WO2021164706A1 - 包含亚甲蓝类染料的药物组合物及其应用 - Google Patents

包含亚甲蓝类染料的药物组合物及其应用 Download PDF

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Publication number
WO2021164706A1
WO2021164706A1 PCT/CN2021/076749 CN2021076749W WO2021164706A1 WO 2021164706 A1 WO2021164706 A1 WO 2021164706A1 CN 2021076749 W CN2021076749 W CN 2021076749W WO 2021164706 A1 WO2021164706 A1 WO 2021164706A1
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WIPO (PCT)
Prior art keywords
methylene blue
tumor
local
blue dye
composition
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PCT/CN2021/076749
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English (en)
French (fr)
Inventor
邹方霖
邹礼常
王建霞
王艺羲
Original Assignee
成都夸常奥普医疗科技有限公司
夸常股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Priority claimed from PCT/CN2020/075765 external-priority patent/WO2021163897A1/zh
Priority claimed from CN202011064448.1A external-priority patent/CN114306392A/zh
Priority claimed from CN202011059746.1A external-priority patent/CN114306612A/zh
Priority claimed from CN202011059699.0A external-priority patent/CN114344333A/zh
Application filed by 成都夸常奥普医疗科技有限公司, 夸常股份有限公司 filed Critical 成都夸常奥普医疗科技有限公司
Publication of WO2021164706A1 publication Critical patent/WO2021164706A1/zh
Priority to PCT/CN2021/122041 priority Critical patent/WO2022068918A1/zh
Priority to EP21874572.7A priority patent/EP4223313A1/en
Priority to JP2023519733A priority patent/JP2023543858A/ja
Priority to US18/247,359 priority patent/US20230414617A1/en
Priority to PCT/CN2022/076823 priority patent/WO2022174812A1/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
    • A61K31/185—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
    • A61K31/21—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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the disclosure of this application relates to a composition containing the methylene blue dye and its local synergist or synergistic drug and its use in the preparation of a drug combination or preparation for cancer.
  • the present invention relates to methylene blue dyes and their local synergistic drugs or synergistic compositions that preferably minimize rather than maximize local effects as active ingredients that can provide local synergistic effects when prepared to provide said local synergistic effects.
  • a topical pharmaceutical composition for the treatment of cancer such as malignant solid tumors, comprising the methylene blue dye and a local synergistic drug or synergist thereof that can provide a local synergistic effect for the treatment of malignant solid tumors Drugs, and methods for treating cancer such as malignant solid tumors that include administering the pharmaceutical composition or drug combination or formulation.
  • 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 physical target tumor body, but has a higher curative effect, higher compliance, or higher compliance than existing drugs (especially pharmaceutical compositions containing methylene blue dyes in the prior art) / And a more specific topical drug, and a method for treating malignant solid tumors including administering the pharmaceutical composition.
  • a pharmaceutical composition comprising a methylene blue dye as a topical active ingredient and a local synergist or a synergistic drug of the methylene blue dye, wherein the synergist or synergistic drug comprises or One or more selected from nutrients, immunomodulators, and conventional anti-tumor drugs.
  • a combination comprising methylene blue dye, a topical synergist or a synergistic drug, and optionally a nutrient or/and an immunomodulator, wherein the synergistic or synergistic drug includes or One or more selected from conventional anti-tumor drugs, wherein in the composition, the concentration (w/v) of the methylene blue dye when administered is ⁇ 2%, preferably 0.35-2% , 0.5-2%, 0.5-1.5% or 0.5-1%.
  • the concentration (w/v) of the methylene blue dye when applied is ⁇ 2%, for example, 0.35%, 0.5%, 0.7%, 0.9%, 1%, 1.25%, 1.5% , 1.8%, 2%, or any range therebetween, preferably 0.35-2%, 0.5-2%, 0.5-1.5%, 0.7-1.5%, 0.7-1.25% or 0.5-1%.
  • composition comprising a methylene blue dye and a topical synergist or a synergistic drug, wherein the synergist or synergistic drug includes or is selected from one or more nutrients, and Optional conventional anti-tumor drugs or/and immunomodulators.
  • composition comprising a methylene blue dye and a local synergist or a synergistic drug, wherein the synergist or synergistic drug includes or is selected from one or more immunomodulators.
  • the synergist or synergistic drug includes or is selected from one or more immunomodulators.
  • a composition comprising methylene blue dye and a topical synergist or a synergistic drug, wherein the synergist or synergistic drug includes or is selected from nutrients, wherein in the composition, the concentration of the methylene blue dye when applied is a concentration (w/v) ⁇ 2%, preferably 0.35-2%, 0.5-2%, 0.5-1.5% or 0.5-1%.
  • the methylene blue dye may include methylene blue and its living dye analogs, preferably, include a compound selected from the group consisting of methylene blue, patent blue, isosulfan blue, Neomethylene blue is more preferably selected from methylene blue and its derivatives.
  • the methylene blue dye may be selected from methylene blue and its living dye analogs, preferably, it may be selected from methylene blue, patent blue, isosulfur blue, neomethylene blue, more preferably Ground is selected from methylene blue and its derivatives.
  • the nutrient includes one or more of the following: amino acid nutrient, carbohydrate nutrient, lipid nutrient.
  • the nutrient is selected from one or more of amino acid nutrients, carbohydrate nutrients, and lipid nutrients.
  • the concentration of the above-mentioned nutrients at the time of administration is 2.5-50%, preferably 4-40%.
  • the nutrient includes DHA.
  • the amino acid nutrient includes or is selected from one or more of the following amino acid compounds with nutritional and health effects: amino acids, amino acid salts, oligopeptides and polypeptides; preferably amino acids or their salts selected from the following Or oligopeptides and polypeptides containing or consisting of the following amino acids: alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, tyrosine, serine, half Cystine, methionine, asparagine, glutamine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, ⁇ -alanine, taurine, ⁇ Aminobutyric acid (GABA), tea polyphenols (theanine), pumpkin seed amino acids (3-amino-3-carboxypyranoic acid), glutamine, citrulline, ornithine; more preferably selected from the following The amino acid or its salt or
  • the concentration of the amino acid compound when administered is 2.5-50%, preferably 4-40%.
  • the amino acid nutrient is selected from amino acids or amino acid salts with nutritional and health effects, and in the composition, the concentration (w/v) of the amino acid or amino acid salt at the time of administration 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 in the composition, the concentration (w/v) of the oligopeptides and polypeptides at the time of administration is greater than ⁇ 5% , Preferably 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 in the pharmaceutical composition, the concentration of the combination at the time of administration (w/v) It is greater than ⁇ 5%, preferably 7.5%-25%, more preferably 10-25%.
  • the oligopeptide includes or is selected from one or more of the following: glycyl-L-tyrosine, glycylalanine, glycylglycine, lysine-glycine dipeptide, Gamma dipeptide, carnosine ( ⁇ -alanine histidine copolymer), glutathione, collagen oligopeptides, casein hydrolyzed peptides, soybean oligopeptides, oligoarginine, oligoglycine, oligolysine
  • the polypeptide is one or more selected from polyaspartic acid, polyglutamic acid, and polylysine.
  • the carbohydrate nutrient includes or is a carbohydrate compound containing one or more of the following sugar units: glucose, ribose, xylose, fructose, galactose, fucose, preferably one selected from the following Species or more: glucose, fructose, chitooligosaccharides, glucosamine, lactulose, sorbitol, ribose, sorbose, mannose, galactose, sucrose, lactose, trehalose, xylo-oligosaccharides, fructo-oligosaccharides, mannose oligosaccharides Sugar, gluconic acid, sodium gluconate, xylitol, mannitol, maltitol, lactitol, more preferably one or more selected from the following: glucose, sodium gluconate, chitooligosaccharide, glucosamine, lactulose , Ri
  • the concentration (w/v) of the carbohydrate nutrient at the time of administration is greater than 5%, preferably ⁇ 10%, 10-40%, 15-50% or 25-50% .
  • the lipid nutrient includes or is selected from one or more of the following: fatty acid, fat milk and lipid, preferably includes or is selected from one or more of the following: vegetable oil, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), long-chain fat emulsion, medium-chain fat emulsion, phospholipid, and in the composition, the concentration of the lipid nutrient when administered is ⁇ 4%, preferably It is 4%-25%.
  • EPA eicosapentaenoic acid
  • DHA docosahexaenoic acid
  • the concentration of the lipid nutrient when administered is ⁇ 4%, preferably It is 4%-25%.
  • the composition of the present invention also includes conventional anti-tumor drugs.
  • the anti-tumor drugs include or are selected from one or more of the following groups: drugs that disrupt the structure and function of DNA, and intervene in DNA to interfere with transcription of RNA. Drugs, drugs that interfere with DNA synthesis, drugs that affect protein synthesis.
  • conventional anti-tumor drugs include or are selected from one or more of the following: uracil derivatives, cyclophosphamides, gemcitabine, epirubicin, anti-tumor antibiotics, tenipo Glycosides, metal platinum complexes, taxanes, preferably one or more selected from the following drugs and their analogous derivatives: 5-fluorouracil, cyclophosphamide, gemcitabine, epirubicin, antitumor antibiotics, and Niposide, metal platinum complex, paclitaxel.
  • composition of the present invention further includes an immunomodulator, which includes or is selected from one or more of antibodies, nucleic acids, and probiotic components.
  • the concentration (w/v) of the methylene blue dye when applied is ⁇ 2%, preferably 0.35-2%, 0.5-2%, 0.5-1.5% or 0.5-1%, so
  • the concentration (w/v) of the nutrient at the time of administration is ⁇ 2%, preferably 3-40%, and the concentration of the conventional anti-tumor drug at the time of administration is greater than 20% or 30% of its saturated concentration, preferably its saturated concentration
  • the concentration is 30%-100%, wherein the saturated concentration refers to the saturated concentration of the conventional anti-tumor drug in the liquid carrier.
  • the present invention includes immunomodulators, such as probiotic components, immunoglobulins, immunomodulatory peptides.
  • immunomodulators such as probiotic components, immunoglobulins, immunomodulatory peptides.
  • the strains of probiotics used are all strains used in Saccharomyces cerevisiae and probiotic preparations approved by the food and drug authorities or contained in the official pharmacopoeia.
  • strains contained in probiotic preparations approved by the Chinese pharmaceutical authority Clostridium butyricum in Bacillus coagulans live tablets, Bacillus licheniformis strains in live Bacillus licheniformis granules (capsules), Oral Bacillus cereus Bacillus cereus strains in live bacteria preparations, Clostridium butyricum strains in live Clostridium butyricum capsules, Saccharomyces boulardii strains in Saccharomyces boulardii powder (capsules), Lactobacillus acidophilus capsules Lactobacillus butyricum strain, Clostridium butyricum in oral live Clostridium butyricum powder, Enterococcus faecium strain, subtilis strain, and Enterococcus butyricum triple viable tablet in Enterococcus subtilis double live bacteria multidimensional granules Lactobacillus strains, Clostridium butyricum, saccharification strains, Lacto
  • composition of the present invention optionally further comprises one or more of the following: analgesic, sustained-release carrier, and C1-10 aliphatic optionally substituted with 1-3 hydroxyl groups as an acidulant carboxylic acid.
  • the analgesic agent is one or more including or selected from the following: benzyl alcohol, procaine hydrochloride, chlorobutanol, and lidoca hydrochloride, and in the composition, the The concentration of the analgesic at the time of administration is 0.1-4% by weight.
  • 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, citric acid, malic acid, tartaric acid, More preferred is acetic acid.
  • the composition of the present invention further includes a pharmaceutically acceptable carrier, such as an aqueous carrier or an alcohol carrier, preferably water and/or ethanol.
  • a pharmaceutically acceptable carrier such as an aqueous carrier or an alcohol carrier, preferably water and/or ethanol.
  • the composition of the present invention may be prepared as an injection dosage form, which includes a liquid injection and a powder injection for injection.
  • the injection powder for injection includes a sterile dry powder and a solvent, and one 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 sterile dry powder.
  • the solvent, and the concentration of the amino acid nutrients and the ineffective absorption compound are their concentrations in the sterile dry powder and the solvent mixture, respectively.
  • the ratio of the amount of the methylene blue dye to the nutrient, immunomodulator, and anti-tumor drug is selected from one or more of the following groups: partially synergistic ratio (W nutrients / W methylene blue dye) is 1/3 ⁇ W nutrient / W methylene blue dye ⁇ 40 / 0.15, preferably 1/3 ⁇ W nutrient / W type methylene blue dye ⁇ 40 / 0.30, or 1 / 1.8 ⁇ W nutrient / W methylene blue dye ⁇ 40 / 0.30; synergistic ratio of the partial immunomodulator / methylene blue dye composition (W immunomodulators / W methylene blue dye) was 0.2 / 3 ⁇ W immunomodulators / W methylene blue dye ⁇ 20 / 0.15, preferably from 0.5 / 3 ⁇ W immunomodulators / W type methylene blue dye ⁇ 20 / 0.50, or 0.5 / 1 ⁇ W immunomodulators / W methylene blue dye ⁇ 20
  • a medical device comprising a composition according to the above is provided.
  • the application of the above-mentioned composition and an anti-tumor drug or immunomodulator in the preparation of a drug combination or preparation for the treatment of cancer is provided.
  • the cancer includes solid tumors, such as sarcoma, head and neck cancer, breast cancer, pancreatic cancer, thyroid cancer, nasopharyngeal cancer, prostate cancer, liver cancer, lung cancer, bowel cancer, oral cancer, esophageal cancer, gastric cancer, Laryngeal cancer, testicular cancer, vaginal cancer, uterine cancer, ovarian cancer, etc.
  • solid tumors such as sarcoma, head and neck cancer, breast cancer, pancreatic cancer, thyroid cancer, nasopharyngeal cancer, prostate cancer, liver cancer, lung cancer, bowel cancer, oral cancer, esophageal cancer, gastric cancer, Laryngeal cancer, testicular cancer, vaginal cancer, uterine cancer, ovarian cancer, etc.
  • composition of the present invention may be used for local administration, for example, local intratumoral administration.
  • a method for treating cancer which includes administering the above-mentioned composition or a pharmaceutical combination or preparation thereof with an anti-tumor drug or an immunomodulator to a patient suffering from cancer.
  • a method for treating cancer which includes intratumorally administering the above-mentioned composition or a pharmaceutical combination or preparation thereof with an antitumor drug or an immunomodulator to a patient suffering from cancer.
  • the cancer includes solid tumors, such as sarcoma, head and neck cancer, breast cancer, pancreatic cancer, thyroid cancer, nasopharyngeal cancer, prostate cancer, liver cancer, lung cancer, bowel cancer, oral cancer, esophageal cancer, gastric cancer, Laryngeal cancer, testicular cancer, vaginal cancer, uterine cancer, ovarian cancer, etc.
  • the solid tumor is preferably selected from malignant solid tumors containing tumor bodies with a mesenchymal ratio ⁇ 20% or/and an average size ⁇ 2.0 cm.
  • a combination of a local synergist or a synergistic drug of a methylene blue dye and the methylene blue dye which is preferably to minimize rather than maximize local effects, is provided as a local synergistic effect.
  • the methylene blue dye is used as the topical active ingredient
  • the local synergist or synergistic drug is selected from the following One or more of the group: nutrients, immunomodulators, conventional anti-tumor drugs.
  • a topical pharmaceutical composition for the treatment of malignant solid tumors that can provide the local synergistic effect, which comprises a methylene blue dye that preferably minimizes rather than maximizes the local effect, and
  • the local synergist or synergistic drug of the methylene blue dye wherein the methylene blue dye is used as a local active ingredient, and the synergist or synergistic drug is selected from one or more of the following groups: nutrients, immunomodulators Agents, conventional anti-tumor drugs.
  • the ratio of the amount of the methylene blue dye to the nutrient, immunomodulator, and anti-tumor drug is selected from one or more of the following groups: partially synergistic ratio (W nutritional factors / W methylene blue dye) is 1/3 ⁇ W nutrient / W methylene blue dye ⁇ 40 / 0.15, preferably 1/3 ⁇ W nutrient / W methylene blue dye ⁇ 40/0.30, or 1/1.8 ⁇ W nutrient /W methylene blue dye ⁇ 40/0.30; the local synergistic amount ratio of the immunomodulator/methylene blue dye composition (W immunomodulator /W sub A blue dye) was 0.2 / 3 ⁇ W immunomodulators / W methylene blue dye ⁇ 20 / 0.15, preferably from 0.5 / 3 ⁇ W immunomodulators / W methylene blue dye ⁇ 20 / 0.50, or 0.5 /1 ⁇ W immunomodulator /W methylene blue dye ⁇ 20/0.50; the local synergistic ratio
  • the above-mentioned topical pharmaceutical composition further comprises a pharmaceutically acceptable liquid carrier when administered, and in the administration composition of the topical pharmaceutical composition, the concentration of the methylene blue dye (w/v ) ⁇ 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 immunomodulator
  • concentration (w/v) is greater than 0.25%, preferably 0.5%-30%
  • the concentration (w/v) of the conventional anti-tumor drug is greater than 20% of its saturated concentration, preferably 30% of its saturated concentration -100%, wherein the saturated concentration refers to the saturated concentration of the conventional anti-tumor drug in the liquid carrier.
  • a topical pharmaceutical composition for the treatment of malignant solid tumors which comprises (preferably to minimize rather than maximize local effects) methylene blue Dyes, local synergists or synergistic drugs of the methylene blue dyes, and pharmaceutically acceptable liquid carriers, wherein the synergists or synergistic drugs include or are selected from one or more of the following: nutrients , Immunomodulators, conventional anti-tumor drugs, and in the administration composition of 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 (w/v) of the immunomodulator is greater than 0.25%, preferably 0.5%-30%, and the concentration (w/v) of the conventional antitumor drug is greater than 20% of its saturated concentration, preferably 30%-100% of
  • the 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, wherein the concentration (w/v) of the methylene blue dye is ⁇ 1.1%, preferably 0.35-1.09%, 0.5-1.09% or 0.5- 1.05%.
  • the above-mentioned nutrients are one or more selected from the following group: amino acid nutrients, carbohydrate nutrients, lipid nutrients, and wherein the concentration of the nutrients is greater than 2.5% to less than 50%, preferably 4-40%.
  • the above-mentioned immunomodulator is one or more selected from the group consisting of antibodies, nucleic acids, and probiotic components, and wherein the concentration (w/v) of the immunomodulator is greater than 0.25%, Preferably it is 0.5%-30%.
  • the above-mentioned conventional anti-tumor drugs are selected from one or more of the following groups: 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 drugs that affect protein synthesis .
  • the above-mentioned pharmaceutical composition is in the form of an injection, and the injection includes a liquid injection and a powder for injection.
  • a topical pharmaceutical composition for treating malignant solid tumors which comprises a dry powder obtained by freeze-drying or semi-freeze-drying part or all of the above-mentioned pharmaceutical composition.
  • the malignant solid tumors include 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, vagina Cancer, uterine cancer, ovarian cancer.
  • the malignant solid tumor is preferably selected from malignant solid tumors containing tumor bodies with a mesenchymal ratio ⁇ 20% or/and an average size ⁇ 2.0 cm.
  • a topical pharmaceutical composition for the treatment of malignant solid tumors which comprises a methylene blue dye, a local synergistic drug or synergist of the methylene blue dye, and pharmacology
  • An acceptable liquid carrier wherein the synergistic drug or synergist is selected from nutrients or/and conventional anti-tumor drugs, and in the topical pharmaceutical composition, the concentration of the methylene blue dye (w/v) ⁇ 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 conventional antibiotic
  • the concentration of the 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.
  • a topical active ingredient as a topical active ingredient and a local synergistic drug or synergist of the methylene blue dye to prepare a topical pharmaceutical composition for the treatment of malignant solid tumors
  • the synergistic drugs or synergists are selected from nutrients or/and conventional anti-tumor drugs.
  • the topical pharmaceutical composition comprises the methylene blue dye, a local synergistic drug or synergist of the methylene blue dye, and a pharmaceutically acceptable liquid carrier, wherein
  • 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 nutrient
  • the concentration (w/v) of ⁇ 2%, preferably 3%-40%, and the concentration of the conventional antitumor drug is greater than 20% or 30% of its saturated concentration, preferably 30%-100% of its saturated concentration
  • 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 disclosed in the present application also referred to as the composition of the invention in the present invention
  • the composition containing the methylene blue dye in the prior art also in the present invention
  • shared pharmacology is more optimized: the former preferentially utilizes the effective local synergy of the composition, while the latter preferentially utilizes the local effect of methylene blue dyes and additives to the local The additive synergistic effect of the effects; 2)
  • the effective technical solution to achieve shared pharmacology is more optimized: the former only needs to be the methylene blue dye that minimizes rather than maximizes the local effect, while the latter needs to be optimized for the maximum
  • the methylene blue dyes that do not minimize local effects are embodied in two opposite directions of preferred dosing concentration.
  • the side effects of the local synergistic drugs required by the former are not stronger (in terms of being selected from conventional antitumor drugs), or even weaker (in terms of being selected from nutrients or/and immunomodulators) than any of the latter.
  • the former can be used preferentially in one or more of the following indications that the latter cannot: a larger stromal ratio tumor that can be administered locally, and a tumor containing the tumor Treatment of tumors; treatment of larger tumors that can be administered locally and tumors containing the tumors; treatment of nodules related to malignant tumors that can be administered locally.
  • 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 localization.
  • 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.
  • 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.
  • the term “pharmaceutical composition” refers to a substance that clarifies the pharmacology and provides the pharmacological methods, pharmacological composition, pharmacological environment and other pharmacological characteristics necessary for realizing the pharmacology in the patient's body.
  • the term “pharmacological method” refers to the method of administration of the pharmaceutical composition necessary for the realization of a specific pharmacology.
  • the pharmacological method necessary for the improvement of the intestinal barrier of probiotics is oral administration
  • the pharmacological method necessary for ethanol chemical ablation is administration in the lesion area.
  • pharmaceutical composition refers to the composition of the pharmaceutical composition necessary for the realization of a specific pharmacology.
  • pharmacological environment refers to the minimization of exogenous interference necessary for the pharmaceutical composition to achieve a specific pharmacology in the target area.
  • additives such as osmotic pressure regulators
  • Negative effects A pharmaceutical composition without a specific pharmacological environment is bound to contain inactive ingredients that enhance administration compliance (for example, flavoring agents for oral drugs) and administration safety (for example, osmotic pressure regulators contained in injections), while local effects Pharmacology must require a limited pharmacological environment to reduce the possible pharmacological interference of inactive ingredients.
  • the pharmacology of pharmaceutical compositions without specific pharmacological methods means that these methods can be achieved (e.g. probiotic injections for immune enhancement, cytotoxic drugs for cell growth inhibition)
  • the drunken reaction of ethanol means that it can only be achieved in this method (for example, the improvement of the intestinal barrier of oral probiotics, the chemical ablation of topical ethanol).
  • the pharmacology of a pharmaceutical composition without a specific pharmacological composition limitation means that the pharmacology of a pharmaceutical composition is in a given active ingredient, active ingredient amount ratio, and It can be achieved after the dosage of the composition (for example, the immune enhancement of probiotic injections, the cell growth inhibition of cytotoxic drugs, and the drunken reaction of ethanol).
  • pharmacology of ethanol under the limits of its pharmacological method (administration within the lesion), pharmacological concentration (for example, high concentration ⁇ 70%), and pharmacological volume (for example, dosing volume/target volume ratio ⁇ 0.25)
  • pharmacological concentration for example, high concentration ⁇ 70%
  • pharmacological volume for example, dosing volume/target volume ratio ⁇ 0.25
  • topical drug is distinguished from conventional drugs (compositions).
  • compositions refers to conventional administration (or systemic administration, such as oral, intravenous, 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).
  • Therapeutic drugs whose activity produces medicinal effects.
  • local activity refers to a pharmacological activity that is preferentially displayed in local tissues rather than tumor cells, and includes, for example, the local action of a single drug and the local shared action of a shared drug.
  • 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 Ya-Africa 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 by providing local Synergistic active ingredients (e.g. 0.5 to 1.5% methylene blue).
  • local synergistic drug (or local synergist) is used to refer to the local active ingredient (for example, local administration and administration concentration ⁇ 2%, preferably 0.5%-1.5%) Methylene blue dyes) share substances that produce local synergistic effects, which are selected from one or more of the following groups: nutrients, immunomodulators, and conventional anti-tumor drugs.
