WO2022068925A1

WO2022068925A1 - Application of animal non-pathogenic cell-related component and pharmaceutical composition comprising component

Info

Publication number: WO2022068925A1
Application number: PCT/CN2021/122135
Authority: WO
Inventors: 邹方霖; 邹礼常; 王建霞; 王艺羲
Original assignee: 成都夸常奥普医疗科技有限公司; 夸常股份有限公司
Priority date: 2020-09-30
Filing date: 2021-09-30
Publication date: 2022-04-07

Abstract

Provided are an application of an animal non-pathogenic cell-related component and a topical pharmaceutical composition comprising the component. The animal non-pathogenic cell-related component is applied as an active ingredient that can provide a therapeutic action in the preparation of the topical pharmaceutical composition for treating a topical lesion disease, and the topical pharmaceutical composition achieves topical treatment comprising a topical action (or a topical synergistic action) that compositions in the prior art fail to perform or/and immunotherapy comprising a secondary immune action related to the topical action (or the topical synergistic action).

Description

Use of animal non-pathogenic cell-related components and pharmaceutical compositions containing the same

technical field

The present invention relates to the technical field of pharmaceutical preparations, in particular to a new application of non-pathogenic animal cell-related components, a pharmaceutical composition comprising the animal non-pathogenic cell-related components, and a method for treating local pathological diseases .

Background technique

Solid tumors are a classic research model for localized disease. Solid tumor is a tumor disease with tumor symptoms, and the tumor body is a characteristic diseased tissue containing tumor cells. Taking pancreatic cancer tumor as an example, pancreatic cancer cells only account for about 30% of the volume. It can be seen that in addition to tumor cells, there are often a larger number of other components (sometimes also referred to as tumor cell microenvironment) in tumor tissue, including various other cells, interstitial cells, and conduits.

Intratumoral administration has the advantage of physically targeting the drug. It has been suggested that intratumoral administration of cytotoxic drugs can increase their intratumoral concentrations, thereby enhancing their efficacy. However, only increasing its intratumoral concentration does not seem to be able to greatly improve its drug effect. In addition to resorting to their sustained-release forms, cytotoxic drugs are still clinically administered almost systemically.

Clinically used chemical ablative agents (high-purity ethanol, high-concentration acid-base, weak-acid-strong-base salt) are not characterized by tumor cell destruction but tumor tissue destruction. However, classical chemical ablatives are very irritating and long-acting is usually not high. This makes the volume of intervention that they can actually apply (for example, the dosage of acid and alkali does not exceed 0.2ml/kg) and the site of intervention are very limited (for example, the limitation of the organ where the tumor is located, the ablation of the tumor edge, etc.), and the recurrence rate is also high. The high level has led to the clinical fading of classic chemical ablative agents from malignant solid tumors in the past decade.

Thus, there is still a need to develop new drugs for the treatment of locally diseased diseases (eg, solid tumors) to meet various clinical needs that cannot be met by existing technologies.

SUMMARY OF THE INVENTION

In view of this, the present invention aims to propose a new pharmacology based on non-pathogenic cell-related components of animals - that is to provide a local action (or local synergy) and its secondary effect (or secondary synergy), thereby preparing The topical pharmaceutical composition can achieve effective treatment of local lesions, especially produce mid- and long-term curative effects, and provide various options for clinical use.

According to one aspect disclosed in the present application, there is provided the use of an animal non-pathogenic cell-related component as an active ingredient that can provide a therapeutic effect in the preparation of a locally administrable pharmaceutical composition for the treatment of local pathological diseases, wherein the The therapeutic effect includes local treatment involving a local effect (or local synergy) or/and immunotherapy associated with the local effect (or local synergy). In one embodiment, the pharmaceutical composition further comprises a chemically active ingredient and/or a biologically active ingredient capable of producing a synergistic effect with the non-pathogenic cell-associated component of the animal, and the cell-associated component and the The amount ratio of chemically active components (cytospecific volume/chemically active component concentration) is (1.5-100)/(0.1-1), and/or the amount ratio of the cell-related components to the biologically active components (cell ratio) volume/concentration of biologically active ingredients) is (60-180)/(0.1-60).

According to another aspect disclosed in the present application, there is provided a topical pharmaceutical composition for the treatment of local pathological diseases, comprising, as a therapeutically active ingredient, an animal non-pathogenic cell-related component, wherein the treatment comprises involving a local action (or local synergy) or/and immunotherapy associated with this local effect (or local synergy). Preferably, the topical pharmaceutical composition further comprises a chemically active ingredient and/or a biologically active ingredient that can synergize with the non-pathogenic cell-related component of the animal, and the cell-related component is associated with the chemically active ingredient. The amount ratio of the components (cytospecific volume/concentration of chemically active components) is (1.5-100)/(0.1-1), and/or the amount ratio of the cell-related components to the biologically active components (cytospecific volume/ Bioactive ingredient concentration) is (60-180)/(0.1-60).

According to yet another aspect disclosed in the present application, there is provided a topical pharmaceutical composition for the treatment of local pathological diseases, comprising, as a therapeutically active ingredient, an animal non-pathogenic cell-related component, wherein the therapeutic activity includes involving a local action ( or local synergy) local therapeutic activity or/and immunotherapeutic activity associated with this local effect (or local synergy). Preferably, the topical pharmaceutical composition further comprises a chemically active ingredient and/or a biologically active ingredient capable of producing a synergistic effect with the non-pathogenic cell-associated component of the animal, and the cell-associated component is associated with the chemically active ingredient. The amount ratio of the components (cytospecific volume/concentration of chemically active components) is (1.5-100)/(0.1-1), and/or the amount ratio of the cell-related components to the biologically active components (cytospecific volume/ Bioactive ingredient concentration) is (60-180)/(0.1-60).

According to yet another aspect disclosed herein, there is provided a method of treating a local lesion, the method comprising the steps of: facilitating a local action (or local synergy) within and/or outside the local lesion in an individual in need thereof )-related immune effects produced by topical administration of a therapeutically effective amount of a pharmaceutical composition according to the disclosure.

According to yet another aspect of the present disclosure, there is provided a pharmaceutical kit comprising one or more individual containers containing a pharmaceutical composition according to the present disclosure. In one embodiment, the pharmaceutical kit further comprises instructions or labels on how to administer the pharmaceutical composition to an individual in need thereof.

In one embodiment, for the above-mentioned use, pharmaceutical composition, method or kit, the locally diseased disease includes tumor, non-tumoral enlargement, local inflammation, secretory gland dysfunction, and skin disease, wherein the tumor includes malignant and Non-malignant solid tumors. In one embodiment, the solid tumor comprises 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, bowel cancer, oral cancer, Esophageal cancer, stomach cancer, throat cancer, testicular cancer, vaginal cancer, uterine cancer, ovarian cancer, brain tumor, lymphoma.

Embodiments according to the present invention have the following advantages over the prior art: a completely new pharmacology (local action or local synergistic action and its secondary immune action) is provided, allowing for inclusion classes that are not possible with existing compositions Local treatment including chemical ablation or/and immunotherapy including secondary immune effects, the local treatment can produce curative effects (such as more than 3 times tumor inhibition rate) and indications (breakthrough Tumor-specific limitations in the prior art, and limitations on individual immune function dependence), this immunotherapy can produce immune efficacy far beyond the prior art (eg secondary immunity within and/or outside the lesion) The immune effect produced or greatly enhanced greatly exceeds the expectation of immune enhancement of the body) and indications. In addition, embodiments of the present invention also greatly reduce the safety risks of prior art solutions that do not limit the mode of administration.

The invention discloses the application of animal non-pathogenic cell-related components as active ingredients that can provide therapeutic effects in preparing topical pharmaceutical compositions for treating local pathological diseases.

Preferably, the pharmaceutical composition further comprises a chemically active ingredient and/or a biologically active ingredient that can synergize with the cell-related component, and the amount ratio of the cell-related component to the chemically active ingredient (cell Specific volume/chemically active ingredient weight-volume percent concentration) is (1.5-100)/(0.1-1), and/or the amount ratio of the cell-related components to the biologically active ingredient (cytospecific volume/biological activity Ingredient weight-volume percent concentration) is (60-180)/(0.1-60).

The present invention provides a topical pharmaceutical composition for treating local pathological diseases, comprising an animal non-pathogenic cell-related component as an active ingredient that can provide a therapeutic effect.

Preferably, it also includes chemically active components or/and biologically active components that can synergize with the non-pathogenic cell-related components of the animal, and the amount ratio of the cell-related components to the chemically active components (cell ratio volume/concentration of chemically active ingredient) is (1.5-100)/(0.1-1), and/or the amount ratio of the cell-related components to the bioactive ingredient (cytospecific volume/concentration of bioactive ingredient) is ( 60-180)/(0.1-60).

Preferably, the therapeutic effect includes a short-acting therapeutic effect and/or a medium and long-acting therapeutic effect, and the short-acting therapeutic effect includes a local effect (or a local synergistic effect); the medium and long-acting therapeutic effect includes the local effect ( or local synergy) secondary effects (or mid- and long-term synergy).

Preferably, the animal is an animal with minimized immunogenicity, preferably an allogeneic animal selected from autologous or/and minimized transplantation reactions; the cells are one or more of somatic cells, immune cells, and stem cells Species; the cell-related components are selected from one or more of animal tissue components or/and animal cell components associated with the cells, wherein the animal tissue components are selected from one of the following groups or Multiple: animal tissue, tissue fragmentation of animal tissue, cell fragmentation of animal tissue; the animal cell component is selected from one or more of the following groups: animal native cells, native cell destruction components, and the Derivatives or engineered analogs of native cells or native cell disrupting components.

Preferably, the cell specific volume of the animal non-pathogenic cell-related components in the topical pharmaceutical composition is >20%, ≥30%, 30-90%, preferably 45-90% or 75-85% %.

Preferably, the animal non-pathogenic cell-related components include animal cell-related semi-fluid-based components that can make the composition a semi-fluid-based composition, wherein the semi-fluid-based components are selected from animal tissue semi-fluid-based components One or more of components or/and animal cell semi-fluid-like components, and wherein said animal tissue semi-fluid-like components are selected from one or more of the following groups: animal tissue semi-fluid, animal tissue tissue Broken material semi-fluid, animal tissue cell broken material and semi-fluid; the animal cell semi-fluid-like component is selected from one or more of the following groups: animal natural cell semi-fluid-like concentrate, natural cell-destroying component semi-fluid Concentrate-like, semi-fluid-like concentrates of native cells or derivatives or engineered analogs of native cell disrupting components.

Preferably, the tissue is selected from one or more of connective tissue or/and non-connective tissue, wherein the connective tissue includes blood, bone marrow, and spinal cord, and the non-connective tissue includes tissue contained in the following organs: intestine, stomach , meat, pancreas, spleen, liver, lung, cartilage, joint, skin, placenta, umbilical cord, preferably spleen, placenta or/and umbilical cord.

Preferably, the cells include one or more of the following group: blood cells, immune organ cells, other cells enriched in the tissue.

Preferably, the blood cells are selected from one or more of the following cells and their derivatives: red blood cells, white blood cells, and platelets; the immune organ cells are selected from one or more of the following cells and their derivatives: Lymphocytes, T cells, CD cells; the other cells are selected from one or more of the following cells and derivatives thereof: skin cells, muscle cells, secretory gland cells, stem cells.

Preferably, the chemically active ingredient includes a weak local action compound and/or a cytotoxic drug; when the chemically active ingredient is a cytotoxic drug, the amount ratio of the animal non-pathogenic cell-related component to the cytotoxic drug (cytospecific volume/cytotoxic drug weight-volume percent concentration) is (2-100)/(0.1-1); when the chemically active ingredient is a weak local action compound, the animal non-pathogenic cell-related group The amount ratio (cytospecific volume/weight-volume percent concentration of weakly locally acting compound) of fraction and weakly locally acting compound was (1.5-40)/(0.1-1).

Preferably, the weak topical compound includes one or more of the following group: amino acid nutrients, vital dyes, quinine compounds, carbohydrate nutrients.

Preferably, the weakly locally acting compound is an amino acid nutrient, and the ratio of the animal cell-related components to the amino acid nutrient (cytospecific volume/amino acid nutrient weight-volume percentage concentration) is (1.5-7)/( 0.1-1), .

Preferably, the weak local action compound is a vital dye, and the ratio of the cell-related components to the vital dye (cytospecific volume/vital dye weight-volume percent concentration) is (2-40)/(0.1 -1).

Preferably, the weakly locally acting compound is a glyconutrient, and the amount ratio of the cell-related component to the glyconutrient (cytospecific volume/weight-volume percent concentration of glyconutrients is (1.5-7)/ (0.3-1).

Preferably, the chemically active ingredients include vital dyes and one or more other such chemically active ingredients.

Preferably, the chemically active ingredients include cytotoxic drugs, amino acid nutrients and/or vital dyes.

Preferably, the amino acid nutrients are selected from 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 nutrients are present in the topical pharmaceutical composition at a concentration of >2.5%, or 5-30%, preferably 5-25%.

Preferably, the vital dye includes 2.5%-20% red Bengal and/or ≥0.25%, or 0.25-2.5%, preferably 0.5-2.5% methylene blue dyes, and the methylene blue dyes include One or more of methyl blue, patent blue, isosulfur blue and new methylene blue.

Preferably, the carbohydrate nutrients are selected from one or more of the following monosaccharides and sugar derivatives containing the following monosaccharides: glucose, fructose, galactose, ribose, xylose, mannose, sorbose, wherein the The sugar derivatives are preferably selected from one or more of the following groups: nutritional polysaccharides, sugar acids, sugar salts, sugar alcohols, and wherein the concentration (w/v) of the sugar nutrients is ≥ 10%, preferably 15-40 % or 25-40%.

Preferably, the cytotoxic drug is selected from one or more of the following groups: drugs that disrupt DNA structure and function, such as cyclophosphamide, carmustine, metal platinum complexes, doxorubicin drugs, Topotecan, irinotecan; drugs that interfere with transcription of RNA intercalated in DNA, such as antitumor antibiotic drugs; drugs that interfere with DNA synthesis, such as 5-fluorouracil (5-Fu), furofluorouracil, difurfluorouracil, cytarabine , cyclocytidine, 5-azacytidine; drugs that affect protein synthesis, such as colchicine-type drugs, vinblastine-type drugs, taxane-type drugs, and in the pharmaceutical composition, the cytotoxic drugs The concentration of ≥0.1%, 0.1-15%.

Preferably, the biologically active ingredients are selected from one or more of the following groups: antigens, immunomodulatory antibodies, cytokines, and adjuvants.

Preferably, the antigens are selected from microbial antigens or tumor antigens, and the microbial antigens are selected from antigens derived from one or more of the following microbial groups: bacteria, such as Streptococcus pyogenes, Serratia serratia, BCG, Clostridium tetanus, Clostridium butyricum, Lactobacillus acidophilus, Bifidobacterium; viruses such as hepatitis B virus, adenovirus, scab virus, vaccinia virus, mumps virus, Newcastle virus, polio virus, measles Viruses, Seneca Valley Virus, Coxsackie Virus, Reovirus; Parasites, such as Plasmodium; said immunomodulatory antibodies are selected from one or more of the following groups: Antibodies against inhibitory receptors; Blocking agents, such as blocking antibodies against CTLA-4 molecules and PD-1 molecules; antibody blocking agents against ligands for inhibitory receptors, activating antibodies against immune-responsive cell surface stimulatory molecules, such as anti-OX40 antibodies , anti-CD137 antibody, anti-4-1BB antibody; neutralizing antibody against immunosuppressive molecules in the solid tumor microenvironment, such as anti-TGF-p1 antibody; the cytokine is selected from one or more of the following: tumor necrosis factor , interferon, interleukin.

A pharmaceutical composition comprising an animal non-pathogenic cell-associated component and a suitable pharmaceutically acceptable carrier for use in the treatment of local disease, including solid tumors.

The present invention also discloses a method for treating a local lesion disease, comprising the steps of: promoting the immune effect related to the local action (or local synergy) within the local lesion or/and outside the lesion of the individual in need thereof. Secondary effects of local action sensitive topical administration of a therapeutically effective amount of a pharmaceutical composition according to any one of claims 3-23.

Preferably, the step includes the step of administering a therapeutically effective amount of the pharmaceutical composition to the individual's local lesion, or both within and outside the local lesion.

Preferably, the individual is selected from one or more of the following: an immunosuppressed individual, an individual with an internally administrable local lesion, an individual with a chemically ablated or chemical-like local diseased tissue, Individuals with local lesions that act to produce in situ antigens or in situ adjuvants, and individuals with local effects in areas of extra-lesion administration that can form primary or secondary antigens or adjuvants.

Preferably, 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.

Preferably, the administration amount of the pharmaceutical composition is ≥1ml, or the administration amount within the local lesion is 10-150ml or/and the administration amount outside the local lesion is 1.5-50ml.

Preferably, one or more treatments of chemotherapy, immunotherapy, radiation therapy, surgery, chemical ablation, and physical ablation are also optionally performed before, during or after the administration of the pharmaceutical composition.

Preferably, the therapeutic action includes local treatment or/and immunotherapy involving local action (or local synergy), wherein the local action (or local synergy) includes local chemical action (or local chemical synergy) and any said local treatment includes chemical-like ablation of one or more local lesions and optionally other chemotherapy; said immunotherapy includes said local effects within and/or outside of said lesions (or local synergy) secondary immunization and optionally other immunizations.

Preferably, suitable individuals for the treatment include one or more of the following groups: immunosuppressed individuals, individuals who can be administered in local lesions, individuals whose local diseased tissue can be chemically ablated, and secondary immunity can be generated in local lesions Individuals of the substance, individuals who can produce secondary immune substances in the area of extra-lesion administration.

The present invention also discloses a pharmaceutical kit comprising one or more individual containers containing the pharmaceutical composition according to any one of claims 3-23. Preferably, instructions or labels on how to administer the pharmaceutical composition to an individual in need thereof may also be included. Preferably, said administering comprises intralesional administration, or both intralesional and extraterrestrial administration, wherein said extralocalized administration, for example, comprises subcutaneous injection under the armpit of said individual. Preferably, 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. Preferably, the administration amount of the pharmaceutical composition is ≥1ml, or the administration amount within the local lesion is 10-150ml or/and the administration amount outside the local lesion is 1.5-50ml.

The locally diseased diseases include tumors, non-tumoral enlargements, local inflammation, secretory gland dysfunction, and skin diseases, wherein the tumors include malignant and non-malignant solid tumors. Preferably, 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, Stomach cancer, throat cancer, testicular cancer, vaginal cancer, uterine cancer, ovarian cancer, brain tumor, lymphoma.

The embodiments according to the present invention have the following advantages compared with the prior art: compared with the existing cytotoxic drugs, show almost non-toxic systemic safety and significantly higher long-term efficacy; compared with the existing molecular targeted drugs Compared with existing chemical ablative agents, showed significantly lower local irritation than existing chemical ablative agents. sex 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. In addition, 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.

Detailed ways

It should be noted that the embodiments of the present invention and the features of the embodiments may be combined with each other under the condition of no conflict. It is well known that a substance is used as an active ingredient in the treatment of local lesions because it can show that therapeutic activity under certain conditions. The same substance can show different activities or different pharmacology under different conditions. The development of new pharmacology of existing substances, as well as the development of active new substances, is all about introducing therapeutic effects that are more beneficial to the individual. For example, ethanol has a long history of being used as a bactericide in drug preparation. However, it was not until the 1980s that scientists discovered that high concentrations of ethanol under specific conditions (intratumoral administration) could show chemical ablation pharmacology against tumor tissue. Therefore, in the next 20 years, high-concentration ethanol was widely used in the chemical ablation treatment of local lesions such as tumors.

The inventors have unexpectedly discovered in the tumor-bearing nude mice experiments that are usually used as a chemotherapy model, animal tissue semi-fluid can produce meaningful local chemical effects, possibly even chemical-like ablation, under certain specific conditions. Animal cell components, such as leukocyte concentrates, which are very different in composition and structure, actually show this new pharmacology. These specific conditions are not the application conditions of the non-pathogenic cell-related components of animals in the prior art, but are as defined below.

The present application discloses the application of animal non-pathogenic cell-related components in the preparation of topical pharmaceutical compositions for the treatment of local pathological diseases. In one embodiment, the pharmaceutical composition may further comprise a chemically active ingredient and/or a biologically active ingredient that synergizes with the non-pathogenic cell-associated component of the animal, and the cell-associated component is associated with the The amount ratio of chemically active components (cytospecific volume/chemically active component concentration) is (1.5-100)/(0.1-1), and/or the amount ratio of the cell-related components to the biologically active components (cell ratio) volume/concentration of biologically active ingredients) is (60-180)/(0.1-60).

