can be beneficial to the control and regulation of the drug delivery process, such as disintegrating the drug first to release pharmacologically active multivalent ions. It first enters the body to increase the concentration in the body, and then chelates with the ion chelating agent released later to produce effects, etc. Therefore coating with liposomes of different sizes is a strategy that can achieve the gradual release of drugs.
  • Exemplary suitable cancers for the pharmaceutical kits and/or pharmaceutical compositions of the present invention include malignant tumors, such as solid tumors or hematological malignancies.
  • Suitable cancers include, but are not limited to, gastric cancer, lung cancer, bladder cancer, breast cancer, pancreas cancer, kidney cancer, colorectal cancer, cervical cancer, ovarian cancer, brain cancer, prostate cancer, liver cancer, melanoma, esophageal cancer, multiple myeloma, and squamous cell carcinoma of the head and neck.
  • the cancer cells of the cancer are resistant to at least one of chemotherapy, radiation therapy, or immunotherapy.
  • triple-negative breast cancer refers to cancer cells that are negative for three hormone receptors: estrogen (ER), progesterone (PR), and human epithelial growth receptor type 2 (HER2), making it difficult to use hormone therapy and target therapy. Treatment options are quite limited.
  • Human breast cancer cell line (MCF7)-resistant type also has similar drug resistance problems.
  • MCF7 Human breast cancer cell line
  • U-87 the human glioblastoma cell line
  • IRESSA Garfitinib
  • ABT-737 Bcl-2 inhibitor
  • Exemplary administration methods of the pharmaceutical kit and/or pharmaceutical composition of the present invention include, but are not limited to, enteral, oral, nasal, parenteral, topical or transmucosal administration, wherein parenteral administration can be intratumoral, intramuscular, Intravenous, intraarterial, subcutaneous, intraperitoneal, intracranial, intracerebroventricular, or intrathecal injection.
  • mice were purchased from the Animal Experiment Center (Taipei, Taiwan, China) and were given water and feed (LabDiet 5058, PMI Nutrition International Inc, MO, USA) every day. ; 21.56% of energy is provided by fat, metabolizable energy 3.46kcal/gm), free intake, temperature controlled at 22 ⁇ 2°C, humidity 55 ⁇ 15%, light/dark ratio of 12/12 hours (lights on at 1 am, Lights out at 1pm). After raising to 6 weeks of age, experiments were started.
  • mice were further divided into 6 groups in each lung cancer tumor group.
  • S1 Control set, no drug treatment
  • S2 Alone (Niclosamide) group
  • S3 Alone (Disulfiram + pharmacologically active multivalent ions) group
  • S4 Target drug group (IRESSA, Gefitinib)
  • S5 This The effective dose group of the pharmaceutical composition of the invention (Niclosamide+Disulfiram+pharmacologically active multivalent ions)
  • S6 the low-dose group of the pharmaceutical composition of the invention (Niclosamide+Disulfiram+pharmacologically active multivalent ions).
  • A549 group 6 rats in each group, orally administered once a day for 25 consecutive days.
  • H520 group 7 rats in each group, administered orally once a day for 13 consecutive days.
  • the pharmacologically active multivalent ion was copper ion, which was used in the form of copper gluconate.
  • the doses administered to each mouse are: S1: no drug; S2: 2.56 mg; S3: Disulfiram 0.77 mg + pharmacologically active polyvalent ions 0.003 mg, total weight 0.773 mg ; (mg) + Disulfiram 0.38 mg (mg) + pharmacologically active polyvalent ions 0.0015 mg (mg), total weight 0.6815 mg (mg), weigh a predetermined amount of the drug, dissolve it in a physiological saline solvent, and the pharmaceutical composition of the present invention is Dissolve together in physiological saline solvent to form a solution before administration.
  • nude mice had free access to feed and drinking water, and an electronic balance was used to record weekly body weight and food intake.
  • Tumor volume inhibition rate% 1-(tumor volume of drug group)/(tumor volume of control group) ⁇ 100%
  • Tumor mass weight inhibition rate% 1-(tumor weight of drug group)/(tumor weight of control group) ⁇ 100%
  • Cell viability rate % (absorbance value of cells after adding drugs)/(absorbance value of cells without adding drugs) ⁇ 100%
  • the experimental dose concentration of the drug administered to cancer cells is in the nM level, and the effective dose range is 0.8-83 nM.
  • Example 1 Testing the efficacy of different pharmacologically active ions in the pharmaceutical composition of the present invention in human non-small cell lung cancer cell line (A549)
  • Example 2 Efficacy of the pharmaceutical composition of the present invention on human non-small cell lung cancer cell line (A549)
  • the cell survival rate of the lung cancer cell line (A549) was first analyzed. The results are listed in Table 3. As shown in Table 3, the inhibitory effect of the pharmaceutical composition of the present invention on the survival rate of human non-small cell lung cancer cell line (A549) is related to the dosage.
  • the pharmacologically active copper ion used in this experiment is in the form of copper gluconate.
  • the experimental results in Table 3 show that when Niclosamide, Disulfiram and pharmacologically active multivalent ion Cu were used alone in cancer cell line experiments, they did not show effective inhibitory effects.
  • the pharmaceutical composition of the present invention has an inhibitory effect at different doses, and when the concentration of Niclosamide is greater than the concentration of Disulfiram and the pharmacologically active multivalent ion Cu, or the concentration of Disulfiram and the pharmacologically active multivalent ion Cu is greater than the concentration of Niclosamide, it will produce A higher degree of cancer cell inhibition effect, which shows that drugs can produce higher synergistic effects with each other.
  • the pharmaceutical composition of the present invention has the effect of inhibiting the survival rate of cancer cells on lung epithelial cell carcinoma cancer cells. Its inhibitory effect on the survival rate of cancer cells can be compared with the dosage of each drug in the pharmaceutical composition of the present invention. The ratio is positively correlated.
  • Example 3 Inhibitory effect of the pharmaceutical composition of the present invention on cancer cell tumors of human non-small cell lung cancer cell line (A549) in animals
  • Tests were conducted to evaluate whether the pharmaceutical composition of the present invention has an inhibitory effect on human non-small cell lung cancer, whether the drug can effectively inhibit lung cancer tumor growth after being absorbed through the gastrointestinal tract, and whether the drug will cause discomfort.
  • nude mice were injected subcutaneously to establish a tumor model. After 14 days of observation, the tumor size was measured. They were divided into 6 groups and started to be administered. The drug use group was continuously orally fed with test drugs. The changes in tumor growth of nude mice in the tumor model after 25 days of administration were evaluated. , and observe the inhibitory effect of the test drugs on tumors. The results are listed in Tables 4 and 5.
  • Figure 1 shows the correlation between the inhibitory efficacy of the pharmaceutical composition of the present invention and the known target drug group on the cancer cell tumor volume of the human non-small cell lung cancer cell line (A549) in animals and the administration time.
  • Figure 2 shows the correlation between the inhibitory efficacy of the pharmaceutical composition of the present invention and the known target drug group on the tumor weight of cancer cells of the human non-small cell lung cancer cell line (A549) in animals and the administration time.
  • Table 4 Correlation between the pharmaceutical composition of the present invention and known target drug groups on the tumor volume of human non-small cell lung cancer (A549) cancer cells and the number of days of administration.
  • the unit of tumor volume of cancer cells listed in the table is mm 3 .
  • days represent the number of days of administration.
  • Table 4 shows the tumor volume inhibition rate of mice with human non-small cell lung cancer cells (tumor volume of each group at the time of sacrifice compared with the S1 group).
  • the experimental results show that the tumor inhibition rate of the S2 group is 12.78%, and the S3 group has a tumor inhibition rate of 12.78%.
  • the tumor inhibition rate of group S4 was 30.39%, the tumor inhibition rate of group S4 was 47.23%, the tumor inhibition rate of group S5, the effective dose group of the pharmaceutical composition of the present invention was 49.51%, and the tumor inhibition rate of group S6, the low-dose group of the pharmaceutical composition of the present invention was The rate is 44.03%.
  • Table 5 Correlation between the pharmaceutical composition of the present invention and the known target drug group on the tumor mass weight and dosage of human non-small cell lung cancer (A549) cancer cells.
  • the unit of weight of cancer cell tumor mass is grams (g).
  • Table 5 shows the average tumor mass weight inhibition rate of mice with human non-small cell lung cancer cells after being sacrificed (compared to the S1 group).
  • the experimental results show that the tumor inhibition rate of the S2 group is 13.5%, and the S3 group has a tumor inhibition rate of 13.5%.
  • the tumor inhibition rate of group S4 was 48.3%
  • the tumor inhibition rate of group S5 was 51.8%
  • the tumor inhibition rate of group S6 was 51.8%. is 47.4%. Therefore, it can be seen from the experimental results that S5 can produce a more effective cancer inhibitory effect on cancer cells than S4, and the effect of S6 is also more effective than S2 or S3, proving that the pharmaceutical composition of the present invention has a better effect on inhibiting the weight of cancer cell tumor masses. .
  • the pharmaceutical composition of the present invention can effectively inhibit the weight of tumor mass and tumor volume in animals.
  • the pharmaceutical composition of the present invention can effectively absorb through the gastrointestinal tract, so that The drug is clearly targeted to cancer cells and tumor tissues, and the animals do not experience any discomfort during the medication.
  • Example 4 The inhibitory effect of the pharmaceutical composition of the present invention on cancer cell tumors of human lung squamous epithelial cell carcinoma cell line (H520) in animals
  • Tests were conducted to evaluate whether the pharmaceutical composition of the present invention has an inhibitory effect on human lung squamous cell carcinoma, whether the drug can effectively inhibit lung cancer tumor growth after being absorbed through the gastrointestinal tract, and whether the drug will cause discomfort.