  • local synergistic effect refers to a synergistic effect that mainly exhibits local activity.
  • the term “synergistic effect” means that the sharing of active ingredients shows a more favorable pharmaceutical effect for treatment than they are used alone, and it includes, for example, synergistic efficacy and synergistic safety.
  • 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.
  • the drug effect is required (for example, the tissue necrosis effect produced by the sharing of two cytotoxic drugs, or the drug effect of the actual/expected ratio q>1.15 agreed in the embodiment of the present invention).
  • 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 or synergist of the methylene blue dye, and a drug A scientifically acceptable liquid carrier, wherein the synergistic drug or synergist is selected from nutrients or/and conventional anti-tumor drugs, and in the topical pharmaceutical composition, the concentration of the methylene blue dye (w/v) ⁇ 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 20% or 30% of its saturated concentration, preferably 30%-100% of its saturated concentration, wherein the saturated concentration refers to the saturation of the conventional anti-tumor drug in the liquid carrier concentration.
  • a topical active ingredient as a topical active ingredient and a local synergistic drug or synergist of the methylene blue dye to prepare a topical pharmaceutical composition for the treatment of malignant solid tumors
  • the synergistic drugs or synergists are selected from nutrients or/and conventional anti-tumor drugs.
  • the topical pharmaceutical composition comprises the methylene blue dye, a local synergistic drug or synergist of the methylene blue dye, and a pharmaceutically acceptable liquid carrier, wherein
  • 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 nutrient
  • the concentration (w/v) of ⁇ 2%, preferably 3%-40%, and the concentration of the conventional antitumor drug is greater than 20% or 30% of its saturated concentration, preferably 30%-100% of its saturated concentration
  • 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.
  • 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.
  • a new application of methylene blue dyes in the prior art involves topical administration.
  • the methylene blue dyes in this application must maximize rather than minimize local effects pharmacologically, and therefore must be a maximum preferred concentration orientation in terms of composition.
  • synergists can be optionally added, which involve systemic administration of effective anti-tumor substances, Systemic administration of low-efficiency but local administration of high-efficiency low-efficiency anti-tumor substances and metal compounds.
  • a high concentration (for example, 5%) methylene blue dye can be used as a chemical ablation agent in this technical solution. 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 the methylene blue dyes provide only conditions that preferably minimize rather than maximize local effects under non-chemical ablative conditions (for example, 1% methylene blue). Blue administration concentration), it can be shared with nutrients, immunomodulators, and/or conventional anti-tumor drugs that are quite different from pharmacologically.
  • the expected local synergy so that a methylene blue dye that is significantly lower than its chemical ablation concentration threshold can be used to obtain a significantly improved therapeutic effect.
  • These unexpected synergistic effects may be the key point of pharmacology, which significantly improves the specificity of methylene blue dyes, nutrients, immunomodulators, and/or 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 used as a local active ingredient that can provide local synergistic effects, rather than being mainly used to maximize local effects
  • the chemical ablation agent is applied 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.
  • a local synergistic drug or synergist of the methylene blue dye in the treatment of malignant solid tumors in the treatment of malignant solid tumors it may be an option.
  • 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 drug or synergist 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 di 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 topical synergistic drug or synergist includes a carbohydrate nutrient, and the concentration (w/v) of the carbohydrate nutrient 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 or synergist 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%.
  • the term "immunomodulator” used means that it has not been used clinically as a vaccine adjuvant and can be used as an immunomodulatory food or immunomodulatory drug, but its molecular structure can be minimized with the preference in the present invention. Instead of maximizing local effects, methylene blue dyes share and provide local synergistic substances. Therefore, the immunomodulator of the present invention is essentially different from the immunoadjuvant in the prior art: 1).
  • the purpose of the composition is different: the purpose of the latter is to promote the relevant and beneficial treatment of tumors in the affected area.
  • the purpose of the former is to provide local synergy; 2).
  • the pharmacological schemes to achieve their goals are different: the latter only involves immune enhancement, while the former must involve chemical enhancement (local synergy); 3).
  • the components (structures) of the (efficiency) scheme are different: the latter is selected from one or more of the following groups commonly used as vaccine adjuvants: inorganic adjuvants, (highly immunogenic) microorganisms and their products, Synthetic adjuvants, oils, cytokines, heterologous antigens, and the former is selected from probiotic components or/and immunomodulatory peptides that minimize the immunogenicity of bacteria that are usually not used as vaccine adjuvants but used as immunomodulatory foods, and usually Immunoglobulin used as an immunomodulator of a vaccine adjuvant that is completely different from the antigen-enhancing effect; 4).
  • the latter s technical effect is mainly beneficial to the immune enhancement of anti-tumor antigens, but Usually there is no tissue destruction effect, and the necessary technical effect of the former is a local synergy that can cause effective tissue destruction, which is mainly a chemical effect that does not depend on immune action. In the following examples, the realization of this local synergy does not depend on the immune function of the animal, and it can even be achieved in nude mice with severe immunodeficiency.
  • probiotics refers to non-pathogenic living microorganisms that can have a beneficial effect on the health of the host.
  • Probiotics include cells, and yeasts are single-celled microorganisms.
  • probiotic components refers to preparations derived from natural probiotics or engineered bacteria (such as wall polysaccharides) or engineered analogs of the preparations (such as synthetic polysaccharides similar to cell wall polysaccharides or other Source polysaccharide).
  • the probiotic components are preferably selected from those that minimize bacterial immunogenicity, and more preferably selected from one or more of the following groups and their engineering analogs: water-soluble components of probiotics , Probiotics components water-insoluble components, inactivated probiotics.
  • bacterial immunogenicity refers to the ability of bacteria as a complete foreign object to produce an immune response in the recipient's body, and different bacteria have different bacterial immunogenicity. Live probiotics have the strongest bacterial immunogenicity, but direct entry into the body also has a strong safety risk.
  • inactivated probiotic bacteria refers to a preparation obtained after a bacterial inactivation engineering process, wherein the bacterial inactivation is, for example, one or more of the following: high temperature inactivation, high temperature and high pressure inactivation, ultraviolet inactivation, chemical Reagent inactivation, radiation inactivation.
  • probiotic water-insoluble component refers to any component obtained from probiotics with a water solubility of ⁇ 0.1% (such as broken probiotic precipitates, water-insoluble probiotic cell wall components, water-insoluble polysaccharides, water-insoluble ⁇ - Dextran, etc.).
  • water-soluble components of probiotics refers to any components obtained from probiotics with a water solubility of ⁇ 0.1% (for example, water-soluble extracts of probiotics, water-soluble polysaccharides, water-soluble ⁇ -glucans, etc. ).
  • the administration concentration of the inactivated probiotic in the pharmaceutical composition is >0.3%, ⁇ 0.75%, 0.75-15 , Preferably 1.5-15% or 5-15%; when the probiotic component includes the probiotic water-soluble component, the administration concentration of the probiotic water-soluble component in the pharmaceutical composition is >0.25, or 0.35-25%, preferably 0.35-15% or 5-15%; when the probiotic component includes the probiotic water-insoluble component, the probiotic water-insoluble component particles are The administration concentration in the pharmaceutical composition is >0.5, or 0.5-15%, preferably 1.5-15% or 5-15%.
  • the probiotic bacteria are selected from one or more of the following groups of natural bacteria or engineered bacteria: probiotic bacillus, probiotic lactic acid bacteria, probiotic bifidobacteria, and probiotic fungi.
  • the probiotic bacteria include one or more selected from probiotic Bacillus. In one embodiment, the probiotic bacteria include one or more selected from probiotic Lactobacillus. In one embodiment, the probiotic bacteria include one or more selected from probiotic bifidobacteria. In one embodiment, the probiotics include one or more selected from probiotic fungi.
  • the Bacillus is selected from one or more of the following: Bacillus cereus, Bacillus licheniformis, Bacillus subtilis, Bacillus megaterium, Bacillus firmus, Bacillus coagulans Bacillus, Bacillus lentus, Bacillus pumilus, Bacillus natto. In one embodiment, the Bacillus is preferably selected from one or more of the following: Bacillus licheniformis, Bacillus subtilis, Bacillus pumilus.
  • the lactic acid bacteria include one or more selected from the group consisting of Lactobacillus or/and Bifidobacterium.
  • the Lactobacillus includes one or more selected from the group consisting of Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus pumilus, and Lactobacillus fermentum.
  • the Lactobacillus is preferably selected from one or more of the following: Lactobacillus casei, Lactobacillus plantarum, Lactobacillus pumilus, and Lactobacillus fermentum.
  • the bifidobacterium includes one or more selected from the group consisting of: Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus brucelli, Swiss Lactobacillus, Lactobacillus thermophilus, Enterococcus faecium, Streptococcus faecalis.
  • the bifidobacterium is preferably selected from one or more of the following: Bifidobacterium longum, Bifidobacterium adolescentis, Enterococcus faecium, and Streptococcus faecalis.
  • the fungus includes one or more selected from yeast or/and Brucella saccharomyces, wherein the yeast includes one or more selected from the group consisting of Saccharomyces cerevisiae, Delta Saccharomyces cerevisiae, Candida, Wickham's yeast, Pichia pastoris, Saccharomyces leucocephalus, Saccharomyces sp , Candida Ruan.
  • the probiotic is preferably one or more selected from yeast or/and Brucella.
  • the probiotics are preferably selected from probiotics that do not contain mycolic acid cell walls.
  • the probiotic is preferably selected from Saccharomyces brucei.
  • the probiotic is preferably selected from Saccharomyces cerevisiae.
  • immunoglobulin is a structural concept that refers to all drugs that are the same or similar to antibody molecules, including natural immunoglobulins and their engineering analogs.
  • the natural immunoglobulins include immunoglobulin G, immunoglobulin A, immunoglobulin M, immunoglobulin D, and immunoglobulin E, which are usually obtained from the separation of animal plasma, such as intravenous immunoglobulin G and intravenous injection. Injection of immunoglobulin A, intravenous injection of immunoglobulin M, and so on.
  • the engineering analogue of natural immunoglobulin refers to an engineering product (including genetic engineering product) that is different from natural immunoglobulin but has all or part of the key structure of natural immunoglobulin, such as immunoglobulin complex, Immunoglobulin Fc fragments, immunoglobulin Fab fragments, immunoglobulin Fab' fragments, immunoglobulin F(ab') fragments, single immunoglobulins, etc.
  • the administration concentration (w/v) of the immunoglobulin in the pharmaceutical composition is >1.5%, ⁇ 2.0, 2.0-35%, preferably 3.0-30%.
  • the term "immunomodulatory peptide” refers to a non-protein polypeptide derived from organisms or food proteins that has an immunomodulatory effect, for example, the CAS number used in the examples of the present invention is 63231-63-0 Ribonucleic acid.
  • the administration concentration (w/v) of the immunomodulatory peptide in the pharmaceutical composition is >1.5%, ⁇ 2.0, 2.0-35%, preferably 3.0-30%.
  • 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, the topical synergistic drug or synergist, and optionally other active ingredients as described above) and a liquid carrier (such as 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, the topical synergistic drug or synergist, and optionally other active ingredients as described above
  • a liquid carrier such as 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 Part or all of the methylene blue dye, the local synergistic drug or synergist, and the pharmaceutical composition of a pharmaceutically acceptable carrier are 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 Dyestuffs, the local synergistic drug or synergist) and optional other components are added to the vehicle to prepare a liquid; 2) other necessary components (such as other nutrients) in the required amount according to the intratumoral administration concentration 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 required amount of methylene blue dye and the local synergistic drug are added according to the intratumoral administration concentration. Or the synergist and optional other components are added to the solvent (or pharmaceutically acceptable liquid carrier) and mixed uniformly and prepared as a sterile liquid I after sterilization; 2) The required amount will be based on the intratumoral 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: The sterile dry powder of the local synergistic drug or synergist; and the preparation of 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 dye, the local synergistic drug or synergist 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 the local synergistic drugs or synergists, containing different types and concentrations of other additives ( For example, analgesics, acidifiers, 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
  • the treatment of malignant solid tumors by the composition of the present invention preferably includes tumor treatment.
  • the composition of the present invention for tumor treatment of malignant solid tumors preferably includes effective destruction of non-malignant tumor cell components in the tumor or tumor-related nodules, especially connective tissue.
  • treatment refers to any reasonable and advantageous medical treatment, which includes post-diagnosis treatment (for example, medical treatment performed after diagnosis) and preventive treatment (for example, medical treatment that can be performed without a diagnosis) .
  • treatment of malignant solid tumors or “treatment of malignant solid tumors” refers to the treatment of patients who have been diagnosed with or are likely to have malignant solid tumors, including tumor treatment and other tissues containing tumor cells (such as lymphatic tissue) the treatment.
  • tumor treatment refers to the treatment of tumors or tumor-related nodules.
  • the preferred solution for the indications of the composition of the present invention is one or more of the following: a larger stromal ratio tumor body that can be administered locally, and the treatment of tumors containing the tumor body ; Treatment of larger tumors that can be locally administered and tumors containing the tumors; treatment of malignant tumor-related nodules that can be administered locally.
  • tumor body refers to a mass formed due to abnormal proliferation of cells or mutated cells.
  • solid tumor (sometimes abbreviated as tumor) refers to a disease in which tumors can appear.
  • malignant solid tumor (sometimes abbreviated as malignant tumor) refers to a malignant disease in which tumors can appear.
  • Tumors include, for example, the following groups classified according to tumor cell types: epithelial cell tumors, sarcomas, lymphomas, germ cell tumors, and blastoma; and include tumors named according to the organ or tissue where the tumor cell concentration area is located, including, for example, according to Tumors named for 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, testis, ovary 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 promote the effective destruction of non-malignant tumor cell components (especially connective tissue) in tumors or tumor-related nodules. At the same time, there is only minimal damage to the patient's normal tissues, so as to achieve safe and effective treatment of malignant solid tumors.
  • the immunomodulators used in the following specific examples can all be obtained from commercial sources.
  • the strains of probiotics used are all strains used in Saccharomyces cerevisiae and probiotic preparations approved by the food and drug authorities or contained in the official pharmacopoeia.
  • strains contained in probiotic preparations approved by the Chinese pharmaceutical authority Clostridium butyricum in Bacillus coagulans live tablets, Bacillus licheniformis strains in live Bacillus licheniformis granules (capsules), Oral Bacillus cereus Bacillus cereus strains in live bacteria preparations, Clostridium butyricum strains in live Clostridium butyricum capsules, Saccharomyces boulardii strains in Saccharomyces boulardii powder (capsules), Saccharomyces boulardii strains in Lactobacillus acidophilus capsules Lactobacillus butyricum strains, Clostridium butyricum strains in oral clostridium butyricum viable bacteria powders, Enterococcus faecium strains, subtilis strains in Bacillus subtilis double live bacteria multi-dimensional granules, and Enterococcus butyricum triple viable bacteria tablets Lactic acid strain
  • 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
  • Test observation, measurement and analysis items including general status, body weight, food intake, tumor volume, tumor weight, etc.
  • 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 inhibition rate R(%) (CW-TW)/CW ⁇ 100%, where TW is the average tumor weight of the study group; CW is the average tumor weight of the negative control group.
  • E i i the drug efficacy referred to as E i i, where E i may be a (100-r i)% or R i%.
  • B/A the combination of A drug and B drug
  • the single-use drug effects of A and B are recorded as E A and E B respectively
  • the actual common drug effects of A/B are recorded as E A+B .
  • test results (such as tumor weight, tumor volume) are expressed as mean ⁇ standard deviation (x ⁇ s), and the difference between the two test animal groups and the group mean uses the statistical software SPSS 13.0 or SPSS The 19.0 software performs a significance test for comparison.
  • the test uses the statistic t to perform the test.
  • the type of drug action (shared pharmacology) is studied through drug effects, especially comparing the drug effects of the same research drug in different technical solutions.
  • the difference in drug effect of drug i between regimens X and Y is not unusually large (for example, E iX /E iY ⁇ 150%), it is likely to be basically the same drug action type (pharmacology) with different dynamics Due to scientific conditions (action concentration); and when the effect of the drug is unusually large (for example, E iX /E iY >150%), the drug effect of drug i in plan X should exceed its effect in plan X
  • the kinetic expected range of the drug action type (pharmacology) in Y which is likely to involve a drug action type (pharmacology) different from that of the scheme Y.
  • the positive controls for chemotherapy include classic cytotoxic drugs (e.g. 0.5-1% 5-fluorouracil, which has a tumor inhibition rate of ⁇ 30% under the conditions of the following examples) and classic chemical ablation agents (e.g. 75- 99% ethanol, its tumor inhibition rate under local administration conditions in the following examples is ⁇ 30%).
  • classic cytotoxic drugs e.g. 0.5-1% 5-fluorouracil, which has a tumor inhibition rate of ⁇ 30% under the conditions of the following examples
  • classic chemical ablation agents e.g. 75- 99% ethanol, its tumor inhibition rate under local administration conditions in the following examples is ⁇ 30%).
  • E A/B is the actual common drug effect of composition A/B (such as tumor inhibition rate)
  • E A and E B are the actual single-drug efficacy of component A and component B of composition A/B, respectively
  • (E A + E B- E A ⁇ E B ) is the expected effect of component A and component B based on the theory of the actual efficacy of the single drug.
  • the actual/expected ratio q is calculated according to the q calculation formula in the above-mentioned Jin Zhengjun's modified method, and the common effect of the combined administration according to the above-mentioned Zhang Xiaowen-Jin Zhengjun method is judged as follows:
  • composition A/B group does not show therapeutically significant pharmacological effects (for example, the tumor inhibition rate is less than 30%), the combined administration of A and B does not show a therapeutically meaningful shared effect, which is regarded as No synergy.
  • the composition group shows therapeutic effect (for example, tumor inhibition rate ⁇ 30%)
  • the actual/expected ratio q is between 0.85 and 1.15
  • the common effect of the composition is additive (the actual effect is in line with the theoretical Simply add up expectations); if the actual/expected ratio q>1.15, the sharing effect of the composition is obviously synergistic (the actual effect exceeds the theoretical simple addition expected); if the actual/expected ratio q ⁇ 0.85, the sharing of the composition
  • the effect is an obvious antagonistic effect (the actual effect is not as good as the theoretical simple addition expectation).
  • composition of part of the composition (X%/Y%) of the present invention prepared in this example is listed in the following table.
  • Test 1A Preparation of liquid injection (1)
  • methylene blue dye e.g. 1g of methylene blue
  • local synergists e.g. 10g of glutathione
  • optional other components e.g. dilute to the total volume according to the required concentration (as described in Table 2)
  • the volume (e.g. 100ml) of liquid carrier e.g. 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
  • Test 1C Preparation of powder injection for injection
  • methylene blue dye e.g. 1g methylene blue
  • local synergists e.g. 20g arginine, 1g 5-fluorouracil
  • optionally other groups according to the required concentration (as described in Table 2)
  • Divide and dilute to the total volume (e.g. 100ml) of the liquid carrier e.g. water for injection
  • the liquid carrier e.g. water for injection
  • %Methylene blue/20%arginine/5-fluorouracil aqueous solution can be used as a liquid drug for intratumoral administration.
  • Test 2A The test animal was a mouse, the modeling cell was a sarcoma S180 cell, and the transplanted tumor was modeled under the skin of the animal's right axillary at 1 ⁇ 10 6 cells/only.
  • Successfully modeled experimental animals (mice bearing malignant sarcoma, with an average tumor volume of 121mm 3 ) were randomly divided into 2 series (series I and II), and each series was divided into a negative control group (I0 and II0) And 11 study groups (I1-I11 and II1-II11).
  • the negative control is normal saline, and the 11 study drugs are shown in the table below, series I for systemic administration (intraperitoneal injection) and series II for local administration (intratumoral injection).
  • 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.
  • the animals were euthanized 10 days after the end of the medication, and the tumor weight (W) was measured after anatomy, and the tumor inhibition rate (R) was calculated from the negative control group of each medication mode. The results are shown in the following table.
  • the intraperitoneal injection group (group I1) and the intratumoral injection group (group II-1) of the positive control substance (5-fluorouracil) showed almost indistinguishable tumor inhibition rates, although some people believe that the intratumoral drug can be used.
  • Increasing its local drug concentration will significantly improve its efficacy.
  • This result 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 systemically rather than locally.
  • the methylene blue used is lower than the concentration of its clinical injection ( ⁇ 1%), and it is considered to be methylene blue that does not produce adverse local effects even if injected intravenously.
  • the difference between the tumor weight of the study group I2 and the negative control group I0 is not statistically significant (P>0.05), and its tumor inhibition rate is at a negligibly low level, indicating that the methylene blue dye
  • the conventional anti-tumor effect of serotonin can not produce tumor growth inhibitory effect.
  • the results of study group II2 are consistent with tumor study group I2, indicating that the methylene blue dye only provides a minimized rather than maximized local effect.
  • each methylene blue dye/nutrient composition group (6-8) in series I is exactly the same as that of methylene blue dye single-drug group 5 and the corresponding nutrient single-drug group (1-4) in the same series. It shows that the conventional combination of the methylene blue dye and the common substance (nutrient) (whether it is a combination of nutrition and conventional anti-tumor effects, or a combination between conventional anti-tumor effects) can not produce tumor growth inhibitory effects, more It is not an effective synergy.
  • the composition group 9 in the series I did not increase the tumor inhibition rate, indicating that any conventional combination of methylene blue dye and conventional anti-tumor drugs in the conventional composition (such as conventional anti-tumor effects) The combination) can not achieve tumor suppression and synergy.
  • the tumor inhibition rate of group II9 is more than 150% higher than that of group I9, indicating that the topical pharmaceutical composition in series II achieves effective tumor treatment through a new pharmacology that is different from the conventional pharmaceutical composition in series I.
  • the tumor inhibition rate of the composition group 9 is also beyond the expected range of the purely additive effect of the two drugs, and its actual/expected ratio (q) is greater than 1.15, showing The above-mentioned new pharmacology should be the local synergistic pharmacology.
  • the tumor inhibition rate of group II11 is more than 150% higher than that of group I11, indicating that the topical pharmaceutical composition in series II achieves effective tumor treatment through a new pharmacology that is different from the conventional pharmaceutical composition in series I.
  • the tumor inhibition rate of the composition group 11 is also beyond the expected range of the theoretical simple additive effect of the two drugs, and its actual/expected ratio (q) is greater than 1.15, showing The above-mentioned new pharmacology should be the local synergistic pharmacology.
  • Test 2B The test animal was a nude mouse, the modeling cell was human pancreatic cancer cell (PANC-1), and the transplanted tumor was modeled at 1 ⁇ 10 6 cells/only under the skin of the animal's right axillary.
  • Successfully modeled test animals (nude mice bearing pancreatic cancer, with an average tumor volume of 147mm 3 ) were randomly divided into 2 series (series I and II), and each series was divided into a negative control group (I0 and II0) And 11 study groups (I1-I11 and II1-II11).
  • the negative control is physiological saline, and the 11 study drugs are shown in the following table.
  • immunoglobulin is human immunoglobulin
  • probiotics are heat-inactivated Saccharomyces boulardii
  • the water-soluble components of probiotics are water-soluble yeast glucosamine.
  • the sugar, water-insoluble component of probiotics is Saccharomyces cerevisiae ⁇ -glucan.
  • the medicines are all liquids and are prepared according to the preparation method of Example 1. Series I line systemic administration (intraperitoneal injection) and series II line local administration (intratumoral injection). In the present invention, all intratumoral injections need to avoid blood vessels.
  • the medicine is administered once every 3 days, a total of 3 times, and the injection volume is 120 ⁇ l/mouse.
  • Ten days after the end of the medication the animals were euthanized, and the tumor weight (W) was measured after anatomy, and the tumor inhibition rate (R) was calculated from the negative control group of each medication method. The results are shown in the following table.
  • the active combination (referred to as conventional combination in the present invention) that can be provided by drug sharing is usually studied through systemic administration like the series I in the above two experiments.
  • the mainstream view is that local administration is just another mode of administration that is different from systemic administration. It may have an increase in kinetic concentration but no increase in technical content, and it will not be expected to improve the kinetics of conventional combination pharmacology.
  • new pharmacology is generated.
  • Methylene blue is mainly used clinically as an antidote or vital dye.
  • it also has properties similar to ethanol: its maximum cytotoxic effect can be used in liquid reactors (cell test or non-solid tumor animal test), but not in non-liquid reactors (animal intratumoral administration test) Shows effective anti-tumor efficacy, and its maximum local effect can show effective anti-tumor efficacy in non-liquid reactors, but not in liquid reactors.