The present application provides a topical pharmaceutical composition for treating local pathological diseases, comprising as a therapeutic active ingredient an animal non-pathogenic cell-related component. In one embodiment, the pharmaceutical composition further comprises a chemically active ingredient and/or a biologically active ingredient capable of producing a synergistic effect with the non-pathogenic cell-associated component of the animal, and the cell-associated component and the The amount ratio of chemically active components (cytospecific volume/chemically active component concentration) is (1.5-100)/(0.1-1), and/or the amount ratio of the cell-related components to the biologically active components (cell ratio) volume/concentration of biologically active ingredients) is (60-180)/(0.1-60).

The present application also discloses a method for the treatment of a local diseased disease, comprising the steps of: promoting an immune effect related to local action (or local synergy) within or/and outside the local lesion of an individual in need thereof. A therapeutically effective amount of a pharmaceutical composition according to the present disclosure is administered topically.

In the context of the present disclosure, the term "cell" refers to native cells and analogs thereof, wherein the analogs include engineered cells that are similar to native cells, as well as derivatives of native cells or engineered cells. The term "non-pathogenic cell" refers to a cell that does not cause a pathogenic effect in an individual. The term "animal non-pathogenic cell-associated component" (abbreviated as animal cell-associated component or cell-associated component in the present invention) refers to a pharmaceutically acceptable component derived from an animal and associated with non-pathogenic cells and analogs thereof, which include non-pathogenic cells, components comprising non-pathogenic cells (such as animal tissues), pharmaceutically acceptable components contained in non-pathogenic cells (such as components contained in animal cells), and their analogs (eg cell derivatives).

The term "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. The term "active ingredient" refers to a substance that can perform a specific pharmacology under specific conditions. The term "pharmacological method" refers to the administration method necessary to achieve a specific pharmacology, eg, the pharmacological method of immune cells as the active ingredient of immune action is intravenous injection. The term "pharmacological 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. Different pharmacology generally requires different preferred pharmacological compositions (active ingredient, active kinetic conditions, dosage form conditions, etc.). The term "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. The term "dosage form conditions" refers to the pharmacological reaction environmental conditions that must be met in order to ensure that the active ingredient achieves specific pharmacology. The same substance used as different active ingredients may need to meet different dosage form conditions.

The term "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). The term "therapeutic effect" is distinguished from 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. The term "obvious 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.

The term "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 following differences: 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.

The term "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. The term "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.

The term "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) 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). In the prior art, the non-pathogenic cell-related components of animals used are mainly used for their conventional activities after entering the blood.

The term "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. The term "topical chemical action" refers to a localized action that includes chemical action. The term "local synergy" refers to a local effect that includes synergy. The term "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. The term "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. The term "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). The term "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.

The term "secondary 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.

Within the scope of the present invention, said cell-associated components are locally or separately localized together with said synergistic co-uses (chemically active and/or biologically active components) capable of producing a synergistic effect with the non-pathogenic cell-associated components of the animal For administration, the separate local administration includes sequential administration of the cell-related components and the synergistic compound or administration in different local areas. In one embodiment, the animal cell-related component is contained in the same medicament as the co-active ingredient.

In the context of the present disclosure, the term "synergistic effect" refers to a particular pharmacological property of a particular active ingredient (eg, an animal cell-related component) under particular conditions, such that the particular active ingredient interacts with other active ingredients ( The combined effects (eg, short-term efficacy and/or mid- to long-term efficacy) of chemically active ingredients, as in this application, exceed the expected sum of their individual effects. The term "local synergy" refers to a local effect that includes synergy. The term "topical chemical synergy" refers to topical chemical effects that include synergy.

In one embodiment, the pharmaceutical compositions disclosed in accordance with the present application have the desired pharmacological composition such that the cell-associated components provide the therapeutic effect. In one embodiment, the pharmaceutical composition comprises an animal non-pathogenic cell-associated component and the pharmacological composition necessary for the cell-associated component to provide a local effect (or/and a local synergistic effect) and optionally other effects , for example, one or more of the following group: preferred pharmacologically active ingredient, active ingredient pharmacological content (pharmacological concentration, or/and pharmacological volume), active ingredient local pharmacological environment. In one embodiment, the cell-associated component is used as an active ingredient in a use as defined in the present disclosure. In one embodiment, the pharmaceutical composition further comprises a liquid carrier, such as water for injection.

In the method for treating local pathological diseases disclosed according to the application, the animal cell-related component and the synergistic compound capable of producing a synergistic effect are contained in different pharmaceutical agents. In one embodiment, administration of the chemically active ingredient precedes administration of the animal cell-associated component.

In the uses, compositions and methods disclosed according to the present application, the therapeutic effect includes local treatment or/and immunotherapy involving local action (or local synergy). In one embodiment, the local effect (or local synergy) includes a local chemical effect (or local chemical synergy) and optionally other effects. In one embodiment, the local treatment includes chemo-like ablation of one or more local lesions and optionally other chemotherapy. In one embodiment, the immunotherapy comprises the locally acting (or locally synergistic) secondary immune effects and optionally other immune effects within the lesion or/and outside the lesion. In one embodiment, the local effect (or local synergistic effect) is preferably a pathogen (cellular, viral or bacterial) proliferation inhibitory effect independent of the local lesion.

Within the scope of the present disclosure, the term "immunotherapy" is distinguished from the term "immunopotentiation", which refers to the immunization of which alone can achieve a therapeutic effect (eg, the immunization of therapeutic vaccines, specific antibodies, etc.), and the latter The first refers to the immune effect that cannot achieve the therapeutic effect but still has an auxiliary effect when used alone (for example, the immune enhancer has the effect of improving the immune function of the body). The term "secondary 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). The term "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. The term "secondary 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. The term "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.).

In the scope of this disclosure, the term "vaccine antigen" is distinguished from the term "antigen", which refers to any substance that can induce an immune response in the body, while the former refers to a substance that can induce the body to produce a specific disease and achieve a therapeutic effect antigens, for example the same substance can be used as vaccine antigen and immunopotentiator antigen through very different technical schemes. The term "adjuvant" refers to a substance in a vaccine that enhances the immunotherapeutic effect of its antigen. The term "antigen in situ" refers to a secondary immune substance in situ that can act as an antigen. The term "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.

In the present disclosure, the applicable subject for the treatment is selected from one or more of the following groups: immunosuppressed subjects, subjects who can be administered in local lesions, subjects in which local lesion tissue can be chemically ablated, subjects in local lesions Individuals who can produce secondary immune substances, and individuals who can produce secondary immune substances in the extra-lesion administration area.

In the scope of the present invention, the term "immunosuppressed individual" refers to any individual who can be represented by the tumor-bearing nude mouse model, such as the composition whose immune function is at a lower level for any reason and is in the technical solution of the present invention Individuals who cannot achieve normal levels within the time period in which local action can be provided (eg, within 7 days of the first administration in Example 2 below) by other methods (eg, immune enhancement), such as those who are immunocompromised and difficult to receive radiation therapy or conventional chemotherapy .

In one embodiment, the therapeutic effect is the local treatment or/and the immunotherapy involving the local effect (or local synergy). In one embodiment, the applicable subject for the treatment is selected from one or more of the group comprising immunosuppressed subjects, intralesional administration and the local lesion tissue can be chemically ablated or/and intralesional Individuals who can produce secondary immune effects, and individuals who can produce secondary immune effects in the extra-lesion administration area. In one embodiment, the composition is a chemoablation-like-in situ and ex situ immunotherapy drug.

In one embodiment, the therapeutic effect comprises the immunotherapy involving the local effect (or local synergistic effect), and the immunotherapy comprises the local effect (or local effect) within the lesion or/and outside the lesion local synergy) secondary immunization and optionally other immunizations. In one embodiment, the subject for treatment is selected from the group comprising one or more of the following: intralesional administration can be performed and the local lesion tissue can be ablated chemically or/and secondary lesions can be generated in the local lesion Individuals with immune effects, individuals with secondary immune effects in the extra-lesion administration area. In one embodiment, the composition is an in situ or/and and an ex situ immunotherapeutic drug.

In one embodiment, said therapeutic effect is a class comprising said local treatment or/and immunotherapy involving said local effect (or local synergistic effect), and wherein said local treatment comprises one or more local lesions Chemical ablation and optionally other chemotherapy; said immunotherapy includes said locally acting (or locally synergistic) secondary immune effects and optionally other immune effects within said lesions. In one embodiment, the subject suitable for the treatment is selected from one or more of the following group: immunosuppressed subjects, subjects subject to local intralesional administration, subjects subject to chemo-like ablation of local lesion tissue, subjects subject to local lesions Individuals with secondary immunity. In one embodiment, the composition is a chemoablation-in situ immunotherapy-like drug.

In one embodiment, the therapeutic effect is a local treatment involving local action (or local synergy), and wherein the local treatment comprises chemo-like ablation of one or more local lesions and optionally other chemotherapy . In one embodiment, the subject for treatment is selected from one or more of the group comprising immunosuppressed subjects, subjects subject to local intralesional administration, subjects subject to chemo-like ablation of local lesion tissue. In one embodiment, the composition is a chemo-like ablative drug.

In one embodiment, the therapeutic effect is an immunotherapy involving a local effect (or local synergy), and wherein the immunotherapy comprises a secondary immunization of the local effect (or local synergy) within the lesion effects and optionally other immune effects. In one embodiment, the subject for the treatment is selected from individuals who can be administered in a localized lesion and where the localized lesion can produce secondary immune effects. In one embodiment, the composition is an in situ immunotherapeutic drug, eg, an immunotherapeutic drug that provides in situ vaccine activation and optionally other immune effects in the target region within the lesion.

In one embodiment, the therapeutic effect is an immunotherapy involving a local effect (or local synergy), and wherein the immunotherapy comprises a secondary immunization of the local effect (or local synergy) outside the lesion effects and optionally other immune effects. In one embodiment, the eligible individuals for the treatment are selected from individuals in the area of extra-lesional administration that develop secondary immune effects. In one embodiment, the composition is an immunotherapeutic agent that provides extra-lesional secondary immunity and, optionally, other immune effects. In one embodiment, the extra-lesional secondary immunization includes the locally acting (or locally synergistic) secondary immunization of abnormal structures (eg, nodules). In one embodiment, the immunotherapeutic drug includes, for example, a vaccine-like drug.

In the use, pharmaceutical composition or method as defined in accordance with the present disclosure, the animal cell-related component is selected from those that provide one or more of the following group of effects: the local effect, the local effect Secondary effects, immune effects of the topical administration. In one embodiment, the secondary effects include immune effects involving secondary immune substances in the administration area.

In one embodiment, the animal is selected from animals with minimized immunogenicity, preferably from autologous or/and allogeneic animals with minimized transplantation responses. In one embodiment, the non-pathogenic cell-associated components are one or more selected from animal tissue components or/and animal cell components, preferably animals, wherein the animal tissue components are selected from the following One or more of the group: animal tissue, tissue fragmentation of animal tissue, cell fragmentation of animal tissue; the animal cell component is selected from one or more of the following groups: animal natural cells, natural cell destruction group components, and derivatives or engineered analogs of said native cells or native cell disrupting components.

Within the scope of the disclosure of the present application, the allograft acceptable for the transplant response is preferably one or more selected from the group consisting of: Severely injured allogeneic in an individual.

In the context of this disclosure, the term "animal cell component" refers to preparations derived from animal cells or analogs thereof, including, for example, animal cells, components comprising animal cells, and derivatives thereof (eg, amplified or/and activated cells, cell disruption components). The term "animal tissue fraction" refers to preparations derived from animal tissue (including cells) or the like, including, for example, animal tissue and derivatives thereof (eg, animal tissue disrupted fractions).

In one embodiment, the tissue is selected from one or more of connective tissues such as blood, bone marrow, ridges, preferably blood.

In one embodiment, the tissue is selected from one or more of one or more of non-connective tissues, such as tissue contained in the following organs: intestine, stomach, meat, pancreas, spleen, liver , lung, cartilage, joint, skin, placenta, umbilical cord. In one embodiment, the non-connective tissue is preferably the spleen, placenta or/and umbilical cord.

In one embodiment, the cells comprise blood cells, the blood cells being one or more selected from the group of cells and derivatives thereof: red blood cells, white blood cells, platelets. In one embodiment, the cells include white blood cells and red blood cells. In one embodiment, the cells comprise leukocytes. In one embodiment, the leukocytes are one or more of the following cells and derivatives thereof: granulocytes, monocytes, lymphocytes. In one embodiment, the cells are selected from one or more including lymphocytes. In one embodiment, the lymphocytes are one or more of the following cells and derivatives thereof: T cells, B cells, null cells. In one embodiment, the blood cell derivative is selected from one or more of the group comprising DC cells, LAK cells, TIL cells generated by in vitro induction, activation, expansion of autologous or allogeneic blood cells , CIK cells, DC-CIK, CTL cells, TCR-T cells, CAR-T cells, NK cells, γδ stem cells, etc.

In one embodiment, the cells comprise one or more selected from the group consisting of skin cells, muscle cells, secretory gland cells, the tissue-rich cells, stem cells. In one embodiment, the stem cells are eg mesenchymal stem cells, hematopoietic stem cells.

In a use, pharmaceutical composition or method as defined in accordance with the present disclosure, the animal cell-associated component is highly deviated from its native state. In one embodiment, the animal tissue semi-fluid-like component is highly deviated from the native state. In one embodiment, the animal cell component is highly deviated from the native state. In one embodiment, the high degree of deviation from the native state includes one or more of the following processed states: a change in the native composition (eg, dissection of blood vessels or fascia within a tissue, a large increase in hematocrit, etc.), Changes in native structure, segmentation of tissue mass), severe damage in native state, concentrations much higher than native concentrations.

In one embodiment, the severe damage is selected from the group consisting of damage caused by one or more of the following treatments: coagulation treatment, mechanical disruption, ultrasonic treatment, heat treatment, freeze-thaw treatment, irradiation treatment. It is well known that treatment of severe tissue damage can alter tissue composition (eg, dissection of blood vessels or fascia within the tissue), structure (eg, marrow or dorsal medullary aspirate), morphology (eg, from fluid or solid to semi-fluid), thereby allowing the tissue to move away from natural state. Severe damage to cells (e.g., mechanical damage to cells, ultrasonic damage, thermal damage, freeze-thaw damage, radiation damage, chemical damage) can also change cell composition, structure, cell aggregate morphology (e.g. from fluid or solid to semi-fluid) ), thereby keeping cells or cell aggregates away from their native state. In addition to the possibility of loss of physiological functions away from the natural state (for example, the tissue can no longer be used for organ transplantation or tissue transplantation, and cell proliferation is weakened), it may also be treated as a major trauma by the body's immune system (for example, its predominant antigenicity is no longer heterogenous). Gene antigenicity) recognition and response (antigenicity).

In the scope of the present disclosure, the term "severe injury" refers to a state of injury that not only loses physiological function, but can be recognized and eliminated by the body's immune system as a major trauma. The term "mechanical disruption" refers to disruption using machinery, including mechanical dissection (eg tissue sampling) and shear disruption (rotational speed > 10 rpm, preferably 10-50,000 rpm). Solids or semi-solids can be turned into granules after mechanical disintegration. In addition, semi-solid and semi-fluid (eg ridges) are also severely damaged by mechanical breakage when entering the syringe or injection. The term "coagulation" refers to a process of converting a liquid into a solid or semi-solid, selected from coagulation processes including any liquid tissue known in the art, such as: self-coagulation (eg, self-coagulation of blood), thermal coagulation (eg, Thermal coagulation of blood), coagulation of coagulants (eg coagulation of blood by coagulants). Among them, thermal coagulation may be performed by heat treatment, and coagulation by a coagulant may be performed by adding a coagulant to a liquid (eg, adding thrombin and calcium chloride to blood). The term "sonication" refers to placing an object to be treated (eg, blood, tissue particles) into an ultrasonic device for sonication (eg, operating frequency of 2-60 kHz) to damage its structure. The term "thermal treatment" refers to heating selected from the group consisting of one or more of: direct heat treatment, steam heat treatment, freeze drying heat treatment, microwave heat treatment, radio frequency heat treatment, laser heat treatment. The heat treatment temperature is ≥40°C, preferably 60°C-115°C. For example, blood can be heat treated as described above. The term "freeze-thaw treatment" refers to treatment including freezing treatment and thawing of frozen objects, wherein the freezing treatment is selected from mechanical refrigeration or/and liquid nitrogen refrigeration, and the refrigeration temperature is ≤-60°C, preferably -60°C -160℃. For example, blood, tissue pellets can be freeze-thawed as described above. The term "irradiation treatment" refers to treatment with an irradiation intensity of 20-100 Gy, which is selected from one or more of the following: X-ray irradiation treatment, γ-ray irradiation treatment, photosensitizing drug+ultraviolet irradiation treatment. For example, the irradiation treatment as described above can be performed on blood and tissue particles.

In the use, pharmaceutical composition or method defined according to the present disclosure, the composition of the composition must satisfy the conditions of the pharmacological concentration required for the animal cell-associated component to provide the effect, wherein the pharmacological concentration (topical The dosing cell volume) is > 20%, > 30%, 30-90%, preferably 45-90% or 75-85%.

In the context of the present disclosure, unless otherwise stated, the term "concentration" refers to the percent concentration of the specified component in the drug (or composition), wherein, for animal components, the concentration of the cell-associated component It refers to the cell specific volume of the cells contained in the component or/and the source cells corresponding to the components contained in the cells in the composition; the concentration of other active components refers to the weight-volume percentage concentration of the component. The term "cytospecific volume" refers to the volume-volume percent concentration of cells contained in an animal cell-related component or/and derived cells in a composition.

The term "pharmacological 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. The term "formulation concentration" refers to the concentration of the specified component in the form of the pharmaceutical formulation (eg, injection or perfusate). The term "administration concentration" refers to the concentration of the specified component in the administered form of the pharmaceutical formulation (eg, a dilution of the formulation). The term "initial intralesion concentration" refers to the concentration of a given component in a medicated medium (eg, medicated blood) when the drug enters the lesion. Even if the administration concentration of the animal cell-related components of the composition of the present invention is the same as the administration concentration of the animal cell-related components in conventional injections, the initial intratumoral concentrations necessary for their respective pharmacology (local chemical action vs immune enhancement) are also the same. can be very different. And one of the technical 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.

According to the local administration concentration of animal cell-related components in the pharmaceutical composition disclosed in the present application, it is usually the animal in the drug at the end point (eg needle hole, catheter outlet, etc.) of the drug delivery device (syringe, trocar, perfusion catheter, etc.) Cell-associated component concentrations. For injection powders, the administration concentration is the concentration of animal cell-related components in the mixture (eg, suspension) of dry powder and liquid carrier.

In one embodiment, the methods disclosed in accordance with the present application comprise administering a therapeutically effective amount of the pharmaceutical composition to the subject within a local lesion, or both within and outside the local lesion. The individual is one or more individuals selected from the group consisting of immunosuppressed individuals, individuals with locally administrable local lesions, individuals with chemically ablated or chemically ablated locally diseased tissue, individuals with locally actionable local lesions Individuals with local lesions that produce in situ antigens or in situ adjuvants, individuals in which local action in the area of extra-lesion administration can form primary or secondary antigens or adjuvants.

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. Alternatively, the administration amount of the pharmaceutical composition is ≥ 1 ml, or the administration amount within the local lesion is 10-150 ml or/and the administration amount outside the local lesion is 1.5-50 ml.

The term "target" refers to the primary target of pharmacology, eg, cytotoxic drugs against tumor cells, immunomodulatory drugs against regulators of the immune system, chemoablative agents against tumor tissue, and the like. The term "target area" refers to the spatial area (eg, tumor or a part thereof) of the target for which the administration is designed. For example, the target area can refer to a tumor that is the target of this treatment (when the diameter of the tumor is small, the required dose is clinically feasible) or a part of the tumor (when the diameter of the tumor is large, the It is not clinically feasible to administer a single dose).

In one embodiment of the use, pharmaceutical composition or method as defined according to the present disclosure, the pharmaceutical composition is a semi-fluid-like composition. In one embodiment, the animal non-pathogenic cell-associated component comprises an animal cell-associated semi-fluid-like component that renders the composition a semi-fluid-like composition, wherein the semi-fluid-like component is selected from animals One or more of tissue semi-fluid-like components or/and animal cell semi-fluid-like components, and wherein the animal tissue semi-fluid-like components are selected from one or more of the following groups: (e.g. bone marrow, spinal cord , coagulated blood), tissue fragmentation semi-fluid of animal tissue (such as placenta fragmentation, umbilical cord fragmentation, coagulated blood fragmentation), cell fragmentation semi-fluid of animal tissue; the animal cell semi-fluid class component is selected from the following One or more of the group: animal natural cell semi-fluid-based concentrate, natural cell-disrupting component semi-fluid-based concentrate, natural cells or a derivative or engineered analog of natural cell-destroying component semi-fluid-based concentrate.