  • nude mice were injected subcutaneously to establish a tumor model. After 14 days of observation, the size of the tumors was measured. They were divided into 6 groups and started administration. The drug use group was continuously orally fed with test drugs. Since H520 tumors grow faster in mice, they were The animals were sacrificed when the tumors grew to a certain extent. The tumor model nude mice were sacrificed 13 days after administration. The changes in tumor growth were evaluated and the inhibitory effect of the test drugs on tumors was observed. The results are listed in Tables 6 and 7.
  • Figure 3 shows the correlation between the inhibitory efficacy of the pharmaceutical composition of the present invention and the known target drug group on the cancer cell tumor volume of the human lung squamous epithelial cancer cell line (H520) in animals and the administration time.
  • Figure 4 shows the correlation between the inhibitory efficacy of the pharmaceutical composition of the present invention and the known target drug group on the cancer cell tumor weight of the human lung squamous epithelial cell line (H520) in animals and the administration time.
  • Table 6 Correlation between the pharmaceutical composition of the present invention and the known target drug group on the cancer cell tumor volume of human lung squamous epithelial cell carcinoma (H520) and the number of days of administration.
  • the unit of cancer cell tumor volume is mm 3 .
  • days represent the number of days of administration.
  • Table 6 shows the tumor volume inhibition rate of mice with human lung squamous cell carcinoma (tumor volume of each group at the time of sacrifice compared with the S1 group).
  • the experimental results show that the tumor inhibition rate of the S2 group is 30.49%.
  • the tumor inhibition rate of group S3 was 32.09%, that of group S4 was 28.38%, that of the effective dose group S5 of the pharmaceutical composition of the present invention was 47.30%, and that of the low-dose group S6 of the pharmaceutical composition of the present invention was The inhibition rate was 51.62%.
  • human lung squamous cell carcinoma is highly resistant to the target drug S4 group, so the S4 group has the smallest tumor inhibition rate.
  • the tumor inhibition rates of groups S2 and S3 were lower than those of the pharmaceutical composition of the present invention, proving that the pharmaceutical composition of the present invention has better tumor volume inhibition effect on cancer cells.
  • the low-dose S6 group had the best inhibitory effect on this lung cancer tumor type.
  • Table 7 Correlation between the pharmaceutical composition of the present invention and the known target drug group on the tumor mass weight and dosage of human lung squamous cell carcinoma (H520) cancer cells.
  • the weight of cancer cell tumor mass is measured in grams.
  • Table 7 shows the tumor mass weight inhibition rate of mice with human lung squamous cell carcinoma (compared to the S1 group).
  • the experimental results show that the tumor inhibition rate of the S2 group is 30.5%, and the tumor inhibition rate of the S3 group is 30.5%.
  • the tumor inhibition rate of group S4 was 28.6%
  • the tumor inhibition rate of group S5 the effective dose group of the pharmaceutical composition of the present invention
  • the tumor inhibition rate of group S6 the low-dose group of the pharmaceutical composition of the present invention
  • target drugs have poor therapeutic effect on human lung squamous cell carcinoma, and tumor tissues will become resistant to target drugs.
  • the pharmaceutical composition of the present invention can produce an effective tumor suppressive effect for this cancer treatment, and the tumor tissue does not develop resistance to the pharmaceutical composition of the present invention.
  • the low-dose S6 group had the best effect on this lung cancer tumor type. inhibitory effect.
  • Example 3 According to the analysis results of Example 3 and Example 4, it can be clearly understood that different types of lung cancer tumors will develop resistance to target drugs, resulting in a decrease in the effect of tumor suppressive treatment.
  • the pharmaceutical composition of the present invention has no problem of resistance to lung cancer tumors in the treatment of different types of lung cancer tumors, and shows obvious inhibitory effect.
  • Example 5 The efficacy of the pharmaceutical composition of the present invention on brain cancer human glioblastoma cell line (T98G)
  • the cell survival rate of the human glioblastoma cell line (T98G) was first analyzed. , the results are listed in Table 8.
  • Table 8 Effect of the pharmaceutical composition of the present invention on the survival rate of human glioblastoma cell line (T98G). This cancer cell line is used in brain cancer research.
  • T98G is a human glioblastoma cell line used in brain cancer research and drug development.
  • the results of this experiment show that in Table 8, when Niclosamide, Disulfiram and pharmacologically active multivalent ion Cu were used alone in cancer cell line experiments, they did not show effective inhibitory effects.
  • the pharmaceutical composition of the present invention has an inhibitory effect under different combinations of concentrations, and when the concentration of Niclosamide is greater than the concentration of Disulfiram and pharmacologically active multivalent ions Cu, or the concentration of Disulfiram and pharmacologically active multivalent ions Cu is greater than the concentration of Niclosamide, it will Producing a higher degree of cancer cell inhibitory effect, it can be seen that drugs can produce higher synergistic effects with each other.
  • the survival rate of the pharmaceutical composition of the present invention on human glioblastoma cell line is related to the dosage.
  • the pharmacologically active copper ions used in this experiment are in the form of copper gluconate.
  • the experimental results show that when Niclosamide, Disulfiram, and copper ions are each applied to the T98G cancer cell line at a concentration of 8 to 83 nM, the survival rate of the cancer cells is maintained at a relatively high level. , ranging from 87.87% to 102.19%, with no obvious effect.
  • the survival rate of cancer cells can be reduced to 59.99%, which is a significant decrease.
  • the concentrations of the three drugs are adjusted relative to each other, the survival rate of T98G cancer cells can be reduced to as low as 23.17%. According to this experiment, it is known that the three drugs in the pharmaceutical composition of the present invention can produce a higher degree of cancer cell inhibitory effect and have synergistic effects.
  • the pharmaceutical composition of the present invention has the effect of inhibiting the survival rate of cancer cells in human glioblastoma. Its inhibitory effect on the survival rate of cancer cells can be compared with the dosage ratio of each drug in the pharmaceutical composition of the present invention. There is a positive correlation, which reduces the survival rate of the cancer cells.
  • Example 6 The efficacy of the pharmaceutical composition of the present invention on brain cancer human glioblastoma cell line (U-87)
  • the pharmaceutical composition of the present invention has the effect of inhibiting the survival rate of cancer cells on the brain cancer human glioblastoma cell line (U-87), the cells of the human glioblastoma cell line (U-87) were Survival rates were analyzed and the results are listed in Table 9.
  • Table 9 Effect of the pharmaceutical composition of the present invention on the survival rate of human glioblastoma cell line (U-87). This cancer cell line is used in brain cancer research and breast cancer research.
  • the experimental results show that in Table 9, when Niclosamide, Disulfiram and pharmacologically active multivalent ion Cu were used alone in cancer cell line experiments, they did not show effective inhibitory effects.
  • the pharmaceutical composition of the present invention has an inhibitory effect under different combinations of concentrations, and when the concentration of Niclosamide is greater than the concentration of Disulfiram and pharmacologically active multivalent ions Cu, or the concentration of Disulfiram and pharmacologically active multivalent ions Cu is greater than the concentration of Niclosamide, it will Producing a higher degree of cancer cell inhibitory effect, it can be seen that the drugs in the pharmaceutical composition of the present invention can produce higher synergistic effects with each other.
  • the inhibitory effect of the pharmaceutical composition of the present invention on the survival rate of human glioblastoma cell line (U-87) is related to the dosage.
  • the pharmacologically active copper ions used in this experiment are in the form of copper gluconate.
  • the experimental results show that when Niclosamide, Disulfiram, and copper ions are each applied to the U-87 cancer cell line at a concentration of 8 to 83 nM, the survival rate of the cancer cells is maintained at 63.98 % ⁇ 103.97%. When 83 nM of Disulfiram was administered alone, the cancer cell survival rate was 63.98%.
  • the drug concentration can be effectively reduced and the cancer cell inhibition rate can be improved.
  • the survival rate of cancer cells can be reduced to 30.45%. Compared with the group using Disulfiram alone, the survival rate of cancer cells is significantly reduced. .
  • the concentrations of the three drugs were adjusted relative to each other, the survival rate of U-87 cancer cells could be reduced to as low as 30.45%. According to this experiment, it is known that the three drugs in the pharmaceutical composition of the present invention can produce a lower and obvious survival rate of cancer cells at specific concentrations, and the combination of the three drugs can produce a higher degree of synergistic effect.

