WO2022068924A1 - 益生菌组分的应用以及包含益生菌组分的药物组合物 - Google Patents

益生菌组分的应用以及包含益生菌组分的药物组合物 Download PDF

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WO2022068924A1
WO2022068924A1 PCT/CN2021/122132 CN2021122132W WO2022068924A1 WO 2022068924 A1 WO2022068924 A1 WO 2022068924A1 CN 2021122132 W CN2021122132 W CN 2021122132W WO 2022068924 A1 WO2022068924 A1 WO 2022068924A1
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Prior art keywords
probiotic
local
component
pharmaceutical composition
concentration
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PCT/CN2021/122132
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English (en)
French (fr)
Inventor
邹方霖
邹礼常
王建霞
王艺羲
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成都夸常奥普医疗科技有限公司
夸常股份有限公司
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Priority claimed from CN202011059746.1A external-priority patent/CN114306612A/zh
Priority claimed from CN202011064448.1A external-priority patent/CN114306392A/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
Priority to EP21874578.4A priority Critical patent/EP4223300A4/en
Priority to JP2023519649A priority patent/JP2023544310A/ja
Priority to US18/247,421 priority patent/US20230405063A1/en
Publication of WO2022068924A1 publication Critical patent/WO2022068924A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/062—Ascomycota
    • A61K36/064—Saccharomycetales, e.g. baker's yeast
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/006—Biological staining of tissues in vivo, e.g. methylene blue or toluidine blue O administered in the buccal area to detect epithelial cancer cells, dyes used for delineating tissues during surgery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/844—Liver

Definitions

  • the present invention relates to the technical field of pharmaceutical preparations, in particular to a new application of probiotic components and a pharmaceutical composition comprising the probiotic components.
  • a solid tumor is a representative model of localized disease; it is a neoplastic disease with symptoms of a tumor that contains tumor tissue that contains tumor cells and a larger number of other components ( Also sometimes referred to as the tumor cell microenvironment), such as various other cells, various intercellular spaces, various conduits, and the like.
  • tumor cell microenvironment Also sometimes referred to as the tumor cell microenvironment
  • pancreatic cancer cells only account for about 30% of the tumor volume.
  • Cytotoxic drugs and chemical ablative agents are common drugs for the treatment of solid tumors in the past century.
  • intratumoral administration of cytotoxic drugs can increase their intratumoral concentration, their efficacy is only slightly increased within the expected range of cancer cell inhibition kinetics (usually less than 200%); considering the compliance of patients, currently Cytotoxic drugs are basically administered systemically orally or intravenously.
  • chemical ablative agents usually do not have tumor cell destruction but tumor tissue destruction as their pharmacological characteristics.
  • the pharmacological effects of intratumoral administration are much greater than the expected range of their cancer cell inhibition kinetics, and their pharmacology is significantly different from systemic administration.
  • the intervention volume and intervention site are very limited, for example, the amount of acid and alkali does not exceed 0.2ml/kg and The limitation of the organ where the tumor is located and the limited ablation of the tumor edge have gradually faded out of the clinic.
  • tumor immunotherapy methods represented by tumor vaccines, cell adoptive therapy and immune checkpoint inhibitors.
  • These therapeutic drugs vary widely, but they have very high requirements on patient gene specificity and tumor specificity.
  • In ordinary animal experiments there is almost no obvious difference between the residual weight of the tumor between the administration group and the negative control group.
  • the clinical adaptation The range of symptoms is very small.
  • probiotics may have three mechanisms of action to support their beneficial effects: (1) inhibit the growth of pathogenic bacteria by binding to intestinal epithelial cells to compete for pathogen attachment sites or deplete their nutrients; (2) improving the intestinal barrier (3) Improve the immune function of the body.
  • probiotics can improve the immune function of the body is still attracting attention.
  • yeast as a feed additive to enhance the immune function of pigs (eg Patent Publication No. CN106387398A).
  • inactivated yeast for enhancing immune function, antibacterial and antiviral (eg Patent Publication No.: CN108524925A).
  • the antitumor effects of probiotics in the prior art are believed to be mainly related to their bacterial immunogenicity.
  • the immunogenicity has not shown the therapeutic effect of the therapeutic vaccine antigen, so it is mainly used to enhance the immunity of the patient's body.
  • Immune heterogeneity is one of the characteristics of tumor heterogeneity. This pharmacology makes the immune enhancement of probiotics only have effect on colorectal cancer, showing strong tumor specificity. Therefore, relative to other drugs, probiotics did not show meaningful tumor growth inhibition in animal experiments (tumor inhibition rate ⁇ 15% or relative tumor proliferation rate ⁇ 85%), and these anti-tumor immune enhancement effects provided are very strong Limited, it only plays the role of adjuvant therapy, and it is far from the role of providing therapy.
  • the present invention aims to propose a new pharmacology based on probiotic components-that is to provide a local action (or local synergy) and its secondary action (or secondary synergy), thereby preparing a topical pharmaceutical composition, Achieving high efficiency and low toxicity, especially in the medium and long term, provides various options for clinical use.
  • a probiotic component as an active ingredient that can provide a therapeutic effect in the preparation of a topical pharmaceutical composition for the treatment of a local pathological disease, wherein the therapeutic effect includes a topical treatment involving a local effect (or a local synergistic effect) or/and Immunotherapy associated with this local effect (or local synergy).
  • the pharmaceutical composition further comprises a chemically active ingredient capable of producing a synergistic effect with the probiotic component, and wherein the amount ratio of the probiotic component to the chemically active ingredient (weight of the probiotic component) Concentration/weight concentration of common substance) is (1-110)/(1-100).
  • a topical pharmaceutical composition for the treatment of local pathological diseases which comprises a probiotic component that can provide a therapeutic effect, and a pharmaceutically acceptable suitable carrier, but does not contain a specific agent for increasing blood osmotic pressure to normal physiological levels. Salts or monosaccharides, wherein the therapeutic effect includes topical therapy or/and immunotherapy involving local action (or local synergy).
  • it also optionally contains chemically active ingredients that can synergize with the probiotic components.
  • a topical pharmaceutical composition for treating local pathological diseases which comprises a probiotic component that can provide a therapeutic effect, a chemically active ingredient capable of producing a synergistic effect with the probiotic component, and a pharmaceutically acceptable suitable carrier , and the amount ratio of the probiotic component to the chemically active ingredient (weight concentration of the probiotic component/weight concentration of the common substance) is (1-110)/(1-100), wherein the therapeutic effect includes involving Locally acting (or locally synergistic) local therapy or/and immunotherapy.
  • the probiotic component is selected from a preparation derived from natural probiotics or engineered bacteria with minimal bacterial immunogenicity or an engineered analog of the preparation, wherein the preparation is preferably selected from It includes one or more of the following groups and derivatives thereof: water-soluble components of probiotics, semi-fluid components of probiotics, water-insoluble particles of probiotic components, and inactivated probiotics.
  • the probiotic water-soluble component is selected from one or more of the following groups and derivatives thereof: probiotic or broken probiotic supernatant component, probiotic extract, probiotic intracellular water-soluble
  • the probiotic semi-fluid component is preferably selected from the group of probiotics whose aqueous mixture can form a semi-fluid composition
  • the water-insoluble particles of the probiotic component are selected from one or more of the following groups and derivatives thereof: crushed probiotic precipitation components, probiotic cell wall polysaccharide particles, probiotic cell wall polysaccharide nanoparticles
  • the inactivated probiotics are preferably selected from inactivated probiotics that are not limited to complete cells;
  • probiotic water-soluble component is selected from one or more of the following groups and derivatives thereof: probiotic water-soluble beta-glucan, preferably water-soluble beta-glucan with a purity greater than 90% Sugar, probiotic ribonucleic acid.
  • the probiotics are selected from one or more of the following groups of natural bacteria or/and engineered bacteria: probiotic bacillus, probiotic lactobacillus, probiotic bifidobacteria, and probiotic fungi.
  • the Bacillus spp comprises one or more selected from the group consisting of: One or more of the following groups: Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus pumilus, and Lactobacillus fermentum;
  • the bifidobacteria include one or more selected from the group consisting of: Bifidobacterium longum Bifidobacterium, Bifidobacterium adolescentis, Bifidobacterium breve, Enterococcus faecium, Streptococcus faecalis;
  • the fungi include one or more selected from the group consisting of: Saccharomyces cerevisiae, Saccharomyces brucei, and Wherein the yeast comprises one or more selected from the group consisting of: Saccharomyces cerevisiae, Saccharomyces cerevisiae, Saccharomyces cerevisiae, Sac
  • the probiotics are selected from Saccharomyces cerevisiae or/and Brucella saccharomyces.
  • the suitable carrier is water;
  • the chemically active ingredient is soluble in water, and the probiotic component is water-insoluble and is one or more selected from the group consisting of: : Inactivated probiotics, water-insoluble granules of probiotic components, semi-fluid components of probiotics.
  • the concentration of the probiotic component in the pharmaceutical composition is >0.1%, ⁇ 0.25%, 0.25-25%, preferably 0.5-15%, more preferably 1-15% or 5-15% %.
  • the 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 concentration of the probiotic water-soluble component in the pharmaceutical composition is >0.1, or 0.15- 25%, preferably 0.35-15% or 5-15%; when the probiotic component includes the probiotic water-insoluble component granules, the probiotic water-insoluble component granules are contained in the pharmaceutical composition.
  • the concentration is >0.5, or 0.5-15%, preferably 1.5-15% or 5-15%; when the pharmaceutical composition is a semi-fluid composition, the probiotic semi-fluid component is in the pharmaceutical composition Concentrations are >2.5%, 2.6-25%, preferably 5-15%.
  • the chemically active ingredients include one or more selected from weak local action compounds and/or cytotoxic drugs
  • the probiotic components The amount ratio (W probiotic component /W cytotoxic drug ) to cytotoxic drug is (1-110)/(1-100); when the chemically active compound includes a weak local action compound, the probiotic
  • the amount ratio of the component and the weakly locally acting compound (V probiotic component /W weakly locally acting compound ) was (1-90)/(1-100).
  • the weak local action compound is selected from one or more of the following groups: amino acid nutrients, vital dyes, quinine drugs, low-concentration acidifiers, low-concentration alkalizers, containing acidifiers or/or A pH buffering system for an alkalizing agent, and wherein when the weak topical compound includes the amino acid nutrient, the amount ratio (W probiotic component /W amino acid nutrient) of the probiotic component to the amino acid nutrient (W probiotic component/W amino acid nutrient ) is ( 1-20)/(1-100); when the weak local action compound includes the vital dye, the amount ratio of the probiotic component to the vital dye (W probiotic component /W vital dye ) is ( 7-90)/(1-100); when the weak local action compound includes the quinine, the amount ratio of the probiotic component to the quinine (W probiotic component /W quinine ) Ninth drugs ) is (2-90)/(1-100); when the weak local action compound includes the acidifying agent
  • the chemically active ingredient is selected from vital dyes and one or more other chemically active ingredients.
  • the chemically active ingredient is selected from the cytotoxic drugs and vital dyes.
  • the amino acid nutrients are amino acids or their salts selected from the group consisting of the following groups, or oligopeptides and polypeptides comprising or consisting of the following amino acids: arginine, lysine, glycine, cysteine, alanine
  • concentration of amino acid, serine, aspartic acid, glutamic acid, and said amino acid nutrients is >2.5%, or 5-30%, preferably 5-25%.
  • the vital dye is one or more selected from the group consisting of Bengal red or/and the following methylene blue dyes: methylene blue, patent blue, isosulfur blue, new methylene blue, and wherein the The concentration of red Bengal is 2.5%-20%; the concentration of the methylene blue dye is ⁇ 0.25%, or 0.25-2.5%, preferably 0.5-2.5%.
  • the acidulant is selected from one or more of strong acid or/and weak acid, and the amount ratio of the probiotic component to the acidulant (weight concentration of probiotic component/weight concentration of acidulant) is (1-20)/(0.5-50), and the concentration of the acidulant is ⁇ 0.5%, 0.5-2% (strong acid), or 2-15% (weak acid), wherein the strong acid is, for example, hydrochloric acid;
  • the weak acid is, for example, oxalic acid, acetic acid, lactic acid, citric acid and malic acid;
  • the amount ratio of the alkalizing agent (weight concentration of probiotic bacteria component/weight concentration of alkalizing agent) is (1-20)/(0.5-50), and the concentration of the alkalizing agent is ⁇ 0.5%, 0.5-5% (strong base), or 2-15% (weak base), wherein the strong base is, for example, sodium hydroxide, potassium hydroxide; the weak base is, for example, sodium dihydrogen phosphate, sodium bicarbonate, and sodium carbonate.
  • the cytotoxic drugs include one or more selected from the group consisting of: drugs that disrupt DNA structure and function, such as cyclophosphamide, carmustine, metal platinum complexes, doxorubicin Class drugs, topotecan, irinotecan; drugs that interfere with transcriptional RNA intercalated in DNA, such as anti-tumor antibiotics; drugs that interfere with DNA synthesis, such as 5-fluorouracil (5-Fu), fur-fluorouracil, difur-fluorouracil, azithromycin Glycocytidine, cyclocytidine, 5-azacytidine; drugs that affect protein synthesis, such as colchicine, vinblastine, taxane, and the concentration of the cytotoxic drug is ⁇ 0.1 %, 0.1-15%.
  • drugs that disrupt DNA structure and function such as cyclophosphamide, carmustine, metal platinum complexes, doxorubicin Class drugs, topotecan, irinotecan
  • the pharmaceutical composition further optionally includes a biologically active ingredient, wherein the biologically active ingredient is selected from one or more of the following groups: antigens, immunomodulatory antibodies, cytokines, and adjuvants.
  • a biologically active ingredient is selected from one or more of the following groups: antigens, immunomodulatory antibodies, cytokines, and adjuvants.
  • a method of treating a local lesion comprising the steps of topically administering a therapeutically effective amount of the above-described pharmaceutical composition to the local lesion and/or extralocally lesions in an individual in need thereof.
  • it comprises the step of administering a therapeutically effective amount of the pharmaceutical composition within the local lesion, or both within the local lesion and outside the local lesion in the individual.
  • it also includes optionally one or more other treatments, such as chemotherapy, immunotherapy, radiation therapy, surgery, chemical ablation, physical ablation, before, during or after administration of the pharmaceutical composition.
  • treatments such as chemotherapy, immunotherapy, radiation therapy, surgery, chemical ablation, physical ablation, before, during or after administration of the pharmaceutical composition.
  • the content of the inactivated probiotic in the pharmaceutical composition must be such that its local administration concentration 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 probiotic water-soluble component in the pharmaceutical composition The content must be such that its topical concentration is >0.1, or 0.15-25%, preferably 0.35-15% or 5-15%; when the probiotic component includes the probiotic water-insoluble component, the The content of the probiotic water-insoluble component in the pharmaceutical composition must be such that its local administration concentration is >0.5, or 0.5-15%, preferably 1.5-15% or 3.5-15%.
  • the content or unit content of the pharmaceutical composition is such that its administration volume is multiplied by the following coefficient multiplied by the volume of the target volume in the local lesion: >0.1, 0.15-1.5, preferably 0.23-1.5 or 0.5-1.5.
  • the content or unit content of the pharmaceutical composition is such that its administration volume is ⁇ 1ml, or 10-150ml for intralesional administration or/and 1.5-50ml for extralocal administration.
  • the local lesions comprise tumor cells or/and fibroblasts.
  • the locally diseased disease includes tumors, non-tumoral enlargement, local inflammation, secretory gland dysfunction and skin diseases, wherein the tumor includes malignant and non-malignant solid tumors.
  • the solid tumor includes one or more of the following tumors and their secondary tumors: breast cancer, pancreatic cancer, thyroid cancer, nasopharyngeal cancer, prostate cancer, liver cancer, lung cancer, intestinal cancer, oral cancer, esophageal cancer , gastric cancer, throat cancer, testicular cancer, vaginal cancer, uterine cancer, ovarian cancer, brain tumor, lymphoma.
  • the local effect includes a local chemical effect (or local chemical synergy) and optionally other effects
  • the local treatment includes chemical-like ablation of one or more local lesions and Optionally other chemotherapy
  • said immunotherapy includes said locally acting (or locally synergistic) secondary immune effects and optionally other immune effects within said lesions or/and outside of said lesions.
  • the applicable patients for the treatment are selected from one or more of the following groups: immunosuppressed patients, patients who can be administered in local lesions, patients whose local diseased tissue can be chemically ablated, and patients who can produce local lesions in local lesions. Patients with secondary immune substances and patients who can produce secondary immune substances in the administration area outside the lesion.
  • a pharmaceutical composition comprising a probiotic component and a suitable pharmaceutically acceptable carrier for the treatment of local disease, wherein the local disease includes solid tumors.
  • a pharmaceutical kit comprising one or more containers containing the above-described pharmaceutical composition.
  • the pharmaceutical kit may also include instructions or labels on how to administer the pharmaceutical composition to an individual in need thereof.
  • said administering comprises intralesional administration, or both intralesional and extralocalized administration, wherein said extralesional administration, for example, comprises subcutaneous injection under the armpit of said individual.
  • a local medicine for the treatment of local lesions comprising a probiotic component and a pharmaceutically acceptable carrier, and the concentration of the probiotic component is 0.25- 25%.
  • the topical drug further includes a weak local action compound and/or a cytotoxic drug, wherein the concentration of the weak local action compound is 1-20%, and the concentration of the cytotoxic drug is 1-5%.
  • the composition of the topical medication includes 0.25%-10% probiotic components, and also includes 0.1-2.5% cytotoxic drugs and/or 5-20% amino acid nutrients and/or 0.5-1% reactive dyes.
  • the composition of the topical medication includes 0.5%-10% probiotic components, and further includes 0.5-1% methylene blue or 5-20% lysine or 0.1-2.5% gemcitabine or 5-fluorouracil; the The composition of the topical application includes 0.25-0.5% probiotic component and 5-10% arginine.
  • the topical medicine is composed of 2.5% probiotic component, 1% 5-fluorouracil, and the balance is water for injection; or it is composed of 2.5% probiotic component, 2.5%, 10% arginine, and the balance is water for injection; Or 2.5% probiotic components, 1% 5-fluorouracil, 10% arginine, and the balance is water for injection; or it consists of 5% heat-killed Saccharomyces boulardii, 1% methylene blue, and the balance is Water for injection; or the composition is 5% heat-killed Saccharomyces boulardii, 1% 5-fluorouracil, and the balance is water for injection; or the composition is 5% heat-killed Saccharomyces boulardii, 1% 5-fluorouracil, 1% % methylene blue, the balance is water for injection; or the composition is 7.5% beta-glucan, 1% methylene blue, 1% 5-fluorouracil in a semi-fluid composition, and the balance is water for
  • the composition of the topical medication includes 10% probiotic components, 1% methylene blue, and 1% 5-fluorouracil.
  • the composition of the topical medication includes 0.5% probiotic component/7% sodium bicarbonate/3% sodium hydroxide or 10% probiotic component/0.7% sodium bicarbonate/0.3% sodium hydroxide.
  • the probiotic components include at least one of inactivated probiotics, water-soluble components of probiotics, particles of water-insoluble components of probiotics, and semi-fluid components of probiotics.
  • the probiotics include at least one of Bacillus, Lactobacillus, Bifidobacterium, and probiotic fungi.
  • the probiotic is Saccharomyces boulardii.
  • the topical medication does not contain osmotic pressure regulators and flavoring agents.
  • the topical drug of the present invention has the following advantages: a completely new pharmacology (local action or local synergistic action and their secondary immune action) is provided, so that the existing probiotic composition can be used to achieve the desired effect.
  • Local treatment including chemical-like ablation or/and immunotherapy including secondary immune effects can be performed, which local treatment can produce curative effects far exceeding existing technical solutions (for example, more than 3 times the tumor inhibition rate)
  • the immunotherapy can also produce immunological efficacy far exceeding prior art solutions (eg, within the lesion area).
  • embodiments of the present invention also have the potential to greatly reduce the safety risks of prior art solutions that do not limit the mode of administration.
  • Embodiments according to the present invention have the following advantages over the prior art of other compositions for the treatment of local pathological diseases: show almost non-toxic systemic safety and significantly higher long-term efficacy compared to existing cytotoxic drugs ; Compared with existing molecular targeted drugs, it shows less stringent indication screening and great potential for fast-growing tumors, large tumors and hypovascular tumors; Compared with existing chemical ablative agents, Shows significantly lower local irritation and better long-term effects.
  • the applications and compositions of the present invention are also not plagued by drug resistance problems encountered with existing cytotoxic drugs and existing molecularly targeted drugs.
  • the application and composition are convenient to prepare and low cost, and are particularly helpful for making safe and effective treatment available to a large number of people who cannot afford high costs.
  • the present invention also provides the use of the above-mentioned topical medication in preparing a topical pharmaceutical composition for treating local pathological diseases, wherein the therapeutic action includes topical treatment or/and immunotherapy involving local action (or local synergistic action). effect.
  • the administration volume V 1 is 1.5-150ml, or if the administration is in the local lesion, the administration volume V 1 is 10-150ml, and if the administration is outside the local lesion, the administration volume V 1 is 1.5- 50ml.
  • the local lesions include tumors, non-tumor enlargements, local inflammation, abnormal secretion of glands, and skin diseases, wherein the tumors include malignant and non-malignant solid tumors.
  • the solid tumor is breast cancer, pancreatic cancer, thyroid cancer, nasopharyngeal cancer, prostate cancer, liver cancer, lung cancer, colon cancer, oral cancer, esophagus cancer, gastric cancer, throat cancer, testicular cancer, vaginal cancer, uterine cancer , ovarian cancer, brain tumor, lymphoma.
  • the present invention also provides a pharmaceutical kit, comprising one or more containers containing the above-mentioned pharmaceutical composition.
  • the pharmaceutical kit further comprises instructions or labels on how to administer the pharmaceutical composition to an individual in need thereof.
  • the administering comprises intralesional administration, or both intralesional and extraterrestrial administration, wherein the extraterrestrial administration is an axillary subcutaneous injection.
  • the present invention also provides a method of treating a local lesion, comprising administering a therapeutically effective amount of the above-mentioned pharmaceutical composition to the local lesion of a patient or to the local lesion and the local lesion.
  • a therapeutically effective amount of the above-mentioned pharmaceutical composition is also performed before and/or during and/or after the administration of the pharmaceutical composition.
  • the local administration concentration of the probiotic component therein is 0.25-25%, preferably 0.5-15%, more preferably 1-15% or 5-15%.
  • the patient is one or more selected from the group consisting of immunosuppressed patients, patients who can be administered in local lesions, patients whose local lesions can be chemically ablated, and secondary immunity can be generated in local lesions Any of a patient who has a substance or a patient who can produce a secondary immune substance in an extra-lesional administration area.
  • L-amino acids are all abbreviated as amino acids, for example, L-arginine is abbreviated as arginine.
  • a substance shows therapeutic activity under specific conditions, it means that it can be used as an active ingredient in the treatment of local pathological diseases.
  • the same substance can show different activities or pharmacology under different conditions, for example, ethanol is usually used as a bactericide, and high concentration of ethanol shows the pharmacology of chemical ablation of tumor tissue by intratumoral administration.
  • the inventors unexpectedly discovered in the tumor-bearing nude mice experiments that inactivated probiotics, which are usually used as immune enhancers, can actually produce meaningful local chemical effects, and possibly even chemical-like ablation effects, under certain specific conditions.
  • the specific conditions are not the application conditions of the probiotic components in the prior art, but are as defined below.
  • An object of the invention of the present application is to provide the use of a probiotic component in the preparation of a topical pharmaceutical composition for the treatment of local pathological diseases, where the probiotic component can be used as an active ingredient with a therapeutic effect.
  • the relevant terms in this application are explained below:
  • probiotics refers to non-pathogenic live bacteria capable of producing beneficial effects on the health of the host.
  • probiotic component refers to a preparation derived from a natural probiotic or its engineered bacteria (eg, muramin) or an analog of the preparation.
  • analog refers to an analogous substance with similar activity to the preparation, including derivatives of the preparation (eg, muramin derivatives that can improve water solubility or activity), synthetics (eg, muramin-like derivatives) Synthetic polysaccharides), preparations from other sources similar to probiotic preparations (eg other sources of polysaccharides similar to muramins).
  • drug or "pharmaceutical composition” refers to a substance that contains an active ingredient and that gives it the pharmacological method, pharmacological composition, and other pharmacological characteristics necessary to achieve its pharmacology in a patient.
  • active ingredient refers to a substance that can perform a specific pharmacology under specific conditions.
  • pharmacological method refers to the method of administration necessary to achieve a particular pharmacology, eg, the pharmacological method for improving the intestinal barrier of probiotics is oral administration.
  • pharmaceutical composition refers to the composition of the components of the pharmaceutical composition necessary to achieve a particular pharmacology, especially the pharmaceutical composition entering the target area.
  • active kinetic conditions refers to the quantitative conditions that must be met for an active ingredient to achieve a particular pharmacology, and the same substance used for different active ingredients may therefore need to satisfy different kinetic conditions.
  • dosage form conditions refers to the pharmacological reaction environmental conditions that must be met in order to ensure that the active ingredient achieves a specific pharmacology, and the same substance used as different active ingredients may need to meet different dosage form conditions.
  • the pharmaceutical composition comprising probiotics and their components
  • medicated blood containing a lot of substances
  • conventional dosage forms as topical active ingredients in the present invention, they are highly sensitive to the reaction environment, and some commonly used additives (such as the following implementations)
  • sodium chloride commonly used as an osmotic pressure regulator, may have a negative effect on the local activity of the probiotic components, so a closed pharmacological formulation (preferably containing only the active ingredient and vehicle) must be used to allow exogenous interference. minimize.
  • therapeutic drug is distinguished from “adjuvant (therapeutic) drug,” which refers to a drug that provides a therapeutic effect, and the latter refers to a drug that provides an adjunctive (therapeutic effect).
  • adjuvant (therapeutic) effect the former refers to the main pharmacological effect (such as local treatment of local lesions or/and immunotherapy) that effectively alleviates, improves or heals the disease, while the latter refers to a disease that does not effectively alleviate A secondary pharmacological effect that improves or cures the disease but is also beneficial to the patient (eg, immune enhancement of the body).
  • Therapeutic effects usually include significant efficacy when used alone, or in combination with other drugs or in combination with other therapeutic methods to provide a major or equivalent effect; and adjuvant (therapy) usually when used alone provides only beneficial but insignificant effects effect, or provide a beneficial but non-primary or equivalent effect in the apparent efficacy of co-administration with other drugs or in combination with other treatments.
  • novel curative effect refers to effectively inhibiting the growth or development of local lesions compared with classical chemotherapeutic drugs or classical chemical ablative agents, such as the clinical pathological response rate (PR+CR) ⁇ 30% for local lesions, and also Or the local lesion growth inhibition in animal experiments has non-negligible pharmaceutical significance, such as tumor proliferation rate ⁇ 85% or tumor inhibition rate ⁇ 15%, preferably tumor proliferation rate ⁇ 60% or tumor inhibition rate ⁇ 40%.
  • the same substance used as different active ingredients may then provide completely different effects.
  • compositions containing probiotics and their components which in the prior art mainly provide auxiliary effects when used as systemically acting active ingredients, such as immune-enhancing effects that are often insufficient to observe tumor suppressive effects;
  • auxiliary effects when used as systemically acting active ingredients, such as immune-enhancing effects that are often insufficient to observe tumor suppressive effects;
  • a topical active ingredient it mainly provides a therapeutic effect.
  • topical drug or “topical drug” is different from “conventional drug” or “conventional drug”, the latter refers to drugs based on the pharmacology of systemic action, while the former refers to drugs based mainly on the pharmacology of local action, and there is a distinction between the two.
  • the former is usually a therapeutic drug, while the latter can be a therapeutic drug or an adjuvant (therapeutic) drug, and their effects on the indications may be completely different;
  • the pharmacological method of the former is local, but it is local lesion administration, while the latter Mainly routine administration (systemic administration), their concerns about administration side effects may be completely different;
  • the pharmacologically active ingredients of the former must provide local effects, while the pharmacologically active ingredients of the latter must provide systemic effects, and their The preferred directions may be completely opposite;
  • the element in the pharmacokinetic composition of the former is the administration concentration, while the latter is concerned with the administration dose, and their drug concentration may have completely different meanings;
  • the pharmacological dosage form of the former is a topical dosage form, while the latter
  • the pharmacological dosage forms of these drugs are conventional administration dosage forms, and their pharmacological dosage forms may have completely different requirements.
  • topical administration is different from conventional administration (systemic administration), and refers to any mode of administration for the purpose of producing a local effect, including intralesional administration or/and extra-lesional favorable localization that can produce a local therapeutic effect.
  • Intradermal administration e.g., subcutaneous injection that produces local effects and facilitates secondary immune effects of local effects
  • conventional administration refers to any mode of administration for the purpose of producing systemic effects, including administration of the drug through the alimentary tract (e.g., Oral) or vascular (eg, intravenous, intraperitoneal) administration followed by blood delivery to the target area.
  • intralesional administration refers to any mode of administration that allows the drug to enter the local lesion directly rather than indirectly into the local lesion by means of the drug-carrying blood, such as intratumoral drug delivery, local lesion implantation, local lesion smear, local lesion spray , subcutaneous or transvascular local intralesional injection, local lesion insertion, and the like.
  • local therapy is distinguished from “systemic therapy”, which refers to the use of systemic pharmacology (conventional activity) primarily in the medicated blood against a patient's systemic lesions (e.g. tumors, areas connected to tumors, tumors contained in other parts of the body)
  • systemic therapy refers to the use of systemic pharmacology (conventional activity) primarily in the medicated blood against a patient's systemic lesions (e.g. tumors, areas connected to tumors, tumors contained in other parts of the body)
  • the former refers to the treatment that mainly uses the local action pharmacology (local activity) of the drug itself to target the local area where the local lesion of the patient is located (such as the local lesion and other lesion areas connected to it).
  • the main function of the probiotic components used in the treatment of local pathological diseases is based on the therapeutic auxiliary effect (such as immune enhancement) that they can provide as a xenogeneic antigen, rather than a therapeutic effect, let alone a local therapeutic effect.
  • therapeutic auxiliary effect such as immune enhancement
  • topical activity is distinguished from conventional activity, which may provide a local effect (local synergy) or/and a secondary effect (intermediate and long-acting synergy) associated with that local effect (local synergy), whereas conventional activity usually Only provide systemic effects (eg cytotoxicity, immune enhancement), which are two completely different pharmacological activities.
  • the term "local action” is different from conventional action, and refers to the pharmacological action, usually including local chemical action, produced in the local area (such as tumor or/and extra-tumoral local) where the drug penetrates into the interstitial space after local administration, while conventional
  • the effect refers to the pharmacological effect of the drug delivered to the target area in the form of blood drug after routine drug administration through the digestive tract or blood vessels.
  • topical chemical action refers to a localized action that includes chemical action.
  • local synergy refers to a local effect that includes synergy.
  • topical chemical synergy refers to topical chemical effects that include synergy. Local activity sometimes produces systemic effects through its secondary effects, such as secondary spanning effects.
  • the topical chemical effects (or topical chemical synergy) described herein pharmacologically include general topical chemical effects, chemical ablation, and chemical-like ablation.
  • ordinary local chemistry refers to a local chemistry where the effect of a drug does not exceed the maximum expected (eg, within 200%) of the kinetic difference in conventional chemistry of the same drug, such as chemotherapy produced by routine administration of cytotoxic drugs.
  • chemical ablation refers to local chemistries in which the effect of a drug exceeds the maximum expected (eg, greater than 200%, preferably greater than 400%) kinetic differences for the same drug's conventional chemical effects (eg, chemotherapy produced by routine administration of high concentrations of ethanol).
  • the effect usually refers to the local chemical effect exhibited by classical chemical ablative agents (such as high concentration of ethanol, high concentration of acid, high concentration of alkali).
  • classical chemical ablative agents such as high concentration of ethanol, high concentration of acid, high concentration of alkali.
  • chemoablation-like refers to the local effect of a drug effect similar to that of chemical ablation (extraordinary effect expected). Although chemical-like ablation is not caused by classical chemical ablation agents, its pharmacological effects are obviously different from ordinary local chemical effects.
  • second effect is different from the term “direct effect”, the latter refers to the effect of the drug directly reacting with the drug target after entering the target area (for example, the local effect of local drugs entering local lesions and the local effect of the diseased tissue), while the former refers to Related to the latter, but not identical, and producing a later effect in time.
  • immunotherapy is different from the term “immunity enhancement”, the former refers to the immune effect that can achieve therapeutic effect when used alone (such as the immune effect of therapeutic vaccines, specific antibodies, etc.), while the latter refers to the immune effect that cannot achieve therapeutic effect when used alone Immune effects that have an auxiliary effect but still have an auxiliary effect (for example, immune enhancers have the effect of improving the immune function of the body).
  • second immune effect is distinguished from the term “drug antigenic effect”, which refers to the antigenic effect of the drug itself (such as the antigenic effect caused by any drug entering the body as a foreign substance), and the former refers to administration-related, But it is different from the immune effect of the drug antigen effect, such as the in situ vaccine effect caused by the local chemical effect of the drug).
  • in situ secondary immunity is distinguished from the term “ex situ secondary immunity”, which refers to secondary immunity within a lesion (eg, a tumor), and the latter, which refers to extra-lesion (eg, axillary intradermal) ) secondary immune effects.
  • second immune substance refers to an immune substance formed in the administration area due to local administration and different from the administered drug itself, such as being released, generated, activated, or/and tentative for any reason after administration. A collection of antigens, adjuvants, or/and other immune molecules.
  • in situ secondary immune substances is different from the term “ex situ secondary immune substances", the former refers to secondary immune substances (such as in situ antigens, in situ adjuvants, or/and other immune molecules that are released, generated, activated, or/and collected within the lesion), while the latter refers to secondary immune substances outside the lesion (eg, intradermal in the armpit) (eg, nodules formed at the site of administration). nodular immune substances, other immune molecules released, generated, activated, or/and actuated by the nodule or/and other drug effects at the site of administration, etc.).
  • vaccine antigen is different from the term "antigen”, which refers to any substance that can induce an immune response in the body, while the former refers to an antigen that can induce the body to produce a specific disease and achieve a therapeutic effect.
  • the same substance can It is used as vaccine antigen and immunopotentiator antigen by very different technical solutions.
  • adjuvant refers to a substance in a vaccine that enhances the immunotherapeutic effect of its antigen.
  • antigen in situ refers to a secondary immune substance in situ that can act as an antigen.
  • vaccine-like drug or “vaccine-like drug” refers to a therapeutic drug that provides secondary immune effects similar to those of vaccines (as distinguished from immune enhancers and conventional vaccines) and optionally, the effects of exogenous antigens.
  • the probiotic component is administered topically together with the chemically active ingredient and/or bioactive ingredient capable of acting synergistically with the probiotic component.
  • the probiotic component is contained in the same medicament as the co-active ingredient.
  • the term “synergistic effect” means that a specific active ingredient (eg, a probiotic component of the present application) interacts with a specific active ingredient (eg, a probiotic component of the present application) under specific conditions (eg, a pharmacological component that provides a preference for minimizing bacterial immune effects and a pharmacological concentration that minimizes local effects).
  • the combined effects (eg, short-term and/or mid- to long-term effects) of other active ingredients eg, weak local-acting compounds and/or cytotoxic drugs) exceed the expected sum of their individual effects.
  • the term “local synergy” refers to a local effect that includes synergy.
  • topical chemical synergy refers to topical chemical effects that include synergy.
  • amount ratio refers to the weight ratio of multiple active ingredients (such as the probiotic components of the present application and their co-uses) in the same composition.
  • concentration ratio refers to the concentration ratio of multiple active ingredients (eg, the probiotic components of the present application and their co-uses) in the same composition.
  • Suitable patients for the treatment are selected from one or more of the following groups: immunosuppressed patients (such as immunocompromised patients, elderly patients), patients who can be administered in local lesions, and patients with local lesions that can be chemically ablated. Patients, patients who can produce secondary immune substances in local lesions, and patients who can produce secondary immune substances in the administration area outside the lesions.
  • the “immunosuppressed patient” refers to any patient represented by the tumor-bearing nude mouse model, such as the time when its immune function is at a low level for any reason, and the composition in the technical solution of the present invention can provide local effects Patients who cannot achieve normal levels by other methods (such as immune enhancement) within (eg, within 7 days of the first administration in Example 2 below), such as patients who are difficult to receive radiotherapy or conventional chemotherapy due to immunocompromised immunity.
  • Said therapeutic effect includes local treatment or/and immunotherapy involving said local effect (or local synergistic effect).
  • the immunotherapy includes secondary immune effects of the local action (or local synergy) and optionally other immune effects within the lesion and/or outside the lesion; the local treatment includes one or more local Chemical-like ablation of lesions and optional other chemotherapy.
  • the pharmaceutical composition is a chemical-like ablation-in situ and ex-situ immunotherapy drug, and the patient is selected from the group that can be administered in a local lesion and the local diseased tissue can be chemically ablated or/and in the local lesion
  • One or more of patients with secondary immune effects and patients with secondary immune effects in the extra-lesional administration area are examples of the composition.
  • the pharmaceutical composition is a chemical-like ablation drug or an in situ immunotherapy drug, and the in situ immunotherapy drug can be an immunotherapy drug that provides in situ vaccine activation of the target area in the lesion and other immune effects optionally present; the The pharmaceutical composition may also be an immunotherapeutic drug, such as a vaccine-like drug, that provides extra-lesional secondary immunity and optionally other immunity.
  • the extra-lesional secondary immune effects include the local effects (or local synergies) secondary to the immune effects of abnormal structures (eg, nodules).
  • the probiotic component can provide at least one of a local effect, a secondary effect of the local effect, and an immune effect of local administration, wherein the secondary effect of the local effect includes secondary immune substances in the administration area Involved in immune function.
  • the probiotic components are selected from one or more of water-soluble components of probiotics, semi-fluid components of probiotics, water-insoluble granules of probiotic components, and inactivated probiotics, and have low bacterial immunogenicity.