In the context of this disclosure, the term "semi-fluid species" 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). The term "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. For example, tissues of certain animal organs (eg, muscle mass, liver, stomach, intestine, heart, lung, pancreas, cartilage, joints, etc.), and certain gels that do not flow under pressure (eg, fibrin glue) are semisolid , rather than semi-fluid. While the cell concentrate is a suspension at a conventional concentration, and a semi-fluidic component at an ultra-normal concentration (eg, a hematocrit ≥ 70%, preferably ≥ 75%).

In one embodiment, the semi-fluid comprises a coagulated semi-fluid of liquid tissue (eg, blood) or animal cell components (eg, blood-containing components). In one embodiment, the semi-fluid-based component may also provide sustained release. This is because the semi-fluid is similar to a gel semi-solid, but is more injectable, and the semi-fluid nodules formed in the administration area can controllably release other active ingredients contained in them, resulting in excess of carrier and Therapeutic effect of single agent additive action of the active ingredients.

In the use, pharmaceutical composition or method defined according to the present disclosure, the composition of the composition must satisfy the local pharmacological environment conditions required for the animal cell-related component to provide the effect, wherein the local pharmacological environment requires Minimize the presence of components other than chemically active ingredients capable of producing a synergistic effect, preferably free of inactive ingredients required for pharmacy or/and administration safety in conventional compositions, such as solid excipients in oral formulations and flavoring agents, as well as osmotic pressure enhancers in conventional injections.

In one embodiment, the animal cell-related component is selected from the cell-related water-soluble components, and the chemically active ingredient capable of producing a synergistic effect is water-soluble. In one embodiment, in the pharmaceutical composition, the chemically active ingredients are water-soluble, and the animal cell-related components are selected from animal cell-related water-insoluble components or/and animal cell-related semi-fluid components. In one embodiment, in the pharmaceutical composition, the chemically active ingredient is water-insoluble, and the cell-related component is selected from animal cell-related water-insoluble components or/and animal cell-related semi-fluidic components.

In the application, pharmaceutical composition or method defined according to the present disclosure, the chemically active ingredients capable of producing a synergistic effect are selected from one or more of compounds comprising weak local action and/or cytotoxic drugs. In one embodiment, the weak topical compound is selected from one or more of the group comprising amino acid based nutrients, vital dyes, quinine based compounds. In one embodiment, the chemically active ingredient is preferably selected from the group consisting of vital dyes and one or more other said chemically active ingredients. In one embodiment, the chemically active ingredient is preferably selected from vital dyes and one or more cytotoxic drugs. In one embodiment, the chemically active ingredient is preferably selected from the group consisting of vital dyes and one or more amino acid nutrients. In one embodiment, the chemically active ingredient is preferably selected from amino acid nutrients and one or more cytotoxic drugs. In one embodiment, the chemically active ingredient is preferably selected from amino acid nutrients, vital dyes and one or more cytotoxic drugs.

In one embodiment, when the chemically active compound includes a cytotoxic drug, the amount ratio of the animal cell-related component to the cytotoxic drug (V _{animal cell-related component} /W _{cytotoxic drug} ) is (2-100 )/(0.1-1); when the chemically active compound includes a weak local action compound, the amount ratio of the animal cell-related component to the weak local action compound (V _{animal cell-related component} /W _{weak local action compound} ) is (1.5-40)/(0.1-1), wherein when the weakly locally acting compound is an amino acid nutrient, the amount ratio (V _{animal cell-related components} /W _{amino acid nutrient} ) is (1.5-7)/(0.1 -1); when the weak local action compound is a vital dye, the amount ratio (V _{animal cell-related components} /W vital _dye ) is (2-40)/(0.1-1); when the weak local action compound is a vital dye When the compound includes the quinine compound, the amount ratio of the animal cell-related component to the quinine compound (V _{animal _cell} -related component/W _{quinine compound} ) is (1-40)/(0.1 -1).

In the disclosure of this application, the term "amino acid nutrient" refers to an amino acid compound with nutritional health care effect, preferably selected from the amino acid nutritional medicines and amino acid auxiliary materials with nutritional health care effect contained in the official pharmacopoeia or guideline of each country.

In one embodiment, the amino acid nutrient is one or more selected from the group consisting of amino acids, amino acid salts, oligopeptides and polypeptides. In one embodiment, the amino acid nutrient is preferably an amino acid or a salt thereof selected from the group consisting of amino acids or oligopeptides and polypeptides comprising or consisting of the following amino acids: alanine, valine, leucine, isoleucine amino acid, phenylalanine, proline, tryptophan, tyrosine, serine, cysteine, methionine, threonine, lysine, arginine, histidine, aspartic acid, Glutamic acid, beta-alanine, taurine, gamma aminobutyric acid (GABA), theanine, citrulline, ornithine. In one embodiment, the amino acid-based nutrients are more preferably amino acids or salts thereof selected from the group consisting of oligopeptides and polypeptides comprising or consisting of the following amino acids: arginine, lysine, glycine, cysteine acid, alanine, serine, aspartic acid, glutamic acid.

In one embodiment, the amino acid nutrient concentration is >2.5%, or 5-30%, preferably 5-25%.

In the disclosure of the present application, 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 animals, but has no unacceptable harm to the whole animal.

In one embodiment, the vital dye may be any suitable one known to those skilled in the art, for example, may be one or more organic dyes and hydrates or derivatives thereof selected from the group consisting of methylene blue , Patent Blue, Isosulfur Blue, Bengal Red, Toluidine Blue, Trypan Blue, Basic Blue, Eosin, Basic Magenta, Crystal Violet, Gentian Violet, Neutral Red, Jenners Green B, Saffron . In one embodiment, the dye is selected from the group of methylene blue dyes. In one embodiment, the methylene blue dye may be selected from the group consisting of, for example, the following compounds and hydrates and derivatives thereof: methylene blue, patent blue, isothiocyanate, neomethylene blue. In one embodiment, the methylene blue dye is preferably selected from methylene blue and its hydrates and derivatives.

In one embodiment, the concentration of the vital dye is > 0.25%, or 0.25-10%, preferably 0.25-1.5% or 2.5%-10%. In one embodiment, the concentration (w/v) of the methylene blue dye is ≥ 0.35%, preferably 0.35-2%, more preferably 0.35-1.5% or 0.5-1%. In one embodiment, the concentration (w/v) of other vital dyes (eg, Bengal Red) other than the methylene blue dyes is 1-10%.

In one embodiment, the chemically active ingredient is selected from one or more compounds including quinines. In one embodiment, the quinine compound is selected from, for example, one or more of the following group and derivatives thereof: quinine, quinine monohydrochloride, quinine dihydrochloride, and the concentration of the quinine compound It is ≥ 0.5%, or 0.5-5%, preferably 1.5-5% or 1.5%-3%.

In one embodiment, the chemically active ingredient is selected from one or more including cytotoxic drugs. In one embodiment, the concentration of the cytotoxic drug is > 0.1%, 0.1-15%.

The term "cytotoxic drug" refers to an active ingredient that mainly targets diseased cells or intracellular structures to achieve its drug effect, such as a substance that exhibits anti-proliferation of cancer cells in cell experiments or in tumor-bearing animals, preferably selected from the group consisting of conventional chemotherapy drugs. The term "conventional chemotherapeutic drugs" refers to drugs that can effectively treat local diseased diseases by conventional drugs at safe doses, which are selected from any pharmaceutically acceptable conventional chemotherapeutic drugs, preferably selected from conventional chemotherapeutic drugs known in the art, more It is preferably selected from conventional chemotherapeutic drugs (such as antitumor drugs) that have been approved or will be approved by the official competent administrative departments of various countries (such as FDA or China Food and Drug Administration), or have been or will be listed in the official pharmacopoeia of various countries.

In one embodiment, the cytotoxic drug may be one or more selected from the group consisting of drugs that disrupt DNA structure and function [eg, alkylating agents (eg, cyclophosphamide, carmustine, etc.), metal Platinum complexes (such as cisplatin, carboplatin, etc.), DNA topoisomerase inhibitors (such as doxorubicin, topotecan, irinotecan, etc.), drugs that interfere with transcriptional RNA intercalation in DNA (such as anti- Tumor antibiotics, such as actinomycins, daunorubicin, doxorubicin, etc.), drugs that interfere with DNA synthesis (such as pyrimidine antagonists, such as uracil derivatives 5-fluorouracil, fur-fluorouracil, difur-fluorouracil, cytosine Pyrimidine derivatives, such as cytarabine, cyclocytidine, 5-azacytidine, etc.), purine antagonists (such as oncolytic, thioguanine, etc.), folic acid antagonists (such as methotrexate, etc.), Drugs that affect protein synthesis (eg colchicines, vinblastines, taxanes such as paclitaxel, docetaxel, etc.).

In the pharmaceutical composition disclosed in the present application, the cytotoxic drug may be one or more selected from the group consisting of uracil derivatives, cyclophosphamide, gemcitabine, epirubicin, anti- Tumor antibiotics, teniposide, metal platinum complexes, taxanes; preferably one or more selected from the following drugs and their similar derivatives: 5-fluorouracil, cyclophosphamide, gemcitabine, epirubicin Star, antitumor antibiotics, teniposide, metal platinum complexes, paclitaxel.

In a specific embodiment, the concentration (w/v) of a cytotoxic drug (eg, cyclophosphamide, carmustine, etc.) selected from the alkylating agent in the pharmaceutical composition is 0.5-6%, It is preferably 0.75-1.5%.

In a specific embodiment, the concentration (w/v) of the cytotoxic drug (eg, cisplatin, carboplatin, etc.) selected from the metal platinum complexes in the pharmaceutical composition is 0.03-0.15%, preferably 0.05-0.15%.

In a specific embodiment, the concentration (w /v) is 0.05-0.20%, preferably 0.075-0.15%.

In a specific embodiment, the concentration (w/v) of cytotoxic drugs (eg actinomycins, daunorubicin, etc.) selected from the antitumor antibiotics in the pharmaceutical composition is 1-4 %, preferably 1-2%.

In a specific embodiment, a cytotoxic drug selected from the group consisting of said pyrimidine antagonists (eg, uracil derivatives 5-fluorouracil, furfluorouracil, difurfluorouracil, cytosine derivatives cytarabine, cyclocytidine, 5-fluorouracil The concentration (w/v) of azacytidine, etc.) in the pharmaceutical composition is 0.5-2%, preferably 0.75-1.5%.

In the application, pharmaceutical composition or method defined according to the disclosure of the present application, the bioactive ingredient capable of producing a synergistic effect is selected from one or more of the following groups: antigen, immunomodulatory antibody, cytokine, adjuvant .

In one embodiment, the other biologically active ingredient is selected from one or more including antigens, wherein the antigens are selected from microbial antigens or tumor antigens, and wherein the microbial antigens are selected from the group consisting of: One or more antigens: Bacteria, such as Streptococcus pyogenes, Serratia serratia, BCG, Clostridium tetani, Clostridium butyricum, Lactobacillus acidophilus; viruses, such as hepatitis B virus, adenovirus, scar simplex Herpes virus, vaccinia virus, mumps virus, Newcastle disease virus, polio virus, measles virus, Seneca Valley virus, Coxsackie virus, reovirus; parasites, such as Plasmodium; the tumor antigens are One or more selected from the group consisting of breast cancer, pancreatic cancer, thyroid cancer, nasopharyngeal cancer, prostate cancer, liver cancer, lung cancer, colon cancer, oral cancer, stomach cancer, colorectal cancer, bronchial cancer, throat cancer, testicular cancer Cancer, vaginal cancer, uterine cancer, ovarian cancer, malignant melanoma, brain tumor, renal cell carcinoma, astrocytoma, glioblastoma.

In one embodiment, the other biologically active ingredient is selected from one or more including immunomodulatory antibodies. In one embodiment, the immunomodulatory antibody is selected from one or more of the following group: antibody blocking agents against inhibitory receptors, such as blocking antibodies against CTLA-4 molecules and PD-1 molecules ; Antibody blockers against ligands of inhibitory receptors, activating antibodies against stimulatory molecules on the surface of immune-responsive cells, such as anti-OX40 antibodies, anti-CD137 antibodies, anti-4-1BB antibodies; Neutralizing antibodies to inhibitory molecules, such as anti-TGF-p1 antibodies. In one embodiment, the content of the immunomodulatory antibody drug in the composition is >0.1%, preferably 0.25-10%.

In one embodiment, the other biologically active ingredient is selected from one or more including immunomodulatory antibodies, wherein the cytokine is selected from one or more of the following: tumor necrosis factor, interferon, interleukin. In one embodiment, the cytokine content in the vaccine is >0.1%, preferably 0.25-3%.

The dosage form of the pharmaceutical composition disclosed according to the present application is a topical dosage form. The pharmaceutical compositions disclosed in accordance with the present application can be in any topical dosage form that can contain an animal cell-related component and satisfy the requisite conditions for the animal cell-related component to provide the desired effect. In one embodiment, the topical dosage forms include injections, smears, or ointments.

In the context of the present invention, the term "injectable" refers to a sterile preparation containing the active ingredient and a liquid carrier for in vivo administration. The injections are classified into local injections, intravenous injections, etc. according to the mode of administration, and local injections can be used as local injections only after a given local administration concentration. Injections are classified into liquid injections, semi-fluid injections, powder injections for injection, etc. A powder for injection contains sterile dry powder and a vehicle, the sterile dry powder contains part or all of the active ingredients, and the vehicle contains all the liquid carriers. The concentration of the active ingredient in the injection is the concentration of the active ingredient in the mixture with all the liquid carriers, usually the end point (such as needle hole, catheter outlet, etc.) of the topical drug delivery device (syringe, trocar, injection catheter, etc.) ) of the active ingredient concentration in the liquid medicine. For injection powders, the concentration of the active ingredient is the concentration of the active ingredient in a mixture of sterile dry powder and a vehicle (eg, a reconstituted solution, or the pharmaceutically acceptable liquid carrier).

The pharmaceutical compositions disclosed in accordance with the present application may further optionally comprise excipients. The excipient may be any suitable one known to those skilled in the art, and may include, for example, one or more of the following: dispersion media, preservatives, stabilizers, wetting and/or emulsifiers, solubilizers, Tackifier, etc. The tackifier is, for example, sodium carboxymethylcellulose, carboxymethylcellulose, polyvinylpyrrolidone or gelatin. Such preservatives are, for example, antioxidants such as ascorbic acid.

The pharmaceutical compositions disclosed in accordance with the present application may further optionally comprise a tracer. The tracer may be any suitable one known to those skilled in the art, which may include, for example, lipiodol.

The present disclosure also provides a pharmaceutical kit comprising one or more individual containers containing a pharmaceutical composition according to the present disclosure. The individual containers may, for example, include ampoules, vials, and the like.

In one embodiment, the pharmaceutical kit may further include instructions or labels on how to administer the pharmaceutical composition to an individual in need thereof. In one embodiment, 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. When administered, the ratio of the administered 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. Alternatively, according to the specific conditions of the above local lesions, the administration amount of the pharmaceutical composition is ≥1ml, or the administration amount within the local lesion is 10-150ml or/and the administration amount outside the local lesion is 1.5-50ml.

The pharmaceutical compositions disclosed herein can be prepared by a method of preparing topical pharmaceutical formulations containing the animal cell-related components, the co-uses, and optionally other substances. The topical drug can be a liquid drug or a semi-fluid drug. And the liquid drug can be a solution (eg, a solution in a hydrophilic vehicle, preferably an aqueous solution), a suspension, or an emulsion.

In the disclosure of the present application, the concentration of the pharmaceutical composition and other drugs is greater than or equal to the administration concentration thereof in the pharmaceutical composition of the present invention. When it is greater than the administration concentration in the pharmaceutical composition of the present invention, it can be further diluted for use.

In one embodiment, the preparation of a pharmaceutical composition comprising an animal tissue semi-fluid-like component comprises the steps of: a. providing animal tissue; b. subjecting said animal tissue to semi-fluidization and/or severe injury treatment to A semi-fluid comprising or formed from the tissue is obtained.

In one embodiment, the semi-fluidization treatment includes one or more of the following: viscosification of liquid tissue, coagulation of liquid tissue, mechanical disruption of non-liquid tissue, mechanical disruption of liquid tissue coagulation, non-liquid tissue coagulation Softening of fluid tissue. In one embodiment, the tissue may be severely damaged to obtain a semi-fluid, or the tissue may be prepared to be semi-fluid and then severely damaged. In one embodiment, the severe injury treatment comprises one or more of the following: mechanical disruption, coagulation, ultrasonic injury, thermal injury, freeze-thaw injury, radiation injury, chemical injury.

In one embodiment, when tissue is provided, the corresponding tissue source may be isolated. In the context of the present invention, the separation includes liquid tissue separation and non-liquid (solid or semi-solid) tissue separation. Liquid tissue separation refers to the separation process of obtaining liquid tissue, such as drawing and obtaining normal blood, and concentrating the drawn normal blood to obtain concentrated blood. Non-liquid tissue separation includes, for example, extracting a desired tissue from an organ, or stripping an unwanted component from a portion containing the desired tissue, wherein the unwanted component includes, for example, one or more of the following: fat, tendon, membranes, blood vessels.

In one embodiment, when the tissue is solid or semi-solid, semi-fluidization according to the present disclosure may be performed, for example, by subjecting the tissue to mechanical disruption as disclosed herein to produce a granulated tissue comprising tissue . In one embodiment, the active ingredients disclosed herein are added prior to or/and during the fluidization and severe injury treatment of the tissue.

According to the preparation method of the present invention, the preparation of the pharmaceutical composition comprising the animal tissue cell component of the present invention comprises the following steps: a. providing animal tissue cells; b. Semi-fluid or liquid of cellular components.

In one embodiment, the treating includes concentration. In one embodiment, the treatment comprises cell disruption.

In one embodiment, the semi-fluid comprising the cells or cellular components may be prepared by one or more of the following methods: viscosification of cell-containing fluid, coagulation of cell-containing fluid, cell-containing coagulation mechanical fragmentation of objects.

Following the principles of these methods described above, one skilled in the art can employ any suitable specific method to prepare a variety of specific dosage forms comprising the compositions of the present invention. For example, variations in the pharmaceutical 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).

In the present disclosure, the locally diseased diseases include solid tumors, non-tumorous enlargements (eg, non-tumoral nodules), local inflammation (eg, cervical erosions), secretory gland dysfunction, and skin diseases.

In the context of the present disclosure, the term "focal disease" refers to a disease with symptoms of local disease. The term "local lesions (also referred to as lesions)" refers to 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. For example, when the local disease is a solid tumor, 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 communicating with the tumor (for example, through lymphatic or vascular communication) If the local disease is a non-neoplastic mass, the local disease is an abnormal non-tumor mass, such as hyperplasia, cyst, nodule, etc.; when the local disease is local inflammation When the local lesion is an inflamed area, such as the inflamed surface or inflamed body; when the local lesion is an abnormal secretion, the local lesion is the abnormal source or the secretory gland where it is located. For another example, when the disease is abnormal insulin secretion, the abnormal source is the pancreatic islet, and the local tissue is the pancreatic islet or the pancreas where the pancreatic 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.

In the context of the present invention, the term "tumor" refers to a mass formed due to abnormal proliferation of cells or mutated cells, which includes solid tumors. The term "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, including, for example, the following groups classified by tumor cell type: epithelial cell tumors, sarcomas, Lymphoma, germ cell tumor, blastoma; and tumors named after the organ or tissue in which the tumor cell concentration is located, including, for example, tumors named after the following organs or tissues: brain, skin, bone, muscle, breast, Kidney, liver, lung, gallbladder, pancreas, brain, esophagus, bladder muscle, large intestine, small intestine, spleen, stomach, prostate, green pill, ovary or uterus.

Specifically, the tumors include malignant tumors and non-malignant tumors. Such malignancies include, for example, 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, ovary cancer Cancer, malignant lymphoma, malignant brain tumor, etc. The non-malignant tumors include, for example, breast tumors, pancreatic tumors, thyroid tumors, prostate tumors, liver tumors, lung tumors, intestinal tumors, oral tumors, esophageal tumors, gastric tumors, nasopharyngeal tumors, laryngeal tumors, testicular tumors, vaginal tumors, Uterine tumor, fallopian tube tumor, ovarian tumor, lymphoma, brain tumor, etc.