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Abstract

一种医药组合物，包含(a)癌细胞线粒体的靶向剂、(b)离子螯合剂、和(c)药理活性多价离子。(a)的重量比按医药组合物的总重量计为64.5％～43.7％，(b)的重量比按医药组合物的总重量计为56.2％～16.1％，(c)的重量比按医药组合物的总重量计为19.4％～0.000116％。

Description

医药组合物及其用途 技术领域
本发明大致上涉及一种医药组合物、包含此医药组合物的试剂盒、以及其用途。具体来说，本发明涉及一种可以治疗动物细胞不正常生长的恶性疾病的医药组合物、包含此医药组合物的试剂盒、以及此医药组合物与此医药试剂盒的用途。
 背景技术
癌症是一个全球性的重大健康问题，不仅影响人类，也影响其他物种。恶性肿瘤的发展是由基因突变导致的异常细胞。这些细胞不断***和增殖，形成肿瘤，有能力侵入并摧毁附近正常的组织和器官。
此外，它们还可以通过血液循环或淋巴***传播到远处的器官，或是产生抗药性，这增加了治疗的难度。恶性肿瘤细胞活动力旺盛、具有破坏性、复发率高且难以治愈。癌症的成因非常复杂，至今仍缺乏完全治愈的方法。因此，开发能有效抑制癌细胞的药物对于病患来说至关重要。
常见的抗癌疗法包含外科手术、放射治疗及化学治疗，例如使用癌症治疗靶标药物。不幸地，除了抑制肿瘤生长，这些熟知的治疗方式往往伴随着伤害或毒杀正常组织的风险。此外，设计用以毒杀癌细胞而不产生副作用的替代性疗法则尚在研究及临床前试验阶段。
目前常见且价格最低的癌症治疗靶标药物为艾瑞莎(Iressa)，其使用于治疗上一年需要花费54万台币，即便要使用保健也只能有条件给付并依照各医院订价为准，因此患者在治疗费用负担上是十分沉重的。因此为患者提供更低廉的价格负担的癌症治疗药物且具备较佳治疗效果的药物是十分重要的。
发明内容
因此，本发明提出一种有效对抗恶性肿瘤的医药组合物、包含此医药组合物的试剂盒、以及其治疗动物细胞不正常生长的恶性疾病的用途。本发明医药组合物可以应用于肿瘤学(Oncology)的治疗领域中。本发明所提出的医药组合物设计通过多种现有药物之间的功能性互动来协同干扰癌细胞的生长机制。本发明所提出的医药组合物的制备简单、价格便宜、又能在抑制恶性肿瘤生长方面表现出统计学上显著的功效。此外，还可以分开施用本发明所提出的医药组合物的成分，并在个体中表现出抑制癌细胞的功效。
在一方面，本发明首先提出一种医药组合物，其包含(a)癌细胞线粒体的靶向剂、(b)离子螯合剂、和(c)药理活性多价离子。癌细胞线粒体的靶向剂为Niclosamide。离子螯合剂为Disulfiram。药理活性多价离子选自由镁离子、钙离子、锰离子、亚铁离子、铜离子、锌离子所组成的群组。(a)的重量比按该医药组合物的总重量计为64.5％～43.7％，(b)的重量比按该医药组合物的总重量计为56.2％～16.1％，(c)的重量比按该医药组合物的总重量计为19.4％～0.000116％。
在本发明的一种实施方式中，0.091≤(a)/((b)+(c))≤15.548。
在另一方面，本发明又提出前述医药组合物用于制备用于治疗有需要的个体的恶性肿瘤的药物的用途。
在本发明的一种实施方式中，恶性肿瘤选自由实体瘤(solid tumor)与血液恶性疾病所组成的群组。
在本发明的一种实施方式中，恶性肿瘤选自由肺癌与脑癌所组成的群组。
在本发明的一种实施方式中，(a)每次的施用量为2000mg～100mg，(b)每次的施用量为500mg～128.6mg，(c)每次的施用量为600mg～2.65×10-4mg。
在本发明的一种实施方式中，(c)的施用顺序不晚于(b)的施用顺序。
在本发明的一种实施方式中，药物1天施用不超过2次。
在本发明的一种实施方式中，药物以口服、注射、经皮、或吸入方式施用。
 附图说明
图1显示了本发明的医药组合物与熟知的靶标药物组对人类非小细胞肺癌细胞株(A549)在动物体内的癌细胞肿瘤体积的抑制功效与给药时间的相关性。
图2显示了本发明的医药组合物与熟知的靶标药物组对人类非小细胞肺癌细胞株(A549)在动物体内的癌细胞肿瘤重量的抑制功效与给药时间的相关性。
图3显示了本发明的医药组合物与熟知的靶标药物组对人类肺鳞状上皮细胞癌细胞株(H520)在动物体内的癌细胞肿瘤体积的抑制功效与给药时间的相关性。
图4显示了本发明的医药组合物与熟知的靶标药物组对人类肺鳞状上皮细胞癌细胞株(H520)在动物体内的癌细胞肿瘤重量的抑制功效与给药时间的相关性。
 具体实施方式
除非另有定义，本文使用的所有技术和科学术语具有本领域技术人员通常理解的含义。本文在说明书中所使用的术语只是为了描述具体的实施方式的目的，而不是旨在限制本发明。除非有相反的陈述，在说明书和申请专利范围中使用的术语具有下述含义。
恶性(malignant)肿瘤(tumor,neoplasm)：也被称为癌症，是一种异常细胞生长和***的疾病，其特点是细胞具有无限增殖的潜力，同时还有侵袭周围组织和器官、转移到其他部位的倾向。恶性肿瘤是一种复杂的疾病，可能涉及多种基因变异和分子机制。早期检测、精准诊断和多学科治疗策略对于控制恶性肿瘤的发展至关重要。治疗方法可以包括手术切除、放疗、化疗、靶向治疗和免疫疗法等，取决于患者的具体情况和癌症类型。恶性肿瘤与良性肿瘤不同，后者通常是局限生长，较少侵犯邻近组织，并且很少转移到其他部位。
恶性肿瘤的主要特征包括：
1.无限增殖：恶性肿瘤的细胞失去了对正常细胞生长的限制，它们可以持续无限地***和增殖，形成肿瘤组织。
2.浸润和侵袭：恶性肿瘤的细胞能够侵入周围正常组织和器官，破坏其结构和功能。这种侵袭性可能使手术切除变得困难，也增加了治疗的挑战。
3.转移：恶性肿瘤的细胞有时会通过血液或淋巴***转移到身体其他部位，形成转移瘤(也称为子宫瘤)。这会导致癌症在身体多个部位同时出现，增加了治疗的难度。
4.异常细胞形态：恶性肿瘤的细胞在形态上常常与正常细胞不同，它们可能不规则、变形、大小不均等。
5.细胞异质性：在同一个恶性肿瘤中，细胞的性质和特点可能会有所不同，这被称为细胞异质性。这使得治疗更加复杂，因为不同的细胞亚群可能对治疗反应不同。
6.血管生成：恶性肿瘤通常会诱导新的血管生成，为其提供养分和氧气，从而维持其不断的生长。
药物协同效果：也被称为药物协同作用，是指两种或更多种药物同时使用时，其效果超过了单独使用每种药物的效果之和。在药物治疗中，药物协同效果可以带来更强大、更持久或更广泛的治疗效果，同时可能降低了使用单一药物时可能出现的副作用。药物协同效果可以在不同类型的药物中观察到，包括抗生素、抗癌药物、抗病毒药物等。药物协同效果的含义可以更具体地描述如下：
1.增强疗效：药物协同作用可能导致治疗效果的增强。这意味着在给予多种药物时，其合并效果可能比单一药物更强大，从而更有效地控制或治疗疾病。
2.降低耐药性风险：经常使用单一药物可能导致细菌、病毒或肿瘤等在疾病治疗中产生耐药性。通过使用多种不同机制的药物，可以降低疾病对其中任何一种药物产生耐药性的风险，从而保持治疗的有效性。
3.减少副作用：在一些情况下，使用低剂量的多种药物可以取代使用单一药物的高剂量，从而降低了可能出现的不良副作用。
4.多靶点干预：不同药物可能通过影响疾病的不同靶点或途径发挥作用，通过多重途径来干预疾病，增加了治疗的多样性和全面性。
5.应对复杂疾病：一些复杂的疾病可能涉及多个病理学机制，单一药物难以全面干预。药物协同作用可以在多个水平上影响疾病的不同方面，从而更有效地应对复杂疾病。
血液恶性疾病是指来自不正常增生的白血球细胞，可以包括但不限于白血病(leukemia)或淋巴瘤(lymphoma)。
癌细胞致死率：是指在一定时间内，癌症患者体内的癌细胞被治疗方法(如化疗、放疗、靶向治疗等)杀死的百分比。它通常用于评估治疗方法的效果，以了解治疗对癌症病情的影响。癌细胞致死率的含义可以解释如下：
治疗效果：癌细胞致死率是评估治疗方法对癌症的影响的重要指标之一。更高的致死率通常意味着治疗方法在杀死癌细胞方面更为有效。
疾病控制：通过监控癌细胞致死率，可以了解治疗是否可以有效地控制或减少癌症的扩散和进展。如果致死率较低，可能需要重新评估治疗方法以达到更好的控制效果。
预测患者预后：更高的癌细胞致死率可能与患者的预后改善相关。癌细胞被有效地杀死可能降低疾病复发和进展的风险，从而提高患者的生存率。
治疗选择：癌细胞致死率可以帮助医疗专业人员评估不同治疗方法的优劣。治疗方法的选择通常会考虑到治疗的致死率以及可能的副作用、耐药性等因素。
治疗监测：跟踪癌细胞致死率可以帮助医疗专业人员监测治疗的进展。如果致死率在一段时间内没有明显改善，可能需要考虑调整治疗方案。
