WO2021163897A1 - 包含亚甲蓝类染料、营养素或/和常规抗肿瘤药的药物组合物及其应用 - Google Patents

包含亚甲蓝类染料、营养素或/和常规抗肿瘤药的药物组合物及其应用 Download PDF

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WO2021163897A1
WO2021163897A1 PCT/CN2020/075765 CN2020075765W WO2021163897A1 WO 2021163897 A1 WO2021163897 A1 WO 2021163897A1 CN 2020075765 W CN2020075765 W CN 2020075765W WO 2021163897 A1 WO2021163897 A1 WO 2021163897A1
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Prior art keywords
methylene blue
concentration
pharmaceutical composition
tumor
cancer
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PCT/CN2020/075765
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English (en)
French (fr)
Inventor
邹方霖
邹礼常
王建霞
王艺羲
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成都夸常奥普医疗科技有限公司
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Priority to PCT/CN2020/075765 priority Critical patent/WO2021163897A1/zh
Priority to PCT/CN2021/076749 priority patent/WO2021164706A1/zh
Publication of WO2021163897A1 publication Critical patent/WO2021163897A1/zh

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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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/255—Esters, e.g. nitroglycerine, selenocyanates of sulfoxy acids or sulfur analogues thereof
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/5415—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with carbocyclic ring systems, e.g. phenothiazine, chlorpromazine, piroxicam
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof

Definitions

  • the disclosure of this application relates to the application of methylene blue dye as a combination of local active ingredients and nutrients or/and conventional anti-tumor drugs in the preparation of topical pharmaceutical compositions for the treatment of malignant solid tumors, comprising the methylene blue dye and nutrients or/ A local pharmaceutical composition for treating malignant solid tumors with conventional antitumor drugs, and a method for treating malignant solid tumors including administering the pharmaceutical composition.
  • anti-solid tumor drugs One of the main problems facing the development of anti-solid tumor drugs is specificity. Since conventional anti-tumor drugs cannot sufficiently distinguish between target cells and normal cells, and the difference between the effective dose and the safety limit is not large enough, they will produce systemic effects (tumor cell inhibitory effects inside and outside the tumor) at the same time. Greater risk of systemic toxicity. In addition, the drug molecule needs to penetrate effectively in the tumor tissue to have an effect on the tumor cells in between. For some tumors with poor blood supply (such as pancreatic cancer), the chance of benefiting the patient is even smaller.
  • Intratumoral administration has the advantage of physically targeting drugs.
  • intratumoral administration of conventional anti-tumor drugs increased the concentration of the target area, it did not show a significant improvement in efficacy.
  • conventional anti-tumor drugs are almost still administered systemically in clinical practice.
  • Conventional chemical ablation agents high-purity ethanol, high-concentration acids and bases
  • the target tissue cannot be sufficiently distinguished from other tissues, the actual intervention volume (for example, the amount of acid and alkali does not exceed 0.2 ml/kg) and the intervention site are very limited. Therefore, chemical ablation agents have gradually faded out of malignant solid tumors in the past ten years. In fact, there are almost no local special drugs with high local safety and high local curative effect in clinical practice.
  • the purpose of the present invention is to provide a topical drug that physically targets tumors, but has higher efficacy, higher compliance, or/and higher specificity than existing drugs, and includes administration of the drug composition A method for treating malignant solid tumors.
  • a topical pharmaceutical composition for the treatment of malignant solid tumors which comprises a methylene blue dye, a local synergistic drug of the methylene blue dye, and a pharmaceutically acceptable Liquid carrier, wherein the synergistic drug is selected from nutrients or/and conventional anti-tumor drugs, and in the topical pharmaceutical composition, the concentration (w/v) of the methylene blue dye is ⁇ 2%, preferably 0.35- 2%, 0.5-2%, 0.5-1.5% or 0.5-1%, the concentration of the nutrient (w/v) ⁇ 2%, preferably 3-40%, and the concentration of the conventional anti-tumor drug is greater than that
  • the saturated concentration is 20% or 30%, preferably 30%-100% of its saturated concentration, wherein the saturated concentration refers to the saturated concentration of the conventional anti-tumor drug in the liquid carrier.
  • methylene blue dye as a topical active ingredient and a local synergistic drug of the methylene blue dye in the preparation of a topical pharmaceutical composition for the treatment of malignant solid tumors ,
  • the synergistic drug is selected from nutrients or/and conventional anti-tumor drugs.
  • the topical pharmaceutical composition comprises the methylene blue dye, the local synergistic drug of the methylene blue dye, and a pharmaceutically acceptable liquid carrier, wherein the topical drug In the composition, the concentration (w/v) of the methylene blue dye is ⁇ 2%, preferably 0.35-2%, 0.5-2%, 0.5-1.5% or 0.5-1%, and the concentration of the nutrient (w/v) w/v) ⁇ 2%, preferably 3%-40%, and the concentration of the conventional anti-tumor drug is greater than 20% or 30% of its saturated concentration, preferably 30%-100% of its saturated concentration, wherein The saturated concentration refers to the saturated concentration of the conventional anti-tumor drug in the liquid carrier.
  • it provides a method for treating malignant solid tumors, which comprises intratumorally administering the pharmaceutical composition disclosed in the present application to an individual in need thereof.
  • composition containing the methylene blue dye and the local synergistic drug disclosed according to the present application has the following advantages compared with a single drug containing the corresponding components: it provides a synergistic effect against malignant solid tumors to improve effectiveness, and at the same time has the advantages of It may provide antagonism against non-specific tissue destruction to improve safety.
  • the embodiment according to the present invention has the following advantages: Compared with the existing cytotoxic drugs and related treatment methods, it shows almost non-toxic systemic safety and significantly higher local treatment Efficacy; Compared with the existing molecular targeted drugs and related treatment methods, it shows less stringent indication screening, and has great potential for reducing the load of fast-growing tumors, large tumors and poor blood donor tumors; Compared with chemical ablation agents and related methods, they show higher compliance (such as irritation, corrosivity) or specificity, so that they can have a larger range of intervention and a higher application volume.
  • the method and composition of the present invention are also not troubled by the drug resistance problems encountered by existing cytotoxic drugs and existing molecular targeted drugs. In addition, the method and composition are convenient in application and low in cost, and are particularly helpful for the general population who cannot afford high expenses to enjoy safe and effective treatment.
  • the inventor of the present invention unexpectedly discovered in a tumor-bearing animal experiment that although the addition of DHA generally cannot, it can form a highly synergistic effect with methylene blue under certain specific conditions, which also makes the use of methylene blue
  • the dose can be reduced exponentially to reduce the risk of side effects.
  • substances selected from other nutrients or/and conventional anti-tumor drugs can also form an unexpected synergistic effect with lower concentrations (for example, ⁇ 1%) of methylene blue under these specific conditions.
  • These specific conditions are not the conditions of methylene blue dyes, nutrients or/and conventional anti-tumor drugs in the existing anti-malignant solid tumor technology (for example, intratumoral administration concentration), but are as defined below.
  • composition in the scope of the present invention, the term “topical drug (composition)” is distinguished from conventional drugs (compositions), which refer to conventional administration (or systemic administration, such as oral administration, intravenous injection, intraperitoneal injection, pleural injection, etc.) ) So that its active ingredients are transported to the tumor through the blood and diffuse and penetrate through the blood vessels (compositions), while the former refers to intratumoral administration and mainly diffuse and penetrate through the spaces of extravascular structures, and mainly Therapeutic drugs (compositions) that produce medicinal effects through local action.
  • local action or “local activity” refers to a pharmacological action or pharmacological activity that preferentially targets tumor tissues rather than tumor cells.
  • the term "locally active ingredient” is distinguished from chemical ablation agents, which refers to chemical substances (such as 50% acetic acid, absolute ethanol, 5% methylene blue) under effective ablation conditions of the tumor (usually exceeding the ablation concentration threshold) , While the former refers to not through its conventional effects (not any administration other than intratumoral administration), nor its chemical ablation effect (used under conditions lower than its chemical ablation concentration), but mainly through providing local synergy Active ingredient (for example 0.5 to 1.5% methylene blue).
  • chemical ablation agents refers to chemical substances (such as 50% acetic acid, absolute ethanol, 5% methylene blue) under effective ablation conditions of the tumor (usually exceeding the ablation concentration threshold) , While the former refers to not through its conventional effects (not any administration other than intratumoral administration), nor its chemical ablation effect (used under conditions lower than its chemical ablation concentration), but mainly through providing local synergy Active ingredient (for example 0.5 to 1.5% methylene blue).
  • local synergistic drug or local synergist refers to the methylene blue that can be combined with the local active ingredient (for example, topical administration and administration concentration ⁇ 2%, preferably ⁇ 1%) Dyestuffs) produce local synergistic effects, which are selected from nutrients or/and conventional anti-tumor drugs.
  • local synergistic effect refers to a synergistic effect that is mainly shown as a local effect.
  • concentration a synergistic effect
  • the term “synergistic effect” refers to the combination of active ingredients exhibiting a higher desired medicinal effect than any component used alone, and/or the combination of any component exhibiting no single use. Need drug effect (for example, the tissue necrosis effect produced by the sharing of two cytotoxic drugs).
  • Need drug effect for example, the tissue necrosis effect produced by the sharing of two cytotoxic drugs.
  • the term “synergistic safety” means that when effective pharmacological effects are obtained, the active components collectively show a higher required safety than any component used alone.
  • a topical pharmaceutical composition for the treatment of malignant solid tumors which comprises a methylene blue dye, a local synergistic drug of the methylene blue dye, and a pharmaceutically acceptable
  • the liquid carrier wherein the synergistic drug is selected from nutrients or/and conventional anti-tumor drugs
  • the concentration (w/v) of the methylene blue dye is ⁇ 2%, preferably 0.35 -2%, 0.5-2%, 0.5-1.5% or 0.5-1%, the concentration of the nutrient (w/v) ⁇ 2%, preferably 3%-40%, and the concentration of the conventional anti-tumor drug It is greater than 20% or 30% of its saturated concentration, preferably 30%-100% of its saturated concentration, wherein the saturated concentration refers to the saturated concentration of the conventional antitumor drug in the liquid carrier.
  • methylene blue dye as a topical active ingredient and a local synergistic drug of the methylene blue dye in the preparation of a topical pharmaceutical composition for the treatment of malignant solid tumors ,
  • the synergistic drug is selected from nutrients or/and conventional anti-tumor drugs.
  • the topical pharmaceutical composition comprises the methylene blue dye, the local synergistic drug of the methylene blue dye, and a pharmaceutically acceptable liquid carrier, wherein the topical drug In the composition, the concentration (w/v) of the methylene blue dye is ⁇ 2%, preferably 0.35-2%, 0.5-2%, 0.5-1.5% or 0.5-1%, and the concentration of the nutrient (w/v) w/v) ⁇ 2%, preferably 3%-40%, and the concentration of the conventional anti-tumor drug is greater than 20% or 30% of its saturated concentration, preferably 30%-100% of its saturated concentration, wherein The saturated concentration refers to the saturated concentration of the conventional anti-tumor drug in the liquid carrier.
  • it provides a method for treating malignant solid tumors, which comprises intratumorally administering a therapeutically effective amount of the methylene blue dye disclosed in the present application to an individual in need thereof.
  • a pharmaceutical composition of a topical active ingredient and the synergistic drug is provided.
  • intramoral administration refers to the injection of drugs (such as injections) into the tumor through devices, such as transcatheter arterial infusion, transcatheter intratumor infusion, intratumoral injection, and the like.
  • therapeutically effective amount refers to the amount of a drug used to treat a disease (such as a tumor) and obtain an effective effect (such as reducing or/and alleviating the symptoms of the disease).
  • concentration refers to the weight/volume percentage concentration% (w/v) of the specified component in the topical pharmaceutical composition.
  • intramoral administration concentration refers to the concentration of the specified component when the drug is administered intratumorally, which may be the concentration of the specified component where the drug contacts the target area (for example, the injection needle hole or the outlet of the perfusion tube).
  • methylene blue dyes at the above concentrations have many applications, such as antidote, analgesic, vital dyes, and so on.
  • high-concentration (for example, 5%) methylene blue dyes can also be used as chemical ablation agents. It is generally believed that the greater the concentration of the chemical ablation agent, the more effective it is.
  • the inventors of the present invention unexpectedly discovered that methylene blue dyes can be very different from conventional pharmacological conditions under non-chemical ablative conditions (for example, 1% methylene blue administration concentration).
  • These unexpected synergistic effects may be the key point of pharmacology, which significantly improves the specificity of methylene blue dyes, nutrients or/and conventional anti-tumor drug compositions against intratumoral tissue destruction.
  • the drug should specify the intratumoral administration concentration of methylene blue chromosome in its instructions to ensure that it is applied as a local active ingredient rather than as a chemical ablation agent to avoid risks.
  • the methylene blue dye is preferably selected from the following compounds and their derivatives: methylene blue, patent blue, isosulfur blue, and neomethylene blue.
  • the methylene blue dye is more preferably selected from methylene blue and its derivatives.
  • the low-concentration methylene blue dye as a local synergistic drug in the treatment of malignant solid tumors may be one or more selected from nutrients and/or conventional anti-tumor drugs.
  • the term "nutrient” refers to organic compounds with nutritional and health effects, which are usually used in the preparation of nutritional health products, traditional diets and functional diets (such as health diets), which mainly include amino acid nutrients , Carbohydrate nutrients and lipid nutrients.
  • the concentration of the nutrient in the composition is 2.5-50%, preferably 4-40%.
  • the local synergistic medicine includes amino acid nutrients, and the concentration (w/v) of the amino acid nutrients in the pharmaceutical composition is ⁇ 2%, ⁇ 2.5, ⁇ 5% , Preferably ⁇ 7.5%, 10-25% or 18-25%, more preferably 15%-25% or 20%-25%.
  • amino acid nutrients refers to amino acid compounds with nutritional and health effects, preferably selected from amino acids, amino acid polymers and amino acid derivatives with nutritional and health effects, more preferably selected from China , Amino acid nutritional drugs and amino acid excipients with nutritional and health effects contained in the official pharmacopoeias or guidelines of the United States or Europe.
  • amino acid-based nutrients amino acids and amino acid derivatives is preferably a polymer selected from the group of amino acids, or an amino acid of the group of oligopeptide and polypeptide, or the group The amino acid salt in the amino acid: protein amino acid and non-protein amino acid.
  • the protein amino acids include amino acids selected from the following group: non-polar amino acids (such as alanine, valine, leucine, isoleucine, phenylalanine , Proline), polar neutral amino acids (such as tryptophan, tyrosine, serine, cysteine, methionine, asparagine, glutamine, threonine), basic amino acids (such as lysine) Acid, arginine, histidine), acidic amino acids (e.g. aspartic acid, glutamic acid). All the above except glycine are L-type ⁇ -amino acids.
  • non-polar amino acids such as alanine, valine, leucine, isoleucine, phenylalanine , Proline
  • polar neutral amino acids such as tryptophan, tyrosine, serine, cysteine, methionine, asparagine, glutamine, threonine
  • basic amino acids such as lysine
  • Acid arg
  • the non-protein amino acids may include the following amino acids: ⁇ -alanine, taurine, ⁇ -aminobutyric acid (GABA), tea polyphenols (theanine), pumpkin seed amino acids (3-amino-3-carboxypyridine) Alkanoic acid), glutamine, citrulline, ornithine, etc.
  • oligopeptide refers to a polymer of amino acids comprises 2 to 10 identical or different amino acids linked by peptide bonds employed; the term “polypeptide” refers to a same or different 11-100 amino acid polymer linked by peptide bonds.
  • the amino acids constituting the oligopeptide or polypeptide may all be one or more of the above-mentioned amino acids, or may additionally include other amino acids.
  • the oligopeptide may be one or more selected from the group consisting of: glycyl-L-tyrosine, glycylalanine, glycylglycine, lysine-glycine two Peptides, glutathione, carnosine ( ⁇ -alanine histidine copolymer), glutathione, collagen oligopeptides, casein hydrolyzed peptides, soybean oligopeptides, oligoarginine, oligoglycine, oligo-lysine Acid.
  • the polypeptide may be one or more selected from the group consisting of polyaspartic acid, polyglutamic acid, and polylysine.
  • amino acid salt refers to the salt formed by the above-mentioned amino acid and acid or base, such as lysine hydrochloride, histidine hydrochloride, glutamic acid hydrochloride , Cysteine hydrochloride, arginine hydrochloride, glycine sulfate, iron glycine sulfate, lysine hydrochloride, aspartic acid hydrochloride, etc.
  • the amino acid nutrient may be one or more of amino acids, amino acid salts, oligopeptides, and polypeptides, for example, 2, 3, 4, or 5 or more. .
  • amino acids, amino acid salts, oligopeptides and polypeptides as the amino acid nutrients are preferably amino acids or their salts selected from the following group, or oligopeptides and polypeptides comprising or consisting of the following amino acids: alanine , Valine, leucine, isoleucine, phenylalanine, proline, tryptophan, tyrosine, serine, cysteine, methionine, threonine, lysine, arginine Acid, histidine, aspartic acid, glutamic acid, ⁇ -alanine, taurine, ⁇ -aminobutyric acid (GABA), theanine, citrulline, ornithine; more preferably selected from Amino acids in the following groups or their salts or oligopeptides and polypeptides comprising or consisting of the following amino acids: arginine, lysine, glycine, cysteine, alanine, serine
  • the amino acid nutrient includes arginine.
  • the amino acid nutrient is selected from compound amino acids including arginine, for example: arginine/tolerant, arginine/serine, arginine/glycine, arginine/cysteine Hydrochloride and so on.
  • the amino acid nutrient includes lysine.
  • the amino acid nutrient includes glycine.
  • the amino acid nutrient includes glutamic acid.
  • the amino acid nutrient is selected from amino acids or amino acid salts with nutritional and health effects, and the concentration (w/v) of the amino acid or amino acid salt in the topical pharmaceutical composition is ⁇ 2%, ⁇ 2.5, ⁇ 5%, ⁇ 7.5%, 10-25% or 18-25%, preferably 15%-25% or 20%-25%.
  • the amino acid nutrients are selected from oligopeptides and polypeptides with nutritional and health effects, and the concentration (w/v) of the oligopeptides and polypeptides in the topical pharmaceutical composition is greater than ⁇ 5%, Preferably it is 7.5-25%, more preferably 10%-25%.