  • the combination (X/Y) of methylene blue (X) and any drug (Y) with minimal local effects can only produce conventional combinations (such as X conventional anti-tumor effects and X/Y) when administered systemically.
  • the combination of Y's immunomodulatory effect, or/and the combination of Y's conventional anti-tumor effect), and X/Y can only show its function in systemic medication when administered locally.
  • the study group 11 in the series I did not have an increase in tumor inhibition rate compared with 1, indicating that the methylene blue dye and the complex (immunomodulator/conventional anti-tumor drug) are any conventional combination in the conventional composition (for example, the combination of immunomodulatory effect and conventional anti-tumor effect, or/and the combination between conventional anti-tumor effect) can not achieve tumor suppression synergistic effect, let alone effective synergistic effect.
  • the tumor inhibition rate of group II11 is more than 150% higher than that of group I11, indicating that the topical pharmaceutical composition in series II achieves effective tumor treatment through a new pharmacology that is different from the conventional pharmaceutical composition in series I.
  • the tumor inhibition rate of the composition group 11 is also beyond the expected range of the purely additive effect of the two drugs, and its actual/expected ratio (q) is greater than 1.15, indicating that the above-mentioned new pharmacological principle should be The local synergistic pharmacology.
  • Drugs are not substances (X) completely defined by chemistry, but active substances (AX) with uniform structure (X) and effect (A).
  • AX active substances
  • different substances (X1 and X2, such as methylene blue dyes and conventional anti-tumor drugs in the above systemic administration series) that provide the same activity (A, such as cytotoxicity) can easily be regarded as different drugs (AX1 and AX2).
  • the same chemical substance (X, such as methylene blue dye) that provides different activities (A1, A2, and A3, such as cytotoxicity, local effect, and local synergy) can easily be prejudiced as the same drug (such as both Treat it as A1X and ignore A2X and A3X).
  • the effective cytotoxic effect of methylene blue dyes is denoted as A1, and the characteristic composition required for the effect is denoted as X1; its effective local effect (hence the local effect that is preferably maximized) is denoted as A2
  • the characteristic composition required for this action is denoted as X2; its effective local synergy is denoted as A3, and the characteristic composition required for this action is denoted as X3.
  • X1 with A1 was found in cell test suspension reactors and non-solid cancers, but A1X1 could not produce effective effects in tumor tissue reactors; in tumor tissue reactors, X1 with A1 was found A2 must be composed of X2 (extra-conventional concentration, such as ⁇ 2%), but the drug efficiency of A2X2 still needs to be improved.
  • the present invention provides the X3 technical solution to produce A3: low concentration ( ⁇ 2%, ⁇ 1%, or ⁇ 1%), A3X3 then only provides the local effect that is preferably minimized rather than maximized, but can provide effective local synergy The role of methylene blue dye.
  • A3X3 can prepare the composition of the present invention that far exceeds the expected technical effects (administration efficiency) of A1X1 and A2X2.
  • the characteristic activities of the methylene blue dye shared substance (Y) (such as the nutritional effect of nutrients, the immunomodulatory effect of immunomodulators, the cytotoxic effect of conventional antitumor drugs) are denoted as B1; nutrients and immunity
  • B1 The conventional anti-tumor activity (cytotoxic effect or other anti-tumor effect that can be exhibited by systemic administration, if any) other than the characteristic activity of the modulator is denoted as B2; its local synergy with the aforementioned methylene blue A3X3 is Denoted as B3, the latter is also known as the local synergist of A3X3 (denoted as B3Y).
  • the functional combination or composition of the composition of the present invention is not 1): the combination of methylene blue dye cytotoxicity (A1) and common compound characteristic activity (B1) (A1/B1) , Or A1X1/B1Y composition, nor 2): The combination (A1/B2) of methylene blue dye cytotoxicity (A1) and common anti-tumor activity (B2), or A1X1/B2Y composition, also Not 3): Combination of methylene blue dye local effect (A2) and common substance characteristic activity (B1) or conventional anti-tumor activity (B2) (A2/B1 or A2/B2), or A2X2/B1Y composition or A2X2 /B2Y composition, but 4): The combination (A3/B3) of the local synergistic effect of methylene blue dye (A3) and the local synergistic effect (B3) of the methylene blue dye, or the A3X3/B3Y composition.
  • this example and the following examples also confirm that the combination (A3/B3) or
  • the functional combination in the methylene blue dye/nutrient composition of the present invention is not the cytotoxic effect of methylene blue dye and the nutritional effect of nutrients (whether positive or side effects) or/and conventional anti-tumor effects (if If it exists) combination (A1/B1 or/and A1/B2), the local effect of non-methylene blue dye and the nutritional effect of nutrients (whether it is a positive or side effect) or/and conventional anti-tumor effect (if it exists) ) Combination (A2/B1 or/and A2/B2), but the local synergy of the methylene blue dyes that can provide nutrients (as chemical substances) and the nutrients that the nutrients can provide to the methylene blue dyes Combination (A3/B3).
  • the functional combination in the methylene blue dye/immunomodulator composition of the present invention is not the cytotoxic effect of the methylene blue dye and the immunomodulatory effect of the immunomodulator (whether it is a positive effect or a side effect) or/and
  • the methylene blue dye/conventional antitumor drug composition of the present invention is not a combination of the cytotoxic effect of methylene blue dye and the cytotoxic effect of conventional antitumor drugs (A1/B1), and it is also non-methylene blue.
  • Methylene blue dyes can provide conventional antitumor drugs (as Chemical substances) and the local synergistic combination (A3/B3) that the conventional anti-tumor drugs can provide to the methylene blue dye.
  • This example further studies the pharmacology of the local synergism of the composition of the present invention by optimizing the local synergy of the methylene blue dye to minimize rather than maximize the local action.
  • Experiment 3A The test animal was a nude mouse, the modeling cell was human pancreatic cancer cell (PANC-1), and the transplanted tumor was modeled at 1 ⁇ 10 6 cells/only under the skin of the animal's right axillary.
  • Successfully modeled test animals (nude mice bearing pancreatic cancer, with an average tumor volume of 218 mm 3 ) were randomly divided into 1 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 100ul/only.
  • groups 3 and 8 a strong stimulus response to ethanol was observed in each animal.
  • groups 4 and 9 each animal observed the most powerful damaging response of acetic acid to the non-tumor tissue at the administration site, although its tumor inhibition rate was lower than that of groups 6 and 7, indicating that the latter had higher For the specificity of tumor tissue and better curative effect.
  • Test 3B The test animal was a nude mouse, the modeling cell was a liver cancer cell (MDA-MB231), and the transplanted tumor was modeled at 1 ⁇ 10 6 cells/only under the skin of the animal's right axillary.
  • the experimental animals (nude mice bearing liver cancer, with an average tumor volume of 127mm 3 ) that were successfully modeled were randomly divided into a negative control group and 14 study groups.
  • the negative control is physiological saline, and the 13 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 drug is administered once every 3 days, a total of 3 times, and the injection volume is 100 ⁇ l/mouse.
  • Ten days after the end of the medication the animals were euthanized, and the tumor weight (W) was measured after dissection, and the tumor inhibition rate (R) was calculated from the negative control group. The results are shown in the table below.
  • low-potency anti-tumor drug refers to drugs that are ineffective in conventional administration and are very effective in local administration, including non-steroidal anti-inflammatory compounds, quinoline compounds, artemisinin derivatives, and phenolic compounds.
  • quinine dihydrochloride is used as a representative of low-efficiency anti-tumor drugs
  • ferric chloride is used as a representative of metal compounds
  • 5-fluorouracil is used as a representative of effective anti-tumor drugs.
  • these drugs can be used as methylene blue dyes to maximize the local effect of further synergy.
  • the tumor inhibition rates of study groups 6, 8, and 10 are all above 115% of study group 4, which is in line with the synergy expectation of the prior art.
  • the tumor inhibition rates of study groups 6, 8, and 10 did not exceed the expected range of the theoretical simple additive effect (the actual/expected ratio q was both less than 1.15), indicating that these drugs ( The common effects of Y) on high-concentration methylene blue (X2) are all additive.
  • the tumor inhibition rates of study groups 7 and 9 are both above 115% of study group 5, and their tumor inhibition rates did not exceed their respective component single-drug groups (groups 2 and 5, respectively, As well as the expected range of the purely additive effect of groups 3 and 5) (the actual/expected ratio q is less than 1.15), it seems to further strengthen the above-mentioned mainstream view: drug Y is compared with low concentration methylene blue (X2) and high concentration sub The sharing of methyl blue (X2) only has a dynamic difference but no sharing mechanism difference, that is, all of the above-mentioned additive effects. Therefore, only by overcoming this mainstream view as a prejudice can we obtain a superadditive composition.
  • the two different methylene blue dyes studied are the prior art methylene blue dye (structure-activity B2X2) and the present invention methylene blue dye (structure-activity B3X3).
  • the prior art methylene blue dyes (B2X2) must be high concentrations (X2, preferably 2-6%) that can provide a high concentration (X2, preferably 2-6%) that preferably maximizes rather than minimizes the local effect (B2) dye.
  • high-concentration methylene blue dyes cannot, while low-concentration (X3, preferably 0.5%-1.5% or 0.5%-1.0%) methylene blue dyes may be
  • the local synergy (B3) is provided in the local sharing with the common object in the embodiment of the present invention, and the latter can only provide the local effect that is preferably minimized rather than maximized.
  • only methylene blue dyes whose local effects are preferably minimized rather than maximized can be used as "local active ingredients that can provide local synergistic effects" in the present invention.
  • the common substances studied include nutrients, immunomodulators, conventional anti-tumor drugs, chemical ablation agents, low-efficiency anti-tumor substances in the prior art, and metal compounds.
  • chemical ablation agents, conventional anti-tumor drugs, and low-efficiency anti-tumor substances all show strong local effects.
  • Nutrients and immunomodulators generally do not show strong local effects, and metal compounds have relatively small local effects.
  • whether these common compounds can provide local synergistic effects (rather than local effects) with methylene blue dyes does not seem to have much relationship with their own local effects. In other words, the local synergy of methylene blue dyes cannot be inferred based on the combination of local effects, and its intricacies are even required to overcome certain inherent concepts to be observed.
  • chemical ablation agents and low-efficiency anti-tumor substances with the strongest local effect can synergize high and low concentrations of methylene blue dyes (X2, X3) (for example, to increase the effect to more than 115%), but its actual effect
  • the /expected ratio q is all ⁇ 1.15
  • the synergistic effect is the additive effect of the local effects of the common components, rather than the local synergistic effect defined in the present invention. Therefore, chemical ablative agents and low-efficiency anti-tumor substances can be used as synergists for methylene blue dyes, but surprisingly they cannot be preferred as local synergists of methylene blue dyes in the present invention.
  • metal compounds with weaker local action is similar to chemical ablation agents and low-efficiency anti-tumor substances. They can also be used as synergists for methylene blue dyes, and they cannot be preferably used as methylene blue dyes in the present invention. Local synergist of dyes. In addition, the use of metal compounds with low-concentration methylene blue dyes even makes it difficult to produce the high efficacy required for the purpose of the present invention.
  • the local action between the chemical ablation agent and the metal compound, and even in many cases, the nutrient and immunomodulator, which is more likely to be the metal compound, and the low concentration methylene blue dye (X3) share the actual/expected ratio q up to Above 1.15, the shared effect is exactly the local synergistic effect not defined in the present invention. Therefore, the local synergists of methylene blue dyes in the present invention can be selected from nutrients and immunomodulators.
  • Example 4 Local synergistic composition (local synergistic amount ratio) is preferred
  • topical pharmaceutical composition a topical pharmaceutical composition
  • the topical pharmaceutical composition of the present invention must be completely different from the compositional characteristics of conventional pharmaceutical compositions: local synergistic dose ratio.
  • the term “synergistic dose ratio” refers to the combination of drugs X and Y necessary to produce a synergistic effect rather than an additive or antagonistic effect (actual/expected ratio q ⁇ 1.15).
  • the weight ratio of component X and component Y (W X :W Y ), the term “local synergistic amount ratio” means that it is not necessary in the conventional pharmaceutical composition (systemic administration), but in the local pharmaceutical composition (local Administering) the necessary synergistic amount ratio.
  • Test 4A The test animal was a mouse, the modeling cell was a sarcoma cell (S180), and the transplanted tumor was modeled at 1 ⁇ 10 6 cells/only under the skin of the animal's right axillary. Successfully modeled experimental animals (mice with sarcoma, with an average tumor volume of 112mm 3 ) 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 the methylene blue concentration), 16 kinds of varying types and concentrations of other component single drugs (Y%, Y is Other component concentration), 20 compositions (X%/Y%) composed of varying concentrations of methylene blue (X%) and varying types and concentrations of other components (nutrients, conventional anti-tumor drugs) (Y%) ), their composition is shown in the table below.
  • 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 the table below.
  • 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.
  • Test 4B The test animal was a nude mouse, the modeling cell was a human liver cancer cell (MDA-MB231), and the transplanted tumor was modeled at 1 ⁇ 10 6 cells/only under the skin of the animal's right axillary.
  • Successfully modeled experimental animals (nude mice bearing liver cancer, with an average tumor volume of 163 mm 3 ) were randomly divided into a negative control group and 12 study groups.
  • the negative control is physiological saline.
  • the composition of the study drug is shown in the following table, including: 4 kinds of methylene blue single agents (X%) with varying concentrations, 4 kinds of immunomodulators (inactivated Saccharomyces cerevisiae) single agents ( Y%), and a combination of 4 different concentrations of immunomodulators and 4 different concentrations of methylene blue (X%/Y%).
  • the medicines are all water-containing liquids, which are prepared according to the preparation method of Example 1. Each group was treated twice, and the second treatment was 7 days after the first treatment, with an injection volume of 150 ⁇ l/mouse. The animals were euthanized 7 days after the end of the second medication. After dissection, the tumor tissue was stripped to determine the tumor weight, and the tumor inhibition rate was calculated from the negative control group.
  • the tumor inhibition rate of each study drug group is shown in the table below.
  • the data in the horizontal 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 10.1%
  • the data without brackets is the average tumor inhibition rate of the X% methylene blue/Y% immunomodulator composition group, for example, the average tumor inhibition rate of the 3% methylene blue/0.2% immunomodulator group containing group The rate is 75.2%.
  • the actual/expected ratio q of the composition did not increase with the increase of the methylene blue dye concentration. Instead, the local synergy ratio q was mainly dependent on the methylene blue dye concentration. The increase and decrease indicate that the local synergistic effect provided by methylene blue dyes is different or even deviated from its local effect. The actual/expected ratio q of the composition did not increase with the increase in the concentration of the shared materials, indicating that the local synergy provided by these shared materials is different from the local effect. Strength is the condition.
  • the local synergy ratio of methylene blue dye and its local synergist is actually what is needed for its local synergy Pharmacological dose ratio.
  • the local synergy ratio is >0.1/3 and ⁇ 40/0.15, preferably >0.1/1.8 and ⁇ 40/0.15, or >0.2/1 and ⁇ 40/0.30. in particular:
  • the local synergistic amount ratio of the nutrient/methylene blue dye composition (W nutrient /W methylene blue dye ) is 1/3 ⁇ W nutrient /W methylene blue dye ⁇ 40/0.15, preferably 1/3 ⁇ W nutrient /W methylene blue dye ⁇ 40/0.30, or 1/1.8 ⁇ W nutrient /W methylene blue dye ⁇ 40/0.30.
  • the local synergistic ratio of methylene blue dye / nutrient composition of amino acids is 1/3 ⁇ W-amino acid nutrient / W type dye methylene blue Dye ⁇ 20/0.15, preferably 1/3 ⁇ W amino acid nutrient /W methylene blue dye ⁇ 20/0.30, or 2/1.8 ⁇ W amino acid nutrient /W methylene blue dye ⁇ 20/0.30; lipid
  • the local synergistic quantity ratio of nutrient-like nutrients/methylene blue dye composition (W lipid nutrient /W methylene blue dye ) is 2/3 ⁇ W lipid nutrient /W methylene blue dye ⁇ 8/0.15, preferably 2/3 ⁇ W lipid nutrient /W methylene blue dye ⁇ 8/0.30, or 3/1.8 ⁇ W lipid nutrient /W methylene blue dye ⁇ 8/0.30; carbohydrate nutrient/methylene blue dye
  • Synergistic ratio topical immunomodulators / methylene blue dye composition was 0.2 / 3 ⁇ W immunomodulators / W methylene blue dye ⁇ 20 / 0.15 , preferably from 0.5 / 3 ⁇ W immunomodulators / W methylene blue dye ⁇ 20 / 0.50, or 0.5 / 1 ⁇ W immunomodulatory agents / W methylene blue dye ⁇ 20 / 0.50.
  • the local synergistic amount ratio of the conventional antitumor drug/methylene blue dye composition (W conventional antitumor drug /W methylene blue dye ) is 0.1/3 ⁇ W conventional antitumor drug /W methylene blue dye ⁇ 1.5 /0.15, preferably 0.3/3 ⁇ W conventional anti-tumor drug /W methylene blue dye ⁇ 1.5/0.15, or 0.3/1.8 ⁇ W conventional anti-tumor drug /W methylene blue dye ⁇ 1.5/0.30.
  • Example 5 The effective local synergistic concentration is optimized
  • the dosage concentration can be adjusted by the clinician (for example, 1-5 times difference), which is not a feature of the pharmaceutical composition, at least not an important feature that can lead to changes in the shared mechanism of action.
  • the topical pharmaceutical composition of the present invention has a composition characteristic completely different from conventional pharmaceutical compositions: local effective synergistic concentration.
  • the term “synergistic concentration” refers to the group in the administration composition necessary for the drugs X and Y to have a synergistic effect instead of an additive or antagonistic effect (actual/expected ratio q ⁇ 1.15).
  • concentration of component X or component Y refers to the sub-necessary synergistic concentration in conventional pharmaceutical compositions (systemic administration) but necessary in local pharmaceutical compositions (local administration).
  • Local effective synergistic concentration refers to the local synergistic concentration required for the composition X/Y to show more competitive efficacy compared with existing drugs.
  • Test 5A The test animal was a BALB/c mouse, the modeling cells were breast cancer 4T1 cells, and the transplanted tumor was modeled at 1 ⁇ 10 6 cells/only under the skin of the animal's right axillary.
  • Successfully modeled experimental animals (mice bearing breast cancer, with an average tumor volume of 119mm 3 ) were randomly divided into 1 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 drug is administered once every 3 days, a total of 3 times, and the injection volume is 100 ⁇ l/mouse.
  • the animals were euthanized 10 days after the end of the medication, and the tumor weight (W) was measured after anatomy, and the tumor inhibition rate (R) was calculated from the negative control group of each medication mode. The results are shown in the following table.
  • the dose ratio of the methylene blue dye/topical synergistic composition pharmaceutical composition used in the composition groups 4 and 9 is the same but the dose concentration is different. They not only show different therapeutic effects, but also show a common effect. Different mechanisms.
  • the actual/expected ratio q of the former is >1.15, showing local synergy; the actual/expected ratio q of the latter is ⁇ 1.15 and >0.85, showing an additive effect.
  • the composition groups 5 and 10 also showed different mechanisms of shared action. It is especially important that the efficacy of the composition obviously depends on whether they show a local synergistic effect.
  • the local synergistic concentration of methylene blue dye is> 0.25%, and its effective local synergistic concentration is preferably ⁇ 0.5%.
  • Test 5B The test animal was a BALB/c mouse, the modeling cells were breast cancer 4T1 cells, and the transplanted tumor was modeled at 1 ⁇ 10 6 cells/only under the skin of the animal's right axillary.
  • Successfully modeled experimental animals (mice bearing breast cancer, with an average tumor volume of 386mm 3 ) were randomly divided into 1 negative control group (01) and 6 drug research groups (1-6).
  • the negative control is physiological saline, and the study drugs are shown in the table below.
  • human immunoglobulin is an immunoglobulin isolated from human plasma for laboratory use, and the main component is IgG.
  • the medicines are all aqueous solutions, which are prepared according to the preparation method of Example 1.
  • Each test group received intratumoral injection, once every 3 days for a total of 3 times.
  • the amount of injection is shown in the table below.
  • the efficacy depends on the dose rather than the concentration of the drug.
  • the clinically safe blood drug concentration is usually very low (for example, less than 10ug/ml), which is much lower than the usual preparation concentration.
  • the dose ratio of the methylene blue dye/immunomodulator composition used in the composition groups 5 and 6 is the same, the dose is the same, but the dose concentration is different. They not only show different therapeutic effects, but also share Different mechanisms of action.
  • the actual/expected ratio q of the former is> 1.15, showing local synergy; the latter does not show meaningful efficacy, let alone local synergy.
  • the tumor inhibition rate of the composition group 5 is 663% of that of the composition group 6, which also shows that the corresponding compositions have completely different common pharmacology.
  • the local synergistic concentration of methylene blue dye is> 0.30%, and its effective local synergistic concentration is preferably ⁇ 0.9%.
  • Test 5C The test animal was a BALB/c mouse, the modeling cell was a sarcoma cell S180 cell, and the transplanted tumor was modeled at 1 ⁇ 10 6 cells/only under the skin of the animal's right axillary.
  • Successfully modeled experimental animals (mice bearing sarcoma, 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 tumor weight (W) was determined after the animal was dissected 10 days after the end of the medication, and the tumor inhibition rate (R) was calculated from the negative control group. The results are shown in the table below.
  • the methylene blue dye in the methylene blue dye/amino acid nutrient composition also shows a characteristic when it meets its local synergistic amount ratio and local synergistic concentration: it makes the drug significantly exceed the conventional antitumor drugs Group (positive control group) the concentration threshold of the drug effect, that is, the effective local synergistic concentration threshold.
  • the concentration threshold of the drug effect that is, the effective local synergistic concentration threshold.
  • the effective local synergistic concentration of methylene blue dye is 0.35%-2%, preferably 0.35%-1.5% or 0.50%-1.5% (w/v).
  • Test 5D The successfully modeled test animals (mice bearing S180 cells, 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 normal saline, and the positive control is 1% 5-fluorouracil.
  • the composition of the study drug is shown in the table below, which contains a local synergist (arginine) of varying concentration (X%, w/v) and a fixed concentration (1%) 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 end of the medication, and the tumor weight (W) was measured after anatomy, and the tumor inhibition rate (R) was calculated from the negative control group. The results are shown in the following table.
  • the amino acid nutrients in the methylene blue dye/amino acid nutrient composition when meeting its local synergistic amount ratio and local synergistic concentration, also show a feature: making the drug significantly exceed the conventional anti-tumor drug group ( Positive control group) the concentration threshold of the drug effect, that is, the effective local synergistic concentration threshold.
  • the concentration threshold of the drug effect that is, the effective local synergistic concentration threshold.
  • the effective local synergistic concentration of amino acid nutrients is ⁇ 5%, preferably 5%-35% or 15%-35% (w/v).
  • a necessary technical condition for the application of the combination of methylene blue dye and its local synergist as a local synergistic active ingredient is: the preparation and composition of the composition must meet the methylene blue dye
  • the local synergistic concentration of its local synergist preferably the effective local synergistic concentration, specifically:
  • the local administration concentration (w/v) of the methylene blue dye is less than or equal to 2%, preferably 0.3%-1.8%, 0.5%-1.0%, or 0.5%-0.9%;
  • the local administration concentration (w/v) of the conventional antitumor drug is greater than 20% of its saturated concentration, preferably 30%-100% of its saturated concentration;
  • the local administration concentration (w/v) of the immunomodulator is greater than 0.25%, preferably 0.5%-30%, wherein when the immunomodulator includes immunoglobulin, the administration concentration (w/v) of the immunoglobulin /v) ⁇ 0.25%, preferably 0.25-30% or 1-30%; when the immunomodulator includes a probiotic component, the administration concentration (w/v) of the probiotic component is ⁇ 0.35%, preferably When the immunomodulator includes an immunomodulatory peptide, the administration concentration (w/v) of the immunomodulatory peptide is ⁇ 3%, preferably 3-30%;
  • the local administration concentration (w/v) of the nutrient is greater than 3%, preferably 3%-45%, wherein when the nutrient includes a carbohydrate nutrient, the administered concentration of the carbohydrate nutrient (w/v) ⁇ 20%, preferably 20-40%; when the nutrient includes lipid nutrient, the administration concentration (w/v) of the lipid nutrient is ⁇ 4%, preferably 4-25%; when the nutrient includes amino acid nutrient
  • the administration concentration (w/v) of the amino acid nutrients is ⁇ 3%, preferably 3-25%, more preferably 5-25% or 15-35%.