“治疗”一词包含部分或完全预防、改善、减轻和/或处理与癌症相关的病征(symptom)、次要病征(secondary disorder)或症状(condition)。“治疗”一词在此说明书中也是指将本发明的一种或多种药物应用或施用于个体，该个体患有与癌症相关的病征、次要病征或症状，以达到部分或完全减轻、减缓、治愈疾病、延迟发病、抑制病程发展、降低疾病严重性，和/或降低一个或多个与癌症相关的病征、症状、或次要病征的发生。与癌症相关的病征、次要病征和/或症状包括，但不限于，发烧、虚弱、疲倦、体重减轻、疼痛、咳嗽、出血、皮肤改变、腹泻或便秘、恶心、呕吐及食欲不振。在此“治疗”也可以是施用于患有早期征或症状的个体，以降低此个体发展成为与癌症相关的病征、次要病征和/或症状的风险。在此“治疗”可以为有效地减少一个或多个病征或临床标记。换句话说，在此“治疗”也可以是降低、减缓或终止疾病病程、病征或症状的发展。
“有效量”(effective amount)在此处是指药物的用量足以产生预定的疗效反应。有效量也是指一种化合物或组合物的量，其治疗利益效果超越其毒性或有害影响。具体的有效量取决于多种因素，如欲治疗的特定状况、患者的生理条件(如，患者体重、年龄或性别)、接受治疗的哺乳动物或动物的类型、治疗持续时间、目前疗法(若适用时)的本质以及所用的具体配方和化合物或其衍生物的结构。举例来说，可将有效量表示为药物的总重量(例如以克、毫克或微克为单位)或表示为药物重量与体重的比例(其单位为毫克/公斤(mg/kg))。或者是，可将有效量表示为活性成分(例如，本发明药剂)的浓度，例如摩尔浓度、重量浓度、体积浓度、重量摩尔浓度、摩尔分数、重量分数及混合比值。具体来说，“治疗有效量”(therapeutically effective amount)一词，是指药物的施用量，其足以减缓或减轻个体与癌症相关的病征。技术人员可依据动物模式的剂量来计算药物(如本发明药剂)的人体等效剂量(human equivalent dose,HED)。举例来说，技术人员可依据美国食品药物管理局(US Food and Drug Administration,FDA)所公告的“估算成人健康志愿者在初始临床治疗试验的最大安全起始剂量”(Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers)来估算人体使用的最高安全剂量。
如本文所用，术语“体外”(in vitro)是指在人工环境中，例如在试管或反应容器中，在细胞培养物中等，而不是在生物体内发生的事件。
如本文所用，术语“体内或者活体内”(in vivo)是指在生物体内，例如人体内细胞和非人动物内细胞内发生的事件。在基于细胞的叙述的表述中，可指在活细胞内发生的事件(与例如体外***相反)。
如本文所用，术语“个体”可以是指例如出于实验目的、诊断目的、预防目的、美容目的和/或治疗目的向其施用所提供的药物的任何生物体。典型的个体可以包括动物，例如哺乳动物，诸如小鼠、大鼠、兔子、非人类灵长类和/或人类。在一些实施方式中，个体可以是人类。
如本文所用，术语“药学上可接受的”或“药理活性”，可以指与合理的受益/风险比相称在合理的医学判断范围内，适用于与人和动物的组织接触，而没有过度毒性、刺激性、过敏反应或其他问题或并发症的物质。
如本文所用，术语“预防有效量”可以指当施用于个体时，基于不同敏感程度的个体或癌细胞足以预防和/或延迟症状发作的量。本领域的一般技术人员可以理解，通常通过包含至少一个单位剂量的给药方案来施用预防有效的剂量。
虽然用以界定本发明较广范围的数值范围与参数都是大致的数值，此处已尽可能精确地呈现具体实施例中的相关数值。除非另有说明，此处所述的数值范围都包含端点。
在本发明中，“周期”(cycle)、“治疗周期”(cycle of treatment或treatment cycle)为可互换的词汇，是指对病患施用治疗的一段时间。一般来说，治疗癌症时，完成一个治疗周期后会接续一段完全不给予任何治疗的休息时间。在此休息时间后，可再次施用一个或多个治疗周期，每个治疗周期后都会接续一段休息时间。
药物再利用是指利用现有的药物进行新的治疗应用。基于这些药物已经获得其他治疗方面的批准，这种方法可以减少开发所需的成本和时间，以及上市后产生不良药物反应的风险。
在癌症治疗中，已知药物再利用结合药物组合策略的方法，相比传统的新靶标药物有显著的优势，主要原因如下：
1.已知药物利用新的药物组合策略模式，产生新的恶性肿瘤治疗应用，药物组合有助于不同药物之间的相互作用。
2.药物组合可以发挥药物之间的协同效应，干扰癌细胞的生长机制。
3.重新利用使用已久的已知临床药物以找出新的治疗应用，这些药物有丰富的临床数据和相对较高的安全性。
4.通过不同的药理机制来抑制与癌症有关的多个信号通路分子。
5.提高治疗效果的同时降低个别药物的原有的使用剂量，使治疗更加安全。
6.组合药物治疗恶性肿瘤的成本远低于靶标药物治疗恶性肿瘤的成本。
本发明药物在治疗癌症上的成本可以远低于目前所有保健给付靶标药物的自费价格。例如以治疗癌症靶标药物目前最低价格的IRESSA(Gefitinib)来说，即使在目前中国台湾全民保健给付的辅助下，对于IRESSA单粒药物患者仍然需要支付573元，并且天天连续使用。而本发明药物用于治疗癌症的单粒药物价格仅需要184元。本发明药物的费用虽尚无保健给付补助，若未来可以获得保健补助，则可以大量减轻患者的医药负担和治疗费用。
出乎意料地，根据本发明的多种实施方式，本发明的发明人提供一种有效对抗恶性肿瘤的医药组合物。本发明的医药组合物中的各有效成分为现有的已知药物，这些已知药物已有丰富的临床数据和相对较高的安全性。与靶标药物相比，利用已知药物再结合药物组合策略的方法具有显著的多种优势。
本发明首先提出一种医药组合物，其包含(a)癌细胞线粒体的靶向剂、(b)离子螯合剂、和(c)药理活性多价离子等药物。三种药物形成一种药物组合，且三种药物的比例设计可以依照不同治疗效果进行剂量调整。(a)、(b)(c)等药物的治疗剂量单位为毫克(mg)。
癌细胞线粒体的靶向剂可以为Niclosamide。Niclosamide可以靶向癌细胞中的线粒体，以诱导细胞周期停滞、抑制细胞生长和诱导细胞凋亡。在GlobalData数据中，在单一使用治疗癌症上，并无任何公司开发完成Niclosamide进入癌症的治疗市场。按医药组合物的总重量计，(a)的重量比为64.5％～43.7％。Niclosamide的剂量范围是，在七天中每日使用，每日使用不超过2次。Niclosamide的有效剂量是857.2mg。Niclosamide的治疗剂量范围为2000mg～100mg。
离子螯合剂可以为Disulfiram。Disulfiram可以诱发内质网应激和自噬以诱导细胞凋亡，减少血管生成，并可作为放射治疗的增敏剂，或离子螯合物。在单一使用治疗癌症上，并无任何公司开发完成Disulfiram进入癌症的治疗市场。按医药组合物的总重量计，(b)的重量比为56.2％～16.1％。Disulfiram每次给药的上限剂量是500mg，下限剂量是128.6mg。Disulfiram的有效剂量是257.2mg。Disulfiram的治疗剂量范围为500mg～128.6mg。
药理活性多价离子为药学上可接受的营养添加剂类型的金属离子及其盐类，例如氯离子、氧离子、氢氧离子、葡萄糖酸根、硫酸根、甘氨酸根、琥珀酸根、硬脂酸根、甘油磷酸根、醋酸根、乳酸根、磷酸根、硼酸根、碳酸根、反丁烯二酸根、戊二酸根、果糖硼酸根、胆酸根、苹果酸根、甘油酸根、草酸根、酒石酸根、柠檬酸根、HAP螯合离子、HVP螯合离子…等，但本发明不以此为限。多价活性离子为二价或以上的典型元素离子，或过渡元素离子。药理活性多价离子例如为镁离子、钙离子、铬离子、锰离子、亚铁离子、铜离子、锌离子中的至少一种。按医药组合物的总重量计，(c)的重量比为19.4％～0.000116％。药理活性多价离子的剂量范围是1800mg～2.65×10-4mg。药理活性多价离子的有效剂量是0.139mg～5.3×10-3mg。例如，作为离子螯合剂时，两个Disulfiram可以与一个药理活性多价离子形成药理活性螯合物。表一列出不同的药理活性多价离子的剂量范围。
表一不同的药理活性多价离子的剂量范围
本发明的医药组合物中的(a)成分、(b)成分和(c)成分，可以视情况需要具有不同的调配剂量比例范围或组合。
在本发明的一种实施方式中，(a)与(b)项的剂量比例可以是 比例范围。
在本发明的另一种实施方式中，(a)与(c)项的剂量比例可以是比例范围。
在本发明的另一种实施方式中，(a)与(b)+(c)项的剂量比例可以是比例范围。
在本发明的另一种实施方式中，(a)+(b)与(c)项的剂量比例可以是比例范围。
在本发明的另一种实施方式中，(a)+(c)与(b)项的剂量比例可以是比例范围。
在本发明的另一种实施方式中，(b)与(a)项的剂量比例可以是 比例范围。
在本发明的另一种实施方式中，(b)与(c)项的剂量比例可以是比例范围。
在本发明的另一种实施方式中，(b)与(a)+(c)项的剂量比例可以是比例范围。
在本发明的另一种实施方式中，(b)+(c)与(a)项的剂量比例可以是比例范围。
在本发明的另一种实施方式中，(c)与(a)项的剂量比例可以是13.35×比例范围。
在本发明的另一种实施方式中，(c)与(b)项的剂量比例可以是5.3×比例范围。
在本发明的另一种实施方式中，(c)与(a)+(b)项的剂量比例可以是比例范围。