  • the amino acid nutrient is a combination of the amino acid and/or amino acid salt and the oligopeptide and/or polypeptide, and the concentration (w/v) of the combination in the topical pharmaceutical composition is More than ⁇ 5%, preferably 7.5%-25%, more preferably 10-25%.
  • the local synergistic medicine includes carbohydrate nutrients, and the concentration (w/v) of the carbohydrate nutrients in the pharmaceutical composition is greater than 5%, preferably ⁇ 10%, 10% -50%, 15-50% or 25-50%.
  • carbohydrate nutrient refers to carbohydrate compounds with nutritional and health effects, preferably selected from monosaccharides, sugar polymers and sugar derivatives with nutritional and health effects, more preferably selected from Carbohydrate nutritional medicines and carbohydrate excipients with nutritional and health effects contained in the official pharmacopoeias or guidelines of China, the United States or Europe.
  • the monosaccharides, sugar polymers, and sugar derivatives as the carbohydrate nutrients are preferably monosaccharides selected from the following groups, sugar polymers containing monosaccharides in the following groups, or their derivatives Substances: glucose, ribose, deoxyribose, xylose, fructose, galactose, fucose.
  • the sugar polymer may be selected from disaccharides, oligosaccharides and polysaccharides containing monosaccharides as described above.
  • disaccharide used refers to a polymer containing two monosaccharides connected by glycosidic bonds
  • oligosaccharide used refers to a polymer containing 3-10 monosaccharides connected by glycosidic bonds.
  • Sugar polymer and the term “polysaccharide” as used refers to a polymer containing more than 10 monosaccharides connected by glycosidic bonds.
  • the monosaccharides constituting the disaccharides, oligosaccharides or polysaccharides may all be one or more of the above-mentioned monosaccharides, or may additionally contain other monosaccharides.
  • the disaccharide may be one or more selected from the group consisting of lactulose, maltose, sucrose, lactose, and trehalose.
  • the oligosaccharide may be one or more selected from the group consisting of chitooligosaccharides, xylo-oligosaccharides, fructooligosaccharides, mannose oligosaccharides, malto-oligosaccharides, and isomalto-oligosaccharides.
  • the polysaccharide may be one or more selected from the group consisting of starch, cellulose, dextran, and glycosaminoglycan.
  • the sugar derivative may be, for example, the following sugar derivatives selected from the above-mentioned monosaccharides or sugar polymers: sugar acid, sugar acid salt, sugar alcohol.
  • sugar acid used refers to acid derivatives of monosaccharides or sugar polymers
  • sugar acid salt refers to salt derivatives of monosaccharides or sugar polymers
  • sugar alcohol refers to monosaccharides or alcohol derivatives of sugar polymers.
  • the sugar acid may be one or more selected from the group consisting of gluconic acid, mannonic acid, and arabinonic acid.
  • the sugar acid salt may be one or more selected from the group consisting of sodium gluconate, sodium mannate, and sodium arabinate.
  • the sugar alcohol may be one or more selected from the group consisting of mannitol, maltitol, lactitol, and xylitol.
  • the carbohydrate nutrient may be one or more of monosaccharides, oligosaccharides, polysaccharides, sugar acids, saccharates, and sugar alcohols, such as 2, 3, 4 Species or 5 species or more.
  • the carbohydrate nutrient is selected from glucose, glucose-containing sugar polymers, or glucose derivatives.
  • the carbohydrate nutrient is selected from ribose, ribose-containing sugar polymers, or ribose derivatives.
  • the carbohydrate nutrient is selected from xylose, xylose-containing sugar polymers, or xylose derivatives.
  • the carbohydrate nutrient is preferably one or more selected from the following: glucose, fructose, chitooligosaccharide, glucosamine, lactulose, sorbitol, ribose, sorbose, mannose, galactose , Sucrose, lactose, trehalose, xylo-oligosaccharides, fructooligosaccharides, mannose oligosaccharides, xylitol, more preferably one or more selected from the following: glucose, sodium gluconate, chitooligosaccharides, glucosamine, Lactulose, ribose, mannose oligosaccharides, xylitol.
  • the concentration (w/v) of the carbohydrate nutrient in the pharmaceutical composition is greater than 5%, preferably ⁇ 10%, 10-40%, 15-50% or 25-50%.
  • the topical synergistic drug includes lipid nutrients, and the concentration (w/v) of the lipid nutrients in the pharmaceutical composition is ⁇ 4%, preferably 4-25% .
  • the lipid nutrient includes any pharmaceutically acceptable lipid nutrient, preferably selected from the group of lipids with nutritional and health effects contained in the official pharmacopoeias or guidelines of China, the United States or Europe.
  • the compound is more preferably one or more selected from the group consisting of fats, fatty acids, fat emulsions and lipids.
  • the lipid nutrient is one or more selected from the group consisting of vegetable oil, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), long-chain fat emulsion, Medium chain fat milk, phospholipids.
  • the concentration (w/v) of the lipid nutrient in the pharmaceutical composition is ⁇ 4%, preferably 4-25%.
  • conventional antineoplastic drug refers to a drug that can effectively inhibit solid tumors by absorption at a safe dose, which is selected from any pharmaceutically acceptable conventional antineoplastic drugs, preferably selected from the present invention.
  • Conventional anti-tumor drugs known in the art are more preferably selected from China, the United States or Europe, which has been approved or will be approved by the official competent administrative department of China, the United States or Europe (such as the FDA or China Food and Drug Administration), or has been included in the official pharmacopoeia of China, the United States or Europe. The anti-tumor drugs to be loaded.
  • absorption refers to the pharmacological effect of the drug being absorbed by the blood to form the drug-carrying blood into the target area.
  • Some conventional anti-tumor drugs have been expected to improve their efficacy through intratumoral administration, but the increase in drug efficacy with the increase in concentration (chemical kinetics) is far lower than its theoretical correlation.
  • different drugs were shared locally in a large number of studies, there was not much local synergy, let alone unexpected synergy, indicating that the synergy of these drugs under special conditions in the tumor (such as the microenvironment of cancer cells) has a high degree of inefficiency. Certainty. .
  • the conventional anti-tumor drugs may be one or more selected from the group consisting of: drugs that disrupt the structure and function of DNA, drugs that intercalate in DNA and interfere with transcription of RNA, drugs that interfere with DNA synthesis, and proteins that affect the structure and function of DNA. Synthetic drugs.
  • the drugs that disrupt DNA structure and function include, for example, alkylating agents (e.g., cyclophosphamide, carmustine, etc.), metal platinum complexes (e.g., cisplatin, carboplatin, etc.), DNA topoisomerase inhibitors ( For example, doxorubicin, topotecan, irinotecan, etc.).
  • the drugs that intercalate DNA to interfere with transcription of RNA include, for example, anti-tumor antibiotics, such as actinomycins, daunorubicin, doxorubicin, and the like.
  • the drugs that interfere with DNA synthesis include, for example, pyrimidine antagonists (such as uracil derivatives 5-fluorouracil, furfurouracil, difurfurouracil, cytosine derivatives cytarabine, cyclocytidine, 5-azacytidine, etc.) , Purine antagonists (e.g. oncolytic, thioguanine, etc.), folic acid antagonists (e.g., methotrexate, etc.).
  • the drugs that affect protein synthesis include, for example, colchicines, vinblastines, taxanes (such as paclitaxel, docetaxel, etc.) and the like.
  • the pharmaceutically acceptable liquid carrier includes water and/or ethanol.
  • the pharmacologically acceptable liquid carrier is mainly selected according to the properties of conventional anti-tumor drugs, so that the drug can reach a corresponding concentration.
  • the conventional anti-tumor drug is selected from the group consisting of water-soluble conventional anti-tumor drugs and alcohol-soluble conventional anti-tumor drugs.
  • the term "alcohol-soluble conventional anti-tumor drugs” refers to conventional anti-tumor drugs whose solubility in ethanol or ethanol aqueous solution at room temperature is greater than or equal to the concentration required for effective local action, which includes, for example, yew Alkanes, vinblastines, etc.
  • water-soluble conventional antitumor drug refers to a conventional antitumor drug whose solubility in aqueous solution at room temperature is greater than or equal to the concentration required for its effective local action, which includes, for example, one or more water-soluble drugs selected from the following groups Sexual compounds: uracil derivatives, cyclophosphamide, gemcitabine (such as gemcitabine hydrochloride), epirubicin (such as epirubicin hydrochloride), antitumor antibiotics (such as doxorubicin, actinomycetes) Vinblastine, etc.), vinblastines (for example, vinblastine sulfate), teniposide, metal platinum complexes, etc.
  • sexual compounds uracil derivatives, cyclophosphamide, gemcitabine (such as gemcitabine hydrochloride), epirubicin (such as epirubicin hydrochloride), antitumor antibiotics (such as doxorubicin, actinomycetes) Vinblastine, etc
  • the conventional anti-tumor drugs may be one or more selected from the following groups: uracil derivatives, cyclophosphamides, gemcitabine, epirubicin, Anti-tumor antibiotics, teniposide, metal platinum complexes, taxanes; preferably one or more selected from the following drugs and their analogous derivatives: 5-fluorouracil, cyclophosphamide, gemcitabine, epirubin Bicin, antitumor antibiotic, teniposide, metal platinum complex, paclitaxel.
  • the concentration of the conventional anti-tumor drug is greater than 30% of its saturated concentration, preferably 50-100%, 60-100%, 70-100%, 80% of its saturated concentration. -100%, or 90-100%, wherein the saturated concentration refers to the saturated concentration of the conventional anti-tumor drug in the liquid carrier.
  • the concentration (w/v) of a conventional anti-tumor drug (such as cyclophosphamide, carmustine, etc.) selected from the alkylating agent in the topical pharmaceutical composition is 0.5-6 %, preferably 0.75-1.5%.
  • the concentration (w/v) of a conventional anti-tumor drug (such as cisplatin, carboplatin, etc.) selected from the metal platinum complex in the topical pharmaceutical composition is 0.03-0.08% , Preferably 0.03-0.06%.
  • the concentration of a conventional anti-tumor drug (such as doxorubicin, topotecan, irinotecan, etc.) selected from the DNA topoisomerase inhibitor in the topical pharmaceutical composition is 0.05-0.20%, preferably 0.75-0.15%.
  • the concentration (w/v) of conventional anti-tumor drugs selected from the anti-tumor antibiotics (such as actinomycins, daunorubicin, etc.) in the topical pharmaceutical composition is 1 -4%, preferably 1-2%.
  • a conventional antitumor drug selected from the pyrimidine antagonist for example, uracil derivative 5-fluorouracil, furfurouracil, difurfurouracil, cytosine derivative cytarabine, cyclocytidine, 5
  • concentration (w/v) of azacytidine, etc.) in the topical pharmaceutical composition is 0.5-2%, preferably 0.75-1.5%.
  • the concentration (w/v) of a conventional anti-tumor drug selected from the taxanes (such as paclitaxel, docetaxel, etc.) in the topical pharmaceutical composition is 0.5-2% , Preferably 0.75-1.5%.
  • composition disclosed according to the present application it also optionally includes one or more selected from the group consisting of analgesics, sustained-release carriers, pH adjusters, and excipients.
  • the pharmaceutical composition disclosed according to the present application may further optionally include an analgesic.
  • the analgesic is used to alleviate the pain of the patient, and it may be any suitable one known to those skilled in the art, such as benzyl alcohol, procaine hydrochloride, chlorobutanol, lidocaine hydrochloride and the like.
  • the concentration of the analgesic in the pharmaceutical composition may be, for example, 0.1-4% by weight.
  • the concentration of benzyl alcohol in the pharmaceutical composition may be 1-4% by weight
  • the concentration of procaine hydrochloride, chlorobutanol, and lidoca hydrochloride in the pharmaceutical composition may be 1- 3% by weight.
  • the pharmaceutical composition disclosed according to the present application may further optionally include a sustained-release carrier.
  • the sustained-release carrier may be any suitable one known to those skilled in the art, including, for example, a gel matrix, a particulate carrier, a micellar matrix, and the like.
  • the concentration (w/v) of the sustained-release carrier in the pharmaceutical composition can be, for example, 0.5-13%, preferably 1-12% or 1-15%.
  • the pharmaceutical composition disclosed according to the present application may further optionally include excipients.
  • the excipient may be any suitable one known to those skilled in the art, which may include, for example, one or more of the following: dispersion medium, preservative, stabilizer, wetting agent and/or emulsifier, solubilizer, Tackifiers, etc.
  • the viscosity increasing agent is, for example, sodium carboxymethyl cellulose, carboxymethyl cellulose, polyvinylpyrrolidone or gelatin.
  • the preservatives are, for example, antioxidants (such as ascorbic acid).
  • the pharmaceutical composition disclosed according to the present application may include active ingredients (the methylene blue dye, nutrients and/or conventional anti-tumor drugs, and optionally other active ingredients as described above) and a liquid carrier (e.g. Water, ethanol, or water/ethanol mixture) any dosage form suitable for intratumoral administration, preferably the following dosage forms: injections (preferably local injections), external liquids, nebulizers, and the like.
  • active ingredients the methylene blue dye, nutrients and/or conventional anti-tumor drugs, and optionally other active ingredients as described above
  • a liquid carrier e.g. Water, ethanol, or water/ethanol mixture
  • any dosage form suitable for intratumoral administration preferably the following dosage forms: injections (preferably local injections), external liquids, nebulizers, and the like.
  • injection refers to a sterile preparation containing an active ingredient and a liquid carrier for in vivo administration.
  • the injections are divided into local injections, intravenous injections, etc. according to the mode of administration, and intravenous injections can be used as local injections only after a given intratumoral administration concentration.
  • Injections are classified into liquid injections, powder injections for injection, etc. according to their commercial form.
  • the injection powder contains sterile dry powder and a solvent, the sterile dry powder contains part or all of the active ingredients, and the solvent contains all the liquid carriers.
  • the concentration of the active ingredient in the injection is the concentration of the active ingredient in the mixture with all the liquid carriers, which is usually the end point of the intratumoral drug delivery device (syringe, puncture, injection catheter, etc.) (such as needle hole, catheter outlet, etc.) Etc.)
  • the concentration of the active ingredient in the liquid medicine is usually the end point of the intratumoral drug delivery device (syringe, puncture, injection catheter, etc.) (such as needle hole, catheter outlet, etc.) Etc.)
  • the concentration of the active ingredient in the liquid medicine is usually the end point of the intratumoral drug delivery device (syringe, puncture, injection catheter, etc.) (such as needle hole, catheter outlet, etc.) Etc.)
  • the concentration of the active ingredient in the liquid medicine is usually the end point of the intratumoral drug delivery device (syringe, puncture, injection catheter, etc.) (such as needle hole, catheter outlet, etc.) Etc.)
  • the concentration of the active ingredient in the liquid medicine is usually the end
  • a topical pharmaceutical composition for the treatment of malignant solid tumors in a freeze-dried or semi-lyophilized form which is lyophilized or semi-lyophilized according to the disclosed Methylene blue dyes, nutrients and/or conventional anti-tumor drugs and a pharmaceutically acceptable carrier are partly or wholly obtained.
  • composition of the present invention should be made into a dosage form that can be administered into the target area intratumorally, preferably a topical pharmaceutical dosage form.
  • the preparation of the pharmaceutical composition of the present invention includes the following steps: preparing a liquid medicine containing the methylene blue dye, nutrients and/or conventional anti-tumor, liquid medium and optionally other substances.
  • the liquid medicine may be a solution (for example, a solution in a hydrophilic vehicle, preferably an aqueous solution), a suspension, or an emulsion containing a topical active ingredient.
  • the dispersion medium therein can be any suitable one known to those skilled in the art, such as micro-materials or nano-materials.
  • the dispersion medium therein can be any suitable one known to those skilled in the art, such as vegetable oil, synthetic oil or semi-synthetic oil that can be used for injection.
  • the vegetable oil may be, for example, cottonseed oil, almond oil, olive oil, castor oil, sesame oil, soybean oil, and peanut oil.
  • the pharmaceutical composition liquid injection of the present invention can be prepared by a method comprising the following steps: 1) The necessary components (such as the methylene blue Dyes, nutrients and/or conventional anti-tumor drugs) and optional other components are added to the solvent to prepare a liquid; 2) other necessary components (such as other nutrients) and Optionally, add the liquid prepared in 1) and mix uniformly to obtain a liquid medicine; 3) sterilize the liquid medicine prepared in 2) and prepare a liquid injection.
  • the sterilizing liquid medicine in the liquid injection can be used directly or diluted as a liquid medicine for intratumoral administration.
  • the pharmaceutical composition liquid injection of the present invention can be prepared by a method including the following steps: 1) The amount of methylene blue dye, nutrients and/or conventional The anti-tumor drug and optional other components are added to the solvent (or pharmaceutically acceptable liquid carrier), mixed uniformly and sterilized, and then prepared as a sterile liquid I; 2) The amount required is based on the intratumor administration concentration The optional other components (such as acidulant) are added to the solvent (or pharmaceutically acceptable liquid carrier), mixed uniformly, and sterilized to prepare the sterilizing liquid II.
  • the sterilizing liquid I and the sterilizing liquid II form a mixed liquid before or after entering the intratumoral administration device, which can be used directly or diluted as a liquid drug for intratumoral administration.
  • the injection powder of the pharmaceutical composition of the present invention can be prepared by a method including the following steps: And/or sterile dry powder of conventional antitumor drugs; and preparing a sterile vehicle containing the required amount of the other components (such as amino acid nutrients, analgesics, etc.) according to the intratumor administration concentration.
  • the sterile dry powder is preferably a sterile freeze-dried dry powder, and its preparation method includes: 1) preparing a solution containing methylene blue dyes, nutrients and/or conventional anti-tumor drugs and optional other components; 2) removing Bacteria filtration and packaging; 3) freeze-drying; 4) plugging and capping.
  • the process conditions of the freeze-drying include, for example, the pre-freezing condition is kept at the pre-freezing temperature -45°C for 4 hours; the sublimation drying condition is that the temperature rise rate is 0.1°C/min and the temperature is raised to -15°C and kept for at least 10 hours; The adsorption drying conditions are 30°C for 6 hours.
  • the sterile dry powder of the injection powder is re-dissolved in a sterile solvent to form a re-solution drug, which can be used directly or diluted as a liquid drug for intratumoral administration.
  • the changes in the composition of the present invention include: containing different types and concentrations of the methylene blue dye, containing different types and concentrations of synergistic drugs (such as nutrients and/or conventional anti-tumor drugs), containing different types and concentrations Concentration of other additives (such as analgesics, acidulants, etc.).