  • the probiotic components are selected from the water-soluble components of probiotics and/or their engineering analogs, and the composition is a solution composition.
  • the probiotic component is selected from bacterial component water-insoluble particles, or/and inactivated probiotics and the composition is a suspension composition.
  • the probiotic component is selected from probiotic semi-fluid-based components and the composition is a semi-fluid-based composition.
  • the composition is preferably a semi-fluid-based composition or an aqueous solution, more preferably a semi-fluid-based composition.
  • the "bacterial immunogenicity” refers to the ability of bacteria as a complete foreign body to generate an immune response in the recipient, and different bacteria have different bacterial immunogenicity. Live probiotics have the strongest bacterial immunogenicity, but also carry strong safety risks directly into the body.
  • the “inactivated probiotics” refer to preparations obtained after inactivation such as high temperature inactivation, high temperature and high pressure inactivation, ultraviolet inactivation, chemical inactivation, radiation inactivation, etc.
  • probiotic water-insoluble component refers to any component with a water solubility of ⁇ 0.1% obtained from probiotics
  • probiotic water-soluble component refers to any component obtained from probiotics with a water solubility ⁇ 0.1% of any component
  • broken probiotics refers to the mixture obtained after the probiotics are processed by the crushing process
  • crushed probiotic precipitation component refers to the separation (such as filtration or/and) from the broken probiotics. The water-insoluble precipitate from centrifugation), its further separated components, and the like.
  • broken probiotic supernatant fraction refers to the supernatant separated (eg, filtered and/or centrifuged) from the broken probiotic, its further isolated components, and the like.
  • probiotic cell wall polysaccharide refers to the polysaccharide contained in the cell wall and its analogs, such as beta-glucan.
  • probiotic semi-fluid-like component refers to a probiotic component that provides a semi-fluid-like form to the compositions of the present invention.
  • semi-fluid refers to the class of physical forms between liquids and semi-solids, which includes semi-fluids and the like.
  • the term “semi-fluid” means no flow visible to the naked eye with no external pressure within the lower limit of room temperature (eg, 20 seconds), and a clinically (at the time of administration) acceptable external pressure (eg, an external pressure that can be applied to a syringe advancer).
  • Objects that can flow and cause irreversible deformation under pressure are distinguished from liquids (which also flow without external pressure) and semi-solids (which only undergo reversible deformation under clinically acceptable external pressure).
  • semi-fluid analog refers to a physical form between a liquid (suspension) and a semi-fluid that is close to the semi-fluid, which does not appear at rest at room temperature for about 1 minute, while the suspension appears significantly Delamination; it occurs within about 1 minute at room temperature without external pressure, and the semi-fluid does not flow visibly to the naked eye.
  • the suspension may be transformed into a semi-fluid object, such as 5% ⁇ -glucan mixed with water to form a suspension, 5% ⁇ -glucan aqueous suspension can be heated to prepare a semi-fluid analog, and 5% ⁇ -glucan - Dextran/1% methylene blue/1% 5-fluorouracil aqueous suspension can be prepared as a semi-fluid by heating.
  • a semi-fluid object such as 5% ⁇ -glucan mixed with water to form a suspension
  • 5% ⁇ -glucan aqueous suspension can be heated to prepare a semi-fluid analog
  • 5% ⁇ -glucan - Dextran/1% methylene blue/1% 5-fluorouracil aqueous suspension can be prepared as a semi-fluid by heating.
  • the probiotic water-soluble component is selected from at least one of crushed probiotic supernatant component, probiotic extract, and probiotic intracellular water-soluble component.
  • the probiotic semi-fluid component is selected from one or more of the probiotic components whose aqueous mixture can form (such as by heating followed by cooling, alkalizing, etc.) a semi-fluid composition, such as polysaccharides and its analogs.
  • the probiotic water-insoluble particles are selected from at least one of crushed probiotic precipitation components, probiotic cell wall polysaccharide particles, and probiotic cell wall polysaccharide nanoparticles.
  • the inactivated probiotics are not limited to inactivated probiotics with complete cells, such as the ratio of incomplete cells in the inactivated probiotics > 20% or > 30%, or the number of complete cells ⁇ 10 5 /ml or ⁇ 0.5 x 105/ml.
  • the probiotic is selected from at least one of bacillus, lactic acid bacteria, bifidobacteria, and fungi, which can be natural bacteria or engineered bacteria.
  • the Bacillus is one or more of Bacillus cereus, Bacillus licheniformis, Bacillus subtilis, Bacillus megaterium, Bacillus firmus, Bacillus coagulans, Bacillus lentus, Bacillus pumilus, and Bacillus natto.
  • the lactic acid bacteria are Lactobacillus and/or Bifidobacterium, wherein the Lactobacillus is selected from at least one of Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus pumilus and Lactobacillus fermentum.
  • the bifidobacteria include Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus brucelli, Lactobacillus helveticus, Lactobacillus thermophilus, Enterococcus faecium, and Streptococcus faecalis. at least one.
  • the fungi include yeasts and/or Brucella, wherein the yeasts include Saccharomyces cerevisiae, Saccharomyces delbrueckii, Candida spp, Wickham Saccharomyces cerevisiae, Pichia spp. At least one of T. leucococcus, S. chevalii, S. crimsonii, S. pombe, S. baumannii, and Candida rugugenens.
  • the pharmacological composition required for the probiotic component to provide the above-mentioned therapeutic effect satisfies that the pharmacological concentration (local administration concentration) required for the probiotic component to provide the above-mentioned effect is >0.1%, ⁇ 0.25%, 0.25-25% , preferably 0.5-15%, more preferably 1-15% or 5-15%.
  • concentration is the weight-volume percent concentration (w/v) of a particular component in the drug.
  • pharmaceutical concentration refers to the concentration of a given component into the target area necessary to achieve its specific pharmacological response, eg, the initial concentration in the lesion.
  • formulation concentration refers to the concentration of the specified component in the form of the pharmaceutical formulation (eg, injection or perfusate).
  • administration concentration refers to the concentration of the specified component in the administered form of the pharmaceutical formulation (eg, a dilution of the formulation).
  • initial intralesion concentration refers to the concentration of a given component in a medicated medium (eg, medicated blood) when the drug enters the lesion.
  • the initial intratumoral concentration necessary for their respective pharmacology can also be larger. Not the same.
  • one of the technical scheme features of the application, composition and method of the present invention is to ensure the pharmacological concentration (local administration concentration) required for the effect, especially the local chemical effect.
  • the administration concentration of the probiotic components in the composition described in this application is the same as the administration concentration of the probiotic components in conventional injections, their respective pharmacology (local chemical effect vs immune enhancement effect) is necessary for the initial intralesion Concentrations can also vary widely.
  • one of the technical solution features of the application, composition and method of the present invention is the initial local concentration (local administration concentration) required to ensure the effect, especially the local chemical effect.
  • the local administration concentration of the probiotic component in the pharmaceutical composition in this application is usually the concentration of the probiotic component in the drug at the end point (such as needle hole, catheter outlet, etc.) .
  • the administration concentration is the concentration of the probiotic component after mixing the dry powder and the liquid carrier.
  • target refers to the primary target of pharmacology, such as cytotoxic drugs targeting tumor cells, immunomodulatory drugs targeting regulators of the immune system, chemical ablative agents targeting tumor tissue, and the like.
  • target volume refers to the spatial extent in which the target is administered at the time of administration, such as a tumor or a portion thereof. For example, when the diameter of the tumor is small and the required one-time dose is clinically feasible, the target area is a tumor that is the target of this treatment; or when the tumor has a large diameter, the required one-time dose is clinically feasible When this is not feasible, the target area is part of the tumor that is the target of this treatment.
  • the composition or unit composition When used to treat a patient, the composition or unit composition must satisfy the pharmacological volume conditions required for the probiotic component to provide the effect, wherein the pharmacological volume (volume for topical administration) is ⁇ 1 ml, or Administer 10-150ml in local lesions or/and 1.5-50ml outside local lesions.
  • the ultra-conventional volume administration is especially required for the local action of the probiotic component.
  • composition of the composition requires the minimization of components other than synergists, so as to satisfy the local pharmacological environmental conditions required to provide the local effect, preferably free from the pharmacy and/or administration safety requirements of conventional compositions.
  • Desired inactive ingredients such as solid excipients in oral formulations, flavoring agents and osmotic pressure enhancers in conventional injections.
  • the carrier of the pharmaceutical composition is water, the co-use is soluble in water, and the probiotic component is a water-soluble component of probiotic or a water-insoluble particle of inactivated probiotic or probiotic component , one or more of the semi-fluid components of probiotics or the water-insoluble components of their derivatives.
  • the carrier of the pharmaceutical composition is water, the co- and/or insoluble in water, and the probiotic component is a probiotic semi-fluid component.
  • the chemically active ingredients described in this application are selected from compounds with weak local action and/or cytotoxic drugs, for example, the combination is at least two chemically active ingredients. If the chemically active ingredient is a cytotoxic drug, the amount ratio of the probiotic component to the cytotoxic drug (W probiotic component /W cytotoxic drug ) is (1-110)/(1-100), If the chemically active compound is a compound with weak local action, the amount ratio of the probiotic component to the compound with weak local action (W probiotic component /W compound with weak local action ) is (1-90)/(1- 100).
  • the term "chemically active ingredient” refers to any active ingredient that provides a chemical action, including cytotoxic drugs and topical chemicals.
  • topical chemical ingredient refers to any active ingredient that provides a local chemical effect when administered topically.
  • the term “poorly locally acting compound” refers to a chemically active ingredient that is less locally active than classical chemical ablatives under certain conditions (eg, concentrations used as a co-use).
  • the weak topical compounds include: amino acid based nutrients, vital dyes, quinine based drugs, low concentration acidulants, low concentration alkalizers, pH buffer systems containing acidifiers or/or alkalizers.
  • the combination can be a vital dye and at least one other chemically active ingredient, or a vital dye and a cytotoxic drug, or a vital dye and an amino acid nutrient, or a cytotoxic drug and at least one other chemically active ingredient.
  • the weak local action compound is an amino acid nutrient
  • the amount ratio of the probiotic component to the amino acid nutrient is (1-20)/(1-100); if When the weak local action compound is a vital dye, the amount ratio of the probiotic component to the vital dye (W probiotic component /W vital dye ) is (7-90)/(1-100);
  • the weak local action compound is a quinine drug
  • the ratio of the probiotic component to the quinine drug is (2-90)/(1-100)
  • the weak local action compound is an acidulant and/or an alkalinizer
  • the amount ratio of the probiotic component to the acidulant and/or alkalinizer is (2-60)/(1-100).
  • amino acid nutrients refers to amino acid compounds with nutritional and health care effects, preferably amino acid nutritional medicines or amino acid excipients with nutritional health care effects listed in official pharmacopoeia or guidelines.
  • the amino acid nutrients include amino acids, amino acid salts, oligopeptides and polypeptides.
  • the amino acid nutrients include amino acids or their salts in the following group or oligopeptides and polypeptides composed of the following amino acids: alanine, valine, leucine, isoleucine, phenylalanine, Proline, tryptophan, tyrosine, serine, cysteine, methionine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, beta-alanine acid, taurine, gamma aminobutyric acid (GABA), theanine, citrulline, ornithine.
  • amino acids or their salts in the following group or oligopeptides and polypeptides composed of the following amino acids: alanine, valine, leucine, isoleucine, phenylalanine, Proline, tryptophan, tyrosine, serine, cysteine, methionine, threonine, lysine, arginine, histidine,
  • the amino acid nutrients include amino acids or their salts in the following group, or oligopeptides and polypeptides comprising or consisting of the following amino acids: arginine, lysine, glycine, cysteine, alanine, serine , aspartic acid, glutamic acid.
  • the amino acid nutrient concentration is >2.5%, or 5-30%, preferably 5-25%.
  • the term "vital dye” refers to an aromatic compound dye that can color tissues, cells, subcellular units and other structures after entering the living tissue of an animal, but has no unacceptable harm to the animal as a whole.
  • the vital dyes include methylene blue, patent blue, isosulfur blue, Bengal red, toluidine blue, trypan blue, basic blue, eosin, basic fuchsin, crystal violet, gentian violet, neutral red, At least one of Jenners Green B and Saffron.
  • the vital dye is a methylene blue dye, such as methylene blue, patent blue, isosulfur blue, and new methylene blue.
  • the concentration of the vital dye is > 0.25%, or 0.25-10%, preferably 0.25-1.5% or 2.5%-10%.
  • the vital dye is a methylene blue dye whose concentration (w/v) is ⁇ 0.35%, preferably 0.35-2.5%, more preferably 0.35-1.5% or 0.5-1%.
  • the vital dye is Red Bengal at a concentration (w/v) of 1-10%.
  • the quinine compounds include at least one of quinine, quinine monohydrochloride and quinine dihydrochloride, and the concentration thereof is ⁇ 0.5%, or 0.5-5%, preferably 1.5-5% or 1.5%-3%.
  • concentration thereof is ⁇ 0.5%, or 0.5-5%, preferably 1.5-5% or 1.5%-3%.
  • acidulant refers to an acid mainly used as an adjuvant in the preparation of pharmaceuticals, more specifically for pH adjustment, and which generally does not introduce specific biological activity other than to provide acidity when used; Strong and/or weak acids approved by the authorities or listed in the official pharmacopoeia or guidelines of each country.
  • the strong acid can be hydrochloric acid, sulfuric acid, nitric acid, perchloric acid, selenic acid, hydrobromic acid, hydroiodic acid, chloric acid, etc., preferably hydrochloric acid.
  • the weak acid can be carbonic acid, boric acid, acetic acid, phosphoric acid, sulfurous acid, pyruvic acid, oxalic acid, tartaric acid, nitrous acid, etc., preferably acetic acid.
  • the amount ratio of the probiotic component to the acidulant - ie the weight concentration (W probiotic component /W acidulant ) is (1-20)/(0.5-50). In one embodiment, the concentration of the acidulant is > 0.5%, 0.5-2% (strong acid) or 2-15% (weak acid).
  • the amount ratio of the probiotic component to the alkalizing agent is (1-20)/(0.5-50). In one embodiment, the concentration of the alkalizing agent is > 0.5%, 0.5-5% (strong base), or/and 2-15% (weak base).
  • alkalizing agent refers to a basic compound mainly used as an excipient in the preparation of pharmaceuticals, more specifically for pH adjustment; preferably selected from the group approved by the official competent administrative department of each country, or included in the official pharmacopoeia or guideline of each country alkalizing agents, including strong bases and weak bases.
  • the strong base is an alkali metal hydroxide or/and an organic strong base, and its concentration (w/v) in the pharmaceutical composition is ⁇ 0.5%, preferably 0.5-7.5% or 0.75-7.5%.
  • the strong base is an alkali metal hydroxide such as sodium hydroxide, potassium hydroxide, calcium hydroxide, etc., more preferably sodium hydroxide.
  • Described weak base includes polybasic weak acid acid inorganic salt, polybasic weak acid base inorganic salt, nitrogenous weak base, wherein polybasic weak acid acid inorganic salt includes sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium bicarbonate, potassium bicarbonate , calcium bicarbonate, sodium hydrogen sulfate, preferably sodium bicarbonate.
  • the concentration (w/v) of the polybasic weak acid inorganic salt in the pharmaceutical composition is ⁇ 1%, preferably 2-10% or 3-10%.
  • the polybasic weak acid and basic inorganic salts include sodium phosphate, sodium carbonate, potassium carbonate, and borax, preferably sodium carbonate; the concentration (w/v) of the polybasic weak acid and basic inorganic salts in the pharmaceutical composition ⁇ 1%, preferably 2-10% or 3-10%.
  • the nitrogen-containing weak base includes ammonia water, ammonium chloride, 2-aminoethanol, tromethamine, triethanolamine, trimethylolaminomethane, 2-aminoethanol, tromethamine, triethanolamine, meglumine, Meglumine; the concentration (w/v) of the nitrogen-containing weak base in the pharmaceutical composition is ⁇ 2%, preferably 2-35% or 3-35%.
  • the alkalizing agent can be sodium hydroxide and sodium bicarbonate, and the concentrations (w/v) of the two in the pharmaceutical composition are 2-5% and 3-10%, respectively.
  • the alkalizing agent can also be sodium carbonate and sodium bicarbonate, and the concentrations (w/v) of the two in the pharmaceutical composition are 3-10% and 3-10%, respectively.
  • the pharmaceutical composition contains an acidifying agent or/and an alkalizing agent and has pH buffering capacity.
  • the chemically active ingredient includes at least one cytotoxic drug, and the cytotoxic drug includes an antitumor drug active ingredient; wherein the concentration of the antitumor drug active ingredient is greater than or equal to 0.1%, 0.1-15%.
  • cytotoxic drug refers to an active ingredient that primarily targets diseased cells or intracellular structures to achieve its drug effect, such as conventional chemotherapeutic drugs.
  • conventional chemotherapeutic drugs refers to drugs that can effectively treat local diseased diseases through conventional drugs at safe doses. Conventional chemotherapeutic drugs (such as antineoplastic drugs) that have been or will be included in the pharmacopoeia.
  • the antitumor drug active ingredients can be selected from 1) drugs that destroy DNA structure and function, such as a) alkylating agents such as cyclophosphamide and carmustine; b) metal platinum complexes, such as cisplatin, carmustine, etc.
  • DNA topoisomerase inhibitors such as doxorubicin, topotecan, irinotecan, etc.
  • drugs that interfere with transcriptional RNA intercalated in DNA such as antitumor antibiotics, such as actinomycetes 3
  • Drugs that interfere with DNA synthesis including a) pyrimidine antagonists, such as 5-fluorouracil, fur-fluorouracil, difur-fluorouracil, cytosine derivatives cytarabine, Uracil derivatives such as cyclocytidine and 5-azacytidine, b) purine antagonists such as oncolysine and thioguanine, c) folic acid antagonists such as methotrexate; 4) drugs that affect protein synthesis, such as Colchicine, vinblastine, and taxanes such as paclitaxel and docetaxel.
  • the active ingredients of the antitumor drugs include uracil derivatives, cyclophosphamide, gemcitabine, epirubicin, antitumor antibiotics, teniposide, metal platinum complexes, and taxanes. At least one; preferably, the antitumor drug active ingredient is selected from at least one of 5-fluorouracil, cyclophosphamide, gemcitabine, epirubicin, antitumor antibiotics, teniposide, metal platinum complexes, and paclitaxel .
  • the active ingredient of the antitumor drug can be an alkylating agent such as cyclophosphamide and carmustine, and the concentration (w/v) in the pharmaceutical composition is 0.5-6%, preferably 0.75-1.5%; or It is a metal platinum complex such as cisplatin and carboplatin, and the concentration (w/v) in the pharmaceutical composition is 0.03-0.15%, preferably 0.05-0.15%; or doxorubicin, topological DNA topoisomerase inhibitors such as tecan and irinotecan, and the concentration (w/v) in the pharmaceutical composition is 0.05-0.20%, preferably 0.075-0.15%; or actinomycins , daunorubicin and other antitumor antibiotics, and the concentration (w/v) in the pharmaceutical composition is 1-4%, preferably 1-2%; or uracil derivatives 5-fluorouracil, fur-fluorouracil , bisfuryl fluorouracil, cytosine derivatives cytar
  • the pharmaceutical composition comprises biologically active components, and the biologically active components include at least one of antigens, immunomodulatory antibodies, cytokines, and adjuvants.
  • the antigen is a microbial antigen or a tumor antigen, wherein the microbial antigen includes 1) non-probiotic bacteria such as Streptococcus pyogenes, Serratia serratia, BCG, Clostridium tetani, Clostridium butyricum, Lactobacillus acidophilus, etc.
  • the tumor antigens are selected from breast cancer, pancreatic cancer, thyroid cancer, nasopharyngeal cancer, prostate cancer, liver cancer, lung cancer, intestinal cancer, oral cancer, gastric cancer, colorectal cancer, bronchial cancer, At least one of throat cancer, testicular cancer, vaginal cancer, uterine cancer, ovarian cancer, malignant melanoma, brain tumor, renal cell carcinoma, astrocytoma, and glioblastoma; the immunomodulatory antibodies include 1 ) Antibody blockers against inhibitory receptors, such as blocking antibodies for CTLA-4 molecules and PD-1 molecules; 2) Antibody blockers against ligands for inhibitory receptors, for immune response cell surface
  • the dosage form of the pharmaceutical composition described in the present application is a topical dosage form
  • the pharmaceutical composition can be any topical that contains a probiotic component and satisfies the necessary conditions for the probiotic component to provide the desired effect Dosage forms such as injections, smears or ointments.
  • injection refers to a sterile preparation containing active ingredients and a liquid carrier for in vivo administration. According to the mode of administration, it is divided into local injections, intravenous injections, etc. Local injections can be used as local injections only after a given local administration concentration. Use; according to the commercial form, it is divided into liquid injection, semi-fluid injection, injection powder for injection, etc., wherein the injection powder for injection contains sterile dry powder and a solvent, the sterile dry powder contains part or all of the active ingredients, and the solvent contains all 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, usually the end point (such as needle hole, catheter outlet, etc.) of local drug delivery devices such as syringes, punctures, and injection catheters Active ingredient concentration in liquid medicines.
  • the concentration of the active ingredient is the concentration of the active ingredient in a mixture of sterile dry powder and a vehicle (such as a reconstituted solution or the pharmaceutically acceptable liquid carrier).
  • the pharmaceutical composition described in the present application further comprises excipients, such as dispersion medium, preservative, stabilizer, wetting agent and/or emulsifier, solubilizer, tackifier, etc.
  • excipients such as dispersion medium, preservative, stabilizer, wetting agent and/or emulsifier, solubilizer, tackifier, etc.
  • the tackifier can be Sodium carboxymethyl cellulose, carboxymethyl cellulose, polyvinylpyrrolidone or gelatin
  • the preservatives can be antioxidants such as ascorbic acid.
  • the pharmaceutical composition of the present application further comprises a tracer such as iodized oil.
  • the application also provides a pharmaceutical kit comprising one or more individual containers containing a pharmaceutical composition according to the disclosure, wherein the individual containers may be ampoules, vials, or the like.
  • the pharmaceutical kit further comprises instructions or labels on how to administer the pharmaceutical composition to an individual in need thereof.
  • the administration includes intralesional administration, or both intralesional and extraterrestrial administration, eg, subcutaneous injection under the armpit.
  • the ratio of the administration amount of the pharmaceutical composition to the volume of the target volume in the local lesion is >0.1, 0.15-1.5, preferably 0.23-1.5 or 0.5-1.5.
  • the preparation of the pharmaceutical composition of the present invention comprises: preparing a topical pharmaceutical formulation containing the probiotic component and optionally other substances or preparing a topical pharmaceutical formulation containing the probiotic component, the combination and optionally other substances of topical pharmaceutical preparations.
  • the drug may be a liquid drug or a semi-fluid drug, and the liquid drug may be a solution (eg, a solution in a hydrophilic vehicle, preferably an aqueous solution), a suspension, or an emulsion.
  • the medicament may be an in vivo administration agent or a body application.
  • the inactivated probiotics can be prepared by high temperature inactivation, high temperature and high pressure inactivation, ultraviolet inactivation, chemical reagent inactivation, radiation inactivation, and the like.
  • the crushed probiotic components in this application can be prepared by the following methods: 1), the live probiotics are crushed by high-pressure homogenization, oscillating bead crushing, high-speed stirring bead grinding and crushing, ultrasonic crushing, impact crushing, Preparation by osmotic shock crushing method, freeze-thaw crushing method, enzymatic crushing method, chemical crushing method, detergent crushing method, etc.; if necessary, 2), the preparation of 1) is subjected to separation engineering such as filtration or/and centrifugation Prepared as broken probiotic isolated components, such as broken probiotic water-insoluble component granules, broken probiotic water-soluble component.
  • the probiotic water-soluble component in the present invention is prepared by the above-mentioned crushed probiotic water-soluble component (eg, crushed probiotic supernatant liquid component) or the probiotic water-soluble component in the prior art (such as the preparation of probiotic extracts).
  • the probiotic water-insoluble component granules in the present invention are prepared by the above-mentioned crushed probiotic water-insoluble component granules (such as crushed probiotic precipitation components) or the probiotic water-insoluble component granules in the prior art (such as muramidan) preparation method.
  • the probiotic semi-fluid component of the present invention can be prepared by a method of heating a mixture of a probiotic polysaccharide or an analog thereof and water.
  • the heating temperature of the probiotic polysaccharide/water mixture is preferably 50-110° C., which is heated by microwave heating, electric furnace heating, steam heating and the like.
  • the preparation of the pharmaceutical composition comprising the probiotic semi-fluidic component and the combination of the present invention comprises: combining the probiotic polysaccharide or its analog, the combination and water The warming is carried out after mixing uniformly.
  • the preparation of the pharmaceutical composition of the present invention further comprises: making the concentration of the probiotic component, the co-substance and optionally other substances in the pharmaceutical formulation greater than or equal to that in the present invention
  • the administration concentration required by the inventive technical solution When it is greater than the administration concentration in the pharmaceutical composition of the present invention, it can be further diluted for use.
  • the preparation of the pharmaceutical composition of the present invention further comprises: making the content of the probiotic component, the common substance and the optional other substances in the pharmaceutical preparation suitable to satisfy the technical solution of the present invention
  • the desired drug volume/target volume ratio for example, drug dispensing and capping are performed according to the drug volume/target volume ratio.
  • the preparation of the pharmaceutical composition of the present invention further comprises: performing high temperature sterilization, high temperature and high pressure sterilization, ultraviolet sterilization, chemical reagent sterilization, radiation sterilization and other sterilization treatments on the preparation.
  • compositions of the present invention include: containing different types and concentrations of the pharmaceutical composition, containing different types and concentrations of other drugs, containing different types and concentrations of other additives (eg, analgesics, activators, etc.) Wait).
  • other additives eg, analgesics, activators, etc.
  • focal disease refers to a disease with local disease symptoms.
  • local lesions also referred to as lesions
  • abnormal parts of the animal (preferably human) body either native or secondary, which include structural (such as diseased tissue), morphological or functional symptom blocks and associated components. abnormal area.
  • Local pathological diseases mentioned in this application include solid tumors, non-tumor swellings, such as non-tumor nodules, local inflammation such as cervical erosion, abnormal secretion gland function and skin diseases.
  • the local disease is the tumor body and its tissue where the tumor cells are located, and the abnormal area that communicates with it is connected to the tumor body (for example, through lymphatic or blood vessels), and Adjacent areas with or suspected of having tumor cells; if the local lesions are non-tumorous, the local lesions are abnormal non-tumor masses such as hyperplasia, cysts, and nodules; polyps of thyroid, breast, liver, lung, and intestines and other non-tumor nodules.
  • the local disease When the local disease is local inflammation, the local disease is an inflamed area such as the inflamed surface or inflamed body; if the local disease is abnormal secretion, the local disease is the abnormal source or the secretory gland where it is located; for example, when the disease is insulin When the secretion is abnormal, the abnormal source is the islet, and the local tissue is the islet or the pancreas where the islet is located; when the disease is a skin disease, the local tissue is the diseased skin or an accessory organ of the diseased skin.
  • tumor refers to a mass formed due to abnormal proliferation of cells or mutated cells, including solid tumors.
  • solid tumor refers to a tumor with a tumor body, which can be due to any pathology (malignant and non-malignant) and at any stage, classified according to tumor cell type as epithelial tumor, sarcoma, lymphoma, germ cell Tumors, blastomas, etc.; tumors named according to the organ or tissue in which the tumor cell concentrated area is located include tumors named after the following organs or tissues: brain, skin, bone, muscle, breast, kidney, liver, lung, Gallbladder, pancreas, brain, esophagus, bladder, large intestine, small intestine, spleen, stomach, prostate, green pill, ovary or uterus.
  • the tumors include malignant tumors and non-malignant tumors
  • the malignant tumors include breast cancer, pancreatic cancer, thyroid cancer, nasopharyngeal cancer, prostate cancer, liver cancer, lung cancer, colon cancer, oral cancer, esophagus cancer, and gastric cancer , laryngeal cancer, testicular cancer, vaginal cancer, uterine cancer, ovarian cancer, malignant lymphoma, malignant brain tumor, etc.
  • the non-malignant tumors include breast tumor, pancreatic tumor, thyroid tumor, prostate tumor, liver tumor, lung tumor, intestinal tumor tumor, oral tumor, esophagus tumor, gastric tumor, nasopharyngeal tumor, laryngeal tumor, testicular tumor, vaginal tumor, uterine tumor, fallopian tube tumor, ovarian tumor, lymphoma, brain tumor, etc.
  • the local lesions may also be skin diseases such as chronic mucocutaneous candidiasis and various ringworms.
  • the pharmaceutical composition described in this application when used to treat diseases, it can also be administered in combination with other interventional therapy, systemic chemotherapy, immunotherapy, photodynamic therapy, sonodynamic therapy, surgical intervention, etc., to further improve the curative effect.
  • the pharmaceutical composition of the present invention has been shown to promote the effective destruction of tumor tissue while minimizing damage to the patient's normal tissue, thereby achieving a safe and effective Pharmacological effects in the treatment of local lesions.
  • probiotics used in this application and some of the probiotic components can be obtained from commercial sources, and other probiotic components (inactivated probiotics, crushed probiotics, supernatant components of crushed probiotics, precipitation of crushed probiotics) use the aforementioned methods (such as (100 ° C, 2 hours of high temperature inactivation, pulverizer at 5000 rpm / min, centrifuge at 10,000 rpm to separate supernatant and precipitate) self-made.
  • mice and nude mice are healthy at 6-8 weeks old. Female, weighing 17.5-20.5g.
  • the animal experiments of subcutaneously transplanted cells to generate local lesions were all carried out in accordance with the test guidelines issued by the drug administration and in accordance with the conventional method of subcutaneous inoculation of cells.
  • the modeling is successful.
  • the animals are divided into several groups of 6 animals in each group. The general state, body weight, food intake, animal graft-versus-host disease, tumor volume, tumor weight, and survival time were observed and determined.
  • the models of respectively, contain tumor cells (representing tumors), fibroblast-containing nodules (representing fibroblast-containing tumors and other local lesions), and normal living tissue (representing local lesions containing ordinary tissues).
  • the volume (V) of local lesions (such as tumors), the relative proliferation rate (R) of local lesions (such as tumors), the inhibition rate (r') of local lesions (such as tumors), and the tumor inhibition rate (r) are respectively as follows:
  • Local lesion (such as tumor) proliferation rate (R) TV/CV ⁇ 100, where TV and CV are the local lesion volume of the study group and the negative control group, respectively;
  • Tumor inhibition rate r (CW-TW)/CW ⁇ 100%, where TW and CW are the average tumor weights of the study group and the negative control group, respectively.
  • the efficacy of drug i is denoted as Ei, which can be represented by ri' or ri.
  • the type of action of a drug i.e. pharmacology, can be studied by the efficacy of the drug, especially the efficacy of the agreed drug in different regimens.
  • experimental results are expressed as mean ⁇ standard deviation (x ⁇ s), and the difference between the two experimental animal groups and the group mean is expressed by statistical software SPSS13.0 or SPSS19.0 was used for significance test.
  • the test was carried out with statistic t.
  • Positive controls for chemotherapy include classical cytotoxic drugs (such as 0.5-1% 5-fluorouracil, whose tumor inhibition rate is ⁇ 30% under the conditions of the following examples) and classical chemical ablative agents (such as 75-99% ethanol, which The tumor inhibition rate under the conditions of the following examples is ⁇ 15%).
  • Positive controls for immune enhancement include cytokines (such as interleukin-12,), bacterial components (such as attenuated bacterial vaccines, etc.), vaccine adjuvants and other immune enhancers.
  • EA and EB such as tumor inhibition rate
  • EA +B the actual shared efficacy of A / B
  • q ⁇ 1 it means that the actual sharing effect is weaker than the theoretical expectation, showing an antagonistic effect
  • q>1 it means that the actual sharing effect exceeds the theoretical expectation. , showing a synergistic effect.
  • the method for judging the effect of concomitant medication in animal experiments includes Burgi's method (Burgi Y. Pharmacology; Drug actions and reactions. Cancerres. 1978, 38(2), 284-285).
  • the shared pharmacodynamic effect of the combined administration of drug A and drug B carried out according to the actual drug effect/expected drug effect ratio q of the Jin Zhengjun method is judged as follows:
  • the combined administration also does not show a meaningful synergistic effect, which is considered in the present invention.
  • compositions of the present invention can be formulated.
  • the compositions of some of the compositions of the present invention prepared in this example are listed in Table 2 (probiotic components as active ingredients) and Table 3 (probiotic components as synergistic active ingredients).
  • Table 2 Composition of probiotic components Aqueous formulations, pharmacological concentrations (>0.1%, ⁇ 0.25%, 0.25-25%, preferably 0.5-15%, more preferably 1-15% or 5-15% %)
  • Preparation method of A1-A8 Measure the probiotics (eg 2.5g dry powder of Saccharomyces cerevisiae), optional other components, and make up to the total volume (eg 100ml) at the desired concentration (eg for local chemical action). ) liquid carrier (such as water for injection), and they are slowly mixed to obtain a probiotic mixture. If the liquid mixture is aliquoted (for example, 10ml/vial) and capped according to the desired drug volume/target volume ratio (for example, the average volume of clinically common solid tumors is 30cm3), it can provide intratumoral volume. Dosage forms and strengths of pharmaceutical compositions for local action.
  • the probiotics eg 2.5g dry powder of Saccharomyces cerevisiae
  • liquid carrier such as water for injection
  • the preparation (eg, 2.5% Saccharomyces cerevisiae) was put into a Pasteur inactivation cabinet for pasteurization (60° C., 48 hours) to obtain A1.
  • the preparation of different inactivated probiotics eg preparations A1-A8 in the above table
  • Preparation method of A9-A16 Measure the probiotics (eg 2.5g dry powder of Saccharomyces cerevisiae), optional other components, and make up to the total volume (eg 100ml) at the concentration required (eg for local chemical action). ) liquid carrier (such as water for injection), they are slowly mixed well and broken up using a homogenizer. Broken probiotics with different degrees of fragmentation (preferably 100% fragmentation) can be obtained by adjusting the homogenization process parameters (for example, the rotation speed is 10000-25000 rpm, the rotation time is 0.5-1 min, and the number of homogenization is 2-10 times).
  • the probiotics eg 2.5g dry powder of Saccharomyces cerevisiae
  • liquid carrier such as water for injection
  • the liquid mixture is aliquoted (for example, 10ml/vial) and capped according to the desired drug volume/target volume ratio (for example, the average volume of clinically common solid tumors is 30cm3), it can provide intratumoral volume.
  • the obtained preparation was A9.
  • the preparation of different compositions eg preparations A9-A16 in the above table
  • a high-concentration broken probiotic suspension eg, 10% broken Saccharomyces cerevisiae suspension can be prepared from probiotics (for example, 10 g of Saccharomyces cerevisiae dry powder and water for injection) with a volume of 100 ml).
  • probiotics for example, 10 g of Saccharomyces cerevisiae dry powder and water for injection
  • a volume of 100 ml Add the suspension to the centrifuge bottle and carry out centrifugation on the centrifuge.
  • the centrifugation time for example, 0.5-30 minutes
  • the number of centrifugal separations for example, 2- 4 times
  • the supernatant is poured out for another use, and the remaining precipitation components are dried (for example, 125 ° C, 90 minutes) It is then prepared as a dry powder of crushed probiotic precipitation components.
  • the difference between the raw probiotics (such as 10 g of Saccharomyces cerevisiae) and the dry powder of crushed probiotic precipitation components (such as 4 g) prepared with this is counted as the dry weight of supernatant components of crushed probiotics (eg 6g).
  • the preparation method of A17-A19 measure the probiotic water-insoluble component particles (for example, 1.4 g of crushed Saccharomyces cerevisiae dry powder prepared by the method of Example 1a), optionally The other components, and a liquid carrier (eg, water for injection) made up to a total volume (eg, 100 ml), are slowly mixed until uniform. If the liquid mixture is dispensed (for example, 10ml/vial) and capped according to the desired drug volume/target volume ratio (for example, the average volume of clinically common solid tumors is 30cm 3 ), a tumor-providing tumor can be obtained. Dosage forms and strengths of locally acting pharmaceutical compositions. The obtained preparation was A17.
  • a liquid carrier eg, water for injection
  • Probiotics can generate semi-fluid components (such as 10 g water-insoluble ⁇ -glucan particles), other components optionally present, at the concentration required (such as required for local chemical action). , and a liquid carrier (such as water for injection) that is made up to a total volume (such as 100ml), slowly mix them into a suspension, and then heat the suspension (such as a temperature of 50-110°C and a time of 0.5-24h) , which can form a semi-fluid after cooling.
  • semi-fluid components such as 10 g water-insoluble ⁇ -glucan particles
  • other components optionally present, at the concentration required (such as required for local chemical action).
  • a liquid carrier such as water for injection
  • a tumor-providing tumor can be obtained. Dosage forms and strengths of locally acting pharmaceutical compositions.
  • the obtained preparation was A16.
  • the preparation of different semi-fluid compositions can be carried out from different probiotic semi-fluidizable components, respectively. Experiments have shown that only when the concentration of the probiotic semi-fluidizable component is greater than a certain threshold (eg ⁇ -glucan ⁇ 2.5%), the liquid containing it can be converted into semi-fluid after warming.
  • a certain threshold eg ⁇ -glucan ⁇ 2.5%)
  • Preparation method of A22-A24 Measure the water-soluble component of probiotics according to the required concentration (for example, required for local chemical action) (for example, 1.1 g or equivalent of the supernatant component of broken Saccharomyces cerevisiae prepared according to the method of A17-A18) liquid 20ml), optional other components, and a liquid carrier (eg, water for injection) made up to a total volume (eg, 100ml), and slowly mixed until uniform. If the liquid mixture is dispensed (for example, 10 ml/bottle) and capped according to the desired drug volume/target volume ratio (for example, the average volume of clinically common solid tumors is 30 cm 3 ), a tumor-providing tumor can be obtained.