  • Low-concentration methylene blue dyes can be used as detoxification active ingredients or conventional anti-tumor ingredients (A1X1) to prepare conventional pharmaceutical compositions, and high-concentration (or local effects maximize rather than minimize) methylene blue dyes can provide effective Locally acting active ingredients (A2X2) should be used in the preparation of locally acting pharmaceutical compositions, while low concentrations (or local effects are minimized rather than maximized) methylene blue dyes alone are not acceptable, only in combination with their local synergists It can be used as a local active ingredient (A3X3) that provides effective local synergistic effect and is applied to prepare a local synergistic pharmaceutical composition. Using the same dose of methylene blue dye, the local synergistic drug composition shows the tumor tissue destruction efficacy, so that the scope of indications exceeds the expectations of the local drug composition and the conventional drug composition.
  • Methylene blue dyes can only provide the above-mentioned local synergistic effects when they are shared with the preferred common substance (its local synergist).
  • the shared effect of methylene blue dyes and their local synergists must be local synergy, not an additive effect of local effects, so it is impossible to find clues taught in the prior art (for example, shared enhancement of local effects) that can provide the local Synergistic commons.
  • the local synergist is preferably selected One or more from the following groups: nutrients, immunomodulators, conventional anti-tumor drugs.
  • compositions of the combination are in addition to the above-mentioned component characteristics (X is selected from the local effect).
  • Minimized but not maximized methylene blue dye, Y is selected from nutrients, immunomodulators, or/and conventional anti-tumor drugs), and also includes the synergistic ratio between X and Y.
  • the compositional characteristics of the locally synergistic pharmaceutical composition may also include an effective synergistic concentration different from its conventional pharmaceutical composition or a locally acting pharmaceutical composition, for example, a methylene blue dye that is different from the effective synergistic concentration of the locally acting pharmaceutical composition. concentration.
  • 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).
  • the combination of the methylene blue and the topical synergist which can be used as a local synergistic active component, has strong coupling properties, and it is difficult to derive from common sense.
  • the following experiments further study the coupling, in order to facilitate the optimization of the essential components of the composition of the present invention.
  • Test 6A The test animal was a BALB/c nude mouse, the modeling cells were melanoma cells, and the transplanted tumor was modeled at 5 ⁇ 10 6 cells/only under the skin of the animal's right axillary.
  • Successfully modeled test animals (nude mice bearing melanoma, with an average tumor volume of 238mm 3 ) were randomly divided into 1 negative control group (0) and 9 drug study groups (1-9).
  • the negative control is physiological saline, and the 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. Each group was given the drug once every 3 days for a total of 3 times, intratumoral injection was performed, and the injection volume was 125 ⁇ l/mouse. Three days after the end of the medication, the animals were euthanized, and the tumor weight (W) was measured after dissection, and the tumor inhibition rate (R) was calculated from the negative control group. The results are shown in the following table.
  • the tumor weights of the composition groups 6-8 are statistically significant compared with the negative control group (all p ⁇ 0.05), and their respective actual/expected ratios q are all> 1.15, all showing Obvious local synergy.
  • the tumor weight of the composition group 9 was not statistically significant (p>0.05), and it did not show significant pharmacological effects (tumor inhibition rate less than 20%), so it did not show obvious local synergy .
  • Test 6B The test animal was a BALB/c mouse, the modeling cell was a sarcoma S180 cell, and the transplanted tumor was modeled at 1 ⁇ 10 6 cells/only under the skin of the animal's right axillary.
  • Successfully modeled test animals (S180 cell-bearing mice with an average tumor volume of 151mm 3 ) were randomly divided into negative control group, positive control group and 15 study groups.
  • the negative control was normal saline
  • the positive control was 1% 5-fluorouracil
  • the study drug was 1% methylene blue/10% amino acid nutrient composition (1% methylene blue/Y%).
  • the amino acid nutrients in the compositions used in study groups 1-14 were: arginine, glycine, cysteine hydrochloride, valine, threonine, proline, histidine hydrochloride, benzene Alanine, lysine, leucine, alanine, glutathione, serine, alanine (aminoacyl)-glutaminamide dipeptide, the study group 15 medication is 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 10 days after the end of the medication, and the tumor weight was measured after anatomy, and the tumor inhibition rate (R) 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 the table below.
  • the amino acid nutrient is preferably selected from the following amino acids and one of the following amino acids Or multiple 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, and glutamic acid.
  • Test 6C The test animal was a BALB/c nude mouse, the modeling cells were human pancreatic cancer PANC-1 cells, and the transplanted tumor was modeled at 1 ⁇ 10 6 cells/only under the skin of the animal's right axillary.
  • Successfully modeled test animals (nude mice bearing pancreatic cancer, with an average tumor volume of 207mm 3 ) were randomly divided into negative control group (group 0), methylene blue single-agent group (group 01), and 8 local synergistic single-agents Groups (groups 1-8) and 8 methylene blue/topical synergistic composition groups (groups 9-16).
  • the negative control is physiological saline
  • the local synergists are as shown in the following table, including: single local synergist (groups 1, 2, 9, 10), acidified nutrients (groups 3, 4, 11, 12), neutral pH Chemical amino acid nutrients (groups 5, 13), nutrients/conventional anti-tumor drug complexes (groups 6, 14), nutrients/immunosuppressive compound (groups 7, 15), immunosuppressant/conventional anti-tumor drug complexes (Group 8, 16).
  • the medicines are all water-containing liquids, which are prepared according to the preparation method of Example 1. Each group was injected intratumorally once, and the injection volume was 170 ⁇ l.
  • the animals were euthanized 7 days after the treatment, and the tumor weight (W) was measured after anatomy, and the tumor inhibition rate (R) was calculated from the negative control group, and the actual/expected ratio (q) was calculated from the tumor inhibition rate.
  • W tumor weight
  • R tumor inhibition rate
  • q actual/expected ratio
  • the tumor weights of 9-16 of each composition group are statistically significant compared with the negative control group (all p ⁇ 0.05), and their actual/expected ratio q are all >1.15, all showing local synergy effect. More specifically, the composition groups 11-16 showed higher efficacy than the composition groups 9 and 10.
  • the mainstream view of anti-tumor drug composition in the prior art is: the probability of synergistic effect between X and a drug Y is very low, while the probability of synergistic effect with drug compound Y/Z is lower, and the compound is on the contrary Increase the risk of drug antagonism. As a result, there is almost no anti-tumor three-component synergistic composition clinically.
  • a preferred technical solution of the topical synergist in the composition of the present invention is: a multi-component complex containing the topical synergist (abbreviated in the present invention) Is a local synergist complex), more preferably the following local synergist complex: homogeneous local synergist complex, different local synergist complex, a single local synergist or a pH adjusting complex of a local synergist complex, wherein :
  • the local synergistic complex of the same type includes multiple local synergists of the same type, including nutrient complexes, immunosuppressant complexes, and conventional anti-tumor drug complexes, wherein the nutrient complexes include complexes of multiple different nutrients Compound (for example, amino acid nutrient/carbohydrate nutrient complex) and a complex of multiple same nutrients (for example, amino acid nutrient complex, carbohydrate nutrient complex), wherein the amino acid nutrient complex includes a compound with acid-basic amino acid nutrient (Eg arginine/acid-resistant complex, glutamate/glycine complex, etc.) and iso-acid-basic amino acid nutrient complex, wherein the iso-acid-basic amino acid nutrient complex is preferably such that The pH of the aqueous liquid tends to be neutralized iso-acid-basic amino acid nutrient complex (e.g.
  • the term "same acid-base complex” refers to a complex of multiple local synergists that are both acidic or the same in a 1% aqueous solution; the term “iso acid-base complex” "Refers to a complex of local synergists of acidity and alkalinity respectively in 1% aqueous solution;
  • the different types of local synergistic complexes include multiple different types of local synergists, including, for example, nutrient/conventional anti-tumor drug complexes (such as arginine/lysine/conventional anti-tumor drugs, arginine/conventional Anti-tumor drugs, etc.), nutrient/immunosuppressant complexes (e.g. arginine/immunoglobulin, arginine/probiotic components, arginine/lysine/RNA, etc.), immunosuppressants/ Conventional anti-tumor drug complexes (such as probiotic components/conventional anti-tumor drugs), etc.
  • nutrient/conventional anti-tumor drug complexes such as arginine/lysine/conventional anti-tumor drugs, arginine/conventional Anti-tumor drugs, etc.
  • nutrient/immunosuppressant complexes e.g
  • the pH-adjusting complex of the local synergist includes the acidification (local synergist/acid) or alkalinization (local synergist/base) complex of the local synergist, preferably so that the local synergist is aqueous liquid
  • the pH change value of is> 1, more preferably a complex (such as arginine/acetic acid complex) that makes the pH of the aqueous liquid of the local synergistic neutralize, wherein the acid in the acidification is preferably an organic acid ( For example, acetic acid), the alkali in the alkalization is preferably a pharmaceutically acceptable pH adjusting alkali (for example, sodium hydroxide, sodium bicarbonate, etc.).
  • 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: A (1% methylene blue/5% DHA/5% acetic acid), B (1% methylene blue/20% arginine), C (1% methylene blue/10% glycine/10% lysine), D (1% methylene blue/30% glucose), E (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.
  • Jinghuai treatment requires precise determination of the relationship between anti-tumor drugs and their indications.
  • the safety dose of methylene blue dye (for example, 1 mg/kg body weight or 80 mg/person in the technical solution for maximum safety) is the decisive factor for the safety dose of the composition containing it. factor.
  • the administration volume (for example, 8 ml of a drug containing 1% (w/v) methylene blue, or 4 ml of a drug containing 2% (w/v) methylene blue) is directly related to the drug effect, which is
  • the technical solution for maximizing the effectiveness is closely related to the characteristics of the tissue within the tumor and the size of the tumor body, for example, it is preferably equal to or greater than the volume of the tumor body to be administered. Therefore, the composition of the present invention is greatly superior to the medicinal effect (also referred to as methylene blue dye drug efficiency in the present invention) provided by a unit methylene blue dye dose (e.g. mg) due to its local synergistic effect
  • methylene blue-containing dye composition of the prior art enables the composition of the present invention to expand new indications.
  • heterogeneity is a core feature of tumors
  • the indications defined by this heterogeneity are then a core feature of tumor therapy drugs. It is generally believed that the heterogeneity of cancer cells is the basis for the indications of anticancer drugs.
  • the following experiments further illustrate the characteristics of the composition of the invention and the indication characteristics corresponding to the characteristics of the composition under completely different conditions of tumor heterogeneity through a comparative study of the composition of the invention and the composition of the prior art.
  • Test 8A The different sharing mechanisms of different topical compositions against diseased tissues of different compositions were studied.
  • the test animals are BALB/c nude mice.
  • the rat benign fibroma tissue fragments (approximately 20 mm 3 /piece) of approximately 300 mm 3 / only under the skin of the animal's right axillary tissue transplantation tumor modeling for series I use.
  • human breast cancer cells (MDA-MB231) were used at 1 ⁇ 10 6 cells/only under the skin of the right axilla of other animals in the same batch to model cell transplantation tumors for use in Series II.
  • the main component of rat breast benign fibroma tissue is fibrous connective tissue.
  • the tumor weights of each series of composition groups 5-7 are statistically significant compared with the negative control group (all p ⁇ 0.05).
  • the actual/expected ratio q of each series of composition groups 5 and 6 are both> 1.15
  • the actual/expected ratio q of each series of composition group 7 are both ⁇ 1.15, indicating that the former pharmaceutical compositions all show local synergy
  • the latter's pharmaceutical composition did not show local synergistic effects.
  • the composition of the present invention can solve the problem that cannot be solved by the methylene blue-containing dye composition of the prior art at the same dose: the effective destruction of tumor tissues rich in non-cancer components.
  • a preferred solution for the indications of the composition of the present invention is the treatment of nodules related to malignant tumors.
  • malignant tumor-related nodules refer to any nodules that should be paid attention to clinically for the treatment of malignant tumors, such as those that have not been detected (for example, undetected due to undetected, misdetected, or missed detection) It is, but cannot be 100% excluded as malignant tumor nodules, or nodules that may develop into malignant tumor tumors.
  • Test 8B The test animals were BALB/c nude mice. According to known techniques, the easy-to-operate human pancreatic cancer tissue is broken into about 50 mm 3 /piece, and then the tissue transplantation tumor is modeled under the skin of the animal's right axillary at a rate of about 300 mm 3 /piece for use in Series II. After the transplantation was successful, human pancreatic cancer cells (PANC-1) were modeled under the skin of the right axillary of the same batch of other animals at 1 ⁇ 10 6 cells/only for use in series I.
  • PANC-1 human pancreatic cancer cells
  • the animals were euthanized 7 days after the treatment, the tumor weight was measured after anatomy, and the tumor inhibition rate (R) was calculated from the negative control group, and the actual/expected ratio (q) was calculated from the tumor inhibition rate.
  • the results of the study group are shown in the table below .
  • the average interstitial ratios of series I and II tumors were 11.3% and 19.7%, respectively.
  • the term "larger interstitial ratio" refers to an interstitial ratio of ⁇ 20%, and an interstitial ratio of ⁇ 20% is referred to as a smaller interstitial ratio.
  • tumor parenchyma such as cancer cells
  • interstitium such as connective tissue, fibrous structure, etc. intercellular substance in non-cancerous cells.
  • the quantity and composition of the stroma is one of the key factors for tumor occurrence and development.
  • Mesenchymal ratio can then characterize this tumor heterogeneity. More and more clinical data show that under roughly the same treatment conditions, the tumor-to-interstitial ratio is an independent factor of tumor treatment efficacy (or prognosis).
  • the heterogeneity based on the interstitial ratio can be so great that the same anti-tumor drug can have very different effects on the same tumor cells but with different interstitial ratios.
  • tumors with different mesenchymal ratios must be regarded as two different tumors at this time.
  • 5-fluorouracil is effective when applied to human pancreatic cancer cell transplantation tumors (usually relatively small in interstitial) in animal experiments (tumor inhibition rate ⁇ 40%), but it is ineffective when applied to clinical human pancreatic pancreatic cancer (usually relatively large in interstitial) (Tumor inhibition rate ⁇ 20%). Therefore, animal human pancreatic cancer cell transplantation tumor is, but clinical human pancreatic pancreatic cancer is not an indication for 5-fluorouracil.
  • the tumor weights of each series of composition groups 5-7 are statistically significant compared with the negative control group (all p ⁇ 0.05).
  • the actual/expected ratio q of each series of composition groups 5 and 6 are both> 1.15
  • the actual/expected ratio q of composition group 7 are both ⁇ 1.15, indicating that the pharmaceutical compositions of the former all show local synergistic effects.
  • the latter pharmaceutical composition did not show local synergy.
  • the methylene blue-containing dye composition of the present invention can solve the problem that cannot be solved by the methylene blue-containing dye composition of the prior art at the same dose: tumor tissue with a larger interstitial ratio Effective destruction.
  • a preferred solution for the indications of the composition of the present invention is the treatment of tumors with larger stromal ratios and tumors containing the tumors.
  • Test 8C The test animals were BALB/c nude mice.
  • the human liver cancer cell MDA-MB231 was modeled under the skin of the animal's right axillary at 1 ⁇ 10 6 cells/only according to known techniques for use in Series II. After the transplantation is successful (the tumor is visible), the hepatoma cell HepG2 is then used at 1 ⁇ 10 6 cells/only under the skin of the right axillary of the same batch of other animals to model the cell transplantation tumor for series I.
  • the tumor weight of each series composition group 9 is not statistically significant compared with the negative control group (all p>0.05), and the tumor weights of each series composition group 8, 10-12 are compared with the negative control group The comparisons are statistically significant (all p ⁇ 0.05).
  • the actual/expected ratio q of each series of composition groups 8 and 11 are both> 1.15, while the actual/expected ratio q of composition groups 10 and 12 is ⁇ 1.15, indicating that the pharmaceutical composition of the former shows that the pharmaceutical composition of the former and the latter The pharmaceutical composition did not show local synergy.
  • the same partial synergistic composition group (groups 8 and 11) showed a higher actual/expected ratio q in series II than in series I.
  • the term "larger tumor” when applied to humans refers to tumors with an average size of ⁇ 2.0cm, while tumors with an average size of ⁇ 2.0cm are called smaller tumors. body.
  • the increase in tumor size is the result of a series of complex living environment evolution that is caused by the proliferation of tumor cells and the growth of non-tumor components in the tumor.
  • tumor cell metabolism requires redistribution of blood vessels, which in turn requires tumor cell proliferation to preferentially proceed on the surface where redistribution is most likely to occur.
  • the enlargement of the tumor also increases the influence of non-tumor components (for example, the inter-fibrous structure) on the growth of the tumor tissue.
  • the average tumor size can then characterize this tumor heterogeneity. More and more clinical data show that under roughly the same treatment conditions, the average tumor size is an independent factor of the therapeutic effect (or prognosis) of local tumor treatment.
  • the heterogeneity based on the average size of the tumor can be so great that the same local drug or physical method can have very different therapeutic effects on tumors of the same tumor cells but of different sizes.
  • tumors of different sizes must be regarded as two different tumors at this time.
  • the classical chemical ablation agent applied to the transplanted tumors of human liver cancer cells (usually small tumors) in animal trials is effective (tumor inhibition rate ⁇ 40%), but it is no longer regarded as clinically larger tumors of human liver cancer (average size). ⁇ 2.0cm) effective drugs. Therefore, the transplanted tumor of animal human liver cancer cells is, but the clinically larger human liver cancer is not an indication for the classic chemical ablation agent.
  • the methylene blue-containing dye composition of the present invention can solve the problem that cannot be solved by the methylene blue-containing dye composition of the prior art at the same dose: effective destruction of tissues in larger tumors.
  • a preferred solution for the indications of the composition of the present invention is the treatment of larger tumor bodies and tumors containing the tumor bodies.