  • the pharmaceutical composition is mainly used to treat malignant solid tumors, especially refractory malignant solid tumors (such as pancreatic cancer) by intratumoral administration.
  • Intratumoral administration requires that the composition of the drug (local active ingredients, composition ratio and component concentration) can be administered into the tumor by interventional means, and produce the desired therapeutic effect in it.
  • the composition of the drug local active ingredients, composition ratio and component concentration
  • tumor refers to a mass formed due to abnormal proliferation of cells or mutated cells, which includes solid tumors.
  • malignant solid tumor refers to malignant tumors with tumor bodies, including, for example, the following groups classified according to tumor cell types: epithelial cell tumors, sarcomas, lymphomas, germ cell tumors, and blastoma; Tumors named after the organ or tissue where the area is located include, for example, tumors named after the following organs or tissues: skin, bone, muscle, breast, kidney, liver, lung, gallbladder, pancreas, brain, esophagus, bladder muscle, large intestine, Small intestine, spleen, stomach, prostate, testes, ovaries or uterus.
  • the malignant solid tumors include, for example, breast cancer, pancreatic cancer, thyroid cancer, nasopharyngeal cancer, prostate cancer, liver cancer, lung cancer, bowel cancer, oral cancer, esophageal cancer, stomach cancer, laryngeal cancer, testicular cancer, and vaginal cancer. , Uterine cancer, ovarian cancer, etc.
  • the local drug in the present invention is a therapeutic drug.
  • it can also be combined with other interventional therapies, systemic chemotherapy, immunotherapy, photodynamic therapy, sonodynamic therapy, surgical intervention or Combinations of such therapies are administered in combination to further improve the efficacy.
  • the pharmaceutical composition is mainly used for the treatment of malignant solid tumors by intratumoral administration.
  • the acidifying agent and the ineffective absorption compound are administered intratumorally at their concentration or amount in the local pharmaceutical composition. This concentration or amount can provide a synergistic effect of local response compared to intratumoral administration.
  • composition of the present invention has been shown to be effective in promoting the relevant structures of the tissue where the local disease is located (such as diseased tissue, diseased cells, and any structure involved in the formation of them) It destroys and minimizes damage to the patient's normal tissues at the same time, so as to achieve a safe and effective pharmaceutical effect for the treatment of malignant solid tumors.
  • L-amino acids are abbreviated as amino acids (for example, L-arginine is abbreviated as arginine), reduced glutathione is abbreviated as glutathione, and alanyl-glutamine dipeptide is abbreviated as For glutathione.
  • the animal tests of subcutaneous transplantation tumors are carried out in accordance with the test guidelines issued by the drug administration authority.
  • the test animals are Balb/c nude mice or mice aged 6-8 weeks and weighing 17.5-20.5 g.
  • the subcutaneous transplantation is carried out according to the conventional method of subcutaneous inoculation of tumor cells.
  • the PEMS 3.2 software compiled by West China School of Public Health, Sichuan University
  • the items to be observed, measured and analyzed in the experiment include general status, body weight, food intake, tumor volume, tumor weight, thymus weight, spleen weight, etc.
  • the tumor volume calculation formula is as follows:
  • Tumor volume (V) 1/2 ⁇ a ⁇ b 2 , where a represents the length of the tumor and b represents the width of the tumor.
  • tumor growth inhibition rate (abbreviated as tumor inhibition rate in the present invention) is as follows:
  • Tumor inhibition rate Y(%) (TW-CW)/CW ⁇ 100%, where TW is the average tumor weight of the study group; CW is the average tumor weight of the negative control group.
  • B/A the combination of A drug and B drug is denoted as B/A.
  • drug A and drug B are methylene blue dyes and other active components, respectively.
  • the single-use efficacy of A and B (denoted as E A and E B , respectively) and the actual combined efficacy of A/B (denoted as E A+B ) are both tumor inhibition rates.
  • One method for judging the effect of combined medication in animal experiments is the Burgi method (Burgi Y. Pharmacology; Drug actions and reactions. Cancer res. 1978, 38(2), 284-285). Jin Zhengjun improved the Burgi method (Jin Zhengjun, Addition in combined medication, Chinese Journal of Pharmacology 1980; 1(2), 70-76), and the formula for q is:
  • compositions of the present invention can be formulated.
  • the compositions of some of the compositions of the present invention prepared in this example are listed in Table 3.
  • methylene blue dye e.g. 1g of methylene blue
  • conventional anti-tumor drugs or/and conventional ineffective compounds e.g. 10g of glutathione
  • optionally other compounds according to the required concentration (as described in Table 2)
  • the components and the liquid carrier such as water for injection
  • the preparation for example, 1% methylene blue/10% glutathione in water
  • Solution I and solution II are mixed evenly according to the required concentration of each component (for example, 8.5ml of solution I and 1.5ml of solution II are mixed) into a mixed solution (for example, 1% methylene blue/30% glucose/5% acetic acid aqueous solution) )
  • a mixed solution for example, 1% methylene blue/30% glucose/5% acetic acid aqueous solution
  • methylene blue dye e.g. 1g methylene blue
  • conventional anti-tumor drugs or/and conventional ineffective compounds e.g. 20g arginine, 30g glucose
  • a liquid carrier such as water for injection
  • a liquid carrier such as water for injection
  • Freeze-drying, stoppering and capping prepare as sterile dry powder for later use.
  • % Methylene blue/20% arginine/30% glucose/5% acetic acid aqueous solution can be used as a liquid drug for intratumoral administration.
  • the experimental animals S180-bearing mice with an average tumor volume of 121mm 3
  • the negative control was normal saline, and the 11 study drugs were as shown in the table below. They were injected intraperitoneally and intratumorally, respectively.
  • the medicines are all aqueous solutions, which are prepared according to the preparation method of Example 1. The medicine is administered once every 3 days, a total of 3 times, and the injection volume is less than or equal to 120 ⁇ l/mouse.
  • Ten days after the end of the medication the animals were euthanized, and the tumor weight was measured after anatomy, and the tumor inhibition rate was calculated from the negative control group of each medication mode. The results are shown in Table 4.
  • the intraperitoneal injection group and intratumor injection group of the positive control substance showed almost the same tumor inhibition rate, although some people believe that the drug in the tumor can increase its local drug concentration and thus significantly increase its tumor inhibition rate.
  • Medicinal effect shows that the drug did not substantially change its targeting (tumor cells) and pharmacology (inhibition of tumor cells) when injected intratumorally. Therefore, unless placed in a sustained-release system, conventional anti-tumor drugs are still mainly administered by absorption rather than intratumoral administration. 2.50 (respectively
  • the tumor inhibition rates of the methylene blue intraperitoneal injection group and the intratumor injection group were both at a negligibly low level, indicating that it did not show any ablation effect when used alone at this concentration.
  • the tumor weight of the methylene blue/nutrient composition group and its component single-drug group (group 6 and groups 5 and 2, group 7 and groups 5 and 3, and group 8 and groups 5 and 4)
  • the difference was not statistically significant in each group of intraperitoneal injection (all p>0.05), and no synergistic efficacy was shown, while there were statistically significant in each group of intratumoral injection (all p ⁇ 0.05), and
  • the q value of the composition calculated based on the tumor inhibition rate of the latter is far greater than 1.00, and even extremely rarely greater than 2.50 (2.81, 3.32, and 4.00, respectively).
  • the intratumor injection group of the same composition showed a tumor inhibition rate of more than 10 times than that of the intraperitoneal injection group, thus showing significantly different targeting and pharmacology.
  • the difference in tumor weight between the methylene blue/conventional antineoplastic drug group and its component single agent group was not statistically significant between the intraperitoneal injection group 9 and group 1 (p>0.05), no synergistic drugs were shown
  • the group 9 injected intratumorally was statistically significant between groups 1 and 5 (all p ⁇ 0.05), and the q value of the composition calculated based on the tumor inhibition rate of the latter was much higher than 1.00 (1.30).
  • the tumor inhibition rate of the intratumor injection group of the same composition was 159% of that of the intraperitoneal injection group, which showed significantly different targeting and pharmacology.
  • the difference in tumor weight between the methylene blue/conventional antitumor drug/nutrient group and its component single agent group was not statistically significant between the intraperitoneal injection group 11 and group 10 (p>0.05).
  • Synergistic drug effect, and the intratumoral injection group 11 was statistically significant between groups 10 and 5 (both p ⁇ 0.05), and the q value of the composition calculated based on the tumor inhibition rate was much higher than 1.00 (1.24).
  • the tumor inhibition rate of the intratumor injection group of the same composition was 175% of that of the intraperitoneal injection group, which showed significantly different targeting and pharmacology.
  • the preferred synergistic pharmacology of the composition of the present invention is not conventional synergistic effects, but local synergistic effects.
  • the experimental animals PANC-1-bearing nude mice with an average tumor volume of 218 mm 3
  • the negative control is physiological saline
  • the 9 study drugs are shown in the table below.
  • the medicines are all aqueous solutions, which are prepared according to the preparation method of Example 1.
  • the drugs are injected intratumorally.
  • the two drugs containing 50% acetic acid are administered once, and the injection volume is 1000ul/only.
  • Other drugs are administered once every 3 days for a total of 3 times, with an injection volume of 100 ⁇ l/only.
  • Tumor inhibition rate 0 Normal saline 2.83 ⁇ 0.22g (0) 1 3% gemcitabine 1.47 ⁇ 0.18 48% 2 30% xylitol 2.32 ⁇ 0.13 18% 3 90% ethanol 0.91 ⁇ 0.21 68% 4 50% acetic acid 0.62 ⁇ 0.19 78% 5 0.7% methylene blue 2.52 ⁇ 0.17 11% 6 3% gemcitabine/0.7% methylene blue 0.42 ⁇ 0.11 85% 7 30% xylitol/0.7% methylene blue 0.34 ⁇ 0.09 88% 8 90% ethanol/0.7% methylene blue 0.82 ⁇ 0.12 71% 9 50% acetic acid/0.7% methylene blue 0.57 ⁇ 0.15 80%
  • composition of the present invention shows certain specificity, and its local synergistic effect shows very different specificity from the usual synergistic effect (same pharmacological synergistic effect).
  • the local synergist of the methylene blue dye in the composition of the present invention is not selected from chemical ablative agents with strong local action, preferably selected from drugs with relatively mild local action, more preferably selected from nutrients or/and conventional anti-inflammatory agents. Oncology medicine.
  • the experimental animals (mice bearing S180 cells with an average tumor volume of 112mm 3 ) that were successfully modeled were randomly divided into a negative control group and 40 study groups.
  • the negative control is physiological saline
  • the study drugs include: 4 kinds of methylene blue single drugs with varying concentrations (X%, X is methylene blue), 16 kinds of varying types and concentrations of other component single drugs (Y%, Y is other Components), 20 compositions (X%/Y%) composed of varying concentrations of methylene blue and varying types and concentrations of other components (nutrients, conventional anti-tumor drugs), and their compositions are shown in the following table.
  • the medicines are all aqueous solutions, which are prepared according to the preparation method of Example 1.
  • All groups were injected intratumorally, once every 3 days for a total of 3 times.
  • the dose of each administration was: methylene blue ⁇ 100mg/kg, lysine ⁇ 1000mg/kg, DHA ⁇ 375mg/kg, glucose ⁇ 2250mg/kg , 5-Fluorouracil ⁇ 50mg/kg, injection volume ⁇ 150 ⁇ l.
  • the animals were euthanized on the 10th day after the treatment, the tumor weight was measured after anatomy, and the tumor inhibition rate was calculated from the negative control group.
  • the tumor inhibition rate of each study drug group is shown in Table 6.
  • the data in the brackets is the average tumor inhibition rate of the X% methylene blue group, for example, the average tumor inhibition rate of the 0.15% methylene blue group is 3%
  • the data in the column bracket is the average tumor inhibition rate of Y% other component groups, for example, the average tumor inhibition rate of 1% lysine group is 5%
  • the data without brackets is the average tumor inhibition rate of X% methylene blue/Y% other component groups, for example, the average tumor inhibition rate of 3% methylene blue/1% lysine group is 45%
  • mice modeled with 1 ⁇ 10 6 breast tumor 4T1 cells per mouse mice modeled with 2 ⁇ 10 6 liver cancer H22 cells per mouse, and 1 ⁇ 10 6 liver cancer H22 cells per mouse.
  • mice modeled with 2 ⁇ 10 6 liver cancer H22 cells per mouse mice modeled with 2 ⁇ 10 6 liver cancer H22 cells per mouse
  • mice modeled with 1 ⁇ 10 6 liver cancer H22 cells per mouse mice modeled with 1 ⁇ 10 6 liver cancer H22 cells per mouse.
  • 106 CT26 mouse colon cancer cells modeling to 2 ⁇ 10 6 mouse B16-f10 th melanoma cells modeling each a similar effect can be observed.
  • the successfully modeled test animals (breast tumor-bearing 4T1 cell mice with an average tumor volume of 119mm 3 ) were randomly divided into a negative control group and 9 study groups.
  • the negative control is physiological saline, and the 9 study drugs are shown in the table below.
  • the medicines are all aqueous solutions, which are prepared according to the preparation method of Example 1.
  • the drugs are injected intratumorally.
  • the two drugs containing 50% acetic acid are administered once, and the injection volume is 1000ul/only.
  • Other drugs are administered once every 3 days for a total of 3 times, with an injection volume of 100 ⁇ l/only.
  • Table 7 The results are shown in Table 7.
  • Tumor inhibition rate 0 Normal saline 1.81 ⁇ 0.22g (0) 1 1% 5-fluorouracil 0.98 ⁇ 0.17 46% 2 30% xylitol 1.57 ⁇ 0.19 13% 3 0.5% methylene blue 1.67 ⁇ 0.20 8% 4 1% 5-fluorouracil/0.5% methylene blue 0.45 ⁇ 0.13 75% 5 30% xylitol/0.5% methylene blue 0.58 ⁇ 0.12 68% 6 0.5% 5-fluorouracil 1.18 ⁇ 0.18 35% 7 15% xylitol 1.61 ⁇ 0.16 11%
  • the difference in tumor weight between methylene blue/conventional anti-tumor drug group 4 and single-agent group 1 and 3 is statistically significant (both p ⁇ 0.05), and is calculated based on the tumor inhibition rate
  • the composition q value of 1.50 is far greater than the theoretical expected value of 1.00, showing a significant synergistic effect.
  • the difference in tumor weight between the methylene blue/nutrient group 5 and the single-agent group 2 and 3 was statistically significant (both p ⁇ 0.05), and the q value of the composition calculated based on the tumor inhibition rate was 3.40 It is much larger than the theoretical expected value of 1.00, showing a clear synergistic effect.
  • the difference in tumor weight between the composition group 9 and the single-agent group 6, and the composition groups 10 and 7) was not statistically significant (p>0.05) and did not show a synergistic drug effect.
  • the synergistic technical solution of the methylene blue dye, nutrient or/and conventional anti-tumor drug composition of the present invention is intratumoral administration, and its synergistic composition is not conventional during intratumoral administration.
  • the amount ratio in the synergistic composition but the concentration relationship as defined below:
  • the intratumoral administration concentration (w/V) of the methylene blue dye is ⁇ 2%, preferably 0.3%-1.8%;
  • intratumoral administration concentration of the conventional antitumor drug is greater than 20% of its saturated concentration, preferably 30%-100% of its saturated concentration; or/and
  • the intratumoral administration concentration of the nutrient is greater than 1%, preferably 2%-40%, wherein:
  • the intratumoral administration concentration of the amino acid nutrients is greater than 2%, preferably 2%-35%;
  • the intratumoral administration concentration of the lipid nutrient is greater than 2%, preferably 3%-25%; or/and
  • the intratumoral administration concentration of the carbohydrate nutrient is greater than 10%, preferably 20%-40%.
  • the composition of the present invention can not only produce a synergistic effect, but the synergistic effect can be further optimized under specific conditions to obtain a super effective synergistic effect. These specific conditions are not the conditions in the prior art for methylene blue dyes and nutrients or/and conventional anti-tumor drugs. The following test example investigated this.
  • Example 5 Further optimization of the intratumoral administration concentration of methylene blue dyestuff in the synergistic composition
  • the successfully modeled test animals (S180 cell-bearing mice with an average tumor volume of 132mm 3 ) were randomly divided into a negative control group, a positive control group and 7 study groups.
  • the negative control is physiological saline
  • the positive control is 1% 5-fluorouracil
  • the study drug contains 20% lysine and methylene blue (20% lysine/X%) with varying concentrations (X%) as shown in the table below. Methylene blue).
  • the medicines are all aqueous solutions, which are prepared according to the preparation method of Example 1. Each group was injected intratumorally, once every 3 days for a total of 3 times.
  • the dose of each administration was: methylene blue ⁇ 75 mg/kg, 5-fluorouracil 50 mg/kg, and injection volume ⁇ 150 ⁇ l.
  • the animals were dissected and the tumor weight was determined, and the tumor inhibition rate was calculated from the negative control group. The results are shown in Table 8.
  • composition of the present invention is: its composition is such that the intratumoral administration concentration of the methylene blue dye provided by the composition is ⁇ 0.35%, preferably 0.35 -1.5%, more preferably 0.5-1.5% or 0.5-1%.
  • the same substance can show completely different pharmacology according to its administration mode, so it can be used as different drugs.
  • systemically administered methylene blue is usually used as an antidote, but cannot be used as the local active ingredient in the composition of the present invention; systematically administered nutrients or/and conventional antitumor drugs are usually used as nutrients and antitumor drugs, but cannot As a local synergist of methylene blue dyes in the composition of the present invention.
  • the same substance can also show completely different pharmacology according to its local administration conditions and be used as different drugs.
  • ultra-high concentration (for example, ⁇ 3%) methylene blue can be used as a chemical ablation agent but not suitable for use in the present invention
  • the local active ingredient in the composition, while low concentration (for example, ⁇ 1%) methylene blue is usually used as a staining agent, but this local active ingredient can produce local synergistic effects with nutrients or/and conventional anti-tumor drugs.
  • Methylene blue dyes show unusual synergistic conditions. For example, it can be used as a chemical ablation agent at a high concentration but does not show local synergy with conventional chemical ablation agents at a low concentration. This is contrary to The prior art shares enhanced teaching about the same pharmacology; it actually produces a high synergy (q ⁇ 2.0) with nutrients that have little damage to tissues (such as lipid nutrients), which is contrary to the prior art’s similar pharmacology.