  • the required concentration for example, required for local chemical action
  • a liquid carrier eg, water for injection
  • compositions A22-A24 in the above table Dosage forms and strengths of locally acting pharmaceutical compositions.
  • the obtained preparation was A22.
  • preparation of different compositions eg preparations A22-A24 in the above table
  • Preparation method of A25-A31 Measure the water-soluble component of probiotics (such as 5 g of water-soluble yeast ⁇ -glucan obtained from commercial sources) at the concentration required (such as required for local chemical action), other optional The components, and a liquid carrier (eg, water for injection) to a total volume (eg, 100 ml), are slowly mixed until uniform. If the liquid mixture is dispensed (for example, 10ml/vial) and capped according to the desired drug volume/target volume ratio (for example, the average volume of clinically common solid tumors is 30cm 3 ), a tumor-providing tumor can be obtained. Dosage forms and strengths of locally acting pharmaceutical compositions. The obtained preparation was A26. Different compositions (eg, preparations A25-A32 in the table above) can be prepared from different probiotic components that are commercially available, respectively, using the same method used for the preparation of A26.
  • probiotics such as 5 g of water-soluble yeast ⁇ -glucan obtained from commercial sources
  • concentration required such
  • composition water preparation obtained by the above preparation method can add chemically active ingredients according to the method in the prior art to prepare the composition water preparation comprising the probiotic component* and the chemically active ingredients.
  • Table 3 Components and proportions of the composition water preparation containing probiotic components* and chemically active ingredients
  • composition water preparation (B1, B5-B7, B9-B18, B25, B26, B29-B32) comprising probiotic components (water-soluble components 1-3) and chemically active ingredients: as required (for example, for local synergy or/and mid- and long-term synergy), the synergistic ratio and synergistic concentration are taken to measure the water-soluble components of probiotics (such as water-soluble yeast ⁇ -glucan 10g), the common drug (such as 20g arginine) , optional other ingredients, and water for injection to make up to a total volume of 100 ml, and slowly mix them well.
  • probiotics such as water-soluble yeast ⁇ -glucan 10g
  • the common drug such as 20g arginine
  • a tumor-providing tumor can be obtained. Dosage forms and strengths of locally acting pharmaceutical compositions.
  • the obtained preparation was B1. Using the same method as B1 preparation, different compositions (such as preparations B1, B5-B7, B9-B18, B25, B26, B29-B32 in the above table) can be prepared from different probiotic water-soluble components and their co-drugs, respectively. ) preparation.
  • composition aqueous B2-B4, B8
  • probiotic components water-insoluble granules/water-soluble components
  • chemically active ingredients as required (eg local synergy or/and mid- to long-term synergy required) synergistic amount ratio and synergistic concentration to measure probiotics (for example 1.5g Saccharomyces cerevisiae), common medicine (for example 20g acid resistance), other components optionally present, and water for injection fixed to a total volume of 100ml, And using the same method as the preparation of A10, they were slowly mixed uniformly and crushed using a homogenizer to obtain a crushed probiotic/shared drug suspension.
  • probiotic components water-insoluble granules/water-soluble components
  • chemically active ingredients as required (eg local synergy or/and mid- to long-term synergy required) synergistic amount ratio and synergistic concentration to measure probiotics (for example 1.5g Saccharomyces cerevisiae), common medicine (for example 20g
  • the suspension can be obtained.
  • the obtained preparation was B2.
  • the preparation of different compositions eg preparations B2-B4, B8 in the above table
  • composition water B19-B22
  • probiotic component si-fluidizable component
  • chemically active ingredient synergistic as required (eg for local synergy or/and for mid- to long-term synergy)
  • Amount Ratio and Synergistic Concentration Measure probiotic semi-fluidizable components (for example, 7.5 g of water-insoluble beta-glucan), co-medicine (for example, 1 g of 5-fluorouracil), other components optionally present, and make up to a total volume.
  • a tumor-providing tumor can be obtained. Dosage forms and strengths of locally acting pharmaceutical compositions.
  • the obtained preparation was B19. Using the same method as for the preparation of B17, the preparation of different semi-fluid compositions (eg B20-B22 in the above table) can be carried out.
  • composition water preparation comprising probiotic components (water-insoluble component particles) and chemically active ingredients: according to the required (for example, local synergistic effect or/and mid- and long-term synergistic effect required) synergistic amount ratio and The synergistic concentration measures the probiotic water-insoluble component granules (such as 10 g of water-insoluble ⁇ -glucan particles in A19), the co-medicine (such as 10 g of reduced glutathione, 1 g of 5-fluorouracil, 1 g of sodium hydroxide), any Select the other ingredients present, and water for injection to make up to a total volume of 100 ml, and mix them slowly until uniform.
  • probiotic water-insoluble component granules such as 10 g of water-insoluble ⁇ -glucan particles in A19
  • co-medicine such as 10 g of reduced glutathione, 1 g of 5-fluorouracil, 1 g of sodium hydroxide
  • any Select the other ingredients present and water for injection to make up to
  • a tumor-providing tumor can be obtained. Dosage forms and strengths of locally acting pharmaceutical compositions.
  • the obtained preparation was B23. Using the same method as for the preparation of B6, different compositions can be prepared from different probiotic components (water-insoluble particles such as probiotic precipitation components) and different co-medicines, respectively.
  • composition aqueous preparation (B24, B27, B28) comprising probiotic components (inactivated probiotics) and chemically active ingredients: synergistic as required (eg for local synergy or/and for mid- and long-term synergy)
  • synergistic concentration Measure probiotics (such as 2.5g dry powder of Saccharomyces cerevisiae), other ingredients (such as 1g methylene blue, 1g 5-fluorouracil), and water for injection with a total volume of 100ml, and mix them slowly and uniformly Get a probiotic/shared medicine mix.
  • a tumor-providing tumor can be obtained.
  • B24 can be obtained by placing the preparation (eg, 2.5% S. cerevisiae) in a Pasteur inactivation cabinet for pasteur inactivation (60°C, 48 hours). Using the same method as the preparation of B24, the preparation of different inactivated probiotics (eg preparations B27, B28 in the above table) can be carried out from different probiotics, respectively.
  • non-inactivated preparations such as A1-B31, B1-B32
  • Pasteur inactivation cabinet machine for pasteurization (60°C, 48 hours)
  • sterilized liquid injections can be obtained .
  • the above-mentioned liquid preparations are respectively freeze-dried.
  • the freeze-drying process conditions are: -45°C for 4 hours for pre-freezing, and the temperature rises to -15°C at a heating rate of 0.1°C/min. Keep at least 10h for sublimation; raise the temperature to 30°C, and keep for 6h for desorption and drying. Divide the water for injection as needed, such as 7.5ml/bottle, and seal it to obtain a solvent bottle for injection.
  • the sterile solvent in the bottle is drawn into the above-mentioned lyophilized powder bottle for injection and mixed uniformly to form a liquid medicine, that is, an injection medicine, such as 1.5% Saccharomyces cerevisiae broken component/20% amino acid.
  • Tumor-bearing nude mice are widely used in drug research for chemotherapy rather than immunotherapy in patients with solid tumors.
  • Nude mice were used as experimental objects, and human liver cancer HepG2 cells were used as model cells. 1 ⁇ 10 5 cells were injected into the right The transplanted tumor was modeled subcutaneously in the lateral axilla. The average volume of the tumor in nude mice was 161.3 mm 3 .
  • the model animals were randomly divided into 17 groups and administered according to the drugs and methods in Table 4. Each group was given medication. 1 time, each injection volume is 150 ⁇ l/only. On the 7th day after administration, the animals were euthanized, and the tumor tissue was removed after dissection to measure the tumor weight, and the tumor inhibition rate (r) was calculated according to the negative control group.
  • intravenous 5-fluorouracil provides conventional chemistries
  • intratumoral injections provide common local chemistries
  • the differences in pharmacodynamics caused by the two modes of administration are related to cytotoxic kinetics.
  • the ratio of tumor inhibition rate E 7 /E 1 ⁇ 200% in group 7 indicates that 5-fluorouracil acts as a cytotoxic drug under different administration conditions (systemic administration vs topical administration).
  • High concentrations (75-99%) of ethanol by intravenous injection provide a drunken response; intratumoral injection provides chemoablation.
  • Interleukin-12 which is commonly used clinically as an immunopotentiator, unexpectedly showed no short-term tumor suppressive effect by intratumoral injection (group 9).
  • probiotics and their components can also be used as auxiliary drugs, and their anti-tumor effects are also based on their immune-enhancing effects.
  • the results of groups 4-6 are consistent with those of group 3. It is worth noting that 1 animal in groups 4 and 5 died after injection, indicating that the suspension of 5% probiotic components has a safety risk through intravenous injection.
  • the results of group 10 should be consistent with the immune enhancement control group 9.
  • the pharmacological behaviors between them are significantly different: the same drug and the same dose of group 9 and group 3 have almost no difference in efficacy; while the same drug and the same dose of the same dose of group 10 and group 4
  • the pharmacodynamic difference exceeds expectations for its kinetic improvement.
  • the limit (E 10 /E 9 > 200%), which is also reflected in the significant difference in efficacy between them (E 10 /E 9 459% > 200%).
  • enteral administration can probiotic components play a role in regulating the bacterial flora in the intestinal tract; and both enteral and parenteral administration can make probiotic components play a role in enhancing immunity. Therefore, when the conventional administration methods such as oral and intravenous injection are excluded, and local administration, especially intralesional administration, can enable the probiotic components to provide new functions that are different from the prior art (E 10 /E 4 >> 200% ).
  • the immune-enhancing effects of probiotics and their components are based on their bacterial immunogenicity. It is generally considered that the order of bacterial immunogenicity decreases from strong to strong: live bacteria, inactivated bacteria that basically maintain the shape of live bacteria, water-insoluble particles that have a somewhat similar shape to live bacteria, semi-fluid that loses the shape of live bacteria, water-soluble components .
  • Pharmacological functions are the fundamental characteristics of drugs, and new pharmacology discovered by old drugs can often creatively produce new applications just like new drugs.
  • the effective application of a drug usually depends on the pharmacological function provided with a comparative advantage, that is, the preferred pharmacological effect, under experimental conditions defined by clinical treatment, such as efficacy and safety under the minimization of dosing frequency and dosage.
  • the same substance may then be used as different active ingredients under different conditions, and the same type of substance may have different preferred solutions suitable for use as different active ingredients.
  • each positive control substance (5-fluorouracil, ethanol, interleukin-12) was shown to meet the expectations of its preferred pharmacology in different pharmacological methods (respectively, cytotoxic pharmacology, immune enhancement pharmacology, local action pharmacology) Pharmacodynamics and pharmacological behavior, and presents a pharmacological comparative study system.
  • the pharmacological effects of probiotic components during intravenous injection may still be in line with their pharmacological (such as immune enhancement) expectations in the prior art, but the pharmacological effects of their intralesional administration clearly far exceed this expectation. Its pharmacological behavior exceeded even the cytotoxic pharmacological (if any) expectations.
  • the probiotic component as an active ingredient that provides a local effect is far more pharmacologically and therapeutically than it is as an active ingredient (non-topical) that provides any non-local effect (such as cytotoxicity or immune enhancement). active ingredient).
  • mice Taking nude mice as experimental objects, using breast cancer cells (MDA-MB231) as modeling cells, 1 ⁇ 10 5 cells per mouse were injected subcutaneously into the right axilla to model transplanted tumors. The average tumor volume was 158.2 mm 3 .
  • the model animals were randomly divided into 16 groups and administered intratumorally according to the drug components and doses in Table 5, and each group was administered once. On the 7th day after administration, the animals were euthanized, and the tumor tissue was removed after dissection to measure the tumor weight, and the tumor inhibition rate (r) was calculated according to the negative control group.
  • the supernatant fraction is the supernatant fraction of broken Saccharomyces boulardii; **: The inactivated bacteria are heat-killed Saccharomyces boulardii; ***; The microparticles are Saccharomyces cerevisiae ⁇ -glucan microparticles; * ***: Broken bacteria is Broken Saccharomyces boulardii
  • the administration concentration of the probiotic component in the composition is the pharmacological concentration required for local effect, not required for the enhancement of rabbit infection.
  • a necessary condition for the probiotic component to provide local action is: the content of the probiotic component in the pharmaceutical composition must make its pharmacological concentration (local administration concentration) > 0.1%, ⁇ 0.25%, 0.25-25%, preferably 0.5-15%, more preferably 1-15% or 5-15%.
  • the probiotic component when the probiotic component is inactivated probiotic, its pharmacological concentration is greater than 0.3%, ⁇ 0.75%, 0.75-15%, preferably 1.5-15% or 5-15%; when the probiotic component is It is a water-soluble component of probiotics, and its pharmacological concentration is greater than 0.1%, such as 0.15-25%, preferably 0.35-25% or 5-25% (wherein, the water-soluble component is the probiotic supernatant component, then 0.35- 3.5%, 2-5% or 5-15% for water-soluble probiotic-derived polysaccharide, and 15-25% for probiotic-derived ribonucleic acid); when the probiotic component is probiotic water-insoluble component particles , then its pharmacological concentration is > 0.5% or 0.5-15%, preferably 1.5-15% or 5-15%; when the probiotic component is a probiotic semi-fluid component, its pharmacological concentration is > 2.5 %, 2.6-25%, preferably 5-15%.
  • the use of the same substance as different active ingredients may need to satisfy different pharmacokinetic characteristics.
  • the results of the dose-dependent comparative study of the above pharmacodynamics further confirmed that the local activity of the probiotic components of the present invention is similar to chemical ablation, and is far from conventional activities (cancer cell inhibition, tumor vascular inhibition, or immune enhancement, etc.) Far.
  • the pharmacological concentrations of the probiotic components described in this application as topical active ingredients cannot be simply compared with the formulation concentrations or administration concentrations of the probiotic components as non-topical active ingredients in the prior art.
  • the formulation concentration of non-locally active drugs is often only limited by the formulation.
  • high-concentration formulations can save transportation and storage costs, and appropriate high-concentration injections can reduce the injection volume and shorten the administration time.
  • formulation concentrations can vary widely, but are essentially different from the administration concentrations required for pharmacological action.
  • concentration of the drug is far lower than the concentration of the preparation by dilution, so as to avoid the safety risk caused by the rapid entry of the drug into the blood.
  • the pharmacological concentration in the composition of the present invention not only limits its composition and preparation as a feature, but also must appear in the new drug application as a pharmacological condition, and must also appear in the instruction manual of the medicine as an application condition.
  • the content of components of different pharmacological pharmaceutical compositions usually needs to be defined by different characteristics.
  • conventional pharmaceutical compositions have no volume-dependent pharmacological volume of the lesion target area outside the administration dose, but the following experiments are studied.
  • Nude mice were used as experimental objects, and human pancreatic cancer cells (PANC-1) were used as modeling cells. 1 ⁇ 10 5 cells per mouse were injected subcutaneously into the right axilla to model transplanted tumors. The modeling was successful in nude mice. The average tumor volume was 213.1 mm 3 . The model animals were randomly divided into 16 groups and administered intratumorally according to the drug components and administration volumes in Table 6, and each group was administered once.
  • PANC-1 human pancreatic cancer cells
  • the medicines of groups 1-12 are all prepared by the method of Example 1, wherein the inactivated probiotics are heat-killed Saccharomyces boulardii, and the water-insoluble component particles of probiotics are ⁇ -glucan particles, probiotics
  • the water-soluble component is the broken Saccharomyces boulardii supernatant component
  • the probiotic water-insoluble component granules and the water-soluble component mixture (abbreviated as the probiotic component mixture) is the broken Saccharomyces boulardii
  • the preparation method is the same as the Example 3: On the 7th day after administration, the animals were euthanized, and the tumor tissue was removed after dissection to measure the tumor weight, and the tumor inhibition rate (r) was calculated according to the negative control group.
  • Ethanol failed to provide chemical ablation, similar to intravenous ethanol; while the tumor inhibition rate of group 02 was significantly different from that of group 01 (E 02 /E 01 >200%), and the tumor inhibition rate of group 02 was similar to that of group 03 Large (E 03 /E 02 < 200%), showing expected chemical ablation. This shows that only if ethanol exceeds a certain dose volume/tumor volume ratio threshold, such as 0.15, it can be used as a chemical ablation agent.
  • a certain dose volume/tumor volume ratio threshold such as 0.15
  • the pharmacodynamic differences of the probiotic components at the same dose but with different administration volume/target volume ratios can far exceed the expected kinetic differences (eg, E 2 ) . /E 1 >200%)), at this time, the administration volume of the probiotic component (administration volume/target volume volume ratio) is not a pharmacodynamic issue, but a pharmacological issue.
  • the content of the probiotic component in the pharmaceutical composition is such that the administration volume/target volume ratio is >0.09, 0.1-1.5, preferably 0.23-1.5 or 0.5-1.5.
  • the administration volume of drugs based on immune-enhancing pharmacology in the tumor area is generally very low (such as cytokines, the volume is less than or equal to 2ml).
  • the pharmaceutical composition described in this application can only be administered under the conditions that the above - mentioned administration volume/target volume ratio is satisfied.
  • the required administration volume is greater than or equal to 6.0 cm 3 , and the specification volume of the pharmaceutical preparation at this time is 6 ml or an integer multiple. It is well known that, in essence, the drug specification can also be one of the commonly required content forms for the active ingredient to achieve the desired pharmacological effect.
  • the above-mentioned preferred regimen of the administration volume according to the lesion volume of the probiotic component as the topical active ingredient described in this application exceeds the expectations of the prior art of the probiotic component.
  • the probiotic components used as non-topical active ingredients (such as immune-enhancing active ingredients) in the prior art may be able to assist in the treatment, but cannot be used as the main drug to treat local diseased diseases. Lesions are treated, so the dose volume is not based on the local lesion volume.
  • the safety volume (such as the administration volume for death) of the same concentration of drug administered in the lesion can be 2 times higher than that of intravenous administration above.
  • the probiotic components described herein as topical active ingredients are particularly suitable for larger (eg, mean diameter greater than 3 cm) lesions.
  • the results of the volume-dependent comparative study of the above drug effects further confirmed that the local activity of the probiotic components of the present invention is similar to chemical ablation, and is far from conventional activities (cancer cell inhibition, tumor vascular inhibition, or immune enhancement, etc.) Far.
  • the pharmacokinetic characteristics of probiotic components as non-topical active ingredients in the prior art are blood concentration (usually very low, such as 0.25 ⁇ 10 -5 %), and the administration volume is only related to the dose required for blood concentration, It has nothing to do with the volume of the target volume of the lesion. Since this dose is associated with systemic safety, the dose administered is broadly diluted from the pharmacokinetic profile and determined by the physician based on the patient's weight and other status.
  • the pharmacological volume of the probiotic components described herein as topical active ingredients is an important pharmacokinetic feature (involving the pharmacological relationship and consequences between the effective penetration zone and the drug-free zone), which not only limits its composition (e.g. unit volume of preparation) and preparation, and it must also appear as a pharmacological condition in the approval of a new drug, and it must also appear as an application condition in the instructions for use of the drug.
  • the pharmacological volume of the probiotic component as a topical active ingredient described in this application cannot be simply compared with the unit formulation volume or administration volume of the probiotic component as a non-topical active ingredient in the prior art.
  • compositions of the present invention comprising a probiotic component outperform non-probiotics of the prior art comprising a probiotic component
  • compositions of the present invention comprising a probiotic component outperform non-probiotics of the prior art comprising a probiotic component
  • the probiotic component in the composition of the present invention is based on the discovery of its new pharmacology-drug penetration zone tissue destruction effect-that is, the local effect as a local active component, and this local effect mainly includes local chemical Effects (eg, chemical-like ablation) that exceed the pharmacological expectations of any non-local activity (eg, immune effects, antiviral effects, tumor cytotoxic effects, or tumor vascular destruction effects, etc.) of prior art probiotic components;
  • the probiotic component in the composition of the present invention must be strictly limited to local administration to enter the target zone reactor to realize its new function, and the probiotic component in the prior art composition.
  • Pharmacological methods are not limited to local administration, preferably systemic administration to achieve its target function;
  • the probiotic component in the prior art immune-enhancing composition is preferably a component with stronger bacterial immunity (such as a live bacteria and a component with the highest possible proximity in morphology. , such as inactivated probiotics that basically retain the form of live bacteria), while the composition of the present invention is preferably a component that minimizes bacterial immunity (components that are as close as possible to live bacteria in morphology, such as water-soluble components of probiotics) , probiotic semi-fluid components).
  • the probiotic component in prior art composition realizes its systemic effect (pharmacological concentration is blood drug concentration) the kinetic condition depends on the administration dose rather than the administration concentration, Not to mention the concentration of the preparation (which is usually prepared as a concentrate for the convenience of preparation and storage and transportation), the preparation is also diluted to avoid local effects such as damage at the injection site.
  • the kinetic conditions for the probiotic components in the composition of the present invention to achieve their local effects are conversely dependent on the administered concentration rather than the administered dose (blood drug concentration). its formulation concentration.
  • the administration volume of the prior art composition is only related to the dose of the blood drug concentration required by its systemic pharmacology, and has nothing to do with the volume of the target area of the lesion, while the administration volume of the composition of the present invention is related to the target area that its local activity needs to penetrate volume related;
  • the reaction environment required for its pharmacological response is beyond expectations: the composition of the present invention is strictly limited to the corresponding composition of the topical dosage form (must not include some excipients for systemic dosage forms, compositions during administration) need to be a sufficient mixture to require a uniform solvent, etc.), etc.;
  • the treatment effect on local lesions exceeds expectations: first, the treatment effect on local lesions exceeds expectations. For example, at least in the area of administration, comparable to the recognized effective drug, far exceeding the short-term expectations of prior art compositions (eg, 21 days after the first dose in the case of multiple doses, or 7 days after the last dose). day) of the drug effect (r composition of the invention /r composition of the prior art >200%, preferably r composition of the invention /r composition of the prior art >400%).
  • the efficacy of the drug also leads to a wider range of indications than expected.
  • the above-mentioned animal models can represent immunocompromised patients, elderly patients, and patients with weakened immunity after various treatments. These also further show that the differences between the two compositions are not kinetic but pharmacological (eg local vs conventional).
  • nude mice As experimental objects, using lung tumor cells (A549) as modeling cells, 1 ⁇ 10 5 cells/mouse was injected subcutaneously into the right axilla to model the transplanted tumor, and the tumor body of nude mice was successfully modeled. The average volume was 153.7 mm 3 .
  • the model animals were randomly divided into 18 groups and administered according to the drug components and administration methods in Table 7. Each group was administered once, and the injection volume was 150 ⁇ l per animal.
  • the probiotic component was heat-killed Saccharomyces boulardii, and the medication for group 16 was intratumoral injection of the probiotic component for about 2 hours, followed by intratumoral injection of 10% arginine; on the 7th day after administration, the animals were treated with Euthanasia was performed, the tumor tissue was removed after dissection to measure the tumor weight, and the tumor inhibition rate (r) was calculated according to the negative control group, and the actual/expected drug efficacy ratio q was also calculated according to the curative effect (tumor inhibition rate).
  • Group 12 of the same probiotic component/chemical active substance (ethanol) mixture with different administration methods showed much higher efficacy than group 5, but their actual combined effect/theoretical pure additive expected effect ratio q were all higher. Not more than 1.00, the high efficacy is mainly due to the local effect of ethanol (group 8). According to this expectation, the same probiotic components and other chemically active substances should also have similar pharmacological behaviors. However, groups 13 and 14 showed significantly higher efficacy than groups 6 and 7 (respectively using the same probiotic component/chemically active mixture), indicating that their higher efficacy is directly related to their local effects related. Moreover, the actual synergistic effect/theoretical simple additive expected effect ratio q of groups 13 and 14 were both greater than 1.00, indicating that the local effect was a local synergistic effect of the probiotic components and these chemical active substances.
  • group 15 had the highest efficacy, and its actual combined effect/theoretical simple additive expected effect ratio q>1.00, indicating that the three-component composition (probiotic component/weak local action compound/cytotoxic drug) actually A synergistic effect can be further produced on the basis of the synergistic effect of its two components (probiotic component/weak local action compound and probiotic component//cytotoxic drug).
  • the pharmaceutical composition of group 16 is not in the same agent despite the same ingredients as group 14, and its actual synergistic effect/theoretical simple additive expected effect ratio q ⁇ 1.00 shows no synergistic effect.
  • probiotic component single drug or non-synergistic drug combination should be used under analgesic conditions to avoid safety risks; No. 13-15, the above-mentioned strong irritant response was not observed, indicating that the synergistic pharmaceutical composition of the probiotic component and the weak local action compound and/or cytotoxic drug has a safety that exceeds the expected safety of the single-drug effect.
  • probiotic semi-fluid components, probiotic water-soluble components, probiotic water-insoluble component particles, etc. with chemical drugs also has the same results, which will not be described in detail here due to space limitations.
  • the applicant has carried out intensive research on the composition of probiotic components, especially the composition formed by the three components.
  • Nude mice were used as experimental subjects, gastric tumor cells (BGC823) were used as modeling cells, and 1 ⁇ 10 5 cells per mouse were injected subcutaneously into the right axilla to model the transplanted tumor.
  • the tumor body of nude mice was successfully modeled.
  • the average volume was 164.4 mm 3 .
  • the model animals were randomly divided into 14 groups, and administered intratumorally according to the drug components in Table 8. Each group was administered once, and the injection volume was 150 ⁇ l per animal; The pharmaceutical composition of the group was semi-fluid, and the rest of the groups were liquid.
  • the tumor inhibition rate (r) was calculated according to the negative control group, and the actual/expected ratio of drug efficacy could also be calculated according to the curative effect (tumor inhibition rate).
  • the animals were euthanized on the 7th day after administration, and the muscle mass of the right leg of the nude mice was dissected out, and gross pathological analysis was carried out.
  • the results showed that the abnormal areas of groups 1, 2 and 3 were 32.24 ⁇ 13.71mm 2 , 46.78 ⁇ 13.64mm 2 and 72.35 ⁇ 23.71mm 2 , respectively, indicating that the local synergy provided by the probiotic components was similar but stronger than Ethanol, which is primarily a topical chemical synergy.
  • mice Taking nude mice as experimental objects, using mouse embryonic fibroblasts 3T3 as modeling cells, 2 ⁇ 10 4 cells/mouse were injected subcutaneously into the right axilla to model the transplanted nodules. The average volume of tumor-like nodules was 171.5 mm 3 .
  • the model animals were randomly divided into 16 groups and administered according to the drugs in Table 9. Except for groups 21-24, each group was given intraperitoneal injection and intra-nodular injection respectively.
  • the medicine used in the group 21-24 is the composition of the non-local administration dosage form (the dosage form in which the probiotic component and the common substance can be administered separately), and the administration mode of the series A is: intraperitoneal injection of the probiotic component + Intra-nodular injection of the combination, the administration mode of series B is: intraperitoneal injection of the combination + intra-nodular injection of the probiotic component.
  • Each group was treated twice with an interval of 2 days, and the injection volume was 150 ⁇ l/piece/time.
  • the local lesion volume (V) containing fibroblasts was measured on the 7th day after the last administration, and the series were calculated according to the negative control group.
  • the relative local lesion inhibition rate (r'A, r'B) of A and series B , and the actual/expected drug effect ratio q can also be calculated according to the efficacy (local lesion inhibition rate).
  • the tumor inhibition rates of the drugs in group 03 in series A and B are both less than 15%, which is in line with the pharmacological expectation that they are only used as adjuvant therapy drugs to enhance immunity.
  • the tumor inhibition rate of group 04 after intraperitoneal injection r ⁇ 15%, and the tumor inhibition rate r>15% after intratumoral injection, has a certain therapeutic effect, which is also in line with its non-specificity in extreme conditions.
  • Pharmacology of cytotoxic action but only showed the expected pharmacology of local action ( EB04 /E A04 > 200%) under normal conditions.
  • the efficacy of the drugs in groups 1-4 by intraperitoneal injection is consistent with that in groups 01-04, and the possible immune-enhancing effects, cytotoxic effects or any other effects provided by the probiotic components by systemic administration do not exceed the control Pharmacological expectations for drugs 01-04.
  • the efficacy of the drugs in groups 1-4 by intratumoral injection is much higher than that by intraperitoneal injection ( EB /EA>200%), showing a distinct new pharmacology.
  • the efficacy of the drugs in groups 1-4 was similar to that of the control drug 01-04, showing only local effects under conventional conditions.
  • the drugs in groups 11 and 12 also showed similar efficacy and pharmacology to the control drug 04.
  • Nude mice were used as experimental objects, and human hepatoma cells (HepG2) were used as model cells. 1 ⁇ 10 5 cells per mouse were injected subcutaneously into the right axilla to model the transplanted tumor. The tumor body of nude mice was successfully modeled. The average volume is 155.7 mm 3 , the model animals are randomly divided into 13 groups, and the drugs are administered according to the drugs in Table 10.
  • the relevant drugs are all aqueous liquids, prepared according to the method of Example 1; each group is administered once and injected The amount was 150 ⁇ l/animal/time, the animals were euthanized on the 7th day after administration, the tumor tissue was removed after dissection to measure the tumor weight, and the tumor inhibition rate was calculated according to the negative control group, and the drug can also be calculated according to the curative effect (tumor inhibition rate). Effective actual/expected ratio q.
  • tumor inhibition rate 01 (saline) 0.261 ⁇ 0.098 - 1 0.2% probiotic component 0.231 ⁇ 0.113 11.5% 2 0.5% probiotic component 0.208 ⁇ 0.109 20.3% 3 10% probiotic component 0.149 ⁇ 0.091 43.1% 4 20% probiotic component 0.127 ⁇ 0.085 51.4% 5 10% sodium bicarbonate/4.3% sodium hydroxide 0.032 ⁇ 0.021 87.6% 6 7% sodium bicarbonate/3% sodium hydroxide 0.064 ⁇ 0.036 75.4% 7 0.7% sodium bicarbonate/0.3% sodium hydroxide 0.175 ⁇ 0.088 33.1% 8 0.2% sodium bicarbonate/0.09% sodium hydroxide 0.228 ⁇ 0.104 12.5%
  • the sodium bicarbonate-sodium hydroxide buffer system reduces a solution of sodium hydroxide alone from pH > 12.5 to around 11, a pH reduction that is generally considered to be detrimental to topical chemistry.
  • the actual synergistic effect/theoretical pure additive expected effect ratio q ⁇ 1.00 of group 9 the pharmaceutical composition in it did not show a synergistic effect
  • the pH of the pharmaceutical composition in group 10, 11, 12 They are pH10 ⁇ 1.0 or pH4 ⁇ 1.0, respectively.
  • Their actual synergistic effect/theoretical simple additive expected effect ratio q are all greater than 1.00, and they all show synergistic effect. (0.2-20)/(0.29-14.3).
  • Nude mice were used as experimental objects, and human hepatoma cells (HepG2) were used as model cells. 1 ⁇ 10 6 cells per mouse were injected subcutaneously into the right axilla to model the transplanted tumor.
  • the tumor body of nude mice was successfully modeled.
  • the average volume is 163.2mm 3
  • the model animals are randomly divided into 13 groups, and the drugs are administered according to the medicines in Table 11.
  • the relevant medicines are all aqueous liquids, prepared according to the method of Example 1, wherein the probiotic components are inactivated Saccharomyces cerevisiae, the weak topical compound was methylene blue; each group was administered twice, with an interval of 7 days, and the injection volume was 150 ⁇ l/animal/time. Animals were euthanized on the 7th day after the last administration, and stripped after dissection. The tumor tissue was removed to measure the tumor weight, and the tumor inhibition rate was calculated according to the negative control group.
  • Nude mice were used as experimental objects, and human hepatoma cells (HepG2) were used as model cells. 1 ⁇ 10 5 cells per mouse were injected subcutaneously into the right axilla to model the transplanted tumor. The tumor body of nude mice was successfully modeled. The average volume is 168.7mm 3 , the model animals are randomly divided into 13 groups, and the medicines are administered according to the medicines in Table 12. The relevant medicines are all aqueous liquids, and are prepared according to the method of Example 1, wherein the probiotic components are broken wine brewing.
  • Yeast supernatant fraction the weak local action compound is lysine; each group was administered twice, the administration interval was 7 days, the injection volume was 150 ⁇ l/only/time, and the animals were euthanized on the 7th day after the last administration. After dissection, the tumor tissue was removed to measure the tumor weight, and the tumor inhibition rate was calculated according to the negative control group.
  • Nude mice were used as experimental objects, and human pancreatic cancer cells (PANC-1) were used as modeling cells. 1 ⁇ 10 5 cells per mouse were injected subcutaneously into the right axilla to model transplanted tumors. The modeling was successful in nude mice. The average volume of the tumor body was 157.3mm 3 .
  • the model animals were randomly divided into 13 groups and administered according to the medicines in Table 13. The relevant medicines were all water-containing liquids, prepared according to the method of Example 1, wherein the probiotic components The supernatant fraction of Saccharomyces cerevisiae was broken, and the chemotherapeutic drug was gemcitabine; each group was administered once, and the injection volume was 100 ⁇ l/animal/time. The animals were euthanized on the 7th day after administration, and the tumor tissue was removed after dissection. The tumor weight was measured, and the tumor inhibition rate was calculated according to the negative control group.
  • compositions in group 9 showed synergy (q ⁇ 1.00), while the compositions in groups 10, 11, 12 showed significant synergy (q >1.00, respectively) and were synergistic
  • the amount ratio (W probiotic component /W common compound ) is (0.2-20)/(0.05-2.5), which also shows that the local chemical synergy provided by the probiotic component has nothing to do with the local chemical effect provided by the weak local action compound. .
  • the synergistic amount ratio of the probiotic components in the pharmaceutical composition is not required for the enhancement of the synergistic effect of the rabbit epidemic, but is required for the local synergistic effect, so it is the pharmacological amount ratio.
  • the pharmacological ratio (W probiotic component /W chemically active compound ) of the probiotic component when used together with the chemically active compound is (1-110)/(1-100).
  • (W probiotic component /W cytotoxic drug ) is (1-110)/(1-100); if the chemically active compound is a weak local action compound , then (W probiotic component /W weak local action compound ) is (1-90)/(1-100); if the weak local action compound is amino acid nutrient, then W probiotic component /W amino acid nutrient (1-20)/(1-100); if the weak local action compound is a vital dye, then the W probiotic component /W vital dye is (7-90)/(1-100); if the When the weak local action compound is an acidulant and/or an alkalinizer, then the W probiotic component /W of the acidulant or/and the alkalinizer is (2-60)/(1-100).
  • Example 8 Pharmacological study of local synergy and optimization of pharmacological concentration
  • Nude mice were used as experimental objects, and human pancreatic cancer cells (PANC-1) were used as model cells. 1 ⁇ 10 6 cells per mouse were injected subcutaneously into the right axilla to model transplanted tumors. The modeling was successful in nude mice. The average volume of the tumor was 152.8mm 3 .
  • the model animals were randomly divided into 10 groups and administered intratumorally according to the drugs and doses in Table 14. The relevant drugs were all liquid preparations, and the probiotic components were broken Saccharomyces cerevisiae The clear component, the chemically active compound is arginine, was prepared according to the method of Example 1; each group was administered once, the animals were euthanized on the 7th day after administration, and the tumor tissue was removed after dissection to measure the tumor weight. The tumor inhibition rate was calculated according to the negative control group.
  • Tumor weight (x ⁇ s_g) tumor inhibition rate 0 normal saline 100 0.246 ⁇ 0.123 - 1 0.1% probiotic component 100 0.218 ⁇ 0.096 11.3%
  • probiotic component 200 0.189 ⁇ 0.069 23.1% 3 0.5% probiotic component 500 0.169 ⁇ 0.088 31.4% 4 2% Arginine 100 0.226 ⁇ 0.104 8.1% 5 5% Arginine 200 0.210 ⁇ 0.113 14.7% 6 10% Arginine 500 0.193 ⁇ 0.075 21.4% 7 0.1% probiotic component/2% arginine 100 0.206 ⁇ 0.082 16.3% 8 0.25% probiotic component/5% arginine 200 0.146 ⁇ 0.091 40.7% 9 0.5% probiotic component/10% arginine 500 0.089 ⁇ 0.020 63.9%
  • groups 7-9 are injections of probiotic components/chemically active compounds with the same component and amount ratio but different concentrations into the same volume of tumor, wherein the pharmaceutical composition of group 7 does not show a synergistic effect ( q ⁇ 1.00), while the pharmaceutical composition of group 8 showed obvious synergy (q>1.00), and the result of group 9 (q>1.00) further indicated that the synergy was dose-dependent.
  • the pharmaceutical composition has the same amount ratio and dosage of active ingredients, its drug co-effects are the same; and the probiotic components can obtain different co-drug effects by providing different shared pharmacology. Even with intratumoral administration, the difference in pharmacodynamics of probiotic components at the same dose with different administration concentrations can even exceed the expected kinetic difference (E 2 /E 1 >200% in Table 13).
  • the above test further confirms that the short-term efficacy of the probiotic component/combination composition of the present invention obtained through local intralesional administration mainly comes from the local synergistic effect produced by their mixed use, wherein the probiotic component provides the above-mentioned synergistic effect.
  • the pharmacological concentration is the concentration that provides the local effect in the above examples, so the pharmacological structure of the probiotic components and their co-uses to produce this local synergy is not the amount ratio between them but the concentration ratio between them (above. Synergistic ratio + probiotic components to provide the concentration required for local action).
  • the concentration of the local synergistic effect is provided, the concentration of methylene blue dyes is the concentration that produces a weak local effect (0.5-1.5%), and other weak local action compounds are the concentrations that produce strong local effects (1 -35%), cytotoxic drugs are produced at concentrations (0.1-5%) that maximize normal local chemical effects.
  • the probiotic component/combination composition of the present invention is also preferably administered at the administration volume/target volume ratio described in Example 4 in relation to its local action.