In one embodiment, the locally diseased disease is selected from non-neoplastic enlargements (eg, non-neoplastic nodules). In one embodiment, the non-neoplastic mass comprises, eg, a secretory gland non-neoplastic nodule. In one embodiment, the secretory glands include, for example, thyroid, breast, liver, lung, intestine (eg, polyps), and the like.

In one embodiment, the local disease is selected from local inflammation (including erosion). In one embodiment, the non-neoplastic enlargement comprises, eg, local inflammation of a secretory gland. In one embodiment, the secretory glands include, for example, the thyroid, breast, liver, lung, intestine, cervix, vagina, and the like.

In one embodiment, the topical disease is selected from skin diseases. In one embodiment, the skin disease includes, eg, chronic mucocutaneous candidiasis, various ringworms.

The pharmaceutical composition disclosed in the present application is a therapeutic drug, and when it is used to treat a disease, it can also be combined with other interventional therapy, systemic chemotherapy, immunotherapy, photodynamic therapy, sonodynamic therapy, surgical intervention or such therapy The combination is administered in combination to further enhance the efficacy.

Based on the studies described in more detail below, although the specific mechanism remains to be further studied, the pharmaceutical composition of the present invention has been shown to promote effective destruction of tumor tissue with minimal damage to individual normal tissue, thereby achieving safe and effective Pharmacological effects in the treatment of solid tumors.

Example

The present invention will be further illustrated by the following specific examples, but not intended to limit the present invention. The embodiments in this application may be combined with each other.

In the following embodiments, the used animal cell-related components are selected from one or more of animal tissue components or/and animal cell components, wherein the animal tissue components are selected from one or more of the following groups: animal Tissue, tissue fragmentation of animal tissue, cell fragmentation of animal tissue; the animal cell component is selected from one or more of the following groups: animal natural cells, natural cell destruction components, natural cells or natural cell destruction groups derivatives or engineered analogs. in:

The experimental animal tissues used were purchased from experimental animal companies or routinely prepared from experimental animals, including: blood, bone marrow, spinal cord, skin, intestine, stomach, meat, pancreas, spleen, liver, lung, cartilage. Human blood and human blood cell concentrates are legally obtained from volunteers. wherein the tissue is preferably selected from autologous or/and allogeneic tissue acceptable for transplantation

The cells or cell derivatives used can be prepared from animal tissue by the prior art or obtained commercially. For example, cell pellets, leukocyte pellets, erythrocyte pellets, platelet pellets obtained from natural blood separation according to the prior art. In another example, hematopoietic stem cells and the like are extracted and prepared from tissues such as bone marrow according to the prior art. For another example, lymphocytes are obtained from the spleen of an animal after lysis and removal of erythrocytes according to the prior art: after the animal is killed, the spleen is taken and gently crushed, and then serum-free DMEM culture medium and erythrocyte lysate (0.0075% ammonium chloride/0.0026 % Tris sterilized water solution and diluted to 500mL), rest for 5 minutes after gentle stirring, centrifuged and washed 3 times (centrifugation condition is 1000rpm/min, the sedimentation resuspended is serum-free DMEM), and finally the lymphocyte pellet is obtained. Another example, by in vitro induction, activation, expansion of autologous or allogeneic blood cells, such as DC cells, LAK cells, TIL cells, CIK cells, DC-CIK, CTL cells, TCR-T cells, CAR-T cells, NK cells, γδ stem cells, etc.

Other compounds used in the following examples, all commercially available, are partially listed in Table 1.

Table 1

In the present invention, L-amino acids are abbreviated as amino acids (for example, L-arginine is abbreviated as arginine).

The experimental animals used in the following examples were purchased from professional experimental animal companies, and all were SPF (Specific Pathogen Free, specific pathogen-free) grade animals. Mice were healthy females 6-8 weeks old, weighing 17.5-20.5 g, unless otherwise stated.

In the following examples, unless otherwise specified, the animal experiments of subcutaneously transplanted cells to generate local lesions (such as tumors) 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. Unless otherwise stated, when the local lesions (such as tumors) grow to the required volume (such as mice bearing tumor 50-500mm ³ ), the modeling is successful, and then the PEMS3.2 software (compiled by West China School of Public Health, Sichuan University) is used. Randomly divided into several experimental groups with 6 animals in each group. Items observed, measured and analyzed in the trial, including general state, body weight, food intake, animal graft-versus-host disease, solid tumor volume, tumor weight, survival time, etc.

The calculation formulas of tumor volume V, relative tumor proliferation rate R, and tumor inhibition rate r are as follows:

Tumor volume V=l/2×a×b ² , where a represents tumor length and b represents tumor width.

Tumor proliferation rate R(%)=TV/CV×100, where TV and CV are the tumor volume of the study group and the negative control group, respectively.

Tumor proliferation inhibition rate r'=100%-R, where R is the tumor proliferation rate.

Tumor inhibition rate r(%)=(CW-TW)/CW×100%, where TW is the average tumor weight of the study group; CW is the average tumor weight of the negative control group.

In the following examples, the efficacy of drug i is denoted as Ei, which can be represented by r'i or ri. The type of action of a drug, i.e. pharmacology, can be studied by its efficacy, especially the efficacy of the same drug in different regimens. For example, when there is little difference in the efficacy of drug i between regimens X and Y (such as Ei _X /Ei _Y <200%), it means that the pharmacology of the drug is the same; When the difference in drug effect is large (eg Ei _X /Ei _Y > 200%), it means that the pharmacology of drug i in scheme X greatly exceeds the expected range of its pharmacology in scheme Y, and it is likely to involve new drugs different from scheme Y. pharmacology. If two drugs show clearly different EiX/EiY relationships, they are likely to involve different pharmacology; if two drugs show similar EiX/EiY relationships, then they are likely to involve the same pharmacology, at least similar pharmacology, Such as chemical ablation pharmacology and chemical ablation pharmacology.

In the following examples, experimental results (such as tumor weight, tumor volume, lesion tissue volume) 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. The test level was α=0.05. P<0.05 indicated a statistically significant difference, while P>0.05 indicated no statistical significance.

Positive controls for chemotherapy include classical cytotoxic drugs (such as 0.5-1% 5-fluorouracil, whose short-term tumor inhibition rate is ≥30% under the conditions of the following examples) and classical chemical ablative agents (such as 75-99% ethanol, Its short-term tumor inhibition rate under the conditions of the following examples is ≥15%).

The pharmacological effects (or efficacy) produced by the combined use of drugs are highly uncertain, and the industry often judges based on the following actual efficacy/expected efficacy ratio q: q=q ₁ /q ₂ , where q ₁ is the actual shared efficacy , q ₂ is the expected additive efficacy derived from the actual single efficacy (EA, EB) of drugs _A and _B. If q=1, it means that the actual shared drug effect is in line with expectations, that is, it shows that the effect is additive; The efficacy of the shared drug exceeds expectations, which means that the drug effect is synergistic.

Among them, there are many calculation methods for q ₂ , such as Burgi method (Burgi Y. Pharmacology; Drug actions and reactions. Cancer res. 1978, 38(2), 284-285); Jin Zhengjun improved the Burgi method (Jin Zhengjun, et al. Probability and curve and "Q50", Journal of Shanghai Second Medical College; 1981, 1, 75-86), where q ₁ =E _A/B is the actual combined efficacy of the combination of drugs A and B (such as tumor inhibition rate ), q ₂ =E _A +E _B -E _A × _EB is the theoretically expected drug effect based on the simple addition of the actual drug effects (EA and _EB ) of drugs _A and B. On the basis of the q calculation of the above-mentioned Jin Zhengjun method, Zhang Xiaowen, Jin Zhengjun, etc. further made the q judgment more suitable for the experimental reality (Zhang Xiaowen et al., A new method for estimating the effect of combined medication with Q value: "Double 30 Method", Journal of Shanghai Second Medical College , 1985, 5, p353-354). They reflect purely additive expected effects with q=0.85-1.15. In the following examples of the present invention, the shared effect of combined administration according to the Jin Zhengjun modified method is judged as follows:

When the efficacy of the combination of drugs A and B is insignificant (eg, r≦15%), the combination shows no synergy, or is considered to have negligible synergy. When the efficacy of the composition is meaningful (for example, r≧15%), if the actual efficacy/expected efficacy ratio q=1.15, it means that the combined efficacy of the composition is the addition of the individual efficacy, which is consistent with the theoretical simple addition Expected; if the actual efficacy/expected efficacy ratio q>1.15, it means that the combined efficacy of the composition has a significant synergistic effect, that is, the actual effect exceeds the theoretical simple additive expectation; if the actual efficacy/expected efficacy ratio q<1.15, then It shows that the combined efficacy of the composition has obvious antagonism, that is, the actual effect is not as expected by the simple addition of the theory.

It is generally accepted that the co-safety expectations of drugs A and B are limited by the least safe drug (A or B) of the co-drugs. The shared safety of the drug A/B composition is judged by the following method: If the actual safety of the A/B composition is consistent with the theoretical pure additive expected safety (the worst safety of A or B), then the composition The shared safety of A/B is the additive effect of safety; if the actual safety of the A/B composition significantly exceeds the theoretical purely additive expected safety (the worst safety of A or B), then the shared safety of the composition If the actual safety of the A/B composition is significantly less than the theoretical pure additive expected safety (the worst safety in A or B), the combined safety of the composition is obviously antagonistic effect.

Example 1: Preparation of the composition

In accordance with the methods for preparing the compositions of the present invention described above, many different 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 (compositions containing animal cell-related components) and Table 3 (combinations containing animal cell-related components and chemically active ingredients or/and biologically active ingredients) thing). The hematocrit of the non-diseased tissue in the table was provided by the veterinarian of the relevant animal testing company, and the hematocrit of the blood and cell concentrates was determined according to the blood routine determination method (for example, using a fully automatic hematology analyzer BC5000).

Table 2 Components, pharmacological concentrations (cytospecific volume) of compositions comprising animal cell-related components > 20%, 21-90%, ≥ 30%, 30-90%, preferably 45-90% or 75- 85%)

Example 1A: Non-diseased tissue (eg, spinal cord) is selected for species immunogenic minimization desired (for example, for local action or/and its secondary effects), and prepared from animal organs or tissues (for example, from membranous spinal cord via Tissue puncture extraction or removal of adventitia from dissection) to obtain the desired tissue (eg, spinal cord tissue). If the liquid mixture is aliquoted (eg 10ml/bottle) and capped according to the desired drug volume/target volume ratio (eg, the average volume of a clinically common solid tumor is 30cm ³ ), lesions that can provide lesions can be obtained. Dosage forms and strengths of locally acting pharmaceutical compositions. This preparation is A1 in the above table.

Example 1B: Obtain 200ml of human blood with sodium citrate preset as an anticoagulant according to the standard method of the blood bank, put it in a plurality of 20ml beakers, seal the cups with tin foil and heat them (for example, place them in steam for 20- 150 minutes) to obtain thermally coagulated blood. If the liquid mixture is aliquoted (eg 10ml/bottle) and capped according to the desired drug volume/target volume ratio (eg, the average volume of a clinically common solid tumor is 30cm ³ ), lesions that can provide lesions can be obtained. Dosage forms and strengths of locally acting pharmaceutical compositions. The obtained preparation is A2 in the above table. Using the same method as the preparation of A6, the preparation of different compositions (eg preparation A3 in the above table) can be carried out separately from the blood of different animals.

Example 1C: Selection of non-diseased tissue (eg, human placental tissue) for desired (eg, local or/and secondary effects) hematocrit, and preparation from animal organs or tissues (eg, by stripping and removing the obtained human placenta) fat, membranes, tendons, blood vessels) to obtain the desired tissue (eg, human placental tissue). Then, the tissue is placed in a mixer for tissue crushing (for example, the rotation speed is 3000-5000 rpm, and the total time is 1-3 minutes). Tissue semifluid formed by differentiated tissue particles. If the tissue semi-fluid is aliquoted (eg 10ml/bottle) and capped according to the desired drug volume/target volume ratio (eg, the average volume of clinically common solid tumors is 30cm ³ ), the available Dosage forms and strengths of pharmaceutical compositions for local action in lesions. The obtained preparation is A4 in the above table. Using the same method as the preparation of A2, the preparation of different compositions (eg preparations A5-A7 in the above table) can be performed separately from different non-diseased tissues.

Example ID: Selection of non-diseased tissue (eg, human placental tissue) for desired (eg, local or/and secondary effects) hematocrit and preparation from animal organs or tissues (eg, by stripping and removing the obtained human placenta) fat, membranes, tendons, blood vessels) to obtain the desired tissue (eg, human placental tissue). Then the tissue is placed in a homogenizer for cell disruption (for example, the rotation speed is 10000-25000 r/min, the rotation time is 0.5-1 min, and the number of homogenization times is 2-10 times), and the average size of the cross section can be obtained. Cell fragments smaller than 0.3mm x 0.3mm are semi-fluid. If the tissue semi-fluid is aliquoted (eg 10ml/bottle) and capped according to the desired drug volume/target volume ratio (eg, the average volume of clinically common solid tumors is 30cm ³ ), the available Dosage forms and strengths of pharmaceutical compositions for local action in lesions. The obtained preparation is A8 in the above table. Using the same method as the preparation of A8, the preparation of different compositions (eg preparations A9-A12 in the above table) can be carried out separately from different non-diseased tissues.

Example 1E: Obtain 200ml of human blood with the anticoagulant sodium citrate preset according to the standard method of the blood bank, carry out the same heating as described in the preparation of A2 in the above table, and then carry out the same as described in the preparation of A9 in the above table. Cell disruption, or performing the same cell disruption as in the preparation of A9 in the table above, followed by the same heating as in the preparation of A2 in the table above, to obtain a cell disruption semi-fluid. If the liquid mixture is aliquoted (eg 10ml/bottle) and capped according to the desired drug volume/target volume ratio (eg, the average volume of a clinically common solid tumor is 30cm ³ ), lesions that can provide lesions can be obtained. Dosage forms and strengths of locally acting pharmaceutical compositions. The obtained preparation is A11 in the above table. Using the same method as the preparation of A11, the preparation of different compositions (eg preparation A12 in the above table) can be carried out separately from the blood of different animals.

Example 1F: After obtaining tissue (such as blood, bone marrow, etc.), different natural cell concentrations can be obtained by existing cell preparation techniques (such as centrifugation) according to the desired (such as local effect or/and its secondary effect required) cell specific volume. host (eg human leukocyte concentrate). If the liquid mixture is aliquoted (eg 10ml/bottle) and capped according to the desired drug volume/target volume ratio (eg, the average volume of a clinically common solid tumor is 30cm ³ ), lesions that can provide lesions can be obtained. Dosage forms and strengths of locally acting pharmaceutical compositions. This preparation is A13 (suspension or precipitate) in the above table. Using the same method as the preparation of A13, the preparation of different compositions (eg preparations A14-A16 in the above table) can be carried out separately from the blood of different animals. When the cell concentrate obtained in the preparation of A13 contains a sufficient amount of plasma (for example, more than 10%), the preparation is heated (for example, the container is placed in steam for more than 10 minutes) or other coagulation treatment, and the above table is obtained. A17 (semi-fluid).

Example 1G: After obtaining tissue (eg blood) by prior art (eg centrifugation) to obtain a cell concentrate (eg human leukocyte concentrate) according to the desired (eg required for local action or/and its secondary effects) hematocrit ), and then place it in a homogenizer for cell disruption (for example, the rotation speed is 10000-25000 r/min, the rotation time is 0.5-1 minute, and the number of homogenization times is 2-10 times), and the average size of the cross section is less than 0.3mm× 0.3mm of disrupted cells. If the liquid mixture is aliquoted (eg 10ml/bottle) and capped according to the desired drug volume/target volume ratio (eg, the average volume of a clinically common solid tumor is 30cm ³ ), lesions that can provide lesions can be obtained. Dosage forms and strengths of locally acting pharmaceutical compositions. This preparation is A18 in the above table. Using the same method as the preparation of A18, the preparation of different compositions (eg preparations A19, A20 in the above table) can be carried out from different animal cells, respectively.

Example 1H: Natural cell derivatives of desired hematocrit (eg for local action or/and secondary action thereof) are commercially available from specialized laboratories. Its preparation method, for example, after obtaining tissue (such as blood, bone marrow, etc.), target natural cells (such as human leukocytes) can be obtained by existing cell preparation technology (such as centrifugation), and then by existing cell expansion technology (such as cell culture) Expansion, then harvesting the cell pellet and adjusting the cell concentration (specific volume) as needed (e.g. for local effects or/and its secondary effects) to obtain a cell concentrate, such as a human leukocyte (in vitro expansion) concentrate (table above). Preparation A21). For another example, after the bone marrow cell suspension extracted from Balb/c mice was treated with erythrocyte lysate, the remaining monocytes were cultured and expanded in RPMI-1640 medium containing 12% FBS, and the cells were added successively according to the prior art. Factors rmGM-CSF, rmIL-4, rmTNF-α are matured, then the cell pellet is collected and the cell concentration (specific volume) is adjusted as required (for example, for local effects or/and its secondary effects) to obtain cell concentrates, For example, in vitro matured dendritic cell (DC) concentrate (preparation A22 in the table above).

The animal cell-related components obtained by using the preparation methods of Examples 1A-1H above can be determined according to the amount of cell-related components/combination required by the composition of the present invention (for example, for local synergy or/and mid- and long-term synergy). The desired amount and desired hematocrit of the above-mentioned cell-related components (eg, 23.75 g of human placental tissue fragment with a 55% hematocrit) and a commercially available Chemically active ingredients or/and bioactive ingredients (such as 1.25g reduced glutathione) are thoroughly mixed to prepare a composition comprising animal cell-related components and chemically active ingredients or/and bioactive ingredients (see table below) If necessary, the liquid mixture can be dispensed (for example, 10ml/bottle) and capped according to the desired drug volume/target volume ratio (for example, the average volume of clinically common solid tumors is 30cm ³ ), that is, The dosage forms and specifications of the pharmaceutical composition that can provide local action in the lesion can be obtained. The obtained preparation is B1 in the table below.

Table 3 Components and ratios of compositions containing animal cell-related components and co-uses (chemically active ingredients or/and biologically active ingredients)

The above preparations are all injectable suspensions or injectable semi-fluids, and all can be prepared as injections that can be administered locally. If the above preparation is put into a Pasteur inactivation cabinet machine for pasteurization (60° C., 48 hours), a sterile liquid injection can be obtained.

If the above preparation is lyophilized, a lyophilized powder injectable vial can be obtained. The freeze-drying process conditions include, for example: the pre-freezing conditions are kept at the pre-freezing temperature -45°C for 4 hours; the sublimation drying conditions are that the heating rate is 0.1°C/min, and the temperature is raised to -15°C and kept for at least 10 hours; The adsorption drying conditions were kept at 30°C for 6 hours. The liquid carrier (for example, water for injection) is sub-packed (for example, 7.5ml/vial) and capped according to the desired concentration of the non-pathogenic animal cell-related components of the animal, and then the injectable vehicle bottle can be obtained. When in use, the sterile solvent in the bottle is drawn into the above-mentioned lyophilized powder bottle for injection and mixed uniformly to form a liquid drug (for example, 1.5% Saccharomyces cerevisiae broken component/20% amino acid), which can be used as an injection drug .

The following Examples 2-8 conducted pharmacological studies on the therapeutic effects provided by animal cell-related components using an immunosuppressive animal model (nude mice). Nude mice are congenital athymic nude mice obtained by introducing a recessive mutation gene "nu" on the 11th pair of chromosomes into BALB/c mice. The thymus has only remnants or abnormal epithelium, which cannot make T cells differentiate normally, lacks the auxiliary, inhibitory and killing functions of mature T cells, and has low immunity. In the prior art, tumor-bearing nude mice are widely used in drug research for chemotherapy rather than immunotherapy.

Example 2: Short-acting pharmacological research and technical solution optimization in immunosuppressive animal models

The experimental animals were nude mice, and the model cells were human hepatoma HepG2 cells, and 1×10 ⁶ cells per animal were subcutaneously transplanted into the right armpit of the animal to model. The successfully modeled experimental animals (average tumor volume 151.3 mm ³ ) were randomly divided into 14 groups as shown in the table below, and were further divided into series 1 (negative control group 01, study drug groups 1-6) and series 2 ( Negative control group 02, drug research group 7-12). The negative control is normal saline, and the research drugs are shown in the table below, which are prepared according to the preparation method of Example 1. Except for the cell-related components, which are semi-fluid, other drugs are liquid. Wherein the animal tissue component (1) is nude mouse meat tissue fragment (such as the preparation of A5 in Table 2, cell specific volume> 55%), and the animal cell component (2) is nude mouse blood cell concentrate (such as Preparation of B4 in Table 3, hematocrit 73%), the animal cell component (3) is a human blood cell concentrate (as in the preparation of B4 in Table 3, hematocrit 75%). The animals in series 1 and 2 were administered intravenously and intratumorally, respectively. Each group was administered once, and the injection volume was 150 μl per animal. 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 from each series of negative control groups. The results are shown in the following table.