  • the teaching of sharing enhancement can produce almost uniform synergy (the q value is close), which is contrary to the prior art teaching about sharing enhancement of the same target.
  • the decisive synergy condition is not only the quantity ratio (concentration ratio) but also the concentration value itself.
  • concentration ratio concentration ratio
  • the intratumoral administration concentration of methylene blue dyestuffs below a certain threshold will not produce the local synergistic effect, which is contrary to the prior art teaching that the synergistic effect of conventional cytotoxic drugs is usually determined by the dosage ratio. .
  • composition of the present invention show higher specificity for tumor tissues.
  • composition of the present invention is compared with a conventional chemical ablation agent (50% acetic acid).
  • Example 6 Optimal intratumoral administration concentration of nutrients in the synergistic composition
  • the successfully modeled test animals (S180 cell-bearing mice with an average tumor volume of 128mm 3 ) were randomly divided into a negative control group (0), a positive control group (7) and 6 study groups (1-6 ).
  • the negative control is physiological saline, and the positive control is 1% 5-fluorouracil.
  • the composition of the study drug is shown in the table below, which contains varying concentrations (X%) of arginine and a fixed concentration (1%) of methylene blue ( X% arginine/1% methylene blue).
  • the medicines are all aqueous solutions, which are prepared according to the preparation method of Example 1. Each group was injected intratumorally, once every 3 days for a total of 3 times.
  • the dose of each administration was: arginine ⁇ 1500 mg/kg, 5-fluorouracil 50 mg/kg, and injection volume ⁇ 150 ⁇ l.
  • the animals were euthanized 5 days after the treatment, and the tumor weight was measured after anatomy, and the tumor inhibition rate was calculated from the negative control group. The results are shown in Table 9.
  • a super-effective and synergistic technical solution of the composition of the present invention is: its composition is such that the intratumoral administration concentration of the nutrient provided by the composition is ⁇ 3%, preferably When the nutrient is selected from carbohydrate nutrients, the intratumoral administration concentration of the nutrient is ⁇ 20%, preferably 20-40%; when the nutrient is selected from lipid nutrients, the lipid The intratumoral administration concentration of the nutrient is ⁇ 4%, preferably 4-25%; when the nutrient is selected from amino acid nutrients, the intratumoral administration concentration of the amino acid nutrient is ⁇ 3%, preferably 3-25%, and more Preferably it is 5-25% or 15-25%.
  • the successfully modeled test animals (S180 cell-bearing mice with an average tumor volume of 151mm 3 ) were randomly divided into a negative control group, a positive control group and 15 study groups.
  • the negative control is physiological saline
  • the positive control is 1% 5-fluorouracil
  • the research drug is 1% methylene blue/10% amino acid nutrient composition.
  • the amino acid nutrients in the compositions used in the study groups 1-13 are: arginine, glycine, cysteine hydrochloride, valine, threonine, proline, histidine hydrochloride, benzene Alanine, lysine, leucine, alanine, glutathione, serine, alanyl-glutamine dipeptide, the study group 15 medication was 1% methylene blue/5% arginine/ 5% glycine.
  • the medicines are all aqueous solutions, which are prepared according to the preparation method of Example 1.
  • Each group was injected intratumorally, once every 3 days for a total of 3 times, and the volume of each injection was ⁇ 150 ⁇ l.
  • the animals were euthanized on the 10th day after the treatment, the tumor weight was measured after anatomy, and the tumor inhibition rate was calculated from the negative control group.
  • the tumor inhibition rate of the positive control group was 51%, and the results of the study group are shown in Table 10.
  • the amino acid nutrient is preferably selected from the following amino acids and contains one of the following amino acids
  • One or more amino acid derivatives arginine, glycine, cysteine, threonine, proline, lysine, leucine, alanine, serine, glutamic acid, more preferably selected From the following amino acids and amino acid derivatives containing one or more of the following amino acids: arginine, glycine, cysteine, lysine, alanine, serine, glutamic acid.
  • the successfully modeled nude mice bearing human cancer cells were randomly divided into a negative control group and 5 study groups (groups A, B, C, D, and E).
  • the corresponding negative control is physiological saline
  • the 5 study drugs are: 1% methylene blue/5% DHA/5% acetic acid, 1% methylene blue/20% arginine, 1% methylene blue/10 % Glycine/10% lysine, 1% methylene blue/30% glucose, 1% methylene blue/1% 5-fluorouracil.
  • the medicines are all aqueous solutions, which are prepared according to the preparation method of Example 1.
  • Each group was injected intratumorally, once every 3 days, a total of 3 times, each time 100-150 ⁇ l/mouse.
  • the animals were euthanized, the tumor weight was measured after anatomy, and the tumor inhibition rate was calculated from the respective negative control group.
  • the successfully modeled nude mice bearing human head and neck cancer cells (F ⁇ da) (the average tumor volume is 169mm 3 ) were randomly divided into a negative control group and 5 study groups (groups A, B, C, D, and E) ).
  • the tumor inhibition rates of groups A, B, C, D, and E were 76%, 82%, 74%, 88%, 70%, respectively, which all met the generally considered effective anti-tumor standards (tumor inhibition rate ⁇ 40%).
  • CNE1 human nasopharyngeal carcinoma cells
  • compositions of the present invention prepared in Example 1 for example, the composition in Table 2
  • similar results can be obtained in the application of the above-mentioned tumor treatments.
  • a topical pharmaceutical composition for the treatment of malignant solid tumors comprising a methylene blue dye, a local synergistic drug of the methylene blue dye, and a pharmaceutically acceptable liquid carrier, wherein the synergistic drug It is selected from nutrients or/and conventional anti-tumor drugs, and in the topical pharmaceutical composition, the concentration (w/v) of the methylene blue dye is ⁇ 2%, preferably 0.35-2%, 0.5-2%, 0.5-1.5% or 0.5-1%, the concentration of the nutrient is greater than 2%, preferably 3%-40%, and the concentration of the conventional antitumor drug is greater than 20% or 30% of its saturated concentration, preferably The saturated concentration is 30%-100%, wherein the saturated concentration refers to the saturated concentration of the conventional anti-tumor drug in the liquid carrier.
  • the topical pharmaceutical composition comprises the methylene blue dye, the local synergistic drug of the methylene blue dye, and a pharmaceutically acceptable liquid carrier, wherein the topical drug
  • the concentration (w/v) of the methylene blue dye is ⁇ 2%, preferably 0.35-2%, 0.5-2%, 0.5-1.5% or 0.5-1%
  • the concentration of the nutrient is Greater than 2%, preferably 3%-40%
  • the concentration of the conventional anti-tumor drug is greater than 20% or 30% of its saturated concentration, preferably 30%-100% of its saturated concentration, wherein the saturated concentration is Refers to the saturated concentration of the conventional anti-tumor drug in the liquid carrier.
  • methylene blue dye is selected from methylene blue and its living dye analogs, preferably selected from the following compounds and their derivatives: methylene blue , Patent blue, isosulfur blue, neomethylene blue, more preferably selected from methylene blue and its derivatives.
  • composition or application according to any one of items 1-3, wherein the nutrient is one or more selected from the group consisting of amino acid nutrients, carbohydrate nutrients, lipid nutrients, and they are in the combination
  • concentration in the product is 2.5-50%, preferably 4-40%.
  • amino acid nutrient includes one or more of the following amino acid compounds with nutritional and health effects: amino acids, amino acid salts, oligopeptides and polypeptides; preferably selected from the following Group of amino acids or their salts or oligopeptides and polypeptides containing or consisting of the following amino acids: alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, Tyrosine, serine, cysteine, methionine, asparagine, glutamine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, ⁇ -alanine Acid, taurine, gamma aminobutyric acid (GABA), tea polyphenols (theanine), pumpkin seed amino acids (3-amino-3-carboxypyranoic acid), glutamine, citrulline, ornithine ; More preferably selected from the group of amino acids, amino acid salts, oligopeptides
  • amino acid nutrient is selected from amino acids or amino acid salts with nutritional and health effects, and the concentration of the amino acid or amino acid salt in the topical pharmaceutical composition (w/v ) Is ⁇ 2%, ⁇ 2.5, ⁇ 5%, ⁇ 7.5%, 10-25% or 18-25%, preferably 15%-25% or 20%-25%.
  • composition or application according to item 6 wherein the amino acid nutrient is selected from oligopeptides and polypeptides with nutritional and health effects, and the concentration of the oligopeptides and polypeptides in the topical pharmaceutical composition (w/v ) Is greater than ⁇ 5%, preferably 7.5-25%, more preferably 10%-25%.
  • concentration (w/v) is greater than ⁇ 5%, preferably 7.5%-25%, more preferably 10-25%.
  • oligopeptide is one or more selected from the group consisting of glycyl-L-tyrosine, glycylalanine, and glycylglycin , Lysine-glycine dipeptide, glutathione, carnosine ( ⁇ -alanine histidine copolymer), glutathione, collagen oligopeptide, casein hydrolyzed peptide, soybean oligopeptide, oligoarginine, Oligoglycine, oligolysine; the polypeptide is one or more selected from the following group: polyaspartic acid, polyglutamic acid, polylysine.
  • carbohydrate nutrient is selected from a carbohydrate compound containing one or more of the following sugar units: glucose, ribose, xylose, fructose, galactose, fucoid
  • the sugar is preferably one or more selected from the following: glucose, fructose, chitooligosaccharides, glucosamine, lactulose, sorbitol, ribose, sorbose, mannose, galactose, sucrose, lactose, trehalose, wood Oligosaccharides, fructooligosaccharides, mannose oligosaccharides, gluconic acid, sodium gluconate, xylitol, mannitol, maltitol, lactitol, more preferably one or more selected from the following: glucose, sodium gluconate, Chitooligosaccharides, glucosamine, lactulose, ribos
  • the lipid nutrient is one or more selected from the group consisting of fatty acids, fat milk and lipids, preferably one or more selected from the group consisting of vegetable oils , Eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), long-chain fat emulsion, medium-chain fat emulsion, phospholipid, and its concentration in the pharmaceutical composition is ⁇ 4%, preferably 4%-25%.