  • Example 9 Short-term single-use/combined pharmacological study and optimization of drug delivery to lesion area in common animal models
  • mice Taking BALB/c mice as experimental objects, using breast cancer 4T1 cells as model cells, 0.5 ⁇ 10 6 cells/mouse was injected subcutaneously into the right axilla to model the transplanted tumor, and the tumor body of nude mice was successfully modeled.
  • the average volume was 160.1 mm 3 , and the model animals were randomly divided into 21 groups, and were administered intravenously (series A) and intratumoral injection (series B) according to the drugs and doses in Table 14, wherein the drugs were all liquid preparations , configured according to the method of Example 1; each group was administered twice, the animals were euthanized on the 7th day after the last administration, and the tumor tissue was stripped off after dissection to measure the tumor weight, and according to the corresponding negative results in the series A and B In the control group, the tumor inhibition rates r A and r B were calculated.
  • the chemotherapeutic drugs can attack their targets when they enter the target area, thus showing the chemotherapeutic efficacy in a short period of time. It can be seen from Table 14 that the therapeutic effects and pharmacological behaviors of the drugs in groups 1-5 in the common animal model are consistent with the results of the corresponding drugs in the immunodeficiency animal model in Experiment 2, which are in line with cytotoxicity, chemical ablation, immune enhancement, and local-acting pharmacology of chemical-like ablation.
  • mice Taking BALB/c mice as experimental objects, the model animals were randomly divided into 4 groups, 6 animals in each group, and the drugs were administered according to the drugs in Table 15; The injection volume was 100ul/mouse, the animals were euthanized on the 7th day after administration, and the muscle mass specimens on the lateral side of the right leg of the mice were dissected out, and gross pathological analysis was performed. Wait.
  • the topical pharmaceutical compositions of the present invention comprising the probiotic components and their co-uses (referred to as the present compositions of the present invention) are in the following aspects Exceeds expectations of any conceivable prior art non-topical pharmaceutical compositions comprising probiotic components/combinations (referred to as prior art compositions):
  • the probiotic component in the composition of the present invention and its co-use are shared between the topical active components, and its shared pharmacology includes the shared use of their respective local effects, and the short-term synergistic effect it produces.
  • the probiotic component in the composition of the present invention and its co-use must be strictly limited to local administration in order to produce short-term synergy, and any predictable probiotic component in the prior art and
  • the shared pharmacological method of the shared substance is not necessarily limited to local administration, preferably systemic administration;
  • the preferred scheme of its active components exceeds expectations: in terms of the number of component types, the composition of the present invention is preferably 3 or more (such as probiotic components/weak local action compounds/cytotoxic drugs), although 2 groups Sub-synergistic compositions (eg, probiotic components/weak topical compounds) have exceeded expectations.
  • the probiotic components surprisingly did not provide local synergy to ethanol, but to weak locally acting compounds or/and cytotoxic drugs.
  • the co-administration also preferably includes one or more drugs of a weak locally acting compound that mainly provides a local effect rather than a systemic effect, such as a weak one or more of topically acting compounds, weakly topically acting compounds and slimming drugs, weakly topically acting compounds and immune enhancers, and the like;
  • the common pharmacological structure between its active components is expected: the composition of the present invention must include the local action pharmacological concentration of the probiotic component, so its common pharmacological structure parameter is the concentration ratio rather than the amount ratio in the prior art composition .
  • the pharmacological concentrations of the probiotic components that provide the synergistic effect are the concentrations that provide the local effect in the above examples.
  • the pharmacological concentrations of the weak local action compounds are respectively 5-25% for amino acid nutrient, 0.5-3% or 0.5-1.5% for methylene blue dye, 3-10% for quinine, and 3-10% for weak acid. 3-20%, 1-10% for strong bases, 1-20% for weak bases, 3-25% total pH buffer system concentration of acidulants or/alkalis;
  • the composition of the present invention can provide local activity (local action or local synergy) that cannot be provided by prior art compositions, at least in the administration area that cannot be provided by prior art compositions. higher local therapeutic effect.
  • the pharmaceutical composition of the present application can be used for destructive treatment (eg, tumor decompression) of any diseased tissue (eg, comprising tumor cells or/and fibroblasts). More preferably, the pharmaceutical composition of the present application can be used for any systemic drugs (cytotoxic drugs, antiviral drugs, antibacterial drugs, vascular inhibitory drugs, immune drugs, etc.) that are difficult to treat, but intralesional administration is feasible. disease treatment.
  • Example 10 Medium- and long-term single-use/combined pharmacological research and optimization of drug delivery to lesion areas in common animal models
  • mice Taking BALB/c mice as experimental objects, using breast cancer 4T1 cells as model cells, 0.5 ⁇ 10 6 cells/mouse was injected subcutaneously into the right axilla to model the transplanted tumor, and the tumor body of nude mice was successfully modeled. The average volume was 107.4 mm 3 , and the model animals were randomly divided into 17 groups;
  • the drugs in Table 16 were administered, and the drugs were prepared according to the method of Example 1; each group was administered twice with an interval of 7 days; the doses of groups 13 and 14 were 300 ⁇ l/piece/time , the other groups were 100 ⁇ l/piece/time; the administration method of group 15 was the right forelimb axillary administration, and the other groups were intratumoral injection administration; group 16 was sequential administration (the first administration was probiotics Fluid components/cytotoxic drugs/weak local action compounds, the last administration was inactivated probiotics), and the other groups were administered the same drug twice.
  • the tumor volume of each group (V 7d and V 21d ) were measured respectively, and the relative tumor proliferation rate ( R 7d , R 21d ) was calculated from the negative control group on the 7th and 21st days after the medication. , so that the short-term efficacy (100% - R 7d ) and the long-term efficacy (100% - R 21d ) can be calculated respectively.
  • the positive controls (5-fluorouracil, ethanol, interleukin-12) in groups 1-3 showed mid- and long-term efficacy in line with the expected effects of their pharmacology (cytotoxic pharmacology, immune-enhancing pharmacology, and local action pharmacology, respectively)
  • Kinetics (R 21 /R 7 ) constructed a comparative study system.
  • the short-term efficacy of the pharmaceutical compositions of groups 1-7 on day 7 was consistent with the results in Example 9.
  • the short-term efficacy of the probiotic components in Groups 5-7 is as expected as a topical active ingredient similar to the chemical ablative agent in Group 2, and it seems reasonable to show that it is consistent with the chemical ablative agent in Group 2.
  • the probiotic components showed completely different effect kinetics (R 21 /R 7 was significantly less than 100%), which greatly exceeded the mid- and long-term pharmacological expectations of all positive controls. Different probiotic components showed nearly identical effect kinetics, independent of their bacterial immunogenicity.
  • the administration concentration of probiotic components in group 13 is not the optimal concentration for its local pharmacology, which shows that the short-term efficacy difference is greater than 200%.
  • the administration concentration of the composition is not the optimal concentration for its local synergy pharmacology, and it also shows that the short-term efficacy difference is greater than 200%.
  • the medium and long-term efficacy (R 21 ) of groups 13 and 14 was also significantly inferior to that of groups 5 and 6.
  • the medium and long-term efficacy of the sequentially administered composition (probiotic semi-fluidic component/cytotoxic drug/weak local-acting compound, inactivated probiotics once each) in group 16 was significantly higher than that of the second administration of two identical drugs ( Inactivated probiotics and probiotic semi-fluidic components/cytotoxic drugs/weak local-acting compounds, which showed different short-term (locally acting) efficacy in groups 5, 12), further illustrating the mid- to long-term effects of this class of drugs Efficacy is related to its short-term (locally acting) efficacy.
  • the short-term effects of the composition of the present application after intralesional administration are mainly local effects (or local synergistic effects), and the long-term effects are mainly secondary effects generated by local effects (or local synergistic effects), such as secondary effects.
  • immune effect The composition of the present application is completely different from immune drugs such as vaccines, wherein the substances that act as antigens include in situ immune substances such as in situ antigens produced after administration.
  • probiotic components like other foreign substances, may also have non-specific antigenic effects for enhancing the efficacy of the in situ immune substances.
  • composition of the present application has chemical/immunopharmacological functions including local effects (or local synergistic effects) and its secondary immune effects, thereby greatly exceeding the pharmacodynamic expectations of the probiotic components in the prior art, and becoming a therapeutic drug rather than an existing Immunopotentiator of technology; can provide effective treatment for any local lesion disease by defining local intralesional administration, local action concentration threshold, local action volume threshold, local synergistic composition, etc.; avoids within a course of treatment necessary for cytotoxic drugs High-frequency high-dose administration, high safety and good compliance.
  • composition of the present application can be used for the treatment of difficult-to-treat local lesions such as cytotoxic drugs, antiviral drugs, antibacterial drugs, vascular inhibitor drugs, immune drugs, such as solid tumors with a stroma ratio greater than 25%.
  • difficult-to-treat local lesions such as cytotoxic drugs, antiviral drugs, antibacterial drugs, vascular inhibitor drugs, immune drugs, such as solid tumors with a stroma ratio greater than 25%.
  • the composition described in this application can also use fibroblasts as a target for the treatment of the above-mentioned indications. It is one of the main features of refractory local disease.
  • Example 11 Single use/combination pharmacological study and optimization of topical administration outside the lesion area in a common animal model
  • Example 9 Although the drug effect by extratumoral administration was not obvious in Example 9, the study of extratumoral administration has not been abandoned. Taking BALB/c mice as experimental subjects, using breast cancer 4T1 cells as model cells, 0.25 ⁇ 10 5 cells/mice were injected subcutaneously into the right axilla to model transplanted tumors.
  • the experimental animals with obvious tumors were randomly divided into 12 groups, with 10 animals in each group, and were administered according to the drugs and doses in Table 17, wherein the drugs were configured according to the method of Example 1; Groups 10 and 11 were administered with different drugs twice a day (the probiotic component/combination composition was administered intratumorally, and the single drug of the probiotic component was intradermally administered to the left armpit of the animal on the same day), The other groups were intradermally administered twice in the armpit of the animals, with an interval of 7 days, and each time was the same drug.
  • the tumor volume (V 7d and V 21d ) were measured respectively from the 7th and 21st days after the last administration of groups 1-11, and the relative tumor proliferation rate on the 7th and 21st days after administration was calculated from the negative control group ( R 7d , R 21d ), the short-term efficacy (100% - R 7d ) and the mid- and long-term efficacy (100% - R 21d ) can be calculated respectively, and the actual/expected ratio of the short-term efficacy can be calculated according to the short-term efficacy and the mid- and long-term efficacy. Actual/expected ratio q 7d and mid- to long-term efficacy q 21d .
  • the intratumoral administration of probiotic components/combination composition was the same day as the extratumoral administration of probiotic components as single drugs.
  • Their short-term efficacy was mainly intratumoral local synergy, while the mid- and long-term efficacy was Co-action of this intratumoral local synergy and secondary effects of the extratumoral local action shown by groups 5 and 6.
  • compositions of the present application under the armpit subcutaneous.
  • mice were randomly divided into two groups, A and B, with 6 mice in each group.
  • Amino acid was administered to groups A and B respectively, and each group was administered once, intradermally to the axilla of the right front leg, with an injection volume of 100 ul/piece.
  • the animals were euthanized, and the intradermal nodules in the right foreleg of the mice were dissected out. After rinsing the slices, the areas of abnormal areas different from normal muscles, such as necrosis and nodules, were measured.
  • the results show that the abnormal areas of A and B are 30.15 ⁇ 11.31 mm 2 and 45.74 ⁇ 13.71 mm 2 , respectively, which are consistent with the results of Example 9.
  • the probiotic component in the technical solution of the present application is similar to high-concentration ethanol, and the first drug effect shown after local administration is mainly local action (or local synergistic effect), which is similar to the intratumoral effect in Examples 2, 5 and 9.
  • the local effects (or local synergy) shown after administration were consistent, mainly similar to the local chemical effects (or local chemical synergies) of chemical ablation.
  • the short-term effect of the composition of the present application after local administration outside the tumor is mainly the destruction of the tissue in the subcutaneous drug penetration zone, and the medium and long-term effect is mainly the secondary effect produced by the destruction.
  • the mid- and long-term spanning effects observed for groups 5-7 in the table above suggest that the secondary effects mainly include vaccine effects. Therefore, the composition of the present application can provide an extra-tumoral vaccine effect that is completely different from classical vaccines, wherein the substance acting as an antigen includes non-specific antigens produced by tissue destruction after administration.
  • probiotic components also have non-specific antigenic effects, which are used to enhance the efficacy of vaccine effects.
  • the composition comprising the probiotic component of the present invention is completely different from the usual systemic action pharmacology (such as the above cytotoxic pharmacology, immune enhancement pharmacology) or Local action pharmacology (such as above chemical ablation) composition (usual composition): 1), target pharmacology: the composition of the present invention can provide a sufficiently strong local action secondary effect or secondary synergistic effect (including but not limited to bad tissue Antigen effect and other secondary immune responses that have yet to be studied), which greatly exceeds the pharmacological expectations of the usual composition (for example, the effect of bad tissue antigens caused by chemical ablation that has little effect); 2), pharmacological composition: the composition of the present invention
  • the conditions that must be met to produce a sufficiently strong local effect outside the local lesion are the same as the pharmacological composition of the local action of the probiotic components in the above example in the local lesion, which greatly exceeds the expected pharmac
  • composition of the present invention can produce an obvious mid- and long-term span inhibiting local lesion effects similar to therapeutic vaccines , which greatly exceeds the expected technical effect of the existing conventional compositions.
  • the dosage is more than 0.01ml/kg person, 0.015-0.25ml/kg person, preferably 0.020-0.25ml/kg person, which requires a drug containing probiotic components for extratumoral local injection in this application
  • the administration volume of the conventional vaccine is 2 times or 3-100 times, preferably 5-100 times that of the conventional vaccine.
  • Example 12 Further research on secondary (synergistic) effects and program optimization
  • BALB/c mice were used as experimental subjects, in which model animals were used as series A, and non-model animals were used as series B.
  • the model animals used breast cancer 4T1 cells as the model cells, and 0.25 ⁇ 10 5 cells/cell were injected subcutaneously into the right axilla to model the transplanted tumor.
  • the average tumor volume of the successfully modeled nude mice was 173.6 mm 3
  • the model animals were randomly divided into 18 groups, 10 animals in each group, and administered by intratumoral injection according to the drugs in Table 18; BALB/c mice were also taken as the experimental objects, and the model animals were directly randomly divided into 18 groups without modeling. , 10 animals in each group, and administered by intramuscular injection of the right hind leg and thigh according to the drugs in Table 18.
  • the research drugs containing probiotic components are all water-containing agents prepared according to the preparation method of Example 1, and the other drugs are aqueous solutions prepared according to the known methods; each group was administered twice, and each time interval On the 3rd, dosage: 50ul/only/time.
  • each group of series A the tumor volume of each group was measured on the 10th and 30th day after the first administration, and the tumor inhibition rate (r' 10d , r' 30d ) was calculated according to the negative control.
  • each group of series A and series B were subcutaneously treated with 1 ⁇ 10 5 breast cancer 4T1 cells per animal and 0.2 ⁇ 10 5 fibroblast 3T3 cells / Only the cells were transplanted, and the 4T1 cell transplanted tumor rate (S 4T1 ) and the 3T3 cell transplanted tumor rate (S 3T3 ) were observed on the 10th day after the cell transplantation.
  • the tumor incidence rate S (the number of tumor-emergent animals/the number of cells transplanted animals) ⁇ 100%.
  • the tumor inhibition rates r' 10d and r' 30d in series A respectively indicate that the drug is administered in the lesion area to produce short-term efficacy and mid-term and long-term efficacy for local lesions; as mentioned above, the probiotic components
  • the short-term efficacy of the drug and its composition is directly related to the local action, while the mid- and long-term efficacy, especially the mid- and long-term efficacy that is significantly higher than the expected mid- and long-term efficacy of the chemotherapeutic drugs, is associated with the secondary action or secondary synergistic effect of the local action, especially related to secondary immunity.
  • the q 30d of the composition formed by the probiotic components/combination in groups 21-26 are all greater than 1.0, even greater than 1.15, and the long-term efficacy is greater than the expected sum of the medium and long-term efficacy of the composition. There is a secondary synergy of local action.
  • the short-term efficacy (r' 10d ) of the group 22-26 was inferior to that of the group 21, but their mid- and long-term efficacy (r' 30d ) was higher.
  • S 3T3 and S 4T1 in Table 18 represent transplant rejection against transplanted cells 3T3 and 4T1, respectively.
  • both S3T3 and S4T1 in series A or B were 100%, indicating that they did not produce immunotherapeutic effects.
  • the probiotic components used in groups 1-4 did not produce sufficient immunity to completely suppress the non-pathogen (100% for S 3T3 ), but were sufficient to clear or at least strongly suppress the pathogen (S 4T1 was much less than 100%) %), with obvious specificity. It shows that the short-term (local) effects of related drugs are limited to local lesions, while the long-term (secondary) effects are not limited to local lesions, but include therapeutic immune effects.
  • the actual/expected ratio q 4T1 S' A/B4T1 /[S' A4T1 +S' B4T1 -(S' A4T1 ⁇ S' B4T1 )] of the immune pooling effect in the combined efficacy of the composition can be calculated.
  • the actual/expected ratio q 4T1 of the compositions in groups 21-26 were all greater than 1.00, showing immune synergy, especially specific immune synergy.
  • the probiotic component and the composition containing it first produce local effects at the lesions, indicating that the immune effect or immune synergy in the medium and long-term efficacy, especially the specific immune effect or the specific immune synergy, should be localized.
  • the topical pharmaceutical composition comprising the probiotic component (referred to as the composition of the present invention) according to the present invention surpasses any non-specific method in the prior art in the following aspects.
  • Expectations of pharmaceutical compositions for heterosexual immunotherapy (referred to as prior art compositions, such as the above-mentioned cytotoxic drugs, immune enhancers, chemical ablative agents, vaccine adjuvants, topical drugs for conditions, etc.):
  • the pharmacology of the composition of the present invention is still the secondary effect (or secondary synergy) of the medium and long-term pharmacology including the special local effect (or local synergy) that prior art compositions do not have, It is pathogen-specific immunity;
  • the local effect provided by the probiotic component may not be maximized, or even preferably minimized, such as the probiotic group.
  • the components are preferably selected from components (water-soluble components and semi-fluidic components) with weak bacterial immunogenicity and local effects, and the pharmacological concentration of probiotic components is preferably selected from lower rather than higher administration Concentration (limited to 3-15% or 3-5% for water-soluble polysaccharide, 10-25% for nucleic acid, 4-12% for semi-fluid components), with specific combinations Multiple (preferably 2 or more) shared drugs in a relationship, etc.;
  • Each group was given intratumoral injections twice, with an interval of 2 days, and the injection volume was 150 ⁇ l/only/time.
  • the animals were euthanized on the 10th day after the first administration, and the tumor tissue was removed after dissection to measure the tumor weight, and the tumor inhibition rate (r) was calculated according to the negative control group.
  • compositions containing probiotic components in the prior art are usually oral dosage forms or conventional injection dosage forms, the former usually needs to contain solid excipients, while the latter usually needs to contain osmotic pressure enhancers (salts or monosaccharides) of the probiotic components To ensure that its liquid and blood osmotic pressure is the same or similar.
  • osmotic pressure enhancers salts or monosaccharides
  • the pharmaceutical composition described in the present application only needs to meet the requirements of providing the local effect, the preferred solution of the pharmaceutical composition may not contain solid excipients or osmotic pressure enhancers, so as to avoid reducing the local effect.
  • the above-mentioned administration forms are also preferred dosage forms for locally acting drugs.
  • mice 8-month-old mice as the experimental object in the model of middle-aged and elderly patients, 0.5 ⁇ 10 5 mouse breast cancer 4T1 cells/mice were injected subcutaneously into the right axilla to model the transplanted tumor, and the tumor in nude mice was successfully modeled.
  • the average body volume was 117.3 mm 3 , and the model animals were randomly divided into 18 groups. Each group was administered once by intratumoral injection according to the drugs listed in Table 19, and the injection volume was 50 ⁇ l per animal; the local lesion volume (V) was measured on the 3rd and 21st days after administration, and the negative control group was calculated according to the time.
  • Tumor inhibition rate r' 3d , r' 21d
  • the medicines used were all prepared according to the conventional aqueous solution preparation method or the preparation method in Example 1.
  • the actual/expected ratio q 3d of short-term efficacy and the actual/expected ratio q 21d of mid- and long-term efficacy were calculated according to the efficacy (r' 3d , r' 21d ) at different times (3rd, 21st).
  • the short-term efficacy (r' 3d ) of the probiotic components (including analogs) combined with the same topical drug was significantly higher, and the difference in the mid- and long-term efficacy (r' 21d ) was even greater significantly expanded.
  • the q 21d of the probiotic components (including analogs) shared with the same topical drug were all greater than 1.00, well beyond the mid- to long-term synergy expected based on the sugar control/topical drug effect.
  • probiotic components/topical agents can produce short-term and mid- to long-term synergies beyond those expected from analogs/topical agents from other sources.
  • probiotic components themselves do not provide mid- and long-term efficacy under the conditions of this example, the shared pharmacology of the above results exceeds the pharmacological expectation of synergy in the prior art; bacterial antigenicity is minimized, local effects Probiotic components (including analogs) that minimize (r' 3d ⁇ 15%) and mid- and long-term efficacy (r' 21d ⁇ 15%) can provide a previously undiscovered but very Useful pharmacological function - that is, to provide mid- to long-term pharmacodynamic synergy for concomitants that do not necessarily exhibit local synergy, or only exhibit weaker local synergy, under conditions that do not necessarily exhibit strong local effects on their own, preferably minimize local effects activity.
  • the indication scope of the mid- and long-term synergistic pharmaceutical composition comprising probiotic components (including analogs) has the following requirements: for middle-aged and elderly patients, its pharmaceutical composition has the following requirements: a probiotic group with minimal local effects components (such as water-soluble components and semi-fluid components), pharmacological concentrations that minimize local effects (such as the administration concentration of water-soluble polysaccharides is limited to 3-15%, the administration concentration of nucleic acids is limited to 10-25%, semi-fluid components Fluid-like components are limited to 4-12%, etc.).
  • a probiotic group with minimal local effects components such as water-soluble components and semi-fluid components
  • pharmacological concentrations that minimize local effects such as the administration concentration of water-soluble polysaccharides is limited to 3-15%, the administration concentration of nucleic acids is limited to 10-25%, semi-fluid components Fluid-like components are limited to 4-12%, etc.
  • the topical pharmaceutical composition comprising the probiotic component of the present invention (referred to as the composition of the present invention) in addition to exceeding the expectations of any non-specific immunotherapy pharmaceutical composition in the above example, according to the above results of this example ( It is the mid- to long-term results) and the results of other similar experiments, which also surpass the topical pharmaceutical compositions (referred to as the present invention) comprising topical drugs of the prior art (e.g., ethanol, conventional glyconutrients, etc.) in the following aspects.
  • topical pharmaceutical compositions e.g., ethanol, conventional glyconutrients, etc.
  • the local synergistic effect of conventional glyconutrients is greater or even maximized
  • the preferred co-pharmacology of the composition of the present invention beyond the existing synergy definition is when the local effect of the components is not significantly beneficial
  • the local effect of probiotic components is small or even minimized, and the mid- and long-term synergy produced under the conditions that probiotic components and their co-uses do not produce short-term synergy, or even short-term antagonism
  • the mid- and long-term synergy is very May include immune effects beyond the local effects of each component;
  • the pharmacological composition of any non-specific immunotherapy pharmaceutical composition is expected.
  • Local effects are generally considered to be positively correlated with local irritation.
  • preferred probiotic semi-fluid or probiotic water-soluble components have significantly lower local irritation (topical administration) than probiotic particulate components (inactivated probiotics, probiotic water-insoluble granules) the proportion of animals struggling violently), the preferred co-use is not ethanol, which is more locally irritating, but a weaker topical compound that is less locally irritating, etc.;
  • composition of the present invention can produce obvious, even effective medium and long-term medicinal effect and systemic medicinal effect (span medicinal effect), greatly exceeding the prior art composition can only produce short-term local medicinal effect. technical effect is expected.
  • the mid- and long-term efficacy and systemic efficacy are particularly suitable for middle-aged and elderly patients.
  • preferred compositions of the present invention exhibit near-no local irritation, whereas the local effects of classical chemical ablative agents (eg, ethanol) are positively correlated with local irritation.
  • the combination in which the probiotic component can provide local synergy or/and mid- to long-term synergy can also be selected from other pharmaceutically suitable drugs (chemically active ingredients or/and biologically active ingredients) or drug combinations.
  • drugs chemically active ingredients or/and biologically active ingredients
  • alkali metal hydroxide selected from the combination comprising alkali metal hydroxide, alkaline inorganic salt, and one or more selected from the following group: methylene blue and its analogs, immunomodulators, cytotoxic drugs, such as the following combinations : Alkali metal whose concentration ratio (W alkali metal hydroxide /W alkaline inorganic salt /W methylene blue and the like ) is (1-5%)/(3-15%)/(0.35-5%) Combination of hydroxide/alkaline inorganic salt/methylene blue and its analogs, the concentration ratio (W alkali metal hydroxide /W basic inorganic salt /W immunomodulator ) is (1-5%)/(3- 15%)/(1-15%) of alkali metal hydroxide/alkaline inorganic salt/immunomodulator combination, the concentration ratio (W alkali metal hydroxide /W basic inorganic salt / cytotoxic drug ) is (1 -5%)/(3-15%)/(0.1-10%) of the
  • the concentration ratio (W basic organic compounds /W basic inorganic salts / methylene blue and the like ) is (5-25%)/(3-15%)/(0.35-5%) basic organic compounds/ Alkaline inorganic salt/methylene blue and its analogs
  • the concentration ratio (W basic organic compound /W basic inorganic salt /W immunomodulator ) is (5-25%)/(3-15%)/( 1-15%) basic organic compound/basic inorganic salt/immunomodulator combination
  • the concentration ratio (W basic organic compound /W basic inorganic salt /W cytotoxic drug ) is (5-25%)/( 3-15%)/(0.1-10%) of basic organic compound/basic inorganic salt/cytotoxic drug combination.
  • cytotoxic drugs be selected from the combination comprising cytotoxic drugs and at least two kinds selected from the following group: basic organic compounds, immunomodulators, polyols, methylene blue and the like, such as the following combinations: concentration ratio (W cells) cytotoxic drug/basic organic compound /w immunomodulator ) is (0.1-10%)/(5-25%)/(1-15%) cytotoxic drug/basic organic compound/immunomodulator combination, Cytotoxic drug / polyol / The combination and concentration ratio of methylene blue and its analogs (W cytotoxic drugs /W methylene blue and its analogs /W immunomodulators ) is (0.1-10%)/(0.35-5%)/(1-15 %) of the cytotoxic drug/methylene blue and its analogs/immunomodulator combination.