Table 4 Tumor inhibition rate data of different groups

It is generally believed that 5-fluorouracil can provide conventional chemical effects under conventional medication conditions (study group 1), and can provide general topical chemical effects under topical administration conditions (study group 7). The difference in efficacy between the two is mainly Differences in cytotoxicity kinetics (low vs high concentrations of drug acting on intratumoral tumor cells). In the above table, compared with study group 1, the tumor inhibition rate of study group 7 did not increase beyond the expected limit of kinetic improvement based on the latter (E ₈ /E ₁ <200%), indicating that the same substance (5- Fluorouracil) can be used as the same active ingredient (cytotoxic drug) under different action conditions (systemic vs topical).

It is generally believed that high concentrations (75-99%) of ethanol under conventional drug conditions (study group 2) can provide a chemical effect of intoxication-like reactions, and under local administration conditions (study group 8) can provide tumorigenic tissue. The chemical ablation effect of necrosis thereby inhibiting tumor. In the above table, more than 50% of the animals in study group 2 could observe a drunkenness-like reaction within 5 minutes after injection, while the expected cytotoxicity results based on the results of cell experiments were not observed during the experimental period. Compared with study group 2, the tumor inhibition rate of study group 8 was improved far beyond the expected limit of kinetic improvement based on the latter (E ₉ /E ₂ >200%), showing pharmacological differences, indicating the same substance (ethanol) It can be used as a different active ingredient (intoxicating agent vs chemical ablative agent) under different action conditions (systemic vs topical).

Interleukin-12 is used clinically as an immune enhancer. It is well known that even in tumor-bearing animal models with normal immune background, the use of immune enhancers alone can hardly observe obvious tumor efficacy. The results for study groups 3 and 9 in the table above are in line with the expectation that immune enhancers may have an adjunctive but no therapeutic effect.

At present, none of the relevant components of animal cells have been used as active components of chemotherapy, and their indications should not include tumor treatment of tumor-bearing nude mice, especially the effective destruction of their tumor bodies. In the above table, study group 4, like study group 3, did not show significant tumor efficacy, which seems to be in line with the expectations of its current technology. Surprisingly, with the same drug, the difference in tumor inhibition rates between study group 10 and study group 4 far exceeded the expected limit of kinetic improvement for the same drug (E ₁₁ /E ₄ >200%). In addition, the tumor inhibition rate of study group 10 was not lower than 50% of that of study groups 7 and 8. In addition, one animal each died shortly after injection in study group 4, indicating that the intravenous injection of relevant components of animal tissue may have non-negligible safety risks.

In the above table, the study group 5, like the study group 4, did not show obvious tumor curative effect; the study group 11, like the study group 10, showed that the tumor inhibition rate was not lower than 50% of that of the study group 8. The study group 6, like the study groups 4 and 5, did not show obvious tumor curative effect; the study group 12, like the study groups 10 and 11, showed that the tumor inhibition rate was not lower than 50% of that of the study group 8. In the prior art, xenogeneic animal cell-related components have stronger immune effects than autologous animal cell-related components. However, the tumor inhibition rate shown by study group 12 was not significantly higher than that of study group 11.

According to the above results, different animal cell-related components of different species of animals that should provide significantly different effects (such as stronger or weaker immune effects) in the existing scheme, resulting in different drug effects, are unexpectedly present in this The technical solution of the invention shows the effect of similar medicines. The above studies show that conventional drug delivery techniques (oral, intravenous, etc.) must be excluded, and only local, especially intralesional, drug delivery techniques are used to make it possible for animal cell-related components to provide the above-mentioned tumor-bearing patients with it. New functions that cannot be provided in the prior art (eg E ₁₀ /E ₄ >200% in the above table). The following examples further investigate this new function.

In one experiment, the experimental animals were nude mice , which were randomly divided into 2 study groups (1 and 2), with 6 mice in each study group. The study drugs for study groups 1 and 2 were chemical ablatives (75% ethanol in water) and animal cell-related fractions (animal tissue fraction 3 in the previous experiment), respectively. Each group was treated once, and the drug was administered to the muscle mass of the right leg, and the injection volume was 100ul/a. The animals were euthanized on the 7th day after the drug was administered, and the muscle mass of the right leg of the nude mice was dissected out. After sectioning and washing, the area of abnormal areas different from normal muscles, such as necrosis and nodules, was measured.

The abnormal area areas of study groups 1 and 2 were 32.27 ± 16.05 mm ² and 33.74 ± 15.71 mm 2 , respectively. The local effects provided are similar to those of ethanol, which mainly or at least not negligibly include local chemical effects, especially chemical-like ablative effects that are independent of conventional effects (immune, cytotoxic, or tumor vascular destruction). effect), which mainly, or at least not negligibly, includes tissue destruction in the drug penetration zone. These results further confirm the local action pharmacology of animal cell-associated components similar to ethanol. As with ethanol, this local action provided by the animal cell-associated component allows it to be used in any local lesion containing diseased tissue.

In the prior art, the preferred solution for cell-related components to provide their pharmacological functions depends on their immune effects, while the preferred solution for the animal cell-related components that provide the above-mentioned local effects in the composition of the present invention is that they can provide local effects including local effects. Including any animal cell-related components of the therapeutic effect. Furthermore, the latter is preferred between xenogeneic and allogeneic, the latter is preferred between allografts with strong and weak grafting responses, the latter is preferred between allogeneic and autologous, and between mass tissue and fragmentation The latter is preferred between tissues, and the latter is preferred between tissues and cells.

Example 3: Pharmacological study of local action and optimization of its pharmacological concentration

In one experiment, the experimental animals were nude mice, and the modeled cells were breast cancer cells (MDA-MB231), and 1×10 ⁶ cells per animal were subcutaneously transplanted into the right armpit of the animal to model. Successfully modeled experimental animals (average tumor volume 157.1 mm ³ ) were randomly divided into 4 control groups (0, 01-03) and 6 study groups (1-6) as shown in the table below. The negative control is normal saline, the positive control is ethanol, and the research drugs are shown in the table below. The medicines were prepared according to the preparation method of Example 1, wherein the animal tissue component was the broken flesh tissue of nude mice (as prepared as A5 in Table 2, the cell specific volume was >55%), and the animal cell component was nude mouse Mouse blood cell concentrates (prepared as B4 in Table 3, hematocrit 63%), and the study drug are their water-for-injection dilutions (hematocrit is shown in the table below). Each group was administered once, and the injection volume was shown in the table below. Seven days after administration, the animals were euthanized, and the tumor tissue was removed after dissection to measure the tumor weight, and the tumor inhibition rate was calculated from the negative control group. The results are shown in the following table.

Table 5 Tumor weight and tumor inhibition rate data of different groups

组别group	药物drug	注射量Injection volume	瘤重(x±sg)Tumor weight (x±sg)	抑瘤率tumor inhibition rate
00	生理盐水normal saline	150ul150ul	0.241±0.1930.241±0.193	--
0101	50％乙醇50% ethanol	150ul150ul	0.225±0.1110.225±0.111	6.8％6.8%
0202	75％乙醇75% ethanol	107ul107ul	0.185±0.0990.185±0.099	23.1％23.1%
0303	无水乙醇anhydrous ethanol	75ul75ul	0.141±0.0840.141±0.084	41.7％41.7%
11	动物细胞相关组分(细胞比容10％)Animal cell-related fractions (10% hematocrit)	300ul300ul	0.223±0.1060.223±0.106	7.6％7.6%
22	动物细胞相关组分(细胞比容20％)Animal cell-related fractions (20% hematocrit)	200ul200ul	0.190±0.0610.190±0.061	21.3％21.3%
33	动物细胞相关组分(细胞比容30％)Animal cell-related fractions (30% hematocrit)	100ul100ul	0.163±0.0720.163±0.072	32.3％32.3%
44	动物组织组分(细胞比容10％)Animal tissue fraction (10% hematocrit)	300ul300ul	0.221±0.1030.221±0.103	8.5％8.5%
55	动物组织组分(细胞比容20％)Animal tissue fraction (20% hematocrit)	200ul200ul	0.182±0.0890.182±0.089	24.3％24.3%
66	动物组织组分(细胞比容30％)Animal tissue fraction (30% hematocrit)	100ul100ul	0.141±0.0920.141±0.092	41.7％41.7%

Research groups 01-03 injected the same dose of ethanol with different concentrations into the same volume of tumor. It is well known that ethanol can only be used as a chemical ablative agent if it exceeds a concentration threshold (eg, 70%). In the table above, the results of Study Group 01 confirm that the ethanol in this technical solution is similar to intravenous ethanol and fails as the active ingredient to provide chemical ablation. The tumor inhibition rates of study group 02 and study group 01 were very different (E ₀₂ /E ₀₁ >200%), but not significantly different from 03 (E ₀₃ /E ₀₂ <200%), showing the expected chemical ablation effect.

Research groups 1-3 injected the same dose of different concentrations of animal cell-related components into the same volume of tumor. In the above table, there was little difference in tumor inhibition rate between study group 2 and study group 02 (E ₃ /E ₀₂ =92%), and there was no significant difference in tumor weight between them (P>0.05), showing that The animal cell-related components provided the local effect required for the therapeutic effect in the technical solution of study group 2, and the local effect was similar to the chemical ablation effect in the positive control group 02. Study Group 5 also achieved similar results to Study Group 02. The results of study groups 3 and 6 further showed that the local effect was dose-dependent.

It is generally believed that the same drug (such as the same drug for cancer cell suppressing drugs, tumor vascular suppressing drugs, and the same drug for immune drugs) has the same drug effect. However, cell-associated components can achieve different pharmacological effects by providing different pharmacological effects in different technical solutions. For example, in the prior art, cell-related components such as red blood cell concentrates can be injected intravenously to achieve their pharmacological functions (such as immune enhancement), and it can be seen that their pharmacological concentrations are not administered concentrations but blood drug concentrations (usually very low, such as 0.25 ×10 ^-5 %), it can provide its pharmacological effect only if the dosage is satisfied. Therefore, its composition must be composed of only its necessary components, and its formulation composition is only limited by the formulations (for example, high-concentration formulations can save transportation and storage costs, and appropriate high-concentration injections can reduce the injection volume and thus shorten the time. )limit. It is well known that formulation concentrations can be broad, but are essentially different from those required for administration for pharmacological effects (and thus indications). For example, in the case of intravenous injection, the concentration of the drug is often made by diluting before use so that the concentration of the drug is much smaller than the concentration of the preparation (for example, more than 5 times dilution), so as to avoid the safety risk caused by the concentration and rapid entry of the drug into the blood.

The use of the same substance as different active ingredients needs to satisfy different pharmacokinetic characteristics. According to the above studies and more similar studies, even with intratumoral administration, the difference in the drug effect of the cell-related components at the same dose with different administration concentrations can even exceed the expected kinetic difference (eg E ₂ /E ₁ > in the above table). 200%). The results of the dose-dependent comparative study of the above pharmacodynamics further confirm that the local activity of the cell-related 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. Thus, the administration concentration of the cell-associated components in the compositions of the present invention is not required for other effects (eg, enhancement of rabbit immunity), but is required for the local effect, and thus the pharmacological concentration for that local effect. A necessary condition for the animal cell-related component to provide a therapeutic effect including the local effect is that it must be present in the composition in such a way that its pharmacological concentration (local administration concentration, cell specific volume) is > 20%, ≥30%, 30-90%, preferably 45-90% or 75-85%. This pharmacological concentration not only limits its composition as a feature, but also must appear in the new drug application as a pharmacological condition, and must also appear in the drug's instructions for use as an application condition. In fact, the compositions of the present invention are more similar to chemical ablative agents than other (eg, cancer cell suppressing drugs, tumor vascular suppressing drugs, or immunologic drugs) as far as the therapeutic effect is necessary for this dosing concentration scheme. ).

Example 4: Pharmacological study of local action and optimization of its pharmacological volume

It is well known that the content of components of pharmaceutical compositions of different pharmacology usually needs to be defined by different characteristics. Although the conventional pharmaceutical composition has no volume-dependent pharmacological volume of the lesion target area outside the administered dose, the following experiments were conducted to study this.

In one experiment, the experimental animals were nude mice, and the model cells were human pancreatic cancer cells (PANC-1), and 1×10 ⁶ cells per animal were subcutaneously transplanted into the right armpit of the animal to model. Successfully modeled experimental animals (average tumor volume 209.2 mm ³ ) were randomly divided into 5 experimental groups (3 control groups and 2 study groups). The negative control is normal saline, and the positive control is 75% ethanol. The research drugs are shown in the table below, which are prepared according to the preparation method of Example 1. The animal cell-related components (cell specificity is shown in the table below) They are nude mouse blood cell concentrate (prepared as B4 in Table 3) and its 2-fold dilution in water for injection. Each group was administered once, and the injection volume was calculated according to the administration volume/tumor volume ratio (V _injection /V _tumor ) shown in the following table. Seven days after administration, the animals were euthanized, and the tumor tissue was removed after dissection to measure the tumor weight, and the tumor inhibition rate was calculated from the negative control group. The results are shown in the following table.

Table 6

组别group	注射浓度Injection concentration	(V _注/V _瘤) (V _Note /V _Tumor )	瘤重(x±sg)Tumor weight (x±sg)	抑瘤率tumor inhibition rate
00	(生理盐水)(saline)	0.500.50	0.249±0.2130.249±0.213	--
0101	75％乙醇75% ethanol	0.10.1	0.230±0.1110.230±0.111	7.6％7.6%
0202	75％乙醇75% ethanol	0.200.20	0.201±0.1050.201±0.105	19.3％19.3%
11	动物细胞相关组分(细胞比容75％)Animal cell related fractions (75% hematocrit)	0.10.1	0.232±0.1060.232±0.106	6.8％6.8%
22	动物细胞相关组分(细胞比容38％)Animal cell-related fractions (cytospecific volume 38%)	0.200.20	0.206±0.0980.206±0.098	17.4％17.4%

Research groups 01 and 02 injected ethanol with different dose/target volume ratios into the same volume of tumor. It is well known that ethanol can only be used as a chemical ablative agent if it exceeds a threshold of the administration volume/tumor volume ratio (eg, 0.15). In the table above, the results of Study Group 01 confirm that the ethanol in this technical solution is similar to intravenous ethanol and fails as the active ingredient to provide chemical ablation. The tumor inhibition rates of study group 02 and study group 01 were very different (E ₀₂ /E ₀₁ >200%), showing the expected chemical ablation effect.

Research groups 1 and 2 injected the same dose of animal cell-related components with different administration volume/target volume ratios into the same volume of tumor. In the above table, there was little difference in tumor inhibition rate between study group 2 and study group 02 (E ₂ /E ₀₂ =90%), and there was no significant difference in tumor weight between them (P>0.05), showing that The animal cell-related components provided the local effect required for the therapeutic effect in the technical solution of study group 2, and the local effect was similar to the chemical ablation effect in the positive control group 02.

It is generally believed that the same drug (such as the same drug for cancer cell suppressing drugs, tumor vascular suppressing drugs, and the same drug for immune drugs) has the same drug effect when administered at the same dose. However, according to the above studies and more similar studies, even if higher concentrations of the composition are administered intratumorally, the differences in the drug effects of animal cell-related components at the same dose with different administration volume/target volume ratios can even exceed kinetics Differences are expected (eg E ₂ /E ₁ >200% in the above table). The results of the volume-dependent comparative study of the above pharmacodynamics further confirm that the local activity of the cell-related 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. Therefore, the ratio of the administration volume of the animal cell-related components to the target volume in the present invention is no longer a matter of pharmacodynamics, but a matter of pharmacology.

The pharmacological effects of animal cell-related components in the prior art can be achieved by their blood concentration (usually very low, such as 0.25×10 ^-5 %), and the administration volume is only related to the dose required for the blood concentration, It has nothing to do with the intratumoral target volume. Therefore, its composition must be composed of only its necessary components, and its formulation composition is only limited by the formulations (for example, high-concentration preparations can save transportation and storage costs, and injection of appropriately high concentrations can reduce the injection volume and thus reduce the time), and there is no restriction on the ratio of volume to be administered to volume of the target volume. It is well known that in clinical practice, the injection time is often minimized to improve patient compliance.

However, a necessary condition for the animal cell-related component to provide the therapeutic effect is that it must be present in the composition such that its administration volume/target volume ratio is >0.1, 0.15-1.5, preferably 0.23-1.5 or 0.5-1.5. The pharmacological volume determined by the administration volume/target volume ratio not only limits its composition (especially its content) as a feature, but this feature must also appear in the new drug application as a pharmacological condition of the composition, and must also be used as a The conditions of its application appear in the instructions for use of the drug. In fact, the compositions of the present invention are more similar to chemical ablatives than others (eg, cancer cell suppressing drugs, tumor vascular suppressing drugs, or immunological drugs) in terms of this pharmacological volume as a component content characteristic.

Clinically, although many tumors have a volume of ≥30 cm ³ , drugs based on immune-enhancing pharmacology (eg, certain cytokines) are generally administered in very low volumes (eg, ≤ 2 ml) within the tumor. Therefore, the size of such pharmaceutical unit preparations (eg, injection bottles, or powder injection bottles) is usually not large. However, the composition of the present invention can only be administered under the condition that the above-mentioned administration volume (or administration volume/target volume ratio) is satisfied. For example, if the volume of the commonly used tumor target volume is ≥30 cm ³ , and the administration volume/target volume ratio is selected to be ≥ 0.2, the required administration volume of the pharmaceutical composition of the present invention is ≥ 6.0 cm ³ , then the unit preparation The volume can be 6ml and its multiples. As is well known, a pharmaceutical specification can also be substantially one of the forms of the active ingredient usually required to achieve the desired pharmacological effect. For example, each tablet contains different levels of aspirin "Bai aspirin" can have different indications.

Pharmacological function is the fundamental characteristic of medicine. Any substance lacking pharmacological function cannot be used as a medicine. However, if a new pharmacological function is discovered in an old medicine, it can creatively generate new applications like new medicines. According to the results of the above Examples 2-4 and more similar studies, the animal cell-related components included in the compositions of the present invention (cell-associated components of the present invention) exceed those contained in prior art non-topical pharmaceutical compositions in the following respects Expectations of animal cell-related fractions (prior art cell-related fractions, such as those available for intravenous injection):

1), its basic pharmacology exceeds expectations: the cell-related components of the present invention are based on the discovery of its new pharmacology-drug penetration zone tissue destruction effect-that is, local effects, mainly including local chemical effects (such as chemical ablation), which exceeds the pharmacological expectations of any non-local activity (eg, immune effects, etc.) of the cell-associated components of the prior art;

2), its pharmacological method is beyond expectation: the composition of the present invention must be strictly limited to local administration to enter the target zone reactor and realize the above-mentioned new function of the cell-related component of the present invention, and the pharmacological method of the prior art composition is not limited to local Administration, preferably intravenous infusion, to achieve its target function;

3), its active ingredient selection scheme exceeds expectations: the animal cell-related components in the prior art immunization composition are preferably composed of stronger immunization effects (such as a live cell ratio of up to 85% or more), and the composition of the present invention is The local effects provided by the animal cell-related components are independent of their immune effects, so the above-mentioned compositions with stronger immune effects are not preferred.

4), its pharmacokinetic scheme exceeds expectations: the kinetic condition of prior art animal cell-related components to achieve its systemic effect (pharmacological concentration is blood drug concentration) depends on the dosage rather than the dosage, let alone the concentration. The concentration of the above formulation (usually prepared as a concentrate for the convenience of preparation and storage and transportation), in order to avoid local action damage such as the injection site, the pharmaceutical agent is also diluted for administration. However, the kinetic conditions for the cell-related components of the present invention to achieve their local effects are oppositely dependent on the administration concentration rather than the administration dose (blood drug concentration), and the local administration concentration is its pharmacological concentration, and often also its formulation concentration. In addition, the administration volume of the cell-related components in the prior art 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 cell-related components of the present invention is required for its local activity. is related to the volume of the target area penetrated;

5), 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 adjuvants used for the systemic dosage form, the composition during administration) need to be a sufficient mixture to require a uniform solvent, etc.), etc.;

6), its technical effect exceeds expectations: First, the treatment effect on local lesions exceeds expectations. For example, a short-term drug effect comparable to that of a recognized effective drug, far exceeding that of a prior art cell-related component (r _{composition of the invention} /r _{prior art composition} > 200%, preferably r, is obtained, at least in the area of administration _{Inventive composition} /r _{prior art composition} >400%). This pharmacodynamic feature also leads to its indication range far exceeding the expectations of the prior art compositions, such as for chemical-like ablation of local diseased tissues including solid tumors to treat any local diseased disease containing the diseased tissue, as it is for Immunodeficiency patients, elderly patients, and patients with reduced immune capacity after various treatments can be represented by the above animal models. These also further show that the differences between the two cell-associated components are not kinetic but pharmacological (eg local vs conventional).