  • the conventional anti-tumor drugs are one or more selected from the group consisting of uracil derivatives, cyclophosphamides, gemcitabine, epirubicin , Anti-tumor antibiotics, teniposide, metal platinum complexes, taxanes, preferably one or more selected from the following drugs and similar derivatives: 5-fluorouracil, cyclophosphamide, gemcitabine, table Rubicin, antitumor antibiotic, teniposide, metal platinum complex, paclitaxel.
  • the conventional anti-tumor drugs are one or more selected from the group consisting of uracil derivatives, cyclophosphamides, gemcitabine, epirubicin , Anti-tumor antibiotics, teniposide, metal platinum complexes, taxanes, preferably one or more selected from the following drugs and similar derivatives: 5-fluorouracil, cyclophosphamide, gemcitabine, table Rubicin, antitumor antibiotic, teniposide,
  • composition or application according to items 1-4 which optionally further comprises one or more of the following groups: analgesics, sustained-release carriers, and C1 optionally substituted by 1-3 hydroxyl groups as an acidulant -10 Aliphatic carboxylic acid.
  • analgesic is one or more selected from the group consisting of benzyl alcohol, procaine hydrochloride, chlorobutanol, and lidocaine hydrochloride, and the analgesic
  • concentration of the agent in the pharmaceutical composition is 0.1-4% by weight.
  • composition or application according to item 15 wherein the acidulant is one or more selected from the group consisting of acetic acid, propionic acid, butyric acid, malonic acid, succinic acid, glycolic acid, lactic acid, lemon Acid, malic acid, tartaric acid, more preferably acetic acid.
  • the acidulant is one or more selected from the group consisting of acetic acid, propionic acid, butyric acid, malonic acid, succinic acid, glycolic acid, lactic acid, lemon Acid, malic acid, tartaric acid, more preferably acetic acid.
  • the injection powder for injection includes a sterile dry powder and a solvent, and part or all of the amino acid nutrients and the ineffective absorption compound are contained in the sterile dry powder, and The liquid carrier is contained in the solvent, and the concentrations of the amino acid nutrients and the ineffective absorption compound are their concentrations in the sterile dry powder and the solvent mixture, respectively.
  • a topical pharmaceutical composition for the treatment of malignant solid tumors which comprises a dry powder obtained by lyophilizing or semi-lyophilizing part or all of the pharmaceutical composition according to one of items 1 and 4-18.
  • a method for preventing and treating malignant solid tumors which comprises administering the pharmaceutical composition according to one of items 1 and 4-21 to an individual in need thereof.
  • malignant solid tumors include breast cancer, pancreatic cancer, thyroid cancer, nasopharyngeal cancer, prostate cancer, liver cancer, lung cancer, colon cancer, oral cancer , Esophageal cancer, stomach cancer, laryngeal cancer, testicular cancer, vagina cancer, uterine cancer, ovarian cancer.

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Abstract

一种亚甲蓝类染料作为局部活性成分与其局部协同药物的组合在制备治疗恶性实体肿瘤的局部药物组合物中的应用，包含该亚甲蓝类染料作为局部活性成分及其局部协同药物的用于治疗恶性实体肿瘤的局部药物组合物。

Description

包含亚甲蓝类染料、营养素或/和常规抗肿瘤药的药物组合物及其应用 技术领域
本申请公开涉及亚甲蓝类染料作为局部活性成分与营养素或/和常规抗肿瘤药的组合在制备治疗恶性实体肿瘤的局部药物组合物中的应用，包含该亚甲蓝类染料与营养素或/和常规抗肿瘤药的用于治疗恶性实体肿瘤的局部药物组合物，以及包括施用该药物组合物的一种治疗恶性实体肿瘤的方法。
背景技术
抗实体肿瘤药物开发所面临的一个主要问题是特异性。由于常规抗肿瘤药不能对靶细胞和正常细胞作足够区别，其有效剂量与安全限量之差不够大，故在产生全身性疗效(瘤体内、外的肿瘤细胞抑制效应)的同时，也会产生全身性毒性的较大风险。此外，药物分子需在瘤体组织内有效渗透才能与其间的肿瘤细胞发生作用。对于某些瘤体乏血供肿瘤(例如胰腺癌)而言，患者受益的机会则更小。
瘤内给药具有将药物进行物理靶向的优点。然而，瘤内给药常规抗肿瘤药虽提高了靶区浓度，却并未显示出疗效的明显提高。除了求助于其缓释形式以外，常规抗肿瘤药在临床上几乎仍全身给药。常规化学消融剂(高纯度乙醇、高浓度酸碱)几乎无靶向癌细胞的全身疗效，却通过组织坏死显示出较高局部疗效。然而，由于不能对靶组织和其它组织作足够区别，它们实际能够应用的介入体积(例如酸碱用量不超过0.2ml/kg)和介入部位非常受限。因而，近十年来化学消融剂以经从恶性实体肿瘤临床上淡出。实际上，目前临床上以几乎没有局部安全性和局部疗效均高的局部专用药物。
因而，仍然需要开发新的治疗恶性实体肿瘤的药物、尤其是特异性高于常规化学剂的局部专用药物，以满足现有技术尚不能满足的各种临床需求。
发明内容
本发明的目的在于提供一种物理地靶向瘤体、但却具有比现有药物更高疗效、更高依从性、或/和更高特异性的局部药物，以及包括施用该药物组合物的一种治疗恶性实体肿瘤的方法。
根据本申请公开的一个方面，其提供一种用于治疗恶性实体肿瘤的局部药物组合物，其包含亚甲蓝类染料、所述亚甲蓝类染料的局部协同药物、以及药物学可接受的液体载体，其中所述协同药物选自营养素或/和常规抗肿瘤药，且在该局部药物组合物中，所述亚甲蓝类染料的浓度(w/v)≤2％、优选为0.35-2％、0.5-2％、0.5-1.5％或0.5-1％，所述营养素的浓度(w/v)≥2％、优选为3-40％，以及所述常规抗肿瘤药的浓度大于其饱和浓度的20％或30％、优选为其饱和浓度的30％-100％，其中所述饱和浓度是指所述常规抗肿瘤药在所述液体载体中的饱和浓度。
根据本申请公开的另一个方面，其提供亚甲蓝类染料作为局部活性成分与所述亚甲蓝类染料的局部协同药物的组合在制备用于治疗恶性实体肿瘤的局部药物组合物中的应用，其中所述协同药 物选自营养素或/和常规抗肿瘤药。在此方面的一个实施方案中，所述局部药物组合物包含所述亚甲蓝类染料、所述亚甲蓝类染料的局部协同药物、以及药物学可接受的液体载体，其中在该局部药物组合物中，所述亚甲蓝类染料的浓度(w/v)≤2％、优选为0.35-2％、0.5-2％、0.5-1.5％或0.5-1％，所述营养素的浓度(w/v)≥2％、优选为3％-40％，以及所述常规抗肿瘤药的浓度大于其饱和浓度的20％或30％、优选为其饱和浓度的30％-100％，其中所述饱和浓度是指所述常规抗肿瘤药在所述液体载体中的饱和浓度。
根据本申请公开的再一个方面，其提供一种治疗恶性实体肿瘤的方法，其包括向有此需要的个体瘤内给药根据本申请公开的药物组合物。
根据本申请公开的包含所述亚甲蓝类染料及局部协同药物的组合物与含相应成分的单药相比具有以下优点：提供了针对恶性实体肿瘤的协同作用从而提高有效性，同时又有可能提供针对非特异性组织破坏的拮抗作用从而提高安全性。
根据本发明的实施方案与恶性实体肿瘤治疗的现有技术相比具有以下优点：与现有细胞毒药物及相关治疗方法相比，显示出几乎无毒的全身安全性和明显较高的局部治疗疗效；与现有分子靶向药物及相关治疗方法相比，显示出不那么苛刻的适应症筛选，以及针对快速生长瘤体、大瘤体和乏血供瘤体的巨大减荷潜力；与现有化学消融剂及相关方法相比，显示出更高的依从性(例如刺激性、腐蚀性)或特异性，从而可以有较大的介入适应范围和较高的应用体积。本发明的方法和组合物也不受现有细胞毒性药物和现有分子靶向药物遭遇的耐药性问题的困扰。此外，该方法和组合物应用方便、成本便宜，特别有助于使难以承受高额费用的广大人群也享受到安全、有效治疗。
具体实施方式
本发明的发明人在一个荷瘤动物实验中意外地发现，DHA的加入虽然一般不能、但在某些特定的条件下却能与亚甲蓝形成高度协同作用，这也使得亚甲蓝的使用剂量可以成倍减小以减小其副作用风险。进一步的研究发现，选自其它营养素或/和常规抗肿瘤药物的物质在这些特定的条件下也能与较低浓度(例如≤1％)的亚甲蓝形成超预期的协同作用。这些特定条件并非亚甲蓝类染料、营养素或/和常规抗肿瘤药在现有抗恶性实体肿瘤技术中的条件(例如瘤内给药浓度)，而是如以下所限定的。
在本发明的范围中，术语“局部药物(组合物)”区别于常规药物(组合物)，后者是指常规给药(或***给药，例如口服、静注、腹腔注射、胸腔注射等)从而其活性成分经血液输运至瘤体并在其中经血管扩散渗透作用的药物(组合物)，而前者则是指瘤内给药并在其中主要经血管外结构间隙扩散渗透、并主要通过局部作用产生药效的治疗药物(组合物)。术语“局部作用”或“局部活性”是指优先靶向瘤体组织而非肿瘤细胞的药理作用或药理活性。术语“局部活性成分”区别于化学消融剂，后者是指在瘤体有效消融条件(通常为超过消融浓度阈值)下的化学物质(例如50％乙酸、无水乙醇、5％亚甲蓝)，而前者则是指並非通过其常规作用(并非瘤内给药之外的任何给药)、也非其化学消融作用(低于其化学消融浓度条件下使用)、而是主要通过提供局部协同作用的活性成分(例如0.5～1.5％亚 甲蓝)。
在本发明的范围中，所用术语“局部协同药物”(或局部协同物是指可与局部活性成分(例如局部给药且给药浓度≤2％、优选≤1％)的所述亚甲蓝类染料)产生局部协同作用的物质，其选自营养素或/和常规抗肿瘤药。术语“局部协同作用”是指主要显示为局部作用的协同作用。术语“协同作用”是指活性组分的共用显示出的比它们分别单用对治疗更为有利的药学效应，其包括例如协同药效和协同安全性。术语“协同药效”是指活性组分共用显示出的比任一组分单用都更高的所需药效和/或该共用显示出的任一组分单用都未显示出的所需药效(例如2个细胞毒药物共用产生的组织坏死效应)。术语“协同安全性”是指获得有效药效时活性组分共用显示出的比任一组分单用更高的所需安全性。
因此，根据本发明的一个方面，其提供一种用于治疗恶性实体肿瘤的局部药物组合物，其包含亚甲蓝类染料、所述亚甲蓝类染料的局部协同药物、以及药物学可接受的液体载体，其中所述协同药物选自营养素或/和常规抗肿瘤药，且在该局部药物组合物中，所述亚甲蓝类染料的浓度(w/v)≤2％、优选为0.35-2％、0.5-2％、0.5-1.5％或0.5-1％，所述营养素的浓度(w/v)≥2％、优选为3％-40％，以及所述常规抗肿瘤药的浓度大于其饱和浓度的20％或30％、优选为其饱和浓度的30％-100％，其中所述饱和浓度是指所述常规抗肿瘤药在所述液体载体中的饱和浓度。
根据本申请公开的另一个方面，其提供亚甲蓝类染料作为局部活性成分与所述亚甲蓝类染料的局部协同药物的组合在制备用于治疗恶性实体肿瘤的局部药物组合物中的应用，其中所述协同药物选自营养素或/和常规抗肿瘤药。在此方面的一个实施方案中，所述局部药物组合物包含所述亚甲蓝类染料、所述亚甲蓝类染料的局部协同药物、以及药物学可接受的液体载体，其中在该局部药物组合物中，所述亚甲蓝类染料的浓度(w/v)≤2％、优选为0.35-2％、0.5-2％、0.5-1.5％或0.5-1％，所述营养素的浓度(w/v)≥2％、优选为3％-40％，以及所述常规抗肿瘤药的浓度大于其饱和浓度的20％或30％、优选为其饱和浓度的30％-100％，其中所述饱和浓度是指所述常规抗肿瘤药在所述液体载体中的饱和浓度。
根据本申请公开的再一个方面，其提供一种治疗恶性实体肿瘤的方法，其包括向有此需要的个体瘤内给药治疗有效量的根据本申请公开的包含所述亚甲蓝类染料作为局部活性成分和所述协同药物的药物组合物。
在本发明的范围内，所用术语“瘤内给药”是指将药物(例如注射剂)通过器械注入瘤内，例如经导管动脉灌注、经导管瘤内灌注、瘤内注射、等等。术语“治疗有效量”是指用于治疗疾病(例如肿瘤)并获得有效效果(例如降低或/和缓解疾病症状)的药物的量。
在本发明的范围中，除非另有说明，术语“浓度”是指所述局部药物组合物中指定组分的重量/体积百分比浓度％(w/v)。术语“瘤内给药浓度”是指指定组分在药物实施瘤内给药时的浓度，其可以是指定组分在药物接触靶区处(例如注射针孔或灌注管出口)的浓度。
在现有技术中，上述浓度的亚甲蓝类染料有多种应用，例如解毒剂、止疼剂、活体染料、等。此外，高浓度(例如5％)亚甲蓝类染料还可以作为化学消融剂应用。通常认为，化学消融剂浓度越大越有效。在本发明的实施例中，本发明的发明人意外地发现，亚甲蓝类染料在非化学消融剂条件(例 如1％亚甲蓝给药浓度)时，其可以与药理大不相同的常规抗肿瘤药和/或营养素形成q判断远超其理论预期、甚至于远超现有技术中常规抗肿瘤药共用预期的局部协同作用，从而可以用明显低于其化学消融浓度阈值的亚甲蓝类染料获得明显提高的疗效。这些超预期的协同作用也许就是那个药理关键点，其明显提高了亚甲蓝类染料、营养素或/和常规抗肿瘤药组合物针对瘤内组织破坏的特异性。根据本发明，所述药物应当在其使用说明书中明确亚甲蓝类染料瘤内给药浓度，以保证其被作为局部活性成分应用，而非作为化学消融剂应用，以避免风险。
根据本发明的药物组合物，其中所述亚甲蓝类染料优选为选自以下化合物及其衍生物：亚甲蓝、专利蓝、异硫蓝、新亚甲蓝。
根据本发明的药物组合物，所述亚甲蓝类染料更优选为选自亚甲蓝及其衍生物。
在本申请公开的药物组合物中，作为所述低浓度亚甲蓝类染料在治疗恶性实体肿瘤的局部协同药物，可以是选自营养素和/或常规抗肿瘤药之一种或多种。
在本发明的范围中，术语“营养素”是指具有营养保健效应的有机化合物，其通常被用于营养保健品、传统饮食及功能性饮食(例如保健饮食)的制备，其主要包括氨基酸类营养素、糖类营养素和脂类营养素。在一个实施方案中，所述营养素在所述组合物中的浓度为2.5-50％，优选为4-40％。
在根据本申请公开的一个实施方案中，所述局部协同药物包括氨基酸类营养素，且所述氨基酸类营养素在该药物组合物中的浓度(w/v)≥2％、≥2.5、≥5％、优选为≥7.5％、10-25％或18-25％，更优选为15％-25％或20％-25％。
在本发明的范围中，所用术语“氨基酸类营养素”是指具有营养保健效应的氨基酸类化合物，优选为选自具有营养保健效应的氨基酸、氨基酸聚合物及氨基酸衍生物，更优选为选自中国、美国或欧洲官方药典或指南所载的氨基酸类营养药和具有营养保健作用的氨基酸类辅料。
在本发明的范围内，作为所述氨基酸类营养素的氨基酸、氨基酸聚合物及氨基酸衍生物优选为选自以下组中的氨基酸、或者包含以下组中的氨基酸的的 寡肽和多肽、或者以下组中的氨基酸的氨基酸盐：蛋白氨基酸和非蛋白氨基酸。
具体而言，在本申请公开中，所述蛋白氨基酸包括选自以下组中的氨基酸：非极性氨基酸(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、脯氨酸)，极性中性氨基酸(例如色氨酸、酪氨酸、丝氨酸、半胱氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、苏氨酸)，碱性氨基酸(例如赖氨酸、精氨酸、组氨酸)，酸性氨基酸(例如天冬氨酸、谷氨酸)。以上除甘氨酸以外均为L型α-氨基酸。所述非蛋白氨基酸可包括以下氨基酸：β-丙氨酸、牛磺酸、γ-氨基丁酸(GABA)、茶多酚(茶氨酸)、南瓜子氨基酸(3-氨基-3-羧基吡烷酸)、谷氨酰胺、瓜氨酸、鸟氨酸等。
在本申请公开中，所用术语“寡肽”是指包含2-10个相同或不同的以 肽键相连的氨基酸的氨基酸聚合物；而术语“多肽”是指包含11-100个相同或不同的以 肽键相连的氨基酸聚合物。对于组成所述寡肽或多肽的氨基酸，可以全部是上述一种或多种氨基酸，也可以额外包含其他氨基酸。在一个实施方案中，所述寡肽可以为选自以下之一种或多种：甘氨酰-L-酪氨酸、甘氨酰丙氨酸、双甘氨肽、赖氨酸-甘氨酸二肽、丙谷二肽、肌肽(β-丙氨酸组氨酸共聚物)、谷胱甘肽、胶原蛋白寡肽、 酪蛋白水解肽、大豆寡肽、寡聚精氨酸、寡聚甘氨酸、寡聚赖氨酸。在一个实施方案中，所述多肽可以为选自以下之一种或多种：聚天冬氨酸、聚谷氨酸、聚赖氨酸。
在本发明的范围中，所用术语“氨基酸盐”是指如上所述之氨基酸与酸或碱所形成的盐，例如赖氨酸盐酸盐、组氨酸盐酸盐、谷氨酸盐酸盐、半胱氨酸盐酸盐、精氨酸盐酸盐、硫酸甘氨酸、硫酸甘氨酸铁、赖氨酸盐酸盐、天冬氨酸盐酸盐等。
在本发明的药物组合物中，所述氨基酸类营养素可以是氨基酸、氨基酸盐、寡肽和多肽中的一种或多种，例如是2种、3种、4种或5种或者更多种。
在本申请公开中，作为所述氨基酸类营养素的氨基酸、氨基酸盐、寡肽和多肽优选为选自以下组中的氨基酸或其盐或者包含或由以下氨基酸构成的寡肽和多肽：丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、脯氨酸、色氨酸、酪氨酸、丝氨酸、半胱氨酸、蛋氨酸、苏氨酸、赖氨酸、精氨酸、组氨酸、天冬氨酸、谷氨酸、β-丙氨酸、牛磺酸、γ氨基丁酸(GABA)、茶氨酸、瓜氨酸、鸟氨酸；更优选为选自以下组中的氨基酸或其盐或者包含或由以下氨基酸构成的寡肽和多肽：精氨酸、赖氨酸、甘氨酸、半胱氨酸、丙氨酸、丝氨酸、天冬氨酸、谷氨酸。
在一个实施方案中，所述氨基酸类营养素包括精氨酸。
在一个实施方案中，所述氨基酸类营养素选自包括精氨酸的复合氨基酸，例如：精氨酸/耐氨酸、精氨酸/丝氨酸、精氨酸/甘氨酸、精氨酸/半胱氨酸盐酸盐等等。在一个实施方案中，所述氨基酸类营养素包括赖氨酸。在一个实施方案中，所述氨基酸类营养素包括甘氨酸。在一个实施方案中，所述氨基酸类营养素包括谷氨酸。在一个实施方案中，所述氨基酸类营养素选自具有营养保健效应的氨基酸或氨基酸盐，且所述氨基酸或氨基酸盐在该局部药物组合物中的浓度(w/v)为≥2％、≥2.5、≥5％、≥7.5％、10-25％或18-25％，优选为15％-25％或20％-25％。
在一个实施方案中，所述氨基酸类营养素选自具有营养保健效应的寡肽和多肽，且所述寡肽和多肽在该局部药物组合物中的浓度(w/v)为大于≥5％、优选为7.5-25％、更优选为10％-25％。
在一个实施方案中，所述氨基酸类营养素为所述氨基酸和/或氨基酸盐与所述寡肽和/或多肽的组合，且该组合在该局部药物组合物中的浓度(w/v)为大于≥5％、优选为7.5％-25％、更优选为10-25％。
在根据本申请公开的一个实施方案中，所述局部协同药物包括糖类营养素，且所述糖类营养素在该药物组合物中的浓度(w/v)大于5％，优选≥10％、10-50％、15-50％或25-50％。
在本发明的范围中，所用术语“糖类营养素”是指具有营养保健效应的糖类化合物，优选为选自具有营养保健效应的单糖、糖聚合物及糖衍生物，更优选为选自中国、美国或欧洲官方药典或指南所载的糖类营养药和具有营养保健作用的糖类辅料。
在本申请公开中，作为所述糖类营养素的单糖、糖聚合物及糖衍生物优选为选自以下组中的单糖、包含以下组中的单糖的糖聚合物、或者它们的衍生物：葡萄糖、核糖、脱氧核糖、木糖、果糖、半乳糖、岩藻糖。
所述糖聚合物可以为选自包含如上所述之单糖的双糖、寡糖和多糖。在本发明的范围中，所用术语“双糖”是指包含有2个通过糖苷键相连的单糖的聚合物；所用术语“寡糖”是指包含有3-10个由糖苷键相连的单糖的聚合物；以及所用术语“多糖”是指包含10个以上由糖苷键相连的单糖的聚合物。对于组成所述双糖、寡糖或多糖的单糖，可以全部是上述一种或多种单糖，也可以额外包含其他单糖。在一个实施方案中，所述双糖可以为选自以下之一种或多种：乳果糖、麦芽糖、蔗糖、乳糖、海藻糖。在一个实施方案中，所述寡糖可以为选自以下之一种或多种：壳寡糖、木寡糖、果寡糖、甘露寡糖、麦芽寡糖、异麦芽寡糖。在一个实施方案中，所述多糖可以为选自以下之一种或多种：淀粉、纤维素、右旋糖酐、糖胺聚糖。
所述糖衍生物例如可以为选自如上所述之单糖或糖聚合物的以下糖衍生物：糖酸、糖酸盐、糖醇。在本发明的范围中，所用术语“糖酸”是指单糖或糖聚合物的酸类衍生物；所用术语“糖酸盐”是指单糖或糖聚合物的盐类衍生物；术语“糖醇”是指单糖或糖聚合物的醇类衍生物。在一个实施方案中，所述糖酸可以为选自以下之一种或多种：葡萄糖酸、甘露糖酸、阿糖酸。在一个实施方案中，所述糖酸盐可以为选自以下之一种或多种：葡萄糖酸钠、甘露糖酸钠、阿糖酸钠。在一个实施方案中，所述糖醇可以为选自以下之一种或多种：甘露醇、麦芽糖醇、乳糖醇、木糖醇。
在本发明的药物组合物中，所述糖类营养素可以是单糖、寡糖、多糖、糖酸、糖酸盐、糖醇中的一种或多种，例如是2种、3种、4种或5种或者更多种。
在一个实施方案中，所述糖类营养素选自葡萄糖、包含葡萄糖的糖聚合物、或葡萄糖衍生物。
在一个实施方案中，所述糖类营养素选自核糖、包含核糖的糖聚合物、或核糖衍生物。
在一个实施方案中，所述糖类营养素选自木糖、包含木糖的糖聚合物、或木糖衍生物。
在一个实施方案中，所述糖类营养素优选为选自以下之一种或多种：葡萄糖、果糖、壳寡糖、氨基葡萄糖、乳果糖、山梨醇、核糖、山梨糖、甘露糖、半乳糖、蔗糖、乳糖、海藻糖、木寡糖、果寡糖、甘露寡糖、木糖醇，更优选为选自以下之一种或多种：葡萄糖、葡萄糖酸钠、壳寡糖、氨基葡萄糖、乳果糖、核糖、甘露寡糖、木糖醇。在一个实施方案中，所述糖类营养素在所述药物组合物中的浓度(w/v)大于5％，优选≥10％、10-40％、15-50％或25-50％。
在根据本申请公开的一个实施方案中，所述局部协同药物包括脂类营养素，且所述脂类营养素在该药物组合物中的浓度(w/v)≥4％、优选为4-25％。
在根据本申请公开的药物组合物中，所述脂类营养素包括药学上可接受的任何脂类营养素，优选与选自中国、美国或欧洲官方药典或指南所载的具有营养保健效应的脂类化合物，更优选为选自以下组之一种或多种：脂、脂肪酸、脂肪乳和类脂。
在一个实施方案中，所述脂类营养素为选自以下之一种或多种：植物油、二十碳五烯酸(EPA)、二十二碳六烯酸(DHA)、长链脂肪乳、中链脂肪乳、磷脂。在一个实施方案中，所述脂类营养素在所述药物组合物中的浓度(w/v)≥4％、优选为4-25％。
在本申请公开中，所用术语“常规抗肿瘤药”是指在安全剂量下可以通过吸收作用有效抑制实体肿瘤的药物，其选自药学上可接受的任何常规抗肿瘤药，优选为选自本领域公知的常规抗肿瘤药， 更优选为选自中国、美国或欧洲官方主管行政部门(例如FDA或中国药监局)己批准或将批准、或经中国、美国或欧洲官方药典己载入或将载入的抗肿瘤药。在此所用术语“吸收作用”是指药物经血液吸收形成带药血液进入靶区所产生的药理作用。某些常规抗肿瘤药曾经被寄望于通过瘤内给药提高药效，但该药效提高随浓度提高(化学动力学)却远低于其理论相关性。尽管在大量研究中局部共用不同药物，却未出现多少局部协同作用、更别说超预期的协同作用，说明在瘤内特殊条件(例如癌细胞微环境)下该类药物的协同作用具有高度不确定性。.