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Abstract

本发明提供了益生菌组分的一种新应用以及包含益生菌组分的局部药物组合物，所述益生菌组分作为可提供治疗作用的活性成分在制备用于治疗局部病变疾病的局部药物组合物中的应用。本发明所述的局部药物提供了全新的药理，从而实现现有益生菌组合物未能进行的包含局部作用(或局部协同作用)的类化学消融局部治疗或/和包含与该局部作用(或局部协同作用)相关的次生免疫作用在内的免疫治疗；同时几乎无毒的全身安全性和明显较高的长期疗效，对快速生长瘤体、大瘤体和乏血供瘤体的巨大潜力，且不存在耐药性问题、制备方便、成本低廉。

Description

益生菌组分的应用以及包含益生菌组分的药物组合物 技术领域
本发明涉及药物制剂技术领域，特别涉及益生菌组分的一种新应用以及包含益生菌组分的药物组合物。
背景技术
从病变组织的角度看，实体肿瘤是局部病变疾病的代表性模型；它是一种具有瘤体症状的肿瘤疾病，瘤体包含瘤体组织，该组织包含肿瘤细胞还存在更大量的其它组成(有时亦被称作肿瘤细胞的微环境)，例如其它多种细胞、多种细胞间质、多种管道等。以胰腺癌瘤体为例，胰腺癌细胞在瘤体中仅占约30％的体积。
细胞毒药物、化学消融剂是近百年来治疗实体肿瘤的常见药物。其中细胞毒药物通过瘤内给药虽然可提高其瘤内浓度，但其药效仅仅是在癌细胞抑制动力学预期范围(通常小于200％)内略有提高；考虑到患者的顺应性，目前细胞毒药物基本都通过口服或静注的方式进行全身给药。而化学消融剂，通常不以肿瘤细胞破坏、而以瘤体组织破坏为药理特征，其瘤内给药的药效远大于其癌细胞抑制动力学预期范围，具有明显不同于全身给药的药理，如高纯度乙醇、高浓度酸化剂或碱化剂等；但存在刺激性大，长效性不高且介入体积和介入部位均非常受限，例如酸碱用量不超过0.2ml/kg以及对瘤体所在器官的限制、瘤体边缘消融受限等，因此已逐渐从临床上淡出。
近年来，以肿瘤疫苗、细胞过继疗法和免疫检查点抑制剂为代表的肿瘤免疫治疗方法有一些进展。这些治疗药物千差万别，但对患者基因特异性和肿瘤特异性均要求非常高，在普通动物实验中几乎观察不到给药组与阴性对照组瘤体残重之间的明显差异，临床上的适应症范围非常小。
世界卫生组织对益生菌的定义为：一种摄入充足数量时会赋予宿主某种健康益处的活的非致病性细菌。研究表明，益生菌可能有三种作用机制来支持其有益性：(1)与肠道上皮细胞结合竞争病原体附着位点或消耗其营养物质从而抑制致病菌的生长；(2)改善肠道屏障功能；(3)提高机体免疫功能。其中，益生菌提高机体免疫功能的研究犹其令人关注。有研究将酵母作为饲料添加剂用于增强猪的免疫功能(例如专利公开号：CN106387398A)。还有研究将灭活酵母菌用于增强免疫功能、抗菌和抗病毒(例如专利公开号：CN108524925A)。
现有技术中益生菌的抗肿瘤作用被认为主要与其细菌免疫原性有关。但该免疫原性并未显示出治疗性疫苗抗原所应有的治疗效应，因而目前主要被用于增强患者机体的免疫力。免疫异质性是肿瘤异质性的特征之一，该药理使益生菌的免疫增强仅对结直肠癌存在作用，显示出强烈的肿瘤特异性。因此，相对于其他药物，益生菌在动物实验中没有显示出有意义的瘤体生长抑制(抑瘤率≤15％或相对肿瘤增值率≥85％)，所提供的这些抗肿瘤免疫增强作用十分有限，仅仅起到辅助治疗作用，远远达不到提供治疗作用。
开发益生菌的新药理来突破现有技术中仅提供免疫功能增强等预期，能够为临床治疗提供新的解决方案。
发明内容
有鉴于此，本发明旨在提出一种基于益生菌组分的新药理-即提供局部作用(或局部协同作用)及 其次生作用(或次生协同作用)，从而制备的局部药物组合物，实现高效低毒，尤其是中长期内，以为临床提供供各种选择。
益生菌组分作为可提供治疗作用的活性成分在制备用于治疗局部病变疾病的局部药物组合物中的应用，其中所述治疗作用包括涉及局部作用(或局部协同作用)的局部治疗或/和与该局部作用(或局部协同作用)相关的免疫治疗。
优选的，其中所述药物组合物还包含能够与所述益生菌组分产生协同作用的化学活性成分，以及其中所述益生菌组分与所述化学活性成分的量比(益生菌组分重量浓度/共用物重量浓度)为(1-110)/(1-100)。
一种用于治疗局部病变疾病的局部药物组合物，其包含可提供治疗作用的益生菌组分、以及药物学可接受的合适载体，但不包含专用于将血液渗透压增加至正常生理水平的盐或单糖，其中所述治疗作用包括涉及局部作用(或局部协同作用)的局部治疗或/和免疫治疗。
优选的，其还任选地包含能够与所述益生菌组分产生协同作用的化学活性成分。
一种用于治疗局部病变疾病的局部药物组合物，其包含可提供治疗作用的益生菌组分、能够与所述益生菌组分产生协同作用的化学活性成分、和药物学可接受的合适载体，且所述益生菌组分与所述化学活性成分的量比(益生菌组分重量浓度/共用物重量浓度)为(1-110)/(1-100)，其中所述治疗作用包括涉及局部作用(或局部协同作用)的局部治疗或/和免疫治疗。
优选的，其中所述益生菌组分为选自细菌免疫原性最小化的源于天然益生菌或其工程菌的制备物或该制备物的工程类似物，其中所述制备物优选为选自包括以下组及其衍生物之一种或多种：益生菌水溶性组分、益生菌半流体类组分、益生菌组分水不溶颗粒、灭活益生菌。
优选的，其中所述益生菌水溶性组分选自包括以下组及其衍生物之一种或多种：益生菌或破碎益生菌上清组分、益生菌抽提物、益生菌细胞内水溶性组分、益生菌细胞壁组分的水溶性成分或非水溶成分的水溶性衍生物；所述益生菌半流体类组分优选为选自其含水混合物可形成半流体类组合物的益生菌组分之一种或多种，例如益生菌聚多糖及其类似物之一种或多种：所述益生菌组分水不溶颗粒选自包括以下组及其衍生物之一种或多种：破碎益生菌沉淀组分、益生菌细胞壁聚多糖颗粒、益生菌细胞壁聚多糖纳米颗粒；所述灭活益生菌优选为选自并不限定其皆为完整菌体的灭活益生菌；
其中所述益生菌水溶性组分选自包括以下组及其衍生物之一种或多种：益生菌水溶性β-葡聚糖、优选为选自纯度大于90％的水溶性β-葡聚糖，益生菌核糖核酸。
优选的，其中所述益生菌选自包括以下组之天然菌或/和工程菌的一种或多种：益生芽抱杆菌、益生乳酸杆菌、益生双歧杆菌、益生真菌。
优选的，其中所述芽抱杆菌包括选自包括以下组之一种或多种：地衣芽抱杆菌、枯草芽抱杆菌、短小芽抱杆菌、纳豆芽抱杆菌；所述乳酸杆菌包括选自包括以下组之一种或多种：嗜酸乳杆菌、干酪乳杆菌、植物乳杆菌、短小乳杆菌和发酵乳杆菌；所述双歧杆菌包括选自包括以下组之一种或多种：长双歧杆菌、青春双歧杆菌、短双歧杆菌、屎肠球菌、粪链球菌；所述真菌包括选自包括以下组包括选自之一种或多种：酵母菌、布魯氐酵母菌，且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、威克汉姆酵母菌、毕赤酵母菌、产阮假丝酵母、乳清酵母。
优选的，其中所述益生菌包括选自酿酒酵母菌或/和布魯氐酵母菌。
优选的，其中所述合适载体为水；所述化学活性成分可溶于水中，而所述益生菌组分为水不溶性的且为选自包括以下组中的一种或多种或其衍生物：灭活益生菌、益生菌组分水不溶颗粒、益生菌半流体类组分。
优选的，其中所述益生菌组分在该药物组合物中的浓度为＞0.1％、≥0.25％、0.25-25％、优选为0.5-15％、更优选为1-15％或5-15％。
优选的，其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浓度为＞0.1、或0.15-25％、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颗粒，则所述益生菌水不溶组分颗粒在该药物组合物中的浓度为＞0.5、或0.5-15％、优选为1.5-15％或5-15％；当该药物组合物为半流体类组合物，则所述益生菌半流体类组分在该药物组合物中的浓度为＞2.5％、2.6-25％、优选为5-15％。
优选的，其中所述化学活性成分为包括选自弱局部作用化合物和/或细胞毒药物之一种或多种，以及其中当所述化学活性化合物包括细胞毒药物时，所述益生菌组分和细胞毒药物的量比(W 益生菌组分/W 细 胞毒药物)为(1-110)/(1-100)；当所述化学活性化合物包括弱局部作用化合物时，所述益生菌组分和弱局部作用化合物的量比(V 益生菌组分/W 弱局部作用化合物)为(1-90)/(1-100)。
优选的，其中所述弱局部作用化合物选自包括以下组之一种或多种：氨基酸类营养素、活体染料、奎宁类药物、低浓度酸化剂、低浓度碱化剂、包含酸化剂或/碱化剂的pH缓冲系统，以及其中当所述弱局部作用化合物包括所述氨基酸营养素时，所述益生菌组分和氨基酸营养素的量比(W 益生菌组分/W 氨基酸营养 素)为(1-20)/(1-100)；当所述弱局部作用化合物包括所述活体染料时，所述益生菌组分和活体染料的量比(W 益生菌组分/W 活体染料)为(7-90)/(1-100)；当所述弱局部作用化合物包括所述奎宁类药物时，所述益生菌组分和奎宁类药物的量比(W 益生菌组分/W 奎宁类药物)为(2-90)/(1-100)；当所述弱局部作用化合物包括所述酸化剂或/和碱化剂时，所述益生菌组分和酸化剂或/和碱化剂的量比(W 益生菌组分/W 酸化剂或/ 和碱化剂)为(2-60)/(1-100)。
优选的，其中所述化学活性成分为选自活体染料和1种或多种其它所述化学活性成分。优选的，其中所述化学活性成分为选自所述细胞毒药物和活体染料。
优选的，其中所述氨基酸类营养素为选自包括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，以及所述氨基酸类营养素的浓度为＞2.5％、或5-30％、优选为5-25％。
优选的，其中所述活体染料为选自包括孟加拉红或/和以下亚甲蓝类染料之一种或多种：亚甲蓝、专利蓝、异硫蓝、新亚甲蓝，且其中所述孟加拉红的浓度为2.5％-20％；所述亚甲蓝类染料的浓度为≥0.25％、或0.25-2.5％、优选为0.5-2.5％。
优选的，其中所述酸化剂选自强酸或/和弱酸之一种或多种，且所述益生菌组分与所述酸化剂的量比(益生菌组分重量浓度/酸化剂重量浓度)为(1-20)/(0.5-50)，以及所述酸化剂的浓度为≥0.5％、0.5-2％(强酸)、或2-15％(弱酸)，其中所述强酸例如为盐酸；所述弱酸例如为乙二酸、乙酸、乳酸、柠 檬酸、苹果酸；所述碱化剂例如选自强碱或/和弱碱之一种或多种，且所述益生菌组分与所述碱化剂的量比(益生菌组分重量浓度/碱化剂重量浓度)为(1-20)/(0.5-50)，以及所述碱化剂的浓度为≥0.5％、0.5-5％(强碱)、或2-15％(弱碱)，其中所述强碱例如为氢氧化钠、氢氧化钾；所述弱碱例如为磷酸二氢钠、碳酸氢钠、碳酸钠。
优选的，其中所述细胞毒药物包括选自包括以下组之一种或多种：破坏DNA结构和功能的药物，例如环磷酰胺、卡莫司汀、金属铂络合物、多柔比星类药物、拓扑替康、伊立替康；嵌入DNA中干扰转录RNA的药物，例如抗肿瘤抗生素药物；干扰DNA合成的药物，例如5-氟尿嘧啶(5-Fu)、呋氟尿嘧啶、双呋氟尿嘧啶、阿糖胞苷、环胞苷、5-氮杂胞苷；影响蛋白质合成的药物，例如秋水仙碱类药物、长春碱类药物、紫杉烷类药物，且所述细胞毒药物的浓度为≥0.1％、0.1-15％。
优选的，其中所述药物组合物还任选地包含生物活性成分，其中所述生物活性成分选自包括以下组之一种或多种：抗原、免疫调节类抗体、细胞因子、佐剂。
一种治疗局部病变疾病的方法，其包括以下步骤：向有此需要的个体的局部病变内或/和局部病变外局部施用治疗有效量的上述药物组合物。
优选得，其包括以下步骤：向所述个体的局部病变内、或局部病变内和局部病变外施用治疗有效量的所述药物组合物。
优选的，其还包括在施用所述药物组合物之前、期间或之后还任选进行一种或多种其它治疗，例如化疗、免疫疗法、放射疗法、手术、化学消融、物理消融。
优选的，其中所述益生菌组分在该药物组合物中的含量使得它的局部给药浓度为＞0.1％、≥0.25％、0.25-25％、优选为0.5-15％、更优选为1-15％或5-15％。
优选的，其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含量必须使得它的局部给药浓度为＞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含量必须使得它的局部给药浓度为＞0.1、或0.15-25％、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在该药物组合物中的含量必须使得它的局部给药浓度为＞0.5、或0.5-15％、优选为1.5-15％或3.5-15％。
优选的，其中所述药物组合物的含量或单位含量使得其给药体积为以下系数乘以局部病变内靶区体积：＞0.1、0.15-1.5、优选为0.23-1.5或0.5-1.5。
优选的，其中所述药物组合物的含量或单位含量使得其给药体积为≥1ml、或局部病变内给药10-150ml或/和局部病变外给药1.5-50ml。
优选的，其中所述局部病变包含肿瘤细胞或/和成纤维细胞。
优选的，其中所述局部病变疾病包括肿瘤、非瘤肿大、局部炎症、分泌腺功能异常和皮肤病，其中所述肿瘤包括恶性和非恶性实体肿瘤。
优选的，其中所述实体肿瘤包括以下肿瘤及其次生肿瘤之一种或多种：乳腺癌、胰腺癌、甲状腺癌、鼻咽癌、前列腺癌、肝癌、肺癌、肠癌、口腔癌、食道癌、胃癌、喉癌、睾丸癌、阴道癌、子宫癌、卵巢癌、脑瘤、淋巴瘤。
优选的，其中所述局部作用(或局部协同作用)包括局部化学作用(或局部化学协同作用)和任选存在的其它作用；所述局部治疗包括一处或多处局部病变的类化学消融和任选存在的其它化疗；所述免疫治疗包括所述病变内或/和病变外所述局部作用(或局部协同作用)的次生免疫作用和任选存在的其它免疫作用。
优选的，其中所述治疗的适用患者选自包括以下组之一种或多种：免疫抑制患者、可局部病变内给药的患者、局部病变组织可类化学消融的患者、局部病变内可产生次生免疫物质的患者、病变外给药区域内可产生次生免疫物质的患者。
包含益生菌组分以及药物学可接受的合适载体的药物组合物，其是用于治疗局部病变疾病，其中所述局部病变疾病包括实体肿瘤。
药物试剂盒，其包括一个或多个装有上述的药物组合物的容器。优选的。所述药物试剂盒还可包括如何向有需要的个体施用所述药物组合物的说明书或标签。优选的其中所述施用包括在所述局部病变内施用，或者在局部病变内和局部病变外施用，其中所述局部病变外施用例如包括在所述个体的腋下皮下注射。
为达到上述目的，本发明的技术方案是这样实现的：一种用于治疗局部病变的局部用药，包含益生菌组分及药学上可接受的载体，所述益生菌组分的浓度为0.25-25％。
优选的，所述局部用药还包括弱局部作用化合物和/或细胞毒药物，其中所述弱局部作用化合物的浓度为1-20％，所述细胞毒药物的浓度为1-5％。
优选的，所述局部用药的组成包括0.25％-10％益生菌组分，还包括0.1-2.5％细胞毒药物和/或5-20％氨基酸类营养素和/或0.5-1％活性染料。
优选的，所述局部用药的组成包括0.5％-10％益生菌组分，还包括0.5-1％亚甲蓝或者5-20％赖氨酸或者0.1-2.5％吉西他滨或5-氟尿嘧啶；所述局部用药的组成包括0.25-0.5％益生菌组分和5-10％精氨酸。
所述局部用药的组成为2.5％益生菌组分、1％5-氟尿嘧啶，余量为注射用水；或者组成为2.5％益生菌组分2.5％、10％精氨酸，余量为注射用水；或者为2.5％益生菌组分、1％5-氟尿嘧啶、10％精氨酸，余量为注射用水；或者组成为5％热灭活布拉氏酵母菌、1％亚甲蓝，余量为注射用水；或者组成为5％热灭活布拉氏酵母菌、1％5-氟尿嘧啶，余量为注射用水；或者组成为5％热灭活布拉氏酵母菌、1％5-氟尿嘧啶、1％亚甲蓝，余量为注射用水；或者组成为半流体组合物中的7.5％β-葡聚糖、1％亚甲蓝、1％5-氟尿嘧啶，余量为注射用水；或者组成为5％破碎布拉氏酵母菌上清组分、1％亚甲蓝、1％5-氟尿嘧啶，余量为注射用水；或者组成为5％破碎布拉氏酵母菌、1％亚甲蓝、1％5-氟尿嘧啶，余量为注射用水；或者组成为5％热灭活布拉氐酵母菌、1％亚甲蓝、1％5-氟尿嘧啶，余量为注射用水；或者组成为5％β-葡聚糖、1％亚甲蓝、1％5-氟尿嘧啶的水悬液经加热后降温处理形成的半流体；或者组成为5％水溶性酿酒酵母菌β-葡聚糖、1％亚甲蓝、1％5-氟尿嘧啶，余量为注射用水；或者组成为20％酿酒酵母菌核糖核酸、1％亚甲蓝、1％5-氟尿嘧啶，余量为注射用水。优选的，所述局部用药的组成包括10％益生菌组分、1％亚甲蓝、1％5-氟尿嘧啶。优选的，所述局部用药的组成包括0.5％益生菌组分/7％碳酸氢钠/3％氢氧化钠或者10％益生菌组分/0.7％碳酸氢钠/0.3％氢氧化钠。优选的，所述益生菌组分包括灭活益生菌、益生菌水溶性组分、益生菌水不溶组分颗粒、益生菌半流体类组分中的至少一种。优选的，所述益生菌包括芽抱杆菌、 乳酸杆菌、双歧杆菌、益生真菌中的至少一种。优选的，所述益生菌为布拉氐酵母菌。优选的，所述局部用药不含有渗透压调节剂、矫味剂。
相对于现有技术，本发明所述的局部用药具有以下优势：提供了一种全新的药理(局部作用或局部协同作用以及它们的次生免疫作用)，从而可以进行现有益生菌组合物未能进行的包含类化学消融在内的局部治疗或/和包含次生免疫作用在内的免疫治疗，该局部治疗可以产生远远超过现有技术方案的疗效(例如3倍以上的抑瘤率)以及适应症(例于突破了现有技术中的肿瘤特异性限制，以及对患者免疫功能的依赖性限制)，该免疫治疗也可以产生远远超过现有技术方案的免疫疗效(例如病变区内或/和病变区外所述次生免疫作用产生或大大加强的免疫疗效大大超过了机体免疫增强的预期)以及适应症。此外，本发明的实施方案还具有大大降低了不限定给药方式的现有技术方案的安全性风险。
根据本发明的实施方案与治疗局部病变疾病的其它组合物的现有技术相比具有以下优点：与现有细胞毒药物相比，显示出几乎无毒的全身安全性和明显较高的长期疗效；与现有分子靶向药物相比，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潜力；与现有化学消融剂相比，显示出明显低得多的局部刺激性以及更好的长期效应。本发明的应用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，该应用和组合物制备方便、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。
本发明还提供了上述述的局部用药在制备用于治疗局部病变疾病的局部药物组合物中的应用，其中所述治疗作用包括涉及局部作用(或局部协同作用)的局部治疗或/和免疫治疗作用。优选的，所述药物组合物的含量或单位含量使给药体积V 1＝(0.15-1.5)×V 2，优选为(0.23-1.5)×V 2或(0.5-1.5)×V 2，其中V 2为局部病变内靶区的体积。优选的，所述给药体积V 1为1.5-150ml，或若局部病变内给药，则给药体积V 1为10-150ml，若局部病变外给药，则给药体积V 1为1.5-50ml。
优选的，所述局部病变包括肿瘤、非瘤肿大、局部炎症、分泌腺功能异常和皮肤病，其中所述肿瘤包括恶性和非恶性实体肿瘤。优选的，所述实体肿瘤为乳腺癌、胰腺癌、甲状腺癌、鼻咽癌、前列腺癌、肝癌、肺癌、肠癌、口腔癌、食道癌、胃癌、喉癌、睾丸癌、阴道癌、子宫癌、卵巢癌、脑瘤、淋巴瘤中的一种。
本发明还提供了一种药物试剂盒，包括一个或多个装有上述药物组合物的容器。优选的，所述药物试剂盒还包括如何向有需要的个体施用所述药物组合物的说明书或标签。优选的，所述施用包括在所述局部病变内施用，或者在局部病变内和局部病变外施用，其中所述局部病变外施用为腋下皮下注射。
本发明还提供了一种治疗局部病变疾病的方法，包括向患者的局部病变内或向局部病变内和局部病变外施用治疗有效量的上述的药物组合物。优选的，所述在施用所述药物组合物前和/或中和/或后还进行化疗、免疫疗法、放射疗法、手术、化学消融、物理消融中的至少一种治疗。优选的，所述药物组合物经施用后，其中的益生菌组分的局部给药浓度为0.25-25％、优选为0.5-15％，更优选为1-15％或5-15％。
优选的，所述患者为选自包括以下组之一种或多种：免疫抑制患者、可局部病变内给药的患者、局部病变组织可类化学消融的患者、局部病变内可产生次生免疫物质的患者、病变外给药区域内可产生次生免疫物质的患者中的任意一种。
具体实施方式
需要说明的是，在不冲突的情况下，本发明中的实施例及实施例中的特征可以相互组合。本申请中L-氨基酸均简写为氨基酸，如L-精氨酸简写为精氨酸。
若物质在特定条件下显示出治疗活性，这说明其作为活性成分可用于局部病变疾病的治疗。同一物质在不同条件下可显示出不同的活性或药理，例如乙醇通常被用作杀菌剂，而高浓度乙醇通过瘤内给药显示出对肿瘤组织的化学消融的药理。
发明人在荷瘤裸鼠实验中意外地发现，通常用作免疫增强剂的灭活益生菌居然可以在某些特定的条件下产生有意义的局部化学作用、甚至可能是类化学消融作用。所述特定条件并非现有技术中益生菌组分的应用条件，而是如以下所限定的。
本申请的一个发明目的是提供益生菌组分在制备用于治疗局部病变疾病的局部药物组合物中的应用，所述益生菌组分可作为具有治疗作用的活性成分。下面对本申请中的相关术语进行说明：
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细菌。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)或该制备物的类似物。术语“类似物”是指与制备物具有类似活性的类似物质,包括制备物的衍生物(例如能够提高水溶性或活性的胞壁聚多糖衍生物)、合成物(例如类似于胞壁聚多糖的合成聚多糖)、类似于益生菌制备物的其它来源制备物(例如类似于胞壁聚多糖的其它来源聚多糖)。
术语“药物”或“药物组合物”是指包含活性组分并给出其在患者体内实现其药理所必须的药理方法、药理组成等药学特征的物质。术语“活性成分”是指可在特定条件下进行某特定药理的物质。术语“药理方法”是指为实现某特定药理所必须的的给药方法，如益生菌肠道屏障改善的药理方法是口服。术语“药理组成”是指实现某特定药理所必须的药物组合物、尤其是进入靶区的药物组合物的组分构成。不同的药理通常需要不同的优选药理组成(活性成分、活性动力学条件、剂型条件、等等)。术语“活性动力学条件”是指使得活性成分实现某特定药理所必须满足的数量条件,同一个物质用作不同活性成分于是有可能需要满足不同动力学条件。术语“剂型条件”是指为保证活性成分实现特定药理所必须满足的药理反应环境条件,同一个物质用作不同活性成分于是有可能需要满足不同剂型条件。以包含益生菌及其组分的药物组合物为例,其在现有技术中用作全身性作用活性成分时以带药血液(含很多物质)进入靶区反应器,因而可以采用开放式组成(如口服剂型的矫味剂、常规注射剂中的渗透压调节剂、等等)的常规剂型；其在本发明中作为局部活性成分则对反应环境高度敏感,某些常用的添加剂(如以下实施例中常用作渗透压调节剂的氯化钠)可能对益生菌组分的局部活性产生负面作用,因而必须采用封闭式的药理剂型组成(优选为仅包含活性成分和溶媒)使得外源性干扰最小化。
术语“治疗药物”区别于“辅助(治疗)药物”，前者是指可提供治疗作用的药物，后者指提供辅助(治疗作用)的药物。术语“治疗作用”区别于辅助(治疗)作用，前者是指使得疾病有效缓解、好转或痊愈的主要药理作用(例如局部病变的局部治疗或/和免疫治疗)，而后者是指虽不能有效缓解、好转或痊愈疾病但却也对患者有利的次要药理作用(例如机体的免疫增强)。治疗作用通常包括单独使用时具有明显疗效、或在与其它药物共用或结合其他治疗方法产生的明显疗效中提供主要或相等作用；而辅助(治疗)通常在单独使用时只提供虽有利但不明显的作用、或在与其它药物共用或结合其他治疗方法所产生的明显疗效中提供虽有利但非主要或同等的作用。术语“明显疗效”是指与经典化疗药物或 经典化学消融剂相比，使局部病变的生长或发展得到有效抑制，例如临床上对局部病变的病理有效率(PR+CR)≧30％，亦或者动物实验中的局部病变生长抑制有不可忽略的药学意义，例如瘤体增值率≦85％或抑瘤率≧15％，优选为瘤体增值率≦60％或抑瘤率≧40％。用作不同活性成分的同一个物质于是可能提供完全不同的作用。以包含益生菌及其组分的药物组合物为例,其在现有技术中用作全身性作用活性成分时主要提供辅助作用,例如免疫增强作用通常不足以观察到抑瘤效果；其在本发明中作为局部活性成分则主要提供治疗作用。
术语“局部药物”或“局部用药”区别于“常规药物”或“常规用药”后者是指基于全身性作用药理的药物,而前者是指主要基于局部作用药理的药物,两者之间有以下区别:前者通常为治疗药物,而后者可以是治疗药物或辅助(治疗)药物,它们对适应症的作用有可能完全不同；前者的药理方法为局部、犹其是局部病变给药,而后者主要是常规给药(全身性给药),它们对给药副作用的关注有可能完全不同；前者的药理活性成分必须能提供局部作用,而后者的药理活性成分必须能提供全身性作用,它们的优选方向可能完全相反；前者的药理动力学组成中的要素是给药浓度,而后者则关注给药剂量,它们的药剂浓度的含义可能完全不同；前者的药理剂型为局部给药剂型,而后者的药理剂型为常规给药剂型,它们的药理剂型组成可能有完全不同的要求。
术语“局部给药”区别于常规给药(全身性给药)，是指以产生局部作用为目的的任何给药方式,包括病变内给药或/和可产生局部治疗作用的病变外有利局部内给药(例如在可产生局部作用且有利于局部作用次生免疫作用的皮下注射)；常规给药是指以产生全身性作用为目的的任何给药方式,包括将药物经消化道(例如口服)或血管(例如静脉注射、腹腔注射)给药后经血液递送至靶区的方式。术语“病变内给药”是指让药物直接进入而非以带药血液的方式间接进入局部病变的任何给药方式，如瘤内给药、局部病变内植入、局部病变涂抹、局部病变喷射、经皮下或经血管局部病变内注射、局部病变插入、等等。
术语“局部治疗”区别于“全身治疗”，后者是指主要利用带药血液的全身性药理(常规活性)针对患者全身病变(例如瘤体、瘤体联通的区域、体内其它部分所含肿瘤细胞)的治疗，而前者是指主要利用药物本身的局部作用药理(局部活性)针对患者局部病变所在的局部区域(例如给药局部病变及与其联通的其它病变区域)的治疗。现有技术中，所用的益生菌组分在治疗局部病变疾病的主要功能基于其作为异种抗原所能提供的治疗辅助作用(例如免疫增强作用)、而非治疗作用、更不是局部治疗作用。
术语“局部活性”区别于常规活性,局部活性可提供局部作用(局部协同作用)或/和与该局部作用(局部协同作用)相关的次生作用(中长效协同作用),而常规活性通常只提供全身性作用(例如细胞毒作用、免疫增强作用),它们是两种完全不同的药理活性。术语“局部作用”区别于常规作用，是指局部给药后药物在组织间隙渗透所及的局部(例如瘤或/和瘤外局部)范围内产生的药理作用、通常包括局部化学作用，而常规作用则是指常规用药后药物经消化道或血管给药后以血药形式递送至靶区所产生的药理作用。术语“局部化学作用”是指包括化学作用的局部作用。术语“局部协同作用”是指包括协同作用的局部作用。术语“局部化学协同作用”是指包括协同作用的局部化学作用。通过其次生作用(如次生的跨距效应),局部活性有时也会产生全身性效果。
本申请所述的局部化学作用(或局部化学协同作用)在药理上包括普通局部化学作用、化学消融和 类化学消融。术语“普通局部化学作用”是指药物效应不超过同一药物的常规化学作用的动力学差异最大预期(例如200％以内)的局部化学作用，如细胞毒药物常规给药产生的化疗作用。术语“化学消融”是指药物效应超过同一药物的常规化学作用(例如高浓度乙醇常规给药产生的化疗作用)的动力学差异最大预期(例如大于200％、优选为大于400％)的局部化学作用，通常指经典化学消融剂(如高浓度乙醇、高浓度酸、高浓度碱)所显示的局部化学作用。术语“类化学消融”是指药物效应类似于化学消融(超常规作用预期)的局部作用。类化学消融虽非经典化学消融剂所致，但其在药理上明显区别于普通局部化学作用。
术语“次生作用”区别于术语“直接作用”,后者是指药物进入靶区后与药靶直接反应的作用(例如局部用药进入局部病变与病变组织发生的局部作用),而前者是指与后者有关但不相同、且产生时间上稍后的作用。
术语“免疫治疗”区别于术语“免疫增强”，前者是指其单独使用便可达到治疗作用的免疫作用(例如治疗疫苗、特异性抗体等的免疫作用)，而后者是指单独使用不能达到治疗作用、但仍有辅助作用的免疫作用(例如免疫增强剂具有提高机体免疫功能的作用)。术语“次生免疫作用”区别于术语“药物抗原作用”，后者是指药物自身的抗原作用(例如任何药物作为外来物进入体内引起的抗原作用)，而前者是指与给药相关的、但区别于药物抗原作用的免疫作用，例如药物局部化学作用导致的原位疫苗作用)。术语“原位次生免疫作用”区别于术语“非原位次生免疫作用”，前者是指病变(例如瘤体)内的次生免疫作用，而后者是指病变外(例如腋下皮内)的次生免疫作用。术语“次生免疫物质”是指在给药区域内由于局部给药形成的、且与所给药物本身不同的免疫物质，例如给药后因任何原因被释放、生成、激活、或/和幕集的抗原、佐剂、或/和其它免疫分子。术语“原位次生免疫物质”区别于术语“非原位次生免疫物质”，前者是指是指病变(例如瘤体)内的次生免疫物质(例如原位抗原、原位佐剂、或/和病变内被释放、生成、激活、或/和幕集的其它免疫分子)，而后者是指病变外(例如腋下皮内)的的次生免疫物质(例如给药处形成的结节性免疫物质、因该结节或/和给药处其它药物效应被释放、生成、激活、或/和幕集的其它免疫分子、等等)。
术语“疫苗抗原”区别于术语“抗原”，后者是指能够诱发机体产生免疫应答的任何物质，而前者是指能够诱发机体产生针对特定疾病、且达到治疗作用的抗原，例如同一种物质可以通过大不相同的技术方案被用作疫苗抗原和免疫增强剂抗原。术语“佐剂”是指在疫苗中可以增强其抗原的免疫治疗作用的物质。术语“原位抗原”是指可作为抗原的原位次生免疫物质。术语“类疫苗药物”或“类疫苗”，是指可提供类似于疫苗作用的次生免疫作用(区别于免疫增强剂和常规疫苗)和任选存在的外源性抗原作用的治疗药物。
在本发明的范围内，所述益生菌组分与所述能够与该益生菌组分产生协同作用的化学活性成分和/或生物活性成分一起局部给药。在一个实施方案中，所述益生菌组分与所述共用活性成分包含在同一个药剂中。
术语“协同作用”是指特定的活性成分(如本申请的益生菌组分)在特定条件下(如可提供最小化细菌免疫作用的药理组分优选和最小化局部作用的药理浓度优选)与另一些活性成分(如弱局部作用化合物和/或细胞毒药物)的共用的作用效果(如短期药效和/或中长期药效)超过各自单独使用的作用效果 的加和预期。术语“局部协同作用”是指包括协同作用的局部作用。术语“局部化学协同作用”是指包括协同作用的局部化学作用。
术语“量比”是指多个活性成分(如本申请的益生菌组分及其共用物)在同一个组合物中的重量比。术语“浓度比”是指多个活性成分(如本申请的益生菌组分及其共用物)在同一个组合物中的浓度比。
所述治疗的适用患者选自包括以下组之一种或多种：免疫抑制患者(如免疫能力低的患者、老年患者)、可局部病变内给药的患者、局部病变组织可类化学消融的患者、局部病变内可产生次生免疫物质的患者、病变外给药区域内可产生次生免疫物质的患者。
所述“免疫抑制患者”是指荷瘤裸小鼠模型所能代表的任何患者，例如其免疫功能因任何原因处于较低水平、且在本发明技术方案中的组合物可提供局部作用的时间内(例如以下实施例2中第一次给药7日内)利用其它方法(如免疫增强)不能达到正常水平的患者例如因免疫为低下而难以进行放疗或常规化疗的患者。
所述治疗作用包括涉及所述局部作用(或局部协同作用)的局部治疗或/和免疫治疗。所述免疫治疗包括在所述病变内和/或病变外所述局部作用(或局部协同作用)的次生免疫作用和任选存在的其它免疫作用；所述局部治疗包括一处或多处局部病变的类化学消融和任选存在的其它化疗。优选的，所述药物组合物为类化学消融-原位和非原位免疫治疗药物，所述患者选自可局部病变内给药且该局部病变组织可类化学消融或/和该局部病变内可产生次生免疫作用的患者、病变外给药区域内可产生次生免疫作用的患者中的一种或多种。所述药物组合物为类化学消融药物或者原位免疫治疗药物，所述原位免疫治疗药物可以是提供病变内靶区原位疫苗激活和任选存在的其它免疫作用的免疫治疗药物；所述药物组合物也可以是可提供病变外次生免疫作用和任选存在的其它免疫作用的免疫治疗药物，如类疫苗药物。所述病变外次生免疫作用包括所述局部作用(或局部协同作用)次生的非正常结构(例如结节)的免疫作用。
所述益生菌组分可提供局部作用、局部作用的次生作用、局部给药的免疫作用中的至少一种作用，其中所述局部作用的次生作用包括给药区域内次生的免疫物质参与的免疫作用。
所述益生菌组分选自益生菌水溶性组分、益生菌半流体类组分、益生菌组分水不溶颗粒、灭活益生菌中的一种或多种，且细菌免疫原性低。作为本发明的一个示例，所述益生菌组分选自益生菌水溶性组分和/或及其工程类似物以及所述组合物为溶液组合物。在一个实施方案中，所述益生菌组分选自菌组分水不溶颗粒、或/和灭活益生菌以及所述组合物为悬浊液组合物。在一个实施方案中，所述益生菌组分选自益生菌半流体类组分以及所述组合物为半流体类组合物。在一个实施方案中，所述组合物优选为半流体类组合物或水溶液组合物、更优选为半流体类组合物。
所述“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。所述“灭活益生菌”是指经如高温灭活、高温高压灭活、紫外线灭活、化学试剂灭活、辐射灭活等灭活处理后获得的制备物。所述“益生菌水不溶组分”是指来源于益生菌获得的水溶度为<0.1％的任意组分，所述“益生菌水溶性组分”是指来源于益生菌获得的水溶度≥0.1％的任意组分，所述“破碎益生菌”是指益生菌经破碎工程处理后获得的混合物，所述“破碎益生菌沉淀组分”是指自破碎益生菌分 离(如过滤或/和离心)出的水不溶沉淀、其进一步分离组分及其类似物。术语“破碎益生菌上清组分”是指自破碎益生菌分离(如过滤和/或离心)出的上清液、其进一步分离组分及其类似物。术语“益生菌细胞壁聚多糖”是指细胞壁所含聚多糖及其类似物，如β-葡聚糖。术语“益生菌半流体类组分”是指为本发明的组合物提供半流体类形态的益生菌组分。
术语“半流体类”是指液体和半固体之间的这样一类物理形态，其包括半流体及其类似物。术语“半流体”是指在室温下限时内(例如20秒)无外压则无肉眼可见的流动、而在临床(施用时)可接受的外压(例如可施加在注射器推进装置上的外压)下可以流动并导致不可逆形变的物体，其区别于液体(无外压时亦有流动性)和半固体(在临床可接受的外压下仅发生可逆形变)。术语“半流体类似物”是指液体(悬浊液)和半流体之间的、与半流体接近的一种物理形态，其在室温下静止约1分钟时不出现、而悬浊液出现明显分层；其在室温下无外压时约1分钟以内可出现、而半流体不会出现肉眼可见的流动。悬浊液有可能转变为半流体类物体，如5％β-葡聚糖加水混合为悬浊液，5％β-葡聚糖水悬浊液可加热制备为半流体类似物，而5％β-葡聚糖/1％亚甲蓝/1％5-氟尿嘧啶水悬浊液可加热制备为半流体。
所述益生菌水溶性组分选自破碎益生菌上清组分、益生菌抽提物、益生菌细胞内水溶性组分中的至少一种。所述益生菌半流体类组分选自其含水混合物可形成(如通过加热后冷却、碱化等形成)半流体类组合物的益生菌组分之一种或多种，如多聚糖及其类似物。
所述益生菌水不溶颗粒选自：破碎益生菌沉淀组分、益生菌细胞壁聚多糖颗粒、益生菌细胞壁聚多糖纳米颗粒中的至少一种。所述灭活益生菌并不限定其皆为完整菌体的灭活益生菌，如灭活益生菌中不完整菌体的量比＞20％或＞30％，或完整菌体的数量<10 5个/ml或<0.5×10 5个/ml。
所述益生菌选自芽孢杆菌、乳酸菌、双歧杆菌、真菌中的至少一种，其可以是天然菌或工程菌。所述芽孢杆菌为蜡样芽孢杆菌、地衣芽孢杆菌、枯草芽孢杆菌、巨大芽孢杆菌、坚强芽孢杆菌、凝结芽孢杆菌、缓慢芽孢杆菌、短小芽孢杆菌、纳豆芽孢杆菌中的一种或多种。所述乳酸菌为乳酸杆菌和/或双歧杆菌，其中所述乳酸杆菌选自嗜酸乳杆菌、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中的至少一种。所述双歧杆菌包括长双歧杆菌、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、粪链球菌中的至少一种。所述真菌包括酵母菌和/或布魯氐酵母菌，其中所述酵母菌包括酿酒酵母菌、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、产阮假丝酵母中的至少一种。
所述益生菌组分提供上述治疗作用所需的药理组成满足使该益生菌组分提供所述作用所需的药理浓度(局部给药浓度)为＞0.1％、≥0.25％、0.25-25％、优选为0.5-15％、更优选为1-15％或5-15％。
术语“浓度”是特定组分在药物中的重量-体积百分比浓度(w/v)。术语“药理浓度”是指指定组分为达成其特定药理反应所必须的进入靶区浓度,例如病变内初浓度。术语“制剂浓度”是指指定组分在药物制剂形态(例如注射剂或灌注液)中的浓度。术语“给药浓度”是指指定组分在药物制剂的给药形态(例如制剂的稀释液)中的浓度。术语“病变内初浓度”是指药物进入病变时指定组分在含药介质(例如含药血液)中的浓度。既使本发明组合物的益生菌组分给药浓度与常规注射剂中的益生菌组分给药浓度相同，它们各自药理(局部化学作用vs免疫增强作用)所必须的瘤内初浓度也可以大不相同。而本发明 的应用、组合物及方法的技术方案特征之一，是要保障所述作用、尤其是局部化学作用所须的药理浓度(局部给药浓度)。