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Abstract

一种包含亚甲蓝类染料作为局部活性成分与其局部协同药物或协同物的组合及其在制备用于治疗癌症如恶性实体肿瘤的局部药物组合物中的应用，以及使用该亚甲蓝类染料作为局部活性成分与其局部协同药物或协同物的组合治疗癌症如恶性实体肿瘤的方法。

Description

包含亚甲蓝类染料的药物组合物及其应用 技术领域
本申请公开涉及包含该亚甲蓝类染料及其局部协同物或协同药物的组合物及其在制备用于癌症的药物组合或制剂中的用途。具体地，本发明涉及优选最小化而非最大化局部作用的亚甲蓝类染料及其局部协同药物或协同物的组合物作为可提供局部协同作用的活性成分在制备可提供所述局部协同作用的用于治疗癌症例如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及其局部协同药物或协同物的可提供局部协同作用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或药物组合或制剂的用于治疗癌症例如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(尤其是现有技术中包含亚甲蓝类染料的药物组合物)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。
在本申请公开的一个方面，提供了包含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物或协同药物的药物组合物，其中所述协同物或协同药物包括或 选自营养素、免疫调节剂、常规抗肿瘤药物中的一种或多种。
在本申请公开的另一个方面，提供了包含亚甲蓝类染料、局部协同物或协同药物、以及任选存在的营养素或/和免疫调节剂的组合，其中所述协同物或协同药物包括或选自常规抗肿瘤药物中的一种或多种，其中在所述组合物中，所述亚甲蓝类染料在施用时的浓度(w/v)为≤2％、优选为0.35-2％、0.5-2％、0.5-1.5％或0.5-1％。在一些实施方式中，所述亚甲蓝类染料在施用时的浓度(w/v)为≤2％，例如，0.35％、0.5％、0.7％、0.9％、1％、1.25％、1.5％、1.8％、2％、或其间的任何范围，优选0.35-2％、0.5-2％、0.5-1.5％、0.7-1.5％、0.7-1.25％或0.5-1％。
在本申请公开的另一个方面，提供了包含亚甲蓝类染料和局部协同物或协同药物的组合物，其中所述协同物或协同药物包括或选自营养素中的一种或多种，以及任选存在的常规抗肿瘤药物或/和免疫调节剂。
在本申请公开的还另一个方面，提供了包含亚甲蓝类染料和局部协同物或协同药物的组合物，其中所述协同物或协同药物包括或选自免疫调节剂中的一种或多种，以及任选存在的营养素或/和常规抗肿瘤药物
在本申请公开的一个方面，提供了包含亚甲蓝类染料和局部协同物或协同药物的组合物，其中所述协同物或协同药物包括或选自营养素，其中在所述组合物中，所述亚甲蓝类染料在施用时的浓度为浓度(w/v)≤2％、优选为0.35-2％、0.5-2％、0.5-1.5％或0.5-1％。
在一些实施方式中，所述亚甲蓝类染料可以包括亚甲蓝及其活体染色类似物，优选地，包括选自以下的化合物及其衍生物：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。
在一些实施方式中，所述亚甲蓝类染料可以选自亚甲蓝及其活体染色类似物，优选地，可以选自亚甲蓝、专利蓝、异硫蓝、新亚甲蓝，更优选地选自亚甲蓝及其衍生物。
在一些实施方式中，所述营养素包括以下的一种或多种：氨基酸类营养素、糖类营养素、脂类营养素。
在一些实施方式中，所述营养素选自氨基酸类营养素、糖类营养素、脂类营养素以下的一种或多种。
在一些实施方式中，上述营养素在施用时的浓度为2.5-50％，优选为4-40％。
在一些实施方式中，所述营养素包括DHA。
在一些实施方式中，氨基酸类营养素包括或选自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。
在一些实施方式中，氨基酸类化合物在施用时的浓度为2.5-50％，优选为4-40％。
在一些实施方式中，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，且在所述组合物中，所述氨基酸或氨基酸盐在施用时的浓度(w/v)为≥2％、≥2.5、≥5％、≥7.5％、10-25％或18-25％，优选为15％-25％或20％-25％。
在一些实施方式中，氨基酸类营养素选自具有营养保健效应的寡肽和多肽，且在所述组合物中，所述寡肽和多肽在施用时的浓度(w/v)为大于≥5％、优选为7.5-25％、更优选为10％-25％。
在一些实施方式中，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，且在药物组合物中，所述组合在施用时的浓度(w/v)为大于≥5％、优选为7.5％-25％、更优所述选为10-25％。
在一些实施方式中，寡肽包括或选自以下的一种或多种：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；所述多肽为选自聚天冬氨酸、聚谷氨酸、聚赖氨酸中的一种或多种。
在一些实施方式中，糖类营养素包括或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。
在一些实施方式中，在所述药物组合中，糖类营养素在施用时的浓度(w/v)为大于5％，优选≥10％、10-40％、15-50％或25-50％。
在一些实施方式中，脂类营养素包括或选自以下的一种或多种：脂肪酸、脂肪乳和类脂，优选包括或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，且在所述组合物中，所述脂类营养素在施用时的浓度≥4％，优选为4％-25％。
在一些实施方式中，本发明的组合物还包括常规抗肿瘤药物，所述抗肿瘤药物包括或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。
在一些实施方式中，常规抗肿瘤药物包括或选自以下的一种或多种：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。
在一些实施方式中，本发明的组合物还包括免疫调节剂，所述免疫调节剂包括或选 自抗体、核酸、益生菌组分中的一种或多种。
在上述组合物中，所述亚甲蓝类染料在施用时的浓度(w/v)≤2％、优选为0.35-2％、0.5-2％、0.5-1.5％或0.5-1％，所述营养素在施用时的浓度(w/v)≥2％、优选为3-40％，以及所述常规抗肿瘤药在施用时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。
在一些实施方式中，本发明包括的免疫调节剂，例如益生菌组分、免疫球蛋白、免疫调节肽。例如，所用益生菌的菌株均选用食品、药品当局批准或官方药典所载的酿酒酵母菌及益生菌制剂中所用的菌株。例如以下中国药品当局批准的益生菌制剂所含菌株：凝结芽抱杆菌活菌片中的酪酸梭菌株、地衣芽抱杆菌活菌颗粒(胶囊)中的地衣芽抱杆菌株、口服蜡样芽抱杆菌活菌制剂中的蜡样芽抱杆菌株、酪酸梭菌活菌胶囊中的酪酸梭菌株、布拉氏酵母菌散(胶囊)中的布拉氏酵母菌株、嗜酸乳杆菌胶囊中的嗜酸乳杆菌株、口服酪酸梭菌活菌散剂中的酪酸梭菌株、枯草杆菌肠球菌二联活菌多维颗粒剂中的屎肠球菌株、枯草杆菌株、酪酸梭肠球菌三联活菌片中的乳酸菌株、酪酸梭菌株、糖化菌株、***用乳杆菌活菌胶囊中的德氏乳杆菌株、双歧杆菌活菌散中的青春型双歧杆菌株、双歧杆菌三联活菌胶囊中的长双歧杆菌活菌株、嗜酸乳杆菌株、粪肠球菌株，等等。
在一些实施方式中，本发明的组合物任选地还包含以下的一种或多种：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。
在一些实施方式中，所述止痛剂为包括或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，且在所述组合物中，所述止痛剂在施用时的浓度为0.1-4重量％。
在一些实施方式中，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。
在一些实施方式中，本发明的组合物还包括药物学可接受的载体，例如水性载体或醇类载体，优选地为水和/或乙醇。在一些实施方式中，本发明的组合物可以被制备为注射剂的剂型，所述注射剂包括液体注射剂和注射用粉针剂。
在一些实施方式中，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。
在一些实施方式中，其中所述亚甲蓝类染料与营养素、免疫调节剂、抗肿瘤药物的量比选自以下组之一种或多种：所述营养素/亚甲蓝类染料组合物的局部协同量比(W 营 养素/W 亚甲蓝类染料)为1/3＜W 营养素/W 亚甲蓝类染料＜40/0.15，优选为1/3＜W 营养素/W 亚甲蓝类染料≤40/0.30、或1/1.8＜W 营养素/W 亚甲蓝类染料≤40/0.30；所述免疫调节剂/亚甲蓝类染料组合物的局部协同量比(W 免疫调节剂/W 亚甲蓝类染料)为0.2/3＜W 免疫调节剂/W 亚甲蓝类染料≤20/0.15，优选为0.5/3＜W 免疫调节剂/W 亚甲蓝类染料≤20/0.50、或0.5/1≤W 免疫调节剂/W 亚甲蓝类染料≤20/0.50；所述常规抗肿瘤药物/亚甲蓝类染料组合物的局部协同量比(W 常规抗肿瘤药物/W 亚甲蓝类染料)为0.1/3＜W 常规抗肿瘤药物/W 亚甲蓝类染料＜1.5/0.15，优选为0.3/3＜W 常规抗肿瘤药物/W 亚甲蓝类染料＜1.5/0.15、或0.3/1.8≤W 常规抗肿瘤药物/W 亚甲蓝类 染料≤1.5/0.30。
在本申请的一个方面，提供包含根据上述组合物的医疗装置。
在本申请公开的一个方面，提供了上述组合物与抗肿瘤药物或免疫调节剂在制备用于治疗癌症的药物组合或制剂中的应用。
在一些实施方式中，所述癌症包括实体肿瘤，例如肉瘤、头颈癌、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、卵巢癌等等。
在一些实施方式中，本发明的组合物可以用于局部施用，例如，局部瘤内施用。
在本申请公开的另一个方面，提供了治疗癌症的方法，包括给患有癌症的患者施用上述组合物或其与抗肿瘤药物或免疫调节剂的药物组合或制剂。
在本申请公开的另一个方面，提供了治疗癌症的方法，包括给患有癌症的患者瘤内施用上述组合物或其与抗肿瘤药物或免疫调节剂的药物组合或制剂。
在一些实施方式中，所述癌症包括实体肿瘤，例如肉瘤、头颈癌、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、卵巢癌等等。在一些实施方式中，所述实体肿瘤优选为选自含间质比≥20％或/和平均尺寸≥2.0cm的瘤体的恶性实体肿瘤。
根据本申请公开的一个方面，提供了优选为最小化而非最大化局部作用的亚甲蓝类染料与所述亚甲蓝类染料的局部协同物或协同药物的组合作为可提供局部协同作用的活性成分在制备可提供所述局部协同作用的用于治疗恶性实体肿瘤的局部药物组合物中的应用，所述亚甲蓝类染料作为局部活性成分，所述局部协同物或协同药物选自以下组之一种或多种：营养素、免疫调节剂、常规抗肿瘤药。
根据本申请公开的另一方面，提供了可提供所述局部协同作用的用于治疗恶性实体肿瘤的局部药物组合物，其包含优选为最小化而非最大化局部作用的亚甲蓝类染料和所述亚甲蓝类染料的局部协同物或协同药物，其中所述亚甲蓝类染料作为局部活性成分，所述协同物或协同药物选自以下组之一种或多种：营养素、免疫调节剂、常规抗肿瘤药。
在一些实施方式中，其中所述亚甲蓝类染料与营养素、免疫调节剂、抗肿瘤药物的量比选自以下组之一种或多种：所述营养素/亚甲蓝类染料组合物的局部协同量比(W 营养 素/W 亚甲蓝类染料)为1/3＜W 营养素/W 亚甲蓝类染料＜40/0.15，优选为1/3＜W 营养素/W 亚甲蓝类染料≤40/0.30、或1/1.8＜W 营养素/W 亚甲蓝类染料≤40/0.30；所述免疫调节剂/亚甲蓝类染料组合物的局部协同量比(W 免疫调节剂/W 亚甲蓝类染料)为0.2/3＜W 免疫调节剂/W 亚甲蓝类染料≤20/0.15，优选为0.5/3＜W 免疫调节剂/W 亚 甲蓝类染料≤20/0.50、或0.5/1≤W 免疫调节剂/W 亚甲蓝类染料≤20/0.50；所述常规抗肿瘤药物/亚甲蓝类染料组合物的局部协同量比(W 常规抗肿瘤药物/W 亚甲蓝类染料)为0.1/3＜W 常规抗肿瘤药物/W 亚甲蓝类染料 ＜1.5/0.15，优选为0.3/3＜W 常规抗肿瘤药物/W 亚甲蓝类染料＜1.5/0.15、或0.3/1.8≤W 常规抗肿瘤药物/W 亚甲蓝类 染料≤1.5/0.30。
在一些实施方式中，上述局部药物组合物在施用时还包含药物学可接受的液体载体，以及在该局部药物组合物的给药组成中，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％，所述免疫调节剂的浓度(w/v)为大于0.25％、优选为0.5％-30％，以及所述常规抗肿瘤药的浓度(w/v)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。
在本申请公开的另一方面，提供了用于治疗恶性实体肿瘤(优选为可破坏瘤体组织)的局部药物组合物，其包含(优选为最小化而非最大化局部作用的)亚甲蓝类染料、所述亚甲蓝类染料的局部协同物或协同药物、以及药物学可接受的液体载体，其中所述协同物或协同药物包括或选自选自以下的一种或多种：营养素、免疫调节剂、常规抗肿瘤药，且在该局部药物组合物的给药组成中，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％，所述免疫调节剂的浓度(w/v)为大于0.25％、优选为0.5％-30％，以及所述常规抗肿瘤药的浓度(w/v)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。
在一些实施方式中，上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，其中所述所述亚甲蓝类染料的浓度(w/v)为＜1.1％、优选为0.35-1.09％、0.5-1.09％或0.5-1.05％。
在一些实施方式中，上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，且其中所述营养素的浓度为大于2.5％至小于50％，优选为4-40％。
在一些实施方式中，上述免疫调节剂为选自以下组之一种或多种：抗体、核酸、益生菌组分，且其中所述免疫调节剂的浓度(w/v)为大于0.25％、优选为0.5％-30％。
在一些实施方式中，上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。
在一些实施方式中，上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。
在本申请公开的另一方面，提供了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上述药物组合物之一部或全部得到的干粉。
在一些实施方式中，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。
在一些实施方式中，所述恶性实体肿瘤优选为选自含间质比≥20％或/和平均尺寸≥2.0cm的瘤体的恶性实体肿瘤。
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或协同物、以及药物学可接受的液体载体，其中所述协同药物或协同物选自营养素或/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或协同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，其中所述协同药物或协同物选自营养素或/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或协同物、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或协同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。
根据本申请公开的包含所述亚甲蓝类染料的组合物(在本发明中也简称为本发明组合物)与现有技术中的包含亚甲蓝类染料的组合物(在本发明中也简称为现有技术组合物)相比具有以下优点：1)、共用药理更优化：前者优先利用组合物的有效局部协同作用、而后者优先利用亚甲蓝类染料的局部作用及添加剂对该局部作用的相加增效作用；2)、实现共用药理的有效技术方案更优化：前者仅需优选为最小化而非最大化局部作用的亚甲蓝类染料，而后者则需优选为最大化而非最小化局部作用的亚甲蓝类染料，具体体现为两种相反的优选给药浓度取向上。此外，前者所需的局部协同药物的副作用不强于(就选自常规抗肿瘤药而言)、甚至于更弱于(就选自营养素或/和免疫调节剂者而言)后者中任选存在的局部作用增强剂；3)、共用药效更高：单位亚甲蓝类染料在前者中的应用效率超过其在后者中的预期；4)、适应症范围更广：在安全性和有效性最大化的技术方案中，前者能够在后者所不能的以下适应症之一种或多种中优先应用：可局部给药的较大间质比瘤体、以及包含该瘤体的肿瘤的治疗；可局部给药的较大瘤体、以及包含该瘤体的肿瘤的治疗；可局部给药的恶性肿瘤相关结节的治疗。
根据本发明的实施方案与恶性实体肿瘤治疗的其它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。
在本申请公开的范围中，术语“药物组合物”是指明确了药理以及给出了在患者体内实现该药理所必须的药理方法、药理组成、药理环境等药学特征的物质。术语“药理方法”是指某特定药理的实现所必须的药物组合物给药方法，例如益生菌肠道屏障改善必须的药理方法是口服，而乙醇化学消融必须的药理方法是病变区内给药。术语“药理组成”是指某特定药理的实现所必须的药物组合物组成，不同的药理往往可能有非常不同的药理组成，例如常规协同作用仅要求活性成分协同量比，而局部常规协同作用还要求协同浓度等协同组成。术语“药理环境”是指药物组合物在靶区内实现某特定药理所必须的外源性干扰的最小化，例如其它原因所须的添加剂(例如渗透压调节剂)可能对该特定药理产生的负面作用。无特定药理环境限定的药物组合物通常必然包含有增强给药顺应性(例如口服药的调味剂)和给药安全性(例如注射剂所包含的渗透压调节剂)的非活性成分，而局部作用药理则必须要求限定药理环境以减少非活性成分的可能的药理干扰。无特定药理方法限定(例如可静注、肌注或其它局部注射)的药物组合物的药理意味着在这些方法中都可以实现者(例如益生菌注射剂的免疫增强、细胞毒药物的细胞增长抑制、乙醇的醉酒样反应)，有特定药理方法限定的药物组合物的药理则意味着仅在该方法中可以实现者(例如口服益生菌的肠道屏障改善、局部给药乙醇的化学消融)。无特定药理组成限定(例如给出制剂浓度而无药理浓度，或/和给出给药剂量而无药理体积)的药物组合物的药理意味着在给定的活性成分、活性成分量比、和组合物剂量后都可以实现者(例如益生菌注射剂的免疫增强、细胞毒药物的细胞增长抑制、乙醇的醉酒样反应)，有特定药理组成限定的药物组合物的药理则 意味着仅在该组成条件下可以实现者，例如乙醇在其药理方法(病变内给药)、药理浓度(例如≥70％的高浓度)、药理体积(例如给药体积/靶区体积比≥0.25)限定下的药理为化学消融。
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(或全身性给药，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)，而前者则是指身体局部组织内(例如瘤内)给药、并主要通过其局部活性产生药效的治疗药物(组合物)。术语“局部活性”是指优先在局部组织内而非肿瘤细胞内显示的药学活性，其包括例如单一药物的局部作用和共用药物的局部共用作用。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)，而前者则是指亚非通过其常规作用(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0.5％-1.5％的所述亚甲蓝类染料)共用产生局部协同作用的物质，其选自以下组之一种或多种：营养素、免疫调节剂、常规抗肿瘤药物。术语“局部协同作用”是指主要显示为局部活性的协同作用。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、或本发明实施例中约定的实际/预期比q＞1.15的药效)。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或协同物、以及药物学可接受的液体载体，其中所述协同药物或协同物选自营养素或/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或协同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，其中所述协同药物或协同物选自营养素或/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或协同物、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或协同物的药物组合物。
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，现有技术中亚甲蓝类染料的一种新应用涉及局部给药。该应用中的亚甲蓝类染料为获得有效(通常为最大化)药效，在药理上必需最大化而非最小化局部作用，从而在组成上必然是一个最大化的优选给药浓度取向。在现有技术中，为使得该亚甲蓝类染料的最大化局部作用有更高药效，还可以任选地加入某些增效剂，它们涉及全身性给药有效的有效抗肿瘤物质、全身给药低效但局部给药高效的低效抗肿瘤物质、以及金属化合物。高浓度(例如5％)亚甲蓝类染料在该技术方案中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、尤其是仅提供优选最小化而非最大化局部作用的条件(例如1％亚甲蓝给药浓度)时，其可以与药理大不相同的营养素、免疫调节剂、和/或常规抗肿瘤药形成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、免疫调节剂、和/或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，以保证其被作为可提供局部协同作用的局部活性成分应用，而非作为主要提供最大化局部作用的化学消融剂应用，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(或提供优选最小化而非最大化局部作用的)亚甲蓝类染料在治疗恶性实体肿瘤的局部协同药物或协同物，可以是选自以下组之一种或多种：营养素、免疫调节剂、常规抗肿瘤药。
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，所述局部协同药物或协同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，所述局部协同药物或协同物包括糖类营养素，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，所述局部协同药物或协同物包括脂类营养素，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“免疫调节剂”是指在临床上未被用作疫苗佐剂而可用作免疫调节食物或免疫调节药物、但其分子结构可与本发明中的优选最小化而非最大化局部作用的亚甲蓝类染料共用并提供局部协同作用的物质。因而，本发明中的免疫调节剂与现有技术中的免疫佐剂具有本质的不同：1).用于组合物的目的不同：后者的目的为促进患病区内相关的有益于***的抗原的活动，而前者的目的是提供局部协同作用；2).实现各自目的的药理方案不同：后者仅涉及免疫增强，而前者必须涉及化学增 強(局部协同作用)；3).实现药理(效)方案的组分(构)方案不同：后者选自通常用作疫苗佐剂的以下组之一种或多种：无机佐剂、(免疫原性较强的)微生物及其产物、合成佐剂、油剂、细胞因子、异种抗原，而前者选自通常不用作疫苗佐剂而用作免疫调节食物的细菌免疫原性最小化的益生菌组分或/和免疫调节肽、以及通常用作完全不同于抗原增强作用的疫苗佐剂的免疫调节剂的免疫球蛋白；4).超现有技术预期的技术效果：后者的技术效果主要为有益于抗肿瘤抗原的免疫增强、但通常并无组织破坏作用，而前者的必须技术效果为可致组织有效破坏的局部协同作用，这主要是不依赖于免疫作用的化学作用。在以下实施例中，该局部协同作用的实现不依赖于动物免疫功能，其甚至于可在免疫严重缺陷的裸鼠中实现。
在本申请公开中，术语“益生菌”(probiotics)是指能够对宿主健康产生有益作用的非致病性活微生物，益生菌包含细胞，酵母更是单细胞微生物。术语“益生菌组分”是指源于天然益生菌或其工程菌的制备物(例如胞壁聚多糖)或该制备物的工程类似物(例如类似于胞壁聚多糖的合成聚多糖或其它来源聚多糖)。在一个实施方案中，所述益生菌组分优选为选自细菌免疫原性最小化者，更优选为选自包括以下组及其工程类似物之一种或多种：益生菌水溶性组分、益生菌组分水不溶组分、灭活益生菌。
在本申请公开中，术语“细菌免疫原性”是指细菌作为一个完整的外来物体在受者体内产生免疫响应的能力，不同的细菌具有不同的细菌免疫原性。活益生菌具有最强的细菌免疫原性，但直接进入体内也有很强的安全性风险。术语“灭活益生菌”是指经细菌灭活工程处理后获得的制备物，其中所述细菌灭活例如以下之一种或多种：高温灭活、高温高压灭活、紫外线灭活、化学试剂灭活、辐射灭活。术语“益生菌水不溶组分”是指自益生菌获得的水溶度为＜0.1％的任意组分(例如破碎益生菌沉淀、水不溶性益生菌细胞壁组分、水不溶性聚多糖、水不溶性β-葡聚糖、等等)。术语“益生菌水溶性组分”是指自益生菌获得的水溶度≥0.1％的任意组分(例如益生菌水溶性抽提物、水溶性聚多糖、水溶性β-葡聚糖、等等)。