本发明的医药组合物，以上述(a)、(b)和(c)的剂量比例范围可以产生对应的癌细胞致死率的功效。
在本发明的一种实施方式中，当小鼠实验的癌细胞致死率为46％。
在本发明的一种实施方式中，当小鼠实验的癌细胞致死率为50％。
在本发明的一种实施方式中，当细胞实验的癌细胞致死率为44.3％。
在本发明的一种实施方式中，当细胞实验的癌细胞致死率为58％。
在本发明的一种实施方式中，当细胞实验的癌细胞致死率为86.5％。
本发明的医药组合物中的各药剂成分可视情况需要安排多种不同的给药顺序。依据本发明的实施方式，第一药剂可为(c)药理活性多价离子，第二药剂可为(b)离子螯合剂，而第三药剂可为(a)癌细胞线粒体的靶向剂。第一药剂(c)药理活性多价离子的给药顺序不晚于第二药剂(b)离子螯合剂的给药顺序，有助于药理活性多价离子提升动物体内离子浓度。第三药剂(a)癌细胞线粒体的靶向剂的给药顺序无特别限制。
在本发明的一些实施方式中，第一药剂(c)、第二药剂(b)、第三药剂(a)的给药顺序可以有下列的可行方式：
1.先(a)后(c)再(b)。
2.先(a)+(c)一起，然后(b)。
3.先(c)后(b)再(a)。
4.先(c)+(b)一起，然后(a)。
5.(a)、(c)、(b)同时给药。
视情况需要，本发明的医药组合物中可以更进一步包含一种或多种稳定剂、结合剂、填充剂、崩散剂、赋形剂、和/或添加剂。
本发明的医药组合物，还可以制成适合不同给药顺序的医药试剂盒(pharmaceutical kit)。例如，将本发明的医药组合物制成一种用于治疗患有或疑似患有癌症的个体的医药试剂盒。本发明的医药试剂盒包含第一容器、视情况需要的第二容器、视情况需要的第三容器、和/或视情况需要的第四容器。第一容器、视情况需要的第二容器与视情况需要的第三容器分别包含第一药剂、第二药剂与第三药剂中的至少一种。依据本发明实施方式，第一药剂可为(c)药理活性多价离子，第二药剂可为(b)离子螯合剂，而第三药剂可为(a)癌细胞线粒体的靶向剂。
本发明的另一方面是关于一种使用本发明医药试剂盒来治疗患有或疑似患有癌症的个体的方法。此方法包含对个体施用第一容器中的本发明药剂；对此个体施用视情况需要的第二容器中的本发明药剂；以及对此个体施用视情况需要的第三容器中的本发明药剂。第一容器包含第一药剂、第二药剂与第三药剂中的至少一种。视情况需要的第二容器包含第一药剂、第二药剂与第三药剂中的至少一种。视情况需要的第三容器包含第一药剂、第二药剂与第三药剂中的至少一种。依据本发明实施方式，第一药剂可为(c)药理活性多价离子，第二药剂可为(b)离子螯合剂，而第三药剂可为(a)癌细胞线粒体的靶向剂。
本发明第一药剂(c)药理活性多价离子的第一有效量的范围可以是1800mg～2.65×10-4mg。第二药剂为(b)离子螯合剂，其第二有效量的范围可以是500mg～128.6mg。本发明第三药剂包含(a)癌细胞线粒体的靶向剂，其第三有效量的范围可以是2000mg～100mg。
可以使用例如玻璃、脂质体或塑料等不同材料来制备适合用以承载本发明药剂的第一容器、视情况需要的第二容器及视情况需要的第三容器。第一容器可承载有效量的本发明药剂或其药学剂型。第二容器可承载有效量的本发明药剂或其药学剂型。第三容器可承载有效量的本发明药剂或其药学剂型。本发明医药试剂盒还可包含随附于第一容器和/或第二容器和/或第三容器的使用说明。此外，本发明医药试剂盒可视情况需要还包含第四容器，其中有药学上可接受的缓冲液，例如磷酸盐缓冲生理食盐水、林格氏液或葡萄糖溶液。其还可包含其他商业或使用者所需要的材料，包含其他缓冲液、稀释液、过滤器、针头及注射器。
本发明的另一方面是关于一种使用本发明医药试剂盒来治疗有需要的个体(例如，患有癌症的个体，或疑似患有癌症的个体)的给药方法。此方法包括：对个体施用第一有效量的本发明第一容器；以及对此个体施用第二有效量的视情况需要的本发明第二容器；或对此个体施用第三有效量的视情况需要的本发明第三容器。对此个体的第一容器的第一施用时间点可以不晚于对此个体的第二容器的第二施用时间点，而对此个体的第三容器的第三施用时间点可以不早于第二容器的第二施用时间点，第三施用时间点可以不早于第二施用时间点10分钟。本发明医药试剂盒中的药物，一天中施用的次数不超过2次。
在第一施用时间点，对此个体施用本发明第一容器，以脂质体(Liposome)、胶囊或片剂形式给药。在第二施用时间点，对此个体施用本发明第二容器，以脂质体、胶囊或片剂形式给药。在第三施用时间点，对此个体施用本发明第三容器，以脂质体、胶囊或片剂形式给药。依据一些实施方式，此个体为人类，例如为体重60公斤的人类，对此个体以每日每公斤个体体重10毫克到4.42×10-6毫克的剂量施用第一药剂，对此个体以每日每公斤个体体重8.33毫克到2.14毫克的剂量施用第二药剂，并对此个体以每日每公斤个体体重33.33毫克到1.67毫克的剂量施用第三药剂。依据本发明的一个操作实施例，以51.67毫克到3.81毫克/公斤/日的剂量施用本发明医药组合物，即足以降低此个体的癌细胞数量，从而表现出本发明医药组合物的抗癌功效。
在本发明的一些实施方式中，第一容器包含第三药剂，第二容器包含第一药剂，第三容器包含第二药剂。在第一施用时间点，对此个体先施用第一容器中的本发明药剂；在第二施用时间点，对此个体施用第二容器中的本发明药剂；以及在第三施用时间点，对此个体施用第三容器中的本发明药剂，使得使用本发明医药试剂盒来治疗患有或疑似患有癌症的个体的方法的药剂施用顺序为先(a)后(c)再(b)。
在本发明的一些实施方式中，第一容器包含第一药剂，第二容器包含第二药剂，第三容器包含第三药剂。在第一施用时间点，对此个体先施用第一容器中的本发明药剂；在第二施用时间点，对此个体施用第二容器中的本发明药剂；以及在第三施用时间点，对此个体施用第三容器中的本发明药剂，使得使用本发明医药试剂盒来治疗患有或疑似患有癌症的个体的方法的药物施用顺序为先(c)后(b)再(a)。
在本发明的一些实施方式中，第一容器包含第一药剂与第三药剂，第二容器包含第二药剂。在第一施用时间点，对此个体先施用第一容器中的本发明药剂；以及在第二施用时间点，对此个体施用第二容器中的本发明药剂，使得使用本发明医药试剂盒来治疗患有或疑似患有癌症的个体的方法的药物施用顺序为(a)与(c)同时施用后再施用(b)。
在本发明的一些实施方式中，第一容器包含第一药剂与第二药剂，第二容器包含第三药剂。在第一施用时间点，对此个体先施用第一容器中的本发明药剂；以及在第二施用时间点，对此个体施用第二容器中的本发明药剂，使得使用本发明医药试剂盒来治疗患有或疑似患有癌症的个体的方法的药物施用顺序为(c)与(b)同时施用后再施用(a)。
在本发明的一些实施方式中，第一容器包含第一药剂、第二药剂与第三药剂。在第一施用时间点，对此个体施用第一容器中的本发明药剂，使得使用本发明医药试剂盒来治疗患有或疑似患有癌症的个体的方法的药物施用顺序为(a)与(b)与(c)同时施用。
关于本发明医药组合物的释放形式，可以以双层或三层片剂进行压片，内层片剂通过缓释赋形剂结合药物压片而制成，外层片剂通过速崩赋形剂结合优先释放药物压片而制成，再用外层包裹内层，制成最终片剂。其释放药物速度为最外层最快，越内层释放速度递减。利用崩解速度不同，可以使药理活性多价离子先进入体内，提高体内浓度，进而再与后崩解释放的离子螯合剂进行螯合以产生作用等。
快速崩解释放剂型的赋形剂
药物的快速崩解释放剂型旨在快速释放药物，以实现迅速的药效。在本发明的一些实施方式中，赋形剂可用于制备快速崩解释放剂型，例如但不限于：
1.乳糖(Lactose)：乳糖是一种常见的辅助赋形剂，常用于制备口服片剂。它具有优异的压片性能和快速崩解特性，可促进药物的快速释放。
2.玉米淀粉(Corn starch)：玉米淀粉是一种常见的天然多糖赋形剂，具有良好的可压性和崩解性能。它可以用于制备快速崩解片剂和颗粒剂。
3.微晶纤维素(Microcrystalline cellulose)：微晶纤维素是一种由纤维素处理得到的微细结晶赋形剂，常用于制备口服片剂。它能增加片剂的机械强度并促进药物的快速释放。
4.水溶性聚合物：一些水溶性聚合物，如羟丙基甲基纤维素(Hydroxypropyl methylcellulose,HPMC)、聚乙二醇(Polyethylene glycol,PEG)等，可以在制备快速崩解剂型时作为崩解剂使用。它们能与水迅速相互作用，从而促使片剂迅速崩解并释放药物。
5.蛋白质赋形剂：某些蛋白质，如明胶(Gelatin)和羧甲淀粉钠(Sodium starch glycolate)，能够在湿润条件下快速膨胀和崩解，从而实现药物的快速释放。这些赋形剂可用于制备各种快速崩解剂型，例如快速崩解片剂、崩解颗粒剂和崩解胶囊等。
缓释剂型的赋形剂
药物的缓释剂型通常使用一些特殊的赋形剂。在本发明的一些实施方式中，这种赋形剂可以在体内延长药物的释放时间，使药物以稳定的速率进入血液或目标组织，例如但不限于：
1.聚合物：聚合物是常见的赋形剂，如聚乙二醇(Polyethylene glycol,PEG)、聚乳酸-共-聚羟基乙酸(Poly(lactic-co-glycolic acid),PLGA)等。这些聚合物可以形成药物载体，使药物以控制的速率释放。