本申请所述组合物中益生菌组分的给药浓度即使与常规注射剂中益生菌组分的给药浓度相同，但它们的各自药理(局部化学作用vs免疫增强作用)所必须的病变内初浓度也可以大不相同。而本发明的应用、组合物及方法的技术方案特征之一是保障所述作用、尤其是局部化学作用所须的局部初浓度(局部给药浓度)。本申请中药物组合物中益生菌组分的局部给药浓度，通常是注射器、穿刺器、灌注导管等给药器械终点(如针孔、导管出口等)的药物中的益生菌组分的浓度。对注射用粉针剂而言，所述给药浓度即为干粉和液体载体的混合后益生菌组分的浓度。
术语“靶”是指药理的主要目标，如细胞毒药物针对肿瘤细胞，免疫调节药物针对免疫系统的调节因子，化学消融剂针对瘤组织等。术语“靶区”是指给药时针对靶所在的空间范围，如瘤体或其一部分。例如，当瘤体直径较小，所需一次给药剂量在临床上可行时，靶区是作为本次治疗目标的一个瘤体；或当瘤体直径较大，所需一次给药剂量在临床上不可行时，靶区是作为本次治疗目标的瘤体中的一部分。
当用于治疗患者时，所述组合物的组成或单位组成必须满足使得该益生菌组分提供所述作用所需的药理体积条件，其中所述药理体积(局部给药体积)≥1ml、或局部病变内给药10-150ml或/和局部病变外给药1.5-50ml。作为本发明的应用、组合物及方法的技术方案特征之一，该超常规的体积给药尤其是所述益生菌组分的局部作用所需。
所述组合物的组成要求除协同物外的其它组分的最小化，从而满足提供所述局部作用所需的局部药理环境条件优选不含常规组合物中药剂学和/或给药安全性所需的非活性成分，如口服制剂中的固体赋形剂、调味剂及常规注射剂中的渗透压提高剂。
在一个实施方案中，所述药物组合物的载体为水，共用物可溶于水且所述益生菌组分为益生菌水溶性组分或者是灭活益生菌、益生菌组分水不溶颗粒、益生菌半流体类组分中的一种或多种或其衍生物水不溶性组分。在一个实施方案中，所述药物组合物的载体为水，所述共用物和/或难溶于水，而所述益生菌组分为益生菌半流体类组分。
优选的，本申请中所述的化学活性成分选自弱局部作用化合物和/或细胞毒药物，如所述共用物为至少两种化学活性成分。若所述化学活性成分为细胞毒药物时，所述益生菌组分和细胞毒药物的量比(W 益生菌组分/W 细胞毒药物)为(1-110)/(1-100)，若所述化学活性化合物为弱局部作用化合物时，所述益生菌组分和弱局部作用化合物的量比(W 益生菌组分/W 弱局部作用化合物)为(1-90)/(1-100)。
术语“化学活性成分”是指任何可提供化学作用的活性成分，包括细胞毒药物和局部化学成分。术语“局部化学成分”是指任何在局部给药时可提供局部化学作用的活性成分。术语“弱局部作用化合物”是指在特定条件(如用作共用物的浓度)下局部作用低于经典化学消融剂的化学活性成分。所述弱局部作用化合物包括：氨基酸类营养素、活体染料、奎宁类药物、低浓度酸化剂、低浓度碱化剂、包含酸化剂或/碱化剂的pH缓冲系统。所述共用物可以是活体染料和至少一种其它化学活性成分，也可以是活体染料和细胞毒药物，或者活体染料和氨基酸类营养素，亦或者细胞毒药物和至少一种其他化学活性成分。
若所述弱局部作用化合物为氨基酸营养素时，则所述益生菌组分和氨基酸营养素的量比(W 益生菌组分/W 氨基酸营养素)为(1-20)/(1-100)；若所述弱局部作用化合物为活体染料时，则所述益生菌组分和活 体染料的量比(W 益生菌组分/W 活体染料)为(7-90)/(1-100)；若所述弱局部作用化合物为奎宁类药物时，则益生菌组分和奎宁类药物的量比(W 益生菌组分/W 奎宁类药物)为(2-90)/(1-100)；若所述弱局部作用化合物为酸化剂和/或碱化剂时，则所述益生菌组分和酸化剂和/或碱化剂的量比(W 益生菌组分/W 酸化剂或/和碱化剂)为(2-60)/(1-100)。
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，优选为官方药典或指南所载的氨基酸类营养药或具有营养保健作用的氨基酸类辅料。所述氨基酸类营养素包括氨基酸、氨基酸盐、寡肽和多肽。优选的，所述氨基酸类营养素包括以下组中的氨基酸或其盐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。优选的，所述氨基酸类营养素包括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的浓度＞2.5％、或5-30％、优选为5-25％。
术语“活体染料”是指进入动物活体组织后能够使组织、细胞、亚细胞单元等结构上色、但对动物整体没有不可接受的危害性的芳香化合物染料。所述活体染料包括亚甲蓝、专利蓝、异硫蓝、孟加拉红、甲苯胺蓝、台盼蓝、碱性蓝、伊红、碱性品红、结晶紫、龙胆紫、中性红、詹纳斯绿B、番红中的至少一种。优选的，所述活体染料为亚甲蓝类染料，如亚甲蓝、专利蓝、异硫蓝、新亚甲蓝。在一个实施方案中，所述活体染料的浓度≥0.25％、或0.25-10％、优选为0.25-1.5％或2.5％-10％。优选的，所述活体染料为亚甲蓝类染料，其浓度(w/v)≥0.35％、优选为0.35-2.5％、更优选为0.35-1.5％或0.5-1％。在一个实施方案中，所述活体染料为孟加拉红，浓度(w/v)为1-10％。
所述奎宁类化合物包括奎宁、一盐酸奎宁、二盐酸奎宁中的至少一种，其浓度≥0.5％、或0.5-5％、优选为1.5-5％或1.5％-3％。术语“酸化剂”是指在药物制备中主要用作辅料、更具体讲用作pH调节的酸，其在使用时除提供酸性外通常并不引入特殊的生物活性；优选为经各国官方主管行政部门批准、或经各国官方药典或指南载入的强酸和/或弱酸。所述强酸可以是盐酸、硫酸、硝酸、高氯酸、硒酸、氢溴酸、氢碘酸、氯酸等，优选为盐酸。所述弱酸可以是碳酸、硼酸、乙酸、磷酸、亚硫酸、丙酮酸、草酸、酒石酸、亚硝酸等，优选为乙酸。在一个实施方案中，所述益生菌组分与所述酸化剂的量比-即重量浓度(W 益生菌组分/W 酸化剂)为(1-20)/(0.5-50)。在一个实施方案中，所述酸化剂的浓度≥0.5％、0.5-2％(强酸)或者2-15％(弱酸)。
在一个实施方案中，所述益生菌组分与所述碱化剂的量比(益生菌组分重量浓度/碱化剂重量浓度)为(1-20)/(0.5-50)。在一个实施方案中，所述碱化剂的浓度为≥0.5％、0.5-5％(强碱)、或/和2-15％(弱碱)。术语“碱化剂”是指在药物制备中主要用作辅料、更具体讲用作pH调节的碱性化合物；优选为选自经各国官方主管行政部门批准、或经各国官方药典或指南载入的碱化剂，包括强碱和弱碱。所述强碱为碱金属氢氧化物或/和有机强碱，其在药物组合物中的浓度(w/v)≥0.5％、优选为0.5-7.5％或0.75-7.5％。优选的，所述强碱为氢氧化纳、氢氧化钾、氢氧化钙等碱金属氢氧化物，更优选为氢氧化纳。所述弱碱包括多元弱酸酸式无机盐、多元弱酸碱式无机盐、含氮弱碱，其中多元弱酸酸式无机盐包括磷酸二氢钠、磷酸氢二钠、碳酸氢钠、碳酸氢钾、碳酸氢钙、氢硫酸钠，优选为碳酸氢钠。在一个实 施方案中，所述多元弱酸酸式无机盐在所述药物组合物中的浓度(w/v)≥1％、优选为2-10％或3-10％。
所述多元弱酸碱式无机盐包括磷酸钠、碳酸钠、碳酸钾、硼砂，优选为碳酸钠；所述多元弱酸碱式无机盐在所述药物组合物中的浓度(w/v)≥1％、优选为2-10％或3-10％。所述含氮弱碱包括氨水、氯化氨、2-氨基乙醇、氨丁三醇、三乙醇胺、三羟甲基氨基甲烷、2-氨基乙醇、氨丁三醇、三乙醇胺、葡甲胺、葡乙胺；所述含氮弱碱在所述药物组合物中的浓度(w/v)≥2％、优选为2-35％或3-35％。所述碱化剂可以是氢氧化钠和碳酸氢钠，且二者在所述药物组合物中的浓度(w/v)分别为2-5％、3-10％。所述碱化剂也可以是碳酸钠和碳酸氢钠，且二者在所述药物组合物中的浓度(w/v)分别为3-10％、3-10％。优选的，所述药物组合物包含酸化剂或/和碱化剂且具有pH缓冲能力。
所述化学活性成分包括至少一种细胞毒药物，所述细胞毒药物包括抗肿瘤药物活性成分；其中所述抗肿瘤药物活性成分的浓度≥0.1％、0.1-15％。
术语“细胞毒药物”指主要以病变细胞或病变细胞内结构为靶实现其药物效应的活性成分，如常规化疗药物。术语“常规化疗药物”是指在安全剂量下可以通过常规用药有效治疗局部病变疾病的药物，优选为各国官方主管行政部门(例如FDA或中国药监局)己批准或将批准、或经各国官方药典己载入或将载入的常规化疗药物(如抗肿瘤药物)。
所述抗肿瘤药物活性成分可以为选自1)破坏DNA结构和功能的药物，如a)环磷酰胺、卡莫司汀等烷化剂；b)金属铂络合物，如顺铂、卡铂等；c)DNA拓扑异构酶抑制剂，如多柔比星类、拓扑替康、伊立替康等；2)、嵌入DNA中干扰转录RNA的药物，如抗肿瘤抗生素，如放线菌素类、柔红霉素、多柔比星等、3)干扰DNA合成的药物，包括a)嘧啶拮抗物，如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-氮杂胞苷等尿嘧啶衍生物、b)溶癌呤、硫鸟嘌呤等嘌呤拮抗物、c)甲氨蝶呤等叶酸拮抗物；4)影响蛋白质合成的药物，如秋水仙碱类、长春碱类以及紫杉醇、多西紫杉等紫杉烷类。
所述抗肿瘤药物活性成分包括尿嘧啶衍生物类、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、紫杉烷类中的至少一种；优选的，抗肿瘤药物活性成分选自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、紫杉醇中的至少一种。
抗肿瘤药物活性成分可以是环磷酰胺、卡莫司汀等烷化剂，且在所述药物组合物中的浓度(w/v)为0.5-6％、优选为0.75-1.5％；也可以是顺铂、卡铂等金属铂络合物，且在所述药物组合物中的浓度(w/v)为0.03-0.15％、优选为0.05-0.15％；或者是多柔比星类、拓扑替康、伊立替康等DNA拓扑异构酶抑制剂，且在所述药物组合物中的浓度(w/v)为0.05-0.20％、优选为0.075-0.15％；或者是放线菌素类、柔红霉素等抗肿瘤抗生素，且在所述药物组合物中的浓度(w/v)为1-4％、优选为1-2％；或者是尿嘧啶衍生物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-氮杂胞苷等嘧啶拮抗物，且在所述药物组合物中的浓度(w/v)浓度为0.5-2％、优选为0.75-1.5％。
优选的，所述所述药物组合物包含生物活性成分，所述生物活性成分包括抗原、免疫调节类抗体、细胞因子、佐剂中的至少一种。所述抗原为微生物抗原或肿瘤抗原，其中所述微生物抗原包括1)化脓性链球菌、豁质沙雷菌、卡介苗、破伤风梭菌、丁酸梭菌、嗜酸乳杆菌等非益生菌细菌；2)乙肝病毒、 腺病毒、单纯疤疹病毒、牛痘病毒、腮腺炎病毒、新城鸡瘟病毒、脊髓灰质炎病毒、麻疹病毒、西尼卡谷病毒、柯萨奇病毒、呼肠孤病毒等病毒；3)疟原虫等寄生虫；所述肿瘤抗原选自乳腺癌、胰腺癌、甲状腺癌、鼻咽癌、前列腺癌、肝癌、肺癌、肠癌、口腔癌、胃癌、结直肠癌、支气管癌、喉癌、睾丸癌、阴道癌、子宫癌、卵巢癌、恶性黑色素瘤、脑瘤、肾细胞癌、星形细胞瘤、胶质母细胞瘤中的至少一种；所述免疫调节类抗体包括1)针对抑制性受体的抗体阻断剂，如CTLA-4分子和PD-1分子的阻断性抗体；2)针对抑制性受体的配体的抗体阻断剂、针对免疫反应细胞表面刺激分子的激活性抗体，如抗OX40抗体、抗CD137抗体、抗4-1BB抗体；3)针对局部病变疾病微环境中免疫抑制性分子的中和抗体，如抗TGF-p1抗体。所述细胞细胞包括肿瘤坏死因子、干扰素、白介素。
优选的，本申请所述药物组合物的剂型为局部给药剂型，所述药物组合物可以是包含益生菌组分、且满足使得所述益生菌组分提供所需作用的必需条件的任何局部给药剂型，例如注射剂、涂抹剂或膏剂。
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，按给药方式分为局部注射剂、静脉注射剂等，局部注射剂只有在给定局部给药浓度后方可作为局部注射剂使用；按商品形式分为液体注射剂、半流体注射剂、注射用粉针剂等，其中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，通常是注射器、穿刺器、注入导管等局部给药器械终点(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(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。
优选的，本申请所述药物组合物还包含赋形剂，例如分散介质、防腐剂、稳定剂、湿润剂和/或乳化剂、增溶剂、增粘剂等，其中所述增粘剂可以为羧甲基纤维素钠、羧甲基纤维素、聚乙烯毗咯烷酮或明胶，所述防腐剂可以是抗坏血酸等抗氧化剂。优选的，本申请所述药物组合物还包含碘化油等示踪剂。
本申请还提供了一种药物试剂盒，其包括一个或多个装有根据本申请公开的药物组合物的单独容器，其中所述单独容器可以是瓿、小玻璃瓶等。优选的，所述药物试剂盒还包括如何向有需要的个体施用所述药物组合物的说明书或标签。所述施用包括在所述局部病变内施用，或者在局部病变内和局部病变外施用，所述局部病变外施用例如腋下皮下注射。在施用时，所述药物组合物的施用量与所述局部病变内靶区体积之比＞0.1、0.15-1.5，优选为0.23-1.5或0.5-1.5。
本发明的药物组合物的制备包含：制备含所述益生菌组分和任选存在的其它物质的局部药物制剂或制备含所述益生菌组分、所述共用物和任选存在的其它物质的局部药物制剂。其中所述药物可以是液体药物或半流体药物，而该液体药物可以是溶液(例如为亲水溶媒的溶液、优选为水溶液)、悬浮液、乳浊液。在一个实施方案中，所述药物可以是体内给药剂或体表涂抹剂。
本申请中灭活益生菌可通过高温灭活、高温高压灭活、紫外线灭活、化学试剂灭活、辐射灭活等方式制备。本申请中的破碎益生菌组分可通过以下方法制备：1)、将活益生菌通过高压匀浆破碎法、振荡珠击破碎法、高速搅拌珠研磨破碎法、超声波破碎法、撞击破碎法、渗透压冲击破碎法、冻融破碎法、酶溶破碎法、化学破碎法、去垢剂破碎法等制备；如有必要，2)、将1)的制备物经过滤或/和离心等分离工程制备为破碎益生菌分离组分，例如破碎益生菌水不溶组分颗粒、破碎益生菌水溶性组分。在一个实施方案中，本发明中的益生菌水溶性组分通过上述破碎益生菌水溶性组分(例如破碎益生菌上清液 组分)的制备或现有技术中益生菌水溶性组分(例如益生菌抽提物)的制备的方法制备。在一个实施方案中，本发明中的益生菌水不溶组分颗粒通过上述破碎益生菌水不溶组分颗粒(如破碎益生菌沉淀组分)的制备或现有技术中益生菌水不溶组分颗粒(如胞壁聚多糖)的制备的方法制备。在一个实施方案中，本发明中的益生菌半流体类组分可通过益生菌聚多糖或其类似物和水的混合物的加温步骤的方法制备。在一个实施方案中，所述益生菌聚多糖/水混合物的加温温度优选为50-110℃，其通过微波致热、电炉加温、蒸汽加温等方法加温。在一个实施方案中，本发明的包含所述益生菌半流体类组分和所述共用物的药物组合物的制备包含：将所述益生菌聚多糖或其类似物、所述共用物和水混合均匀后进行所述加温。在一个实施方案中，本发明的药物组合物的制备还包含：使得所述药物制剂中的所述益生菌组分、所述共用物和任选存在的其它物质的浓度大于或等于其在本发明技术方案所需的给药浓度。当大于在本发明之药物组合物中的给药浓度时，其可进一步被稀释使用。
优选的，本发明的药物组合物的制备还包括：使得所述药物制剂中的所述益生菌组分、所述共用物和任选存在的其它物质的含量宜于满足其在本发明技术方案所需的药物体积/靶区体积比，如根据该药物体积/靶区体积比对进行药物分装和封盖。优选的，本发明的药物组合物的制备还包括：对制备物进行高温灭菌、高温高压灭菌、紫外线灭菌、化学试剂灭菌、辐射灭菌等灭菌处理。
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，本发明的药物组合物中的变化包括：含不同种类和浓度的所述药物组合物、含不同种类和浓度的其它药物、含不同种类和浓度的其他添加剂(例如止痛剂、增活剂等)。
术语“局部病变疾病”是指具有局部病变症状的疾病。术语“局部病变(亦简称病变)”是指动物(优选人类)身体原生或继生的不正常局部，其包括结构(例如病变组织)、形态或功能上的症状区块及与之相联通的不正常区域。在本申请中所述局部病变疾病包括实体肿瘤、非瘤肿大，如非瘤结节、宫颈糜烂等局部炎症、分泌腺功能异常和皮肤病。若局部病变疾病为实体肿瘤时，局部病变为肿瘤细胞所在的瘤体及其组织，而与之相连通的不正常区域为与该瘤体相联通(例如通过淋巴管或血管联通)的、且有或疑似有肿瘤细胞的相邻区域；若局部病变疾病为非瘤肿大时，局部病变为增生、囊肿、结节等异常的非瘤肿块；甲状腺、乳腺、肝、肺、肠若的息肉等非瘤结节。局部病变疾病为局部炎症时，则局部病变为发炎面或发炎体等发炎区；若局部病变疾病为分泌异常时，则局部病变为异常源或其所在的分泌腺体；例如，当疾病为胰岛素分泌异常时，异常源在胰岛，局部组织则为胰岛或胰岛所在的胰腺；当疾病为皮肤病时，局部组织为病变皮肤或病变皮肤的附属器官。
术语“肿瘤”是指由于细胞或变异的细胞异常增殖形成的肿块，其包括实体肿瘤。术语“实体肿瘤”是指具有瘤体的肿瘤，其可以是由于任何病理(恶性和非恶性)和处于任何阶段的肿瘤，若按照肿瘤细胞类型分为上皮细胞肿瘤、肉瘤、淋巴瘤、生殖细胞肿瘤、胚细胞瘤等；若按肿瘤细胞集中区所在的器官或组织来命名的肿瘤，包括按以下器官或组织来命名的肿瘤：脑、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、前列腺、翠丸、卵巢或子宫。具体的，所述肿瘤包括恶性肿瘤和非恶性肿瘤，其中所述恶性肿瘤包括乳腺癌、胰腺癌、甲状腺癌、鼻咽癌、前列腺癌、肝癌、肺癌、肠癌、口腔癌、食道癌、胃癌、喉癌、睾丸癌、阴道癌、子宫癌、卵巢癌、恶性淋巴瘤、恶性脑瘤等；所述非恶性肿瘤包括乳腺瘤、胰腺瘤、甲状腺瘤、前列腺瘤、肝瘤、肺瘤、肠瘤、 口腔瘤、食道瘤、胃瘤、鼻咽瘤、喉瘤、睾丸瘤、阴道瘤、子宫瘤、输卵管瘤、卵巢瘤、淋巴瘤、脑瘤等。所述局部病变疾病也可以是慢性粘膜皮肤念珠菌病、各种癣等皮肤病。
当本申请所述药物组合物用于治疗疾病时，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、手术干预等进行组合施用，以进一步提高疗效。
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，本发明的药物组合物显示出促进瘤体组织的有效破坏、同时对患者正常组织仅有最小化的损害，从而达到安全、有效治疗局部病变疾病的药学效果。
本申请所用益生菌以及部分的益生菌组分(酵母菌水不溶β-葡聚糖微粒、酵母菌水溶性β-葡聚糖、酵母菌羟甲基葡聚糖、酵母菌羧甲基葡聚糖、酵母菌核糖核酸、等)均可从商业途径得到，其他益生菌组分(灭活益生菌、益生菌粉碎物、益生菌粉碎物上清成分、益生菌粉碎物沉淀)利用前述方法(如(100℃、2小时的高温灭活、转速5000转/分的粉碎机粉碎、10000转/分的离心机分离上清液和沉淀)自制。
以下实施例中所用的实验动物均由专业实验动物公司购入的SPF(Specific Pathogen Free，无特定病原体)级动物，除非另有说明，其中小鼠和裸小鼠均为6-8周龄健康雌性、体重17.5-20.5g。
在以下实施例中，除非另有说明，皮下移植细胞生成局部病变(如瘤体)的动物试验均依据药管当局颁发的试验指南、按常规的细胞皮下接种方法进行。除非另有说明，当局部病变(如瘤体)长至所需体积(如小鼠荷瘤为50-500mm 3)为建模成功，建模后将动物分成若干组，每组6只，定期观察测定一般状态、体重、摄食量、动物移植物抗宿主病、瘤体体积、瘤重、生存时间等。的模型分别含肿瘤细胞的瘤体(代表瘤体)、含成纤维细胞的结节(代表含成纤维细胞的瘤体和其它局部病变)、正常活组织(代表含普通组织的局部病变)。
局部病变(例如瘤体)体积(V)、相对局部病变(例如瘤体)增殖率(R)、局部病变(例如瘤体)抑制率(r’)、抑瘤率(r)分别按照下述公式计算：
局部病变(例如瘤体)体积V＝0.5×a×b 2，其中a、b分别代表局部病变的长、宽；
局部病变(例如瘤体)增值率(R)＝TV/CV×100，其中TV和CV分别为研究组和阴性对照组的局部病变体积；
局部病变(例如瘤体)抑制率(r’)＝100％-R，其中R为局部病变增殖率。
抑瘤率r＝(CW-TW)/CW×100％，其中TW、CW分别为研究组、阴性对照组的平均瘤重。
在下述实施例中，药物i的药效记为Ei，可以用ri’或ri表示。药物的作用类型即药理可通过药效，尤其是同意药物在不同方案中的药效来研究。如当药物i在方案X、Y之间的药效差异不大时(如Ei X/Ei Y＜200％)，其说明药物的药理是相同的；当药物i在方案X、Y之间的药效差不大时(如Ei X/Ei Y＞200％)，说明药物i在方案X中的药理大大超过了其在方案Y中药理的动力学预期范围，很可能涉及与方案Y不同的新药理。若两个药物显示出显然不同的Ei X/Ei Y关系，则很可能涉及不同的药理；若两个药物显示出相似的Ei X/Ei Y关系，则它们很可能涉及相同的药理，至少涉及相似的药理，如类化学消融药理和化学消融药理。
在以下实施例中，实验结果(例如瘤重、瘤体积、病变组织体积)采用均数±标准差(x±s)表示，两个实验动物组与组均数之间的差别采用统计学软件SPSS13.0或SPSS19.0进行显著性检验，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。
化疗的阳性对照包括经典的细胞毒药物(如0.5-1％5-氟尿嘧啶，其在下述实施例条件下的抑瘤率为≥30％)和经典化学消融剂(如75-99％乙醇，其在下述实施例条件下的抑瘤率为≥15％)。免疫增强阳性对照包括细胞因子(如白细胞介素-12，)、细菌组分(如减毒细菌疫苗等)、疫苗佐剂等免疫增强剂。
在本发明的范围内，A药和B药的组合物记为B/A，其共用作用通过q＝实际共用作用/理论单纯相加预期作用判断进行，其中A、B的单用药效分别记为E A和E B，如如抑瘤率，A/B的实际共用药效记为E A+B。当q＝1时说明实际共用作用符合理论预期，显示为相加作用；当q＜1时说明实际共用作用弱于理论预期，显示为拮抗作用；当q＞1时说明实际共用作用超理论预期，显示为协同作用。
判断动物实验中合并用药效应的方法有Burgi法(Burgi Y.Pharmacology；Drug actions and reactions.Cancerres.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)。在本发明以下实施例中,根据金正均法的实际药效/预期药效比q进行的药物A和药物B联合给药的共用药效作用判断如下：
当药物A和药物B的组合物组并不显示有意义的药效((例如r或r’≦15％)时,该联合给药也就未显示有意义的共用作用,在本发明中视作可忽略不计的共同作用。当组合物组显示有意义的药效(例如抑瘤率≧15％)时,如果实际药效/预期药效比q＝1.00则该组合物的共用药效作用为药效相加作用(实际作用符合理论单纯相加预期)；如果实际药效/预期药效比q>1.00则该组合物的共用药效作用为明显药效协同作用(实际作用超理论单纯相加预期)；如果实际药效/预期药效比q<1.00则该组合物的共用药效作用为明显药效拮抗作用(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的组成列于表2(益生菌组分作为活性成分)和表3(益生菌组分作为协同活性成分)。
表2包含益生菌组分的组合物水剂的组分、药理浓度(＞0.1％、≥0.25％、0.25-25％、优选为0.5-15％、更优选为1-15％或5-15％)
Figure PCTCN2021122132-appb-000001
Figure PCTCN2021122132-appb-000002
以下列出表2中几个制备物的制备试验例子。
A1-A8的制备方法：按所需(例如局部化学作用所需)浓度量取益生菌(例如2.5g酿酒酵母菌干粉)、任选存在的其他组分、以及定容至总体积(例如100ml)的液体载体(例如注射用水)，将它们缓慢混合均匀获得益生菌混合液。如果按所需药物体积/靶区体积比(例如临床上常见的实体肿瘤的平均体积为的30cm3)对该液体混合物进行分装(例如10ml/瓶)和封盖，即可获得可提供瘤内局部作用的药物组合物剂型和规格。将该制备物(例如2.5％酿酒酵母菌)放入巴斯德灭活櫃式机中进行巴斯德灭活(60℃，48 小时)，即可获得A1。使用与A1制备相同的方法，可分别从不同益生菌进行不同灭活益生菌(例如上表中制备物A1-A8)的制备。
A9-A16的制备方法：按所需(例如局部化学作用所需)浓度量取益生菌(例如2.5g酿酒酵母菌干粉)、任选存在的其他组分、以及定容至总体积(例如100ml)的液体载体(例如注射用水)，将它们缓慢混合均匀并使用匀浆机对其进行破碎。通过调整匀浆工艺参数(例如转速10000-25000转/分钟，转时0.5-1分钟，匀漿次数2-10次)可以获得不同破碎程度(优选为100％破碎)的破碎益生菌。如果按所需药物体积/靶区体积比(例如临床上常见的实体肿瘤的平均体积为的30cm3)对该液体混合物进行分装(例如10ml/瓶)和封盖，即可获得可提供瘤内局部作用的药物组合物剂型和规格。所获制备物为A9。使用与A9制备相同的方法，可分别从不同益生菌进行不同组合物(例如上表中制备物A9-A16)的制备。
此外，使用上述制备中制备破碎益生菌的方法，可从益生菌(例如10g酿酒酵母菌干粉以及定容至100ml的注射用水制备高浓度破碎益生菌悬浊液(例如10％破碎酿酒酵母菌悬浊液)。将该悬浊液加入离心瓶在离心机上进行离心。通过调整离心机转速(例如1000-25000转/分钟)、离心时间(例如0.5-30分钟)和离心分离次数(例如2-4次)可以获得不同离心程度的破碎益生菌上清成分和破碎益生菌沉淀组分成分。离心后倾出上清液另用，所剩沉淀组分经干燥处理(例如125℃，90分钟)后制备为破碎益生菌沉淀组分干粉。原料益生菌(例如10g酿酒酵母菌)和以此制备的破碎益生菌沉淀组分干粉(例如4g)之差被计作破碎益生菌上清成分干重(例如6g)。
A17-A19的制备方法：按所需(例如局部化学作用所需)浓度量取益生菌水不溶组分颗粒(例如按实施例1a方法制备的破碎酿酒酵母菌干粉1.4g)、任选存在的其他组分、以及定容至总体积(例如100ml)的液体载体(例如注射用水)，将它们缓慢混合均匀。如果按所需药物体积/靶区体积比(例如临床上常见的实体肿瘤的平均体积为的30cm 3)对该液体混合物进行分装(例如10ml/瓶)和封盖，即可获得可提供瘤内局部作用的药物组合物剂型和规格。所获制备物为A17。使用与A17制备相同的方法，可分别从不同益生菌进行不同破碎益生菌沉淀组分(例如上表中制备物A17和A18)和从商业途径得到的水不溶β-葡聚糖微粒(例如上表中制备物A19)的制备。
A20-A21的制备方法：按所需(例如局部化学作用所需)浓度量取益生菌可生成半流体类组分(例如10g水不溶β-葡聚糖微粒)、任选存在的其他组分、以及定容至总体积(例如100ml)的液体载体(例如注射用水)，将它们缓慢混合均匀为悬浊液，然后将该悬浊液加热(例如温度50-110℃和时间0.5-24h)，冷却后可形成半流体。如果按所需药物体积/靶区体积比(例如临床上常见的实体肿瘤的平均体积为的30cm 3)对该半流体进行分装(例如10ml/瓶)和封盖，即可获得可提供瘤内局部作用的药物组合物剂型和规格。所获制备物为A16。使用与A16制备相同的方法，可分别从不同益生菌可半流体化组分进行不同半流体组合物的制备。实验显示，只有当益生菌可半流化组分的浓度大于某个阀值(例如β-葡聚糖≥2.5％)，方可使得包含它的液体在加温后转化为半流体类。
A22-A24的制备方法：按所需(例如局部化学作用所需)浓度量取益生菌水溶性组分(例如按A17-A18方法制备的破碎酿酒酵母菌上清成分1.1g或等量上请液20ml)、任选存在的其他组分、以及定容至总体积(例如100ml)的液体载体(例如注射用水)，将它们缓慢混合均匀。如果按所需药物体积/靶区体积比(例如临床上常见的实体肿瘤的平均体积为的30cm 3)对该液体混合物进行分装(例如10ml/瓶)和封盖， 即可获得可提供瘤内局部作用的药物组合物剂型和规格。所获制备物为A22。使用与A12制备相同的方法，可分别从不同益生菌进行不同组合物(例如上表中制备物A22-A24)的制备。
A25-A31的制备方法：按所需(例如局部化学作用所需)浓度量取益生菌水溶性组分(例如从商业途径得到的水溶性酵母β-葡聚糖5g)、任选存在的其他组分、以及定容至总体积(例如100ml)的液体载体(例如注射用水)，将它们缓慢混合均匀。如果按所需药物体积/靶区体积比(例如临床上常见的实体肿瘤的平均体积为的30cm 3)对该液体混合物进行分装(例如10ml/瓶)和封盖，即可获得可提供瘤内局部作用的药物组合物剂型和规格。所获制备物为A26。使用与A26制备相同的方法，可分别从商业途径得到的不同益生菌组分制备不同组合物(例如上表中制备物A25-A32)。
使用上述制备方法获得的组合物水剂，可按现有技术的方法加入化学活性成分，制备出包含益生菌组分*和化学活性成分的组合物水剂。
表3包含益生菌组分*和化学活性成分的组合物水剂的组分、配比
Figure PCTCN2021122132-appb-000003
Figure PCTCN2021122132-appb-000004
*：益生菌组分的类型及其浓度为表2中的类型及其浓度
以下列出表3中几个制备物的制备试验例子。
包含益生菌组分(水溶性组分1-3)和化学活性成分的组合物水剂(B1、B5-B7、B9-B18、B25、B26、B29-B32)的制备方法：按所需(例如局部协同作用或/和中长期协同作用所需)协同量比和协同浓度量取益生菌水溶性组分(例如水溶性酵母β-葡聚糖10g)、共用药(例如20g精氨酸)、任选存在的其它成分、以及定容至总体积100ml的注射用水，并将它们缓慢混合均匀。如果按所需药物体积/靶区体积比(例如临床上常见的实体肿瘤的平均体积为的30cm 3)对该液体混合物进行分装(例如10ml/瓶)和封盖，即可获得可提供瘤内局部作用的药物组合物剂型和规格。所获制备物为B1。使用与B1制备相同的方法，可分别从不同益生菌水溶性组分及其共用药进行不同组合物(例如上表中制备物B1、B5-B7、B9-B18、B25、B26、B29-B32)的制备。
包含益生菌组分(水不溶颗粒/水溶性组分)和化学活性成分的组合物水剂(B2-B4、B8)的制备方法：按所需(例如局部协同作用或/和中长期协同作用所需)协同量比和协同浓度量取益生菌(例如1.5g酿酒酵母菌)、共用药(例如20g耐氨酸)、任选存在的其它成分、以及定容至总体积100ml的注射用水，并使用与A10制备相同的方法将它们缓慢混合均匀并使用匀浆机对其进行破碎并获得破碎益生菌/共用药 悬浊液。如果按所需药物体积/靶区体积比(例如临床上常见的实体肿瘤的平均体积为的30cm 3)对该悬浊液进行分装(例如10ml/瓶)和封盖，即可获得可提供瘤内局部作用的药物组合物剂型和规格。所获制备物为B2。使用与B2制备相同的方法，可分别从不同益生菌或/和不同共用药进行不同组合物(例如上表中制备物B2-B4、B8)的制备。
包含益生菌组分(可半流体化组分)和化学活性成分的组合物水剂(B19-B22)的制备方法：按所需(例如局部协同作用或/和中长期协同作用所需)协同量比和协同浓度量取益生菌可半流体化组分(例如7.5g水不溶β-葡聚糖)、共用药(例如1g5-氟尿嘧啶)、任选存在的其他组分、以及定容至总体积(例如100ml)的液体载体(例如注射用水)，将它们缓慢混合均匀为悬浊液，然后将该悬浊液加热(例如温度50-110℃和时间0.5-24小时)，冷却后可形成半流体。如果按所需药物体积/靶区体积比(例如临床上常见的实体肿瘤的平均体积为的30cm 3)对该半流体进行分装(例如10ml/瓶)和封盖，即可获得可提供瘤内局部作用的药物组合物剂型和规格。所获制备物为B19。使用与B17制备相同的方法，可进行不同半流体组合物(例如上表中B20-B22)的制备。
包含益生菌组分(水不溶组分颗粒)和化学活性成分的组合物水剂(B23)的制备方法：按所需(例如局部协同作用或/和中长期协同作用所需)协同量比和协同浓度量取益生菌水不溶组分颗粒(例如A19中的水不溶性β-葡聚糖微粒10g)、共用药(例如10g还原型谷胱甘肽、1g5-氟尿嘧啶、1g氢氧化钠)、任选存在的其它成分、以及定容至总体积100ml的注射用水，并将它们缓慢混合均匀。如果按所需药物体积/靶区体积比(例如临床上常见的实体肿瘤的平均体积为的30cm 3)对该液体混合物进行分装(例如10ml/瓶)和封盖，即可获得可提供瘤内局部作用的药物组合物剂型和规格。所获制备物为B23。使用与B6制备相同的方法，可分别从不同益生菌组分(益生菌沉淀组分等水不溶颗粒)和不同共用药进行不同组合物的制备。
包含益生菌组分(灭活益生菌)和化学活性成分的组合物水剂(B24、B27、B28)的制备方法：按所需(例如局部协同作用或/和中长期协同作用所需)协同量比和协同浓度量取益生菌(例如2.5g酿酒酵母菌干粉)、其它成分(例如1g亚甲蓝、1g5-氟尿嘧啶)、以及定容至总体积100ml的注射用水，并将它们缓慢混合均匀获得益生菌/共用药混合液。如果按所需药物体积/靶区体积比(例如临床上常见的实体肿瘤的平均体积为的30cm 3)对该液体混合物进行分装(例如10ml/瓶)和封盖，即可获得可提供瘤内局部作用的药物组合物剂型和规格。将该制备物(例如2.5％酿酒酵母菌)放入巴斯德灭活櫃式机中进行巴斯德灭活(60℃，48小时)，即可获得B24。使用与B24制备相同的方法，可分别从不同益生菌进行不同灭活益生菌(例如上表中制备物B27、B28)的制备。
如果将上述未灭活制备物(例如A1-B31、B1-B32)放入巴斯德灭活櫃式机中进行巴斯德灭菌(60℃，48小时)，即可获得灭菌液体注射剂。
需要说明的是，A1-16、B1-B19的制备中，所述灭菌采用60℃，48小时进行巴斯德灭菌，得到灭菌液体注射剂。
若制备冻干粉制剂时，则分别将上述液体制剂进行冷冻干燥，所述冷冻干燥的工艺条件为：-45℃保持4h进行预冻，以0.1℃/分钟的升温速率升至-15℃时至少保持10h进行升华；升温至30℃，6h保持进行解吸附干燥，即得。按需对注射用水进行分装如7.5ml/瓶、封盖，即得注射用溶媒瓶。使用时， 将瓶中的无菌溶媒抽入上述注射用冻干粉瓶中混合均匀形成液体药物，即注射药物，如1.5％酿酒酵母菌破碎组分/20％氨基酸。
实施例2免疫抑制动物模型中的药理研究
荷瘤裸小鼠被广泛用于实体瘤患者化疗而非免疫治疗的药物研究，以裸小鼠为实验对象，采用人肝癌HepG2细胞为建模细胞，将1×10 5个细胞/只注入右侧腋部皮下进行移植瘤建模，建模成功裸小鼠的瘤体平均体积为161.3mm 3，将模型动物随机分为17组，按表4的药物、用药方式给药，每组均用药1次，每次注射量150μl/只。用药后第7日，对动物进行安乐死，解剖后剥离出肿瘤组织测定瘤重，并根据阴性对照组计算抑瘤率(r)。
表4不同组别的抑瘤率数据
Figure PCTCN2021122132-appb-000005
Figure PCTCN2021122132-appb-000006
通常认为，5-氟尿嘧啶静脉注射可提供常规化学作用，而通过瘤内注射可提供普通的局部化学作用，两种给药方式引起的药效差异与细胞毒动力学相关。组7与组1相比，其抑瘤率之比E 7/E 1＜200％，说明5-氟尿嘧啶在不同给药条件(全身性用药vs局部用药)下均作为细胞毒药物发挥作用。高浓度(75-99％)乙醇通过静脉注射可提供醉酒样反应；而通过瘤内注射可提供化学消融作用。组8与组2相比，其抑瘤率提高远远超过基于后者的动力学提高预期极限(E 8/E 2＞200％)，说明乙醇在不同给药条件(全身性用药vs局部用药)下可作为不同的活性成分(醉酒剂vs化学消融剂)应用。
白细胞介素-12通常在临床上作为免疫增强剂使用，其通过瘤内注射(组9)合乎预期地未显示短期抑瘤效果。而现有技术中益生菌及其组分也可作为辅助药物,其抗肿瘤作用也是基于其免疫增強作用，组别4-6的结果与组别3一致。值得注意的是,组别4、5在注射后均有1只动物死亡，说明5％益生菌组分的悬浊液通过静注存在安全性风险。
根据现有技术的免疫增强预期,组别10的结果应当与免疫增强对照组9一致。出人意料的是，它们之间的药理行为明显不同:相同药物相同剂量的组9与组3几无药效差异；而相同药物相同剂量的组10与组4的药效差异超过其动力学提高预期极限(E 10/E 9＞200％),这也体现为它们之间的药效明显不同(E 10/E 9＝459％＞200％)。相反,组10与药理预期之外的组7、组8的抑瘤率差异却不大(例如E 7/E 10＝133％<200％),而且组10与组8的药理行为也明显一致:相同药物相同剂量的组8与组2的抑瘤率差异巨大(E 10/E 9＞200％)。而组别11、12存在与组10相同的药效和药理行为。
实际上，只有通过肠道内给药才能使益生菌组分起到调节肠道内细菌群作用；而肠道内、外给药均可使益生菌组分起到免疫增强的作用。因此，当排除口服、静注等常规给药方式，而通过局部，尤其是病变内给药，才能使益生菌组分提供区别于现有技术的新功能(E 10/E 4＞＞200％)。