在一个实施方案中，当所述益生菌组分包括所述灭活益生菌，则所述灭活益生菌在该药物组合物中的给药浓度为＞0.3％、≥0.75％、0.75-15、优选为1.5-15％或5-15％；当所述益生菌组分包括所述益生菌水溶性组分，则所述益生菌水溶性组分在该药物组合物中的给药浓度为＞0.25、或0.35-25％、优选为0.35-15％或5-15％；当所述益生菌组分包括所述益生菌水不溶组分，则所述益生菌水不溶组分颗粒在该药物组合物中的给药浓度为＞0.5、或0.5-15％、优选为1.5-15％或5-15％。
在一个实施方案中，所述益生菌选自包括以下组天然菌或工程菌之一种或多种：益生芽抱杆菌、益生乳酸杆菌、益生双歧杆菌、益生真菌。
在一个实施方案中，所述益生菌包括选自益生芽抱杆菌之一种或多种。在一个实施方案中，所述益生菌包括选自益生乳酸杆菌之一种或多种。在一个实施方案中，所述益生菌包括选自益生双歧杆菌之一种或多种。在一个实施方案中，所述益生菌包括选自益 生真菌之一种或多种。
在一个实施方案中，所述芽抱杆菌选自包括以下之一种或多种：有蜡样芽抱杆菌、地衣芽抱杆菌、枯草芽抱杆菌、巨大芽抱杆菌、坚强芽抱杆菌、凝结芽抱杆菌、缓慢芽抱杆菌、短小芽抱杆菌、纳豆芽抱杆菌。在一个实施方案中，所述芽抱杆菌优选为选自包括以下之一种或多种：地衣芽抱杆菌、枯草芽抱杆菌、短小芽抱杆菌。
在一个实施方案中，所述乳酸菌包括选自乳酸杆菌或/和双歧杆菌之一种或多种。在一个实施方案中，所述乳酸杆菌包括选自包括以下之一种或多种：嗜酸乳杆菌、唾液乳杆菌、干酪乳杆菌、植物乳杆菌、短小乳杆菌和发酵乳杆菌。在一个实施方案中，所述乳酸杆菌优选为选自包括以下之一种或多种：干酪乳杆菌、植物乳杆菌、短小乳杆菌和发酵乳杆菌。在一个实施方案中，所述双歧杆菌包括选自包括以下之一种或多种：长双歧杆菌、青春双歧杆菌、短双歧杆菌、婴儿双歧杆菌、布魯氐乳杆菌、瑞士乳杆菌、嗜热乳杆菌、屎肠球菌、粪链球菌。在一个实施方案中，所述双歧杆菌优选为选自包括以下之一种或多种：长双歧杆菌、青春双歧杆菌、屎肠球菌、粪链球菌。
在一个实施方案中，所述真菌包括选自酵母菌或/和布魯氐酵母菌之一种或多种，其中所述酵母菌包括选自包括以下之一种或多种：酿酒酵母菌、德尔布有抱圆酵母菌、假丝酵母菌、威克汉姆酵母菌、毕赤酵母菌、白球拟酵母菌、薛瓦酵母菌、深红酵母菌、粟酒裂殖酵母菌、鲍氏酵母菌、产阮假丝酵母。在一个实施方案中，所述益生菌优选为选自酵母菌或/和布魯氐酵母菌之一种或多种。
在一个实施方案中，所述益生菌优选为选自不含分枝菌酸细胞壁的益生菌。在一个实施方案中，所述益生菌优选为选自布魯氐酵母菌。在一个实施方案中，所述益生菌优选为选自酿酒酵母菌。
在本申请公开中，所用术语“免疫球蛋白”是一个结构概念，指所有与抗体分子相同或相似的药物，其包括天然免疫球蛋白及其工程类似物。其中所述天然免疫球蛋白包括免疫球蛋白G、免疫球蛋白A、免疫球蛋白M、免疫球蛋白D、免疫球蛋白E，通常从动物血漿的分离获得，例如静注免疫球蛋白G、静注免疫球蛋白A、静注免疫球蛋白M、等等。其中所述天然免疫球蛋白的工程类似物是指不同于天然免疫球蛋白、但具有天然免疫球蛋白的全部或部分关健结构的工程制品(包括基因工程制品)，例如免疫球蛋白复合物、免疫球蛋白Fc片断、免疫球蛋白Fab片断、免疫球蛋白Fab’片断、免疫球蛋白F(ab’)片断、单健免疫球蛋白、等等。在一个实施方案中，所述免疫球蛋白在该药物组合物中的给药浓度(w/v)为＞1.5％、≥2.0、2.0-35％，优选为3.0-30％。
在本申请公开中，所用术语“免疫调节肽”是指源自生物体或食物蛋白的、具有免疫调节作用的非蛋白质多肽，例如本发明实施例中使用的CAS号为63231-63-0的核糖核酸。在一个实施方案中，所述免疫调节肽在该药物组合物中的给药浓度(w/v)为＞1.5％、≥2.0、2.0-35％，优选为3.0-30％。
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、所述局部协同药物或协同物、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、所述局部协同药物或协同物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、所述局部协同药物或协同物)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、所述局部协同药物或协同物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、所述局部协同药物或协同物的无菌干粉；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、所述局部协同药物或协同物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所述局部协同药物或协同物、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。
根据本发明的一个实施方案，本发明的组合物对恶性实体肿瘤的治疗优选为包括瘤体治疗。根据本发明的一个实施方案，本发明的组合物对恶性实体肿瘤的瘤体治疗优选为包括瘤体或瘤体相关结节内的非恶性肿瘤细胞成分、尤其是***的有效破坏。
在本发明的范围内，术语“治疗”是指任何合理且有利的医学处理，其包括诊断后治疗(例如诊断后进行的医学处理)和预防性治疗(例如不必确诊即可进行的医学处理)。术语“治疗恶性实体肿瘤”或“恶性实体肿瘤的治疗”是指针对经诊断为、或有可能为恶性实体肿瘤的患者的治疗，包括瘤体治疗和其它包含肿瘤细胞的组织(例如淋巴组织)的治疗。术语“瘤体治疗”是指针对瘤体或瘤体相关结节的治疗。
根据本发明的一个实施方案，本发明的组合物的适应症的优选方案为以下之一种或多种：可局部给药的较大间质比瘤体、以及包含该瘤体的肿瘤的治疗；可局部给药的较大瘤体、以及包含该瘤体的肿瘤的治疗；可局部给药的恶性肿瘤相关结节的治疗。
在本发明的范围内，术语“瘤体”是指由于细胞或变异的细胞异常增殖形成的肿块。术语“实体肿瘤”(有时亦简记为肿瘤)是指可出现瘤体的疾病。术语“恶性实体肿瘤”(有时亦简记为恶性肿瘤)是指可出现瘤体的恶性疾病。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瘤体或瘤体相关结节内的非恶性肿瘤细胞成分(尤其是***)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。
以下具体实施例中所用的免疫调节剂(益生菌组分、免疫球蛋白、免疫调节肽)，均可从商业途径得到。例如，所用益生菌的菌株均选用食品、药品当局批准或官方药典所载的酿酒酵母菌及益生菌制剂中所用的菌株。例如以下中国药品当局批准的益生菌制剂所含菌株：凝结芽抱杆菌活菌片中的酪酸梭菌株，地衣芽抱杆菌活菌颗粒(胶囊)中的地衣芽抱杆菌株，口服蜡样芽抱杆菌活菌制剂中的蜡样芽抱杆菌株，酪酸梭菌活菌胶囊中的酪酸梭菌株，布拉氏酵母菌散(胶囊)中的布拉氏酵母菌株，嗜酸乳杆菌胶囊中的嗜酸乳杆菌株，口服酪酸梭菌活菌散剂中的酪酸梭菌株，枯草杆菌肠球菌二联活菌多维颗粒剂中的屎肠球菌株、枯草杆菌株，酪酸梭肠球菌三联活菌片中的乳酸菌株、酪酸梭菌株、糖化菌株，***用乳杆菌活菌胶囊中的德氏乳杆菌株，双歧杆菌活菌散中的青春型双歧杆菌株，双歧杆菌三联活菌胶囊中的长双歧杆菌活菌株、嗜酸乳杆菌株、粪肠球菌株，等等。
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 PCTCN2021076749-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裸小鼠或小鼠。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 50-700mm 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、瘤重等。
在以下实施例中，肿瘤体积(V)、相对肿瘤增殖率(R)、抑瘤率(r)的计算公式分别如下：
肿瘤体积V＝l/2×a×b 2，其中a表示肿瘤长，b表示肿瘤宽。
相对肿瘤增殖率r(％)＝T RTV/C RTV×100，其中T RTV和C RTV分别为研究组和阴性对照组的相对肿瘤体积，相对肿瘤体积＝V t/V 0，其中V 0为分组给药当天(即第一天)测得的平均肿瘤体积；V t为分组给药后第t天测得的平均肿瘤体积。
抑瘤率R(％)＝(CW-TW)/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，药物i的药效记为E i，其中E i可以为(100-r i)％或R i％。在本发明的范围內，A药和B药的组合物记为B/A。A、B的单用药效分别记为E A和E B，A/B的实际共用药效记为E A+B。
在以下实施例中，试验结果(例如瘤重、瘤体积)采用均数±标准差(x±s)表示，两个试验动物组与组均数之间的差别采用统计学软件SPSS 13.0或SPSS 19.0软件进行显著性检验来比较，检验选用统计量t来进行，检验水准α＝0.05，P＜0.05表示差异有统计学意义，P＞0.05则无统计学意义。
在以下实施例中，药物作用类型(共用药理)通过药物效应、尤其是比较同一研究药物在不同技术方案中的药物效应来进行研究。例如，当药物i在方案X和Y之间的药物效应差异并非不寻常地大(例如E iX/E iY＜150％)时，其很可能是基本相同的药物作用类型(药理)中不同动力学条件(作用浓度)所致；而当该药物效应差异超乎寻常地大(例如E iX/E iY＞150％)时，药物i在方案X中的药物效应之大应当超过了其在方案Y中的药物作用类型(药理)的动力学预期范围，从而很可能涉及与方案Y不同的药物作用类型(药理)。当两个药物显示出显然不同的E iX/E iY关系，则它们很可能涉及显然不同的药理；而当两个药物显示出相似的E iX/E iY关系，则它们很可能涉及相同的药理、至少涉及相似的药理(例如类化学消融药理和化学消融药理)。
在以下实施例中，化疗阳性对照物包括经典细胞毒药物(例如0.5-1％5-氟尿嘧啶，其在以下实施例条件下的抑瘤率为≥30％)和经典化学消融剂(例如75-99％乙醇，其在以下实施例局部给药条件下的抑瘤率为≥30％)。
药物联合使用产生的作用具有高度的不确定性，业内往往依据以下实际/预期比q判断进行：
q＝实际共用作用/理论单纯相加预期作用。
q计算式中理论单纯相加预期药效的计算方法很多，大多针对细胞试验效应。通常认为，当实际/预期比q＝1时，实际共用作用符合理论预期，显示为相加作用；当实际/预期比q＜1时，实际共用作用弱于理论预期，显示为拮抗作用；当实际/预期比q＞1时，实 际共用作用超理论预期，显示为协同作用。
一种判断动物试验中药物共用效应的方法是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，等概率和曲线和“Q50”，上海第二医学院学报；1981，1，75-86)，其q计算式为：
q＝E A/B/(E A+E B-E A·E B)，
其中：E A/B为组合物A/B的实际共用药效(例如抑瘤率)，E A和E B分别为组合物A/B的组分A和组分B的单药实际药效，(E A+E B-E A·E B)为组分A和组分B的根据其单药实际药效的理论单纯相加预期效应。
通常认为q＝1.00反映单纯相加预期作用。
在上述金正均法的q计算基础上，张效文、金正均等进一步使q判断更适合试验实际(张效文等，用Q值估计合并用药效果的新方法：“双30法”，上海第二医学院学报，1985，5，p353-354)。他们以q＝0.85-1.15反映单纯相加预期作用。在本发明以下实施例中，根据上述金正均改良法中的q计算式计算实际/预期比q，以及根据上述张效文-金正均法进行联合给药的共用作用判断如下：
当组合物A/B组并不显示有治疗意义的药效(例如抑瘤率＜30％)时，A和B联合给药也就未显示有治疗意义的共用作用，在本发明中视作无共同作用。当组合物组显示有治疗意义的药效(例如抑瘤率≥30％)时，如果实际/预期比q在0.85与1.15之间则该组合物的共用作用为相加作用(实际作用符合理论单纯相加预期)；如果实际/预期比q＞1.15则该组合物的共用作用为明显协同作用(实际作用超理论单纯相加预期)；如果实际/预期比q＜0.85则该组合物的共用作用为明显拮抗作用(实际作用不及理论单纯相加预期)。
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(X％/Y％)的组成列于下表。
表2
Figure PCTCN2021076749-appb-000002
Figure PCTCN2021076749-appb-000003
以下列出本发明的组合物的制备试验的几个例子。
试验1A：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)、局部协同物(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。
试验1B：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)、局部协同物(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)量取酸化剂或碱化剂(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。
试验1C：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)、局部协同物(例如20g精氨酸、1g5-氟尿嘧啶)、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/5-氟尿嘧啶水溶液)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
营养素注射在肿瘤治疗中的意义一直存在争议，值得研究。在我们的一个试验中，将DHA(5％)加入少量亚甲蓝(0.5％)注射到瘤内，其中的DHA被用作营养剂，而局部作用最小化的亚甲蓝用作瘤内给药分布的染色指示剂。明显看到100ul该营养液分布在约200mm 3的整个瘤内。通常认为，瘤体的超正常生长需要更多的营养支持，例如胰腺癌瘤体生长对半胱氨酸的更多需求。然而，接下来却意外地观察到瘤体并非出现增长而是明显缩小。该发现打破了若干固有观念的束缚。在接下来的一系列试验中，进一步研究了亚甲蓝类染料、尤其是局部作用最小化的亚甲蓝类染料与常规活性大不相同的物质的功能组合，并获得本发明的基本技术方案。
试验2A：试验动物为小鼠，建模细胞为肉瘤S180细胞，以1×10 6个细胞/只在动 物右侧腋部皮下进行移植瘤建模。成功建模的试验动物(荷载恶性肉瘤的小鼠，瘤体平均体积121mm 3)随机分为2个系列(系列I和II)，每个系列各分为1个阴性对照组(I0和II0)和11个研究组(I1-I11和II1-II11)。阴性对照物为生理盐水，11个研究药物如下表所示，系列I行全身性给药(腹腔注射)和系列II行局部给药(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(W)，并从各用药方式的阴性对照组计算抑瘤率(R)，结果示于下表。
表3
Figure PCTCN2021076749-appb-000004
通常认为，同一组合物在B技术方案中的抗肿瘤药效超过A方案中的药效动力学提高预期极限(一般为＜150％，或从无作用变为有效作用，或瘤重差异有统计学意义)，就很可能是不同药理的药物效应。
在上表中，阳性对照物(5-氟尿嘧啶)的腹腔注射组(组I1)和瘤内注射组(组II-1)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全身性给药而非局部给药。
在本试验中，所用亚甲蓝低于其临床注射剂的浓度(≤1％)，被认为是即使静注也不 会产生不良局部作用的亚甲蓝。在上表中，研究组I2的瘤重与阴性对照组I0之间的差异无统计学意义(P＞0.05)，且其抑瘤率在可忽略不计的低水平，说明该亚甲蓝类染料的常规抗肿瘤作用并不能产生瘤体生长抑制作用。研究组II2的结果与瘤研究组I2一致，说明该亚甲蓝类染料只提供了最小化而非最大化的局部作用。
对于与本试验中的普通动物一致的患者(并不特别缺乏所给药营养素的患者)而言，临床上并不推荐提供营养素。向瘤内肿瘤直接提供营养素似乎更加提高了肿瘤组织生长风险。在本试验中，在对动物进行安乐死前，系列I和II中各组动物的体重均未观察到异常，说明本试验中低频次给药并未显示出营养作用的影响，无论是正影响还是副影响。
系列I中各营养素单药组(1-4)的瘤重与其阴性对照组之间的差异均无统计学意义(均为P＞0.05)，且它们的抑瘤率均在可忽略不计的低水平，说明营养素所提供的营养作用或常规抗肿瘤作用(假如有的话)并不能产生瘤体生长抑制药效。系列II中的各营养素单药组(1-4)与其阴性对照组之间的瘤重差异也都无统计学意义(均为P＞0.05)，且它们的抑瘤率仍然不高，说明局部给药营养素仅可产生全身性给药营养素所不能产生的弱局部作用。
系列I中各亚甲蓝类染料/营养素组合物组(6-8)的药效与同系列中亚甲蓝类染料单药组5和相应的营养素单药组(1-4)完全一致，说明该亚甲蓝类染料和该共用物(营养素)的常规组合(无论是营养作用与常规抗肿瘤作用的组合、还是常规抗肿瘤作用之间的组合)并不能产生瘤体生长抑制作用，更说不上有效的协同作用。然而，系列II组合物组6-8与其系列I中的相应组6-8比较，瘤重差异均有统计学意义(p＜0.05)，抑瘤率差异均在10倍以上，说明系列II中的局部药物组合物通过不同于系列I中的常规药物组合物的新药理实现了有效瘤体治疗。与其相应组成单药组相比较，系列II组合物组6-8的抑瘤率也超出理论单纯相加效应的预期范围，其实际/预期比(q)均大于1.15，显示出上述新药理应为该局部协同药理。
系列I中的组合物组9与单药组1相比较并无抑瘤率提高，说明亚甲蓝类染料和常规抗肿瘤药在该常规组合物中的任何常规组合(例如常规抗肿瘤作用的组合)都不能够实现抑瘤增效。然而，组II9比组I9的抑瘤率高出150％以上，说明系列II中的局部药物组合物通过不同于系列I中的常规药物组合物的新药理实现了有效瘤体治疗。与系列II中相应组成单药组1、5相比较，组合物组9的抑瘤率也超出该两药物理论单纯相加效应的预期范围，其实际/预期比(q)大于1.15，显示出上述新药理应为该局部协同药理。
系列I中的研究组11与10相比较并无抑瘤率提高，说明该亚甲蓝类染料和营养素/常规抗肿瘤药复合物在该常规组合物中的任何常规组合(例如常规抗肿瘤作用的组合)都不能够实现抑瘤增效。然而，组II11比组I11的抑瘤率高出150％以上，说明系列II中的局部药物组合物通过不同于系列I中的常规药物组合物的新药理实现了有效瘤体治疗。与系列II中相应组成单药组10、5相比较，组合物组11的抑瘤率也超出该两药 物理论单纯相加效应的预期范围，其实际/预期比(q)大于1.15，显示出上述新药理应为该局部协同药理。
以上结果、尤其是上述亚甲蓝类染料与营养素的非常规功能组合的新药理(局部协同药理)的发现，使得我们进一步解放思想。以下试验研究了亚甲蓝类染料与免疫调节剂的非常规功能组合。
试验2B：试验动物为裸小鼠，建模细胞为人胰腺癌细胞(PANC-1)，以1×10 6个细胞/只在动物右侧腋部皮下进行移植瘤建模。成功建模的试验动物(荷载胰腺癌裸小鼠，瘤体平均体积147mm 3)随机分为2个系列(系列I和II)，每个系列各分为1个阴性对照组(I0和II0)和11个研究组(I1-I11和II1-II11)。阴性对照物为生理盐水，11个研究药物如下表所示，其中：免疫球蛋白为人免疫球蛋白，益生菌为热灭活布拉氐酵母菌，益生菌水溶性组分为水溶性酵母葡聚糖，益生菌水不溶性组分为酿酒酵母菌β-葡聚糖。药物均为液体，按实施例1的制备方法配置而成。系列I行全身性给药(腹腔注射)和系列II行局部给药(瘤内注射)。在本发明中，瘤内注射均需避开血管。每3日用药一次，一共3次，注射量为120μl/只。用药结束后10日，对动物进行安乐死，解剖后测定瘤重(W)，并从各用药方式的阴性对照组计算抑瘤率(R)，结果示于下表。
表4
Figure PCTCN2021076749-appb-000005
在现有技术中，通常通过如以上两个试验中系列I那样的全身性给药来研究药物共用所能提供的活性组合(在本发明中称作常规组合)。主流观点是，局部给药只是不同于全身性给药的另一种给药方式而己，或有动力学浓度的提高，却无技术含量的提高，不会在常规组合药理的动力学提高预期之外产生新药理。
在试验2A中，常规抗肿瘤药物(5-氟尿嘧啶)的应用合乎上述主流观点的预期：局部用药对全身性用药的药效提高在动力学提高预期范围内(＜150％)。在本试验上表中，益生菌/5-氟尿嘧啶复合物的局部给药组(系列II之组1)和全身性给药组(系列I之组1)的抑瘤率差异也为＜150％，似乎也在后者的动力学提高预期范围内。这些结果似乎进一步加强了上述主流观点：在相同化学组成的组合物(X/Y)中，局部用药该组合物也只能产生X/Y在全身性用药中的功能组合(常规组合)。于是，只将该主流观点当作偏见加以克服，才能获得超常规组合。
在上表中，系列I中各亚甲蓝类染料/免疫调节剂组合物组7-10的瘤重与其阴性对照组之间的差异均无统计学意义(均为P＞0.05)，且它们的抑瘤率也都在可忽略不计的低水平，说明亚甲蓝类染料和这些免疫调节剂的任何常规组合(例如免疫调节作用与常规抗肿瘤作用的组合、或/和常规抗肿瘤作用之间的组合)都不能够实现抑瘤增效，更说不上有效的协同作用。
亚甲蓝在临床上主要用作解毒剂或活体染料。此外，它还具有类似于乙醇的性质：其最大化细胞毒效应可在液体反应器(细胞试验或非实体肿瘤动物试验)中、而不能在非液体反应器(动物瘤内给药试验)中显示出有效抑瘤药效，以及其最大化局部作用可在非液体反应器中、而不能在液体反应器中显示出有效抑瘤药效。按照主流的药学观点，局部作用最小化的亚甲蓝(X)与任一药物(Y)的组合物(X/Y)在全身性用药时只能产生常规组合(例如X常规抗肿瘤作用与Y免疫调节作用的组合、或/和Y常规抗肿瘤作用之间的组合)，而X/Y在局部用药时也只能显示其在全身性用药中的功能。
然而，系列II组合物组7-10与其系列I中的相应组7-10比较，瘤重差异均有统计学意义(p＜0.05)，抑瘤率差异均有10倍以上，说明系列II中的局部药物组合物通过不同于系列I中的常规药物组合物的新药理实现了有效瘤体治疗。与其相应组成单药组相比较，系列II组合物组7-10的抑瘤率也超出理论单纯相加效应的预期范围，其实际/预期比(q)均大于1.15，显示出上述新药理应为该局部协同药理。
系列I中的研究组11与1相比较并无抑瘤率提高，说明该亚甲蓝类染料和该复合物(免疫调节剂/常规抗肿瘤药)在该常规组合物中的任何常规组合(例如免疫调节作用与常规抗肿瘤作用的组合、或/和常规抗肿瘤作用之间的组合)都不能够实现抑瘤增效，更说不上有效的协同作用。然而，组II11比组I11的抑瘤率高出150％以上，说明系列II中的局部药物组合物通过不同于系列I中的常规药物组合物的新药理实现了有效瘤体治疗。与其组成单药组1、5相比较，组合物组11的抑瘤率也超出该两药物理论单纯相加效应的预期范围，其实际/预期比(q)大于1.15，显示出上述新药理应为该局部协同药理。
众所周知，药学并非化学，药物并非完全由化学定义的物质(X)，而是构(X)效(A)统一的活性物质(AX)。传统观念中，提供相同活性(A，例如细胞毒作用)的不同物质(X1和X2，例如上述全身性给药系列中的亚甲蓝类染料和常规抗肿瘤药)很容易被视作不同药物(AX1和AX2)。然而，提供不同活性(A1、A2和A3，例如细胞毒作用、局部作用和局部协同作用)的相同化学物质(X，例如亚甲蓝类染料)则很容易被偏见视作相同药物(例如都当作A1X，而忽视A2X和A3X)。
在本发明中，亚甲蓝类染料的有效细胞毒作用被记作A1、该作用所需的特征组成被记作X1；其有效局部作用(因而是优选最大化的局部作用)被记作A2、该作用所需的特征组成被记作X2；其有效局部协同作用被记作A3、该作用所需的特征组成被记作X3。从药学而非化学的角度，这是可提供不同的活性(A1、A2、A3)、从而也具有不同给药组成特征(X1、X2、X3)的构效不同的三种亚甲蓝类染料(A1X1、A2X2、A3X3)。
在现有技术中，在细胞试验悬浮液反应器和非实体癌中发现了具有A1的X1，但A1X1却不能在瘤体组织反应器中产生有效效应；在瘤体组织反应器中发现了具有A2的必须组成X2(超常规浓度，例如≥2％)，但A2X2的药物效率仍待提高。本发明提供了产生A3的X3技术方案：低浓度(＜2％、≤1％、或＜1％)，A3X3于是就是仅提供优选为最小化而非最大化局部作用、但可提供有效局部协同作用的亚甲蓝类染料。并且，在本实施例和以下实施例还证实，应用A3X3可以制备出远远超过A1X1和A2X2的预期技术效果(给药效率)的本发明组合物。
在本发明中，亚甲蓝类染料共用物(Y)的特征活性(例如营养素的营养作用、免疫调节剂的免疫调节作用、常规抗肿瘤药的细胞毒作用)被记作B1；营养素和免疫调节剂特征活性之外的常规抗肿瘤活性(细胞毒作用或其它可通过全身性给药显示的抗肿瘤作用，假如存在的话)被记作B2；其与上述亚甲蓝A3X3的局部协同作用被记作B3，后者也因之被称作A3X3的局部协同物(记作B3Y)。
以上研究以及其它类似研究说明，本发明的组合物的功能组合或组合物，并非1)：亚甲蓝类染料细胞毒作用(A1)和共用物特征活性(B1)的组合(A1/B1)，或A1X1/B1Y组合物，也非2)：亚甲蓝类染料细胞毒作用(A1)和共用物常规抗肿瘤活性(B2)的组合(A1/B2)，或A1X1/B2Y组合物，也非3)：亚甲蓝类染料局部作用(A2)和共用物特征活性(B1)或常规抗肿瘤活性(B2)的组合(A2/B1或A2/B2)，或A2X2/B1Y组合物或A2X2/B2Y组合物，而是4)：亚甲蓝类染料局部协同作用(A3)和共用物局部协同作用(B3)的组合(A3/B3)，或A3X3/B3Y组合物。并且，在本实施例和以下实施例还证实，该组合(A3/B3)或该组合物(A3X3/B3Y)产生了其它几种组合或组合物所不能达到的技术效果(药效)。