在本申请公开中，所述常规抗肿瘤药可以为选自以下组之一种或多种：破坏DNA结构和功能的药物、嵌入DNA中干扰转录RNA的药物、干扰DNA合成的药物、影响蛋白质合成的药物。所述破坏DNA结构和功能的药物包括例如烷化剂(例如环磷酰胺、卡莫司汀等)、金属铂络合物(例如顺铂、卡铂等)、DNA拓扑异构酶抑制剂(例如多柔比星类、拓扑替康、伊立替康等)等。所述嵌入DNA中干扰转录RNA的药物包括例如抗肿瘤抗生素，如放线菌素类、柔红霉素、多柔比星等。所述干扰DNA合成的药物包括例如嘧啶拮抗物(例如尿嘧啶衍生物5-氟尿嘧啶、呋氟尿嘧啶、双呋氟尿嘧啶，胞嘧啶衍生物阿糖胞苷、环胞苷、5-氮杂胞苷等)、嘌呤拮抗物(例如溶癌呤、硫鸟嘌呤等)、叶酸拮抗物(例如甲氨蝶呤等)等。所述影响蛋白质合成的药物包括例如秋水仙碱类、长春碱类、紫杉烷类(例如紫杉醇、多西紫杉等)等。
在本申请公开的药物组合物中，所述药物学可接受的液体载体包括水和/或乙醇。该药物学可接受的液体载体主要是根据常规抗肿瘤药的性质进行选择，以使该药物能够达到相应的浓度。
在本申请公开的药物组合物中，所述常规抗肿瘤药选自水溶性常规抗肿瘤药和醇溶性常规抗肿瘤药。在本发明的范围中，术语“醇溶性常规抗肿瘤药”是指在常温下在乙醇或乙醇水溶液中的溶解度大于或等于其有效局部作用所需浓度的常规抗肿瘤药，其包括例如紫杉烷类、长春碱类等。所用术语“水溶性常规抗肿瘤药”是指在常温下在水溶液中的溶解度大于或等于其有效局部作用所需浓度的常规抗肿瘤药，其包括例如选自以下组之一种或多种水溶性化合物：尿嘧啶衍生物类、环磷酰胺类、吉西他滨类(例如盐酸吉西他滨)、表柔比星类(例如盐酸表柔比星)、抗肿瘤抗生素类(例如多柔比星、放线菌素等)、长春碱类(例如硫酸长春碱)、替尼泊苷、金属铂络合物等。
在根据本申请公开的药物组合物中，所述常规抗肿瘤药可以为选自以下组之一种或多种：尿嘧啶衍生物类、环磷酰胺类、吉西他滨类、表柔比星类、抗肿瘤抗生素类、替尼泊苷、金属铂络合物、紫杉烷类；优选为选自以下药物及其类似衍生物一种或多种：5-氟尿嘧啶、环磷酰胺、吉西他滨、表柔比星、抗肿瘤抗生素、替尼泊苷、金属铂络合物、紫杉醇。
在根据本申请公开的局部药物组合物中，所述常规抗肿瘤药的浓度大于其饱和浓度的30％、优选为其饱和浓度的50-100％、60-100％、70-100％、80-100％、或90-100％，其中所述饱和浓度是指所述常规抗肿瘤药在所述液体载体中的饱和浓度。
在一个具体实施方案中，选自所述烷化剂的常规抗肿瘤药(例如环磷酰胺、卡莫司汀等)在所述局部药物组合物中的浓度(w/v)为0.5-6％、优选为0.75-1.5％。
在一个具体实施方案中，选自所述金属铂络合物的常规抗肿瘤药(例如顺铂、卡铂等)在所述 局部药物组合物中的浓度(w/v)为0.03-0.08％、优选为0.03-0.06％。
在一个具体实施方案中，选自所述DNA拓扑异构酶抑制剂的常规抗肿瘤药(例如多柔比星类、拓扑替康、伊立替康等)在所述局部药物组合物中的浓度(w/v)为0.05-0.20％、优选为0.75-0.15％。
在一个具体实施方案中，选自所述抗肿瘤抗生素的常规抗肿瘤药(例如放线菌素类、柔红霉素等)在所述局部药物组合物中的浓度(w/v)为1-4％、优选为1-2％。
在一个具体实施方案中，选自所述嘧啶拮抗物的常规抗肿瘤药(例如尿嘧啶衍生物5-氟尿嘧啶、呋氟尿嘧啶、双呋氟尿嘧啶，胞嘧啶衍生物阿糖胞苷、环胞苷、5-氮杂胞苷等)在所述局部药物组合物中的浓度(w/v)浓度为0.5-2％、优选为0.75-1.5％。
在一个具体实施方案中，选自所述紫杉烷类的常规抗肿瘤药(例如紫杉醇、多西紫杉等)在所述局部药物组合物中的浓度(w/v)为0.5-2％、优选为0.75-1.5％。
在根据本申请公开的药物组合物中，其还任选包含选自以下组中的一种或多种：止痛剂、缓释载体、pH调节剂、以及赋形剂。
根据本申请公开的药物组合物还可进一步任选地包含止痛剂。所述止痛剂用以减轻患者的疼痛感，其可以是本领域技术人员已知的任意合适者，例如苯甲醇、盐酸普鲁卡因、三氯叔丁醇、盐酸利多卡等。所述止痛剂在所述药物组合物中的浓度例如可以是0.1-4重量％。例如苯甲醇在所述药物组合物中的浓度可以为1-4重量％，以及盐酸普鲁卡因、三氯叔丁醇、盐酸利多卡在所述药物组合物中的浓度可以分别为1-3重量％。
根据本申请公开的药物组合物还可进一步任选地包含缓释载体。所述缓释载体可以是本领域技术人员已知的任意合适者，包括例如凝胶基质、微粒载体、胶束基质等。所述缓释载体在所述药物组合物中的浓度(w/v)例如可以为0.5-13％、优选为1-12％或1-15％。
根据本申请公开的药物组合物还可进一步任选地包含赋形剂。所述赋形剂可以是本领域技术人员已知的任意合适者，其可包括例如以下之一种或多种：分散介质、防腐剂、稳定剂、湿润剂和/或乳化剂、增溶剂、增粘剂等。所述增粘剂例如为羧甲基纤维素钠、羧甲基纤维素、聚乙烯毗咯烷酮或明胶。所述防腐剂例如抗氧化剂例(如抗坏血酸)。
根据本申请公开的药物组合物可以是可包含活性成分(所述亚甲蓝类染料、营养素和/或常规抗肿瘤药、以及任选存在的如上所述的其他活性成分)和液体载体(例如水、乙醇、或者水/乙醇混合物)的任何适用于瘤内给药的剂型，优选为以下剂型：注射剂(优选为局部注射剂)、外用液剂、雾化剂等。
在本发明的范围中，所用术语“注射剂”是指含活性成分和液体载体并供体内给药的无菌制剂。所述注射剂按给药方式分为局部注射剂、静脉注射剂等，静脉注射剂只有在给定瘤内给药浓度后方可作为局部注射剂使用。注射剂按商品形式分为液体注射剂、注射用粉针剂等。注射用粉针剂包含无菌干粉和溶媒，无菌干粉中包含部分或全部活性成分，溶媒中包含全部液体载体。注射剂中所述活性成分的浓度均为其与全部所述液体载体的混合物中的活性成分浓度，通常是瘤内给药器械(注射器、穿刺器、注入导管等)终点(例如针孔、导管出口等)的液体药物中的活性成分浓度。对注 射用粉针剂而言，所述活性成分的浓度即为无菌干粉和溶媒的混合物(例如复溶液，或所述药物学可接受的液体载体)中的活性成分浓度。
根据本申请公开的另一个方面，其提供一种冻干或半冻干形式的用于治疗恶性实体肿瘤的局部药物组合物，其是通过冻干或半冻干根据本申请公开的包含所述亚甲蓝类染料、营养素和/或常规抗肿瘤药以及药物学可接受的载体的药物组合物之一部分或全部而得到的。
本领域技术人员会理解，根据本发明的技术方案，本发明的组合物应被制成可向所述靶区瘤内给药的剂型、优选为局部药物剂型。
根据本发明的制备方法，本发明的药物组合物的制备包含以下步骤：制备含所述亚甲蓝类染料、营养素和/或常规抗肿瘤、液体介质和任选存在的其它物质的液体药物。该液体药物可以是包含局部活性成分的溶液(例如为亲水溶媒的溶液、优选为水溶液)、悬浮液、或乳浊液。当所述液体药物为悬浊液时，其中的分散介质可以本领域技术人员已知的任意合适者，例如微米材料或纳米材料。当所述液体药物为乳浊液时，其中的分散介质可以本领域技术人员已知的任意合适者，例如可用于注射的植物油、合成油或半合成油。其中所述植物油可以是例如棉籽油、杏仁油、橄榄油、蓖麻油、芝麻油、大豆油和花生油。
根据本发明制备方法的一个实施方案，本发明的药物组合物液体注射剂可通过包含以下步骤的方法制备：1)将根据瘤内给药浓度所需量的必须组分(例如所述亚甲蓝类染料、营养素和/或常规抗肿瘤药)以及任选存在的其他组分加入溶媒中制备为液体；2)将根据瘤内给药浓度所需量的其它必须组分(例如其它营养素)以及任选存在的加入在1)中制备的液体混合均匀获得液体药物；3)将在2)中制备的液体药物除菌后制成液体注射剂。使用时，液体注射剂中的除菌液体药物可直接或稀释后用作瘤内给药液体药物。
根据本申请公开的一个实施方案，本发明的药物组合物液体注射剂可通过包括以下步骤的方法制备：1)将根据瘤内给药浓度所需量的亚甲蓝类染料、营养素和/或常规抗肿瘤药以及任选存在的其他组分加入溶媒(或者药物学可接受的液体载体)中混合均匀并经除菌后制备为除菌液体I；2)将根据瘤内给药浓度所需量的任选存在的其他组分(例如酸化剂)加入溶媒(或者药物学可接受的液体载体)中混合均匀并经除菌后制备为除菌液体II。使用时，除菌液体I和除菌液体II在进入瘤内给药器械前或后形成混合液，其即可直接或稀释后用作瘤内给药液体药物。
根据本申请公开的一个实施方案，本发明的药物组合物之注射用粉针剂可通过包括以下步骤的方法制备：制备含根据瘤内给药浓度所需量的所述亚甲蓝类染料、营养素和/或常规抗肿瘤药的无菌干粉；和制备包含根据瘤内给药浓度所需量的所述其它组分(例如氨基酸类营养素、止痛剂等)的无菌溶媒。所述除菌干粉优选为除菌冻干干粉，其制备方法包括：1)制备包含亚甲蓝类染料、营养素和/或常规抗肿瘤药以及任选存在的其他组分的溶液；2)除菌过滤和分装；3)冷冻干燥；4)压塞、轧盖。所述冷冻干燥的工艺条件例如包括：预冻条件为在预冻温度-45℃保持4小时；升华干燥条件为升温速率为0.1℃/分钟、且升至-15℃时至少保持10小时；解吸附干燥条件为30℃保持6小时。使用时，将注射用粉针剂的无菌干粉复溶于无菌溶媒中形成复溶液体药物，其即可直接或 稀释后用作瘤内给药液体药物。
按上述这些方法的原则，本领域技术人员可以采用任意合适的具体方法制备多种包含本发明组合物的具体剂型。例如，本发明的组合物中的变化包括：含不同种类和浓度的所述亚甲蓝类染料、含不同种类和浓度的协同药物(如营养素和/或常规抗肿瘤药)、含不同种类和浓度的其他添加剂(例如止痛剂、酸化剂等)。
在本申请公开中，所述药物组合物主要是用于通过瘤内给药治疗恶性实体肿瘤、尤其是难治性恶性实体肿瘤(例如胰腺癌)。
瘤内给药要求药物组成(局部活性成分、组成比及组分浓度)可通过介入手段给药于瘤内、并在其中产生所需要的疗效。
在本发明的范围内，术语“肿瘤”是指由于细胞或变异的细胞异常增殖形成的肿块，其包括实体肿瘤。术语“恶性实体肿瘤”是指具有瘤体的恶性肿瘤，包括例如按照肿瘤细胞类型进行分类的以下组：上皮细胞肿瘤、肉瘤、淋巴瘤、生殖细胞肿瘤、胚细胞瘤；以及包括按照肿瘤细胞集中区所在的器官或组织来命名的肿瘤，包括例如按照以下器官或组织来命名的肿瘤：皮肤、骨、肌肉、乳腺、肾、肝、肺、胆囊、胰腺、脑、食道、膀肌、大肠、小肠、脾、胃、***、睾丸、卵巢或子宫。
具体而言，所述恶性实体肿瘤包括例如乳腺癌、胰腺癌、甲状腺癌、鼻咽癌、***癌、肝癌、肺癌、肠癌、口腔癌、食道癌、胃癌、喉癌、睾丸癌、***癌、子宫癌、卵巢癌等。
本发明中的所述局部药物是一种治疗药物，当其用于预防和治疗恶性实体肿瘤时，还可与其它介入疗法、全身化疗、免疫疗法、光动力疗法、声动力疗法、手术干预或此类疗法的组合相组合施用，以进一步提高疗效。
在本申请公开中，所述药物组合物主要是用于通过瘤内给药治疗恶性实体肿瘤。
在根据本申请公开的局部治疗恶性实体肿瘤的应用和方法中，所述酸化剂和无效吸收化合物以它们在所述局部药物组合物中的浓度或量比瘤内给药。该浓度或量比瘤内给药能够提供局部反应的协同作用。
基于在下文中更详细描述的研究，尽管具体机理尚待进一步研究，本发明的组合物显示出促进局部病变所在组织的相关结构(例如病变组织、病变细胞及参与构成它们的任一结构)的有效破坏、同时对患者正常组织仅有最小化的损害，从而达到安全、有效治疗恶性实体肿瘤的药学效果。
实施例
通过以下具体实施例对本发明作进一步的说明，但不作为对本发明的限制。在以下实施例中，所有的试验动物均依照相关法规及行业自律进行。如无特殊说明，所有试验均按常规方法进行。
以下具体实施例所用的材料、试剂等，如无特殊说明，均可从商业途径得到。以下实施例中所用的部分亚甲蓝类染料、营养素和常规抗肿瘤药列于表1中。
表1
Figure PCTCN2020075765-appb-000001
在本发明中，L-氨基酸均简写为氨基酸(例如L-精氨酸均简写为精氨酸)，还原型谷胱甘肽简写为谷胱甘肽，丙氨酰-谷氨酰胺二肽简写为丙谷二肽。
在以下实施例中，除非另有说明，皮下移植瘤动物试验均按药管当局颁发的试验指南进行。试验动物为6-8周龄、体重17.5-20.5g的Balb/c裸鼠或小鼠。皮下移植均按常规的肿瘤细胞皮下接种方法进行。除非另有说明，待肿瘤长至所需体积(例如75-500mm 3)，采用PEMS 3.2软件(四川大学华西公共卫生学院编制)随机分为若干个组，每组6只动物。试验观察、测量和分析的项目，包括一般状态、体重、摄食量、肿瘤体积、瘤重、胸腺重量、脾脏重量等。
肿瘤体积计算公式如下：
肿瘤体积(V)＝l/2×a×b 2，其中a表示肿瘤长，b表示肿瘤宽。
肿瘤生长抑制率(本发明中简记为抑瘤率)计算公式如下：
抑瘤率Y(％)＝(TW-CW)/CW×100％，其中TW为研究组的平均瘤重；CW为阴性对照组的平均瘤重。
在以下实施例中，试验均采用重复测量方差分析(Repeated Measμres ANOVA)分别对指标均数的组别差异进行统计学检验。组别差异有统计学意义时(P≤0.05)，采用最小显著差异法对各组与阴性对照组间差异进行比较。定量指标采用均数±标准误(X±SEM)描述。当LEVENE方差齐性检验提示方差不齐时(P≤0.05)，采用Mann-WhitneyΜ秩和检验(M-W法)比较组间差异。所有的统计分析，均在SPSS for Windows 13.0软件下完成。
在本发明的范围內，A药和B药的组合物记为B/A。以下实施例中，除非另有说明，A药和B药分别为亚甲蓝类染料和其它活性组分。
药物联合使用产生的作用(包括药效和安全性)，理论上可依据于q判断进行：
q＝实际合用作用/理论单纯相加预期作用。
当q＝1时，实际合用作用符合理论预期，显示为相加作用；当q<1时，实际合用作用弱于理论预期，显示为拮抗作用；当q>1时，实际合用作用超理论预期，显示为协同作用。除非另有说明，A、B的单用药效(分别记作E A、E B)和A/B的实际合用药效(记作E A+B)均为抑瘤率。一种判断动物实验中合并用药效应的方法是Burgi法(Burgi Y.Pharmacology；Drug actions and reactions.Cancer res.1978，38(2)，284-285)。金正均对Burgi法进行了改进(金正均，合并用药中的相加，中国药理学报1980；1(2)，70-76)，其q计算式为:
q＝E A+B/(E A+E B-E A·E B)，
其中(E A+E B-E A·E B)为A药和B药理论单纯相加预期效应。
另外，改善抗肿瘤药物的药效一直是世界最大的医药难题。哪怕百分之几的药效提高也难之又难，以致于动物实验中联合用药的理论预期通常不高，而一旦实现就意义重大。动物实验文献中常见的一种抗肿瘤联合用药药效判断方法(W判断)，基于假设:实际合用作用能明显超单药作用便是超理论预期。具体判断为:(W A+B<E A和W A+B<E B)且组合物组与各组分组的瘤重差异均有统计学意义(均为p<0.005)。
在以下抗肿瘤动物实验中，药物联合使用产生的作用的判断如表2所示:
表2
Figure PCTCN2020075765-appb-000002
Figure PCTCN2020075765-appb-000003
实施例1：组合物的制备
按照上述本发明的组合物的制备方法，可以配制出本发明众多不同的组合物，本实施例制备的部分本发明的组合物的组成列于表3。
表3
Figure PCTCN2020075765-appb-000004
Figure PCTCN2020075765-appb-000005
以下列出本发明的组合物的制备试验的几个例子。
1、液体注射剂的制备(1)
按所需浓度(如表2所述)量取亚甲蓝类染料(例如1g亚甲蓝)、常规抗肿瘤药或/和常规无效化合物(例如10g谷胱甘肽)、任选存在的其他组分及定容至总体积(例如100ml)的液体载体(例如注射用水)，并将它们缓慢混合均匀，除菌过滤后分装为所需量(例如10ml/瓶)储存备用。该制备物(例如1％亚甲蓝/10％谷胱甘肽水溶液)可作为液体药物瘤内给药。
2、液体注射剂的制备(2)
1)、按所需浓度(如表2所述)量取亚甲蓝类染料(例如1g亚甲蓝)、常规抗肿瘤药或/和常规无效化合物(例如30g葡萄糖)、任选存在的其他组分及定容至总体积(例如85ml)的液体载体(例如注射用水)，并将它们缓慢混合均匀，除菌过滤后分装为所需量(例如8.5ml/瓶)储存备用，此为溶液I。