2.硅酸盐：硅酸盐赋形剂，如二氧化硅(Silica dioxide)和氢氧化铝(Aluminum hydroxide)的复合物，可以通过形成药物包覆层或网络结构，推迟药物的释放速度。
3.聚乳酸(Polylactic acid,PLA)和聚乳酸-共-聚羟基乙酸(Polylactic-co-glycolic acid,PLGA)微球：这些微球可以作为缓释剂型的载体被注射或口服。可以根据不同的需求调整微球的大小和结构，从而控制药物的释放速率。
4.脂质体：脂质体是由脂质双层组成的微小颗粒，可以包裹药物并形成稳定的载体。脂质体可以用于口服、注射或局部应用，以实现药物的缓慢释放。
5.聚合脂质体：聚合脂质体是脂质体的改进型，通过添加聚合物或高分子物质，可以进一步调节药物的释放速率和稳定性。
本发明医药组合物的释放形式不限于前述可用于制备缓释剂型的药物的赋形剂和技术。可以视药物的特性、治疗需求和给药途径等因素选择合适的赋形剂。
在本发明的一些实施方式中，本发明的医药组合物在电中性、负电性、或正电性的脂质体影响下对于癌症或肿瘤细胞的作用可能有差异。从两个方面讨论不同带电性的脂质体作用于细胞的差异：
一、脂质体与细胞的相互作用，
二、脂质体对细胞(药物)递送(delivery)的影响。
这些差异及影响分为以下几个方面说明。
相互作用机制：带正电的脂质体与带负电的细胞膜之间存在静电相互吸引力，使得正电性脂质体更容易与细胞膜结合，而负电性脂质体则相对较难。电中性脂质体没有明显的静电相互作用。这种相互作用机制决定了不同带电性脂质体与细胞之间的亲和性和摄取(uptake)效率。
细胞摄取效率：正电性脂质体由于与细胞膜的负电荷相互作用，能够促进脂质体的细胞内摄取。细胞摄取主要通过内吞作用进行，正电性脂质体与细胞膜结合后能够被主动或被动地摄入细胞内。相比之下，负电性脂质体由于与细胞膜的静电排斥作用，摄取效率相对较低。
细胞内递送效率：正电性脂质体能够促进药物或基因等载荷物质的释放和递送到细胞内部，因为它们可以与细胞内的负电荷分子相互作用，如核酸、蛋白质等。负电性脂质体也可以在一定程度上递送药物或载荷物质，但相对于正电性脂质体来说，负电性脂质体更适合用于稳定和保护药物，而不太适合直接递送到细胞内。电中性脂质体与细胞膜之间没有电荷相互作用，因此在某些参数下可以降低与细胞的非特异性相互作用，减少吸附和被吞噬的可能性。这有助于提高脂质体在体内的稳定性和生物可用性，并通常具有较高的药物载荷能力，可以更有效地包覆和稳定各种类型的药物(水溶性和脂溶性)。
在本发明的一些实施方式中，制备正电性脂质体的材料，例如但不限于DOTAP(Dioleoyl-3-trimethylammonium propane)。
在本发明的一些实施方式中，制备负电性脂质体的材料，例如但不限于DOPG(dioleoylphosphatidylglycerol，二油酰磷脂酰甘油)。
在本发明的一些实施方式中，制备电中性脂质体的材料，例如但不限于DSPC(Distearoylphosphatidylcholine，二硬脂酰磷脂酰胆碱)。
在本发明的一些实施方式中，其他用于制备脂质体的成分，例如但不限于胆固醇、聚乙二醇(PEG)、表面活性剂(如Tween,Span)、辅助剂(如卵磷脂酰丝胺酸、卵磷脂酸)、脂质体包覆剂(如卵磷脂酸胆碱)、靶向配体(如抗体、配体)、染料(如荧光染料、核酸染料)、脂质体稳定剂(如脂质体结构调节剂、防腐剂)。
在本发明的一些实施方式中，可以以大小球(ball-in-ball)进行药剂的包覆。例如，先以小球脂质体包覆两种药物：离子螯合剂+癌细胞线粒体的靶向剂，再将该小球脂质体与药理活性多价离子以大球脂质体包覆，形成两层式脂质体。大球可以先崩解使药理活性多价离子先释放出以提升浓度，之后小球再崩解释放出离子螯合剂+癌细胞线粒体的靶向剂，其中的离子螯合剂可以与早先释放出的药理活性多价离子产生螯合进而产生治疗作用。例如大球先崩解10分钟，然后小球再崩解。
在本发明的一些实施方式中，双层或三层脂质体指利用双层或三层脂质体来同时包覆和递送三种不同药物的工艺，具有多个脂质层的结构。例如，可增强各个药物的稳定性：每种药物都被封装在自己的脂质体层内，保护其免受降解、化学相互作用和酶降解的影响；或者是控制药物释放：双层脂质体结构允许对每种药物分别释放并进行控制。通过调整脂质体的成分和结构，可以实现各药物的独立释放，从而精确控制药物的释放速率和时间。这将有助于在储存和运输过程中维持药物的稳定性和完整性。适用于药物递送，以增加药物的稳定性，减少药物分解或失活的可能性，并提高药物生物利用率。因此，通过调整内外层的脂质成分的制备工艺所制成的双层或三层脂质体，可有利于药物递送过程的控制和调节，例如使药物先崩解释放出药理活性多价离子，使其先进入体内提高体内浓度，再与后释放的离子螯合剂进行螯合以产生作用等。因此使用不同大小的脂质体进行包覆是一种策略，可以实现药物的逐步释放。通过先用小的脂质体包覆其中一种药物，再用大粒径的脂质体包覆药理活性多价离子和小粒径的脂质体。在释放的过程中，大的脂质体可以先崩解，使其中的药理活性多价离子发挥提升体内浓度作用，再与小的脂质体稍后崩解释放出的离子螯合剂发生螯合反应产生作用，延迟药物的释放作用，以优化治疗效果和减轻药物副作用。
本发明医药试剂盒和/或医药组合物的例示性适合的癌症包括恶性肿瘤，例如实体瘤或血液恶性疾病。适合的癌症例如但不限于，胃癌、肺癌、膀胱癌、乳癌、胰脏、肾脏癌、结肠直肠癌、子***、卵巢癌、脑癌、***癌、肝癌、黑色素瘤、食道癌、多发性骨髓瘤，以及头颈部鳞状细胞癌。依据本发明的某些实施方式，此癌症的癌细胞对于化学治疗、放射治疗或免疫治疗中的至少一种具有抗性。例如，三阴性乳癌是指癌细胞对***(ER)、黄体素(PR)和第二型人上皮生长受体(HER2)三种激素受体呈现阴性，而难以采用激素疗法以及靶标治疗，治疗方式相当有限。人类乳腺癌细胞株(MCF7)-耐药型也有类似的抗药性问题。或者是，已知人类胶质母细胞瘤细胞株(U-87)对于细胞凋亡的抗性较高，对于靶标药物IRESSA(Gefitinib)及ABT-737(Bcl-2抑制剂)等的抗性较高(Chang et al.,2011；Cristofanon and Fulda,2012；Jane et al.,2013)。还有，人类肺鳞状上皮细胞癌(H520)的肿瘤组织对于靶标药物IRESSA会产生抗性，所以靶标治疗的效果较差。
本发明医药试剂盒和/或医药组合物的示例性给药方式包括，但不限于，肠内、口服、鼻腔、非口服、局部或经黏膜施用，其中非口服可以是肿瘤内、肌肉内、静脉内、动脉内、皮下、腹腔内、颅内、脑室内或鞘内注射。
实验步骤
动物试验材料与方法
实验动物
[根据细则26改正 05.09.2023]
5周龄的雌性、Balb/c CAnN.Cg-Foxnlnu/CrlNarl裸鼠，购自动物实验中心(中国台湾，台北市)，每日给予水及饲料(LabDiet 5058,PMI Nutrition International Inc,MO,USA；21.56％能量由脂肪提供，代谢能为3.46kcal/gm)，自由摄取，温度控制于22±2℃，湿度55±15％，光照/黑暗比例为12/12小时(上午1时开灯，下午1时关灯)。饲养至6周龄后，开始进行实验。
诱导癌症肿瘤动物模型
对于6周龄的裸鼠，开始建立两种肺上皮细胞癌肿瘤及动物模型。本实验使用食品工业发展研究所(中国台湾，新竹市)提供人类非小细胞肺癌细胞株(A549)、人类肺鳞状上皮细胞癌细胞株(H520)，诱导方式为将100μL的含有1×107A-549细胞悬浮液或H520细胞悬浮液皮下注射至右后肢近背侧部分，给予A549细胞与给予H520细胞后2周，观察肿瘤至直径5mm至6mm时，进行分组并开始给药，即成功建立诱导肺上皮细胞癌肿瘤小鼠模型。
试验产品给药
[根据细则26改正 05.09.2023]
建立两类肺癌肿瘤即人类非小细胞肺癌细胞株(A549)、人类肺鳞状上皮细胞癌细胞株(H520)小鼠模型后，在各个肺癌肿瘤组中，将小鼠再分成6组。S1：为对照组(Control set)，不使用药物治疗；S2：单独(Niclosamide)组；S3：单独(Disulfiram+药理活性多价离子)组；S4：靶标药物组(IRESSA,Gefitinib)；S5：本发明医药组合物有效剂量组(Niclosamide+Disulfiram+药理活性多价离子)；S6：本发明医药组合物低剂量组(Niclosamide+Disulfiram+药理活性多价离子)。A549组：每组6只，口服，一天一次，连续25天。H520组：每组7只，口服，一天一次，连续13天。在本实验中药理活性多价离子为铜离子，以葡萄糖酸铜形式使用。每只小鼠给药剂量分别为：S1：不使用药物；S2：2.56毫克(mg)；S3：Disulfiram 0.77毫克(mg)+药理活性多价离子0.003毫克(mg)，总重0.773毫克(mg)；S4：0.6毫克(mg)；S5：Niclosamide 2.57毫克(mg)+Disulfiram 0.77毫克(mg)+药理活性多价离子0.003毫克(mg)，总重3.343毫克(mg)；S6：Niclosamide 0.3毫克(mg)+Disulfiram 0.38毫克(mg)+药理活性多价离子0.0015毫克(mg)，总重0.6815毫克(mg)，称取预定量的药物，溶于生理盐水溶剂中，本发明医药组合物为共同溶解生理食盐水溶剂中，形成溶液后给药。