益生菌及其组分的免疫增强作用基于其细菌免疫原性。通常认为的细菌免疫原性由强减弱的次序为:活菌、基本保持活菌形状的灭活菌、多少有一些类似活菌形状的水不溶颗粒、失去活菌形状的半流体、水溶性成分。然而,局部给药益生菌及其组分的各组的药效由强减弱的次序却为:半流体类组分(组13)、并不要求基本保持活菌形状的灭活益生菌(不完整菌体的量比>20％的灭活益生菌,组10)、水不溶组分/水溶性组分混合物(组14)、水溶性组分(组12)、水不溶组分(尽管其与半流体类组分的化学组成相同)(组11)、活菌(组15)。这进一步说明益生菌组分新功能并不依赖于其在现在技术中的功能(如细菌免疫原性)。因此，益生菌组分在药物组合物中的浓度可用重量浓度而非单位体积内的完整菌体数目来表示。
药理功能是药物的根本特征，老药物发现的新药理通常可创造性地产生与新药物一样的新应用。药物的有效应用通常取决于在临床治疗限定的实验条件下，如最小化给药频次和剂量下的有效性和安全性，提供的具有相对优势的药理功能，也就是优选的药理作用。同一个物质于是在不同条件下有可能被用作不同活性成分,而同一类物质更是可能有适于用作不同活性成分的不同优选方案。在上述实验中，各阳性对照物(5-氟尿嘧啶、乙醇、白细胞介素-12)均显示合乎其在不同药理方法中的优选药理(分别为细胞 毒药理、免疫增强药理、局部作用药理)预期的药效和药理行为，并给出了一个药理比较研究系统。益生菌组分在静注时显示的药效或许还合乎其在现有技术中的药理(例如免疫增强)预期,但其在病变内给药产生的药效则显然远远超过了该预期。其药理行为甚至也超过细胞毒药理(假如有的话)预期。其在不同给药方式中显示的药理差异导致巨大的药效差异,这种药理行非常类似于局部作用阳性对照物乙醇，也说明其在病变内给药时的优选药理为局部作用。总之，益生菌组分作为提供局部作用的活性成分(局部活性成分)在药理、疗效上大大超出了其作为提供任何非局部作用(如细胞毒作用或免疫增强作用)的活性组分(非局部活性成分)的预期。
为深入研究上述新功能，申请人进行以动物正常组织为局部病变模型(该模型为化学消融剂的经典研究模型)的下述对比实验——以裸小鼠为实验动物，随机分成A、B两组，每组6只，分别以75％乙醇水溶液、5％灭活益生菌为实验药物，注射至右腿外侧肌肉块内，注射量100ul/只；用药后第7日对动物进行安乐死，解剖取出裸小鼠右腿外侧肌肉块标本，切片洗涤后测量区别于正常肌肉的异常区域面积，如坏死、结节等。结果显示：A组、B组的异常区域面积分别为35.14±15.92mm 2和34.12±15.81mm 2，且两者之间无统计学差异(P＞0.05)。这些结果进一步证实了益生菌组分类似于乙醇的局部作用药理。如同乙醇,益生菌组分所提供的该局部作用使得它可以被用于包含病变组织的任何局部病变。
实施例3局部作用的药理研究及其药理浓度优化
以裸小鼠为实验对象，采用乳腺癌细胞(MDA-MB231)为建模细胞，将1×10 5个细胞/只注入右侧腋部皮下进行移植瘤建模，建模成功裸小鼠的瘤体平均体积为158.2mm 3，将模型动物随机分为16组，按表5的药物组份、剂量进行瘤内注射给药，每组均用药1次。用药后第7日，对动物进行安乐死，解剖后剥离出肿瘤组织测定瘤重，并根据阴性对照组计算抑瘤率(r)。
表5不同药物组分、剂量对抑瘤率的影响
Figure PCTCN2021122132-appb-000007
Figure PCTCN2021122132-appb-000008
*:上清组分为破碎布拉氏酵母菌上清组分；**:灭活菌为热灭活布拉氏酵母菌；***；微粒为酿酒酵母β-葡聚糖微粒；****:破碎菌为破碎布拉氏酵母菌
由表5可知，组别01的技术方案中乙醇未提供化学消融活性，仅类似于静注乙醇；组别02、03的抑瘤率接近(E 03/E 02＜200％)，但远高于组别01(E 02/E 01＞200％)，显示出预料中的化学消融；说明只有乙醇浓度高于某一阀值(如70％)才能作为化学消融剂使用。
组别1-3是将相同剂量不同浓度的益生菌组分注射入相同体积瘤体，其中组3的抑瘤率与组02相差不大(E 3/E 02＝79％)，说明其显示出类似局部作用。组别5、8、11存在与组02相似的局部作用，而组3、6、9、12的抑瘤率数据说明该局部作用存在给药浓度依赖性。
组别1、2虽然为相同剂量的瘤内给药，不同给药浓度的益生菌组分的药效差异超过了动力学差异预期(E 2/E 1＞200％)；因此本申请的药物组合物中益生菌组分的给药浓度是局部作用所需的药理浓度，而非兔疫增强所需。
益生菌组分提供局部作用的一个必要条件是：益生菌组分在药物组合物中的含量须使其药理浓度(局部给药浓度)＞0.1％、≥0.25％、0.25-25％，优选为0.5-15％，更优选为1-15％或5-15％。其中当所述益生菌组分为灭活益生菌，则其药理浓度＞0.3％、≥0.75％、0.75-15％，优选为1.5-15％或5-15％；当所述益生菌组分为益生菌水溶性组分，则其药理浓度＞0.1％，如0.15-25％、优选为0.35-25％或5-25％(其中,水溶性组分为益生菌上清组分则0.35-3.5％、为水溶性益生菌源聚多糖则2-5％或5-15％、为益生菌源核糖核酸则15-25％)；当所述益生菌组分为益生菌水不溶组分颗粒，则其药理浓度为＞0.5％或0.5-15％、优选为1.5-15％或5-15％；当所述益生菌组分为益生菌半流体类组分，则其药理浓度为＞2.5％、2.6-25％、优选为5-15％。
同一个物质用作不同活性成分有可能需要满足不同药理动力学特征。上述药效的给药浓度依赖性比较研究结果进一步证实本发明所述益生菌组分的局部活性类似于化学消融，而与常规活性(癌细胞抑制、肿瘤血管抑制、或免疫增强等)相去甚远。本申请所述作为局部活性成分的益生菌组分的药理浓度,完全不能简单地与现有技术中作为非局部活性成分的益生菌组分的制剂浓度或给药浓度进行比较。非局部活性药物的制剂浓度往往仅受制剂学限制，如高浓度制剂可节约运输、储存成本，适当高浓度的注射可减小注射体积以缩短给药时间。众所周知，制剂浓度可较大范围，但其在本质上不同于药理作用所需的给药浓度。例如静注时通过稀释使给药浓度远低于制剂浓度，以避免药物快速进入血液产生安全性风险。本发明组合物中的药理浓度不仅作为一个特征限制了其组成和制备，且其还必须作为药理条件出现 在新药报批中，也必须作为应用条件出现在药物的使用说明书之中。
实施例4：局部作用药理研究及其药理体积优化
不同药理的药物组合物的组分含量通常需要用不同的特征来限定，通常常规的药物组合物在给药剂量外并无病变靶区体积依赖性的药理体积，以下实验却对此研究。
以裸小鼠为实验对象，采用人胰腺癌细胞(PANC-1)为建模细胞，将1×10 5个细胞/只注入右侧腋部皮下进行移植瘤建模，建模成功裸小鼠的瘤体平均体积为213.1mm 3，将模型动物随机分为16组，按表6的药物组份、给药体积进行瘤内注射给药，每组均用药1次。其中组别1-12的药物均采用实施例1的方法制备，其中的灭活益生菌为热灭活布拉氏酵母菌、益生菌水不溶组分颗粒为β-葡聚糖颗粒、益生菌水溶性组分为破碎布拉氏酵母菌上清组分、益生菌水不溶组分颗粒和水溶性组分混合物(简记为益生菌组分混合物)为破碎布拉氏酵母菌，制备方法同实施例3；用药后第7日，对动物进行安乐死，解剖后剥离出肿瘤组织测定瘤重，并根据阴性对照组计算抑瘤率(r)。
表6不同注射浓度、体积的抑瘤率数据
由表6可知，组别01-03将不同给药体积/靶区体积比的乙醇注射入相同体积瘤体，其中组01中的
Figure PCTCN2021122132-appb-000009
乙醇未能提供化学消融作用，类似于静注乙醇；而组02的抑瘤率与组01差异极大(E 02/E 01＞200％)、 而组02的抑瘤率与组03的相差不大(E 03/E 02＜200％)，显示出预料中的化学消融。由此表明，只有乙醇超过某一给药体积/瘤体体积比阀值，如0.15，才能作为化学消融剂应用。
组别1-3将相同剂量不同给药体积/靶区体积比的的益生菌组分注射入相同体积瘤体，其中组别2的抑瘤率与组别02的抑瘤率相近(E 3/E 02＝105％)，显示出与之类似的局部作用；而组别5、8、11也存在类似于组别02的局部作用，而组别3、6、9、12则进一步说明该局部作用对靶区体积的相关性和对给药体积的依赖性。综上，即使是瘤内给药的浓度较高的药物组合物，相同剂量但不同给药体积/靶区体积比的益生菌组分的药效差异可远超动力学差异预期(如E 2/E 1＞200％))，此时益生菌组分的给药体积(给药体积/靶区体积比)已经不是药效动力学问题，而是药理问题。
优选的，益生菌组分在药物组合物中的含量使给药体积/靶区体积比＞0.09、0.1-1.5，优选为0.23-1.5或0.5-1.5。临床上尽管很多瘤体体积≥30cm 3，但基于免疫增强药理的药物在瘤区内的给药体积普遍很低(如细胞因子，体积≤2ml)；这类药物制剂的规格体积通常不大，如注射液瓶、或粉针剂瓶。然而本申请所述的药物组合物只能在满足上述给药体积/靶区体积比的条件下给药，如肿瘤靶区体积为≥30cm 3、给药体积/靶区体积比为0.2，则所需给药体积≥6.0cm 3，此时药物制剂的规格体积为6ml或整数倍数。众所周知，实质上药物规格也可以是活性成分实现所需药理作用的常用所需含量的形式之一，如每片包含不同含量阿司匹林的“拜阿司匹灵”有不同的适应症范围。
本申请所述作为局部活性成分的益生菌组分的上述根据病变体积的给药体积的优选方案超过益生菌组分现有技术的预期。具体的，现有技术中用作非局部活性成分(如免疫增强活性成分)的益生菌组分也许可以辅助治疗、却不能作为主药治疗局部病变疾病,犹其是可降低局部病变体积的局部病变治疗,因而其给药体积并不依据局部病变体积。此外,根据以上实施例2中的实验及其它类似实验的结果,相同浓度药物在病变内给药与静注给药比较,其安全性体积(例如出现死亡的给药体积)可以高出2倍以上。因而,本申请所述作为局部活性成分的益生菌组分特别适用于较大(例如平均直径大于3cm)病变。
上述药效的给药体积依赖性比较研究结果进一步证实本发明所述益生菌组分的局部活性类似于化学消融，而与常规活性(癌细胞抑制、肿瘤血管抑制、或免疫增强等)相去甚远。现有技术中作为非局部活性成分的益生菌组分的药理动力学特征为血药浓度(通常非常低，如0.25×10 -5％)，给药体积仅与血药浓度所需剂量有关,而与病变靶区体积无关。由于该剂量与全身性安全性相关,给药体积于是广泛地从药理动力学特征中淡化,而由医生根据病人体重等状态决定。本申请所述作为局部活性成分的益生菌组分的药理体积是一个重要的药理动力学特征(涉及有效渗透区与无药区之间的药理关系及后果),其不仅限制了其组成(例如单位制剂体积)和制备，且其还必须作为药理条件出现在新药报批中，也必须作为应用条件出现在药物的使用说明书之中。本申请所述作为局部活性成分的益生菌组分的药理体积,完全不能简单地与现有技术中作为非局部活性成分的益生菌组分的单位制剂体积或给药体积进行比较。
根据上述实施例2-4以及更多的类似研究的结果，包含益生菌组分的本发明局部药物组合物(本发明组合物)在以下方面超过包含益生菌组分的现有技术中的非局部药物组合物(现有技术组合物,例如可用于口服或静注的益生菌组分)的预期：
1)、其基础药理超预期:本发明组合物中的益生菌组分作为局部活性组分是基于其新药理-药物渗透区组织破坏作用-即局部作用的发现，该局部作用主要包括局部化学作用(例如类化学消融)，超过了现 有技术益生菌组分的任何非局部活性(例如免疫作用、抗病毒作用、肿瘤细胞毒作用、或瘤体血管破坏作用等)的药理预期；
2)、其药理方法超预期:本发明组合物中的益生菌组分必须严格限定于局部给药才能进入靶区反应器实现其新功能，而现有技术组合物中的益生菌组分的药理方法则不限于局部给药、优选为全身性给药去实现其目标功能；
3)、其活性成分选择方案超预期:现有技术免疫增强组合物中的益生菌组分优选为细菌免疫作用较强组分(如活菌和在形貌上接近度尽可能高的组分,如基本保留活菌形态的灭活益生菌),而本发明组合物优选细菌免疫作用最小化组分(与活菌形貌上接近度尽可能低的组分,如益生菌水溶性组分、益生菌半流体组分)。
4)、其药理动力学方案超预期:现有技术组合物中的益生菌组分实现其全身性作用(药理浓度为血药浓度)的动力学条件依赖于给药剂量而非给药浓度、更谈不上制剂浓度(为制备和储运方便通常制备为浓缩剂),为避免出现例如注射部位的局部作用损伤还将药剂稀释给药。然而本发明组合物中的益生菌组分实现其局部作用的动力学条件则相反地依赖于给药浓度而非给药剂量(血药浓度),局部给药浓度即其药理浓度,而且往往也是其制剂浓度。此外,现有技术组合物给药体积仅与其全身性药理所需血药浓度的剂量有关,而与病变靶区体积无关,而本发明组合物给药体积则与其局部活性所需渗透的靶区体积有关；
5)、其药理反应所需的时间超预期:现有技术组合物的全身性活性必须在靶区进行长时间高频次(例如3个月内数十次)的药理反应来实现,相应地必须高频次和长时间供药,而本发明组合物的局部活性类似于化学消融,其在给药区域内仅需少数次(例如3个月内≦3次)药理反应即可实现。这也为剂型(例如数十针剂vs数针剂)和制备方案带来很大的不同；
6)、其药理反应所需的反应环境要求超预期:本发明组合物严格限定于局部给药剂型的相应组成限定(不得包含某些用于全身型给药剂型的辅料、给药时组合物需为充分混合物从而要求统一的溶媒、等等)、等等；
7)、其技术效果超预期:首先，对局部病变的治疗效果超预期。例如至少在给药区域内获得可与公认的有效药物相比、大大超过超过现有技术组合物短期预期(例如多次给药时第一次给药后21天、或最后一次给药后7天)的药物效应(r 本发明组合物/r 现有技术组合物＞200％，优选r 本发明组合物/r 现有技术组合物＞400％)。该药效特点也导致其适应症范围超预期,例如，上述动物模型所能代表的免疫缺陷患者、老龄患者、经多种治疗后免疫能力下降的患者。这些也进一步显示两种组合物之间的差异并非动力学差异而是药理差异(例如局部作用vs常规作用)。
实施例5：免疫抑制动物模型中的共用药理研究及技术方案优选
以裸小鼠为实验对象，采用肺肿瘤细胞(A549)为建模细胞，将1×10 5个细胞/只注入右侧腋部皮下进行移植瘤建模，建模成功裸小鼠的瘤体平均体积为153.7mm 3，将模型动物随机分为18组，按表7的药物组份、给药方式进行给药，每组均用药1次，注射量为150μl/只。其中益生菌组分为热灭活布拉氐酵母菌，组别16的用药方式为瘤内注射益生菌组分约2h后再瘤内注射10％精氨酸；用药后第7日，对动物进行安乐死，解剖后剥离出肿瘤组织测定瘤重，并根据阴性对照组计算抑瘤率(r),还可根据疗效(抑瘤率)计算药效实际/预期比q。
表7不同组分、给药方式对抑瘤率的影响
Figure PCTCN2021122132-appb-000010
由上表可知，组别5、6、7的实际共用作用/理论单纯相加预期作用比q均不大于1.00，说明此时益生菌组分与化学活性物质在静注条件下共用未能产生任何协同作用。
相同益生菌组分/化学活性物质(乙醇)混合物不同给药方式的组别12显示出远远高于组别5的药效,但它们的实际共用作用/理论单纯相加预期作用比q均不大于1.00,该高药效主要是因为乙醇的局部作用(组别8)。按此预期,相同益生菌组分与其它化学活性物质的共用也应有类似药理行为。然而,组别13和14却分别显示出明显高于组别6和7(分别使用相同益生菌组分/化学活性物质混合物)的药效,说明 它们的较高药效直接与它们的局部作用相关。而且组别13和14的实际共用作用/理论单纯相加预期作用比q均大于1.00,说明该局部作用为益生菌组分和这些化学活性物质的局部协同作用。
进一步的，组别15的药效最高，且其实际共用作用/理论单纯相加预期作用比q＞1.00,说明三组分组合物(益生菌组分/弱局部作用化合物/细胞毒药物)居然还可以在其二组分(益生菌组分/弱局部作用化合物和益生菌组分//细胞毒药物)协同作用的基础上进一步产生协同作用。最后，组别16的药物组合物尽管与组别14的成分相同但却不在同一个药剂之中，其实际共用作用/理论单纯相加预期作用比q＜1.00,未显示出协同作用。
实验中观察发现，组别11、12中大部分实验动物在注射后产生较强烈反应，说明益生菌组分单药或非协同药物组合物应在止痛条件下使用以避免安全性风险；然而组别13-15则未观察到上述强刺激反应，说明，益生菌组分与弱局部作用化合物和/或细胞毒药物的协同药物组合物具有超单药效应预期的安全性。其它益生菌组分，如益生菌半流体类组分、益生菌水溶性组分、益生菌水不溶组分颗粒等与化学药物的共用也存在相同的结果，限于篇幅在此不进行详述。
申请人对益生菌组分的组合物，尤其是三组分形成的组合物进行深入研究。
以裸小鼠为实验对象，采用胃肿瘤细胞(BGC823)为建模细胞，将1×10 5个细胞/只注入右侧腋部皮下进行移植瘤建模，建模成功裸小鼠的瘤体平均体积为164.4mm 3，将模型动物随机分为14组，按表8的药物组份进行瘤内注射给药，每组均用药1次，注射量为150μl/只；其中组别5、11的药物组合物为半流体，其余组别均为液体。用药后第7日，对动物进行安乐死，解剖后剥离出肿瘤组织测定瘤重，并根据阴性对照组计算抑瘤率(r),还可根据疗效(抑瘤率)计算药效实际/预期比q。
表8不同药物组合的抑瘤结果
Figure PCTCN2021122132-appb-000011
Figure PCTCN2021122132-appb-000012
经瘤内注射时，组别8-13的实际共用作用/理论单纯相加预期作用比q均大于1.00，都显示出明显的协同作用。申请人对该新功能的药物效应进行进一步研究，以裸小鼠为实验对象，随机分为3组，每组6只小鼠，分别以75％乙醇水溶液、5％热灭活布拉氏酵母菌/1％亚甲蓝、半流体组合物中的7.5％β-葡聚糖/1％亚甲蓝/1％5-氟尿嘧啶作为研究药物，各组均用药1次，给药至右腿外侧肌肉块内，注射量100ul/只。用药后第7日对动物进行安乐死，解剖取出裸小鼠右腿外侧肌肉块标本，并进行大体病理分析，切片测量区别于正常肌肉的异常区域面积，如坏死、结节等。结果表明：组别1、2、3的异常区域面积分别为32.24±13.71mm 2、46.78±13.64mm 2和72.35±23.71mm 2，说明益生菌组分所提供的的局部协同作用类似但强于乙醇，其主要是局部化学协同作用。
实施例6：非瘤局部病变模型中的药理研究及优化
以裸小鼠为实验对象，采用小鼠胚胎成纤维细胞3T3为建模细胞，将2×10 4个细胞/只注入右侧腋部皮下进行移植结节建模，建模成功裸小鼠的类瘤结节体平均体积为171.5mm 3，将模型动物随机分为16组，按表9的药物进行给药，除组别21-24外各组别分别进行腹腔注射、结节内注射给药，在组别21-24中所用药物为非局部给药剂型(益生菌组分和共用物可分别给药的剂型)的组合物,系列A的给药方式为：腹腔注射益生菌组分+结节内注射共用物，系列B的给药方式为：腹腔注射共用物+结节内注射益生菌组分。各组均用药2次，每次间隔2天，注射量为150μl/只/次，末次给药后第7天测量包含成纤维细胞的局部病变体积(V)，并根据阴性对照组分别计算系列A和系列B的相对局部病变抑制率(r’ A、r’ B),还可根据疗效(局部病变抑制率)计算药效实际/预期比q。
表9不同药物、给药方式的相对局部病变抑制率数据
Figure PCTCN2021122132-appb-000013
Figure PCTCN2021122132-appb-000014
本实施例中组别02中5-氟尿嘧啶以针对肿瘤瘤体有效的剂量给药作为细胞毒阳性对照物；组别02中香菇多糖注射液以10倍的临床最大剂量给药作为免疫增强对照物，组别04中无水乙醇作为非特异性细胞毒阳性/局部作用阳性对照物，所述无水乙醇在非临床条件下显示非特异性细胞毒阳性，而在临床条件下只显示局部作用阳性，所述临床条件是指临床安全性和顺应性上可接受的常规条件，如一个疗程(40天)中注射次数小于10次，而非临床条件是指远离临床安全性和顺应性的常规条件，如细胞试验条件或一个疗程(40天)中注射次数大于10次。
由上表可知，组别03中的药物在系列A、B中的抑瘤率均小于15％，，符合其仅用作辅助治疗药物 起到免疫增强的药理预期。组别04的药物经腹腔注射后的抑瘤率r＜15％，而经瘤内注射的抑瘤率r＞15％，存在一定的治疗作用，也合乎其虽可能在极端条件下显示非特异性细胞毒作用药理、但在通常条件下只显示局部作用药理(E B04/E A04＞200％)的预期。组别02的药物经腹腔注射后的抑瘤率r＜15％，明显小于其在实施例2中的药效，符合细胞毒作用的药理预期，但经瘤内注射后的药效却明显更高(E B02/E A02＞200％，显示出类似于乙醇的局部作用且超出细胞毒作用的药理预期。
组别1-4中的药物经腹腔注射的药效与组别01-04一致，益生菌组分可能存在的免疫增强作用、细胞毒作用或其它经全身给药提供的任何作用均未超出对照药物01-04的药理预期。然而组别1-4中的药物经瘤内注射的药效远高于腹腔注射的药效(E B/E A＞200％)，呈现出明显不同的新药理。实际上组别1-4中药物的药效与对照药物01-04相类似，在常规条件下仅显示局部作用。同样的，组别11、12的药物也显示出与对照药物04相类似的药效和药理。
在组别21-24的组合物经不同给药方式后显示的瘤内注射、腹腔注射的q都不大于1.0，说明非局部给药剂型中的益生菌组分(X)与共用物(Y)以不同方式给药形成的组合物(X+Y)的共用药效并未超过其单用药效的加和预期，二者共用时不存在协同作用；而在组别31-34中，当使用相同组成物(X、Y)的局部给药剂型(混合共用药物)经腹腔注射后q均都不大于1.0，而经病变内注射后q均大于1.00，显示出局部协同作用。
实施例7：局部协同作用药理研究及量比优化
以裸小鼠为实验对象，采用人肝癌细胞(HepG2)为建模细胞，将1×10 5个细胞/只注入右侧腋部皮下进行移植瘤建模，建模成功裸小鼠的瘤体平均体积为155.7mm 3，将模型动物随机分为13组，按表10的药物进行给药，相关药物均为含水液体，按实施例1的方法配置而成；各组均用药1次，注射量为150μl/只/次，给药后第7天对动物进行安乐死，解剖后剥离出肿瘤组织测定瘤重，并根据阴性对照组计算抑瘤率,还可根据疗效(抑瘤率)计算药效实际/预期比q。
表10不同组别的瘤重、抑瘤率数据
组号 研究药物 瘤重(x±s_g) 抑瘤率
01 (生理盐水) 0.261±0.098 -
1 0.2％益生菌组分 0.231±0.113 11.5％
2 0.5％益生菌组分 0.208±0.109 20.3％
3 10％益生菌组分 0.149±0.091 43.1％
4 20％益生菌组分 0.127±0.085 51.4％
5 10％碳酸氢钠/4.3％氢氧化钠 0.032±0.021 87.6％
6 7％碳酸氢钠/3％氢氧化钠 0.064±0.036 75.4％
7 0.7％碳酸氢钠/0.3％氢氧化钠 0.175±0.088 33.1％
8 0.2％碳酸氢钠/0.09％氢氧化钠 0.228±0.104 12.5％
9 0.2％益生菌组分/10％碳酸氢钠/4.3％氢氧化钠 0.038±0.044 85.6％
10 0.5％益生菌组分/7％碳酸氢钠/3％氢氧化钠 0.013±0.012 95.1％
11 10％益生菌组分/0.7％碳酸氢钠/0.3％氢氧化钠 0.048±0.018 81.7％
12 20％益生菌组分/0.2％碳酸氢钠/0.09％氢氧化钠 0.096±0.079 63.4％
碳酸氢钠-氢氧化钠缓冲系统使单独氢氧化钠的溶液由pH＞12.5降至11左右，通常认为pH降低不利于局部化学作用。由上表可知，组别9的实际共用作用/理论单纯相加预期作用比q＜1.00,其中的药物组合物未显示出协同作用，而组别10、11、12中的药物组合物的pH分别为pH10±1.0或pH4±1.0，它们的实际共用作用/理论单纯相加预期作用比q均大于1.00,均显示出协同作用，其协同量比(W 益生菌 组分/W 共用物)为(0.2-20)/(0.29-14.3)。
以裸小鼠为实验对象，采用人肝癌细胞(HepG2)为建模细胞，将1×10 6个细胞/只注入右侧腋部皮下进行移植瘤建模，建模成功裸小鼠的瘤体平均体积为163.2mm 3，将模型动物随机分为13组，按表11的药物进行给药，相关药物均为含水液体，按实施例1的方法配置而成，其中益生菌组分为灭活釀酒酵母菌，弱局部作用化合物为亚甲蓝；各组均用药2次，用药间隔为7天，注射量为150μl/只/次，末次给药后第7天对动物进行安乐死，解剖后剥离出肿瘤组织测定瘤重，并根据阴性对照组计算抑瘤率。
表11不同组别的瘤重、抑瘤率数据
组号 研究药物 瘤重(x±s_g) 抑瘤率
01 (生理盐水) 0.253±0.117 -
1 0.2％益生菌组分 0.240±0.109 5.3％
2 0.5％益生菌组分 0.207±0.105 18.1％
3 10％益生菌组分 0.155±0.070 38.6％
4 20％益生菌组分 0.144±0.081 43.1％
5 3％亚甲蓝 0.073±0.033 71.1％
6 1％亚甲蓝 0.186±0.096 26.4％
7 0.50％亚甲蓝 0.208±0.115 17.7％
8 0.15％亚甲蓝 0.227±0.107 10.1％
9 0.2％益生菌组分/3％亚甲蓝 0.063±0.026 75.2％
10 0.5％益生菌组分/1％亚甲蓝 0.112±0.094 55.9％
11 10％益生菌组分/0.50％亚甲蓝 0.071±0.031 71.8％
12 20％益生菌组分/0.15％亚甲蓝 0.111±0.062 56.1％
由上表可知，组别9中的组合物均未显示出协同作用(q<1.00)，而组10、11、12中的组合物却显示出协同作用(q分别为>1.00),且其协同量比(W 益生菌组分/W 共用物)为(0.2-20)/(0.15-1.0)，说明益 生菌组分提供局部化学协同作用与弱局部作用化合物提供的局部化学作用强弱无关。
以裸小鼠为实验对象，采用人肝癌细胞(HepG2)为建模细胞，将1×10 5个细胞/只注入右侧腋部皮下进行移植瘤建模，建模成功裸小鼠的瘤体平均体积为168.7mm 3，将模型动物随机分为13组，按表12的药物进行给药，相关药物均为含水液体，按实施例1的方法配置而成，其中益生菌组分为破碎釀酒酵母菌上清组分，弱局部作用化合物为赖氨酸；各组均用药2次，用药间隔为7天，注射量为150μl/只/次，末次给药后第7天对动物进行安乐死，解剖后剥离出肿瘤组织测定瘤重，并根据阴性对照组计算抑瘤率。
表12不同组别的瘤重、抑瘤率数据
组号 研究药物 瘤重(x±s_g) 抑瘤率
01 (生理盐水) 0.238 ±0.191 -
1 0.2％益生菌组分 0.230±0.106 3.2％
2 0.5％益生菌组分 0.199±0.099 16.4％
3 10％益生菌组分 0.126±0.061 47.1％
4 20％益生菌组分 0.111±0.065 53.4％
5 25％赖氨酸 0.109±0.074 54.1％
6 20％赖氨酸 0.134±0.073 43.6％
7 5％赖氨酸 0.211±0.091 11.3％
8 1％赖氨酸 0.226±0.112 5.2％
9 0.2％益生菌组分/25％赖氨酸 0.093±0.020 61.1％
10 0.5％益生菌组分/20％赖氨酸 0.076±0.038 67.9％
11 10％益生菌组分/5％赖氨酸 0.065±0.026 72.6％
12 20％益生菌组分/1％赖氨酸 0.094±0.055 60.3％
由上表可知，组别9中的组合物均未显示出协同作用(q>1.00)，而组10、11、12中的组合物却显示出明显的协同作用(q分别为>1.00)且其协同量比(W 益生菌组分/W 共用物)为(0.2-20)/(1-25)，同样说明益生菌组分提供局部化学协同作用与弱局部作用化合物提供的局部化学作用强弱无关。
以裸小鼠为实验对象，采用人胰腺癌细胞(PANC-1)为建模细胞，将1×10 5个细胞/只注入右侧腋部皮下进行移植瘤建模，建模成功裸小鼠的瘤体平均体积为157.3mm 3，将模型动物随机分为13组，按表13的药物进行给药，相关药物均为含水液体，按实施例1的方法配置而成，其中益生菌组分为破碎釀酒酵母菌上清组分，化疗药物为吉西它滨；各组均用药1次，注射量为100μl/只/次，用药后第7天对动物进行安乐死，解剖后剥离出肿瘤组织测定瘤重，并根据阴性对照组计算抑瘤率。
表13不同组别的瘤重、抑瘤率数据
组号 研究药物 瘤重(x±s_g) 抑瘤率
01 (生理盐水) 0.294±0.189 -
1 0.2％益生菌组分 0.284±0.115 3.3％
2 0.5％益生菌组分 0.278±0.109 5.6％
3 10％益生菌组分 0.161±0.080 45.1％
4 20％益生菌组分 0.140±0.096 52.4％
5 5％吉西它滨 0.191±0.077 35.1％
6 2.5％吉西它滨 0.204±0.103 30.4％
7 0.1％吉西它滨 0.271±0.114 7.8％
8 0.05％吉西它滨 0.278±0.101 5.6％
9 0.2％益生菌组分/5％吉西它滨 0.172±0.062 41.4％
10 0.5％益生菌组分/2.5％吉西它滨 0.107±0.069 63.7％
11 10％益生菌组分/0.1％吉西它滨 0.101±0.070 65.6％
12 20％益生菌组分/0.05％吉西它滨 0.117±0.073 60.2％
同样的，组别9中的组合物均未显示出协同作用(q<1.00)，而组10、11、12中的组合物却显示出明显的协同作用(q分别为>1.00)且其协同量比(W 益生菌组分/W 共用物)为(0.2-20)/(0.05-2.5)，也说明益生菌组分提供局部化学协同作用与弱局部作用化合物提供的局部化学作用强弱无关。
所述药物组合物中益生菌组分协同量比不是兔疫增强协同所需，而是局部协同作用所需，因此是药理量比。优选的，所述益生菌组分与所述化学活性化合物共用时的药理量比(W 益生菌组分/W 化学活性化合物)为(1-110)/(1-100)。若所述化学活性化合物为细胞毒药物时，则(W 益生菌组分/W 细胞毒药物)为(1-110)/(1-100)；若所述化学活性化合物为弱局部作用化合物时，则(W 益生菌组分/W 弱局部作用化合物)为(1-90)/(1-100)；若所述弱局部作用化合物为氨基酸营养素时，则W 益生菌组分/W 氨基酸营养素(1-20)/(1-100)；若所述弱局部作用化合物为活体染料时，则W 益生菌组分/W 活体染料为(7-90)/(1-100)；若所述弱局部作用化合物为酸化剂和/或碱化剂时，则W 益生菌组分/W 酸化剂或/和碱化剂为(2-60)/(1-100)。
实施例8：局部协同作用药理研究及药理浓度优化
以裸小鼠为实验对象，采用人胰腺癌细胞(PANC-1)为建模细胞，将1×10 6个细胞/只注入右侧腋部皮下进行移植瘤建模，建模成功裸小鼠的瘤体平均体积为152.8mm 3，将模型动物随机分为10组，按表14的药物、剂量进行瘤内注射给药，相关药物均为液体制剂，益生菌组分为破碎酿酒酵母菌上清组分，化学活性化合物为精氨酸，按实施例1的方法配置而成；各组均用药1次，用药后第7天对动物进行安乐死，解剖后剥离出肿瘤组织测定瘤重，并根据阴性对照组计算抑瘤率。
表13不同药物、剂量的瘤重、抑瘤率数据
组别 药物 注射剂量μl 瘤重(x±s_g) 抑瘤率
0 生理盐水 100 0.246±0.123 -
1 0.1％益生菌组分 100 0.218±0.096 11.3％
2 0.25％益生菌组分 200 0.189±0.069 23.1％
3 0.5％益生菌组分 500 0.169±0.088 31.4％
4 2％精氨酸 100 0.226±0.104 8.1％
5 5％精氨酸 200 0.210±0.113 14.7％
6 10％精氨酸 500 0.193±0.075 21.4％
7 0.1％益生菌组分/2％精氨酸 100 0.206±0.082 16.3％
8 0.25％益生菌组分/5％精氨酸 200 0.146±0.091 40.7％
9 0.5％益生菌组分/10％精氨酸 500 0.089±0.020 63.9％
由上表可知，组别7-9是将相同组分、量比但浓度不同的益生菌组分/化学活性化合物注射入相同体积瘤体内，其中组别7的药物组合物未显示协同作用(q＜1.00)，而组别8的药物组合物则显示出明显的协同作用(q＞1.00)，组别9的结果(q＞1.00)则进一步表明该协同作用存在给药浓度依赖性。
通常若药物组合物具有相同的活性成分的量比、给药剂量，则其药物共用效应相同；而益生菌组分可通过提供不同共用药理而获得不同共用药效。既使瘤内给药，相同剂量不同给药浓度的益生菌组分的药效差异甚至可以超过动力学差异预期(表13中E 2/E 1＞200％)。
以上试验进一步证实,本发明益生菌组分/共用物组合物通过局部病变内给药获得的短期药效主要来自它们混合共用产生的局部协同作用,其中所述益生菌组分提供上述协同作用的药理浓度为其在以上实施例中提供局部作用的浓度,因而所述益生菌组分与其共用物产生该局部协同作用的药理结构并非它们之间的量比而是它们之间的浓度比(上述协同量比+益生菌组分提供局部作用所须浓度)。
以上实施例中提供该局部协同作用的浓度,亚甲蓝类染料为其产生较弱局部作用的浓度(0.5-1.5％),其它弱局部作用化合物则为其产生较强局部作用的浓度(1-35％),细胞毒药物为其产生最大化普通局部化学作用的浓度(0.1-5％)。
与其局部作用相关，本发明益生菌组分/共用物组合物也优选按实施例4中所述给药体积/靶区体积比给药。
实施例9：普通动物模型中病变区给药的短期单用/共用药理研究及优化
以BALB/c小鼠为实验对象，采用乳腺癌4T1细胞为建模细胞，将0.5×10 6个细胞/只注入右侧腋部皮下进行移植瘤建模，建模成功裸小鼠的瘤体平均体积为160.1mm 3，将模型动物随机分为21组，按表14的药物、剂量分别进行静脉注射(系列A)、瘤内注射(系列B)给药，其中所给药物均为液体制剂，按实施例1的方法配置而成；各组均用药2次，末次用药后第7天对动物进行安乐死，解剖后剥离出肿瘤组织测定瘤重，并根据系列A和系列B中相应的阴性对照组计算抑瘤率r A和r B。
表14不同药物组合、给药方式对瘤重、抑瘤率的影响
Figure PCTCN2021122132-appb-000015
Figure PCTCN2021122132-appb-000016
实体肿瘤的药物治疗中，化疗药物进入靶区就可以攻击其靶，从而在短期内即显示出化疗药效。由表14可知，组别1-5的药物在普通动物模型中显示的疗效和药理行为与实验2中相应药物在免疫缺陷动物模型的结果一致，分别符合细胞毒作用、化学消融、免疫增强、和类化学消融的局部作用药理。
组别9的药物在普通动物模型中显示的疗效(r 9B>r 4B和r 9B>r 5B)、共用作用(q>1.00)和共用行为(r 9B/r 9A>200％)与实验5中相应药物在免疫缺陷动物模型的结果一致，即益生菌组分在上述提供局部作用的条件下还可以向弱局部作用化合物提供局部药效协同作用和局部安全性协同作用。组别10-13的结果对此进一步证实。
下述实验给出一个以动物正常组织为局部病变模型的研究例：
以BALB/c小鼠为实验对象，将模型动物随机分为4组，每组6只，按表15的药物进行给药；各组均用药1次，给药至右腿外侧肌肉块内，注射量100ul/只，给药后第7天对动物进行安乐死，解剖取出小鼠右腿外侧肌肉块标本，并进行大体病理分析，切片测量区别于正常肌肉的异常区域面积，如坏死、结节等。
表15各组的用药组分
Figure PCTCN2021122132-appb-000017
Figure PCTCN2021122132-appb-000018
上述试验和其它类似试验的短期药效结果显示,本申请所述益生菌组分作为局部活性成分在普通动物模型中与在实施例2-8的免疫缺陷动物模型中有一致的药理,均是通过提供局部作用或局部协同作用对药理靶(药物渗透区内的组织)进行破坏。
根据以上实施例2-9的结果以及其它类似试验的结果，用于局部病变内时,包含益生菌组分及其共用物的本发明局部药物组合物(简称现本发明组合物)在以下方面超过现有技术中任何可预期的包含益生菌组分/共用物的非局部药物组合物(简称现有技术组合物)的预期：
1)、其共用药理超预期:本发明组合物中的益生菌组分与其共用物是局部活性组分之间的共用,其共用药理包括它们各自局部作用的共用，其所产生的短期协同作用主要为局部协同作用，超过了现有技术中益生菌组分与其共用物之间的任何涉及与其非局部活性(例如免疫作用、抗病毒作用、肿瘤细胞毒作用、或瘤体血管破坏作用等)共用的药理预期；
2)、其药理方法超预期:本发明组合物中的益生菌组分与其共用物必须严格限定于皆为局部给药才能产生短期协同作用，而现有技术任何可预期的益生菌组分与共用物的共用药理方法则不必限于局部给药、优选为全身性给药；
3)、其活性组分的优选方案超预期:在组分种类数目优选上,本发明组合物优选3或更多(例如益生菌组分/弱局部作用化合物/细胞毒药物)，尽管2组分协同组合物(例如益生菌组分/弱局部作用化合物)已超出预期。在共用物种类的选择上,益生菌组分出人意料地并未向乙醇、却可以向弱局部作用化合物或/和细胞毒药物提供局部协同作用。在具体药物优选上,除了益生菌组分为提供局部作用的前述优选方案外,共用物也优选包含主要提供局部作用而非全身性作用的弱局部作用化合物的一种或多种药物,例如弱局部作用化合物中的一种或多种、弱局部作用化合物和细肥毒药物、弱局部作用化合物和免疫增强剂、等等；
4)、其活性组分间的共用药理结构预期:本发明组合物必须包括益生菌组分的局部作用药理浓度,因而其共用药理结构参数为浓度比而非现有技术组合物中的量比。益生菌组分提供共用作用的药理浓度为其在以上实施例中提供局部作用的浓度。所述弱局部作用化合物的药理浓度分别为:氨基酸类营养剂为5-25％、亚甲蓝类染料为0.5-3％或0.5-1.5％、奎宁类药物为3-10％、弱酸为3-20％、强碱为1-10％、弱碱为1-20％、酸化剂或/碱化剂的pH缓冲系统总浓度为3-25％；