2)、按所需浓度(如表2所述)量取酸化剂(例如5g乙酸)、任选存在的其他组分及定容至总体积(例如15ml)的溶媒(例如注射用水)，并将它们缓慢混合均匀，除菌过滤后分装为所需量(例如1.5ml/瓶)储存备用，此为溶液II。
3)、溶液I和溶液II按各组分所需浓度混合均匀(例如将8.5ml溶液I和1.5ml溶液II混合)为混合液(例如1％亚甲蓝/30％葡萄糖/5％乙酸水溶液)后即可作为液体药物瘤内给药。
3、注射用粉针剂的制备
1)、按所需浓度(如表2所述)量取亚甲蓝类染料(例如1g亚甲蓝)、常规抗肿瘤药或/和常规无效化合物(例如20g精氨酸、30g葡萄糖)、任选存在的其他组分及定容至总体积(例如100ml)的液体载体(例如注射用水)，并将它们缓慢混合均匀，除菌过滤后分装为所需量(例如10ml/瓶)进行冷冻干燥及压塞、轧盖，制备为除菌干粉备用。
2)、按所需浓度(如表2所述)量取剩余任选存在的其他组分(假如还有的话，例如5g乙酸)、及定容至总体积(例如100ml)的液体载体(例如注射用水)，并将它们缓慢混合均匀，除菌过滤后分装为所需量(例如10ml/瓶)，制备为除菌液体备用。
3)、按各组分所需浓度将所需量的除菌干粉(例如1瓶上述干粉)复溶于所需量的除菌液体(例如1瓶上述溶媒)为所需复溶液(例如1％亚甲蓝/20％精氨酸/30％葡萄糖/5％乙酸水溶液)后即可作为液体药物瘤内给药。
实施例2：组合物协同药理优选
本研究试验中，成功建模的试验动物(荷S180小鼠，瘤体平均体积121mm 3)随机分为2个阴性对照组和22个研究组。阴性对照物为生理盐水，11个研究药物如下表所示，它们分别行腹腔注射和行瘤内注射。药物均为水溶液，按实施例1的制备方法配置而成。每3日用药一次，一共3次，注射量≤120μl/只。用药结束后10日，对动物进行安乐死，解剖后测定瘤重，并从各用药方式的阴性对照组计算抑瘤率，结果示于表4。
表4
Figure PCTCN2020075765-appb-000006
在上表中，阳性对照物(5-氟尿嘧啶)的腹腔注射组和瘤内注射组显示出几无区别的抑瘤率，尽管有人认为:瘤体内用药可提高其局部药物浓度从而会明显提高其药效。该结果说明该药瘤内注射时并未从本质上改变其靶向性(肿瘤细胞)和药理(抑制肿瘤细胞)。因而，除非置之于缓释***中，常规抗肿瘤药迄今仍然主要是吸收给药而非瘤内给药。2.50(分别为
在上表中，亚甲蓝腹腔注射组和瘤内注射组的抑瘤率均在可忽略不计的低水平，显示其在该浓度单用时并未显示任何消融效应。
在上表中，亚甲蓝/营养素组合物组与其组分单药组(组6分别与组5和2、组7分别与组5和3、组8分別与组5和4)的瘤重差异，在腹腔注射各组中，均无统计学意义(均为p>0.05)未显示出协同药效，而在瘤内注射各组中均有统计学意义(均为p<0.05)，且后者的根据抑瘤率计算出的组合物q值均远大1.00、甚至极其罕见地大于2.50(分别为2.81、3.32和4.00)。实际上同一组合物的瘤 内注射组比腹腔注射组显示出10倍以上的抑瘤率，从而显示出明显不同的靶向和药理。
在上表中，亚甲蓝/常规抗肿瘤药组与其组分单药组的瘤重差异，在腹腔注射的组9与组1之间无统计学意义(p>0.05)未显示出协同药效，而在瘤内注射的组9分别与组1、5之间均有统计学意义(均为p<0.05)，且后者的根据抑瘤率计算出的组合物q值远大1.00(1.30)。实际上同一组合物的瘤内注射组的抑瘤率为腹腔注射组的159％，从而显示出明显不同的靶向和药理。在上表中，亚甲蓝/常规抗肿瘤药/营养素组与其组分单药组的瘤重差异，在腹腔注射的组11与组10之间无统计学意义(p>0.05)未显示出协同药效，而在瘤内注射的组11分别与组10、5之间均有统计学意义(均为p<0.05)，且后者的根据抑瘤率计算出的组合物q值远大1.00(1.24)。实际上同一组合物的瘤内注射组的抑瘤率为腹腔注射组的175％，从而显示出明显不同的靶向和药理。
根据这些结果，本发明的组合物的优选的协同药理并非常规协同作用、而是局部协同作用。
实施例3：局部协同药理的优选
本研究试验中，成功建模的试验动物(荷PANC-1裸鼠，瘤体平均体积218mm 3)随机分为1个阴性对照组和9个研究组。阴性对照物为生理盐水，9个研究药物如下表所示。药物均为水溶液，按实施例1的制备方法配置而成。药物均为瘤内注射。包含50％乙酸的两种药物用药一次，注射量1000ul/只。其它药物每3日用药一次，一共3次，每次注射量100μl/只。用药结束后10日，对动物进行安乐死，解剖后测定瘤重，并从各用药方式的阴性对照组计算抑瘤率，结果示于表5。
表5
组別 研究药物 瘤重(g) 抑瘤率
0 生理盐水 2.83±0.22g (0)
1 3％吉西他滨 1.47±0.18 48％
2 30％木糖醇 2.32±0.13 18％
3 90％乙醇 0.91±0.21 68％
4 50％乙酸 0.62±0.19 78％
5 0.7％亚甲蓝 2.52±0.17 11％
6 3％吉西他滨/0.7％亚甲蓝 0.42±0.11 85％
7 30％木糖醇/0.7％亚甲蓝 0.34±0.09 88％
8 90％乙醇/0.7％亚甲蓝 0.82±0.12 71％
9 50％乙酸/0.7％亚甲蓝 0.57±0.15 80％
在上表中，亚甲蓝/常规化学消融剂组(组8和9)与常规化学消融剂单药组〔组3和4〕的瘤重差异之间无统计学意义(p>0.05)未显示出协同药效。而亚甲蓝/营养素组7和亚甲蓝/常规抗肿瘤药组 6与它们各自的单药组(分别为组5和2；组5和1)之间的瘤重差异均有统计学意义(均为p<0.05)，且根据抑瘤率计算出的组合物q值分别为3.26(组7)和1.58)均远大于理论预期值1.00，显示出明显的协同效应。此外，在组3和8中，每只动物均察到乙醇的强剌激反应。而在组4和9中，每只动物均察到乙酸对组织的最强大破坏反应，尽管其抑瘤率还比组6和7更低。
根据上述结果，本发明的组合物显示出—定的特异性、且其局部协同作用显示出与通常的协同作用(相同药理增效)很不相同的特殊性。
于是，本发明的组合物中亚曱蓝类染料的局部协同物并非选自局部作用強烈的化学消融剂，优选为选自局部作用比较温和的药物，更优选为选自营养素或/和常规抗肿瘤药。
实施例4、局部协同组成优选
在一个试验中，成功建模的试验动物(荷S180细胞小鼠，瘤体平均体积112mm 3)随机分为阴性对照组和40个研究组。阴性对照物为生理盐水，研究药物包括：4种变化浓度亚甲蓝单药(X％，X为亚甲蓝)、16种变化种类及浓度的其它组分单药(Y％，Y为其它组分)、20个分别由变化浓度亚甲蓝和变化种类及浓度的其它组分(营养素、常规抗肿瘤药)构成的组合物(X％/Y％)，它们的组成如下表所示。药物均为水溶液，按实施例1的制备方法配置而成。各组均瘤内注射，每3日用药一次，一共3次，每次用药剂量为：亚甲蓝≤100mg/kg、赖氨酸≤1000mg/kg、DHA≤375mg/kg、葡萄糖≤2250mg/kg、5-氟尿嘧啶≤50mg/kg，注射体积≤150μl。用药结束后10日对动物进行安乐死，解剖后测定瘤重，并从阴性对照组计算抑瘤率。各研究药物组抑瘤率示于表6。
表6
Figure PCTCN2020075765-appb-000007
Figure PCTCN2020075765-appb-000008
*：横栏括号中的数据为X％亚甲蓝组的平均抑瘤率，例如0.15％亚甲蓝组的平均抑瘤率为3％
**：纵栏括号中的数据为Y％其它组分组的平均抑瘤率，例如1％赖氨酸组的平均抑瘤率为5％
***：不带括号的数据为X％亚甲蓝/Y％其它组分组的平均抑瘤率，例如3％亚甲蓝/1％赖氨酸组的平均抑瘤率为45％
在其它荷瘤小鼠模型(例如，以每只1×10 6个乳腺瘤4T1细胞建模的小鼠，以每只2×10 6个肝癌H22细胞建模的小鼠，以每只1×10 6个结肠癌CT26细胞建模的小鼠，以每只2×10 6个黑色素瘤B16-f10细胞建模的小鼠)，也可以观察到类似效应。
在另一个试验中，成功建模的试验动物(荷乳腺瘤4T1细胞小鼠，瘤体平均体积119mm 3)随机分为1个阴性对照组和9个研究组。阴性对照物为生理盐水，9个研究药物如下表所示。药物均为水溶液，按实施例1的制备方法配置而成。药物均为瘤内注射。包含50％乙酸的两种药物用药一次，注射量1000ul/只。其它药物每3日用药一次，一共3次，每次注射量100μl/只。用药结束后10日，对动物进行安乐死，解剖后测定瘤重，并从各用药方式的阴性对照组计算抑瘤率，结果示于表7。
表7
组別 研究药物 瘤重(g) 抑瘤率
0 生理盐水 1.81±0.22g (0)
1 1％5-氟尿嘧啶 0.98±0.17 46％
2 30％木糖醇 1.57±0.19 13％
3 0.5％亚甲蓝 1.67±0.20 8％
4 1％5-氟尿嘧啶/0.5％亚甲蓝 0.45±0.13 75％
5 30％木糖醇/0.5％亚甲蓝 0.58±0.12 68％
6 0.5％5-氟尿嘧啶 1.18±0.18 35％
7 15％木糖醇 1.61±0.16 11％
8 0.25％亚甲蓝 1.82±0.23 5％
9 0.5％5-氟尿嘧啶/0.25％亚甲蓝 1.16±0.14 36％
10 15％木糖醇/0.25％亚甲蓝 1.57±0.15 13％
在上表中，亚甲蓝/常规抗肿瘤药组4分别与单药组1和3之间的瘤重差异之间均有统计学意义(均为p<0.05)，且根据抑瘤率计算出的组合物q值为1.50远大于理论预期值1.00，显示出明显的协同效应。亚甲蓝/营养素组5分别与单药组2和3之间的瘤重差异之间均有统计学意义(均为p<0.05)，且根据抑瘤率计算出的组合物q值为3.40远大于理论预期值1.00，显示出明显的协同效应。然而，使用相同的组合物量比，组合物组9与单药组6、组合物组10与7)之间的瘤重差异无统计学意义(p>0.05)未显示出协同药效。
利用本发明的其它组合物(例如表2中的组合物)的实验，也可以获得类似结果。
根据上述研究及更多的类似研究，本发明的亚甲蓝类染料、营养素或/和常规抗肿瘤药组合物的协同技术方案为瘤内给药、且瘤内给药时其协同组成并非常规协同组成中的量比，而是如以下所限定的浓度关系：
所述亚甲蓝类染料的瘤内给药浓度(w/V)≤2％、优选为0.3％-1.8％；
所述常规抗肿瘤药的瘤内给药浓度大于其饱和浓度的20％、优选为其饱和浓度的30％-100％；或/和
所述营养素的瘤内给药浓度为大于1％，优选为2％-40％，其中:
所述氨基酸类营养素的瘤内给药浓度为大于2％、优选为2％-35％；
所述脂类营养素的瘤内给药浓度为大于2％、优选为3％-25％；或/和
所述糖类营养素的瘤内给药浓度为大于10％、优选为20％-40％。本发明的组合物不仅可以产生协同作用，而且该协同作用通过在特定条件下还可以进一步优化以获得超有效协同作用。而这些特定条件并非亚甲蓝类染料与营养素或/和常规抗肿瘤药在现有技术中的条件。以下试验例对此进行了研究。
实施例5：协同组合物中亚甲蓝类染料瘤内给药浓度的进一步优选
本研究试验中，成功建模的试验动物(荷S180细胞小鼠，瘤体平均体积132mm 3)随机分为阴性对照组、阳性对照组和7个研究组。阴性对照物为生理盐水，阳性对照物为1％5-氟尿嘧啶，研究药物含20％赖氨酸和如下表所示的变化浓度(X％)的亚甲蓝(20％赖氨酸/X％亚甲蓝)。药物均为水溶液，按实施例1的制备方法配置而成。各组均瘤内注射，每3日用药一次，一共3次，每次用药剂量为：亚甲蓝≤75mg/kg、5-氟尿嘧啶50mg/kg，注射体积≤150μl。用药结束后10日对动物进行解剖后测定瘤重，并从阴性对照组计算抑瘤率，结果示于表8。
表8
Figure PCTCN2020075765-appb-000009
在上表中，在满足实施例4给出的协同作用技术方案时，只有亚甲蓝瘤内给药浓度≥0.35％的各组显示出超有效协同药效(与阳性对照组比较)。当亚甲蓝瘤内给药浓度大于或等于这个阈值后，超有效协同药效对其瘤内给药浓度变得敏感，然后又重新变得不那么敏感。令人吃惊的是，当亚甲蓝瘤内给药浓度大于2％后，抑瘤率出现明显下降。在另一组试验中，当使用1％5-氟尿嘧啶与X％亚甲蓝的组合物时，也获得类似结果。
根据这些结果，本发明的组合物的一个超有效协同药效技术方案为：其组成使得所述组合物可以提供的所述亚甲蓝类染料的瘤内给药浓度≥0.35％、优选为0.35-1.5％、更优选为0.5-1.5％或0.5-1％。
根据以上多个实施例的研究和其它更多研究揭示:
1)、同一物质可以根据其给药方式显示出完全不同的药理从而作为不同药物使用。例如，***给药的亚甲蓝通常作为解毒剂，却不能作为本发明组合物中的局部活性成分；糸统给药的营养素或/和常规抗肿瘤药通常作为营养素和抗肿瘤药，却不能作为本发明组合物中亚甲蓝类染料的局部协同物。
2)、同一物质也可以根据其局部给药条件显示完全不同的药理从而作为不同的药物使用，例如，超高浓度(例如≥3％)的亚甲蓝可以作为化学消融剂却不宜作为本发明组合物中的局部活性成分，而低浓度(例如≤1％)亚甲蓝通常作为染色剂却可与营养素或/和常规抗肿瘤药产生局部协同作用的该局部活性成分。
3)、亚甲蓝类染料显示出异乎寻常的协同条件，例如，其在高浓度时可作为化学消融剂却在低浓度时并未显示出与常规化学消融剂的局部协同作用，这有违现有技术关于相同药理共用增强的教导；其与对组织破坏性很小的营养素(例如脂类营养素)居然产生出很高的协同作用(q≥2.0)，这有违 现有技术关于相近药理共用增强的教导能产生几乎一致的协同作用(q值接近)，这有违现有技术关于相同靶点共用增强的教导。
4)、决定性的协同条件不仅是量比(浓度比)而且还包括浓度值本身。例如，相同量比时，亚甲蓝类染料瘤内给药浓度低于某个阈值不会产生所述局部协同作用，这有违常规细胞毒药物协同作用通常由量比决定的现有技术教导。
5)、该局部协同作用使得本发明的组合物显示出针对肿瘤组织的较高特异性。例如，本发明的组合物与常规化学消融剂(50％乙酸)比较。
实施例6、协同組合物中营养素瘤内给药浓度优选
本研究试验中，成功建模的试验动物(荷S180细胞小鼠，瘤体平均体积128mm 3)随机分为阴性对照组(0)、阳性对照组(7)和6个研究组(1-6)。阴性对照物为生理盐水，阳性对照物为1％5-氟尿嘧啶，研究药物的组成如下表所示，其含变化浓度(X％)的精氨酸和固定浓度(1％)的亚甲蓝(X％精氨酸/1％亚甲蓝)。药物均为水溶液，按实施例1的制备方法配置而成。各组均瘤内注射，每3日用药一次，一共3次，每次用药剂量为：精氨酸≤1500mg/kg、5-氟尿嘧啶50mg/kg，注射体积≤150μl。用药结束后5日对动物进行安乐死，解剖后测定瘤重，并从阴性对照组计算抑瘤率，结果示于表9。
表9
Figure PCTCN2020075765-appb-000010
在上表中，在满足实施例4给出的协同作用技术方案时，精氨酸只有在瘤内给药浓度超过某个阈值时其组合物才突然显示出超有效协同药效(与阳性对照组比较)。当精氨酸瘤内给药浓度大于或等于这个阈值(3％)后，超有效协同药效对其瘤内给药浓度变得敏感，然后又重新变得不那么敏感，甚至于抑瘤率可能出现下降。
根据这些结果以及更多的类似研究，本发明的组合物的一个超有效协同药效技术方案为：其组 成使得所述组合物可以提供的所述营养素的瘤内给药浓度≥3％，优选为3％-45％，其中当营养素选自糖类营养素时，所述营养素的瘤内给药浓度≥20％、优选为20-40％；当营养素选自脂类营养素时，所述脂类营养素的瘤内给药浓度≥4％，优选为4-25％；当营养素选自氨基酸类营养素时，所述氨基酸类营养素的瘤内给药浓度≥3％，优选为3-25％，更优选为5-25％或15-25％。
实施例7、协同组合物中的营养素优选