体重及摄食量观察
实验期间裸鼠饲料和饮水自由摄取，利用电子天平记录每周体重、食物摄取量。
肿瘤体积、重量及抑制率测量评估
利用肿瘤测量仪(TM900,Peira,Turnhout,Belgium)每周测量肿瘤体积，给药五周后将裸鼠处死，取出右后肢处肿瘤，肿瘤体积大小以测量外观最长径及最短径，所采用的肿瘤体积计算公式：体积＝0.5×最长径×最短径2，来计算肿瘤实际体积，再将肿瘤团块称重并记录，以对照组为基准，计算各组肿瘤抑制率。
肿瘤体积抑制率％＝1-(药物组肿瘤体积)/(对照组肿瘤体积)×100％
肿瘤团块重量抑制率％＝1-(药物组肿瘤重量)/(对照组肿瘤重量)×100％
癌细胞试验材料与方法
细胞培养(Cell culture)
将人类非小细胞肺癌细胞株(A549)、人类胶质母细胞瘤细胞株(U87)、人类胶质母细胞瘤细胞株(T98G)、人类永生化骨髓性白血病细胞株(K-562)、人类单核白血球细胞株(THP-1)、人类肝癌细胞株(HepG2)、人类大肠直肠癌细胞株(HCT116)、人类卵巢癌细胞株(TOV-21G)、人类三阴性乳癌细胞株(MDA-MB-231)或人类乳腺癌细胞株(MCF7)-耐药型，培养在含10％(v/v)胎牛血清(Fetal Bovine Serum,Penicillin-Streptomycin Solution)(100X)(ACE Biolabs,CC1009)及含有Glutamine的Nutrient Mixture F-12 Ham Kaighn’s Modification(F-12/K)(SIGMA-ALDRICH,USA)以及RPMI-1640(HiMedia Laboratories LLC,USA)培养基中，之后将各个细胞株放入5％CO2、37℃的培养箱(Astec-SCA-165DS)。
细胞活力测验(Cell Viability)
将不同癌细胞株接种到96孔(well)中，使细胞贴附于底盘24小时后，经由三种药物联合处理，无药物处理的组别为对照组，加入20μL MTS试剂(Progema,USA)，在37℃下放置1小时，使用ELISA检测仪(BioTeck EPOCH2)，在490nm的波长下检测吸光值，评估细胞活性及增殖能力。细胞活力计算公式如下：
细胞活力率％＝(加入药物后的细胞吸光值)/(没有加药物的细胞吸光值)×100％
本发明药物施用于癌细胞株的实验剂量(Drug Dosage)
在本发明的一些实施例中，药物施用于癌细胞的实验剂量浓度为nM级别，其中该有效剂量范围为0.8～83nM。
实施例1在人类非小细胞肺癌细胞株(A549)中测试本发明医药组合物中不同的药理活性离子的功效
为评估本发明医药组合物内不同的药理活性离子是否能够有效产生药物协同作用，在人类非小细胞肺癌细胞株(A549)中进行测试，观察是否有抑制癌细胞存活率的功效，分析结果列于表二中，表二中的剂量单位是nM。本实验所使用的药理活性离子的形式均为葡萄糖酸根离子。
表二、含有不同的药理活性多价离子的本发明医药组合物对人类非小细胞肺癌细胞株(A549)的细胞存活率的影响
各表中的符号“-”表示未添加该成分。
表二中的实验结果显示，当Niclosamide、Disulfiram和药理活性多价离子各自单独用于癌细胞株实验时，其并未展现处有效的抑制效果。在本发明药组合物更换不同离子的组合下，可以对癌细胞产生明显的抑制效果，依据实验结果，在与药理活性多价离子Cu进行组合时，可以产生最佳抑制功效。
表二中的实验结果显示，本发明医药组合物的单个成分各自对于人类非小细胞肺癌细胞(A549)存活率并无明显抑制效果。但本发明医药组合物使用不同的离子都可以产生癌细胞抑制效果，使得癌细胞存活率明显下降。
实施例2本发明医药组合物对人类非小细胞肺癌细胞株(A549)的功效
为评估本发明医药组合物不同的给药剂量是否会对人类非小细胞肺癌细胞株(A549)都具有抑制癌细胞存活率的功效，先对肺癌细胞株(A549)细胞存活率进行分析，结果列于表三中。如表三所示，本发明医药组合物对人类非小细胞肺癌细胞株(A549)存活率的抑制作用与给药剂量相关。本实验所使用的药理活性铜离子为葡萄糖酸铜形式。
表三、本发明医药组合物对人类非小细胞肺癌细胞株(A549)细胞存活率的影响
表三中的实验结果显示，当Niclosamide、Disulfiram和药理活性多价离子Cu各自单独用于癌细胞株实验时，其并未展现出有效的抑制效果。本发明医药组合物在不同的剂量下，都具有抑制效果，并且当Niclosamide浓度大于Disulfiram和药理活性多价离子Cu的浓度，或Disulfiram和药理活性多价离子Cu的浓度大于Niclosamide浓度时，会产生更高程度的癌细胞抑制效果，由此可知药物彼此间可产生更高的协同功效。
实验结果显示，在Niclosamide、Disulfiram、铜离子各自以2～80nM的浓度施用于A549癌细胞株时，并未产生抑制癌细胞存活率的效果。当仅同时施用各25nM浓度的Disulfiramc和铜离子时，其产生抑制癌细胞株存活率的效果，癌细胞存活率下降至47.63％。当本发明医药组合物同时施用于癌细胞株时，具有显著较高的抑制癌细胞存活率的效果，其A549癌细胞存活率最低可以下降至13.46％。依本实验得知本发明医药组合物中的三种药物可以产生更高程度的癌细胞抑制效果，具有协同功效。
本实验结果显示，本发明医药组合物对肺上皮细胞癌的癌细胞具有抑制癌细胞存活率的功效，其对癌细胞存活率的抑制作用可以与本发明医药组合物中各药物之间的剂量比例呈正相关。
实施例3本发明医药组合物对人类非小细胞肺癌细胞株(A549)在动物体内的癌细胞肿瘤的抑制功效
为评估本发明医药组合物是否会对人类非小细胞肺癌具有抑制效果的功效、药物经由胃肠道吸收后是否能够有效抑制肺癌肿瘤生长、药物是否会造成不适性，而进行了试验。先对裸鼠皮下注射建立肿瘤模型，观察14天后测量肿瘤大小，分成6组并开始给药，药物使用组连续以口服管饲试验药品，评估肿瘤模型裸鼠在给药25天后肿瘤生长的变化，并观察试验药品对肿瘤的抑制效果，结果列于表四与表五中。图1显示了本发明医药组合物与已知靶标药物组对人类非小细胞肺癌细胞株(A549)在动物体内的癌细胞肿瘤体积的抑制功效与给药时间的相关性。图2显示了本发明医药组合物与已知靶标药物组对人类非小细胞肺癌细胞株(A549)在动物体内的癌细胞肿瘤重量的抑制功效与给药时间的相关性。
表四、本发明医药组合物与已知靶标药物组对人类非小细胞肺癌(A549)的癌细胞肿瘤体积与给药天数的相关性，表中所列的癌细胞肿瘤体积单位为mm3。
在表四中，天所代表的为给药天数。
在表四中显示带有人类非小细胞肺癌细胞的小鼠的肿瘤体积抑制率(在处死时的各组肿瘤体积对比S1组)，实验结果显示，S2组的肿瘤抑制率为12.78％，S3组的肿瘤抑制率为30.39％，S4组的肿瘤抑制率为47.23％，本发明医药组合物有效剂量组S5组的肿瘤抑制率为49.51％，本发明医药组合物低剂量组S6组的肿瘤抑制率为44.03％。因此根据实验结果可以得知S5对于癌症细胞可以产生比S4更加有效的癌症抑制效果，并且S6效果也比S2或S3更佳，证明本发明医药组合物具有较佳的癌细胞肿瘤体积抑制功效。
表五、本发明医药组合物与已知靶标药物组对人类非小细胞肺癌(A549)的癌细胞肿瘤团块重量与给药剂量的相关性。癌细胞肿瘤团块重量的单位为克(g)。
在表五中显示带有人类非小细胞肺癌细胞的小鼠在被处死后的平均肿瘤团块重量抑制率(对比S1组)，实验结果显示，S2组的肿瘤抑制率为13.5％，S3组的肿瘤抑制率为37.3％，S4组的肿瘤抑制率为48.3％，本发明医药组合物有效剂量组S5组的肿瘤抑制率为51.8％，本发明医药组合物低剂量组S6组的肿瘤抑制率为47.4％。因此在实验结果可以得知S5对于癌症细胞可以产生比S4更加有效的癌症抑制效果，并且S6效果也比S2或S3更加，证明本发明医药组合物具有较佳的癌细胞肿瘤团块重量抑制功效。
与目前的肺癌靶标药物比较，根据本实验结果，可以清楚了解本发明医药组合物在动物体内可以有效抑制肿瘤团块重量和肿瘤体积，本发明医药组合物能够有效通过胃肠道吸收后，使药物明确靶向至癌细胞肿瘤组织中，并且动物在用药过程中并无任何不适性。
实施例4本发明医药组合物对人类肺鳞状上皮细胞癌细胞株(H520)在动物体内的癌细胞肿瘤的抑制功效
为评估本发明医药组合物是否对人类肺鳞状上皮细胞癌具有抑制效果的功效、药物经由胃肠道吸收后是否能够有效抑制肺癌肿瘤生长、药物是否会造成不适性，而进行了试验。先对裸鼠皮下注射建立肿瘤模型，观察14天后测量肿瘤大小，分成6组并开始给药，药物使用组连续以口服管饲试验药品，由于H520肿瘤在小鼠体内成长速度较快，因此在肿瘤成长至一定程度时将动物处死，肿瘤模型裸鼠在给药13天后被处死，评估肿瘤生长的变化，并观察试验药品对肿瘤的抑制效果，结果列于表六与表七中。图3显示了本发明医药组合物与已知靶标药物组对人类肺鳞状上皮细胞癌细胞株(H520)在动物体内的癌细胞肿瘤体积的抑制功效与给药时间的相关性。图4显示了本发明医药组合物与已知靶标药物组对人类肺鳞状上皮细胞癌细胞株(H520)在动物体内的癌细胞肿瘤重量的抑制功效与给药时间的相关性。