5)、其技术效果超预期:本发明组合物可提供现有技术组合物所不能提供的局部活性(局部作用或局部协同作用),至少在给药区域内提供现有技术组合物所不能提供的较高局部治疗效果。优选的，本申请的药物组合物可用于任何病变组织(如包含肿瘤细胞或/和成纤维细胞)的破坏性治疗(如瘤体减荷)。更优选的，本申请的药物组合物可用于任何全身性药物(细胞毒药物、抗病毒药物、抗细菌药物、血管抑制药物、免疫药物等)难以治疗、而病变内给药又切实可行的局部病变的治疗。
实施例10：普通动物模型中病变区给药的中长期单用/共用药理研究及优化
以BALB/c小鼠为实验对象，采用乳腺癌4T1细胞为建模细胞，将0.5×10 6个细胞/只注入右侧腋部皮下进行移植瘤建模，建模成功裸小鼠的瘤体平均体积为107.4mm 3，将模型动物随机分为17组；
按表16的药物进行给药，其中的药物按实施例1的方法配置而成；各组均用药2次，每次间隔7日；组别13、14的给药量为300μl/只/次，其它组为100μl/只/次；组别15的给药方式为右前肢腋下给药，其它组为瘤内注射给药；组别16为序贯给药(首次给药为益生菌半流体类组分/细胞毒药物/弱局部作用化合物，末次给药为灭活益生菌)，其它组两次给药均为同一药物。末次用药后第7、21天分别测量各组瘤体体积(V 7d、V 21d)，并从阴性对照组分别计算用药后第7、21天的相对瘤体增值率( R 7d 、 R 21d ),从而可分别算出短期疗效(100％- R 7d )和中长期疗效(100％- R 21d )。
表16不同药物用药后第7、21天的抑瘤结果
Figure PCTCN2021122132-appb-000019
众所周知，化疗药物的效应动力学特征为药物在攻击在、药物代谢后则攻击不再,而免疫增强剂的药物效应几乎显示不出瘤体变化。组别1、2在第21、7天的相对瘤体增值率R的比值(R 21/R 7)大于1.50，表明短期药物效应的抑瘤效果明显减小。组别3的相对瘤体增值率一直很高，符合单独使用免疫增强剂 并不显示明显疗效的预期。组别1-3中的阳性对照物(5-氟尿嘧啶、乙醇、白细胞介素-12)显示的中长期药效合乎其药理(分别为细胞毒药理、免疫增强药理、局部作用药理)预期的效应动力学(R 21/R 7)构建了一个比较研究系统。
组别1-7的药物组合物在第7日的短期药效与实施例9中的结果一致,。组别5-7中的益生菌组分的短期药效合乎其作为类似于组别2中的化学消融剂的局部活性成分的预期,似乎也理应显示与组别2中的化学消融剂一致的中长期药效。出人意料地,益生菌组分显示出完全不同的效应动力学(R 21/R 7明显小于100％),大大地超出所有阳性对照物药理的中长期药效预期。不同益生菌组分显示出几乎相同的效应动力学,独立于它们的细菌免疫原性。具有类似情况的还有组别8-12,其中的益生菌组分/共用物显示出局部协同作用(q 7d>1.00)、中长期协同作用(q 21d>1.00)、和完全不同于上述阳性对照物的效应动力学(R 21/R 7<100％)。
组别13与组别5(给药组分、剂量相同)相比,益生菌组分给药浓度不是其局部作用药理的优选浓度，显示为短期药效差异大于200％。组别14与组别6(给药组分及量比、剂量相同)相比,组合物给药浓度不是其局部协同作用药理的优选浓度，也显示为短期药效差异大于200％。而组别13、14的中长期药效(R 21)也大大不如组别5、6。更进一步,组别13、14的效应动力学(R 21/R 7近似或大于100％)明显不同于组别5、6(R 21/R 7明显小于100％)。组别15的结果则类似于组别13,似乎益生菌组分在病变外的局部作用(短期药效)及其次生作用(中长期药效、效应动力学)同样太弱。组别16序贯给药的组合物(益生菌半流体类组分/细胞毒药物/弱局部作用化合物、灭活益生菌各一次)的中长期药效在二次给药二种相同药物(灭活益生菌和益生菌半流体类组分/细胞毒药物/弱局部作用化合物,它们在组5、12中显示不同的短期(局部作用)疗效)之间,进一步说明这类药物的中长期疗效与其短期(局部作用)疗效相关。
上述结果表明，组别5-12的益生菌组分显示出超出组别1-3药理、甚至于超出其简单局部作用药理预期的中长期作用，也就是与化学作用或免疫增强作用不同的新药理。该新药理的产生至少须满足上述实施例2-4中局部作用药理条件(包括优选细菌免疫原性最小化的益生菌组分、等),因而可以合理推出其为该局部作用的次生(协同)作用。
综上，本申请的组合物经病变内给药后的短期作用主要为局部作用(或局部协同作用)，长期作用则主要为局部作用(或局部协同作用)产生的次生作用，例如次生免疫作用。本申请的组合物完全不同于疫苗等免疫药物，其中起抗原作用的物质包括给药后产出的原位抗原等原位免疫物质。此外，益生菌组分如同其它外源物质一样还可能具有非特异性抗原作用，用于加强该原位免疫物质的药效。
本申请的组合物具有包括局部作用(或局部协同作用)及其次生免疫作用的化学/免疫药理功能，从而大大超过现有技术关于益生菌组分的药效预期，成为治疗药物而非现有技术的免疫增强剂；通过限定局部病变内给药、局部作用浓度阈值、局部作用体积阈值、局部协同组合物等可对任何任何局部病变疾病提供有效治疗；避免细胞毒药物所必须的一个疗程内高频次大剂给药，安全性高和顺应性好。本申请的组合物可用于细胞毒药物、抗病毒药物、抗细菌药物、血管抑制药物、免疫药物等难以治疗的局部病变疾病的治疗，例如间质比大于25％的实体肿瘤。相对于其他药物，本申请所述的组合物除用作组织毒/免疫药物，还可以将成纤维细胞作为作用靶点用于治疗上述适应症，而成纤维细胞及其产生的纤维等沉积物往往是难治性局部病变疾病的主要特点之一。
实施例11：普通动物模型中病变区外局部给药的单用/共用药理研究及优化
尽管在实施例9中通过瘤外给药的药效不明显，但瘤外给药的研究仍未放弃。以BALB/c小鼠为实验对象，采用乳腺癌4T1细胞为建模细胞，将0.25×10 5个细胞/只注入右侧腋部皮下进行移植瘤建模。建模后第5日，将目测明显出瘤的试验动物随机分为12组，每组10只，按表17的药物、剂量进行给药，其中的药物按实施例1的方法配置而成；组别10、11为一日二处给药不同药物(益生菌组分/共用物组合物进行瘤内给药,同日将益生菌组分单药于动物左侧腋下皮内给药)，其它组均两次于动物腋下皮内给药，每次间隔7日,每次均为同一药物。自组别1-11的末次给药后第7、21日分别测量瘤体体积(V 7d、V 21d)，并从阴性对照组分别计算用药后第7、21天的相对瘤体增值率( R 7d 、 R 21d ),从而可分别算出短期疗效(100％- R 7d )和中长期疗效(100％- R 21d ),以及根据短期疗效和中长期疗效分别计算短期药效的实际/预期比q 7d和中长期药效的实际/预期比q 21d。
表17不同药物用药后第7、21天的抑瘤结果
Figure PCTCN2021122132-appb-000020
*:剂量(μl/只/次)
由表17可知，组别1-3中的阳性对照物(5-氟尿嘧啶、乙醇、白细胞介素-12)显示的结果合乎其药理(分别为细胞毒药理、免疫增强药理、局部作用药理)预期,均无法提供有意义的中长期药效。组别5-7的短期药效主要是局部作用或局部协同作用，其无意义结果与组别2一致。出人意料地，组别5-7观察到不可忽略的中长期药效，显示出大大不同于对照物组别1-3任何药理的新药理。使用常规疫苗剂量(20mg/kg)的组别8、9未能观察到与超常规疫苗剂量(240mg/kg)组别组5、6相同的中长期药效,显示出 后者的新药理与局部作用的关系更加密切。组别10、11瘤内给药益生菌组分/共用物组合物的同日瘤外给药益生菌组分单药,它们的短期药效主要是瘤内局部协同作用,而中长期药效则为该瘤内局部协同作用和组别5、6显示的瘤外局部作用的次生作用的共作用。
实验中观察发现，组别5、6的在注射时大多数实验动物产生较强挣扎反应，说明益生菌组分单药应在止痛条件下使用以避免安全性风险；而组别7则难以解释地未观察到上述强烈反应，说明益生菌组分与弱局部作用化合物和/或细胞毒药物的协同药物组合物具有超单药效应预期的安全性。
以下实施例对本申请的组合物在腋下皮下的局部作用进行进一步研究。
以BALB/c小鼠为实验对象，随机分为A、B两组，每组6只；以10％热灭活布拉氏酵母菌和10％热灭活布拉氏酵母菌/20％耐氨酸分别对A、B组用药，各组均用药1次，给药至右前腿腋下皮内，注射量100ul/只。用药后7日，对动物进行安乐死，解剖取出小鼠右前腿腋下皮内节结标本，切片冲洗后测量区别于正常肌肉的异常区域的面积，如坏死、结节等的横切面。
结果表明，A、B的异常区域面积分别为30.15±11.31mm 2和45.74±13.71mm 2，这与实施例9的结果相一致。本申请技术方案中的益生菌组分类似于高浓度乙醇，经局部给药后首先显示的药效主要为局部作用(或局部协同作用),其与实施例2、5和9中经瘤内给药后显示的局部作用(或局部协同作用)相一致,主要是类似于化学消融的局部化学作用(或局部化学协同作用)。本申请的组合物经瘤外局部给药后的短期作用主要为对皮下药物渗透区组织的破坏，中长期作用则主要是该破坏产生的次生作用。上表中组别5-7所观察到的中长期跨距作用说明该次生作用主要包括疫苗作用。因此，本申请的组合物可提供完全不同于经典疫苗的瘤外疫苗作用，其中起抗原作用的物质包括给药后组织破坏产出的非特异性抗原。此外，与其它外源物质一样，益生菌组分还具有非特异性抗原作用，用于加强疫苗作用的药效。
由上可知，用于局部病变外适当局部时,本发明包含益生菌组分的组合物(本发明组合物)完全不同于通常的全身性作用药理(如以上细胞毒药理、免疫增强药理)或局部作用药理(如以上化学消融)组合物(通常组合物):1)、目标药理:本发明组合物可以提供足够强的局部作用的次生作用或次生协同作用(包括但不限于坏组织抗原作用及其它尚待研究的次生免疫反应),这大大超过通常组合物的作用药理预期(例如化学消融所致几无影响的坏组织抗原作用)；2)、药理组成:本发明组合物在局部病变外为产生足够强局部作用所必须满足的条件与以上实施例益生菌组分在局部病变内的局部作用药理组成相同,这大大超过通常组合物的药理组成预期(如免疫原性较强甚至最大化的优选免疫增强组分、大剂量细胞毒药物优选、等等)；3)、技术效果:本发明组合物可产生类似于治疗性疫苗的明显的中长期跨距抑制局部病变作用,大大超过现有通常组合物的技术效果预期。
此外，尽管目前尚无瘤外给药体积和瘤内靶区体积关系的发现，但其给药体积的一个必要条件是能够形成作为抗原所需的结节，如当瘤体总体积≥2.85cm 3，则用药量＞0.01ml/kg人、0.015-0.25ml/kg人，优选为0.020-0.25ml/kg人，这就要求本申请中用于瘤外局部注射的包含益生菌组分的药物的给药体积为常规疫苗的给药体积的2倍或3-100倍，优选5-100倍。
实施例12：次生(协同)作用进一步研究及方案优化
以BALB/c小鼠为实验对象，其中以建模动物作为系列A，以不造模动物作为系列B。建模动物采用乳腺癌4T1细胞为建模细胞，将0.25×10 5个细胞/只注入右侧腋部皮下进行移植瘤建模，建模成功裸 小鼠的瘤体平均体积为173.6mm 3，将模型动物随机分为18组，每组10只，并按表18的药物经瘤内注射给药；另取BALB/c小鼠为实验对象，不建模直接将模型动物随机分为18组，每组10只，并按表18的药物经右后肢大腿肌肉内注射给药。含益生菌组分研究药物均为按实施例1的制备方法配置而成的含水剂，其它药物均为按现有公知的方法配置而成的水溶液；各组均给药2次，每次间隔3日，用药量：50ul/只/次。
系列A各组在第1次给药后第10、30日分别测量各组的瘤体体积并根据阴性对照计算瘤体抑制率(r’ 10d、r’ 30d)。系列A和系列B各组在第1次给药后第30日均在动物左侧前后腋部皮下分别以1×10 5个乳腺癌4T1细胞/只和0.2×10 5个成纤维3T3细胞/只进行细胞移植,并在该细胞移植后第10日分别观测各组4T1细胞移植出瘤率(S 4T1)和3T3细胞移植出瘤率(S 3T3)。其中出瘤率S＝(出瘤动物数目/细胞移植动物数目)×100％。
表18不同组别的抑瘤率、出瘤率数据
Figure PCTCN2021122132-appb-000021
Figure PCTCN2021122132-appb-000022
由表18可知，系列A中的抑瘤率r’ 10d、r’ 30d分别表示药物在病变区给药产生针对局部病变的短期药效和中长期药效；如前所述，益生菌组分及其组合物的短期药效直接与局部作用有关，而中长期药效，尤其是明显高于化疗药物预期的中长期药效，则与局部作用的次生作用或次生协同作用，尤其是次生免疫作用有关。
在系列A中,组别02-06中各对照物经瘤内注射给药后显示的药效均合乎它们各自的药理预期,分别为细胞毒作用、免疫增强作用、化学消融作用、疫苗佐剂作用、条件局部作用(聚多糖),其中的细胞毒作用(组别02)和化学消融作用(组别04)显示、而其它作用未显示短期药效(r’ 10d),而所有作用均未显示中长期药效(r’ 30d)。组别1-4中采用的益生菌组分单药则显示出明显的短期和中长期治疗作用。出人意料的是，尽管其中的灭活益生菌的短期药效(r’ 10d)最高,但其中长期药效(r’ 30d)却最低。
根据系列A中组合物(A/B)与相应组成物(A、B)的药效(r’ 10d、r’ 30d)，可计算出组合物共用的短期、中长期药效的实际/预期比(q 10d、q 30d)，其中q＝r’ A/B/[r’ A+r’ B-(r’ A×r’ B)]。组别21-26中的益生菌组分/共用物形成的组合物的q 30d均大于1.0，甚至于大于1.15，其中长期药效大于组成物的中长期药效的加和预期，药物共用时存在局部作用的次生协同作用。与上述组别3-4一致,组别22-26的短期药效(r’ 10d)逊于组别21,它们的中长期药效(r’ 30d)却更高。
此外，表18中S 3T3和S 4T1分别代表针对移植细胞3T3和4T1的移植排斥。组别01-06的对照物无论系列A或B中的 S3T3和S 4T1均为100％，说明均未产生免疫治疗作用。在系列A中，组别1-4中使用的益生菌组分产生的免疫作用尚不足以完全抑制非病原(S 3T3为100％)，但足以清除或至少强烈抑制病原(S 4T1远小于100％)，具有明显的特异性。说明相关药物生成的短期(局部)作用局限于局部病变，其中长期(次生)作用却并非局限于局部病变，而是包括了治疗免疫作用。组别21-26的药物组合物产生的免疫作用分别与组别1-4中相应的益生菌组分一致，而针对病原(4T1)的兔疫作用却更强，具有更低的出瘤率。 在系列B中，组别1-4中的益生菌组分以及组别21-26中的组合物产生的非特异性免疫作用也都不足以强烈抑制任何移植细胞(4T1和3T3)。
通过比较表中，尤其是系列A的组合物(A/B)及相应的组成物(A、B)针对同种病原的移植排斥作用[S 4T1或S' 4T1＝(1-S 4T1)]，可计算组合物共用药效中的免疫共用作用的实际/预期比q 4T1＝S’ A/B4T1/[S’ A4T1+S’ B4T1-(S’ A4T1×S’ B4T1)]。其中组别21-26中组合物的实际/预期比q 4T1均大于1.00，显示出免疫协同作用，尤其是特异性免疫协同作用。益生菌组分及包含其的组合物首先在病变处产生局部作用，说明中长期药效中的免疫作用或免疫协同作用，尤其是特异性免疫作用或特异性免疫协同作用，应当是局部作用的一种环境依赖型的次生作用。
根据以上结果以及其它类似试验的结果，用于局部病变治疗时,本发明所述的包含益生菌组分的局部药物组合物(简称本发明组合物)在以下方面超过现有技术中的任何非特异性免疫治疗的药物组合物(简称现有技术组合物,例如上述细胞毒药物、免疫增强剂、化学消融剂、疫苗佐剂、条件局部作药物、等等)的预期：
1)、其药理超预期:本发明组合物的药理犹其是中长期药理包括现有技术组合物所没有的特殊局部作用(或局部协同作用)的次生作用(或次生协同作用)、犹其是病原特异性免疫作用；
2)、其药理组成超预期:在以上实施例益生菌组分局部作用药理组成基础上,可以并不需要益生菌组分提供的局部作用最大化、甚至优选局部作用最小化,如益生菌组分优选为选自细菌免疫原性和局部作用都很微弱的组分(水溶性组分和半流体类组分)、益生菌组分药理浓度优选为选自较低而非较高的给药浓度(水溶性聚多糖的给药浓度限制在3-15％或3-5％、核酸的给药浓度限制在10-25％、半流体类组分限制在4-12％)、具有特定组合关系的多种(优选为2种或2种以上)共用药物等等；
3)、其技术效果超预期:如在现有技术组合物并不产生的条件(例如并无短期协同药效、甚至于为短期拮抗药效)下可产生明显、甚至有效的中长期药效和跨距药效,犹其是类似于治疗性疫苗的明显的特异性免疫作用,使其适应症范围大大超过现有技术组合物。
实施例13：剂型研究及优化
以BALB/c裸小鼠为实验对象，采用乳腺癌4T1细胞为建模细胞，将0.25×10 5个细胞/只注入右侧腋部皮下进行移植瘤建模，建模成功裸小鼠的瘤体平均体积为291.7mm 3，将模型动物随机分为3组，其中A、B、C分别给以生理盐水、5％灭活布拉氐酵母菌水悬液、5％灭活布拉氐酵母菌加入生理盐水混合所得水悬液作为研究药物，上述药物均按实施例1的制备方法配置而成。每组均瘤内注射给药2次，每次间隔2天，注射量为150μl/只/次。第一次给药后第10日对动物进行安乐死，解剖后剥离出肿瘤组织测定瘤重，并根据阴性对照组计算抑瘤率(r)。
结果表明，B组的药物为局部剂型且不含渗透压调节剂，而C组的药物为非局部剂型且含渗透压调节剂-氯化钠，其中B组、C组的抑瘤率分别为19.8％、41.3％，C组较B组明显更低，说明渗透压调节剂会干扰组合物的免疫增强作用，从而降低局部作用。在本实验条件下更是使益生菌组分从B组中提供有效治疗作用变为C组的无治疗作用。由上可知，本申请组合物的剂型选择与现有技术组合物有着本质差异。具体的。现有技术中包含益生菌组分的组合物通常为口服剂型或常规注射剂型，前者通常需包含固体赋形剂，而后者通常需包含益生菌组分的渗透压提高剂(盐或单糖)以保证其药液与血液渗透压相 同或相近。而本申请所述的药物组合物仅须满足可提供该局部作用的要求，其优选方案可不包含固体赋形剂或渗透压提高剂，以避免降低局部作用。实际上，上述给药剂型也是局部作用药物的优选剂型。
实施例14中老年患者模型中的比较研究及进一步优化
以8月龄小鼠作为中老年患者模型中的实验对象，将0.5×10 5个小鼠乳腺癌4T1细胞/只注入右侧腋部皮下进行移植瘤建模，建模成功裸小鼠的瘤体平均体积为117.3mm 3，将模型动物随机分为18组。每组通过瘤内注射按照表19的药物分组给药1次，注射量50μl/只；用药后在第3日、第21日测局部病变体积(V)，并根据阴性对照组计算各时间的瘤体抑制率(r’ 3d、r’ 21d)，结果见下表。所用药物均按常规的水溶液制备方法或实施例1中的制备方法制备。根据不同时间(3日、21日)的药效(r’ 3d、r’ 21d)分别计算短期药效的实际/预期比q 3d和中长期药效的实际/预期比q 21d。
表19不同组别用药后的短效、长效数据
Figure PCTCN2021122132-appb-000023
Figure PCTCN2021122132-appb-000024
中老年患者模型与通常使用的年青患者模型在肿瘤形成和免疫能力方面并不一致。由表19可知，组别1-3中的糖对照物在很高浓度(30％)才显示出短期药效(r’ 3d>15％),而任何浓度均未显示中长期药效(r’ 21d＜15％),合乎糖营养素只有在很高浓度(30-50％)才可能提供局部作用、但不能提供有意义的次生作用的预期。组别4-9中的益生菌组分(包括类似物)未观察到有意义的短期药效(r’ 3d＜15％)和中长期药效(r’ 21d＜15％)，说明老年患者中局部给药时任何可产生短期药效和中长期药效的全身性机理(如细胞毒等化疗机理)所起作用可忽略不计，而其局部作用也为最小化的局部作用。组别10中药物组合的局部作用明显有效(r 3d＞40％)，但其局部作用的有效性随时间衰减(r’ 21d＜2/3×r’ 3d)。
根据不同时间(3日、21日)的药效(r’ 3d、r’ 21d)分别计算短期药效的实际/预期比q 3d和中长期药效的实际/预期比q 21d。组别11-13中的糖对照物在很高浓度(30％)才显示出局部协同作用(q 3d＞
1.00),而任何浓度均未显示中长期协同作用(q 21d均为小于1.00),合乎糖营养素只有在很高浓度(30-50％)才可能提供局部协同作用、但不能提供有意义的次生协同作用的预期。组别14-19中的益生菌组分及其类似物与组别11、12中的糖对照物的浓度类似,局部作用也都最小化，它们与相同局部药物的共用似乎也应当产生类似结果。然而与相同浓度的糖对照物比较，益生菌组分(包括类似物)与相同局部药物共用的短期药效(r’ 3d)明显较高,中长期药效(r’ 21d)的差异更是明显扩大。实际上，益生菌组分(包括类似物)与相同局部药物共用的q 21d均大于1.00，大大超出根据糖对照物/局部药物作用预期的中长期协同作用。此外,尽管结构非常相似，益生菌组分/局部药物可以产生超出其它来源类似物/局部药物预期的短期作用和中长期协同作用。
考虑到益生菌组分(包括类似物)本身在本实施例条件下并未提供中长期药效，以上结果的共用药理超过现有技术中协同作用的药理预期；细菌抗原性最小化、局部作用最小化(r’ 3d＜15％)、中长期药效最小化(r’ 21d＜15％)的益生菌组分(包括类似物)在相关技术方案中可提供一种先前未经发现却很有用的药理功能-即在自身不一定显示较强、优选为最小化局部作用的条件下,为不一定发生局部协同作用、或只发生较弱局部协同作用的共用物提供中长期药效协同作用的活性。在相关技术方案中，包含益生菌组分(包括类似物)的中长期协同药物组合物的适应症范围有以下要求:中老年患者，其药物组成有以下要求：局部作用最小化的益生菌组分(如水溶性组分和半流体类组分)、局部作用最小化的药理浓度(如水溶性聚多糖的给药浓度限制在3-15％、核酸的给药浓度限制在10-25％、半流体类组分限制在4-12％等)。
本发明所述的包含益生菌组分的局部药物组合物(简称本发明组合物)除了在以上实施例中超过任何非特异性免疫治疗的药物组合物的预期之外,根据本实施例以上结果(犹其是中长期结果)以及其它类似试验的结果，其还犹其是在以下方面超过现有技术中的包含局部作用药物(例如乙醇、常规糖营养素等等)的局部药物组合物(简称现有技术组合物)的预期：
1)、其优选共用药理超预期:现有技术组合物的基于现有协同定义(协同作用为多个特定活性成分的类似作用通过共用互相增强)的优选共用药理是在有利于组分局部作用(例如常规糖营养素局部作用 较大、甚至于最大化)的条件下进行的局部协同作用,而本发明组合物超出现有协同定义的优选共用药理是在并不明显有利于组分局部作用(例如益生菌组分局部作用较小、甚至于最小化,益生菌组分与其共用物不产生短期协同作用、甚至产生短期拮抗作用)的条件下产生的中长期协同作用,该中长期协同作用很可能包括超出各组分局部作用的免疫作用；
2)、其药理组成超预期:如在以上实施例中所述超任何非特异性免疫治疗的药物组合物的药理组成预期。通常认为局部作用与局部刺激性正相关。然而,优选的益生菌半流体类组分或益生菌水溶性组分与益生菌颗粒组分(灭活益生菌、益生菌水不溶颗粒)相比具有明显较低的局部刺激性(局部给药时动物剧烈挣扎的比例),优选的共用物也并非局部刺激性较强的乙醇、而是局部刺激性较弱的弱局部作用化合物,等等；
3)、其技术效果超预期:本发明组合物可产生明显、甚至有效的中长期药效和全身性药效(跨距药效),大大超过现有技术组合物只能产生短期局部药效的技术效果预期。该中长期药效和全身性药效特别适应中老年、犹其是老年患者。此外,优选的本发明组合物显示几近于无的局部刺激性,而经典化学消融剂(如乙醇)的局部作用与局部刺激性正相关。
其中所述益生菌组分可提供局部协同作用或/和中长期协同作用的共用物还可以为包含选自其它药学上适宜的、可提供有意义的局部作用的药物(化学活性成分或/和生物活性成分)或药物组合。例如：
1)、选自包含碱金属氢氧化物、碱性无机盐、和选自以下组之一种或多种的组合：亚甲蓝及其类似物、免疫调节剂、细胞毒药物，如以下组合：浓度比(W 碱金属氢氧化物/W 碱性无机盐/W 亚甲蓝及其类似物)为(1-5％)/(3-15％)/(0.35-5％)的碱金属氢氧化物/碱性无机盐/亚甲蓝及其类似物组合、浓度比(W 碱金属氢氧化物/W 碱性无机盐/W 免疫调节剂)为(1-5％)/(3-15％)/(1-15％)的碱金属氢氧化物/碱性无机盐/免疫调节剂组合、浓度比(W 碱金属氢氧化物/W 碱性无机盐/ 细胞毒药物)为(1-5％)/(3-15％)/(0.1-10％)的碱金属氢氧化物/碱性无机盐/细胞毒药物组合。
2)、选自包含碱性有机化合物、碱性无机盐、和选自以下组之一种或多种的组合：亚甲蓝及其类似物、免疫调节剂、细胞毒药物，如以下组合：浓度比(W 碱性有机化合物/W 碱性无机盐/ 亚甲蓝及其类似物)为(5-25％)/(3-15％)/(0.35-5％)的碱性有机化合物/碱性无机盐/亚甲蓝及其类似物组合、浓度比(W 碱性有机化合物/W 碱性无机盐/W 免疫调节剂)为(5-25％)/(3-15％)/(1-15％)的碱性有机化合物/碱性无机盐/免疫调节剂组合、浓度比(W 碱性有机化合物/W 碱性无机盐/W 细胞毒药物)为(5-25％)/(3-15％)/(0.1-10％)的碱性有机化合物/碱性无机盐/细胞毒药物组合。
3)、选自包含碱性有机化合物、亚甲蓝及其类似物、和选自以下组之一种或多种的组合：免疫调节剂、细胞毒药物、非碱性氨基酸及其酸式盐，例如浓度比(W 碱性有机化合物/W 亚甲蓝及其类似物/ 免疫调节剂)为(5-25％)/(0.35-3.5％)/(1-15％)的碱性有机化合物/亚甲蓝及其类似物/免疫调节剂组合、浓度比(W 碱性有机化合 物/W 亚甲蓝及其类似物/W 细胞毒药物)为(5-25％)/(0.35-3.5％)/(0.1-10％)的碱性有机化合物/亚甲蓝及其类似物/细胞毒药物组合、浓度比(W 碱性有机化合物/W 亚甲蓝及其类似物/W 非碱性氨基酸及其酸式盐)为(5-25％)/(0.35-3.5％)/(10-25％)的碱性有机化合物/亚甲蓝及其类似物/非碱性氨基酸及其酸式盐组合。
4)、选自包含细胞毒药物和选自以下组之至少二种的组合：碱性有机化合物、免疫调节剂、多元醇、亚甲蓝及其类似物，例如以下组合：浓度比(W 细胞毒药物/W 碱性有机化合物/W 免疫调节剂)为(0.1-10％)/(5-25％) /(1-15％)的细胞毒药物/碱性有机化合物/免疫调节剂组合、浓度比(W 细胞毒药物/W 多元醇/W 亚甲蓝及其类似物)为(0.1-10％)/(5-25％)/(0.35-3.5％)的细胞毒药物/多元醇/亚甲蓝及其类似物组合、浓度比(W 细胞毒 药物/W 亚甲蓝及其类似物/W 免疫调节剂)为(0.1-10％)/(0.35-5％)/(1-15％)的细胞毒药物/亚甲蓝及其类似物/免疫调节剂组合。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (25)

  1. 益生菌组分作为可提供治疗作用的活性成分在制备用于治疗局部病变疾病的局部药物组合物中的应用，其中所述治疗作用包括涉及局部作用(或局部协同作用)的局部治疗或/和免疫治疗。
  2. 一种用于治疗局部病变疾病的局部药物组合物，其包含可提供治疗作用的益生菌组分、以及药物学可接受的合适载体，但不包含专用于将血液渗透压增加至正常生理水平的盐或单糖，其中所述治疗作用包括涉及局部作用(或局部协同作用)的局部治疗或/和免疫治疗。
  3. 根据权利要求2或3的应用或药物组合物，其中所述药物组合物还还包含能够与所述益生菌组分的所述治疗作用产生协同作用的化学活性成分或/和生物活性成分，以及在所述药物组合物中，所述益生菌组分与所述化学活性成分的量比(益生菌组分重量浓度/化学活性成分重量浓度)为(1-110)/(1-100)或(1.5-100)/(0.1-1)，或/和所述益生菌组分与所述生物活性成分的量比(益生菌组分浓度重量/生物活性成分重量浓度)为(60-180)/(0.1-60)。
  4. 一种用于治疗局部病变疾病的局部药物组合物，其包含可提供治疗作用的益生菌组分、能够与所述益生菌组分的所述治疗作用产生协同作用的化学活性成分或/和生物活性成分，以及在所述药物组合物中，所述益生菌组分与所述化学活性成分的量比(益生菌组分重量浓度/化学活性成分重量浓度)为(1-110)/(1-100)或(1.5-100)/(0.1-1)，或/和所述益生菌组分与所述生物活性成分的量比(益生菌组分浓度重量/生物活性成分重量浓度)为(60-180)/(0.1-60)，其中所述治疗作用包括涉及局部作用(或局部协同作用)的局部治疗或/和免疫治疗。
  5. 根据权利要求1-4之一的应用或药物组合物，其中所述益生菌组分为选自细菌免疫原性最小化的源于天然益生菌或其工程菌的制备物或该制备物的工程类似物，其中所述制备物优选为选自包括以下组及其衍生物之一种或多种：益生菌水溶性组分、益生菌半流体类组分、益生菌组分水不溶颗粒、灭活益生菌。
  6. 根据权利要求5的应用或药物组合物，其中所述益生菌水溶性组分选自包括以下组及其衍生物之一种或多种：益生菌或破碎益生菌上清组分、益生菌抽提物、益生菌细胞内水溶性组分、益生菌细胞壁组分的水溶性成分或非水溶成分的水溶性衍生物、益生菌核糖核酸；所述益生菌半流体类组分优选为选自其含水混合物可形成半流体类组合物的益生菌组分之一种或多种，例如益生菌聚多糖及其类似物之一种或多种：所述益生菌组分水不溶颗粒选自包括以下组及其衍生物之一种或多种：破碎益生菌沉淀组分、益生菌细胞壁聚多糖颗粒、益生菌细胞壁聚多糖纳米颗粒；所述灭活益生菌优选为选自并不限定其皆为完整菌体的灭活益生菌；
    其中所述益生菌水溶性组分选自包括以下组及其衍生物之一种或多种：益生菌水溶性β-葡聚糖、优选为选自纯度大于90％的水溶性β-葡聚糖，益生菌β-葡聚糖的水溶性衍生物、益生菌核糖核酸。
  7. 根据权利要求1-6之一的应用或药物组合物，其中所述益生菌选自包括以下组之天然菌或/和工程菌的一种或多种：益生芽抱杆菌、益生乳酸杆菌、益生双歧杆菌、益生真菌，其中:所述芽抱杆菌包括选自包括以下组之一种或多种：地衣芽抱杆菌、枯草芽抱杆菌、短小芽抱杆菌、纳豆芽抱杆菌；所述乳酸杆菌包括选自包括以下组之一种或多种：嗜酸乳杆菌、干酪乳杆菌、植物乳杆菌、短小乳杆菌和发酵乳杆菌；所述双歧杆菌包括选自包括以下组之一种或多种：长双歧杆菌、青春双歧杆菌、短双歧杆菌、屎肠球菌、粪链球菌；所述真菌包括选自包括以下组包括选自之一种或多种：酵母菌、布魯氐酵母菌， 且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、威克汉姆酵母菌、毕赤酵母菌、产阮假丝酵母、乳清酵母。
  8. 根据权利要求1-7之一的应用或药物组合物，其中所述益生菌包括选自酿酒酵母菌或/和布魯氐酵母菌。
  9. 根据权利要求1-8之一的应用或药物组合物，其中所述益生菌组分在该药物组合物中的浓度为＞0.1％、≥0.25％、0.25-25％、优选为0.5-25％、更优选为1-5％、5-15％或15-25％。
  10. 根据权利要求9的应用或药物组合物，其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浓度为＞0.1、或0.15-25％、优选为0.35-5％、5-15％或15-25％；当所述益生菌组分包括所述益生菌水不溶组分颗粒，则所述益生菌水不溶组分颗粒在该药物组合物中的浓度为＞0.5、或0.5-15％、优选为1.5-15％或5-15％；当该药物组合物为半流体类组合物，则所述益生菌半流体类组分在该药物组合物中的浓度为＞2.5％、2.6-25％、优选为5-15％。
  11. 根据权利要求3-10之一的应用或药物组合物，其中所述化学活性成分为包括选自弱局部作用化合物和/或细胞毒药物之一种或多种，以及其中当所述化学活性化合物包括细胞毒药物时，所述益生菌组分和细胞毒药物的量比(W 益生菌组分/W 细胞毒药物)为(1-110)/(1-100)；当所述化学活性化合物包括弱局部作用化合物时，所述益生菌组分和弱局部作用化合物的量比(V 益生菌组分/W 弱局部作用化合物)为(1-90)/(1-100)。
  12. 根据权利要求11的应用或药物组合物，其中所述弱局部作用化合物选自包括以下组之一种或多种：氨基酸类营养素、活体染料、奎宁类药物、低浓度酸化剂、低浓度碱化剂、包含酸化剂或/碱化剂的pH缓冲系统，以及其中当所述弱局部作用化合物包括所述氨基酸营养素时，所述益生菌组分和氨基酸营养素的量比(W 益生菌组分/W 氨基酸营养素)为(1-20)/(1-100)；当所述弱局部作用化合物包括所述活体染料时，所述益生菌组分和活体染料的量比(W 益生菌组分/W 活体染料)为(7-90)/(1-100)；当所述弱局部作用化合物包括所述奎宁类药物时，所述益生菌组分和奎宁类药物的量比(W 益生菌组分/W 奎宁类药物)为(2-90)/(1-100)；当所述弱局部作用化合物包括所述酸化剂或/和碱化剂时，所述益生菌组分和酸化剂或/和碱化剂的量比(W 益生菌组分/W 酸化剂或/和碱化剂)为(2-60)/(1-100)。
  13. 根据权利要求12的应用或药物组合物，其中所述氨基酸类营养素为选自包括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，以及所述氨基酸类营养素的浓度为＞2.5％、或5-30％、优选为5-25％。
  14. 根据权利要求12的应用、药物组合物，其中所述活体染料为选自包括孟加拉红或/和以下亚甲蓝类染料之一种或多种：亚甲蓝、专利蓝、异硫蓝、新亚甲蓝，且其中所述孟加拉红的浓度为2.5％-20％；所述亚甲蓝类染料的浓度为≥0.25％、或0.25-2.5％、优选为0.5-2.5％。
  15. 根据权利要求12的应用或药物组合物，其中所述酸化剂选自强酸或/和弱酸之一种或多种，且所述益生菌组分与所述酸化剂的量比(益生菌组分重量浓度/酸化剂重量浓度)为(1-20)/(0.5-50)，以及所述酸化剂的浓度为≥0.5％、0.5-2％(强酸)、或2-15％(弱酸)，其中所述强酸例如为盐酸；所述弱酸例如为乙二酸、乙酸、乳酸、柠檬酸、苹果酸；所述碱化剂例如选自强碱或/和弱碱之一种或多种，且所 述益生菌组分与所述碱化剂的量比(益生菌组分重量浓度/碱化剂重量浓度)为(1-20)/(0.5-50)，以及所述碱化剂的浓度为≥0.5％、0.5-5％(强碱)、或2-15％(弱碱)，其中所述强碱例如为氢氧化钠、氢氧化钾；所述弱碱例如为磷酸二氢钠、碳酸氢钠、碳酸钠。
  16. 根据权利要求11的应用或药物组合物，其中所述细胞毒药物包括选自包括以下组之一种或多种：破坏DNA结构和功能的药物，例如环磷酰胺、卡莫司汀、金属铂络合物、多柔比星类药物、拓扑替康、伊立替康；嵌入DNA中干扰转录RNA的药物，例如抗肿瘤抗生素药物；干扰DNA合成的药物，例如5-氟尿嘧啶(5-Fu)、呋氟尿嘧啶、双呋氟尿嘧啶、阿糖胞苷、环胞苷、5-氮杂胞苷；影响蛋白质合成的药物，例如秋水仙碱类药物、长春碱类药物、紫杉烷类药物，且所述细胞毒药物的浓度为≥0.1％、0.1-15％。
  17. 根据权利要求1-16之一的应用或药物组合物，其中所述生物活性成分选自包括以下组之一种或多种：抗原、免疫调节类抗体、细胞因子、佐剂。
  18. 一种治疗局部病变疾病的方法，其包括以下步骤：向有此需要的个体的局部病变内或/和局部病变外有利于产生局部作用(或局部协同作用)相关的次生免疫作用的局部施用治疗有效量的根据权利要求3-22之一的药物组合物。
  19. 根据权利要求18的方法，其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含量必须使得它的局部给药浓度为＞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含量必须使得它的局部给药浓度为＞0.1、或0.15-25％、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在该药物组合物中的含量必须使得它的局部给药浓度为＞0.5、或0.5-15％、优选为1.5-15％或3.5-15％。
  20. 根据权利要求1-19之一的应用、药物组合物或方法，其中所述局部病变包含肿瘤细胞或/和成纤维细胞。
  21. 根据权利要求1-20之一的应用、药物组合物或方法，其中所述局部病变疾病包括肿瘤、非瘤肿大、局部炎症、分泌腺功能异常和皮肤病，其中所述肿瘤包括恶性和非恶性实体肿瘤。
  22. 根据权利要求21的应用、药物组合物或方法，其中所述实体肿瘤包括以下肿瘤及其次生肿瘤之一种或多种：乳腺癌、胰腺癌、甲状腺癌、鼻咽癌、前列腺癌、肝癌、肺癌、肠癌、口腔癌、食道癌、胃癌、喉癌、睾丸癌、阴道癌、子宫癌、卵巢癌、脑瘤、淋巴瘤。
  23. 根据权利要求1-22之一的应用、药物组合物或方法，其中所述治疗的适用患者选自包括以下组之一种或多种：免疫抑制患者、可局部病变内给药的患者、局部病变组织可类化学消融的患者、局部病变内可产生次生免疫物质的患者、病变外给药区域内可产生次生免疫物质的患者。
  24. 包含益生菌组分以及药物学可接受的合适载体的药物组合物，其是用于治疗局部病变疾病，其中所述局部病变疾病包括实体肿瘤。
  25. 药物试剂盒，其包括一个或多个装有根据权利要求2-22之一所述的药物组合物的容器，其中还可包括如何向有需要的个体施用所述药物组合物的说明书或标签。
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