Example 5: Common pharmacological research and technical solution optimization in immunosuppressive animal models

In one experiment, the experimental animals were nude mice, the model cells were lung tumor cells (A549), and 1×10 ⁶ cells per animal were subcutaneously transplanted into the right armpit of the animal to model the tumor. The successfully modeled experimental animals (average tumor volume 154.7mm ³ ) were randomly divided into 18 groups as shown in the table below, and were further divided into series 1 (negative control group 01, study drug groups 1-7) and series 2 ( Negative control group 02, drug research group 8-16). The negative control is normal saline, and the research drugs are shown in the table below. The medicines are all liquid preparations, which are configured according to the preparation method of Example 1, and the animal cell-related components are the preparation of nude mouse blood cell concentrate such as the preparation of A16 in Table 2 (hematocrit 63%), and the animal cell-related components are as follows: Components/combinations were prepared as B4 in Table 2. The animals in series 1 and 2 were administered intravenously and intratumorally, respectively, while the medication in study group 16 was injected with animal cell-related components for about 2 hours and then injected with 10% arginine. Each group was administered each drug once, and the injection volume was 150 μl per drug. The animals were euthanized 7 days after administration, the tumor tissue was removed after dissection to measure the tumor weight, and the tumor inhibition rate was calculated from each series of negative control groups. The results are shown in the following table.

Table 7

In the above table, when administered intravenously, composition study groups 5 and 7 did not show therapeutically meaningful efficacy, and these compositions are thus not therapeutic pharmaceutical compositions. Study group 6 showed similar efficacy to study group 2, but did not show significant synergy (q=1.05). These results suggest that co-administration with chemically active substances does not appear to produce local synergistic effects when animal cell-related components cannot provide local effects, including chemical effects.

During intratumoral injection, compared with the positive control group 8-chemical ablation agent, the study group 12-16 showed therapeutic effects; among them, the animal cell-related components in the study group 12 were used as chemical ablation drugs (refer to the implementation of The use of Example 2) and diluted chemical ablative agent did not show synergy as expected (q ₁₁ <1.15). Co-use of animal cell-related components and cytotoxic drugs in study group 13 showed a synergistic effect (q ₁₃ >1.15). Co-administration of animal cell-related components and weakly locally acting compounds in study group 14 also showed synergy (q ₁₄ >1.15). In particular, there was a statistically significant difference in tumor weight between the study group 15 and the negative control group, and this animal cell-related component was shared with three components of two different chemically active compounds (weak local-acting compounds and cytotoxic drugs). Actually, it can further synergize on the basis of the synergistic effect of its two components (q ₁₅ >1.15). In addition, the co-drug of study group 16 was not in the same agent despite the same drug composition as study group 14, and showed no significant synergy (q ₁₆ <1.15).

In other experiments, the use of other animal cell-related components (such as other cell components, tissue components) and chemically active compounds also yielded results consistent with the above table, which will not be repeated here. The following experiments conducted more research on the composition of animal cell-related components, especially the three-component composition.

In one experiment, the experimental animals were nude mice, the model cells were human gastric cancer cells (BGC823), and 1×10 6 cells per animal were subcutaneously transplanted into the right armpit of the animal to model. In this research experiment, the successfully modeled experimental animals (average tumor volume 161.3 mm ³ ) were randomly divided into 12 experimental groups (1 negative control group and 11 research groups). The negative control is normal saline, and the study drugs 1-11 are shown in the table below. The medicines are prepared according to the preparation method of Example 1. For example, the composition of the cell component (or tissue component) and the chemically active compound is obtained by adding the latter dry powder to the former and stirring, wherein: the cell component is human leukocyte concentrate (cytospecific volume 73%), the tissue component is human placenta fragmented granules and semi-fluid (cytospecific volume>55%), common drug 1 is 1% methylene blue, common drug 2 is 1% 5-fluorouracil, and common drug 3 is 1% methylene blue/1% 5-fluorouracil composition. Each group was administered once, and the injection volume was 150 μl per animal. The animals were euthanized 7 days after administration, and the tumor tissue was removed after dissection to measure the tumor weight, and the tumor inhibition rate was calculated from the negative control group. The results are shown in the following table.

Table 8 Effects of different drugs on tumor weight and tumor inhibition rate

组别group	药物drug	瘤重(x±sg)Tumor weight (x±sg)	抑瘤率tumor inhibition rate
0101	生理盐水normal saline	0.239±0.1970.239±0.197	--
11	共用药1shared medicine 1	0.207±0.1090.207±0.109	13.2％13.2%
22	共用药2shared medicine 2	0.146±0.0850.146±0.085	39.1％39.1%
33	共用药3shared medicine 3	0.083±0.0110.083±0.011	65.3％65.3%
44	细胞组分cellular components	0.157±0.0870.157±0.087	34.2％34.2%
55	细胞组分/共用药1Cell Components/Common Drug 1	0.087±0.0300.087±0.030	63.4％63.4%

66	细胞组分/共用药2Cell Components/Common Drug 2	0.069±0.0390.069±0.039	71.2％71.2%
77	细胞组分/共用药3Cell Components/Common Drug 3	0.012±0.0280.012±0.028	95.1％95.1%
88	组织组分tissue components	0.148±0.0640.148±0.064	38.2％38.2%
99	组织组分/共用药1Tissue Components/Common Drugs 1	0.092±0.0330.092±0.033	61.4％61.4%
1010	组织组分/共用药2Tissue Components/Common Drugs 2	0.060±0.0160.060±0.016	74.8％74.8%
1111	组织组分/共用药3Tissue Components/Common Drugs 3	0.005±0.0700.005±0.070	97.9％97.9%

During intratumoral injection, both study groups 5 and 6 with the two-component composition as the investigational drug showed significant synergy (both q ₅ and q ₆ were greater than 1.15), and the study group with the three-component composition as the investigational drug 7 also showed significant synergy (q ₇ >1.15) when viewed as a combination of cellular components and the chemically active compound 1/chemically active compound 2 combination (co-drug 3). When they are regarded as a combination of cellular component/chemically active compound 1 (combination drug 1) synergistic composition and chemically active compound 2 (combination drug 2), or cellular component/chemically active compound 2 (combined drug 2) synergistic composition, respectively The composition and the composition of chemically active compound 1 (combination drug 1) also all showed obvious synergistic effect (all q>1.15). The results of composition study groups 9-11 were completely consistent with composition study groups 5-7.

According to the results of the above studies and many more similar studies, the drug co-action provided by the animal cell-related components is closely related to the dosing technology scheme using it: the conventional dosing technology scheme (oral, intravenous, etc.) must be excluded, And only by using the intratumoral drug delivery technology, it is possible to make the animal cell-related components provide the above-mentioned tumor-bearing patients with new drug-combination effects that it cannot provide in the prior art. The following examples further investigate the drug effect of this new function.

In one experiment, the experimental animals were nude mice , which were randomly divided into 3 study groups (1, 2 and 3) with 6 mice in each study group. The study drugs of study groups 1, 2 and 3 were chemical ablative agents (75% ethanol in water), animal cell-related components/weak local action compounds (cellular components/combined drug 2 in the previous experiment), weak local action compounds Compound/Cytotoxic Drug Composition (Cell Component/Combined Drug 3 from previous experiment). Each group was treated once, and the drug was administered to the muscle mass of the right leg, and the injection volume was 100ul/a. Seven days after administration, the animals were euthanized, and the muscle mass of the right leg of the nude mice was dissected out. Gross pathological analysis was performed, and the area of abnormal (such as necrosis, nodule, etc.) areas that were different from normal muscles was measured by sectioning.

The abnormal area areas of study groups 1, 2 and 3 were 35.37±13.84mm ² , 48.37±13.91mm ² and 73.46±23.91mm ² , respectively, indicating that the local synergy provided by animal cell-related components was similar to but stronger than ethanol , which mainly or at least not negligibly include local chemical synergy. This local chemical synergy appears to be a chemoablation-like effect, which mainly, or at least not negligibly, involves tissue destruction (tissue toxicity) in the subcutaneous drug penetration zone independent of conventional effects.

It is well known that obtaining a synergistic composition (animal cell-related component/chemically active compound composition) has exceeded expectations in the field of solid tumors. It is particularly rare that the two-component synergistic composition of the present invention can actually form a further synergistic composition with the chemically active compound, namely, animal cell-related component/chemically active compound 1 (such as a weak local action compound)/chemically active compound 2 (eg cytotoxic drugs) compositions. This is also a preferred version of the composition of the present invention. A more preferred embodiment of the composition of the present invention is animal cell-related components/methylene blue dyes/other chemically active compounds, wherein the cell specific volume of the animal cell-related components is 30-85%, and the methylene blue The concentration of the quasi-dye is 0.5-1.5% and the concentration of the other chemically active compounds is 0.1-35%.

Example 6: Pharmacological study of local synergistic effect and optimization of its pharmacological dose ratio

In one experiment, the experimental animals were nude mice, the model cells were human hepatoma cells (HepG2), and 1×10 ⁶ cells per animal were subcutaneously transplanted into the right armpit of the animal to model. Successfully modeled experimental animals (average tumor volume 152.9 mm ³ ) were randomly divided into negative control group and 12 study groups. The negative control is normal saline, and the composition of the study drugs is shown in the table below: 4 kinds of single drugs of animal cell-related components, 4 kinds of single drugs of weak local action compounds, and 4 kinds of different concentrations of animal cells A composition of related components and 4 varying concentrations of a weakly locally acting compound, wherein the weakly locally acting compound is methylene blue. The medicine was prepared according to the preparation method of Example 1, wherein the animal cell-related components were nude mouse blood cell concentrates prepared as A16 in Table 2 (cytospecific volume 63%), and the animal cell-related components 1-4 were nude mice Water-for-injection dilutions of murine blood cell concentrates, animal cell-related components/complexes are prepared as B4 in Table 2. Each group was administered once, and the injection volume was 150 μl per animal. The animals were euthanized on the 7th day after administration, and the tumor tissue was removed after dissection to measure the tumor weight, and the tumor inhibition rate was calculated from the negative control group.

Table 9 Tumor weight and tumor inhibition rate data in different groups

In the table above, composition groups 9 and 12 did not show synergy (q were <1.15, respectively), while composition groups 10 and 11 showed synergy (q were >1.15, respectively), the synergistic amount ratio (V _{animal cell related Component} /W vital _dye ) was between 69.9/0.15 and 4.8/3, indicating that the local chemical synergy provided by the animal cell-related components does not appear to be conditioned on the strength of the local chemical effect provided by the weakly locally acting compounds.

In one experiment, the experimental animals were nude mice, the model cells were human hepatoma cells (HepG2), and 1×10 ⁶ cells per animal were subcutaneously transplanted into the right axilla of the animals to model. Successfully modeled experimental animals (average tumor volume 162.1 mm ³ ) were randomly divided into negative control group and 12 study groups. The negative control is normal saline, and the composition of the study drugs is shown in the table below: 4 kinds of single drugs of animal cell-related components, 4 kinds of single drugs of weak local action compounds, and 4 kinds of different concentrations of animal cells A composition of related components and 4 varying concentrations of a weakly locally acting compound, wherein the weakly locally acting compound is lysine. The medicine was prepared according to the preparation method of Example 1, wherein the animal cell-related components were nude mouse blood cell concentrates prepared as A16 in Table 2 (cytospecific volume 63%), and the animal cell-related components 1-4 were nude mice Water-for-injection dilutions of murine blood cell concentrates, animal cell-related components/complexes are prepared as B4 in Table 2. Each group was administered once, and the injection volume was 150 μl per animal. The animals were euthanized on the 7th day after administration, and the tumor tissue was removed after dissection to measure the tumor weight, and the tumor inhibition rate was calculated from the negative control group.

Table 10 Tumor weight and tumor inhibition rate data in different groups

In the above table, composition groups 9 and 12 did not show obvious synergy (q were <1.15), but composition groups 10 and 11 showed obvious synergy (q were >1.15), and the synergistic amount ratio ( V _{animal cell-related components} /W co _-use ) between 12.8/15 and 69/1, indicating that animal cell-related components provide local chemical synergy that does not appear to be conditioned on the strength of the local chemical effect provided by weakly locally acting compounds .

In one experiment, the experimental animals were nude mice, and the model cells were human pancreatic cancer cells (PANC-1), and 1×10 ⁶ cells per animal were subcutaneously transplanted into the right armpit of the animal to model. Successfully modeled experimental animals (with an average tumor volume of 106.5 mm ³ ) were randomly divided into a negative control group and 12 study groups. The negative control is normal saline, and the composition of the study drugs is shown in the following table, including: 4 different concentrations of animal cell-related component single drugs, 4 different concentrations of chemotherapeutic drugs, and 4 different concentrations of animal cell-related groups. A composition of 4 different concentrations of chemotherapeutic drugs, wherein the chemotherapeutic drug is gemcitabine. The medicine was prepared according to the preparation method of Example 1, wherein the animal cell-related components were nude mouse blood cell concentrates prepared as A16 in Table 2 (cytospecific volume 63%), and the animal cell-related components 1-4 were nude mice Water-for-injection dilutions of murine blood cell concentrates, animal cell-related components/complexes are prepared as B4 in Table 2. Each group was administered once, and the injection volume was 100 μl per animal. The animals were euthanized on the 7th day after administration, and the tumor tissue was removed after dissection to measure the tumor weight, and the tumor inhibition rate was calculated from the negative control group.

Table 11 Tumor weight and tumor inhibition rate data in different groups

In the above table, composition groups 9 and 12 did not show obvious synergy (q were <1.15), but composition groups 10 and 11 showed obvious synergy (q were >1.15), and the synergistic amount ratio ( V _{animal cell-related components} /W _{cytotoxic drugs} ) between 70/0.05 and 4.8/5, indicating that the local chemical synergy provided by animal cell-related components does not seem to be conditioned on the strength of the local chemical effects provided by cytotoxic drugs .

Cell-associated components can provide different pharmacological effects, eg, different synergistic effects, in different technical scenarios, as described above. The synergistic amount ratio of the cell-related components in the composition of the present invention is not required for other synergistic effects (eg, enhanced synergy of rabbit disease), but is required for the local synergistic effect, and thus is the pharmacological amount ratio. In the application, composition, or method of the present invention, a necessary condition of the technical solution for the local synergistic effect of the animal cell-related components is: the pharmacological ratio of the animal cell-related components and the chemically active compound co-drugs ( V _{animal cell-related components} /W _{chemically active compounds} ) is (1.5-100)/(0.1-1), wherein: when the chemically active compounds include cytotoxic drugs, the animal cell-related components and cytotoxic drugs The pharmacological ratio of (V _{animal cell-related components} /W _{cytotoxic drugs} ) is preferably (2-100)/(0.1-1); when the chemically active compound includes a weak local action compound, the animal cell-related group The pharmacological ratio (V _{animal cell-related} _component /W _{weak local action compound} ) of the compound to the weak local action compound is preferably (1.5-40)/(0.1-1), wherein when the weak local action compound is an amino acid nutrient , the pharmacological ratio (V _{animal cell-related components} /W _{amino acid nutrients} ) is preferably (1.5-7)/(0.1-1); when the weak local action compound is a vital dye, the pharmacological ratio (V _{animal Cell-related component} /W _{vital dye} ) is preferably (2-40)/(0.1-1), wherein V _{animal cell-} related component is the cytospecific volume of the animal cell-related component in the composition.

Example 7: Pharmacological study of local synergy and optimization of its pharmacological concentration

In one experiment, the experimental animals were nude mice, the model cells were human pancreatic cancer cells (PANC-1), and 1 x 10 ⁵ cells per animal were subcutaneously transplanted into the right armpit of the animal to model. Successfully modeled experimental animals (tumor-bearing mice , with an average tumor volume of 156.4 mm ³ ) were randomly divided into 1 control group (0) and 9 study groups (1-9) as shown in the table below. The negative control is normal saline, and the research drugs are shown in the table below. The medicines are all liquid preparations, configured according to the preparation method of Example 1, wherein the common medicine is arginine. The medicine is prepared according to the preparation method of Example 1, wherein the animal cell-related components are human erythrocyte concentrates such as the preparation of A14 in Table 2, the animal cell-related components 1-3 are the water-for-injection dilutions of the human erythrocyte concentrates, and the animal cell-related components Components/combinations were prepared as B7 in Table 2. Each group was administered once, and the injection volume was shown in the table below. The animals were euthanized on the 7th day after administration, and the tumor tissue was removed after dissection to measure the tumor weight, and the tumor inhibition rate was calculated from the negative control group. The results are shown in the following table.

Table 12 Tumor weight and tumor inhibition rate data in different groups

组别group	药物drug	细胞比容cell volume	注射量Injection volume	瘤重(x±sg)Tumor weight (x±sg)	抑瘤率tumor inhibition rate
00	生理盐水normal saline	--	100ul100ul	0.261±0.2150.261±0.215	--
11	动物细胞相关组分animal cell related components	16.3％16.3%	300ul300ul	0.242±0.1030.242±0.103	7.4％7.4%
22	动物细胞相关组分animal cell related components	32.6％32.6%	150ul150ul	0.195±0.0990.195±0.099	25.3％25.3%
33	动物细胞相关组分animal cell related components	48.9％48.9%	100ul100ul	0.164±0.0840.164±0.084	37.2％37.2%
44	4％精氨酸4% Arginine	--	300ul300ul	0.246±0.1070.246±0.107	5.6％5.6%
55	8％精氨酸8% Arginine	--	150ul150ul	0.245±0.1060.245±0.106	6.1％6.1%
66	12％精氨酸12% Arginine	--	100ul100ul	0.197±0.0820.197±0.082	24.5％24.5%
77	动物细胞相关组分/4％精氨酸Animal cell related fractions/4% arginine	15.6％15.6%	300ul300ul	0.237±0.0850.237±0.085	9.1％9.1%
88	动物细胞相关组分/8％精氨酸Animal cell related fractions/8% arginine	30％30%	150ul150ul	0.136±0.0610.136±0.061	47.8％47.8%
99	动物细胞相关组分/12％精氨酸Animal cell related fractions/12% arginine	43％43%	100ul100ul	0.097±0.0230.097±0.023	62.9％62.9%

In the above table, study groups 7-9 injected the same components, the same amount ratio and the same dose but different concentrations of animal cell-related components/chemically active compounds into the same volume of tumor. Of these, composition group 7 did not show therapeutically meaningful results, whereas composition group 8 showed a clear synergistic effect (q>1.15), and the results of study group 9 (q>1.15) further demonstrated the administration of this synergistic effect concentration-dependent.

It is generally believed that the same pharmaceutical composition (such as the same pharmaceutical composition of cancer cell suppressing drugs, tumor vascular suppressing drugs, and immunological drugs) has the same amount of active ingredients and the same dosage, and then the drug sharing effect is the same. However, it is possible for the cell-associated components to obtain different shared pharmacological effects in different technical solutions by providing different shared pharmacological effects. In the prior art, the sharing (if any) of animal cell-related components with other drugs can achieve its pharmacological effect by intravenous injection of animal cell-related components, and it can be seen that what is required is its blood concentration (usually very low, For example, 0.25×10 ^-5 %), the pharmacological effect can be provided only by satisfying the dosage and dosage ratio. Therefore, its composition must be composed only of its necessary components (eg, animal cell-related components, co-drugs, necessary excipients, necessary osmotic pressure enhancers) and necessary amount ratios. As described in Example 3, its formulation composition is then limited only by the formulation (eg, high-concentration formulations can save transportation, storage costs, and appropriately high-concentration injections can reduce injection volume and thus time).

In addition, more studies have shown that even in the dosing technical scheme of the present invention, even in addition to the quantity ratio technical scheme and dosing concentration technical scheme of the present invention, animals with the same dose of different administration volume/target volume ratio Cell-associated components may also provide different shared pharmacological effects. In the application, composition, or method of the present invention, one condition of the technical solution for the local synergistic effect of cell-related components is that the composition of the composition containing it must be such that its administration volume/target volume ratio is > 0.1 , 0.15-1.5, preferably 0.23-1.5 or 0.5-1.5. The pharmacological volume determined by the administration volume/target volume ratio not only limits its composition (especially its content) as a feature, but this feature must also appear in the new drug application as a pharmacological condition of the composition, and must also be used as a The conditions of its application appear in the instructions for use of the drug. In fact, the compositions of the present invention are more similar to chemical ablative agents than others (eg, cancer cell suppressing drugs, tumor vascular suppressing drugs, or immunologic drugs) in terms of this pharmacological volume as a component content characteristic.