例如，本发明的亚甲蓝类染料/营养素组合物中的功能组合，并非亚甲蓝类染料的细胞毒作用与营养素的营养作用(无论是正作用还是副作用)或/和常规抗肿瘤作用(假如存在的话)的组合(A1/B1或/和A1/B2)，也非亚甲蓝类染料的局部作用与营养素的营养作用(无论是正作用还是副作用)或/和常规抗肿瘤作用(假如存在的话)的组合(A2/B1或/和A2/B2)，而是亚甲蓝类染料所能够提供给营养素(作为化学物质)的、以及 该营养素所能够提供给亚甲蓝类染料的局部协同作用组合(A3/B3)。
又例如，本发明的亚甲蓝类染料/免疫调节剂组合物中的功能组合，并非亚甲蓝类染料的细胞毒作用与免疫调节剂的免疫调节作用(无论是正作用还是副作用)或/和常规抗肿瘤作用(假如存在的话)的组合(A1/B1或/和A1/B2)，也非亚甲蓝类染料的局部作用尤其是最大化的局部作用与免疫调节剂的免疫调节作用(无论是正作用还是副作用)或/和常规抗肿瘤作用(假如存在的话)的组合(A2/B1或/和A2/B2)，而是亚甲蓝类染料所能够提供给免疫调节剂(作为化学物质)的、以及该免疫调节剂所能够提供给亚甲蓝类染料的局部协同作用组合(A3/B3)。
又例如，本发明的亚甲蓝类染料/常规抗肿瘤药组合物，并非亚甲蓝类染料的细胞毒作用与常规抗肿瘤药的细胞毒作用的组合(A1/B1)，也非亚甲蓝类染料的局部作用与常规抗肿瘤药的局部细胞毒作用(A2/B1)的组合，而是优选最小化而非最大化局部作用亚甲蓝类染料所能够提供给常规抗肿瘤药(作为化学物质)的、以及该常规抗肿瘤药所能够提供给该亚甲蓝类染料的局部协同作用组合(A3/B3)。
实施例3：局部协同药理的优选
本实施例通过对优选最小化而非最大化局部作用亚甲蓝类染料的局部协同物的优选进一步研究本发明组合物的局部协同作用药理。
试验3A：试验动物为裸小鼠，建模细胞为人胰腺癌细胞(PANC-1)，以1×10 6个细胞/只在动物右侧腋部皮下进行移植瘤建模。成功建模的试验动物(荷载胰腺癌裸小鼠，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100ul/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(W)，并从阴性对照组计算抑瘤率(R)，结果示于下表。
表5
Figure PCTCN2021076749-appb-000006
按照现有共用技术的主流习惯，局部增效共用物往往选自局部作用较强的药物。90％乙醇和50％乙酸是经典化学消融剂，可提供有效局部作用。在上表中，亚甲蓝类染料/经典化学消融剂组合物组(组8和9)与各组成单药组〔组3-5〕的抑瘤率相比较，它们的实际/预期比(q)均小于1.15，未显示出协同药效。而亚甲蓝类染料/营养素组合物组(组7)和亚甲蓝类染料/常规抗肿瘤药组合物组(组6)与它们各自的组成单药组〔组1、2、5〕的抑瘤率相比较，它们的实际/预期比(q)均大于1.15，显示出协同药效。此外，在组3和8中，每只动物均观察到乙醇的强剌激反应。而在组4和9中，每只动物均观察到乙酸对给药处非瘤组织的最强大破坏反应，尽管其抑瘤率还比组6和7更低，显示后者具有较高的针对瘤体组织的特异性和更好的疗效。
试验3B：试验动物为裸小鼠，建模细胞为肝癌细胞(MDA-MB231)，以1×10 6个细胞/只在动物右侧腋部皮下进行移植瘤建模。成功建模的试验动物(荷载肝癌裸小鼠，瘤体平均体积127mm 3)随机分为1个阴性对照组和14个研究组。阴性对照物为生理盐水，13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。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(W)，并从阴性对照组计算抑瘤率(R)，结果示于下表。
表6
Figure PCTCN2021076749-appb-000007
在本发明中，术语“低效抗肿瘤药物”是指常规给药无效局部给药很有效的药物，其包括非甾体抗炎化合物、喹啉类化合物、青蒿素衍生物、酚类化合物。在本实施例中，二盐酸奎宁作为低效抗肿瘤药物的代表，二氯化铁作为金属化合物的代表，5-氟尿嘧啶作为有效抗肿瘤药物的代表。在现有技术中，这些药物均可用作亚甲蓝类染料的最大化局部作用的进一步增效。在上表中，研究组6、8、10的抑瘤率均为研究组4的115％以上，合乎现有技术的增效预期。然而，与它们各自组分单药组比较，研究组6、8、10的抑瘤率均未超出理论单纯相加效应的预期范围(实际/预期比q均为小于1.15)，说明这些药物(Y)对高浓度亚甲蓝(X2)的共用作用都是相加作用。
通常认为，浓度仅仅是一个动力学因素，增效药物Y与不同浓度药物X的共用于是只会产生动力学预期范围内的同种共用效应，而不会产生异种共用效应。按照这种主流观点，Y与低浓度亚甲蓝(X2)的共用不应当超出Y与高浓度亚甲蓝(X2)的共用作用预期，在己对后者研究后再研究前者毫无意义。
在上表中，研究组7和9的抑瘤率均为研究组5的115％以上，且它们的抑瘤率也未均未超出它们各自组分单药组(分别为组2和5，以及组3和5)理论单纯相加效应的预期范围(实际/预期比q均为小于1.15)，似乎进一步加强了上述主流观点：药物Y分别与低浓度亚甲蓝(X2)和高浓度亚甲蓝(X2)的共用只有动力学差异而无共用机理差异，即均为上述相加作用。于是，只将该主流观点当作偏见加以克服，才能获得超相加作用组合物。
然而，在上表中，研究组13与其组分单药组(组12和5)比较，其抑瘤率超出理论单纯 相加效应的预期范围(实际/预期比q为大于1.15)，显示出协同作用。
此外，按照公认的增效原理，与相同的有效抗肿瘤药物(Y1)共用，如单药X4的疗效高于单药X5，则药合物X4/Y1的疗效理应高于X5/Y1。组7与组6、或组9与组8的抑瘤率差与组5和组4的抑瘤率差正相关，符合该主流观点。然而，组11与组10的抑瘤率差和组5和组4的抑瘤率差却并未显示出正相关，说明很可能组11中本发明的亚甲蓝类染料组合物提供了组6、8、10中现有技术的亚甲蓝类染料组合物所不能提供的新药理作用。实际上，研究组11与其组分单药组(组1和5)比较，其抑瘤率超出理论单纯相加效应的预期范围(实际/预期比q为大于1.15)，显示出协同作用。该协同作用应为上述新药理作用的主要作用。在以上二个实施例及以下实施例的部分局部给药试验中，可以观察到亚甲蓝类染料与其它药物的共用的复杂性。
所研究的二种不同亚甲蓝类染料分别为现有技术亚甲蓝类染料(构效B2X2)和本发明亚甲蓝类染料(构效B3X3)。为提供有效药效，现有技术亚甲蓝类染料(B2X2)必须是可提供优选最大化而非最小化局部作用(B2)的高浓度(X2，优选为2-6％)亚甲蓝类染料。而根据本发明实施例的结果，高浓度亚甲蓝类染料(X2)不能、而低浓度(X3，优选为0.5％-1.5％或0.5％-1.0％)亚甲蓝类染料却有可能在与本发明实施例中的共用物的局部共用中提供其局部协同作用(B3)，而后者仅可以提供优选最小化而非最大化的局部作用。换言之，只有局部作用为优选最小化而非最大化的亚甲蓝类染料才可作为本发明中的“可提供局部协同作用的局部活性成分”。
所研究的共用物包括营养素、免疫调节剂、常规抗肿瘤药、化学消融剂、现有技术中的低效抗肿瘤物质、金属化合物。其中，化学消融剂、常规抗肿瘤药和低效抗肿瘤物质均显示较强的局部作用，营养素和免疫调节剂营养素总体上并未显示较强局部作用，金属化合物的局部作用也较小。根据本发明实施例的试验结果，这些共用物能否与亚甲蓝类染料互相提供局部协同作用(而非局部作用)似乎与它们本身的局部作用大小的关係不大。换言之，亚甲蓝类染料的局部协同物不可能根据局部作用组合来推测，其错综复杂性甚至于要求克服某些固有观念才能观察到。
例如，具有最强局部作用的化学消融剂和低效抗肿瘤物质可以对高浓度和低浓度亚甲蓝类染料(X2、X3)进行增效(例如增效至115％以上)，但其实际/预期比q均为≤1.15，该增效作用为各共用组分局部作用的相加作用，而非本发明中定义的局部协同作用。因而，化学消融剂和低效抗肿瘤物质可以用作亚甲蓝类染料的增效剂，但出人意外地不能优选为本发明中亚甲蓝类染料的局部协同物。而具有较弱局部作用的金属化合物的情况居然也和化学消融剂和低效抗肿瘤物质相似，也可以用作亚甲蓝类染料的增效剂，也不能优选为本发明中亚甲蓝类染料的局部协同物。此外，金属化合物与低浓度亚甲蓝类染料共用甚至于难以产生本发明目的所需的高药效。
然而，局部作用在化学消融剂和金属化合物之间、甚至于在很多情况更趋于金属化合物的营养素和免疫调节剂与低浓度亚甲蓝类染料(X3)共用的实际/预期比q可达1.15以上，该共用作用正是非本发明中定义的局部协同作用。因而，本发明中亚甲蓝类染料的 局部协同物居然可以从营养素和免疫调节剂优选。
更为复杂的情况出现在常规抗肿瘤药中，它们的局部作用通常也在化学消融剂和金属化合物之间。常规抗肿瘤药(B1Y)与高浓度亚甲蓝类染料(X2)的共用可以对后者的最大化局部作用(A2)增效(例如增效至115％以上)，但其实际/预期比q均为≤1.15。按照主流观点，事情似乎到此为止。出乎意料的是，常规抗肿瘤药与低浓度亚甲蓝类染料的组合物(A3X3/B1Y)的实际/预期比却较大(q≥1.15)。尤其奇怪的是，这两种组合物的药效差异并未反映其中两种亚甲蓝类染料的局部作用差异水平。这说明，不同浓度亚甲蓝类染料与常规抗肿瘤药的共用具有完全不同的共用药理(局部协同作用vs局部相加作用)。常规抗肿瘤药对高浓度亚甲蓝类染料的增效作用应为局部作用的相加作用，而非本发明中定义的协同作用；而常规抗肿瘤药与低浓度亚甲蓝类染料的共用作用超过它们局部作用的相加作用的预期，显示为本发明中定义的协同作用。亚甲蓝类染料于是在本发明的组合物中显示出更高的药理效率。
众所周知，药物反应是错综复杂的，包括必须反应和可能存在的其它反应(例如共用物各自的特征活性)，很多时候甚至还包括可以接受的副反应。以上这些结果进一步说明，本发明组合物的必须组合并非其局部作用增效剂的局部作用对亚甲蓝类染料最大化局部作用的加和增效，而是其局部协同物与优选最小化而非最大化局部作用的亚甲蓝类染料之间的局部协同作用组合。于是，是否可以提供该局部协同作用、而非是否可以提供该相加作用才是本发明中的局部协同物的基本特征。换言之，本发明组合物的必要药理为局部协同作用。
实施例4、局部协同组成(局部协同量比)优选
通常认为，有了常规药物组合物，简单地将其进行局部给药便是局部药物组合物，后者于是不应当有与前者不同的组成特征。然而，本发明的局部药物组合物却必须完全不同于常规药物组合物的组成特征：局部协同量比。
在本发明的范围内，术语“协同量比”是指在使得药物X和Y共用产生协同作用而非相加作用或拮抗作用(实际/预期比q≥1.15)所必需的给药组成中的组分X和组分Y的重量比(W X∶W Y)，术语“局部协同量比”是指在常规药物组合物(全身性给药)亚不必须、而在局部药物组合物(局部给药)中必须的的协同量比。
试验4A：试验动物为小鼠，建模细胞为肉瘤细胞(S180)，以1×10 6个细胞/只在动物右侧腋部皮下进行移植瘤建模。成功建模的试验动物(荷载肉瘤小鼠，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为亚甲蓝浓度)、16种变化种类及浓度的其它组分单药(Y％，Y为其它组分浓度)、20个分别由变化浓度亚甲蓝(X％)和变化种类及浓度的其它组分(营养素、常规抗肿瘤药)(Y％)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下表。
表7
Figure PCTCN2021076749-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。
试验4B：试验动物为裸小鼠，建模细胞为人肝癌细胞(MDA-MB231)，以1×10 6个细 胞/只在动物右侧腋部皮下进行移植瘤建模。成功建模的试验动物(荷载肝癌裸小鼠，瘤体平均体积163mm 3)随机分为阴性对照组和12个研究组。阴性对照物为生理盐水，研究药物的组成如下表所示包括：4种变化浓度的亚甲蓝单药(X％)、4种变化浓度的免疫调节剂(灭活釀酒酵母菌)单药(Y％)、以及4种变化浓度的免疫调节剂和4种变化浓度的亚甲蓝的组合物(X％/Y％)。药物均为含水液体，按实施例1的制备方法配置而成。各组均用药2次，第2次用药在第1次用药后7日，每次注射量150μl/只。第2次用药结束后7日对动物进行安乐死，解剖后剥离出肿瘤组织测定瘤重，并从阴性对照组计算抑瘤率。各研究药物组抑瘤率示于下表。
表8
Figure PCTCN2021076749-appb-000009
*：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10.1％
**：纵栏括号中的数据为Y％免疫调节剂组的平均抑瘤率，例如0.2％免疫调节剂组的平均抑瘤率为5.3％
***：不带括号的数据为X％亚甲蓝/Y％免疫调节剂组合物组的平均抑瘤率，例如3％亚甲蓝/0.2％免疫调节剂组含物组的平均抑瘤率为75.2％。
根据以上2表的结果计算，组合物的实际/预期比q并未随亚甲蓝类染料浓度的升高而升高，该q反而在局部协同量比主要是随亚甲蓝类染料浓度的升高而下降，说明亚甲蓝类染料所提供的局部协同作用与其局部作用的不同、甚至于背离。组合物的实际/预期比q也未随共用物浓度的升高而升高，说明这些共用物所提供的局部协同作用与其局部作用的不同，该局部协同作用的大小并不以该局部作用的強弱为条件。
根据以上研究及更多的类似研究的结果，亚甲蓝类染料及其局部协同物的局部协同量比(W 局部协同物/W 亚甲蓝类染料)其实就是其局部协同作甪所需的药理量比。该局部协同量比为＞0.1/3和＜40/0.15，优选为＞0.1/1.8和＜40/0.15，或＞0.2/1和≤40/0.30。具体而言：
营养素/亚甲蓝类染料组合物的局部协同量比(W 营养素/W 亚甲蓝类染料)为1/3＜W 营养素/W 亚甲蓝类 染料＜40/0.15，优选为1/3＜W 营养素/W 亚甲蓝类染料≤40/0.30、或1/1.8＜W 营养素/W 亚甲蓝类染料≤40/0.30。 更具体而言，亚甲蓝类染料/氨基酸类营养素组合物的局部协同量比(W 氨基酸类营养素/W 亚甲蓝类染 料)为1/3＜W 氨基酸类营养素/W 亚甲蓝类染料＜20/0.15，优选为1/3＜W 氨基酸类营养素/W 亚甲蓝类染料≤20/0.30、或2/1.8≤W 氨基酸类营养素/W 亚甲蓝类染料≤20/0.30；脂类营养素/亚甲蓝类染料组合物的局部协同量比(W 脂类营养素/W 亚甲蓝类染料)为2/3＜W 脂类营养素/W 亚甲蓝类染料＜8/0.15，优选为2/3＜W 脂类营养素/W 亚甲蓝类染料≤8/0.30、或3/1.8≤W 脂类营养素/W 亚甲蓝类染料≤8/0.30；糖类营养素/亚甲蓝类染料组合物的局部协同量比(W 糖类营养素/W 亚甲蓝类染料)为5/3＜W 糖类营养素/W 亚甲蓝类染料＜40/0.15，优选为10/3＜W 糖类 营养素/W 亚甲蓝类染料≤40/0.30、或10/1.8≤W 糖类营养素/W 亚甲蓝类染料≤40/0.30。
免疫调节剂/亚甲蓝类染料组合物的局部协同量比(W 免疫调节剂/W 亚甲蓝类染料)为0.2/3＜W 免 疫调节剂/W 亚甲蓝类染料≤20/0.15，优选为0.5/3＜W 免疫调节剂/W 亚甲蓝类染料≤20/0.50、或0.5/1≤W 免疫调 节剂/W 亚甲蓝类染料≤20/0.50。
常规抗肿瘤药物/亚甲蓝类染料组合物的局部协同量比(W 常规抗肿瘤药物/W 亚甲蓝类染料)为0.1/3＜W 常规抗肿瘤药物/W 亚甲蓝类染料＜1.5/0.15，优选为0.3/3＜W 常规抗肿瘤药物/W 亚甲蓝类染料＜1.5/0.15、或0.3/1.8≤W 常规抗肿瘤药物/W 亚甲蓝类染料≤1.5/0.30。
实施例5、有效局部协同浓度优选
通常认为，在相同剂量条件下，协同量比确定之后，给药浓度可由临床医生调整(例如1-5倍差异)，其并非药物组合物特征、至少并非可导致共用作用机理改变的重要特征。然而，本发明的局部药物组合物却具有完全不同于常规药物组合物的组成特征：局部有效协同浓度。
在本发明的范围内，术语“协同浓度”是指在使得药物X和Y共用产生协同作用而非相加作用或拮抗作用(实际/预期比q≥1.15)所必需的给药组成中的组分X或组分Y的浓度，术语“局部协同浓度”是指常规药物组合物(全身性给药)亚不必须、而在局部药物组合物(局部给药)中必须的协同浓度，术语“局部有效协同浓度”是指使得组合物X/Y显示与现有药物相比更有竞争力的药效所需的局部协同浓度。
试验5A：试验动物为BALB/c小鼠，建模细胞为乳腺癌4T1细胞，以1×10 6个细胞/只在动物右侧腋部皮下进行移植瘤建模。成功建模的试验动物(荷载乳腺癌小鼠，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。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(W)，并从各用药方式的阴性对照组计算抑瘤率(R)，结果示于下表。
表9
组别 研究药物 W(x±s，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 45.9％
2 30％木糖醇 1.57±0.19 13.3％
3 0.5％亚甲蓝 1.67±0.20 7.7％
4 1％5-氟尿嘧啶/0.5％亚甲蓝 0.45±0.13 75.1％
5 30％木糖醇/0.5％亚甲蓝 0.58±0.12 68.0％
6 0.5％5-氟尿嘧啶 1.18±0.18 34.8％
7 15％木糖醇 1.61±0.16 11.0％
8 0.25％亚甲蓝 1.82±0.23 -0.6％
9 0.5％5-氟尿嘧啶/0.25％亚甲蓝 1.16±0.14 35.9％
10 15％木糖醇/0.25％亚甲蓝 1.57±0.15 13.3％
在上表中，组合物组4和9所用亚甲蓝类染料/局部协同物组合物药物组合物的给药量比相同但给药浓度不同，它们不仅显示出不同疗效，还显示出共用作用的不同机理。前者的实际/预期比q为＞1.15，显示出局部协同作用；后者的实际/预期比q为＜1.15和＞0.85，显示出相加作用。同样地，组合物组5和10也显示出共用作用的不同机理。尤其重要的是，组合物的药效高低显然决定于它们是否显示出局部协同作用。根据上表，亚甲蓝类染料的局部协同浓度为＞0.25％，其有效局部协同浓度优选为≥0.5％。
试验5B：试验动物为BALB/c小鼠，建模细胞为乳腺癌4T1细胞，以1×10 6个细胞/只在动物右侧腋部皮下进行移植瘤建模。成功建模的试验动物(荷载乳腺癌小鼠，瘤体平均体积386mm 3)随机分为1个阴性对照组(01)和6个药物研究组(1-6)。阴性对照物为生理盐水，研究药物如下表所示。其中的人免疫球蛋白为从人血浆分离的供实验室用的免疫球蛋白，主要成分为IgG。药物均为水溶液，按实施例1的制备方法配置而成。各试验组均行瘤内注射，每3日用药一次，一共3次，注射量如下表所示。用药结束后3日，对动物进行安乐死，解剖后测定瘤重(W)，并从阴性对照组计算抑瘤率(R)，结果示于下表。
表10
组别 药物 注射量/只 W(x±s，g) 抑瘤率
01 生理盐水 100μl 2.46±0.26 0
1 3％人免疫球蛋白 100μl 2.56±0.21 -4.1％
2 1％人免疫球蛋白 300μl 2.40±0.25 2.4％
3 0.9％亚甲蓝 100μl 1.99±0.23 19.1％
4 0.3％亚甲蓝 300μl 2.24±0.19 8.9％
5 3％人免疫球蛋白/0.9％亚甲蓝 100μl 0.66±0.13 73.2％
6 1％人免疫球蛋白/0.3％亚甲蓝 300μl 2.19±0.24 11.0％
通常认为，药效取决于剂量而非用药浓度。临床上安全的血药浓度通常非常之低(例如小于10ug/ml)，远低于通常的制剂浓度。在上表中，组合物组5和6所用亚甲蓝类染料/免疫调节剂组合物的给药量比相同、剂量相同、但给药浓度不同，它们不仅显示出不同疗效，还显示出共用作用的不同机理。前者的实际/预期比q为＞1.15，显示出局部协同作用；后者则未显示出有意义的药效、更别说局部协同作用。组合物组5的抑瘤率是组合物组6的663％，也说明相应组合物具有完全不同的共用药理。根据上表，亚甲蓝类染料的局部协同浓度为＞0.30％，其有效局部协同浓度优选为≥0.9％。
试验5C：试验动物为BALB/c小鼠，建模细胞为肉瘤细胞S180细胞，以1×10 6个细胞/只在动物右侧腋部皮下进行移植瘤建模。成功建模的试验动物(荷载肉瘤小鼠，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(W)，并从阴性对照组计算抑瘤率(R)，结果示于下表。
表11
Figure PCTCN2021076749-appb-000010
在上表中，在满足其局部协同量比和局部协同浓度时，亚甲蓝类染料/氨基酸类营养素组合物中的亚甲蓝类染料还显示出一个特征：使得药物明显超过常规抗肿瘤药组(阳性对照组)药效的给药浓度阈值，即有效局部协同浓度阀值。当亚甲蓝瘤内给药浓度大于或等于这个阈值后，该有效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。合乎本发明特征的是，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。根据上表，亚甲蓝类染料的有效局部协同浓度为0.35％-2％、优选为0.35％-1.5％或 0.50％-1.5％(w/v)。
试验5D：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％，w/v)的局部协同物(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(W)，并从阴性对照组计算抑瘤率(R)，结果示于下表。
表12
Figure PCTCN2021076749-appb-000011
在上表中，在满足其局部协同量比和局部协同浓度时，亚甲蓝类染料/氨基酸类营养素组合物中的氨基酸类营养素还显示出一个特征：使得药物明显超过常规抗肿瘤药组(阳性对照组)药效的给药浓度阈值，即有效局部协同浓度阀值。当氨基酸类营养素瘤内给药浓度大于或等于这个阈值后，该有效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。根据上表，氨基酸类营养素的有效局部协同浓度为≥5％、优选为5％-35％或15％-35％(w/v)。
根据以上研究及更多的类似研究，亚甲蓝类染料及其局部协同物的组合作为局部协同活性成分应用的一个必要技术条件是：所述组合物的制备和组成必须满足亚甲蓝类染料及其局部协同物的局部协同浓度、优选为有效局部协同浓度，具体而言：
所述亚甲蓝类染料的局部给药浓度(w/v)≤2％、优选为0.3％-1.8％、0.5％-1.0％、或0.5％-0.9％；
所述常规抗肿瘤药的局部给药浓度(w/v)大于其饱和浓度的20％、优选为其饱和浓度的30％-100％；
所述免疫调节剂的局部给药浓度(w/v)为大于0.25％，优选为0.5％-30％，其中当免疫调节剂包括免疫球蛋白时，所述免疫球蛋白的给药浓度(w/v)≥0.25％、优选为 0.25-30％或1-30％；当免疫调节剂包括益生菌组分时，所述益生菌组分的给药浓度(w/v)≥0.35％，优选为0.5-30％；当免疫调节剂包括免疫调节肽时，所述免疫调节肽的给药浓度(w/v)≥3％，优选为3-30％；
所述营养素的局部给药浓度(w/v)为大于3％，优选为3％-45％，其中当营养素包括糖类营养素时，所述糖类营养素的给药浓度(w/v)≥20％、优选为20-40％；当营养素包括脂类营养素时，所述脂类营养素的给药浓度(w/v)≥4％，优选为4-25％；当营养素包括氨基酸类营养素时，所述氨基酸类营养素的给药浓度(w/v)≥3％，优选为3-25％，更优选为5-25％或15-35％。
根据以上实施例2-5的研究和其它更多研究揭示：
1)、某一物质在药物制备而非其它制备中的应用，应用的是其构(X)效(A)统一体AX。最通用的说法是，X作为可提供A的活性成分在...中的应用。同一物质(X)进入反应器的初始状态(给药方式)不同，则可在其中进行不同反应(提供不同作用A1、A2...)，从而作为可提供不同效(A1、A2...)的活性成分(A1X、A2X...)应用。如果提供不同效(A1、A2...)还需不同组成特征(不同构X1、X2...)，则是不同构效统一体(A1X1、A2X2、A3X3)的不同应用。
2)、以不同组成特征提供不同药理作用的亚甲蓝类染料自然有不同应用。低浓度亚甲蓝类染料可以作为解毒活性成分或常规抗肿瘤成分(A1X1)应用于制备常规药物组合物，高浓度(或局部作用最大化而非最小化)亚甲蓝类染料可以作为提供有效局部作用的活性成分(A2X2)应用于制备局部作用药物组合物，而低浓度(或局部作用最小化而非最大化)亚甲蓝类染料单药不行、只有在与其局部协同物的组合中才可以作为提供有效局部协同作用的局部活性成分(A3X3)应用于制备局部协同作用药物组合物。使用相同的亚甲蓝类染料剂量，其局部协同作用药物组合物显示出的瘤体组织破坏疗效、从而适应症范围超过其局部作用药物组合物和常规药物组合物预期。
3)、亚甲蓝类染料只能在和优选的共用物(其局部协同物)的共用中才可以提供上述局部协同作用。亚甲蓝类染料及其局部协同物的共用作用必须是局部协同作用、而非局部作用的相加作用，因而不能按现有技术教导的线索(例如局部作用共用增强)寻找到可提供该局部协同作用的共用物。在不同局部作用的不同种类(化学消融剂、现有技术中局部作用强大的常规低效抗肿瘤物质、金属化合物、营养素、免疫调节剂、常规抗肿瘤药)中，该局部协同物优选为选自以下组之一种或多种：营养素、免疫调节剂、常规抗肿瘤药。