表六、本发明医药组合物与已知靶标药物组对人类肺鳞状上皮细胞癌(H520)的癌细胞肿瘤体积与给药天数的相关性，癌细胞肿瘤体积的单位为mm3。
在表六中天所代表的为给药天数。
在表六中显示带有人类肺鳞状上皮细胞癌的小鼠的肿瘤体积抑制率(在处死时的各组肿瘤体积对比S1组)，实验结果显示，S2组的肿瘤抑制率为30.49％，S3组的肿瘤抑制率为32.09％，S4组的肿瘤抑制率为28.38％，本发明医药组合物有效剂量组S5组的肿瘤抑制率为47.30％，本发明医药组合物低剂量组S6组的肿瘤抑制率为51.62％。根据实验分析结果得知，人类肺鳞状上皮细胞癌对于靶标药物S4组有较高抗性，因此S4组的肿瘤抑制率最小。S2和S3组的肿瘤抑制率较本发明医药组合物低，证明本发明医药组合物具有较佳的癌细胞肿瘤体积抑制功效。另外，在本实验中意外发现低剂量S6组对于此肺癌肿瘤类型反而有最佳的抑制效果。
表七、本发明医药组合物与已知靶标药物组对人类肺鳞状上皮细胞癌(H520)的癌细胞肿瘤团块重量与给药剂量的相关性。癌细胞肿瘤团块重量的单位为克。
在表七中显示患有人类肺鳞状上皮细胞癌的小鼠的肿瘤团块重量抑制率(对比S1组)，实验结果显示，S2组的肿瘤抑制率为30.5％，S3组的肿瘤抑制率为32.1％，S4组的肿瘤抑制率为28.6％，本发明医药组合物有效剂量组S5组的肿瘤抑制率为42.7％，本发明医药组合物低剂量组S6组的肿瘤抑制率为52.0％。根据实验分析结果可以得知，不论在肿瘤体积还是重量的分析上，此肿瘤类型对于靶标药物S4组均有较高抗性，在肿瘤团块重量抑制率同样为低剂量S6组具有最佳的功效。
根据实验结果显示，靶标药物对于人类肺鳞状上皮细胞癌的治疗效果较差，肿瘤组织对于靶标药物会产生抗性。但本发明医药组合物对于此癌症治疗能够产生有效的肿瘤抑制效果，肿瘤组织对本发明医药组合物没有产生抗性，并且在本实验中意外发现低剂量S6组对于此肺癌肿瘤类型反而具有最佳的抑制效果。
根据实施例3和实施例4的分析结果，可以清楚知道不同类型的肺癌肿瘤对于靶标药物会产生抗性，使得肿瘤抑制治疗效果下降。本发明医药组合物在不同类型的肺癌肿瘤治疗上对肺癌肿瘤无抗性问题，表现出明显的抑制功效。
实施例5本发明医药组合物对脑癌人类胶质母细胞瘤细胞株(T98G)的功效
为评估本发明医药组合物是否会对脑癌人类胶质母细胞瘤细胞株(T98G)具有抑制癌细胞存活率的功效，先对人类胶质母细胞瘤细胞株(T98G)细胞存活率进行分析，结果列于表八中。
表八、本发明医药组合物对人类胶质母细胞瘤细胞株(T98G)细胞存活率的影响，此癌细胞株应用于脑癌研究
T98G是应用于脑癌研究和药物开发的人类胶质母细胞瘤细胞株。本实验结果显示，在表八中，当Niclosamide、Disulfiram和药理活性多价离子Cu各自单独用于癌细胞株实验时，其并未展现出有效的抑制效果。本发明医药组合物在不同浓度的组合下，都具有抑制效果，并且当Niclosamide浓度大于Disulfiram和药理活性多价离子Cu的浓度，或Disulfiram和药理活性多价离子Cu的浓度大于Niclosamide浓度时，会产生更高程度的癌细胞抑制效果，由此可知药物彼此间可产生更高的协同功效。
如表八所示，本发明医药组合物对人类胶质母细胞瘤细胞株(T98G)存活率与给药剂量相关。本实验所使用的药理活性铜离子为葡萄糖酸铜形式，实验结果显示，在Niclosamide、Disulfiram、铜离子各自以8～83nM的浓度施用于T98G癌细胞株时，癌细胞存活率维持相对高的水平，范围为87.87％～102.19％，未产生明显效果。当本发明医药组合物(其中Niclosamide 4nM、Disulfiram 8nM、铜离子8nM)同时施用于癌细胞株时，癌细胞存活率可以降至59.99％，具有显著下降。当调整三个药物彼此之间的浓度时，可以使T98G癌细胞存活率最低可以下降至23.17％。根据本实验得知本发明医药组合物中的三个药物可以产生更高程度的癌细胞抑制效果，具有协同功效。
本实验结果显示，本发明医药组合物对人类胶质母细胞瘤具有抑制癌细胞存活率的功效，其对癌细胞存活率的抑制作用可以与本发明医药组合物中各药物之间的剂量比例呈正相关，使得该癌细胞存活率下降。
实施例6本发明医药组合物对脑癌人类胶质母细胞瘤细胞株(U-87)的功效
为评估本发明医药组合物是否会对脑癌人类胶质母细胞瘤细胞株(U-87)具有抑制癌细胞存活率的功效，对人类胶质母细胞瘤细胞株(U-87)的细胞存活率进行分析，结果列于表九中。
表九、本发明医药组合物对人类胶质母细胞瘤细胞株(U-87)细胞存活率的影响，此癌细胞株应用于脑癌研究也用于乳腺癌研究
已知人类胶质母细胞瘤细胞株(U-87)对于细胞凋亡的抗性较高，对于靶标药物IRESSA(Gefitinib)及ABT-737(Bcl-2抑制剂)等的抗性较高(Chang et al.,2011；Cristofanon and Fulda,2012；Jane et al.,2013)。
实验结果显示，表九中，当Niclosamide、Disulfiram和药理活性多价离子Cu各自单独用于癌细胞株实验时，其并未展现出有效的抑制效果。本发明医药组合物在不同浓度的组合下，都具有抑制效果，并且当Niclosamide浓度大于Disulfiram和药理活性多价离子Cu的浓度，或Disulfiram和药理活性多价离子Cu的浓度大于Niclosamide浓度时，会产生更高程度的癌细胞抑制效果，由此可知本发明医药组合物中的药物彼此间可产生更高的协同功效。
如表九所示，本发明医药组合物对人类胶质母细胞瘤细胞株(U-87)存活率的抑制作用与给药剂量相关。本实验所使用的药理活性铜离子为葡萄糖酸铜形式，实验结果显示，在Niclosamide、Disulfiram、铜离子各自以8～83nM的浓度施用于U-87癌细胞株时，癌细胞存活率维持再63.98％～103.97％。当单独施用83nM的Disulfiram时，癌细胞存活率为63.98％。运用本发明医药组合物，可以有效降低药物浓度并且提高癌细胞抑制率。当本发明医药组合物(其中Niclosamide 4nM、Disulfiram 41nM、铜离子41nM)同时施用于癌细胞株时，癌细胞存活率可以降至30.45％，对比于单独使用Disulfiram组，其癌细胞存活率显著下降。当调整三个药物彼此之间的浓度时，可以使U-87癌细胞存活率最低下降至30.45％。根据本实验得知，本发明医药组合物中的三个药物在特定浓度下可以产生较低且明显的癌细胞存活率，三个药物的组合可产生更高程度的协同功效。
以上所述仅为本发明较佳实施例，凡依本发明申请专利范围所做的等同变化与修饰，都应落在本发明涵盖的范围内。

Claims (10)

  1. 一种医药组合物，包含：
    (a)癌细胞线粒体的靶向剂；
    (b)离子螯合剂；以及
    (c)药理活性多价离子；
    其中，该癌细胞线粒体的靶向剂为Niclosamide；该离子螯合剂为Disulfiram；该药理活性多价离子选自由镁离子、钙离子、锰离子、亚铁离子、铜离子、锌离子所组成的群组，(a)的重量比按该医药组合物的总重量计为64.5％～43.7％，(b)的重量比按该医药组合物的总重量计为56.2％～16.1％，(c)的重量比按该医药组合物的总重量计为19.4％～0.000116％。
  2. 如权利要求1所述的医药组合物，其中(a)与(b)+(c)的剂量比例为0.091≤(a)/((b)+(c))≤15.548。
  3. 如权利要求1所述的医药组合物，其中(b)与(c)的剂量比例是0.214≤(b)/(c)≤1.8867×104。
  4. 权利要求1或权利要求2或权利要求3所述的医药组合物用于制备用于治疗有需要的个体的恶性肿瘤的药物的用途。
  5. 如权利要求4所述的用途，其中该恶性肿瘤选自由实体瘤与血液恶性疾病所组成的群组。
  6. 如权利要求5所述的用途，其中该恶性肿瘤选自由肺癌与脑癌所组成的群组。
  7. 如权利要求4所述的用途，其中(a)每次的施用量为2000mg～100mg，(b)每次的施用量为500mg～128.6mg，(c)每次的施用量为600mg～2.65×10-4mg。
  8. 如权利要求7所述的用途，其中(c)的施用顺序不晚于(b)的施用顺序。
  9. 如权利要求4所述的用途，其中该药物1天施用不超过2次。
  10. 如权利要求4所述的用途，其中该药物以口服、注射、经皮、或吸入方式施用。
PCT/CN2023/115614 2022-08-29 2023-08-29 医药组合物及其用途 WO2024046332A1 (zh)

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