Example 8: Short-term single-pharmacological/combined pharmacological research and technical scheme optimization of drug delivery to lesion area in common animal models

In one experiment, the experimental animals were BALB/c mice, the model cells were breast cancer 4T1 cells, and 0.5×10 ⁶ cells per animal were subcutaneously transplanted into the right armpit of the animal to model. Successfully modeled experimental animals (average tumor volume 153.2 mm ³ ) were randomly divided into 21 groups as shown in the table below, and these groups were further divided into series 1 (negative control group 01, study drug groups 1-6) and series 2 (Negative control group 02, drug research group 7-19). The negative control is normal saline, and the research drugs are shown in the table below, which are prepared according to the preparation method of Example 1. Among them: animal cell-related fraction 1 (human leukocyte concentrate, hematocrit 73%), animal cell-related fraction 2 (mouse blood cell concentrate, hematocrit 75%), animal cell-related fraction 3 (human placenta Tissue fragmentation, cell specific volume is greater than 55%), animal cell-related component 4 (mice meat tissue fragmentation, cell specific volume is more than 55%) are prepared as the preparation methods of A13, A16, A4, A7 in Table 2; Each animal cell-related component/combination is prepared by the preparation methods of B8 (adding methylene blue dry powder to the animal cell-related component) and B17 (adding 5-chlorouracil and other dry powder to the animal cell-related component) in Table 3 respectively. . The animals in series 1 and 2 were administered by intraperitoneal injection and intratumoral injection, respectively. Each group was treated twice, and the second dose was 7 days after the first dose, with an injection volume of 150 μl per animal. The animals were euthanized on the 7th day after the second administration, the tumor tissue was removed after dissection to measure the tumor weight, and the tumor inhibition rate was calculated from each series of negative control groups. The results are shown in the following table.

Table 13 Data of tumor weight and tumor inhibition rate under different groups in different groups

组别group	药物drug	瘤重(x±sg)Tumor weight (x±sg)	抑瘤率tumor inhibition rate
0101	生理盐水normal saline	0.371±0.1960.371±0.196	--
11	1％5-氟尿嘧啶1% 5-fluorouracil	0.236±0.1140.236±0.114	36.3％36.3%
22	75％乙醇75% ethanol	0.361±0.1050.361±0.105	2.6％2.6%
33	4×104U/ml白细胞介素-124×104U/ml interleukin-12	0.359±0.1030.359±0.103	3.1％3.1%
44	10％精氨酸10% Arginine	0.347±0.1000.347±0.100	6.5％6.5%
55	动物细胞相关组分1animal cell related fraction 1	0.350±0.0970.350±0.097	5.6％5.6%
66	动物细胞相关组分1/1％亚甲蓝Animal cell related fractions 1/1% methylene blue	0.356±0.1070.356±0.107	4.1％4.1%
0202	生理盐水normal saline	0.367±0.2370.367±0.237	--
77	1％5-氟尿嘧啶1% 5-fluorouracil	0.231±0.0950.231±0.095	37.1％37.1%
88	75％乙醇75% ethanol	0.244±0.0930.244±0.093	33.4％33.4%
99	4×10 ⁴U/ml白细胞介素-12 4×10 ⁴ U/ml interleukin-12	0.354±0.1120.354±0.112	3.6％3.6%
1010	10％精氨酸10% Arginine	0.326±0.0870.326±0.087	11.2％11.2%
1111	动物细胞相关组分1animal cell related fraction 1	0.215±0.0910.215±0.091	41.3％41.3%
1212	动物细胞相关组分2animal cell related fraction 2	0.212±0.0880.212±0.088	42.1％42.1%

1313	动物细胞相关组分3animal cell related fraction 3	0.194±0.0620.194±0.062	47.2％47.2%
1414	动物细胞相关组分4animal cell related fraction 4	0.197±0.0750.197±0.075	46.4％46.4%
1515	动物细胞相关组分1/1％5-氟尿嘧啶Animal cell related fraction 1/1% 5-fluorouracil	0.091±0.0790.091±0.079	75.1％75.1%
1616	动物细胞相关组分2/1％5-氟尿嘧啶Animal cell related fraction 2/1% 5-fluorouracil	0.079±0.0630.079±0.063	78.6％78.6%
1717	动物细胞相关组分3/1％5-氟尿嘧啶Animal cell related fraction 3/1% 5-fluorouracil	0.077±0.0200.077±0.020	79.1％79.1%
1818	动物细胞相关组分4/1％5-氟尿嘧啶Animal cell related fraction 4/1% 5-fluorouracil	0.083±0.0340.083±0.034	77.4％77.4%
1919	动物细胞相关组分1/1％5-氟尿嘧啶/10％精氨酸Animal cell related fractions 1/1% 5-fluorouracil/10% arginine	0.019±0.0580.019±0.058	94.8％94.8%

In the table above, study groups 1-3 and 7-9 are in full agreement with their treatment results in immunosuppressive animal models (Examples 2 and 5), as expected for cytotoxicity, chemical ablation, and immune enhancement, respectively . The tumor inhibition rates of study groups 5 and 6 were consistent with those of study group 3, which seemed to be in line with the expectations of the existing technology (non-chemotherapy drugs) for animal cell-related components. The difference in tumor inhibition rate between study group 11 and study group 5 was far beyond the expected limit of kinetic improvement for the same drug (E ₁₁ /E ₅ >200%), and was significantly different from that of study group 8 (classical chemical ablation agent group). There was little difference in tumor inhibition rate (E ₁₁ /E ₈ <200%). Study Groups 12-14 are in the same situation as Study Group 11. The tumor inhibition rates of the compositions in the study groups 15-18 were significantly greater than their respective animal cell-related component single-drug groups 11-14, and all showed significant synergistic effects (all q were greater than 1.15). Animal cell-related components that formed a synergistic composition (study group 15) with one chemically active compound further formed a three-component composition (study group 19) with another chemically active compound (study group 7) also showed significant Synergy (q>1.15). These results are fully consistent with the treatment results in Example 5.

According to the results of Examples 2, 5 above and the study of this example and more similar studies, the animal cell-related components can provide short-term drug effects in common animal models as well as in immunosuppressed animal models and use it. It is closely related to the dosing technology of the patient: conventional dosing technology (oral, intravenous, etc.) must be excluded, and only the intratumoral dosing technology is used to make it possible for animal cell-related components to provide tumor-bearing patients with its New functions that cannot be provided in the prior art (eg E ₁₁ /E ₅ >200% in the above table). The following examples further investigate this new function.

In one experiment, the experimental animals were BALB/c mice and were randomly divided into 4 study groups (1, 2, 3 and 4) with 6 mice in each study group. The study drugs of study groups 1, 2, 3 and 4 were chemical ablative agents (75% ethanol in water), animal cell-related components (animal cell-related components 1 in the previous experiment), animal cell-related components/chemical Active substance (animal cell-related fraction 1/1% 5-fluorouracil in the previous experiment), and animal cell-related fraction/chemically active substance 1/chemically active substance 2 (animal cell-related fraction 1 in the previous experiment /1% 5-fluorouracil/10% arginine). Each group was treated once, and the drug was administered to the muscle mass of the right leg, and the injection volume was 100ul/a. The animals were euthanized on the 7th day after administration, and the muscle mass samples of the right leg of the mice were dissected out, and gross pathological analysis was performed.

The abnormal areas of study groups 1-4 were 31.24±13.75mm ² , 32.84±15.61mm ² and 53.18±23.07mm ² , 75±4.21mm ² , respectively. These results are in good agreement with those of similar experiments in Examples 2 and 5.

According to the results of the studies of Examples 2-9 above and more similar studies, the animal cell-related components in the technical solution of the present invention are similar to high-concentration ethanol, and whether for immunosuppressed patients or general patients, firstly after administration The displayed drug effect mainly or at least not negligibly includes a local effect (or local synergy), and the local effect (or local synergy) mainly or at least not negligibly includes a local chemical effect (or local chemical synergy), especially Local chemical effects (or local chemical synergy) analogous to chemical ablation, which mainly, or at least not negligibly, include subcutaneous drug penetration zone tissue destruction independent of conventional effects. The pharmacological function of this local action (or local synergy) allows the composition of the present invention to provide a drug effect that greatly exceeds that expected in the prior art of cell-associated components, at least in any area of patient administration, and thus becomes a therapeutic agent rather than prior art This pharmacological function also allows the compositions of the present invention to provide medicinal uses that greatly exceed those expected in the prior art of cell-associated components, such as for chemical-like ablation of localized diseased tissue to treat any localized diseased disease containing the diseased tissue . Furthermore, the composition of the present invention can be used for local lesions (such as cytotoxic drugs, antiviral drugs, antibacterial drugs, vascular inhibitor drugs, immune drugs, etc.) tumor) treatment. For example, the compositions of the present invention can be used as histotoxic drugs for the treatment of the above-mentioned indications, relative to these anti-local disease drugs, which are mainly based on anti-pathogens.

Achieving this local action (or local synergy) also requires that the compositions of the present invention have pharmacological properties different from those of the prior art. In addition to the above-mentioned special pharmacological methods, special pharmacological compositions are also required, such as the preferred technical solutions for pharmacological compositions in Examples 2-7 (for example, the selection of cell-related components, the selection of common substances, the ratio of pharmacological doses, the pharmacological concentration , pharmacological volume, etc.).

According to the results of Examples 2-8 above, as well as the results of other similar experiments, the topical pharmaceutical compositions of the present invention (referred to as the present compositions of the present invention) comprising cell-associated components and their co-uses outperform any available in the prior art in the following respects Expected non-topical pharmaceutical compositions (referred to as prior art compositions) comprising cell-associated components and their co-uses:

1), its shared pharmacology exceeds expectations: the cell-related component in the composition of the present invention is a local active component, and its shared pharmacology is the shared use including its local action, and the short-term synergistic effect it produces is mainly local synergy, more than Any pharmacological expectations in the prior art between cell-associated components and their co-uses involving co-use with their non-local activities (eg, immune effects, etc.);

2), its pharmacological method exceeds expectations: the cell-related components in the composition of the present invention and their co-uses must be strictly limited to local administration in order to produce short-term synergy, and any predictable cell-related components in the prior art and The shared pharmacological method of the shared substance is not necessarily limited to local administration, preferably systemic administration;

3), 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 cell-related components/weak local action compounds/cytotoxic drugs), although 2 groups Group compositions (eg, cell-associated components/weak local-acting compounds) synergistically have exceeded expectations. In the choice of concomitant species, cell-associated components surprisingly did not provide local synergy to ethanol, but to weak locally acting compounds or/and cytotoxic drugs. In terms of specific drug preferences, in addition to the aforementioned preferred embodiments in which the probiotic component provides a local effect, 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 the topically acting compounds, weakly topically acting compounds and slimming drugs, weakly topically acting compounds and immune enhancers, and the like. Furthermore, the synergistic effect can be provided only when the animal cell-related components are administered in combination with their co-drugs in one pharmaceutical agent;

4), 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 cell-related components, so its common pharmacological structure parameter is the concentration ratio rather than the amount ratio in the prior art composition . The pharmacological concentrations at which the cell-associated components provide a synergistic effect are those at which they provide a local effect in the above examples. The pharmacological concentrations of the weak local action compounds are respectively: amino acid nutrient is 5-25%, methylene blue dye is 0.5-3% or 0.5-1.5%, quinine is 3-10%, carbohydrate is 3-10%. 3-35% of nutrients;

5), its technical effect exceeds expectations: 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. Preferably, 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 9: Medium- and long-term single-pharmacological/combined pharmacological research and technical scheme optimization for drug delivery to lesion areas in common animal models

In one experiment, the experimental animals were BALB/c mice, and the model cells were breast cancer 4T1 cells, and 0.5×10 ⁵ cells per animal were subcutaneously transplanted into the right armpit of the animal to model. Successfully modeled experimental animals (average tumor volume 115.6 mm ³ ) were randomly divided into 1 negative control group (01) and 16 drug study groups (1-16). The negative control is normal saline, and the research drugs are shown in the table below. The medicines are prepared according to the preparation method of Example 1, wherein the animal cell-related component 1 is human leukocyte concentrate (cytospecific 75%), and the animal cell-related component 2 is mouse blood cell concentrate (cytospecific 75%). %), animal cell-related component 3 is mouse meat tissue fragment (cell specific volume > 55%), and the composition of each animal cell-related component and the common component is the dry powder of the common component added to the animal cell-related component Stir well, and the dilution of each drug is a 3-fold dilution of 1 volume of drug added to 2 volumes of water for injection. Each group was injected twice, and the second dose was before the tumor volume measurement on the 7th day from the first dose. The doses of study groups 13 and 14 were 300 μl/acupuncture each time, and the doses of other groups were 100 μl/acupuncture each time. The study group 15 was administered under the armpit of the right forelimb, and the other groups were administered intratumorally. In study group 16, the first intratumoral administration of animal cell-related component 3/20% amino acid resistance, the second intratumoral administration of animal cell-related component 1, and the other groups were administered the same drug twice. The tumor volume (V ₇ , V ₂₁ ) of each group was measured from the 7th and 21st day of the second administration, and the relative tumor proliferation rate (R) was calculated from the negative control group. The results are shown in the following table.

Table 14 Tumor volume and tumor inhibition rate of different groups at different times

In the above table, on the 7th day, the drug effects of study groups 1-12 were completely consistent with those of study groups 7-18 in the table of the previous example, further illustrating that the animal cell-related components in the technical solution of the present invention were administered after administration. That is, it is unavoidable and even possibly mainly to provide the local effect (study groups 5-7) or local synergy (study groups 8-12). The doses of study groups 13 and 14 did not meet their pharmacologically preferred concentrations, so that the pharmacodynamic differences were very large (both >200%) with study groups 5 and 6 (the same dose of the same components administered). The dosing mode of study group 15 did not meet the required dosing mode for local synergy, so that its pharmacodynamic difference was very large (E ₈ /E ₁₅ >200%) compared with study group 8 (the same drug was administered). The first dose of drug in study group 16 was the same as in study group 11 and showed similar local synergy as study group 11.

It is well known that chemotherapeutic pharmacokinetics are characterized by a drug that is attacked and no longer attacked after the drug is metabolized. Compared with the results on day 21 in the above table, the relative tumor proliferation rates of study groups 1 and 2 increased significantly (for example, R on day 21/R on day 7 were both greater than 1.50), indicating that the effect of short-term drug effects was significantly reduced. . The relative tumor proliferation rate in study group 3 remained high, in line with the expectation that immunopotentiators alone did not show significant mid-term efficacy. The relative tumor proliferation rate increase in study groups 5-7 was small (for example, R on 21st/7th was less than 1.25), which was significantly different from that in study groups 1-3. Study groups 8-12 also had smaller increases in relative tumor proliferation rates (eg, 21-day R/7-day R were both less than 1.25), similar to study groups 5-7. Relative tumor proliferation rates for study groups 13-15 remained high, similar to study group 3. The rise in relative tumor proliferation rate was also smaller in study group 16. Similar to study group 11.

The above results show that the study groups 5-12 and 16 of the compositions of the technical solution of the present invention exhibited mid-to-long-term effects that were not shown in the study groups 1-3, i.e., new pharmacology different from chemical effects or immune-enhancing effects. According to the significant differences between study groups 5-12 and 16 and study groups 13-15, the new pharmacology is obviously related to the local action or local synergy provided by the animal cell-related components in the technical solution of the present invention.

The results of the above studies and more similar studies demonstrate that the short-term effects following intralesional administration of the compositions of the present invention mainly or at least non-negligibly include local effects (or local synergistic effects), while the long-term effects are predominantly or at least non-negligible Secondary effects resulting from this local effect (or local synergistic effect) are included, and secondary effects mainly or at least not negligibly include immune effects (eg vaccine effects). Thus, the compositions of the present invention are quite different from classical immunization drugs (eg vaccines), in which the antigenic substance is likely to mainly, or at least insignificantly, comprise the in situ immunizing substance (eg in situ antigen) produced after administration. In addition, animal cell-related components, like other foreign substances, may also have non-specific antigenic effects, which may also enhance the efficacy of the in situ vaccine effect.

Example 10: Concentration-dependent study of single pharmacology/combined pharmacology in common animal model and technical solution optimization

In one experiment, the experimental animals were BALB/c mice, and the model cells were breast cancer 4T1 cells, and 0.5×10 ⁵ cells per animal were subcutaneously transplanted into the right armpit of the animal to model. The average tumor volume of nude mice with successful modeling was 92.7 mm ³ . The model animals were randomly divided into 8 groups, and each group was administered in two ways: A, conventional administration (intraperitoneal injection); B, intratumoral injection. Each single group was administered twice, with an interval of 3 days, and the dose of each injection was 50 μl × 60% (volume ratio) cell fractions of cell derivatives, or/and 50 μl × (1% 5-fluorouracil) per animal. +10% arginine) of which the cellular component (cell derivative) is a concentrate of mouse in vitro matured dendritic cells (DC). On the 3rd and 21st days after the second treatment, the local lesion volume (V) was measured, and the short-term efficacy (3-day growth inhibition rate r _3d ) and medium- and long-term efficacy (21 days) were calculated according to the negative control group of series A and B Growth inhibition rate r _21d '), the actual/expected ratio of short-term efficacy q _3d and the actual/expected ratio of mid- and long-term efficacy q _21d can also be calculated according to short-term efficacy and mid- and long-term efficacy, respectively. The results are shown in the following table.

The pharmaceutical compositions used in each group were prepared according to the conventional aqueous solution preparation method or the preparation method in Example 1.

Table 15 Short-term and mid- and long-term growth inhibition rates in different groups

In the above table, the cell components of the same dose and different concentrations in study groups 1-3 failed to show in series A, while in series B they showed short-term drug effects under the concentration conditions (study groups 1, 2). and mid- and long-term drug effects, further confirming that the concentration-dependent local effects (short-term effects) and secondary effects (medium- and long-term effects) of cellular components in the compositions of the present invention exceed any of their systemic effects in the prior art The pharmacological expectations (neither the hypothesized short-term chemical effects nor the mid- and long-term immune effects produced r'>15% tumor-suppressive effect in series A), and the technical effect expectations (study groups 1 and 2 in two The short-term and mid- and long-term efficacy differences between series were >200%).

Corresponding to the results of study groups 1-3, the cell components/chemically active ingredient compositions of the same dose and different concentrations in study groups 5-7 failed to be displayed in series A, while in series B, they could be displayed under the concentration conditions. (Study groups 5 and 6) showed a mid- and long-term actual efficacy/expected efficacy ratio q _21d of >1.15, further confirming that the mid- and long-term synergistic effect is precisely based on the concentration-dependent local action of the cellular components in the composition of the present invention (short-term effects) secondary effects (medium and long-term effects). This mid- and long-term synergy also exceeds its pharmacological expectations for any systemic synergy in the prior art (assuming that any systemic effects present do not result in mid- to long-term synergy in Series A), and the technical effect expectations ( The differences in mid- and long-term shared drug efficacy between the two series of study groups 5 and 6 were both >200%).