4)、亚甲蓝类染料及其局部协同物的组合(X/Y)作为可提供局部协同作用(A)的局部活性应用，该组合的组成特征除了上述组分特征(X选自局部作用最小化而非最大化的亚甲蓝类染料，Y选自营养素、免疫调节剂、或/和常规抗肿瘤药)之外，还包括X和Y之间的协同量比。此外，该局部协同作用药物组合物的组成特征还可包括不同于其常规药物组合物或局部作用药物组合物的有效协同浓度，例如不同于其局部作用药物组合物的亚甲蓝类染料有效协同浓度。
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、局部协同物优选
如上所述，可作为局部协同活性组分的所述亚甲蓝类和局部协同物的组合的偶合性很强，很难按常理推导。以下试验进一步研究该偶合性，以便于本发明组合物的必要组分的优选。
试验6A：试验动物为BALB/c裸小鼠，建模细胞为黑色素瘤细胞，以5×10 6个细胞/只在动物右侧腋部皮下进行移植瘤建模。成功建模的试验动物(荷载黑色素瘤裸小鼠，瘤体平均体积238mm 3)随机分为1个阴性对照组(0)和9个药物研究组(1-9)。阴性对照物为生理盐水，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。各组均每3日用药一次，一共3次，行瘤内注射，注射量125μl/只。用药结束后3日，对动物进行安乐死，解剖后测定瘤重(W)，并从阴性对照组计算抑瘤率(R)，结果示于下表。
表13
组别 药物 W(x±s，g) 抑瘤率
0 生理盐水 2.38±0.26 0
1 4％人免疫球蛋白 2.50±0.24 -5.0％
2 1％亚甲蓝 2.02±0.29 15.1％
3 1％新亚甲蓝 2.04±0.23 14.3％
4 1％专利蓝 1.93±0.22 18.9％
5 1％孟加拉红 2.14±0.19 10.1％
6 4％人免疫球蛋白/1％亚甲蓝 0.69±0.14 71.0％
7 4％人免疫球蛋白/1％新亚甲蓝 0.62±0.12 73.9％
8 4％人免疫球蛋白/1％专利蓝 1.09±0.17 54.2％
9 4％人免疫球蛋白/1％孟加拉红 1.95±0.21 18.1％
在上表中，组合物组6-8各自的瘤重与阴性对照组相比均有统计意义(均为p＜0.05)，且它们各自的实际/预期比q均为＞1.15，均显示出明显局部协同作用。然而，组合物组9的瘤重与阴性对照组相比无统计意义(p＞0.05)，且未显示出有意义的药效(抑瘤率小于20％)，故未显示出明显局部协同作用。以上结果说明，可与本发明优选的局部协同物进行局部协同作用组合者，并非可以是任何活体染料，也并非选自局部作用最大化的亚甲蓝类染料，而是优选自最小化而非最大化局部作用的亚甲蓝类染料。
试验6B：试验动物为BALB/c小鼠，建模细胞为肉瘤S180细胞，以1×10 6个细胞/只在动物右侧腋部皮下进行移植瘤建模。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(1％亚甲蓝/Y％)。研究组1-14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(R)。阳性对照组抑瘤率为51％，研究组结果示于下表。
表14
Figure PCTCN2021076749-appb-000012
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。
试验6C：试验动物为BALB/c裸小鼠，建模细胞为人胰腺癌PANC-1细胞，以1×10 6个细胞/只在动物右侧腋部皮下进行移植瘤建模。成功建模的试验动物(荷载胰腺癌裸小鼠，瘤体平均体积207mm 3)随机分为阴性对照组(组0)、亚甲蓝单药组(组01)、8个局部协同物单药组(组1-8)和8个亚甲蓝/局部协同物组合物组(组9-16)。阴性对照物为生理盐水，局部协同物分别为如下表所示，包括：单一局部协同物(组1、2、9、10)、酸化营养素(组3、4、11、12)、pH中性化的氨基酸营养素(组5、13)、营养素/常规抗肿瘤药物复合物(组6、14)、营养素/免疫抑制剂复合物(组7、15)、免疫抑制剂/常规抗肿瘤药物复合物(组8、16)。药物均为含水液体，按实施例1的制备方法配置而成。各组均瘤内注射1次，注射体积为170μl。用药结束后7日对动物进行安乐死，解剖后测定瘤重(W)，并从阴性对照组计算抑瘤率(R)，以及从抑瘤率计算实际/预期比(q)，研究组结果示于下表。
表15
Figure PCTCN2021076749-appb-000013
在上表中，各组合物组9-16的瘤重与阴性对照组相比均有统计意义(均为p＜0.05)，它们的实际/预期比q均为＞1.15，均显示出局部协同作用。更具体而言，组合物组11-16显示出较组合物组9和10更高的药效。
现有技术中的关于抗肿瘤药物组合物的主流观点为：X与一种药物Y产生协同作用的几率已经非常之低，而与药物复合物Y/Z产生协同作用几率更低，复合物反而增加了药物拮抗的风险。其结果是，临床上几乎没有抗肿瘤三组分协同组合物。然而，根据以上结果以及更多的类似研究的结果，本发明的组合物中的所述局部协同物的一个优选技术方案却为：包含局部协同物的多组分复合物(在本发明中简称为局部协同物复合物)，更优选为以下局部协同物复合物：同类局部协同物复合物，不同类局部协同物复合物，单一局部协同物或局部协同物复合物的pH调节复合物，其中：
1、所述同类局部协同物复合物包含多个同类局部协同物，包括营养素复合物、免疫抑制剂复合物、常规抗肿瘤药物复合物，其中所述营养素复合物包括多个不同种营养素的复合物(例如氨基酸营养素/糖类营养素复合物)和多个同种营养素的复合物(例如氨基酸营养素复合物、糖类营养素复合物)，其中所述氨基酸营养素复合物包括同酸碱性 氨基酸营养素复合物(例如精氨酸/耐氨酸复合物、谷氨酸盐/甘氨酸复合物、等等)和异酸碱性氨基酸营养素复合物，其中所述异酸碱性氨基酸营养素复合物优选为使得其含水液体的pH趋于中性化的异酸碱性氨基酸营养素复合物(例如精氨酸/甘氨酸复合物、精氨酸/耐氨酸/甘氨酸复合物、精氨酸/赖氨酸/甘氨酸/半胱氨酸盐/丙氨酸/丝氨酸复合物、等等)。在本发明的范围内，术语“同酸碱性复合物”是指其在1％水溶液中同为酸性或同为碱性的多个局部协同物的复合物；术语“异酸碱性复合物”是指其在1％水溶液中分别为酸性和碱性的局部协同物的复合物；
2、所述不同类局部协同物复合物包含多个不同类局部协同物，包括例如营养素/常规抗肿瘤药物复合物(例如精氨酸/赖氨酸/常规抗肿瘤药物、精氨酸/常规抗肿瘤药物、等)、营养素/免疫抑制剂复合物(例如精氨酸/免疫球蛋白、精氨酸/益生菌组分、精氨酸/赖氨酸/RNA、等)、免疫抑制剂/常规抗肿瘤药物复合物(例如益生菌组分/常规抗肿瘤药物)、等等。
3、局部协同物(单一或复合物)的pH调节复合物包括局部协同物的酸化(局部协同物/酸)或碱化(局部协同物/碱)复合物，优选为使得局部协同物含水液体的pH变化值为＞1、更优选为使得局部协同物含水液体的pH趋于中性化的复合物(例如精氨酸/乙酸复合物)，其中所述酸化中的酸优选为有机酸(例如乙酸)，所述碱化中的碱优选为药学允许的pH调节碱(例如氢氧化钠、碳酸氢钠、等)。
实施例7：更多的抗肿瘤应用
在以下试验中，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：A(1％亚甲蓝/5％DHA/5％乙酸)、B(1％亚甲蓝/20％精氨酸)、C(1％亚甲蓝/10％甘氨酸/10％赖氨酸)、D(1％亚甲蓝/30％葡萄糖)、E(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(Hep62)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(B6C823)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。
实施例8：局部协同作用的进一步研究和抗肿瘤适应症优选
精淮治疗要求精准确定抗肿瘤药物及其适应症的关系。综合以上实施例的结果，由于亚甲蓝类染料的安全性剂量(例如在安全性最大化的技术方案中为1mg/kg体重或80mg/人)是包含它的组合物的安全性剂量的决定性因素。对于局部药物而言，给药体积(例如含1％(w/v)亚甲蓝的药物8ml、或含2％(w/v)亚甲蓝的药物4ml)与药效直接 相关，其在有效性最大化的技术方案中与瘤内组织特征和瘤体大小紧密相关，例如其优选为等于或大于给药瘤体的体积。本发明组合物于是因其局部协同作用而在单位亚甲蓝类染料剂量(例如mg)所能提供的药效(在本发明中也称作亚甲蓝类染料药物效率)方面大幅度优于现有技术的含亚甲蓝类染料组合物，这使得本发明组合物可以拓展新的适应症。
由于异质性是肿瘤的一个核心特征，由该异质性限定的适应症于是便是肿瘤治疗药物的一个核心特征。通常认为，癌细胞异质性是抗癌药物适应症的基础。以下试验通过本发明组合物与现有技术组合物的比较研究，进一步说明本发明组合物的作用特征，以及在完全不同的肿瘤异质性条件下与该作用特征相应的适应症特征。
试验8A：研究了不同局部组合物针对不同组成的病变组织的不同共用机理。试验动物为BALB/c裸小鼠。按公知技术，将大鼠乳腺良性纤维瘤组织碎片(约20mm 3/个)以约300mm 3/只在动物右侧腋部皮下进行组织移植瘤建模，供系列I使用。该移植成功后，将人乳腺癌细胞(MDA-MB231)以1×10 6个细胞/只在同一批次其它动物右侧腋部皮下进行细胞移植瘤建模，供系列II使用。成功建模的系列I试验动物(良性纤维瘤患者，瘤体平均体积238mm 3)和系列II试验动物(乳腺癌患者，瘤体平均体积231mm 3)各自随机分为阴性对照组和7个研究组，阴性对照物为生理盐水，研究药物如下表所示。药物均为含水液体，按实施例1的制备方法配置而成。各组均瘤内注射1次，注射体积为100μl。用药结束后7日对动物进行安乐死，解剖后测定瘤重(W)，并从阴性对照组计算抑瘤率(R)，以及从抑瘤率计算实际/预期比(q)，研究组结果示于下表。
表16
Figure PCTCN2021076749-appb-000014
众所周知，大鼠乳腺良性纤维瘤组织的主要成分为纤维***。在上表中，各系列组合物组5-7的瘤重与阴性对照组相比均有统计意义(均为p＜0.05)。其中，各系列组合物组5和6的实际/预期比q均为＞1.15，而各系列组合物组7的实际/预期比q均为＜1.15， 说明前者的药物组合物均显示出局部协同作用，而后者的药物组合物未显示出局部协同作用。
尤其值得注意的是，相同药物在系列I中有效性低于在系列II中，以及相同局部协同组合物(组5或6)在系列I中显示的实际/预期比q大于在系列II中。该结果说明，本发明组合物的局部协同性、从而其中亚甲蓝类染料的药物效率在富含非癌细胞成分的瘤体组织中大于在富含癌细胞成分的瘤体组织中，这使得本发明组合物有可能通过提高给药体积来提高其在前者中的药物效应。
根据以上研究及其它研究，本发明组合物可以解决同剂量的现有技术的含亚甲蓝类染料组合物所不能解决的问题：富含非癌细胞成分的瘤体组织的有效破坏。本发明组合物的适应症的一个优选方案为：恶性肿瘤相关结节的治疗。在本发明中，“恶性肿瘤相关结节”是指临床上为治疗恶性肿瘤而应当关注的任何结节，例如具有虽未检出(例如因未检、误检或漏检的未检出)为、但不能100％排除为恶性肿瘤瘤体的节结，或具有可发展为恶性肿瘤瘤体的风险的结节。
试验8B：试验动物为BALB/c裸小鼠。按公知技术将易操作的较软人胰腺癌组织碎为约50mm 3/个，再将其以约300mm 3/只在动物右侧腋部皮下进行组织移植瘤建模，供系列II使用。该移植成功后，将人胰腺癌细胞(PANC-1)以1×10 6个细胞/只在同一批其它动物右侧腋部皮下进行细胞移植瘤建模，供系列I使用。成功建模的系列I试验动物(胰腺癌患者，瘤体平均体积213mm 3)和系列II试验动物(胰腺癌患者，瘤体平均体积234.6mm 3)随机各取3只动物进行安乐死，获取瘤体后按公知的病理分析技术测定各系列的平均间质比。同时将各系列成模动物随机分为阴性对照组和7个研究组，阴性对照物为生理盐水，研究药物如下表所示。药物均为含水液体，按实施例1的制备方法配置而成。各组均瘤内注射1次，注射体积为160μl。用药结束后7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(R)，以及从抑瘤率计算实际/预期比(q)，研究组结果示于下表。
表17
Figure PCTCN2021076749-appb-000015
Figure PCTCN2021076749-appb-000016
根据病理分析测定，系列I和II的瘤体平均间质比分别为11.3％和19.7％。在本发明的范围内，除非另有说明，术语“较大间质比”是指≥20％的间质比，而＜20％的间质比则被称为较小间质比。肿瘤实质(例如癌细胞)与间质(例如非癌细胞成分中的***、纤维结构、等等细胞间质)之间存在着一系列复杂的动态演化关系。间质的数量和构成是肿瘤发生、发展的关健因素之一。间质比于是可以表征这种肿瘤异质性。越来越多的临床数据显示，在大致相同的治疗条件下，瘤体间质比是肿瘤治疗疗效(或预后)的一个独立因素。
实际上，以间质比为基础的异质性可以是如此之大，可使得同一种抗肿瘤药物对相同肿瘤细胞但不同间质比的瘤体有大不相同的疗效。从适应症的角度，不同间质比瘤体的肿瘤在这时必须被视作两种不同的肿瘤。例如，5-氟尿嘧啶应用于动物试验的人胰腺癌细胞移植瘤(通常间质比较小)有效(抑瘤率≥40％)、但应用于临床人胰胰癌(通常间质比较大)却无效(抑瘤率≤20％)。于是，动物人胰腺癌细胞移植瘤是、而临床人胰胰癌却不是5-氟尿嘧啶的适应症。
在上表中，各系列组合物组5-7的瘤重与阴性对照组相比均有统计意义(均为p＜0.05)。其中，各系列组合物组5和6的实际/预期比q均为＞1.15，而组合物组7的实际/预期比q均为＜1.15，说明前者的药物组合物均显示出局部协同作用，而后者的药物组合物未显示出局部协同作用。
尤其值得注意的是，相同药物在系列II中有效性低于在系列I中，以及相同局部协同组合物(组5或6)在系列II中显示的实际/预期比q大于在系列I中。该结果说明，本发明组合物的局部协同性、从而其中亚甲蓝类染料的药物效率在间质比较大的瘤体组织中大于在间质比较小的瘤体组织中，这使得本发明组合物有可能通过提高给药体积来提高其在前者中的药物效应。
根据以上研究及其它研究，本发明的含亚甲蓝类染料组合物可以解决同剂量的现有技术的含亚甲蓝类染料组合物所不能解决的问题：较大间质比的瘤体组织的有效破坏。本发明的组合物的适应症的一个优选方案为：较大间质比瘤体、以及包含该瘤体的肿瘤的治疗。
试验8C：试验动物为BALB/c裸小鼠。按公知技术将人肝癌细胞MDA-MB231以1×10 6个细胞/只在动物右侧腋部皮下进行细胞移植瘤建模，供系列II使用。移植成功后(瘤体凸起可见)，再将肝癌细胞HepG2以1×10 6个细胞/只在同一批其它动物右侧腋部皮下进行细胞移植瘤建模，供系列I使用。成功建模的系列I试验动物(肝癌患者，瘤体平均体积205mm 3)和系列II试验动物(肝癌患者，瘤体平均体积461mm 3)各随机分为阴性对照组和12个研究组，阴性对照物为生理盐水，研究药物如下表所示。药物均为含水液体，按实施例1的制备方法配置而成。各组均瘤内注射1次，系列1注射体积为100μl，系列2注射体积为180μl。用药结束后7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(R)，以及从抑瘤率计算实际/预期比(q)，研究组结果示于下表。
表18
Figure PCTCN2021076749-appb-000017
在上表中，各系列组合物组9的瘤重与阴性对照组相比均无统计意义(均为p＞0.05)，而各系列组合物组8、10-12的瘤重与阴性对照组相比均有统计意义(均为p＜0.05)。其中，各系列组合物组8和11的实际/预期比q均为＞1.15，而组合物组10和12的实际/预期比q为＜1.15，说明前者的药物组合物均显示出、而后者的药物组合物未显示出局部协同作用。尤其值得注意的是，相同的局部协同组合物组(组8和11)在系列II中显示的实际/预期比q大于在系列I中。
尤其值得注意的是，相同药物在系列II中有效性低于在系列I中，以及相同局部协同组合物(组8或10)在系列II中显示的实际/预期比q大于在系列I中。该结果说明，本发明组合物的局部协同性、从而其中亚甲蓝类染料的药物效率在较大瘤体的组织中大于在较小瘤体的组织中，这使得本发明组合物有可能通过提高给药体积来提高其在前者中的药物效应。
在本发明的范围内，除非另有说明，术语“较大瘤体”应用于人时是指平均尺寸≥2.0cm的瘤体，而平均尺寸＜2.0cm的瘤体则被称为较小瘤体。瘤体尺寸增大是肿瘤细胞增殖以及与瘤体中非瘤成分增长的一系列复杂的生存环境演化的结果。例如，肿瘤细 胞代谢动力学需要血管的重新分布，这反过来又需要肿瘤细胞的增殖优先在该重新分布最易发生的表层进行。此外，瘤体增大也使得非瘤成分(例如纤维间隔结构)对于瘤体组织生长的影响增大。瘤体平均尺寸于是可以表征这种肿瘤异质性。越来越多的临床数据显示，在大致相同的治疗条件下，瘤体平均尺寸是肿瘤局部治疗疗效(或预后)的一个独立因素。
实际上，以瘤体平均尺寸为基础的异质性可以是如此之大，可使得同一种局部药物或物理方法对相同肿瘤细胞但不同尺寸的瘤体有大不相同的疗效。从适应症的角度，不同尺寸瘤体的肿瘤在这时必须被视作两种不同的肿瘤。例如，经典化学消融剂应用于动物试验的人肝癌细胞移植瘤(通常瘤体较小)有效(抑瘤率≥40％)、但却不再被视作临床上瘤体较大人肝癌(平均尺寸≥2.0cm)的有效药物。于是，动物人肝癌细胞移植瘤是、而临床瘤体较大人肝癌却不是经典化学消融剂的适应症。
根据以上研究及其它研究，本发明的含亚甲蓝类染料组合物可以解决同剂量的现有技术的含亚甲蓝类染料组合物所不能解决的问题：较大瘤体内组织的有效破坏。本发明的组合物的适应症的一个优选方案为：较大瘤体、以及包含该瘤体的肿瘤的治疗。
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 (26)

  1. 组合物，其包含亚甲蓝类染料、局部协同物、以及任选存在的营养素或/和免疫调节剂，其中所述协同物包括或选自常规抗肿瘤药物中的一种或多种，其中在所述组合物中，所述亚甲蓝类染料在施用时的浓度(w/v)为≤2％、优选为0.35-2％、0.5-2％、0.5-1.5％或0.5-1％。
  2. 组合物，其包含亚甲蓝类染料和局部协同物，其中所述协同物包括或选自营养素中的一种或多种，以及任选存在的常规抗肿瘤药物或/和免疫调节剂。
  3. 组合物，其包含亚甲蓝类染料和局部协同物，其中所述协同物包括或选自免疫调节剂中的一种或多种，以及任选存在的营养素或/和常规抗肿瘤药物。
  4. 根据上述权利要求中任一项所述的组合物，其中所述亚甲蓝类染料包括或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. 根据上述权利要求中任一项所述的组合物，其中所述协同物包括或选自以下的一种或多种：同类局部协同物复合物，不同类局部协同物复合物，单一局部协同物或局部协同物复合物的pH调节复合物，其中：
    所述同类局部协同物复合物包括或选自以下的一种或多种：营养素复合物、免疫抑制剂复合物、常规抗肿瘤药物复合物；
    所述不同类局部协同物复合物包括或选自以下的一种或多种：营养素/常规抗肿瘤药物复合物、营养素/免疫抑制剂复合物、免疫抑制剂/常规抗肿瘤药物复合物；
    所述pH调节复合物包括局部协同物的酸化(局部协同物/酸)或碱化(局部协同物/碱)复合物，优选为使得局部协同物含水液体的pH变化值为＞1、更优选为使得局部协同物含水液体的pH趋于中性化的复合物。
  6. 根据权利要求2或4所述的组合物，其中所述营养素包括或选自以下的一种或多种：氨基酸类营养素、糖类营养素、脂类营养素。
  7. 根据权利要求6所述的组合物，其中所述氨基酸类营养素包括或选自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。
  8. 根据权利要求7所述的组合物，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，且在所述组合物中，所述氨基酸或氨基酸盐在施用时的浓度(w/v)为≥2％、≥2.5、≥5％、≥7.5％、10-25％或18-25％，优选为15％-25％或20％-25％。
  9. 根据权利要求7所述的组合物，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，且在所述组合物中，所述寡肽和多肽在施用时的浓度(w/v)为大于≥5％、优选为7.5-25％、更优选为10％-25％。
  10. 根据权利要求7所述的组合物，其中所述氨基酸类营养素选自所述氨基酸和/或氨基酸盐与所述寡肽和/或多肽的组合，且在药物组合物中，所述组合在施用时的浓度(w/v)为大于≥5％、优选为7.5％-25％、更优所述选为10-25％。
  11. 根据权利要求7所述的组合物，其中所述寡肽包括或选自以下的一种或多种：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；所述多肽为选自聚天冬氨酸、聚谷氨酸、聚赖氨酸中的一种或多种。
  12. 根据权利要求6所述的组合物，其中所述糖类营养素包括或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。
  13. 根据权利要求12所述的组合物，其中在所述药物组合中，所述糖类营养素在施用时的浓度(w/v)为大于5％，优选≥10％、10-40％、15-50％或25-50％。
  14. 根据权利要求6所述的组合物，其中所述脂类营养素包括或选自以下的一种或多种：脂肪酸、脂肪乳和类脂，优选包括或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，且在所述组合物中，所述脂类营养素在施用时的浓度≥4％，优选为4％-25％。
  15. 根据权利要求1-3中任一项所述的组合物，其中所述抗肿瘤药物包括或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。
  16. 根据权利要求15所述的组合物，其中所述常规抗肿瘤药物包括或选自以下的一种或多种：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。
  17. 根据权利要求1或2所述的组合物，其还包括免疫调节剂，所述免疫调节剂包括或选自抗体、免疫调节肽、益生菌组分中的一种或多种。
  18. 根据权利要求5所述的组合物，其中所述酸选自药学上可接受的pH调节酸，优选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；所述碱选自药学上可接受的pH调节碱，优选为选自以下之一种或多种：氢氧化钠、碳酸氢钠、氢氧化钾。
  19. 根据权利要求1-18中任一项所述的组合物，其中所述组合物被制备为注射剂的剂型，所述注射剂包括液体注射剂和注射用粉针剂。
  20. 根据权利要求19所述的组合物，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. 根据权利要求15-20中任一项所述的组合物，其中所述亚甲蓝类染料与营养素、免疫调节剂、抗肿瘤药物的量比选自以下的一种或多种：
    所述营养素/亚甲蓝类染料组合物的局部协同量比(W 营养素/W 亚甲蓝类染料)为1/3＜W 营养素/W 亚甲蓝类染料＜40/0.15，优选为1/3＜W 营养素/W 亚甲蓝类染料≤40/0.30、或1/1.8＜W 营养素/W 亚甲蓝类染料≤40/0.30；
    所述免疫调节剂/亚甲蓝类染料组合物的局部协同量比(W 免疫调节剂/W 亚甲蓝类染料)为0.2/3＜W 免疫调节剂/W 亚甲蓝类染料≤20/0.15，优选为0.5/3＜W 免疫调节剂/W 亚甲蓝类染料≤20/0.50、或0.5/1≤W 免疫调节剂/W 亚甲蓝类染料≤20/0.50；
    所述常规抗肿瘤药物/亚甲蓝类染料组合物的局部协同量比(W 常规抗肿瘤药物/W 亚甲蓝类染料)为0.1/3＜W 常规抗肿瘤药物/W 亚甲蓝类染料＜1.5/0.15，优选为0.3/3＜W 常规抗肿瘤药物/W 亚甲蓝类染料＜1.5/0.15、或0.3/1.8≤W 常规抗肿瘤药物/W 亚甲蓝类染料≤1.5/0.30。
  22. 包含根据权利要求1-12中任一项所述的组合物的医疗装置。
  23. 根据权利要求1-12中任一项所述的组合物在制备用于治疗癌症的药物组合或制剂中的应用。
  24. 根据权利要求21所述的应用，其中所述癌症包括实体肿瘤，例如肉瘤、头颈癌、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。
  25. 根据权利要求23或24所述的应用，其中所述亚甲蓝类染料与营养素、免疫调节剂、抗肿瘤药物的量比选自以下组之一种或多种：
    所述营养素/亚甲蓝类染料组合物的局部协同量比(W 营养素/W 亚甲蓝类染料)为1/3＜W 营养素/W 亚甲蓝类染料＜40/0.15，优选为1/3＜W 营养素/W 亚甲蓝类染料≤40/0.30、或1/1.8＜W 营养素/W 亚甲蓝类染料≤ 40/0.30；
    所述免疫调节剂/亚甲蓝类染料组合物的局部协同量比(W 免疫调节剂/W 亚甲蓝类染料)为0.2/3＜W 免疫调节剂/W 亚甲蓝类染料≤20/0.15，优选为0.5/3＜W 免疫调节剂/W 亚甲蓝类染料≤20/0.50、或0.5/1≤W 免疫调节剂/W 亚甲蓝类染料≤20/0.50；
    所述常规抗肿瘤药物/亚甲蓝类染料组合物的局部协同量比(W 常规抗肿瘤药物/W 亚甲蓝类染料)为0.1/3＜W 常规抗肿瘤药物/W 亚甲蓝类染料＜1.5/0.15，优选为0.3/3＜W 常规抗肿瘤药物/W 亚甲蓝类染料＜1.5/0.15、或0.3/1.8≤W 常规抗肿瘤药物/W 亚甲蓝类染料≤1.5/0.30。
  26. 根据权利要求22-24中任一项所述的应用，其中所述恶性实体肿瘤优选为选自以下的一种或多种：含间质比≥20％瘤体的肿瘤、含平均尺寸≥2.0cm的瘤体的肿瘤、恶性肿瘤相关结节。
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