本研究试验中，成功建模的试验动物(荷S180细胞小鼠，瘤体平均体积151mm 3)随机分为阴性对照组、阳性对照组和15个研究组。阴性对照物为生理盐水，阳性对照物为1％5-氟尿嘧啶，研究药物为1％亚甲蓝/10％氨基酸类营养素组合物。研究组1-13所用组合物中的氨基酸类营养素分别为：精氨酸、甘氨酸、半胱氨酸盐酸盐、缬氨酸、苏氨酸、脯氨酸、组氨酸盐酸盐、苯丙氨酸、赖氨酸、亮氨酸、丙氨酸、谷胱甘肽、丝氨酸、丙氨酰-谷氨酰胺二肽，研究组15用药为1％亚甲蓝/5％精氨酸/5％甘氨酸。药物均为水溶液，按实施例1的制备方法配置而成。各组均瘤内注射，每3日用药一次，一共3次，每次注射体积≤150μl。用药结束后10日对动物进行安乐死，解剖后测定瘤重，并从阴性对照组计算抑瘤率。阳性对照组抑瘤率为51％，研究组结果示于表10。
表10
组别 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Y(％) 95 81 92 43 56 33 5 48 90 74 83 89 91 83 63
根据这些结果以及更多的类似研究，本发明的氨基酸类营养素/亚甲蓝类染料组合物的一个超有效协同药效技术方案为：氨基酸类营养素优选为选自以下氨基酸和包含以下氨基酸之一种或多种的氨基酸衍生物：精氨酸、甘氨酸、半胱氨酸、苏氨酸、脯氨酸、赖氨酸、亮氨酸、丙氨酸、丝氨酸、谷氨酸，更优选为选自以下氨基酸和包含以下氨基酸之一种或多种的氨基酸衍生物：精氨酸、甘氨酸、半胱氨酸、赖氨酸、丙氨酸、丝氨酸、谷氨酸。
实施例8：更多的抗肿瘤应用
在以下实验中，成功建模的荷人癌细胞裸鼠均随机分为1个阴性对照组和5个研究组(A、B、C、D、E组)。所对应的阴性对照物为生理盐水，5个研究药物分别为：1％亚甲蓝/5％DHA/5％乙酸、1％亚甲蓝/20％精氨酸、1％亚甲蓝/10％甘氨酸/10％赖氨酸、1％亚甲蓝/30％葡萄糖、1％亚甲蓝/1％5-氟尿嘧啶。药物均为水溶液，均按实施例1的制备方法配置而成。各组均瘤内注射，每3日用药一次，一共3次，每次100-150μl/只。在用药结束后次日，对动物进行安乐死，解剖后测定瘤重，并从各自阴性对照组计算抑瘤率。
1)、在乳腺肿瘤治疗中的应用
本研究试验中，成功建模的荷人乳腺癌细胞(MDA-MB231)裸鼠(瘤体平均体积173mm 3)随机分为一个阴性对照组和5个研究组(A、B、C、D、E组)。A、B、C、D、E组的抑瘤率分别为：81％、76％、73％、91％、77％，均合乎通常认为的有效抗肿瘤标准(抑瘤率≥40％)。
2)、在肺肿瘤治疗中的应用
本研究试验中，成功建模的荷人肺癌细胞(A549)裸鼠(瘤体平均体积181mm 3)随机分为一个阴性对照组和5个研究组(A、B、C、D、E组)。A、B、C、D、E组的抑瘤率分别为：75％、73％、81％、87％、74％，均合乎通常认为的有效抗肿瘤标准(抑瘤率≥40％)。
3)、在甲状腺肿瘤治疗中的应用
本研究试验中，成功建模的荷人甲状腺癌细胞(SW579)裸鼠(瘤体平均体积172mm 3)随机分为一个阴性对照组和5个研究组(A、B、C、D、E组)。A、B、C、D、E组的抑瘤率分别为：74％、83％、71％、86％、76％，均合乎通常认为的有效抗肿瘤标准(抑瘤率≥40％)。
4)、在***肿瘤治疗中的应用
本研究试验中，成功建模的荷人***癌细胞(LNCaP/AR)裸鼠(瘤体平均体积191mm 3)随机分为一个阴性对照组和5个研究组(A、B、C、D、E组)。A、B、C、D、E组的抑瘤率分别为：73％、71％、75％、81％、69％，均合乎通常认为的有效抗肿瘤标准(抑瘤率≥40％)。
5)、在肝肿瘤治疗中的应用
本研究试验中，成功建模的荷人肝癌细胞(HepG2)裸鼠(瘤体平均体积191mm 3)随机分为一个阴性对照组和5个研究组(A、B、C、D、E组)。A、B、C、D、E组的抑瘤率分别为：72％、81％、77％、89％、71％，均合乎通常认为的有效抗肿瘤标准(抑瘤率≥40％)。
6)、在头颈肿瘤治疗中的应用
本研究试验中，成功建模的荷人头颈癌细胞(Fμda)裸鼠(瘤体平均体积169mm 3)随机分为一个阴性对照组和5个研究组(A、B、C、D、E组)。A、B、C、D、E组的抑瘤率分别为：76％、82％、74％、88％、70％，均合乎通常认为的有效抗肿瘤标准(抑瘤率≥40％)。
7)、在鼻咽肿瘤治疗中的应用
本研究试验中，成功建模的荷人鼻咽癌细胞(CNE1)裸鼠(瘤体平均体积182mm 3)随机分为一个阴性对照组和5个研究组(A、B、C、D、E组)。A、B、C、D、E组的抑瘤率分别为：71％、72％、72％、83％、74％，均合乎通常认为的有效抗肿瘤标准(抑瘤率≥40％)。
8)、在胃肿瘤治疗中的应用
本研究试验中，成功建模的荷人胃癌细胞(BGC823)裸鼠(瘤体平均体积181mm 3)随机分为一个阴性对照组和5个研究组(A、B、C、D、E组)。A、B、C、D、E组的抑瘤率分别为：69％、80％、79％、85％、72％，均合乎通常认为的有效抗肿瘤标准(抑瘤率≥40％)。
9)、在卵巢肿瘤治疗中的应用
本研究试验中，成功建模的荷人卵巢癌细胞(PA1)裸鼠(瘤体平均体积151mm 3)随机分为一个阴性对照组和5个研究组(A、B、C、D、E组)。A、B、C、D、E组的抑瘤率分别为：77％、70％、69％、82％、73％，均合乎通常认为的有效抗肿瘤标准(抑瘤率≥40％)。
利用实施例1制备的一些其它本发明的组合物(例如表2中的组合物)，在上述各肿瘤治疗中的应用也可以获得类似结果。
本公开涉及以下项目：
1、一种用于治疗恶性实体肿瘤的局部药物组合物，其包含亚甲蓝类染料、所述亚甲蓝类染料的局部协同药物、以及药物学可接受的液体载体，其中所述协同药物选自营养素或/和常规抗肿瘤药，且在该局部药物组合物中，所述亚甲蓝类染料的浓度(w/v)≤2％、优选为0.35-2％、0.5-2％、0.5-1.5％或0.5-1％，所述营养素的浓度为大于2％、优选为3％-40％，以及所述常规抗肿瘤药的浓度大于其饱和浓度的20％或30％、优选为其饱和浓度的30％-100％，其中所述饱和浓度是指所述常规抗肿瘤药在所述液体载体中的饱和浓度。
2、亚甲蓝类染料作为局部活性成分与所述亚甲蓝类染料的局部协同药物的组合在制备用于治疗恶性实体肿瘤的局部药物组合物中的应用，其中所述协同药物选自营养素或/和常规抗肿瘤药。
3、根据项目2的应用，其中所述局部药物组合物包含所述亚甲蓝类染料、所述亚甲蓝类染料的局部协同药物、以及药物学可接受的液体载体，其中在该局部药物组合物中，所述亚甲蓝类染料的浓度(w/v)≤2％、优选为0.35-2％、0.5-2％、0.5-1.5％或0.5-1％，所述营养素的浓度为大于2％、优选为3％-40％，以及所述常规抗肿瘤药的浓度大于其饱和浓度的20％或30％、优选为其饱和浓度的30％-100％，其中所述饱和浓度是指所述常规抗肿瘤药在所述液体载体中的饱和浓度。
4、根据项目1-3之一的药物组合物或应用，其中所述亚甲蓝类染料选自亚甲蓝及其活体染色类似物，优选为选自以下化合物及其衍生物：亚甲蓝、专利蓝、异硫蓝、新亚甲蓝，更优选为选自亚甲蓝及其衍生物。
5、根据项目1-3之一的药物组合物或应用，其中所述营养素为选自以下组之一种或多种：氨基酸类营养素、糖类营养素、脂类营养素，且其在所述组合物中的浓度为2.5-50％，优选为4-40％。
6、根据项目5的药物组合物或应用，其中所述氨基酸类营养素包括具有营养保健效应的以下氨基酸类化合物之一种或多种：氨基酸、氨基酸盐、寡肽和多肽；优选为选自以下组中的氨基酸或 其盐或者包含或由以下氨基酸构成的寡肽和多肽：丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、脯氨酸、色氨酸、酪氨酸、丝氨酸、半胱氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、苏氨酸、赖氨酸、精氨酸、组氨酸、天冬氨酸、谷氨酸、β-丙氨酸、牛磺酸、γ氨基丁酸(GABA)、茶多酚(茶氨酸)、南瓜子氨基酸(3-氨基-3-羧基吡烷酸)、谷氨酰胺、瓜氨酸、鸟氨酸；更优选为选自以下组中的氨基酸或其盐或者包含或由以下氨基酸构成的寡肽和多肽：精氨酸、赖氨酸、甘氨酸、半胱氨酸、丙氨酸、丝氨酸、谷氨酸，且其在该药物组合物中的≥2％、≥2.5、≥5％、优选为≥7.5％、10-25％或18-25％，更优选为15％-25％或20％-25％
7、根据项目6的药物组合物或应用，其中所述氨基酸类营养素选自具有营养保健效应的氨基酸或氨基酸盐，且所述氨基酸或氨基酸盐在该局部药物组合物中的浓度(w/v)为≥2％、≥2.5、≥5％、≥7.5％、10-25％或18-25％，优选为15％-25％或20％-25％。
8、根据项目6的药物组合物或应用，其中所述氨基酸类营养素选自具有营养保健效应的寡肽和多肽，且所述寡肽和多肽在该局部药物组合物中的浓度(w/v)为大于≥5％、优选为7.5-25％、更优选为10％-25％。
9、根据项目6的药物组合物或应用，其中所述氨基酸类营养素为所述氨基酸和/或氨基酸盐与所述寡肽和/或多肽的组合，且该组合在该局部药物组合物中的浓度(w/v)为大于≥5％、优选为7.5％-25％、更优选为10-25％。
10、根据项目6的药物组合物或应用，其中所述寡肽为选自以下组之一种或多种：甘氨酰-L-酪氨酸、甘氨酰丙氨酸、双甘氨肽、赖氨酸-甘氨酸二肽、丙谷二肽、肌肽(β-丙氨酸组氨酸共聚物)、谷胱甘肽、胶原蛋白寡肽、酪蛋白水解肽、大豆寡肽、寡聚精氨酸、寡聚甘氨酸、寡聚赖氨酸；所述多肽为选自以下组之一种或多种：聚天冬氨酸、聚谷氨酸、聚赖氨酸。
11、根据项目5的药物组合物或应用，其中所述糖类营养素为选自包含以下糖单元之一种或多种的糖类化合物：葡萄糖、核糖、木糖、果糖、半乳糖、岩藻糖，优选为选自以下之一种或多种：葡萄糖、果糖、壳寡糖、氨基葡萄糖、乳果糖、山梨醇、核糖、山梨糖、甘露糖、半乳糖、蔗糖、乳糖、海藻糖、木寡糖、果寡糖、甘露寡糖、葡萄糖酸、葡萄糖酸钠、木糖醇、甘露醇、麦芽糖醇、乳糖醇，更优选为选自以下之一种或多种：葡萄糖、葡萄糖酸钠、壳寡糖、氨基葡萄糖、乳果糖、核糖、低聚甘露糖、木糖醇，且其在该药物组合物中的浓度(w/v)大于5％，优选≥10％、10-40％、15-50％或25-50％
12、根据项目5的药物组合物或应用，其中所述脂类营养素为选自以下的一种或多种：脂肪酸、脂肪乳和类脂，优选为选自以下之一种或多种：植物油、二十碳五烯酸(EPA)、二十二碳六烯酸(DHA)、长链脂肪乳、中链脂肪乳、磷脂，且其在该药物组合物中的浓度≥4％，优选为4％-25％。
13、根据项目1-3之一的药物组合物或应用，其中所述常规抗肿瘤药选自以下组之一种或多种：破坏DNA结构和功能的药物、嵌入DNA中干扰转录RNA的药物、干扰DNA合成的药物、影响蛋白质合成的药物。
14、根据项目13的药物组合物或应用，其中所述常规抗肿瘤药为选自以下组之一种或多种： 尿嘧啶衍生物类、环磷酰胺类、吉西他滨类、表柔比星类、抗肿瘤抗生素类、替尼泊苷、金属铂络合物、紫杉烷类，优选为选自以下药物及其类似衍生物一种或多种：5-氟尿嘧啶、环磷酰胺、吉西他滨、表柔比星、抗肿瘤抗生素、替尼泊苷、金属铂络合物、紫杉醇。
15、根据项目1-4的药物组合物或应用，其还任选包含以下组之一种或多种：止痛剂、缓释载体、作为酸化剂的任选被1-3个羟基取代的C1-10脂族羧酸。
16、根据项目15的药物组合物或应用，其中所述止痛剂为选自以下的一种或多种：苯甲醇、盐酸普鲁卡因、三氯叔丁醇和盐酸利多卡，且所述止痛剂在所述药物组合物中的浓度为0.1-4重量％。
17、根据项目15的药物组合物或应用，其中所述酸化剂为选自以下之一种或多种：乙酸、丙酸、丁酸、丙二酸、丁二酸、羟基乙酸、乳酸、柠檬酸、苹果酸、酒石酸，更优选为乙酸。
18、根据项目1-14之一的药物组合物或应用，其中所述药物学可接受的液体载体为水和/或乙醇。
19、根据项目1-18之一的药物组合物或应用，其中所述药物组合物为注射剂的剂型，所述注射剂包括液体注射剂和注射用粉针剂。
20、根据项目19的药物组合物或应用，其中所述注射用粉针剂包括无菌干粉和溶媒，且所述氨基酸类营养素和无效吸收化合物之一部或全部包含于所述无菌干粉，以及所述液体载体包含于所述溶媒，而且所述氨基酸类营养素和无效吸收化合物的浓度分别为它们在无菌干粉和溶媒混合物中的浓度。
21、一种用于治疗恶性实体肿瘤的局部药物组合物，其包含通过冻干或半冻干根据项目1和4-18之一的药物组合物之一部或全部得到的干粉。
22、一种预防和治疗恶性实体肿瘤的方法，其包括向有此需要的个体瘤内给药根据项目1以及4-21之一的药物组合物。
23、根据项目1-22之一的药物组合物、应用或方法，其中所述恶性实体肿瘤包括乳腺癌、胰腺癌、甲状腺癌、鼻咽癌、***癌、肝癌、肺癌、肠癌、口腔癌、食道癌、胃癌、喉癌、睾丸癌、***癌、子宫癌、卵巢癌。
24、根据项目23的药物组合物、应用或方法，其中所述恶性实体肿瘤包括胰腺癌。
除本文中描述的那些外，根据前述描述，本发明的多种修改对本领域技术人员而言会是显而易见的。这样的修改也意图落入所附权利要求书的范围内。本申请中所引用的各参考文献(包括所有专利、专利申请、期刊文章、书籍及任何其它公开)均以其整体援引加入本文。

Claims (10)

  1. 一种用于治疗恶性实体肿瘤的局部药物组合物，其包含亚甲蓝类染料、所述亚甲蓝类染料的局部协同药物、以及药物学可接受的液体载体，其中所述协同药物选自营养素或/和常规抗肿瘤药，且在该局部药物组合物中，所述亚甲蓝类染料的浓度(w/v)≤2％、优选为0.35-2％、0.5-2％、0.5-1.5％或0.5-1％，所述营养素的浓度为大于2％、优选为3％-40％，以及所述常规抗肿瘤药的浓度大于其饱和浓度的20％、优选为其饱和浓度的30％-100％，其中所述饱和浓度是指所述常规抗肿瘤药在所述液体载体中的饱和浓度。
  2. 亚甲蓝类染料作为局部活性成分与所述亚甲蓝类染料的局部协同药物的组合在制备用于治疗恶性实体肿瘤的局部药物组合物中的应用，其中所述协同药物选自营养素或/和常规抗肿瘤药。
  3. 根据权利要求2的应用，其中所述局部药物组合物包含所述亚甲蓝类染料、所述亚甲蓝类染料的局部协同药物、以及药物学可接受的液体载体，其中在该局部药物组合物中，所述亚甲蓝类染料的浓度(w/v)≤2％、优选为0.35-2％、0.5-2％、0.5-1.5％或0.5-1％，所述营养素的浓度为大于2％、优选为3％-40％，以及所述常规抗肿瘤药的浓度大于其饱和浓度的20％、优选为其饱和浓度的30％-100％，其中所述饱和浓度是指所述常规抗肿瘤药在所述液体载体中的饱和浓度。
  4. 根据权利要求1-3之一的药物组合物或应用，其中所述亚甲蓝类染料选自亚甲蓝及其活体染色类似物，优选为选自以下化合物及其衍生物：亚甲蓝、专利蓝、异硫蓝、新亚甲蓝，更优选为选自亚甲蓝及其衍生物。
  5. 根据权利要求1-3之一的药物组合物或应用，其中所述营养素为选自以下组之一种或多种：氨基酸类营养素、糖类营养素、脂类营养素，且其在所述组合物中的浓度为大于2.5％至小于50％，优选为4-40％。
  6. 根据权利要求1-3之一的药物组合物或应用，其中所述常规抗肿瘤药选自以下组之一种或多种：破坏DNA结构和功能的药物、嵌入DNA中干扰转录RNA的药物、干扰DNA合成的药物、影响蛋白质合成的药物。
  7. 根据权利要求1-6之一的药物组合物或应用，其中所述药物组合物为注射剂的剂型，所述注射剂包括液体注射剂和注射用粉针剂。
  8. 一种用于治疗恶性实体肿瘤的局部药物组合物，其包含通过冻干或半冻干根据权利要求1和4-6之一的药物组合物之一部或全部得到的干粉。
  9. 根据权利要求1-8之一的药物组合物或应用，其中所述恶性实体肿瘤包括乳腺癌、胰腺癌、甲状腺癌、鼻咽癌、***癌、肝癌、肺癌、肠癌、口腔癌、食道癌、胃癌、喉癌、睾丸癌、***癌、子宫癌、卵巢癌。
  10. 根据权利要求1-8之一的药物组合物或应用，其中所述恶性实体肿瘤包括胰腺癌。
PCT/CN2020/075765 2020-02-18 2020-02-18 包含亚甲蓝类染料、营养素或/和常规抗肿瘤药的药物组合物及其应用 WO2021163897A1 (zh)

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