According to the results of the above Examples 2-10 and the results of other similar experiments (administration in the lesion area), the topical pharmaceutical composition containing animal cell-related components (referred to as the composition of the present invention) according to the present invention is in addition to the above The non-topical pharmaceutical compositions containing animal cell-related components in the prior art (referred to as prior art compositions for short, such as the prior art cellular drugs for intravenous injection of blood cells, intravenous immune cells, etc.) are described in the examples. )), and exceed the latter's medium- and long-term expectations in the following areas:

1) The basic pharmacology in the medium and long term exceeds expectations: the cell-related components are used as conventional active components in the pharmaceutical compositions of the prior art, and the long-term pharmacology is completely aimed at targeting the immune system to participate in the systemic immune response, while the cell-related components are in The composition of the present invention is used as a local active component, wherein the long-term pharmacology is the secondary effect of the local action of the targeted drug delivery to the local diseased tissue, and the secondary immune response included is mainly the in situ secondary immune response;

2), the pharmacology of medium and long-term sharing is beyond expectation: the cell-related components and their synergistic combination can be used as local active components in the composition of the present invention, and the long-term sharing is the local sharing effect of the targeted drug delivery to the local diseased tissue. Secondary synergy, including the secondary immune response is mainly in situ synergistic immune response;

3), the pharmacological composition exceeds expectations, such as: completely different principles for the optimization of pharmacologically active ingredients, completely different pharmacological content (pharmacological concentration, or/and pharmacological volume), different pharmacological environments (such as rejection of salt osmotic pressure regulators) ), etc.; first, the local activity of cell-related components can be independent of their immune activity, and the cell-related components used as different active components can have different or even opposite preferred schemes. For example, the prior art based on immunization expects that xenogeneic is stronger than allogeneic, allogeneic is stronger than autologous, bulk tissue is stronger than fragmented tissue, tissue is stronger than cells, and living cells are stronger than dead cells. On the other hand, the selection scheme based on local action exceeded the expectations of the above selection order: the latter between xenogeneic and allogeneic, the latter between allogeneic with strong and weak graft response, and allogeneic and autologous. The latter is preferred between the bulk tissue and the broken tissue, the latter is preferred between the tissue and the cells, and it is not necessary to use a very high proportion of viable cells (for example, the prior art cell drugs often require more than 85%). Quality Standard. Second, the local activity of cell-related components can be independent of their systemic activities, and cell-related components used as different active ingredients can have different or even opposite pharmacokinetic preferences. For example, the local active pharmacological concentration far exceeds the systemic pharmacological concentration, the local activity is dose-dependent and the systemic effect is dose-dependent, and the therapeutic dose using local activity is often related to the size of the diseased tissue (pharmacological volume) and Treatments utilizing systemic effects are often more related to the patient's body weight, immune capacity, genotype and other systemic conditions, etc.;

4), the medium and long-term technical effect exceeded expectations. The secondary effects, including in situ secondary immune responses, of the compositions of the present invention show mid- and long-term efficacy not achieved by the systemic immune responses of prior art compositions. The composition of the present invention exhibits a mid- and long-term therapeutic effect on local lesions, even solid tumors, which the prior art compositions cannot. The efficacy characteristics of the drug also lead to a wider range of indications than expected. For example, the weak immune effect of the prior art cellular drugs does not meet the standard of efficacy in solid tumors, which limits the application, while the composition of the present invention can effectively treat solid tumors. The weak immune effect of the prior art cell drug itself is less effective in patients with poor immunity (for example, immunodeficiency patients represented by the above animal models, elderly patients, and patients with reduced immune ability after various treatments), while the present The inventive composition as described above also achieves better efficacy in such patients.

Since local effects can also occur in other regions, some of which may also be sensitive to secondary effects, this is investigated in the following examples.

Example 11: Single Pharmacology/Combined Pharmacology Study and Technical Scheme Optimization of Local Administration Outside the Lesion Area in Common Animal Models

In one experiment, the experimental animals were BALB/c mice, and the model cells were breast cancer 4T1 cells, and 0.25×10 ⁶ cells per animal were subcutaneously transplanted into the right armpit of the animal to model. Five days after modeling, the experimental animals with obvious tumors were randomly divided into 1 negative control group (01) and 11 drug study groups (1-11), with 10 animals in each group. The negative control is normal saline, and the research drugs of drug research groups 1-11 are shown in the table below, which are prepared according to the preparation method of Example 1. The compositions containing cell-related components are semi-fluid except for the dilution. , other drugs are liquid. The animal cell-related component 1 is the flesh tissue fragment of nude mice (prepared as A5 in Table 2, cell specific volume > 55%), and the animal cell-related component 2 is mouse blood cell concentrate (cytospecific volume 75%). %), the composition of each animal cell-related component and the common compound is formed by adding the dry powder of the common compound to the animal cell-related component and stirring evenly, and the dilution of each drug is 4 times that of 1 volume of drug added to 3 volumes of water for injection dilution. Except for the study group 11, the other groups were injected twice, and the second dose was before the tumor volume measurement on the 7th day from the first dose. The doses of study groups 9 and 10 were 400 μl per animal each time, and the doses of other groups were 100 μl per animal each time. Study group 11 was given intratumoral administration of 1/10% arginine of animal cell-related components for the first time, and animal cell-related component 1 was intradermally administered to the left armpit of animals at the same time. the same drug. The tumor volume (V ₇ , V ₂₁ ) of each group was measured on the 7th and 21st day of the second administration of the study group 1-10, and the relative tumor proliferation rate (R) was calculated from the negative control group. The results are shown in the following table .

Table 16 Relative tumor proliferation rates of different groups in the short and medium term

In the table above, on day 7, the results of study groups 1 and 2 met the pharmacological expectations for cytotoxic and chemoablative agents, respectively, and the results of study group 3 were consistent with the immunopotentiators that did not provide therapeutically meaningful In anticipation of effect, the results of study groups 5-10 were similar to those of study groups 2 or 3, and the results of study group 11 were in line with the expected efficacy of intratumoral local administration of chemoablation-like drugs. These results further demonstrate that the primary cause of the short-term efficacy of the compositions of the present invention (study groups 5-8) is local action (or local synergy) rather than other effects (eg immune effects). This local action (or local synergy) can show short-term efficacy when it is intratumoral (study group 11), and is not in line with the technical scheme (pharmacological concentration) of the composition of the present invention when it is outside the tumor (study group 5-8). The compositions (study groups 9 and 10) showed no short-term efficacy as well.

In the above table, the results on day 21 were compared with those on day 7. The chemotherapy effect of study group 1 was significantly reduced, the treatment effect was still not observed in study groups 2 and 3, and study groups 9 and 10 were still similar to study groups 2 and 10. 3. Unexpectedly, the relative tumor proliferation rates of study groups 5-8 were significantly decreased (eg, the relative tumor proliferation rate of study group 5 decreased from 97% to 78%), showing a difference from study groups 1-3, 9 and 10. A new pharmacological effect. The relative tumor proliferation rate of study group 11 is still at a high level of efficacy, showing the intratumoral local synergy provided by the relevant components of animal cells and its secondary effects (such as in situ vaccine effects) and drugs used in combination with the above new pharmacological effects effect.

In the above table, the difference in dosing between study groups 5 and 9, 7 and 10 is the dosing concentration, which in turn is the short-term effect including the local effect (or local synergy) in Examples 2-10 required conditions. The new pharmacological effects shown by the mid-term drug effects of study groups 5 and 7 (and 6 and 8) should then include, and possibly even be primarily secondary effects of this short-term effect. The following examples further investigate this short-term effect.

In one experiment, the experimental animals were BALB/c mice , which were randomly divided into 2 study groups (1 and 2), with 6 mice in each study group. The research drugs of research groups 1 and 2 were animal cell-related fraction 1 and animal cell-related fraction 1/10% arginine in the previous experiment, respectively. Each group was treated once, and the drug was intradermally administered to the axilla of the right front leg, with an injection volume of 100 ul/piece. 7 days after administration, the animals were euthanized, and the intradermal nodules in the right foreleg of the mice were dissected out. After the slices were washed, the abnormal (such as necrosis, nodules, etc.) areas (such as the cross section of the nodules) that were different from normal muscles were measured. area.

The abnormal areas of study groups 1 and 2 were 37.27±12.14mm ² and 47.14±12.26mm ² , respectively. These results are in good agreement with the results of similar experiments in Example 9. These results show that the animal cell-related components in the technical solution of the present invention are similar to high-concentration ethanol, and the first drug effect shown after local administration is mainly local action (or local synergy), and the local effect (or local synergy) effect) is mainly, or at least not negligible, includes local chemical effects (or local chemical synergies), especially local chemical effects (or local chemical synergies) similar to chemical ablative effects, which are mainly, or at least local chemical synergies Non-negligible effects of tissue destruction in the subcutaneous drug penetration zone independent of these effects are included. These local effects (or local synergies) are fully consistent with the local effects (or local synergies) shown by the intratumoral administration in Examples 2, 5 and 9.

The results of the above studies and more similar studies demonstrate that the short-term effects following extratumoral (preferably immunologically favorable) administration of the compositions of the invention are predominantly, or at least not negligible, local effects (or local synergistic effects) ), wherein the long-term efficacy is mainly, or at least not negligible, the secondary effects produced by the local effect (or local synergy), and the secondary effects are mainly, or at least not negligibly including the vaccine effect. Thus, the compositions of the present invention that provide extra-tumoral vaccine action are quite different from classical vaccines in that the antigenic substance is likely to be predominantly, or at least not negligibly, nodular antigens produced after administration. In addition, animal cell-related components, like other foreign substances, may also have non-specific antigenic effects, which may also enhance the efficacy of the vaccine effect.

According to the above studies, the immunopharmacological function including this secondary immune effect enables the composition of the present invention to provide a drug effect that greatly exceeds the expectations of the prior art for cell-related components, thereby becoming a therapeutic drug (vaccine-like) rather than an immune system in the prior art. Enhancers; this pharmacological function also allows the compositions of the present invention to provide medicinal uses well beyond those contemplated by the prior art for cell-associated components, such as for any locally pathological disease, preferably secondary to local action (or local synergy) Immune-related localized disease treatable by immune stimulation of the body. Furthermore, the composition of the present invention can be used for local lesions (for example, cytotoxic drugs, antiviral drugs, antibacterial drugs, vascular inhibitor drugs, immune drugs, etc.) such as solid tumors). For example, the compositions of the present invention can be used as immunopharmaceuticals (vaccine-like) for the treatment of the above-mentioned indications, as opposed to these mainly antipathogen-based anti-topical disease drugs.

The realization of this secondary immune effect also requires that the compositions of the present invention have pharmacological properties different from those of the prior art. In addition to the above-mentioned special pharmacological methods, special pharmacological compositions are also required, such as their local action (or local synergistic effects) in Examples 2-8. Preferred technical solutions for pharmacological compositions (for example, selection of cell-related components, combination of selection, pharmacological ratio, pharmacological concentration, pharmacological volume, etc.). For example, although there is no discovery of the relationship between the administration volume and the intratumoral target volume, a necessary condition for the administration volume is obviously the formation of nodules required as antigens, for example: when the total tumor volume ≥ 2.85cm ³ , The dosage is greater than 0.01ml/kg human, 0.015-0.25ml/kg human, preferably 0.020-0.25ml/kg human, which requires the animal cell-related component (or containing it) of the present invention for local injection outside the tumor The administration volume of the pharmaceutical composition) is >2 times, or 3-100 times, preferably 5-100 times (eg ≥ 1 ml, or 1.5-50 ml) compared to the conventional vaccine volume administration volume.

Some other compositions of the invention prepared by the method of Example 1 can also obtain similar results in similar experiments of the above examples.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the scope of the present invention. within the scope of protection.

Claims

The use of animal non-pathogenic cell-related components as active ingredients that can provide therapeutic effects in the preparation of topical pharmaceutical compositions for the treatment of local pathological diseases.
The use according to claim 1, characterized in that the pharmaceutical composition further comprises a chemically active ingredient and/or a biologically active ingredient capable of producing a synergistic effect with the cell-related component, and the cell-related component is associated with the The amount ratio of the chemically active ingredient (cytospecific volume/weight-volume percent concentration of the chemically active ingredient) is (1.5-100)/(0.1-1), and/or the amount of the cell-related component and the biologically active ingredient The ratio (cytospecific volume/weight-volume percent concentration of bioactive ingredient) was (60-180)/(0.1-60).
A topical pharmaceutical composition for treating local pathological diseases, characterized by comprising, as an active ingredient that can provide a therapeutic effect, an animal non-pathogenic cell-related component.
The topical pharmaceutical composition according to claim 3, further comprising a chemically active ingredient or/and a biologically active ingredient capable of producing a synergistic effect with the non-pathogenic cell-related component of the animal, the cell-related component and The amount ratio of the chemically active component (cytospecific volume/concentration of the chemically active component) is (1.5-100)/(0.1-1), and/or the amount ratio of the cell-related component to the biologically active component ( Cytospecific volume/bioactive ingredient concentration) was (60-180)/(0.1-60).
The application or topical pharmaceutical composition according to any one of claims 1-4, characterized in that the therapeutic effect includes a short-acting therapeutic effect and/or a medium-long-acting therapeutic effect, and the short-acting therapeutic effect includes a local effect (or a local synergistic effect); the mid- and long-term therapeutic effect includes the secondary effect (or mid- and long-term synergistic effect) of the local effect (or local synergistic effect).
The application or topical pharmaceutical composition according to any one of claims 1 to 5, wherein the animal is an animal with minimized immunogenicity, preferably an allogeneic animal selected from autologous or/and minimized transplantation reactions; The cells are one or more of somatic cells, immune cells, and stem cells; the cell-related components are one or more selected from animal tissue components or/and animal cell components related to the cells. Species, wherein the animal tissue component is selected from one or more of the following groups: animal tissue, tissue fragmentation of animal tissue, cell fragmentation of animal tissue; The animal cell component is selected from one of the following groups or more: animal native cells, native cell disrupting components, and derivatives or engineered analogs of said native cells or native cell disrupting components.
The application or topical pharmaceutical composition according to any one of claims 1 to 6, wherein the pharmacological concentration (cytospecific volume) of the animal non-pathogenic cell-related component in the topical pharmaceutical composition is >20 %, ≥30%, 30-90%, preferably 45-90% or 75-85%.
The use or pharmaceutical composition according to any one of claims 1 to 7, wherein the animal non-pathogenic cell-related components include animal cell-related semi-fluid-like groups that can make the composition a semi-fluid-like composition wherein the semi-fluid-like component is selected from one or more of animal tissue semi-fluid-like components or/and animal cell semi-fluid-like components, and wherein the animal tissue semi-fluid-like component is selected from the following One or more of the group: animal tissue semi-fluid, animal tissue tissue fragmentation semi-fluid, animal tissue cell fragmentation semi-fluid; the animal cell semi-fluid-like component is selected from one or more of the following groups : Semi-fluid concentrates of animal natural cells, semi-fluid concentrates of natural cell destruction components, semi-fluid concentrates of natural cells or derivatives or engineering analogs of natural cell destruction components.
The use or pharmaceutical composition according to claim 8, wherein the cells comprise one or more of the following groups: blood cells, immune organ cells, other cells enriched in the tissue, wherein: the blood cells It is selected from one or more of the following cells and their derivatives: red blood cells, white blood cells, and platelets; the immune organ cells are selected from one or more of the following cells and their derivatives: lymphocytes, T cells, CD cells; the other cells are selected from the group consisting of one or more of the following cells and derivatives thereof: skin cells, muscle cells, secretory gland cells, stem cells.
The application or pharmaceutical composition according to any one of claims 2 or 4-9, wherein the chemically active ingredient comprises a weak local action compound and/or a cytotoxic drug; when the chemically active ingredient is a cytotoxic drug, The amount ratio of the non-pathogenic cell-related components of the animal to the cytotoxic drug (cytospecific volume/cytotoxic drug weight-volume percentage concentration) is (2-100)/(0.1-1); when the chemical activity When the component is a weak local action compound, the amount ratio of the non-pathogenic cell-related component of the animal to the weak local action compound (cytospecific volume/weak local action compound weight-volume percentage concentration) is (1.5-40)/( 0.1-1).
The use or pharmaceutical composition according to claim 10, wherein the weakly locally acting compound comprises one or more of the following group: amino acid nutrients, vital dyes, quinine compounds, carbohydrate nutrients.
The use or pharmaceutical composition according to claim 10 or 11, wherein the weakly locally acting compound comprises a vital dye, and the cell-associated component and the vital dye are in an amount ratio (cytospecific volume/vital dye weight) - volume percent concentration) is (2-40)/(0.1-1).
The application or pharmaceutical composition according to claim 10, wherein the chemically active ingredients include cytotoxic drugs, amino acid nutrients and/or vital dyes.
The application or pharmaceutical composition according to claim 11, wherein the amino acid nutrients are selected from 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 pharmacological concentration of the amino acid nutrients in the topical pharmaceutical composition is >2.5%, or 5-30%, It is preferably 5-25%.
The application or pharmaceutical composition according to claim 11, wherein the vital dye comprises red Bengal with a pharmacological concentration of 2.5%-20% and/or a pharmacological concentration of ≥ 0.25%, or 0.25-2.5%, preferably 0.5-2.5% methylene blue dyes, the methylene blue dyes include one or more of methylene blue, patent blue, isosulfur blue and new methylene blue.
The use or pharmaceutical composition according to claim 10, wherein the cytotoxic drug is selected from one or more of the following groups: drugs that disrupt DNA structure and function, such as cyclophosphamide, carmustine, metal Platinum complexes, doxorubicin-like drugs, topotecan, irinotecan; drugs that interfere with transcription of RNA intercalated in DNA, such as antitumor antibiotics; drugs that interfere with DNA synthesis, such as 5-fluorouracil (5-Fu) , furofluorouracil, difurfluorouracil, cytarabine, cyclocytidine, 5-azacytidine; drugs that affect protein synthesis, such as colchicines, vinblastines, taxanes, and in In the pharmaceutical composition, the concentration of the cytotoxic drug is ≥0.1%, 0.1-15%.
The application or pharmaceutical composition according to any one of claims 2 or 4-11, wherein the biologically active ingredient is selected from one or more of the following groups: antigens, immunomodulatory antibodies, cytokines, and adjuvants.
The use or pharmaceutical composition according to claim 17, wherein the antigen is selected from microbial antigens or tumor antigens, the microbial antigens are selected from antigens derived from one or more of the following microbiome: bacteria, such as purulent Streptococcus, Serratia serratia, BCG, Clostridium tetanus, Clostridium butyricum, Lactobacillus acidophilus, Bifidobacterium; viruses such as hepatitis B virus, adenovirus, scab virus, vaccinia virus, mumps virus, Newcastle disease virus, polio virus, measles virus, Seneca Valley virus, Coxsackie virus, reovirus; parasites, such as Plasmodium; the immunomodulatory antibody is selected from one of the following groups or more: antibody blockers against inhibitory receptors, such as blocking antibodies against CTLA-4 molecules and PD-1 molecules; antibody blockers against ligands of inhibitory receptors, against immune-reactive cells Activating antibodies to surface stimulatory molecules, such as anti-OX40 antibodies, anti-CD137 antibodies, anti-4-1BB antibodies; neutralizing antibodies to immunosuppressive molecules in the solid tumor microenvironment, such as anti-TGF-p1 antibodies; One or more of the following: tumor necrosis factor, interferon, interleukin.
A pharmaceutical composition comprising an animal non-pathogenic cell-associated component and a suitable pharmaceutically acceptable carrier for use in the treatment of local disease, including solid tumors.
A method for the treatment of local diseased diseases, characterized in that it comprises the steps of: promoting local action (or local synergy) related immune effects to the local lesions of an individual in need thereof or/and outside the lesions. Secondary effects of action sensitive topical administration of a therapeutically effective amount of a pharmaceutical composition according to any one of claims 3-19.
21. The method of claim 20, comprising the step of administering a therapeutically effective amount of said pharmaceutical composition to said individual within a local lesion, or within a local lesion and outside said local lesion.
The method according to claim 20 or 21, wherein the ratio of the administered 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 method according to any one of claims 20-22, characterized in that the administration amount of the pharmaceutical composition is ≥ 1ml, or the administration amount within the local lesion is 10-150ml or/and the administration amount outside the local lesion is 1.5- 50ml.
The method according to any one of claims 20-23, characterized in that, further comprising optionally performing chemotherapy, immunotherapy, radiation therapy, surgery, chemical ablation, and physical ablation before, during or after the administration of the pharmaceutical composition. one or more treatments.
The use, pharmaceutical composition or method according to any one of claims 1 to 24, characterized in that the applicable individual for the treatment includes one or more of the following group: an immunosuppressed individual, an individual that can be administered in local lesions, Individuals whose local diseased tissue can be chemically ablated, individuals who can produce secondary immune substances in local lesions, and individuals who can produce secondary immune substances in the drug delivery area outside the lesions.
A pharmaceutical kit comprising one or more individual containers containing the pharmaceutical composition of any one of claims 3-19.
The pharmaceutical kit of claim 26, further comprising instructions or labels on how to administer the pharmaceutical composition to an individual in need thereof, wherein the administration comprises administration within the local lesion, or within the Local intralesional and local extralesional administration, wherein the local extralesional administration, for example, comprises subcutaneous injection in the armpit of the individual.
The application, pharmaceutical composition, method or kit according to any one of claims 1 to 27, wherein the local pathological disease includes tumor, non-tumor swelling, local inflammation, abnormal secretion gland function and skin disease, wherein the Such tumors include malignant and non-malignant solid tumors.
The application, pharmaceutical composition, method or kit according to claim 28, wherein the solid tumor comprises one or more of the following tumors and secondary tumors: breast cancer, pancreatic cancer, thyroid cancer, and nasopharyngeal cancer , prostate cancer, liver cancer, lung cancer, colon cancer, oral cancer, esophagus cancer, stomach cancer, throat cancer, testicular cancer, vaginal cancer, uterine cancer, ovarian cancer, brain tumor, lymphoma.